27131

UNIVERSIDADE FEDERAL FLUMINENSE
FACULDADE DE VETERINÁRIA
PROGRAMA DE PÓS-GRADUAÇÃO EM MEDICINA VETERINÁRIA
(CLÍNICA E REPRODUÇÃO ANIMAL)
ISABEL OLIVEIRA COSENTINO
USO DE PROTOCOLOS DE INDUÇÃO DO ESTRO SINCRONIZADO SEGUIDOS

DE PROTOCOLOS DE RESSINCRONIZAÇÃO PRECOCE DO ESTRO EM
PEQUENOS RUMINANTES
Niterói – RJ
2022

PEQUENOS RUMINANTES
Tese apresentada à Universidade

Federal Fluminense, como parte das
exigências do Programa de Pós-Graduação
em Medicina Veterinária para obtenção do
título de Doutora.
Área de Concentração: Clínica e

Reprodução Animal.
Orientador: Prof. Dr. Felipe Zandonadi

Brandão.
Coorientador: Dr. Mario Felipe Alvarez

Balaro.
Niterói, RJ
2022
Ficha catalográfica automática - SDC/BFV
Gerada com informações fornecidas pelo autor
C834u Cosentino, Isabel Oliveira

DE PROTOCOLOS DE RESSINCRONIZAÇÃO PRECOCE DO ESTRO EM PEQUENOS
RUMINANTES / Isabel Oliveira Cosentino. - 2022.
193 f.: il.
Orientador: Felipe Zandonadi Brandão.

Coorientador: Mario Felipe Alvarez Balaro.
Tese (doutorado)-Universidade Federal Fluminense, Faculdade
de Veterinária, Niterói, 2022.
1. Reprodução animal. 2. Controle hormonal do ciclo

estral. 3. Caprinocultura. 4. Ovinocultura. 5. Produção
intelectual. I. Brandão, Felipe Zandonadi, orientador. II.
Balaro, Mario Felipe Alvarez, coorientador. III. Universidade
Federal Fluminense. Faculdade de Veterinária. IV. Título.
CDD - XXX
Bibliotecário responsável: Debora do Nascimento - CRB7/6368


PEQUENOS RUMINANTES
Tese apresentada à Universidade Federal

Fluminense, como parte das exigências
do Programa de Pós-Graduação em
Medicina Veterinária para obtenção do
título de Doutora.
Área de Concentração: Clínica e
Reprodução Animal.
BANCA EXAMINADORA
Prof. Dr. Felipe Zandonadi Brandão – UFF

Presidente
Prof. José Nélio de Sousa Sales - UFJF
Profa. Dra. Raquel Pérez Clariget - Udelar
Dr. Luiz Gustavo Bruno Siqueira - Embrapa Gado de Leite
Dr. Pedro Henrique Nicolau Pinto - UFF
Niterói
2022
À minha família
AGRADECIMENTOS
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil
(CAPES) pelo financiamento da bolsa de estudo durante o doutorado.
Aos meus pais, Valéria e Giovanni, que me acompanham em cada sonho e

cada vitória, como deles próprios. Obrigada pelos sermões incansáveis, pelas
palavras e gestos de carinho, pelas noites mal dormidas e por todo o sacrifício que
vocês fizeram para que eu realizasse meus sonhos. Amo vocês mais do que consigo
expressar.
Agradeço à minha família, em especial os meus avós, José, Iliana e Magaly,

que sempre me serviram de exemplo de força, determinação e perseverança.
Obrigada por todo o carinho e suporte que vocês me deram toda a minha vida,
vocês são e sempre serão motivos de orgulho para mim.
Às amigas fora da pós-graduação, Julia Zordan, Maria Fernanda Nunes, que

me acompanharam e deram apoio ao longo destes últimos anos, durante as
atividades experimentais e comemorações. Um agradecimento especial aos amigos
da Marinha do Brasil, que me acompanharam neste último ano e meio, dando
suporte à “quase interminável” fase de escrita e revisão dos artigos. Obrigada a
todos por tornar a vida mais divertida.
Aos amigos da pós-graduação e do GEPECO, pelos momentos que

passamos juntos, pelo apoio, amizade e carinho, além de toda ajuda necessária
para realização dos experimentos. Em especial Augusto, Ana Clara e Juliana, sem
vocês meu último experimento não seria possível. E Pedro, pelo suporte nos
experimentos realizados no Uruguai e escrita.
Aos meus orientadores Prof. Felipe Zandonadi Brandão e Mario Felipe

Alvarez Balaro, por todas as oportunidades e confiança recebida desde a
graduação. Obrigada por confiarem a mim uma linha de pesquisa pela qual sou
apaixonada e com a qual me identifico; pela paciência e por todo o cuidado e
atenção que recebi; e por fazer esse sonho se tornar possível. Agradeço também
pelo suporte e compreensão dedicada no último ano e meio, ao me permitir trilhar
novos caminhos e conquistar novos sonhos servindo à Marinha do Brasil.
Aos professores Rodolfo Ungerfeld e Raquel Pérez Clariget, pela parceria
realizada com a Universidad de la República do Uruguai. Agradeço imensamente
por abrirem as portas de seu grupo de pesquisa para nós e compartilharem ótimos
momentos durante o período de experimento e escrita.
Aos funcionários e colaboradores da Estación Experimental Bernardo

Rosengurtt da Facultad de Agronomía, Universidad de la República, em Cerro
Largo, pelo suporte técnico e manejo de nossos animais experimentais.
Ao proprietário e colaboradores do Capril Vale das Amalthéias por nos

receberem tão bem e permitirem a realização de nossos experimentos em suas
instalações.
Aos funcionários da Fazenda Escola de Cachoeiras de Macacu da UFF, pelo

suporte técnico e manejo de nossos animais experimentais.
Aos professores do doutorado por dividirem comigo seu conhecimento e me

permitirem subir mais um degrau na escada do conhecimento.
Às cabras e ovelhas, nossos objetos de estudo, sem elas nada seria

possível.
“Por vezes sentimos que aquilo que fazemos não é senão uma gota de água
no mar. Mas o mar seria menor se lhe faltasse uma gota”.
– Madre Teresa de Calcuta

RESUMO
O objetivo deste estudo foi avaliar distintos protocolos de indução do estro

sincronizado e de ressincronização precoce do estro nas espécies caprina e ovina
em diferentes categorias de ordem de parto. Foram realizados três experimentos na
espécie caprina e quatro na espécie ovina.
No primeiro experimento na espécie caprina, foi avaliado o efeito do momento da

aplicação de 25 μg de lecirelina (28 ou 34h e controle com aplicação de solução
salina às 28h), após a retirada da esponja de medroxiprogesterona (MAP) sintética,
visando a sincronização da ovulação. Os resultados demonstraram que a lecirelina
usada às 28 ou 34 h é eficiente para a sincronizar a ovulação. Não foram
encontradas diferenças (P > 0,05) entre os grupos tratados com GnRH e o grupo
controle quanto a apresentação de cio e média das ovulações. Entretanto, os
intervalos entre a retirada da esponja e a ovulação, e da apresentação do cio até a
ovulação apresentaram menor variação (P < 0,05) nos grupos tratados. No segundo
estudo, foi avaliado o efeito do uso de uma esponja de MAP sintética durante a fase
luteal (D16-D21) sobre a funcionalidade do corpo lúteo (CL) de cabras gestantes e
não gestantes. Para isso, todas as fêmeas receberam um protocolo de indução do
estro sincronizado e foram inseminadas. Na sequência, foram divididas
aleatoriamente em dois grupos experimentais (com e sem esponja) durante o D16
até D21 do ciclo estral. Foram realizadas dosagens séricas de P4 e avaliação
ultrassonográfica do CL existente durante o período de permanência da segunda
esponja. Após o diagnóstico de gestação (D30) as fêmeas foram divididas em
gestantes e não gestantes e se receberam ou não a segunda esponja para a análise
dos dados. Não foram encontradas diferenças nos níveis séricos de P4 entre os
grupos ou interações entre tratamentos e tempo. Assim, o uso da segunda esponja
de MAP, para a ressincronização do estro, não interferiu na produção de P4
endógena nem na viabilidade luteal.
No terceiro experimento, as cabras passaram por protocolo de indução do estro

sincronizado, sem que fossem inseminadas ou acasaladas. No momento da
ressincronização (D21; momento da retirada da segunda esponja), receberam
solução salina ou 100 UI de eCG visando acompanhar a dinâmica folicular e
ovulação subsequente. Também foi avaliado o efeito do manejo reprodutivo (monta
natural x inseminação artificial) neste protocolo de ressincronização. Em caprinos
não foram encontradas diferenças entre os grupos quanto ao tempo de ovulação,
sincronização da ovulação e tamanho do folículo. Entretanto, a associação de eCG
com monta natural apresentou maior taxa de apresentação de cio e gestação
quando comparado com inseminação artificial sem eCG. Em ovinos, o primeiro
estudo teve metodologia similar ao segundo experimento caprino, com segunda
esponja sendo mantida pelo período de D12-D17, e diagnóstico precoce da
gestação sendo realizado no D17. Assim como em caprinos, após a segunda
esponja de MAP, utilizada para a ressincronização do estro, não houve diferença na
concentração sérica de P4 nem a viabilidade luteal para a espécie ovina,
independente do grupo. O segundo estudo teve metodologia similar ao terceiro
caprino comparando três doses de eCG (0 UI, 200 UI e 300 UI), sem avaliar o
método de acasalamento. Para ovelhas, não houve diferença entre as médias dos
grupos, entretanto, 200 UI de eCG na ressincronização tornou o momento de
ovulação mais homogêneo. No terceiro estudo, os animais foram sincronizados e
ressincronizados, e avaliadas pelo diagnóstico precoce de gestação. As fêmeas
foram divididas aleatoriamente em dois grupos: fêmeas re-inseminadas
considerando o diagnóstico de gestação (apenas fêmeas vazias foram
inseminadas), enquanto o outro grupo não (independente do diagnóstico, todas as
fêmeas foram inseminadas). Nesse estudo foi avaliado o efeito de uma nova IA em
fêmeas gestantes, se levaria a perdas gestacionais, mostrando que não houve
diferença entre os grupos, e que o protocolo em si não leva a perdas gestacionais,
mas que o diagnóstico precoce auxilia na diminuição de manejo de fêmeas já
gestantes. No quarto experimento, o protocolo de ressincronização foi testado em
fêmeas 30 dias pós-parto, fêmeas secas na estação e nulíparas. O protocolo de
ressincronização foi eficiente em ovelhas nulíparas e multíparas, apresentando
diferenças apenas na fecundidade, que foi maior nas multíparas. Entretanto, o
tratamento não foi eficiente para ovelhas no pós-parto, visto que poucas
responderam ao protocolo e ficaram gestantes. Assim, os protocolos de
ressincronização precoce em pequenos ruminantes podem ser realizados com
sucesso, desde que consideradas as particularidades de cada espécie e categoria.
Palavras-Chave: Corpo lúteo, perfusão luteal, ciclo estral, controle hormonal,

caprinos e ovinos.
ABSTRACT
The aim of this study was to evaluate different protocols for estrus synchronization
and early estrus resynchronization in sheep and goats, in different calving order
categories. Three experiments were carried out with goats and four with sheep. In
the first experiment in goats, the effect of the moment of application of 25 μg of
lecirelin (28 or 34h and control with application of saline solution at 28h) was
evaluated after a synthetic medroxyprogesterone (MAP) sponge withdrawal, aiming
ovulation synchronization. The results showed that lecirelin used at 28 or 34h is
efficient to synchronize ovulation. There were no differences (P > 0.05) among
GnRH-treated and control goats in estrus responses and mean ovulation rates.
However, the intervals from MAP sponge withdrawal to ovulation and from the estrus
onset to ovulation were less variable (P < 0.05) in both GnRH-treated groups. In the
second study, the effect of using a MAP sponge during the luteal phase (D16-D21)
on the functionality of the corpus luteum (CL) of pregnant and non-pregnant goats
was evaluated. For this, all females passed through a synchronization protocol and
were inseminated. Subsequently, they were randomly divided into two experimental
groups (with and without sponge) during D16 to D21 of the estrous cycle. Serum
dosages of P4 and ultrasound evaluation of the existing CL were performed during
the period of permanence of the second sponge. After pregnancy diagnosis (D30)
the females were divided into pregnant and non-pregnant and whether or not they
received the second sponge for data analysis. No P4 differences were found
between groups or for the interaction of group and time. Thus, the use of the second
MAP sponge for estrus resynchronization did not interfere with endogenous P4
production or luteal viability. In the third experiment, the goats underwent another
synchronization protocol, without being inseminated or mated. At the time of
resynchronization (D21; time of removal of the second sponge), they received saline
solution or 100 IU of eCG to monitor follicular dynamics and subsequent ovulation.
The effect of reproductive management (natural mating x artificial insemination) in
this resynchronization protocol was also evaluated. In goats, no differences were
found regarding ovulation time, synchronization and follicle size. However, the
association between natural breeding and eCG presented a greater estrus
manifestation and pregnancy rates when compared to artificial insemination without
eCG. In sheep, the first study had a similar methodology to the second goat
experiment, with the second sponge being maintained for the period from D12-D17,
and early diagnosis of pregnancy being performed on D17. As in goats, after the
second MAP sponge, used for estrus resynchronization, there were no differences in
serum P4 values or luteal viability for ewes, regardless the group. The second study
had a similar methodology to the third goat comparing three doses of eCG (0 UI,
200 UI and 300 UI), without evaluating the mating method. For sheep, there were no
difference between group means, however, 200 IU of eCG in resynchronization
induced a more homogeneous ovulation. In the third study, the animals were
synchronized and resynchronized, and evaluated for early pregnancy diagnosis
(regardless of the diagnosis, all females were inseminated). In this study, the effect of
a new AI in pregnant females was evaluated, whether it would lead to pregnancy
losses, showing that there was no difference between the groups, and that the
protocol itself does not lead to pregnancy losses, but that early diagnosis helps to
reduce management of pregnant females. In the fourth experiment, the
resynchronization protocol was tested in females 30 days postpartum, dry females in
estrous season. The resynchronization protocol was efficient in nulliparous and
multiparous ewes, groups presented differences only in fecundity, which was higher
in multiparous. However, the treatment was not efficient in postpartum ewes, since
few responded to the protocol and became pregnant. Thus, early resynchronization
protocols in small ruminants can be successfully performed, if the particularities of
each species and category are considered.
Keywords: Corpus luteum, luteal perfusion, estrous cycle, synchronization,

resynchronization
LISTA DE FIGURAS
CAPÍTULO I
2.1 ARTIGO REVISÃO DE LITERATURA: REPRODUÇÃO EM PEQUENOS

RUMINANTES – APLICAÇÕES PRÁTICAS
Figura 1. Esquema das fases do ciclo estral em pequenos ruminantes. ................. 31
Figura 2. Representação da duração de cada etapa para a execução de três partos

em dois anos em sistemas de produção de cordeiros. PVE – período
voluntário de espera (período de involução uterina e retorno da ciclicidade);
P. de Serviço – Período de serviço (janela de tempo em que deve ocorrer a
concepção). ................................................................................................. 32
Figura 3. Representação gráfica do sistema Cornel STAR©, onde cada ponta da

estrela equivale ao período de desmame e separação dos filhotes de suas
mães. No esquema, cada cor representa um lote de fêmeas, de modo que
haja coincidência ente as estações de monta natural ou inseminação artificial
(MN/IA) de um lote com a estação de parição (parto) de outro lote. No
esquema também são representados o período de aleitamento (lactação) e
desmame (DSM). Adaptado do site do sistema Cornel STAR©23 ................. 33
Figura 4. Representação de um protocolo de estimulação por luz em pequenos

ruminantes................................................................................................... 35
Figura 5. Representação de um protocolo de estimulação por efeito macho em

pequenos ruminantes. ................................................................................. 36
Figura 6. Representação de um protocolo curto baseado em progestágenos em

pequenos ruminantes. A duração da exposição ao progestágeno pode variar,
assim como os outros hormônios utilizados em associação. Os protocolos
podem ser curtos (6 dias; conforme ilustrado nesta figura), médios (9 dias)
ou longos (a partir de 12 dias), onde o uso de prostaglandinas pode ser
suprimido. O uso dos progestágenos estimula o crescimento folicular e a
ovulação. Nesses protocolos a aplicação da eCG e PGF pode ser feita no
dia anterior ou no dia da retirada do progestágeno...................................... 39
Figura 7. Representação de um protocolo baseado em análogos das
prostaglandinas em pequenos ruminantes. O intervalo entre as aplicações
pode variar entre 7 a 11,5 dias, levando à luteólise e ovulação após a
aplicação da prostaglandina. ....................................................................... 42
Figura 8. Representação esquemática comparando protocolos de indução do estro

sincronizado tradicionais (A) e de ressincronização precoce em pequenos
ruminantes (B e C), onde as datas são as sugeridas para caprinos (B) e para
ovinos (C). ................................................................................................... 46
Figura 9. A) Tinta xadrez colorida para pintar o peito do macho B) Monta natural,
onde vê-se o macho com o peito pintado e a fêmea com o dorso colorido de
monta anterior. ............................................................................................ 47
Figura 10. Comparação entre cérvix caprina (esquerda) e ovina (direita). Cérvix
caprina apresentando maior alinhamento cervical, enquanto que na espécie
ovina visualiza-se uma cérvix mais tortuosa. ............................................... 48
Figura 11. Esquema de local de inseminação, dose inseminante e volume

recomendado para inseminação artificial. A) deposição vaginal; B) deposição
cervical superficial; C) deposição cervical profunda; D) deposição intrauterina
transcervical; E) deposição intrauterina laparoscópica. ............................... 49
Figura 12. A-B) Apoio bipedal da fêmea para inseminação cervical superficial. Com
apoio em uma superfície emborrachada (A), ou apenas com a sustentação
do colaborador (B). C) Material utilizado para inseminação artificial
superficial .................................................................................................... 50
Figura 13. A) Imagem de ovário vista pela videolaparoscopia, com um folículo em

evidência; B) Fêmea em posição de Trendelenburg, em um ângulo de
aproximadamente 45 a 60º com os posteriores para cima, com o útero sendo
localizado por videolaparoscopia para inseminação. ................................... 52
Figura 14. Momento da Inseminação em caprinos, baseado no tipo de sêmen e

62. .................. 53
característica do muco cervical. Adaptado de Fonseca et al.
Figura 15. A) Coleta de sêmen através de vagina artificial com fêmea no cio como
manequim; B) Coleta de sêmen através de eletro ejaculação: C) Material
utilizado para coleta por vagina artificial; D) Eletroejaculador. ..................... 54
Figura 16. Planilha de coleta de dados de Rufiação e Inseminação Artificial. ......... 56
Figura 17. A) Avaliação em Modo-B da morfologia luteal em escores de 1 a 3, onde

o escore 1 é considerado para CL em luteólise; B) Avaliação doppler do fluxo
sanguíneo luteal em escores de 1 a 4, onde o escore 1 é considerado para
56. ............................ 57
CL em luteólise. Imagens cedidas por Cosentino et al
2.2 ARTIGO REVISÃO DE LITERATURA: RECENTES AVANÇOS NOS

TRATAMENTOS DE RESSINCRONIZAÇÃO DA OVULAÇÃO EM
PEQUENOS RUMINANTES: UMA REVISÃO
Figure 1. A) Resynchronization protocol for ewes adapted from Cosentino et al.

(2019); B) Resynchronization protocol for does adapted from Cosentino et al.
(2022); C) 75 days traditional breeding season calendar D) Proposed
successive resynchronizations for ewes E) Proposed successive
resynchronizations for goats. ....................................................................... 84
CAPÍTULO II
1 ARTIGO: ATIVIDADE OVARIANA EM CABRAS SAANEN LEITEIRAS SUJEITAS

A PROTOCOLO INDUÇÃO DE OVULAÇÃO E INJEÇÃO ÚNICA DE LECIRELINA
(ANÁLOGO GNRH) 28 OU 36 H APÓS O TRATAMENTO CURTO DE
PROGESTERONA
Figure 1. Experimental proceedings timeline. ........................................................ 102
Figure 2. Small (detectable follicles ≤3.0 mm), medium-sized (˃3.0 and ˂6.0 mm)
and large (≥6.0 mm) antral follicle numbers determined ultrasonographically
in Saanen does subjected to short-term estrus synchronization and receiving
a saline solution (Gcon) or lecirelin dose at 28 h (G28h) or 34 h (G34h) after
sponge withdrawal from 52 to 156 h after sponge withdrawal (from 8 h before
to 96 h after it). .......................................................................................... 104
2 ARTIGO: PROTOCOLOS DE RESSINCRONIZAÇÃO PRECOCE EM CABRAS:
PROGESTÁGENOS PODEM SER USADOS ANTES DE UM DIAGNÓSTICO DE
GESTAÇÃO PRECOCE SEM AFETAR A FUNCIONALIDADE DO CORPO LÚTEO
Figure 2. Serum progesterone concentration (ng/mL) from Day 16 to Day 21 and on

Day 24, in pregnant (n=12) and non-pregnant (n=14) does. An intravaginal
sponge impregnated with medroxyprogesterone acetate was inserted in does
from the GNPSP and GPSP groups. ......................................................... 121
3 ARTIGO: INFLUÊNCIA DO ECG E MANEJO REPRODUTIVO NA

RESSINCRONIZAÇÃO DA OVULAÇÃO EM CABRAS LEITEIRAS
Figure 2. Reproductive outcomes per female compared within and between groups:
(A) estrus duration; (B) beginning of estrus to ovulation; (C) sponge
withdrawal to ovulation. ............................................................................. 136
and large (≥6.0 mm) antral follicle numbers determined ultrasonographically
in Saanen does subject to short-term estrus resynchronization and receiving
or not 100 IU of eCG in different breeding systems (natural mating or artificial
A,B,C,D,E
insemination). Different letters denote means with significant
differences over time (considering all treatments together; Fisher LSD test,
P<0.05)...................................................................................................... 137
CAPÍTULO III
1 ARTIGO: PROTOCOLOS HORMONAIS PARA RESSINCRONIZAÇÃO PRECOCE

DA OVULAÇÃO EM OVELHAS: USO DE PROGESTÁGENOS, ECG, E INCLUSÃO
DO DIAGNÓSTIOCO PRECOCE COM ULTRASSONOGRAFIA DOPPLER
COLORIDO
Figure 1. (I) Study 1 methodology: A) GPSP: pregnant animals with sponge; B)

GPNSP: pregnant animals without sponge; C) GNPSP: non-pregnant
animals with sponge; and D) GNPNSP: non-pregnant animals without
sponge. (II) Study 2 methodology: A) GroupCon; B) GroupeCG200; C)
GroupeCG300. (III) Study 3 methodology: A) GroupEPD; B) GroupNEP. .. 154
Figure 2. Serum progesterone concentration (ng/mL) from Day 12 to Day 17, in

pregnant (A) and non-pregnant (B) ewes. An intravaginal sponge
impregnated with medroxyprogesterone acetate was inserted in ewes from
the GNPSP and GPSP groups. ................................................................. 160
2 ARTIGO: INSEMINAÇÃO EM TEMPO FIXO E RESSINCRONIZAÇÃO PRECOCE

DA OVULAÇÃO DE OVELHAS EM DIFERENTES CATEGORIAS EM SISTEMA DE
PASTEJO: RESPOSTA DE MULTÍPARAS, NULÍPARAS E OVELHAS LACTANTES
DURANTE A ESTAÇÃO DE MONTA
Figure 1. Schematic representation of the experimental groups, hormonal protocols

used for estrous synchronization and resynchronization (when applied), with
indication of the moments where fixed time artificial inseminations, Doppler
ultrasonography (US) and B-Mode US were performed. ............................ 175
LISTA DE TABELAS
CAPÍTULO I

Tabela 1. Principais protocolos utilizados para induzir fêmeas caprinas e ovinas .... 40
Tabela 2. Principais protocolos utilizados para sincronizar fêmeas caprinas e ovinas.

.................................................................................................................... 43
Tabela 3. Características desejáveis para o sêmen do pequeno ruminante fresco,

refrigerado ou congelado. ............................................................................ 55
Tabela 4. Principais afecções reprodutivas possíveis de serem detectadas através

da ultrassonografia e os principais manejos empregados ........................... 58
CAPÍTULO II

PROGESTERONA
Table 1. Reproductive outcomes (mean ± SD) recorded in Saanen does subjected to

short-term estrus synchronization and receiving a saline solution (Gcon) or
lecirelin treatment at 28 h (G28h) or 34 h (G34h) after sponge withdrawal. 103
Table 1. Estrus response and manifestation (mean ± standard error) recorded in

Saanen does subject to short-term estrus resynchronization and receiving or
not 100 IU of eCG in different breeding systems (natural mating or artificial
insemination). ............................................................................................ 135
Table 2. Ovulation parameters (mean ± standard error) recorded in Saanen does

subject to short-term estrus resynchronization and receiving or not 100 IU of
eCG in different breeding systems (natural mating or artificial insemination).
.................................................................................................................. 135
Table 3. Post protocol ovarian parameters and pregnancy diagnosis (mean ±

standard error) recorded in Saanen does subject to short-term estrus
resynchronization and receiving or not 100 IU of eCG in different breeding
systems (natural mating or artificial insemination)...................................... 138
CAPÍTULO III

COLORIDO
Table 1. Time from sponge withdrawal to ovulation, and from estrous onset to
ovulation, in ewes that received 0 (group Con), 200 (group eCG200), or 300
(group eCG300) IU of eCG during a treatment for resynchronization of
ovulations (LSmean ± SEM). ..................................................................... 161
Table 2. Pregnancy rate and accumulated pregnancy in ewes subjected to

treatments for synchronization and resynchronization of ovulation with or
without pregnancy diagnosis with color DUS 17 days after first insemination.
.................................................................................................................. 161
DURANTE A ESTAÇÃO DE MONTA
Table 1. Rates of estrous presentation, pregnancy, pregnancy losses in ewes

submitted to the synchronization in the first FTAI moment (multiparous
ewes). ........................................................................................................ 176

submitted to the resynchronization in both FTAI moments for each category
(multiparous and nulliparous ewes). .......................................................... 177

(multiparous and post-partum ewes).......................................................... 178
LISTA DE SÍMBOLOS E ABREVIAÇÕES
CEUA Comitê de Ética do Uso de Animais

CL Corpo Lúteo
D Profundidade (do inglês: Depth)
D(nº) Dia (número)
eCG Gonadotrofina Coriônica equina (do inglês: equine Chorionic
Gonadotropin)
FPS Quadros por segundo
FRQ Frequência
g Gravidade (força da = 10 m/s²)
GN Ganho (do inglês: Gain)
GnRH Hormônio Liberador de Gonadotrofinas (do inglês: Gonadotropin
Releasing Hormone)
h Hora
i.m. Intramuscular
IA Inseminação Artificial
mg Miligrama
MHz Mega hertz
min Minutos
mm Milímetro
MN Monta Natural
n Número amostral
ºC Graus Celsius
P Probabilidade de significância
P4 Progesterona
PGF2α Prostaglandina F2α
PRF frequência de repetição de pulso
RIA radioimunoensaio em fase líquida
UI Unidades Internacionais
WF Filtro de parede (do inglês: wall filter)
SUMÁRIO
AGRADECIMENTOS ........................................................................................ 5
RESUMO .......................................................................................................... 8
ABSTRACT ..................................................................................................... 10
LISTA DE FIGURAS ....................................................................................... 12
LISTA DE TABELAS ....................................................................................... 17
LISTA DE SÍMBOLOS E ABREVIAÇÕES ....................................................... 20
SUMÁRIO ....................................................................................................... 21
CAPÍTULO I
CONSIDERAÇÕES INICIAIS
1 INTRODUÇÃO ............................................................................................. 25
2 REVISÃO DE LITERATURA ........................................................................ 27
2.1 ARTIGO REVISÃO DE LITERATURA: REPRODUÇÃO EM PEQUENOS RUMINANTES –

APLICAÇÕES PRÁTICAS.................................................................................................................... 27
2.2 ARTIGO REVISÃO DE LITERATURA: RECENTES AVANÇOS NOS TRATAMENTOS DE

RESSINCRONIZAÇÃO DA OVULAÇÃO EM PEQUENOS RUMINANTES: UMA REVISÃO .............. 76
3 HIPÓTESES ................................................................................................ 93
4 OBJETIVOS ................................................................................................. 94
4.1 OBJETIVO GERAL ............................................................................................................ 94
4.2 OBJETIVOS ESPECÍFICOS.............................................................................................. 94
5 METODOLOGIAS ........................................................................................ 95
CAPÍTULO II
CAPRINOS

PROGESTERONA .......................................................................................... 97

.......................................................................................................................115

RESSINCRONIZAÇÃO DA OVULAÇÃO EM CABRAS LEITEIRAS...............128
CAPÍTULO III
OVINOS

COLORIDO ....................................................................................................150

DURANTE A ESTAÇÃO DE MONTA .............................................................168
CAPÍTULO IV
CONSIDERAÇÕES FINAIS
1 CONCLUSÕES ...........................................................................................187
2 REFERÊNCIAS BIBLIOGRÁFICAS ............................................................188
3 ANEXOS .....................................................................................................190
3.1 PROTOCOLO CEUA CAPRINOS – 1021/2017 .............................................................. 190
3.2 PROTOCOLO CEUA OVINOS – 923/2017 ..................................................................... 191
3.3 TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO ............................................ 192

CAPÍTULO I
CONSIDERAÇÕES INICIAIS
25
1 INTRODUÇÃO
O uso de protocolos hormonais para a sincronização/indução do estro em

pequenos ruminantes permite que os partos sejam planejados e que ocorram de
forma concentrada em épocas desejáveis, facilitando o manejo dentro da
propriedade e permitindo a padronização de lotes, agregando valor no momento da
comercialização (SIMPLÍCIO; FREITAS; FONSECA, 2007). Sua aplicação em
pequenos ruminantes se destaca, pois em tais espécies a estacionalidade
reprodutiva (BALARO et al., 2014, 2018), influenciada por uma série de fatores
extrínsecos e intrínsecos, torna-se um viés no sistema produtivo. Assim, o uso de
protocolos hormonais permite a reprodução de cabras e ovelhas durante o anestro
estacional, aumentando a rentabilidade do sistema de criação e, igualmente,
distribuindo a oferta do produto, carne ou leite, ao longo do ano.
Visando o aumento dos índices de eficiência reprodutiva no plantel,

protocolos de ressincronização têm sido estudados e realizados com sucesso em
bovinos desde o início do século (BARTOLOME et al., 2005; SANI et al., 2011;
STEVENSON et al., 2003). Com variações no tempo e nos procedimentos, tais
protocolos são usados principalmente para se reduzir o intervalo de partos,
induzindo ovulações mais sincrônicas durante a estação reprodutiva. Ademais, tais
protocolos, se associados a métodos de diagnóstico de gestação eficientes e
precoces (SCULLY et al., 2014), podem elevar a eficiência reprodutiva do plantel, já
que permitem o uso de protocolos de ressincronização do estro tão cedo quanto aos
22 dias após a inseminação prévia e redução do tempo improdutivo dos animais no
rebanho (SÁ FILHO et al., 2014; SANI et al., 2011). Além disso, pode ser feita uma
nova inseminação artificial em tempo fixo (IATF) em intervalos semelhantes ao ciclo
estral natural (PALHÃO et al., 2020; PUGLIESI et al., 2019), para isso, um novo
dispositivo de progesterona (P4) deve ser inserido antes do diagnóstico de gestação.
Outrossim, estudos em novilhas de corte (KELLEY et al., 2016) e vacas leiteiras pós-
parto (WIJMA et al., 2018), demonstraram que o protocolo de ressincronização é
eficaz em melhorar as respostas reprodutivas em fêmeas de diferentes categorias.
Embora o diagnóstico precoce da gestação já seja utilizado em ovinos

(ARASHIRO et al., 2018) e caprinos (COSENTINO et al., 2018), o uso destes em
26
protocolos de ressincronização ainda não foi avaliado. Além disso, estudos sobre
protocolos de ressincronização em ovinos são escassos (MIRANDA et al., 2018) e
inexistentes em caprinos. Assim, há a necessidade de estudo dos efeitos e
viabilidade de tais protocolos na espécie caprina e ovina visando a melhoria da
produtividade e índices reprodutivos em diferentes categorias dentro do sistema de
produção.
27
2 REVISÃO DE LITERATURA

Reprodução em pequenos ruminantes – Aplicações práticas
Artigo submetido na Revista Brasileira de Buiatria

28
Reprodução em pequenos ruminantes – Aplicações práticas
Reproduction in small ruminants – Practical applications
Isabel Oliveira Cosentino1*; Pedro Henrique Nicolau Pinto1; Mário Felipe Alvarez
Balaro1; Felipe Zandonadi Brandão1
1Faculdade de Veterinária, Universidade Federal Fluminense, Niterói, Rio de Janeiro,

Brazil * Corresponding author. Rua Vital Brazil Filho, 64. Niterói, Rio de Janeiro,
24230-340, Brazil. isabelcosentino@id.uff.br
Resumo
A criação de pequenos ruminantes no Brasil vem crescendo e ganhando

destaque nas últimas décadas, mas ainda existe a necessidade de melhorias na
eficiência reprodutiva dos plantéis. Nesse contexto, as biotécnicas da reprodução e
as técnicas de manejo reprodutivo são aplicáveis aos cenários mais distintos
encontrados pelo país e são estratégias que permitem melhorar o desempenho dos
sistemas de criação. Nesta revisão, é abordado de forma objetiva as principais
ferramentas e biotécnicas reprodutivas voltadas aos pequenos ruminantes, tais
como as principais metodologias hormonais ou naturais utilizadas para a
sincronização e indução do estro em fêmeas; métodos de manejo reprodutivo;
biotécnicas como a inseminação artificial (IA) e o uso de outras ferramentas como o
diagnóstico ultrassonográfico da gestação, enfermidades reprodutivas e avaliação
da qualidade seminal.
Palavras-chave: biotécnica reprodutiva, caprinos, ovinos, ultrassonografia, manejo

reprodutivo.
Abstract
Small ruminants’ production in Brazil has been growing and gaining visibility
every year, but there is still a need for improvements in herds’ reproductive efficiency.
In this context, reproduction biotechniques and reproductive management are
applicable to the most different scenarios found cross country and are strategies that
allow improving the performance of breeding systems. In this review, it is highlighted
29
the main reproductive tools and biotechniques used in small ruminants’ reproduction,
such as the main hormonal or natural methodologies used for estrus synchronization
and induction in females; reproductive management methods; biotechniques such as
artificial insemination (AI) and the use of other tools such as ultrasound diagnosis of
pregnancy and reproductive pathologies and seminal quality assessment.
Keywords: reproductive biotechnology, goats, sheep, ultrasound, reproductive

management.
1. Introdução
O rebanho nacional de pequenos ruminantes (corte e leite) saiu de 22 milhões

de animais em 2013, para mais de 31 milhões em 2019 1,2. A região nordeste
concentra o maior efetivo do rebanho nacional, com impressionantes 94% do
rebanho caprino e 68% do rebanho ovino. A região Sul ocupa o segundo lugar com
1,8% dos caprinos e 20% dos ovinos 1.
De forma geral, os sistemas de criação nordestinos são menos tecnificados e

de característica familiar 3. Esta realidade fica bem ilustrada quando comparamos a
média de produção de leite caprino da região nordeste, 9,2 L/animal/ano, com a
média de produção da região Sudeste, 21,4 L/animal/ano 4. Em relação aos ovinos,
a produção de carne nacional vem aumentando, principalmente na região Sul. No
entanto, a produção nacional ainda não supre a demanda do mercado interno,
sendo necessária a importação de países vizinhos como o Uruguai 3,5.
Diante de diferentes realidades socio regionais, as biotécnicas da reprodução

e as técnicas de manejo reprodutivo são estratégias aplicáveis aos cenários mais
distintos e permitem melhorar a eficiência reprodutiva na ovino e caprinocultura 6.
Nesta revisão será abordado de forma objetiva as principais ferramentas e
biotécnicas reprodutivas voltadas aos pequenos ruminantes.
2. Fisiologia reprodutiva
As ovelhas e cabras são consideradas poliéstricas estacionais de dias curtos,

ou seja, manifestam estro repetidas vezes principalmente durante o outono e
inverno. O quão marcante é a estacionalidade varia com a raça, latitude e o tipo de
30
criação. Animais mantidos mais ao norte do país e próximos da linha do equador

tendem a manifestar estacionalidade menos marcante do que animais mantidos ao
sul do país 7. Com relação a espécie ovina na região Sudeste, Balaro et al. 8,9
demonstraram que ovelhas da raça Santa Inês apresentaram baixo grau de
8
estacionalidade, com poucas fêmeas apresentando anestro durante a primavera e
níveis de progesterona que indicam funcionalidade ovariana ao longo do ano 9.
Quanto aos machos, apesar de apresentarem diminuição na qualidade seminal e
10–13.
libido, continuam férteis ao longo do ano Em cabras, primíparas e multíparas
apresentaram estro durante outono e inverno, enquanto nulíparas concentraram a
apresentação de estro durante o inverno, e independente da ordem de parto, todas
14
apresentaram anestro durante a primavera . Na região Nordeste, no estado da
15,
Bahia, cabras Anglo-Nubianas demonstraram anestro no verão enquanto cabras
Saanen não demonstraram estro no Ceará 16; enquanto ovinos apresentam o estro
17
marcado na região sul do país, com estação reprodutiva ocorrendo no outono .
Além das condições estacionais impostas pela latitude, é importante destacar que
raças adaptadas aos trópicos tendem a ter a estacionalidade menos marcadas que
as raças importadas europeias.
O ciclo estral na espécie caprina dura em média 21 dias (podendo variar entre
18,19
17 a 25 dias) e, nas ovelhas, oscila entre 16 - 17 dias (Figura 1). Cabras tem
duração de estro em torno de 36 h (24 – 48 horas) e ovelhas 30 horas 19,20. O
período gestacional tanto para ovinos quanto caprinos é de aproximadamente 150
dias (5 meses) 19,21.
31
Figura 1. Esquema das fases do ciclo estral em pequenos ruminantes.
A foliculogênese ovariana ocorre ao longo do ciclo estral, de forma resumida,

o processo inicia com uma fase independente da ação das gonadotrofinas, onde há
19.
transformação de alguns folículos primordiais em primário Com o aumento das
concentrações séricas de estradiol há a inibição da secreção de Hormônio Folículo
Estimulante (FSH) pelo eixo hipotalâmico-hipofisário, iniciando a fase de seleção,
onde os folículos maiores e com maior número de receptores para FSH continuam
seu desenvolvimento enquanto os outros entram em atresia, dando sequência à fase
de dominância. Assim, ocorrem sucessivas ondas de emergência folicular enquanto
a fase folicular final e o pico ovulatório estiverem suprimidos pela ação inibitória da
progesterona (P4) sobre o Hormônio Luteinizante (LH), até que ocorra a luteólise e
22.
consequente queda da concentração de P4 O número de ondas foliculares varia
de acordo com a raça, fotoperíodo, estado nutricional e até por fatores inerentes ao
indivíduo, variando entre duas a seis ondas em caprinos e três a quatro ondas em
ovinos 18,19.
32
3. Sistemas de produção
Para os sistemas de produção de cordeiros de corte, consideram-se como

eficientes fêmeas que conseguem parir três vezes em um intervalo de dois anos
(Figura 2). Quando há necessidade de fornecer animais para o mercado durante o
ano todo e acelerar a produção de cordeiros, é possível adotar o sistema Cornel
STAR© (Figura 3), que permite chegar a cinco partos em três anos 23. Deve-se,
portanto, acompanhar de perto a condição de escore corporal das fêmeas e o
manejo nutricional, de forma a garantir que ela terá o suporte nutricional necessário
para atingir tal marco.
Figura 2. Representação da duração de cada etapa para a execução de três partos

em dois anos em sistemas de produção de cordeiros. PVE – período voluntário de
espera (período de involução uterina e retorno da ciclicidade); P. de Serviço –
Período de serviço (janela de tempo em que deve ocorrer a concepção).
33
Figura 3. Representação gráfica do sistema Cornel STAR©, onde cada ponta da

estrela equivale ao período de desmame e separação dos filhotes de suas mães. No
esquema, cada cor representa um lote de fêmeas, de modo que haja coincidência
ente as estações de monta natural ou inseminação artificial (MN/IA) de um lote com
a estação de parição (parto) de outro lote. No esquema também são representados
o período de aleitamento (lactação) e desmame (DSM). Adaptado do site do sistema
Cornel STAR©23
Atualmente o modelo para pequenos ruminantes é similar ao utilizado em

bovinos, chegando a um parto por ano com número de filhotes variando com a
prolificidade da fêmea. Em sistemas de produção de leite caprino, esta alta
frequência de partos pode não ser desejada em função da maior demanda de
atividade como secagem, cobertura e cuidados com os cabritos. Além disso, de
forma a manter a oferta de leite ao longo do ano, as fêmeas podem ser divididas em
lotes de manejo, onde algumas serão reproduzidas na estação e outras na contra
estação. Nestes sistemas há ainda a possibilidade de selecionar fêmeas com maior
24,25,
persistência de lactação o que pode ser mais interessante do que aumentar o
número de partos.
34
4. Controle do ciclo estral
Devido à estacionalidade reprodutiva, em algumas regiões do país, há a

necessidade do uso de biotécnicas que permitam induzir e sincronizar o estro
durante o anestro. Podem ser adotados diferentes métodos (naturais, artificiais ou a
associação destes), tais como o efeito macho, a manipulação do fotoperíodo e o uso
de protocolos hormonais – mais comumente utilizados 26,27. Tais protocolos podem
ser utilizados durante a estação reprodutiva para a sincronização do estro e/ou
ovulação com uso de análogos das prostaglandinas, ou durante o período de
28.
anestro, onde será realizada a indução do estro sincronizado No material
suplementar número 1 pode ser visto o comportamento de cabras no cio, sozinhas
ou junto ao macho.
4.1 Métodos não farmacológicos
Protocolos de luz (controle artificial do fotoperíodo) são provavelmente os

mais utilizados entre os métodos naturais, para a indução de estro. Baseia-se na
exposição dos animais, geralmente a fêmea e reprodutores, a 16h de luz (98 luxes
29.
na altura dos olhos) e 8h de escuridão, diariamente, por 60 dias Deste modo,
podem ser utilizados acionadores automáticos que mantém as luzes acessas das 04
às 08 e das 16 às 20 horas, totalizando 16 h de luz. Caso os animais fiquem em
galpões fechados ou com pouca luz natural, a luz ambiente pode ficar acessa
durante todo o período das 04 às 20h. Para garantir que essa quantidade mínima
de lux chegue na altura dos olhos, os dispositivos utilizados são de pelo menos 200
30.
luxes Transcorridos mais 60 dias, as fêmeas começam a apresentar sinais de
estro por aproximadamente 60 dias. 31–33. A queda abrupta da iluminação artificial
induz as fêmeas a retornarem a ciclicidade, da mesma forma que aconteceria com a
diminuição natural das horas de luz na entrada da estação reprodutiva. (Figura 4).
35
Figura 4. Representação de um protocolo de estimulação por luz em pequenos

ruminantes.
D – Dia; LH – Hormônio Luteinizante; FSH – Hormônio Folículo Estimulante.
Interações sócio-sexuais como os efeitos macho e fêmea são a estimulação

34,35,
sexual que um animal sexualmente ativo realiza sobre animais em anestro e
ocorrem de forma natural na natureza, principalmente no período de transição, os
que começam a ciclar antes induzem outros animais a entrarem no período
reprodutivo. Normalmente, fêmeas em anestro, que ficaram sem contato visual ou
olfativo com machos, por períodos em torno de 60 dias, quando expostas a machos
ativos têm sua secreção de LH e ovulação induzida e sincronizada nos primeiros
36,37.
dias de contato com o macho A primeira ovulação, que ocorre
aproximadamente 20h após a introdução do macho é comumente seguida por um
CL de curta duração, que sofre luteólise em até 48h, e depois uma segunda
ovulação ocorre, em torno de 6 a 9 dias após a introdução do macho, e dá origem a
38.
um CL de vida útil normal Quando se trata do efeito fêmea, o contato com fêmeas
ativas induz no macho um aumento na secreção de LH, acompanhado de um
34,39.
aumento no teor de fluido e concentração de espermatozoides na ejaculação.;
O efeito fêmea também é observado entre fêmeas, com fêmeas ativas sexualmente
induzindo ciclicidade em outras do rebanho 40.
36
Figura 5. Representação de um protocolo de estimulação por efeito macho em

pequenos ruminantes.
D – Dia; LH – Hormônio Luteinizante; FSH – Hormônio Folículo Estimulante.
Outro método não farmacológico de melhorar os resultados reprodutivos em

pequenos ruminantes é o Flushing, podendo este ser trabalhado de forma contínua,
estática ou dinâmica. A alimentação por curtos períodos com uma dieta rica em
proteínas e energia, fornecida para ovelhas cíclicas aumenta as concentrações de
insulina e glicose no sangue dessas fêmeas, além das concentrações de glicose no
41.
fluido folicular Além disso, as alterações na dieta causam ações diretas sobre o
folículo, levando a um feedback negativo reduzido para o sistema hipotálamo-
hipofisário e um consequente aumento da secreção de FSH, levando a uma
42.
estimulação da foliculogênese Ainda, resultados podem ser positivos quando os
métodos são utilizados em conjunto, dependendo da raça. Em ovelhas Santa Inês o
efeito macho foi capaz de induzir atividade cíclica, e associação com o flushing
melhorou os resultados na contra estação, já ovelhas Suffolk e Romney Marsh
nenhuma das técnicas induziu a ciclicidade 37.
37
4.2 Métodos farmacológicos
4.2.1 Indução do estro
Protocolos hormonais de indução do estro são utilizados durante o anestro

estacional na ausência de um CL ativo. São baseados no uso de progestágenos,
tais como: (1) dispositivo intravaginal com molécula de P4 idêntica à original
[Controlled Internal Drug Release (CIDR) and Dispositivo Intravaginal Caprino Ovino
43–45,
(DICO)] (2) esponjas intravaginais com análogos da progesterona como o
46,47
acetado de medroxiprogesterona e a flurogestona e a (3) progesterona injetável
48–50. Os progestágenos exógenos foram propostos inicialmente como forma de
mimetizar o efeito do CL no organismo. Os protocolos de longa exposição ao
progestágeno (9-14 dias) são tradicionalmente usados e geram os efeitos desejados
27 51
independente da fase estral ou estação do ano , , entretanto os folículos ovulados
acabam sendo estimulados por um longo tempo, perdendo a qualidade do oócito
ovulado. Assim, visando diminuir o tempo de duração, ocorrência de vaginites em
resposta ao dispositivo/esponja, custo dos protocolos e melhoria de resultados,
51,52.
protocolos mais curtos (5-7 dias) foram avaliados obtendo bons resultados Já
em caprinos, o uso de diferentes períodos de exposição aos progestágenos (seis,
nove ou 12 dias) foram igualmente eficientes para indução de estro sincronizado,
apresentando apenas diferença quanto a apresentação de estro quando comparada
a categoria animal, onde nulíparas apresentaram o comportamento sexual antes das
53,54.
pluríparas lactantes, independentemente do tratamento Em ovinos Santa Inês,
foram encontrados resultados similares para seis, nove ou 12 dias, porém com uma
55.
concentração maior da ovulação com o protocolo de nove dias Assim, a escolha
pelo uso de um ou outro protocolo deve ser feita baseada no estado cíclico das
fêmeas e grau de estacionalidade apresentada, de modo a promover os estímulos
mais adequados ao momento fisiológico reprodutivo e endócrino do animal. Um
exemplo desta afirmação foi visto pelo nosso grupo em um estudo recente realizado
em um rebanho caprino comercial, onde, inicialmente, tentou-se o protocolo curto,
que não apresentou resultado satisfatório e ao tentar um estímulo mais prologando
56.
ao progestágeno, o protocolo foi bem sucedido
38
Com relação aos tipos de dispositivos intravaginais utilizados, a escolha pelo

tipo a ser utilizado pode ser feita considerando custo-benefício, além do
progestágeno que ele contém. Os dispositivos plásticos (CIDR e DICO), apesar de
maior custo de aquisição podem ser autoclavados e utilizados por duas ou três
indução, a depender do tempo de exposição, além de causar menor incidência de
vaginite; entretanto autoclavar os dispositivos pode não ser um procedimento
57–59.
acessível a todas as propriedades Os métodos de inserção e retirada da
esponja de progesterona podem ser vistos no material suplementar número 2.
O uso dos análogos da prostaglandina (PG) deve ser feito em tratamentos de

curta exposição, pois, caso haja um CL, este ainda poderá estar funcional ao final do
tratamento com progestágeno, e a PG causará sua lise 27,51. Para concluir o
protocolo, uma gonadotrofina, como a gonadotrofina coriônica equina (eCG; de 50
60 61
até 300 UI) ou a gonadotrofina coriônica humana (hCG; de 50 até 300 UI) , ,
podem ser aplicadas, de 48 h antes até o momento da retirada do
dispositivo/esponja, para que ocorra um estímulo mais sincrônico do crescimento
27,51.
folicular, manifestação do estro e ovulação no lote As doses utilizadas podem
variar de acordo com o grau de estacionalidade que a fêmea se encontra, assim
como a raça e a localização geográfica. Outros hormônios que também podem ser
associados são os análogos sintéticos do Hormônio liberador de gonadotrofina
61,
(GnRH) como estímulo ao pico pré ovulatório do LH ou o análogo do FSH retirado
da hipófise de suínos (pFSH – FSH suína), para melhoria dos folículos estimulados
62. Na figura 6 é demonstrado um esquema simplificado de um protocolo baseado
em progestágenos associado a um análogo da PG e gonadotrofina. Na tabela 1 são
apresentados os principais protocolos para cada espécie.
39
Figura 6. Representação de um protocolo curto baseado em progestágenos em

pequenos ruminantes. A duração da exposição ao progestágeno pode variar, assim
como os outros hormônios utilizados em associação. Os protocolos podem ser
curtos (6 dias; conforme ilustrado nesta figura), médios (9 dias) ou longos (a partir de
12 dias), onde o uso de prostaglandinas pode ser suprimido. O uso dos
progestágenos estimula o crescimento folicular e a ovulação. Nesses protocolos a
aplicação da eCG e PGF pode ser feita no dia anterior ou no dia da retirada do
progestágeno.
D – Dia; P4 – progestágeno; eCG – Gonadotrofina Coriônica Equina; PGF – prostaglandina; IA –
inseminação artificial; NM – acasalamento natural; LH – Hormônio Luteinizante; FSH – Hormônio
Folículo Estimulante.
40
Tabela 1. Principais protocolos utilizados para induzir fêmeas caprinas e ovinas
Protocolo Início do estro (h) Momento da ovulação (h) Fêmeas gestantes Referência
Caprinos
54
MAP (6 dias) + d-cloprostenol + 200 UI eCG 26,7±5,6 44,6±2,2 60% (6/10)
54
54
63
MAP (6 dias) + d-cloprostenol + 200 UI eCG + 300 IU N/R N/R 74,4% (32/43)
hCG (7 dias depois)
63
MAP (6 dias) + d-cloprostenol + 200 UI eCG + saline (7 N/R N/R 90,7% (39/43)
dias depois)
62
MAP (6 dias) + d-cloprostenol + 20 UI FSH 56,0±19,6 93,5±30,3 52,2% (12/23)
62
MAP (6 dias) + d-cloprostenol + 200 UI eCG 43,0±20,5 72,7±19,9 72,0% (18/25)
62
48
P4 --- --- 0% (0/10)
48
P4 + 50 UI hCG 60±13 depois do hCG 90±12 depois do hCG 67% (6/9)
48
48
53
CIDR (6 dias) + dinoprost + 200 UI eCG 29,5±9,7 50,5±11,4 N/R
53
53
Ovinos
47
MAP (9 dias) 27,06±17,46 N/R 70% (21/30)
47
MAP (9 dias) + 400 UI eCG 37,76±13,39 N/R 86% (26/30)
47
MAP (9 dias) + 400 UI eCG + 200 UI hCG 40,83±23,45 N/R 56% (17/30)
64
MAP +300 UI eCG + cloprostenol N/R N/R 37,4% (74/198)
64
CIDR (6 dias) +300 UI eCG + cloprostenol N/R N/R 55,8% (110/197)
64
DICO (6 dias) +300 UI eCG + cloprostenol N/R N/R 55,7% (97/174)
55
55
55
65
MAP (6 dias) + d-cloprostenol + 300 UI eCG 37,3±3,2 64,1±3,0 N/R
41
65
MAP (6 dias) + d-cloprostenol + 300 UI eCG + GnRH 24h --- 48,0±3,2 N/R
65
MAP (6 dias) + d-cloprostenol + 300 UI eCG + GnRH 36h 31,4±3,8 56,7±1,9 N/R
66
MAP (6 dias) + d-cloprostenol + 200 UI eCG + hCG (7,5 N/R N/R 60,0% (33/55)
dias depois)
66
MAP (6 dias) + d-cloprostenol + 200 UI eCG + saline (7,5 N/R N/R 47,1% (25/53)
dias depois)
67
MAP (14 dias) + 200 UI eCG N/R N/R 76,4% (126/165)
68
MAP (14 dias) + delprostenate + 380 UI eCG N/R N/R 66,2% (49/74)
69
CIDR (5 dias) + dinoprost + eCG 30,1±1,5 66,3±3,8 N/R
69
CIDR (5 dias) + dinoprost + GnRH 24h (diluição em 30,0±2,5 68,0 N/R
propileno glicol)
69
CIDR (5 dias) + dinoprost + GnRH 36h (diluição em 40,0±2,4 74,5±2,3 N/R
propileno glicol)
69
CIDR (5 dias) + dinoprost + GnRH 56h (diluição em água 47,0±3,2 79,5±2,2 N/R
destilada)
70
CIDR (5 dias) + 400 UI eCG + dinoprost 39,2±4,0 72,0±3,6 N/R
70
CIDR (5 dias) + dinoprost 44,4±2,8 74,8±2,6 N/R
70
CIDR (14 dias) + 400 UI eCG 34,0±5,0 58,0±3,7 N/R
70
CIDR (14 dias) 33,7±3,0 64,7±3,2 N/R
71
CIDR (5 dias) + dinoprost+ 400 UI eCG 34,1±2,0 65,8±2,3 68,4% (13/19)
71
CIDR (5 dias) + dinoprost + GnRH 56h 39,3±2,0 68,4±2,5 57,9% (11/19)
71
GnRH + CIDR (5 dias) + dinoprost + GnRH 56h 39,8±2,2 73,8±2,1 68,4% (13/19)
72
CIDR (5 dias) + dinoprost + 400 UI eCG 40,8±8,0 71,8±7,3 66,7% (n=6)
72
CIDR (5 dias) + dinoprost + solução salina 45,0±5,7 72,6±5,8 83,3% (n=6)
72
72
CIDR (6 dias) + dinoprost + solução salina 52,4±14,5 69,0±7,1 33,3% (n=6)
72
72
CIDR (7 dias) + dinoprost + solução salina 58,2±8,9 77 16,7% (n=6)
72
CIDR (14 dias) + 400 UI eCG 30,9±9,5 56,7±6,9 83,3% (n=12)
72
CIDR (14 dias) + solução salina 45,3±16,3 66,7±8,3 60,0% (n=10)
N/R – Não Relatado: dados não disponíveis
42
4.2.2 Sincronização
Quando há presença de um CL, ou seja, as fêmeas estão cíclicas, protocolos

de sincronização podem ser utilizados baseados no uso de prostaglandinas F2α
(PGF) ou seus análogos (d-cloprostenol, cloprostenol, delprostenato, dinoprost –
73.
doses de acordo com o análogo utilizado), que induzem a luteólise Os protocolos
são baseados em duas administrações consecutivas, de modo que as fêmeas que
não apresentassem CL na primeira administração (proestro/estro), ou estes não
estivessem responsivos (metaestro), teriam o efeito luteolítico desejado na segunda
aplicação 74 (Figura 7).
Assim como protocolos de indução, os de sincronização não tem uma data

fixa entre as aplicações, tendo sido conduzidos diversos estudos para determinar o
melhor intervalo entre aplicações (Tabela 2). Em caprinos, intervalos de 7 e 11,5
dias foram mais eficazes em relação ao intervalo de 10 dias ao usar d-cloprostenol
75
. Em ovinos, administrações em intervalos de 9 e 11,5 dias promoveram maior
76.
sincronia de ovulação do que o protocolo de 7 dias usando cloprostenol
Resultados semelhantes foram encontrados ao comparar intervalos longos entre as
administrações em ovelhas, ao usar delprostenato, onde não foram observadas
diferenças em intervalo de ovulação, taxa de ovulação e prolificidade, para os
77.
intervalos entre 7, 10, 12, 14 ou 16 dias
Figura 7. Representação de um protocolo baseado em análogos das

prostaglandinas em pequenos ruminantes. O intervalo entre as aplicações pode
variar entre 7 a 11,5 dias, levando à luteólise e ovulação após a aplicação da
prostaglandina.
IA – inseminação artificial; NM – acasalamento natural.
43
Tabela 2. Principais protocolos utilizados para sincronizar fêmeas caprinas e ovinas.
Protocolo Início do estro (h) Momento da ovulação (h) Fêmeas gestantes Referência
Caprinos
Duas doses de d-cloprostenol (10 dias de intervalo) com 78
47±10,1 86,6±11,4 61% (14/23)
ou sem hCG no início do estro
55,6% (10/18) 1º
ensaio 75
Duas doses de d-cloprostenol (7 dias de intervalo) 43,8±10,7 N/R
85,2% (23/27) 2º
ensaio
18,8% (3/16) 1º
75
Duas doses de d-cloprostenol (10 dias de intervalo) 50,6±13,7 N/R ensaio
N/R 2º ensaio
26,7% (4/15) 1º
ensaio 75
Duas doses de d-cloprostenol (11,5 dias de intervalo) 47,3±5,8 N/R
93,6% (29/31) 2º
ensaio
Ovinos
76
Duas doses de cloprostenol (7 dias de intervalo) 36,5±6,2 56,8±6,2 66,7% (22/33)
76
Duas doses de cloprostenol (9 dias de intervalo) 45,8±18,9 67,9±24,4 71,0% (22/31)
76
Duas doses de cloprostenol (11,5 dias de intervalo) 43,7±16,7 78,7±9,4 71,0% (22/31)
77
Duas doses de delprostenato (7 dias de intervalo) N/R N/R 28,8% (21/73)
77
77
77
77
68
68
68
68
68
69
Duas doses de dinoprost (7 dias de intervalo) + água 48,0±0,1 N/R N/R
44
destilada 32h
Duas doses de dinoprost (7 dias de intervalo) + GnRH 69
40,8±4,3 N/R N/R
32h (diluição em propileno glicol)
Duas doses de dinoprost (7 dias de intervalo) + GnRH 69
42,0±4,8 N/R N/R
32h (diluição em água destilada)
67
Duas doses de cloprostenol (14 dias de intervalo) N/R N/R 52,0% (52/100)
Duas doses de cloprostenol (14 dias de intervalo) + 67
N/R N/R 62,5% (65/104)
200 UI eCG nas duas administrações
N/R – Não Relatado: dados não disponíveis
45
4.2.3 Ressincronização
Além dos protocolos de indução e sincronização do estro, protocolos de

ressincronização precoce da ovulação, já amplamente descritos em bovinos, vêm
79,80,80–82.
sendo estudados em pequenos ruminantes Tais protocolos podem ser
82
utilizados com manejo de monta natural ou Inseminação Artificial em Tempo Fixo
79,
(IATF), com o uso de diferentes doses de eCG apresentando bons resultados em
ovelhas nulíparas e pluríparas, mas com resultados limitados para fêmeas no pós-
parto 81.
Em cabras, resultados preliminares mostram que uma segunda esponja de

MAP inserida do meio para o fim do ciclo (D16-D21) não interfere no CL ou na
83
produção de progesterona endógena, esteja a fêmea gestante ou não , da mesma
forma que não interfere para a espécie ovina (D12-D17) 79,82.
Assim, tais protocolos possuem potencial de trazer melhorias para o sistema

de produção, visto que diminuem o tempo improdutivo de uma fêmea no rebanho e
concedem novas chances de gestação para a mesma (Figura 8). Protocolos de
ressincronização precoce associados ao uso do diagnóstico precoce da fêmea não
gestante permitem: (1) diminuir o uso de hormônios desnecessários, como eCG,
GnRH e PGF; (2) prevenir a perda de doses de sêmen, possibilitando o investimento
em reprodutores mais caros; e (3) melhorar os resultados gerais dos programas de
ressincronização, encurtando o período total de trabalho e a simplificação das
práticas de manejo. Deste modo, as fêmeas precocemente detectadas como
gestantes podem ter o manejo nutricional balanceado prontamente, além de evitar
manejos desnecessários - incluindo administração de hormônios e inseminação -, o
que tem implicações importantes para o bem-estar animal.
46
Figura 8. Representação esquemática comparando protocolos de indução do estro

sincronizado tradicionais (A) e de ressincronização precoce em pequenos
ruminantes (B e C), onde as datas são as sugeridas para caprinos (B) e para ovinos
(C).
5. Manejo reprodutivo
O manejo reprodutivo pode ser realizado de diferentes formas, entre elas a

inseminação artificial em suas diferentes técnicas e a monta natural, de forma livre,
guiada ou controlada, tal como apresentado a seguir.
5.1 Monta Natural
A monta natural, que ocorre pelo acasalamento do macho com a fêmea, pode
ser realizada de forma livre ou guiada. O macho pode se deixado a pasto ou na baia
84,85
junto com as fêmeas, geralmente a uma proporção de 1 macho: 25 a 30 fêmeas
(podendo variar de acordo com a qualidade seminal, idade e libido do macho e se as
fêmeas foram ou não sincronizadas). Nesse sistema os machos podem ter o peito
pintado 84,85 (tinta xadrez colorida – Figura 9), ou pode ser utilizado um dispositivo
que solta tinta, o bursal marcador, de forma a marcar as fêmeas que já foram
cobertas. Pode-se também não realizar marcações nas fêmeas e apenas ser
realizado o diagnóstico de gestação, iniciando 30 dias após o início das coberturas e
repetindo a cada semana. Podendo também estar associado ao uso de protocolos
ou sem nenhuma interferência durante a estação de monta.
A monta natural pode também ser realizada de forma direcionada, geralmente

após a realização de um protocolo de indução ou sincronização do estro. Nesse
47
sistema as fêmeas tem a apresentação do estro avaliada constantemente, deixando

um rufião vasectomizado no com peito pintado ou buçal marcador no lote das
84,85.
fêmeas De acordo com a apresentação do estro, as fêmeas são cobertas,
seguindo, em média a proporção 1 macho: 6 a 8 fêmeas por vez. Enquanto
persistirem os sinais de estro as fêmeas podem continuar sendo cobertas,
entretanto, pode ser dada a preferência das primeiras ejaculações para fêmeas
começando a apresentar estro, de forma a possibilitar maiores chances de
emprenhar para todas, visto que a qualidade seminal decai a cada ejaculado
seguido. Além disso, a cada turno de monta, as fêmeas que foram cobertas por
último no turno anterior terão prioridade no turno seguinte, de forma a realizar um
rodízio entre as fêmeas e dar oportunidades similares para todas.
Figura 9. A) Tinta xadrez colorida para pintar o peito do macho B) Monta natural,
onde vê-se o macho com o peito pintado e a fêmea com o dorso colorido de monta
anterior.
48
5.2 Inseminação Artificial
Para selecionar uma técnica de inseminação artificial adequada aos

pequenos ruminantes é preciso levar em conta as peculiaridades anatômicas da
cérvix de caprinos e ovinos. Caprinos possuem os anéis cervicais mais alinhados e
maior diâmetro da luz do órgão, já os ovinos possuem um canal cervical mais
86
estreito e menor grau de alinhamento dos anéis cervicais (Figura 10). Essas
características tornam a transposição cervical em caprinos mais fácil de ser
realizada do que na espécie ovina. Assim, para caprinos, independentemente do tipo
87.
de sêmen, recomenda-se a inseminação pela via transcervical Em ovinos sugere-
se o uso de inseminação artificial por via laparoscópica ao trabalhar com sêmen
congelado e cervical superficial para sêmen fresco ou resfriado 88. De acordo com o
local de inseminação, diferentes doses inseminantes também são recomendadas
(Figura 11).
Figura 10. Comparação entre cérvix caprina (esquerda) e ovina (direita). Cérvix
caprina apresentando maior alinhamento cervical, enquanto que na espécie ovina
visualiza-se uma cérvix mais tortuosa.
49
Figura 11. Esquema de local de inseminação, dose inseminante e volume

recomendado para inseminação artificial. A) deposição vaginal; B) deposição
cervical superficial; C) deposição cervical profunda; D) deposição intrauterina
transcervical; E) deposição intrauterina laparoscópica.
5.2.1 Cervical superficial
A técnica de inseminação pela via cervical superficial caracteriza-se pela

deposição de sêmen no óstio cervical com o auxílio de pistolas específicas ou
pipetas adaptadas. Assim, o sêmen é depositado no orifício cervical externo,
podendo a fêmea estar em estação na linha de ordenha ou em algum local em que a
a vulva da fêmea fique na altura dos olhos do profissional inseminador, ou
preferencialmente em apoio bipedal, com o posterior elevado, de modo que a
gravidade auxilie a entrada do sêmen na cérvix (Figura 12). A dose de sêmen
recomendada para sêmen fresco gira em torno de 100 – 200 x 106 e para sêmen
resfriado doses de 200 x 106 têm sido utilizadas 89. Associada à sincronização de cio
espera-se taxa de concepção próxima a 60% com a utilização de sêmen fresco e
88
40% com sêmen resfriado . Para diluição sem resfriamento é possível utilizar leite
desnatado UHT. Outra opção para aplicação em ovinos seria a utilização de
diluidores comerciais para bovinos. É importante lembrar que o plasma seminal
50
caprino possui duas enzimas secretadas pelas glândulas bulbo uretrais a EYCE e a
BUS gp60 que reagem com a gema de ovo e com diluidores a base de leite, por isso
90,91.
devem ser evitados para esta espécie Assim, para caprinos uma alternativa
92.
seria a utilização de água de coco como diluidor Em ovinos, a gema de ovo e óleo
88.
de soja são opções viáveis de diluidor
Figura 12. A-B) Apoio bipedal da fêmea para inseminação cervical superficial. Com
apoio em uma superfície emborrachada (A), ou apenas com a sustentação do
colaborador (B). C) Material utilizado para inseminação artificial superficial
51
5.2.2 Laparoscopia
A técnica de inseminação artificial por laparoscopia exige maior investimento

93,94.
com equipamentos e maior grau de treinamento do técnico envolvido Sua
aplicação é justificada quando for utilizado sêmen ovino congelado. Previamente ao
procedimento, os animais precisam passar por jejum hídrico de 12 horas e alimentar
de 24 horas. É necessário que ocorra sedação, por exemplo com associação de
94.
acepromazina (0,1 mg/Kg) e diazepan (0,4 mg/kg) As fêmeas são posicionadas
em macas específicas para a alocação na posição de Trendelenburg (Figura 13), ou
seja, em um ângulo de aproximadamente 45º com os posteriores para cima, de
modo a permitir que o efeito da gravidade, junto com o ar inserido na cavidade
abdominal, permita que o útero seja melhor destacado das outras vísceras,
localizado e fixado para a inseminação.
Animais com estro sincronizado, inseminados por laparoscopia e utilizando

93 95
sêmen congelado apresentam em torno de 50 a 60% de taxa de concepção , .
Alguns técnicos optam por aplicar este método de IA com sêmen fresco ou resfriado
96.
e atingem resultados ainda maiores de concepção Apesar do possível incremento
com a associação destas técnicas, sua aplicação deve ser avaliada para cada
cenário devido aos riscos e custos inerentes ao procedimento.
52
Figura 13. A) Imagem de ovário vista pela videolaparoscopia, com um folículo em

evidência; B) Fêmea em posição de Trendelenburg, em um ângulo de
aproximadamente 45 a 60º com os posteriores para cima, com o útero sendo
localizado por videolaparoscopia para inseminação.
5.2.3 Transcervical
Atualmente, em nossa concepção, a técnica mais apropriada para IA em

87,97.
caprinos é o método de fixação cervical desenvolvido pela EMBRAPA Esta
53
técnica consiste em, com o animal em estação e em um piso elevado, inserir um

espéculo do tipo Collins para a visualização do óstio cervical auxiliado por uma fonte
de luz. Este deve ser pinçado utilizando uma pinça de Allis (com suas proeminências
para fixação lixadas), ou com uso de pinças especificas tais como as desenvolvidas
97,
pela Embrapa este procedimento irá permitir que a cérvix não se mova. Com a
cérvix fixada é possível introduzir o aplicador de sêmen e, com movimento
oscilatório, transpor os anéis cervicais. A perda de resistência à progressão do
87.
aplicador indicará que a transposição foi total e o sêmen pode ser depositado
Associada à sincronização de estro e utilizando sêmen congelado, espera-se

98.
taxas de concepção em torno de 50% Entre um animal e outro, é importante
higienizar os instrumentos para evitar, principalmente, a disseminação das
lentiviroses dos pequenos ruminantes (LVPR). Água em temperatura elevada e
87 97
amônia quaternária são opções eficientes para este procedimento , .
Além disso, o momento da inseminação em caprinos pode ser melhor

75,
determinado ao associar o momento da entrada ao estro e as características do
62. 75,
muco cervical Resumidamente, de acordo com Maia et al as fêmeas que
apresentam estro 36h, 48h e 60h após a remoção da esponja são inseminadas às
24h, 18h e 10h após o início dos sinais, respectivamente. Com relação ao muco
cervical, este pode ser classificado em cinco graus: (I) cristalino; (II) cristalino-
estriado; (III) estriado; (IV) estriado-caseoso; e (V) caseoso (Figura 14).
Figura 14. Momento da Inseminação em caprinos, baseado no tipo de sêmen e

62.
característica do muco cervical. Adaptado de Fonseca et al.
54
5.3 Coleta e avaliação seminal
Quando é feita a escolha pela inseminação artificial podem ser utilizados

sêmen fresco, refrigerado ou congelado, nestes casos deve ser realizada a coleta
dos ejaculados, podendo ser por eletroejaculação ou vagina artificial, ambos os tipos
de coletas carregam vantagens e desvantagens no processo89. A coleta através da
vagina artificial (Figura 15.A), apesar de mais natural, demanda que o reprodutor
seja treinado a coletar com um manequim ou que tenha uma fêmea no cio para
estimular, além da habilidade de quem for coletar em desviar o pênis no momento
adequado. A coleta por eletroejaculação (Figura 15.B) é baseada no estímulo
elétrico crescente por via transretal das glândulas acessórias do macho (vesícula
seminal, próstata e bulbouretral), de forma a induzir a ejaculação. Esse método além
de demandar experiência de quem for coletar, necessita de atenção à relação
estímulo-desconforto do animal, além de gerar um ejaculado mais líquido devido ao
estímulo nas glândulas sexuais acessórias.
1Figura 15. A) Coleta de sêmen através de vagina artificial com fêmea no cio como
manequim; B) Coleta de sêmen através de eletro ejaculação: C) Material utilizado
para coleta por vagina artificial; D) Eletroejaculador.
Assim, após coletado o sêmen deve ser avaliado, visto que para se obter
resultados satisfatórios com a inseminação artificial, não apenas o momento da
55
fêmea deve ser avaliado. De igual importância a qualidade do sêmen utilizado,

devem ser levados em consideração também as características, aspectos e forma
de uso (Tabela 3). Além disso, o controle dos dados da inseminação pode ser
realizado através de planilhas, contendo dados da inseminação, reprodutor,
momento da inseminação entre outros (Figura 16).
Tabela 3. Características desejáveis para o sêmen do pequeno ruminante fresco,

refrigerado ou congelado.
Caraterística Valor para caprinos Valor para ovinos

Fresco
Volume 0,5 – 1,5 mL 0,5 – 3,0 mL
Aspecto Cremoso
Cor Branco ou amarelo marfim
Odor “sui generis”
Movimento de massa ≥4 ≥3
Motilidade espermática 70 – 90 % 80 %
Vigor ≥3 ≥3
Concentração 2 – 5 x 109/mL 1 – 3 x 109/mL
Espermatozoides/ejaculad
3 – 5 x 109 3 – 5 x 109
o
Patologias espermáticas < 20% < 20%
Refrigerado
Motilidade espermática ≥ 60% ≥ 60%
Vigor ≥3 ≥3
Defeitos maiores ≤ 10% ≤ 10%
Dose inseminante 1,5 x 108 sptz/palheta 1,5 x 108 sptz/palheta
Congelado
Motilidade espermática ≥ 30% ≥ 30%
Vigor ≥2 ≥3
Defeitos maiores ≤ 10% ≤ 10%
Dose inseminante 4 x 107 sptz/palheta 4 x 107 sptz/palheta
Adaptado de CBRA 89.
56
RUFIAÇÃO
Rufiação
DD/MM/AAAA DD/MM/AAAA DD/MM/AAAA DD/MM/AAAA
Nº Animal Baia ECC
manhã tarde manhã tarde manhã tarde manhã tarde
(08 h) (20 h) (08 h) (20 h) (08 h) (20 h) (08 h) (20 h)
1
2
3
4
5
INSEMINAÇÃO ARTIFICIAL
Sêmen Inseminação
(Fresco, DD/MM/AAAA
Nº Animal Baia ECC Macho
Resfriado, 1ª 2ª
Congelado) Muco Profund Hora Muco Profund Hora
1
2
3
4
5
Figura 16. Planilha de coleta de dados de Rufiação e Inseminação Artificial.
6. Ultrassonografia e reprodução
6.1 Diagnóstico de gestação por ultrassonografia
A US tem sido utilizada para diagnósticos do trato reprodutivo de ruminantes

99–102.
desde os anos 80 Na década de 90 o diagnóstico de gestação em caprinos
era realizado com exames abdominais US modo-A (50-120 dias após cobertura), US
modo-B (40-75 dias após cobertura) e Doppler (75-150 dias após cobertura); além
disso, o diagnóstico por via transretal era dado por achado uterinos, tais como
distensão uterina, conteúdo anecóico, vesícula embrionária e batimentos cardíacos,
103
utilizando o US modo-B associado, ou não, ao Doppler, 30 dias após a cobertura .
Em pequenos ruminantes, a presença de batimentos cardíaco no concepto é

104,
detectável a partir de 21 dias de gestação em algumas fêmeas sendo
usualmente feito em torno de 30 dias de gestação. Além da avaliação da presença
do concepto, através da ultrassonografia é possível avaliar a sua viabilidade e
estimar a idade gestacional 105–107.
Além da avaliação uterina do concepto, através da ultrassonografia pode ser

feita avaliação da ecogenicidade e ecotextura luteal (Figura 17.A). Em pequenos
ruminantes já foi demonstrada que a avaliação doppler do CL (Figura 17.B) permite
a identificação precoce da luteólise e com isso a identificação precoce de fêmeas
57
56,108.
não gestantes Para isso, deve-se conhecer o momento da ovulação
108 56,
(protocolos de indução), e assim, aos 17 dias em ovinos e 21 dias em caprinos
é possível tal avaliação. Ainda, em caprinos, com acurácia de 98,59% a partir dos 23
dias, foi possível o diagnóstico de gestação com a avaliação conjunta, em modo-B,
56.
da morfologia luteal e a presença de conteúdo anecóico no útero
Figura 17. A) Avaliação em Modo-B da morfologia luteal em escores de 1 a 3, onde

o escore 1 é considerado para CL em luteólise; B) Avaliação doppler do fluxo
sanguíneo luteal em escores de 1 a 4, onde o escore 1 é considerado para CL em
luteólise. Imagens cedidas por Cosentino et al 56.
6.2 Diagnóstico e tratamento de afecções reprodutivas
Além do diagnóstico gestacional, a avaliação do trato reprodutivo permite a

identificação de afecções reprodutivas, tanto no macho como na fêmea, de forma
105,109,
pouco invasiva e rápida permitindo assim o início do tratamento e manejo
mais adequado para cada tipo de situação. Através da tabela 4 são demonstradas
algumas afecções reprodutivas possíveis de serem detectadas através da
ultrassonografia e os principais manejos corretivos empregados.
58
Tabela 4. Principais afecções reprodutivas possíveis de serem detectadas através da ultrassonografia e os principais manejos
empregados
Afecção Achados ultrassonográficos Referência Manejo Referência

Ovários e Trompas Uterinas
Cistos foliculares Estrutura esférica formada pelo acúmulo 110 Administração de um análogo sintético 105
de líquido anecóico, pertencente ao do GnRH associado ao uso de

ovário. Podem medir de 10 a 30 mm de prostaglandinas
diâmetro. Apresentam pouca perfusão ao
doppler
Cistos luteínicos Estrutura esférica formada pelo acúmulo 110,111 Administração de prostaglandinas 105
de líquido anecóico, com espessada e

ecogênica parede, pertencente ao ovário.
Podem medir de 10 a 30 mm de
diâmetro. Apresentam acentuada
perfusão ao doppler
Hipoplasia Presença de pequena estrutura 112 Descarte 105
ecogênica esférica na posição anatômica

do ovário, de contorno regular sem
presença de estruturas ovarianas, tais
como folículos ou corpos lúteos
Neoplasia Sem uma caracterização única, 105,112 Ovariectomia ou descarte 105
ovarianas neoplasias podem ser uni ou bilaterais,

focais ou difusas, com perda das
características morfológicas do órgão,
podendo apresentar contorno liso ou
grosseiro, regular ou não, aumento ou
diminuição da ecogenicidade, de forma
homogênea ou não. Cistos podem estar
presentes de forma única ou múltipla.
Geralmente apresenta aumento da
59
perfusão tecidual no doppler

Hidrossalpinge Estrutura esférica formada pelo acúmulo 113 Descarte 105,113
de líquido anecóico próximo a, mas não

parte do útero nem ovário
Útero e Cérvix
Hidrometra Acúmulo de conteúdo anecóico no 114 Aplicação de 2 a 3 doses de d- 114
interior do útero, podendo formar cloprostenol (10 dias de intervalo), de

trabéculas de acordo com a gravidade acordo com a gravidade
(Grau 1 a 4)
Piometra Acúmulo de conteúdo líquido 105 Aplicação de prostaglandina associada 115,116
heterogêneo hiperecóico no interior do a antibioticoterapia sistêmica e/ou

útero lavagem uterina
Hiperplasia Presença de cistos no endométrio com 112 Correção da causa primária a descarte 105
endometrial cerca de 1 cm de diâmetro, preenchidos

cística por um conteúdo anecóico, em casos
graves podem formar imagens com
aspecto de teia de aranha
Endometrite e Espessamento do endométrio podendo 105,112 Correção da causa primária, aplicação 105
Metrite apresentar conteúdo líquido ecogênico de prostaglandina associada a

no útero. Em casos de metrite há antibioticoterapia sistêmica e/ou
também o acometimento do miométrio., lavagem uterina a descarte
podendo apresentar partículas
hiperecóicas suspensas no conteúdo
líquido na cavidade uterina. A avaliação
doppler demonstra aumento da
vascularização da parede uterina
Neoplasias Sem uma caracterização única, 117 Retirada da massa a descarte 117
uterinas neoplasias podem ser focais ou difusas,

com perda das características
morfológicas do órgão, podendo
apresentar contorno liso ou grosseiro,
regular ou não, aumento ou diminuição
60
da ecogenicidade, de forma homogênea

ou não. Cistos podem estar presentes de
forma única ou múltipla. Geralmente
apresenta aumento da perfusão tecidual
no doppler
Cervicite Espessamento da parede cervical e 105 Antibioticoterapia sistêmica e 105
presença de conteúdo ecóico antiinflamatórios sistêmicos

Testículos, Epidídimos e Bolsa escrotal
Orquite Em casos agudos há aumento do 105,112,118 Antibioticoterapia sistémica prolongada 105
testículo e diminuição da ecogenicidade quando a causada por bactérias

de forma difusa
Em casos crônicos pode haver aumento
da ecogenicidade e presença de pontos
hiperecóicos formadores de sombra
acústica (focos de mineralização)
Anorquidia, Ausência de um (monorquidia) ou ambos 105,119 Descarte 105
Monorquidia e (anorquidia) testículos, sendo o

Criptorquidia criptorquida o que possui o testítulo, mas
não na bolsa escrotal. Quando
visualizável o testículo fora da bolsa
encontra-se muitas vezes degenerado ou
hipoplásico
Degeneração Perda da arquitetura testicular 112 Tratamento da causa primária em 105
testicular dimininuição do órgão. Aumento casos iniciais a descarte

progressivo e difuso da ecogenicidade,
levando a formação de sombras
acústicas por pontos de mineralização do
parênquima testicular
Hipoplasia Atrófico, hipoecóico e de ecotextura 105,119 Descarte 105
testicular geralmente homogênea

Neoplasia Sem uma caracterização única, 105,120 Orquiectomia ou descarte 105,120
testicular neoplasias podem ser uni ou bilaterais,

61
focais ou difusas, com perda das

características morfológicas do órgão,
podendo apresentar contorno liso ou
grosseiro, regular ou não, aumento ou
diminuição da ecogenicidade, de forma
homogênea ou não. Cistos podem estar
presentes de forma única ou múltipla.
Geralmente apresenta aumento da
perfusão tecidual no doppler
105,112,118 105
Epididimite Espessamento da parede cervical e Antibioticoterapia sistémica prolongada
presença de conteúdo ecoico quando a causada por bactérias
119 121
Hidrocele Espaço entre bolsa escrotal e testículo Orquiectomia unilateral em casos
preenchida por fluido anecóico de forma iniciais a descarte
focal ou difusa
112 121
Varicocele Presença de áreas hipoecoicas Orquiectomia unilateral em casos
irregulares no cordão espermático, sem iniciais a descarte
sinais detectáveis de fluxo sanguíneo,
alargamento dos vasos sanguíneos com
padrão laminar e ecogenicidade
aumentada
Adaptado de Balaro et al. 109
62
7. Considerações finais
O uso de biotécnicas para o controle da reprodução em pequenos ruminantes

é uma estratégia amplamente difundida. Variadas são as técnicas e protocolos
existentes para o manejo reprodutivo em pequenos ruminantes, desde protocolos de
sincronização e indução da ovulação e estro baseados em métodos farmacológicos
ou naturais, até o manejo reprodutivo em si, que pode seguir pela monta natural ou
por diferentes técnicas para inseminação artificial, finalizando com o diagnóstico
gestacional que pode ser realizado de forma precoce, avaliando o corpo lúteo, ou
tardia, avaliando o concepto. Independente do manejo, o uso dessas ferramentas
pode aumentar eficiência reprodutiva do rebanho, sendo indicado considerar custos,
estrutura e manejo associado a cada técnica antes de sua escolha.
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ORCID dos autores
Isabel Oliveira Cosentino (https://orcid.org/0000-0002-1059-1831), Pedro Henrique

Nicolau Pinto (https://orcid.org/0000-0001-5137-0106), Mário Felipe Alvares Balaro
(https://orcid.org/0000-0002-8198-7964), Felipe Zandonadi Brandão
(https://orcid.org/0000-0003-4027-5562).
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2.2 ARTIGO REVISÃO DE LITERATURA: RECENTES AVANÇOS NOS

TRATAMENTOS DE RESSINCRONIZAÇÃO DA OVULAÇÃO EM PEQUENOS
RUMINANTES: UMA REVISÃO
Recent advances on treatments for resynchronization of ovulation in small

ruminants: a review
Artigo submetido na Revista Animal Reproduction

77
Resynchronization of ovulation in small ruminants
Recent advances on treatments for resynchronization of ovulation in small
ruminants: a review
Isabel Oliveira Cosentino1* (https://orcid.org/0000-0002-1059-1831), Mário Felipe
Alvarez Balaro1 (https://orcid.org/0000-0002-8198-7964), Alejo Menchaca2,3
(https://orcid.org/0000-0002-2494-9574), Raquel Perez Clariget4 (https://orcid.org/
0000-0003-3998-3449), Rodolfo Ungerfeld5 (https://orcid.org/ 0000-0003-4685-2105),
Felipe Zandonadi Brandão1 (https://orcid.org/0000-0003-4027-5562)
Brazil
2Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Montevideo, Uruguay
3Plataforma de Investigación en Salud Animal, Instituto Nacional de Investigación
Agropecuaria (INIA), Montevideo, Uruguay
4Departamento de Producción Animal y Pasturas, Facultad de Agronomía,
Universidad de la República, Avenida Garzón 780, Montevideo, 12900, Uruguay
5Departamento de Biociencias Veterinarias, Facultad de Veterinaria, Universidad de
la República, Ruta 8 km 18, Montevideo 13000, Uruguay
* Corresponding author. Rua Vital Brazil Filho, 64. Niterói, Rio de Janeiro, 24230-
340, Brazil. isabelcosentino@id.uff.br

78
Abstract
Hormonal methodologies to control small ruminants’ estrous cycle are worldwide

used and evolved adjusting the application to the precise female physiological
moments to enhance reproductive performance. The estrous cycle can be induced
and/or synchronized, aiming for fixed-time artificial insemination or based on estrus
behavior signs for insemination, natural or guided mating. For increasing
reproductive outcomes, even more, successive protocols can be performed to
resynchronize ovulation. The objective of these treatments recently developed is to
resynchronize the ovulation as earlier as non-pregnancy is detected. In this sense,
the present review aimed to summarize the recent advances and main findings
regarding resynchronization protocols used in small ruminants. Lastly, future
perspectives and new paths to be studied in the present theme are presented. The
resynchronization treatment is still a growing field in small ruminant reproduction,
nevertheless, some enhancements are found in the reproductive outcome, showing
that such protocols can be successfully used in sheep and goat production.
Key words: estrous synchronization, estrous induction, ewes, does, hormonal

protocols, insemination.
1. Introduction
Although research in techniques for handling the estrous cycles began more
than 70 years ago, protocols more frequently used for estrous synchronization in
small ruminants still provide variable results. The studies directed to understand the
physiology of the estrous cycle in small ruminants started during the1920s
(Drummond-Robinson & Asdell, 1926), but the pharmacological attempts to control
the reproductive physiology began later, in the 1940s. Initially, the studies began with
the use of gonadotrophins for the induction of estrus in anestrous ewes and goat
does (Folley et al., 1949; Van Der Noot et al., 1946), the subsequent studies tried to
mimic the luteal phase with progestogens (Dutt & Casida, 1948; O’mary et al., 1950;
Robinson, 1956; Denny & Hunter, 1958), and later studies aimed to induce the
luteolysis with prostaglandins (Douglas & Ginther, 1973; Ott et al., 1980; Serna et al.,
1978; Thorburn & Nicol, 1971). In small ruminants, it is important to carefully consider
the physiology of each species and breed, as the degrees of reproductive seasonality
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vary widely, so, while some breeds present pronounced seasonality, others are
almost non-seasonal (Gómez-Brunet et al., 2011; Ramírez et al., 2021). In this
sense, hormonal protocols can be used for estrous synchronization (during the
breeding season) or induction of synchronized estrus (outbreeding season, during
the postpartum or before puberty).
One of the major advances in reproductive technologies in livestock in the last

years has been the new protocols for Fixed Time Artificial Insemination (FTAI). From
the understanding of follicular dynamics in the 1990s and 2000s, novel strategies
were developed to improve the implementation of FTAI, in cattle (Mapletoft et al.,
2018) as in sheep and goats (Menchaca & Rubianes, 2004). In order to minimize the
incidence of non-productive intervals when FTAI programs are implemented, it is
required to apply successive protocols to unpregnant females, resynchronizing their
ovulation during the shortest period possible (Cosentino et al., 2019). This requires a
precise diagnosis of non-pregnant females the earliest as possible to apply a second
treatment, or the application of blind treatments to all females ensuring that these
treatments do not affect the establishment of pregnancies or the early embryo
development in pregnant females.
Hence, the aim of this review was to update and summarize the knowledge on
hormonal treatments used for resynchronization of ovulation in sheep and goats.
Therefore, this review highlights the main concerns regarding the resynchronization
protocols in small ruminants and the main studies related. Although the state of the
art is still limited, is important to also highlight the advantages/disadvantages of the
on-farm application of these protocols, which is also briefly presented at the end of
this review.
2. General aspects
Hormonal protocols are wildly used to manipulate small ruminants’ estrous

cycles. Progestogens are usually administered by intravaginal devices, including
silicone-based devices impregnated with progesterone (P4), such as Controlled
Internal Drug Release (CIDR) and Dispositivo Intravaginal Caprino Ovino (DICO)
(Vilariño et al., 2010; Wheaton et al., 1993), or, intravaginal sponges (IVS)
impregnated with progesterone analogues, as medroxyprogesterone acetate (MAP)
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or flurogestone acetate (FGA) (Barrett et al., 2008; Santos et al., 2010). There are
some preliminary studies on the administration of long-acting injectable progesterone
(Alvarado-Espino et al., 2016, 2019; Rodríguez-Martínez et al., 2018), but the results
are still inconsistent. Until now, for the resynchronization protocol sponges
impregnated with MAP have been mainly used (Cosentino et al., 2019, 2021, 2022;
Miranda et al., 2018), but to the best of our knowledge, there are no studies with
other progestogens.
An important risk for resynchronization protocols was the possible negative

effect of the insertion of a progestogen device on the functionality of the endogenous
corpus luteum, and therefore, possible fetal loss. In this sense, even though the
increase in serum P4 levels induced by CIDR-type treatments administered early in
the luteal phase (i.e., 0 to 3 days after ovulation) could have a positive effect on the
uterine environment and conceptus development (Pugliesi et al., 2014), other studies
reported that it can affect the luteal function. In effect, Menchaca & Rubianes (2001)
and Pugliesi et al. (2014) reported that the insertion of a novel device in some
conditions can affect the functionality of the corpus luteum in goats and cattle,
respectively. Discarding this risk is essential, as for early resynchronization protocols
in small ruminants, the second device should be inserted later in the luteal phase
(e.g., 12 days after ovulation).
In sheep, protocols based on synthetic progestogens led to a reduction of the

endogenous P4 serum values but did not affect pregnancy nor trigger luteolysis if the
devices are inserted 12 d after ovulation (Miranda et al., 2018). Consistently,
Cosentino et al. (2019) corroborated that the insertion of a second MAP device 12 d
after ovulation does not affect the endogenous P4 serum concentration despite
whether ewes are pregnant or not. In goats, a first attempt performed in a
commercial flock showed that similarly to sheep, the second progesterone device did
not interfere with the gestational CL (Cosentino et al., 2020). In these studies, the
insertion of a second MAP sponge did not affect the luteal blood flow nor the P4
production during the following days, and these results did not differ in pregnant or
non-pregnant females. Therefore, the proposed protocols are proven to be secure for
application in females in which pregnancies were still not diagnosed.
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3. Resynchronization of ovulation
Resynchronization protocols have been developed to shorten the females'

unproductive time in the flock, mainly when it is intended to carry out sequential FTAI
in the same females. These treatments have been successfully developed in bovines
since the beginning of the century (Bartolome et al., 2005; Sani et al., 2011;
Stevenson et al., 2003), once the FTAI technology was implemented in that species.
Therefore, treatments in small ruminants are based on what has been previously
proposed in cattle. In general, resynchronization protocols in cattle repeat similar
treatments to those previously used for FTAI, over-imposed during the following
luteal phase. Treatments used for resynchronization of the ovulation without knowing
pregnancy status should not include prostaglandin analogues as this would induce
luteal regression, and thus, pregnancy loss. To minimize those risks, beef cattle
protocols are mainly based on GnRH analogues or estradiol (Bó et al., 2016).
If prostaglandins are used in the protocol, as early as the pregnancy diagnosis

is done, the earlier that the resynchronization treatments can be applied. This is of
practical importance, as the inclusion of these analogues is useful to synchronize
better the luteolysis and indirectly the ovulation, so an alternative recently developed
is the early pregnancy diagnosis based on the luteal blood flow. Although this
technique does not allow discarding the occurrence of premature luteal regressions,
provides a great accuracy on that corpus luteum that effectively, are not functional,
and therefore, the administration of prostaglandin analogues would not be a problem.
This allowed the development of resynchronization protocols that include those
analogues, without changing the conception rate when compared to the use of other
hormones (Sani et al., 2011). Other protocols, based on the application of eCG at the
time of removal of the P4 device, promote the growth of the dominant follicle, and,
consequently, an increase in the ovulation rate, the ovulation of larger follicles with
greater production of endogenous estradiol, better oocyte competence, greater luteal
diameter and, therefore, a greater P4 values in the following luteal phase (Bó et al.,
2016).
Originally, the application of these treatments in cattle was delayed, as began

after not identifying the conceptus in the uterine environment (Pereira et al., 2013).
82
However, recently these protocols included an early pregnancy diagnosis based on

luteal blood flow evaluation (Correa-Calderón et al., 2016; Siqueira et al., 2013).
Early pregnancy diagnosis allowed to apply resynchronization treatments as early as
22 to 28 days after the first artificial insemination (AI) (Pessoa et al., 2018; Sinedino
et al., 2014), or even earlier to allow FTAI at intervals similar to the natural estrous
cycle (Palhão et al., 2020; Pugliesi et al., 2019). This requires that the second
progestogen should be inserted from 14 to 21 days after the first AI, without harming
early pregnancy not yet diagnosed by ultrasound.
3.1. Treatments in sheep
In sheep, initially, Miranda et al. (2018) presented a protocol that consisted of

a traditional estrus synchronization treatment followed by AI or natural mating. The
treatment includes the insertion of a new P4 intravaginal device fourteen days later,
remaining in situ for six days. After its removal, all ewes were submitted once again
to AI or NM without a previous pregnancy diagnosis, thus, even being pregnant.
Pregnancy rates were similar for resynchronized ewes at the second service,
indicating that resynchronization of the ovulation can be effectively combined with
FTAI. However, this treatment implied that all ewes should be retreated, involving
great costs of hormonal treatments, losing semen doses in pregnant ewes,
increasing the environmental negative impacts, and requiring extra handling during
animals' early pregnancy, with the increase in the risk of pregnancy losses.
Therefore, similar to what was reported in cattle for sequential FTAI, Arashiro et al.
(2018) validated in sheep the use of US doppler scan for pregnancy diagnosis 17
days after FTAI, and therefore, decide to continue the hormonal treatments only in
non-pregnant ewes. This diagnosis is based on the study of the corpus luteum blood
perfusion by US doppler, and in sheep allows effectively to determine which females
are not pregnant as early as 17 days after ovulation (Arashiro et al., 2018).
Cosentino et al. (2019) also proposed a protocol using a lower dose of eCG
for the resynchronization of ovulations, as the use of 200 IU synchronized better the
second ovulation than 300 IU, the dose used for the initial FTAI in ewes. Thus, with
this information, treatments were combined, beginning with the insertion of an IVS
impregnated with MAP for six days, and one day before sponge removal, females
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received 300 IU of eCG and 0.24 mg of cloprostenol sodium. Finally, 24 h after

sponge withdrawal, ewes received 0.025 mg of lecirelin acetate (GnRH analogue)
and were inseminated 27 – 30 h after lecirelin administration with fresh semen
(Figure 1A). Twelve days later, a new IVS was inserted, remaining in situ for five
days, when the early pregnancy diagnosis was performed, allowing to separate those
ewes that had a functional or a non-functional corpus luteum. Immediately after, the
IVS was removed and 200 IU of eCG were administrated, followed by the
administration of a GnRH analogue 36 h later. The second AI was performed 9 – 12
h later, and the final pregnancy diagnosis was performed 40 days later, 59 days after
the initial AI (Cosentino et al., 2019). Results showed an increase from 19.4% of
pregnant ewes in the first AI to 41.9% in the second with 47.8% of accumulated
confirmed pregnancy.
This treatment was tested also in different categories of sheep (Cosentino et

al., 2021) and nulliparous ewes can respond similarly to multiparous ewes to
resynchronization. However, the response of early postpartum ewes during the
breeding season was strongly affected resulting in very low outcomes. In these
studies the protocol used was adapted from Cosentino et al. (2019), only delaying
the time of the first GnRH administration (36 h after the IVS removal). By comparing
the results of females that did and did not receive the second protocol, it was
confirmed that the resynchronization protocol did not affect results from the first FTAI
by inducing premature luteal regression or fetal losses (Cosentino et al., 2021).
3.2. Treatments in goats
In general, these treatments are less advanced in goats. Similarly to what was
reported in cattle and sheep, it was demonstrated in goats that the insertion of a
second progesterone device did not interfere with the gestational corpus luteum
(Cosentino et al., 2020). Therefore, it was proposed a resynchronization protocol in
which the does were synchronized by the insertion of a MAP containing IVS for six
days, followed by the administration of 200 IU of eCG and 0.12 mg of cloprostenol
sodium one day before sponge withdrawal (Figure 1B). Thirty-four hours after the
sponge removal, does received a GnRH analogue (Cosentino et al., 2020). Sixteen
days later, a new IVS was inserted, remaining in situ for five days, when early
84
pregnancy diagnosis was performed according to luteal blood flow (Cosentino et al.,
2018). At IVS removal, the use of saline or 100 IU of eCG was compared, with no
administration of GnRH analogue. Also, the breeding management was assessed:
natural mating was performed from the sponge removal for 4 days, and AI was
performed following estrous behavior and ovulation detection. There were no
differences in using or not of eCG in pregnancy outcomes (Cosentino et al., 2022).
Figure 1. A) Resynchronization protocol for ewes adapted from Cosentino et al.

(2019); B) Resynchronization protocol for does adapted from Cosentino et al. (2022);
C) 75 days traditional breeding season calendar D) Proposed successive
resynchronizations for ewes E) Proposed successive resynchronizations for goats.
D – Day; eCG – equine Chorionic Gonadotrophin; PGF – prostaglandin; GnRH – Gonadotrophin

Releasing Hormone; P4 – progestogen device; AI – artificial insemination; NM – natural mating; EPD
– early pregnancy diagnosis (luteal evaluation); PD – pregnancy diagnosis (uterine/fetal evaluation).
4. Perspectives
In sheep and goats, resynchronization protocols appear as a promising tool to

improve females’ reproductive performance after FTAI is implemented in these
species. Besides reducing the unproductive time of a ewe/doe in the flock and
granting new chances of becoming pregnant, resynchronization protocols associated
with the use of early diagnosis of the non-pregnant female allow the reduction of
85
using unnecessary hormones, (2) prevention of the semen doses loss, enabling
investment in more expensive breeders, and (3) improvement of overall results of
reproductive programs by applied sequential FTAI, shortening the total work period
and simplifying practices. In addition, females early detected as pregnant can have
nutritional management balanced promptly, in addition to avoiding unnecessary
animal handling during early pregnancy, decreasing the risks of early pregnancy
losses. Also, even though not yet studied, successive resynchronization treatments
enhance the number of chances given for a female to get pregnant compared to
traditional protocols for FTAI that require waiting for pregnancy diagnosis around 30
days after insemination (Figure 1C to 1E).
However, it should be considered that protocols for resynchronization of

ovulation in small ruminants are still being developed. More attempts should be
performed to improve reproductive indices and identify the best treatments to
enhance each category's outcomes. In sheep, studies are a bit more advanced, even
though such techniques should be tested in different breeds, climates, and different
reproductive statuses (multiparous, nulliparous, primiparous, early post-partum,
lactating, and seasonal anestrus) and with different synchronization protocols
(progestogens vs prostaglandin-based protocol, and different sources of
progestogens). In goats, a preliminary protocol should be still settled. Considering
results obtained until the present moment, a protocol similar to that used in ewes
could be applied to goats, whether considering the similarities and differences
between the species. The association of treatments for resynchronization of the
ovulation with early pregnancy diagnosis allows for reducing the use of unnecessary
hormones, avoids the loss of sperm doses, and shortens the lambing/kidding interval,
which concentrates births and homogenizes the lambs/kids batch. Also, more
opportunities to impregnate are given (two FTAI within about 20 days), with the aid of
the identification of early pregnant ewes/does prevent unnecessary management and
implementing adequate nutrition management of the pregnant females.
Acknowledgments
The present article was supported by Fundação Carlos Chagas Filho de

Amparo à Pesquisa do Estado do Rio de Janeiro - FAPERJ, Pró-Reitoria de
86
Pesquisa Pós-Graduação - Proppi/UFF (UFF/Proppi/Infra-LabPesq) and was

financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior - Brasil (CAPES) - Finance Code 001. FZB is a fellow of the CNPq. IOC was
a recipient of a scholarship provided by CAPES.
Authors’ contributions
IOC: Conceptualization, Data curation, Formal Analysis, Investigation,

Methodology, Visualization, Writing – original draft, Writing – review & editing; MFAB:
Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation,
Methodology, Project administration, Resources, Supervision, Validation
Visualization, Writing – original draft, Writing – review & editing; AM:
Conceptualization, Data curation, Formal Analysis, Investigation, Methodology,
Visualization, Writing – original draft, Writing – review & editing; RPC:
Visualization, Writing – original draft, Writing – review & editing; RU:
Visualization, Writing – original draft, Writing – review & editing; FZB:
Visualization, Writing – original draft, Writing – review & editing.
Conflict of interest statement
None of the authors has any conflict of interest to declare.
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3 HIPÓTESES
• A ovulação é adequadamente sincronizada com a administração de lecirelina

(análogo do GnRH) em caprinos;
• Um dispositivo de progestágeno exógeno (Medroxiprogesterona - MAP) ao
final do ciclo estral não interfere na funcionalidade do Corpo Lúteo (CL) de
pequenos ruminantes;
• A redução da dose de eCG não compromete a sincronização da ovulação no
protocolo de ressincronização precoce do estro em pequenos ruminantes;
• Em caprinos o manejo reprodutivo associado ao uso de eCG na
ressincronização precoce melhora os resultados obtidos com o protocolo;
• O diagnóstico precoce da gestação ao ser utilizado como ferramenta aumenta
a eficiência dos protocolos de ressincronização precoce em pequenos
ruminantes;
• O uso de protocolo de ressincronização precoce do estro é efetivo e melhora
o desempenho reprodutivo em ovelhas nulíparas, multíparas e lactantes.
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4 OBJETIVOS
4.1 OBJETIVO GERAL
• Desenvolver e avaliar a eficiência de protocolos de ressincronização precoce

do estro em cabras e ovelhas.
4.2 OBJETIVOS ESPECÍFICOS
• Testar diferentes momentos de aplicação de lecirelina para a sincronização

da ovulação de caprinos para melhor eficiência do protocolo de sincronização
e ressincronização;
• Avaliar o efeito da MAP exógena sobre a dinâmica luteal e produção de P4
endógena na gestação inicial em caprinos (D16-D21) e ovinos (D12-D17);
• Testar diferentes doses de eCG para a ressincronização precoce de caprinos
e ovinos;
• Desenvolver e avaliar a eficiência de protocolos de sincronização e de
ressincronização precoces do estro em caprinos e ovinos na estação e contra
estação;
• Avaliar e comparar o efeito do protocolo de ressincronização precoce em
ovinos no desempenho reprodutivo de nulíparas, multíparas e fêmeas pós-
parto.
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5 METODOLOGIAS
Este projeto foi aprovado pelo Comitê de Ética em Pesquisa com Animais da
Universidade Federal Fluminense (Projeto CEUA 923/2017 e 1021/2017 – anexos 1
e 2).
No presente estudo foram utilizados animais adultos da espécie caprina e

ovina. As etapas referentes aos caprinos deste estudo foram realizadas em uma
propriedade particular a partir da assinatura do termo de consentimento livre e
esclarecido (anexo 3), por parte do proprietário e pesquisador, após o mesmo ter
sido completamente informado sobre o referido estudo e as eventuais dúvidas terem
sido sanadas. Os ovinos da raça Santa Inês foram oriundos da Unidade de Pesquisa
Experimental em Caprinos e Ovinos da UFF e os da raça Corriedale da Fazenda da
Faculdade de Veterinária da Universidade da República Uruguaia.
Os seguintes fatores foram considerados, preferencialmente, como critérios

de seleção das fêmeas: (1) condição sanitária, (2) escore da condição corporal, (3)
desempenho produtivo e reprodutivo. Exame clínico-ginecológico foi efetuado nas
fêmeas e apenas animais em estado de saúde adequado foram utilizados.
As metodologias utilizadas em cada experimento foram detalhadas nos

artigos apresentados a seguir.
CAPÍTULO II
CAPRINOS
97
1 ARTIGO: ATIVIDADE OVARIANA EM CABRAS SAANEN LEITEIRAS

SUJEITAS A PROTOCOLO INDUÇÃO DE OVULAÇÃO E INJEÇÃO ÚNICA DE
LECIRELINA (ANÁLOGO GNRH) 28 OU 34 H APÓS O TRATAMENTO CURTO DE
PROGESTERONA
Ovarian activity in dairy Saanen goats subjected to a short-term ovulation

induction protocol and a single injection of lecirelin (GnRH analog) given 28 h
or 34 h after progestin pre-treatment
Artigo publicado no periódico: Small Ruminant Research v.191 (2020)
DOI: 10.1016/j.smallrumres.2020.106214
98
Ovarian activity in dairy Saanen goats subjected to a short-term ovulation

induction protocol and a single injection of lecirelin (GnRH analog) given 28 h
or 34 h after progestin pre-treatment
Isabel Oliveira Cosentinoa*, Mario Felipe Alvarez Balaroa, Felipe Seabra Cardoso
Leala, Ana Luiza Cunha Badea, Lucas de Figueiredo Cardoso Barbosaa, Fernanda
Martins Gonçalvesa, Paula Renata Cortat de Souzaa, Paulo Victor dos Santos
Pereiraa, Pawel M. Bartlewskib, Felipe Zandonadi Brandãoa
aVeterinarian College, Universidade Federal Fluminense, Niterói, Rio de Janeiro,

24230-340, Brazil
bUniversity of Guelph, Ontario Veterinary College, Department of Biomedical

Sciences, Guelph, Ontario, Canada N1G 2W1
* Corresponding author. Rua Vital Brazil Filho, 64. Niterói, Rio de Janeiro, 24230-
340, Brazil; E-mail addresses: isabelcosentino@id.uff.br (I.O. Cosentino)
Abstract
Fixed-time artificial insemination (FTAI) and hormonal superovulation (SOV)

are instrumental in enhancing genetic gain and productivity of commercial goat
herds. The goal of this study was to document changes in antral follicle populations
and ovulation in dairy Saanen does. Fifty-six animals received MAP sponges that
were left in place for 6 days as well as 200 IU of eCG and 120 μg of cloprostenol i.m.
24 h before sponge withdrawal. Does were randomly divided into three groups: G con
(n = 18) received 1 mL of saline 28 h after, whereas G 28h (n = 19) and G34h (n = 19)
received 25 μg of lecirelin i.m. 28 h or 34 h after sponge removal. Estrus was
detected with intact bucks every 12 h and transrectal ovarian ultrasonography was
carried out from the time of sponge removal until ovulation detection (8/8 h – 60 h
after sponge removal) and then until 156 h after sponge withdrawal (12/12 h). There
were no differences (P > 0.05) among GnRH-treated and control goats in estrus
responses (G28h–57.9%; G34h–84.2%; Gcon–83.3%) and mean ovulation rates (G28h–
100%; G34h–91.7%; Gcon–81.8%), but the intervals from MAP sponge withdrawal to
ovulation and from the estrus onset to ovulation were less variable (P < 0.05) in both
99
GnRH-treated groups. The number of large antral follicles (≥6 mm) decreased (P <
0.05) from 52 h to 72 h after sponge removal and then rose (P < 0.05) to 144 h and
122 h in G28h and G34h, respectively. Synchronous ovulation and distinctive pattern of
antral follicle growth after short-term estrus synchronization with lecirelin injections at
28 h or 34 h after MAP sponge removal can pave the way for improving FTAI and
SOV yields in goats.
Key Words: Estrus; Ovulation; Ultrasonography; Superovulation; Fixed time artificial

insemination
1. Introduction
The application of assisted reproductive techniques in small ruminant farming

has significantly improved animal genetic gains and production efficiency, especially
in difficult natural and climatic conditions (Menchaca and Ungerfeld, 2017).
Ultrasonography is an useful diagnostic and research tool, which paved the way for
the development or improvement of assisted reproductive technologies. The use of
transrectal ovarian ultrasonography in small ruminants has been instrumental in
understanding the mechanisms governing antral follicular development and ovulation
(Soboleva et al., 2000; Riesenberg et al., 2001; Esteves et al., 2013; Pietroski et al.,
2013; Alvarado-Espino et al., 2016; Balaro et al., 2016; Texeira et al., 2016;
Menchaca and Ungerfeld, 2017; Almeida et al., 2018; Nascimento-Penido et al.,
2018; Hameed et al., 2020).
In the last decade, hormonal induction and synchronization of estrus, followed

by natural mating or artificial insemination (AI), have been extensively tested in dairy
goats (Abecia et al., 2012; Jeferson Ferreira Fonseca et al., 2019; González-Bulnes
et al., 2020). In cattle, fixed-time artificial insemination (FTAI) has been widely used
around the world (Bó et al., 2018; Feyjoo et al., 2019; Thatcher and Santos, 2020),
but due mainly to a lack of synchronous ovulations after estrus synchronization
protocols currently used, FTAI is not commonly performed in goats (Maffili et al.,
2006; Holtz et al., 2008; Esteves et al., 2013; Fonseca et al., 2017). Determining
optimal time for insemination in small ruminants typically requires estrus detection,
which is cumbersome and time-consuming, especially in large flocks, and not
sufficiently accurate without appropriate personnel training (Fonseca et al., 2017).
100
Therefore, several attempts have been made to improve the synchrony of ovulations
in small ruminants undergoing estrus induction protocols, and the most promising
results were obtained using GnRH-based protocols with (Husein and Kridli, 2003;
Pierson et al., 2003; Luther et al., 2007; Titi et al., 2010) or without (Holtz et al., 2008;
Al Yacoub et al., 2011; Martemucci and D’Alessandro, 2011; Nur et al., 2013) pre-
treatment with progesterone-releasing intravaginal devices.
The Multiple Ovulation and Embryo Transfer (MOET) program is important for
attaining genetic improvement in dairy goat operations. However, the outcome of
MOET in small ruminants is highly variable due mainly to multiple intrinsic and
extrinsic influencing factors (Candappa and Bartlewski, 2011; Ledda and González-
Bulnes, 2018; Fonseca et al., 2019). The best superovulatory results were obtained
when superovulatory (SOV) treatments were initiated in presence of large numbers
of small antral follicles and in the absence of a large/dominant follicle(s) (Guilbault et
al., 1991; Huhtinen et al., 1992; Nasser et al., 1993; Menchaca et al., 2002, 2007b,
2009;). Considering that supposition, Menchaca et al. (2007b) devised the “Day 0
superovulatory protocol” for goats and Balaro et al. (2016) for ewes, wherein a GnRH
agonist (buserelin or lecirelin respectively) is given to synchronize ovulation after a
progestogen treatment and the SOV protocol commences 84 h or 80 h after
progestin device withdrawal in goats and ewes, respectively; consequently, the
superovulatory FSH regimen begins in the absence of dominant ovarian follicles.
Since the preovulatory LH peak heralding ovulation occurs in goats

approximately 35 h after progestin pre-treatment (Pierson et al., 2003; Titi et al.,
2010; Zarazaga et al., 2014), in the present study we decided to examine and
compare the effects of lecirelin administered 28 h or 34 h after the end of progestin
priming. Those times were chosen to induce a synchronous LH discharge prior to or
around the time of the expected endogenous rise in LH secretion in most animals.
The main objective of this study conducted in Saanen goats kept under tropical
conditions was to employ transrectal ovarian ultrasonography to determine the time
of ovulations and changes in numbers of ovarian antral follicles following lecirelin
injections at 28 h or 34 h after progestogen sponge removal. We anticipate that the
present results may pave the way for improving the synchrony of ovulation and
hence conception rates during the FTAI procedure, and for maximizing ovarian
101
responses and embryo yields in goats subjected to the “Day 0 protocol” in MOET
programs.
2. Materials and methods
2.1 Location and experimental animals
All experimental procedures described in this section had been approved by

the Ethical Committee for Animal Use at the Universidade Federal Fluminense
(protocol 1021), and complied with the guidelines of the Brazilian Society of Animal
Experimentation and of the Animal Research: Reporting of In vivo Experiments
(Kilkenny et al., 2010). The present study was conducted in a dairy goat farm located
in Rio de Janeiro state, Brazil (22°07ʹ50.2ʹʹS), during the transition to the breeding
season (January–February; Balaro et al., 2019). It used 56 Saanen goats [2.9 ± 0.5
years old, body condition score: 2.9 ± 0.3 (scale 1–5; Suiter, 1994) (mean ± SD)].
According to Köppen (1948), the local climate is a tropical hot-humid type (Aw). None
of the goats had any reproductive abnormalities detected by ultrasonography or
clinical examination. Throughout the entire experiment, the does were kept in group
pens and fed twice a day with corn silage and concentrate according to their
maintenance requirement [16% crude protein; (National Research Council (NCR),
2007)]. Water and mineral salt licks (Caprinofós, Tortuga, São Paulo, Brazil) were
provided ad libitum.
2.2 Experimental procedures
All goats received intravaginal sponges containing 60 mg of

medroxyprogesterone acetate (MAP; Progespon; Schering Plough, SP, Brazil) for 6
days. One day before sponge withdrawal, 200 IU of equine chorionic gonadotropin
(eCG; Folligon, MSD, São Paulo, SP, Brazil) and 120 μg of cloprostenol sodium
(Estron, Agner União, São Paulo, SP, Brazil) were administered i.m. After sponge
withdrawal, the does were divided into three groups: Gcon (n = 18) received 1 mL of
saline i.m. 28 h after sponge withdrawal; G28h (n = 19) received 25 μg of lecirelin i.m.
28 h after sponge withdrawal; and G34h (n = 19) received 25 μg of lecirelin i.m. 34 h
after sponge withdrawal. Estrus was detected every 12 h with sexually mature, intact
bucks for 96 h after sponge withdrawal.
102
Transrectal ovarian ultrasonography was performed using a portable scanner

(Sonoscape S6, Shenzhen, China) equipped with a 7.5 MHz linear rectal transducer
adapted for use in small ruminants. Animals were restrained and examined in a
standing position. Eleven, 12 and 12 goats from Gcon, G28h and G34h, respectively,
were used for ultrasonographic examinations carried out every 8 h from sponge
withdrawal until ovulation detection (Day 0) and then every 12 h until Day 4 of the
estrous cycle studied (96 h after ovulation). All detectable antral follicles were
categorized into the following 3 size classes: small follicles (≤3.0 mm), medium-sized
follicles (˃3.0 and ˂6.0 mm), and large follicles (≥6.0 mm) (Ginther and Kot, 1994; De
Castro et al., 1999; González-Bulnes et al., 1999). The preovulatory follicle diameter
was defined as the last diameter recorded prior to ovulation detection. Ovulation time
was defined as the mid-way time between the last ultrasonographic detection of the
preovulatory follicle(s) and first time where such a follicle(s) was/were no longer
recorded. Figure 1 shows the experimental proceedings.
Figure 1. Experimental proceedings timeline.
2.3 Statistical analyses
Data were analyzed using SAEG 9.0 statistical program (Universidade Federal
de Viçosa, Minas Gerais, Brazil). The following variables were determined: (1) rate of
estrus response, (2) duration of estrus, (3) interval from sponge withdrawal to the
beginning of estrus, (4) interval from sponge withdrawal to ovulation, (5) interval from
103
the beginning of estrus to ovulation, (6) number of ovulations, and (7) number of
follicles in different size classes. Lilliefors's test was used to verify the normality of
variables and Bartlett's test was used to see if the data were from populations with
equal variances. The F variance test was used to examine the differences in
variability among experimental groups. Parametric data (e.g., follicle growth–data
presented in Fig. 2) were analyzed by one-way analysis of variance and Fisher’s
least significant difference (LSD) test for comparison on individual mean values. Non-
parametric data (e.g., reproductive responses–data presented in Table 1) were
analyzed using Kruskal-Wallis test and Dunn's test. For all tests, P value < 0.05 was
considered statistically significant.
3. Results
There were no significant differences among the three groups of goats for
various intervals between MAP sponge removal, the onset of estrus and ovulation or
for the duration of behavioral estrus, ovulation rates and preovulatory follicle diameter
(Table 1). However, the standard deviation values for the mean interval from sponge
withdrawal to ovulation and from the estrus onset to ovulation were lower (P < 0.05)
in both GnRH-treated groups (G28h and G34h) than in the saline group (Gcon).
Table 1. Reproductive outcomes (mean ± SD) recorded in Saanen does subjected to

short-term estrus synchronization and receiving a saline solution (Gcon) or lecirelin
treatment at 28 h (G28h) or 34 h (G34h) after sponge withdrawal.
Variables G28h G34h Gcon Total
84.2 83.3
Rate of estrus manifestation (%)* 57.9 (11/19) 75.0 (42/56)
(16/19) (15/18)
Duration of estrus (h)* 32.5 ± 17.1 26.8 ± 12.2 36.6 ± 19.6 31.8 ± 16.6
Sponge withdrawal to the beginning
26.4 ± 9.2 29.4 ± 9.5 28.7 ± 11.3 28.4 ± 9.9
of estrus manifestation (h)*
59.6 ±
Sponge withdrawal to ovulation (h)** 52.7 ± 4.2B 53.8 ± 5.5B 55.0 ± 8.5
13.6A
24.5 ± 23.6 ± 35.3 ±
Beginning of estrus to ovulation (h)** 26.9 ± 14.5
10.4AB 10.2B 22.2A
Largest follicle diameter (mm)** 7.1 ± 0.9 7.9 ± 1.4 7.9 ± 1.3 7.6 ± 1.2
Number of ovulations** 2.0 ± 0.4 1.8 ± 0.7 2.3 ± 0.5 2.1 ± 0.6
91.7
Ovulatory response (%)** 100 (12/12) 81.8 (9/11) 91.4 (32/35)
(11/12)
A,B
Different letters within rows denote a significant difference (F test, P < 0.05).
* Gcon–n = 18; G28h–n = 19; G34h–n = 19 ** Gcon–n = 11; G28h–n = 12; G34h–n = 12
104
and large (≥6.0 mm) antral follicle numbers determined ultrasonographically in
Saanen does subjected to short-term estrus synchronization and receiving a saline
solution (Gcon) or lecirelin dose at 28 h (G28h) or 34 h (G34h) after sponge
withdrawal from 52 to 156 h after sponge withdrawal (from 8 h before to 96 h after it).
Each column represents the mean and the error line presents the SD (±).
a, b
Different letters within the chart area represent a significant difference between treatments (Fisher
LSD test, P < 0.05).
A,B
Different letters denote means with significant differences over time (for medium-sized and large
antral follicles; Fisher LSD test, P < 0.05).
105
Mean numbers of small follicles did not differ (P > 0.05) over time or among
treatment groups (Fig. 2). In general, the number of medium-sized follicles increased
(P < 0.05) from 8 h before to 24 h after ovulation, and then began to decline with
slight differences over time within each group (Fig. 2). The G 28h group exceeded (P <
0.05) G34h and Gcon in medium-sized follicle numbers at 52 h, 60 h, 72 h and 96 h,
and Gcon animals at 60 h after sponge withdrawal. In both GnRH-treated groups,
large follicle numbers decreased (P < 0.05) from 52 h to 72 h after sponge removal
and then rose (P < 0.05) to 144 h and 122 h in G28h and G34h, respectively. In Gcon, a
minimum number of large antral follicles was recorded at 96 h and then increased (P
< 0.05) to 144 h after sponge removal. The mean number of large follicles remained
greater (P < 0.05) in Gcon compared with both GnRH treatment groups from 52 h to
72 h after sponge removal.
4. Discussion
To the best of authors’ knowledge, this is the first study of ovulatory responses
and changes in antral follicular numbers after the progestin-based estrus
synchronization protocol combined with the GnRH analog lecirelin treatment in goats.
It is evident that lecirelin synchronized the occurrence of ovulation since both G 28h
and G34h groups exhibited less variable ovulation times than Gcon. Similar results
were obtained in cyclic ewes receiving lecirelin 24 h or 36 h after progestin-sponge
withdrawal (Balaro et al., 2016). Therefore, the lecirelin injections given at 28 h or 34
h after sponge withdrawal can potentially be used as a method to induce
synchronous ovulation for FTAI in small ruminants.
In a previous study in cyclic Alpine goats subjected to the 5-day progesterone

treatment and induced to ovulate with estradiol benzoate (EB) or eCG (Menchaca et
al., 2007a) given at the time of eCG or 24 h after EB CIDR withdrawal, ovulations
occurred 60 h and 70 h after CIDR removal in the treatment group and saline-
injected controls, respectively. Variability in the mean ovulation times observed by
Menchaca et al. (2007a) was 50% less than in the present study (Table 1), but it can
be attributed to synchronizing Alpine goats during the breeding season. However, the
potential differences due to an application of either natural progesterone (CIDR ) or
106
synthetic progestin (medroxyprogesterone acetate) and the duration of the treatment

with progestogen may also play a role and remain to be elucidated.
In the present study, the number of does in estrus was lower for animals that
received lecirelin injections 28 h after MAP sponge withdrawal than in goats injected
34 h after MAP treatment. These results are similar to those reported for ewes that
received lecirelin injections 24 h or 36 h after MAP sponge removal (Balaro et al.,
2016). Diminished manifestations of estrous signs may be problematic if natural
breeding is used, but a lack of behavioral estrus during application of the FTAI
protocol is somewhat less important. The suitability of the presently used GnRH
treatments for FTAI and superovulatory protocols in goats has yet to be evaluated. In
a recent trial conducted in our facility, the pregnancy rate in Saanen does subjected
to a short-term estrus synchronization protocol and a single dose of lecirelin 34 h
after sponge withdrawal was 56% (20/36; unpublished results); the experiment was
carried out during the early anestrous period (August–October) and the does were
inseminated 48 h after MAP sponge withdrawal. Those results were better compared
with a trial by Nur et al. (2013) using buserelin before sponge insert at 48 h after
sponge withdrawal in Saanen goats (24%–38%–breeding season at 40.19°N), but
similar to those obtained by Al Yacoub et al. (2011) (50%) also using buserelin at 48
h after prostaglandin treatment (does with at least 5 ng/mL of serum progesterone) in
Boer goats (October–January, breeding season at 51°46ʹN). Therefore, lecirelin
appears to be equally or more effective than currently used buserelin for
synchronizing ovulation during AI protocols in goats.
Based on the number of large antral follicles detected using ultrasonography,

the optimal time to begin the superovulatory regimen with exogenous FSH in
lecirelin-treated goats (both 28 h and 34 h after sponge removal) would be within 12
h of ovulation or between 60–72 h after MAP sponge removal. Due mainly to highly
synchronous ovulation times, detectable numbers of large antral follicles are
consistently low during that time window. During that period, there is also a gradual
(numerical) increase in small follicle numbers and a significant rise in the numbers of
medium-sized follicles. The latter may indicate an increasing responsiveness of
ovarian follicles to FSH stimulation. Bartlewski et al. suggested that the number of
medium-sized antral follicles in superovulated ewes is a better predictor of ovulatory
107
responses in SOV ewes than small follicle numbers (Bartlewski et al., 2016, 2008).
This suggested time to commence the SOV treatment in goats differs from the
original protocol developed by Menchaca et al. (2007b), wherein FSH administration
begins 84 h after sponge withdrawal. According to our study, at 84 h after sponge
withdrawal the numbers of large antral follicles are still relatively low, but the numbers
of medium-sized antral follicles begin to decline in GnRH treatment groups. In control
goats, however, the 84-h mark appears to be optimal due to the lowest number of
large and peak number of medium-sized antral follicles. As mentioned earlier, more
confirmatory studies are needed to corroborate the effectiveness of this treatment in
superovulated goats.
5. Conclusion
The administration of 25 μg of GnRH analog lecirelin, either 28 h or 34 h after

the removal of MAP-soaked intravaginal sponges inserted for 6 days, improves the
synchrony of ovulations in Saanen goats raised in a tropical climate. Therefore, this
treatment can potentially be used for FTAI programs. In addition, ultrasonographic
observations revealed that the highest numbers of small antral follicles and the
absence of large, ostensibly dominant, follicles occurred between 60 h and 72 h after
sponge withdrawal in GnRH-treated animals, suggesting that this would be an
optimal period to start a SOV treatment (Day 0 protocol) in Saanen goats under
tropical conditions.
Acknowledgments
The present study was supported by Fundação Carlos Chagas Filho de

Pesquisa Pós-Graduação - Proppi/UFF (UFF/Proppi/Infra-LabPesq). This study was
financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior - Brasil (CAPES) - Finance Code 001. FZB is fellow of the CNPq. IOC is a
receipient of a scholarship provided by CAPES. The authors would like to thank
Enago (www.enago.com) for the English language review. We also thank the dairy
goat farm: Capril Vale das Amalthéias by their animals and management support for
this research.
108
IOC, MFAB, FZB co-designed the present study and organized the
experiment. IOC, MFAB, FSCL, ALCB, LFCB, FMG, PRCS, PVSP, FZB collected
and analyzed the data as well as wrote and revised the manuscript. PMB helped with
data interpretation and critical review of the present paper. All authors approved its
final version.
Conflict of interest statement
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Early resynchronization protocols for goats: Progestogens can be used prior

to an early pregnancy diagnosis without affecting corpus luteum functionality
Artigo publicado no periódico: Reproduction in Domestic Animals v. 55 (2020)

p.1655-1659
DOI: 10.1111/rda.13830
116
Early resynchronization protocols for goats: progestogens can be used prior to

an early pregnancy diagnosis without affecting corpus luteum functionality
P4 in unknown pregnancy status goats: effect on CL function
Isabel Oliveira Cosentinoa*, Mario Felipe Alvarez Balaroa, Felipe Seabra Cardoso
Leala, Lucas de Figueiredo Cardoso Barbosaa, Fernanda Martins Gonçalves a,
Gabriel Feliciano Felizardo a, Marina Monteiro Nettoa, Felipe Zandonadi Brandãoa
aFaculdade de Veterinária, Universidade Federal Fluminense, Niterói, Rio de Janeiro,

24230-340, Brazil
* Corresponding author. Rua Vital Brazil Filho, 64. Niteroi, Rio de Janeiro, 24230-
340, Brazil. E-mail addresses: isabelcosentino@id.uff.br (I.O. Cosentino),

fzbrandao@id.uff.br (F.Z. Brandão).
Summary
This study aimed to evaluate the exogenous progesterone (P4) effect on the
luteal function from Day 16 to Day 21 of the estrous cycle in inseminated goats with
unknown pregnancy status. A total of 54 does passed through a short progestin-
based synchronization protocol and, on Day 16 of the following estrous cycle, 27
does received a new P4 device which was retained until Day 21. Blood samples were
collected daily from all does during this period, as well as on Day 24. Pregnancy
diagnoses were performed on Day 30. Serum P4 values from 26 animals (G NPSP:
Group of non-pregnant does with second sponge: n=8; GNPNSP: Group of non-
pregnant does without second sponge: n=6; G PSP: Group of pregnant does with
second sponge: n=5; GPNSP: Group of pregnant does without second sponge: n=7)
were determined by radioimmunoassay commercial kits. No P4 differences were
found between groups (GNPSP: 3.1 ± 2.8; 1.7 ± 1.8; 0.4 ± 1.0; and 0.0 ± 0.0 vs.
GNPNSP: 4.4 ± 1.8; 3.0 ± 2.2; 0.8 ± 0.8; and 0.0 ± 0.0 or GPSP: 4.2 ± 1.0; 3.4 ± 0.6; 3.3
± 1.6; 3.2 ± 0.9; 3.6 ± 1.2; 3.5 ± 1.3; 2.7 ± 1.3 vs. G PNSP: 4.4 ± 1.6; 3.6 ± 1.5; 3.7 ±
1.5; 3.8 ± 1.4; 3.2 ± 1.2; 3.1 ± 1.2; 3.6 ± 1.1; D16, D17, D18, D19, D20, D21, D24,
respectively) or for the interaction of group and time. In conclusion, a second
117
progestogen device had no effect on luteolysis or early pregnancy in the following

estrous cycle.
Keywords: Serum P4, ultrasound, medroxyprogesterone acetate
1 Introduction
Resynchronization protocols have been studied and successfully performed in

cattle since the beginning of the century (Bartolome et al., 2005; Sani et al., 2011;
Stevenson et al., 2003). With variations in timing and procedures, such protocols are
primarily used to reduce the calving interval by inducing more synchronic ovulations
during the breeding season. Hence, early resynchronization protocols are being
studied to allow fixed time artificial insemination (FTAI) at intervals similar to the
natural estrous cycle (Palhão et al., 2020; Pugliesi et al., 2019), for which a new
progesterone (P4) device should be inserted before the pregnancy diagnosis.
The corpus luteum (CL) is a temporary endocrine gland of high importance in

the maintenance of pregnancy, especially for goats, since ewes and cows can
sustain late pregnancy without it (Casida & Warwick, 1945; Drummond-Robinson &
Asdell, 1926; Estergreen et al., 1967; Meites et al., 1951). Knowledge of its
functionality has led to the implementation of new hormonal protocols. However,
studies in which exogenous P4 administration is used indicate concerns about its
effect on CL in early pregnancy: a P4 device inserted as early as three days after
estrous manifestation in cows may lead to both better conceptus development and
early luteolysis (O’Hara et al., 2014; Pugliesi et al., 2014). However, when it is used
at the end of the estrous cycle, aiming at a resynchronization protocol, no effect on
CL has been detected (Sani et al., 2011; Stevenson et al., 2003).
In ewes, even though Miranda et al. (2018) demonstrated a negative effect on

P4 production, Cosentino et al. (2019) did not. Both studies concluded that the use of
a second progestogen device from Day 12 to Day 17 of the cycle had no effects on
pregnancy or luteolysis, enabling its use in the early resynchronization protocol.
However, it has not been studied for goats, and we hypothesize that, as with ewes, a
new P4 device inserted early in does would not have a negative effect on CL viability
and functionality. Therefore, this study aimed to evaluate the effect of a second
118
progestogen device inserted late in the estrous cycle on goats’ CL functionality, and
consequently in their not yet diagnosed pregnancy or sub-sequential luteolysis.
2 Materials and methods
All procedures performed in this study were given prior approval by the Ethical
Committee for Animal Use of the Universidade Federal Fluminense (Protocol 1021)
and were carried out under the ethical principles of the Sociedade Brasileira de
Experimentação Animal. Moreover, this manuscript followed the guidelines laid out in
Animal Research: Reporting of In Vivo Experiments (Kilkenny et al., 2010).
This study was carried out in a dairy goat farm located in the state of Rio de
Janeiro, Brazil (22°07'50.2"S, 42°47'47.1"W) during the transition season (Jan –
Feb/2019). According to Köppen (1948), the local climate is tropical hot-humid (Aw).
For this study, a total of 54 primiparous and multiparous Saanen goats {2.7 ± 0.5
years old; body condition score: 2.9 ± 0.3 [scale 1-5; (Suiter, 1994)]; bodyweight 60 ±
5 kg; 7.7 ± 3.6 months post-partum (mean ± SD)} were used. All animals had
previously undergone a gynecological exam and only does without reproductive
abnormalities detected by ultrasonography (US) or clinical exam were used.
Throughout the study, does were kept confined in collective pens and fed twice a day
with corn silage, as well as concentrate according to their maintenance requirements
[16% crude protein; National Research Council (NCR), 2007]. Water and mineral salt
for goats (Caprinofós, Tortuga, São Paulo, Brazil) were provided ad libitum.
Intravaginal sponges containing 60 mg of medroxyprogesterone acetate

(MAP: Progespon; Schering Plough, SP, Brazil) were used for six days. One day
before sponge withdrawal, 200 IU of equine chorionic gonadotropin (eCG, Folligon,
MSD, São Paulo, Brazil) and 0.24 mg of cloprostenol sodium (Estron, Agner União,
São Paulo, Brazil) were administered intramuscularly (i.m.). Thirty-four hours after
sponge withdrawal, the females received 0.025 mg of lecirelin (GnRH: Gestran Plus,
Tecnopec, São Paulo, Brazil) and 52 h after the sponge withdrawal, they were
inseminated with a 0.25 mL straw of commercial frozen semen (5.0 x 107 alive
spermatozoa after defrost). From Day 16 to Day 21 of the following estrous cycle,
half the does received a new MAP device. General proceedings are described in
Figure 1.
119
Ovarian US was conducted every 8 h after sponge withdrawal until the

moment of ovulation, which was taken to be Day 0 (55.0 ± 8.5 h after sponge
withdrawal) of the estrous cycle. US scans were performed using a portable device
(Sonoscape S6, Shenzhen, China) with a 7.5 MHz linear rectal transducer adapted
for use in small ruminants, with does in a standing position. All does were re-
examined on Days 16, 21, and 24 for assessment of luteal morphology (score 1: near
anechoic, heterogeneous, and coarse granulation; 2: hypoechoic, homogeneous,
and fine granulation; and 3: echogenic, homogeneous, and fine granulation) and
blood flow (score 1: 0 to 25% vascularization; 2: 25 to 50%; 3: 50 to 75%; 4: 75 to
100%), with scores above 2 being related to pregnancy (Cosentino et al., 2018). The
first evaluation (Day 16) aimed to count the viable CLs before the second P4 device;
the second evaluation (Day 21) was for early pregnancy diagnosis at time of sponge
removal; and the third evaluation (Day 24) was to evaluate possible early pregnancy
losses and further effects of the second P4 device. The last US scan was performed
on Day 30 for pregnancy diagnosis by evaluation of the embryo vesicle and
heartbeat. After the Day 30 pregnancy diagnosis, the does were classified in four
groups: GNPSP: Group of non-pregnant does with second sponge; GNPNSP: Group of
non-pregnant does without second sponge; GPSP: Group of pregnant does with
second sponge; GPNSP: Group of pregnant does without second sponge.
120
Blood samples were collected daily from Day 16 to Day 21 and on Day 24 by
jugular venipuncture using tubes (without anti-coagulant) with a vacuum system.
Blood samples were centrifuged at 1000 x g for 15 min; serum was separated and
stored at - 20 ºC until analysis. The serum P4 concentration from 26 animals (G NPSP
= 8; GNPNSP = 6; GPSP = 5; GPNSP = 7) was determined by radioimmunoassay using
commercial kits (MP Biomedicals, LLC, Diagnostics Division, Orangeburg, NY, USA).
Sensitivity and intra-assay coefficient of variation were 0.05 ng/mL and 12%,
respectively. All data were within minimum and maximum points of the curve. CL
were considered functional when production was above 1.0 ng/mL.
Data were analyzed using a statistical program for statistical analyses

(BioEstat®). Lilliefors and Bartlett tests were used to verify the normality and
homoscedasticity of variables, respectively. P4 values were analyzed using a mixed
model procedure including the group (treated or not treated), time (from Day 16 to
Day 21, and Day 24), and their interaction (groups vs. time) as main effects in the
model. Fisher’s LSD test was used for individual mean comparisons. Score data (CL
evaluation) were assessed by Fisher’s exact test. For all tests, P<0.05 was
considered significant.
3 Results
A total of 15/54 females did not present signs of estrous, of which only four did
not present mucus production and cervix dilatation at the moment of FTAI. On Day
16 eight females did not present CL, of which only one did not present mucus at the
moment of FTAI. Of the 14 does diagnosed pregnant on Day 21, 13 were confirmed
on Day 24 (93%), and 12 on Day 30 (86%), both from the group without a second
sponge (22% of pregnancy: 12/54). No differences were found between groups
(GNPSP vs. GNPNSP or GPSP vs. GPNSP: P < 0.05) both for P4 values (Figure 2) and the
presence of vascularized CL on Days 16, 21, 24, and 30. Pregnant does presented
100% of vascularized CL on all days (5/5 GPSP and 7/7 GPNSP); non-pregnant does
GNPSP presented 72.7% (16/22) on Day 16 and 0% on the others; and G NPNSP
presented 90% (18/20), 10% (2/20), 5% (1/20), and 0%, respectively. The CL
vascularization was related to P4 values above 1.0 ng/mL for all does. Nor did the
interaction between group and time show any differences for the P4 analysis.
121
However, P4 values differ over time, when considering the treatments together
(Figure 2).
Figure 2. Serum progesterone concentration (ng/mL) from Day 16 to Day 21 and on

Day 24, in pregnant (n=12) and non-pregnant (n=14) does. An intravaginal sponge
impregnated with medroxyprogesterone acetate was inserted in does from the
GNPSP and GPSP groups.
GNPSP: Group of non-pregnant does with second sponge: n=8; GNPNSP: Group of non-pregnant
does without second sponge: n=6; GPSP: Group of pregnant does with second sponge: n=5; GPNSP:
Group of pregnant does without second sponge: n=7
A,B different letters over time when considering the treatments together (silver line) by Student’s T test
(p<0,05).
122
4 Discussion
To the author’s knowledge, this is the first study performed in goats which
aims to investigate the effect of a second P4 device on CL functionality for further
application in early resynchronization protocols. Our current results show that the
second P4 device has no effect on luteolysis or early pregnancy from the first FTAI in
Saanen goats. Considering mucus production and cervix dilatation, it is possible to
say that the majority of does responded to the synchronization protocol, even though
not all of them presented signs of estrous, which can be explained by social ranking
(Zuñiga-Garcia et al., 2020) or a silent heat. However, when analyzing the presence
of CL on Day 16, it is not possible to say whether those females did not respond at
all, or whether the absence of CL was due to early regression. Since none of the
females without mucus at FTAI became pregnant, both are possible.
Miranda et al. (2018) proposed a negative effect of MAP devices on P4

production in ewes (5.8 ± 0.5 ng/mL for control group vs. 4.7 ± 0.5 ng/mL for MAP
group on Day 15), although without negative effects on the final percentage of
pregnant females. A later study found no effect on P4 values either on pregnancy or
luteolysis in sheep species (Cosentino et al., 2019). The results of the present study
indicate that the use of a second P4 device does not imply negative effects on
ovarian activity, since no difference was found between P4 values in does that
received the second device and those which did not (GNPSP vs. GNPNSP or GPSP vs.
GPNSP). The differences found over time were observed in both groups for each
category, indicating that these were physiological and not induced by the MAP
device. Unlike in heifers, when the progestogen device is used during early
metoestrous (Day 3 to 7 after ovulation), during which the presence of exogenous P4
is enough to affect the CL development and therefore decrease its size and lifespan
(O’Hara et al., 2014), the use in mid/late dioestrus does not affect the functionality
and viability of the mature CL (Burke et al., 1999). Neither does it enhance the
conceptus development or τ-interferon production (Mann et al., 2006). It could be
expected that the exogenous progestogen would downregulate or LH release and
therefore impact the P4 production, since its production is partially dependent on LH
levels (Wiltbank et al., 2012). In this sense, we feared that, by the time of sponge
withdrawal, the CL from pregnant does could have been affected and would suffer
123
luteolysis. However, the Day 24 P4 serum concentration and Day 30 ultrasound

suggest that the MAP device had no deleterious effect.
Additionally, both females diagnosed as pregnant on Day 21 by luteal

evaluation and finally diagnosed as non-pregnant on Day 24 and Day 30 did not
receive the second MAP device. In this case, the change in diagnosis may be
explained by the fact that those does presented long lifespan CL or early fetal loss;
however, neither was caused by the use of external progestogen. Also, the P4 value
of above 1ng/mL and vascularization above score 2 (Cosentino et al., 2018) confirm
luteal functionality even after the second progestogen device. These results endorse
the finding that the second P4 device had no negative effects on CL function and
viability, which enables its use in resynchronization protocols.
In conclusion, a second P4 device can be used in goats with unknown

pregnancy status without affecting early pregnancy, luteolysis, or endogenous P4
production. Therefore, a second P4 device can be used for resynchronization
protocols in goats.
Acknowledgments

Pesquisa Pós-Graduação - Proppi/UFF (UFF/Proppi/Infra-LabPesq). FZB is fellow of
the CNPq. IOC had a scholarship provided by CAPES. This study was financed in
part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil
(CAPES) - Finance Code 001. We also thank the dairy goat farm: Capril Vale das
Amalthéias by their animals and management support for this research.
IOC discussed the general study design, organized the experimental

procedures, collected data, revised and worked on the preparation of the manuscript,
and approved the final version. MFAB discussed the general study design, collected
data, revised and worked on the preparation of the manuscript, and approved the
final version. FSCL, LFCB, FMG, GFF and MMN collected data, revised and worked
124
on the preparation of the manuscript, and approved the final version. FZB discussed
the general design, revised and worked on the preparation of the manuscript, and
approved the final version.
Competing interests
Data availability statement
No additional data are available.
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ovulation in dairy goats
Artigo publicado no periódico: Animal Reproduction v. 19 (2022)
DOI: 10.1590/1984-3143-AR2021-0112
129
eCG use and breeding system at does’ resynchronization

ovulation in dairy goats
Isabel Oliveira Cosentino1* (https://orcid.org/0000-0002-1059-1831), Mário Felipe

Alvares Balaro1 (https://orcid.org/0000-0002-8198-7964), Polyanne Martins da Silva2
(https://orcid.org/ 0000-0002-5893-7862), Augusto Ryonosuke Taira1
(https://orcid.org/0000-0001-5919-6405), Juliana Dantas Rodrigues Santos1
(https://orcid.org/0000-0001-8324-1309), Ana Clara Sarzedas Ribeiro1
(https://orcid.org/0000-0003-0747-4826), Bruna Ramalho Rigaud de Figueiredo1
(https://orcid.org/0000-0001-9873-4472), Marta Maria Campos Pereira da Costa1
(https://orcid.org/0000-0003-3898-8539), Bruno Ribeiro Vieira1
(https://orcid.org/0000-0001-6414-9021), Felipe Zandonadi
Brandão1(https://orcid.org/0000-0003-4027-5562)

24230-340, Brazil
2Capril Vale das Amalthéias, Sapucaia, Rio de Janeiro, Brazil, 25780-000
* Corresponding author. Rua Vital Brazil Filho, 64. Niteroi, Rio de Janeiro, 24230-
340, Brazil.
E-mail addresses: isabelcosentino@id.uff.br (I.O. Cosentino), fzbrandao@id.uff.br

(F.Z. Brandão).
Abstract
Resynchronization protocols have been proposed as a way of shortening females’

unproductive time in the flock, with good results in cattle and sheep. In goats, initial
studies have shown that a second progestogen device inserted before luteolysis and
pregnancy diagnosis does not interfere with the corpus luteum lifespan or
functionality. This study aimed to evaluate the follicular growth, ovulation pattern and
pregnancy rate after insertion of a second and new progestogen device for
resynchronizing, with or without equine Chorionic Gonadotrophin (eCG), submitted to
130
natural mating (NM) or artificial insemination (AI) to propose a viable

resynchronization protocol for dairy goats. A total of 38 multiparous Saanen goats
underwent a short-term progesterone protocol [six days exposed to
medroxyprogesterone acetate (MAP) intravaginal sponges + 200 IU eCG and 0.12
mg of cloprostenol sodium on the 5th day + 0.025 mg of lecirelin 34 hours after
sponge withdrawal] and, on day 16th after the ovulation, received a new MAP device
which was retained until day 21. At this moment females were split into four groups:
GeCG+NM – 100 IU eCG with NM; GSal+NM – saline solution with NM; GeCG+AI –
100 IU eCG with AI; and GSal+AI – saline solution with AI. Ultrasound scans were
performed every 12 h from sponge withdrawal (day 21) until 108 h after sponge
withdrawal (day 25) for follicular dynamics evaluation, at 240 h (day 31) for assessing
the presence of active corpus luteum, and on day 60 for pregnancy diagnosis. No
differences were found regarding ovulation time, synchronization and follicle size.
However, GeCG+NM presented a greater estrus manifestation rate (100%) and
pregnancy rate (62.5%) when compared to GSal+AI. In conclusion, resynchronization
protocols in dairy goats may present satisfactory results.
Keywords: follicle dynamics, ultrasound, artificial insemination, natural mating.
Introduction
Seasonality is recognized as an important factor that interfere in goats’

reproduction. Adult Saanen goats living in tropical condition present accentuated
anestrus during spring; however, during early summer, social interactions with males
or cycling females may induce, in different degrees, the return to cyclicity (Balaro et
al., 2019). During this transition season, the use of hormonal protocols leads to better
results in reproductive rates, since cyclicity is homogenized (Alvarado-Espino et al.,
2016; Véliz-Deras et al., 2020).
Resynchronization protocols have been studied and proposed as a way of

reducing females´ unproductive time in the flock. In cattle, it has successfully been
used in different protocols (Bartolome et al., 2005; Bó et al., 2016; Sani et al., 2011),
including the ones that use Fixed Time Artificial Insemination (FTAI) at intervals that
come closer to their natural estrous cycle (Palhão et al., 2020; Pugliesi et al., 2019).
In small ruminants such protocols are still being studied for ewes under management
131
with natural mating (NM) (Miranda et al., 2018) or FTAI, using different doses of eCG:
saline, 200IU, or 300IU (Cosentino et al., 2019), with promising results in nulliparous
and pluriparous ewes, but with limited results for post-partum females (Cosentino et
al., 2021). In goat, preliminary findings have shown that a second P4 device inserted
before luteolysis and pregnancy diagnosis does not interfere with corpus luteum (CL)
lifespan or P4 production (Cosentino et al., 2020b), as it was defined for ewes
(Cosentino et al., 2019; Miranda et al., 2018).
eCG is a gonadotrophin often used in synchronization and induction of

ovulation protocols. It acts in the ovary promoting the expression of angiogenic
factors, and, as a consequence, causing neovascularization of follicles, thus
increasing the ovarian response to LH (Honnens et al., 2009; Mattioli et al., 2001)
and enhancing synchronization results. However, the successive use of eCG in goats
leads to antibodies production and the decrease of efficiency of the protocol
(Chemineau et al., 1999; Roy et al., 1999). Therefore, we hypothesized that, in goats,
the second protocol that does not use eCG may lead to the resynchronization of
ovulation without losing the efficiency. That is, the dose used in the first
synchronization would be enough for both protocols.
In ewes, the resynchronization protocol has been used with two reproductive
managements – NM and FTAI – with successful results (Cosentino et al., 2021,
2019; Miranda et al., 2018). However, the presence of the male is known to influence
the moment of ovulation. Also, Muñoz et al. (2019) showed that the presence of the
sexually active male was enough to induce a second ovulation after an induced
luteolysis in estrous induced does. During transition season, Véliz-Deras et al. (2020)
demonstrated that NM presented higher pregnancy rates (42% x 20%) than Artificial
Insemination (AI) even when it was performed with fresh semen. That being so, the
presence of an active male during the second synchronization may improve
synchronization results, especially for females that did not undergo eCG treatment.
This study aimed to evaluate the follicular growth and ovulation pattern after
the second progestogen device in resynchronized does, with or without eCG,
submitted to NM or AI to propose a viable resynchronization of ovulation protocol for
dairy goats.
132
Methods
Procedures performed in this study were approved by the Ethical Committee

for Animal Use of Universidade Federal Fluminense (protocol 1021) and carried out
under the ethical principles of Sociedade Brasileira de Experimentação Animal. Also,
the study followed the guidelines of Animal Research: Reporting of in vivo
experiments (Kilkenny et al., 2010).
Experiment location and animals
This study was carried out in a dairy goat farm located in Rio de Janeiro state,
Brazil (22°07'50.2”S, 42°47'47.1”W) during the transition season (Dez/2020 –
Feb/2021), under a tropical hot-humid type (Aw) local climate (Köeppen, 1948). A
total of 38 multiparous Saanen goats were used [3.3±1.4 years-old; body condition
score: 3.2±0.2 (scale 1-5; (Suiter, 1994))]. All does were previously submitted to a
gynecological exam and only females without reproductive abnormalities detected by
ultrasound (US) or clinical exam were studied. Throughout the study, animals were
kept confined in collective pens and were fed twice a day with corn silage and
concentrate, all provided according to their maintenance requirements (16% crude
protein). Water and mineral salt for goats (Caprinofós, Tortuga, São Paulo, Brazil)
were provided ad libitum.
Estrus synchronization and resynchronization
Females had their estrus initially synchronized as described by Cosentino et

al. (2020b): using intravaginal sponges containing 60mg of medroxyprogesterone
acetate (MAP: Progespon; Schering Plough, SP, Brazil) for six days. One day before
sponge withdrawal, 200 IU of equine chorionic gonadotropin (eCG, Folligon, MSD,
São Paulo, Brazil) and 0.12 mg of cloprostenol sodium (Estron, Agner União, São
Paulo, Brazil) were administered intramuscularly (i.m.). Thirty-four hours after sponge
withdrawal, females also received 0.025 mg of lecirelin (GnRH: Gestran Plus,
Tecnopec, São Paulo, Brazil) i.m. (Figure 1).
133

For the resynchronization protocol, a second sponge was used from day 16 to
day 21 (Cosentino et al., 2020b). At sponge withdrawal, half of the does received
100IU of eCG i.m., while the other half received 0.5mL of saline solution i.m. At this
moment, estrous signs evaluation begun, being performed every 12h for five days in
all does, or until the ovulation was detected by ultrasound (US). Females were
considered in heat when accepting mating (which was not allowed in the AI group),
and not running from the teaser male. After sponge removal, half of the does were
kept constantly with fertile male bucks (previously assessed by andrological
examination) for NM, while the others were kept apart in pens distant (100m) from
the male presence for AI. Four groups were formed: GeCG+NM – 100 IU eCG with
NM (n=8); GSal+NM – saline solution with NM (n=10); GeCG+AI – 100 IU eCG with
AI (n=10); and GSal+AI – saline solution with AI (n=10). Does were randomly divided
into groups, so that ages would be equally distributed among the groups. Bucks kept
with GeCG+NM and GSal+NM had their chest painted in a different color each day,
as a way to identify females that accepted mating. Females from GeCG+AI and
GSal+AI groups were artificially inseminated with commercially frozen semen in
accordance with the follicular growth (above 8 mm for animals that did not present
estrous signs) and estrous signs beginning (Maia et al., 2017). Females that
presented estrous behavior 36 h, 48 h and 60 h after the sponge removal had AI
performed at 24 h, 18 h and 10 h after the beginning of the signs, respectively. AI
was performed once per female, transcervical (Fonseca et al., 2017), using 0.25 mL
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commercial straws (concentration of approximately 300 x 106 sperm/mL) from three

different bucks, by the same technician. Two fertile bucks were kept with the females
from groups GeCG+NM and GSal+NM (8:1 female:male), from sponge withdrawal
until two days after the end of US examinations.
Ultrasound evaluation and pregnancy diagnosis
US scans were performed using a portable device (Mindray Z5, China) with a
7.5MHz linear rectal transducer, adapted for transrectal use in small ruminants, with
does in a standing position. Ovarian US was conducted every 12h from sponge
withdrawal (Day 21) until 108h after sponge withdrawal (Day 25) to evaluate follicular
dynaics. Follicles were classified in small-sized (<3.0mm), medium-sized (3-6mm)
and large-sized (>6.0mm), and were considered ovulated when the large-sized
dominant follicle disappeared (Cosentino et al., 2020a; Castro et al., 1999; Ginther
and Kot, 1994; Gonzalez de Bulnes et al., 1999). Another US scan was conducted at
240h (day 31) to assess ovulation by the presence of an active CL. The last US scan
was performed transrectally on day 60 for pregnancy diagnosis to evaluate embryo
vesicle and heartbeats.
Statistical analysis
To perform the analysis, SAEG (UFV, 2007) was used. Lilliefors test was used
to verify the normality of the variables, and Bartlett test to verify whether the data will
be obtained from populations with equal variances. The F variance test was used to
examine differences in variability between experimental groups. Parametric data
were analyzed by unilateral analysis of variance and Fisher's least significant
difference test (LSD) to compare individual mean values. Nonparametric data were
analyzed using both Kruskal-Wallis test and Dunn test. For all tests, a P<0.05 value
was considered statistically significant.
Results
Reproductive outcomes are shown in Tables 1, 2 and 3. Estrus duration and

the interval from sponge withdrawal to the beginning of estrus did not differ between
treatments, but GSal+AI presented an estrus response rate lower than GeCG+NM
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(P< 0.05), and GSal+NM and GeCG+AI were similar in all groups (Table 1).
Regarding ovulation time, synchronization indices and follicle size, no differences
were found among groups (Table 2).
Table 1. Estrus response and manifestation (mean ± standard error) recorded in

Saanen does subject to short-term estrus resynchronization and receiving or not 100
IU of eCG in different breeding systems (natural mating or artificial insemination).
Parameters GeCG+NM GSal+NM GeCG+AI GSal+AI Total

Estrus response 100.0%a 70.0%ab 70.0%ab 50.0%b 71.1%
(%) (8/8) (7/10) (7/10) (5/10) (27/38)
Estrus duration (h) 30.0±5.6 37.7±8.0 44.6±4.3 40.8±8.1 37.8±3.2
Sponge withdrawal
to the beginning of 60.0±5.6 48.9±6.9 45.4±4.3 56.4±4.5 52.7±2.9
estrus (h)
a, b
Different letters within rows denote a significant difference Kruskal-Wallis test (P<0.05) or Fisher’s
test (P< 0.05). Sal – saline; NM–natural mating; AI–artificial insemination; eCG-equine chorionic
gonadotropin; CL–corpus luteum.
Table 2. Ovulation parameters (mean ± standard error) recorded in Saanen does

subject to short-term estrus resynchronization and receiving or not 100 IU of eCG in
different breeding systems (natural mating or artificial insemination).

Sponge withdrawal to
79.8±5.0 74.8±5.5 74.7±5.7 93.0±4.9 79.4±3.0
ovulation (h)
80.1–
Ovulation window (h) 69.7–89.9 61.3–88.2 60.7–88.7 65.3–91.6
102.7
Beginning of estrus to
22.8±6.8 30.8±4.8 32.7±3.6 41.4±2.5 31.4±2.6
ovulation (h)
Interval between first NM/AI
16.8±6.8 26.3±5.6 12.2±5.4 23.1±8.2 19.2±3.2
and ovulation (h)
Largest follicle diameter
7.1±0.4 7.1±0.2 7.5±0.4 7.6±0.2 7.3±0.2
(mm)
50.0% 60.0% 60.0% 40.0% 52.6%
Ovulation detected rate (%)
(4/8) (6/10) (6/10) (4/10) (20/38)
No differences were found at Kruskal-Wallis test (P<0.05) or Fisher’s test (P< 0.05). Sal – saline; NM–
natural mating; AI–artificial insemination; eCG-equine chorionic gonadotropin; CL–corpus luteum.
Looking for individual outcomes for estrus duration, interval from the beginning
of estrus to ovulation, and interval from sponge withdrawal to ovulation, comparisons
between each female and the group mean were made (Figure 2), showing variances
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of individual responses. No significant differences were found between groups

(Tables 1 and 2), but saline groups presented numerically lower homogeneity in
estrus duration than eCG induced groups (P< 0.05) (Table 1 and Figure 2). The
follicular dynamics performed showed no differences between treatments. The only
differences found were in time, as expected to a follicular wave. Considering all
groups together large follicles are largely found at 48h and 72h, medium-sized from
24 to 60h after sponge withdrawal, and small follicles at sponge removal and 12h
after it (Figure 3).
Figure 2. Reproductive outcomes per female compared within and between groups:
(A) estrus duration; (B) beginning of estrus to ovulation; (C) sponge withdrawal to
ovulation.
137
and large (≥6.0 mm) antral follicle numbers determined ultrasonographically in
Saanen does subject to short-term estrus resynchronization and receiving or not 100
IU of eCG in different breeding systems (natural mating or artificial insemination).
A,B,C,D,E
Different letters denote means with significant differences over time (considering all treatments
together; Fisher LSD test, P<0.05).
No differences were found between groups for presence and number of CL

seven days after ovulation (Table 3). However, GeCG+AI and GSal+AI presented
lower pregnancy rate than GeCG+NM (P< 0.05); and GSal+NM was similar to all
groups (Table 3). Regarding the evaluation of ovulation, considering the number of
CLs seven days after, there was no significant difference between groups:
GeCG+NM had 5 does with single ovulation, and 1 with double ovulation; GSal+NM
138
had 2 does with single ovulation and 4 with double ovulation; GeCG+AI had 4 does
with single ovulation, 2 with double ovulation and 1 with triple ovulation; and GSal+AI
had 3 does with single ovulation and 2 with double ovulation.
Table 3. Post protocol ovarian parameters and pregnancy diagnosis (mean ±

standard error) recorded in Saanen does subject to short-term estrus
resynchronization and receiving or not 100 IU of eCG in different breeding systems
(natural mating or artificial insemination).

Ovarian evaluation 7 days after ovulation
Presence of CL
75.0% (6/8) 70.0% (7/10) 70.0% (7/10) 50.0% (5/10) 65.8% (25/38)
(%)
Number of CL
1.2±0.2 1.7±0.2 1.6±0.3 1.4±0.2 1.5±0.1
detected
Pregnancy diagnosis
Pregnant does 30.0%ab 10.0%b
62.5%a (5/8) 10.0%b (1/10) 26.3% (10/38)
(%) (3/10) (1/10)
Pregnant/ovulatio
83.3%a (5/6) 42.9%ab (3/7) 14.3%b (1/7) 20.0%ab (1/5) 40.0% (10/25)
n (%)
CL presence during pregnancy diagnosis in non-pregnant does*
Non-pregnant 88.89%A 78.57%A
33.33% (1/3) 71.43% (5/7) 88.89%A (8/9)
without CL (%) (8/9) (22/28)
B
Non-pregnant 11.11%
66.67% (2/3) 28.57% (2/7) 11.11%B (1/9) 21.43%B (6/28)
with CL (%) (1/9)
a, b
Different letters within rows denote a significant difference Kruskal-Wallis test (P<0.05) or Fisher’s
test (P< 0.05). *A,B Different letters within columns denote a significant difference in Fisher’s test (P<
0.05). Sal – saline; NM–natural mating; AI–artificial insemination; eCG-equine chorionic gonadotropin;
CL–corpus luteum.
After pregnancy diagnosis, the ovulation data were compared, showing that
not all females ovulated after the protocol, and considering it, GeCG+AI presented
lower pregnancy rate than GeCG+NM (P< 0.05); while GSal+NM and GSal+AI were
similar to all groups for pregnancy/ovulation (Table 3). Also, during pregnancy
diagnosis, it was defined weather non pregnant does were cycling or not, considering
the presence of a new CL. No differences were found comparing the treatments for
the presence of a CL from the following cycle. However, differences were found
inside GeCG+AI and GSal+AI. groups when comparing females that presented a
new CL or not. It is, in these groups there were more females without CL than with a
139
new one (P< 0.05 – Table 3). This difference is also present when considering all
groups together.
Discussion
To the authors’ knowledge, this is the first study performed aiming to

investigate follicular growth patterns and reproductive parameters after
resynchronization of ovulation in goats. Even though results may be limited by the
number of animals used in each group, they fit as a primary study and guide for other
trials. The study found few differences between groups regarding estrus
manifestation, and although not significant, results suggest that the use of eCG and
NM will provide better outcomes. Contrary to our hypothesis, the use of an eCG dose
in the first synchronization may not be enough to provide appropriate results in the
resynchronization.
The main differences were found in estrus manifestation, in which GeCG+NM

does reached better results than GSal+AI, indicating that the combined use of NM
with the use of eCG at transition period may improve behavior results for
resynchronization protocols in goats. However, not only the estrus response
determines the results of a hormonal protocol, it must also be associated with good
ovulation and pregnancy rates.
Although follicular dynamics and luteal presence did not show difference
among groups in the present study, resynchronization protocol with eCG application
associated to NM reached better outcomes than AI with or without eCG as observed
by the pregnancy rates obtained. When analyzing pregnancy rate over ovulation, the
difference was between NM and AI, both eCG treated groups (83.3% for NM x 14.3%
for AI), it is, considering females that actually ovulated in eCG treated groups, 83.3%
of females that were submitted to NM got pregnant, while only 14.3% of the females
that were artificially inseminated were got pregnant. Véliz-Deras et al. (2020) also
showed that during the transition season, NM presented better pregnancy rates
(42%) than AI (20%), even though (differently from the current study) the authors
performed AI with fresh semen, and, yet lower pregnancy rates were obtained.
Likewise, difference between AI and MN may be explained by the fact that AI was
performed once. Even though AI was performed in accordance with the ovulation and
140
estrus behavior, females were given only one chance to get pregnant with frozen
semen, which has lower fertility potential than the fresh. While for NM groups,
females were kept constantly with the bucks, and the number of mounts were not
recorded, thus it may probably have happened more than once. Moreover, AI groups
presented inside groups differences when considering the presence or not of a new
CL at pregnancy diagnosis time (Table 3). This data suggests that females from AI
groups did not keep cycling after the resynchronization protocol, returning to an
anestrus condition, while NM females presented other ovulation after the protocol
was performed. However, since the ultrasound evaluation was not sequentially
performed, it is possible that the does presented an early CL regression and were in
the follicular phase during pregnancy diagnosis. Also, since no differences were
found between groups, it can be suggested, however not stated, that the constant
presence of male may have an influence on future estrous cycles occurred naturally
after the resynchronization protocol, which should be studied further.
Regarding the use or not of eCG, even though the present study did not find
differences between groups, Andrade et al. (2021) demonstrated that even though
the protocol without this hormone was able to induce estrus response, the absence
of eCG was associated with a decreased ovulation rate (96.4% with eCG x 67.9%
without eCG). The decrease may be due to the increased quality of oocyte ovulated
from a better follicle blood flow in the eCG treated groups, since follicular fluid is
mainly derived from blood plasma and its content influences oocyte quality (Kumar et
al., 2015, 2014). Also, eCG effect in the final maturation of follicles, longer half-life
(when compared to other gonadotropins) and stimulation for estradiol production
(Bottino et al., 2021; Bukar et al., 2012; Hosseini et al., 2018; Riesenberg et al.,
2001) may also be considered relevant factors for the better results found by
Andrade et al. (2021) for eCG treated group.
When looking at the resynchronization protocol, results are comparable to a

59.3% pregnancy at the second service and 62.3% of cumulative pregnancy found
by Miranda et al. (2018) in sheep (cumulative pregnancy of 67.3% for sync + MN
without resync x 62.3% for sync + resync + MN), even though, in the present study
only the second ovulation was used for mating/insemination. This must be taken into
account since the first protocol usually presented better results than the second one,
141
considering that more fertile females got pregnant before the others, and
resynchronization tends to present lower rates of pregnancy, also due to conception
failures on the first attempt (Cosentino et al., 2021).
Yet, previous studies in ewes show that resynchronization protocol enhances

pregnancy rates from 58.3% at the first service to 73.3% in the accumulated
pregnancy (Cosentino et al., 2021), and from 19.4% at the first service to 47.8% in
the accumulated pregnancy (Cosentino et al., 2019), with the females being kept with
the teaser male, and inseminated 36h after GnRH administration. The present study
observed only resynchronization parameters, since during synchronizations females
were not mated. Therefore, a full resynchronization protocol with mating/inseminating
in both attempts should be studied.
Current does presented a large ovulation window from 65 to 91h after sponge
withdrawal (Figure 3). In ewes, when using 200UI of eCG at the sponge removal and
GnRH 36h after sponge removal, females ovulated earlier on 56.2±3.8h after second
sponge withdrawal (Cosentino et al., 2019). In goats, GnRH administration at 28 and
34h after the first sponge removal induced ovulation around 48 to 58h after sponge
withdrawal (Cosentino et al., 2020a). For that matter, even if not tested in the present
study, GnRH could also be administered in does 50h after removing the intravaginal
device, to induce and reduce the ovulation window around 70h after MAP removal
and enhance the protocol response allowing an FTAI with greater outcomes.
Accordingly, regardless of the treatment, 48h and 72h are the moments where the
large follicles are most expressive (as settled by the follicular dynamics – Figure 3),
suggesting that GnRH administration would be able to induce a more synchronous
ovulation during this period.
Thus, current results lead the way for new studies. In the light of the present
study, future essays may go further in the use of GnRH and mating management at
both synchronization and resynchronization protocols. About the influence of the
male, although results are better linked to the higher number of mating, compared to
the AI, whether the bare presence of a teaser would be enough to increase
pregnancy results could be tested.
142
Conclusion
In conclusion, resynchronization protocols in does at the transition season

may present satisfactory results. This study found promising, although not significant,
results in favor of the use of eCG in association with NM. Therefore, further studies
are needed to settle a resynchronization protocol for does, aiming to improve
pregnancy outcomes.
Acknowledgements
This study was supported by Fundação Carlos Chagas Filho de Amparo à

Pesquisa do Estado do Rio de Janeiro (FAPERJ). MFAB and FZB are fellow of
FAPERJ. FZB is fellow of Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq). This study was partially financed by Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code
001. IOC had a scholarship provided by CAPES. We also thank dairy goat farm
Capril Vale das Amalthéias for the animals and management support for this
research.
Author contributions
IOC: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology,

Visualization, Writing – original draft, Writing – review & editing; MFAB:
Visualization, Writing – original draft, Writing – review & editing; PMS: Data curation,
Formal Analysis, Investigation, Writing – review & editing; ART: Data curation,
Formal Analysis, Investigation, Writing – review & editing; JDRS: Data curation,
Formal Analysis, Investigation, Writing – review & editing; ACSR: Data curation,
Formal Analysis, Investigation, Writing – review & editing; BRRF: Data curation,
Formal Analysis, Investigation, Writing – review & editing; MMCPC: Data curation,
Formal Analysis, Investigation, Writing – review & editing; BRV: Data curation,
Formal Analysis, Investigation, Writing – review & editing; FZB: Conceptualization,
Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology,
143
Project administration, Resources, Supervision, Validation Visualization, Writing –

original draft, Writing – review & editing.
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CAPÍTULO III
OVINOS
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1 ARTIGO: PROTOCOLOS HORMONAIS PARA RESSINCRONIZAÇÃO

PRECOCE DA OVULAÇÃO EM OVELHAS: USO DE PROGESTÁGENOS, ECG, E
INCLUSÃO DO DIAGNÓSTIOCO PRECOCE COM ULTRASSONOGRAFIA
DOPPLER COLORIDO
Hormonal protocols for early resynchronization of ovulation in ewes: the use of

progestagens, eCG, and inclusion of early pregnancy diagnosis with color
Doppler ultrasound
Artigo publicado no periódico: Theriogenology, v. 133 (2019) p.113-118
DOI: 10.1016/j.theriogenology.2019.04.033
151
Hormonal protocols for early resynchronization of ovulation in ewes: the use of

progestagens, eCG, and inclusion of early pregnancy diagnosis with color
Doppler ultrasound
Isabel Oliveira Cosentino1; Mario Felipe Alvarez Balaro1; Eduardo Kenji Nunes
Arashiro1; Juliana Dantas Rodrigues Santos1; Ana Beatriz da Silva Carvalho1;
Raquel Perez Clariget2; Rodolfo Ungerfeld3; Felipe Zandonadi Brandão1

24230-340, Brazil
2Departamento de Producción Animal e Pastura, Facultad de Agronomía,

3Departamento de Fisiología, Facultad de Veterinaria, Universidad de la República,

Lasplaces 11 1550, Montevideo, 11600, Uruguay
*Corresponding author: isabelcosentino@id.uff.br; fzbrandao@id.uff.br
Abstract
This study aimed to evaluate 1) the effect of inserting an intravaginal sponge

containing medroxyprogesterone during the late luteal phase on the corpus luteum
(CL) function and endogenous production of P4; 2) the effectiveness of two different
equine chorionic gonadotrophin (eCG) doses on synchronization of ovulations for a
resynchronization treatment; and 3) the inclusion of an early pregnancy diagnosis in
an early resynchronization protocol for ovulation in ewes. For all studies, the
synchronization protocol was based on a short-term protocol of six days of
progestogen treatment plus one dose of prostaglandin F2alpha, one dose of eCG,
and gonadorelin acetate after sponge withdrawal. For the first study, the ewes were
mated with fertile rams; a second sponge was inserted in half of these ewes 12 days
later, and blood samples were collected daily for six days, until sponge withdrawal.
For the second study, the ewes were not mated, and received a second sponge
during the same period, after which they were divided into three groups according to
eCG dose (0, 200, or 300 IU). In the third study, all ewes were artificially inseminated
and received the second sponge during the same period. At sponge withdrawal,
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pregnancy was diagnosed by color Doppler ultrasonography (DUS) of the CL, and
only nonpregnant ewes were re-inseminated two days later. In the first study, serum
progesterone values were similar regardless of whether an intravaginal sponge had
been inserted. In the second study, the ovulation time was more concentrated in
those ewes which received 200 IU of eCG. In the third study, there was no difference
between the experimental groups (with or without a previous pregnancy diagnosis) in
pregnancy rate at the first insemination, accumulated pregnancy rate, and pregnancy
loss. The insertion of an intravaginal sponge impregnated with medroxyprogesterone
acetate did not affect the endogenous production of P4. The application of 200 IU of
eCG provided the best result with regard to the synchronization of ovulations in the
resynchronization treatment. Also, the inclusion of an early pregnancy diagnosis with
DUS is useful and improves the general results of resynchronization programs,
shortening the total working period.
Key words: sheep, Doppler ultrasound, artificial insemination, pregnancy diagnosis,

progesterone.
1. Introduction
An early pregnancy diagnosis increases reproductive efficiency, especially in

that non-pregnant females can be identified and re-inseminated as early as possible,
thus reducing inter-insemination and lambing intervals [1]. In this sense, pregnancy
can be accurately diagnosed as soon as 17 days after mating in ewes by assessing
the luteal blood flow using color Doppler ultrasonography (DUS). This technique may
be included in treatments for estrous resynchronization, reducing the time required
between synchronization and re-synchronization of ovulations [2–5]. Hormonal
protocols for resynchronizing ovulation have been applied to all previously
inseminated ewes without knowing which were pregnant and which were not [3].
However, administering these treatments to the whole flock is costly, time-
consuming, and laborious, and increases the use of unnecessary hormones as many
treated ewes are pregnant from the previous insemination. Moreover, pregnant ewes
are not unnecessarily moved for the re-insemination treatment to be administered,
thus avoiding any decrease in grazing time. Therefore, if an early pregnancy
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diagnosis with DUS is included, treatments can be continued in non-pregnant ewes

only to improve artificial insemination programs.
In cattle, treatments to resynchronize the ovulation after an initial Fixed-Time

Artificial Insemination (FTAI) or natural breeding have already been used
successfully [2,5,6]. These hormonal protocols require a combination of different
drugs (P4, GnRH analogue) differing in dose and/or time of administration [2,4,6].
However, it is not known if the application of such hormonal treatments, especially an
exogenous source of P4, could affect the functionality and maintenance of the corpus
luteum (CL) in early-pregnant ewes. In cows, an exogenous source of P4
administered through intravaginal devices before pregnancy diagnosis did not
produce luteolysis, and thus did not affect the conception rate of resynchronization
treatments [4]. This could have happened due to the negative feedback of external
P4 on the functionality and viability of the CL, which did not occur in that study [4].
Equine chorionic gonadotropin (eCG) is widely used in hormonal protocols to

induce the final follicular growth and ovulation [7,8]. In FTAI protocols, eCG may be
included to synchronize ovulations. In this sense, greater doses of eCG associated
with progesterone devices increase pregnancy rates in synchronization protocols
[9,10]; however, little is known about the relationship between eCG dose and the
synchronization of ovulations in a second eCG application administered for this
purpose.
Therefore, this study aimed to determine in ewes: (1) whether the insertion of
an intravaginal sponge containing medroxyprogesterone during the late luteal phase
affects the functionality of the CL and the endogenous production of P4; (2) the
effectiveness of two different eCG doses on synchronization of ovulations for a
resynchronization treatment; and (3) whether an early pregnancy diagnosis should
be included in the protocol for early resynchronization of ovulations.
The procedures of Studies 1 and 2 were approved by the Comissão de Ética

no Uso de Animais of the Universidade Federal Fluminense (Brazil) (protocol
#923/2017). These trials were carried out according to the ethical principles of the
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Colégio Brasileiro de Experimentação Animal (COBEA). The animal management

and experimental procedures of the third study were approved by the Comité de
Ética en el Uso de Animales de Experimentación of the Facultad de Agronomía,
Universidad de la República (Uruguay) (protocol 021130-000373-18).
Figure 1. (I) Study 1 methodology: A) GPSP: pregnant animals with sponge; B)

GPNSP: pregnant animals without sponge; C) GNPSP: non-pregnant animals with
sponge; and D) GNPNSP: non-pregnant animals without sponge. (II) Study 2
methodology: A) GroupCon; B) GroupeCG200; C) GroupeCG300. (III) Study 3
methodology: A) GroupEPD; B) GroupNEP.
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2.1 Study 1
The study was conducted at Unidade de Pesquisa Experimental em Caprinos

e Ovinos of the Universidade Federal Fluminense, located in Cachoeiras de Macacu
(22°S, 42°W), Rio de Janeiro, Brazil (tropical hot-humid climate type – Aw [11]). A
total of 42 Santa Ines x Dorper adult ewes with a body condition score of 3.0 ± 0.3
(mean ± SD) (scale 1-5, [12]) were used. All ewes were subjected to a gynecological
examination before the study to ensure that none had reproductive abnormalities
detectable by ultrasound or clinical examination. Throughout the study, ewes were
kept in a semi-intensive system under grazing supplemented with chopped grass
(Pennisetum purpureum) at 2.5 kg DM/kg of their live weight. Also, a concentrate at
0.5 kg DM/kg of their live weight (12% of crude protein) was provided as needed [13].
Water and mineral salt were provided ad libitum.
2.1.1 Estrous synchronization treatments
All ewes were treated with an estrous synchronization protocol adapted from
Balaro et al. [7] (Figure 1.I). Briefly, a sponge impregnated with 60 mg of
medroxyprogesterone acetate (Progespon, Schering Plough, São Paulo, Brazil) was
inserted in each animal, and remained in situ for six days. Twenty-four hours before
sponge withdrawal, all animals received 0.24 mg of cloprostenol sodium (Estron,
Agner União, São Paulo, Brazil) and 300 IU of eCG (Novormon, Schering Plough,
São Paulo, Brazil). All ewes also received 0.025 mg of gonadorelin acetate (GnRH –
Gestran Plus, Tecnopec, São Paulo, Brazil) 36 h after sponge withdrawal. To ensure
a similar number of pregnant and non-pregnant animals, only 24 ewes were mated
with fertile rams. The estrous behavior of those ewes was checked twice a day, and
they were mated once while receptive.
A second intravaginal sponge was inserted on Day 12 (Day 0 = 56 h after

sponge withdrawal) in 12 animals submitted for natural breeding and in nine of those
that were not bred. Sponges remained in situ until Day 17, when the early pregnancy
diagnosis was performed with DUS according to Arashiro et al. [14]. Briefly, CL blood
perfusion was assessed and classified in scores 1-4, where 1 corresponded to non-
pregnant ewes, and 2 to 4 to pregnant ewes. The ultrasound scan was performed
with a portable device (Sonoscape S6, Shenzhen, China) coupled to a 7.5 MHz
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linear rectal transducer adapted for use with small ruminants. The Doppler settings
used in the luteal assessments follow in sequence: 20% color gain, pulse repetition
frequency (PRF) of 1.0 kHz, 7 cm of depth, and wall filter (WF) of 75 MHz.
After a final pregnancy diagnosis on Day 30 by uterus scan using B-Mode

ultrasound (gold-standard method), the animals were retrospectively categorized in
groups as follows: GPSP: pregnant animals with sponge (n=10); GPNSP: pregnant
animals without sponge (n=9); GNPSP: non-pregnant animals with sponge (n=11),
and GNPNSP: non-pregnant animals without sponge (n=12). In non-pregnant
animals, the onset of luteolysis was considered to be the moment when plasma
progesterone concentration decreased more than 50% when compared to the
previous day, followed by a decrease to values lower than 1 ng/mL on the
subsequent day.
2.1.2 Blood sample collection and serum P4 concentration
Blood samples were collected daily from Day 12 to Day 17 by jugular

venipuncture, using tubes (without anti-coagulant) with a vacuum system. Blood
samples were centrifuged at 1000 x g for 15 min; serum was separated, and stored
at -20ºC until analysis. Serum P4 concentration was determined by
radioimmunoassay using commercial kits (MP Biomedicals, LLC, Diagnostics
Division, Orangeburg, NY, USA). Sensitivity and intra-assay coefficient of variation
were 0.05 ng/mL and 12%, respectively. All data were within minimum and maximum
points of the curve.
2.2. Study 2
The second study (Figure 1.II) was conducted at the same experimental farm,
and under the same nutritional conditions, using 30 adult Santa Ines x Dorper ewes
(body weight: 47.8 ± 5.6 kg; body condition score: 2.9 ± 0.3).
2.2.1 Estrous synchronization, resynchronization treatments, and ultrasound

assessment
All ewes were treated with the same estrous synchronization treatment used in
the first study. However, ewes were not mated with the ram. Then, a second
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intravaginal sponge impregnated with 60 mg of medroxyprogesterone was inserted

on Day 12 (Day 0 = 56 h after sponge withdrawal) and maintained in situ until Day 17
in all the animals. On Day 17, ewes were randomly allocated to three experimental
groups (n=10 each) according to the eCG (Novormon, Schering Plough, São Paulo,
Brazil) dose administered i.m. simultaneously with the sponge withdrawal. Ewes from
GroupCon received 1.0 mL of saline solution, and those from GroupeCG200 and
GroupeCG300 received 200 IU and 300 IU of eCG, respectively. Thirty-six hours
after sponge withdrawal, all ewes received 0.025 mg of gonadorelin acetate (Gestran
Plus, Tecnopec, São Paulo, Brazil).
The ovaries of all ewes were observed by ultrasound scan every 12 h from the
second sponge withdrawal until ovulation (confirmed by the disappearance of a
previous dominant follicle(s) greater than 5 mm). Estrous behavior was also
determined every 12 h by fertile rams.
2.3. Study 3
The third study was conducted at Estación Experimental Bernardo Rosengurtt

of Facultad de Agronomía, Universidad de la República, located in Cerro Largo
(32°S, 54°W), Uruguay (humid subtropical climate type – Cfa [11]). A total of 188
adult Corriedale ewes (body weight: 49.2 ± 6.2 kg; body condition score: 3.27 ± 0.03)
were used during the breeding season (April). Animals grazed under extensive
conditions on natural grassland at approximately 3.0 kg DM/kg of their live weight,
with free access to water.
2.3.1 Estrous synchronization and artificial insemination
All ewes were treated with the same protocol used in the first and second
studies with the exception of the GnRH analogue (gonadorelin, Gonasyn, Zoetis,
Montevideo, Uruguay), which was administered 24 h after sponge withdrawal (0.050
mg i.m.) (Figure 1.III). Estrous behavior was recorded twice daily with vasectomized
rams, and ewes were inseminated 27-30 h after GnRH administration with fresh
semen collected from two fertile Corriedale rams. Semen doses, which had
approximately 2 x 108 spermatozoa in 0.2 mL, determined by a photometer, were
inserted using a speculum equipped with a light source and a multidose insemination
158
gun (Walmur Veterinary Instrument, Montevideo, Uruguay), which provided a

superficial cervical insemination.
2.3.2 Ultrasonographic procedures and resynchronization of ovulation
A second intravaginal sponge (Progespon, Syntex, Buenos Aires, Argentina)

was inserted on Day 12 (Day 0 = 51-54 h after sponge withdrawal) and maintained in
situ until Day 17 in all animals. Pregnancy was determined on Day 17 according to
Arashiro et al. [14] using a portable DUS device (Esaote, MyLabOne, Genoa, Italy)
equipped with a 7.5 MHz linear rectal transducer. The Doppler settings used in the
luteal assessments follow in sequence: 32% color gain, PRF of 2.0 kHz, 7 cm of
depth, and WF of 90 MHz.
The females were randomly divided in two groups: 1) GroupEPD: the early
pregnancy diagnosis was performed, and therefore, only non-pregnant ewes were
resynchronized; and 2) GroupNEP: all ewes were treated regardless of gestational
status.
All sponges were removed on Day 17, and non-pregnant ewes from
GroupEPD and all ewes from GroupNEP received 200 UI of eCG i.m. (Novormon,
Zoetis, Montevideo, Uruguay) according to the results of the second study. The same
ewes received 0.050 mg of gonadorelin 36 h after sponge withdrawal, and then
underwent a second insemination 9 to 12 h later (Day 19). Forty days after the
second insemination, pregnancy diagnosis was performed by transrectal ultrasound.
After the final pregnancy diagnosis was performed, the results of the first and
second AI were compared in each experimental group. In sequence, the
accumulated pregnancy (ewes diagnosed as pregnant at the first and second),
accumulated non-pregnant (ewes that were not pregnant at both moments), and
pregnancy losses (ewes that were diagnosed as pregnant at the first but non-
pregnant at the second) were calculated.
2.4 Data Analysis
The Lilliefors test was used to verify data normality. Data were analyzed with
SAS (University Edition version). In the first study, progesterone values were
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analyzed separately for the ewes that were or were not pregnant using a mixed
model procedure including the group (treated or not treated), time, and their
interaction as main effects in the model. LS means were compared with the pdiff
option of the mixed model. In the second study, dispersion of the data
(homoscedasticity) was compared by using Bartlett’s test. The time from sponge
withdrawal to ovulation, and that from estrus onset to ovulation, were compared with
a mixed model including the treatments as main factors. The frequency of ewes with
single or multiple ovulations was compared with Fisher’s exact probability test. In the
third study, pregnancy rate, accumulated pregnancy rate, and possible pregnancy
losses were compared among groups using the chi-square test. Differences were
considered as statistically significant when P ≤ 0.05.
3. Results
3.1 Study 1
From Day 12 to Day 17, serum progesterone values did not differ between
pregnant ewes with or without the second sponge (GPSP and GPNSP, respectively;
Fig.2A). In non-pregnant animals, the luteolysis time did not differ between groups
(Days 14.6 ± 1.1 and 14.6 ± 0.9 for GNPNSP and GNPSP, respectively), and the
serum progesterone did not differ before luteolysis in non-pregnant ewes (Fig.2B).
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Figure 2. Serum progesterone concentration (ng/mL) from Day 12 to Day 17, in

pregnant (A) and non-pregnant (B) ewes. An intravaginal sponge impregnated with
medroxyprogesterone acetate was inserted in ewes from the GNPSP and GPSP
groups.
3.2 Study 2
There were no differences among groups (GCon, GeCG200, and GeCG300)

in the time from sponge withdrawal to ovulation, or time from estrus onset to
ovulation (Table 1). However, ovulation time was more concentrated in GeCG200
than in GCon and GeCG300 treated ewes. The frequency of ewes with single or
double ovulations did not differ among groups (8/10, 8/9, and 6/7 ewes with single
ovulations from GCon, GeCG200, and GeCG300, respectively).
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Table 1. Time from sponge withdrawal to ovulation, and from estrous onset to
ovulation, in ewes that received 0 (group Con), 200 (group eCG200), or 300 (group
eCG300) IU of eCG during a treatment for resynchronization of ovulations (LSmean ±
SEM).
Con eCG200 eCG 300

Sponge withdrawal to ovulation (h) 46.8 ± 11.5 56.2 ± 3.8 54.1 ± 14.9a
a b
Estrous onset to ovulation (h) 19.2 ± 14.7 25.8 ± 10.0 20.9 ± 14.1
a,b
means a significant difference in dispersion among treatments.
3.3 Study 3
There was no difference between experimental groups (with or without

previous pregnancy diagnosis) in pregnancy rate at the first insemination,
accumulated pregnancy rate, and pregnancy losses (Table 2).
Table 2. Pregnancy rate and accumulated pregnancy in ewes subjected to

treatments for synchronization and resynchronization of ovulation with or without
pregnancy diagnosis with color DUS 17 days after first insemination.
Pregnancy at D17 Non-pregnancy at D17 Accumulated

Accumulated
Non- Non- non-
Pregnancy Pregnancy Pregnancy pregnancy at
pregnancy pregnancy pregnancy at
at D17 at D59 at D59 D59
at D59 at D59 D59
21.7% 75.0% 25.0% 45.8% 54.1% 52.1% 47.9%
GroupEPD
(20/92) (15/20) (5/20) (33/72) (39/72) (48/92) (44/92)
19.1% 66.7% 33.3% 38.1% 61.8% 43.6% 56.4%
GroupNEP
(18/94) (12/18) (6/18) (29/76) (47/76) (41/94) (53/94)
19.4% 71.1% 28.9% 41.9% 58.1% 47.8% 52.2%
Total
(38/186) (27/38) (11/38) (62/148) (86/148) (89/186) (97/186)
GroupEPD: the early pregnancy diagnosis was performed and, therefore, only non-pregnant ewes
continued with the treatment for resynchronization of ovulation; Group NEP: all ewes continued with the
treatment regardless of gestational status.
Accumulated pregnancy (females that were diagnosed as pregnant at the first and second pregnancy
diagnosis), accumulated non-pregnant (ewes that were not pregnant at both moments), and
pregnancy loses (females that were diagnosed as pregnant at the first but non-pregnant at the second
pregnancy diagnosis).
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4. Discussion
To the best of our knowledge, these were the first studies directed at the
implementation of protocols of resynchronization of ovulations for FTAI in ewes
including early pregnancy diagnosis with DUS. In general, our results indicate that,
the second sponge did not negatively influence early pregnancy, and it was possible
to determine that 200 IU was the most effective dose of eCG to synchronize the
ovulation. The use of DUS was useful in determining which ewes should undergo a
second hormone protocol and FTAI, and thus simplify treatments and avoid the
unnecessary movements of animals, with practical implications for simplifying the use
of these techniques, decreasing cost, increasing the grazing time of pregnant ewes,
and preventing them from being subjected to stressors during this key period.
Our results demonstrated clearly that the use of a second sponge does not
interfere with the luteolysis or pregnancy from the first FTAI. Although Miranda et al.
[3] proposed a negative effect of medroxyprogesterone devices on progesterone
production, in that study there were no negative effects on the final percentage of
pregnant ewes. In line with this, in our study progesterone concentration patterns
were similar in ewes that received or did not receive a second sponge. Therefore,
although the use of a sponge in those ewes in which pregnancy had not yet been
detected implies the inclusion of an unnecessary step for the final result, it does not
have negative effects on the ovarian activity of the ewes, and thus allows the
treatment to be applied.
The administration of 200 IU was the most effective dose for synchronizing the
ewes’ ovulation. Greater doses of eCG may be more effective for induction of estrus
and ovulation in non-cycling ewes [9,10], but there is scarce information on the
effects of the dose on the dispersion of the ovulations. A mild eCG dose such as 200
IU may produce less interference in the endogenous LH peak and, subsequently,
obtain a more homogenous hormonal response and ovulation in ewes. In this sense,
greater doses of eCG, such as those used for superovulatory treatments, advance
the release of the endogenous LH peak, and may cause greater dispersion of
ovulation among different animals [15,16]. It is also important to highlight that the
ewes had their ovarian cycle pre-synchronized due to the first protocol, and therefore
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a low dose might have been enough to obtain a good synchronization in the second
protocol.
The inclusion of the early pregnancy diagnosis did not modify the final
pregnancy rate. Doppler examination is increasingly being used in livestock for
ovarian assessment for early pregnancy diagnosis in cows [17], ewes [14], and does
[18]. This is a very important result as, at the same time, the treatments ended for all
the pregnant ewes, thus decreased the management of the animals, the costs, the
loss of an important number of semen doses, and the use of unnecessary hormones.
Moreover, ewes were not unnecessarily moved, allowing them to graze longer and
preventing stressors, thus improving their welfare. This decrease of hormones has a
further practical impact on ewes’ welfare, as no pregnant animal received more
hormonal treatments or was subjected to management for artificial insemination while
pregnant. Changing the moment at which GnRH was administered in the first FTAI of
this study meant the number of pregnant ewes was low compared to that traditionally
obtained. This explains the low number of ewes that were not pregnant at the second
ultrasound although they had been considered pregnant at the first. That slight
difference can be due to early pregnancy losses, which can occur in ewes until the
65th day [19], or to errors in the interpretation of the Doppler images, as there may
be 8.6 – 14.3 % of false positives when the early pregnancy diagnosis is carried out
17 days after mating [14]. Overall, it is important to highlight that the inclusion of the
early pregnancy diagnosis does not undermine the results of the first FTAI, with
several practical advantages.
5. Conclusions
The insertion of an intravaginal sponge impregnated with

medroxyprogesterone acetate did not affect the endogenous production of P4. The
application of 200 IU of eCG proved the best means to synchronize the ovulation of
the resynchronization treatment, leading to a better determination of the time for
FTAI. Also, the inclusion of an early pregnancy diagnosis with DUS is useful to
decrease the use of unnecessary hormones, prevent the loss of sperm doses, and
improve the general results of resynchronization programs, shortening the total
working period. The simplification of practices, allowing early pregnant ewes to graze
164
longer and preventing unnecessary management – including administration of

hormones and insemination – has important welfare implications.
Acknowledgments

Pesquisa Pós-Graduação - Proppi/UFF (UFF/Proppi/Infra-LabPesq). The authors
acknowledge Dr. Javier Meilán, from Zoetis (Uruguay), for his partial collaboration
with the hormonal treatments in Study 3. EKNA had a postdoctoral scholarship from
FAPERJ. FZB is fellow of the CNPq. IOC had a scholarship provided by CNPq. The
authors also acknowledge Alvaro López, Ignacio Sosa, Nicolás Zunnino, Lorena
Lacuesta, Noelia Zambra, and Juan Bottino for their support with the animal
management in Study 3.
IOC collected data, revised and worked on the preparation of the manuscript,
and approved the final version. RU proposed the initial hypothesis, organized the
study, analyzed the data, and wrote the first draft, revised and worked on the
preparation of the manuscript, and approved the final version. MFB discussed the
general study design, collected data, revised and worked on the preparation of the
manuscript, and approved the final version. EKNA organized the experimental
procedures, collected data, revised and worked on the preparation of the manuscript,
and approved the final version. JDRS collected data, revised and worked on the
preparation of the manuscript, and approved the final version. ABSC collected data,
revised and worked on the preparation of the manuscript, and approved the final
version. RPC collected data, revised and worked on the preparation of the
manuscript, and approved the final version. FZB discussed the general design,
collected data, revised and worked on the preparation of the manuscript, and
approved the final version.
Competing interests

165
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168

PASTEJO: RESPOSTA DE MULTÍPARAS, NULÍPARAS E OVELHAS
LACTANTES DURANTE A ESTAÇÃO DE MONTA
Fixed timed artificial insemination and early resynchronization of ovulation in

different categories of ewes in grazing system: response of multiparous,
nulliparous, and lactating ewes during the breeding season
Artigo publicado no periódico: Livestock Science v. 247 (2021)
DOI: 10.1016/j.livsci.2021.104461
169
Fixed timed artificial insemination and early resynchronization of ovulation in

different categories of ewes in grazing system: response of multiparous,
nulliparous, and lactating ewes during the breeding season
Isabel Oliveira Cosentino1*; Felipe Zandonadi Brandão1; Pedro Henrique Nicolau

Pinto1; Raquel Perez Clariget2; Rodolfo Ungerfeld3

24230-340, Brazil
2Departamento de Producción Animal e Pastura, Facultad de Agronomía,

3Departamento de Biociencias Veterinarias, Facultad de Veterinaria, Universidad de

la República, Lasplaces 1620, Montevideo, 11600, Uruguay
*Corresponding author: isabelcosentino@id.uff.br
Abstract
The aims of this study were to: 1) determine if the insertion of a second MAP
sponge in ewes 12 days after the first FTAI affects the pregnancy rate; 2) compare
the responses to resynchronization protocol in multiparous and nulliparous ewes; and
3) compare the responses to resynchronization protocol in multiparous and
postpartum lactating ewes. Three trials were performed with 186 Corriedale ewes:
111 multiparous [(control – CON (n = 60) and synchronization – SYNC groups (n =
51)], forty-two 1.5 yr-old nulliparous (group NUL), and 33 lactating ewes with at least
28 postpartum (group PtP). All ewes were treated with a MAP sponge for 6 days.
Twenty-four hours before the sponge withdrawal, all animals received 0.24 mg of
cloprostenol sodium and 300 IU of eCG, and 0.05 mg of gonadorelin 36 h after
sponge withdrawal and artificially inseminated 27-30 h after the GnRH administration
(Day 0). A second intravaginal sponge was inserted to all groups (except for SYNC
group), on Day 12. At Day 17, early pregnancy diagnosis was performed with doppler
ultrasound in all ewes. Sponge withdrawal occurred at Day 18, and a new GnRH
dose (0.05 mg) was administrated 36 h after it. Once again, artificially inseminated
27-30 h after the GnRH administration. A final pregnancy diagnosis on Day 42 was
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performed in all ewes for confirmation of first diagnosis and the data was confirmed
by parturition. Ewes in all groups presented estrus signs (SYNCH – 94.1%; CON –
71.7%; NUL – 83.3%; PtP – 39.4% at first insemination and CON – 65.2%; NUL –
76.5%; PtP – 62.5% at the second). The pregnancy rate and pregnancy loss were
not different between synchronized and resynchronized multiparous ewes (SYNCH –
56.9% vs. CON – 58.3% pregnant at D42 and SYNCH – 5.9% vs. CON – 7.1% fetal
loss at D42). No differences were found between resynchronized multiparous and
nulliparous ewes at the first FTAI, only fecundity was higher in multiparous ewes
[CON – 0.88 vs. NUL – 0.62 (P = 0.04)]. Resynchronized multiparous ewes had
higher reproductive performance than postpartum ewes that only presented
pregnancy after the second FTAI [CON – 73.3% vs. PtP – 15.1% total pregnancy (P
= 0.003)]. Overall, we concluded that the synchronization-resynchronization FTAI
treatments may be included in reproductive programs in nulliparous ewes, but the
treatments cannot superimpose to the limiting conditions imposed by postpartum
lactating status. The resynchronization treatments allow to include practical
managements with no deleterious effects in the results of the first FTAI.
Key words: sheep, doppler ultrasound, artificial insemination, pregnancy diagnosis,

progesterone.
1. Introduction
In sheep, intensive reproductive managements as those needed in

accelerated reproductive systems, require the inclusion of techniques to advance
postpartum rebreeding, as well as first estrus and ovulation. However, seasonality
and age of puberty are main limiting factors, especially in extensive sheep systems.
The age at which females achieve puberty depends on the time of the year of birth
(autumn or spring), since it will reflect the nutrition condition and consequently body
weight gain, and the photoperiod (Foster, 1981; Foster et al., 1985; Pool et al., 2020),
all what are affected by several intrinsic/genetic factors (Hileman et al., 2020). In
general, in extensive grazing systems based in natural pastures, ewes do not
achieve enough body weight to be breed during their first autumn (Asadi-Fozi et al.,
2020; Foster et al., 1985), so their first breeding is delayed until the following autumn
(Kenyon et al., 2004). Therefore, although puberty is achieved earlier, the first
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breeding is delayed until females are 1.5 yr-old, or in many farms even to when ewes
are 2.5 yr-old, loosing productivity and generating high costs due to prolonged
rearing. Therefore, it would be important to advance the first breeding period, but in
general, the reproductive results of female hoggets are poorer than those of adult
ewes (Edwards and Juengel, 2017), even when they are 1 year-old (Ungerfeld,
2016).
Also, there is scarce information on how handling postpartum to improve the

results, achieving high variability in the results of reproductive managements, at least
in grazing systems (Araújo et al., 2019; Sharpe et al., 1986; Ungerfeld and Sánchez-
Dávila, 2012). If ewes are induced to ovulate during spring, they lamb in autumn, and
thus, postpartum anestrous occurs during the period in which ewes normally cycle.
During the postpartum period, ewes secrete very low amounts of LH, and therefore
do not present new ovulations or estrus signs (Dobek et al., 2013; Lewis and Bolt,
1987). Therefore, it would be important to develop standardized techniques, with
predictable results, that can be included in accelerated reproductive systems.
In this sense, treatments to synchronize and early resynchronize ovulations

open an interesting possibility to overcome limitations of parity and postpartum
status. In this sense, it has been recently demonstrated that the doppler observation
of the corpus luteum (CL) blood flow 17 days after insemination can be used to
disentangle which ewes are pregnant or not (Arashiro et al., 2018), and this
information might be included in treatments for early resynchronization of the
ovulation (Cosentino et al., 2019). However, to include these treatments in practical
applications, there is still scarce data to know if the insertion of an intravaginal device
containing progestogens even before knowing if the ewe is pregnant or not may
affect the results, so this should be determined before aiming to include these
treatments in other physiological situations.
We hypothesized that the insertion of an intravaginal sponge 12 days after

estrus does not affect the pregnancy, and that the use of hormonal programs to
synchronize the ovulations, and resynchronize them again in non-pregnant ewes,
enhance the reproductive response of nulliparous and postpartum ewes. Therefore,
the aims of this studies were to: 1) determine if the insertion of a second MAP
172
sponge in ewes 12 days after the first FTAI affects the pregnancy rate; 2) compare
the responses to FTAI and resynchronization of ovulation in multiparous and
nulliparous ewes; and 3) determine if the postpartum status of multiparous lactating
ewes affects the responses to FTAI and resynchronization of ovulation during the
breeding season.
2.1. Animals and general procedures
Procedures were approved by the Comité de Ética en el Uso de Animales de

Experimentación of the Facultad de Agronomía, Universidad de la República
(Uruguay). The study was conducted at Estación Experimental Bernardo Rosengurtt
of Facultad de Agronomía, Universidad de la República, located in Cerro Largo
(32°S, 54°W), Uruguay [humid subtropical climate type – Cfa (Köeppen, 1948)].
Animals remained grazing under extensive conditions on natural grassland at

approximately 3.2 kg DM/kg LW, with free access to water. Forage allowance [kg
DM/ kg animal BW; Sollenberger et al. (2005)] was 2.9 kg dry matter (DM)/kg BW. At
the beginning of the experiment, the forage availability was 1620 kg DM/ha (9.9% of
crude protein, 74% NDF and 36% ADF which should cover 137% of the required
energy and 110% of the protein requirement). The animals were previously
dewormed (0.2 mL/kg; Startect, Zoetis, Montevideo, Uruguay), vaccinated against
clostridiums (Clostrisan, Lab Virbac-Santa Elena, Uruguay), checked for any foot
infection and received a foot bath with Zinc Sulfate. Females were checked for the
health of the teeth and the mammary gland. The wool of the perineal region and the
tail was sheared to facilitate the AI.
Three trials were performed simultaneously with 186 Corriedale ewes. From
this, 111 were multiparous ewes, aged 3.5 to 7.5 yr, weighing (LW) 50.7 ± 7.8 kg
(mean ± SD), and with body condition score (BCS) of 3.2 ± 0.4 [scale 1-5, (Suiter,
1994)]. These multiparous groups belonged to two treatments: 60 were controls for
the three trials (group CON), and 51 were only included in the first trial (group
SYNC). There were also 42 nulliparous ewes, 1.5 yr-old, 40.2 ± 3.5 kg of LW and 3.3
± 0.3 of BCS, respectively (group NUL), and 33 postpartum ewes, that remained
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nursing their lambs during the whole study (4.5 to 7.5 yr-old, 48.7 ± 5.6 kg of LW and
2.7 ± 0.5 of BCS– group PtP). The studies were performed during the mid-breeding
season (April-May, autumn in the South Hemisphere).
2.2. Trial 1: Effect of resynchronization treatments on pregnancy rate
The 111 multiparous ewes were used in this study, including the 60 CON and
the 51 SYNC ewes. While the CON ewes were subjected to the synchronization and
resynchronization protocol (see Section 2.6), the SYNC ewes received only the
synchronization treatment. This design allowed to determine if the resynchronization
treatment affects the pregnancy rate.
2.3. Trial 2: Multiparous vs nulliparous ewes
Nulliparous ewes received the same treatment and handling than the CON
ewes, thus, comparing the reproductive results of the synchronization-
resynchronization treatment in both categories.
2.5. Trial 3: Multiparous vs lactating ewes
The reproductive response of CON and PtP ewes to the same

synchronization-resynchronization treatments was compared. The PtP ewes were
induced to ovulate during the previous spring (during the non-breeding season) using
the ram effect (Ungerfeld et al., 2005, 2002). Briefly, an intravaginal sponge
impregnated with 30 mg of acetate of medroxyprogesterone was inserted and
withdrawn 6 days later, and thereafter adult marking rams were acutely joined with
the ewes (Ungerfeld et al., 2005). These females gave birth during February-March
and remained with their single lambs, suckling freely, before and during the
experiment period. The first FTAI was applied when they had 28 to 32 days of
postpartum.
2.6. Hormonal treatments, insemination, and pregnancy diagnosis
All ewes were treated with an estrous synchronization protocol adapted from
Balaro et al. (2016), followed by the resynchronization protocol described by
Cosentino et al. (2019) (Figure 1). Briefly, a sponge impregnated with 60 mg of
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medroxyprogesterone acetate (Progespon, Syntex, Buenos Aires, Argentina) was

inserted to each animal, and remained in situ for 6 days. Twenty-four hours before
the sponge withdrawal, all animals received 0.24 mg of cloprostenol sodium (Ciclase
DL, Syntex, Buenos Aires, Argentina) and 300 IU of equine chorionic gonadotropin
(eCG, Novormon, Zoetis Syntex, Buenos Aires, Argentina). All ewes also received
0.05 mg of an analogue of GnRH (gonadorelin, Gonasyn, Zoetis, Montevideo,
Uruguay) 36 h after sponge withdrawal. From sponge withdrawal until FTAI (Day 0)
ewes remained in contact with marking vasectomized rams for detection of ewes
coming into estrous. Estrous ewes were detected twice daily according to the marks
of the rumps. All ewes were gently immobilized, with their hind legs lifted and a
trained technician cervically inseminated all the ewes 27-30 h after GnRH
administration with undiluted fresh semen. Semen was collected with artificial vagina
from two previously proven fertile and trained Corriedale rams, according to López-
Pérez and Pérez-Clariget (2012). Semen doses, which had approximately 1.2 x 108
spermatozoa in 0.04 mL [estimated with a photometer (SDM1, Minitub, Germany)],
were inserted using a speculum equipped with a light source and a multidose
insemination gun (Walmur Veterinary Instrument, Montevideo, Uruguay).
A second intravaginal sponge was inserted to all, except those of group

SYNC, on Day 12. At Day 17, early pregnancy diagnosis was performed with doppler
ultrasound according to Arashiro et al. (2018). Briefly, ewes were gently immobilized
and remained standing up to assess their CL blood perfusion, which was classified in
scores 1-4, where 1 corresponded to non-pregnant ewes, and 2 to 4, to pregnant
ewes (Arashiro et al., 2018). It was used a portable ultrasound equipment (United
Imaging, IUStar 160 Vet, Beijing, China) equipped with a 7.5 MHz linear rectal
transducer adapted for small ruminant rectal evaluation. The doppler was set in 32%
color gain, pulse repetition frequency (PRF) of 2.0 kHz, 7 cm of depth, and wall filter
(WF) of 90 MHz.
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Figure 1. Schematic representation of the experimental groups, hormonal protocols

used for estrous synchronization and resynchronization (when applied), with
indication of the moments where fixed time artificial inseminations, Doppler
ultrasonography (US) and B-Mode US were performed.
CON – multiparous ewes from control group; SYNCH – multiparous ewes from only synchronization
group; NUL – Nulliparous ewes; PtP – Post-partum ewes; eCG – equine Chorionic Gonadotrophin;
GnRH – Gonadotrophin Releasing Hormone; MAP – medroxyprogesterone acetate; US – ultrasound;
FTAI – Fixed Time Artificial Insemination.
The sponges were withdrawn after the doppler observation, and in those
diagnosed as non-pregnant, a new GnRH dose (0.05 mg) was administrated 36 h
after it. Once again, ewes were kept with vasectomized rams, from sponge
withdrawal until FTAI, for estrous behavior analysis twice daily. The ewes were
inseminated 27-30 h after GnRH administration with undiluted fresh semen, collected
from two fertile Corriedale rams (semen doses with approximately 1.2 x 108
spermatozoa in 0.04 mL). A final pregnancy diagnosis on Day 42 were performed in
all ewes by uterus scan using B-Mode transrectal ultrasound (gold-standard method),
and this data was confirmed by lambing. To calculate the pregnancy rate of the
second FTAI only ewes that did not lamb from the first one were considered. The
pregnancies rate and pregnancy loss between the early (Day 17) and late (Day 42)
pregnancy diagnosis of CON and SYNC ewes was compared to determine if the
application of the resynchronization treatment affect the pregnancy rate.
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2.7. Statistical analyses
Two ewes from each group (total of six ewes) were not used at the second
FTAI due to sanitary reasons. In CON group there is a difference of 7 ewes between
the early and the late diagnosis confirmed by date of lambing due to practical
difficulties in the management of the flock. Four NUL ewes were not diagnosed as
pregnant at Day 17, but on Day 42 and according to date of lambing were pregnant
on Day 17, so for their data were analyzed considering them as pregnant at that
moment.
Data were analyzed using generalized models (GENMOD procedure, SAS

University Edition) specifying the binomial distribution of the variables and logit
transformation of the data. In each trial the models included the effect of the Group.
When pregnancy rates’ data were compared, the ram was included in the model, but
as it was no significant (P = 0.6), it was removed from it. Differences were considered
as statistically significant when P ≤ 0.05, and as tendencies when 0.05 < P ≤ 0.10.
3. Results
The results of the trials are presented in Tables 1, 2 and 3. The pregnancy
rate did not differ between the CON and the SYNC ewes, without differences in the
pregnancy loss (Table 1).

submitted to the synchronization in the first FTAI moment (multiparous ewes).
Variables CON* SYNCH P

1st FTAI
Number of ewes 60 51
Ewes with estrus sighs 43/60 (71.7%) 48/51 (94.1%) 0.003
Pregnant ewes at 17 days 28/60 (46.7%) 32/51 (62.8%) 0.06
Pregnant ewes at 42 days** 35/60 (58.3%) 29/51 (56.9%) ns
Fetal loss from 17 to 42 days 2/28 (7.1%) 3/51 (5.9%) ns
CON – multiparous ewes from control group; SYNCH – multiparous ewes from only synchronization
group.
* CON data are the same in the three Tables as the three trials were done simultaneously, and thus, it
was the same control group.
** Ewes with the presence of conceptus at Day 42 were considered pregnant regardless the diagnosis
at Day 17, so 7/60 CON ewes were misdiagnosed at Day 17 by the luteal blood flow evaluation.
177

(multiparous and nulliparous ewes).
Variables CON* NUL P

st
1 FTAI
Ewes with estrus sighs 43/60 (71.7%) 35/42 (83.3%) ns
Pregnant ewes at 17 days 28/60 (46.7%) 19/42 (45.2%) ns
Pregnant ewes at 42 days** 35/60 (58.3%) 23/42 (54.8%) ns
Lambing 34/60 (56.7%) 21/42 (50.0%) ns
Double lambing (%) 8/34 (23.5%) 3/21 (14.3%) ns
Fetal loss from 17 to 42 days 2/28 (7.1%) 2/19 (10.5%) ns
Fetal loss from 42 days to birth 2/34 (5.8%) -
2nd FTAI
Number of ewes*** 23 17
Lambing 8/23 (34.7%) 2/17 (11.8%) 0.09
Fetal loss from 42 days to birth 2/10 (20.0%) 1/3 (33.3%)
TOTAL RESULTS
Total pregnancy (FTAI 1 + FTAI 2) 44/60 (73.3%) 26/42 (61.9%) ns
Total lambing (FTAI 1 + FTAI 2) 43/60 (71.7%) 23/42 (54.8%) ns
Prolificacy (Lambs/ ewe that lambed) 1.23 ± 0.43 1.13 ± 0.35 ns
Fecundity (Lambs/treated ewes) 0.88 0.62 0.04
CON – multiparous ewes from control group; NUL – Nulliparous ewes.
at Day 17, so 7/60 CON ewes and 4/42 NUL ewes were misdiagnosed at Day 17 by the luteal blood
flow evaluation.
*** The number of ewes considered for the second FTAI is according to the non-pregnant ewes at Day
42 diagnosis (CON: n = 25; NUL: n = 19). However, two ewes from each group were disregarded due
to sanitary reasons.
In general, the CON ewes had a higher reproductive performance than the PP
ewes (Table 3). In fact, the percentage of ewes that came into estrus and got
pregnant at the first FTAI was greater in the CON than in the PtP group (P = 0.001
and P = 0.05). Moreover, all the PtP ewes that were pregnant at the first FTAI lost the
pregnancies before Day 42, as no ewes from this group were detected pregnant on
Day 42 examination. However, ewes coming into estrus after the resynchronization
treatment did not differ between groups. At the second FTAI, pregnancy rate tended
to be higher in CON than in PtP group (P = 0.06), without differences in the
178
proportion of lambing ewes. The fecundity of the CON group was also greater than
that of the PP group (P <0.0001).

(multiparous and post-partum ewes).
Variables CON* PtP P

st
1 FTAI
Ewes with estrus sighs 43/60 (71.7%) 13/33 (39.4%) 0.001
Pregnant ewes at 42 days** 35/60 (58.3%) 0/33 (0.0%) <0.0001
Lambing 34/60 (56.7%) 0/33 (0.0%) <0.0001
Double lambing (%) 8/34 (23.5%) - ns
Fetal loss from 17 to 42 days 2/28 (7.1%) 7/7 (100%) <0.0001
Fetal loss from 42 days to birth 2/34 (5.8%) -
2nd FTAI
Number of ewes*** 23 24
Lambing 8/23 (34.7%) 5/24 (20.8%) ns
Fetal loss from 42 days to birth 2/10 (20.0%) 0/5 (0.0%)
TOTAL RESULTS
Total pregnancy (FTAI 1 + FTAI 2) 44/60 (73.3%) 5/33 (15.1%) 0.0028
Total lambing (FTAI 1 + FTAI 2) 43/60 (71.7%) 5/33 (15.1%) 0.005
Prolificacy (Lambs/ ewe that lambed) 1.23 ± 0.43 1.0 ± 0.0 0.11
Fecundity (Lambs/treated ewes) 0.88 0.15 <0.0001
CON – multiparous ewes from control group; PtP – Post-partum ewes.
at Day 17, so 7/60 CON ewes were misdiagnosed at Day 17 by the luteal blood flow evaluation.
*** The number of ewes considered for the second FTAI is according to the non-pregnant ewes at Day
42 diagnosis (CON: n = 25; PtP: n = 26). However, two ewes from each group were disregarded due
to sanitary reasons.
4. Discussion
In agreement with previous studies in which there was no deleterious effect of

inserting an intravaginal sponge on CL activity (Cosentino et al., 2019), the results of
the first trial confirmed that beginning a resynchronization treatment before the early
diagnosis of CL perfusion done on Day 17 does not affect the pregnancy rate of the
first FTAI. This expands previous information, in which it was reported that the
insertion of the sponge does not affect the ovarian dynamics (Cosentino et al., 2019;
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Miranda et al., 2018), to the practical in-farm application of this technique. This
confirms the innocuity of introducing an exogenous intravaginal device to ewes which
pregnancy status is still unknown. Therefore, for accelerated reproductive programs
that require an advancement of first pregnancies and effective induction of ovulations
during the early postpartum the technique might have positive impacts, as it also
allows a simple process with concentrated activities, allowing the application of two
FTAIs in less than 20 days. However, the results of Trials 2 and 3 demonstrate that
other factors might limit the results of the treatment itself, as the synchronization and
resynchronization treatments per se could not overcome the restrictive conditions of
the animals to achieve a good reproductive response.
The resynchronization protocol was effective to induce ovulations and

pregnancies in 1.5 yr-old nulliparous ewes, achieving similar results than in CON
ewes, and similar to those previously reported with similar treatments (Cosentino et
al., 2019). The general conditions were similar to that of other studies, as although
NUL ewes were 1.5 yr-old, they were still lighter than CON ewes (Ungerfeld and
Sánchez-Dávila, 2012). Although in general, 1.5 yr-old ewes are less fertile than
adult ewes (Aktaş et al., 2015), their reproductive performance at the first FTAI did
not differ from the adult multiparous ewes. However, at the second FTAI, even with
similar number of ewes in heat, pregnancies and lambing rates tended to be lower
than those of CON ewes. These tendencies could be explained by embryo losses,
but more probably the fertilization rate in this ovulation was lower in NUL than CON
ewes. The results at the first FTAI did not differ with that of CON ewes, so probably
the more fertile NUL ewes got pregnant at the first FTAI, and thus the results of the
resynchronized FTAI tended to be lower. This explanation agrees with the wider
variation of nulliparous ewes to respond to reproductive treatments, which is
consistent with what frequently happens in their first reproductive challenge. Overall,
the results open interesting possibilities to include synchronization-resynchronization
hormonal treatments associated with FTAI in nulliparous ewes breeding programs.
Otherwise, the protocol failed to induce fertile estrus in autumn lambing ewes.
Indeed, ewes suckling their lambs showed poorer results, with no ewe pregnant after
the first FTAI. This result is striking, as some ewes came into estrus, ovulated, were
inseminated, and had an active CL on Day 17, meaning that they probably lost the
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embryo between Day 17 and 42. In fact, the few PtP ewes that resulted pregnant did
it from the second FTAI. As the protocols were the same, the lactating condition, the
presence of the lamb, and the metabolic status were probably the main limiting
factors. Uterine involution seems not to be a limitation, as autumn lambing Corriedale
ewes complete it around 17 days after parturition (Rubianes and Ungerfeld, 1993).
However, milk production implies a high energy demand for the females, which
coincides with the approximately 20% lower BCS of PtP than CON ewes (section 2.1
from material and methods). Low body reserves are associated with low ovulatory,
conception and pregnancy rates, and with high embryo loss (Diskin and Morris,
2008). Also, Sharpe et al (1986) showed that those CLs originated after a
pretreatment with progesterone produce more progesterone, which may also explain
the pregnancies achieved at the second FTAI. Considering whether the presence of
lambs would have affected the pregnancy rate, Ungerfeld and Sánchez-Dávila (2012)
did not find differences in the reproductive response of autumn-lambing ewes to the
ram effect when lambs were weaned or not 27 days after parturition, and ewes were
stimulated 3 days later. Although the conditions were clearly different to those of the
present study, this suggests that the general postpartum metabolism after a late
gestation period occurring during summer is the main limiting factor in grazing
systems. The findings of the present work showed that although some PtP ewes
(21%) had a functional CL 17 days after the first FTAI, probably the metabolic status
limited its’ functionality, as happens in ewes and cows with low body conditions
(LeBlanc, 2010; Rutter and Manns, 1986; Zurek et al., 1995). Therefore, further
studies aiming to breed postpartum autumn-lambing ewes should study deeply the
relationship between the metabolic status and the ovarian functionality.
The implementation of synchronization-resynchronization FTAI programs

implies an increase in the costs of hormones, human resources, and technical work.
Moreover, as the initial pregnancy diagnosis is performed as early as 17 days after
de first FTAI, it should be performed by a trained technician. However, it also
simplifies planning, management, and reduces the unproductive time of the females.
It also simplifies handlings of the animals during the process, as animals should be
moved to the facilities less times, and only those animals that still should be treated
are moved. This also allows to avoid moving early pregnant animals, with the risks
that stress might induce in pregnancy results (Romo-Barron et al., 2019). Moreover,
181
since ewes lamb more concentrated, it is easier to maintain homogeneous groups in

pregnancy status, and then of lambs and postpartum females.
5. Conclusions
Overall, we concluded that the synchronization-resynchronization FTAI

treatments may be included in reproductive programs in nulliparous ewes, but the
treatments cannot superimpose to the limiting conditions imposed by postpartum
lactating status. The resynchronization treatments allow to include practical
managements with no deleterious effects in the results of the first FTAI.
Acknowledgments
PHNP had a postdoctoral scholarship from FAPERJ. FZB is fellow of the CNPq. IOC
had a scholarship provided by CAPES. The authors also acknowledge Alvaro López-
Pérez Ignacio Sosa, and Nicolás Zunnino for their support with the animal
management. This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code
001.
RU, RPC and FZB proposed the initial hypothesis, organized the study, discussed
the general study design. IOC, PHNP, FZB, RPC and RU collected the data. RPC
analyzed the data. IOC wrote the first draft. IOC, PHNP, FZB, RPC and RU revised
and worked on the preparation of the manuscript and approved the final version.
Competing interests
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CAPÍTULO IV
CONSIDERAÇÕES FINAIS
187
1 CONCLUSÕES
Com os estudos realizados concluímos que os protocolos de ressincronização

precoce em pequenos ruminantes são possíveis, desde que consideradas as
particularidades de cada espécie e categoria. Em caprinos, os estudos demonstram
inicialmente que o protocolo em si não causa perdas reprodutivas, necessitando
ainda de ajustes em doses e momentos para se chegar a um protocolo com
resultados satisfatórios. Em ovinos os estudos apresentaram sucesso,
principalmente com multíparas, onde não houve perdas reprodutivas induzidas pelo
protocolo e os resultados foram aceitáveis. Quando avaliado em outras categorias
os resultados em nulíparas se mostraram promissores, entretanto, para fêmeas pós-
parto, não foi possível se sobrepor às condições limitantes impostas pelo estado de
lactação pós-parto com o protocolo testado.
Assim, o estudo continuado e o uso da técnica em pequenos ruminantes

busca trazer melhorias para o sistema de produção, visto que, além de diminuir o
tempo improdutivo de uma fêmea no rebanho e conceder novas chances de
gestação para a fêmea, protocolos de ressincronização precoce associado ao uso
do diagnóstico precoce da fêmea não gestante permitem: (1) diminuir o uso de
hormônios desnecessários, (2) prevenir a perda de doses de sêmen, possibilitando o
investimento em reprodutores mais caros, e (3) melhorar os resultados gerais dos
programas de ressincronização, encurtando o período total de trabalho e a
simplificação das práticas. Desse modo, as fêmeas precocemente detectadas como
gestantes podem ter o manejo nutricional balanceado prontamente, além de evitar
manejos desnecessários - incluindo administração de hormônios e inseminação -, o
que tem implicações importantes para o bem-estar dos animais.
188
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190
3 ANEXOS
3.1 PROTOCOLO CEUA CAPRINOS – 1021/2017

191
3.2 PROTOCOLO CEUA OVINOS – 923/2017

192
3.3 TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO

193

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UNIVERSIDADE FEDERAL FLUMINENSE

ISABEL OLIVEIRA COSENTINO

USO DE PROTOCOLOS DE INDUÇÃO DO ESTRO SINCRONIZADO SEGUIDOS

USO DE PROTOCOLOS DE INDUÇÃO DO ESTRO SINCRONIZADO SEGUIDOS

Tese apresentada à Universidade

Área de Concentração: Clínica e

Orientador: Prof. Dr. Felipe Zandonadi

Coorientador: Dr. Mario Felipe Alvarez

C834u Cosentino, Isabel Oliveira

Orientador: Felipe Zandonadi Brandão.

1. Reprodução animal. 2. Controle hormonal do ciclo

Bibliotecário responsável: Debora do Nascimento - CRB7/6368

USO DE PROTOCOLOS DE INDUÇÃO DO ESTRO SINCRONIZADO SEGUIDOS

Tese apresentada à Universidade Federal

Prof. Dr. Felipe Zandonadi Brandão – UFF

Prof. José Nélio de Sousa Sales - UFJF

Profa. Dra. Raquel Pérez Clariget - Udelar

Dr. Luiz Gustavo Bruno Siqueira - Embrapa Gado de Leite

Dr. Pedro Henrique Nicolau Pinto - UFF

Aos meus pais, Valéria e Giovanni, que me acompanham em cada sonho e

Agradeço à minha família, em especial os meus avós, José, Iliana e Magaly,

Às amigas fora da pós-graduação, Julia Zordan, Maria Fernanda Nunes, que

Aos amigos da pós-graduação e do GEPECO, pelos momentos que

Aos meus orientadores Prof. Felipe Zandonadi Brandão e Mario Felipe

Aos funcionários e colaboradores da Estación Experimental Bernardo

Ao proprietário e colaboradores do Capril Vale das Amalthéias por nos

Aos funcionários da Fazenda Escola de Cachoeiras de Macacu da UFF, pelo

Aos professores do doutorado por dividirem comigo seu conhecimento e me

Às cabras e ovelhas, nossos objetos de estudo, sem elas nada seria

– Madre Teresa de Calcuta

O objetivo deste estudo foi avaliar distintos protocolos de indução do estro

No primeiro experimento na espécie caprina, foi avaliado o efeito do momento da

No terceiro experimento, as cabras passaram por protocolo de indução do estro

Palavras-Chave: Corpo lúteo, perfusão luteal, ciclo estral, controle hormonal,

Keywords: Corpus luteum, luteal perfusion, estrous cycle, synchronization,

2.1 ARTIGO REVISÃO DE LITERATURA: REPRODUÇÃO EM PEQUENOS

Figura 1. Esquema das fases do ciclo estral em pequenos ruminantes. ................. 31

Figura 2. Representação da duração de cada etapa para a execução de três partos

Figura 3. Representação gráfica do sistema Cornel STAR©, onde cada ponta da

Figura 4. Representação de um protocolo de estimulação por luz em pequenos

Figura 5. Representação de um protocolo de estimulação por efeito macho em

Figura 6. Representação de um protocolo curto baseado em progestágenos em

Figura 8. Representação esquemática comparando protocolos de indução do estro

Figura 11. Esquema de local de inseminação, dose inseminante e volume

Figura 13. A) Imagem de ovário vista pela videolaparoscopia, com um folículo em

Figura 14. Momento da Inseminação em caprinos, baseado no tipo de sêmen e

Figura 17. A) Avaliação em Modo-B da morfologia luteal em escores de 1 a 3, onde

2.2 ARTIGO REVISÃO DE LITERATURA: RECENTES AVANÇOS NOS

Figure 1. A) Resynchronization protocol for ewes adapted from Cosentino et al.

1 ARTIGO: ATIVIDADE OVARIANA EM CABRAS SAANEN LEITEIRAS SUJEITAS

Figure 1. Experimental proceedings timeline. ........................................................ 102

Figure 1. Experimental proceedings timeline. ........................................................ 119

Figure 2. Serum progesterone concentration (ng/mL) from Day 16 to Day 21 and on

3 ARTIGO: INFLUÊNCIA DO ECG E MANEJO REPRODUTIVO NA

Figure 1. Experimental proceedings timeline. ........................................................ 133

1 ARTIGO: PROTOCOLOS HORMONAIS PARA RESSINCRONIZAÇÃO PRECOCE

Figure 1. (I) Study 1 methodology: A) GPSP: pregnant animals with sponge; B)

Figure 2. Serum progesterone concentration (ng/mL) from Day 12 to Day 17, in

2 ARTIGO: INSEMINAÇÃO EM TEMPO FIXO E RESSINCRONIZAÇÃO PRECOCE

Figure 1. Schematic representation of the experimental groups, hormonal protocols

2.1 ARTIGO REVISÃO DE LITERATURA: REPRODUÇÃO EM PEQUENOS

Tabela 2. Principais protocolos utilizados para sincronizar fêmeas caprinas e ovinas.

Tabela 3. Características desejáveis para o sêmen do pequeno ruminante fresco,

Tabela 4. Principais afecções reprodutivas possíveis de serem detectadas através