Effect of milking frequency on oxytocin release

and milk production in dairy ewes

J.A. Negrao
a,b,*
, P.G. Marnet
b
, J. Labussiere
b
a
USP/FZEA, CNPq, FAPESP, Cx. P. 23, 13630-970 Pirassununga/SP, Brazil
b
Equipe Associee INRA/ENSAR de Recherches sur la Traite, 65, rue de Saint Brieuc,
35042 Rennes Cedex, France
Accepted 27 June 2000
Abstract
The experiment was conducted to investigate the effectiveness of milking stimulus on oxytocin release and to compare the
effect of milking frequency on plasma levels of oxytocin and milk parameters. Twelve Lacaune ewes were subjected to six
treatments (T1, T2, T3, T4, T5 and T7 daily milkings) during 6 days. At each milking, blood was sampled and plasma
oxytocin levels were determined by enzyme immunoassay. Baseline levels of oxytocin were similar for all milking
frequencies. The start of milking was followed by a signicant increase in oxytocin levels for all milking frequencies. One
daily milking induced signicantly higher oxytocin levels than 2, 3, 4, 5 and 7 daily milkings. Milk yield was signicantly
increased between 4 (1787X0 141X5 ml) and 7 (1780X0 53X6 ml) daily milkings compared to 1 (1104X0 81X2 ml) daily
milking. Total concentration of milk protein did not change, but the total milk fat yield for 5 (73X0 2X0 gal) and 7
(72X8 1X4 gal) daily milkings were signicantly higher than for 1 (58X1 4X3 gal) daily milking. This study conrmed milk
yield gains caused by frequent milk ejection and also showed that oxytocin release was not a limiting factor for milk yield gain
when daily milking frequency was increased. # 2001 Elsevier Science B.V. All rights reserved.
Keywords: Milking frequency; Oxytocin; Ewes; Milk yield; Dairy ewes
1. Introduction
The effect of the number of the daily milkings
depends on the cisternal compartment size of the
ruminant udder (Bruckmaier et al., 1994a; Knight
and Dewhurst, 1994). It is well established that
between milkings, there is an accumulation of milk
in the udder. This causes an intramammary pressure
increase and milk synthesis decreases (Labussiere,
1988). Previous study demonstrated that animals with
large cistern size tolerate once daily milking, but do
not respond well to higher milking frequency (Labus-
siere, 1988). Recently, a study showed that the accu-
mulation of a protein (FIL: feedback inhibitor of
lactation) synthesized by the mammary gland, reduced
milk synthesis in the alveolar cells (Knight et al.,
1996). Removing these physical and chemical inhi-
bitors during milk ejection, could explain the favor-
able effect on milk secretion frequency.
A complete milk ejection depends on the release of
neurohypophyseal oxytocin during milking (Schams
et al., 1984; Gorewit et al., 1992; Bruckmaier et al.,
1994b). Some authors observed that small quantities
Small Ruminant Research 39 (2001) 181187
*
Corresponding author. Present address: USP/FZEA, CNPq,
FAPESP, Cx. P. 23, 13630-970 Pirassununga/SP, Brazil.
Tel.: 55-19-561-6122; fax: 55-19-561-8606.
E-mail address: jnegrao@usp.br (J.A. Negrao).
0921-4488/01/$ see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S0 9 2 1 - 4 4 8 8 ( 0 0 ) 0 0 1 8 6 - 3
of oxytocin released during milking resulted in incom-
plete milk ejection (Denamur and Martinet, 1961;
Carruthers et al., 1993; Bruckmaier et al., 1994a,b).
Other authors demonstrated that the exogenous
administration of oxytocin can increase milk yield
and decrease the milk yield loss during incomplete
milkings (Gorewit and Sagi, 1983; Carruthers et al.,
1993) or disturbed milk removal (Bruckmaier et al.,
1994b).
However, these studies used two daily milkings, and
collected blood samples only during one of the milk-
ings. For these reasons, the present study was under-
taken to determine if oxytocin could be a limiting
factor on milk yield when increased milking fre-
quency is practised.
2. Materials and methods
Twelve multiparous Lacaune ewes (from 3 to 5
lactations) between 60 and 65 days postpartum were
used for this experiment. At the beginning of the
experiment, the ewes were milked twice per day,
and milk yield measured was 1636X6 103X0 ml.
Experimental ewes were in loose housing and fed
according to production level (INRA, 1990). The ewes
were weighted in the rst and last experimental day.
The effect of the number of daily mechanical
milkings was studied using a 6 6 Latin Square
experimental design (Table 1). Twelve homogeneous
Lacaune ewes were distributed into six groups and
subjected to six treatments (T): 1, 2 (control), 3, 4, 5
and 7 milkings per day, for 6 days (Table 2).
The settings for the lowline milking machine (Alfa-
Laval, VP 77) were vacuum of 44 kPa, pulsation ratio
of 50% and pulsation rate of 120 c/min. Silicon liners
(n10000080, Alfa-Laval) and plastic clusters
(n9610361, Alfa-Laval) were used. The teat cups were
attached at time 0 (without cleaning or massaging the
teats and udder) and detached after 90 s. Hand-milk-
ing and stripping were not applied. Milk yield, and
milk composition were evaluated at each milking.
Milk protein and milk fat were determined by infra-
red Milkoscan 605 (Foss Electric).
At 72 h before the beginning of the experiment, a
silicon catheter (Nutricath, Vigon) was implanted into
the jugular vein. At each milking a blood sample was
taken 30 s before milking (baseline levels) and 30, 60
and 120 s after the starting of milking. These samples
were collected in chilled heparinized tubes and imme-
diately centrifuged at 48C and 3000g for 15 min.
Plasma was preserved at 208C for later determina-
tion of oxytocin by enzyme immunoassay (Marnet
et al., 1994).
The milk ow was monitored and recorded as
previously described by Labussiere and Martinet
(1964), the milk kinetics were monitored on the
second and fourth experimental days.
The SAS software (SAS, 1990) was used for sta-
tistical evaluation of the data by analysis of variance
Table 1
Experimental design for oxytocin study: daily milking frequency
and treatments (milking time)
Daily milking
frequency
Treatments
(T)
Time of the
day (h)
1 T1 15
2 T2 5, 17
3 T3 7, 15, 23
4 T4 5, 11, 17, 23
5 T5 5, 10, 15, 20, 24
7 T7 5, 8, 11, 14, 18, 21, 24
Table 2
Organization of the Latin Square: ewes, groups and experimental days
Ewes Groups (G) Experimental days
First Second Third Fourth Fifth Sixth
1, 2 G1 T4 T5 T2 T1 T3 T7
3, 4 G2 T1 T3 T4 T2 T7 T5
5, 6 G3 T5 T1 T7 T3 T2 T4
7, 8 G4 T2 T7 T1 T4 T5 T3
9, 10 G5 T7 T4 T3 T5 T1 T2
11, 12 G6 T3 T2 T5 T7 T4 T1
182 J.A. Negrao et al. / Small Ruminant Research 39 (2001) 181187
and correlation. Mean comparisons were performed
using the Student t-test and the NewmanKeuls test at
a probability level of 5% (P ` 0X05). The independent
variables were: ewes, treatment and period.
3. Results
Results of oxytocin release are shown in Figs. 13.
The baseline data of oxytocin levels (30 s before
milking) were similar for all milking frequencies.
The milking machine caused signicant increase in
oxytocin levels for all milking frequencies at 30 s after
the beginning. The milk ow stopped at 60 20 s
after the start of milking, however, at 120 s the oxy-
tocin levels were still higher than the baseline levels
(time, 30 s).
The milking time did not signicantly inuence
oxytocin release (Fig. 4). However, the mean oxytocin
release for T1 was signicantly higher than oxytocin
release for the other treatments. For the treatments
with more than one milking, the prior oxytocin release
did not affect the following release. Since each milk-
ing caused an oxytocin release, the total secretion of
this hormone per treatment increased with the milking
frequency (Table 3).
Fig. 1. Average (means SEM) released oxytocin (pg/ml) during 1 (T1), 2 (T2) and 3 (T3) daily milkings.
Fig. 2. Average (means SEM) released oxytocin (pg/ml) during 4 (T4) and 5 (T5) daily milkings.
J.A. Negrao et al. / Small Ruminant Research 39 (2001) 181187 183
The milk yield, protein and fat for each treatment,
are shown in Table 3. The increase in milking fre-
quency signicantly increased total milk production
and milk fat, but did not affect milk protein.
For treatments 3 (1755X0 116X7 ml), 4
(1787X0 141X5 ml), 5 (1642X0 140X9 ml) and 7
(1780X0 53X6 ml) the milk production was not dif-
ferent, but their production was higher than T1
(1104X0 81X2 ml). Milk yield increased between 1
and 3 daily milkings, and plateaued between 3 and 7
daily milkings. The milk fat yield did not differ for T5
(73X0 2X0 gal) and T7 (72X8 1X4 gal) but was
higher than T1 (58X1 4X3 gal).
There was no statistical difference for milk produc-
tion between T1 and T2, although milk production for
the T1 was 15.4% less than T2. At the same time, the
Fig. 3. Average (means SEM) released oxytocin (pg/ml) during 7 (T7) daily milkings.
Fig. 4. Average (means SEM) released oxytocin (pg/ml) during
the different milking times, independent of the treatments.
Table 3
Means and their standard error (SEM) for the total oxytocin released, milk yield, milk fat and milk protein measured during the different
treatments
Daily milking frequency
(treatment)
Oxytocin
d
(pg/ml)
Milk yield
(ml)
Milk fat
(g/l)
Milk protein
(g/l)
Milk yield
(%)
1 (T1) 213.5
a
34.3 1104.0
a
81.2 58.1
a
4.3 45.5
a
1.2 15.4
2 (T2) 242.4
a
30.1 1305.0
a,b
61.5 65.0
a,b
2.8 44.1
a
1.0 Control
3 (T3) 278.1
a,b
42.1 1755.0
b,c
116.7 70.2
a,b
3.1 43.3
a
0.7 34.5
4 (T4) 447.0
b,c
53.2 1787.0
c
141.5 71.0
a,b
2.1 43.7
a
0.7 37.0
5 (T5) 503.4
b,c
55.0 1642.0
b,c
140.9 73.0
b
2.0 43.3
a
0.6 25.8
7 (T7) 676.3
c
56.8 1780.0
c
53.6 72.8
b
1.4 42.8
a
0.5 37.9
a,b,c
Means on the same column with different superscripts differ (P ` 0X05).
d
Total oxytocin released, i.e. T2 = [first milking (samples 30 60 120 s) second milking (30 60 120 s)[, etc.
184 J.A. Negrao et al. / Small Ruminant Research 39 (2001) 181187
increase on daily milkings from treatments 2 to 3, 2 to
4, 2 to 5 and 2 to 7 caused gains of 34.5, 37.0, 25.8 and
37.9% of milk yield, respectively. The correlations
between oxytocin level, and milk yield, protein and fat
were not signicant (Table 4).
The body weight of the ewes did not change
between the beginning (62X0 3X2 kg) and end
(60X8 3X0 kg) of the experiment.
4. Discussion
In this experiment, baseline levels of oxytocin were
not inuenced by hour of the day nor by milking
frequency, and when compared with those of other
ewes (Fuchs et al., 1987; Mayer et al., 1989) and cows
(Sagi et al., 1980; Schams et al., 1984; Gorewit and
Gassman, 1985), were similar or slightly higher.
At the same time, our data showed that indepen-
dently of the number of milkings, the milking machine
induced stimuli (Figs. 13), and a signicant release of
oxytocin was detected, which conrms that the milk-
ing machine induces oxytocin release (Gorewit and
Gassman, 1985; Mayer et al., 1989, 1991; Gorewit
et al., 1992). Nevertheless, a higher oxytocin dis-
charge was recorded in T1. Probably, this high dis-
charge was caused by the accumulation of oxytocin, in
the neurohypophysis, due to long intervals between
milkings. It is also possible that ewes, as others
species, rats and women, have an enormous storage
of oxytocin in the neurohypophysis (Lincoln and
Paisley, 1982), probably higher than the amount
required to elicit milk ejection.
It is interesting to observe that on the other treat-
ments with more than one daily milking, oxytocin
release was similar between the rst and the last
milking during the same day (i.e. milkings of
Fig. 3), which suggests an increase on oxytocin synth-
esis. At the same time, the efciency of the stimulus
caused by milking machine seems to be the same,
because during high milking frequency the ewes pre-
sented a fast (from 30 to 60 s after the start of all
milkings) and signicant increase in oxytocin levels.
These results suggest, that frequent activation of the
nervous inux of the udder by the milking machine,
increased oxytocin synthesis in the magnocellular
neurons of the hypothalamus, and also induced the
oxytocin release by the neurohypophysis. Other
authors (Gorewit and Sagi, 1983; Bruckmaier et al.,
1994a,b) already demonstrated that the increase of the
oxytocin levels in blood could facilitate milk transfer
from the alveoli to the cistern, and inuenced posi-
tively the milk yield.
However, Schams et al. (1984) suggested that
small increases in circulating oxytocin (35 pg/ml)
were effective to induce milk ejection in cows. In this
study, oxytocin release (higher level observed during
milking baseline levels) was between 19.4 and
74.7 pg/ml, and probably caused a complete ejection
of the alveolar milk. This effect was also conrmed
by the milkers, since they did not need to strip the
ewes.
Comparing twice to once daily milkings there was a
decrease of 15.4% in milk production, in the present
study. In similar conditions, Sardinian and Prealpine
ewes milked twice versus once daily, had a decrease of
5.2% (Casu and Labussiere, 1972) and 34.8% (Labus-
siere et al., 1974a,b), respectively, in milk production.
It is known that Sardinian ewes have a larger cistern
when compared to other breeds. For this reason,
Sardinian ewes produced more milk with once daily
milking than Lacaune (intermediate cistern) and Pre-
alpine ewes (little cistern) (Casu and Labussiere,
1972; Labussiere et al., 1974a,b). The physical
Table 4
Correlation coefcients between milk yield, milk protein, milk fat, oxytocin levels and total oxytocin release
Milk fat Milk protein Oxytocin release during sample (s) Total oxytocin
a
0 30 60 120
Milk yield 0.72 (P ` 0X05) 0.91 (P ` 0X05) 0.07 (NS
b
) 0.14 (NS) 0.01 (NS) 0.12 (NS) 0.14 (NS)
Milk fat 0.84 (P ` 0X05) 0.12 (NS) 0.12 (NS) 0.11 (NS) 0.22 (NS) 0.16 (NS)
Milk protein 0.08 (NS) 0.05 (NS) 0.13 (NS) 0.11 (NS) 0.15 (NS)
a
Total oxytocin released, i.e. T2 = [first milking (samples 30 60 120 s) second milking (30 60 120 s)[, etc.
b
Not signicant.
J.A. Negrao et al. / Small Ruminant Research 39 (2001) 181187 185
capacity of the udder cistern is limited, and prolonged
milking intervals increase the intramamary pressure,
decreasing, consequently, the milk synthesis effective-
ness by the alveolar cells.
In this study, the increase from twice to thrice daily
milkings caused a milk yield gain of 34.5%. In a
similar experiment, with Prealpine du Sud breed
(Labussiere et al., 1978), the increase from 2 to 3
daily milkings caused a gain that varied from 15 to
26%. These differences could be attributed to the
genetic potential of the studied breeds.
It seems that continuous milk synthesis and alveolar
milk ejection into the cistern were optimized with 3
daily milkings. Some authors suggested that the
increase observed during multiple milkings was pro-
voked by cell proliferation (Wilde et al., 1987; Sven-
nersten et al., 1990; Wilde and Knight, 1990; Gorewit
et al., 1992). However, in our experiment, the short
duration of the treatments (6 days) and the use of Latin
Square experimental design diminished the possibility
of a cell proliferation. Indeed, the modest gain
observed from 4 up to 7 daily milkings reinforced
this observation.
Generally, the increase of milk production is asso-
ciated with a decrease of the milk constituents (Hen-
derson and Peaker, 1987; Barnes et al., 1990),
however, total milk fat yield was signicantly higher
for 5 and 7 daily milkings in this study. It is also
possible that frequent milkings could stimulate local
synthesis of the milk fat within the mammary gland
(Barnes et al., 1990; Svennersten et al., 1990; Wilde
and Knight, 1990). It is more probable that frequent
milk ejections increased the proportion of fat alveolar
milk, which increased total milk fat content (Labus-
siere, 1988; Knight and Dewhurst, 1994).
Some authors (Hillerton et al., 1990; Svennersten
et al., 1990; Wilde et al., 1989) suggested that this
increase in milk production and milk fat was caused by
a metabolic adaptation. In other researches, the
increase in milk production and milk fat was explained
by reduction in body weight (Barnes et al., 1990;
Svennersten et al., 1990; Wilde et al., 1989). In this
study, body weight did not change between the begin-
ning (62X0 3X2 kg) and the end (60X8 3X0 kg) of the
experiment. However, hay and concentrate were fed in
accordance to milk production (20%), since there
was no leftover, we can assume that the feed intake
increased.
In conclusion, for treatments with 1 and 2 milkings
daily, the limiting factor for milk yield should be the
capacity of the cisternal storage. However, oxytocin
release did not seem to be a limiting factor for milk
production when high milking frequencies were used.
When 5 and 7 milkings daily were studied, there were
modest milk yield gains, probably, the limiting factor
was the milk synthesis rate. Better interaction between
continuous milk synthesis and alveolar milk ejection
was observed during 3 milkings daily, consequently an
important milk yield gain was measured. In many
countries milk from dairy sheep is of great economic
importance, our results suggest that the increase of
milking frequency could be used to improve ef-
ciently milk production of the many breeds.
Acknowledgements
The authors thank M. Beauls, A. Briand, F. De La
Chevalerie, J.F. Combaud, J. De Saint Jan, P. Lam-
bion, M. Chorho, J. Le Bail, C. Yard, P. Rolland and
E. Siroux, for technical assistance.
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