This article was downloaded by: [Central Institute for Brackishwater Aquaculture-ICAR]

On: 26 August 2014, At: 23:46

Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Animal Biotechnology
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/labt20
Copy Number Differences of Y Chromosomal Genes
Between Superior and Inferior Quality Semen
Producing Crossbred (Bos taurus Bos indicus) Bulls
Ayan Mukherjee
a

b
, Gulshan Dass
a
, Jagan Mohanarao G.
a

c
, Vinay Kumar Katneni
d
, Dipak
Banerjee
a

e
, Tapan Kumar Das
a

f
, Moloya Gohain
a
, A. K. Chakrabarty
g
, Tirtha Kumar
Datta
a
& Sachinandan De
a
a
Animal Genomics Lab, Animal Biotechnology Center , National Dairy Research Institute ,
Karnal , India
b
Department of Molecular Immunology and Microbiology , National Institute for Research in
Reproductive Health , Mumbai , India
c
Department of Veterinary Biochemistry, College of Veterinary Sciences & Animal
Husbandry , Central Agricultural University, Selesih , Aizawl , Mizoram , India
d
Genetics and Biotechnology Unit , Central Institute of Brackishwater Aquaculture ,
Chennai , India
e
Department of Veterinary Physiology , West Bengal University of Animal & Fishery
Sciences , Kolkata , India
f
Department of Instructional Livestock Farm Complex (Animal Nutrition) , College of
Veterinary Sciences & Animal Husbandry , Agartala , Tripura , India
g
Artificial Breeding Research Center , National Dairy Research Institute , Karnal , India
Published online: 25 Aug 2014.
To cite this article: Ayan Mukherjee , Gulshan Dass , Jagan Mohanarao G. , Vinay Kumar Katneni , Dipak Banerjee , Tapan
Kumar Das , Moloya Gohain , A. K. Chakrabarty , Tirtha Kumar Datta & Sachinandan De (2015) Copy Number Differences of Y
Chromosomal Genes Between Superior and Inferior Quality Semen Producing Crossbred (Bos taurus Bos indicus) Bulls, Animal
Biotechnology, 26:1, 65-72, DOI: 10.1080/10495398.2014.887020
To link to this article: http://dx.doi.org/10.1080/10495398.2014.887020
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions
D
o
w
n
l
o
a
d
e
d

b
y

[
C
e
n
t
r
a
l

I
n
s
t
i
t
u
t
e

f
o
r

B
r
a
c
k
i
s
h
w
a
t
e
r

A
q
u
a
c
u
l
t
u
r
e
-
I
C
A
R
]

a
t

2
3
:
4
6

2
6

A
u
g
u
s
t

2
0
1
4

Copy Number Differences of Y Chromosomal Genes
Between Superior and Inferior Quality Semen Producing
Crossbred (Bos taurus Bos indicus) Bulls
Ayan Mukherjee,
1,2
Gulshan Dass,
1
Jagan Mohanarao G.,
1,3
Vinay Kumar Katneni,
4
Dipak Banerjee,
1,5
Tapan Kumar Das,
1,6
Moloya Gohain,
1
A. K. Chakrabarty,
7
Tirtha Kumar Datta,
1
and
Sachinandan De
1
1
Animal Genomics Lab, Animal Biotechnology Center, National Dairy Research Institute,
Karnal, India
2
Department of Molecular Immunology and Microbiology, National Institute for Research in
Reproductive Health, Mumbai, India
3
Department of Veterinary Biochemistry, College of Veterinary Sciences & Animal Husbandry,
Central Agricultural University, Selesih, Aizawl, Mizoram, India
4
Genetics and Biotechnology Unit, Central Institute of Brackishwater Aquaculture, Chennai, India
5
Department of Veterinary Physiology, West Bengal University of Animal & Fishery Sciences,
Kolkata, India
6
Department of Instructional Livestock Farm Complex (Animal Nutrition), College of Veterinary
Sciences & Animal Husbandry, Agartala, Tripura, India
7
Articial Breeding Research Center, National Dairy Research Institute, Karnal, India
The removal of crossbred bulls from semen collection programs due to the production of poor
quality semen causes substantial monetary losses to the dairy industry. Seminal quality, a
quantitative trait, is greatly inuenced by genome level variations. Deletion and/or duplication
of Y chromosomal genes and subsequent changes in gene copy number have a major role in
determining spermatogenic efciency and, therefore, seminal quality. In this study, copy numbers
of three Y chromosomal genes TSPY, DDX3Y, and USP9Y in genomic DNA were estimated and
compared in two groups of crossbred (Bos taurus Bos indicus) bulls of ten each, superior
and inferior quality semen producing bulls, which were classied based on their seminal quality
parameters. For TSPY gene, the inferior quality semen donor group has signicantly lower copy
number than superior quality semen donor group (p <0.05). No signicant difference was found in
DDX3Y and USP9Y gene copy numbers between two groups (p >0.05). In conclusion, this study
demonstrates that the copy number of TSPY, a Y chromosomal spermatogenesis related gene,
may be an important determinant to predict the quality of bull semen, facilitating better selection
of bulls in a herd for semen collection program.
Keywords Absolute copy number; Crossbred bull; DDX3Y gene; TSPY gene; USP9Y gene;
Y chromosome
Crossbreeding of indigenous stocks with elite exotic bulls
through articial insemination is popular practice in the
dairy industry. The unavailability of quality bulls and poor
quality semen production of crossbred bulls have become
major constraints to implementing a crossbreeding program.
Semen production is a quantitative trait governed by several
genetic and nongenetic factors. Mutation screening and
association studies have previously revealed substantial
genetic basis behind seminal quality parameters and fertility
(1). Genetic factors involved in poor quality semen
production include chromosomal aberrations, monogenic
disorders, and endocrine factors of genetic origin. Structural
abnormalities in genes of Y chromosomal origin are also of
special importance in this aspect as most of the genes on this
chromosome are exclusively expressed in the testis and their
dosage is associated with spermatogenesis (2).
The mammalian Y chromosome has long been inappro-
priately ignored as a gene-poor chromosome. However,
human studies have indicated the presence of 78 protein
Address correspondence to Ayan Mukherjee, Animal
Genomics Lab, Animal Biotechnology Center, National Dairy
Research Institute, Karnal, India. E-mail: ayanabtc@gmail.com
Color versions of one or more of the gures in the article can
be found online at www.tandfonline.com/labt.
Animal Biotechnology, 26:6572, 2015
Copyright # Taylor & Francis Group, LLC
ISSN: 1049-5398 print=1532-2378 online
DOI: 10.1080/10495398.2014.887020
65
D
o
w
n
l
o
a
d
e
d

b
y

[
C
e
n
t
r
a
l

I
n
s
t
i
t
u
t
e

f
o
r

B
r
a
c
k
i
s
h
w
a
t
e
r

A
q
u
a
c
u
l
t
u
r
e
-
I
C
A
R
]

a
t

2
3
:
4
6

2
6

A
u
g
u
s
t

2
0
1
4

coding genes on the Ychromosome that encode 27 (18 single
copy genes and nine gene families) distinct proteins (2).
These nine gene families are all multi-copied, localized in
the eight palindromes of the ampliconic sequences and are
expressed only in human testis, suggesting their possible role
in spermatogenesis and fertility (3). The TSPY gene encodes
a testis-specic protein that interacts with type B cyclins
and activates cyclin B-CDK complexes and the activated
complex, in turn, impacts on biological machineries in
spermatogonial cell renewal and in prophase I spermatocyte
differentiation (4). The unique feature of this gene is the wide
range of copy number variations among mammals. Mice
have an inactive copy (5, 6), whereas rats have one func-
tional copy (5). Cattle are estimated to have 50 to 200 copies
of the TSPY gene (7). The TSPY copy number on the
human Y chromosome varies from 23 to 64 (7). In humans,
a decrease in the TSPY copy number has been linked to
prostate cancer and an increase in copy number to male
infertility (810). DEAD box polypeptide 3 (DDX3) is
ATP-dependent helicase, and involved in unwinding
double-stranded RNA structures and remodeling RNA pro-
tein interactions. Ubiquitin specic peptidase 9, Y-linked
(USP9Y) gene encodes a protein that increases the efciency
of spermatogenesis. DDX3Y an USP9Y are both considered
as regulator of spermatogenesis (11).
Crossbreeding is performed to increase milk yield by
improving the genetic merit of the indigenous livestock
breeds. The use of genetically superior bulls in a breeding
system expedites the propagation of superior germplasm,
and the breeding bull is of the utmost importance to fulll
the stated objectives of crossbreeding. In particular, poor
seminal prole (12) and semen freezability are major rea-
sons for disposing of breeding crossbred bulls. Several
studies have shown that the fertility potential of crossbred
bulls is poor compared to that of purebred bulls (1315).
Chacon et al. (16) reported the higher culling of crossbred
bulls than Bos taurus and Bos indicus bulls in extensively
managed bull farms in Costa Rica. In a study population
of 751 bulls in southern Brazil, the culling rate due to com-
promised semen quality was 47% for Braford (crossbred of
Hereford bull and Brahman cow) bulls compared to 18%
for Hereford bulls maintained under the same conditions
(17). In a study on Frieswal bulls (a crossbred produced
in India), Tyagi et al. (18) found that 55% of the bulls
produced poor quality semen. Long term records of Karan
Fries (KF; a crossbred produced through crossbreeding
program initiated in 1971 at the National Dairy Research
Institute (NDRI), India and Sahiwal bulls reared at the
National Dairy Research Institute herd showed that the dis-
posal of bulls due to subfertility problem was much higher
in KF (43%) than in Sahiwal (29%) (19). Considering the
higher incidences of poor seminal quality in crossbred bulls
and a pivotal role played by Y chromosomal genes to deter-
mine spermatogenic efciency, that is, seminal quality, the
present study was conducted to nd out whether genomic
abundances of three important Y-chromosome genes
TSPY, DDX3Y, and USP9Y are associated with seminal
quality parameters in KF bulls.
MATERIALS AND METHODS
Animals and Semen Collection
The animals were reared at the Articial Breeding
Research Complex (ABRC), National Dairy Research Insti-
tute, Karnal, India and Frozen Semen Station, Uttarakhand
Livestock Development Board (ULDB), Rishikesh under
stall-fed management. Semen samples were collected from
bulls by articial vagina (IMV, LAigele cedex, France).
The volume of the semen was measured in a graduated conical
tube. Mass motility was evaluated by light microscopy. Sperm
concentration was estimated using a hemocytometer (20).
A systematic and rigorous procedure is followed for the
selection of bulls based on the seminal quality parameters at
ABRC herd of NDRI and ULDB Frozen Semen Station.
Seminal attributes, evaluated with fresh semen included
progressive motility, membrane integrity, and acrosomal
integrity of the sperm. Based on 3035 observations of semi-
nal quality over one year, 40 bulls were arranged in descend-
ing order of value for each semen quality parameter and 10
bulls of extreme phenotypes (superior and inferior quality
semen producer) from both ends were selected as subjects
of the present study. The bulls with extreme phenotypes
for seminal quality formed an ideal sample for association
studies that were planned to establish a link between copy
number variations and seminal quality traits.
Assessment of Sperm Quality Parameters
Estimation of Individual Motility Parameters
The percentage of motile sperm was determined by mixing
100 mL of undiluted semen into pre-warmed tubes containing
900 mL of Tris buffer. A thin drop of diluted semen was
placed on a prewarmed glass slide (37

C) and allowed to
spread uniformly under the cover slip (18 18mm). Amicro-
scopic eld was randomly chosen and motile sperm showing
any movement of the agellum and nonmotile sperm with no
agellar movement were counted at 37

C with an Olympus
BX51 microscope (Olympus America, Center Valley, PA,
USA) at 400. Another eld was chosen after counting in
the rst eld. The setting of the eld has always been fromleft
side of the slide to right side. Likewise, 300 cells were counted
in at least ve elds per slide. Minimum three slides were
evaluated per ejaculate per bull. The mean of the three
estimations was used as the nal motility score (21).
Assessment of Membrane Integrity
The plasma membrane of the sperm is heterogeneous in
nature and several domains divide it into compartments.
66 A. MUKHERJEE ET AL.
D
o
w
n
l
o
a
d
e
d

b
y

[
C
e
n
t
r
a
l

I
n
s
t
i
t
u
t
e

f
o
r

B
r
a
c
k
i
s
h
w
a
t
e
r

A
q
u
a
c
u
l
t
u
r
e
-
I
C
A
R
]

a
t

2
3
:
4
6

2
6

A
u
g
u
s
t

2
0
1
4

Hence domain-specic assay is often performed to evaluate
the membrane integrity of sperm. For example, the
integrity of membrane wrapping the sperm head is assessed
after staining the cells with uorescent dyes such as the
combination of 6-carboxyuoroscein diacetate (CFDA)
with propidium iodide (PI), or SYBR-14 with PI (22) while
the hypo-osmotic swelling test, in which sperm are incu-
bated in hypo-osmotic media, assess the plasma membrane
over the principal piece (2224).
6-carboxyuorescein Diacetate (CFDA) and Propidium
Iodide (PI) Staining
6-carboxyuorescein diacetate (CFDA) and propidium
iodide (PI) staining was used to assess the sperm membrane
integrity as described by Selvaraju et al. (25). Integrity of
the plasma membrane is reected by the ability of a viable
cell to exclude the PI dye. Briey, 80 mL of semen sample
was incubated with 100 mL CFDA=PI staining solution at
37

C for 15 minutes. CFDA=PI staining solution contained

0.8 mL of CFDA stock solution (4 mg=mL in DMSO) and
5 mL of PI stock solution (0.27 mg=mL in PBS). After
incubation, 10 mL of 0.2% glutaraldehyde was added and
4 mL of the stained sample was placed on a warm slide, a
cover slip was applied, and the preparations were examined
at 400 using an Olympus BX51 microscope equipped
with a CFDA lter set (Excitation, 460490 nm; Emission,
510550 nm; Dichromatic mirror, 505 nm). A minimum of
200 cells were counted per slide, and a minimum of three
slides were evaluated per ejaculate per bull. Sperm with
complete green uorescence were considered plasmalemma
intact, while those showing partial or complete red nuclei
were considered plasmalemma-decient.
Hypoosmotic Swelling Test (HOST)
The hypoosmotic swelling test was performed for assess-
ment of sperm membrane integrity based on curled and
swollen tails. The assay was performed by mixing 10 mL of
neat semen with 990 mL of 150 mOsM hypoosmotic solution
(13.51 g fructose 7.35 g tri sodium citrate per liter of
distilled water) and incubated at 38.5

C for 1 hour (26).

After incubation, a small drop was placed on a clean, dry,
and grease-free glass slide and covered with a cover slip.
The slide was examined at 200 with a bright eld micro-
scope. A minimum of 200 sperm were counted per slide to
assess different types of swelling patterns.
Assessment of Acrosome Integrity
Acrosomal integrity of the sperm was assessed by
staining air-dried smears with uorescein isothiocyanate-
conjugated pisum sativum agglutinin (FITC-PSA) staining
(27). Air-dried smear was ooded with the FITC-PSA for
30 minutes in the dark. Slides were then rinsed in distilled
water and mounted with glycerol and covered with a cover
slip. The uorescence pattern of 200 sperm in randomly
selected elds was assessed with an Olympus BX51
microscope at 400. The acrosomal status of sperm was
classied according to three typical lectin staining patterns:
(1) intact acrosome, complete staining of acrosome; (2)
reacting acrosome, partial or patchy staining of acrosome;
and (3) reacted acrosome, complete staining of the equa-
torial segment only or no staining of the whole sperm head.
The proportions of intact, reacting, and reacted acrosome
were expressed as percentages of the respective patterns in
the total number of sperm counted (28).
Collection of Blood and Isolation of Genomic DNA
The 10 mL of blood was collected from each animal in a
sterile Vaccutainer (Beckton-Dickinson, Franklin Lakes,
NJ, USA) containing sodium heparin. The collected sam-
ples were stored at 4

C and transported to the laboratory.

Lymphocytes population from blood was isolated by
Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and
counted with a hemocytometer. The genomic DNA was
extracted from white blood cells using standard phenol-
chloroform procedure (29). The DNA samples were dis-
solved in Tris-EDTA buffer (pH 8.0); their concentrations
were determined by optical density at 260 nm using a Nano-
drop1000 Spectrophotometer (Thermo Fisher Scientic,
Wilmington, DE, USA) and stored at 20

C for sub-
sequent experiments of absolute gene quantication.
Real Time Absolute Quantication
Construction of Standard Curve
Quantication of USP9Y, DDX3Y, and TSPY genes was
carried out by real-time PCR. Unlike endpoint measurement
where a slight difference in any of the limiting components
might have an effect on the amount of PCRproduct, the cal-
culation of the gene copy number was performed based on a
point threshold cycle (Cp), when PCR amplication was still
in the exponential phase in real-time PCR. This makes
real-time PCR more advantageous than endpoint measure-
ments. USP9Y (1646 base-pair), DDX3Y (2010 base-pair)
full length coding sequences were amplied from cDNA of
lymphocytes and that of TSPY (974 base-pair) from the tes-
ticular tissue of a bull. The amplicons were cloned separately
into pcr12.1 vector (3.9 kb) using the TOPO TA cloning
system (Invitrogen Corporation, Carlsbad, CA, USA).
Concentration of plasmids carrying USP9Y, DDX3Y, and
TSPY inserts were adjusted at 100 ng=mL using Nano-
drop1000 Spectrophotometer (Thermo Fisher Scientic,
Wilmington, DE, USA). The concentration of the plasmid
was converted to corresponding copy concentration using
the following equation (30):
DNA copy

6:023 10
23
copies=mol DNA amount g
Plasmid + Insert length bp 660 gm=mol=bp
SEMEN QUALITY OF PRODUCING CROSSBRED BULLS 67
D
o
w
n
l
o
a
d
e
d

b
y

[
C
e
n
t
r
a
l

I
n
s
t
i
t
u
t
e

f
o
r

B
r
a
c
k
i
s
h
w
a
t
e
r

A
q
u
a
c
u
l
t
u
r
e
-
I
C
A
R
]

a
t

2
3
:
4
6

2
6

A
u
g
u
s
t

2
0
1
4

A tenfold dilution series of each of the plasmid constructs,
ranging from 10
6
copies=mL to 10
1
copies=mL, 1.90 10
9
copies=mL to 1.90 10
4
copies=mL, and 10
11
copies=mL to
10
7
copies=mL were used to construct the standard curves
of USP9Y, DDX3Y, and TSPY, respectively. Each standard
dilution for three genes under study was assayed in tripli-
cate; qPCR was performed using 2 mL template. The Cp
values were plotted against the logarithm of their initial
template copy concentrations. Each standard curve was
generated by a linear regression of the plotted points. From
the slope of each curve, PCRamplication efciency (E) was
calculated according to the following equation (31):
E 10
1=slope
1
Primers and Conditions for Real-Time PCR
Real-time PCR reaction was carried out in a Lightcycler
480 instrument with software version 1.5 (Roche Diagnos-
tics, Mannheim, Germany) and Lightcycler Multiwell plate
96 (Roche Diagnostics, Mannheim, Germany). All the
DNA samples used as template for real time PCR ampli-
cation were adjusted to the concentration of 10 ng=mL using
nanodrop. The crossing point or Cp values were deter-
mined by second-derivative max method in the software
and extrapolated in the previously constructed standard
curve to determine the copy numbers of TSPY, DDX3Y,
and USP9Y genes in the genomic DNA. The reaction was
performed in a total volume of 10 mL containing 20 ng
of genomic DNA, 5 mL 2 KAPA SYBR FAST qPCR
Master Mix (Kapa Biosystems, Woburn, MA, USA), 0.5
pMol=mL primer pairs and 2 mL PCR-grade water
(Sigma-Aldrich, St. Louis, MO, USA). The cycling con-
ditions employed for all the genes were: preincubation at
95

C for 3 minutes, followed by 40 cycles of 10 seconds at

95

C, 20 seconds at 60

C, and 1 second at 72

C. The uor-
escence signal was measured at the end of each extension
step at 72

C. After the amplication, a melting peak analy-

sis with a temperature gradient of 0.1

C=s from 65

C to
95

C was performed to conrm that only the specic

products were amplied. Finally, the samples were cooled
to 40

C for 10 seconds. The following oligonucleotides

were used for the analysis: TSPY forward primer:
AGTTGTGAGCCCAGTTGTCA; TSPY reverse primer:
CACCTCCTCCACGATGTCTT; DDX3Yforward primer:
GTTAGATTTCTGCAAATACTTGGTGTT; DDX3Y
reverse primer: GCATAGTGTCTTGTTCAATTATACGAC;
USP9Y forward primer: GTACACAGTGGTCAAGC
AAGTGGTG; USP9Y reverse primer: CTTCTCCCATGT
ACTCTCCACCAAA.
Statistical Analysis
Data were analyzed using Statistical Product and Service
Solutions, Version 17.0.1 software (SPSS Inc., Chicago, IL,
USA). A difference with p <0.05 was considered statisti-
cally signicant. Percentage data of seminal attributes
were arcsine transformed before analysis to maintain the
homogeneity of variances. Students t-test was employed
to study the signicance of the difference in the mean copy
numbers of three genes between two groups of bulls. Copy
number determination experiments by real time PCR were
replicated three times. The results are expressed as mean
standard error of the mean (mean SEM).
RESULTS
Evaluation of Seminal Attributes
Long term evaluation of semen parameters vis-a`-vis pro-
gressive sperm motility, plasmalemma and acrosomal integ-
rity in the bulls under study showed signicant differences
FIG. 1. Seminal quality parameters of superior and inferior quality
semen producers of Karan Fries, a crossbred bull. The sperm progressive
forward motility, viability, membrane integrity (HOST) and acrosomal
integrity were assessed in superior (n10) and inferior (n10) quality
semen producer of Karan Fries, a crossbred bull. Representative photo-
graphs of viability (A), HOST (B), and acrosomal integrity (C) have been
shown. The percentages of progressive motility (D), viability (E), HOST-
reacted sperm (F), and acrosomal integrity (G) were compared between
two groups of bulls. Values are the means S.E.M. of 3035 observations
performed in two groups of bulls. Different superscripts indicate signicant
difference (p <0.05) between two groups.
68 A. MUKHERJEE ET AL.
D
o
w
n
l
o
a
d
e
d

b
y

[
C
e
n
t
r
a
l

I
n
s
t
i
t
u
t
e

f
o
r

B
r
a
c
k
i
s
h
w
a
t
e
r

A
q
u
a
c
u
l
t
u
r
e
-
I
C
A
R
]

a
t

2
3
:
4
6

2
6

A
u
g
u
s
t

2
0
1
4

between those producing superior and inferior quality
semen. Representative pictures of sperm viability, HOST,
and acrosomal integrity are shown in Fig. 1 AC. As evi-
denced from Fig. 1 DG, the progressive motility, sperm
viability, HOST, and acrosomal integrity are signicantly
higher in bulls producing superior quality semen than that
producing inferior quality semen (81.24 0.93 vs. 73.35
3.71, 73.59 0.74 vs. 51.79 2.75, 97.14 0.26 vs. 93.69
0.99, respectively).
Determination of TSPY, DDX3Y, and USP9Y Copy
Number
The absolute copy numbers of three Y-chromosome
specic genes were determined using SYBR Green real-time
quantication assay and mentioned in Table 1. Standards
curves developed for quantication of TSPY, DDX3Y,
and USP9Y genes have been presented in Fig. 2 AC. Gene
copy number per diploid cell of the individual bull was
determined by dividing the absolute copy number of each
gene with previously counted lymphocyte number. As
shown in Fig. 2D, the TSPY copy number in diploid
genome content of superior semen producing bulls was
signicantly higher than that of inferior semen producing
bulls (165.03 1.77 vs. 118.76 2.72). No signicant differ-
ences (Fig. 2E and F) were observed between superior and
inferior semen producing bulls in terms of USP9Y and
DDX3Y gene copy numbers (1.99 0.03 vs. 2.01 0.03
and 2.02 0.04 vs. 2.04 0.03, respectively).
DISCUSSION
Present study investigated copy number differences of
three important Y chromosomal genes TSPY, DDX3Y,
and USP9Y in superior and inferior quality semen produc-
ing bulls and revealed that the copy number of TSPY gene
varies signicantly between two groups of bulls. Poor semi-
nal quality and subfertility in domestic animals disrupt the
breeding system and cause huge economic losses. Molecular
mechanisms and role of different genes regulating semen
prole and fertility status in farm animals is a vast area
to be explored properly. Our knowledge on the genetic
component behind these important traits especially that is
TABLE 1
Number of copies of USP9Y, DDX3Y, and TSPY copies per single cell quantied by real-time PCR
Bulls
Cell
count
a
TSPY absolute copy
d
(Mean SEM)
TSPY
copy=cell
DDX3Y absolute
copy
c
(Mean SEM)
DDX3Y
copy=cell
USP9Y absolute
copy
b
(Mean SEM)
USP9Y
copy=cell
Superior Quality Semen Producing Bulls
KF1 35847 5909736 1329 165 68467 560 1.91 77787 567 2.17
KF2 107894 16900516 1567 156 225498 948 2.09 224419 1092 2.08
KF3 78832 13302112 1685 169 176583 980 2.24 156087 324 1.98
KF4 32622 5637082 865 172 60676 745 1.86 65570 589 2.01
KF5 70455 11864622 1234 168 151478 469 2.15 133864 856 1.90
KF6 42568 7100342 768 166 88967 326 2.09 79602 386 1.87
KF7 56827 9456013 965 166 109107 580 1.92 114790 542 2.02
KF8 51017 8285161 943 162 101013 764 1.98 96422 340 1.89
KF9 74112 11479949 1326 154 155635 680 2.10 140812 982 1.90
KF10 45690 7690541 876 168 86354 650 1.89 96405 459 2.11
Inferior Quality Semen Producing Bulls
KF1 21018 2691565 708 128 44137 456 2.10 40985 234 1.95
KF2 39227 5325458 986 136 81984 763 2.09 77669 578 1.98
KF3 34112 4197140 543 123 72317 256 2.12 69588 756 2.04
KF4 32849 3641969 654 111 68325 563 2.08 68654 934 2.09
KF5 20690 2471421 1092 119 42414 487 2.05 42414 256 2.05
KF6 50423 6083031 1187 121 105384 785 2.09 97820 489 1.94
KF7 142128 16293554 1121 115 284256 679 2.00 279992 886 1.97
KF8 84832 9906681 876 117 172209 902 2.03 160332 843 1.89
KF9 132622 14442536 1062 109 265244 904 2.00 291768 456 2.20
KF10 107374 11757453 1321 110 195420 542 1.82 217969 678 2.03
a
Number of cells=reaction genomic DNA.
b
Number of USP9Y copies=reaction genomic DNA.
c
Number of DDX3Y copies=reaction genomic DNA.
d
Number of TSPY copies=reaction genomic DNA.
SEMEN QUALITY OF PRODUCING CROSSBRED BULLS 69
D
o
w
n
l
o
a
d
e
d

b
y

[
C
e
n
t
r
a
l

I
n
s
t
i
t
u
t
e

f
o
r

B
r
a
c
k
i
s
h
w
a
t
e
r

A
q
u
a
c
u
l
t
u
r
e
-
I
C
A
R
]

a
t

2
3
:
4
6

2
6

A
u
g
u
s
t

2
0
1
4

regulated by Y chromosome is minimal. The singular Y
chromosome is haploid in nature and it escapes recombi-
nation with the X chromosome for most of its length
(2, 32). In absence of interchromosomal recombination
the repeated gene sequences within a chromatid or between
sister chromatids act as substrates for recombination. This
mechanism of intrachromsomal recombination leads to
high proportion of segmental duplications and consequent
generation of copy number variations (2, 33). Several
studies in human have been targeted to nd out the relation-
ship between Y chromosomal gene copy number and
seminal quality. Copy numbers of TSPY, DDX3Y, and
USP9Y were determined by standard curve based real-time
quantication assay and their relationship with seminal
quality was investigated such that quantities of these genes
in genomic DNA become useful marker for seminal quality
in crossbred bulls.
Present study demonstrates that copy number of TSPY
gene per cell is signicantly higher in superior quality
groups than bulls producing inferior quality semen.
Previous studies have estimated that bulls have between
50 and 200 copies (7) and wide range of variation prevails
within the breeds of Bos taurus (34). To the authors
knowledge, this is the rst study to relate the copy number
information of this gene with seminal quality in bulls. The
question whether the copy number of TSPY affects the
seminal quality has been addressed by several studies in
humans. Vodicka et al. (9) reported that higher copy
number of TSPY gene results in spermatogenic impairment.
Contrary to this result, absolute TSPY copy number deter-
mination by Giachini et al. (10) revealed that low TSPY
copy number is associated with lower sperm production.
TSPY is a multi-copied gene that is arranged in cluster in
a conserved manner across many mammalian species. The
protein product of the gene interacts with type B cyclins
and activates cyclin B-CDK complexes and the activated
complex, in turn, impacts on biological machineries in
spermatogonial cell renewal and in prophase I spermatocyte
differentiation (4). Therefore, it may be plausible that lower
copy number of TSPY may be one of the reasons that lead
to inferior quality semen production in subfertile bulls. In
the present study, DDX3Y copy numbers per cell did not
FIG. 2. Copy number differences of Y chromosomal genes between superior and inferior quality semen producers of Karan Fries, a crossbred
bull. The standard curves for TSPY (A), DDX3Y (B) and USP9Y (C) absolute copy number determinations together with their respective line equation
are shown here. Set of serial 10-fold dilutions was made for each gene. Each of these dilutions (standard dilutions) had known copies of the plasmid
construct harboring the respective gene fragment as an insert. X-axis represents log transformed values of the standard copy number and Y-axis
represents the crossing point (Cp) values, that is, the fractional cycle number required for the uorescence to cross the threshold. Copy number
differences for TSPY (D), DDX3Y (E), and USP9Y (F) have been represented. Copy number differences were calculated by dividing absolute copy
number of each gene by number of lymphocytes used for genomic DNA isolation.
70 A. MUKHERJEE ET AL.
D
o
w
n
l
o
a
d
e
d

b
y

[
C
e
n
t
r
a
l

I
n
s
t
i
t
u
t
e

f
o
r

B
r
a
c
k
i
s
h
w
a
t
e
r

A
q
u
a
c
u
l
t
u
r
e
-
I
C
A
R
]

a
t

2
3
:
4
6

2
6

A
u
g
u
s
t

2
0
1
4

vary signicantly between two groups of bulls. DDX3Y is a
single copy gene in the genome in most of the mammals
including cattle (35) and this may be the reason behind no
difference in DDX3Y copy number between two groups
of bulls. USP9Y is a single copy gene in most of the
mammalian species the exact copy number of the gene in
the cattle genome is unknown (35). No signicant difference
in absolute copy number of this gene between crossbreed
and indicine bulls was observed and quantity of this gene
per cell is almost equal to DDX3Y gene copy number.
Therefore, it can be assumed that like other mammalian
species cattle Y chromosome also harbors single copy of
this gene.
Development of biomolecular markers to establish com-
prehensive evaluation scheme for predicting bull fertility
potential is essential. This will expand our understanding
of the complexity of male fertility as well as develop means
of diagnosing and preventing suboptimum fertility. The
present study revealed that lower copy number of TSPY
per diploid cell in KF bulls producing inferior quality
semen. Hence, TSPY gene may be an important diagnostic
indicator of bull fertility. DNA screening at preliminary
level with standard curve based quantitative real time
PCR may help to anticipate the quality of semen, facilitat-
ing better selection of bulls in a herd for semen collection
program. To the best of our knowledge this is the rst
study aimed at nding out the association between copy
number variation of Y chromosomal genes and seminal
quality in farm animals. In future, identication of single
nucleotide polymorphisms, analysis of transcriptome and
endocrinological assays can be envisaged to enhance our
understanding of the phenotypic consequences of these
gene copy number variations.
ACKNOWLEDGMENT
The authors thank the Director, National Dairy
Research Institute, Karnal, India for all the necessary
facilities during the course of the study.
FUNDING
This study was supported by the World Bank funded
National Agricultural Innovation Project (C4=C30015).
REFERENCES
1. Visser L, Repping S. Unravelling the genetics of spermato-
genic failure. Reproduction 2010; 139:303307.
2. Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, et al. The
male-specic region of the human Y chromosome is a mosaic
of discrete sequence classes. Nature 2003; 423:825837.
3. Liu WS, Ponce de Leo n FA. Mapping of the Bovine Y
chromosome. Electronic J Biol 2007; 3:512.
4. Lau YFC, Li Y, Kido T. Role of the Y-located putative
gonadoblastoma gene in human spermatogenesis. Syst Biol
Reprod Med 2011; 57(12):2734.
5. Mazeyrat S, Mitchell MJ. Rodent Y chromosome TSPY gene
is functional in rat and non-functional in mouse. Hum Mol
Genet 1998; 7:557562.
6. Vogel T, Boettger-Tong H, Nanda I, et al. A murine TSPY.
Chromosome Res 1998; 6:3540.
7. Repping S, van Daalen SK, Brown LG, et al. High mutation
rates have driven extensive structural polymorphism among
human Y chromosomes. Nat Genet 2006; 38:463467.
8. Vijayakumar S, Hall DC, Reveles XT, et al. Detection of
recurrent copy number loss at Yp11.2 involving TSPY gene
cluster in prostate cancer using array-based comparative
genomic hybridization. Cancer Res 2006; 66:40554064.
9. Vodicka R, Vrtel R, Dusek L, et al. TSPY gene copy number
as a potential new risk factor for male infertility. Reprod
Biomed Online 2007; 14:579587.
10. Giachini C, Nuti F, Turner DJ, et al. TSPY1 copy number vari-
ation inuences spermatogenesis and shows differences among
Y lineages. J Clin Endocrinol Metab 2009; 94:40164022.
11. Krausz C, DeglInnocenti S, Nuti F, et al. Natural
transmission of USP9Y gene mutations: A new perspective
on the role of AZFa genes in male fertility. Hum Mol Genet
2006; 15:26732681.
12. Mandal DK, Tyagi S, Kumar M. Sexual behavior in
Holstein Friesian Sahiwal crossbred bulls. Indian Vet J
2008; 85:636639.
13. Sethi RK, Raina VS, Joshi BK, et al. Multistage selection of
crossbred males and effect of their age and body weight on
semen quality and freezability. Indian J Anim Sci 1989;
59(1):171174.
14. Rao KR, Sremanarayana O, Mukundarao R. Breeding life
and disposal patterns of breeding bulls. Indian Vety J 1995;
72:883888.
15. Chenoweth PJ, Chase Jr. CC, Larsen RE, et al. The
assessment of sexual performance in young Bos taurus and
Bos indicus beef bulls. Appl Anim Behav Sci 1996; 48:225.
16. Chacon J, Perez E, Muller E, et al. Breeding soundness
evaluation of extensively managed bulls in Costa Rica.
Theriogenology 1999; 52:221231.
17. Moraes JCF, Horn MM, Rosado A, et al.
Avaliac aoandrolo gicaemtouros: Qualidade dos indicadores
da aptidaoreprodutivaemdistintosgruposraciais. Ciencia Rural
1998; 28:647652.
18. Tyagi S, Mathur AK, Agarwal SC. Semen production perfor-
mance of Frieswal bulls. Indian J AnimSci 2000; 70:10321034.
19. Mukhopadhyay CS, Gupta AK, Yadav BR, et al. Subfertility
in males: An important cause of bull disposal in bovines.
Asian-Aust J Anim Sci 2010; 23:450455.
20. Salisbury GW, Van Denmark NL, Lodge J. Physiology of
Reproduction and Articial Insemination of Cattle. Delhi:
CBS Publishers and Distributors, 1985.
21. Tomar NS. Articial Insemination and Reproduction of Cattle
and Buffaloes. Allahabad: Saroj Prakashan, 1997.
22. Colenbrander B, Gadella BM, Stout TAE. The predictive
value of semen analysis in the evaluation of stallion fertility.
Reprod Domest Anim 2003; 38:305311.
SEMEN QUALITY OF PRODUCING CROSSBRED BULLS 71
D
o
w
n
l
o
a
d
e
d

b
y

[
C
e
n
t
r
a
l

I
n
s
t
i
t
u
t
e

f
o
r

B
r
a
c
k
i
s
h
w
a
t
e
r

A
q
u
a
c
u
l
t
u
r
e
-
I
C
A
R
]

a
t

2
3
:
4
6

2
6

A
u
g
u
s
t

2
0
1
4

23. Jeyendran RS, van der Ven HH, Perez-Pelaez M, Craboand
BJ, Zaneveld LJD. Development of an assay to assess the
functional integrity of the human sperm membrane and its
relationship to other semen characteristics. J Reprod Fertil
1984; 70:219228.
24. Neild DM, Chaves MG, Flores M, et al. The HOS test and its
relationship to fertility in the stallion. Andrologia 2000;
32:351355.
25. Selvaraju S, Ravindra JP, Ghosh J, et al. Evaluation of sperm
functional attributes in relation to in vitro sperm-zona
pellucida binding ability and cleavage rate in assessing
frozen thawed buffalo (Bubalus bubalis) semen quality. Anim
Reprod Sci 2008; 106:311321.
26. Revell SG, Mrode RA. An osmotic resistance test for bovine
semen. Anim Reprod Sci 1994; 36:7786.
27. Cross NL, Morales P, Overstreet JW, et al. Two simple
methods for detecting acrosome-reacted human sperm.
Gamete Res 1986; 15:213226.
28. Perry RL, Naeeni M, Barratt CL, et al. A time course study
of capacitation and the acrosome reaction in human sperma-
tozoa using revised chlortetracycline pattern classication.
Fertil Steril 1995; 64:150159.
29. Sambrook J, Russell D. Molecular Cloning: A Laboratory
Manual. New York: Cold Spring Harbor Press, 2001.
30. Whelan JA, Russel NB, Whelan MA. A method for the
absolute quantication of cDNA using real time PCR. J
Immunol Method 2003; 278:261269.
31. Rasmussen R. Quantication on the light cycler. In Rapid
Cycle Real-Time PCR: Methods and Applications; Meuer S,
Wittwer C, Nakagawara K, Eds; Berlin: Springer, 2001.
32. Pecon Slattery J, Sanner-Wachter L, OBrien SJ. Novel
gene conversion between X-Y homologues located in the
non-recombining region of the Y chromosome in Felidae
(Mammalia). Proc Natl Acad Sci USA 2000; 97:53075312.
33. Jobling MA. Copy number variation on the human Y
chromosome. Cytogenet Genome Res 2008; 123:253262.
34. Hamilton CK, Favetta LA, Di Meo GP, et al. Copy number
variation of testis-specic protein, Y-encoded (TSPY) in
14 different breeds of cattle (Bos taurus). Sex Dev 2009;
3:205213.
35. Paria N, Raudsepp T, Pearks Wilkerson AJ, et al. A gene
catalogue of the euchromatic male-specic region of the
horse Y chromosome: comparison with human and other
mammals. PLoS ONE 2011; 6:e21374.
72 A. MUKHERJEE ET AL.
D
o
w
n
l
o
a
d
e
d

b
y

[
C
e
n
t
r
a
l

I
n
s
t
i
t
u
t
e

f
o
r

B
r
a
c
k
i
s
h
w
a
t
e
r

A
q
u
a
c
u
l
t
u
r
e
-
I
C
A
R
]

a
t

2
3
:
4
6

2
6

A
u
g
u
s
t

2
0
1
4