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The major limitations of goat production are the lack of
good breeding bucks and the seasonal nature of semen
production and quality (Kridli et al. 2007). Black Bengal
breed of goat is known for its prolificacy and excellent meat
and skin quality in India (Singh et al. 1991). The acute
shortage of genetically superior buck is one of the major
constraints for the efficient propagation of this breed. Body
weight and growth rate may serve as a useful predictor of
live weight since Black Bengal goats are being reared
primarily for meat production (Islam 2001). The most
accurate way to test the genetic worth of a sire is to perform
breeding soundness evaluation (BSE) i.e., fertility potential
of a buck based on an examination that includes tests for
physical soundness, testicular size, semen quality, and in
some cases libido/mating ability (Okere et al. 2011). In the
present study we reported the testicular biometry and the
change in seminiferous epithelium of testis at different ages
and the seasonal variations in semen parameters of Black
Bengal goat.
MATERIALS AND METHODS
Animals: Male Black Bengal goats (19), maintained in
the experimental goat farm of the laboratory at the Institute,
were included in the study. All the measurements (scrotal
and testicular biometry) were initiated at the birth and
continued till 20 months of age. For biometrical studies,
animals were classified in to 6 different age groups: A, at
birth, B, up to 4 months, C,5 to 8 months, D, 9 to 12 months,
E, 13 to 16 months and F, 17 to 20 months. To commensurate
the results of biometry, testes of 4 male kids of different
age groups were subjected to histological examination. For
studying the semen parameters, adult bucks (3) of 2 years
of age were included.
Recording of age and body weight (BW), Scrotal and
testicular biometry: The age of goats were calculated from
the date of birth. The body weight (kg) of each animal was
recorded at 30 days interval. Scrotal circumference (SC)
was measured with a measuring tape in centimeters (cm)
and the testicular diameter (mm) was measured with a
vernier caliper (Raji et al. 2008). All these measurements
Present address: 1Ph.D. Scholar (rakesh05vet@gmail.com),
DCB Division, NDRI, Karnal. 2 Ph.D. Scholar (drapramodr
@gmail.com); 3Senior Research Fellow (rohit2005biotec
@gmail.com); 4Senior Research Fellow (mamtaanegi
@gmail.com), 6Principal Scientist (rajendra_singh5747
@rediffmail.com), Department of Pathology, 7National Fellow
(drabhijitmitra@gmail.com), Genome Analysis Laboratory,
Animal Genetics Division. 5Assistant Professor (satsinpal21
@gmail.com), Department of Animal Genetics and Breeding,
College of Veterinary Science, DUVASU, Mathura
Indian Journal of Animal Sciences 84 (6): 37–00, June 2014/Article
Testicular biometry and seasonal variations in semen
parameters of Black Bengal goats
RAKESH KUMAR1, R KUMAR PRAMOD2, ROHIT KUMAR3, MAMTA NEGI4, SATYENDRA PAL SINGH5,
RAJENDRA SINGH6 and ABHIJIT MITRA7
Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243 122 India
Received: 20 August 2013; Accepted: 30 January 2014
ABSTRACT
Body weight, growth rate and testicular biometry of Black Bengal bucks (19) belonging to different age groups
were studied. Further, this study was extended to delineate the histological changes in the testes with advancement
of ages and to evaluate the seasonal variations in the important semen parameters. The body weight differed
significantly across the age groups but the body weight gain (g/d) was higher in the age groups of 0–4 and 17–20
months. The mean scrotal circumference increased with the advancement of age and attained maximum circumference
at the age of 12 months. The mean testicular diameter increased significantly till 10th month of age. All these
biometrical parameters were highly correlated with each other, and with age and body weight. The histological
examination of testes revealed an effect of age on the seminiferous tubules and stages of the spermiogenesis. At 3.5
months of age, lumen of seminiferous tubules became distinct with the abundance of spermatozoa. These observations
along with testicular biometry indicated the age at which bucks attained puberty. Significant effect of the seasons
(e.g., spring and summer) was observed on the semen parameters with the improvement in the progressive motility,
percentage of live spermatozoa, chromatin integrity and a drop in membrane integrity during summer. These results
will provide an important impetus in selection of breeding bucks of Black Bengal breed.
Key words: Acrosome integrity, Histology of testis, HOST, Membrane integrity, Progressive motility,
Semen parameter, Testis
38 KUMAR ET AL. [Indian Journal of Animal Sciences 84 (6)
x
were taken on the same day when body weight was
recorded.
Evaluation of semen parameters: Ejaculates were
collected from the trained bucks (3) using an artificial
vagina (AV). In order to assess the effects of seasons on
semen parameters, semen was collected during two
consecutive seasons: spring (February) and summer (May).
Semen was evaluated for different parameters. To assess
progressive motility, semen was diluted with sodium citrate
glucose buffer (1:10). One drop of the diluted semen was
placed on a glass slide and after placing a cover slip it was
immediately examined high power (40×) objective of phase
contrast microscope. To calculate the live/dead percentage
of sperms, 2 methods namely, Eosin-Nigrosin (Rodríguez-
Martínez 2000) and carboxyfluorescein diacetate/propidium
iodide (CFDA/PI; Peòa et al. 1998) based differential
staining were used. Acrosomal integrity was determined
by using both Giemsa (Kumar and Singh 2003) and
Fluorescein isothiocyanate coupled with peanut agglutinin
(FITC-PSA; Sukardi et al. 1997) based staining methods.
Hypo-osmotic swelling test (HOST) was used to assess the
functional integrity of the sperm tail membrane (Jeyendran
et al. 1984). Sperm chromatin structure assay was
performed using chromomycin A3 according to Bianchi et
al. (1996).
Histological study: Male kids (4) of different ages (e.g.,
3 days, 1, 3.5 and 7 months) were included. Under local
anesthesia, one of the testes was removed. Tissue sections
were fixed and stained with haematoxylin and eosin. Slides
were examined under 10× and 40× magnifications of a
compound microscope.
Statistical analysis: Mean and standard error for body
weight and testicular biometric data was analyzed using
SPSS software program (ver. 16). Testicular biometry with
relation to age was studied by performing One-way ANOVA
analysis. Duncan multiple range test (DMRT) was done to
observe significant difference among mean values.
Pearson’s correlation analysis was performed to study
correlation among different traits. To study the seasonal
difference in semen characteristics t-test was applied.
RESULTS AND DISCUSSION
Body weight and growth rate: Mean body weights (Table
1) increased steadily from birth till 20 months of age. The
body weight also differed significantly across the age groups
(P<0.01). However, the body weight gain (g/d) was
significantly high in the age group of 0–4 and 17–20 months
and almost same in remaining age groups The body weight
of Black Bengal bucks significantly increased with the
advancement of age which is in agreement with previous
studies (Rahman 2007, Kabiraj et al. 2011) in Black Bengal
bucks.
Scrotal circumference: The mean SC increased
significantly with the advancement of age (P<0.01; Table
2) and attained maximum value after 12 month of age. The
SC is an indirect measurement of testicular weight and a
reliable indicator of testicular growth and spermatogenic
capacity of the testis. Scrotal circumference, being the most
heritable component of fertility, is proposed to be included
in breeding soundness evaluations (Bailey et al. 1996). It
was extensively used in predicting the reproductive capacity
of male domestic animals. Shamsuddin et al. (2000) and
Kabiraj et al. (2011) observed the significant increase in
SC of Black Bengal bucks also with the advancement of
age.
Testicular diameter: The mean testicular diameter
increased till 10th month of age and did not increase further
significantly (Table 1). The testicular diameters of age group
B to E were significantly higher than that of group A
(P<0.01).
Correlation coefficients (r) between age, BW, SC and
average testicular diameter: All the biometrical parameters
studied were significantly correlated with each other and
age (P<0.01; Table 2). Body weight showed high (>0.8)
correlation with age, SC and testicular diameter. In the
present study, all the parameters of testicular biometry
showed a significant correlation among themselves as well
Table 2. Correlation coefficients (r) between age,
body weight, scrotal circumference and testicular
diameter in Black Bengal buck (19)
Parameter Age Body Scrotal
weight circumference
Body weight 0.838** ––
Scrotal circumference 0.763** 0.784** –
Testicular diameter 0.681** 0.827** 0.923**
**P< 0.01.
Table 1. Comparative studies of testicular biometry of Black Bengal bucks with respect to age and body weight (mean±SE)
Age group No. of kids Body weight Average Scrotal Average testicular Body weight
(kg)** circumference diameter gain (g/d)*
(cm)** (mm)**
Group-A (At birth) 11 1.86a±0.11 5.50a±0.23 8.55a±0.38 –
Group- B (0 to 4 m) 19 6.23b±0.32 11.71b±0.59 24.1b±1.30 32.57b±3.58
Group- C (5 to 8 m) 13 8.20c±0.53 16.90c±0.41 34.1c±1.08 19.79a±1.7
Group- D (9 to 12 m) 11 10.56d±0.46 18.29cd±0.26 36.4c±1.18 19.79a±2.0
Group-E (13 to 16 m) 11 12.86e±0.41 18.67d±0.28 37.0c±0.93 20.83a±1.5
Group–F (17 to 20 m) 11 15.31f±0.79 – – 27.97b±3.8
Means with different superscripts within the column differ significantly (**P<0.01, *P<0.05);SE, standard error.
June 2014] TESTICULAR BIOMETRY AND SEASONAL VARIATIONS IN SEMEN OF GOATS 39
x
with the age and body weight. Similar findings were
reported in Black Bengal (Kabiraj et al. 2011) and other
breeds (Raji et al. 2008) of goat.
Histology of testes: The histological examinations (Fig.1)
revealed the effect of age on the seminiferous tubules (ST)
and stages of the spermiogenesis. Increase in tubular
convolution and decrease of inter-tubular space was marked
as the age of the animals advanced. Transverse section of
the testes from 3– day old kid showed smaller sized ST
amidst connective tissue (Fig.1a), which were lined by
undifferentiated germ cells or spermatogoinal stem cells
interspersed with primitive polygonal or cone-shaped sertoli
cells (Fig.1a). The lumens of the ST were indistinct
because of long cytoplasmic process of the sertoli cells,
referred as sex cords and there was no evidence of
spermatogenesis (Fig.1a). However, in the testes of 1- month
old kid, STs were larger in which abundant germ cells,
prespermatogonia, started migrating towards the basement
membrane of the sex cords and differentiating into primary/
secondary spermatocytes and spermatids (Fig.1b). The
lumens of the ST were distinct and filled with detached
spermatids (Fig.1b). By the age of 3 months (Fig.1c) the
spermatozoal bunches were prominently seen embedded
in the cytoplasmic process of the sertoli cells and the lumen
of the tubules was broader and contained moderate number
of spermatozoa. In seven months, the ST have well
developed series of different cell types and the lumen
contained plenty number of spermatozoa along with few
detached degenerated/apoptotic cells (Fig.1d). It was
observed that undifferentiated germ cells (i.e., pre-
spermatogonia) started migrating towards the basement
membrane of the sex cords as early as 1 month of age (Sarma
and Devi 2012). Further, increase in tubular convolution
and decrease of intertubular space which is indicative of
well formed reproductive stage of an adult animal was
marked as the age of the animals advanced. These
microscopic changes were well corroborated with the
testicular morphometry. Histological examination and
testicular biometry indicated that bucks attained puberty at
3–4 months age and became reproductively active at 7
months of age as indicated by the abundance of spermatozoa
in the lumen of ST.
Effect of seasons on semen characteristics: Semen
parameters like progressive motility, live/dead count and
membrane and chromatin integrity varied significantly
between the seasons (Table 3; Fig.2). The progressive
motility improved (P<0.01) in spring to summer. The
percentage of live spermatozoa, as estimated using eosin-
nigrosin staining, also increased significantly (P<0.05) from
spring to summer. The mean value of percent spermatozoa
with abnormal chromatin differed (P<0.01) between these
2 seasons while the integrity of membrane lowered (P<0.05)
during summer than that in spring. However, remaining
parameters did not show any significant changes.
Fig. 1. Photomicrograph (H & E stain) of testis of Black Bengal bucks at different ages: (a); 3rd day (b); 1 month (c); 3 months and
(d); 7 months.
Table 3. Comparative study for seminal parameter in Black
Bengal bucks (n=3) during spring and summer months.
Descriptive Seasons Significance
Spring Summer
Progressive motility (%) 65.00±1.17 70.55±1.30 **
Live (%), Eosin- 61.11±0.60 64.44±1.33 *
Nigrosin method
Live (%), CFDA/ 49.44±1.23 49.44±1.75 NS
PI method
Acrosome Integrity 84.72±0.27 83.33±0.33 NS
(%), Giemsa
Acrosome Integrity 88.05±0.55 86.11±1.38 NS
(%), FITC-PSA
HOST positive (%) 82.77±0.27 76.66±2.35 *
Chromomycin A370.00±0.00 62.77±2.22 **
positive (%)
**P<0.01, * P< 0.05, NS, not significant.
40 KUMAR ET AL. [Indian Journal of Animal Sciences 84 (6)
x
We evaluated different semen parameters of Black
Bengal bucks using different laboratory staining techniques.
In this study, in comparison to eosin-nigrosin staining, live
count of spermatozoa was under estimated by 10% while
using CFDA/PI. This difference between eosin-nigrosin and
CFDA/PI might be due to the difference in time of exposure
to the stain, which is only a few seconds for eosin-nigrosin
and 10–30 min for PI (Brito et al. 2003). Acrosomal integrity
provides an indicator for fertilizing capability of
spermatozoa. The results obtained using FITC-PSA based
staining was highly correlated with those obtained using
Giemsa staining in the present study. Hypo osmotic swelling
test (HOST) is used for identification of viable sperm in a
non-destructive manner (Jeyendran et al. 1984). The
chromatin integrity of spermatozoa was evaluated using
chromomycin A3 test, an indirect indicator of protamine
deficiency and fertility status of spermatozoa (Foresta et
al. 1992).
In the present study, a significant variation was observed
in the semen parameters of Black Bengal bucks between
the seasons. Others also reported significant seasonal
variation in the semen production in goats (Webb et al. 2004,
Zarazaga et al. 2009) and bulls (Sarder 2007). However,
some studies could not observe any seasonal variation in
the semen quality of Angora and Boer bucks (Greyling and
Grobbelaar 1983) and buffalo bulls (Koonjaenak et al.
2007). In contrast to our result, Elsheikh et al. (2013)
reported a decrease in livability of crossbred goat sperm in
Fig. 2. Assessment of semen parameters in Black Bengal bucks: Live and dead count (%) of spermatozoa using (a) Eosin-nigrosin
method (black and white arrows indicate live and dead sperm, respectively) (b) CFDA/PI method (live and dead sperms stained green
and red, respectively); acrosome integrity using (c) Giemsa staining (black and white arrows indicate intact and reacted acrosome,
respectively), (d) FITC-PSA method (white and red arrows indicate intact and reacted acrosome, respectively); membrane integrity of
sperm using (e) HOST (white arrow indicate HOST+ve and black arrow indicate HOST–ve sperm); sperm chromatin integrity using
(f) chromycin A3 (CMA3) assay (black and white arrows indicate CMA3+ve and –ve sperm, respectively).
summer season.
In conclusion, we demonstrated a strong association of
testicular biometry with age and body weight in Black
Bengal breed of goat. All the parameters of testicular
biometry increased significantly with the advancement of
age. Histological studies also indicated that SC may serve
as an accurate predictor of the puberty. Our results on
testicular biometry and the seasonal variations of semen
characteristics would be helpful in selection of breeding
buck in Black Bengal goat.
ACKNOWLEDGEMENTS
This work was carried out under the project supported
by Competitive Grant of National Agricultural Innovation
Project (NAIP) and National Fellow Scheme, Indian
Council of Agricultural Research (ICAR), Ministry of
Agriculture, Government of India. Financial help in the form
of Institute Scholarship to RK and ICAR SRF to PK during
their research programme is duly acknowledged. The
authors are grateful to Dr Reema Gupta, Research Associate
for critical reading of the manuscript.
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