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Postpubertal changes in gonadal and extragonadal sperm reserves in Bos indicus strain bulls
THERIOGENOLOGY
POSTPUBERTAL
CHANGES
RESERVES
IN
GONADAL
IN EQS
INDICUS
AND
EXTRAGONADAL
STRAIN
SPERM
BULLS
S. Wildeus and K.W. Entwistle Department of Tropical Veterinary Science James Cook University of North Queensland Townsville, QLD. 4811 Australia
Received
for
vublication: Accepted:
Januarv 4. 1982 April 1, i982
Abstract Testes and epididymides were collected from 136 young (1.5 to 3.5 years of age) and 33 mature &,s J_ndlcus strain bulls, reared under extensive conditions in range the tropics. Testicular and epididymal sperm reserves and different rates in the weights increased at Daily sperm production per young bulls. gram (DSPG) increased of testis significantly before and epididymal sperm reserves after 2.5 Testicular years of age. and epididymal weights increased linearly with bodyweight. cross bulls had higher testicular and Sahiwal epididymal weights, and greater sperm reserves did Brahman cross bulls at the same age. than Testicular and epididymal weights, daily sperm epididymal sperm reserves for production and Brahman bulls were 67Og, mature 61g. 4.9 billion and 25.5 billion respectively. Testis weight was highly correlated with total daily sperm
production
(DSP)
and
epididymal
weight.
Introduction
upon Beef cattle reproductive performance is dependent female and male and thus the from both the contributions herd sex influence fertility in either level of may
Acknowledgements: Queensland Department of Primary Holroyd, thank Mr R. We and Mr H.R. Research Station Lagoon" "Swan's Industries for access to Research Station "Belmont" Christensen, CSIRO Miss J. The technical assistance of material. experimental These acknowledged. Bradshaw is gratefully Prideaux and Mr I. from the Australian Meat financed by a grant studies were Research
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655
THERIOGENOLOGY performance and hence economic returns in a beef cattle enterprise. Estimates spermatozoa1 production of daily (DSP) and extra-gonadal sperm reserves (EGR) are essential for the efficient utilization of available sperm resources (1) and have been determined for &s w dairy (2) and beef bulls (3) and Br;a e bulls (4). Seebeck (5) and Seifert .& a. (6) indicated in studies done in Central Queensland that &S i&&&u,8 bulls showed reduced fertility when compared to contemporary m taurus bulls. This reduced fertility may be a reflection of smaller testicular size higher proportion of (7), reproductive tract abnormalities, particularly testicular hypoplasia (8), delayed onset of puberty (9,lO) and smaller gonadal (11) and epididymal sperm reserves (4). It is not clear however to what extent these factors are inherent to this genotype or are caused by seasonal periods of undernutrition, internal and external parasite burdens, and a slower rate of maturity. The development of DSP precedes the establishment of EGR in Friesian bulls (12.13) and only small numbers of spermatozoa are available for ejaculation in young bulls. The delayed reproductive development occurs together with a slow rate of maturity in m indicus bulls, it was necessary to determine whether a combination of both may be contributing to the fertility reduced in &S indicus strains. Hence the establishment of gonadal and extra gonadal sperm reserves in half and three-quarter ins indicus strain bulls was examined in this study.
Materials
and Methods
Animals: Material for this study was obtained from clinically normal E&8 indicus strain bulls which had been raised on Bulls native pasture in tropical Australia. between 1.5 and 3.5 years of age originated either from the Lagoon" Queensland Department of Primary Industries "Swan's Long 147 E) or from the CSIRO Research Station (Lat 19 S; National Cattle Breeding Station "Belmont" (Lat 23 S; Long 151 E) and detailed production records (growth rates and circumference (SC)) reproductive traits, including scrotal Testes and epididymides were available. on these animals castration. Genitalia from were obtained at slaughter or mature as such from dentition bulls (>5 years), classified These bulls (14), were collected at two local abattoirs. were selected as high grade Brahman on phenotypic appearance and sampled at random, with no more than five animals from a single herd. Laboratory transported
656
were After collection the testes procedures: on ice to the laboratory and processed within 4
JUNE 1982 VOL. 17 NO. 6
THERIOGENOLOGY hours of slaughter. The epididymis was dissected from the testis and divided into head, body and tail by the same operator and the segments weighed. Care was taken to avoid sperm loss through leaking. The testis was freed of the pampiniform plexus and weighed before and after removal of the tunica albuginea and a representative subsample of approximately 25 g of testicular parenchyma was weighed. All samples were stored at -15'C until homogenization. Tissue homogenization was carried out according to the methods of Amann and Lambiase (15) with minor modifications to allow the efficient processing of large numbers of Preliminary tests showed that homogenization over samples. 2 minutes, subsample size greater than one ml and storage of 4 C for more than 24 hours had no effect on subsamples at final spermatid counts and therefore these minimum times and volumes were adopted. Spermatid numbers were determined from haemocytometer appropriate dilution to provide counts of counts after approximately 100 spermatids per chamber. Gonadal spermatid counts per gram testicular parenchyma were divided by a time divisor of 5.11 (16) to calculate daily sperm production Estimates rate per gram (DSPG). of DSP were derived by multiplying DSPG by 0.99 times testis parenchyma weight (mediastinal weight representing 1% of parenchymal weight). Epididymal sperm reserves were calculated from spermatozoa counts in epididymal homogenates. Statistical Analysis: The data were analyzed by least of variance with age, bodyweight, herd of squares analysis and breed as variables. For origin independent the was excluded from description of the data by age, bodyweight considered when data were the model, while age was not Analysis of variance of in bodyweight categories. analyzed variable breed effects included age as an independent and this being the model with the body-weight as a covariate, Data from mature bulls were analyzed least residual effect. a model corrected only for herd of origin, separately with previous since accurate information on age, bodyweight, and reproductive history was not available.
Results
combined testis 20% had a In bulls aged 1.5 years, of less than 100 grams and 30% had sperm reserves of weight However, data from these less than one billion spermatozoa. cases were considered these since included, were bulls were weights for this breed (11) and testis normal distributed continuously.
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THERIOGENOLOGY
1
Table
Least squares means for paired testicular and epididymal characteristics in B.OS ;bndicu strain bulls between 1.5 and 3.5 years of age. Data on mature bulls are included for comparison, but were analyzed separately
Age
(years)
Characteristics 1.5
Age
(days)
Bodyweight SC
(kg)
(cm)
Testis DSPG DSP
wt.
(g)
(~10~) (x10')
Epid. wt. (9) caput corpus cauda Epid. sperm res. (x10') caput corpus cauda Rel. sperm distribution caput corpus cauda Epid. transit time (days)* caput corpus cauda +
3.5
,5.0
23
33
33
575
874
1328
236.ga+
341.5h
498.gc
23.Ea
28.Zb
33.5c
210.3a
302.8'
473.2'
80
n
2.5
5.93a
9.1Eb
11.13h
8.03
1.4Ea
2.68'
5.01c
4.90
18.5Sa 10.53a 2.67a 5.34a
27.17b 1S.87b 3.77a 7.5Zb
42.09c 22.95c 6.14b 12.99=
60.82 33.56 8.81 18.45
6.36a 2.50a 0.34a 3.5Za
8.8ga 3.07a 0.8Za 5.07a
24.2433 8.lsh 1.79h 14.3oh
25.46 9.72 2.44 13.30
33.6 7.4 59.0
38.2 9.6 52.2
(%) 39.3 5.4 55.3
1.69 0.23 2.38
34.6 9.2 57.2
1.38 0.31 1.89
Means in the same row with different significantly (Pc.01) ++ Calculated after Amann _& ti. (17)
658
669.6
1.63 0.36 2.85
1.98 0.50 2.71
superscripts
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differ
1982 VOL. 17 NO. 6
THERIOGENOLOGY
(soil
satl
epne3
Epfdldymal
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1982 VOL. 17 NO. 6
i
sa’d
Wt
plds
/
dsa
ig)
659
THEFUOGENOLOGY Table
2
Correlation epididymal
coefficients characteristics
for testicular and within age groups
Age
(years)
Variables 1.5
2.5
3.5
80
24
33
wt. res.
.78*** .93x** .90*** .71***
.21 .79*x* .84*** .58***
.35* .87*** .77*** *69***
p-04 .62X"* .61*** .15
and DSP Epid. res. Caput res. Cauda res.
,88*** :76*** .69*** .77*x*
.75*x .39* .41* .34
.74*** .45** .43** .37*
.76*X* .03 -.ll .08
.78*x* .68+x* .80*X*
.61** .67*** .52**
.73*** .72*** .66***
.08 -.15 -.14
.80*"* .72*** .81***
*77*x* .86*** .67*X*
.73*** .81**" .64***
.12 -.09 -.21
_I_
15.0
~_-----_
n
Testis
wt.
DSPG DSP Epid. Epid.
33
and
DSPG
DSP and Epid. Caput Cauda Epid.
res. res. res.
wt.
and
Epid.
res.
Caput Cauda
res. res.
* (P<.O5) ** (PC.01) *** (P<.OOl)
Scrotal circumference in young bulls, 1.5 to 3.5 years, highly correlated with testis weight (.96), with DSP was (.89) and with bodyweight (.86). chronological Data on were with a development analyzed least squares model corrected for breed and property of Most origin. characteristics were significantly different (Pc.01) between Testis weight, DSP and groups (table 1). DSPG, age epidldymal weights and reserves progressively increased with age in the young bulls, although the increases In DSPG 3.5 years were smaller. Epididymal sperm between 2.5 and differ between but reserves did not 1.5 and 2.5 year. There were minor increases increased in 3.5 year old bulls. to total cauda reserves with age in the contribution of between reserves. Correlations sperm epididymal
660
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THERIOGENOLOGY
reproductive tract characteristics were DsP was only less correlated with (n.s.) and 3.5 (Pc.05) year old bulls.
high (table 2) and testis weight in 2.5
Analysis of the data after ranking into bulls bodyweight categories (100 kg range, e.g. 350 - 450) showed that testicular and epididymal weights increased in a linear with fashion increasing bodyweight (figure la). Maximum DSPG were values for reached at a bodyweight of 400 kg (figure while increased approximately DSP lb), moderately with increasing bodyweight. were marked There in reserves as increases epididymal sperm bodyweight increased from 200 to 700 kg (figure lb). Breed and genotype data were analyzed for corrected both weight and age. Between breed comparison (Brahman vs. Sahiwal) Indicated that Sahiwal bulls had significantly higher (Pc.001) values for testicular and epididymal weights significant and sperm reserves. There was no difference between genotypes (half vs. three-quarter breds) (table 3).
Table
Least squares means for breed and genotype effects on paired testicular and epididymal weights, DSPG, DSP and epididymal reserves of bulls aged 1.5 to 3.5 years of age.
3
Genotype Characteristic
n Testis DSPG DSP
wt.
(g)
(~10~) (~10~)
Epid.
wt.
(g)
Epid. res.
sperm (~10~)
3/4 Brahman
3/4 Sahiwal
l/2 Sahiwal
45
25
32
18
235.5a+
266.6ab
295.5h
330.2'
5.86a
7.57ah
g.o&
9.46b
1.88a
2.38ab
2.96h
3.o+
22.86a
23.69ah
25.9&
29.3ob
9.25a
9.71ah
14.59h
14.16h
common
superscript
+ Means in the same row without differ significantly (P<.OS)
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l/2 Brahman
1982 VOL. 17 NO. 6
661
I-HERIOGENOLOGY on gross examination the testes of mature were normal, morphologically DSPG was reduced to 8 as compared to 11 million in 3.5 year old bulls (table 1). The left testis was 5% heavier than the right in these animals. Analysis of the data from mature bulls indicated significant differences in DSP for bulls from
Although
bulls million
different billion.
herds, estimates ranging from 3.6 billion to 6.2 Testis weight was significantly correlated with epididymal weight (.61) and DSP (.62) (table Z), but little correlation existed between weights epididymal and epididymal sperm reserves. Sperm reserves in both testis and epididymis were similar to those of 3.5 year old bulls, but the testis and epididymal weights were heavier in mature bulls. The relative contribution of the cauda to the epididymal sperm reserve was smaller in mature bulls, but calculated epididymal transit time (17) was longer than in younger animals.
Discussion
Body weight was found to be better correlated to the stage of development of gonadal and extragonadal sperm reserves than was age. This weight dependence has to be viewed in relation to the large environmental differences these animals are to feed intake,
subjected to during development in regard climate and parasite burden. The patterns of development of DSP and EGR were found to be similar to that reported for Bes &a.~~3 bulls (13) with gonadal reserves being well established before extragonadal reserves. Comparable stages of development however were reached much later in animals in the present study. Sperm 16.5 billion numbers of 4.2 and for DSP and EGR respectively, reported in twelve months old dairy bulls had not been reached by 30 months in &.s ind,&&? (12), comparable strain bulls, a time when they had reached a bodyweight (340.5 vs 341.5 kg). The shift in the relative distribution of epididymal sperm numbers in favor of the cauda, storage portion storage
lends support to the suggestion that further capacity is still developing until 3.5 years; of the epididymis being the major site of (18).
sperm this sperm
weight The strong correlation between and within most in young bulls and the characteristics and sperm reserve adult animals these correlations in complete absence of (table 2) further suggests that reproductive growth is, with The rate the exception of DSPG, not completed by 3.5 years. of growth however varied and the relative increase in epididymal sperm numbers exceeded that of epididymal weight. DSP increased at the same rate as testis weight and reflects about 400 kg rates achieved at mature sperm production The delayed these BQS a genotypes. bodyweight in
662
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THERIOGENOLOGY development of EGR is also evident from the data in table 1. significant increases NO in epididymal sperm reserves occurred between 1.5 and 2.5 years, there but were significant increases between 2.5 and 3.5 years of age. The data from mature bulls not be compared could directly with that of the younger animals, for the reasons outlined. Inclusion of the data was considered warranted however, since no published information is available on EGR the breeds for examined here, and it was considered necessary to provide an estimate of the degree of maturity reached in 3.5 year old bulls. Fibrosis and epithelial occurs in testis and epididymis with advancing degeneration age (19) and the decline in DSPG from 11 to 8 million spermatozoa confirm that these observed per gram histological differences have a direct effect on sperm production rates, and perhaps on subsequent fertility.
Table
Testicular and epididymal characteristics and BQ~ faurus strain bulls
4
in EQS
lndlcus
Characteristic
n Age
range
Testis DSPG DSP
(years)
wt.
(g)
(x106) (X10')
Epid.
wt.
Epld.
res.
(g) (x10')
Rel.sperm distribution Caput Corpus Cauda 1) 2) 3) 4) 5) 6)
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Data Data Data Data Time Time
Holstein Friesianl
Hereford, Angus'
African Zebu3
Brahman cross 4
15
14
30
33
2-12
7
mature
>5
768
686
354
670
10.35
10.05
12.e6
8.06
7.2
6.4
4.0
4.9
84.4
68.1
44.0
60.8
61.8
41.0
12.6
25.5
31.4 7.6 61.0
31.1 3.7 65.2
21.4 6.4 72.2
38.2 9.6 52.2
(%)
calculated calculated calculated from mature divisor of divisor of
1982 VOL. 17 NO. 6
from Amann and Almquist (2) from Weisgold and Almquist (3) from Igboeli and Rakha (4) bulls in present study 5.32 days for EQS taurus bulls (20) bulls (16) 5.11 days for EQ8 w
663
THERIOGENOLOGY The similar (3), but
lndlcus breeds
average testis weight in the mature bulls was to that reported for mature &S taurus beef well above that reported for Central African
bulls (4) (table 4). Differences found in this study confirm earlier
very bulls w
within ELLS indicus work by Entwistle
(11) who reported greater testis weight and higher .& al. DSP in Sahiwal than in Brahman bulls. The apparent breed differences in DSPG found in this study (table 3) are, however, more likely to be a reflection of a different level testicular development than of evidence of an inherently lower sperm production rate in Brahman cross compared to Sahiwal cross bulls. There was a consistent, but not significant, trend within breeds in favor of the half grade genotypes over high grade animals. While epididymal weights were also similar to that of Hereford and Angus bulls (3) the epididymal sperm reserves were lower. Caution has to be exercised however in comparing data from different studies using different strains, and in interpreting these results since cauda storage is influenced by the sexual activity of the male (la), and in the present study the previous sexual status of the mature bulls prior to slaughter was not known. The relative contribution of the cauda reserve to total epididymal sperm reserve had decreased to compared to 52% 59% in rested 3.5 year old bulls, suggesting loss due to ejaculation. Additionally progressing fibrosis in these animals could reduce the distensibility of the cauda (16) and thus decrease storage capacity. for significant correlations found in mature bulls with DSP (.62) and with epididymal weight weight (.61) are in agreement with reports for dairy bulls (2) and E$.Qs taurus beef bulls (3). The absence, in this study, of a significant correlation between epididymal weight and epididymal sperm numbers in mature bulls confirms data for with data Angus and Hereford bulls (3). but is at variance Hence the absence from dairy (2) and Charolais bulls (3). ratio of weight to sperm numbers in the of a fixed The
testis
epididymis
would not enable a prediction of EGR from testis despite the good correlation as measured by SC, These findings are testis and epididymal weights. correlations between in the nonsignificant reflected Differences epididymal reserves and testis weight and DSP. in the rate of sperm resorption (21) may account for some of the variation in EGR and influence these correlations.
weight, between
days Transit time through the caput and corpus is 2.5 when calculated from DSP (17). and agrees with (table l), that calculated for dairy (17) and &S tauruS beef bulls the rate of passage through the The consistency in (3). proximal regions of the epididymis, which is the site of suggests that sperm maturation in the bull sperm maturation, is
time
dependent and occurs at a fixed rate, independent calculated estimates of the duration However passage are shorter than have been determined appearance of isotope labeled spermatozoa in
genotype. epididymal the first
664
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of of by the
1982 VOL. 17 NO. 6
THERIOGENOLOGY ejaculate
(22).
The results of this study indicate that some of the differences between &S w and &B indicus genotypes in reproductive capacity can be attributed to a slower rate of maturity in the latter, Weights of testis and epididymis in mature EQs LL&_Gus cross bulls were found to be similar to those reported for E@s m beef bulls while EGR was found to be lower and this factor could be contributing to a lowered fertility. The extremely low epididymal sperm numbers reported in Be9. &%~.!&z bulls by Igboeli and Rakha could not be confirmed and may reflect the different (4) techniques used to liberate the sperm. The results suggest that some of the reproductive deficiencies in m indicus bulls can be overcome by improving nutritional status to increase bodyweight gain and hence gonadal development, and exploiting breed differences within BQS i&&d8 by genotypes.
References
1.
Almquist,J.O. Amann,P.R. and Bull management to 6th Techn. maximize sperm output, Proc. Conf. Anim. Reprod. Artif. Insem., Milwaukee,l976.
2.
and Amann, R.P. Almquist, J.O. Reproductive capacity Gonadal and extragonadal reserves of dairy bulls. II. counts as determined by direct and depletion trials; dimensions and weight and genitalia, J. Dairy Sci. a: 1668-1678(1961).
3.
Almquist, J.O. Weisgold, A.D. and Reproductive beef bulls. IV. capacity of Daily spermatozoa1 reserves production, spermatozoa1 and dimensions and organs, J. reproductive Sci. 48: weight of Anim. 351-358 (1979).
4.
and Rakha, A.M. Gonadal Igboeli, G. reserves of indigenous Central sperm 107-109 (1971). Reprod. Fert. 25:
5.
Sources of variation Seebeck, R.M. zebu British and herd of zebu, agric. Northern Australia, J. 253-262 (1973).
6.
and Christensen, G.W., Bean, K.G. Seifert, Calving performance of reciprocally mated Africander Aust. Brahman crossbred cattle at Belmont, ProC. 62-64 (1980). Prod. U: Anim.
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1982 VOL. 17 NO. 6
extragonadal and African bulls, J.
in fertility of a x British cattle in Sci., Camb., 81:
H.R. and
sot .
665
THERIOGENOLOGY
7.
Seifert, G.W. Christensen, H.R. Endo, Y., and Differences in testicle shape and weight of different breeds of cattle at weaning, Proc. Anim. Aust. sot . Prod. Xi: 190 (1978).
8.
Chenoweth, P.J. Breed differences in and Osborne, H.G. abnormalities of the reproductive organs of young beef bulls, Aust. vet. J. 14: 463-467 (1978).
9.
Christensen, H.R., Seifert, G.W. and Parsonson, Onset of spermatogenesis in Ba$! indicus genotypes, Aust. sot. Anim. Prod. ;L3: 67-68 (1980).
I.M. Proc.
10.
Aire, T.A. and Akpokodje, in white Fulani (I~.QSm) 146-150 (1975). U:
11.
Entwistle, K.W., Winantea, A. and Holroyd, R.G. production rates in Bsa indicus strain bulls, Aust. sot . Anim. Prod. s: 68-70 (1980).
12.
MacMillan, K.L. and Hafs, H.D. Gonadal and extra gonadal sperm numbers during reproductive development of Holstein bulls, J. Anim. Sci. 22: 697-700 (1968).
13.
MacMillan, K.L., Newhook, c. and Watson, J.D. Testicular and epididymal sperm numbers in young Friesian bulls, N.Z. J. agric. Res. ;L5: 255-259 (1972).
14.
Ensminger, Interstate p.106.
15.
Amann, R.P. The male rabbit. III. and Lambiase, J.T. Determination of sperm production by means of daily Sci. 28: 369-374 testicular homogenates, J. Anim. (1969).
16.
Salim, B. Aspects of strain bulls, M.Sc. 1980, pp.73.
17.
and Pickett, Amann, R.P., Johnson, L., Thompson, D.L. epididymal production, Daily spermatozoa1 B.W. spermatozoa time of spermatozoa1 reserves and transit of the Rhesus monkey, Biol. through the epididymis 586-592 (1976). Reprod. L5:
18.
capacity Reproductive and Almquist, J.O. Amann, R.P. Effect of unilateral vasectomy and of dairy bulls. VI. aspects of reserves; sperm ejaculation frequency on 260-268 Fert. 2: Reprod. epididymal physiology, J. (1962).
666
J.U.
Development of puberty bull calf, Br. vet. J.
M.E. Beef Cattle Science, Printers and Publishers Inc.,
spermatogenesis Thesis, James
Sperm Proc.
4th ed., The Illinois, 1968,
in &S indicus Cook University,
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THERIOGENOLOGY
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19.
Humphrey, J.D. and Ladds, histological study of changes epididymis associated with age, 135-141 (1975).
20.
Amann, R.P., Kavanaugh, J.F., Griel, L.C. and Voglmayr, Sperm production of Holstein bulls determined from J.K. testicular spermatid reserves, after cannulation of the testis or vas deferens, and by daily ejaculation, rete Dairy Sci. z: 93-99 (1974). J.
21.
Crabo, B., Gustafsson, B., Nicander, L. and Rao, A.R. Subnormal testicular function in a bull concealed by phagocytosis of abnormal spermatozoa in the efferent ductules, J. Reprod. Fert. 24: 393-396 (1971).
22.
and Entwistle, K.W. The effect of scrotal Ross, A.D. spermatozoa1 morphology and the rates of insulation on spermatogenesis and epididymal passage of spermatozoa in the bull, Theriogenology ;u: 111-129 (1979).
1982 VOL. 17 NO. 6
P.W. in the Res.
A quantitative bovine testis and vet. Sci. U:
667
POSTPUBERTAL
CHANGES
RESERVES
IN
GONADAL
IN EQS
INDICUS
AND
EXTRAGONADAL
STRAIN
SPERM
BULLS
S. Wildeus and K.W. Entwistle Department of Tropical Veterinary Science James Cook University of North Queensland Townsville, QLD. 4811 Australia
Received
for
vublication: Accepted:
Januarv 4. 1982 April 1, i982
Abstract Testes and epididymides were collected from 136 young (1.5 to 3.5 years of age) and 33 mature &,s J_ndlcus strain bulls, reared under extensive conditions in range the tropics. Testicular and epididymal sperm reserves and different rates in the weights increased at Daily sperm production per young bulls. gram (DSPG) increased of testis significantly before and epididymal sperm reserves after 2.5 Testicular years of age. and epididymal weights increased linearly with bodyweight. cross bulls had higher testicular and Sahiwal epididymal weights, and greater sperm reserves did Brahman cross bulls at the same age. than Testicular and epididymal weights, daily sperm epididymal sperm reserves for production and Brahman bulls were 67Og, mature 61g. 4.9 billion and 25.5 billion respectively. Testis weight was highly correlated with total daily sperm
production
(DSP)
and
epididymal
weight.
Introduction
upon Beef cattle reproductive performance is dependent female and male and thus the from both the contributions herd sex influence fertility in either level of may
Acknowledgements: Queensland Department of Primary Holroyd, thank Mr R. We and Mr H.R. Research Station Lagoon" "Swan's Industries for access to Research Station "Belmont" Christensen, CSIRO Miss J. The technical assistance of material. experimental These acknowledged. Bradshaw is gratefully Prideaux and Mr I. from the Australian Meat financed by a grant studies were Research
JUNE
Commitee.
1982 VOL. 17 NO. 6
655
THERIOGENOLOGY performance and hence economic returns in a beef cattle enterprise. Estimates spermatozoa1 production of daily (DSP) and extra-gonadal sperm reserves (EGR) are essential for the efficient utilization of available sperm resources (1) and have been determined for &s w dairy (2) and beef bulls (3) and Br;a e bulls (4). Seebeck (5) and Seifert .& a. (6) indicated in studies done in Central Queensland that &S i&&&u,8 bulls showed reduced fertility when compared to contemporary m taurus bulls. This reduced fertility may be a reflection of smaller testicular size higher proportion of (7), reproductive tract abnormalities, particularly testicular hypoplasia (8), delayed onset of puberty (9,lO) and smaller gonadal (11) and epididymal sperm reserves (4). It is not clear however to what extent these factors are inherent to this genotype or are caused by seasonal periods of undernutrition, internal and external parasite burdens, and a slower rate of maturity. The development of DSP precedes the establishment of EGR in Friesian bulls (12.13) and only small numbers of spermatozoa are available for ejaculation in young bulls. The delayed reproductive development occurs together with a slow rate of maturity in m indicus bulls, it was necessary to determine whether a combination of both may be contributing to the fertility reduced in &S indicus strains. Hence the establishment of gonadal and extra gonadal sperm reserves in half and three-quarter ins indicus strain bulls was examined in this study.
Materials
and Methods
Animals: Material for this study was obtained from clinically normal E&8 indicus strain bulls which had been raised on Bulls native pasture in tropical Australia. between 1.5 and 3.5 years of age originated either from the Lagoon" Queensland Department of Primary Industries "Swan's Long 147 E) or from the CSIRO Research Station (Lat 19 S; National Cattle Breeding Station "Belmont" (Lat 23 S; Long 151 E) and detailed production records (growth rates and circumference (SC)) reproductive traits, including scrotal Testes and epididymides were available. on these animals castration. Genitalia from were obtained at slaughter or mature as such from dentition bulls (>5 years), classified These bulls (14), were collected at two local abattoirs. were selected as high grade Brahman on phenotypic appearance and sampled at random, with no more than five animals from a single herd. Laboratory transported
656
were After collection the testes procedures: on ice to the laboratory and processed within 4
JUNE 1982 VOL. 17 NO. 6
THERIOGENOLOGY hours of slaughter. The epididymis was dissected from the testis and divided into head, body and tail by the same operator and the segments weighed. Care was taken to avoid sperm loss through leaking. The testis was freed of the pampiniform plexus and weighed before and after removal of the tunica albuginea and a representative subsample of approximately 25 g of testicular parenchyma was weighed. All samples were stored at -15'C until homogenization. Tissue homogenization was carried out according to the methods of Amann and Lambiase (15) with minor modifications to allow the efficient processing of large numbers of Preliminary tests showed that homogenization over samples. 2 minutes, subsample size greater than one ml and storage of 4 C for more than 24 hours had no effect on subsamples at final spermatid counts and therefore these minimum times and volumes were adopted. Spermatid numbers were determined from haemocytometer appropriate dilution to provide counts of counts after approximately 100 spermatids per chamber. Gonadal spermatid counts per gram testicular parenchyma were divided by a time divisor of 5.11 (16) to calculate daily sperm production Estimates rate per gram (DSPG). of DSP were derived by multiplying DSPG by 0.99 times testis parenchyma weight (mediastinal weight representing 1% of parenchymal weight). Epididymal sperm reserves were calculated from spermatozoa counts in epididymal homogenates. Statistical Analysis: The data were analyzed by least of variance with age, bodyweight, herd of squares analysis and breed as variables. For origin independent the was excluded from description of the data by age, bodyweight considered when data were the model, while age was not Analysis of variance of in bodyweight categories. analyzed variable breed effects included age as an independent and this being the model with the body-weight as a covariate, Data from mature bulls were analyzed least residual effect. a model corrected only for herd of origin, separately with previous since accurate information on age, bodyweight, and reproductive history was not available.
Results
combined testis 20% had a In bulls aged 1.5 years, of less than 100 grams and 30% had sperm reserves of weight However, data from these less than one billion spermatozoa. cases were considered these since included, were bulls were weights for this breed (11) and testis normal distributed continuously.
JUNE
1982 VOL. 17 NO. 6
657
THERIOGENOLOGY
1
Table
Least squares means for paired testicular and epididymal characteristics in B.OS ;bndicu strain bulls between 1.5 and 3.5 years of age. Data on mature bulls are included for comparison, but were analyzed separately
Age
(years)
Characteristics 1.5
Age
(days)
Bodyweight SC
(kg)
(cm)
Testis DSPG DSP
wt.
(g)
(~10~) (x10')
Epid. wt. (9) caput corpus cauda Epid. sperm res. (x10') caput corpus cauda Rel. sperm distribution caput corpus cauda Epid. transit time (days)* caput corpus cauda +
3.5
,5.0
23
33
33
575
874
1328
236.ga+
341.5h
498.gc
23.Ea
28.Zb
33.5c
210.3a
302.8'
473.2'
80
n
2.5
5.93a
9.1Eb
11.13h
8.03
1.4Ea
2.68'
5.01c
4.90
18.5Sa 10.53a 2.67a 5.34a
27.17b 1S.87b 3.77a 7.5Zb
42.09c 22.95c 6.14b 12.99=
60.82 33.56 8.81 18.45
6.36a 2.50a 0.34a 3.5Za
8.8ga 3.07a 0.8Za 5.07a
24.2433 8.lsh 1.79h 14.3oh
25.46 9.72 2.44 13.30
33.6 7.4 59.0
38.2 9.6 52.2
(%) 39.3 5.4 55.3
1.69 0.23 2.38
34.6 9.2 57.2
1.38 0.31 1.89
Means in the same row with different significantly (Pc.01) ++ Calculated after Amann _& ti. (17)
658
669.6
1.63 0.36 2.85
1.98 0.50 2.71
superscripts
JUNE
differ
1982 VOL. 17 NO. 6
THERIOGENOLOGY
(soil
satl
epne3
Epfdldymal
JUNE
1982 VOL. 17 NO. 6
i
sa’d
Wt
plds
/
dsa
ig)
659
THEFUOGENOLOGY Table
2
Correlation epididymal
coefficients characteristics
for testicular and within age groups
Age
(years)
Variables 1.5
2.5
3.5
80
24
33
wt. res.
.78*** .93x** .90*** .71***
.21 .79*x* .84*** .58***
.35* .87*** .77*** *69***
p-04 .62X"* .61*** .15
and DSP Epid. res. Caput res. Cauda res.
,88*** :76*** .69*** .77*x*
.75*x .39* .41* .34
.74*** .45** .43** .37*
.76*X* .03 -.ll .08
.78*x* .68+x* .80*X*
.61** .67*** .52**
.73*** .72*** .66***
.08 -.15 -.14
.80*"* .72*** .81***
*77*x* .86*** .67*X*
.73*** .81**" .64***
.12 -.09 -.21
_I_
15.0
~_-----_
n
Testis
wt.
DSPG DSP Epid. Epid.
33
and
DSPG
DSP and Epid. Caput Cauda Epid.
res. res. res.
wt.
and
Epid.
res.
Caput Cauda
res. res.
* (P<.O5) ** (PC.01) *** (P<.OOl)
Scrotal circumference in young bulls, 1.5 to 3.5 years, highly correlated with testis weight (.96), with DSP was (.89) and with bodyweight (.86). chronological Data on were with a development analyzed least squares model corrected for breed and property of Most origin. characteristics were significantly different (Pc.01) between Testis weight, DSP and groups (table 1). DSPG, age epidldymal weights and reserves progressively increased with age in the young bulls, although the increases In DSPG 3.5 years were smaller. Epididymal sperm between 2.5 and differ between but reserves did not 1.5 and 2.5 year. There were minor increases increased in 3.5 year old bulls. to total cauda reserves with age in the contribution of between reserves. Correlations sperm epididymal
660
JUNE
1982 VOL. 17 NO. 6
THERIOGENOLOGY
reproductive tract characteristics were DsP was only less correlated with (n.s.) and 3.5 (Pc.05) year old bulls.
high (table 2) and testis weight in 2.5
Analysis of the data after ranking into bulls bodyweight categories (100 kg range, e.g. 350 - 450) showed that testicular and epididymal weights increased in a linear with fashion increasing bodyweight (figure la). Maximum DSPG were values for reached at a bodyweight of 400 kg (figure while increased approximately DSP lb), moderately with increasing bodyweight. were marked There in reserves as increases epididymal sperm bodyweight increased from 200 to 700 kg (figure lb). Breed and genotype data were analyzed for corrected both weight and age. Between breed comparison (Brahman vs. Sahiwal) Indicated that Sahiwal bulls had significantly higher (Pc.001) values for testicular and epididymal weights significant and sperm reserves. There was no difference between genotypes (half vs. three-quarter breds) (table 3).
Table
Least squares means for breed and genotype effects on paired testicular and epididymal weights, DSPG, DSP and epididymal reserves of bulls aged 1.5 to 3.5 years of age.
3
Genotype Characteristic
n Testis DSPG DSP
wt.
(g)
(~10~) (~10~)
Epid.
wt.
(g)
Epid. res.
sperm (~10~)
3/4 Brahman
3/4 Sahiwal
l/2 Sahiwal
45
25
32
18
235.5a+
266.6ab
295.5h
330.2'
5.86a
7.57ah
g.o&
9.46b
1.88a
2.38ab
2.96h
3.o+
22.86a
23.69ah
25.9&
29.3ob
9.25a
9.71ah
14.59h
14.16h
common
superscript
+ Means in the same row without differ significantly (P<.OS)
JUNE
l/2 Brahman
1982 VOL. 17 NO. 6
661
I-HERIOGENOLOGY on gross examination the testes of mature were normal, morphologically DSPG was reduced to 8 as compared to 11 million in 3.5 year old bulls (table 1). The left testis was 5% heavier than the right in these animals. Analysis of the data from mature bulls indicated significant differences in DSP for bulls from
Although
bulls million
different billion.
herds, estimates ranging from 3.6 billion to 6.2 Testis weight was significantly correlated with epididymal weight (.61) and DSP (.62) (table Z), but little correlation existed between weights epididymal and epididymal sperm reserves. Sperm reserves in both testis and epididymis were similar to those of 3.5 year old bulls, but the testis and epididymal weights were heavier in mature bulls. The relative contribution of the cauda to the epididymal sperm reserve was smaller in mature bulls, but calculated epididymal transit time (17) was longer than in younger animals.
Discussion
Body weight was found to be better correlated to the stage of development of gonadal and extragonadal sperm reserves than was age. This weight dependence has to be viewed in relation to the large environmental differences these animals are to feed intake,
subjected to during development in regard climate and parasite burden. The patterns of development of DSP and EGR were found to be similar to that reported for Bes &a.~~3 bulls (13) with gonadal reserves being well established before extragonadal reserves. Comparable stages of development however were reached much later in animals in the present study. Sperm 16.5 billion numbers of 4.2 and for DSP and EGR respectively, reported in twelve months old dairy bulls had not been reached by 30 months in &.s ind,&&? (12), comparable strain bulls, a time when they had reached a bodyweight (340.5 vs 341.5 kg). The shift in the relative distribution of epididymal sperm numbers in favor of the cauda, storage portion storage
lends support to the suggestion that further capacity is still developing until 3.5 years; of the epididymis being the major site of (18).
sperm this sperm
weight The strong correlation between and within most in young bulls and the characteristics and sperm reserve adult animals these correlations in complete absence of (table 2) further suggests that reproductive growth is, with The rate the exception of DSPG, not completed by 3.5 years. of growth however varied and the relative increase in epididymal sperm numbers exceeded that of epididymal weight. DSP increased at the same rate as testis weight and reflects about 400 kg rates achieved at mature sperm production The delayed these BQS a genotypes. bodyweight in
662
JUNE
1982 VOL. 17 NO. 6
THERIOGENOLOGY development of EGR is also evident from the data in table 1. significant increases NO in epididymal sperm reserves occurred between 1.5 and 2.5 years, there but were significant increases between 2.5 and 3.5 years of age. The data from mature bulls not be compared could directly with that of the younger animals, for the reasons outlined. Inclusion of the data was considered warranted however, since no published information is available on EGR the breeds for examined here, and it was considered necessary to provide an estimate of the degree of maturity reached in 3.5 year old bulls. Fibrosis and epithelial occurs in testis and epididymis with advancing degeneration age (19) and the decline in DSPG from 11 to 8 million spermatozoa confirm that these observed per gram histological differences have a direct effect on sperm production rates, and perhaps on subsequent fertility.
Table
Testicular and epididymal characteristics and BQ~ faurus strain bulls
4
in EQS
lndlcus
Characteristic
n Age
range
Testis DSPG DSP
(years)
wt.
(g)
(x106) (X10')
Epid.
wt.
Epld.
res.
(g) (x10')
Rel.sperm distribution Caput Corpus Cauda 1) 2) 3) 4) 5) 6)
JUNE
Data Data Data Data Time Time
Holstein Friesianl
Hereford, Angus'
African Zebu3
Brahman cross 4
15
14
30
33
2-12
7
mature
>5
768
686
354
670
10.35
10.05
12.e6
8.06
7.2
6.4
4.0
4.9
84.4
68.1
44.0
60.8
61.8
41.0
12.6
25.5
31.4 7.6 61.0
31.1 3.7 65.2
21.4 6.4 72.2
38.2 9.6 52.2
(%)
calculated calculated calculated from mature divisor of divisor of
1982 VOL. 17 NO. 6
from Amann and Almquist (2) from Weisgold and Almquist (3) from Igboeli and Rakha (4) bulls in present study 5.32 days for EQS taurus bulls (20) bulls (16) 5.11 days for EQ8 w
663
THERIOGENOLOGY The similar (3), but
lndlcus breeds
average testis weight in the mature bulls was to that reported for mature &S taurus beef well above that reported for Central African
bulls (4) (table 4). Differences found in this study confirm earlier
very bulls w
within ELLS indicus work by Entwistle
(11) who reported greater testis weight and higher .& al. DSP in Sahiwal than in Brahman bulls. The apparent breed differences in DSPG found in this study (table 3) are, however, more likely to be a reflection of a different level testicular development than of evidence of an inherently lower sperm production rate in Brahman cross compared to Sahiwal cross bulls. There was a consistent, but not significant, trend within breeds in favor of the half grade genotypes over high grade animals. While epididymal weights were also similar to that of Hereford and Angus bulls (3) the epididymal sperm reserves were lower. Caution has to be exercised however in comparing data from different studies using different strains, and in interpreting these results since cauda storage is influenced by the sexual activity of the male (la), and in the present study the previous sexual status of the mature bulls prior to slaughter was not known. The relative contribution of the cauda reserve to total epididymal sperm reserve had decreased to compared to 52% 59% in rested 3.5 year old bulls, suggesting loss due to ejaculation. Additionally progressing fibrosis in these animals could reduce the distensibility of the cauda (16) and thus decrease storage capacity. for significant correlations found in mature bulls with DSP (.62) and with epididymal weight weight (.61) are in agreement with reports for dairy bulls (2) and E$.Qs taurus beef bulls (3). The absence, in this study, of a significant correlation between epididymal weight and epididymal sperm numbers in mature bulls confirms data for with data Angus and Hereford bulls (3). but is at variance Hence the absence from dairy (2) and Charolais bulls (3). ratio of weight to sperm numbers in the of a fixed The
testis
epididymis
would not enable a prediction of EGR from testis despite the good correlation as measured by SC, These findings are testis and epididymal weights. correlations between in the nonsignificant reflected Differences epididymal reserves and testis weight and DSP. in the rate of sperm resorption (21) may account for some of the variation in EGR and influence these correlations.
weight, between
days Transit time through the caput and corpus is 2.5 when calculated from DSP (17). and agrees with (table l), that calculated for dairy (17) and &S tauruS beef bulls the rate of passage through the The consistency in (3). proximal regions of the epididymis, which is the site of suggests that sperm maturation in the bull sperm maturation, is
time
dependent and occurs at a fixed rate, independent calculated estimates of the duration However passage are shorter than have been determined appearance of isotope labeled spermatozoa in
genotype. epididymal the first
664
JUNE
of of by the
1982 VOL. 17 NO. 6
THERIOGENOLOGY ejaculate
(22).
The results of this study indicate that some of the differences between &S w and &B indicus genotypes in reproductive capacity can be attributed to a slower rate of maturity in the latter, Weights of testis and epididymis in mature EQs LL&_Gus cross bulls were found to be similar to those reported for E@s m beef bulls while EGR was found to be lower and this factor could be contributing to a lowered fertility. The extremely low epididymal sperm numbers reported in Be9. &%~.!&z bulls by Igboeli and Rakha could not be confirmed and may reflect the different (4) techniques used to liberate the sperm. The results suggest that some of the reproductive deficiencies in m indicus bulls can be overcome by improving nutritional status to increase bodyweight gain and hence gonadal development, and exploiting breed differences within BQS i&&d8 by genotypes.
References
1.
Almquist,J.O. Amann,P.R. and Bull management to 6th Techn. maximize sperm output, Proc. Conf. Anim. Reprod. Artif. Insem., Milwaukee,l976.
2.
and Amann, R.P. Almquist, J.O. Reproductive capacity Gonadal and extragonadal reserves of dairy bulls. II. counts as determined by direct and depletion trials; dimensions and weight and genitalia, J. Dairy Sci. a: 1668-1678(1961).
3.
Almquist, J.O. Weisgold, A.D. and Reproductive beef bulls. IV. capacity of Daily spermatozoa1 reserves production, spermatozoa1 and dimensions and organs, J. reproductive Sci. 48: weight of Anim. 351-358 (1979).
4.
and Rakha, A.M. Gonadal Igboeli, G. reserves of indigenous Central sperm 107-109 (1971). Reprod. Fert. 25:
5.
Sources of variation Seebeck, R.M. zebu British and herd of zebu, agric. Northern Australia, J. 253-262 (1973).
6.
and Christensen, G.W., Bean, K.G. Seifert, Calving performance of reciprocally mated Africander Aust. Brahman crossbred cattle at Belmont, ProC. 62-64 (1980). Prod. U: Anim.
JUNE
1982 VOL. 17 NO. 6
extragonadal and African bulls, J.
in fertility of a x British cattle in Sci., Camb., 81:
H.R. and
sot .
665
THERIOGENOLOGY
7.
Seifert, G.W. Christensen, H.R. Endo, Y., and Differences in testicle shape and weight of different breeds of cattle at weaning, Proc. Anim. Aust. sot . Prod. Xi: 190 (1978).
8.
Chenoweth, P.J. Breed differences in and Osborne, H.G. abnormalities of the reproductive organs of young beef bulls, Aust. vet. J. 14: 463-467 (1978).
9.
Christensen, H.R., Seifert, G.W. and Parsonson, Onset of spermatogenesis in Ba$! indicus genotypes, Aust. sot. Anim. Prod. ;L3: 67-68 (1980).
I.M. Proc.
10.
Aire, T.A. and Akpokodje, in white Fulani (I~.QSm) 146-150 (1975). U:
11.
Entwistle, K.W., Winantea, A. and Holroyd, R.G. production rates in Bsa indicus strain bulls, Aust. sot . Anim. Prod. s: 68-70 (1980).
12.
MacMillan, K.L. and Hafs, H.D. Gonadal and extra gonadal sperm numbers during reproductive development of Holstein bulls, J. Anim. Sci. 22: 697-700 (1968).
13.
MacMillan, K.L., Newhook, c. and Watson, J.D. Testicular and epididymal sperm numbers in young Friesian bulls, N.Z. J. agric. Res. ;L5: 255-259 (1972).
14.
Ensminger, Interstate p.106.
15.
Amann, R.P. The male rabbit. III. and Lambiase, J.T. Determination of sperm production by means of daily Sci. 28: 369-374 testicular homogenates, J. Anim. (1969).
16.
Salim, B. Aspects of strain bulls, M.Sc. 1980, pp.73.
17.
and Pickett, Amann, R.P., Johnson, L., Thompson, D.L. epididymal production, Daily spermatozoa1 B.W. spermatozoa time of spermatozoa1 reserves and transit of the Rhesus monkey, Biol. through the epididymis 586-592 (1976). Reprod. L5:
18.
capacity Reproductive and Almquist, J.O. Amann, R.P. Effect of unilateral vasectomy and of dairy bulls. VI. aspects of reserves; sperm ejaculation frequency on 260-268 Fert. 2: Reprod. epididymal physiology, J. (1962).
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Development of puberty bull calf, Br. vet. J.
M.E. Beef Cattle Science, Printers and Publishers Inc.,
spermatogenesis Thesis, James
Sperm Proc.
4th ed., The Illinois, 1968,
in &S indicus Cook University,
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1982 VOL. 17 NO. 6
THERIOGENOLOGY
JUNE
19.
Humphrey, J.D. and Ladds, histological study of changes epididymis associated with age, 135-141 (1975).
20.
Amann, R.P., Kavanaugh, J.F., Griel, L.C. and Voglmayr, Sperm production of Holstein bulls determined from J.K. testicular spermatid reserves, after cannulation of the testis or vas deferens, and by daily ejaculation, rete Dairy Sci. z: 93-99 (1974). J.
21.
Crabo, B., Gustafsson, B., Nicander, L. and Rao, A.R. Subnormal testicular function in a bull concealed by phagocytosis of abnormal spermatozoa in the efferent ductules, J. Reprod. Fert. 24: 393-396 (1971).
22.
and Entwistle, K.W. The effect of scrotal Ross, A.D. spermatozoa1 morphology and the rates of insulation on spermatogenesis and epididymal passage of spermatozoa in the bull, Theriogenology ;u: 111-129 (1979).
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