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Gonadal and extragonadal sperm reserves and testicular histometric characteristics in Zebu and crossbred bulls: Effect of dry season nutritional supplementation
AnimalReproduction Science, 29 (1992) 25-33
25
Elsevier Science Publishers B.V., Amsterdam
Gonadal and extragonadal sperm reserves and testicular histometric characteristics in Zebu and crossbred bulls: effect of dry season nutritional supplementation A z a g e T e g e g n e a,b, K . W . E n t w i s f l e b a n d E. M u k a s a - M u g e r w a a
alnternational Livestock Centre for Africa, P.O. Box 5689, ,4ddis Ababa, Ethiopia bGraduate School of Tropical Veterinary Science and Agriculture, James Cook University, Townsville, Qld, 481L Australia (Accepted 28 November 1991 )
ABSTRACT Tegegne, A., Entwistle, K.W. and Mukasa-Mugetwa, E., 1992. Gonadal and extragonadal sperm reserves and testicular histometric characteristics in Zebu and crossbred bulls: effect of dry season nutritional s~applementation. Anim. Reprod. Sci., 29: 25-33. A total of 64 bull calves, composed of 27 Boran and 37 Boran×Friesian, were weaned at 6 months of age and divided into supplement or control groups within breed. All bulls were kept in one paddock and grazed on natural pasture. Supplement groups received 1.5 kg per head day- 1of a diet containing 16% crude protein and 8 MJ kg- I dry matter for two dry seasons until castrated at 21 months of age. Testes weight was heavier in supplemented than in control bulls (285 vs. 242 g ) and in Boran X Friesian than in Boran bulls (300 vs. 215 g). Epididymal weight differed between supplemented and control bulls (30.4 vs. 25.1 g) and between Boran and Boran × Friesian (22.1 vs. 31.9 g) bulls. Supplemented bulls had 27% greater daily sperm production (2.65 X 109 vs. 2.08 × 109) and 36% greater epididymal sperm reserves (4.05 X 109 vs. 2.99 × 109) than control bulls. Sperm reserves in the caput and cauda segments were also greater in supplemented than in control bulls. BoranxFriesian bulls had 32% greater daily sperm production ( 2.63 X 109 vs. 1.99 × 109) than Boran bulls, but epididymal sperm reserves did not differ between the two breeds. Seminiferous tubule diameter was larger by 9% in supplemented than in control and by 14% in Boran X Friesian than in Boran bulls. Supplementary feeding to young Boran and Boran × Friesian bulls during dry seasons resulted in heavier testes and epididymis weights, larger seminiferous tubule diameter and higher gonadal and extragonadal sperm reserves.
INTRODUCTION T h e p u r p o s e o f p r o p e r m a n a g e m e n t o f a y o u n g b u l l is t o i m p r o v e g r o w t h r a t e a n d t o e n a b l e it t o p r o d u c e o p t i m u m l e v e l s o f g o o d q u a l i t y s e m e n a t t h e
Correspondence to."A. Tegegne, International Livestock Centre for Africa, P.O. Box 5689, Addis Ababa, Ethiopia.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0378-4320/92/$05.00
26
A. TEGEGNE ET AL.
earliest age possible. The amount and efficiency of sperm producing testicular parenchymal tissue play an important role in influencing the amount and quality of spermatozoa produced by a bull. Bos indicus bulls have been reported to have lower fertility than Bos taurus bulls (Seifertet al., 1980) and this is attributed to smaller testis size (Entwistle, 1983) and fewer gonadal and extragonadal sperm reserves (Igboeli and Raldaa, 1971; Entwistle et al., 1980; Wildeus and Entwistle, 1982 ). However, none of these studies has directly dealt with the effects of supplementary feeding on reproductive potential of young bulls. The most likely occurrence of nutritional deficiency in cattle in tropical regions is during the dry season. Strategic intervention to avert this adverse effect, with supplementary feeding, is likely to have its greatest impact during this period. This study was designed to investigate the influence of dry season nutritional supplementation on gonadal and eXtragonadal sperm reserves and on histometric characteristics of testicular and epididymal tissues in young Boran and Boran × Friesian bulls in Ethiopia. MATERIALS AND METHODS
The study was undertaken at Abernossa ranch in Ethiopia and involved 2 7 Boran and 37 Boran × Friesian bulls. Immediately after weaning at 6 months of age, bulls were divided randomly to either receive a supplement ration or to serve as controls with grazing onlyl Supplemented and control bulls were identified with ear tags of different colours, kept in one paddock and grazed on natural pasture. The predominant grass species were Hyparrhenia rufa, Chlorius gayanaand Cynodon dactylon. Bulls had access to clean w a t e r a n d mineral licks. Supplemented groups were drafted into an enclosure and provided with 1.5 kg per head d a y - 1of aconcentrate mixture composed of wheat bran and Noug cake (Guizotia abyssinica) at a ratio of 2: 1. The mixture contained 16% crude protein and provided 8 MJ kg- 1 dry matter. Supplementary feeding was restricted to two dry seasons of 6 months duration each, December-May and October-March, divided by a wet season of 4 months when all bulls were offered grazing only. Body weight and scrotal circumference (SC) measurements were recorded when bulls were castrated at the end of the second supplementation period at an average age of 21 months. Each testis was dissected, the epididymis separated and divided into caput, corpus and cauda segments. Testis weight and epididymal segment weights were recorded, Testicular parenchymal samples and epididymal segments were taken from the right testis and kept frozen until use for determination of daily sperm production (DSP) and epididymal sperm reserves (ESR) by the homogenization technique (Almquist and Amann, 1961; Amann and Lambiase, 1969). Samples were also taken from the left testis and epididymal
SUPPLEMENTARY NUTRITION AND SPERM PRODUCTION IN BULLS
27
segments and were transferred into Bouin's fixative. The fixed samples were routinely processed, cut at 7/tm and stained with hematoxylin and eosin for histometric determinations. Seminiferous tubule diameter (STD), epididymal tubule diameter and epithelial height measurements were taken from 20 circular tubules on each animal. Data on body and testicular traits were analyzed by analysis of variance using the General Linear Models (GLM) procedure of the Statistical Analysis System (SAS, 1987) with nutrition, genotype and their interactions included in the model. RESULTS
Results on body weight, testis and epididymis weights are presented in Table 1. At castration, supplemented bulls were heavier (41 kg) than control bulls (P<0.001) while Boran×Friesian were heavier (56 kg) than Boran bulls (P< 0.001 ). A difference of 19% in body weight between supplemented and control bulls was associated with an 18% increase in paired testes weight. Paired testes and epididymides were significantly (P< 0.01 ) heavier by 18 and 21% in supplemented than in control groups, respectively. The difference in epididymal weight was due to differences in caput and cauda segments. Paired testes and epididymal weights were heavier by 40 and 46%, respectively in Boran×Friesian than in Boran bulls. A difference of 28% in body weight between Boran X Friesian and Boran bulls resulted in a 40% difference in paired testes weight. There were also significant (P< 0.001 ) differences in all epididymal segment weights between the two breeds. TABLE1 Body weight, paired testes and epididymal weights in Boran and Boran X Friesian (Bo X Fr) bulls reared with or without dry season supplementation 1 Treatments
n
Body weight (kg)
Testes weight (g)
Epididymal weight (g) Total
Caput
Corpus
Cauda
Breed Boran BoXFr
27 35
203+6 259-+5
215+14 300+ 11
22+1 32-+ 1
12-+0.8 18-+0.6
3.9-+0.3 5.3_+0.3
6.0+0.4 8.9+0.4
Nutrition Supplement Control
30 32
251+5 210_+5
285+12 242_+ 16
30+1 25_+ 1
17+0.8 14_+0.8
5,0+0.3 4.4_+0.3
8.6_+0.5 6.7_+0.4
~Entries are means + standard error. **P<0.01; ***P< 0.001.
28
A. TEGEGNE ET AL.
Data on gonadal and extragonadal sperm reserves are presented in Table 2. Neither supplementary feeding nor breed differences influenced daily sperm production per gram (DSPG). DSP, being dependent on testes size, however, was significantly ( P < 0.05 ) higher by 27% in supplemented than in control bulls. ESR were significantly ( P < 0.01 ) higher by 36% in supplemented than in control bulls. Supplemented bulls also had 28 and 51% higher sperm reserves in the caput and cauda segments than control bulls, respectively. Sperm contributions of the cauda segment to epididymal sperm reserves were 52 and 47% in supplemented and control bulls, respectively. Boran× Friesian had 32% higher DSP than Boran bulls (P<0.01). There were no differences in ESR between the two breeds. The sperm contribution of the cauda epididymis to ESR was about 50% in both breeds. Results on histometric characteristics are presented in Table 3. Mean STD was 9% larger in supplemented than in control bulls. Supplemented bulls also had larger caput, corpus and cauda tubule diameters than control bulls. However, epithelial heights in all segments were not affected by supplementation. Boran×Friesian bulls had significantly larger (P<0.001) STD, caput and corpus segment tubule diameters and epithelial height measurements than Boran bulls. Within breed correlation coefficients between body weight and scrotal circumference, body weight and testes weight, scrotal circumference and testes TABLE 2
Daily sperm production per gram ( D S P G ) , daily sperm production ( D S P ) and epididymal sperm reserves (ESR) in Boran and Boran×Friesian ( B o X F r ) bulls reared with or without dry season supplementation ~ Variables
D S P G ( × 104) DSP ( × 107) ESR ( × 107) Caput ( X 107)
Corpus(X10 7) 107)
Cauda ( ×
Nutrition
Breed
Supplement (n=30)
Control (n=32)
Boran (n=27)
Bo × Fr (n=35)
929 + 33 265 ___18 405 + 26 159+ 11 35 + 4 211 _+ 15
823 + 42 208 + 19* 299 + 28** 124+ 12" 32+ 5 140+ 14"*
887 + 27 199 _+ 19 320 -+ 19 130_+ 8 25_+ 4 161 + 10
870 + 44 263 + 19** 375 + 32 149_+ 14 40+ 5* 186_+ 19
Relative epididymal sperm distribution (%) Caput Corpus Cauda
40 9 51
1Entries are mean + standard error. *P<0.05; **P< 0.01.
42 11 47
41 9 50
41 11 48
SUPPLEMENTARY NUTRITION AND SPERM PRODUCTION IN BULLS
29
TABLE3 Least squares means ( + standard error) for histometric measurements of seminiferous tubule diameter, epididymal segment tubules and epithelial heights in Boran and Boran X Friesian (Bo X Fr) bulls reared with or without dry season supplementation Variables
Nutrition
Seminiferous tubule diameter ( a m )
Breed
Supplement (n=30)
Control (n=32)
Boran (n=27)
Bo × Fr (n=35)
209 + 4
191_+ 5**
186_+ 5
213_+ 4***
368+ 8 66_+ 1
346+12 63_+ 2
332___11 61_+ 2
375_+ 8*** 68_+ 1"**
340_+ 9 75_+ 1
308_+ 9** 75+ 2
300_+11 70_+ 2
343_+ 7*** 79_+ 1"**
538+20 54_+ 2
482_+22 51+ 2
486_+22 53+ 2
528_+21 52+ 2
Epididymis (am) Caput Diameter(/tm) Epithelialheight(#m) Corpus Diameter(/lm) Epithelialheight ( a m ) Cauda Diameter ( a m ) Epithelialheight ( # m ) **P< 0.01 ; ***P< 0.001. TABLE4 Correlation coefficients between body weight (BWT), scrotal circumference (SC), paired testes weight ( P T W ) , daily sperm production (DSP) and seminiferous tubule diameter (STD) in Boran (below diagonal) and Boran × Friesian (above diagonal) bulls
BWT SC PTW DSP STD
BWT
SC
PTW
DSP
STD
0.84"** 0.76*** 0.60** 0.60**
0.66*** 0.87*** 0.75*** 0.67***
0.56*** 0.90"** 0.90*** 0.59**
0.67.** 0.76*** -
0.46"* 0.47** -
**P
weight and scrotal circumference and daily sperm production were high in both Boran and Boran X Friesian bulls (Table 4 ). DISCUSSION
The association between nutritional level, body weight, testes and epididymal weights is shown in the present study where testes weight was greater in supplemented than in control bulls. Similar results have been reported earlier (Lindsay, 1976; Coulter and Kozub, 1984; Venter et al., 1984; Coulter et al.,
30
A. TEGEGNE ET AL.
1987). In contrast, however, feeding high or medium energy diets to young Bos taurus beef bulls was reported to have no effect on paired testes and epi-
didymal weights (Coulter and Bailey, 1988 ). This disparity in results could be caused by differences in age and breed of animals, in the quality and quantity of supplementary feed and in the duration of feeding. The breed difference in paired testes weight is in agreement with a number of other reports (Entwistle, 1988 ). Testes weight in Boran bulls was heavier than reported for other Bos indicus bulls (Osman and E1-Azab, 1974; Aire and Akpokodje, 1975; Kxishnalingam et al., 1982). Testes weight in the crossbred bulls compared well with other Bos indicus cross breeds (Wildeus and Entwistle, 1982), but was lighter than values reported for Bos taurus beef bulls (Coulter and Bailey, 1988 ). Breed differences in paired testes weight could be associated with breed differences in body weight for a given age. In the present study, the percentage difference in paired testes weight (40%) was higher than the percentage difference in body weight (28%) between the two breeds. Estimates of gonadal and extragonadal sperm reserves compare well with earlier reports as summarized by Entwistle ( 1988 ). In these young bulls, neither supplementation nor breed affected DSPG. This could vary with the efficiency of spermatogenesis, but is considered to be a biological constant in a particular species (Courot et al., 1970). Estimates of DSP however, being mainly dependent on the amount of sperm producing testicular parenchymal tissue, were significantly influenced by supplementation and breed. This was a direct reflection of differences in testes weight, as correlations between testes weight and DSP are high (Weisgold and Almquist, 1979; Wildeus and Entwistle, 1982). The significant influence of nutrition on ESR is in agreement with earlier reports (Coulter and Kozub, 1984; Coulter et al., 1987; Coulter and Bailey, 1988). The cauda epididymis, being a major site of sperm storage, contributed the largest percentage of sperm reserves to ESR (Wildeus and Entwistle, 1982; Coulter and Bailey, 1988 ). Estimates of DSP in Boran bulls compare well with estimates for Brahman cross bulls (Wildeus and Entwistle, 1982) and for Nelore bulls (Cardoso and Godinho, 1985), but were higher than those for Balady bulls (Osman and E1-Azab, 1974). The percentage differences in paired testes and epididymal weights between supplemented and control bulls were similar. However, the associated differences in ESR and DSP were not consistent; the difference for ESR being higher than for DSP. The distensibility of the cauda epididymis is related to its storage capacity (Amann and Almquist, 1962; Wildeus and Entwistle, 1982) and no significant correlations exist between epididymal weight and epididymal sperm reserves (Weisgold and Almquist, 1979; Wildeus and Entwistle, 1982), making prediction of ESR from epididymal weight difficult. Therefore, the disparity in differences between organ weights and sperm re-
SUPPLEMENTARYNUTRITIONAND SPERMPRODUCTIONIN BULLS
31
serves could be due to the accumulation of relatively large reserves in the cauda epididymis without a marked change in weight. The influences of supplementary feeding and breed differences on STD were a reflection of the differences in testes weight and in gonadal and extragonadal sperm reserves. Increases in testes size have been associated with increases in the proportion and length of seminiferous tubules in the testis, in seminiferous tubule diameter and in the number of germ cells in the seminiferous tubules (Curtis and Amann, 1981; Amann, 1983 ). In the present study, a 9% difference in STD between supplemented and control bulls was associated with an 18% increase in paired testes weight and a 27% increase in DSP while a difference of 14% in STD between crossbred and Boran bulls resulted in a difference of 40% in paired testes weight and 32% in DSP. The high correlation coefficients between scrotal circumference and body weight, body weight and paired testes weight and paired testes weight and daily sperm production in both genotypes are in agreement with Wildeus and Entwistle ( 1982, 1983) and indicate that measurement of scrotal circumference could be used to estimate testes weight and sperm production rates in young Boran and Boran × Friesian bulls. In conclusion, supplementary feeding to young Boran and Boran X Friesian bulls during two consecutive dry seasons increased paired testes and epididymal weights. Histometric characteristics showed that STD increased significantly through supplementation. Gonadal and extragonadal sperm reserves were also higher in supplemented than in control bulls. Distinct breed differences in paired testes weight, epididymal weight, STD and in gonadal and extragonadal sperm reserves were also observed. The results showed that the reproductive potential of young Boran and Boran X Friesian bulls could be improved through strategic supplementary feeding during dry seasons. ACKNOWLEDGMENTS
The authors thank the Ethiopian Ministry of Agriculture for providing animals and facilities at Abernossa ranch, Kassahun Zewdie for technical assistance, and Solomon Zewdie and G. De'ath for statistical analyses.
REFERENCES Aire, T.A. and Akpokodje, J.U., 1975. Development of puberty in the White Fulani (Bos indicus) bull calf. Br. Vet. J., 131: 146-151. Almquist, J.O. and Amann, R.P., 1961. Reproductive capacity of dairy bulls. II. Gonadal and extragonadal sperm reserves as determined by direct counts and depletion trials; dimensions and weight of genitalia. J. Dairy Sci., 44: 1668-1678. Amann, R.P., 1983. Endocrine changes associated with onset of spermatogenesis in Holstein bulls. J. Dairy Sei., 66: 2606-2622.
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A.TEGEGNEETAL.
Amann, R.P. and Almquist, J.O., 1962. Reproductive capacity of dairy bulls. VI. Effect of unilateral vasectomy and ejaculation frequency on sperm reserves; aspects of epididymal physiology. J. Reprod. Fertil., 3: 260-268. Amann, R.P. and Lambiase, J.T., 1969. The male rabbit. III. Determination of daily sperm production by means of testicular homogenates. J. Anim. Sci., 28: 369-374. Cardoso, F.M. and Godinho, H.P., 1985. Daily sperm production of Zebus (Bos indicus) estimated by quantitative histology of the testes. Theriogenology., 23:841-847. Coulter, G.H. and Bailey, D.R.C., 1988. Epididymal sperm reserves in 12-month-old Angus and Hereford bulls: effects of bull strain plus dietary energy. Anim. Reprod. Sci., 16:169-175. Coulter, G.H. and Kozub, G.C., 1984. Testicular development, epididymal sperm reserves and seminal quality in two year old Hereford and Angus bulls: Effects of two levels of dietary energy. J. Anim. Sci., 59: 432-440. Coulter, G.H., Carruthers, T.D., Amann, R.P. and Kozub, G.C., 1987. Testicular development, daily sperm production and epididymal sperm reserves in 15-month-old Angus and Hereford bulls: Effects of bull strain plus dietary energy. J. Anim. Sci., 64: 254-260. Courot, M., Hochereau de Reviers, M.T. and Ortavant, R., 1970. Spermatogenesis. In: A.D. Johnson, W.R. Gomes and N.L. VanDemark (Editors), The Testis. Vol. 1. Academic Press, New York, pp. 339-432. Curtis, S.K. and Amann, R.P., 1981. Testicular development and establishment of spermatogenesis in Holstein bulls. J. Anim. Sci., 3: 1645-1657. Entwistle, K.W., 1983. Factors influencing reproduction in beef cattle in Australia. Australian Meat Research Council, Review No. 43 Sydney, N.S.W., pp. 1-30. Entwistle, K.W., 1988. Sperm production rate in Bos indicus bulls. Int. J. Anim. Prod., 3: 1728. Entwistle, ICW., Winantea, A.. and Holroyd, R.G., 1980. Sperm production rate in Bos indicus strain bulls. Proc. Aust. Soc. Anim. Prod., 13: 68-72. Godinho, H.P. and Cardoso, F.M., 1984. Gonadal and extragonadal sperm reserves of the Brazilian Nelore Zebu (Bos indicus). Andrologia, 16: 131-134. Igboeli, G. and Rakha, A.M., 1971. Gonadal and extragonadal sperm reserves of indigenous central African bulls. J. Reprod. Fertil., 25: 107-109. Krishnalingam, V., Ladds, P.W., Entwistle, K.W. and Holroyd, R.G., 1982. Quantitative macroscopic and histological study of testicular hypoplasia in Bos indicus strain bulls. Res. Vet. Sci., 32: 131-139. Linsday, D.R., 1976. The usefulness to the animal producer of research findings in nutrition on reproduction. Proc. Aust. Soc. Anim. Prod., 11: 217-224. Osman, A.M. and E1-Azab, E.A., 1974. Studies on gonadal and extragonadal sperm reserves in Balady bulls. Anita. Breed. Abstr., 43: 457-461. Seifert, G.W., Bean, K.G. and Christensen, H.R., 1980. Calving performance of reciprocally mated Africander and Brahman crossbred cattle at Belmont. Proc. Aust. Soc. Anim. Prod., 13: 62-64. Statistical Analysis Systems Institute, 1987. SAS User's Guide: Statistics, Version 5. SAS Institute Inc., Cary, NC. Venter, H.A.W., Van Zyl, J.G.E. and Tami Vasconcellos, O.A.D.R., 1984. Comparative testicular development in young beef bulls. Proc. 2nd. World Congr. on Sheep and Beef Cattle Breeding, June 1984, Vienna, 2: 1-6. Weisgold, A.D. and Almquist, J.O., 1979. Reproductive capacity ofbeefbuUs: Daily spermatozoal production, spermatozoal reserves and dimensions and weights of reproductive organs. J. Anim. Sci., 48: 351-358. Wildeus, S. and Entwistle, K.W., 1982. Postpubertal changes in gonadal and extragonadal sperm reserves in Bos indicus strain bulls. Theriogenology, 17: 655-668.
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Wildeus, S. and Entwistle, K.W., 1983. A quantitative histological study of testicular and epididymal development in Bos indicus cross bulls. Anim. Reprod. Sci., 6: 1-10.
25
Elsevier Science Publishers B.V., Amsterdam
Gonadal and extragonadal sperm reserves and testicular histometric characteristics in Zebu and crossbred bulls: effect of dry season nutritional supplementation A z a g e T e g e g n e a,b, K . W . E n t w i s f l e b a n d E. M u k a s a - M u g e r w a a
alnternational Livestock Centre for Africa, P.O. Box 5689, ,4ddis Ababa, Ethiopia bGraduate School of Tropical Veterinary Science and Agriculture, James Cook University, Townsville, Qld, 481L Australia (Accepted 28 November 1991 )
ABSTRACT Tegegne, A., Entwistle, K.W. and Mukasa-Mugetwa, E., 1992. Gonadal and extragonadal sperm reserves and testicular histometric characteristics in Zebu and crossbred bulls: effect of dry season nutritional s~applementation. Anim. Reprod. Sci., 29: 25-33. A total of 64 bull calves, composed of 27 Boran and 37 Boran×Friesian, were weaned at 6 months of age and divided into supplement or control groups within breed. All bulls were kept in one paddock and grazed on natural pasture. Supplement groups received 1.5 kg per head day- 1of a diet containing 16% crude protein and 8 MJ kg- I dry matter for two dry seasons until castrated at 21 months of age. Testes weight was heavier in supplemented than in control bulls (285 vs. 242 g ) and in Boran X Friesian than in Boran bulls (300 vs. 215 g). Epididymal weight differed between supplemented and control bulls (30.4 vs. 25.1 g) and between Boran and Boran × Friesian (22.1 vs. 31.9 g) bulls. Supplemented bulls had 27% greater daily sperm production (2.65 X 109 vs. 2.08 × 109) and 36% greater epididymal sperm reserves (4.05 X 109 vs. 2.99 × 109) than control bulls. Sperm reserves in the caput and cauda segments were also greater in supplemented than in control bulls. BoranxFriesian bulls had 32% greater daily sperm production ( 2.63 X 109 vs. 1.99 × 109) than Boran bulls, but epididymal sperm reserves did not differ between the two breeds. Seminiferous tubule diameter was larger by 9% in supplemented than in control and by 14% in Boran X Friesian than in Boran bulls. Supplementary feeding to young Boran and Boran × Friesian bulls during dry seasons resulted in heavier testes and epididymis weights, larger seminiferous tubule diameter and higher gonadal and extragonadal sperm reserves.
INTRODUCTION T h e p u r p o s e o f p r o p e r m a n a g e m e n t o f a y o u n g b u l l is t o i m p r o v e g r o w t h r a t e a n d t o e n a b l e it t o p r o d u c e o p t i m u m l e v e l s o f g o o d q u a l i t y s e m e n a t t h e
Correspondence to."A. Tegegne, International Livestock Centre for Africa, P.O. Box 5689, Addis Ababa, Ethiopia.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0378-4320/92/$05.00
26
A. TEGEGNE ET AL.
earliest age possible. The amount and efficiency of sperm producing testicular parenchymal tissue play an important role in influencing the amount and quality of spermatozoa produced by a bull. Bos indicus bulls have been reported to have lower fertility than Bos taurus bulls (Seifertet al., 1980) and this is attributed to smaller testis size (Entwistle, 1983) and fewer gonadal and extragonadal sperm reserves (Igboeli and Raldaa, 1971; Entwistle et al., 1980; Wildeus and Entwistle, 1982 ). However, none of these studies has directly dealt with the effects of supplementary feeding on reproductive potential of young bulls. The most likely occurrence of nutritional deficiency in cattle in tropical regions is during the dry season. Strategic intervention to avert this adverse effect, with supplementary feeding, is likely to have its greatest impact during this period. This study was designed to investigate the influence of dry season nutritional supplementation on gonadal and eXtragonadal sperm reserves and on histometric characteristics of testicular and epididymal tissues in young Boran and Boran × Friesian bulls in Ethiopia. MATERIALS AND METHODS
The study was undertaken at Abernossa ranch in Ethiopia and involved 2 7 Boran and 37 Boran × Friesian bulls. Immediately after weaning at 6 months of age, bulls were divided randomly to either receive a supplement ration or to serve as controls with grazing onlyl Supplemented and control bulls were identified with ear tags of different colours, kept in one paddock and grazed on natural pasture. The predominant grass species were Hyparrhenia rufa, Chlorius gayanaand Cynodon dactylon. Bulls had access to clean w a t e r a n d mineral licks. Supplemented groups were drafted into an enclosure and provided with 1.5 kg per head d a y - 1of aconcentrate mixture composed of wheat bran and Noug cake (Guizotia abyssinica) at a ratio of 2: 1. The mixture contained 16% crude protein and provided 8 MJ kg- 1 dry matter. Supplementary feeding was restricted to two dry seasons of 6 months duration each, December-May and October-March, divided by a wet season of 4 months when all bulls were offered grazing only. Body weight and scrotal circumference (SC) measurements were recorded when bulls were castrated at the end of the second supplementation period at an average age of 21 months. Each testis was dissected, the epididymis separated and divided into caput, corpus and cauda segments. Testis weight and epididymal segment weights were recorded, Testicular parenchymal samples and epididymal segments were taken from the right testis and kept frozen until use for determination of daily sperm production (DSP) and epididymal sperm reserves (ESR) by the homogenization technique (Almquist and Amann, 1961; Amann and Lambiase, 1969). Samples were also taken from the left testis and epididymal
SUPPLEMENTARY NUTRITION AND SPERM PRODUCTION IN BULLS
27
segments and were transferred into Bouin's fixative. The fixed samples were routinely processed, cut at 7/tm and stained with hematoxylin and eosin for histometric determinations. Seminiferous tubule diameter (STD), epididymal tubule diameter and epithelial height measurements were taken from 20 circular tubules on each animal. Data on body and testicular traits were analyzed by analysis of variance using the General Linear Models (GLM) procedure of the Statistical Analysis System (SAS, 1987) with nutrition, genotype and their interactions included in the model. RESULTS
Results on body weight, testis and epididymis weights are presented in Table 1. At castration, supplemented bulls were heavier (41 kg) than control bulls (P<0.001) while Boran×Friesian were heavier (56 kg) than Boran bulls (P< 0.001 ). A difference of 19% in body weight between supplemented and control bulls was associated with an 18% increase in paired testes weight. Paired testes and epididymides were significantly (P< 0.01 ) heavier by 18 and 21% in supplemented than in control groups, respectively. The difference in epididymal weight was due to differences in caput and cauda segments. Paired testes and epididymal weights were heavier by 40 and 46%, respectively in Boran×Friesian than in Boran bulls. A difference of 28% in body weight between Boran X Friesian and Boran bulls resulted in a 40% difference in paired testes weight. There were also significant (P< 0.001 ) differences in all epididymal segment weights between the two breeds. TABLE1 Body weight, paired testes and epididymal weights in Boran and Boran X Friesian (Bo X Fr) bulls reared with or without dry season supplementation 1 Treatments
n
Body weight (kg)
Testes weight (g)
Epididymal weight (g) Total
Caput
Corpus
Cauda
Breed Boran BoXFr
27 35
203+6 259-+5
215+14 300+ 11
22+1 32-+ 1
12-+0.8 18-+0.6
3.9-+0.3 5.3_+0.3
6.0+0.4 8.9+0.4
Nutrition Supplement Control
30 32
251+5 210_+5
285+12 242_+ 16
30+1 25_+ 1
17+0.8 14_+0.8
5,0+0.3 4.4_+0.3
8.6_+0.5 6.7_+0.4
~Entries are means + standard error. **P<0.01; ***P< 0.001.
28
A. TEGEGNE ET AL.
Data on gonadal and extragonadal sperm reserves are presented in Table 2. Neither supplementary feeding nor breed differences influenced daily sperm production per gram (DSPG). DSP, being dependent on testes size, however, was significantly ( P < 0.05 ) higher by 27% in supplemented than in control bulls. ESR were significantly ( P < 0.01 ) higher by 36% in supplemented than in control bulls. Supplemented bulls also had 28 and 51% higher sperm reserves in the caput and cauda segments than control bulls, respectively. Sperm contributions of the cauda segment to epididymal sperm reserves were 52 and 47% in supplemented and control bulls, respectively. Boran× Friesian had 32% higher DSP than Boran bulls (P<0.01). There were no differences in ESR between the two breeds. The sperm contribution of the cauda epididymis to ESR was about 50% in both breeds. Results on histometric characteristics are presented in Table 3. Mean STD was 9% larger in supplemented than in control bulls. Supplemented bulls also had larger caput, corpus and cauda tubule diameters than control bulls. However, epithelial heights in all segments were not affected by supplementation. Boran×Friesian bulls had significantly larger (P<0.001) STD, caput and corpus segment tubule diameters and epithelial height measurements than Boran bulls. Within breed correlation coefficients between body weight and scrotal circumference, body weight and testes weight, scrotal circumference and testes TABLE 2
Daily sperm production per gram ( D S P G ) , daily sperm production ( D S P ) and epididymal sperm reserves (ESR) in Boran and Boran×Friesian ( B o X F r ) bulls reared with or without dry season supplementation ~ Variables
D S P G ( × 104) DSP ( × 107) ESR ( × 107) Caput ( X 107)
Corpus(X10 7) 107)
Cauda ( ×
Nutrition
Breed
Supplement (n=30)
Control (n=32)
Boran (n=27)
Bo × Fr (n=35)
929 + 33 265 ___18 405 + 26 159+ 11 35 + 4 211 _+ 15
823 + 42 208 + 19* 299 + 28** 124+ 12" 32+ 5 140+ 14"*
887 + 27 199 _+ 19 320 -+ 19 130_+ 8 25_+ 4 161 + 10
870 + 44 263 + 19** 375 + 32 149_+ 14 40+ 5* 186_+ 19
Relative epididymal sperm distribution (%) Caput Corpus Cauda
40 9 51
1Entries are mean + standard error. *P<0.05; **P< 0.01.
42 11 47
41 9 50
41 11 48
SUPPLEMENTARY NUTRITION AND SPERM PRODUCTION IN BULLS
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TABLE3 Least squares means ( + standard error) for histometric measurements of seminiferous tubule diameter, epididymal segment tubules and epithelial heights in Boran and Boran X Friesian (Bo X Fr) bulls reared with or without dry season supplementation Variables
Nutrition
Seminiferous tubule diameter ( a m )
Breed
Supplement (n=30)
Control (n=32)
Boran (n=27)
Bo × Fr (n=35)
209 + 4
191_+ 5**
186_+ 5
213_+ 4***
368+ 8 66_+ 1
346+12 63_+ 2
332___11 61_+ 2
375_+ 8*** 68_+ 1"**
340_+ 9 75_+ 1
308_+ 9** 75+ 2
300_+11 70_+ 2
343_+ 7*** 79_+ 1"**
538+20 54_+ 2
482_+22 51+ 2
486_+22 53+ 2
528_+21 52+ 2
Epididymis (am) Caput Diameter(/tm) Epithelialheight(#m) Corpus Diameter(/lm) Epithelialheight ( a m ) Cauda Diameter ( a m ) Epithelialheight ( # m ) **P< 0.01 ; ***P< 0.001. TABLE4 Correlation coefficients between body weight (BWT), scrotal circumference (SC), paired testes weight ( P T W ) , daily sperm production (DSP) and seminiferous tubule diameter (STD) in Boran (below diagonal) and Boran × Friesian (above diagonal) bulls
BWT SC PTW DSP STD
BWT
SC
PTW
DSP
STD
0.84"** 0.76*** 0.60** 0.60**
0.66*** 0.87*** 0.75*** 0.67***
0.56*** 0.90"** 0.90*** 0.59**
0.67.** 0.76*** -
0.46"* 0.47** -
**P
weight and scrotal circumference and daily sperm production were high in both Boran and Boran X Friesian bulls (Table 4 ). DISCUSSION
The association between nutritional level, body weight, testes and epididymal weights is shown in the present study where testes weight was greater in supplemented than in control bulls. Similar results have been reported earlier (Lindsay, 1976; Coulter and Kozub, 1984; Venter et al., 1984; Coulter et al.,
30
A. TEGEGNE ET AL.
1987). In contrast, however, feeding high or medium energy diets to young Bos taurus beef bulls was reported to have no effect on paired testes and epi-
didymal weights (Coulter and Bailey, 1988 ). This disparity in results could be caused by differences in age and breed of animals, in the quality and quantity of supplementary feed and in the duration of feeding. The breed difference in paired testes weight is in agreement with a number of other reports (Entwistle, 1988 ). Testes weight in Boran bulls was heavier than reported for other Bos indicus bulls (Osman and E1-Azab, 1974; Aire and Akpokodje, 1975; Kxishnalingam et al., 1982). Testes weight in the crossbred bulls compared well with other Bos indicus cross breeds (Wildeus and Entwistle, 1982), but was lighter than values reported for Bos taurus beef bulls (Coulter and Bailey, 1988 ). Breed differences in paired testes weight could be associated with breed differences in body weight for a given age. In the present study, the percentage difference in paired testes weight (40%) was higher than the percentage difference in body weight (28%) between the two breeds. Estimates of gonadal and extragonadal sperm reserves compare well with earlier reports as summarized by Entwistle ( 1988 ). In these young bulls, neither supplementation nor breed affected DSPG. This could vary with the efficiency of spermatogenesis, but is considered to be a biological constant in a particular species (Courot et al., 1970). Estimates of DSP however, being mainly dependent on the amount of sperm producing testicular parenchymal tissue, were significantly influenced by supplementation and breed. This was a direct reflection of differences in testes weight, as correlations between testes weight and DSP are high (Weisgold and Almquist, 1979; Wildeus and Entwistle, 1982). The significant influence of nutrition on ESR is in agreement with earlier reports (Coulter and Kozub, 1984; Coulter et al., 1987; Coulter and Bailey, 1988). The cauda epididymis, being a major site of sperm storage, contributed the largest percentage of sperm reserves to ESR (Wildeus and Entwistle, 1982; Coulter and Bailey, 1988 ). Estimates of DSP in Boran bulls compare well with estimates for Brahman cross bulls (Wildeus and Entwistle, 1982) and for Nelore bulls (Cardoso and Godinho, 1985), but were higher than those for Balady bulls (Osman and E1-Azab, 1974). The percentage differences in paired testes and epididymal weights between supplemented and control bulls were similar. However, the associated differences in ESR and DSP were not consistent; the difference for ESR being higher than for DSP. The distensibility of the cauda epididymis is related to its storage capacity (Amann and Almquist, 1962; Wildeus and Entwistle, 1982) and no significant correlations exist between epididymal weight and epididymal sperm reserves (Weisgold and Almquist, 1979; Wildeus and Entwistle, 1982), making prediction of ESR from epididymal weight difficult. Therefore, the disparity in differences between organ weights and sperm re-
SUPPLEMENTARYNUTRITIONAND SPERMPRODUCTIONIN BULLS
31
serves could be due to the accumulation of relatively large reserves in the cauda epididymis without a marked change in weight. The influences of supplementary feeding and breed differences on STD were a reflection of the differences in testes weight and in gonadal and extragonadal sperm reserves. Increases in testes size have been associated with increases in the proportion and length of seminiferous tubules in the testis, in seminiferous tubule diameter and in the number of germ cells in the seminiferous tubules (Curtis and Amann, 1981; Amann, 1983 ). In the present study, a 9% difference in STD between supplemented and control bulls was associated with an 18% increase in paired testes weight and a 27% increase in DSP while a difference of 14% in STD between crossbred and Boran bulls resulted in a difference of 40% in paired testes weight and 32% in DSP. The high correlation coefficients between scrotal circumference and body weight, body weight and paired testes weight and paired testes weight and daily sperm production in both genotypes are in agreement with Wildeus and Entwistle ( 1982, 1983) and indicate that measurement of scrotal circumference could be used to estimate testes weight and sperm production rates in young Boran and Boran × Friesian bulls. In conclusion, supplementary feeding to young Boran and Boran X Friesian bulls during two consecutive dry seasons increased paired testes and epididymal weights. Histometric characteristics showed that STD increased significantly through supplementation. Gonadal and extragonadal sperm reserves were also higher in supplemented than in control bulls. Distinct breed differences in paired testes weight, epididymal weight, STD and in gonadal and extragonadal sperm reserves were also observed. The results showed that the reproductive potential of young Boran and Boran X Friesian bulls could be improved through strategic supplementary feeding during dry seasons. ACKNOWLEDGMENTS
The authors thank the Ethiopian Ministry of Agriculture for providing animals and facilities at Abernossa ranch, Kassahun Zewdie for technical assistance, and Solomon Zewdie and G. De'ath for statistical analyses.
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