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Effect of treatment with recombinant bovine somatotropin on responses to superovulatory treatment in swamp buffalo (Bubalus bubalis)
EFFECT OF TREATMENT WITH RECOMBINANT BOVINE SOMATOTROPLN ON RESPONSES TO SUPEROVULATORY TREATMENT IN SWAMP BUFFALO (Bubalus bubalis) N. Songsasen,la V. Yiengvisavakul,’ B. Buntaracha,’ S. Pharee,* M. Apimeteetumrong’ and Y. Sukwongs’ ‘Artificial Insemination Division, Department of Livestock Development Tiwanon Rd., Bangkadee, Meung District, Pathumthani, Thailand 12000 *Surin Livestock Breeding Station, Department of Livestock Development Meung District, Surin, Thailand 32000 Received for publication: Accepted:
28
December
3 March
1998 1999
ABSTRACT The objective of this study was to determine the effect of treatment with recombinant bovine somatotropin (rBST) on the response to superovulatory treatment in swamp buffalo. Estrous cycles of 16 buffalo cows were synchronized by intravaginal administration of progesterone and estradiol benzoate, and the cows were then randomly divided into 2 groups. The rBST-treated group received 250 mg of a sustained-release formula of rBST on Day 4 after progesterone implantation, whereas the control group did not receive rBST. Both groups were then given a superovulatory regimen of twice daily injections of FSH for 3.5 d (total dose of 260 mg, im), between Days 9 and 11 after administration of progesterone. The cows were bred naturally 1 d after the last FSH injection, then 6 d after breeding they were slaughtered, and their reproductive tracts were removed. The numbers of corpora lutea (CL) and follicles were recorded, and embryos were flushed out of the uterine horns. There were no significant differences between the rBST-treated and control cows for the mean numbers (? SEM) of CL (6.0 + 2.2 vs 4.3 + l.l), follicles (15.9 * 4.1 vs 19.8 + 2.9), or total embryos recovered per collection (4.5 + 1.6 vs 2.3 f 1.0). However, there were significant differences between rBST-treated and control cows for the numbers of transferable embryos per collection (3.0 + 1.O vs 0.8 + 0.3; P 5 0.05) and the overall proportion of transferable embryos (75 vs 33%; P 5 0.01). The results of this study show that pretreatment of swamp buffalo with rBST significantly increases the production of transferable embryos in response to superovulation, 0 1999 by &evier sdenceInc. Key words: recombinant bovine somatotropin, swamp buffalo, superovulation
Acknowledgments The authors thank Dr. N. Saito, Dr. M. Techakumphu and Mrs. J. Indaramongkol for assistance with this study. We also thank Dr. D. Rieger, University of Guelph, Guelph, Ontario, Canada, for a critical reading of our manuscript. This research is funded by the Ministry of Agriculture and Cooperatives. a Correspondence and reprint requests. Theriogenology 52:377-384, 0 1999 by Elsevier Science
1999 Inc.
009~691 W99$-66e front PII S0093-691x(99)00136-3
matter
378 INTRODUCTION A major limitation of the use of embryo transfer as a tool for genetic improvement in buffalo is the extremely low response to superovulatory treatment (1, 5, 8, 24, 27). An average number of transferable embryos collected from a single cow is 1 to 2 embryos for river buffalo (8, 24) and 1 embryo for swamp buffalo (5, 6, 27-29). This is probably due to the lower number of follicles (21) and higher rate of follicular atresia in buffalo than in cattle (8, 25). Attempts to improve the superovulatory response by using different gonadotrophin preparations, treatment regimens, and/or pretreatment with GnRH have failed to increase ovulation rates and the number of transferable embryos (28,29). In the present study, an attempt was made to improve the superovulatory response of swamp buffalo by administration of recombinant bovine somatotropin (rBST) prior to gonadotrophin treatment. In the human, treatment with recombinant human somatotropin reduces the incidence of anovulation (19X significantly improves follicular development and permits the use of lower doses of menopausal gonadotrophins in woman undergoing superovulation (2, 7). Treatment with rBST has also been shown to improve superovulatory response in cattle (12, 15, 18, 20, 26) and in Mediterranean buffalo (34). Webb et al. (33) suggested that pretreatment with rBST may reduce variability in the ovulatory response between animals and may enable the quantity of gonadotrophins required to be reduced. It was suggested that such a beneficial effect of rBST is mediated through increases in concentration of insulin-like growth factor I (IGF-I) and insulin (11, 13, 14, 16, 33). Recently, Gong et al. (16) demonstrated that the number of ovarian follicles < 5 mm in diameter was increased in response to rBST treatment. Development of such follicles to preovulatory follicles is dependent on the amount of gonadotrophin in the circulation (10). Thus, it has been suggested that an increase in the number of follicles at the time of gonadotrophin treatment is responsible to enhancement of superovulatory responses in rBST-treated cattle (11, 16, 18). The objective of this study was to determine superovulatory response of swamp buffalo. MATERIALS
effect of pretreatment with rBST on
AND METHODS
Sixteen swamp buffalo cows, 5 to 17 yr of age, were used in this study. The buffalo were housed at the Surin Livestock Breeding Station, the Department of Livestock Development. During the day, the buffalo were allowed to graze in a pasture. At night, roughage, concentrate and minerals were provided to the animals. Fresh water was provided ad libitum. Estrus Synchronization
and Superovulation
The estrous cycles of 16 buffalo cows were synchronized by intravaginal administration of 1.9 g progesterone in inert silicone elastomer (EAZI-BREED CIDR-B,Q InterAg, Hamilton, New Zealand) and 10 mg estradiol benzoate (CIDIROL@ capsule, InterAg, Hamilton, New Zealand). The buffalo COWS were then randomly divided into 2 groups of 8 animals each: rBST pretreated and control animals.
379
Theriogenology
The rBST-pretreated animals received a subcutaneous injection of 250 mg sustainedrelease formula of rBST (Boostin-250@, LG Chemical Ltd., Dai Jeon, Korea) on Day 4 after the administration of progesterone. The controls buffalo cows received no rBST injection. Superovulation treatment of the cows in both groups was initiated between Days 9 and 11 after the administration of progesterone. Superovulatory treatment consisted of decreasing dosages of intramuscular injections of FSH (Folltropin,B Vetrepharm, Victoria, Australia) twice daily for 3.5 d (48 to 44 , 40 to 40 , 32 to 32 and 24 mg for a total of 260 mg). Synthetic prostaglandin Fza (500 kg; EstrumateB, Coopers Animal Health Ltd., Berkhamsted, England) was administered intramuscularly on the morning of Day 3 of the superovulatory treatment, followed by removal of progesterone in the afternoon of the same day. Approximately 24 to 36 h after the last FSH injection, the cows were naturally bred with bulls of proven fertility. Embryo Collection Six days after the buffalo cows were bred, they were sent to a local slaughterhouse and their reproductive tracts were removed. Ovulation rate was determined by counting the number of CL on both ovaries. The numbers of follicles on both ovaries were also recorded according to 4 size categories: < 2 mm, 2 to 6 mm, 6 to 10 mm, and > 10 mm. The embryos were flushed from the uterine horns using Vigro uterine-base solution8 supplemented with 50X Dulbecco’s modified supplement (AB Technology Jnc, Pullman, WA, USA) and were evaluated according to morphological criteria (22). Embryos classified as excellent, good and fair were considered to be transferable. Statistical Analysis All the data are presented as mean k SEM unless otherwise specified. Comparisons between rBST-treated and control groups for the number of cows with no response were performed by Fisher’s exact probability test. Statistical differences between the 2 groups for the numbers of CL, follicles in each size category, and total and transferable numbers of embryos were analyzed by Student’s t- test. Comparison between rBST-treated and control groups for the percentage oftransferable embryos per collection were performed using Student’s t-test following arcsin transformation of the proportional data. Chi-square analysis was used to compare the overall percentage of transferable embryos between the 2 groups. Differences were considered significant at P < 0.05. RESULTS Table 1 shows the numbers of superovulated buffalo cows that did not respond to superovulation and the mean number of CL. Pretreatment with rBST had no significant effect on the number of cows with a good response to superovulatory treatment. The mean number of CL was greater in the rBST-treated group than in the control group (6.0 vs 4.3), but the difference was not statistically significant. There were no significant differences in the number of follicles between the 2 groups. Most of the follicles on the pair of ovaries observed 6 d after estrus were 2 to 6 mm in diameter for both groups of buffalo cows (Table 2).
Theriogenology
380 Table 1.
The effect of treatment with a single 250 mg injection of rBST on the number of buffalo cows that did not respond to superovulation, and on the mean number of corpora lutea
No. of No. of donors with No. of corpora donors lutea b no responsea 8 2 4.3 It 1.1 FSH 1 FSH + rBST 8 6.0 zk 2.2 a Cows that had less than 2 corpora lutea. b Corpora lutea from cows with no responses were included into the calculation. Treatment
Table 2. Number of follicles in buffalo cows following without rBST treatment
Treatment
Total
FSH FSH + rBST
19.8 + 2.9 15.9k4.1
superovulatory
treatment with and
<2mm
No. of follicles 2-6 mm 6-10 mm
> 1omm
0.1 It-0.1 2.8 + 2.0
10.0 + 1.8 6.3 i 2.0
3.4 rt 0.8 4.1 f 1.0
6.3 + 1.7 3.8 ltl.4
Table 3. The effect of pretreatment with 250 mg rBST on numbers of total and transferable embryos, percentage of transferable per collection, and the overall percentage of transferable embryos collected from superovulated buffalo cows Treatment
Total no. of embryos
No. of transferable embryos
Overall no. of % transferable embryosf
0.8 f 0.3b
Percentage of transferable embryos per collectione 53.3 f 17.0
FSH
2.3 i 1.0
FSH + rBST
4.5 zk 1.6
3.0 * l.oa
77.0 zk 8.9
75.0 (24132)’
a, b: P < 0.05; c, d: P < 0.01. e Mean percentage per cow. f Percentage of transferable embryos obtained from each group. transferable/total embryos collected from each group.
33.3 (6/18)d
Numbers in parentheses are
The numbers of ova and embryos collected from superovulated buffalo cows with and without rBST treatment are shown in Table 3. Treatment with rBST had no significant effect on the total number of collected ova and embryos, but it significantly increased the number of
Theriogenology transferable embryos (range 0 to 8 vs 0 to 2; P < 0.05) and the overall percentage of transferable embryos (75 vs 33%; P< 0.01). DISCUSSION The results of this study demonstrate that treatment with rBST prior to superovulatory treatment with FSH significantly increases the number of transferable embryos collected from Thai swamp buffalo. However, rBST treatment did not increase the number of cows that responded to superovulatory treatment, the ovulation rate, or the total number of ova and embryos collected. The superovulatory response of swamp buffalo has been reported to be extremely low, and this has limited the efficiency of embryo transfer because of the lack of a predictable and reliable supply of embryos. The average number of transferable embryos collected from swamp buffalo following superovulation is 0.5 to 1 embryo per cow (5, 6, 27-29) which is only 10 to 20% of that expected for that of cattle (3,4). It has been suggested that a low oocyte population (21, 23) and a high frequency of follicular atresia (8, 25) could contribute to the poor response to superovulation in the buffalo. Studies on the effect of rBST on the superovulatory response in humans and cattle have yielded divergent results (2, 12, 15, 18, 20, 26). However, treatment with rBST in a superovulation regimen has been reported to increase the proportion of cows that responded to superovulatory treatment as well as the mean number of transferable embryos (12, 18, 20, 26). In Mediterranean buffalo, it was shown that the treatment of cows with rBST decreased the percentage of superovulated cows with ovarian cysts and increased the mean numbers of recovered and transferable embryos (34). The enhancing effect of rBST on the superovulatory response of swamp buffalo observed in the present study agrees with results previously reported in cattle and Mediterranean buffalo. Treatment with rBST in superovulation program clearly increased the mean number of transferable embryos (3.0 vs 0.8 embryos/cow) and the overall percentage of transferable embryos. Although the differences were not statistically significant, rBST-treated cows yielded an average of approximately 2 more CL (6.0 vs 4.3) and recovered embryos (4.5 vs 2.3) than the control cows. It has been shown that rBST pretreatment enhances the recruitment of small ovarian follicles in cattle rather than increasing the growth rate or reducing the rate of follicular atresia (1 l-13, 16, 18, 32-33). This effect is probably responsible for the increase in superovulatory responses of cattle and Mediterranean buffalo. Gong et al. (13, 16) demonstrated that the stimulatory effect of rBST treatment on ovarian follicle development is mediated by increased serum concentration of IGF-1 and insulin. Increases in IGF-1 enhances proliferation of granulosa cells, especially those of small follicle (< 5 mm in diameter). This results in an increased number of follicles being present on the day of superovulation treatment. Uoc et al. (30) demonstrated that pretreatment with E2 prior to administration gonadotrophin improved superovulatory response in swamp buffalo. Enhancement
of of
Theriogenology
382
proliferation of granulosa cells in rBST-treated cows may affect amounts of estrogen secreted This may explain the enhancing effect of rBST on the superovulation into the circulation. response of swamp buffalo observed in the present study. It has been demonstrated that grandosa cells play an important role in regulation of oocyte growth. They regulate oocyte metabolic processes that are essential for the growth, development and maturation of mammalian oocytes (for review see 9, 17, 31). This essential role of granulosa cells may affect the quality of oocytes in rBST-treated buffilo, resulting in the significant increase in number of transferable embryos observed in the present study. The mean number of transferable embryos obtained in our study is greater than that reported elsewhere (5, 6, 27-29). The mean number of unovulated follicles (> 10 mm in diameter) on the pairs of ovaries of buffalo cows from both groups in our study was lower than that reported previously (28, 29). This may due to the low dose of FSH (260 mg) used in our study. Techakumphu et al. (29) reported that lower FSH concentration (280 vs 400 mg) in a superovulation regimen resulted in decreases in the number of unovulated follicles. In the present study, the mean numbers of unovulated follicles were 3.4kO.8 and 4. lkl .O in the FSH- and FSH + rBST-treated cows, respectively. Although the results obtained in the present study cannot be directly compared to those of previous studies, it is reasonable to suggest that using a low dose of FSH may attenuate the problem of high numbers of unovulated follicles in superovulated swamp buffalo (28, 29). In conclusion, the results of the present study demonstrate that treatment of buffalo with rBST prior to superovulation increases the number of transferable embryos. Further investigation will be necessary to establish the optimum dosages and intervals of rBST and FSH treatments for the superovulation of swamp buffalo. Nevertheless, the results of this study suggest that rBST treatment of superovulated cows may optimize embryo transfer for genetic improvement and the conservation of swamp buffalo in Thailand. REFERENCES 1. Beg MA, Sanwal PC, Yadav MC. Ovarian response and endocrine changes in buffalo superovulated at midluteal and late luteal stage of the estrous cycle: a preliminary report, Theriogenology 1997; 47: 423-432. 2. Blumenfeld Z, Lumenfeld B. The potential effect of growth hormone on follicle stimulation with human menopausal gonadotropin in a panhypopituitary patient. Fertil Steril 1989; 82: 328-331. 3. Bo GA, Adams GP, Pierson RA, Mapletoft RJ. Exogenous control of follicular wave emergence in cattle. Theriogenology 1995; 43: 31-40. 4. Boni R. In vivo collection of oocytes and embryos in bovine and buffalo species. Buffalo J 1994; 2 (Suppl2): 161-171. 5. Chantaraprateep P, Lohachit C, Virakul P, Kobayashi G, Techakumphu M, Kunayongkrit A, Prateep P. Ovarian responses to gonadotrophin stimulation in swamp buffalo. Buffalo Bulletin 1988; 7: 82-86.
6. Chantaraprateep P, Lohachit C, Techakumphu M, Kobayashi G, Virakul P, Kunayongkrit A, Prateep P, Limskul A. Early embryonic development in Thai swamp buffalo (Bubalus bubalis). Theriogenology 1989; 31: 1131-1139. 7. Christman GM, Halme JK. Growth hormone: revisited. Fertil Steril 1992; 57: 12- 14. 8. Crux LC. Embryo transfer in water buffaloes. Proc 8” AAAP Animal Sci Congr 1996: 142152. 9. Eppig JJ, Intercommunication between mammalian oocytes and companion somatic cells. BioEssays 1991; 13: 569-574. 10. Fortune JE. Ovarian follicular growth and development in mammals. Biol Reprod 1994; 50: 225-232. 11. Gong JG, Bramley T, Webb R. The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and peripheral hormones. Biol Reprod I991 ; 45: 941-949. 12. Gong JG, Wilmut I, Bramley TA, Webb R. Effects of recombinant bovine somatotropin (BST) on superovulatory response in heifers. Proc 12”’ Int Congr Anim Reprod 1992; 2 14216. 13. Gong JG, McBride D, Bramley TA, Webb R. Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on the proliferation of bovine granulosa cells in vitro. J Endocrinol 1993; 139: 67-75. 14. Gong JG, Bramley TA, Webb R. The effect of recombinant bovine somatotrophin on ovarian follicular growth and development in heifers. J Reprod Fertil 1993; 97: 247-254. 15. Gong JG, Bramley TA, Wilmut I, Webb R. Effect of recombinant bovine somatotropin on the superovulatory response to pregnant mare serum gonadotropin in heifers. Biol Reprod 1993; 48: 1141-l 149. 16. Gong JG, Baxter G, Bramley TA, Webb R. Enhancement of ovarian follicle development in heifers by treatment with recombinant bovine somatotrophin: a dose-response study. J Reprod Fertil 1997; 110: 91-97. 17. Gordon I. Laboratory Production of Cattle Embryos. Oxon: CAB International 1994; 116121. 18. Herrler A, Einspanier R, Schams D, and Niemann H. Effect of recombinant bovine somatotropin (rBST) on follicular IGF-1 contents and the ovarian response following superovulatory treatment in dairy cows: a preliminary study. Theriogenology 1994; 4 1: 60 l611. 19. Humburg R, West C, Torresani T, Jacobs HS. Co-treatment with human growth hormone and gonadotropins for induction of ovulation: a controlled clinical trial. Fertil Steril 1990; 53: 254-260. 20. Kuehner LF, Rieger D, Walton JS, Zhao X, Johnson WH. The effect of a depot injection of recombinant bovine somatotropin on follicular development and embryo yield in superovulated Holstein heifers. Theriogenology 1993; 40: 1003- 10 13. 2 1. Le Van TY, Chupin D, Driancourt MA. Ovarian follicular population in buffaloes and cows. Anim Reurod Sci 1989: 19:171-178. 22. Lindner GM, Wright RW. Bovine embryo morphology and evaluation. Theriogenology 1983; 20: 407-416. 23. Madan ML, Singla SK, Chauhan, MB, Manik RS. In vitro production and transfer of embryos in buffaloes. Theriogenology 1994; 4 1: 139- 143.
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Theriogenology
24. Misra AK, Kasiraj R, Mutha Rao M, Ranga Reddy NS, Joshi BV. Embryo transfer in buffalo in India: progress in the last five years. Proc 4ti World Buffalo Congr 1994; 3:501-504. 25. Ocampo MB, De Asis AT, Gcampo LC, Kanagawa H. Histological observation of follicular atresia in swamp buffalo. Buffalo Bulletin 1994; 13: 5 l-55. 26. Rieger D, Walton JS, Goodwin ML, Johnson WH. The effect of co-treatment with recombinant bovine somatotrophin on plasma progesterone concentration and number of embryos collected from superovulated Holstein heifers. Theriogenology 1991; 35: 863868. 27. Sophon S, Panpai R, Kamonpatana M. Multiple ovulation leading to embryo transfer in buffalo; using PMSG and FSH under oestrous regulation by norgestomet. Buffalo J 1989; 5: 85-98. 28. Techakumphu M, Sukawong Y, Jntaramongkol J, Phari S, Boontarath B. Effect of gonadotropin releasing hormone in superovulation program in swamp buffalo. Proc 24” Thai Vet Med Assoc 1998; 81-87. 29. Techakumphu M, Sukawong Y, Apimethithumrong M, Jntaramongkol S, Jntaramongkol J, Phari S, Lohachit C, Chantaraprateep P. Use of pregnant mare serum gonadotropin and follicle sitmulating hormone for superovulation in Thai swamp buffalo (Bubalus bubalis). Proc 24” Thai Vet Med Assoc 1998; 88-96. 30. Uoc NT, Nguyen BX, TY LV, Chupin D, Beckers JF, Renard JP. Effect of estradiol supplementation on superovulation in swamp buffalo. Theriogenology 1992; 38: 471-478. 31. Wassarman PM, Albertini DF. The mammalian ovum. In: Knobil E, Neil1 JD (eds), The Physiology of Reproduction. New York: Raven Press 1994; 79-122. 32. Webb R, Gong JG, Law AS, Rusbridge SM. Control of ovarian function of cattle. J Reprod Fertil 1992; 45 (Suppl45): 141-156. 33. Webb R, Gong JG, Bramley TA. Role of growth hormone and intrafollicular peptides in follicle development in cattle. Theriogenology 1994; 41: 25-30. 34. Zicarelli L. Embryo transfer in Mediterranean buffaloes. Buffalo J 1994; 2 (Suppl 2): 87101.
28
December
3 March
1998 1999
ABSTRACT The objective of this study was to determine the effect of treatment with recombinant bovine somatotropin (rBST) on the response to superovulatory treatment in swamp buffalo. Estrous cycles of 16 buffalo cows were synchronized by intravaginal administration of progesterone and estradiol benzoate, and the cows were then randomly divided into 2 groups. The rBST-treated group received 250 mg of a sustained-release formula of rBST on Day 4 after progesterone implantation, whereas the control group did not receive rBST. Both groups were then given a superovulatory regimen of twice daily injections of FSH for 3.5 d (total dose of 260 mg, im), between Days 9 and 11 after administration of progesterone. The cows were bred naturally 1 d after the last FSH injection, then 6 d after breeding they were slaughtered, and their reproductive tracts were removed. The numbers of corpora lutea (CL) and follicles were recorded, and embryos were flushed out of the uterine horns. There were no significant differences between the rBST-treated and control cows for the mean numbers (? SEM) of CL (6.0 + 2.2 vs 4.3 + l.l), follicles (15.9 * 4.1 vs 19.8 + 2.9), or total embryos recovered per collection (4.5 + 1.6 vs 2.3 f 1.0). However, there were significant differences between rBST-treated and control cows for the numbers of transferable embryos per collection (3.0 + 1.O vs 0.8 + 0.3; P 5 0.05) and the overall proportion of transferable embryos (75 vs 33%; P 5 0.01). The results of this study show that pretreatment of swamp buffalo with rBST significantly increases the production of transferable embryos in response to superovulation, 0 1999 by &evier sdenceInc. Key words: recombinant bovine somatotropin, swamp buffalo, superovulation
Acknowledgments The authors thank Dr. N. Saito, Dr. M. Techakumphu and Mrs. J. Indaramongkol for assistance with this study. We also thank Dr. D. Rieger, University of Guelph, Guelph, Ontario, Canada, for a critical reading of our manuscript. This research is funded by the Ministry of Agriculture and Cooperatives. a Correspondence and reprint requests. Theriogenology 52:377-384, 0 1999 by Elsevier Science
1999 Inc.
009~691 W99$-66e front PII S0093-691x(99)00136-3
matter
378 INTRODUCTION A major limitation of the use of embryo transfer as a tool for genetic improvement in buffalo is the extremely low response to superovulatory treatment (1, 5, 8, 24, 27). An average number of transferable embryos collected from a single cow is 1 to 2 embryos for river buffalo (8, 24) and 1 embryo for swamp buffalo (5, 6, 27-29). This is probably due to the lower number of follicles (21) and higher rate of follicular atresia in buffalo than in cattle (8, 25). Attempts to improve the superovulatory response by using different gonadotrophin preparations, treatment regimens, and/or pretreatment with GnRH have failed to increase ovulation rates and the number of transferable embryos (28,29). In the present study, an attempt was made to improve the superovulatory response of swamp buffalo by administration of recombinant bovine somatotropin (rBST) prior to gonadotrophin treatment. In the human, treatment with recombinant human somatotropin reduces the incidence of anovulation (19X significantly improves follicular development and permits the use of lower doses of menopausal gonadotrophins in woman undergoing superovulation (2, 7). Treatment with rBST has also been shown to improve superovulatory response in cattle (12, 15, 18, 20, 26) and in Mediterranean buffalo (34). Webb et al. (33) suggested that pretreatment with rBST may reduce variability in the ovulatory response between animals and may enable the quantity of gonadotrophins required to be reduced. It was suggested that such a beneficial effect of rBST is mediated through increases in concentration of insulin-like growth factor I (IGF-I) and insulin (11, 13, 14, 16, 33). Recently, Gong et al. (16) demonstrated that the number of ovarian follicles < 5 mm in diameter was increased in response to rBST treatment. Development of such follicles to preovulatory follicles is dependent on the amount of gonadotrophin in the circulation (10). Thus, it has been suggested that an increase in the number of follicles at the time of gonadotrophin treatment is responsible to enhancement of superovulatory responses in rBST-treated cattle (11, 16, 18). The objective of this study was to determine superovulatory response of swamp buffalo. MATERIALS
effect of pretreatment with rBST on
AND METHODS
Sixteen swamp buffalo cows, 5 to 17 yr of age, were used in this study. The buffalo were housed at the Surin Livestock Breeding Station, the Department of Livestock Development. During the day, the buffalo were allowed to graze in a pasture. At night, roughage, concentrate and minerals were provided to the animals. Fresh water was provided ad libitum. Estrus Synchronization
and Superovulation
The estrous cycles of 16 buffalo cows were synchronized by intravaginal administration of 1.9 g progesterone in inert silicone elastomer (EAZI-BREED CIDR-B,Q InterAg, Hamilton, New Zealand) and 10 mg estradiol benzoate (CIDIROL@ capsule, InterAg, Hamilton, New Zealand). The buffalo COWS were then randomly divided into 2 groups of 8 animals each: rBST pretreated and control animals.
379
Theriogenology
The rBST-pretreated animals received a subcutaneous injection of 250 mg sustainedrelease formula of rBST (Boostin-250@, LG Chemical Ltd., Dai Jeon, Korea) on Day 4 after the administration of progesterone. The controls buffalo cows received no rBST injection. Superovulation treatment of the cows in both groups was initiated between Days 9 and 11 after the administration of progesterone. Superovulatory treatment consisted of decreasing dosages of intramuscular injections of FSH (Folltropin,B Vetrepharm, Victoria, Australia) twice daily for 3.5 d (48 to 44 , 40 to 40 , 32 to 32 and 24 mg for a total of 260 mg). Synthetic prostaglandin Fza (500 kg; EstrumateB, Coopers Animal Health Ltd., Berkhamsted, England) was administered intramuscularly on the morning of Day 3 of the superovulatory treatment, followed by removal of progesterone in the afternoon of the same day. Approximately 24 to 36 h after the last FSH injection, the cows were naturally bred with bulls of proven fertility. Embryo Collection Six days after the buffalo cows were bred, they were sent to a local slaughterhouse and their reproductive tracts were removed. Ovulation rate was determined by counting the number of CL on both ovaries. The numbers of follicles on both ovaries were also recorded according to 4 size categories: < 2 mm, 2 to 6 mm, 6 to 10 mm, and > 10 mm. The embryos were flushed from the uterine horns using Vigro uterine-base solution8 supplemented with 50X Dulbecco’s modified supplement (AB Technology Jnc, Pullman, WA, USA) and were evaluated according to morphological criteria (22). Embryos classified as excellent, good and fair were considered to be transferable. Statistical Analysis All the data are presented as mean k SEM unless otherwise specified. Comparisons between rBST-treated and control groups for the number of cows with no response were performed by Fisher’s exact probability test. Statistical differences between the 2 groups for the numbers of CL, follicles in each size category, and total and transferable numbers of embryos were analyzed by Student’s t- test. Comparison between rBST-treated and control groups for the percentage oftransferable embryos per collection were performed using Student’s t-test following arcsin transformation of the proportional data. Chi-square analysis was used to compare the overall percentage of transferable embryos between the 2 groups. Differences were considered significant at P < 0.05. RESULTS Table 1 shows the numbers of superovulated buffalo cows that did not respond to superovulation and the mean number of CL. Pretreatment with rBST had no significant effect on the number of cows with a good response to superovulatory treatment. The mean number of CL was greater in the rBST-treated group than in the control group (6.0 vs 4.3), but the difference was not statistically significant. There were no significant differences in the number of follicles between the 2 groups. Most of the follicles on the pair of ovaries observed 6 d after estrus were 2 to 6 mm in diameter for both groups of buffalo cows (Table 2).
Theriogenology
380 Table 1.
The effect of treatment with a single 250 mg injection of rBST on the number of buffalo cows that did not respond to superovulation, and on the mean number of corpora lutea
No. of No. of donors with No. of corpora donors lutea b no responsea 8 2 4.3 It 1.1 FSH 1 FSH + rBST 8 6.0 zk 2.2 a Cows that had less than 2 corpora lutea. b Corpora lutea from cows with no responses were included into the calculation. Treatment
Table 2. Number of follicles in buffalo cows following without rBST treatment
Treatment
Total
FSH FSH + rBST
19.8 + 2.9 15.9k4.1
superovulatory
treatment with and
<2mm
No. of follicles 2-6 mm 6-10 mm
> 1omm
0.1 It-0.1 2.8 + 2.0
10.0 + 1.8 6.3 i 2.0
3.4 rt 0.8 4.1 f 1.0
6.3 + 1.7 3.8 ltl.4
Table 3. The effect of pretreatment with 250 mg rBST on numbers of total and transferable embryos, percentage of transferable per collection, and the overall percentage of transferable embryos collected from superovulated buffalo cows Treatment
Total no. of embryos
No. of transferable embryos
Overall no. of % transferable embryosf
0.8 f 0.3b
Percentage of transferable embryos per collectione 53.3 f 17.0
FSH
2.3 i 1.0
FSH + rBST
4.5 zk 1.6
3.0 * l.oa
77.0 zk 8.9
75.0 (24132)’
a, b: P < 0.05; c, d: P < 0.01. e Mean percentage per cow. f Percentage of transferable embryos obtained from each group. transferable/total embryos collected from each group.
33.3 (6/18)d
Numbers in parentheses are
The numbers of ova and embryos collected from superovulated buffalo cows with and without rBST treatment are shown in Table 3. Treatment with rBST had no significant effect on the total number of collected ova and embryos, but it significantly increased the number of
Theriogenology transferable embryos (range 0 to 8 vs 0 to 2; P < 0.05) and the overall percentage of transferable embryos (75 vs 33%; P< 0.01). DISCUSSION The results of this study demonstrate that treatment with rBST prior to superovulatory treatment with FSH significantly increases the number of transferable embryos collected from Thai swamp buffalo. However, rBST treatment did not increase the number of cows that responded to superovulatory treatment, the ovulation rate, or the total number of ova and embryos collected. The superovulatory response of swamp buffalo has been reported to be extremely low, and this has limited the efficiency of embryo transfer because of the lack of a predictable and reliable supply of embryos. The average number of transferable embryos collected from swamp buffalo following superovulation is 0.5 to 1 embryo per cow (5, 6, 27-29) which is only 10 to 20% of that expected for that of cattle (3,4). It has been suggested that a low oocyte population (21, 23) and a high frequency of follicular atresia (8, 25) could contribute to the poor response to superovulation in the buffalo. Studies on the effect of rBST on the superovulatory response in humans and cattle have yielded divergent results (2, 12, 15, 18, 20, 26). However, treatment with rBST in a superovulation regimen has been reported to increase the proportion of cows that responded to superovulatory treatment as well as the mean number of transferable embryos (12, 18, 20, 26). In Mediterranean buffalo, it was shown that the treatment of cows with rBST decreased the percentage of superovulated cows with ovarian cysts and increased the mean numbers of recovered and transferable embryos (34). The enhancing effect of rBST on the superovulatory response of swamp buffalo observed in the present study agrees with results previously reported in cattle and Mediterranean buffalo. Treatment with rBST in superovulation program clearly increased the mean number of transferable embryos (3.0 vs 0.8 embryos/cow) and the overall percentage of transferable embryos. Although the differences were not statistically significant, rBST-treated cows yielded an average of approximately 2 more CL (6.0 vs 4.3) and recovered embryos (4.5 vs 2.3) than the control cows. It has been shown that rBST pretreatment enhances the recruitment of small ovarian follicles in cattle rather than increasing the growth rate or reducing the rate of follicular atresia (1 l-13, 16, 18, 32-33). This effect is probably responsible for the increase in superovulatory responses of cattle and Mediterranean buffalo. Gong et al. (13, 16) demonstrated that the stimulatory effect of rBST treatment on ovarian follicle development is mediated by increased serum concentration of IGF-1 and insulin. Increases in IGF-1 enhances proliferation of granulosa cells, especially those of small follicle (< 5 mm in diameter). This results in an increased number of follicles being present on the day of superovulation treatment. Uoc et al. (30) demonstrated that pretreatment with E2 prior to administration gonadotrophin improved superovulatory response in swamp buffalo. Enhancement
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proliferation of granulosa cells in rBST-treated cows may affect amounts of estrogen secreted This may explain the enhancing effect of rBST on the superovulation into the circulation. response of swamp buffalo observed in the present study. It has been demonstrated that grandosa cells play an important role in regulation of oocyte growth. They regulate oocyte metabolic processes that are essential for the growth, development and maturation of mammalian oocytes (for review see 9, 17, 31). This essential role of granulosa cells may affect the quality of oocytes in rBST-treated buffilo, resulting in the significant increase in number of transferable embryos observed in the present study. The mean number of transferable embryos obtained in our study is greater than that reported elsewhere (5, 6, 27-29). The mean number of unovulated follicles (> 10 mm in diameter) on the pairs of ovaries of buffalo cows from both groups in our study was lower than that reported previously (28, 29). This may due to the low dose of FSH (260 mg) used in our study. Techakumphu et al. (29) reported that lower FSH concentration (280 vs 400 mg) in a superovulation regimen resulted in decreases in the number of unovulated follicles. In the present study, the mean numbers of unovulated follicles were 3.4kO.8 and 4. lkl .O in the FSH- and FSH + rBST-treated cows, respectively. Although the results obtained in the present study cannot be directly compared to those of previous studies, it is reasonable to suggest that using a low dose of FSH may attenuate the problem of high numbers of unovulated follicles in superovulated swamp buffalo (28, 29). In conclusion, the results of the present study demonstrate that treatment of buffalo with rBST prior to superovulation increases the number of transferable embryos. Further investigation will be necessary to establish the optimum dosages and intervals of rBST and FSH treatments for the superovulation of swamp buffalo. Nevertheless, the results of this study suggest that rBST treatment of superovulated cows may optimize embryo transfer for genetic improvement and the conservation of swamp buffalo in Thailand. REFERENCES 1. Beg MA, Sanwal PC, Yadav MC. Ovarian response and endocrine changes in buffalo superovulated at midluteal and late luteal stage of the estrous cycle: a preliminary report, Theriogenology 1997; 47: 423-432. 2. Blumenfeld Z, Lumenfeld B. The potential effect of growth hormone on follicle stimulation with human menopausal gonadotropin in a panhypopituitary patient. Fertil Steril 1989; 82: 328-331. 3. Bo GA, Adams GP, Pierson RA, Mapletoft RJ. Exogenous control of follicular wave emergence in cattle. Theriogenology 1995; 43: 31-40. 4. Boni R. In vivo collection of oocytes and embryos in bovine and buffalo species. Buffalo J 1994; 2 (Suppl2): 161-171. 5. Chantaraprateep P, Lohachit C, Virakul P, Kobayashi G, Techakumphu M, Kunayongkrit A, Prateep P. Ovarian responses to gonadotrophin stimulation in swamp buffalo. Buffalo Bulletin 1988; 7: 82-86.
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