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Use of steroid hormone treatments prior to superovulation in Nelore donors
Animal Reproduction Science 69 (2002) 9–14
Use of steroid hormone treatments prior to superovulation in Nelore donors J.C.O. Andrade a , M.A.L. Oliveira a,c,∗ , P.F. Lima a,c , A.S. Santos Filho a , V.M.R. Pina b a
Programa de Pós-Graduação em Ciência Veterinária - UFRPE, Av. D. Manoel de Medeiros s/n, Dois Irmãos, CEP 56500.000 Recife, PE, Brazil b Curso de Graduação em Medicina Veterinária - UFRPE, Av. D. Manoel de Medeiros s/n, Dois Irmãos, CEP 56500.000 Recife, PE, Brazil c Departmento de Medicina Veterinaria - UFRPE, Av. D. Manoel de Medeiros s/n, Dois Irmãos, CEP 56500.000 Recife, PE, Brazil
Received 20 January 2001; received in revised form 4 September 2001; accepted 17 September 2001
Abstract The objective of this study was to evaluate the effectiveness of synchronization of follicular wave emergence using steroid hormone treatments in Nelore cows. Donors were placed into three groups. Those that were between days 9 and 12 of their cycle (estrus = day 0) formed the TI group (n = 60), whilst those that were in any other stages of their estrus cycle constituted groups TII (n = 60) and TIII (n = 60). TI donors were submitted to a standard protocol of superovulation, however, TII and TIII donors were treated with the Syncro-Mate-B (SMB) or Controlled Internal Drug Releasing Device (CIDR-B) programs, respectively. Superovulation was induced with p-FSH, divided into eight decreasing doses at intervals of 12 h. The donors received cloprostenol 48 h after the beginning of the treatment and progestagens were removed 12 h later. Artificial inseminations (AI) were done at 12 and 22 h after the initiation of estrus and the embryo collections were done 7 days after AI. In the donors which displayed behavioral estrus, mean (±S.E.M.) total ova and viable (transferable) embryos were 15.8 ± 1.4 and 8.3 ± 1.0 (TI, n = 56); 15.6 ± 1.3 and 8.9 ± 1.0 (TII, n = 56); 17.3 ± 1.0 and 9.9 ± 0.9 (TIII, n = 57), respectively, with no significant difference (P ≥ 0.05) among groups. In those animals that did not displayed behavioral estrus, the mean values of total ova and viable embryos were 3.5 ± 1.6 and 0.7 ± 0.5 (TI, n = 4); 11.5 ± 3.9 and 9.0 ± 4.4 (TII, n = 4); 8.7 ± 5.0 and 5.0 ± 2.9 (TIII, n = 3), respectively, with no significant differences (P ≥ 0.05) among groups. Pregnancy rates of 62.2% (TI, n = 235); 66.4% (TII, n = 284) and 65.1% (TIII, n = 244) were obtained with embryos transferred from these collections and did not differ significantly (P ≥ 0.05) among groups. It was concluded that the synchronization
∗ Corresponding author. E-mail address: [email protected] (M.A.L. Oliveira).
0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 1 4 9 - X
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J.C.O. Andrade et al. / Animal Reproduction Science 69 (2002) 9–14
of the emergence of follicular waves in Nelore donors is usable and does not harm the efficiency of embryo transfer programs. In addition, in contrast to the standard superovulation protocol, this method permits the use of a large number of donors in a short time period, at any stage of the estrus cycle, minimizing the costs of embryo transfer. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Bos indicus; Embryo; Syncro-Mate-B; Controlled Internal Drug Releasing Device
1. Introduction The advent of ultrasonography has led to a better understanding of diverse reproductive events, in particular, follicular dynamics in cattle. Follicular development in cattle is characterized by the occurrence of two or three growth waves during the estrus cycle (Fortune, 1993). In Nelore cows, Figueiredo et al. (1997) observed a predominant pattern of two ovarian follicular growth waves, while Bo et al. (1993) reported that a pattern of three follicular growth waves prevails over that of two ‘such waves’ in Brahman cows. Superovulation, a factor which may limit the efficiency of embryo transfer, continues to be a common topic for research, particularly in relation to the time of initiating exogenous hormonal stimulation. Superovulation must be initiated on the day prior to follicular wave emergence (Adams, 1994) or on the day of follicular wave emergence (Adams, 1994; Bo et al., 1995), before the subordinate follicles begin the atresie process (Bo et al., 1995). The synchronization of follicular wave emergence is done by the removal of the suppressive effect of the dominant follicle over the growth of the next wave (Bo et al., 1995). Several techniques have been used to promote the emergence of a new follicular wave at a specific time including the administration of hormones such as estrogens (Bo et al., 1993, 1994; Carriere et al., 1995), estrogens and progesterone (Bo et al., 1996) because these steroid hormones are capable of promoting the emergence of a new follicular wave by causing regression of antral follicles. Combinations of between progesterone/estrogen or progestagen/estrogen have gained special attention in protocols for superovulation in cattle (Bo et al., 1995, 1996; Broadbent et al., 1995). When progesterone or progestagen are used in combination with a 17-estradiol, the emergence of a new follicular wave occurs around 4 days after treatment but occurs 5 days after treatment, when estradiol benzoate and estradiol valerate are used (Bo et al., 1995, 1996; Caccia and Bo, 1998; Mapletoft et al., 1999). In Brazil, the size of the country and the lack of technicians trained in the basics of embryo transfer have limited the diffusion of this technology in some regions. Elaborate protocols which allow the use of a larger number of donors per program, such as the synchronization of follicular wave emergence using steroid hormones, has minimized the routine problem of control of the estrus cycle in large herds in the northern region of the country. This type of protocol allows the veterinarian, even when far from the property to determine the most suitable period for the performance of the embryo transfer program (Andrade and Oliveira, 1998). Thus, the lack of superovulatory protocols for the synchronization of the emergence of follicular waves in females of the Nelore breed motivated this study, which aimed to compare the superovulatory response, ova/embryo recovery and number of transferable embryos, in donors superovulated by the conventional protocol on days 9–12 of cycle,
J.C.O. Andrade et al. / Animal Reproduction Science 69 (2002) 9–14
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those submitted to the synchronization of follicular wave emergence with steroid hormones prior to gonadotropin treatment.
2. Materials and methods One hundred and eighty superovulation treatments were analyzed in females of the Nelore breed. Animals presented regular estrus cycles and were between 6 and 10 years of age, with body condition scores ranging from 3 to 4 on a scale of 1–5. All animals were submitted to clinical-gynecological examinations before the experiment. Animals were kept on pasture, where they were fed on Braquiaria brisanta grass and had free access to water and mineral salts. The 180 donors were equally distributed into three groups according to the stage of their estrus cycle. Donors between the 9th and 12th days of their cycle formed the TI group (standard superovulatory protocol), while the remaining 120 donors, at all other stages of the estrus cycle, were randomly distributed into two groups, TII and TIII (submitted to protocols for synchronization of the emergence of the follicular wave). The TII group was submitted to the SMB program (Rhodia–Mérieux—Veterinary LTDA), which consisted of a subcutaneous auricular implant of 6 mg norgestomet and an intramuscular injection of 3 mg norgestomet and 5 mg estradiol valerate. TIII animals were submitted to the CIDR-B program (InterAg—Edvet HD), consisting of an intravaginal device, containing 1.9 g progesterone, and an intramuscular injection of 4 mg estradiol benzoate (Estrogin—Farmavet Veterinary Products LTDA) administered 24 h after the introduction of the vaginal insertion. Gonadotropin treatment began between the 9th and 12th days of the estrus cycle in those animals undergoing the standard superovulatory protocol (TI) and 5 days after the administration of the SMB implant (TII) and the insertion of the CIDR-B device (TIII). In all the groups, superovulation included intramuscular injections of 400 p-FSH (Pluset—Serono Pharmaceutical Products LTDA) administered in eight decreasing doses (80, 60, 40, and 20 BID) at intervals of 12 h. After the 48 h of beginning of this treatment, donors received 1 mg cloprostenol (PGF2␣ -Ciosin-Coopers, Mallinckrodt Vet LTD) via intramuscular injection and, 12 h later, implants in the TII and devices in the TIII groups were removed. Artificial inseminations were performed with commercial semen at 12 and 22 h after the detection of estrus by teaser animals using a chin ball. Donors that did not display behavioral estrus within 54 h of PGF2␣ injection were treated intramuscularly with 0.02 mg busereline acetate (Conceptal—Hoechst Roussel Vet) and inseminated at 6, 12 and 18 h later. Embryos were collected 7 days after the first artificial insemination (AI) and were then evaluated and classified according to development and morphology (Robertson and Nelson, 1998), then transferred immediately to recipients by via transcervical method. The collected embryos not used were frozen. The recipient synchrony, obtained with PGF2␣ administration, was maintained between (−) 2 days before and (+) 2 days after the estrus cycles of the donors. Data were analyzed using the PROC GLM option of the Statistical Analysis Systems (1988). The numbers of total ova, viable (transferable) embryos, degenerate embryos and unfertilized ova were compared using the Kruskal–Wallis test because the data were not distributed normality, which was verified using the Shapiro Wilks test. Pregnancy rates were
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J.C.O. Andrade et al. / Animal Reproduction Science 69 (2002) 9–14
analyzed with the contingency table for the Chi-squared test (χ 2 ). A confidence interval of 5% or less was considered to be statistically significant.
3. Results In the females, which displayed behavioral estrus, mean values for total ova, viable and degenerate embryos and unfertilized ova are presented in Table 1, differences among the three groups were not detected (P ≥ 0.05). Large variations in these parameters were observed both between the animals of the same group and between animals in different groups of the viable embryos alone, among from 0–28 (TI), 0–30 (TII) and 0–28 (TIII) (Tables 2 and 3). Table 1 Mean (±S.E.M.) values for the total embryos/ova, viable and degenerate embryos and number of unfertilized ova after superovulation, using the standard protocol (TI) or by the synchronization of follicular wave emergence using SMB (TII) or CIDR-B (TIII) Groups
TI TII TIII
Donors (n)
Total of ova
15.8 ± 10.8 15.6 ± 9.7 17.3 ± 8.8
60 60 60
Embryos
Unfertilized ova
Viable
Degenerate
8.3 ± 7.4 8.9 ± 7.5 9.9 ± 7.0
3.0 ± 4.2 2.7 ± 3.3 3.0 ± 2.6
4.4 ± 5.7 3.9 ± 5.1 4.4 ± 5.0
Table 2 Mean (±S.E.M.) values of total viable and degenerate embryos and the total number of unfertilized ova in the females which did no show estrus after superovulation, using the standard protocol (TI) and following synchronization of follicular wave emergence with SMB (TII) or CIDR-B (TIII) Groups
TI TII TIII
Donors (n)
Total of ova
3.5 ± 3.3 11.5 ± 7.8 11.7 ± 10.2
4 4 3
Embryos
Unfertilized ova
Viable
Degenerate
0.7 ± 1.0 9.2 ± 8.4 6.7 ± 5.8
1.0 ± 1.1 1.0 ± 0.8 2.3 ± 2.5
1.7 ± 2.1 1.2 ± 1.3 2.7 ± 2.3
Table 3 Pregnancy rates with embryos collected from Nelore cows submitted to a standard superovulatory protocol (TI) and submitted to synchronization of follicular wave emergence with SMB (TII) or CIDR-B (TIII) Group
TI TII TIII
Embryos
Pregnancy, n (%)
Viable (n)
Transferred (n)
Frozen (n)
501 534 593
378 428 375
123 106 218
235 (62.2) 284 (66.4) 244 (65.1)
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4. Discussion Results support the hypothesis, that the use of steroid hormone combinations for the synchronization of ‘ovarian’ follicular wave emergence in Nelore donors does not result in an increase in the number of degenerate embryos and unfertilized ova. Both the protocols employed for the synchronization of follicular wave emergence in the donors eliminated the need to know the exact stage of each animal’s estrus cycle at the time gonadotrophin treatments were initiated, an indispensable condition that must be calculated when using the conventional superovulation protocol. Thus, these techniques permit the superovulation of large numbers of donors in a reduced period of time, making them more convenient for the veterinarian and the owner. Furthermore, these methods may allow the collection and transfer of embryos in large herds in a practical manner, since they may be implemented in separate properties almost simultaneously. As a consequence, the costs of an embryo transfer program can be reduced due to the reduced time involved. In addition, the interval between embryo collections in the same donor may be decreased since the protocol does not depend on the time of the previous estrus (Andrade and Oliveira, 1998). A possible justification for the results of this study, previously hypothesized by Bo et al. (1996), is that the steroid hormones promote the emergence of a follicular wave after treatment in a previously known time, a situation which occurs naturally in animals between the 9th and the 12th days of detection of estrus. Another relevant aspect of the protocols that utilize combinations of steroid hormones is the fact that the females which do not undergo estrus after treatment produced a similar number of viable embryos than the females which demonstrated estrus. However, it does not occur with the treated donors using a conventional protocol of superovulation. The percentage of females which did not undergo estrus was less than previously reported by Azevedo and Coelho (1991) and Tahira and Hackett (1993a,b), but was similar to those obtained by Butzke and Cardoso (1996). The greatest problem with superovulation is the large degree of variation in superovulatory response between individuals of the same species (Bowen and Pineda, 1989; Mapletoft et al., 1991). Despite the variation in response between the donors observed in this study, we may suggest both the CIDR and SMB programs efficiently synchronized the emergence of follicular waves, thus making their use in Nelore (Bos indicus) embryo transfer programs viable, a theory previously defended by Bo et al. (1995), Broadbent et al. (1995) and Bo et al. (1996) with females of European races (Bos taurus). Pregnancy rates obtained are comparable with those reported by Azevedo and Coelho (1991) and Andrade and Oliveira (1998) and support the hypothesis that the synchronization of follicular wave emergence, with a combination of progestagen/estrogen, does not interfere with the morphology of the embryo, nor with its developmental capacity in vivo. The results of this study allow us to conclude that a combination of steroid hormones may be used to synchronize the emergence of follicular waves in embryo transfer programs of Nelore donors for superovulation. These treatment protocols also minimizes costs of embryo transfer by permitting the use of a large number of donors in a short period of time, making it a valuable tool for animal production programs that use this technology.
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References Adams, G.P., 1994. Control of ovarian follicular wave dynamics in cattle: implications for synchonization and superstimulation. Theriogenology 41, 19–24. Andrade, J.C.O., Oliveira, M.A.L., 1998. Synchronization of the superovulation of zebu donors through association between steroid hormones. Arq. Fac. Vet. UFRGS 26, 217. Azevedo, N.S., Coelho, E.N., 1991. Superovulation e sincronização de cio em bovinos. In: Proceedings of the 9th Congresso Brasileiro de Reprodução Animal, Belo Horizonte, pp. 46–59. Bo, G.A., Adams, G.P., Nasser, L.F., Pierson, R.A., Mapletoft, R.J., 1993. Effect of estradiol valerate on ovarian follicles, emegence of follicular waves and circulating gonadotropins in heifers. Theriogenology 40, 225–239. Bo, G.A., Martinez, M., Nasser, L.F., Caccia, M., Tribulo, H., Mapletoft, R.J., 1993. Follicular dynamics of Bos indicus and Bos taurus beef cattle under pasture conditions in Argentina. In: Proceedings of the 10th Congresso Brasileiro de Reprodução Animal, Belo Horizonte, 221 pp. Bo, G.A., Adams, G.P., Pierson, R.A., Tribulo, H.E., Caccia, M., Mapletoft, R.J., 1994. Follicular waves dynamics after estradiol-17 treatment of heifers with or without a progestogen implant. Theriogenology 41, 1555–1569. Bo, G.A., Adams, G.P., Pierson, R.A., Mapletoft, R.J., 1995. Exogenous control of follicular wave emergence in cattle. Theriogenology 43, 32–40. Bo, G.A., Bergfelt, D.R., Mapletoft, R.J., 1996. Follicle wave dynamics and superovulation in cattle: recent advances and practical experience. Arq. Fac. Vet. UFRGS 24, 31–52. Bowen, R.A., Pineda, M.H., 1989. Embryo transfer in domestic animals. In: McDonald, L.E., Pineda, M.H. (Eds.), Veterinary Endocrinology and Reproduction. Lea & Febiger, Philadelphia, pp. 526–555. Broadbent, P.J., Gebbie, F.E., Dolman, D.F., Watt, R.G., King, M.E., Higgins, L.C., 1995. Superovulatory responses in cattle pre-treated with estradiol and progestagen. Theriogenology 43, 176. Butzke, G., Cardoso, C.R.S., 1996. Comparação dos resultados obtidos com o uso de três produtos comerciais para superovulação de vacas e novilhas. Arq. Facul. Vet. UFRGS 24, 198–203. Caccia, M., Bo, G.A., 1998. Follicle emergence following treatment of CIDR-B implanted beef cows with estradiol benzoate and progesterone. Theriogenology 49, 341. Carriere, P.D., Amaya, D., Lee, B., 1995. Ultrasonography and endocrinology of ovarian disfunctions induced in heifers with Estradiol Valerate. Theriogenology 43, 1061–1076. Figueiredo, R.A., Barros, C.M., Pinheiro, O.L., Soler, J.M.P., 1997. Ovarian follicular dynamics in Nelore breed (Bos indicus) cattle. Theriogenology 47, 1489–1505. Fortune, J.E., 1993. Follicular dynamics during the bovine estrus cycle: a limiting factor in improvement of fertility. Anim. Reprod. Sci. 33, 111–125. Mapletoft, R.J., Bo, G., Murphy, B.D., 1991. The effect of biological activity of gonadotrophins on superovulation in the cow. Rev. Bras. Reprod. Anim. 15, 74–92. Mapletoft, R.J., Martinez, M.F., Adams, G.P., Kastelic, J., Burnley, C.A., 1999. The effect of estradiol on follicular wave emergence and superovulatory response in norgestomet-impanted cattle. Theriogenology 51, 411. Robertson, I., Nelson, R.E., 1998. Certificação e identificação de embriões. In: Stringfellow, D.A., Seidel, S.M. (Eds.), Manual da Sociedade Internacional de Transferência de Embriões, pp. 109–140. Statistical Analysis Systems Institute, 1988. SAS user’s guide: statistics. SAS Institute, Cary, NC. Tahira, J.K., Hackett, A.J., 1993a. Superovulação de vacas Canchin com FSH-p. Rev. Bras. Reprod. Anim. 17, 33–39. Tahira, J.K., Hackett, A.J., 1993b. Superovulação de vacas Canchin com folltropin. Rev. Bras. Reprod. Anim. 17, 41–47.
Use of steroid hormone treatments prior to superovulation in Nelore donors J.C.O. Andrade a , M.A.L. Oliveira a,c,∗ , P.F. Lima a,c , A.S. Santos Filho a , V.M.R. Pina b a
Programa de Pós-Graduação em Ciência Veterinária - UFRPE, Av. D. Manoel de Medeiros s/n, Dois Irmãos, CEP 56500.000 Recife, PE, Brazil b Curso de Graduação em Medicina Veterinária - UFRPE, Av. D. Manoel de Medeiros s/n, Dois Irmãos, CEP 56500.000 Recife, PE, Brazil c Departmento de Medicina Veterinaria - UFRPE, Av. D. Manoel de Medeiros s/n, Dois Irmãos, CEP 56500.000 Recife, PE, Brazil
Received 20 January 2001; received in revised form 4 September 2001; accepted 17 September 2001
Abstract The objective of this study was to evaluate the effectiveness of synchronization of follicular wave emergence using steroid hormone treatments in Nelore cows. Donors were placed into three groups. Those that were between days 9 and 12 of their cycle (estrus = day 0) formed the TI group (n = 60), whilst those that were in any other stages of their estrus cycle constituted groups TII (n = 60) and TIII (n = 60). TI donors were submitted to a standard protocol of superovulation, however, TII and TIII donors were treated with the Syncro-Mate-B (SMB) or Controlled Internal Drug Releasing Device (CIDR-B) programs, respectively. Superovulation was induced with p-FSH, divided into eight decreasing doses at intervals of 12 h. The donors received cloprostenol 48 h after the beginning of the treatment and progestagens were removed 12 h later. Artificial inseminations (AI) were done at 12 and 22 h after the initiation of estrus and the embryo collections were done 7 days after AI. In the donors which displayed behavioral estrus, mean (±S.E.M.) total ova and viable (transferable) embryos were 15.8 ± 1.4 and 8.3 ± 1.0 (TI, n = 56); 15.6 ± 1.3 and 8.9 ± 1.0 (TII, n = 56); 17.3 ± 1.0 and 9.9 ± 0.9 (TIII, n = 57), respectively, with no significant difference (P ≥ 0.05) among groups. In those animals that did not displayed behavioral estrus, the mean values of total ova and viable embryos were 3.5 ± 1.6 and 0.7 ± 0.5 (TI, n = 4); 11.5 ± 3.9 and 9.0 ± 4.4 (TII, n = 4); 8.7 ± 5.0 and 5.0 ± 2.9 (TIII, n = 3), respectively, with no significant differences (P ≥ 0.05) among groups. Pregnancy rates of 62.2% (TI, n = 235); 66.4% (TII, n = 284) and 65.1% (TIII, n = 244) were obtained with embryos transferred from these collections and did not differ significantly (P ≥ 0.05) among groups. It was concluded that the synchronization
∗ Corresponding author. E-mail address: [email protected] (M.A.L. Oliveira).
0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 1 4 9 - X
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J.C.O. Andrade et al. / Animal Reproduction Science 69 (2002) 9–14
of the emergence of follicular waves in Nelore donors is usable and does not harm the efficiency of embryo transfer programs. In addition, in contrast to the standard superovulation protocol, this method permits the use of a large number of donors in a short time period, at any stage of the estrus cycle, minimizing the costs of embryo transfer. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Bos indicus; Embryo; Syncro-Mate-B; Controlled Internal Drug Releasing Device
1. Introduction The advent of ultrasonography has led to a better understanding of diverse reproductive events, in particular, follicular dynamics in cattle. Follicular development in cattle is characterized by the occurrence of two or three growth waves during the estrus cycle (Fortune, 1993). In Nelore cows, Figueiredo et al. (1997) observed a predominant pattern of two ovarian follicular growth waves, while Bo et al. (1993) reported that a pattern of three follicular growth waves prevails over that of two ‘such waves’ in Brahman cows. Superovulation, a factor which may limit the efficiency of embryo transfer, continues to be a common topic for research, particularly in relation to the time of initiating exogenous hormonal stimulation. Superovulation must be initiated on the day prior to follicular wave emergence (Adams, 1994) or on the day of follicular wave emergence (Adams, 1994; Bo et al., 1995), before the subordinate follicles begin the atresie process (Bo et al., 1995). The synchronization of follicular wave emergence is done by the removal of the suppressive effect of the dominant follicle over the growth of the next wave (Bo et al., 1995). Several techniques have been used to promote the emergence of a new follicular wave at a specific time including the administration of hormones such as estrogens (Bo et al., 1993, 1994; Carriere et al., 1995), estrogens and progesterone (Bo et al., 1996) because these steroid hormones are capable of promoting the emergence of a new follicular wave by causing regression of antral follicles. Combinations of between progesterone/estrogen or progestagen/estrogen have gained special attention in protocols for superovulation in cattle (Bo et al., 1995, 1996; Broadbent et al., 1995). When progesterone or progestagen are used in combination with a 17-estradiol, the emergence of a new follicular wave occurs around 4 days after treatment but occurs 5 days after treatment, when estradiol benzoate and estradiol valerate are used (Bo et al., 1995, 1996; Caccia and Bo, 1998; Mapletoft et al., 1999). In Brazil, the size of the country and the lack of technicians trained in the basics of embryo transfer have limited the diffusion of this technology in some regions. Elaborate protocols which allow the use of a larger number of donors per program, such as the synchronization of follicular wave emergence using steroid hormones, has minimized the routine problem of control of the estrus cycle in large herds in the northern region of the country. This type of protocol allows the veterinarian, even when far from the property to determine the most suitable period for the performance of the embryo transfer program (Andrade and Oliveira, 1998). Thus, the lack of superovulatory protocols for the synchronization of the emergence of follicular waves in females of the Nelore breed motivated this study, which aimed to compare the superovulatory response, ova/embryo recovery and number of transferable embryos, in donors superovulated by the conventional protocol on days 9–12 of cycle,
J.C.O. Andrade et al. / Animal Reproduction Science 69 (2002) 9–14
11
those submitted to the synchronization of follicular wave emergence with steroid hormones prior to gonadotropin treatment.
2. Materials and methods One hundred and eighty superovulation treatments were analyzed in females of the Nelore breed. Animals presented regular estrus cycles and were between 6 and 10 years of age, with body condition scores ranging from 3 to 4 on a scale of 1–5. All animals were submitted to clinical-gynecological examinations before the experiment. Animals were kept on pasture, where they were fed on Braquiaria brisanta grass and had free access to water and mineral salts. The 180 donors were equally distributed into three groups according to the stage of their estrus cycle. Donors between the 9th and 12th days of their cycle formed the TI group (standard superovulatory protocol), while the remaining 120 donors, at all other stages of the estrus cycle, were randomly distributed into two groups, TII and TIII (submitted to protocols for synchronization of the emergence of the follicular wave). The TII group was submitted to the SMB program (Rhodia–Mérieux—Veterinary LTDA), which consisted of a subcutaneous auricular implant of 6 mg norgestomet and an intramuscular injection of 3 mg norgestomet and 5 mg estradiol valerate. TIII animals were submitted to the CIDR-B program (InterAg—Edvet HD), consisting of an intravaginal device, containing 1.9 g progesterone, and an intramuscular injection of 4 mg estradiol benzoate (Estrogin—Farmavet Veterinary Products LTDA) administered 24 h after the introduction of the vaginal insertion. Gonadotropin treatment began between the 9th and 12th days of the estrus cycle in those animals undergoing the standard superovulatory protocol (TI) and 5 days after the administration of the SMB implant (TII) and the insertion of the CIDR-B device (TIII). In all the groups, superovulation included intramuscular injections of 400 p-FSH (Pluset—Serono Pharmaceutical Products LTDA) administered in eight decreasing doses (80, 60, 40, and 20 BID) at intervals of 12 h. After the 48 h of beginning of this treatment, donors received 1 mg cloprostenol (PGF2␣ -Ciosin-Coopers, Mallinckrodt Vet LTD) via intramuscular injection and, 12 h later, implants in the TII and devices in the TIII groups were removed. Artificial inseminations were performed with commercial semen at 12 and 22 h after the detection of estrus by teaser animals using a chin ball. Donors that did not display behavioral estrus within 54 h of PGF2␣ injection were treated intramuscularly with 0.02 mg busereline acetate (Conceptal—Hoechst Roussel Vet) and inseminated at 6, 12 and 18 h later. Embryos were collected 7 days after the first artificial insemination (AI) and were then evaluated and classified according to development and morphology (Robertson and Nelson, 1998), then transferred immediately to recipients by via transcervical method. The collected embryos not used were frozen. The recipient synchrony, obtained with PGF2␣ administration, was maintained between (−) 2 days before and (+) 2 days after the estrus cycles of the donors. Data were analyzed using the PROC GLM option of the Statistical Analysis Systems (1988). The numbers of total ova, viable (transferable) embryos, degenerate embryos and unfertilized ova were compared using the Kruskal–Wallis test because the data were not distributed normality, which was verified using the Shapiro Wilks test. Pregnancy rates were
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J.C.O. Andrade et al. / Animal Reproduction Science 69 (2002) 9–14
analyzed with the contingency table for the Chi-squared test (χ 2 ). A confidence interval of 5% or less was considered to be statistically significant.
3. Results In the females, which displayed behavioral estrus, mean values for total ova, viable and degenerate embryos and unfertilized ova are presented in Table 1, differences among the three groups were not detected (P ≥ 0.05). Large variations in these parameters were observed both between the animals of the same group and between animals in different groups of the viable embryos alone, among from 0–28 (TI), 0–30 (TII) and 0–28 (TIII) (Tables 2 and 3). Table 1 Mean (±S.E.M.) values for the total embryos/ova, viable and degenerate embryos and number of unfertilized ova after superovulation, using the standard protocol (TI) or by the synchronization of follicular wave emergence using SMB (TII) or CIDR-B (TIII) Groups
TI TII TIII
Donors (n)
Total of ova
15.8 ± 10.8 15.6 ± 9.7 17.3 ± 8.8
60 60 60
Embryos
Unfertilized ova
Viable
Degenerate
8.3 ± 7.4 8.9 ± 7.5 9.9 ± 7.0
3.0 ± 4.2 2.7 ± 3.3 3.0 ± 2.6
4.4 ± 5.7 3.9 ± 5.1 4.4 ± 5.0
Table 2 Mean (±S.E.M.) values of total viable and degenerate embryos and the total number of unfertilized ova in the females which did no show estrus after superovulation, using the standard protocol (TI) and following synchronization of follicular wave emergence with SMB (TII) or CIDR-B (TIII) Groups
TI TII TIII
Donors (n)
Total of ova
3.5 ± 3.3 11.5 ± 7.8 11.7 ± 10.2
4 4 3
Embryos
Unfertilized ova
Viable
Degenerate
0.7 ± 1.0 9.2 ± 8.4 6.7 ± 5.8
1.0 ± 1.1 1.0 ± 0.8 2.3 ± 2.5
1.7 ± 2.1 1.2 ± 1.3 2.7 ± 2.3
Table 3 Pregnancy rates with embryos collected from Nelore cows submitted to a standard superovulatory protocol (TI) and submitted to synchronization of follicular wave emergence with SMB (TII) or CIDR-B (TIII) Group
TI TII TIII
Embryos
Pregnancy, n (%)
Viable (n)
Transferred (n)
Frozen (n)
501 534 593
378 428 375
123 106 218
235 (62.2) 284 (66.4) 244 (65.1)
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4. Discussion Results support the hypothesis, that the use of steroid hormone combinations for the synchronization of ‘ovarian’ follicular wave emergence in Nelore donors does not result in an increase in the number of degenerate embryos and unfertilized ova. Both the protocols employed for the synchronization of follicular wave emergence in the donors eliminated the need to know the exact stage of each animal’s estrus cycle at the time gonadotrophin treatments were initiated, an indispensable condition that must be calculated when using the conventional superovulation protocol. Thus, these techniques permit the superovulation of large numbers of donors in a reduced period of time, making them more convenient for the veterinarian and the owner. Furthermore, these methods may allow the collection and transfer of embryos in large herds in a practical manner, since they may be implemented in separate properties almost simultaneously. As a consequence, the costs of an embryo transfer program can be reduced due to the reduced time involved. In addition, the interval between embryo collections in the same donor may be decreased since the protocol does not depend on the time of the previous estrus (Andrade and Oliveira, 1998). A possible justification for the results of this study, previously hypothesized by Bo et al. (1996), is that the steroid hormones promote the emergence of a follicular wave after treatment in a previously known time, a situation which occurs naturally in animals between the 9th and the 12th days of detection of estrus. Another relevant aspect of the protocols that utilize combinations of steroid hormones is the fact that the females which do not undergo estrus after treatment produced a similar number of viable embryos than the females which demonstrated estrus. However, it does not occur with the treated donors using a conventional protocol of superovulation. The percentage of females which did not undergo estrus was less than previously reported by Azevedo and Coelho (1991) and Tahira and Hackett (1993a,b), but was similar to those obtained by Butzke and Cardoso (1996). The greatest problem with superovulation is the large degree of variation in superovulatory response between individuals of the same species (Bowen and Pineda, 1989; Mapletoft et al., 1991). Despite the variation in response between the donors observed in this study, we may suggest both the CIDR and SMB programs efficiently synchronized the emergence of follicular waves, thus making their use in Nelore (Bos indicus) embryo transfer programs viable, a theory previously defended by Bo et al. (1995), Broadbent et al. (1995) and Bo et al. (1996) with females of European races (Bos taurus). Pregnancy rates obtained are comparable with those reported by Azevedo and Coelho (1991) and Andrade and Oliveira (1998) and support the hypothesis that the synchronization of follicular wave emergence, with a combination of progestagen/estrogen, does not interfere with the morphology of the embryo, nor with its developmental capacity in vivo. The results of this study allow us to conclude that a combination of steroid hormones may be used to synchronize the emergence of follicular waves in embryo transfer programs of Nelore donors for superovulation. These treatment protocols also minimizes costs of embryo transfer by permitting the use of a large number of donors in a short period of time, making it a valuable tool for animal production programs that use this technology.
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