Doi:10.1016/s0378-4320(03)00036-8

Animal Reproduction Science 77 (2003) 117–125 The use of steroid hormones in superovulation of Nelore donors at different stages of estrous cycle J.C.O. Andrade , M.A.L. Oliveira , P.F. Lima , S.I. Guido , C.C. Bartolomeu , F. Tenório Filho , V.M.R. Pina , T.C. Iunes-Souza , N.R.O. Paula , J.C.C. Freitas a Programa de Pós-Graduação em Ciˆencia Veterinária, UFRPE, Av. D. Manoel de Medeiros s/n, Dois Irmãos, CEP 52171-900 Recife-PE, Brazil b Curso de Graduação em Medicina Veterinária, UFRPE, Av. D. Manoel de Medeiros s/n, Dois Irmãos, CEP 52171-900 Recife-PE, Brazil c Curso de Graduação em Medicina Veterinária, UECE, Av. Piranjana, 1700 Parangaba, CEP 60740-000 Fortaleza-CE, Brazil d Departmento de Medicina Veterinária, UFRPE, Av. D. Manoel de Medeiros s/n, Dois Irmãos, e Departamento de Ciˆencias Agrárias e Ambientais, UESC, Rodovia Ilhéus-Itabuna, km 16, Salobrinho, CEP 45660-000 Ilhéus-BA, Brazil Received 14 October 2002; received in revised form 28 December 2002; accepted 13 January 2003 Abstract
The objective of the present study was to evaluate the superovulatory response and ova/embryo recovery from Nelore donors following treatment with a controlled internal drug releasing deviceand estradiol benzoate (CIDR-B program) at different stages of the estrous cycle. The control group(TI; n = 40) received a standard superovulation protocol with females of this group being betweendays 9 and 12 of the estrous cycle (estrus = day 0). The donors that received a CIDR-B programcontaining 1.9 g progesterone and an intramuscular injection of estradiol benzoate (2 mg) were atday 0 (TII; n = 30), between days 2 and 6 (TIII; n = 30), days 7 and 12 (TIV; n = 30), days 13and 16 (TV; n = 30) and days 17 and 20 (TVI; n = 30) of the estrous cycle. Superovulation wasinduced with 400 IU of p-FSH, divided into eight decreasing doses (80/80; 60/60; 40/40; 20/20) atintervals of 12 h. The donors received PGF2␣ (Cloprostenol) 48 h after beginning the treatment andCIDRs were removed 12 h later. Artificial inseminations (AI) were performed 12 and 22 h after theinitiation of estrus and embryos were collected 7 days after AI. The mean numbers (±S.E.M.) oftotal ova and embryos, viable (transferable) and degenerated embryos were 14.2±11.3, 7.4 ± 6.9and 3.2 ± 3.5 (TI), 13.3 ± 10.4, 7.1 ± 6.2 and 3.3 ± 4.3 (TII), 13.5 ± 7.0, 8.1 ± 6.7 and 2.3 ± 3.0(TIII), 17.4 ± 9.9, 9.4 ± 6.9 and 4.0 ± 4.4 (TIV), 16.9 ± 8.8, 9.8 ± 8.1 and 2.7 ± 2.5 (TV) and E-mail address: maloufrpe@uol.com.br (M.A.L. Oliveira).
0378-4320/03/$ – see front matter 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-4320(03)00036-8 J.C.O. Andrade et al. / Animal Reproduction Science 77 (2003) 117–125 13.0 ± 7.8, 7.2 ± 6.9 and 2.3 ± 2.5 (TVI), with no significant differences (P ≥ 0.05) among groups.
Pregnancy rates of 67.1% (TI; n = 86/128), 60.8% (TII; n = 59/97), 62.5% (TIII; n = 73/115),64.1% (TIV; n = 84/131), 72.3% (TV; n = 81/112) and 60.6% (TVI; n = 63/104) were obtainedwith embryos transferred from these collections and did not differ significantly (P ≥ 0.05) amonggroups. The results of the present study allow us to conclude that a combination of steroid hormonesmay be used prior to superovulation in Nelore donors, at any stage of the estrous cycle withoutaffecting the efficiency of embryo transfer programs.
2003 Elsevier Science B.V. All rights reserved.
Keywords: Zebu; Progesterone; Estrogen; Embryo 1. Introduction
Superovulation is an efficient technique for obtaining progeny from genetically valuable females. The ovarian response of each female depends on the number of gonadotropin-sen-sitive follicles present at the time that treatment is initiated. Identification of the number ofsuch follicles in each female would improve the efficacy of superovulation, by allocatingpotential non-responders to groups where other techniques are used for superovulation.
One of the main factors influencing response to superovulation is stage of the follicularwave when gonadotropin treatments are given. Treatment in the absence of a dominantfollicle generally enhances the response to superovulation. The development of practicalapproaches to achieve enhanced responses to superovulation protocols still require furtherresearch ( Superovulation in cattle is a factor, which may limit the efficiency of embryo transfer and must be initiated on the day prior or on the day, of follicular wave emergencethe subordinate follicles begin the process of atresiaDifferences in the stage of follicle and oocyte maturation are oftenobserved at the beginning of superovulatory treatments (may resultin the presence of follicles at different stages of development at the time of the preovulatoryLH surge.
Experiments and field studies performed during the 1980s concluded that superovulatory treatments initiated 9–10 days after estrous detection resulted in a greater superovulatoryresponse as compared with those initiated 2–6 or 12–13 days after estrous detection This is due to the occurrence of two to three (and even four in zebu) follicularwaves during the estrous cycle and, in the majority of these animals, the second follicularwave begins between days 9 and 10 of the estrous cycle ). In Nelore cows, reporteda predominant pattern of two ovarian follicular growth waves. The synchronization offollicular wave emergence is achieved by the removal of the suppressive effect of thedominant follicle over the growth of the next follicular wave (One of themost promising strategies is the use of hormone treatments to synchronize the follicularwave so that it begins at the start of superovulation (Administration of steroidhormones has been used to promote the emergence of a new follicular wave at a specific timebecause these hormones are capable ofpromoting the emergence of a new follicular wave by causing regression of antral follicles.
J.C.O. Andrade et al. / Animal Reproduction Science 77 (2003) 117–125 The steroid hormones also have a luteolytic action, particularly when estradiol valerate isused (probably as a result of increasing prostaglandin secretion), as well as acting directlyon the CL, making it more susceptible to luteolysis ( Combinations of progesterone/estrogen have gained special attention in protocols for superovulation in cattle (; When progesterone is used in combination with estradiolbenzoate, the emergence of a new follicular wave occurs around 5 days after treatment(The progesterone/estrogen combination used in the cattle super-ovulatory protocol has enhanced the efficiency of the embryo transfer program, minimizingthe routine problem of control of the estrous cycle in large herds ( In recent years, a number of studies determined the efficiency of steroid hormones in the superovulatory protocols of Nelore cattle (However, because the administration of steroid hormones was performed withoutcontrol of estrous cycle stage, it remains necessary to determine if the effectiveness of thistreatment is equivalent at all stages of the estrous cycle (the aimof the present study was to observe the efficiency of steroid hormones on the superovulatoryresponse of Nelore donors when administered at different stages of the estrous cycle. Thesuperovulatory response, as measured by ova/embryo recovery and number of transferableembryos was the variable used to evaluate the effect of a progesterone/estrogen combinationadministered before gonadotrophin treatment.
2. Materials and methods
Superovulation treatments were analyzed in females (n = 180) of the Nelore breed.
Animals had presented regular estrous cycles and were between 5 and 10 years of agewith 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 grazed Braquiaria brisanta grass and had freeaccess to water and mineral salts.
The 180 donors, submitted to a daily control of estrous cycle using sterilized males equipped with a chin ball-marking device, were equally distributed into six groups accordingto the stage of their estrous cycle. Donors between the 9th and 12th day (n = 30) of theircycle (estrus = day 0) formed the control group (TI), while the remaining 150 donors (30 pergroup), at all other stages of the estrous cycle, were distributed into five treatment groups.
Donors at day 0 (TII), between days 2 and 6 (TIII), days 7 and 12 (TIV), days 13 and 16 (TV),as well as between days 17 and 20 (TVI) of their estrous cycle were treated using a protocolwhich has been reported to synchronize emergence of a new follicular wave. The donorstreated with the CIDR-B program (InterAg, Edvet HD) received an intravaginal devicecontaining 1.9 g of progesterone and an intramuscular injection of 4 mg estradiol benzoate(Estrogin, Farmavet Veterinary Products LTDA) administered 24 h after the introduction ofthe CIDR device.
Gonadotrophin treatment began between on days 9 and 12 of the estrous cycle in those animals undergoing the standard superovulatory protocol (TI) and 5 days after the insertion J.C.O. Andrade et al. / Animal Reproduction Science 77 (2003) 117–125 of the CIDR-B device in the other groups (TII, TIII, TIV, TV, TVI). In all groups, su-perovulation was induced by intramuscular injections of 400 IU of p-FSH (Pluset, SeronoPharmaceutical Products LTDA) administered in eight decreasing doses (80/80; 60/60;40/40; 20/20) at intervals of 12 h. Forty-eight hours after the beginning of this treatment,donors received 1 mg Cloprostenol (PGF2␣, Ciosin, Coopers, Mallinckrodt Vet LTD) viaintramuscular injection and, 12 h later, CIDR devices were removed from groups TII, TIII,TIV, TV and TVI.
Two trained technicians performed the artificial inseminations (AI) with commercial semen from three bulls, equally distributed into six experimental groups, at 12 and 22 hafter detection of estrus by sterilized males equipped with a chin ball-marking device.
Donors that did not display estrus behavior within 54 h after the 1 mg of PGF2␣ injectionadministered intramuscularly were treated intramuscularly with 0.02 mg buserelin acetate(Conceptal, Hoechst Roussel Vet) and inseminated 6, 12 and 18 h later.
Embryos were collected 7 days after the first AI and were then evaluated and classified according to development and morphology Only blastocystof grade I were transferred to recipients by the transcervical method and the others viableembryos which were not transferred were frozen. The recipient estrous synchrony, obtainedusing PGF2␣ administration, was between (−) 2 days before and (+) 2 days after the timeof insemination.
Data were analyzed using the PROC GLM option of The numbers of total ova, viable (transferable) embryos, degenerate embryos and unfer-tilized ova were compared using the Kruskal–Wallis test since data were not distributednormally (verified using the Shapiro–Wilks test). Pregnancy rates were analyzed using thecontingency table for the χ2-test. A confidence interval of 5% or less was considered to bestatistically significant.
3. Results
Data from the females that displayed or did not display behavioral estrus, mean numbers of total ova and embryos, viable and degenerate embryos and unfertilized ova are presentedin ferences among the six groups were not detected (P ≥ 0.05). The females that Table 1Mean (±S.E.M.) numbers of total embryos/ova, viable and degenerated embryos and number of unfertilized ovain the females which displayed estrus or did not display estrus after superovulation, using the standard protocol(TI) or using a steroid hormone combination prior to superovulation (TII, TIII, TIV, TV, TVI) J.C.O. Andrade et al. / Animal Reproduction Science 77 (2003) 117–125 Table 2Mean (±S.E.M.) numbers of total embryos/ova, viable and degenerated embryos and the total number of unfer-tilized ova in the females which did not display estrus after superovulation, using the standard protocol (TI) orusing a steroid hormone combination before superovulation (TII, TIII, TIV, TV, TVI) Table 3Pregnancy rates with embryos collected from Nelore cows submitted to a standard superovulatory protocol (TI)or submitted to a treatment with a steroid hormone combination prior to superovulation (TII, TIII, TIV, TV, TVI) did not display estrus total embryos/ova, viable embryos, degenerated embryosand unfertilized ova also did not differ (P ≥ 0.05) among groups. Large variations in thenumber of viable embryos were observed both among animals of the same group and amonganimals in different groups; these ranged from 0 to 33 (TI), 0 to 31 (TII), 0 to 28 (TIII), 0to 29 (TIV), 0 to26 (TV) and 0 to 30 (TVI).
Some embryos were transferred fresh and others were frozen for transfer at a later time (There were no differences in pregnancy rates with embryos collected from thesix superovulatory groups.
4. Discussion
The data from the present study indicate that the combination of progesterone/estrogen treatment can be effectively used in superovulatory protocol in Nelore donors probably asa consequence of the synchronization of follicular wave emergence. Previous authors (ave hypothesized that the steroid hormones promotethe emergence of a follicular wave after treatment at a previously known time, a situation,which occurs naturally in animals between the days 9 and the 12 after detection of estrus.
Another relevant aspect of the superovulatory protocol using the progesterone/estrogen J.C.O. Andrade et al. / Animal Reproduction Science 77 (2003) 117–125 combination, is the fact that it eliminates the need to know the exact stage of the estrouscycle of each animal at the start of gonadotropin treatments, a factor that must be calculatedwhen using the conventional superovulation protocol. Thus, this technique allows the su-perovulation of a large number of donors in a reduced period of time, making it convenientfor the veterinarian and the farmer. Furthermore, these methods may allow the collectionand transfer of embryos in large herds in a practical manner since they may be implementedon separate properties almost simultaneously. As a consequence, the costs of an embryotransfer program can be reduced due to the reduced time involved. In addition, the intervalbetween embryo collections in the same donor may be decreased becasue the protocol doesnot depend on the time of the previous estrus ( The use of steroid hormones prior to superovulation of Nelore donors does not harm the efficiency of embryo transfer program because the number of degenerated embryos andunfertilized ova are not increased (Results of the present study agree with the previous data and it isimportant to emphasize that pregnancy rates obtained are comparable with those reported byand support the hypothesis that the synchronization of follicular wave emergence, witha combination of progesterone/estrogen, does not interfere with the morphology of theembryo, nor with its developmental capacity in vivo.
that both progesterone and estrogen can regulate the emergence of the ovulatory follicle and prevent the ovulation of dominant ovarian folliclespresent at the time of treatment in Bos indicus cows. These results are consistent withthe findings of others who have used either estrogen or progesteron to regulate ovarianfolliclar development in Bos taurus females The ability of the combination of progesterone and estrogen to promote the regression of the dominant follicle is based on the fact that lesser mean concentrations of LH and greatermean concentrations of FSH are detected 24 h after treatment Asdominant follicles are thought to be critically dependent on LH for continued survivalincreasing LH pulse frequency can prolong the life of dominantfollicles whereas a reduction in LH pulse frequency is associated with the induction ofatresia among dominant follicles a greater effect of progesterone compared with estrogen in suppressing LH pulse frequency, whilst that estrogen functionsdirectly at the ovarian follicle to cause atresia. These findings are consistent with those of thecurrent study where treatments occurred at different stages of the estrous cycle in animalspresenting different follicle categories. The treatment efficiently promoted the regression ofdominant follicles and the start of a new follicle wave so that the superovulatory treatmentwas started a time when no dominant follicle was present, resulting in acceptable embryorecovery in all females at different stages of follicle development.
As seen in the present study, the use of a protocol employing the combination of proges- terone and estrogen at different controlled stages of estrous cycle did not interfere with therecovery of viable embryos. This finding demonstrates the efficiency of estrogen in pro-moting the synchronous emergency of a new follicular wave, independently of the statusof follicular development at the start of treatment. This result corroborates those reported J.C.O. Andrade et al. / Animal Reproduction Science 77 (2003) 117–125 by relevant aspect of the protocols utilizing combi-nations of steroid hormones is the fact that those females that did not display estrus aftertreatment produced a similar number of viable embryos to those that demonstrated estrus.
However, this outcome is not observed in donors treated using the conventional superovu-lation protocol. The percentage of females which did not display estrus was less than thepercentages previously reported by and but was similar to those obtained by result was expected, since the ability of estradiol benzoate to inducebehavioral estrus in cattle is independent of ovarian follicular events.
A problem with regard to superovulation is the large degree of variation in superovulatory responses among individuals of the same species Despite the variation in responses between donors observed in the present study,results demonstrate that the CIDR-B program makes Nelore (Bos indicus) embryo transferprograms viable, a theory previously defended by and females of European races (Bos taurus).
Pregnancy rates obtained are compatible with those reported by support the hypothesisthat the superovulatory protocol using a combination of progesterone and estrogen, doesnot interfere with the morphology of the embryo, nor with its developmental capacity invivo.
The results of the present study allow us to conclude that a combination of steroid hor- mones may be used before superovulation in Nelore donors, at any stage of the estrouscycle without affecting efficiency of embryo transfer programs.
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