Superovulation and embryo transfer in Bos indicus cattle

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1 Theriogenology 65 (2006) Superovulation and embryo transfer in Bos indicus cattle Pietro S. Baruselli a, *, Manoel F. de Sá Filho a, Claudiney M. Martins a, Luiz F. Nasser a, Marcelo F.G. Nogueira b, Ciro M. Barros b, Gabriel A. Bó c a Departament of Animal Reproduction, FMVZ-USP, Rua Prof. Orlando Marques de Paiva, 87, CEP , Sao Paulo-SP, Brazil b Departamento de Farmacología, Instituto de Biociências, UNESP, Botucatu-SP, Brazil c Instituto de Reproducción Animal Córdoba, Universidad Católica de Córdoba, Córdoba, Argentina Abstract Compared to Bos taurus breeds, Bos indicus breeds of cattle present several differences in reproductive physiology. Follicular diameter at deviation and at the time of ovulatory capability are smaller in B. indicus breeds. Furthermore, B. indicus breeds have a greater sensitivity to gonadotropins, a shorter duration of estrus, and more often express estrus during the night. These differences must be considered when setting up embryo transfer programs for B. indicus cattle. In recent studies, we evaluated follicular dynamics and superovulatory responses in B. indicus donors with the objective of implementing fixed-time AI protocols in superstimulated donors. Protocols using estradiol and progesterone/progestrogen releasing devices to control follicular wave emergence were as efficacious as in B. taurus cattle, allowing the initiation of superstimulatory treatments (with lower dosages of FSH than in B. taurus donors) at a self-appointed time. Furthermore, results presented herein indicate that delaying the removal of progesterone/progestogen-releasing devices, combined with the administration of GnRH or plh 12 h after the last FSH injection, results in synchronous ovulations, permitting the application of fixed-time AI of donors without the necessity of estrus detection and without compromising the results. # 2005 Elsevier Inc. All rights reserved. Keywords: Superstimulation; Embryo transfer; Bos indicus; Fixed-time AI; Ultrasonography * Corresponding author. address: baruselli@usp.br (P.S. Baruselli) X/$ see front matter # 2005 Elsevier Inc. All rights reserved. doi: /j.theriogenology

2 78 P.S. Baruselli et al. / Theriogenology 65 (2006) Introduction Bovine embryo transfer has been applied widely around the world. This technology increases the number of offspring obtained from donors with high genetic value and is used to disseminate desirable genetics around the world. In Brazil and in other tropical countries, there has been an increasing demand to multiply the genetics of valuable Bos indicus females. However, there are important differences in the physiology and the reproductive behavior between B. indicus and Bos taurus cattle that can affect the efficiency of superstimulation programs. Traditional superstimulation protocols have some limitations: (1) necessity of handling and detecting estrus to establish the base heat, (2) inability to start superstimulatory treatments at the optimal time of follicular development, (3) necessity to detect estrus to determine time of AI, (4) high variability in embryo production per donor, and (5) 20 30% of unresponsive donors that do not produce embryos. 2. Factors that influence superovulatory response Variability in superovulatory responses after gonadotropin treatments continues to be the greatest problem for commercial embryo transfer [1 3]. Individual variation in superovulatory response has also been observed in Nelore cattle using a cross-over experimental design [4]. Numbers of CL, ova/embryos and embryos suitable for freezing varied significantly among donors. A recent study involving high producing Holstein cows in a cross-over experimental design in a tropical environment also reported significant individual variation in the number of follicles >8 mm in diameter at the time of estrus and in the number of CL at the time of ova/embryo collection [5]. In the conventional protocol for superstimulation, gonadotropin treatments are initiated during mid-cycle (8 12 d post-ovulation). This approach presents difficulties because it requires estrus detection prior to initiation of gonadotropin treatments, and because there is a great individual variation in the day of emergence of the second follicular wave. These difficulties can adversely affect superovulatory responses [3]. Several studies have demonstrated the importance of initiating gonadotropin treatments at the time of follicular wave emergence. The absence of a dominant follicle at the beginning of treatment increased the efficacy of the superstimulatory treatments [3,6]. Nasser et al. [7] obtained a higher superstimulatory response when gonadotropin treatments were initiated on the day of follicle wave emergence than when treatments were initiated 1 or 2 d later. Therefore, alternatives to control follicular wave emergence at random stages of the estrus cycle, without necessity to detect estrus to establish a base heat, would facilitate management of B. indicus donors, and possibly increase the efficiency of embryo transfer programs in cattle of Zebu breeding. 3. Control of follicular dynamics for superstimulation Mechanical (follicle ablation) [8] or pharmacological (GnRH) [9], LH, hcg or estradiol plus progesterone (P4) [10,11] methods of controlling follicular wave emergence have

3 P.S. Baruselli et al. / Theriogenology 65 (2006) been developed in the last 10 year. Treatment with GnRH resulted in ovulation of the dominant follicle and emergence of a new follicular wave 1 2 d later [12]. In B. taurus B. indicus heifers, the administration GnRH at random stages of the estrus cycle induced ovulation in only 45.7% (16/35) of heifers [13]. Low pregnancy rates following administration of GnRH have been observed in B. indicus cattle kept on tropical regions [14]. The results of these studies raise doubts about the efficacy of GnRH to synchronize wave emergence in B. indicus cattle. In B. indicus cattle, an elective treatment of induction of follicular wave emergence is the association between estradiol and P4. The efficacy of this association has been demonstrated in several studies in B. taurus cattle [10,11,15 17]. We have also studied the effects of estradiol and P4 treatment for the synchronization of follicular wave emergence in B. indicus, B. indicus B. taurus crosses, and B. taurus heifers that were kept under the same tropical conditions [18]. There were no differences in the mean interval from treatment to wave emergence between B. indicus, B. taurus or B. taurus B. indicus heifers. However, B. indicus heifers recruited more follicles into the wave than B. taurus heifers, suggesting the probability of a higher superstimulatory response following gonadotropin treatments in B. indicus cattle (Table 1). Different esters of estradiol, including estradiol benzoate (EB), estradiol valerate (EV), and estradiol cypionate (ECP) are commercially available in South America. All have been reported to induce follicle regression when administered in the presence of elevated plasma P4 concentrations [11]. Both EV and ECP have a long half-life, resulting in delayed and more variable intervals to follicular wave emergence [11,19,20] than the shorter-acting estradiol-17b [11] or EB [21]. We have recently evaluated the effect of administration of 2 mg EB at the time of insertion of a 3 mg norgestomet ear implant (Crestar; Intervet, Sao Paulo, Brazil) or an intravaginal insert (CIDR; Pfizer Animal Health, Sao Paulo, Brazil) in cycling B. indicus heifers [22]. The interval from treatment to follicular wave emergence was not difference between heifers treated with Crestar ear implants ( d) and those treated with CIDR inserts ( d). In a more recent experiment [23], we evaluated the effect of EB and two different doses of EV on the time and synchrony of follicular wave emergence in B. indicus cows and heifers receiving Crestar ear implants. There were significant effects of treatment (Table 2), but no treatment-by-class interaction on the mean interval to, and synchrony of, wave emergence. We have recently evaluated the effect of administering 50 mg of P4 intramuscularly along with EB at the time of insertion of a DIB vaginal device (1 g of progesterone; Syntex, Buenos Aires, Argentina) on the interval to, and synchrony of, follicular wave emergence Table 1 Mean (S.E.M.) interval from treatment to follicular wave emergence and number of follicles recruited per wave in Bos indicus, B. indicus Bos taurus, and B. taurus heifers treated with 2 mg estradiol benzoate and CIDR insert Heifers N Interval to follicular wave emergence (d) Number of follicles recruited per wave B. indicus a B. indicus B. taurus ab B. taurus b Adapted from [18]. Means within the same column with different superscripts (a, b) differ (P < 0.05).

4 80 P.S. Baruselli et al. / Theriogenology 65 (2006) Table 2 Mean (S.E.M.) interval (d), and range, from treatment to follicular wave emergence in B. indicus cows and heifers treated with 2 mg estradiol benzoate (EB), 2.5 mg EV plus 1.5 mg norgestomet (1/2 EV + Nor), or 5 mg of estradiol valerate plus 3 mg norgestomet (EV + Nor) at the time of insertion of a Crestar ear implant Class EB 1/2 EV + Nor EV + Nor P-value N treated animals Heifers a b c < x 3 6 xy 3 8 y <0.05 N treated animals Cows a ab b < x 2 6 xy 2 6 y <0.05 Adapted from [23]. in B. indicus cattle [24]. Treatment with P4 plus EB at the time of DIB insertion resulted in a delayed and more synchronous wave emergence ( d) than treatment with EB alone at DIB insertion ( d). These results are in agreement with those previously reported in B. taurus cattle [25]. In yet another experiment [26], we examined the effect of different doses of EB (2 or 3 mg), time of EB administration and the addition of a P4 injection in 50 Nelore cows treated with DIB devices (n = 10/group). Treatment with EB plus P4 on the day of DIB insertion resulted in more synchronous emergence of a new follicular wave ( d) than 2 mg EB given alone at DIB insertion ( d) or 1 d later ( d), or 3 mg EB given alone on the day of DIB insertion ( d) or 1 d later ( d). In conclusion, treatment with EB plus injectable P4 at the time of insertion of a progesterone releasing intravaginal device induced the most synchronous follicular wave emergence in B. indicus cattle. 4. Fixed-time AI in superstimulated donors Although the control of follicular development allows for the elective initiation of gonadotropin treatments, the time of AI continues to be dependent on estrus detection. Unfortunately, estrus detection in B. indicus cattle is difficult, variable and subject to errors. Estrus is shorter than that in B. taurus cattle and there is a greater tendency for B. indicus cattle to show estrus during the night [27]. Therefore, several studies have been conducted to investigate the pharmacological control of the time of ovulation in superstimulated B. indicus cattle, with the objective of developing a fixed-time AI protocol. A superstimulation protocol (named P-36) has been developed in Brazil [28]. In this protocol the progesterone/progestogen-releasing device is removed 36 h after PGF treatment (thus, P-36) and ovulation is induced with plh (Lutropin-V; Bioniche Animal Health, Belleville, Ont., Canada), administered 12 h after progesterone/progestogenreleasing device removal (i.e., 48 h after PGF administration). Since ovulation occurs between 24 and 36 h after plh treatment [28], fixed-time AI is done 12 and 24 h after plh injection, avoiding the inconvenience of estrus detection. Recently, the effectiveness of the P-36 protocol was tested in a commercial herd of Nelore cattle. In 136 superstimulations and embryo collections performed with the P-36 protocol (CIDR or DIB and 3 mg EB on

5 P.S. Baruselli et al. / Theriogenology 65 (2006) Table 3 Mean (S.E.M.) number of ova and embryos and transferable embryos in Nelore cows superstimulated 5 d after synchronization of follicular wave emergence with 3 mg estradiol benzoate and a progesterone-releasing intravaginal device (CIDR or DIB) Progesterone device Dose of Plh CIDR DIB 25.0 mg 12.5 mg Number of treated cows Total ova/embryos Transferable embryos The progesterone-releasing intravaginal device was removed 36 h after PGF treatment (P-36 protocol) and cows were treated with plh (25.0 or 12.5 mg) 12 h later. Fixed-time AI was done 12 and 24 h after plh treatment. Adapted from [29]. Means did not differ. Day 0 and superstimulation initiated on Day 5), the number of ova/embryos and transferable embryos and pregnancy rates following nonsurgical transfer of fresh embryos were , and 43.5% (528/1213), respectively [29]. Additionally, the type of progesterone-releasing intravaginal device (DIB versus CIDR) and dose of plh (12.5 mg versus 25.0 mg) did not influence embryo yield significantly (Table 3). These results were comparable those reported in studies in which Nelore cows were inseminated 12 and 24 after onset of behavioral estrus [28 31]. Another experiment was designed to evaluate the effect of the time of removal of the progesterone-releasing device in superstimulated B. indicus cattle that were fixed-time inseminated [32]. Ten Nelore cows were placed at random into one of two groups in a cross-over experimental design and were superstimulated twice 35 d apart with Folltropin-V (Bioniche Animal Health). The cows received a CIDR and were treated with 2.5 mg EB plus 50 mg of P4 at random stages of the estrus cycle (Day 0). Superstimulation treatments (133 mg NIH-FSH-P1 of Folltropin-V) divided into eight decreasing doses 12 h apart were initiated on Day 4. Cows received PGF on the morning of Day 6. CIDR were removed in the morning (P-24) or afternoon (P-36) of Day 7. Cows also received 25 mg plh (Lutropin-V) in the morning of Day 8 (12 h after the last Folltropin-V treatment or 48 h after PGF) and were fixed-time inseminated 12 and 24 h later. No differences in superovulatory responses or ova/embryo quality were observed between Groups P-24 and P-36 (Table 4). These results demonstrate that the progesterone-releasing device can be Table 4 Mean (S.E.M.) number of ova/embryo, fertilized ova and transferable embryos in superstimulated Nelore donors treated with progesterone releasing devices (CIDR) for 7 (P-24) or 7.5 d (P-36), plh on Day 8 a.m. and AI 12 and 24 h later P-24 protocol P-36 protocol Number of treated cows Total ova/embryos Fertilized ova Transferable embryos Adapted from [31]. Means did not differ (P > 0.05).

6 82 P.S. Baruselli et al. / Theriogenology 65 (2006) removed either 24 h (P-24) or 36 h (P-36) after PGF treatment without affecting superovulatory response or ova/embryo quality following for fixed-time AI in superstimulated B. indicus donors. 5. Optimum time for induction of ovulation for fixed-time AI in superstimulated B. indicus donors Currently, we are investigating the most appropriate time for induction of ovulation for fixed-time AI in superstimulated Nelore (B. indicus) and Holstein (B. taurus) donors. Physiological differences between Nelore and Holstein cows, especially the diameter of the dominant follicle at the time of deviation, have been reported. The diameter of the dominant follicle at the time of deviation has been reported to be 6.0 mm [33] or 6.3 mm [34] in Nelore and 8.5 mm in Holstein [35] cattle. A study was designed to determine the diameter at which the dominant follicle acquired the capacity to ovulate in response to treatment with plh in Nelore heifers [36]. Theonsetof ovulatory capacity of the dominant follicle was found to be between 7 and 8.4 mm (30%; 3/9), and there was a linear increase in response from 8.5 to 10 mm (80.0%; 8/ 10). In heifers with follicles >10 mm, 90% ovulated in response to plh treatment. These results differed from those reported in Holstein cows where ovulatory capacity was found to begin at 10 mm [37]. In a recent study, we evaluated the effect of delaying the induction of ovulation in superstimulated Nelore donors by 12 h (i.e., 12 or 24 h after the last Folltropin-V injection) [38]. The objective of this experiment was to improve ovulation rates by increasing follicle diameter (and ovulatory capacity) at the time of plh treatment. Twenty Nelore cows were placed at random into one of two experimental groups and superstimulated twice utilizing a cross-over experimental design. All cows received a DIBplus2.5mgEBand50mgP4onDay0,andonDay4,weresuperstimulatedwitha total of 100 mg Folltropin-V in a twice-daily decreasing dose schedule over 4 d (Days 4 7). On Day 6, PGF was given, and in the morning of Day 7, DIB were removed (P-24). Cows were subdivided to receive 25 mg plh 12 or 24 h after the last Folltropin-V injection (Day 8 a.m. and p.m.) and were fixed-time inseminated 12 and 24 h later with the same batch of frozen-thawed semen. Timing of ovulations was determined by twice daily ultrasonography and on Day 15, ova/embryos were collected and classified according to the IETS criteria. Delaying treatment with plh from 12 to 24 h after the last Folltropin-V injection increased (P < 0.01) the number of degenerated embryos, and decreased (P < 0.005) the number transferable and freezable embryos (Table 5). The decreased embryo quality following the delay in induction of ovulation may be explained by the increased (P < 0.05) interval between first to last ovulation and the tendency (P =0.08) for increased variability in ovulation times after treatment at 24 h. These results indicate that the optimum time for treatment with plh for fixed-time AI in superstimulated Nelore donors is 12 h after the last Folltropin-V treatment (i.e. the morning of Day 8 in the EB + P4/P-24 superstimulation protocol). The results obtained in Nelore donors differ from those observed in Holstein donor cows. In high producing Holstein cows, plh treatment 24 h after the last Folltropin-V

7 P.S. Baruselli et al. / Theriogenology 65 (2006) Table 5 Mean (S.E.M.) number of ova/embryo, fertilized ova and transferable embryos in superstimulated Nelore donors treated with progesterone releasing devices (DIB) for 7 d (P-24), plh 12 (Day 8) or 24 h (Day 8.5) after the last Folltropin-V injection and AI 12 and 24 h later plh 12 h (Day 8) plh 24 h (Day 8.5) P-value Treatment Donor Number of treated cows Number of follicles >8 mm at plh injection Number of CL at embryo collection (Day 15) Total ova/embryos collected Unfertilized ova Degenerate embryos Transferable embryos (Grades 1, 2 and 3) Freezable embryos (Grades 1 and 2) Interval first to the last ovulation (h) Interval last FSH injection to ovulation (h) Interval-pLH injection to ovulation (h) Adapted from [38]. injection resulted in an improved superovulatory response and higher numbers of transferable embryos [5,38,39]. Rodrigues et al. [39] also observed a reduction in the number of unovulated follicles (>10 mm) at ova/embryo collection and a numerically higher number of transferable embryos when GnRH was administered 24 h after the last FSH injection, compared to GnRH administered at 12 h after the last FSH treatment. Martins et al. [5], utilizing a cross-over experimental design and the same batch of semen, also observed an increase in the number of transferable embryos when plh was administered 24 h after the last FSH treatment in Holstein cows rather than 12 h after the last FSH. Based on these results, it was concluded that 24 h after the last FSH injection (60 h after treatment with PGF) was the optimum time for induction of ovulation with plh for fixed-time AI in superstimulated high-producing Holstein cows, whereas a 12 h interval from the last treatment with FSH (48 h after PGF treatment) was more appropriate for Nelore cows. Based on the results of the previous study in Nelore cows, we have recently performed an experiment to test the hypothesis that it is possible to use a single fixedtime insemination in B. indicus donors without adversely affecting fertilization rates or ova/embryo quality. Ten Nelore cows were subdivided into two experimental groups utilizing a cross-over experimental design. Treatments were similar to those in the previous study except that DIB were removed in the afternoon of Day 7 (P-36 protocol). All cows received plh 12 h after the last Folltropin-V injection (Day 8a.m.;12hafter DIB removal or 48 h after PGF treatment) and cows were fixed-time inseminated with the same batch of frozen-thawed semen twice, 12 and 24 h after plh, or only once, 16 h after plh treatment. There was no difference in superovulatory response or ova/embryo quality between one or two fixed-time inseminations (Table 6). Results indicate that it is possible to perform a single fixed-time AI 16 h after plh in superstimulated B. indicus donors using the P-36 treatment protocol without compromising ova/embryo production.

8 84 P.S. Baruselli et al. / Theriogenology 65 (2006) Table 6 Effect of number of fixed-time inseminations (1 vs. 2 FTAI) on embryo production in Nelore donors superstimulated with Folltropin-V and induced to ovulate with Lutropin-V 12 h after DIB removal and last FSH injection (mean S.E.M.) 1FTAI(n = 10) 2 FTAI (n = 10) P-value Number of follicles >8 mm at plh Number of CL at ova/embryo collection Total ova/embryos collected Unfertilized ova Degenerated embryos Transferable embryos (Grades 1, 2 and 3) Freezable embryos (Grades 1 and 2) Interval from the first to the last ovulation (h) Use of different dosages of FSH for superstimulation of B. indicus cattle B. indicus breeds have been shown to have a reduced capacity for LH secretion and a greater sensitivity to exogenous gonadotropins than B. taurus cattle [40]. Baruselli et al. [4] evaluated superovulatory response in 23 Nelore cows to three different doses of Folltropin- V (100, 133 or 200 mg) in a cross-over experimental design. All cows received 2.5 mg EB and 50 mg P4 at the time of CIDR insertion (Day 0) and superstimulatory treatments (twice daily im injections over 4 d) were initiated on Day 4. PGF was given in the morning and afternoon of Day 6, CIDR were removed in the afternoon of Day 7 and plh was administered in the morning of Day 8. All cows were fixed-time AI, utilizing the same batch of frozen-thawed semen, 12 and 24 h after plh and ova/embryos were collected on Day 15. There were no significant differences in any of the parameters evaluated (Table 7). Results indicate that it is possible to reduce the dose of Folltropin-V to 100 mg in a fixedtime AI, superstimulatory protocol in Nelore cattle without compromising superovulatory response and ova/embryo quality. 7. Superstimulatory protocols for in vitro embryo production Ovarian superstimulation with gonadotropins has been reported to increase the number of oocytes retrieved by ultrasound-guided oocyte aspiration as compared to nonstimulated B. taurus cattle [41 44]. However, forcing a population of follicles to continue in the growth phase by administering FSH may not provide an ideal follicular environment for cumulus-oocyte complexes (COC) to acquire developmental competence [45]. There is evidence in B. taurus cattle that a coasting period between hormonal stimulation and slaughterhouse ovary collection [46] or from ovary collection to oocyte aspiration [47] will affect the in vitro developmental potential of COC. In both situations, follicles in phases of pseudodominance or early atresia provided COC with an ideal environment in which to acquire developmental competence [45]. Blondin et al. [45] achieved a surprisingly high blastocyst rate (80%), following superstimulation of Holstein heifers with FSH and delaying COC retrieval for 48 h i.e., a stage of early atresia was induced in these follicles by depriving them of FSH for the 48 h

9 P.S. Baruselli et al. / Theriogenology 65 (2006) Table 7 Means (S.E.M.) superovulatory response and ova/embryo production in Nelore cows utilizing three different doses of Folltropin-V Total dose of Folltropin-V 100 mg 133 mg 200 mg Number of treated cows Number of CL Total ova/embryos Transferable embryos (Grades 1, 2 and 3) Freezable embryos (Grades 1 and 2) Adapted from [4]. Means did not differ. period. Additionally, LH was administered 6 h before oocyte aspiration in an attempt to render the COC more competent. Recently, the same protocol was evaluated against other protocols used for oocyte aspiration and in vitro embryo production (IVP) in Nelore cattle [48]. Eighteen Nelore cows were randomly placed into the following three groups: Group 1: oocyte aspiration was performed without superstimulation; Group 2: superstimulation was induced before oocyte aspiration; Group 3: superstimulation and FSH deprivation were induced before oocyte aspiration. Three oocyte aspiration sessionswereperformedoneachcowina cross-over designsothatallcowswere subjected to all three-treatment protocols. At random stages of the estrus cycle, all follicles 6 mm were aspirated to induce a new follicular wave 2 d later (Day 0). In Group 1, oocyte aspiration was performed on Day 2. In Group 2, cows were superstimulated with twice daily injections of 30 mg Folltropin-V for 3 d (starting on Day 0), 12.5 mg plh 6 h after the last FSH treatment, and oocyte aspiration 6 h later (12 h after the last FSH treatment). In Group 3, cows received the same treatment as in Group 2, except that plh was administered 36 h after the last treatment with FSH and oocyte aspiration was performed 6 h later. In this group, follicles were deprived of exogenous FSH for 48 h before oocyte aspiration. Regular in vitro production procedures were used to mature and fertilize COC and culture presumptive zygotes. Results indicate that ovarian superstimulation associated with deprivation of FSH prior to oocyte aspiration did not increase in vitro embryo production in Nelore cattle (Table 8). On the contrary, the highest rate of hatched blastocysts was observed in oocytes from nonstimulated cows (Group 1). Table 8 Number of oocytes collected, cleavage, blastocyst and hatched blastocyst rates in Nelore cows treated with three different protocols prior to oocyte aspiration: Group 1 (control), Group 2 (superstimulation and oocyte aspiration), and Group 3 (superstimulation, FSH deprivation and oocyte aspiration) Number of oocytes Cleavage (%) Blastocysts (%) Hatched blastocysts (%) Group a Group b Group b Adapted from [48]. Percentages within the same column with different superscripts (a, b) differ (P < 0.01).

10 86 P.S. Baruselli et al. / Theriogenology 65 (2006) Fig. 1. Treatment protocol for superstimulation, induction of ovulation, and fixed-time AI in Nelore embryo donors. Treatment consists of insertion of progesterone releasing intravaginal device and administration of estradiol benzoate (EB) and progesterone (P4) im on Day 0. Superstimulatory treatments are initiated on Day 4, with FSH given twice daily over 4 d. Donors receive PGF treatment in the a.m. and p.m. of Day 6 and progesterone devices are removed with the last FSH, in the p.m. of Day 7. Donors also receive plh in the a.m. of Day 8 and are inseminated without estrus detection 12 and 24 h later, or once on Day 8 (16 h after plh). Ova/embryos are collected nonsurgically on Day Conclusion The results of the studies presented herein demonstrate that it is possible to synchronize follicular wave emergence and initiate superstimulatory treatments at random stages of the estrus cycle in B. indicus cattle. Moreover, treatment with plh 12 h after the last FSH treatment synchronized ovulations, allowing for the application of fixed-time AI protocols in superstimulated B. indicus cattle. Finally, the number of inseminations was also reduced to only one (16 h after plh treatment) without adversely affecting ova/embryo quality (Fig. 1). References [1] Barros CM, Nogueira MFG. Superovulação em zebuínos de corte. In: Proceedings of the first simpósio internacional de reprodução animal aplicada Londrina; p [2] Nogueira MFG, Barros BJP, Teixeira AB, Trinca LA, D Occhio MJ, Barros CM. Embryo recovery and pregnancy rates after the delay of ovulation and fixed-time insemination in superstimulated beef cows. Theriogenology 2002;57: [3] Mapletoft RJ, Steward KB, Adams GP. Recent advances in the superovulation in cattle. Reprod Nutr Dev 2002;42: [4] Baruselli PS, Marques MO, Reis EL, Nasser LFT, Silva RCP, Menegatti JA, et al. Adequação da dose de FSH (Folltropin-V) em protocolos de superovulação de vacas nelore (Bos taurus indicus) com inseminação artificial em tempo fixo. Acta Sci Vet 2003;31(Suppl 1): [5] Martins CM, Castricini ESC, Reis EL, Torres-Júnior JRS, Gimenes LU, Sá Filho MF, et al. Produção embrionária de vacas holandesas a diferentes protocolos de superovulação com inseminação artificial em tempo fixo. Acta Sci Vet 2005;3(Suppl 1):286 (abstract). [6] Adams GP. Control of ovarian follicular wave dynamics in cattle: implications for synchronization and superstimulation. Theriogenology 1994;54: [7] Nasser L, Adams GP, Bó GA, Mapletoft RJ. Ovarian superstimulatory response relative to follicular wave emergence in heifers. Theriogenology 1993;40: [8] Bergfelt DR, Bó GP, Mapletoft RJ, Adams GP. Superovulatory response following ablation-induced follicular wave emergence at random stages of the oestrous cycle in cattle. Anim Reprod Sci 1997; 49:1 12.

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