New approaches to superovulation in the cow

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1 CSIRO PUBLISHING Reproduction, Fertility and Development, 2010, 22, New approaches to superovulation in the cow Gabriel A. Bó A,E, Daniel Carballo Guerrero A,B, Andrés Tríbulo A,B, Humberto Tríbulo A,B, Ricardo Tríbulo A,B, Dragan Rogan C and Reuben J. Mapletoft D A Instituto de Reproducción Animal Córdoba (IRAC), Zona Rural General Paz, 5145 Córdoba, Argentina. B Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Avenida Valparaíso y Rogelio Martínez, Ciudad Universitaria, 5000 Córdoba, Argentina. C Bioniche Life Sciences, 231 Dundas Street, East Belleville, ON K8N 5J2, Canada. D Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan S7N 5B4, Canada. E Corresponding author. gabrielbo@iracbiogen.com.ar Abstract. There is continuing need to simplify bovine superovulation protocols without compromising embryo production. The control of follicular wave emergence and ovulation has facilitated donor management, but the most commonly used treatment, oestradiol, cannot be used in many parts of the world and mechanical removal of the dominant follicle is difficult to apply in the field. Other alternatives include gonadotrophin-releasing hormone (GnRH) or LH, but efficacy in groups of randomly cycling animals is variable. Another alternative is to increase the response to GnRH by inducing a persistent follicle and initiating FSH treatments following GnRH-induced ovulation. The number of transferable embryos following superovulation during the first follicular wave did not differ from that achieved 4 days after oestradiol benzoate and progesterone. To further simplify superovulation, FSH has been administered as a single intramuscular injection. Superovulation of beef donors with a single intramuscular injection of Folltropin-V (Bioniche Animal Health, Belleville, ON, Canada) diluted in a slow-release formulation resulted in embryo production comparable to that obtained using the traditional twice-daily protocol. The single intramuscular injection has the potential to reduce labour and handling and may be useful when handling stress is an impediment to success. These alternatives provide ways of facilitating widespread application of embryo transfer technologies. Additional keywords: first follicular wave, FSH, GnRH, progesterone releasing device, slow release formulation. Introduction Variability in the response to superovulation protocols and the time and effort required to administer treatments have affected the widespread application of embryo transfer (ET) in genetic improvement programmes (Bó et al. 2008). Although research efforts in recent years have resulted in no increase in the number of transferable embryos per treatment, protocols that control emergence of the follicular wave (Bó et al. 1995, 2002) and the timing of ovulation (Baruselli et al. 2006; Bó et al. 2006) have allowed the treatment of groups of donors, regardless of the stage of the oestrous cycle, and permitted the fixed-time AI (FTAI) of donors without the need to detect oestrus. This has had a positive impact on commercial ET, because it has facilitated the scheduling of working protocols without being dependent on the knowledge and skill of personnel to detect oestrus. However, the complexity of protocols can lead to errors that can affect superovulatory response and embryo production. In this regard, the need to inject FSH twice daily is a factor of great concern (Bó et al. 1994). Moreover, the most commonly used treatment for synchronisation of follicular wave emergence for superovulation involves the use of a progestogen/progesterone (P4)-releasing device and 17β-oestradiol or its esters (E2; Mapletoft et al. 2002), which cannot be used in many countries because of concerns about the effects of steroid hormones in the food chain (Lane et al. 2008). The intention of this paper is to present progress in the development of superovulation protocols using gonadotrophin-releasing hormone (GnRH) as a replacement for E2 to synchronise follicular development and the administration of FSH in a single injection as an alternative to twice-daily treatments. Manipulation of the follicular wave for superovulation Traditionally, gonadotrophin treatments were initiated during the mid-luteal phase, approximately 9 11 days after oestrus (Lindsell et al. 1986), around the time of emergence of the second follicular wave (Ginther et al. 1989). However, a greater superovulatory response occurred when treatments were initiated on IETS /RD /10/010106

2 Superovulation in the cow Reproduction, Fertility and Development 107 the day of follicular wave emergence, rather than 1 day before or 1 2 days later (Nasser et al. 1993;Adams et al. 1994).Therefore, conventional treatment protocols have two drawbacks: (1) the requirement to have trained personnel dedicated to the detection of oestrus; and (2) the need to have all donors in oestrus at the same time in order to initiate treatments at the same time. Progestins and oestradiol In the 1990s, we reported on the use of P4 and E2 to synchronise follicular wave emergence, on average, 4 days after treatment (Bó et al. 1995, 2006). This treatment has been used by practitioners around the world (Mapletoft et al. 2002; Baruselli et al. 2006; Bó et al. 2006), but its use has been restricted recently in several countries. This restriction leaves many ET practitioners with a serious dilemma and has created the need to develop treatments that do not involve the use of E2. Follicle ablation One alternative is to eliminate the suppressive effect of the dominant follicle by ultrasound-guided follicle aspiration and to initiate superstimulatory treatments 1 or 2 days later (Bungartz and Niemann 1994; Bergfelt et al. 1997). The disadvantage is that this requires ultrasound equipment and trained personnel, which is only appropriate for embryo production centres where donors are held; it is very difficult to apply in the field. GnRH or LH Another alternative to synchronise follicular wave emergence is to use LH or GnRH to induce ovulation of the dominant follicle (Macmillan and Thatcher 1991), which is followed by wave emergence 1 2 days later (Pursley et al. 1995). However, this occurs by chance (stage of the cycle) or only when the treatment results in ovulation (Martinez et al. 1999). Pursley et al. (1995) reported that the administration of GnRH at random stages of the cycle induced ovulation in approximately 85% of dairy cows, but, more recently, Colazo et al. (2009) reported an ovulation rate of 62.4% in lactating dairy cows receiving 25 mg porcine (p) LH (Lutropin-V; Bioniche Animal Health, Belleville, ON, Canada) and 44.3% in those receiving 100 µg GnRH. In another study, plh and GnRH induced ovulation in 78% and 56% of beef heifers, respectively (Martinez et al. 1999). The occurrence of ovulation following administration of GnRH or plh in beef cows appears to be approximately 60% (Small et al. 2009). Not surprisingly, treatment with GnRH at random stages of the oestrous cycle before initiation of superstimulatory treatments resulted in lower responses than treatments initiated after follicular aspiration or E2 treatment (Deyo et al. 2001). However, in a retrospective analysis of commercial data, R. Hinshaw (pers. comm.) found no differences in the number of transferable embryos between donors superstimulated 4 days after treatment with E2 and P4 (7.8 transferable embryos; n = 1136) and those superstimulated 2 days after treatment with GnRH (7.7 transferable embryos; n = 56). In another recent study (Wock et al. 2008), dairy cows (n = 411) treated with a P4-releasing device (Eazy-Breed CIDR; Pfizer Animal Health, NewYork, NY, USA) were superstimulated 4 days after receiving E2 or 2 days after GnRH to synchronise follicular wave emergence; there was no significant difference in the number of ova/embryos or transferable embryos between groups. In another retrospective analysis of commercial data, dairy donors superstimulated 60 h after the administration of GnRH (n = 245) produced similar numbers of transferable embryos as those superstimulated 4 days after receiving E2 (n = 691; Steel and Hasler 2009). Controlled studies with the use of GnRH must be conducted to validate these promising results. Superovulation during the first follicular wave Follicular wave emergence occurs consistently at the time of ovulation (Ginther et al. 1989) and experiments performed in cattle (Nasser et al. 1993) and sheep (Menchaca et al. 2002) have indicated that it is possible induce superovulation of the first follicular wave. Adams et al. (1994) also reported no difference in superovulatory response when FSH treatments were initiated at the time of emergence of the first or second follicular wave. However, success relies upon successful determination of the time of ovulation or accurate oestrus detection with ovulation expected to occur 1 day after the onset of oestrus. To avoid the need to observe oestrus and ovulation in Nelore (Bos indicus) donors, Nasser et al. (2003) induced synchronous ovulation with a protocol that involved the administration of E2 at the time of CIDR insertion (Day 0), prostaglandin (PG) F 2α at CIDR removal (Day 8) and plh 24 h later. FSH treatments were initiated 24 h after plh (i.e. the expected time of ovulation and emergence of the first follicular wave). Superovulatory response and the number of transferable embryos did not differ from a contemporary group superstimulated 4 days after treatment with E2. However, the number of transferable embryos was reduced in cows superstimulated during the first follicular wave without accompanying use of a CIDR. Recently, we conducted a series of five experiments (see below), with the overall objective of developing a protocol for superovulation during the first follicular wave, using P4- releasing devices but not E2. We considered previous reports indicating that ovulatory response to GnRH could be increased by the administration of PGF 2α to regress the corpus luteum (CL) at the time of insertion of a P4-releasing device that remained in place for 7 10 days (Small et al. 2009); ovulation and wave emergence occurred 1 2 days later (Small et al. 2009). In the first experiment (Carballo Guerrero et al. 2008a), 70 Bonsmara donors (29 cows and 41 heifers) were randomly assigned to one of two treatment groups. Donors in the first wave group received a P4-releasing device (1.56 g P4; Cue- Mate; Bioniche Animal Health) along with a dose of PGF 2α (0.150 mg D (+) cloprostenol; Bioprost-D; Biotay, Buenos Aires, Argentina) at random stages of the oestrous cycle. The Cue-Mate device was removed 10 days later and a second dose of PGF 2α was administered, followed by GnRH (0.050 mg lecirelin; Biosin-OV; Biotay) 36 h later. Ovulation was expected to occur within 36 h after GnRH. On Day 0 (36 h after GnRH), donors received a new Cue-Mate device and treatment with a total dose of mg (heifers) or 320 mg (cows) NIH-FSH-P1 of Folltropin-V (Bioniche Animal Health), in twice-daily decreasing doses over 5 days, was initiated. Donors received PGF 2α with

3 108 Reproduction, Fertility and Development G. A. Bó et al. the last two Folltropin-V treatments and the Cue-Mate device was removed with the last Folltropin-V dose. All donors received 12.5 mg Lutropin-V 24 h after Cue-Mate removal and were subjected to FTAI 12 and 24 h later. Ova/embryos were collected 7 days later. Donors in the control group received a Cue-Mate and E2 (Bioestradiol; Biotay) and 50 mg P4 (Progesterona Río de Janeiro; Laboratorios Allignani Hnos, Santa Fe, Argentina) and Folltropin-V treatments were initiated 4 days later, with similar doses and treatment protocols as described for the first wave group. The mean (± s.e.m.) number of transferable embryos did not differ significantly between the control and first wave groups (5.1 ± 0.9 v. 3.7 ± 0.8, respectively). Although that study indicated that superovulation during the first follicular wave was as efficacious as the standard E2+P4 treatment protocol in beef cattle, the duration of the protocol (26 days v. 15 days from Cue-Mate insertion until embryo collection) made it time consuming and difficult to implement. Therefore, a series of studies was designed to shorten and simplify the protocol. In the first of those studies (Carballo Guerrero et al. 2008b), superovulatory response was compared between donors pretreated with a Cue-Mate for 10 days and those pretreated with a Cue-Mate for 5 days. Ovulation rate and the interval from GnRH administration to ovulation were 81.8% (9/11) and 32.0 ± 2.8 h, respectively, in the former group and 100% (11/11) and 36.0 ± 0.0 h, respectively, in the latter group. The number to transferable embryos did not differ between groups pretreated with a Cue-Mate for 10 or 5 days (5.5 ± 1.6 v. 5.2 ± 2.6, respectively) or from donors superstimulated 4 days after E2+P4 (5.4 ± 1.6). Another experiment (Carballo Guerrero et al. 2009) determined whether it was necessary to remove the P4-releasing device during the superovulation protocol. Angus cows (n = 27) and heifers (n = 10) were superovulated by two treatments in a cross-over design. All donors received a Cue-Mate along with PGF 2α at random stages of the oestrous cycle; those in Group 1 received a second PGF 2α dose at the time of Cue-Mate removal 5 days later, followed by GnRH 36 h later. On Day 0 (36 h after GnRH), donors received a new Cue-Mate and superovulation treatments were initiated with a total dose of 400 mg NIH-FSH- P1 of Folltropin-V in twice-daily decreasing doses over 5 days. PGF 2α administration, Cue-Mate removal, plh administration, FTAI and embryo collection were as described for the previous studies. Donors in Group 2 were treated similarly to those in Group 1, except that the Cue-mate device was not removed, but remained in place for 13 days (i.e. it was removed with the last FSH and PGF 2α injections). The ovulation rate and the interval from GnRH treatment to ovulation in Group 1 were 86.5% (64/74) and 35.6 ± 1.6 h, respectively, compared with 89.2% (33/37) and 37.5 ± 0.7 h, respectively, for Group 2. The mean (± s.e.m.) number of ova/embryos and transferable embryos was 8.2 ± 1.0 and 4.1 ± 0.6, respectively, for Group 1 and 9.8 ± 0.9 and 5.7 ± 0.7, respectively, for Group 2 (P > 0.2). It was not necessary to remove the P4-releasing device to synchronise ovulation (and follicle wave emergence for superovulation) with GnRH. In addition, Carballo Guerrero et al. (2009) evaluated the efficacy of shortening the protocol one more day, by giving Folltropin-V for 4 rather than 5 days, as in the previous three experiments. Simmental (n = 18) and Angus (n = 6) cows were superstimulated by the two treatment protocols in a cross-over design. Cows in both groups were treated similarly to those in Group 2 in the previous experiment (i.e. Cue-Mates were not replaced during treatment). Cows in Group 1 (control) received FSH over 5 days, whereas those in Group 2 received the same dose of Folltropin-V, but given in twice-daily decreasing doses over 4 days (Cue-Mates were removed with the last FSH and PGF 2α injections). The mean (± s.e.m.) number of ova/embryos and transferable embryos was 13.5 ± 2.4 and 6.6 ± 1.1, respectively, for Group 1 and 12.0 ± 1.9 and 5.8 ± 1.0, respectively, for Group 2 (P > 0.6). A final experiment determined whether it was possible to reduce the number of PGF 2α treatments during the pretreatment, further simplifying the first wave protocol (Carballo Guerrero et al. 2010). A second objective of this study was to confirm the effectiveness of the first wave protocol by comparing it with the P4+E2 protocol to synchronise follicle wave emergence. Simmental donors cows (n = 14) were assigned randomly to three treatment groups in a cross-over design so that all animals received all treatments. Donors in Groups 1 and 2 received Cue-Mates as in the previous experiments; however, those in Group 1 received PGF 2α at the time of Cue-Mate insertion and 5 days later (as in previous experiments), whereas those in Group 2 received PGF 2α at Cue-Mate insertion only (i.e. eliminating the need to handle animals on Day 5). Cows in Groups 1 and 2 received GnRH 7 days after Cue-Mate insertion to induce ovulation and treatments with Folltropin-V were initiated 36 h later (Day 0; as in previous experiments). Donors in Group 3 received a Cue-Mate plus P4+E2 at random stages of the oestrous cycle and FSH treatments were initiated 4 days later. All cows received 400 mg Folltropin-V in twice-daily injections over 4 days, as described in the previous experiment (Carballo Guerrero et al. 2009). The mean (± s.e.m.) number of ova/embryos, fertilised ova and transferable embryos did not differ among groups (12.9 ± 2.0, 9.8 ± 1.7 and 6.6 ± 1.2, respectively, for Group 1; 11.5 ± 1.7, 9.3 ± 1.5 and 7.7 ± 1.6, respectively, for Group 2; and 14.5 ± 2.8, 9.4 ± 2.3 and 6.8 ± 1.7, respectively, for Group 3). In conclusion, the results from these studies indicate that superovulation during the first follicular wave can be used successfully in groups of randomly cycling donors without the need for oestrus detection or E2 to synchronise follicular wave emergence. The protocol is easy to follow and embryo production is comparable with that of the E2+P4 protocol. The recommended treatment protocol is outlined in Fig. 1. Superovulation using a single administration of FSH Traditional superstimulatory treatments consist of a single administration of equine chorionic gonadotrophin (ecg) or twice-daily injections of pituitary extracts containing FSH over 4 or 5 days (Mapletoft et al. 2002). The ecg is a complex glycoprotein that has a long half-life (over 40 h), which represents a practical advantage because a single administration will induce ovarian superovulation (Schams et al. 1977; Murphy and Martinuk 1991). However, it is necessary to neutralise ecg with antibodies at the time of insemination to avoid the adverse

4 Superovulation in the cow Reproduction, Fertility and Development 109 PGF 2α D3 and D4 PGF 2α GnRH FSH plh AI AI Embryo collection Cue-Mate D-8 D-1 D0 D3 D4 D5 D5 D6 D12 Fig. 1. Treatment schedule for superovulation of donor cows during the first follicular wave. Donors receive a progesterone-releasing device (Cue-Mate; Bioniche Life Sciences, Belleville, ON, Canada) along with prostaglandin (PG) F 2α, followed by gonadotrophin-releasing hormone (GnRH) 7 days later. On Day 0 (36 h after GnRH), superovulation with Folltropin-V (Bioniche Animal Health, Belleville, ON, Canada) (FSH) is initiated (twice-daily decreasing doses over 4 days). PGF 2α is administered with the last two FSH injections and Cue-Mate devices are removed with the last FSH injection. Ovulation is induced with Lutropin-V (Bioniche Animal Health; porcine (p) LH) 24 h after Cue-Mate removal, donors are subjected to fixed-time AI 12 and 24 h later and ova/embryos are collected 7 days after plh treatment. Adapted from Carballo Guerrero et al. (2010). effects of continuing ovarian stimulation (large unovulated follicles at the time of embryo collection and decreased embryo collection efficiency and quality; for a review, see Dieleman et al. 1993). In contrast, the half-life of FSH is 5 h in the cow (Laster 1972; Demoustier et al. 1988) and frequent applications are required to induce superovulation (Bellows et al. 1969; Monniaux et al. 1983). Twice-daily treatments with FSH have resulted in a greater superovulatory response than once daily administration (Looney et al. 1981; Monniaux et al. 1983; Walsh et al. 1993). The need to inject FSH twice a day requires frequent attention by farm personnel and increases the possibility of failures due to mishandling and errors in the administration of treatments. In addition, twice-daily treatments may cause undue stress in donor cows, with a subsequent decreased superovulatory response (Edwards et al. 1987; Bó et al. 1994) and/or altered preovulatory LH surge (Stoebel and Moberg 1982). Therefore, simplified protocols of superovulation may be expected to reduce donor-handling costs and improve responses, particularly in less-tractable animals. More than 10 years ago, we reported that a single subcutaneous injection of 400 mg NIH-FSH-P1 of Folltropin-V in beef cows with a good body condition score (>3 out of 5), resulted in a superovulatory response equivalent to the traditional treatment protocol of twice-daily injections over 4 days (Bó et al. 1994). However, the results could not be repeated in Holstein cows, which had less subcutaneous fat (Hockley et al. 1992). In a subsequent study in Holstein cows, the single injection was split into two, with 75% of the dose of Folltropin-V administered subcutaneously on the first day of treatment and the remaining 25% administered 48 h later, when PGF 2α is normally administered (Lovie et al. 1994). Superovulatory response was intermediate, between that with the traditional protocol (the highest response) and a single subcutaneous injection (the lowest response). An alternative to induce a consistent superovulatory response with a single injection of FSH would be to combine the pituitary extract with agents that cause the hormone to be released slowly over several days. Yamamoto et al. (1994) reported that FSH in a 30% solution of polyvinylpolypyrrolidone (PVP) and administered in a single intramuscular injection resulted in a comparable superovulatory response to twice-daily treatments. However, Callejas et al. (2002) and G. A. Bó and R. J. Mapletoft (unpubl. obs.) were unable to induce a satisfactory superovulatory response with this compound. Kimura et al. (2007) reported that a single injection of FSH in aluminium hydroxide gel was effective in inducing superovulation in cattle, but aluminium hydroxide is commonly used as a vaccine adjuvant (Baylor et al. 2002), which may preclude its use for superovulation. In another study, FSH dissolved in polyethylene glycol (PEG) resulted in a satisfactory superovulatory response (Choi et al. 2002). We have recently completed a series of experiments in which Folltropin-V diluted in a slow release formulation (SRF; Bioniche Animal Health) was administered in a single injection. In the first experiment (Tríbulo et al. 2010), a single intramuscular injection of Folltropin-V diluted in SRF (n = 29) was compared with the traditional twice-daily intramuscular injection protocol over 4 days (n = 29) in RedAngus donors. On Day 0 (beginning of treatment), all cows received a Cue-Mate and the P4+E2 injection and on Day 4 treatments with 400 mg NIH- FSH-P1 Folltropin-V were initiated. Cows in Group 1 received twice-daily intramuscular injections over 4 days and cows in Group 2 received a single intramuscular injection in the neck. The single injection was prepared by diluting the Folltropin-V lyophilised powder in 1 ml saline for injection and then mixed with 9 ml SRF in the syringe, immediately before administration. In the morning and evening of Day 6, all cows received PGF 2α and Cue-Mates were removed in the evening. In the morning of Day 8, cows received 12.5 mg plh and were FTAI 12 and 24 h later. Ova/embryos were collected non-surgically

5 110 Reproduction, Fertility and Development G. A. Bó et al. Table 1. Mean (± s.e.m.) ova/embryo production in Angus, Brangus, Braford and Bonsmara donors treated with Folltropin-V (Bioniche Animal Health, Belleville, ON, Canada) given by twice-daily intramuscular injections over 4 days (control) or diluted in a slow-release formulation and given by a single intramuscular injection Adapted from Tríbulo et al. (2010) and Rogan et al. (2010). There were no significant differences in mean values or percentages (P > 0.1) Treatment n No. ova/embryos No. fertilised ova No. transferable embryos No. cows with 0 transferable embryos Control (twice-daily injections) ± ± ± (8.2%) Single injection ± ± ± (13.0%) on Day 15 and evaluated. The mean (± s.e.m.) number of total ova/embryos collected, fertilised ova and transferable embryos did not differ between the twice-daily (12.3 ± 1.5, 7.2 ± 1.1 and 4.9 ± 0.8, respectively) and single-injection (13.7 ± 2.1, 8.4 ± 1.4 and 6.4 ± 1.3, respectively; P > 0.4) groups. One cow in each treatment group had 2 CL at the time of ova/embryo collection and one cow in the control group and two cows in the single-injection group did not produce any transferable embryos. Additional experiments were conducted in several different breeds of donors to confirm the effectiveness of this protocol and to determine the appropriate dose of Folltropin-V for administration as a single intramuscular injection (Rogan et al. 2010;Tríbulo et al. 2010). Overall, the single-injection protocol resulted in a similar number of ova/embryos collected as the traditional twice-daily FSH protocol (Table 1). In Angus donors (138 superovulations), the number transferable embryos did not differ between treatment with 300 and 400 mg Folltropin-V (6.1 ± 0.7 v. 6.5 ± 0.7, respectively), but was higher than in the 200 mg Folltropin-V treatment group (4.0 ± 0.5; P < 0.02). In Brangus donors (69 superovulations), the number of transferable embryos did not differ between groups treated with either 260 or 300 mg Folltropin-V (9.5 ± 1.6 v. 7.9 ± 1.5, respectively), but tended to be higher than that in groups treated with 200 mg Folltropin-V (5.2 ± 0.8; P < 0.1). In Bonsmara donors (64 superovulations), the number of transferable embryos did not differ between groups treated with either 200 or 300 mg Folltropin-V (7.2 ± 0.8 v. 7.6 ± 1.0, respectively). Summary and conclusions Incorporation of protocols that control follicular dynamics and ovulation has the advantage of being able to schedule the treatments quickly and without the need for the detection of oestrus in donor cows. These treatments are practical and easy to perform by farm staff and, more importantly, they do not depend on the skill and accuracy of oestrus detection. However, E2, which has been most useful in synchronising follicle wave emergence, is not available in many countries around the world. Although the administration of GnRH to synchronise follicular wave emergence yields variable results, presynchronisation with a P4-releasing device improves the response to GnRH and allows for superovulation during the first follicular wave after ovulation, with results that do not differ from those obtained using E2. Recent studies with a proprietary SRF have shown that it is possible to induce a consistent superovulatory response following administration of a single injection of Folltropin-V without adversely affecting the number of transferable embryos. Acknowledgements The authors research reported herein was supported by the Instituto de Reproducción Animal Córdoba (IRAC) and Bioniche Animal Health (Canada). The authors thank Biotay SA (Argentina) for the provision on PGF 2α and GnRH and for facilitating the importation of the other hormones used in these trials. The authors extend special thanks to colleagues at IRAC for technical assistance. References Adams, G. P., Nasser, L. F., Bó, G. A., Garcia, A., Del Campo, M. R., and Mapletoft, R. J. (1994). Superovulatory response of ovarian follicles of wave 1 versus wave 2 in heifers. Theriogenology 42, doi: / x(94) Baruselli, P. S., Sá Fhilo, M., Matins, C. M., Naser, L. F., Nogueira, M. F. G., Barros, C. M., and Bó, G. A. (2006). Superovulation and embryo transfer in Bos indicus cattle. Theriogenology 65, doi: /j.theriogenology Baylor, N. W., Egan, W., and Richman, P. (2002). Aluminum salts in vaccines: US perspective. 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