Treatment alternatives to induce follicular wave emergence for timed-AI in lactating dairy Cows.

Two experiments evaluated the effect of different hormonal treatments to synchronize follicle wave emergence on follicle dynamics and pregnancies per AI (P/AI) in estradiol (E2)/progesterone (P4) timed-AI (TAI) protocols in lactating dairy cows. In Experiment 1, lactating, primiparous Holstein cows (n = 36) received a P4 releasing device (Day 0) and were allocated at random to one of the following three treatment groups: Group EB received 2 mg E2 benzoate (EB) intramuscularly (i.m.), Group EB + GnRH received 2 mg EB+20 µg buserelin (GnRH) i.m., or Group EB + P4 received 2 mg EB + 100 mg of injectable P4 (iP4) in oil i.m. All cows received 0.150 mg D-Cloprostenol on Days 7 and 8 followed by P4 device removal, 400 IU eCG and 1 mg ECP on Day 8. Daily ultrasound examinations revealed that although the interval from P4 device removal to ovulation was not affected by treatment, cows that received EB + GnRH had an earlier (P < 0.05) emergence of the new follicular wave (Day 2.6 ± 0.2) than the other two treatment groups (Days 3.5 ± 0.3 and 6.1 ± 0.3, for EB and EB + P4, respectively). In Experiment 2, 808 lactating cows were assigned randomly to the three treatments evaluated in Experiment 1, and all the cows were TAI to determine P/AI. Cows in the EB + GnRH group had greater P/AI (57.4 %, P < 0.01) than those in the EB (44.6 %) or EB + P4 (45.7 %) groups. In conclusion, the administration of GnRH, but not iP4, on the day of insertion of a P4 device improves P/AI in lactating dairy cows synchronized for TAI with an estradiol/P4-based protocol.

Effect of the addition of GnRH and a second prostaglandin F2α treatment on pregnancy per artificial insemination in lactating dairy cows submitted to an estradiol/progesterone-based timed-AI protocol.

Two experiments determined whether the addition of GnRH at the beginning of an estradiol (E2)/progesterone (P4)- based synchronization protocol and/or a second dose of prostaglandin F2α (PGF2α) the day before P4 device removal improves pregnancy rate in lactating dairy cows. On Day 0, all cows received a CIDR-B device and 2 mg i.m. estradiol benzoate, and half received 200 µg i.m. gonadorelin acetate (GnRH). On Day 7, cows were further subdivided to receive PGF2α (500 µg i.m. cloprostenol) or no PGF2α treatment. On Day 8, CIDR-B were removed, and all cows received PGF2α, 1 mg estradiol cypionate and 400 IU eCG i.m., and an estrus detection aid. Experiment 1 was designed to evaluate the effect of treatments on follicular development from P4 device removal to ovulation, expression of estrus, time of ovulation and serum P4 concentrations. Cows (n = 76) were examined by ultrasonography and bled for serum P4 determinations every 12 h from the time of P4 device removal but were not inseminated. In Experiment 2, all cows (n = 1036) were inseminated based on estrus detection using tail-paint. Cows with >50% of the paint rubbed-off by 48 h after P4 device removal were inseminated at that time, whereas those not in estrus received 100 µg i.m. of GnRH and were inseminated 12 h later. In Experiment 1, the interval from P4 device removal to ovulation was 71.7 ± 1.5 h and did not differ among groups. However, cows that received 2 injections of PGF2α had a greater (P < 0.01) estrus rate and lower (P < 0.01) P4 concentrations at 48 h after P4 device removal than those that received 1 PGF2α (estrus rate: 86.8% vs 68.4% and P4 concentration: 0.12 ± 0.01 vs 0.36 ± 0.07, for 2 and 1 PGF2α, respectively). In Experiment 2, estrus rate was also influenced by the number of PGF2α treatments, regardless of whether cows received or did not receive GnRH on Day 0 (2 PGF2α: 84.7%, 438/517 vs 1 PGF2α: 65.7%, 341/519; P < 0.01). Furthermore, there was a GnRH treatment by number of PGF2α treatments interaction (P < 0.05) on P/AI that was attributed to greater (P < 0.05) P/AI in cows that received GnRH on Day 0 and 2 PGF2α than in the other three treatment groups (EB+1 PGF2α: 45.2%, 119/263; EB+2 PGF2α: 45.8%, 119/260; EB + GnRH + 1 PGF2α: 45.7%, 117/256 and EB + GnRH + 2 PGF2α: 57.2%, 147/257). It was concluded that the addition of GnRH on Day 0 and a second dose of PGF2α the day before P4 device removal improves P/AI in lactating dairy cows synchronized with an estradiol/P4-based protocol.

Antral follicle counts and association with ovarian superstimulatory response to gonadotropins in prepubertal calves.

Antral follicle count (AFC) repeatability at the time of follicular wave emergence and duration of gonadotropin treatment in calves with small and large AFC affects the superstimulatory response of follicles. In Study I, the individual AFC was determined, calves were ranked as having a small, medium or large AFC, and a second count was performed prior to FSH treatments. There was a positive association between the number of follicles ≥1 mm after the first and second counts (r = 0.4; P = 0.003). In Study II, calves with small and large AFC were administered pFSH for 4 or 7 days, pLH 20 h after last pFSH administration and cumulus-oocyte-complexes (COC) were collected. In calves having large as compared with small AFC, number of follicles ≥6 mm were greater (P = 0.01) and COC collected were greater (P = 0.001). The proportion of large-sized follicles (>9 mm) was greater in the 7-day than in the 4-day gonadotropin treatment group (56.4 ±â€¯8.3 and27.8 ±â€¯7.5 %, respectively; P = 0.01). In Study III, there was a positive association between AFC and number of follicles ≥6 mm at the time of COC collection (r = 0.6; P = 0.003). In summary, the number of follicles at the time of follicular wave emergence was associated with the number of follicles recruited during subsequent waves of follicular development and ovarian response following gonadotropin superstimulation. Calves with a large AFC had more COC collected than calves with a small AFC.

Effect of expression of estrus and treatment with GnRH on pregnancies per AI in beef cattle synchronized with an estradiol/progesterone-based protocol.

Three experiments evaluated the effects of expression of estrus and gonadotropin releasing hormone (GnRH) treatment on pregnancies per AI (P/AI) in beef cattle that were treated with an estradiol/progesterone (P4)-based protocol for fixed-time artificial insemination (FTAI). In Experiment 1, 20 non-lactating beef cows were treated with 2 mg estradiol benzoate (EB) and an intravaginal device containing 0.5 g of P4. Seven days later, P4 devices were removed and all cows received prostaglandin F2 alpha (PGF2α) and 0.5 mg estradiol cypionate (ECP). Estrus was detected using tail paint and cows that did not show estrus by 48 h after P4 device removal were randomized to receive GnRH or no treatment. Ovulation, as determined by ultrasonography, occurred earlier in cows that showed estrus (68.0 ± 2.5 h) than in cows that did not (82.0 ± 2.1 h, P < 0.05). Furthermore, cows that received GnRH ovulated earlier (78.0 ± 2.6 h) than those that did not (86.0 ± 2.0, P < 0.05). Experiment 2 determined whether expression of estrus and the administration of GnRH to animals that did not show estrus increased P/AI. Non-lactating beef cows and heifers (n = 1356) were treated as in Experiment 1 (P4 device removal, PGF2α and ECP administration on Day 7) or extended until Day 8. All animals in estrus by 48 h after P4 device removal were inseminated and those not showing estrus received GnRH or no treatment and were FTAI 8 h later (i.e., at 56 h). P/AI were greater (P < 0.01) in animals that were observed in estrus by 48 h (56.4%) than in those that did not show estrus (46.5%). Likewise, animals that did not show estrus but were treated with GnRH had greater P/AI (53.8%, P < 0.04) than those that did not receive GnRH (37.9%). Experiment 3 was designed to determine the effect of delaying GnRH treatment to the time of FTAI (at 56 h) in cows not showing estrus by 48 h after P4 device removal. Suckled beef cows (n = 969) were treated as in Experiment 1, except that all cows also received 400 IU of eCG at the time of P4 device removal on Day 7. Cows that showed estrus by 48 h or 56 h had greater P/AI (62.3%, P < 0.05) than those did not show estrus (51.5%). Furthermore, when cows that did not show estrus by 48 h were analyzed separately, P/AI were greater (P < 0.05) in those that received GnRH at 48 h and were FTAI by 56 h (64.9%) than in those that received GnRH concurrent with FTAI by 56 h after device removal (54.6%). In summary, expression of estrus was associated with earlier ovulations and resulted in greater P/AI in cows and heifers treated with an estradiol/P4-based protocol for FTAI. Furthermore, GnRH treatment in animals that did not show estrus hastened the time of ovulation and increased P/AI.

Effect of estrus expression or treatment with GnRH on pregnancies per embryo transfer and pregnancy losses in beef recipients synchronized with estradiol/progesterone-based protocols.

Two experiments were designed to determine the effect of expression of estrus or GnRH treatment on pregnancies per embryo transfer (P/ET) and pregnancy losses in beef recipients that were synchronized with estradiol/progesterone based protocols for fixed-time embryo transfer (FTET). Experiment 1 evaluated the effect of expression of estrus and GnRH treatment in the absence of estrus on P/ET. Beef cows (n = 729) were treated with 2 mg estradiol benzoate (EB) and an intravaginal device containing 0.5 g of progesterone. Devices were removed 8 d later and all cows received prostaglandin F2α (PGF2α), 400 IU eCG, and 0.5 mg estradiol cypionate (ECP) at that time. Expression of estrus was determined at 48 and 56 h after device removal using tail-paint and cows that did not show positive signs of estrus by 48 h received GnRH or no treatment at random. The overall estrus rate was 76.0% (554/729); 68.0% had positive signs of estrus by 48 h after progesterone device removal and 28.0% of those not in estrus by 48 h showed estrus by 56 h. The proportion of recipients receiving in vivo-derived (IVD) or in vitro-produced (IVP) embryos and P/ET were greater in recipients that showed estrus by 48 and 56 h (94.0% and 48.4%, respectively) than in those that did not show estrus 41.0% and 29.0%, respectively; P < 0.01). However, GnRH treatment of recipients not showing estrus by 48 h did not improve P/ET. Experiment 2 evaluated the effect of expression of estrus on P/ET and pregnancy loses up to parturition in recipients synchronized with two estradiol-based protocols. Beef cows (n = 403) were divided at random to receive the same synchronization protocol as in Experiment 1 (ECP) or a J-Synch protocol (device removal on day 6 and without using estradiol cypionate to induce ovulation). In this experiment, pregnancy was determined at 30 and 60 d by ultrasonography, and all pregnant recipients were followed until parturition to determine pregnancy losses during gestation. Although the number of recipients receiving IVP embryos was greater in the ECP group (90.5% vs. 83.5%; P = 0.03), P/ET did not differ (ECP: 37.0% and J-Synch: 39.0%; P = 0.43). Overall, 88.0% (357/407) of the recipients synchronized showed estrus and a greater P/ET (P = 0.05) was found in the recipients that showed estrus (39.0%) vs. those that did not show estrus (26.0%), regardless of treatment group. Pregnancy losses were lower (P = 0.004) and the calving rate was higher (P = 0.01) in recipients that showed estrus (25.0% and 29.3%, respectively) than in those that did not (88.8% and 2.9%, respectively). In summary, expression of estrus was associated with a greater P/ET in recipients treated with two different estradiol/P4-based synchronization protocols. The expression of estrus was associated with a greater proportion of recipients receiving embryos, P/ET and calving rate. Treatment with GnRH did not improve P/ET in the recipients that did not show estrus, questioning the its use in recipients synchronized with estradiol/progesterone based FTET protocols.

Effect of dose and duration of FSH treatment on ovarian response in prepubertal calves.

The objective of the study was to determine relative effects of dose (200 or 350 mg) and duration (4 or 7 days) of superstimulatory treatment on the ovarian response in prepubertal calves. Calves with similar antral follicular counts at wave emergence (n = 24) were given eight doses of either 25 or 44 mg pFSH every 12 h for 4 days or 14 doses of either 14 or 25 mg pFSH for 7 days beginning at the time of follicular wave emergence and 12.5 mg of pLH im 12 h after the last FSH treatment. On Day 4 of pFSH treatment, calves given 14 mg had fewer follicles ≥3 mm than those given 25 mg (15.1 ± 1.9 and 27.9 ± 3.3, respectively; P = 0.04). At the end of treatment (24 h post-LH), number of follicles ≥9 mm was greater in calves of groups treated with 350 than 200 mg (13.5 ± 1.8 and 8.8 ± 1.3, respectively; P = 0.02) and calves of groups treated for 7 than 4 days (13.3 ± 1.8 and 9.0 ± 1.3, respectively; P = 0.03). The number of spontaneous ovulations was greater in calves of groups treated for 7 than 4 days as was the total number of ovulations (9.7 ± 0.9 and 6.9 ± 1.0, respectively; P ≤ 0.05). In summary, a dose of 25 mg of pFSH per treatment given twice daily for 7 days resulted in a greater ovarian response than other superstimulatory treatments in prepubertal calves.

An attempt to potentiate the ovarian superstimulatory response in cattle by co-treatment with an aromatase inhibitor.

Letrozole is used for the treatment of subfertility in women undergoing ovarian superstimulation, but the mechanism of action has not been investigated critically. The objective was to test the hypothesis that treatment with letrozole will potentiate the superstimulatory response following gonadotropin treatment by increasing the number of follicles present at ovarian follicular wave emergence in cattle. In Experiment 1, ovarian follicular wave emergence was synchronized among beef heifers (n = 8) by transvaginal ultrasound-guided follicle ablation. On Day 0 (wave emergence), a letrozole-releasing device (LRD) was placed intravaginally for 5 days, followed again by transvaginal follicle ablation on Day 5. The number of follicles ≥3 mm was recorded by transrectal ultrasonography on Days 0 and 6.5 (i.e., pre- vs. post-LRD treatment). In Experiment 2, non-lactating dairy cows were assigned randomly to one of two groups (n = 15/gp) after follicle ablation-induced synchronization of wave emergence (Day 0), and given either an LRD or sham device for 5 days. Superstimulatory treatment was initiated on Day 0, consisting of 8 doses of 50 mg of porcine FSH im at 12 h intervals, and luteolytic doses of prostaglandin on Days 3 and 3.5. The LRD/sham devices were removed on Day 3.5, GnRH was given im on Day 5, estrus response was determined on Days 5 and 6, and the ovarian response was recorded by ultrasonography on Days 0, 3.5, 5, 6.5, and 12. In Experiment 1, no difference was detected in the number of antral follicles at wave emergence pre- vs. post-LRD treatment (23.2 ± 3.2 vs. 23.5 ± 3.8 follicles; P = 0.67; mean ± SEM). In Experiment 2, the interval from prostaglandin treatment to estrus was longer (50.3 ± 1.1 vs. 40.7 ± 2.0 h; P < 0.001) and less variable (residuals: 3.1 ± 0.5 vs. 6.7 ± 0.9 h; P < 0.01) in the LRD vs. sham group. The proportion of ovulations (number of CL on Day 12 over the number of follicles ≥3 mm on Day 0) did not differ (0.65 ± 0.02 vs. 0.70 ± 0.02; P = 0.15) nor did the number of CL on Day 12 (15.9 ± 2.5 vs. 19.0 ± 2.0; P = 0.32) between the LRD and sham groups. In summary, treatment with letrozole did not increase the number of antral follicles at wave emergence or the superstimulatory response, but increased precision in the interval to estrus and may be useful for artificial insemination at a fixed time in superstimulatory protocols.

Superstimulation of ovarian follicles in cattle: Gonadotropin treatment protocols and FSH profiles.

The objective of ovarian superstimulatory treatments in cattle is to obtain the maximum number of viable embryos by stimulating growth of antral follicles and ovulation of competent oocytes. While factors inherent to the donor animal are critical, an increased knowledge of ovarian physiology, gonadotropin biochemistry and the ability to manipulate ovarian function have provided alternatives for the design of simple and successful protocols for superovulation in cattle. Recent protocols have also been made more user-friendly and allowed for the grouping of donors for successful superovulation. Although the number of reports associating FSH profiles with superovulatory response is limited, studies designed to reduce the number of FSH treatments necessary to induce superstimulation may provide guidance for the development of optimized gonadotropin treatment protocols. Although high peak levels of circulating FSH following a single administration of Folltropin-V have been shown to be associated with a reduced superstimulatory response, the ideal treatment protocol would seem to be to increase circulating FSH levels to values comparable to those required for the induction of follicle wave emergence, and to maintain these levels for at least 72 h (or 36 h for superstimulation prior to ovum pick-up) to allow follicles to reach an ovulatory size and acquire the capacity to ovulate.

Effect of season and superstimulatory treatment on in vivo and in vitro embryo production in wood bison (Bison bison athabascae).

Two experiments were done using a two-by-two design to determine the effects of season and superstimulatory protocol on embryo production in wood bison. In Experiment 1 (in vivo-derived embryos), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with either two or three doses of FSH given every-other-day (FSH × 2 vs. FSH × 3, respectively). Bison were given hCG to induce ovulation, inseminated 12 and 24 hr after hCG, and embryos were collected 8 days after hCG (n = 10 bison/group). In Experiment 2 (in vitro embryo production), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with two doses of FSH, and in vivo maturation of the cumulus-oocyte complexes (COC) was induced with hCG at either 48 or 72 hr after the last dose of FSH. COC were collected 34 hr after hCG, and expanded COC were used for in vitro fertilization and culture. In Experiment 1, the number of follicles ≥9 mm, the proportion of follicles that ovulated, the number of CL, and the total number of ova/embryos collected did not differ between seasons or treatment groups, but the number of transferable embryos was greater (p < .05) in the ovulatory season. In Experiment 2, no differences were detected between seasons or treatment groups for any end point. The number of transferable embryos produced per bison was greatest (p < .05) using in vitro fertilization and was unaffected by season (1.5 ± 0.2 and 1.1 ± 0.3 during anovulatory and ovulatory seasons, respectively), in contrast to in vivo embryo production which was affected by season (0.1 ± 0.01 and 0.7 ± 0.2 during anovulatory and ovulatory seasons, respectively). Results demonstrate that transferable embryos can be produced throughout the year in wood bison by both in vivo and in vitro techniques, but the efficiency of embryo production of in vivo-derived embryos is significantly lower during the anovulatory season.

Strategies to increment in vivo and in vitro embryo production and transfer in cattle.

Knowledge of follicular wave dynamics obtained through the use of real-time ultrasonography and the development of the means by which follicular wave dynamics can be controlled have provided practical approaches for the in vivo and in vitro production and transfer of embryos in cattle. The elective control of follicular wave emergence and ovulation has had a great impact on the application of on-farm embryo transfer, especially when large groups of donors need to be superstimulated at the same time. Although estradiol and progestins have been used for many years, practitioners in countries where estradiol cannot be used have turned to alternative treatments, such as mechanical follicle ablation or the administration of GnRH for the synchronization of follicle wave emergence. In vitro embryo production also benefits from the synchronization of follicle wave emergence prior to Cumulus Oocyte Complexes (COCs) recovery. As Bos indicus cattle have high antral follicle population, large numbers of oocytes can be obtained by ovum pick-up (OPU) without superstimulation. However, synchronization of follicular wave emergence and superstimulation is necessary to obtain high numbers of COCs by OPU and blastocysts following in vitro fertilization in Bos taurus donors. Finally, embryos can now be transferred in commercial beef or dairy herds using efficacious synchronization and re-synchronization protocols that are easily implemented by farm personnel. These technologies can also be used to resolve reproductive problems such as the reduced fertility observed during summer heat stress and/or in repeat-breeder cows in commercial dairy herds.

Effect of injectable progesterone on follicular development in lactating beef cows treated with estradiol plus a low-concentration progesterone device / Efeito da progesterona injetável no desenvolvimento folicular em vacas de corte lactantes tratadas com estradiol e dispositivo de progesterona de baixa concentração

The effect of injectable progesterone was evaluated along with estradiol benzoate (EB) on the fate of the dominant follicle (DF) present in the ovary at the beginning of low progesterone-based TAI protocol. All cattle were given 500 µg cloprostenol im (PGF; Schering-Plough Animal Health for Estrumate, Pointe-Claire, QC, Canada) twice, 11 d apart, and allocated into two groups: Estradiol group (E group, n = 11) and Estradiol-Progesterone group (EP group, n = 11). Ten days after the second PGF (Day 0), all cattle were given an intravaginal progesterone device with half progesterone concentration (Cue-Mate with a single pod containing 0.78 g progesterone). Concurrently, all cattle were given 1.5 mg im of estradiol benzoate in 3 mL of canola oil and PGF im on Day 0 of the protocol in a crossover design, in which each cow received both treatments. Cows in the EP group also received 100 mg im progesterone (Sigma) in 2 mL of canola oil. On Day 8, progesterone devices were removed and all cattle were given PGF im. All statistical analyses were performed with SAS 9.0. The DF present on Day 0 ovulated in 76% (16/21) of cows from E group and 28.6% (6/21) of cows from EP group (P = 0.002). After progesterone device removal, the size of ovulatory follicle did not differ between groups (E group, 15.5 ± 0.43 mm vs EP group, 15.8 ± 0.98 mm; P = 0.82). These follicles ovulated in 81.3 ± 3.1 h in E group and 71.0 ± 6.1 h in EP group (P = 0.13). In conclusion, injectable progesterone reduced the proportion of cows that ovulate the dominant follicle present in the ovary at the beginning of estradiol-progesterone-based protocols. However, no difference was detected on time of ovulation after progesterone device removal between groups.(AU)

Foi avaliado o efeito da progesterona injetável e do benzoato de estradiol (BE) no destino do olículo dominante (FD) presente no ovário no início do protocolo de IATF. Todas as vacas receberam duas injeções de 500 µg de cloprostenol im (PGF; Schering-Plough Animal Health for Estrumate, Pointe-Claire, QC, Canadá) em um intervalo de onze dias e foram alocadas em dois grupos: Estradiol (grupo E, n = 11) e Estradiol-Progesterona (grupo EP, n = 11). Dez dias após a segunda injeção de PGF (Dia 0), elas receberam um implante intravaginal de progesterona com metade da concentração hormonal (Cue-Mate com apenas uma haste contendo 0,78 g de progesterona). Além disso, todas vacas receberam 1,5 mg im de BE dissolvido em óleo de canola e PGF im no Dia 0 do protocolo, em um delineamento em crossover no qual cada vaca recebeu ambos tratamentos. Vacas do grupo EP ainda receberam uma injeção de 100 mg im de progesterona (Sigma) em 2 mL de óleo de canola no Dia 0. No Dia 8, os dispositivos de progesterona foram removidos e todas as vacas receberam PGF im. A análise estatística foi realizada por meio do pacote estatístico SAS 9.0. O FD presente no Dia 0 ovulou em 76% (16/21) das vacas do grupo E e em 28,6% (6/21) das vacas do grupo EP (P = 0,002). Após a remoção do dispositivo de progesterona, o diâmetro do folículo ovulatório não apresentou qualquer diferença entre os grupos (grupo E, 15,5 ± 0,43 mm; grupo EP, 15,8 ± 0,98 mm; P = 0,82). Esses folículos ovularam em 81,3 ± 3,1 h no grupo E e em 71,0 ± 6,1 h no grupo EP (P = 0,13). A conclusão obtida foi que o uso de progesterona injetável reduziu a proporção de vacas que ovularam o folículo dominante presente no ovário no início do protocolo à base de estradiol e progesterona. No entanto, entre os grupos não houve diferença no momento da ovulação após a remoção do dispositivo de progesterona.(AU)

Pursuit of a method for single administration of pFSH for superstimulation in cattle: What we have learned.

A single dose protocol of FSH for superstimulation in cattle may improve compliance and superovulatory response. A single subcutaneous (sc) administration of pFSH was efficacious, but response depended on body condition and injection site; the adipose tissue pad behind the shoulder was most efficacious. Inconsistent results in Holsteins were partially overcome by sc administration of 75% of the total pFSH dose behind the shoulder on the first day followed by 25% 48 h later. An alternative would be to combine FSH with polymers that cause it to be released slowly over several days. Hyaluronan is found normally in most animal tissues and is nonreactive when administered parentally. A single intramuscular (im) administration of pFSH in a 2.0% hyaluronan induced a superovulatory response that did not differ from twice daily im administration over 4 d. However, 2.0% hyaluronan was viscous and difficult to mix with FSH. Although solutions of 1.0 and 0.5% hyaluronan were less viscous, they lacked efficacy as a single im administration. However, superovulatory response was high when either 1.0 or 0.5% hyaluronan was used in a two-dose im protocol; two-thirds on the first day and one-third 48 h later. A single im administration of FSH in 0.5% hyaluronan effectively induced superstimulation for OPU in cattle. Successful superovulation in the cow was associated with circulating FSH levels that were similar to endogenous FSH levels prior to follicular wave emergence; however, levels must be maintained above baseline for at least 72 h, or 36 h for OPU. Circulating FSH levels following a single sc administration of 400 mg NIH-FSH-P1 behind the shoulder in beef cows increased to 1.0 or 1.2 ng/mL at 12 h and were back near baseline in approximately 60 h, while FSH levels following im administration of 200 mg NIH-FSH-P1 in 0.5% hyaluronan into Holstein donors reached 1.5 ng/mL at 12 h and returned to baseline in approximately 36 h.

Evolution of knowledge on ovarian physiology and its contribution to the widespread application of reproductive biotechnologies in South American cattle.

As our understanding of ovarian function in cattle has improved, our ability to control it has also increased. Luteal function in cattle has been studied in detail, and prostaglandin F2α has been used for several years for the elective induction of luteal regression. More recently, follicle wave dynamics has been studied and protocols designed to induce follicular wave emergence and ovulation have reduced, and even eliminated, the need for estrus detection. The addition of progestin-releasing devices, estradiol, GnRH and equine chorionic gonadotropin (eCG) have provided opportunities for fixed-time AI (FTAI) and possibilities for increased pregnancy rates. In embryo transfer programs, these same treatments have eliminated the need for estrus detection, permitting fixed-time embryo transfer and the initiation of superstimulatory treatments without regard to day of the estrous cycle. Collectively, new treatment protocols have facilitated the application of assisted reproductive technologies, and this is especially true in South America. Over the last 20 years, the use of AI in South America has increased, due largely to the use of FTAI. There has been more than a 10-fold increase in the use of FTAI in Brazil with more than 11 million treatments in 2016, representing 85% of all AI. Similar trends are occurring in Argentina and Uruguay. Production of in vivo-derived (IVD) embryos has remained relatively stable over the years, but in vitro embryo production (IVP) has increased dramatically over the past 10 to 15 years, especially in Brazil where more than 300,000 IVP embryos were produced in 2010. World-wide, more than 666,000 bovine IVP embryos were produced in 2016, of which more than 57% were produced in South America. The use of assisted reproductive technologies has facilitated the dissemination of improved genetics and increased reproductive performance; other South American countries are now following suit.

Effect of extending FSH treatment on superovulation and embryo production in wood bison (Bison bison athabascae).

The effect of extending the length of the FSH treatment protocol on superovulatory response and embryo production was investigated in wood bison during the anovulatory and ovulatory seasons. In Experiment 1 (anovulatory season), follicular wave emergence was synchronized by follicular ablation (Day -1) and bison were assigned randomly to two groups (n = 14/group) and given 200 mg FSH on Day 0 and Day 2 (non-extended group), or 133 mg FSH on Days 0, 2, and 4 (extended group). Human chorionic gonadotropin (hCG; 3000 IU) was given on Day 5 and Day 6 in the non-extended and extended groups, respectively, and bison were inseminated 12 and 24 h later. Ova/embryos were collected 8 days after hCG treatment. In Experiment 2 (ovulatory season), bison were synchronized and superstimulated as in Experiment 1 (n = 12/group), but prostaglandin was given to control CL development. Data were compared by t-test and Chi-square test. In Experiment 1, no differences in ovarian response or embryo production between groups were detected. In Experiment 2, there was no difference in the ovarian response between groups, however, a greater number of ova/embryos (4.3 ± 0.8 vs. 2.3 ± 0.4; P ≤ 0.05) and freezable embryos (2.5 ± 0.6 vs. 1.2 ± 0.4; P ≤ 0.05) were obtained in the extended group. The number of freezable embryos was greater during the ovulatory vs anovulatory season (1.8 ± 0.4 vs. 0.3 ± 0.2; P ≤ 0.05). In conclusion, extending the FSH treatment in wood bison did not improve the superovulatory response during the anovulatory season, but resulted in twice as many freezable embryos during the ovulatory season. The number of freezable embryos collected during the anovulatory season was <20% that of the ovulatory season.

Pregnancy per AI in Holstein heifers inseminated with sex-selected or conventional semen after estrus detection or timed-AI.

This study compared pregnancy per AI (P/AI) of heifers inseminated with sex-selected or conventional semen after estrus detection (ED) or timed-AI (TAI). Heifers in the ED group received 2 treatments with prostaglandin F2α 14 d apart and those in the TAI group received a modified 5-day Co-synch protocol plus an intravaginal progesterone releasing insert device (PRID) and were inseminated 72 h after PRID removal. Overall P/AI were 69.2% (74/107) and 64.1% (75/117) for conventional and sex-selected semen (P > 0.05). Although P/AI in ED heifers following the use of conventional semen were numerically higher (75.0% versus 63.6%), P/AI with sex-selected semen were almost identical (65.0% versus 63.2%) for ED and TAI heifers. Pregnancy losses from first pregnancy diagnosis to term did not differ between ED and TAI heifers (6.0% versus 11.3%). In summary, heifers subjected to TAI with sex-selected semen following the application of a modified 5-day Co-synch plus PRID protocol had acceptable P/AI.

Gestation par IA chez les génisses Holstein inséminées avec du sperme déterminant le sexe ou un sperme conventionnel après la détection de l'oestrus ou l'IA à temps prédéterminé. Cette étude a comparé la gestation par IA (G/IA) des génisses inséminées avec du sperme déterminant le sexe ou du sperme conventionnel après la détection de l'oestrus (DOE) ou l'IA à temps déterminé (IATD). Les génisses dans le groupe DOE ont reçu 2 PGF à un intervalle de 14 jours et celles dans le groupe IATD ont reçu un protocole modifié 5-d Co-synch et PRID et ont été inséminées 72 heures après l'enlèvement de PRID. Dans l'ensemble, le taux de G/IA était de 69,2 % (74/107) et de 64,1 % (75/117) pour le sperme conventionnel et celui déterminant le sexe (P > 0,05). Même si le taux de G/IA chez les génisses DOE après l'utilisation de sperme conventionnel était numériquement supérieur (75,0 % c. 63,6 %), le taux de G/IA avec le sperme déterminant le sexe était presque identique (65,0 % c. 63,2 %) pour les génisses DOE et IATD. La perte de gestations entre le premier diagnostic de gestation jusqu'à la parturition ne différait pas entre les génisses DOE et TAI (6,0 c. 11,3 %). En résumé, les génisses soumises à l'IATD avec du sperme déterminant le sexe après l'application d'un protocole modifié 5-d Co-synch et PRID avaient une G/IA acceptable qui confirmait notre hypothèse.(Traduit par Isabelle Vallières).

Effects of eCG and progesterone on superovulation and embryo production in wood bison (Bison bison athabascae).

Experiments were done to determine if inclusion of eCG and progesterone in the superstimulation protocol will increase the ovarian response and embryo production in wood bison, and to provide preliminary information regarding the effect of season. In Experiment 1 (anovulatory season), bison (n=26) were synchronized by follicular ablation (Day -1) and given FSH on Days 0 and 2, and assigned to 3 groups: Progesterone (Days 0-4), eCG (Day 3), or progesterone+eCG. On Day 5, bison were given hCG and inseminated 12 and 24h later. Ova/embryos were collected 8days after hCG. In Experiment 2 (ovulatory season), bison (n=24) were synchronized and assigned randomly to two groups in which superstimulation was induced with FSH, either with or without eCG, as in Experiment 1. No differences among groups were found in ovarian response or embryo production in either experiment. The follicular count at wave emergence was positively correlated with the number of large follicles at the end of superstimulation in all groups. A significantly greater number of follicles present at wave emergence in the anovulatory vs. ovulatory season was associated with a greater number of CL at the time of embryo collection, but only half the number of freezable embryos. In conclusion, the number of transferable embryos collected (1-2/bison) was higher than in any previous report, but was not attributable to the inclusion of eCG or progesterone in the superovulatory protocol. The apparent effect of season on oocyte competence, and not superovulatory response, is worthy of further investigation.

In vitro-production of embryos using immature oocytes collected transvaginally from superstimulated wood bison (Bison bison athabascae).

Two experiments were done to test the hypothesis that morphologic characteristics of wood bison cumulus-oocyte complexes (COC) are reflective of the ability of the oocyte to develop to an advanced embryonic stage after in vitro maturation, fertilization and culture, and to determine the effect of prolonging the interval from the end of superstimulation treatment to oocyte collection (FSH starvation period). Experiments were done during the anovulatory season. In Experiment 1, ovarian superstimulation was induced in 10 bison with two doses of FSH given at 48 h intervals beginning at the time of follicular wave emergence. COC were collected 3 days (72 h) after the last dose of FSH by follicular aspiration and classified as compact, expanded or denuded. The COC were matured in vitro for 24 h before fertilization in vitro (Day 0). Embryo development was assessed on Days 3, 7 and 8. The blastocyst rate was 7/34, 2/10 and 0/3 in COC classified as compact, expanded and denuded, respectively; however, only compact COC resulted in embryos that reached the expanded blastocyst stage. In Experiment 2, COC were collected at either 3 or 4 days (72 or 96 h) after the last dose of FSH (n = 16 bison/group) to determine the effect of the duration of FSH starvation on oocyte competence. The COC were classified as compact good (>3 layers of cumulus cells), compact regular (1-3 layers of cumulus cells), expanded or denuded, and then matured, fertilized and cultured in vitro. Although follicles were larger (P < 0.05) in the 4-day FSH starvation group, there was no effect of starvation period on the distribution of COC morphology; overall, 112/194 (57.7%) were compact, 29/194 (26.3%) were expanded, 39/194 (20.1%) were denuded, and 14/194 (7.2%) were degenerated (P < 0.05). Similarly, there was no effect of starvation period on embryo development. Compact good COC had the highest cleavage (88%) and blastocyst rates (54%; P < 0.05), followed by compact regular COC at 73% and 25%, respectively. Expanded and denuded COC had low cleavage (40% vs. 59%, respectively) and blastocyst rates (5% vs. 8%, respectively). We conclude that morphologic characteristics of wood bison COC are reflective of the ability of the oocyte to develop into an embryo in vitro. Importantly, oocytes collected from superstimulated bison during the anovulatory season were competent to develop to the blastocyst stage following in vitro maturation, fertilization and culture.

In vitro embryo production in wood bison (Bison bison athabascae) using in vivo matured cumulus-oocyte complexes.

Experiments were conducted in wood bison to determine the effect of additional maturation time on embryo development of in vivo matured oocytes. In experiment 1, cumulus-oocyte complexes (COC) were collected 30 hours after hCG treatment in superstimulated wood bison, and expanded COC were fertilized immediately or after 4 hours of additional in vitro maturation. Embryo development was assessed on Days 3, 7, and 8 (Day 0 = day of fertilization). No difference in cleavage rate was detected (55.3% vs. 60.5%, P = 0.82), but the Day 8 blastocyst rate was higher after an additional 4 hours of in vitro maturation time (44.7 vs. 18.4%, P = 0.03). In experiment 2, COC were collected at either 30 hours or 34 hours after hCG treatment. Expanded COC from the 30 hours group were fertilized after 4 hours of in vitro maturation, whereas those from the 34 hours group were fertilized immediately. A higher cleavage rate (74.3 vs. 57.0%) and blastocyst rate (54.1 vs. 37.2%) were found in the 34 hours group versus the 30 hours group (P < 0.05). In conclusion, an additional short period of in vitro maturation, or an extended period of in vivo maturation are beneficial for in vitro embryo production in wood bison.

In vivo and in vitro maturation of oocytes collected from superstimulated wood bison (Bison bison athabascae) during the anovulatory and ovulatory seasons.

Experiments were done to compare the in vivo and in vitro maturational characteristics of cumulus-oocyte complexes (COC) collected from live wood bison. In Experiment 1 (anovulatory season), follicular ablation was done to synchronize follicle wave emergence among bison on Day -1, and FSH was given on Days 0 and 2. Bison were then assigned to 5 groups (n=5/group) in which COC were collected by transvaginal follicle aspiration on Day 4 and either fixed immediately with no maturation (control), matured in vitro for 24 or 30h, or collected on Day 5 after in vivo maturation for 24 or 30h (i.e., after hCG treatment). In Experiment 2 (ovulatory season), bison were treated as described for Experiment 1, but PGF2α (cloprostenol) was given to control the luteal phase on Days -9 and 3. In both experiments, cumulus cell expansion was more extensive following in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30h groups. Nuclear maturation occurred more rapidly in vitro; 60-70% of oocytes were at the MII stage 24h after in vitro maturation while only 25-27% of oocytes had reached the MII stage after 24h of in vivo maturation. In conclusion, nuclear maturation occurred more rapidly during in vitro vs. in vivo maturation, but was associated with less cumulus expansion than in vivo maturation. In vivo oocyte maturation was more complete at 30 vs. 24h after hCG treatment. Season had no effect on the maturational capacity of wood bison oocytes.

Superovulation in wood bison (Bison bison athabascae): Effects of progesterone, treatment protocol and gonadotropin preparations for the induction of ovulation.

Experiments were done to determine the ovarian response and embryo production following superstimulation of wood bison. In Experiment 1 (Anovulatory season), the efficacy of pLH vs. hCG for inducing ovulation was compared in wood bison superstimulated with a single dose of pFSH in 0.5% hyaluronan and the effect of exogenous progesterone (PRID) on superovulatory response and embryo quality was examined. In Experiment 2 (Ovulatory season), the efficacy of pLH vs. hCG for the induction of ovulation was compared in wood bison superstimulated with pFSH in a single intramuscular dose vs. a two-dose regimen 48 h apart (split dose) in 0.5% hyaluronan. In Experiment 1, the number of CL was greater (P < 0.05) in bison treated with hCG than pLH (6.6 ± 1.8 vs. 2.8 ± 0.8) and in those that were not given PRID (6.0 ± 1.5 vs. 2.7 ± 1.0). There was no effect of progesterone treatment on embryo quality. In Experiment 2, the number of CL was greater (P < 0.05) in bison treated with hCG than with pLH (6.3 ± 0.8 vs. 3.8 ± 1.2) and in bison superstimulated with split dose vs. single dose of FSH (7.1 ± 0.9 vs. 3.0 ± 0.8). The number of ova/embryos and freezable embryos did not differ among groups in either experiment. In conclusion, hCG induced a greater ovulatory response than pLH in both seasons. Two doses of FSH induced the greatest superovulatory response during the ovulatory season. Exogenous progesterone did not improve embryo quality during the anovulatory season.