The 6-d timed artificial insemination protocol has been designed to advance luteolysis after the first administration of GnRH so that the preovulatory follicular diameter at second GnRH is reduced and thereby pregnancy outcome may be improved. To achieve an earlier and complete luteolysis (5 to 6 d after the first GnRH treatment), an extra PGF(2α) treatment must be administered to cows 24 h after the initial PGF(2α) treatment. Although the use of 2 PGF(2α) treatments increases labor costs resulting from the increased handling of cows, no alternative and efficient protocol with a single PGF(2α) treatment has been found to date. The objective of this study was to compare the effect of a modified 6-d synchronization protocol on the luteolytic response and final preovulatory follicle diameter. The study followed a crossover design: 14 nonlactating dairy cows were included in 2 treatment doses. All cows received a presynchronization treatment consisting of 2 administrations of a PGF(2α) analog (PGF) 14 d apart followed by treatment with GnRH 11 d later. After the first GnRH administration, one treatment consisted of 150 µg of d-cloprostenol 5 and 6 d later (split dose) and the other treatment consisted of 375 µg of d-cloprostenol as a single dose 6 d after the first GnRH (single large dose). All cows were then treated with a second GnRH 8 d after the first. The luteolytic response to treatment was evaluated by blood progesterone concentration and CL area regression -1 to 3 d relative to the last PGF treatment obtained by ELISA and ultrasonography, respectively. Fewer cows of the split dose tended to have complete luteolysis 3 d after the last PGF treatment compared with the cows of the single large dose (35.7 and 64.3%, respectively). The final preovulatory diameter of the dominant follicle was similar in cows from the split dose and single large dose (13.7 ± 0.3 and 13.1 ± 0.5mm, respectively). Our results support the modification of the 6-d synchronization protocol by administering a single high dose of PGF 6 d after GnRH (with the subsequent reduction in labor resulting from reduced handling of animals) without detrimental effects on the luteolytic response of dairy cows and preovulatory diameter of the dominant follicle compared with the original protocol. However, this modification of the 6-d synchronization protocol should be tested in a large field study involving fertility data with lactating cows before its use can be recommended.

A study was designed to determine the effect of stage of the estrous cycle on the proportion of animals that ovulated and the synchrony of ovulation of heifers treated with an aromatase inhibitor-based protocol. Forty-eight heifers were treated intramuscularly with 500 μg of cloprostenol (PGF) followed by 100 μg of GnRH 24 hours later to serve as control data for comparison of the ovulatory response to a subsequent aromatase inhibitor protocol. Daily ultrasound examinations were done to determine the incidence of and interval to ovulation. At the time of ovulation (Day 0), heifers were assigned randomly to five day-groups (n = 8-11/group) and given an intravaginal device containing 3 g of letrozole for 4 days starting on Day 0, 4, 8, 12, or 16. At the time of device removal, heifers were given PGF followed by GnRH 24 hours later. Ultrasound examinations were done daily from 2 days before device insertion to 9 days after the posttreatment ovulation. The preovulatory follicle diameter after letrozole treatment was larger in the Day 4 group compared to the Day 0 and 16 groups and intermediate in the Day 8 and 12 groups (P < 0.001). Compared to control data, the percentage of heifers that ovulated after letrozole treatment was greater (87.1% vs. 69.4%, respectively; P < 0.05) as was the synchrony of ovulation (residuals: 0.24 ± 0.07 vs. 0.68 ± 0.13; P < 0.01). The day on which letrozole treatment was initiated did not affect the proportion of heifers that ovulated or the interval to ovulation. Plasma estradiol concentrations at the time of removal of the letrozole device in the Day 0 and 4 groups was lower (P < 0.05) than in the corresponding controls. Estradiol concentrations in the Day 8 and 12 groups did not differ from already low concentrations in the respective controls. Corpus luteum diameter profiles and progesterone production were not affected by day-group although reduced luteal lifespan after letrozole treatment was observed and requires further investigation. In summary, a protocol involving a letrozole-impregnated intravaginal device for 4 days, PGF treatment at device removal, and GnRH 24 later resulted in a greater ovulation rate and greater synchrony of ovulation than in heifers not given letrozole. Results suggest that the protocol may be initiated effectively at random stages of the estrous cycle and may provide impetus for further studies to assess the efficacy of a letrozole-based synchronization protocol for fixed-time insemination.

Two experiments were conducted to assess a hormonal strategy developed to reduce animal handling for timed artificial insemination (TAI) with sex-sorted semen. Four-hundred ninety-one (491) suckled beef cows received a progesterone (P4) intravaginal device and 2 mg intramuscular (im) injection of estradiol benzoate (EB) on a randomly chosen day of the estrus cycle (Day 0) in Experiment 1. Cows were treated with 500 μg of sodic cloprostenol (PGF2α) and with 300 IU of eCG at P4 device removal (Day 8); these cows were also randomly assigned to receive 1 mg of estradiol cypionate (EC) administered at P4 device removal (treatment EC-0h) or 1 mg of EB 24 h after P4 device removal (treatment EB-24h). Both treatments were timed inseminated (TAI) with sex-sorted semen 60 h after P4 device removal. Cows treated with EC-0h presented higher pregnancy rate per AI (P/AI) [45.0% (113/251)] than the ones treated with EB-24h [35.4% (85/240); P = 0.03)]. A subset of cows (n = 26) were subjected to ultrasound examination every 12 h after P4 device removal for 96 h in the row in order to determine the time of ovulation. Similar interval between device removal and ovulation was recorded for EB-24h = 70.0 ± 2.9 h vs. EC-0h = 66.0 ± 2.8 h (P = 0.52). Five-hundred ninety-one (591) cows were subjected to the same synchronization protocols and treatments (EC-0h or EB-24h). In addition, they were randomly assigned to a 2 × 2 factorial arrangement aiming at determining the effects of treatment with estradiol (EC-0h vs. EB-24h) and of semen type (Sex-sorted vs. Non-sex-sorted semen). All animals were timed inseminated 60 h after P4 device removal. There was no interaction (P = 0.07) between the ovulation inducer and semen type. The EC protocol led to greater P/AI than EB (P = 0.03). Greater (P = 0.01) P/AI was achieved through treatments with non-sex-sorted semen rather than with sex-sorted semen [sex-sorted (EB-24h = 49.0%; EC-0h = 51.0%) vs. non-sex-sorted semen (EB-24h = 52.4%; EC-0h = 68.2%)]. Therefore, EC administered at P4 device removal resulted in greater P/AI. Furthermore, the EC-0h protocol allowed reducing suckled beef cow handing for timed artificial insemination with sex-sorted semen.

The aim of the present study was to determine the recovery of embryonic structures (ova/embryos) and fertilization rate in superovulated buffaloes treated with PGF_2α during the periovulatory period. On day 0 (D0), buffaloes at random stages of the estrous cycle were treated with an intravaginal progesterone device (P4; 1.0 g) and estradiol benzoate (EB, 2.0 mg i.m.). From D4 to D7, all buffaloes received i.m. FSH (200 mg total) twice-daily over 4 days in decreasing doses. On D6 and D7, the animals were given PGF_2α analogue (0.53 mg i.m. sodium cloprostenol) and the P4 device was removed on D7. On D8, all buffaloes received GnRH (20 μg i.m. buserelin acetate). Buffaloes were then randomly allocated to one of three groups: control (Group C, n = 18), no further treatment; PGF_2α analogue injection (Group IM-PGF; n = 18), four injections (0.53 mg i.m. sodium cloprostenol) 12 h apart, from D8 to D10; PGF_2α analogue osmotic pump (Group OP-PGF; n = 18), s.c. osmotic mini-pump (2.12 mg sodium cloprostenol) from D8 to D10. The study had a crossover design (three treatments x three replicates). All animals underwent timed AI, 12 and 24 h after treatment with GnRH. Embryonic structures were recovered on D14. Ovarian ultrasonography was used on D8 and D14 to record follicular superstimulation and superovulatory responses. Blood samples were obtained on Days 7, 8, 9 and 10 to measure circulating concentrations of P4, E2 and PGFM. Data were analyzed by GLIMMIX procedure of SAS®. There was no effect (P = 0.58) of treatment on the total number of embryonic structures (Group C, 2.1 ± 0.8; Group IM-PGF, 2.1 ± 0.6; Group OP-PGF, 1.4 ± 0.4). There was also no effect (P = 0.93) of treatment on the recovery rate of embryonic structures (oocytes and embryos D14/CL D14). The fertilization rate was higher (P = 0.04) in Groups IM-PGF (84.6%) and OP-PGF (88.0%), which did not differ, than Group C (63.2%). The viable embryos rate was greater (P < 0.01) for Groups IM-PGF (82.0%) and OP-PGF (88.0%) than Group C (52.6%). There was no interaction between treatment and time and treatment effects for P4, E2 and PGFM concentrations. The findings showed that treatment with PGF_2α during the periovulatory period has potential to increase fertilization rate and embryo production in superovulated buffaloes.

The aim of this study was to evaluate the effect of eCG or hCG on the final growth of the dominant follicle in Nelore (Bos indicus) cows submitted to fixed-time AI (FTAI). Eighty-four lactating cows with body condition score (BCS) of 2.9 (range 1-5) were used. At a random day of the estrous cycle (D0) cows received 2 mg estradiol benzoate and a reused intravaginal progesterone device (1.9 g). At D8, when the device was removed, 0.5 mg cloprostenol and 1 mg estradiol cypionate was given i.m., and cows were randomly assigned to receive on D8 one of the following treatments: Control (no treatment; n = 17), eCG (300 IU i.m.; n = 17), hCG 300 (300 IU i.m.; n = 18), hCG 200 IM (200 IU i.m.; n = 16) and hCG 200 SC (200 IU s.c.; n = 16). On the same day and 2 days later, cows were subjected to ovarian ultrasonography to evaluate the diameter of the largest follicle and to calculate follicular growth rate (D8 to D10). No differences were observed for the diameter of the largest follicle on D8 (P = 0.3) or D10 (P = 0.4) among treatments. However, the growth rate of the dominant follicle between D8 and D10 was greater for the groups eCG and hCG 300 and there were no differences between the other treatments (Control = 1.1 mm/day; eCG = 1.8 mm/day; hCG 300 = 1.8 mm/day; hCG 200 IM = 1.3 mm/day; hCG 200 SC = 1.3 mm/day; P = 0.02). In addition, more cows from the Group hCG 300 presented premature ovulation (44.4%) than cows from Control (5.8%), eCG (0%), or hCG 200 IM (12.5%), but did not differ from Group hCG 200 SC (18.7%). Regardless of treatment, the size of the largest follicle on D8 was different between cows that presented premature ovulation vs. cows that did not ovulate prematurely (11.3 mm vs. 9.9 mm, respectively; P = 0.01). Treatment with different hCG doses on D8 of a FTAI protocol failed to produce similar effects compared to eCG in terms of final follicular growth support and greater ovulatory follicle size. In addition, the groups hCG 300 and hCG 200 SC induced premature ovulation in a greater portion of cows. Thus, a single administration of hCG on D8 does not appear to be a reliable alternative to eCG treatment in FTAI protocols.

This study compared estradiol cypionate (ECP) or GnRH as ovulation inducers at the end of a timed AI (TAI) protocol in Angus heifers. On day 0, heifers (n = 415), between 22 and 24 months of age, were treated with an intravaginal 1 g progesterone (P4) insert and 2 mg of estradiol benzoate. On day 8, heifers had P4 removed, received 500 μg cloprostenol, and were randomized into two groups: ECP [n = 214; 0.5 mg of ECP on day 8] or GnRH [n = 201; 25 μg of GnRH analog licerelin acetate on day 10]. All heifers received TAI on day 10; 48-50 h after P4 insert withdrawal. Estrus was determined by removal of tail paint. Ovaries of heifers were evaluated by ultrasound on day 0 to determine CL presence (with CL = 213, without CL = 202) and on day 10 to measure preovulatory follicle size. Heifers were divided into three categories based on preovulatory follicle diameter: <8.5 mm (smaller than deviation), 8.5-10.9 mm, or ≥11 mm. Pregnancy diagnosis was performed 32 days after TAI. Heifers treated with ECP had greater expression of estrus than GnRH-treated heifers (93.9% vs 67.7%; P < 0.0001), regardless of CL presence at beginning of protocol. Heifers with CL at beginning of protocol had larger preovulatory follicle diameter (10.4 mm vs 9.6 mm; P = 0.0058) and greater pregnancy per AI (P/AI; 61.0% vs 50.5%; P = 0.032) than heifers without CL at day 0. In heifers with CL at day 0, GnRH treatment increased P/AI compared to ECP treatment (68.0% vs 54.9%; P = 0.0498). Expression of estrus was greater in ECP-treated than GnRH-treated heifers that had small (<8.5 mm; 77.1% vs 5.6%; P < 0.001) or medium-sized (8.5-10.9 mm; 98.4% vs. 61.7%) follicles, but not in heifers with large follicles (≥11 mm; 97.9% vs 98.3%). The P/AI was very low in both treatments for heifers with follicles <8.5 mm (ECP-14.3% vs GnRH-16.7%). In heifers with medium-sized follicles (8.5-10.9 mm), ECP treatment tended to increase P/AI compared with GnRH-treated heifers (62.9 vs 46.7%; P = 0.074). In contrast, P/AI was greater for GnRH-treated than ECP-treated heifers with large preovulatory follicles (≥11 mm; 79.7% vs 60.4%; P = 0.032). Thus, the optimal inducer of ovulation in a TAI protocol for beef heifers appears to depend on the presence of a CL at beginning of protocol (GnRH>> ECP if CL present) and size of the preovulatory follicle, with ECP increasing expression of estrus and tending to increase fertility in heifers with medium-sized follicles but GnRH increasing fertility in heifers having large preovulatory follicles.

Fixed-time and split-time AI were compared following the melengestrol acetate (MGA^®) prostaglandin F_2α (Experiment 1) and 7-d CO-Synch + controlled internal drug release (CIDR^®) protocols (Experiment 2). Heifers in Experiments 1 (n = 524) and 2 (n = 456) were assigned within pen to balanced treatments based on weight and reproductive tract score (RTS; Scale 1-5). In Experiment 1, MGA^® (0.5 mg∙animal^-1∙d^-1) was fed for 14 d, and prostaglandin F_2α (PG; 250 μg im cloprostenol sodium) was administered 19 d after MGA^® withdrawal. In Experiment 2, gonadotropin-releasing hormone (GnRH; 100 μg gonadorelin acetate) was administered coincident with CIDR^® (1.38 g progesterone [P₄]) insertion. Inserts were removed after 7 d, and PG (250 μg im cloprostenol sodium) was administered at CIDR^® removal. In both experiments, estrus detection aids (Estrotect^®) were applied at the time of PG administration. Estrous status was recorded at FTAI or STAI. Estrus was defined as removal of ≥ 50% of the grey coating from the Estrotect^® patch. Heifers assigned to FTAI treatments received GnRH and were artificially inseminated at the standard time for FTAI for each protocol: 72 or 54 h after PG administration for the MGA-PG or 7-d CO-Synch + CIDR^® protocol, respectively. In the STAI treatments, only heifers that expressed estrus prior to the standard time of FTAI were artificially inseminated at that time. For heifers failing to express estrus, AI was postponed 24 h. Only heifers that failed to exhibit estrus by the delayed time received GnRH concurrent with AI. In both experiments, estrous response prior to the standard time of FTAI did not differ between treatments. Total estrous response was increased (P < 0.01) among heifers assigned to STAI in Experiment 1 (88%, STAI; 72%, FTAI) and 2 (74%, STAI; 47%, FTAI). In Experiment 1, pregnancy rates resulting from AI were greater (P < 0.04) for heifers assigned to STAI compared with FTAI (55% vs 46%, respectively). In Experiment 2, pregnancy rates resulting from AI were similar between treatments (48% and 46%, respectively; P = 0.6). In summary, when compared with FTAI, STAI resulted in greater estrous response following both the MGA^®-PG and 7-d CO-Synch + CIDR^® protocols. The increased estrous response through use of STAI was associated with a corresponding increase in pregnancy rates to AI following the MGA^®-PG protocol; however, a similar improvement in pregnancy rates was not observed following the 7-d CO-Synch + CIDR^® protocol.

The influence of ovarian follicle status and follicle dominance on the response to superstimulatory treatment with FSH was examined in cows. In Experiment 1, oestrus was synchronised using Crestar and on Days 4-6 of the ensuing oestrous cycle cows were assigned to: Group NO (n = 9), control, endogenous CL and no treatment; Group N1 (n = 15), injected with a luteolytic dose of cloprostenol (500 micrograms) and implanted with one implant (3 mg) of the synthetic progestogen, norgestomet; Group N8 (n = 18), injected with 500 micrograms cloprostenol and implanted with eight (24 mg) implants of norgestomet. On Days 9-11, seven implants were removed from six cows in Group N8 and these cows, plus eight Group N1 and all Group N0 cows, were superstimulated with porcine FSH (Folltropin-V) over 4 days (360 mg total dose). The remaining implants were removed from cows in Groups N1 and N8 on Days 11-13, and all cows received 500 micrograms cloprostenol. Numbers and sizes of ovarian follicles, and CL, were recorded by trans-rectal ultrasonography; the largest follicle>> 10 mm in diameter was considered morphologically dominant (DF). On Days 9-11, the proportions of cows with a DF were: Group N0, 3/9; Group N1, 14/15; Group N8, 0/18. Total follicles on the 4th day of FSH treatment were greater (P < 0.05) for cows in Group N1 (21.6 +/- 4.2) compared with Group N0 (10.9 +/- 2.4), with cows in Group N8 (13.2 +/- 0.9) not different from the other two groups. Subsequent numbers of CL were lower (P < 0.05) for cows in Group N1 (5.0 +/- 1.3) compared with Group N0 (9.4 +/- 2.0), with cows in Group N8 (8.5 +/- 1.0) not different from the other two groups. In Experiment 2, oestrus was synchronised in cows and on Days 4-6, cows were assigned to: Group C0 (n = 7), control, endogenous CL and no treatment; Group C3 (n = 6), received three CIDR-B intra-vaginal devices that delivered progesterone. On Days 9-11, two CIDR-B were removed from cows in Group C3 and all cows treated with FSH as in Experiment 1. The remaining CIDR-B was removed from cows in Group C3 on Days 11-13 and all cows injected with 500 micrograms cloprostenol. Proportions of cows with a DF on Days 9-11 and diameter of largest follicle were: Group C0, 6/7 and 12.6 +/- 0.9 mm; Group C3, 2/6 and 9.6 +/- 0.8 mm. Numbers of CL on Day 8 after oestrus were: Group C0, 20.0 +/- 7.1; Group C3, 14.8 +/- 4.8 (P>> 0.05). Exposure to low dose norgestomet allowed development of a persistent dominant follicle, resulting in a reduced response to superstimulation with FSH. High dose progestogen restricted follicle growth without apparent effects on responses to superstimulation.

The present study tested the hypothesis that administration of GnRH on day 5 of the estrous cycle in embryo transfer (ET) recipients would increase progesterone (P4) concentrations, embryo size, and improve fertility. Holstein and cross-bred Holstein heifers (n = 1562) were synchronized using a modified 5-day CIDR-Synch protocol as follows (All AM treatments): D-8, CIDR inserted; D-3, CIDR removed and PGF2α (500 μg cloprostenol) treatment; D-2, second PGF2α; D0, GnRH (G1, 100 μg gonadorelin acetate) to induce ovulation. On D5 in the afternoon, heifers were assigned in a completely randomized design to one of two treatments: Control (untreated) or GnRH (200 μg). Transfer of day 7 fresh IVP embryos was performed between D6 and D8 after G1. Data collected from each heifer included: embryo stage and quality, body condition score, technician performing ET, interval from G1 to ET, and number of previous transfers. All heifers were evaluated by transrectal ultrasonography on D5, D33, and D60 and a subset of heifers was scanned on D12 (n = 718; to determine ovulation to treatment) and another subset on D33 (n = 295; 16 s video to determine embryo and amniotic vesicle size). Serum P4 was determined from a subset of heifers on D12 (n = 467) and on D21 (n = 837) and pregnancy specific protein B (PSPB) on D28 (n = 843). Pregnancies per ET (P/ET) were analyzed by logistic regression and continuous outcomes by ANOVA. Ovulation to D5 GnRH, defined by the presence of an accessory CL on D12, was 83.9% (302/360) in GnRH-treated heifers vs. 3.3% (12/358) in Controls (P < 0.001). On D12, P4 was greater (P < 0.001) in GnRH-treated heifers (7.2 ± 0.1 ng/ml) vs Controls (6.0 ± 0.1 ng/ml). There was greater P/ET at D33 and D60 of pregnancy for Stage 7 than Stage 6 embryos. Treatment with GnRH did not alter P/ET with either embryo stage but decreased pregnancy loss between D33 and D60 in heifers receiving Stage 7 embryos. Presence of an accessory CL at the D33 pregnancy diagnosis was associated with a larger reduction in pregnancy loss from D33 to D60 in recipients of Stage 7 embryos (11.6 vs 27.6%). Although there was no GnRH effect on embryo size, the presence of an accessory CL was associated (P < 0.05) with larger amniotic vesicle volume in recipients of Stage 7 embryos. In addition, greater PSPB was linked to greater amniotic vesicle volume (P = 0.01) and to reduced pregnancy loss (P < 0.0001). In conclusion, treatment with GnRH on D5 caused ovulation and formation of an accessory CL, increased circulating P4, and reduced pregnancy loss in heifers receiving a Stage 7 but not a Stage 6 IVP embryo.

An experiment was designed to evaluate the effect of extending duration of the presynchronization treatment in a long-term progestin-based estrus synchronization protocol. Heifers were assigned to either an 18 d (Day 0-18) or 14 d (Day 4 to Day 18) CIDR^® treatment (1.38 g progesterone controlled internal drug release insert; Zoetis, Madison, NJ), with prostaglandin F_2α (PG; 250 μg im cloprostenol sodium) administered 16 d after CIDR^® removal (Day 34). Heifers at two locations (location one, n = 193; location two, n = 649) were assigned to treatment based on reproductive tract score (RTS; Scale 1-5) and body weight. Heifers that were assigned RTS 1 were not retained for the trial (n = 6). Estrus detection aids (Estrotect^®) were applied at PG. Split-time artificial insemination (STAI) was utilized and AI performed based on expression of estrus at 66 h. Expression of estrus was defined as removal of ≥50% of the grey coating from the Estrotect^® patch. Heifers that expressed estrus at 66 h were inseminated then and heifers that had not expressed estrus were inseminated at 90 h. Only heifers that failed to express estrus by 90 h received gonadotropin-releasing hormone (GnRH; 100 μg im gonadorelin acetate) at the time of AI. At location one, blood samples were collected at PG and AI (66 h or 90 h) from all heifers to determine E₂ concentration by radioimmunoassay, and transrectal ovarian ultrasound was performed to detail ovarian structures on a subset of heifers (n = 73) at both time points. The proportion of heifers expressing estrus did not differ between treatments, either by 66 h (60%) or in total by 90 h (84%) after PG. Pregnancy rate to STAI did not differ between treatments (P = 0.3; 52%, 14-d CIDR^®-PG; 50%, 18-d CIDR^®-PG), or at the end of the 60 d breeding season (P = 0.2; 86%, 14-d CIDR^®-PG; 82%, 18-d CIDR^®-PG). No differences were detected in mean diameter of the dominant follicle at PG (P = 0.6; 10.9 ± 0.4 mm, 14-d CIDR^®-PG; 11.0 ± 0.4 mm, 18-d CIDR^®-PG) or at STAI (P = 0.3; 12.6 ± 0.4 mm, 14-d CIDR^®-PG; 13.2 ± 0.4 mm, 18-d CIDR^®-PG), nor were any differences observed between treatments in concentrations of E₂ at PG (P = 0.8; 1.1 ± 0.19 pg/ml, 14-d CIDR^®-PG; 1.1 ± 0.19 pg/ml, 18-d CIDR^®-PG) or STAI (P = 0.6; 3.8 ± 0.19 pg/ml, 14-d CIDR^®-PG; 3.6 ± 0.19 pg/ml, 18-d CIDR^®-PG). These data indicate that duration of CIDR^® treatment can be extended from 14 to 18 d, thus providing flexibility in scheduling without compromising reproductive outcomes.

The induction of optimal synchrony of estrus in cows requires synchronization of luteolysis and of the waves of follicular growth (follicular waves). The aim of this study was to determine whether hormonal treatments aimed at synchronizing follicular waves improved the synchrony of prostaglandin (PG)-induced estrus. In Experiment 1, cows were treated on Day 5 of the estrous cycle with saline in Group 1 (n = 25; 16 ml, i.v., 12 h apart), with hCG in Group 2 (n = 27; 3000 IU, i.v.), or with hCG and bovine follicular fluid (bFF) in Group 3 (n = 21; 16 ml, i.v., 12 h apart). On Day 12, all cows were treated with prostaglandin (PG; 500 micrograms cloprostenol, i.m.). In Experiment 2, cows were treated on Day 5 of the estrous cycle with saline (3 ml, i.m.) in Group 1 (n = 22) or with hCG (3000 IU, i.v.) in Group 2 (n = 20) and Group 3 (n = 22). On Day 12, the cows were treated with PG (500 micrograms in Groups 1 and 2; 1000 micrograms in Group 3). Blood samples for progesterone (P4) determination were collected on Day 12 (Experiment 1) or on Days 12 and 14 (Experiment 2). Cows were fitted with heat mount detectors and observed twice a day for signs of estrus. Four cows in Experiment 1 (1 cow each from Groups 1 and 2; 2 cows from Group 3) had plasma P4 concentrations below 1 ng/ml on Day 12 and were excluded from the analyses. In Experiment 1, cows treated with hCG or hCG + bFF had a more variable (P = 0.0007, P = 0.0005) day of occurrence of and a longer interval to estrus (5.9 +/- 0.7 d, P = 0.003 and 6.2 +/- 0.8 d, P = 0.005) than saline-treated cows (3.4 +/- 0.4 d). The plasma P4 concentrations on Day 12 were higher (P < 0.0001) in hCG- and in hCG + bFF-treated cows than in saline-treated cows (9.4 +/- 0.75 and 8.5 +/- 0.75 vs 4.1 +/- 0.27 ng/ml), but there was no correlation (P>> 0.05) between plasma P4 concentrations and the interval to estrus. In Experiment 2, cows treated with hCG/500PG and hCG/1000PG had a more variable (P = 0.0007, P = 0.002) day of occurrence of and a longer interval to estrus (4.2 +/- 0.4 d, P = 0.04; 4.1 +/- 0.4 d, P = 0.03) than saline/500PG-treated cows (3.2 +/- 0.1 d). The concentrations of plasma P4 on Days 12 and 14 of both hCG/500PG- and hCG/1000PG-treated cows were higher (P < 0.05) than in saline/500PG-treated cows (7.3 +/- 0.64, 0.7 +/- 0.08 and 7.7 +/- 0.49, 0.7 +/- 0.06 vs 5.3 +/- 0.37, 0.5 +/- 0.03 ng/ml). The concentrations of plasma P4 on Days 12 or 14 and the interval to estrus were not correlated (P>> 0.05) in any treatment group. The concentrations of plasma P4 on Days 12 and 14 of hCG/500PG- or hCG/1000PG-treated cows were correlated (r = 0.65, P < 0.05; r = 0.50, P < 0.05). This study indicated that treatment of cows with hCG on Day 5 of the estrous cycle reduced the synchrony of PG-induced estrus and that this reduction was not due to the failure of luteal regression.

The variability between animals in the timing of oestrus after administration of a synchronization treatment seems to explain the low rate of fertility in goats inseminated at a predetermined time after progesterone withdrawal. Two experiments were performed during the breeding season to test whether the variation was due to the exogenous hormone regime or to the endogenous physiology of the animals. Twenty-one goats were given a synchronization treatment consisting of a vaginal sponge impregnated with 45 mg of fluorogestone acetate (FGA) for 11 days associated with intramuscular injection of 400 I.U. of equine chorionic gonadotrophin (eCG) and 50 microg of cloprostenol 48 h before sponge removal. Progesterone concentrations were measured during the subsequent cycle and the patterns were modelled to allow precise determination of the onset of luteolysis. Oestrus and the luteinizing hormone (LH) surge began 33.0+/-6.8 h and 76.0+/-33.0 h after sponge withdrawal, v. 43.4+/-5.7 h and 90.0+/-36.0 h after natural luteolysis. For both observations, the between-goat variability was larger during the natural than during the synchronized oestrus (P < 0.05). The duration of the oestrous cycle was independent of the number of corpora lutea (CL), whereas the duration of luteal phase was shorter in goats with 2-3 CL (16.4+/-0.9 day than in those with 1 CL: 17.7+/-1.3 day; P < 0.05). In the second experiment, 20 goats were ovariectomized and given a vaginal sponge as described above. Sixteen h after sponge removal, they were injected with 50 microg of oestradiol benzoate (ODB). This treatment was repeated with the second sponge being inserted 1-2 days after observation of oestrus. Oestrus and LH surge were observed: 32.8+/-6 8 h v. 27.8+/-7.8 h after the first ODB injection, and 36.6+/-7.3 h v. 34.3+/-4.8 h after the second ODB injection. No relationship was observed between data of the two experiments. In both cases, the variability in the occurrence of oestrus and LH surge was of the same order as observed in the first experiment. This study shows that the timing of oestrus and LH surge is less variable after progestagen treatment than during a natural oestrous cycle. Moreover, a significant proportion of variability is inherent in the delays following the oestradiol peak, suggesting that further improvement in the synchronizing capacity of treatment based on progestagen administration is unlikely.

The aim of this study was to determine if treatment with estradiol cypionate (EC) at the time of P4 withdrawal induced ovulation in a synchronization/timed-AI (TAI) protocol in buffalo. In Experiment 1, 56 buffaloes received an intravaginal P4 device (1.0 g) plus estradiol benzoate (EB, 2.0 mg im) on Day 0 (D0). On Day 9, the P4 device was removed and buffaloes were given PGF_2α (0.53 mg im sodium cloprostenol) plus eCG (400 IU im). Buffaloes were then randomly allocated to one of two groups: Group GEC (n = 29), treated with EC (1.0 mg im) at P4 device removal; Group GEB (n = 27), treated with EB (1.0 mg im) 24 h after P4 device removal. Ovarian ultrasound was undertaken on: D0, to ascertain general ovarian status; D9 to D11 (every 24 h), to measure diameter of the largest follicle (LF) and follicular growth rate; D11 to D13 (every 12 h for 72 h), to determine the time of ovulation and ovulation rate. Following P4 device removal, Groups GEC and GEB had a similar follicular growth rate (0.9 ± 0.1 and 1.1 ± 0.1 mm/day, respectively; P = 0.15) and similar LF diameter on D11 (11.4 ± 0.6 and 12.5 ± 0.5 mm; P = 0.12). Groups GEC and GEB also had a similar diameter of the ovulatory follicle (13.0 ± 0.5 and 13.4 ± 0.6 mm; P = 0.52), interval from P4 device removal to ovulation (68.2 ± 2.8 and 71.1 ± 1.4 h; P = 0.41) and ovulation rate (62.1% and 70.4%; P = 0.44). In Experiment 2, 199 buffaloes were assigned to the two treatments in Experiment 1 (GEC, n = 100; GEB, n = 99). All animals underwent TAI 56 h after P4 device removal and pregnancy diagnosis was preformed on D41. The pregnancy rate was similar for Groups GEC and GEB (50.0 and 45.5%, respectively; P = 0.45). The findings indicate that treatment with EC at the time of P4 withdrawal induces ovulation and achieves the same pregnancy rate to TAI as treatment with EB 24 h after P4 removal. The use of EC requires one less handling which is highly important in facilitating practical adoption of TAI in assisted breeding and genetic improvement in buffalo.

Keywords: Buffalo; Estradiol cypionate; Ovulation synchronization; Pregnancy; Timed-AI.

Primiparous and multiparous lactating crossbred dairy cows with a mature corpus luteum and a follicle with >10 mm in diameter were treated with cloprostenol. Those cows that showed oestrus within 5 days after treatment were inseminated (Group P). The other cows (Group PG) were treated with GnRH 2 days after cloprostenol treatment and timed artificial insemination (AI) was performed on the consecutive day, or were inseminated (Group G) after detected oestrus and treated with GnRH immediately after AI. The control cows (Group C) after detected oestrus were only inseminated. All of the AIs using frozen semen were done between 6 and 7 a.m. while the ultrasonographic examinations after AI were performed between 4 to 6 p.m. The ovaries of each cow were scanned by means of transrectal ultrasonography from the day of AI until ovulation. Daily blood samples were collected for progesterone measurements. The ovulation and pregnancy rates among the groups changed between 84.6% and 95.5%, as well as 44.4% and 60%, respectively, however the differences were not statistically significant. All the cows were evaluated according to date of ovulation after AI and the pregnancy rate was 55.4% (Group 1: ovulation occurred between AI and 9-11 h after AI), 54.5% (Group 2: ovulation occurred between 9-11 h and 33-35 h after AI) and 35.5% (Group 3: ovulation occurred between 33-35 h and 57-59 h after AI), respectively. There was a trend (P=0.087) for 2.2 greater odds of staying open among cows inseminated between 33 to 35 h and 57 to 59 h before ovulation compared to cows inseminated within 9 to 11 h before ovulation. If ovulation occurred before AI, the pregnancy rate was only 22.2%, therefore determination of optimal time for AI is of great importance.

We hypothesized that: (i) repeated GnRH treatments would increase the magnitude and duration of the LH surge and would increase progesterone (P4) concentrations after ovulation; and (ii) the release of pituitary LH would be greater in response to larger doses of GnRH. In Experiment 1, ovary-intact cows were given an intravaginal P4 (1.9g) insert (CIDR) for 10 d and 500 microg cloprostenol (PGF) at CIDR removal to synchronize estrus. On Days 7 or 8 after estrus, cows received two PGF treatments (12h apart) and 100 microg GnRH at 36 (Control), 36 and 38 (GnRH38), or 36 and 40h (GnRH40) after the first PGF. Mean plasma LH concentration (ng/mL) was greater (P<0.05) in GnRH38 (8.8+/-1.1) than in Control (5.1+/-1.3), with that in GnRH40 (5.8+/-1.3) being intermediate. Although the duration (h) of the LH surge was longer in GnRH40 (8.0+/-0.4) than in either GnRH38 (P<0.05; 7.0+/-0.3) or Control (P<0.09; 7.1+/-0.4), mean postovulatory P4 (ng/mL) was greater (P<0.01) in Control (4.2+/-0.7) than in GnRH38 (2.9+/-0.6) or GnRH40 (3.0+/-0.7) cows. In Experiment 2, ovariectomized cows were given a CIDR for 10 d and 2mg of estradiol cypionate im at CIDR insertion. Thirty-six hours after CIDR removal, cows received, 50, 100, or 250 microg of GnRH. Cows given 250 microg GnRH released more LH (9.4+/-1.4ng/mL) than those given 50 or 100 microg (6.1+/-1.3 and 5.4+/-1.4ng/mL, respectively), and had an LH surge of longer duration than those given 50 microg (6.8+/-0.4 vs. 5.1+/-0.3h). In summary, ovary-intact cows in the GnRH38 group had greater mean and peak LH concentrations, but subsequent plasma P4 concentrations were lower than in Control cows. Ovariectomized cows given 250 microg GnRH had a greater pituitary release of LH.

The aim of this study was to evaluate whether an additional intramuscular (im) injection of pFSH would increase the embryo production in zebu cows superovulated with a single subcutaneous (sc) pFSH injection. Twenty-one Nelore cows were treated with a progesterone vaginal implant (Controlled Internal Drug Relased - CIDR B) and injected im with 2.5 mg estradiol benzoate. Four days later, cows were assigned randomly into three groups and superovulated with pFSH. Groups A and B received single sc injections of 400 and 320 IU, respectively; Group C received multiple im injections of 400 IU in decreasing doses at 12-h intervals over 4 days. In the morning (07:00 am) of day 3 after starting superovulation, cows received im 150 mcg cloprostenol and Group B was additionally injected im with 80 IU of pFSH. CIDR-B was withdrawn in the afternoon (07:00 pm). Cows were inseminated 48 and 62 h after the cloprostenol injection. Embryo collection and corpora lutea (CL) estimation were done 7 days after insemination. Alternation of treatments (crossover design) occurred at a 60-day interval. There was no significant difference (p>> 0.05) of CL counts among treatments. The total (transferable and no transferable) number of recovered embryos from Group A (6.9 +/- 1.5) was not different from Group C (9.8 +/- 1.2), whereas Group B (5.7 +/- 1.5) was lower than Group C (p < 0.05). The number of transferable embryos from Groups A (2.4 +/- 0.7) and B (1.7 +/- 0.6) was lower (p < 0.05) than Group C (4.6 +/- 1.2). Lesser (p < 0.05) embryo production from Group B was related to lower recovery rate (46.4%), compared with Groups A (65.1%) and C (81.7%). It was concluded that an additional im subdose of pFSH, injected 48 h after a single subcutaneous (sc) dose of pFSH, does not improve the embryo production in zebu cows.

This study evaluated the effect of the protected fatty acid inclusion during estrus synchronization on reproductive parameters. Goats (n = 32) received progestagen sponges for 6 days and 200 IU equine chorionic gonadotropin and 30 µg d-cloprostenol were given on Day 5. No difference was found among control (C), 1% protected fatty acid inclusion (C + 1%) or 4% protected fatty acid inclusion (C + 4%) groups, respectively, in estrus (100.0, 100.0 or 90.9%), estrus duration (31.6 ± 12.3; 43.2 ± 12.9 or 40.8 ± 14.1 h), animals ovulating (100.0, 90.0 or 100.0%) or ovulation rate (1.3 ± 0.5; 1.1 ± 0.3 or 1.2 ± 0.4). The interval from sponge removal to ovulation and from estrus to ovulation, respectively, were shorter for C + 4% (45.2 ± 8.0 h; 18.3 ± 11.0 h) compared with C (56.3 ± 12.6 h; 30.6 ± 10.5 h) or C + 1% (57.7 ± 8.7 h; 30.3 ± 11.1 h). The average ovulatory follicle diameter was smaller for C + 4% (6.2 ± 0.7 mm) than C (7.5 ± 0.8 mm), but similar to C + 1% (7.0 ± 1.5 mm). Insulin, insulin-like growth factor 1, glucose and progesterone concentrations were similar among groups. The inclusion of protected fatty acid during synchronization treatment promoted no benefits on ovulation rate, but 4% anticipated the ovulation time.

This study evaluated effects of equine chorionic gonadotropin (eCG) on fertility of 679 crossbred (Bos taurus x Bos indicus) lactating grazing cows synchronized for fixed-time AI (FTAI). At a random day of the estrous cycle cows received an intravaginal progesterone (P4) implant, 2 mg estradiol benzoate (EB) and 100 μg gonadorelin (D0-AM). On D7-AM, cows received 0.5 mg sodium cloprostenol and were randomly assigned into two treatments: eCG (n = 340; 400 IU eCG on D7), or Control (n = 339; no eCG). On D8-PM, P4 implants were removed and cows received 0.5 mg sodium cloprostenol and 1 mg EB. Insemination was performed on D10-AM. Pregnancy was diagnosed 30 and 60 d after AI. Treatment with eCG tended to increase pregnancy per AI (P/AI) compared to Control at 30 (37.8 vs. 30.2%; P = 0.06) and 60 (31.9 vs. 25.1%; P = 0.08) d. Pregnancy loss and twinning did not differ between groups. Treatment with eCG increased (P < 0.05) P/AI at 30 (39.0 vs. 25.2%) and 60 (32.8 vs. 21.3%) d for cows inseminated at ≤ 70 d in milk (DIM) but had no effect in cows receiving AI after 70 DIM. Thus, eCG on D7 of a FTAI protocol increased fertility of crossbred dairy cows inseminated in the early postpartum period.

Two experiments were conducted to compare pregnancy rates when GnRH or estradiol were given to synchronize ovarian follicular wave emergence and ovulation in an MGA-based estrus synchronization program. Crossbred beef cattle were fed melengestrol acetate (MGA, 0.5 mg per day) for 7 days (designated days 0-6, without regard to stage of the estrous cycle) and given cloprostenol (PGF; 500 microg intramuscular (im)) on day 7. In Experiment 1, lactating beef cows (n=140) and pubertal heifers (n=40) were randomly allocated to three groups to receive 100 microg gonadorelin (GnRH), 5 mg estradiol-17beta and 100 mg progesterone (E+P) in canola oil or no treatment (control) on day 0. All cattle were observed for estrus every 12 h from 36 to 96 h after PGF. Cattle in the GnRH group that were detected in estrus 36 or 48 h after PGF were inseminated 12 h later; the remainder were given 100 microg GnRH im 72 h after PGF and concurrently inseminated. Cattle in the E+P group were randomly assigned to receive either 0.5 or 1.0 mg estradiol benzoate (EB) in 2 ml canola oil im 24 h after PGF and were inseminated 30 h later. Cattle in the control group were inseminated 12 h after the first detection of estrus; if not in estrus by 72 h after PGF, they were given 100 microg GnRH im and concurrently inseminated. In the absence of significant differences, all data for heifers and for cows were combined and the 0.5 and 1.0 mg EB groups were combined into a single estradiol group. Estrus rates were 57.6, 57.4 and 60.0% for the GnRH, E+P and control groups, respectively (P=0.95). The mean (+/-S.D.) interval from PGF treatment to estrus was shorter (P<0.001) and less variable (P<0.001) in the E+P group (49.0+/-6.1 h) than in either the GnRH (64.2+/-15.9 h) or control (66.3+/-13.3 h) groups. Overall pregnancy rates were higher (P<0.005) in the GnRH (57.6%) and E+P (55.7%) groups than in the control group (30.0%) as were pregnancy rates to fixed-time AI (47.5, 55.7 and 28.3%, respectively). In Experiment 2, 122 crossbred beef heifers were given either 100 microg GnRH or 2 mg EB and 50 mg progesterone in oil on day 0 and subsequently received either 100 microg GnRH 36 h after PGF and inseminated 14 h later or 1 mg EB im 24 h after PGF and inseminated 28 h later in a 2 x 2 factorial design. Pregnancy rates were not significantly different among groups (41.9, 32.2, 33.3 and 36.7% in GnRH/GnRH, GnRH/EB, EB/GnRH and EB/EB groups, respectively). In conclusion, GnRH or estradiol given to synchronize ovarian follicular wave emergence and ovulation in an MGA-based synchronization regimen resulted in acceptable pregnancy rates to fixed-time insemination.

Analogous to the study of Onclin and Verstegen of 1996 the combination of a dopamine agonist, cabergoline, and a synthetic analogue of prostaglandin F2 alpha, cloprostenol or dinoprost, was used to induce termination of pregnancy 25 (+/- 2) days after the first mating. The study was done in six bitches. Cabergoline was administered at a dose of 5 micrograms/kg daily over a period of nine days and cloprostenol was injected subcutaneously at 1-2 micrograms/kg (dinoprost at a dose of 25 micrograms/kg) every other day. Treatment efficacy was 100%. No side effects were observed. Pregnancy and its termination was diagnosed by ultrasonographic examination. The combination of cabergoline and cloprostenol offers a simple and safe procedure for the termination of unwanted pregnancy.

This study aimed to evaluate the effect of recombinant bovine somatotropin (bST) in combination with progesterone (P4) and estradiol benzoate (EB) on ovarian follicular dynamics using a protocol for estrus and ovulation synchronization in crossbred Bos taurus taurus cows. Twenty-four non-lactating multiparous cows were randomly assigned to two groups: the recombinant bovine somatotropin group (GbST; n&#x202F;=&#x202F;11) received an intravaginal P4 device (1.5&#x202F;g), estradiol benzoate (EB&#x202F;=&#x202F;1.0&#x202F;mg IM), bST (500&#x202F;mg SC), and an ovarian ultrasonography (US) on day zero (d0&#x202F;=&#x202F;beginning of the study); d-cloprostenol (150&#x202F;μg, IM), US, and P4 removal on d8; 1.0&#x202F;mg of EB (IM) on d9; and US on d10 and d15. On the other hand, to the control group (GC; n&#x202F;=&#x202F;13), the same protocol as the GbST was applied, except for the non-receipt of bST on d0. The follicles were measured and evaluated on d0, d8, and d10, as were the corpora lutea (CL) on d15 (using ultrasonography). The effect of the two treatments (GbST vs. GC) on the follicle size, CL (F-test), and ovulation rate (logistic regression) were evaluated. The GbST showed a greater follicle diameter on d10 (14.5&#x202F;mm) than the GC (12.1&#x202F;mm; P&#x202F;<&#x202F;0.03), as well as a greater diameter of CL on d15 (19.7 vs. 16.9&#x202F;mm, P&#x202F;<&#x202F;0.01). In addition, in the former, the ovulation rate (90.9 vs. 69.2%, P&#x202F;=&#x202F;0.09) was observed to be greater. It was concluded that the combination of bST, P4, and EB in synchronization for estrus and ovulation protocols significantly increased the diameter of the preovulatory follicle, produced a higher follicular growth rate, and a greater diameter of the corpus luteum. Additionally, there was a higher percentage of cows with ovulation compared to the group that did not receive bST.