Comparison of gonadotropin-releasing hormone and estradiol benzoate plus gonadotropin-releasing hormone to initiate a progesterone-based timed artificial insemination resynchronization protocol in lactating dairy cows.

The present study compared 2 strategies to initiate a progesterone (P4)-based timed artificial insemination (TAI) protocol for lactating dairy cows: only GnRH or estradiol benzoate (EB) plus GnRH (EB+GnRH). Lactating Holstein cows (n = 487; 184 primiparous and 303 multiparous) from 2 commercial dairy herds were used for their second or greater services postpartum. Each week, cows that were nonpregnant at the pregnancy diagnosis 32 d after a previous AI were randomly assigned to 1 of 2 experimental groups that differed only in the strategy to initiate (d 0) the TAI protocol. On d 0, every cow received a 2.0-g P4 implant; in the EB+GnRH group, cows were treated with 2.0 mg i.m. of EB and 16.8 µg i.m. of the GnRH analog buserelin acetate, whereas in the GnRH group, cows received only 16.8 µg i.m. of GnRH. On d 7 after the initial treatment, 0.530 mg i.m. of cloprostenol sodium (PGF) was administered in all cows, followed by a second dose on d 8, concomitant with 1.0 mg i.m. of estradiol cypionate and P4 implant withdrawal. The TAI was performed on d 10 (48 h after P4 device withdrawal) in both experimental groups. Only conventional Holstein semen was used throughout the study. The percentage of cows with corpus luteum (CL) on d 0 (73%) and overall ovulation rate after d 0 (54%) did not differ between groups. The CL regression between d 0 and the first PGF treatment was greater in the EB+GnRH group than the GnRH group (42% vs. 31%). Consequently, the proportion of cows with CL at PGF was greater when only GnRH was used on d 0 compared with EB+GnRH (86% vs. 82%), and the mean number of CL at PGF was greater (1.23 vs. 1.11). The expression of estrus near TAI was greater in GnRH group (84% vs. 77%), and cows showing estrus had greater (44% vs. 10%) pregnancy per AI (P/AI) on d 32 for both treatments. We found no effect of the presence of CL on d 0 or at PGF, nor of ovulation after d 0 or CL regression between d 0 and d 7 on fertility. However, fertility was critically impaired when cows did not have CL at both times, d 0 and at PGF treatment. We did not observe any interaction between treatment and other variables, and the P/AI was similar in cows receiving EB+GnRH or only GnRH on d 0 (37.8% vs. 36.6%). In summary, although there was no detectable difference in P/AI between treatments, this study demonstrated potential negative physiological outcomes caused by EB treatment on d 0 (greater incidence of luteolysis after d 0 and fewer cows with CL at PGF treatment). Overall, we found no benefit of adding EB at the initiation of a P4-based TAI protocol on fertility compared with using GnRH alone, despite differences in ovarian dynamics and expression of estrus.

Effect of progesterone from corpus luteum, intravaginal implant, or both on luteinizing hormone release, ovulatory response, and subsequent luteal development after gonadotropin-releasing hormone treatment in cows.

This study aimed to determine the effect of circulating progesterone (P4) concentrations produced by a corpus luteum (CL) or released by an intravaginal P4 implant (IPI) on GnRH-induced LH release, ovulatory response, and subsequent CL development, after treatment with 100 µg of gonadorelin acetate (GnRH challenge). Nonlactating multiparous Holstein cows were synchronized and GnRH was used to induce ovulation (d -7). Over 4 replicates, cows that ovulated (n = 87) were randomly assigned to a 2 × 2 factorial arrangement (presence or absence of CL and insertion or not of an IPI at GnRH challenge), creating 4 groups: CL_IPI, CL_NoIPI, NoCL_IPI, and NoCL_NoIPI. On d -1.5, NoCL_IPI and NoCL_NoIPI received 2 doses of 0.53 mg of cloprostenol sodium (PGF2α), 24 h apart to regress CL. On d 0, cows were treated with 100 µg of GnRH and, simultaneously, cows from IPI groups received a 2-g IPI maintained for the next 14 d. Diameter of dominant follicle, ovulatory response, and subsequent CL volume were assessed by ultrasonography on d -1.5, 0, 2, 7, and 14. Blood samples were collected on d -1.5, 0, 1, 2, 3, 5, 7, and 14 for analysis of circulating P4 and at 0, 1, 2, 4, and 6 h after GnRH challenge for analysis of circulating LH. In a subset of cows (n = 34), the development of the new CL was evaluated daily, from d 5 to 14. The presence of CL at the time of GnRH challenge affected the LH peak and ovulatory response (CL: 5.3 ng/mL and 58.1%; NoCL: 13.2 ng/mL and 95.5%, respectively). However, despite producing a rapid increase in circulating P4, IPI insertion did not affect LH concentration or ovulation. Regardless of group, ovulatory response was positively correlated with LH peak and negatively correlated with circulating P4 on d 0. Moreover, new CL development and function were negatively affected by the presence of CL and by the IPI insertion. In summary, circulating P4 produced by a CL exerted a suppressive effect on GnRH-induced LH release and subsequent ovulation of a 7-d-old dominant follicle, whereas the IPI insertion at the time of GnRH had no effect on LH concentration or ovulation. Finally, elevated circulating P4, either from CL or exogenously released by the IPI, compromised the development and function of the new CL, inducing short cycles in cows without CL at the time of GnRH treatment.

Accessory corpus luteum regression during pregnancy I: timing, physiology, and P4 profiles.

Inappropriate corpus luteum (CL) regression can produce pregnancy loss. An experimental model was utilized to investigate regression of accessory CL during pregnancy in dairy cows. Cows were bred (day 0) and treated with gonadotrophin-releasing hormone 6 days later to form accessory CL. Transrectal ultrasound (every other days) and blood samples for progesterone (P4; daily) were performed until day 56 of pregnancy. On day 28, 13 cows were confirmed pregnant, and accessory CL were found contralateral (n = 9) or ipsilateral (n = 4) to previous ovulation. On day 18, CL biopsy was performed to analyze mRNA expression for interferon-stimulated genes (ISGs). Luteolysis occurred more frequently in cows that had contralateral accessory CL (88.9% (8/9)) than in cows with ipsilateral accessory CL (0% (0/4)). Luteolysis of contralateral accessory CL occurred either earlier (days 19-23; 2/8) or later (days 48-53; 6/8) in pregnancy and occurred rapidly (24 h), based on daily P4. After onset of earlier or later accessory CL regression, circulating P4 decreased by 41.2%. There was no difference in luteal tissue mRNA expression for ISGs on day 18 between accessory and original CL and between CL that subsequently regressed or did not regress. On day 56, an oxytocin challenge dramatically increased prostaglandin F2α metabolite (PGFM) in all cows but produced no pregnancy losses, although cows with previous accessory CL regression had greater PGFM. In summary, ipsilateral accessory CL did not regress during pregnancy, whereas most contralateral CL regressed by 63 days of pregnancy, providing evidence for local mechanisms in regression of accessory CL and protection of CL during pregnancy.

Interactions of circulating estradiol and progesterone on changes in endometrial area and pituitary responsiveness to GnRH†.

Changes in circulating progesterone (P4) and estradiol (E2) during proestrus produce dynamic changes in endometrial function and pituitary release of gonadotropins. Independent and combined effects of P4 and E2 on endometrium and pituitary were evaluated. In a preliminary study, an exogenous hormone model of proestrus was created by removal of corpus luteum and follicles ≥5 mm followed by gradual removal of intravaginal P4 implants during 18 h and treatment with increasing doses of estradiol benzoate during 48 h to mimic proestrus using high E2 (n = 9) or low E2 (n = 9). Decreased P4, increased E2, and increased endometrial area (EA) simulated proestrus in high-E2 cows and this was used subsequently. The main experiment used a 2 × 2 factorial design with: high E2 and low P4 (n = 11); high E2 and high P4 (n = 11); low E2 and high P4 (n = 11); low E2 and low P4 (n = 10). At 48 h, gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) and follicle stimulating hormone (FSH) release was determined. Variables were analyzed using PROCMIXED of Statistical Analysis System. The EA increased dramatically during 48 h only in high-E2 and low-P4 cows. For FSH, high-E2 cows had greater area under the curve (AUC) and FSH peak after GnRH than low E2, with mild negative effects of high P4. For LH, concentration at peak and AUC were 2-fold greater in high E2 compared to low-E2 groups, with low P4 also 2-fold greater than high-P4 groups. Thus, maximal changes in uterus and pituitary during proestrus depend on both low P4 and high E2, but different physiologic responses are regulated differently by E2 and P4. Changes in endometrium depend on low P4 and high E2, whereas GnRH-induced FSH secretion primarily depends on high E2, and GnRH-induced LH secretion is independently increased by high E2 or reduced by high P4.

Mechanisms for rescue of corpus luteum during pregnancy: gene expression in bovine corpus luteum following intrauterine pulses of prostaglandins E1 and F2α.

In ruminants, uterine pulses of prostaglandin (PG) F2α characterize luteolysis, while increased PGE2/PGE1 distinguish early pregnancy. This study evaluated intrauterine (IU) infusions of PGF2α and PGE1 pulses on corpus luteum (CL) function and gene expression. Cows on day 10 of estrous cycle received 4 IU infusions (every 6 h; n = 5/treatment) of saline, PGE1 (2 mg PGE1), PGF2α (0.25 mg PGF2α), or PGE1 + PGF2α. A luteal biopsy was collected at 30 min after third infusion for determination of gene expression by RNA-Seq. As expected, IU pulses of PGF2α decreased (P < 0.01) P4 luteal volume. However, there were no differences in circulating P4 or luteal volume between saline, PGE1, and PGE1 + PGF2α, indicating inhibition of PGF2α-induced luteolysis by IU pulses of PGE1. After third pulse of PGF2α, luteal expression of 955 genes were altered (false discovery rate [FDR] < 0.01), representing both typical and novel luteolytic transcriptomic changes. Surprisingly, after third pulse of PGE1 or PGE1 + PGF2α, there were no significant changes in luteal gene expression (FDR > 0.10) compared to saline cows. Increased circulating concentrations of the metabolite of PGF2α (PGFM; after PGF2α and PGE1 + PGF2α) and the metabolite PGE (PGEM; after PGE1 and PGE1 + PGF2α) demonstrated that PGF2α and PGE1 are entering bloodstream after IU infusions. Thus, IU pulses of PGF2α and PGE1 allow determination of changes in luteal gene expression that could be relevant to understanding luteolysis and pregnancy. Unexpectedly, by third pulse of PGE1, there is complete blockade of either PGF2α transport to the CL or PGF2α action by PGE1 resulting in complete inhibition of transcriptomic changes following IU PGF2α pulses.

Continuous infusion of propofol in calves: bispectral index and hemodynamic effects.

OBJECTIVE: To assess the bispectral index (BIS) and the hemodynamic effects of propofol administered by continuous infusion at different rates in calves. STUDY DESIGN: Experimental crossover study. ANIMALS: Eight intact male Dutch calves, aged 6-12 months and weighing 84-124 kg. METHODS: The calves were anesthetized with propofol (5 mg kg(-1) ) intravenously (IV), and after endotracheal intubation, positioned in right lateral recumbency and allowed to breathe ambient air. Anesthesia was maintained by continuous infusion of propofol, administered IV with an infusion pump at 0.6 mg kg(-1)  minute(-1) (treatment G6) or 0.8 mg kg(-1)  minute(-1) IV (treatment G8), for 60 minutes. The eight animals were anesthetized twice, 1 week apart. The following hemodynamic variables and BIS were assessed before the induction of anesthesia (baseline) and 15, 30, 45, and 60 minutes after beginning the infusion of propofol: heart rate, systolic, diastolic and mean arterial pressures, cardiac output, mean pulmonary artery pressure, cardiac index, stroke index, pulmonary vascular resistance index, and systemic vascular resistance index, BIS, electromyography, and signal quality index. RESULTS: The continuous infusions of propofol at different rates did not alter BIS variables during the infusion time between dose rates, and no clinically significant hemodynamic changes were observed. CONCLUSIONS AND CLINICAL RELEVANCE: A continuous infusion of propofol at 0.6 or 0.8 mg kg(-1)  minute(-1) caused minimal hemodynamic changes without clinical relevance in calves. BIS could not be reliably used to discriminate the anesthetic depth during the two propofol infusion rates.

Synchronization of follicle wave emergence before ovarian superstimulation with FSH and ovum pick-up improves in vitro embryo production in pregnant heifers.

The objective of the present study was to evaluate effects of synchronization of timing of follicle wave emergence, before ovarian superstimulation and ovum pick-up (OPU), on ovarian response and embryo production in pregnant heifers. Pregnant (47-69 days of gestation) Holstein heifers (n = 64), 19.0 ± 0.3 months of age, were assigned in a completely randomized design to one of two groups: synchronization of follicular wave emergence using follicle ablation (Synchronized) or untreated control (Non-synchronized). Superstimulatory treatments consisting of 160 mg (280 IU) of porcine follicle stimulating hormone (p-FSH), administered in four decreasing dose treatments 12 h apart, were initiated 36 h after follicle ablation or at random stages of the follicular wave in heifers of the Synchronized and Non-synchronized group, respectively. Ovum pick-up was performed in all heifers 40 h after the last p-FSH administration and retrieved cumulus oocyte complexes (COCs) were subjected to in vitro embryo production (IVEP) procedures. Ultrasonography was performed immediately before OPU to determine number and size of ovarian follicles. Differences in treatment responses between groups were evaluated using generalized linear mixed models. Total number of follicles at the time of OPU was not different between treatment groups (P = 0.61), however, the number of small follicles (<6 mm) was greater (P = 0.05) in heifers of the Non-synchronized group, whereas number of medium size follicles (6-10 mm) tended (P = 0.09) to be greater in heifers of the Synchronized group. Heifers in the Synchronized group had a greater (P = 0.01) percentage of medium sized follicles and a lesser (P = 0.01) percentage of smaller sized follicles than heifers in the Non-synchronized group. There were no differences (P > 0.15) in total number of recovered COCs, or number of viable COCs between groups. Cleavage percentage (84.5% and 72.8%) and blastocyst percentage (48.2% and 33.4%) were greater (P < 0.01) in heifers of the Synchronized than Non-synchronized group, respectively. As a result, mean number of blastocysts per OPU/heifer was greater (P = 0.006) in the heifers of the Synchronized (8.9 ± 1.0) than the Non-synchronized (5.5 ± 0.9) group. In conclusion, synchronizing the time of follicle wave emergence in pregnant heifers, prior to ovarian superstimulation with FSH and OPU results in a greater superstimulatory response and oocyte competence leading to greater embryo production.

Postovulatory treatment with GnRH on day 5 reduces pregnancy loss in recipients receiving an in vitro produced expanded blastocyst.

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 ±â€¯0.1 ng/ml) vs Controls (6.0 ±â€¯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.

Progesterone-based timed AI protocols for Bos indicus cattle II: Reproductive outcomes of either EB or GnRH-type protocol, using or not GnRH at AI.

The aim of these experiments was to study ovarian dynamics and fertility of Bos indicus beef cattle submitted to 7-d progesterone (P4)-based fixed-time AI (FTAI) protocols using different hormonal treatments. In Exp. 1, 2 yr old Nelore heifers (n = 973) were randomly assigned to one of four treatments: EB-0 (estradiol benzoate, EB on D0 and no GnRH at AI), EB-G (EB on D0 and GnRH at AI), G-0 (GnRH on D0 and no GnRH at AI), or G-G (GnRH on D0 and at AI). On D0, heifers received an intravaginal P4 implant (0.5 g) for 7 d and EB (1.5 mg) or GnRH (16.8 µg). On D7, the P4 implant was withdrawn and heifers received cloprostenol (PGF; 0.5 mg) and estradiol cypionate (EC, 0.5 mg). Heifers in G groups also received PGF and eCG (200 IU) on D6, whereas EB heifers received eCG on D7. At FTAI on D9, only EB-G and G-G groups received GnRH (8.4 µg). In Exp. 2, Nelore cows (n = 804) received the same treatments (EB-0, EB-G, G-0, or G-G) using a 1.0 g P4 implant, 2.0 mg EB, and 300 IU eCG. Effects were considered significant when P ≤ 0.05. After treatment on D0, G had more ovulations than EB in heifers (60.3 [287/476] vs. 12.7% [63/497]) and cows (73.7 [83/112] vs. 24.4% [28/113]). Luteolysis after D0 was greater in EB than G in heifers (39.2 [159/406] vs. 20.0% [77/385]) and cows (25.5 [14/55] vs. 1.6% [1/64]). Heifers in G had larger follicles (mm) than EB on D7 (10.3 ± 0.2 vs. 9.2 ± 0.2) and at AI (11.9 ± 0.2 vs. 11.3 ± 0.2). Cows had larger follicles in G than EB on D7 (11.0 ± 0.3 vs. 9.9 ± 0.3) but not at AI. More estrus was observed in G than EB for heifers (80.3 [382/476] vs. 69.6% [346/497]) and cows (67.6 [270/400] vs. 56.2% [227/404]). There was no interaction between D0 and D9 treatments on pregnancy per AI (P/AI) in heifers (EB-0: 56.7 [139/245], EB-G: 53.6 [135/252], G-0: 52.6 [127/241], and G-G: 57.5% [135/235]). However, cows from EB-G had greater P/AI than EB-0 (69.5 [142/204] vs. 60.2% [120/200]), whereas P/AI for G-0 (62.7% [127/203]) was similar to G-G (60.9% [120/197]). In heifers, there was no interaction of GnRH at AI with estrus, however, cows that did not display estrus had greater P/AI if they received GnRH at AI (GnRH = 59.1 [91/154] vs. No GnRH = 48.2% [78/162]). Thus, protocols initiated with EB or GnRH for Bos indicus heifers and cows had differing ovarian dynamics but similar overall fertility, enabling their use in reproductive management programs. Treatment with GnRH at time of AI increased fertility in some instances in Bos indicus cows but not in heifers.

Progesterone-based timed AI protocols for Bos indicus cattle I: Evaluation of ovarian function.

Three experiments evaluated ovarian dynamics and circulating progesterone (P4) during P4-based protocols initiated with GnRH, estradiol benzoate (EB), or no additional treatment in Nelore (Bos indicus) cattle. In Exp 1 (n = 59 cows), a 5-d P4-only protocol (P-5d; D0: P4 implant alone (1g); D5: P4 removal, 0.5 mg estradiol cypionate [EC], 0.526 mg cloprostenol [PGF], and 300 IU equine chorionic gonadotropin [eCG]; D7: 8.4 µg buserelin acetate [GnRH]) was compared to a 9d protocol initiated with EB (EB-9d; D0: 2 mg EB + P4; D9: P4 removal + EC + PGF + eCG), and to a 7d GnRH protocol (G-7d; D0: 16.8 µg GnRH + P4; D6: PGF + eCG; D7: P4 removal + PGF; D9: GnRH). Exp 2 (n = 55 cows) compared G-7d and EB-7d protocols (similar to EB-9d, but D9 treatments were done on D7). Exp 3 (n = 64 heifers) compared EB-7d, G-7d, and P-5d protocols. For all experiments, daily ovarian ultrasonography was done from D0 until 4d after implant withdrawal and blood samples were collected at D0 and first PGF. Follicle dynamics were determined for each individual animal, analyzed within individual experiments, and afterwards combined to determine overall effects of treatments. The protocol that began with GnRH, G-7d, had greater ovulation rate after D0 with subsequently greater number of CL and circulating P4 at time of PGF (52.8%, 1.0 ± 0.1 CL, 4.0 ± 0.4 ng/mL) than for EB protocols (12.1%, 0.4 ± 0.05 CL, 2.0 ± 0.2 ng/mL), or P-5d (2.5%, 0.6 ± 0.09 CL, 2.6 ± 0.3 ng/mL). The G-7d and EB protocols had synchronized follicle wave emergence in 92.1% of animals but with distinct patterns. For the G-7d group, wave emergence occurred earlier in ovulating than non-ovulating animals (1.4 ± 0.2 d vs 2.5 ± 0.4 d). By comparison, most animals in EB-7d or EB-9d (80.3%) displayed atresia of the dominant follicle, followed by wave emergence 2-3 d after EB treatment. In contrast, P-5d protocol synchronized wave emergence in only 30.0% of cows. Nevertheless, no differences among treatments were detected for ovulation at end of the protocol (85.7%). In conclusion, the P-5d protocol did not synchronize follicle wave emergence but produced similar final ovulation, whereas, GnRH and EB protocols had follicle dynamics synchronized by distinct mechanisms that produced differences in CL number and P4 at the time of PGF treatment but similar final ovulation. Based on ovarian function, each of these synchronization methods are promising for use in FTAI, although fertility still needs to be evaluated.

Ovulation rate, antral follicle count, and circulating anti-Müllerian hormone in Trio allele carriers, a novel high fecundity bovine genotype.

High fecundity genotypes in sheep are a valuable model to study the physiological mechanisms underlying follicle selection and the control of ovulation rate. Similar genotypes in cattle had not been described until the recent identification of a major bovine allele, termed Trio, which had a large effect on ovulation rate. The present study was designed to evaluate ovulation rate, antral follicle count (AFC), circulating ant-müllerian hormone (AMH), and the association among these measures in unstimulated and superstimulated Trio carrier cattle. We hypothesized that AFC and AMH would be variable among individual cows but would be similar between Trio carriers and non-carrier control cows and that there would be no association between these measures of follicle numbers and ovulation rate. In experiment 1, ovulation rate was determined during 4 consecutive estrous cycles in Trio carriers (n = 34) and non-carrier controls (n = 27). Ovulation rate, on average, was greater (P < 0.01) in Trio carriers (3.5 ± 0.2) compared to non-carrier controls (1.1 ± 0.1) with ∼70% of carrier cycles (n = 136) having 3-4 ovulations while only ∼5% had single ovulations. In contrast, non-carrier cycles (n = 108) were mostly single ovulation (89%) with none having more than two ovulations. In experiment 2, AFC, determined at wave emergence, was not different (P = 0.54) between Trio carriers (24.5 ± 1.3; n = 45) and non-carrier controls (23.1 ± 0.9; n = 37), and no correlation was found between AFC and mean ovulation rate in either genotype (r = -0.009 and r = -0.07; P > 0.70, respectively). In Experiment 3, circulating AMH was also not different between genotypes (P = 0.65) while correlations were found between AFC and AMH in Trio carriers (r = 0.43; P = 0.05; n = 27) and non-carrier controls (r = 0.78; P < 0.01; n = 19). In experiment 4, AFC and AMH were determined in Trio-carriers (n = 9) in relation to a synchronized follicular wave which was unstimulated or stimulated with exogenous FSH. Stimulation with FSH increased ovulation rate, compared to unstimulated Trio carriers, however no association was found between AFC or AMH and ovulation rate regardless of whether superstimulation with exogenous FSH was used. In conclusion, the novel high fecundity bovine genotype Trio, results in consistent multiple ovulations despite having similar AFC and AMH. Therefore, our results suggest that differences in antral follicle numbers during the final stages of follicle development are not a key component of the mechanism underlying multiple ovulations in Trio carriers.