Selecting oocyte donors based on anti-Müllerian hormone (AMH) concentrations: A critical analysis of using cutoff values as exclusion criterion for an in vitro embryo production program in Gir cattle.

The aims of this study were to determine anti-müllerian hormone (AMH) cutoff values for selecting Gir (Bos taurus indicus) oocyte donors and estimate the impact of using AMH concentrations as a selection criterion. In Exp. 1, Gir heifers (n=120) were sampled for AMH analysis and submitted to ovum pick-up and in vitro embryo production (OPU-IVEP). AMH cutoff values were calculated using ROC analysis or, alternatively, by the successive exclusion of heifers with the lowest AMH values. The correlations between AMH and OPU-IVEP outcomes were significant (P<0.001), though low or moderate (r= 0.34-0.52). We estimated an improvement (P<0.05) after the use of AMH cutoff values to select donors of +15.3% for total oocyes, +19.4% for viable COC, and +23.4% for blastocysts. This selection pressure, however, led to the exclusion of 32.8%, 37.9%, and 50.0% of the initial potential donors, respectively. In Exp. 2, we analyzed data from OPU-IVEP sessions of 658 Gir donors with known genomic values for predicted transmitting ability for milk (GPTAm) and age at first calving (GPTAafc). The selection based on the number of oocytes recovered had no effect (P>0.05) on the average GPTAm nor GPTAafc values of the remaining donors. In summary, plasma AMH ≥700 pg/mL is a cutoff value that can be used to select Gir heifers with a greater potential as oocyte donors. Nevertheless, this selection leads to the exclusion of up to 50% of potential donors. Finally, exclusion of poor responders had no effect on mean genomic estimates for milk production or age at first calving in the selected subset of donors.

Timing of early resynchronization protocols affects subsequent pregnancy outcome in dairy cows.

The aim of this study was to evaluate the outcomes of an early resynchronization protocol (Resynch) initiated at different timepoints after timed artificial insemination (TAI) and with unknown pregnancy status. Holstein cows (n = 164) were submitted to the following TAI protocol: D0, insertion of an intravaginal progesterone (P4) device and 2 mg im estradiol benzoate (EB); D8, removal of P4 device and treatment with 0.5 mg im sodium cloprostenol (PGF); D9, 0.1 mg im Lecirelin (LEC); and D10, TAI1. Cows were then randomly assigned to Resynch protocols starting either on day 20 (Resynch20D, n = 82) or 25 after TAI1 (Resynch25D, n = 82) with the insertion of a new P4 device and EB treatment. In both groups, P4 device was removed on day 8 after the beginning of Resynch, the same day of pregnancy diagnosis by ultrasonography. In pregnant cows there was no further action. Non-pregnant cows were treated with 0.5 mg im PGF, had a blood sample collected for serum P4 analysis and we measured and recorded the size of the largest follicle and the presence or absence of a corpus luteum (CL). One day later, cows were treated with 0.1 mg im LEC and TAI2 occurred 12-14 h later. The diameter of the largest follicle and serum P4 were compared between groups by ANOVA for the main effects of treatment, presence of a CL, and their interaction, whereas pregnancy per artificial insemination (P/AI) and the percentage of cows with a CL on the day of ultrasonography were analyzed using the Chi-square test. Follicle diameter on day 8 of Resynch was greater for cows in the Resynch20D group compared with Resynch25D (15.9 ± 3.9 vs 12.2 ± 2.5 mm, respectively; P = 0.046). The Resynch25D group had a greater percentage of cows with a CL (51.9 vs 18.9%, respectively; P = 0.0008) and higher serum P4 (2.8 ± 1.1 vs 1.7 ± 0.8 ng/mL; P = 0.041) at the end of the protocol compared with Resynch20D. P/AI at TAI1 was 35.4 and 36.6% (P > 0.10) for cows enrolled in Resynch20D and Resynch25D groups, respectively. P/AI to TAI2, after Resynch protocols, was greater in Resynch25D than Resynch20D (44.2 vs 22.6%, respectively; P < 0.05). In conclusion, starting an early resynchronization protocol 25 days after TAI increases P/AI compared with starting 20 days after TAI, and this was associated with a presumed greater proportion of cows with a functional CL at the moment of P4 device removal.

Likelihood of pregnancy after the transfer of embryos derived from follicle aspiration and in vitro embryo production sessions with different relative efficiencies.

The aim of the present study was to evaluate the likelihood of pregnancy of in vitro-produced (IVP) embryos from batches with distinct relative efficiencies. Data were retrospectively analyzed from 605 transvaginal ultrasonic-guided follicle aspiration sessions (OPU) followed by in vitro embryo production (IVEP) and 2456 fresh embryo transfers (ET), performed between 2008 and 2012 in individuals of the Gir (dairy Bos indicus) breed. The OPU and IVEP were performed using standard procedures by a single group of technicians at the same laboratory facility. Records were stratified into quartiles (I to IV) according to the total of cumulus-oocytes complexes (COC) produced per donor, or in percentile ranges (0%-25%, 26%-50%, 51%-75%, and 76%-100%) for endpoints related to COC quality or efficiency of embryo production. Pregnancy per embryo transfer (P/ET) was compared among quartiles or ranges using the chi-squared test. Donors producing a greater number of total COC (quartile I) also had more viable and grade I COC, and a greater number of embryos than donors ranked in quartiles II, III or IV, respectively (P < 0.0001). Nevertheless, P/ET did not differ (P > 0.05) among embryos produced by donors ranked in Quartiles I to IV. Similarly, there was no difference (P > 0.05) in P/ET for embryos derived from OPU sessions with a relatively greater or lesser percentage of viable or Grade I COC. Cleavage and blastocyst rates within each IVEP batch had no effect (P > 0.05) on P/ET. In conclusion, data suggest that there is no relationship among oocyte yield after OPU, or efficiency of IVEP, and the likelihood of pregnancy after ET of fresh IVP embryos.

Safety of vaccination against brucellosis with the rough strain in pregnant cattle.

Brucellosis is an infectious and contagious disease that profoundly impacts public health. However, in many countries, disease prevention is restricted to the vaccination of calves, and there is no prophylactic strategy for pregnant heifers and cows. The aim of this study was to evaluate the safety of the rough strain vaccine against brucellosis in pregnant cattle. Crossbred cows (N = 96) at three gestational periods (early, mid, or late pregnancy) were randomly allocated into the vaccine treatment group or to the control group. We then compared the percentage of pregnancies reaching full term, live calves 60 days after delivery, and seropositive calves. There was no effect of vaccination in any of the gestational periods on the evaluation endpoints. In conclusion, vaccination against brucellosis with the rough strain is safe for pregnant cattle at all gestational periods.

Characterization of blood flow and the effects of exogenous estradiol benzoate on residual follicles formed after ultrasound-guided transvaginal follicle aspiration in cattle.

BACKGROUND: Removal of the follicular content by ultrasound-guided transvaginal follicle aspiration (TVFA) may fail to induce immediate atresia and loss of function, resulting in the occurrence of residual follicles (RF). The aims of this study were to characterize the blood flow in RF and to determine the effects of the treatment with estradiol benzoate on RF fate. Lactating, cyclic Holstein-Gir crossbred cows were used. In Experiment 1, follicular wave emergence (D0) was synchronized in cows (n = 10) and follicular growth was then monitored by transrectal ultrasonography from D0 to D8, followed by TVFA of the largest follicle present on the ovaries 24 h later. Color Doppler ultrasound imaging was used to examine blood flow on the follicular wall, which was recorded immediately before and every 12 h after TVFA, up to 72 h. In experiment 2, cows (n = 22) were randomly allocated to receive either 2 mL of saline i.m. (Control group, n = 11) or 2 mL estradiol benzoate i.m. (EB group, n = 11) immediately after TVFA. Ovaries were scanned every 12 h to confirm the presence and to measure the diameter of RF. The contents of the RF, if present, were collected 72 h after the first TVFA, using the same aspiration procedures. Follicular fluid from original follicles and RF were stored at -20 °C until hormonal assays. RESULTS: In Experiment 1, there was no reduction (P > 0.05) of blood flow in the remaining follicle walls after TVFA and maximum blood flow values were observed at 49.5 ± 19.7 h post-TVFA. In Experiment 2, formation of RF after TVFA was proportionally similar between Controls (5/9) and EB (5/10) cows. Also, RF diameter did not differ between groups (P > 0.05). Nonetheless, the content of RF from cows in the EB group had lower (P = 0.0004) estradiol (E2) concentration and lower (P = 0.0005) E2:P4 ratio compared with Controls. CONCLUSIONS: In conclusion, 1) the persistence of vascularization in the remaining follicle wall may contribute to the formation of RF after follicle aspiration, and 2) the treatment with estradiol benzoate does not prevent formation of RF, but does reduce their estradiol production.

Weight gain potential affects pregnancy rates in bovine embryo recipients raised under pasture conditions.

The aim of the present study was to evaluate the effect of differences in body weight gain after embryo transfer on the pregnancy rates of crossbred heifers used as recipients and raised under a grazing system. The study was performed during the dry (April to September) and the rainy (October to March) seasons. The embryos transferred were produced by in vitro fertilization. The body weight of each recipient was measured immediately before the embryo transfer and 23 to 25 days later, when the diagnosis of pregnancy was performed by ultrasonography. The associations among initial body weight (IBW), daily body weight gain (DWG), season, and pregnancy rate were evaluated using a logistic procedure that included the effect of the IBW, season, and linear and quadratic effects of the DWG. Altogether, there was no effect of season and pregnancy rates did not change between the dry and rainy seasons (42.3 vs. 45.8%, respectively; P > 0.05). However, the pregnancy rate was greater in the recipients with daily body weight gains over 250 g/day, regardless of the season. In addition, the pregnancy rate of the recipients was better (P < 0.04) explained by a logistic regression model that included the linear and quadratic effects of the DWG. The probability of each heifer to become pregnant according to DWG is explained by the follow equation: P(y = 1) = (Exp((-1.06703 + 0.0108 * DWG - 0.00002 * DWG ^ 2)))/(1 + Exp((-1.6703 + 0.0108 * DWG - 0.00002 * DWG ^ 2))). In conclusion, body weight gain potential is a critical factor for the pregnancy rates of in vitro embryo recipients managed under grazing systems.

Sincronização folicular e vascularização do folículo dominante em novilhas mestiças tratadas com estradiol / Follicle synchronization and changes in vascularization of the dominantfollicle in crossbred heifers after estradiol treatment

Avaliou-se a sincronização folicular e vascularização na parede do folículo dominante (FD) após o tratamento com Cipionato (CE) ou Benzoato de estradiol (BE). No Experimento 1, 45 novilhas receberam implante de P4 (1,0g) e foram distribuídas em 3 grupos de acordo com éster de estradiol administrado pela via intramuscular: 1mL salina; 0,5mg CE ou 2,0mg BE. O diâmetro do FD e a emergência folicular (EF) foram monitorados por 5 dias. No experimento 2, D0 (dia 0): 30 novilhas receberam implante de P4 e 2,0mg BE; D8, retirada do implante e 0,5mg de prostaglandina (cloprostenol); D9: tratamentos: 1mL salina; 0,5mg CE ou 1,0mg BE. O diâmetro e a vascularização do FD foram monitorados do D8 ao D10. Os dados foram analisados por ANOVA ou Kruskal-Wallis, e as médias comparadas por Tukey (5%). No experimento 1, a EF ocorreu mais tarde (P 0,0013) no grupo BE (4,3±0,8 vs. 3,5±0,8d). No experimento 2, o fluxo sanguíneo do FD aumentou (P 0,001) 24h após o tratamento com BE. A taxa de ovulação foi semelhante (P>0,05) entre os grupos (80,0, 70,0 e 55,5%, BE, CE e salina). Conclui-se que nas concentrações utilizadas, o BE, no início do protocolo, retardou a EF e, no final do protocolo, promoveu uma melhor sincronia do FD demonstrada pela elevação do fluxo sanguíneo.

The study aimed to evaluate synchronization and blood flow of the dominant follicle (DF) after estradiol cypionate (EC) or benzoate(EB) treatment. In experiment 1, progesterone (P4) implants (1.0g) were inserted in 45 heifers, which were given 1mL saline,0.5mg EC or 2.0mg EB. The diameter of the DF and follicle emergence (FE) were recorded for 5 days. For experiment 2, 30 heiferswere given: D0 (day 0): P4 implant and 2.0mg EB; D8: implant removal and 0.5mg of prostaglandin (cloprostenol). On D9, theywere assigned in 1mL saline, 0.5mg EC or 1.0mg EB. From D8 to D10, the diameter and blood flow of the DF was monitored.Data were analyzed by ANOVA or Kruskal-Wallis test, and compared by Tukey (5%). In experiment 1, the FE occurred later(P0.05) between groups (80.0, 70.0 and 55.5%, EB, EC and saline). The interpretation was that,in these concentrations, the EB, at the beginning, delayed FE and, at the end of the protocol, promoted a better synchrony of theDF, confirmed by the increase of the blood flow.