E. O. S. Batista

Anti-Mullerian Hormone Concentration and Antral Ovarian Follicle Population in Murrah Heifers Compared to Holstein and Gyr Kept Under the Same Management

This study was performed to evaluate plasma concentrations of anti-Mullerian hormone (AMH) and the ovarian antral follicle population (AFP) in different genetic groups. Cyclic heifers (13 Bubalus bubalis [Murrah]; 15 Bos taurus [Holstein] and 10 Bos indicus [Gyr]) were maintained under the same management and were synchronized with two doses of 150 μg IM d-cloprostenol administered 14 days apart. After the second d-cloprostenol treatment, heifers had their ovaries scanned daily by ultrasound to define the day of ovulation. On the same day, the AFP was determined and a plasma sample was collected to measure AMH. Murrah heifers had less AFP (25.6 ± 2.1 follicles; p = 0.01) and plasma AMH concentration (0.18 ± 0.03 ng/ml; p < 0.001) than Gyr (60.0 ± 12.2 follicles and 0.60 ± 0.12 ng/ml of AMH); however, data were similar when compared to Holstein (35.9 ± 6.8 follicles and 0.24 ± 0.06 ng/ml of AMH) heifers. Regardless of genetic background, there was a positive relationship between the AFP and plasmatic AMH concentration (Murrah [r = 0.62; p < 0.01], Holstein [r = 0.66; p < 0.001] and Gyr [r = 0.88; p < 0.001]). Also, when heifers were classified according to high- or low-AMH concentration based on the average within each genetic group, high-AMH heifers had greater (p < 0.0001) AFP than low-AMH heifers. In conclusion, both Murrah and Holstein heifers presented lower plasma AMH concentration and AFP when compared to Gyr.

Plasma anti-Müllerian hormone as a predictive endocrine marker to select Bos taurus (Holstein) and Bos indicus (Nelore) calves for in vitro embryo production

This study evaluated the association between plasma anti-Müllerian hormone (AMH) concentrations and in vitro embryo production in Bos indicus (Nelore; experiment 1) and Bos taurus (Holstein; experiment 2) calves superstimulated or not with 140 mg of porcine follicle-stimulating hormone (pFSH; 4 decreasing doses twice daily). Oocytes were recovered from calves aged 2 to 4 mo after receiving gonadotropin stimulation (Nelore, n = 15; Holstein, n = 12) or not (Nelore, n = 15; Holstein, n = 12). Cycling heifers formed a positive control group (n = 15 for Nelore [aged 18-24 mo], n = 10 for Holstein [aged 14-16 mo]). All the calves underwent laparoscopic ovum pickup, and cycling heifers underwent a regular transvaginal ultrasound-guided ovum pickup for oocyte recovery. Immediately before oocyte retrieval, blood samples were taken for subsequent AMH determination (ng/mL). Regardless of the genetic group, calves that received pFSH (3.6 ± 1.1 in Nelore and 4.6 ± 1.2 in Holstein) or did not receive pFSH (3.2 ± 1.0 in Nelore and 2.5 ± 0.8 in Holstein) had greater plasma AMH concentrations (P = 0.01 in Nelore and P = 0.003 in Holstein) than cycling heifers (1.1 ± 0.2 in Nelore and 0.6 ± 0.07 in Holstein). AMH concentrations in calves with or without pFSH were similar in both genetic groups (3.6 ± 1.1 vs 3.2 ± 1.0 in Nelore; 4.6 ± 1.2 vs 2.5 ± 0.8 in Holstein). In calves, positive correlations were observed between plasma AMH concentrations and the numbers of follicles >2 mm (r = 0.86, P < 0.0001 in Nelore; r = 0.78, P < 0.0001 in Holstein), cumulus-oocyte complexes (COCs) retrieved (r = 0.91, P < 0.0001 in Nelore; r = 0.82, P < 0.0001 in Holstein), COCs cultured (r = 0.71, P < 0.0001 in Nelore; r = 0.79, P < 0.0001 in Holstein), and blastocysts produced (r = 0.62, P = 0.0003 in Nelore; r = 0.58, P = 0.009 in Holstein), and these results were independent of pFSH treatment. In conclusion, calves had greater plasma AMH concentrations than cycling heifers. In addition, treatment with pFSH did not influence AMH concentrations in calves, regardless of the genetic group. More importantly, plasma AMH concentrations were positively correlated with the antral follicle population and the number of COCs retrieved, COCs cultured, and blastocysts produced in B indicus and B taurus calves. Therefore, AMH is a promising tool for selecting oocyte donor calves to maximize results during in vitro embryo production.

Number of oocytes retrieved per donor during OPU and its relationship with in vitro embryo production and field fertility following embryo transfer

The association of OPU-IVEP is an important instrument to drive genetic progress. In vitro embryo production (IVEP) has remarkably expanded in the last decade compared to in vivo embryo production. Because of the high repeatability of oocyte retrieval within oocyte-donors, studies exploring the relationship between the number of oocytes recovered per OPU section with IVEP efficiency, as well as with field fertility (pregnancy results following embryo transfer; P/ET) are extremely important to guide cow-donor selection and optimize field reproduction efficiency and the herd’s genetic gain. Based on this rationale, our group conducted a retrospective analysis of a large database comprising IVEP records from several cattle breeds, including Bos indicus and Bos taurus for either beef or dairy purposes. A total of 205,140 oocytes recovered from 7,906 OPU procedures of 6,902 donors (5,227 beef and 1,675 dairy) of Brazilian farms were analyzed. Beef breeds analyzed were Nelore (Bos indicus) and Senepol (Bos taurus) and dairy breeds were Gyr (Bos indicus) and Holstein (Bos taurus). According to our analysis, the IVEP in beef cattle had a great improvement throughout the last years, with a remarkable increase in numbers of pregnancies per OPU compared to late 90’s (averaging only 1 pregnancy per OPU in 1998 vs 2,4 in 2014). As for the distribution of oocytes retrieved, both Bos indicus beef (Nelore = 27.2) and dairy (Gyr = 23.8) breeds seem to yield greater average numbers of oocytes per OPU compared to Bos taurus (Senepol = 21.8; Holstein = 19.3). Despite these differences across genetic groups, outstanding donors can be found in all breeds and the number of oocytes retrieved per donor seems consistent across time. For both beef cattle breeds studied, it appears that number of oocytes retrieved at OPU had a negative but minor effect on both cleavage and blastocyst rates, especially for Senepol breed. Conversely, in dairy breeds the number of oocytes recovered per OPU had essentially no effect on cleavage rates, but we captured a trend for lower blastocyst rates with greater numbers of oocytes per OPU. For both, beef and dairy breeds the number of blastocyst per OPU was greater when higher number of oocytes were recovered per OPU, regardless of genetic group. Pregnancy rate following ET in Nelore breeds was lower in donors with greater amounts of oocytes retrieved per OPU. In contrast, in the Senepol breed and both dairy breeds (Gyr and Holstein) pregnancy rates after ET seems to increase when the number oocytes recovered per OPU increases. In addition, the semen utilized had a major impact of IVEP efficiency: top ranking sires yielded outstanding blastocyst rates, while poor performers produced very low blastocyst rates. The season of the year also had effect on IVEP, with Bos indicus breeds showing less variation in IVEP results throughout the year. In conclusion, despite the evolution of IVEP in the last two decades, the number of oocytes recovered per OPU had a minor effect both on blastocyst rate and pregnancy rates after ET. However as more oocytes are collected, the number of produced blastocysts improves. Thus, it seems important to identify donors with greater oocyte recovery-per-OPU potential, especially in cattle breeds yielding fewer oocytes per OPU, such as Holstein, to assure greater IVEP efficiency. It is also clear that cattle breed, semen used during IVEP and season of the year can potentially influence IVEP and field fertility results. A holistic approach controlling the quality of the performed OPU, consistency in lab routines, as well as selecting donors with high genetic value (through genomics) and greater oocyte population (through AMH assays or ultrasound) are highly advisable.

Prostaglandin treatment at the onset of norgestomet and estradiol-based synchronization protocols did not alter the ovarian follicular dynamics or pregnancy per timed artificial insemination in cyclic Bos indicus heifers

The aim of the present study was to evaluate the effects of the PGF2α treatment given at the onset of a synchronization of ovulation protocol using a norgestomet (NORG) ear implant on ovarian follicular dynamics (Experiment 1) and pregnancy per AI (P/AI; Experiment 2) in cyclic (CL present) Bos indicus heifers. In Experiment 1, a total of 46 heifers were presynchronized using two consecutive doses of PGF2α 12 days apart. At first day of the synchronization protocol the heifers received implants containing 3mg of NORG and 2 mg of estradiol benzoate (EB). At the same time, heifers were randomly assigned to receive 150 mg of D-cloprostenol (n=23; PGF2α) or no additional treatment (n=23; Control). When the ear implants were removed 8 days later, all heifers received a PGF2α treatment and 1 mg of EB was given 24 h later. The follicular diameter and interval to ovulation were determined by transrectal ultrasonography. No effects of PGF2α treatment on the diameter of the largest follicle present were observed at implant removal (PGF2α=9.8±0.4 vs. Control=10.0±0.3 mm; P=0.73) or after 24 h (PGF2α=11.1±0.4 vs. Control=11.0±0.4 mm; P=0.83). No differences in the time of ovulation after ear implant removal (PGF2α=70.8±1.2 vs. Control=73.3±0.9 h; P=0.10) or in the ovulation rate (PGF2α=87.0 vs. Control=82.6%; P=0.64) between treatments were observed. In Experiment 2, 280 cyclic heifers were synchronized using the same experimental design described above (PGF2α; n=143 and Control; n=137), at random day of the estrous cycle. All heifers received 300 IU of equine chorionic gonadotropin (eCG) and 0.5 mg of estradiol cypionate (as ovulatory stimulus) when the NORG ear implants were removed. Timed artificial insemination (TAI) was performed 48 h after implant removal and the pregnancy diagnosis was conducted 30 days later. No effects on the P/AI due to PGF2α treatment were observed (PGF2α=51.7 vs. Control=57.7%; P=0.29). In conclusion, PGF2α treatment at the onset of NORG-based protocols for the synchronization of ovulation did not alter the ovarian follicular responses or the P/AI in cyclic Bos indicus beef heifers synchronized for TAI.

Paradoxical effects of bovine somatotropin treatment on the ovarian follicular population and in vitro embryo production of lactating buffalo donors submitted to ovum pick-up

The aim of the present study was to evaluate the effect of bovine somatotropin (bST; 500mg) administration on lactating buffalo donors submitted to two different ovum pick-up (OPU) and in vitro embryo production schemes with a 7 or 14d intersession OPU interval. A total of 16 lactating buffalo cows were randomly assigned into one of four experimental groups according to the bST treatment (bST or No-bST) and the OPU intersession interval (7 or 14d) in a 2×2 factorial design (16 weeks of OPU sessions). The females submitted to OPU every 14d had a larger (P<0.001) number of ovarian follicles suitable for puncture (15.6±0.7 vs. 12.8±0.4) and an increased (P=0.004) number of cumulus-oocyte complexes (COCs) recovered (10.0±0.5 vs. 8.5±0.3) compared to the 7d interval group. However, a 7 or 14d interval between OPU sessions had no effect (P=0.34) on the number of blastocysts produced per OPU (1.0±0.1 vs. 1.3±0.2, respectively). In addition, bST treatment increased (P<0.001) the number of ovarian follicles suitable for puncture (15.3±0.5 vs. 12.1±0.4) but reduced the percentage (18.9% vs. 10.9%; P=0.009) and the number (1.4±0.2 vs. 0.8±0.1; P=0.003) of blastocysts produced per OPU session compared with the non-bST-treated buffaloes. In conclusion, the 14d interval between OPU sessions and bST treatment efficiently increased the number of ovarian follicles suitable for puncture. However, the OPU session interval had no effect on embryo production, and bST treatment reduced the in vitro blastocyst outcomes in lactating buffalo donors.

Short communication: Field fertility in Holstein bulls: Can type of breeding strategy (artificial insemination following estrus versus timed artificial insemination) alter service sire fertility?

The aim of this study was to compare pregnancy per artificial insemination (P/AI) from service sires used on artificial insemination after estrus detection (EAI) or timed artificial insemination (TAI) breedings. Confirmed artificial insemination outcome records from 3 national data centers were merged and used as a data source. Criteria edits were herds overall P/AI within 20 and 60%, a minimum of 30 breedings reported per herd-year, service sires that were used in at least 10 different herds with no more than 40% of the breedings performed in a single herd, breeding records from lactating Holstein cows receiving their first to fifth postpartum breedings occurring within 45 to 375 d in milk, and cows with 1 to 5 lactations producing a minimum of 6,804 kg. Initially 1,142,859 breeding records were available for analysis. After editing, a subset of the data (n=857,539) was used to classify breeding codes into either EAI or TAI based on weekly insemination profile in each individual herd. The procedure HPMIXED of SAS was used and took into account effects of state, farm, cow identification, breeding month, year, parity, days in milk at breeding, and service sire. This model was used independently for the 2 types osires f breeding codes (EAI vs. TAI), and service sire P/AI rankings within each breeding code were performed for sires with >700 breedings (94 sires) and for with >1,000 breedings (n=56 sires) following both EAI and TAI. Correlation for service sire fertility rankings following EAI and TAI was performed with the PROC CORR of SAS. Service sire P/AI rankings produced with EAI and TAI were 0.81 (for sires with >700 breedings) and 0.84 (for sires with >1,000 breedings). In addition, important changes occurred in service sire P/AI ranking to EAI and TAI for sires with less than 10,000 recorded artificial inseminations. In conclusion, the type of breeding strategy (EAI or TAI) was associated with some changes in service sire P/AI ranking, but ranking changes declined as number of breedings per service sire increased. Future randomized studies need to explore whether changes in P/AI ranking to EAI versus TAI are due to specific semen characteristics.

Factors that interfere with oocyte quality for in vitro production of cattle embryos: effects of different developmental & reproductive stages

The success of IVP is ultimately dependent on the number and quality of the cumulus-oocyte complexes (COC) harvested during the OPU procedure. Several factors appear to be critical to oocyte quality including follicle size, environment factors such as heatstress, genetic background, age and lactation status of donor animals, all having a remarkable influence on the results of IVP. The aim of this review is to highlight some critical areas that can help veterinary practitioners to enhance OPU efficiency and successfully implement IVP into their routine practice. Focus will be given to recent findings in the literature and underlying physiological aspects that may be interfering with the quality of oocytes addressed to IVP in cattle at younger ages (calves and prepubertal heifers), pregnant vs nonpregnant status, and possible interactions with lactation and days postpartum during OPU.