The dataset was obtained from 201 ovum pick-up (OPU) sessions and 184 ETs carried out at Cenatte Embriões LTDA, located in the city of Pedro Leopoldo, Minas Gerais, Brazil (latitude: 19°37′12″ south, longitude: 44°2′38″ west).

A total of 73 crossbred Murrah buffalo females (23 nulliparous and 50 multiparous), healthy, nonpregnant, and cycling were used as oocyte donors and embryo recipients. The animals were kept under grazing of Brachiaria decumbens and B. brizantha with free access to water and mineral salt. During the autumn and winter periods, all animals were supplemented with elephant grass silage to meet the demand of each category (17).

All females were used as oocyte donors along the experimental period, and groups of 6–36 animals were submitted to 11 replicates with a minimum interval of 19 days, for 8 months, without any hormonal stimulation.

The animals were contained and submitted to epidural anesthesia, using 3 ml of 2% lidocaine (Lidovet®, Bravet). OPU procedure was performed using transvaginal ultrasound equipment (Aloka 500®, Tokyo, Japan; frequency 5 MHz) coupled to a system with an 18G disposable hypodermic needle (BD) and a 1.7-mm-diameter and 80-cm-long Teflon aspiration line (WTA, Watanabe Applied Technology). The vacuum pressure used was 60–65 mmHg (10 and 12 ml of water/minute). A warm solution (37°C) containing PBS with sodium heparin (5,000 UI/L; Parinex®, Hypolabor), 1% fetal bovine serum (FBS, Nutricell), and 0.1% antibiotic solution (amikacin) was used to collect the aspirated oocytes from follicles larger than 2 mm.

After OPU, cumulus-oocyte complexes (COCs) were searched and classified under a stereomicroscope. TCM 199 with 5% FBS, pyruvate (0.2 mM), and amikacin (83.4 mg/ mL) was used to wash the COCs collected. The criteria used for the evaluation of COC were the presence, the number of layers, and the degree of compaction of Cumulus cells, as well as the appearance of the cytoplasm in color, homogeneity, and integrity. Up to their parameters, the COCs were classified as follows: grade I (three or more layers of cells of the compact cumulus and homogeneous cytoplasm), II (one to three layers of cumulus cells covering totally or partially the oocyte and with homogeneous or heterogeneous cytoplasm), and III (one layer of cumulus cells covering the oocyte totally or partially and with cytoplasm homogeneous or heterogeneous), denuded, degenerated, expanded, and atretic (18). For in vitro embryo production (IVEP), only oocytes grades I, II, and III (viable oocytes) were used.

Viable oocytes were maintained in 1.2 ml cryovials (Corning) containing an oocyte maturation medium, inside an incubator for embryo transport (WTA) at 38.5°C with gas mixture (5.5% CO₂, 5.0% O₂, and 89.5% N₂), until the arrival to the laboratory. The maximum transport time to the laboratory was 6 h after the beginning of OPU.

In the laboratory, groups of 10 to 12 oocytes were transferred to 50-μL drops of a maturation medium, composed of TCM199 with sodium bicarbonate supplemented with 10% FBS, pyruvate (0.2 mM), amikacin (83.4 mg/ mL), 0.5 μg of FSH (Folltropin), 5 μg of LH (Lutropin), 1 μg of 17 α-estradiol, and supplemented with 50 μM of 2-mercaptoethanol, covered with sterile mineral oil. Oocytes were incubated for 20–24 h, at 38.7°C, with 5.5% CO₂ in air and saturated humidity.

One hour before the end of the maturation period, the semen from a tested bull was thawed at 35°C for 20 s and deposited on a Percoll gradient (30, 60, and 90%) for sperm selection. Centrifugation was performed at 9,000 × g for 2 min, and, subsequently, the pellet was washed twice in a total alkaline phosphatase medium (TALP), at 4,500 × g for 1 min. The inseminating dose was adjusted to 1.2 million sperm/mL. After IVM, the oocytes were washed in a TCM199 HEPES medium containing 0.3% BSA, pyruvate (0.2 mM), and amikacin (83.4 mg/mL). Then they were washed in a TALP fertilization medium with 0.5% BSA, pyruvate (0.2 mM), amikacin (83.4 mg/ mL), heparin (20 μg/ mL), and PHE solution (2 μM of penicillamine, 1 μM of hypotaurine, and 0.25 μM of epinephrine), and groups of 10–12 oocytes were co-cultured with sperm in 50 μL drops of this same medium, under the same conditions of atmosphere, humidity, and temperature used for IVM for 18–20 h.

After fertilization (D0), the presumptive zygotes were denuded, and groups of 10–12 were transferred to 100 μL drops containing a SOFaa medium with 3% FBS under mineral oil, during 5–7 days at 38.7°C, with 5.5% CO₂, 5.0% O₂, and 89.5% N₂ in saturated humidity. Forty-eight hours after fertilization, the embryo cleavage was evaluated. There was no change of medium throughout the in vitro culture. On 5th to 7th days after fertilization (D5–D7, respectively), the blastocyst rate (blastocysts/viable oocytes) was calculated.

Embryos were morphologically evaluated and classified according to the IETS manual (19), in terms of developmental stage: early blastocyst, blastocyst, expanded blastocyst, and hatched blastocyst; and quality: grade I (excellent or good), II (fair), III (poor), or IV (degenerated).

Only grade I D5 and D6 embryos, varying from early to hatched blastocysts, were transferred. The embryos were loaded in 0.25-mL straws containing a maturation washing medium (TCM 199 HEPES, 5% FBS, pyruvate, and amikacin) and were maintained in the portable incubator until transfer to recipients.

Vitrification was performed when the number of embryos exceeded the number of recipients. Only grade I D5, D6, and D7 embryos, varying from expanded to hatched blastocysts, were vitrified on Cryotop® according to the method described by De Rosa et al. (20). Briefly, the embryos were placed in a solution containing 7.5% ethylene glycol and 7.5% dimethyl sulfoxide (DMSO), prepared in TCM 199 HEPES supplemented with 20% FBS, for 3 min and then were transferred to a second solution containing 16.5% ethylene glycol, 16.5% DMSO prepared in FBS with 0.5 M sucrose. After 25 s, the embryos were kept in a volume of <0.1 μL of vitrification solution and loaded on Cryotop®, which was immediately immersed in liquid nitrogen. The warming procedure was carried out by immersing the top of Cryotop® containing the embryos in a 0.25 M sucrose solution. After 1 min, the embryos were transferred to a 0.15 M sucrose solution for 5 min and then loaded in 0.25-mL straws for transfer to recipients.

All females previously used as oocyte donors were also used as recipients, along 24 months, totalizing 184 procedures divided in 18 replicates (fresh: n = 10; vitrified: n = 8 replicates).

All recipients were submitted to one of the two following synchronization protocols: Ovsynch (21) or estradiol plus progesterone based (22). Briefly, the Ovsynch protocol consisted of 0.5 mg of lecirelin (Gestran Plus®, Tecnopec) on a random day of estrous cycle. Seven days later, 0.5 mg of cloprostenol (Sincrosin®, MSD Animal Health) was administered. A second dose of lecirelin (0.25 mg) was injected 48 h later. The second protocol consisted in the insertion of an intravaginal progesterone device, used for 8 days (multiparous) or 16 days (nulliparous) associated to 2 mg of estradiol benzoate (Estrogin®, Biofarm) and 530 mg of cloprostenol, on a random day of estrous cycle. Nine days later, the devices were removed and 400 IU of eCG (Folligon®, MSD Animal Health) and PGF2α were administered to all recipients. Finally, after 48 h, the recipients were treated with 0.5 mg of lecirelin (Gestran Plus®, Tecnopec).

Six or seven days later, ovulation was evaluated by the presence of a CL, which was measured by ultrasound (Aloka 500®, Tokyo, Japan; frequency 5 MHz), and fresh or vitrified embryos were transferred in the uterine horn ipsilateral to CL, by a nonsurgical method. Only one embryo was transferred in each recipient. Asynchrony between embryos and recipients was ±1 day.

Pregnancies were monitored by ultrasound (Aloka 500, Tokyo, Japan; frequency 5 MHz) 30 and 60 days after the expected ovulation. At the due day, the animals were evaluated for labor stages, dystocia, and placental retention. The newborns were weighed, the sex determined, and suckling reflex and umbilicus were examined.

P/ET was calculated as the percentage of pregnant females, diagnosed on days 30 (P/ET 30) and 60 (P/ET 60), on the total number of the recipients. The calving rate was calculated as the percentage of females calving out on the total number of recipients. PL was calculated as the percentage of nonpregnant females, from days 30 to 60, day 60 to calving, and day 30 to calving, in relation to females pregnant on days 30, 60, and 30, respectively.

Data concerning in vitro embryo production were shown according to descriptive statistics, except by analysis of birth weight associated to sex of calves, which was analyzed by logistic regression, by using the GLIMMIX procedure in SAS for Windows (23). Data regarding recipients were analyzed by univariate and multivariate logistic regression, using the LOGISTIC procedure (23). All groups with fewer than 10 recipients were excluded from the analysis. For pregnancy and calving rate analysis, the following were excluded: asynchrony (−1 day, n = 7), day of embryo development (day 7, n = 7), and embryo stage (hatched blastocyst, n = 9). For pregnancy loss, the following were excluded: asynchrony (−1 day, n = 2), day of embryo development (day 7, n = 2), and embryo stage (early blastocyst, n = 5; blastocyst, n = 8; hatched blastocyst, n = 5). Adjusted odds ratio (AOR) and 95% confidence interval (CI) were generated by logistic regression. Odds ratio is a measure of association between a treatment and an outcome and represents the odds that an outcome will occur given a particular treatment compared to the odds of the outcome occurring in the absence of that treatment or another treatment (24). Results are presented as percentages and AOR. P ≤ 0.05 were considered significant, and those 0.05 ≤ P ≤ 0.10 were considered as tendency.

A total of 998 oocytes were recovered from 201 OPU sessions (5.0 ± 0.5/donor), of which 584 were viable oocytes (2.9 ± 0.3/donor). A total of 318 structures cleaved (1.6 ± 0.2/donor), resulting in a cleavage rate of 54.5%. The blastocyst rate was 44.9% producing 262 embryos (1.3 ± 0.1/donor), of which 229 were grade I, 5 grade II, and 28 grade III.

From the total embryos produced, 114 were transferred fresh resulting in P/ET 30, P/ET 60, and a calving rate of 43.0% (49/114), 41.2% (47/114), and 35.1% (40/114), respectively. Pregnancy loss between days 30 and 60 was 4.1% (2/49), between day 60 and calving was 14.9% (7/47), and between day 30 and calving was 18.4% (9/49). The mean gestation length was 310.2 ± 0.5 days. All calvings were eutocic, and the average weight of calves born was 32.6 ± 0.2 kg, being lower for females (31.4 ± 0.4 kg; n = 20) than for males (33.8 ± 0.6 kg; n = 20; P < 0.01). Concerning postpartum occurrences, two females showed retained placenta (5%), two cervix prolapse (5%), and one rejected her calf (2.5%).

For vitrified embryos, 70 were transferred and P/ET 30, P/ET 60, and the calving rate were, respectively, 37.1% (26/70), 31.4% (22/70), and 24.3% (17/70). Pregnancy loss between days 30 and 60 was 15.4% (4/26), between day 60 and calving was 22.7% (5/22), and between day 30 and calving was 34.6% (9/26). The mean gestation length was 314.5 ± 0.9 days, and one of the animals presented dystocia. The average weight of calves born was 33.8 ± 0.2 kg, being similar in females (33.6 ± 0.7 kg; n = 10) and males (34.0 ± 0.4 kg; n = 7; P = 0.33). In the postpartum period, retained placenta was not observed in any recipient, and one of the calves had suckling difficulty in the first 24 h (5.9 %).

Pregnancy and calving rates were significantly associated to the synchronization protocol and the CL diameter. Reproductive season, parity, asynchrony, day of recipient estrus, type of embryo, and day or stage of embryo development did not affect P/ET 30, P/ET 60, or the calving rate (Table 1).

When progesterone associated to estradiol plus the eCG-based protocol was compared to Ovsynch, there was a tendency of greater P/ET 30 (41.9 vs. 36.1%; P = 0.07; AOR = 1.28) and P/ET 60 (37.8 vs. 36.1%; P = 0.09; AOR = 1.08).

Recipients with a CL diameter larger than or equal to 14.5 mm had increased P/ET 30 (47.4 vs. 32.5%; P < 0.01; AOR = 1.87), P/ET 60 (45.3 vs. 27.7%; P < 0.01; AOR = 2.16), and a calving rate (37.9 vs. 21.7%; P < 0.01; AOR = 2.20), when compared to those with CL <14.5 mm.

Pregnancy loss (PL) was associated only to the season of the embryo transfer. No other factor influenced PL from days 30 to 60 (PL 30–60), day 60 to calving (PL 60-C), or day 30 to calving (PL 30-C; Table 2).

PL was greater when ET was performed in the nonbreeding season compared to the breeding season (PL 30–60: 12.8 vs. 0.0%, P = 0.01; AOR > 999.99; PL 60-C: 26.8 vs. 3.6%, P = 0.03; AOR = 9.90; and PL 30-C: 36.2 vs. 3.6%, P = 0.01; AOR = 15.30).