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The objective was to clarify in vitro production of bovine embryos in Brazil. Data from 656 ovum pick-up/in vitro production (OPU/IVP) procedures, performed on 317 Nelore (Bos indicus) donors, without hormone stimulation or control of ovarian follicular waves, were analysed. Donors were subjected to OPU from one to nine times (no specific schedule), with < 15 d between consecutive procedures. There were 20,848 oocytes, of which 15,747 (75.53%) were considered viable, 5,446 embryos were obtained, 5,398 embryos were immediately transferred, resulting in 1,974 pregnancies (36.57%) at Day 30 and 1,788 (33.12%) pregnancies at Day 60. The average number of total and viable oocytes produced per OPU session was (mean ± SEM) 30.84 ± 0.88 and 23.35 ± 0.7 (average of 8.1 ± 0.3 embryos and 3.0 ± 0.1 pregnancies per OPU-IVP procedure). Since oocyte production varied widely among donor, they were designated as very high, high, intermediate, and low, with 58.94 ± 2.04, 32.61 ± 0.50, 22.13 ± 0.50, and 10.26 ± 0.57 oocytes, respectively, produced by 78, 80, 79, and 80 donors. The number of viable oocytes recovered ranged from 0 to 128; since donors with numerous viable oocytes produced many viable embryos and pregnancies, oocyte production was useful for donor selection. However, there was no significant effect of the number of OPU sessions per donor on mean numbers of oocytes produced. In conclusion, we confirmed field reports of high oocyte production by some Nelore donors and demonstrated individual variation in oocyte yield, which was associated with embryo production and pregnancy rates., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The objective of the present study was to evaluate the effects of double uterine flushing on the recovery of embryos/ova in cattle. Two hundred and ten embryo recovery procedures were conducted using a double uterine flushing method, and the results were compared with 432 conventional single-flushing procedures. Cyclic Limousin (n = 403) and Guzera (n = 239) donor cows received an intravaginal progesterone releasing device and 2 mg of estradiol benzoate on Day 0. Between Days 5 and 9, donors received decreasing doses of FSH, which ranged from 200 to 300 IU (Bos indicus) and 300 to 500 IU (Bos taurus). On the afternoon of Day 7, donors received an injection of 500 microg cloprostenol and progesterone implants were removed 12 h later (morning of Day 8). Artificial insemination was performed between 14 and 26 h after first detection of behavioral estrus. Cows were randomly assigned to have embryos recovered by a double-flushing method (n = 210) or the conventional single-flushing procedure (n = 432). For the double-flushing procedure, after first flushing the whole uterus with 1L of Dubelco's Phosphate Buffered Saline (DPBS), a Foley catheter was positioned in the uterine body to permit refilling of the uterus with fresh DPBS (80-150 mL). The catheter was closed with the plunger of a disposable 5 mL syringe, and the donors were allowed to rest in a holding area for 30 min. Thereafter, a second flush was performed to recover the solution remaining in the uterus. Animals from the control group were subjected to a single uterine flush. From 210 double-flushing procedures, 1409 viable embryos were recovered. In comparison, from 432 cows receiving the single-flushing procedure, 1993 embryos were recovered. Double flushing increased (P < 0.05) the number of embryos recovered per procedure compared to single flushing (6.7 +/- 0.4 versus 4.6 +/- 0.2, respectively; mean +/- S.E.M.). When double flushing was performed, average recovered embryos/ova increased (P < 0.05) from 8.3 +/- 0.4 to 12.7 +/- 0.7 in Limousin and from 7.9 to 11.5 in Guzera. Also, utilization of double flushing increased (P < 0.05) the number of viable embryos from 4.7 +/- 0.3 to 6.9 +/- 0.5 in Limousin and from 4.5 +/- 0.4 to 6.4 +/- 0.7 in Guzera. Mean total embryos/ova was similar (P > 0.05) between the control group and after the first uterine flushing in the double-flushing group; therefore, both flushings were conducted efficiently. In conclusion, double uterine flushing increased embryo recovery in cattle.

The objective of experiment 1 was to evaluate the influence of follicular wave moment on the quality and number of recovered oocytes through ovum pick-up (OPU), as well as blastocyst rate, in Nelore cows. Two hypotheses were tested: (1) the number of recovered viable COC would be higher at Days 3 and 5 than at Days 7 and 9; (2) the COC aspirated at the beginning of the follicular wave (Day 3 or Day 5) would be more competent and would produce more blastocysts than COC obtained at the end of the wave (Day 7 and Day 9), because these would be in a certain degree of advanced atresia. Cows (n= 10) received 2 mL of estradiol benzoate (Estrogin®), an auricular progestagen implant (Crestar®) for 7 days, and 2 mL of PGF2 analog (Preloban®) at the moment of implant retrieval. After 24 h animals were submitted to aspiration of the dominant follicle (ADF), this moment being considered Day 0. After ADF the donors were submitted to OPU on Days 3, 5, 7, and 9 (groups D3, D5, D7, and D9, respectively) of the new follicular wave. Each animal was submitted to all treatments. In each phase the protocol began with a 2-day interval from the group D9, D7, D5, and D3. Recovered COC went to IVM, IVF, and IVC in the same session of IVP. Statistical analysis were performed with SAS software (SAS Institute, Cary, NC, USA), using ANOVA at a 5% level of significance. There was no difference in the total number of follicles visualized at the moment of OPU among groups (D3:195, D5:189, D7:161, D9:151) or in the recovery rate (77.94; 78.30; 72.04; 73.50) and number of recovered total structures (152, 148, 116, 111). There was no difference in the total number of viable COC (133, 137, 103, 97) or in cleavage rate of COC in the 4 groups (79.69; 83.45; 71.84; 76.28). The total number and the blastocyst rates were higher in groups D3 and D5 (66, 49%, and 62, 45.25%) than in groups D7 and D9 (34, 33%, and 35, 36.08%). Statistical analysis was performed with the chi-square test, with a significance level of 5%. In experiment 2 nuclear and cytoplasmatic maturation were evaluated in COC recovered at Days 4 and 8 regarding nuclear maturation (progression to metaphase II stage) and cortical granule migration, respectively. The hypothesis tested was that COCs recovered at the beginning of the follicular wave would have a higher competence and therefore higher maturation rate compared with those the aspirated at the end. There was no difference in the number of viable COC (78 v.97), percentages of nuclear maturation (74.35 v.53.6), and cytoplasmatic maturation (48.71 v.30.92). The results of the 2 experiments demonstrated that the beginning wave (Days 3 to 5) is the most appropriate moment for obtaining oocytes destined for IVP because of the greater oocyte competence and consequently, larger number and increased blastocyst production rates. Moreover, the results obtained in experiment 2 indicate that the low rate of cytoplasmic maturation can be a major limiting factor of IVP in Nelore cows. FAPESP. [ABSTRACT FROM AUTHOR]

In recent years, Brazil has become the leading country in the world for the number of embryos produced in vitro(Thibier M 2009 IETS Embryo Transfer Newsletter 22, 12-19). This is partly due to the large numbers of Bos indicusanimals in Brazil, making up about 80% of the total cattle. The mean oocyte production per ultrasound-guided follicular aspiration from Bos indicusis higher than those for European breeds (Pontes JHF et al.2009 Theriogenology 71, 690-697). In the present study, we analyzed 5407 ovum pick ups (OPU) and compared the average production of total (n= 90,086) and viable (n= 64,826) oocytes and the number of embryos produced in vitrofrom Gir (Bos taurus indicus), Holstein (Bos taurus taurus), 1/4 Holstein × 3/4 Gir, and 1/2 Holstein-Gir crossbreed cows. To obtain oocytes, OPU was repeated from 4 to 7 times (mean = 5.7 ± 2.4) in each donor cow aged from 3 to 7 years (mean = 5.0 ± 2.3) during a 12-mo period. COCs (n= 90,086) obtained were classified according to the presence of cumulus cells and the oocyte cytoplasm aspect (homogeneous or heterogeneous/fragmented). The viable oocytes (n= 64,826) were in vitromatured for 24 h at 38.8°C in an atmosphere of 5% CO2in air. Since this was a commercial programm, frozen sexed semen (2 × 106mL-1) from Gir (n= 8) or Holstein (n= 7) sires previously tested for high efficiency was used for IVF. Fertilization was carried out (18-20 h) and the presumed embryos were cultured for 7 days in the same conditions as were used for IVM. Data were analyzed by ANOVA. On average, 16.7 ± 6.2 oocytes were obtained per OPU/IVF procedure and 71.96% were considered viable. The mean numbers of total oocytes per OPU/IVF procedure were 17.1 ± 4.4 for Gir cows (n= 617), 11.4 ± 3.9 for Holstein cows (n= 180), 20.4 ± 5.8 for 1/4 Holstein × 3/4 Gir (n= 44), and 31.4 ± 5.6 for 1/2 Holstein-Gir crossbreed females (n= 37, P< 0.01). The mean numbers of viable oocytes per OPU/IVF procedure were 12.1 ± 3.8 for Gir cows, 8.0 ± 2.6 for Holstein cows, 16.8, ± 5.0 for 1/4 Holstein × 3/4 Gir, and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbreed females (P< 0.01). The average number of embryos produced by OPU/IVF were 3.2 (n= 12,243/3378) for Gir cows, 2.2 (n= 2426/1138) for Holstein cows, 3.9 (n= 1033/267) for 1/4 Holstein × 3/4 Gir, and 5.5 (n= 1222/224) for 1/2 Holstein-Gir. The average number of embryos produced per IVF session from 1/2 taurus × indicusdonor cows was greater (P< 0.01) than from Bos indicuscows. The number of recoverable and viable oocytes and the number of embryos produced in vitrofrom Bos indicusdonors were higher than from Bos taurusfemales. Therefore, the highest oocyte yield and the greatest embryo production were obtained from 1/2 taurus × indicusfemales. This work was supported by In Vitro Brasil. [ABSTRACT FROM AUTHOR]