Efeito da utilização de diferentes diluidores para a produção in vitrode embriões bovinos
Within an in vitro production controlled system, bulls differ with respect to their semen potential in generating embryos when the variables of maternal effect are minimized (Marquant-le-Guienne and Humblot 1992 Ann. Zootech. 41, 361-370). We have tested the hypothesis that even with this variation among bulls, there is also an intra-bull variation, according to the frozen semen batch used in the in vitro fertilization, identified with the date of ejaculate and its freezing. In an embryo commercial production system, over 12 months, 10 619 viable oocytes were obtained by ultrasound-guided follicular aspiration from 642 Nelore cows (Bos indicus). The oocytes were matured in vitro for 24 h in TCM-199 supplemented with 0.5 μg mL-1 FSH, 50 μg mL-1 LH, and 10% fetal bovine serum. They were then inseminated for 18 hours in IVF-TALP medium, using the semen from 4 bulls (A to D) subdivided into 4 frozen batches (I to IV) and selected by 45/90% Percoll gradient. Putative zygotes surrounded in cumulus cells were transferred in CR2aa medium drops (Rosenkrans and First 1994 J. Anim. Sci. 72, 434-437) for 163 h at 39°C in a humidified atmosphere of 5% CO2 in air. The oocyte distribution, the total number of blastocysts, and the embryo development rate by each bull and respective batch are described in Table 1. The chi-square test was measured with a significance level of P < 0.05 and showed that there is a difference between the used batches of each bull regarding the development rate of blastocysts 163 h after IVF Therefore, there is intra-bull variation in the ability to develop in vitro embryos according to the batch of frozen semen. Table 1.Viable oocytes (VO), total blastocysts (TB), and embryo development rate (%E) by bull and batch used in IVF
Within an in vitro production system, bulls differ with respect to their semen potential in generating embryos when the variables of maternal effects are minimized (Marquant-le-Guienne and Humblot 1992 Ann. Zootech. 41, 361-370). We have tested the hypothesis that even with this variation among bulls, there is also an intra-bull variation among frozen sexed semen batches (Sexing Technologies, Navasota, TX, USA; personal communication) when used with IVF. In an embryo commercial production system, 5058 viable oocytes obtained by ovum pick-up with ultrasound from 193 Nelore cows (Bos indicus) over a 12-month period were matured in vitro for 24 h in TCM-199 (Gibco, Life Technologies, Carlsbad, CA, USA) containing 0.5 μg mL-1 FSH (Bioniche Animal Health, Belleville, Ontario, Canada), 50 μg mL-1 LH (Intervet, Boxmeer, the Netherlands) and 10% fetal bovine serum (Gibco). Mature oocytes were inseminated in vitro for 18 h in IVF-Talp medium (BSA-FAF 6 mg mL-1; 10 ng of heparin, Sigma, France), using 3 different batches (I, II, III) of frozen-thawed sexed semen from 4 bulls (A, B, C, D), separated with a Percoll gradient (45:90; Sigma, St-Quentin Fallavier, France). Putative zygotes surrounded in cumulus cells were transferred in CR2aa medium droplets (Rosenkrans and First 1994 J. Anim. Sci. 72, 434-437) with 3 mg mL-1 BSA-FV (Sigma) for 163 h in a humidified incubator at 39°C, with an atmosphere of 5% CO2 in air. Total number of oocytes, total number of blastocysts, and embryonic developmental rates for each bull and respective batch are reported in Table 1. The chi-square test was measured with a significance level of P < 0.05 and showed that there is difference between the batches used with respect to developmental rates of blastocysts. Therefore, there is intra-bull variation in the ability to produce in vitro embryos according to the batch of frozen sexed semen. Table 1.Viable oocytes (VO), total blastocysts (TB), and embryo development rate (%E) by bull and batch used in IVF
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