Lucio Pereira Rauber

Desenvolvimento embrionário in vitro de oócitos bovinos mantidos em líquido folicular ou TCM-hepes

In order to evaluate the effect of a transport medium on the rate of in vitro embryonic development, 1381 Cumulus-oocyte Complexes (COC) were obtained by aspiration of 2-8mm diameter follicles witch were randomly divided in 4 treatment groups. The Control group was formed by oocytes matured in modified TCM-199 for 24h, incubated at 39°C and 5,00% CO2 with saturated humidity. The group 1 (WB24h), included oocytes matured in 1.0mL tubes containing TCM-HEPES (5.95mg/mL), in water bath (WB) at 39°C for 24h. The group 2 (FFb6C18h), included oocytes kept in bovine follicular fluid (FFb) for 6h at 30°C followed by a period of 18h maturation under the same conditions as the Control group and with the oocytes maintained in FFb followed by 18h IVM under the same conditions as the group 1, group 3 (FFb6WB18h). Fertilization was performed in FERT-TALP for 18h. Zygotes were cultured in SOFaaci under mineral oil within gasified bags. The cleavage rate differed (P<0.05) between the Control and FFb6BM18h groups. However, there was no difference on the D7 and D9 blastocyst rates and on the percentage of blastocyst ecloded. It was concluded that it is possible to maintain the oocytes in FFb for 6h at 30°C before 18h IVM, or to proote the transport and maturation of the oocytes for 24h, in TCM-HEPES and water-bath at 39°C, without compromising embryonic development. The simplification of MIV showed in this experiment through of tubes (1.0mL) replete with TCM-HEPES and holding in water bath at 39°C, could be a viable and practice for the bovine programs of OPU/PIV.

Cultivo individual de blastocistos bovinos produzidos in vitro

In vitro produced bovine embryos were individually cultured from D7 to D9 to observe their development. Forty-nine blastocysts (early blastocyst, blastocyst and expanded blastocyst) were individually cultured, from D7 to D9 (D0= fertilization), in 50ml SOF medium plus 5% OCS. Between D7 and D8, blastocysts were cultured in straws (SC) or in plates (PC) and from D8 up to D9, they were cultured on plates. The ratio of early blastocyst, blastocyst, and expanded blastocyst advancing at least one stage of development were, respectively, 71%, 37%, 44% in PC and 100%, 66%, 36% in SC (P>;0.05). Overall development rates on D8 were not significantly different (P>;0.05) for PC (50%) and SC (60%). At D9, both culture systems produced similar (P>;0.05) hatching rates (29% and 24% for PC and SC, respectively). After fluorescent nuclei staining, the average number of cells counted in the hatched (182.7 vs. 202.8) and expanding (94.5 vs. 88.0) blastocysts were similar (P>;0.05) for PC and SC, respectively. In vitro produced bovine blastocysts can be individually cultured from D7 to D9, and the culture system employed (dishes or straws) has no effect on hatching rates and cell number of developed blastocysts.

Produção in vitro de embriões bovinos com soro de égua ou de vaca em estro com ou sem a adição de LH/FSH

A thousand two hundread and seventy-one oocytes were allocated in four treatments, in order to evaluate the influence of the addition of FSH and LH on in vitro production of bovine embryos, with oestrous cow serum (OCS) or mare´s serum obtained at the first day of estrus (OMS). In all treatments oocytes were matured with TCM199 + 5.95mg/ml Hepes and 0.025mg/ml sodium pyruvate and 2.2mg/ml of sodium bicarbonate, supplemented with 10% OMS (ES), 10% of OCS (VS), 10% of OMS + LHb + rFSHh (EH) and 10% of OCS + LHb + rFSHh (VH). All the treatments groups were matured in a controlled incubator at 39oC with 5% CO2 and saturated humidity for 22-24h. IVF was performed in TALP-FERT for 18-20h, with a pool of Bos taurus semen selected by swim up procedure in TALP-SPERM with 1x106 spermatozoa/ml dose, and cultured for 8 days under SOF medium + 5% OMS (ES and EH) or OCS (VS and VH). The 72% of cleavage rate obtained for treatment VH and 61% obtained in the VS group were significantly less (p<0.05) than treatments EH (80%), ES (80%). Blastocyst rates at D7 after insemination for treatments ES (32%), EH (28%) and VH (27%) were significantly superior than the 20% obtained on VS group (p<0.05). Evaluations at D9 demonstrated higher blastocyst rates in treatment ES (31%) and EH (29%) when compared with treatment VS (22%), but no differences were detected when compared to the VH (24%). These results suggest that the utilization of OCS, the supplementation with FSH and LH renders an increase on blastocyst rates and, that the utilization of MS, even without FSH and LH, shows similar results as those obtained with OCS plus hormones.

Desenvolvimento embrionário de oócitos bovinos mantidos em fluido folicular bovino de folículos de diferentes diâmetros

Bovine oocytes have been maintained in the follicular fluid to be transported and to increase their competence, before maturation. Eight hundred eighty-one (881) oocytes, aspirated from bovine slaughterhouse ovaries, were used to evaluate the effect of holding bovine oocytes in follicular fluid (FF) of bovine follicles of different diameters on the rate of embryo development. The oocytes were randomly distributed in four treatments with seven replicates each: The control group (n=217) was constituted by oocytes matured for 24h in modified TCM-199 with Estrus Mare Serum (EMS), pyruvate and rFSH-h in incubator with 5,00% CO2, 39°C and saturated humidity. In the FFsmall group (3 to 5mm follicles; n=216), the oocytes were held for 6h in follicular fluid at 30°C and matured for 18h in the same conditions of the Control-group. The oocytes of the FFmedium group (5,1-8mm follicles; n=226) and of the FFlarge group (>8,1mm follicles; n=222) were held in follicular fluid and matured like FFsmall. Fertilization was accomplished during 18h and, after this, the zygotes were cultured for 8 days in SOFaaci medium + 5,00% EMS in incubator at 39°C using plastic bags gasified with 5,00%CO2, 5,00%O2 and 90,00%N2. FFsmall oocytes produced a lower (P 0,05) between the groups. Follicular fluid of medium and large follicles could be used to hold for 6h at 30°C bovine oocytes before their maturation for 18h.

Influence of climatic conditions on in vitro production of bovine embryos

Temperature and rainfall were analyzed daily during six years to evaluate their influence on in vitro production of bovine embryos. Weekly replications (n=480) were performed on 14,778 ovaries collected at slaughterhouses. Cumulus oocyte complexes (n=19,180) were fertilized with a pool of Bos taurus taurus semen in one incubator with 5% CO2. Presumable zygotes were cultured in gasified plastic bags with 5% CO2, 5% O2, and 90% N2. In the first year, cleavage and embryo yield were 60.3% and 15.6%, respectively, being lower (P<0.05) than in the following years. Average cleavage rates were always lower in winter (P<0.0001), thus producing less embryos. Winter climatic conditions had a negative influence on in vitro production, when cleavage and embryo yield declined, possibly because of reduced availability and growth of native pasture.