RESUMO.-[Acúmulo de lipídios intracitoplasmáticos, desenvolvimento e criotolerância de embriões bovinos produzidos in vitro e tratados com diferentes concentrações de forskolin antes da vitrificação.] Os embriões foram produzidos a partir de ovários obtidos em abatedouro e foram alocados em quatro grupos experimentais. Nos grupos tratados, o forskolin foi adicionado ao meio de cultivo in vitro no dia 6 do cultivo e os embriões foram incubados durante 24 horas com uma das seguintes concentrações: 2,5μM (grupo Forsk 2,5), 5,0μM (grupo Forsk 5,0) ou The aim of the present study was to evaluate the intracytoplasmic lipid content, development and cryotolerance of in vitro-produced bovine embryos treated with different concentrations of forskolin before vitrification. Embryos were produced from abattoir-derived ovaries and allocated into four groups. In the treatment groups, forskolin was added to the in vitro culture medium on Day 6 and incubated for 24 hours in one of the following concentrations: 2.5μM (Forsk 2.5 group), 5.0μM (Forsk 5.0 group) or 10.0μM (Forsk 10.0 group). Embryos from the control group were cultured without forskolin. On Day 7 of culture, the expanded blastocysts were stained with the lipophilic dye Sudan Black B for determination of the intracytoplasmic lipid content or were cryopreserved via the Vitri--Ingá ® procedure. Although there were no significant differences (P>0.05) in the blastocyst rates between the Control group (44.9%) and the other treatments, the embryo production was lower (P<0.05) in Forsk 10.0 group (38.8%) compared to Forsk 2.5 (50.5%) and Forsk 5.0 (54.7%) groups. The intracytoplasmic lipid content (expressed in arbitrary units of pixels) in blastocysts from the Control group (1.00±0.03) was similar (P>0.05) to that found in Forsk 2.5 (0.92±0.03) and Forsk 10.0 groups (1.06±0.03) groups; however the lipid accumulation in blastocysts from Forsk 5.0 group (0.82±0.04) was lower than in the Control group (P<0.05). Based on these results, Forsk 5.0 treatment was tested for cryotolerance and it was observed that the blastocoel re-expansion rate evaluated 24 hours after warming was greater (P<0.05) in Forsk 5.0 group (72.2%) compared to the Control group (46.2%). In conclusion, pre-treatment with forskolin at a concentration of 5.0 μM for 24 hours before vitrification is effective in reducing the intracytoplasmic lipid content and, consequently, improves cryotolerance of IVP bovine embryos.
The aim of this study was to evaluate if blastocysts arising from in vitro culture of Grade 3 bovine morulae produced in vivo can promote acceptable pregnancy rates when transferred into recipients. Embryos of different stages and qualities were recovered from superovulated Bos taurus and B. indicus donors. Grade 3 morulae were cultured in either Holding Plus or TCM-199 (supplemented with 10% bovine fetal serum) media for 24 h at 38.5 degrees C. After this culture period, the resulting blastocysts were morphologically classified (Grades 1, 2 and 3) and transferred into recipients previously synchronized with the donors. Non-cultured Grades 1 and 3 morulae were used as control. Pregnancy diagnosis was carried out 60 days after embryo transfer and the data were analysed by logistic regression, considering variables, such as embryo quality (Grade), donor breed, culture medium, donor-recipient synchrony and seasonality. Embryo quality was the only variable, showing significant effect on the pregnancy rate. Pregnancy rates for non-cultured Grade 1 and 3 morulae, and blastocysts arising from cultured Grade 3 morulae were 58.1% (n = 31), 17.1% (n = 35) and 51.1% (n = 47), respectively (p < 0.05). There were no statistical differences between non-cultured Grade 1 morulae and cultured blastocysts. Pregnancy rates for Grades 1 (65.0%) and 2 (60.0%) were higher than Grade 3 (29.4%) cultured blastocysts (p < 0.05). It was concluded that short-term in vitro culture is a very convenient method of identifying morphologically low quality morulae with higher chances of continuing development after the transfer into recipients.
In the present study, the superstimulatory protocol coined P-36 (Nogueira et al. 2007 Theriogenology 67, 655–660) was modified in order to test if replacement of pFSH by eCG in the last day of superstimulatory treatment would improve follicular growth, ovulation rate, and embryo yield. Nelore cows (n = 20) were randomly allocated to two groups: P-36/LH48 and P-36/LH48/eCG. Each female received both treatments in a cross-over design; the first experimental period was in October (Spring 2006, both groups) and the second in February (Summer 2007, both groups). At a random stage of the estrous cycle (D0), animals received a progesterone intravaginal device (DIB�, 1.0 g; Syntex S.A., Buenos Aires, Argentina) plus estradiol benzoate (EB, Estrogin�, 2.5 mg IM; Farmavet, Sao Paulo, Brazil). The animals were superstimulated with pFSH (Folltropin-V�, Bioniche Animal Health, Ontario, Canada), administered twice daily in decreasing doses of 53.2, 39.9, 26.6, and 13.3 mg (total dose = 133 mg), from Day 5 to Day 8, except the P-36/LH48/eCG group, where the last two doses of pFSH were replaced by two doses (200 IU) of eCG (Novormon�, Syntex, Buenos Aires, Argentina). All cows were treated with D-cloprostenol (150 µg IM;Veteglan�, Calier, Barcelona, Spain) on Day 7 at 7:00 h, and DIBs� were removed 36 h after PGF2alpha administration. On Day 9, ovulation was induced by pLH administration (Lutropin�, 12.5 mg IM; Bioniche Animal Health) at 7:00 h. The animals were inseminated at 12 and 24 h after pLH administration, without estrus detection. Embryos were recovered on Day 16 or 17. Data were analyzed by ANOVA (Proc Mixed, SAS; SAS Institute, Inc., Cary, NC, USA), and the difference was considered significant when P < 0.05 or not significant (NS). Results for animals from groups P-36/LH48 and P-36/LH48/eCG were compared and are reported in this order. The number of follicles with diameter larger than 6 mm at the time of pLH administration (15.25 � 2.06 and 21.05 � 2.76; P < 0.01), the ovulation rate observed up to 48 h after pLH administration (77.7 � 5.6 and 83.9 � 2.6; NS), the total number of oocytes/embryos recovered (6.65 � 1.18 and 10.0 � 1.48; P < 0.03), the number of embryos recovered (6.05 � 1.24 and 8.35 � 1.30; NS), and the number of viable embryos (5.10 � 1.10 and 7.30 � 1.20; NS) are reported. The embryo quality (excellent, good, fair, and poor) was NS among groups P-36/LH48 and P-36/LH48/eCG. It is concluded that replacement of pFSH by eCG, on the last day of the superstimulatory protocol P-36, was beneficial since there was a significant increase in the number of follicles (>6 mm at the time of pLH administration) and the total number of structures recovered. Experiments are in progress to confirm these beneficial effects of eCG on the P-36 protocol. This work was supported by FAPESP (Sao Paulo, Brazil). A. C. Z. Barcelos received a fellowship from CAPES (Brazil).
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