Effects of Gonadotropins on In Vitro Maturation and of Electrical Stimulation on Parthenogenesis of Canine Oocytes

The objective of this study was to determine the effects of gonadotropins on in vitro maturation (IVM) and electrical stimulation on the parthenogenesis of canine oocytes. In experiment I, cumulus oocyte complexes were collected from ovaries at a random phase of the oestrus cycle and cultured on maturation medium treated with hCG or eCG for 48 or 72 h. There were no significant differences in the effects on the metaphase II (MII) rate between the hCG and eCG treatment groups over 48 h (5.4% vs 5.5%). The MII rate in the co-treatment group of hCG and eCG for 48 h was higher than in each hormone treated group (15.5%, p < 0.05). In experiment 2, the parthenogenetic effect on oocyte development, at various electrical field strengths (1.0, 1.5, 2.0 kV/cm DC) for 60 or 80 μs with a single DC pulse after IVM on the co-treatment of hCG and eCG, was examined. The rate of pronuclear formation (37.1%) in electrical activation at 1.5 kV/60 μs without cytochalasin B (CB) was higher than that of oocytes activated in the other groups (p < 0.05). However, we did not observe the cleavage stages. Also, CB did not influence parthenogenesis of canine oocytes. The results showed that the pronucleus formation rate, indicative of the parthenogenesis start point, could be increased by electrical stimulation. Therefore, these results can provide important data for the parthenogenesis of canine oocytes and suggest the probability of parthenogenesis in canines.     

Differential expression of insulin‐like growth factor family members in immature cumulus–oocyte complexes from dairy cows with different genotypes

It has been evident the improvement of in vitro embryo production (IVEP) in dairy cows. Nevertheless, it is known that differences in the number and quality of oocytes between taurine and zebu females impact the efficiency and economic viability of IVEP. As the insulin‐like growth factor (IGF) system is related to follicular and oocyte development, we aimed to quantify mRNA abundance of IGF system members and pregnancy‐associated plasma protein‐A (PAPPA) in the cumulus–oocyte complexes (COCs) of Gir, 1/2 Holstein × 1/2 Gir and Holstein cows. Four pools of 30 immature COCs from Gir, 1/2 Holstein × 1/2 Gir and Holstein cows were obtained by ovum pickup (OPU), and the oocytes and cumulus cells (CC) were mechanically separated and stored at ?80°C. Total RNA was extracted from pools of 30 oocytes and their respective CC. Expression of target genes was assessed by real‐time RT‐PCR. In oocytes, the abundance of IGFR1 mRNA was higher (p < .05) in Gir cows compared with the other breeds. In contrast, in CC, mRNA encoding IGF2 (p < .05), IGFR2 (p < .05) and IGFBP4 (p < .01) was higher in Holstein donors compared with Gir and 1/2 Holstein × 1/2 Gir cows. Additionally, the abundance of PAPPA mRNA was higher in oocytes (p < .001) and CC (p < .01) in Gir and 1/2 Holstein × 1/2 Gir cows compared with the Holstein donors. In conclusion, the higher abundance of PAPPA mRNA in the oocytes and CC from Gir and cross‐breed donors combined with the low expression of IGFBP4 in the CC suggests an enhancement of the bioavailability of IGF‐free when compared with Holstein COCs.     

Intracytoplasmic Glutathione Levels in Heifer Oocytes Cultured in different Maturation Media and its Effect on Embryo Development

The present study was carried out to study the effect of different maturation media on embryo development of heifer oocytes and on their glutathione (GSH) synthesis during in vitro maturation (IVM). Immature heifer oocytes were matured in parallel in one of four maturation media: (i) Tissue Culture Medium (TCM)-199 supplemented with 10 ng/ml of epidermal growth factor (EGF); (i) TCM-199 supplemented with 10 ng/ml of EGF plus 1 microg/ml of FSH; (iii) TCM-199 supplemented with 10% of foetal bovine serum (FBS) and (iv) TCM-199 supplemented with 10% of FBS plus 1 microg/ml of FSH. Cow oocytes were used as control and were matured in TCM-199 supplemented with 10 ng/ml of EGF. No differences were observed in blastocyst rate among the different heifer oocyte groups (8.8, 7.5. 8.4 and 6.8%, respectively) however, the percentage of blastocysts obtained from cow oocytes was significantly higher (30%; p < 0.01) than those obtained from heifer oocytes. De novo GSH synthesis during oocyte maturation of heifer and cow oocytes was detected. No significant differences in intracytoplasmic GSH levels were observed among the experimental heifer oocyte groups or between heifer and cow oocytes both before and after IVM. In conclusion, the blastocyst yield obtained from heifer oocytes was lower than that from cow oocytes and this fact could not be explained by significant differences in intracytoplasmic GSH contents of oocytes before or after IVM.     

Resveratrol–cyclodextrin complex affects the expression of genes associated with lipid metabolism in bovine in vitro produced embryos

Antioxidants have been widely used during in vitro production to decrease the negative effect of reactive oxygen species. It was reported that the complex resveratrol–methyl β‐cyclodextrin (RV ‐CD ) improves resveratrol's stability and bioavailability and increases its antioxidant activity. This study evaluates the effect of RV ‐CD during in vitro oocyte maturation (IVM ) or in vitro embryo culture (IVC ) on developmental competence and quantitative changes in gene expression of developmental important genes. In experiment 1, RV ‐CD was added to IVM media and maturation level, embryo development and oocytes, cumulus cells, and blastocysts gene expression by RT ‐qPCR were examined. In experiment 2, presumptive zygotes were cultured in SOF supplemented with RV ‐CD and embryo development and blastocysts gene expression by RT ‐qPCR were studied. A group without RV ‐CD (control^?) and a group with cyclodextrin (control⁺) were included. No differences were found in cleavage rate or blastocyst yield between groups. However, the expression of LIPE was higher in blastocysts derived from oocytes treated with resveratrol compared with control groups (p  <  .05). Blastocysts produced by IVC with resveratrol showed that RV ‐CD could modify the expression of genes related to lipid metabolism (CYP 51A1 , PNPLA 2 and MTORC 1 ) compared with control groups (p  < .05). RV ‐CD in the IVM and IVC media could reduce accumulated fat by increasing lipolysis and suppressing lipogenesis of blastocysts.     

Subtropical goats ovulate in response to the male effect after a prolonged treatment of artificial long days to stimulate their milk yield

The objectives of this study were to determine (i) if in subtropical goats that gave birth during mid‐December, the exposition to an artificial long‐day photoperiod consisting in only 14 hr of light per day can increase the milk yield and (ii) to test whether these females can respond to the male effect at the end of the prolonged photoperiodic treatment. In experiment 1, 17 lactating goats were maintained under natural short days (control group), while another 22 goats were maintained under artificial long days (treated group) consisting in 14 hr light and 10 hr darkness starting at day 10 of lactation. The continuous exposition to an artificial long‐day photoperiod produced an increase in the milk yield level during the first 110 days of lactation (time × treatment interaction; p = .01), while none of the milk components were modified due to the photoperiodic treatment (p > .05). In experiment 2, all control and treated anovulatory goats were submitted to the male effect using photostimulated males. All females showed oestrous behaviour within the first 10 days that were in contact with males (100% in both groups; p > .05). Thus, the latency to onset of oestrus did not differ between females from control (58.2 ± 3.0 hr) and treated (62 ± 4.6 hr) groups. Male exposition provoked ovulation independently if females were previously under long days or natural photoperiod (96 vs 100%, respectively; p = .79). It was concluded that exposure to 14 hr of light per day in subtropical goats that gave birth in late autumn stimulates milk yield without preventing the ovulation in response to the male effect at the end of the prolonged photoperiodic treatment.