Housekeeping genes for RT-qPCR in ovine preimplantation embryos.

Housekeeping genes (HKG) are paramount for accurate gene expression analysis during preimplantation development. Markedly, quantitative reverse transcription polymerase chain reaction (RT-qPCR) in ovine embryos currently lacks HKGs. Therefore, we tested 11 HKGs for RT-qPCR normalization during ovine parthenogenetic preimplantation development. Seven HKGs reached the qPCR efficiency threshold (97.20-105.96%), with correlation coefficients ranging from -0.922 to -0.998 and slopes from -3.22 to -3.59. GeNorm ranked glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-binding protein (TBP) as the best HKG pair, while H3 histone, family 3A (H3F3A) was the third HKG. Relative gene expression was measured for zinc finger protein X-linked (ZFX) and developmental pluripotency-associated 3 (DPPA3) transcripts during ovine parthenogenetic preimplantation development. ZFX did not show any transcript abundance fluctuation among oocytes, cleavage-stage embryos, and morulae. DPPA3 transcript abundance was also similar among all developmental stages, therefore suggesting that it may not display a maternal gene expression profile. In silico analysis of ovine DPPA3 mRNA and protein showed high conservation to bovine orthologues. However, DPPA3 orthologues differed in regulatory motifs. In conclusion, GAPDH, TBP and H3F3A are stable HKGs in ovine parthenogenetic embryos and allow accurate RT-qPCR-based gene expression analysis.

Application of platelet-rich plasma in the in vitro production of bovine embryos.

The aim of this study was to replace fetal bovine serum (FBS) with platelet-rich plasma (PRP) for in vitro production of bovine embryos. The maturation media (TCM-199 medium) for the cumulus-oocyte complexes (COCs) was supplemented with 5% (G5) and 10% (G10) PRP or 10% FBS (GC). After fertilization, the presumed zygotes were randomly distributed in culture medium supplemented with 5% (G5) and 10% (G10) PRP or 10% FBS (GC) for 7 days. Cumulus cell (CC) expansion was greater (P < 0.05) in the GC (88.9%) group than in G5 (34.1%) or G10 (50.0%). Nevertheless, the expansion of CCs in group G10 was greater than in G5 (P < 0.05). Cleavage was higher in group G5 (86.0%) than in G10 (79.0%) (P < 0.05) and did not differ from group GC (82.0%). The percentage of blastocysts in group G5 (50.0%) was higher than in CG (40.2%) and G10 (34.2%) (P < 0.05). In addition, the number of blastomeres was higher in G5 (159.0 ± 4.18) than in GC (132.4 ± 4.11) and in G10 (127.1 ± 5.88) (P < 0.05). The addition of PRP into the oocytes maturation medium is not beneficial. On the other hand, the PRP addition into the embryo culture medium at 5% concentration is recommended where it increased the quantity and quality of in vitro-produced bovine embryos.

Use of retinoids during oocyte maturation diminishes apoptosis in caprine embryos.

Exposure of caprine oocytes and embryos to retinoids enhances embryonic development, but the mechanisms governing this phenomenon have not been characterised. The aim of the present study was to evaluate if the incidence of apoptosis is affected by the addition of retinyl acetate (RAc) and 9-cis-retinoic acid (RA) during in vitro maturation (IVM) of caprine oocytes. Embryonic development was recorded on days 3 and 8 post-fertilisation, and apoptosis was measured by caspase activity and DNA fragmentation (TUNEL assay). Control zygotes had lower capacity to cleave and reach the blastocyst stage (24.45 ± 2.32 and 5.32 ± 0.81, respectively) than those of RAc- (29.96 ± 1.62 and 7.94 ± 0.93, respectively) and RA-treated groups (30.12 ± 1.51 and 7.36 ± 1.02, respectively). Oocytes and blastocysts positive for TUNEL assay were more frequent, respectively, in the controls (8.20 ± 0.78, 8.70 ± 1.05) than in RAc (5.60 ± 0.52, 4.80 ± 0.51) and RA (6.40 ± 0.69, 5.40 ± 0.69). Caspase activity did not differ between control oocytes (7.20 ± 0.91), RAc (6.60 ± 0.68) and RA (7.30 ± 0.67), but it was reduced in RAc- (5.05 ± 0.62) and RA-treated blastocysts (5.75 ± 0.22) compared to controls (8.35 ± 0.71). These results indicate that the addition of retinoids during IVM increases the developmental potential of goat embryos with a concomitant reduction in apoptosis rates.