Superstimulation prior to the ovum pick-up improves the in vitro embryo production in nulliparous, primiparous and multiparous buffalo (Bubalus bubalis) donors.

The aim of this study was to evaluate the ovarian follicular population, the oocyte yield and the in vitro embryo production (IVEP) of nulliparous (NU), primiparous (PR) and multiparous (MU) buffalo donors submitted to the superstimulation with FSH prior to the ovum pick-up (OPU). A total of 54 buffalo donors (18 NU, 15 PR and 21MU) received an intravaginal progesterone device (1.0 g) plus estradiol benzoate [2.0 mg, intramuscular (im)] at random stage of the estrous cycle (Day 0) during the breeding season (autumn and winter). Buffaloes from different categories were then randomly allocated to one of two groups (Control or FSH), in a cross-over experimental design. Buffalo donors in the Control group received no further treatment, whereas buffalo donors in the FSH group received a total dosage of 200 mg im of FSH on Days 4 and 5, in four decreasing doses 12 h apart (57, 57, 43 and 43 mg). On Day 7, the progesterone device was removed and the OPU procedure was performed in both groups. The same semen was used across all replicates and donor category. Data were analyzed by the GLIMMIX procedure of SAS 9.4®. There was no interaction between FSH treatment and animal category for all analyzed variables. Furthermore, no differences between animal category (P = 0.73) and FSH treatment (P = 0.53) were observed regarding the total follicles aspirated. However, the FSH treatment increased (P < 0.001) the proportion of large (>10 mm; FSH = 16.2% and Control = 2.0%) and medium-sized follicles (6-10 mm; FSH = 36.3% and Control = 6.1%) available for the OPU procedure. The total of recovered oocytes was greater in NU than in MU, and PR were similar to NU and MU (P = 0.05). No effect of FSH treatment was observed (P = 0.85) for this variable. Buffalo donors treated with FSH had a greater viable oocytes rate (P = 0.03), blastocyst rate (P = 0.03) and embryo yield per OPU-IVEP session (P = 0.07), however, no category effects were observed for these variables. These results provided evidence that superstimulation with FSH increased the proportion of large and medium-sized follicles available for the OPU procedure. Consequently, the FSH treatment enhanced the proportion of viable oocytes for culture and resulted in greater blastocyst rates and embryo yield per OPU-IVEP session in all buffalo donors categories.

Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer.

Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis.