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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.

Two experiments were conducted to evaluate the effects of equine chorionic gonadotropin (eCG) treatment on ovarian follicular response, luteal function, and pregnancy in buffaloes subjected to a timed artificial insemination (TAI) protocol during the nonbreeding season. In experiment 1, 59 buffalo cows were randomly assigned to two groups (with and without eCG). On the first day of the synchronization protocol (Day 0), cows received an intravaginal progesterone (P4) device plus 2.0 mg estradiol benzoate im. On Day 9, the P4 device was removed, all cows were given 0.150 mg PGF(2α) im, and half were given 400 IU eCG im. On Day 11, all cows were given 10 μg of buserelin acetate im (GnRH). Transrectal ultrasonography of the ovaries was performed on Days 0 and 9 to determine the presence and diameter of the largest follicle; between Days 11 and 14 (12 hours apart), to evaluate the dominant follicle diameter and the interval from device removal to ovulation; and on Days 16, 20, and 24 to measure CL diameter. Blood samples were collected on Days 16, 20, and 24 to measure serum P4. In experiment 2, 256 buffaloes were assigned to the same treatments described in experiment 1, and TAI was performed 16 hours after GnRH treatment. Pregnancy diagnosis was performed by ultrasonography 30 days after TAI. Treatment with eCG increased the maximum diameter of dominant follicles (P = 0.09), ovulation rate (P = 0.05), CL diameter (P = 0.03), and P4 concentrations (P = 0.01) 4 days after TAI, and pregnancy per AI (52.7%, 68/129 vs. 39.4%, 50/127; P = 0.03). Therefore, eCG improved ovarian follicular response, luteal function during the subsequent diestrus, and fertility for buffalo subjected to a TAI synchronization protocol during the nonbreeding season.

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Our expanding knowledge of ovarian function during the buffalo estrous cycle has given new approaches for the precise synchronization of follicular development and ovulation to apply consistently assisted reproductive technologies (ART). Recent synchronization protocols are designed to control both luteal and follicular function and permit fixed-time AI with high pregnancy rates during the breeding (autumn-winter) and nonbreeding (springsummer) seasons. Additionally, allow the initiation of superstimulatory treatments at a self-appointed time and provide opportunities to do fixed-time AI in donors and fixed-time embryo transfer in recipients. However, due the scarce results of in vivo embryo recovery in superovulated buffaloes, the association of ovum pick-up (OPU) with in vitro embryo production (IVEP) represents an alternative method of exploiting the genetics of high yeld buffaloes. Nevertheless, several factors appear to be critical to OPU/IVEP efficiency, including antral follicle population, follicular diameter, environment, farm and category of donor. This review discusses a number of key points related to the manipulation of ovarian follicular growth to improve assisted reproductive technologies in buffalo.

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