J. G. Soares

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.

Timing of insemination using sex-sorted sperm in embryo production with Bos indicus and Bos taurus superovulated donors.

Two experiments were designed to evaluate the effect of different insemination times (12 and 24h or 18 and 30h) and different types of semen (sex-sorted or non-sorted sperm) on embryo production in Nelore (Bos indicus) and Holstein (Bos taurus) superstimulated donors. In the first experiment, hormonal superstimulation of ovarian follicular development in Nelore donors (n=71) was performed in randomly allocated animals to one of the three treatment groups, and they were inseminated at 12 and 24h after an ovulatory stimulus with pLH treatment was applied, either with sex-sorted (4.2×10(6) sperm/insemination; S12/24; n=17) or non-sorted sperm (20×10(6) sperm/insemination; NS12/24; n=18), or they were inseminated at 18 and 30h using sex-sorted sperm (4.2×10(6) sperm/insemination; S18/30; n=19). A greater number of transferable embryos were found when sex-sorted sperm was used to inseminate the animals at 18 and 30h (4.5±3.0) compared to insemination at 12 and 24h (2.4±1.8; P<0.001). However, a greater embryo production (6.8±2.6) was obtained with non-sorted sperm. In the second experiment, the same insemination times and semen types were used in lactating high-production Holstein cows (n=12). A crossover design was employed in this trial. A lesser embryo production (P=0.007) was found in Holstein donors that were inseminated using sex-sorted sperm at 12 and 24h (4.6±3.0) compared to non-sorted sperm (8.7±2.8). However, intermediate results were obtained when the inseminations with sex-sorted sperm were performed at 18 and 30h (6.4±3.1). These results supported the current hypothesis that it is possible to improve embryo production using sex-sorted sperm in B. indicus and B. taurus superstimulated donors when the inseminations are performed near the same time as time-synchronized ovulations. However, the embryo production for timed artificial insemination (TAI) with sex-sorted sperm was still less than the production with non-sorted sperm.

Different circulating progesterone concentrations during synchronization of ovulation protocol did not affect ovarian follicular and pregnancy responses in seasonal anestrous buffalo cows

Three experiments were designed to evaluate the effect of different circulating progesterone (P4) concentrations during synchronization of ovulation protocol for timed artificial insemination of seasonal anestrous buffalo cows. In the first trial, ovariectomized cows were randomly allocated into one of three groups: using new P4 devices (G-New; n = 8), using devices previously used for 9 days (G-Used1x; n = 8), and using devices previously used for 18 days (G-Used2x; n = 8). The P4 device was maintained for 9 days, and the circulating P4 concentration was measured daily. The circulating P4 concentrations during the P4 device treatment were the lowest for G-Used2x (1.10 ± 0.04 ng/mL), intermediate for G-Used1x (1.52 ± 0.05 ng/mL), and the highest for G-New (2.47 ± 0.07 ng/mL; P = 0.001). In the second trial, 31 anestrous cows had their ovarian follicular dynamics evaluated after receiving the treatments described previously (G-New [n = 10], G-Used1x [n = 11], and G-Used2x [n = 10]). At insertion of the P4 device, cows were administered 2.0 mg of estradiol benzoate. Nine days later, the P4 device was removed and cows were administered 0.53 mg of cloprostenol sodium plus 400 IU of eCG. Forty-eight hours after P4 device removal, 10 μg of buserelin acetate was administered. There were no differences among the groups (G-New vs. G-Used1x vs. G-Used2x) in diameter of the largest follicle at P4 device removal (9.0 ± 0.8 vs. 10.1 ± 0.9 vs. 8.6 ± 0.8 mm; P = 0.35), in interval from P4 device removal to ovulation (77.1 ± 4.5 vs. 76.5 ± 4.7 vs. 74.0 ± 4.4 hours; P = 0.31), or in ovulation rate (80.0% vs. 81.8% vs. 60.0%; P = 0.51). In experiment 3, 350 anestrous cows were randomly assigned into one of the three treatments described previously (G-New, n = 111; G-Used1x, n = 121; G-Used2x, n = 118) and received a timed artificial insemination for 16 hours after buserelin treatment. The 30-day pregnancy rates did not differ among groups (55.9% vs. 55.4% vs. 48.3%; P = 0.39). Thus, the low circulating P4 concentrations released from a used P4 device efficiently control the ovarian follicular growth and had no detrimental effect on the pregnancy rates of the seasonal anestrous buffalo cows.

Strategies to overcome seasonal anestrus in water buffalo

Reproductive seasonality in buffalo (Bubalus bubalis) is characterized by behavioral, endocrine, and reproductive changes that occur over distinct periods of the year. During the nonbreeding season (spring and summer), the greater light-dark ratio (long days) suppresses estrus behavior and the occurrence of ovulation. Anestrous buffaloes have insufficient pulsatile of LH to support the final stages of follicular development, and subsequently, estrus behavior and ovulation do not occur, limiting reproductive efficiency, especially in artificial insemination (AI) programs. A number of therapeutic strategies designed to synchronize follicular wave emergence and ovulation have allowed for the use of AI throughout the year, overcoming seasonal anestrus in buffalo. These therapies also improve reproductive performance by increasing the service rate and pregnancy per AI in buffalo herds, regardless of reproductive seasonality.

Timed artificial insemination should be performed early when used norgestomet ear implants are applied for synchronizing ovulation in beef heifers.

The present study evaluated the effect of the type of norgestomet ear implant (new vs. used) on the ovarian follicular response (experiment 1) and pregnancy per artificial insemination (AI) (P/AI; experiment 2) of beef heifers subjected to an estradiol plus progestin timed artificial insemination (TAI) program. In experiment 1, 57 cyclic beef heifers were randomly assigned to one of two groups according to the type (new or previously used for 9 days) of norgestomet ear (NORG) implant. At the time of NORG implant insertion, the heifers were treated with 2 mg of intramuscular estradiol benzoate. Eight days later, the NORG implants were removed, and the heifers received an intramuscular administration of 150 μg of d-cloprostenol, 300 IU of equine chorionic gonadotropin, and 0.5 mg of estradiol cypionate. The heifers had their ovaries scanned every 12 hours from the time of NORG implant removal to 96 hours after verifying the occurrence and timing of ovulation. No difference (P = 0.89) was observed in the ovulation rates between the two treatments (new = 80.0%; 24/30 vs. used = 81.5%; 22/27). However, the heifers treated with a used NORG implant had (P = 0.04) higher proportion (36.4%; 8/22) of early ovulation (between 36 and 48 hours after NORG implant removal) compared with the heifers treated with a new NORG implant (8.3%; 2/24). In experiment 2, at the beginning of the synchronization protocol, 416 beef heifers were randomly assigned into two groups, as described in the experiment 1. Two days after the NORG implant removal, the heifers were reassigned to be inseminated at 48 or 54 hours after NORG implant removal. There was an interaction (P = 0.03) between the type of NORG implant and the timing of TAI on P/AI. The timing of insemination only had an effect (P = 0.02) on the P/AI when the heifers were treated with a used NORG implant [(TAI 54 hours = 41.9% (44/105) vs. TAI 48 hours = 58.6% (58/99)]. In conclusion, beef heifers synchronized with a used NORG implant plus estradiol exhibited a higher proportion of earlier ovulations, and TAI in these heifers should be performed 48 hours after removal of used NORG implants.