N.A.T. Carvalho

Follicle deviation and ovulatory capacity in Bos indicus heifers

The objectives of Experiment 1 were to determine the interval from ovulation to deviation, and diameter of the dominant follicle (DF) and largest subordinate follicle (SF) at deviation in Nelore (Bos indicus) heifers by two methods (observed and calculated). Heifers (n = 12) were examined ultrasonographically every 12 h from ovulation (Day 0) to Day 5. The time of deviation and diameter of the DF and largest SF at deviation did not differ (P>0.05) between observed and calculated methods. Overall, deviation occurred 2.5+/-0.2 d (mean +/- S.E.M.) after ovulation, and diameters for DF and largest SF at deviation were 6.2+/-0.2 and 5.9 +/- 0.2 mm, respectively. Experiment 2 was designed to determine the size at which the DF acquires ovulatory capacity in B. indicus heifers. Twenty-nine heifers were monitored every 24 h by ultrasonography, from ovulation until the DF reached diameters of 7.0-8.4 mm (n=9), 8.5-10.0 mm (n=10), or >10.0 mm (n=10). At that time, heifers were treated with 25 mg of pLH and monitored by ultrasonography every 12 h for 48 h. Ovulation occurred in 3 of 9, 8 of 10, and 9 of 10 heifers, respectively (P<0.05). In summary, there was no significant difference between observed and calculated methods of determining the beginning of follicle deviation. Deviation occurred 2.5 d after ovulation when the DF reached 6.2 mm, and ovulatory capacity was acquired by DF as small as 7.0 mm.

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.

Ultrasonographic and endocrine aspects of follicle deviation, and acquisition of ovulatory capacity in buffalo (Bubalus bubalis) heifers

The objectives of this study were to determine the interval from ovulation to deviation and the diameter of the dominant (DF) and largest subordinate (SF) follicles at deviation in buffalo (Bubalus bubalis) heifers. Two methods of evaluation (observed vs. calculated) were used. FSH and LH profiles encompassing follicle deviation (Experiment 1), and the follicular diameter when the DF acquired ovulatory capacity (Experiment 2) were also determined. The time of deviation and the diameter of the DF and the largest SF at deviation did not differ between observed and calculated methods. Overall, follicle deviation occurred 2.6 ± 0.2d (mean ± SEM) after ovulation, and the diameters of the DF and SF at deviation were 7.2 ± 0.2 and 6.4 ± 0.2mm, respectively. No changes in plasma levels of FSH or LH were observed (P=0.32 and P=0.96, respectively). Experiment 2 was conducted in two phases according to the diameter of the DF during the first wave of follicular development at the time of LH challenge (25mg of pLH). In the first phase, follicles ranging from 5.0 to 6.0mm (n=7), 6.1 to 7.0mm (n=11), or 7.1 to 8.0mm (n=9) were used, and in the second phase, follicles ranging from 7.0 to 8.4mm (n=10), 8.5 to 10.0mm (n=10), or 10.1 to 12.0mm (n=9) of diameter were used. After the pLH treatment, the DF was monitored by ultrasonography every 12h for 48h. No ovulations occurred in heifers in the first phase. However, in the second phase, an effect of follicular diameter was observed on ovulation rate [7.0-8.4mm (0.0%, 0/10), 8.5-10.0mm (50.0%, 5/10), and 10.0-12.0mm (55.6%, 5/9)]. In summary, follicle deviation occurred 2.6d after ovulation in buffalo (B. bubalis) heifers, when the diameters of the DF and SF were 7.2 and 6.4mm, respectively. No significant changes in plasma concentrations of FSH or LH were detected. Finally, the acquisition of ovulatory capacity occurred when the DF reached 8.5mm in diameter.

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.

Follicle selection by ultrasonography and plasmatic characteristics and ovulatory capacity in buffaloes

Abstract The objectives of the present work were to determine follicle deviation assessed by ultrasonography and profile of plasmatic gonadotrophins, and ovulatory capacity in buffalo species, since these information are not available in the literature. The knowledge of follicular development and gonadotrophins profile during deviation and the follicular response to an exogenous ovulation inducer can be useful tools for follicular manipulation of estrous cycle.

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.

Ovulation and conception rates according intravaginal progesterone device and hCG or GnRH to induce ovulation in buffalo during the off breeding season

Abstract The objective of this study was to evaluate the effect of intravaginal progesterone device (P4; first or second use) of different ovulatory inductors on ovulation and conception rates in buffaloes during the off breeding season. Two hundred and forty two buffaloes were allocated in four groups and received P4 device of first or second use plus estradiol benzoate on Day 0 (D0). The P4 device was removed and a dose of PGF2α and eCG was administered on D9. On D11, buffaloes received hCG or GnRH and 16hs after the animals were inseminated. The ultrasound examination was performed on D0 to verify the ovarian status, from D9 to D14 to establish the moment of ovulation and on D40 for pregnancy diagnosis. Data were analyzed by ANOVA and Chi-square test. There was no effect of interaction. The ovulation and conception rate were similar for P4 device of first and second use, for hCG and GnRH. Results indicate that the use of P4 device for two times and the use of GnRH instead of hCG provide satisfactory ovulation and conception rate in buffalo during the off breeding season and might reduce the cost of the protocol for artificial insemination.

Use of different doses of rBST associated to a protocol for multiple ovulation and embryo transfer in buffalo (Bubalus bubalis)

Abstract The efficiency of different doses of rBST in a MOET protocol was verified in buffaloes. The animals received an intravaginal progesterone device (DIB) plus 2.0 mg of estradiol benzoate (Ric-BE, i.m.) and 0 mg, 250 mg or 500 mg of rBST. Ovarian follicular growth was stimulated by treatment with 200 mg of FSH in decreasing amount (12/12hs). Buffaloes received injections of PGF2α on D6 and on D7. The DIB was removed on D7. On D8, 24 hours after DIB removal, ovulation was induced with GnRH. The follicular emergence and the response to FSH treatment were evaluated by ultrasound on D4 and D8. The artificial insemination (AI) was performed 12 and 24 hours after the GnRH administration. The embryonic structures were collected 5.5 days after the first AI. Data were analyzed by ANOVA. No statistical differences were found between groups. The results indicated that rBST, associated to a MOET protocol at the different dosages used, does not to improve the efficiency of the technique in buffalo.

Detection of estrous behavior in buffalo heifers by radiotelemetry following PGF2α administration during the early or late luteal phase.

This study examined the usefulness of radiotelemetry for estrous detection in buffalo heifers and the impact of prostaglandin F2α (PGF2α) administration during the early or late luteal phase on estrous behavior and ovulatory follicle variables. Induction of estrus with PGF2α at a random stage of the estrous cycle was followed by the arbitrary division of heifers into groups receiving a second dose of PGF2α during either the early (n=33) or late (n=17) luteal phase (6-9 or 11-14 days after estrus, respectively) for the induction of synchronized estrus. The electronic detection of synchronized estrus by radiotelemetry was confirmed using ultrasonography every 6h until ovulation. Radiotelemetry was 90% efficient and 100% accurate for estrous detection. Intervals between the PGF2α dose and the beginning of synchronized estrus (40.7 ± 10.9 vs. 56.7 ± 12.8h) or ovulation (70.0 ± 11.3 vs. 85.6 ± 12.5h) were shorter (P<0.05) for heifers receiving PGF2α during the early luteal phase. PGF2α administration during the early or late luteal phase produced similar (P>0.05) results for the duration of estrus, the intervals from the beginning or end of estrus to ovulation, the number and duration of mounts per estrus, the duration of mounts, the diameter of the ovulatory follicle and the luteal profile on day 5 after estrus. In conclusion, radiotelemetry was a suitable tool for the efficient and accurate detection of estrus in buffalo heifers. Treatment with PGF2α during the early luteal phase had a shorter interval to synchronized estrus and ovulation; however, estrous behavior, ovulatory follicle dynamics and subsequent luteal activity were similar following PGF2α administration during the early or late luteal phase.