E. H. Madureira

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.

Ovarian follicular dynamics during the estrous cycle in buffalo ( Bubalus bubalis )

The growth, selection, regression and ovulation of ovarian follicles was ultrasonically monitored in 30 Murrah buffalo throughout a spontaneous estrous cycle during the breeding season (autumn). Examinations revealed that follicular growth during the estrous cycle occurs in waves; the buffalo showed 1-wave (3.3%, n = 1), 2-wave (63.3%, n = 19) or 3-wave (33.3%, n = 10) follicular growth. The first wave began at 1.00, 1.16 +/-0.50 and 1.10 +/- 0.32 d in buffalo with 1, 2 and 3 waves, respectively (ovulation = Day 0). The second wave appeared at 10.83 +/- 1.09 and 9.30 +/- 1.25 d (P < 0.01) for the 2 and 3 wave cycle animals, respectively. The third wave started at 16.80 +/- 1.22 d. Structural persistence of the first dominant follicle was longer in the 2- than 3-wave cycles (20.67 +/- 1.18 vs 17.90 +/- 3.47 d ; P < 0.05). The duration of the growth and static phases of the first dominant follicle differed between the 2 and 3 wave cycles (P < 0.05), whereas there were no differences in linear growth rates (cm/d). Two and three wave cycles differed (P < 0.05) with respect to the maximum diameter of both the first dominant follicle (1.51 +/- 0.24 vs 1.33 +/- 0.18 cm) and the ovulatory follicles (1.55 +/- 0.16 vs 1.34 +/- 0.13 cm). No relationship was found between dominant follicle development and the presence of either a CL or a previous dominant follicle in either ovary. Two and three wave cycles also differed with respect to the mean length of intervals between ovulation (22.27 +/- 0.89 vs 24.50 +/- 1.88 d; P < 0.01) and the mean length of luteal phases (10.40 +/- 2.11 vs 12.66 +/- 2.91 d; P < 0.05). These results demonstrate that buffalo have estrous cycles with 1, 2 or 3 follicular waves; that 2-wave cycles are the most common; and that the number of waves in a cycle is associated with the luteal phase and with estrous cycle length.

Equine chorionic gonadotropin and gonadotropin-releasing hormone enhance fertility in a norgestomet-based, timed artificial insemination protocol in suckled Nelore (Bos indicus) cows

Two experiments were conducted to investigate the effects of equine chorionic gonadotropin (eCG) at progestin removal and gonadotropin-releasing hormone (GnRH) at timed artificial insemination (TAI) on ovarian follicular dynamics (Experiment 1) and pregnancy rates (Experiment 2) in suckled Nelore (Bos indicus) cows. Both experiments were 2x2 factorials (eCG or No eCG, and GnRH or No GnRH), with identical treatments. In Experiment 1, 50 anestrous cows, 134.5+/-2.3 d postpartum, received a 3mg norgestomet ear implant sc, plus 3mg norgestomet and 5mg estradiol valerate im on Day 0. The implant was removed on Day 9, with TAI 54 h later. Cows received 400 IU eCG or no further treatment on Day 9 and GnRH (100 microg gonadorelin) or no further treatment at TAI. Treatment with eCG increased the growth rate of the largest follicle from Days 9 to 11 (means+/-SEM, 1.53+/-0.1 vs. 0.48+/-0.1mm/d; P<0.0001), its diameter on Day 11 (11.4+/-0.6 vs. 9.3+/-0.7 mm; P=0.03), as well as ovulation rate (80.8% vs. 50.0%, P=0.02), whereas GnRH improved the synchrony of ovulation (72.0+/-1.1 vs. 71.1+/-2.0 h). In Experiment 2 (n=599 cows, 40 to 120 d postpartum), pregnancy rates differed (P=0.004) among groups (27.6%, 40.1%, 47.7%, and 55.7% for Control, GnRH, eCG, and eCG+GnRH groups). Both eCG and GnRH improved pregnancy rates (51.7% vs. 33.8%, P=0.002; and 48.0% vs 37.6%, P=0.02, respectively), although their effects were not additive (no significant interaction). In conclusion, eCG at norgestomet implant removal increased the growth rate of the largest follicle (LF) from implant removal to TAI, the diameter of the LF at TAI, and rates of ovulation and pregnancy rates. Furthermore, GnRH at TAI improved the synchrony of ovulations and pregnancy rates in postpartum Nelore cows treated with a norgestomet-based TAI protocol.

Comparison of two Ovsynch protocols (GnRH versus LH) for fixed timed insemination in buffalo (Bubalus bubalis)

We evaluated the efficiency of replacing GnRH with LH in the ovulation synchronization protocol in buffaloes. Buffaloes received GnRH on Day 0, (Buserelin; Conceptal, 20 microg), PGF2alpha (Luprostiol; Prosolvin, 15 mg) on Day 7 and GnRH (Buserelin; Conceptal, 10 microg; Group 1) or porcine LH (LH; Lutropin-V, 12.5 mg; Group 2) on Day 9. In Experiment 1, we studied the follicular dynamics of 30 buffaloes (Group 1, n = 15 and Group 2, n = 15). We performed ultrasonography every 12 h from Days 0 to 2, then on Day 7 and then every 6 h from the time of GnRH or LH treatment (Day 9) until the time of ovulation. All females not ovulating by 48 h after the second GnRH or LH injection were considered as nonresponders. In Experiment 2, we evaluated 305 buffaloes (Group 1, n = 154; Group 2, n = 151), using the same two treatments studied in Experiment 1. We also recorded and evaluated aspects like parity, lactational status, the presence of mucus, and uterine tone at the time of artificial insemination (Al). In Experiment 1, ovulation rate after the first GnRH was 86.6% (26/30). Ovulation rates were 93.3% (14/15; Group 1) after the second dose of GnRH and 93.3% (14/15) after LH (Group 2). Ovulation occurred 36.4+/-10.4 h after the first GnRH. The interval for treatment to ovulation was 26.5+/-9.6 h for buffaloes treated with GnRH (Group 1) and 24.4+/-7.9 h for buffaloes treated with LH (Group 2); the time of ovulation did not differ statistically between the two groups (GnRH versus LH; P > 0.05). In Experiment 2, conception rates of the animals AI in the field were 56.5% (Group 1) and 64.2% (Group 2), respectively (P = 0.08). The response to the treatment with LH was not different to the treatment with GnRH; however, multiparous buffaloes had higher conception rates than the primiparous buffaloes in both groups (P > 0.05). Buffaloes with mucus at the time of AI in Group 2 had higher conception rates than the buffaloes that had mucus in Group 1 (P < 0.05). Uterine tone and lactational status did not influence conception rates (P > 0.05). In summary, the results showed that both treatments resulted in synchronization of ovulation and acceptable conception rates. Therefore, the exogenous injection of LH can substitute the GnRH injections in the Ovsynch program in buffaloes.

Evaluation of synchronization of ovulation for fixed timed insemination in buffalo (Bubalus bubalis)

The objective of this study was to evaluate the efficiency of synchronization ovulation protocol (GnRH/PGF2a/GnRH) for fixed timed insemination in buffalo. In Experiment 1, thirty-three female buffaloes with 60 days postpartum, were divided in two groups for evaluation of the follicular dynamics (Group 1, n = 16, 10mg of GnRH/15mg of PGF2a/10mg of GnRH; Group 2, n = 17, 20mg of GnRH/15mg of PGF2a/10mg of GnRH). Ultrasonography exams were performed every 12 hours to verify the ovarian response to the first GnRH injection. Another evaluations were performed on PGF2a injection and second GnRH injection. After last hormonal injection, ultrasonography exams were performed every 6 hours to verify the moment of the ovulation. Blood samples were collected on Days 0, 7 and 9 to evaluate the progesterone concentrations at the moment of the hormonal injections. In Experiment 2, the efficiency of the synchronization of ovulation was verified for fixed timed insemination (16 hours after second GnRH injection, n=1053), during the favorable and unfavorable reproductive season, in the years of 1998 and 1999. Animals received 20mg of GnRH/15mg of PGF2a/10mg of GnRH. Besides, also recorded and evaluated aspects like body condition score, parity, postpartum period, year and the reproductive season (favorable or unfavorable season), on the conception rate. In Experiment 1, it was verified that 33.0 ± 8.3h after the first GnRH injection, 60.60% of the animals ovulated (Group 1 = 70.5 vs. Group 2 = 50.00%; P>0.05). The dose of GnRH (10mg vs. 20mg) and the progesterone concentration did not interfere in ovulation in the beginning of the treatment (P>0.05). Animals that ovulated (n = 20) after the first GnRH injection presented larger follicular diameter (P 0.05). In Experiment 2, it was observed conception rate of 45.40% (n = 1053). Animals synchronized during the favorable reproductive season presented better conception rate than the unfavorable season (48.80% vs. 6.90%; P 0.05). In summary, the Experiments indicated that the use of synchronization of the ovulation for fixed timed insemination, during the favorable reproductive season, presents satisfactory results in buffalo and could still be better when used multiparous buffaloes with good body conditon score in the beginning of the treatment.

LC-MS/MS quantitation of plasma progesterone in cattle

Quantitation of progesterone (P(4)) in biological fluids is often performed by radioimmunoassay (RIA), whereas liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been used much less often. Due to its autoconfirmatory nature, LC-MS/MS greatly minimizes false positives and interference. Herein we report and compare with RIA an optimized LC-MS/MS method for rapid, efficient, and cost-effective quantitation of P(4) in plasma of cattle with no sample derivatization. The quantitation of plasma P(4) released from three nonbiodegradable, commercial, intravaginal P(4)-releasing devices (IPRD) over 192 h in six ovariectomized cows was compared in a pairwise study as a test case. Both techniques showed similar P(4) kinetics (P > 0.05) whereas results of P(4) quantitation by RIA were consistently higher compared with LC-MS/MS (P < 0.05) due to interference and matrix effects. The LC-MS/MS method was validated according to the recommended analytical standards and displayed P(4) limits of detection (LOD) and quantitation (LOQ) of 0.08 and a 0.25 ng/mL, respectively. The high selective LC-MS/MS method proposed herein for P(4) quantitation eliminates the risks associated with radioactive handling; it also requires no sample derivatization, which is a common requirement for LC-MS/MS quantitation of steroid hormones. Its application to multisteroid assays is also viable, and it is envisaged that it may provide a gold standard technique for hormone quantitation in animal reproductive science studies.

Superovulação de novilhas da raça Nelore com diferentes doses de FSH/LH e congelação de embriões pelo método one-step com etilenoglicol

The objetive of this study was to identify the better dose between 300 (n = 20), 400 (n = 21) and 500 IU (n = 21) of FSH/LH to stimulate Nelore heifers. The superovulation treatment started on day 10 (D0 = estrous) of the estrous cycle in 8 decreasing aplications for 4 days. The embryo recovery was achieved on day 6.5 after the first artificial insemination. The superovulatory response for 300, 400 and 500 IU FSH/LH was follicles (15.12, 15.76 and 14.94); corpus luteum (10.68, 11.55 and 10.81) and transferable embryos (5.20, 1.81 and 2.76). The 300 IU of FSH/LH group presented the best results in regard to transferable embryos. The transferable embryos were cryopreserved by one-step method with 1.5 M of ethylene-glycol, resulting in 8 pregnancies (7.5%) of 106 embryos transferred by non-curgical method. The 300 IU of FSH/LH presented better superovulatory response in comparison with 400 and 500 IU in Nelore heifers. The transfer of Bos taurus indicus embryos cryopreserved by one-step method in 1.5 M of ethilene glycol was not efficient.

Half dose of prostaglandin F2a is effective to induce luteolysis in the synchronization of ovulation protocol for fixed-time artificial insemination in buffalo (Bubalus bubalis)

The objective of this study was to evaluate the efficiency of half dose of PGF2a in the synchronization of ovulation protocol (GnRH/PGF2a/GnRH) for fixed-time artificial insemination in buffalo. Two hundred and sixty two buffaloes (³40 days postpartum) were divided in 2 groups (Group 1, n = 125, GnRH/ ½ dose de PGF2a/GnRH; Control Group, n = 137, GnRH/PGF2a/GnRH). Animals in Group 1 received 25mg of GnRH IM (Lecirelin, Gestran-plus®) and, seven days later, half dose of PGF2a IM (75mg d-cloprostenol, Preloban®). Two days after PGF2a injection, all animals received 25mg of GnRH IM (Lecirelin). Animals in Control Group received the same treatment, except for the dose of PGF2a(150mg, IM, d-cloprostenol, Preloban®). All animals were fixed-time inseminated 16 hours after the second GnRH injection. Blood samples were collected from 35 buffaloes in Group 1 and 30 buffaloes in Control Group on day of PGF2a and second GnRH injections to measure plasma progesterone concentrations. Conception rate was not influenced by PGF2a dosage (P>0.05). Conception rates were 48.00% (60/125) and 40.90% (56/137) in Group 1 and Control Group, respectively. It was observed that 88.60% (n = 35) of the buffaloes in Group 1 and 90.00% (n = 30) in Control Group presented luteolysis 48 hours after the PGF2a injection (P>0.05). In summary, present data show that both treatments result in acceptable conception rates. Therefore, the recommended dose of PGF2a can be reduced in 50.00% in the synchronization of ovulation protocol for fixed-time artificial insemination in buffalo.

Comparação dos índices reprodutivos com inseminação artificial ou cobertura natural sob influências sazonais em suínos

Os resultados das taxas de fertilidade e dos tamanhos das leitegadas foram analisados na granja de suinos da Faculdade de Medicina Veterinaria e Zootecnia da Universidade de Sao Paulo no Campus de Pirassununga, durante o periodo de 4 anos (1992 a 1995), com o objetivo de comparar a inseminacao artificial (IA) e a cobertura natural (CN) em diferentes epocas do ano, procurando estudar as influencias sazonais sobre os indices reprodutivos. Foram avaliadas 799 coberturas em femeas das racas Landrace (L), Large White (LW) e mesticas, sendo 539 de IA e 260 de CN. Reprodutores (L e LW) de comprovada fertilidade foram utilizados tanto para CN quanto para IA. As doses de semen apresentavam concentracao minima de 3 bilhoes de espermatozoides em volume de 100 ml. As IA foram realizadas as 12 e as 24 horas apos reflexo positivo de tolerância ao macho, enquanto as montas foram realizadas no momento e as 24 horas apos o diagnostico do cio. Os indices de fertilidade foram de 72,9% e 75,8% e o numero de leitoes nascidos de 12,4 e 12,1, respectivamente, para inseminacao artificial e monta natural, nao mostrando diferenca significativa. A estacao climatica influenciou a taxa de paricao (71,2%, 81,4%, 76,9% e 66,4%, para verao, outono, inverno e primavera, respectivamente; p < 0,05), mas nao mostrou efeito sobre o tamanho da leitegada. Nao houve influencia do tipo de cobertura (IA ou CN) e da interacao entre estacao climatica e tipo de cobertura sobre as taxas de paricoes e os tamanhos das leitegadas. As medias de temperatura maxima e minima se correlacionaram negativamente com a taxa de paricao.