N. A. T. Carvalho

Control of ovulation with a GnRH agonist after superstimulation of follicular growth in buffalo: fertilization and embryo recovery

The potential to use a GnRH agonist bioimplant and injection of exogenous LH to control the time of ovulation in a multiple ovulation and embryo transfer (MOET) protocol was examined in buffalo. Mixed-parity buffalo (Bubalus bubalis; 4-15-year-old; 529 +/- 13 kg LW) were randomly assigned to one of five groups (n = 6): Group 1, conventional MOET protocol; Group 2, conventional MOET with 12 h delay in injection of PGF2alpha; Group 3, implanted with GnRH agonist to block the preovulatory surge release of LH; Group 4, implanted with GnRH agonist and injected with exogenous LH (Lutropin, 25 mg) 24 h after 4 days of superstimulation with FSH; Group 5, implanted with GnRH agonist and injected with LH 36 h after superstimulation with FSH. Ovarian follicular growth in all buffaloes was stimulated by treatment with FSH (Folltropin-V, 200 mg) administered over 4 days, and was monitored by ovarian ultrasonography. At the time of estrus, the number of follicles >8 mm was greater (P < 0.05) for buffaloes in Group 2 (12.8) than for buffaloes in Groups 1(8.5), 3 (7.3), 4 (6.1) and 5 (6.8), which did not differ. All buffaloes were mated by Al after spontaneous (Groups 1-3) or induced (Groups 4 and 5) ovulation. The respective number of buffalo that ovulated, number of corpora lutea, ovulation rate (%), and embryos + oocytes recovered were: Group 1 (2, 1.8 +/- 1.6, 18.0 +/- 13.6, 0.2 +/- 0.2); Group 2 (4,6.1 +/- 2.9, 40.5 +/- 17.5, 3.7 +/- 2.1); Group 3 (0, 0, 0, 0); Group4 (6, 4.3 +/- 1.2, 69.3 +/- 14.2, 2.0 +/- 0.9); and Group 5 (1, 2.5 +/- 2.5, 15.5 +/- 15.5, 2.1 +/- 2.1). All buffaloes in Group 4 ovulated after injection of LH and had a relatively high ovulation rate (69%) and embryo recovery (46%). It has been shown that the GnRH agonist-LH protocol can be used to improve the efficiency of MOET in buffalo.

Growth, metabolic status and ovarian function in buffalo (Bubalus bubalis) heifers fed a low energy or high energy diet

The aim was to establish the capacity of buffalo heifers to adapt their metabolic requirements to a low energy diet. Murrah buffalo (Bubalus bubalis) heifers undergoing regular estrous cycles were randomly assigned by age, live weight (LW) and body condition score (BCS) to a high energy group (HE, 5.8 milk forage units (MFU)/day, n=6) or low energy group (LE, 3.6 MFU/day, n=6). Circulating concentrations of metabolic substrates, metabolic hormones and reproductive hormones were determined weekly for 19 weeks. Ovarian follicular characteristics and oocyte parameters were also ascertained weekly. Heifers fed the LE diet had a better dry matter conversion than heifers fed the HE diet and the calculated daily energy provision was negative for heifers fed the LE diet (-0.248 MFU) and positive for heifers fed the HE diet (5.4 MFU). Heifers fed the HE diet had an increase in 50 kg LW over the duration of the study whereas LW remained constant for heifers fed the LE diet. The BCS of heifers fed the HE diet (4.2) was greater (P<0.05) than the BCS for heifers fed the LE diet (3.4). Heifers fed the HE diet had greater (P<0.05) circulating concentrations of metabolic substrates (glucose, total cholesterol and HDL cholesterol) and metabolic hormones (insulin, glucagon, leptin and T3) compared with heifers fed the LE diet. There were no significant differences in circulating reproductive hormones between the two groups of heifers. Ovarian follicular characteristics were similar for the two groups of heifers while heifers fed the LE diet tended to have oocytes of reduced quality compared with heifers fed the HE diet. The most notable finding was that heifers fed the LE diet had a negative calculated daily energy provision but were able to maintain LW and reproductive activity. It was concluded that buffalo heifers may potentially have the capacity to undergo metabolic adjustment and reduce their energy requirements when dietary energy is limiting. This adaptive capacity would explain why buffaloes remain productive in environments that are limiting to other ruminants.

Dinâmica folicular e taxa de prenhez em novilhas receptoras de embrião (Bos taurus indicus x Bos taurus taurus) tratadas com o protocolo "Ovsynch" para inovulação em tempo fixo

The aim of this study was to evaluate the efficacy of the synchronization of ovulation for fixed-time embryo transfer in crossbred recipient heifers (Bos taurus indicus x Bos taurus taurus). At Experiment 1, ovarian ultrasonography was performed to evaluate follicular dynamics during the Ovsynch protocol (G1; n=35) or single PGF2a injection (G2; n=34). At Experiment 2, the same treatments were used at field conditions in 168 (G1) and 177 (G2) heifers. On D6, ovarian ultrasonography and blood collection for P4 assay were performed. On D7, a embryo was transferred. At Experiment 1, 45.7% of the heifers ovulated after the 1st and 74.3% after the 2nd GnRH injection (P 0.05). Thus, the pregnancy rate in Gl was 35.7% and 25.4% in G2 (P<0.05). Estrus was detected in 53.7% of the recipients in G2 and 33.3% in those of the Gl (P<0.05). It was verified that CL with larger area induced an increase on P4 concentration and on the conception rate of both groups (P<0.05). The synchronization of ovulation for fixed-time embryo transfer increased the ovulation, the selection and the pregnancy rates in recipient heifers.

Evidence of congenital transmission of Neospora caninum in naturally infected water buffalo ( Bubalus bubalis ) fetus from Brazil

The aim of this study was to determine the congenital infection by Neospora caninum in the water buffalo (Bubalus bubalis), a natural intermediate host. Nine pregnant water buffalos, raised under free-grazing condition, were slaughtered, and their fetuses were collected. Samples of brain and thoracic fluid were obtained from those fetuses, with gestational ages ranging from 2 to 5xa0months. The DNA of N. caninum was detected and identified in the brain of one of those fetuses, using two PCR assays, one directed to the Nc5 gene and the other, to the common toxoplasmatiid ITS1 sequence. The DNA fragments produced on PCR were sequenced, and N. caninum was confirmed in the samples. No antibodies to N. caninum were detected on any sample of thoracic fluid by immunofluorescent antibody test (IFATu2009<u200925). This is the first confirmation of congenital transmission of N. caninum in water buffalos.

Effect of recombinant bovine somatotropin (bST) on follicular population and on in vitro buffalo embryo production

The objective of this study was to evaluate the effect of bovine somatotropin (bST) on ovarian follicular population in buffalo heifers and its influence on oocyte quality, recovery rates and in vitro embryo production. We tested the hypothesis that bST treatment in buffalo females submitted to an ovum pick-up (OPU) program would improve the number of follicles recruited, oocyte quality and in vitro embryo production. A total of 10 heifers were assigned into two treatment groups: group bST (n=5; receiving 500 mg of bST in regular intervals) and control group (n=5; without additional treatment). Both groups were subjected to OPU sessions twice a week (every 3 or 4 days), for a total of 10 sessions per female, although due to procedural problems, only the first five OPU sessions produced embryos. The number of follicles and the diameters were recorded at all OPU sessions. The harvested oocytes were counted and classified according to their quality as either A, B, C, D or E, with A and B considered good quality. Cleavage and blastocyst production rates were evaluated 2 and 7 days after in vitro fertilization, respectively. The bST treatment increased the total number of antral follicles (>3mm in diameter; 12.2 compared with 8.7; p<0.05) and of small antral follicles (<5mm; 9.1 compared with 6.5; p<0.05) per OPU session. The bST also tended to increase the number of oocytes recovered per session (5.2 compared with 4.1; p=0.07), and enhanced the percentage of good quality oocytes (48.8% compared with 40.6%; p=0.07). bST showed no effect on cleavage and blastocyst production rates (p>0.05). The significant effects of performing repeated OPU sessions were decreasing the follicular population (p<0.001) as well as the number of follicles aspirated (p<0.001), and oocytes recovered (p<0.02). In conclusion, bST treatment improves the follicular population, demonstrating its possible application in buffalo donors submitted to OPU programs.

Ovarian responses of dairy buffalo cows to timed artificial insemination protocol, using new or used progesterone devices, during the breeding season (autumn–winter)

This study evaluated the effect of new or used P4 devices on the ovarian responses of dairy buffalo that were administered an estradiol (E2) plus progesterone (P4)-based timed artificial insemination (TAI) protocol during the breeding season. On the first day of the TAI protocol, 142 cows were randomly assigned to receive one of the following: a new device (New; 1.0u2009g of P4; nu2009=u200948); a device that had previously been used for 9 days (Used1x, nu2009=u200947); or a device that had previously been used for 18 days (Used2x, nu2009=u200947). Ultrasound was used to evaluate the following: the presence of a corpus luteum (CL); the diameter of the dominant follicle (ØDF) during protocol; ovulatory response; and pregnancies per AI (P/AI). Despite similar responses among the treatments, there was a significant positive association of the ØDF during TAI protocol with ovulatory responses and number of pregnancies. In conclusion, satisfactory ovarian responses and a satisfactory pregnancy rate were achieved when grazing dairy buffalo were subjected to the TAI protocol in breeding season, independent of whether a new or used P4 device was used. Furthermore, the presence of the larger follicle was associated with a higher ovulation rate and higher P/AI following TAI.

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.

Effect of season on dairy buffalo reproductive performance when using P4/E2/eCG-based fixed-time artificial insemination management

This study aimed to compare the reproductive efficiency of dairy buffaloes subjected to TAI protocols based on progesterone, estrogen, and equine chorionic gonadotrophin (P4/E2+eCG) during the fall/winter (nxa0=xa0168) and spring/summer (nxa0=xa0183). Buffaloes received an intravaginal P4 device (1.0xa0g) plus estradiol benzoate (EB; 2.0xa0mg im) at a random stage of the estrous cycle (D-12). Nine days later (D-3), the P4 device was removed and buffaloes were given PGF2α (0.53xa0mg im sodium cloprostenol) plus eCG (400 IU im). GnRH (10xa0μg im buserelin acetate) was administered 48xa0h after P4 device removal (D-1). All animals were subjected to TAI 16xa0h after GnRH administration (D0). Frozen-thawed semen from one bull was used for all TAI, which were all performed by the same technician. Ultrasound examinations were performed on D-12 and D-3 to ascertain cyclicity (presence of CL), D-3 and D0 to measure the diameter of the dominant follicle (ØDF), D+10 to verify the ovulation rate and diameter of the corpus luteum (ØCL), and D+30 and D+45 to detect pregnancy rate (P/AI 30d and 45d, respectively) and embryonic mortality (EM). Fetal mortality (FM) was established between 45 days and birth, and pregnancy loss between 30 days and birth. There were significant differences between fall/winter and spring/summer only for cyclicity rate [76.2% (128/168) vs. 42.6% (78/183); Pxa0=xa00.02]. The others variables did not differ between the seasons: ØDF on D-3 (9.6xa0±xa00.2xa0mm vs. 9.8xa0±xa00.2xa0mm; Pxa0=xa00.35); ØDF on D0 (13.1xa0±xa00.2xa0mm vs. 13.2xa0±xa00.2xa0mm; Pxa0=xa00.47); ovulation rate [86.9% (146/168) vs. 82.9% (152/182); Pxa0=xa00.19]; ØCL on D+10 (19.0xa0±xa00.3xa0mm vs. 18.4xa0±xa00.3xa0mm, Pxa0=xa00.20); P/AI on D+30 [66.7% (112/168) vs. 62.7% (111/177); Pxa0=xa00.31]; P/AI on D+45 [64.8%% (107/165) vs. 60.2% (106/176); Pxa0=xa00.37]; EM [1.8% (2/111) vs. 3.6% (4/110); Pxa0=xa00.95]; FM [21.9% (18/82) vs. 8.0% (7/87); Pxa0=xa00.13]; and PL [23.8% (20/84) vs. 12.1% (11/91); Pxa0=xa00.13]. In conclusion, dairy buffaloes present similar reproductive efficiency in fall/winter and spring/summer when subjected to P4/E2/eCG-based protocol for TAI.