Wanderlei Ferreira de Sá

Gestation length, birth weight and offspring gender ratio of in vitro-produced Gyr (Bos indicus) cattle embryos

In vitro embryo production (IVP) has been suggested to result in a greater proportion of male calves, longer gestation and heavier offspring than artificial insemination in Bos taurus cattle. Despite the increasing use of IVP in tropical countries, its effects upon these traits in Bos indicus have not been conclusively investigated. Gyr is a B. indicus dairy breed with known physiological differences from B. taurus, such as a longer gestation period and lighter offspring. The aim of this study was to evaluate the effect of IVP on gestation length, birth weight and gender ratio in Gyr offspring. Oocytes were recovered from Gyr cows by ovum pick-up and were matured and fertilized with thawed Gyr semen in vitro. Embryos were cultured in CR2aa medium with cumulus cells and 10% fetal calf serum under 5% CO(2) at 38.5 degrees C in air. Seven- to eight-day blastocysts were transferred to synchronized recipients. Data on gestation length and birth weight of calves from in vitro-produced embryos were compared to data obtained from Gyr calves produced by artificial insemination (AI) and natural breeding (NB) during the same period using analysis of variance, and the gender ratio was compared to the expected 1:1 ratio using a chi-square test. IVP increased (P<0.01) the percentage of male offspring (76.9%) compared to the expected 1:1 ratio, while no difference (P>0.05) was observed in the AI and NB groups. Gestation length was similar (P>0.05) between the IVP and AI groups, but IVP-derived offspring were heavier (P<0.05) than AI- and NB-derived ones, mainly for male calves (P<0.05). These data show that in vitro production affects the subsequent development of Gyr embryos, resulting in a skewed sex ratio and increased birth weight.

Post-biopsy bovine embryo viability and whole genome amplification in preimplantation genetic diagnosis.

OBJECTIVE To evaluate the effect of the biopsy of 8-cell to 16-cell bovine embryos on their subsequent development and the effect of whole genome amplification (WGA) on removed blastomeres. DESIGN Randomized study. SETTING Molecular genetics and animal reproduction laboratories. PATIENT(S) Cow ovaries obtained from slaughterhouses. INTERVENTION(S) The ovaries were punctured, and the oocytes were matured and fertilized in vitro. On the fourth day after fertilization, 8-cell to 16-cell bovine embryos were biopsied, one quarter of each embryo being removed. The blastomeres were submitted to WGA followed by polymerase chain reaction (PCR). The embryos were returned to culture for evaluation of their development. MAIN OUTCOME MEASURE(S) Subsequent rate of blastocyst development, embryo cell number, WGA efficiency, and sex determination. RESULT(S) A total of 92 embryos were submitted to biopsy. The blastocyst production was 53.3%, with 44.9% of hatching rate. These results were similar to those of the control group (66.0% and 42.6%) of 103 embryos. Overall, no impact was detected on embryo quality in blastocyst cell number between the two groups. Removed blastomeres were submitted to WGA, resulting in 98.2% of efficiency. However, only 59% of the samples were sexed by PCR. CONCLUSION(S) Biopsy of 8-cell to 16-cell bovine embryos did not affect their subsequent development. WGA was successful in removed blastomeres.

Características andrológicas de touros da raça Gir

Gyr bulls were ranked regarding their reproductive potential, following evaluations through the score-based andrological classification system (ACP). For the examinations, three animal age groups were considered: 18 to 24 month-old (G1, n=33), 25 to 31 month-old (G2, n=24), and 32 to 38 month-old (G3, n=12). Scrotal circumference was lower in G1, as compared to G2 and G3 (P<0.05). Percent motility and vigor were not affected by the age groups. Percent rates of major and total defects were higher (P<0.05) in G1 than in G2 or G3. No significant differences were observed when G2 and G3 were compared. Regarding ACP data, G1, G2 and G3 yielded, respectively, 25.7, 58.3 and 61.5% of adequate breeders; 11.4, 20.8 and 15.4% of bulls with undefined breeding capacity, and 57.1, 12.5 and 15.4% of inapt breeders or immature animals. Age group affected scrotal circumference and major and total defects, but did not influence motility or vigor. The highest proportion of adequate breeders was obtained with 24-month-old bulls and older.

Avaliação ultra-sonográfica da dinâmica folicular e lútea em vacas da raça Guzerá

The follicular growth pattern and luteal function during the estrous cycle were studied using multiparous, non-lactating Guzera cows (n=5). The animals presented similar age, body score condition, and mean body weight of 518 ± 48.5kg. Follicular dynamics was daily monitored between ovulations during two consecutive estrous cycles, using an ultrasound device equipped with a linear rectal 5MHz transducer. Blood samples were collected each 48h after ovulation, during the evaluated cycles. The first cycle was synchronized using a luteolytic agent (cloprostenol), and the second estrous cycle was natural. Follicles were identified and measured, and data were individually recorded according to the day of the cycle. The mean length of the cycles was 19.10 ± 1.86 days. There was a higher incidence of cycles presenting three follicular growth waves (50%), but cycles presenting two (37.5%) or three (12.5%) waves were also observed. The maximum diameter of non-ovulatory follicles was 11.60± 2.37mm, and that of ovulatory follicles was 14.40± 0.50mm. The growth rate of dominant follicles during the first, second, third and fourth waves were 1.48 ± 0.60; 0.81 ± 0.13; 1.10 ± 0.27 and 1.33mm/day, respectively. Progesterone maximum concentration during diestrus was 5.50± 0.92ng/ml. These results show that the Guzera breed presents characteristics of the follicular dynamics similar to those observed in other Zebu breeds, like the trend to a higher number of follicular waves associated with lower growth rate, maximum diameter and persistence of the dominant follicles emerging during non-ovulatory waves.

Taurina no desenvolvimento de embriões bovinos fecundados in vitro

The effect of different taurine concentrations on bovine embryo development in medium supplemented with different serum sources was studied. In the first experiment, in vitro fertilized zygotes (n=440) were divided into treatments with 0, 3, 7 or 14 mM of taurine in culture medium supplemented with 10% of fetal calf serum (FCS) and 3g/l of bovine serum albumin (BSA). In the second experiment, zygotes (n=940) were divided into treatments with 0, 3 or 14 mM of taurine in cultured medium supplemented with 10% of FCS or 3g/l of BSA. In the third experiment, zygotes (n=191) were divided into treatments with 0 or 3 mM of taurine in culture medium without serum source, even so supplemented with 3g/l of polyvinyl alcohol. In the first and second experiments no differences (P>0.05) in cleavage rate, blastocyst production and cells number among the concentrations of taurine were observed. In the third experiment, taurine increased (P<0.05) cleavage rate (68.5% vs. 16.9%) and blastocysts production (8.3% vs. 0%). The culture of zygotes in medium supplemented with FCS produced more (P<0.01) blastocyst in the seventh (25.6% vs. 6.7%) and eighth (30.8% vs. 13.9%) day post-fertilization and total cells number/blastocysts (104.8± 2.63 vs. 84.7± 3.86) than in medium with BSA, despite lower (P<0.01) cleavage rate (58.1% vs. 71.3%). In conclusion, taurine only has a beneficial effect in the embryo development in culture medium in the absence of FCS and BSA. Fetal calf serum decreases cleavage rate, however, it improves the embryo development after the early cleavage.

Características morfológicas e funcionais do corpo lúteo durante o ciclo estral em vacas da raça Gir

Luteal development, function and regression were studied in Gir cattle. Morphologic characteristics of corpora lutea were evaluated during the estrous cycle (n=15), using a portable ultrasound device. Luteal activity was evaluated by serum progesterone. The corpus luteum was first identified at 3.28±0.19 days after ovulation. There was a day effect on corpus luteum cross-section area (P<0.0001), on luteal tissue cross-section area (P<0.0001) and on progesterone levels (P<0.0001). Maximum corpus luteum cross-section area occurred between days 7 and 16 of the estrous cycle. During this period, mean corpus luteum area was 3.21±0.05cm2, mean luteal cavity area was 0.42±0.04cm2, luteal tissue was 3.06±0.05cm2, and mean progesterone concentration was 4.61±0.17ng/ml. Luteal growth rate between first detection and day 7 was 0.42±0.05cm2/day, and regression rate between days 16 and 21 was -0.36±0.04cm2/day. Corpora lutea characteristics in Gir cattle were similar to those observed in other cattle breeds.

Regressão luteal e dinâmica folicular após luteólise natural ou induzida por cloprostenol em vacas da raça Gir

A regressao luteal e a dinâmica folicular foram avaliadas durante o periodo de regressao luteal natural (n=14) ou apos a inducao artificial da luteolise pela aplicacao de 500 g de cloprostenol (n=13), utilizando-se um aparelho portatil de ultra-som. Apos a inducao da luteolise foi detectada maior taxa de regressao luteal em 24 (0,89± 0,13´0,24± 0,17cm2/dia; P 0,05). Nao houve diferenca (P>0,05) nas caracteristicas da dinâmica folicular entre os dois grupos. No momento da luteolise, quando havia um foliculo dominante funcional, observou-se reducao na duracao do crescimento folicular (3,71±0,56´5,26±0,34 dias; P<0,05) e no intervalo luteolise-estro (85,71±14,68´121,33±8,34 horas; P<0,05). Os resultados demonstram que a regressao funcional do corpo luteo e a dinâmica folicular sao semelhantes apos a luteolise natural ou induzida em vacas da raca Gir, e que o fator determinante no intervalo luteolise-estro e o estadio fisiologico dos foliculos presentes.

Protocolos de produção in vitro de embriões na raça Gir

Avaliaram-se os efeitos de dois protocolos de puncao folicular na quantidade/qualidade dos oocitos e na producao in vitro de embrioes, em vacas da raca Gir, nao-lactantes. O ciclo estral foi sincronizado com cloprostenol e ao longo do experimento os animais receberam implantes auriculares de norgestomet, renovados a cada 14 dias. Os animais foram submetidos aos protocolos I (sem estimulacao hormonal, com puncao folicular duas vezes/semana) e II (pre-tratamento com 250 UI de FSH em doses decrescentes, tres dias antes da puncao folicular). Os oocitos recuperados foram levados ao laboratorio em meio TALP-Hepes e submetidos a maturacao. Na fecundacao in vitro utilizou-se semen de touro Gir, previamente capacitado. Apos 22 horas de fecundacao, os provaveis zigotos foram co-cultivados com celulas da granulosa em CR2aa acrescido de 10% de soro fetal bovino. A taxa de clivagem foi avaliada 72 horas pos-fecundacao e a de blastocisto 192 horas pos-fecundacao. O numero total de foliculos foi maior (P 0,05). O numero de oocitos de grau I e a taxa de clivagem foram maiores (P<0,05) e o numero de degenerados menor protocolo II. A pre-estimulacao ovariana com FSH pode melhorar a qualidade e a taxa de clivagem dos oocitos recuperados por puncao folicular em animais Gir.

Fecundação in vitro com sêmen de bovinos da raça Gir

The aim of this study was to evaluate the effects of differents sperm concentration and incubation time during in vitro fecundation on sperm penetration, monospermic fecundation and cleavage rates, using sperm of Gyr bulls. In vitro matured oocytes (n=817) were distributed into treatments to in vitro fertilization (IVF) in a factorial design, with two sperm concentration (2 and 4´106 spermatozoa/ml), two incubation time (12 and 18h) and two bulls (A and B). Viable spermatozoa were obtained by swin-up. The IVF was realized in Fert-Talp medium with heparin, on incubator with 5% CO2 and 95% humidity in air, at 39oC. After insemination, 359 oocytes were fixed and stained to determine penetration and polyspermy rates. The remainder were co-cultivated in bovine oviduct epithelial cells and TCM-199, for 72h, to evaluate cleavage rate. The penetration, monospermic fecundation, and cleavage rates were not affected (P>0.05) by sperm concentration and incubation time. Higher (P<0.05) penetration and cleavage rates were observed for bull B (83.3 and 81.8%, respectively) than for bull A (66.3 and 64%, respectively). Bull B showed a tendency to present higher (P<0.07) polyspermy than bull A (16.4 and 6.25, respectively). No interactions among treatments were observed.

Efeito do citrato e taurina em meio CR2aa no desenvolvimento de embriões bovinos fecundados in vitro

The effect of citrate added to CR2aa medium supplemented with fetal calf serum (FCS) or serum-proteinfree and its association with taurine on the development of in vitro-fertilized bovine embryos was evaluated. Embryos were cultured with 0, 0.5, 1.0, and 3.0mM citrate, in CR2aa supplemented with 10% FCS (experiment 1), or polyvinyl alcohol (PVA; experiment 2). In experiment 3, embryos were cultured with 0.5mM citrate, 7.0mM taurine or with association of both, in medium supplemented with FCS. Embryo culture was performed with cumulus cells at 38.8oC in 5% CO2 under air for all experiments. Positive effect on embryo development was only observed with 0.5 and 1.0mM citrate in FCS-free CR2aa (P 0.05) embryo rate nor total cell number. Citrate in CR2aa medium can be an alternative for serumfree embryo culture under 5% CO2 in air, absence of serum protein.