Luiz Altamiro Garcia Nogueira

Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro

The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900 mOsm) for 5 min following 120 min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (P<0.05) dehydration. Embryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P<0.05). In the experiment 2, the amount of Aqp3 and ATPase1 transcripts were quantified in blastocysts with high or low rehydration after exposure to hypertonic medium. No difference (P>0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72 h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (P<0.05) embryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (P<0.01) amount of Aqp3 transcripts was found in the vitrified-warmed embryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system.

Effects of GnRH administration on ovulation and fertility in ewes subjected to estrous synchronization

The objective of this study was to verify the effects of GnRH on ovulation and pregnancy of ewes subjected to a short-term synchronization of estrus. Santa Ines and crossbred Santa Ines/Dorper ewes received 60 mg MAP sponges during 6 days plus 300 IU eCG and 30 μg d-cloprostenol 24 h prior to sponge withdrawal (SW). Ewes were assigned to receive 0.9% NaCl solution (T control ; n = 32) or 25 μg GnRH (licerelin, T GnRH ; n = 34) 24 hours after SW. Each group was assigned to intrauterine insemination by laparoscopy (n = 25) or to natural mating (n = 41). Artificial insemination was performed with a single dose of fresh semen. For controlled mating, females were exposed to males 12, 24, 36 and 48 hours after SW. Ten females per treatment were subjected to transrectal ultrasound examination at 12-hour intervals (SW to 60 hours after). Estrous response (100.0% vs 95.2%), interval from SW to estrus (32.9±7.4 vs 29.8±6.9 hours), estrous length (37.4±9.0 vs 31.5±10.4 hours), pregnancy rates (57.0% vs 41.0%), ovulation rate (100.0% vs 90.0%), number of ovulations/ewe (1.1±0.3 vs 1.2±0.4), maximum follicular diameter (6.4±0.7 vs 6.1±0.6 mm), interval from SW to ovulation (59.1±3.5 vs 58.4±3.5 hours) did not differ between T control and T GnRH , respectively. Administration of GnRH 24 hours after SW does not improve ovulation or pregnancy rate in estrous synchronization in ewes.

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.

Associação entre o perfil andrológico e a congelação de sêmen de touros da raça Nelore aos dois anos de idade, pré-selecionados pela classificação andrológica por pontos (CAP)

Andrologic (n=163) and semen freezing (n=55) parameters from young Nelore bulls were evaluated for two consecutive years, as well as their associations searching for andrologic markers for high fertility and semen freezing. Differences in andrologic profiles (P<0.05) between years were registered, reflecting the success of semen freezing, even after andrologic selection. Percentages of 26.6 and 19.5 of discharged ejaculates, pre and post-freezing, respectively, were recorded, due to physical semen parameters. Overall sperm motility, vigor, and viability means were, respectively, 66.4±5.7%, 4.8±0.4, and 76.3±8.5% pre-freezing; and 29.4±8.5%, 4.6±0.6, and 34.5±11.1% post-freezing. The post-freezing recovery mean was 44.5±13.4%. Significant correlations (P<0.05) between body weight and pre-freezing sperm motility (0.43) and post-freezing sperm recovery standard (-0.50) were estimated. Correlations were also observed (P<0.05) between volume of ejaculate and age (0.50), sperm motility (0.28), total sperm defects (-0.32), and post-freezing sperm recovery standard (-0.37). The correlation between sperm concentration and post-freezing sperm recovery standard was 0.42. Significant correlations (P<0.05) between testes shape and scrotal circumference (0.58), and total sperm defects (0.33) and vesicular gland area (0.43) were also observed.

Concentração espermática e tempo de incubação na fecundação in vitro usando-se sêmen de touros da raça Guzerá

Estudou-se o efeito da concentracao espermatica e periodo de incubacao e da interacao dessas caracteristicas sobre a fecundacao in vitro (FIV) usando-se semen de touros Guzera. Oocitos (n=1146) maturados in vitro foram divididos em tratamentos objetivando a FIV, em esquema fatorial 3×2×2 (tres touros - A, B e C, duas concentracoes espermaticas - 2 e 4×106 espermatozoides/ml e dois tempos de incubacao 12 e 18 horas). Utilizaram-se espermatozoides viaveis obtidos por swin-up. A FIV foi realizada em meio fert-talp com heparina, em incubadora com 5% de CO2 e 95% de umidade, a 38,5°C. Apos incubacao, 50% dos oocitos foram fixados e corados para determinacao das taxas de penetracao, fecundacao monoespermatica e poliespermia. O restante foi co-cultivado com celulas da granulosa em meio CR2aa por oito dias, avaliando-se a taxa de clivagem e a producao de blastocisto. Houve maior taxa de penetracao (P 0,05) entre os demais tratamentos. A taxa de poliespermia foi maior (P<0,05) na concentracao espermatica de 4×106, independente do tempo de incubacao. Na concentracao espermatica mais alta, a taxa de poliespermia foi maior no tempo de incubacao de 18 horas (P<0,05). O touro A apresentou menor (P<0,05) taxa de poliespermia em relacao aos demais. Ainda, no touro A a taxa de clivagem foi maior (P<0,05) que no touro B, enquanto o C mostrou-se semelhante tanto ao A quanto ao B. O tempo de incubacao, a concentracao espermatica e a interacao das variaveis influenciaram as taxas de penetracao e poliespermia, sem interferir na taxa de fecundacao monoespermatica e na producao de blastocistos.

Evaluation of the simulated physiological oocyte maturation (SPOM) system on F1 Gyr × Holstein oocytes and embryos

The present study aimed to evaluate the effect of the simulated physiological oocyte maturation (SPOM) system on F1 Gyr × Holstein oocytes and embryos by evaluating the meiotic arrest, embryo production rates, total number of cells and lipid score. Three experiments were conducted and the following three experimental groups were formed according to in vitro maturation (IVM) treatments: CONTROL 1 (TCM 199 medium without FBS), CONTROL 2 (commercial medium) and SPOM (TCM 199 medium with forskolin and 3-isobutyl-1-methylxanthine (IBMX) in pre-IVM and extended IVM with cilostamide). In the first experiment (ovum pick-up), a significant (P 0.05) difference in total number of cells among the groups. No difference (P > 0.05) was found on lipid score among the groups at Day 7 of development, in both Experiments 2 and 3. At Day 9 (Experiment 2), only the CONTROL 2 showed a significant increase (P > 0.05) compared with the other treatments. It was concluded that under our conditions, the SPOM system was efficient in prolonging meiotic arrest on Gyr × Holstein oocytes, offering the oocytes in vitro conditions more similar to those found in vivo; however, it adversely affected embryo production rates and promoted no beneficial effect on the total number of cells and the lipid score.

Efeito do transporte no desenvolvimento de embriões bovinos cultivados in vitro a fresco ou reaquecidos após vitrificação

Avaliou-se a viabilidade de embrioes bovinos cultivados in vitro, a fresco ou reaquecidos apos vitrificacao, depois de transportados por 6 ou 12 horas. Oocitos obtidos de foliculos de ovarios coletados em matadouro foram maturados, fecundados e cultivados in vitro. Apos sete dias de cultivo, blastocistos com grau de qualidade I e II (segundo o manual da IETS-1998) foram selecionados, envasados em OPS (open pulled straws) e vitrificados em nitrogenio liquido. O reaquecimento foi realizado a 39oC pela passagem em solucoes de HM com concentracoes decrescentes de sacarose (0,25M - 0,15M) por cinco minutos em cada solucao. Foram avaliados tres tratamentos - V0: embrioes vitrificados, reaquecidos e cultivados in vitro (n=25); V6: embrioes vitrificados, transportados por 6 horas (simulacao em palhetas), reaquecidos e cultivados in vitro (n=29); e V12: embrioes vitrificados, transportados por 12 horas, reaquecidos e cultivados in vitro - comparados, cada um, a um tratamento controle, com embrioes a fresco-C0: embrioes a fresco cultivados in vitro (n=26); C6: embrioes a fresco cultivados in vitro apos 6 horas de transporte (n=30); e C12: embrioes a fresco cultivados in vitro apos 12 horas de transporte (n=30). Os embrioes foram co-cultivados com celulas da granulosa em microgotas de TCM 199 acrescido de SFB. Foram avaliadas as taxas de re-expansao e eclosao apos 48 horas de cultivo. A analise foi realizada pelo teste do qui-quadrado. As taxas de re-expansao entre os grupos V0, V6 e V12 nao diferiram, assim como as taxas de eclosao entre os embrioes vitrificados e os controles. As taxas de eclosao, no entanto, diferiram entre os embrioes submetidos a vitrificacao e os controles. Embrioes bovinos produzidos in vitro podem ser transportados a fresco ou vitrificados por periodos de ate 12 horas, pois possibilitam taxas de eclosao satisfatorias.

Concentração de progesterona peripuberal em novilhas mestiças

O presente estudo reporta observacoes sobre idade,peso e concentracao plasmatica de progesterona em novilhasmesticas (zebu x holandes) durante o periodo peripuberal. As medias de idade e peso na puberdade foram29,1-2,0 meses e 326-19,2 kg respectivamente. Doscinco animais acompanhados, dois apresentaram elevacoestransitorias de progesterona (entre 0,5 e 1 ,O ng/ml)ante~iores a puberdade. Dentre os que nao apresentaramelevacao transitoria, dois tiveram corpo luteo de baixa producaode progesterona (1 ,2 e 1 ,6 ng/ml) apos a primeiraovulacao e um apresentou corpo luteo de longa duracao(acima de 28 dias). Os dados observados indicam que aselevacoes transitorias de progesterona anteriores a puberdadepodem ocorrer ocasionalmente, no entanto pareceser necessaria a instalacao de uma funcao luteal competente apos o primeiro cio.