RESUMO
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.
Assuntos
Blastocisto/fisiologia , Criopreservação , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária/veterinária , Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos , Vitrificação , Animais , Aquaporina 3/genética , Aquaporina 3/metabolismo , Biomarcadores/metabolismo , Bovinos , Criopreservação/métodos , Criopreservação/veterinária , Células do Cúmulo/fisiologia , Dessecação , Desenvolvimento Embrionário , Feminino , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Expressão Gênica , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Oócitos/fisiologia , Osmose , Salinidade , Sêmen/fisiologia , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Zigoto/fisiologiaRESUMO
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.
Assuntos
Peso ao Nascer/fisiologia , Bovinos/embriologia , Técnicas de Cultura Embrionária/veterinária , Fertilização in vitro/veterinária , Prenhez , Razão de Masculinidade , Animais , Animais Recém-Nascidos , Embrião de Mamíferos , Desenvolvimento Embrionário , Feminino , Masculino , Gravidez , Fatores de TempoRESUMO
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.
Assuntos
Blastocisto/fisiologia , Blastômeros/fisiologia , Bovinos , Fertilização in vitro/veterinária , Técnicas de Amplificação de Ácido Nucleico/veterinária , Diagnóstico Pré-Implantação/veterinária , Animais , Biópsia , Blastocisto/citologia , Blastômeros/citologia , Técnicas de Cultura Embrionária/veterinária , Feminino , Genoma , Oócitos/citologia , Reação em Cadeia da Polimerase/veterinária , Gravidez , Análise para Determinação do Sexo/veterináriaRESUMO
The estrus behavior was evaluated during an induced and natural subsequent estrus in twelve Guzera breed cows, in the winter and summer seasons in two consecutive years. The proportion of cows that responded to estrus synchronization was 84.8 percent. The effect of the interaction season x year of experiment on estrous cycle length was observed. Pro-estrus was longer in the summer (57.69±4.72h) and in the natural estrus (74.23±4.41h) than in the winter (38.95±4.02h) and in the induced estrus (22.40±4.36h). Estrus length was similar in the winter (11.48±0.70h) and in the summer seasons (13.40±0.82h) as well as among cows with induced (12.47±0.75h) and natural estrus (12.41±0.76h). The number of mounts accepted during estrus and the number of mounts accepted/ hour in estrus were similar in winter (29.17±2.86 and 2.59±0.22, respectively) and summer (31.45±3.36 and 2.42±0.26, respectively) as well as between induced (30.23±3.10 and 2.54±0.24, respectively) and natural estrus (30.40±3.14 and 2.47±0.24, respectively). The length and intensity of estrus were not influenced by synchronization or season of the year. Longer period of pro-estrus in summer may favor the identification of sexually active animals in this season.
A manifestação do comportamento estral foi avaliada durante um estro induzido e o estro subseqüente, em 12 vacas da raça Guzerá, no inverno e no verão em dois anos consecutivos. A proporção de vacas que respondeu à sincronização de estros foi de 84,8 por cento. Observou-se efeito de interação entre estação e ano sobre a duração do ciclo estral. O proestro foi maior no verão (57,69±4,72h) e no estro natural (74,23±4,41h), que no inverno (38,95±4,02h) e no estro induzido (22,40±4,36h). A duração do estro foi similar no inverno (11,48±0,70h) e no verão (13,40±0,82h), assim como entre as vacas com estro induzido (12,47±0,75h) e natural (12,41±0,76h). O número de montas recebidas durante o estro e o número de montas recebidas/hora em estro foram similares no inverno (29,17±2,86 e 2,59±0,22, respectivamente) e no verão (31,45±3,36 e 2,42±0,26, respectivamente), assim como entre o estro induzido (30,23±3,10 e 2,54±0,24, respectivamente) e o natural (30,40±3,14 e 2,47±0,24, respectivamente). A duração e a intensidade do estro não foram influenciadas pela sincronização ou pela estação do ano. O maior período de proestro no verão pode favorecer a identificação dos animais sexualmente ativos.
RESUMO
A importância do (F1) para o Brasil é notória, associando rusticidade e produtividade, porém é grande a dificuldade do produtor em manter seu rebanho nesse grau de sangue. No laboratório da Embrapa Gado de Leite em Juiz de Fora, MG foram produzidos in vitro 266 embriões (F1), a partir de complexos cumulus-oócitos (CCO's) de vacas Gir puncionadas e fecundadas com sêmen de touro Holandês. Destes, 53 embriões foram transferidos a fresco para receptoras de rebanhos comerciais de uma Cooperativa da Zona da Mata, MG, enquanto 72 embriões foram inovulados em rebanho controlado da Embrapa Campo Experimental de Coronel Pacheco. A produção de embriões F1 por fecundação in vitro apresentou taxa de produção de blastocisto de 15,58%. A taxa de gestação obtida foi de 26,4% no rebanho comercial e 40,2% no rebanho da Embrapa. Considerando-se o nascimento de 50% de machos e fêmeas, conclui-se que em rebanhos de 100 vacas seriam obtidas 13 fêmeas (13% de reposição) nos rebanhos comerciais e 20 fêmeas (20% de reposição) no rebanho da Embrapa. Foram muitas as dificuldades de operacionalização para o uso de dessa biotecnologia que requer um mínimo de escrituração zootécnica, controle sanitário, observação de cios e manejo reprodutivo.(AU)
The importance of (F1) to Brazil is notable, associating productivity and rusticity, it is difficult to the producer maintaining the herd in this blood degree. In Embrapa's Gado de Leite, laboratory in the city of Juiz de Fora, M.G., 266 embryos (F1) were produced in vitro from the oocytes of Gir cows, punched and fecundated with Holstein Friesan bull semen. Out of these, 53 embryos were transferred at natural into recipient cows in the commercial herds indicated by the technicians of a great cooperative of Zona da Mata's area, 72 embryos were transferred into recipients of Embrapa's herds under nutritional and sanitary controls. The remainder 139 embryos were frozen due to the lack of adequate recipient cows. The production of F1 embryos through in vitro fecundation of oocytes from Gir cows with semen from Holstein Friesan bull blastocist production rate of 15,58 %. The pregnance rate obtained was 26.4% in the cows from the commercial herds and 40.2% in the herds of Embrapa Gado de Leite. Considering the birth of 50% female cows, we can estimate a replacement annual rate of 13% (26.2% for gestation) for commercial herds and 20% (40% for pregnancy) for the controlled herd. There were many difficulties encountered in the operation, such as (i) the low level of adoption of technology; (ii) little investment in feeding, handling and sanity conditions of the herds; (iii) conditions capable to compromising the utilization of embryos, which requires a minimum of structure in terms of zoo-technical documentation, observation of rutting and reproduction management.(AU)
Assuntos
Animais , Bovinos , Embrião de Mamíferos/embriologia , Indústria Agropecuária/análise , Fertilização in vitro , Comércio/estatística & dados numéricos , Vigor Híbrido/genética , OócitosRESUMO
A importância do (F1) para o Brasil é notória, associando rusticidade e produtividade, porém é grande a dificuldade do produtor em manter seu rebanho nesse grau de sangue. No laboratório da Embrapa Gado de Leite em Juiz de Fora, MG foram produzidos in vitro 266 embriões (F1), a partir de complexos cumulus-oócitos (CCO's) de vacas Gir puncionadas e fecundadas com sêmen de touro Holandês. Destes, 53 embriões foram transferidos a fresco para receptoras de rebanhos comerciais de uma Cooperativa da Zona da Mata, MG, enquanto 72 embriões foram inovulados em rebanho controlado da Embrapa Campo Experimental de Coronel Pacheco. A produção de embriões F1 por fecundação in vitro apresentou taxa de produção de blastocisto de 15,58%. A taxa de gestação obtida foi de 26,4% no rebanho comercial e 40,2% no rebanho da Embrapa. Considerando-se o nascimento de 50% de machos e fêmeas, conclui-se que em rebanhos de 100 vacas seriam obtidas 13 fêmeas (13% de reposição) nos rebanhos comerciais e 20 fêmeas (20% de reposição) no rebanho da Embrapa. Foram muitas as dificuldades de operacionalização para o uso de dessa biotecnologia que requer um mínimo de escrituração zootécnica, controle sanitário, observação de cios e manejo reprodutivo.
The importance of (F1) to Brazil is notable, associating productivity and rusticity, it is difficult to the producer maintaining the herd in this blood degree. In Embrapa's Gado de Leite, laboratory in the city of Juiz de Fora, M.G., 266 embryos (F1) were produced in vitro from the oocytes of Gir cows, punched and fecundated with Holstein Friesan bull semen. Out of these, 53 embryos were transferred at natural into recipient cows in the commercial herds indicated by the technicians of a great cooperative of Zona da Mata's area, 72 embryos were transferred into recipients of Embrapa's herds under nutritional and sanitary controls. The remainder 139 embryos were frozen due to the lack of adequate recipient cows. The production of F1 embryos through in vitro fecundation of oocytes from Gir cows with semen from Holstein Friesan bull blastocist production rate of 15,58 %. The pregnance rate obtained was 26.4% in the cows from the commercial herds and 40.2% in the herds of Embrapa Gado de Leite. Considering the birth of 50% female cows, we can estimate a replacement annual rate of 13% (26.2% for gestation) for commercial herds and 20% (40% for pregnancy) for the controlled herd. There were many difficulties encountered in the operation, such as (i) the low level of adoption of technology; (ii) little investment in feeding, handling and sanity conditions of the herds; (iii) conditions capable to compromising the utilization of embryos, which requires a minimum of structure in terms of zoo-technical documentation, observation of rutting and reproduction management.
Assuntos
Animais , Bovinos , Embrião de Mamíferos/embriologia , Fertilização in vitro , Indústria Agropecuária/análise , Comércio/estatística & dados numéricos , Oócitos , Vigor Híbrido/genéticaRESUMO
Apresenta uma revisão sobre as técnicas de reprodução assistida.
Assuntos
Humanos , Masculino , Feminino , Infertilidade , Técnicas Reprodutivas , Fertilização in vitro , Inseminação ArtificialRESUMO
The aim of the present study was to evaluate oocyte recovery and embryo yield using two different ovarian follicular aspiration schedules in donor cows of the Gir breed. Pluriparous, non-lactating Gir cows (n = 14) were randomly allocated to one of two groups, one of which had aspirations of ovarian follicular contents conducted once a week (TVFA-1x) and the other twice weekly (TVFA-2x), for nine consecutive weeks. Before follicle aspiration, follicular population was recorded in three classes according to size (> 6 mm, 6-9 mm and > 9 mm). The cumulus-oocyte complexes (COCs) recovered were identified, morphologically classified and in vitro matured, fertilized with Gir sperm and cultured in CR2 medium for 7 days. There was no difference (P > 0.05) in the size of the largest follicle, number of follicles identified or follicular content aspirations between TVFA-1x and TVFA-2x groups. Large follicles (> 9 mm) were observed for all the aspiration intervals considered (3, 4 or 7 days). More oocytes were recovered per session in TVFA-1x as compared with TVFA-2x (8.9 +/- 0.8 versus 7.0 +/- 0.7, P < 0.01), resulting in a greater recovery rate in this group (74.3% versus 58.7%, P < 0.01). More COCs of Grade I were recovered from TVFA-2x (22.6% versus 13.3%, P < 0.01). There was no difference in cleavage rate between groups, but the percentage of embryos that reached the blastocyst stage was greater in TVFA-2x as compared with the TVFA-1x (31.8% versus 21.6%, P < 0.01). The greater in vitro performance qualities of TVFA-2x oocytes compensates for the greater oocyte recovery rate in TVFA-1x, demonstrating a greater embryo production potential. Despite showing uncommon follicular dynamics characteristics when subjected to follicular aspiration, Gir cows can be successfully used as oocyte donors for in vitro embryo production.