Contrasting effects of heat shock during in vitro maturation on development of in vitro-fertilized and parthenogenetic bovine embryos.

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus-oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro-fertilized or activated with ionomycin and cultured in vitro for 192 hr post-in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat-shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down-regulated the expression of AQP3 (p < .01) and up-regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down-regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat-shocked oocytes.

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.

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.

Técnica de tunel em embriões de ratas wistar: avaliação da qualidade e da capacidade de desenvolvimento dos embriões / Tunel technique in wistar rats embryos: evaluation of qualify and development capacity of embryos

A técnica do TUNEL tem se mostrado eficiente como indicadora de qualidade embrionária em embriões bovinos e de camundongos. Entretanto, em ratos, a técnica não é amplamente utilizada. Objetivou-se avaliar a qualidade do desenvolvimento de embriões de ratas Wistar e o uso da técnica de TUNEL. Animais (n=8) foram superovulados através da injeção intraperitoneal de 150 UI/kg de peso via intraperitoneal (IP) de PMSG (Pregnant Mare Gonadotropin) e de 75 UI/kg de hCG 48h após. Fêmeas superovuladas foram colocadas para acasalar. 107 embriões de 4-8 células foram coletados 72 horas após hCG e cultivados em meio KSOM com 5% albumina sérica humana (BSA), em incubadora com 5% de CO2, 95% de umidade a 37,0ºC, por 48 horas. Após o cultivo, foi calculada a taxa de blastocisto e realizada a técnica do TUNEL. A taxa de blastocisto foi de 83,17% (89/107), com 19,62% (21/107) de blastocisto inicial, 14,01% (15/107) de blastocisto, e 49,53% (53/107) de blastocisto expandido. O número total de blastômeros foi de 28,82±4,76. A taxa apoptótica foi de 10.32 ± 8.91% e 94% (31/34) dos embriões apresentavam pelo menos uma célula apoptótica. Em conclusão, a técnica de TUNEL se mostrou viável na avaliação da qualidade embrionária de embriões de ratas Wistar.

The TUNEL technique has proven to be efficient as indicator of embryo quality in cattle and mice. Athough the technique is not yet widely used in rats. Thus the aim of this study was to evaluate the quality of Wistar rat embryos and the use of the TUNEL technique. Animals (n=8) were superovulated by intraperitoneal injection of 150 IU/kg PMSG (Pregnant Mare Gonadotropin) and 75 IU/kg 48h after hCG. Superovulated females were placed with males at a ratio of 1/1. 107 4-8 cells embryos were collected 72 hours after hCG and were cultured in KSOM medium with 5% bovine serum albumin (BSA), for 48h ,with 5% CO2, 95% humidity at 37.0ºC. After cultured, the blastocyst rate was calculated and the TUNEL technique was performed. The blastocyst rate was 83.17% (89/107), with 19.62% (21/107) of initial blastocyst, 14.01% (15/107) of blastocyst and 49.53% (53 / 107) of expanded blastocyst. The total number of blastomeres was 28.82 ± 4.76. The apoptotic cells rate was 10.32 ± 8.91% and 94% (31/34) of embryos had at least one apoptotic cell. In conclusion, the TUNEL technique showing that it can be used to evaluation embryo quality, in Wistar rats embryos.