Pre-incubation of porcine semen reduces the incidence of polyspermy on embryos derived from low quality oocytes / Pré-incubação dos espermatozoides suínos diminui polispermia e aumenta a produção embrionária em oócitos de baixa qualidade

ABSTRACT: The main cause of low efficiency of in vitro produced porcine embryos is the high polyspermic penetration rates at fertilization, which is aggravated in low quality oocytes. Experiment 1 evaluated the embryo development in high and low quality oocytes. Experiment 2 evaluated the embryo development and quality of low quality oocytes fertilized with sperm pre-incubated during 0h (control), 0.5h, 1h and 1.5h. Experiment 3 investigated fertilization and monospermic rates of the same groups of Experiment 2. Experiment 4 evaluated embryo development, cell density, fertilization and monospermic rates of high quality oocytes using semen pre incubated during the best time observed in the previous experiments. Cleavage and blastocyst rates were analyzed by chi-square test, and remaining data by ANOVA and Tukey test (P≤0.05). The cleavage (74.8 vs 51.7%) and blastocyst (33.7 vs 9.8%) rates were greater in oocytes of high versus low quality, with no differences in cell density. Fertilization rates (65.6 to 79.5%) were not influenced by pre-incubation time. However, semen pre-incubation during 1.5h increased monospermic penetration (53.3%) and cleavage rates (92.5%) in low quality oocytes. Blastocyst rate was improved with 1.5h of semen pre incubation; however they were still lower than that observed with high quality control oocytes. Ultimately, pre-incubation did not influence fertilization, monospermic penetration, embryo development rates, nor cell density in oocytes of high quality. Low-quality porcine oocytes resulted in better rates of embryo development if in vitro fertilized with sperm pre-incubated for 1.5 hour.

RESUMO: A principal causa da baixa eficiência na PIV de embriões suínos é a elevada taxa de polispermia, que é exacerbada em oócitos de baixa qualidade. O experimento 1 avaliou o desenvolvimento embrionário de oócitos de baixa e alta qualidade. O experimento 2 avaliou a qualidade e o desenvolvimento embrionário de oócitos de baixa qualidade fecundados com sêmen pré-incubado por 0h (controle), 0,5h, 1h e 1,5h. O experimento 3 investigou a fecundação e as taxas de monospermia dos mesmos grupos do experimento 2. O experimento 4 avaliou o desenvolvimento embrionário, a densidade celular, a fecundação e as taxas de monospermia de oócitos de alta qualidade, fecundados com sêmen pre-incubado com o melhor tempo observado nos experimentos anteriores. As taxas de clivagem e de blastocistos foram submetidas ao teste de Qui-quadrado e os demais dados submetidos à ANOVA e teste de Tukey (P≤0,05). As taxas de clivagem (74,8 vs 51,7%) e de blastocistos (33,7 vs 9,8%) foram superiores nos oócitos de alta qualidade, comparados aos de baixa qualidade, não havendo diferenças na quantidade de células embrionárias. As taxas de fecundação (65,6 vs 79,5%) não foram influenciadas pelo tempo de pré-incubação. Todavia, a pré-incubação do sêmen por 1,5h aumentou a penetração monospérmica (53,3%) e a taxa de clivagem (92,5%), nos oócitos de baixa qualidade. As taxas de blastocisto aumentaram com sêmen pré-incubado por 1,5h, que foram ainda inferiores às obtidas dos oócitos de alta qualidade do grupo controle. Finalmente, a pré-incubação do sêmen não influencia na fecundação, na penetração monospérmica, no desenvolvimento embrionário, nem na quantidade de células embrionárias com oócitos de alta qualidade. Oócitos suínos de baixa qualidade produzem melhores taxas de desenvolvimento embrionário se fecundados in vitro com sêmen pré-incubado por 1,5 horas.

Production of bovine hand-made cloned embryos by zygote-oocyte cytoplasmic hemi-complementation.

The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; hand-made cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

Developmental potential of bovine hand-made clone embryos reconstructed by aggregation or fusion with distinct cytoplasmic volumes.

Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 x 50%); aggregation of two (2 x 50%), three (3 x 50%), or four (4 x 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 x 100%); or aggregation of two clone or parthenote embryos (2 x 100%). Control zona-intact parthenote or IVF embryos were in vitro cultured in four-well dishes. Results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate, and cell density. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies and the birth of a viable clone calf after transfer to recipients. However, embryo aggregation did not improve development on a hemicytoplast basis, except for the aggregation of two clone embryos.