In vitro and in vivo embryo production efficiency in Flemish and Holstein donor females.

The aim of this study was to compare embryo production efficiency in Flemish and Holstein donor females using ovum pick-up and in vitro fertilization (OPU-IVF) or in vivo production (superovulation; SOV) procedures. The study was conducted using a split-plot design, with eight Flemish and eight Holstein non-lactating cycling females. Females were subjected to ten weekly OPU/IVF sessions and/or two SOV/embryo collections sessions at a 63-day interval, for a total of 160 OPU-IVF and 32 SOV sessions. Mean numbers of follicles and corpora lutea, and cumulus-oocyte complex (COC) recovery rates were similar between breeds after the OPU and SOV sessions. However, Flemish donors yielded better quality grade II COCs (301, 41.9%) than Holstein females (609, and 202, 33.1%). Also, cleavage and blastocyst rates, and the total number and the mean number of viable embryos obtained after OPU-IVF were higher in Flemish (49.6% and 11.8%, and 63 and 11.8 per donor, respectively) than in Holstein (32.8% and 7.2%, and 34 and 7.2 per donor, respectively) females. Flemish females were also more efficient in yielding viable embryos after SOV (111, 7.3 per donor) than Holstein (48, 3.3 per donor) females. Overall, Flemish donor females had better responses to OPU-IVF or SOV procedures than Holstein counterparts. Irrespective of the breeds, SOV procedures were more efficient than OPU-IVF in yielding more viable embryos, under the conditions of this study. Both reproductive procedures were useful tools for the genetic conservation of the Flemish cattle breed in Southern Brazil.

Effect of culture media on porcine embryos produced by in vitro fertilization or parthenogenetic activation after oocyte maturation with cycloheximide.

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 µg/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX- blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

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.

Criopreservação e desenvolvimento in vitro de embriões bovinos / Cryopreservation and in vitro survival of bovine embryos

O objetivo deste trabalho foi estudar a remoção do crioprotetor, em duas ou três etapas, em embriões bovinos produzidos in vitro após a congelação em vapor de Nirtogênio. Blastocistos expandidos (1329) foram mantidos em co-cultivo (controle) ou criopreservados em 3 protocolos de congelação em vapor de nitrogênio. Os embriões foram equilibrados na solução de 10% de EG por 10 minutos e em 17%, 22% ou 28% de EG por 30 segundos. Após o envase, as palhetas foram mantidas em vapor de nitrogênio por 2 minutos e armazenadas em nitrogênio líquido. Após a descongelação, os crioprotetores foram diluídos em duas etapas, usando 0,3M de sacarose e solução isotônica ou em três etapas usando 0,3M de sacarose + 10% de EG; 0,3M de sacarose e solução isotônica. Os embriões foram co-cultivados com células da granulosa, avaliando as taxas de re-expansão após 24 horas e de eclosão após 24, 48, 72 e 96 horas. Para os grupos congelados no vapor e diluição do crioprotetor em duas etapas, as taxas de eclosão foram de 1,94; 11,88 e 6,06% para EG17, EG 22 e EG 28 respectivamente. Já para os grupos com diluição do crioprotetor em três etapas, as taxas de eclosão foram de 4,67; 9,90 e 10,78% para EG17, EG22 e EG28 respectivamente.

The aim of this study was to evaluate the dilution of cryoprotectant in 2 or steps of bovine in vitro-produced embryos after quick-freezing. A total of 1329 expanded blastocyst were kept in co-culture as control group or cryopreserved by 3 quick-freezing protocols. The embryos were exposed to 10% EG for 10 minutes then to 17%, 22% or 28% for 30 seconds. After loading, the straws were held in nitrogen vapor for 2 minutes and then plunged and stored in liquid nitrogen. After warming, cryoprotectants were diluted in two steps using 0.3 M sucrose and isotonic solution, or three steps using 0.3 M sucrose + 10% EG then 0.3 M sucrose and isotonic solution. Embryos were co-cultured on a granulosa cell monolayer and evaluated after 24 hours for re-expansion and at 24, 48, 72 and 96 hours of co-culture for hatching rates. The in vitro survival rates of embryos cryopreserved by the quick-freezing method and two-step cryoprotectant dilution were 1.94; 11.88 and 6.06%, for EG17, EG22 and EG28 groups, respectively. At the three step dilution, the in vitro survival rates were 4.67; 9.90 and 10.78% for EG17, EG22 and EG28 groups, respectively.