Morphometric developmental pattern of bovine handmade cloned concepti in late pregnancy.

Cloning procedures often interfere with conceptus growth and life ex utero, in a set of symptoms known as abnormal offspring syndrome (AOS). The aim of the present study was to compare the developmental pattern of in vivo-derived (IVD), IVF-derived and handmade cloning-derived (NT-HMC) Day 225 bovine concepti using established procedures. Pregnancy diagnosis was performed on Day 30 following blastocyst transfer on Day 7. Conceptus morphometry was assessed by ultrasonography on Day 51, and on Day 225 pregnant cows were killed for morphological examination of concepti. Pregnancy outcome was similar between groups, with greater pregnancy losses in the first trimester (70.6%) and smaller fetuses on Day 51 in the NT-HMC group than in the IVD (14.3%) and IVF (20.0%) groups. However, NT-HMC-derived concepti were twofold larger on Day 225 of gestation than controls. A higher frequency (63.5%) of placentomes larger than the largest in the IVD group was observed in the NT-HMC group, which may be relevant to placental function. Conceptus traits in the IVF group were similar to the IVD controls, with only slight changes in placentome types. Morphological changes in cloned concepti likely affected placental function and metabolism, disrupting the placental constraining mechanism on fetal growth in mid- to late pregnancy.

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

Calves born after direct transfer of vitrified bovine in vitro-produced blastocysts derived from vitrified immature oocytes.

Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super-cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non-vitrified control group, (ii) vitrified in normal (-196 degrees C) liquid nitrogen (LN(2)) and (iii) vitrified in super-cooled LN(2) (< or =-200 degrees C). Open-pulled glass micropipettes were used as vitrification containers. Immature oocytes were in vitro-matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN(2) state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super-cooled LN(2), resulted in viable blastocysts and live calves following in vitro embryo production.

In-straw cryoprotectant dilution of IVP bovine blastocysts vitrified in hand-pulled glass micropipettes.

The aim of this study was to determine the influence of two ethylene glycol-based vitrification solutions on in vitro and in vivo survival after in-straw cryoprotectant dilution of vitrified in vitro-produced bovine embryos. Day-7 expanded blastocysts were selected according to diameter (> or = 180 microm) and osmotic characteristics and randomly assigned to one of three groups (i) VSa: vitrification in 40% EG+17.1% SUC+0.1% PVA; (ii) VSb: vitrification in 20% EG+20% DMSO; (iii) control: non-vitrified embryos. Vitrification was performed in hand-pulled glass micropipettes (GMP) and cryoprotectant dilution in 0.25 ml straws after warming in a plastic tube. Embryo viability was assessed by re-expansion and hatching rates after 72 h of IVC and by pregnancy rates after direct transfer of vitrified embryos. No differences in re-expansion rates were observed between vitrified groups after 24 h in culture (VSa=84.5%; VSb=94.8%). However, fewer VSa embryos (55.2%, P<0.05) hatched after 72 h than the VSb (75.8%) and control embryos (80.0%). To evaluate in vivo viability, vitrified embryos (VSa=20; VSb=21) were warmed under field conditions and individually transferred to synchronous recipients. Pregnancy rates (day 60) were similar between groups (VSa=20%; VSb=19%). Greater hatching rates occurred after 72 h of IVC for EG+DMSO than EG+SUC+PVA vitrification solutions. However, using a GMP vitrification container and in-tube warming, both solutions provided similar pregnancy rates after the in-straw cryoprotectant dilution and direct embryo transfer.

Current status of sperm cryopreservation: why isn't it better?

Cryopreservation extends the availability of sperm for fertilization; however, the fertilizing potential of the frozen-thawed sperm is compromised because of alterations in the structure and physiology of the sperm cell. These alterations, characteristics of sperm capacitation, are present in the motile population and decrease sperm life-span, ability to interact with female tract, and fertilizing ability. The etiology of such alterations may represent a combination of factors, such as inherited fragility of the sperm cell to withstand the cryopreservation process and the semen dilution. Although the former is difficult to address, approaches that make-up for the dilution of seminal fluid may be sought. The aim of this work is to review aspects of sperm cryopreservation paralleled by events of capacitation and evaluate the possible roles of sperm membrane cholesterol, reactive oxygen species, and seminal plasma as mediators of cryopreservation effects on sperm function.

Splitting and biopsy for bovine embryo sexing under field conditions.

Improvements on embryo micromanipulation techniques led to the use of embryo bisection technology in commercial embryo transfer programs, and made possible the direct genetic analysis of preimplantation bovine embryos by biopsy. For example, aspiration and microsection, allow bovine embryos sexing by detection of male-specific Y-chromosome in a sample of embryonic cells. We report on the application of the methodologies of splitting and biopsy of bovine embryos in field conditions, and on the results of embryo sex determination by the polymerase chain reaction (PCR). Pregnancy rates achieved with fresh bisected or biopsied embryos (50 to 60%) were similar to the fresh intact embryos (55 to 61%). The PCR protocol used for embryo sexing showed 92% to 94% of efficiency and 90 to 100% of accuracy. These results demonstrate these procedures are suitable for use in field conditions.