We have investigated whether culture in glycine-containing medium affects subsequent glycine transport by the specific transport system, GLYT1, which is the sole glycine transporter in fertilized mouse ova. When fertilized ova were maintained for 6 h in culture with a physiological level of glycine (1 mM), subsequent transport of radiolabelled glycine was decreased by 40% compared with fertilized ova that had been maintained in glycine-free medium. Kinetic measurements showed that the apparent glycine affinity was decreased after culture with glycine (Km increased from 0.20 to 0.41 mM), but maximal transport rate was unchanged (similar Vmax of 20 and 23 fmol/fertilized ovum/min). These findings could have reflected activation of GLYT1 by prolonged substrate starvation, similar to some other amino acid transport systems. However, our findings were instead consistent with the alteration in glycine transport being due to trapping of glycine within the zona pellucida resulting in competitive transport inhibition even after ova were removed from glycine-containing media. First, even very brief exposures to glycine resulted in decreased subsequent glycine transport rates, with a maximal effect apparent within ~6 min. Second, extensive washing (at least six) reversed the effect. Third, the effect was absent when zona-free fertilized ova were used. Thus, it appears that components of the external environment of preimplantation embryos may continue to affect transport kinetics for a period even after embryos are removed from environments that contain them.

Oocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.

The identification of early changes in the sperm plasmalemma is currently a factor in the improvement of freezing protocols. We analysed the presence of active caspases in freeze–thawed (FT) dog spermatozoa, and evaluated straws from eight dogs using flow cytometry and fluorescence microscopy with fluorescein isothyocyanate–Val–Ala–Asp–fluoromethylketone (FITC–VAD–fmk) combined with ethidum homodimers. Apoptotic-like changes were evaluated using the YO–PRO-1/ethidium homodimer combination, and changes in mitochondrial membrane potential were monitored with JC-1. Sperm motility post-thaw was evaluated using a CASA system. FITC–VAD–fmk stained sperm cells in situ and the subcellular labelling pattern was consistent with known localization of caspases. On average, a high proportion of FT canine sperm showed caspase activity, ranging from 30.2 to 70.7% of the live sperm compared with 7.3 to 24.0% in dead spermatozoa. This observed differentiation between caspase activity in dead and live spermatozoa may be a simple method to disclose subtle differences in sperm quality, since this staining allowed us to find statistically significant differences among dogs. Notably, the sperm sample with overall better results in all sperm parameters studied after thawing had a lower percentage of active caspases in both dead and live spermatozoa.

In fully grown pig oocytes, meiotic maturation in vitro is retarded by inhibition of histone deacetylases by trichostatin A (TSA). In growing oocytes with partial meiotic competence, culture with TSA has no significant effect on the meiotic maturation. Growing oocytes treated with TSA mature mainly to metaphase I. The ratio of oocytes that mature to metaphase II is very limited. After transient exposure to TSA, the maturation of growing oocytes with partial meiotic competence takes a different course. When these oocytes are first cultured in a TSA-free medium, then cultured for another 24 h with 100 nM TSA and finally again in a TSA-free medium for 24 h, the ratio of oocytes that mature to metaphase II significantly increases reaching 59%. When oocytes were cultured for the same length of time without transient exposure to TSA, only 19% matured to metaphase II. Those oocytes that matured to metaphase II after transient exposure to TSA were successfully activated using calcium ionophore. However, the subsequent cleavage was very limited. We can conclude that transient exposure of growing pig oocytes with partial meiotic competence to TSA increases oocyte meiotic competence, but it does not enhance developmental competence after parthenogenetic activation.

The aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 × 106/ml with or without plasmid pEYFP–NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC–PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.9% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

Aiming to improve in vitro production of bovine embryos and to obtain supplements to replace serum for in vitro maturation (IVM), this study evaluated the effects of macromolecular supplementation of IMV medium (bovine serum albumin – BSA, polyvinyl alcohol – PVA, polyvinyl pyrrolidone – PVP, Ficoll, KnockoutSR, or fetal calf serum – FCS) and oxygen tension [5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2 (5% O2)] on oocyte maturation and embryo development. Nuclear progression to germinal vesicle breakdown, metaphase I and metaphase II stages were evaluated and overall results revealed that undefined (FCS) and semi-defined (BSA) media gave better results at 20% O2 and defined media (PVA, PVP and Ficoll) at 5% O2. Independent of macromolecule supplement, IVM at 20% O2 was considered optimal for nuclear maturation. To evaluate embryo development, oocytes matured in the previously described conditions were fertilized and cultured at the same oxygen tension used for IVM and assessed for cleavage (43.0 to 74.8%) and development to morulae (16.4 to 33.8%), blastocyst (7.7 to 52.9%) and hatched blastocyst (9.6 to 48.1%). Apart from oxygen tension, all treatments, except Knockout (22.7%), gave similar results for blastocyst development (26.5 to 38.7%). Independently of macromolecule supplement, higher development rates were obtained in an oxygen tension of 20% O2 (67.4% cleavage, 29.2% morulae, 40.8% blastocyst and 34.0% hatched blastocyst) when compared with 5% O2 (52.5, 21.8, 18.2 and 15.6%, respectively). This study indicates that BSA, PVA, PVP and Ficoll can replace serum during IVM and that the optimal atmospheric condition for in vitro production of bovine embryos is 5% CO2 and 20% O2.

Bufo arenarum oocytes are oviposited surrounded by jelly coats, one component of the extracellular matrix required for fertilization. The secretion, released to the oviductal lumen, was analysed by SDS-PAGE. The coomassie blue staining evidenced an electrophoretic pattern with molecules ranging between 300 and 19 kDa that showed variations in their secretion profiles during the sexual cycle. In the preovulatory period the densitometric analysis showed the presence of nine peaks with marked predominance of the 74 kDa molecule. Once ovulation has occurred, the jelly coats become arranged around the oocytes during their transit throughout the oviductal pars convoluta (PC), revealing the addition of three proteins only observed during this period, which suggests a differential secretion. Some of these proteins could not diffuse under any extraction treatment, indicating for them a structural or in situ function. Proteins of low molecular mass diffused totally while others showed a partial diffusing capacity. After ovulation a marked decrease in the relative amount of all the proteins released to the lumen, especially the 74 kDa protein, could be detected. During this period, unlike the other stages of the sexual cycle, a differential secretion pattern was observed along the PC. The histochemical analysis performed during the ovulatory period showed the presence of glycoconjugates including both acidic and neutral groups. The present results are in agreement with previous ultrastructural and histochemical studies that describe the role of Bufo arenarum jelly coats in fertilization.

Usually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 °C/min in a low-temperature (−80 °C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; −20 °C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type.

Elongated embryos provide a unique source of information about trophoblastic differentiation, gene expression and maternal-embryonic interactions; however they are difficult and costly to obtain, especially elongated cloned embryos. One alternative is their production in heterologous temporary recipients such as sheep and goats. We aimed to produce elongated bovine cloned embryos using heterologous transfer to temporary recipients. Day-7 cloned cattle blastocysts were transferred to the uteri of ewes and goats and recovered as elongated structures at day 17. We evaluated elongation, length, presence of embryonic disc and expression of several important genes for embryonic development. We also produced homologous (cloned cattle embryos transferred into cattle uteri). Cloned bovine blastocysts were able to proceed with preimplantation development through elongation with high efficiency despite the species to which they were transferred. In qualitative and quantitative RT-PCR experiments we found differences in the pattern of gene expression among embryos recovered from different species. Sox2, Nanog and FGF-4 were markedly deregulated. No previous reports about the expression pattern of the studied genes had been published for elongated bovine cloned embryos produced in intermediate recipients, furthermore, the pattern of expression of Nanog, Oct4, Eomes, Cdx2, IFN-tau, Dicer, FGF-4 and Sox2 shown here are novel for elongated cloned bovine embryos created by hand-made cloning. Our data confirmed that sheep and goats can be used as temporary recipients. This model could serve as a basis for further research on gene expression and cellular changes during bovine peri-implantation development.