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In vitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of in vivo -matured oocytes has yielded better results, but is less practical. The presence of the corona radiata seems to partially protect those factors that are necessary for the construction of the normal spindle and for chromosome alignment, but multiple layers of cumulus cells may impair permeation of cryoprotective agents. In addition to the spindle, the oolemma and mitochondria are also particularly sensitive to vitrification damage, which should be minimised in future vitrification procedures. This review presents promising protocols and novel strategies in equine oocyte vitrification, with a focus on blastocyst development and foal production as most reliable outcome parameters. Cryopreservation of a mare's oocytes could become an important strategy in horse breeding if the foaling rates were better. Here we discuss the most important technical and biological factors affecting the success of oocyte vitrification in the horse, referring to novel approaches in other species. Further research is needed to optimise oocyte vitrification in order to achieve high blastocyst and foaling rates. [ABSTRACT FROM AUTHOR]

Platelet-activating factor (PAF) is a well-known marker for embryo quality and viability. For the first time, we describe an intracellular localisation of PAF in oocytes and embryos of cattle, mice and humans. We showed that PAF is represented in the nucleus, a signal that was lost upon nuclear envelope breakdown. This process was confirmed by treating the embryos with nocodazole, a spindle-disrupting agent that, as such, arrests the embryo in mitosis, and by microinjecting a PAF-specific antibody in bovine MII oocytes. The latter resulted in the absence of nuclear PAF in the pronuclei of the zygote and reduced further developmental potential. Previous research indicates that PAF is released and taken up from the culture medium by preimplantation embryos in vitro , in which bovine serum albumin (BSA) serves as a crucial carrier molecule. In the present study we demonstrated that nuclear PAF does not originate from an extracellular source because embryos cultured in polyvinylpyrrolidone or BSA showed similar levels of PAF in their nuclei. Instead, our experiments indicate that cytosolic phospholipase A2 (cPLA2) is likely to be involved in the intracellular production of PAF, because treatment with arachidonyl trifluoromethyl ketone (AACOCF3), a specific cPLA2 inhibitor, clearly lowered PAF levels in the nuclei of bovine embryos. Platelet-activating factor (PAF) is a well-known growth factor, modulating various physiological processes, including reproduction. For many years, PAF was believed to reside solely at the plasma membrane. Here, we describe for the first time its nuclear origin in oocytes and embryos of cattle, mice and humans. This distinct location hints towards unique subcellular functions, left to be characterised. [ABSTRACT FROM AUTHOR]

Platelet-activating factor (PAF) is a well-described autocrine growth factor involved in several reproductive processes and is tightly regulated by its hydrolysing enzyme, PAF acetylhydrolase 1B (PAFAH1B). This intracellular enzyme consists of three subunits: one regulatory, 1B1, and two catalytic, 1B2 and 1B3. PAFAH1B3 has remained uncharacterised until now. Here, we report that PAFAH1B3 is present during the different stages of the first meiotic division in bovine, murine and human oocytes. In these species, the PAFAH1B3 subunit was clearly present in the germinal vesicle, while at metaphase I and II, it localised primarily at the meiotic spindle structure. In cattle, manipulation of the microtubules of the spindle by nocodazole, taxol or cryopreservation revealed a close association with PAFAH1B3. On the other hand, disruption of the enzyme activity either by P11, a selective inhibitor of PAFAH1B3, or by PAFAH1B3 antibody microinjection, caused arrest at the MI stage with defective spindle morphology and consequent failure of first polar body extrusion. In conclusion, our results show that one of the catalytic subunits of PAFAH1B, namely PAFAH1B3, is present in bovine, murine and human oocytes and that it plays a functional role in spindle formation and meiotic progression during bovine oocyte maturation. Maturation of mammalian eggs is highly prone to chromosome segregation errors that markedly reduce female fertility. In the present study, we showed that platelet-activating factor acetylhydrolase 1B3 is localised at the meiotic spindle structure and that functional inhibition of this enzyme leads to defective spindle morphology and failure of polar body extrusion. These findings help us to better understand the origin of errors of female meiosis, causing miscarriages and congenital defects. [ABSTRACT FROM AUTHOR]

The equine oviduct plays a pivotal role in providing the optimal microenvironment for early embryonic development, but little is known about the protein composition of the oviducal fluid in the horse. The aim of the present study was to provide a large-scale identification of proteins in equine oviducal fluid and to determine the effects of ovulation and pregnancy. Four days after ovulation, the oviducts ipsilateral and contralateral to the ovulation side were collected from five pregnant and five non-pregnant mares. Identification and relative quantification of proteins in the oviducal fluid of the four groups was achieved by isobaric tags for relative and absolute quantification (iTRAQ) labelling and HPLC-tandem mass spectrometry. The presence of an embryo in the ipsilateral oviducal fluid of pregnant mares induced upregulation of 11 and downregulation of two proteins compared with the contralateral side, and upregulation of 19 proteins compared with the ipsilateral side of non-pregnant mares. Several of these upregulated proteins are related to early pregnancy in other species. The present study represents the first high-throughput identification of proteins in the oviducal fluid of the mare. The results support the hypothesis that the equine embryo interacts with the oviduct, affecting the maternal secretion pattern of proteins involved in pregnancy-related pathways. [ABSTRACT FROM AUTHOR]

In humans, there is evidence that metabolic diseases occurring in later life arise in utero as a result of programming of key endocrine systems during suboptimal intrauterine conditions. The process by which prenatal insults lead to permanent changes in tissue structure and function, and finally to low birthweight (BW), is known as developmental programming. Poor nutrition, environmental temperature, oxygen availability and overnutrition all have been shown to significantly affect intrauterine development. Because the placenta is the organ for communication between mother and fetus, placental insufficiency invariably affects embryonic development and health in later life. In order to optimise their income, dairy farmers inseminate their nulliparous heifers at adolescent age, and subsequently strive for calving intervals not longer than 380 days. Hence, heifers are still growing and multiparous animals are still yielding large quantities of milk while pregnant. Dairy cows heavily selected for milk yield have specific endocrinological characteristics, like low peripheral insulin levels and low peripheral insulin sensitivity, both contributing to safeguard glucose for milk production. The reverse of this advanced selection is the high incidence of a wide range of metabolic diseases. Evidence from epidemiological studies is now available demonstrating that milk yield during gestation and environmental factors, such as season of pregnancy and parturition, affect both the size and the intermediary metabolism of the neonatal calf. The latter suggests that further optimisation in terms of production, reproduction, general health and longevity in the dairy sector may be feasible by taking into account environmental factors occurring during pregnancy. [ABSTRACT FROM AUTHOR]

In vitro, efficient communication between mammalian embryos in groups or between embryos and cocultured somatic cells implies that there is a sender, a message and a receiver that is able to decode the message. Embryos secrete a variety of autocrine and paracrine factors and, of these, extracellular vesicles have recently been implicated as putative messengers in embryo--embryo communication, as well as in communication of the embryo with the maternal tract. Extracellular vesicles (EVs) are membrane-bound vesicles that are found in biofluids and in culture media conditioned by the presence of embryos or cells. EVs carry and transfer regulatory molecules, such as microRNAs, mRNAs, lipids and proteins. We conducted a systematic search of the literature to review and present the currently available evidence regarding the possible roles of EVs in in vitro embryo communication and embryo development. It is important to note that there is limited information available on the molecular mechanisms and many of the biologically plausible functions of EVs in embryo communication have not yet been substantiated by conclusive experimental evidence. However, indirect evidence, such as the use of media conditioned by embryos or by somatic cells with improved embryo development as a result, may indicate that EVs can be an important asset for the development of tailor-made media, allowing better embryo development in vitro, even for single embryo culture. [ABSTRACT FROM AUTHOR]

The oviduct undergoes dramatic functional and morphological changes throughout the oestrous cycle of the mare. To unravel the effects of steroids on the morphology, functionality and gene expression of the equine oviduct, an in vitro oviduct explant culture system was stimulated with physiological concentrations of progesterone and 17boestradiol. Four conditions were compared: unsupplemented preovulatory explants, preovulatory explants that were stimulated with postovulatory hormone concentrations, unsupplemented postovulatory explants and postovulatory explants that were stimulated with preovulatory hormone concentrations. The modulating effects of both steroids on oviduct explants were investigated and the following parameters examined: (1) ciliary activity, (2) glucose consumption and lactate production pattern, (3) ultrastructure, (4) mRNA expression of embryotrophic genes, (5) steroidogenic capacities of oviductal explants and (6) progesterone receptor expression. The present paper shows that the equine oviduct is an organ with potential steroidogenic capacities, which is highly responsive to local changes in progesterone and 17βoestradiol concentrations at the level of morphology, functionality and gene expression of the oviduct. These data provide a basis to study the importance of endocrine and paracrine signalling during early embryonic development in the horse. [ABSTRACT FROM AUTHOR]

Steroids play an important role in mammalian reproduction and early pregnancy. Although systemic changes in steroid concentrations have been well documented, it is not clear how these correlate with local steroid concentrations in the genital tract. We hypothesised that, in the horse, the preimplantation embryo may be subjected to high local steroid concentrations for several days. Therefore, we measured progesterone, 17-hydroxyprogesterone, 17b-oestradiol, testosterone and 17a-testosterone concentrations in equine oviductal tissue by ultra-HPLC coupled with tandem mass spectrometry, and progesterone, 17b-oestradiol, oestrone and testosterone concentrations in oviduct fluid by radioimmunoassay, with reference to cycle stage and side of ovulation. Progesterone concentrations were high in oviductal tissue and fluid ipsilateral to the ovulation side during dioestrus, whereas other steroid hormone concentrations were not influenced by the side of ovulation. These results suggest that the high ipsilateral progesterone concentration is caused by: (1) contributions from the follicular fluid in the oviduct and diffusion of follicular fluid steroids after ovulation; (2) local transfer of steroids via blood or lymph; (3) local synthesis of progesterone in the oviduct, as evidenced by the expression of steroidogenic enzymes; and (4) a paracrine contribution from follicular cells. These data provide a basis for the study of the importance of endocrine and paracrine signalling during early embryonic development in the horse. [ABSTRACT FROM AUTHOR]

Retrotransposons are transposable elements that insert extra copies of themselves throughout the genome via an RNA intermediate using a 'copy and paste' mechanism. They account for more than 44% of the bovine genome and have been reported to be functional, especially during preimplantation embryo development. In the present study, we tested whether high oxygen tension (20% O2) influences global DNA methylation analysed by immunofluorescence staining of developing bovine embryos and whether this has an effect on the expression of some selected retrotransposon families. High oxygen tension significantly increased global DNA methylation in 4-cell embryos and blastocysts. A significant expression difference was observed for ERV1-1-I_BT in female blastocysts, but no significant changes were observed for the other retrotransposon families tested. Therefore, the study indicates that global DNA methylation is not necessarily correlated with retrotransposon expression in bovine preimplantation embryos. [ABSTRACT FROM AUTHOR]

Many studies have shown that in vitro culture can negatively impact preimplantation development. This necessitates some selection criteria for identifying the best-suited embryos for transfer. That said, embryo selection after in vitro culture remains a subjective process in most mammalian species, including cows, mice and humans. General consensus in the field is that embryos that develop in a timely manner have the highest developmental competence and viability after transfer. Herein lies the key question: what is a timely manner? With emerging data in bovine and mouse supporting increased developmental competency in embryos with moderate rates of development, it is time to question whether the fastest developing embryos are the best embryos for transfer in the human clinic. This is especially relevant to epigenetic gene regulation, including genomic imprinting, where faster developing embryos exhibit loss of imprinted methylation, as well as to sex selection bias, where faster developmental rates of male embryos may lead to biased embryo transfer and, in turn, biased sex ratios. In this review, we explore evidence surrounding the question of developmental timing as it relates to bovine embryo quality, mouse embryo quality and genomic imprint maintenance, and embryo sex. Many studies have shown that in vitro culture can negatively impact preimplantation development, which necessitates some selection criteria to identify the best-suited embryos for transfer. With emerging data in bovine and mouse supporting increased developmental competency in embryos with moderate rates of development, it is time to question whether the fastest developing embryos are the best embryos for transfer in the human clinic. In this review we explore evidence surrounding the question of developmental timing as it relates to bovine embryo quality, mouse embryo quality and genomic imprint maintenance, and embryo sex. [ABSTRACT FROM AUTHOR]

Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n = 1239) were collected at three different time points (12, 18 and 24 h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P < 0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential. Shortly after fertilisation, methylation of both maternal and paternal DNA is reprogrammed for further embryo development. The objectives of this study were to investigate DNA methylation reprogramming following fertilisation of oocytes with heat-stressed spermatozoa and the results show that the normal pattern of DNA methylation reprogramming is perturbed in the paternal genome, leading to a lower fertilisation rate. Our data can be used to address lower fertilisation potential of spermatozoa with suboptimal quality. [ABSTRACT FROM AUTHOR]

Equine embryos remain for 6 days in the oviduct and thus there is a need for an in vitro model to study embryo-oviductal interactions in the horse, since this subtle way of communication is very difficult to analyse in vivo. Until now, no equine oviduct explant culture model has been characterised both morphologically and functionally. Therefore, we established a culture system for equine oviduct explants that maintained epithelial morphology during 6 days of culture, as revealed by light microscopy and transmission electron microscopy. We demonstrated the presence of highly differentiated, tall columnar, pseudostratified epithelium with basal nuclei, numerous nucleoli, secretory granules and apical cilia, which is very similar to the in vivo situation. Both epithelium and stromal cells originating from the lamina propria are represented in the explants. Moreover, at least 98% of the cells remained membrane intact and fewer than 2% of the cells were apoptotic after 6 days of culture. Although dark-cell degeneration, which is a hypoxia-related type of cell death, was observed in the centre of the explants, quantitative real-time PCR failed to detect upregulation of the hypoxia-related marker genes HIF1A, VEGFA, uPA, GLUT1 and PAI1. Since the explants remained morphologically and functionally intact and since the system is easy to set up, it appears to be an excellent tool for proteome, transcriptome and miRNome analysis in order to unravel embryo-maternal interactions in the horse. The subtle manner of embryo-oviductal communications in the horse is very difficult to analyse in vivo. Therefore, we developed a system sustaining equine oviduct explants, bordered by highly differentiated, functional and intact epithelial cells, with only a negligible percentage of apoptotic or necrotic cells and so mimicking the in vivo situation very closely. Since the system is easy to set up, it appears to be an excellent tool for proteome, transcriptome and miRNome analysis, in order to unravel embryo-maternal interactions in the horse. [ABSTRACT FROM AUTHOR]

Individual culture of bovine embryos is usually associated with low blastocyst development. However, during preliminary experiments in our laboratory we observed high blastocyst development after individual embryo culture in a serum-free culture system. We therefore hypothesised that serum has a negative effect on embryos cultured individually whereas embryos in groups can counteract this. First, we determined whether the timing of removal of serum (during maturation or culture) had an influence on individual embryo development. The results clearly showed that removal of serum during embryo culture was the main contributing factor since high blastocyst development was observed after individual culture in synthetic oviductal fluid supplemented with bovine serum albumin (BSA) and insulin, transferrin and selenium (ITS), independent of the maturation medium. Second, we investigated whether an individual factor of the ITS supplement was essential for individual embryo development. We demonstrated that repeatable high blastocyst percentages were due to the synergistic effect of ITS. Finally, we investigated if a group-culture effect can still be observed under serum-free conditions. Group culture generated blastocysts with higher total cell numbers and less apoptosis. These data show that individual culture in serum-free conditions leads to high blastocyst development, but group culture still improves blastocyst quality. Most attempts to introduce individual culture in cattle embryos have shown a reduction in development from a typical 25-35% blastocysts in group culture to less than 10% in individual culture. However, we observed that the very simple act of using serum-free culture medium based on BSA and ITS unexpectedly led to high blastocyst development in individual culture. The development of a semi-defined individual culture system is not only an important tool for research laboratories and practitioners working with bovine IVF; in addition this study upgraded the value of bovine embryos as a model for human IVF. [ABSTRACT FROM AUTHOR]

Heat stress has long been recognised as a cause of subfertility in farm animals. The objectives of the present study were to elucidate the effect of heat stress on sperm function and involvement of the mitogen-activated protein kinase (MAPK) 14 signalling pathway. Spermatozoa incubated for 4 h at a physiological temperature (38.5°C) exhibited significantly (P < 0.05) reduced motility, plasma membrane integrity and mitochondrial potential compared with non-incubated spermatozoa; the reductions in these parameters were more severe following incubation at a hyperthermic (41 °C) temperature (P < 0.01). Percentages of fertilisation and embryo development were highly affected in spermatozoa incubated at 41 °C compared with non-incubated spermatozoa (P < 0.01). Similarly, embryo quality was adversely affected by sperm incubation at 41 °C, as indicated by a higher apoptotic cell ratio in Day 7 blastocysts compared with that in the non-incubated control group (14.6% vs 6.7%, respectively; P < 0.01). Using SB203580 (10 μgmL[sup -1]), a specific inhibitor of the p38 MAPK pathway, during sperm hyperthermia reduced MAPK14 activation (24.9% vs 35.6%), increased sperm motility (45.8% vs 26.5%) and reduced DNA fragmentation (16.9% vs 23.4%) compared with the untreated control group, but did not improve subsequent fertilisation and embryo development. In conclusion, heat stress significantly affects the potential of spermatozoa to penetrate oocytes, as well as subsequent embryo development and quality. Notably, the data show that the MAPK14 signalling pathway is largely involved in heat-induced sperm damage. However, further research is needed to elucidate other signalling pathways possibly involved in heat-induced sperm damage. [ABSTRACT FROM AUTHOR]

During mammalian preimplantation development, two successive differentiation events lead to the establishment of three committed lineages with separate fates: the trophectoderm, the primitive endoderm and the pluripotent epiblast. In the mouse embryo, the molecular mechanisms underlying these two cell fate decisions have been studied extensively, leading to the identification of lineage-specific transcription factors. Species-specific differences in expression patterns of key regulatory genes have been reported, raising questions regarding their role in different species. The aim of the present study was to characterise the gene expression patterns of pluripotency (OCT4, SOX2, NANOG) and differentiation (CDX2, GATA6)-related markers during feline early development using reverse transcription-quantitative polymerase chain reaction. In addition, we assessed the impact of in vitro development on gene expression by comparing transcript levels of the genes investigated between in vitro and in vivo blastocysts. To normalise quantitative data within different preimplantation embryo stages, we first validated a set of stable reference genes. Transcript levels of all genes investigated were present and changed over the course of preimplantation development; a highly significant embryo-stage effect on gene expression was observed. Transcript levels of OCT4 were significantly reduced in in vitro blastocysts compared with their in vivo counterparts. None of the other genes investigated showed altered expression under in vitro conditions. The different gene expression patterns of OCT4, SOX2, CDX2 and GATA6 in cat embryos resembled those described in mouse embryos, indicative of a preserved role for these genes during early segregation. However, because of the absence of any upregulation of NANOG transcription levels after embryonic genome activation, it is unlikely that NANOG is a key regular of lineage segregation. Such results support the hypothesis that the behaviour of early lineage markers can be species specific. The present study also revealed a pool of maternal NANOG mRNA transcripts, the role of which remains to be elucidated. Comparing transcription levels of these genes between in vivo and in vitro blastocysts revealed low levels of OCT4 mRNA in the latter, which may contribute to the reduced developmental competence of embryos under suboptimal conditions. A basic understanding of the molecular pathways that regulate developmental events during early feline embryogenesis is still lacking. The aim of the study was to quantify mRNA profiles of pluripotency and differentiation-related markers during preimplantation development and results showed that these genes are expressed and undergo species-specific changes during early embryonic development. These findings add evidence to the hypothesis that behavior of these markers may differ among species. [ABSTRACT FROM AUTHOR]

Exposure of gametes to specific stressors at sublethal levels can enhance the gametes' subsequent performance in processes such as cryopreservation. In the present study, bull spermatozoa were subjected to H[sub 2]O[sub 2] for 4 h at 100-, 200- and 500-μM levels; computer-assisted sperm analysis (CASA) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay were used for evaluation of subsequent sperm motility and DNA integrity, respectively. Exposure of spermatozoa to H[sub 2]O[sub 2] did not affect sperm motility but DNA integrity was negatively affected by 500 μM H[sub 2]O[sub 2] compared with mock-exposed spermatozoa, whereas both motility and DNA integrity were affected compared with untreated spermatozoa. Nevertheless, insemination of oocytes with spermatozoa exposed to 200 μM H[sub 2]O[sub 2] increased fertilisation, cleavage and blastocyst rates (P < 0.05). Furthermore, the higher blastocyst yield after fertilisation of oocytes with spermatozoa exposed to 200 μM H[sub 2]O[sub 2] was related to oocyte diameter, with large-medium oocytes yielding higher blastocyst rates, while small-diameter oocytes consistently failed to develop into blastocysts. In conclusion, the results indicate that exposure of spermatozoa to 200 μM H[sub 2]O[sub 2] before sperm-oocyte interaction may enhance in vitro embryo production in cattle. However, this increased embryo production is largely dependent on the intrinsic quality of the oocytes. Oxidative stress is, in general, pernicious for cells, but specific stressors, such as hydrogen peroxide, may have a beneficial effect on cell function, depending on the concentration used. In this study we exposed bovine sperm cells to different levels of hydrogen peroxide before fertilisation and found that spermatozoa exposed to sublethal levels of hydrogen peroxide can produce more blastocysts of better quality, depending on the diameter of the fertilised oocyte. This implies that stressed spermatozoa may improve subsequent embryo development in combination with careful selection of oocytes. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the editor discusses various reports within the issue related to topics which include recognition of pregnancy in cattle and pigs, fertilization in horses and placenta ion in cattle and pigs.

In vitro-produced (IVP) equine blastocysts can give rise to successful pregnancies, but their morphology and developmental rate differ from those of in vivo-derived equine blastocysts. The aim of the present study was to evaluate this difference at the genetic level. Suppression subtractive hybridisation (SSH) was used to construct a cDNA library enriched for transcripts preferentially expressed in in vivo-derived equine blastocysts compared with IVP blastocysts. Of the 62 different genes identified in this way, six genes involved in embryonic development (BEX2, FABP3, HSP90AA1, MOBKL3, MCM7and ODC) were selected to confirm this differential expression by reverse transcription–quantitative real-time polymerase chain reaction (RT-qPCR). Using RT-qPCR, five genes were confirmed to be significantly upregulated in in vivo-derived blastocysts (i.e. FABP3, HSP90AA1(both P<0.05), ODC, MOBKL3and BEX2(P<0.005 for all three)), confirming the results of the SSH. There was no significant difference in MCM7expression between IVP and in vivo-derived blastocysts. In conclusion, five genes that are transcriptionally upregulated in in vivo-derived equine blastocysts compared with IVP blastocysts have been identified. Because of their possible importance in embryonic development, the expression of these genes can be used as a marker to evaluate in vitroembryo production systems in the horse. [ABSTRACT FROM AUTHOR]

Although the transfer of embryos is much less likely to result in disease transmission than the transport of live animals, the sanitary risks associated with embryo transfer continue to be the subject of both scientific investigations and adaptations of national and international legislation. Therefore, the implications are important for veterinary practitioners and livestock breeders. In vivo-derived and in vitro-produced embryos are widely used in cattle and embryos from other species, such as sheep, goats, pigs and horses, are also currently being transferred in fairly significant numbers. Bearing in mind the wide variety of embryos of different species and the correspondingly large number of viruses that are of concern, it is expedient at this time to look again at the importance of the zona pellucida (ZP) as a barrier against viruses and at the susceptibility or otherwise of embryonic cells to viral infection if ever they are exposed. For embryos with an intact ZP, viral infection of the embryo is unlikely to occur. However, the virus may stick to the ZP and, in this case, International Embryo Transfer Society (IETS) washing procedures in combination with trypsin treatment are mandatory. A caveat is the fact that currently more and more types of embryos are becoming available for transfer and scientific data cannot be extrapolated from one species to another. These topics are discussed in the present review. [ABSTRACT FROM AUTHOR]

An introduction to the journal is presented in which the editor discusses various reports within the issue on topics including the different aspects of epigenetic mechanisms and how they are influenced by the periconception environment.

The recent blue tongue virus serotype-8 (BTV-8) epidemic in central Western Europe has been associated with field fertility problems (De Clercq et al.2008 Transbound. Emerg. Dis. 55, 352–359). Previous research clearly showed that in vitroproduced bovine hatched blastocysts are susceptible for BTV-8 infection (Vandaele et al.2010 Reprod. Fertil. Dev. 22, 254 abst.). The aim of the present study was to investigate the effect of a BTV-8 infection on the occurrence of apoptosis in embryos in order to gain a clear insight into the role BTV-8 might play in early embryonic death. Immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes were denuded 24h post-insemination and cultured in 50-μL droplets of modified SOF medium with 10% fetal calf serum (tested negative for BTV antibodies) at 38.5°C in 5% CO2, 5% O2, and 90% N2. At 7 days post-insemination (dpi), blastocysts were grouped to enhance hatching. At 8.5 dpi, 4 to 8hatched embryos were placed in 800μL of minimum essential medium (MEM), containing 103.8to 104.950% tissue culture infectious doses (TCID50) of BTV-8 (Bel 2006/3, VAR, Brussels, Belgium) and incubated for 1h at 38.5°C in an atmosphere of 5% CO2in air. Two groups of hatched control embryos were incubated under the same circumstances in 800μL of SOF and 800μL of MEM, respectively. After inoculation, embryos were washed according to IETS guidelines with the exception that they were not ZP-I and cultured in new SOF. At 48 and 72h post-inoculation (hpi), half of the embryos of each group were fixed in 4% paraformaldehyde for 12 to 24h and subsequently stained for BTV-8 and apoptosis with a double immunofluorescent staining using a BTV-8 monoclonal antibody (8A3B.6, ID-Vet, Montpellier, France) in combination with TUNEL (Roche Diagnostics, Mannheim, Germany). All mock-inoculated embryos were negative for BTV-8 virus antigen. At 48 and 72hpi, respectively, 45 and 38% of the embryos were BTV-8 positive in all embryonic cells, and all remaining embryos had at least some BTV-8 blastomeres. The overall apoptotic cell ratio in infected embryos (17.9±1.14% at 48hpi and 15.3±1.16% at 72hpi) was significantly higher than in noninfected, mock-inoculated embryos (10.4±0.57% at 48hpi and 4.3±0.30% at 72hpi) (P<0.01). Furthermore, total cell number was substantially lower in infected embryos (103±14.2 at 48hpi and 120±17.5 at 72hpi) compared with noninfected, mock-inoculated embryos (258±47.4 at 48hpi and 348±64.1 at 72hpi) (P<0.05). This study indicates that embryonic apoptosis after BTV-8 infection results in embryonic arrest and early embryonic death and thus might be involved in herd fertility problems during a BTV epidemic. The first author is supported by Research Foundation Flanders (Grant number G.0210.09). [ABSTRACT FROM AUTHOR]

The hyperthermia associated with summer heat stress (HS) is a major cause of reduced fertility in high yielding dairy cows in the tropics and subtropics. The hyperthermia directly affects reproductive function. Furthermore, HS induces indirect effects (e.g. through a dropped dry matter intake and thus an altered energy balance), which may also have an impact on cow fertility. The extent of exposure to HS and HS-associated metabolic changes, such as hypoglycemia and high levels of non-esterified fatty acids (NEFA), may affect cattle fertility by impairing the developmental capacity of the oocyte. It is known that changes in glucose and NEFA concentrations in the blood may be reflected in the follicular fluid of the dominant follicle (Leroy et al.2004). Therefore, the aim of the current study was to estimate to what extent oocytes may be exposed to HS-associated metabolic changes in dairy cattle during summer by assessing the diurnal changes in plasma. Respiration rate (RR) and rectal temperature (RT) along with plasma concentrations of glucose and NEFA were examined in 10 lactating Holstein cows at 4-h intervals from 7:00 AM to 3:00 AM for three successive days in August 2007. Temperature humidity indices (THI) were calculated as well. Linear mixed effect models with cow as random factor including the effect of daytime were used to investigate possible differences among studied parameters at different daytimes. At 3:00 PM, THI and RT were the highest (83.7 ± 0.6 and 40.1 ± 0.1, respectively) and the lowest value for THI recorded at 3:00 AM (74.2 ± 1.4) and for RT recorded at 7:00 AM (39.04 ± 0.08). RR increased gradually from 66.1 ± 3.1 resp min-1at 3:00 AM to 93.0 ± 4 resp min-1at 11:00 AM. Glucose concentrations decreased significantly (P< 0.001) at 11:00 AM and 3:00 PM. Decreased glucose concentrations coincided with increased NEFA concentrations at 11:00 AM and 3:00 PM (P< 0.001). The NEFA concentrations measured at 3:00 PM were significantly higher than at all other time points (Table 1). In conclusion, high producing dairy cows may be exposed to diurnal fluctuation of HS-associated metabolic changes such as significantly decreased glucose and increased NEFA concentrations in plasma for about 12 h per day, which may be reflected in the follicular fluid of the dominant follicle and as such directly affect oocyte and granulosa cell quality.Table 1.Mean ± SEM of glucose and non-esterified fatty acids (NEFA) concentrations (mmol L-1) in blood plasma of 10 lactating Holstein cows at 6 different daytimes during August 2007 in Egypt [ABSTRACT FROM AUTHOR]

High concentrations of non-esterified fatty acids (NEFA) during negative energy balance (NEB) in high yielding dairy cows have been proven to be partially responsible for reduced fertility. This hypothesis has been tested by the addition of NEFAs to in vitro maturation medium at concentrations present in follicular fluid during NEB. We aimed to evaluate whether high concentrations of palmitic acid (C16:0) (PA), stearic acid (C18:0) (SA), or oleic acid (C18:1) (OA) during oocyte maturation could have a carry-over effect on embryo quality and could subsequently affect embryo cryotolerance. Cumulus?oocyte complexes (n = 4600) were matured in serum-free TCM199 plus epidermal growth factor (EGF, 20 ng mL?1; negative control), supplemented with ethanol alone (positive control) or with 0.133 mmol L?1 PA, 0.067 mmol L?1 SA, or 0.200 mmol L?1 OA (NEFAs dissolved in ethanol). The three NEFAs were tested separately in 4 replicates for PA and 5 replicates for OA or SA. Each fatty acid tested per replicate including a negative and a positive control group. After the embryos were cultured for 7 days in SOF medium, the number of blastocysts was recorded and classified as expanded, hatching, or hatched. Then, blastocysts were cryopreserved by open pulled straw vitrification using the two-step approach described by Vajta et al. (1998 Mol. Reprod. Dev. 51, 53?58). Vitrified warmed embryos were cultured in groups of <25 per 50-?L droplet of modified SOF medium with 5% fetal calf serum (FCS) under mineral oil for 48 h and examined for re-expansion and hatching. The percentages of survival in the different treatment groups were analyzed using logistic regression analyses, including the effect of replicates. Survival or not was included as the dependent variable and group was the independent variable. For every fatty acid a separate model was used. For all analyses, differences were considered to be statistically significant at the P < 0.05 level. Addition of OA to in vitro maturation media had no significant effects on cryotolerance of embryos. However, addition of PA or SA to in vitro maturation media (Table 1) significantly (P < 0.05) decreased the survival of bovine blastocysts from 79% in the positive control to 57% in PA and from 61% to 53% in SA. The results of the present study indicate that maturation of oocytes in the presence of NEB-associated concentrations of PA and SA can have carry-over effects on embryo quality, leading to reduced cryotolerance. We suggest that elevated NEFA concentrations in the follicular fluid may be one of the factors through which NEB exerts its negative effects on fertility in high yielding dairy cows. [ABSTRACT FROM AUTHOR]

Bovine viral diarrhea virus (BVDV) causes serious economic losses in the cattle industry. Evidence exists that only zona pellucida (ZP)-free bovine embryos are susceptible to BVDV infection (Vanroose et al. 1998 Biol. Reprod. 58, 857?866); however, BVDV can adhere to and therefore ?infect'' both in vivo-(Waldrop et al. 2004 Theriogenology 62, 387?397) and in vitro-produced ZP intact embryos (Stringfellow et al. 2000 Theriogenology 53, 827?839). To eliminate these sanitary risks, pre-treatment of embryos with antiviral compounds may be a promising approach (Givens et al. 2006 Theriogenology 65, 344?355). BPIP (5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine) has been reported to display antiviral activity against BVDV, with a 50? effective inhibition of BVDV-induced cytopathic effect formation at a concentration of 0.04 M (Paeshuyse et al. 2006 J. Virol. 80, 149?160). However, since the short- and long-term effects of BPIP have not been described, the aim of the current study was to assess whether addition of BPIP for 2 days at a concentration of 5 M is toxic for ZP-free cattle embryos. Oocytes were aspirated from 3?6-mm follicles of cattle ovaries, matured for 24 h, and subsequently co-incubated with 1 106 sperm cells mL-1 in IVF-TALP with 20 g/mL-1 heparin for 24 h at 39C and 5? CO2 in air. After fertilization, presumptive zygotes were put in groups of 25 into 50-L droplets of SOF under oil in 5? CO2, 5? O2, and 90? N2 for 6 days. Afterwards, morulae and blastocysts were collected, rendered ZP-free by means of pronase treatment, and divided into 4 groups: (i) ZP-free control group, (ii) ZP-free control group treated with a volume of DMSO equal to condition (iv), (iii) ZP-free group treated with 5 M BPIP in DMSO, and (iv) ZP-free group treated with 10 M BPIP in DMSO. Because BPIP is a fat-soluble molecule, embryos were cultured in 0.5 mL SOF without oil for 2 days. At Day 8, all embryos were fixed, TUNEL-stained, and analyzed for total cell number and percentage of apoptotic cells. Three independent replicates were performed. Results are shown in Table 1 and were analyzed by means of ANOVA. Only group iv showed a significant decrease in total cell number, indicating that at 10 M BPIP may negatively influence embryo development. At both 5 and 10 M, BPIP treatment resulted in an increase in percentage of apoptotic cells compared to the control group. However, a similar increase was observed using DMSO alone (group ii), indicating that the apoptotic effect may be due solely to the DMSO. In conclusion, BPIP does not appear to cause embryo toxicity at 5 M, but an alternative, less toxic, dissolving agent may be considered. [ABSTRACT FROM AUTHOR]

An abstract of the study "Prevalence of Membrane-Damaged Cells in Bovine Embryos After Culture to the Blastocyst Stage in Defined Medium," by S. Heras and colleagues is presented.

An abstract of the study "Steroid-Regulated mRNA Expression in Oviduct Epithelial Cells in the Mare," by H. M. Nelis et al. is presented.

An abstract of the study "Bovine Embryo Culture in the Modified Well of the Well System Increases Blastocyst Rate But Lowers Blastocyst Quality," by S. Heras and colleagues is presented.

An abstract of the study "Altered Chromatin Conformation in Bovine Spermatozoa Perturbs Dynamic DNA Methylation in the Male Pronucleus After Fertilization in Vitro," by M. B. Rahman et al is presented.

Heat stress can cause bulls to start producing sub- or even infertile semen. This is usually not noticed immediately during quality control while processing semen. Sometimes, a heat period is already over before an increase of rejection ejaculates is noticed in the laboratory. This delayed effect, but also because a heat period is often not discrete or clear-cut, makes it difficult to relate heat stress with semen quality. The recent study of Rahman et al. (Theriogenology, in press) is therefore of interest, given that heat stress was mimicked by elevation of testicular temperature (~2°C, 48 h) of 2 Holstein Friesian bulls in a controlled experiment. Analyses of semen, collected 1 week before until 9 weeks after the experiment, revealed that specific stages in spermatogenesis, meiosis and spermiogenesis are susceptible to heat stress. During these stages, the percentages of morphologically normal, progressive motile and membrane-intact cells reduced by approximately 33, 25 and 20%, respectively. The objective of this study was to model how these reductions would affect rejection rates for these parameters in a moderate climate, such as the Netherlands. The standards of the 3 evaluated quality parameters, which are used by many AI stations, are per ejaculate more than 75% morphologically normal cells, more than 35% progressive motile and 50% membrane intact cells after thawing. Normal rejection rates were determined from original scores of at least 2 years from 2 AI stations (CRV, the Netherlands). That is, 11 453 morphology scores of fresh semen, 10 805 progressive motility and 3741 membrane integrity scores of thawed semen from 2065, 492 and 1488 bulls, respectively. To estimate rejection rates as if ejaculates were affected by heat stress, each morphology, progressive motility and membrane integrity score was reduced by 33, 25 and 20%, respectively. Normal vs estimated heat stress rejection rates, based on original and modeled scores, respectively, were 50 vs 100% for morphology, 2 vs 17% for progressive motility and 9 vs 61% for membrane integrity. Rahman showed that already a short period of heat stress can lead to reduced sperm quality for over a month. This study indicates that this effect can be very important for AI stations. In the case of progressive motility, which in the Netherlands is scored for each ejaculate, the estimated higher rejection rate of 17% could lead to a considerable increase of production costs. Membrane integrity is occasionally scored (when requested for export of semen), but morphology is always scored when bulls start to produce semen until ejaculates are produced in succession with more than 75% normal cells. High rejection rates for morphology are typical when bulls (~11 months old) start, but this decreases quickly for most bulls to 0% within a few months as they mature. When these young bulls suffer heat stress before production, it is possible that the rejection rate will stay at 100% for a long time. When this is suspected, a prolonged test period should be considered to avoid wrongful culling of bulls. [ABSTRACT FROM AUTHOR]

Bluetongue virus (BTV) has been categorized by the OIE as a category 1 disease agent, for which proper handling between collection and transfer is thought to be sufficient to prevent transmission through embryo transfer. For bovine viral diarrhoea virus, it was shown that effectiveness of washing procedures depends on virus strains (Waldrop et al. 2004 Theriogenology 62, 45-55). Also BTV-8 has unique characteristics in comparison with other strains (De Clercq et al. 2008 Transbound. Emerg. Dis. 55, 352-359). The aim here was to investigate whether embryo transfer of in vivo-derived bovine embryos after in vitro exposure to BTV-8 can be performed without risk for infection of the recipients if IETS washing and trypsin treatment procedures are followed. Donor cows (n = 2) were synchronized and superovulated using Stimufol® (Ulg, Liége, Belgium) and subsequently inseminated. At 6.5 days post-insemination (dpi), flushed embryos (n = 14 and n = 3) were placed in 800 μL of minimal essential medium (MEM), containing 104.9 50% tissue culture infectious doses (TCID50) of BTV-8 (Bel 2006/2 P5, VAR, Brussels, Belgium) and incubated for 1 h at 39°C in 5% CO2 in air (Vandaele et al. 2011 Vet. Res. 42, 14-21). Next, embryos were washed in pairs in 5 consecutive Petri dishes containing PBS with antibiotics and 0.4% BSA, w/o Ca and Mg. Then, embryos were exposed to 2 consecutive trypsin (Invitrogen, Carlsbad, CA, 25050-014) washes of 45 s each at 39°C in 5% CO2 in air and finally, another 5 consecutive washes in PBS with 2% FCS. Each Petri dish contained at least 2 mL of medium and was gently agitated between washes. Embryos were transferred in a maximum of 7 μL of medium and a new tip was used after every wash step. Washes 1 to 5 and washes 6 to 10 were pooled and analysed for BTV-8 (RT-qPCR). After these washes, 3 pairs of embryos (n = 6) were loaded in straws and transferred to 3 BTV-8 negative recipients. Two sentinel cows served as control. Cows were bled twice weekly and blood and serum samples were analysed for BTV-8 (RT-qPCR) and BTV-8 antibodies. Viral BTV-RNA was detected in all 3 recipient cows at 7 days after transfer and viraemia was confirmed by the establishment of high antibody titers at 14 days after transfer. Viral BTV-RNA was detected in washes 1 to 5 for each pair of embryos (Cp-value around 29), whereas washes 6 to 10 had Cp-values around the cut-off value (40), indicating that probably the last wash was BTV-8 negative. None of the recipients was pregnant at 28 days post-transfer. In conclusion, washing and trypsin treatment did not succeed in removing BTV-8 from in vitro-spiked in vivo-derived bovine embryos. These unexpected results stress the need for further in vivo research, e.g. what is the virus load in vivo embryos may be exposed to in utero during viraemia? Does BTV-8 react differently with the zona compared with other strains? Are alternative washing procedures needed to remove BTV-8 from the zona? [ABSTRACT FROM AUTHOR]

In pigs, the exact role of the cumulus oöphurus during IVF still needs to be clarified. Indirect evidence exists that the rate of cumulus expansion is positively correlated with the defense against polyspermy. Epidermal growth factor (EGF) acts synergistically with FSH in the synthesis of hyaluronic acid, the deposition of which in the extracellular matrix is a prerequisite for cumulus expansion. Therefore, we aimed to evaluate the importance of cumulus expansion for fertilization results of porcine cumulus–oocyte complexes (COC) by using different EGF concentrations in the maturation medium. Cumulus–oocyte complexes were matured in vitroin NCSU23 medium supplemented with 10% follicular fluid (FF, obtained from 6- to 10-mm follicles) or 10% serum and 10, 20, or 50ngmL-1of EGF (n=480 per experiment). In vitromaturation (IVM) in the presence of 10% FF and 10ngmL-1of EGF served as the control group. At 0, 22, 36, and 44h of IVM, 20 COC of each group were selected for evaluation of cumulus expansion by measuring the maximum distance across the cumulus matrix (3 replications). Matured COC were co-incubated with frozen–thawed semen (6000 spermatozoa per oocyte) for 6h. Subsequently, oocytes were cultured for 18h. Zygotes were stained with 10μgmL-1of bis-benzimide (Hoechst) to assess the fertilization rate, polyspermy, and sperm penetration index (sp index, mean number of penetrated spermatozoa per fertilized oocyte; 2 replications). Differences in cumulus diameter were analyzed by one-way ANOVA. Fertilization parameters were analyzed by applying a logistic regression model to the results. Cumulus–oocyte complexes selected for IVM had a mean diameter of 240μm. After 22h of IVM in 10% FF, the mean diameter of COC was 336, 313, and 300μm for 10, 20, and 50ngmL-1of EGF, respectively. After 44h of IVM, these diameters had increased to 425, 388, and 397μm. Twenty-two hours of IVM in 10% serum resulted in a COC diameter of 296, 305, and 276μm for 10, 20, and 50ngmL-1of EGF. After 44h of IVM, these diameters reached 330, 325, and 275μm, respectively. Only 10% serum with 50ngmL-1of EGF proved unfavorable for cumulus expansion (P<0.05). In vitromaturation for 44h in 10% serum resulted in a smaller rate of cumulus expansion compared with IVM in 10% FF (P<0.05), irrespective of EGF concentration. Penetration rate fluctuated between 84 and 100%, with no significant differences. Monospermic fertilization was lower in COC matured in the presence of 50ngmL-1of EGF compared with the control group (P<0.05). The sp index increased in parallel with EGF concentrations and was higher after IVM in 10% serum than in 10% FF. Oocytes were penetrated by 3.1, 4.3, and 6.0 spermatozoa after IVM in serum with 10, 20, and 50ngmL-1of EGF, respectively. Results showed a tendency toward a lower rate of cumulus expansion concomitant with higher EGF concentrations. Follicular fluid was superior to serum in supporting cumulus expansion. Oocytes were penetrated by more spermatozoa when matured in 10% serum and 20 or 50ngmL-1of EGF compared with 10ngmL-1of EGF. Thus, the degree of cumulus expansion appears to be related to the sp index, confirming that the cumulus matrix may play a role in the polyspermy defense. This study was supported by Research Foundation-Flanders. [ABSTRACT FROM AUTHOR]

Isolation of pure inner cell mass (ICM) and trophoblast samples from a single blastocyst is necessary to obtain accurate information on the transcriptome of these cells. Microsurgical techniques have been described to separate the ICM and trophoblast, but unfortunately, contamination of the ICM cell population with trophoblastic cells is inevitable with these methods. Alternatively, immunosurgery has been described as a valuable technique to obtain a pure ICM sample, although this technique seems to alter the normal gene expression pattern. Laser capture microdissection (LCM) provides the possibility of isolating small tissue fractions from heterogeneous tissue sections, without contamination by the surrounding tissue and without changing the gene expression pattern of the cells. In this study, a protocol is described for the application of LCM to isolate homogeneous ICM and trophoblast samples from single bovine blastocysts for downstream gene expression analysis. The absence of contaminating trophoblastic fractions in the isolated ICM cells was controlled with primers for the keratin 18 (KRT18) gene, which is considered a trophoblast-specific marker in bovine blastocysts. Expanded blastocysts were produced by routine in vitromethods described by (Vandaele et al.2010 Reproduction 139, 505–511) and fixed in a modified methacarn solution for 24h. After fixation, the blastocysts were embedded in RNase-free soluble agarose 2%, processed in an STP 420D Tissue Processor, embedded in paraffin, cut in serial sections, and adhered to glass slides, followed by deparaffinization in xylene and staining of the sections with 0.1% cresyl violet in a 85% ethanol solution. Laser capture microdissection was performed as described previously by (De Spiegelaere et al.2008 Anal. Biochem. 382, 72–74). The ICM was isolated by placing the same cap over 3 to 4 serial sections of one blastocyst. Subsequently, the same procedure was performed with a second cap to isolate the trophoblast. Total RNA was isolated from the LCM-derived ICM and trophoblast on the caps and converted into cDNA. Gene-specific primers for KRT18 (5′-GCAGACCGCTGAGATAGGA-3′ and 5′-GCATATCGGGCCTCCACTT-3′) and for 18S rRNA, a commonly used reference gene (5′-AGAAACGGCTACCACATCCA-3′ and 5′-CACCAGACTTGCCCTCCA-3′), were used and PCR was carried out. Expression of the control gene 18S rRNA was readily detectable in all cell samples. Keratin 18 was detectable in LCM-derived trophoblast, but was absent in the LCM-derived ICM cells, indicative of the successful isolation of ICM cells without contaminating trophoblastic cells. This study demonstrates a novel approach for the application of LCM on small tissue samples that are difficult to handle and which can be used for molecular analysis of specific cell lineages within embryos of different species. Supported by the Fund for Scientific Research–Flanders, Belgium, aspirant 1.1.477.07N00. [ABSTRACT FROM AUTHOR]

Semen can be collected from tomcats by urethral catheterization (CT) after medetomidine administration, offering a novel approach to obtain sperm for in vitrofertilization. This study was designed to determine motion characteristics of CT sperm samples by means of computer-assisted sperm analysis (CASA) and to compare sperm quality parameters and in vitrofertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in 17 adult cats by urethral catheterization as reported by Zambelli et al.(2008 Theriogenology 69, 485-490), after which the cat was orchidectomized. Motility (subjective and objective by means of CASA), morphology [eosin/nigrosin (E/N)], plasma membrane integrity (E/N and SYBR14-PI staining), and acrosomal status (FITC-PSA staining) of fresh CT and EP samples were evaluated and compared between both methods with a paired t-test or the Wilcoxon Rank test, dependent on normality of the data. In vitromaturation (24 h), fertilization (18 h), and culture (6h) of grade I to III oocytes were carried out as described by Pope et al.(2009 Theriogenology 71, 864-871). Twenty-four hours after in vitroinsemination, fertilization rates were assessed for group 1 (CT; n= 148) and group 2 (EP; n= 159) presumptive zygotes. The distribution of presumptive zygotes between CT and EP over the different developmental stages was compared using Pearson chi-square test. Results showed that total and progressive motility as well as the percentage of normal spermatozoa were higher for EP sperm compared with CT sperm (P 0.05) between collection techniques. In group 1, 84% of in vitro-matured oocytes (metaphase II) were penetrated (40.2% cleaved, 24.4% with 2 pronuclei, 12.2% with >2 pronuclei, 7.3% with expanded sperm head), whereas in group 2, penetration rate was 88.5% (42.7% cleaved, 21.8% with 2 pronuclei, 16.7% with >2 pronuclei, 7.3% with expanded sperm head). No difference (P> 0.05) in in vitrofertilizing capacity between spermatozoa collected by means of the 2 methods was found. In conclusion, semen collection by means of CT yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Because CT is repeatable and easy to perform and does not require a trained male/queen in heat, it may be preferable for routine IVF experiments with fresh spermatozoa. The first author is a research fellow of the Fund for Scientific Research-Flanders (Belgium). [ABSTRACT FROM AUTHOR]

Although in vitroproduction of equine embryos has greatly evolved in recent years, there are still substantial differences between in vitro-produced and in vivo-derived equine embryos. Fundamental insight into these differences could lead to optimization of equine assisted reproductive techniques. Reverse transcription quantitative real-time PCR (RT-qPCR) is a highly specific and sensitive tool to compare mRNA expression levels of specific genes and was used in this study to determine differences in gene expression between equine in vivoand in vitroembryos. In vivoembryos (n= 8) were derived by uterine flushing of artificially inseminated mares at 7 days after ovulation. For the production of the in vitroembryos (n= 8), oocytes from slaughtered mares were matured in DMEM-F12-based medium (Galli et al.2007 Anim. Reprod. Sci. 98, 39-55) in 5% CO2in air (maturation rate: 57%), fertilized by intracytoplasmic sperm injection, and cultured in DMEM-F12 with 10% fetal calf serum in 5% CO2, 5% O2, and 90% N2for 9.5 days (cleavage rate: 74%; blastocyst rate: 7%). RNA was extracted from single early to expanded blastocysts and amplified and converted into cDNA with the WT-Ovation RNA Amplification System (NuGEN, San Carlos, CA, USA). Based on the presumed gene functions and differential gene expression as determined in a previously performed suppression subtractive hybridization (SSH; Smits et al.2009 Reprod. Dom. Anim. 44, 75), 5 genes [brain expressed X-linked 2 (BEX2), Mps one binder kinase activator-like 3 (MOBKL3), fatty acid binding protein 3 (FABP3), minichromosome maintenance complex component 7 (MCM7), and ornithine decarboxylase (ODC)] were selected for quantification by RT-qPCR with the KAPA SYBR®FAST qPCR Kit (Kapa Biosystems, Belgium) on the iCycler iQ Real-Time PCR Detection System (Bio-Rad, Nazareth, Belgium). All data were normalized with previously determined stable reference genes (beta actin, ubiquitin C, ribosomal protein L32, and glyceraldehyde-3-phosphate dehydrogenase) and statistically analyzed by means of a Mann-Whitney test. The fact that all genes were expressed at greater levels in the in vivo-derived blastocysts than in the in vitro-produced blastocysts confirmed the results of the SSH. This difference was highly significant for MOBKL3, BEX2, and ODC (P< 0.005), significant for FABP3 (P< 0.05), and not significant for MCM7. These genes have already been shown to be important for embryonic cell survival (ODC), oocyte maturation and pre- implantation development (MOBKL3) in mice, regulation during embryonic development (BEX2) and fetal development (FABP3) in human, and genome replication in eukaryotes (MCM7) (Pendeville et al.2001 Mol. Cell Biol. 21, 6549-6558; Han et al.2005 Nucleic Acids Res. 33, 6555-6565). In conclusion, 4 genes (MOBKL3, BEX2, ODC, and FABP3) with greater expression levels in in vivo-derived equine blastocysts have been identified. Whether the up-regulation of these genes is important for normal embryonic differentiation in the horse embryo is currently under investigation. [ABSTRACT FROM AUTHOR]

In 2006 and 2007, Bluetongue virus serotype 8 (BTV-8) caused devastating outbreaks in Northern Europe; the outbreaks were controlled in 2008 and 2009 by an international vaccination policy. Remarkably, BTV-8 differs from other serotypes in that it spread transplacentally (De Clercq K et al.2008 Transboundary and Emerging Diseases 55, 352-359). Apart from the transplacental spreading, a significant increase in the incidence of abortions was reported in Belgium (Meroc E et al.2009 Transboundary and Emerging Diseases 56, 39-48). The aim of the present study was to investigate the susceptibility of bovine-hatched, in vitro-produced blastocysts to BTV-8. A total of 1390 immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes (n= 1148) were denuded 24 h post-insemination and cultured in 50-μL droplets of modified synthetic oviduct fluid (SOF) medium with 10% fetal calf serum (tested negative for BTV antibodies) at 39.0°C in 5% CO2, 5% O2, and 90% N2. At 7 days post-insemination (dpi), blastocysts were grouped to enhance hatching. For virus incubation, BTV-8 Bel 2006/2 from Veterinary and Agrochemical Research Centre (VAR, Brussels, Belgium) was used. At 8.5 dpi, hatched embryos were placed in 800μL of minimum essential medium (MEM) containing 103.850% tissue culture infectious doses (TCID50) of BTV-8 and incubated for 1 h at 39°C in an atmosphere of 5% CO2in air. At the same time, 2 groups of hatched control embryos were incubated under the same circumstances in 800 μL of SOF and 800 μL of MEM, respectively. After infection, all embryos were washed according to IETS guidelines with the exception that they were not zona pellucida intact and cultured in new SOF. At 48, 60, 72, and 96 h post-infection (hpi), one-fourth of the embryos of each group were fixed in 4% paraformaldehyde for 12 to 24 h and subsequently stained for BTV-8 with double immunofluorescent staining using a BTV-8 monoclonal antibody (8A3B.6, ID-Vet, Montpellier, France). All control embryos (CTRL and MEM) were negative for BTV-8 virus antigen at all time points. At 48 hpi, only 1 out of 7 infected embryos was positive for virus antigen (in all its cells). At 60 hpi, all remaining embryos (n= 6) were negative, whereas at 72 hpi and 96 hpi all embryos had 25% to 100% BTV-8-positive cells (n= 6 at 72 hpi and n= 7 at 96 hpi). Furthermore, 1 embryo at 72 hpi and 2 embryos at 96 hpi showed morphological signs of degeneration. This study has showed for the first time that hatched in vitro-produced blastocysts are susceptible for BTV-8 virus infection and replication in vitro. The relatively long time between virus infection and detection of viral antigen is in accordance with the slow replication cycle of the virus. Further research is ongoing to investigate the importance of BTV-8 infection in early embryonic death. The first author is supported by Research Foundation-Flanders. [ABSTRACT FROM AUTHOR]

In in vitroexperiments, the percentage of bovine oocytes that develops to the blastocyst stage is much lower compared with the in vivocounterparts. The quality of the oocyte is the main factor affecting blastocyst yield. Moreover, in vitro-produced bovine embryos are more sensitive to cryo-injuries than those produced in vivo. Exposure of oocytes to sub-lethal concentrations of stressors may enhance their quality through upregulation of intracellular shock proteins. We aimed to evaluate whether addition of ethanol at low concentrations (0.27 or 0.53%) during oocyte maturation could have a carry-over effect on embryo quality and could subsequently affect embryo cryotolerance. Cumulus-oocyte complexes (n= 934) were matured in serum-free TCM199 plus 20 ng mL-1of epidermal growth factor (control), supplemented with ethanol 0.27% (treatment 1) or 0.54% (treatment 2), in 3 replicates. After fertilization, the presumptive zygotes were cultured for 6 days in modified SOF medium supplemented with 5% fetal calf serum (FCS); the number of blastocysts was recorded and classified. Then, expanded blastocysts were cryopreserved by open pulled straw vitrification using the 2-step approach described by Vajta G et al.(1998 Mol. Reprod. Dev. 51, 53-58). After 24 h, vitrified embryos were warmed and cultured in groups of <25 per 50 μL droplet of modified SOF medium with 5% FCS under mineral oil for 48 h and examined for re-expansion and hatching. Differences between the groups in blastocyst yield were analyzed by ANOVA; differences in survival rates between the groups were analyzed using logistic regression analysis. For all statistical models, group was included as fixed effect, and also the effect of replicate was included. Differences were considered to be statistically significant when P< 0.05. Addition of ethanol to in vitromaturation media had no significant effects on blastocyst yield on 7 dpi. Also, addition of ethanol at 0.27% did not affect blastocyst cryotolerance. However, addition of ethanol 0.54% to in vitromaturation media significantly increased the survival of bovine blastocysts after vitrification (P< 0.01; Table 1). The results of the present study indicate that maturation of oocytes under ethanol stress at low concentrations has carry-over effects on embryo quality, leading to improved cryotolerance. Table 1.Survival percentage (mean ± SE) of vitrified expanded bovine blastocysts matured in the presence of sub-stress concentrations of ethanolThe authors thank I. Lemahieu and P. Vandamme for their excellent technical support. This research was supported by the special research fund, Ghent University (Grant, BOF/DOS No. 01W05706). [ABSTRACT FROM AUTHOR]

The isolation, preservation, and identification of hematopoietic and mesenchymal stem cells from fresh umbilical cord blood (UCB) has been extensively reported in humans. Although both types of stem cells may be of therapeutic interest in horses, data on equine UCB cells are scarce. In the present study, two separation methods to isolate stem and progenitor cells from equine UCB and two cryoprotectant solutions for their subsequent freezing were compared. Characterization of the isolated cells was evaluated flow cytometrically, based on the presence of the cytosolic enzyme aldehyde dehydrogenase (ALDH), which has been shown to be highly expressed in primitive hematopoietic cells in a number of species. Cord blood was collected from 15 foals immediately after birth. While the placenta was still in utero, the umbilical cord was clamped and disinfected. A sterile blood bag collection system containing citrate-phosphate-dextrose-adenine anticoagulant was used to collect the UCB by gravity. The UCB units were stored at 4C and processed within 36 h. Percoll density gradient separation and rouleaux formation induced by hydroxyethyl starch (HES) were tested in parallel on equal volumes of each UCB unit. The enriched progenitor cell fraction was cryopreserved at 10 106 nucleated cells mL–1 using two cryoprotectant solutions based on plasma or RPMI 1640, and both containing 10% DMSO and DNase I (20 IU mL–1). Before and after thawing, cells were labeled using a fluorescent ALDH substrate (Aldefluor, StemCell Technologies SARL, Grenoble, France) including a negative control. Cell viability was simultaneously evaluated by means of exclusion of propidium iodide. Cryopreservation was performed using a programmable freezer (–1C/min–1 until –70C, then –10C/min–1 until –140C) prior to storage in liquid nitrogen. Results were analyzed statistically with a nonparametric Mann-Whitney test. The concentration of the isolated UCB cells ranged from 0.3 to 4 106 cells mL–1 for Percoll and from 0.4 to 7.3 106 cells mL–1 for HES. The average viability before and after freezing was 94% and 93% for Percoll-, and 93% and 94% for HES-separated cells, respectively. No significant differences in concentration or in viability were observed between both isolation procedures and both cryoprotectant solutions. Before freezing, the proportion of Aldefluor-positive cells after Percoll and HES isolation ranged between 0.5 and 38% and between 1 and 60.5%, respectively. No significant differences were found. In conclusion, the percentage of ALDH-positive cells as determined by flow cytometry was highly variable between foals, but was independent of the isolation procedures used. Whether the isolated cells represent true progenitor cells remains to be confirmed. Ongoing, flow cytometrical experiments showed that the isolated cells are CD29+ and CD44+, which may be indicative for their mesenchymal origin. [ABSTRACT FROM AUTHOR]

Feline epididymal sperm is commonly used for in vitro fertilization. It also yields the opportunity to conserve genetic material from valuable males that suddenly die. Epididymal sperm quality parameters vary considerably among laboratories, implicating the need for objective evaluation methods. The aim of the present study was to describe reference values of computer-assisted sperm analysis (CASA) parameters of fresh epididymal cat sperm and to assess the effect of prolonged cooled storage (4C) on various sample characteristics. Epididymides obtained from tomcats after routine orchiectomy (2–4 pairs/replicate) were sliced to release spermatozoa. The sperm suspension was placed on a 2-layer gradient and, after centrifugation, the sperm pellet was recovered. In Experiment 1 (20 replicates), sperm motility parameters were assessed immediately after retrieval (T0) using the Hamilton Thorne analyzer Ceros 12.1 (HTR; Hamilton Thorne Biosciences, Beverly, MA, USA). In Experiment 2, fresh (T0) sperm samples (4 replicates) were evaluated for motility parameters (HTR), acrosomal status (FITC-Pisum sativum agglutinin staining), morphology (eosin/nigrosin (E/N) staining), and membrane integrity (E/N and SYBR-14-propidium iodide staining; Molecular Probes, Inc., Eugene, OR, USA). After addition (1:2) of a Tris-glucose-citrate diluent containing 20% egg yolk, samples were cooled and reassessed on Days 1 (T1), 3 (T3), 5 (T5), 7 (T7), and 10 (T10). Results were analyzed in a mixed linear model, with replicate as random factor and time as fixed effect (S-PLUS 7.0; Insightful Corp., Seattle, WA, USA). Results of Experiment 1 were as follows (mean SD): motility (MOT): 80.8% 23.5; progressive motility (PMOT): 69.9% 23.2; velocity average pathway (VAP): 98.7 µm s–1 24.2; velocity straight line (VSL): 89.3 µm s–1 25.4; velocity curved line (VCL): 134.8 µm s–1 31.9; amplitude lateral head (ALH): 4.3 µm 2.0; beat cross frequency (BCF): 34.6 Hz 7.0; and straightness (STR): 89.6% 6.6. In Experiment 2, MOT, PMOT, VAP, VSL, VCL, BCF, and the percentage of normal spermatozoa showed a decrease over time (P 0.05) by cooled storage. To summarize, we have presented a set of reference values for CASA-parameters of fresh, epididymal cat spermatozoa. Cooled storage impaired most motility parameters and lowered the percentage of normal spermatozoa, but did not influence membrane integrity or acrosomal status. The effect of cooled storage on DNA fragmentation of sperm and its subsequent influence on in vitro embryo development require further investigation. [ABSTRACT FROM AUTHOR]

Although cryopreservation of epididymal sperm has been studied extensively in several species, some factors that could negatively influence its quality are still unknown, such as the storage conditions of the epididymides prior to sperm collection. Studies indicate that the lower the storage temperature, the better the sperm quality after collection (Kaabi et al. 2003 Theriogenology 60, 1249–1259). An additional factor is lipid peroxidation in which sperm membrane resistance against reactive oxygen species (ROS) attacks is an important component. The objective of this experiment was to study whether the epididymis storage temperature following slaughter, as well as the intrinsic resistance against oxidative stress, affects the fertilizing capacity of cryopreserved epididymal bull sperm in vitro. Twelve epididymides (6 bulls) were collected after slaughter and divided into 2 groups, (stored at either 4 or 37C for 2), after which semen was collected from the caudae epididymides and cryopreserved. Subsequently, one aliquot of the frozen–thawed semen samples was subjected to induced lipid peroxidation with ferrous sulfate and ascorbate (37C; 2 h), after which tiobarbituric acid-reactive substances (TBARS), as an index of lipid peroxidation, were measured according to a method previously described (Beorlegui et al. 1997 Andrologia 29, 37–42). A second aliquot was used for in vitro fertilization in a routine IVF–IVC setup in duplicate (24-h maturation, SOF culture medium in 5% CO2, 5% O2, and 90% N2). In vitro embryo production results at Day 7 and TBARS levels were statistically analyzed using SAS (SAS Institute, Inc., Cary, NC, USA). No influence of storage temperature was observed at either TBARS level (4C: 943.6 173.4; 37C: 751.4 136.2 ng of TBARS/108 spermatozoa; P = 0.3) or on blastocyst rates (4C: 23.0 2.8; 37C: 18.7 3.6% of blastocysts; P = 0.2). However, the percentage of hatched blastocysts tend to be higher for epididymides stored at 4C when compared to those stored at 37C (6.4 1.8 and 2.3 0.9, respectively; P = 0.06). In addition, a negative correlation was found between TBARS concentrations and blastocyst rates (R = –0.57; P < 0.05). Compared to fresh samples collected from epididymides under the same conditions (unpublished data), levels of TBARS were two- to threefold higher for the cryopreserved sperm, indicating that lipid peroxidation appears to play a role in the decrease in quality of cryopreserved epididymal sperm. In conclusion, temperature during the epididymides short-term storage prior to sperm cryopreservation did not seem to influence the sperms'' in vitro fertilizing capacity. On the other hand, an alternative to improve cryopreserved epididymal sperm in vitro fertility (or fertilizing capacity) could be the addition of antioxidants to semen extenders. Further studies are necessary to confirm this hypothesis. [ABSTRACT FROM AUTHOR]