First results about ProAKAP4 concentration in stallion semen after cryopreservation in two different freezing media.

The quality of fresh or thawed sperm in stallions has been generally determined by the viability and total and progressive motility of the sperm. Today, the expression of ProAKAP4, a protein present in the flagellum of spermatozoa, appears to be an innovative and relevant functional marker to assess semen quality and male fertility. This study aims to compare the concentration of ProAKAP4 in the semen from 5 stallions frozen with two different extenders immediately after thawing (T0) and 4 h post-thawing (T4). Viability, total and progressive motility were measured in parallel. Significant differences for sperm viability and total motility were observed between the two extenders, as was the concentration of ProAKAP4 both at T0 and T4. At T4, all quality parameters and ProAKAP4 content significantly decreased compared to T0, but with a considerably slower decrease in one extender than the other. These preliminary results suggest that measuring the concentration of ProAKAP4 is a promising tool for the comparison of different extenders and the selection of the optimal freezing medium for each stallion ejaculate.

Short-term exposure to hydrogen peroxide during oocyte maturation improves bovine embryo development.

Recent studies have shown that short-term exposure of oocytes to a stressor such as hydrostatic pressure or osmotic stress might induce stress tolerance in embryos. The aim of the present study was to investigate the consequences of short-term hydrogen peroxide (H(2)O(2)) exposure to bovine in vitro matured cumulus-oocyte complexes (COCs) on subsequent preimplantation embryo development and apoptosis. In the first experiment, mature COCs were incubated in H(2)O(2) at concentrations ranging between 0.01 and 100 micromol/l, and subsequently fertilized and cultured. Oocyte incubation with 50-100 micromol/l of H(2)O(2) resulted in a significantly higher blastocyst yield (47.3%) in comparison with control medium (31.8%), while apoptotic cell ratio was inversely related with H(2)O(2) concentration. In the second experiment, we showed that the stress tolerance after H(2)O(2) exposure was not mediated by increased glutathione content in treated oocytes nor by enhanced fertilization or penetration. Further research should concentrate on the potential role of players that have been associated with stress tolerance in somatic cell lines.

Plant protein hydrolysates (plant peptones) as substitutes for animal proteins in embryo culture medium.

The aim of the present study was to improve the sanitary quality of in vitro-produced bovine embryos by using plant protein hydrolysates (plant peptones) as substitutes for animal proteins. Peptones were compared with bovine serum albumin (BSA) as the protein source in synthetic oviduct fluid medium and the quality of the resulting embryos was determined. Two batches of peptones (wheat and cotton) were selected on the basis of their anti-oxidant properties. When added to the culture medium, both peptones (at 0.56 mg mL(-1) for cotton peptone and at 0.18 mg mL(-1) for wheat peptone) led to similar developmental and hatching rates compared with 4 mg mL(-1) BSA and embryos were equally resistant to freezing and able to elongate after transfer. Surprisingly, a significant decrease in reduced glutathione (GSH) content was observed when embryos were produced with plant peptone instead of BSA. Supplementation of the culture medium with precursors of GSH (cysteine and beta-mercaptoethanol) significantly increased the GSH content. A shift of the sex ratio towards male embryos was seen for Day 8 embryos cultured with wheat peptone, whereas no shift was observed for embryos cultured in the presence of BSA or polyvinylpyrrolidone. In conclusion, culture with plant peptones enables embryos to be obtained at a similar rate and of similar quality to that seen following the use of BSA. The use of the plant peptones increased the sanitary quality of the embryos and decreased the cost of embryo production.

Expression, localization, and concentration of A-kinase anchor protein 4 (AKAP4) and its precursor (proAKAP4) in equine semen: Promising marker correlated to the total and progressive motility in thawed spermatozoa.

A-kinase anchor protein 4 (AKAP4) is playing a central role in flagellar structure, chemotaxis, capacitation and sperm motility. In mammals, AKAP4 is expressed during spermatogenesis. AKAP4 is synthesized as a precursor, proAKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. The proAKAP4 is a good indicator of sperm quality in humans and boars. The aims of this work were to study the expression, the localization and the concentration of proAKAP4 and AKAP4 in equine semen, and to evaluate the possible correlation between the total and progressive motility and the concentration of proAKAP4 measured by ELISA in post-thawed semen. Frozen sperm from 13 different stallions were used. Semen samples (n = 17) were prepared using the INRA Freeze medium to reach a concentration of 150 million spermatozoa/mL, packaged and frozen in 0.5 mL straws. The precursor proAKAP4 and the mature protein AKAP4 both localize to the fibrous sheath of the principle piece of equine sperm flagellum. The concentrations of proAKAP4 were determined in the post-thawed semen using ELISA method (Horse 4MID® kits, 4BioDx, France). The mean concentration of proAKAP4 was then of 7.372 ±â€¯0.79 ng/µL and was significantly correlated with the post-thawed total motility (Pearson coefficient r = 0.66, p = 0.002) and progressive motility (Pearson coefficient r = 0.76, p = 0.0002) and the amount of proAKAP4 represent the amount of spermatozoa that expressed proAKAP4. Taken together, these preliminary results confirm the interest to use proAKAP4 concentrations as a promising marker of stallion sperm quality as close correlation was observed between the proAKAP4 concentration and sperm motility parameters.

Kinetics of fertilization and development, and sex ratio of bovine embryos produced using the semen of different bulls.

The between bulls variation in in vitro fertility and the shift of sex ratio towards male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the kinetics of fertilization, embryo development and the sex ratio of the resulting embryos using the frozen/thawed semen of four different bulls. In a first experiment, the kinetics of pronucleus (PN) formation was evaluated at 8, 12 and 18 h post-insemination (hpi). Based upon the pronuclei sizes and the distance between the two pronuclei, inseminated oocytes were classified in three PN stages. Differences between bulls were observed at each time point, but were more important at 12 hpi. At 8 and 12 hpi bull III showed a significantly faster PN evolution by comparison with the three other bulls (P<0.05), while at 18 hpi, the proportion of the three PN stages was similar to those of bulls I and IV, bull II being delayed. In a second experiment, the kinetics of in vitro embryo development was compared using time-lapse cinematography. The analysis of embryos reaching the blastocyst stage revealed significant differences in the mean time of first cleavage (range of 22.7-25.6h, P<0.05), while the lengths of the subsequent three cell cycles did not differ between bulls. The early mean time of first cleavage with bull III was associated with an early blastulation and a high blastocyst rate at Day 7, in opposition to what was observed with bull II showing a later timing of first cleavage (first cleavage 22.1 hpi versus 25.5 hpi; blastulation 140.4 hpi versus 152.5 hpi; D7 blastocyst rates: 31.3% versus 21.9%; P<0.05). In a third experiment, 65-76 Day 8 blastocysts per bull were sexed by PCR. Only blastocysts obtained with bull III showed a shift in sex ratio towards male embryos (76% male embryos; P<0.05). Such shift was already observed at the 2-cell and morula stages. In conclusion, the bull influences the kinetics of PN formation, of embryo development and the sex ratio of the embryos. Moreover, those parameters might be related.

Set up of a serum-free culture system for bovine embryos: embryo development and quality before and after transient transfer.

It is well known that serum in culture medium negatively affects blastocyst quality. The objective of this work was to develop and test a serum-free culture medium which could improve embryo quality, measured by the resistance to freezing, lipid and glutathione content of the resulting blastocysts, as well as the ability of the blastocysts to elongate after transient transfer to recipient cows. In a first experiment we showed that adding a mixture of insulin, transferrin and selenium to serum-free Synthetic Oviduct Fluid medium (SOF-ITS) improved embryo development and quality. In the second experiment, the addition of BSA to SOF-ITS further improved blastocyst development. Moreover, a reduction in lipid content of morulae was observed in SOF-ITS-BSA by comparison with morulae cultured with serum (SOF-FCS). The resistance to freezing measured by hatching rates 24h post-thawing was also improved for blastocysts with a diameter between 160 and 180 microm cultured in SOF-ITS-BSA by comparison to those produced with serum. In order to evaluate the redox potential of the embryos, reduced glutathione content (GSH) was evaluated both before and after cryopreservation. A significant decrease in glutathione was observed after freezing, whatever the culture medium, but no difference was observed between culture conditions. Transient transfers were performed and elongated D-13 embryos were recovered. Elongation was more pronounced and the embryonic disk more often visible in embryos cultured in SOF-ITS-BSA than in embryos cultured with FCS. In conclusion, the serum-free system we developed to produce in vitro bovine embryos meets the developmental and qualitative requirements for a large-scale use.

Analysing motility parameters on fresh bull semen could help to predict resistance to freezing: a preliminary study.

Batches of straws often need to be thrown away after freezing because of a too few number of motile or progressive sperm cells after thawing. Our objective was to evaluate the possibility to predict before freezing the quality of the semen after freezing/thawing. Computer-Aided Sperm Analysis was performed on motility parameters both before and after freezing of 20 ejaculates from different bulls. Significant variation between bulls was observed both before and after freezing for all the analysed traits (anova2; p < 0.001): proportion of motile (%mot) and progressive (%prog) sperm, velocity on the curved line (VCL), velocity on the straight line (VSL), velocity on the average path (VAP), linearity (VSL/VCL), beat cross frequency and average orientation change of the head. Freezing significantly altered the motility parameters and correlations were found between samples analysed before and after freezing (Pearson coefficient: R = 0.43-0.72; p < 0.05). The mean VAP, VSL and the %prog obtained before freezing were highly correlated to the %mot and %prog observed after freezing (R = 0.75-0.82; p < 0.001). Applying thresholds on mean VAP and VSL values allowed us to predict respectively 6 and 7 of nine batches that would be rejected after freezing due to a too low %prog (<15%). Combining different traits did not add to the precision. In conclusion, analysis of velocity traits on fresh sperm seems more efficient than analysis of %mot or %prog to discard batches that will be of poor quality after freezing.

Assessment of sperm quality parameters of six bulls showing different abilities to promote embryo development in vitro.

Experiments were conducted to investigate the possible origins of variation between six bulls showing various blastocyst rates after in vitro fertilisation. No significant difference was observed for the rates of cleavage and 5-8 cell stages, whereas blastocyst yields at Day 6, 7 and 8 post insemination were significantly different between bulls (P < 0.05). Fertilisation rates ranged from 59.5 to 79.3% (P < 0.05), with no difference in the incidence of polyspermy. The proportions of motile and progressive spermatozoa before and after Percoll separation were analysed. A positive effect of Percoll was noted on both parameters (P < 0.05), leading to the absence of difference between bulls after the separation process. Sperm viability and spontaneous acrosome reaction were assessed during 18 h incubation in fertilisation medium. A sharp decrease in sperm viability was observed for all bulls after 2 h incubation, with only 12.6-21.7% of spermatozoa still viable at 18 h. In contrast, the proportion of reacted acrosomes was low in five out of six bulls (<15% at 18 h). In conclusion, the fertilisation rate was the only parameter to show some correlation with blastocyst rate for all bulls.

Freezing of in vitro produced bovine embryos in animal protein-free medium containing vegetal peptones.

Successful cryopreservation is essential for a large-scale dispersal of bovine in vitro produced (IVP) embryos that have been shown to be more sensitive to cryopreservation than their in vivo counterparts. On the other hand, the use of animal proteins in freezing media increases sanitary risks. We first replaced animal proteins, such as bovine serum albumin (BSA) in the freezing medium by plant-derived peptides (vegetal peptones). A batch of wheat peptones was selected after a preliminary experiment showing the absence of toxicity of concentrations<18 mg/mL on in vitro bovine blastocysts. Increasing concentrations of peptones were then added in the freezing medium. The surviving and hatching rates were not affected by comparison with those observed with BSA. No significant difference was observed between groups either for the total number of cells or for the ratio ICM/Total cell, nor for the rate of apoptosis in surviving embryos. When embryos were cryopreserved in 1.8 mg/mL peptone, the hatching rate and embryo quality as assessed at 48 h post-thawing were not significantly different from those of unfrozen embryos. In a second experiment two additives were added in this animal protein-free freezing medium containing 1.8 mg/mL peptones. No beneficial effect of adding 1 mg/mL sodium hyaluronate or 100 microM beta-mercaptoethanol was observed on embryo survival or quality. In conclusion, we have demonstrated that vegetal peptones can replace BSA in freezing media without affecting blastocyst survival and quality.

Apoptosis at the time of embryo implantation in mouse and rat.

The aim of this review is to summarize the information currently available regarding the occurrence of apoptosis in the developing embryo and in the receptive uterus during the peri-implantation period of gestation. Cell death is detected in the inner cell mass of late pre-implantation embryos as the result of an eliminative process that helps trim the embryonic cell lineages of surplus or dysfunctional stem cells. Cell death is also detected in the epiblastic core of early post-implantation embryos, where the process is implicated in the formation of the pro-amniotic cavity. On the maternal side, uterine epithelial cells situated around the attachment site undergo cell death during the initial phase of implantation in order to facilitate embryo anchorage and access to maternal blood supply. Uterine stromal cells closest to the implantation chamber first transform into decidual cells and then commit suicide to make room for the rapidly growing embryo. Although apoptosis is well recognized as a crucial determinant of successful peri-implantation development, our understanding of the cellular and molecular mechanisms regulating this process clearly lags behind the comprehension of cell death control in other systems.

Expression and role of Bcl-2 in rat blastocysts exposed to high D-glucose.

Bcl-2 mRNA expression was detected in rat blastocysts by in situ hybridization. The distribution of mRNA expression was rather heterogenous, with approximately 2% of high-expressing cells. In vitro exposure to 28 mmol/l D-glucose for 24 h resulted in a significant increase in the proportion of these cells compared with control embryos in either 6 mmol/l D-glucose or 28 mmol/l D+L-glucose. Heterogeneity in the expression of Bcl-2 was also observed at the protein level by immunocytochemistry. Exposure to 28 mmol/l D-glucose significantly increased the incidence of chromatin degradation (karyolysis) and nuclear fragmentation (karyorhexis), two nuclear markers of apoptosis in rat blastocysts. When two different antisense oligodeoxynucleotides designed to block Bcl-2 expression were added to 28 mmol/1 D-glucose, the incidence of karyolysis (but not karyorhexis) was increased compared with embryos in 28 mmol/l D-glucose alone. These data suggest that Bcl-2 is involved in the protective response against the induction of karyolysis in blastocysts on their exposure to high concentrations of D-glucose in vitro, whereas karyorhexis appears to result from the activation of an intracellular pathway that is independent of Bcl-2.

Phorboid 20-homovanillates induce apoptosis through a VR1-independent mechanism.

BACKGROUND: Vanilloids, such as capsaicin and resiniferatoxin (RTX), are recognized at the cell surface by vanilloid receptor type 1 (VR1), which has recently been cloned. VR1 mediates the effects of capsaicin and RTX in VR1-expressing cells, but vanilloids can induce apoptosis through a pathway not mediated by VR1. Phorboid 20-homovanillates can be used to investigate cell death induced by vanilloids. RESULTS: 12,13-Diacylphorbol-20 homovanillates were prepared by the sequential esterification of the natural polyol. Phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) induced apoptosis in Jurkat cells to the same extent as RTX. Apoptosis was preceded by an increase in intracellular reactive oxygen species and by the loss of mitochondrial transmembrane potential. PPAHV-induced apoptosis was mediated by a pathway involving caspase-3 activation and was initiated at the S phase of the cell cycle. The cell-death pathway triggered by VR1 activation was studied in 293T cells transfected with the cloned rat vanilloid receptor. In this system, capsaicin and PPAHV induced cell death by an apparent necrotic mechanism, which was selectively inhibited by the competitive vanilloid receptor antagonist capsazepine. Interestingly, phorbol-12, 13-bisnonanoate-20-homovanillate, an analogue of PPAHV, induced cell death in VR1-transfected cells but could not trigger apoptosis in the Jurkat cell line. CONCLUSIONS: Vanilloids can induce cell death through different signalling pathways. The cell death induced in a VR1-independent manner has the hallmark of apoptosis, whereas the cell death mediated by vanilloids binding to VR1 is seemingly necrotic. Phorboid homovanillates that have antitumour and anti-inflammatory activities but lack the undesirable side effects of the natural vanilloids could be developed as potential drugs.

The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes.

This study aimed to investigate the use of Nile red, a fluorescent dye specific for intracellular lipid droplets, to quantify the lipid content of single mammalian oocytes. It was hypothesized that a higher amount of lipid present in lipid droplets in an oocyte would result in a higher amount of emitted fluorescent light. Following fixation and subsequent staining of denuded oocytes, the fluorescence of the whole oocyte was visualized by fluorescence microscopy and quantified with a photometer and photomultiplier connected to the microscope. The peak of fluorescence was observed in the yellow spectrum (590 nm) and the fluorescence was restricted to the lipid droplets corresponding to apolar lipids. Nile red concentrations ranging from 0.1 to 10 microg/ml yielded similar results. After fixation, a minimum of 2 h staining was necessary to reach maximal fluorescence which remained stable for several hours. The position of the microscopic focus within the oocyte had no influence on the amount of measured fluorescence. Successive measurements of the same oocyte yielded very similar results indicating the repeatability of the method. Finally, the technique was validated by comparing the lipid content of bovine, porcine and murine immature oocytes, which are known to contain different amounts of lipids. After staining, the fluorescence of murine oocytes was 2.8-fold lower than the fluorescence of bovine oocytes which in turn were 2.4 times less fluorescent than porcine oocytes. Based on this study, it can be said that this rather fast and easy technique allows for the relative quantification of the lipid content (present in the lipid droplets) of one single oocyte. The different amounts of emitted fluorescent light in bovine, porcine and murine oocytes correlated with the known lipid contents in these three species. This technique could be used to compare the lipid content of oocytes originating from different donors, from different sized follicles or cultured in various conditions.

Growth factor-like activity of phenol red preparations in the MCF-7 breast cancer cell line.

Hormonal responsiveness of the estrogen-sensitive MCF-7 human breast cancer cell line is known to vary between laboratories although the causes and implications of these variations remain unclear. Our findings lead us to conclude that the pH indicator phenol red (PHR) has growth factor-like effects in addition to its well known estrogen-like effects. To demonstrate this hypothesis, we have assessed the importance of PHR either in the absence or in the presence of the estrogens contained in the serum added to the culture medium. The basal growth rate of MCF-7 cells was significantly reduced by short-term or long-term withdrawal of PHR. The stimulatory effects of estradiol and the inhibitory effects of the antiestrogen 2-CH3,4-OH-tamoxifen (MHT) were not significantly affected by long-term withdrawal of the dye. Moreover, long-term cell maintenance without PHR alone or in complete estrogen-depleted medium did not change their basal steroid receptor content. The molecular structure of the estrogen receptor which is usually modified under estrogenic stimulation remained identical whether or not the cells were maintained in the presence of the dye. Maintaining cells without the dye in the presence of serum estrogens led to the death of the cell line after 50 transfers. Lastly, addition of PHR had clearcut growth stimulatory effects on the hormono-independent cell line Evsa-T.

Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification.

The ability of bovine blastocysts to recover after cryopreservation and thawing procedures is often assessed by evaluating their re-expansion during in vitro co-culture. However, the influence of factors such as feeder cell type and gas atmosphere on blastocyst survival and evolution have never been considered. This study therefore compared two cell co-culture systems and two different gas atmospheres to assess survival of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n = 181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured either on Buffalo rat liver cells (BRL CC cell line) or on granulosa cells (GR CC primary culture) in TCM 199 supplemented with 10% FCS and under an atmosphere of 5% or 20% O2. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, surviving blastocysts were treated for differential counting of inner cell mass (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O2: 62% vs. 5% O2: 25%, P < 0.0001). However, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O2 than under 5% O2 as indicated by lower total and trophectoderm cell numbers (respectively 79 +/- 6 and 56 +/- 6 at 20% O2 vs. 100 +/- 10 and 74 +/- 10 at 5% O2, P < 0.05), by an altered ICM/trophectoderm ratio (20% O2: 28% vs. 5% O2: 23%, P < 0.05), by a higher total nuclear fragmentation (20% O2: 3.7% vs. 5% O2: 1.5%, P < 0.05) and a trend to decreased hatching (20% O2: 32% vs. 5% O2: 81%, P = 0.07). Whereas, for BRL co-culture, 20% O2 yielded higher quality blastocysts than 5% O2 as evaluated by higher ICM and trophectoderm cell numbers (19 +/- 1 and 71 +/- 5 at 20% O2 vs. 15 +/- 2 and 48 +/- 9 at 5% O2, respectively, P < 0.05), by lower nuclear fragmentation in the ICM (20% O2: 2.2% vs. 5% O2: 6.7%, P < 0.05). In conclusion, co-culture conditions may influence blastocysts survival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O2 seems to be the best combination to evaluate blastocyst survival and quality after vitrification.

Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity.

Methodological studies were undertaken to test the validity of a three-step vitrification procedure for bovine in vitro produced embryos using glycerol and ethylene glycol as cryoprotectants. Embryos were produced in a low-phosphate culture system (medium VT1 + 10% foetal calf serum) and vitrified at day 7 post-insemination either in a mixture of 25% glycerol--25% ethylene glycol or a mixture of 10% glycerol--40% ethylene glycol. In the first mixture 67% (n = 283) of blastocysts were re-expanded after 72 h of culture and 53% were hatched while in the second one (n = 65) only 5% survived. The mean number of cells of the surviving blastocysts was correlated with the rate of survival (R2 = 0.47; P = 0.0024). Embryo size (diameter < or > to 180 microm) did not influence blastocyst survival or cell number, but hatching rate was higher for embryos > 180 microm. Embryo survival, hatching rate and cell number 72 h post-warming were not affected by the mode of vitrification (direct plunging into nitrogen liquid or vitrification into nitrogen liquid vapour), the mode of preparation of the vitrification solutions (molar or molal basis) or by the concentration of galactose used as a diluent (0 to 0.85 M). Only one calf was born after transfer of 22 vitrified blastocysts. These results confirm the apparent lack of correlation for cryopreserved embryos between in vitro survival or hatching and viability after transfer.

Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitro-produced bovine embryos.

Embryo quality of in vitro-produced bovine blastocysts was assessed at several steps of a vitrification procedure in which glycerol and ethylene glycol were used as cryoprotectants (3-step equilibration with cryoprotectants followed by vitrification, dilution of the cryoprotectants in 0.85 M galactose then in embryo transfer freezing medium [ETF], and finally co-culture for periods). To visualize cell membrane alterations, double staining was performed using a cell permeant fluorochrome (bisbenzimide--BIS) and a nonpermeant one (propidium iodide--PI). In Experiment 1, the effect of the vitrification procedure on the hatching rate and total cell number was assessed 72 h after treatment. Hatching rate and the number of stained nuclei were decreased in comparison with untreated embryos when blastocysts were exposed to the whole procedure with or without vitrification (respectively 42 and 53% vs 76% for hatching and 128 +/- 17 and 141 +/- 17 vs 226 +/- 13 for stained nuclei). In Experiment 2, the effect of cryoprotectants and their dilution was evaluated on membrane permeability and total cell numbers at various steps of the vitrification procedure. Blastocysts exposed only to cryoprotectant solutions and stained immediately after dilution in galactose showed no modification. After dilution in ETF, the total number of stained nuclei decreased, and the number of blastomeres showing membrane permeabilization (PI-stained) increased (respectively, 74 +/- 5 vs 110 +/- 5 and 32 +/- 2% vs 0.1 +/- 1.8%). In Experiment 3, we demonstrated that the total number of stained nuclei after ethanol fixation (membrane permeabilization) was higher when embryos treated up to dilution in ETF were stained with PI than when the same embryos were stained with BIS. This suggests that, for unknown reasons, some nuclei of the treated embryos were not stained with BIS. Membrane permeabilization and inability of BIS to stain some nuclei were the most obvious alterations probably induced by osmotic shock at dilution. This hypothesis is supported by the fact that the introduction of a further dilution step in 0.42 M galactose (Experiment 4) before dilution in ETF decreased the proportion of cells permeant to PI and increased the hatching rate after 72 h of co-culture. In conclusion, double staining with BIS and PI allowed for discrimination between different types of cellular injuries after the various steps of our vitrification protocol. It represents a useful tool for adjusting equilibration and dilution conditions during a cryopreservation procedure.

Effect of high molecular weight factors present in bovine oviduct-conditioned medium on in vitro bovine embryo development.

To investigate the presence of embryotrophic factors in bovine oviduct-conditioned medium (BOCM), the high molecular weight fraction (> 10 KDa) from BOCM was added to 3 chemically defined embryo culture media (TCM199, DMEM/F12 and modified synthetic oviduct fluid [mSOF]). Zygotes were obtained by in vitro maturation and fertilization of oocytes. Conditioning of TCM199 with oviduct cells increased both cleavage to the 5- to 8-cell stage (59 vs 37%) and further development to the blastocyst stage (19 vs 4%). The low molecular weight fraction (< 10 KDa) of BOCM maintained development to the 5- to 8-cell stage but did not allow development to the blastocyst stage. Adding the high molecular weight fraction to the inactive low molecular weight fraction restored bovine embryo development up to the blastocyst stage. This embryotrophic effect of the high molecular weight fraction was not observed when this fraction was added to TCM199 or DMEM/F12 medium. Whereas adding this fraction to mSOF medium significantly (P<0.05) increased embryo development up to the blastocyst stage (36%) in comparison with that of mSOF (15%) or BOCM (14%). These results show that BOCM contains high molecular weight factors promoting embryo development up to the blastocyst stage. Some chemically defined media mask the effect of these embryotrophic factors.

Embryo production by ovum pick up in unstimulated calves before and after puberty.

The possibility of producing embryos from oocytes repeatedly collected from unstimulated calves was tested, and results obtained before and after puberty were compared in the same animals. Ovum pick-up (OPU) coupled with in vitro embryo production was used on 2 sets of 7 and 9 calves, aged 7 to 10 m.o. at the start of the experiment. The oocytes were collected twice a week during a 2-m.o. period before puberty and a 1-m.o. period after puberty. Oocytes were fertilized and co-cultured with cumulus cells in modified synthetic oviduct fluid (SOF) up to Day 7 post insemination. Some Day 7 blastocysts were vitrified and transferred to recipient heifers. An average of 3.8 to 6.8 follicles was punctured per OPU session; 1.9 to 3.1 oocytes were collected, of which more than 60% were of good quality. The number of punctured follicles and collected oocytes varied between donors. Blastocyst rates of 19 to 27% were obtained for the 2 sets. Three normal calves were born from the transfer of 20 vitrified embryos. While no significant difference was observed for the first set of calves, a significant decrease in the number of punctured follicles was observed after puberty in the second set. A direct correlation was also obtained between the number of follicles punctured before and after puberty in the same animal. In conclusion, oocytes can be collected by repeated OPU in calves 7 to 10 m.o. old without affecting their growth or the onset of puberty. An average of 5 to 11 (range 0 to 16) blastocysts per donor was produced over 2 month. However, important variations were found between donors. The correlation observed for the number of follicles punctured before and after puberty suggests that this parameter is determined before puberty.

Effects of co-culture and embryo number on the in vitro development of bovine embryos.

It is generally accepted that culturing embryos in groups or with somatic cells improves both the yield and quality of the blastocysts obtained. The aims of this study were 1) to compare the yield and quality of the embryos obtained after culture in several number conditions and in several culture systems and 2) to assess the effect of co-culture started at various stages of embryo development. Under cell-free culture conditions (modified synthetic oviduct fluid [mSOF] supplemented with 10% fetal calf serum [FCS] 48 h post insemination, the rate of Day 10 blastocysts was lower when embryos were cultured in small groups (1 to 6 per drop) than in large groups (4 versus 23% ; P < 0.01). There was no group effect when embryos were co-cultured either with Buffalo rat liver (BRL) cells in TCM 199, or in a culture system allowing the progressive development of cumulus cells in mSOF, even if co-culture started at 66 or 114 h post insemination. However, embryos cultured singly had lower cell numbers than embryos cultured in large groups when co-culture started at 114 h post insemination. This suggests that 1) somatic cells improve the development of singly cultured bovine embryos up to the blastocyst stage after the 9-16 cell stage; 2) co-culture affects blastocyst cell number of singly cultured embryos by acting roughly between the 5-8 and the 9-16 cell stage; and 3) cooperation between embryos could replace the effect of co-culture either on the yield of blastocysts or on blastocyst cell number. Blastocysts appeared significantly earlier in co-culture with cumulus cells in mSOF than in co-culture with BRL cells in TCM 199 (detection of the blastocysts: 7.3 +/- 0.1 d post insemination with cumulus cells versus 8.1 +/- 0.1 d with BRL cells; P < 0.001) and had a significant higher number of cells (143 +/- 9 versus 85 +/- 11; P < 0.001). This system thus seems suitable for the culture of small numbers of embryos resulting from in vitro maturation and fertilization of oocytes from individual donor cows.