Cushioned centrifugation during sperm selection increases the fertilization and cleavage rates of cattle embryos produced in vitro.

This study was conducted to evaluate the effect of utilization of an iodixanol-based solution as a cushioning method during the sperm selection utilizing discontinuous Percoll gradient centrifugation in in vitro production (IVP) of cattle embryos. In Experiment I, all aliquots of thawed semen were subjected to sperm selection using the same discontinuous Percoll® gradients, except for the following four conditions: presence of cushioning solution (Cushion Fluid, Minitube) during the first centrifugation process (C1), presence of cushioning solution during the second centrifugation process (C2), inclusion of cushioning solution in both centrifugation steps (C1-2), and no addiction of cushioning solution (C; control group). Recovery rates, sperm kinetics, and reactive oxygen species (ROS) production were evaluated. In Experiment II, sperm cells were processed using sperm selection conditions C and C1, and fertilization rates and embryonic development kinetics were compared between experimental groups. With use of condition C1, there was improvement in fertilization and cleavage rates when compared to use of condition C (56.4% compared with 45.5% and 80.0% compared 64.7%, respectively). In conclusion, results indicate the use of a cushioning solution during sperm selection positively affects the developmental potential of embryos.

Analysis of endometrial thickness threshold and optimal thickness interval in cleavage embryo hormone replacement freeze-thawed embryo transfer (HRT-FET).

To investigate the effect of endometrial thickness on the clinical outcome of cleavage embryo HRT-FET on the day of embryo transfer and analyzed the threshold and optimal thickness interval corresponding to ideal clinical pregnancy rate by statistical method. A total of 5861 HRT-FET cycles with cleavage embryo transferred from January 2013 to December 2017 in the Reproductive Medicine Center of Henan Provincial People's Hospital were studied retrospectively．Fifth-order grouping of endometrial thickness (EMT) on embryo transfer day as a continuous variable by statistical software, they were divided into five subgroups: Q1 (EMT:4.0-7.9 mm), Q2 (EMT: 8.0-8.9 mm), Q3 (EMT: 9.0-9.5 mm), Q4 (EMT: 9. 6-10.9 mm), Q5 (EMT: 11.0-19.0 mm). After adjusting for confounding factors, the clinical pregnancy rate and live birth rate in other groups were higher than Group Q1 significantly (p < .05). The cutoff value of the endometrial thickness was 8.6 mm, When endometrial thickness was less than 8.6 mm, with each additional 1 mm of endometrial thickness, clinical pregnancy rate increased by 49% (OR = 1.49, 95%CI (1.35, 1.66), p < .001), the live birth rate increased by 59% (OR= 1.59, 95%CI (1.42, 1.78), p < .001), When the endometrial thickness was thicker than the threshold, clinical pregnancy rate (OR = 1.02, 95%CI (0.97, 1.07), p = .398) and the live birth rate (OR = 1.00, 95%CI (0.96, 1.05), p = .398) remained stable. In the cleavage embryo HRT-FET cycle, endometrial thickness is a curvilinear relationship with clinical outcome, the optimal endometrial thickness interval for ideal clinical outcome was 8.6-15mm.

Detection of CD9 and CD81 tetraspanins in bovine and porcine oocytes and embryos.

Tetraspanins are multifunctional molecules located in specific microdomains on the plasma membrane. Thanks to their ability to form networks with other proteins they can participate in many cellular functions. Tetraspanins are part of the interactive network in gametes; however, their precise role in fertilization is not yet clear. The aim of this study was to compare the localization of CD9 and CD81 tetraspanins during oocyte maturation and early development of the embryos in bovine and porcine model. CD9 was detected on the oocyte plasma membrane and vesicles in the perivitelline space of bovine oocytes and embryos. We suggest that CD9 could be a component involved in transzonal projections. Based on the results of in vitro fertilization assay, CD9 and CD81 seem to be part of a more complex fusion network on the plasma membrane of bovine oocytes. On the other hand, both tetraspanins showed a clustered expression pattern on the plasma membrane and inner margin of zona pellucida (ZP) in porcine oocytes and embryos. We found a new species-specific pattern of CD9 and CD81 distribution in ZP which could reflect their specialized role in processes associated with cell adhesion and intercellular communication upon fertilization.

Crocin supplementation during oocyte maturation enhances antioxidant defence and subsequent cleavage rate.

The purpose of the present study was to assess the effect of crocin supplementation during oocyte maturation on the antioxidant defence and anti-apoptotic ability and subsequent developmental competence of porcine oocytes. Oocytes were cultured in media containing 0, 300, 400 or 500 µg/ml of crocin. Upon maturation, the maturation rates, reactive oxygen species (ROS) and glutathione (GSH) levels, mRNA expression of genes (SOD, CAT, GPx, Bcl-2, BAX and Caspase3), expression of cleaved caspase3 and subsequent embryo cleavage rates were measured. Results indicated that the maturation rate of the 400 µg/ml group was 86.80% (p < 0.01). The ROS concentration of the 500 µg/ml group was the lowest (p < 0.01). The GSH concentration of the 400 µg/ml group was the highest (p < 0.01). The SOD, CAT and GPx mRNA expression levels were the highest in the 300, 400 and 500 µg/ml groups, respectively, with the expression levels of all genes being significantly higher than that of the control group (p < 0.01). The Bcl-2/BAX mRNA expression ratio in 400 and 500 µg/ml groups significantly higher than other groups and significantly decreased caspase3 expression level (p < 0.01). The expression level of cleaved caspase3 in the 500 µg/ml treatment group was the lowest, significantly lower than that of the control group (p < 0.01). The cleavage rate of the 400 µg/ml group was 62.50% (p < 0.01). These experimental results show that the supplementation of in vitro culture medium with 400 µg/ml of crocin significantly enhanced the antioxidant defence and anti-apoptotic ability and subsequent cleavage rate of porcine embryo.

Exogenous glutathione improves intracellular glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos.

Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0-32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.

Hormonal stimulation in 4 to 7 months old Nelore (Bos taurus indicus) females improved ovarian follicular responses but not the in vitro embryo production.

The inclusion of pre-pubertal bovine females in reproductive management could allow in vitro embryo production and reduce generation interval, thereby causing faster genetic gain of the herd. However, oocytes of pre-pubertal females have lower competence, blastocyst production, and pregnancy rates than those collected from pubertal animals. This study aimed to evaluate the effect of an induced hormonal stimulation on the serum concentrations of Anti-Mullerian hormone (AMH) and FSH, ovarian responses, ovum pick up (OPU), and in vitro produced embryos (IVP) from oocytes obtained from four-to seven-months old Nelore female cattle. In a crossover design, these females were randomly allocated into: 1) Treated Group (TG, n = 9): the animals were subjected to a hormonal protocol (implanted progesterone device, estradiol benzoate, LH, and FSH) from Day 0 (the start of the treatment) to Day 7 (OPU day), and 2) Control Group (CG, n = 9): the females did not receive any hormonal stimulation, but they had ablation of their largest follicles on Day 2 of experiment. Blood collection for serum FSH measurements was done on Days 5, 6, 7, and 8, and collection for serum AMH measurements was done on Days 5 and 8. As hypothesized, TG had higher serum FSH concentrations (p < 0.05) on Day 5 (1.16 ±â€¯0.31 ng/mL), Day 6 (1.21 ±â€¯0.45 ng/mL), and Day 7 (0.95 ±â€¯0.26 ng/mL) than CG (0.56 ±â€¯0.17 ng/mL on Day 5, 0.60 ±â€¯0.25 ng/mL on Day 6, and 0.60 ±â€¯0.14 ng/mL on Day 7). However, serum AMH concentrations were neither significantly different (p > 0.05) between CG and TG, nor between the collection days. Hormonal stimulation also increased (p < 0.05) total follicular population (20.0 ±â€¯4.95 CG vs 26.66 ±â€¯4.24 TG), ovarian diameter (13.08 ±â€¯1.0 mm CG vs 14.81 ±â€¯1.38 mm TG) and number of follicles ≥2.5 mm (6.88 ±â€¯2.14 CG vs 11.55 ±â€¯4.09 TG). In TG, grades I and II oocytes predominated, whereas, in CG grades III and IV oocytes were more abundant (p < 0.05). No significant increases (p > 0.05) in the cleavage (49.33% CG vs 51.42% TG), cleavage > 4 cells (9.33% CG vs 16.19% TG), and blastocysts rates (1.33% CG vs 8.57% TG) were seen in TG. This hormonal protocol increased serum FSH concentrations that possibly contributed to increases in the observed follicle, as well as improving oocyte quality. This exogenous hormonal stimulation increased available oocytes numbers for IVP, despite no increase in the in vitro embryo production efficiency.

Embryonic early-cleavage rate is decreased with aging in GnRH agonist but not inantagonist protocols.

PURPOSE: The aim of this study was to evaluate the correlation between embryonic early-cleavage status and the age of patients receiving either a GnRH agonist long protocol or a GnRH antagonist protocol. METHODS: This retrospective study included 534 patients undergoing a fresh cycle of oocyte retrieval and day-3 embryo transfer. Of the 534 patients treated, 331 received a GnRH agonist long stimulation protocol (GnRH agonist group) for ovarian stimulation and 203 patients received a GnRH antagonist protocol (GnRH antagonist group). RESULTS: By logistic regression analysis, the rate of embryonic early-cleavage was significantly decreased with increasing age of women in the agonist (P < 0.001) but not in antagonist groups (P = 0.61). Based on the results of this study, maternal age is a critical factor for embryonic early-cleavage in agonist protocol but not in antagonist protocol. The results also showed that early-cleavage embryos were of better quality and resulted in a higher pregnancy rate than late-cleavage embryos in the GnRH agonist group. However, embryo quality and pregnancy rate was not significantly different between early and late cleavage embryos in the GnRH antagonist group. CONCLUSIONS: We conclude that embryonic early-cleavage status is negatively correlated with aging in women receiving GnRH agonist long down-regulation but not in GnRH antagonist protocols. We also conclude that early cleavage of the zygote is not a reliable predictor for pregnancy potential using the GnRH antagonist protocol.

Reduction of Mitochondrial Function by FCCP During Mouse Cleavage Stage Embryo Culture Reduces Birth Weight and Impairs the Metabolic Health of Offspring.

The periconceptual environment represents a critical window for programming fetal growth trajectories and susceptibility to disease; however, the underlying mechanism responsible for programming remains elusive. This study demonstrates a causal link between reduction of precompaction embryonic mitochondrial function and perturbed offspring growth trajectories and subsequent metabolic dysfunction. Incubation of embryos with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), which uncouples mitochondrial oxidative phosphorylation, significantly reduced mitochondrial membrane potential and ATP production in 8-cell embryos and the number of inner cell mass cells within blastocysts; however, blastocyst development was unchanged. This perturbed embryonic mitochondrial function was concomitant with reduced birth weight in female offspring following embryo transfer, which persisted until weaning. FCCP-treated females also exhibited increased adiposity at 4 wk, increased adiposity gain between 4 and 14 wk, glucose intolerance at 8 wk, and insulin resistance at 14 wk. Although FCCP-treated males also exhibited reduced glucose tolerance, but their insulin sensitivity and adiposity gain between 4 and 14 wk was unchanged. To our knowledge, this is one of the first studies to demonstrate that reducing mitochondrial function and, thus, decreasing ATP output in the precompacting embryo can influence offspring phenotype. This is of great significance as a large proportion of patients requiring assisted reproductive technologies are of advanced maternal age or have a high body mass index, both of which have been independently linked with perturbed early embryonic mitochondrial function.

Effects of metformin on fertilisation of bovine oocytes and early embryo development: possible involvement of AMPK3-mediated TSC2 activation.

Studies on bovine oocytes have revealed that the activation of adenosine monophosphate activated protein kinase (AMPK) by millimolar concentrations of metformin controls nuclear maturation. Tuberous sclerosis complex 2 (TSC2) has been identified as a downstream target of AMPK. The objective of this study was to investigate the effects of addition of low concentrations of metformin (1 nM to 10 µM) on the percentage of cultured cumulus-oocyte complexes (COC) giving rise to cleavage-stage embryos and AMPK-mediated TSC2 activation. Metformin was supplemented either throughout in vitro embryo production (IVP) or only during in vitro fertilization (IVF). COC were matured in vitro, inseminated, and presumptive zygotes cultured for a further 72 h post insemination before the percentage of COC that gave rise to zygotes and early embryo development was assessed. The presence of TSC2 in bovine embryos and its possible AMPK-induced activation were assessed by immunocytochemistry. Metformin had a dose-dependent effect on the numbers of cultured COC that gave rise to embryos. Drug treatment either throughout IVP or only during IVF decreased the percentage of ≥ 8-cell embryos (1 µM, P < 0.05; 10 µM, P < 0.01; and 0.1 µM, 10 µM, P < 0.01, respectively) and increased the percentage of 2-cell embryos (10 µM, P < 0.01 and P < 0.05 respectively). The percentage of cultured COC that gave rise to zygotes was not affected by metformin. TSC2 is expressed in early embryos. Metformin (10 µM) either throughout IVP or during IVF only, increased AMPK-induced PhosphoS1387-TSC2 immunoreactivity (P < 0.01) and this increase corresponded to the total TSC2 protein levels expressed in cells. Our results suggest that there is a dose-dependent negative effect of metformin on the ability of oocytes to cleave following insemination, possibly mediated through an AMPK-induced activation of TSC2.

Vitrification of in vitro mature alpaca oocyte: effect of ethylene glycol concentration and time of exposure in the equilibration and vitrification solutions.

The effect of different ethylene glycol concentrations, times of exposure and vitrification procedure on viability, cleavage and blastocyst rate of in vitro matured alpaca oocytes chemically activated after vitrification was analyzed. In Experiment 1, oocytes were incubated for 12-15 min with different concentrations of ethylene glycol (EG) in the equilibration solution (ES) followed by chemical activation and in vitro cultured for 8 days to determine oocyte viability, cleavage and blastocyst rates. In Experiment 2, oocytes were incubated in the equilibration solution containing 4% of EG for 12-15 min and then randomly assigned to vitrification solutions containing 25, 35 or 45% of EG for 30s, vitrified and stored at -196°C. In Experiment 3, oocytes were incubated in the equilibration solution containing 4% of EG for 12-15 min and then randomly assigned to the vitrification solution containing 35% of EG for 15, 30 or 45s, vitrified and stored at -196°C. For Experiments 2 and 3, non-vitrified and vitrified oocytes were activated and cultured in vitro. In Experiment 1, oocyte viability was lowest at concentrations of 6 or 8%, intermediate at 2 or 4% and highest at 0% of EG. Oocyte viability and cleavage rate were affected by EG concentration, time of exposure in the vitrification solution or vitrification procedure in Experiment 2 and 3. Alpaca oocytes were viable after vitrification, given that oocyte viability, cleavage and blastocyst rate were affected by the vitrification procedure, EG concentration and time of exposure in the equilibration and vitrification solutions.

The type of GnRH analogue used during controlled ovarian stimulation influences early embryo developmental kinetics: a time-lapse study.

OBJECTIVE: To explore if the GnRH analogue used for controlled ovarian stimulation (COS) and the ovulation triggering factor (GnRH agonist + hCG triggering versus GnRH antagonist + GnRH agonist triggering) affect embryo development and kinetics. STUDY DESIGN: In a retrospective cohort study in the Instituto Valenciano de Infertilidad (IVI) Alicante and the Instituto Universitario-IVI Valencia, Spain, 2817 embryos deriving from 400 couples undergoing oocyte donation were analysed. After controlled ovarian stimulation and IVF/intracytoplamic sperm injection, the timing of embryonic cleavages was assessed by a video time-lapse system. The results were analysed using Student's t test for comparison of timings (hours) and Chi-squared test for comparison of proportions. A p-value < 0.05 was considered to be statistically significant. RESULTS: Embryos from cycles co-treated with GnRH antagonist + GnRH agonist (n = 2101) cleaved faster than embryos deriving from patients co-treated with GnRH agonist + hCG (n = 716): these differences were significant at the first stages of development but they disappeared as long as the embryo developed. Assessing embryo quality in terms of morphokinetic characteristics, we did not find significant differences between the two groups. CONCLUSION(S): By adopting a time-lapse video system, we can suggest that the type of protocol used for controlled ovarian stimulation influences embryo kinetics of development but these variations are not reflected in embryo quality.

A marked animal-vegetal polarity in the localization of Na(+),K(+) -ATPase activity and its down-regulation following progesterone-induced maturation.

The stage-VI Xenopus oocyte has a very distinct animal-vegetal polarity with structural and functional asymmetry. In this study, we show the expression and distribution pattern of Na(+),K(+) -ATPase in stage-VI oocytes, and its changes following progesterone-induced maturation. Using enzyme-specific electron microscopy phosphatase histochemistry, [(3) H]-ouabain autoradiography, and immunofluorescence cytochemistry at light microscopic level, we find that Na(+),K(+) -ATPase activity is mainly confined to the animal hemisphere. Electron microscopy histochemical results also suggest that polarized distribution of Na(+),K(+) -ATPase activity persists following progesterone-induced maturation, and it becomes gradually more polarized towards the animal pole. The time course following progesterone-induced maturation suggests that there is an initial up-regulation and then gradual down-regulation of Na(+),K(+) -ATPase activity leading to germinal vesicle breakdown (GVBD). By GVBD, the Na(+),K(+) -ATPase activity is completely down-regulated due to endocytotic removal of pump molecules from the plasma membrane into the sub-cortical region of the oocyte. This study provides the first direct evidence for a marked asymmetric localization of Na(+),K(+) -ATPase activity in any vertebrate oocyte. Here, we propose that such asymmetry in Na(+),K(+) -ATPase activity in stage-VI oocytes, and their down-regulation following progesterone-induced maturation, is likely to have a role in the active state of the germinal vesicle in stage-VI oocytes and chromosomal condensation after GVBD.

Differential gene expression profile in bovine blastocysts resulting from hyperglycemia exposure during early cleavage stages.

To understand the compromised survival of embryos derived from assisted reproductive techniques, transcriptome survey of early embryonic development has shown the impact of in vitro culture environment on gene expression in bovine or other living species. However, how the differentially expressed genes translate into developmentally compromised embryos is unresolved. We therefore aimed to characterize transcriptomic markers expressed by bovine blastocysts cultured in conditions that are known to impair embryo development. As increasing glucose concentrations has been shown to be stressful for early cleavage stages of mammalian embryos and to decrease subsequent blastocyst survival, in vitro-matured/fertilized bovine zygotes were cultured in control (0.2 mM) or high-glucose (5 mM) conditions until the 8- to 16-cell stage, and then transferred to control media until they reached the blastocyst stage. The concentration of 5 mM glucose was chosen as a stress treatment because there was a significant effect on blastocyst rate without the treatment's being lethal as with 10 mM. Microarray analysis revealed gene expression differences unrelated to embryo sex or hatching. Overrepresented processes among differentially expressed genes in treated blastocysts were extracellular matrix signalling, calcium signaling, and energy metabolism. On a pathophysiological level, higher glucose treatment impacts pathways associated with diabetes and tumorigenesis through genes controlling the Warburg effect, i.e., emphasis on use of anaerobic glycolysis rather than oxidative phosphorylation. These results allowed us to conclude that disruption of in vitro preattachment development is concomitant with gene expression modifications involved in metabolic control.

Antioxidants rescue stressed embryos at a rate comparable with co-culturing of embryos with human umbilical cord mesenchymal cells.

PURPOSE: During laboratory manipulations, oocytes and embryos are inevitably exposed to suboptimal conditions that interfere with the normal development of embryos. MATERIALS AND METHODS: In this study, we examined the effects of antioxidants, feeder cells and a conditioned medium on embryo development and cleavage rate following exposure of the embryos to suboptimal conditions. We exposed mouse two-cell embryos to visible light and divided them into four groups: control (E-ctr), co-culture (Co-c), conditioned medium (Cndm) and antioxidant-plus medium (Aopm). We used human umbilical cord matrix-derived mesenchymal cells for co-culture. A group of embryos was not exposed to visible light and served as the non-exposed control (NE-ctr) group. RESULTS: The developmental rate was higher in NE-ctr embryos than in the E-ctr group. Exposed embryos in the various groups showed a comparable developmental rate at different stages. Blastomere number significantly increased (P < 0.05) in the Co-c and Aopm groups compared with the E-ctr and Cndm groups. No significant difference was observed between the Co-c and Aopm groups. CONCLUSIONS: Our data indicate that in suboptimal conditions, antioxidants could improve the embryo cleavage rate in the same way as feeder cells. Antioxidants probably improve embryo quality through their ability to scavenge reactive oxygen species.

A prospective randomized sibling-oocyte study of two media systems for culturing cleavage-stage embryos-impact on fertilization rate.

PURPOSE: Although several media systems have been developed, data from prospective randomised clinical studies are still lacking. In the present study we compared the effects of 2 different media systems on embryo morphology and development at days 2/3 using sibling oocytes. METHODS: In this prospective sibling-split trial, 1206 oocytes from 110 women were divided via alternate allocation to fertilization and culture in media system A (G-IVF (TM) v5 PLUS/ G-1(TM) v5 PLUS) or for fertilization and culture in media system B (Universal IVF medium/EmbryoAssist (TM)). RESULTS: The use of media system A significantly increased the normal fertilization rate (73.5% versus 67.2%; p = 0.030) and embryo utilization rate (55.5% versus 42.9%; p = 0.001), whereas polyploidy and embryo quality were similar in the two groups. CONCLUSION: The different impacts on fertilization and early embryo development between the two commercially available and commonly used media systems show the importance of evaluation of the efficacy of existing sequential culture media and the need to further improve media for in vitro development of human embryos.

Fyn kinase is involved in cleavage furrow ingression during meiosis and mitosis.

Fertilization of mammalian oocytes triggers their exit from the second meiotic division metaphase arrest. The extrusion of the second polar body (PBII) that marks the completion of meiosis is followed by the first mitotic cleavage of the zygote. Several lines of evidence in somatic cells imply the involvement of Fyn, an Src family kinase (SFK), in cell cycle control and actin functions. In this study, we demonstrate, using live cell confocal imaging and microinjection of Fyn cRNAs, the recruitment of Fyn to the oocyte's cortical area overlying the chromosomes and its colocalization with filamentous actin (F-actin) during exit from the meiotic metaphase. Fyn concentrated asymmetrically at the cortical site designated for ingression of the PBII cleavage furrow, where F-actin had already been accumulated, and then redispersed throughout the entire cortex only to be recruited again to the cleavage furrow during the first mitotic division. Although microinjection of dominant negative Fyn did not affect initiation of the cleavage furrow, it prolonged the average duration of ingression, decreased the rates of PB extrusion and of the first cleavage, and led to the formation of bigger PBs and longer spindles. Extrusion of the PBII was blocked in oocytes exposed to SU6656, an SFK inhibitor. Our results demonstrate, for the first time, a continuous colocalization of Fyn and F-actin during meiosis and imply a role for the SFKs, in general, and for Fyn, in particular, in regulating pathways that involve actin cytoskeleton, during ingression of the meiotic and mitotic cleavage furrows.

Methionine requirements for the preimplantation bovine embryo.

The early embryo's nutritional environment plays an important role in establishing its developmental potential. However, little is known about the specific nutrient requirements of the embryo. The objective of the present study was to determine requirements of the in vitro produced bovine embryo for the essential amino acid methionine. In addition to serving as a precursor for polypeptides, methionine plays roles in regulation of translation, DNA methylation, and antioxidant balance. In the first experiment, embryos were cultured in potassium simplex optimized medium - bovine embryo modification 2 containing 0, 35, 50, 100, 200 or 400 µmol/l L-methionine for 8 days. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 than other groups but was similar for embryos cultured with 35-400 µmol/l. Neither total cell number, allocation of cells to trophectoderm or inner cell mass, or frequency of apoptosis was affected by methionine concentration. In the second experiment, embryos were cultured with 0, 7, 14, 21, 28 or 35 µmol/l methionine. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 but was not different between embryos cultured with 7-35 µmol/l methionine. However, the proportion of blastocysts that were expanded, hatching or hatched on Day 7 was reduced at lower concentrations of methionine (7 and 14). DNA methylation of blastocyst nuclei was unaffected by methionine concentration but intracellular glutathione content was higher for embryos cultured without methionine. In conclusion, the methionine requirement for preimplantation development is between 14 and 21 µmol/l. These concentrations are lower or similar to those found in the reproductive tract and suggest that methionine deficiency is not a common cause of embryonic mortality.

Impact of linoleic acid on bovine oocyte maturation and embryo development.

Linoleic acid (LA; 18:2 n-6) is the most abundant fatty acid in bovine follicular fluid, and it was previously reported that LA concentration significantly decreases when follicle size increases. This suggests that LA may have a role in the regulation of oocyte maturation. The present study investigated the effect of LA supplementation on bovine oocyte maturation and early embryo development in vitro. Treatment of cumulus-oocyte complexes (COCs) with LA significantly inhibited cumulus cell expansion and retarded development of the oocytes to the metaphase II (MII) stage in a dose-dependent manner. This effect was reversible, and the oocytes developed to the MII stage after extended culture in the absence of LA. Treatment of COCs with LA also resulted in a significantly lower percentage of cleaved embryos and blastocyst yield. Furthermore, COCs treated with LA had significant effects compared with controls in i) increasing prostaglandin E(2) concentration in the medium, ii) decreasing intracellular cAMP at 6 and 24 h of maturation and iii) decreasing phosphorylation of the MAPK1 and 3 at 24 h, and AKT at 6 h of maturation. In conclusion, LA supplementation to bovine oocytes during maturation altered the molecular mechanisms regulating oocyte maturation and resulted in decreased percentage of oocytes at MII stage and inhibition of the subsequent early embryo development. These data provide evidence for adverse effects of LA on oocyte development, which can be associated with dietary increased level of LA in the follicular fluid and the decline in fertility in farm animals and human.

Effect of follicular wave synchronization on in vitro embryo production in heifers.

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7d, followed by the administration of 150microg d-cloprostenol. On Day 7, all follicles >3mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14d. Each experiment had three treatment groups. In Experiment 1 (n=12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28d. Heifers from Group 1X-EB also received 2mg EB i.m. immediately after each OPU session. In Experiment 2 (n=11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos.

Metabolic and mitochondrial dysfunction in early mouse embryos following maternal dietary protein intervention.

Dietary supply of nutrients, both periconception and during pregnancy, influence the growth and development of the fetus and offspring and their health into adult life. Despite the importance of research efforts surrounding the developmental origins of health and disease hypothesis, the biological mechanisms involved remain elusive. Mitochondria are of major importance in the oocyte and early embryo, particularly as a source of ATP generation, and perturbations in their function have been related to reduced embryo quality. The present study examined embryo development following periconception exposure of females to a high-protein diet (HPD) or a low-protein diet (LPD) relative to a medium-protein diet (MPD; control), and we hypothesized that perturbed mitochondrial metabolism in the mouse embryo may be responsible for the impaired embryo and fetal development reported by others. Although the rate of development to the blastocyst stage did not differ between diets, both the HPD and LPD reduced the number of inner cell mass cells in the blastocyst-stage embryo. Furthermore, mitochondrial membrane potential was reduced and mitochondrial calcium levels increased in the 2-cell embryo. Embryos from HPD females had elevated levels of reactive oxygen species and ADP concentrations, indicative of metabolic stress and, potentially, the uncoupling of oxidative phosphorylation, whereas embryos from LPD females had reduced mitochondrial clustering around the nucleus, suggestive of an overall quietening of metabolism. Thus, although periconception dietary supply of different levels of protein is permissive of development, mitochondrial metabolism is altered in the early embryo, and the nature of the perturbation differs between HPD and LPD exposure.