Bovine Embryo Culture Medium Research Articles

MicroRNA-146b negatively affects bovine embryo development and quality

In brief MicroRNAs (miRNAs) carried in extracellular vesicles are one of the essential factors for embryo development. This study demonstrates that miRNA-146 b has negative impacts on the quality and development of bovine embryos and also shows its higher expression in non-blastocyst embryos and related EVs. Abstract MicroRNAs (miRNAs), which can be carried inside extracellular vesicles (EVs), play a crucial role in regulating embryo development up to the blastocyst stage. Yet, the molecular mechanisms underlying blastocyst development and quality are largely unknown. Recently, our group identified 69 differentially expressed miRNAs in extracellular vesicles (EVs) isolated from culture medium conditioned by bovine embryos that either developed to the blastocyst stage or did not (non-blastocysts). We found miR-146b to be more abundant in the EVs derived from media conditioned by non-blastocyst embryos. Using RT-qPCR, we here confirmed the upregulation of miR-146b in non-blastocyst (arrested at two- to four-cell and morula stage) embryos compared to blastocysts (P < 0.005), which coincides with the upregulation of miR-146b in EVs derived from the medium of these non-blastocysts. To evaluate a functional effect, bovine embryo culture media were supplemented with miR-146b mimics, resulting in significantly decreased embryo quality, with lower blastocyst rates at day 7 and lower total cell numbers, while the opposite was found after supplementation with miR-146b inhibitors, which resulted in reduced apoptosis rates (P < 0.01). Transcriptomic analysis of embryos treated with miR-146b mimics or inhibitors showed differential expression (P < 0.01) of genes associated with apoptosis, cell differentiation, and the RNA Pol II transcription complex, including WDR36, MBNL2, ERCC6l2, PYGO1, and SNIP1. Overall, miR-146b is overexpressed in non-blastocyst embryos and in EVs secreted by these embryos, and it regulates genes involved in embryo development and apoptosis, resulting in decreased embryo quality.

Effect of bovine endometrial mesenchymal cell conditioned medium on bovine embryo development

Endometrial mesenchymal stromal cell (eMSCs) secretes bioactive molecules such as cytokines and growth factors. MSCs conditioned media (CM) maintains the immunomodulation and regenerative potential properties of the cells that produced it. In this work CM was used during IVC as an alternative to deliver growth factors in bovine embryo culture medium to induce embryo development and reduce apoptosis. The percentage of embryos that underwent cleavage was similar (P>0.05) among CM, BSA and FBS groups, but blastocyst formation was higher (P<0.05) in FBS group. The total cell number was higher in CM group, but there was no significant differences in cell numbers or apoptotic index among the 3 experimental groups (P>0.05). The relative mRNA expression of ELOVL6 was higher in the CM group, of CASP3 in the BSA group and of ACSL3 and VEGF in the FBS group. Taken together, these data suggest that CM can be used as an alternative supplement in bovine IVF. The gene expression profile suggested that the use of CM inhibited an increase in the relative mRNA levels for CASP3. Moreover, the CM favored the total number cells, inhibited the percentage of cells in apoptosis and produces better quality embryo.

Oxygen tension modulates extracellular vesicles and its miRNA contents in bovine embryo culture medium.

The biotechnology for in vitro embryo production is becoming increasingly popular, being applied to humans and domestic animals. Embryo development can be achieved with either 20% or 5% oxygen tension. The extracellular vesicles (EVs) are secreted by different cell types and carry bioactive materials. Our objective was to determine the secretion pattern and micro RNA (miRNA) contents of EVs released in the bovine embryo culture environment-embryo and cumulus cell monolayer-on Days 3 and 7 of in vitro culture under two different oxygen tensions: High (20%) and low (5%). The EVs were isolated from the medium and analyzed to determine size, concentration, and miRNA levels. EVs concentration in low oxygen tension increased on Day 3 and decreased on Day 7. Additionally, altered EV miRNAs derived from the embryo-cumulus culture medium were predicted to regulate survival and proliferation-related pathways on Days 3 and 7. Moreover, miR-210 levels decreased in EVs isolated from the culture medium under high oxygen tension suggesting that this miRNA can be used as a marker for normoxia since it is associated with low oxygen tension. In summary, this study provides knowledge of the oxygen tension effects on EVs release and content, and potentially, on cell-to-cell communication during in vitro bovine embryo production.

49 EFFECTS OF DIETARY CONJUGATED LINOLEIC ACID SUPPLEMENTATION ON BOVINE OOCYTE LIPID METABOLISM, LIPID COMPOSITION, AND EMBRYO CRYOTOLERANCE

Reduced tolerance to chilling and cryotolerance of oocytes and embryos has been associated with greater cytoplasmic lipids. Previous studies in the cow have demonstrated nutrition-induced modification of follicular components. trans-10, cis-12 conjugated linoleic acid (CLA) was identified as a potent inhibitor of milk fat synthesis in lactating cows, and inclusion of CLA in bovine embryo culture medium reduced embryo lipid content and improved post-thaw embryo survival. Dietary supplementation of cows with CLA could alter oocyte fatty acid metabolism, oocyte lipid composition, and embryo cryotolerance, and responses may be different between Bos indicus and Bos taurus cattle. Therefore, a series of experiments were conducted to evaluate effects of dietary CLA on bovine oocyte lipid content and lipid metabolism and cryosurvival of in vitro-produced embryos from CLA-supplemented oocyte donor cows. Lactating Holstein cows (n = 39) were supplemented 100 g per head/d CLA or Ca salts of palm oil to determine effects of dietary CLA on milk production and milk composition. Nonlactating Holstein (n = 8) and Brahman (n = 17) cows were individually supplemented with 150 g per head/d CLA or no lipid supplement. Cumulus-oocyte complexes (COC) were collected after 41 ± 1 day of CLA supplementation, and mRNA was isolated for qPCR. Relative gene expression in COC from CLA- and control-fed cows was evaluated for genes involved in lipid metabolism (CPT1, FADS2, and PPARα). Crossbred (Angus × Red Angus × Brangus) cows (n = 28) were randomly allotted to a 2 × 2 factorial experiment and fed 150 g per head/d CLA or no lipid supplement. Oocytes were collected (Day 129 and 143 of CLA supplementation), matured, fertilized, and cultured in vitro for 7 days in serum-free culture medium. Embryos were cryopreserved in individual 0.25-mL plastic straws containing 1.5 M ethylene glycol using a slow-cooled method. Post-thaw survival and hatching were evaluated using a 24-h in vitro culture (mSOF with 5% FBS) assay. Milk yield, milk composition, and Nile Red intensity were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). Follicle and oocyte responses were analysed with the Mixed procedure of SAS. Relative gene expression of COC was evaluated using the REST 2009 Software. In vitro embryo production, post-thaw survival, and hatching rates were analysed using Chi Square. Milk fat was depressed (P < 0.001) by 10.1% in lactating Holstein cows fed CLA. Dietary supplementation of Holstien and Brahman cows with CLA did not alter expression of genes (CPT1, FADS2, and PPARα) in COC. Dietary supplementation of crossbred cows with CLA before oocyte collection did not influence cryotolerance of in vitro-produced embryos. Lipid content of oocytes (measured by Nile Red florescence) and follicle, oocyte, and embryo production was not influenced by CLA supplementation of Holstein and Brahman cows. The highly regulated mechanisms involved in fatty acid uptake by ovarian components may help explain the lack of ovarian response to dietary CLA in the current study.

Supplementation of bovine embryo culture medium with L-arginine improves embryo quality via nitric oxide production.

Nitric oxide (NO) is a cell-signaling molecule that regulates a variety of molecular pathways. We investigated the role of NO during preimplantation embryonic development by blocking its production with an inhibitor or supplementing in vitro bovine embryo cultures with its natural precursor, L-arginine, over different periods. Endpoints evaluated included blastocyst rates, development kinetics, and embryo quality. Supplementation with the NO synthase inhibitor N-Nitro-L-arginine-methyl ester (L-NAME) from Days 1 to 8 of culture decreased blastocyst (P < 0.05) and hatching (P < 0.05) rates. When added from Days 1 to 8, 50 mM L-arginine decreased blastocyst rates (P < 0.001); in contrast, when added from Days 5 to 8, 1 mM L-arginine improved embryo hatching rates (P < 0.05) and quality (P < 0.05) as well as increased POU5F1 gene expression (P < 0.05) as compared to the untreated control. Moreover, NO levels in the medium during this culture period positively correlated with the increased embryo hatching rates and quality (P < 0.05). These data suggest exerts its positive effects during the transition from morula to blastocyst stage, and that supplementing the embryo culture medium with L-arginine favors preimplantation development of bovine embryos.

133 EVALUATION OF OOCYTE DONOR SOURCE AND CULTURE MEDIUM ON DOMESTIC CAT EMBRYO DEVELOPMENT RATES

The use of assisted reproductive technology (ART), including in vitro maturation (IVM) and embryo culture, is well established in several species, including canine and feline culture systems. Embryo production conditions tend to be specific for each species and prepared in unique formulations by laboratory. However, the increasing numbers of commercially available media allows for new comparisons in companion animal systems. Therefore, a goal of this study was to compare the development rates of feline parthenotes cultured in a commercially available bovine embryo culture medium with those cultured in a published 3-step domestic cat-specific system. In addition, the source of ovaries utilised for oocyte retrieval was evaluated as a factor in development rates. Ovaries from 2 locations (L1 and L2) were collected on the same day, and harvested oocytes were held in meiotic arrest medium containing 25 μM roscovitine for 14 to 18 h. Oocytes were incubated in maturation medium for 24 h before cumulus cell removal with vigorous pipetting in 0.4% hyaluronidase, and a subset of each group was fixed and stained to determine meiotic maturation rates (n = 76 and 55 for L1 and L2, respectively). Following activation (day 0) by a single course of three 50-μs electric pulses at 1.2 kV cm–1 in 0.3 M mannitol, 0.1 mM CaCl2, and 0.1 mM MgSO4, parthenotes from each source were randomly divided to culture medium treatment of Bovine Evolve medium (Zenith Biotech, Guilford, CT, USA) with 4 mg mL–1 BSA (n = 209) or IVC-1 medium n = 269; (Pope et al. 2009 Theriogenology 71, 864–871), each containing 10 μg mL–1 cycloheximide and 7.5 μg mL–1 cytochalasin B. After a 4-h activation treatment, parthenotes were moved to culture media without cycloheximide and cytochalasin B for embryo development. All parthenotes in IVC-1 medium were moved to IVC-1a medium on day 2. On day 5, both sets of parthenotes were moved to culture media containing 10% heat-inactivated FBS instead of BSA. On day 7, all parthenotes were fixed and stained with Hoechst to determine cell number. No differences were seen in maturation rates between L1 and L2 (56.3 ± 9.5 v. 54.7 ± 9.5, respectively). However, cleavage rates tended to differ, and proportion of embryos greater than 64 cells was different (60.7 ± 5.8 v. 78.3 ± 5.8, P = 0.056 and 3.0 ± 3.1 v. 19.7 ± 3.1, P &lt; 0.005; respectively). We hypothesised that the physical condition of the ovary donors may have affected development rates because cats from L1 tended to be feral animals, whereas cats from L2 were mostly privately owned. Bovine Evolve was similar to IVC-1 medium for cleavage, 32-cell, and 64-cell development rates (74.2 ± 6.7 v. 64.8 ± 6.7; 24.0 ± 7.5 v. 31.8 ± 7.5; 10.7 ± 4.8 v. 12.0 ± 4.8, respectively; P &gt; 0.05). These results indicate that commercially available culture medium can support in vitro development, even if the commercial medium is developed for a different species, but that source of cat ovaries should be considered in feline ART.

41 TRANSMISSION OF A PARASITE-RESISTANT PHENOTYPE FROM A DONOR RAM TO ITS CLONED OFFSPRING

A 9-year-old parasite-resistant superfine Merino ram was cloned to study the inherited phenotype in cloned offspring. This donor ram was &amp;gt;3 standard deviations below the mean for internal parasite fecal egg counts in a 10 000 sheep flock. Two viable rams were produced from 3 days of NT. Their health status was studied from birth to maturity and their fertility and progeny via an AI program. Their mucosal immune system was indirectly assessed through their ability to cope with high internal parasite challenges. Oocytes were extracted from abattoir ovaries in late autumn. Serum-starved fibroblasts were combined with enucleated oocytes and fused; the couplets wereactivated at 25–26 h postmaturation with ionomycin and then DMAP. Embryos were cultured in a 2-stage bovine embryo culture medium (Cook) for 6 days in 5% oxygen. From 3 days of NT, all 37 embryos reaching blastocyst or compact morula stages were transferred into 14 ewes. At Day 30, 6/14 ewes were pregnant with single fetuses and 1 fetus died by Day 60. Each of the 5 lambs had breathing difficulties at birth and 3 died. For 2 weeks prior to term, maternal serum levels (collected every other day) of progesterone and estradiol were not different between viable and non-viable pregnancies. In the 2 viable pregnancies, cortisol levels peaked (&amp;gt;10 ng mL–1), then dropped sharply (&amp;lt;2 ng mL–1 for 4 days), and then rose to the time of birth (&amp;gt;7 ng mL–1). This cortisol profile may help explain why the 2 viable lambs acquired sufficient lung capacity by the time of birth to survive. Samples for fecal egg counts and complete blood counts were collected monthly for 12 months on the 2 surviving ram lambs. Fecal egg counts were 2–5 times higher than for 2 control animals, with peak counts reaching 3000–4000 eggs per gram. Larval culture showed the eggs were predominantly Hemonchus. Clone 2 had consistently higher egg counts than clone 1. Both clones had persistent loose feces for the first 6 months and Clone 2 continues to have diarrhea. Red blood cell counts were consistently low normal (8–11 million/�L). White cell counts were normal, other than when high counts (16 000 per �L) corresponded with high fecal egg counts. Body weight, wool quantity, and fiber diameter at 1 year of age were slightly different between clones (59 kg; 5.3 kg; 16.4 �m vs. 54 kg; 4 kg; 16 �m, respectively). Scrotal circumference was larger for clone 1 than for clone 2 (29 cm vs. 24.5 cm). Semen from the rams was cryopreserved for use in an AI program (50 ewes per sire) to progeny-test lambs produced by the 2 rams, the original donor ram, and a link ram, with lambing due in October 06. Overall, clone 1 has remained consistently healthier than clone 2. High fecal egg counts indicated significant internal parasite challenge during their first year of life, which resulted in elevated white blood cell responses. The obvious clinical effect of high egg counts was diarrhea, which may indicate a beneficial response to parasite infection. Continued studies will further document their parasite resistance, immune responses, and progeny characteristics.

Amino acid metabolism of preimplantation bovine embryos cultured with bovine serum albumin or polyvinyl alcohol

Bovine serum albumin (BSA) is an embryotrophic macromolecule used in embryo culture media, which is commonly replaced with synthetic compounds, such as polyvinyl alcohol (PVA). This study compared the effect of BSA and PVA on the development, blastocyst cell number and amino acid metabolism of preimplantation bovine embryos in vitro. Embryos were produced by in vitro maturation and fertilization of immature oocytes from abattoir-derived ovaries. Zygotes were cultured in synthetic oviduct fluid with either 4 mg/ml BSA (SOFaaBSA) or 1 mg/ml PVA (SOFaaPVA) in microdrops with a mineral oil overlay at 39 °C under a 5% O 2/5% CO 2/90% N 2 atmosphere. Blastocyst rate and cell numbers were determined after 123 h of culture. In parallel, single expanding blastocysts grown in either medium were incubated in microdrops for 12 h. Amino acid profile of spent drops was determined by high performance liquid chromatography. Replacing BSA with PVA depressed blastocyst rate and cell numbers, and led to quantitative and qualitative differences in amino acid appearance, disappearance and turnover. These differences could partly be due to an increase in free intracellular amino acid concentration in SOFaaBSA embryos derived from hydrolysis of endocytosed BSA, and argue against the inclusion of PVA in bovine embryo culture media.

Exogeneous substances affecting development of in vitro-derived bovine embryos before and after embryonic genome activation

Three experiments were conducted to evaluate how exogenous substances [fetal bovine serum (FBS), arachidonic acid (AA), glutathione (GSH), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF), fibroblast growth factor (FGF), insulin, transferrin and selenium (ITS)] affect preimplantation bovine embryo development. Cumulus-oocyte complexes (COC) were matured and fertilized in vitro, and their development was monitored up to 192 h post insemination in a two-step culture system. In Experiment 1, inseminated oocytes were cultured in modified bovine embryo culture medium (mBECM) supplemented with FBS or BSA for up to 60 h post insemination, and the resultant 8-cell embryos were then cultured singly in mBECM supplemented with AA+GSH+PDGF+TGF. More (P<0.005) blastocysts were derived from 8-cell embryos produced in media containing FBS than BSA. In Experiment 2, the 8-cell embryos produced in mBECM supplemented with FBS were cultured singly in mBECM as follows: 1) no supplementation; 2) AA and GSH or 3) AA, GSH, PDGF and TGF. Compared with no supplementation, a significant (P<0.05) increase in the proportion of 16-cell embryos and morulae was obtained after the addition of either AA+GSH or AA+GSH+PDGF+TGF. In Experiment 3, oocytes were cultured singly in mBECM as follows: 1) no supplementation; 2) AA+GSH+PDGF+TGF; 3) AA+GSH+PDGF+TGF and FGF; 4) AA+GSH+PDGF+TGF and ITS; 5) AA+GSH+PDGF+TGF, FGF and ITS or 6) FBS. Eight-cell embryos grown in each system were subsequently cultured singly in mBECM with AA+GSH+PDGF+TGF. More (P<0.05) 16-cell embryos were obtained in medium supplemented with either AA+GSH+PDGF+TGF and ITS or FBS than in unsupplemented medium. Fewer (P<0.05) oocytes developed to the 8-cell stage with the addition of AA+GSH+PDGF+TGF and FGF than without. In conclusion, embryo development to the blastocyst stage is regulated by exogenous AA, GSH, PDGF, FGF and ITS in a stage-specific manner.

Development of bovine ivf oocytes cultured in medium supplemented with a nitric oxide scavenger or inhibitor in a co-culture system

Bovine IVF oocytes were cultured in modified bovine embryo culture medium (mBECM) supplemented with either a nitric oxide (NO) scavenger, hemoglobin (Hb, 1 μg/mL) and/or a NO synthesis inhibitor, Lω-nitro-L-arginine methyl ester (L-NAME, 1 or 1000 nM) in a cumulusgranulosa cell co-culture system. In Experiment 1, a total of 1,675 cumulus-oocytes complexes was collected for 7 mo and cultured to the blastocyst stage in mBECM with or without Hb after IVM and IVF. There were significant (P<0.0024) model effects of Hb addition and month of oocyte collection on embryo development. A significant (P<0.0023) monthly variation was detected in all developmental stages. However, addition of Hb to mBECM consistently enhanced embryo development to the blastocyst stage over all months. No statistical differences were found in the interaction between Hb addition and month except for the cleavage rate. Overall, a greater percentage of oocytes developed to the 8-cell (P<0.0459), 16-cell (P<0.001), morula (P<0.0013) and blastocyst (P<0.0024) stages after the addition of Hb. In Experiment 2, addition of L-NAME to mBECM supplemented with Hb did not further stimulate prehatched development. In conclusion, the promoting effect of Hb on in vitro development of embryos is highly repeatable over an extended period of time.

Precursors of taurine in female genital tract: effects on developmental capacity of bovine embryo produced in vitro.

Two precursors of taurine have been studied: cysteamine and hypotaurine. Cysteamine has been quantified in genital secretions and found in follicular fluids of all species tested. On the contrary cysteamine was not detected (or traces) in tubal fluids of the same species. Addition of 50, 100 or 250 microM of cysteamine to the maturation medium used in the culturing of bovine oocytes did not improve the cleavage rate nor the embryo's developmental potential in vitro. Furthermore, at 250 microM, cysteamine seems to be toxic to the embryo. Addition of 0.5-1 mM hypotaurine to the bovine embryo culture medium improved significantly blastocyst production and quality. The respective roles of these 2 taurine precursors on maturation and embryo development are discussed.

Effects of sperm treatments on the in vitro development of bovine oocytes in semidefined and defined media

Bovine in vitro matured oocytes were inseminated with frozen-thawed spermatozoa prepared by A) swim-up through Fert-TALP supplemented with hyaluronic acid (HYA, 1 mg/ml), heparin (5.0 μg/ml) and bovine serum albumin (BSA, 6 mg/ml) or B) washing by centrifugation in modified Brackett-Oliphant medium (mBO) supplemented with 10 mM caffeine-sodium benzoate. For Method A, in vitro fertilization (IVF) was performed in Fert-TALP supplemented with 6 mg/ml BSA, 5.0 μg/ml heparin, 20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine. For Method B it was performed in mBO medium supplemented with 10 mg globulin-free BSA/ml and 10 μg heparin/ml. Presumptive zygotes were cultured in 1 of 3 culture media: 1) BSAITS — TCM 199 supplemented with 10 mg/ml BSA and ITS (5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml sodium selenite); 2) BECM — bovine embryo culture medium; and 3) BECM supplemented with ITS. Altogether, a significantly higher proportion of oocytes developed to the blastocyst stage after insemination with spermatozoa prepared by Method A than by Method B (17.9 vs 7.1%, respectively; P < 0.001). For Method A, the cleavage rate and the proportion of zygotes with >2 cells 48 h after insemination did not differ significantly between any of the 3 culture media assayed, but blastocyst formation was significantly stimulated in BSAITS and BECMITS compared with that in BECM (20.7 and 22.1% vs 10.7%, respectively; P < 0.05). For Method B, the cleavage rate and the proportion of zygotes with >2 cells were significantly lower in BSAITS than in BECM and BECMITS (56.4 and 28.7% vs 71.6 and 42.1%; and 70.2 and 51.1%, respectively; P < 0.05). However, no significant differences were recorded in blastocyst development rates between any of the culture media assayed (6.4 to 7.4%; P > 0.05).

A continuous flow, perifusion culture system for 8- to 16-cell bovine embryos derived from in vitro culture

Eight- to 16-cell stage bovine embryos derived from in vitro maturation and in vitro fertilization were obtained after static culture until 80 to 88 h post insemination. The embryos were cultured in a dynamic (continuous flow) perifusion culture system for 24 h and were further co-cultured on a cumulus cell monolayer in a static CO 2 incubator for an additional 80 to 88 h. Based on preliminary experiments, a serum-free bovine embryo culture medium (BECM) supplemented with 25 mM HEPES was used as the perifusion culture medium (PCM), and the flow rate was fixed within the range of 30 to 38 μl/min in subsequent experiments. In Experiment 1, the combined effects of tubing (silicone vs Tygon ™) and/or culture chamber size (0.2 vs 1.5 ml) on prehatching development of the embryos following 24 h perifusion culture were evaluated in an experiment with a 2 × 2 factorial arrangement. More (P < 0.005) embryos developed to the blastocyst stage with Tygon ™ than with silicone tubing, regardless of chamber size. A higher percentage of blastocysts (29%) was obtained following the perifusion culture by the combination of Tygon ™ tubing and the 1.5-ml culture chamber than by any other combination. In addition, a significantly (P < 0.001) lower mean pH of the perifusate was obtained with Tygon ™ (7.57 to 7.58) than with silicone (7.78 to 7.79) tubing, regardless of chamber size. In Experiment 2, embryo development was compared after culture in the perifusion system and in a static culture system using either serum-free medium (control) or serum-free medium plus a cumulus cell monolayer (co-culture). Percentages of morulae (49 vs 57%) and blastocysts (31 vs 33%) obtained from the control system were not significantly (P > 0.4) higher than the percentages obtained from the perifusion system. However, these proportions were significantly (P < 0.01) increased by co-culturing the embryos on a cumulus cell monolayer in the static incubator for the same intervals (71% morulae and 44% in blastocysts). In conclusion, 8- to 16-cell bovine embryos can develop to the blastocyst stage following 24 h perifusion in the dynamic culture system using a serum-free medium. Physical stress due to flow rate within the range of 30 to 38 μl/min in an air atmosphere has little effect on the development of perifused embryos cultured under this system.

Intracytoplasmic glutathione concentration and the role of β-mercaptoethanol in preimplantation development of bovine embryos

In vitro-matured/in vitro-fertilized bovine oocytes were cultured on cumulus cell layers in a serum-free medium (bovine embryo culture medium; BECM) supplemented with 3 mg/ml fatty acid-free BSA. The intracytoplasmic glutathione concentration of embryos was found to change significantly (P < 0.008) during the preimplantation stages, beginning to increase at the 9- to 16-cell stage (20.7 pM/embryo) and reaching the highest (P < 0.03) level at the hatched-blastocyst stage (36.7 pM/embryo). A significantly (P < 0.06) lower concentration of glutathione was obtained at the 2- to 8-cell stage (7.1 pM/embryo) than at any other stage. When inseminated oocytes were cultured in BECM supplemented with different concentrations of β-mercaptoethanol (2-ME) to promote glutathione synthesis, higher (P < 0.05) percentages of embryos developed to the 9- to 16-cell, morula and blastocyst stages at 96, 144 and 192 h post insemination, following the addition of 6.25 and 12.5 μM than after no supplementation with 2-ME. However, when 16-cell embryos were cultured in BECM supplemented with 6.25 and 12.5 μM of 2-ME, blastocyst formation was not significantly (P > 0.9) increased. When the combined effects of 2-ME and/or cumulus cells were compared in a 2 × 2 factorial design, there was a significant (P < 0.03) effect of 2-ME on the development of oocytes to blastocysts. The presence of cumulus cells significantly (P < 0.001) affected development after the fourth cleavage (morula compaction and blastocyst formation), but there was no significant (P > 0.11) interaction between 2-ME and cumulus cells. In conclusion, intracytoplasmic glutathione concentration of bovine embryos derived from in vitro-culture increases during preimplantation development. The glutathione synthesis promoter 2-ME exerts its embryotropic role on the development before the fourth cleavage, thus yielding an improvement in blastocyst formation.

A serum-free medium for use in a cumulus cell co-culture system for bovine embryos derived from in vitro maturation and in vitro fertilization

The objective of this study was to develop a serum-free medium for the co-culture of bovine embryos that would yield a percentage of blastocysts equal to that obtained with fetal bovine serum (FBS)-supplemented medium. Cumulus cell-enclosed oocytes (CEO) matured and inseminated in vitro were cultured in a tissue culture medium (TCM)-199 or in a serum-free medium (bovine embryo culture medium; BECM) until 240 h post insemination. Replacement of 10% (v/v) FBS with either 3 mg crystallized bovine serum albumin (BSA)/ml or 3 mg fatty acid-free BSA/ml in TCM-199 had no effect (P > 0.14) on embryo development to the ≥ 2-cell (51 to 60%), ≥ 8-cell (24 to 33%), blastocyst (16 to 19%) and hatched-blastocyst (7 to 10%) stages at 48, 96, 192 and 240 h post insemination, respectively. Oocyte-enclosing cumulus cells in BSA-supplemented medium grew in clusters rather than in layers, as was noted in FBS-supplemented medium. When CEO were cultured in fatty acid-free BSA-supplemented media (TCM-199 and BECM), a significantly (P < 0.001) higher percentage of oocytes developed to blastocysts after culture with (22%) or without (18%) a cumulus cell monolayer than after denuding the oocytes (7%). Glucose in concentrations of 0 to 5.56 mM added for periods of 18 and 120 h post-insemination had neither a stimulatory nor a deleterious effect on preimplantation development. In conclusion, a serum-free medium supplemented with BSA can be successfully used in a cumulus cell co-culture system for bovine embryos.

Bovine inner cell mass cells as donor nuclei in the production of nuclear transfer embryos and calves.

Bovine inner cell mass (ICM) cells were used as donor nuclei in nuclear transfer procedures to determine their totipotency. ICMs were isolated by immunosurgery from expanded Day 7-9 blastocysts that had been produced in vitro. Each individual ICM cell was transferred into an enucleated oocyte. Oocytes were checked for enucleation with Hoechst dye to ensure that all DNA was removed, thus eliminating the possibility of parthenogenetic development. ICM cell-oocyte units were fused by a brief electrical pulse (110 V DC, 15 microseconds in a 500-microns chamber), and the resulting zygotes were placed into CR1 bovine embryo culture medium supplemented with amino acids and fetal calf serum. The nuclear transfer embryos were scored for development to the blastocyst stage on Day 7 (day of fusion = 0). A total of 948 nuclear transfers were completed in 25 trials. In 12 of the trials, development to the blastocyst stage (5%, 30 of 629) was observed. This resulted in an overall developmental rate of 3% for all trials. Twenty-six of the ICM-derived blastocysts were transferred. The initial pregnancy rate at 30 days was 23% with six pregnant recipients. Two pregnancies were lost after 60 days, and four calves were born, two of which were stillborn. These results demonstrated that nucleic of ICM cells from expanded bovine blastocysts were pluripotent, if not in fact totipotent, since these nuclei after nuclear transfer to enucleated oocytes could direct embryonic and fetal development resulting in live offspring.

Effects of fetal calf serum in culture medium on development of bovine oocytes matured and fertilized in vitro

Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined bovine embryo culture medium (BECM) or polyvinylalcohol (PVA)-free BECM supplemented with 10% heat-treated or unheated fetal calf serum (FCS). When PVA in BECM was replaced with FCS 8 h postinsemination, the proportion of ≥-cell-stage embryos 48 h postinsemination was significantly lower in heat-treated FCS (74%) than in PVA (91%) medium, but the value (84%) in unheated FCS was not different from the values in PVA and heat-treated FCS. However, the addition of either heat-treated FCS or unheated FCS to PVA-free BECM did not depress or increase the proportions (63 to 70%) of ≥-cell-stage embryos 96 h postinsemination and stimulated blastocyst formation 144 (21 to 28%) and 192 (47 to 54%) h postinsemination. Blastocyst formation was also stimulated when heattreated FCS or unheated FCS was added to PVA-free BECM 120 h postinsemination. The mean cell number of blastocysts developed in PVA-free BECM with heat-treated FCS (150 cells) or unheated FCS (152 cells) significantly increased compared with that (119 cells) developed without FCS. These results indicate that, although BECM can support bovine embryo development, a high proportion (about 50%) of morphologically normal blastocysts can be produced from oocytes matured and fertilized in vitro when they are cultured in PVA-free BECM containing FCS.

Assay of embryo-derived platelet activating factor (EDPAF) by an equine platelet aggregation assay: Preliminary data concerning its presence in bovine embryo culture media

This study was designed to establish a sensitive bioassay for bovine platelet-activating factor (PAF), to determine if the bovine embryo secretes PAF in vitro and if PAF release is correlated with the embryo's potential to establish a pregnancy. Using an equine platelet aggregation assay, lipid extracted culture media from 33 Day-7 embryos (individually cultured for 18 hours in 1 ml of Ham's F10 containing 0.4% BSA at 37°C in an air: CO 2 mixture of 95:5 prior to their transfer to recipient heifers) and from control media (n=15, Ham's F10 + 0.4% BSA incubated simultaneously without embryos) were investigated. In addition, culture media from Day-6 (n=6) and Day-1 (2-cell, n=12) bovine embryos that were cultured for 4 hours but not transferred were examined. The aggregation assay proved to be sensitive to 5 pg of PAF. The assay proved to be specific, since the PAF receptor antagonist SRI 63–441 inhibited platelet aggregation induced by culture media in dosages comparable to aggregation induced by synthetic PAF 18. From the 15 Day-7 embryos that established a pregnancy 2 contained measurable amounts of PAF in their culture media. No PAF was detected in the culture media from 13 embryos that succeeded, in the 18 embryos that failed to establish a pregnancy, or in the control media. One of 6 Day-6 embryos and 3 of 12 Day-1 (2-cell) embryos secreted detectable amounts of PAF into the culture media. Although the results indicate that some bovine embryos release PAF or a PAF-like substance in vitro, PAF measurements in the culture medium seem not to be a suitable method for the evaluation of bovine embryos prior to transfer.

Addition of bovine oviductal epithelial cells (BOEC) and/or penicillamine, hypotaurine and epinephrine (PHE) to bovine in vitro fertilization (IVF) medium increases the subsequent embryo cleavage rate

The objective of this study was to develop a serum-free medium for the co-culture of bovine embryos that would yield a percentage of blastocysts equal to that obtained with fetal bovine serum (FBS)-supplemented medium. Cumulus cell-enclosed oocytes (CEO) matured and inseminated in vitro were cultured in a tissue culture medium (TCM)-199 or in a serum-free medium (bovine embryo culture medium; BECM) until 240 h post insemination. Replacement of 10% (v/v) FBS with either 3 mg crystallized bovine serum albumin (BSA)/ml or 3 mg fatty acid-free BSA/ml in TCM-199 had no effect (P > 0.14) on embryo development to the ≥ 2-cell (51 to 60%), ≥ 8-cell (24 to 33%), blastocyst (16 to 19%) and hatched-blastocyst (7 to 10%) stages at 48, 96, 192 and 240 h post insemination, respectively. Oocyte-enclosing cumulus cells in BSA-supplemented medium grew in clusters rather than in layers, as was noted in FBS-supplemented medium. When CEO were cultured in fatty acid-free BSA-supplemented media (TCM-199 and BECM), a significantly (P < 0.001) higher percentage of oocytes developed to blastocysts after culture with (22%) or without (18%) a cumulus cell monolayer than after denuding the oocytes (7%). Glucose in concentrations of 0 to 5.56 mM added for periods of 18 and 120 h post-insemination had neither a stimulatory nor a deleterious effect on preimplantation development. In conclusion, a serum-free medium supplemented with BSA can be successfully used in a cumulus cell co-culture system for bovine embryos.