A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.     

Effect of cysteine,glutamate and glycine supplementation to in vitro fertilization medium during bovine early embryo development

Glutathione (GSH) is an antioxidant synthesized from three constitutive amino acids (CAA): cysteine (Cys), glycine (Gly) and glutamate (Glu). Glutathione plays an important role in oocyte maturation, fertilization and early embryo development. This study aimed to investigate the effect of Cys (0.6 mM), Gly (0.6 mM) and Glu (0.9 mM) supplementation during in vitro fertilization (IVF) of cattle oocytes. In a Pilot Experiment, de novo synthesis of GSH in bovine zygote was evaluated using a modified TALP medium prepared without MEM-essential and MEM-non-essential amino acids (mTALP): mTALP + CAA (constitutive amino acids); mTALP + CAA+5 mMBSO (buthionine sulfoximide); mTALP + Cys + Gly; mTALP + Cys + Glu and mTALP + Gly + Glu. This evidence led us to investigate the impact of CAA supplementation to TALP medium (with essential and non-essential amino acids) on zygote viability, lipid peroxidation, total intracellular GSH content (include reduced and oxidized form; GSH-GSSG), pronuclear formation in zygotes and subsequent embryo development. IVF media contained a) TALP; b) TALP + Cys + Gly + Glu (TALP + CAA); c) TALP + Cys + Gly; d) TALP + Cys + Glu; e) TALP + Gly + Glu, were used. Total GSH-GSSG concentration was increased in TALP, TALP + CAA, and TALP + Cys + Gly. The viability of zygote was similar among treatments. Lipid peroxidation was increased in zygote fertilized with TALP + Cys + Gly; TALP + Cys + Glu; TALP + Gly + Glu and TALP + CAA. The percentage of penetrated oocytes decreased in TALP + CAA and TALP + Cys + Gly. The cleavage rate was lower in TALP + CAA and TALP + Gly + Glu. The percentage of embryos developing to the blastocyst stage was lower in TALP + Cys + Glu and TALP + CAA. In conclusion, we have demonstrated the synthesis of GSH during IVF. However, Cys, Gly and Glu supplementation to TALP medium had negative effects on embryonic development.     

The role of the oviduct and extracellular vesicles during early embryo development in bovine

The oviduct is an important reproductive structure that connects the ovary to the uterus and takes place to important events such as oocyte final maturation, fertilization and early embryonic development. Thus, gametes and embryo can be directly influenced by the oviductal microenvironment composed by epithelial cells such secretory and ciliated cells and oviductal fluid. The oviduct composition is anatomically dynamic and is under ovarian hormones control. The oviductal fluid provides protection, nourishment and transport to gametes and embryo and allows interaction to oviductal epithelial cells. All these functions together allows the oviduct to provides the ideal environment to the early reproductive events. Extracellular vesicles (EVs) are biological nanoparticles that mediates cell communication and are present at oviductal fluid and plays an important role in gametes/embryo - oviductal cells communication. This review will present the ability of the oviducts based on its dynamic and systemic changes during reproductive events, as well as the contribution of EVs in this process.     

Porcine oviductal cells support in vitro bovine embryo development

This study was designed to investigate the developmental competency of in vitro-matured and in vitro-fertilized bovine embryos co-cultured with a) medium alone, b) bovine oviductal cells (BOC), c) bovine conditioned medium (BCM), d) porcine oviductal cells (POC), and porcine conditioned medium (PCM). Follicular oocytes collected from cattle at local slaughterhouses were matured and fertilized in vitro. Epithelial cells were scraped from the luminal surface tissue of either bovine or porcine oviducts collected after ovulation, cultured in TALP + 10% heat-treated fetal calf serum, and the conditioned media were collected following a 3- to 5-d incubation period. After 18 to 22 h of sperm-ova co-incubation, the fertilized and/or cleaved ova were randomly assigned to 1 of 5 co-culture groups. The results revealed that the efficiency of medium alone in supporting embryo development from the 16- to 32-cell stage up to the blastocyst stage was significantly (P<0.01) lower than of embryos co-cultured with either bovine or porcine epithelial cells, or with conditioned media from such cells. Epithelial cell co-culture, regardless of cell source, was more effective (P<0.01) than culture with conditioned medium. Co-culture in medium containing or conditioned by porcine cells was more effective in supporting bovine embryo development than co-culture with bovine-derived cells or conditioned medium. These data support the concept that oviductal cells produce a soluble component which enhances embryo development to the blastocyst stage in vitro and that the effect is not species-specific.     

Cumulus cell function during bovine oocyte maturation,fertilization, and embryo development in vitro

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures. Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.     

Culture of in vitro produced bovine zygotes in vitro vs in vivo: implications for early embryo development and quality

The objectives of this study were to examine the effect of culture system on bovine blastocyst formation rates and quality. Presumptive IVM/IVF bovine zygotes were cultured either in vitro in synthetic oviduct fluid (SOF, 25 embryos/25 microL in 5% CO2, 5% O2, 90% N2 at 39 degrees C) or in vivo in the ewe oviduct (approximately 100 embryos per oviduct). The recovery rate after in vivo culture was 53% (813/1,530). The blastocyst rate on Day 7 was significantly higher for the in vitro system (28%, 362/1,278 vs 17%, 37/813; P< 0.0001). However, after culture in vitro for a further 24 h, there was no difference in Day 8 yields (36%, 457/1,278 vs 32%, 258/813, for in vitro and in vivo culture, respectively). There was no difference in blastocyst cell number between treatments (Day 7: 96 vs 103; Day 8: 78 vs 85 for in vitro and in vivo culture, respectively). Irrespective of culture system, Day 7 blastocysts had a significantly higher cell number than those appearing on Day 8. There was no difference in pregnancy rate at Day 35 after fresh transfer of a single Day 7 blastocyst (37.5%, 21/56 vs 45.3%/, 24/53 for in vitro and in vivo culture, respectively). After cryopreservation by freezing in 10% glycerol, VS3a vitrification or solid surface vitrification, the survival of in vitro cultured embryos was significantly lower than survival of embryos cultured in the ewe oviduct or those produced by superovulation of donors. In conclusion, these findings demonstrate that while bovine zygotes cultured in vitro are capable of rates of development similar to those of their in vivo cultured counterparts (in terms of Day 8 blastocyst yield, cell number and early pregnancy rate), there are significant differences in embryo cryosurvival. This suggests that current in vitro culture systems need to be improved to optimize embryo quality and pregnancy rates.     

Interaction between embryos and culture conditions during in vitro development of bovine early embryos

Various factors such as embryo density and substances in the medium can influence embryo development in vitro. These factors and the embryos probably interact with each other, however the interactions are not fully understood. To investigate the interactions, we examined the effects of the number of embryos, drop size, oxygen concentration and glucose and inorganic phosphate in the medium during protein-free culture of bovine IVM/IVF embryos. In Experiment 1, different numbers of embryos were cultured in a 50 microl drop of medium. The frequencies of blastocyst development in the groups with 25, 50 and 100 embryos per drop were higher than in the other groups. One, five and 25 embryos were cultured in different drop sizes (Experiment 2), a 50 microl drop of medium at different O2 concentrations (Experiment 3) and a 50 microl drop of medium excluding glucose and/or inorganic phosphate (Experiment 4). In Experiment 2, the size of the medium drops did not improve blastocyst development. In Experiment 3, the highest frequency of blastocyst development for one, five and 25 embryos per drop was obtained at 1, 2.5 and 5% O2, respectively. In Experiment 4, blastocyst development for one and five embryos per drop were improved in the medium excluded inorganic phosphate. These results indicate that there is a cooperative interaction among embryos during culture and that this interaction may be mediated by reduction of toxic factors in the medium. At low embryo density, reduced oxygen concentration or the exclusion of inorganic phosphate enhanced blastocyst development.     

RNA polymerase during early development in mouse embryo

RNA polymerase activity in mouse embryo homogenates has been measured at various stages of pre-implantation development. The amount of enzyme/embryo appears to increase in the period under consideration. On a per cell basis a decline in the level of polymerase was, however, observed from the 2-cell to the early blastocyst stages.     

Ammonia concentration in bovine follicular fluid and its effect during in vitro maturation on subsequent embryo development

10 mm) to determine ammonia concentration. Ammonia concentration was greater (P<0.001) in the bFF from smaller follicles compared with bFF from larger follicles, and ammonia concentration decreased (P10 mm follicles, respectively. In Experiment 2, abattoir oocytes (n=2885) were matured in vitro in media containing ammonia at the concentrations of 0 microM (Control), 29 microM (A1), 88 microM (A2), 132 microM (A3), 176 microM (A4) and 356 microM (A5), followed by in vitro fertilization and culture. There was no difference (P0.05) in cleavage rates and morula or blastocyst development between treatments, indicating that in vitro exposure of bovine oocytes to ammonia does not adversely influence subsequent embryonic development.     

Effects of glucose and protein sources on bovine embryo development in vitro

Oviductal factors may be obtained by ultrafiltration of conditioned medium, added to a simple media and used in bovine embryo culture. In this study, we aimed to analyze the development of bovine embryos produced with oviductal factors compared to those cultured in the presence of BSA or serum, the effects of glucose in presence of these protein supplements, and the ability of oviductal factors to support embryo development during the entire culture period. In vitro produced bovine zygotes from slaughterhouse ovaries were cultured in modified-synthetic oviduct fluid (mSOF) alone or supplemented with (1) oviductal factors, (2) BSA and (3) FCS. Oviductal factors showed embryotrophic activity, although with blastocyst rates lower than those in BSA and FCS. Glucose (1.5 mM) added at Day 2 of culture did not affect development in the presence of oviductal factors. The number of cells in expanded blastocysts was unaffected by the presence of glucose or any of the protein supplements used. Both BSA and FCS, respectively, improved blastocyst rates of Day 6 embryos produced with oviductal factors. The effect of oviductal factors was masked by the presence of BSA during the entire culture. FCS promoted an earlier appearance of blastocysts. It is concluded that the effect of glucose on in vitro embryo development depends upon the source of protein. Oviductal factors are not an appropriate supplement for embryos beyond Day 6 of culture in SOF, although blastocyst rates of such embryos may be increased by culturing them in the presence of FCS or BSA.     

Effects of resazurin on bovine oocyte fertilization and embryo development in vitro

Resazurin is a redox dye (7-hydroxy-3H-phenoxazin-3-one-10-oxide) used for assessing potential fertility of spermatozoa and functional status of eukaryotic cells. In this study, the fertilizing capacity of spermatozoa treated with resazurin and effects of resazurin on bovine embryo development in vitro was examined. Abattoir-derived bovine oocytes were collected and subjected to in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 1, bovine oocytes (n=2767) were fertilized with spermatozoa exposed to resazurin (17.6 μg/ml) for 0, 15, 30, 60 min, respectively. There was no significant (P>0.05) difference with respect to oocyte cleavage, morula and blastocyst production between treatments. In Experiment 2, oocytes (n=1671) were treated with resazurin (1.8 μg/ml) during IVM, IVF, IVC, respectively, or during the entire IVM, IVF and IVC procedures. There was no significant (P>0.05) difference in cleavage rates. However, the proportion of embryos that developed into blastocysts, expanded and hatched blastocysts in those groups in which oocytes/embryos were treated with resazurin during IVC or IVM/IVF/IVC was significantly (P<0.05) less than those exposed to resazurin during IVM only, or during IVF only. We conclude that resazurin did not have significant adverse effects on fertilizing capability of bovine spermatozoa; however, extended treatment of embryos with resazurin may be detrimental to embryonic development.     

Telomerase activity in bovine embryos during early development

The telomere is the end structure of the DNA molecule. Telomerase is the ribonuclear enzyme that helps the cell's telomere to elongate; otherwise, the telomere will shorten with each cell division through conventional DNA replication. In most mammalian species, telomerase activity is present in germ cells but not in somatic cells. Recent research shows that telomerase activity is also present in early embryos, but to our knowledge, the dynamics of this enzyme during early embryo development have not been studied. In the present work, we conducted telomerase activity assays on bovine embryos fertilized in vitro and harvested at different stages from zygote to blastocyst. A polymerase chain reaction-based assay (Telomeric Repeat Amplification Protocol) was used to detect the telomerase activity in these embryos. We demonstrated that the telomerase activity is present in the early embryos, but that its level varies with the different developmental stages. The activity was relatively low in mature oocytes. It increased after in vitro fertilization and then decreased gradually until the embryo reached the eight-cell stage. After the eight-cell stage, the telomerase activity increased again and reached its highest level in the blastocyst stage. This study provides insight regarding how telomerase activity and, possibly, the length of the telomere are reprogrammed during early embryo development.