Changes in ribosomal ribonucleic acid content within in vitro-produced bovine embryos

The abundance of 28S, 18S, and 5S rRNA was measured by Northern blot techniques applied to RNA samples extracted from bovine oocytes and preattachment embryos produced by in vitro procedures. Total RNA content was estimated by comparing the intensity of hybridization signals of 28S and 18S rRNA probes to embryo RNA samples and to standard curves generated from bovine ovary or bovine oviduct cell RNA. RNA content declined from the oocyte to the morula stage (2.4 +/- 0.3 ng/oocyte, 1.7 +/- 0.5 ng/1-cell embryo, 2.2 +/- 0.9 ng/2- to 4-cell embryo, 0.8 +/- 0.2 ng/6- to 8-cell embryo, and 0.7 +/- 0.2 ng/morula). A marked increase in RNA content, based on levels of hybridization to 28S and 18S rRNA, was observed in blastocysts, in which values averaged 5.3 +/- 0.6 ng/embryo. On a relative basis, 5S rRNA abundance followed a pattern similar to that of 28S and 18S rRNA across the early development period to the blastocyst stage.     

Hypertrophic cardiomyopathy. Role of Doppler echocardiography in the diagnosis and therapeutic approach

The present study was undertaken with the aim to study the role of isologous and heterologous (buffalo) oviductal cell to co-culture on in vitro development of goat embryos. The oocytes were collected by puncturing the goat ovaries obtained from slaughterhouse. The oocyte recovery rate per ovary was 3.0. The media used for oocyte maturation and embryo development was TCM-199 + 10 percent buffalo estrus serum. A total of 79.8 percent oocytes got matured out of 1056 oocytes. The oocytes were inseminated with epididymal buck spermatozoa capacitated in Brackett and Oliphant media. In group I without oviductal cells co-culture only 13.6 percent matured oocytes cleaved and 3.3 and 0.0 percent reached the morula and blastocyst stage. In group II and III having goat and buffalo oviductal cells the cleavage was 57.6 and 59.2 percent respectively. The percentage of morula, blastocyst and those embryos arrested between 2-16 cells were 26.3, 10.2, 63.5 and 26.6, 8.9 and 64.5 in goat and buffalo oviductal cell groups. The results indicated that the oviductal cell co-culture had a marked effect on cleavage and development of goat IVF embryos. Buffalo oviductal cells can be used well for goat embryo development.     

Development of in vitro derived bovine embryos following pronuclear transplantation and in vitro culture

This study was designed to evaluate the survival and development of in vitro derived bovine embryos following pronuclear transplantation and in vitro embryo culture. Bovine zygotes were produced by in vitro maturation and in vitro fertilization. Pronuclei were removed by micromanipulation and either transferred back to the same cell (Group 1) or into a previously enucleated zygote (Group 2) by electrofusion. Micromanipulated and non-micromanipulated (Group 3, control) zygotes were co-cultured with oviductal cells in a sealed modular chamber filled with 5% CO2, 5% O2 and 90% N2 at 39 degrees C for 7-8 days. Fusion rates were similar for Groups 1 and 2 (90.7 and 85.1%, respectively, P > 0.05). The percentage of embryos that cleaved was not different for Groups 1 (82.0%), 2 (90.0%) and 3 (76.9%, P > 0.05). Also, the percentage of embryos developing to the compact morula or blastocyst stage was similar (25.6, 22.5 and 22.3%, respectively, for Groups 1, 2 and 3, P > 0.05). The results of this experiment are the first to demonstrate that pronuclear transfer can be carried out successfully using bovine embryos derived from in vitro oocyte maturation and in vitro fertilization. In addition, pronuclei can be transferred from one bovine embryo to another and the reconstructed embryos develop to the compact morula and blastocyst stage in vitro. This technique, used in combination with oocyte retrieval by ultrasound-guided follicular aspiration and embryo transfer, offers the potential to study cytoplasmic inheritance in cattle directly, and to evaluate the effect of cytoplasmic inheritance on traits of economic importance.     

Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability

Glutathione (GSH) has been shown to play an important role in embryo development. In a previous study, we demonstrated that cysteamine supplementation of in vitro maturation (IVM) medium increased the intracellular GSH content in bovine oocytes and improved subsequent embryo development to the blastocyst stage. The present study was carried out to evaluate the effect of inhibition by buthionine sulfoximide (BSO) of GSH synthesis during IVM in the presence of cysteamine, on subsequent embryo development, and the effect of cysteamine during IVM on the survival of blastocysts following freezing. The effect of beta-mercaptoethanol and cysteine added to the maturation medium on GSH levels in bovine oocytes, as well as the effect of these compounds on de novo GSH synthesis by oocytes during in vitro maturation, was also studied. The inhibitory effect of BSO during in vitro maturation on GSH synthesis was also evaluated. Evidence was found confirming that GSH synthesis occurs intracellularly during IVM of oocytes and is stimulated by cysteamine, beta-mercaptoethanol and cysteine. Moreover, the present results suggest that the increase in the rate of embryo development exerted by cysteamine, when present during IVM, was due to its stimulatory effect on GSH synthesis. This increase in GSH levels during IVM improves embryo development and quality, producing more embryos reaching the blastocyst stage on day 6, those most suitable for freezing.     

Leigh syndrome: clinical features and biochemical and DNA abnormalities

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5-9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 +/- 21.4, which significantly decreased to 85.6 +/- 17.4, 62.0 +/- 14.5, and 17.0 +/- 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5-5.5. Blastocysts developed, at the earliest, between days 5.0-6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 +/- 11.3, including 59.2 +/- 8.4 cells of TE and 42.1 +/- 6.9 cells of ICM.     

Trophic effects of myeloid leukaemia inhibitory factor (LIF) on mouse embryos

Myeloid leukaemia inhibitory factor (LIF) is expressed at highest concentrations in the maternal endometrial glands at about the stage of blastocyst implantation. LIF is also expressed by the extraembryonic membranes of the early mouse embryo. Embryos of different ages were cultured with, or without, LIF, and embryo growth in vivo and in vitro was examined to determine whether LIF is important for embryo development. Supplementing embryo culture media with 1000 U recombinant human LIF ml-1 increased the number of eight-cell mouse embryos developing beyond the hatched blastocyst stage in vitro from 62.1% to 85.1% (P < 0.05). LIF significantly increased the number of embryos hatching (33.8% versus 7.65% for controls 96 h after hCG injection, P < 0.001), completely hatching (85.1% versus 62.1%, P < 0.05), and exhibiting trophoblast outgrowth (13.5% versus 0% 120 h after hCG treatment, 85.1% versus 47.0% 144 h after hCG treatment, P < 0.001) in vitro. LIF-treated embryos also displayed a significantly greater area of trophoblast outgrowth than did controls as early as day 5 in culture (P < 0.005). These data show that LIF enhances mouse eight-cell embryo development in vitro, as seen by the accelerated rate of embryo hatching and trophoblast outgrowth. In addition, enhanced embryo survival in vivo is shown, following the transfer of LIF-treated embryos into a pseudopregnant recipient female. Expression of mRNA encoding LIF was detected in endometrial cells cultured in monolayer from uteri of day 3 pregnant females, explaining the known embryotrophic effects of endometrial coculture. This expression was not enhanced significantly by treatment with oestradiol (3.7 x 10(-5) mol l-1) or progesterone (3.2 x 10(-6) mol l-1) or both hormones. These results indicate that LIF could have a dual action in early embryogenesis as an embryotrophin and as a factor required for embryo implantation. Multiple roles for LIF are consistent with the expression of this factor at embryonic, extraembryonic and maternal sites during early embryogenesis.     

Developmental potential of embryos produced by in-vitro fertilization from gonadotrophin-releasing hormone antagonist-treated macaques stimulated with recombinant human follicle stimulating hormone alone or in combination with luteinizing hormone

We previously demonstrated, in luteinizing hormone (LH)-deficient macaques, that follicular growth and maturation occurred with administration of exogenous (recombinant human) follicle stimulating hormone (r-hFSH) alone, and that the oocytes recovered fertilized at a notably higher rate than their counterparts from animals receiving both r-hFSH and r-hLH (Zelinski-Wooten et al., 1995). Here, the developmental potential of embryos produced from animals treated with r-hFSH alone or in combination with r-hLH was evaluated. Embryos (n = 127) were cryopreserved, thawed and either co-cultured on buffalo rat liver cells until the hatched blastocyst stage or transferred to synchronized recipients. Although embryos from each treatment group demonstrated a similar ability to develop to hatched blastocysts with a definitive inner cell mass, a significant difference was seen in cryosurvival (56 versus 78%) and in developmental rate to the hatched blastocyst (12 versus 10 days) between embryos from the r-hFSH alone and the combination group respectively. Pregnancies resulted following oviductal embryo transfers in both groups, with corpus luteum rescue occurring on days 12-16 of the luteal phase. In summary, r-hFSH alone during the pre-ovulatory interval is adequate for the gametogenic events required to produce embryos that develop either in vitro or in vivo; however, exposure to r-hLH may improve embryo viability and the rate of development.     

An EORTC international multicenter randomized trial (EORTC number 19923) comparing two dosages of liposomal amphotericin B for treatment of invasive aspergillosis

Experiments were performed to determine the actions of recombinant bovine interleukin-1beta (IL-1beta) on the growth of preimplantation embryos. In the first series of studies, IL-1beta was added at 8-10 h after insemination, and the percentage of oocytes developing to the blastocyst stage was evaluated. IL-1beta increased development to the blastocyst stage when embryos were cultured at high density ( approximately 25-30 embryos/drop) but decreased or had no effect on development when cultured at low density ( approximately 10 embryos/drop). Thus, the positive effect of IL-1beta depends upon some other embryo-derived product. The effect of IL-1beta on embryonic development was maintained in completely denuded embryos, indicating that cumulus cells do not mediate the actions of IL-1beta. Maximum development of embryos cultured at approximately 25-30/drop occurred at 0.1-1 ng/ml; 10 ng/ml was less effective. Addition of IL-1beta to groups of approximately 25-30 embryos/drop at 8-10 h after insemination also increased embryo cell number at Day 5 postinsemination by increasing the proportion of embryos that reached the 9- to 16-cell stage. However, IL-1beta had no effect on the proportion of blastocysts when added at Day 5 postinsemination. Thus, IL-1beta probably acts to increase blastocyst numbers by exerting actions on embryo growth before Day 5. In contrast to its effect on embryos, addition of IL-1beta during oocyte maturation did not affect cumulus expansion, cleavage rate of oocytes, or subsequent development to the blastocyst stage. In conclusion, IL-1beta can modulate growth of bovine embryos at early stages of development in a manner dependent upon embryo density.     

Expression of leukaemia inhibitory factor (LIF) receptor in human placenta: a possible role for LIF in the growth and differentiation of trophoblasts

Leukaemia inhibitory factor (LIF) is a cytokine that displays multiple activities in various tissues and is essential for blastocyst implantation in mice. In the human uterus, LIF is expressed in endometrial tissue and the decidua. To elucidate the role it plays, the mRNA levels for two LIF receptor (R) subunits, LIF-R and gp130, were examined in human endometrium, placenta and decidua by Northern blot hybridization. The expression of LIF-R gene was detected in the chorionic villus during the first trimester, in term placenta, and at lower levels in the decidua. The expression of LIF-R gene was not detectable in non-pregnant endometrium. The expression of the gp130 gene was detected in all tissues examined. During pregnancy, there was no significant change in the mRNA concentration of LIF-R in the placenta, while that of gp130 increased after the second trimester. The human choriocarcinoma cell line, BeWo, was found to express LIF-R and gp130. LIF inhibited forskolin-induced human chorionic gonadotrophin (HCG)-beta production by BeWo in a dose-dependent manner, and it ameliorated forskolin-induced growth suppression. These findings suggest that LIF plays a regulatory role in trophoblast growth and differentiation during pregnancy in human placenta.     

Embryo cloning in cattle: the use of in vitro matured oocytes

In vitro matured (IVM) bovine oocytes were examined to determine their potential viability in embryo cloning. Activation competence, as monitored by pronuclear formation, increased with oocyte age. Oocytes readily formed a pronucleus when challenged with an electrical pulse 30 h after the onset of maturation. Developmental competence of IVM oocytes tended to increase with oocyte age (P = 0.079). Selection of IVM oocytes on the basis of the presence of a polar body 24 h after the onset of maturation and the size of the follicle from which the oocyte was derived improved development of nuclear transfer embryos (polar body positive 25% versus polar body negative 10%, P < 0.05; large follicle oocytes 31% versus small follicle oocytes 14%, P < 0.05). When selected, IVM oocytes were compared with in vivo matured oocytes recovered from superovulated cows and heifers; no difference was detected for the frequency of embryos produced, pregnancies confirmed between days 50 and 60 of gestation, or the number of calves born. We conclude that selected IVM oocytes are equivalent to in vivo matured oocytes when used for bovine embryo cloning.     

Reproductive wastage in the androstenedione-immune ewe

An experiment was carried out using 320 adult Merino ewes to examine the effects of immunization against an androstenedione human serum albumin conjugate (Fecundin) on ovulation rate, fertilization rate and embryo viability at days 2, 9 and 13-14 after fertilization. The ovulation rate of immunized ewes (2.19 +/- 0.06) was greater (P < 0.001) than that of control ewes (1.43 +/- 0.04). The recovery rate of embryos or of unfertilized oocytes on day 2 was reduced in immunized ewes, but fertilization rate of recovered oocytes was unaffected by immunization. The mean number of normal embryos per ewe pregnant (prolificacy) was higher and the proportion of ewes pregnant (fertility) was lower in immunized than in the control ewes. The distribution of the number of cells per embryo showed no differences in developmental age over the period of sampling, the majority of embryos at this time being at the two- to four-cell stage of development. At day 9 of pregnancy, blastocyst recovery rates were lower in immunized than in control ewes. About 90% of blastocysts recovered were developing normally in control ewes compared with 64% in immunized ewes. The majority of blastocysts recovered on day 9 had hatched from the zona pellucida prior to recovery (mean values were 94.2% and 87.8% for control and immunized groups, respectively). In control ewes single blastocysts were larger than twin blastocysts, but for the immunized ewes this difference was not significant. Both single blastocysts (P < 0.01) and twin blastocysts (P < 0.05) from immunized ewes were smaller than those from control ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of pig oocytes activated by stimulation of an exogenous G protein-coupled receptor

This study determined whether stimulation of a G protein-coupled receptor could initiate the events that occur at fertilization in pig oocytes and, if so, whether the activated oocytes were competent to form blastocysts. After maturation for 30 h, oocytes received microinjections of mRNA encoding the rat M1 muscarinic receptor, a G protein-coupled acetylcholine (ACh) receptor. Oocytes were then incubated for an additional 15 h to complete maturation of oocytes and translation of microinjected mRNA, and they were subsequently cultured in the presence of ACh. ACh treatment of these oocytes triggered pronuclear formation (50.4%) as well as cortical granule exocytosis. SDS-PAGE showed that mRNA-microinjected oocytes treated with ACh were activated (61.1%), as characterized by the appearance of the 22-kDa polypeptide derived from dephosphorylation of the 25-kDa precursor. Furthermore, after being cultured in a ligated pig oviduct for 6 days, 17.4% of treated oocytes developed to the compact morula or blastocyst stage. Transmission electron microscopy revealed that blastocysts recovered from ligated oviducts contained reticulated nucleoli with fibrillar cores surrounded by fibrillar and granular components. In addition, mitochondria in the blastocysts were dispersed throughout the cytoplasm and contained numerous transverse cristae. These results show that pig oocyte activation mediated by a G protein-coupled signal transduction system can signal a series of intracellular changes that lead to activation events associated with fertilization. Furthermore, oocytes activated through this pathway showed preimplantation development consistent with normal development.     

Polyvinyl alcohol as a defined substitute for serum in vitrification and warming solutions to cryopreserve ovine embryos at different stages of development

The purpose of this study was to assess the viability of ovine embryos after replacing fetal calf serum (FCS) with polyvinyl alcohol (PVA) in vitrification and warming solutions. Ovine embryos were obtained from superovulated Sardinian breed ewes at 4, 5, 6, and 7 days after insemination. All vitrification and warming solutions were prepared using buffered saline solution with 20% FCS (group a) or 0.1% PVA (group b). Embryos were vitrified in 20 microliters of glycerol 3.4 M + ethylene glycol 4.6 M and loaded into the centre of 0.25 ml straws between two columns of sucrose solution (0.5 M), and plunged immediately into liquid nitrogen. After being warmed in a water bath at 35 degrees C for 10 s, the vitrified embryos were moved to 0.25 M sucrose solution for 3 min. Embryos were cultured in TCM-199 after washing with 10% FCS and sheep oviductal epithelial cells up to hatching or re-expansion of the blastocoelic cavity. No significant difference in the viability rates was observed between embryos vitrified/warmed in PVA or FCS solutions. In both groups, the rate of in vitro viability was (P < 0.01) lower at the precompacted and compacted morula stages than at the expanded, hatching or hatched blastocyst stage. In both groups, early blastocysts were less viable than expanded (P < 0.01), hatching or hatched blastocyst (P < 0.05). There was no significant difference in survival rates at days 14 (79 and 76%) and 45 (63 and 59%) after transfer into sychronised recipients between vitrified expanded blastocysts of groups a and b, respectively. These results suggest that it is possible replace serum with PVA in vitrification and warming solutions without reducing in vivo and in vitro viability.     

Three year results of in vitro production of bovine embryos in serum-poor bovine oviduct conditioned medium. An overview

This paper presents a synthesis of 3 year results of in vitro production of bovine embryos in medium previously conditioned by bovine oviduct epithelial cells. In Louvain-la-Neuve, Belgium, a total of 18356 oocytes were matured and inseminated in vitro: 13967 (76%) had cleaved at 3 days post-insemination and 3593 (26%) became blastocysts using this culture system. Our data show that conditioned medium can be stored frozen for up to 3 years without significant loss of activity and is resistant to lyophilization. One single batch of conditioned medium was tested within the same period in four different laboratories and yielded variable results: 27 and 37% blastocysts/cleaved embryos in two of them and only 7 and 0% in the two others whereas in each case more than 30% blastocysts were obtained with the local reference co-culture system. In one laboratory, the batch of oil used to overlay the culture drops had a detrimental effect on the blastocyst rate in conditioned medium but not in co-culture.     

Influence of light intensity on methanotrophic bacterial activity in Petit Saut Reservoir, French Guiana

We have determined that the tolerance of in vitro matured/in vitro fertilized (IVM/IVF) bovine embryos to cryopreservation at the pre-morula stage can be improved by removal of cytoplasmic lipid droplets by centrifugation. Nucleus transfer was also performed using cryopreserved, delipated (lipid droplets removed) 8- to 16-cell-stage blastomeres of IVM/IVF embryos as donor nuclei. In vitro developmental ability of the delipated embryos to the blastocyst stage (20 of 126) was found to be equal to that of undelipated embryos (35 of 176); and of 53 delipated embryos cryopreserved at the 8- to 16-cell stage, 12 developed into blastocysts in vitro after thawing. On the other hand, only 2 of 43 undelipated embryos and 5 of 59 sham-operated embryos survived (p < 0.05). When blastomeres isolated from cryopreserved, delipated 8- to 16-cell-stage embryos were used for nucleus transfer, 57 of 80 successfully fused with enucleated oocytes, which was significantly lower than the fusion rate obtained with blastomeres of unfrozen, undelipated embryos (93 of 104, p < 0.01). However, the developmental rate to the blastocyst stage for nucleus transfer embryos reconstituted with frozen, delipated blastomeres (9 of 57) was not different from that of the nucleus transfer embryos with unfrozen, undelipated embryos (23 of 93). These results confirm that removal of cytoplasmic lipid droplets from bovine IVM/IVF zygotes allows for successful cryopreservation at the 8- to 16-cell stage and that blastomeres from these embryos can be used as donors of karyoplasts for nucleus transfer.     

Current and future methodology for monitoring sleep

The present study examined the effect of follicular shell pieces (FSP) during in vitro maturation (IVM) of porcine oocytes on 1) in vitro fertilization (IVF) parameters, 2) subsequent embryo development, 3) oocyte glutathione (GSH) concentration, and 4) viability after embryo transfer. Cumulus-oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, and hormonal supplements and with or without FSP for 20-22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20-22 h. After culture, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 5-6 h. Putative zygotes were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. In comparisons between the presence and absence of FSP, no differences were observed in fertilization parameters. At 48 h, no mean differences were found in cleavage rates. However, at 144 h, the proportion of embryos that developed to the blastocyst stage was significantly (p < 0.01) higher (18% vs. 36%) for oocytes cocultured with FSP. A significantly (p < 0.05) higher GSH concentration was found in oocytes matured with FSP as determined by dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Transfer of embryos to 9 recipients resulted in 5 pregnancies with the birth of 18 live piglets. The results provide clear evidence of the beneficial effect of FSP during IVM of pig oocytes cultured in the presence of cysteine on subsequent embryo development to the blastocyst stage. The birth of piglets confirms the viability of IVM-IVF-derived embryos.     

Complete activation of porcine oocytes induced by the sulfhydryl reagent, thimerosal

Thimerosal (200 microM) triggered Ca2+ oscillations in 56 of 56 mature porcine oocytes. The Ca2+ oscillations were blocked by the sulfhydryl-reducing agent dithiothreitol (DTT), thus supporting the hypothesis that thimerosal acts by oxidizing critical sulfhydryl groups on intracellular Ca2+-release proteins. Thimerosal treatment alone arrested the oocytes in metaphase, probably by oxidizing tubulin sulfhydryl groups and thus destroying the spindle. However, a 10-min exposure to 200 microM thimerosal followed by a 30-min incubation in 8 mM DTT induced complete activation, as 73.8% of the oocytes formed pronuclei. The second polar body was visible in 73.3% (55 of 75) of the activated oocytes. Combined thimerosal/DTT treatment of the oocytes also induced cortical granule exocytosis, as revealed by confocal microscopy, and the subsequent hardening of the zona pellucida. After activation, some oocytes were incubated in vitro, or in vivo in a ligated porcine oviduct, for 6 days. When cultured in vitro, 42.0% (37 of 88) of the oocytes developed to the compact morula or blastocyst stage; the average number of inner cell mass (ICM) and trophectoderm (TE) nuclei in the blastocysts was 8.6 +/- 0.7 and 20.1 +/- 1.3, respectively. Culture in a ligated oviduct resulted in 42.9% development to the compact morula or blastocyst stage, with the blastocysts having a mean number of 12.5 +/- 1.0 ICM and 63.6 +/- 9.2 TE nuclei.     

Antigens recognized by T-lymphocytes on human tumours

Some of the factors influencing the success of a nuclear transfer procedure are the quality of the recipient oocyte and the efficiency of the method of artificial activation. In this study we evaluated the ability of an electrical pulse to stimulate in vitro-matured porcine oocytes to develop. Maturation in Waymouth's medium resulted in significantly greater development than maturation in TCM-199 (p < 0.05), while there was no significant difference between degrees of development in Waymouth's medium and Whitten's medium. Oocytes matured in Waymouth's medium and electrically stimulated at 36 h (young oocytes) developed to the same degree as oocytes stimulated at 48 h (aged oocytes). Oocytes matured in Waymouth's medium and treated with cytochalasin B showed significantly greater development (p < 0.10) in response to electrical activation than controls. Staurosporine activation of oocytes resulted in significantly (p < 0.05) fewer morulae and blastocysts when compared to electrical stimulation. Development of parthenogenic embryos to the elongated filamentous stage (10% development beyond blastocyst) was obtained by maturing oocytes in Waymouth's medium and electrically stimulating them to develop. By obtaining development of porcine parthenotes beyond the blastocyst stage, we have identified an efficient method of oocyte maturation and oocyte activation for use in a system for nuclear transfer.     

Effect of free amino acids and vitamins on cleavage and developmental rate of bovine zygotes in vitro

Due to the complicated media used for culturing bovine embryos, most of the nutrient requirements are unknown. Recently, we developed a simple, serum-free medium (CR1) that allows bovine embryos to develop in vitro. Therefore, our objective was to determine whether development of bovine embryos would be improved by the addition of free amino acids and vitamins to CR1. Oocytes were recovered from slaughterhouse ovaries and matured 22 +/- 2 h, following which the oocytes were randomly allotted to treatment. The experiment was a randomized block design with a 2 x 5 factorial treatment structure. The oocytes were fertilized with or without cumulus cells intact. The five fertilization media were 1) Control (CR1 +/- 10 micrograms/mL of phenol red); 2) control + basal medium Eagle (BME) essential amino acids (EAA) + minimum essential medium (MEM) nonessential amino acids (NEA) + MEM vitamins (VIT); 3) control + EAA + NEA; 4) control + EAA + VIT; and 5) control + NEA + VIT. Cleavage rate was greater (P < .001) when cumulus cells remained on the oocytes during fertilization (51.7 vs 73.2% without and with cumulus cells, respectively). The frequency of blastocysts was increased (P < .001) when EAA or NEA were added to CR1; however, adding VIT had no effect or tended (P = .12) to decrease the frequency of embryos attaining the blastocyst stage. This experiment demonstrates that development of bovine embryos in vitro can be improved by the addition of free amino acids to a simple medium. Contrary to work in rodents, the mixture of vitamins in MEM was not beneficial for bovine embryos.