Parthenogenetic development of activated in vitro matured bovine oocytes

Bovine oocytes matured in vitro for 26 hours were electrically stimulated 1) by a single pulse (Treatment A); 2) by 3 pulses 30 minutes apart (Treatment B); 3) by a single pulse followed by 5 minutes of incubation in the stimulation medium (Treatment C); or 4) by a single pulse at 27 hours of maturation (Treatment D). The oocytes were then cultured for up to 8 days to assess parthenogenetic activation and development. Each electrical stimulation consisted of a 60-mus square wave pulse of 2.5 or 3.6 kV/cm. Treatment A was less effective than the other treatments (P<0.05), activating 47 or 59% of oocytes at 2.5 or 3.6 kV/cm, respectively. However, there were no differences due to voltage nor among the other treatments, which activated 64 to 78% of the oocytes. The cleavage rate, 28 to 38%, was not affected by the activation treatment, but development to the 8-cell stage or beyond was greater after activation with the higher voltage. While the numbers of morulae or blastocysts resulting from any given treatment were too small to support meaningful statistical comparison, the results indicate that bovine parthenogenotes produced in vitro are capable of development to the blastocyst stage.     

Synergistic effect of ethanol and cycloheximide on activation of freshly matured bovine oocytes

Bovine follicular oocytes were collected from ovarian antral follicles (2 to 7 mm in diameter) from slaughtered cattle. They were matured in vitro (IVM) for 23 to 24 h and then activated. In Experiment 1, 4 concentrations of ethanol were compared. The activation rates of oocytes were 4, 12, 36 and 27%, respectively, following exposure for 7 min to 0, 5, 7 and 10% ethanol. In Experiment 2, 7% ethanol was tested with exposure times of 0, 5, 7.5 and 10 min, and 6, 32, 27 and 33% of the oocytes were activated, respectively. In Experiment 3 the synergistic effect of ethanol and electric pulse was compared within 4 treatments: A) 7% ethanol alone, B) electric pulse alone, C) ethanol first and then electric pulse treatment, and D) electric pulse first followed by ethanol exposure. Of the oocytes activated, 37, 31, 28 and 51%, respectively, were from Treatments A through D. In Experiments 4 and 5 the possible synergistic effect of ethanol and a protein synthesis inhibitor, cycloheximide, was studied within 4 treatments: A) parthenogenetic control with no activation treatment, B) ethanol alone, C) cycloheximide alone, and D) ethanol treatment followed by cycloheximide. The oocyte activation rates in Experiment 4 in Treatments A through D, respectively, were 9, 44, 43 and 84%. Corresponding values for development of oocytes to the 2 to 8-cell stage after culture for 3 d (Experiment 5) were 9, 20, 14 and 45%, respectively (P<0.05). In conclusion, exposure to 7% ethanol for 5 min followed by incubation with cycloheximide was the best activation treatment for bovine IVM oocytes.     

Optimized combined electrical-chemical parthenogenetic activation for in vitro matured bovine oocytes

Sperm-mediated oocyte activation is a complex procedure, both in steps and duration, not yet been completely mimicked during in vitro studies, e.g., parthenogenesis or somatic cell nuclear transfer. Furthermore, parthenogenetic studies have been recognized as a suitable model for studying activation efficiency for nuclear transfer cloning. This study, therefore, was conducted to develop an optimized artificial activation method, based on bovine cloning. In vitro matured bovine oocytes were initially exposed to electrical pulse, used for cell fusion during cloning, and then treated with 15 temporal sequential combinations of 3 chemical activators [calcium ionophore (CI), strontium (SR) and ethanol (ET)], followed by exposure to a protein kinase inhibitor or used for in vitro fertilization as control group. Treated and naturally fertilized oocytes were further cultured for up to 8 days. Embryo development was scored daily and blastocyst cell counting was carried out using differential staining at day 8 of culture. Among 15 temporal sequential combinations of three chemical activators, the best cleavage rates were associated with double (SR-CI, 84.4%), triple (CI-SR-ET, 79.4%) and single (CI, 73.7%) compounds, respectively, which were not significantly different with each other and with in vitro fertilized (IVF) (85.5%). The highest blastocyst rates were gained with ET-SR (24.5%), SR-CI-ET (20.4%) and CI (24.5%) accordingly which were not significantly different with each other but significantly lower than IVF (47%). Embryo cell counting further confirmed reasonably better quality of blastocysts produced using double, triple and single compounds. Although most of the sequential artificial activation compounds induced high cleavage rate, close to IVF, but this did not assure comparable further embryo development to the blastocyst stage. Nevertheless, the results suggest exposure of in vitro matured bovine oocytes to electrical pulse, followed by exposure to CI-6-dimethylaminopurine (6-DMAP) or ET-SR-6-DMAP could be regarded as the optimal artificial activation protocol for in vitro development of parthenogenic bovine oocytes or as a step for activation protocol in cloning procedure.     

Parthenogenetic development of bovine oocytes treated with ethanol and cytochalasin B after in vitro maturation.

The present study was conducted to investigate the effects of different culture durations (24-36 hr) on bovine oocyte maturation in vitro and the effect of the presence or absence of cumulus cells at the time of treatment to induce parthenogenetic activation (exposure to ethanol and cytochalasin B; CB) (experiment I). The effects of dosage (2.5 or 5.0 micrograms/ml) and incubation time (2.5, 5, or 10 hr) in CB (experiment II) on the subsequent development to the blastocyst stage in vitro was also investigated. In experiment I, cleavage and development to the blastocyst stage were not affected by the presence or absence of cumulus cells at the time of parthenogenetic activation. However, the 24-hr culture duration for in vitro maturation had a significantly lower rate of development to the blastocyst stage than the longer culture durations (27-36 hr). In experiment II, treatment with 5 micrograms/ml CB for 5 hr showed the highest percentage of development to blastocyst in the oocytes matured for both 27 and 30 hr. To determine the viability of the parthenogenetic embryos (morulae and blastocysts), four recipient heifers received two embryos each, and one heifer was found to be pregnant on day 35 following transfer. Although fetal heartbeat was not observed, the subsequent estrus was prolonged in all heifers. The present results demonstrate development of in vitro-matured, parthenogenetically activated bovine embryos up to the preimplantation stage.     

Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP

The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1 h, followed by parthenogenetic activation. Vitrified-warmed oocytes had a morphologically normal rate similar to that of controls (nonvitrified oocytes cultured in vitro for 24 h; 98.6% vs. 100%, P > 0.05). When vitrified-warmed oocytes were first activated with 7% ethanol for 5 min and then incubated in 6-dimethylaminopurin (6-DMAP) for 4 h, cleavage and blastocyst rates were 41.2% and 23.2%, respectively, which were lower than those of controls (77.5% and 42.0%, P < 0.05). Subsequently, we varied the ethanol concentration to increase the effectiveness of parthenogenetic activation. When either 5%, 6%, 7%, 8%, 9%, 10%, or 11% ethanol alone (for 5 min) or in combination with 6-DMAP (4 h) was used to activate vitrified-warmed oocytes, cleavage rates ranged from 22.3% to 61.1% and blastocyst rates ranged from 1.1% to 30.6%. These rates were optimized when oocytes were treated with 9% ethanol plus 6-DMAP; this was verified in experiments evaluating other activation protocols with 9% ethanol, calcium ionophore A23187, or ionomycin alone, or in combination with DMAP or cycloheximide (CHX). In conclusion, the oocyte activation protocol affected developmental capacity of vitrified bovine oocytes; 9% ethanol (5 min) followed by 6-DMAP (4 h) promoted optimal parthenogenetic activation.     

Fine structure of equine oocytes matured in vitro for 15 hours

Transmission electron microscopy (TEM) was used to evaluate the fine structure of equine oocytes cultured in vitro. Oocytes obtained by follicular aspiration were cultured for either zero or 15 hr. After treatment oocytes were processed either by light microsocopy (nuclear evaluation) or TEM (cytoplasmic evaluation). Those oocytes cultured for 15 hr were incubated in modified TCM 199 with 15% (v/v) mare serum (day of ovulation) at 39 ± 0.2°C. Evaluation using TEM revealed that cortical granules were present in all oocytes. However, zero-time oocytes contained few cortical granules, and these were scattered throughout the cytoplasm, whereas 15 hr oocytes contained numerous cortical granules primarily found in very close proximity to the oolemma. Further ultrastructural analysis of both groups revealed organelle structure similar to that previously described for in vivo matured equine oocytes. Evaluation of nucelar maturity (lacmoid stain) showed that 15 hr of culture resulted in significant numbers of oocytes at metaphase II (8/17; 47%). These data demonstrate that oocytes cultured for 15 hr in modified TCM 199 with 15% mare serum (day of ovulation) are mature with respect to nuclear configuration and cortical granule migration and, therefore, would be appropriate candidates for in vitro fertilization. © 1994 Wiley-Liss, Inc.     

Activation and early parthenogenesis of bovine oocytes treated with ethanol and strontium

Efficient artificial activation is indispensable for the success of cloning programs. Strontium has been shown to effectively activate mouse oocytes for nuclear transfer procedures, however, there is limited information on its use for bovine oocytes. The present study had as objectives: (1). to assess the ability of strontium to induce activation and parthenogenetic development in bovine oocytes of different maturational ages in comparison with ethanol; and (2). to verify whether the combination of both treatments improves activation and parthenogenetic development rates. Bovine oocytes were in vitro matured for 24, 26, 28, and 30 h, and treated with ethanol (E, 7% for 5 min) or strontium chloride (S, 10mM SrCl(2) for 5h) alone or in combination: ethanol+strontium (ES) and strontium+ethanol (SE). Activated oocytes were cultured in vitro in synthetic oviductal fluid (SOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage (D7). Treatment with ethanol and strontium promoted similar results regarding pronuclear formation (E, 20-66.7%; S, 26.7-53.3%; P>0.05) and cleavage (E, 12.8-40.6%; S, 16.1-41.9%; P>0.05), regardless of oocyte age. The actions of both strontium and ethanol were influenced by oocyte age: ethanol induced greater activation rates after 28 and 30 h of maturation (48.4 and 66.7% versus 20.0 and 23.3% for 24 and 26 h, respectively; P<0.05) and strontium after 30 h (53.3%) was superior to 24 and 26 h (26.7% for both). Blastocyst development rates were minimal in all treatments (0.0-6.3%; P>0.05), however, when the mean (+/-S.D.) cell number in blastocysts at the same maturational period was compared, strontium treatment was superior to ethanol for activation rates (82+/-5.7 and 89.5+/-7.8 versus 54 and 61, at 28 and 30 h, respectively). Improved results were obtained by combined treatments. The combination of ethanol and strontium resulted in similar pronuclear formation (ES, 36.7-83.9%; SE, 53.1-90.3%) and cleavage rates (ES, 31.3-81.3%; SE, 65.6-80.7%). Regarding embryo development, there was no difference (P>0.05) between treatments, and blastocysts were only obtained in treatment SE at 24 and 26 h (6.5% for both). It is concluded that, SrCl(2) induces activation and parthenogenetic development in bovine oocytes.     

Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro

The role of follicle cells in the acrosome reaction of frozen-thawed bovine spermatozoa, in vitro fertilization, cleavage, and development in vitro was investigated. Cumulus-oocyte complexes were cocultured and matured in vitro with additional granulosa cells for 24 hr. Immediately before in vitro insemination, the oocytes were divided into three types with different follicle cells: denuded and corona- and cumulus-enclosed oocytes. The proportion of live, acrosome-reacted spermatozoa significantly increased at 3 and 6 hr after insemination in all types of oocytes. However, the mean proportion of live, acrosome-reacted spermatozoa that inseminated cumulus-enclosed oocytes at 6 hr after insemination was significantly higher than that of spermatozoa inseminating denuded oocytes (18.3% and 13.3%, respectively). The frequency of in vitro fertilization was significantly higher for cumulus-enclosed oocytes (65.4%) than for denuded and corona-enclosed oocytes (30.8% and 39.4%, respectively). Cumulus-enclosed oocytes when cocultured with oviduct epithelial cells also had significantly higher rates of cleavage (two- to eight-cell, 59.8%; eight-cell, 22.4%) and blastocyst formation (7.7%) than denuded and corona-enclosed oocytes. No eight-cell embryos or more advanced stages of embryonic development were observed in either denuded or corona-enclosed oocytes without the coculture. The present results indicate that cumulus cells at fertilization play an important role in inducing the acrosome reaction and promoting a high fertilization rate, cleavage, and development into blastocysts in vitro.     

Penetration of intact bovine ova with ram sperm in vitro

In culture, mature bovine ovarian oocytes were fertilized in vitro with freshly ejaculated ram spermatozoa treated with heparin. The zona pellucida does not prevent penetration of ram spermatozoa. The penetration rate varied between 10 and 84%, and in most instances, after 24 hr of culture, two normal-looking pronuclei and sperm tail were present in the cytoplasm. These results suggest that the zona pellucida of bovine oocytes does not represent a barrier for the penetration of ram spermatozoa.     

Laparoscopic oviductal transfer of in vitro matured and in vitro fertilized bovine oocytes

The objective of this study was to obtain normal pregnancy following laparoscopic oviductal transfer of in vitro matured and fertilized bovine oocytes. Methods for in vitro maturation and in vitro fertilization were similar to those previously reported (1). Primary oocytes judged to be potentially viable were cultured for 26 h in modified TCM 199 supplemented with heat-treated fetal calf serum (20% v/v), 5mug/ml FSH (USDA-bFSH-B-1), and 1mug/ml estradiol 17-beta. Oocyte cumulus complexes were microscopically evaluated for maturation (first polar body formation) following a brief treatment with hyaluronidase. Mature oocytes were inseminated with heparin-treated spermatozoa and incubated at 39 degrees C under paraffin oil and moist 5% CO(2), 5% O(2), 90% N(2). In this work, 450 oocytes were recovered at slaughter from ovaries of 42 random cows of unknown reproductive status and 336 oocytes (74.7%) with compact cumulus were selected for culture. Of these, 322 (95.4%) matured in vitro. Of 218 inseminated oocytes, 198 (90.8%) were penetrated by sperm and 83 (38.1%) cleaved, with 102 (46.6%) of the embryos reaching four- to eight-cell stages. None of 40 oocytes not exposed to sperm and none of 30 oocytes inseminated with untreated sperm showed signs of activation. In a control experiment with hormones added, 105 of 115 (91.3%) oocytes matured in vitro and 20 of 105 (19.5%) cleaved following in vitro insemination. Laparoscopy was performed on four synchronized recipients under local anesthesia. A catheter containing three embryos in the two to four cell stages was passed through the operating channel of a direct viewing bronchoscope for deposition in the oviduct ipsilateral to the recipients developing corpus luteum while the fimbria and the mesovarium were manipulated with Semm's forceps. A normal term pregnancy confirmed in vitro fertilization and provides feasibility data for use of laparoscopic methodology developed in this work for testing viability of bovine oocytes and embryos. These results are encouraging for the application of in vitro maturation and in vitro fertilization for overcoming infertility in domestic and endangered species.     

In vitro fertilization and cleavage of in vivo matured bovine oocytes

The effect of the interval between onset of estrus and oocyte collection on in vitro fertilization (IVF) rates has been investigated. The oocytes were surgically collected 6-18 h (Group I), 19-24 h (Group II), 25-29 h (Group III) and 30-36 h (Group IV) after the beginning of estrus. Recognizable stages of nuclear maturation were identified in 54.9% of the oocytes used for IVF (5.9% at germinal vesicle, 31.4% at metaphase I, 17.6% at metaphase II); the other 45.1% were degenerate. Considerable between- and within-cow variation in oocyte morphology, oocyte maturation and IVF results was observed. The cverall fertilization and cleavage rates (to four-cell stages) were 26.5 and 6.0%, respectively. The fertilization rate increased as the interval between onset of estrus and collection increased and was optimal 30-36 h after onset. Thus, onset of estrus proved an effective means of timing oocyte collection for IVF.     

Development of in vitro matured bovine oocytes after cryopreservation with different cryoprotectants

In vitro matured bovine oocytes at the metaphase-II stage were slowly frozen in phosphate buffered saline (PBS) containing 1.0 M glycerol, 1.0 M dimethylsulfoxide (DMSO) or 1.0 M propylene glycol (PROH). When thawed rapidly, more (P<0.05) oocytes were morphologically normal after being frozen with DMSO (86%) or PROH (83%) than with glycerol (62%). When inseminated in vitro with frozen-thawed bull spermatozoa, higher (P<0.05) penetration rates were observed in DMSO (79%) or PROH (76%) than in glycerol (48%). The percentages of oocytes developing to the 2-cell stage at 48 h postinsemination were also significantly (P<0.05) higher in DMSO (51%) and PROH (54%) than in glycerol (33%). However, a significant increase in the proportions of 8-cell embryos (46 vs 21 to 26%; P<0.05) at 72 h postinsemination and morulae (14 vs. 6 to 8%; P<0.05) was derived from oocytes frozen with PROH than with DMSO or glycerol. In conclusion, the type of cryoprotectant used is one of the critical factors affecting developmental competence of bovine oocytes frozen at the metaphase-II stage. For this stage of oocytes, PROH was the most effective, yielding a large number of 8-cell embryos and morulae than either glycerol or DMSO in a slow freezing method combined with a 3-step thawing protocol.     

Developmental competence of in vivo and in vitro matured porcine oocytes after subzonal sperm injection

In vivo and in vitro matured porcine oocytes were fertilized by subzonal sperm injection (SUZI), and their subsequent development in vitro was examined to determine whether ooplasmic incompetence is the major cause of limited developmental ability of in vitro matured/fertilized porcine oocytes (Experiment 1). There was no significant difference in rates of fertilization (61% vs. 70%), monospermy (37% vs. 45%), and male pronuclear formation (77% vs. 61%) between in vivo and in vitro matured oocytes. Blastocyst formation rate was significantly lower for in vitro matured oocytes (11% vs. 42%; P < 0.001). Forty-six percent of in vivo matured oocytes cleaved to the 2-4 cell stage by 24 hr in culture after SUZI, compared with 3% of in vitro matured oocytes (P < 0.01). In experiment 2, in vitro development of in vitro matured oocytes with evenly and unevenly granulated cytoplasm were compared after SUZI to examine whether developmentally competent in vitro matured oocytes can be identified on the basis of morphological appearance. Most of the blastocysts obtained developed from oocytes with unevenly granulated cytoplasm (7/56 vs. 1/45; P > 0.05). Experiment 3 revealed that the proportion of oocytes with evenly granulated cytoplasm was originally low (11%) in the population of oocytes used for in vitro maturation, and it increased approximately 3-fold (36%; P < 0.001) after maturation. These results suggest that ooplasmic incompetence in porcine in vitro matured oocytes is the major cause of their limited developmental competence. Cytoplasmic maturation measured by male pronucleus formation does not directly reflect developmental competence of the oocytes. It was also shown that evenness of granulation of the cytoplasm is not a useful morphological indicator of developmental competence. © 1996 Wiley-Liss, Inc.     

Method for producing transgenic bovine embryos from in vitro matured and fertilized oocytes

Microinjection and in vitro culture procedures were developed to produce transgenic bovine embryos after in vitro fertilization of in vitro matured oocytes. In Experiment I, zygotes were subjected to pronuclear microinjection of DNA 18 or 24 h following addition of spermatozoa to oocytes. Microinjections were performed in either Hepes-buffered TCM-199 or modified Dulbecco's phosphate-buffered saline without glucose. Viability of embryos was similar at both injection times and for both media, as determined by morphological evaluation after culturing embryos in vitro for 10 d. In Experiment II, microinjected embryos were cultured 1) in rabbit oviducts, 2) in vitro in a 5% CO(2) in air, or 3) in a 5% CO(2) / 5% O(2) / 90% N(2) incubator. There were no significant differences between the 2 in vitro culture environments. The in vitro culture systems supported development of embryos significantly better than the rabbit oviducts; 33% of cleaved ova developed to blastocysts in vitro vs 10% in vivo; 98% of transferred ova were recovered from the rabbit oviducts. From both experiments, 6 of 92 blastocysts were positive for the microinjected DNA as determined by a polymerase chain reaction followed by gel electrophoresis.     

Relationship between equilibration times and the presence of cumulus cells, and effect of taxol treatment for vitrification of in vitro matured porcine oocytes

The effects of the presence or absence of cumulus cells and equilibration times (1 and 4 min) with cryoprotectant, and Taxol treatment before vitrification (0.5-5.0 microM) on development of in vitro matured porcine oocytes after vitrification were examined. Ethylene glycol (30%) and sucrose (0.5M) was used as a vitrification solution (39 degrees C), and cryotop was used for cryo-container. There was a significant relationship (F value: 6.077, P<0.05) in the rate of morphologically normal oocytes after vitrification between the equilibration times and the presence or absence of cumulus cells. The blastocyst rates were not significantly different between Taxol (1.4-5.5%) and non-treated control (8.8%). The results show that the optimal exposure time to achieve survival after vitrification depends on the presence or absence of cumulus cells, and that Taxol has no positive effect on the developmental capacity of vitrified, in vitro matured porcine oocytes.     

In vitro fertilization and development of bovine oocytes matured in serum-free medium

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.     

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO₂ in air and heparin.

In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.     

Development of in vitro matured and fertilized bovine oocytes co-cultured with Buffalo Rat Liver cells

This study was designed to evaluate the efficacy of Buffalo Rat Liver cells (BRLC) monolayers in supporting the development of in vitro matured and fertilized (IVM/IVF) bovine oocytes through to the hatched blastocyst stage compared to the commonly used co-culture system of bovine oviduct epithelial cells (BOEC). Cumulus oocyte complexes (COCs) obtained from 2- to 6-mm ovarian follicles at slaughter were matured for 24 h in TCM-199 supplemented with FBS and hormones (FSH, LH and estradiol 17-beta). In vitro fertilization (IVF) was performed using 1 x 10(6) percoll separated frozen-thawed spermatozoa in 1 ml of IVF-TL medium containing 18 to 20 matured oocytes. After 20 to 22 h of sperm exposure, 584 presumptive zygotes in 2 separate trials were randomly assigned to 3 treatment groups (BRLC co-culture, BOEC co-culture and control, consisting of medium alone). Zygotes were cultured in CZB media, a simple semi-defined medium, without glucose for the first 2 d, transferred to M199/FBS (TCM-199-HEPES supplemented with 20% HTFBS, 1 mM Sodium pyruvate), and cultured for an additional 8 days. Cleavage and development to morula and various blastocyst stages were recorded between d 3 and 11 after the start of IVF. Overall average cleavage rate was 75% (440 584 ) and did not vary across the treatments or trials. The proportion of embryos that reached the morula stage in both co-culture systems did not differ (P > 0.05) and was significantly higher (P > 0.05) compared to the control group. However, the percentage of the number of blastocysts, expanded blastocysts and hatched blastocysts varied across the treatment groups (P < 0.05), with the highest results obtained in the BRLC co-culture system. The production of blastocysts in BOEC co-culture was inconsistent between the 2 trials where a significant difference (40.6 vs 53.0%; P > 0.05) was observed. Rate of development to the blastocyst stage was similar between the 2 co-culture systems, with most of the embryos reaching the blastocyst stage by d 8 post insemination. The results of this study show that BRLC from a commercially available established cell line offer a more reliable alternative to a BOEC co-culture system for in vitro maturation, fertilization and culture of bovine embryos.     

Full-term development of bovine follicular oocytes matured in culture and fertilized in vitro

Follicular oocytes were cultured for 28h in vitro and 91% of the oocytes reached the the second metaphase in culture. The penetration rate after insemination in vitro using frozen-thawed spermatozoa was 81%. After cultivation for 48h in vitro, 18% of the in vitro fertilized oocytes developed to the three- to four-cell stages and 21% of these developed to the six- to eight-cell stages. Following in vivo culture in the rabbit oviduct, 18% of six- to eight-cell and 5% of three- to four-cell embryos developed to the blastocyst stage. To confirm the full developmental competence, 11 blastocysts were transferred to recipient cows, and six (55%) cows became pregnant or delivered calves.