Collection of oocytes through transvaginal ultrasound-guided aspiration of follicles in an Indian breed of cattle

The present study was undertaken in Karan Fries, an Indian breed of cattle to (1) determine the number of follicles available for puncture and (2) explore the potential of this breed as a donor of developmentally competent oocytes. Ovum pick-up (OPU) was performed using an ultrasound machine with a transvaginal convex transducer (5 MHz) with a needle guide, single lumen 19-gauge 60 cm long needle and a vacuum pressure of 90 mmHg. The number and size of follicles in each ovary was determined before puncture. The follicles were characterized on the basis of their diameter as small (3-5 mm), medium (6-9 mm) and large (>/=10 mm). The oocytes recovered were classified by quality. They were matured in vitro, irrespective of their grade, in 50 microl droplets of the in vitro maturation (IVM) medium (TCM-199+10% fetal bovine serum(FBS)+5 microg/ml follicle stimulating hormone (folltropin)+1 microg/ml estradiol-17beta+0.2 mM sodium pyruvate), covered with paraffin oil, in 35 mm petridish for 24 h in a CO(2) incubator (5% CO(2) in air) at 38.5 degrees C. The cleavage rate was recorded at day 2 post-insemination after subjecting the oocytes to in vitro fertilization (IVF). The differences in follicular populations of all size categories among individual donors were not significant. A total of 92 oocytes were recovered by aspiration of 157 follicles, with an overall recovery rate of 59% (range 35-79%). Of these, 32% were of grades A and B and the rest of grades C and D. The mean numbers of total follicles and the oocytes recovered per session did not differ significantly among individual donors. Out of the 73 oocytes subjected to IVM and IVF, 24 reached 2-4 cell stage at day 2 post-fertilization, with a cleavage rate of 33%. The total number of oocytes recovered was correlated with the number of small (R=0.54, P<0.01) but not with the number of medium and large follicles. This study demonstrates the use of OPU as a means of obtaining developmentally competent oocytes from an Indian breed of cattle for obtaining cattle oocytes in India where cow slaughter is not allowed for religious reasons.     

Parthenogenetic activation of cattle follicular oocytes in vitro with ethanol

Cattle follicular oocytes cultured in vitro for 24–33 h were treated with ethanol to induce artificial activation. When oocytes were cultured for 27–33 h before ethanol treatment, 60–68% of oocytes were activated and were found to have a female pronucleus(ei). In contrast, maturation culture of oocytes for 24–26 h resulted in low activation rates (25–38%). The female pronucleus was formed in the activated oocytes within 8–10 h of incubation after ethanol treatment. And it became visible under interference-contrast microscope by centrifugation for 3 min at 15,000g and 10 min at 20,000g. These results indicate that ethanol treatment is effective for activation of cattle follicular oocytes and that the pronucleus formed in the activated oocyte can be visualized by centrifugation.     

In vitro maturation and fertilization of follicular oocytes in cattle

This work aims towards developing research concerning the improvement of animal reproduction, embryo development and genetic engineering. In our laboratory, an attempt has been made to standardize in vitro conditions able to optimally support bovine oocyte maturation and fertilization in order to yield viable embryos. Ovaries from cows and heifers, obtained from local slaughter-house, were used for recovery of oocytes from antral follicles. Cumulus-oocyte complexes were statically cultured for 24h at 39 degrees C in medium TCM 199 supplemented with fetal calf serum inactivated, hormones, glucose and granulosa cells under a 5% CO2 and 95% humidity atmosphere. A first group of oocytes was used for fixing and staining procedure for evidence of in vitro maturation. After culture 69.4% (77/111) of oocytes reached full maturation showing cumulus expansion, first polar body extrusion and the 2nd metaphase plate. A 2nd group was used for in vitro fertilization. In vitro semen capacitation was obtained with swim-up system (8.9) with separation of high motility fraction in Talp Hepes medium. Oocytes and spermatozoa were coincubated for 18-20h in Talp medium at 39 degrees C with 5% CO2 and 95% humidity. At the end of culture stereoscope and microscope observations were made for evidence of fertilization. After IVF 67.4% (58/86) resulted fertilized. Most of them showed two pronuclei and residual sperm tail. In few cases oocytes with 1 pronucleus and the swollen sperm head or with syngamy or polyspermic were found. In these experiments high percentages of in vitro matured and in vitro fertilized oocytes have been obtained. These bovine zygotes can be considered an essential step to develop new technologies in cattle breeding.     

Gonadotropin stimulation regimens for follicular aspiration and in vitro embryo production from calf oocytes

Crossbred beef x dairy calves were randomly allocated at 3 wk of age to different gonadotropin treatment regimens for stimulation of follicle development and induction of oocyte maturation in vivo. Follicular responses were assessed laparoscopically, and oocytes were aspirated for assessment of maturational state or for in vitro fertilization (IVF) and culture to determine developmental capacity. Follicle-stimulating Hormone (FSH), administered in a single subcutaneous injection together with a low dosage of PMSG, was as effective as the same total dosage of FSH administered in 6 injections over a 3-d period. Without accompanying PMSG, this dose of FSH was ineffective in stimulating follicle development. The mean number of preovulatory follicles (> 5mm, with hyperemic appearance) doubled with each successive stimulation at 3-wk intervals, reaching 35 follicles per calf at 9 wk of age. Oocyte yields ranged from 55 to 81% of follicles aspirated, and did not differ significantly among age, FSH regimen and oocyte maturation stimulus. A combination of LH + FSH was more effective in stimulating cumulus cell expansion than LH by itself (73 vs 22% of recovered oocyte-cumulus cell complex (OCC) respectively; P<0.05). Of 33 unselected immature oocytes (cumulus unexpanded) subjected to in vitro maturation (IVM) and IVF, 30% developed to blastocysts during co-culture with bovine oviduct epithelial cells, which was not significantly different from 25% of 36 oocytes from adult ovaries which reached the blastocyst stage under similar conditions. The results indicate that follicle responses of calf ovaries to FSH stimulation increase progressively from 3 to 9 wk of age, and that oocytes recovered laparoscopically from these follicles produce blastocysts in culture at rates similar to oocytes from adult cattle ovaries collected at slaughter. The approach offers promise for embryo production from donor calves of superior genetic merit for embryo transfer, thereby enhancing the rate of genetic gain above that attainable by conventional breeding or by embryo transfer in adult cattle.     

Effect of different stages of the follicular wave on in vitro developmental competence of bovine oocytes

This study aimed to investigate the developmental competence of ovum pick-up collected oocytes on three stages of the follicular wave: Days 2, 5 and 8. A group of 11 cows was used in successive cycles to perform ovum pick-up on either Day 2, 5 or 8 of an induced follicular wave (three sessions per stage). Follicular waves were initiated by puncturing the dominant follicle and all other follicles sized > or = 5 mm at Days 5-7 of the cycle. The plasma progesterone concentrations did not differ between the days of ovum pick-up: 4.0 +/- 1.8, 5.1 +/- 1.6 and 5.2 +/- 1.7 ng/ml for Days 2, 5 and 8, respectively. The proportion of oocytes with three or more layers of non-expanded cumulus cells was higher for Day 5 than Day 8, while Days 2 and 5 did not significantly differ from each other (85, 96 and 68% of 113, 60 and 101 oocytes for Days 2, 5 and 8, respectively). The proportion of oocytes competent to develop a blastocyst in an in vitro production system was higher for Days 2 and 5 than for Day 8: 27, 29 and 15% for the oocytes with fair to good cumulus investment and 23, 27 and 11%, respectively, when all oocytes were taken in account. This indicates that the dominant follicle reduces the developmental competence of oocytes from subordinate follicles at a relatively late stage of dominance. This finding has practical consequences for the handling of cows that undergo ovum pick-up only once or very irregularly. The embryo yield can then be improved by performing the ovum pick-up at Days 2-5 of the cycle or 2-5 days after ablation of the large follicles.     

Alteration of follicular dynamics and superovulatory responses by gonadotropin releasing hormone and follicular puncture in cattle: a field trial

A field experiment was conducted to determine the influence of follicular alteration on superovulatory responses. Ultrasonography was performed once daily over 4 d prior to gonadotropin treatment (Day 0), on the day of estrus during superstimulation, and on the day of embryo collection to monitor follicular development. Animals were superstimulated between Days 8 and 12 of the estrous cycle. Follicular status was altered 2 d prior to initiation of superstimulation (Day 0) with GnRH (Cystorelin, 200 micrograms i.m.) administered with (GnRH-puncture group, n = 31) or without (GnRH-no puncture group, n = 52) concomitant removal of the largest follicle by follicular aspiration. Responses were compared with those of an untreated control group superovulated 8 to 12 d after estrus (n = 102). The proportion of animals with a high number (> or = 2) of large follicles (> = 7 mm) on Day 0 was lower (P < 0.001) in the 2 GnRH-treated groups than in the control group, while the increase in the number of medium size follicles (4 to 6 mm) on Day 0 was greater (P < 0.02) in the GnRH-puncture group. During superstimulation, the proportion of superovulatory cycles with a high follicular (> or = 10 follicles) response was similar in the control and GnRH-no puncture groups. Within the GnRH-treated animals, follicular and ovulatory responses were greater in the GnRH-puncture than in the GnRH-no puncture group (P < 0.001 to P < 0.02). Despite these changes in follicular and ovulatory responses, however, the mean number of embryos produced did not differ (P < 0.1) among treatments (4.3 +/- 0.4, 3.7 +/- 0.7, and 5.4 +/- 0.8 in control, GnRH-no puncture, and GnRH-puncture groups, respectively). This was due primarily to an increase in the mean numbers of unfertilized ova (P < 0.005) and in degenerated embryos (P < 0.06) in the GnRH-puncture group. Results indicate that the beneficial effects of treatment with GnRH and follicular puncture 2 d prior to superstimulation on follicular and ovulatory responses were limited by an increase in the number of unfertilized ova and degenerated embryos.     

Ovarian superstimulation after follicular wave synchronization with GnRH at two different stages of the estrous cycle in cattle

The objective of this study was to evaluate superovulatory programs based on synchronization of follicular waves with GnRH at 2 different stages of the estrous cycle. Sixteen Holstein cows were randomly assigned to 1 of 3 groups and administered GnRH (Cystorelin, 4 ml i.m.) between Days 4 and 7 (Groups 1 and 3) or between Days 15 and 18 (Group 2) of the estrous cycle (estrus = Day 0). Four days after GnRH treatment, > or = 7-mm follicles were punctured in Groups 1 (n = 6) and 2 (n = 6) or were left intact in Group 3 (n = 4). All cows were superstimulated 2 d later (i.e., from Days 6 to 10 after GnRH treatment) with a total of 400 mg NIH-FSH (Folltropin-V) given twice daily in decreasing doses. The GnRH treatment caused a rapid disappearance of large follicles (P < 0.005), rapid decrease in estradiol concentrations (P < 0.003), and increase in the number of recruitable follicles (4 to 6 mm; P < 0.04), indicative of the emergence of a new follicular wave within 3 to 4 d of treatment. Between 4 and 6 d after GnRH treatment, the mean number of 4- to 6-mm follicles decreased (4.7 +/- 1.8 to 1.5 +/- 3.3) in the nonpunctured group but increased (3.9 +/- 1.0 to 7.3 +/- 1.9) in the punctured group of cows (P < 0.05). In response to FSH treatment, the increase in the number of > or = 7-mm follicles was delayed by approximately 2 d in the nonpunctured group (P < 0.006). Moreover, the mean number of > or = 7-mm follicles at estrus was higher (16.9 +/- 1.7 vs 11.5 +/- 3.0; P < 0.1) in the punctured than the nonpunctured group. The increase in progesterone concentration after estrus was delayed in the nonpunctured group (P < 0.1) compared with the punctured follicles. Mean numbers of CL as well as freezable (Grade 1 and 2) and transferable (Grade 1, 2 and 3) embryos were similar (P > 0.1) in punctured and nonpunctured groups. Spontaneous estrus did not occur prior to cloprostenol-induced luteolysis in any group, and stage of the estrous cycle during which GnRH was given did not affect (P > 0.1) hormonal and follicular responses in the punctured groups. In conclusion, GnRH given at different stages of the estrous cycle promotes the emergence of a follicular wave at a predictable time. Puncture of the newly formed dominant follicle increases the number of recruitable follicles (4 to 6 mm) 2 d later and, in response to superstimulation with FSH, causes a greater number and faster entry of recruitable follicles into larger classes (> or = 7 mm) and a faster postovulatory increase in progesterone concentrations.     

Induction of maturation in follicle-enclosed oocytes: the response to gonadotropins at different stages of follicular development

Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.     

Endoscopy in cattle by the paralumbar route: Technique for ovarian examination and follicular aspiration

The ovary and follicular aspiration, or both, were observed in 50 heifers by endoscopy via the right paralumbar fossa. A total of 129 laparoscopies were performed. Eight animals underwent more than four interventions. Occasionally adhesions were observed, but they never interfered with ovary examination and follicular aspiration. The rate of ovum recovery was higher when a suction device was used rather than a syringe system (P < 0.01). The bore size of the needles influenced the proportion of oocytes obtained from the follicular aspirates. The use of a 19-G (1.00 mm) needle with a 45-degree bevel and a vacuum pressure of 250 mmHg gave the best results (recovery rate: 72%). This was significantly higher (P < 0.01) than the results obtained with a 21-G needle and a vacuum pressure of 500 mmHg (recovery rate: 53%).     

In vitro maturation of cattle oocytes taken from ovaries with different morphofunctional states

We studied the capacity of cattle oocytes taken from ovaries with different morphofunctional state for development to metaphase 2 in vitro. A classification of ovaries has been proposed according to their morphofunctional state: (1) ovaries with a yellow body from the last cycle, without dominating follicle, with many follicles of varying diameter; (2) ovaries with a yellow body from the last cycle, with dominating follicle (from 10 mm in diameter); (3) ovaries with a large functioning yellow body and follicles of varying diameter; (4) ovaries with a follicular cystoid formation (more than 25 mm in diameter); (5) ovaries with a yellow body from past cycles and small (1-2 mm) follicles, supposedly with a weakened hormonal function. It was shown that the morphofunctional state of ovaries determined the total number of oocytes isolated from an ovary and number of morphologically normal oocytes feasible for cultivation. At the same time, no reliable differences in the capacity for extrusion of the first polar body between the oocytes from the ovaries of different types were found in the experiments on in vitro oocytes maturation. Since the coefficient of correlation between the extrusion of the first polar body and maturation to metaphase 2 was in 0.95, there is every reason to believe that the capacity for development to metaphase 2 does not depend on the morphofunctional state of ovaries.     

Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation

We conducted this study to examine whether serum starvation in culture contributes to better development of bovine reconstructed oocytes and to evaluate which serum-starved somatic cell is the most effective for cloned calf production. In Experiment 1, donor cells of four different types (cumulus cells, ear fibroblasts, oviduct cells and uterine cells) were either serum-starved or not before fusion with enucleated oocytes, and reconstructed oocytes were further cultured for 168 h. Regardless of serum starvation, cumulus cells or ear fibroblasts yielded higher (P < 0.05) rates of fusion than other cells (62.6-69.3 versus 33.3-38.7%). In the serum-starved group, the first cleavage after reconstruction was significantly increased in cumulus cells and ear fibroblasts, compared with oviduct cells (93.4-94.3 versus 78.8-86.0%), and oocytes reconstructed with either of these yielded more blastocysts than oocytes reconstructed with oviduct or uterine cells (40.6-43.8 versus 20.3-19.0%). We observed a similar pattern in the non-starved group, but we found a significant increase in blastocyst formation was found only in cumulus cells compared with other donor cells (42.6 versus 15.4-27.7%). Overall comparison showed that serum starvation increased the rates of cleavage and development to the blastocyst stage, but we found a statistical significance only in the cleavage rate (80.0 versus 89.5%). In Experiment 2, we transferred randomly selected 59 blastocysts that were developed from oocytes reconstructed with serum-starved cells to 44 synchronised recipients. Of those recipients, 23 became pregnant on Day 60 after transfer (52.3%) and 12 (27.3%) delivered cloned calves. The mean gestation length and birth weight was 275 +/- 8 days and 39.6 +/- 15.6 kg, respectively. Although there was no significant difference among donor cells, blastocysts that were derived from oocytes reconstructed with ear fibroblasts yielded the highest rates of pregnancy (50.0%) and delivery (27.3%). In conclusion, serum starvation is effective for improving preimplantation development of oocytes reconstructed with cumulus or ear fibroblast cells and it may positively influence on obtaining better pregnancy outcome.     

Bovine and buffalo in vitro embryo production using oocytes derived from abattoir ovaries or collected by transvaginal follicle aspiration

This study was undertaken in order to evaluate the effect of oocyte source (live animals and abattoir ovaries) on subsequent embryo development in buffalo (Bubalus bubalis). Cow ovaries were also collected as oocyte donors for in vitro embryo production (IVEP).Three hundred thirty-eight oocytes were recovered by ovum pick up (OPU, Group A) from 8 pluriparous buffalo cows, while 1127 and 1457 oocytes were aspirated, respectively, from buffalo (Group B) and bovine (Group C) slaughterhouse ovaries. Cumulus enclosed oocytes (COCs) suitable for IVEP were in vitro matured (IVM), fertilized (IVF) and cultured (IVC) to the tight morula (Tm) and blastocyst (Bl) stage.Within buffalo species Group A had a higher Bl yield (29.7 % versus 19.9%; P<0.05) and a lower proportion of embryos arrested at Tm stage (11.1% versus 22.3%; P<0.05) than Group B.Within slaughterhouse groups cattle oocytes had a higher cleavage rate (83.9% versus 64.8%; P<0.05) and yielded 49.2% more blastocysts than buffalo. However, when data are related to the total number of cleaved oocytes, only 13.7% more blastocysts were produced in cattle than in buffalo.In conclusion, in buffalo species the source of oocytes significantly affected post-fertilization embryo development, as demonstrated by the higher Bl yields derived from OPU-derived oocytes.A higher overall IVEP efficiency, mainly related to the higher cleavage rate, was recorded in cattle compared with buffalo when ovaries from an abattoir were used as oocyte donors.     

Mycoflora of cervicovaginal fluids in dairy cows with or without reproductive disorders

Fungal infection of reproduction system of dairy cattle have not been received much more attention. The aim of this study was to determine the fungal infection of Holstein dairy cows with reproductive disorders or healthy. Fungal isolates of cervicovaginal fluids of 176 Holstein dairy cows were collected by using the double rod swabs for cervix and the sterilized cotton swabs for discharges of vagina. They were evaluated for fungal infections. The treatment group included 70 dairy cows with reproductive diseases, such as abortion, repeat breeder, endometritis, metritis, retention of fetal membrane, dystocia, cervicitis, and vaginitis. The control groups were included 42 healthy non-pregnant cows and 64 pregnant cows. Isolates of fungi were obtained from cervix and vagina of 27.1% and 28.6% of treatment group, 26.7% and 31.2% of pregnant cows, and 33.3% and 21.4% of healthy non-pregnant cows, respectively, indicating no significant differences. It is showed that the cervix and vagina of the treatment group have been infected by six different mycotic isolates. However, the cervix and vagina of pregnant and non-pregnant healthy cows in control group were infected with 5, 6 and 5, 4 different fungal agents, respectively. Penicillium and yeast were the most common isolated agents. Regarding to the result of this study, it is concluded that fungal infections can occur in cervicovaginal cavity of Holstein dairy cows with or without reproductive diseases.     

Optimization of biogas production from cattle manure by pre-treatment with ultrasound and co-digestion with crude glycerin

Biogas production by co-digestion of cattle manure with crude glycerin obtained from biodiesel production was studied after pre-treatment of the cattle manure or mixtures of cattle manure with different amounts of added glycerin with ultrasound. Batch experiments with 1750 mL of medium containing 1760 g of screened cattle manure or mixtures of cattle manure (screened or ground) and 70-140 mL or crude glycerin were incubated under mesophilic and thermophilic condition in stirred tank reactors. Under mesophilic conditions, the addition of 4% glycerin to screened manure increased biogas production by up to 400%. Application of sonication (20 kHz, 0.1 kW, and 4 min) to a mixture of manure + 4% glycerin increased production of biogas by up to 800% compared to untreated manure. The best results were obtained under thermophilic conditions using sonicated mixtures of ground cattle manure with 6% added glycerin (348 L methane/kg COD removed were obtained).     

Pre-treatment of cattle semen or oocytes with purified milk osteopontin affects in vitro fertilization and embryo development

This study was designed to investigate the effects of pre-incubating cattle spermatozoa or matured oocytes with purified osteopontin (OPN) from cattle milk on fertilization in cattle and embryonic development in vitro. There were two different experiments, semen from six mature Holstein bulls (Bos Taurus) was frozen with different concentrations of OPN (0, 1, 10, 100 μg/mL). Matured cattle oocytes were also pre-treated with OPN (0, 10, 100 μg/mL). In both experiments, pre-treated oocytes or frozen semen, was processed for in vitro fertilization and embryo development. Significantly more oocytes were fertilized when using frozen semen with 10 μg/mL OPN (bull 2 = 85 ± 4% and bull 5 = 78 ± 4%) than without OPN (bull 2 = 75 ± 4% and bull 5 = 69 ± 4%). Those bulls also had increase in cleavage and embryo development (bull 2 = 85 ± 3%, 41 ± 1.9%; bull 5 = 76 ± 2%, 37 ± 1.8%) compared with control (bull 2 = 75 ± 3%, 30 ± 2%; bull 5 = 68 ± 2%, 29 ± 2%). Incubating matured oocytes in 10 μg/mL OPN (87 ± 3%) and 100 μg/mL OPN (88 ± 3%) significantly increased fertilization than control (73 ± 3%). OPN also improve cleavage, and embryo development in treatments with 10 μg/mL OPN (82.7 ± 1.3%; 31.7 ± 1.4%) and 100 μg/mL OPN (85.8 ± 1.3%; 33.8 ± 1.5%) when compared with control (74.1 ± 1.3%; 24.2 ± 1.2%). These data suggest that both, spermatozoa from some bulls and oocytes may associate with OPN, suggesting a facilitory role on in vitro fertilization and embryo development.     

Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.     

In vitro development up to hatching of bovine in vitro-matured and fertilized oocytes with or without support from somatic cells

To verify the importance of somatic cells upon in vitro embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 supplemented with estrous cow serum (10% v/v) and 0.25 mM sodium pyruvate (ECSTCM) under the following treatments: 1) ECSTCM alone; 2) together with bovine oviduct epithelial cells (BOEC); 3) with cumulus cells (CC); 4) in fresh BOEC conditioned ECSTCM; or 5) in frozen-thawed BOEC conditioned ECSTCM. Culturing zygotes encased in cumulus cells significantly reduced the cleavage rate (P<0.05). There was no difference between culture systems in the proportions of embryo development through the 8-cell stage (P=0.42) up to the morula/blastocyst stages (P=0.50) at Day 7 post insemination. However, co-culture with BOEC yielded the highest percentage (21.2% of zygotes; P<0.05) of quality Grade-1 and Grade-2 embryos with the number of blastomeres per embryo (114.4) comparable to that of 7-day-old in vivo-developed embryos of similar grades (102.5), and higher (P<0.05) than those of the other treatments. The ratio of blastocysts to total morulae/blastocysts obtained from frozen-thawed conditioned medium was lower (P<0.05) than that from ECSTCM or after co-culture with BOEC at Day 7 post insemination. On average, 7.5 to 17.5% of the zygotes developed to blastocyst, expanded blastocyst and hatched blastocyst stages by Day 10 post insemination, depending upon the culture system. The difference between treatments, however, was not significant (P=0.68). The results indicate that chronological development up to hatching of bovine IVM-IVF embryos is not favored by somatic cells; however, the presence of viable oviduct epithelial cells in culture significantly improves the quality of 7-day-old embryos.