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.     

Role of cumulus cells and serum on the in vitro maturation, fertilization, and subsequent development of rat oocytes

Immature oocytes were collected from immature female rats (60-65 g) 40 h after injection with 6 IU pregnant mare's serum gonadotropin (PMSG). Oocytes were matured cumulus-intact (CI) or cumulus-free (CF) in medium supplemented with 0.5% bovine serum albumin (BSA) or 5-20% serum for periods of up to 24 h. After assessment for nuclear maturation, the oocytes were exposed to epididymal sperm for fertilization in vitro. In vitro-matured and ovulated oocytes undergoing fertilization were transferred to unilaterally pregnant recipients for embryonic and fetal development. The presence of cumulus cells and serum shortened (by 2 h) the time required for polar body emission by in vitro-matured oocytes and also helped to increase significantly the penetrability of the oocytes by spermatozoa. A high proportion (45.6%) of fertilized oocytes showed evidence of abnormal fertilization following maturation in the absence of cumulus cells. Oocytes matured CI before fertilization were able to develop to viable fetuses (57.8%) in proportions similar to ovulated oocytes (55.0%) after in vitro fertilization. These findings indicate an essential role for cumulus cells in promoting normal cytoplasmic maturation of oocytes necessary for pronuclear formation and subsequent developmental capability.     

Effects of media and the presence of bovine oviduct epithelial cells during in vitro fertilization on fertilizability and developmental capacity of bovine oocytes

The effect of the presence of bovine oviduct epithelial cells (BOEC; Experiment 1) as well as the effects of media (Tyrode fertilization medium: TFM vs synthetic oviduct fluid: SOF), fertilization containers (drops in petri dish vs 96-wells), and the number of oocytes per drop and well (5 vs 10) for in vitro fertilization (Experiment 2) on the fertilizability and in vitro development of bovine oocytes were investigated. Immature oocytes with cumulus cells were cultured in TCM199 supplemented with 10% ECS and 2.5x10(6) granulosa cells for 24 hours at 39 degrees C under 5% CO(2) in air. In vitro fertilization was performed with frozen-thawed, heparin-treated spermatozoa (100 mug/ml, 15 minutes) and with BOEC (Experiment 1). In Experiment 2, in vitro fertilization was performed with two different media (TFM and SOF) and various conditions (culture dish and different number of oocytes). Cleavage, development to the blastocyst stage were evaluated on Day 2 and Day 7 after the start of culture. Effect of the presence of BOEC on fertilizability and developmental capacity (Experiment 1) was not significantly different. In Experiment 2, alterations in media, containers and number of oocytes during in vitro fertilization had no affect. The SOF medium showed results similar to those of TFM (normal fertilization rate: 63.2 vs 64%; cleavage: 69.3 vs 73.9%; development to the blastocyst stage: 14 vs 15%; and mean number of nuclei per blastocyst: 80.5 vs 86.6). The results indicate that the presence of BOEC during in vitro fertilization did not improve fertilizability, and that SOF as well as TFM medium can be utilized as a simple fertilization medium.     

The effects of estrous cow serum on the in vitro maturation and fertilization of the bovine follicular oocyte

The effect of serum obtained from a cow at the time of standing estrus (serum A), at ovulation (serum B), and at 24 h after ovulation (serum C) on the in vitro maturation and fertilization of bovine oocytes was examined. Of 144 (Group A), 159 (Group B), and 158 (Group C) oocytes, 77 (53.4%), 82 (51.6%) and 82 (51.9%) oocytes were characterized by expansion of cumulus cells, respectively. There was no significant difference in the effect of the three types of cow serum on the cumulus expansion (P < 0.05). Of 461 oocytes, 316 oocytes were cultured with sperm for fertilization, and 145 oocytes were cultured without sperm for evidence of parthenogenetic development. Of 56 (Group A), 56 (Group B), and 62 (Group C) oocytes with expanded cumulus cells, 19 (33.9%), 7 (12.5%), and 11 (17.7%) oocytes were cleaved, respectively, after exposed to the sperm for 24 h. There was a significant difference in the effect of the three types of cow serum on the fertilization rate (P < 0.05). A total of 145 oocytes was cultured in the absence of sperm and no evidence of parthenogenetic division was observed. The effect of the three types of serum obtained from the cow on the maturation of oocytes was not significant, but a significant difference did exist in the fertilization rate of oocytes. Cow serum obtained at the time of standing estrus had a beneficial effect on the fertilization rate of oocytes in vitro.     

In vitro fertilization and development of llama (Lama glama ) oocytes using epididymal spermatozoa and oviductal cell co-culture

A study was designed to determine the feasibility of developing in vitro maturation, fertilization and culture systems utilizing follicular oocytes and epididymal spermatozoa collected from llamas at slaughter. From a total of 1324 cumulus oocyte complexes (COCs) recovered, 972 were cultured in 50-ul drops of TCM-199 medium with 10% heat inactivated steer serum (DBS) and hormones for 30 h. After maturation, the oocytes were randomly allocated into 4 groups in a 2x2 factorial design: cumulus-enclosed oocytes, 2 ug/ml heparin (Group 1); cumulus-enclosed oocytes, 5 ug/ml heparin (Group 2); denuded oocytes, 2 ug/ml heparin (Group 3); and denuded oocytes, 5 ug/ml heparin (Group 4). Denuded oocytes were obtained for groups 3 and 4 by vortexing. Epididymides were also collected at slaugther and fresh spermatozoa (for each replicate) were obtained by mincing the cauda epididymis with a scalpel blade. A total of 721 oocytes were inseminated with 2-3 x 10(6) epididymal spermatozoa/ml in a 50-ul drop of FERT-TALP medium. After 18 h of in vitro insemination, 234 oocytes were placed in a llama oviductal epithelial cell (LLOEC) co-culture in TCM-199 for 9 d. All cultures were done at 38.5 degrees C under 5% CO(2) in air with high humidity. The rate of fertilization, initial cleavage and development in co-culture were evaluated and compared. Of 192 oocytes examined for signs of fertilization, 56 (29.2%) were penetrated by spermatozoa with 57.1% (32 56 ) of the penetrated oocytes having a male and female pronucleus. There were no differences among treatment groups in total fertilization. However, the frequency of oocytes fertilized normally tended to be higher in the denuded oocytes 67.7% (21 31 ) than the oocytes inseminated with cumulus cells 44.0% (11 25 ) independent of heparin concentration (P<0.06). The total embryo development rate to the 2 cells to blastocyst stage was 32.1% (75 234 ). There was no difference in development rate between groups. From the 234 oocytes co-cultured in LLOEC for 9 d, 15.8% developed into 2 to 16 cells, 5.6% into morulae, 6.0% into early/expanded blastocysts and 4.7% into hatching/hatched blastocysts. The results indicate that an in vitro fertilization system is possible in the llama utilizing slaughterhouse material and that llama oocytes can be fertilized in the presence of heparin and epididymal spermatozoa.     

An essential role for functional telomeres in mouse germ cells during fertilization and early development

Late generations of telomerase-null (TR(-/-)) mice exhibit progressive defects in highly proliferative tissues and organs and decreased fertility, ultimately leading to sterility. To determine effects of telomerase deficiency on germ cells, we investigated the cleavage and preimplantation development of embryos derived from both in vivo and in vitro fertilization of TR(-/-) or wild-type (TR(+/+)) sperm with either TR(-/-) or TR(+/+) oocytes. Consistently, fertilization of TR(-/-) oocytes with either TR(+/+) or TR(-/-) sperm, and TR(-/-) sperm with TR(+/+) oocytes, resulted in aberrant cleavage and development, in contrast to the normal cleavage and development of TR(+/+) oocytes fertilized by TR(+/+) sperm. Many (>50%) of the fertilized TR(-/-) eggs developed only one pronucleus, coincident with increased incidence of cytofragmentation, in contrast to the normal formation of two pronuclei and equal cleavage of wild-type embryos. These results suggest that both TR(-/-) sperm and oocytes contribute to defective fertilization and cleavage. We further found that a subset (7-9%) of telomeres was undetectable at the ends of some metaphase I chromosomes from TR(-/-) spermatocytes and oocytes, indicating that meiotic germ cells lacking telomerase ultimately resulted in telomere shortening and loss. Dysfunction of meiotic telomeres may contribute to aberrant fertilization of gametes and lead to abnormal cleavage of embryos, implying an important role of functional telomeres for germ cells undergoing fertilization and early cleavage development.     

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium,duration of maturation,and sperm calcium ionophore treatment,and comparison with rates of fertilization in vivo after oviductal transfer

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.     

Improved development of ovine matured oocyte following solid surface vitrification (SSV): effect of cumulus cells and cytoskeleton stabilizer

The objective of the present study was to examine the effects of cumulus cells, cytochalasin B (CB), and taxol on the development of ovine matured oocyte following solid surface vitrification (SSV). In experiment 1, effects of cumulus cells during the vitrification were examined. Survival rates after warming were not different between ovine mature oocytes with cumulus cells and without cumulus cells. After in vitro fertilization, rates of embryonic cleavage and development to blastocyst were not different between these two groups. In experiment 2, the effects of cytochalasin B (CB) on vitrification of ovine matured oocytes were examined. The rates of survived ovine matured oocytes were not significantly different among the treatment with 0, 2.5, 5.0, 7.5 and 10.0 microg/mL CB. After in vitro fertilization, the rate of cleavage was not different between the five treatment groups. However, vitrified oocytes treated with 7.5 or 10.0 microg/mL CB resulted in a higher (8.1+/-4.6% and 7.8+/-2.4% respectively, P<0.05) blastocyst development rate than those of oocytes treated with lower CB concentrations. In Experiment 3, the effects of taxol on vitrification of ovine matured oocytes were examined. The rate of survived oocytes was not significantly different among the taxol treatment group with 0, 0.5, 1.0, and 5.0 microM taxol. After in vitro fertilization, the rates of embryos that reached cleavage were not different between the four treatment groups. However, vitrified oocytes treated with 0.5 microM taxol resulted in a higher blastocyst (10.1%+/-6.3, P<0.05) development rate compared to other treatment groups. In conclusion, no effect of cumulus cells on vitrification of ovine matured oocytes was detected in this study. Pretreatment of ovine matured oocytes with cytoskeletal inhibitor cytochalasin B or taxol have a positive effect and helps to reduce the damage induced by vitrification and is a potential way to improve the development of vitrified/warmed ovine matured oocytes.     

In vitro maturation and fertilization of prepubertal goat oocytes

The aim of this work was to study the IVM-IVF of prepubertal goat oocytes collected from a slaughterhouse as an alternative source of oocytes to those of FSH-primed adult goats. In Experiment 1, IVM of prepubertal goat oocytes in co-culture with granulosa cells were compared with IVM in 50 microl microdrops of medium. There was no significant difference in the percentage of maturation (72.0 vs 76.9%) between the 2 groups. In Experiment 2, a low percentage of normal fertilization (24.4%) was observed for prepubertal goat oocytes matured with granulosa cells from prepubertal goats. This result was significantly lower than that obtained for ovulated (62.2%) or in vitro-matured (48.7%) oocytes from adult goats. There were no significant differences with respect to the oocytes from adult goats matured in vitro when prepubertal goat oocytes were cultured with adult goat granulosa cells (33.3%) or in microdrops (29.7%). No differences were observed among the treatments in the percentage of oocytes showing evidence of fertilization (normal fertilization + abnormal fertilization + polyspermy). In Experiment 3, it was shown that there were no differences in the percentage of normally fertilized oocytes after in vitro maturation in microdrops containing oocytes with 1 to 2 and 3 or more complete layers of cumulus cells (32.1 and 33.3% respectively). In conclusion, the ovaries of prepubertal slaughterhouse goats were found to be an economical alternative for an abundant source of oocytes for IVM-IVF research. In vitro maturation of oocytes in microdrops yielded maturation and fertilization rates comparable to those obtained with oocytes from FSH-primed adult goats. Moreover, similar maturation and fertilization rates were obtained using oocytes with 1 to 2 layers or 3 or more layers of cumulus cells.     

In vitro fertilization of bovine follicular oocytes obtained by laparoscopy

Bovine follicular oocytes (n = 454), obtained after laparoscopy, were used to study in vitro capacitation, fertilization, and embryo development. Capacitation was accomplished by treating bovine spermatozoa with high ionic strength medium. Maturation, fertilization, and development studies were carried out in Brackett's defined medium or in Ham's F-10. In vitro fertilization rates, ranging from 14% to 55%, were found to be influenced by individual variations among males. Brackett's defined medium was found to be superior to Ham's F-10 for oocyte maturation, fertilization, and growth, these media giving cleavage rates of 60% and 32%, respectively. Oocytes with expanded cumuli at the time of recovery cleaved at a rate of 43%, which is significantly different from oocytes recovered without granulosa cells (22%) or oocytes with compact cumuli and corona cells (5%). The in vitro development pattern of the in vitro-fertilized embryos was found to be similar to that observed in vivo. Embryos were observed at the 2-cell stage 44.5 +/- 6.3 h after in vitro insemination, 4-cell after 59.0 +/- 9.4 h, 8-cell after 74.8 +/- 12.7 h, and 16-cell after 96.2 +/- 13.9 h (observations at 12-h intervals). The procedures described here resulted in cleavage rates of up to 60% using follicular oocytes embedded in expanded cumuli cells and semen samples from selected males.     

Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection.

The objective of this study was to assess fertilization and embryonic development following intracytoplasmic sperm injection (ICSI) of oocytes from porcine preantral follicles matured in vitro. Also, another aim was to describe actin filament distribution during fertilization and embryonic development of those oocytes after ICSI as one of the factors assessed. Preantral follicles isolated from prepubertal porcine ovaries were cultured in a system that supports follicular development. After in vitro maturation, the oocytes were fertilized by ICSI or conventional fertilization in vitro (IVF). Actin filaments of the fertilized oocytes and embryos produced by ICSI or IVF were stained by rhodamine-phalloidin and visualized by fluorescence microscopy. ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro. Of those, 51% of the fertilized oocytes cleaved and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45% and 16%, respectively). Actin filament distribution during fertilization and embryonic development of ICSI- or IVF-fertilized oocytes from porcine preantral follicles was similar to that of oocytes derived from antral follicles and fertilized by standard IVF. These results indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development.     

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.     

Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes

Sera (fetal calf serum: FCS; and oestrous cow serum: ECS), hormones (2.5 FSH micrograms/ml + 5 micrograms LH/ml + 1 microgram oestradiol/ml) and granulosa cells (5 x 10(6)/ml) were added to culture medium to determine the frequencies of in-vitro maturation, fertilization, cleavage (2- to 8-cell) and development into blastocysts of bovine follicular oocytes. The maturation rates after 24 h in culture were not significantly different among the three factors tested (56-72%). The fertilization rates were significantly affected by serum type and the addition of granulosa cells. FCS gave significantly higher rates of fertilization (57-71%) than did ECS (34-52%), but the proportions of polyspermic fertilization were significantly higher in the former (8-19%) than in the latter (2-3%). The addition of hormones did not affect fertilization, cleavage and development. Neither type of serum affected cleavage and development. The highest rates of blastocyst formation were obtained when granulosa cells alone were added (FCS, 17%; ECS, 16%). The cell numbers of the blastocysts obtained were 100-150, similar to those of blastocysts developed in vivo. Transfer of 6 blastocysts to 3 cows resulted in 1 pregnancy. The present results indicate that the co-culture with granulosa cells is the most important factor for in-vitro fertilization to development into blastocysts of bovine oocytes matured in vitro.     

Role of cumulus cells during maturation of porcine oocytes in the rise in intracellular Ca2+ induced by inositol 1,4,5-trisphosphate

At the time of fertilization, release of inositol 1,4,5-trisphosphate (IP3) into the cytoplasm of oocytes is said to be induced by hydrolysis of phosphatidylinositol bis phosphate (PI2) via activation of phospholipase C and is responsible for the Ca2+ oscillation in oocytes immediately after sperm penetration. On the other hand, cumulus cells have been reported to play an important role in cytoplasmic maturation of mammalian oocytes and to affect embryonic development after fertilization. To obtain more information on the role of cumulus cells in cytoplasmic maturation of oocytes, the effects of cumulus cells on the rise in [Ca2+]i and the rates of activation and development of porcine mature oocytes induced by IP3 injection were investigated. Mature porcine oocytes that had been denuded of their cumulus cells in the early stage of the maturation period had a depressed rise in [Ca2+]i (4.0-6.0) and reduced rates of activation (31.4-36.8%) and development (10.0-24.4%) induced by IP3 injection compared with those of their cumulus-enclosed counterparts (7.3, 69.1% and 43.8%; P < 0.05). The [Ca2+]i rise and the rates of activation and development depressed by the removal of cumulus cells were restored by adding pyruvate to the maturation medium. Furthermore, the IP3 injection-induced depression of [Ca2+]i rise in mature oocytes derived from cumulus-denuded oocytes (DOs) was restored when they were cultured in a medium with pyruvate (3.9-6.3, P < 0.05). Also, mature oocytes from cumulus-oocyte complexes (COCs) cultured in a medium without glucose had a lower rise in [Ca2+]i than that in mature oocytes from COCs cultured with glucose (7.4-6.0, P < 0.05). Cumulus cells supported porcine oocytes during maturation in the rise in [Ca2+]i induced by IP3 and the following activation and development of porcine oocytes after injection of IP3. Moreover, we inferred that a function of cumulus cells is to produce pyruvate by metabolizing glucose and to provide oocytes with pyruvate during maturation, thereby promoting oocyte sensitivity to IP3.     

In vivo and in vitro embryo production in goats

Assisted reproductive technologies (ART) such as artificial insemination (AI) and multiple ovulation and embryo transfer (MOET) have been used to increase reproductive efficiency and accelerate genetic gain. The principal limitations of MOET are due to variable female response to hormonal treatment, fertilization failures and premature regression of Corpora luteum. The in vitro production (IVP) of embryos offers the possibility of overcoming MOET limitations. The method of IVP of embryos involves three main steps: in vitro maturation of oocytes (IVM), in vitro fertilization of oocytes (IVF) with capacitated sperm and in vitro culture (IVC) of embryos up to blastocyst stage. Recovering oocytes from live selected females by laparoscopic ovum pick-up (LOPU) and breeding prepubertal females by juvenile in vitro embryo technology (JIVET) will allow a greater production of valuable goats. Also, IVP of goat embryos will provide an excellent source of embryos for basic research on development biology and for commercial applications of transgenic and cloning technologies. Different protocols of IVP of embryos have been used in goats. However oocyte quality is the main factor for embryos reaching blastocyst stage from IVM/IVF/IVC oocytes. One of the principal determinant factors in the results of blastocyst development is the age of the oocyte donor females. In goats, oocytes from prepubertal and adult females do not show differences in in vitro maturation and in vitro fertilization; however the percentage of oocytes reaching blastocyst stage ranges from 12 to 36% with oocytes from prepubertal and adult goats, respectively.     

Efficiency and kinetics of the in vitro fertilization process in bovine oocytes with different meiotic competence

The aim of the study was to investigate the efficiency and kinetics of fertilization in oocytes with different meiotic competence, as defined by the phase of the follicular wave and follicle size. Oocytes were recovered from cows with synchronized estrus cycles, slaughtered in either the growth (day 3) or the dominant (day 7) phase, separately from large, medium and small follicles. The oocytes were matured and fertilized by a standard protocol. Twenty-four hours after fertilization, the oocytes were denuded from cumulus cells, fixed and stained with bisbensimid Hoechst-PBS. Fertilization was more efficient and the first cleavage was accelerated in growth phase-derived oocytes, as shown by significantly higher (p < or = 0.01) proportions of both normally fertilized and cleaved oocytes (68.8 and 25.1%), in comparison with dominant phase-derived oocytes (44.2 and 10.3%). In the growth-phase derived oocytes, proportions of normally fertilized and cleaved oocytes were significantly higher (p < or = 0.01) in oocytes from large (100.0 and 36.4%) and medium (83.3 and 36.5%) follicles than in those from small (54.8 and 14.6%) follicles. The dominant phase-derived oocytes showed higher proportions of normally fertilized and cleaved oocytes in the populations recovered from small (51.5 and 10.0%) and medium (43.1 and 12.0%) follicles than in those from large (25.0 and 0%) follicles; however, the differences were not significant. It can be concluded that: (i) efficiency and kinetics of fertilization differ in relation to oocyte's meiotic competence; (ii) improved development of embryos from oocytes with greater meiotic competence is associated with a more effective fertilization process.     

High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer

Successful cryopreservation of mammalian oocytes would provide a steady source of materials for nuclear transfer and in vitro embryo production. Our goal was to develop an effective vitrification protocol to cryopreserve bovine oocytes for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. Bovine oocytes matured in vitro were placed in 4% ethylene glycol (EG) in TCM 199 plus 20% fetal bovine serum (FBS) at 39 degrees C for 12-15 min, and then transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose in TCM 199 and 20% FBS). Oocytes were vitrified in microdrops on a precooled (-150 degrees C) metal surface (solid-surface vitrification). The vitrified microdrops were stored in liquid nitrogen and were either immediately thawed or were thawed after storage for 2-3 wk. Surviving oocytes were subjected to 1) parthenogenetic activation, 2) in vitro fertilization, or 3) nuclear transfer with cultured adult fibroblast cells. Treated oocytes were cultured in KSOM containing BSA or FBS for 9 to 10 days. Embryo development rates were recorded daily and morphologically high-quality blastocysts were cryopreserved for nuclear transfer-derived embryos at Day 7 or Day 8 of culture. Immediate survival of vitrified/thawed oocytes varied between 77% and 86%. Cleavage and blastocyst development rates of vitrified oocytes following in vitro fertilization or activation were lower than those of the controls. For nuclear transfer, however, vitrified oocytes supported embryonic development as equally well as fresh oocytes.     

Effects of oviductal and cumulus cells on in vitro fertilization and embryo development of porcine oocytes fertilized with epididymal spermatozoa

This study was designed to evaluate the effects of adding porcine oviductal epithelial cell (POEC) monolayers before or during the fertilization of denuded or cumulus-enclosed oocytes, in terms of fertilization results and subsequent embryo development. The variables determined were: penetration rate, mean number of spermatozoa per oocyte, male pronucleus formation rate, monospermy rate, cleavage rate after 48 h of fertilization, blastocyst rate, and mean number of nuclei per blastocyst. We used cumulus-free and cumulus-enclosed oocytes preincubated or fertilized in the presence of POEC, once the purity in epithelial cells of these cultures had been assessed. All the experiments involved the use of frozen-thawed epididymal spermatozoa to avoid replicate variability. The POEC cultures prepared showed a high proportion of epithelial cells (over 95%). Preincubation of oocytes with POEC before fertilization showed no effects on the fertilization variables determined. In contrast, during IVF under our experimental conditions, these cells attached to the cumulus cells and their interaction had a significant effect on some of the fertilization variables analyzed. The presence of POEC and cumulus cells during IVF increased oocyte penetrability. Moreover, in the absence of POEC, cumulus cells resulted in a reduced monospermy rate. On subsequent embryo culture, a lower cleavage and blastocyst formation rate were recorded when the oocytes had been preincubated with POEC before IVF.     

Influence of sperm:oocyte ratio during in vitro fertilization of in vitro matured cumulus-intact pig oocytes on fertilization parameters and embryo development

The present study was conducted to evaluate the influence of sperm:oocyte ratio during in vitro fertilization (IVF) of in vitro matured cumulus-intact oocytes on fertilization parameters and embryo development in pigs. In vitro matured oocytes surrounded by intact cumulus cells (COC) were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000:1, 3000:1, 4000:1, 6000:1, and 8000:1). Denuded oocytes inseminated with 2000 frozen-thawed spermatozoa:oocyte were the control group. A total of 2546 oocytes in five replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture (EC) medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The penetration rate increased in the COC groups with the sperm:oocyte ratio, reaching the highest rates with 8000:1 spermatozoa:oocyte (72.1 +/- 6.5%), similar to the control (73.5 +/- 3.5%). However, the monospermy was highest with the lower spermatozoa:oocyte rates (82.6-94.8%) and decreased drastically (P<0.05) in the COC group fertilized with 8000 sperm:oocyte (36%). The efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) showed no difference among the COC groups (20-30%) but they were significantly lower (P<0.007) than those obtained by the control group (43.7 +/- 2%). Embryo development was highest in the control group (58% for cleavage and 23% for BF) but not significantly different with the 6000 and 8000 sperm:oocyte COC groups (47 and 50% for cleavage and 19 and 17% for BF, respectively). These results indicate that the use of COC for IVF involves a drop in the efficiency of the fertilization and the necessity to increase the frozen-thawed sperm:oocyte ratio three to four times more to obtain similar embryo development to denuded oocytes.     

Improvement on in vitro maturation, fertilization and development of minke whale (Balaenoptera acutorostrata) oocytes

The aims of the present study were to improve in vitro maturation, fertilization and subsequent development of minke whale oocytes. We investigated the effects of different concentrations (0, 10 and 20%) of fetal whale serum (FWS) in maturation medium on nuclear maturation, morphological grade (A or B) of cumulus-oocyte complexes (COC) obtained from prepubertal and adult minke whales. Grade A (> or = 5 layers of cumulus cells) COC collected from the adult whales and cultured in the medium with 20% FWS had a higher (P < 0.05) maturation rate (31.8%) than those in the medium without FWS (0%). Adding FWS to the maturation medium significantly (P < 0.01) improved the proportion of oocytes at Metaphase II (M-II): without FWS (7.9%), with 10% (19.4%) and 20% (21.4%) FWS. However, sexual maturity of whales and COC grades were not significantly affected by M-II oocytes. When in vitro fertilization of matured oocytes was performed in the presence of 20% FWS or 0.6% BSA in the fertilization medium, the proportions of sperm penetration and two-pronuclei formation in matured oocytes were not significantly different. Grade A COC cultured in a culture medium supplemented with 10% FWS cleaved at a higher rate (15.4%, P < 0.05) than did Grade A and B COCs cultured in the medium without FWS (0%). Neither Grade A nor B COCs cleaved when the medium was without FWS. The proportions of cleaved oocytes increased (P < 0.05) with FWS supplementation (6.9% and 8.1% for 1.0% FWS and 20% FWS, respectively). Grade A COC was significantly (P < 0.05) superior in its ability to cleave (14.5%) and develop to morula (4.2%) compared with that of the oocytes from Grade B COC (2.5% and 0%). Coculture with granulosa cells during in vitro culture did not significantly affect cleavage and development to the morula stage. These results indicate that FWS addition in the maturation medium improved the rate of in vitro maturation and cleavage after insemination of minke whale oocytes. The BSA supplementation in fertilization medium was as effective as FWS supplementation for in vitro fertilization of matured oocytes. In vitro embryo production beyond the morula stage of minke whale oocytes could be possible, if Grade A COC was selected and cultured in the maturation medium supplemented with 10% or 20% FWS.