Y.H. Choi

In vitro maturation of equine oocytes collected by follicle aspiration and by the slicing of ovaries

The aim of this study was to examine 2 techniques for oocyte recovery from equine ovaries at slaughter: by aspiration of follicles and by additional slicing of ovaries. The morphology and nuclear configuration of oocytes recovered with either technique, and the time course of nuclear maturation during in vitro maturation were evaluated. Recovery rates were 1.75 and 4.14 oocytes per ovary for aspiration and slicing (total 145 and 344 oocytes from 83 ovaries), respectively. The oocytes were classified according to their cumulus/ooplasm morphology into 4 groups: compact/circular(A), compact/semicircular(B), expanded(C) and others(D). The percentages of oocytes in Groups A, B, C and D were 34, 38, 25 and 3% (aspiration) and 55, 26, 17 and 3% (slicing), respectively. The proportions of oocytes with a germinal vesicle in Groups A, B, C and D were 28 29 (97%), 23 35 (66%), 11 23 (48%) and 2 4 (50%) in oocytes from aspiration and 91 100 (91%), 52 65 (80%), 15 29 (52%) and 1 2 (50%) in oocytes from slicing, respectively. Group A and B oocytes recovered by aspiration (n=212) and slicing (n=312) were cultured in TCM199 supplemented with 10% fetal bovine serum, 1 mug/ml estradiol-17beta, and 0.02 AU/ml FSH at 38.5 degrees C in 5% CO(2) in air (5 to 10 oocytes per 50- mu l microdrop). At 8, 16, 24, 32 and 40 h of culture, the oocytes were fixed and stained. There were no significant differences in the percentages of Metaphase II stage (MII)-oocytes between recovery techniques at any time points examined. The proportions of MII-oocytes were 1 42 (2%), 4 43 (9%), 21 42 (50%), 28 45 (62%), and 28 40 (70%) at the respective time point in oocytes from aspiration and 0 51 (0%), 3 54 (6%), 22 59 (37%), 43 72 (60%), and 51 76 (67%) in oocytes from slicing, respectively. In most of the oocytes, resumption of meiosis occurred between 8 and 16 h of culture. The proportions of MII-oocytes increased significantly between 16 and 24 h and between 24 and 32 h of culture.

In vitro fertilization rate of horse oocytes with partially removed zonae

Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 micro M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under x 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (2/57), 12 (7/58), 52 (31/60), and 86% (44/51) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (2/2), 57 (4/7), 58 (18/31), and 34% (15/44), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 11/49), and increased to 38% (21/55) at 5 h, to 46% (23/50) at 10 h, and to 56% (27/48) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.

Effect of ovarian steroids and oxytocin on the production of prostaglandin E2, prostaglandin F2α and endothelin-1 from cow oviductal epithelial cell monolayers in vitro

Cyclic physio-anatomical variation in the oviducts is mediated by the local countercurrent transfer of ovarian products. Thus, in this study cow oviductal epithelial cells (COEC) culture were utilized to investigate the effects of ovarian products such as progesterone (P4), estradiol 17beta (E2) and oxytocin (OT) on local oviductal prostaglandin E2 (PGE2), F2alpha (PGF2alpha) and endothelin-1 (ET-1) production. COEC were collected from non-pregnant Holstein cows (n = 8) during the follicular phase and cultured in M199 under standard culture conditions until monolayer formation. Cells in first passage were incubated for 24 or 48 h with P4 (500 ng/ml), E2 (1 ng/ml), OT (10(-9) M) or combination of E2 + P4. Administration of E2 significantly increased the production of PGE2, PGF2alpha and ET-1. However, simultaneous administration of P4 blocked the effect of E2. OT did not show any effect on oviductal productions of either PGs or ET-1. The results of this study show that E2 stimulates PG and ET-1 production by COEC in vitro. Thus, it can be suggested that locally transferred E2 from the ovarian follicles may be important for oviductal contraction and gamete/zygote transport during the peri-ovulatory period.

DEVELOPMENTAL CAPACITY OF BOVINE OOCYTES MATURED IN TWO KINDS OF FOLLICULAR FLUID AND FERTILIZED IN VITRO

This study was conducted to assess the ability of the follicular fluid derived from large and small follicles to support the in vitro oocyte maturation and subsequent fertilization and developmental capacity. Oocytes were cultured in bovine follicular fluid aspirated from small (SFF; 2-5 mm in diameter), large (LFF; 10 to 20 mm in diameter) follicles and TCM199 as a control under 5% CO2 in air. All maturation media were supplemented with 1 IU ml-1 pregnant mare serum gonadotropin. After 24 h culture, oocytes were fertilized in vitro with frozen-thawed and heparin-treated (10 micrograms ml-1, 15 min) bull spermatozoa and cultured in TCM199 with bovine oviductal epithelial cells (BOEC) for 7 days. Maturation of bovine oocytes cultured in LFF was inhibited and the low of male pronucleus formation was observed when compared with that of SFF (maturation rate: 69 vs. 78%; P < 0.05; male pronucleus formation rate: 58 vs. 80%; P < 0.05). Developmental capacity of bovine oocytes cultured in SFF was significantly (P < 0.05) higher than that of LFF (15 vs. 5%), but significantly (P < 0.05) lower than that of the control. There were no differences in the number of nuclei per blastocyst obtained after each treatment. These results indicate that the inhibitory action of follicular fluid on in vitro maturation, male pronucleus formation and developmental capacity of bovine oocytes is dependent on the developmental stage of the follicles from which fluid was obtained.

Cryopreservation of equine oocytes by 2-step freezing

Immature equine oocytes were frozen-thawed with ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GL) in PBS and cultured to assess the rate of in vitro maturation (Experiment 1). Compact-cumulus oocyte complexes were collected from slaughterhouse ovaries and equilibrated for 10 min in the freezing medium containing 10% (V/V) cryoprotectant and 0.1 M sucrose. The 0.25-ml straws, loaded with 10 to 30 oocytes, were seeded at -6 degrees C and cooled to -35 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. The straws were thawed rapidly in a 37 degrees C waterbath for 20 sec. The proportions of frozen-thawed oocytes reaching Metaphase II (MII) stage after in vitro maturation of 32 h were 15.8% (EG), 5.8% (PD) and 0% (GL), while 63.3% of the nonfrozen control oocytes matured in vitro. The fertilizing ability of immature and mature oocytes after freezing in EG was tested by the insemination of zona-free oocytes with stallion spermatozoa (Experiment 2). Spermatozoa were preincubated for 3 h with 5 mM caffeine, treated with 0.1 mu M ionophore A23187, and inseminated for 20 h at the concentration of 1 to 2 x 10(7)/ml with 6 to 10 oocytes in 50 mu l of Brackett and Oliphant (BO) medium. Immature oocytes (Group 1) were matured in vitro after thawing and then their zona pellucida removed using 0.5% protease. The zona of mature oocytes were removed immediately after thawing (Group 2) or maturation (nonfrozen controls). The oocytes, which had mechanically damaged plasma membrane or lost by artifact, were not examined for insemination. Significantly more control oocytes exhibited a polar body at the time of insemination (53.5%) than either frozen-thawed immature or mature oocytes (25.8 and 27.3%, respectively). Similar proportion of frozen-thawed and control oocytes were penetrated by spermatozoa (71.8 to 79.1%) and exhibited 2 or more pronuclei (73.6 to 80.8%). The mean numbers of spermatozoa per penetrated oocyte were 1.9, 3.0 and 2.5, respectively, for Groups 1 and 2 and for the control oocytes. These results indicate that immature equine oocytes mature to the MII stage in vitro following freezing and thawing in EG or PD but not in GL. Stallion spermatozoa can penetrate zona-free immature and mature oocytes following freezing/thawing in EG and form morphologically normal pronuclei.

Evaluation of fluids from cystic follicles for in vitro maturation and fertilization of bovine oocytes

Follicular cysts are defined as cystic structures derived from unovulated follicles. The formation of the cysts appears to be related to failure of the oocyte to resume meiosis. The aim of this study was to evaluate in the bovine: 1) the ability of the fluid from cystic follicles to promote in vitro oocyte maturation and fertilization, 2) the predictive value of the morphology of oocytes derived from cystic follicles on the ability of the follicular fluid to promote in vitro maturation/fertilization as well as the oocytes to undergo maturation and fertilization. In Experiment 1, the ability of fluid from cystic (and normal) follicles from live and slaughtered cows (to promote) in vitro maturation and fertilization of bovine cumulus-oocyte-complexes (COCs) was assessed by cumulus expansion, sperm penetration, male pronucleus formation and polyspermy rates. Concentrations of progesterone (P4) and estradiol-17 beta (E2) were measured in the fluid from cystic follicles collected from live and slaughtered cows. In Experiment 2, we investigated the relationship of the morphology of COCs from cystic follicles, and the effect of the follicular fluids on oocyte maturation as well as P4 and E2 concentrations. In Experiment 1, although sperm penetration and male pronucleus formation were inhibited significantly by fluid from some cystic follicles collected from live and slaughtered cows, there were no significant differences in sperm penetration, male pronucleus formation and polyspermy rates between fluid from cystic follicles collected from live cows, from slaughtered cows and from control groups, regardless of the P4/E2 ratio. In Experiment 2, the morphology of cumulus-oocyte complexes from cystic follicles varied and the pronucleus formation of oocytes after in vitro fertilization was abnormal. On the other hand, the male pronucleus formation rates were not significantly different between the cystic follicular fluids and control, regardless, of the P4/E2 ratio. The results of this study suggest that many of the bovine follicular fluids from cystic follicles possess the ability to induce cumulus expansion, nuclear maturation and male pronucleus formation following in vitro maturation and fertilization of bovine oocytes. The morphology of the cumulus-oocytes complexes from cystic follicles seems not to relate to the ability of the cystic follicular fluids to induce oocyte maturation, and oocytes from cystic follicles possess the ability to form male pronucleus even though most were abnormal after in vitro fertilization.

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.

Development of in vitro matured and fertilized bovine embryos cocultured with bovine oviductal epithelial cells obtained from oviducts ipsilateral to cystic follicles

The present experiment was conducted to clarify the effect of bovine oviductal epithelial cells (BOEC) collected from oviducts ipsilateral to cystic follicles (CFs) using an in vitro coculture system on the development of in vitro matured/fertilized (IVM/IVF) bovine embryos. In the first comparison, the effect of the presence of CF on the development of the embryos cocultured with BOEC derived from the cows with CF (n = 18) and corpus hemorrhagicum (CH, n = 10) was examined. In the second comparison, the effect of the type of cyst [progesterone (P4)-dominant; n = 9, estradiol-17beta (E2)-dominant; n = 5] on the development of the embryos cocultured with BOEC derived from the cystic cows was examined. No difference was observed between CF and CH (control) groups in the mean developmental rates of embryos developed to > or =2-cell (86.3% vs. 86.4%), 8-16 cells (53.0% vs. 56.2%), blastocyst (24.2% vs. 24.8%) and hatched blastocyst (12.0% vs. 14.6%). However, the blastocyst production rate was significantly different (P<0.05) between the P4-dominant (19.8%) and E2-dominant (32.6%) groups. The rate of development from cleavage stage embryo to blastocyst was significantly different between P4-dominant (22.9%) and E2-dominant (37.9%) groups. Moreover, the blastocyst rate from 8-16 cells of E2-dominant group (61.6%) was significantly higher than that of P4-dominant one (39.5%). These results indicate that the effects of BOEC collected from oviduct ipsilateral to CFs on embryo development are variable, and the variability is closely associated with the steroid hormone profiles of the follicular fluid.

Effects of follicular fluid on fertilization and embryonic development of bovine oocytes in vitro.

This study was conducted to evaluate the effect of bovine follicular fluid (BFF) on fertilizability and developmental capacity of bovine oocytes matured in vitro. Oocytes were collected from slaughterhouse ovaries, and matured in TCM199 supplemented with 5% superovulated cow serum (SCS), 2 mM pyruvate and 1 IU/mL PMSG. BFF was aspirated from small follicles (1 to 5 mm in diameter). In Experiment 1, BFF was added to the Brackett and Oliphant (BO) fertilization medium at concentrations of 0, 1, 5, 10 and 20%. After insemination with frozen-thawed and heparin-treated (10 micrograms/mL, 15 min) bull spermatozoa for 18 h, some of the oocytes were fixed and stained to evaluate the fertilization rate. The rest of the oocytes were co-cultured in serum-free embryo culture medium (ECM; TCM199 supplemented with 5% SCS, 2 mM pyruvate and 5 micrograms/mL insulin) with bovine oviductal epithelial cells (BOEC) at 38.5 degrees C under 5% CO2 in air, and the developmental capacity of embryos was examined at 2, 7 and 9 d. In Experiment 2, BFF was added to the serum-free ECM with BOEC at 0, 5, 10 and 20% concentrations, and embryos were cultured for 9 d. Fertilization rates and blastocyst rates in low (1 and 5%) BFF in fertilization medium were not significantly different from the control (without BFF). However, high concentrations of BFF (10 and 20%) in the fertilization medium suppressed both fertilization rates and development. Large vesicles with fast monolayer formation were observed at all concentrations of BFF added to ECM with BOEC. There were no significant differences in cleavage or development to blastocyst in different concentrations of BFF added to ECM. However, the rate of development to hatched blastocysts in 20% BFF was significantly lower than that of the control (P < 0.05). The results of the present study indicate that BFF addition to fertilization medium and ECM with BOEC does not improve fertilizability or developmental capacity and that high concentrations of BFF reduce the rate of both fertilization and development.

In vitro deveropment of porcine oocytes fertilized in vitro with spermatozoa preincubated in two different media

This study was conducted to clarify the effects of sperm concentration and media during preincubation on fertilization and development of porcine oocytes fertilized in vitro (IVF). The effect of porcine oviduct epithelial cell aggregates (POECA) on in vitro development of IVF embryos was also examined. Oocytes matured in vitro for 48 to 50 h were inseminated with epididymal spermatozoa preincubated at 2 sperm concentrations (1 - 2 x 10(8)/ ml vs 4 - 5 x 10(8)/ ml) for 3 h in either Dulbeccos phosphate buffered saline (PBS) or Brackett and Oliphant medium (BO). For capacitation, spermatozoa were treated with heparin (100microg / ml) for 15 min at 38.5 degrees C under 5% CO(2) in air. Cleavage and development to the blastocyst stage were evaluated on Day 3 and Day 8 after culture with or without POECA. The effect of sperm concentration on preincubation did not affect the fertilization rate, but preincubation in PBS medium did result in a higher fertilization rate (P < 0.05) than did the BO medium. The proportion of embryos undergoing cleavage and development to the blastocyst stage was significantly higher (P < 0.05) in the POECA co-culture group than in the group without POECA co-culture. The present results indicate that PBS medium can be utilized as a simple preincubation medium for porcine spermatozoa and that the presence of POECA during in vitro culture improved the development of IVF oocytes to the blastocyst stage.