FSH-stimulated follicular secretions enhance oocyte maturation in pigs

Follicular secretions can support cytoplasmic maturation in vitro in the pig. The effects of follicular secretions stimulated in vitro by different combinations of gonadotropins and over different culture periods on cytoplasmic maturation of the pig oocyte were studied. In Experiment 1, follicular shells (including theca and mural granulosa cells) from 5 to 7-mm follicles were cultured in vitro under the stimulation of different combinations of gonadotropins for 48 h, and then the obtained conditioned media were used for oocyte maturation. Oocytes cultured in conditioned medium harvested after treatment of follicular shells with 2.5 mug/ml FSH (FSH-stimulated conditioned medium) yielded a higher percentage of male pronuclear formation than those matured in conditioned medium harvested after culture of follicular shells with a combination of hormones (2.5mug/ml FSH, 2.5 mug/ml LH and 20 ng/ml PRL, FSH-LH-PRL-stimulated conditioned medium; 54.1 vs 28.5%; P=0.001). Addition of the combination of FSH, LH and PRL during the period of oocyte maturation marginally improved male pronuclear formation rates (41.3 vs 55.6%; P=0.06). In Experment 2, follicular shells were cultured under the stimulation of FSH only. Conditioned media were harvested after the first 24 h and the second 24 h of culture. The rates of male pronuclear formation in oocytes matured in these 2 conditioned media did not differ (P=0.65), but were higher than those of oocytes matured in fresh control medium (P<0.03). It is concluded that factors secreted by follicular cells stimulated by FSH alone provide better support for full oocyte maturation in the pig than by combined FSH, LH and PRL treatment.     

Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor

This study examined the ability of epidermal growth factor (EGF) to improve the developmental competence of pig oocytes matured in a protein-free (PF) in vitro maturation (IVM) system. Oocyte maturation was done in one of three media: 1. PF-TCM: tissue culture medium (TCM) 199 + 0.1% polyvinylalcohol (PVA); 2. PF-TCM+EGF: PF-TCM + 10 ng/ml EGF; and 3. +ve CONT: North Carolina State University (NCSU) 23 medium + 10% porcine follicular fluid. All media contained 0.57 mM cysteine. Hormonal supplements, 0.5 microg/mL LH and 0.5 microg/mL FSH, were present only for the first half (20 to 22 h) of the culture period. After maturation, oocytes were co-incubated with frozen-thawed spermatozoa for 5 to 6 h and transferred to embryo culture medium, NCSU 23 containing 0.4% BSA, for 144 h. In Experiment 1, differences in cumulus expansion were observed for oocytes matured in +ve CONT (Category 4), PF-TCM (Category 2) and PF-TCM+EGF (Category 3). However, no significant differences in nuclear maturation to metaphase II stage were observed. In Experiment 2, no differences in fertilization parameters were observed. Significant (P < 0.01) differences in cleavage rates were observed among the three media for a proportion of the oocytes matured (52, 60 and 69% in PF-TCM, PF-TCM+EGF, and +ve CONT, respectively). Oocytes matured in PF-TCM showed the lowest (P < 0.01) blastocyst development (22%). However, the same rate of blastocyst development was obtained for +ve CONT (37%) and PF-TCM+EGF (37%). Blastocyst cell numbers were significantly higher when oocytes were matured in the presence of EGF (26 vs. 37 to 41). In Experiment 3, oocytes matured in PF-TCM+EGF had a significantly (P < 0.05) higher intracellular glutathione (GSH) concentration (5.9 vs. 11.4 pmol/oocyte) compared with PF-TCM. Twenty-two of 25 embryo transfer recipients became pregnant (Experiment 4). Four animals returned to estrus in within 60 days. Six pregnant animals slaughtered at 26 to 45 days had 43 fetuses (range: 4 to 12) and the remaining 12 animals farrowed 82 piglets (range: 3 to 12). These results indicate that EGF enhances the developmental competence of pig oocytes matured in a protein-free culture medium which is correlated with higher GSH level in oocytes. Birth of piglets indicate that embryos derived from oocytes matured in the presence of EGF are viable.     

Maturity at collection and the developmental potential of rhesus monkey oocytes

The purpose of this study was to evaluate the in vitro fertilizability of rhesus monkey oocytes and the developmental capacity of the resulting embryos as they relate to oocyte maturation at the time of follicular aspiration. Animals were hyperstimulated with human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH), with follicular aspiration performed 27 h after administration of an ovulatory stimulus (1000 IU human chorionic gonadotropin [hCG] or 3 x 100 micrograms gonadotropin-releasing hormone [GnRH]). In 7 animals exhibiting a continuously rising pattern of serum estradiol through Day 10 of hyperstimulation, 45 germinal vesicle-intact (GV), 106 metaphase I (MI), and 24 metaphase II (MII) oocytes were collected and cultured in vitro. Upon reaching MII, oocytes were inseminated with 5 x 10(4) motile sperm/ml. Twenty-four percent of GV oocytes cultured in vitro matured to MII with 11 inseminated and none fertilized. Seventy-three percent of MI oocytes matured to MII in vitro with 50% inseminated and 32% fertilized. Oocytes collected at MII stage and inseminated underwent fertilization at a high rate of efficiency (93%). Pronuclear to 8-cell stage embryos were frozen and, upon thawing, 67% (10/15) survived with all blastomeres intact. Frozen-thawed embryos (2- to 6-cell) were transferred to the oviducts of 4 recipients (2 embryos/recipient) during the early luteal phase (1-3 days post LH surge) of natural menstrual cycles. Three twin pregnancies resulted. Thus, a positive correlation exists between the degree of nuclear maturation of rhesus monkey oocytes at collection and their potential for fertilization in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of epidermal growth factor during in vitro maturation of pig oocytes and embryo culture can modulate blastocyst development after in vitro fertilization

The present study examined the effect of epidermal growth factor (EGF) during in vitro maturation (IVM) and embryo culture on blastocyst development in the pig. In experiment 1, cumulus oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, hormonal supplements, and with or without EGF (0–40 ng/ml) for 20–22 hr. They then were cultured for an additional 20–22 hr without hormones. After maturation, cumulus-free oocytes were co-incubated with frozen-thawed spermatozoa for 5–6 hr. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 hr. In experiment 2, oocytes were matured in medium containing 10 ng/ml EGF, inseminated, and putative embryos were cultured in the presence of 0–40 ng/ml EGF. In experiment 3, oocytes were cultured in the presence of 0, 10 and 40 ng/ml EGF to examine the kinetics of meiotic maturation. In experiment 4, 2- to 4-cell and 8-cell to morula stage embryos derived from oocytes matured with 10 ng/ml EGF were transferred to the oviduct and uterus, respectively, of each of three recipient gilts (3 and 4 days post-estrus, respectively). The presence or absence of EGF during IVM did not affect cumulus expansion, nuclear maturation, fertilization parameters, or cleavage rate. However, compared to no addition (21%), presence of 1 (33%) and 10 ng/ml EGF (42%) during IVM increased (P < 0.01) the rate of blastocyst development in a concentration-dependent manner. Compared to 10 ng/ml EGF, higher concentrations (20 and 40 ng/ml) reduced (P < 0.01) blastocyst development in a concentration-dependent manner (35% and 24%, respectively). No difference was observed between no addition and 40 ng/ml EGF (22%). Compared to no addition and 10 ng/ml EGF, a significantly (P < 0.001) higher proportion (25% vs. 55%) of oocytes reached metaphase II stage 33 hr after IVM with 40 ng/ml EGF. However, no difference was observed at 44 hr. Transfer of embryos to six recipient gilts resulted in three pregnancies and birth of 18 piglets. The results show that EGF at certain concentrations in IVM medium can influence the developmental competence of oocytes. However, addition of EGF during the culture of pig embryos derived from oocytes matured in the presence of EGF is without effect. Birth of piglets provides evidence that embryos derived from oocytes matured in a medium containing EGF are viable. Mol. Reprod. Dev. 51:395–401, 1998. © 1998 Wiley-Liss, Inc.     

Effects of bovine follicular fluid on maturation of bovine oocytes

Three experiments were conducted to determine the effects of follicular fluid and media on bovine oocyte maturation. Experiments 1 and 3 test the effects of follicular fluid obtained at different times after the LH surge on bovine oocyte maturation in vitro, while Experiment 2 was designed to compare TALP and Medium 199 as serum-free maturation media. Bovine follicular fluid (BFF) was obtained from preovulatory follicles either before (0 h BFF) or at 4, 8, 12 or 20 h after a GnRH-induced LH surge. Oocytes were obtained from follicles 1 to 6 mm in diameter from ovaries retrieved from a slaughterhouse. In Experiment 1, both 0 h and 4 h BFF inhibited resumption of meiosis, whereas BFF collected at 8, 12 and 20 h did not. When oocytes were cultured in media that contained equal portions of 0 and 8 h BFF, meiosis was not inhibited. In Experiment 2, Medium 199 supplemented with bovine serum albumin (BSA) was superior to Tyrode's medium with albumin, lactate and pyruvate for oocyte maturation. In Experiment 3, a higher percentage (P<0.05) of oocytes cultured for 18 h in 40% 20 h BFF in Medium 199 reached Metaphase-II (64%) than those cultured in 0 h BFF (41%) or control medium (39%). There was a transient meiotic arrest due to 0 h BFF as evidenced by the higher percentage of oocytes with germinal vesicles at 8 h of incubation (35% with 0 h vs 20% with 20 h; P<0.05). Furthermore, expansion of cumulus cells was induced in 8 and 20 h BFF, but not 0 h BFF.     

Developmental potential of rabbit oocyte matured in vitro: the possible contribution of prolactin

The present study was undertaken to define hormonal conditions for in vitro maturation that support subsequent fertilization and embryonic development. Follicular oocytes were recovered from nonstimulated rabbit ovaries and cultured for 12 h in Brackett's medium supplemented with or without hormones. Matured oocytes were inseminated in vitro and transferred 12 h later to Ham's F-10 medium supplemented with 20% fetal calf serum. The initial cleavage frequency of matured oocytes in Brackett's medium was comparable to the frequency of development for in vitro-matured oocytes under various hormonal conditions. However, the addition of estradiol (E2, 1 microgram/ml) to incubation medium containing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) increased significantly (p less than 0.001) the percentage of embryos achieving morula or blastocyst formation (16/98, 16.3%), as compared to the mature oocytes in medium containing LH, LH plus FSH, or no hormone. The addition of prolactin (PRL) to the maturation medium increased the percentage of development to organized embryos in a dose-dependent manner. In vitro-matured oocytes in medium containing LH, FSH, and PRL exhibited a significantly (p less than 0.001) lower incidence of developmental competence (5/95, 5.3%) than oocytes matured in the presence of E2 in conjunction with pituitary hormones (43/89, 48.3%). These results demonstrate that hormonal composition in the environment of the oocyte is critical for acquisition of developmental capacity. PRL as well as E2 appears to be an important constituent in the process of oocyte maturation, promoting preimplantation embryonic development.     

IN VITRO FERTILIZATION OF IN VITRO MATURED MINKE WHALE (BALAENOPTERA ACUTOROSTRATA) FOLLICULAR OOCYTES

In vitro fertilization of follicular oocytes harvested from ovaries and matured in vitro was attempted for 55 minke whales ( Balaenoptera acutorostrata ) captured for Japanese research purposes in the Antarctic Ocean during the period from November 1995 to March 1996. In Experiment 1, effects of culture duration (96 h or 120 h) on maturation of follicular oocytes and addition of caffeine (5 mM) and/or heparin (100 pg/ml) on sperm penetration and pro-nuclear formation were investigated. Spermatozoa recovered from the vasa deferentia of four mature males were diluted (5-fold) and frozen at - 80°C. The post-thawed and pooled spermatozoa were used for in vitro insemination. A higher ( P < 0.05) proportion of the oocytes cultured for 120 h (34.2% of 260) progressed beyond the second metaphase stage than of the oocytes cultured for 96 h (26.0% of 262). For the matured oocytes, higher rates of penetration ( P < 0.05) and pronuclear formation ( P < 0.01) were obtained in the oocytes cultured for 120 h (55.1% and 40.4%) than in those cultured for 96 h (32.4 % and 20.6%). Addition of caffeine and heparin did not show a significant effect. In Experiment 2, follicular oocytes matured for 120 h and then inseminated were cultured to examine the subsequent development in two culture systems (with and without co-cultured cumulus cells). Of 448 inseminated oocytes, cleaved embryos (2–16 cells) were observed with (5.8%) and without (4.9%) co-cultured systems. No cleavage was observed in 54 ova without insemination. These results indicate that in vitro fertilization of minke whale in vitro matured follicular oocytes with cryopreserved spermatozoa is possible, yielding cleaved embryos.     

Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry

The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris-buffered medium containing 2.5 mM caffeine and 0.4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium containing 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, no difference was observed between cleavage rates at 48 h after IVF (X, 49 vs Y, 45%). Transfer of embryos derived from X-bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex.     

Maturation of pig oocytes: observations on membrane potential

The membrane-potential changes of pig oocytes during maturation are described. Cumulus-enclosed oocytes have a resting potential of -41.81 +/- 0.60 mV; the removal of cumulus cells caused this potential to drop to -30.95 +/- 0.43 mV. Adding LH to the culture medium did not influence the potential of denuded oocytes but depolarized the potential of cumulus-enclosed oocytes to -32.90 +/- 0.43 mV. FSH did not affect the membrane potential of denuded or cumulus-enclosed oocytes, but significantly reduced the amplitude of the depolarization induced by LH. The effect of gonadotropins on cultured granulosa cells was also investigated. Plated granulosa cells have a resting potential of -45.21 +/- 0.72 mV, similar to that of cumulus-enclosed oocytes. As recorded in cumulus-enclosed oocytes, LH depolarized granulosa cell membrane potential (-30.33 +/- 0.69 mV) and FSH reduced this effect. To evaluate if oocyte maturation in vivo is accompanied by membrane-potential depolarization, follicular growth and oocyte maturation were induced in 6 prepubertal gilts by using an eCG-hCG treatment. Twenty hours after the beginning of oocyte maturation in vivo (induced by hCG), the membrane potential of the oocyte was depolarized to -28.84 +/- 1.01 mV, a value similar to that observed in vitro. These data indicate that both LH and FSH can influence the membrane potential of follicular somatic cells and, consequently, that of the oocyte. The electrical coupling between somatic cell and oocyte may represent a means by which the gonadotropin message is passed to the germinal cell by the somatic compartment.     

Effect of angiotensin II with follicle cells and insulin-like growth factor-I or insulin on bovine oocyte maturation and embryo development

The objective was to evaluate the effects of angiotensin II (Ang II), insulin-like growth factor-I (IGF-I) and insulin on the nuclear and cytoplasmic maturation of bovine oocytes in the presence of follicular cells. Cumulus-oocyte complexes (COCs) were cultured for 22h in the presence of follicular cells (control with cells) and Ang II, IGF-I or insulin (treatments), or in the absence of follicular cells (control without cells). Using these five groups, Experiment 1 was conducted with and without the addition of gonadotrophins. Only oocytes in the Ang II group resumed meiosis at rates (88.2+/-1.8% and 90.7+/-4.3% for oocytes cultured in the absence or presence of LH/FSH, respectively) similar to those observed for oocytes cultured in the absence of follicular cells (89.7+/-0.3% and 92.6+/-2.6%; P<0.01). In Experiment 2, the effect of Ang II alone and in combination with IGF-I or insulin on oocyte maturation for 7h (germinal vesicle breakdown), 12h (metaphase I) and 22h (metaphase II) was evaluated in a design similar to that of the first experiment. Ang II plus IGF-I or insulin induced the resumption of meiosis, irrespective of the presence of gonadotrophins (P<0.01). Experiment 3 used groups similar Experiment 2 to determine the rate of subsequent embryo development, using fetal calf serum (FCS) in the culture medium. The COCs were cultured in maturation medium for 1h (1+23h), 12h (12+12h) or 24h in the presence of follicular cells and the respective treatments and for the remaining period in the absence of follicular cells to complete 24h. In Experiment 4, BSA was used in lieu of serum in the maturation medium in a 12+12h maturation system. Oocytes matured using the 12+12h system with BSA or FCS in the presence of Ang II+IGF-I had higher rates of blastocyst formation than the other treatments (P<0.05). In conclusion, Ang II reversed the inhibitory effect of follicular cells on nuclear maturation of bovine oocytes, irrespective of the presence of gonadotrophins, IGF-I and insulin. However, oocyte cytoplasmatic maturation (i.e., subsequent embryo development), was higher when Ang II and IGF-I were present in the maturation medium containing follicular cells cultured for 12+12h.     

Developmental competence of somatic cell nuclear transfer embryos reconstructed from oocytes matured in vitro with follicle shells in miniature pig

The developmental competence of domestic pig oocytes that were transferred to somatic cell nuclei of miniature pig was examined. A co-culture system of oocytes with follicle shells was used for the maturation of domestic pig oocytes in vitro. Co-cultured oocytes progressed to the metaphase II stage of meiosis more quickly and more synchronously than non co-cultured oocytes. Oocytes were enucleated and fused with fibroblast cells of Potbelly miniature pig at 48 h of maturation. The blastocyst formation rate of nuclear transfer (NT) embryos using cocultured oocytes (24%) was significantly higher (p < 0.05) than that of non-co-cultured oocytes (13%). Cleaved embryos at 48 h after nuclear transfer using co-cultured oocytes were transferred to the oviducts of 14 G?ttingen miniature pigs and four Meishan pigs. Estrus of all G?ttingens returned at around 20-31 days of pregnancy. Two of the four Meishans became pregnant. Three and two cloned piglets were born after modest number of embryo transfer (15 and 29 embryos transferred), respectively. These results indicated that oocytes co-cultured with follicle shells have a high developmental competence after nuclear transfer and result in full-term development after embryo transfer.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

Evidence that Meishan and Large-White hybrid preovulatory follicles may differentially affect oocyte in vitro maturation and fertilization

Two experiments were carried out to test the hypothesis that follicles recovered from Meishan animals may provide a more favourable environment for oocyte maturation in vitro than follicles recovered from Large-White hybrid animals. In Experiment 1, all follicles ≥4 mm were recovered from six Meishan and seven Large-White hybrid gilts in the late follicular phase and healthy oocyte cumulus complexes recovered. Cumulus oocyte complexes were randomly divided into two groups, and each group cultured for 27 or 34 h (62 and 64; 56 and 56 for Meishan and Large-White hybrid, respectively) in defined medium in the presence of either of the two largest follicle shells per animal. Subsequent examination of oocyte nuclear maturation showed that although maturation did not differ significantly between the breeds after 27 h, more (P<0.01) Meishan oocytes co-cultured with Meishan follicles developed to metaphase II stage than Large-White hybrid oocytes co-cultured with Large-White hybrid follicles after 34 h. The next eight largest follicles per animal were cultured for 34 h to produce conditioned media. In Experiment 2, oocytes recovered from the slaughterhouse were matured for 46 h in the presence of conditioned media from Meishan (612 oocytes) or Large-White hybrid (731 oocytes) follicles, or in fresh medium in the presence of a follicle shell from slaughterhouse ovaries. Oocytes were then inseminated and 12 h later examined for penetration and male pronuclear formation. A higher (P<0.05) percentage of oocytes cultured in Meishan follicle conditioned medium underwent sperm penetration and male pronuclear formation than oocytes cultured in conditioned media from Large-White hybrid animals. Concentrations of oestradiol and progesterone in the conditioned media did not differ between the breeds (P>0.1). In conclusion, these results suggest that (1) Meishan oocytes have advanced maturational capacity when cultured with Meishan preovulatory follicle shells and (2) differences in follicle maturation in the Meishan compared to the Large-White hybrid pig may result in an improved ability of the follicles, via conditioned media, to support oocyte maturation and fertilization in vitro.     

In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium

The effect of BSA, caffeine and calcium was studied on the penetration of pig oocytes by frozen-thawed spermatozoa in a modified Tris-buffered medium (mTBM) without added bicarbonate. Pig cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22 h. The COC were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with spermatozoa for 6 h in mTBM containing caffeine (5 mM) and 0.1 or 0.4% BSA (Experiment 1). In Experiment 2, oocytes were inseminated in mTBM containing 0.1% BSA and various concentrations of caffeine (0 to 5 mM). In Experiment 3, insemination was carried out in mTBM containing 0.1% BSA, 1 mM caffeine and various concentrations of Ca(2+) (0.5 to 10 mM). Supplementation of mTBM with either 0.1 or 0.4% BSA resulted a high penetration rate with a high polyspermy rate. However, the mean number of spermatozoa per oocyte was significantly higher at 0.4% than at 0.1% BSA. The penetration rate, polyspermy rate and mean number of spermatozoa per oocyte were all significantly higher when 1 to 5 mM caffeine were added to the medium than in caffeine-free medium. No penetration was observed in the presence of 0.5 mM Ca(2+). The penetration rate was significantly increased from 12 to 92% at 2.5 to 10 mM Ca(2+). The mean number of spermatozoa per oocyte did not differ between 2.5 and 5 mM Ca(2+) but increased significantly at 7.5 and 10 mM. These results show the successful in vitro penetration of pig oocytes in a chemically semidefined medium without added bicarbonate. Although BSA and caffeine can modulate the rate of sperm penetration, calcium seems to be an important regulatory ion.     

Gamete recovery and follicular transfer (graft) using transvaginal ultrasonography in cattle

Current in vitro culture systems may not be adequate to support maturation, fertilization and embryo development of calf oocytes. Thus, we initiated a study to investigate an alternative method of assessing oocyte competence in vivo, initially using oocytes from adults. Experiment 1 was done to determine if follicle puncture would alter subsequent follicle development, ovulation and CL formation. In control (no follicle puncture, n = 3) and treated (follicle puncture, n = 3) heifers, ultrasound-guided transvaginal follicle aspiration was used to ablate all follicles > or = 5 mm at random stages of the estrous cycle to induce synchronous follicular wave emergence among heifers; PGF2 alpha was given 4 d later. Three days after PGF2 alpha, the preovulatory follicle in treated heifers was punctured with a 25-g needle between the exposed and nonexposed portions of the follicular wall, and 200 microL of PBS were infused into the antrum. There was no significant difference between control and treated heifers for mean diameter of the dominant follicle prior to ovulation, the interval to ovulation following PGF2 alpha, or first detection and diameter of the CL. Experiment 2 was designed to assess multiple embryo production following interfollicular transfer of oocytes (i.e., transfer of multiple oocytes from donor follicles to a single recipient preovulatory follicle). Follicular wave emergence was synchronized among control (no follicle puncture, n = 5), oocyte recipient (n = 7) and oocyte donor (n = 5) heifers as in Experiment 1. In control and oocyte recipient heifers, a norgestomet ear implant was placed at the time of ablation and removed 4 d later, at the second PGF2 alpha treatment. In oocyte donor heifers, FSH was given the day after ablation, and, 4 d later, oocytes were collected by transvaginal follicle aspiration, pooled and placed in holding medium. Five or 6 oocytes were loaded into the 25-g needle of the follicle infusion apparatus with < or = 200 microL of transfer medium. Puncture of the preovulatory follicle of recipient heifers was done as in Experiment 1. Immediately thereafter, LH was given to control and oocyte recipient heifers, but only the recipients were inseminated. Ovarian function was assessed by transrectal ultrasonography and control and oocyte recipient heifers were sent to the abattoir 2 or 3 d after ovulation, where excised oviducts were flushed. The interval between LH administration and ovulation (33 to 36 h) was highly synchronous within and among control and oocyte recipient heifers. Four of 5 (80%) ova were collected from controls and 16 of a potential 43 (37%) ova/embryos were recovered from oocyte recipients; 8 embryos from 3 heifers. Thus, the gamete recovery and follicular transfer procedure (GRAFT) did not alter ovulation or subsequent CL formation, and resulted in the recovery of multiple ova/embryos in which a total of 19 oocytes yielded as many as 8 early embryos, a 42% embryo production rate.     

Histone H1 kinase activity during in vitro fertilization of pig follicular oocytes matured in vitro

The present study was conducted to clarify the relationship between histone H1 kinase (H1K) activity and events associated with in vitro fertilization of pig follicular oocytes matured in vitro. Histone H1 kinase has been shown to be homologous with a maturation promoting factor (MPF). Cumulus-oocyte complexes obtained from prepubertal gilts were cultured for 46 h in a modified Waymouth's MB752/1 medium and were then inseminated in vitro with frozen-thawed and preincubated epididymal boar spermatozoa. At 4, 6, 8 and 10 h post insemination, the oocytes were stained with 10 microg/ml Hoechst-33342 and examined under a fluorescent microscope for the stage of fertilization, according to morphological changes of oocyte nuclear chromatin and the extent of sperm penetration. Sperm penetration was observed to occur within 4 h post insemination (20.5%), and the percentage of fertilized oocytes increased (P < 0.01) to 72.9% at 8 h post insemination. Pronuclear formation was observed from 6 h post insemination (3.3%) and the percentage increased (P < 0.01) to 46.8% at 10 h post insemination. In each examination period, H1K activities in unfertilized oocytes at metaphase-II remained unchanged (112.0 fmol/h/oocyte) and were higher (P < 0.01) than those in fertilized oocytes (30.1 fmol/h/oocyte). The H1K activity in fertilized oocytes such as oocytes emitting a second polar body, oocytes with an enlarging sperm head(s) and oocytes with multiple pronuclei did not differ significantly. These results suggest that MPF in pig oocytes is inactivated shortly after sperm penetration and is maintained at the basal level throughout pronuclear formation.     

Nuclear and cytoskeletal dynamics during oocyte maturation and development of somatic cell cloned pig embryos injected with membrane disintegrated donor cells

The objectives of this study were to characterize the nuclear and cytoskeletal changes of pig oocytes during in vitro maturation (IVM) and the development of the reconstructed embryos after injection with membrane intact or disintegrated donor cells. Cumulus-oocyte complexes (COCs) were collected from abattoir ovaries by follicle (2-8mm) aspiration. In Experiment 1, COCs were cultured in NCSU-23 medium for 0, 11, 22, 33, and 44 h. Oocytes were fixed at different time points for nuclear and cytoskeletal labeling. Forty-three percent and 75% oocytes progressed to MII stage at 33 and 44 h after IVM culture, respectively. Dynamic shift of spindle and cytoplasmic microtubules was evident. In Experiment 2, matured oocytes were injected with either the whole cumulus cell with or without intact cell membranes after enucleation. The reconstructed oocytes were fixed at 0, 2, or 4 h after cell injection for nuclear and cytoskeletal evaluation. When an intact cumulus cell was injected, the injected cell remained intact within 4h after injection. When a cell with disintegrated membrane was injected, 59-63% (n=146) of the injected cell underwent premature chromosome condensation (PCC). In Experiment 3, the reconstructed pig oocytes received membrane-disintegrated cumulus cells or fetal fibroblasts were cultured in PZM medium. The blastocyst rate of the fibroblast-injected embryos was 10%, which was lower than the non-cloned parthenotes (33%, P<0.05) but higher than the cumulus cell-injected embryos (2.7%). These results suggest that pig oocytes are subjected to nuclear and cytoskeletal reorganization during maturation. Pig oocytes injected with membrane-disintegrated fibroblast cells support better blastocyst development of the cloned embryos.     

Nuclear maturation of canine oocytes cultured in protein-free media

The objective of this study was to determine the ability of canine oocytes to complete nuclear maturation in a protein-free medium. Oocytes obtained from ovaries of bitches aged 6 months to 2 years were cultured either in TCM199 or CMRL1066 medium without protein supplementation in 5% or 20% O(2). Sixteen of 121 (13%) oocytes cultured in TCM199 reached metaphase II, but only 1 of 135 oocytes cultured in CMRL1066 did so (P < 0.05). Oxygen concentration did not affect nuclear maturation. An additional 103 oocytes were cultured in TCM199 for 48 hr, inseminated with chilled ejaculated spermatozoa, fixed in 1:3 acetic acid-ethanol and then stained with aceto-orcein; 34% of these oocytes were penetrated by spermatozoa. To determine developmental competence of oocytes cultured in a protein-free medium, 85 oocytes were cultured in TCM 199 for 48 hr, inseminated and then cultured; 7 early stage embryos were produced. The effects of growth hormone, beta-mercaptoethanol (betaME), luteinizing hormone (LH) and energy substrates, alone or in combination, on nuclear maturation of oocytes cultured in a protein-free medium were also determined. Growth hormone enhanced cumulus expansion, but did not improve nuclear maturation. beta-mercaptoethanol had no effect on nuclear maturation. However, percentages of MII oocytes significantly decreased when the oocytes were cultured for 48 hr in the medium containing LH or a high concentration of glucose (P < 0.05). In conclusion, canine oocytes are able to complete nuclear maturation in a protein-free medium. The specific type of medium and other supplements significantly influence the meiotic maturation of canine oocytes.     

Fertilizability and developmental capacity of individually cultured bovine oocytes

Culture of single oocytes throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC) provides detailed information on maturity, fertilizability and developmental capacity of individual bovine oocytes and embryos. In the present study, effects of sperm concentration (Experiment 1), microdrop size (Experiment 2), and the addition of hypotaurine (HT) or glutathione (GSH; Experiment 3) during IVF were investigated. In Experiment 4, in vitro maturity and developmental capacity of bovine oocytes cultured for IVM in a medium supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) during IVM were investigated. In Experiments 1 to 3, the percentages of normal (2 pronuclei with a spermtail) and polyspermic fertilization in singly cultured oocytes were similar to those of group IVF culture (5 oocytes/drop). The addition of GSH during single oocyte IVF significantly increased the proportion of normal fertilization and decreased the polyspermic fertilization compared with addition of HT or of the control. The rates of mature oocytes (62.4 and 67.7%) and blastocyst development (12.9 and 15.2%) for single oocyte IVM cultures (Experiment 4) were also similar compared with the group culture; PVA supplementation significantly increased the matured oocyte rate, but decreased blastocyst development significantly (7.1%) as compared with FCS (19.5%) or BSA (15.6%). These results indicate that a single oocyte culture system throughout in vitro production of bovine embryos provides similar maturity, fertilizability and developmental capacity to oocytes cultured in groups.     

Effect of protein synthesis inhibition before or during in vitro maturation on subsequent development of bovine oocytes

The overall objective of this study was to assess the effect of maintaining meiotic arrest in bovine oocytes in vitro on developmental competence. In Experiment 1 the effect of inhibition of meiotic resumption using cycloheximide (CX), on subsequent was examined. Immature cumulus oocyte complexes (COCs, n = 804) were cultured in the absence (24 h) or presence of CX for 6, 12, 18 or 24 h. The control was inseminated 24 h later, while CX-treated oocytes were cultured for a further 24 h before insemination. In Experiment 2 the effect of exposing the oocyte (n = 1239) during meiotic arrest to putative stimulatory substances (pFSH and FCS) was examined. In Experiment 3, to study the importance of protein synthesis during maturation, synthesis was blocked for a 6-h period at various times (6, 12, 18 h) after start of culture (n = 1117). In Experiment 1, there was no difference in cleavage rate between treatments. However, the percentage of 5 to 8 cell embryos at 72 h post insemination was significantly lower after CX treatment (64 vs 42 to 51%; P < 0.05). This was reflected in a lower rate of blastocysts at Day 6 (9 to 15 vs 31%, P < 0.002). While the blastocyst rate at Day 8 was lower in CX-treated oocytes, the effect was only significant when CX was present for longer than 12 h. A marked decrease in development was noted following inhibition for 18 h or more compared with the control (17 to 19 vs 40%; P < 0.0002). In Experiment 2, addition of either FSH or FCS to oocytes in the presence of CX had no effect on any of the parameters studied, even though there was a positive effect in control oocytes. In Experiment 3, treatment with CX after the oocytes had matured for varying periods resulted in decreased blastocyst rates at Days 6 and 8 of culture. The most significant drop in development occurred when oocytes were cultured for 12 h before exposure to CX (15 vs 40%; P < 0.0001). In conclusion, CX-blocked oocytes retained their developmental competence, although final blastocyst yields were reduced.