Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration

Oocytes were recovered by laparoscopic aspiration from 3- to 8-week-old calves treated with follicle-stimulating hormone (FSH) followed by human chorionic gonadotropin (hCG) to induce follicular growth and oocyte maturation in vivo. Most of the recovered oocytes either had resumed meiotic maturation at the time of aspiration or were competent to undergo maturation during subsequent culture in vitro. Oocytes matured in vivo following FSH and hCG treatment underwent in vitro fertilization (70%) at rates not significantly different from those of control oocytes recovered from adult cow ovaries at abattoirs and matured in vitro (75%). Calf oocytes that were immature at aspiration exhibited lower fertilization rates after in vitro maturation (36%) but their rate of development to morulae and blastocysts did not differ from that of mature oocytes at aspiration. A total of 91% of the zygotes produced from calf oocytes developed to morula and 27% to blastocyst stages during 6 days of culture. The proportion developing to morulae was significantly higher (P<0.05) than that observed for zygotes resulting from in vitro maturation and fertilization of oocytes recovered from cow ovaries obtained at an abattoir and processed concomitantly (59% to morulae and 18% to blastocysts). Morulae or blastocysts developed from oocytes from 5 to 6-week-old calves, when transferred to synchronized recipient heifers, resulted in 2 confirmed pregnancies, one of which produced a single full-term live calf. The ability to produce embryos from oocytes recovered from newborn or prepubertal calves offers the potential for markedly reducing the generation interval in cattle, thereby substantially accelerating the rate of genetic gain that can be achieved through embryo transfer.     

In vitro embryo production after microinjection and ovarian dynamics following transvaginal follicular oocyte aspiration

Ultrasound-guided transvaginal follicular aspiration of oocytes from live cows combined with IVM, IVF and in vitro culture (IVC) is a procedure for producing preimplantation-stage bovine embryos and a source of oocytes for pronuclear microinjection of DNA for producing transgenic cattle. This experiment was designed to compare in vitro embryo development rates between oocytes derived from transvaginal follicular aspiration and those obtained from cows at slaughter. Nine cows were subject to a twice-weekly aspiration. Oocytes were aspirated with a 5 MHz ultrasound transducer packaged in a vaginal probe equipped with a dorsal-mounted needle guide (16-ga). All visible follicles (>2 mm) were punctured with a 17-ga, 55-cm needle at each aspiration session and the contents removed under vacuum suction. Oocytes underwent IVM/IVF/IVC. Microinjection of DNA was performed during the pronuclear stage of development, and the zygotes were co-cultured on Buffalo Rat Liver (BRL) cells in modified M199 at 39 degrees C in 5% CO2 and air. After 7 d in culture, embryos were removed and scored for development. A Chi-square analysis was used to compare transvaginal follicular-derived oocytes (microinjected and not) and slaughterhouse-derived, matured in transit oocytes (SHDMT; microinjected and not). Nonmicroinjected embryos resulting from IVF of transvaginal aspiration-derived oocytes developed to blastocysts at a higher rate than SHDMT oocytes (40.0 vs 30.8%; P < 0.05). There was no difference in development rates between the microinjected groups (aspiration = 15.9% vs SHDMT = 12.8%). Higher proportions of the embryos generated from the aspirated oocytes were of excellent or good quality following culture (P < 0.05). In the present experiments the effects of microinjection may overshadow some effects of ova source, but transvaginal follicular aspiration may provide a more consistent, synchronous population of oocytes than those derived from commercial slaughter house sources for use with in vitro systems.     

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

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

Individualized gonadotropin regimens for follicular stimulation in macaques during in vitro fertilization (IVF) cycles

Follicular stimulation was compared in macaques receiving sequential gonadotropin treatment which was terminated after seven, eight, or nine days depending on the time required to attain preselected criteria of follicular maturation. Although estradiol levels and follicle sizes varied, the number of follicles and oocytes/animal, oocyte nuclear maturity, IVF rates and progesterone levels during the luteal phase were similar among groups. Reducing the duration of gonadotropin treatment to individualize follicular stimulation regimens does not compromise follicle or gamete quality.     

Developmental competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vitro embryo production

Developmental competence of bovine oocytes collected from follicles of different size categories (in either the growth or the dominant phase of the first follicular wave) was studied, with the aim of improving in vitro embryo production. Estrus and ovulation of 39 cyclic Holstein dairy cows were synchronized by two prostaglandin F2alpha treatments at 11-day intervals and one hCG treatment on the day of onset of estrus (Day 0). Cows with follicles in either the growth (Day 3, n=25) or the dominant phase (Day 7, n=14) were slaughtered, and follicles >5 mm were counted. Three oocyte populations were recovered separately from large (11-15 mm), medium (6-10 mm) and small (2-5 mm) follicles in both follicular phases. All collected cumulus-oocyte complexes (COC), except for markedly atretic oocytes without cumulus cells, were used in experiments. Oocytes were matured, fertilized and cultured by standard methods. There were no significant differences between the growth and the dominant phases for mean numbers of large follicles, usable oocytes and embryos per donor. Generally, those numbers were low, but the development rates of oocytes into blastocysts were high, particularly in the growth phase (60.0%). Mean (+/- S.E.M.) numbers of medium follicles, oocytes and embryos per donor were higher in the growth as compared with the dominant phase; in the usable oocytes and embryos, this difference was significant (9.6 +/- 1.4 and 3.5 +/- 0.6 versus 3.9 +/- 0.6 and 1.1 +/- 0.3; P<0.01). The development rates of oocytes into blastocysts, however, did not differ significantly between the growth and the dominant phases (36.7% versus 27.8%). Mean numbers of usable oocytes and embryos per donor recovered from small follicles in both follicular wave phases were similar. The development rate of oocytes into blastocysts was generally low, but higher (P<0.01) in the growth than in the dominant phase (24.5% versus 11.7%). Comparison between the two phases showed that mean number of all counted follicles and all usable oocytes collected per donor were similar, but the mean number of embryos per donor and the development rate of oocytes into blastocysts were higher in the growth phase than in the dominant phase (8.0 +/- 1.2 versus 3.8 +/- 2.4; P=0.012 and 30.3% versus 14.9%; P<0.01). The interaction between follicle size and the phase of follicular wave affected the efficiency of embryo production. The yield of embryos was primarily influenced by the number of oocytes collected from medium follicles and the developmental competence of oocytes from small follicles. The growth phase was more effective for oocyte collection; the number of oocytes from medium follicles and the developmental competence of oocytes from small follicles decreased in the dominant phase.     

Effect of follicular wave synchronization on in vitro embryo production in heifers

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7 d, followed by the administration of 150 μg d-cloprostenol. On Day 7, all follicles >3 mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14 d. Each experiment had three treatment groups. In Experiment 1 (n = 12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28 d. Heifers from Group 1X-EB also received 2 mg EB i.m. immediately after each OPU session. In Experiment 2 (n = 11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5 mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28 d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos.     

GnRH improves the recovery rate and the in vitro developmental competence of oocytes obtained by transvaginal follicular aspiration from superstimulated heifers

In this study we assessed the effect of GnRH on the recovery rate, meiotic synchronization and in vitro developmental competence of oocytes recovered close to the expected time of ovulation. Twenty-three heifers were superstimulated with FSH, and luteolysis was induced by PGF(2alpha) injection 48 h after the start of treatment Twelve heifers received 200 microg GnRH at 34 h after PGF(2alpha) treatment, Blood samples were collected between 35 to 47 h after PGF(2alpha) administration to determine the time of the LH surge. Transvaginal follicular aspiration was performed at 60 h after PGF(2alpha), and the recovered oocytes were fertilized or fixed either immediately or after 24 h of maturation in vitro. GnRH-treated heifers showed an LH surge within 3 h after treatment, while only 4 of the 10 heifers in the control group exhibited an LH surge by 47 h after treatment with PGF(2alpha). The average number of large follicles (> 10 mm) was 21.3 +/- 2.3 and 19.3 +/- 2.4 for GnRH-treated and control heifers, respectively. The oocyte recovery rate was 87.7 and 63.1% (P < 0.05), respectively, and most of the cumulus-oocyte-complexes (COC) recovered from the 2 groups had an expanded cumulus (80.4 and 80.5%, respectively). Oocytes with an expanded cumulus from the GnRH group had completed meiotic maturation at higher rate than the controls (97 vs 20%;P < 0.05). In vitro development to the blastocyst stage of cumulus-expanded oocytes fertilized immediately after recovery was higher in GnRH-treated than in control heifers (60.3 vs 40.0%; P < 0.05). No difference was observed when oocytes with compact or expanded cumulus were matured in vitro for 24 h before fertilization. These results indicate that GnRH injections improve the oocyte recovery rate and that oocytes have a higher development competence than those obtained from non-GnRH-treated animals. We propose that this higher in vitro developmental competence may result from a more synchronous or further advanced meiotic maturation. However, due to the small number of oocytes in our study, we must emphasize that our findings on meiotic resumption are of preliminary nature.     

In vitro fertilization of horse follicular oocytes matured in vitro

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 x 10(6) sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophore-treated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.     

Carbohydrate and amino acid requirements and ammonia production of rabbit follicular oocytes matured in vitro.

Rabbit follicular oocytes were cultured at 37 °C for 18–24 h in a basic salt medium containing 0.4% bovine serum albumin (BSA), carbohydrates and amino acids in various combinations. Osmolarity of the medium was maintained at 308 mOsm. The carbohydrates, pyruvate, lactate and glucose were all about equally beneficial, but not essential for rabbit oocyte maturation. Glutamine and proline, but not methionine or phenylalanine stimulated oocyte development. Glutamine stimulated more follicular oocytes to develop to prophase and metaphase II than did any of the three carbohydrates tested alone or in combination. Ammonia production after 24 h of culture was highest in media containing glutamine (15.2 μg/ml) but this was not inhibitory to maturation. Negligible amounts of ammonia were found with the other amino acids added. With 0, 0.08, 0.4, 2, 10 and 50 mM of glutamine in the basic salt medium, plus 0.4 % BSA, but without carbohydrates, 30, 73, 70, 71, 59 and 45 % of the follicular oocytes developed to the prophase or metaphase II stage. It is concluded that the optimum level of glutamine ranged from 0.08 to 2 mM and that no carbohydrate need be added to the medium for culturing oocytes when glutamine is included.     

Methods for collecting follicular oocytes from mares

A series of experiments was conducted to develop a procedure for consistent, repeatable collection of oocytes from the preovulatory follicle of the mare. In one experiment, in situ follicular aspiration with a needle and syringe was performed on 19 mares. From 37 aspirations, four oocytes were recovered (10% recovery rate). In a second experiment, ovaries were visualized via standing flank laparotomy during which two different aspiration techniques were used. Use of a needle and syringe as in the first experiment resulted in successful oocyte recovery in one of seven (14%) attempts. Aspiration via a continuous irrigation vacuum system (CIV), developed for use during laparotomy, resulted in collection of oocytes from six of 10 (60%) attempts. In the third experiment, oocytes were recovered from seven of 18 (38%) attempts at in situ follicular aspiration using a double-lumen needle attached to the CIV. In each experiment, some mares were subjected to stimulation of follicular maturation by exogenous hormones. Oocyte recovery was significantly increased in treated mares as compared with nontreated mares. Results indicate that collection of equine follicular oocytes by in situ aspiration is possible with moderate success. Oocytes apparently are not physically damaged by the procedure, as most retained either the corona radiata or the entire cumulus cell mass.     

Factors affecting recovery and quality of oocytes for bovine embryo production in vitro using ovum pick-up technology

In Experiment 1, different vacuum pressures (30, 50, 70 and 90 mm Hg) were used to aspirate 4156 bovine follicles in vitro, to assess their effect on flow rate and the recovery, morphology and blastocyst formation of the recovered oocytes. Cumulus oocyte complexes (COCs) were classified according to the morphology of the cumulus cells. Data were analyzed using Chi Square analysis. Overall recovery rate declined as the aspiration pressure increased above 50 mm Hg (P<0.05). The recovery rate of Grade 1 oocytes decreased significantly (P<0.05) as the vacuum pressure increased with a corresponding increase in the number of denuded oocytes recovered (P<0.05). The blastocyst yield, expressed as a percentage of recovered COCs decreased significantly as the aspiration pressure increased beyond 50 mm Hg (P<0.05). In Experiment 2, the holding media (hepes- or bicarbonate-buffered TCM 199) and holding time (1 h or 5 h) did not affect the blastocyst formation of the oocytes (P>0.05). In Experiment 3, it was found that individual culture of the oocyte during fertilization or culture had a detrimental effect on the oocytes blastocyst formation (8.8% to 16% blastocyst yield on Day 8) when compared to control (31.3%). In Experiment 4, groups of 5, 10 and 25 oocytes were compared with singly cultured oocytes. There were no significant differences (P<0.05) in the blastocyst formation rate among groups of 5, 10, or 25 oocytes, but there was a significant difference between oocytes processed in groups and those processed individually. In Experiment 5, when groups of 10 oocytes were cultured in different drop sizes, there was no significant difference in cleavage rates between oocytes cultured in 100 microL (85.0%, n = 280) and in 10 microL (86.8%, n = 280) of media, but culture in 50 microL (79.3%, n = 280) resulted in cleavage rates significantly lower (P<0.05) than culture in 10 microL drops. There was no significant difference in the blastocyst formation. However there was a significant difference (P<0.05) in cell numbers of Day 8 blastocvsts, with oocytes cultured in 100 microL drops having significantly lower cell counts than oocytes cultured in 50 or 10 microL drops.     

In vitro fertilization analysis of squirrel monkey oocytes produced by various follicular induction regimens and the incidence of triploidy

An in vitro fertilization system utilizing squirrel monkeys was used to compare follicle-stimulating hormone, clomiphene citrate and prostaglandin E₁ as follicular induction regimens, analyze culture medium characteristics, and examine the physiological phenomenon of polyspermy. Induction of follicular growth was poor with clomiphene citrate when compared to the control group and increased the incidence of atretic follicles at all levels tested. When prostaglandin E₁ was administered, larger numbers of mature oocytes were recovered at laparoscopy. There was no difference in fertilization rate between the treatment and control groups. Homologous serum was an adequate protein source in TC-199 fertilization medium for squirrel monkey oocytes. Although the rate of triploidy was increased with in vitro fertilization, there was no relationship between sperm concentration and the incidence of polyspermy. These findings demonstrate that the squirrel monkey is a valuable primate system for studies of in vitro fertilization and preimplantation development. © 1993 Wiley-Liss, Inc.     

In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up

The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly) ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF, using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P=0.357) and blastocyst development rate (16.0 vs. 23.9%, P=0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%, P=0.978) or blastocyst development (15.3 vs. 19.1%, P=0.291) after IVF using OPU-derived oocytes. Of the embryos produced in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%) transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for further research and wider field application of these technologies in buffalo.     

In vitro maturation and fertilization of follicular oocytes in cattle

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

Rescue and maturation in vitro of follicular oocytes collected from nondomestic felid species.

The potential for rescuing immature oocytes from the ovaries of females of rare felid species which die or undergo medical ovariohysterectomy was evaluated. Ovaries were recovered from 13 species representing 35 individuals in good-to-poor health. Although the majority of females were 10 yr of age or older and in fair-to-poor health, a total of 846 oocytes were recovered of which 608 (71.9%) were classified as fair-to-excellent quality. One hundred of these oocytes were used for initial maturation classification and as parthogenetic controls. Overall, of the 508 fair-to-excellent quality oocytes placed in culture, 164 (32.3%) matured to metaphase II in vitro. For species in which 3 or more individuals yielded oocytes, mean oocyte maturation rates were as follows: 36.2%, tiger; 27.9% leopard; and 8.3%, cheetah. In vitro insemination of oocytes resulted in fertilization (2 polar bodies, 2 pronuclei, or cleavage) rates of 9.1% to 28.6% (leopard) using homologous fresh spermatozoa and 4.0% (lion) to 40.0% (puma) using homologous frozen-thawed spermatozoa. Inseminations using heterologous (domestic cat) spermatozoa also resulted in fertilized oocytes in the tiger, leopard, snow leopard, puma, serval, and Geoffroy's cat (range in fertilization rate, 5.0% for leopard to 46.2% for puma). Cleaved embryos resulted from the insemination of leopard oocytes with homologous sperm (n = 1 embryo) and puma oocytes with domestic cat sperm (n = 3 embryos). These results demonstrate that immature ovarian oocytes from rare felid species can be stimulated to mature in vitro despite an excision-to-culture interval as long as 36 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative analysis of calf and cow oocytes during in vitro maturation

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3–5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 ± 1.15 versus 122.83 ± 0.74 μm). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [³⁵S]methionine and [³⁵S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence. Mol. Reprod. Dev. 49:168–175, 1998. © 1998 Wiley-Liss, Inc.     

Improvement in bovine embryo production in vitro by treatment with green tea polyphenols during in vitro maturation of oocytes

The present study examined the effect of green tea polyphenols (GTP) during in vitro maturation (IVM) of bovine oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration and subsequent embryo development. Cumulus-oocyte complexes were aspirated from the ovaries derived from slaughterhouse and cultured in modified synthetic oviduct fluid (m-SOF) supplemented with 0-25 microM GTP for 24h. After IVM, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 15-18 h. Putative embryos were transferred to m-SOF and cultured for 8 days (Experiment 1). In comparison with the absence of GTP, treatment with GTP at a concentration of 15 microM showed a significant increase in the proportion of pronuclear (PN) formation after sperm penetration (65% versus 80%, P<0.05). No significant differences in the rates of sperm penetration and polyspermic fertilization were found among treatments. The cleavage rate at 48 h of in vitro insemination showed no difference in oocytes matured with or without GTP. However, compared to no addition (23.5%), the presence of 15 and 20 microM GTP during IVM significantly (P<0.05) increased the proportion of blastocysts (38.1% and 36.4%) on day 9 of in vitro insemination. A further increase from 20 to 25 microM GTP reduced (P<0.05) the proportion of blastocysts. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P<0.05) level of GSH was found in oocytes matured with 15 microM GTP and compared with 15 microM GTP, GSH was low (P<0.05) at 20 and 25 microM GTP. The results suggest that at certain concentrations of GTP (15 microM) in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in bovine oocytes.     

Parthenogenetic activation of cattle follicular oocytes in vitro with ethanol

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

Developmental competence of juvenile calf oocytes in vitro and in vivo: influence of donor animal variation and repeated gonadotropin stimulation

Juvenile calf oocytes represent an untapped source of germ plasm for reproduction. Reports on the developmental competence of calf oocytes have been controversial. In this research, oocytes were recovered after gonadotropin stimulation from Holstein calves (N = 10) at 2-3 mo of age (2-mo cycle) and again at 4-5 mo of age (4-mo cycle). The in vitro developmental competence was measured, and prestimulation follicle numbers (for 2-mo cycle) and poststimulation follicle numbers (both cycles) were obtained. The number of antral follicles doubled after stimulation (23.4 +/- 6.1 vs. 55.1 +/- 16.1) for the 2-mo cycle and for the 4-mo cycle (47.4 +/- 12.4). The number of follicles observed prior to stimulation in the 2-mo cycle was found to be highly correlated with the poststimulation oocyte recovery for both collection cycles (r = 0.95, 2-mo cycle; r = 0.81, 4-mo cycle). The majority (90-96%) of recovered oocytes were found to be usable for in vitro maturation and fertilization; of these, 41-42% cleaved and 10-11% developed to morulae or blastocysts. Eighty-four in vitro-produced embryos were transferred to synchronized recipients and resulted in 11 pregnancies, leading to 7 live (4 males, 3 females) and 2 dead (one male, one female) calves at full term. No significant differences were observed between the 2-mo and 4-mo collection cycles; however, 73% of the total pregnancies resulted from the 2-mo cycle. All pregnancies resulted from embryos of high-responding donors. The high correlation between the number of follicles prior to stimulation and the poststimulation response suggests the possibility of screening calves prior to stimulation for routine embryo production.