Maturity and fertility of rhesus monkey oocytes collected at different intervals after an ovulatory stimulus (human chorionic gonadotropin) in in vitro fertilization cycles

In rhesus monkeys undergoing ovarian stimulation for in vitro fertilization (IVF), a midcycle injection of human chorionic gonadotropin (hCG) substitutes for the LH surge and induces preovulatory oocyte maturation. The time interval between injection and oocyte collection, ideally, allows for the completion of oocyte maturation without ovulation, which would reduce the number of oocytes available for harvest. To evaluate the influence of this time interval on oocyte parameters following hCG administration, we conducted a series of gonadotropin treatment protocols in 51 animals in which the interval from hCG administration to follicular aspiration was systematically varied from 27 to 36 hr. Follicle number and size, evaluated prior to hCG administration by sonography, did not vary significantly or consistently with preovulatory maturation time. Oocytes were harvested by laparotomy or laparoscopy, and scored for maturity before insemination. The percentage of mature, metaphase II (MII) oocytes at recovery increased significantly with increasing preovulatory time and was inversely proportional to that of metaphase I (MI) oocytes. However, oocyte yield tended toward a progressive decrease with increasing preovulatory maturation times from a high of 27 oocytes at 27 hr to a low of 17 oocytes/animal at the 36 hr time interval. Fertilization levels declined significantly from a high of 50% at 27 hr to a low of 30% at 36 hr. Thus, although higher percentages of mature oocytes were recovered at the longer time intervals, optimal oocyte/embryo harvests were realized after the shorter time intervals (27 and 32 hr) and are most compatible with the goal of achieving high yields of fertile oocytes and embryos following gonadotropin stimulation in rhesus monkeys. © 1996 Wiley-Liss, Inc.     

不同取卵时间对兔ICSI胚胎体外发育的影响

目的探讨不同取卵时间对兔ICSI胚胎体外发育的影响。方法采用Piezo操作系统对实验兔进行辅助体外受精。结果hCG注射后14、16、18h取卵,ICSI后的受精率分别为82.2％、75.9％和0.0％,对受精卵进行体外发育培养,桑椹胚的发育率分别为72.9％、70.0％、0.0％,囊胚的发育率分别为62.2％、53.3％、0.0％。14h和16h之间受精率、桑椹胚率、囊胚率差异不显著（P〉0.05）,但是14h采卵比16h要好;18h和14h、16h之间差异显著（P〈0.05）。结论不同取卵时间影响实验兔的ICSI体外受精率及胚胎的体外发育率,hCG注射后14h取卵最有利于兔ICSI胚胎的发育。     

FSH priming improves oocyte maturation,but priming with FSH or hCG has no effect on subsequent embryonic development in an in vitro maturation program

AIM: To determine whether maturation and subsequent blastocyst development of in vitro matured oocytes can be improved by in vivo follicle stimulating hormone (FSH) or human chorionic gonadotrophin (hCG) priming, using a mouse model. EXPERIMENTAL DESIGN: Five groups of oocytes were used: in vivo control, in vitro matured (IVM) control, IVM after 24 h in vivo priming with FSH, IVM after 48 h in vivo priming with FSH and IVM after 16 h in vivo priming with hCG. In vitro fertilization (IVF) was performed on all groups.Oocyte maturation, fertilization, blastocyst development rates and blastocyst cell numbers were assessed for all groups. RESULTS: Significant improvement in oocyte maturation was observed in the two FSH priming groups compared with the IVM control group (P<0.005 and P<0.001, respectively). There were no significant differences in fertilization between all five groups. Blastocyst development was significantly higher in the in vivo control compared to the IVM groups (P<0.001). No significant differences were observed in blastocyst cell numbers among all five groups. CONCLUSIONS: While FSH priming improves the maturation rate of IVM oocytes, FSH or hCG priming does not improve development to the blastocyst stage.     

Fertilization and ovum recovery rates in superovulated ewes following cervical insemination or laparoscopic intrauterine insemination at different times after progestagen withdrawal and in one or both uterine horns

In Exp. 1, 40 ewes were used in a 2 x 2 factorial design to investigate the effects of intrauterine versus cervical insemination and superovulation using pig FSH or PMSG and GnRH on egg recovery and fertilization rate. Cervical inseminations were carried out at 48 and 60 h (N = 20 ewes) and intrauterine insemination at 52 h (N = 20 ewes) after progestagen pessary withdrawal. Eggs were recovered on Day 3 of the oestrous cycle. Ovulation, egg recovery and fertilization rates were independent of the type of superovulatory hormone used. Fertilization rate was high irrespective of insemination site but intrauterine insemination at 52 h was associated with a significant (P less than 0.01) decrease in egg recovery of over 40% compared with cervically inseminated ewes. In Exp. 2 ewes were inseminated at 36 (N = 5), 48 (N = 6) or 60 (N = 6) h after pessary withdrawal to determine the optimum intrauterine insemination time to maximize both fertilization rate and egg recovery. Egg recovery per ewe flushed was 23, 59 and 67% after intrauterine insemination at 36, 48 and 60 h respectively. Correspondingly, 0, 85 and 100% of the eggs recovered were fertilized. The results of Exps 1 and 2 suggest that when intrauterine insemination occurs before or during ovulation it interferes with oocyte collection by the fimbria. In Exp. 3 egg recovery and fertilization rates were determined after cervical insemination at 48 and 60 h (N = 8) or intrauterine insemination at 48 (N = 9) or 60 (N = 8) h after progestagen withdrawal. Ewes in the last two groups were subdivided and inseminated unilaterally or bilaterally. Egg recovery was high after cervical insemination (95%) but only 36% of these eggs were fertilized. Unilateral intrauterine insemination was as effective as bilateral in ensuring high fertilization rates (100 versus 97%). Intrauterine insemination at 48 h compared with 60 h resulted in a significantly lower (P less than 0.05) percentage of eggs recovered (42 versus 90% respectively). However, reducing the degree of interference by adopting unilateral rather than bilateral insemination did not alleviate the detrimental effects of the 48-h insemination time on egg recovery. From these results we advocate the adoption of intrauterine insemination at 60 h after progestagen withdrawal to maximize fertilization rate and egg recovery in superovulated ewes.     

Maturation and fertilization of porcine oocytes in vitro

Four experiments were conducted to study 1) factors affecting porcine oocyte maturation in culture medium and 2) a new method for oocyte maturation outside the porcine body. In Experiment 1, five groups of oocytes were cultured in m-TCM199 or m-KRB medium for 24 to 28, 32 to 36 or 40 to 42 hours and then were fertilized in vitro. The cleavage rate (two to four-cell stage) of oocytes cultured for 32 to 36 hours was significantly higher than those of the other oocytes. The results indicate that a suitable culture period for the in vitro maturation of porcine oocytes is 32 to 36 hours. In Experiment 2, four groups of oocytes were cultured in m-KRB or m-KRB supplemented with PFF, PMSG or FSH for in vitro maturation, and the cleavage rates of oocytes were 7.94, 22.56, 30.23 and 23.26%, respectively, after in vitro fertilization. The results show that porcine follicular fluid (PFF) and gonadotrophins added to the culture medium promote porcine oocyte maturation in vitro. In Experiment 3, oocytes were cultured in m-KRB or m-TCM199, supplemented with both gonadotrophin and pocine folliclar fluid for maturation in vitro. After fertilization in vitro, the cleavage rates of oocytes were 26.32 and 27.93% for the two media. The results indicate that the difference between m-KRB and m-TCM199 was insignificant when the media were used to culture porcine oocytes. But there was a significant difference when PFF and gonadotrophins were added to the basic media. In Experiment 4, porcine oocytes were transferred into the reproductive tracts of other animals for maturation. After 34 to 36 hours, the oocytes were collected and fertilized in vitro. The cleavage rates of oocytes were 10.42, 28.45, 3.33 and 36.36%, respectively, for the oocytes matured in mouse uterine horns, rat uterine horns, rat oviducts or rabbit oviducts. The results show that porcine oocytes can be matured in the reproductive tracts of other animals.     

Effect of hCG priming on embryonic development of immature oocytes collected from unstimulated women with polycystic ovarian syndrome

ABSTRACT: Backgroud: The effect of hCG priming on oocyte maturation and subsequently outcome in IVM cycles has remained a debated issue. A randomized controlled study was performed to investigate whether or not hCG priming prior to oocyte aspiration can improve the developmental competence of immature oocytes from unstimulated ovaries in women with polycystic ovarian syndrome (PCOS). MethodsEighty two patients with PCOS underwent IVM cycles. Each patient was randomly assigned to the hCG-primed (10,000 IU) or non-primed groups 36-38 hours before oocyte retrieval depending on the computerized random table. After the oocytes had in vitro matured, fertilizationculture and embryo transfer were performed. ResultsThe average number of cumulus-oocyte complexes (COCs) recovered was 13.80 and 14.35 in the hCG-primed and non-primed groups, respectively (p>0.05). The maturation rate of COCs was significantly improved in the hCG-primed group (55.43% vs. 42.29%; p<0.05). The fertilization and cleavage rates were comparable between the groups. The hCG-primed and non-primed groups did not differ with respect to the clinical pregnancy (37.50% vs. 50.00%), live birth (22.50% vs. 30.95%), and implantation rates (32.86% vs. 32.56%). The pregnancy losses was 6 (40.00%) of 15 clinical pregnancies in the hCG-primed groupand 8 (38.10%) of 21 clinical pregnancies in the non-primed group. CONCLUSIONS: While a significant improvement in the nuclear maturation rate of immature oocytes was observed in hCG-primed IVM cycles with PCOS patients, the use of hCG prior to oocyte retrieval did not improve the subsequent embryo developmental competence. The high rate of pregnancy loss in IVM cycles should receive more attention.     

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

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

Prolonging the interval from ovarian hyperstimulation to laparoscopic ovum pick-up improves oocyte yield, quality, and developmental competence in goats

The objective was to evaluate the effect of the interval between ovarian hyperstimulation and laparoscopic ovum pick-up (LOPU) on quality and developmental competence of goat oocytes before and after in vitro maturation (IVM) and intracytoplasmic sperm injection (ICSI). Estrus was synchronized with an intravaginal insert containing 0.3g progesterone (CIDR) for 10d, combined with a luteolytic treatment of 125 microg cloprostenol 36 h prior to CIDR removal. Ovaries were hyperstimulated with 70 mg FSH and 500 IU hCG given im 36, 60, or 72 h prior to LOPU (n=15, 16, and 7 does, respectively). For these groups, oocyte retrieval rates (mean+/-S.E.M.) were 24.7+/-2.9, 54.5+/-4.7, and 82.8+/-4.6% (P<0.001), and the proportions of cumulus-oocyte complexes (COC) with more than five layers of cumulus cells were 29.7+/-8.3, 37.6+/-6.9, and 37.3+/-7.0% (P<0.001). The proportion of IVM oocytes was highest at 72 h (82.1+/-2.8%; P<0.05), with no significant difference between 36 and 60 h (57.3+/-8.9% and 69.0+/-8.4%). Cleavage rates of ICSI embryos were 4.2+/-4.2, 70.9+/-8.4, and 78.9+/-8.2% with LOPU 36, 60, and 72 h post FSH/hCG (P<0.01), with a lower proportion of Grade-A embryos (P<0.05) following LOPU at 36 h compared to 60 and 72 h (29.7+/-8.3%, 37.6+/-6.9%, and 37.3+/-7.0%). In summary, a prolonged interval from FSH/hCG to LOPU improved oocyte retrieval rate and oocyte quality. Therefore, under the present conditions, LOPU 60 or 72 h after FSH/hCG optimized yields of good-quality oocytes for IVM and embryo production in goats.     

Effects of insemination-ovulation interval on fertilization rates and embryo characteristics in dairy cattle

The objective of this study was to examine effects of the interval between insemination and ovulation on fertilization and embryo characteristics (quality scored as good, fair, poor and degenerate; morphology; number of cell cycles and accessory sperm number) in dairy cattle. Time of ovulation was assessed by ultrasonography (every 4h). Cows were artificially inseminated once between 36h before ovulation and 12h after ovulation. In total 122 oocytes/embryos were recovered 7d after ovulation. Insemination-ovulation interval (12h-intervals) affected fertilization and the percentages of good embryos. Fertilization rates were higher when AI was performed between 36-24 and 24-12h before ovulation (85% and 82%) compared to AI after ovulation (56%). AI between 24 and 12h before ovulation resulted in higher percentages of good embryos (68%) compared to AI after ovulation (6%). Insemination-ovulation interval had no effect on number of accessory sperm cells and number of cell cycles when corrected for embryo quality. This study showed that the insemination-ovulation interval with a high probability of fertilization is quite long (from 36 to 12h before ovulation). However, the insemination-ovulation interval in which this fertilized oocyte has a high probability of developing into a good embryo is shorter (24-12h before ovulation).     

In vitro fertilization and cleavage of in vivo matured bovine oocytes

The effect of the interval between onset of estrus and oocyte collection on in vitro fertilization (IVF) rates has been investigated. The oocytes were surgically collected 6-18 h (Group I), 19-24 h (Group II), 25-29 h (Group III) and 30-36 h (Group IV) after the beginning of estrus. Recognizable stages of nuclear maturation were identified in 54.9% of the oocytes used for IVF (5.9% at germinal vesicle, 31.4% at metaphase I, 17.6% at metaphase II); the other 45.1% were degenerate. Considerable between- and within-cow variation in oocyte morphology, oocyte maturation and IVF results was observed. The cverall fertilization and cleavage rates (to four-cell stages) were 26.5 and 6.0%, respectively. The fertilization rate increased as the interval between onset of estrus and collection increased and was optimal 30-36 h after onset. Thus, onset of estrus proved an effective means of timing oocyte collection for IVF.     

In vitro fertilization of bovine follicular oocytes obtained by laparoscopy

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

Chromosomal analyses after in vitro fertilization of squirrel monkey (Saimiri sciureus) oocytes

The effect of 1 microM dibutyril cAMP (cAMP media) on the chromosomal normality of in vitro fertilized squirrel monkey oocytes was examined. A total of 877 oocytes were collected laparoscopically 15-16 h after hCG injection from hormonally-induced follicles of 65 squirrel monkeys. Of these, 330 (37.6%) were prepared between 6 and 30 h after insemination and analyzed. Sperm penetration and the presence of a swollen sperm head in the ooplasm were observed by 6 h, and the nucleate stage by 10 h. Addition of 1 microM dbcAMP did not affect the oocyte maturation rate, however the in vitro fertilization rate was increased 26.5% over controls (46.3 vs. 36.6%). The first cleavage metaphase was found at 16 h after insemination. Between 24 and 30 h post-insemination, 76.6% of fertilized oocytes had reached the first cleavage metaphase. An incidence of triploidy (including dispermy) of 11.7% was observed. After chromosomal analysis of metaphase I, univalents were observed in 16.7% of the cases. Aneuploidy (8.9%) was also found in metaphase II. Of the first cleavage metaphases, 88.0% had the diploid number of chromosomes. Aneuploidy occurred 12.0% of the time and 5 cases of triploidies were observed.     

The chromosomal normality of in vitro-fertilized rabbit oocytes

The chromosomal normality of rabbit oocytes fertilized in vitro was examined. Ovum donors were superovulated with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). Follicular oocytes were collected laparoscopically 9-10 h after hCG treatment and incubated in vitro with spermatozoa capacitated in vivo. Of 267 aspirated oocytes, 191 (71.4%) were fertilized in vitro and developed to the 2- to 8-cell stage 24-48 h after insemination. In the chromosomal studies, 121 (63.4%) were examined. Of these, 94 (77.7%) had a normal diploid complement of chromosomes (2n = 44) and 6 (5%) showed aneuploidy. Of the remaining 21, 20 were triploid and one was tetraploid. The incidence of triploid oocytes after in vitro fertilization was higher than the rate in vivo (16.5% vs. 9.0%, respectively). These triploid oocytes were suspected to be the result of polyspermic fertilization in vitro. In addition, at the Metaphase II stage, 62 (89.9%) of 69 induced, preovulatory oocytes had a normal number of chromosomes.     

A study of factors affecting the success of human fertilization in vitro. II. Influence of semen quality and oocyte maturity on fertilization and cleavage

The incidence of in vitro fertilization was analyzed with respect to the degree of cumulus dissociation (expansion) at the time of oocyte recovery and also the semen quality. Of the oocytes surrounded by perfectly ("++") or moderately ("+") dissociated cumuli, 78.6% and 30.8%, respectively (P less than 0.001), were fertilized when the husband's semen analysis was in the normal range. The proportion of fertilized oocytes was not decreased in cases of polyzoospermia (greater than 130 X 10(6) spermatozoa/ml), but was decreased (P less than 0.05) when the semen analysis revealed other anomalies: oligozoospermia (less than 15 X 10(6) spermatozoa/ml), asthenozoospermia (less than 50% motile cells) or teratozoospermia (greater than 50% abnormal spermatozoa). The proportion of fertilized eggs cleaving in vitro was unaffected by semen quality but was lower when "+" cumulus oocytes were collected than when "++" cumulus oocytes were obtained (58.3% vs. 87.0%, P less than 0.02). In vitro incubation of the oocyte prior to insemination increased the incidence of fertilization by about 28% for both "+" (22.2 to 50.0%) and "++" (65.7 to 93.9%) cumulus oocytes. Finally, 67.6% of "++" cumulus oocytes developed into embryos when the insemination with spermatozoa from normal semen samples was delayed by several hours, compared with only 29.0% when the conditions were suboptimal ("+" cumulus oocyte, abnormal semen analysis or no delay prior to insemination). Eight pregnancies began following the replacement of 38 embryos in 34 patients. Six spontaneous abortions occurred, and chromosomal abnormalities were proven in the two cases analyzed. Two pregnancies continued for more than 3 months, resulting in term deliveries of two normal babies.     

Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes

The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm^?1 AC followed by a 30 μsec pulse at 120 V mm^?1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo-matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88–100%) and polyspermic rates (79–100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 ± 0.5 vs. 4.6 ± 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. © 1993 Wiley-Liss, Inc.     

Nuclear and Cytoplasmic Maturation of Bovine Oocytes Cultured with dbc AMP,FSH AND hCG

The present investigation was undertaken to study the effect of addition of dbc AMP on bovine oocyte maturation and fertilization in vitro. The bovine oocytes isolated from 2–8 mm follicles were cultured for 26 h in TCM-199. The maturation rate (71.4 %) did not significantly increase after supplementation of the culture medium with dbc AMP (86.3 %.) or FSH + hCG (86.3 %). The in vitro fertilization rate of oocytes based on sperm penetration and presence of sperm tail in the ooplasm increased significantly in the dbc AMP (34.7 %) and the dbc AMP + FSH + hCG (33.9 %) treated groups when compared with untreated controls (17.9 %). However, dbc AMP treated oocytes were not able to secure the formation of male pronucleus 20 h after in vitro fertilization, while in oocytes matured in dbc AMP free medium both pronuclei were present in approximately 15 % of the penetrated oocytes. Also, the sperm head decondensation was blocked or slowed down by the dbc AMP treatment. It is concluded (1) that dbc AMP may improve the condition for the interaction of oocytes with spermatozoa, and (2) that the ooplasm of such dbc AMP treated oocytes apparently is not able to decandense the sperm head and transform it to the male pronucleus.     

Comparison of culture and insemination techniques for equine oocyte transfer

This study was designed to test 3 approaches for insemination and transfer of oocytes to recipient mares. Oocytes were recovered transvaginally from naturally cycling donor mares 24 to 26 h after an intravenous injection of 2500 IU of hCG when follicles reached 35 mm in diameter. Multiple oocytes (1 to 4) were transferred surgically into the oviducts of 4 or 5 recipient mares per group. Three groups of transfers were compared: 1) transfer of oocytes cultured in vitro for 12 to 14 h postcollection with insemination of the recipient 2 h postsurgery; 2) transfer of oocytes into the oviduct within 1 h of collection, with completion of oocyte maturation occurring within the oviduct, and insemination of the recipient 14 to 16 h postsurgery; and 3) transfer of spermatozoa and oocytes (cultured 12 to 14 h in vitro) into the oviduct. Numbers of embryos detected by Day 16 of gestation were not different (P>0. 1) for groups 1, 2, and 3 (57%, 43% and 27%). Therefore, equine oocytes successfully completed the final stages of maturation within the oviduct, and sperm deposited within the oviduct were capable of fertilizing oocytes.     

In vitro fertilization with normal development in the sheep

Ovine tubal (n = 87) and ovarian in vitro matured oocytes (n = 99) were fertilized in vitro with ejaculated spermatozoa capacitated for 8 h in modified defined medium buffered with Hepes. High levels of fertilization were obtained as assessed by development to two-to six-cell stage within 40 h (75. 8% for ovulated and 62. 6% for in vitro matured oocytes). Electron microscope analysis of oocytes 20–22 h after insemination indicated that in vitro fertilization approximated the in vivo events. Embryos (two- to six-cell) were transferred surgically to the oviducts of pseudopregnant rabbits. Three days later, 42 (from ovulated oocytes) and 15 (from in vitro matured oocytes) embryos were recovered; 26 (61. 9%) and 10 (66. 6%), respectively, had cleaved at least once. Embryos incubated in vivo (n = 20 from ovulated oocytes; n = 9 from in vitro matured oocytes) were transferred surgically to the uteri of seven and four recipient ewes resulting in four and two pregnancies, respectively, from which three and one, respectively, have been maintained ( > 3 months). The first lamb resulting from the in vitro fertilization of an ovulated oocyte was born. In addition, six embryos (two- to four-cell) from tubal oocytes and ten embryos (two- to six-cell) from in vitro matured oocytes were directly transferred to the oviducts of two and three ewes, respectively. Two pregnancies resulting from in vitro matured fertilized oocytes are in progress ( > 3 months).     

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.     

The difference in embryo quality between Belclare and Suffolk ewes is not due to differences in oocyte quality

We have previously reported that the percentage of fertilized oocytes which reached the blastocyst stage by Day 6 after AI with frozen-thawed semen was higher for Belclare (94%) than Suffolk (59%) ewes. This may reflect differences in the timing of fertilization (Experiment 1) or differences in oocyte quality (Experiments 2 and 3). In Experiment 1, oocytes recovered from slaughterhouse ovaries were matured in vitro for 18, 20, 24, 28 or 30 h prior to fertilization and were then cultured in vitro. In Experiment 2, Belclare (n = 69) and Suffolk (n = 71) ewes were laparoscopically inseminated using frozen-thawed semen. Presumptive zygotes were recovered between 23 and 47 h post-insemination and cultured in vitro (grouped by breed). In Experiment 3, immature oocytes from Suffolk and Belclare ewes, were matured, fertilized and cultured in vitro (grouped by breed). Cleavage rate and blastocyst development was assessed. There was no effect of time of fertilization on cleavage rate, however, a lower proportion of cleaved oocytes reached the blastocyst stage after insemination at 30h compared to 24 h (P < 0.001). Ewe breed did not affect cleavage rate of oocytes matured and fertilized in vivo (41+/-9.6 and 47+/-10.1) or in vitro (47+/-9.4 and 52+/-9.4) for Belclare and Suffolk ewes, respectively (P > 0.05; %+/-S.E.). Likewise, ewe breed had no effect on the percentage (+/-S.E.) of cleaved oocytes developing to the blastocyst stage for in vivo (29+/-7.2 and 25+/-7.9) or in vitro matured and fertilized oocytes (29+/-6.1 and 36+/-5.9) from Belclare and Suffolk ewes, respectively (P>0.05). Based on this study oocyte quality does not differ between the breeds and in addition a 4h difference in the timing of fertilization, reflective of the breed difference in the timing of the LH surge in vivo, would not affect early embryo development.