Characterization of the maturational changes induced by a GnRH analogue in the rat ovarian follicle

The GnRH analogue [D-Ser(t-Bu)6]des-Gly10-GnRH-N-ethylamide (GnRHa, 2 micrograms/rat) or hCG (4 i.u./rat) was administered to hypophysectomized, PMSG-primed immature female rats. Oocyte maturation was initially detected by 2 h after GnRHa administration but the response to hCG was observed only after 4 h. Initiation of GnRHa-induced ovulation also preceded the response to hCG by 2 h. Maximal response to both these hormones was obtained at 10 and 14 h after hormone administration for oocyte maturation and ovulation respectively. The number of oocytes ovulated after GnRHa was significantly lower than that with hCG (29 +/- 4 and 50 +/- 7 per rat respectively; P less than 0.05). Expansion of the cumulus mass and secretion of mucoid material, which are characteristic responses to LH, were also observed after GnRHa administration. However, while the action of 5 micrograms ovine LH/ml on the cumulus cells was mediated by cAMP, no accumulation of the nucleotide could be detected in follicles exposed to GnRHa (10(-7) M). We conclude that even though GnRHa and LH/hCG seem to elicit similar responses in the ovarian follicle they differ in their kinetics, their efficiency and the mediator of their action.     

Embryonic development of fertilized rat oocytes induced to mature by an analogue of gonadotrophin-releasing hormone

A gonadotrophin-releasing hormone analogue (GnRHa) was administered to hypophysectomized immature rats. Postovulatory mature oocytes obtained under these conditions were exposed in vitro to a sperm suspension for fertilization. Developmental ability of the fertilized ova was studied by transfer of the 2-cell stage embryos to oviducts of foster mothers. The potential of oocytes, undergoing maturation in response to GnRHa, to develop into 2-cell embryos was similar to that of oocytes stimulated by hCG (76.4% and 83.1% respectively). The 2-cell stage embryos obtained from such oocytes were equally able to implant in the uteri of foster mothers (25.7% and 21.2% respectively) and subsequently develop into live embryos (15.3% and 15.2%, respectively, at Day 20 of pregnancy).     

In vitro maturation and fertilization of bovine oocytes and in vitro culture of presumptive zygotes in the presence of bovine pestivirus

A pathogen which has been shown to commonly contaminate in vitro bovine embryo production system is bovine pestivirus (bovine viral diarrhea virus). Three experiments were designed to evaluate the in vitro maturation (experiment I), fertilization (experiment II) and embryo development (experiment III) of immature oocytes, inseminated oocytes and presumptive zygotes in the presence of a bovine pestivirus (non-cytopathic, nCP type 1). The virus inoculum used was derived from a persistently infected cow. In experiment I, follicular oocytes (n=1257) recovered from slaughterhouse derived ovaries were randomly assigned to either a control group (n=578) which did not become exposed to bovine pestivirus and a treatment group (n=679) which was inoculated with bovine pestivirus (2.20-3.69 log(10) TCID(50)/50 microl) at the time of commencement of in vitro maturation. Overall, there was no significant difference between the control and pestivirus inoculated oocytes in either the cumulus cell expansion rate (79+/-7.5% versus 74+/-10.7%) or the nuclear maturation rate (89+/-4.8% versus 85+/-7.4%), respectively. In experiment II, in vitro matured oocytes (n=607) were inseminated either in the absence (control; n=301) or the presence of bovine pestivirus (4-4.6 log(10) TCID(50)/50 microl; n=306). A significant (P<0.01) reduction in the overall number of fertilized oocytes with two well formed male and female pronuclei was observed in the treatment group compared to the control group (58.5+/-5.8% versus 73.3+/-3.6%, respectively). In experiment III, after in vitro maturation and fertilization, presumptive zygotes were randomly assigned to either a control group (n=139) which was not exposed to bovine pestivirus or a treatment group which was inoculated with bovine pestivirus (2.97-4.47 log(10) TCID(50)/30 microl; n=139). The zygotes were then cultured under mineral oil in an atmosphere of 88% N(2), 7% O(2) and 5% CO(2) at 39 degrees C. The morphologic appearance of the embryos was assessed 48 h after the commencement of culture, and then every 48 h up to days 7-8 after insemination. The 22% (31/139) and 3.6% (5/139) of the presumptive zygotes developed to the morula or blastocyst stage in the control and the bovine pestivirus inoculated groups, respectively (P<0.001). This study demonstrates that bovine pestivirus has a significant detrimental effect on in vitro fertilization and early in vitro embryo development.     

Effects of LH and FSH on the maturation of pig oocytes in vitro

This research was designed to investigate the effects of LH and FSH (50 ng/ml) on pig oocyte maturation in vitro. The following parameters were studied: a) the degree of heterologous coupling between cumulus cells and oocytes, evaluated by measuring the (3)H-uridine and (3)H-choline uptake in cumulus enclosed oocytes; b) meiotic maturation; c) cytoplasmatic maturation, evaluated by analyzing the ability of the oocytes to promote male pronucleus formation after in vitro fertilization. Despite the marked cumuli expansion induced by gonadotropins, uridine uptake was not influenced by LH or FSH. By contrast, choline uptake in LH-treated oocytes was significantly higher than in FSH-treated or control oocytes (3199 cpm +/- 251 vs 1686 cpm +/- 142, P<0.01). Gonadotropins accelerated meiotic progression, and after 30 hours of culture the percentage of oocytes at the germinal vesicle stage was significantly lower (P<0.01) in LH-(24%, 24 102 ) and FSH-(20%, 18 90 ) treated oocytes than in control oocytes (76%, 64 84 ). After 44 hours of culture, the percentage of oocytes reaching the MII stage was significantly higher (P<0.01) in the presence of LH (76%, 92 120 ) and FSH (86%, 92 108 ) than in the controls (35%, 40 116 ). The percentage of oocytes capable of sustaining male pronucleus formation was similar in the control (48.4%, 63 132 ) and FSH-treated oocytes (44.3%, 51 116 ), while it was markedly increased (P<0.01) by the addition of LH (72.7%, 143 197 ). The data reported indicate that in vitro pig oocytes tend to undergo meiotic maturation even in the absence of hormones. However, in our in vitro system, LH and FSH accelerated and facilitated meiotic progression, and LH selectively improved cytoplasmic maturation which is required to promote the formation of a male pronucleus.     

Peritoneal fluid from rabbits or goats as media for in vitro maturation, fertilization and initial culture of caprine oocytes

The efficacy of peritoneal fluid from rabbit and goat for in vitro maturation, fertilization and initial culture of embryos from caprine oocytes was evaluated. Peritoneal fluid was collected from adult female goats (n = 9) or rabbits (n = 9). Good quality oocytes were subjected to in vitro maturation and fertilization in three different media viz. Tissue Culture Medium (TCM-199), goat Peritoneal Fluid (gPF) and rabbit Peritoneal fluid (rPF). Maturation rates were 74.7+/-2.07% and 63.6+/-5.28% in TCM-199, gPF 65.8+/-2.54% and 55.6+/-3.79%, and rPF 57.7+/-1.78% and 44.6+/-3.01% when evaluated on the basis of cumulus cell expansion and the achievement of metaphase-II stage, respectively. However, no significant differences were observed in respect of maturation rate between the control and gPF and between gPF and rPF groups. Freshly ejaculated buck semen was treated with heparin (10 microg/ml) and after 45 min incubation with heparin, 8.0% sperm were live and acrosome reacted. The proportions of fertilized oocytes based on male and female pronuclei formation or on cleavage development were 50.5+/-5.03, 42.3+/-3.15 and 34.2+/-1.98%; 31.0+/-2.80, 27.9+/-2.12 and 21.8+/-1.69% for TCM, gPF and rPF, respectively. It was concluded that peritoneal fluids either from goats or rabbits could be used as an alternative medium to TCM-199. However, further research is required to confirm its efficacy for embryo development up to the blastocyst stage.     

Live offspring by in vitro fertilization of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation and in vitro maturation

The objectives of the present study were to achieve 1) oocyte maturation, 2) oocyte competence of fertilization, and 3) oocyte competence of embryogenesis with oocytes from primordial follicles obtained from cryopreserved newborn mouse ovaries by using a two-step method. In the first step, frozen-thawed newborn mouse ovaries were transplanted under the kidney capsule of recipients for the initiation of growth from the primordial follicle stage on. In the second step, growing preantral follicles in the ovarian grafts were recovered and cultured. The results demonstrated that primordial follicles were able to be recruited to preantral follicles during the period of transplantation, and preantral follicles could be mechanically isolated from ovarian grafts. Under the present in vitro culture conditions, 85.8% of the isolated follicles (n = 332) from ovarian grafts survived the 12-day in vitro culture process, 84.9% of the recovered oocytes (n = 285) were germinal vesicle breakdown (GVBD)-competent, and 76% of the oocytes that underwent GVBD (n = 242) developed to the metaphase II (MII) stage. In the in vitro fertilization experiments, 75.4% of 142 inseminated MII oocytes underwent fertilization and cleavage to the 2-cell stage. Subsequently, 79.7% of the 2-cell-stage embryos (n = 69) progressed to the late morula-early blastocyst stage. Transfer of late morula-early blastocyst embryos resulted in the production of live offspring. From our experiments, it may be concluded that in vivo maturation by grafting followed by in vitro maturation of frozen-thawed primordial follicles can restore fertility in mice. This model could be useful for a similar application in the human.     

Oocyte recovery, maturation and fertilization in vitro in the puma (Felis concolor)

Eight female pumas were treated i.m. with 1000 (N = 5) or 2000 (N = 3) i.u. PMSG followed 84 h later by 800 i.u. hCG. Eggs were recovered 24-26 h after hCG from ovarian follicles by using laparoscopy and transabdominal aspiration. Mature eggs were inseminated in vitro 4-6 h later whereas immature eggs were cultured for 24 h and then inseminated. Electroejaculates from 3 pumas were diluted with mKRB before insemination to evaluate the influence of sperm concentration on fertilization. Seven of 8 pumas responded with follicle development, and 140 eggs were recovered from 145 follicles (96.6%; 77 mature, 43 immature, 20 degenerate eggs; mean +/- s.e.m., 20.0 +/- 5.9 eggs/female). Overall fertilization rate was 43.5% (total eggs fertilized = 40) despite using inseminates containing 82-99% pleiomorphic spermatozoa. Of the 36 immature oocytes matured in vitro and inseminated, 12 were fertilized even though 50% of the inseminating spermatozoa contained an acrosomal defect. Fertilization rate of mature oocytes collected from follicles appeared unrelated (P greater than 0.05) to PMSG dose or number of spermatozoa/inseminate. This study demonstrates that a high proportion of follicular eggs can be recovered laparoscopically from adult pumas treated with PMSG and hCG. These gametes are capable of being fertilized in vitro (immediately or after maturation in vitro) even with low quality semen with a high incidence of sperm pleiomorphisms.     

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.     

Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory LH surge

In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n = 417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n = 219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P < 0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 +/- 6.1 (SEM; n = 10 heifers) vs 26.4 +/- 1.0% (n = 2 replicates), and 39.1 +/- 5.1% vs 20.6 +/- 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences.     

In vitro oocyte maturation, fertilization and culture after ovum pick-up in an endangered gazelle (Gazella dama mhorr)

The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of biological material of great genetic value for the establishment of genetic resource banks of endangered species. Studies exist on sperm cryopreservation of endangered Mohor gazelle (Gazella dama mhorr), but no work has been carried out yet on oocyte collection, fertilization and culture in this or related species. The purpose of this study was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC in the Mohor gazelle using frozen-thawed spermatozoa. Ovum pick-up was performed after ovarian stimulation with a total dose of 5.28 mg of ovine FSH. A total of 35 oocytes were recovered from 56 punctured follicles (62%) (N=6 females). Out of 29 cumulus-oocyte complexes matured in vitro, 3% were found at germinal vesicle stage, 7% at metaphase I, 21% were degenerated, and 69% advanced to metaphase II. Fertilization and cleavage rates of matured oocytes were 40 and 30%, respectively. Embryos cleaved in vitro up to the 6-8 cell stage but none progressed to the blastocyst stage, suggesting the existence of a developmental block and the need to improve culture conditions. Although more studies are needed to improve hormonal stimulation and oocyte harvesting, as well as IVMFC conditions, this study demonstrates for the first time the feasibility of in vitro fertilization with frozen-thawed semen of in vitro matured oocytes collected by ovum pick-up from FSH-stimulated endangered gazelles.     

小鼠卵母细胞体外成熟、体外受精的效果观察

目的　研究不同培养条件对小鼠卵母细胞体外成熟及体外受精率的影响。方法　小鼠卵母细胞分别在含有FSH、BSA和胰岛素的培养液中体外成熟,在Whitten 氏液中体外受精,比较体外成熟率、体外受精率。结果　1- 裸卵(DO) 的体外成熟率、体外受精率(81-4% ,31-0 % ) 均高于卵丘卵母细胞复合体(COC)(48-6 % ,27-1% ) 。2- 在培养液中添加FSH、胰岛素和BSA,卵母细胞的体外成熟率为77-9 % ,82-3% 、60-7% ;体外受精率为77-2 % 、72-6 % 、26-7% ;2 - 细胞率为49-2 % 、34-2 % 、10-0% 。胰岛素组的卵母细胞IVM 率最高,但IVF率、2 - 细胞率低于FSH 组。3- 添加BSA的两组的体外受精率只有26-7 % 、25-8 % ,显著低于其他组,其体外成熟率也较添加FSH 和胰岛素的组成。4- 排出第一极体(PbI) 的卵母细胞的体外受精率和2 - 细胞率(85-9 % ,22-4% ) 均高于GV期卵母细胞(71-1 % ,12-9 % ) 。结论　1- 卵丘卵母细胞(COC) 较裸卵(DO) 的体外成熟率、体外受精率都低,差异显著(P成熟< 0-01;P受精< 0-05) 。2-FSH 和胰岛素均能提高小鼠卵母细胞的体外成熟率、体外受精率。3-BSA可以降低小鼠卵母细胞体外受精率,差异极显著。4-GV 期卵母细胞的体外受精率显著低于体外培养的排出第一极体的卵母细胞(P2 - cell < 0-05,P受精<0-05)     

Effects of follicle-stimulating hormone and ovarian steroids during vitro meiotic maturation on fertilization of rat oocytes

The present study examined the effects of gonadotropins and ovarian steroids during in vitro meiotic maturation of rat oocytes on their ability to undergo in vitro fertilization. Fully grown oocytes were isolated from antral follicles of immature rats and cultured as oocyte-cumulus cell complexes (OCC) under conditions in which completion of meiotic maturation occurs spontaneously. They were then exposed to spermatozoa under conditions in which oocytes matured in vivo exhibit high fertilization rates. Compared with oocytes from pregnant mare's serum gonadotropin (PMSG) or follicle-stimulating hormone (FSH)-treated rats, a simiiar proportion of the oocytes (>80%) from untreated rats underwent germinal vesicle breakdown, but such oocytes had a lower rate of fertilization (70% vs. 20%). The presence of FSH during in vitro maturation restored the fertilization rate for oocytes from untreated rats, while a cytochrome P450 inhibitor, aminoglutethimide phosphate abolished this beneficial effect of FSH. The addition of progesterone during the in vitro maturation period duplicated the beneficial effect of FSH on fertilization rate of oocytes from untreated rats; oestradiol-17β was less effective in this regard, and 5α-dihydrotestosterone was ineffective. These findings indicate that FSH and progesterone, although having no apparent effect on nuclear maturation of the oocyte, play an important role during oocyte maturation in enabling normal fertilization to occur.     

Superovulation and in vitro oocyte maturation in three species of mice (Mus musculus, Mus spretus and Mus spicilegus)

Mouse oocytes can be obtained via superovulation or using in vitro maturation although several factors, including genetic background, may affect response. Our previous studies have identified various mouse species as models to understand the role of sexual selection on the evolution of sperm traits and function. In order to do comparative studies of sperm-oocyte interaction, we sought reliable methods for oocyte superovulation and in vitro maturation in mature females of three mouse species (genus Mus). When 5IU pregnant mare's serum gonadotrophin (PMSG) and 5IU human chorionic gonadotrophin (hCG) were injected 48h apart, and oocytes collected 14h post-hCG, good responses were obtained in Mus musculus (18+/-1.3oocytes/female; mean+/-S.E.M.) and Mus spretus (12+/-0.8), but no ovulation was seen in Mus spicilegus. Changes in PMSG or hCG doses, or longer post-hCG intervals, did not improve results. Use of PMSG/luteinizing hormone (LH) resulted in good responses in M. musculus (19+/-1.2) and M. spretus (12+/-1.1) but not in M. spicilegus (5+/-0.9) with ovulation not increasing with higher LH doses. Follicular puncture 48h after PMSG followed by in vitro maturation led to a high oocyte yield in the three species (M. musculus, 23+/-0.9; M. spretus, 17+/-1.1; M. spicilegus, 10+/-0.9) with a consistently high maturation rates. In vitro fertilization of both superovulated and in vitro matured oocytes resulted in a high proportion of fertilization (range: 83-87%) in the three species. Thus, in vitro maturation led to high yields in all three species. These results will allow future studies on gamete interaction in these closely related species and the role of sexual selection in gamete compatibility.     

GnRH antagonist enhance follicular growth in FSH-treated sheep but affect developmental competence of oocytes collected by ovum pick-up

This study evaluates the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from sheep treated with GnRH antagonists (GnRHa) and high doses of FSH. Eighteen Sarda ewes were treated with progestagen sponges (day 0). On day 7, 10 ewes received 3 mg of GnRHa s.c., while 8 served as control receiving saline. On day 10, all animals were treated with 96 IU of ovine FSH in four equal doses given i.m. every 12 h. We monitored follicular development by ultrasonography, twice daily from day 7 to 11, and found that GnRHa induced a significant increase in the number of total follicles in 72 h (11.7+/-0.9 to 21+/-2.4, r(2)=0.598, P<0.0001), while this number remained stable in control sheep. We found that FSH induced a significant rise in the number of follicles in both groups; but always higher (P<0.05) in GnRHa treated sheep, confirming that GnRHa enhances ovarian response to exogenous FSH stimulation. Twelve hours after the last FSH dose, oocytes were collected by OPU. Recovery percentage, morphological quality, ability to resume meiosis, fertilization and cleavage were similar in oocytes from treated and untreated sheep. However, the final blastocysts output was lower in GnRHa group (10.1% versus 27.4% in control group; P<0.05). In addition, re-expansion rates after vitrification, thawing and in vitro culture were lower in GnRHa treated ewes, although differences did not reach statistical significance (55.5% versus 74.1% in GnRHa treated and in control sheep, respectively).     

In-vivo and in-vitro maturation rate of oocytes from two strains of mice

Female mice of the KE and CBA strains were used to examine the rate of oocyte maturation in vivo and in vitro. In CBA females killed just before ovulation most preovulatory oocytes were already in the metaphase II stage, while the oocytes of KE mice were arrested at metaphase I until the time of ovulation, and further stages of maturation occurred in the oviduct, reaching the metaphase II stage 3-5 h later. A similar strain difference in oocyte maturation rate was observed from in-vitro culture of cumulus-free oocytes, isolated from the ovaries of PMSG-primed females and intact females killed at the metoestrous phase of the cycle. This indicates that the strain-specific course of maturation is determined in the oocyte by a few days before ovulation. Therefore, if the rate of oocyte maturation is influenced by somatic components of the follicle, this must occur at some earlier stages of follicle development.     

Conditions for in vitro maturation and artificial activation of ferret oocytes

The ferret represents an attractive species for animal modeling of lung diseases because of the similarity between ferret and human lung biology and its relatively small size and short gestation time. In an effort to establish experimental protocols necessary for cloning ferrets, optimized conditions for in vitro maturation and artificial activation of ferret oocytes were examined. Cumulus-oocyte complexes were harvested from ovaries of superovulated ferrets, and in vitro maturation was evaluated in three different culture media: medium 1 (TCM-199 + 10% FBS), medium 2 (TCM-199 + 10% FBS with eCG [10 IU/ml] and hCG [5 IU/ml]), or medium 3 (TCM-199 + 10% FBS with eCG, hCG, and 17beta-estradiol [2 microg/ml]). After 24 h of maturation in vitro, the maturation rate of oocytes cultured in medium 2 (70%, n = 79) was significantly greater (P < 0.01) than those of oocytes cultured in the other two media (27%-36%, n = 67-73). At 48 h, similar maturation rates (56%-69%, n = 76-87) were observed for all three types of media. For activation experiments, oocytes cultured in medium 2 were stimulated with electrical and chemical stimuli either individually or in combination. Treatment with cycloheximide and 6-dimethylaminopurine (6-DMAP) following electrical stimulation resulted in 43% (n = 58) of the oocytes developing to the blastocyst stage. Such an activation rate represented a significant improvement over those obtainable under other tested conditions, including individual treatment with electrical pulses (10%, n = 41), cycloheximide (3%, n = 58), or 6-DMAP (5%, n = 59). Blastocysts derived from in vitro activation appeared to be normal morphologically and were composed of an appropriate number of both inner cell mass (mean +/- SEM, 10.3 +/- 1.1; n = 11) and trophectoderm (60.8 +/- 2.9, n = 11) cells. These results have begun to elucidate parameters important for animal modeling and cloning with ferrets.     

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

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

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.     

The equine oocyte: Factors affecting meiotic and developmental competence

There is currently much interest in assisted reproduction techniques in the horse, however, many aspects of oocyte maturation, fertilization, and embryo development in the horse differ from those in other species. Because of the close attachment of the equine oocyte to the follicle wall, scraping of the follicle is the most effective method for oocyte recovery. A notable feature of equine oocytes is that those with expanded cumuli (Ex oocytes), which originate from atretic follicles, have higher meiotic competence (ability to mature to metaphase II in vitro) than do oocytes with compact cumuli (Cp oocytes). Cp oocytes originate in viable follicles but are largely juvenile. Recovery and culture of equine oocytes immediately after slaughter yields a higher maturation rate than that obtained from oocytes after ovary storage; this is related to damage to chromatin in Cp oocytes during storage. In contrast, developmental competence (rate of blastocyst development in vitro) is higher in oocytes recovered from the ovary after a delay. The optimum duration of maturation varies based on cumulus morphology and time of recovery from the ovary, but there is no difference in developmental competence between Ex and Cp oocytes. Because standard in vitro fertilization is not repeatable in the horse, oocyte transfer (surgical transfer of oocytes to the oviducts of inseminated mares) has been developed to allow fertilization of isolated oocytes. Fertilization in vitro may be achieved using intracytoplasmic sperm injection; culture of injected oocytes in a medium with high glucose can yield over 30% blastocyst development. Mol. Reprod. Dev. 77: 651–661, 2010. © 2010 Wiley‐Liss, Inc.     

Preliminary results on variability in oocyte recovery and developmental competence in cattle derived from embryonic cloning: work in progress

To investigate female gamete developmental competence and variability in cloned cattle, we performed ovum pick-up and in vitro fertilization in four sets of cloned heifers (n = 10, two sets of triplets and two sets of twins), and four groups of non-genetically related control animals (n = 13). A total of 304 OPU were performed and 1798 oocytes were recovered. Mean oocyte production per female per OPU (+/-S.D.) was similar for clone or control animals (5.7+/-2.9 versus 6.1+/-4.5, respectively), however, in two sets of clones variance for the number of oocytes recovered differed significantly (7.1 versus 23.9 and 7.3 versus 26.7, respectively P<0.001) between clone groups and their respective controls, cloned animals being more homogenous. After in vitro maturation, fertilization with semen from the same bull, and culture, the proportion of oocytes from cloned animals that developed into blastocysts was 35.0+/-29.2% and was not significantly different from controls (29.4+/-30.9). The CV for oocyte recovery, and blastocyst rates was lower in all groups of cloned animals than in controls. Nevertheless, within each set of clones, CV values indicated some degree of variability between animals, thus confirming that cloned cattle are not the exact phenotypic copy of each other. Despite the large number of oocytes analyzed, results should be interpreted with caution due to the limited number of cloned animals.