Use of a neuroleptic in assisted reproduction of the critically endangered Mohor gazelle (Gazella dama mhorr)

Stress is a limiting factor in assisted reproduction in wild animals maintained in captivity and measures to reduce it should improve reproductive success. The effect of the long-acting neuroleptic (LAN) perphenazine enanthate was assessed on ovarian stimulation for the recovery of immature oocytes from Mohor gazelle (Gazella dama mhorr) and their subsequent in vitro maturation, fertilization and embryo culture. The viability of embryos after transfer was also examined. Perphenazine enanthate decreased activity levels and facilitated handling of treated animals when compared to controls. LAN-treated animals showed a more regular pattern of respiratory and heart rates and body temperature than controls; no major differences were found in hematological and biochemical parameters between groups. Perphenazine-treated females had lower plasma cortisol levels during the days of intense handling. No significant differences were found in the number of punctured follicles and recovered oocytes between groups. The percentage of mature oocytes per female was significantly higher in the LAN-group. Fertilization and cleavage rates were not significantly different between groups. Embryos developed in culture but none reached the blastocyst stage, and those transferred to the oviduct of synchronized recipients did not develop to term. In conclusion, treatment of females with perphenazine enanthate during ovarian stimulation did not have negative effects on maturation, fertilization and embryo development in vitro. Moreover, an increase in oocyte maturation rate per female was observed. Thus, the use of LANs could be useful to alleviate the effects of handling-stress during assisted reproductive procedures in wild ungulates.     

The endocannabinoid system modulates the ovarian physiology and its activation can improve in vitro oocyte maturation

The cannabinoid (CB) system has been involved in many aspects of reproduction and it is known that the systemic chronic use of exogenous CBs are deleterious to reproductive processes. Even so, it is not known what happens in relation to the physiology of the ovary when CB receptors are absent. The present study investigated the effect of the lack of CB1 and CB2 receptors in mice ovarian morphology, folliculogenesis, oocyte retrieval, and oocyte maturation and evaluated the use of Δ9-tetrahydrocannabinol (THC) on oocyte in vitro maturation (IVM) by comparing classical IVM and two-step IVM by analyzing the meiotic competence of the oocytes and their evolution toward embryos. Thus, when CB1 and CB2 receptors were missed, the ovary area and volume was significantly less and the action of the equine chorionic gonadotropin (eCG) hormone was diminished. In addition, the mutant genotypes had fewer ovarian follicles and they were less competent after eCG administration compared with wild-type mice, and this lack of CB receptors showed a mismatch of oocyte maturation. However, the in vitro use of THC showed improvements in oocytes IVM after a Pre-IVM step for 48 hr, as those oocytes reached a significantly higher polar body rate, a larger diameter and the best result on blastocysts rate was achieved when THC was used during the IVM step.     

In vitro oocyte maturation and preantral follicle culture from the luteal-phase baboon ovary produce mature oocytes

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.     

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.     

Current progress on assisted reproduction in dogs and cats: in vitro embryo production

The objective of the development of assisted reproduction techniques in dogs and cats is their application to non-domestic canine and feline species, most of which are considered threatened or endangered. Among these techniques, an entirely in vitro system for embryo production is effectively an important tool for conservation of wildlife. In the last decade, progress has been made in embryo production in carnivores. It has been shown that canine oocytes can resume meiosis in vitro and that these oocytes can be fertilized and developed in vitro, although at a much lower rate than most other domestic animal oocytes. The reason lies in the dissimilarities of reproductive physiology of the dog compared to other species and the lack of precise information concerning the oviductal environment, in which oocyte maturation, fertilization and early embryonic development take place. Successful in vitro embryo production in the domestic cat has been attained with oocytes matured in vitro, and kittens were born after transfer of IVM/IVF derived embryos. On the basis of these results the in vitro fertilization of oocytes has also been applied in several non-domestic feline species. The effectiveness of such protocols in the preservation of genetic material of rare species can be improved by developing better techniques for long-term storage of gametes. In dogs and cats sperm cells have been successfully frozen and the cryopreservation of oocytes would greatly increase their availability for a range of reproductive technologies. Cryopreserved cat oocytes can be fertilized successfully and their development in vitro after fertilization is enhanced when mature oocytes are frozen. Thus refined techniques of oocyte maturation and fertilization in vitro coupled with oocyte cryopreservation could allow for an easy establishment of genetic combinations when male and female gametes in the desired combination are not simultaneously available, and the propagation of endangered carnivores would be facilitated.     

Effect of vitrification on human oocyte maturation rate during in vitro maturation procedure: A systematic review and meta-analysis

Combination of in vitro maturation (IVM) and cryopreservation offers new opportunities for women with contraindication in ovarian stimulation, and females who desire to postpone the childbearing due to different problems. There are still controversies regarding IVM procedure and its impact on oocytes fertilization capability. This systematic review and meta-analysis were conducted to evaluate the impact of vitrification on human oocyte maturation rate during IVM procedure. In this review, we searched Medline, Embase, Scopus and ISI web of science to identify English-language studies. The last search was implemented on 3 February 2018. The original articles which assessed maturation rate after vitrification of MI or GV oocytes were included. Animal trials and the studies that performed cryopreservation using slow-freeze method were excluded. Bias and quality assessments were performed. 2476 articles were screened primarily. After duplication removing and the application of inclusion and exclusion criteria, 14 studies included for the analysis. All studies compared maturation rate between the oocytes that were vitrified at the GV or MI stage before maturation and oocytes which were matured in vitro without vitrification. Meta-analysis showed that oocyte vitrification at GV stage had a significant negative impact on maturation rate (RR = 0.76, 95% CI: 0.66–0.88); I² = 85.2%; P = 0.000). Finally, based on our results, oocyte vitrification decreases the maturation rate by 24%.     

Effects of in vitro maturation of monkey oocytes on their developmental capacity

The study of in vitro maturation (IVM) of rhesus monkey oocytes has important implications for biomedical research and human infertility treatment. In vitro-matured rhesus monkey oocytes show much less developmental potential than IVM oocytes of other species. Since about 1980 when rhesus monkey IVM, in vitro fertilization (IVF) and in vitro embryo culture (IVC) systems were established, numerous efforts have been made to improve the developmental competence of oocytes and to understand the mechanisms regulating oocyte maturation. This review describes recent progress in this area, particularly the effects of factors such as steroid hormones, energy substrates, amino acids, ovarian follicle status, maternal age and breeding season on the developmental competence, gene expression patterns and genome integrity of rhesus IVM oocytes.     

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.     

In vitro maturation of human primordial ovarian follicles: clinical significance, progress in mammals, and methods for growth evaluation

As cancer treatment improves, more young women of reproductive age are surviving, but they suffer from infertility as a consequence of the radiation and chemotherapy. Human ovarian tissue containing immature primordial follicles has been successfully cryopreserved. The ultimate aim of this technique is to induce ovarian function by re-plantation of ovarian tissue or, further into the future, by in vitro maturation (IVM) of the oocytes derived from the cryopreserved-thawed ovarian tissue, followed by routine in vitro fertilization. IVM of primordial follicles from young cancer survivors would avoid the risk of cancer re-transmission by the ovarian grafts. The present review discusses the current achievements in IVM of female germ cells and primordial ovarian follicles and the attempts to improve their development by adding various factors to the culture medium. The established methods for the evaluation of survival and growth in culture are also discussed: follicular counts, immunocytochemical methods, transmission electron microscopy, fluorescent viability markers and endocrine assays. Although the development of IVM systems is still in its infancy, researchers need to pursue their approach step-by-step, especially with regard to factors that might be involved in the activation of the ovarian follicles or female germ cells. The final measure of success will be the ability of the in vitro matured oocytes to fertilize and produce healthy offsprings. The availability of such treatment will probably lead to its demand not only by cancer patients but by other women as well.     

Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey

Somatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 degrees C) without CO(2) supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.     

Analysis of different factors influencing the intracytoplasmic sperm injection (ICSI) yield in pigs

Intracytoplasmic sperm injection (ICSI) in pigs is a technique with potential application in diverse fields of animal production and biomedicine. Even though there are some cases of live offspring resulting from this technique, its yield is still quite low compared to other species. The aim of this study was to evaluate different factors affecting the ICSI performance. This was done by studying (1) the sequence of culture media for the oocytes after injection; (2) modifications in the in vitro maturation system (IVM) through meiotic inhibitors such as roscovitine, and changes in the IVM time; (3) oocyte activation through injection of inositol triphosphate (InsP(3)) together with the sperm. In vitro matured oocytes were employed. All the ICSI experiments were performed with fresh ejaculated semen. Results showed that porcine ICSI zygotes give an improved proportion of two-cell embryos using the sequence IVF medium-embryo culture medium (NCSU-23) rather than transferring directly to NCSU-23. Pronuclear formation ability was not affected by prematuration, but a faster embryo development was observed in roscovitine treated oocytes. In relation to IVM times, oocytes matured for 36 h can achieve better fertilization percentages than oocytes matured for 44 h. These results were independent of the roscovitine treatment. Finally, no influence on embryo development was observed until the blastocyst stage with the use of the InsP(3) as an exogenous activating factor.     

The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol, cysteine and cystine

Supplementation of IVM medium with cysteamine, beta-mercaptoethanol, cysteine and cystine induced bovine oocyte glutathione (GSH) synthesis, but only the effect of cysteamine on the developmental competence of these oocytes was tested. During IVM of sheep oocytes, cysteamine but not beta-mercaptoethanol increased embryo development. However, it is not known how long the high intracellular oocyte GSH levels obtained after IVM with thiol compounds, can be maintained. Thus, the present study was carried out to evaluate the effects of supplementing maturation medium with 100 microM beta-mercaptoethanol, 0.6 mM cysteine and 0.6 mM cystine on 1) intracellular GSH level after IVM, 2) after IVF, 3) in 6 to 8-cell embryos and 4) on embryo development. In oocytes after IVM and in presumptive zygotes after IVF, intracellular GSH levels were significantly higher in the treated groups (P < 0.05). While, GSH content in 6 to 8-cell embryos was similar among treatment groups (P > 0.05). Differences in cleavage rates and the percentage of embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) for treated oocytes than for those matured in the control medium. We conclude from the results that the high intracellular GSH levels after induction of GSH synthesis in bovine IVM by thiol compounds remain during IVF and are still present at the beginning of IVC, improving developmental rates. Moreover, the results indicate that this metabolic pathway is an important component of the cytoplasmic maturation process that affects the subsequent steps of in vitro embryo production.     

Estradiol and its membrane-impermeable conjugate (estradiol-bovine serum albumin) during in vitro maturation of bovine oocytes: effects on nuclear and cytoplasmic maturation, cytoskeleton, and embryo quality

In various cell types, there is increasing evidence for nongenomic steroid effects, i.e., effects that are not mediated via the classical steroid receptors. However, little is known about the involvement of the nongenomic pathway of estradiol (E2) on mammalian oocyte in vitro maturation (IVM). The aim of this study was to investigate whether the effects of E2 on bovine oocyte IVM are mediated via a plasma membrane receptor (nongenomic). First, we investigated the expression of estradiol (classical) receptor alpha (ERalpha) and beta (ERbeta) mRNA in oocytes and cumulus cells (CC). We also studied the effects of different exposure times to E2 (before and after germinal vesicle breakdown, GVBD) on nuclear maturation. To study the possible involvement of the putative estradiol plasma membrane receptor on the IVM of oocytes, we used E2 conjugated with bovine serum albumin (E2-BSA), which cannot cross the plasma membranes. Our results demonstrate that oocytes expressed ERbeta mRNA, while CC expressed both ERalpha and ERbeta mRNA. Exposure to E2 during the first 8 h of culture (before GVBD) induced a block at the metaphase I stage (MI). However, the presence of E2 after GVBD induced an increase of oocytes with nuclear aberrations. Meiotic spindle organization was severely affected by E2 during IVM and multipolar spindle was the most frequently observed aberration. Exposure of oocytes to E2-BSA did not affect nuclear maturation, blastocyst formation rate, nor embryo quality. Our results suggest that the detrimental effects of E2 on in vitro nuclear maturation of bovine oocyte are not exerted via a plasma membrane receptor.     

Xenogenous fertilization of equine oocytes following recovery from slaughterhouse ovaries and in vitro maturation

The in vitro production (IVP) of equine embryos using currently available protocols has met limited success; therefore investigations into alternative approaches to IVP are justified. The objective of this study was to evaluate the feasibility of xenogenous fertilization and early embryo development of in vitro matured (IVM) equine oocytes. Follicular aspirations followed by slicing of ovarian tissue were performed on 202 equine ovaries obtained from an abattoir. A total of 667 oocytes (3.3 per ovary) were recovered from 1023 follicles (recovery rate, 65%). Oocytes underwent IVM for 41 +/- 2 h (mean +/- S.D.), before being subjected to xenogenous gamete intrafallopian transfer (XGIFT). An average of 13 +/- 0.8 oocytes and 40x10(3) spermatozoa per oocyte were transferred into 20 oviducts of ewes. Fourteen percent of transferred oocytes (36/259) were recovered between 2 and 7 days post-XGIFT and 36% of those recovered displayed embryonic development ranging from the 2-cell to the blastocyst stage. Fertilization following XGIFT was also demonstrated by the detection of zinc finger protein Y (ZFY) loci. Ligation of the uterotubal junction (UTJ), ovarian structures, or the duration of oviductal incubation did not significantly affect the frequency of embryonic development or recovery of oocytes/embryos after XGIFT. In conclusion, equine embryos can be produced in a smaller non-equine species that is easier for handling.     

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.     

Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development

alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.     

Influences of zinc on fertilisation and development of bovine oocytes in vitro.

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 micrograms added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 micrograms zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 micrograms added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 micrograms per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 microgram zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 microgram/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 microgram ml of zinc in vitro than in vivo because a concentration of 3.0 micrograms/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 micrograms/ml of zinc; however, higher concentrations were inhibitory.     

Effect of oocyte diameter on meiotic competence, embryo development, p34 (cdc2) expression and MPF activity in prepubertal goat oocytes

The aim of this study was to analyze the relationship between oocyte diameter, meiotic and embryo developmental competence and the expression of the catalytic subunit of MPF, the p34(cdc2), at mRNA, RNA and protein level, as well as its kinase activity, in prepubertal (1-2 months old) goat oocytes. MPF is the main meiotic regulator and a possible regulator of cytoplasmic maturation; therefore, it could be a key factor in understanding the differences between competent and incompetent oocytes. Oocytes were classified according to oocyte diameter in four categories: <110, 110-125, 125-135 and >135 microm and matured, fertilized and cultured in vitro. The p34(cdc2) was analyzed in oocytes at the time of collection (0 h) and after 27 h of IVM (27 h) in each of the oocyte diameter categories. The oocyte diameter was positively related to the percentage of oocytes at MII after IVM (0, 20.7, 58 and 78%, respectively) and the percentage of blastocysts obtained at 8 days postinsemination (0, 0, 1.95 and 12.5%, respectively). The expression of RNA and mRNA p34(cdc2) did not vary between oocyte diameters at 0 and 27h. Protein expression of p34(cdc2) increased in each oocyte category after 27 h of maturation. MPF activity among diameter groups did not vary at 0h but after IVM there was a clear and statistically significant increase of MPF activity in the biggest oocytes.