In vitro developmental competence of buffalo oocytes collected at various stages of the estrous cycle

The objective was to determine the in vitro developmental competence of buffalo oocytes collected from abattoir-derived ovaries at various stages of the estrous cycle and follicular status. In Experiment 1, ovaries (n=476 pairs) were collected and divided into the following five groups: (a) ovaries with a corpus hemorragicum and no dominant follicle (CH-NO-DF); (b) ovaries with a mature functional corpus luteum (CL) and a dominant follicle (CL-DF); (c) ovaries with a mature functional CL and no dominant follicle (CL-NO-DF); (d) ovaries with a regressing CL and a dominant follicle (RCL-DF); and (e) ovaries without any luteal structures and only small follicles (ANEST). In Experiment 2, 144 pairs of ovaries with a CL (or regressing CL) and a dominant follicle were collected and follicles were classified as dominant, largest subordinate, and subordinate. In both experiments, the dominant follicle was defined as any follicle >10mm in diameter that exceeded the diameter of all other (subordinate) follicles. Although oocytes were collected from each group of ovaries, only Grades A or B oocytes were used for in vitro embryo production. Cleavage rates were higher (P<0.05) from oocytes collected from ovaries in the CH-NO-DF (59.6%) and CL-NO-DF (59.2%) groups than those collected from CL-DF (52.2%) and ANEST (43.6%) groups. The yield of transferable embryos was higher (P<0.05) from oocytes collected from CH-NO-DF (27.4%) and CL-NO-DF (24.0%) ovaries than from CL-DF (16.2%), RCL-DF (15.4%), and lowest (P<0.05) from ANEST (8.8%). In Experiment 2, oocytes from the dominant follicle had a higher (P<0.05) cleavage rate (65.2 %) and transferable embryo yield (30.2%) than those collected from the largest subordinate and subordinate follicles. In conclusion, oocyte competence depended on the morphofunctional state of ovaries. Oocyte development was maximal in pairs of ovaries with a corpus hemorragicum or CL and no dominant follicle; in paired ovaries with a CL and a dominant follicle, development was maximal in oocytes derived from the dominant follicle.     

Changes in ovarian, follicular, and oocyte morphology immediately after the onset of puberty are not accompanied by an increase in oocyte developmental competence in the pig

This study was conducted to determine whether ovarian morphology and developmental competence of in vitro-matured (IVM) oocytes is immediately affected by the onset of puberty in the pig. Ovaries of peri-pubertal pigs were sorted into two groups according to the presence or absence of corpora lutea presence (CL and NCL, respectively. Ovary dimensions, follicle diameter and number, and oocyte diameter (with and without zona pellucidae) were determined. The developmental competence of in vitro-matured oocytes from these two groups was evaluated following parthenogenetic activation and culture in vitro. CL ovaries were significantly (P<0.01) larger than NCL ovaries (width: 22.3+/-0.9 mm versus 15.9+/-0.4 mm, length: 33.2+/-1 mm versus 24.1+/-0.4 mm). Although CL ovaries had fewer antral follicles in total compared with NCL ovaries (21.1+/-1.8 mm versus 46.8+/-2.2 mm), they had a similar number of follicles 3-8mm in diameter. The mean diameter of follicles that were aspirated was greater for CL ovaries than for NCL ovaries (4.5+/-0.1 mm versus 3.3+/-0.02 mm). Oocytes from CL ovaries were greater in diameter compared with those from NCL ovaries (zona retained: 159+/-1.3 microm versus 146.1+/-1.5 microm, zona free: 124.7+/-1.8 microm versus 113.1+/-1.6 microm). No differences were found between oocytes from CL and NCL ovaries for rates of meiotic maturation (91.6+/-3.2% versus 92.4+/-3.2%), cleavage (88.4+/-11% versus 90.7+/-2.6%) and blastocyst formation (21.0+/-3.7% versus 23.7+/-5.7%). Therefore, the onset of puberty coincides with immediate changes in ovarian morphology, increased ovary size, follicle and oocyte diameter, but not with improved oocyte developmental competence. This suggests that the higher developmental competence usually observed in adult oocytes is acquired gradually and requires exposure to multiple estrus cycles.     

Effects of exogenous melatonin on in vivo embryo viability and oocyte competence of undernourished ewes after weaning during the seasonal anestrus

This study investigated the effects of exogenous melatonin on embryo viability and oocyte competence in post-partum undernourished ewes during the seasonal anestrus. At parturition (mid-Feb), 36 adult Rasa Aragonesa ewes were assigned to one of two groups: treated (+MEL) or not treated (−MEL) with a subcutaneous implant of melatonin (Melovine®, CEVA) on the day of lambing. After 45 d of suckling, lambs were weaned, ewes were synchronized using intravaginal pessaries, and fed to provide 1.5× (Control, C) or 0.5× (Low, L) times daily maintenance requirements. Thus, ewes were divided into four groups: C−MEL, C+MEL, L−MEL, and L+MEL. At estrus (Day=0), ewes were mated. At Day 5 after estrus, embryos were recovered by mid-ventral laparotomy and classified based on their developmental stage and morphology. After embryo collection, ovaries were recovered and oocytes were classified and selected for use in in vitro fertilization (IVF). Neither diet nor melatonin treatment had a significant effect on ovulation rate and on the number of ova recovered per ewe. Melatonin treatment significantly improved the number of fertilized embryos/corpus luteum (CL) (−MEL: 0.35 ± 0.1, +MEL: 0.62 ± 0.1; P = 0.08), number of viable embryos/CL (−MEL: 0.23 ± 0.1, +MEL: 0.62 ± 0.1; P < 0.01), viability rate (−MEL: 46.6%, +MEL: 83.9%; P < 0.05), and pregnancy rate (−MEL: 26.3%, +MEL: 76.5%; P < 0.05). In particular, exogenous melatonin improved embryo viability in undernourished ewes (L−MEL: 40%, L+MEL: 100%, P < 0.01). Neither nutrition nor exogenous melatonin treatments significantly influenced the competence of oocytes during IVF. Treatment groups did not differ significantly in the number of healthy oocytes used for IVF, number of cleaved embryos, or number of blastocysts and, consequently, the groups had similar cleavage and blastocyst rates. In conclusion, melatonin treatments improved ovine embryo viability during anestrus, particularly in undernourished post-partum ewes, although the effects of melatonin did not appear to be mediated at the oocyte competence level.     

Effect of the presence and location of corpus luteum on competence of bovine cumulus-oocyte complexes

This study aimed to determine the effect of presence of the corpus luteum (CL) and its influence on cumulus–oocyte complexes (COCs) obtained from the ipsilateral or contralateral ovary in bovine on the recovery and capacity of the oocytes to sustain mono-spermic fertilization, undergo preimplantation development, and develop to the blastocyst stage. Ovaries were collected at a local slaughterhouse and kept in pairs corresponding to the same animal. In the first experiment the variables evaluated were compared between cows with (CCL⁺) and without (CCL^-) CL, and for the second experiment, comparisons were made between ovaries with an ipsilateral (CL⁺), contralateral (CL⁻), and no (NCL). The recovery rate of COCs was higher in ovaries from CCL⁻ cows, and a higher proportion of grade 1 COCs were recovered from this group. A higher proportion of metaphase I oocytes at 7 h of maturation, and a higher rate of cleavage were observed in the CCL⁺ group; however, a higher proportion of embryos were obtained from the CCL⁻ group. Besides, COCs from the CL⁺ group had a lower proportion of grades 1 and 2 morphological qualities, lower rate of metaphase II oocytes at 22 h of maturation, and lower rate of formation of two pronuclei, whereas a higher proportion of unfertilized oocytes after in vitro fertilization. On the other hand, the COCs from the CL⁻ group displayed a lower proportion of oocytes with more than two pronuclei, higher cleavage rate, and higher final blastocyst production were obtained when compared to CL+. Thus, the effects of CL on the competence of bovine COCs are different depending on the anatomical proximity of their location in the animal, negatively affecting the quality of COCs located in the same ovary, but not having negative effects on the competence of COCs in the ovaries contralateral to their location.     

Seasonal effects on oocyte developmental competence in sows experiencing pregnancy loss

Recently, oocyte quality in sows culled for reasons unrelated to fertility was found to decline during the period of seasonal infertility. Wean-to-service interval (WSI) has also been associated with pregnancy loss in sows mated during the period of seasonal infertility. The aims of this study were to determine whether WSI and season are associated with changes in oocyte developmental competence in sows experiencing early (before Day 35 of gestation) and late (after Day 35 of gestation) pregnancy loss. Ovaries were collected in pairs from sows sourced from commercial piggeries that were culled for reasons related to infertility after being mated in summer and winter/spring. Sows were grouped according to their pregnancy loss type, their previous WSI and the presence or absence of corpora lutea (CL) on their ovaries. Oocyte developmental competence was assessed following in vitro maturation, artificial activation and parthenote development in vitro. In sows culled for early-pregnancy loss, there was a greater number of CL present on ovaries collected in spring compared to those collected in summer (11.57±3.3 vs. 9.26±0.99; P<0.05). Also, the proportion of oocytes developing to the blastocyst stage was greater in summer than in spring (55.9±5.2% vs. 31.2±6.4%; P<0.05). In sows culled for late-pregnancy loss, a greater proportion of oocytes developed to the blastocyst stage in winter compared with late-spring (64.3±7.0% vs. 34.1±6.6%; P<0.05). In addition, the blastocyst formation rate of oocytes was lower in sows that displayed a WSI≤6 days than in sows that displayed a WSI>6 days (37.8±7.3% vs. 62.2±6.9%; P<0.05). The results of the present study indicate that sows culled for pregnancy loss exhibit seasonal changes in oocyte developmental competence. The mechanism which causes WSI to be prolonged does not appear to result in reduced oocyte developmental competence. While poor oocyte quality and the mechanism that increases WSI may contribute to pregnancy loss during the seasonal infertility period, the findings suggest that these factors are not the main drivers of early and late pregnancy loss throughout the year.     

Transport of equine ovaries for assisted reproduction

Use of assisted reproduction to obtain foals from valuable mares post-mortem typically necessitates holding of ovaries during shipment to a laboratory. The present study evaluated whether holding ovaries briefly at a warm ( approximately 30 degrees C) temperature improves meiotic and developmental competence of oocytes, as determined after maturation in vitro and intracytoplasmic sperm injection. Ovaries were packaged in pairs in insulated containers, and held either at 24 or 25-35 degrees C for 4h, followed by cooling. Ovaries in both treatments were held for either a short (mean, 7-7.4h) or long (mean, 20.6-20.7h) duration before oocyte recovery. Control ovaries were collected en masse at the abattoir. The ovary temperature in this treatment slowly decreased to approximately 27 degrees C; oocyte recovery was performed after 3.5-7h total holding. There was no effect of temperature on oocyte meiotic or developmental competence within either treatment time period. Oocytes in the short duration holding group had similar meiotic competence to controls, but had a significantly decreased rate (P<0.05) of blastocyst development. Oocytes in the long duration holding group had decreased (P<0.05) meiotic competence and blastocyst development compared to controls. These findings indicate that storage of equine ovaries for only 7h may decrease blastocyst development, and that longer storage reduces both rate of oocyte maturation and blastocyst development. Further work is needed to determine if there is a critical time before 7h post-mortem by which equine oocytes should be recovered to maximize developmental competence.     

Disturbances of nuclear maturation in BCB positive oocytes collected from peri-pubertal gilts

The developmental competence (quality) of oocytes is affected by several factors linked to their intrinsic properties and also to growth and maturation environment. Donor puberty and chromosomal complement are one of the main factors influencing oocyte quality. A high rate of porcine oocytes matured in vitro is chromosomally imbalanced. Moreover, there is no published data on chromosomal aberrations in oocytes selected by the brilliant cresyl blue (BCB) test. Therefore, the aim of this study was to analyze whether BCB positive (BCB+) oocytes derived from ovaries of peripubertal gilts (prepubertal NCL and cyclic CL) differ with respect to the incidence of numerical chromosome aberrations. COCs collected from NCL and CL ovaries were selected by the BCB test. Only BCB+ oocytes were matured in vitro and subjected to FISH analysis using molecular probes for chromosome pairs 1 and 10. The rate of BCB+ oocytes was similar for both groups of ovaries (NCL 80%, CL 92%). Altogether 554 oocytes were fixed and 471 oocytes at the MII stage were analyzed cytogenetically. Diploid (2MII) and aneuploid oocytes were detected. The contribution of MII oocytes was similar for NCL (85%) and CL (90%) group. Chromosomally aberrant BCB+ oocytes accounted for 18.0% and ranged from 13.7% for CL and 22.2% for NCL ovaries. Diploidy was a predominant anomaly observed (11.2%) with a significantly higher frequency in BCB+ oocytes of pre-pubertal (16.7%) than cyclic gilts (5.6%, P < 0.05). Aneuploid oocytes occurred with similar rate in NCL (6.7%) and CL (8.5%) females. The majority of aneuploid spreads (72.2%; P < 0.01) concerned the chromosome pair 10. The overall rate of disomy (56%) and nullisomy (44.4%) was similar. We have shown that donor puberty affects the incidence of chromosomal abnormalities in porcine oocytes matured in vitro. Significantly more diploid oocytes was derived from prepubertal ovaries, whereas the frequency of aneuploidy was similar in NCL and CL gilts.     

Effects of oocyte quality, semen donor and embryo co-culture system on the efficiency of blastocyst production in goats

The aim of the study was to determine whether the selection of immature oocytes by a combination of cumulus-oocyte-complexes (COCs) morphology and staining with brilliant cresyl blue (BCB) would be helpful in selecting developmentally competent oocytes, and thereby increase the efficiency of blastocyst production from ovarian oocytes of FSH-primed, adult goats. In a second experiment the interaction between oocyte quality and semen donor was assessed. In a third experiment the usefulness of Vero cells for co-culture with goat embryos was investigated. In the pool of morphologically normal COCs recovered from ovaries following slicing (21.9+/-11.0), the mean rate of COCs classified as BCB+ was 85.6%, and the BCB- was approximately 11%. Oocytes classified as grade 1 and BCB+ exhibited the highest developmental competence (P<0.001) after in vitro maturation and fertilization compared with oocytes of grade 1 BCB- and grade 2 BCB+ or BCB-. There were no significant differences in developmental competence in grade 2 oocytes, regardless of BCB coloration. No significant differences in embryo cleavage and blastocyst formation rates among three bucks were observed when morphologically normal, BCB+ oocytes were used. For all tested bucks, differences in embryo production efficiency were related only to the oocyte quality. Similar blastocyst rates were developed from embryos co-cultured with goat oviduct epithelial cells (34.3%) and with Vero cells (33.3%). These results show that the most important criterion for selection of COCs before maturation is the visual assessment of morphological features. Staining with BCB of COCs recovered from adult goats does not enhance efficiency of selection of developmentally competent oocytes for IVF.     

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.     

Evaluation of activation treatments for blastocyst production and birth of viable calves following bovine intracytoplasmic sperm injection

The objective of this study was to compare the effectiveness of different methods of bovine oocyte activation following intracytoplasmic sperm injection (ICSI) in terms of oocyte cleavage and blastocyst rates, and calf production. Oocytes were harvested, post mortem, from the ovaries of Japanese Black heifers or cows. ICSI was carried out using a piezo-electric actuator. The injected or sham-injected oocytes that were assigned to three activation treatments, each replicated three times, were studied: (1) exposure to 5 microM ionomycin for 5 min (ionomycin); (2) exposure to 5 microM ionomycin for 5 min followed by culture in TCM199 for 3 h and a further 3h culture in 1.9 mM 6-dimethylaminopurine (DMAP-ionomycin+DMAP); (3) exposure to 7% ethanol in TCM199 for 5 min, 4 h after ICSI (ethanol). One or two blastocysts from the ionomycin+DMAP (8 recipients) and ethanol (17 recipients) oocyte activation treatments were non-surgically transferred into Holsteins for the study of calf production. The highest cleavage and blastocyst production rates were observed in the ionomycin+DMAP treatment (83.9% and 40.1%) by the ICSI. These rates were significantly (P<0.05) higher than those for the ionomycin oocyte activation treatment (57.6% and 18.2%) but did not differ from the ethanol treatment (75.6% and 29.4%). In the sham-injected, the highest blastocyst production rates were observed for the ionomycin+DMAP and ethanol treatments (10.7% and 11.3%). Pregnancy and birth rates for blastocysts derived from the ethanol oocyte activation treatment (58.8% and 47.4%) were significantly higher (P<0.05) than those of the ionomycin+DMAP treatment (12.5% and 9.2%). The results showed that post-ICSI oocyte activation with ethanol is more effective than activation with ionomycin alone or with ionomycin+DMAP for the production of viable blastocysts and calves.     

Effect of meiotic stages and maturation protocols on bovine oocyte's resistance to cryopreservation

We investigated the effect of meiotic stages and two maturation protocols on bovine oocyte's resistance to cryopreservation. Oocytes at germinal vesicle breakdown (GVBD) and metaphase II (MII) stage as well as oocytes matured for 22 h in media supplemented with FSH or LH were vitrified by the open pulled straw method. After warming, oocytes underwent additional 16 h (GVBD group) or 2 h (MII group) maturation. Then they were subjected to in vitro fertilization and culture. Some oocytes that matured in the medium supplemented with LH were subjected to parthenogenetic activation after vitrification to determine their developmental potential in absence of fertilization. Survival of oocytes after vitrifying/warming was determined after 22 h in fertilization medium. Cleavage and blastocyst formation rates were used to assess their developmental competence. In both experiments, a portion of unvitrified MII oocytes were subjected to in vitro fertilization and culture as control groups. In Experiment 1, similar cleavage rates were obtained for both GVBD and MII oocytes (53.56 versus 58.01%, P > 0.05). However, significantly higher proportion of cleaved embryos from vitrified MII oocytes developed into blastocysts than those from vitrified GVBD oocytes (1.06 versus 8.37%, respectively, P < 0.01). In Experiment 2, vitrified MII oocytes matured in medium supplemented with LH were superior to vitrified MII oocytes matured in FSH supplementation not only in cleavage rates (61.13 versus 50.33%), but in blastocyst formation rates (11.79 versus 5.19%, P < 0.01) as well. Cleavage and blastocyst formation rates of parthenogenetically activated oocytes were similar to those that were fertilized. Nevertheless, the vitrifying/ warming process significantly compromised the oocytes' developmental capacity since the vitrified oocytes showed significant reduction in both cleavage and blastocyst rates compared to those of not vitrified controls in both experiments (P < 0.01). We showed that oocytes at different maturation stages respond to cryopreservation differently and MII stage oocytes have better resistance to cryopreservation than GVBD stage oocytes. The maturation protocols also influence oocyte's ability to survive cryopreservation. Poor developmental potential after vitrification seem to have resulted from the cryodamage to the oocyte itself. These results suggested the importance of maturation on the developmental competence of cryopreserved oocytes.     

Comparisons between nulliparous heifers and cows as oocyte donors for embryo production in vitro

The aims of the present study were to compare (1) Holstein-Friesian heifers versus early postpartum lactating cows, and (2) different age categories of crossbred beef heifers versus cows, in terms of oocyte yield, morphological quality and developmental competence. Four experiments were designed to test the associated hypotheses. In Experiment 1, ovum pick up was carried out twice weekly for a period of 5 weeks on Holstein-Friesian heifers (n = 8) and early postpartum cows (n = 8). Oocytes were submitted to in vitro maturation (IVM), fertilization and culture. Significantly more follicles were punctured on the ovaries of heifers than cows (10.4 versus 7.8, P < 0.001). This was reflected in a significantly higher number of total oocytes (4.7 versus 2.8, P < 0.001) and grade 1-2 oocytes recovered/animal from heifers than from cows (3.0 versus 1.8, P < 0.05). There was no significant difference in the percentage of oocytes cleaving after fertilization, or in the percentage reaching the blastocyst stage between heifers and cows. In Experiment 2, oocytes were obtained by manual aspiration from the ovaries of slaughtered crossbred beef heifers (under 30 months, n = 1241) and cows (over 4 years old, n = 1125), and processed in vitro as above. No significant difference was observed between the two groups in terms of the number of aspirated follicles or oocytes recovered. A significantly higher proportion (P < 0.01) of cow oocytes than heifer oocytes reached the blastocyst stage (Day 8: 46.5% versus 33.4%). In Experiment 3, ovaries were separated according to age of heifer into three groups: (1) 12-18 months, (2) 19-24 months and (3) 25-30 months, and compared with cow oocytes. There was no significant difference in the blastocyst yield between the different age groups of heifers. Irrespective of heifer age, the blastocyst yield on Day 8 was significantly lower than that from cow oocytes (35.0, 35.2, 36.5 and 48.3%, respectively, P < 0.05). In Experiment 4, a significantly higher proportion (P < 0.001) of presumptive zygotes derived from abattoir-derived cow oocytes reached the blastocyst stage following culture in vivo in the ewe oviduct than those derived from heifer oocytes (Day 8: 53.1% versus 25.2% for cow and heifer oocytes, respectively). In conclusion, the origin of the oocyte has a significant impact on its subsequent developmental potential. These results would suggest that in an in vitro production system, cow oocytes should be preferentially used over those from heifers in order to maximize blastocyst development.     

Oocyte-secreted factors enhance oocyte developmental competence

The capacity of fully grown oocytes to regulate their own microenvironment by paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) may in turn contribute to oocyte developmental competence. Here, we investigated if OSFs have a direct influence on oocyte developmental competence during in vitro maturation (IVM). Bovine cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries and matured in serum-free medium. COCs were either co-cultured with denuded oocytes (DOs) or treated with specific OSFs: recombinant bone morphogenetic protein 15 (BMP15) and/or growth differentiation factor 9 (GDF9). Following maturation, embryos were fertilized and cultured in vitro and blastocyst development and cell number were assessed on day 8. Co-culturing intact COCs with DOs did not affect cleavage rate, but increased (P<0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination from 39% to 51%. OSFs also altered blastocyst cell allocation as co-culture of COCs with DOs significantly increased total and trophectoderm cell numbers, compared to control COCs. BMP15 alone, GDF9 alone or the two combined all (P<0.05) increased the proportion of oocytes that reached the blastocyst stage post-insemination from 41% (controls) to 58%, 50% and 55%, respectively. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which antagonize BMP15 and GDF9, respectively, neutralized the stimulatory effects of the exogenous growth factors and impaired the developmental competence of control COCs. These results demonstrate that OSFs, and particularly BMP15 and GDF9, enhance oocyte developmental competence and provide evidence that OSF regulation of the COC microenvironment is an important determinant of oocyte developmental programming.     

Effect of follicular size on meiotic and developmental competence of porcine oocytes

In several species, the developmental competence of the oocyte is acquired progressively during late follicular growth, after the acquisition of the competence to resume and complete meiosis. In the pig, full meiotic competence of the oocyte is reached in ovarian follicles with a diameter of 3 mm or more. However, there is no information about developmental competence acquisition. We analyzed the ability of oocytes from three foll icular size classes to resume and complete meiosis, to be fertilized, and to develop in vitro to the blastocyst stage. A total of 941 follicles were dissected from slaughterhouse gilt ovaries and classified as small (<3 mm, n = 330), medium (3-5 mm, n = 373), or large (>5 mm, n = 238). The cumulus-oocyte complexes recovered from these follicles were submitted to in vitro maturation for 44 h in TCM199 supplemented with 10 ng/ml EGF, 400 ng/ml pFSH and 570 microM cysteamine; in vitro fertilized for 18 h in mTBM with 10(5) frozen-thawed percoll-selected sperms/ml; and developed for 7 days in mSOF. Samples of oocytes or presumptive zygotes were fixed and stained at the end of maturation and fertilization. Groups of oocytes were cultured for 3 h in the presence of 35S-methionine before or after maturation for SDS-PAGE analysis of protein neosynthesis. More oocytes originating from medium and large follicles were competent for maturation than oocytes from small follicles (77 and 86% of metaphase II, respectively, versus 44%, P < 0.05). More oocytes from medium and large follicles werepenetratedby spermatozoa during in vitro fertilization, resulting in significantly more oocytes presenting two or more pronuclei at the end of fertilization (73 and 77% for medium and large follicles, respectively, versus 53% for small follicles, P < 0.05). More oocytes from medium and large follicles developed to the blastocyst stage (14 and 23%, respectively) than those from small follicles (3%, P < 0.05), even if the development rates were corrected by the maturation or fertilization rates. It is concluded that a high proportion of oocytes harvested from follicles of less than 3 mm in the pig are not fully competent for meiosis and are cytoplasmically deficient for development.     

In vitro maturation,fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle

This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.     

Effect of different parthenogenetic activation methods on the developmental competence of in vitro matured porcine oocytes

The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% approximately 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 muM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% approximately 57.3%; 22.3% vs 7.4% approximately 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% approximately 46.2%; 18.0% vs 7.1% approximately 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8-74.4% vs 56.5-57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% approximately 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3-27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.     

Developmental competence of oocytes from prepubertal Bos indicus crossbred cattle

The aim of the present study was to evaluate the effect of age on embryogenic competence of oocytes recovered from Bos indicus crossbred calves and heifers. Cumulus-oocyte complexes (COCs) were collected from 4- to 7-month-old calves (experiment 1) and from 9- to 14-month-old heifers (experiment 2) during processing at an abattoir. In both experiments cow COCs were used as control. COCs were in vitro matured and fertilized, and the presumptive zygotes co-cultured with cumulus cells until 224 h post insemination (hpi). In experiment 1, the development rate during the first 68-72 hpi was similar (P > 0.05) between embryos derived from calves and cows. Fewer embryos from calves developed to the blastocyst stage, resulting in a lesser blastocyst production as well as lesser hatching rate (P < 0.05). The embryo development after blastocyst stage was, nevertheless, similar (P > 0.05) between blastocysts derived from calves and cows, suggesting that the development after blastocoele formation is not compromised in embryos derived from calves. In experiment 2, there were no differences (P > 0.05) on cleavage, blastocyst and hatching rates between embryos derived from prepubertal heifers and cows. The rate of blastocyst development until hatching was also similar (P > 0.05). These results indicate that oocytes from 9- to 14-month-old B. indicus crossbred heifers have the same developmental competence as oocytes derived from cows, while ocytes derived from 4- to 7-month-old B. indicus crossbred calves are less competent in developing to the blastocyst stage in vitro. It suggests that oocyte competence in B. indicus crossbred cattle is achieved around 9-14 months of age.     

Effects of culturing bovine oocytes either singly or in groups on development to blastocysts

In vitro maturation, fertilization and culture (IVM/IVF/IVC) of cattle oocytes from individual cows requires adapting existing culture protocols so that small numbers of oocytes can be cultured. The culture of single oocytes is desirable for correlating the relationship between follicular properties with oocyte developmental competence or for facilitating ovum pick-up procedures. In Experiment 1 we compared group and single culture under cell-free conditions on embryo development; significantly higher (P<0.001) rates of cleavage (66.4 vs 47.6%) and blastocyst formation (7.5 vs 0.5%) were observed in the group cultured oocytes. In Experiment 2 we compared group and single oocyte co-culture with granulosa cells. Although there was no effect of oocyte number on the percentage cleaving (73.1 vs 66.6%), there were significantly higher blastocyst yields (37.4 vs 10.1%) and blastocyst cell numbers (91.6 vs 66.2) in group-cultured oocytes. In Experiment 3 we examined the effect of group size (1, 5, 10, 20 and 40 oocytes) in a co-culture system using granulosa cell monolayers. The results show a difference in cleavage rates between the single cultured oocytes (66.8%) and each group of cultured oocytes, with the highest cleavage rate (81.5%) obtained in the 20-oocyte group. The blastocyst yield from both cleaved and total oocytes showed that group culture of 20 or 40 oocytes resulted in the highest number of blastocysts (32.5%), with smaller group sizes yielding significantly (P<0.05) fewer blastocysts. In Experiment 4 we examined the effects of co-culture on the development of single vs group-cultured oocytes. The results showed no significant difference (P>0.05) in the cleavage rate between single and group culture systems. No blastocysts were formed with single oocytes cultured without monolayers, while the blastocyst formation rate for those co-cultured with granulosa cells was 12.4%. Blastocyst formation was significantly higher (P < 0.006) in group co-culture on monolayers (24.2 vs 8.5%). These data indicate that oocytes cultured in groups are developmentally more competent and suggest that for optimum development oocytes need some undefined paracrine activity that is absent from the culture medium in addition to coculture with granulosa cells, which enhances development to the blastocyst stage of both group and singly cultured oocytes.     

The beneficial effects of insulin and metformin on in vitro developmental potential of porcine oocytes and embryos

To investigate whether insulin and/or metformin improve the developmental competence of porcine oocytes and embryos, insulin (100 ng/ml) and/or metformin (10(-5) M) were supplemented during in vitro maturation (IVM) and/or in vitro culture (IVC) periods. Insulin (33 to 34% vs. 21%) or insulin plus metformin (27 to 39% vs. 21%) significantly (P < 0.01) increased the rates of blastocyst formation, whereas metformin alone had no effect when added during the first half (0-22 h), the latter half (22-44 h), or the entire (0-44 h) maturation period. No additional effect of insulin plus metformin on increasing blastocyst formation was observed compared to insulin alone. When supplemented during IVC, insulin (34% vs. 23%) or insulin plus metformin (35% vs. 23%) significantly (P < 0.05) increased the rates of blastocyst formation, whereas metformin alone had no effect. Compared to insulin alone, no additional effect of insulin plus metformin on increasing blastocyst formation was observed. When added during the entire IVM and IVC, insulin (40% vs. 24%) or insulin plus metformin (52% vs. 21%) significantly increased the rates of blastocyst formation, whereas metformin alone had no effect. In addition, the effect of insulin was enhanced when supplemented with metformin compared to insulin alone (52% vs. 40%). After IVM, oocyte glutathione (GSH) content and tyrosine kinase activity were measured. Insulin significantly (P < 0.01) increased oocyte GSH content (6.2 pmol vs. 4.3 pmol) and metformin significantly (P < 0.01) enhanced the action of insulin on GSH content (7.3 pmol vs. 6.2 pmol) and tyrosine kinase activity (1.9 arbitrary units [AU] vs. 1.5 AU) when compared to insulin alone. In conclusion, insulin increased the developmental potential of porcine oocytes and embryos, and metformin enhanced the action of insulin when supplemented during the entire IVM and IVC. The effects of insulin and metformin were associated with oocyte GSH content and tyrosine kinase activity.