Effect of administering a crude equine gonadotrophin preparation to mares on follicular development, oocyte recovery rate and oocyte maturation in vivo

In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all follicles >/= 4 mm were aspirated from 19 pony mares (first aspiration: A1). Over the next 8 days, the mares were treated daily with either 25 mg crude equine gonadotrophins (n = 10) or physiological saline (n = 9). Between day 1 and day 8, follicular growth was monitored by ultrasonography. On day 8, all follicles >/= 4 mm were evacuated (second aspiration: A2) and nuclear maturation of the recovered oocytes was assessed after orcein staining. Follicular growth between A1 and A2, as well as the number and size of follicles at A2 were similar for control mares and mares treated with crude equine gonadotrophins. The oocyte recovery rates at A1 and A2 were similar. At A2, the oocyte recovery rate and oocyte maturation in vivo were not affected by treatment with crude equine gonadotrophins. The number of expanded cumulus oophorus complexes recovered from follicles 相似文献   

Structural and endocrine aspects of equine oocyte maturation in vivo

The objectives were to describe the ultrastructure of equine oocytes aspirated from small and preovulatory follicles, and to relate the ultrastructural features to follicle size and follicular fluid steroid concentrations. Mares were examined every second day by transrectal ultrasonography, and follicles measuring ≤30 mm were aspirated (in vivo) using a 20-cm-long 12-gauge needle through the flank. Following slaughter, both large and small follicles were aspirated (in vitro) from six mares. The oocytes were isolated under a stereomicroscope and processed for transmission electron microscopy, and the follicular fluid was assayed for progesterone (P4) amd estradiol-17β (E2). A total of 29 oocytes (32% recovery rate) were aspirated in vivo, and 15 oocytes were recovered in vitro. According to the stage of nuclear maturation, the oocytes could be divided into the following six categories: 1) the central oocyte nucleus (CON) stage, 2) the peripheral spherical oocyte nucleus (PON-I) stage, 3) the peripheral flattened oocyte nucleus (PON-II) stage, 4) the oocyte nucleus breakdown (ONBD) stage, 5) the metaphase I (M-I) stage, and 6) the metaphase II (M-II) stage. The maturation of the preovulatory follicle was reflected by alterations in the follicular fluid concentrations of steroid hormones. E2 was high in all preovulatory follicles, whereas P4 concentration exhibited a 10-fold increase during follicle maturation, particularly associated with the progression from M-I-to M-II-stage oocytes. The nuclear oocyte maturation included flattening of the spherical oocyte nucleus, followed by increasing undulation of the nuclear envelope, formation of the metaphase plate of the first meiotic division, and, finally, the extrusion of the first polar body and the subsequent formation of the metaphase plate of the second meiotic division. The cytoplasmic oocyte maturation changes comprised breakdown of the intermediate junctions between the cumulus cell projections and the oolemma, enlargement of the perivitelline space, the formation and arrangement of a large number of cortical granules immediately beneath the oolemma, the rearrangement of mitochondria from a predominantly peripheral distribution to a more central or semilunar domain, and the rearrangement of membrane-bound vesicles and lipid droplets from an even distribution to an often semilunar domain, giving the ooplasm a polarized appearance. It is concluded that the final equine oocyte maturation includes a series of well-defined nuclear and cytoplasmic changes that are paralleled by an increase in P4 concentration in the follicular fluid, whereas E2 concentration remains constantly high. © 1995 wiley-Liss, Inc.     

Embryo development rates after transfer of oocytes matured in vivo, in vitro, or within oviducts of mares

Objectives of the present study were to use oocyte transfer: 1) to compare the developmental ability of oocytes collected from ovaries of live mares with those collected from slaughterhouse ovaries; and 2) to compare the viability of oocytes matured in vivo, in vitro, or within the oviduct. Oocytes were collected by transvaginal, ultrasound-guided follicular aspiration (TVA) from live mares or from slicing slaughterhouse ovaries. Four groups of oocytes were transferred into the oviducts of recipients that were inseminated: 1) oocytes matured in vivo and collected by TVA from preovulatory follicles of estrous mares 32 to 36 h after administration of hCG; 2) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and matured in vitro for 36 to 38 h; 3) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and transferred into a recipient's oviduct <1 h after collection; and 4) im mature oocytes collected from slaughterhouse ovaries containing a corpus luteum and matured in vitro for 36 to 38 hours. Embryo development rates were higher (P < 0.001) for oocytes matured in vivo (82%) than for oocytes matured in vitro (9%) or within the oviduct (0%). However, neither the method of maturation nor the source of oocytes affected (P > 0.1) embryo development rates after the transfer of immature oocytes.     

Content of meiosis activating sterols in equine follicular fluids: correlation to follicular size and dominance

Meiosis activating sterols (MAS) are pre-cholesterol sterols that can be isolated from follicular fluid (FF-MAS) or testes (T-MAS). Meiosis activating sterols trigger the resumption of meiosis in cultured meiotically competent oocytes. In the present work MAS, cholesterol and progesterone were assayed by HPLC in follicular fluids collected from pony mares at fixed days after the last ovulation. Follicles were divided into two groups according to whether they were aspirated before or after Day 17 after the last ovulation. The latter group was further divided according to whether the follicle diameter was < or = 22 mm or > 27 mm. Both FF-MAS and T-MAS were detected in almost all samples. Overall, the total amount of MAS in the follicular fluids increased with the size of the follicles but was accompanied by a decrease in the amount of free cholesterol. The amounts of MAS and progesterone in > 27 mm follicles aspirated after Day 17 were significantly higher as compared to the other groups. A transversal cohort analysis showed that the largest follicle at the time of aspiration had the highest level of MAS after day 17 of the cycle, which was not always true for follicle samples aspirated before Day 17 of the cycle. The study demonstrates that the content of MAS in equine follicular fluids increased during follicular maturation concomitant with a decrease in the concentration of free cholesterol. Moreover, MAS concentration is higher in dominant follicles than in subordinate follicles. The MAS may therefore play an as yet unknown physiological role during pre-ovulatory maturation.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

In vitro maturation of bovine cumulus-oocyte complexes in undiluted follicular fluid: effect on nuclear maturation,pronucleus formation and embryo development

Since resumption of meiosis and cytoplasmic maturation of bovine oocytes takes place in close association with follicular fluid, it would be logical to assume that this might be a perfect maturation medium. To test the hypothesis, abattoir-derived cumulus-oocyte complexes (COCs) were in vitro matured in undiluted (i) mixed follicular fluid (FF) from 3 to 15 mm follicles from abattoir ovaries, (ii) preovulatory follicular fluid (POF) from the dominant follicle from a cyclic unstimulated heifer, (iii) preovulatory follicular fluid (OPU) from synchronised and superovulated heifers 60 h after prostaglandin and 20 h after GnRH treatment, and in (iv) TCM-199 with 5% serum. Subsequent to IVM, the COC were subjected to IVF and IVC, and embryo development was followed until the blastocyst stage at Day 8 after insemination. The MII rates in the TCM-199 (69%), POF (69%) and OPU (72%) groups were not different from each other but different from the FF (41%) group (P<0.05). In spite of the high MII rates, none of the follicular fluids supported embryo development: the FF, POF and OPU blastocyst rates were alike (3%, 3%, 2%) and different (P<0.05) from the rates in the TCM-199 (19%). During IVM in follicular fluids but not in TCM-199, the expanded cumulus masses became trapped in a coagulum. Although it could be prevented by the presence of heparin during IVM, it did not improve the blastocyst rates. In conclusion, undiluted preovulatory follicular fluids supported nuclear maturation but not further embryonic development as judged by the high MII and low blastocyst rates.     

Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection

Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2 g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to 9.1) or oocytes recovered (4.8 to 5.0) per mare per aspiration session. Of 314 oocytes recovered, 180 (57%) matured in culture. Thirty-six mature oocytes were subjected to ICSI; 33% formed blastocysts (63% per mare per aspiration session). In Experiment 2, the preovulatory follicle was aspirated every 14 d for three to four cycles. Prostaglandin F_2α was given on Days 6 and 7 after aspiration. A follicle ≥25 mm in diameter was present on Day 13, the day of deslorelin administration, in 23 of 24 cycles, and ovulatory response (granulosa expansion) was seen in 24 of 25 follicles aspirated. Blastocyst development after ICSI was 41% per injected oocyte, or an estimated 33% per mare per aspiration session. We concluded that both aspiration of immature follicles and aspiration of the preovulatory follicle can be performed effectively every 14 d without monitoring ovarian follicular growth. As performed in these separate experiments, aspiration of immature follicles provided more blastocysts per aspiration session.     

M-phase promoting factor (MPF) and mitogen activated protein kinases (MAPK) activities of domestic cat oocytes matured in vitro and in vivo

This work was undertaken in order to examine M-phase promoting factor (MPF) and mitogen-activated protein kinases (MAPK) activities during meiotic progression of cat oocytes cultured in two different media for two different incubation times and preovulatory cat oocytes that reached MII in vivo. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 or SOF for 24 h and 40 h. In vivo matured oocytes were recovered by follicular aspiration from ovaries of domestic cats ovariectomized 24 h to 26 h after hormonal treatment. Results showed that the kinetic of MPF and MAPK activity was similar during meiotic progression of cat oocytes matured in TCM 199 and SOF. After 24 h of incubation, MII oocytes had significantly (p < 0.001) higher MPF and MAPK levels than MII oocytes cultured for 40 h in both culture media. MPF and MAPK activity was significantly (p < 0.01) lower in the oocytes matured in vitro than in those matured in vivo. This study provides evidence that the two different maturation media did not determine differences in MPF and MAPK fluctuations and levels during meiotic progression of cat oocytes and that the time of maturation influenced the level of the two kinases. Moreover, it shows that MPF and MPK activity is higher in in vivo matured oocytes than in in vitro matured oocytes, suggesting a possible incomplete cytoplasmic maturation after culture.     

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 maturation and early developmental capacity of bovine oocytes cultured in pure follicular fluid and supplementation with follicular wall

Mammalian oocytes mature in follicular fluid (FF), surrounded by follicular cells. In the present study, in vitro maturation of bovine oocytes cultured in FF from dominant follicles 15-17mm in diameter (with various forms of heat pretreatment) and supplementation with follicular wall from follicles 3-5mm in diameter (FW1) were examined. Heat pretreatment of FF was as follows: (1) no treatment (FF1); (2) 56 degrees C for 30min (FF2); and (3) 100 degrees C for 20s (FF3). After IVM in FF1, oocytes underwent IVF and IVC and embryo development was assessed (up to the morula stage). The rate of oocyte maturation was decreased in pure FF1 versus control (44.5% versus 62.8%, P<0.001). In the control medium, FW1 did not significantly affect nuclear maturation. By contrast, the addition of FW1 to FF1 increased the rate of matured oocytes approximately two-fold (85.9% versus 45.6%, P<0.001). Furthermore, the maturation rate in the FF+FW1 system declined (from 85.9 to 71.0%, P<0.001), whereas that in the FF system increased (from 45.6 to 71.6%, P<0.001) with increased temperature of the FF treatment. Supplementation of the control medium with FW1 increased the yield of morulae (42.6% versus 13.7%, P<0.001). However, the stimulatory effect of FW1 on the morula rate was much higher in pure FF1 (72.5% versus 31.7%, P<0.001). These findings indicated, for the first time, the stimulatory impact of FW1 on in vitro maturation and early developmental capacity of bovine oocytes cultured in pure FF from dominant follicles. We also inferred that bovine FF constituents affecting bovine oocyte maturation and the meiosis-promoting ability of the FW were heat-labile.     

Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro

This study examines the effect of sheep and human follicular fluid on the in vitro maturation (IVM) of sheep follicular oocytes. Oocyte cumulus complexes recovered post mortem were matured for 24 to 26 h at 38.6 degrees C, 5% CO(2) in air, in TCM-199 bicarbonate medium supplemented with 20% fetal calf serum (FCS) and, where stated, with maturation hormones, including FSH (5.0 ug/ml), LH (5.0 ug/ml) and estradiol (1 ug/ml), or with sheep follicular fluid recovered from large (>5mm) or small (2 to 5mm) ovarian follicles post mortem, or with human periovular follicular fluid obtained during routine IVF procedures. The matured oocytes were then denuded, and their maturation stage and developmental capacity were assessed by in vitro fertilization (IVF) and culture (IVC). It was found that inclusion of sheep or human follicular fluid or hormone supplements in the IVM media more than doubled the number of oocytes completing maturation (FCS alone 33%, compared with 76.2% for maturation hormones, 84.2% for fluid from large and 69.6% for fluid from small sheep follicles and 82.6% for human follicular fluid), and significantly increased fertilization rates (FCS alone 51.6%, compared with 71.9% for maturation hormones, 78.4% for fluid from the large and 75.7% for fluid from small sheep follicles and 73.1% for human follicular fluid) without discernible adverse effects on the development of the cleaving embryos to the morula or blastocyst stage in culture. Omission of FCS and supplements from the IVM medium resulted in a marked reduction (56%) in the number of oocytes maturing. This reduction could be offset to a large part, but not completely, by inclusion of human follicular fluid or human follicular fluid plus LH (5 ug/ml) in the medium. The results of this study show that addition of sheep or human follicular fluid to maturation medium can enhance rather than inhibit the maturation and fertilizability of sheep follicular oocytes in vitro.     

In vitro maturation and intracytoplasmic sperm injection of oocytes collected from hormonally stimulated common wombats, Vombatus ursinus

Porcine FSH/LH stimulation successfully induced development of multiple large (>or=4mm) antral follicles in 10 of 11 common wombats. A mean of 5.5 metaphase II (MII) oocytes were aspirated from wombats that were stimulated during the follicular phase of the oestrous cycle (n=3) or after pouch young removal (n=3). Three subadults (n=3) and two anoestrus adults did not produce MII oocytes despite pFSH/pLH administration. In vitro maturation of immature oocytes at the time of aspiration doubled the number of MII oocytes that could be collected from pFSH/pLH stimulated wombats. Immature oocytes with cumulus attached, matured more readily to the MII stage than immature oocytes without cumulus. Following intracytoplasmic sperm injection (ICSI), approximately 5% of the oocytes that were MII at the time of collection cleaved. Approximately 5% of those that were matured by in vitro maturation (IVM) formed two polar bodies following ICSI, although they not cleave. Parthenogenesis cannot be excluded. This demonstrates that assisted reproductive technologies may be applicable to the common wombat.     

Effect of homologous preovulatory follicular fluid on in vitro maturation of equine cumulus-oocyte complexes

The objective of this study was to test the hypothesis that incubating equine cumulus-oocyte complexes (COCs) in medium containing 50% or 100% homologous preovulatory follicular fluid would improve cumulus expansion and nuclear maturation. Oocytes were incubated in one of three media: 1) supplemented TCM-199 (control), 2) 50% (v/v) follicular fluid in control medium or 3) 100% follicular fluid. Cumulus expansion was evaluated subjectively, and nuclear maturation was evaluated by staining oocytes with Hoechst 33258. The hypothesis that incubating COCs in medium containing follicular fluid would improve cumulus expansion was supported. More (P < 0.05) compact COCs incubated in 50% or 100% follicular fluid developed a moderately to completely expanded cumulus after 24 and 36 h of incubation and more (P < 0.05) expanded COCs incubated in 100% follicular fluid developed a moderately to completely expanded cumulus after 36 h of incubation compared to control medium. The hypothesis that incubating COCs in medium containing follicular fluid would improve nuclear maturation was not supported. Although more (P < 0.05) compact COCs incubated in 50% follicular fluid reached polar body-stage compared to those in control medium, the nuclear maturation rate in the control medium was lower than it was when the same medium was used in a preliminary experiment (described in main text); therefore, the apparent superiority of 50% follicular fluid must be interpreted cautiously. Based on these results, future studies are warranted to further address the value of adding preovulatory follicular fluid to equine IVM culture systems.     

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

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

Effect of stage of follicular growth during superovulation on developmental competence of bovine oocytes

The final steps of oocyte capacitation and maturation are critical for embryonic development but detailed information is scarce on how the oocyte is affected during this period. In this study, 2033 oocytes were collected from 106 superovulated cattle at four different time points before ovulation. Follicular characteristics were measured and oocyte quality was assessed by morphology, mRNA expression of eight marker genes or developmental ability after in vitro/in vivo maturation and subsequent in vitro fertilization and culture. Approaching ovulation, expected increases in follicular size and cumulus expansion suggested progression of oocyte maturation. No differences were found in the expression patterns of analyzed genes, except for heat-shock-protein (Hsp) that was lower in in vivo matured oocytes collected shortly before ovulation. Oocytes collected at this time also had higher developmental ability measured as blastocyst rates (57.6%) after in vitro production while no differences were found between oocytes recovered earlier at the first three time points (39.3-41.5%). We conclude that oocytes recovered late in the preovulatory period are more developmentally competent than oocytes recovered at the pre-capacitation and the capacitation period, probably due to the former having matured in vivo. However, a precisely defined time for aspirating immature oocytes for subsequent in vitro development seems not to be crucial.     

No single way to explain cytoplasmic maturation of oocytes from prepubertal and cyclic gilts

The objective of this study was to evaluate selected aspects of cytoplasmic maturation in oocytes from prepubertal and cyclic crossbred gilts before and after in vitro maturation. For this purpose, cortical granule redistribution, mitochondrial DNA content and mitochondria translocation were analyzed. Moreover, for the first time the fatty acid profiles in follicular fluid (FF) of both gilt categories was evaluated. The nuclear maturation (the percentage of metaphase II oocytes was 83% in prepubertal gilts compared with 87% in cyclic gilts), cortical granule relocation from the cortex to peripheral ooplasm (98.7% vs. 98.8% of oocytes, respectively) and mitochondrial DNA content (227 543 vs. 206 660, respectively) was not affected by sexual maturity of the donor gilt. However, the redistribution of active mitochondria during in vitro maturation was observed only in the oocytes of cyclic gilts. With regard to FF analysis, saturated, unsaturated, and monounsaturated fatty acids were significantly more abundant in the FF of prepubertal females. In particular, stearic (C18:0) and palmitic (C16:0) fatty acids had significantly higher concentrations in the FF of prepubertal gilts. In conclusion, although the oocytes of prepubertal gilts matured in vitro at a rate similar to those of cyclic gilts, they differed with respect to the selected factors attributed to cytoplasmic maturation. We suggest that the higher content of particular fatty acids, which is known to have a negative influence on oocyte maturation, as well as impaired mitochondria redistribution are factors limiting the maturation potential of oocytes from prepubertal gilts.     

Effects of fetuin on zona pellucida hardening and fertilizability of equine oocytes matured in vitro.

In vitro fertilization (IVF) has had poor success in the horse, a situation related to low rates of sperm penetration through the zona pellucida (ZP). Zona pellucida hardening (ZPH) is seen in mouse and rat oocytes cultured in serum-free medium. The hardened ZP is refractory to sperm penetration. Fetuin, a component of fetal calf serum, inhibits ZPH and allows normal fertilization rates in oocytes cultured in the absence of serum. We evaluated whether fetuin is present in horse serum and follicular fluid (FF) and whether fetuin could inhibit ZPH in equine oocytes matured in vitro, thus increasing sperm penetration during IVF. The presence of fetuin in equine serum and FF was confirmed by immunoblotting. Oocytes submitted to in vitro maturation (IVM) in medium containing fetuin were used for ZPH assay or IVF. Intracytoplasmic sperm injection (ICSI) was carried out as a control procedure. The presence of fetuin during IVM did not affect the rate of maturation to metaphase II. Maturation of oocytes in the presence of fetuin reduced ZPH in a dose-dependent manner. After both IVF and ICSI, there was no significant difference in oocyte fertilization between fetuin-treated and untreated oocytes. The fertilization rate was significantly higher after ICSI than after IVF, both in fetuin-treated and in untreated oocytes. In conclusion, fetuin reduced ZPH in equine oocytes but did not improve sperm penetration during IVF. This implies that, in the horse, "spontaneous" ZPH is unlikely to be the major factor responsible for inhibiting sperm penetration in vitro.     

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium,duration of maturation,and sperm calcium ionophore treatment,and comparison with rates of fertilization in vivo after oviductal transfer

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.