Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 microM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm.     

马卵母细胞胞质内精子注射后体外发育能力的研究

本研究在非繁殖季节评估卵丘形态(松散型、致密型)、成熟培养体系(TCM 199、NCSU 23)、体外成熟时间(34、38 h)和离子霉素结合6-DMAP激活对马卵母细胞胞质内精子注射(ICSI)后体外发育能力的影响。从屠宰场采集马卵巢,获得的卵母细胞进行体外成熟,然后注射马冷冻解冻精液,统计分裂情况。试验结果表明,①马松散型卵母细胞成熟率显著高于致密型卵母细胞(P<0.05),分别为61.09%和41.24%,但ICSI后36 h分裂率无显著差异(P>0.05),分别为47.34%和44.92%;②两种培养体系对马松散型或致密型卵母细胞成熟率及ICSI后36 h分裂率无显著影响(P>0.05),但相同成熟体系培养松散型卵母细胞成熟率均显著高于致密型卵母细胞(P<0.05),然而ICSI后36 h分裂率差异不显著(P>0.05);③松散型或致密型卵母细胞在TCM 199或NCSU 23中成熟38 h成熟率均高于34 h成熟率,分别为44.43%～68.87%和34.52%～58.90%,松散型卵母细胞在TCM 199体系中成熟34 h、ICSI后激活组或对照组的分裂率显著高于成熟38 h、ICSI后激活组的分裂率(P<0.05),以及致密型卵母细胞在TCM 199体系中成熟34 h、ICSI后激活组的分裂率(P<0.05),而且显著高于松散型卵母细胞在NCSU 23体系中成熟38 h、ICSI后对照组的分裂率(P<0.05);④ICSI后用离子霉素结合6-DMAP激活对马卵母细胞ICSI后36 h分裂无显著影响(P>0.05)。因此,马松散型和致密型卵母细胞的成熟能力存在差异,TCM 199和NCSU 23成熟体系对这2种类型卵母细胞的发育能力无显著影响(P>0.05),马卵母细胞成熟38 h成熟率高于34 h成熟率,TCM 199成熟体系培养松散型卵母细胞34 h进行ICSI后的分裂率最高。离子霉素结合6-DMAP激活对TCM 199或NCSU 23体系成熟马卵母细胞ICSI后的体外发育能力无显著影响(P>0.05)。     

Effect of the in vitro maturation medium on equine oocytes: comparison of follicular fluid and oestrous mare serum

The present study evaluated the effect of supplementing the medium used to mature equine oocytes in vitro with oestrous mare serum (EMS) or horse follicular fluid (HFF). To this end, 144 ovaries were obtained from mares aged 16-21 months and transported to the laboratory in Dulbecco's phosphate buffered saline (D-PBS) at 30 degrees C. Oocytes were harvested from the ovaries by slicing, and then selected for in vitro maturation (IVM) according to the number of cumulus cell layers and the characteristics of the cytoplasm. The selected oocytes were washed three times in TCM199 medium plus HEPES (TCM-199H) or in the same medium plus glutamine (TCM-199G), then matured in vitro in six study groups established according to the in vitro maturation (IVM) treatment to see possible interactions between HEPES and glutamine on other supplements: Ten percent EMS was added to two of these media (TCM-199H+EMS and TCM-199G+EMS) and 10% HFF was added to the media in two other groups (TCM-199H+HFF and TCM-199G+HFF). IVM was performed at 38.5 degrees C for 40 h in a controlled atmosphere (5% CO2, 95% relative humidity). The findings indicate that the presence of EMS or HFF in the TCM-199H medium gives rise to the best results in terms of the proportions of oocytes reaching maturity (37.7% and 36.8%, respectively). The values obtained with EMS and HFF were statistically similar to each other but differed from the other treatments. The media containing glutamine led to the highest proportions of degenerated oocytes.     

Sperm Pretreatment with Dithiothreitol Increases Male Pronucleus Formation Rates After Intracytoplasmic Sperm Injection (ICSI) in Swamp Buffalo Oocytes

Failure of male pronucleus formation has hampered the success of intracytoplasmic sperm injection (ICSI) in swamp buffalo. The aim of the present study was to improve male pronucleus formation by pretreating sperm with various chemicals before ICSI. In Experiments1 and 2, sperm were treated according to one of the following protocols: (1) 0.1% Triton-X 100 (TX) for 1 min, (2) 10 µM calcium ionophore (CaI) for 20 min, (3) freezing and thawing (FT) without any cryoprotectant, or (4) no treatment (control). These sperm treatment groups then either did or did not receive additional sperm treatment with 5 mM dithiothreitol (DTT) for 20 min. Acrosomal integrity (Experiment 1) and DNA fragmentation (Experiment 2) were evaluated in the sperm before ICSI. In Experiment 3, oocytes matured in vitro were subjected to ICSI using pretreated sperm as described above and then were cultured either with or without activation. The TX- and CaI-treated sperm caused an increase in the number of acrosome-loss sperm, whereas the FT treatment and control increased the proportion of acrosome-reacted sperm (P<0.05). The DNA fragmentation did not differ among treatments (P>0.05). At 18 h post-ICSI, pronucleus (PN) formation was found only in activated oocytes. The majority of the activated ICSI oocytes contained intact sperm heads. Normal fertilization was observed in the CaI and FT treatment groups and control group when sperm were treated with DTT before ICSI. In conclusion, DTT treatment of sperm with reacted acrosomes before ICSI together with activation of the ICSI oocytes is important for successful male pronucleus formation.     

Electrical activation enhances pre-implantation embryo development following sperm injection into in vitro matured pig oocytes

The objective of this study was to evaluate the effect of electrical stimulation (EST) on pronuclear formation, chromosomal constitution, and developmental capability among in vitro matured pig oocytes following intracytoplasmic sperm injection (ICSI). After ICSI, the oocytes were randomly distributed and cultured into 3 groups: the EST activated ICSI group, non-activation ICSI group, and in vitro fertilization (IVF) group. The proportion of oocytes in which 2 pronuclei were formed in ICSI groups was significantly higher in the former groups than in the IVF group (96.2 and 93.5 vs. 64.5%, respectively, P<0.05). The cleavage rate was significantly higher in EST activated ICSI group (78.6%) than in the IVF and non-activated ICSI groups (51.8 and 46.0%, respectively, P<0.05), as was the proportion of oocytes that developed to the blastocyst stage at day 7 (18.9 vs. 11.6 and 9.1%, respectively, P<0.05). Diploid blastocysts were observed in 52.4, 63.0, and 65.2% of oocytes in the IVF, activated, and non-activated ICSI groups, respectively. Eight out of 23 gilts (34.8%) were confirmed to be pregnant in activated ICSI groups, but none of these pregnancies were carried to term. These results show that oocyte activation after ICSI is effective in elevating the cleavage rate and blastocyst development, while ensuring normal chromosome composition. Further research is needed to determine the pregnancy maintenance requirements for ICSI-embryos in pigs.     

Heterogenous and xenogenous fertilization of in vivo matured equine oocytes

Forty-five in vivo matured equine oocytes were recovered from 63 follicular aspiration attempts (71.4%). HCG did not improve recovery rate (65% — 24/37 for treated vs 81% — 21/26 for nontreated mares). Fifteen oocytes were transferred into the oviduct of inseminated recipient mares (heterogenous fertilization) and 15 oocytes plus equine spermatozoa were transferred into rabbit oviducts (xenogenous fertilization). Ten oocytes (3 fertilized) were recovered from recipient mare oviducts following removal and flushing two days after transfer. Eight oocytes (nonfertilized) were recovered from rabbit oviducts. Oviductal transfer into separate recipient mares of three embryos produced from heterogenous fertilization resulted in two pregnancies. One mare produced a normal live foal and the other mare aborted at 20 days of gestation. Results from these studies suggest that: 1) a reliable method for collection of in vivo matured oocytes has been established, and 2) heterogenous fertilization is a technique that with refinement should be immediately applicable to obtain foals from valuable infertile mares that fail to get pregnant or produce embryos by standard methods.     

Nuclear and cytoplasmic maturation of bovine oocytes cultured with dbc AMP, FSH and hCG

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

Selection of In Vitro-Matured Porcine Oocytes Based on Localization Patterns of Lipid Droplets to Evaluate Developmental Competence

Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.     

Collection and in Vitro Maturation of Equine Oocytes from Estrus,Diestrus and Pregnant Mares

Two experiments were conducted to evaluate oocyte collection rates and in vitro nuclear maturation rates of equine oocytes obtained during diestrus and pregnancy, and to compare these rates with maturation rates in oocytes derived from preovulatory follicles. In Experiment I, transvaginal ultrasound-guided aspiration of follicle was performed during estrus and diestrus in 14 mares over four consecutive cycles. Follicular aspirations during estrus were performed 24 to 27 hours after injection of 2500 IU of hCG given when the largest follicle reached 35 mm in diameter. Oocyte recovery rate from preovulatory follicles was 51% (33/65) in 49 aspiration sessions. Cumulus-oocyte complexes from preovulatory follicles were cultured for 12-15 hours in TCM199 + 10% (NCS) at 38.5°C in 5% CO₂ in air, and 22/33 (67%) were in metaphase II. During diestrus, mares were treated (Group I) or not treated (Group II) daily with equine pituitary extract (EPE) during alternate cycles from days 1 to 14 after the preovulatory aspiration. Diestrous follicles were aspirated when four or more follicles greater than 12 mm in diameter were present. EPE had no effect on the number of follicles that developed during estrus or diestrus (p>0.05). Oocytes were recovered from 119 of 383 diestrous follicles (31 %)in 75 sessions. There was no difference in recovery rates between Groups I and II (p>0.05). Maturation rates for oocytes collected during diestrus, after 42 hours of culture in TCM 199 + 1 μg/ml of Estradiol and lnl/ml of EPE, were not significantly different (p>0.05) between Groups I and II (49% vs. 53%). In Experiment II, mares between 50 and 85 days of pregnancy were used as oocyte donors. The oocyte recovery rate was 53% (66/125). After in vitro maturation for 40 hours (compact COC) or 15 hours (expanded COC), 22% (7/32) and 22% (7/32), respectively, of the oocytes were in metaphase II. It was concluded that: 1) Preovulatory follicles yield a higher percentage of oocytes with a higher rate of maturation to metaphase II than follicles of diestrus and pregnant mares. 2) Diestrous follicles yielded fewer oocytes than follicles of pregnant mares but with a higher percentage of oocyte maturation. Further studies are necessary to determine if oocytes recovered from diestrous follicles and matured in vitro can be fertilized successfully.     

In vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection

In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates of metaphase II-stage oocytes, normal fertilization and cleavage were not significantly different between the two oocyte categories (38.5, 70.0 and 48.7% for CP and 43.5, 60.0 and 58.8% for Ex, respectively). However, the blastocyst development rate of Ex was significantly (P<0.05) higher than that of Cp (25.5 vs. 7.7%). Three Cp-derived and 12 Ex-derived early blastocysts were cryopreserved using the slow cooling protocol, and all of them developed to hatching blastocysts after thawing. These results suggest that equine oocytes fertilized by ICSI can develop to the preimplantation stage in culture conditions similar to those used in the bovine. Furthermore, the Ex oocytes had higher developmental competence than the Cp oocytes, and the in vitro-produced blastocysts had high viability after freezing and thawing.     

Optimization of in vitro embryo production and zygote vitrification for the indigenous Vietnamese Ban pig: The effects of different in vitro oocyte maturation systems

The Vietnamese Ban pig is a precious genetic resource that needs to be preserved. In vitro embryo production from in vitro matured (IVM) oocytes is an important tool for the utilization of cryopreserved porcine sperm. The aim of this study was to compare two media for the IVM of Ban pig oocytes. Immature oocytes were subjected to IVM either in a non‐defined (TCM‐199 + pig follicular fluid) or in a defined base medium (POM + epidermal growth factor). At the end of IVM, the oocytes were in vitro fertilized (IVF) with frozen Ban sperm. Ten hours after IVF, the oocytes were either subjected to orcein staining to check fertilization and maturation status or cultured in vitro for 7 days. There was no difference between the two IVM media in terms of percentages of oocyte maturation and blastocyst production. However, the percentage of male pronuclear formation after IVF and the total cell numbers in blastocysts were higher with the defined system. Zygotes obtained by the two IVM systems survived vitrification at similar rates. In conclusion, the two IVM systems were both effective for the production of Ban pig embryos; however, better embryo quality was achieved with the defined one.     

Influence of Cysteamine on In Vitro Maturation, In Vitro and In Vivo Fertilization of Equine Oocytes

The effect of cysteamine on in vitro nuclear and cytoplasmic maturation of equine oocytes collected by transvaginal ultrasound guided follicular aspiration was assessed. Oocytes were matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining, penetration rates after two different in vitro fertilization (IVF) techniques (IVF media with ionophore and Hepes buffer with heparin) and the embryo yield following oocyte intra-oviductal transfer were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, IVF or in vivo embryonic development under our conditions. Ovum pick up yields (52%) and maturation rates (control group: 47% and cysteamine group: 55%) were similar to those previously reported. From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and five in the cysteamine group (9%). Those two percentages were not statistically different (p > 0.05). No effect of IVF technique was seen on the success rate (6%) in each group.     

海狸鼠卵母细胞体外成熟培养初探

实验用ＰＭＳＧ或ＰＭＳＧ＋ＨＣＧ处理或未经激素处理的海狸鼠８只，共获卵巢卵母细胞１３８枚。激素处理对获取卵巢卵母细胞的数量没有影响，而对体外成熟发育至卵丘扩展和半成熟阶段有促进作用。三种不同培养液（Ｗｈｉｔｅｎ＋ＦＣＳ；ＴＣＭ１９９＋ＰＭＳＧ＋ＦＣＳ；ＴＣＭ１９９＋ＨＣＧ＋ＦＣＳ）共培养１２５枚卵母细胞，培养后卵丘扩展率及半成熟率分别为５６．５％，４５．７％，４７．６％和２１．７％，１２．３％，９．５％，以Ｗｈｉｔｅｎ液较高（分别为５６．５％和２１．７％），但只有ＴＣＭ１９９＋ＰＭＳＧ＋ＦＣＳ组有２枚卵母细胞出现第一极体。结果表明海狸鼠卵母细胞与其它啮齿动物的卵母细胞一样，能够在体外培养成熟，完成第一次减数分裂，排出第一极体     

Improved embryo development in Japanese black cattle by in vitro fertilization using ovum pick-up plus intracytoplasmic sperm injection with dithiothreitol

The purpose of this study was to determine whether dithiothreitol (DTT) treatment of sperm and ethanol activation improve embryo production by intracytoplasmic sperm injection (ICSI). Further, we compared ICSI with standard in vitro fertilization (IVF) in oocytes obtained from cattle. We demonstrated that DTT reduced the disulfide bond in the bovine sperm head. Using oocytes obtained from a slaughterhouse, ICSI-DTT treatment without ethanol showed the highest rate of blastocyst formation. We applied these results to fertilization using ovum pick-up (OPU). Eleven Japanese black cattle served as donors for OPU plus standard IVF (OPU-IVF). Of them, four donors with low embryo development rates were selected to determine whether embryo development was enhanced by OPU plus ICSI (OPU-ICSI). We assessed effects on embryo development following IVF and ICSI in oocytes obtained using OPU. Blastocyst rates were significantly higher for OPU-ICSI than for OPU-IVF. Our results suggest that OPU-ICSI improves the blastocyst development rate in donors with low embryo production compared with the standard OPU-IVF.     

Cleavage capability of water buffalo follicular oocytes classified by cumulus cells and fertilized in vitro.

Water buffalo (Murrah) oocytes were collected from ovaries obtained from the slaughter house. They were classified according to the character of the cumulus cells under a stereomicroscope, and cultured in 25 mM Hepes buffered Tissue Culture Medium-199 (TCM-199) supplemented with 5% estrous water buffalo serum in an atmosphere containing 5% CO2 in air at 39 degrees C. After 20-24 hr of in vitro maturation, the oocytes were fertilized using capacitated sperm obtained from 4 different bulls. For cleavage the oocytes were cultured at 39 degrees C in TCM-199 supplemented with 1% estrous water buffalo serum and in an atmosphere containing 5% CO2 in air. The good oocytes, with compact and dense cumulus cells cleaved significantly higher (p less than 0.01, 67.3%), than those of fair. partially naked oocytes with thin cumulus layers (27.5%, 25/91) or small remnants of cumulus cells and poor naked oocytes (3/100). A substantial variation cumulus layers (27.5% 25/91) or small remnants of cumulus cells and poor naked oocytes (3/100). A substantial variation in fertilization and developmental rates (16.0% to 43.8%) was observed among 4 different bulls. Late non-surgically into 14 buffalo recipients on day 6 or 7 of their estrous cycle. One recipient was diagnosed to be pregnant by rectal palpation on day 60 and confirmed to be so on day 90 post-estrus.     

Intracytoplasmic Glutathione Levels in Heifer Oocytes Cultured in different Maturation Media and its Effect on Embryo Development

The present study was carried out to study the effect of different maturation media on embryo development of heifer oocytes and on their glutathione (GSH) synthesis during in vitro maturation (IVM). Immature heifer oocytes were matured in parallel in one of four maturation media: (i) Tissue Culture Medium (TCM)-199 supplemented with 10 ng/ml of epidermal growth factor (EGF); (i) TCM-199 supplemented with 10 ng/ml of EGF plus 1 microg/ml of FSH; (iii) TCM-199 supplemented with 10% of foetal bovine serum (FBS) and (iv) TCM-199 supplemented with 10% of FBS plus 1 microg/ml of FSH. Cow oocytes were used as control and were matured in TCM-199 supplemented with 10 ng/ml of EGF. No differences were observed in blastocyst rate among the different heifer oocyte groups (8.8, 7.5. 8.4 and 6.8%, respectively) however, the percentage of blastocysts obtained from cow oocytes was significantly higher (30%; p < 0.01) than those obtained from heifer oocytes. De novo GSH synthesis during oocyte maturation of heifer and cow oocytes was detected. No significant differences in intracytoplasmic GSH levels were observed among the experimental heifer oocyte groups or between heifer and cow oocytes both before and after IVM. In conclusion, the blastocyst yield obtained from heifer oocytes was lower than that from cow oocytes and this fact could not be explained by significant differences in intracytoplasmic GSH contents of oocytes before or after IVM.     

Effect of cumulus cell removal and sperm pre‐incubation with progesterone on in vitro fertilization of equine gametes in the presence of oviductal fluid or cells

In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre‐incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre‐incubation with progesterone. In experiments 1 and 2, IVF was performed in the presence of porcine oviduct epithelial cells. The removal of cumulus cells from equine oocytes after in vitro maturation tended to increase the percentage of fertilization when fresh sperm was used (1/33 vs. 4/31, p > 0.05) but had no effect when frozen sperm was used (1/32 vs. 1/32). Equine sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/14 vs. 2/18 for fresh, 1/29 vs. 1/25 for frozen). In experiments 3 and 4, IVF was performed after pre‐incubation of sperm with porcine oviductal fluid. The removal of cumulus cells tended to increase the percentage of fertilization when fresh sperm was used (1/24 vs. 3/26, p > 0.05). Sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/39 vs. 2/36 for fresh, 2/37 vs. 1/46 for frozen), but two 3–4 cell stage zygotes were obtained with fresh sperm pre‐incubated with progesterone. This is an encouraging result for the setting up of an efficient IVF procedure in equine.     

Effect of Different Media and Protein Source on Equine Gametes: Potential Impact During In Vitro Fertilization

Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18–20 h (no sperm added). In Ex oocytes, TCM‐199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%) or FBS (52.2%; p < 0.05); this was not observed for Cp oocytes. Equine sperm were capacitated in the same media at 10 × 10⁶ sperm/ml for 4 h at 37°C; total motility and protein tyrosine phosphorylation (PY) were evaluated. While motility remained unchanged, TCM or MW added with FBS enhanced the number of sperm showing PY‐stained tails (25 ± 4.8% and 31 ± 6.6%; mean ± SEM, respectively) over BSA supplemented media (3 ± 1.2% and 11.7 ± 1.1%) for TCM and MW (p < 0.05). In view of the previous results, sperm were capacitated in TCM + FBS and MW + BSA (control); IVF trials were run in the same media supplemented with 200 ng/ml of progesterone, but no fertilization occurred. Our results show that TCM + FBS enhances Ex equine oocyte's meiotic competence over MW + BSA and TCM or MW added with FBS successfully induce equine PY over media supplemented with BSA.     

Selected Aspects of Advanced Porcine Reproductive Technology

In vitro fertilization (IVF) of in vitro matured (IVM) oocytes in pigs has become the most popular method of studying gametogenesis and embryogenesis in this species. Furthermore, because of recent advances in in vitro culture (IVC) of IVM–IVF embryos, in vitro production (IVP) of embryos now enables us to generate viable embryos as successfully as for in vivo -derived embryos and with less cost and in less time. These technologies contribute not only to developments in reproductive physiology and agriculture but also to the conservation of porcine genetic resources and the production of cloned or genetically modified pigs. However, in IVP, there still remains the problem of abnormal ploidy, which is caused by performing procedures under non-physiological conditions. In recent years, unique technologies such as intracytoplasmic sperm injection (ICSI) or xenografting of gonadal tissue into immunodeficient experimental animals have been developed to help conserve gamete resources. These technologies combined with IVP are expected to be useful for the conservation of gametes from important genetic resources. Here, we discuss the developmental ability and normality of porcine IVP embryos and also the utilization of ICSI and xenografting in advancing biotechnology in pigs.