In vitro fertilization of pig oocytes matured in vitro

The development of in vitro fertilization (IVF) techniques in pigs as well as in other species is of great importance because of the possible applications of this technology in different research fields. Methods of IVF vary in different incubation periods and temperatures, in the hormone concentrations used, and in the treatment of the sperm samples. It has been particularly difficult to succeed in the achievement of fertilization in the pig. In the present study we used FSH and LH concentrations of 2 IU/ml for oocyte maturation, an incubation temperature of 37°C, and dilution of spermatozoa for capacitation, and we achieved a high fertilization rate (50 to 75%) with no cases of polyspermy.     

In vitro fertilization of horse follicular oocytes matured in vitro

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 x 10(6) sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophore-treated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.     

In vitro fertilization of sheep oocytes matured in vivo

Incubating washed ram spermatozoa in a modified Brackett's defined medium buffered with Hepes (DM-H) containing 20% of heat-inactivated sheep serum appears to be a reliable method of capacitating sperm for in vitro fertilization. Raising the Ca(++) concentration in the fertilization medium (DM-H-SS) to 10 mM stabilized the fertilization rate of various rams (2). This study was designed to determine if the developmental competence of the oocytes fertilized under such conditions was normal. Thirty-seven ewes, treated with progestagen sponges, were superovulated with porcine follicle stimulating hormone (pFSH: 16 mg). An intramuscular injection of gonadotropin releasing hormone (GnRH: 100 mug); given 24 to 26 h after sponge removal, induced the synchronization of ovulations 24 h later. Ovulated oocytes (n = 229) recovered with flushing of the oviducts were inseminated in vitro and 17 h later either fixed in acetic/alcohol (n = 115) to evaluate fertilization or transferred (n = 114) into 38 synchronized recipients (three oocytes/recipient) to evaluate their developmental competence. Of the fixed oocytes, 82.6% were fertilized and 61.7% were monospermic. Nineteen of the recipient ewes (50%) were pregnant at Day 18, and 16 ewes produced a total of 26 live young (mean: 1.63/ewe). The results showed a high efficiency of in vitro fertilization of ovulated oocytes in sheep following a pFSH-GnRH treatment and the in vivo developmental competence of oocytes fertilized in the presence of elevated Ca(++) concentration.     

In vitro fertilization and cleavage of in vivo matured bovine oocytes

The effect of the interval between onset of estrus and oocyte collection on in vitro fertilization (IVF) rates has been investigated. The oocytes were surgically collected 6-18 h (Group I), 19-24 h (Group II), 25-29 h (Group III) and 30-36 h (Group IV) after the beginning of estrus. Recognizable stages of nuclear maturation were identified in 54.9% of the oocytes used for IVF (5.9% at germinal vesicle, 31.4% at metaphase I, 17.6% at metaphase II); the other 45.1% were degenerate. Considerable between- and within-cow variation in oocyte morphology, oocyte maturation and IVF results was observed. The cverall fertilization and cleavage rates (to four-cell stages) were 26.5 and 6.0%, respectively. The fertilization rate increased as the interval between onset of estrus and collection increased and was optimal 30-36 h after onset. Thus, onset of estrus proved an effective means of timing oocyte collection for IVF.     

In vitro fertilization and development of bovine oocytes matured in serum-free medium

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.     

In vitro fertilization of in vitro matured canine oocytes using frozen-thawed dog semen

Experiments were conducted to evaluate in vitro fertilization (IVF) of in vitro matured (IVM) bitch oocytes using dog spermatozoa frozen in three different extenders. Sperm-rich fraction from eight ejaculates of five dogs was frozen in each one of three egg yolk Tris extenders with additional: (A) 1.4 g citric acid and 0.8 g glucose; (B) 0.7 g citric acid and 3.5 g glucose; or (C) 1.4 g citric acid and 0.8 g fructose (all with 5% glycerol in 100 mL milliQ water). Thawed sperm were co-incubated with IVM bitch oocytes for 6 h. Oocytes were fixed and evaluated under an epifluorescence microscope; penetrated oocytes were defined as those having sperm heads in the perivitelline space or in the oocyte cytoplasm. Higher penetration rates (P < 0.05) were obtained in oocytes cultured with spermatozoa frozen in extenders B and C than those frozen in extender A (33.1, 34.2 and 26.4%, respectively).     

Blastocyst formation by pig embryos resulting from in-vitro fertilization of oocytes matured in vitro

Follicular oocytes collected from prepubertal gilts at a local slaughter house were matured (36 h), fertilized and developed in vitro. Of 785 embryos, 190 (24%) embryos cleaved to the 2-4 cell stages with blastomeres of regular size by 33 h after insemination. These cleaved embryos were surgically transferred into the oviducts of 4 synchronized recipient gilts and recovered from the uterine horns 4 or 7 days later: 13 morulae, 2 blastocysts and 1 expanded blastocyst were recovered after 4 days and 3 hatched blastocysts were recovered 7 days after transfer. Re-culture in vitro sustained further development of morulae recovered 4 days after transfer: 11 of 13 morulae had developed to the blastocyst/hatched blastocyst stages. Overall, 17 of 190 (9%) embryos developed to the blastocyst stage. The results indicate that pig oocytes can be matured and fertilized in vitro, and subsequently develop to the blastocyst stage.     

Histone H1 kinase activity during in vitro fertilization of pig follicular oocytes matured in vitro

The present study was conducted to clarify the relationship between histone H1 kinase (H1K) activity and events associated with in vitro fertilization of pig follicular oocytes matured in vitro. Histone H1 kinase has been shown to be homologous with a maturation promoting factor (MPF). Cumulus-oocyte complexes obtained from prepubertal gilts were cultured for 46 h in a modified Waymouth's MB752/1 medium and were then inseminated in vitro with frozen-thawed and preincubated epididymal boar spermatozoa. At 4, 6, 8 and 10 h post insemination, the oocytes were stained with 10 microg/ml Hoechst-33342 and examined under a fluorescent microscope for the stage of fertilization, according to morphological changes of oocyte nuclear chromatin and the extent of sperm penetration. Sperm penetration was observed to occur within 4 h post insemination (20.5%), and the percentage of fertilized oocytes increased (P < 0.01) to 72.9% at 8 h post insemination. Pronuclear formation was observed from 6 h post insemination (3.3%) and the percentage increased (P < 0.01) to 46.8% at 10 h post insemination. In each examination period, H1K activities in unfertilized oocytes at metaphase-II remained unchanged (112.0 fmol/h/oocyte) and were higher (P < 0.01) than those in fertilized oocytes (30.1 fmol/h/oocyte). The H1K activity in fertilized oocytes such as oocytes emitting a second polar body, oocytes with an enlarging sperm head(s) and oocytes with multiple pronuclei did not differ significantly. These results suggest that MPF in pig oocytes is inactivated shortly after sperm penetration and is maintained at the basal level throughout pronuclear formation.     

Ram-specific effects on in-vitro fertilization and cleavage of sheep oocytes matured in vitro

The present experiments were designed to identify possible male-specific effects on early embryonic development in vitro: Sheep oocytes were matured in vitro for 24-26 h and then fertilized in vitro using equal numbers of viable spermatozoa from 1 of 6 Clun Forest rams. At 15-18 h after insemination, oocytes were either fixed and examined for fertilization and polyspermy or further cultured in modified M 199 medium for 3 days in an oviduct epithelial co-culture system. There were significant differences in 5 separate trials between the rams with respect to the rate of fertilization, degree of polyspermy and cleavage rate after monospermic fertilization. The mean rate of fertilization varied from 89% in Ram B to 43% in Ram C while the percentage of polyspermic eggs varied from 5 to 34%. Both the absolute number of embryos cleaving to the 16-cell stage and the calculated percentage of monospermic eggs reaching the 16-cell stage differed markedly between groups of eggs fertilized by different rams. The results indicate that the development of sheep eggs in vitro is differentially affected by the ram from which the spermatozoa are collected.     

Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture

Two experiments were conducted to assess the effects of caffeine and casein phosphopeptides (CPPs). One experiment tested the ability of frozenthawed epididymal spermatozoa from boar (A, B, C), of proven low in vitro fertilization rates, to penetrate pig follicular oocytes. The other experiment tested the ability of ejaculated spermatozoa to uptake Ca²⁺. In Experiment 1, oocytes matured in vitro were inseminated with spermatozoa (Boar A) in medium that contained 0, 2, 5, 10, 15, and 20 mM caffeine and CPPs (1 mg/ml), or in medium that contained the same caffeine concentrations without CPPs. When CPPs were added to the caffeine-containing medium, significantly higher penetration rates were obtained than when the oocytes were inseminated in the CPPs-free medium. When the oocytes were inseminated with the spermatozoa (Boar A, B, C) in medium that contained 5 mM caffeine and dephosphorylated CPPs (dCPP:1 mg/ml), the penetration rate was significantly lower than when the oocytes were inseminated with the spermatozoa in medium containing 5 mM caffeine and CPPs (1 mg/ml). In Experiment 2, the concentration of Ca²⁺ in ejaculated spermatozoa of proven low in vitro fertilization rates during incubation in the fertilization medium was determined with fluorescence, Fura2/AM. When the medium contained CPPs, the intracellular concentration of Ca²⁺ in spermatozoa increased with a peak of 113 nM after 90 min of incubation. The concentration of Ca²⁺ was gradually decreased in the medium without CPPs. However, addition of CPPs in the medium had no effect on the motility of spermatozoa in Experiments 1 and 2. These results indicate that CPPs promote Ca²⁺ uptake by spermatozoa and are effective for capacitation and/or acrosome reaction of spermatozoa leading to sperm penetration when caffeine is present in the medium and that the effect is reduced by dephosphorylation of CPPs. © 1994 Wiley-Liss, Inc.     

Influence of sperm:oocyte ratio during in vitro fertilization of in vitro matured cumulus-intact pig oocytes on fertilization parameters and embryo development

The present study was conducted to evaluate the influence of sperm:oocyte ratio during in vitro fertilization (IVF) of in vitro matured cumulus-intact oocytes on fertilization parameters and embryo development in pigs. In vitro matured oocytes surrounded by intact cumulus cells (COC) were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000:1, 3000:1, 4000:1, 6000:1, and 8000:1). Denuded oocytes inseminated with 2000 frozen-thawed spermatozoa:oocyte were the control group. A total of 2546 oocytes in five replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture (EC) medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The penetration rate increased in the COC groups with the sperm:oocyte ratio, reaching the highest rates with 8000:1 spermatozoa:oocyte (72.1 +/- 6.5%), similar to the control (73.5 +/- 3.5%). However, the monospermy was highest with the lower spermatozoa:oocyte rates (82.6-94.8%) and decreased drastically (P<0.05) in the COC group fertilized with 8000 sperm:oocyte (36%). The efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) showed no difference among the COC groups (20-30%) but they were significantly lower (P<0.007) than those obtained by the control group (43.7 +/- 2%). Embryo development was highest in the control group (58% for cleavage and 23% for BF) but not significantly different with the 6000 and 8000 sperm:oocyte COC groups (47 and 50% for cleavage and 19 and 17% for BF, respectively). These results indicate that the use of COC for IVF involves a drop in the efficiency of the fertilization and the necessity to increase the frozen-thawed sperm:oocyte ratio three to four times more to obtain similar embryo development to denuded oocytes.     

Chromosome abnormalities in secondary pig oocytes matured in vitro

Abnormalities of chromosome segregation during in vitro maturation of oocytes cause failure of in vitro fertilization. Oocytes collected from pig ovaries after slaughter were matured in vitro (IVM) for 30-48 h. In total, 1144 secondary oocytes were studied cytogenetically. An unreduced (diploid) chromosome set was identified in 146 spreads (12.8 %). A higher proportion of diploidy was noticed in secondary oocytes matured for 40 h and longer (15.0 %) than in the groups matured for 30 and 36 h (9.0 %). Among 998 secondary oocytes with the reduced chromosome number, 612 could be analyzed in detail. Hypohaploidy (n=19-1) was identified in 22 cells (3.59 %) and a hyperhaploid (n=19+1) set of chromosomes was identified in 15 cells (2.45 %). The rate of aneuploidy, estimated by doubling the rate of hyperhaploidy was 4.9 %. It was also found that aneuploid spreads occurred more frequently in the group of oocytes matured for 40 h and longer. Small acrocentrics were mostly found as an extra chromosome in the hyperhaploid spreads. Our study indicates that to avoid an excess of chromosomally abnormal secondary oocytes, IVM duration of pig oocytes should not exceed 40 h.     

In vitro fertilization of pig oocytes after different coincubation intervals

The effects of gamete coincubation time on porcine in vitro fertilization for 4, 6 or 8 hours in Trial 1 or for 1, 2, 3 or 4 hours in Trial 2 were studied. The 876 oocytes were recovered from oviducts of prepubertal gilts. Ovulation was induced. Excessive spermatozoa attached to zona pellucida were removed by pippeting after coincubation (1 to 8 hours). Optimum results were obtained after 3 to 4 hours of coincubation, when 23 to 29% of the oocytes were fertilized by a single spermatozoa. Shorter intervals of coincubation were characterized by reduced percentages of fertilized oocytes and longer intervals of coincubation by increased rates of polyspermic fertilization. The employed sperm concentration was deliberately high (2 x 10(6) sperm/ml). The results show that a coincubation time of 4 hours may be optimal for pig in vitro fertilization. Under these assay conditions, modification of other factors such as sperm concentration, medium volume and physiological environment may increase the percentage of monospermic fertilization.     

Effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes

This study was carried out to investigate the effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes. The concentrations of frozen-thawed sperm were 0.2 x 10(7), 2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml, respectively. Culture media were NCSU-23, HEPES-buffered (25 mM) NCSU-23, PZM-3 and PZM-4, respectively. Increasing the sperm concentration from 0.2 x 10(7) to 2 x 10(7)/ml, significantly increased the penetration rate. Also, increasing the sperm concentration from 20 x 10(7) to 200 x 10(7)/ml increased the penetration rate from 62.1% to 69.9%, respectively, with no differences between these two concentrations. A similar pattern was observed for polyspermic penetration and male pronucleus formation. The mean number of sperm per oocyte significantly increased in the 20 x 10(7)/ml and again in the 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes at the 2 x 10(7)/ml sperm concentration was significantly higher than that at the 0.2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes and the cell numbers per blastocyst were significantly higher in the HEPES-buffered NCSU-23 culture medium than in the NCSU-23, PZM-3 and PZM-4 culture media under a gas atmosphere of 5% CO2 in air.     

Developmental competence of pig oocytes matured and fertilized in vitro

Pig follicles 3 to 6 mm in diameter were everted and matured for 44 h. The oocytes were then collected and exposed to capacitated boar sperm purified by centrifugation in a two step (65 and 70%) Percoll gradient. Of 110 ova fixed 14 h after in vitro fertilization, 78% were penetrated and 47% were monospermic. Next, 681 oocytes were cultured in vitro for 44 h after in vitro fertilization and the 266 embryos which had reached the two- to four-cell stage were transferred into the oviducts of 12 synchronized recipient gilts. Four days later, 211 embryos (79%) were recovered by uterine flushing. 40.7% of these were at the blastocyst stage, and 20% were at the morula stage. In a final experiment, four out of eight gilts which had received 40 to 50 two- to four-cell embryos, were diagnosed pregnant 30 and 37 d after in vitro fertilization. One sow farrowed nine live piglets and one stillborn, two pregnancies were in progress, while one sow returned to estrus 47 d after in vitro fertilization. These results demonstrate that pig oocytes matured and fertilized in vitro can develop to the blastocyst stage and establish a normal pregnancy resulting in the birth of live piglets.     

In vitro fertilization and cleavage capability of bovine follicular oocytes classified by cumulus cells and matured in vitro

Bovine oocytes were collected from ovaries obtained from an abattoir. They were classified according to the character of the cumulus cells using a stereomicroscope, and cultured in 25 mM Hepes buffered Tissue Culture Medium 199 supplemented with 10% fetal calf serum at 39 degrees C and inseminated by capacitated sperm. Maturation rates of Class A oocytes, with compact, dense cumulus cells; Class B, partially naked oocytes with thin cumulus layers or small remnants of cumulus cells and Class C, naked oocytes were 97.4% (38/39), 89.8% (106/118) and 52.9% (45/85), respectively. The fertilization rates for the three classes were 86.8%, 85.8% and 53.3%, respectively. The naked oocytes had a significantly lower fertilization rate than oocytes of the other two classes. Significantly more Class A oocytes cleaved (63.7%, 232/364) than those of Class B (29.5%, 36/122) and Class C (17.7%, 28/158).     

In vitro culture of sheep oocytes matured and fertilized in vitro

Sheep oocytes that matured and fertilized in vitro were cultured to evaluate their cleavage to the 8- to 16- cell stage and further development in five different media as follows: 1) CPMW (TCM199 + 20% ewe serum + 0.4% BSA), 2) Ham's F-10 + 10% ewe serum, 3) Brinster's pyruvate medium + 0.1% glucose (BPM-G), 4) co-culture with sheep oviduct epithelial cells in TCM199 + 10% fetal calf serum, and 5) co-culture with sheep granulosa cells in the same medium as 4. The culture duration was 4 or 7 d for 8- to 16-cell or further development. The proportions of 8- to 16-cell eggs were 1) 16% (8 49 ), 2) 25% (12 49 ), 3) 52% (58 112 ), 4) 63% (105 167 ) and 5) 45% (27 60 ). The co-culture with sheep oviduct cells resulted in a significantly (P < 0.05) higher rate of cleavage than the other media, except BPM-G. The proportion of noncompacted morula (35%, 24 68 ) was also significantly (P < 0.05) higher in the co-culture of sheep oviduct cells than the other media. The 8- to 16-cell eggs produced by BPM-G (n=38) and the co-culture with sheep oviduct cells (n=42) were transferred into the uterus of recipient ewes, but no elongated blastocysts were obtained 13 d later. On the other hand, 8 out of 55 one-cell eggs (15 to 18 h after in vitro insemination) transferred to the oviduct of recipient ewes were elongated blastocysts (24% of 34 recovered eggs). The data show that the co-culture of in vitro fertilized eggs with sheep oviduct epithelial cells could support development of 8- to 16-cell embryos or early morula, but their viability is still questionable.