In-vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa.

Pig oocytes matured in culture were inseminated with frozen-thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85-89%) and increased incidence of polyspermy were obtained at 25-100 x 10(6) spermatozoa/ml. Wide variation in penetration rates (16-89%) was observed in oocytes inseminated in medium containing 5mM caffeine and at 25-50 x 10(6) spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25-50 x 10(6) spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mM caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5-40 micrograms/ml. When heparin was included in the medium with 5mM caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.     

Effects of caffeine and/or heparin in a chemically defined medium with or without glucose on in vitro penetration of bovine oocytes and their subsequent development

Bovine cumulus-enclosed oocytes were matured in culture for 22 to 24 h, freed from cumulus cells and inseminated with frozen-thawed spermatozoa in a chemically defined medium containing 1 mg polyvinylalcohol/ml with or without 5 mM caffeine and/or 10 micrograms heparin/ml and 13.9 mM glucose. Penetration of oocytes was observed only in the medium containing caffeine and/or heparin. Regardless of the presence of glucose, similar proportions of oocytes were penetrated in the medium containing heparin with (73 and 83%) or without (36 and 41%) caffeine. However, when the medium was supplemented with caffeine only, a higher penetration rate was observed in the presence (41%) than in the absence (27%) of glucose. When oocytes inseminated in medium containing caffeine and heparin with or without glucose were cultured in a chemically defined, protein-free medium, 72 and 90% and 9 and 21% of inseminated oocytes developed to the > or = 2-cell and blastocyst stages 48 and 192 h post insemination, respectively. These results, obtained using chemically defined conditions, indicate that glucose is required for stimulating fertilization in vitro of bovine oocytes and that synergistic action of caffeine and heparin appears independently of the reversing activity of caffeine on the inhibition of heparin-induced sperm capacitation by glucose.     

Penetration of bovine follicular oocytes by frozen-thawed spermatozoa in the presence of caffeine and heparin

Bovine follicular oocytes matured in culture were inseminated with frozen-thawed spermatozoa which were either preincubated for 5-5.5 h or not preincubated in a medium with caffeine (5 mM) and heparin (10 micrograms/ml). When the oocytes with cumulus and corona cells were inseminated, spermatozoa started to penetrate oocytes 3 h later regardless of whether spermatozoa were preincubated or not. However, a significantly higher proportion of oocytes was penetrated by preincubated than non-preincubated spermatozoa. When the oocytes were freed from cumulus and corona cells, penetration was observed to start 1 h after insemination and there were no differences in penetration rates 1-5 h after insemination between preincubated and non-preincubated spermatozoa. This study demonstrates that capacitation and the acrosome reaction of bovine spermatozoa can be induced within 1 h in a medium containing both caffeine and heparin when denuded oocytes are inseminated.     

In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium

The effect of BSA, caffeine and calcium was studied on the penetration of pig oocytes by frozen-thawed spermatozoa in a modified Tris-buffered medium (mTBM) without added bicarbonate. Pig cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22 h. The COC were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with spermatozoa for 6 h in mTBM containing caffeine (5 mM) and 0.1 or 0.4% BSA (Experiment 1). In Experiment 2, oocytes were inseminated in mTBM containing 0.1% BSA and various concentrations of caffeine (0 to 5 mM). In Experiment 3, insemination was carried out in mTBM containing 0.1% BSA, 1 mM caffeine and various concentrations of Ca(2+) (0.5 to 10 mM). Supplementation of mTBM with either 0.1 or 0.4% BSA resulted a high penetration rate with a high polyspermy rate. However, the mean number of spermatozoa per oocyte was significantly higher at 0.4% than at 0.1% BSA. The penetration rate, polyspermy rate and mean number of spermatozoa per oocyte were all significantly higher when 1 to 5 mM caffeine were added to the medium than in caffeine-free medium. No penetration was observed in the presence of 0.5 mM Ca(2+). The penetration rate was significantly increased from 12 to 92% at 2.5 to 10 mM Ca(2+). The mean number of spermatozoa per oocyte did not differ between 2.5 and 5 mM Ca(2+) but increased significantly at 7.5 and 10 mM. These results show the successful in vitro penetration of pig oocytes in a chemically semidefined medium without added bicarbonate. Although BSA and caffeine can modulate the rate of sperm penetration, calcium seems to be an important regulatory ion.     

The effect of preincubation of frozen-thawed spermatozoa with oviductal cells on the in vitro penetration of porcine oocytes

Porcine follicular oocytes matured in culture were inseminated with frozen-thawed boar spermatozoa preincubated for 0, 1, 2, 3 and 4 h. The penetration rate was higher at Time 0 (59.5%) than with preincubation of spermatozoa for 1 to 4 h in the control medium (19.7 to 23.8%). When the oocytes were inseminated with spermatozoa incubated with oviductal vesicles, no decrease in penetration rates was observed for up to 4 h of preincubation. When spermatozoa were incubated with oviductal vesicles for 1 and 2 h, the penetration rates were significantly higher (P < 0.05) in those with (57.0 and 50.6% for 1 and 2 h) than without (39.5 and 30.8% for 1 and 2 h) caffeine. In a second experiment, the penetration rates were significantly (P < 0.05) higher in medium with (64.5%) than without (39.1%) caffeine when oocytes were inseminated with spermatozoa preincubated for 2 h in presence of oviductal epithelial cell monolayer. The rate of polyspermy in penetrated oocytes in medium without cells decreased with the period of sperm preincubation (54.5, 30.0, 10.5, 13.5 and 0% for 0, 1, 2, 3 and 4 h, respectively). Despite higher penetration rates with cells, no differences were observed in polyspermy rates in the presence of oviductal vesicles or epithelial cell monolayer compared to caffeine alone. These results indicate the significant advantages of preincubating spermatozoa with oviductal vesicles and epithelial cell monolayer for 1 and 2 h to maintain penetration potential without increased polyspermy rates during in vitro fertilization in the pig.     

Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes

Pig follicular oocytes cultured in a defined medium for 28-29 h were inseminated in vitro by epididymal or ejaculated boar spermatozoa that were preincubated in a modified KRB solution at various sperm concentrations for 4 h at 37 degrees C. Sperm concentration at insemination was 2 X 10(6) cells/ml. When epididymal spermatozoa were preincubated at concentrations of 4-16 X 10(8) cells/ml, 71-75% of oocytes were penetrated. In contrast, preincubation at a low concentration (0.8 X 10(8) cells/ml) resulted in a low penetration rate (11%). Epididymal spermatozoa preincubated at a concentration of 4 X 10(8) cells/ml could also penetrate denuded oocytes. None of the oocytes were penetrated by epididymal spermatozoa that were exposed to seminal plasma before preincubation or by ejaculated spermatozoa. After preincubation, whiplash motility was observed in the epididymal spermatozoa, but not in the ejaculated spermatozoa.     

Synergistic effect of caffeine and heparin on in-vitro fertilization of cattle oocytes matured in culture

Frozen-thawed spermatozoa obtained from six different bulls were suspended in Brackett and Oliphant's (BO) medium (14), with or without 10 mM caffeine, after washing. A 50-mul aliquot of the sperm suspension was added to the 50-mul BO medium supplemented with bovine serum albumin (BSA, 20 mg/ml) and heparin (20 mug/ml) in which the bovine follicular oocytes matured in culture had been introduced previously. The proportion (35%) of oocytes penetrated in the presence of heparin alone 20 to 24 h after insemination was not significantly different from those (32%) penetrated in the presence of caffeine alone as reported previously (1). When heparin was added to the caffeine in the fertilization medium, the penetration rate of oocytes increased significantly to 68% (P < 0.001), indicating that both chemicals act sinergistically to induce capacitation and/or acrosome reaction of spermatozoa and stimulate in vitro fertilization of cattle oocytes. However, great variation in penetration rates (35 to 96%) was observed among the different bulls. The optimal concentration of heparin in the suspension medium in which the highest rate of oocyte penetration took place was 10 mug/ml.     

Effect of oviductal epithelial cells on fertilization of pig oocytes in vitro

The incidence of polyspermy is reduced by co-culture of pig oocytes with oviductal cells. It is not known whether the effect is due to soluble factors secreted into the medium. Oviductal epithelial cell monolayers and cell-conditioned media were prepared and their effects on fertilization of pig oocytes were examined. In vitro matured pig oocytes were inseminated with ejaculated boar spermatozoa at a concentration of 1 x 10(5) or 1 x 10(6) cells/ml and co-cultured in one of 5 culture systems: an oviductal epithelial cell monolayer, a fibroblast monolayer, an oviductal epithelial cell-conditioned medium, or a fibroblast-conditioned medium, and medium alone (modified-TCM199). In all 5 systems, the majority (range 85 to 100%) of the oocytes were penetrated by sperm. When oocytes were inseminated with spermatozoa at a concentration of 1 x 10(5) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with oviductal epithelial cells and fibroblasts than that of the oocytes cultured without these cells. In contrast, when oocytes were inseminated with spermatozoa at a concentration of 1 x 10(6) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with epithelial cells than those cultured with the fibroblasts and in the control medium. The suppressive effect on polyspermy was observed in the oviductal epithelial cells-conditioned medium when oocytes were inseminated with spermatozoa at both concentrations of 1 x 10(5) and 1 x 10(6) cells/ml. The effect was absent in the fibroblasts-conditioned medium. Moreover, the effect of the epithelial cells was maintained during the culture period, whereas the proportion of monospermic oocytes co-cultured with fibroblasts showed a gradual decrease, reaching 0% after 16 h. These results suggest that a soluble factor(s) derived from the oviductal epithelial cells decreased the number of spermatozoa penetrating the oocytes without suppressing the high rate of fertilization.     

In vitro penetration of zona pellucida of salt-stored bovine oocytes before and after maturation by frozen-thawed spermatozoa

Bovine oocytes, before and after maturation in culture, were stored in PBS with 2 M-(NH(4))(2)SO(4) + 0.1% dextran or 2 M-(NH(4))(2)SO(4) + 40 mM-Hepes + 0.5% dextran and were inseminated with frozen-thawed spermatozoa in BO medium with caffeine (5 mM) and heparin (10 mug/ml). The penetration rates of mature oocytes were very low (19 to 24%) and not significantly different between the two salt solutions in which the oocytes were stored for 2 to 89 days. Significantly lower (P < 0.01) penetration rates were observed in immature (7 to 8%) than in mature (20 to 21%) oocytes stored in the two solutions. The synergistic effect of caffeine and heparin was observed in the penetration rate of fresh mature oocytes but not in the stored oocytes, indicating the difficulty of assessing sperm capacitation and/or acrosome reaction of salt-stored mature bovine oocytes under the present condition. Using 0.1% protease the solubility of the zonae decreased in salt-stored but not in fresh oocytes, but there was no significant difference between the immature and mature oocytes regardless of storage in the salt solutions. It appears from these results that some alteration was induced in the nature of zona glycoprotein by ammonium sulfate solution.     

Evaluation of boar spermatozoa penetrating capacity using pig oocytes at the germinal vesicle stage

We evaluated the ability of immature pig oocytes (at germinal vesicle stage) to detect differences in the in vitro penetration rates of boar spermatozoa. In Experiment 1, immature and ovulated oocytes (n=303) were exposed to capacitated boar spermatozoa to determine if the penetrability of immature pig oocytes was comparable to that of ovulated oocytes. The percentages of penetrated oocytes and the mean number of spermatozoa per oocyte were similar for immature (88.82 and 7.42+/-0.41) and ovulated oocytes (90.97 and 7.95+/-0.34, respectively). In Experiment 2, immature oocytes (n=1230) were inseminated with semen from 2 boars (A and B) with satisfactory semen characteristics to establish the variability of in vitro penetrating capacity between the boars. Semen was examined for motility, movement quality, acrosome integrity and plasma membrane integrity at various stages of the in vitro procedure. Although the sperm evaluation results were similar between boars, Boar A exhibited a significantly higher (P<0.001) penetration rate (91.49%) and number of spermatozoa penetrated per oocyte (5.90+/-0.25) than Boar B (52.87% and 2.03+/-0.12, respectively). Increasing the sperm concentration at insemination from 1x10(6) to 10x10(6) cells/ml resulted in an increased penetrating capacity for both boars, and the differences in the number of spermatozoa per oocyte between boars also increased. These results indicate that immature pig oocytes can be used in a homologous in vitro fertilization assay, and that despite similarities in semen characteristics a significant boar effect is evident for parameters of in vitro penetration of oocytes.     

Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen-thawed spermatozoa in Medium BO with caffeine (5 mM) and heparin (10 micrograms/ml). Very high penetration rates (95-100%) were obtained in all oocytes which had been cultured for 0-20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20-22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.     

IN VITRO FERTILIZATION OF IN VITRO MATURED MINKE WHALE (BALAENOPTERA ACUTOROSTRATA) FOLLICULAR OOCYTES

In vitro fertilization of follicular oocytes harvested from ovaries and matured in vitro was attempted for 55 minke whales ( Balaenoptera acutorostrata ) captured for Japanese research purposes in the Antarctic Ocean during the period from November 1995 to March 1996. In Experiment 1, effects of culture duration (96 h or 120 h) on maturation of follicular oocytes and addition of caffeine (5 mM) and/or heparin (100 pg/ml) on sperm penetration and pro-nuclear formation were investigated. Spermatozoa recovered from the vasa deferentia of four mature males were diluted (5-fold) and frozen at - 80°C. The post-thawed and pooled spermatozoa were used for in vitro insemination. A higher ( P < 0.05) proportion of the oocytes cultured for 120 h (34.2% of 260) progressed beyond the second metaphase stage than of the oocytes cultured for 96 h (26.0% of 262). For the matured oocytes, higher rates of penetration ( P < 0.05) and pronuclear formation ( P < 0.01) were obtained in the oocytes cultured for 120 h (55.1% and 40.4%) than in those cultured for 96 h (32.4 % and 20.6%). Addition of caffeine and heparin did not show a significant effect. In Experiment 2, follicular oocytes matured for 120 h and then inseminated were cultured to examine the subsequent development in two culture systems (with and without co-cultured cumulus cells). Of 448 inseminated oocytes, cleaved embryos (2–16 cells) were observed with (5.8%) and without (4.9%) co-cultured systems. No cleavage was observed in 54 ova without insemination. These results indicate that in vitro fertilization of minke whale in vitro matured follicular oocytes with cryopreserved spermatozoa is possible, yielding cleaved embryos.     

Effects of caffeine,Ca++, capacitation time,and strain on in vitro fertilization in mice

Oocytes (N=2922) were collected from superovulated female C57B16/J X DBA2/J (B6D2F₁) mice and distributed among 48 treatments consisting of a 2×3×2×2×2 factorial design. The factors were strain of spermatozoa, B6D2F₁ or SJL/J; caffeine concentration in the fertilization medium, 0,2, or 6 mM; time oocytes were exposed to sperm, 1 or 2 hours; Ca⁺⁺ concentration in the capacitation medium, 0 or 1.8 mM; and capacitation time, 1 or 2 hr. Ova were observed 400 min after they were initially exposed to 10⁵ spermatozoa per ml. Ova with two or more pronuclei and a second polar body were considered fertilized, In vitro embryonic development was monitored for 5 days. B6D2F₁ spermatozoa resulted in consistently higher rates of fertilization than SJL spermatozoa, 77.5% vs 38.7% when averaged over other treatments. Caffeine concentrations of 0,2, and 6 mM resulted in respective mean fertilization rates of 50.1%, 58.8%, and 65.4% (P<0.005) when averaged over other factors. Fertilization rates of ova exposed 1 and 2 hr to sperm were 53.0% and 63.3% (P<0.005). B6D2F₁ spermatozoa capacitated in medium with 1.8 mM Ca⁺⁺ fertilized more ova (P<0.01), 83.1%, than when no Ca⁺⁺ was present, 71.9%; this effect was absent with SJL spermatozoa. The effect of capacitation time depended on strain. Fertilization rates with B6D2F₁ spermatozoa were higher, 80.1%, with a 2-hour capacitation time than with a 1-hour capacitation time, 75.0%. Exactly the opposite was true for the SJL spermatozoa; 43.4% for the 1-hour and 34.1% for 2-hour capacitation (P<.01). Development to the blastocyst stage was significantly greater (P<0.025) for ova fertilized by B6D2F₁ (26.8%) than by SJL spermatozoa (17.7%).     

In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis).

Cumulus-oocyte complexes, 5596, were cultured for 24 h in either TCM-199 or Ham's F-10 with or without gonadotrophins and supplemented with either 20% buffalo oestrous serum (BES) or fetal calf serum (FCS). The maturation rates of oocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20% BES were 47.4 +/- 17.8 and 44.8 +/- 25.6, respectively. Addition of luteinizing hormone (LH) (5 micrograms ml-1) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20% BES (76.8 +/- 18.3), but follicle-stimulating hormone (FSH) (0.5 micrograms ml-1) and oestradiol (1 microgram ml-1) failed to synergize with LH (71.7 +/- 19.5). In the TCM-199 system, LH failed to enhance the maturation rate but the addition of FSH and oestradiol significantly enhanced the proportion of mature oocytes (42.7 +/- 1.4 and 81.7 +/- 14.5, respectively; P less than 0.05). Frozen-thawed spermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine 1(-1) + 10 micrograms heparin showed a higher fertilization rate (29.8%) than those treated in Hepes-Talp and treated with 10 micrograms heparin ml-1 (19.6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine 1-1 and 10 micrograms heparin in BO medium (50%) was used. Rate of cleavage and development were also higher when in vitro fertilization was carried out with fresh ejaculated spermatozoa treated with caffeine and heparin (34.1 and 36.8%, respectively) than with frozen-thawed spermatozoa (27.0 and 22.0%, respectively). Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28.0%) than when co-cultured on oviductal cell monolayers (8.2%). The results indicate that oocytes cultured in medium supplemented with BES and gonadotrophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa.     

Molitity and acrosomal changes in ionophore-treated bovine spermatozoa and their relationship with in vitro penetration of zone-free hamster oocytes

Frozen-thawed spermatozoa from Friesian bulls held at stud in Ireland were used to assess the effect of ionophore on motility, acrosome reaction and heterologous in vitro fertilization. Bovine spermatozoa penetrated zone-free hamster oocytes following treatment with calcium ionophore in the absence of bovine serum albumin (BSA) and in the presence of 10 mM caffeine. Sperm velocity was stimulated in concentrations of caffeine <2.5 mM following dilution with medium containing BSA. Sperm attachment to the plasmalemma showed no association with penetration rates of zona-free hamster oocytes. Penetrated oocytes in regimens with >0.1 mM ionophore did not progress through Meiosis II. Increasing concentrations of ionophore induced the acrosome reaction more rapidly, although this was associated with reduced motility. Hyperactive motility was observed in calcium ionophore-treated spermatozoa which were capable of penetrating zona-free hamster oocytes. Sperm velocity remained unchanged. whereas the track:vector ratio, a measurement of curvilinear movement, was reduced. This work may have important implications for the assessment of bovine fertility and cytogenetic analysis of bovine sperm.     

Brief coincubation of gametes in porcine in vitro fertilization: role of sperm:oocyte ratio and post-coincubation medium

In this study, a short coincubation time of 10 min was used to determine the effect of different sperm:oocyte ratios during in vitro fertilization (IVF), and different periods of post-coincubation in a medium that is not appropriate for IVF, on fertilization parameters. In the first experiment, a total of 1624 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000, 1500, 1000 and 500 sperm:oocyte) and coincubated for 10 min or 6 h. The oocytes from 10 min of coincubation were washed in IVF medium to remove spermatozoa not bound to the zona pellucida and transferred to another droplet of the same medium (containing no spermatozoa) for 6h. The oocytes from the other group remained with the spermatozoa for 6h. Oocytes from both groups were then cultured in embryo culture medium (IVC) for 12h to assess fertilization parameters. In the second experiment, 1872 in vitro matured oocytes, in 3 replicates were inseminated with frozen-thawed spermatozoa using the same sperm:oocyte ratios as in the first experiment. The oocytes were coincubated for 10 min and transferred directly to IVC medium for 18 h (group A), to IVF medium (containing no sperm) only for 2h and then to IVC medium for 16 h (group B), or to IVF medium (containing no sperm) for 6h and then to IVC medium for 12 h (group C or control). There was an effect of sperm:oocyte ratio on all fertilization parameters in experiment 1. The efficiency of IVF (number of monospermic oocytes/total number inseminated) was higher (P<0.05) for oocytes coincubated with spermatozoa for 10 min and inseminated with 1500 and 1000 sperm:oocyte (35.8+/-3 and 37.6+/-2.7%, respectively) and for those coincubated for 6h with 500 spermatozoa per oocyte (37.2+/-3.1%). In experiment 2, the penetration and efficiency rates obtained in group A were poor (between 3 and 15%) irrespective of the sperm:oocyte ratio. However, in group B the fertilization parameters were similar to the controls and were also affected by the sperm:oocyte ratio. These results demonstrate that coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocyte ratio, and that the spermatozoa bound to the zona pellucida require a maximum of 2h in an appropriate medium to penetrate the oocytes.     

Adjustments in IVF system for individual boars: value of additives and time of sperm-oocyte co-incubation

In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5,943 COC's were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa from 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2mM), hyaluronic acid (HA; [0.5mg/mL]) and adenosine (10 microM), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5mg/ml) and adenosine (0, 10, 20 and 40 microM) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 min or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 12-15 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P<0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9+/-3.9% versus 62.7+/-3.9% and 1.5+/-3.2 versus 1.3+/-3.5 for 10 min or 6h, respectively), but reduced mono-spermy (P<0.001, 57.9+/-2.5% versus 70.0+/-2.8%) when boar A was used. However, such effects were not seen with boar B, in which sperm-oocyte co-incubation time did not affect the efficiency of fertilization. In view of the present results, a preliminary screening for each individual male is required to select optimal conditions for IVF.     

In vitro capacitation of boar ejaculated spermatozoa: Effect of conditioned media prepared from preincubated sperm suspension

The fertilizing ability of boar ejaculated spermatozoa was examined in vitro after prcincubation at a concentration of 2.5 × 10⁸/ml for 4 hr in several conditioned media (CM). For preparation of CM, boar spermatozoa were incubated in a modified Krebs-Ringer bicarbonate solution (TYH) at concentrations of 20 to 40 × 10⁸/ml for several hours up to 4 hr; then their supernatant fluids were collected by centrifugation. When boar ejaculated spermatozoa were preincubated in TYH alone, 14.1% of oocytes were penetrated by them as we reported previously. On the other hand, preincubating them with CM, their fertilizing ability was elevated according as the incubation time of CM preparation was lengthened. The fertilization rate reached 75.0%, using 4 hr-incubated CM for the preincubation medium. The effect of CM was not deteriorated by heat treatments (56°C, 30 min, or 100°C, 5 min). The components of CM were separated at a molecular weight of 25,000 by ultrafiltration, and high fertilization rate (69.8%) was obtained when low molecular weight fraction was used for the preincubation medium. Sperm extracts prepared from directly frozen-thawed sperm suspension and 0.1–10 mM of taurine or hypotaurine had no effect on the fertilizing ability of boar spermatozoa. These results suggest that substances stimulating boar sperm capacitation were accumulated from viable spermatozoa into the medium during incubation and that the effective substances were heat-stable and of low molecular weight and were not taurine and hypotaurine.     

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.     

Effect of the volume of medium and number of oocytes during in vitro fertilization on embryo development in pigs

The present study was designed to determine the effect of the volume of medium (VM) and the number of oocytes (NOOC) during in vitro fertilization (IVF) on embryo development in pigs. Groups of 15, 30 and 50 in vitro matured oocytes were transferred to 2, 1 and 0.1 ml of modified Tris-buffered medium (mTBM) and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) in a 3 x 3 factorial experiment. A total of 2739 oocytes from four replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The efficiency of fertilization (EF: number of monospermic oocytes/total number of inseminated oocytes) and BF decreased (P<0.03) as the VM increased (EF: 45.9+/-2.2, 43.8+/-2.6 and 36.9+/-1.6% and BF: 29.4+/-2.7, 23.2+/-1.8 and 19.9+/-2.1% for VM 0.1, 1 and 2 ml, respectively). The BF, but not EF, was also affected (P<0.04) by NOOC (19.8+/-1.6, 28.1+/-2.3 and 24.6+/-2.9% for groups of 15, 30 and 50 oocytes, respectively). The effect of the interaction VM x NOOC on EF and BF was not significant. These results indicate that when 2000 spermatozoa/oocyte were used, a low volume of IVF medium (0.1 ml) and the number of oocytes during IVF (30-50) can improve the in vitro embryo production in pigs.