In vitro fertilization of in vitro matured pig oocytes: effects of boar and ejaculate fraction

Ejaculates from 3 young boars were collected on 4 occasions as a series of separate 15-ml fractions. The contribution of different fractions of these ejaculates to observed variability in the quality of the semen when used for IVF was then determined. On the basis of sperm concentration, 3 fractions representing the first peak concentration (Fraction 1), the lowest sperm concentration after Fraction 1 (Fraction 2), and the second peak concentration (Fraction 3) were selected for analysis in vitro. Oocyte-cumulus-granulosa cell complexes were obtained by dissection from slaughterhouse ovaries. In vitro matured oocytes were randomly assigned for fertilization by the 3 semen samples from each boar. Sperm concentration was the same in all the samples during prefertilization incubation, while the final concentration for fertilization was 5 x 10(5) sperm/ml. Data were analysed using ANOVA for a split-plot design. In the presence of fraction effects, Student-Newman-Keuls (SNK) test was used for multiple comparison of treatment means. Oocyte penetration rates differed among fractions (P = 0.001) and varied from 69 to 100% (mean 95.7%) for Fraction 1, from 0 to 100% (mean 53.3%) for Fraction 2, and from 50to 100% (mean 89.9%) for Fraction 3. There were also differences in male pronuclear formation rate (P = 0.028; mean 27.6, 9.3 and 16.4% for Fractions 1, 2 and 3, respectively); in the rate of polyspermy (P = 0.0001; mean 92.3, 31.9 and 76.3% for Fractions 1, 2 and 3, respectively); and in the number of penetrated spermatozoa per oocyte P = 0.002; mean 5.58, 1.94 and 4.07 for Fractions 1, 2, and 3, respectively). The first peak concentration of semen (Fraction 1) showed superiority in fertilizing ability and less variability in penetration rate from replicate to replicate compared with the other 2 fractions. By multiple comparison, Boar 1 showed higher rates of penetration (P < 0.05), male pronuclear formation (P < 0.05) and polyspermy (P < 0.05) than the other 2 boars. There was no fraction-by-boar interaction. The IVM-IVF system adopted proved to be a promising method for boar semen evaluation.     

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.     

Does multivariate analysis of post-thaw sperm characteristics accurately estimate in vitro fertility of boar individual ejaculates?

The objective of this study was to determine if a multivariate pattern analysis of frozen-thawed sperm characteristics of boar semen of unknown fertility, thus identifying groups of ejaculates as "good" or "bad" freezers, would estimate their fertilizing potential in an in vitro embryo production (IVP) system. Frozen-thawed spermatozoa from a single ejaculate collected from 46 boars were evaluated for sperm motility and kinematic patterns, for sperm viability and for early changes in sperm membrane stability. All data generated were used for a multivariate pattern analysis (PATN; CSIRO, Canberra, Australia) which objectively classified all ejaculates within a data set in to one of two groups, categorised as "good" (n = 25) or "bad" (n = 21) according with their freezability. In vitro matured oocytes were exposed to 2000 or 4000 frozen-thawed spermatozoa per oocyte for 6h and then cultured in embryo culture medium for either 6h (assurance of fertilization) or 7 days (to collect data on embryo development). Rates of sperm oocyte penetration and of embryo development significantly (p < 0.05) increased in a sperm:oocyte ratio-dependent manner. A similar pattern was observed when sperm characteristics were grouped. Indeed, ejaculates classified as "good" showed significantly (p < 0.05) higher rates of oocyte penetration, cleavage and of blastocyst formation than those classified as "bad". However, variation was still present among individuals (ejaculates, boars) in their ability to produce blastocysts in vitro. It is therefore concluded that despite the presence of a relationship for ejaculates with good semen quality post-thaw (thus grouped as "good") to higher IVP-results, the presence of individual variation does not allow for an accurate estimation of in vitro fertility based solely on the frozen-thawed semen quality parameters of a single ejaculate from a given boar.     

Acrosome reaction of boar spermatozoa in homologous in vitro fertilization

Ejaculated spermatozoa from four different boars were used to evaluate the acrosome reaction during in vitro fertilization with homologous ovulated oocytes. The acrosome reaction was assessed according to a peroxidase-labeling peanut agglutinin method and a triple-stain technique. An increase in the proportion of living sperm with reacted acrosomes was observed after preincubation and 2 hr of coincubation (P < 0.05). The percentage of true acrosome-reacted sperm remained reasonably constant throughout coincubation. In vitro penetration rates of oocytes varied among boars, but no relationship was found between fertilization rates of oocytes and maximum percentages of acrosome reacted living sperm. © 1993 Wiley-Liss, Inc.     

In vitro sperm penetration through the zona pellucida of immature and in vitro matured oocytes using fresh, chilled and frozen canine semen

The aim of this study was to evaluate the effect of sperm cryopreservation and the maturation state of the oocyte on the time course of canine gamete interaction during co-culture for periods of 1-10 h. Semen samples were obtained by digital stimulation and ejaculates processed as fresh, chilled and frozen samples. Sperm were co-cultured with immature or in vitro mature bitch oocytes for up to 10 h. At hourly intervals, oocytes were evaluated for sperm penetration with epifluorescence microscopy. The results were analyzed statistically using generalized linear models. Spermatozoa treatments had a significant effect on the total percentage of oocyte penetration for both types of oocytes; fresh spermatozoa showed the highest average penetration rate, while frozen sperm showed the lowest value (p<0.05). At the 1st hour of co-culture, chilled and frozen dog sperm had a higher penetration percentage (p<0.05) of in vitro matured canine oocytes (43.6% and 45.7%, respectively) than the fresh sperm had (33.8%). Sperm penetration was directly proportional to the time of incubation, when fresh or chilled sperm were used (P<0.05); in contrast, frozen dog sperm did not change penetration rates with either immature or in vitro matured oocytes over time. There was a significant difference in the average of penetration rate between immature (47.3%) and in vitro matured oocytes (56.6%) throughout the 10h of culturing; irrespective of sperm treatment. The optimal incubation time in terms of maximizing penetration rates probably are dependent on how spermatozoa were processed prior to fertilization.     

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.     

Effect of short periods of sperm-oocyte coincubation during in vitro fertilization on embryo development in pigs

The present study was conducted to determine if the efficiency of in vitro pig embryo production could be improved by a reduction in the period of time that oocytes are exposed to sperm during in vitro fertilization. A total of 1596 immature cumulus-oocyte complexes from five replicates were matured in vitro and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) for 10, 30, 60 min or 6h (control group). The oocytes from short coincubation times were washed three times in fertilization medium to remove spermatozoa not bound to the zona and transferred to another droplet of the same medium (containing no sperm) for 6h. After 6h, the oocytes from each group were cultured in embryo culture medium for another 6h to assess fertilization parameters and for 7 days to assess embryo development. After each period of coincubation, some oocytes were stained with Hoechst-33342 to count zona-bound sperm. Although the number of zona-bound sperm increased with the coincubation time (34.1 +/- 1.7, 46.8 +/- 2.8, 62.8 +/- 3.8 and 139.5 +/- 6.1 for 10, 30, 60 min and 6h, respectively, P < 0.02), the penetration rate was not significantly different among groups (61.3-68.2%). However, the efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) increased (P < 0.04) as the coincubation time was increased (26.6 +/- 2.9%, 29.0 +/- 4.4%, 39.5 +/- 6.2%, and 49.3 +/- 3.0% for 10, 30, 60 min and 6h, respectively). Nevertheless, there were no significant differences among groups in blastocyst formation rates (17.5-25.5%). These results demonstrate that although a sperm-oocyte coincubation time of as little as 10 min results in fertilization rates similar to a 6-h coincubation, the reduction in the period of time of sperm-oocyte coincubation does not improve the efficiency of in vitro pig embryo production.     

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).     

In vitro penetration of fresh and vitrified swine oocytes by homologous spermatozoa using different incubation systems

The present study consisted of two experiments. In the first one, ejaculates from four boars were used to compare in vitro penetration (IVP) rates of fresh and vitrified swine oocytes by homologous spermatozoa in four treatments: fresh oocytes in conventional incubation (CO2 incubator) (FC), vitrified oocytes in conventional incubation (VC), fresh oocytes in submarine (bag) incubation (FS) and vitrified oocytes in submarine incubation (VS). The IVP rates for FC, VC, FS and VS were 46.5, 44.3, 36.9 and 33.1%, respectively. Analysis through Chi-square tests identified no differences in IVP rates between FC and VC and between FS and VS (P > 0.05), but IVP rate for FC was greater (P < 0.05) than those for both FS and VS. Besides IVP rate for VC did not differ (P > 0.05) from those for FC and FS, but it was greater than that for VS (P < 0.05). Logistic regression analysis identified differential effects of treatments dependant on individual boars. The second experiment evaluated the influence of semen storage period on the semen quality of the two boars associated with greater IVP rate in the first experiment. Semen quality was estimated by IVP rate using the VC treatment and by the following methods: sperm motility, sperm morphology, hypoosmotic swelling test (HOST) and thermal stress test (TST). According to analysis using Chi-square tests, IVP rate did not differ (P > 0.05), for the first boar, between 0 (100.0%) and 24 h of semen storage (98.1%) nor after 48 and 72 h (66.0 and 59.3%, respectively), but IVP rates were greater during the 0-24 h period compared with the 48-72 h period (P < 0.05). For the second boar, IVP rate at 0 h (50.6%) was greater (P < 0.05) than at 24, 48 and 72 h of semen storage (34.3, 28.3 and 24.0%, respectively), with no further differences observed after 24 h (P > 0.05). Logistic regression analysis identified that the effect of storage on IVP rate was influenced by the effect of individual boars. No differences in semen quality during the storage period were identified by conventional methods of semen evaluation, for either boar (P > 0.05) using analysis of variance with repeated measures. These results indicate that IVP test can be used to estimate boar fertility, even when vitrified oocytes are used (if using conventional CO2 incubators) or using an alternative submarine incubation system (if using fresh oocytes). The IVP test was the only method of semen evaluation that identified the reduction in semen quality up to 72 h of storage.     

Evaluation of fertility status of Murrah buffalo bulls in vitro using zona-free hamster eggs

Cryopreserved semen samples from 10 Murrah buffalo bulls were used for sperm penetration bioassay using zona-free hamster oocytes. The samples were evaluated for sperm motility, viability and acrosome integrity. Actively motile spermatozoa recovered by the swim-up technique were capacitated using calcium ionophore A(2 3 1 8 7). Mature female golden hamsters were superovulated with 50 IU PMSG followed 56 h later by 75 IU hCG. Cumulus mass, recovered by puncture of oviducts at the infundibulum region, was treated with 0.1% hyaluronidase and 0.1% trypsin to obtain zona-free oocytes. After coincubation of zona-free oocytes with capacitated buffalo spermatozoa, scoring was done as fertilization percentage and fertilization index. The correlation coefficients with conception rate were statistically significant with fertilization percentage (r = 0.588, P < 0.05) and fertilization index (r = 0.660, P < 0.01). However, conventional parameters like viability, motility and acrosome integrity showed poor correlation with conception rate.     

Effects of maturational stage, cumulus cells and coincubation of mature and immature cumulus-oocyte complexes on in vitro penetrability of porcine oocytes

The in vitro penetrability of porcine oocytes is conditioned by several factors, some of which remain unclear. Knowledge of the different effects of the cellular components involved in penetrability would no doubt serve to simplify laboratory IVF methods. This study was designed to evaluate the effects of the following factors on penetrability: oocyte maturational stage, the presence of isolated or oocyte-attached cumulus cells, and coincubation of in vitro-matured and immature oocytes. Immature oocytes and oocytes matured in Waymouth medium were obtained from non atretic follicles and fertilized in TCM 199 medium. Sperm-rich fractions were collected by the gloved hand method and semen was used for IVF at a final concentration of 1 x 10(6) cells/mL in all experiments. Under the same conditions of IVF, the penetrability of the immature cumulus-oocyte complexes (COCs) was significantly lower than that of mature COCs, in terms of penetration rate and mean number of sperm per penetrated oocyte. This difference was abolished when the oocytes were denuded, leading to similar penetration rates. Coincubation of mature and immature COCs reduced the penetrability of immature COCs compared with that observed when these were incubated in isolation. However, neither the addition of isolated cumulus cells from decumulated mature oocytes nor the addition of denuded mature oocytes to immature COCs modified the penetration rate. These findings suggest that the presence of surrounding cumulus cells is mainly responsible for the differences observed in penetrability, regardless of the maturational stage of the oocyte. Moreover, when mature and immature COCs are coincubated, penetrability of immature COCs is diminished by the effects of the mature COC and not by the independent actions of the cellular components.     

Variability in relationships between semen quality and estimates of in vivo and in vitro fertility in boars

The present experiment was designed to characterize relationships between common semen quality and fertility estimates for three boars known to differ in farrowing rate, number of pigs born alive, and monospermic penetration rate. The approach chosen to accomplish this was to monitor semen quality from these boars and use their semen alternately for either artificial insemination or in vitro fertilization for 40 weeks. This strategy relied on the variability in semen quality parameters that normally occurs in an individual boar over time. When comparisons were made among boars, farrowing rates, numbers of pigs born alive, and monospermic penetration rates were significantly different, but progressive motility, normal head and tail morphology, and acrosome morphology were not. However, when comparisons were made among ejaculates within individual boars, there were significant effects of semen quality on both in vivo and in vitro fertility. For boar 3495, the proportion of spermatozoa exhibiting progressive motility and distribution of spermatozoa in a percoll gradient had a positive linear effect on number born alive and monospermic penetration rate, respectively. For boar 2901, quadratic equations best described changes in litter size as a function of progressive motility and normal acrosomes. In addition, monospermic penetration rate increased linearly as normal acrosomes and the proportion of spermatozoa recovered from a percoll gradient increased. For boar 4291, the relationship between progressive motility and number born alive and between normal acrosomes and number of pigs born alive were also quadratic. However, a significant linear relationship was present only between normal acrosomes and monospermic penetration rate. These results demonstrate that simply relying on the means of common semen quality estimates from some boars has limited value in terms of being used as a prospective indicator of their in vivo or in vitro fertility. In contrast, characterization of relationships between semen quality and fertility estimates is useful for estimating differences in the fertility of ejaculates from individual boars. However, both quantitative and qualitative differences in these relationships among boars are present and a given semen quality estimate that is a good predictor of in vivo or in vitro fertilization for one boar, may not be applicable for others.     

Prognostic value of canine frozen-thawed semen parameters on in vitro sperm-oocyte interactions

Combining the data from conventional semen analysis with oocyte penetration assays should improve the assessment of the fertilizing ability of a semen sample. Thus, the objective of the present study was to evaluate the prognostic value of various semen parameters on the in vitro interactions between frozen-thawed canine sperm and homologous oocytes. Ten ejaculates from five stud dogs (two ejaculates/dog) were collected by digital manipulation. Semen samples were evaluated, extended in Tris-egg yolk-glycerol, frozen and stored in liquid nitrogen, and thawed several weeks later. Samples were evaluated for motility and sperm populations by computer-aided semen analysis (CASA), plasma membrane integrity (carboxy-fluorescein diacetate and propidium iodide), and sperm morphology (Bengal Rose). Thawed spermatozoa were also incubated with homologous oocytes for 18 h in an atmosphere of 5% CO(2) and 95% air at 38 degrees C and sperm-oocyte interactions were evaluated. Simple linear regression models were calculated, with sperm parameters as independent variables and sperm-oocyte interactions as the dependent variable. There were significant associations between: percentage of oocytes bound to spermatozoa and beat cross frequency (BCF; R(2)=63%); percentage of oocytes that interacted with spermatozoa and BCF (R(2)=73%); and number of penetrated spermatozoa and velocity average pathway (VAP; R(2)=64%) and velocity straight line (VSL; R(2)=64%). Although plasma membrane integrity and sperm morphology had little prognostic value for in vitro interactions between canine frozen-thawed sperm and homologous oocytes, some motility patterns (evaluated by CASA) were predictive of in vitro sperm-oocyte interactions.     

The effect of sperm preparation and co-incubation time on in vitro fertilization of Bos indicus oocytes.

The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm-oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18h. After co-incubation oocytes were transferred to the culture medium and culture for 44h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P<0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm-oocyte co-incubation time was observed (P>0.05). However, sperm-oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P<0.05) progressively at 6h (63.3 and 54.4%) and 12h (77.6 and 67.6%). No differences (P>0.05) were observed between 12 and 18h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P>0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12h, and the prolongation of that time for up to 18h had no detrimental effect on fertilization.     

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.     

Correlation of sperm viability with gamete interaction and fertilization in vitro in the cheetah (Acinonyx jubatus).

Sperm-oocyte interaction in vitro was studied in the cheetah, a species known to produce poor quality ejaculates and to experience low rates of fertility. Twelve female cheetahs were injected (i.m.) with eCG followed by hCG 84 h later. Twenty-four to 26 h post hCG, each was subjected to laparoscopic oocyte aspiration. A sperm motility index (SMI) was calculated for each of 9 cheetah sperm donors that produced ejaculates averaging 41.3 +/- 22.9 x 10(6) motile sperm and 28.4 +/- 4.9% structurally normal sperm. Each ejaculate was used to inseminate cheetah oocytes from 1 or 2 females and salt-stored, domestic cat oocytes. The presence of ovarian follicles (greater than or equal to 1.5 mm in diameter) showed that all females responded to exogenous gonadotropins (range, 11-35 follicles/female). A total of 277 cheetah oocytes was collected from 292 follicles (94.9% recovery; 23.1 +/- 2.2 oocytes/female). Of these, 250 (90.3%) qualified as mature and 27 (9.7%) as degenerate. Of the 214 mature oocytes inseminated, 56 (26.2%) were fertilized, and 37 (17.3%) cleaved to the 2-cell stage in vitro; but the incidence of in vitro fertilization (IVF) varied from 0 to 73.3% (p less than 0.001) among individual males. When oocytes from individual cheetahs (n = 5) were separated into two groups and inseminated with sperm from a male with an SMI greater than 0 after 6 h coincubation versus an SMI = 0 at this time, the mean fertilization rates were 28/44 (63.6%) and 0/37 (0%), respectively (p less than 0.05). Of the 117 domestic cat oocytes coincubated with cheetah sperm, 96.6% contained 1 or more cheetah sperm in the outer half of the zona pellucida (ZP). Although the mean number of cheetah sperm penetrating the outer ZP of the cat oocyte was similar (p greater than 0.05) among all males, there was a positive correlation between the number of sperm reaching the inner half of the ZP and fertilization rate in vitro (r = 0.82; p less than 0.05). Compared to IVF efficiency in the domestic cat and tiger as reported in earlier studies, IVF efficiency in the cheetah is low. Because oocytes from 11 of 12 cheetahs were fertilized in vitro, there is no evidence that the female gamete is incompetent. Although sperm pleiomorphisms may contribute to poor reproductive performance, examination of the data on the basis of individual sperm donors reveals that effective gamete interaction in the cheetah is dictated, in part, by sperm motility.     

Effects of the 7 21 Robertsonian translocation on fertilization rates and preimplantation development of bovine oocytes in vitro

A study was conducted to investigate the effect of the 7/21 Robertsonian translocation on fertilization and subsequent development of bovine oocytes matured in vitro. Semen from Japanese Black bulls, 2 with a normal karyotype (Bulls A and B) and 2 that were heterozygous for the 7/21 translocation (Bulls C and D), was used in this study. In vitro matured bovine oocytes were inseminated with frozen-thawed sperm capacitated with heparin. After insemination, oocytes were cultured at 38.5 degrees C on a monolayer of cumulus cells in TCM-199 supplemented with 5% superovulated cow serum and 0.5 mM sodium pyruvate in an atmosphere of 2% CO2 in air. Cleavage rate was evaluated at 54 h after insemination, and development of embryos to the blastocyst stage was observed 7 to 10 d post insemination. There was no difference in the fertilization rate among the 4 bulls. Although the cleavage rate of oocytes inseminated with semen from Bull C (heterozygote) was lower (P < 0.05) than that obtained with semen from Bull B (normal), the blastocyst formation rate did not differ among the 4 bulls. These results indicate that the 7/21 Robertsonian translocation had no effect on the fertilization and blastocyst formation rates of bovine in vitro-matured oocytes.     

Effect of semen preparation on IVF of prepubertal goat oocytes

The aim of these experiments was to study the effects of different methods of washing and selection of spermatozoa on the IVF of IVM oocytes from prepubertal goats. Fresh ejaculates from 3 males of proven fertility were processed according to the following treatments: 1) centrifugation in TALP, 2) centrifugation in sucrose-based Ficoll medium, 3) centrifugation in Percoll gradients at 40 and 80%, 4) by swim-up and 5) by dilution of spermatozoa (1:40) in (1:1) TALP. In all 5 treatments spermatozoa were incubated for 45 min with 100 microg/mL of heparin and then added to Fert-TALP. Oocytes were matured for 27 h in TCM-199 supplemented with 20% estrous goat serum (EGS), FSH, LH and estradiol-17beta. Spermatozoa (4x10(6) cells/mL) were coincubated with oocytes in 100 microL of Fert-TALP with hypotaurine for 24 h, after which the oocytes were transferred to a granulosa cells monolayer in TCM-199 plus 10% of EGS for 24 h (48 h post insemination). At 17 h post insemination a sample of sperm-exposed oocytes was taken and stained in lacmoid to observe sperm penetration and the formation of pronuclei. At 48 h post insemination the cleavage rate of oocytes was evaluated. Motility, viability and acrosome status of the spermatozoa were evaluated immediately after the mixing of the ejaculates, after washing and selection treatments, and after incubation with heparin and at 17 h post insemination. The different ejaculate treatments did not affect the penetration and cleavage rates of oocytes. At 48 h post insemination the cleavage rate was 46.9, 36.6 and 29.0% for dilution, Ficoll and swim-up preparations, respectively. Only the swim-up protocol improved sperm motility and viability compared with that of the initial semen sample and with the other sample treatments. At 17 h post insemination the semen parameters were the same for all sperm sample treatments.     

Zona-penetration in vitro test for evaluating boar sperm fertility

We investigated the capacity of porcine sperm-zona binding and penetration by using bioassay to differentiate between spermatozoa from fertile and subfertile boars. Semen was collected from Large White boars grouped into categories of fertile and subfertile (n=5 per each group) according to the results of artificial insemination. Boars in both groups showed similarly hyperactivated sperm motility at insemination (44.72 and 43.03% respectively) regardless of the lower percentage of progressive motility observed in the ejaculates of subfertile boars. At in vitro insemination, a high proportion of the sperm population (43.76%) in the subfertile boars was without acrosomes, while in the fertile boars this proportion was only 24.35%. The sperm penetration rate of fertile boars reached 66.03% while that of subfertile boars was only 25.08%. In conclusion, the results of our study showed that the penetration rate by boar spermatozoa of the zona pellucida can be used to predict fertility and/or as an in vitro standard for describing porcine semen characteristics.