Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development

The objectives of these experiments were to characterize separation of frozen-thawed bovine spermatozoa on a Percoll gradient and then to compare sperm separation by either a swim-up or Percoll gradient procedure for the ability of spermatozoa to fertilize oocytes in vitro. The Percoll gradient was a 45 and 90% discontinuous gradient. Initial experiments found that centrifugation of semen on the Percoll gradient for 15 min at 700 g was sufficient to obtain optimal recovery of motile spermatozoa. Most of the nonmotile spermatozoa were recovered at the interface of the 45 and 90% Percoll layers, while the motile spermatozoa were primarily in the sperm pellet at the bottom of the gradient. When frozen-thawed semen from each of 7 bulls was separated by swimup, a mean +/- SEM of 9% +/- 1 of the motile spermatozoa were recovered after the procedure. In contrast, more spermatozoa were recovered after Percoll gradient separation (P < 0.05), with 40% +/- 4 of the motile spermatozoa recovered. The effect of separation procedure on in vitro fertilization found swim-up separated spermatozoa penetrated a mean +/- SEM of 74% +/- 5 of the oocytes, while fewer oocytes were penetrated by Percoll separated spermatozoa at 52% +/- 8 (P < 0.05). There was no effect of the separation procedure on the rates of polyspermy as measured by sperm/penetrated ova, with a mean +/- SEM of 1.25 +/-.09 for swim-up separated spermatozoa and 1.14 +/-.07 for Percoll separated spermatozoa (P>0.05). A carry over of Percoll into the fertilization medium with the Percoll separated spermatozoa was found not the cause for the decreased penetration of oocytes by these spermatozoa. In 2 of 3 bulls tested, the decreased penetration of oocytes by Percoll separated spermatozoa could be overcome by increasing the sperm concentration during fertilization from 1 x 10(6) to 5 x 10(6)/ml. When development of embryos fertilized by either swim-up or Percoll separated spermatozoa was compared for the semen from 2 bulls, a difference in cleavage rate was found in favor of swim-up separated spermatozoa (P < 0.05), but there was no effect of separation procedure on development (Day 7) to the morula + blastocyst or blastocyst stage (P>0.05). The disadvantages of the Percoll procedure could easily be overcome and the procedure was faster and yielded a six-fold greater recovery of motile spermatozoa than the swim-up method.     

Examination of some processing methods for freezing boar semen.

Five experiments were conducted to examine the effect of processing methods and diluents on survival and morphology of boar spermatozoa after freezing. Post-thawing survival of spermatozoa was better for Beltsville-F3 (BF3) than for tris-fructose-EDTA freezing diluent when the seminal plasma and glycerol were removed prior to freezing (method A). Both freezing diluents yielded similar viability results when the spermatozoa were frozen in the presence of siminal plasma and glycerol (method B). Viability of spermatozoa after thawing was better when glycerol concentration in the prefreezing diluent (method A) or in the freezing medium (method B) was 2-5 and 5-0 rather than 7-5%. Cooling of diluted semen to 5 degrees C beyond 4 h decreased the post-thawing survival of spermatozoa. The proportion of spermatozoa with undamaged acrosomes after processing and thawing by different methods was indistinguishable and relatively low. When the semen was frozen at cell concentrations ranging from 0-25 to 2-0 X 10(9)/ml, the viability of spermatozoa declined with increasing concentration following freezing in BF3, and S-1 diluents. Viability results were very similar for all cell concentrations examined when tris-fructose-EDTA diluent was used, indicating the possibility of freezing boar semen in a concentrated state.     

In vitro fertilizing capacity and chromatin condensation of deep frozen boar semen packaged in 0.5 and 5 ml straws

The effect of the straw volume employed for semen freezing was studied in 14 ejaculates from seven boars, by evaluating the viability, IVF capacity and chromatin state of spermatozoa. Frozen-thawed semen from 0.5 and 5 ml straws was compared to fresh semen. The chromatin condensation degree was determined by flow cytometry, using propidium iodide as fluorochrome, and the chromatin stability was evaluated by inducing its decondensation with SDS and EDTA. The results obtained for IVF, motility and normal apical ridge (NAR) were: 91.64, 78.14 and 81.47% sperm penetration, 80.78, 68.38 and 70.83% monospermy, 10.86, 9.76 and 10.64% polyspermy, 87.14, 50.71 and 47.86% motility, 79.14, 56.14 and 53.36% NAR, for fresh semen, thawed semen in 0.5 and 5 ml straws, respectively. Frozen-thawed spermatozoa showed significantly increased (P < 0.05) chromatin compactness compared to fresh spermatozoa (55.42, 48.41 and 47.08 fluorescence units (MIFU), for fresh semen, thawed semen in 0.5 and 5 ml straws, respectively). Chromatin was significantly more unstable (P < 0.05) in spermatozoa frozen in 0.5 ml straws (174.7 MIFU) compared to those frozen in 5 ml straws (155.53 MIFU) or to those in fresh semen (149.74 MIFU).     

Effect of co-culture of porcine sperm and oocytes with porcine oviductal epithelial cells on in vitro fertilization

This study was designed to determine the effect of co-culture with porcine oviductal epithelial cell (POEC) monolayers on in vitro fertilization of pig oocytes. The in vitro penetrability of mature (experiment 1) or immature (experiment 2) oocytes was studied in presence or absence of POEC during IVF with fresh semen. In experiment 3, boar and POEC effects were analyzed but in this case with frozen-thawed spermatozoa. In experiment 4, the spermatozoa were pre-incubated before IVF with or without POEC in order to assess their effect on IVF sperm-related parameters. In experiment 5, the effect of POEC was studied by co-culturing them with oocytes before IVF to determine if monospermy was improved. The results showed that high sperm concentration and POEC increase oocyte penetrability (P<0.01) and decrease monospermy rate (P<0.01), in both mature and immature oocytes (P<0.01) with fresh semen and a 18 h culture time. With frozen semen was detected a boar and POEC effect (P<0.01) on penetration rate. The sperm pre-culture 2 h with POEC also resulted in an increase of sperm penetration in terms of number of sperm per oocyte (P<0.01) and this treatment did not increase monospermy when contact time between gametes was limited to 6 h although monospermy was higher when POEC were present during IVF. Finally, exposure of oocytes to POEC for 4 h before IVF facilitated monospermic penetration to over 70% (P<0.01). In conclusion, the use of POEC in porcine IVF systems provides the possibility of working with low sperm concentrations and the effect of POEC on monospermy depends on sperm concentration, boar and contact time between gametes. Moreover, the exposure of oocytes to POEC before IVF improves the rate of monospermy.     

Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen.

The influence oftemperature, addition of glycerol, initial freezing temperature, method of dilution, level of glycerol in the diluted semen, equilibration time and type of diluent on the survival and fertilizing capacity of deep-frozen according to the best conditions was compared with that of "fresh" semen. The addition of glycerol at plus30 degrees C resulted in a highly significant decrease in the mean proportion of motile spermatozoa immediately after thawing compared with the effect of addition at plus 4 degrees C. The immersion of the straws at minus55 degrees C significantly reduced the revival of the spermatozoa compared with initial freezing at lower temperatures. The exposure time to glycerol had no significant effect on the survival of spermatozoa after thawing and incubation, but fertility was significantly higher with 4% than with 2% glycerol. The I. N. R. A. diluent provided better sperm survival and a significantly higher conception rate than did lactose-egg yolk extender. The semen frozen according to the best conditions (about 50% of the samples) had a fertilizing ability similar to that of "fresh" semen when the proportion of motile spermatozoa before, and after 1 or 3 hr of incubation was equal to or above 45, 40 and 30% respectively.     

Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The aim of this study was to evaluate the effects of the addition of different sugars (lactose, trehalose and glucose) on boar spermatozoa cryopreserved in an egg yolk based extender. Ejaculates were collected from a boar previously selected and semen samples were processed using the straw freezing procedure. In experiment 1, subsamples of semen were frozen in three different extenders: recommended lactose egg yolk extender (LEY); trehalose egg yolk extender (TEY) and glucose egg yolk extender (GEY). Sperm quality was assessed for motility, viability, acrosome integrity and hypoosmotic swelling test response upon collection, after freezing and thawing and then every hour for 3 h. Results showed that total motility at 1 and 3 h, progressive motility at 3 h, positive hypoosmotic response at 2 and 3 h and acrosome integrity at all times were significantly improved when trehalose was added to the extender. In experiment 2, sugar influence was also demonstrated in vitro fertilization. A total of 1691 oocytes were in vitro matured and inseminated with frozen-thawed sperm at 2000:1 sperm:oocyte ratio and coincubated for 6 h. Presumptive zygotes were cultured in NCSU-23 medium to assess fertilization parameters and embryo development. Both penetration and monospermy rates were significantly higher for trehalose frozen semen. A significant increase was observed in efficiency and blastocyst formation rates from TEY to the other groups. Our results demonstrated that trehalose extender enhances spermatozoa viability and its in vitro fertilization parameters in boar ejaculates with good sperm freezability. Further studies are necessary to assess the impact of sugars on the entire population.     

Fertilization and early cleavage in vitro of ageing bovine oocytes after maturation in culture

Bovine follicular oocytes cultured for 20 to 48 hours were inseminated with frozen thawed spermatozoa. Significantly higher penetration rates (94 to 100% vs 86 to 94%: P < 0.05) and proportions of polyspermy (35 to 78% vs 22 to 45%: P < 0.01) were obtained for oocytes cultured continuously for 24 hours with spermatozoa than for those separated from spermatozoa 8 hours after insemination. The most prominent effect of ageing of oocytes on early events of penetration was observed in the incidence of polyspermy rather than in the penetration rate and the proportion of pronuclear plus cleaved oocytes: the proportion of polyspermic oocytes significantly increased (P < 0.05) in oocytes inseminated after 28 to 48 hours of culture (36 to 78%) compared with those cultured for 20 to 24 hours (22 to 35%) for maturation. Culture experiments for early development of penetrated oocytes indicated that no significant differences were observed in the proportions of oocytes cleaved to the two- to four-cell stage 48 hours after insemination among those cultured for 20 to 40 hours for maturation. However, further cleavage to the four- to sixteen-cell stage 72 to 96 hours after insemination was greatly inhibited as ageing of oocytes proceeded from 28 hours in culture for maturation.     

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.     

Prediction of fertility of bovine semen: Preliminary studies with the hamster egg penetration test

The ability of liposome-treated fresh and frozen spermatozoa from two bulls to interact with zona-free hamster oocytes was examined to show whether the in vitro test results would correspond with in vivo fertility as indicated by the 60 to 90 d nonreturn to service rates which, using frozen semen, were 77 and 59%, respectively. The motility of spermatozoa in washed suspensions was also rated. Hamster test results were obtained using three ejaculates from each bull both as fresh and frozen semen. The results with frozen semen corresponded with fertility. The averages of three hamster tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte comparing spermatozoa from the bull with the higher fertility with spermatozoa from the bull with the lower fertility were 91% and 2.7 versus 56% and 1.4, respectively. Spermatozoa washed from frozen semen from the bull with the higher fertility interacted with hamster oocytes at the higher rate even when sperm motility was rated the same for both bulls. By contrast, fresh spermatozoa from the lower fertility bull interacted with hamster oocytes at a higher rate than spermatozoa from the higher fertility bull in six tests, comparing six ejaculates of fresh semen from both bulls. Comparing the higher fertility bull with the lower fertility bull, the average of six tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte were 60% and 1.6 versus 89% and 3.0, respectively. This suggests that this hamster test cannot be used with fresh semen to predict relative levels of fertility of frozen semen. Also, the subjective rating of sperm motility did not correspond with the in vitro oocyte penetrating ability of the spermatozoa.     

Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws

The interaction of glycerol concentrations of 0-10% and cooling rates from 1 to 1,500 degrees C/min with boar spermatozoa motility and acrosomal integrity (proportion of spermatozoa with normal apical ridge) was studied after thawing 0.5 ml straws at a constant rate. While increasing the glycerol concentration from 0 to 4% progressively improved motility, the percentage of spermatozoa with a normal apical ridge gradually decreased. The magnitudes of the respective changes depended on cooling rate. A peak value of 48.1% and rating 3.8 were obtained in semen protected with 4% glycerol, frozen at 30 degrees C/min. Increasing the glycerol levels above 6% resulted in a gradual decrease in motility. The proportion of spermatozoa with normal apical ridge was highest in semen protected with 0-1% glycerol after cooling at 30 degrees C/min (64.4% and 66.1%, respectively), but at these glycerol concentrations the percentage of motile spermatozoa was low. At the 30 degrees C/min cooling rate, the decline in the proportion of cells with normal apical ridge due to increasing the glycerol levels to 3 and 4% was relatively slow (57.3% and 49.4%, respectively). Cooling at 1 degrees C/min was detrimental to acrosomal integrity, which decreased with increasing glycerol concentration, in contrast to increasing motility, which even at its maximum, remained low. The direct plunging of straws into liquid nitrogen (1,500 degrees C/min) resulted in damaged acrosomes in all spermatozoa with the total loss of motility. Balancing motility and acrosomal integrity, freezing boar semen protected with 3% glycerol by cooling at 30 degrees C/min resulted in optimal survival for boar semen frozen in 0.5 ml French straws.     

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.     

Freezing rabbit semen by the use of BF5 diluent

Three experiments were carried out to find the optimal concentration of DMSO and glycerol in BF5 diluent for freezing rabbit spermatozoa. Semen was diluted 1:1 with diluent A (BF5 + DMSO) at 25 degrees C and diluted further 1:1 with diluent B (diluent A + glycerol) after cooling down to 5 degrees C. Diluted semen was frozen immediately and stored in liquid nitrogen. Maximum percentages for motility and normal acrosomes were obtained in the presence of 12% DMSO (as expressed in diluent A) and 3% glycerol (final concentration) after thawing.     

The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level

The effect of thawing velocities ranging from 10°C/min to 1.800°C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1°C/min (suboptimal), 5°C/min, and 30°C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1°C/min, the percentage of motile sperm (FMP) initially fell to 3.5–4.0% when the thawing rose to 200°C/ min, but, with further increases in thawing rate, increased and reached peak values (10.3–11.0% FMP) after thawing at 1,800°C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1°C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800°C/min. In semen frozen at the optimal rate of 30°C/min, the increases in thawing rates above 200°C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200°C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration. The peak value 53% NAR was recorded in semen protected by 2% glycerol, frozen at 30°C/min, and thawed at 1,200°C/min. In view of the inverse relationship between FMP and NAR, selection of optimal conditions from among the interacting variables, freezing rate, glycerol concentration, and thawing rate requires compromising between maximal FMP and maximal NAR. Accordingly, we have adopted as optimal a protocol with a thawing rate of 1,200°C/min, a freezing rate of 30°C/min and concentrations of 3% glycerol. © 1993 Wiley-Liss, Inc.     

Effect of N-acetyl-D-glucosamine, and glycerol concentration and equilibration time on acrosome morphology and motility of frozen-thawed boar sperm.

A series of experiments were conducted to determine the effect of N-acetyl-D-glucosamine, glycerol concentration and equilibration time for the freezing of boar spermatozoa in 5 ml maxi-straws. The optimum final glycerol concentration in the diluent with 0.05% N-acetyl-D-glucosamine in the first diluent was 2-3% and the optimum glycerol equilibration time was 2-3h. In conclusion, we recommend the first diluent containing 11% lactose hydrate, 20% egg yolk and 0.05% N-acetyl-D-glucosamine in 100ml distilled water, and the second diluent containing 11% lactose hydrate, 20% egg yolk, 4% glycerol and 1% orvus es paste for the diluents of boar sperm freezing. Also, we found out that 0.05% soluble N-acetyl-D-glucosamine was the optimum concentration in the first diluent and a concentration of 0.05% soluble N-acetyl-D-glucosamine significantly enhanced the cryopreservation of boar spermatozoa.     

Caprine blastocyst development after in vitro fertilization with spermatozoa frozen in different extenders

The feasibility of using frozen-thawed semen in caprine IVF outside the breeding season was investigated. Electroejaculated spermatozoa from a Nubian buck were washed twice and then frozen in skim milk- or in egg yolk-based extenders. Goat oocytes were matured and inseminated by frozen-thawed spermatozoa selected by swim-up. In vitro fertilization was performed in a modified defined medium (mDM), altered experimentally, for 24 h. Embryos were cultured in 50 microL of c-SOF + NEA for 9 d. The percentages of oocytes exposed to heparin-capacitated spermatozoa, (previously cryopreserved in skim milk-based extender) that cleaved, reached morula, blastocyst and expanded blastocyst stages were 82.8, 57.1, 35.7 and 30.0%, respectively. Without heparin treatment the rates for cleavage, morula, blastocyst and expanded blastocyst stages were 44.3, 31.4, 18.6 and 8.6%, respectively. Therefore, heparin treatment was included in sperm capacitation. Use of spermatozoa with BSA in the IVF medium yielded no cleavage. Although extenders containing 8 to 20% egg yolk enabled good sperm motility after cryopreservation, in vitro fertilizing ability was compromised under our conditions. By contrast, semen commercially processed in season in an egg yolk-based diluent remained effective for IVF. The highest proportion of blastocysts resulted from the use of spermatozoa diluted in a skim milk extender, heparin capacitation, and insemination in medium containing lamb serum.     

An effective method for freezing White Italian gander semen

Efficiency of freezing method, worked out for the White Italian gander semen was evaluated by comparing motility, morphology and fertilizing ability of spermatozoa in fresh and frozen-thawed semen. A part of pooled semen, collected from 25 White Italian ganders by dorso-abdominal massage was used immediately for artificial insemination of 10 geese (the control group) with a dose of 80 microl. This insemination was performed six times at weekly intervals. The remainder of the semen was diluted 1:0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with 6% (v/v) of dimethylformamide (DMF) and frozen to -140 degrees C at a rate of 60 degrees C/min. Frozen semen was thawed in a 60 degrees C water-bath and inseminated twice weekly in a dose of 100 microl (10 females of the experimental group, 12 inseminations were made). The freezing process affected spermatozoa motility and morphology, but had no effect on their fertilizing ability. Positive movement was observed in 50-60% of the spermatozoa in fresh semen and about 40% of the frozen-thawed cells. The average percentage of total live and live normal spermatozoa decreased due to freezing from 92.2 to 68.4% and from 34.7 to 14.1%, respectively. After the fresh semen insemination with average 12 million of the live normal spermatozoa per week average fertility was 88.24%; hatchability of set eggs was 80.88% and hatchability of fertile eggs was 91.67%. For frozen-thawed semen inseminated with average 9.5 million of the undamaged spermatozoa per week, the average fertility and hatchability rate was 83.78, 73.87, and 88.17%, respectively. Fecundity rates obtained after insemination with the frozen-thawed gander semen allow for the application of the freezing technique into breeding practice, in place of natural mating or to assist natural mating in periods of lowered fertility level.     

Effect of osmolality of skim-milk diluents and thawing rate on cryosurvival of ram spermatozoa

The effect of osmolality of skim-milk diluents (200, 320, 450, 600, and 750 mOsm/kg water) on the survival of ram spermatozoa frozen in straws were investigated after thawing in 39 °C water or in 20 °C air.Spermatozoa motility improved with increasing osmolality of the freezing diluent, irrespective of thawing rate. Diluents of 600 and 750 mOsm resulted in highest motility immediately after thawing and after 60 min incubation at 39 °C. A significant decrease in spermatozoa motility was observed when straws were thawed at 20 °C air with the magnitude of decrease inversely related to osmolality of the freezing diluent. Fertility of progestagen synchronized ewes inseminated with semen frozen in the 600 mOsm hypertonic skim-milk diluent was comparable to that obtained with fresh semen.     

Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4 degrees C

The influence of prolonged storage of boar epididymides on post-thaw sperm motility, and in vitro fertilization was evaluated. Twenty pairs of epididymides were obtained from Large White boars, and spermatozoa from one of each of the pairs were immediately collected and frozen (control group). The remaining epididymides were cooled to 4 degrees C and stored for 1, 2 or 3 d, after which spermatozoa were collected and frozen (experimental groups Day 1, 2 and 3, respectively). Sperm motility was maintained throughout the dilution procedure and then dropped (P < 0.01) after freezing and thawing. During storage the motility of nonfrozen spermatozoa decreased significantly (P < 0.01), reaching a value equal to that of frozen-thawed spermatozoa on Day 3. In vitro fertilization experiments revealed significantly (P < 0.05) lower penetration rates using Day 1, 2 and 3 stored spermatozoa (12, 13 and 2%, respectively) than that of the control group (40%). Oocyte penetration ability seemed to be reflected by acrosome integrity. However, the motility of spermatozoa with the ability to penetrate oocytes in Day 1 and Day 2 groups did not differ from that of the controls. The motility of spermatozoa lacking penetration ability, on the other hand, gradually decreased as the storage period was prolonged. This suggests that the sperm motility and penetration ability are affected by different mechanisms during the cold storage of epididymides. Finally, control and experimental groups exhibited high incidences of monospermic penetration (64 to 90%) and of male pronuclear formation (67 to 71%). These data suggest that cryopreservation of spermatozoa from boar epididymides stored at 4 degrees C for 1 to 2 d can be used for conserving male germ cells when epididymal spermatozoa can not be collected immediately and cryopreserved.     

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 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.