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.     

Evaluation of a cushioned method for centrifugation and processing for freezing boar semen

The purpose of this investigation was to evaluate the use of an iodixanol cushion during centrifugation on sperm recovery and yield after centrifugation (sperm recovery, sperm motility, viability, membrane lipid disorder, acrosome reaction and ROS generation); and to investigate how this procedure affects sperm function after freezing-thawing (sperm motility, membrane lipid disorder, acrosomal status and homologous in vitro penetration test). The sperm-rich fractions from fertile boars were centrifuged under two centrifugation régimes: 800xg for 10min (standard method) and 1000xg for 20min with an iodixanol (60% w/v) cushion at the bottom of the centrifuge tubes (Cushion method). The highest recovery was achieved using the cushion method (sperm loss for cushion method was 0.50%+/-0.18 versus 2.97%+/-0.43 for standard method, P<0.01) and sperm quality was not significantly affected by the centrifugation régime. The motion parameters (% progressive motility, % motility, VCL, VSL, VAP, ALH, BCF, P<0.05) of frozen-thawed samples showed higher values using the standard method. However, a higher number of viable spermatozoa with lower lipid disorders were found in spermatozoa processed with the cushion method. The in vitro penetration assay showed that the individual boar influenced the parameters studied but there were no differences between the two centrifugation régimes used. Our results support the hypothesis that the proportion of sperm loss in frozen-thawed semen was significantly influenced by the centrifugation régime. Therefore, the iodixanol cushion method is a suitable tool for cryopreservation of boar semen in order to reduce sperm loss without affecting sperm quality.     

Effects of semen plasma from different fractions of individual ejaculates on IVF in pigs

We applied IVM/IVF techniques to investigate effects of preincubation of sperm with different fractions of semen plasma harvested from fresh ejaculates on in vitro penetration and fertilization of in vitro matured oocytes. Three fractions of semen plasma were separated from the complete ejaculate of three Landrace boars and used to coincubate sperm obtained from the first sperm-rich fraction of the same ejaculates. After 14 to 16 h coincubation at room temperature, sperm were preincubated in capacitation medium and then inseminated into fertilization medium containing porcine oocytes matured in vitro. The semen plasma used for coincubation affected penetration rate (P < 0.001); Sperm coincubated with Fraction 1 semen plasma had a higher penetration rate compared with sperm coincubated with Fraction 2 (P < 0.05), but not with Fraction 3. Boar affected male pronucleus formation rates after insemination (P < 0.05), but no difference among boars was found in monospermy rate, average number of sperm penetrating into each fertilized oocyte, or the average number of sperm attached. No boar by fraction interaction was found for any parameters studied.     

Sperm chromatin structural integrity in normospermic boars is not related to semen storage and fertility after routine AI

Standard semen parameters are limited in their capacity to distinguish subfertile boars and to assess storage influences on liquid preserved boar semen. The evaluation of sperm chromatin structural integrity could have potential as a diagnostic tool in AI practice. This study assessed whether the determination of sperm DNA integrity adds a useful diagnostic tool for selection of boar ejaculates in routine AI procedure and assessment of storage effects in diluted semen. Special emphasis was laid on the standard spermatological characterization of semen samples in parallel with the determination of the DNA fragmentation index (DFI) through the sperm chromatin structure assay (SCSA). Six hundred ninety two (692) ejaculates from 79 Piétrain boars in an AI center were analyzed for motility, morphology and DFI over a period of 24 weeks. 95.5% of the semen samples had a DFI < 5% with low distribution of variation for DFI due to boar and ejaculate (< 5%). 61.3% of ejaculates with DFI > 5% showed values below thresholds for sperm motility or morphology. Based on field data from 13,239 inseminations, fertility of boars with temporarily elevated DFI was not impaired (P > 0.05). 24 randomly selected diluted semen samples did not show a significant increase of DFI during 168 h storage (P > 0.05). In a further experiment, 42 diluted semen samples from 14 normospermic boars were assessed for motility, membrane integrity (PI, FITC-PNA) and SCSA parameters. Three single ejaculates showed an increase of DFI at 120 and 168 h storage time. This was accompanied by a pronounced loss of both motility and membrane integrity. In conclusion, the evaluation of sperm chromatin structural integrity by the SCSA has only limited value for identifying sperm deficiencies in normospermic fresh or stored boar semen. Temporarily elevated DFIs seem not to be indicative of subfertility in normospermic boars.     

Semen changes in boars after experimental infection with porcine reproductive and respiratory syndrome (PRRS) virus

Eleven boars seronegative to porcine reproductive and respiratory syndrome virus (PRRSV) were trained for semen collection: five boars were inoculated intranasally with 6 x 10(6)TCID(50)/ml of PRRSV (Group A); four boars were inoculated intranasally with 6 x 10(4)TCID(50)/ml (Group B); and two boars were used as uninfected control (Group C). Semen samples were collected at 7-d intervals from 49 d prior to experimental inoculation with PRRSV to 70 d after inoculation, and were examined for sperm volume, sperm concentration, sperm morphology, sperm motility and for the presence of PRRSV. The infection in boars was demonstrated by the reisolation of PRRSV from the serum of all inoculated boars. Rectal temperatures and general health of the boars were clinically normal throughout the trial. Differences were observed in the quality of semen collected from boars after experimental infection with PRRSV. This infection induced a significant decrease in sperm motility and in spermatozoa with normal acrosomes. Of the semen samples tested for virus isolation in swine alveolar macrophages PRRSV was only isolated in 1 boar from Group B. The virus was detected in an additional semen sample in Group A by the production of an antibody titer in a biological assay. All attempts to detect PRRSV by RT-PCR in semen samples were unsuccessful. Nevertheless, from our study it is possible to suggest that the PRRSV can occasionally be transmitted in the semen during the initial phase of the disease.     

Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility

Extended semen doses from some boars used for AI have been shown to develop high levels of sperm DNA fragmentation during storage. Studies in other animals and humans have shown that if DNA damage is present in a certain percentage of the sperm cells the fertility potential of the semen sample is reduced. The objectives of the present study was to determine the relationship between sperm DNA fragmentation measured using the sperm chromatin structure assay (SCSA) in extended stored semen and field fertility in the boar. Three ejaculates from each of 145 boars were collected. Preparation of the semen doses included dilution with an EDTA extender and storage for up to 72 h post collection. The semen doses were assessed using flow cytometric methods for the percentage of viable sperm (PI/SYBR-14) and sperm DNA fragmentation (SCSA) at 0, 24, 48, and 72 h. A total of 3276 experimental inseminations in Danish breeding herds were conducted. The results showed that for 11 (7.6%) of the boars at least one of the three samples showed a value of DNA fragmentation index (DFI) above 20% within the storage period. Total number of piglets born (litter size) for Hampshire, Landrace and Danish Large White boars was, respectively, 0.5, 0.7 and 0.9 piglets smaller per litter when DFI values were above 2.1% as opposed to below this value. In conclusion the SCSA technique appears to be able to identify individuals with lower fertility with respect to litter size, and could in the future be implemented by the pig industry after a cost-benefit analysis.     

Toxicity effects of latex gloves on boar spermatozoa

It is known that several materials used in semen collection have been found to be detrimental to spermatozoal motility. In this study, examinations for toxic effects of latex and vinyl gloves, used with and without talcum powder on boar spermatozoa, were performed. Ten boars of known fertility with >/=80% sperm motility were divided into two groups (n = 5 boars each) for in vitro and in vivo studies. In the in vitro study, semen was collected from each of the five boars and was divided into five separate aliquots (5 ml each). One aliquot from each of the boars remained as the control, while the remaining aliquots were divided into individual treatments exposing the semen to a l cm(2) piece of latex or vinyl glove with or without talcum powder. In the in vivo experiment, semen from each of the five boars was collected using a gloved hand. During collection, the first half of the sperm-rich fraction was collected into a filtered sterile container, while the second half of the fraction was allowed to run through the palm of either a latex or vinyl powdered glove prior to collection in the container. In both experiments, semen sample motility was assessed by two independent observers at 1 minute after exposure. Results of both experiments consistently showed a significant (P<0.05) effect of latex gloves (with or without talcum powder) on boar semen when compared with the control semen. Motility was at or near 0% at 1 min after exposure to latex. No significant difference (P>0.05) in motility was observed between the control semen and the semen exposed to talcum powdered vinyl gloves. These results show that latex gloves are detrimental to boar spermatozoa. Therefore, it is suggested that when collecting boar semen vinyl gloves should be used.     

An update on North American boar stud practices

This survey included 44 boar studs from Canada and the USA with a total of approximately 10,000 boars. Studs with 51-500 boars accounted for 84% of respondents. More than 90% of boars were housed in stalls. Evaporative and mechanical cooling systems predominated and boars were typically fed based on body condition. The predominant age of boars was 1-2 years with annual culling rates between 20 and 70%. The primary reasons for culling included genetic improvement, semen quality and feet and leg issues. Collection occurred commonly on Mondays and Thursdays and boars were rested 3-7 days between collections. The average sperm produced per boar per week was 51-150 billions and resulted in 21-40 doses per boar per week. Most studs collected boars using double gloves and disposable cups or liners and used pre-warmed containers. Ejaculate pooling was practiced by >60% of studs. Evaluation of semen for motility was performed with 0-5min of warming in extender with viewing at 100-400x magnification. Concentration estimation occurred by photometer and CASA for 88% of studs. Ejaculate discard occurred for reasons of poor motility, abnormal sperm and bacteria. Most studs retained extended samples for 3-7 days for quality control. Discard rates were most common between 1 and 10% and were related to individual boar and season. Doses of semen contained 2-4 billion sperms, with final sperm numbers adjusted for fertile sperm and packaged as doses in tubes and bags with 60-100mL.     

Increasing storage time of extended boar semen reduces sperm DNA integrity

There is an extensive use of artificial insemination (AI) in the pig industry. Extended liquid boar semen may be used for insemination for up to 5 days after collection. The objective of this study was to determine the changes in sperm quality, when boar semen was extended and stored at 18 degrees C for up to 72 h post-collection. The study included three ejaculates from five boars, for each of the four breeds: Duroc, Hampshire, Landrace and Danish Large White (n=60 ejaculates). The sperm chromatin structure assay (SCSA) showed an increase in DNA fragmentation index (DFI) after 72 h of incubation (P<0.001), with no differences between breeds (P=0.07). For two Hampshire boars, all ejaculates had a large increase in DFI after 24 h of incubation. The standard deviation of DFI (SD-DFI) differed between breeds, with the SD-DFI for Hampshire being significantly greater than for the other breeds. The SD-DFI did not change during the 72 h of storage. Sperm viability was determined using SYBR-14 and propidium iodide in combination with flow cytometry. The sperm viability did not differ between breeds (P=0.21), but a difference in viability during storage (P<0.001) was detected. In conclusion, the SCSA cytogram patterns were consistent for different ejaculates within boars and storage of extended boar semen at 18 degrees C for 72 h significantly decreased the integrity of sperm DNA.     

Porcine sperm zona binding ability as an indicator of fertility

The escalated use of artificial insemination in swine has increased the importance of determining fertility of a semen sample before it is used. Multiple laboratory assays have been developed to assess fertilizing potential but they have yielded inconsistent results. This experiment sought to determine the relationship between in vitro competitive zona binding ability and in vivo fertility based on heterospermic inseminations and paternity testing. The zona pellucida binding ability and fertility of sperm from 15 boars was assessed by comparing sperm from one boar with sperm from other individual boars in a pairwise fashion using four ejaculates. The relationship of zona binding ability to the mean number of piglets sired per litter for each boar as well as historic fertility data (litter size and farrowing rate) was assessed. The in vitro competition assay consisted of labeling sperm from each boar of the pair with a different fluorophore and incubating an equal number of sperm from each boar in the same droplet with porcine oocytes. The competitive assay was highly effective in ranking boars by zona binding ability (R2=0.94). Paternity testing using microsatellite markers was used to determine the mean number of piglets sired per litter for each boar during heterospermic inseminations. The pairwise heterospermic insemination assay was effective in ranking boar fertility (R2=0.59). Using historical data from these boars, average litter size and farrowing rate were correlated (r=0.81, p<0.001). However, zona binding ability was not significantly correlated with historic farrowing rate data or historic average litter size. Boar sperm zona binding ability was also not correlated significantly with the mean number of piglets sired per litter following heterospermic insemination. But the number of piglets sired by each boar was related to a combination of zona binding ability, sperm motility, normal morphology, acrosomal integrity, and the presence of distal droplets (R2=0.70). These results suggest that zona binding ability is not an accurate predictor of fertilizing ability when used alone; however, when coupled with other sperm assessments, fertility may be predicted successfully.     

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.     

B-mode ultrasonographic evaluation of paired testicular diameter of mature boars in relation to average total sperm numbers

Crossbred, meat-type terminal sire boars (n = 215) were randomly assigned by age group (240-300, 301-360, 361-420, 421-480, 481-540, 541-600, and >721 days). Stud boars were on a once or twice weekly semen collection schedule. Testis diameters, in duplicate, were obtained using B-mode ultrasonography. Summation of average left and right testis diameter within boar gave the paired testicular diameter (PTD). Average ejaculate volume, sperm concentration (sperm/ml), and total sperm numbers for each boar were determined using composite data (average values) obtained from the last four semen collections. There was a <0.5cm difference between left and right testis diameters, with the left testis being the larger of the two testes (P = 0.03). There was no difference in PTD found between age groups in this study. Conversely, a dramatic increase in average total sperm numbers (ATSN) was observed between boars of 240-300 days (57.0+/-27.4 x 10(9) sperm) and up to 420 days (118.6+/-33.6 x 10(9) sperm) of age. The ATSN (127+/-32.5 x 10(9) sperm) remained constant for the 421-480 to >721-day age groups. The correlation between PTD and ATSN was low (r = 0.24) in this study. The results of this study demonstrate that normal boars should exhibit <0.5cm diameter difference between testes. As observed in other studies, the left testis was usually larger than the right testis. Correlation of total sperm numbers in a boar ejaculate using a composite ejaculate score (average values) and PTD measurements obtained using B-mode ultrasonography was poor when used in boars >8 months of age.     

Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility

Boar semen from a heterospermic mating trial and semen cryopreserved by various methods were evaluated by the flow cytometric sperm chromatin structure assay (SCSA), which measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ. Spermatozoa were treated with a pH 1.4 buffer and then stained with the metachromatic dye acridine orange. Acridine orange intercalated into double-stranded DNA (native) fluoresces green while single-stranded DNA (denatured) fluoresces red when excited with 488 nm light. The ratio of red to total fluorescence provides an index of normality/abnormality. The SCSA data on neat boar semen or semen in either Kiev-Merck or Pursel-Johnson extender and frozen directly on dry ice blocks or plunged into LN(2) did not differ within individual boars. Therefore, chromatin structure, as measured by the SCSA, was not influenced differently by these 2 methods of semen cryopreservation. When semen from 6 boars was mixed in equal sperm numbers in six 3-way combinations and inseminated into at least 3 Duroc gilts per combination, 4 of the 6 combinations yielded 2 litters, while the remaining 2 combinations yielded 3 litters. The SCSA correctly predicted both the high and low fertility boars based on a ratio of offspring as deviated from the theoretical percentage. Thus, the SCSA was found to be a valuable adjunct method for evaluating boar cemen quality.     

Apoptotic-like changes in the spermatozoa of fresh and stored boar semen and the quality of embryos produced in vivo

The aim of this study was to determine the apoptotic-like changes in the spermatozoa of fresh and stored boar semen and to investigate the relationship between this phenomenon and the quality of embryos produced in vivo. The experiments were divided into two series. In the first series, ten ejaculates were collected from five boars, which were crossbreeds of the Polish Landrace and Large White breeds. The semen was stored as a liquid until Day A (the day on which sperm motility decreased to 30%). Three fluorescence methods were used to evaluate semen quality: an assay to assess the early changes in sperm membrane integrity using the fluorophore YO-PRO-1, an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled annexin-V and the mitochondrial-specific probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) for measuring changes in mitochondrial membrane potential. Our results showed that liquid preservation of boar semen causes apoptotic-like changes in the sperm, and a significant increase in both: apoptotic sperm (YO-PRO-1(+)/PI(-)) and early apoptotic sperm (annexin-V(+)/PI(-)) were observed between Day 0 (fresh semen) and Day A only in semen from three of the five boars. In the second series of experiments, the semen from boar nos. 1, 2, and 3 was selected for insemination of superovulated gilts. The fertilizing capacity of fresh and stored semen with different levels of apoptotic spermatozoa was measured based on the morphology and the number of cells of embryos that were obtained after insemination with this semen. Our studies indicated no significant differences in the fertilization rate of gilts after insemination with fresh and stored semen with increased levels of apoptotic spermatozoa. After insemination with stored semen, a significantly greater number of degenerated embryos were observed, but the morphologically normal blastocysts obtained after insemination with either fresh or stored semen had a similar number of nuclei.     

Assessment of sperm viability in boar,rabbit and rooster: a modification of the fluorometric ethidium bromide exclusion procedure

The ethidium bromide (EtBr) exclusion procedure, a fluorometric method for measuring sperm cell viability, was studied to optimize the use of this technique on boar, rabbit and rooster semen. Diluted semen was used for boars and roosters. Diluted rabbit semen did not allow for reliable fluorescence readings; the interference of granules characteristic of rabbit seminal plasma was suggested as its cause. Therefore, rabbit semen was washed on several Percoll and Optiprep density gradients, with the aim of removing the granules from the sperm suspension. The complete absence of granules was not obtained, however, the best result was provided by the 35/70% Percoll density gradient. Most spermatozoa formed a loose pellet with low contamination. Although the washing procedure resulted in a selective action, Percoll washed semen was used to assess the EtBr procedure. The fluorescence intensities of stained fresh and stained digitonin-permeabilized samples were corrected, respectively, for the nonspecific fluorescence measures of fresh and digitonin-permeabilized samples both unstained. The contribution of the dye was subtracted from the corrected values, then the ratio between the corrected values of fresh and permeabilized cells provided the proportion of damaged cells in the sample. The working cell concentration range giving a constant proportion of damaged cells was set using diluted semen for boars and roosters (8-32 x 10(6) cell/ml) and Percoll washed semen for rabbits (4-16 x 10(6) cell/ml). The reliability of the fluorometric method was compared with the traditional nigrosin-eosin (NE) staining technique. The intactness of sperm samples containing known proportions of fresh and killed cells was measured in defined working cell ranges. For boars and roosters the values determined by fluorometry agreed closely with those determined using the NE method.     

Liquid storage of miniature boar semen.

The effects of liquid storage at 15 degrees C on the fertilizing ability of miniature pig semen were investigated. Characterization of ejaculated semen from 3 miniature boars was carried out. Semen volume and pH were similar among these boars. In one of the boars, sperm motility was slightly low, and sperm concentration and total number of sperm were significantly lower than in the others (P < 0.01). Seminal plasma of the semen was substituted with various extenders (Kiev, Androhep, BTS and Modena) by centrifugation and semen was stored for 7 days at 15 degrees C. Sperm motility was estimated daily at 37 degrees C. For complete substitution of seminal plasma, Modena was significantly more efficient than the other extenders (P < 0.001) in retaining sperm motility. Semen from each of the 3 miniature boars that had been stored for 5 to 7 days at 15 degrees C in Modena was used for artificial insemination of 15 miniature sows. The farrowing rates were 100, 100 and 60%, and litter sizes were 6.4 +/- 1.5, 5.8 +/- 0.8 and 5.0 +/- 1.0 for each boar semen, respectively. The boar that sired the smallest farrowing rate was the same one that showed lower seminal quality with respect to sperm motility, sperm concentration and total number of sperm. These results suggest that miniature boar semen can be stored for at least 5 days at 15 degrees C by the substitution of seminal plasma with Modena extender.     

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.     

Assessment of sexual behavior and effect of semen collection pen design and sexual stimulation of boars on behavior and sperm output--a review

The importance of sexual behavior and factors influencing sexual behavior of AI boars has received minimal study. The majority of studies reviewed used a very small number of boars. A sexual behavior index (SBI) has been developed for naturally mating boars but not for AI boars. Some studies have reported significant correlations between sexual behavior traits and semen characteristics; while other studies did not find significant correlations. A new semen collection pen design (Reicks Design) has reduced the duration of time a boar requires to mount a dummy sow after entering the collection pen and the duration of time needed to exit the collection pen after ejaculation. In general, the observation of another boar mounted on the dummy sow prior to collection, releasing the penis after extension, exposing boars to non-estrous gilts for 2 days before collecting semen, placing a non-estrous gilt underneath a dummy, and removing the boar for 2 min after first mount did not enhance the number of sperm cells collected. Treatment of boars with PGF2alpha has facilitated the training of sexually experienced boars to mount a dummy sow but not that of sexually inexperienced boars. In general, the treatment of boars with PGF2alpha did not increase the total number of spermatozoa ejaculated.     

Identifying useable semen

The "predictors of useable semen" used in most commercial AI centers provide a very conservative estimate of the relative fertility of individual boars. Furthermore, the relatively high sperm numbers used in commercial AI practice (usually >3 x10(9) total sperm per dose of extended semen) usually compensate for reduced fertility, as can be demonstrated in some boars when lower numbers of sperm are used for AI. Differences in relative boar fertility are also masked by the widespread use of pooled semen for commercial AI in many countries. However, the need to continually improve the efficiency of pork production, suggests that commercial AI practice should involve increased use of boars with the highest genetic merit for important production traits. Necessarily, this must be linked to the use of fewer sperm per AI dose, fewer inseminations per sow bred, and hence more sows bred by these superior sires. In turn, this requires improved techniques for evaluating semen characteristics directly related to the fertilization process, such as IVM-IVF assays, analysis of seminal plasma protein markers, more discriminatory tests of sperm motility and morphology, with the goal of identifying high-index boars whose fertility is sustained when low numbers of sperm are used for AI. This paper reviews the current status of laboratory-based boar semen evaluation techniques that meet these criteria.     

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.