Effects of cooling rate and storage temperature on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storage at 4 degrees C, and 5) fast cooling rate to and storage at 4 degrees C. Total spermatozoal motility (TSM), progressive spermatozoal motility (PSM), and spermatozoal velocity (SV) were estimated at 6, 12, 24, 48, 72, 96 and 120 h postejaculation. The longevity of spermatozoal motility was greatly reduced when spermatozoa were stored at 37 degrees C as compared to lower spermatozoal storage temperatures. At 6 h postejaculation, TSM values (mean % +/- SEM) of semen stored at 37 degrees C, slowly cooled to and stored at 25 degrees C or slowly cooled to and stored at 4 degrees C were 5.4 +/- 1.1, 79.8 +/- 1.6, and 82.1 +/- 1.6, respectively. Mean TSM for semen that was cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a moderate rate for four of seven time periods (6, 24, 72 and 120 h), and it was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a fast rate for five of seven time periods (6, 12, 24, 72 and 120 h). Mean TSM of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 25 degrees C for five of seven time periods (24 to 120 h). A similar pattern was found for PSM. Mean SV of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean SV of semen cooled to 25 degrees C for all time periods. A slow cooling rate (initial cooling rate of -0.3 degrees /min) and a storage temperature of 4 degrees C appear to optimize liquid preservation of equine spermatozoal motility in vitro.     

Equine spermatozoal motility and fertility associated with the incorporation of d-(+)-mannose into semen extender

Mannose is capable of decreasing bacterial attachment to the uterine mucosa in mares. Bacteria gain entry into the mare's uterus during breeding; therefore, a practical method to deliver mannose to the uterus is to incorporate it into semen extenders. The effect of mannose on spermatozoal motility and subsequent sperm fertilizing capability is unknown. The present study evaluated progressive spermatozoal motility in semen extender formulations incorporating mannose and assessed the fertility of mares inseminated with a mannose-containing semen extender. In Experiment 1, progressive spermatozoal motility in extender mixtures containing 0 mannose (control), 25, 37 or 49 mg/mL mannose was evaluated at 20 degrees C or 5 degrees C holding temperatures for 0, 12, 24 and 48 h post-dilution. Measures were repeated three times using five stallions of proven fertility. High concentrations of mannose in the extender affected progressive motility beyond the time and temperature effects noted in the controls. Extender containing only mannose sugar (49 mg/mL) displayed an immediate depression in progressive motility compared with controls (45.5% versus 62.9%, respectively; P<0.001). The 37 mg/mL mannose extender had a less dramatic decrease in motility (P<0.05) and only after storage at 5 degrees C for > or =12h (48.7% versus 58.0%, respectively). Extender with 25 mg/mL mannose performed no differently than the control formulation under all conditions. In Experiment 2, two groups of mares (n=11 each) were inseminated with 500 x 10(6) progressively motile spermatozoa extended in a traditional skim milk (control) extender or the 37 mg/mL mannose extender preparation. A single-cycle pregnancy rate of 72% was achieved by both groups. Present data suggest that a semen extender containing up to 37 mg/mL mannose could maintain motile spermatozoa for on-farm use and 25 mg/mL mannose concentrations preserved motility during long-term cooling. Likewise, sperm extended with up to 37 mg/mL of mannose had the same fertilizing capability as sperm in traditional extender mixtures.     

Influence of extender, cryoperservative and seminal processing procedures on postthaw motility of canine spermatozoa frozen in straws

Experiments were conducted to evaluate two extenders (egg-yolk Tris and egg-yolk lactose), varying concentrations of two cryopreservatives (glycerol and dimethyl sulfoxide), and rates for cooling to 5 degrees C, cooling from 5 to -100 degrees C, and warming for canine spermatozoa packaged in 0.5-ml French straws. At optimal concentrations of glycerol, egg-yolk Tris extender was superior to egg-yolk lactose in preserving spermatozoal motility. Addition of dimethyl sulfoxide, alone or in combination with glycerol in either extender, was not beneficial to spermatozoal survival after thawing. Canine spermatozoa withstood a range of cooling and equilibration times with no detrimental effect on spermatozoal motility prior to freezing. However, there were differences in spermatozoal motility immediately after thawing; these differences were variable, resulting in a cooling time by equilibration time interaction. Spermatozoal motility after thawing was best preserved by freezing in egg-yolk Tris extender containing 2-4% glycerol, using a moderate rate of cooling from 5 to -100 degrees C (-5 degrees C/min from 5 to -15 degrees C, then -20 degrees C/min from -15 to -100 degrees C). Three of 12 bitches inseminated intravaginally with semen frozen using this protocol became pregnant.     

Effects of dead spermatozoa on motion characteristics and membrane integrity of live spermatozoa in fresh and cooled-stored equine semen

The aim of this study was to determine if dead spermatozoa reduced motility or membrane integrity of live spermatozoa in fresh and cooled-stored equine semen. Three ejaculates from each of three stallions were centrifuged and virtually all seminal plasma was removed. Spermatozoa were resuspended to 25 x 10(6) spermatozoa/ml with EZ-Mixin CST extender and 10% autologous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50, or 75% (v/v) dead spermatozoa were added. Dead spermatozoa preparations contained 25 x 10(6) spermatozoa/ml and 10% seminal plasma from pooled ejaculates of the three stallions, in EZ-Mixin CST extender. Spermatozoa were killed in the pooled ejaculates by repeated freezing and thawing, then stored at -20 degrees C until warmed to 37 degrees C and mixed with aliquots of fresh spermatozoa to be cooled and stored in an Equitainer for 24h. Motion characteristics (% total motility (MOT), % progressive motility (PMOT), and mean curvilinear velocity (VCL)) for fresh and 24h cooled samples were determined using a computerized spermatozoal motion analyzer. The presence of up to 75% dead spermatozoa did not adversely affect MOT or PMOT of live spermatozoa in either fresh or cooled-stored semen. However, VCL and the percentage of membrane-intact spermatozoa were reduced compared to control samples when 75% (v/v) dead spermatozoa were added. Membrane integrity, as assessed by staining with carboxyfluoresein diacetate-propidium iodide, was highly correlated (r>0.8; P<0.001) with MOT and PMOT in both fresh and cooled-stored semen samples. Results of this study have application to the processing of both cooled and frozen equine semen.     

Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing

The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoal motility significantly better throughout storage at 5 degrees C. Addition of 5 or 25% seminal plasma to perfused epididymal spermatozoa (0% seminal plasma) resulted in a significant stimulation of spermatozoal motility by 25% seminal plasma at 0 h (P<0.05) and to a lesser extent at 24 and 48 h. Post-thaw motility of ejaculated as well as epididymal spermatozoa was not influenced by slow cooling to 15 degrees or 5 degrees C with or without glycerol prior to rapid freezing in liquid nitrogen vapor. During cooled storage, seminal plasma had a stimulatory effect on epididymal spermatozoa and depressed motility in ejaculated spermatozoa. Results on cryopreservation indicate that freezability of equine spermatozoa is already determined when spermatozoa leave the tail of the epididymis.     

Effect of storage on sperm membrane integrity and other functional characteristics of canine spermatozoa: In vitro bioassay for canine semen

The effect of storage of canine semen on sperm membrane integrity, as determined by the hypoosmotic swelling test, and on other functional characteristics of the canine spermatozoa was evaluated by established procedures. The results of this study indicated that storage of canine semen at a chilling temperature of 5 degrees C for 24 h did not significantly impair the physical and functional characteristics of the canine spermatozoa. The overall mean percentage of motility, hypo-osmotic swelling response, which assessed sperm membrane integrity, acrosome-reacted spermatozoa, acrosomal defects, and the percentage of live spermatozoa, did not significantly differ between the fresh and chilled semen samples. However, storage altered the rate of motility and acrosome reaction. The percentage of acrosome reaction in the canine capacitating medium peaked earlier in chilled than in fresh semen. It is probable that storing semen at 5 degrees C initiated/triggered the acrosome reaction. This did not amount to impairment of functional properties. Significant correlations were observed between hypo-osmotic swelling vs motility (r=0.98, P<0.002); hypo-osmotic swelling vs acrosome reaction (r=0.83, P<0.08); and acrosome reaction vs motility (R=0.89, P<0.04) in the fresh semen, and between hypo-osmotic swelling vs motility (r=0.87, P<0.05) and hypo-osmotic swelling vs acrosome reaction (r=0.56, P<0.05) in the chilled semen. It was concluded: that 1) storage of canine semen at 5 degrees C for 24 h did not significantly impair the physical and functional integrity of the spermatozoa; 2) the significant association between motility or acrosome reaction vs hypo-osmotic swelling indicates their value in assessing sperm viability; and 3) the hypo-osmotic swelling assay could have predictive value in screening out subfertile males with apparently normal spermiograms.     

Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa

A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested.     

Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa

This study was conducted to compare the cooling rates and storage temperatures within equine semen transport containers exposed to different ambient temperatures, and to evaluate the ability of these containers to preserve spermatozoal motility following 24 h of storage under these conditions. In Experiment 1, nonfat dried milk solids, glucose, sucrose, equine semen extender was divided into seven 40-mL aliquots and loaded into seven different semen transport containers: Equitainer I, Equitainer II, Equitainer III, ExpectaFoal, Bio-Flite, Lane STS, and Equine Express. After containers were loaded, they were subjected to one of three ambient storage temperatures: 1) 22 degrees C for 72 h, 2) -20 degrees C for 6 h followed by 22 degrees C for 66 h, or 3) 37 degrees C for 72 h. Cooling rates and storage temperatures of semen extender in each container were monitored with thermocouples and a chart recorder. In Experiment 2, semen from each of three stallions (3 ejaculates per stallion) was diluted to 25 x 10(6) spermatozoa/mL with semen extender, divided into 40 mL aliquots and loaded into transport containers as in Experiment I. Containers were subjected to one of three ambient storage conditions: 1) 22 degrees C for 24 h, 2) -20 degrees C for 6 h, followed by 22 degrees C for 18 h, or 3) 37 degrees C for 24 h. After 24 h of storage, spermatozoal motion characteristics (percentage of motile spermatozoa; MOT, percentage of progressively motile spermatozoa; PMOT, and mean curvilinear velocity; VCL) were evaluated using a computerized spermatozoal motion analyzer. Significant interactions were detected among storage conditions and semen transport containers for the majority of the temperature endpoints measured. When exposed to temporary ambient freezing conditions, the lowest temperatures attained by samples in containers ranged from -2.8 to 0.8 degrees C. Lowest temperature samples attained was not correlated (P > 0.05) with spermatozoal motility under any ambient condition. However, time below 4 degrees C was highly correlated (P < 0.05) with a reduction in spermatozoal motility. Mean cooling rates from 20 degrees C to 8 degrees C did not correlate with spermatozoal motility, except when containers were exposed to temporary freezing conditions. No container cooled samples below 6 degrees C in 22 degrees C or 37 degrees C environments except for the ExpectaFoal, in which samples fell below 4 degrees C under all ambient conditions. Ambient temperature affected MOT, PMOT and VCL of semen stored in all containers (P < 0.05) except for the Equitainer II in which motion characteristics remained high and were similar among all ambient temperatures (P > 0.05). Results suggest that stallion semen may be able to tolerate a wider range of cooling rates and storage temperatures than previously considered safe.     

Effect of spermatozoal concentration and number on fertility of frozen equine semen

Information on the number of motile spermatozoa needed to maximize pregnancy rates for frozen-thawed stallion semen is limited. Furthermore, concentration of spermatozoa per 0.5-mL straw has been shown to affect post-thaw motility (7). The objectives of this study were 1) to compare the effect of increasing the concentration of spermatozoa in 0.5-mL straws from 400 to 1,600 x 10(6) spermatozoa/mL on pregnancy rate of mares, and 2) to determine whether increasing the insemination dose from approximately 320 to 800 million progressively motile spermatozoa after thawing would increase pregnancy rates. Several ejaculates from each of 5 stallions were frozen in a skim milk-egg yolk based freezing medium at 2 spermatozoal concentrations in 0.5-mL polyvinyl-chloride straws. Half of each ejaculate was frozen at 400 x 10(6) cells/mL and half at 1,600 x 10(6) cells/mL. Insemination doses were based on post-thaw spermatozoal motility and contained approximately 320 x 10(6) (320 to 400) motile spermatozoa or approximately 800 x 10(6) (800 to 900) motile spermatozoa. Sixty-three mares were assigned to 1 of 4 spermatozoal treatments (1--low spermatozoal number, low concentration; 2--low spermatozoal number, high concentration; 3--high spermatozoal number, low concentration; 4--high spermatozoal number, high concentration) and were inseminated daily. Post-thaw spermatozoal motility was similar for cells frozen at both spermatozoal concentrations (P > 0.1). One-cycle pregnancy rates were 15, 40, 28 and 33%, respectively, for Treatments 1, 2, 3 and 4. Packaging spermatozoa at the high concentration tended to increase pregnancy rates vs packaging at the low concentration (37 vs 22%; P = 0.095). Furthermore, when the lower spermatozoal number was used, there tended (P < 0.1) to be a higher pregnancy rate if spermatozoa were packaged at the higher concentration. There was no increase in pregnancy rates when higher numbers of motile spermatozoa were inseminated (27 vs 31%; P > 0.1). Based on these results, a single 0.5-mL straw dose containing 800 x 10(6) spermatozoa should be used and each insemination dose should contain approximately 320 x 10(6) motile spermatozoa. Fertility trials utilizing other freezing extenders are necessary before recommending a single 0.5-mL insemination dose for all freezing extenders.     

Motility and fertility of alginate encapsulated boar spermatozoa

Ejaculated boar spermatozoa are vulnerable to cold shock. Prolonged storage of boar spermatozoa at low temperatures reduces survival rate, resulting in a bottleneck for the extension of artificial insemination in pig husbandry. This study evaluated whether alginate microencapsulization processing can improve the longevity of boar spermatozoa stored at 5 degrees C and the fertility of microencapsulated spermatozoa in vivo. Sperm-rich fraction semen from three purebred boars were concentrated and microencapsulated using alginate at 16-18 degrees C, and then were stored at 5 degrees C. Following storage for 1, 3 and 7 days, the microcapsule was taken out to assess sperm release under 37 degrees C incubation with or without 110 rpm stirring. The percentage of sperm released from microcapsules with 110 rpm stirring was higher than without stirring (81 versus 60%) after 24h of incubation. In another experiment, semen was also microencapsulated to evaluate the sperm motility. The motility of spermatozoa was assessed at 10 min, 8, 24, 32, 48, 56 and 72 h following incubation at 37 degrees C for nine consecutive days. The fertility of the free and microencapsulated semen was assessed by inseminating sows, and the reproductive traits (conception rate, farrowing rate, and litter size) were recorded. The motility of encapsulated spermatozoa was significantly higher than that of free semen after 8h incubation at 37 degrees C after storing for over three days (P<0.05). No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and non-encapsulated semen after four days of storage. In conclusion, microencapsulation can increase the longevity of boar spermatozoa and may sustain in vivo ova fertilization ability.     

Effect of milk fractions on survival of equine spermatozoa

Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage. Due to complex composition of milk, the components which are beneficial or harmful to spermatozoa are unknown. To address these unknowns the effect of various milk fractions on motility of stallion spermatozoa was evaluated. The fractions tested were native phosphocaseinate (NPPC), beta-casein, whey protein concentrate (WPC), alpha-lactalbumin, beta-lactoglobulin, microfiltrate, and ultrafiltrate. The standard reference diluents were INRA 82, commercial skim milk, and Hank's salts solution supplemented with Hepes, glucose, lactose (HGLL) supplemented with BSA. After 48 and 96 h storage at 4 or 15 degrees C some milk fractions (ultrafiltrate, microfiltrate, and alpha-lactalbumin fraction) decreased spermatozoal survival. Others (beta-lactoglobulin (BL) and native phosphocaseinate) were protective. Native phosphocaseinate (NPPC) at milk concentration afforted better protection than did the standard reference diluents. The optimal concentration of beta-lactoglobulin afforted significantly better protection than did BSA. The protection afforded by native phophocaseinate was not synergistic with beta-lactoglobulin. This implies a similar mechanism of protective action of these two components. Semen diluted in HGLL supplemented with NPPC (HGLL-NPPC) or in INRA 82 and stored 24 h at 15 degrees C or 4 degrees C, respectively, produced no difference of spermatozoal motility. However, fertility of semen stored in HGLL-NPPC (60%) was higher (p < 0.05) than that stored in INRA 82 (36%).     

Effects of Equex STM Paste on the quality of frozen-thawed epididymal dog spermatozoa

This study was carried out to investigate the cryoprotective efficacy of Equex STM Paste on the quality of canine post-thaw epididymal spermatozoa. Following castration, spermatozoa were flushed from the cauda epididymides. Epididymal spermatozoa from 13 of 16 dogs with a sperm motility of >70% were frozen in an egg yolk-Tris extender, supplemented with Equex STM Paste (0.5%, v/v); the extender free of Equex STM Paste served as a control cryoprotective diluent. The quality of spermatozoa, judged by its motility, plasma membrane integrity and acrosome integrity, was evaluated on four occasions, immediately after collection, after equilibration and at 0 and 2h post-thaw. Reducing the temperature to 4 degrees C for 2h prior to freezing decreased sperm motility (P=0.001), but had no effects on membrane integrity or acrosome integrity. Immediately after thawing, the percentage of acrosome-intact spermatozoa significantly decreased in samples frozen without Equex STM Paste compared to freshly collected or Equex-treated samples. After incubation at 37 degrees C for 2h post-thaw, a greater percentage of motile spermatozoa (P=0.018) and spermatozoa with intact acrosomes (P=0.001) were observed in Equex-treated samples compared with the control. The percentage of membrane-intact spermatozoa did not differ significantly between Equex-treated and control samples at any time. Supplementation with Equex STM Paste in the semen extender was effective for freezing canine epididymal spermatozoa because it protected acrosome integrity against damage induced by cryopreservation and it prolonged post-thaw sperm motility during in vitro incubation at 37 degrees C.     

Correlations between heterospermic fertility and assays of porcine semen quality before and after cryopreservation

The relative fertilizing potential of frozen-thawed semen from four black and four white boars was determined following heterospermic insemination. A heterospermic index (HI) was computed for each of the 16 possible pairs of black and white boars. Correlation coefficients were computed between the HI and several in vitro tests of semen quality before and afttr cryopreservation of the semen. For the in vitro tests before cryopreservation, the HI were negatively correlated (-0.57) with spermatozoal motility before cooling the semen, but they were not correlated with spermatozoal motility after cooling to 5 degrees C. After freezing and thawing, the HI were correlated with the following in vitro tests: spermatozoal motility (0.50), spermatozoa with either normal or damaged apical ridges (0.31), spermatozoa with missing apical ridges (-0.51), spermatozoa filtered through sephadex columns (0.32), spermatozoa with acrosin-activity (0.38), percentage of maximal releasable glutamic oxalacetic transaminase (GOT) present extracellularly (0.54), spermatozoal intracellular GOT (-0.57), spermatozoa bound per zona-free hamster oocyte (0.64), and percentage of zona-free hamster oocytes penetrated (0.75). The HI were not correlated with the following in vitro tests after freezing and thawing: spermatozoa with normal apical ridges, damaged apical ridges and loose acrosomal caps, extracellular and maximal releasable GOT, and the number of penetrations per zona-free hamster oocyte. The multiple regression correlation coefficient between the HI and four selected variables from three in vitro tests was 0.94. This high correlation indicated that the fertilizing potential of the semen could be accurately predicted with four variables that appeared to measure different properties of the spermatozoa.     

Assessment of fresh and stored duck spermatozoa quality via in vitro sperm-egg interaction assay

An in vitro sperm-egg interaction assay was used to measue the quality of duck spermatozoa in fresh and stored semen. The inner perivitelline layer (IPVL), which had been separated from laid duck eggs, was incubated with spermatozoa in vitro. The number of points of sperm hydrolysis in the IPVL in vitro was logarithmically correlated with the fertility of the eggs laid by inseminated females, for both fresh semen (r = 0.85, P < 0.001) and stored semen at 5 degrees C for 24 h (r = 0.84, P < 0.001). After semen storage, the ability of spermatozoa to hydrolyze the IPVL decreased by 67.4% compared with the values for fresh semen, whereas egg fertility and sperm motility decreased by 47.8% and 15.2%, respectively. These results suggest that the in vitro sperm-egg interaction assay accurately reflects the fertilizing ability of fresh and stored duck spermatozoa and detects spermatozoal damage due to semen storage more sensitively than motility or fertility tests.     

Advances in cryopreservation of stallion semen in modified INRA82.

In the procedure used in this paper, semen was first diluted in INRA82+2% egg yolk (E1) at 37 degrees C. Before or after cooling to 4 degrees C, semen was centrifuged and diluted in E1+2.5% glycerol (E2). Cooled semen was frozen in 0.5-ml straws. Straws were thawed at 37 degrees C for 30s. For fertility trials, frozen ejaculates were used only if total post-thaw motility was above 35%. Most mares were inseminated two times before ovulation with 400 x 10(6) total spermatozoa every 24h. This paper presents post-thaw motility (CASA) and fertility results obtained when some steps of the procedure were evaluated.Use of the first three jets of ejaculate before the centrifugation did not improve post-thaw motility compared to use of the whole semen (25% versus 25%, 2 stallions x 12 ejaculates, P>0.80). When the first dilution was performed in E2 at 22 degrees C instead of in E1 at 37 degrees C, motility was slightly improved (38% versus 36%, n>283 ejaculates per group, P<0.04) but fertility was similar (51% versus 58%, n>196 cycles per group, P>0.10). Coating the spermatozoa with 0.5, 1, 2, 4 and 8mM of Concanavalin A resulted in unchanged post-thaw motility (6 stallions x 3 ejaculates, P>0.05). The extender E2 was modified or supplemented with different substances. Increasing egg yolk concentration from 2 to 4% (v/v) did not increase post-thaw motility (42% versus 34%, 6 stallions x 2 ejaculates, P>0.05). Different glycerol concentrations (range: 1.7-3.7%) had no significant effect on post-thaw motility even though 2.4-2.8% resulted in a nonsignificant higher motility (7 stallions x 2 ejaculates, P>0.05). Glutamine at 50mM in E2 improved post-thaw motility compared with no glutamine (49% versus 46%, n>584 ejaculates per group, P<0.0001) but not fertility (53% versus 54%, n>451 cycles per group, P>0.80). Thawing at 75 degrees C for 10s slightly increased motility after 120 min at 37 degrees C (6 stallions x 1 ejaculate, P<0.05) but no effect on per-cycle fertility was noted (32% (19 cycles) versus 41% (17 cycles), P>0.50). When post-thaw dilution was performed using a fixed molarity multi-step system (25 mOsm per step) from various osmolarities (900-690 mOsm) to 365 mOsm, motility was unaffected compared with dilution in one step (36% versus 38%, 6 stallions x 1 ejaculate, P>0.20).     

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.     

Effect of cryopreservation of zona-binding capacity of canine spermatozoa in vitro

The hemizona assay (HZA) was used as a functional test for zona pellucida binding capacity of fresh and frozen-thawed canine spermatozoa. We investigated 30 ejaculates from 3 dogs with sperm motility > 70% and sperm concentration > 5.10(8) cells per ejaculate with up to 20% abnormal and dead spermatozoa. Fifteen ejaculates were each divided into 2 portions: one portion was used for analysis of fresh semen, the other for cryopreserved semen. On the day of the experiments, in vitro-matured canine oocytes were bisected into 2 equal hemizonae. One half of the hemizonae were coincubated with fresh capacitated (control) spermatozoa, the other half of the hemizonae were coincubated with frozen-thawed (tested) spermatozoa at final concentration of 1 to 2 x 10(6) cells/mL in 200 microL droplets of BSA-supplemented Toyoda, Yokojama and Hoshi (TYH) medium at 37 degrees C, 5%, CO2 for 1 h. Sperm suspensions were examined kinesigraphically for post capacitation type of movement. The Student's t-test was used to compare differences between semen parameters. The data on HZA binding activity of fresh and frozen-thawed canine semen were analyzed by ANOVA and then by the Newman-Keuls multiple range method. The results showed no differences in the initial semen quality parameters among the 3 dogs. After thawing, the semen from Dog 1 and Dog 2 demonstrated relatively uniform sperm parameters, while in Dog 3 sperm motility, and viability and the percentage of morphologically normal spermatozoa were significantly decreased. The binding activity of frozen-thawed spermatozoa from the 3 dogs was significantly reduced (29.40 +/- 9.02, 18.60 +/- 3.30, 8.20 +/- 4.49) compared with that (107.20 +/- 19.22, 109.80 +/- 20.75, 78.20 +/- 12.47; P < 0.01) of fresh spermatozoa. The results showed that semen samples with similar sperm parameters prior to cryopreservation displayed different sperm zona-binding capacity after freezing. The HZI (value of sperm binding capacity of frozen-thawed vs fresh semen samples) was higher in Dog 1 (27.43) than in Dog 2 (16.90) or Dog 3 (10.40), and thus confirmed the variation of zona binding activity after thawing between dogs. The freezability of individual dog semen is discussed. In conclusion HZA may be a valuable tool for evaluating the post-thaw fertilizing ability of canine spermatozoa.     

Effect of blood admixture on in vitro survival of chilled and frozen-thawed canine spermatozoa

Hematospermia in the dog usually occurs secondary to benign prostatic hypertrophy or trauma of the penis or prepuce during semen collection. Regarding the difficulty of removing blood cells from a hematospermic sample, the present study was performed to determine whether blood contaminated ejaculates can still be chilled (4 degrees C) or frozen (-196 degrees C) without an additional decrease in sperm quality. In the first experiment, blood additions of up to 10% exerted no negative effects on the functional characteristics of canine spermatozoa cooled (4 degrees C) and stored for 4 days in an egg-yolk-Tris extender. In contrast, in experiment 2, blood admixtures of 4% or more clearly caused negative effects on cryopreserved (-196 degrees C) spermatozoa, mainly on the motility parameters, on the membrane integrity and on the acrosomal status of the spermatozoa. In experiment 3, we showed that these negative effects of blood admixture on cryopreserved spermatozoa were mainly associated with the red blood cells (RBCs) whereas the addition of plasma, serum or inactivated serum exerted little or no negative effect. Moreover, in experiment 4, we showed that 58.3+/-11.6% of the RBCs hemolysed after a freeze-thaw process. In experiment 5, a clear and negative effect of hemoglobin on cryopreserved canine spermatozoa was observed. We conclude that the presence of up to 10% blood is not detrimental for the storage of chilled canine spermatozoa and that the detrimental effects of blood on cryopreserved spermatozoa are at least partly attributable to the high amount of hemoglobin originating from the RBC hemolysis observed after freezing and thawing.     

Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoal motility

Ejaculates were collected form three mixed-breed male dogs daily for 3 d. The semen was diluted in either a nonfat dried milk solid-glucose (NFDMS-G) or egg yolk citrate (EYC) extender at a concentration of 25 x 10(6) sperm/ml. The diluted samples were exposed to three different storage temperatures (35, 22 and 4 degrees C). Three cooling rates (-1.0, -0.3 and -0.1 degrees C/min) were also investigated at the lowest storage temperature (4 degrees C). The semen was evaluated for total motility, progressive motility and velocity at 0, 6, 12, 24, 48, 72, 96 and 120 h after collection by two independent observers. Interactions between extenders, temperatures and time after collection were found for each of the variables. Nonfat dried milk solid-glucose diluent was superior to EYC (P<0.05) in preservating sperm motility parameters that were evaluated for most of the observations. The evaluated sperm motility parameters were also significantly superior (P<0.05) in semen stored at 4 degrees C than at 35 or 22 degrees C for most of the observations. The progressive motility and velocity of sperm in semen cooled at 4 degrees C in NFDMS-G were higher (P<0.05) at the fast and medium cooling rates (-1.0 and -0.3 degrees C) than at the slow cooling rate (-0.1 degrees C/min) at 24 and 72 h, and at 48 h, respectively. In conclusion, the present study suggests that canine spermatozoal motility is well preserved when a NFDMS-glucose extender is added to the semen and the semen is cooled at a medium or fast rate to a storage temperature of 4 degrees C. Additional studies are needed to evaluate the fertility of semen stored in this manner.