Identification of anti-agglutinin for spermatozoa in epididymal boar plasma

The present report identifies epididymal boar anti-agglutinin and examines its effect on sperm motility. Boar spermatozoa from the cauda epididymidis were washed and incubated in modified Krebs–Ringer bicarbonate at 37°C (5% CO₂ in air). In the samples washed three or five times and then incubated for 3–5 h, higher rates (72–79%) of spermatozoa were associated with one another at the acrosomal region, mainly in groups of 2–5 cells (head-to-head agglutination), and many cells exhibited intensively flagellant and/or circular types of movement but rarely progressive motility. The addition of epididymal plasma or 25 kDa protein purified from it markedly inhibited the occurrence of head-to-head agglutination in washed spermatozoa, whereas heat treatment and subsequent removal of insoluble materials reduced the anti-agglutination activity of epididymal plasma. The percentages of progressively motile cells in the samples incubated with epididymal plasma or 25 kDa epididymal protein rose coincident with the reduction of sperm agglutination. These findings demonstrate that the 25 kDa epididymal protein is an anti-agglutinin for the cauda spermatozoa and that it effectively functions to maintain progressive motility of the cells in vitro. © 1994 Wiley-Liss, Inc.     

Effects of spermatozoal concentration and post-thaw dilution rate on survival after thawing of dog spermatozoa

The objectives of this study were to evaluate the effects and interactions of freezing dog semen using 4 different sperm concentrations (50 x 10(6), 100 x 10(6), 200 x 10(6) and 400 x 10(6) spermatozoa/mL) in 0.5-mL straws and diluting the thawed semen at 4 different rates (1:0, 1:1, 1:2 and 1:4) on post-thaw survival and longevity of dog spermatozoa during incubation at 38 degrees C. Fifteen ejaculates were collected from 12 dogs and pooled. The semen pool was divided into 4 aliquots containing respectively 4,200 x 10(6), 2,100 x 10(6), 1,050 x 10(6) and 525 x 10(6) spermatozoa, which were centrifuged. Sperm pellets were rediluted with TRIS-glucose-egg yolk extender containing 5% glycerol and 0.5% of Equex STM Paste to obtain the designated sperm concentrations. The semen was frozen in 0.5-mL straws 4 cm above liquid nitrogen (LN2). The straws were thawed at 70 degrees C for 8 sec and the contents of each straw were divided into 4 aliquots and diluted with TRIS buffer at 38 degrees C at rates of 1:0, 1:1, 1:2 and 1:4 (semen:buffer), respectively, making a total of 16 treatments. Sperm motility was subjectively evaluated after thawing and at 1-h intervals during 8 h of incubation at 38 degrees C. Plasma membrane integrity and acrosomal status were evaluated at 1, 3, 6, 12 and 18 h post-thaw using a triple-staining procedure and flow cytometry. For data pooled across the post-thaw dilution rate, motility was higher (P< 0.001) in samples frozen with 200 x 10(6) spermatozoa/mu. The integrity of sperm plasma membranes after 18 h incubation was higher (P<0.05) in samples frozen with 200 x 10(6) and 400 x 10(6) spermatozoa/mL. For data pooled across sperm concentration, samples diluted at a rate of 1:2 or 1:4 had better (P<0.001) motilities after 8 h of incubation than undiluted samples or those diluted at 1:1. The integrity of the sperm plasma membranes was higher (P<0.001) at increasing dilution rates. When the 16 treatments were compared, the best longevity was obtained when semen packaged at a concentration of 200 x 10(6) spermatozoa/mL was diluted immediately after thawing at 1:4 dilution rate.     

Effect of sodium and potassium concentrations and pH on the maintenance of motility of rabbit and rat epididymal spermatozoa.

Spermatozoa were collected from the caput and cauda epididymidis of rabbits and rats and diluted in Hank's solution containing BSA, with various concentrations of Na+ and K+. Ionic strength and osmolarity were kept constant. Motility was assessed at various intervals during incubation at 25 degrees C. In the pH range 7.05--7.20, the motility of rabbit spermatozoa was not affected by changes in the ratio of K+ to Na+. Similarly, the motility of rat cauda spermatozoa was not altered, but that of caput spermatozoa was slightly depressed by a high K+/Na+ ratio. In the pH range 5.45--5.85, rabbit cauda and caput spermatozoa had much greater motility in media with a high K+/Na+ ratio. The reverse result was obtained for the rat. These findings indicate that the motility of epididymal spermatozoa is influenced by external Na+ and K+ concentrations and that this phenomenon is pH-dependent.     

Development of a chemically defined ram semen diluent (RSD-1)

A chemically defined ram semen diluent (RSD-1) has been developed. RSD-1 maintained spermatozoal motility of diluted semen containing approximately 800 million spermatozoa ml⁻¹ during cooling to 15°C and its storage for 1 h. Motility was further maintained when the cooled semen was diluted to 100 million spermatozoa ml⁻¹ and incubated at 38°C for about 24 h. In contrast, a conventional milk-based diluent supported motility for less than 6 h at 38°C. Spermatozoal motility was influenced by the buffering capacity, osmolarity and the presence or absence of macromolecules and calcium in the chemically defined diluent. Among the organic buffers tested, MOPS (3-(N-morpholino)propanesulphonic acid) had a marked influence on the maintenance of spermatozoal motility. The presence of MOPS also overcame the detrimental effects of 2 mM calcium in Krebs Ringer improved (KR-I) buffer.     

Improvements and short-term viability of mouse epididymal spermatozoa recovered through the Spermprep filtration method

This study was designed to determine the effects of Sephadex filtration (Spermprep(trade mark)I method) on the separation of motile, morphologically normal, mouse epididymal spermatozoa and to study the viability of the recovered spermatozoa over a 3-h incubation period. Spermatozoa were harvested from the caudae epididymie (5 animals per run or replication; n=10) following bilateral testicular excision, after which they were incubated in 2-ml of Test-Yolk buffer (TYB) at 37 degrees C for 15-min. The specimens were then split into 2 1-ml aliquots, with Aliquot 1 as the control and Aliquot 2 as the filtered sample. The Spermprep(trade mark)I column was employed according to the manufacturer's specifications using TYB. During filtration (10-min), 2 different fractions were obtained: first 5-min (Sample 1) and second 5-min (Sample 2). The 2 fractions were evaluated and incubated at 37 degrees C and assessed for percentage of motility and grade of motility (0 to 4) every 30-min for 3-h. Filtration resulted in a significant improvement in the percentage and grade of motility (91.5% and 3.0 vs 76.5% and 2.5, respectively). The results indicate that filtration with the Spermprep(trade mark)I method improved the percentage and grade of motility (P<0.05) but not the percentage of normal morphology of the spermatozoa. In addition, the Spermprep(trade mark)I method enabled the recovery of 45% (8.3 x 10(6) spermatozoa recovered) of the total number of spermatozoa processed in the control aliquot (18.4 x 10(6) spermatozoa), which is consistent with previous observations. Most importantly, filtered spermatozoa incubated for 3-h showed a greater percentage and grade of motility than the control spermatozoa (63% and 1.66 vs 39% and 0.82, respectively. The Spermprep(trade mark)I filtration method selected a higher proportion of quality spermatozoa, which also displayed significant long-term motility (longevity) during in vitro incubation.     

Different inhibitors of the gibberellin biosynthesis pathway elicit varied responses during in vitro culture of aspen (<Emphasis Type="Italic">Populus tremula</Emphasis> L.)

Several synthetic plant growth regulators (PGRs), including prohexadione-calcium (ProCa), paclobutrazol (PBZ), and chlormequat chloride (CCC), known for their ability to inhibit gibberellin (GA) biosynthesis, were investigated for their influence on Populus tremula L. (aspen) shoots grown in vitro. Changes in plant growth induced by these inhibitors were compared to the effects of exogenous gibberellins (GA₃ and GA_4/7). All PGRs were added to the nutrient medium at concentrations of either 1 or 5 μM. Stem segments with and without apical buds were excised from in vitro-grown shoot culture, and these explants were incubated either in test tubes or Petri dishes. In the presence of 5 μM ProCa, shoot growth and rooting were inhibited when grown in test tubes, while shoots grown in Petri dishes exhibited strongly enhanced shoot and root growth. PBZ suppressed shoot development both in test tubes and Petri dishes, although 1 μM PBZ promoted adventitious root formation when shoots were grown in test tubes. Five micromolars CCC suppressed shoot and root development in test tubes, but promoted shoot growth in Petri dishes.     

Detrimental Effect of Water Submersion of Stools on Development of Strongyloides stercoralis

Strongyloidiasis is prevalent in Thailand, yet its prevalence in the south is lower than in other parts of the country. This might be due to the long rainy season in the south resulting in stool submersion in water inhibiting worm development. In this study, the effect of water submersion of fecal samples on development of Strongyloides stercoralis was investigated. Ten ml of a 1∶5 fecal suspension were placed in 15-ml tubes, 35-mm dishes, and 90-mm dishes producing the depths of 80 mm, 11 mm and 2 mm-suspensions, respectively. The worm development was followed at 1/6, 4, 6, 8, 10, 12, 14, 16, 24, and 36 h, by determining the number of filariform larva (FL) generated from agar-plate cultures (APC). Fecal suspensions kept in tubes and 35-mm dishes showed a decline in FL yield relative to incubation time and reached zero production 14 h after incubation. In contrast, the number of FL generated from the suspension kept in 90-mm dishes remained stable up to 36 h. Cumulatively, all tubes and 35-mm dishes became negative in APC after 14 h while 90-mm dishes remained APC-positive up to 36 h. Adding more water or stool suspension to dishes resulted in a decreased number of FL. Mechanical aeration of the suspensions in tubes restored an almost normal FL yield. It appears that the atmospheric air plays a significant role in growth and development of S. stercoralis in the environment and may be one of factors which contribute to a lower prevalence of human strongyloidiasis in the south of Thailand.     

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.     

Effect of seminal plasma on the cryopreservation of equine spermatozoa

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.     

Bovine oviduct-specific glycoprotein: A potent factor for maintenance of viability and motility of bovine spermatozoa in vitro

In the cow, a specific glycoprotein-bovine oviduct-specific glycoprotein (BOGP)-is secreted by the epithelial cells of the oviduct at the follicular stage of the estrous cycle. In this study, we examined the effects of purified BOGP on the viability and motility of bovine spermatozoa in culture in vitro. Frozen-thawed bovine spermatozoa were incubated in modified Tyrode's solution (TALP) that contained purified BOGP (TALP-BOGP). In TALP-BOGP, both the viability and motility of bovine spermatozoa were more effectively maintained than in the control medium without any added protein. The increases in both the viability and motility of spermatozoa were dose-dependent. Spermatozoa were also incubated in TALP medium supplemented with bovine serum albumin, egg albumin, lactalbumin, or gastric mucin, and their viability and motility in these media were compared with that in TALP-BOGP. Both the viability and motility of spermatozoa were more effectively maintained in TALP-BOGP throughout a 12-hr incubation than in other media tested. An immunolabeling study demonstrated that a monoclonal antibody specific for BOGP reacted with the posterior region of the head, the middle portion, and the tail of spermatozoa that had been incubated with TALP-BOGP, suggesting that BOGP becomes specifically associated with particular regions of the spermatozoon. These results suggest that BOGP is a potent factor for maintenance of the viability and motility of sperm. On the basis of the present results, we also propose that BOGP may play an important role in sperm functions during the reproductive process. © 1995 wiley-Liss, Inc.     

Effect of Hoechst 33342 on stallion spermatozoa incubated in KMT or Tyrodes modified INRA96

The only known means of effectively separating populations of X and Y bearing sperms is the Beltsville sexing technology. The technology implies that each individual sperm is interrogated for DNA content, measuring the intensity of the fluorescence after staining the spermatozoa with Hoechst 33342. Because there are no data regarding the effect of the staining on stallion sperm, ejaculates were incubated up to 90 min in presence of 0, 4.5, 9, 22.5, 31.5, 45, 54, 67.5, 76.5 and 90 μM of Hoechst 33342, in two media, KMT or INRA-Tyrodes. After 40 and 90 min of incubation, motility (CASA) and membrane integrity (flow cytometry after YoPro-1/Eth staining) were evaluated. In KMT extender sperm motility significantly decreased after 45 min of incubation when sperm were incubated in the presence of concentrations of Hoechst of 45 μM or greater (P<0.05). When incubated in modified INRA96, stallion spermatozoa tolerated greater concentrations of Hoechst, because sperm motility only decreased when incubated in presence of 90 μM (P<0.05) and membrane integrity was not affected. After 90 min of incubation the same effect was observed, but in this case at concentrations over 45 μM the percentage of total motile sperm was also reduced although only in samples incubated in KMT. To produce this effect in samples incubated in Tyrodes modified INRA 96, Hoechst had to be present at concentrations over 67.5 μM. Apparently, the detrimental effect of Hoechst to stallion spermatozoa varies depending on the media, and INRA modified extender may be an alternative to KMT.     

Influence of porcine spermadhesins on the susceptibility of boar spermatozoa to high dilution

The effect of heparin-binding and non-heparin-binding spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa at the high dilution (300,000 sperm/ml) to which sperm are exposed during the process of sex sorting by flow cytometry was investigated. Incubation of spermatozoa with heparin-binding spermadhesins caused a time- and dose-dependent decrease in the percentage of functional spermatozoa. The percentage of viable spermatozoa incubated at 38 degrees C with heparin-binding spermadhesins diluted in PBS (1 mg/ml) dropped from 75% (0.5 h) to 4% (5 h), whereas the percentage of viable spermatozoa incubated in PBS without proteins (control) decreased from 85% (0.5 h) to 19% (5 h). Addition of non-heparin-binding PSP-I/PSP-II spermadhesin to the PBS resulted in a concentration-dependent increment of the percentage of viable cells (65% after 5-h incubation), with maximum effect at 1.5 mg/ml. The heparin-binding spermadhesins totally suppressed sperm motility and mitochondrial activity after 5 h of incubation. The same parameters of sperm incubated in the presence of 1.5 mg/ml of PSP-I/PSP-II were 50% and 58%, respectively, and the percentages of control sperm displaying motility and mitochondrial activity were 21% and 26%, respectively. Moreover, the viability, motility, and mitochondrial activity all decreased on incubation of spermatozoa with mixtures of PSP-I/PSP-II and heparin-binding spermadhesins as the concentration of the latter increased. We conclude that PSP-I/PSP-II and the heparin-binding spermadhesins exert antagonistic effects on the functionality of highly diluted boar spermatozoa. The finding that PSP-I/PSP-II contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 h at physiological temperature points to its potential use as an additive for sperm preservation, specifically of highly diluted, flow-sorted spermatozoa for sex preselection.     

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.     

Preliminary study on the cryopreservation of tropical bagrid catfish (Mystus nemurus) spermatozoa; the effect of extender and cryoprotectant on the motility after short-term storage

The effects of different extenders, and cryoprotectants on the motility of tropical bagrid catfish (Mystus nemurus) spermatozoa were evaluated after short-term storage. Three extenders, physiological saline, Ringer or saline at three levels of sperm to extender dilutions (1:20, 1:30, or 1:40) and four cryoprotectants (DMSO, ethanol, glycerol or methanol) at three concentrations (5, 10, or 15%) were examined in two separate experiments. In the first experiment, milt was suspended in the respective extender at the three milt to extender dilution ratios in two sets of tubes. Extended milt in the first set of tubes was stored at -4 degrees C, and motility assessed after 24h, while the second set was kept at 23 degrees C and sperm motility was assessed immediately and at 30-min intervals thereafter. Ringer retained sperm motility better than the other extenders at all dilution levels at temperatures of 23 and -4 degrees C respectively. At 23 degrees C, the sperm motility was almost completely lost after 150 min except for those in Ringer at 1:20 dilution level which still had a motility of 18% (compared to those kept at -4 degrees C for 24, which had motility from 39 to 71%, regardless of extender). In the second experiment, various cryoprotectants were added to solutions of milt (that was diluted in Ringer at 1:20 ratio and cryopreserved in liquid nitrogen for 15 days). Sperm cryopreserved in 10% methanol had the highest motility (58%) compared with those in the other cryoprotectants at all concentrations.     

Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen

Cryopreservation is associated with the production of reactive oxygen species which lead to lipid peroxidation of sperm membranes. The objective was to determine an alpha-tocopherol concentration capable of improving the quality of cryopreserved porcine semen. Boar spermatozoa frozen with 200, 500 or 1000 microg/mL alpha-tocopherol were thawed and incubated at 37 degrees C for 4 h. Routine parameters of semen quality, susceptibility to lipid peroxidation 2-thiobarbituric acid (TBARS) and oxygen uptake were evaluated. Motility was higher (P<0.05) in samples treated with different concentrations of alpha-tocopherol up to 2 h of incubation. Viability and acrosome integrity significantly decreased during incubation (no significant differences between treatments). Two hundred micrograms per milliliter alpha-tocopherol protected spermatozoa against lipid peroxidation during incubation, but 1000 microg/mL failed to protect after 2 h of incubation. There was a negative association between TBARS and motility, suggesting that lipid peroxidation affected sperm motility. Both control and 200 microg/mL alpha-tocopherol samples preserved the capacity to generate oxidative energy up to 1 h of incubation. The addition of 200 microg/mL alpha-tocopherol in the semen extender could be useful to preserve boar spermatozoa against the oxidative stress generated by cryopreservation.     

Effects of bovine oviductal proteins on bull spermatozoal function

The effects of bovine oviductal proteins on bull sperm viability, acrosome reaction and motility were studied. Motile frozen/thawed spermatozoa from Percoll gradients were incubated with 1.0 mg/mL oviductal proteins (>8 kDa) extracted by ammonium sulphate precipitation from oviductal extract (OE) or serum-free oviductal epithelial cell-conditioned media (CM), treated in the presence (CM+) or absence (CM-) of 1 microg/mL 17beta-estradiol. Inclusion of oviductal proteins had a significant beneficial effect on sperm viability (76.3 to 80.6%+/-5.3) compared with the control (without oviductal proteins; 57.8%+/-5.3) immediately after the commencement of incubation. After 5 h, viability was significantly higher for CM- and OE treatments than for the control, although no differences were observed at 24 h. Acrosomal status only differed among treatments after 24 h, when higher percentages of acrosome- reacted spermatozoa were found in the control (46.0%+/-2.5) than in the oviductal protein treatments (33.1 to 38.2%+/-2.5). No differences in percentages of motile spermatozoa occurred within the first hour of incubation, although inclusion of CM proteins decreased sperm velocities, beat cross frequency, linearity, and straightness but increased values for mean angular displacement. These findings suggest that proteins secreted by oviductal epithelium promote viability, delay the acrosome reaction and suppress the motion of spermatozoa.     

The effects of different types of syringes on equine spermatozoa

Control extender was incubated at 4 degrees C for 24 hours. Rubber or plastic syringe plungers were separately incubated in semen extender for 24 hours at 4 degrees C. Following incubation, the extender was stored at -20 degrees C until the time of semen collection. The treatments consisted of the following: Group A = equine semen plus control extender; Group B=equine semen plus extender incubated with rubber plungers and Group C=equine semen plus extender incubated in plastic plungers; Group D=equine semen plus control extended in rubber plunger syringes and Group E=equine semen plus control extender in plastic plunger syringer. Each group contained a 5-ml volume of semen and extender at a concentration of 1.0 x 10(8) sperm/ml. The number of live spermatozoa, percentage of progressively motile spermatozoa and rate of progressive motility were taken following collection and every 15 minutes for 1 hour following application of treatments. In experiment 2, treatments were allowed to incubate with semen for 45 minutes, then the extender was removed and was replaced with fresh extender. The rate of progressive motility and the percentage of progressively motile spermatozoa were taken immediately, at 45 minutes, and then every 15 minutes for 1 hour. In experiment 1, the number of live spermatozoa was not affected among the 5 groups. However, there was a decrease (P<0.01) in the rate of progressive motility and in the percentage of progressively motile spermatozoa in Group B compared with the remaining 4 treatment groups at 30, 45 and 60 minutes, with no differences noted when semen was held in syringes with a rubber or a plastic plunger. In experiment 2, the percentage of progressively motile spermatozoa increased after the addition of the control extender.     

Single-layer centrifugation with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed easily

The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P < 0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 15 mL of colloid of a reduced density, Androcoll-E-Large (SLC-Large), in 50-mL tubes were both found to give similar yields of motile spermatozoa as that of the SLC-Small method (SLC-Small, 49.7 ± 18.6%; SLC-Inc, 53.3 ± 17.1%; SLC-Large, 44.9 ± 18.3%) and were found to be equivalent in quality (motility: 88.0 ± 8.8%, 84.0 ± 3.5%, 90.0 ± 5.4%; normal morphology: 69.4 ± 17.0%, 69.4 ± 12.7%, 63.9 ± 15.6%; viability: 78 ± 16.7%, 83.8 ± 12.5%, 80.05 ± 14.6%; DNA fragmentation index: 14.7 ± 10.9%, 12.8 ± 8.1%, 11.6 ± 7.6%, respectively). The processing of an “average” stallion Equus caballus ejaculate in approximately twenty-seven 10-mL tubes (SLC-Inc) or eight 50-mL tubes (SLC-Large) is feasible, the latter being considered more practical for on-stud use.     

Motility and penetration competence of frozen-thawed miniature pig spermatozoa are substantially altered by exposure to seminal plasma before freezing

The objective was to determine if exposure of spermatozoa to seminal plasma before freezing decreases its freezability, assessed by percentage motile cells (using computer-assisted semen analysis) and in vitro penetration ability (using in vitro fertilization and chlortetracycline fluorescence assessment). Ejaculated spermatozoa from miniature pigs were washed by centrifugation within 20 min after collection, then incubated in seminal plasma or modified Hulsenberg VIII diluents (mHM). When the spermatozoa were cryopreserved, spermatozoa incubated in seminal plasma before freezing had significantly lower post-thaw motility than spermatozoa incubated in mHM. The incubation of spermatozoa in seminal plasma also significantly prevented frozen-thawed spermatozoa from penetrating the oocytes. The second experiment, using unfrozen spermatozoa, was to determine if the incubation of spermatozoa with seminal plasma reduced penetration ability before freezing, resulting in a significantly lower penetration rate after freezing (compared with spermatozoa incubated without seminal plasma). The penetration competence of unfrozen spermatozoa was significantly decreased by incubation in seminal plasma, but no difference in motility was observed between spermatozoa exposed to seminal plasma versus mHM. We concluded that ejaculated seminal plasma contained some factor(s) that modified the sperm before freezing and reduced the freezability and post-thaw penetration competence of spermatozoa.     

Postthaw evaluation of in vitro function of epididymal spermatozoa from four species of free-ranging African bovids

An improved understanding of reproductive physiology in nondomestic bovids is necessary for the development of assisted reproductive technologies (ARTs) for use in the conservation of endangered bovids. In this study, epididymal spermatozoa were recovered from blesbok (Damaliscus dorcas phillipsi), African buffalo (Syncerus caffer), springbok (Antidorcas marsupialis), and black wildebeest (Connochaetes gnou) following organized culls in South Africa. Our objectives were 1) to characterize the quality of epididymal spermatozoa, 2) evaluate the effectiveness of a cryopreservation protocol, and 3) compare postthaw sperm longevity (motility, viability, and acrosomal integrity) and functionality in two culture media with two capacitation reagents (caffeine and heparin). Following recovery, spermatozoa were diluted in EQ extender, slow-cooled, and frozen in the presence of 5% glycerol. Thawed spermatozoa were separated on a Percoll gradient and diluted in fertilization media (SOF for fertilization [SOFfert]; 0.6% BSA, 0.0 mM glucose, 25.0 mM NaHCO(3)) or modified SOFfert (1.2% BSA, 1.5 mM glucose, 37.0 mM NaHCO(3)) and either heparin or caffeine, and incubated for 6 h. Spermatozoa from these species maintained an average of 64% initial motility after thawing. Incubation medium and capacitation reagent had species-specific effects on the motility, viability, and acrosomal integrity of spermatozoa, suggesting ART procedures need to be optimized for each species. Springbok spermatozoa were also shown to be competent for in vitro fertilization. Information from this study concerning sperm physiology in blesbok, African buffalo, springbok, and black wildebeest will be useful in the development of ART for the conservation of these and other species of bovids.