Response of spermatozoa from the emu (Dromaius novaehollandiae) to rapid cooling, hyperosmotic conditions and dimethylacetamide (DMA)

Three experiments conducted to improve the survival of emu sperm during cryopreservation aimed to: (1) minimize chilling injury during the cooling phase; (2) determine the osmotic effects of dimethylacetamide (DMA), sucrose and trehalose; and (3) investigate the timing and nature of cryoprotectant toxicity. We measured sperm membrane integrity, motility, morphology and egg membrane penetration. In Experiment 1, semen diluted 1:1 with a pre-cooled diluent (5°C) prevented chilling injury. In Experiment 2, semen was diluted with DMA, trehalose or sucrose (300-2400mOsm/L) in deionized water. Only added DMA decreased the percentage of morphologically normal sperm. The percentage of motile sperm was higher with DMA than with the sugars, but membrane intact sperm were comparable amongst all cryoprotectants. As for the osmotic effects, the percentage of membrane intact sperm decreased with 2400mOsm/L and sperm motility decreased with 1200-2400mOsm/L, but sperm morphology was similar at all osmolarities. In Experiment 3, sperm membrane integrity, motility and morphology were comparable at all DMA osmolarities between sperm equilibrated for 0 and 15min, and remained unchanged after removal of DMA. We conclude that: (a) loss of sperm function during the cooling phase can be avoided by using a diluent maintained at 5°C; (b) emu spermatozoa tolerate upto 1400mOsm/L; (c) DMA results in a permanent change in sperm morphology when it is dissolved in deionized water, but does not alter sperm membrane integrity and motility; and (d) equilibration time of sperm with DMA can be less than 10min.     

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.     

Measurement of the sperm motility index via the sperm quality analyzer and its relationship to other qualitative sperm parameters

Sperm parameters such as the concentration and percentage of motile spermatozoa are commonly used to assess semen quality. The sperm quality analyzer (SQA) is a device that detects variations in the optical density of motile spermatozoa, providing a sperm motility index (SMI) that is based on various sperm parameters including the concentration, morphology and acrosomal status of motile spermatozoa. The relationship between SMI values of frozen-thawed bovine spermatozoa undergoing swelling in a hypoosmotic medium (100 mOsm/L) and other sperm parameters were evaluated. Frozen semen specimens from 3 bulls were thawed and washed with Ham's F-10 supplemented with 3% BSA and split into 3 (0.2 mL) aliquots. The aliquots were diluted with 1.0 mL of Ham's F-10 (Aliquot 1), isotonic sodium citrate (Aliquot 2), and hypotonic sodium citrate (Aliquot 3). The osmotic pressure of the media used for dilution of Aliquots 1 and 2 was 300 mOsm/L, while that for Aliquot 3 was 100 mOsm/L. Following dilution, the aliquots were incubated for 30 min and manually assessed at 5-min intervals for the percentage and grade of motility (Grades 0 to 4) as well as for the percentage of swollen spermatozoa. Sperm samples were simultaneously evaluated by SQA to obtain the SMI values at the same 5-min intervals during the 30-min incubation. Significant correlations were observed between SMI values and other sperm parameters in Aliquot 3 (P < 0.05). The results indicated that the SMI values obtained from frozen-thawed bovine spermatozoa exposed to a 100 mOsm/L diluent, which causes optimal swelling of spermatozoa, are highly correlated to other sperm parameters. The SQA unit, as applied in this study, can be used for rapid and reliable screening of sperm samples.     

Assessment of sperm survival and functional membrane integrity of the six-banded armadillo (Euphractus sexcinctus)

The objective was to evaluate sperm survival in the six-banded armadillo, using a thermoresistance test, and to compare sugar solutions with varying osmolarities to analyze the integrity of the functional sperm plasma membrane in this species. Twelve ejaculates were obtained from four mature males by electroejaculation and evaluated for sperm motility, vigor, live sperm, and morphology. Sperm survival was evaluated during a thermoresistance test at 34 °C (the body temperature of this species). The functional integrity of the plasma membrane was evaluated by means of the hypo-osmotic swelling test (HOST), using solutions of varying osmolarities (0, 50, 100, and 150 mOsm/L). During the thermoresistance test, at each evaluation, there was a reduction (P < 0.05) in mean values for sperm motility, sperm vigor, and percentage of live sperm (no movement was observed at 360 min). Sperm survival varied among individual armadillos (P < 0.05). In two individuals, sperm vigor was significantly enhanced when semen was diluted in Tris extender. The response of armadillo sperm to the HOST varied among individuals (P < 0.05). On average, maximal values (P < 0.05) of reactive sperm (59%) were detected with 50 mOsm/L solution; furthermore, this concentration had the largest significant positive correlation (r = 0.84) to live sperm percentage. In conclusion, six-banded armadillos had significant individual variation with regard to sperm survival in a thermoresistance test at 34 °C; in some individuals, sperm survived until 360 min. The use of a 50 mOsm/L fructose solution was recommended for conducting a HOST in this species.     

Sperm viability in the black-footed ferret (Mustela nigripes) is influenced by seminal and medium osmolality

Fundamental knowledge of spermatozoa cryobiology can assist with optimizing cryopreservation protocols needed for genetic management of the endangered black-footed ferret. Objectives were to characterize semen osmolality and assess the influence of two media at various osmolalities on sperm viability. We examined the influence of Ham's F10 +Hepes medium (H) at 270, 400, 500 or 700 mOsm (adjusted with sucrose, a nonpermeating cryoprotectant) and TEST Yolk Buffer (TYB) with 0% (300 mOsm) versus 4% (900 mOsm) glycerol (a permeating cryoprotectant). Electroejaculates (n=16) were assessed for osmolality using a vapor pressure osmometer. For media comparison, semen (n=5) was collected in TYB 0%, split into six aliquots, and diluted in H270, H400, H500, H700, and TYB 0% or TYB 4%. Each sample was centrifuged (300 g, 8 min), resuspended in respective medium, and maintained at 37 degrees C for 3h. Sperm motility and forward progression were monitored every 30 min for 3h post-washing. Acrosomal integrity was monitored at 0 and 60 min post-washing. Results demonstrated that black-footed ferret semen has a comparatively high osmolality (mean+/-SEM, 513.1+/-32.6 mOsm; range, 366-791 mOsm). Ferret spermatozoa were sensitive to hyperosmotic stress. Specifically, sperm motility was more susceptible (P<0.01) to hyperosmotic conditions than acrosomal integrity, and neither were influenced (P>0.05) by hypotonic solutions. Exposure to TYB 4% glycerol retained more (P<0.01) sperm motility than a hyperosmotic Ham's (700 mOsm). These findings will guide the eventual development of assisted breeding with cryopreserved sperm contributing to genetic management of this rare species.     

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.     

Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo (Bubalus bubalis)

The study was carried out to evaluate the potential impact of butylated hydroxytoluene (BHT) on the frozen-thawed semen quality of Nili-Ravi buffalo bulls. Ejaculated bull semen was extended in a Tris-citrate egg yolk extender containing various concentrations of BHT (0.5, 1.0, 2.0 and 3.0 mM). Semen was frozen at −196 °C using 50 × 10⁶ spermatozoa per 0.5 mL straws. Five straws from each treatment were thawed to assess the semen quality in terms of sperm motility, viability, plasma membrane integrity and acrosomal integrity. Post-thawed sperm motility was determined using a phase-contrast microscope. Viability, plasma membrane integrity and acrosomal integrity were evaluated by the supravital staining, hypo-osmotic swelling test and normal acrosomal reaction, respectively. The highest (P < 0.05) motility, acrosomal integrity and hypo-osmotic swelling response of spermatozoa was achieved by addition of 1.0 and 2.0 mM BHT to semen extender. However, highest (P < 0.05) viability of spermatozoa was achieved by inclusion of 2.0 mM BHT. The higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of the spermatozoa compared to other concentration used. In conclusion, BHT when added in the semen extender can improve the semen quality of buffalo bulls.     

Comparative semen cryopreservation in ferrets (Mustela putorius furo) and pregnancies after laparoscopic intrauterine insemination with frozen-thawed spermatozoa

A study was conducted to determine an optimum technique for semen cryopreservation and the biological competence of frozen-thawed ferret spermatozoa. Fifty-two fresh electroejaculates from 4 males were evaluated for sperm percentage motility, forward progressive motility, motility index (SMI) and acrosomal integrity. To determine the optimum temperature for maintaining sperm motility in vitro and the influence of glycerol on sperm motility, seminal aliquants were diluted in TEST diluent (containing either 0 or 4% glycerol) and maintained at 25 degrees or 37 degrees C. For cryopreservation, semen was diluted in each of 3 cryodiluents (TEST, PDV, BF5F), cooled for 30 min at 5 degrees C and pelleted on solid CO2 or frozen in 0.25 ml straws (20 degrees C/min to -100 degrees C). Following thawing, SMI and acrosomal integrity were determined. Ten females with maximum vulval swelling were given 90 i.u. human chorionic gonadotrophin and laparoscopically inseminated in utero with spermatozoa previously frozen using the optimum diluent and freeze-thaw method. The maintenance temperature of 25 degrees C was superior (P less than 0.05) to 37 degrees C for sustaining sperm motility, and glycerol did not influence (P greater than 0.05) motility for up to 11 h of culture. After thawing, motile spermatozoa were recovered in all treatment groups, but sperm motility and normal acrosomal ratings were highest using the PDV diluent, the pelleting method and thawing at 37 degrees C (P less than 0.05). Seven of the 10 ferrets (70%) inseminated with spermatozoa frozen by this approach became pregnant and produced 31 kits (mean litter size 4.4; range 1-9 kits).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of solid storage of sheep spermatozoa at 15 degrees C on their survival and penetrating capacity

This study was designed to evaluate the possible benefits of adding gelatin to a standard milk extender, for solid storage of sheep semen at 15 degrees C. Solid storage was assessed in terms of effects on sperm motility and membrane integrity up to 2 days (Study 1), and on in vitro penetration capacity after storage for 24h (Study 2). In both studies, semen was diluted in CONTROL (standard milk extender) and GEL (1.5 g gelatin/100ml extender) diluents to a final concentration of 400 x 10(6)sperm/ml. In Study 1, semen samples were stored at 15 degrees C, and sperm quality variables analyzed after 2, 24 and 48 h of storage. Motility and viability values were significantly lowered using the liquid compared to the gel extender for all storage periods, except for motility after 2h of storage, whose values were similar. After 2h of incubation at 37 degrees C, motile cell percentages and membrane integrity were significantly lower in the CONTROL group than in the GEL group for all storage periods. In Study 2, in vitro matured lamb oocytes were randomly divided into three groups and fertilized with CONTROL diluted semen stored for 2h or 24h, or with GEL diluted semen stored for 24h. After co-incubation, oocytes were evaluated for signs of penetration. Storage of semen in the GEL diluent for 24h gave rise to increased in vitro fertilization rates in comparison with the CONTROL diluent. Our findings indicate that the solid storage at 15 degrees C of ram spermatozoa by adding gelatin to the extender leads to improved survival and in vitro penetrating ability over the use of the normal liquid extender. A solid diluent could thus be a useful option for the preservation of fresh ovine semen for extended periods.     

Effects of different extenders on DNA integrity of boar spermatozoa following freezing-thawing

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100 mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing–thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P < 0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100 mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100 mM trehalose, but cryopreservation could increase the degree of DNA damage (P < 0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.     

The effect of supplementation of cryopreservation diluents with sugars on the post-thawing fertility of ram semen

This study was conducted to elucidate the effect of increasing the osmolality of a basic Tris, extender supplemented with sucrose, trehalose or raffinose on post-thawing ram semen quality (sperm motility, viability, acrosome integrity, total sperm abnormalities and membrane integrity). After primary evaluation of the collected ejaculates, only semen samples with more than 70% motile sperm, and a sperm concentration of higher than 3 × 10⁹ sperm/ml were used for cryopreservation. The semen samples were pooled and diluted (1:4) with a Tris-citric acid-fructose-yolk extender, supplemented with different concentrations (50, 70 or 100 mM) of sucrose, trehalose or raffinose. As control, semen was diluted and frozen in the base diluent, without additional sugars. Pooled semen samples were aspirated into 0.25 ml straws, cooled to 5 °C within 90 min and frozen by exposure to liquid nitrogen vapor (4-5 cm above the liquid nitrogen surface) for 10 min - before plunging into liquid nitrogen, for storage. After 24 h, straws were thawed in a water bath (37 °C) for 30 s. The frozen-thawed sperm characteristics were improved significantly (P < 0.05) by increasing the level of the sugars. Optimal results being obtained with 70 and 100 mM trehalose or raffinose. All extenders containing supplemental sugars were superior in terms of sperm quality to the control (P < 0.01) group. The highest sperm motility (60.6 ± 1.9%), viability (60.6 ± 2.5%) and membrane integrity (58.2 ± 2.1%) were recorded using 100 mM trehalose and the lowest with 50 mM sucrose (48.6 ± 1.9%, 51.4 ± 2.5% and 47.9 ± 2.1%, respectively). All sugar concentrations decreased the percentage of acrosomal and total sperm abnormalities (P < 0.05). The extenders containing 100 mM trehalose or raffinose significantly (P < 0.05) decreased the occurrence of sperm abnormalities, compared to the other treatments. The fertility rates obtained after cervical insemination of the frozen-thawed sperm were 46.8%, 44.1% and 16.7% for 100 mM trehalose, 100 mM raffinose and the control with supplementation of the diluents, respectively. The study showed that ram sperm can tolerate hyperosmotic diluents, and that a range of sugar concentrations (50-100 mM) may successfully be incorporated in the ram semen cryopreservation diluents, although further research is warranted.     

Changes in extracellular osmolality initiate sperm motility in freshwater teleost rosy barb Puntius conchonius

The objective was to investigate the effects of extracellular osmolality and membrane osmotic-sensitive channels on the initiation of sperm motility and to explore mechanisms of sperm initiation in rosy barb (Puntius conchonius). We found that (1) sperm were immotile in seminal plasma and remained quiescent in electrolyte or nonelectrolyte solutions isotonic to seminal plasma; (2) sperm movement was initiated when the sperm were exposed to hypo-osmotic electrolyte or hypo-osmotic nonelectrolyte solutions, and that the responsiveness of sperm to changes in the extracellular osmolalities (100, 200, 250, 270, and 300 mOsm/kg) differed among sperm cells (P < 0.05); (3) sperm movement could be initiated and terminated repeatedly by decreasing and increasing the osmolality (in increments of 100 and 300 mOsm/kg) of a nonelectrolyte mannitol solution, respectively (P < 0.05); (4) gadolinium (20, 40, and 80 μM) inhibited the initiation of sperm motility and abolished the sperm activation caused by the hypo-osmotic media treatment in dose- and time-dependent manners (P < 0.05); and (5) sperm activation in a hypo-osmotic medium and inhibition in an isotonic solution were associated with swelling and shrinkage of the sperm sleeves, respectively. Therefore, we concluded that osmolality was a critical physiologic signal in regulating the initiation and termination of sperm motility in freshwater teleost rosy barb. Furthermore, we inferred that rosy barb sperm were hypo-osmotic–dependent conformers, and the osmotic-sensitive channel could be involved in the mechanism of sperm initiation.     

Effect of oviductal fluid proteins on buffalo sperm characteristics during cryopreservation

The objective was to determine the effects of oviductal proteins on sperm function. Abbatoir-derived buffalo oviducts were flushed with PBS; the fluid recovered (protein concentration, 2.3 mg/mL; average of 3.5 mg protein/oviduct) was centrifuged, dialyzed, and clarified, and the supernatant applied to a Heparin-Sepharose affinity column. Unbound fractions were collected and bound proteins were separately eluted (with elution buffer). Eight distinct protein bands (from 12 to 177 kDa) in the H-unbound fraction and 15 distinct protein bands (from 12 to 165 kDa) in the H-bound fraction were detected in SDS-PAGE. Semen from four buffalo bulls was divided into three parts: Parts 1 and 2 were treated with the heparin binding (H-bound) and non-heparin binding (H-unbound) oviductal proteins, respectively, whereas Part 3 remained as an untreated control. Equilibrated and frozen-thawed semen was assessed for motility, viability, intact acrosome percentage, mucus penetration distance, and hypo-osmotic swelling test. The H-bound oviductal fluid proteins enhanced (P<0.05) the proportion of sperm that were progressively motile, alive, had an intact acrosome and functional plasma membrane (hypo-osmotic swelling test), as well as the distance covered in the cervical mucus sperm penetration test during cryopreservation. Addition of the H-unbound oviductal protein fraction did not increase sperm motility and penetration distance but increased (P<0.05) the proportion of sperm that were live, had an intact acrosome, and functional plasma membrane (hypo-osmotic swelling test). We concluded that the H-bound fraction of buffalo oviductal fluid protein(s) maintained sperm motility, viability and membrane integrity during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity.     

Hypo-osmotic swelling can accurately assess the viability of nonmotile sperm

Viable, healthy sperm are preferred for oocyte fertilization with intracytoplasmic sperm injection. Currently, motility is the most widely applied measure of sperm viability. However, with this criterion, viable but immotile sperm are overlooked as candidates for micromanipulation. Supravital stains identify viable sperm but may be toxic to the gamete or embryo. We tested the hypothesis that hypo-osmotic swelling, developed to assess sperm membrane integrity, can accurately determine sperm viability. Thawed sperm from 12 fertile donors were exposed to a hypo-osmotic solution and to two supravital stains. A total of 2,010 sperm were assessed for tail coiling after a 30-min exposure to hypo-osmotic solution. By supravital stains, 31% of thawed sperm were viable; 23% showed tail coiling. Among coiled-tail sperm, 100% were viable by supravital stain. As a measure of viability, tail coiling exhibited a sensitivity of 30%, specificity of 100%, and positive predictive value of 100% compared to supravital stains. With a 60-min hypo-osmotic incubation, the specificity (89%) and positive predictive value (78%) decreased significantly (P < 0.05). Therefore, hypo-osmotic swelling accurately detects viable sperm among a nonmotile population. Assay accuracy, however, is very sensitive to the incubation time in hypo-osmotic solution. Mol. Reprod. Dev. 47:200–203, 1997. © 1997 Wiley-Liss, Inc.     

Effect of dialysis on quality characteristics of turkey semen during liquid storage

Low molecular weight substances such as zinc and peroxides are present in seminal plasma and are responsible for deleterious effects in stored semen. On the contrary, molecules larger than 50 kDa are beneficial to in-vitro storage of spermatozoa. Since the effects of different seminal plasma fractions in turkey semen are not completely known, the purpose of the study was to determine the effects of turkey semen dialysis with a 12-14 kDa cut-off on viability, hypo-osmotic membrane integrity, or sperm motility of turkey spermatozoa stored up to 48 h at 5 degrees C. Twelve pools of semen, each pool originating from four toms, were used. Each pool was divided into two aliquots, one of which was dialyzed while the other represented the control. Each semen aliquot was evaluated for sperm viability, membrane integrity and motility after 6, 24 and 48 h of in-vitro storage. Cold storage of turkey semen for 48 h significantly worsened (P<0.01) sperm viability, hypo-osmotic membrane integrity, and sperm motility index of both control and dialyzed samples. After 24 and 48 h sperm viability, membrane integrity and sperm motility index were better (P<0.01) in dialyzed semen compared to the control.     

Membrane changes during different stages of a freeze-thaw protocol for equine semen cryopreservation

Many theories have been postulated concerning the possible effects of cryopreservation on spermatozoa, including suggestions the freeze-thawing process produces membranes that have greater fluidity and are more fusogenic, thus inducing changes similar to those of capacitation. The main objectives of this study were to determine at what stage of the freeze-thaw process membrane changes occur and whether evaluation with chlortetracycline (CTC) stain could predict the freezability of stallion sperm. Sperm viability and state of capacitation were simultaneously evaluated using CTC and Hoechst 33258 (H258) techniques. Membrane function was evaluated using the hypo-osmotic swelling test (HOS) and progressive motility (PM) was evaluated under light microscopy at each stage of a freeze-thaw protocol. Evaluated were raw semen; after dilution and centrifugation; after redilution and equilibration at room temperature; after cooling to 5 degrees C; after super cooling to -15 degrees C; and after thawing. The most pronounced functional damage to membranes and the greatest decrease in PM occurred in samples of all stallions after thawing (P<0.05). Cryopreservation, as evaluated by CTC/H258 staining, significantly (P<0.05) affected sperm membrane integrity after centrifugation, after redilution and equilibration at room temperature and after cooling to 5 degrees C. The HOS and H258 tests gave similar results (R values of approximately 0.75) and correlated inversely with the number of live noncapacitated sperm cells (R values of approximately -0.75). Remarkably, the subpopulation of capacitated live cells was unaffected in all freeze-thawing steps and the number of live acrosome reacted cells increased by a factor of 4. However, it was not possible to determine whether the changing CTC patterns reflect a true capacitation phenomenon or an intermediate destabilized state of the sperm cell membrane. This increase may indicate that the subpopulation of functional sperm cells capable of binding to the zona pellucida increases after freeze-thawing despite the deteriorative effect of this procedure for the entire live sperm population.     

Effects of osmotic pressure on motility, plasma membrane integrity and viability in fresh and frozen-thawed buffalo spermatozoa

In the first experiment, osmotic pressure of semen and seminal plasma in a semen sample from each of the 20 mature Nili-Ravi buffalo bulls was determined. In the second experiment, effects of osmotic pressure on motility (%), plasma membrane integrity (%) and viability (%) in fresh and frozen-thawed semen samples from each of the seven mature Nili-Ravi buffalo bulls was determined. In the first experiment, seminal plasma was harvested by centrifuging semen at 400 × g for 10 min at 37°C and osmotic pressure was determined using an osmometer. In the second experiment, motility (%) was assessed in fresh and frozen-thawed (37°C for 30 s) semen samples using a phase-contrast microscope (×400). Plasma membrane integrity (%) was determined by mixing 50 μl each of fresh and frozen-thawed semen with 500 μl of solution having an osmotic pressure of 50, 100, 150, 190 or 250 mOsm/l (hypotonic treatments of fructose + sodium citrate) and incubating at 37°C for 1 h. Viability (%) of fresh and frozen-thawed spermatozoa before and after challenging them to osmotic pressure (hypotonic treatments) was assessed using supravital stain under a phase-contrast microscope (×400). In the first experiment, the mean ± s.e. osmotic pressures of the buffalo semen and seminal plasma were 268.8 ± 1.17 and 256.0 ± 1.53 mOsm/l, respectively. In the second experiment, motility (%) decreased (P < 0.05) in frozen-thawed semen samples as compared with fresh semen (60.1 ± 1.34 v. 81 ± 1.57, respectively). The plasma membrane integrity (%) and magnitude of osmotic stress in fresh and frozen-thawed semen samples was higher (P < 0.05) at 50, 100, 150 and 190 mOsm/l as compared with 250 mOsm/l. Loss of viability (%) in fresh and frozen-thawed semen samples was higher (P < 0.05) at 50 mOsm/l (59% in fresh, 70% frozen thawed) as compared with other osmotic pressures, while it was lowest at 250 mOsm/l (4.1% for fresh, 9.7% frozen thawed). In conclusion, osmotic pressure of Nili-Ravi buffalo semen and seminal plasma is determined. Furthermore, variation in osmotic pressure below 250 mOsm/l is not favorable to fresh and frozen-thawed buffalo spermatozoa.     

In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions

In this study, environmental conditions mimicking those prevailing in the epididymis were used for storing ejaculated bull spermatozoa in vitro during 4 days at ambient temperature. These conditions were low pH, high osmolarity, high sperm concentration and low oxygen tension. Hepes-TALP was used as basic storage medium. Fresh spermatozoa were stored at a concentration of 10 x 10(6)spermatozoa/ml in Hepes-TALP of different pH (pH 4, 5, 6, 7 or 8), and osmolarity (100, 300, 400, 500, 600 or 800 mOsm/kg), and under different atmospheric conditions (nitrogen gassed or aerobic). Spermatozoa were also stored undiluted or at different concentrations: 10x 10(6), 100 x 10(6), 500 x 10(6) or 1 x 10(9)spermatozoa/ml. Sperm parameters such as membrane integrity, motility, mitochondrial membrane potential or DNA fragmentation were used to assess semen quality after storage. Adjustment of the pH of Hepes-TALP to pH 6 yielded significantly better results than storage at all other pH values. Isotonic Hepes-TALP (300 mOsm/kg) had a less detrimental effect on spermatozoa than hypo- and hyperosmotic versions. No differences in sperm parameters were observed when spermatozoa were incubated under aerobic or under nitrogen gassed storage conditions. Optimal sperm concentration in vitro is 10 x 10(6)spermatozoa/ml. This is in contrast with the in vivo situation, where spermatozoa are stored at high concentration. However, better results at high sperm concentrations were obtained when spermatozoa were diluted for less than 5 min in Triladyl-egg yolk-glycerol diluent immediately after ejaculation.     

Trehalose-enhanced fluidity of the goat sperm membrane and its protection during freezing

In an attempt to find a suitable freezing method for goat semen, two experiments were conducted to study the influence of trehalose on the cryopreservation of goat spermatozoa. In experiment 1, goat spermatozoa were frozen in trehalose extender (0.375 M) alone (100%) or at different combinations of trehalose with Tris-citric acid-glucose (TCG) extender (0%, 25%, 50%, 75%). Final concentrations of 20% (v:v) egg yolk and 4% (v:v) glycerol were employed in the extenders (osmolality = 370, pH = 7). Sperm motility was assessed using a computer-assisted sperm analysis system and acrosome integrity was assessed using fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA). The sperm-motility parameters improved significantly by increasing the concentration of trehalose (P < 0.05) and significantly high recovery rates for the motility parameters were also achieved by a high concentration of trehalose (P < 0.05). Motility of the frozen-thawed spermatozoa after a 3-h incubation improved significantly with increasing concentrations of trehalose in the extender (P < 0.05). The 75% and 100% trehalose extenders yielded a significant increase in the percentage of spermatozoa with intact acrosome (P < 0.05). In experiment 2, merocyanine 540/Yo-Pro staining was used to study the influence of trehalose on membrane fluidity compared with that of sucrose and TCG. Percentage of cells with high merocyanine fluorescence was significantly higher in spermatozoa treated with trehalose than sucrose or TCG (P < 0.05), indicating a significantly highest membrane fluidity of sperm samples extended with trehalose solution. We thus conclude that the substitution of a Tris-citric acid diluent composition with trehalose significantly improves the freezability of goat spermatozoa. Furthermore, the cryoprotective effects of trehalose observed in this study may be due to enhanced sperm membrane fluidity before freezing.     

Effects of different extenders on DNA integrity of boar spermatozoa following freezing–thawing

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100 mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing–thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P < 0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100 mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100 mM trehalose, but cryopreservation could increase the degree of DNA damage (P < 0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.