Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose

Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding numbers of lines of mutant mice. However, successful and reproducible cryopreservation has proven to be a difficult problem. Furthermore, the underlying factors responsible for success or failure are mostly obscure. Several contributors to these difficulties have been identified. Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses associated with pipetting, mixing, and centrifugation, and others have found that they are severely limited in their tolerance to osmotic volume changes. We have hypothesized two other contributors to the difficulties. One is that the concentrations of glycerol used in published protocols are substantially lower than those found to be optimal for most mammalian cells. The other hypothesis relates to the fact that mouse sperm membranes are especially susceptible to damage from oxygen-derived free radicals. That damage may reduce their ability to survive freezing. If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure. To achieve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media. A previous report showed a protective effect. That is confirmed here under a broader range of conditions. The conditions studied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility after freezing at 21 degrees C/min to -70 degrees C. Cryoprotection increased with increasing raffinose concentration, provided that the concentration of PBS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits. Surprisingly, the best results were achieved in the total absence of glycerol. The highest motilities to date (68 +/- 8%) after freezing to -70 degrees C have been achieved using media containing Oxyrase, 0 M glycerol, and 18% raffinose in 14x strength modified PBS. We also determined the motility loss after freezing to intermediate temperatures, i.e., -10 and -30 degrees C. The major motility loss occurred by -10 degrees C, especially in the absence of Oxyrase. These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation. Oxyrase appears to lessen that damage substantially.     

Mouse Spermatozoa in High Concentrations of Glycerol: Chemical Toxicity vs Osmotic Shock at Normal and Reduced Oxygen Concentrations

The cryobiological preservation of mouse spermatozoa has presented difficulties in the form of poor motilities or irreproducibility. We have identified several likely underlying problems. One is that published studies have used concentrations of the cryoprotectant glycerol that are substantially lower (0.3 M) than the ∼1 M concentrations that are optimal for most cells. Another may arise from the known high susceptibility of mouse sperm to free radical damage. We have identified two contributors to damage from higher concentrations of glycerol, namely, chemical toxicity proportional to concentration and exposure time and osmotic damage arising from too rapid an addition and removal of the glycerol. When toxicity is minimized by restricting the exposure time to 1 or 5 min and osmotic shock is minimized by adding and removing the glycerol stepwise, relatively high percentages of the sperm survive contact with 0.8 M glycerol. Free-radical damage in mouse sperm is known to be proportional to the oxygen concentration. We have determined the consequences of reducing the oxygen to <3% of atmospheric by the use of a bacterial membrane preparation, Oxyrase. Oxyrase reduced damage from centrifugation and substantially reduced damage from osmotic shock; however, it did not significantly reduce glycerol toxicity.     

Effects of cooling and warming rate to and from -70 degrees C, and effect of further cooling from -70 to -196 degrees C on the motility of mouse spermatozoa

We have previously reported high survival in mouse sperm frozen at 21 degrees C/min to -70 degrees C in a solution containing 18% raffinose in 0.25 x PBS (400 mOsm) and then warmed rapidly at approximately 2000 degrees C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to -70 degrees C at rates ranging from 0.3 to 530 degrees C/min. The survival curve was an inverted "U" shape, with the highest motility occurring between 27 and 130 degrees C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130 degrees C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was -70 degrees C. When sperm were frozen to -70 degrees C at optimum rates, lowering the temperature to -196 degrees C had no adverse effect.     

Effects of centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and thawing rate on the post-thaw motility of equine sperm

Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm motility between semen cooled slowly in extender with or without glycerol to 5 degrees C prior to freezing to -120 degrees C and semen cooled continuously from 20 degrees C to -120 degrees C. From these experiments, a new procedure for processing, freezing and thawing semen evolved. The new procedure involved dilution of semen to 50 x 10(6) sperm/ml in centrifugation medium and centrifugation at 400 x g for 15 min, resuspension of sperm in lactose-EDTA-egg yolk extender containing 4% glycerol, packaging in 0.5-ml polyvinyl chloride straws, freezing at 10 degrees C/min from 20 degrees C to -15 degrees C and 25 degrees C/min from -15 degrees C to -120 degrees C, storage at -196 degrees C, and thawing at 75 degrees C for 7 sec. Post-thaw motility of sperm averaged 34% for the new method as compared to 22% for the old method (P<0.01).     

Sperm cryopreservation of a live-bearing fish, the platyfish Xiphophorus couchianus

The objectives of this study were to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio, as well as somatic relationships of body length, body weight, and testis weight to sperm density in the platyfish Xiphophorus couchianus. Sperm motility and survival duration after thawing were significantly different between cryopreservation with dimethyl sulfoxide (DMSO) and glycerol, with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as cryoprotectant, the highest motility after thawing was observed with Hanks' balanced salt solution (HBSS) across a range of 240-300 mOsm/kg. Samples cooled from 5 to -80 degrees C at 25 degrees C/min yielded the highest post-thaw motility, although no significant difference was found for cooling rates across the range of 20-30 degrees C/min. In addition, the highest motility after thawing was found in samples equilibrated from 10 to 30 min with 14% glycerol and cooled at 25 degrees C/min. The post-thaw motility declined rapidly with use of 10% glycerol and cooling at 5 degrees C/min across the equilibration range of 10 min to 2h. Sperm motility with a dilution ratio of sperm to extender of 1:10 was not different at 10 min after thawing with those samples at greater dilutions, but declined significantly from Day 1 after thawing and showed lower survival duration when stored at 4 degrees C. However, the additional dilution of sperm solutions with HBSS (300 mOsm/kg) immediately after thawing significantly slowed the decline of motility and prolonged the duration of survival. Based on the above findings, the highest average sperm motility (78+/-3 %) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsm/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS-glycerol of 1:20, equilibrated for 10 min, cooled at 25 degrees C/min from 5 to -80 degrees C before plunging into liquid nitrogen, and thawed at 40 degrees C in a water bath for 7 s. If diluted within 5 h after thawing, sperm frozen by the above protocol retained continuous motility for 15 days when stored at 4 degrees C.     

Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates?

Mouse spermatozoa in 18% raffinose and 3.8% Oxyrase in 0.25 x PBS exhibit high motilities when frozen to -70 degrees C at 20-130 degrees C/min and then rapidly warmed. However, survival is <10% when they are frozen at 260 or 530 degrees C/min, presumably because, at those high rates, intracellular water cannot leave rapidly enough to prevent extensive supercooling and this supercooling leads to nucleation and freezing in situ (intracellular ice formation [IIF]). The probability of IIF as a function of cooling rate can be computed by coupled differential equations that describe the extent of the loss of cell water during freezing and from knowledge of the temperature at which the supercooled protoplasm of the cell can nucleate. Calculation of the kinetics of dehydration requires values for the hydraulic conductivity (Lp) of the cell and for its activation energy (Ea). Using literature values for these parameters in mouse sperm, we calculated curves of water volume versus temperature for four cooling rates between 250 and 2000 degrees C/min. The intracellular nucleation temperature was inferred to be -20 degrees C or above based on the greatly reduced motilities of sperm that underwent rapid cooling to a minimum temperature of between -20 and -70 degrees C. Combining that information regarding nucleation temperature with the computed dehydration curves leads to the conclusion that intracellular freezing should occur only in cells that are cooled at 2000 degrees C/min and not in cells that are cooled at 250-1000 degrees C/min. The calculated rate of 2000 degrees C/min for IIF is approximately eightfold higher than the experimentally inferred value of 260 degrees C/min. Possible reasons for the discrepancy are discussed.     

Sperm survival during short-term storage and after cryopreservation of semen from striped trumpeter (Latris lineata)

Methods of short-term storage and cryopreservation were examined for semen from striped trumpeter (Latris lineata). For fresh semen at 18 degrees C, the percentage of motile sperm declined rapidly from over 80% immediately after activation with sea water to less than 2% within 9 min after activation. The motility after activation of undiluted fresh sperm stored at 5 degrees C was maintained for two days and then declined markedly so that by the eighth day, sperm were mostly immotile after activation. The post-thawing motility was higher for sperm frozen with a non-activating diluent containing 2.84 M DMSO in saline (117 mM NaCl) than in an activating glycerol (2 M) medium in dilute sea water (300 mOsm). Post-thawing motility was higher for a dilution rate of 1:5 (semen:diluent) than 1:2 or 1:11 but was similar when frozen semen was thawed at 10 degrees, 20 degrees or 30 degrees C. For semen stored at a range of volumes as pellets frozen on dry ice (0.2 to 2.0 mL) or straws frozen in liquid nitrogen vapor (0.25 to 0.5 mL) and thawed in a waterbath at 20 degrees C, the post-thawing motilities were similar even though the patterns of cooling and thawing differed markedly between methods of freezing and sizes of pellets and straws.     

Sensitivity of domestic cat (Felis catus) sperm from normospermic versus teratospermic donors to cold-induced acrosomal damage.

Freeze-thawing cat sperm in cryoprotectant results in extensive membrane damage. To determine whether cooling alone influences sperm structure and viability, we compared the effect of cooling rate on sperm from normospermic (N; > 60% normal sperm per ejaculate) and teratospermic (T; < 40% normal sperm per ejaculate) domestic cats. Electroejaculates were divided into raw or washed (Ham's F-10 + 5% fetal calf serum) aliquots, with the latter resuspended in Ham's F-10 medium or Platz Diluent Variant Filtered without glycerol (20% egg yolk, 11% lactose). Aliquots were 1) maintained at 25 degrees C (no cooling; control), 2) cooled to 5 degrees C in a commercial refrigerator for 30 min (rapid cooling; approximately 4 degrees C/min), 3) placed in an ice slush at 0 degrees C for 10 min (ultrarapid cooling; approximately 14 degrees C/min), or 4) cooled to 0 degrees C at 0.5 degrees C/min in a programmable alcohol bath (slow cooling); and aliquots were removed every 4 degrees C. All samples then were warmed to 25 degrees C and evaluated for percentage sperm motility and the proportion of intact acrosomes using a fluorescein-conjugated peanut agglutinin stain. In both cat populations, sperm percentage motility remained unaffected (p > 0.05) immediately after exposure to low temperatures and after warming to 25 degrees C. However, the proportion of spermatozoa with intact acrosomes declined (p < 0.05) after rapid cooling ( approximately 4 degrees C/min) to 5 degrees C (N, 65.6%; T, 27.5%) or ultrarapid cooling ( approximately 14 degrees C/min) to 0 degrees C (N, 62.1%; T, 23.0%) in comparison to the control value (N, 81.5%; T, 77.5%). Transmission electron microscopy of cooled sperm revealed extensive damage to acrosomal membranes. In contrast, slow cooling (0.5 degrees C/min) to 5 degrees C maintained (p > 0.05) a high proportion of spermatozoa with intact acrosomes (N, 75.5%; T, 68.3%), which also remained similar (p > 0.05) between cat populations (N, 64.7%; T, 56.8%) through continued cooling to 0 degrees C. Results demonstrate that 1) rapid cooling of domestic cat sperm induces significant acrosomal damage without altering sperm motility, 2) spermatozoa from teratospermic males are more susceptible to cold-induced acrosomal damage than normospermic counterparts, and 3) reducing the rate of initial cooling markedly decreases sperm structural damage.     

Development of a simple sperm cryopreservation model using a chemically defined medium and goat cauda epididymal spermatozoa

This investigation was carried out to develop a simple sperm cryopreservation model using a chemically defined synthetic medium (modified Ringer's solution) and mature goat cauda epididymal sperm as the model system. Rates of cooling, freezing, and maximum freezing temperature were manipulated with the help of a computer-controlled programmable biofreezer. Highly motile goat cauda sperm dispersed in a modified Ringer's solution was subjected to the freezing protocol: cooling 0.25 degrees C min(-1) to 5 degrees C, 5 degrees C min (-1) to -20 degrees C, 20 degrees C min(-1) to -100 degrees C, prior to plunging into liquid nitrogen. In the absence of any cryoprotective agent, all of the spermatozoa lost their motility. Addition of glycerol (0.22 to 0.87 M) caused a dose-dependent increase of sperm motility recovery. The highest recovery of forward and total motility was (32 and 35%, respectively) at 0.87 M. Further increase of the glycerol concentration caused a marked decrease in motility. Changes in the cooling rate particularly before and during freezing had a notable effect on the sperm motility recovery. There was no or low recovery (0-18%) of sperm motility when the cells were transferred directly to liquid nitrogen from the initial two cooling stages. The data demonstrate the importance of all of the cooling stages in the cryopreservation of the cells. Like glycerol, dimethyl sulfoxide (Me(2)SO) and ethylene glycol also showed a dose-dependent increase in motility recovery as well as a biphasic curve of cryoprotection. At optimal concentrations, dimethyl sulfoxide (1.00 M) and ethylene glycol (1.29 M) were effective in recovering sperm motility to the extent of 20 and 13%, respectively. Thus these reagents have markedly lower cryoprotection potential than glycerol.     

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of egg yolk, glycerol and cooling rate

Two experiments were conducted to determine the effects of egg yolk (EY), glycerol, and cooling rate on the cryosurvival of red deer epididymal spermatozoa. The aim of Experiment 1 was to examine the effects of two EY types (clarified EY, CE, prepared by centrifugation, and whole EY, WE), and four EY concentrations (0, 5, 10 and 20%) on cryosurvival of red deer epididymal spermatozoa. Sperm samples were diluted to a final sperm concentration of approximately 200 x 10(6)spermatozoa/ml with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility, viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Cryopreservation of red deer epididymal spermatozoa frozen in a clarified EY extender, and with a 20% EY resulted in more vigorous post-thaw and post-incubation motilities (P<0.0001). Moreover, our results showed that regardless of the egg yolk concentration tested, the best sperm quality was obtained with the use of CE. Therefore, the objective of Experiment 2 was to explore the post-thaw effects of four clarified egg yolk concentrations (0, 5, 10 and 20%), two final glycerol concentrations (3 and 6%), and two cooling rates from 22 to 5 degrees C (slow: 0.23 degrees C/min; rapid: 4.2 degrees C/min) on red deer epididymal spermatozoa. At thawing, the effects of CE and glycerol concentrations, and cooling rate, all independently affected post-thaw sperm quality, while there were no effects of interactions on post-thawing sperm quality. Therefore, we studied each variable separately. Differences (P<0.05) for most of the semen parameters evaluated were found between the two final glycerol concentrations tested, with the high values after thawing found with the use of 6% glycerol (58.8+/-1.4 versus 46.2+/-1.4, for sperm motility). Moreover, the cooling rate did not have an effect on the semen characteristics, except for NAR (P<0.05), with the high values after thawing found with the use of the rapid protocol (64.5+/-1.4 versus 59.9+/-1.4). In conclusion, the use of 20% CE and 6% glycerol in combination with a rapid cooling rate, significantly improved red deer epididymal spermatozoa freezability.     

Cryopreservation of yellowfin seabream (Acanthopagrus latus) spermatozoa (Teleost, Perciformes, Sparidae)

The effects of both osmolality and cation in the initiation of sperm motility were examined in yellowfin seabream, Acanthopagrus latus. Various factors involved in the cryopreservation of yellowfin seabream spermatozoa on motility are discussed. Extender containing only glucose proved to be a suitable medium for freezing yellowfin seabream spermatozoa to -196 degrees C. Glycerol seems to have a direct osmotic effect on yellowfin seabream sperm cells, and it induced sperm motility before freezing and during thawing. However, this exhausted the energy needed for sperm motility for fertilization. Dimethyl sulfoxide (DMSO) proved superior to ethylene glycerol, propylene glycerol, glycerol and methanol as a cryoprotectant. Prolonged equilibration time had a detrimental effect on both prefreezing and post-thawing sperm motility. The estimated optimum freezing rate was in the range of -20 to -154 degrees C/min. More frozen-thawed than fresh spermatozoa are required to achieve comparable fertilization rates.     

Sperm cryopreservation of green swordtail Xiphophorus helleri, a fish with internal fertilization

Sperm cryopreservation for fishes with internal fertilization is essentially unexplored although many species of these fishes are valuable biomedical research models. To explore methods for sperm cryopreservation within the live-bearing genus Xiphophorus, this study used X. helleri to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio. Sperm motility and survival duration after thawing showed significant differences among different cryoprotectants with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as the cryoprotectant, the highest motility after thawing was observed with Hanks' balanced salt solution (HBSS) at 300 mOsmol/kg. Samples cooled from 5 to -80 degrees C at 20 degrees C/min yielded the highest post-thaw motility although no significant difference was found in the first 4h after thawing for cooling rates across the range of 20-35 degrees C/min. Evaluation of equilibration time revealed no significant difference between 20 min and 2h, but the highest motility at 10 min after thawing was found with a 20-min equilibration. Dilution ratios of sperm-to-extender at 1:20, 1:60, and 1:120 showed no significant differences in motility and survival duration after thawing, but the dilution of sperm solutions with HBSS (320 mOsmol/kg) immediately after thawing reduced the decline of sperm motility, and significantly prolonged the survival duration. Based on these findings, the highest average sperm motility (77%) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsmol/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS-glycerol of 1:20, equilibrated for 10 min, cooled at 20 degrees C/min from 5 to -80 degrees C before being plunged in liquid nitrogen, and thawed in a 40 degrees C water bath for 7s. If diluted immediately after thawing, sperm frozen by the protocol above retained continuous motility after thawing for more than 8 days when stored at 4 degrees C.     

Effect of osmotic immobilization on refrigerated storage and cryopreservation of sperm from a viviparous fish, the green swordtail Xiphophorus helleri

In this study, refrigerated storage and cryopreservation of sperm from the green swordtail Xiphophorus helleri were investigated. Previous cryopreservation research in this species utilized motile sperm because unlike in most fish species, Xiphophorus sperm can remain continuously motile after collection for a week with refrigerated storage. However, this species reproduces by internal fertilization, and given the significant requirements for motility within the female reproductive tract and potential limitations on sperm energetic capacities, immobilization of sperm prior to insemination could be used to improve fertilization success. Thus, the goal in this study was to use osmotic pressure to inhibit the motility of sperm after collection from X. helleri, and to test the effect of immobilization on refrigerated storage and cryopreservation. The objectives were to: (1) estimate the motility of sperm at different osmotic pressures, and determine an osmotic pressure suitable for immobilization; (2) cryopreserve the immobilized sperm, and estimate the motility after thawing with or without dilution, and (3) compare motility of non-immobilized and immobilized sperm after thawing, centrifugation, and washing to remove cryoprotectant. Motility was determined when sperm were suspended in 11 different osmotic pressures (24-500 mOsmol/kg) of Hanks' balanced salt solution (HBSS). Motility was observed between 116 and 425 mOsmol/kg. Sperm were not motile when the osmolality was lower than 116 or higher than 425 mOsmol/kg. Motility of the immobilized (non-motile) sperm could be activated by changing the osmotic pressure to 291-316 mOsmol/kg, and motility of immobilized sperm from hypertonic HBSS (425 mOsmol/kg) was significantly higher than that from hypotonic HBSS (145 mOsmol/kg) after 48 h of storage. At an osmolality of 500 mOsmol/kg, HBSS was used as extender to maintain immobilized sperm during cryopreservation with glycerol as the cryoprotectant. High motility (approximately 55%) was obtained in sperm after thawing when cryopreserved with 10-15% glycerol, and dilution of thawed sperm in fresh HBSS (1:4; V:V) was found to decrease the motility significantly. No difference was found in the motility of thawed sperm cryopreserved with 14% glycerol and extended in 310 and 500 mOsmol/kg HBSS. Washing by centrifugation prolonged the motility of thawed sperm from 24 to 72 h in HBSS at 310 and 500 mOsmol/kg. This study showed that sperm from X. helleri could be immobilized by use of specific osmotic pressures, and that the immobilization did not affect sperm motility after thawing. The immobilization of sperm by osmotic pressure could minimize reduction of the energetic capacities necessary for insemination, traversal, and residence within the female reproductive tract, and fertilization.     

Quantification of damage at different stages of cryopreservation of endangered North American bison (Bison bison) semen and the effects of extender and freeze rate on post-thaw sperm quality

Semen cryopreservation is an important technique for the banking of animal germplasm from endangered species and exploitation of genetically superior sires through artificial insemination. Being a member of bovidae family, bison semen has poor freezing ability as compared to dairy and beef bulls' semen. This study was designed to quantify the damage to bison sperm at different stages of cryopreservation, and to determine the effects of extender (commercial Triladyl(?) vs. custom made tris-citric acid [TCA]) and freeze rate (-10, -25 and -40°C/min) on post-thaw quality of bison semen. Semen was collected from five bison bulls (three woods and two plains) via electroejaculation. In Experiment 1, semen was diluted in Triladyl? extender and frozen with freeze rate -10°C/min. Sperm motility characteristics were recorded in fresh, diluted, cooled (4°C) and freeze-thawed semen using computer-assisted sperm analyzer (CASA). In Experiment 2, semen was diluted in Triladyl? or TCA extender, and frozen with three different freeze rates, i.e. -10, -25 or -40°C/min. Thawing was performed at 37°C for 60s. Post-thaw sperm motility characteristics were assessed using CASA, and sperm structural characteristics (plasma membrane, mitochondrial membrane potential and acrosomes) were evaluated using flow cytometer, at 0 and 3h while incubating semen at 37°C. In Experiment 1, total and progressive motilities did not differ among pre-freeze stages of cryopreservation (P>0.05). However, sperm total and progressive motilities declined (P<0.001) in freeze-thawed semen by 35% and 42%, respectively, compared to after cooling (pre-freeze) semen. In Experiment 2, Triladyl?, as compared to TCA, yielded greater (P<0.05) post-thaw sperm total motility (41% compared to 36%) and progressive motility (34% compared to 29%) at 0h, respectively. The percent change in post-thaw sperm total and progressive motilities, VAP, VCL, VSL, IPM-high ΔΨm and IPM-IACR during 3h incubation at 37°C, was less (P<0.05) in TCA than in Triladyl?. There was an effect of freeze rate on post-thaw sperm average path velocity at 0h, and total motility, progressive motility, VCL, IPM and IPM-IACR at 3h were the greatest (P<0.05) when bison semen was frozen at -40°C/min. Likewise, the percent change in post-thaw sperm total and progressive motilities, during 3h incubation at 37°C, was less (P<0.05) in bison semen frozen at -40°C/min. All post-thaw bison sperm characteristics decreased (P<0.05) from 0h to 3h, during incubation at 37°C. In conclusion, the maximum damage to bison sperm occurred during freeze-thaw processes. Post-thaw total and progressive motilities of bison sperm were greater in Triladyl? at 0h whereas sperm survival was greater in TCA extender during 3h post-thaw incubation. Bison sperm had greater survival (P<0.05) when frozen at -40°C/min freeze rate.     

Thawing and processing of cryopreserved bovine spermatozoa at various temperatures and their effects on sperm viability, osmotic shock and sperm membrane functional integrity

The objective of this study was to evaluate the effects of thawing and processing temperatures on post-thaw sperm viability, occurrence of osmotic shock and sperm membrane functional status. The occurrence of osmotic shock, characterized by increased spermatozoa with coiled tails, eventually results in reduced sperm viability and sperm membrane integrity. The effects of different thawing temperatures were assessed by thawing frozen specimens at 37, 21 or 5 degrees C for 1 to 2-min, followed by processing at these temperatures. A subset of frozen specimens were thawed at 37 degrees C for 10 to 15-sec and transferred to a water bath at 21 or 5 degrees C for 1 to 2-min to complete thawing, followed by processing at these temperatures. Sperm processing (washing) consisted of dilution, centrifugation and resuspension to remove glycerol from the medium and to gradually return the spermatozoa to isotonic conditions. Post-thawed specimens (0.5 mL) were slowly diluted 1:1 (v/v) at a rate of 0.1 mL/min, centrifuged, and resuspended to 0.5 mL (37 degrees C). Diluted specimens were equilibrated for 1 to 2-min after dilution and for 5-min after resuspension. The specimens were then incubated for 2-h (37 degrees C) and assessed at 60-min intervals for the percentage of motility, for progressive motility (Grades 0 to 4), for the percentage of spermatozoa with coiled tails, and for the percentage of swollen spermatozoa. The percentage of swollen spermatozoa (measurement of sperm membrane integrity) was assessed by exposing spermatozoa to a modified hypoosmotic swelling (HOS) test. The results obtained seem to indicate that physiological thawing and processing temperatures (37 degrees C) are required to maintain sperm motility. However, thawing and processing at lower temperatures (< 37 degrees C) seems to prevent the occurrence of osmotic shock and to maintain sperm membrane functional integrity. In this study, thawing at 37 degrees C (10 to 15-sec) and transfer to a water bath at 21 degrees C (1-min) to complete thawing, followed by processing at 21 degrees C, yielded better results in terms of increased sperm viability, reduced occurrence of osmotic shock and higher reactivity to the HOS test.     

Effect of some permeating cryoprotectants on CASA motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility because they provide objective results for thousands of mammalian spermatozoa. Mammalian spermatozoa experience osmotic stress when the glycerol is added to the cells prior to freezing and removal from the cells after thawing. In order to minimize osmotic damage, cryoprotectants having lower molecular weights and greater membrane permeability than glycerol, were evaluated to determine their effectiveness for cryopreserving bull spermatozoa. The aim of this study was to compare the cryopreservation effects of low molecular weight cryoprotectants (ethylene glycol and methanol) to glycerol, on post-thaw CASA sperm parameters. Bull semen was diluted with tris-egg yolk extender containing 3% glycerol, 3, 2 and 1% ethylene glycol or 3, 2 and 1% methanol. Bull semen was frozen in 0.5 straws. Bull spermatozoa exhibited higher percentages (p<0.01) for total (Mot, 72.4%) and progressively (Prog, 29.5%) motilities when frozen in extender containing 3% glycerol compared to 3, 2 and 1% ethylene glycol or 3, 2 and 1% methanol. In conclusion, no advantages were found in using ethylene glycol or methanol to replace glycerol in bull semen freezing. Glycerol provided the best sperm characteristics for bull spermatozoa after freezing and thawing. The possibility of using ethylene glycol or methanol as permeating cryoprotectants for bull semen deserves further investigation, and these cryoprotectants should also be evaluated in extenders that contain disaccharides or cholesterol.     

Evaluation of amides and centrifugation temperature in boar semen cryopreservation

Two experiments were conducted to evaluate the use of amides as cryoprotectants and two centrifugation temperatures (15 or 24 degrees C) in boar semen cryopreservation protocols. Semen was diluted in BTS, cooled centrifuged, added to cooling extenders, followed by the addition of various cryoprotectants. In experiment 1, mean (+/-S.E.M.) sperm motility for 5% dimethylformamide (DMF; 50.6+/-1.9%) and 5% dimethylacetamide (DMA; 53.8+/-1.7%) were superior (P<0.05) to 5% methylformamide (MF; 43.2+/-2.4%) and 3% glycerol (GLY; 38.1+/-2.3%), with no significant difference between MF and GLY. Sperm membrane integrity was higher (P<0.05) for DMA than for MF or GLY (50.9+/-1.9, 43.3+/-2.5, and 34.5+/-2.8%, respectively). Sperm membrane integrity was higher in DMF (47.9+/-2.1%) than in glycerol (34.5+/-2.8%, P<0.05), but was similar to other treatments (P>0.05). In experiment 2, we tested MF, DMF, and DMA at 3, 5, and 7%. Sperm motility and membrane integrity were higher for 5% DMA (53.8+/-1.7 and 50.9+/-1.9%) and 5% DMF (50.6+/-1.9 and 47.9+/-2.1%), in comparison with 7% DMF and all MF concentrations (P<0.05). For sperm motility and membrane integrity, 5% DMA exceeded (P<0.05) 3% DM, with greater membrane integrity than 3% DMF (P<0.05). In both experiments, sperm motility and membrane integrity were superior at 15 degrees C versus 24 degrees C (P<0.05), with no interaction between centrifugation temperature and treatments (P>0.05). In conclusion, boar semen was successfully cryopreserved by replacement of glycerol with amides (especially 5% DMA) and centrifugation at 15 degrees C, with benefits for post-thaw sperm motility and membrane integrity.     

Frozen-thawed bovine spermatozoa diluted by slow or rapid dilution method: measurements on occurrence of osmotic shock and sperm viability

This study was undertaken to investigate the occurrence of osmotic shock, sperm viability and membrane functional status of frozen-thawed bovine spermatozoa during a short-term incubation period (2 h) in vitro after dilution by 2 methods. Frozen semen from 10 bulls (0.5-ml plastic straws, 7% glycerol) was thawed and diluted by slow or rapid dilution method with Ham's F-10 medium containing 0 or 7% glycerol and assessed for sperm motion parameters, percentage of spermatozoa with coiled tails and reactivity to the hypoosmotic swelling (HOS; percentage of spermatozoa swelling) test at 60 min intervals during a 2 h incubation period (37 degrees C). Post-thaw sperm viability, as reflected by percentage and grade of motility (0 to 4) did not differ between the 2 dilution methods (P > 0.05) at the beginning of incubation (Time 0). However, differences were apparent (P < 0.05) as the incubation time increased. Slow dilution with medium containing 0% glycerol caused less increase (P < 0.05) in percentage of spermatozoa with coiled tails; Moreover, these spermatozoa showed greater reactivity to the HOS test. When contrasting slow vs rapid dilution methods, the occurrence of osmotic shock was less frequent, and response to the HOS test was greater for spermatozoa diluted slowly, regardless of the glycerol content of the incubation medium. Rapid deglycerolization of frozen-thawed bovine spermatozoa in a single step, induces damage which is not detected on the basis of spennatozoal motility but is clearly evident after several hours of incubation by using the HOS test to detect damage.     

Preliminary studies of sperm cryopreservation in the mushroom coral, Fungia scutaria

Coral species throughout the world are facing severe environmental pressures. Because of this, we began cryobiological studies on the sperm of the mushroom coral, Fungia scutaria. We determined that F. scutaria sperm had a mean length of 56 microm and head diameter of 2.5 microm, and a mean spontaneous ice nucleation temperature of -37.2 +/- 1.7 degrees C. When the sperm were exposed to the cryoprotectant glycerol for 5 or 20 min (at 10% v/v), no fertilized larvae were produced. However, when sperm were exposed for 20 min to propylene glycol (10% v/v), fertilizations were produced at the same rate as untreated control eggs and sperm (P > 0.05), but slightly less for dimethyl sulfoxide (10% v/v) (P < 0.05). Regardless, dimethyl sulfoxide caused less osmotic damage to the sperm membrane than did propylene glycol. Therefore, we used the dimethyl sulfoxide (10% v/v) to develop cryopreservation protocols that yielded good post-thaw morphology and motility (>95%) for coral sperm.     

Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system

Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25 degrees C x min(-1) to 5 degrees C, 5 degrees C x min(-1) to -20 degrees C, and 20 degrees C x min(-1) to -100 degrees C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, l-alanine showed maximal cryoprotection potential. l-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 +/- 2% and 19 +/- 2%, respectively. l-Glutamine, l-proline, and glycine at optimum concentration (100-150 mM) cryopreserved approx. 11-17% total motility of the sperm cells, whereas amino acids such as l-arginine, l-lysine, and l-histidine offered little cryoprotection (0-5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7-10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100-150 mM to 20-70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.