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.     

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

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

Cryopreservation of microencapsulated canine sperm

The objective was to develop a method for cryopreserving microencapsulated canine sperm. Pooled ejaculates from three beagle dogs were extended in egg yolk tris extender and encapsulated using alginate and poly-L-lysine at room temperature. The microcapsules were cooled at 4 °C, immersed in pre-cooled extender (equivalent in volume to the microcapsules) to reach final concentration of 7% (v/v) glycerol and 0.75% (v/v) Equex STM paste, and equilibrated for 5, 30 and 60 min at 4 °C. Thereafter, microcapsules were loaded into 0.5 mL plastic straws and frozen in liquid nitrogen. In Experiment 1, characteristics of microencapsulated canine sperm were evaluated after glycerol addition at 4 °C. Glycerol exposure for 5, 30 and 60 min did not significantly affect progressive motility, viability, or acrosomal integrity of microencapsulated sperm compared with pre-cooled unencapsulated sperm (control). In Experiment 2, characteristics of frozen-thawed canine microencapsulated sperm were evaluated at 0, 3, 6, and 9 h of culture at 38.5 °C. Pre-freeze glycerol exposure for 5, 30, and 60 min at 4 °C did not influence post-thaw quality in unencapsulated sperm. Post-thaw motility and acrosomal integrity of microencapsulated sperm decreased more than those of unencapsulated sperm (P < 0.05) following glycerol exposure for 5 min. However, motility, viability and acrosomal integrity of microencapsulated sperm after 30 and 60 min glycerol exposure were higher than unencapsulated sperm cultured for 6 or 9 h (P < 0.05). In conclusion, since microencapsulated canine sperm were successfully cryopreserved, this could be a viable alternative to convention sperm cryopreservation in this species.     

The effect of straw size, freezing rate and thawing rate upon post-thaw quality of dog semen

Optimal freeze-thaw processes for dog semen will yield a maximal number of insemination doses from an ejaculate. The objectives of this study were to compare the effects of two straw sizes (0.25- and 0.5-mL French), two freezing rates (straws suspended 3.5 and 8 cm above liquid nitrogen) and two thawing rates (in water at 37 and 70 degrees C) upon post-thaw quality of dog semen, and to determine the best treatment combination. Quality was expressed in terms of the percentage progressively motile sperm 5 and 60 min after thawing and the percentage of abnormal acrosomes 5 min after thawing. One ejaculate from each of eight dogs was frozen. Two straws from each ejaculate were exposed to each of the eight treatment combinations. Data were analyzed by means of a repeated measures factorial analysis of variance and means compared using Bonferroni's test. Dog affected each response variable (P < 0.01). Neither straw size, nor freezing rate, nor thawing rate affected motility 5 min after thawing (P > 0.05). Half-milliliter straws resulted in 5.7% more progressively motile sperm 60 min after thawing and 6.5% fewer abnormal acrosomes than 0.25-mL straws (P < 0.05, n = 64). The percentage progressively motile sperm 60 min after thawing tended to be higher for semen thawed at 70 degrees C compared to 37 degrees C (P < 0.06, n = 64). Semen thawed in water at 70 degrees C had 6.6% fewer abnormal acrosomes than semen thawed in water at 37 degrees C (P < 0.05, n = 64). Freezing rate interacted with thawing rate (P < 0.05) in their effects upon acrosomal morphology and freezing 8 cm above liquid nitrogen and thawing in water at 70 degrees C was best. Dog semen should be frozen in 0.5-mL straws, 8 cm above liquid nitrogen and thawed in water at 70 degrees C.     

Influence of cryoprotective diluent on post-thaw viability and acrosomal integrity of spermatozoa of the African elephant (Loxodonta africana)

Electroejaculates from free-ranging, African elephants were frozen to test various seminal diluents, freezing methods and thawing media on post-thaw sperm viability and structural integrity. In Study I, each ejaculate was tested with each of 7 cryoprotective diluents. After cooling to 5 degrees C and equilibration on ice (4 degrees C) for 120 min, each aliquant was pellet frozen on solid CO2, stored in liquid nitrogen and thawed (37 degrees C) in saline or tissue culture solution. Amongst all diluents, post-thaw sperm motility, motility duration in vitro (37 degrees C) and acrosomal integrity were greatest (P less than 0.05) when diluent BF5F was used. Thawing medium had no effect on results. In Study II, the optimal diluent from Study I (BF5F) was compared with the diluent SGI. Results were not affected by a 90- or a 150-min cooling-equilibration interval in an electronic cooler (5 degrees C); however, post-thaw sperm motility rating and duration of motility in vitro were greater (P less than 0.01) with the pellet than the straw container freezing method. When the pelleting method was used, diluents BF5F and SGI provided comparable cryoprotection. Duration of post-thaw motility was enhanced 2-fold and up to 12 h by maintaining thawed semen at 21 rather than 37 degrees C (P less than 0.05). All diluents provided some protection on acrosomal integrity, but the overall proportion of intact acrosomes after thawing was markedly less in Study II, apparently as a result of the slower initial cooling rate (approximately 1.5 degrees C/min) compared to that of Study I (approximately 6.5 degrees C/min). This study demonstrates the feasibility of cryopreserving semen from free-ranging African elephants and indicates that spermatozoa must effectively survive freezing when the BF5F or SGI diluent is used in conjunction with the pelleting method.     

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.     

Influence of extender, freezing rate, and thawing rate on post-thaw motility, viability and morphology of coyote (Canis latrans) spermatozoa

The objective of this study was to examine the post-thaw effects of three cryoprotective extenders (Tris-fructose-citric acid extender, Tris-glucose-citric acid extender, and lactose extender), three linear freezing rates (-1, -6, and -20 degrees C/min), and three thawing rates (37 degrees C water bath for 120s, 60 degrees C water bath for 30s, and 70 degrees C water bath for 8s) on coyote spermatozoa. After thawing, the findings supported that cryopreservation of coyote (Canis latrans) spermatozoa frozen at a moderate freezing rate (-6 degrees C/min), in either a Tris-fructose or Tris-glucose extender, and thawed at a slow rate (37 degrees C water bath for 120s) or moderate rate (60 degrees C water bath for 30s), resulted in a more vigorous post-thaw motility (range, 57.5-44.0%) and viability (range, 64-49.6%) with the least amount of morphological and acrosomal abnormalities.     

A simple, inexpensive and successful freezing method for curimba Prochilodus lineatus (Characiformes) semen

The cryopreservation of fish sperm provides a tool by which reproduction is optimized and thereby larval production is increased. The aims of this study were to evaluate the effects of cryosolutions, motility-activation media, straw volumes and thawing temperatures on the post-thaw motility of curimba semen. Furthermore, semen cryopreserved in a simple and inexpensive cryosolution and that yielded excellent post-thaw motility was tested for fertility. Semen was diluted in each of the eight cryosolutions in a factorial of two cryoprotectants (DMSO and methylglycol) x four extenders (0.9% NaCl, 5% glucose, BTS and M III). Diluted semen was frozen in 0.5-mL straws in a nitrogen vapor vessel. Sperm motility was evaluated after thawing (60 degrees C water bath for 8s) and activation with a total of four different activation media (distilled water, 0.15% NaCl, 0.29% NaCl or 1% NaHCO(3)). To evaluate straw volume and thawing temperature, semen was diluted in 5% glucose and methylglycol and frozen in 0.5- and 4.0-mL straws. Half of the 0.5-mL straws were thawed in a water bath at 60 degrees C for 8s and the other half at 30 degrees C for 16s. The 4.0-mL straws were thawed at 60 degrees C for 24s only. In the last experiment, semen cryopreserved in 5% glucose and methylglycol, 0.5-mL straws, and thawed at 60 degrees C for 8s was tested for fertility. The results of these comparisons are presented and show that curimba semen can be successfully cryopreserved in a simple glucose solution combined with methylglycol as cryoprotectant, in 0.5-mL straws, yielding motility rates between 86% and 95% and fertilization rates between 47% and 83%.     

Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate freezers: a comparison

This study compared variation in the quality of cryopreserved boar spermatozoa and the control and accuracy of cooling rates between three semen freezers (CryoLogic Freeze Control CL3000, Planer Products Kryo Save Compact KS1.7/Kryo 10 Control module and a controlled rate 'Watson' freezing machine developed within our laboratory). Five ejaculates were collected from each of 15 boars (five boars from each of three breeds). Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% v/v glycerol) and placed into 0.5 ml straws. Three straws per treatment, from each ejaculate were cooled to -5 degrees C at 6 degrees C/min, held at -5 degrees C for 30s while ice crystal formation was induced, then further cooled from -5 to 80 degrees C at either 40 degrees C/min (Kryo Save Compact KS1.7 and Watson) or 6 degrees C/min (Freeze Control CL3000). Precise measurements of temperature fluctuations during the programmed cooling curves were made by inserting thermocouples into the semen filled straws. Semen was assessed for %motile cells, motility characteristics using computer-assisted semen analysis (CASA), plasma membrane integrity (%SYBR-14 positive stained spermatozoa) and acrosome integrity (%FITC-PNA positive stained spermatozoa). Spermatozoa cryopreserved using the Freeze Control CL3000 system (maximum rate of 6 degrees C/min) exhibited reduced post-thaw viability (14.2+/-2.8% mean plasma membrane intact spermatozoa) when compared to both the KS1.7 and Watson freezers (optimal rate of 40 degrees C/min) (18.4+/-3.2 and 25.7+/-3.7% mean plasma membrane intact spermatozoa, respectively). Differences in motility characteristics were observed between spermatozoa cryopreserved at 40 degrees C/min with the Watson apparatus preserving a larger proportion of sperm with progressive motility. Cooling curves in the CL3000 and KS1.7 were interrupted by a pronounced increase in temperature at -5 degrees C that corresponded with the latent heat of fusion released with ice crystal formation. This temperature change was significantly reduced in the cooling curves produced by the Watson freezer. These findings suggest that preserving spermatozoa using the Watson freezer improved post-thaw semen quality, with regard to sperm motility characteristics. Furthermore, that post-thaw semen viability was enhanced by minimising temperature fluctuations resulting from the release of the latent heat of fusion at ice crystal formation.     

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)     

Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing

Influence of different sugars supplemented to the extender on the motility, viability and intact acrosome rates of dog spermatozoa during dilution, equilibration and freezing was studied. The ejaculate was divided into 10 aliquots, which were diluted 1:3 with TRIS-citric acid extender containing 240 mMTRIS, 63 mM citric acid, 8% (v/v) glycerol, 20% (v/v) egg yolk and 70 mM sugar, which was either fructose, galactose, glucose, xylose (monosaccharide), lactose, trehalose, maltose, sucrose (disaccharide) or raffinose (trisaccharide). No sugar was added to the extender in the control group. Extended semen samples were cooled to 5 degrees C over 45 min, packaged in 0.25-mL straws, equilibrated for 2 h at 5 degrees C and frozen in liquid nitrogen vapor. Samples were thawed by placing straws into 37 degrees C water for 30 sec. Motility, viable sperm and intact acrosome rates decreased gradually in all groups after equilibration and consecutively freezing (P<0.001). The type of sugar significantly effected motility, viability and acrosomal integrity during equilibration and freezing (P<0.05). Galactose, lactose, trehalose, maltose and sucrose reduced damaged acrosome percentages in equilibrated samples (P<0.05). Sugar supplementation did not enhance motility and viability during equilibration. The disaccharides, except lactose, reduced post-thaw dead sperm and/or damaged acrosome percentages without promoting post-thaw motility (P<0.01), whereas monosaccharides, especially fructose and xylose, improved motility (P<0.05) along with viability and intact acrosome rates (P<0.05). Trehalose, xylose and fructose significantly increased total active sperm rates (motility x live sperm rate x normal acrosome rate) compared to other sugars (P<0.01) and control (P<0.0001) in frozen thawed samples. Therefore, sugar supplementation of the extender influenced post-equilibration and post-thaw sperm quality, and the type or locality of protective impact of the sugar on dog spermatozoa vary according to type of the sugar.     

Modulation of post-thaw sperm functions with oviductal proteins in buffaloes

A study was undertaken to determine the effects of oviductal proteins obtained from various stages of the estrous cycle on spermatozoa characteristics in buffaloes. Oviducts were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle) and separated into isthmus and ampulla. Each segment of oviduct (nonluteal and luteal) was flushed with PBS (pH 7.4). The flushing obtained was centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal isthmic and ampullary and luteal isthmic and ampullary fluids were precipitated overnight using ammonium sulphate, centrifuged (10000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored in frozen form at -20 degrees C. Six pooled good-quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was splited into five parts and extended in Tris-egg yolk-citrate extender (20% egg yolk; 7% glycerol), so that final dilution yielded approximately 60 million sperm cells per ml, and cryopreserved in 0.5 ml French straws (30 million sperm cells/straw) in LN(2) (-196 degrees C). Before freezing, nonluteal isthmic and ampullary and luteal isthmic and ampullary proteins were incorporated at the rate of 1mg/ml of extended semen. The equilibrated and frozen-thawed (37 degrees C for 30 s) semen was evaluated for motility, live %, acrosomal integrity percentage, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides this, spermatozoa from treatment and control groups were incubated at 37 degrees C for 6 h in sperm TALP. Among the nonluteal and luteal oviductal proteins, the former maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity than the control group. Between the isthmic and ampullary proteins, the isthmic proteins incorporated group maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity. Similarly, higher sperm penetration distance in cervical mucus was recorded in nonluteal isthmic proteins incorporated group. But, irrespective of the stage of an estrous cycle, isthmic proteins included group maintains higher sperm membrane integrity as revealed by higher (P < 0.05) swollen sperm percentage in response to hypo-osmotic solution than the ampullary proteins included and control groups. Similarly, at any time during incubation the sperm motility and viability was higher (P < 0.05) in isthmic proteins treated group than the ampullary and control group. But, the same trend was not observed in terms of acrosomal integrity percentages. It is inferred that inclusion of oviductal proteins in the extender prior to freezing improved post-thaw semen quality. Oviductal proteins differentially affected sperm function depending upon the region of oviduct and the stage of estrous cycle at which the proteins were obtained.     

Cryopreservation of spermatozoa from wild-born Namibian cheetahs (Acinonyx jubatus) and influence of glycerol on cryosurvival

Sperm cryopreservation is a valuable tool for the genetic management of ex situ populations. This study was conducted to assess: (1) semen characteristics of wild-born cheetahs; and (2) the impact of three types of glycerol influence (duration of exposure, temperature, and method of addition) on sperm cryosensitivity. To evaluate the impact of duration of glycerol exposure, spermatozoa were incubated in Test Yolk Buffer (TYB) with 4% glycerol at ambient temperature (approximately 22 degrees C) for 15 vs. 60 min before cryopreservation. To evaluate the influence of temperature and method of glycerol addition, spermatozoa were resuspended at ambient temperature either in TYB with 0% glycerol followed by addition of 8% glycerol (1:1 v/v; at ambient temperature vs. 5 degrees C) or directly in TYB with 4% glycerol. All samples were cryopreserved in straws over liquid nitrogen vapor and evaluated for sperm motility and acrosomal integrity after thawing. Semen samples (n = 23; n = 13 males) contained a high proportion (78%) of pleiomorphic spermatozoa. Ejaculates also contained a high proportion of acrosome-intact (86%) and motile spermatozoa (78%). Immediately after thawing, a significant proportion of spermatozoa retained intact acrosomes (range, 48-67%) and motility (range, 40-49%). After thawing, incubation in glycerol for 60 min at ambient temperature before freezing decreased (p < 0.05) sperm motility and acrosomal integrity at one time-point each (pre-centrifugation and post-centrifugation, respectively). However, method or temperature of glycerol addition had no (p > 0.05) impact on sperm cryosurvival. In summary, (1) wild-born cheetahs produce high proportions of pleiomorphic spermatozoa but with a high proportion of intact acrosomes; and (2) resuspension in 4% glycerol, followed by exposure for up to 60 min at ambient temperature, had minimal effect on sperm motility and acrosomal integrity after cryopreservation. Results indicate the feasibility of cryopreserving cheetah spermatozoa under field conditions, providing a user-friendly method to capture and store gametes to enhance genetic management.     

Effect of different glycerol treatments on frozen-thawed dog sperm longevity and acrosomal integrity

Four different concentrations of glycerol in a Tris-fructose-citric acid extender for frozen dog semen and the effects of adding glycerol at 37 degrees C or 4 degrees C to the extender were studied by monitoring the post-thaw sperm longevity and acrosomal integrity during incubation at 39 degrees C. In the first part of this study, ejaculates from 13 dogs were pooled and divided into 4 aliquots, which were centrifuged and the sperm pellets rediluted with a Tris-fructose-citric acid extender containing 2, 4, 6 and 8% (v/v) glycerol, respectively. Progressive motility by subjective estimation, live:dead spermatozoa ratio using eosin-nigrosin staining, and acrosomal integrity using phase contrast microscopy were evaluated before processing and at 0, 0.5, 1, 2 and 4 hours post-thawing incubating the semen samples in the dark at 39 degrees C. The experiment was performed using seven replicates and it was found that sperm motility and acrosomal integrity were superior following the use of 8% glycerol in the extender. In Experiment 2, 13 ejaculates from the same dogs used in the first experiment were pooled and divided into 3 aliquots, and an 8% glycerol diluent was added at 37 degrees C and 4 degrees C after 1 h of cooling or at 4 degrees C after 2 h of cooling, respectively. After freezing and thawing the same parameters as studied in the first experiment were assessed. The experiment was performed in 7 replicates, and no difference was found between treatments.     

In vitro characteristics of canine spermatozoa subjected to two methods of cryopreservation

The in vitro viability of canine spermatozoa was evaluated after freezing-thawing using the Andersen method, and the commercial CLONE method. These methods differ in the extenders used, number of dilution steps, and equilibration times as well as in both freezing and thawing techniques and rates. Insemination with semen frozen-thawed by either method gives high whelping rates in practice, implying that dog spermatozoa can retain their fertilizing ability after being subjected to widely different preservation methods. The in vitro viability of spermatozoa processed by these methods has not been previously evaluated in detail. Three ejaculates were collected from each of 5 fertile dogs. Each ejaculate was divided into 2 parts and frozen in medium straws according to the 2 methods. Two straws were thawed and examined from each freezing batch. Sperm motility was assessed in the undiluted semen, and in frozen-thawed semen immediately after thawing, and after storage for 3, 6 and 24 h at room temperature (Straw 1) or 1, 2 and 3 h at 37 degrees C (Straw 2, thermoresistance test). The integrity of the sperm plasma membrane was evaluated in undiluted, in equilibrated (diluted and chilled), and in frozen-thawed spermatozoa using fluorophore probes. The acrosome morphology of frozen-thawed spermatozoa was assessed using a commercial stain (Spermac). Motility immediately after thawing was significantly higher with the CLONE method (75.3% [SD = 4.0] for Straw 1 and 73.7% [SD = 3.2] for Straw 2) than with the Andersen method (70.0% [SD = 5.1] and 69.7% [SD = 3.2]). Motility decreased during storage after thawing. Spermatozoa frozen-thawed using the CLONE method showed a significantly lower thermoresistance. The proportion of spermatozoa with intact plasma membrane was not affected by the equilibration procedure used with either method but was significantly decreased (P < 0.001) after thawing with both methods. The percentage of spermatozoa exhibiting changes thought to represent different stages of acrosomal degradation, was 45.7% (SD = 5.3) using the Andersen method and 44.1% (SD = 9,4) using the CLONE method. Both cryopreservation methods thus resulted in high initial post-thaw sperm motility and membrane integrity but low thermoresistance, and under both methods a large proportion of sperm cells were undergoing acrosomal degradation. The methods differed significantly in terms of their effect on sperm motility but not on plasma membrane integrity or acrosomal morphology.     

Evaluation of seasonal variations of semen freezability in Leccese ram

The experiment was carried out in Southern Italy (41 degrees N latitude) to examine the effects of seasonal variations of semen freezability in Leccese ram. Semen from five rams, collected every 2 weeks for a whole year, was frozen in straws, using a system based on Tris-fructose egg yolk as extender to constitute semen doses of 100x10(6) spermatozoa. Post-thaw survival and acrosomal status of cells were assessed by dual staining by Hoechst 33258 and FITC-PSA. Three different forms of fluorescence distribution were displayed indicating sperm without acrosome (unstained cells), sperm with damaged acrosome (cells with incomplete fluorescence over the head), sperm with widespread fluorescence (cells completely fluorescent). Motility and kinetic rating at thawing and after 1 and 3h incubation (37 degrees C) were also assessed.Semen frozen in summer and autumn, corresponding to the breeding season, showed the highest (P<0.01) post-thaw survival of spermatozoa (41.7%) and the lowest (P<0.01) incidence of spermatozoa with damaged acrosome. The positive influence of the summer-autumn period was expressed also on motility and kinetic rating of spermatozoa at thawing. The integrity of the acrosomal membrane was positively correlated (P<0.01) with sperm viability before processing (r=0.32) and after thawing (r=0.51).In conclusion, the results show that season exerts a significant influence on semen freezability in Leccese ram, with the best performance occurring the summer and autumn period, corresponding to the reproductive season in temperate zones.     

Sperm cryopreservation in the spermcasting Australian flat oyster Ostrea angasi by a programmable freezing method

Cryopreservation offers long-term storage of gametes without constraint from seasonal gamete maturation, provides opportunities to improve the efficiency of breeding and genetic programs, and protects endangered species from extinction due to epidemic diseases and natural disasters. In this study, a protocol for cryopreserving sperm of the spermcasting Australian flat oyster Ostrea angasi was developed by optimizing key factors influencing the quality of cryopreserved sperm. Dimethyl sulfoxide (DMSO) was non-toxic to sperm within the concentration and duration assessed in the toxicity experiment whereas 10% methanol or a higher concentration was toxic to sperm from the exposure duration of 30 min onwards. DMSO produced higher post-thaw sperm motility among the treatments with a single cryoprotectant. The inclusion of trehalose or glucose with DMSO further increased the post-thaw sperm motility (%) and plasma membrane integrity (PMI). Sperm equilibrated for 30 min showed higher post-thaw motility and PMI than those for 10 or 50 min. Higher post-thaw sperm motility and PMI were achieved at the freezing rate of −3 °C/min than at −7 °C/min. Sperm packaged in 0.5 ml straws had a higher post-thaw motility and PMI than those packaged in 0.25 ml straws. In this study, 44.4% post-thaw sperm motility and 49.2% PMI were achieved when sperm were equilibrated in 10% DMSO +0.45 M trehalose for 30 min, packaged in 0.5 ml straws, frozen at −3 °C/min from 4 °C to −80 °C, and thawed at 40 °C for 8 s. The availability of viable cryopreserved sperm would open an option for future breeding and genetic improvement programs for the spermcasting Australian flat oyster.     

Different patterns of metabolic cryo-damage in domestic cat (Felis catus) and cheetah (Acinonyx jubatus) spermatozoa

Felid spermatozoa are sensitive to cryopreservation-induced damage, but functional losses can be mitigated by post-thaw swim-up or density gradient processing methods that selectively recover motile or structurally-normal spermatozoa, respectively. Despite the importance of sperm energy production to achieving fertilization, there is little knowledge about the influence of cryopreservation or post-thaw processing on felid sperm metabolism. We conducted a comparative study of domestic cat and cheetah sperm metabolism after cryopreservation and post-thaw processing. We hypothesized that freezing/thawing impairs sperm metabolism and that swim-up, but not density gradient centrifugation, recovers metabolically-normal spermatozoa. Ejaculates were cryopreserved, thawed, and processed by swim-up, Accudenz gradient centrifugation, or conventional washing (representing the 'control'). Sperm glucose and pyruvate uptake, lactate production, motility, and acrosomal integrity were assessed. Mitochondrial membrane potential (MMP) was measured in cat spermatozoa. In both species, lactate production, motility, and acrosomal integrity were reduced in post-thaw, washed samples compared to freshly-collected ejaculates. Glucose uptake was minimal pre- and post-cryopreservation, whereas pyruvate uptake was similar between treatments due to high coefficients of variation. In the cat, swim-up, but not Accudenz processing, recovered spermatozoa with increased lactate production, pyruvate uptake, and motility compared to controls. Although confounded by differences in non-specific fluorescence among processing methods, MMP values within treatments were positively correlated to sperm motility and acrosomal integrity. Cheetah spermatozoa isolated by either selection method exhibited improved motility and/or acrosomal integrity, but remained metabolically compromised. Collectively, findings revealed a metabolically-robust subpopulation of cryopreserved cat, but not cheetah, spermatozoa, recovered by selecting for motility rather than morphology.     

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).