Optimal physicochemical conditions for the manipulation and short-term preservation of koala (Phascolarctos cinereus) spermatozoa

Protocols for the successful manipulation and preservation of semen in a given species depend upon a fundamental knowledge of how spermatozoa respond to the physicochemical conditions of the extension media; methods developed for the preservation of eutherian spermatozoa may not necessarily be suitable for marsupial semen. The aim of this study was to investigate the effects on koala sperm motility of serial dilution, changes in temperature, diluent pH and osmolality to establish the optimal physicochemical conditions for short-term semen storage. This study showed that electroejaculated koala semen diluted 1∶1 (v/v) with PBS frequently coagulated after incubation at 35 degrees C, but that further dilution and incubation resulted in a corresponding increase in the percentage of spermatozoa swimming in a non-linear trajectory. The effect of rapid temperature change on the motility of koala spermatozoa was investigated by exposing semen, initially diluted at 35 degrees C, to temperatures of 45, 25, 15 and 5 degrees C. Although sperm motility was reduced after incubation at 45 degrees C, a rapid decrease in temperature of up to 20 degrees C did not result in a significant reduction in sperm motility. However, contrary to evidence in other marsupials, there was a small but significant decrease in sperm motility after rapid cooling of diluted semen from 35 to 5 degrees C. The effects of diluent pH and osmolality on the motility of koala spermatozoa were investigated. These experiments indicated that diluents for koala sperm manipulation should buffer in a pH range of 7-8 and have an osmolality of approximately 300 mmol kg(-1). The final experiment compared the relative effectiveness of Tris-citrate buffer (1% glucose) and PBS to maintain koala sperm motility over a range of incubation temperatures (5-35 degrees C) for up to 8 days. Reduction in sperm motility was directly related to temperature, and motility was sustained for the longest duration when stored at 5 degrees C. The Tris-citrate buffer solution was superior to PBS as a preservation diluent at all temperatures, and koala spermatozoa remained motile even after 42 days storage at 5 degrees C. Spermatozoa diluted in PBS (with Ca(2+) or Mg(2+)) and cooled to 5 degrees C showed evidence of an unusual motility pattern, similar to that of hyperactivated eutherian spermatozoa. This study showed that koala spermatozoa respond to different physicochemical conditions associated with short-term liquid storage in essentially the same way as the spermatozoa of eutherian mammals, although koala spermatozoa appear to be more tolerant of rapid temperature shock. The results of this study can be used to make informed selections with regard to appropriate diluent composition and improved short-term sperm preservation protocols and represent the first such database for any species of marsupial.     

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.     

Effects of low-molecular-weight fractions (LMWF) from milk, egg yolk, and seminal plasma on freezability of bovine spermatozoa

The effects of the dialyzable fractions from bovine seminal plasma, egg yolk, and milk and of two buffer systems (TEST and sodium citrate) on post-thaw sperm motility were studied. Each basic salt solution was used in the experimental design. These solutions were used as extender systems in combination with egg yolk and glycerol. After collection, semen samples were extended (1:20), cooled to 5 degrees C in 1.5 hr, and frozen in 0.5-cc French straws after 3 hr of equilibration. Post-thaw samples were assayed for percentage of motile cells immediately after thawing and after 4 hr of incubation at room temperature (22 degrees C). Egg yolk (25%) provided the same protection as did the combination of colloidal material present in the skim milk-yolk extenders. The use of TEST as a buffer provided significantly higher (P less than 0.01) sperm post-thaw motility than milk salts or Na citrate. Sperm survival in extenders containing high concentrations of seminal plasma and/or egg yolk salts was significantly lower (P less than 0.01). Spermatozoa frozen in the presence of 6% glycerol resulted in sperm motility significantly (P less than 0.05) higher than that of spermatozoa frozen with 3% glycerol. However, no difference was observed between these two concentrations when TEST solution was used.     

Effects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved buffalo spermatozoa.

The principal objective of this study was to derive an improved procedure for cryopreservation of swamp buffalo (Bubalus bubalis) spermatozoa. Experiments were conducted to determine effects of cooling rate, intermediate plunge temperature and warming rate on motility and acrosome integrity of spermatozoa. Spermatozoa were obtained from three bulls (three ejaculates/bull) and were subjected to nine cooling conditions before being frozen in liquid nitrogen: cooling at 10, 20, or 30 degrees C/min each to -40, -80, or -120 degrees C before being plunged into liquid nitrogen. The spermatozoa frozen under a given condition were then thawed either at 1000 or 200 degrees C/min. Cooling rate, intermediate temperature and warming rate significantly affected survival of spermatozoa obtained from the three bulls. Cooling spermatozoa from 4 to -120 degrees C either at 20 or 30 degrees C/min yielded better progressive motility compared to other cooling conditions (50 versus 30%). Rapid warming was superior to slow warming. In an additional study, motility and fertility of spermatozoa frozen after being cooled to -120 degrees C at 20 degrees C and 30 degrees C/min and those frozen by a standard protocol used routinely for semen processing were assessed. Progressive motility of cryopreserved spermatozoa cooled at 20 degrees C and 30 degrees C/min was 40%, while that of spermatozoa cryopreserved using a standard protocol was 25%. A total of 178 buffalo cows were inseminated with cryopreserved spermatozoa obtained from one bull, and their pregnancy status was assessed 60 days later by rectal palpation. Out of the 60, 26 (43%) and 23 of 58 (40%) cows inseminated with sperm cooled at 20 and 30 degrees C/min, respectively, became pregnant, whereas 17 of 60 (28%) cows inseminated with sperm frozen by a standard protocol became pregnant. This study demonstrates that an effective cryopreservation procedure for buffalo spermatozoa can be derived by systematic examination of various cryobiological factors.     

Effect of cooling on the motility and function of human spermatozoa

Human spermatozoa were cooled from 37 to 0 degrees C at 10 degrees C min(-1) in 5 degrees C steps with 1 min equilibration at each step, the temperature control was +/- 0.1 degrees C. Spermatozoa were held at 0 degrees C for 5 min and then rewarmed at the same rate. No significant effect of cooling on the straight-line velocity was found using computer-aided semen analysis. The physiological function of spermatozoa was also examined before and after cooling using hypoosmotic swelling, ionophore-provoked acrosome reaction, and binding to fragments of human zonae pellucidae. Spermatozoa were cooled either in seminal plasma or in conventional IVF medium with or without fractionation by centrifugation through a discontinuous Percoll gradient. When spermatozoa were cooled and rewarmed in seminal plasma there was no significant change in either the ionophore-induced acrosome reaction or the binding to zona pellucida fragments. When spermatozoa were fractionated by centrifugation through Percoll an increased response in both was seen. However, following cooling and rewarming, a significant decline in the response of both occurred. We suggest that motility alone is not a reliable predictor of changes in other physiological functions of spermatozoa following cooling. Furthermore, short-term cooling appears to have no significant detrimental effect on normozoospermic samples and cold shock may be avoided in the clinical context by controlled cooling and warming.     

Characteristics of Iberian red deer (Cervus elaphus hispanicus) spermatozoa cryopreserved after storage at 5 degrees C in the epididymis for several days

The aim of this study was to assess the influence of prolonged cold storage of Iberian red deer epididymides on post-thaw sperm characteristics. Thirty-seven pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control group). The remaining epididymides were cooled to 5 degrees C and stored for 12, 24, 48, 72 and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. After thawing, sperm motility, membrane and acrosome integrities, mitochondrial function and DNA damage were evaluated. The motility of spermatozoa stored in the epididymis for up to 96 h did not decrease significantly (P>0.05) but, after cryopreservation, a decline in sperm motility was seen in spermatozoa stored for 48 h, or later. A slower decrease in sperm membrane and acrosome integrities after cryopreservation were seen as storage time progressed. Some differences were seen when different methods were used to assess the same sperm parameter although changes followed similar patterns. This was the case for acrosome integrity (phase contrast microscopy versus fluorescent lectin) or membrane integrity (hypo-osmotic swelling test or nigrosin-eosin stain versus propidium iodide). We conclude that frozen-thawed spermatozoa of Iberian red deer recovered from epididymides stored at 5 degrees C have a good sperm quality (including motility) during less than 48 h of storage for most of the sperm parameters assessed.     

Effects of long-term chilled storage of red deer epididymides on DNA integrity and motility of thawed spermatozoa

We have carried out a study on the influence of prolonged cold storage (5 degrees C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5 degrees C and stored for 24, 96 and 192 h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37 degrees C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96 h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192 h at 5 degrees C (P<0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96 h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192 h.     

Cryopreservation of the sperm of the Japanese bitterling

Sperm of the Japanese bitterling Tanakia limbata that had been cryopreserved with 5 or 10% methanol plus 95 or 90% foetal bovine serum (FBS) showed higher percentage and longer duration of motility than those that had been cryopreserved with 90% FBS and 10% DMSO, glycerol, N,N-dimethylacetamide or N, N-dimethylformamide. Foetal bovine serum, used as extender, had some cryoprotective effects when spermatozoa were cooled either with 10% methanol or without methanol. Spermatozoa, cooled to −40° C and then immersed in liquid nitrogen, had greater post-thaw motility than those cooled to −20, −60, or −80° C. The post-thaw percentage of motile spermatozoa increased significantly ( P < 0·001) with decreases in the freezing rate from 60 to 5°C min⁻¹. These results indicate that 10% methanol plus 90% foetal bovine serum is a suitable diluent for cryopreservation of bitterling spermatozoa and that samples should be cooled to -40°C at a low freezing rate for effective storage.     

Refrigerated storage and cryopreservation of black drum (Pogonias cromis) spermatozoa

Procedures were developed for the collection, refrigerated storage and cryopreservation of black drum spermatozoa. Sperm samples were collected by removing and slicing the testis, and suspending the spermatozoa in Hanks' balanced salt solution (HBSS) at 200 mOsm/kg. Threshold activation (10%) of black drum spermatozoa occurred at 370 mOsm/kg, and complete activation occurred at 580 mOsm/kg in HBSS. Sperm cells activated in artificial seawater had higher motility than those activated in HBSS at osmolalities from 350 to 500 mOsm/kg. Spermatozoa stored at 4 degrees C in HBSS or artificial seawater at osmolalities from 202 to 290 mOsm/kg retained motility longer than did those stored at other osmolalities Dilution rate had no effect on sperm storage time at 4 degrees C. Four chemicals were evaluated as cryoprotectants: dimethyl sulfoxide (DMSO), n,n-dimethyl acetamide (DMA), methanol, and glycerol. Glycerol and DMA at concentrations of 10% significantly reduced motility within 52 min. Spermatozoa were cryopreserved at 3 freezing rates (-27, -30, or -45 degrees C/min) in a nitrogen vapor shipping dewar or a computer-controlled freezer. Spermatozoa frozen using 10% DMSO had the highest post-thaw motility at a freezing rate of -27 or -30 degrees C/min. Spermatozoa frozen using 5% glycerol, 5% DMSO, or 10% DMSO had the highest post-thaw motility at a freezing rate of -45 degrees C/min.     

Semen cryopreservation of small abalone (Haliotis diversicolor supertexa)

Methods for cryopreserving spermatozoa and maximizing fertilization rate in Taiwan small abalone, Haliotis diversicolor supertexa, were developed. The gametes (spermatozoa and eggs) of small abalone were viable 3 h post-spawning, with fertilization, and development rate decreasing with time. A minimum of 10(2) cell/ml sperm concentration and a contact time of 2 min between gametes is recommended for artificial insemination of small abalone eggs. Eight cryoprotectants, dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), ethylene glycol (EG), propylene glycol (PG), butylene glycol (BG), polyethylene glycol, glycerol and methanol, were tested at concentrations between 5 and 25% to evaluate their effect on motility of spermatozoa exposed to cryoprotectant for up to 60 min at 25 degrees C before freezing. The least toxic cryoprotectant, 10% DMSO, was added to artificial seawater (ASW) to formulate the extender for freezing. Semen was diluted 1:1 with the extender, inserted into 1.5 ml microtubes and frozen using a cooling rate between -3.5 and -20 degrees C/min to various transition temperatures (0, -30, -60, -90 and -120 degrees C), followed by transfer and storage in liquid nitrogen (-196 degrees C). The microtubes were thawed from +45 to +145 degrees C/min. Spermatozoa, cooled to -90 degrees C at a cooling rate of -12 or -15 degrees C/min and then immersed in liquid nitrogen, had the best post-thaw motility. Post-thaw sperm motility was markedly reduced compared to fresh sperm. More frozen-thawed spermatozoa are required to achieve fertilization rates comparable to those achieved using fresh spermatozoa.     

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

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

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.     

Effects of temperature on the immobilization and the initiation of motility of spermatozoa in the male reproductive tract of the domestic fowl, Gallus domesticus

1. The motility of undiluted fowl spermatozoa taken from testis, epididymis and ductus deferens was negligible at 40 degrees C, around the normal avian body temperature. 2. The immobilization was not permanent and motility was restored by decreasing the temperature to 30 degrees C or by suspending in a NaCl/TES buffer with 2 mM Ca2+, 2 mM HCO3- or 10% seminal plasma at 40 degrees C. 3. Demembranated spermatozoa taken from testis, epididymis and ductus deferens were also immotile at 40 degrees C. However, these spermatozoa were restored the motility at 30 degrees C except testicular spermatozoa. 4. These results suggest that the capacity of movement of fowl spermatozoa can be readily obtained from testis, but that these spermatozoa are immotile due to temperature-dependent immobilization in the male reproductive tract. 5. Furthermore, it is possible that changes in environmental temperature at ejaculation are one of the important exogenous physiological factors of the initiation of fowl sperm motility.     

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

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

Liquid storage of flow cytometrically sorted ram spermatozoa

This study investigated the optimum short-term storage conditions for ram spermatozoa before and after flow cytometric sorting. Prior to sorting, semen from four rams (n = 3 ejaculates per ram) was diluted in either a Tris-based diluent (TRIS) or AndroHep (AH) and stored at 5, 15 or 21 degrees C for 0, 6 or 24h. Sperm characteristics were assessed during storage and after sorting, freeze-thawing and incubation (6h, 37 degrees C). Functional capacity and migration ability in artificial cervical mucus (sperm migration test (SMT)) of stored, sorted and non-sorted (control) spermatozoa were assessed after freeze-thawing. After sorting, semen from three rams (n = 3 ejaculates per ram) was diluted in four different extenders: ultra-heat-treated (UHT) long life milk, TRIS containing 10% (v/v) egg yolk (TRIS-EY), AH (pH 7.4), or TEST buffer containing 10% (v/v) egg yolk (TYB). Sorted and non-sorted (control) spermatozoa were stored at 15 degrees C for 24h or 5 degrees C for 6 days. Sperm characteristics were evaluated at 0, 6 and 24h for samples stored at 15 degrees C and daily for samples stored at 5 degrees C. The SMT was performed on sorted and non-sorted (control) spermatozoa after 6h and 3 days storage at 15 and 5 degrees C, respectively. Spermatozoa stored in TRIS were sorted more efficiently, had higher motility after sorting, freezing, thawing and incubation and had greater numbers of spermatozoa penetrating into the SMT than spermatozoa stored in AH prior to sorting. Spermatozoa stored in UHT at both temperatures had higher motility, acrosome integrity and traveled greater distances in the SMT than spermatozoa stored in all other diluents. In summary, storage in TRIS at 21 degrees C was optimal for transport of ram spermatozoa to the sorting site, and storage of spermatozoa in UHT diluent (after sorting) preserved sperm viability and migration ability best at both 15 and 5 degrees C.     

Effects of cryopreservation methods on post-thaw motility of spermatozoa from the Japanese pearl oyster, Pinctada fucata martensii

In order to develop cryopreservation techniques for Japanese pearl oyster spermatozoa, the effects of various cryopreservation conditions on post-thaw motility were examined. Spermatozoa cryopreserved with 10% methanol (MET), dimethylformamide or dimethylacetamide plus 90% diluent comprising 80% seawater and 20% fetal bovine serum (FBS) showed higher percentages of post-thaw motility than those cryopreserved with 10% dimethylsulfoxide or glycerol. When spermatozoa were cryopreserved with various concentrations (0-20%) of MET and 100-80% diluent, 10% MET showed the highest percentages of post-thaw motility. When spermatozoa were cryopreserved with 10% MET and 90% diluent comprising various concentrations (0-100%) of FBS or Ringer solution mixed with seawater, the percentages of post-thaw motility peaked at 20% FBS or Ringer solution, and were significantly higher for 20% FBS than for 20% Ringer solution. The percentages of post-thaw motility increased with increasing dilution ratios from 2.5- to 50-fold. Spermatozoa cooled to -50 degrees C and then immersed in liquid nitrogen (LN) showed higher post-thaw motility than those cooled to -30 degrees C or -40 degrees C. When spermatozoa were cryopreserved to -50 degrees C at various cooling rates by changing the sample height above the LN surface, the post-thaw motilities of spermatozoa cooled at 10 cm (cooling rate: -21.3 degrees C/min) and 12.5 cm (-15.6 degrees C/min) from the LN surface were higher than those at 5, 7.5 or 15 cm. These results indicate that 10% MET plus 90% diluent comprising 80% seawater and 20% FBS is a suitable extender for cryopreservation of Japanese pearl oyster spermatozoa and that samples should be cooled to -50 degrees C at a cooling rate between -15 and -20 degrees C/min for efficient storage.     

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)     

Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem

Post-mortem spermatozoa recovery is an important technique for obtaining germplasm reserves from genetically valuable animals or endangered species. However, there are many factors that influence the outcome of this technique. We have studied the effect of the interval between animal's death and sperm recovery (0, 24 or 48 h) on the quality and freezability of ram spermatozoa from cauda epididymidis. Storage temperature of epididymis (room temperature or 5 degrees C) was also analysed. Spermatozoa were diluted with Tes-Tris-Fructose solution supplemented with egg yolk (10%) and glycerol (4%), and frozen using a programmable biofreezer (-20 degrees C/min). Pre-freeze and post-thaw sperm samples showed viable spermatozoa up to 48 h after the animal's death, although their quality declined significantly as post-mortem storage time increased. Epididymis sperm stored at 5 degrees C showed better motility and a lower percentage of abnormal forms than epididymis stored at room temperature after 24 and 48 h. The fertilizing ability of cauda epididymis ram spermatozoa obtained at 0 and 24h after the animal's death is similar to that of ejaculated spermatozoa. Therefore, a good protocol for post-mortem semen collection in rams when epididymal spermatozoa cannot be collected immediately, is to preserve the epididymis at 5 degrees C and process the samples in the first 24h after the animal's death.     

Membrane status of boar spermatozoa after cooling or cryopreservation

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.