Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

Cryoprotective effect of different glycerol concentrations on domestic cat spermatozoa

Cryopreservation of spermatozoa is a pivotal tool in assisted reproduction, and studies aiming to establish optimal freezing/thawing protocols are essential to enhance sperm survival. The objectives of the present study were to (1) compare the cryoprotective efficiency of three different glycerol concentrations (3%, 5%, and 7%) on the basis of post-thaw sperm quality and (2) investigate whether the incidence of morphologically abnormal sperm in fresh samples is related to cryodamage sensitivity. Semen was collected from six tomcats using an artificial vagina (total 18 ejaculates). Each ejaculate was diluted using Tris-egg yolk–based extender (TEY), evaluated, equally divided into three aliquots, and rediluted using TEY with and without glycerol to achieve final concentrations of 3%, 5%, and 7%. Samples were loaded into 0.25 mL straws, equilibrated for 60 minutes at 5 °C, frozen, and then thawed at 46 °C for 12 seconds. Fresh and frozen-thawed samples were evaluated for sperm motion parameters (computer-assisted sperm analysis), plasma membrane integrity (PMI; propidium iodide and carboxyfluorescein diacetate), and DNA integrity (acridine orange). Plasma and acrosomal membrane integrity were assessed by flow cytometry (propidium iodide and fluorescein isothiocyanate–conjugated pea (Pisum sativum) agglutinin) immediately after thawing. Sperm motion parameters were also evaluated at 30 and 60 minutes of postincubation. For all treatment groups, cryopreservation significantly impaired the PMI and sperm motion parameters, except for straightness and amplitude of lateral head displacement. DNA integrity showed a slight reduction (P < 0.05) when 3% glycerol was used. The percentage of total motility, progressive motility, and rapid spermatozoa were significantly lower immediately after thawing and up to 60 minutes of incubation for the 3% glycerol group when compared with 5% and 7%. No difference (P > 0.05) was found for PMI, acrosome integrity, and DNA integrity among post-thaw groups. However, higher (P < 0.05) incidence of viable cells with reacted acrosome and dead cells with intact acrosome were observed with 7% and 3% glycerol, respectively. Percentage of morphologically abnormal spermatozoa in fresh sample was positively correlated with PMI only in the 3% glycerol group and negatively correlated with sperm motility in the 5% and 7% groups. In conclusion, the final concentration of 5% glycerol offered better cryoprotective effect for ejaculated cat sperm, and the relationship found between prefreezing sperm morphology and post-thaw sperm quality showed to be dependent on final glycerol concentration.     

Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria

Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75-90% motility and 14-16 μm s⁻¹ swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 °C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 °C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40-45% viability and 30-35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5-6 × 10⁶/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 °C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.     

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.     

Improvement of post-thaw sperm survivals using liquid nitrogen vapor in a spermcasting oyster Ostrea angasi

Low survival of cryopreserved sperm impedes the application of cryopreservation technique in spermcasting oyster species. This study developed a simple method of liquid nitrogen vapor freezing to improve post-thaw sperm survival in the spermcasting oyster Ostrea angasi. The results indicate that the permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were non-toxic to sperm up to 20% concentration and 90 min exposure whereas methanol at 10% or higher was toxic to sperm for any exposure over 30 min. Among the treatments with permeable cryoprotectants, 15% EG produced the highest post-thaw sperm motility. Sperm motility was further improved by the addition of non-permeable cryoprotectants (trehalose and glucose), with 15% EG + 0.2 M trehalose resulting in the highest post-thaw sperm motility among all the combinations evaluated. The durations of 20, 30 and 60 min equilibrations produced a higher post-thaw sperm motility and plasma membrane integrity (PMI) than 10 min. Higher post-thaw motility and PMI were achieved by freezing sperm at the 8 cm height from the liquid nitrogen surface than at the 2, 4, 6, 10 or 12 cm height. Holding sperm for 10 min in liquid nitrogen vapor produced higher post-thaw motility and PMI than for 2, 5 or 20 min. The cryopreservation protocol developed in this study improved both post-thaw motility and PMI of O. angasi sperm at least 15% higher than those cryopreserved using programmable freezing method. Liquid nitrogen vapor freezing might have greater applicability in improving post-thaw sperm quality of spermcasting oyster species.     

Effect of basic factors of extender composition on post-thawing quality of brown bear electroejaculated spermatozoa

The improvement of freezing extenders is critical when defining sperm cryopreservation protocols for wild species, in order to create germplasm banks. The aim of this study was to evaluate the effect of additives (Equex Paste and EDTA) supplementation, egg-yolk (10 and 20%) and glycerol (4 and 8%) concentrations and extender osmolality (300 and 320 mOsm/kg) on the post-thawing quality of brown bear semen. Semen was obtained from 20 adult males by electroejaculation, and centrifugated individually (600 × g for 6 min). The pellets were diluted 1:1 in the corresponding extender TTF (TES-Tris-Fructose with the aforementioned variants) and cooled to 5 °C. Then, it was diluted down to 100 × 10⁶ spz/mL, loaded in 0.25 mL straws and frozen at −20°C/min. After thawing (in water at 65 °C for 6s), the semen samples were assessed for motility (CASA), viability (SYBR-14 with propidium iodide), acrosomal status (PNA-FITC with propidium iodide) and mitochondrial activity (JC-1). Extender supplementation with additives rendered significantly higher results for these sperm parameters. Comparing the two percentages of egg yolk, 20% egg yolk showed the highest motility results, percentages of viable spermatozoa and viable spermatozoa with intact acrosome. No differences were detected among samples frozen using 4 or 8% glycerol. For extender osmolality, 300 mOsm/kg showed higher values of VAP, VCL, VSL, and ALH than 320 mOsm/kg. Based on the best performance of sperm motility, viability and acrosome status, we conclude that the most suitable extender to cryopreserve brown bear spermatozoa was TTF adjusted to 300 mOsm/kg, supplemented with 20% egg yolk, 4-8% glycerol, and the additives 1% Equex paste and 2% EDTA.     

Ultrastructural observations of cryoinjury in kangaroo spermatozoa

Macropod spermatozoa have proven difficult to cryopreserve such that empirical studies using high concentrations of glycerol and/or DSMO have resulted in only 10% post-thaw motility. We examined the ultrastructure and freeze-fracture of caput and cauda epididymal macropod spermatozoa at 35, 4 degrees C and following cryopreservation with and without 20% glycerol. The addition of 20% glycerol resulted in significant damage to the sperm plasma membrane and mitochondria compared to no glycerol at the same temperatures (P<0.05). Following cryopreservation, 20% glycerol significantly improved the preservation of the cauda epididymal sperm plasma membrane and mitochondria and reduced the incidence of axonemal damage and axonemal spaces. For caput epididymal spermatozoa, glycerol only improved the preservation of the plasma membrane following cryopreservation (P<0.05). Freeze fracture microscopy revealed a pattern of helically wound intramembranous particles in the plasma membrane over the fibre network of the mid piece of the sperm tail. The fibre network is an interconnecting cytoskeletal structure found underneath the plasma membrane of the kangaroo sperm midpiece and is thought to add rigidity to the proximal portion of the sperm tail. After thawing, the plasma membrane was damaged such that this structure was missing in patches, and the helical rows of particles were mal-aligned. On the principal piece, particles were arranged randomly at physiological temperatures; however, upon cooling to 4 degrees C with 20% glycerol, the particles become aggregated. Once rewarmed (35 degrees C), particles over the principal piece resumed their random organisation. This finding is further evidence of a reversible phase transition of the macropod sperm plasma membrane during cooling that is not associated with a loss of motility or membrane integrity.     

Sperm cryopreservation of the critically endangered olive barb (Sarpunti) Puntiussarana (Hamilton, 1822)

The present study focused on development of a sperm cryopreservation protocol for the critically endangered olive barb Puntiussarana (Hamilton, 1822) collected from two stocks within Bangladesh and reared in the Fisheries Field Laboratory, Bangladesh Agricultural University (BAU). The sperm were collected in Alsever’s solution prepared at 296 mOsmol kg⁻¹. Sperm were activated with distilled water (24 mOsmol kg⁻¹) to characterize motility. Maximum motility (90%) was observed within 15 s after activation, and sperm remained motile for 35 s. Sperm activation was evaluated in different osmolalities and motility was completely inhibited when osmolality of the extender was ?287 mOsmol kg⁻¹. To evaluate cryoprotectant toxicity, sperm were equilibrated with 5%, 10% and 15% each of dimethyl sulfoxide (DMSO) and methanol. Sperm motility was noticeably reduced within 10 min, when sperm were equilibrated with 15% DMSO, indicating acute toxicity to spermatozoa and therefore this concentration was excluded in further trials. Sperm were cryopreserved using DMSO at concentrations of 5% and 10% and methanol at 5%, 10% and 15%. The one-step freezing protocol (from 5 °C to −80 °C at 10 °C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia) and 0.25-ml straws containing spermatozoa were stored in liquid nitrogen for 7–15 days at −196 °C. The highest motility in thawed sperm 61 ± 8% (mean ± SD) was obtained with 10% DMSO. The fertilization and hatching rates were 70% and 37% for cryopreserved sperm, and 72% and 62% for fresh sperm. The protocol reported here can be useful for hatchery-scale production of olive barb. The use of cryopreserved sperm can facilitate hatchery operations, and can provide for long-term conservation of genetic resources to contribute in the recovery of critically endangered fish such as the olive barb.     

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.     

La Cryoconservation de la laitance de la carpe, Cyprinus carpio

Cryo-preservation of carp, Cyprinus carpio, sperm Deep-freezing trials of carp sperm were carried out by varying several factors such as the basic saline solution, the cryoprotectors added (glycerol, propanediol, ethylene glycol and DMSO), the media (Menezo-INRA B², egg yolk, urea) and the deep-freezing and dilution rates. The success of deepfreezing was judged by the percentage of motile spermatozoa, intensity of motility, fertilizing ability and morphological integrity of the spermatozoa studied under the scanning electron microscope. DMSO was the best cryoprotector and the mineral composition of the dilution medium the least important factor, but there was noticeable improvement after organic compounds were added. The following mixture has been proposed: NaCl 100 mM + KC1100 mM, Tris 20 mM, pH 8: 37%, Menezo medium B² INRA: 15%, urea 5%, DMSO: 10%, fresh sperm: 33%. Optimal deep-freezing rate was: 5°C/min from 2 to-7°C and 25°C/min from-7 to-70°C. In these conditions, about 70 to 80% of the spermatozoa were motile after thawing compared to fresh control sperm, but fertilizing ability was not more than 30 to 40% of that with fresh sperm. The percentage of spermatozoa considered intact was 66% after thawing as against 83% for fresh control sperm. The motility and fertilizing ability of deep-frozen sperm were significantly improved when the dilution rate at insemination was reduced from ¹/₁₀₀ to 1/2.     

Cryopreservation of sperm from farmed Pacific abalone,Haliotis discus hannai

This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me₂SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me₂SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me₂SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN₂) vapor for 10 min at 5 cm above the LN₂ surface and then submerging them in LN₂ for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me₂SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me₂SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me₂SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.     

Influence of the thawing rate on the cryopreservation of semen from collared peccaries (Tayassu tajacu) using Tris-based extenders

The objective was to evaluate the influence of the thawing rate on the quality of frozen-thawed (cryopreserved in Tris-based extenders) semen obtained from collared peccaries (Tayassu tajacu). Semen from 13 sexually mature collared peccaries males were collected by electroejaculation, and evaluated for motility, vigor, sperm viability, membrane integrity, and sperm morphology. Semen was divided in two equal portions: the first was diluted in Tris-fructose and the other in Tris-glucose, with egg yolk (20%) and glycerol (3%) added to each portion. Extended semen was frozen in liquid nitrogen and thawed using two thawing protocols (37 °C for 1 min or 55 °C for 7 s, followed by an additional 30 s at 37 °C). There were no significant differences between the two extenders after extension, chilling, or glycerol addition. After thawing at 37 °C, there were 37.9 ± 4.2% and 28.5 ± 5.1% motile spermatozoa for samples extended in Tris-fructose and Tris-glucose, respectively, with 33.8 ± 3.7% and 28.2 ± 3.5% motile spermatozoa after thawing at 55 °C (no significant differences). Furthermore, there were no significant interactions between extenders and thawing protocols for any semen end point. In conclusion, semen from collared peccaries was successfully cryopreserved in Tris-based extenders and thawed with two protocols (37 °C for 1 min or 55 °C for 7 s).     

Changes in rat spermatozoa function after cooling,cryopreservation and centrifugation processes

Rat sperm cryopreservation is an effective method of archiving valuable strains for biomedical research and handling of rat spermatozoa is very important for successful cryopreservation. The aim of this study was to evaluate changes in rat sperm function during cryopreservation and centrifugation. Epididymal rat spermatozoa were subjected to cooling and freezing–thawing processes and then motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were compared before and after minimum centrifugation force (200×g). Cryopreservation decreased sperm motility, PMI, and MMP (P < 0.05). Basal (without ROS inducer, tert-butyl hydroperoxide [TBHP] treatment) and stimulated ROS (with TBHP treatment) were increased in viable cooled spermatozoa compared to viable fresh spermatozoa (P < 0.01), with equal susceptibility to TBHP among fresh, cooled, and frozen–thawed spermatozoa. Centrifugation decreased motility and PMI of frozen–thawed spermatozoa (P < 0.05). Centrifugation decreased basal ROS of all spermatozoa (P < 0.01), while it led to higher susceptibility to TBHP in viable cooled spermatozoa, showing higher increased fold in ROS and decreased rate in viability by TBHP in viable cooled spermatozoa (P < 0.05). Cooling process was the major step of ROS generation, with loss in sperm motility, PMI, and MMP. Centrifugation affected function of cryopreserved spermatozoa. These data suggest that centrifugation makes rat spermatozoa susceptible to external ROS source, in particular during cooling process. Thus, protection from ROS damage and minimizing centrifugation should be considered during cryopreservation and post-thaw use of cryopreserved epididymal rat spermatozoa.     

The use of cryomicroscopy in guppy sperm freezing

The present study employed cryomicroscopy to derive an optimal sperm freezing protocol for guppy (Poecilia reticulata) sperm. Evaluation criteria during the freezing-thawing process were assessed for nucleation temperature (Tn), temperature when more than 50% of sperm display bending mid-piece (Tb), temperature when more than 80% of sperm stop moving (Tm), thawing temperature (Tt), and post-thaw motility. We compared four different cryoprotectants: 5% N-dimethyl formamide (DMF), 6% methanol (MEOH), 10% dimethyl sulfoxide (DMSO), and 14% glycerol, as well as glycerol at different concentrations of 7-50%; cooling and rewarming rates ranged from 5 to 100 °C/min. The protocol that yielded the highest post-thaw motility was samples suspended in 14% glycerol, cooled at 25 °C/min, and thawed at 100 °C/min, which was in complete agreement with our previous findings derived from a controlled-rate freezer. In addition, Tb and Tm were found to be negatively correlated with post-thaw motility, suggesting their possible role in predicting freezing success. The present study for the first time demonstrated the usefulness of cryomicroscopy in deriving an optimal sperm freezing protocol for aquatic species.     

Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury,but respond favorably to dimethyl sulfoxide

We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham’s F10 medium (isotonic control) or to this medium plus NaCl (350–1000 mOsm), sucrose (369 and 479 mOsm), 1 M glycerol (1086 mOsm) or dimethyl sulfoxide (Me₂SO, 1151 mOsm) for 10 min. Each sample then was diluted back into Ham’s medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me₂SO had no influence (P > 0.05), NaCl and sucrose solutions affected sperm motility (P < 0.05), but not membrane integrity. Motility of sperm exposed to <600 mOsm NaCl or sucrose was less (P < 0.05) than fresh ejaculate, but comparable (P > 0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to <5%. In a separate study, ejaculates were diluted in Test Yolk Buffer containing 1 M glycerol or Me₂SO and cooled from 5 °C to −120 °C at −57.8 °C, −124.2 °C or −67.0 °C/min, frozen in LN₂, thawed in a water bath for 30 s at 37 °C or 10 s at 50 °C, and then assessed for motility, plasma- and acrosomal membrane integrity. Cryopreservation markedly (P < 0.05) reduced sperm motility by 70% compared to fresh samples. Higher (P < 0.05) post-thaw motility (20.0 ± 1.9% versus 13.5 ± 2.1%) and membrane integrity (51.2 ± 1.7% versus 41.5 ± 2.2%) were observed in samples cryopreserved in Me₂SO than in glycerol. Cooling rates influenced survival of sperm cryopreserved in glycerol with −57.8 °C/min being advantageous (P < 0.05). The findings demonstrate that although maned wolf spermatozoa are similar to domestic dog sperm in their sensitivity to osmotic-induced motility damage, the plasma membranes tolerate dehydration, and the cells respond favorably to Me₂SO as a cryoprotectant.     

Cryopreservation of epididymal sperm collected postmortem in the Tasmanian devil (Sarcophilus harrisii)

Effective sperm cryopreservation protocols are limited to a small number of marsupial species. In this study, postmortem gamete rescue (PMGR) epididymal sperm samples from Tasmanian devils (N = 34) euthanized due to the fatal Devil Facial Tumor Disease were used to develop long-term sperm storage techniques for the species. Cryoprotectant toxicity associated with equilibration of sperm samples in a TEST yolk diluent (TEST; 189 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 85 mM Trizma base [Tris], 11 mM glucose, 20% vol/vol egg yolk; pH 7.1, and 315.0 ± 5.0 mOsm/kg) with a final concentration of 0.06 M trehalose, or 4%, 10%, and 18% vol/vol of either glycerol or dimethyl sulfoxide (DMSO), was examined over 12 h at 15 °C. Trehalose supplementation resulted in an immediate decline (P < 0.05) of total motility. After 12 h, total motility was reduced (P < 0.05) in treatments containing 18% glycerol, and 10% and 18% dimethyl sulfoxide. The effects of final glycerol concentration (4% and 10%), glycerol equilibration duration (10 min 1 h, or 3 h) prefreeze, freezing rate and the addition of 0.10 M lactose or a combination of 0.10 M lactose and 0.11 M raffinose were assessed during three experiments on the cryopreservation of postmortem gamete rescue samples in TEST. In all experiments, motility and viability were reduced (P < 0.01 postthaw). Samples cryopreserved in TEST supplemented with lactose or lactose with raffinose using a fast freezing rate (−8 °C/min from 4 to −40 °C, then −65 °C/min until −165 °C) produced the highest (P < 0.05) postthaw motility (18.6 ± 5.5% and 16.9 ± 8.5%, respectively), which represented 35% to 48% retention of prefreeze motility. These results apparently were the best postthaw results of dasyurid sperm reported to date and will help lay the foundations for developing assisted reproductive technologies for marsupial species.     

The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm

Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10 to 12 weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode’s lactate (TL-HEPES), modified Kreb’s Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon’s Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1 M raffinose or 0.1 M sucrose. The sperm samples in each extender were cooled to 4 °C and held for 45 min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of −150 °C by using various cooling rates (10, 40, 70, and 100 °C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37 °C slide warmer and held for 1 min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P < 0.05). Sperm motility increased as cooling rate was increased for both strains (P < 0.05). Highest cryosurvival was achieved when 100 °C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1 M sucrose or raffinose (P < 0.05). This study showed that TES extender containing 0.1 M raffinose or sucrose with 70 °C/min and 100 °C/min cooling rate improved post-thaw motility of rat sperm.     

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)     

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.