Cryopreservation of sperm in farmed blacklip abalone (Haliotis rubra Leach, 1814)

This study developed a technique of sperm cryopreservation using liquid nitrogen (LN) vapour in farmed blacklip abalone Haliotis rubra through evaluating the following five key factors: (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature; (3) thawing temperature; (4) sperm to egg ratio and (5) sugar addition, using sperm motility or fertilization rate as quality assessment indicators. The results demonstrated that 6% dimethyl sulfoxide (DMSO) was the best single CPA for sperm cryopreservation in this species. The highest post‐thaw sperm motility was achieved when sperm were exposed to LN vapour for 10 min at 5.2 cm above the LN surface and thawed at 60°C and recovered at 16°C in seawater baths. Post‐thaw sperm motility was found to be significantly higher when 6% DMSO was used in combination with 1% or 2% glucose than 6% DMSO alone. Further evaluation of fertilization rate between these CPAs showed that 6% DMSO+2% glucose achieved the highest fertilization rate of 70% at a sperm to egg ratio of 10 000:1.     

Effect of Dimethyl sulfoxide (DMSO) and egg yolk on sperm motility,fertility and hatching rates of depik Rasbora tawarensis (Pisces: Cyprinidae) eggs after short‐term cryopreservation

Cryoprotectant is the crucial factor in the cryopreservation process. In general, there are two types of cryoprotectant, permeating and non‐permeating cryoprotectants. Dimethyl sulfoxide (DMSO) and egg yolk are common permeating and non‐permeating cryoprotectants respectively. Hence, the objective of the present study was to determine the best proportion of DMSO and egg yolk for the cryopreservation of Rasbora tawarensis sperm. A completely randomized experimental design was used in this study which involves two types of cryoprotectant and their combination at different concentrations, namely 5% DMSO, 5% egg yolk, 5% DMSO + 5% egg yolk and 2.5% DMSO + 2.5% egg yolk. Every treatment was conducted in three replicates. Combination of 5% DMSO + 5% egg yolk gave the best results cryoprotectant treatment had significant effects on sperm motility, fertilization and hatching rate of the R. tawarensis eggs (p < .05). It is concluded that the best proportion of cryoprotectants for sperm cryopreservation in this species is 5% DMSO + 5% egg yolk.     

Fertilizing capacity and motility of tench Tinca tinca (L., 1758) sperm following cryopreservation

Experiments were carried out to develop an optimal cryopreservation protocol for tench sperm by testing the fertilizing capacity and motility parameters including progressive motility, curvilinear velocity (VCL) and linearity (LIN) of cryopreserved sperm. Three experiments were designed to this aim: first experiment where we tested the effects of two extenders (sugar‐based Grayling and ion‐based Kurokura 180) and two cryoprotectants (DMSO and methanol) on fertilization and hatching success; second where we tested the effect of cryoprotectant type (methanol or DMSO) in different concentrations (5%, 10% and 15%) on fertilization and hatching success; and third where we tested the effect of two cryoprotectants (methanol and DMSO) on sperm motility parameters (progressive motility, VCL and LIN) after 4 h post‐thaw storage (4°C). Sperm prepared with the sugar‐based Grayling extender displayed better fertilization and hatching rates independently of the applied cryoprotectant most likely due to glucose present which acted as an external cryoprotectant. Concerning cryoprotectant concentrations, the use of 10% methanol yielded the highest fertilization (85 ± 15%) and hatching (80 ± 13%) rates, which were significantly higher than in all other groups. During the post‐thaw storage time, 5% methanol, 10% methanol and 5% DMSO groups had significantly higher motility parameters than other groups and we observed no significant decline in any of the parameters during the storage time. Overall, we found that a sugar‐based extender in combination with methanol as cryoprotectant is suitable for the cryopreservation of tench sperm and allows storage of cryopreserved sperm for up to 4 h post thaw.     

Effect of cryoprotectants, extenders and freezing rates on the fertilization rate of frozen striped catfish, Pangasius hypophthalmus (Sauvage), sperm

The effects of four cryoprotectants (methanol, MeOH; dimethyl sulphoxide, DMSO; dimethyl acetamide, DMA; and ethylene glycol, EG), three extenders (calcium‐free Hanks' balanced salt solution, C‐F HBSS, Hanks' balanced salt solution, HBSS and sodium chloride, NaCl) and two different freezing procedures (one‐ and two‐step) on the cryopreservation of striped catfish (Pangasius hypophthalmus (Sauvage)) sperm were investigated. Sperm were frozen using a controlled‐rate freezer in 250 μL straws and stored for 2 weeks in a liquid nitrogen (LN₂) container. They were then airthawed at room temperature, and fertilization, motility and viability were assessed. The highest fertilization rate of 41% (81% of control) was achieved with the combination of 12% DMSO and 0.9% NaCl using a one‐step freezing procedure (10°C min^?1). Also, DMA resulted in a higher fertilization rate (30% or 51% of the control) than MeOH (18% or 38% of the control) or EG (8% or 12% of the control). In addition, the three extenders used did not affect fertilization rates after cryopreservation with each cryoprotectant. There were no significant differences among the three cryoprotectant concentrations and between the one‐ and two‐step freezing procedures. However, fertilization rates of cryopreserved sperm were significantly lower than the controls (P<0.05). The results of this study indicate that high fertilization rates of striped catfish eggs can be achieved using cryopreserved sperm when frozen at 10°C min^?1 in DMSO or DMA with either 0.9% NaCl or C‐F HBSS.     

Effect of storage time and cryoprotectant concentrations on the fertilization rate and hatching rate of cryopreserved sperm in red seabream (Pagrus major Temminck & Schlegel, 1843)

This study examined the effects of storage time and cryoprotectant concentrations on the post‐thaw sperm of red seabream, Pagrus major. Sperm treated with 12%, 15%, 18% and 21% DMSO were cryopreserved for 10, 30, 60 and 360 days, and fertilization and hatching rates were analysed. For all groups, there were no differences in the fertilization rates and hatching rates between sperm cryopreserved for <60 days and fresh sperm (98.8±0.8%, 96.4±1.3%). However, for sperm cryopreserved for 360 days, both fertilization rates (88.6±3.0% to 7.0±1.9%) and hatching rates (79.4±7.2% to 3.3±0.8%) decreased drastically. Furthermore, the cryoprotectant concentrations affected sperm quality significantly (P<0.05). When cryopreserved for 360 days, sperm treated with 15% DMSO obtained the best results compared with other concentrations. We suggest that 15% DMSO may be an effective cryoprotectant for long‐term sperm cryopreservation of red seabream.     

Cryopreservation of sperm from spotted wolffish

The effects of different concentrations of cryoprotectant (dimethyl sulfoxide; DMSO), cooling rate and straw size on the post-thaw motility of frozen sperm from spotted wolffish, Anarhichas minor, were studied. There was no significant difference in the post-thaw motility of sperm treated with three different concentrations of DMSO (10, 20 and 30%). Similarly, there was no significant difference in the post-thaw motility of spermatozoa when using different freezing rates (i.e. distance of straws from the surface of liquid N₂, 4.7, 5.5 and 7.1°C min⁻¹) and the straw size (0.5 and 1.0 ml) did not affect survival. The cryopreservation of sperm can be used to make up for the frequent lack of sperm and/or the unsynchronised timing of sperm production in spotted wolffish males and the ovulation time in females. The results show that sperm from spotted wolffish can be frozen to secure access to viable sperm, but further experiments are needed in order to reveal the effect of different parameters on the post-thawing mortality and define the optimum conditions for cryopreservation.     

Effect of extenders on sperm cryopreservation of African catfish, Clarias gariepinus (Burchell)

The effect of extenders was studied on the cryopreservation of sperm from African catfish, Clarias gariepinus (Burchell). The following six basic extenders were tested: fructose, glucose, sucrose, NaCl, KCl solutions and the artificial seminal plasma of the African catfish. Each of these extenders was tested both with and without buffer systems (i.e. NaHCO₃-CO₂ and Tris-HCl) by using 10% dimethyl-sulphoxide (DMSO) as a cryoprotectant. The two-step freezing was carried out in a programmable freezer by using the following freezing rates: (1) 4 °C min^–1 between 3 and –4 °C; (2) and 11 °C min^–1 between –4 and –80 °C. The best post-thaw motility (25%) was achieved with 333 mmol L^–1 fructose solution and NaHCO₃ buffer. The fertilization experiments were carried out with unbuffered fructose and glucose extenders using various amounts of sperm and two fertilization methods: (1) dry and (2) wet. The best fertilization rates were achieved with 75 μL of sperm and wet fertilization with glucose extender, or 100 μL of sperm and dry fertilization in case of fructose – both methods fertilized 96% of all eggs.     

Suitable methods for cryopreservation of semen from Atlantic halibut, Hippoglossus hippoglossus L.

Decrease in the quality and quantity of Atlantic halibut, Hippoglossus hippoglossus L., semen towards the end of the reproductive season hampers production of good-quality embryos. Therefore, cryopreservation of spermatozoa is a method showing potential to facilitate controlled reproduction in Atlantic halibut. The present study aimed at establishing the appropriate cryopreservation procedure. We tested 20 extenders composed of four various diluents and five cryoprotectants (DMSO, DMA, methanol, propylene glycol, and glycerol) to determine the best extender. Then, we examined cryopreservation quality using various methods of loading and various volumes of cryopreserved samples. In most of the tested variants, sperm diluted with an extender showed high motility after 24-h incubation despite the high osmotic pressure of the extender. Modified turbot extender (MTE) was the best of the tested diluents, securing the highest post-thaw motility (P < 0.05), and DMSO, DMA, and methanol were the best cryoprotectants (P < 0.05). There was no significant effect of 15-min equilibration of semen in MTE-based extenders prior to freezing on post-thaw motility (P > 0.05). MTE-based extender was chosen as the most suitable. Semen cryopreserved in straws, Eppendorfs or Ziploc bags in volumes ranging from 0.25 to 20 ml showed similar high fertilization ability. Survival of larvae produced with the cryopreserved sperm did not differ from controls produced with freshly collected sperm. Motility 3 h after thawing was high but depended on the type of cryoprotectant and the volume of cryopreserved sperm (P < 0.05). The developed cryopreservation procedure has been applied at our Atlantic halibut breeding station for seed production.     

Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post‐thaw sperm motility was achieved with the sperm being frozen at a cooling rate of ?5°C min^?1 to ?30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post‐thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L‐ascorbic acid, on the other hand, significantly improved the post‐thaw sperm motility. However, only the addition of 0.6% glycine improved the post‐thaw sperm fertilization rate, which was further confirmed by the improvement of the post‐thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.     

Development of a simple method for sperm cryopreservation of Scombridae fishes in outdoor environments

We developed a simple dry shipper method for cryopreserving the sperm of Scombridae fish in outdoor environments. First, we undertook a preliminary study to optimize the sperm cryopreservation conditions using bullet tuna, Auxis rochei (Risso, 1810) sperm. We found that the optimum cryomedium contained 90% foetal bovine serum (FBS) or 300 mM trehalose as an external cryoprotectant and 10% dimethyl sulfoxide (DMSO) as an internal cryoprotectant. Under these optimized conditions, the post‐thaw sperm had a duration of motility of 500 s and a motility rate of >70%. We then performed practical trials of the optimized protocol in various outdoor environments (e.g., fishing boats and ports) using the sperm of five Scombridae species: chub mackerel, Scomber japonicus (Houttuyn, 1782); blue mackerel, S. australasicus (Cuvier, 1832); skipjack tuna, Katsuwonus pelamis (Linnaeus, 1758); longtail tuna Thunnus tonggol, (Bleeker, 1851) and Pacific bluefin tuna, T. orientalis (Temminck & Schlegel, 1844). The post‐thaw sperm of all five of these species had a duration of motility of 650 s and a motility rate of >70%, indicating that this simple method can be used to obtain high‐quality cryopreserved sperm of various Scombridae species in outdoor environments.     

Transport and cryopreservation of sperm of the common snook, Centropomus undecimalis (Bloch)

Sperm were collected in Florida from wild common snook, Centropomus undecimalis (Bloch), and were shipped to Louisiana State University for analysis and cryopreservation. Threshold activation of sperm (10% motility) occurred at 370 mOsmol kg^?1, and complete activation occurred at 680 mOsmol kg^?1. These values were significantly different. Sperm samples stored at 1°C in Hanks' balanced salt solution (HBSS) or in 0.6% NaCl solution at 200 mOsmol kg^?1 retained motility for as long as 22 days. Mean motility remained above 50% for 9 days for sperm stored in HBSS and for 7 days for sperm stored in NaCl solution. Sperm exposed to 5% dimethyl acetamide (62±10%; mean±SD), 10% dimethyl sulphoxide (DMSO) (39±16%), 5% glycerol (26±5%) or 10% glycerol (6±2%) for 30 min had significantly lower motility than did unexposed sperm (89±9%). When used as a cryoprotectant, samples frozen with 5% or 10% DMSO or 5% methanol had significantly higher post‐thaw motility than did samples frozen with other cryoprotectants. Sperm cryopreserved with 10% DMSO (38±12%) had significantly higher post‐thaw motility than did sperm cryopreserved with 15% DMSO (19±10%) or 20% DMSO (4±4%). There were no significant differences in hatch rates of eggs fertilized with fresh sperm (54±29%) or cryopreserved sperm (41±35%). Survival to first feeding was not different between fish produced with fresh sperm (37±30%; range, 0–86%) or with thawed sperm (24±29%; 0–77%). Transport of sperm to a cryopreservation laboratory and back to a hatchery for thawing and use enabled collaboration between groups with specific expertise and provides a model for the application of cryopreservation by transport of fresh and frozen samples.     

Cryopreservation of silver barb Puntius gonionotus (Bleeker) spermatozoa: effect of extender composition,cryoprotective agents and freezing rate on their postthawing fertilization ability

The effects of extender composition, cryoprotectant concentration and freezing and thawing on the fertilization efficiency of cryopreserved spermatozoa of Puntius gonionotus were evaluated. Computer‐aided motility analysis of semen was conducted to check the suitability of spermatozoa for cryopreservation after mixing with different extenders and cryoprotective agents (CPAs). Extender‐4 with an osmolality 260 mOsmol kg⁻¹and pH 7.6 was used for the cryopreservation study. Among the CPAs, dimethyl sulphoxide (DMSO) was least toxic and more than 60% fertilization was achieved when used at 1.4 M at 0 °C for 10 and 30 min, whereas the toxicity of all CPAs to spermatozoa was evident when tested at 30 °C. Semen frozen at −16 °C min⁻¹ with 1.4 M DMSO showed 70% fertilization, which was significantly higher (P<0.05) than other freezing rates. Samples thawed at 35 °C water showed a fertilization rate comparable with that of fresh semen. Computer‐assisted semen analysis of fresh and frozen semen after thawing showed variations in different types of motility in spermatozoa and in their class. There was no significant difference in motility before or after cryopreservation; however, significant differences could be observed in the average path velocity (VAP), straight line velocity (VSL) and curve linear velocity (VCL). Semen of silver barb could be cryopreserved with extender‐4 by addition of 1.4 M DMSO to a final cryopreservation medium (MED 2) cooled at a rate of −16 °C min⁻¹, stored in liquid nitrogen (−196 °C) and utilized after thawing at 35±2 °C.     

The effect of cryoprotectants on the motility and fertilizing capacity of cryopreserved African catfish Clarias gariepinus (Burchell 1822) sperm

The effect of six cryoprotectants was investigated on the cryopreservation of African catfish Clarias gariepinus (Burchell) sperm. Fructose (6%) solution buffered with NaHCO₃‐CO₂ was used as the diluent in the experiments. Glycerol (5–11%), ethylene glycol, methanol and propylene glycol (5–15%) and, finally, dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA) (10%) were tested using various equilibration times (2–30 min). Sperm was frozen in 250‐μL straws in a programmable freezer (Cryocell‐15, BLS, Hungary) from 3 °C to ?4 °C at 4 °C/min and from ?4 °C to ?80°C at 11 °C/min. Thawing was carried out in a 40 °C water bath for 5 s. Fertilization and hatching trials were performed only with DMSO and DMA using 200 and 100 μL of diluted sperm (100 and 50 μL of pure sperm) and the dry and the wet fertilization methods. Ethylene glycol, glycerol, methanol and propylene glycol yielded poor results. An average post‐thaw motility rate of 44.0 ± 9.7% and 22.6 ± 18.1% was achieved after 10 min equilibration using DMSO and DMA respectively. Highest average fertilization (86.8 ± 3.1%) and hatching (67.1 ± 11.9%) rates were achieved with DMA and DMSO, respectively, 200 μL of diluted sperm and the wet fertilization technique. The use of cryoprotectants increased the percentage of malformed larvae compared with the control groups. We found that DMA at a 10% concentration was equally as suitable for the cryopreservation of African catfish sperm as DMSO.     

日本蟳精子超低温冷冻保存技术的研究

以精子存活率作为评价指标,采用两步降温法研究了稀释剂、抗冻保护剂和预冷时间对日本蟳精子超低温冷冻保存效果的影响。结果表明:以采用稀释剂II、15%二甲基亚砜(DMSO)作为抗冻保护剂的保存效果最佳。在液氮中保存24 h后,精子存活率可达83.76%,保存一年后可达73.81%。精液的第一次预冷时间以25 min为宜。     

Cryopreservation of Mekong catfish,Pangasius bocourti Sauvage, 1880 spermatozoa

The effects of three extenders (Ginzburg fish ringer, Calcium‐free Hank's balanced salt solution, C‐F HBSS and sodium chloride, 0.9% NaCl) and four cryoprotectants (dimethyl sulphoxide, DMSO; dimethyl acetamide, DMA; methanol, MeOH and glycerol) in different concentrations (5%, 10% and 15%) on the motility, viability and fertilization rates of Mekong catfish (Pagasius bocourti) sperm were investigated. Sperm samples were transferred into 250‐μL French straws and sealed with a heated haemostat. The straws were then placed in a cryochamber. A computer‐controlled rate freezer (CL 3300) and programmable Cryogenesis, version 4 were used to regulate the freezing rate. The sperm samples were frozen at a rate of 10°C min^?1 from 4 to ?80°C and then evaluated after 72 h. Of the three extenders used with each cryoprotectant, C‐F HBSS had the highest fertilization rate of 75% (93% of control). This was not significantly different from the control treatment (fresh sperm) when tested with DMSO as the cryoprotectant. The lowest fertilization rate of 27% (38% of control) was resulting from the combination of 15% glycerol and C‐F HBSS. This study found that fertilization, motility and viability rates in all of the experiments had a positive significant correlation (P < 0.001).     

Development of cryopreservation for maintaining yellow catfish Pelteobagrus fulvidraco sperm

Yellow catfish (Pelteobagrus fulvidraco) is a candidate freshwater fish for aquaculture in China with its high consumer demand. The aim of this study was to examine the possibility of storage of the sperm of yellow catfish by cryopreservation in liquid nitrogen. Experiments were designed to investigate the effects of the different combinations of three extenders (Ringer extender, Kurokura-1 extender and D-15 extender) and three cryoprotectants (DMSO, Glycerol and Methanol) on the cryopreservation of yellow catfish sperm. Post-thaw sperm motility, fertilization and hatching rate were detected to evaluate the reliability of sperm cryopreservation. The results demonstrated that Ringer extender and 10% methanol was the best combination for protecting the sperm during freezing in liquid nitrogen by a three-step method and thawing in a water bath at 37 °C for 60 s. In this combination for cryopreservation, sperm maintained the highest post-thaw motility (65 ± 5%), fertilization (90.47 ± 3.67%) and hatching rate (88 ± 4%). And more interestingly, the fertilization and hatching rate were similar to those of fresh sperm (97.55 ± 2.74% and 92 ± 5%). Successful sperm cryopreservation techniques for yellow catfish have been developed for hatchery purpose.     

Cryopreservation of common carp sperm

Experiments were carried out to investigate the effect of five extenders (sucrose, glucose, fructose, KCl and a saline carp sperm extender) and two cryoprotectants (dimethyl-sulfoxide (DMSO) and methanol) on the cryopreservation of common carp sperm. Freezing of sperm using glucose extender and methanol as cryoprotectant resulted in the highest post-thaw motility, fertilization as well as hatching rates (63 ± 9%, 74 ± 15% and 67 ± 17% vs. 87 ± 5%, 84 ± 14% and 69 ± 14% using fresh sperm, respectively). In general, sugar-based extenders combined with methanol as cryoprotectant yielded higher motility, fertilization and hatching rates than ionic extenders in combination with DMSO. The jelly-like agglutination observed after thawing in samples frozen with sugar-based extenders did not reduce fertilization and hatching rates. Frozen–thawed sperm samples were able to successfully fertilize 10 g (8000) eggs.     

Short-term storage and cryopreservation of Urechis unicinctus (Echiura: Urechidae) sperm

In the present study, attempts were made to preserve Urechis unicinctus sperm at 4°C. Cryopreservation procedures were optimized for various cryoprotectants and freezing rates, equilibration times and dilution ratios. During short‐term storage, the motility of undiluted sperm was extended for 6 days of cold storage,and in 70% and 100% artificial seawater only persisted for 2 and 4 days respectively. The survival rate of undiluted sperm was maintained at a high level accordingly. After cryopreservation, the highest motility and survival rate (41.5±2.2%) were obtained in 15% dimethyl sulphoxide (Me₂SO) using a freezing rate of 30°C min^?1. After thawing the sperm cryopreserved in glycerol lost almost all motility. The motility and survival rate of post‐thawing sperm did not show significant differences after 8 and 15 min equilibration using 15% Me₂SO as cryoprotectant; the values were significantly higher than those of 2 min equilibration. Comparisons of motility and survival rate between treatments pooled by dilution ratio showed that the effect of 1:1 ratio (sperm volume to cryoprotectant volume) was best. There was no difference between 1:3 and 1:5, and other ratioswere significantly worse.     

Factors Affecting Cryopreservation Efficiency and Enzyme Activity in Northern Pike,Esox lucius,Sperm

Abstract

Two freezing techniques (straws and pellets), three cryo-protectants (DMSO, glycerol, and DMA) in four concentrations, and several extenders were tested to determine their suitability for cryopreservation of northern pike, Esox iucius, sperm. Activity of aspar-tate aminotransferase (AspAT) and acid phosphatase (AcP) in cryo-preserved milt was determined. Fertilization ability of cryopreserved milt was affected by the freezing technique, by type and concentration of cryoprotectant, as well as by the kind of extender used. These factors also influenced AspAT and AcP activity assayed in cryopreserved sperm. Extender containing 0.6 M sucrose + 15% DMSO + 10% egg yolk was most suitable for cryopreservation of pike sperm in pellet form (90.5% of eyed eggs, as compared to control group, which was 89.1%).     

Cryopreservation of summer flounder,Paralichthys dentatus L., sperm

The summer flounder, Paralichthys dentatus L., is a high‐value species and considerable research has been conducted to determine practices conducive for its culture. As milt can be limited in this species, experiments were conducted to develop a practical sperm cryopreservation protocol for hatchery use. Two dilution ratios (1:2 and 1:4; sperm:extender), 2 diluents (saline and sucrose‐based), 2 cryoprotectants (10% DMSO and 12% glycerol) and 3 freezing rates (?5, ?10 and ?15°C min^?1) were evaluated using differential staining to assess post‐thaw sperm survival. Seven combinations of the factors examined reduced post‐thaw viability by less than 30%. The average viability of sperm from fresh, pooled flounder milt (67.2 ± 2.9%) was not different from that of thawed milt diluted 1:4 with sucrose diluent (10% DMSO) frozen at ?5°C min^?1 (38.4 ± 7.7%) and fertilization and hatch success were not different in trials using fresh or thawed, cryopreserved sperm. From these experiments a practical sperm cryopreservation method was developed, but further refinement of the freezing protocol is necessary to optimize results.