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

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

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.     

Cryopreservation of the milt of the northern pike

Seven published extenders, three thawing media and two thawing temperatures were tested in order to determine their suitability for cryopreservation of northern pike ( Esox lucius L.) sperm. Sperm motility during successive steps of cryopreservation was evaluated. Erdahl and Graham's extender with the addition of egg yolk proved to be the most efficient (maximum hatching rate of 74.5%) when semen was thawed in 120 m m NaCl solution warmed up to 30° C. No correlations between motility of sperm (diluted in extenders or diluted in extenders and then activated with thawing solution) and the subsequent hatching success were observed. The relationship between motility of thawed sperm and its fertilization ability was considerable, but correlation was not significant. Spermatozoa frozen in some extenders were frequently motile after thawing but they were not able to fertilize the eggs, this resulted in a poor hatching rate. Depending on the extender, the addition of yolk induced either positive or detrimental effects on fertilization success.     

The effects of extenders and sperm‐egg ratios on fertilizing ability of cryopreserved testicular sperm of northern pike (Esox lucius L.)

The effects of four different extenders and two sperm‐to‐egg ratios on fertilizing ability of cryopreserved testicular sperm of northern pike (Esox lucius L.) were tested in the present study. Testicular sperm was diluted with each of the four extenders (0.6 m sucrose + 15% DMSO, 0.3 m glucose + 15% DMSO, 0.6 m sucrose + 10% methanol and 0.3 m glucose + 10% methanol, all supplemented with 10% egg yolk and 1.7 g KCl L^?1) and frozen in 0.5‐ml straws at 2 cm above the surface of liquid nitrogen and thawed at 25°C for 30 s. Then 125 μl or 50 μl of frozen‐thawed testicular sperm was poured onto about 1250 eggs for fertilization. The results showed that both sperm‐to‐egg ratio and diluent had no significant influence on cryopreservation efficiency of testicular sperm, whereas cryoprotectant had a significant influence. The highest fertilization rate (92.2%) was obtained from testicular sperm cryopreserved in glucose‐based extender containing 10% methanol at a sperm‐to‐egg ratio of 1 × 10⁶ spermatozoa per egg. The results indicated that glucose‐based extender containing 10% methanol, 10% egg yolk and 1.7 g KCl L^?1 was a useful combination.     

Cryopreservation of epididymal stallion sperm

Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion’s breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen–thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose–egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters.     

In vitro assessment of soybean lecithin and egg yolk based diluents for cryopreservation of goat semen

Soybean lecithin is a suitable plant-based cryoprotectant for freezing ruminant sperm. Optimum level of lecithin was not clear for goat semen cryopreservation. The objective of this study was to investigate the effects of different levels of soybean lecithin in semen extender on post-thaw sperm quality including CASA-motion parameters, viability, plasma membrane integrity and lipid peroxidation. Semen samples were collected from 4 Mahabadi bucks using an artificial vagina. Different concentrations of soy lecithin (SL, 0.5%, 1%, 1.5%, 2% and 2.5% w/v) were compared to 15% (v/v) egg yolk-based extender (TR-EY). No significant difference was observed for sperm progressive motility, viability or plasma membrane integrity in 1.5% SL media (33.8%, 66%, and 62.7%, respectively) and TR-EY medium (35.4%, 67.2%, and 64.9%, respectively). Sperm motion characteristics (VAP, VSL, VCL, ALH and LIN) and rapid spermatozoa were improved with extender containing 1% and 1.5% SL, compared to TR-EY extender. Furthermore, egg yolk produced significantly higher malondialdehyde (4.02 ± 0.21) than other groups. Results suggest that the optimal lecithin concentration in the semen extender was 1.5% and also soy lecithin can substitute for egg yolk during cryopreservation for caprine sperm.     

几种冷冻稀释液与单胺类防冻剂对中缅树鼩精子冷冻存活率的影响

该文实验精子采自昆明地区经笼养驯化的树鼩(Tupaia belangeri),检测其冷冻前后运动度、顶体完整率以及检测部分冷冻精子的受精能力。实验一:选用8种已报道的冷冻稀释液TTE、TCG、TCF、TTG、BWW、BTS、DM、SR稀释鲜精,并添加0.4mol/LDMSO,4℃预冷平衡2h后,TTE、DM和SR稀释液的精子的运动度与鲜精无差别(P>0.05),其余处理组均显著下降(P<0.05)。冷冻复苏后,各组的运动度显著低于预冷平衡处理后的运动度(P<0.05);DM组的复苏运动度显著高于其他稀释液组(P<0.05),BWW组最低(P<0.05)。对于顶体完整率,与鲜精相比,4℃平衡2h后,TTE和DM组精子的顶体完整率显著高于BWW、BTS和SR组(P<0.05)。冷冻复苏后,DM组精子的顶体完整率显著高于其它(除了TTE)冷冻组(P<0.05)。实验二:在DM稀释液基础上分别添加4种浓度0.2、0.4、0.8和1.2mol/L的二甲基甲酰胺(DF)、甲酰胺(F)、二甲乙酰胺(DA)和乙酰胺(A)以及0.4mol/LDMSO,经过预冷平衡处理后,与鲜精相比,各防冻剂组的精子运动度没有下降(P>0.05);冷冻解冻后,各冷冻组精子的运动度显著低于预冷平衡处理后的精子运动度(P<0.05);0.8mol/LDF和0.4mol/LDMSO组精子的运动度显著高于其它冷冻组(P<0.05)。对于顶体完整率,预冷平衡处理后各高浓度组的比率显著下降;冷冻复苏后,0.4mol/LF和0.4mol/LDF组精子的顶体完整率相对较高。实验三,人工授精实验中,DM+0.8mol/LDF冷冻精子的受精率为16.7%,DM+0.4mol/LDMSO的受精率为50.0%。以上实验结果提示,含卵黄的非离子冷冻稀释液对树鼩精子冷冻保护效果好,但单胺类防冻剂的防冻效果还需进一步深入研究。     

Effects of glycerol additions on post-thaw fertility of frozen rainbow trout sperm, with an emphasis on interaction between extender and cryoprotectant

The study was conducted to evaluate the effects of different extenders, cryoprotectants and glycerol additions on the post‐thaw fertility and interactions between extenders and cryoprotectants during cryopreservation. Semen was collected by abdominal stripping from 30 adult male rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and diluted with three different extenders (Erdahl–Graham, Lahnsteiner, glucose‐based) containing 15% DMA, 15% DMSO, 15% DMA + 1% glycerol and 15% DMSO + 1% glycerol at a ratio of 1 : 2. Diluted samples were frozen as 0.1 ml pellets directly on dry ice (solid CO₂, −79°C). Eggs were pooled from 10 females. Fertilization was applied in plastic dishes and 600 eggs were used in each fertilization trial. Pellets were thawed in their own extenders (30°C) at a ratio of 1 : 10. 0.3% NaCl was used for activating motility. Sperm–egg ratio was approximately 3 × 10⁶ sp per egg. Experimental success was determined as the percentage of eyed‐eggs 25 days after fertilization. The highest eyed‐egg rate (49.3%) was obtained from semen frozen with glucose‐based extender containing 15% DMSO + 1% glycerol. Our results indicate that the glucose‐based extender containing DMSO is a useful combination, but that the addition of glycerol does not have a positive effect on post‐thaw fertility, and that interaction of the extender‐cryoprotectant is also important in the cryopreservation of rainbow trout semen.     

Rhesus monkey sperm cryopreservation with TEST-yolk extender in the absence of permeable cryoprotectant

Recently, there has been increased interest in ultra-rapid freezing with mammalian spermatozoa, especially for vitrification in the absence of cryoprotectants. Sperm cryopreservation in non-human primates has been successful, but the use of frozen-thawed sperm in standard artificial insemination (AI) remains difficult, and removal of permeable cryoprotectant may offer opportunities for increased AI success. The present study intended to explore the possibility of freezing rhesus monkey sperm in the absence of permeable cryoprotectants. Specifically, we evaluated various factors such as presence or absence of egg yolk, the percentage of egg yolk in the extenders, and the effect of cooling and thawing rate on the success of freezing without permeable cryoprotectants. Findings revealed that freezing with TEST in the absence of egg yolk offers little protection (<15% post-thaw motility). Egg yolk of 40% or more in TEST resulted in decreased motility, while egg yolk in the range of 20-30% yielded the most motile sperm. Cooling at a slow rate (29 °C/min) reduced post-thaw motility significantly for samples frozen with TEST-yolk alone, but had no effect for controls in the presence of glycerol. Similarly, slow thawing in room temperature air is detrimental for freezing without permeable cryoprotectant (<2% motility). In addition to motility, the ability of sperm to capacitate based on an increase in intracellular calcium levels upon activation with cAMP and caffeine suggested no difference between fresh and frozen-thawed motile sperm, regardless of treatment. In summary, the present study demonstrates that ejaculated and epididymal sperm from rhesus monkeys can be cryopreserved with TEST-yolk (20%) in the absence of permeable cryoprotectant when samples were loaded in a standard 0.25-mL straw, cooled rapidly in liquid nitrogen vapor at 220 °C/min, and thawed rapidly in a 37 °C water bath. This study also represents the first success of freezing without permeable cryoprotectant in non-human primates.     

Evaluation of extenders and cryopreservatives for cooling and cryopreservation of spermatozoa from the western spotted skunk (Spilogale gracilis)

Experiments were conducted to develop a suitable protocol for cryopreservation of spotted skunk semen. Semen was collected by electroejaculation of captive male skunks (n = 16) from late January through late November. In the first experiment, fresh semen was diluted in either TEST (n = 10), TRIS (n = 9), or BF5F (n = 7) extenders and maintained at 4°C for 16 hr. Sperm motility in these extenders was not significantly different before cooling (P = 0.71), but samples diluted with BF5F exhibited significantly lower sperm motility than the other extenders at all time points after cooling (P < 0.05). In the second experiment, fresh semen was diluted in TEST containing either 3, 5, or 10% DMSO or 3, 5, or 10% glycerol as a cryopreservative. These samples were cooled to 4°C and frozen in 0.25 ml French straws on dry ice. Some samples containing 5% DMSO or 5% glycerol (n = 4), were also frozen on dry ice as pellets. Frozen samples were maintained in liquid nitrogen. Fresh samples had significantly greater sperm motility in dimethyl sulfoxide (DMSO) than in glycerol (P < 0.05), while frozen and thawed samples had the highest motility in 5 or 10% DMSO or 10% glycerol. Samples frozen in French straws had significantly greater sperm motility after freezing and thawing than those frozen by the pellet method (P < 0.05). Optimum cryoprotection was achieved with the TEST extender containing 5 or 10% DMSO, when used in conjunction with French straws. © 1992 Wiley-Liss, Inc.     

Effect of cryoprotectants on release of various enzymes from buck spermatozoa during freezing

Semen samples from 12 bucks Were extended with 10 different extenders containing glycerol, DMSO, glycerol + DMSO, and glycerol + lactose in varying concentrations as cryoprotective agents. The activities of acrosin, hyaluronidase, alkaline phosphatase (AKP), aspartate aminotransferase (AST), alanine amino transferase (ALT) and lactic dehydrogenase (LDH) were assayed in equilibrated (Prefreeze) and frozen thawed (Postfreeze) semen samples. Significantly (P < 0.01) higher intracellular activity of acrosin was recorded in semen samples extended with lactose than with the other extenders, with the maximum being with Tris yolk glycerol lactose (TYGL(180)). Effects of extenders on acrosin activity were significant (P < 0.01) at both of the pre-and postfreeze stages. However, extracellular activities of hyaluronidase, alkaline phosphatase, transaminases (AST and ALT), and lactic dehydrogenase were significantly higher in extenders containing DMSO than lactose. Leakage of these enzymes was found to increase from the prefreeze to the post freeze stage.     

Gene banking of the neotropical fish Leporinus obtusidens (Valenciennes, 1836): a protocol to freeze its sperm in the field

A practical sperm cryopreservation protocol using a dry-shipper and a diluent of simple composition is described for the neotropical fish Leporinus obtusidens (Valenciennes, 1836). The cooling rate of the dry-shipper and its period of useful time, established under laboratory conditions, were respectively 25.7-30.8 degrees C/min (between 0 and -60 degrees C) and 9 days after charging. Sperm donors were selected on the basis of their hyperemic genital papilla and the ability to ooze milt under gentle manual pressure, during the reproductive months of November to January. Milt volume (1.3+/-0.3 mL; n=9 fish), fresh sperm motility rate (93.3+/-2.5%; n=6 fish), and sperm concentration (10.9+/-3.0 x 10(9)spermatozoa/mL of milt) were obtained. The sperm cryopreservation experiments were conducted with the following cryoprotectants (all at 10%, before mixing with milt): dimethyl sulphoxide (DMSO; n=10 fish), methanol (n=6 fish), propanediol (n=6 fish) and ethylene glycol (n=5 fish). Glucose (5%) and hen's egg yolk (10%) made up the diluents containing DMSO, ethylene glycol or propanediol. Milk powder (10%) replaced hen's egg yolk in the diluent containing methanol. Distilled water (up to 100%) completed the diluent solutions. Milt freezing (in 0.5-mL straws) was performed in the dry-shipper after 1:5 (milt:diluent) dilution. Thawed sperm cryopreserved in DMSO-containing diluent and activated by 119 mM NaHCO(3) gave the highest motility rate (62+/-14%). The fertilizing capacity of L. obtusidens sperm was tested using the combination of DMSO-containing diluent as the cryoprotectant and 119 mM NaHCO(3) as the activating solution. Oocytes were obtained from artificial spawning and fertilized with different proportions of spermatozoa. The greatest rate of fertilization (74%) occurred when the ratio of about 112,000 motile spermatozoa:oocyte was used. Thus, a protocol to freeze L. obtusidens sperm can be elaborated as follows. Milt (<1.5 mL fish(-1)) was readily available only in November to January; a simple solution, composed of 10% DMSO (concentration before adding milt), 5% glucose, and 10% hen yolk egg, in distilled water, was used as sperm diluent; cooling rate of 25-30 degrees C/min, yielded in a portable dry-shipper, was adequate to freeze diluted milt (1:5; milt:diluent), in 5-mL straws; about 112,000 thawed motile spermatozoa:oocyte activated by 119 mM NaHCO(3) assured a fertilization rate of 74%.     

Usefulness of powdered and fresh egg yolk for cryopreservation of Zebu bull spermatozoa

This experiment was designed to compare powdered egg yolk with fresh egg yolk in an extender for cryopreservation of Zebu bull semen. Sperm motility, plasma membrane integrity and viability were assessed at different stages of cryopreservation (post-dilution, pre-freezing and post-thawing). Sperm plasma membrane integrity remained similar at all the stages of cryopreservation. Sperm motility and viability were significantly higher after thawing in the extender containing powdered egg yolk. In conclusion, powdered egg yolk may be used in an extender for the cryopreservation of Zebu bull spermatozoa.     

Cryopreservation of collared peccaries (Tayassu tajacu) semen using a powdered coconut water (ACP-116c) based extender plus various concentrations of egg yolk and glycerol

The objective was to determine the effectiveness of a powdered coconut water-based extender (ACP-116c), plus various concentrations of egg-yolk and glycerol, as an alternative for cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were apportioned into aliquots that were diluted in Tris plus 10% egg yolk and 3% glycerol, or in ACP-116c plus 10 or 20% egg yolk and 1.5 or 3% glycerol. Samples were frozen in liquid nitrogen and, after 1 mo, thawed at 37 °C for 1 min. After thawing, samples were evaluated as reported for fresh semen, and also for sperm membrane integrity (fluorescent probes) and kinematic parameters (computerized analysis). Results were presented as means ± SEM. Freezing and thawing decreased sperm characteristics relative to fresh semen. Overall, ACP-116c plus 20% egg yolk and 3% glycerol provided better (P < 0.05) sperm motility and kinetic rating (48 ± 6.1% and 2.8 ± 0.2, respectively) after thawing than Tris extender (30.4 ± 5.7% and 2.4 ± 0.2). However, there were no differences (P > 0.05) among treatments with regard to the other sperm characteristics. Based on computerized motion analysis, total (26.5 ± 5.9%) and progressive (8.1 ± 2.2%) motility were best preserved (P < 0.05) with the above-mentioned treatment. In conclusion, a coconut water-based extender, ACP-116c, plus 20% egg yolk and 3% glycerol, was effective for cryopreservation of semen from collared peccaries.     

Effects of Soy Lecithin Extender on Dog Sperm Cryopreservation

Semen cryopreservation is an essential biotechnology in canine reproduction and during the cryopreservation process commonly egg yolk are used. The discrepancy in the egg yolk composition and the potential risk of disease dissemination are obstacles for semen exportation and use. Therefore, studies aiming to substitute egg yolk are extremely important. In this context, soy lecithin contains a low-density lipoprotein fraction, is an interesting alternative. Thus, the objective of this study was to compare extenders based on soy lecithin (several concentrations and forms) with egg yolk during the cryopreservation process of dog sperm. For this purpose, we used twelve dogs. Semen was evaluated at different time points (after refrigeration, glycerolization, and thawing), by motility analysis (CASA) and functional tests (e.g., membrane integrity—eosin/nigrosin, acrosome integrity—fast green/Bengal rose, mitochondrial activity—3’3 diaminobenzidine, Chromatin susceptibility to acid-induced denaturation—SCSA, and susceptibility to oxidative stress—thiobarbituric acid reactive substances). The results indicated that egg yolk and lower concentrations of lecithin had similar effects on mitochondrial activity and motility. Thus, soy lecithin is a potentially viable alternative to egg yolk for the cryopreservation of dog semen.     

Semen characteristics, cryopreservation, and successful artificial insemination in the Blue rock pigeon (Columba livia)

The present study was undertaken in the Blue rock pigeon (Columba livia) to evaluate the annual semen characteristics, to identify a suitable extender for semen short-term storage, to determine a protocol for cryopreservation of semen and finally to check whether intracloacal insemination would lead to the birth of a chick. Semen characteristics such as semen volume, sperm concentration, sperm motility, and percentage of normal spermatozoa were maximum during the monsoon season. TALP was observed to be the most suitable semen extender and the sperm survived best at 37 degrees C at a dilution of 1:100 in TALP. Further, cryopreservation studies on pigeon semen indicated that 8% DMSO with or without egg yolk (20%) proved to be a better cryoprotectant compared to glycerol and polyethylene glycol. In addition, the slow freezing protocol was better than the fast-freezing protocol and about 40% of the cryopreserved spermatozoa were motile following thawing. Computer-aided semen analysis indicated that pigeon spermatozoa were extremely active immediately after dilution in TALP and exhibited linear trajectories persisting up to 9h. But, with time there was a time-dependent decrease in the velocity parameters (VAP, VSL, and VCL). Cryopreserved spermatozoa following thawing also exhibited linear trajectories but had reduced velocity as evident from the significant decrease in VAP, VSL, and VCL. Further, artificial inseminations using fresh semen resulted in 45% fertilization and birth of a live chick.     

Cryopreservation of domestic animal sperm cells

Sperm cells are the endpoint of male spermatogenesis and have particular anatomic and metabolic features. Sperm cryopreservation and storage currently require liquid nitrogen or ultralow refrigeration methods for long or short term storage, which requires routine maintenance and extensive space requirements. Conserving sperms have several purposes such as artificial reproductive technologies (ART), species conservation and clinical medicine. The combinations of storage temperature, cooling rate, chemical composition of the extender, cryoprotectant concentration, reactive oxygen species (ROS), seminal plasma composition and hygienic control are the key factors that affect the life-span of spermatozoa. Sperm preservation protocols vary among animal species owing to their inherent particularities that change extenders used for refrigeration and freezing. Extenders for freezing sperm cells contain buffers, carbohydrates (glucose, lactose, raffinose, saccharose and trehalose), salts (sodium citrate, citric acid), egg yolk and antibiotics. The use of different cryoprotectants, like trehalose or glycerol, as well as different concentrations of egg yolk and other constituents in semen extenders are being studied in our laboratory. Several cooling rates have been tested to freeze sperm cells. The use of faster rates (15–60°C/min) gives rise to best sperm survivals after freezing–thawing, but more studies are needed to find the adequate cooling rates for each animal species. Sheep and goat males of some native breeds are being used in studies performed in EZN. Semen from those males has been frozen and stored as part of the Portuguese Animal Germplasm Bank. In small ruminants, individual variations in the quality of frozen semen have been observed, suggesting specific differences in sperm susceptibility to freezing methods, particularly obvious in goat males. Best quality frozen semen from small ruminants is being used in cervical artificial insemination studies aiming to increase productive parameters in selected flocks. Presented at the International Consensus Meeting “New Horizons in Cell and Tissue Banking” on May 16–20, 2007, Vale de Santarém, Portugal.     

Effect of relaxin on cryopreserved beef bull semen characteristics

This study aimed to improve the quality of cryopreserved beef bull (Piedmontese) semen by incorporation of relaxin in diluted semen before cryopreservation procedures. Semen samples were collected from 4 proven fertile bulls, using artificial vagina, once per week for 8 consecutive weeks and pooled together then diluted with Bullxcell® extender, and supplemented with different concentrations of relaxin (0 (control), 25, 50 and 100 ng/ml) before cooling, equilibration and freezing procedures. Frozen semen was thawed and assessed for motility by Computer-Assisted Sperm Analysis and vitality parameters such as acrosome, plasma membrane and DNA integrities, apoptosis, mitochondrial membrane potential, mucus penetration and SOD activity. The developmental potential of bovine embryos produced in vitro by using relaxin-treated was also investigated. In the present study, 50 and 100 ng/ml relaxin incorporation in extended bull semen before cryopreservation induced a reduction of sperm motility immediately after thawing (0h), whereas, during long incubation periods (1–2 h), relaxin showed a significant positive effect on sperm quality by improving the sperm motility and velocity parameters. Interestingly, sperm vitality was improved by 25 and 100 ng/ml relaxin and the blastocyst developmental rate was significantly increased in the 25 ng/ml relaxin group compared with controls (52/118, 44.0% vs. 32/116, 27.6%, respectively). These findings suggest a potential use of relaxin at the doses tested in the present study as an additive in the cryopreservation media of bull semen to improve sperm quality.     

The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability

The Sperm Class Analyzer was used to investigate the effect of freeze-thawing procedure on Florida buck sperm head morphometry, and to relate possible changes in sperm head dimensions to cryopreservation success. Semen samples (n=76) were frozen with tris and milk-based extenders and thawed. Sperm quality samples (motility, morphology, acrosome), and sperm head morphometric values (length, width, area, perimeter, ellipticity) were compared between fresh and frozen-thawed samples. Sperm freezability was judged according to the sperm quality parameters assessed. Fertility data was obtained after artificial insemination with cryopreserved semen. Cryopreservation success was different between freezing methods. Sperm head dimensions were significantly (p<0.05) smaller in cryopreserved tris and milk spermatozoa respectively than in those of the fresh samples. The sperm head morphometric parameters that had changed after cryopreservation were lower in suitable semen samples after thawing and with successful pregnancies after artificial insemination. These data suggest that changes in sperm head morphometry might reflect spermatozoa injury occurred during cryopreservation.     

Comparison of extenders,dilution ratios and theophylline addition on the function of cryopreserved walleye semen

Walleye (Stizostedion vitreum) is a species of interest for the diversification of North American aquaculture production, and semen cryopreservation is of particular value to this effort. To test the hypothesis that adjusting semen extender composition and dilution ratio increases sperm quality after thawing, three extenders (Ext1, Ext2, Ext3; all with DMSO as a cryoprotectant) and three dilution ratios (semen/extender: 1:5, 1:9, 1:15) were screened. The best results were obtained when semen was diluted at a 1:15 ratio with Ext 1, Rathbun extender supplemented with 7% DMSO, 4 mg/ml BSA and 7.5 mg/ml ProFam, a soy-based protein (P = 0.05, n = 6). This method resulted in 46 +/- 3% motility of the thawed spermatozoa and a mortality rate of 39 +/- 4% whereas Ext2 and Ext3 resulted in motility rates of only 10 and 5%. respectively. To test an additional hypothesis that phosphodiesterase inhibition improves sperm function, we assessed the fertility of sperm frozen in optimal conditions and thawed in the presence or absence of 5 mM theophylline (n = 5). The best result was achieved in water without theophylline, with fertilization rates ranging from 28.51 +/- 6.84 to 59.02 +/- 1.06% eyed-up stage, and theophylline reduced fertility (P < 0.05). Our data show that Ext1 at a dilution ratio of one part semen to 15 parts extender should be used for walleye semen cryopreservation and that the fertilizing media does not benefit from theophylline supplementation.