Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific Oysters, Crassostrea gigas

Despite some 26 published reports addressing oyster sperm cryopreservation, systematic factor optimization is lacking, and sperm cryopreservation has not yet found application in aquaculture on a commercial scale. In this study, the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization of shipped sperm samples from diploid oysters. Evaluation of cooling rates revealed an optimal rate of 5 degrees C/min to -30 degrees C followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations suggested that a low concentration (2%) of polyethylene glycol (FW 200) was effective in retaining post-thaw motility and fertilizing capability when combined with permeating cryoprotetcants such as dimethyl sulfoxide (DMSO), methanol (MeOH), and propylene glycol (P-glycol). However, polyethylene glycol alone was not as effective as MeOH, DMSO, and P-glycol when using the same methods. The highest post-thaw motility (70%) and percent fertilization (98%) were obtained for samples cryopreserved with 6% MeOH. However, this does not exclude other cryoprotectants such as DMSO or P-glycol identified as effective agents in other studies. There was no significant difference in post-thaw motility between straw sizes of 0.25- and 0.5-ml. Equilibration time (exposure to cryoprotectant) of 60 min could be beneficial when the cryoprotectant concentration is low and solution is added in a step-wise fashion at low temperature. Differences in post-thaw sperm quality (e.g., motility or percent fertilization) among individual males were evident in this research. As a consequence, a generalized classification describing males with different tolerances (broad, intermediate, and narrow) to cryopreservation was developed. This classification could be applied to strain or species differences in tolerances to the cryopreservation process. The present study demonstrated that oyster sperm could be collected and shipped chilled to another facility for cryopreservation, and that it could be shipped back to the hatchery for fertilization performed at a production scale yielding live larvae with >90% fertilization. Given the existence of facilities for commercial-scale cryopreservation of dairy bull sperm, the methods developed in the present study for oysters provide a template for the potential commercialization of cryopreserved sperm in aquatic species.     

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: (1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me₂SO), N,N-dimethylacetamide (DMA), N,N-dimethyl formamide (DMF), and glycerol with concentrations of 5%, 10%, and 15% for 60 min of incubation at 4 °C; (2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and (3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5% and 10%) did not change the sperm motility after 30 min; Me₂SO, DMA, and DMF (10% and 15%) and glycerol (5%, 10% and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 to 25 °C/min) and were compared to Me₂SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ? 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ?10% which was significantly lower than that of methanol and ME. With Me₂SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ? 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.     

Systematic factor optimization for sperm cryopreservation of tetraploid Pacific oysters, Crassostrea gigas

The availability of tetraploid Pacific oysters provides a unique opportunity for comparative studies of sperm cryopreservation between diploids and tetraploids. In parallel to studies with sperm from diploid oysters, this study reports systematic factor optimization for sperm cryopreservation of tetraploid oysters. Specifically, this study evaluated the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), and straw size. Similar to sperm from diploids, the optimal cooling rate was 5 degrees C/min to -30 degrees C, followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations showed that a combination of the cryoprotectants 6% polyethylene glycol/4% propylene glycol and 6% polyethylene glycol/4% dimethyl sulfoxide yielded consistently high post-thaw motility. A long equilibration (60 min) yielded higher percent fertilization, and confirmed that extended equilibration could be beneficial when low concentrations of cryoprotectant are used. There was no significant difference in post-thaw motility between straw sizes of 0.25 and 0.5 mL. Despite low post-thaw fertilization (<10%) in general for sperm from tetraploids, optimized protocols in the present study effectively retained post-thaw motility for sperm from tetraploid oysters. This study confirmed that sperm from tetraploid Pacific oysters were more negatively affected by cryopreservation than were those of diploids. One possible explanation is that sperm from these two ploidies are different in their plasma membrane properties (e.g., structure, permeability, and elasticity), and the plasma membrane of sperm from tetraploids is more sensitive to cryopreservation effects. The fact that combinations of non-permeating and permeating cryoprotectants improved post-thaw motility in sperm from tetraploids provided presumptive evidence for this interpretation.     

Development of a spermatogonia cryopreservation protocol for blue catfish,Ictalurus furcatus

Sustainability of channel catfish, Ictalurus punctatus ♀ × blue catfish, Ictalurus furcatus ♂ hybrid aquaculture relies on new innovative technologies to maximize fry output. Transplanting spermatogonial stem cells (SSCs) from blue catfish into channel catfish hosts has the potential to greatly increase gamete availability and improve hybrid catfish fry outputs. Cryopreservation would make these cells readily accessible for xenogenesis, but a freezing protocol for blue catfish testicular tissues has not yet been fully developed. Therefore, the objectives of this experiment were to identify the best permeating [dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, methanol] and non-permeating (lactose or trehalose with egg yolk or BSA) cryoprotectants, their optimal concentrations, and the best freezing rates (−0.5, −1.0, −5.0, −10 °C/min until −80 °C) that yield the highest number of viable type A spermatogonia cells. Results showed that all of these factors had significant impacts on post-thaw cell production and viability. DMSO was the most efficient permeating cryoprotectant at a concentration of 1.0 M. The optimal concentration of each cryoprotectant depended on the specific cryoprotectant due to interactions between the two factors. Of the non-permeating cryoprotectants, 0.2 M lactose with egg yolk consistently improved type A spermatogonia production and viability beyond that of the 1.0 M DMSO control. The overall best freezing rate was consistent at −1 °C/min, but similar results were obtained using −0.5 °C/min. Overall, we recommend cryopreserving blue catfish testicular tissues in 1.0 M DMSO with 0.2 M lactose and egg yolk at a rate of either -0.5 or −1 °C/min to achieve the best cryopreservation outcomes. Continued development of cryopreservation protocols for blue catfish and other species will make spermatogonia available for xenogenic applications and genetic improvement programs.     

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.     

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.     

Semen cryopreservation in the Indian rhinoceros (Rhinoceros unicornis)

The objective was to identify an extender and cryoprotectant combination for Indian rhinoceros (Rhinoceros unicornis) sperm that yielded high post-thaw sperm quality. Male Indian rhinoceroses (n = 6; 7.5-34 yr old) were anesthetized and subjected to a regimented electroejaculation procedure (75-100 mAmps; 4-10 volts; 7-150 stimuli; total of 10 electroejaculation procedures). High quality semen fractions from each ejaculate were divided into four aliquots and a 2 x 2 factorial design used to compare the effect of two sperm extenders (standard equine [EQ] and skim milk-egg-yolk-sugar [SMEY]), and two cryoprotectants (glycerol and dimethylsulfoxide [DMSO]). Cyropreserved samples were thawed and assessed for motility, viability and acrosome integrity over time. Electroejaculate fractions processed for cryopreservation had high sperm concentration (516 × 10⁶/mL) and motility (79%). Post-thaw sperm characteristics were higher (P < 0.05) when semen was cryopreserved in EQ versus SMEY. Post-thaw motility of sperm cyropreserved in EQ averaged 50-55% compared to 22-37% in SMEY, with no significant differences in sperm characteristics of samples cyropreserved in glycerol and DMSO. In conclusion, sperm collected from Indian rhinoceroses via electroejaculation were cryopreserved using EQ extender with either glycerol or DMSO; post-thaw quality was adequate for use in assisted reproductive procedures.     

Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution

The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs–Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3 h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1 M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm.     

Effect of glycerol and dimethyl sulfoxide on cryopreservation of rhesus monkey (Macaca mulatta) sperm

Glycerol and dimethyl sulfoxide (DMSO) are widely used as penetrating cryoprotectants in the freezing of sperm, and various concentrations are applied in different species and laboratories. The present study aimed to examine the effect of these two cryoprotectants at different concentrations (2%, 5%, 10%, and 15% glycerol or DMSO) on rhesus monkey sperm cryopreservation. The results showed that the highest recovery of post-thaw sperm motility, and plasma membrane and acrosome integrity was achieved when the sperm was frozen with 5% glycerol. Spermatozoa cryopreserved with 15% DMSO showed the lowest post-thaw sperm motility, and spermatozoa cryopreserved with 15% glycerol and 15% DMSO showed the lowest plasma membrane integrity among the eight groups. The results achieved with 5% glycerol were significantly better for all parameters than those obtained with 5% DMSO. The functional cryosurvival of sperm frozen with 5% glycerol was further assessed by in vitro fertilization (IVF). Overall, 85.7% of the oocytes were successfully fertilized, and 51.4% and 5.7% of the resulting zygotes developed into morulae and blastocysts, respectively. The results indicate that the type and concentration of the penetrating cryoprotectant used can greatly affect the survival of rhesus monkey sperm after it is frozen and thawed. The suitable glycerol level for rhesus monkey sperm freezing is 5%, and DMSO is not suitable for rhesus monkey sperm cryopreservation.     

Cryopreservation effects on sperm function and fertility in two threatened crane species

The capacity to cryopreserve semen from captive cranes facilitates production of offspring from behaviorally incompatible or geographically separated pairs, and allows for long-term preservation of valuable genetic materials. The present study sought to develop effective cryopreservation protocols for whooping (Grus americana) and white-naped (Grus vipio) cranes, through examining the influences of two permeating (DMA and Me₂SO) and one non-permeating (sucrose) cryoprotectants, as well as vitamin E on post-thaw sperm survival. In Study 1, ejaculates (whooping: n = 10, white-naped: n = 8) were collected and cryopreserved in one of six cryo-diluents (crane extender with: DMA; DMA+0.1M sucrose; Me₂SO; Me₂SO+0.1M sucrose; 0.1M sucrose; 0.2M sucrose) using a two-step cooling method. Frozen samples were thawed and assessed for overall motility, motion characteristics, membrane integrity, morphology, and ability to bind to the inner perivitelline membrane (IPVM). In Study 2, whooping crane ejaculates (n = 17) were frozen in crane extender containing Me₂SO alone or with vitamin E (5 μg/mL or 10 μg/mL). Frozen samples were thawed and assessed as in Study 1, except the binding assay. White-naped crane sperm were more tolerant to cryopreservation than whooping crane (15% vs 6% post-thawed motility). In both species, sperm cryopreserved in medium containing Me₂SO alone displayed higher post thaw survival and ability to bind to IPVM than the other cryodiluent treatments. Vitamin E supplementation exerted no benefits to post thaw motility or membrane integrity. The findings demonstrated that there was species specificity in the susceptibility to cryopreservation. Nevertheless, Me₂SO was a preferred cryoprotectant for sperm from both whooping and white-naped cranes.     

Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski’s horse (Equus ferus przewalskii)

Two studies were conducted to understand sperm cryosensitivity in an endangered equid, the Przewalski’s horse (Equus ferus przewalski), while testing the cryoprotectant ability of formamides. The first assessed the toxicity of permeating cryoprotectants (glycerol, methylformamide [MF] and dimethylformamide [DMF]) to Przewalski’s horse spermatozoa during liquid storage at 4 °C. The second examined the comparative influence of three diluents (with or without formamides) on cryosurvival of sperm from the Przewalski’s versus domestic horse. When Przewalski’s horse spermatozoa were incubated at 4 °C in INRA 96 with differing concentrations of glycerol, MF or DMF or a combination of these amides, cells tolerated all but the highest concentration (10% v/v) of MF alone or in combination with DMF, both of which decreased (P < 0.05) motility traits. There was no effect of cryoprotectants on sperm acrosomal integrity. In the cryosurvival study, average sperm motility and proportion of cells with intact acrosomes in fresh ejaculates were similar (P > 0.05) between the Przewalski’s (67%, 84%, respectively) and domestic (66%, 76%) horse donors. Sperm from both species were diluted in lactose–EDTA–glycerol (EQ), Botu-Crio (BOTU; a proprietary product containing glycerol and MF) or SM (INRA 96 plus 2% [v/v] egg yolk and 2.5% [v/v] MF and DMF) and then frozen over liquid nitrogen vapor. After thawing, the highest values recovered for total and progressive sperm motility, acrosomal integrity and mitochondrial membrane potential were 42.4%, 21.8%, 88.7% and 25.4 CN (CN = mean JC-1 fluorescence intensity/cell on a channel number scale), respectively, in the Przewalski’s and 49.3%, 24.6%, 88.9% and 25.8 CN, respectively, in the domestic horse. Although sperm progressive motility and acrosome integrity did not differ (P > 0.05) among treatments across species, mitochondrial membrane potential was higher (P < 0.05) in both species using EQ compared to BOTU or SM media. Additionally, Przewalski’s stallion sperm expressed higher (P < 0.05) post-thaw total motility in BOTU and SM compared to EQ, whereas there were no differences among freezing diluents in the domestic horse. In summary, Przewalski’s stallion sperm benefit from exposure to either MF or DMF as an alternative cryoprotectant to glycerol. Overt sperm quality appears similar between the Przewalski’s and domestic horse, although the total motility of cells from the former appears more sensitive to certain freezing diluents. Nonetheless, post-thaw motility and acrosomal integrity values for Przewalski’s horse spermatozoa mimic findings in the domestic horse in the presence of INRA 96 supplemented with 2% (v/v) egg yolk and a combined 2.5% concentration of MF and DMF.     

Oxidative stress in zebrafish (Danio rerio) sperm

Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line.     

Cryopreservation of sperm of two European percid species, the pikeperch (Sander lucioperca) and the Volga pikeperch (S. volgensis)

Experiments were carried out on sperm cryopreservation of two European percid fish species, the pikeperch (Sander lucioperca) and the Volga pikeperch (S. volgensis). Two experiments were conducted on pikeperch sperm. In the first, the effects of three extenders (Glucose, KCl, Sucrose) and two cryoprotectants (dimethyl-sulfoxide: DMSO, methanol: MeOH) were tested on motility and fertilization. In the second, the effects of two dilution ratios (1 : 1, 1: 9) and two cryoprotectants (DMSO, MeOH) on hatching were investigated. In the experiment on Volga pikeperch the suitability of using cryopreservation for fertilization was investigated. In the first experiment on pikeperch the highest post-thaw motility (28 +/- 21%) and fertilization rate (43 +/- 12%) was found with DMSO as cryoprotectant in combination with Glucose extender. In the second, the highest hatch rate (41 +/- 22%) was observed with MeOH as cryoprotectant and 1 : 1 sperm dilution ratio, however no significant difference was found among the results. In the experiment on Volga pikeperch hatch rates with cryopreserved sperm (60 +/- 2%) did not significantly differ from the control (60 +/- 6%). Contamination of sperm with urine seems to be a key problem in the success of sperm cryopreservation of these species.     

Cryopreservation of rabbit spermatozoa using acetamide in combination with trehalose and methyl cellulose

Glycerol is not an effective cryoprotectant for rabbit spermatozoa; therefore, rabbit spermatozoa were used as a model for developing cryopreservation procedures for other cell types which also freeze poorly when glycerol is used as the cryoprotectant. Experiments were conducted to 1) compare several published protocols for cryopreserving rabbit spermatozoa; 2) determine if removal of seminal granules, required for flow cytometry analysis, affects the motility of rabbit spermatozoa; and 3) determine if using a combination of cell permeating cryoprotectants (acetamide) with cell nonpermeating cryoprotectants (trehalose and methyl cellulose; MC), can increase the recovery of viable rabbit spermatozoa after cryopreservation. Media containing acetamide as a cryoprotectant were found to be most effective for rabbit spermatozoa. The cryoprotectants ethylene glycol, dimethylsulfoxide and glycerol were not effective for cryopreserving rabbit spermatozoa. Second, rabbit spermatozoa could be centrifuged through a Percoll gradient composed of equal volumes of Prcoll and a HEPES-buffered sperm medium. This centrifugation removed all seminal granules without affecting the percentage of motile spermatozoa after initial sperm dilution (85 vs 74%) or after cryopreservation (35 vs 30%), when sperm were either centrifuged or not centrifuged, respectively. The substitution of trehalose in the cryopreservation medium for raffinose did not improve recovery of motile cells following cryopreservation (P > 0.05). However, addition of MC resulted in higher percentages of motile sperm after cryopreservation (43 vs 31%; P < 0.05). In addition, sperm viability and acrosomal integrity were simultaneously evaluated using flow cytometry. The addition of both trehalose and MC to media containing acetamide resulted in higher percentages of live acrosome-intact cells than acetamide alone (53 vs 37%; P < 0.05). These results indicate that a combination of permeating and nonpermeating cryoprotectants (acetamide, trehalose and MC) were more effective in preserving rabbit spermatozoa than acetamide alone and that analyzing multiple sperm characteristics, by flow cytometry, can assess sperm damage not detected by analyzing sperm motion characteristics.     

Sperm cryopreservation of Prochilodus brevis using different concentrations of non-permeable cryoprotectants

The action of substances with non-permeable cryoprotectant potential, besides glucose, has not yet been studied for the species Prochilodus brevis. The objective of this work was to evaluate the action of four non-permeable cryoprotectants on this species sperm cryopreservation. Five pools were cryopreserved in a solution of 5% glucose and 10% dimethyl sulfoxide (Me₂SO) associated or not (control) with cryoprotectants egg yolk (5, 10 or 12%), soy lecithin (2.5, 7.5 or 10%), sucrose (5, 10 or 20%) and lactose (5, 8 or 15%). After thawing, samples were evaluated for sperm kinetics (total motility, motility duration, velocities, and wobble - WOB), morphology and membrane and DNA integrity. The treatments containing egg yolk improved significantly (P<0.05) results when compared the control for the membrane integrity parameter. When compared to other treatments, egg yolk, at any concentration, presented higher results (P<0.05) for membrane integrity, total motility, curvilinear velocity (VCL) and average path velocity (VAP) parameters. Egg yolk also showed the best results for WOB, but it did not differ from 5% and 8% lactose and 5% and 20% sucrose. Soy lecithin had the lowest percentages of morphologically normal sperm (P<0.05), while the other treatments did not differ from each other. There was no difference regarding DNA integrity data. Thus, 5% egg yolk is indicated as a non-permeable cryoprotectant for P. brevis, in association with 5% glucose and 10% Me₂SO.     

Can amides be alternative cryoprotectors for the preservation of feline semen?

Sperm cryopreservation is a tool for the conservation of the genetic material of animals of genetic importance or for species preservation. In the case of domestic cats, this can be used to generate information about seminal harvest, evaluation and preservation, which is especially important due to its applicability to wild felids. This study evaluated seminal samples harvested by urethral catheterisation from 13 adult domestic cats. Samples were cryopreserved with experimental groups of extenders were defined by the penetrating cryoprotectant: 6% glycerol (GLY6%), 3% dimethylacetamide (DMA3%) and 3% dimethylformamide (DMF3%). The samples were thawed and evaluated by conventional microscopy and by computer-assisted sperm analysis (CASA). The structural and functional membrane integrity was assessed by supravital tests (EOS), hypoosmotic swelling tests (HOST) and flow cytometry (FC). There was a correlation (P < 0.05) between total motility and EOS (r = 0.54), HOST and FC (r = −0.62) and total motility and flow cytometry (r = 0.63), indicating that these are complementary parameters that increase the accuracy of the feline sperm quality evaluation post-thaw. The results regarding the structural and functional integrity of the sperm plasma membrane did not differ (P > 0.05) among groups. However, the DMA3% group had a lower (P < 0.05) percentage of morphological changes in the sperm tail compared to samples cryopreserved with GLY6% and DMF3%. Additionally, DMA3% provided lower values of immobile sperm post-thaw when compared to DMF3%. DMA is an interesting alternative to GLY and superior to DMF for the cryopreservation of feline semen at the studied concentrations.     

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.     

Altering fish embryos with aquaporin-3: an essential step toward successful cryopreservation

Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.     

Effect of cryopreservant combinations on the motility and morphology of curimba (Prochilodus lineatus) sperm

This study investigated the application of intra- and extra-cellular cryoprotectant combinations on the quality of curimba Prochilodus lineatus semen subjected to cryopreservation. Semen treatments were tested with 8% DMSO or methanol as intracellular cryoprotectant, 5% egg yolk or lactose as extracellular cryoprotectant and 5% BTS. These cryoprotectant combinations are suitable for curimba but have not been tested at the lesser concentrations proposed or in combination with BTS. Semen samples collected from 19 curimbas were diluted into one of four cryoprotectant combinations: DMSO+yolk; DMSO+lactose; methanol+yolk; and methanol+lactose. After dilution, semen samples were cryopreserved in 0.5 mL straws for 10 days in a liquid nitrogen tank. Semen was thawed in a water bath at 60°C for 8s. We evaluated the quality of fresh, diluted (pre-freezing) and post-freezing semen according to sperm motility rate (%) and duration (s). Sperm morphology was also analyzed in thawed semen. Sperm motility rate decreased progressively after dilution and thawing. The motility rate in post-freezing semen was higher in the treatments using DMSO+lactose and methanol+yolk. Sperm motility duration in post-freezing sperm was greater in the treatments using methanol rather than DMSO as intracellular cryoprotectant, irrespective of the extracellular cryoprotectant used. Abnormality frequency in thawed sperm was less in semen treated with egg yolk than with lactose. Thus the use of methanol intracellular cryoprotectant is recommended along with yolk extracellular cryoprotectant in the cryopreservation process for curimba semen.     

The proteomic characterization of ram sperm during cryopreservation analyzed by the two-dimensional electrophoresis coupled with mass spectrometry

The aim of this study was to analyze the effects of the cryopreservation process on the protein profile of ram sperm using two-dimensional electrophoresis (2-DE) coupled with mass spectroscopy. Semen was collected from five rams and cryopreserved in a Tris-based extender supplemented with glycerol and egg yolk as the main cryoprotectants. The fresh and post-thaw sperm total proteins were extracted and purified, followed by the 2-DE. The differential proteins in the stained gel were determined by mass spectrometry. The results indicated that there were 39 differential proteins between fresh sperm and frozen-thawed sperm. Among these proteins, the abundance of 28 proteins in fresh sperm was higher than those in post-thaw sperm (P < 0.05). However, 11 proteins in post-thaw sperm were up-regulated instead. The gene ontology (GO) analysis showed that most of differential proteins were implicated in cellular process, metabolism and regulation of the biological process. The networks of protein-protein interaction indicated a strong interaction among these differential proteins, which may be involved in sperm metabolism, acrosomal function, sperm motility, and reducing ROS level. In conclusion, the cryopreservation process modifies the proteome of ram sperm, which may be directly associated with ram sperm cryodamage, consequently influencing their fertility. Additionally, these differential proteins can be used as biomarkers for evaluation of frozen ram semen quality.