Semen from rainbow trout produced using cryopreserved spermatozoa is more suitable for cryopreservation

Oocytes from three female rainbow trout Oncorhynchus mykiss were inseminated separately with untreated or cryopreserved semen, which had been produced using either untreated (three males) or cryopreserved (three males) spermatozoa. In half of variants, the cryopreservation did not significantly affect fertilization efficiency. Regardless of whether the sperm donors were produced from cryopreserved or intact semen, insemination of oocytes with their intact sperm resulted in the same percentage of eyed embryos (94.4 and 94.3%, respectively). When eggs were inseminated with cryopreserved semen, the use of sperm from males produced with cryopreserved spermatozoa resulted in a significantly higher percentage of eyed eggs than in case of donors produced with intact sperm (89.6 and 81.7%, respectively). The production of rainbow trout using cryopreserved sperm does not appear to negatively affect reproductive abilities of male progeny and semen from donors, which were produced using cryopreserved sperm, is more suitable for cryopreservation than the semen from donors produced with intactspermatozoa.     

Effect of sperm cryopreservation on sperm DNA stability and progeny development in rainbow trout

This study was carried out to test how sperm cryopreservation affected nuclear DNA stability and whether progeny development was modified when eggs were fertilized with cryopreserved spermatozoa. The "comet assay" (alkaline single-cell gel electrophoresis assay) was adapted to trout spermatozoa to estimate DNA stability as measured by alkali-induced DNA strand break formation. Because trout eggs develop in water after fertilization (oviparous species) and that eggshell is easy to clear up after fixative treatment, progeny development was assessed from the blastodisc flattening stage of the embryos to the first feeding stage of the hatched fries by direct observation. All parameters under study were analyzed on each sperm and comparisons between parameters were made using paired data. Freeze-thawing of sperm slightly but significantly increased the percentage of nuclei showing altered DNA after comet assay. This increase was correlated to the decrease in fertilization rates of sperm, but the absolute percentage of altered nuclei was not predictive of the absolute fertilization ability of sperm. Assessment of progeny development showed that survival rate and abnormality rate obtained after fertilization with cryopreserved sperm were not different from those obtained with fresh sperm. It is concluded that trout sperm cryopreservation only slightly affected sperm DNA stability and that the use of cryopreserved spermatozoa did not impair offspring survival and quality.     

Effect of DNA repair inhibitor (3-aminobenzamide) on genetic stability of loach (Misgurnus fossilis) embryos derived from cryopreserved sperm

Semen cryopreservation is widely used in clinical medicine, agriculture, aquaculture and biomedical research, but it is an inefficient technique that induces extensive cytoplasmic damage and loss of fertilising ability. Whether any genetic damage (i.e. DNA strand breakage or mutation) is also induced is still unclear. However, previous data has indicated that this is likely. The present study was designed to explore this possibility further by using inhibitors of the DNA repair system to block DNA repair in embryos derived from cryopreserved spermatozoa. If cryopreservation causes strand breaks in sperm DNA it might be expected that inhibition of a repair enzyme such as poly(ADP-ribose) polymerase (PARP) would enhance any such negative effect of cryopreservation. To check this hypothesis 3-aminobenzamide (3-AB) was used as an inhibitor of PARP. Weather loach (Misgurnus fossilis) eggs were fertilised using cryopreserved as well as fresh spermatozoa. Embryos derived from cryopreserved spermatozoa were exposed to 10 mM 3-AB for 2 h after fertilisation. The experiments were carried out using 43,544 embryos from 5 females and 10 males. Embryo survival was evaluated at different stages until the hatching stage. Sperm cryopreservation significantly decreased embryo survival (53.6+/-2.79% compared to 76.97+/-2.79% of control; P<0.01). The addition of 3-AB to the medium with embryos derived from cryopreserved sperm further decreased embryo survival from 53.6+/-2.79% to 46.1+/-2.79% (P<0.01) whereas there was no adverse effect of 3-AB exposed embryos derived from fresh sperm (76.97+/-2.79% of control compared to 74.8+/-2.79% of control+3-AB). The effect of 3-AB provides indirect evidence that cryopreservation might induce instability in sperm DNA, and that such damage can be repaired by the oocyte repair system after fertilisation.     

Spermatozoa concentration influences cryopreservation success in sea trout (Salmo trutta m. trutta L.)

Variation among individuals is substantial for spermatozoa concentration in fresh milt in sea trout (Salmo trutta m. trutta L.). The objective of the present study was to examine effects of spermatozoa concentration in this species on subsequent cryopreservation success. Milt with high spermatozoa concentration was diluted with seminal plasma to obtain concentrations ranging between 6 and 24 × 10⁹ mL⁻¹ with steps of 2 × 10⁹ mL⁻¹. Diluted milts were cryopreserved in 0.25-mL straws with extender (0.3 M glucose) containing 10% methanol and 10 % (vol/vol) supplement of hen egg yolk. The dilution ratio was 1:3 (milt:cryomedium). Cryopreservation efficacies were assessed according to evaluation of motility of frozen/thawed spermatozoa and quantification of fertilizing ability. Percentage of motility of frozen/thawed spermatozoa was influenced by spermatozoa concentration in the cryomedium (P < 0.05). The highest motility was observed in samples with 3.0 to 4.0 × 10⁹ spermatozoa per mL of cryomedium, which corresponds to 12 to 16 × 10⁹ spermatozoa per mL in fresh milt. Higher sperm concentrations and lower sperm concentrations in cryomedium reduced the effectiveness of cryopreservation when compared with the optimum. Cryopreservation success measured according to fertilization rate was in agreement with results for motility of frozen/thawed spermatozoa, but the optimum could not be determined with statistical precision because of differences in fertilization rate among individual donor males. However, a significant positive correlation was found between postthaw motility and fertilization rate and between cryopreserved spermatozoa velocity and fertilization rate (P < 0.05). In sea trout, cryopreservation efficiency is influenced by spermatozoa concentration in cryomedium. Individual adjustment of the dilution ratio, based on initial spermatozoa density, is recommended in the freezing protocol. Maximum cryoresistance of the cell was obtained when spermatozoa concentration in cryomedium ranged from 3.0 to 4.0 × 10⁹ mL⁻¹.     

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage.The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2 h (fresh) or 5 days at 4 °C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.     

Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm

Cryopreservation causes several types of damage to spermatozoa, such as loss of plasma membrane integrity and functionality, loss of motility, and ATP content, resulting in decrease of fertility rates. This spermatozoal damage has been widely investigated for several marine and freshwater fish species. However, not much attention has been paid to the nuclear DNA. The objective of this study was to determine the degree to which cryopreservation induces spermatozoal DNA damage in two commercially cultured species, rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), both of which could benefit from the development of cryopreservation strategies on a large scale. We have used the single-cell gel electrophoresis, commonly known as Comet assay to detect strand breaks in DNA. This technique was performed on fresh and cryopreserved sperm from both species. In rainbow trout there was a significant increase in the averages of fragmented DNA and Olive tail moment after cryopreservation (11.19-30.29% tail DNA and 13.4-53.48% Olive tail moment in fresh and cryopreserved sperm, respectively), as well as in the proportion of cells with a high percentage of DNA fragmentation. For gilthead sea bream there were no significant differences in the percentage of tail DNA between the control samples and sperm diluted 1:6 and cryopreserved (28.23 and 31.3% DNA(t), respectively). However, an increase in the sperm dilution rate produced an increase in the percentage of DNA fragmentation (41.4%). Our study demonstrates that cryopreservation can induce DNA damage in these species, and that this fact should be taken into account in the evaluation of freezing/thawing protocols, especially when sperm cryopreservation will be used for gene bank purposes.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.     

Microsatellite genotyping of cryopreserved spermatozoa for the improvement of whitefish semen cryobanking

The cryobanking of semen is recognized as an emerging tool for the conservation of fish biodiversity. Microsatellite analysis of the DNA of cryopreserved sperm would facilitate the assessment of genetic variability of cryobanked semen specimens. The aim of this study was to compare microsatellite profiles of DNA extracted from adipose fins and cryopreserved semen collected from eleven male whitefish (Coregonus lavaretus L.). The following microsatellite loci were employed: Cocl–Lav-8, Cocl–Lav-18, Cocl–Lav-28, Cocl–Lav-80, Str-73 and Sfo-292. The chelex 100 method was used for the successful isolation of DNA from somatic tissue, and the DNeasy method with additional modifications was used for the successful isolation of DNA from sperm. Genotyping was possible with the use of a very low number of spermatozoa (5 × 10⁶ which is less than 0.1% of spermatozoa in standard 250 μL straw). The results of the DNA analysis from both the adipose tissue and spermatozoa were identical. Therefore, microsatellite analysis of cryopreserved spermatozoa can be recommended for future whitefish sperm banking.     

Microsatellite genotyping of cryopreserved spermatozoa for the improvement of whitefish semen cryobanking

The cryobanking of semen is recognized as an emerging tool for the conservation of fish biodiversity. Microsatellite analysis of the DNA of cryopreserved sperm would facilitate the assessment of genetic variability of cryobanked semen specimens. The aim of this study was to compare microsatellite profiles of DNA extracted from adipose fins and cryopreserved semen collected from eleven male whitefish (Coregonus lavaretus L.). The following microsatellite loci were employed: Cocl–Lav-8, Cocl–Lav-18, Cocl–Lav-28, Cocl–Lav-80, Str-73 and Sfo-292. The chelex 100 method was used for the successful isolation of DNA from somatic tissue, and the DNeasy method with additional modifications was used for the successful isolation of DNA from sperm. Genotyping was possible with the use of a very low number of spermatozoa (5 × 10⁶ which is less than 0.1% of spermatozoa in standard 250 μL straw). The results of the DNA analysis from both the adipose tissue and spermatozoa were identical. Therefore, microsatellite analysis of cryopreserved spermatozoa can be recommended for future whitefish sperm banking.     

Post-thawed motility and fertility from Atlantic salmon (Salmo salar L.) sperm frozen with four cryodiluents in straws or pellets

The cryopreservation of salmonid sperm is a complex process involving the interplay of many factors. Although cryopreservation protocols can be evaluated through a range of responses at various stages in the process, the number of progeny is the ultimate indicator of success. We compared reproductive success from freezing Atlantic salmon (Salmo salar L.) sperm using the eight combinations of (1) the penetrating cryoprotectants, 10% dimethyl sulfoxide (DMSO) or methanol (MeOH); (2) the nonpenetrating cryoprotectants glucose (0.3 M) or sucrose (0.6 M), and freezing in 0.1 mL pellets or 0.25 mL straws. All cryodiluents were supplemented with 10% (v/v) of hen's egg yolk. Response variables were the percentage and degree of motility of thawed and activated sperm using computer assisted sperm analysis (CASA), and rates of eyed embryos, hatch and egg sac larvae. Growth rates of alevins were assessed to two months post hatch. Atlantic salmon milt cryopreserved in straws had higher spermatozoa motility and fertilization success than milt cryopreserved in pellets (P < 0.05). Type of sugar tested did not significantly affect the response variables. In the MeOH treatment, thawed spermatozoa achieved higher speed and a higher fertilization rate evaluated at the eyed embryo stage than spermatozoa subjected to the DMSO treatment. Higher mortality rate (especially before hatching) of MeOH offspring than DMSO offspring led to equal numbers of progeny for the two treatments from the swimming stage to the end of the study. Moreover, during feeding fish from the MeOH group produced significantly lower weight larvae than the DMSO and control groups. Even so, the weight of the MeOH group was satisfactory. Length and the condition factors did not differ significantly among the larvae groups. Significant positive correlations were found between fertilization success (measured in number of eyed eggs) and both motility (rs = 0.81), and velocity (rs = 0.49). Freezing in straws gave betters results than freezing in pellets for cryopreservation of salmon milt; whereas type of sugar tested (glucose vs sucrose) did not have significant effects. Penetrating cryoprotectants DMSO and MeOH differed in their effect on post-thawed sperm velocity, fertilization rate and mortality rate of progeny, suggesting the need for further research on the influence of these cryoprotectants on frozen sperm and and post-fertilization devopmental processes.     

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

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

Effect of freezing–thawing process and quercetin on human sperm survival and DNA integrity

We aimed in the first part of our work to study the effect of cryopreservation on the human sperm DNA integrity and the activation of caspase 3, the main apoptosis indicator. In the second part, we were interested in testing the effect of quercetin, as an antioxidant, in preventing sperm damage during the freeze–thawing process. Seventeen semen samples were obtained from 17 men recruited for infertility investigations. Liquefied sperm was cryopreserved using spermfreeze®. Nine of the used samples were divided into two aliquots; the first one was cryopreserved with spermfreeze only (control) and the second one was cryopreserved with spermfreeze supplemented with quercetin to a final concentration of 50 μM. Sperm motility and viability were assessed according to WHO criteria. We used TUNEL assay and the Oxy DNA assay to assess sperm DNA integrity. Activated caspase 3 levels were measured in spermatozoa using fluorescein-labeled inhibitor of caspase (FLICA). Cryopreservation led to a significant increase in sperm DNA fragmentation, DNA oxidation and caspase 3 activation (p < 0.01). Supplementation of the cryopreservation medium with quercetrin induced a significant improvement in post thaw sperm parameters, compared to those of control, regarding sperm motility (p = 0.007), viability (p = 0.008) and DNA integrity (p = 0.02); however, it had no effect on caspase 3 activation (p = 0.3). We conclude that oxidative stress plays a major role in inducing sperm cryodamage but implication of apoptosis in this impairment requires further investigations. Quercetin could have protective effect during cryopreservation but further research is needed to confirm this effect.     

DNA methylation stability in fish spermatozoa upon external constraint: Impact of fish hormonal stimulation and sperm cryopreservation

Highly differentiated mature spermatozoa carry not only genetic but also epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark toward reproductive biotechnologies is a major issue in animal production. The present work explored the impact of hormonal induction of spermiation and sperm cryopreservation in two cyprinids, the goldfish (Carassius auratus) and the zebrafish (Danio rerio), using LUminometric Methylation Assay (LUMA). We showed that while goldfish hormonal treatment did increase sperm production, it did not alter global DNA methylation of spermatozoa. Different sperm samples repeatedly collected from the same males for 2 months also showed the same global DNA methylation level. Similarly, global DNA methylation was not affected after cryopreservation of goldfish spermatozoa with methanol, whereas less efficient cryoprotectants (dimethylsulfoxide and 1,2‐propanediol) decreased DNA methylation. In contrast, cryopreservation of zebrafish spermatozoa with methanol induced a slight, but significant, increase in global DNA methylation. In the less compact nuclei, that is, goldfish fin somatic cells, cryopreservation did not change global DNA methylation regardless of the choice of cryoprotectant. To conclude, global DNA methylation is a robust parameter with respect to biotechnologies such as hormonal induction of spermiation and sperm cryopreservation, but it can be altered when the best sperm manipulation conditions are not met.     

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

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

Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa

The effects of extender composition and equilibration time on fertilizing ability of cryopreserved spermatozoa from rainbow trout, Oncorhynchus mykiss, were investigated. In addition, enzyme activity in supernatants from thawed sperm was assessed. The use of the two extenders: Erdahl & Graham's + 10% DMA (dimethyl acetamide) + 10% egg yolk and 0.3 M glucose + 10% DMA yielded the highest post-thaw fertilization rates. We observed interactions between extender constituents and the equilibration of diluted semen. This indicates a multifactorial effect of the extender constituents on spermatozoal resistance against injuries. The 10-min equilibration of spermatozoa in extender before freezing generally lowered the fertilization ability of spermatozoa, except for DMA-based extenders. The addition of egg yolk to the extender was generally beneficial, especially in DMA- and DMSO-based extenders. The use of low-density lipoprotein fraction showed no advantage to full-yolk or free-of-yolk extenders. Aspartate aminotransferase and lactate dehydrogenase leakage from damaged spermatozoa correlated negatively with the ability of cryopreserved spermatozoa to fertilize eggs. Each factor tested, when analyzed separately, did not give general information about its effect on the fertilization ability of cryopreserved sperm. The multifactorial analysis of the important factors in cryopreservation of trout spermatozoa showed their cumulative effect. This is the most likely reason for divergent information reported elsewhere on the effect of various factors in the cryopreservation of rainbow trout spermatozoa.     

Ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, Cyprinus carpio L

Damage to spermatozoa during cryopreservation is regarded as a major obstacle to the expansion of sperm storage technology. The authors used two-dimensional polyacrylamide gel electrophoresis and matrix-associated laser desorption/ionization time-of-flight mass spectrometry to explore whether the protein profile of common carp (Cyprinus carpio) spermatozoa is affected by cryopreservation. Fourteen protein spots were significantly altered following cryopreservation. Eleven of these were identified: three as specific membrane proteins (N-ethylmaleimide-sensitive fusion protein attachment protein alpha, cofilin 2, and annexin A4) involved in membrane trafficking, organization, and cell movement; six as cytoplasmic enzymes (S-Adenosylhomocysteine hydrolase, Si:dkey-180p18.9 protein, lactate dehydrogenase B, phosphoglycerate kinase 1, transaldolase 1, and esterase D/formylglutathione hydrolase) involved in cell metabolism, oxidoreductase activity, and signal transduction; and two as transferrin variant C and F. Based on these findings, the authors hypothesize that transferrin in cryopreserved sperm may protect spermatozoa against oxidative damage during the freeze-thaw process. Cryopreservation caused changes in spermatozoa protein profiles that may lead to decreased spermatozoa velocity, motility, and fertilization success, and to subsequent ova hatching rate.     

A preliminary study on epigenetic changes during boar spermatozoa cryopreservation

Artificial insemination (AI) with cryopreserved boar semen is limited to no more than 1% of the total number of inseminations due to low conception rates and litter sizes. Cryopreservation causes a dramatic decrease in the viability, motility and fertility of spermatozoa, but the underlying mechanism remains unknown. In this study, mRNA expression and protein levels of epigenetic-related genes (Dnmt3a, Dnmt3b, Jhdm2a, Kat8, Prm1, Prm2 and IGF2) in fresh and cryopreserved boar spermatozoa were evaluated using qRT-PCR and ELISA. The results showed that cryopreservation or freezing, which drastically alter the environmental stimuli, can induce epigenetic changes of boar spermatozoa. Dramatic changes of mRNA expression of epigenetic-related genes were observed before and after cryopreservation, and low protein levels of multiple genes were mainly found in program freezing groups with or without LEY. The addition of different cryoprotective agents to the freezing extender can provide better protective effects for boar spermatozoa to avoid freezing or cryopreservation-induced expression changes of epigenetic-related genes.     

Acrosome staining and motility characteristics of sterlet spermatozoa after cryopreservation with use of methanol and DMSO

In this study we describe acrosome staining and motility characteristics of fresh and cryopreserved sterlet (Acipenser ruthenus L.) spermatozoa using soybean trypsin inhibitor-Alexa conjugate fluorescent staining and computer-aided sperm analysis (CASA), respectively. Methanol or dimethylsulfoxide (DMSO) were used as cryoprotectants. After cryopreservation a decline in sperm motility characteristics occurred, but no differential effect between cryoprotectant was observed. Cryopreservation caused a significant increase in the percentage of spermatozoa with acrosome stained by SBTI-Alexa for samples cryopreserved using DMSO compared to methanol. These data suggest that the low usefulness of DMSO for cryopreservation of sturgeon spermatozoa is related to its harmful specific effect towards the acrosome, probably by causing its precocious triggering, much before any egg contact.     

Gamete cryobanks for laboratory research: Developing a rapid and easy-to-perform protocol for the cryopreservation of the sea urchin Paracentrotus lividus (Lmk, 1816) spermatozoa

Gamete cryopreservation is a biotechnology that can guarantee a continuous supply of gametes, regardless of the seasonal reproductive cycle. In this study we developed a protocol for the cryopreservation of the sea urchin Paracentrotuslividus spermatozoa, with a view to the creation of cryobanks of semen to be used as a model system in laboratory research and ecotoxicological tests. All the key phases of the procedure were separately considered and the effect on sperm motility was evaluated by means of computer assisted analysis. The best results were obtained using 7% dimethylsulfoxide in 1% NaCl plus 0.04 M trehalose as the extender, at a freezing rate of −20 °C/min. On thawing, in semen samples cryopreserved in accordance with this protocol the velocity parameters of the sub-population of rapid sperm (best performing spermatozoa) did not significantly differ from semen on collection; in addition also the fertilization ability was restored, and about 50% of normal developed plutei larvae were obtained by thawed semen. The developed protocol is rapid and easy-to-perform; moreover, the use of gametes from reared urchins makes it unnecessary to continuously collect specimens from natural populations, making this procedure a promising starting point for the creation of alternative and more sustainable methodologies in laboratory research on sea urchin gametes and embryos.