Fish Sperm Cryopreservation Research Articles

Cryopreserved sperm does not affect larval ontogeny and quality in Rhamdia quelen.

Fish sperm cryopreservation is an important technique for optimizing juvenile production in aquaculture stations and laboratories and contributing to the conservation of endangered species. Despite its benefits, the cryopreservation process can cause cellular damage, affecting spermatozoa quality and offspring viability. This study aimed to evaluate the larval development of jundiá Rhamdia quelen originating from cryopreserved sperm. Larvae were obtained from artificial reproduction using oocyte samples from four females combined with fresh (Control) or cryopreserved/thawed sperm. The semen was diluted in the cryoprotective solution (1:3 ratio) consisting of skimmed milk powder (5%), methanol (10%), and fructose (5%), and was packaged into 0.25mL straws. The straws were then stored and cooled in liquid nitrogen vapor for 18h. The straws were individually warmed in a water bath at 25°C for 10s to thaw the samples. The experiments were performed in triplicates. Sperm quality, fertilization, hatching, and larval development were evaluated. After larval hatching, six larval collections were performed (5, 10, 15, 20, and 25days after hatching), and 15 larvae were sampled per collection per treatment. Cryopreservation reduced sperm motility (70.48 ± 7.70 fresh to 41.36 ± 4.80 cryopreserved semen), progressivity (3874 fresh to 2505 cryopreserved semen), and beat cross frequency (55.83 ± 155 fresh to 50.22 ± 190 cryopreserved semen). Increased the percentage of sperm with abnormal morphology and increased most sperm pathologies. Furthermore, the fertilization rate was lower in the cryopreserved group (63.1 ± 18, and 83.72 ± 7.59 for fresh semen), while hatching was not different between groups (65.3 ± 18.05 fresh, 48.89 ± 21.77 cryopreserved semen) Otherwise, the initial larval development morphology showed no difference in the appearance of structures such as the presence of the vitelline structure, pigmentation pattern, development of the anal pore, embryonic membrane, eye, barbells, notochord flexion, and fin rays, for both treatments. There was no significant difference in the frequency of structures between larvae from fresh and cryopreserved/thawed sperm, revealing a similar developmental pattern in both treatments. In conclusion, the cryopreservation protocol affects sperm quality; however, those sperm able to fertilize the oocytes originate normal larvae with regular larval development of R. quelen up to 25days old.

Effects of Long-Term Cryopreservation on the Transcriptomes of Giant Grouper Sperm.

The giant grouper fish (Epinephelus lanceolatus), one of the largest and rarest groupers, is a fast-growing economic fish. Grouper sperm is often used for cross-breeding with other fish and therefore sperm cryopreservation is important. However, freezing damage cannot be avoided. Herein, we performed a transcriptome analysis to compare fresh and frozen sperm of the giant grouper with frozen storage times of 0, 23, 49, and 61 months. In total, 1911 differentially expressed genes (DEGs), including 91 in El-0-vs-El-23 (40 upregulated and 51 downregulated), 251 in El-0-vs-El-49 (152 upregulated and 69 downregulated), and 1569 in El-0-vs-El-61 (984 upregulated and 585 downregulated), were obtained in the giant grouper sperm. DEGs were significantly increased at 61 months of cryopreservation (p < 0.05). GO and KEGG enrichment analyses of the DEGs revealed significant enrichment in the pilus assembly, metabolic process, MAPK signaling pathway, apoptosis, and P53 signaling pathway. Time-series expression profiling of the DEGs showed that consistently upregulated modules were also significantly enriched in signaling pathways associated with apoptosis. Four genes, scarb1, odf3, exoc8, and atp5f1d, were associated with mitochondria and flagella in a weighted correlation network analysis. These genes may play an important role in the response to sperm freezing. The experimental results show that long-term cryopreservation results in freezing damage to the giant grouper sperm. This study provides rich data for studies of the mechanism underlying frozen fish sperm damage as well as a technical reference and evaluation index for the long-term cryopreservation of fish sperm.

Endemic Fish Conservation: Utilization of Cryopreservation Technology with Fructose in Red Bader Fish (Puntius Bramoides) Sperm

Cryopreservation in red bader fish is needed for conservation and development of gamete cell storage. It is a chemical compound that can prevent cell or tissue damage due to freezing. In addition, dimethyl sulfoxide can penetrate cells quickly during equilibration. This research aimed to study reproductive biology and analyze the fructose ability as a extender in the Red Bader fish sperm cryopreservation process. The cryopreservation process was conducted at the Artificial Insemination Center, Singosari. The test fish were obtained from the Freshwater Cultivation Development Center, Umbulan then they were reared for 2 months to get the level of gonad maturity. This study was designed using a completely randomized design (CRD) which consisted of three treatments and three replications. The treatment given was the use of fructose extenders with different percentages i.e., 0.2%, 0.4%, and 0.6%. The results showed that the sperm characteristics of the red bader fish (Puntius bramoides) had a volume of 3.18 mL, a pH value of 7.39, a milky white sperm color, a sperm concentration of 3.5x109 cells/mL, a motility value of 81.67%, and a viability of 85 %. The best type of fructose extender with 0,6% dimethylsulfoxide concentration has a motility value of 38,33% post-cryopservation and 36,67% post-cryopreservation viability. The type of extender affects the sperm quality of angry bader fish during the cryopreservation process, the type of extender obtained was fructose with the best concentration of 0,6% dimethylsulfoxide with the highest motility and viability values.

Effect of Different Plant Extracts on Microbial Population in The Frozen African Catfish (Clarias gariepinus) Semen.

Fish sperm cryopreservation has been attempted on roughly freshwater and marine species since 1953. This study sought to assess the potential of various plant extracts to function as natural antimicrobial agents in the frozen semen of African catfish (Clarias gariepinus). Diluted sperm was packaged in 0.25ml straws and left for 10min equilibration at 4°C. Following equilibration, the straws were exposed to liquid nitrogen vapor for 10 min and plunged into the liquid nitrogen (-196°C) and then thawed in a water bath at 35°C for 20s. Sperm samples were put into sterile 1.5 ml tubes immediately after thawing and the microbial count was detected with classical microbiological culture method. In the results of microbiological analyses, these tree plant extracts especially Echinacea purpurea were found highly effective for decreasing bacterial contamination levels of African catfish (C. gariepinus) semen. These plant extracts may have the potential for antibacterial effect, and they can be useful for the dilution of semen.

Fish sperm cryopreservation using biodegradable containers: New low-cost and environment-friendly methodology.

The containers used in cell cryopreservation are essential to maintain cell integrity and viability after thawing. This paper reveals the methodology of using biodegradable containers for fish sperm cryopreservation. Cryopreserved sperm in biodegradable containers showed high fertility capability. Biodegradable capsules could be alternative containers to plastic straws for sperm cryopreservation. Containers used to cryopreserve sperm are made with non-biodegradable plastic compounds, having a high monetary and environmental cost. Therefore, the development of biodegradable alternative containers for cell cryopreservation is necessary. Thus, this study aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as low-cost and biodegradable alternative containers for sperm cryopreservation. Sperm from 12South American silver catfish Rhamdia quelen were individually cryopreserved in plastic straws 0.25 mL (as control), hard-gelatin, and hard-HPMC capsules. The quality of post-thaw sperm cryopreserved in the different containers was checked by measuring spermatozoa membrane integrity, kinetic parameters, mitochondrial activity, fertilization, hatching, and normal larvae rates. The samples cryopreserved in straws showed a higher percentage of membrane integrity (68%) than those frozen in hard-gelatin (40%) and hard-HPMC capsules (40%). However, we did not observe differences between the samples stored in straws and hard capsules for the rest of the tested sperm parameters. Thus, based on the high sperm fertility capability, both capsules were efficient as cryopreservation containers for maintaining sperm functionality.

Use of Powdered Milk in Semen Cryopreservation Protocols for Fish: A Systematic Review.

This systematic review provides an overview of the history and current status of cryopreservation of fish sperm and a detailed evaluation of cryoprotocols using powdered milk. A literature search was performed in PubMed, Scopus, Web of Science, and SciELO databases. Twenty-nine articles were selected after excluding duplicate articles or articles that did not meet the eligibility criteria. Rhamdia quelen and Danio rerio were the most studied species. Slow freezing method, dry-shipper, freezing rate of -35.6°C/min, thawing in water bath (35.93°C ± 10°C), and 0.25 and 0.5 mL plastic straws were the main approaches evaluated. Methanol was the most used permeable cryoprotectant in combination with powdered milk, yielding the best results at 10% concentration. Motility rate was the main analysis performed after cryopreservation in virtually all studies, being subjectively evaluated by most authors. Powdered milk at 15% promoted the best results in the analyzed studies. For motility rate, the gains with the addition of powdered milk were observed in the orders Perciformes (Oreochromis mossambicus), Siluriformes (Pangasius pangasius, Pseudoplatystoma corruscans, and Pseudoplatystoma mataense), and Cypriniformes (Tor soro and Barbonymus gonionotus). For fertilization, gains were observed in the order Siluriformes (P. mataense) and Cypriniformes (T. soro). Sperm viability gains were observed in the orders Siluriformes (P. pangasius), Characiformes (Piaractus brachypomus), and Cypriniformes (B. gonionotus). The scientific evidence we present in this study may contribute and serve as a starting point for new and more refined studies to be developed in the field.

Investigation of ice particle formation points during fish sperm cryopreservation

This study was conducted in order to investigate of the points of formation of ice particles during cooling of fish sperm. The object of the study was spermatozoa of the Russian sturgeon (Acipenser gueldenstaedtii Brandt, 1833), hybrid Acipenser ruthenus × Huso huso, the spiny-tailed sturgeon Acipenser nudiventris Lovetsky, 1828, and the inconnu Stenodus leucichthys Güldenstädt, 1772. The growth of ice microparticles is formed at the perturbation temperature of the corresponding aqueous suspension, with the concentration of dissolved substances increasing in the non-freezing phase. The process of ice formation and the increase concentration of the remaining liquid continues as it cools to form amorphous (glassy) ice. It is interesting to bring the mass of ice with a further decrease in temperature to (-196) ° C. Cooling causes compression of the formed ice crystals.

Improvement of Sperm Quality of the Depik Fish, Rasbora tawarensis, After Cryopreservation Using Antioxidant

BACKGROUND: The cryopreservation of the sperm of the depik fish, Rasbora tawarensis, has previously been developed. However, the quality of the sperm post cryopreservation was not satisfactory and might be improved through the application of antioxidants. OBJECTIVE: To determine the most suitable antioxidant for the cryopreservation of the depik fish spermatozoa. MATERIALS AND METHODS: A completely randomized design with a non-factorial experiment was used and the tested antioxidants were glutathione, β-carotene, ascorbic acid, and butylated hydroxytoluene (BHT) at 6% concentrations. All treatments had three replications. The sperms were collected from 10 male fishes and diluted with Ringer solution in a ratio of 1: 20 (v/v, sperm: Ringer solution). Then 5% DMSO and 5% egg yolk were added to the diluted sperms. Furthermore, 6% of the tested antioxidants were added to the diluents, and then, cryopreservation was carried out in liquid nitrogen for 14 days. RESULTS: The ANOVA test showed that the application of antioxidants significantly affected the sperm motility, fertility, and hatching rates of the eggs (P&gt; 0.05). Furthermore, the antioxidants also protected the sperm cells during cryopreservation, with glutathione being the best antioxidant. CONCLUSION: The application of antioxidants during the cryopreservation of depik fish sperm had a significant effect on motility, fertility and hatchability of eggs post-cryo. Furthermore, glutathione was the most suitable antioxidant.

Оптимизированные технологии крупномасштабной криоконсервации спермы карповых рыб

The need for optimization of technologies of fish sperm cryopreservation is conditioned by instability of fresh sperm quality. Presented protocols of cyprinids sperm cryopreservation have been optimized following the results of experiments carried out on varied-quality biomaterial. These protocols ensure with high effectiveness both freezing big volumes of sperm under laboratory and field conditions. Effectiveness of the protocol with two-stage freezing regime in addition has been confirmed by results of cryopreservation of cyprinids sperm samples for addition to the collection of low-temperature gene bank of VNIIPRKH for the last four years.

Effect of antioxidants in cryopreservation media on spotted halibut (Verasfer variegatus) sperm quality during cryopreservation

In fish hatcheries, sperm cryopreservation is a helpful tool for managing broodstock and artificial fertilization. However, cold shock can cause sperm injury resulting in decreasing sperm motility, low cell viability, structural damage to the plasma membrane and DNA fragmentation. This parameters are critical factor for the assessment of cryopreserved sperm for commercial scale application. In recent years, antioxidants have been increasingly useful for treating physiological issues in fish, including in reproduction and cryopreservation procedures. The purpose of this study was to examine the effects of supplementation of various antioxidants, including trehalose, reduced glutathione (GSH), Mito-TEMPO (MT), and rosmarinic acid (RA), during fish sperm cryopreservation on sperm motility, cell survival rate, and DNA damage. One-way ANOVA and Duncan's multiple range tests (p > 0.05) were used to compare the parameters. Sperm from adult fish were cryopreserved in 10% DMSO with 300 mM sucrose (1:1) supplemented with different concentrations of trehalose (0, 50, 100, 150, 200, and 250 mM), GSH (0, 2, 4, 6, 8, and 10 mM), MT (0, 25, 50, 75, 100, 125, 150, 175, and 200 μM), or RA (0, 25, 50, 75, and 100 μM). Compared with the control groups, antioxidant supplementation in sperm was more efficient in increasing post-thaw motility, maintaining cell survival rates, and inhibiting the increase in DNA damage that occurs during non-activated sperm during storage. The results demonstrated that the post-thaw motility of sperm was higher in 8 mM GSH (85.71 ± 0.04%), followed by 6 mM GSH (82.97 ± 1.30%), 75 and 25 μM RA (82.93 ± 0.93% and 82.76 ± 1.42%, respectively) and 100 mM trehalose (80.39 ± 0.61%) with not statistically different. The cell survival rate, VCL, and VSL of frozen–thawed sperm were higher in 100 mM trehalose resulted 84.22 ± 2.24%, 114.72 ± 2.00 μm/s, and 95.49 ± 0.70 μm/s, respectively. Mito-TEMPO at concentrations 100 μM resulted lower percentages of tail DNA (1.60 ± 0.08%) with not statistically different with 100 mM trehalose. In conclusion, the present study indicated that antioxidant 100 mM trehalose provided the most pronounced protective effect in improving frozen-thawed quality of frozen–thawed spotted halibut sperm.

Effect of Fetal Bovine Serum on the Sperm Quality of Depik (Rasbora tawarensis) after Short-term Cryopreservation

Background: Sperm cells are susceptible to oxidative stress during cryopreservation. Therefore, an antioxidant is necessary to protect them from damages. Fetal bovine serum (FBS) is one of potent antioxidants for fish sperm cryopreservation. Hence, the aims of this study are to examine the effect of FBS on sperm quality after a short period and to determine its optimum concentration on depik (Rasbora tawarensis). Methods: Depik fish were obtained from the Fish hatchery of Lukup Badak, Aceh Tengah District, Indonesia. Sperms collected from the fish were diluted in Ringer extenders containing FBS concentration of 10% (P1), 20% (P2), 30% (P3), 40% (P4), 50% (P5) and 60% (P6), filled into 2 ml cryotubes and equilibrated prior immersed into liquid nitrogen for 15 days. The parameters observed were sperm motility, consistency, pH, fertilization and hatching rates and DNA fragmentation post-thawing. Result: The ANOVA test indicates that the application of FBS in Ringer had a significant effect on sperm motility, fertilization and hatching rates (P less than 0.05). The highest motility (58.33%) was recorded at FBS 60% and significantly different from those at other concentrations. The laddering analysis showed that applying FBS protected the integrity of depik sperms DNA. It is concluded that the optimum concentration of FBS on depik sperm led to a short-term cryopreservation of 60%.

A short review of discovery and development of fish sperm cryopreservation

Global biodiversity, especially fish, has experienced a decline, this occurs as a result of over-exploitation, the presence of introduced fish species and climate change. This condition makes researchers look for solutions to overcome these problems by using cryopreservation techniques. The main purpose of cryopreservation is to store, maintain, and ensure the survival of genetic material, so that using cryopreservation techniques can maintain the viability and function of gamete cells both immunologically, biologically and physiologically. The success of the cryopreservation technique has made this technique widely developed in various species of living organism including fish. This article summarizes and reviews the history of the development of cryopreservation of animal species with specific focus on fish.Keywords:CryopreservationHistoryDepikEndemic species

Effects of cryoprotective agents and treatment methods on sperm cryopreservation of stone flounder, Kareius bicoloratus

This study was performed as part of the effort to develop artificial seed production techniques for stone flounder Kareius bicoloratus, which has a great potential for farming in northeast Asian countries. To develop a sperm cryopreservation protocol that can be applied for artificial fertilization, we carried out experiments on the key factors for successful fish sperm cryopreservation, including cryoprotective agents (CPAs), diluents, equilibration times, and dilution ratios. The CPAs tested in this study were dimethyl sulfoxide (DMSO), glycerol, methanol, and ethylene glycol, and the diluents were glucose and sucrose. The equilibration times tested in the experiments ranged from 0 to 210 s and the dilution ratios ranged from 1:1 to 1:1000. According to the results of our experiments, the optimal CPA for stone flounder sperm cryopreservation was DMSO in concentrations ranging from 7.5% to 10% when 300 mM glucose was used as diluent and DMSO in concentrations ranging from 15% to 17.5% when 300 mM sucrose was used as diluent. Post-thaw sperm motility was high, with equilibration times up to 150 s, and dilution ratios up to 1:10 (semen:CPA + diluent) showed no significant differences in the effect on post-thaw sperm motility. The cryopreserved sperm showed more significant DNA damage than the fresh sperm with the severity of DNA damage being in inverse proportions to post-thaw sperm motility and survival rate. It is expected that these results will be useful for applications of artificial fertilization at stone flounder hatchery farms.

Sperm motility modulated by Trpv1 regulates zebrafish fertilization

By responding to environmental and intracellular stimuli, ion channels play critical roles in sperm function regulation. Although the importance of ion channel in male reproduction has drawn increasing attention in many species, the knowledge about ion channels in zebrafish sperm is limited. Here, we show zebrafish sperm motility could be suppressed by general calcium channel blockers rather than by general potassium channel blockers. Further investigation found that sperm motility was not only suppressed by antagonist for the transient receptor potential vanilloid channel, subtype 1 (Trpv1), but also restored by its agonist, suggesting functional presence of Trpv1 in zebrafish spermatozoa. As a consequence, the suppression of sperm motility by Trpv1 antagonist could reduce in vitro fertilization rate. Western blot and immunofluorescence analysis proved that Trpv1 was mainly distributed in the neck and tail regions of zebrafish sperm. Additionally, neither antagonist nor agonist of Trpv1 exhibited effect on the motility of trpv1−/− zebrafish sperm. To our knowledge, this is one of the limited studies showing the importance of ion channels in regulating zebrafish sperm function, and may enrich our understanding on male reproductive physiology of zebrafish and offer novel regulatory target for fish breeding and sperm cryopreservation.

Spice up your life!

Cryptic female choice is the ability of a polyandrous mating female to favour - or hinder - the sperm of a particular male from fertilizing her ova, under sperm competition. It operates under varied mechanisms and occurs in both internal and external fertilizers. In fishes, a key process is driven by the effect of ovarian fluid - the fluid released with the ova at spawning - on the swimming characteristics of sperm. Due to the limited functioning life-span of fish gametes, researchers have previously had to use fresh ovarian fluid and sperm, thus limiting the geographical and temporal range of their studies, or freeze one, or the other, or both. Much work has been done on the cryo-preservation of fish sperm; however, it was unclear - until now - whether the freezing of ovarian fluid affects its sperm-influencing properties. This is where Craig Purchase and Anna Rooke of Memorial University of Newfoundland, Canada come in (Purchase & Rooke, 2020). They took ovarian fluid and semen from three species of salmonids - lake tout, brown trout and Atlantic salmon - and tested both frozen and fresh ovarian fluid on the swimming characteristics of sperm. They found quite clearly that freezing did not reduce the effect of ovarian fluid on the motility, swimming speed or linearity of conspecific sperm. This is good news for researchers; it allows them to build up collections of ovarian fluids from different females - the “spice-rack” of the title - in the knowledge that freezing them will not alter the very properties that they are investigating. Now, studies can include different populations, even different years, to investigate how ovarian fluids influence the sperm of different males, and thus the extent and importance of this effect in reproductive isolation, for example. It will also help in the ultimate identification of the proximate factor(s) involved, but this is still some way ahead.

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.

Molecular and subcellular cryoinjury of fish spermatozoa and approaches to improve cryopreservation

AbstractThe quality of frozen/thawed fish sperm is generally lower than that of fresh sperm. Extremely low temperatures are associated with damage to fish spermatozoon subcellular compartments and associated molecules such as DNA and proteins. Cryodamage can negatively affect DNA integrity, spermatozoon metabolism and motility, consequently impairing fertilization and embryo development. To preserve sperm efficiently, the addition of proteins including seminal plasma proteins, antioxidants, antifreeze proteins and antifreeze glycoproteins, and bovine serum albumin before and/or after freezing is suggested to minimize cryoinjury. An appropriate quantity of seminal plasma components in cryopreservation medium can prevent spermatozoon membrane damage by maintaining antioxidant enzymes and their proper distribution on the spermatozoon surface. Under storage conditions, the semen antioxidant system is ineffective in protecting spermatozoa from reactive oxygen species. The addition of antioxidants can provide protection for spermatozoa and reduce cryoinjuries during storage, but the effect of a given antioxidant is species‐specific. Antifreeze proteins and antifreeze glycoproteins can stabilize the cell membrane via interaction with phospholipid components and inhibit ice‐crystal growth to reduce damage related to osmotic stress and ice crystallization. However, they may exhibit a cytotoxic effect. This review summarizes the sources and characteristics of subcellular spermatozoon cryoinjury and discusses approaches to improve outcome of fish sperm cryopreservation.

Potential biomarkers of DNA quality in cryopreserved fish sperm: impact on gene expression and embryonic development

AbstractSperm DNA quality and gene expression are crucial for early embryo development. Abnormal genomic processes can cause irreversible damage to totipotent cells, thereby altering the capacity for cell differentiation. Cryopreservation is a complex procedure that exposes cells to extreme conditions; it is therefore important to determine whether cryopreservation affects genomic stability. Despite this, few studies have focused on the effects of cryopreservation on the genomic stability of fish sperm. Thus, information is lacking to determine how the cryopreservation of fish sperm affects embryo quality and subsequently aquaculture. The aim of this review was to compile background information pertaining to the study of genomic stability in cryopreserved sperm. In this way, we hope to characterize more clearly the embryonic development of fish that are of biological and economic interest. We also discuss the potential use of antioxidants in cryopreservation to help preserve DNA integrity, gene expression and embryo quality. There is a current need to evaluate the use of potential biomarkers of genomic integrity to establish the effects of cryopreservation on the DNA quality of fish gametes and embryos.

Impact of cryopreservation on sterlet, Acipenser ruthenus sperm motility and proteome

Fish sperm cryopreservation is a well-established technique allowing for artificial insemination on a commercial scale. The extent of proteome alterations in seminal plasma and sperm due to cryopreservation, however, is not known. This study was conducted to evaluate the effect of cryopreservation on motility variables of sterlet Acipenser ruthenus sperm and to detect the differences in protein profiles of fresh and cryopreserved sterlet sperm and seminal plasma. Fresh sperm had 89 ± 3% motility and 160 ± 14 μm/s curvilinear velocity at 15 s post-activation. The motility rate of cryopreserved sperm (37 ± 5%) was less at 15 s post-activation. No difference (ANOVA; P > 0.05) in mean curvilinear velocity of fresh and cryopreserved sperm was detected. The protein profiles of seminal plasma and sperm were characterized using comparative proteomics to determine the influence of cryopreservation. Six altered protein spots in seminal plasma and thirteen altered spots in sperm were detected in fresh and thawed sperm. Subsequent protein characterization suggested that the proteins identified were involved in sperm metabolism, cytoskeleton, and stress response. The results broaden the understanding of the effects of cryopreservation and identify the proteins associated with cryo-injury. These data may help to determine the function of altered proteins and provide new insights into improving sperm cryopreservation.

Solea senegalensis sperm cryopreservation: New insights on sperm quality.

Cryopreservation of Senegalese sole sperm can represent an alternative to overcome some reproductive problems of this species. However, it is important to guarantee the safe use of cryopreserved sperm by selecting an appropriate protocol according to a high demand quality need to be ensured. It has been demonstrated that traditional assays such as motility and viability do not provide enough information to identify specific damage caused by cryopreservation process (freezing and thawing). Specific tests, including lipid peroxidation and DNA damage, should be performed. In the present study, motility and lipid peroxidation were performed as specific tests allowing us to discard cryopreservation conditions such as methanol as internal cryoprotectant and bovine serum albumin as external cryoprotectant. In addition, a caspase 3/7 detection by flow cytometry was performed to analyze apoptosis activity in the best selected conditions. Moreover, new highly sensitive tests based on transcript number detection have recently been described in fish sperm cryopreservation. For this reason, a transcript level detection assay was performed on certain oxidative and chaperone genes related to fertilization ability and embryo development (hsp70, hsp90BB, hsp90AA, gpx) to select the best cryopreservation conditions. DMSO+ egg yolk proved to be the best cryoprotectant combination in terms of transcript level. This study describes an optimized cryopreservation protocol for Solea senegalensis sperm demonstrating for the first time that transcript degradation is the most sensitive predictor of cell status in this species after cryopreservation.