Impact of absolute food deprivation on the reproductive system in male goldfish exposed to sex steroids.

There is a link between metabolism and reproduction as metabolic hormones affect hypothalamus-pituitary-testis (HPT) hormonal functions and vice versa. The aim of the present study was to investigate the effects of negative energy balance on the reproductive system in male goldfish exposed to testosterone (T) and 17ß-estradiol (E2). Following 7 days of food deprivation (FD), ANOVA models showed significant FD × sex steroid interactions on sperm quality and circulating sex steroid levels. When FD effects were investigated, 11-ketotestosterone (11-KT) level and sperm motility and velocity decreased in food-deprived goldfish in the control group. In E2-exposed goldfish, FD decreased sperm production in addition to sperm motility and velocity that coincided with an elevation of circulating E2 level. However, FD did not significantly impact sex steroids and sperm quality in T-exposed goldfish. ANOVA models showed non-significant FD × sex steroid interactions for HSI, GSI, circulating luteinizing hormone (Lh) level, and metabolic (preproghrelin, goat and nucb2) and reproductive (kiss1, gpr54 and gnrh3) mRNAs. Furthermore, results showed that FD decreased HSI, and increased Lh levels and testicular preproghrelin and goat mRNAs, while sex steroids increased mid-brain nucb2, kiss1 and gpr54 mRNAs. Together, our results suggest that FD-induced inhibition of androgenesis resulted in diminished sperm quality associated with activation of the testicular ghrelinergic system, and negative feedback of 11-KT increased Lh level. The FD-induced testicular metabolic and hormonal system was impacted in goldfish exposed to sex steroids. However, the negative effects of FD on sperm quality were accelerated in E2-exposed goldfish due to estrogenic activity. This study provides novel information to better understand metabolic-associated reproductive disorders in fish.

In vitro post-ovulatory oocyte ageing in grass carp Ctenopharyngodon idella affects H4K12 acetylation pattern and histone acetyltransferase activity.

Delayed fertilization leads to the ageing of post-ovulatory oocytes and reduces the developmental competence of arising embryos. Little information is available about the molecular processes during fish oocyte ageing. The current study investigated the functional consequences of oocyte ageing in grass carp Ctenopharyngodon idella embryos. In addition, the dynamics of selected post-transcriptionally modified histones (acetylation of H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16) were analyzed during oocyte ageing. Ovulated oocytes were aged in vitro for 4 h in the laboratory incubator at 20 °C and studied for selected post-translational modification of histones. In addition, histone acetyltransferase activity was investigated as an important regulator of histone acetylation modification. The results indicated a significant decrease in oocyte fertilizing ability through 1 h of post-ovulatory ageing, and a complete loss of egg fertilizing abilities was detected at 4-h aged oocytes. Furthermore, post-ovulatory oocyte ageing for 1 and 4 h led to decreased levels of H4K12 acetylation. The activity of histone acetyltransferases increased significantly after ageing of the oocytes for 30 h in vitro. This modification may partly contribute to explaining the failures of egg viability and embryo development in the offspring from the aged oocytes. The results are the first to report histone modifications as a crucial epigenetic regulator during oocyte ageing in fish and might also benefit other vertebrates.

In vivo and in vitro aging of common carp Cyprinus carpio sperm after multiple hormonal application and stripping of males.

The present study was designed to evaluate sperm phenotypic variables during in vivo and in vitro storage following multiple sperm stripping in common carp (Cyprinus carpio L.). Each male was injected 3 times with carp pituitary 3 days apart. Sperm was stored in vivo in the body cavity for 0.5 days (Fresh sperm) and 3 days (Old sperm) after hormonal stimulation. Then sperm was collected and diluted with a carp extender at a ratio of 1:1, and stored in vitro on ice for 0, 3, and 6 days. The phenotypic parameters, including the number of total motile spermatozoa, number of fast motile spermatozoa, number of motile spermatozoa, percentage of fast motile spermatozoa, and percentage of spermatozoa motility were the major components of principal component analysis (PCA). In general, Fresh sperm from the first stripping showed slightly better quality than Old sperm from the second and third stripping, especially in the phenotypic parameters of a number of total spermatozoa and a number of total motile spermatozoa (P < 0.05). The highest kinetic and quantitative spermatozoa variables were obtained in Fresh and Old sperm just after sperm collection (0-day storage in vitro), and then they were decreased during the period of in vitro storage up to 6 days (P < 0.05). However, the fertilization, hatching, and malformation rates from Fresh sperm were similar compared with the Old sperm. Sperm could be stripped 0.5 days post hormonal treatment and stored in vitro up to 6 days with good fertilization performance (fertility, hatching, and malformation rates were 92.5%, 91.5%, and 1.3%, respectively). Therefore, our results suggested that multiple hormonal treatments with multiple stripping could be used in artificial reproduction in common carp.

Common carp (Cyprinus carpio) sperm reduction during short-term in vitro storage at 4 °C.

This study was designed to optimize a short-term storage protocol for common carp sperm at 4 °C under aerobic condition. Sperm from individual males were collected directly with or without extenders. The results demonstrated that in general, it was similar effect to collect sperm directly in extenders and keeping sperm for 0.5 h after collection without extenders. Sperm was diluted with eight selected extenders (sperm: extender = 2:1, 1:1 and 1:9) and undiluted sperm was used as a control. Sperm and seminal plasma parameters (sperm motility, velocity, membrane integrity, sperm concentration, osmolality and pH in seminal plasma) were evaluated in sperm stored on ice under aerobic conditions at 0, 2, 4, 6 and 8 days post stripping (DPS) using the computer- assisted sperm analysis system. Results showed that 1:1 and 2:1 dilution maintained higher sperm function and more sperm for a longer period. After 8 DPS, the best sperm quality and quantity was recorded in the common carp seminal plasma supplemented with 50 mM NaCl, Cejko extender (2 mM CaCl2, 1 mM MgSO4, 20 mM Tris, 110 mM NaCl, 40 mM KCl, pH 7.5 and 310 mOsm/kg) supplemented with/without 25 mM KCl/NaCl. The reduction of spermatozoa number with time during short-term storage but varied according to different dilution ratios and extenders. At 8 DPS, sperm count has dropped by 22.9 % in a dilution of 1:1 compared to 50.3 % in sperm without dilution. Extenders with diluted 1:1, especially Cejko solution, largely postponed sperm reduction, 21.3 % compared to 55.5 % for sperm stored without extenders.

A chronic exposure to bisphenol A reduces sperm quality in goldfish associated with increases in kiss2, gpr54, and gnrh3 mRNA and circulatory LH levels at environmentally relevant concentrations.

The bisphenol A (BPA)-disrupted reproductive functions have been demonstrated in male animals. In fish, it has been shown that environmentally relevant concentrations of BPA decrease sperm quality associated with inhibition of androgen biosynthesis. However, BPA effects on neuroendocrine regulation of reproduction to affect testicular functions are largely unknown. In the present study, reproductive functions of hypothalamus and pituitary were studied in mature male goldfish exposed to nominal 0.2, 2.0 and 20.0 µg/L BPA. At 90 d of exposure, sperm volume, velocity, and density and motility were decreased in goldfish exposed to 0.2, 2.0, and 20.0 µg/L BPA, respectively (p < 0.05). At 30 d of exposure, there were no significant changes in circulatory LH levels and mRNA transcripts of kiss1, Kiss2, gpr54, and gnrh3. At 90 d of exposure, circulatory LH levels showed trends toward increases in BPA exposed goldfish, which was significant in those exposed to 2.0 µg/L (P < 0.05). At this time, Kiss2, gpr54, and gnrh3 mRNA levels were increased in goldfish exposed to any concentrations of BPA (p < 0.05). This study shows that BPA-diminished sperm quality was accompanied by an increase in circulatory LH levels associated with increases in mRNA transcripts of upstream neuroendocrine regulators of reproduction in goldfish. Further, this is the first study to report circulatory levels of LH in fish exposed to BPA.

A Review on Environmental Contaminants-Related Fertility Threat in Male Fishes: Effects and Possible Mechanisms of Action Learned from Wildlife and Laboratory Studies.

Increasing global rates of diminished fertility in males has been suggested to be associated with exposure to environmental contaminants (ECs). The aquatic environments are the final repository of ECs. As the reproductive system is conserved in vertebrates, studies on the effects of ECs on fertility endpoints in fishes provide us with valuable information to establish biomarkers in risk assessment of ECs, and to understand the ECs-related fertility threat. The aim of the present review was to evaluate associations between ECs and fertility determinants to better understand ECs-related male fertility threat in male fishes. Wildlife studies show that the reproductive system has been affected in fishes sampled from the polluted aquatic environment. The laboratory studies show the potency of ECs including natural and synthetic hormones, alkylphenols, bisphenols, plasticizers, pesticides, pharmaceutical, alkylating, and organotin agents to affect fertility determinants, resulting in diminished fertility at environmentally relevant concentrations. Both wildlife and laboratory studies reveal that ECs adverse effects on male fertility are associated with a decrease in sperm production, damage to sperm morphology, alternations in sperm genome, and decrease in sperm motility kinetics. The efficiency of ECs to affect sperm quality and male fertility highly depends on the concentration of the contaminants and the duration of exposure. Our review highlights that the number of contaminants examined over fertility tests are much lower than the number of contaminants detected in our environment. The ECs effects on fertility are largely unknown when fishes are exposed to the contaminants at early developmental stages. The review suggests the urgent need to examine ECs effects on male fertility when a fish is exposed at different developmental stages in a single or combination protocol. The ECs effects on the sperm genome are largely unknown to understand ECs-related inheritance of reproductive disorders transmitted to the progeny. To elucidate modes of action of ECs on sperm motility, it is needed to study functional morphology of the motility apparatus and to investigate ECs-disrupted motility signaling.

Histone Acetylation Dynamics during In Vivo and In Vitro Oocyte Aging in Common Carp Cyprinus carpio.

Aging is the most critical factor that influences the quality of post-ovulatory oocytes. Age-related molecular pathways remain poorly understood in fish oocytes. In this study, we examined the effect of oocyte aging on specific histone acetylation in common carp Cyprinus carpio. The capacity to progress to the larval stage in oocytes that were aged for 28 h in vivo and in vitro was evaluated. Global histone modifications and specific histone acetylation (H3K9ac, H3K14ac, H4K5ac, H4K8ac, H4K12ac, and H4K16ac) were investigated during oocyte aging. Furthermore, the activity of histone acetyltransferase (HAT) was assessed in fresh and aged oocytes. Global histone modifications did not exhibit significant alterations during 8 h of oocyte aging. Among the selected modifications, H4K12ac increased significantly at 28 h post-stripping (HPS). Although not significantly different, HAT activity exhibited an upward trend during oocyte aging. Results of our current study indicate that aging of common carp oocytes for 12 h results in complete loss of egg viability rates without any consequence in global and specific histone modifications. However, aging oocytes for 28 h led to increased H4K12ac. Thus, histone acetylation modification as a crucial epigenetic mediator may be associated with age-related defects, particularly in oocytes of a more advanced age.

Optimization of Sperm Management and Fertilization in Zebrafish (Danio rerio (Hamilton)).

The aim of the present study was to investigate the spontaneous motility of spermatozoa and to optimize sperm collection, short-term sperm storage, and fertilization in zebrafish Danio rerio. The movement of spermatozoon in water was propagated along the flagellum at 16 s after sperm activation then damped from the end of the flagellum for 35 s and fully disappeared at 61 s after activation. For artificial fertilization, milt must be added to an immobilizing solution, which stops the movement of sperm and keeps the sperm motionless until fertilization. E400 and Kurokura as isotonic solutions were shown to be suitable extenders to store sperm for fertilization for 6 h. E400 stored sperm for 12 h at 0-2 °C. Sperm motility decreased only to 36% at 12 h post stripping for the E400 extender and to 19% for the Kurokura extender. To achieve an optimal level of fertilization and swim-up larvae rates, a test tube with a well-defined amount of 6,000,000 spermatozoa in E400 extender per 100 eggs and 100 µL of activation solution has proven to be more successful than using a Petri dish. The highest fertilization and swim-up larvae rates reached 80% and 40-60%, respectively, with milt stored for 1.5 h in the E400 extender at 0-2 °C.

Changes in Phenotypes and DNA Methylation of In Vitro Aging Sperm in Common Carp Cyprinus carpio.

The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.

Influence of Environmental Temperature and Hormonal Stimulation on the In Vitro Sperm Maturation in Sterlet Acipenser ruthenus in Advance of the Spawning Season.

Sturgeon sperm maturation occurs outside the testes during the transit of testicular spermatozoa (TS) through the kidneys and the Wolffian ducts. A method of in vitro TS maturation in sterlet Acipenser ruthenus was used to investigate the effects of temperature and hormonal stimulation of spermiation on the ability of TS to complete this process. Spermatozoa motility parameters after in vitro maturation of testicular sperm, concentrations of sex steroid hormones and testis morphology were studied in three groups of sterlet: (1) after overwintering in ponds (OW), (2) adapted to spawning temperature (ST), and (3) adapted to spawning temperature with hormonal induction of spermiation (ST-HI). Blood plasma concentrations of testosterone, 11-ketotestosterone and 17,20ß-dihydroxy-pregnenolone increased significantly after hormonal induction of spermiation (group ST-HI). In all groups, TS were not motile. After in vitro sperm maturation, motility was up to 60% only in group ST-HI. The data suggest that the ability of TS to be matured in vitro was not related to the environmental temperature, while hormonal stimulation of spermiation during the spawning season was an absolute requirement for optimal in vitro maturation.

Oocyte Ageing in Zebrafish Danio rerio (Hamilton, 1822) and Its Consequence on the Viability and Ploidy Anomalies in the Progeny.

Fish egg quality can be markedly influenced by the oocyte age after ovulation. In this study, we examined the duration of oocyte ageing in the zebrafish (Danio rerio) and whether prolonged ageing is associated with the incidence of ploidy anomalies in the resulting embryos. Oocytes were incubated in vitro for 6 h post-stripping (HPS) at 26 °C and fertilized at 2-h intervals. Meanwhile, for eggs fertilized immediately after stripping, the fertilization, embryo survival, and hatching rates started at ~80%; these rates decreased to 39%, 24%, and 16%, respectively, for oocytes that had been stored for 4 h (p ˂ 0.05), and there was an almost complete loss of egg viability at 6 HPS. Furthermore, almost 90% of the embryos derived from 6-h aged oocytes died prior to hatching, and all larvae originating from 4- and 6-h aged oocytes showed malformations. The proportion of ploidy abnormal embryos was significantly greater at 4 HPS (18.5%) than at either 0 or 2 HPS (4.7% and 8.8%, respectively). The results revealed that zebrafish oocytes retained their fertilization potential for up to 2 h after stripping at 26 °C and indicated the contribution of post-ovulatory oocyte ageing in the occurrence of ploidy anomalies in the resulting embryos.

Level of in vitro storage of the European catfish (Silurus glanis L.) eggs at different temperatures.

European catfish (Silurus glanis) is a commercially important freshwater fish originating from Eastern Europe. The objective of this study was to examine the short-term storage of its eggs especially in relation to maintaining a low level of malformation in newly hatched fry. The eggs from freshly spawned individuals were stored separately in cell incubators at 17 and 22 °C under aerobic conditions. Changes in fertilization, hatching, and malformation were examined in eggs stored at 1, 3, 5, and 7 h post-stripping. The sperm used for fertilization showed very good motility rates (84-90%) and curvilinear velocity (110-125 µm/s), and straight-line velocity did not drop below 77 µm/s. For all females, a temperature of 17 °C was better than 22 °C for egg storage in vitro. Egg fertilization and total hatching decreased rapidly after 7 h storage at 17 °C. The storage time of eggs in vitro to fertilization should therefore not exceed 5 h at 17 °C. In all females, there was no difference in the total number of eggs hatching between 1 and 3 h of egg storage at 17 °C. The storage time of eggs did not correlate with the level of malformations of the fry. However, the level of hatching and malformations was clearly affected by the storage temperature of eggs when it was > 17 °C. Analysis showed that the storage time of eggs, temperature of storage, and individual females had a significant influence on fertilization and total hatching rates. Regression analysis confirmed a low correlation of fertilization and hatching rates with storage time of eggs.

Improving motility and fertilization capacity of low-quality sperm of sterlet Acipenser ruthenus during storage.

Improvement of sperm quality with low motility by storage could ensure higher success of fertilization and maintain higher genetic diversity, especially for sturgeons, which as endangered species have limited broodstock and gametes. Sperm was collected from mature male sterlet Acipenser ruthenus and motility was evaluated using the CASA system; samples were categorized as GS 'good sperm' (>80%) or BS 'bad sperm' (<20%). Samples from both groups were incubated with seminal plasma from good- (GSP) and bad-quality sperm (BSP), respectively for 15 min, 6 h, 24 h and 96 h at 4 °C. Motility of BS incubated in GSP increased after different storage times compared to BS incubated in BSP, while the motility and velocity of GS incubated in BSP decreased compared to GS incubated in GSP. Fertilization rates were evaluated with samples stored for 15 min and 6 h post-stripping; fertilization and hatching rate of BS after incubation in GSP increased significantly compared to the BS incubated in BSP. Inorganic ion (Na+, K+, Cl-) concentrations and osmolality of BSP were significantly lower than that of GSP. These results indicated that sterlet sperm quality can be revitalized by incubation with GSP. Further, fertilization capacity of BS after incubation in GSP can reach similar levels to the good quality sperm (∼70%). Low ion concentration and osmolality in BSP may be a partial cause of low sperm quality. The current study is the first report on the capability to revitalize low quality sterlet sperm by storage in GSP.

Optimization of sterlet (Acipenser ruthenus) egg incubation.

Sterlet Acipenser ruthenus was used to assess egg and embryo development when incubated at 17 °C in Petri dishes placed in a hatchery tank (300 L recirculating dechlorinated water) with incubation occurring in a static tabletop system in an air-conditioned laboratory, or in a 700 L Q-cell incubator. Eggs in each dish were placed in a plastic box with 300 mL dechlorinated water. Separated eggs from three individual females were fertilized using pooled sperm from four males with there being four replicates. There were no differences (P > 0.05) in mean percentages of neurulation and embryos undergoing cleavage for eggs incubated in the hatchery tank and with use of the static tabletop system. Furthermore, there were no differences (P >  0.05) in percentage of embryos undergoing cleavage, neurulation and hatching for each female when eggs were incubated using the two systems. Results indicate a Petri dish placed in a small plastic box with 300 mL of dechlorinated water was adequate for incubation of sterlet eggs. Results of the study also indicate that with the static system: 1) eggs should be fertilized from each female to retain individual identity; 2) eggs should be dispersed in Petri dishes to avoid clumping; 3) water should be changed at 24 h, but not at 48 h (neurulation) post-fertilization; and 4) embryos that do not optimally develop should be removed the day after neurulation (72 h of post-fertilization period) and water should be exchanged every day subsequent to the 48 h time-point post-fertilization.

Transferrin Identification in Sterlet (Acipenser ruthenus) Reproductive System.

Transferrins are a superfamily of iron-binding proteins and are recognized as multifunctional proteins. In the present study, transcriptomic and proteomic methods were used to identify transferrins in the reproductive organs and sperm of out-of-spawning and spermiating sterlet (Acipenser ruthenus) males. The results showed that seven transferrin transcripts were identified in the transcriptome of sterlet, and these transcripts were qualified as two different transferrin genes, serotransferrin and melanotransferrin, with several isoforms present for serotransferrin. The relative abundance of serotransferrin isoforms was higher in the kidneys and Wolffian ducts in the spermiating males compared to out-of-spawning males. In addition, transferrin was immunodetected in sterlet seminal plasma, but not in sterlet spermatozoa extract. Mass spectrometry identification of transferrin in seminal plasma but not in spermatozoa corroborates immunodetection. The identification of transferrin in the reproductive organs and seminal plasma of sterlet in this study provides the potential function of transferrin during sturgeon male reproduction.

Sperm motility in fishes: (III) diversity of regulatory signals from membrane to the axoneme.

Fish spermatozoa acquire potential for motility in the sperm duct where they are immotile. Osmolality of the seminal plasma is a key factor to maintain spermatozoa in the quiescent state in either freshwater or marine fishes. However, potassium (K+) ions prevent spermatozoa motility in salmonid and sturgeon fishes, while CO2 inhibits spermatozoa motility in flatfishes. Once, spermatozoa are released at spawning, their motility is initiated in hypo-osmotic and hyper-osmotic environments in freshwater and marine fishes, respectively. Some substances produced by the testes (a progestin), or released from oocytes (peptides) induce spermatozoa hypermotility in some marine fishes including the Atlantic croaker and Pacific herrings, respectively. Duration of spermatozoa motility is short, lasting for a few seconds to few minutes in most fishes due to rapid depletion of energy required for the beating of the motility apparatus called axoneme. In the osmotic-activated spermatozoa, K+ and water effluxes occur in freshwater and marine fishes, respectively, which trigger spermatozoa motility signaling. In general, initiation of axonemal beating is associated with an increase in intracellular calcium (Ca2+) ions in spermatozoa of both freshwater and marine fishes and a post- or pre-increase in intracellular pH, while cyclic adenosine monophosphate (cAMP) remains unchanged. However, axonemal beating is cAMP-dependent in demembranated spermatozoa of salmonid and sturgeon fishes. Calcium from extracellular environment or intracellular stores supply required Ca2+ concentration for axonemal beating. Several axonemal proteins have been so far identified in fishes that are activated by Ca2+ and cAMP, directly or mediated by protein kinase C and protein kinase A, respectively. The present study reviews differences and similarities in complex regulatory signals controlling spermatozoa motility initiation in fishes, and notes physiological mechanisms that await elucidation.

Effects of antifreeze proteins on cryopreserved sterlet (Acipenser ruthenus) sperm motility variables and fertilization capacity.

The effect of antifreeze proteins on sterlet, Acipenser ruthenus sperm motility variables and fertilization rate were investigated after cryopreservation. Two types of antifreeze proteins (AFPI or AFPIII) were used at concentrations of 0.1, 1, 10 and 100 µg/mL. The motility variables of fresh and cryopreserved sperm with and without addition of antifreeze proteins were evaluated by the Computer Assisted Semen Analyzer (CASA). The fertilization rate using about 200,000 spermatozoa per egg was evaluated after 54 h incubation at 17 °C during the early stage of organogenesis. The motility, curvilinear velocity and straight-line velocity of fresh sperm was 93 ± 5%, 128 ± 13 µm/s and 89 ± 9 µm/s, respectively. There was a significant decrease of sperm motility rate between fresh sperm and cryopreserved sperm with/without addition of antifreeze proteins. The greatest motility among thawed samples was in the sperm cryopreserved with 10 µg/mL of AFPI (56 ± 20%), however, these data were not different compared to the sperm without antifreeze proteins (49 ± 14%). No statistical variations were detected in curvilinear velocity nor straight-line velocity. The fertilization rate with fresh sperm was 67 ± 7%. No significant differences were detected in fertilization rate between fresh and cryopreserved spermatozoa with/without addition of antifreeze proteins, except the sperm cryopreserved with 100 µg/mL of AFPIII (39 ± 14%). Thus, it is concluded that addition of antifreeze proteins to cryopreservation medium do not improve nor have toxicity effects on the quality and fertilization capacity of sterlet sperm after thawing.

Protective role of antifreeze proteins on sterlet (Acipenser ruthenus) sperm during cryopreservation.

The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100 µg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85 ± 4% motility and 160 ± 2 µm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44 ± 9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1 µg/mL of AFPIII (58 ± 14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5 ± 6% live cells, while the cryopreserved sperm only contained 26.6 ± 14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1 µg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10 µg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.

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 ±â€¯3% motility and 160 ±â€¯14 µm/s curvilinear velocity at 15 s post-activation. The motility rate of cryopreserved sperm (37 ±â€¯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.

Progress and challenges of fish sperm vitrification: A mini review.

To survive low temperature is required for a long-term storage (cryopreservation), cells should be vitrified to a state in which intracellular water is solidified without ice crystal formation. Two different approaches are described for fish sperm cryopreservation: 1) sperm conventional cryopreservation, in which extracellular water is partially crystallized and 2) sperm vitrification, in which both intra- and extra-cellular liquids are vitrified. Sperm vitrification has been applied to some fish species with limited success. Traditional vitrification requires rapid cooling/warming rates, small sample carriers, and using high permeable cryoprotectant concentrations. The latter cause cytotoxic effects which must be well managed and will require continuous effort to match an appropriate cryoprotectant with suitable apparatus and warming methods. Novel cryoprotectant-free sperm vitrification approach has been applied to several fishes. This review summarizes development of basic procedures and discusses advantages and disadvantages of vitrification when applied it to fish sperm.