Sturgeon gut development: a unique yolk utilization strategy among vertebrates.

In vertebrates, maternally supplied yolk is typically used in one of two ways: either intracellularly by endodermal cells or extracellularly via the yolk sac. This study delves into the distinctive gut development in sturgeons, which are among the most ancient extant fish groups, contrasting it with that of other vertebrates. Our observations indicate that while sturgeon endodermal cells form the archenteron (i.e., the primitive gut) dorsally, the floor of the archenteron is uniquely composed of extraembryonic yolk cells (YCs). As development progresses, during neurulation, the archenteric cavity inflates, expands laterally, and roofs a semicircle of YCs. By the pharyngula stage, the cavity fully encompasses the YC mass, which begins to be digested at the hatching stage. This suggests a notable deviation in sturgeon gut development from that in other vertebrates, as their digestive tract initiates its function by processing endogenous nutrition even before external feeding begins. Our findings highlight the evolutionary diversity of gut development strategies among vertebrates and provide new insights into the developmental biology of sturgeons.

How do suboptimal temperatures affect polyploid sterlet Acipenser ruthenus during early development?

Sturgeons are ancient fish exhibiting unique genome plasticity and a high tendency to produce spontaneously autopolyploid genome states. The temperature profiles of the rivers in which sturgeon live and reproduce have been severely altered by human intervention, and the effect of global warming is expected to cause further temperature shifts, which may be detrimental for early developmental stages with narrow windows of thermal tolerance. The comparison of the performance of diploid and autopolyploid sturgeon kept at unfavourable temperatures contributes to scientific knowledge of the effects of polyploid genome states on organisms and can shed light on the ability of polyploids to cope with human-induced alterations to natural conditions. Using the sterlet Acipenser ruthenus as a model species, we carried out conventional artificial fertilization, as well as the induction of the second polar body retention (SPBR), of the first mitotic division suppression (FMDS) and of the second polar body retention followed by the first mitotic division suppression (SPBR+FMDS). Two experiments were conducted to evaluate the effect of polyploidy on two basic performance parameters, survival and growth. In Experiment 1, fish belonging to untreated, SPBR-, FMDS- and SPBR+FMDS-induced groups were kept at 10, 16 and 20°C from the neurula stage until the end of endogenous feeding. In Experiment 2, larvae from the untreated and SPBR-induced groups were reared at 10, 16 and 20°C after their endogenous feeding transition for 3 weeks. Based on our findings, we report that the embryos, prelarvae and larvae of triploid A. ruthenus do not differ from diploids in their ability to survive, grow and develop under suboptimal temperature conditions, while the survival of tetraploids was significantly reduced even at the optimal temperature and even more so at temperatures far from the optimum. This was also the case in the 2n/4n mosaics observed in FMDS-induced group. Thus, we assume that in tetraploid and 2n/4n individuals, the limits of thermal tolerance are closer to the optimum than in diploids. We also conclude that the hexaploid genome state is probably lethal in A. ruthenus since none of the hexaploids or 3n/6n mosaics arising from the SPBR+FMDS induction survived the prelarval period.

Intracytoplasmic sperm injection in sturgeon species: A promising reproductive technology of selected genitors.

Sturgeons are the most endangered species group and their wild populations continue to decrease. In this study, we apply intracytoplasmic sperm injection (ICSI), an assisted reproductive technology, for the first time in endangered and critically endangered sturgeons. Using various egg-sperm species combinations we performed different ICSI experiments with immobilized pre- or non-activated spermatozoa, single or many, fresh or cryopreserved. Then we evaluated the fertilization success as well as the paternity of the resultant embryos and larvae. Surprisingly, all experimental groups exhibited embryonic development. Normal-shaped feeding larvae produced in all egg-sperm species-combination groups after ICSI using single fresh-stripped non-activated spermatozoa, in one group after ICSI using single fresh-stripped pre-activated spermatozoa, and in one group after ICSI using multiple fresh-stripped spermatozoa. ICSI with single cryopreserved non-activated spermatozoa produced neurula stage embryos. Molecular analysis showed genome integration of both egg- and sperm-donor species in most of the ICSI transplants. Overall, ICSI technology could be used as an assisted reproduction technique for producing sturgeons to rescue valuable paternal genomes.

Induction of Spermiation in Sterlet Acipenser ruthenus by PLGA Microparticle Delivery with Sustained Alarelin Release.

Carp pituitary treatment versus poly (lactiac-co-glycolic acid) microparticles with slow release of Alarelin at 35 µg kg-1 or 200 µg kg-1 body weight to induce spermiation was compared in sterlet Acipenser ruthenus. All hormone treatments initially increased testosterone and 11-ketotestosterone, with a subsequent decline in testosterone but consistent high levels of 11-ketotestosterone at 48 and 72 h post-treatment. Spermiation did not differ between hormone-treated groups, and was not detected in controls receiving saline solution. Administration of the carp pituitary led to maximum sperm production 24 h post-treatment, followed by a decrease at 48 h post-treatment, with no sperm obtained at 72 h. The effect of Alarelin at 35 µg kg-1 bw and carp pituitary did not differ at 24 and 48 h post-treatment, whereas 200 µg kg-1 bw Alarelin was associated with significantly lower spermatozoon concentration 24 h post-treatment compared to carp pituitary, with no difference in milt volume. Higher relative sperm production was observed 48 h after injection of Alarelin at 200 µg kg-1 bw compared to carp pituitary. Spermatozoon motility was significantly higher in fish receiving Alarelin at 35 µg kg-1 bw than 200 µg kg-1 bw. The treatment with optimal effect on inducing spermiation was poly (lactic-co-glycolic acid) microparticles with slow release of Alarelin at 35 µg kg-1 bw.

Does the Rainbow Trout Ovarian Fluid Promote the Spermatozoon on Its Way to the Egg?

The fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid are likely the major sources of these triggers, which we confirmed here for rainbow trout (Oncorhynchus mykiss). The ovarian fluid affected significantly spermatozoa performance: it supported high velocity for a longer period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout ovarian fluid induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating medium. The interaction of the spermatozoa with the attracting agents was accompanied by the "turn-and-run" behavior involving asymmetric flagellar beating and Ca2+ concentration bursts in the bent flagellum segment, which are characteristic of the chemotactic response. Ovarian fluid created the optimal environment for rainbow trout spermatozoa performance, and the individual peculiarities of the egg (ovarian fluid)-sperm interaction reflect the specific features of the spawning process in this species.

Bioenergetic Pathways in the Sperm of an Under-Ice Spawning Fish, Burbot (Lota lota): The Role of Mitochondrial Respiration in a Varying Thermal Environment.

Regarding the sperm of cold-water fish, the contributions of different bioenergetic pathways, including mitochondrial respiration, to energy production at the spawning temperature and its adaptation at the maximum critical temperature (CTmax) are unclear. The roles of glycolysis, fatty acid oxidation, oxidative phosphorylation (OXPHOS) at 4 °C, and OXPHOS at 15 °C for energy production in burbot (Lota lota) spermatozoa were studied by motility and the oxygen consumption rate (OCR) (with and without pathway inhibitors and the OXPHOS uncoupler). At both temperatures, the effects of the inhibitors and the uncoupler on the motility duration, curvilinear velocity, and track linearity were insignificant; in addition, the OCRs in activation and non-activation media differed insignificantly and were not enhanced after uncoupler treatment. After inhibitor treatment in both media, OXPHOS was insignificantly different at the 2, 30, and 60 s time points at 4 °C but was reduced significantly at the 30 and 60 s time points after treatment with sodium azide at 15 °C. In conclusion, for burbot sperm at both the spawning temperature and the CTmax, the energy synthesized via OXPHOS during motility was insufficient. Therefore, the majority of the energy required to sustain motility was derived from pre-accumulated energy produced and stored during the quiescent state of the spermatozoa.

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.

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.

Ploidy Levels and Fitness-Related Traits in Purebreds and Hybrids Originating from Sterlet (Acipenser ruthenus) and Unusual Ploidy Levels of Siberian Sturgeon (A. baerii).

The present study aimed to investigate and compare fitness-related traits and ploidy levels of purebreds and hybrids produced from sturgeon broodstock with both normal and abnormal ploidy levels. We used diploid Acipenser ruthenus and tetraploid A. baerii males and females to produce purebreds and reciprocal hybrids of normal ploidy levels. Likewise, we used diploid A. ruthenus and tetraploid A. baerii females mated to pentaploid and hexaploid A. baerii males to produce hybrids of abnormal ploidy levels. Fertilization of ova of A. ruthenus and A. baerii of normal ploidy with the sperm of pentaploid and hexaploid A. baerii produced fully viable progeny with ploidy levels that were intermediate between those of the parents as was also found in crosses of purebreds and reciprocal hybrids of normal ploidy levels. The A. ruthenus × pentaploid A. baerii and A. ruthenus × hexaploid A. baerii hybrids did not survive after 22 days post-hatch (dph). Mean body weight and cumulative survival were periodically checked at seven-time intervals. The recorded values of mean body weight were significantly higher in A. baerii × pentaploid A. baerii hybrids than other groups at three sampling points (160, 252 and 330 dph). In contrast, the highest cumulative survival was observed in A. baerii × A. ruthenus hybrids at all sampling points (14.47 ± 5.70 at 497 dph). Overall, most of the studied sturgeon hybrids displayed higher mean BW and cumulative survival compared to the purebreds. The utilization of sturgeon hybrids should be restricted to aquaculture purposes because they can pose a significant genetic threat to native populations through ecological interactions.

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.

Sperm antioxidant system in ocellate river stingray Potamotrygon motoro at transition from seminal vesicle to cloaca.

The importance of reactive oxygen species and the antioxidant system in sperm biology has been recognized for different bony fishes but nothing is known in this regard for chondrichthyans. For the first time for cartilaginous fishes, the enzymatic antioxidant system was shown herein to be present in both fractions of sperm (spermatozoa and seminal fluid) collected from two different places (seminal vesicle and cloaca). In internally fertilizing freshwater ocellate river stingray, Potamotrygon motoro, the activity of superoxide dismutase and glutathione peroxidase was not changed upon sperm transition from the seminal vesicle to the cloaca. The activity of catalase was significantly increased for both sperm fractions at transition from the seminal vesicle to the cloaca (1.6 times for spermatozoa and 1.9 times for seminal fluid). The role of the sperm antioxidant system for different aspects of internal fertilization is discussed. The presented results are the initiatory step in uncovering the biochemical events of internal reproduction in Chondrichthyes.

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.

Delivery of Iron Oxide Nanoparticles into Primordial Germ Cells in Sturgeon.

Nanoparticles are finding increasing applications in diagnostics, imaging and therapeutics in medicine. Iron oxide nanoparticles (IONs) have received significant interest of scientific community due to their distinctive properties. For the first time, we have delivered IONs into germ cells in any species. Our results showed that sturgeon primordial germ cells (PGCs) delivered with IONs could be detected until seven days post fertilization (dpf) under fluorescent microscope and at 22 dpf by micro-CT. Delivery of IONs into cells could be helpful for studying germ cell biology and the improvement of germ cell-based bio-technologies as isolation of PGCs using magnetic activated cell sorting or application of hyperthermia for a host sterilization purpose. Intriguingly, in our study, we did not find any toxic effects of IONs on the survival and hatching rates of sturgeon embryos when compared with embryos injected with FITC-dextran only.

Dnd1 Knockout in Sturgeons By CRISPR/Cas9 Generates Germ Cell Free Host for Surrogate Production.

Sturgeons also known as living fossils are facing threats to their survival due to overfishing and interference in natural habitats. Sterlet (Acipenser ruthenus) due to its rapid reproductive cycle and small body size can be used as a sterile host for surrogate production for late maturing and large sturgeon species. Dead end protein (dnd1) is essential for migration of Primordial Germ Cells (PGCs), the origin of all germ cells in developing embryos. Knockout or knockdown of dnd1 can be done in order to mismigrate PGCs. Previously we have used MO and UV for the aforementioned purpose, and in our present study we have used CRISPR/Cas9 technology to knockout dnd1. No or a smaller number of PGCs were detected in crispants, and we also observed malformations in some CRISPR/Cas9 injected embryos. Furthermore, we compared three established methods to achieve sterility in sterlet, and we found higher embryo survival and hatching rates in CRISPR/Cas9, UV and MO, respectively.

Sperm motility and lipid composition in internally fertilizing ocellate river stingray Potamotrygon motoro.

All extant groups of Elasmobranches have internal fertilization and the structure of the male reproductive organs is very specific: sperm passes from the internal organs via the cloaca, but the male copulating organ (clasper) is distant from the cloaca. This suggests that sperm can contact the surrounding medium before fertilization. Because of this involvement with the environment, external signaling in sperm motility activation could occur in these species even though their fertilization mode is internal. In this case, spermatozoa of Elasmobranches should hypothetically possess a specific structure and membrane lipid composition which supports physiological functions of the sperm associated with environmental tonicity changes occurring at fertilization. Additionally, sperm motility properties in these taxa are poorly understood. The current study examined sperm lipid composition and motility under different environmental conditions for the ocellate river stingray, Potamotrygon motoro, an endemic South America freshwater species. Sperm samples were collected from six mature males during the natural spawning period. Sperm motility was examined in seminal fluid and fresh water by light video microscopy. Helical flagellar motion was observed in seminal fluid and resulted in spermatozoon progression; however, when diluted in fresh water, spermatozoa were immotile and had compromised structure. Lipid class and fatty acid (FA) composition of spermatozoa was analyzed by thin layer and gas chromatography. Spermatozoa FAs consisted of 33 ±â€¯1% saturated FAs, 28 ±â€¯1% monounsaturated FAs (MUFAs), and 41 ±â€¯1% polyunsaturated FAs (PUFAs), and a high content of n-6 FAs (32 ±â€¯2%) was measured. These results allowed us to conclude that sperm transfer from P. motoro male into female should occur without coming into contact with the hypotonic environment so as to preserve potent motility. In addition, this unusual reproductive strategy is associated with specific spermatozoa structure and lipid composition. Low level of docosahexaenoic acid and relatively low PUFA/MUFA ratio probably account for the relatively low fluidity of freshwater stingray membrane and can be the main reason for its low tolerance to hypotonicity.

Cryopreservation effects on a viable sperm sterlet (Acipenser ruthenus) subpopulation obtained by a Percoll density gradient method.

In many fish species, sperm cryopreservation has deleterious effects and leads to a significant decrease in spermatozoa viability. However, the effect of cryopreservation on sperm cells that survive this process and are still viable is not fully understood. The objective of this study was to compare the viability and proteomes of fresh and cryopreserved sterlet (Acipenser ruthenus) sperm samples before and after live-dead cell separation using Percoll density gradient centrifugation. Both fresh and cryopreserved sperm samples were divided into two groups (with or without application of Percoll separation). At each step of the experiment, sperm quality was evaluated by video microscopy combined with integrated computer-assisted sperm analysis software and flow cytometry for live-dead sperm viability analysis. Sperm motility and the percentage of live cells were reduced in the cryopreserved group compared to the fresh group from 89% to 33% for percentage of motility and from 96% to 70% for live cells. Straight line velocity and linearity of track were significantly lower in cryopreserved samples than in those separated by Percoll before and after cryopreservation. However, the percentages of motile and live spermatozoa were higher than 90% in samples subjected to Percoll separation. Proteomic analysis of spermatozoa by two-dimensional differences in-gel electrophoresis coupled with matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that 20 protein spot abundances underwent significant changes in cryopreserved samples compared to fresh ones. However, only one protein spot was significantly altered when samples before and after cryopreservation followed by Percoll separation were compared. Thus, the results of this study show that cryopreservation leads to minimal proteomic changes in the spermatozoa population, retaining high motility and viability parameters. The results also suggest that global differences in protein profiles between unselected fresh and cryopreserved samples are mainly due to protein loss or changes in the lethal and sublethal damaged cell subpopulations.

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.