Post-testicular sperm maturation in ancient holostean species.

Fish speciation was accompanied by changes in the urogenital system anatomy. In evolutionarily modern Teleostei, male reproductive tracts are fully separated from the excretory system, while in evolutionarily ancient Chondrostei and Holostei, the excretory and reproductive tracts are not separated. Sturgeon post-testicular sperm maturation (PTSM) occurring as a result of sperm/urine mixing is phenomenologically well described, while, in holosteans, functional intimacy of seminal ducts with kidney ducts and the existence of PTSM still need to be addressed. In Lepisosteus platostomus (Holostei), sperm samples were collected from testes (TS), efferent ducts (EDS), and Wolffian ducts (WDS). While WDS was motile, no motility was found in TS and EDS. The existence of PTSM was checked by in vitro PTSM procedure. After TS and EDS incubation in seminal fluid from WDS, no more than 5% motile spermatozoa were observed in TS, whereas in EDS the motility percentage was up to 75%. Experimental dyeing of urogenital ducts in gars and sturgeons revealed some differences in the interconnection between sperm ducts and kidneys. It is concluded that post-testicular sperm maturation occurs in gars and suggests that infraclass Holostei occupies an intermediate evolutionary position between Teleostei and Chondrostei in the anatomical arrangement of the urogenital system.

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.

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.

Egg-sperm interaction in sturgeon: role of ovarian fluid.

Fertilization of freshwater fish occurs in the environment which negatively affects a lifespan of gametes mostly due to the osmotic shock; therefore, male gametes should reach the female gamete, as soon as possible. The existence of mechanisms controlling the encounter of gametes would be highly expedient in this case. By analogy with other species for which guidance was demonstrated, it is likely that this control may be performed by ovarian fluid or substances released by eggs. The aim was to study the effect of ovarian fluid and egg-released substances on spermatozoa behavior in sterlet. It was found that the presence of a particular concentration of ovarian fluid (30% solution in water) had an inhibiting effect on spermatozoa motility initiation. Lower concentrations of the ovarian fluid improved the longevity of spermatozoa and did not affect their trajectories. Test of chemotactic response (using a microcapillary injection of fluids into the suspension of motile spermatozoa) showed no effect of ovarian fluid on spermatozoa behavior, while at the same time, the attracting effect of the egg-conditioned medium was evident (i.e., due to some substances released from the eggs during their contact with freshwater). The results of the fertilization test showed that the presence of ovarian fluid prevented the eggs from losing the fertilizing ability due to the contact with water, as well as promoted the spermatozoa to fertilize the eggs during a longer period of time. Thus, the combined physicochemical action of "female factors" affects sterlet gametes during fertilization and may be involved in the guidance and selection mechanisms.

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.

Different glycolipids in sperm from different freshwater fishes - A high-performance thin-layer chromatography/electrospray ionization mass spectrometry study.

RATIONALE: Glycolipids play important roles in many physiological processes - despite their commonly low abundance. This study summarizes selected data on the (glyco)lipid composition of sperm from different fish species. METHODS: Lipid extraction of fish sperm was performed according to the procedure by Bligh and Dyer. The lipid composition of the organic extracts was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization ion trap (ESI-IT)MS coupled to high-performance thin-layer chromatography (HPTLC). RESULTS: It was shown that sperm from carp, northern pike, rainbow trout and burbot contain high amounts of neutral and acidic glycosphingolipids as well as sulfoglycolipids. These particular lipids are presumably involved in reproduction requirements. CONCLUSIONS: Phospholipids and glycolipids in crude lipid extracts can be analyzed in parallel by MS coupled to TLC. The direct application of tandem mass spectrometry (MS/MS) helps to elucidate the glycolipid structure. Thus, compositional analysis can be performed very rapidly.

Energy pathways associated with sustained spermatozoon motility in the endangered Siberian sturgeon Acipenser baerii.

Sturgeon spermatozoa are unique for their sustained motility. We investigated the relative importance of bioenergetic pathways in the energy supply of Siberian sturgeon Acipenser baerii spermatozoa during motile and immotile states. Spermatozoon motility and oxygen consumption rate (OCR) were analysed following exposure to inhibitors of oxidative phosphorylation (sodium azide, NaN3 ), glycolysis (2-deoxy-D-glucose, DOG) and ß-oxidation of fatty acids (sodium fluoride, NaF), and to an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenyl hydrazine, CCCP). No significant difference in curvilinear velocity was observed after addition of these reagents to activation medium (AM) or nonactivation medium (NAM) for incubation. Incubation of spermatozoa in NAM containing CCCP or NaN3 resulted in significantly decreased motility duration compared to controls. The OCR of sturgeon spermatozoa in AM (11.9 ± 1.4 nmol O2 min-1 (109 spz)-1 ) was significantly higher than in NAM (8.2 ± 1.5 nmol O2 min-1 (109 spz)-1 ). The OCR significantly declined with addition of NaN3 to AM and NAM. No significant difference in motility parameters or OCR was observed with NaF or DOG. These results suggest active oxidative phosphorylation in both immotile and motile spermatozoa. Nevertheless, mitochondrial respiration occurring during motility is not sufficient to meet the high energy demands, and the energy required for sustained motility of Siberian sturgeon spermatozoa is derived from adenosine triphosphate accumulated during the quiescent state.

Sperm Lipid Composition in Early Diverged Fish Species: Internal vs. External Mode of Fertilization.

The lipid composition of sperm membranes is crucial for fertilization and differs among species. As the evolution of internal fertilization modes in fishes is not understood, a comparative study of the sperm lipid composition in freshwater representatives of externally and internally fertilizing fishes is needed for a better understanding of taxa-specific relationships between the lipid composition of the sperm membrane and the sperm physiology. The lipidomes of spermatozoa from stingray, a representative of cartilaginous fishes possessing internal fertilization, and sterlet, a representative of chondrostean fishes with external fertilization, have been studied by means of nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), electrospray MS, gas chromatography-(GC) MS, and thin-layer chromatography (TLC). NMR experiments revealed higher cholesterol content and the presence of phosphatidylserine in stingray compared to sterlet sperm. Unknown MS signals could be assigned to different glycosphingolipids in sterlet (neutral glycosphingolipid Gal-Cer(d18:1/16:0)) and stingray (acidic glycosphingolipid sulpho-Gal-Cer(d18:1/16:0)). Free fatty acids in sterlet sperm indicate internal energy storage. GC-MS experiments indicated a significant amount of adrenic acid, but only a low amount of docosahexaenoic acid in stingray sperm. In a nutshell, this study provides novel data on sperm lipid composition for freshwater stingray and sterlet possessing different modes of fertilization.

Ultrastructural feature of spermatogenic cells and spermatozoon in cultured burbot Lota lota.

Testis development and ultrastructure of spermatogenic cells and spermatozoa of burbot Lota lota, a commercially important cold freshwater fish, were studied by light and transmission electron microscopy. Spermatogonia, spermatocytes, spermatids, and spermatozoa are distributed along the seminiferous tubules. Electron-dense bodies appear in germ cells from primary spermatogonia to secondary spermatocytes. We identified three distinct stages of spermatid cell differentiation based on chromatin condensation, development of the flagellum, formation of a nuclear fossa, and elimination of excess cytoplasm. Spermatozoa were anacrosomal and characterized by location of the centrioles outside the nuclear fossa and incomplete perpendicular arrangement of the centrioles. The sperm flagellum displayed an axoneme with nine doublets of peripheral microtubules and two central microtubules. These results provide valuable information for burbot taxonomy and may clarify the process of spermatogenesis for this species.

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.

In vitro antioxidant enzyme activity and sperm motility at different temperatures in sterlet Acipenser ruthenus and rainbow trout Oncorhynchus mykiss.

Influence of in vitro temperature on sperm antioxidant enzyme activity, thiobarbituric acid-reactive substance (TBARS) content and motility parameters was evaluated in sterlet Acipenser ruthenus and rainbow trout Oncorhynchus mykiss. Sperm activation was conducted at 4, 14 and 24 °C in both species. Duration of motility was significantly longer at 4 °C than at 14 and 24 °C in both species. At 60 s post-activation, the velocity of sterlet spermatozoa was highest at 24 °C. This trend continued to 420 s post-activation. In rainbow trout, at 10 s post-activation, the highest velocity was observed at 14 °C. Significantly higher catalase activity was seen at 4 °C in both species. No significant difference in spermatozoon superoxide dismutase activity among temperatures was observed. In sterlet, TBARS content was significantly higher at 24 °C compared to other temperatures, but, in rainbow trout, it was highest at 4 °C. The results presume species-specific level of antioxidant enzyme activity and TBARS content at studied temperatures.

Effects of temperature on sperm motility of burbot Lota lota: spontaneous activation and calcium dependency.

Several factors regulating activation of spermatozoon motility in Eurasian burbot, Lota lota, including osmolality, calcium (Ca2+ ) ions, and temperature were investigated. Spermatozoon motility in Eurasian burbot, Lota lota was assessed at 4 and 30°C in seminal fluid, isotonic media (with and without Ca2+ ) and hypotonic media (with and without Ca2+ ). Spermatozoa were spontaneously activated in seminal fluid at 20°C and the maximum motility was recorded at 30°C, which is out of the spawning temperature range, indicating that no risk of activation occurs during routine semen handling in artificial insemination. Initiation of spermatozoon motility in L. lota is mediated by Ca2+ and sensitivity to Ca2+ is dependent on temperature.

Fish sperm biology in relation to urogenital system structure.

Morphology of the urogenital system has evolved during fish speciation. Chondrostei (sturgeons and paddlefishes) possess an excretory system which is called "primitive" in that the sperm ducts enter the kidneys and share the excretory ducts where sperm is mixed with urine before it is released into the spawning environment. Further, in this group of fishes there are also physiological characteristics which are associated with these anatomical features where the mixing of sperm and urine is a prerequisite for the final sperm maturation rather than contamination. In the Holostei (gars and bowfins) which are closely related to the Chondrostei, sperm also naturally mixed with urine, but the physiological role of such mixing for sperm biology has not been described. In contrast, urinary and sperm ducts in the more evolved Teleostei are completely separate, and sperm and urine are not mixed before being released during spawning. Thus, urine constitutes an inappropriate environment which can be a source of problems when sperm is collected during fisheries practices. In this review, the consequences of such divergent conditions in the urogenital anatomy will be considered in relation to general features of fish sperm biology and in relation to aquaculture and fisheries practices.

Swimming at different temperatures: The lipid composition of sperm from three freshwater fish species determined by mass spectrometry and nuclear magnetic resonance spectroscopy.

The spawning behavior of different fish species is as diverse as their habitats. A lot of factors influence the (phospho)lipid composition of fish sperm, including the water temperature at which spawning takes place. Therefore, this study aimed on the elucidation of the phospholipid composition of sperm from three fish species from different orders (common carp - Cyprinus carpio, northern pike - Esox lucius and burbot - Lota lota) with different spawning temperatures by nuclear magnetic resonance spectroscopy (NMR), matrix-assisted laser desorption/ionization mass spectrometry and thin-layer chromatography (TLC) coupled to electrospray ionization mass spectrometry as well as gas chromatography. Next to the lipid composition that was different for carp, northern pike and burbot, regarding the moieties of the different (phospho)lipid classes (particularly sphingomyelin and acidic phospholipids) and the saturation degree of the fatty acyl residues, there were differences observed depending on the analytical method that was used. The results from TLC and NMR investigations differed regarding the amounts of the different phospholipids. Reasons for these discrepancies are discussed in detail.

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.

Sperm motility of the Nile tilapia (Oreochromis niloticus): Effects of temperature on the swimming characteristics.

Results of previous studies with different fish species, mostly from temperate- or cold-water habitats, indicate a species-specific diversity regarding the relationship between environmental temperature and values for sperm motility variables. In the current study, there was appraisal of environmental temperature effects on sperm motility of tilapia Oreochromis niloticus, a tropical fish species selected because of its aquaculture importance and capacity to reproduce in a broad range of water temperatures. Effects of environmental temperature on the spermatozoa motility characteristics were studied by temperature-controlled video-microscopy and CASA analysis at temperature range from 5 to 50 °C. It appeared that the Nile tilapia spermatozoa exhibit an unexpected capacity to express very different velocity characteristics over this temperature range. In the lower temperature range (5-10 °C), the percentage of motile cells was markedly variable among males. An abrupt increase in the linearity index was observed between 15 and 20 °C suggesting a physiological threshold in sperm movement at about 20 °C which is the minimum temperature for reproduction in the Nile tilapia. With faster spermatozoa velocity, there was a reduction of the motility duration at the greater temperatures. Initially, there is an increase in sperm velocity as the temperature increased until the maximal velocity occurred at 40 to 50 °C which is a temperature beyond that which occurs in natural spawning conditions. Results of the present study clearly indicate the importance of considering ambient temperature when charactering sperm motility and in determining optimal temperature conditions for fertilization in fish.

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.

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.