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.

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.

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 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.

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.

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.

Fish sperm motility analysis: the central role of the flagellum.

Motility analysis of spermatozoa relies on the investigation of either head trajectories or flagellum characteristics. Those two sets of parameters are far from being independent, the flagellum playing the role of motor, whereas the head plays a passive role of cargo. Therefore, quantitative descriptions of head trajectories represent a simplification of the complex pattern of whole sperm cell motion, resulting from the waves developed by the flagellum. The flagellum itself responds to a large variety of signals that precisely control its axoneme to allow activation, acceleration, slowing down or reorientation of the whole spermatozoon. Thus, it is obvious that analysis of flagellum characteristics provides information on the original source of movement and orientation of the sperm cell and presents additional parameters that enrich the panoply of quantitative descriptors of sperm motility. In this review, we briefly describe the methodologies used to obtain good-quality images of fish spermatozoa (head and especially flagellum) while they move fast and the methods developed for their analysis. The paper also aims to establish a link between classical analyses by computer-aided sperm analysis (CASA) and the descriptors generated by fish sperm flagellum analysis, and emphasises the information to be gained regarding motility performance from flagellum motion data.

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.

Role of Ca2+ in the IVM of spermatozoa from the sterlet Acipenser ruthenus.

The role of Ca2+ in sturgeon sperm maturation and motility was investigated. Sperm from mature male sterlets (Acipenser ruthenus) were collected from the Wolffian duct and testis 24h after hormone induction. Testicular spermatozoa (TS) were incubated in Wolffian duct seminal fluid (WDSF) for 5min at 20°C and were designated 'TS after IVM' (TSM). Sperm motility was activated in media with different ion compositions, with motility parameters analysed from standard video microscopy records. To investigate the role of calcium transport in the IVM process, IVM was performed (5min at 20°C) in the presence of 2mM EGTA, 100µM Verapamil or 100µM Tetracaine. No motility was observed in the case of TS (10mM Tris, 25mM NaCl, 50mM Sucr with or without the addition of 2mM EGTA). Both incubation of TS in WDSF and supplementation of the activation medium with Ca2+ led to sperm motility. The minimal Ca2+ concentration required for motility activation of Wolffian duct spermatozoa, TS and TSM was determined (1-2nM for Wolffian duct spermatozoa and TSM; approximately 0.6mM for TS). Motility was obtained after the addition of verapamil to the incubation medium during IVM, whereas the addition of EGTA completely suppressed motility, implying Ca2+ involvement in sturgeon sperm maturation. Further studies into the roles of Ca2+ transport in sturgeon sperm maturation and motility are required.

Protein profile of seminal plasma and functionality of spermatozoa during the reproductive season in the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss).

The purpose of the study was to analyze seminal plasma composition, sperm production, and sperm motility over the course of the spawning season in the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). The highest mean percent of motile sperm (carp, 98.3 ± 1.6%; rainbow trout, 92.8 ± 5.6%) and highest spermatozoon velocity (carp, 286.3 ± 7.8 µm sec-1 ; rainbow trout, 245.3 ± 8.3 µm sec-1 ) were observed in the middle phase of the spawning period, at 5 sec post-activation. Sperm volume and concentration increased in the early spawning period, then decreased significantly toward the end of the season in both species. Seminal plasma osmolality was 262-270 mOsmol kg-1 from carp and 232-248 mOsmol kg-1 from rainbow trout, with little variation over the spawning period. Protein concentration in carp seminal plasma was stable throughout the reproductive season, whereas the highest protein level (2.1 ± 0.3 mg mL-1 ) in rainbow trout was observed in the middle phase of the spawning period. Seminal plasma composition in both species was analyzed using two-dimensional polyacrylamide gel electrophoresis combined with mass spectrometry. Thirteen differentially expressed protein spots in carp seminal plasma and sixteen spots in rainbow trout seminal plasma varied over the course of the reproductive season. The unique proteins identified are involved in the regulation of spermatogenesis, sperm motility, maintenance of sperm membrane lipid stability, and antioxidant protection. These results provide a deeper understanding of the role of fish seminal plasma proteins as well as a list of novel markers of sperm quality. Mol. Reprod. Dev. 83: 968-982, 2016 © 2016 Wiley Periodicals, Inc.

The in vitro effect of temperature on motility and antioxidant response of common carp Cyprinus carpio spermatozoa.

The effect of temperature on Cyprinus carpio spermatozoa in vitro was investigated with spermatozoa activated at 4, 14, and 24°C. At 30s post-activation, motility rate was significantly higher at 4°C compared to 14 and 24°C, whereas highest swimming velocity was observed at 14°C. The thiobarbituric acid-reactive substance (TBARS) content was significantly higher at 14°C and 24°C than at 4°C in motile spermatozoa. No significant differences in catalase and superoxide dismutase activity relative to temperature were observed. This study provides new information regarding effect of temperature on lipid peroxidation intensity and spermatozoon motility parameters in carp. The elevation of TBARS seen at higher temperatures could be due to inadequate capacity of antioxidant enzymes to protect the cell against the detrimental effects of oxidative stress induced by higher temperatures.

The antioxidant system of seminal fluid during in vitro storage of sterlet Acipenser ruthenus sperm.

The role of the seminal fluid antioxidant system in protection against damage to spermatozoa during in vitro sperm storage is unclear. This study investigated the effect of in vitro storage of sterlet Acipenser ruthenus spermatozoa together with seminal fluid for 36 h at 4 °C on spermatozoon motility rate and curvilinear velocity, thiobarbituric acid reactive substance level, and components of enzyme and non-enzyme antioxidant system (superoxide dismutase and catalase activity and uric acid concentration) in seminal fluid. Spermatozoon motility parameters after sperm storage were significantly decreased, while the level of thiobarbituric acid reactive substances, activity of superoxide dismutase and catalase, and uric acid concentration did not change. Our findings suggest that the antioxidant system of sterlet seminal fluid is effective in preventing oxidative stress during short-term sperm storage and prompt future investigations of changes in spermatozoon homeostasis and in spermatozoon plasma membrane structure which are other possible reasons of spermatozoon motility deterioration upon sperm storage.

Characterization of proteolytic and anti-proteolytic activity involvement in sterlet spermatozoon maturation.

In sturgeon, the acquisition of the potential for motility activation called spermatozoon maturation takes place outside testes. This process can be accomplished in vitro by pre-incubation of immature testicular spermatozoa in seminal fluid collected from fully mature Wolffian duct sperm. Addition of trypsin inhibitor to the pre-incubation medium disrupts spermatozoon maturation. There are no available data for the role of proteolysis regulators in fish spermatozoon maturation, while their role is recognized in mammalian sperm maturation. The present study evaluated the involvement of seminal fluid proteases and anti-proteolytic activity in the sterlet spermatozoon maturation process. Casein and gelatin zymography and quantification of amidase and anti-proteolytic activity were conducted in sturgeon seminal fluid from Wolffian duct sperm and seminal fluid from testicular sperm, along with spermatozoon extracts from Wolffian duct spermatozoa, testicular spermatozoa, and testicular spermatozoa after in vitro maturation. We did not find significant differences in proteolytic profiles of seminal fluids from Wolffian duct sperm and ones from testicular sperm. Zymography revealed differences in spermatozoon extracts: Wolffian duct spermatozoon extracts were characterized by the presence of a broad proteolytic band ranging from 48 to 41 kDa, while testicular spermatozoon extracts did not show such activity until after in vitro maturation. The differences in amidase activity coincided with these results. It may not be the levels of proteolytic and anti-proteolytic activity per se, but the alterations in their interactions triggering a cascade of signaling events, that is crucial to the maturation process.

The role of Ca(2+) and Na (+) membrane transport in brook trout (Salvelinus fontinalis) spermatozoa motility.

The role of environmental ion composition and osmolality in Ca(2+) signaled activation was assessed in spermatozoa of brook trout Salvelinus fontinalis. Milt from ten mature males was obtained by abdominal massage. Spermatozoa motility was evaluated in 0, 100, and 300 mOsm/kg NaCl or sucrose solutions, buffered by 10 mM Tris-HCl pH 8.5. For investigation of spermatozoa reaction to external Ca(2+) concentration, 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the activation media as a calcium ions chelator. For investigation of the effect of external Na(+) concentration in conditions of low external Ca(2+), 100 µM amiloride was added to the EGTA-containing solutions as a Na(+) transport blocker. Low motility was observed in sucrose (Na(+) free) solutions containing 2 mM EGTA but not in Na(+) solutions containing 2 mM EGTA. Addition of amiloride led to significantly increased motility (P < 0.05) compared with sucrose (Na(+) free) solutions containing 2 mM EGTA. We conclude that Na(+) transport in Ca(2+)-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na(+) and Ca(2+) transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.

The antioxidant system of sterlet seminal fluid in testes and Wolffian ducts.

Oxidative stress is a possible source of spermatozoa function deterioration. Seminal fluid (SF) protects spermatozoa against reactive oxygen species (ROS) attack during development in testes and transit through the reproductive tract. Spermatozoa curvilinear velocity and percent of motile cells as well as changes in thiobarbituric acid-reactive substance (TBARS) content, superoxide dismutase, and catalase activity, and uric acid concentration in SF were evaluated in sterlet sperm collected from testes 24 h after hormone induction of spermiation and from Wolffian ducts at 12, 24, 36, and 60 h after hormone injection (HI). While testicular spermatozoa motility was not initiated in activating medium, Wolffian duct sperm showed low motility at 12 h, significant increase at 24 and 36 h, and decrease at 60 h. Testicular SF was characterized by the highest level of TBARS and activity of studied enzymes compared with SF from Wolffian duct sperm at 24 h post-HI. In fluid from Wolffian duct sperm, a significant increase in TBARS content was shown at 36-60 h post-HI. In contrast to testicular SF, in SF from Wolffian duct sperm, this increase was not counterbalanced by changes in the studied variables of antioxidant system. This may be the source of the observed decrease in spermatozoa motility parameters 60 h post-HI. The results may confirm a dual role of ROS in fish sperm physiology. The data with respect to decrease in sturgeon spermatozoa motility parameters at 60 h post-HI should be taken into account in artificial sturgeon propagation.

Hypotonic treatment prior to freezing improves cryoresistance of common carp (Cyprinus carpio L.) spermatozoa.

Post-thaw motility rate, curvilinear velocity (VCL), and fertilizing ability of carp spermatozoa can be improved by short-term treatment with moderately hypotonic media prior to freezing. Before cryopreservation, carp sperm samples were treated with NaCl solutions of differing osmolalities, ranging from 100 to 300mOsmkg(-1) for 10s, after which final osmolality was adjusted to 300mOsmkg(-1). The resulting sperm suspension was diluted 1:1 with cryoprotective medium and frozen using conventional techniques. Control samples were treated in the same way, without the pre-dilution step. Post-thaw motility rate in samples pretreated with 200mOsmkg(1) NaCl was significantly higher (44±10%) than in controls (21±15%) and samples pretreated with 100mOsmkg(-1) (25±15%) and 300mOsmkg(-1) (25±12%) NaCl. Significantly higher mean VCL were observed in samples pretreated with 100, 150, and 200mOsmkg(-1) (119±24, 118±22, and 115±32µms(-1), respectively) compared to controls (92±27µms(-1)). Fertilization rate of frozen-thawed sperm treated with 200mOsmkg(-1) solution of 2M NaCl was significantly higher (25±18%) than that of sperm treated with 300mOsmkg(-1) NaCl solutions (12±7%) and the control (9±6%).

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.

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.

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.