Seminal Plasma Osmolality Research Articles

Do milt storage techniques affect the quality of carp spermatozoa during short-term storage?

Short-term storage and management of milt are crucial parts of hatchery practice. There is speculation that sperm stored in a small Eppendorf could not retain their optimal performance compared to those stored in a 50 ml cell culture flask (CCF), thus raising the question of discrepancy between laboratory and hatchery conditions. Therefore, this study aimed to compare sperm quality parameters, such as osmolality and the ionic composition of seminal plasma, spermatozoa motility and its ability to maintain the volume under various osmotic conditions (310, 230, 150, 90, and 20 mOsm/kg) in fresh sperm and after its storage for 24 h in 1.5 ml Eppendorf or 50 ml CCF at 4 °C without any extender. Freshly collected sperm had higher motility percentages at each osmotic condition than 24 h stored sperm in Eppendorf and CCF. There was no significant difference in spermatozoa motility at various osmotic conditions except 90 mOsm/kg osmolality. Besides, VCL, VSL, VAP, and LIN of spermatozoa, osmolality, and ionic composition of seminal plasma did not significantly differ between these two containers. This study suggested no difference between sperm stored in an Eppendorf or CCF regarding sperm functionality.

Effect of Phenolic Extract of Cress Seed on Cations and Osmolality in Rabbit Seminal Plasma

This study aimed to investigated the effect of cress seeds phenolic extract as an antioxidant on cations and osmolality of rabbit seminal plasma. Four groups (24 male and 10 female) of rabbits were taken (N=6). First group was control (Distilled water) and other groups received orally phenol extract (30, 60 and 90 mg/kg), once daily for 60 days. The information of cations concentrations and osmolality of seminal plasma were collected and evaluated. The concentrations of copper, cadmium, lead and potassium were significantly declined (p0.05). While the concentration of iron, sodium, calcium and osmolality increased significantly in the seminal plasma of experimental groups compared to control. Through the results, the appropriate dose of phenolic extract from local plant Rashad can be used as a medicine in improving human fertility through its role in reducing the damage of trace elements and improving semen osmolality, and as results of its antioxidant function.

Dietary Moringa oleifera leaves to male rainbow trout (Oncorhynchus mykiss) broodstock: Effects on sperm quality and reproductive performance

Moringa oleifera leaves (MOL), a superfood rich in protein and vitamins, is native to the Indian subcontinent. Moringa were added to the broodstock trout diet to examine their influence on the reproductive performance of male broodstock rainbow trout (Oncorhynchus mykiss). Females were fed with a commercial broodstock trout diet without any inclusion of moringa leaves. In 12 circular fiberglass tanks, 36 male rainbow trout aged 3+ (1786.36 ± 35.36 g; 56.08 ± 0.94 cm) were divided into four groups (n = 9) (control, MOL-1, MOL-2, and MOL-3) with three parallels each. Fourteen weeks of feeding trials were performed where powdered moringa leaf was used in the diets at various levels: 0% (control), 4% (MOL-1), 8% (MOL-2), and 16% (MOL-3). Sperm quality parameters and fertilization success were investigated. Sperm motility parameters were examined at 10.5 °C using a computer-aided sperm analysis (CASA) system, and all samples were analyzed on the same day. Sperm volume and seminal plasma osmolality did not differ significantly across groups (p > 0.05). Spermatozoa concentration (6.71 ± 0.25, 7.54 ± 0.31, 8.44 ± 0.55, and 7.66 ± 0.35 × 109 cells ml−1 in control, MOL-1, MOL-2, and MOL-3 groups, respectively), spermatocrit ratio (18 ± 2.55%, 25 ± 3.16%, 38 ± 6.44%, and 28 ± 5.15%), and motility duration differed significantly (p < 0.05) among groups. Total motility percentage did not change, but progressive motility (74.72 ± 5.94%, 88.52 ± 4.35%, 89.36 ± 1.76%, and 90.47 ± 2.67%) was considerably higher in all three moringa groups than in control one (p < 0.05). Average Path Velocity (VAP), Straight Line Velocity (VSL), and Curvilinear Velocity (VCL) significantly differ between control and treatment groups (p < 0.05). The fertilization rate in the MOL-2 group was similar to the control and MOL-3 groups, while significant differences were observed from the eyeing stage, with the highest result observed in the MOL-2 group (p < 0.05). Hatching rates were also highest in the MOL-2 group (81.65 ± 0.49%), followed by MOL-3 (79.08 ± 1.27%) and MOL-1 (74.40 ± 2.32%), with the control group having the lowest hatching rate (74.24 ± 0.98%) (p < 0.05). Progressive motility, VAP, VSL, and VCL have significantly enhanced in the moringa-treated groups than the control one, hence boosting the fertilization success. The present study revealed that moringa leaves in the diet improve the sperm quality parameters and enhance the reproductive performance in broodstock male rainbow trout (Oncorhynchus mykiss). Therefore, 8% moringa leaf can be recommended as a supplement diet for male rainbow trout broodstock.

Effects of dietary supplementation of multi-strain probiotics on semen quality, seminal plasma compositions, and fertilization ability of rainbow trout (Oncorhynchus mykiss) broodstock spermatozoa

The present experiment aims to study the effects of dietary multi-strain probiotics on semen quality, seminal plasma compositions, and fertilization ability of male rainbow trout. For this purpose, a total of 48 broodstocks with an average initial weight of 1366.1±33.8g were divided into 4 groups and 3 replicates. Fish were fed with diets containing 0 (control), 1×109 (P1), 2×109 (P2), and 4×109 (P3) CFU probiotic kg-1 diet for 12 weeks. Results showed that dietary supplementation of probiotics significantly increased plasma testosterone level, motility time of spermatozoa, sperm density, and spermatocrit value in P2 and P3 treatments and Na+ level in P2 treatment compared to the control group (P<0.05). The activities of aspartate aminotransferase and lactate dehydrogenase had significantly decreased in the P2 treatment compared to the control group (P<0.05). No considerable variations were observed between control fish and treatment groups (P>0.05) in semen biochemical parameters, percentage of motile spermatozoa, osmolality, and pH of seminal plasma. Based on the results, the highest fertilization rate (97.2±0.9%) and eyed egg survival (95.7±1.6%) were observed in the P2 treatment, and those values showed remarkable differences with the control group (P<0.05). The results indicated that multi-strain probiotics have potential efficacy on semen quality and fertilization ability of rainbow trout broodstock spermatozoa.

Cryopreservation of sperm in the spotted scat, Scatophagus argus (Linnaeus, 1766) - Conservation and aquaculture perspectives

This study emphasizes the need for sperm quality assessment and cryopreservation strategy in the spotted scat Scatophagus argus as the occurrence of male population of this species is limited. Analyses of sperm quality were made including ionic content and osmolality of seminal plasma and blood serum. Sodium chloride (0.9%) was optimized as the extender with a dilution ratio of 1:3 (milt:cryomedium) for all experiments. Cryoprotectants including dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), glycerol (GLY), propylene glycol (PG), ethylene glycol (EG) and methanol (MeOH) were screened at various concentrations and equilibration times. Cryopreservation was performed using the cryoprotectants DMSO, GLY, EG, and MeOH individually as well as in combinations. Experiment 1 was done with DMSO, GLY, EG and MeOH (5, 10 and 15%); experiment 2 was done with DMSO (5%) + GLY (5%), DMSO (5%) + EG (5%), DMSO (5%) + MeOH (5%). Simultaneously, experiment 3 was done with both cryoprotectants and co-cryoprotectants viz. DMSO (5%) + egg yolk (5%), DMSO (5%) + coconut water (5%), DMSO (5%) + tomato water (5%) and DMSO (5%) + honey (5%). The cooling sample was optimized and performed with a one-step freezing protocol of −5 °C/min from 25 °C to −150 °C. The results indicated that samples subjected to experiment 2 using DMSO (5%) + EG (5%) for 60 days of storage registered maximum motility (82.33 ± 2.51%), duration time of sperm motility (10.45± 1.01 min) and viability (84.14 ± 5.29%) of post-thaw cryopreserved sperm. Morphology and ultrastructure of post-thaw sperm showed smooth surface topography and normal architecture of cellular organelles. Biochemical analysis indicated a reduction in the protein content of post-thaw cryopreserved sperm. DNA integrity (Comet assay) showed 6.30 ± 1.00% of tail DNA in post-thaw sperm, whereas fresh sperm registered 2.41 ± 1.85%. Functional integrity using the HOST of post-thaw sperm showed a maximum of 82.66 ± 1.15% with DMSO (5%) + EG (5%). Finally, fertility of post-thaw sperm using mature eggs of wild-caught females indicated the entry of sperm by breaking the vitelline membrane. Overall it was demonstrated that the DMSO (5%) + EG (5%) was amenable to the long-term preservation of sperm. Therefore, this protocol can be used as an alternative for male broodstock development under captive breeding program.

Spermatozoa motility traits of chub (Squalius cephalus L.) under the influence of various water factors

Spermatozoa of different species are characterised by specific traits of movement and sensitivity spectrum to individual environmental factors. The present study aimed to evaluate spermatozoa motility traits and the effect of some water factors such as osmolality, sodium, potassium concentration, and pH on chub spermatozoa movement characteristics. Motility parameters characterising spermatozoa motility were determined using computer-assisted sperm analysis (CASA) and standardised conditions. Other features were also determined, such as the composition of seminal plasma and purine and pyridine nucleotide concentration in spermatozoa. The milt volume of chub from a natural watercourse ranged from 0.5 to 3.5 mL, and the mean spermatozoa concentration was 21.5 × 109 mL−1. Percentage of motile spermatozoa varied from 70% to 99%. Spermatozoa achieved VCL velocity of 53.7 µms−1, LIN of 75.0%, ALH of 0.85 µm, BCF of 15.4 Hz at 10 s post-activation and motility duration of 40 s in distilled water at 15°C and pH 8. The increasing osmolality of the external environment increased motility duration and slightly improved velocity. Motility duration was prolonged in sodium and potassium chloride solutions compared to non-ionic sugar solutions. In order to trigger the best chub’s spermatozoa motility trait, an ionic solution of osmolality of 150–200 mOsm kg−1 and pH 7–8 should be used. Osmolality close to the osmolality of seminal plasma 280 mOsm kg−1 immobilises spermatozoa. In seminal plasma, higher dispersion between the Na+ and K+ concentration was detected in chub compared to other representatives of Cypriniformes. The mean ATP concentration in immotile spermatozoa was 37.9 pmol 10−6 cells. Although some peculiarities in milt were detected, the spermatozoa motility traits of chub and its sensitivity to the environment are typical for Cypriniformes. Research in fish from different taxa according to their sensitivity to environmental components should continue as they differ from each other.

Seasonal changes in sperm freezability of sex-reversed female rainbow trout

In order to obtain sex-reversed females of Salmonid fish (SRF; neomales, masculinized females) the hormonal treatment of fertilized eggs or larvae is used. As a consequence, masculinized fish possess less-developed testes without functional spermatic ducts. Therefore, the semen of sex-reversed females is generally characterized by low or highly variable quality and reflects testicular rather than ejaculated semen. The aim of our study was to evaluate the alterations in sperm freezability of sex-reversed female rainbow trout during the progression of the spawning season. Sperm concentration, seminal plasma osmolality, semen pH, gonadosomatic index (GSI) and total sperm production (TSP) were monitored in fresh semen. The sperm motility parameters, adenosine triphosphate (ATP) production and sperm viability were examined throughout the spawning season in fresh and frozen/thawed semen. The day of the spawning season did not affect sperm motility of fresh semen. During the equilibration of semen, 2–3-fold increase in the sperm motility compared to fresh semen on each day of the spawning season was recorded. A decrease in the sperm motility after cryopreservation compared to equilibrated semen on each day of the spawning season was recorded. The highest ATP concentrations in fresh semen were noted on the 21st day of the spawning season, whereas in cryopreserved semen, these values were the highest on the 16th and 21st days of the spawning season. The viability of fresh semen did not change during the spawning season; however, the highest sperm viability after cryopreservation was observed between the 11th and 21st days of the spawning season. These results should be taken into account during the planning of cryopreservation of semen of valuable SRF individuals, which will be further used for fertilization of eggs under hatchery conditions.

Seasonal changes in the milt quality of spotted scat, Scatophagus argus: Implications for artificial propagation

The current study investigated the correlation among sperm production (milt volume, spermatocrit and sperm count), sperm motility characters and biochemical composition of seminal plasma during different seasons to estimate the quality of semen in the Spotted scat (Scatophagus argus). The sperm of 120 males were collected over a period of 2 years seasonally viz., summer, pre-monsoon, monsoon and post-monsoon. During the collection period, it was observed that there were significant (p < 0.05) seasonal variations in the semen quality including sperm motility and production more in the summer season than other seasons. The osmolality of seminal plasma varied from 343.33 to 370.33 mOsm/kg. The pH of seminal plasma elevated from 7.37 to 7.63 during monsoon and summer seasons. The motility of S. argus sperms correlated with the concentration of biochemical compounds viz., Ca2+, Mg+ and Cl− during the summer season. Based on the present observation it could be highlighted that sodium and potassium were negatively correlated (p < 0.01 and p < 0.05) effect on total protein (r = −0.826; −0.956), glucose (r = −0.819; −0.717), cholesterol (r = −0.978; −0.849), AST (r = −0.853; −0.897), ALT (r = −0.970; −0.967), calcium (r = -0.850; −0.697), magnesium (r = −0.852; −0.843) and chloride (r = −0.906; −0.926). In conclusion, many physical and biochemical compositions of S. argus semen improved during that the summer and pre-monsoon season as compared to that of monsoon and post-monsoon. Fertility tests were performed to determine the ability of spermatozoa to fertilize an egg. Highest fertilization (79.92 ± 4.60%) and hatching (80.75 ± 3.89%) rates were observed during summer, while the rates were lowest during monsoon (40.50 ± 4.52%; 53.92 ± 7.38%). This work forms a compiled data for standard semen quality, which in turn will help to select good milters for hatchery production, cryopreservation and assisted breeding programs of this fish.

Spermatology and sperm ultrastructure in farmed coho salmon (Oncorhynchus kisutch)

Fish sperm quality assessment is an essential process for commercial production. The goal of this study was to evaluate the quality of fresh milt of farmed coho salmon using a functional and structural approach. A total of 37 males were used to determined parameters such as sperm concentration, pH and osmolality of seminal plasma, viability, mitochondrial membrane potential and fertility. In addition, ultrastructure and morphology were assessed through transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The results show that coho salmon release a large volume of milt (55.08 mL ± 20.14 mL) with a mean concentration of 13.45 ± 3.40 × 109 spermatozoa mL−1. A positive correlation was found between sperm concentration and spermatocrit (P < 0.05; r = 0.8447). Milt contained a high percentage of motile sperm (85.00–95.00%) with a mean motility period of 45.25 ± 2.56 s, viability of 95.24 ± 2.60% and mitochondrial membrane potential of 91.33 ± 4.03%. The fertility rate was also high (96.00 ± 4.16%). SEM and TEM images revealed that mature spermatozoa of coho salmon have a total length of 32.85 ± 1.25 μm, are anacrosomal, and consist of an ovoid head, a short cylindrical mid-piece and a long flagellum which can be differentiated into a long main-piece and a short end-piece. The posterior part of the nucleus contains the nuclear fossa in which are located the distal and proximal centrioles, perpendicular to each other. There is only one mitochondrion, positioned as a ring in the mid-piece. The flagellum has two lateral side-fins, apparently of unequal length, and shows the typical “9 + 2” axoneme. These findings indicate that most of milt and sperm traits are within the range reported for other salmonids. Similarly, the results of morphology and ultrastructure are consistent with other salmonids, especially rainbow trout and brown trout.

Sperm cryopreservation of Prochilodus lineatus throughout the same reproductive season

The aim of this study was to determine the effect of seasonality on post-thaw sperm quality in Prochilodus lineatus. Sperm from 43 males was cryopreserved throughout the reproductive season (November–March). Fresh sperm quality, concentration, volume, seminal plasma pH, osmolality and ion concentration were analysed. Post-thaw sperm was analysed for its motility rate, velocities, beat-cross frequency, cell morphology, oxidative stress and fertilization capacity. Prochilodus lineatus fresh sperm yielded high-quality during the reproductive period (motility >80%). Post-thaw sperm quality changed throughout the season, with higher motility (63.2%–72.3%) and curvilinear velocity (VCL) (55.9–59.2 µm/s) from December to March. Sperm concentration was negatively correlated with post-thaw sperm motility and VCL, and positively correlated with seminal plasma Ca2+ concentration, catalase activity (CAT) and reactive oxygen species (ROS). Seminal plasma Ca2+ was positively correlated with ROS and negatively correlated with post-thaw sperm motility. Post-thaw sperm CAT and fertility correlated positively. Although post-thaw sperm quality was reduced in November, the increased CAT was efficient in maintaining its fertilization capacity. In order to face seasonal influence, the optimal period to cryopreserve P. lineatus spermatozoa is from December to March, when sperm exhibits characteristics which make spermatozoa more prone to resist cryodamage.

Multiple sperm collection as an effective solution for gamete management in pikeperch (Sander lucioperca)

The aims of this study were to investigate (a) the effects of single or multiple sperm collection with catheter, and (b) impacts of multiple sampling method (catheter or syringe) on sperm quality in pikeperch (Sander lucioperca). To compare the effects of sampling method, sperm was collected repeatedly from the same fish using catheter (n = 6) or syringe (n = 5) at 48, 52 and 56 h post-hormonal injection. For the single collection group as control, sperm was collected with catheter from 12 fish at 48 h post-injection. The results showed that multiple sperm collection with catheter does not significantly (P ˃ 0.05) influence sperm volume and density compared to single sperm collection. However, multiple collection with syringe led to drastic reduction of sperm density at 56 h post-injection. Moreover, sperm volume was influenced by post-injection time where the highest volume was obtained at 48 h post-injection. Furthermore, sperm density and osmolality of seminal plasma were significantly affected by sampling method and post-injection time where higher values were found for the samples collected with catheter at 48 h post-injection. Number of males producing milt with motile spermatozoa decreased along with multiple sperm collection. Sperm motility at 60 s post-activation was affected by the interaction of sampling method and post-injection time, and the highest value was obtained for the sperm collected with syringe at 48 h post-injection. Sperm curvilinear velocity (VCL) at 15 and 45 s post-activation was affected by sampling method and post-injection time, respectively. These findings contribute to the pikeperch hatchery practices, as multiple sperm collection could be applied during artificial reproduction if limited number of males are available.

Toward controlled breeding of the blackfin icefish Chaenocephalus aceratus (Lönnberg 1906): determination of spermatozoa concentration and evaluation of short- and long-term preservation of semen

Efforts to study the effects of environmental change on early development of Antarctic fishes would benefit from the ability to control reproduction by in vitro fertilization. Here, we established a spectrophotometric method to determine spermatozoa concentration in the semen of the blackfin icefish Chaenocephalus aceratus and we evaluated the potential for short (refrigeration) and long-term (cryopreservation) storage. The former will maximize the use of semen over several hours and the latter alleviate the risk that semen is unavailable when a spawning female is available. Seminal plasma osmolality and pH of GnRH-injected male semen were 423.65 ± 35.6 mOsm kg−1 and 8.05 ± 0.11, respectively. The predictive power of regression models between spermatozoa concentration and absorbance was determined from 200 to 600 nm and revealed a best-fitted correlation at 505 nm. The standard curve was tested against measured concentrations and the average difference between measured and estimated concentrations was 0.27 ± 10.23%. Semen could be kept on ice for up to 6 h with minimal loss ( 90% post-thawing motility using a salt-based extender (pH 7.5–7.85 and ~ 280 mOsm kg−1) and 10% DMSO after 72 h immersed in liquid nitrogen. Spermatozoa morphometry revealed significant inter-individual variability. Average head length, head width, and flagella length were 3.78 ± 1.03, 2.87 ± 0.78, and 17.75 ± 7.15 µm, respectively. Spermatids composed 9.7 ± 0.8% of total spermatogenic cells, probably indicating active spermatogenesis, but perhaps resulting to some degree from the semen extraction technique used. Variations on these baseline methods may facilitate semen preservation and ultimately in vitro fertilization in this and other notothenioid species, thereby enhancing our understanding of the responses of early-life stages of Antarctic fishes to future warming of the Southern Ocean.

Effects of constant and changing water temperature on sperm quality of the endangered Çoruh trout, Salmo coruhensis

ABSTRACT In this study, the cumulative influence of male acclimation temperature on sperm quality of the Çoruh trout Salmo coruhensis was compared during the sperm production period. Broodstocks were kept in constant (12.8ºC), and changing (8.4ºC to 12.8ºC) water temperature using well water and stream water until first spawning and sperm quality traits were compared. 20 individuals three-year-old males with a mean weight of 2147 ± 647 g and a mean length of 56 ± 2 cm were used for the two groups in this study. Total sperm motility (%), progressive motility (%), sperm volume (ml), seminal plasma pH, osmolality (mOsm/kg), spermatozoa concentration (cells/ml), and spermatozoa duration (sec) were investigated. Total sperm motility (96.40% ± 25.06) and sperm volume (13 ± 5.97 ml) in the stream water group were significantly higher than in the well water group (82.68% ± 11.52 and 6.2 ± 2.48 ml) (P < .05). These results indicate that S. coruhensis prefers changing and colder stream water during the sperm production period to produce a good quality gamete.

Effects of seminal plasma ionic content, pH and osmolality on spermatozoa motility in bester (Female Huso huso × Male Acipenser ruthenus) sturgeon

The ionic contents, pH and osmolality of seminal plasma and their relationships to sperm motility indices (sperm motility duration and percentage of motile sperm) were evaluated in bester. Semen was collected from six bester specimens and their seminal plasma was evaluated. The seminal plasma contained 54.5±2.8 mM sodium, 22.33±2.5 mM chloride, 7.1±1.2 mM potassium, 3.55±0.6 mM calcium and 0.58±0.09 mM magnesium. The result shows that Na+, Cl- and K+ (54.5±2.8, 22.33±2.5 and 7.1±1.2 mM, respectively) were predominant ions in bester seminal plasma like other sturgeons. However the concentrations of K+, Ca2+ and Cl- in bester seminal plasma were higher than those reported in beluga and sterlet as parental species. The mean pH and osmolality of seminal plasma were 8.09±0.34 and 127.6±20.8 mOsm kg-1, respectively. A significant negative regression was observed between osmolality and percentage motility (r=- 0.893).

Sperm quality in fish: Determinants and affecting factors

Fish sperm quality assessment is helpful for optimizing production and for monitoring the environmental state. Sperm can be monitored relatively easy and, to date, various analyses have been applied and proven to be helpful in this task. Among them, sperm motility parameters such as sperm speed are one of the main performance traits during assisted fish reproduction. Apart from motility the sperm concentration, volume, and seminal plasma pH and osmolality are also frequently evaluated and are the main sperm quality indicators measured in fish sperm. However, other parameters also determine sperm fertilization potential. Recent knowledge reveals several additional parameters of high importance for sperm function. Among them are DNA integration, membrane stability, mitochondria status and enzymatic activity. Measuring all these parameters in fish sperm provides complex knowledge regarding male fertility and helps to improve broodstock maintenance protocols as well as gamete handling and fertilization processes. This review focuses on the presentation of the sperm quality measures for freshwater and marine species of the fish and provides information regarding recent methods of sperm quality evaluation.

Hormonal stimulation of carp is accompanied by changes in seminal plasma proteins associated with the immune and stress responses

Hormonal stimulation in common carp is a routine practice to enhance sperm production and control gamete maturation. This study aimed to compare the proteome of carp seminal plasma between control and Ovopel-induced males using two-dimensional differential in-gel electrophoresis. Ovopel induction increased sperm volume, total sperm count, seminal plasma osmolality, and pH and decreased seminal plasma protein concentration. In total, 36 spots were identified (23 up- and 13 downregulated), corresponding to 23 proteins differentially abundant in seminal plasma after Ovopel induction (p < .05; fold change 1.2). The majority of proteins were associated with the immune and stress responses including the transport protein (hephaestin), antiproteases (fetuin, α2-macroglobulin, TIMP2), complement components (C3, complement factor B/C2A), regulator of the coagulation cascade (plasminogen), modulators of the innate immune response, such as intelectin, ApoA and ApoE, and the cathepsin/cystatin system, and stress response (enolase1). In addition, hormonal stimulation seems to be related to the proteins involved in lipid metabolism, signal transduction, and tissue remodeling. Our results suggest that hormonal stimulation is not just concomitant with the hydration of testis but also induces the synthesis and secretion of seminal plasma proteins involved in sperm maturation and protection against stress induced by administration of the exogenous hormone. SignificanceIt is well known that hormonal stimulation of male fish induces the final maturation of spermatozoa. However, molecular and biochemical basis underlying hormone-induced changes in semen is unknown at present. This study for the first time reveals, using proteomic approach, that hormonal stimulation in addition to hydration of testis is accompanied by significant changes in seminal plasma proteins related mainly to immune and stress response, lipid metabolism, signal transduction and tissue remodeling. These changes are associated with gene expression and synthesis and secretion of seminal plasma proteins by reproductive tissues. Overall, our results provide a framework for understanding the molecular mechanism responsible for hormonal stimulation in the reproductive tract of fish males.

Sperm quality analysis of normal season (NG) and out-season by photoperiod manipulation (PG) of male rainbow trout broodstock (Oncorhynchus mykiss)

Sperm quality parameters in rainbow trout (Oncorhynchus mykiss) were investigated during normal season spawning (November-January) and out-season spawning (July-August) treated with artificial photoperiod manipulation. Normal spawning males (n = 15) were kept in an open concrete pond under natural condition. Out-season spawning males (n = 15) were treated with artificial LED light (50lm/m2) in a closed concrete pond. In these two experimental groups, five fish were used in each of three spawning periods. The mean weight and body length of males (2 + years, n = 30) were 1213.43 ± 39.43g and 45.08 ± 0.62cm, respectively. Sperms were collected from July to August 2016 in the out-season spawning or photoperiod-manipulated group (PG) (water temperature 14.21 ± 0.31°C) and from December 2016 to January 2017, in the normal season spawning group (NG) (water temperature 8.81 ± 1.03°C). Volume of sperm, osmolality of seminal plasma, density of sperm, percentage of motile spermatozoa (MOT), curvilinear velocity (VCL), and duration of motility were measured for each male. Seminal plasma osmolality, density of sperm, and the motility of duration were 358.47 ± 37.24 and 308.87 ± 44.09, 4.37 ± 2.10 and 9.8 ± 1.56, and 8.8 ± 2.42 and 24.6 ± 6.76 in PG and NG, respectively. Fertilization rate was 37.79 ± 9.37% and 94.51 ± 1.33% in PG and NG, respectively. Sperm quality parameters showed significant differences in most of the cases (p < 0.05) and fertilization rate at eyed egg stage (150-160 degree-days) was significantly higher in normal season spawning group than the photoperiod-manipulated group (p < 0.05). Though the rate of fertilization was low in out-season, it was able to get enough gametes in summer using only artificial light having no changes in other parameters.

Effect of urine contamination on semen quality variables in Eurasian perch Perca fluviatilis L.

The objectives of the present study were to determine values for semen quality variables in the Eurasian perch (i.e., osmolality of seminal plasma as well as sperm motility characteristics analyzed with CASA system) in response to (1) the method of milt collection (stripping or catheterization) and (2) experimental contamination of catheterized semen with urine (0%, 5%, 10%, 20%, 30% and 50% of contamination). Additionally, the effect of short-term chilled storage of experimentally contaminated semen (during the 24 h post semen collection period) on motility characteristics was investigated. Use of a typical stripping procedure resulted in about 5%–10% contamination of semen with urine, what is much less compared with other species. Markedly lesser values of straight line velocity (VSL) and consequently less linearity of spermatozoa movement (LIN) in perch semen, however, occurred as a result of stripping (46 ± 4 μm/s and 38 ± 4% for VSL and LIN, respectively), when compared to sperm collected by catheterization (87 ± 5 μm/s and 77 ± 2% for VSL and LIN, respectively), indicate that even a 10% contamination of semen with urine may have negative effects on quality. Exposure of semen to urine resulted in a significant dose-dependent decrease in the percentage of motile spermatozoa (MOT) and both velocity variables (VSL and VCL). Amount of urine contamination also affected MOT, VCL, VSL and LIN value during short-term storage. In conclusion, it is important to avoid semen contamination by urine when using the stripping procedure in the Eurasian perch, either for controlled reproduction or sperm preservation.

Inhibitory effect of K+ ions and influence of other ions and osmolality on the spermatozoa motility of European burbot (Lota lota L.).

BackgroundIn fish with external fertilization, two main start-up mechanisms of the path that blocks or activates the spermatozoan motility apparatus are known. The main factor managing the path is osmolality or potassium ion. In burbot from the European and North American population, contradictory findings regarding the factors influencing the onset of spermatozoa motility were reported. The objective of the current study was to determine the effect of potassium and osmolality on the spermatozoa activation of European burbot, Lota lota (Actinopterygii, Gadiformes, Lotidae). Moreover, the influence of pH, as well as sodium ion concentrations on spermatozoa motility was investigated. Seven parameters characterising motility were traced by means of computer-assisted sperm analysis (CASA).Principal findingsThe spermatozoa of European burbot are K+ ion-sensitive. A 6-mM KCl solution significantly decreased motility, and above 12-mM (50 mOsm kg-1) totally ceased spermatozoa movement. Sucrose and Na+ solutions inhibited spermatozoa movement only at concentrations > 450–480 mOsm kg-1. Greater differences in the percentage of motile sperm between individuals were noted in solutions containing high concentrations of chemicals triggering sperm motility. The optimum osmolality for spermatozoa motility is in the range of 100–200 mOsm kg-1. The burbot spermatozoa were motile over a wide range of pH values with the best activation at pH 9.ConclusionIt was demonstrated that the spermatozoa of European burbot are inhibited by K+ ions similarly as in North American burbot. Other electrolyte and non-electrolyte solutions inhibit spermatozoa movement only if their osmolality is greater than that of the physiological osmolality of seminal plasma. The data provided on basic knowledge of burbot spermatozoa allow to ensure appropriate conditions during artificial reproduction and scientific research.

Estudio «in vitro» de la influencia de la osmolaridad del plasma seminal en la morfología del espermatozoide

IntroductionSperm morphology is a parameter closely related to the fertility of the male gamete. Various infertility-causing disorders have been associated with anomalous sperm sub-populations that may be accompanied by an altered osmolality. A pilot study was conducted to determine whether the increase or decrease in in vitro osmolality can alter the original morphology. Material and methodsThe study included 42 randomly selected patients who had been classified as normal according to WHO criteria. The samples were processed by a CASA - SCA 5.4 (Sperm Class Analyzer) (Microptic S.L, Barcelona, Spain). Seminal plasma osmolality was measured using an Advanced Osmometer 2020 3MO. ResultsThe in vitro assays carried out on 30 patients showed that an osmolality increase up two times the reference value (880mOsm/kg), caused a progressive loss of sperm viability and a significant increase in macrocephalic sperm heads, with an increase in sperm volume and area. An osmolality decrease (half of the reference value, 228 mOsmol/kg) in a sample of 12 patients had no effect on sperm viability, although the sperm sub-population study showed increased micro-cephalic sperm with lower volume and cephalic area. DiscussionSperm morphology is acquired by sperm during the epididymal maturation. It was found that changing in vitro seminal plasma osmolality did not seem to affect it. Only extremely high values of osmolality cause the formation of macrocephalic sperm heads and a progressive loss of viability. These in vitro assays can help us to understand the biochemical seminal plasma effect in the male gamete.