Andrzej Ciereszko

Estimation of sperm concentration of rainbow trout, whitefish and yellow perch using a spectrophotometric technique

Abstract Milt was collected individually from rainbow trout ( Oncorhynchus mykiss ), whitefish ( Coregonus clupeaformis ) and yellow perch ( Perca flavescens ) and sperm density measured by three different methods: measuring spermatocrit value, counting sperm using a Neubauer counting chamber, and by measuring absorbance at 505 nm. Sperm concentration significantly differed between yellow perch (41.58±3.5×10 9 ml −1 ) and the two other species, rainbow trout (11.80±6.19×10 9 ) and whitefish (7.95±2.57×10 9 ). In diluted yellow perch sperm and directly sampled sperm of whitefish and rainbow trout, correlation coefficients were highly significant between spermatocrit, counting, and the spectrophotometric method of measurement. It is suggested that sperm concentration measured by optical density offers a quick and accurate method of determining sperm concentration.

Effects of season and breed on sperm acrosin activity and semen quality of boars

Acrosin activity and semen quality (sperm concentration, ejaculate volume and number of spermatozoa) were assessed from March 1997 to March 1998 in semen of Large White, Pietrain and Duroc x Pietrain boars. Semen quality varied with season, including high production of spermatozoa in autumn and winter and low production in summer. Semen quality also differed across breeds. Acrosin activity of boar spermatozoa was not affected by breed (range 3.16-3.32 mU/10(6) spermatozoa), but exhibited distinct seasonal changes. Monthly changes in acrosin activity were parallel to changes in number of sperm in the ejaculate from November to March. On the other hand, dramatic changes in acrosin activity between July and October (range 1.85-4.59 mU/10(6) spermatozoa) were not paralleled by similar changes in number of ejaculated sperm. These fluctuations in acrosin activity may reflect either changes in sperm acrosin production or disturbances to sperm membranes, probably related to effects of high summer temperatures during spermatogenesis. Results confirmed seasonal and breed-related differences in boar semen quality characteristics.

Fertilization rate of Siberian sturgeon (Acipenser baeri, Brandt) milt cryopreserved with methanol

Milt obtained from three Siberian sturgeon males (Acipenser baeri, Brandt) were cryopreserved using three extenders: Tris–sucrose–KCl (30 mM Tris, 23.4 mM sucrose, 0.25 mM KCl, pH 8.0), Tris–NaCl (10 mM Tris, 25 mM NaCl, pH 8.5), and Tris–sucrose (20 mM Tris, 400 mM sucrose, pH 8.0) supplemented with 10% methanol. Semen was diluted 1:1 with appropriate extender and frozen in liquid nitrogen vapor. After fertilization with cryopreserved milt, hatching rates of 29.6±5.0%, 18.2±2.4%, and 6.0±3.0% were recorded for Tris–sucrose–KCl, Tris–NaCl, and Tris–sucrose extender, respectively. Rates for the first two extenders were similar to data of fresh semen obtained from two males(17.9% and 26.0% for male #1 and #2, respectively). Our results indicate that Tris–sucrose–KCl and Tris–NaCl are useful extenders and methanol is a useful cryoprotectant for cryopreservation of sturgeon semen.

Effects of diets containing gossypol on reproductive capacity of rainbow trout (Oncorhynchus mykiss).

Abstract We evaluated five practical diets in which 0%, 25%, 50%, 75%, and 100% (dietary treatments 1–5) of fish meal protein was replaced by solvent-extracted cottonseed meal protein. Adult rainbow trout (initial average weight 247 ± 8 g) were fed the diets over a period of 131 days during which a general 2-fold body weight increase occurred. The total diet gossypol concentration (free and protein-bound) showed a gradual increase with increased cottonseed meal substitution. Blood samples were collected on Days 0, 64, 112, and 131 for hematological and steroid hormone determination in plasma of males and females. Hemoglobin content was significantly reduced in fish from treatment 5 (7.9 ± 0.3 g/dl) in comparison to treatments 1–3 (10.3–10.9 g/dl). After 112 and 131 days of feeding, testis weights, concentrations of testosterone, and 11-ketotestosterone were elevated in fish from dietary treatments 2 and 3 in comparison to control and diets 4 and 5. On Day 71, sperm were collected from 6 fish per dietary treatment to assess sperm quality. No significant differences in sperm concentrations (7.2–9.8 × 109/ml), motility (78–89%), and standardized (300 × 105 sperm/egg) fertilizing ability (18.9–22.6% hatched embryos) were found. Total gossypol concentrations in blood plasma differed significantly among treatments, and the levels were among the highest ever recorded in animals fed cottonseed-supplemented diets (2.9 ± 0.2, 11.7 ± 4.1, 21.7 ± 1.4, and 29.9 ± 3.9 μg/ml, for treatments 2–5, respectively). The major portion of gossypol in blood plasma was protein-bound (81–93%). This was in contrast to minute amounts of gossypol present in seminal plasma, mostly in free form (0.02–0.18 μg/ml), which indicates the presence of a barrier between general circulation and the testis with respect to gossypol distribution in lower vertebrates. Thus, the reproductive parameters of male rainbow trout examined in this study were not significantly affected by feeding cottonseed meal for 131 days.

Gossypol isomers bind specifically to blood plasma proteins and spermatozoa of rainbow trout fed diets containing cottonseed meal

We investigated the role of gossypol isomers binding to blood plasma, seminal plasma and spermatozoa to elucidate gossypol anti-fertility action in the teleost fish, rainbow trout (Oncorhynchus mykiss). Growth and hematological indicators of males were depressed when fish meal protein in diets was completely replaced with cottonseed meal. The cottonseed meal contained equal proportions of (-) (47.8+/-1.6%) and (+) gossypol isomers. Concentrations of spermatozoa were decreased with increasing proportions of gossypol in diets (from 0.22% to 0.95%); however, sperm motility and fertilizing ability were not affected. In contrast to mammals, steroid hormone concentrations were not suppressed in fish given diets with gradual increase of gossypol level. Gossypol concentrations were 100-fold higher in blood plasma than in seminal plasma, confirming a barrier in gossypol transfer between the general circulation and the testis. Spermatozoa accumulated predominantly (+) enantiomer (65-75%) with decreasing proportions as dietary gossypol concentrations increased. Spermatozoa bound most of the gossypol contained in the semen; however, this did not result in impairment of the sperm motility apparatus. Teleost fish sperm rely on ATP stores that accumulate during maturation as a source of energy during activation. In addition, the duration of sperm movement is short in these fish. As such, we hypothesize that the major action of gossypol on mammalian sperm, which is uncoupling of oxidative phosphorylation, does not impair the energy supply required for flagellar beating in fish spermatozoa.

Effect of feeding cottonseed meal-containing diets to broodstock rainbow trout and their impact on the growth of their progenies

Rainbow trout Oncorhynchus mykiss broodstocks were fed five experimental diets in which fish meal protein was gradually replaced with cottonseed meal (CS) protein (0%, 25%, 50%, 75%, and 100%; diets 1–5, respectively) during a 22-month period. The effect of increasing dietary levels of CS on reproductive performance was gender specific. Sperm fertilizing ability significantly decreased when CS exceeded 50% protein replacement (72.6±2.0%, 73.6±2.0%, 69.0±3.3%, 43.3±5.1%, and 36.8±4.7%, for diets 1–5, respectively). In contrast, in females, the viability of embryos was only significantly affected at 25% and 50% replacement levels (56.7±15.6%, 20.5±24.9%, 11.4±18.6%, 48.8±25.9%, and 39.6±31.1%, for diets 1–5, respectively). Progenies from multiple parents per dietary treatment were combined and reared on a commercial diet over a 2- or 3-month period fed at a rate of 4% of their body weight. The paternal origin (fresh sperm, experiment 1) had a highly significant effect on growth performance of progenies, and progenies from males fed with 25%, 50%, and 75% CS grew significantly (P<0.05) faster than progenies from males fed with 0% and 100% CS. Growth performance of progenies produced using cryopreserved sperm (experiment 2) was not affected regardless of the CS levels fed to male rainbow trout. Progenies from females (experiment 3) fed a diet containing 50% CS grew significantly (P<0.05) slower than the other groups. Sex ratio was examined histologically after completion of feeding experiments with progenies. Regardless of maternal or paternal origin, males dominated among the progenies. Thus, we postulated that other substances such as flavonoids, present in the CS and possibly transferred to yolk sac reserves, might affect the sex ratio in favor of males.

Different computer-assisted sperm analysis (CASA) systems highly influence sperm motility parameters.

In this study, we examined different computer-assisted sperm analysis (CASA) systems (CRISMAS, Hobson Sperm Tracker, and Image J CASA) on the exact same video recordings to evaluate the differences in sperm motility parameters related to the specific CASA used. To cover a wide range of sperm motility parameters, we chose 12-second video recordings at 25 and 50 Hz frame rates after sperm motility activation using three taxonomically distinct fish species (sterlet: Acipenser ruthenus L.; common carp: Cyprinus carpio L.; and rainbow trout: Oncorhynchus mykiss Walbaum) that are characterized by essential differences in sperm behavior during motility. Systematically higher values of velocity and beat cross frequency (BCF) were observed in video recordings obtained at 50 Hz frame frequency compared with 25 Hz for all three systems. Motility parameters were affected by the CASA and species used for analyses. Image J and CRISMAS calculated higher curvilinear velocity (VCL) values for rainbow trout and common carp at 25 Hz frequency compared with the Hobson Sperm Tracker, whereas at 50 Hz, a significant difference was observed only for rainbow trout sperm recordings. No significant difference was observed between the CASA systems for sterlet sperm motility at 25 and 50 Hz. Additional analysis of 1-second segments taken at three time points (1, 6, and 12 seconds of the recording) revealed a dramatic decrease in common carp and rainbow trout sperm speed. The motility parameters of sterlet spermatozoa did not change significantly during the 12-second motility period and should be considered as a suitable model for longer motility analyses. Our results indicated that the CASA used can affect motility results even when the same motility recordings are used. These results could be critically altered by the recording quality, time of analysis, and frame rate of camera, and could result in erroneous conclusions.

Characterization of carp seminal plasma proteome in relation to blood plasma.

UNLABELLED The present study for the first time characterizes a diverse cohort of carp seminal and blood plasma proteins using the combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry. Using this approach, we identified 137 proteins in carp seminal plasma and 88 proteins in carp blood plasma, most of which were newly identified in fish. Transferrin, serine proteinase inhibitors, apolipoproteins, complement C3 and Wap65 were present in high abundance in carp seminal plasma. In carp blood plasma, besides these proteins, immunoglobulins and macroglobulins were identified as major proteins. Comparative analysis of carp seminal and blood plasma proteome performed using 2D-DIGE revealed that in contrast to mammals the majority (1014 from 1240 spots) of carp seminal plasma proteins are blood proteins. Moreover, proteins more abundant in seminal plasma (99 from 1240 spots) were identified, including parvalbumin, isoforms of apolipoproteins, heat shock proteins, components of antioxidative system, matrix metalloproteinases, cathepsin D, enzymes of glycolysis and sperm structural proteins. These proteins are involved in the regulation of sperm motility, spermatogenesis, maintenance of sperm membrane lipid stability and antioxidant protection. This study enhances the basic knowledge concerning fish seminal plasma protein composition and their potential role in fish reproduction. BIOLOGICAL SIGNIFICANCE Proteins similar or identical to blood plasma components are important for male reproductive physiology. Comparative study of blood and seminal plasma is especially justified in fish. Using 2D-DIGE we indicated that, in contrast to mammals, in carp seminal plasma most proteins are common for blood and seminal plasma, which possibly is related to a lack of accessory glands in reproductive tract of most fish. The proteins present in higher abundance in seminal plasma can be related to physiology of fish male reproduction including regulation of sperm motility, spermatogenesis, maintenance of sperm surface composition and antioxidant protection. Application of proteomics analysis to identify carp seminal and blood plasma proteins significantly extends current knowledge regarding the composition of fish seminal and blood plasma proteins and their relationship to higher vertebrates. Moreover, proteomic profiling of carp seminal plasma appears to be helpful for further understanding of the role of fish seminal plasma proteins in male reproductive tract as well as for identification of novel biomarkers for sperm quality.

Semen characteristics and their ability to predict sperm cryopreservation potential of Atlantic cod, Gadus morhua L.

There is a lack of biomarkers or indices that can be used to predict the quality of fish semen samples following the freezing and thawing cycle. In the present study, a series of semen indices were tested to assess if they could accurately forecast the cryopreservation potential of Atlantic cod (Gadus morhua) semen. Fresh and frozen-thawed sperm activity variables were compared, and relationships between frozen-thawed sperm activity and fertilization success were examined. In comparison with fresh sperm, activity variables of frozen-thawed spermatozoa were reduced. Of the 18 males examined, mean (± SEM) spermatocrit of fresh sperm was 40.72 ± 4.23%, osmolality of the seminal plasma 366.32 ± 4.95 mOsmol/kg, pH 8.32 ± 0.04, protein concentration 1.05 ± 0.08 mg/mL, anti-trypsin activity 153.83 ± 19.25 U/L, and total antioxidant capacity 0.15 ± 0.03 μmol Trolox equivalents/mL. Frozen-thawed fertilization success was highly variable among males with values ranging from 18.5 to 90.2%. Regressions yielded significant positive relationships between frozen-thawed motility, velocity, track crossing frequency, and subsequent fertilization success. Sequential multiple regressions explained up to 95% of the variation in frozen-thawed sperm activity. Spermatocrit and pH of fresh semen were negatively related, whereas osmolality and antioxidant capacity were positively related to frozen-thawed motility and velocity. Each of these indices can be measured within minutes of collecting a fresh sample of semen and are thus early indicators of the capacity of semen samples to withstand cryopreservation. These results have many benefits for conservation of wild stocks, aquaculture production, and for understanding semen biology and cryobiology of fishes.

Isolation and characterization of transferrin from common carp (Cyprinus carpio L) seminal plasma.

Transferrin (Tf) in fish is recognized as a component of non-specific humoral defense mechanisms against bacteria. It is a major protein of common carp seminal plasma but its structure and localization in carp testis is unknown. In this study we developed a simple and efficient three-step purification procedure consisting of affinity chromatography (Con A-Sepharose), hydrophobic interaction chromatography (Phenyl Sepharose) and gel filtration (Superdex 200). The molecular mass of Tf has been determined to be 73.6 kDa and isoelectric point 5.1. The peculiar characteristics of carp transferrin were the lack of carbohydrate component and binding of iron ions by only one functional iron-binding site. Western blot analysis revealed a strong similarity of carp seminal plasma Tf to carp blood Tf and Tf from seminal plasma of other cyprinids but a lower similarity to salmonid and percid fishes. Tf was localized to the blood vessels of the carp testis which strongly suggest that most Tf of carp seminal plasma originates from blood. In conclusion, seminal plasma Tf has a unique structure and is similar or identical to blood Tf.