Biochemical and physiological characteristics of semen of sex-reversed female rainbow trout (Oncorhynchus mykiss, Walbaum)

This works studies the biochemical (protein concentration, osmolality, antitrypsin activity, lactate dehydrogenase activity) and physiological characteristics (sperm motility characteristics) of semen of sex-reversed female rainbow trout (n = 42) obtained with the application of 11β-hydroksyandrostendione for sex reversal. All data were arbitrarily divided into three classes depending on the percentage of sperm motility: I XX < 25%; II XX 25-50% and III XX > 50%. The average percentage of sperm motility was 18 ± 7% n = 12 (group I XX); 42 ± 6% n = 15 (group II XX) and 65 ± 12% n = 15 for group III XX, respectively) to link the values of semen parameters to the maturation stage of semen. Semen from 12 normal males of the same age was used as a reference group. Sperm concentration as well as protein concentration, osmolality, antitrypsin activity, and lactate dehydrogenase activity in seminal plasma of sex-reversed females were higher compared with the values obtained for normal male rainbow trout. The values of these parameters declined with the increasing percentage of sperm motility toward values established for normal males. The fertilization success of semen (3 × 10⁶ spermatozoa/egg) of sex-reversed females was very high (above 90%) for both the percentage of eyed embryos and hatched larvae and was related to sperm motility classes. Correlations between the quality parameters of sex-reversed females semen corresponded to those established previously for the semen of normal male rainbow trout. Antitrypsin activity, lactate dehydrogenase, protein concentration, and osmolality were found to be characteristic of seminal plasma of sex-reversed females. The maturity of sex-reversed female spermatozoa seems to be associated with the decline in the values of those parameters toward the values characteristic for seminal plasma of normal males.     

Blood cells in rainbow trout Oncorhynchus mykiss milt: relation to milt collection method and sampling period

The presence of blood cells in milt of rainbow trout (Oncorhynchus mykiss) collected every week between the middle at the end of the spawning season, either by stripping or by catheterization was investigated. Basic sperm biological and biochemical characteristics were also evaluated. Because milt often becomes contaminated with blood during collection, we also studied the influence of experimental blood contamination on sperm motility and biochemical parameters of seminal plasma. We demonstrated the presence of blood cells (erythrocytes, lymphoid, and phagocytes) in rainbow trout milt collected by both methods. Both sampling period and collection method influenced sperm characteristics, however the relationship between these characteristics and blood cells are not clear at present. A high number of blood cells in milt was found in some samples, possibly due to inflammation, because at the same time we observed bacteria and elevated levels of protein and antiproteinase activity in contaminated samples. Experimental contamination of milt with blood did not influence sperm motility, protein concentration and LDH activity of the 5-day-stored semen. Our study demonstrated that blood cells were present in rainbow trout milt. Blood cells may also appear in milt as a result of bleeding and their elevated levels are present during inflammation.     

Quantitative characteristics of Atlantic halibut, Hippoglossus hippoglossus L., semen throughout the reproductive season

The overall objective of the study was to investigate changes in quantitative parameters of Atlantic halibut (Hippoglossus hippoglossus L.) semen throughout the reproductive season in order to systematize the knowledge about biology of Atlantic halibut spermatozoa. Semen samples were collected from February to May from broodstock males kept under either a natural or 3-month advanced photoperiod regime. Spermatozoa concentration, semen pH and osmolality, as well as spermatozoa motility parameters were investigated. The use of catheterization of sperm was examined. Also, fertilization tests were performed. We found that spermatozoa concentration increases in a linear-like mode towards the end of the spawning season, which correlated with a decrease in a number of spermatozoa motility parameters, including actual percentage of motile spermatozoa (MOT), curvilinear velocity (VCL) and straight-line velocity (VSL) of spermatozoa. A breakpoint in MOT occurred when spermatozoa reached a concentration in the range of 17-20 x 10(9) spermatozoa/mL. The fertilization ability of sperm from males kept under natural photoperiod decreased in April. Survival of embryos at 80 degrees days produced by fertilizing eggs of single female with sperm from natural photoperiod males was 88, 76 and 41% on April 09 and 17, and May 01, respectively, whereas using sperm from 3-month delayed photoperiod males for fertilizing eggs from the same female on April 27 resulted in 80% of surviving embryos, not differing significantly from the data from April 09. Physical decomposition of spermatozoa was observed towards the end of the season and it was related to an increase in the whole semen osmolality. Catheterization of semen did not improve spermatozoa motility parameters, however, it reduced the variation in recorded values, especially in the case of pH, caused by contamination with feces or urine. Post-seasonal decrease in spermatozoa concentration was likely related to intensive ageing processes. Based on the present study and available data by other researchers, a model of changes of quantitative parameters in Atlantic halibut semen throughout the reproductive season is proposed.     

The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation

The influence of permeating and of non-permeating cryoprotectants on motility, eosin permeability (sperm cells viability test) and leaking lactate dehydrogenase activity of spermatozoa of the rainbow trout ( Oncorhynchus mykiss ) was investigated. Correlations between sperm motility rate, percentage of eosinimpermeable (viable) cells and LDH activities established the three parameters as indicators for damages to rainbow trout spermatozoa. High sperm motility rates, velocities, and numbers of linear swimming sperm cells, high numbers of eosinimpermeable spermatozoa and a low extracellular LDH activity were evaluated as positive quality characteristics of semen. With a buffered physiological saline solution as basic extender we found a mixture of 0.67 mol L (5%) dimethylsulfoxide (DMSO) and 0.13 mol/L (1%) glycerol to be the most effective cryoprotectant, followed by DMSO (0.67 mol/L [5%]-1.85 mol/L [15%]) and glycerol (0.65 mol/L [5%]-1.78 mol/L [15%]) and propanediol (1.24mol L [10%]-1.78 mol/L [15%]). Addition of hen egg yolk (7%, 20%) and of 15 mmol/L (0.5%) sucrose significantly increased the semen quality in comparison to the same extender without these additives. Bovine serum albumin (1.5%, 3%) had no influence on the investigated semen parameters.     

Chemical composition of seminal and ovarian fluids of chinook salmon (Oncorhynchus tshawytscha) and their effects on sperm motility traits

The relationships between the compositions of ovarian, seminal fluids and sperm function are not well known in teleostean fish species. The objective of the present study was to determine the concentration of the major inorganic ions (Na(+), K(+), Ca(2+), Mg, Cl(-)), osmolality, and pH of ovarian and seminal fluid of sexually mature chinook salmon (Oncorhynchus tshawytscha), and to determine if the composition of these fluids influences sperm motility traits (swimming speed, duration of forward mobility, swimming path trajectory, and percent motility). Cation concentrations and osmolality were significantly different in the two fluids. The ionic composition of ovarian fluid differed among individual females, and also among samples collected at different times through the spawning season. Carbonate and bicarbonate were the principal buffer ions in ovarian fluid, and its viscosity was considerably greater than that of water and was shear-dependent. The duration of forward motility (longevity) of spermatozoa, swimming speed, percent motility, and path trajectory were measured using milt from 10 males activated in the ovarian fluid from 7 females whose ion concentrations were known. No significant correlations were observed between the composition of the seminal fluid and sperm traits. However, in ovarian fluid, sperm longevity was negatively correlated with variation in [Ca(2+)] and [Mg(2+)], while percent motility increased with increasing [Mg(2+)]. These observations provide a possible chemical basis for cryptic female mate choice whereby female ovarian fluid differentially influences the behaviour of sperm from different males, and thus their fertilization success.     

Monthly fluctuations during the breeding season of sperm density,volume, motility,and composition of seminal and coelomic fluid in broodfish of Persian sturgeon,Acipenser persicus Borodin, 1897

Obtaining knowledge on the gamete quality of certain species is essential for appropriate management and conservation of wild stocks of the species concerned. In the present study, eighteen breeders (nine males and nine females) of Acipenser Persicus Borodin, 1897 were selected for recording breeding season changes in gamete quality in terms of the parameters: spermatozoa motility indices, sperm volume and density, ionic composition and osmolality of seminal and coelomic fluids. Stripping was performed at the beginning of March and during April and May. Results indicated that sperm volume (ml per male) and density (×10⁹ spz ml^?1) decreased towards the end of the spawning season. There was no significant change in osmolality (mOsmol kg^?1) or in the concentrations (as mm ) of sodium, potassium, magnesium or calcium ions in either the seminal or the coelomic fluids during the breeding season. A significant difference in chloride ion concentration in the coelomic fluid was noted at different times during the spawning season. The percentage of motile spermatozoa at 45 s post‐activation was not significantly different for samples taken at different dates, but the maximum duration of spermatozoa movement, at 15 s post‐activation, was observed in March. This value decreased significantly towards the end of the reproductive season. In conclusion, changes observed in A. persicus sperm parameters during the breeding season suggest an association between such changes and the sperm aging processes.     

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.     

Seasonality of LH,testosterone and sperm parameters in spider monkey males (Ateles geoffroyi)

There are no reported data on hormonal fluctuations in black‐handed spider monkey males. On previous research about the reproductive physiology of this monkey we have found that during the dry season females show ovulatory estrogen peaks and males present the best quality semen. As part of an ongoing research, in this study we assessed seasonal variations in the concentration of serum luteinizing hormone (LH) and testosterone (T) in three adult spider monkey males to corroborate the seasonal reproductive synchrony. At the same time sperm count and motility were evaluated to search for any correlation between those sperm parameters and hormonal concentrations. We took blood and semen samples (by electroejaculation) of anesthetized males throughout the rainy (June–September) and dry (October–May) months. Our results revealed that T and LH were higher throughout the dry season and there was a significant correlation between T concentration and sperm count. Although higher during the dry season, sperm motility tended to correlate with testosterone and LH levels. These results demonstrated that black‐handed spider monkeys have a tendency to show a seasonal pattern of reproduction being the dry season the most likely time to achieve fertilization. Am. J. Primatol. 71:427–431, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Improvement of Sperm Motility of Sex-Reversed Male Rainbow Trout, Oncorhynchus mykiss, by Incubation in High-pH Artificial Seminal Plasma

Changes in the motility time of spermatozoa collected from the testes and the sperm duct of normal and sex-reversed male (XX) rainbow trout in physiological balanced salt solution were examined after incubation in artificial seminal plasmas of various pHs. Although untreated spermatozoa from the sperm duct retained motility for 60–90 s in the balanced salt solution, the spermatozoa collected from the testes were immotile. During the incubation in artificial seminal plasma of pH 7.0, the spermatozoa from the sperm duct hardly moved, similar to the testicular spermatozoa in the balanced salt solution. By suspending and incubating the testicular spermatozoa in artificial seminal plasma of pH 9.9 for 2 h at 4°C, the percentage of motile spermatozoa increased from 0–5% to 80%. The spermatozoa remained motile for at least 2 min after long-term incubation (12 h). When the full-sib eggs were inseminated with untreated testicular spermatozoa or testicular sperm treated for 2 h at high pH, the percentage survival increased from 5.5% to 53.8% at the eyed stage due to the high-pH treatment. The incubation of the spermatozoa in high-pH artificial seminal plasma improved the motility of the spermatozoa from the testes of the sex-reversed male that had lost its sperm duct. By this treatment, it is possible to markedly increase the mass production efficiency of all-female or all-female triploid sterile progenies.     

Prostaglandins in rainbow trout (Oncorhynchus mykiss Walbaum, 1792) sperm biology – searching for answers

The purpose of this study was to determine the concentrations of prostaglandins E₂ and F_2α (PGE₂ and PGF_2α) in the blood, testis and seminal plasma of mature male rainbow trout and in the ovarian fluid to assess the effects of these prostaglandins on sperm motility parameters when present in activation media. Also prolonged incubation with prostaglandins on sperm motility and calcium influx were studied. The profile of PGE₂ and PGF_2α differed in concentration between blood, testicular supernatant and seminal plasma. PGE₂ was predominant in the blood sample (0.29 ng ml^?1) and testicular supernatant (3.1 ng ml^?1) whereas their level in seminal plasma was lower than PGF_2α (0.23 ng ml^?1). The concentrations of PGF_2α in blood, testis and seminal plasma were 0.04, 0.99, 1.3 ng ml^?1, respectively. In the ovarian fluid the concentrations of both prostaglandins were higher than in the male reproductive tract. Adding both prostaglandins to activation buffer (at concentrations 15 and 70 ng ml^?1) had no effect on any CASA parameters. Calcium influx related to rainbow trout sperm incubations with PGE₂, and PGF_2α was not detected. After 24 h incubation of sperm in artificial seminal plasma solution without and with prostaglandins all sperm samples increased their motility potential and intracellular calcium concentration. Therefore, this effect was not related to the presence of prostaglandins. In summary PGE₂, and PGF_2α were present in the rainbow trout male reproductive tract, and their profile varies from that of blood, testis and seminal plasma. The specific role of both prostaglandins in salmonid sperm biology remains unclear.     

Changes in Solea senegalensis sperm quality throughout the year

Some of the Senegalese sole (Solea senegalensis) broodstock reproductive constraints are related to sperm quality. Although they present two defined spawning season (spring and autumn), males gave semen during all the year thus an exhaustive annual sperm analysis is important to determine the seasonal changes in semen quality. Sampling was performed monthly during one year, analyzing different cellular parameters to better understand sperm quality limitations obstructing sole mass production. The percentage of progressive motile cells and their linear velocity showed a decrease from March (beginning of the first spawning season) to July (when the highest temperatures were observed), followed by a slight increase in August and October (second spawning season). DNA fragmentation values showed highest values between the two spawning seasons and decreased to the end of the year. The percentage of apoptotic cells was lowest in March (beginning of the first spawning season) and the highest in November. The percentage of cells resistant to seawater exposure presented two peaks related with both spawning seasons. There was a tendency for the semen to attain a quality peak between the beginning and the middle of the first spawning season (March-May), followed by a pronounced decrease, achieving the lowest values during the months with the highest temperature. Also, the different males present in the broodstocks reach their sperm quality peak at different times, which will result in an unequal contribution for the next generation.     

Polymorphism of transferrin of carp seminal plasma: relationship to blood transferrin and sperm motility characteristics

Transferrin (Tf) is a major protein of carp (Cyprinus carpio) seminal plasma. Its relationship with milt quality is unknown. In this study, we sought to determine if Tf is polymorphic in carp seminal plasma and if this polymorphism is related to sperm motility characteristics. We screened males of purebred common carp line (Polish line R6) for Tf polymorphism in blood plasma. The majority of Tf genotypes represented only DD and DG variants. We then collected milt from preselected DD and DG genotypes and tested their sperm motility characteristics using computer-aided sperm analysis (CASA). Tf polymorphism in seminal plasma was found to be identical with that of blood. However, the relationships between Tf polymorphism and iron metabolic parameters were different for blood and semen. These data suggest different regulation of Tf in liver and testis. We found substantial differences in sperm motility characteristics between both genotypes. Spermatozoa of DG males were characterized by lower curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), higher linearity (LIN) and straightness (STR) of movement as compared to DD males. No differences were found in other sperm characteristics such as sperm concentration and percentage of sperm motility. Our results suggest that sperm motility parameters are related to Tf polymorphism and therefore this polymorphism may be related to sperm competitive ability.     

Assessment of ability of endangered Caspian brown trout, <Emphasis Type="Italic">Salmo trutta caspius</Emphasis> (Salmonidae) in production of semen with desirable quality

To assay the effects of stripping frequency on semen characteristics of Caspian brown trout, Salmo trutta caspius, the chemical composition of seminal fluid, sperm production (semen volume, sperm density, spermatocrit,) and sperm motility characteristics (percentage and duration of motility) were investigated over four times stripping during the spawning season. According to data, semen volume, sperm density, osmolality and the concentrations of Na⁺, Cl⁻, K⁺, Ca²⁺, Mg²⁺ and total protein gradually decreased by increasing of stripping frequency. The values of glucose and triglyceride had no significant changes over four times stripping. Also, the values of semen pH, the percentage (5 s after activation) and duration of motility were statistically stable until third stripping but a decrease was recorded for these parameters in the fourth stripping. As well as, significant positive correlations were found for sperm density vs. K⁺, Cl⁻, Na⁺, Ca²⁺, Mg²⁺, total protein, spermatocrit; the percentage of motile spermatozoa vs. Ca²⁺, Mg²⁺, K⁺, Cl⁻, Na⁺, total protein, and also the duration of motility vs. K⁺, Cl⁻, total protein and pH. In text, specific objectives of this study have been expressed.     

Seasonal timing of sperm production in roe deer: interrelationship among changes in ejaculate parameters,morphology and function of testis and accessory glands

Roe deer are seasonal breeders with a short rutting season from mid-July to mid-August. The seasonality of reproductive activity in males is associated with cyclic changes between growth and involution of both testes and the accessory sex glands. This study characterizes morphological and functional parameters of these organs prior to, during and after breeding season in live adult roe deer bucks. Size and morphology of the reproductive tract was monitored monthly by transcutaneous (testes, epididymis) and transrectal (accessory glands) ultrasonography. Semen was collected by electroejaculation. Concentration, motility and morphological integrity of spermatozoa as well as the content of proteins and testosterone in semen plasma were evaluated. Proportions of haploid, diploid and tetraploid cells were estimated by flow cytometry in testicular tissue biopsies. Serum testosterone was measured by enzyme immunoassay. Most parts of the male reproductive tract showed distinct circannual changes in size and texture. These changes were most pronounced in the testes, seminal vesicles, and prostate. All reproductive organs were highly developed during the rut only. The volume of ejaculates, total sperm number and percentages of motile and intact spermatozoa also showed a maximum during this period and corresponded with high proportions of haploid cells in the testis. The highest percentages of tetraploid cells were found in the prerutting period. The production of motile and intact spermatozoa correlated with both the protein content of semen plasma and the concentration of testosterone in semen plasma and blood serum. These results suggest the importance of combined actions of the testes and accessory sex glands and the crucial role of testosterone in facilitating the optimal timing of intensified semen production to ensure sufficient numbers of normal spermatozoa in seasonal breeders.     

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30 sec post-activation) increased from 78.9 ± 6.5 to 128.2 ± 6.5 μm/sec (mean ± SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6 ± 2.4 to 48.9 ± 3.1 μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6 ± 4.2% in January to 40.5 ± 4.4% in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na⁺ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), whereas Ca²⁺ increased then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.     

The quantification of lipid and protein oxidation in stallion spermatozoa and seminal plasma: seasonal distinctions and correlations with DNA strand breaks, classical seminal parameters and stallion fertility

The goal of this work was to correlate oxidative stress caused by reactive oxygen species (ROS) and DNA damage with classic semen parameters in spermatozoa and seminal plasma of fertile and subfertile stallions. Oxidation was measured in both lipids and proteins, using the thiobarbituric acid reactive species (TBARS) assay and the DNPH carbonyl groups assay, respectively. Sperm DNA damage was monitored using the TUNEL assay. These parameters were monitored in samples obtained during the breeding and the non-breeding seasons. In general, fertile stallions showed better classical semen parameters, and those parameters improved from the non-breeding to the breeding season, although an increase in sperm production was accompanied by a decrease in the semen quality from subfertile stallions in the breeding season. In terms of oxidation levels we found that there were clear differences whether lipids or proteins were considered. In the breeding season there seemed to be a tendency towards normalizing lipid oxidation in spermatozoa and seminal plasma, and protein oxidation in the seminal plasma, of both fertile and subfertile animals. Thus, differences monitored in the non-breeding season were no longer visible. Interestingly, a higher level of protein oxidation was found in the sperm of fertile animals in the breeding season. Considering that there were positive correlations between sperm protein oxidation and sperm motility and vitality, these results suggests that the oxidation of semen proteins may be important for sperm function. On the other hand, lipid oxidation in the seminal plasma seemed to be a general indicator for sperm damage. In the non-breeding season positive correlations between lipid and protein oxidation levels in both sperm and seminal plasma and several defects in sperm function were found, but only for subfertile animals, thus suggesting that lipid and protein oxidation may aid in the identification of subfertile stallions during the non-breeding season. Levels of ROS production never seemed to result in compromised sperm DNA integrity, indicating that measurements were within physiological levels and/or that there is an efficient antioxidant activity in stallion sperm cells.     

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.     

Adenosine triphosphate concentration and beta-D-glucuronidase activity as indicators of sea bass semen quality

The most common parameters used to evaluate sperm quality are motility rate and duration and fertilization ability. In this study, chemical and biochemical parameters of sea bass (Dicentrarchus labrax) sperm were investigated to find an alternative method for evaluating sperm fertilization ability before and after cryopreservation. The biochemical and chemical analyses were performed with fresh and frozen-thawed sperm and seminal plasma. To cryopreserve sperm, 250-microl straws were used. Fertilization ability was evaluated by inseminating eggs (obtained from hormonally stimulated females) with fresh and cryopreserved sperm. The results revealed a linear relationship (P < 0.05) between semen fertilization capacity and some seminal plasma (beta-D-glucuronidase activity, potassium concentration) and sperm (ATP concentration, aspartate aminotransferase activity) parameters. Variations in semen fertilization rate could be best described by two multiple regression models: one including the sperm parameters and another including the seminal plasma parameters. For practical application, the use of simple regression models is of value. Fertilization rate in both fresh and cryopreserved sperm was reliably predicted by determining the ATP concentration or the beta-D-glucuronidase activity or both.     

Effects of the pathogenic haemoflagellate, <Emphasis Type="Italic">Cryptobia salmositica</Emphasis> on brood fish, <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

Sexually matured rainbow trout, Oncorhynchus mykiss, were experimentally infected with the pathogenic Cryptobia salmositica. Spawning female trout were more susceptible to cryptobiosis than sexually mature males. Most infected females (seven of nine) with eggs died before or shortly after spawning while all (nine) infected males survived the disease. Also, none of the uninfected females died. Males initially increased milt production and sperm concentration; however semen production declined as the disease progressed. Sperm from infected males fertilized more eggs than those from non-infected males. No differences in weight and survival were observed between progeny of infected and uninfected males.