Ian A.E. Butts

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 microm/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 microm/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 Ca(2+) 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.

Ovarian fluid enhances sperm velocity based on relatedness in lake trout, Salvelinus namaycush

Studying mate choice at the gamete level can provide valuable insights into proximate mechanisms that underlie the evolution of mating systems. The objective was to assess whether ovarian fluid enhances sperm performance based on relatedness of mates in lake trout, Salvelinus namaycush, an iteroparous salmonid. Twelve trios were used, each composed of a female and two male fish; one male was related (full sibling) to the female, whereas the other was unrelated. Sperm from each male was activated in hatchery water or ovarian fluid from each corresponding female. No significant difference in sperm velocity was detected between the related and unrelated male fish when activated in hatchery water. However, when sperm was activated in ovarian fluid, sperm velocity from the related male was significantly higher than that of the unrelated male fish. Overall, ovarian fluid enhanced sperm performance of related male fish and might act as part of a recognition system to select sperm of a specific genotype.

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.

Primary and secondary sexual characters in alternative reproductive tactics of Chinook salmon: Associations with androgens and the maturation-inducing steroid

The proximate mechanisms that underlie the evolution of within-sex variation in mating behavior, sexual characters and reproductive investment patterns are still poorly understood. Species exhibiting alternative reproductive tactics (ARTs) are ideal model systems to examine these mechanisms. Chinook salmon (Oncorhynchus tshawytscha) exhibits two distinct ARTs: hooknoses, which are large males that establish spawning dominance hierarchies via intense male-male competition and jacks, which are smaller precocious sneaking males that steal fertilizations via sperm competition. In this study, we examine plasma testosterone (T), 11-ketotestosterone (11-KT) and maturation-inducing steroid (MIS; 17α,20β-dihydroxy-4-pregnen-3-one) profiles of spawning hooknoses and jacks. Furthermore, we examine relationships between androgens and primary (gonad mass, gonadosomatic index and sperm traits) and secondary (total mass, body size, hump depth and kype length) sexual characters. Relationships between MIS and sperm traits are also examined. We found that hooknoses and jacks did not significantly differ in terms of plasma T, 11-KT or MIS concentrations. Moreover, we found significant positive relationships between levels of both androgens within each ART. There were no significant relationships between androgens, MIS and sperm traits. T and 11-KT concentrations co-varied positively with gonad investment and kype length in jacks. In hooknoses, 11-KT concentration was positively related to total mass, hump depth and condition factor. Overall, these findings suggest that there are differential androgen effects for each of the ARTs in Chinook salmon.

Mechanism of action of mercury on sperm morphology, adenosine triphosphate content, and motility in Perca fluviatilis (Percidae; Teleostei).

The main objectives of the present study were to investigate the performance of mercury chloride (HgCl2 ) on sperm function and structure, identify sites of action of HgCl2 , and investigate the mechanism of action of HgCl2 on fish (Perca fluviatilis L.) spermatozoa. Direct exposure of nonincubated sperm decreased sperm motility and velocity in a dose-dependent manner and was totally suppressed at 250u2009µM HgCl2 . Adenosine-5-triphosphate (ATP) content of sperm after activation in an activation medium (AM) containing more than 25u2009µM HgCl2 did not differ compared with nonactivated sperm. Motility and velocity of demembranated sperm decreased after activation in an AM containing 62u2009µM HgCl2 , and was totally suppressed at 250u2009µM HgCl2 . Incubation of sperm in an immobilizing medium (IM) containing HgCl2 enhanced HgCl2 effects after sperm activation in an AM containing HgCl2 . Sperm motility of incubated sperm in an IM without HgCl2 was totally suppressed at 125u2009µM HgCl2 after 3u2009h incubation. In case of incubated sperm in an IM containing HgCl2 , sperm motility was totally suppressed at 31u2009µM HgCl2 . Adenosine-5-triphosphate content of sperm was significantly lower in an IM containing HgCl2 greater than 3u2009µM compared with those of the control (no HgCl2 ) and lower HgCl2 concentrations. Damage to the plasma membrane and axoneme were observed in sperm incubated in an IM containing HgCl2 compared with the control, when HgCl2 concentration and incubation time increased. In conclusion, HgCl2 acts on sperm through disruption of function of the plasma membrane, axoneme, and ATP content.

Cryopreservation of Atlantic cod Gadus morhua L. spermatozoa: Effects of extender composition and freezing rate on sperm motility, velocity and morphology.

Broodstock selection programs are currently underway for Atlantic cod (Gadus morhua). To complement and further these selection programs we need to develop sperm cryopreservation procedures. This will allow genomic DNA from males from selected individuals or stocks to be frozen and conserved in perpetuity. In our study we used a full factorial ANOVA design to examine the effects of diluent (Mounibs sucrose-based diluent, Hanks Balanced Salt Solution, Mounibs sucrose-based diluent+hens egg yolk, and Hanks Balanced Salt Solution+hens egg yolk), cryoprotectant (propylene glycol, dimethyl sulphoxide, and glycerol), and freezing rate (-2.5, -5.0, -7.5, and -10.0°C/min) on motility of cod frozen-thawed sperm. Sperm velocity and morphometric analyses of sperm heads and flagella were also assessed. We found that sperm motility-recovery index was strongly influenced by the presence of higher-order interactions of the factors we tested. The best cryoprotection used diluents that contained hens egg yolk. Generally, extenders containing propylene glycol yielded higher post-thaw sperm motilities than those with dimethyl sulphoxide or glycerol. In comparison to sperm from other frozen-thawed extenders, sperm from extenders supplemented with propylene glycol had significantly higher curvilinear velocity. Cryopreservation showed no impact on sperm head morphology parameters, however, considerable damage to frozen-thawed sperm flagella was observed. We believe that our experimental/statistical approach and our results add significantly new information to the study of semen biology/cryobiology in fishes. Our findings are also highly relevant to the development of cod mariculture and for aiding in conservation efforts of this very important marine species.

Seminal plasma biochemistry and spermatozoa characteristics of Atlantic cod (Gadus morhua L.) of wild and cultivated origin.

Our objectives were to compare spermatozoa activity, morphology, and seminal plasma (SP) biochemistry between wild and cultivated Atlantic cod (Gadus morhua). Swimming velocities of wild cod spermatozoa were significantly faster than those of cultivated males. Wild males had a significantly larger spermatozoa head area, perimeter, and length, while cultivated males had more circular heads. Total monounsaturated fatty acids and the ratio of n-3/n-6 were significantly higher in sperm from wild males, while total n-6 from cultivated males was significantly higher than the wild males. Significantly higher concentrations of the fatty acids C14:0, C16:1n-7, C18:4n-3, C20:1n-11, C20:1n-9, C20:4n-3, C22:1n-11, and C22:6n-3 were observed in wild males, while significantly higher concentrations of C18:2n-6, C20:2n-6, and C22:5n-3 occurred in cultivated males. Osmolality, protein concentration, lactate dehydrogenase and superoxide dismutase activity of SP of wild males were significantly higher than the cultivated males. Antioxidant capacity of SP was significantly higher in cultivated males, while pH and anti-trypsin did not differ between fish origins. Four bands of anti-trypsin activity and nine protein bands were detected in SP. Performing a discriminant function analysis, on morphology and fatty acid data showed significant discrimination between wild and cultivated fish. Results are relevant to breeding programs and aquaculture development.

Automated sperm head morphology analyzer for open-source software

Sperm head morphology has been identified as a characteristic that can be used to predict a males semen quality. In the present study, we have developed an automated sperm head morphology analysis (ASMA) plug-in for open-source ImageJ software (http://rsbweb.nih.gov/ij/). We describe the plug-ins functionality, and confirm its validity for sperm head morphology analysis using fish sperm. Sperm head morphological measurements (length and width) made with the ASMA plug-in did not differ from manual measurements. Using the plug-in to measure sperm head-shaped objects of known size, the associated plug-in error rate was < 0.5%. Brightness and contrast ratios influenced sperm head measurements, suggesting the need for standardized protocols. This plug-in was effective at measuring elliptical (i.e., Atlantic cod) as well as slightly irregular (i.e., Chinook salmon) shaped sperm heads. In conclusion, our ASMA plug-in represents a versatile alternative to costly sperm morphology software.

Ovarian fluid influences sperm performance in lake trout, Salvelinus namaycush

The objectives of this study were to determine whether (i) the presence and concentration of ovarian fluid (OF) affects sperm performance traits, and (ii) variation in sperm performance traits is due to male identity, female identity, and/or male×female interactions in lake trout, Salvelinus namaycush. Spermatozoa from four males were activated in river water and OF from four females at two concentrations (10 and 15%). Presence of ovarian fluid influenced sperm traits; no differences were detected between 10 and 15% OF. Sperm traits varied depending on parental identity, such that sperm of some males perform better in the OF of all females and that in OF of some females sperm traits are higher for all males.

Sperm morphology, ATP content, and analysis of motility in Atlantic halibut (Hippoglossus hippoglossus)

Spermatozoon of Atlantic halibut (Hippoglossus hippoglossus (L., 1758)) is uniflagellated, lacks an acrosome, and is differentiated into a head, midpiece, and flagellum. There are two to five mitochondria in the midpiece, as well as proximal and distal centrioles. The flagellum consisted of 9 + 2 microtubules surrounded by plasma membrane, which is extended at the proximal part of the flagellum owing to the presence of vacuoles. After sperm activation in seawater, sperm motility and velocity decreased from 98.4% ± 3.4% and 170.3 ± 8.9 mms -1 at 15 s after sperm activation to 4.8% ± 4.7% and 9.2 ± 8.9 mms -1 at 120 s after sperm activation, respectively. ATP content (nmolL -1 ATP per 10 8 spermatozoa) significantly decreased at 60 s after sperm activation (5.9 ± 1.5) compared with at 0 and 30 s after sperm activation (14.9 ± 1.5 and 14.5 ± 1.5, respectively). Beating waves propagated along the full length of the flagellum after sperm acti- vation, whereas waves were restricted to the proximal section during the latter motility period. Wave amplitude signifi- cantly decreased at 45 s after sperm activation, but wavelength did not differ. The present study showed associations among sperm morphology, ATP content, flagellar wave parameters, and sperm velocity, which could be used in compara- tive spermatology. Resume´ : Le spermatozoo¨de du fletan atlantique (Hippoglossus hippoglossus (L., 1758)) est uniflagelle ´, sans acrosome et differencieen tete, corps median et flagelle. Il y a deux acinq mitochondries dans le corps median, ainsi que des centrio- les proximaux et distaux. Le flagelle consiste en 9 + 2 microtubules entoures dune membrane plasmatique qui se prolonge dans la partie proximale du flagelle acause de la presence de vacuoles. Apres lactivation des spermatozoo¨des dans leau de mer, la motiliteet la vitesse des spermatozoo¨des diminuent respectivement de 98,4 % ± 3,4 % et de 170,3 ± 8,9 mms -1 a ` 15 s apres lactivation a 4,8 % ± 4,7 % et 9,2 ± 8,9 mms -1 a 120 s apres lactivation. Le contenu en ATP (nmolL -1 ATP par 10 8 spermatozoo¨des) diminue significativement 60 s apres lactivation (5,9 ± 1,5) par comparaison a ` 0e t 30 s apres lactivation (respectivement 14,9 ± 1,5 et 14,5 ± 1,5). Les ondulations de battement se propagent sur toute la lon- gueur du flagelle apres lactivation, mais sont restreintes ala partie proximale durant la periode de motilitesubsequente. Lamplitude des ondulations diminue significativement a 45 s apres lactivation, mais leur longueur donde ne change pas. Notre etude met en lumiere des associations entre la morphologie des spermatozoo¨des, leur contenu en ATP, les variables ondulatoires des flagelles et la vitesse des spermatozoo¨des qui pourraient servir en spermatologie comparee. (Traduit par la Redaction)