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 male's 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-in's 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.

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.

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 (Mounib's sucrose-based diluent, Hanks' Balanced Salt Solution, Mounib's sucrose-based diluent+hen's egg yolk, and Hanks' Balanced Salt Solution+hen's 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 hen's 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.

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.

Behaviour and performance of juvenile shortnose sturgeon Acipenser brevirostrum at different water velocities.

Critical swimming speeds (mean +/-s.e.) for juvenile shortnose sturgeon Acipenser brevirostrum were 34.4 cm s(-1)+/- 1.7 (2.18 +/- 0.09 body lengths, BL s(-1)). Swimming challenges at 10, 20 and 30 cm s(-1) revealed that juvenile A. brevirostrum are relatively poor swimmers, and that the fish did not significantly modify their swimming behaviour, although they spent more time substratum skimming (i.e. contact with flume floor) at 30 cm s(-1) relative to 10 cm s(-1). When present, these behavioural responses are probably related to morphological features, such as flattened rostrum, large pectoral fins, flattened body shape and heterocercal tail, and may be important to reduce the costs of swimming.

Juvenile sturgeon exhibit reduced physiological responses to exercise.

Experiments were conducted to determine the physiological responses to exercise of Atlantic sturgeon (Acipenser oxyrhynchus) and shortnose sturgeon (A. brevirostrum). We measured the rates of oxygen consumption and ammonia excretion in both species and a variety of physiological parameters in both muscle (e.g. lactate, glycogen, pyruvate, glucose and phosphocreatine concentrations) and blood (e.g. osmolality and lactate concentration) in juvenile shortnose sturgeon following 5 min of exhaustive exercise. In both species, oxygen consumption and ammonia excretion rates increased approximately twofold following exhaustive exercise. Post-exercise oxygen consumption rates decreased to control levels within 30 min in both sturgeon species, but post-exercise ammonia excretion rates remained high in Atlantic sturgeon throughout the 4 h experiment. Resting muscle energy metabolite levels in shortnose sturgeon were similar to those of other fish species, but the levels decreased only slightly following the exercise period and recovery occurred within an hour. Under resting conditions, muscle lactate levels were low (<1 micromol g(-1)) but they increased to approximately 6 micromol g(-1) after exercise, returning to control levels within 6 h. Unlike similarly stressed teleost fish, such as the rainbow trout, plasma lactate levels did not increase substantially and returned to resting levels within 2 h. Plasma osmolality was not significantly affected by exercise in shortnose sturgeon. Taken together, these results suggest that shortnose and Atlantic sturgeon do not exhibit the physiological responses to exhaustive exercise typical of other fish species. They may possess behavioural or endocrinological mechanisms that differ from those of other fishes and that lead to a reduced ability to respond physiologically to exhaustive exercise.