No abstract

© 1998 Blackwell Science Ltd. 267 than did sperm thawed at 10, 20, 30, 40, 50 or 60°C in a water bath.

The endangered razorback sucker Xyrauchen texanus is endemic to the Colorado River system in western North America and is threatened with extinction because of limited recruitment. To assist in management and recovery efforts, we developed methods for the cryopreservation of sperm, evaluated the influence of various factors on motility of thawed sperm, and examined the effect on fertilization of cooling rate and the addition of caffeine. Sperm samples cryopreserved with 10% methanol (MeOH) had significantly higher postthaw motility than did samples preserved with 5% or 20% MeOH or with 5% or 10% dimethyl sulfoxide, N, N‐dimethylacetamide, glycerol, propylene glycol, or ethylene glycol. Sperm samples cryopreserved in 0.5‐mL and 2.5‐mL straws had significantly higher postthaw motility than did samples cryopreserved in 0.25‐mL straws. Exposure to 10% MeOH for up to 30 min did not significantly influence sperm motility before freezing or after thawing. Cooling rate (−21°C/min or −91°C/min) did not significantly influence sperm motility. Samples thawed in a water bath at 20°C, 30°C, or 40°C had significantly higher motility than did samples thawed on the laboratory bench (19°C). Refrigerated sperm had significantly higher motility after the addition of 0.005 M caffeine; however, caffeine did not increase the motility of thawed sperm. Fertilization percentage was 41 ± 31% for the egg quality control treatments (fresh sperm) in the freezing rate study. The freezing rate of −91°C/min yielded 66% fertilization relative to the control (actual value, 27 ± 26%), which was significantly higher than the 12% fertilization (actual, 5 ± 3%) yielded by the freezing rate of −21°C/min. Fertilization percentage was 25 ± 24% for the egg quality control treatments in the caffeine study. Caffeine‐treated sperm yielded 60% fertilization relative to the control (actual, 15 ± 13%), which was significantly higher than the 16% fertilization (actual, 4 ± 4%) yielded by sperm without caffeine treatment.

Populations of sturgeon across the globe are threatened due to unregulated harvest and habitat loss, and the status varies among species across North America. Ready access to viable and functional sperm would contribute to recovery programmes for these species. In this study, we examined the motility, viability (cell membrane integrity) of cryopreserved sperm from three North American acipenseriform species and fertilizing capacity. Milt samples were collected from captive shortnose sturgeon (Acipenser brevirostrum), wild paddlefish (Polyodon spathula) and pallid sturgeon (Scaphirhynchus albus) and cryopreserved using combinations of Modified TsvetkovaÕs (MT) extender, Original TsvetkovaÕs extender, and modified HanksÕ balanced salt solution, along with the cryoprotectants methanol (MeOH) or dimethyl sulfoxide (DMSO). A dual-staining technique using the fluorescent stains SYBR-14 and propidium iodide was employed with flow cytometry to determine the percentages of spermatozoa that were viable by virtue of having intact membranes. The percentage of viable spermatozoa ranged from 5% to 12% in shortnose sturgeon, 30-59% in paddlefish, and 44-58% in pallid sturgeon. In the first experiment with shortnose sturgeon sperm, methanol allowed for higher values for dependent variables than did DMSO, and sperm viability generally correlated with post-thaw motility. However, fertilization rate, neurulation, or hatching rates were independent from these factors. In the second experiment with shortnose sturgeon, 5% MeOH combined with MT yielded higher values for all parameters tested than the other combinations: viability was correlated with motility, fertilization rate, and hatching rate. Overall, viability and post-thaw motility was not affected by the use of hyperosmotic extenders (OT) or cryoprotectants (DMSO), but their use decreased fertilization percentages. For paddlefish sperm (experiment 3), MT combined with 10% MeOH was clearly a good choice for cryopreservation; viability and motility results were correlated, but independent of fertilization. For pallid sturgeon sperm (experiment 4), MT with 5-10% MeOH showed significantly higher sperm quality and fertilization parameters. Membrane integrity can be used as a predictor of fertilization by cryopreserved sperm, however additional sperm quality parameters, supplementary to motility and membrane integrity, would be useful in the refining and optimizing cryopreservation protocols with acipenseriform sperm.

Artificial spawning of channel catfish Ictalurus punctatus relies on the removal of testis and suspension of sperm in an extender solution for storage and use in fertilization. Little is known about the relationships among osmotic pressure, sperm activation, motility, and storage. Our objectives were to (1) estimate motility of channel catfish sperm diluted in solutions ranging in osmotic pressure from 8 to 295 milliosmols (mosmol)/kg, (2) identify the osmotic pressure that induces threshold activation (10% motility) and the highest pressure that induces complete activation, (3) determine the role of ionic dilution in activation by use of ion‐deficient solutions, and (4) evaluate the effect of osmotic pressure on the retention of motility during storage. Motility (percentage of actively swimming sperm) was estimated in diluted Hanksˈ balanced salt solution (HBSS) and sucrose solutions over a range of osmotic pressures. The HBSS osmotic pressures of threshold and complete activation were 218 ± 15 mosmol/kg and 132 ± 9 mosmol/kg, respectively. We found that osmotic pressures above 220 mosmol/kg induced minimal (<10%) activation and pressures below 130 mosmol/kg induced complete activation. Within the zone of incomplete activation (220–130 mosmol/kg) a decrease of 15 mosmol/kg in osmotic pressure increased motility about 10%. We used sucrose solutions to provide an osmotic environment with minimal ionic influence for the testing of activation. Osmotic pressures for threshold (214 ± 1 mosmol/kg) and complete (125 ± 2 mosmol/kg) activation were consistent with those obtained with diluted HBSS, suggesting that reduction in osmotic pressure plays a major role in the activation of channel catfish sperm. Sperm stored in 122 mosmol/kg HBSS lost 82% of initial motility after 2.5 h. However, sperm stored in solutions with higher osmolalities retained motility significantly longer (P = 0.0017). A minimum reduction of osmotic pressure to below 130 mosmol/kg is essential to induce complete motility of channel catfish sperm for motility estimates and artificial fertilization.