Our goal was to develop a standardized approach for sperm vitrification of marine fishes that can be applied generally in aquatic species. The objectives were to: 1) estimate acute toxicity of cryoprotectants over a range of concentrations; 2) evaluate the properties of vitrification solutions (VS); 3) evaluate different thawing solutions, and 4) evaluate sperm quality after thawing by examination of motility and membrane integrity. Sperm were collected from red snapper (Lutjanus campechanus), spotted seatrout (Cynoscion nebulosus), and red drum (Sciaenops ocellatus). A total of 29 combinations of cryoprotectants were evaluated for toxicity and glass formation. Samples were loaded onto 10-µL polystyrene loops and plunged into liquid nitrogen. There was a significant difference (P < 0.05) in post-thaw motility among VS and among species when using the same VS. The sperm in VS of 15% DMSO + 15% ethylene glycol + 10% glycerol + 1% X-1000™ + 1% Z-1000™ had an average post-thaw motility of 58% and membrane integrity of 19% for spotted seatrout, 38% and 9% for red snapper, and 30% and 19% for red drum. Adaptations by marine fish to high osmotic pressures could explain the survival in the high cryoprotectant concentrations. Vitrification offers an alternative to conventional cryopreservation.

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This study reports the first production of offspring with vitrified sperm from a live-bearing fish Xiphophorus hellerii. The overall goal of this study was to develop streamlined protocols for integration into a standardized approach for vitrification of aquatic species germplasm. The objectives were to (1) estimate acute toxicity of cryoprotectants, (2) evaluate vitrification solutions, (3) compare different thawing methods, (4) evaluate membrane integrity of post-thaw sperm vitrified in different cryoprotectants, and (5) evaluate the fertility of vitrified sperm. Nine cryoprotectants and two commercial vitrification additives were tested for acute toxicity and glass forming ability, alone and in combination. Two vitrification solutions, 40% glycerol (Gly) and 20% Gly + 20% ethylene glycol (EG) in 500 mOsmol/kg Hanks' balanced salt solution (HBSS), were selected for vitrification of 10 lL sperm samples using inoculating loops plunged into liquid nitrogen. Samples were thawed at 24°C (one loop in 5 lL of HBSS or three loops in 500 lL of HBSS). Samples thawed in 500 lL were concentrated by centrifugation (1000 g for 5 min at 4°C) into 5 lL for artificial insemination. Offspring were produced from virgin females inseminated with sperm vitrified with 20% Gly + 20% EG and concentrated by centrifugation.

Sex control can solve the problem of stunted black crappie populations in small impoundments. The main objectives of the present study were (1) to identify sex-reversed males of black crappie from a previously obtained androgen-treated group using test crosses, and (2) to develop broodstock of sex-reversed males by masculinization of fish from those crosses. An additional objective of the study was to try to identify sex-specific RAPD markers, which might be used for identification of sex-reversed males. The progenies resulting from test crosses were divided into two groups: Group I (control) fish were raised without hormonal treatment, while Group II fish were subjected to androgen (MT) treatment. Seven progenies were obtained from the cross of preliminary androgen-treated males with normal females. Six of seven progenies had sex ratio in Groups I close to 1:1, and one progeny consisted of females only. This shows that only one male, which generated this progeny, was a sex-reversed homogametic fish (XX) while the other males were heterogametic normal (XY) fish. In Group II the androgen 17α-methyltestosterone (MT) was orally administered to crappie with an artificial diet (30 mg/kg) for 40 days beginning 35 days post hatching; androgen-treated groups consisted of 95-100% males. Sex-specific random-amplified polymorphic DNA markers were not identified in black crappie.