7819 - Laserfiche WebLink

Home Browse Search

Garrett et al. 29 lion and sperm production by injecting fish with human chorionic gonadotropin at 4,000 I.U./kg (Stevens 1970). Ripe eggs were strip- ped from females into 1.8-I dishes. Milt was stripped from males, collected by pipette, and mixed with the eggs. Eggs were fertilized in enough ambient water (approximately 22°C) to just cover them. Gametes obtained from two females and two males were mixed and apportioned evenly among treatments. Eggs were then sub- jected to six different pressure treatments, 5 minutes after fertiliza- tion. ' In each hydrostatic pressure treatment, 100-500 eggs were gently poured into a mesh basket and subjected to high pressure in a pres- sure chamber. The pressure chamber was similar to that described in Curtis et al. (1987). Pressure treatments ranged from 4,000 p.s.i. for 3 minutes to 8,300 p.s.i. for 1 minute (Table 1). After treatment, eggs were removed to aquaria fnr hatching. All pressure treatments occurred under ambient temperature conditions (21-25°C). Controls were groups of fertilized eggs obtained in the same way, at the same time, and from the same adult fish as above, but not subjected to pressure. In control and experimental aquaria, dead eggs were removed daily and counted. Aquaria were cleaned daily, and 50% of the water in each aquarium was replaced weekly with fresh water. Upon hatching, which occurred after approximately b0 hours, all larvae were fed live brine shrimp, Artemia gracilis, twice daily to satiation. Ploidy levels from a subset of control and experimental individ- uals were assayed using flow cytometric analysis following the pro- cedures of Gold et al. (in press). Briefly, approximately 5 mm3 of tissue (internal organs and/ar muscle) were taken from I- or 2- month-old fish (15-20 mm standard length) and placed in a cryovial containing 60-80 µl of storage buffer. The storage buffer contained 85.5 g sucrose (250 mM), 11.7b g citric acid-trisodium dihydrate (40 mM), and 50 ml dimethylsulfoxide (DMSO) in 1 liter of dis- tilled water at pli 7.60. The cryovials were then immersed in liquid nitrogen, transported for approximately 4 hours (to College Station, TX) and maintained at - 80°C until they were analyzed. Samples were prepared for flow cytometry by thawing the mate- rial atroom temperature, douncing the tissue firmly in a 2-ml Kontes