A protocol for the on-site use of frozen milt from rainbow trout (Oncorhynchus mykiss Walbaum) applied to the production of progeny groups: comparing males from different populations

Progeny testing could have a potential in aquaculture particularly in species where the males spawn over several years, and also in the monitoring of genetic changes between generations and sub-populations of salmonids, where the males are used for only one season. But these potentials can only be realised by the use of frozen sperm. Cryopreserved sperm could also be a means of exchange of genetic material between locations and populations when transportation of live fish is restricted. With the present protocol for the on-site use of frozen milt, post-thaw fertilization rates were >80% of controls with Norwegian rainbow trout males at a mean number of motile spermatozoa to egg ratio of 3.8x10(6). Milt samples were diluted either 1:3 or 1:5 in an ice cold extender containing methanol as cryoprotectant and aliquots were transferred to 0.5 ml straws, which were equilibrated at 4 °C for 10 min and then frozen on racks in a programmable freezer unit; the freezing curve had a freezing rate of 30 °C min-1 from +4 to -110 °C. Fertilization rates differed between sires within groups (Italian sires in Italy, Norwegian sires in Norway). Norwegian sires also gave significantly higher fertilization rates than did the Italian sires. Milt was sampled in the Italian stock rather late in the season, which is known to influence sperm quality in a negative way. But differences may also have been as much a function of quantity of the spermatozoa. The Italian males produced on average only half the number of spermatozoa of the Norwegian sires per unit volume, 704 compared with 1633, and had half the fraction of post-thaw motile spermatozoa (17.9% compared with 35.1%). Thus, there was four times the number of motile spermatozoa per cryopreserved dose from Norwegian sires. The between groups and sires within group differences in fertilization rates were substantiated with the production of the progeny groups. The fertilization rates obtained with Italian males and females decreased substantially when the numbers of eggs increased even when mean spermatozoa to egg ratio was kept constant: with 150 eggs the mean fertilization rate was (64.8±3)% or about 70% of controls, whereas with 1000 eggs it was (40.9±5.6)% or about 45% of controls. A minor reduction in mean fertilization rate to (69.9±1.2)% was observed with Norwegian males and females when the number of eggs was raised from 150 to 1000 at constant spermatozoa to egg ratio. The findings indicate that the protocol can be applied on a semi-commercial scale. But the mixing of eggs with spermatozoa could indeed be a crucial variable, and particularly so when milt is not used in excess. Results further indicate that the same sires perform either good or bad in both environments.