CSUS: Biological Sciences: Stephen Stringer

Optimization of hydrostatic pressure treatment to induce triploidy in American River Chinook salmon (Oncorhynchus tshawytscha)

A potential solution that would allow continued planting of Chinook salmon into reservoirs for angling and eliminate genetic concerns is the planting of sterile Chinook into reservoirs in drainages where they are not indigenous. The induction of triploidy is currently the best method for producing sterile salmonids meant for human consumption and hydrostatic pressure treatment is the most effective method for inducing triploidy. Tiploidy has been successfully induced in several species of salmonids using hydrostatic pressure treatments and triploid embryos of salmonid species such as rainbow trout (Oncorhynchus mykiss) are available from commercial hatcheries. Triploid Chinook salmon have been studied far less than other salmonids and methods of inducing triploidy in them are not well published. Triploid Chinook salmon are not commercially available in California and DFG would like to develop methods for producing them.

Triploidy is induced in salmonids by applying hydrostatic pressure treatment to the fertilized eggs within the first hour post-fertilization, which prevents exclusion of the second polar body during the second phase of meiosis and results in retention of an extra set of maternal chromosomes. The intensity and duration of hydrostatic pressure, as well as the time after fertilization that the hydrostatic pressure is applied, likely affects the probability that an egg will become triploid and may also affect the likelihood of survival of an egg. The objective of this study was to optimize hydrostatic pressure treatment to induce triploidy in American River Chinook salmon to produce the maximum percentage of surviving triploids. Four different hydrostatic pressure treatments plus a control group (no pressure treatment) were evaluated in this study. Three replicates of each hydrostatic pressure treatment were conducted. To do this, Chinook eggs were spawned, fertilized and pressure treated on three separate days with one replicate of each of the four hydrostatic pressure treatments and a control group being conducted each day. To facilitate discussion, each set of four hydrostatic pressure treatments and its associated control group is referred to as a Replicate. Replicate 1 was conducted on 6 December 2005, Replicate 2 was conducted on 12 December 2005, and Replicate 3 was conducted on 13 December 2005.

American River Chinook salmon were spawned, and the resulting eggs were fertilized and pressure treated at the DFG Nimbus Salmon and Steelhead Hatchery (Hatchery) in Rancho Cordova, CA. For each Replicate, eggs were collected from seven females and milt was collected from 15 males (with the exception of Replicate #1, see note below). Eggs collected from teh seven females were pooled and used to produce all five experimental groups in that Replicate. Milt was not pooled. Milt from three males was used to fertilize each experimental group, with the exception of the 300oC minute treatment group in Replicate 1. Five males were used to fertilized this group due to low milt output from two of the males. The measurement "oC minutes" is dependant on water temperature in degrees Celsius and time elapsed in minutes. For instance, 300oC minutes could be 30 minutes at 10oC or any other combination of minutes and temperature in degrees Celsius that equal 300 when multiplied. Each hydrostatic pressure treatment was subjected to 9,500 pounds per square inch (psi) of pressure for five minutes in a hydrostatic pressure treatment device manufactured by TRC Hydraulics, Inc. The time after fertilization at which pressure was applied was varied for each of the fou hydrostatic pressure treatments. The first, second, third, and fourth hydrostatic pressure treatment groups were pressure treated at 150oC minutes, 200oC minutes, 250oC minutes, and 300oC minutes are fertilization respectively.

The resulting offspring were raised separately by experimental group at the Hatchery until 4 May 2006. The approximately five month time frame allowed the eggs to hatch and the fish to grow to a sufficient size to obtain blood samples for the purpose of verifying triploidy in the pressure treatment groups. Survival was monitored throughout the experiment. Dead individuals (e.g., eggs, alevins, and fry) were removed periodically and counted. Water temperature was recorded at the time of stripping and fertilization and daily throughout the experiment. The length of time necessary for each treatment group to go from fertilization to hatching, and from hatching to the swim up stage was recorded. On 4 May 2006, a sample of fish from each experimental group was sacrificed and blood samples were collected to verify ploidy level. Length and weight measurements were taken at this time. Ploidy level was verified on a Becton Dickinson FACScan flow cytometer in the Optical Biology Lab Shared Resource Center (Room 3435, Tupper Hall) at the University of California, Davis. Analysis of variance (ANOVA) was used to analyze the affect of each hydrostatic pressure treatment on percent triploidy, survival, dry weight, and total length.

Of the four different hydrostatic pressure treatments evaluated in this study, it appears that the best time after fertilization to begin pressure treatment with American River Chinook salmon in our hatchery is approximately 300oC minutes. The 300oC minute treatment achieved the highest combined survival rate and triploidization success in all three replicates.