Production of Triploid Rainbow Trout OVA By BC Freshwater Finfish Farmers Using Hydrostatic Pressure

Final Report 28 MAY 2010

Table of contents

• EXECUTIVE SUMMARY
• THE PROBLEM
• THE SOLUTION
• THE RESULTS
• CONCLUSION

EXECUTIVE SUMMARY

Three of the member farms of the Freshwater Aquaculture Association of British Columbia – FAABC have adopted a hydrostatic pressure system for producing cultured triploid Rainbow trout. A standard practice of a 30 minute post-fertilization incubation time (at 8 to 10 C) followed by a pressure application at 10,000 psi for 5 minutes was found to be the most appropriate way of consistently inducing triploidy in farmed Rainbow trout.

When appropriate, these triploid trout will be sold to other member farms of FAABC for ongoing culture to larger sizes.

With the use of triploid trout, the inefficiencies that previously occurred in the freshwater trout culture farms can now be avoided. I.e. premature males will not mature in the second fall of culture and the entire cohort of trout will not sexually mature in the third fall of culture. This will allow all trout to be marketed as non-sexually mature fish with the superior pricing that occurs with a bright integumant, firm fleshed trout. In addition, when the farmer so desires, the triploid trout can be ongrown to a size that exceeds 2 – 3 lb since vegetative growth continues to occur without resources being devoted to sexual maturation in the second and third fall of the trout culture.

This AIMAP grant has been used to enhance the efficiency of Rainbow trout production in the freshwater finfish aquaculture industry of British Columbia. The financial assistance of the Canada Department of Fisheries and Oceans is very much appreciated. Without this funding, these efficiencies would not be attained.

THE PROBLEM

Diploid (2N) Rainbow trout become sexually mature in the third fall post egg-take for a total of approximately 36 months to maturity in coastal BC freshwaters. In the interior of BC, the 2N cultured trout become sexually mature in the third year post egg-take in freshwater in the spring. In addition, approximately 5 % of the total population of 2N trout displays sexual maturity of the male fish two-years post-egg-take in both the coast and interior of BC.

Sexual maturity of the precocious males at year 2 and the total population at year 3 causes significant difficulties to the freshwater Rainbow trout farmer. The main difficulties are:

1. The trout cannot attain a size significantly greater than 2 – 3 lb before becoming sexually mature. Yet, the market is ‘asking’ for more and more trout with weights of 4 – 6 lb for slicing for fillets. The BC freshwater trout farmers of FAABC cannot fill this market.
   
   
2. For those individuals growing 2 - 3 lb trout, significant difficulty is experienced October – January each year as the larger trout become sexually mature (and hence are unsuitable for the food fish markets) whereas the new crop of trout have not yet attained this size range.
   
   
3. Once the trout becomes sexually mature it cannot be readily sold to the food fish market. At best, it can be used to produce a smoked product. In many cases, it dies and the carcass is rendered for protein and lipid. If the trout survives the spawning cycle it is not reconditioned sufficiently until 6 months post at which time it can be sold into the foodfish market.
   
   
4. Many eyed trout ova purchasers wish to buy only triploid (3 N) rainbow trout ova and fry. Yet, the four main producers of eyed trout ova and fry in BC cannot produce these ova and fish since they do not have the equipment and experience in its use for the production of these sterile trout.
   
   
5. The smaller BC freshwater trout farmers do not have access to 3 N trout since they cannot import them into BC since they do not have access to a required quarantine facility.

THE SOLUTION

The four FAABC farms of (1) Aquafarms JV, (2) Campbell Lake Trout Farm, (3) Miracle Springs Inc. and (4) N’quatqua Springs Farm asked to be part of this study. Accordingly 4 pressure vessels for production of triploid trout ova were purchased along with 4 specially constructed bench presses. Figure 1 shows a pressure unit and a short technical description of it as purchased from Aquatic Eco-Systems, Inc., 2395 Apopka Blvd. Suite 100, Apopka, FL, USA.

Hydrostatic Pressure Chamber

Reproductive sterilization (triploidy) and sex selection in some species can be induced by subjecting fertilized eggs to high pressures. Triploid fish typically have increased growth rates since energy is not expended on gonad development and breeding behavior (i.e., teenagers). Our HPC is the right tool for the job. This specially designed, high-pressure vessel will pressurize approximately 24 oz of eggs up to 10,000 psi. This precision-made HPC is made from a solid piece of stainless steel with a double O-ring brass piston. Requires a 5-ton press with 23" minimum working height. Weighs 40 lbs. Made in USA.

The unit requires a 20 Tonne bottle jack to attain the pressures of 9,500 to 10,000 psi required to induce triploidy in the trout ova.

Initial tests with the unit showed that the available commercial bench presses were deformed/destroyed at cylinder pressures greater than 8,500 psi. Accordingly, 4 bench presses were constructed to accommodate the pressures unit such that the units could withstand cylinder pressures up to 10,000 psi.

Each of the 4 member farmers of FAABC noted above was provided with a pressure unit and a specially constructed bench press.

Several attempts at organizing a workshop to discuss the use of this equipment for inducing triploidy in trout ova were attempted. The member farmers of FAABC displayed a distinct lack of interest in such a workshop because (1) all had lack of time due of farm business – all are single operators of their farm (2) the technology for inducing triploidy by pressure is relatively straightforward and (3) all were little interested in the scientific reason as to why triploidy is induced by pressure.

Accordingly, workshops were not held. Instead, L.J. Albright explained the use of the equipment to each user and specific parameters of time and pressures that should be used.

THE RESULTS

AQUAFARMS JV

1. In the fall of 2009, Aquafarms JV pressure-treated several lots of fertilized rainbow trout ova (9,000 ova each lot) in a factorial pattern of from 20 to 38 minutes post-fertilization and 5 to 10 minutes pressure treatments of 9,5000 to 10,000 psi each at a mean water temperature of 8.5 C.

Each lot of pressure-treated trout was then hatched separately and the fish grown to 2 g each, at which time 6 individuals from each treatment were sacrificed and analyzed for mean length of the red blood cells – RBCs  nuclei using light microscipy at 1000X.

All samples of pressure-treated trout showed nuclei longer than the non-pressure treated control trout. On average, the pressure-treated nuclei were 29 % longer than the non-pressure treated control nuclei.

Conclusion: The post-fertilization time at 8.5 C water temperature can vary from 20 to 38 minutes before pressure application of between 9,500 and 10,000 psi for a duration of between 5 to 10 minutes to successfully produce triploid rainbow trout.

2. In the fall of 2010, Aquafarms JV pressure-treated 15 lots of 9,000 ova each at 30 minutes post-fertilization with 9,500 psi pressure for 5 minutes at a temperature of 8.5 C. Several lots of control ova were treated in the same manner but without pressure application.

All samples of ova that were pressure-treated and hatched had mean nuclei lengths approximately 27 % longer than the unpressurized control trout.

Conclusion: A post-fertilization time of 30 minutes at 8.5 C followed by a hydrostatic pressure application of 9,500 psi for 5 minutes is sufficient to induce triploidy in the rainbow trout.

CAMPBELL LAKE TROUT FARM

1. In the summer of 2010, Campbell Lake Trout farm treated an unknown number of fertilized trout ova with hydrostatic pressure. L.J. Albright recommend the use of 30 minutes post-fertilization followed by 5 minutes of pressure application at 10,000 psi.

In spite of several requests to this farm to provide data on the incubation and pressure conditions used, Campbell Lake Trout Farm has not provided these data, nor juvenile control and pressure-treated trout to be analyzed by L.J. Albright.

Because of the above, no conclusion can be reached regarding the efficacy of the procedure used by Campbell Lake Trout Farm in its production of triploid trout.

MIRACLE SPRINGS INC.

1. In the fall of 2009, this farm treated 2 lots of 9,000 rainbow trout ova each at 8.5 C for between 25 and  30 minutes post-fertilization for 5 minutes at 10,000 psi.

Samples of these resulting progeny from these ova showed nuclei that were 43 % longer than the control untreated diploid trout.

2. In the fall of 2010, this farm treated lots 1 and 2 of 9,000 ova each with 30 and 35 minutes of pre-incubation respectively before pressurizing each lot to 10,000 psi for 5 minutes.

Of the 10 trout pressured-treated trout fry and the 10 control trout fry provided by this farm to L.J. Albright, the pressure-treated fry had RBC nuclei that were 26 % larger than the control non-pressurized control fry.

Conclusion: A post-fertilization time of 30 or 35 minutes followed by a hydrostatic pressure application of 10,000 psi for 5 minutes is sufficient to induce triploidy in the rainbow trout.

N’QUATQUA SPRINGS FARM

1. Four trips were made to the this farm by L.J. Albright to train the First Nation farm operators in the use of the pressure equipment and carry out trials using the farm’s trout ova.

2. This farm had rainbow trout broodstock in very poor shape and not at all appropriate for production of triploid trout, much regular less diploid trout.

Accordingly, the time of my trips ended up showing basic husbandry and culture methods rather than the triploidy procedure. In addition, the farm manager, Ralph Thevarge was taken on a trip to the annual meeting of the Aquaculature Association of Canada in Nanaimo  to introduce him to other aquaculaturists.

Conclusion: N’quatqua will have to produce better broodstock trout before producing triploid ova.

CONCLUSION

BC farmed Rainbow trout are readily triploided when a 30 to 35 minutes post-fertilization time is used before pressurizing the ova to between 9,500 and 10,000 psi for 5 minutes, when analyzed by microscopic examination of Wright Giemsa stained red blood cells - RBCs. The RBCs from pressurized ova were between 26 and 43 % longer than those of the untreated control trout.

A standard practice of a 30 minute post-fertilization incubation time (at 8 to 10 C) followed by a pressure application at 10,000 psi for 5 minutes is recommended for consistently inducing triploidy in farmed Rainbow trout.

Definite proof of triploidy will occur in the third fall of the culture of these pressure-treated trout. If these pressure-treated trout fail to become sexually mature (under normal conditions cultured trout spawn in the third year post-fertilization), the procedure is successful. If the trout become sexually mature, the procedure is defective and other conditions of post-fertilization time and pressure treatment will have to be determined.

For, this reason, it is unfortunate that this final report has to be handed in at this time. However, the microscopic examination of the red blood cellss indicate that the conditions recommended above are appropriate for press-induced triploid in the BC farmed rainbow trout.