Purification of multiple types of vitellogenins from plasma of flatfishes for use as standards in quantitative assays to assess their contribution to egg quality.

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1 Purpose
The main purpose of this study is to assess the contribution of multiple forms of Vitellogenins (Vtgs) to egg quality in flatfishes such as the Atlantic halibut and the European plaice. The study involves purification of multiple types of vitellogenins (Vtgs; VtgAa, VtgAb, VtgC) from plasma of estradiol (E2) induced juveniles. Purified Vtgs will be utilised as standards in quantitative assays to measure the levels of these multiple Vtgs in plasma from mature females during three consecutive reproductive seasons (2021-2023).

2 Distress
Juvenille fish wil be injected intraperitoneally with estradiol-17beta (E2), using a standardised protocol that has not been seen to have a negative effect on fish health. Blood samples will be taken from juvenile and mature fish. All individuals except halibut broodstock females will be anaesthetised during sampling. Halibut broodstock females >20 kg will not be anaesthetised. In our experience, this cannot be done without creating a large stress load on the fish and may interrupt spawning. Blood samples from the halibut females will only be taken once per year, and will be done during normal handling of the fish during the spawning period, either when checking for maturity status or stripping of eggs.

3 Expected benefit
The study is expected to assist in determining potential solutions to solve identified cause(s) of poor egg and offspring quality in flatfishes that can serve as models to other farmed fishes. Results will be of critical importance to global aquaculture and food security.

4 Number of animals, and what kind
Fifteen juveniles from each fish species, Atlantic halibut and European plaice, will be injected with E2, and 15 juveniles will be kept as controls for this experiment. In addition 10 mature females of halibut and plaice will be sampled for blood once every reproductive season during the period of 2021-2023.

5 How to adhere to 3R
Replacement of the chosen models for this study is not possible as the target is to study the contribution of the multiple Vtgs system to egg quality in fish. The number of individuals is held as low as possible for valid statistic calculations. E2 induction method has been chosen to boost the synthesis of Vtgs to minimise the number of fish to be used in this study. Fish are to be anaesthetised where possible (during injections and blood collection in juveniles), and rapid handling practices are to be applied where the use of anaesthesia is not possible (in large mature females).