Introduction
Atlantic halibut is a batch spawning fish delivering up to 16 batches of pelagic eggs in intervals of 70-90 hours during one reproductive season. While established for decades, aquaculture of Atlantic halibut is challenged by many obstacles one of which is insufficient production of juveniles (Engelsen et al., 2004). Post ovulatory ageing is a process of degeneration on eggs which begins right after ovulation. In rainbow trout, incorrect stripping time can result in very low egg quality, changes in morphology, composition and maternal gene expression due to post ovulatory ageing (Aegerter et al., 2005) . Mating of Atlantic halibut in captivity has only been observed once and hence eggs and milt need to be stripped for artificial fertilisation (Holmefjord , Lein, 1990). Decades ago, wild fish were used for broodstock, but some hatcheries have since bred up to F3 generation fish which are in production today. Wi ld females were thought to spontaneously release their eggs in captivity shortly after ovulation . Females were typically closely monitored by hatchery personnel to estimate the optimal stripping time (Norberg et al., 1991). With farmed broodstock new challenges arose, typically lower fertilisation rates compared to wild fish, higher variability and on many individuals the lack of spontaneous egg release , however growth of farmed fish increased (personal communication with hatchery personnel) . Today, in three of the four Atlantic halibut hatcheries in Norway technicians are stripping females based on how much their ovaries are visually built up and on experience. If no eggs are obtained, the fish are checked again after 12-80 hours. Fish that gave eggs are stripped again in 81-84 hour intervals. This study is aimed at understanding the implications of post ovulatory ageing on egg quality of Atlantic halibut during fertilisation, development and hatching.
Materials and methods
Experiments were conducted at Nordic Halibut AS, Midsund, Norway during Summer 2020 and Spring 2021 spawning seasons. The natural spawning season for Atlantic halibut is Spring but spawning in summer is achieved by photomanipulation. All broodstock were stripped by hatchery personnel, and the stripping time was estimated based on the fertilisation rates of the previous batches and stripping attempts. Batches with low fertilisation rates were stripped more frequently than common hatchery practice. Only gentle pressure was applied to strip the eggs from the females, if the ovaries were hard and eggs could not be obtained the fish were checked later. Six egg batches of good quality were obtained this way. Six fractions (50-150 mL each) of each batch were taken and stored in a concealed container in absence of air at 6°C. To simulate post ovulatory ageing, o ne of each fractions was fertilised using cryopreserved milt (Cryogenetics AS) 0, 1, 2, 4, 6 and 12 hours after stripping to simulate the process of post ovulatory ageing . Between 200-400 eggs were distributed to three petri dishes and twelve 800 mL cell culture flasks containing 1 L of seawater with 0.25mL /L solution of terramycin (100mg/mL) and incubated at 6°C. Eggs were sampled 14h post fertilisation (hpf) for 8 cell stage and fertilisation rates (from petri dishes), 48-50 hpf for blastula stage, 300 hpf and 540 hpf for hatching rate (from cell culture flasks).
Results
Data evaluation for Spring 2021 is still in progress. However, in Summer 2020, fertilisation rates were slightly but not significantly decreasing up to 12 hours past stripping. Survival was constant for four hours after stripping and at 6 hours past stripping it decreased dramatically from 70 % to 30 % in one of the batches. Other observed trends were higher mortality during incubation with longer post ovulatory ageing, a higher percentage of eggs lacking activation during the fertilisation process as well as higher numbers of failures in body axis formation after gastrulation. Most females appear to have different intervals between the batches and following up those times is incredibly difficult without stringent record of temperatures. Small temperature fluctuations will delay or accelerate ovulation.
Discussion and conclusion
Fertilisation and hatching rate have been positively correlated in turbot eggs (Kjørsvik et al., 2003 ). Whilst this correlation can be observed in artificially aged Atlantic halibut eggs over a longer period , this study shows that post ovulatory ageing has a higher influence on survival than on fertilisation rate. This indicates that previous findings based only on fertilisation rate may have underestimated the problem of overripening in Atlantic halibut broodstock ( for example Norberg et al., 1991). Thus, highlighting the importance of a flexible stripping protocol that is suitable to avoid overripening of the eggs as much as possible. Comparisons of low versus good quality eggs in turbot revealed complications such as reduced ability to cope with environmental stress, abnormal development, deformities and incomplete eye migration in low quality batches (Kjørsvik et al., 2003) . It is likely that similar to turbot, fry from low quality batches in Atlantic halibut will suffer higher mortalities and more deformations during development and metamorphosis. Low fertilisation rates and high variability in egg quality in Atlantic halibut can at least to an extend be explained by post ovulatory ageing due to inadequate stripping protocols for farmed broodstock. This study shed light on how to improve batch quality by modifying the established stripping protocols based on the fertilisation rates of previous batches, which not only dramatically increased the quality of successive batches , but also enables researchers to obtain batches that are less affected by post ovulatory ageing when exploring differences in egg quality between fish and batches.
References
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