Evaluation of gamete quality indicators for Arctic charr Salvelinus alpinus

1. EVALUATION OF GAMETE QUALITY INDICATORS FOR ARCTIC CHARR Salvelinus alpinus JEUTHE ET AL. AQUACULTURE 504 (2019)

2. The Arctic charr ( The Arctic charr (Salvelinus Salvelinus alpinus alpinus L.) L.) Holarctic, landlocked and anadromous, Holarctic, landlocked and anadromous, most cold most cold- -adapted freshwater fish adapted freshwater fish Reproductive performance of Reproductive performance of farmed Arctic farmed Arctic charr charr ??? unknown reasons for substantial variations between individual fish in broodstock commercially commercially farmed since the farmed since the early 90s ( early 90s (Iceland Sweden and Sweden and Norway), Norway), successful successful selective breeding selective breeding programs programs Iceland, , farming is predicted to grow farming is predicted to grow - - supply of sufficient quantities of good quality offspring sufficient quantities of good quality offspring to meet the increasing demand for juveniles to meet the increasing demand for juveniles supply of

3. EVALUATIONS AND TESTED CORRELATIONS AIM OF THE STUDY • SEX HORMONE STATUS OF THE BROODSTOCK (blood samples - hormone analyses, immunoassay) ? OFFSPRING LOSS EGG VIABILITY VARIABLES failed fertilisation or mortality? 1. 1. Fertilisation rate Fertilisation rate : proportion of fertilised eggs • EGG TRAITS (egg size and wateriness of egg batches, post-ovulatory aging of eggs, symmetry of early cell division) 2. 2. Mortality Mortality : live and dead eggs after eye stage connected to any specific parent or gamete traits? • MILT TRAITS (volume, density, and sperm motility characteristics – CASA- computer assisted sperm analysis) 3. 3. Proportion of eyed eggs Proportion of eyed eggs maternal or paternal factor more predominant?

4. HYPOTHESIS 1. 1. WATERINESS OF EGG BATCHES WATERINESS OF EGG BATCHES WOULD INDICATE LEVEL OF POST-OVULATION AGING AND HENCE BE ASSOCIATED WITH VARIATIONS IN EGG VIABILITY (DRIVER FOR REPRODUCTIVE IMPAIRMENT) 2. 2. EGG SIZE EGG SIZE COULD BE USED AS AN INDICATOR OF QUALITY 3. 3. SPERM SPERM QUALITY QUALITY AFFECTS FERTILISATION RATES AND EMBRYO SURVIVAL 4. POSITIVE CORRELATION BETWEEN MATERNAL MATERNAL 17,20Β 17,20Β- -P LEVELS P LEVELS AND OFFSPRING VIABILITY 5. 5. MALE MALE HORMONAL STATUS HORMONAL STATUS WOULD BE ASSOCIATED WITH REPRODUCTIVE SUCCESS

5. EGG & SPERM QUALITY EGG & SPERM QUALITY SPERM QUALITY INDICATORS : • POST-OVULATORY AGING OF EGGS, or overripening - eggs are retained in the fish after ovulation their quality and viability deteriorates with time ~ 5 days / 5 °C SUCCESSFUL SUCCESSFUL FERTILISATION FERTILISATION EMBRYONIC EMBRYONIC SURVIVAL RATES SURVIVAL RATES • DAMAGED DNA OR RNA IN THE SPERMATOZOA - still be able to fertilise an oocyte → disturb early embryonic development • EGG SIZE range of contradictory results have been published • WATERINESS OF THE EGG BATCH - amount of fluid surrounding the stripped eggs, ovarian or coelomic fluid; “dry egg batches”, i.e. accompanied by limited amount of fluid, are considered of good quality

6. BROODSTOCK BROODSTOCK HORMONAL STATUS HORMONAL STATUS Gamete development : (1) Gamete growth , (2) Gamete maturation In female levels of 17,20β (maturation-inducing hormone), increase during final oocyte maturation final oocyte maturation (FOM) and peak at ovulation 17,20β- -P P In male 17,20β rises prior to spawning 17,20β- -P P sharply maturation-inducing hormone 17,20β-P 17,20β-dihydroxy-4prehnen-3-one

7. MATERIALS AND METHODS • BROODSTOCK : 45 PAIRS OF ARCTIC CHARR • AGE 3+ YEARS • fish reared in indoor tanks • water at ambient temp from a lake via flow-through system (summer 11°C - 13°C, followed by a steady autumn cooling that started in mid-september) 4 °C and 2.5 °C Stripping and fertilisation on 5 & 12 Nov Water temperatures were 4 °C and 2.5 °C • egg incubations initiated at ambient w. temp. and then increased gradually to 6°c Milt from each of the males (n=23), except one, Milt from each of the males (n=23), except one, was used to fertilise the eggs from two females was used to fertilise the eggs from two females (n=45) (n=45)

8. CATEGORISING CATEGORISING EGG LOSS EGG LOSS SAMPLING AND ANALYSIS EGG VIABILITY VARIABLES 1. 1. Fertilisation rate Fertilisation rate : proportion of fertilised eggs, day after fertilisation (eight cell stage, +examination of cell symmetry), 100 eggs from each egg batch 2. 2. Mortality Mortality : after completion of the eyed stage, live and dead eggs were sorted and counted 3. 3. Proportion of eyed eggs Proportion of eyed eggs : number of eyed eggs divided by the total number of stripped eggs Total Total egg volume egg volume from each female and mean egg size were measured and used to estimate total number of number of eggs eggs Egg size Egg size - counting the eggs in a row of 25 cm Wateriness Wateriness (the amount of ovarian fluid surrounding the stripped eggs) - three total three- -grade scale grade scale ruptured eggs ruptured eggs were removed and counted (damaged eggs; not included in the fertilisation and mortality estimates)

9. Egg loss in hatchery production of Arctic charr (n=43) presented as proportion of original (stripped) egg batches RESULTS RESULTS parts of the egg loss are explained by post-ovulation aging of the oocytes * it is difficult to accurately pinpoint the time of stripping in relation to ovulation WATERINESS varied between individual egg batches Overall reproductive success The proportion of damaged eggs (white & ruptured eggs removed) Fertilisation rates (of remaining eggs) Survival rate from fertilisation to the eyed stage 0-12.6%, median 2.7%, in individual egg batches 59% - 100%, median 93% 9% - 98%, median 70% no correlation with egg viability

10. RESULTS RESULTS SPERM QUALITY the strongest the strongest relationship relationship among results results - - BFC among BFC NO CORRELATION BETWEEN EGG SIZE AND VIABILITY BCF factor beating frequency of the sperm tail, and of head wobbling fertilisations occurred at 4°C and 2.5°C high temp. during summer (11-13C) & early autumn →stress first time spawning salmonids - more sensitive to thermal stress more sensitive to thermal stress during vitellogenesis - reduced investment into oocytes, →smaller and less viable eggs all egg batches in the study were at the lower end of the size range (description in study in 2013) BUT study in 2013 shown that small eggs from this strain have BUT study in 2013 shown that small eggs from this strain have poor viability poor viability → BUT BUT positive relationship between egg size and viability may be effect of broodstock broodstock age Here : single age group used • • • • Mean beat cross frequency (BCF) of sperm from individual males (n=20) and proportion of eyed eggs in egg batches fertilised • age rather than egg size relevance of BCF as a sperm quality indicator is relevance of BCF as a sperm quality indicator is debatable (software developers do not debatable (software developers do not recommend to use it), but correlation with recommend to use it), but correlation with offspring viability presented in the present study offspring viability presented in the present study is too strong to ignore is too strong to ignore completely completely • possible that a significant part of the egg mortality can be due to first time spawners being exposed to supra-optimal rearing temperature •

11. RESULTS RESULTS HORMONE STATUS Positive trend between 17,20β-P levels and fertilisation rates Blood plasma hormone levels in female Arctic charr (n=40) at the time of egg stripping. E2 E2 levels were significantly higher in the second sampling levels were significantly higher in the second sampling; no significant differences in T and 17,20β-P levels were found between samplings high fertilisation rates in whole conc. range No significant differences were found in male hormone levels between samplings. low fertilisation rates in low levels of 17,20β-p males males with higher 17,20β with higher 17,20β- -P levels showed high fertilisation rates fertilisation rates, no significant correlation between the two variables P levels showed high

12. CONCLUSIONS CONCLUSIONS • MAJORITY OF OFFSPRING LOSS: embryo mortality + fertilisation failure • fertilisation and mortality rates were not inter-correlated • none of the evaluated egg traits proved to be useful indicators of egg quality • SPERM SPERM PARAMETERS WERE CORRELATED WITH REPRODUCTIVE PARAMETERS WERE CORRELATED WITH REPRODUCTIVE PERFORMANCE AND COULD POTENTIALLY BE USED AS QUALITY PERFORMANCE AND COULD POTENTIALLY BE USED AS QUALITY INDICATORS INDICATORS • for Arctic charr – MALE 17,20Β variation in proportion of successfully eyed eggs (fertilisation rates) MALE 17,20Β- -P LEVELS P LEVELS and BCF explained 69.5% of

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