Jacques Rinchard

Reproductive cycle and plasma levels of sex steroids in female Eurasian perch Perca fluviatilis

Abstract From April 1995 to April 1996, the annual reproductive cycle of the Eurasian perch Perca fluviatilis was studied at the Fishfarming Lindre Center (Moselle, France). At monthly intervals (at intervals of 10 days during the periovulatory period), 5 females were caught and dissected. From sampled organs, the gonado-, hepato- and viscerosomatic indexes (GSI, HSI, VSI) were calculated, oocyte diameters (OD) and the plasma levels of testosterone (T), 17β-estradiol (E 2 ), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and protein-phosphorus (PPP) were measured. After the sexual resting period observed from May to August (GSI 2 , 17,20β-P and PPP were low during the sexual resting period. E 2 and PPP levels increased significantly at the onset of the oogenesis in September, then the E 2 level raised abruptly in November (3–4 ng · mL −1 ). In December, the T level increased rapidly to 15–20 ng · mL −1 . The testosterone, E 2 and PPP levels remained very high until spawning, indicating the existence of active vitellogenesis. The highest HSI (2.1–2.2 %) recorded in winter confirmed this. During the periovulatory period, a peak of E 2 (4 ng · mL −1 ) appeared, whereas T level diminished. In this study, 17,20β-P levels remained low (0.2–0.6 ng · mL −1 ) and relatively constant. No 17,20β-P peak was observed during the periovulatory period. Sampling at 10 day intervals was probably inadequate to specify the hormonal variations related to the final oocyte maturation and the ovulation.

Effects of diets containing gossypol on reproductive capacity of rainbow trout (Oncorhynchus mykiss).

Abstract We evaluated five practical diets in which 0%, 25%, 50%, 75%, and 100% (dietary treatments 1–5) of fish meal protein was replaced by solvent-extracted cottonseed meal protein. Adult rainbow trout (initial average weight 247 ± 8 g) were fed the diets over a period of 131 days during which a general 2-fold body weight increase occurred. The total diet gossypol concentration (free and protein-bound) showed a gradual increase with increased cottonseed meal substitution. Blood samples were collected on Days 0, 64, 112, and 131 for hematological and steroid hormone determination in plasma of males and females. Hemoglobin content was significantly reduced in fish from treatment 5 (7.9 ± 0.3 g/dl) in comparison to treatments 1–3 (10.3–10.9 g/dl). After 112 and 131 days of feeding, testis weights, concentrations of testosterone, and 11-ketotestosterone were elevated in fish from dietary treatments 2 and 3 in comparison to control and diets 4 and 5. On Day 71, sperm were collected from 6 fish per dietary treatment to assess sperm quality. No significant differences in sperm concentrations (7.2–9.8 × 109/ml), motility (78–89%), and standardized (300 × 105 sperm/egg) fertilizing ability (18.9–22.6% hatched embryos) were found. Total gossypol concentrations in blood plasma differed significantly among treatments, and the levels were among the highest ever recorded in animals fed cottonseed-supplemented diets (2.9 ± 0.2, 11.7 ± 4.1, 21.7 ± 1.4, and 29.9 ± 3.9 μg/ml, for treatments 2–5, respectively). The major portion of gossypol in blood plasma was protein-bound (81–93%). This was in contrast to minute amounts of gossypol present in seminal plasma, mostly in free form (0.02–0.18 μg/ml), which indicates the presence of a barrier between general circulation and the testis with respect to gossypol distribution in lower vertebrates. Thus, the reproductive parameters of male rainbow trout examined in this study were not significantly affected by feeding cottonseed meal for 131 days.

Gossypol isomers bind specifically to blood plasma proteins and spermatozoa of rainbow trout fed diets containing cottonseed meal

We investigated the role of gossypol isomers binding to blood plasma, seminal plasma and spermatozoa to elucidate gossypol anti-fertility action in the teleost fish, rainbow trout (Oncorhynchus mykiss). Growth and hematological indicators of males were depressed when fish meal protein in diets was completely replaced with cottonseed meal. The cottonseed meal contained equal proportions of (-) (47.8+/-1.6%) and (+) gossypol isomers. Concentrations of spermatozoa were decreased with increasing proportions of gossypol in diets (from 0.22% to 0.95%); however, sperm motility and fertilizing ability were not affected. In contrast to mammals, steroid hormone concentrations were not suppressed in fish given diets with gradual increase of gossypol level. Gossypol concentrations were 100-fold higher in blood plasma than in seminal plasma, confirming a barrier in gossypol transfer between the general circulation and the testis. Spermatozoa accumulated predominantly (+) enantiomer (65-75%) with decreasing proportions as dietary gossypol concentrations increased. Spermatozoa bound most of the gossypol contained in the semen; however, this did not result in impairment of the sperm motility apparatus. Teleost fish sperm rely on ATP stores that accumulate during maturation as a source of energy during activation. In addition, the duration of sperm movement is short in these fish. As such, we hypothesize that the major action of gossypol on mammalian sperm, which is uncoupling of oxidative phosphorylation, does not impair the energy supply required for flagellar beating in fish spermatozoa.

Effects of dietary cottonseed meal protein level on growth, gonad development and plasma sex steroid hormones of tropical fish tilapia Oreochromis sp

Five experimental diets containing increasing proportions of cottonseedmeal (CSM) protein (0, 25, 50, 75 and 100%; diets 1 to 5, respectively) toreplace fish meal (FM) protein were formulated for intensive culture of tilapiaOreochromis sp. Each diet was fed to three replicategroupsof fish (mean weight ± SE = 11.3 ± 3.9 g) in30L aquaria connected as a closed recirculating-water system andmaintained at 27 ± 1 °C. Fish were fed three times adayby hand at a rate of 3% of body weight during four weeks, after which thefeeding rate was gradually decreased to reach 1.5% at 16 weeks. Thesubstitutionof 75 and 100% of FM proteins by CSM proteins resulted in significantly lowerbody weights in both sexes. In both sexes, gonadosomatic indexes and plasmaconcentrations of sex steroids (testosterone, 11-ketotestoterone,estradiol-17β and 17,20β-dihydroxy-4-pregnen-3-one) were notsignificantly different among dietary treatments. The concentration ofgossypol,an antifertility agent contained in CSM, was measured in reproductive tissues.The total gossypol concentration in the testis was consistently lower than thatmeasured in the ovaries of the same group. Moreover, in both sexes, theconcentration of the (+)isomer of gossypol was always higher than that of(−)isomer. The total gossypol concentrations in testes increasedsignificantly with the increase of CSM in the diet. The highest levels of the(+)isomer (7.64 ± 0.62 μg g−1)were found in the testes of fish fed diet 4, whereas the (−)isomerreached its highest values in the testes of fish fed diet 5. The highest levelsof both enantiomers of gossypol were found in the ovaries of fish fed diet 4(14.2 ± 2.7 and 5.6 ± 1.5 μg g−1for (+) and (−)isomers, respectively). In both sexes, thehistological analysis of the gonads did not reveal differences among the fishfed different levels of CSM. Although CSM at any levels did not affect thereproductive parameters examined in this study, it cannot be used to substitutemore than 50% of FM since at higher levels growth of tilapia was compromised.

Effect of feeding cottonseed meal-containing diets to broodstock rainbow trout and their impact on the growth of their progenies

Rainbow trout Oncorhynchus mykiss broodstocks were fed five experimental diets in which fish meal protein was gradually replaced with cottonseed meal (CS) protein (0%, 25%, 50%, 75%, and 100%; diets 1–5, respectively) during a 22-month period. The effect of increasing dietary levels of CS on reproductive performance was gender specific. Sperm fertilizing ability significantly decreased when CS exceeded 50% protein replacement (72.6±2.0%, 73.6±2.0%, 69.0±3.3%, 43.3±5.1%, and 36.8±4.7%, for diets 1–5, respectively). In contrast, in females, the viability of embryos was only significantly affected at 25% and 50% replacement levels (56.7±15.6%, 20.5±24.9%, 11.4±18.6%, 48.8±25.9%, and 39.6±31.1%, for diets 1–5, respectively). Progenies from multiple parents per dietary treatment were combined and reared on a commercial diet over a 2- or 3-month period fed at a rate of 4% of their body weight. The paternal origin (fresh sperm, experiment 1) had a highly significant effect on growth performance of progenies, and progenies from males fed with 25%, 50%, and 75% CS grew significantly (P<0.05) faster than progenies from males fed with 0% and 100% CS. Growth performance of progenies produced using cryopreserved sperm (experiment 2) was not affected regardless of the CS levels fed to male rainbow trout. Progenies from females (experiment 3) fed a diet containing 50% CS grew significantly (P<0.05) slower than the other groups. Sex ratio was examined histologically after completion of feeding experiments with progenies. Regardless of maternal or paternal origin, males dominated among the progenies. Thus, we postulated that other substances such as flavonoids, present in the CS and possibly transferred to yolk sac reserves, might affect the sex ratio in favor of males.

Delivering bioactive compounds to fish larvae using microencapsulated diets

The efficient delivery of nutrients and hormones has special relevance to the development of rearing technologies for fish larvae and juveniles. The main aim is to find an effective and measurable way to administer them into the body of small aquatic animals. In this study, three different compounds (hormones, amino acids, and vitamins) were incorporated into protein-walled microencapsulated diets. Specifically these microencapsulated diets were examined for (a) the kinetics of incorporation of estradiol in Sparus aurata larvae, (b) absorption and leaching patterns of the free amino acids (FAA), and (c) growth results and tissue incorporation of vitamins in relation to the supplementation of vitamin C in larvae of S. aurata and Solea senegalensis. The efficiency of inclusion was relatively low, but the capsules were able to retain enough of these compounds when immersed in water and to deliver them into the digestive tract of the larvae. There are noticeable differences among the nominal amount of a given substance in the ingredient mixture, the actual amount in the microparticle and the amount delivered in the larval gut. It is therefore necessary to examine carefully whether the ingredient is reaching the digestive tract for achieving suitable conclusions in nutritional studies. These results indicate the applicability of these microencapsulated particles in nutritional studies of small aquatic animals.

Effects of gossypol on sperm viability and plasma sex steroid hormones in male sea lamprey, Petromyzon marinus.

Male sea lampreys Petromyzon marinus were injected with different doses of gossypol acetic acid in an attempt to sterilize them for use in a program for controlling the sea lampreys through the release of sterile males. Two lots of sea lamprey were used in these experiments. The first lot was divided into three groups and fish were injected intraperitoneally (i.p.) with 0.2 ml 50% ethanol as a control group or with gossypol suspended in ethanol at 100 and 200 mg/kg. The second lot was also divided into three groups and fish were either injected i.p. with vehicle as controls or gossypol at 25 and 50 mg/kg. Sperm weight, concentrations and motility were recorded after 31, 36 and 40 days or 24, 28 and 33 days in lots 1 and 2, respectively. Blood was collected from the caudal vessel prior to injections with gossypol and after 40 or 33 days in lots 1 and 2, respectively. Plasma levels of estradiol-17beta (E2), testosterone (T), progesterone (P) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20betaP) were measured by radioimmunoassay. At the end of the experiment, the testis were removed and fixed in Bouins solution for histological examination. High mortality was observed at the day of injection in the group treated with 200 mg/kg (84.6%), 100 mg/kg (41.7%), and 50 mg/kg (25%). Sperm concentrations were higher in control fish in comparison to most of the treated groups during the first sperm sampling (day 31 or 24), but then differences disappeared. At each sampling, sperm motility was higher in control groups than in treated groups and significant differences were observed (e.g. between control and 50 mg gossypol/kg). Fertility, evaluated at optimized sperm/egg ratio (5 x 10(4) sperm/egg) did not differ among treatments and controls. Changes in mean plasma sex steroid levels in the various treated groups were not significant, but a trend of decreasing plasma E2 was observed with increasing dose of gossypol. The structure of the testis was examined at the end of the experiment using light microscopy and appeared to be unchanged in gossypol-treated lampreys as compared to that of control animals. These findings suggest that gossypol injection into lamprey interferes with some of the reproductive parameters. Although high mortality rate precludes a single i.p. injection of gossypol as a favorable mode of chemosterilization in the lamprey, it would be useful to reassess the means of transfer of gossypol to reproductive tissues. This notion is further supported by the results of an in vitro sperm toxicity assay which revealed that gossypol, at a dose of 100 microM, has an antifertility action in this species.

The effect of fasting, prolonged swimming, and predator presence on energy utilization and stress in juvenile walleye (Stizostedion vitreum).

We evaluated how two diets with different lipid levels (4% vs. 10%) influenced nutrient expenditure in juvenile walleye Stizostedion vitreum subjected to starvation, prolonged swimming, and predator presence. We also determined how exercise and predator presence influenced stress indicators such as blood plasma cortisol and glucose. Groups of six fish were placed in triplicate rectangular wire cages per treatment and submerged randomly in three artificial stream compartments at a water temperature of 14 degrees C. Three treatments were established: (A) no water current and no predator, (B) water current (1.5 body lengths s(-1)) and no predator, (C) water current and predator. Six 1-year-old muskellunge (Esox masquinongy, 28.4+/-2.3 cm) were used as predators. They were allowed to swim freely outside walleye cages and were fed with walleye. To simulate poststocking period of fasting, walleyes were exposed to experimental treatments for 6 weeks and were deprived of food. Regardless of the prior diet, fish weight significantly declined in all treatments but no differences were found among them. Lipids declined in both dietary groups of fish; in turn, body moisture increased. Protein levels were only affected in fish fed with high-lipid diet prior to the experiment and subjected to current and predator presence. Plasma glucose gradually declined in fish fed with high-lipid diet prior to the study; whereas in the fish fed with low-lipid diet before the experiment, it remained low regardless of the treatment severity. Plasma cortisol concentrations never exceeded 30 ng ml(-1) indicating no signs of acute stress during the experiment. We concluded that fish fed with diet containing higher lipid levels prior to stocking would possess better potential of withstanding adverse conditions and better survival once released to the wild.

Linking Egg Thiamine and Fatty Acid Concentrations of Lake Michigan Lake Trout with Early Life Stage Mortality

The natural reproduction of lake trout Salvelinus namaycush in Lake Michigan is thought to be compromised by nutritional deficiency associated with inadequate levels of thiamine (vitamin B1) in their eggs. However, mortality driven by thiamine deficiency (commonly referred to as early mortality syndrome [EMS]) is not the only significant cause of low lake trout survival at early life stages. In this study, we sought to better understand the combined effects of variable levels of thiamine and fatty acids in lake trout eggs on prehatch, posthatch, and swim-up-stage mortality. We sampled the eggs of 29 lake trout females from southwestern Lake Michigan. The concentrations of free thiamine and its vitamers (e.g., thiamine monophosphate [TMP] and thiamine pyrophosphate [TPP]) as well as fatty acid profiles were determined in sampled eggs. Fertilized eggs and embryos were monitored through the advanced swim-up stage (1,000 degree-days). Three distinct periods of mortality were identified: prehatch (0-400 degree-days), immediately posthatch (401-600 degree-days), and swim-up (601-1,000 degree-days). Stepwise multiple regression analysis revealed (1) that cis-7-hexadecenoic acid in both neutral lipids (NL) and phospholipids (PL) correlated with prehatch mortality, (2) that docosapentaenoic acid in PL and docosahexaenoic acid in NL correlated with posthatch mortality, and (3) that total lipids, TPP, and palmitoleic acid in NL, linoleic acid, and palmitic acid in PL correlated with the frequency of EMS. These results indicate the complexity of early life stage mortality in lake trout and suggest that inadequate levels of key fatty acids in eggs, along with variable thiamine content, contribute to the low survival of lake trout progeny in Lake Michigan.

Spawning migrations, sexual maturity and sex steroid levels in female roach Rutilus rutilus from the River Meuse

Abstract: Relationships between sexual maturity, steroid hormone levels and spawning migrations of female roach Rutilus rutilus were investigated in the River Meuse (Belgium). Levels of plasma testosterone (T), estradiol-17 β (E2) and 17,20 β-dihydroxy-4-pregnen-3-one (17,20 βP) were measured in three groups of fish: the roach swimming up the fish pass, those entering the backwaters and those caught in the main channel. Gonadosomatic index (GSI) and relation between stages of maturity and hormone concentrations were also determined. The migration can be divided into a prespawning migration from early February to late March limited to an intense moving of roach to the backwaters and a spawning migration in April and May. In the former period, all fish in exogenous vitellogenesis presented high levels of E2 (1.30 ± 0.64 ng/ml early March) and low levels of other steroid hormones. During the spawning period, roach migrating to the backwaters had higher GSI (18 % in the females caught in mid-May) than those migrating upstream through the fish pass (about 15 % in April). High levels of T (maximum 2.71 ng/ml and 1.06 ng/ml, in backwater and fish pass samples respectively) and 17,20 βP (maximum 19.2 ng/ml and 12.7 ng/ml in backwater and fish pass samples, respectively) were measured during the spawning season. However, T and E2 levels (up to 5.5 ng/ml for both hormones) were significantly higher in fish caught in the main channel of the river at about 2 kms from the migration sites (fish pass and backwater inlet).