Does spatial variation in egg thiamine and fatty-acid concentration of Lake Michigan lake trout Salvelinus namaycush lead to differential early mortality syndrome and yolk oedema mortality in offspring?

Individual variation in fatty-acid and thiamine concentrations were determined in lake trout Salvelinus namaycush eggs collected at two spawning grounds in Lake Michigan. A suite of predictor variables, including spawning location, egg fatty-acid and thiamine concentrations, were used to attempt to explain cause-and-effect in early life stage mortality among S. namaycush families. Lipid and fatty-acid composition of S. namaycush eggs differed between spawning locations. Salvelinus namaycush offspring from south-western Lake Michigan were affected by a high occurrence of yolk oedema, whereas a higher frequency of early mortality syndrome (EMS) was observed among offspring from the north-western part of the lake. Random-forest regressions revealed location as the most influential predictor of yolk oedema mortality, whereas thiamine level in eggs was the strongest predictor of EMS-related mortality. Several polyunsaturated fatty acids were also found to be predictors of both mortalities. There is evidence of spatial variability in egg fatty-acid concentration among S. namaycush in Lake Michigan that, together with diminished thiamine concentration, contribute to low survival of S. namaycush progeny.

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.

Uptake and depletion of plasma 17alpha-methyltestosterone during induction of masculinization in muskellunge, Esox masquinongy: effect on plasma steroids and sex reversal.

Oral administration of 17alpha-methyltestosterone (MT) was used to induce masculinization of sexually undifferentiated muskellunge, Esox masquinongy. Three groups of muskellunge (mean weight, 2.5 +/- 0.6 g) were submitted to MT treatment (15 mg of MT/kg) for 60 days. An additional one group was used as a control (hormone-free diet). Food was distributed over a 10-h period by using automatic belt feeders. Blood was sampled in both control and treated fish at different intervals during and after feeding: before (0 h), at 3 h, 6 h, and cessation of feeding (10 h), and after a fast of 22 h (32 h). MT had no significant effect on growth and survival in muskellunge 6 months after the treatment. Concentrations of plasma MT increased during the feeding period and reached their maximum levels 6 or 10 h after starting feeding. This rapid increase of MT indicated a rapid absorption of this steroid. Plasma MT levels then declined and reached a radir by 22 h after cessation of feeding, suggesting that MT is rapidly metabolized and excreted. The profiles of plasma testosterone during the MT treatment did not differ significantly between control and MT-treated groups. During and after the MT treatment, the concentration of plasma testosterone did not differ significantly between control and MT-treated groups. Moreover, no sexual dimorphism of testosterone levels was observed. Six months after treatment, the sex ratio in MT-treated groups (33% males, 62% females, and 5% intersex) was opposite to control (70% and 30%, respectively) and differed significantly. This suggests that at 15 mg of MT/kg over 60 days, a paradoxical feminization took place.

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.

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 Bouin's 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.

Reproductive efficiency and maternal-offspring transfer of gossypol in rainbow trout (Oncorhynchus mykiss) fed diets containing cottonseed meal.

In a preceding study, complete substitution of fish meal protein with cottonseed meal (CM) protein did not affect the survival or growth rate of adult rainbow trout over a 6-mo period. Gossypol, a naturally occurring compound in cottonseeds, has an antifertility effect in terrestrial animals, but information regarding salmonid fish is lacking. Female rainbow trout in this experiment were fed diets with either 0, 25, 50, 75, or 100% (diets 1 to 5) of the fish meal protein replaced with CM protein until first maturation and spawning to study long-term effects on growth and reproduction. Feeding diets containing CM over a total period of 10 mo did not result in differences in growth and mortality compared to the control group (P > 0.05). Increased CM incorporation levels resulted in decreased (P < 0.05) blood hemoglobin (10.6 +/- 1.3, 8.4 +/- 1.8, 7.3 +/- 1.1, 6.9 +/- 0.8, and 5.6 +/- 1.4 g/dL) and hematocrit (49.6 +/- 3.9, 38.5 +/- 9.3, 34.4 +/- 3.7, 34.8 +/- 4.9 and 28.0 +/- 6.8%) levels in diets 1 to 5, respectively. The CM incorporation level had no effect (P > 0.05) on the number of eggs produced per female but led to a reduction (P < 0.05) in egg weight. Eyed stage survival of embryos was low in all dietary groups and did not show differences (P > 0.05). However, an increasing CM incorporation level led to a linear increase (P < 0.05) in the number of females that produced no viable embryos (23.1, 37.5, 42.9, 60.0, and 71.4%). Gossypol in the diet was absorbed by the female trout and transferred to the eggs (0, 2.2 +/- 0.5, 6.7 +/- 1.6, 10.6 +/- 4.2, and 20.0 +/- 2.6 micrograms/g in diets 1 to 5, respectively). A high concentration of gossypol remained in the juveniles at the swim-up stage (endogenous yolk-absorbed) (0.6 +/- 0.3, 2.4 +/- 0.3, 3.4 +/- 0.0, and 4.7 +/- 1.0 micrograms/g, diets 2 to 5, respectively). The findings suggest that replacement of the dietary fish meal protein with CM protein has no effect on fish growth and mortality but may lead to a reduction in reproductive performance in female rainbow trout.

Sex steroids in plasma of lake whitefish Coregonus clupeaformis during spawning in Lake Erie.

Lake whitefish, Coregonus clupeaformis, were collected from the Western basin of Lake Erie during spawning. Free and conjugated (sulfated and glucuronidated) steroids including testosterone (T), 11-ketotestosterone (11-kT), estradiol-17beta (E2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20betaP) were measured in the plasma by radioimmunoassay. In males, the progression of spermiation was characterized by a significant decrease in plasma free steroids, whereas the levels of conjugated steroids remained similar and low, except for sulfated and glucuronidated testosterone. Plasma sex steroids did not correlate with the density or the motility of the spermatozoa. In females, the concentration of plasma T was significantly higher in preovulating than in ovulating females. The levels of E2 and 17,20betaP in ovulating lake whitefish exhibited large variations ranging from below detection limit to 0.9 ng ml(-1) and from 0.2 to 13 ng ml(-1), respectively. Analysis of conjugated steroids revealed high levels of glucuronidated and sulfated 17,20betaP and glucuronidated T in females ovulating in December. However, no significant differences in the proportion of the conjugated steroids were observed.

Seasonal changes in plasma levels of steroid hormones in an asynchronous fish the gudgeon Gobio gobio L. (Teleostei, Cyprinidae).

Annual changes in plasma of estradiol-17 beta, testosterone, and 17,20 beta-dihydroxy-4-pregnen-3-one concentrations were measured, by radioimmunoassay, in female gudgeon Gobio gobio a fish which has an asynchronous-type ovary containing oocytes at various stages of development and spawns several times during the reproductive period. The gonadosomatic index and the relation between stages of maturity and steroid concentrations were also followed during the reproductive cycle. Plasma levels of estradiol-17 beta, testosterone, and 17,20 beta-dihydroxy-4-pregnen-3-one were low from October to April and increased rapidly in May to reach 0.61 +/- 0.31; 2.3 +/- 0.42; and 3.17 +/- 0.68 ng/ml, respectively. Elevated levels were maintained during spawning when vitellogenic oocytes are present alongside oocytes in final maturation. Histological analysis of the ovary indicated that an important number of spawnings has occurred since the proportion of oocytes in final maturation stage was very low (less than 1%). Fish in the regressive phase also presented high steroid levels. The vitellogenic oocytes in preovulatory atresia and the postovulatory follicles may be responsible for these events.

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

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 x 10(9)/ml), motility (78-89%), and standardized (300 x 10(5) 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 microg/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 microg/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.

Effect of diets containing gossypol on blood parameters and spleen structure in tilapia, Oreochromis sp., reared in a recirculating system.

The high cost of fish meal in tilapia diets warrants the potential use of cottonseed meal (CSM) as an alternative source of high quality protein. The effects of varying levels of CSM (0, 25, 50, 75 and 100%) as fish meal protein replacement on growth, blood parameters, spleen characteristics, free and bound gossypol in blood plasma, haemoglobin and haematocrit were determined in tilapia. Gossypol (C(30)H(30)O(8)) is a polyphenolic substance found in cottonseed that has known toxic effects in fish. Tilapias (n = 219, average weight = 11.3 +/- 3.9 g) were randomly distributed into 15, 32-L glass aquaria, representing five dietary treatments and three replicates per treatment. Each aquarium containing 13-16 fish was supplied with thermoregulated, recirculating water (27 +/- 1 degrees C) at 1 L min(-1) flow rate and photoperiod was constant (12 h L/12 h D). Fish fed 25-50% CSM protein replacement showed similar body weights and total lengths as the controls at the completion of the 16-week trial. Fish fed 75 and 100% CSM protein replacement showed a significant decline in body weight and total length. Fish fed 25-100% CSM protein replacement had significantly lower haematocrit and haemoglobin (ANOVA/LSD, P < 0.05) compared with levels in controls. The decline was most prominent in groups fed diets with 50-100% CSM protein replacement. Total and free gossypol concentrations of blood plasma significantly increased with increasing levels of CSM replacement (P < 0.05). No gossypol was found in blood plasma of fish from the control group. The occurrence of immature and abnormal erythrocytes was significantly greater among fish fed 75 and 100% CSM diets compared with fish fed 0-50% CSM diets. Spleen-somatic index (spleen weight/body weight x 100) did not differ between control fish and fish fed 50-100% CSM diets. Spleen abnormalities, such as large depositions of haemosiderin and melanin pigments and proliferation of melano-macrophage centres, lymphocytic depletion of the white pulp areas (hypocellularity), and presence of vacuoles and necrotic areas were observed among fish fed 50-100% CSM protein diets. In general, the pathological effects of gossypol in tilapia (low haemoglobin and haematocrit levels, abundance of immature red blood cells or polychromatocytes, abnormal spleen morphology) were similar to the effects of vitamin E and/or vitamin C deficiencies observed in other studies.

Induction of gynogenesis in muskellunge with irradiated sperm of yellow perch proves diploid muskellunge male homogamety.

Diploid gynogenesis was induced in muskellunge Esox masquinongy using UV-irradiated muskellunge sperm as the first step in producing monosex females. In this approach, we have to rely on negative controls as an indirect reference for sperm genetic material destruction. In the first experiment, equal proportions of gynogenetic females and males were produced. Negative controls, UV-irradiated sperm without heat shock, yielded some normal hatching larvae, described as spontaneous diploids. In the second experiment, muskellunge eggs were activated using sperm from yellow perch. Because hybrids between these species are not viable, we produced unambiguous gynogens. When UV-irradiated yellow perch sperm was used to inseminate muskellunge eggs, haploids resulted (22.5% +/- 2.8% survival to the eyed stage). To produce diploid gynogens, a heat shock of 31 degrees C was applied to inseminated eggs 20 min after activation for a duration of 6 min. This process yielded several hundreds of gynogens for rearing. Several treatments of masculinizing hormone, 17alpha-methyltestosterone (MT), were carried out. Fish were dissected and gonads examined histologically for sex determination. Gynogens produced using yellow-perch sperm confirmed the presence of males in the control group, whereas the MT bath treatment (400 microg/liter) resulted in the production of fish with ovotestis. These results provide evidence for male homogamety in muskellunge and imply that a change of strategy is needed to produce monosex populations.