Terence P. Barry

Validation of a microtitre plate ELISA for measuring cortisol in fish and comparison of stress responses of rainbow trout (Oncorhynchus mykiss) and lake trout (Salvelinus namaycush)

A microtitre plate enzyme-linked immunoassay (ELISA) was validated for measuring serum cortisol in rainbow trout (Oncorhynchus mykiss) and lake trout (Salvelinus namaycush). The ELISA uses a highly specific rabbit antibody raised against cortisol-3-carboxymethyloxime (CMO)/bovine serum albumin (BSA), and a conjugate of horseradish peroxidase (HRP) also linked to cortisol in the 3-position by a CMO bridge. The chromatogenic substrate is 3,3′,5,5′-tetramethylbenzidine (TMB). The standard curve was linear (logit/log) from the lower limit of sensitivity of the assay (2 pg/well = approx. 100 pg/ml serum) to approximately 1000 pg/well. The ELISA satisfied the strictest criteria of specificity, reproducibility (interassay coefficient of variation 98%). Changes in serum cortisol, glucose and chloride were measured in rainbow trout and lake trout following an acute 1-min handling stressor. In both species, cortisol levels rose rapidly to peaks of approximately 300 ng/ml by 1 h post-stress, and slowly returned to baseline over the next 48 h. Glucose levels rose faster in rainbow trout than in lake trout but reached similar peak levels of approximately 135 mg/dl at 6 h post-stress in both species. Chloride levels declined between 1 and 3 h in both species, although rainbow trout showed a greater and more prolonged hypochloremia than lake trout. Within 1 week following administration of the stressors, 19 rainbow trout ( ∼ 17% of the fish in the experiment) and 2 lake trout ( ∼ 2%) died. Most of the fish which died had been sampled when their serum chloride levels were low, suggesting that the survival rates of stocked trout may be increased if they are rehandled and stocked before or after this period of hypochloremia.

Endocrine and gonadal changes during the annual reproductive cycle of the freshwater teleost,Stizostedion vitreum.

The annual reproductive cycle of walleye (Stizostedion vitreum) was characterized by documenting changes in gonadal development and serum levels of estradiol-17β (E2), testosterone (T), 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P), and 11-ketotestosterone (11-KT) in wild fish captured from upper midwestern lakes and rivers throughout the year. Fish from the populations used in this study spawn annually in early- to mid-April. Walleye showed group synchronous ovarian development with exogenous vitellogenesis beginning in autumn. Oocyte diameters increased rapidly from ∼ 200 μm in October to ∼ 1,000 μm in November, and reached a maximum of 1,500 μm just prior to spawning. Changes in gonadosomatic indices (GSIs) paralleled changes in oocyte diameters. Serum E2 levels in females increased rapidly from low values in October (< 0.1 ng ml−1) to peak levels of 3.7 ng ml−1 in November, coinciding with the period of the most rapid ovarian growth. Subsequently, E2 levels decreased from December through spawning. Serum T levels exhibited a bimodal pattern, increasing to 1.6 ng ml−1 in November, and peaking again at 3.3 ng ml−1 just prior to spawning. We detected 11-KT in the serum of some females at concentrations up to 5.6 ng ml−1, but no seasonal pattern was apparent. In this study (unlike our results in a related study) 17,20-P was not detected. In males, differentiation of spermatogonia began in late August, and by January the testes were filled (> 95% of germ cells) with spermatozoa. Mature spermatozoa could be expressed from males from January through April. GSIs ranged from 0.2% (post-spawn) to 3.2% (pre-spawn). Serum T levels rose from undetectable levels in post-spawn males to 1.6 ng ml−1 by November, remained elevated throughout the winter, and peaked at 2.8 ng ml−1 I prior to spawning. Levels of 11-KT in males remained low (< 10 ng ml−1, from post-spawning through January, then increased significantly by March and peaked just prior to spawning at 39.7 ng ml−1. Our results indicate that vitellogenesis and spermatogenesis are complete or nearly so, in walleye by early winter, and suggest that it may be possible to induce spawning in this species several months prior to the normal spawning season by subjecting fish to relatively simple environmental and hormonal treatments.

Effects of progesterone on reproduction and embryonic development in the fathead minnow (Pimephales promelas)

High concentrations (375 ng/L) of the steroid hormone progesterone (P4) were measured in snowmelt runoff associated with large livestock-feeding operations in Wisconsin. To gain insight into the potential endocrine-disrupting effects of P4 in fish, experiments were conducted to evaluate the effects of short-term exposure to environmentally relevant concentrations of P4 on reproduction and embryonic development in the fathead minnow (Pimephales promelas). For the reproduction assay, groups of reproductively mature fish were exposed for 21 d to nominal concentrations of 0, 10, 100, and 1,000 ng/L P4 in a flow-through system, and various key reproductive endpoints (e.g., egg number, fertilization success) were quantified throughout the exposure period. The embryonic development assay consisted of incubating fathead minnow eggs in static culture to quantify the effects of P4 on early development and hatching success. Progesterone caused dose-dependent decreases in fecundity and fertility and significantly reduced gonadosomatic index and vitellogenin gene expression in females. There were no effects of P4 on early embryonic development or hatching success. Progesterone may be a significant endocrine-disrupting chemical in fish.

Fast growth in rainbow trout is correlated with a rapid decrease in post-stress cortisol concentrations

Abstract Growth rates of rainbow trout (Oncorhynchus mykiss) were assessed with respect to serum concentrations of cortisol, glucose, and chloride following exposure to a standardized handling stressor. Individually tagged rainbow trout (N=400, 90–250 g) were reared for 10 months and the handling stressor was administered three times at approximately 3-month intervals. Fish were bled at either 1-, 3-, or 6-h post-stress for measurement of serum cortisol, glucose, and chloride, with individuals always being bled at the same time post-stress. For cortisol, 42% of the fish had a consistent response, defined as being ranked in the upper or lower 50th percentile for all three samples collected, whereas for glucose and chloride, 35% and 37% had consistent responses. Among all the fish, growth was best correlated (P

Manipulation of ploidy in yellow perch (Perca flavescens) by heat shock, hydrostatic pressure shock, and spermatozoa inactivation

Abstract Heat shocks, hydrostatic pressure shocks, and ultraviolet radiation were evaluated for their efficacy as methods of manipulating ploidy in yellow perch (Perca flavescens). The most effective methods of inducing triploidy were heat shocks of 28–30°C applied at a time of initiation (TI) of 5 min postfertilization for durations of 10 or 25 min, and hydrostatic pressure shocks of 9000 or 11 000 psi applied at a TI of 5 min for a duration of 12 min. These treatments resulted in triploidy induction rates that ranged from 54–100%, and embryonic survival rates of 16–80%. Cold shocks of 0°C had no effect on the ploidy or survival of embryos. For perch, hydrostatic pressure shock offered several advantages over heat shock as a method of manipulating ploidy. The most effective methods of inducing tetraploidy were hydrostatic pressure shocks of 9000 psi applied at a TI of 192 min for durations of 16 or 24 min. Ultraviolet radiation of perch sperm with doses of 3240–6480 ergs/mm2 resulted in 100% inactivation of paternal chromosomes, and perch eggs fertilized with inactivated sperm had survival rates of > 50%, thereby establishing methods for producing gynogenetic perch. Studies comparing the growth and performance of diploid vs. triploid perch are underway. Tetraploid perch are being reared to sexual maturity to evaluate their potential as brood fish.

Effects of tributyltin (TBT) on Xenopus tropicalis embryos at environmentally relevant concentrations

Tributyltin (TBT) has been widely used as a biocide in antifouling paints and is a known endocrine disrupting chemical. In this paper, we exposed embryos of Xenopus tropicalis to 50-400ngL(-1) tributyltin chloride. TBT significantly decreased the survival rate, reduced the body length and retarded the development of embryos after 24, 36 and 48h of exposure. These effects of TBT were concentration- and time-dependent. Embryos treated with TBT showed multiple malformations. The most obvious alterations were abnormal eyes, enlarged proctodaeum, narrow fins, and skin hypopigmentation. Enlarged proctodaeum and narrow fins were mainly observed after 36 and 48h of exposure. The loss of eye pigmentation or the absence of external eyes occurred after 24 and 36h of exposure, while extended lenses or edemas of eyes were more commonly observed after 48h of exposure. Additional malformations included: small anterior region of heads, pericardial edemas, enlarged trunks, and bent tails. These results suggested that TBT is very toxic to X. tropicalis embryos at environmentally relevant concentrations.

Effects of progesterone on sperm motility in fathead minnow (Pimephales promelas)

The steroid hormone progesterone (P4) is found at relatively high concentrations (∼300 ng/L) in association with concentrated animal feeding operations (CAFOs). In an effort to better understand the potential endocrine disrupting effects of P4 in male fish, computer assisted sperm analysis (CASA) was used to evaluate the effects of this steroid on sperm motility in the fathead minnow (Pimephales promelas). The rationale for focusing on sperm motility is that certain progestins have been shown to bind to surface membrane receptors on fish spermatozoa and increase sperm swimming velocity. It was hypothesized, therefore, that sperm swimming velocity might be a useful indicator of progestin exposure in fish. Adult male fathead minnows (ages 6-12 months) were exposed to environmentally relevant doses of P4, both longer-term (1 week, in vivo exposure) and short-term (minutes, in vitro exposure). Sperm were then video recorded and analyzed by CASA. When fathead minnows were continuously exposed for 1 week to low levels of progesterone in vivo there was a significant dose-dependent reduction in sperm motility. There was no effect of short-term P4 exposure on fathead minnow sperm swimming characteristics. Additional research is required to elucidate the mechanism by which progesterone alters sperm swimming in the fathead minnow. With further validation, the fathead minnow sperm motility assay may be a useful tool to rapidly screen for endocrine disrupting chemicals in the aquatic environment.

Effects of selected hormones and male cohorts on final oocyte maturation, ovulation, and steroid production in walleye (Stizostedion vitreum)

Abstract A series of in vivo and in vitro experiments were conducted to evaluate the effects of selected hormones and male cohorts on oocyte maturation and ovulation in walleye captured from the wild. In one experiment conducted 2 weeks prior to the normal spawning season, single intramuscular injections of human chorionic gonadotropin (hCG, 500 IU kg −1 ) and des-Gly 10 [D-Ala 6 ] LHRH-ethylamide (LHRHa, 100 μg kg −1 ) stimulated final oocyte maturation and ovulation. LHRHa induced oocyte maturation faster than hCG. The presence of spermiating males had a slight stimulatory effect on oocyte maturation in non-injected fish, but did not potentiate the effects of LHRHa. In a second experiment conducted 3 weeks prior to normal spawning, hCG (500 IU kg −1 ), LHRHa (100 μg kg −1 ), and 17,20-P (100 μg kg −1 ) all induced final oocyte maturation. In this experiment, however, hCG was more effective than LHRHa, and there was no male cohort effect. In maturing females, oestradiol-17β levels declined, and testosterone levels increased transiently prior to final oocyte maturation and ovulation. Levels of 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P) were significantly elevated 2 and 3 days prior to ovulation. Cortisol levels were high (50–100 ng ml −1 ) in newly captured fish and remained elevated during the experimental period. No control fish in either experiment underwent final oocyte maturation. These findings suggest that capture and confinement stress may inhibit oocyte maturation in walleye. In vitro, human chorionic gonadotropin (hCG) was a potent inducer of final oocyte maturation whereas LHRHa had no effect. Of various steroids tested in vitro, 17,20-P and 17α,20β,21 -trihydroxy-4-pregnen-3-one were the two most effective inducers of final oocyte maturation and ovulation, and the lowest dose of 17,20-P tested (0.01 ng ml −1 ) was a consistent and potent inducer of final oocyte maturation. These data, together with our in vivo results, support the hypothesis that 17,20-P may be the maturation-inducing steroid in walleye. Cortisol, alone or in combination with hCG, had no effect on oocyte maturation or ovulation in vitro, indicating that any negative effects of cortisol on oocyte maturation in walleye probably occurs at higher levels of the hypothalamic-pituitary-gonadal axis.

Production of a recombinantly derived growth hormone antibody and the characterization of growth hormone levels in yellow perch

Abstract Because of restrictions on commercial fishing and declining wild populations, there has been increased interest in the aquaculture of yellow perch. However, the slow growth of yellow perch has been an impediment to the development of this species for aquaculture. Using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), a growth hormone (GH) cDNA was isolated from yellow perch pituitaries that was 935 bp, coding for a protein of 204 amino acids. Yellow perch GH is most similar to sea bream GH at the amino acid level (88% identical). The cDNA was used to produce a recombinant protein in the pBAD TOPO vector, and the protein was used as an antigen to generate a polyclonal antibody to yellow perch GH. The antibody recognizes a 21-kDa protein in yellow perch pituitaries and also recognizes a purified sea bream GH preparation. However, it did not recognize any bands on Western blots of brook trout pituitaries. The antibody was used to measure the levels of GH immunoreactive protein during the year. The highest levels of GH were observed during May and the lowest levels in January. In yellow perch that were treated with estradiol-17β (E 2 ) in the diet for 46 days, there were significantly elevated levels of GH compared to controls and these results are consistent with past published reports on the stimulatory effects of E 2 on growth in yellow perch. The antibody that was generated in this study can be used to measure GH in experiments aimed at understanding the factors that affect yellow perch growth. This should facilitate the optimization of these factors for aquaculture production.

Effects of Anti-phospholipase A2 on the Growth of Rainbow Trout

Abstract Ingestion of a meal stimulates an inflammatory response in the gut of most vertebrates as the first line of defense against food-borne pathogens. In terrestrial farm animals, blocking gut inflammation with antibodies to the phospholipase A2 (PLA2) enzyme results in improved growth and feeding efficiency. Phospholipase A2 is a rate-limiting enzyme in the biochemical pathway that leads to the production of prostaglandins and leukotrienes, which are key mediators of the gut inflammatory response. The purpose of the present investigation was to test the effects of anti-phospholipase A2 (aPLA2) on the growth of rainbow trout Oncorhynchus mykiss. The antibody was mass-produced by injecting laying hens of the domestic chicken Gallus gallus domesticus with PLA2, collecting the antibody-rich eggs, and preparing an egg powder that was then top-dressed onto standard fish diets. There were three treatment groups: Control, 0.15%, and 0.30% aPLA2 egg powder added to an extruded Silver Cup steelhead (anadromous...