J.G. Cloud

Genetic conservation of salmonid fishes

The problem is that, with the continuing degradation of water and habitat quality, selective harvesting, genetic enhancement programs, and the introduction of hatchery fish into wild populations, the genetic makeup of fish is changing radically. In 27 papers from a workshop held in Moscow, Idaho, an

Single pair mating indicates maternal effects on embryo survival in rainbow trout, Oncorhynchus mykiss

Abstract The maternal and paternal influences on early embryo survival in rainbow trout are not established. The purpose of this study was to determine whether variability in the survival of rainbow trout embryos could be attributed to either the female or male parent. Gametes from individual female and male rainbow trout were used in single pair matings to produce families whose survival was followed from fertilization to the time of swim-up (i.e., ∼7 weeks post-fertilization). Survival was assessed at 0.5, 9, 19, 33, and 48 days post-fertilization, corresponding to second cleavage, embryonic keel formation, retinal pigmentation, hatch, and swim-up, respectively. The variability of survival at all times was significantly ( P P >0.05). Therefore, in rainbow trout embryo survival can be equated with the quality of the egg. To predict survival at swim-up (i.e., after 48 days) it was found that embryonic keel formation, measured 9 days after fertilization, was the earliest time at which a highly significant positive correlation ( r =0.889, P

Rainbow trout (Salmo gairdneri) semen: Effect of non-motile sperm on fertility

Abstract In salmonids, low fertility has been correlated with semen containing a low percentage of motile spermatozoa when diluted with an activating medium. This low motility, low fertility correlation does not appear to be due solely to an inadequate number of motile sperm per egg. Non-motile sperm may affect the interaction of motile sperm with the oocyte. To investigate the possible interference of non-motile spermatozoa on fertilization, oocytes of Salmo gairdneri were fertilized with varying proportions of non-motile (heat-inactivated) sperm before, after or simultaneously with control (non-heated) sperm. Fertility was assayed by monitoring the resultant embryonic development to the eyed stage. The motility of heat-inactivated sperm was zero and their mean fertility was negligible. Addition of heat-inactivated sperm in any proportion after control sperm did not affect fertility. Fertility of motile sperm was reduced when the heat-inactivated sperm were added first and their final concentration was equal to or greater than 90%, and when heat-inactivated sperm at a final concentration of 99% were added simultaneously with the motile sperm. Taken together, these data suggest that non-motile sperm can reduce the fertility of normal motile sperm, but this effect does not play a major role in the determination of the level of fertility in fish semen unless the concentration of motile sperm is less than 10%.

Regulation of hepatic estrogen receptor isoform mRNA expression in rainbow trout (Oncorhynchus mykiss)

The complete nuclear estrogen receptor family in rainbow trout consists of two subtypes (ERalpha and ERbeta) each of which consists of two isoforms (alpha1/alpha2 and beta1/beta2). Transcription rate and mRNA stability of ERalpha1 is affected by 17beta-estradiol (E2) but no information on estrogen regulation exists for the other isoforms. The objective of this study was to compare the mRNA expression patterns of the four ER isoforms in the liver of male trout and in immortalized trout hepatocyte lines (RTH-149 and SOB-15) treated with E2 or 17alpha-ethynylestradiol (EE2) using quantitative RT-PCR. To determine the in vivo dose-response, isogenic male trout were injected intra-peritoneally with 0, 1.5, 15 or 150 microg E2 or an equimolar amount of EE2 and the liver sampled 24 h later. Treatment with either E2 or EE2 significantly (p<0.05) increased the level of ERalpha1 mRNA at all doses tested compared to vehicle, while the response of mRNAs for the other three isoforms did not change. The in vitro dose-response was tested by treating both cell lines with 0, 0.1, 1.0 or 10.0 microM E2 for 48 h. In RTH-149 cells, ERalpha1, ERalpha2 and ERbeta2 mRNAs were significantly higher in cells incubated with 10 microM E2 as compared to cells treated with only vehicle (p<0.05). In SOB-15 cells, ERalpha2 and ERbeta1 mRNAs were significantly higher in cells incubated with 1.0 microM E2 as compared to cells incubated with only vehicle (p<0.05). These results support the conclusion that the mRNAs for the four ER isoforms respond differentially to estrogen regulation.

Short-Term Storage of Salmonid Sperm in Air versus Oxygen

Abstract We evaluated the effects of different gaseous environments on the short-term storage of semen samples from hatchery-reared chinook salmon Oncorhynchus tshawytscha, commercial rainbow trout O. mykiss, and steelhead (anadromous rainbow trout). Within each species examined, sperm motility declined steadily over time and was generally about 50% of the initial value after 72 h; however, there was no difference in motility between samples maintained under either ambient air or 100% O2. The motility of samples incubated under either exhaled air or 95% O2 plus 5% CO2, as well as samples incubated under 100% N2, was significantly decreased after 24 h. Sperm samples demonstrating a significant loss of motility following incubation under exhaled air recovered much of the motility after subsequent incubation under ambient air for 24 h. Sperm viability was not altered after 24 h under any incubation condition but was decreased significantly after 72 h under 100% N2. Finally, the spermatocrits of samples maint...

Aneuploid sperm formation in rainbow trout exposed to the environmental estrogen 17α-ethynylestradiol

Environmental contaminants that mimic native estrogens (i.e., environmental estrogens) are known to significantly impact a wide range of vertebrate species and have been implicated as a source for increasing human male reproductive deficiencies and diseases. Despite the widespread occurrence of environmental estrogens and recognized detrimental effects on male vertebrate reproduction, no specific mechanism has been determined indicating how reduced fertility and/or fecundity is achieved. Previous studies show that male rainbow trout, Oncorhynchus mykiss, exposed to the environmental estrogen 17α-ethynylestradiol (EE2) before gamete formation and fertilization produce progeny with significantly reduced embryonic survival. To determine whether this observed decrease results from sperm chromosome alterations during spermatogenesis, male rainbow trout were exposed to 10 ng of EE2/l for 50 days. After exposure, semen was collected and sperm aneuploidy levels analyzed with two chromosome markers by fluorescent in situ hybridization. In vitro fertilizations were also conducted by using control and exposed sperm crossed to eggs from an unexposed female for offspring analysis. Evaluations for nucleolar organizer region number and karyotype were performed on developing embryos to determine whether sperm aneuploidy translated into embryonic aneuploidy. Results conclusively show increased aneuploid sperm formation due to EE2 exposure. Additionally, embryonic cells from propagated progeny of individuals possessing elevated sperm aneuploidy display high levels of embryonic aneuploidy. This study concludes that EE2 exposure in sexually developing male rainbow trout increases levels of aneuploid sperm, providing a mechanism for decreased embryonic survival and ultimately diminished reproductive success in EE2 exposed males.

Gonadal sex reversal in triploid rainbow trout (Oncorhynchus mykiss)

Sex determination in salmonids is primarily governed by sex chromosomes; however, phenotypic expression and successful development of the gonads may be influenced by additional factors. Exposure to exogenous steroids during the critical period of gonadal differentiation will reverse the expected phenotypic sex of both female and male trout. Triploidy, a viable condition in rainbow trout (RBT), alters the degree of gonadal development in a gender-specific manner. Males produce testes with similar morphology and function as diploid fish, but females produce underdeveloped ovaries devoid of growing oocytes. One possible explanation for this observed gender difference is that the timing of meiotic initiation may influence ovarian/testicular development in triploid RBT. To determine whether the early entrance of germ cells into meiosis results in the lack of ovarian development in triploid females, the objective of this study was to sex-reverse genotypic triploid female RBT (XXX) into phenotypic males and genotypic triploid male RBT (XXY) into phenotypic females. Male fish were exposed to estradiol-17beta (E(2)) and females were exposed to the non-aromatizable androgen 17alpha-methyldihydrotestosterone (MDHT). Over 90% of the male fish treated with exogenous E(2) developed gonadal structures indistinguishable from the gonads of triploid females. Triploid female RBT treated with MDHT developed testes; however, not all fish treated with this androgen were completely sex reversed. The results of this investigation are consistent with the hypothesis that the failure of ovarian development in triploid RBT is due to the early onset of meiosis and does not appear to be due to genotypic sex. J. Exp. Zool. 284:466-472, 1999.

Does Co2 enhance short-term storage success of chinook salmon ( Oncorhynchus tshawytscha ) milt?

Successful short-term storage of salmonid milt depends on numerous factors, including temperature, fluid volume, and gaseous environment, with storage at low temperatures under an atmosphere of 100% O2 being the most common method. Salmonid sperm maintained in a storage environment with elevated carbon dioxide (CO2) levels, such as the approximately 4% CO2 in exhaled air, are not motile when activated. While these modest levels of CO2 inhibit sperm motility, the effect is reversible within hours after exposure to a CO2-free oxygenated environment. Therefore, the effect of CO2 (as a component gas in the storage environment) on chinook salmon (Oncorhynchus tshawytscha) sperm motility and viability was examined. The hypothesis of the current investigation was that CO2-exposure with subsequent CO2 removal would be beneficial during short-term chinook salmon milt storage. Milt samples were collected from mature (adult) and precocious (jack) male chinook salmon and stored under various CO2 and O2 levels at 3 to 4 degrees C for up to 14 days. Milt samples were then removed from the incubation environments and maintained under CO2-free humidified air with continuous mixing for 4 h at 10 degrees C before analysis of motility. The resultant motility of samples incubated under 3.5% or less CO2 was not different than controls during the 14 d incubation period; motility of samples stored under higher CO2 tensions were significantly lower. The motility of samples incubated under 3.5% CO2 reached the maximum recovered motility after 2 h exposure to CO2-free humidified air, while the motility of sperm incubated under 13.4% CO2 levels recovered no motility even after 6 h exposure to CO2-free humidified air. The motility of samples incubated under normoxia was significantly greater than that of samples incubated under hyperoxia (approximately 90% O2) at both 7 and 14 d, regardless of the CO2 level. Sperm viability was relatively unaltered by any of the incubation conditions examined. The results of this investigation suggest that there is no apparent advantage to storage of chinook salmon sperm in the presence of CO2 and that storage under hyperoxia negatively affects sperm function compared to storage under normoxia.

Gonadal morphology of female diploid gynogenetic and triploid rainbow trout.

Chromosome sets of fishes can be manipulated; this practice includes the production of triploid and gynogenetic salmonids. Such chromosomal modifications often result in abnormal ovarian development. In rainbow trout (RBT), triploid females have string-like gonads lacking significant developing oocytes and are suggested to be sterile due to the odd set of chromosomes disrupting oogenesis. Aberrant ovarian development is reported to occur in about 30% of gynogenetic females. It has been suggested that gynogenetic fish are more prone to expressing developmental abnormalities due to either increased homozygosity or to incomplete inactivation of the paternal chromatin. This investigation was done to compare the ovarian morphology of female triploid and induced gynogenetic diploid RBT. The objective was to determine whether the presence of supernumerary chromosomal fragments, potentially generated during the process of sperm genome inactivation, would result in abnormal gonadal development in gynogens comparable to that observed in triploid females. Gonadal morphology was observed and karyotypical analysis was completed on 21 gynogenetic fish. In 90% of the fish examined, the presence of chromosomal fragments was positively correlated with irregular ovarian development. The atypical gonadal morphology observed in the gynogens resembled triploid RBT ovarian morphology. The results of this investigation support the hypothesis that disruption of the normal diploid chromosomal complement alters germ cell development in gynogenetic female RBT due to the unbalanced nature of the genome. J. Exp. Zool. 286:505-512, 2000.

Testis Transplantation in Male Rainbow Trout (Oncorhynchus mykiss)

Abstract The objective of the present study was to establish a procedure for the transplantation of an intact testis from one male rainbow trout (Oncorhynchus mykiss) to another individual and evaluate the reproductive function of the transplanted testis at sexual maturity. Isogenic (cloned) male rainbow trout were produced by crossing a completely homozygous male (YY) with a homozygous female (XX) to eliminate any problem of tissue rejection. Transplantation was performed on four pairs of sexually immature animals (n = 8); each served both as a donor and recipient. The left testis was removed by making a ventral midline incision to expose the body cavity and gonads. The left testis was disconnected at the anterior and posterior points of attachment and transferred to the recipient fish where it was placed in position adjacent to the pyloric cecae. The right testis was left intact. After 4 wk, the fish were injected (i.p.) twice weekly for 8 or 9 wk with salmon pituitary extract (1.5 mg/kg) to induce precocious sexual maturation. A similar number of untreated fish were maintained as controls. Following this treatment, all the fish were killed, and the right (intact) and left (transplanted) testes were removed, weighed, and sampled for sperm. Although the mean weights of the left, transplanted testes were significantly (P < 0.05) smaller than the intact testes (transplants = 1.2 g; intact = 3.9 g), transplanted testes were present in all animals, had increased in mass, and were sexually mature containing sperm. The mean fertility, as measured by the proportion of eggs completing first cleavage, of sperm derived from transplanted testes (92%) was no different from the sperm obtained from intact testes (84%). Similarly, there was no difference in the number of embryos attaining the eyed stage of development, after 18 days of incubation, that were derived from transplanted (84%) or intact testes (85%).