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ARS Home » Northeast Area » Leetown, West Virginia » Cool and Cold Water Aquaculture Research » Research » Research Project #418730

Research Project: UTILIZING GENETICS AND PHYSIOLOGY FOR ENHANCING COOL AND COLD WATER AQUACULTURE PRODUCTION

Location: Cool and Cold Water Aquaculture Research

2015 Annual Report


1a. Objectives (from AD-416):
1: Define phenotypic measures and estimate genetic and phenotypic parameters and correlations for production, product quality, and reproductive quality traits. • 1.a. Estimate genetic and phenotypic parameters and correlations for production and product quality traits. • 1.b. Estimate genetic parameters and correlations for reproduction traits. • 1.c. Evaluate accuracy of live-animal ultrasound measures to predict body composition and fillet quality. 2: Identify physiological basis for variation in, and strategies to improve growth and nutrient utilization in rainbow trout. • 2.a. Identify regulatory mechanisms for nutrient utilization in muscle and liver. • 2.b. Identify genetic variation in expression of regulatory proteins within nutrient signaling pathways. • 2.c. Identify and select on genetic markers for growth (feed) efficiency. 3: Identify physiological basis for variation in, and strategies to improve reproductive performance in rainbow trout. • 3.a. Identify growth factors that affect final maturation and their signaling pathways. • 3.b. Identify effects of the maturation-inducing hormone MIH, candidate growth factors and signaling pathways on translation of maternal proteins during follicle maturation and in response to fertilization. • 3.c. Identify changes in germ cell transcription of TGF-beta superfamily growth factors during oogenesis and oocyte recruitment with the aid of transgenic trout carrying a green fluorescent protein gene driven by the vasa gene promoter (GFP-vasa). 4: Improve procedures for natural triploid (2Nx4N) production and evaluate their performance. • 4.a. Evaluate performance of natural triploid (2Nx4N). • 4.b. Improve procedures for natural triploid (2Nx4N) production.


1b. Approach (from AD-416):
A comprehensive multidisciplinary strategy utilizing quantitative genetic, physiological and molecular biological approaches is being used to produce genetically superior strains of rainbow trout for release to trout producers, and to develop the technologies for rapid and continued innovation and improvement. As part of this research we will continue to evaluate and characterize the broodstock established at the NCCCWA selected for improved growth performance. Offspring from this line of rainbow trout will be evaluated for important aquaculture production traits e.g., growth, feed efficiency, and reproductive development. These data will yield estimates of additive genetic variation among and within families of rainbow trout and provide guidance for designing selection and breeding programs for genetic improvement, including strategies that employ the use of molecular markers. Physiological research will focus on defining critical pathways, and molecular components in those pathways, for economically important traits. Furthermore, animals with extreme phenotypes, identified by quantitative genetic analyses, will be used in physiological studies to define the critical physiological differences. Procedures for tetraploid induction will be improved for the development of natural triploids and this technology will be applied to standard and improved lines to evaluate its potential to provide additional benefits to rainbow trout aquaculture.


3. Progress Report:
Five generations of selection for improved growth to and beyond typical market body weight have been completed. Each generation, approximately 100 full-sib families from the growth-selected line, and additional families from multiple randomly-mated control lines, were evaluated for growth performance to 13 months of age. As a result of selection, body weight at 9 months of age (approximately 450 grams) increased by an average of 48 grams per generation in the selected line. Similarly, body weight at 13 months of age (approximately 1 kilogram) increased by an average of 101 grams per generation. Compared to mean body weights in the initial 2004 base population, these gains represent an improvement of approximately 13% per generation for each trait. Heritability estimates for body weight at ages between 5 and 13 months, growth rate between 9 and 13 months, and conformation traits (i.e., condition factor) were moderate to large (0.21 to 0.51). Genetic correlations among all of the body weight, growth rate, and confirmation traits were generally favorable. There was no evidence that selection for improved growth performance adversely affected female reproduction and egg quality traits. The growth-selected line was also evaluated for an array of body composition and fillet quality traits over two generations. Fillet yield, despite being an economically-important production trait, is difficult to measure and thus has not traditionally been included as a breeding objective. Fillet yield was found to be highly heritable (0.49) in this study, and thus has the potential to be improved through selective breeding. This hypothesis will be tested, and selection response will be estimated, in the subsequent 5-year project plan by selectively breeding for improved fillet yield and using the growth-selected line as the base population. Furthermore, USDA ARS National Cool and Coldwater Aquaculture (NCCCWA) scientists are currently evaluating fillet yield in a commercial breeding nucleus with the aim of identifying approaches to improve fillet yield in that population. This multi-generation selection program has provided important resource materials and phenotypic and pedigree data that are continuing to be used in collaborative research aimed at identifying genetic and physiological factors that affect growth performance, fillet yield and quality traits, and stress response. Researchers at NCCCWA in collaboration with researchers at West Virginia University (WVU) and The Conservation Fund’s Freshwater Institute have characterized the effects of sexual maturation and triploidy on growth, physiology, nutrient partitioning, and fillet quality traits. Most rainbow trout harvested as foodfish are sexually immature females, although there is increasing production of larger fish for fillet markets. Production of larger fish benefits from induced sterility which mitigates negative effects of gonadal development on growth and fillet quality traits. Sterility also allows genetic containment to prevent breeding with wild fish populations. A genetic manipulation common in the industry that induces sterility is triploid induction, which is traditionally accomplished by exposing newly fertilized eggs to a pressure or temperature shock which causes them to have three sets of chromosomes (3N, triploids) instead of the normal two sets (2N, diploids). Triploids do not grow ovaries or produce eggs, and have very low sex steroid hormone levels. We have shown differences in growth performance, nutrient utilization, and fillet quality traits between diploids and triploids at most market sizes, however, we have also shown most of the more important negative effects of ovarian development on growth and fillet quality traits are not observed in current rainbow trout lines used for food production, that have been selected for improved growth, until the fish reach about 22 months of age at which time the fish can reach over 3 kg. Little is known about how triploidy affects growth physiology and nutrient utilization, particularly in lines of fish selected for improved growth. Physiological and biochemical pathways have been characterized in diploid and triploid fish and differences in protein turnover in muscle and growth-related mechanisms were identified as partially due to differences in estrogen levels and its negative effects on insulin-like growth factor-I and protein turnover in muscle. In collaboration with scientists from National Institute of Agricultural Research (INRA) (France), protein turnover mechanisms have been extensively characterized, including identifying biological factors that regulate these proteolytic pathways. Our research suggests differences between diploids and triploids, specific to life stages, should be considered when designing diets and feeding strategies for the markets specific goals. Furthermore, we have shown that phytoestrogens, which are estrogenic substances found in plants used in aquafeeds, may have similar or opposite actions on growth as the fish’s own estrogens, depending on the concentration of the phytoestrogens. These findings provide information to facilitate diets, feeding strategies, and husbandry practices tailored to different fish and production goals. This research has led to collaboration with scientists at the ARS Grand Forks Human Nutrition Research Center to evaluate the impacts of rainbow trout consumption of markers of heart health in overweight adults. This project is critical to develop nutritional guidelines that emphasize positive effects of consuming fish rich in omega-3 fatty acids, such as rainbow trout, and support production strategies that maximize omega-3 content in fish. Although triploidy is increasing in importance in rainbow trout aquaculture, the procedure has drawbacks. Current procedures for making triploid (3N) fish using physical shocking of embryos (3NP) are not 100% effective and may negatively impact fish performance. As previously reported, our research has progressed an alternative approach which involves making tetraploid (4N) animals that can then be bred with normal diploid (2N) fish to produce triploid offspring (natural triploids, 3NC). Taking advantage of this increase in success, we were then able to show; 1) growth performance of 3NC fish is superior to that of 3NP fish; 2) growth performance of a family as a diploid is more highly correlated to its performance as 3NC than as 3NP fish supporting greater potential for improving 3NC fish than 3NP fish by genetic selection; 3) genetic improvement for bacterial cold water disease resistance in diploid fish also benefits triploid fish production. We have also shown procedures developed for tetraploid induction in rainbow trout work for brook trout and Atlantic salmon, and most recently, that tetraploid brook trout are fertile. The production of tetraploid lines for natural triploid production is being attempted by a salmon producer we trained in this technology, and a state hatchery producing brook trout and rainbow trout for restocking, with whom we are collaborating. Good egg quality is a foundation for seedstock production, however, factors that lead to poor egg quality are poorly understood. Proper progression through final stages of follicle maturation has long been recognized as a major cause for poor egg quality and failure to ovulate. Sex steroids, gonadotropins and growth factors work in synergy to orchestrate follicle maturation, although, little is known of the regulation or actions of growth factors in these physiological processes. We have shown sex steroids and gonadotropins affect expression of genes associated with the Insulin-like growth factor (IGF) and transforming growth factor-(TGFbeta) super family systems but found little effect of ligands from these systems, at least on processes associated with late stages of egg development. In addition, we found little change in protein expression in response to a progestin regulator of final stages of oocyte maturation. However, switching to RNA-seq analysis, we identified thousands of sequences in the follicle cells and oocyte that respond to a gonadotropin and a progestin that may affect egg quality. Our preliminary data on the use of RNA-seq to identify differences in the transcriptomes of late stage follicles, together with establishment of multiplex (GeXP) arrays that can measure approximately 30 different transcripts in a single egg sample, has led to a planned collaboration with a commercial egg producer to use these approaches to diagnose the basis for differences in egg quality among female broodstock. A major impediment to determining the causes of failed progression through follicle maturation has been the inability to identify fish experiencing this reproductive problem early in the process. We have shown ultrasonography to be a useful tool to identify which females are close to spawning based on a dramatic change in echogenicity due to yolk vesicle fusion, approximately three days before eggs are ovulated. We have also found oocyte development is arrested or delayed in some females after this change in echogenicity and this delay is associated with a decrease in egg quality. Thus, ultrasonography can be used to investigate follicle maturation failure and used by hatchery managers to cull females that will provide poor quality eggs. This research on the use of ultrasonography was recently profiled in a trade journal, Hatchery International. Egg quality can also be affected post ovulation. In collaboration with a commercial egg producer, we have identified effects of different temperature treatments of developing embryos used to synchronize hatching, on eyeing and hatching rates, particularly showing low temperatures should be avoided the first day after fertilization. We have also worked with WVU to show negative impacts of post-ovulatory aging on egg quality are associated with changes in ovum mRNA and microRNA expression.


4. Accomplishments
1. Improved growth performance in rainbow trout would enhance industrial trout production. ARS researchers at Leetown, West Virginia, selected a pedigreed rainbow trout line for improved growth performance over five generations and achieved an average improvement of approximately 13% per generation in body weight at both 9 and 13 months of age. The line was also characterized for genetic control of an array of production, fillet quality, and reproductive traits. Ultrasound was verified as a non-invasive approach to characterize carcass composition and quality traits. Favorable results from efforts with the largest domestic producer of rainbow trout to improve fillet yield in their population. Results have led to ongoing, large-scale field trials to provide further characterization of its performance in production settings in anticipation of its industry release and an ongoing collaboration with the largest domestic producer of rainbow trout to improve fillet yield in their population.

2. Phytoestrogens are plant-derived isoflavones that may affect growth performance of rainbow trout. Phytoestrogen content of aquafeeds is increasing due to higher inclusion levels of soy and other legumes rich in these compounds. It is unknown whether phytoestrogens affect growth-related processes in a manner similar to the catabolic effects of estradiol. ARS researchers in Leetown, West Virginia, conducted a series of in vitro and in vivo studies to determine effects of phytoestrogens on metabolic and physiological mechanisms in rainbow trout. Results indicated that genistein, the phytoestrogen of greatest abundance in soy, increases protein degradation and reduces protein synthesis and cell proliferation in cells from white muscle. Furthermore, when injected into fish, genistein affected expression of genes related to growth and protein and lipid metabolism in a manner similar to estradiol, supporting that these compounds negatively affect growth performance, however results suggested that at low levels of genistein may be beneficial for muscle growth. Collectively, these results suggest that dietary phytoestrogens affect rainbow trout growth performance, although whether the response is positive or negative may be concentration dependent.


Review Publications
Cleveland, B.M., Weber, G.M. 2014. Effects of sex steroids on expression of genes regulating growth-related mechanisms in rainbow trout (Oncorhynchus mykiss). General and Comparative Endocrinology. DOI:10.1016/j.ygcen.2014.11.018.

Manor, M.L., Cleveland, B.M., Kenney, P., Leeds, T.D. 2015. Differences in growth, fillet quality, and fatty acid metabolism-related gene expression between juvenile male and female rainbow trout. Fish Physiology and Biochemistry Journal. 41(2):533-547.

Sieliez, I., Dias, K., Cleveland, B.M. 2014. Contribution of the autophagy-lysosomal and ubiquitin-proteasomal proteolytic systems to total proteolysis in rainbow trout (Oncorhynchus mykiss) myotubes. American Journal of Physiology - Regulatory Integrative & Comparative Physiology. 307(11):R1330-R1337. DOI: 10.1152/ajpregu.00370.2014.

Manor, M.L., Weber, G.M., Cleveland, B.M., Yao, J., Kenney, P. 2015. Expression of genes associated with fatty acid metabolism during maturation in diploid and triploid female rainbow trout. Aquaculture. 435:178-186.

Cleveland, B.M., Manor, M.L. 2015. Effects of phytoestrogens on growth-related and lipogenic genes in rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 170:28-37. DOI: 10.1016/j.cbpc.2015.02.001.

Weber, G.M., Davidson, J.W., Kenney, P.B., Good, C.M., Manor, M.L., Welsh, C., Aussanasuwannakul, A., Summerfelt, S.T. 2015. Changes in sex steroids, growth hormone, and insulin-like growth factor-I during ovarian development in Rainbow Trout cultured within a recirculating system with 24-hour Light. North American Journal of Aquaculture. 77:186-194. DOI: 10.1080/15222055.2014.987933.

Manor, M.L., Cleveland, B.M., Weber, G.M., Kenney, P. 2014. Effects of feeding level and sexual maturation on fatty acid metabolism gene expression in muscle, liver, and visceral adipose tissue of diploid and triploid rainbow trout, Oncorhynchus mykiss. Comparative Biochemistry and Physiology. 179:17-26.

Ma, H., Weber, G.M., Hostuttler, M.A., Wei, H., Wang, L., Yao, J. 2015. MicroRNA expression profiles from eggs of different qualities associated with post-ovulatory ageing in rainbow trout (Oncorhynchus mykiss). Biomed Central (BMC) Genomics. 16(201).1-9. DOI: 10.1186/s12864-015-1400-0.

Weber, G.M., Hostuttler, M.A., Semmens, K.J., Beers, B.A. 2015. Induction and viability of tetraploids in brook trout (Salvelinus fontinalis). Canadian Journal of Fisheries and Aquatic Sciences. DOI: 10.1139/cjfas-2014-0536.

Good, C., Weber, G.M., May, T., Davidson, J., Summerfelt, S. 2015. Reduced photoperiod (18 h light vs 24 h light) during first-year rearing associated with increased early male maturation in Atlantic salmon Salmo salar cultured in a freshwater recirculation aquaculture system. Aquaculture Research. DOI: 10.1111/are.12741.

Liu, S., Vallejo, R.L., Gao, G., Palti, Y., Weber, G.M., Hernandez, A., Rexroad III, C.E. 2015. Identification of single nucleotide polymorphism markers associated with cortisol response to crowding in Rainbow Trout. Marine Biotechnology. DOI: 10.1007\s10126-015-9621-4.