Standardizing and optimizing rearing methods of animal models: Influence of diet on growth and reproduction in xiphophorus maculatus and oryzias latipes
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In research, reproducibility is the ability to duplicate the results of a previous study using the same materials and methods as the original researchers. Fish models of human diseases have become more popular in the last thirty years. However, husbandry practices affect the reproducibility of a study. In most laboratories, fish are reared on a custom blend of live feed and other ingredients or a commercial diet that discloses minimal nutritional information. Further, researchers rarely publish the nutritional content of feeds with findings. Overall, this lack of standardization and reporting may confound research outcomes and lead to unreliable inter-laboratory reproducibility. Accordingly, the objective of our study was to determine how diet affects research outcomes. To achieve this, two biomedical fish models, Xiphophorus maculatus (platy fish) and Oryzias latipes (medaka), were used to evaluate dietary effects on growth and reproductive performance. Platy fish (n =120) and medaka (n =300) were reared from birth to one month of life on Ziegler Aquatox flakes and live Artemia nauplii, then randomly divided into groups fed one of three diets for six months: a control diet (CON), consisting of Ziegler Aquatox flakes, live Artemia nauplii, and beef liver paste (liver paste excluded for Medaka); a commercially available zebrafish diet (GEM); or a laboratory defined reference diet (WAT). Fish were fed 3% body weight/day and grouped at similar stocking densities. At baseline and in monthly increments thereafter, individual fish weight, total length, and width were assessed. Beginning at 3 months of age, broods were raised from platy fish as they were born and eggs were collected from medaka bi-weekly. Data were analyzed in RStudio using a mixed model ANOVA with diet as a fixed factor and tanks nested as random factors within diet. If data were normally distributed (P > 0.01 on Shapiro-Wilks test), a parametric ANOVA test was performed and post hoc analyses and pair-wise analyses were conducted using t-tests. Non-normally distributed data (P ≤ 0.01 on Shapiro-wilks test) were evaluated using the Kruskal Wallis rank sum test. In case of significant results, post hoc testing using the Nemenyi tests for multiple comparisons of rank sums was performed. Diet affected growth measures for both platy fish and medaka. We observed significant differences in percent change of length, weight, and width from baseline through 6-months of age for both species of fish. However, there were not many differences between dietary groups in body condition factor at baseline, 3-months, or 6-months for either species. Significant differences were also observed in reproductive measures for both fish species. In platy fish, only one dietary treatment (CON) produced fry. In medaka, there were dietary group differences in the number of eggs collected and percent survival of hatched eggs. These data indicate that diet affects growth and reproductive outcomes even when feeding protocols are otherwise identical, hindering reproducibility of research if diet is not controlled or reported. Collectively, our data emphasize the need for research laboratories to develop standardized feeding practices, report dietary data, and define reference diets based on fish species.