Effects of Thyroid Powder on Tadpole (Lithobates catesbeiana) Metamorphosis and Growth: The Role of Lipid Metabolism and Gut Microbiota

Abstract

A low metamorphosis rate of amphibian larvae, commonly known as tadpoles, limits the farming production of bullfrogs (Lithobates catesbeiana). This study aimed to examine the effects of processed thyroid powder as a feed additive on tadpole metamorphosis, lipid metabolism, and gut microbiota. Five groups of tadpoles were fed with diets containing 0 g/kg (TH0), 1.5 g/kg (TH1.5), 3 g/kg (TH3), 4.5 g/kg (TH4.5), and 6 g/kg (TH6) thyroid powder for 70 days. The results showed that TH increased the average weight of tadpoles during metamorphosis, with the TH6 group having the highest values. The TH4.5 group had the highest metamorphosis rate (p < 0.05). Biochemical tests and Oil Red O staining showed that the lipid (triglyceride) content in the liver decreased after TH supplementation, especially at doses higher than 1.5 g/kg. RT-qPCR revealed that TH at doses higher than 4.5 g/kg significantly up-regulated the transcriptional expression of the pparα, accb, fas, fadd6, acadl, and lcat genes, which are related to lipid metabolism (p < 0.05). These results showed that TH seems to simultaneously promote the synthesis and decomposition of lipid and fatty acids, but ultimately show a decrease in lipids. As for the gut microbiota, it is noteworthy that Verrucomicrobia increased significantly in the TH4.5 and TH6 groups, and the Akkermansia (classified as Verrucomicrobia) was the corresponding genus, which is related to lipid metabolism. Specifically, the metabolic pathways of the gut microbiota were mainly enriched in metabolic-related functions (such as lipid metabolism), and there were significant differences in metabolic and immune pathways between the TH4.5 and TH0 groups (p < 0.05). In summary, TH may enhance lipid metabolism by modulating the gut microbiota (especially Akkermansia), thereby promoting the growth of tadpoles. Consequently, a supplementation of 4.5 g/kg or 6 g/kg of TH is recommended for promoting the metamorphosis and growth of tadpoles.

Keywords: amphibians; bullfrog; gut microbiota; lipid metabolism; metamorphosis.

Figure 1

Number ratio, average weight, and weight gain ratio of the tadpoles at different developmental periods on day 70. NR-IM (number ratio of tadpoles in metamorphosis), AW-IM (average weight of tadpoles in metamorphosis), WGR-IM (weight gain ratio of tadpoles in metamorphosis), NR-AM (number ratio of tadpoles after metamorphosis), AW-AM (average weight of tadpoles after metamorphosis), WGR-AM (weight gain ratio of tadpoles after metamorphosis), AW-Sum (average weight of all tadpoles), and WGR-Sum (weight gain ratio of all tadpoles). Here, the average weight of tadpoles was used to calculate the weight gain ratio. Groups possessing the same letter indicate that they are not significantly different (p > 0.05).

Figure 2

Tadpole stage metamorphosis rate. The red line is the metamorphosis rate on day 59, the green line is the metamorphosis rate on day 60 to day 70, and the purple line is the total metamorphosis rate. Groups having the same letter in the same line indicate no significant difference (p > 0.05).

Figure 3

Liver lipid-related biochemical indicators. TG (triglyceride), T-CHO (total cholesterol), LDL (low-density lipoprotein), TBA (total bile acid), and GLU (glucose). Groups possessing the same letter indicate that they are not significantly different (p > 0.05).

Figure 4

Liver hematoxylin-eosin (HE) staining sections. The 100× indicates that the magnification of the optical microscope is 100, and 400× indicates that the magnification is 400.

Figure 5

Liver oil red O staining sections. The 100× indicates that the magnification of the optical microscope is 100, and 400× indicates that the magnification is 400.

Figure 6

Transcription level of genes related to liver lipid metabolism. hmgcr (3-hydroxy-3-methylglutaryl-CoA reductase), ldlr1 (low-density lipoprotein receptor 1), cds1 (CDP-diacylglycerol synthase), pemt (phosphatidylethanolamine N-methyltransferase), pparα (peroxisome proliferator-activated receptor alpha), fas (fatty acid synthase), accb (acetyl-CoA carboxylase beta), fadd6 (fatty acid desaturase delta-6), acadl (long-chain-acyl-CoA dehydrogenase), cpt1a (carnitine palmitoyltransferase 1a), lcat (lecithin-cholesterol acyltransferase), and gk (glycerol kinase). Groups possessing the same letter indicate that they are not significantly different (p > 0.05).

Figure 7

The α diversity index and β diversity index of gut microbiota. (a) α diversity index (contains goods_coverage, chao1, observed_species, shannon, simpson indices). (b) β diversity index (principal co-ordinates analysis of gut microbiota).

Figure 8

Composition of the gut microbiota (phylum and genus levels). (a) Column chart of the top 10 species in relative abundance (phylum level). (b) Column chart of the top 10 species in relative abundance (genus level).

Figure 9

LDA effect size analysis and the clade map of the gut microbiota. The rings represent species, genus, family, order, class, and phylum from outside to inside. The species with an LDA SCORE > 3 were defined as statistically different biomarkers.

Figure 10

The difference in predicted functional metabolisms in gut bacterial Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. (a) The main enriched functional pathways. (b) Differential metabolic pathways in the 0 g/kg group and the 4.5 g/kg group.