Schizochytrium Supplementation in Compound Feed: Effects on Growth, Metamorphosis, Intermediate Metabolism, and Intestinal Health of Bullfrogs (Lithobates catesbeianus)
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Diets, Bullfrog, and Sampling
2.3. Analytic Procedures
2.3.1. Analysis of Proximate Composition and Intestinal Histomorphometry
2.3.2. Analysis of Intestinal Biochemical Indicators and Microbiota
2.3.3. Analysis of Polymerase Chain Reaction (PCR)
2.4. Statistical Analysis
3. Results
3.1. Growth Performance, Feed Utilization, and Metamorphosis Rate
3.2. Intestinal Biochemical Indicators and Histomorphometry
3.3. Analyses of Intestinal Microbiota
3.4. The Expression of Genes Related to Growth Performance, Energy Metabolism, and Immune Response
3.4.1. Expression of the Genes Involved in GH-IGF-1 Axes
3.4.2. Expression of the AMPK Gene
3.4.3. Expression of the Genes Involved in Glycolipid Metabolism
3.4.4. Expression of the Genes Involved in Intestinal Barrier Function
3.4.5. Expression of the Genes Involved in Inflammation
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
GH | Growth hormone |
AMPK | Amp-activated protein kinase |
IGF-1 | Insulin-like growth factor 1 |
IGF-1R | Insulin-like growth factor 1 receptor |
IGF-2 | Insulin-like growth factor 2 |
PK | Pyruvate kinase |
G6PC2 | Glucose-6-phosphatase catalytic, 2 |
PYGL | Glycogen phosphorylase, liver |
GK | Glycerol kinase |
SREBP1 | Sterol-regulatory element binding protein 1 |
ACC1 | Acetyl-coa carboxylase 1 |
FAS | Fatty acid synthase |
PPARα | Peroxisome proliferator-activated receptor alpha |
CPT1α | Carnitine palmitoyl transferase 1α |
ACAA2 | Acetyl-coa acyltransferase 2 |
LYZ1 | Lysozyme 1 |
TJP1 | Tight junction protein 1 |
TJP2 | Tight junction protein 2 |
CLDN1 | Claudin 1 |
OCLN | Occluding |
RPL17 | Ribosomal protein L17 |
NF-KB | Nuclear factor kappa B |
TLR4 | Toll-like receptor 4 |
NLRP3 | NACHT, LRR, and PYD domains-containing protein 3 |
TNF-α | Tumor necrosis factor alpha |
IL-1β | Interleukin 1β |
IL-8 | Interleukin 8 |
IL-10 | Interleukin 10 |
IL-17 | Interleukin 17 |
WGR | Weight gain rate |
SGR | Specific growth rate |
FCR | Feed conversion ratio |
FI | Feed intake |
CF | Condition factor |
PPR | Post-premetamorphosis rate |
MR | Metamorphosis rate |
Na+, K+-ATPase | Sodium-potassium adenosine triphosphatase |
AKP | Alkaline phosphatase |
γ-GT | γ-glutamyl transferase |
CK | Creatine kinase |
SOD | Superoxide dismutase |
CAT | Catalase |
T-AOC | Total antioxidant capacity |
MDA | Malondialdehyde |
ASV | Amplicon sequence variant |
CT | Connective tissue |
ME | Mucosal epithelial cells |
M | Microvilli |
N | Cell nucleus |
PCoA | Principal co-ordinates analysis |
NMDS | Non-metric multidimensional scaling |
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Groups | ||||||
---|---|---|---|---|---|---|
Items | S0 | S1 | S2 | S3 | S4 | S5 |
Formulation (%) | ||||||
Fish meal | 10.0 | 10.0 | 10.0 | 10.0 | 10.0 | 10.0 |
Chicken meal | 8.0 | 8.0 | 8.0 | 8.0 | 8.0 | 8.0 |
Dephenolized cottonseed protein | 15.0 | 15.0 | 15.0 | 15.0 | 15.0 | 15.0 |
Rapeseed meal | 16.0 | 16.0 | 16.0 | 16.0 | 16.0 | 16.0 |
Soybean meal | 30.0 | 30.0 | 30.0 | 30.0 | 30.0 | 30.0 |
Corn starch | 14.5 | 14.5 | 14.5 | 14.5 | 14.5 | 14.5 |
Schizochytrium (g/kg) | - | 2.0 | 5.0 | 10.0 | 15.0 | 20.0 |
Fish oil | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
Soybean oil | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
Calcium biphosphate | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
Choline chloride | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
Premix * | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
DL-lysine | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
DL-methionine | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
Proximate analysis (% dry matter basis) | ||||||
Moisture | 8.14 | 7.87 | 8.03 | 7.93 | 8.01 | 7.90 |
Crude protein | 29.17 | 28.14 | 28.9 | 29.16 | 28.77 | 29.13 |
Crude lipid | 4.23 | 4.53 | 4.51 | 4.43 | 4.49 | 4.52 |
Ash | 15.53 | 15.01 | 14.95 | 14.90 | 15.17 | 15.01 |
Target Genes | Forward (5′-3) | Reverse (5′-3) | Accession Numbers or References |
---|---|---|---|
GH | TAGACCTCCTCCGCTTCTCC | ACCGTAGTTCCGGACGTTTC | AY251538.1 |
AMPK | GGAGGTGCTCAGCTGCTTGT | AATGAATCGGGCGGGCTTGT | PIO34006.1 |
IGF-1 | CAGTTTGTATGTGGAGACAGAGGCT | CAGTACATCTCTAGCCTCCTCAGA | XM040344286.1 |
IGF-1R | TCCGTCTGTTAGGCGTTGT | AGGGTTGTTCTCGGCATCT | KF819507 |
IGF-2 | TGCCACTGCATTGCCATCTCT | TCTATTTGCCTCACCCACCGC | XM040328472.1 |
PK | TCCTTCATTCGCAAGGCAGC | TGATATTCTTTCCTTTCTCCCCGA | XM040343042.1 |
G6PC2 | CTGCTCCAGTCATTGAACAGTTTC | CCCCATAGCGTGACCTGAAG | LH203031 |
PYGL | CTCGAGTGCTCTATCCCAATGAT | ACCACAAAGTACTCCTGCTTCAGA | LH181312 |
GK | AACGCTTTGAGCCACAGATTAAT | CTGCTTTTTTCCATCGAGCAT | LH193866 |
SREBP1 | CCACCGCCTGCACCAA | CACTCAGCGCCATGTTGATG | LH362962 |
ACC1 | GTTAAAGCTGCCATCCTCACTGT | TGTCCGTCTGGCTAAGATGGT | LH212450 |
FAS | CCTCCACGCCAGAACAAGAT | GATATTTTTATGAGTGGACATTGTATCGA | LH228595 |
PPARα | CCCGACATTCGATGTTTAGAGATT | CCAGCCCATCTTCTATCACCTT | LH193621 |
CPT1α | TGATTGGCAAAATCAAAGAACATC | AATGCTCTGACCCTGGTGAGA | LH022414 |
ACAA2 | TGTTAGCAGGAGGTAAAAGGAAAGTC | ATGCGTTCAACAGTTTTTGGAA | LH119205 |
LYZ1 | ATGGAAAGACAGCAGGAG | TCCAGAAGTATACGATCC | [25] |
TJP1 | TACGAGAAAGTGGTGCTC | CCTCACTCACAAGACTTG | [25] |
TJP2 | GACCGTAGAAGTGCATAC | CCTGATCTCTTCCATAGC | [25] |
CLDN1 | TTGTCCCTCCTGTCTGTCATCT | ACTGGAGAGGTGAAGTAGTGG | XM040320982.1 |
OCLN | TGCAACGATCGTGTACAC | GTGAATAGAACTGGTTGC | [25] |
RPL17 | CAAACAGTGGGACTGGACACA | TTCTTCAGCATGTGCAGGAGGAAT | XM040336449.1 |
NF-KB | AGGAACAAGATCATCATAGAGGAC | TCAGAAAGAGGGGCCAAGTG | XM040329914.1 |
TLR4 | ACACCTTCAAGGCTCGCTAC | ATGATGGGTCGCCATCTTCC | XM040322748.1 |
NLRP3 | ATCACCACAAGGTCACTGGC | TGATTGGCCAGAGCACACAGC | XM040352091.1 |
TNF-α | ACAGACTTGATGGACCTC | AGTTCCAGCTTCGATGTG | [25] |
IL-1β | TCATTCGGGACAGCAGGCAGAA | GCTTCACTGGCACGGTTGTTCT | [14] |
IL-8 | ACCCTTACCCTCTTCCTGCT | TCAGCTTCACACACTGGCA | XM040335899.1 |
IL-10 | GGAAGTTGTCAGCGGGCTAT | GCCCTCTTGTGCATGTGGTA | XM040339663.1 |
IL-17 | TGATAGTCACGCACTGAGTCCG | ATGTTCACCAGCCAGTCAATGC | [14] |
β-actin | CATCCTTCTTGGGTATGGAATCA | TGGCATACAGGTCCTTACGGATA | AB094353 |
Groups | Polynomial Contrasts (p, R2) | |||||||
---|---|---|---|---|---|---|---|---|
Items | S0 | S1 | S2 | S3 | S4 | S5 | Linear | Quadratic |
Initial weight (g) | 0.04 ± 0.00 | 0.04 ± 0.00 | 0.04 ± 0.00 | 0.04 ± 0.00 | 0.04 ± 0.00 | 0.04 ± 0.00 | 0.524, 0.026 | 0.707, 0.045 |
Final weight (g) | 4.69 ± 0.02 e | 5.34 ± 0.03 d | 5.41 ± 0.05 c,d | 5.66 ± 0.06 b | 6.15 ± 0.06 a | 5.58 ± 0.05 b,c | 0.001, 0.522 | 0.000, 0.826 |
WGR † (%) | 13,066.15 ± 148.16 c | 15,028.36 ± 197.53 b | 15,127.35 ± 85.10 b | 15,469.35 ± 64.80 b | 16,173.45 ± 202.30 a | 15,405.86 ± 83.80 b | 0.002, 0.474 | 0.000, 0.775 |
SGR § (%/day) | 5.42 ± 0.01 c | 5.58 ± 0.01 b | 5.58 ± 0.01 b | 5.61 ± 0.00 a,b | 5.66 ± 0.01 a | 5.60 ± 0.01 b | 0.002, 0.467 | 0.000, 0.768 |
FCR ¶ | 2.55 ± 0.04 a | 2.51 ± 0.05 a | 2.47 ± 0.04 a,b | 2.44 ± 0.03 a,b | 2.24 ± 0.05 c | 2.31 ± 0.02 b,c | 0.000, 0.654 | 0.000, 0.670 |
FI (g/bullfrog tadpoles/d) # | 0.13 ± 0.00 c | 0.15 ± 0.00 a,b | 0.15 ± 0.00 a,b | 0.15 ± 0.00 a | 0.15 ± 0.00 a | 0.14 ± 0.00 b | 0.151, 0.124 | 0.000, 0.697 |
CF ∫ | 0.96 ± 0.02 | 0.97 ± 0.03 | 0.92 ± 0.03 | 0.98 ± 0.02 | 0.98 ± 0.04 | 0.91 ± 0.03 | 0.725, 0.008 | 0.195, 0.196 |
PPR ‽ (%) | 79.17 ± 2.20 b | 80.83 ± 3.00 a,b | 82.50 ± 3.82 a,b | 86.67 ± 3.33 a,b | 93.33 ± 1.67 a | 85.00 ± 1.44 a,b | 0.017, 0.307 | 0.011, 0.452 |
MR1 ↑ (%) | 8.33 ± 2.20 b | 10.00 ± 1.44 b | 13.33 ± 0.83 b | 17.50 ± 2.89 a,b | 23.33 ± 1.67 a | 17.50 ± 2.50 a,b | 0.001, 0.533 | 0.000, 0.679 |
Proximate analysis (% dry matter basis) | ||||||||
Moisture | 80.16 ± 2.64 | 76.69 ± 2.90 | 77.02 ± 1.64 | 75.23 ± 2.49 | 76.62 ± 1.53 | 79.15 ± 1.88 | 0.882, 0.001 | 0.224, 0.181 |
Crude protein | 76.53 ± 0.91 c | 81.84 ± 0.21 a,b | 80.68 ± 1.03 b,c | 84.02 ± 0.08 a,b | 84.56 ± 1.46 a,b | 85.19 ± 0.78 a | 0.000, 0.627 | 0.000, 0.712 |
Crude lipid | 14.56 ± 0.14 a,b | 14.4 ± 0.22 b | 14.51 ± 0.21 b | 15.91 ± 0.33 a | 15.03 ± 0.15 a,b | 15.5 ± 0.52 a,b | 0.015, 0.317 | 0.034, 0.362 |
Ash | 12.37 ± 0.51 | 12.08 ± 0.85 | 12.98 ± 0.39 | 11.9 ± 1.54 | 12.3 ± 1.34 | 12.93 ± 1.63 | 0.783, 0.005 | 0.907, 0.013 |
Groups | Polynomial Contrasts (p, R2) | |||||||
---|---|---|---|---|---|---|---|---|
Items | S0 | S1 | S2 | S3 | S4 | S5 | Linear | Quadratic |
Amylase (U/mg protein) | 0.24 ± 0.01 d | 0.27 ± 0.00 c,d | 0.28 ± 0.01 c | 0.29 ± 0.01 c | 0.38 ± 0.01 a | 0.33 ± 0.01 b | 0.000, 0.563 | 0.000, 0.601 |
Lipase (U/g protein) | 8.20 ± 0.30 e | 11.17 ± 0.39 d | 11.65 ± 0.12 d | 13.38 ± 0.14 c | 19.6 ± 0.25 a | 15.8 ± 0.34 b | 0.000, 0.852 | 0.000, 0.806 |
Protease (U/mg protein) | 1.03 ± 0.03 e | 1.36 ± 0.04 d | 1.72 ± 0.03 c | 1.99 ± 0.03 b | 2.46 ± 0.03 a | 2.01 ± 0.02 b | 0.000, 0.686 | 0.000, 0.919 |
Na+, K+-ATPase (U/g protein) | 44.89 ± 1.81 d | 59.55 ± 2.78 cd | 78.19 ± 2.77 c | 121.00 ± 5.03 b | 159.59 ± 12.80 a | 143.56 ± 3.67 a,b | 0.000, 0.767 | 0.000, 0.827 |
AKP (U/g protein) | 55.28 ± 0.18 c | 54.10 ± 0.78 b,c | 60.34 ± 0.75 b,c | 63.28 ± 0.91 a,b | 68.83 ± 0.57 a | 60.34 ± 0.85 b,c | 0.002, 0.446 | 0.000, 0.766 |
γ-GT (U/g protein) | 79.18 ± 4.85 c | 76.79 ± 1.66 c | 99.78 ± 3.53 b | 122.77 ± 4.97 a | 140.00 ± 5.37 a | 73.09 ± 3.04 c | 0.251, 0.081 | 0.000, 0.709 |
CK (U/mg protein) | 1.47 ± 0.08 c | 1.85 ± 0.02 b | 1.95 ± 0.03 a,b | 1.99 ± 0.02 a,b | 2.12 ± 0.03 a | 2.06 ± 0.04 a | 0.000, 0.476 | 0.000, 0.638 |
SOD (U/mg protein) | 555.75 ± 5.33 c | 576.85 ± 17.78 b,c | 600.13 ± 15.64 b,c | 629.39 ± 9.12 a,b | 669.74 ± 11.9 a | 580.32 ± 7.12 b,c | 0.064, 0.202 | 0.000, 0.666 |
CAT (U/mg protein) | 10.35 ± 0.17 d | 11.32 ± 0.35 c | 11.89 ± 0.16 c | 12.92 ± 0.16 b | 14.01 ± 0.07 a | 11.35 ± 0.07 c | 0.000, 0.215 | 0.000, 0.703 |
T-AOC (mmol/g protein) | 0.03 ± 0.00 | 0.03 ± 0.00 | 0.03 ± 0.00 | 0.03 ± 0.00 | 0.03 ± 0.00 | 0.03 ± 0.00 | 0.703, 0.009 | 0.762, 0.036 |
MDA (nmol/mg protein) | 4.59 ± 0.05 a | 4.39 ± 0.08 a,b | 4.28 ± 0.04 b,c | 4.17 ± 0.07 b,c | 3.68 ± 0.05 d | 4.10 ± 0.06 c | 0.000, 0.461 | 0.000, 0.765 |
Groups | Polynomial Contrasts (p, R2) | |||||||
---|---|---|---|---|---|---|---|---|
Items | S0 | S1 | S2 | S3 | S4 | S5 | Linear | Quadratic |
ASV | 248.67 ± 3.84 a,b | 271.33 ± 38.92 a,b | 182.00 ± 25.32 b | 216.67 ± 19.97 a,b | 352.00 ± 31.34 a | 234.00 ± 41.14 a,b | 0.457, 0.035 | 0.761, 0.036 |
Ace | 250.33 ± 3.18 a,b | 274.33 ± 39.96 a,b | 182.33 ± 25.12 b | 216.67 ± 19.97 a,b | 353.67 ± 31.86 a | 235.00 ± 41.04 a,b | 0.474, 0.033 | 0.774, 0.034 |
Chao1 | 250.33 ± 3.18 a,b | 274.33 ± 39.96 a,b | 182.33 ± 25.12 b | 216.67 ± 19.97 a,b | 353.67 ± 31.86 a | 235.00 ± 41.04 a,b | 0.474, 0.033 | 0.774, 0.034 |
Shannon | 2.98 ± 0.12 | 2.34 ± 0.31 | 2.21 ± 0.34 | 2.46 ± 0.18 | 3.31 ± 0.13 | 2.62 ± 0.55 | 0.483, 0.031 | 0.699, 0.047 |
Simpson | 0.14 ± 0.04 | 0.28 ± 0.07 | 0.28 ± 0.07 | 0.18 ± 0.01 | 0.10 ± 0.02 | 0.22 ± 0.13 | 0.600, 0.018 | 0.874, 0.018 |
Shannoneven | 0.54 ± 0.02 | 0.42 ± 0.04 | 0.42 ± 0.05 b | 0.46 ± 0.03 | 0.56 ± 0.02 a | 0.48 ± 0.09 | 0.541, 0.024 | 0.730, 0.041 |
Simpsoneven | 0.03 ± 0.01 | 0.01 ± 0.00 | 0.02 ± 0.00 | 0.03 ± 0.00 | 0.03 ± 0.01 | 0.03 ± 0.01 | 0.387, 0.047 | 0.527, 0.082 |
Phylogenetic diversity | 28.64 ± 0.11 | 31.04 ± 5.40 | 21.61 ± 2.32 | 25.54 ± 1.52 | 35.86 ± 3.03 | 26.28 ± 3.72 | 0.719, 0.008 | 0.918, 0.011 |
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Ding, H.; He, Y.; Song, Y.; Liang, J.; Li, W.; Xu, C.; Yang, H. Schizochytrium Supplementation in Compound Feed: Effects on Growth, Metamorphosis, Intermediate Metabolism, and Intestinal Health of Bullfrogs (Lithobates catesbeianus). Antioxidants 2025, 14, 1208. https://doi.org/10.3390/antiox14101208
Ding H, He Y, Song Y, Liang J, Li W, Xu C, Yang H. Schizochytrium Supplementation in Compound Feed: Effects on Growth, Metamorphosis, Intermediate Metabolism, and Intestinal Health of Bullfrogs (Lithobates catesbeianus). Antioxidants. 2025; 14(10):1208. https://doi.org/10.3390/antiox14101208
Chicago/Turabian StyleDing, Hao, Yinglin He, Yujian Song, Jingjing Liang, Woxing Li, Chao Xu, and Huirong Yang. 2025. "Schizochytrium Supplementation in Compound Feed: Effects on Growth, Metamorphosis, Intermediate Metabolism, and Intestinal Health of Bullfrogs (Lithobates catesbeianus)" Antioxidants 14, no. 10: 1208. https://doi.org/10.3390/antiox14101208
APA StyleDing, H., He, Y., Song, Y., Liang, J., Li, W., Xu, C., & Yang, H. (2025). Schizochytrium Supplementation in Compound Feed: Effects on Growth, Metamorphosis, Intermediate Metabolism, and Intestinal Health of Bullfrogs (Lithobates catesbeianus). Antioxidants, 14(10), 1208. https://doi.org/10.3390/antiox14101208