饲料中维生素D3的添加水平对黄颡鱼幼鱼生长和Toll样受体TLR18、TLR19和TLR21的影响

郭勋; 程珂; 马春松; 陈昕; 王春芳

doi:10.7541/2020.056

饲料中维生素D₃的添加水平对黄颡鱼幼鱼生长和Toll样受体TLR18、TLR19和TLR21的影响

华中农业大学水产学院, 淡水水产健康养殖湖北省协同创新中心, 池塘健康养殖湖北省工程实验室, 武汉 430070

基金项目: 国家自然科学基金(31672667)资助

详细信息

作者简介:
郭勋(1994—), 男, 硕士; 主要研究方向为鱼类营养与免疫。E-mail: 847175531@qq.com

通信作者:
王春芳(1975—), 主要研究方向为鱼类营养与免疫。E-mail: cfwang@mail.hzau.edu.cn

中图分类号: S965.1
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出版历程
- 收稿日期: 2019-04-10
- 修回日期: 2019-08-16
- 网络出版日期: 2020-05-18
- 发布日期: 2020-04-30

EFFECTS OF DIETARY VITAMIN D₃ ON THE GROWTH AND EXPRESSION OF TLR18, TLR19 AND TLR21 IN THE JUVENILE YELLOW CATFISH PELTEOBAGRUS FULVIDRACO

Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Hubei Provincial Engineering Laboratory for Pond Aquaculture, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China

Funds: Supported by the National Natural Science Foundation of China (31672667)

Corresponding author:

摘要

摘要: 为了探讨饲料中维生素D₃添加水平对黄颡鱼(Pelteobagrus fulvidraco)生长和Toll样受体的影响, 研究设计了5个不同浓度梯度的维生素D₃饲料(1120、2260、3950、8030和16600 IU/kg), 对体重为(5.0±0.2) g的黄颡鱼进行了为期12周的生长实验, 并在生长实验结束后进行鮰爱德华氏菌(Edwardsiella ictaluri)攻毒72h。于攻毒前(0)和攻毒后(72h)采样, 每个饲料组分别取6尾鱼的脾脏、头肾、肝脏和前肠四个组织, 检测不同浓度维生素D₃处理对攻毒前和攻毒后TLR18、TLR19和TLR21基因表达量的影响。同时另取6条新鲜黄颡鱼的肌肉、头肾、肾脏、皮肤、脑、鳃、脾脏、胃上皮、小肠和肝脏, 检测TLR18、TLR19和TLR21基因在黄颡鱼中的组织分布。结果表明: 不同的维生素D₃添加水平会显著影响黄颡鱼幼鱼的生长性能; TLR18、TLR19和TLR21基因在所检测的组织中均有表达, 但在脾脏中表达量最高; 饲料中不同维生素D₃含量在攻毒前后均会显著影响TLR18、TLR19和TLR21在头肾、脾脏、肝脏和前肠中的表达, 攻毒后基因的表达显著高于攻毒前; TLR18、TLR19和TLR21在不同组织中的表达和饲料中维生素D₃的浓度相关, 研究结果表明饲料中添加合适剂量的维生素D₃, 可以促进相关免疫基因的表达, 从而增强黄颡鱼对病原微生物的抵抗力。
- 黄颡鱼 /
- 维生素D₃ /
- TLR18 /
- TLR19 /
- TLR21
Abstract: This experiment was conducted to study the effects of dietary vitamin D₃ levels on the growth performance and the expression of TLR18, TLR19 and TLR21 in Pelteobagrus fulvidraco. A total of 800 healthy Pelteobagrus fulvidraco with an average body weight of (5.0±0.2) g were randomly assigned to 5 groups with 4 replicates per group and 40 fishes per replicate. Juvenile yellow catfish were fed with the basal diet supplemented with 1120, 2260, 3950, 8030 and 16600 IU/kg vitamin D₃. After a 12-week growth trial, 50 fish from each treatment were injected with Edwardsiella ictaluri, and the fish were challenged for 72h. The spleen, head kidney, liver and anterior intestine from fish in each group were collected at 0 and 72h after the Edwardsiella ictaluri challenge and were used to detect the expression of three toll-like receptors, i.e., TLR18, TLR19 and TLR21. Meanwhile, the muscles, head kidney, kidney, skin, brain, gill, spleen, stomach, anterior intestine and liver were collected from fresh nonexperimental yellow catfish to detect the tissue distribution of TLR18, TLR19 and TLR21. The results showed that different levels of dietary vitamin D₃ can significantly affect growth performance. The TLR18, TLR19 and TLR21 genes were expressed in all tested tissues, but the highest expression was found in the spleen. The Edwardsiella ictaluri challenge significantly increased the expression of TLR18, TLR19 and TLR21. Different levels of dietary vitamin D₃ had a significant influence on TLR18, TLR19 and TLR21 gene mRNA expression in the head kidney, spleen, liver and anterior intestine both before and after the pathogen challenge. These results showed that appropriate supplementation with dietary vitamin D₃ can stimulate the growth and pathogen resistance of juvenile yellow catfish.
- Pelteobagrus fulvidraco /
- Vitamin D₃ /
- TLR18 /
- TLR19 /
- TLR21

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图 1 黄颡鱼TLR18 (A)、TLR19 (B)和TLR21 (C)基因mRNA组织表达分布

Muscle. 肌肉; Head kidney. 头肾; Kidney. 肾脏; Skin. 皮肤; Brain. 脑; Gill. 鳃; Spleen. 脾脏; Stomach. 胃; Anterior intestine. 前肠; Liver. 肝脏. 字母代表显著性差异(Duncan检验, P<0.05). Letters indicate significant difference (Duncan test, P<0.05)

Figure 1. Tissue distribution of TLR18(A), TLR19(B), and TLR21(C) mRNA in Pelteobagrus fulvidraco

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图 2 攻毒前后TLR18 mRNA在黄颡鱼头肾(A)、脾脏(B)、肝脏(C)和前肠(D)中相对表达量的变化

Figure 2. Changes in the relative mRNA expression levels of the TLR18 gene in the head kidney, spleen, liver and anterior intestine after the Edwardsiella ictaluri stimulation

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图 3 攻毒前后TLR19 mRNA在黄颡鱼头肾(A)、脾脏(B)、肝脏(C)和前肠(D)中相对表达量的变化

Figure 3. Changes in the relative mRNA expression levels of the TLR19 gene in the head kidney, spleen, liver and anterior intestine after the Edwardsiella ictaluri stimulation

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图 4 攻毒前后TLR21 mRNA在黄颡鱼头肾(A)、脾脏(B)、肝脏(C)和前肠(D)中相对表达量的变化

Figure 4. Changes in the relative mRNA expression levels of the TLR21 gene in the head kidney, spleen, liver and anterior intestine after the Edwardsiella ictaluri stimulation

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表 1 实验饲料组成和营养成分

Table 1 Formulation and proximate composition of experimental diets

项目Item	饲料Ⅰ Diet I	饲料Ⅱ Diet II	饲料Ⅲ Diet III	饲料Ⅳ Diet IV	饲料Ⅴ Diet V
原料(%饲料干重)Ingredients (% dry matter)
血粉	10	10	10	10	10
脱脂奶粉	5	5	5	5	5
玉米蛋白粉	2	2	2	2	2
面粉	10	10	10	10	10
酪蛋白	30	30	30	30	30
豆粕	10	10	10	10	10
菜粕	10	10	10	10	10
大豆油	10	10	10	10	10
赖氨酸	1	1	1	1	1
蛋氨酸	0.1	0.1	0.1	0.1	0.1
氯化胆碱	0.5	0.5	0.5	0.5	0.5
维生素C	0.5	0.5	0.5	0.5	0.5
三氧化二钇	0.1	0.1	0.1	0.1	0.1
抗氧化剂	0.02	0.02	0.02	0.02	0.02
矿物盐	2	2	2	2	2
无VD₃-维生素预混料	0.5	0.5	0.5	0.5	0.5
维生素D₃	0.2	0.4	0.8	1.6	3.2
麸皮	8.08	7.88	7.48	6.68	5.08
营养成分(%饲料干重)Proximate composition (% dry matter)
水分	5.45	6.00	5.23	5.52	5.55
粗蛋白	47.60	47.25	46.44	46.96	46.12
粗脂肪	4.53	4.57	4.56	4.40	4.48
灰分	3.30	3.45	3.47	3.36	3.37
维生素D	1120	2260	3950	8030	16600
注: (1) 矿物盐预混料(每千克饲料): 氯化钠198 mg; 硫酸镁2995 mg; 硫酸钠4560 mg; 硫酸钾4098.5 mg; 氯化钙2340 mg; 硫酸亚铁499.5 mg; 乳酸钙700.5 mg; 硫酸锌70.6 mg; 硫酸镁32.5 mg; 硫酸铜6.2 mg; 硫酸钴0.2 mg; 碘化钾0.6 mg; 麸皮4498.4 mg; (2) 无VD₃的维生素预混物(每千克饲料): 维生素A 2750 IU, 维生素E 25 IU; 维生素K₃ 5 mg, 维生素B₁ 10 mg; 维生素B₆ 10 mg; 维生素B₁₂ 10 mg; 尼克酸50 mg; 核黄素10 mg; 泛酸钙25 mg; 叶酸5 mg; 肌醇50 mg; 抗坏血酸50 mg; 脱脂米糠1757.43 mg. 由广州市联鲲生物科技有限公司定制; (3) 维生素D₃预混物: 包膜饲料级别的维生素D₃ (500000 IU/kg). 由广州市联鲲生物科技有限公司提供Note: (1) Mineral premix (mg per kg diet): NaCl 198 mg; MgSO₄·7H₂O 2995 mg; Na₂SO₄ 4560 mg; K₂SO₄ 4098.5 mg; CaCl₂·2H₂O 2340 mg; FeSO₄ 499.5 mg; Calcium lactate 700.5 mg; ZnSO₄·7H₂O 70.6 mg; MnSO₄·4H₂O 32.5 mg; CuSO₄·5H₂O 6.2 mg; CoSO₄ 0.2 mg; KI 0.6 mg; Bran 4498.4 mg; (2) Vitamin premix (per kg diet): Vitamin A 2750 IU; Vitamin E 25 IU; Vitamin K₃ 15 mg; Vitamin B₁ 20 mg; Vitamin B₆ 20 mg; Vitamin B₁₂ 20 mg; Niacin 50 mg; Riboflavin 10 mg; D-calcium pantothenate 25 mg; Biotin 5 mg; Inositol 100 mg; Ascorbic acid 500 mg; Bran 1757.43 mg. This premix was specially made by Nutriera; (3) Vitamin D₃ premix: Coated vitamin D₃ (500000 IU/kg), which was provided by Nutriera

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表 2 Real-time PCR所用引物

Table 2 Primers used for real-time PCR

名称Name	序列Sequence (5′－3′)
TLR18 Forward	GGCACAGCTAAGGAGTGTCATGG
TLR18 Reverce	GGCCAGCTCAATCACAGACAGAG
TLR19 Forward	TCCGAGGCTGGAGGAACAAGG
TLR19 Reverce	GCACCAGTCGCTTCGCAGATAC
TLR21 Forward	CCAGGCCGCAACATAGTCGATAAC
TLR21 Reverce	AGCACCACTCACTACGGAGGAAG
β-actin Forward	GGATTCGCTGGAGATGATG
β-actin Reverce	TCGTTGTAGAAGGTGTGATG

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表 3 在饲料中添加维生素D₃对黄颡鱼幼鱼生长的影响

Table 3 Effects of dietary vitamin D₃ levels on the growth performance of juvenile P.fulvidraco

指标Index	饲料维生素D₃含量Dietary vitamin D₃ level (IU/kg)
指标Index	1120	2260	3950	8030	16600
初始体重IBW (g)	5.53±0.08	5.51±0.07	5.55±0.03	5.56±0.06	5.32±0.15
终末体重FBW (g)	26.15±0.63	24.00±0.66	24.93±0.60	25.91±0.99	26.99±1.37
增重率WG (%)	373.2±9.2^ab	335.9±12.6^b	349.2±11.6^ab	365.5±16.0^ab	408.3±27.0^a
热积温生长系数TGC (%)	0.060±0.001	0.056±0.001	0.058±0.001	0.060±0.002	0.063±0.003
饲料效率FE (%)	0.63±0.02^ab	0.58±0.01^b	0.62±0.02^ab	0.65±0.35^ab	0.71±0.42^a
饲料系数FCR	1.60±0.05^ab	1.74±0.28^b	1.63±0.05^ab	1.55±0.08^ab	1.43±0.09^a
存活率SUR (%)	93.33±1.36	95.00±0.96	95.00±0.96	95.84±0.84	95.84±0.84
注: 同一行中数值后的上标字母不同代表差异显著(P<0.05)Note: Means with different letters in the same row are significantly different (P<0.05)

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表 4 在饲料中维生素D₃添加水平对黄颡鱼疾病抗性的影响

Table 4 Effects of vitamin D₃ supplementation on the disease resistance of P. fulvidraco

项目Item	时间Time (d)	饲料维生素D₃含量Dietary vitamin D₃ level (IU/kg)
项目Item	时间Time (d)	1120	2260	3950	8030	16600
攻毒后死亡数(尾)Death number after challenge tail	1	12	10	9	8	9
	2	5	5	5	4	3
	3	2	2	1	2	1
累积死亡总数(尾)Cumulative death number tail		19	17	15	14	13
累积死亡率Cumulative mortality (%)		34.54	30.90	27.27	25.45	23.63
免疫保护率Immune protective rate (%)			10.54	21.05	26.32	31.59

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