Chinese Journal of Animal Nutrition >

2024 , Vol. 36 >Issue 5: 3219 - 3230

DOI: https://doi.org/10.12418/CJAN2024.276

Original article

Effects of Dietary Alanyl-Glutamine Supplementation on Growth Performance,Serum Biochemical Indices, Liver Antioxidant Capacity, Intestinal Digestive Enzyme Activities and Morphological Structure of Paramisgurnus dabyranus

  • LI Youjie , 1, 2 ,
  • CHEN Qi 1, 2 ,
  • YUAN Zhiwen 1, 2 ,
  • PAN Jie 1, 2 ,
  • LI Yaping 1, 2 ,
  • ZHANG Yazhou 1, 2 ,
  • ZHOU Qiubai , 1, 2, * ,
  • WANG Zirui , 1, 2, *
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  • 1 College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China
  • 2 Key Laboratory of Featured Hydrobios Nutritional Physiology and Healthy Breeding, Nanchang 330045, China
* ZHOU Qiubai, professor, E-mail: ;
WANG Zirui, professor, E-mail:

Received date: 2023-11-20

  Online published: 2024-05-15

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Abstract

This experiment was conducted to investigate the effects of alanyl-glutamine (Ala-Gln) on growth performance, serum biochemical indices, liver antioxidant capacity, intestinal digestive enzyme activities and morphological structure of Paramisgurnus dabyranus. A total of 1 440 seedlings with an initial body weight of (4.07±0.01) g were randomly divided into 6 groups with 4 replicates per group, and with 60 fish in each replicate. Diets containing 0 (control), 0.25%, 0.50%, 0.75%, 1.00% and 1.25% Ala-Gln were prepared, respectively. The animals were fed for 8 weeks. Results showed that the weight gain rate (WGR), specific growth rate (SGR) and protein efficiency ratio of Paramisgurnus dabyranus which were significantly increased in 0.25% to 1.00% groups compared with control group (P<0.05), and reached the maximum value in 0.75% group. The hepatosomatic ratio and viscerosomatic ratio of Paramisgurnus dabyranus were significantly increased in 0.75% group compared with control group (P<0.05). Serum total antioxidant capacity (T-AOC) and immunoglobulin M (IgM) content in 0.75% group were significantly higher than those in control group (P<0.05); dietary supplementation of 0.25% to 1.00% Ala-Gln significantly increased liver superoxide dismutase (SOD) activity (P<0.05), dietary supplementation of 0.50% to 1.00% Ala-Gln significantly increased catalase (CAT) activity (P<0.05), supplementation with 0.50% Ala-Gln significantly increased glutathione (GSH) content (P<0.05), supplementation with 0.75% and 1.00% Ala-Gln significantly increased glutathione peroxidase (GPX) activity (P<0.05). The activities of SOD and GPX were the highest in 1.00% group, and the activity of CAT was the highest in 0.75% group. The GSH content was the highest in 0.50% group (P<0.05). The MDA content in liver of all groups was significantly decreased after adding Ala-Gln (P<0.05), and 0.75% group was the lowest. In intestinal digestive enzymes, trypsin activity in 0.75% and 1.00% groups was significantly increased compared with control group(P<0.05), and reached the maximum in 0.75% group. Compared with the control group, dietary supplementation of 0.75% to 1.25% Ala-Gln significantly increased intestinal villus height (P<0.05), dietary supplementation of 0.25% to 1.25%Ala-Gln significantly increased villus width (P<0.05), and both reached the maximum when the supplemental level was 0.75%. In conclusion, the supplements of Ala-Gln in diets can promote the growth of Paramisgurnus dabyranus, improve antioxidant capacity and immune capacity, improve intestinal morphology and promote digestion and absorption, among which 0.75% supplementation has the best effect.

Key words： alanyl-glutamine; Paramisgurnus dabyranus; growth performance; antioxidant capacity; intestinal morphology and structure

Cite this article

LI Youjie , CHEN Qi , YUAN Zhiwen , PAN Jie , LI Yaping , ZHANG Yazhou , ZHOU Qiubai , WANG Zirui . Effects of Dietary Alanyl-Glutamine Supplementation on Growth Performance,Serum Biochemical Indices, Liver Antioxidant Capacity, Intestinal Digestive Enzyme Activities and Morphological Structure of Paramisgurnus dabyranus[J]. Chinese Journal of Animal Nutrition, 2024 , 36(5) : 3219 -3230 . DOI: 10.12418/CJAN2024.276

目前,在高密度养殖的情况下,由于受到环境等因素的影响,动物容易发生应激从而导致机体损伤,降低养殖效益[1]。为了满足大规模养殖的需求,且在促生长类饲料抗生素禁用的大背景下,在饲料中合理添加免疫增强剂显得尤为重要。绿色饲料添加剂尤其是微生物制剂和植物提取物备受人们青睐,且在各种动物身上取得良好效果。马郁兰作为一种植物提取物被发现能刺激鲤鱼的生长、抗氧化和免疫系统,抑制气单胞菌败血症期间的鱼类死亡率,是一种适合鲤鱼的饲料添加剂[2]。Liu等[3]在克氏原螯虾试验中发现适量的甘草酸对小龙虾生长性能的提高以及免疫应答、免疫相关基因表达和抗病能力增强有促进作用。周东来等[4]在饲料中添加桑叶提取物投喂鳜鱼的试验中发现桑叶提取物能够在不影响鳜鱼生长的情况下,提高鱼体抗氧化、免疫功能,改善肝脏、肠道健康。谷氨酰胺(glutamine,Gln)是血液中最丰富的游离氨基酸,是动物机体的条件必需氨基酸,在供能、蛋白质和核苷酸合成、免疫以及抗氧化等生理活动中发挥着重要的作用[5⇓-7]。在机体发生应激、生病、受伤时,机体内的Gln供应不足,此时需添加外源Gln,然而Gln稳定性差,受热、高压条件下易分解成有毒的焦谷氨酸和氨,此外,Gln溶解度低,在饲料中的添加量高,极大地影响了其在动物中的应用[8-9],而以其二肽丙氨酰-谷氨酰胺(Ala-Gln)作为供体能够克服这些缺陷[10]。大鳞副泥鳅(Paramisgurnus dabyranus)隶属于鲤形目、鳅科、副泥鳅属[11],其适应性强、生长速度快,且营养价值高,是我国重要的特种经济鱼类。近年来,人们对于Ala-Gln在水产动物的研究还较少,尤其是在泥鳅上的研究甚少。因此,本研究旨在探讨Ala-Gln对大鳞副泥鳅生长性能、血清生化指标、肝脏抗氧化能力以及肠道消化酶活性和形态结构的影响,研究结果可为Ala-Gln在泥鳅饲料中的科学应用提供重要的理论依据。

1 材料与方法

1.1 试验设计

试验配制6种等氮等脂的饲料,对照组饲喂基础饲料,试验组饲喂在基础饲料中分别添加0.25%、0.50%、0.75%、1.00%、1.25%的Ala-Gln(纯度99%)的试验饲料(丙氨酸配平)。试验原料粉碎过80目筛,经逐级混合、制粒膨化成1 mm粒径的颗粒饲料,制成的饲料超过1/2浮在水面,并且在每个网箱同一位置都统一放置了食台,饲料均投放在食台处。饲料组成及营养水平见表1。
表1 饲料组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of diets (air-dry basis) %

项目
Items		 丙氨酰-谷氨酰胺添加量Ala-Gln addition levels/%
0(对照Control) 0.25 0.50 0.75 1.00 1.25
原料Ingredients
鱼粉Fish meal 20.00 20.00 20.00 20.00 20.00 20.00
豆粕Soybean meal 22.00 22.00 22.00 22.00 22.00 22.00
菜籽粕Rapeseed meal 7.00 7.00 7.00 7.00 7.00 7.00
大豆浓缩蛋白Soy protein concentrate 16.00 16.00 16.00 16.00 16.00 16.00
小麦粉Wheat flour 24.35 24.35 24.35 24.35 24.35 24.35
豆油Soybean oil 2.50 2.50 2.50 2.50 2.50 2.50
鱼油Fish oil 2.50 2.50 2.50 2.50 2.50 2.50
大豆卵磷脂Soybean lecithin 1.00 1.00 1.00 1.00 1.00 1.00
磷酸二氢钙Ca (H2PO4)2 2.00 2.00 2.00 2.00 2.00 2.00
胆碱Choline 0.40 0.40 0.40 0.40 0.40 0.40
预混料Premix1) 1.00 1.00 1.00 1.00 1.00 1.00
丙氨酸Alanine 1.25 1.00 0.75 0.50 0.25
丙氨酰-谷氨酰胺Ala-Gln 0.25 0.50 0.75 1.00 1.25
合计Total 100.00 100.00 100.00 100.00 100.00 100.00
营养水平Nutrient levels2)
粗蛋白质CP 39.91 39.98 40.04 40.10 39.99 40.11
粗脂肪EE 7.67 7.58 7.54 7.55 7.63 7.56
粗灰分Ash 7.97 8.01 7.99 7.96 7.97 7.99

1)预混料为每千克饲料提供The premix provided the followings per kg of diets:VA 5 000 IU,VB1 25 mg,VB2 45 mg,VB6 20 mg,VB12 0.1 mg,VK3 10 mg,VE 200 mg,VC 200 mg,VD3 2 500 IU,肌醇 inositol 200 mg,泛酸 pantothenic acid 60 mg,烟酸 nicotinic acid 200 mg,叶酸 folic acid 10 mg,生物素biotin 1.5 mg,NaSeO3·5H2O 0.3 mg,CoCl2·6H2O 0.4 mg,KI 0.8 mg,CuSO4·5H2O 10 mg,MnSO4·4H2O 20 mg,ZnSO4·H2O 50 mg,FeSO4·7H2O 150 mg,MgSO4·7H2O 500 mg,NaCl 1 000 mg。

2)营养水平为测定值。Nutrient levels were measured values.

1.2 试验鱼

试验鱼购于江西省丰城市春鳅养殖专业合作社。养殖试验前将大鳞副泥鳅放置在室内养殖蓝桶中暂养2周并进行饲喂驯化,试验开始前禁饲24 h,随后挑选出1 440尾体质健壮、规格均一且初始均质量为(4.07±0.01) g的健康大鳞副泥鳅,试验鱼分为6组,每组4个重复,每个重复60尾。

1.3 饲养管理

试验地点为江西农业大学水产养殖基地。养殖试验在室内进行,养殖蓝桶的规格为80 cm×66 cm×64 cm。养殖用水为提前曝气的自来水,水体24 h不间断增氧,并且定期对养殖用水进行更换,每隔3 d统一更换养殖用水,每次换水量大约为2/3。养殖周期为8周,每天08:30和17:30各投喂1次,每天观察试验泥鳅摄食情况和死亡情况并记录,每周对大鳞副泥鳅摄食量进行称重测定,根据泥鳅摄食情况对投喂量及时调整,并且对每次投喂后残饵进行打捞,烘干后称重,粪便则通过换水进行处理。同时记录每天的水温。养殖期间水温25~30 ℃,pH 6.8左右,溶解氧含量大于5.0 mg/L。

1.4 样品采集及指标测定

1.4.1 生长性能

养殖试验结束后,饥饿24 h,打捞泥鳅进行称重、计数,计算增重率(WGR)、特定生长率(SGR)、饲料系数(FCR)、存活率(SR)、脏体比(VSI)、肝体比(HSI)、肥满度(CF)、蛋白质效率(PER)。计算公式如下:
WGR(%)=100×(终末体质量-
初始体质量)/初始体质量;
SGR(%/d)=100×(ln终末体质量-
ln初始体质量)/饲喂时间;
FCR=摄入干饲料的量/(鱼体终末体质量-
鱼体初始体质量);
SR(%)=100×终末鱼尾数/初始鱼尾数;
VSI(%)=100×内脏质量/体质量;
HSI(%)=100×肝脏质量/体质量;
CF(g/cm3)=100×体质量/体长3;
PER=(终末体质量-初始体质量)/
蛋白质摄入量。

1.4.2 饲料营养成分

采用国标法测定粗蛋白质(GB/T 6432—2018)、粗脂肪(GB/T 6433—2006)和粗灰分(GB/T 6438—2007)含量。粗蛋白质含量采用凯氏半自动定氮仪测定,粗脂肪含量采用索氏提取器测定,粗灰分含量在马弗炉中550 ℃灼烧测定。

1.4.3 血清生化指标与免疫指标

从每个重复中随机取鱼10尾,用一次性1 mL注射器从尾静脉取血,置于0.2 mL离心管中,于冰箱中静置后,用离心机1 006.2×g离心15 min,分离血清,并把每个重复血清混合后进行测定。使用试剂盒测定血清谷草转氨酶(AST)、谷丙转氨酶(ALT)、溶菌酶(LZM)、碱性磷酸酶(AKP)活性,总抗氧化能力(T-AOC)及免疫球蛋白M(IgM)含量,其中AST、ALT活性、T-AOC采用微板法测定,所用试剂盒由南京建成生物工程研究所提供;LZM、AKP活性、IgM含量采用酶联免疫吸附试验试剂盒测定,所用试剂盒由上海优选生物科技有限公司提供。

1.4.4 肝脏抗氧化指标和肠道消化酶活性

解剖取肠道、肝脏立即冷冻,于-80 ℃冰箱保存备用。称取待测样品,按1∶9(w/v)加入冰冻的生理盐水,冰水浴中用高速组织匀浆机匀浆,冷冻离心机离心(4 ℃,1 006.2×g,15 min),吸取上清液为组织液用于检测。采用试剂盒测定肝脏ALT、AST、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPX)活性及丙二醛(MDA)、还原型谷胱甘肽(GSH)含量;测定肠道胰蛋白酶、脂肪酶、淀粉酶活性。所用试剂盒由南京建成生物工程研究所提供。

1.4.5 肠道组织形态结构

对试验鱼进行解剖,取出其内脏团,将肠道分离出来并剔除表面脂肪组织,取长度为1 cm左右的前肠,于磷酸盐缓冲液(PBS)中浸泡冲洗,除去血液、粪便等杂质,置于10%甲醛固定,用于制作组织切片。固定好的肠道经石蜡切片脱蜡至水、苏木精染色、伊红染色和脱水封装制成石蜡切片。每组选取4张不连续切片,每张切片选取4个视野,在光学显微镜下观察肠道切片(100×),并测定肠道绒毛高度、绒毛宽度、肌层厚度,绒毛宽度通过Image-Pro Plus软件测量每根绒毛的平均宽度,计算其平均值作为测定数据。

1.5 数据分析

试验数据采用SPSS 25.0软件进行单因素方差分析(one-way ANOVA),并作Duncan氏多重比较。试验数据以平均值及其相应的标准误表示,P<0.05表示差异显著。

2 结果与分析

2.1 Ala-Gln对大鳞副泥鳅生长性能的影响

由表2可知,Ala-Gln能显著影响大鳞副泥鳅终末体质量、WGR、SGR、PER、VSI、HIS,其中,0.25%~1.00%组WGR、SGR、PER显著高于对照组(P<0.05),且均在0.75%组达到最大。在饲料中添加Ala-Gln后,0.50%~1.00%组HSI显著高于对照组(P<0.05),在添加量0.75%时达到最大;同时,0.75%组VSI也显著高于对照组(P<0.05)。大鳞副泥鳅各组间SR、FCR、CF均无显著差异(P>0.05)。由图1可知,基于WGR进行二次回归分析发现,饲料中Ala-Gln最适添加量为0.72%。
表2 Ala-Gln对大鳞副泥鳅生长性能的影响

Table 2 Effects of Ala-Gln on growth performance of Paramisgurnus dabyranus

项目
Items		 丙氨酰-谷氨酰胺添加量Ala-Gln addition levels/%
0(对照Control) 0.25 0.50 0.75 1.00 1.25
初始体质量IBW/g 4.11±0.01 4.06±0.02 4.07±0.04 4.04±0.01 4.05±0.03 4.08±0.02
终末体质量FBW/g 10.68±0.39b 11.66±0.17a 11.41±0.22ab 11.74±0.04a 11.51±0.04a 11.21±0.28ab
增重率WGR/% 160.27±10.49b 186.52±5.74a 181.73±3.74a 190.82±1.40a 185.29±3.43a 175.71±8.34ab
特定生长率SGR/(%/d) 1.71±0.07b 1.88±0.04a 1.85±0.02a 1.91±0.01a 1.87±0.02a 1.81±0.05ab
存活率SR/% 88.33±6.74 92.78±4.34 93.33±0.96 96.11±0.56 90.56±1.47 90.00±4.41
饲料系数FCR 2.16±0.14 1.94±0.32 2.04±0.06 1.95±0.01 2.09±0.02 2.17±0.10
蛋白质效率PER 1.00±0.02c 1.18±0.02a 1.13±0.03ab 1.19±0.01a 1.10±0.01b 1.07±0.05bc
脏体比VSI/% 5.30±0.18b 5.57±0.04ab 5.65±0.11ab 5.76±0.12a 5.71±0.14ab 5.70±0.18ab
肝体比HSI/% 0.90±0.02b 0.99±0.03ab 1.07±0.03a 1.08±0.07a 1.04±0.04a 1.01±0.05ab
肥满度CF/(g/cm3) 0.89±0.02 0.88±0.01 0.88±0.01 0.89±0.01 0.91±0.02 0.87±0.01

同行数据肩标不同字母表示差异显著(P<0.05),相同字母或无字母差异不显著(P>0.05)。下表同。

Values in the same line with different letter superscripts mean significant difference (P<0.05), while with the same letter or no letter superscripts mean no significant difference (P>0.05). The same as below.

图1 基于WGR、肝脏GSH含量进行二次回归分析

Fig.1 Quadratic regression analysis based on WGR and GSH content in liver

2.2 Ala-Gln对大鳞副泥鳅血清生化指标和免疫指标的影响

由表3可知,饲料中添加一定量的Ala-Gln对血清T-AOC、IgM含量有显著影响(P<0.05),其中0.75%组血清T-AOC显著高于对照组、0.25%和1.25%组(P<0.05),其余各组间无显著差异(P>0.05);0.50%~1.00%组血清IgM含量较对照组显著升高(P<0.05),在0.75%组达到最大,且0.75%组显著高于除0.50%、1.00%组外其余各组(P<0.05)。各组间血清AST、ALT、LZM、AKP活性均差异不显著(P>0.05)。
表3 Ala-Gln对大鳞副泥鳅血清生化指标和免疫指标的影响

Table 3 Effects of Ala-Gln on serum biochemical and immune indices of Paramisgurnus dabyranus

项目
Items		 丙氨酰-谷氨酰胺添加量Ala-Gln addition levels/%
0(对照Control) 0.25 0.50 0.75 1.00 1.25
谷草转氨酶AST/(U/L) 4.26±0.24 4.97±1.36 5.53±0.48 4.32±0.59 3.58±0.58 3.92±0.77
谷丙转氨酶ALT/(U/L) 1.37±0.04 1.72±0.23 1.49±0.17 1.37±0.08 1.32±0.07 1.49±0.12
总抗氧化能力
T-AOC/(mmol/L)		 0.56±0.01b 0.58±0.02b 0.63±0.02ab 0.69±0.05a 0.63±0.02ab 0.56±0.03b
溶菌酶LZM/(U/L) 10.81±1.02 10.85±0.36 11.75±1.06 11.73±0.50 12.16±0.33 12.49±0.35
碱性磷酸酶
AKP/(金氏单位/dL)		 1.30±0.13 1.54±0.23 1.52±0.11 1.62±0.04 1.40±0.12 1.31±0.23
免疫球蛋白M
IgM/(μg/mL)		 169.51±4.10c 181.84±2.15bc 188.48±2.49ab 200.11±7.13a 187.71±3.49ab 176.52±7.95bc

2.3 Ala-Gln对大鳞副泥鳅肝脏抗氧化指标的影响

由表4可知,饲料中添加Ala-Gln对大鳞副泥鳅肝脏抗氧化指标有显著影响(P<0.05),随着Ala-Gln添加量的升高,肝脏SOD活性先增后减,与对照组相比,在0.25%~1.00%组显著上升(P<0.05),GPX活性在0.75%组、1.00%组显著升高(P<0.05),SOD、GPX活性均在1.00%组有最大值;0.50%~1.00%组肝脏CAT活性相较于对照组显著升高(P<0.05),在添加量为0.75%时最高;与对照组相比,肝脏GSH含量只在0.50%组显著上升(P<0.05),其余各组间均无显著差异(P>0.05)。由图1可知,基于肝脏GSH含量进行二次回归分析发现,饲料中Ala-Gln最适添加量为0.69%。各组肝脏MDA含量均显著降低(P<0.05),且在0.75%组达到最低。
表4 Ala-Gln对大鳞副泥鳅肝脏抗氧化指标的影响

Table 4 Effects of Ala-Gln on liver antioxidant indices of Paramisgurnus dabyranus

项目
Items		 丙氨酰-谷氨酰胺添加量Ala-Gln addition levels/%
0(对照Control) 0.25 0.50 0.75 1.00 1.25
谷丙转氨酶
ALT/(U/g prot)		 4.46±1.05 4.85±0.80 5.63±0.46 4.09±0.88 5.16±0.68 4.28±1.41
谷草转氨酶
AST/(U/g prot)		 51.17±4.51 46.84±7.91 60.74±4.88 60.00±4.67 56.49±6.91 52.62±7.81
超氧化物歧化酶
SOD/(U/mg prot)		 39.61±0.54d 45.00±1.30bc 46.79±0.11abc 48.09±0.10ab 50.85±2.56a 42.99±2.34cd
过氧化氢酶
CAT/(U/g prot)		 213.88±14.66c 236.25±13.76bc 279.74±7.55ab 326.20±17.03a 297.09±10.10ab 254.40±34.59bc
丙二醛
MDA/(nmol/mg
prot)		 0.87±0.07c 0.54±0.02ab 0.52±0.03ab 0.44±0.05a 0.68±0.07b 0.65±0.08b
谷胱甘肽
GSH/(μmol/g prot)		 11.23±1.82b 14.21±0.79ab 18.66±2.07a 15.33±0.62ab 14.46±2.62ab 14.07±1.85ab
谷胱甘肽过
氧化物酶
GPX/(U/mg prot)		 92.12±7.18c 101.84±10.81c 108.60±4.13bc 140.76±9.01ab 163.55±18.37a 118.20±11.68bc

2.4 Ala-Gln对大鳞副泥鳅肠道消化酶活性的影响

由表5可知,与对照组相比,饲料中添加0.75%、1.00%的Ala-Gln能显著提高大鳞副泥鳅肠道胰蛋白酶活性(P<0.05),且在添加0.75%时活性达到最大。各组间肠道脂肪酶、淀粉酶活性均不存在显著差异(P>0.05)。
表5 Ala-Gln对大鳞副泥鳅肠道消化酶活性的影响

Table 5 Effects of Ala-Gln on intestinal digestive enzyme activities of Paramisgurnus dabyranus

项目
Items		 丙氨酰-谷氨酰胺添加量Ala-Gln addition levels/%
0(对照Control) 0.25 0.50 0.75 1.00 1.25
胰蛋白酶
Trypsin/(U/mg prot)		 1 114.35
±52.65bc		 924.99
±11.48c		 1 300.38
±95.71b		 1 773.43
±76.38a		 1 691.13
±104.07a		 1 191.42
±14.74b
脂肪酶
Lipase/(U/g prot)		 0.60
±0.08		 0.59
±0.08		 0.57
±0.02		 0.63
±0.07		 0.60
±0.05		 0.73
±0.02
淀粉酶
Amylase/(U/mg prot)		 1.21
±0.10		 1.77
±0.41		 1.36
±0.31		 1.43
±0.07		 1.36
±0.20		 1.69
±0.53

2.5 Ala-Gln对大鳞副泥鳅肠道形态指标的影响

由表6可知,0.75%~1.00%组绒毛高度较对照组显著升高(P<0.05),0.25%~1.25%组绒毛宽度均显著增加(P<0.05),且均在0.75%组数值达到最大。各组间肌层厚度差异不显著(P>0.05)。由图2可知,肠道形态结构绒毛形状类似叶状。
表6 Ala-Gln对大鳞副泥鳅肠道形态指标的影响

Table 6 Effects of Ala-Gln on intestinal morphology indexes of Paramisgurnus dabyranus μm

项目
Items		 丙氨酰-谷氨酰胺添加量Ala-Gln addition levels/%
0(对照Control) 0.25 0.50 0.75 1.00 1.25
绒毛高度VH 768.55±34.27c 811.90±46.33bc 871.33±46.82bc 1 024.40±72.83a 935.24±27.98ab 934.39±29.18ab
绒毛宽度VW 147.89±6.57b 178.40±8.89a 181.25±9.86a 196.95±9.16a 180.12±4.92a 185.25±6.66a
肌层厚度MT 324.60±12.34 326.94±13.44 355.03±4.54 354.94±9.44 338.65±11.93 348.08±16.01
图2 Ala-Gln对大鳞副泥鳅肠道组织形态的影响

AG0、AG1、AG2、AG3、AG4、AG5分别代表对照组及0.25%、0.50%、0.75%、1.00%、1.25%组;VH表示绒毛高度,VW表示绒毛宽度,MT表示肌层厚度。AG0, AG1, AG2, AG3, AG4 and AG5 represented the control group, 0.25%, 0.50%, 0.75%, 1.00% and 1.25% groups, respectively. VH represented villus height, VW represented villus width, and MT represented muscle thickness.

Fig.2 Effects of Ala-Gln on intestinal tissue morphology of Paramisgurnus dabyranus

3 讨论

3.1 Ala-Gln对大鳞副泥鳅生长性能的影响

Ala-Gln是Gln与丙氨酸形成的二肽,在体内能快速分解成Gln,Gln通过为机体提供氮源来调节氨基酸代谢、促进蛋白质合成,从而促进机体生长发育[12]。在本试验中,与对照组相比,饲料中添加Ala-Gln显著影响大鳞副泥鳅WGR、SGR、PER,生长性能得到显著改善,与在建鲤[13]、军曹鱼[14]、斑马鱼[5]上的研究结果一致。在衡量鱼类经济价值时,形体指数常被作为重要指标之一,同时也能反映器官发育情况[15]。肝脏是一个独特的器官,在消化系统和免疫系统中都起着重要的作用。肝脏含有特化的免疫细胞,也是补体蛋白生物合成的主要部位[16]。肠道不仅是水生动物重要的消化、吸收营养物质的器官,同时也是免疫器官,能作为屏障抵御外界病菌入侵进入血液循环,对机体生长发育及维持机体健康至关重要[17]。Gln被证明能引起机体器官变化甚至影响器官发育。有研究指出,Gln对幼建鲤HSI、肠体指数、肠长指数和脾体指数有显著的影响,可显著提高幼建鲤肝胰脏、肠道和脾脏的重量,且这些参数变化与饲料中Gln添加水平呈正相关[18-19];李源等[20]发现,在泥鳅饲料中添加0.8%外源性Gln可显著或极显著提高泥鳅肠体指数和肝胰脏指数。在本研究中,饲料中添加适量的Ala-Gln显著提高了大鳞副泥鳅HSI、VSI,表明Ala-Gln能影响大鳞副泥鳅机体免疫器官的发育。

3.2 Ala-Gln对大鳞副泥鳅血清生化指标和免疫指标的影响

血液生化指标能较大程度地反映鱼类生理和健康状况[21]。T-AOC是衡量动物机体抗氧化能力的重要指标,反映了动物的整体抗氧化能力[22]。据报道,饲料中补充Gln可以调节血清T-AOC,通过影响抗氧化相关酶活性,进而影响养殖鱼的抗氧化能力[6]。本研究发现,当饲料中Ala-Gln添加量为0.75%时,血清T-AOC显著升高,说明Ala-Gln能提高鱼体抗氧化能力。免疫球蛋白是体液免疫的重要组成部分,其中IgM是一种重要的体液免疫指标,用于评估鱼类的体液免疫功能和健康状况,有助于保护鱼类免受病原体的侵害[23-24]。研究表明,Gln能促进IgM和免疫球蛋白T(IgT)合成和分泌,免疫球蛋白通过补体途径进一步激活补体蛋白,对机体起免疫保护作用[24]。在本研究中,添加0.50%~1.00% Ala-Gln显著提高了血清IgM含量,在0.75%时达到最大,同时该组存活率最高,相对于对照组提高了7.78%,这一结果可能与血清IgM含量升高有关。

3.3 Ala-Gln对大鳞副泥鳅肝脏抗氧化指标的影响

机体抗氧化能力的提高有助于动物的健康和生长。机体抗氧化系统主要包括抗氧化酶(SOD、CAT等)和非酶类抗氧化剂(GSH、维生素E等)[25⇓-27]。SOD是内源性抗氧化系统的第1道屏障,它可以清除体内的自由基,将O2-还原为H2O2,分解超氧化物,保护细胞和组织免受氧化损伤[28]。CAT可以清除体内多余的H2O2,将H2O2转化为H2O,也能抑制自由基的形成[29]。GPX、GSH在水生生物防御脂质过氧化过程中发挥重要作用[30]。在抗氧化过程中,GPX能将GSH作为辅酶,与过氧化物反应,生成氧化型谷胱甘肽(GSSG),同时还原过氧化物[27]。MDA是活性氧自由基(ROS)产生的脂质过氧化产物,能间接反映ROS对细胞的损伤程度[31]。本研究发现,饲料中添加0.25%~1.00%的Ala-Gln,肝脏SOD、CAT、GPX活性及GSH含量分别在不同添加量时得到显著提升,MDA含量呈相反趋势。类似的研究结果在其他鱼类中也有报道。温震威等[14]研究发现,Ala-Gln可能通过提高血清SOD、CAT、GPX活性,降低MDA含量,从而缓解低氧胁迫造成的脂质损伤。Xu等[32]发现,Ala-Gln能显著提高低温环境和正常环境下罗非鱼幼鱼血清、肌肉、肝脏SOD、CAT、GPX活性、T-AOC、GSH/GSSG,并且显著降低MDA含量,同时促进热休克蛋白70(HSP70)、过氧化物酶体增殖激活受体α(PPARα)、GPX基因表达,Ala-Gln通过上调抗氧化相关酶活性、基因表达发挥抗氧化作用,从而调节低温下的应激。此外,有研究表明,饲料中添加12 g/kg Gln可提高草鱼幼鱼血清SOD、CAT、GPX活性、GSH含量和T-AOC,这一结果表明了Gln能对草鱼的抗氧化能力产生影响[33]。

3.4 Ala-Gln对大鳞副泥鳅肠道消化酶活性、肠道形态指标的影响

肠道是鱼类重要的消化、吸收营养物质的器官,而鱼类的消化能力往往通过肠道消化酶活性来反映[34]。Qu等[33]发现,饲料中添加适当水平的Gln可显著提高肠道胰蛋白酶和脂肪酶活性,表明Gln能提高草鱼消化酶活性。据报道,随着饲料中谷氨酰胺二肽(GDP)含量的增加,凡纳滨对虾肠道蛋白酶活性呈先升高后降低的趋势,脂肪酶和淀粉酶活性随着饲料中GDP添加量的增加而升高,表明添加适量GDP可促进蛋白质、脂肪和淀粉的消化[35]。与上述结果相似,在本研究中,饲料中添加0.75%~1.00% Ala-Gln显著提高了肠道胰蛋白酶活性,从而促进蛋白质的消化,提高大鳞副泥鳅的消化能力。肠绒毛高度、绒毛宽度是衡量肠道消化吸收功能的重要指标,肠绒毛高度、绒毛宽度与细胞数量相关,绒毛增高、宽度增大能增大肠吸收表面积,促进肠道对营养物质的吸收[36-37]。本研究中,添加0.75% Ala-Gln能增加肠绒毛高度、绒毛宽度,从而提高肠道消化吸收功能,与在黄颡鱼[38]、罗非鱼[39]、石斑鱼[40]上的研究结果一致。在本研究中,Ala-Gln对大鳞副泥鳅肠道消化酶、肠道形态结构产生了明显的正向作用,促进了大鳞副泥鳅消化吸收能力,从而使生长性能得到改善。

4 结论

在本试验条件下,饲料中添加0.75%的Ala-Gln对大鳞副泥鳅生长性能、血清生化指标、肝脏抗氧化能力、肠道消化酶活性以及肠道形态结构具有明显改善作用,以WGR为评价指标时,通过二次回归分析得出在饲料中添加0.72%的Ala-Cln效果最佳。

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