Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 4: 2503 - 2513

DOI: https://doi.org/10.12418/CJAN2023.234

RESEARCH PAPER

Effects of L-Akanyl-L-Glutamine on Growth Performance, Serum Biochemical and Antioxidant Indexes of Juvenile Cobia (Rachycentron canadum) after Hypoxia Stress

  • WEN Zhenwei , 1 ,
  • HUANG Jiansheng 1 ,
  • CHEN Youming 2 ,
  • LI Yu 1 ,
  • HUANG Jianpeng 1 ,
  • OU Guanghai 1 ,
  • XIE Ruitao 3, 4 ,
  • JIANG Xintao 1 ,
  • CHEN Gang , 1, 5, *
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  • 1 College of Fisheries, Guangdong Ocean University, Zhanjiang 524025, China
  • 2 Guangdong Blue Granary Seed Industry Co., Ltd., Zhanjiang 524025, China
  • 3 Guangdong Evergreen Feed Industry Co., Ltd., Zhanjiang 524000, China
  • 4 Key Laboratory of Aquatic, Livestock and Poultry Feed Science and Technology in South China, Ministry of Agriculture and Rural Affairs, Zhanjiang 524000, China
  • 5 Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, Zhanjiang 524025, China
*professor, E-mail:

Received date: 2022-09-06

  Online published: 2023-04-12

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Abstract

This experiment aimed to investigate the effects of L-akanyl-L-glutamine (Ala-Gln) on growth performance, serum biochemical and antioxidant indexes of juvenile cobia after hypoxia stress. After 14 days of hypoxia stress with an average body weight of (105.16±6.01) g, the juvenile cobias were selected and divided into 3 groups with 3 replicates per group and 30 fish per replicate and they were fed five isonitrogenous and isolipidic diets which contained 0 (control group), 0.50%, 1.00% and 1.50% Ala-Gln, respectively. The serum samples were sampled randomly for index analysis on days 7, 14 and 28 of trial. The results showed as follows: 1) in 1.0% and 1.5% groups, the weight gain rate (WGR)and specific growth rate (SGR) were significantly higher than those in the control group (P<0.05), while the feed conversion ratio (FCR) was significantly lower than that in the control group at 14 and 28 d of feeding (P<0.05). 2) Compared with the control group, the serum triglyceride (TG) and total cholesterol (TC) contents in the 1.0% and 1.5% groups decreased significantly (P<0.05), the serum glucose (GLU), total protein (TP) and albumin (ALB) contents increased significantly in the all Ala-Gln supplementation groups (P<0.05), and the serum aspartate aminotransferase (AST) and alanine transaminase (ALT) activities in the 1.5% group increased significantly at 28 d of feeding (P<0.05). 3) In 1.0% and 1.5% groups, the serum superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were significantly higher than those in the control group (P<0.05), and serum malondialdehyde (MDA) content was significantly lower than that in the control group at 28 d of feeding (P<0.05). The aforementioned findings demonstrate that adding 1.0% Ala-Gln to the diet can enhance the contents of TP and ALB in serum, decrease the buildup of TC and TG in the serum, enhance the growth performance and antioxidant capacity, and lessen the oxidative damage of juvenile cobia caused by hypoxia stress.

Key words： L-alanyl-L-glutamine; cobia; hypoxia stress; growth performance; serum biochemical indexes; antioxidant capacity

Cite this article

WEN Zhenwei , HUANG Jiansheng , CHEN Youming , LI Yu , HUANG Jianpeng , OU Guanghai , XIE Ruitao , JIANG Xintao , CHEN Gang . Effects of L-Akanyl-L-Glutamine on Growth Performance, Serum Biochemical and Antioxidant Indexes of Juvenile Cobia (Rachycentron canadum) after Hypoxia Stress[J]. Chinese Journal of Animal Nutrition, 2023 , 35(4) : 2503 -2513 . DOI: 10.12418/CJAN2023.234

军曹鱼(Rachycentron canadum)又称海鲡、海龙鱼等,隶属鲈形目鲈亚目军曹鱼科军曹鱼属,主要分布于太平洋(东太平洋除外)、大西洋和印度洋等热带水域,我国沿海也有分布,为暖水性海洋鱼类[1]。由于军曹鱼的生长速度极快,抗病力强,鱼肉中不饱和脂肪酸含量较丰富,因此军曹鱼有较高的经济价值。随着大规模种苗生产技术的成熟,军曹鱼已逐渐成为我国南方海水养殖中最重要经济鱼类之一[2]。水体溶氧对鱼类生长、发育、代谢、行为等具有重要影响。鱼类暴露在低氧环境时,机体会产生大量的活性氧(reactive oxygen species,ROS),如不及时消除,则会诱导机体发生细胞凋亡、脂质氧化、组织损伤、DNA和蛋白质降解,造成氧化损伤。近年来,随着气候变化和环境污染加剧,水体富营养化频频发生,水体缺氧问题日益严重,军曹鱼养殖海区常因风和潮汐以及水体温度、季节等因素的影响而出现缺氧现象[3],这严重制约了军曹鱼养殖产业的发展,而有关如何改善低氧对鱼类造成的损伤的研究还未见报道。
谷氨酰胺(glutamine,Gln)是动物血液中含量最多的游离氨基酸,作为前体物质参与嘌呤、蛋白质、嘧啶核苷酸和氨基酸等分子的合成,是鱼类肝脏、肾脏、肠道和骨骼肌组织中的主要能量来源[4],在维持水产动物正常生理活动方面发挥着重要的作用。近二十几年来,Gln作为免疫调节剂,因其独特而复杂的生理功能逐渐成为水产动物营养学、生理学、免疫学等水产学科领域的研究热点。研究表明,Gln可促进鱼肠上皮细胞的增殖和分化[5],在培养皿中添加Gln可维持建鲤(Cyprinus carpio var. Jian)肠道上皮细胞中抗氧化酶活性,使肠道上皮细胞免受过氧化氢(H2O2)诱导所带来的氧化应激损伤[6],而在饲料中补充Gln可提高水产动物的生长性能和抗氧化能力,降低饲料效率,促进肠道发育的同时提高肠内消化酶活性,提高动物机体免疫性能[7⇓⇓-10]。研究显示,在饲料中添加Gln可缓解豆粕和β-伴大豆球蛋白所引起的生长抑制和肠道氧化损伤引发的肠道炎症,增强鱼体免疫系统,促进大菱鲆(Scophthalmus maximus)和杂交石斑鱼(Epinephelus fuscoguttatus♀×Epinephelus lanceolatus ♂)的生长[11-12]。然而,在使用Gln制作成水产饲料的加热过程中和储存过程中容易形成焦谷氨酸等有毒物质,因此目前普遍使用具有高溶解度和高稳定性的L-丙氨酰-L-谷氨酰胺(Ala-Gln)和L-甘氨酰-L-谷氨酰胺(Gly-Gln)替代Gln。研究表明,Ala-Gln与猪肠上皮细胞中的游离Gln具有非常相似的功能[13],可以代替Gln作为水产动物肠道中能量和蛋白质的来源。
根据本实验室前期研究结果,低氧胁迫可导致军曹鱼幼鱼氧化损伤,糖代谢和脂代谢紊乱,蛋白质代谢受阻,抗氧化能力下降,生长受到抑制[14⇓-16]。当前Gln在水产动物中的研究只局限于日常添加,又或者是在胁迫发生的同时添加Gln。由于Gln价格较高,所以将其直接用于日常补充并不符合经济效益;同时,水产养殖中的胁迫应激事件往往是突发的,因此在胁迫发生的同时立即补充Gln也不符合现实。因此,本试验旨在探究在短期内使用Ala-Gln对低氧胁迫后军曹鱼幼鱼生长、血清生化与抗氧化指标的影响,采用先低氧胁迫后进行添加处理的方式,以期为实际生产提供参考,并为Ala-Gln在水产动物生产中的合理运用提供理论依据。

1 材料与方法

1.1 试验饲料

本试验所用Ala-Gln纯度为98%,购于上海某生化科技有限公司。依据Ding等[17]的研究结果,正常情况下军曹鱼饲料中Ala-Gln最适添加量为0.5%,结合本试验胁迫因素与前期预试验结果,按照Ala-Gln添加量分别为0(对照)、0.5%、1.0%、1.5%配制4种等氮等脂的试验饲料。试验饲料参考Li等[18]、吴桐强等[19]的方法选用微晶纤维素作为缓冲物质,其组成及营养水平见表1。饲料原料粉碎后经60目筛,按照配方要求准确称量,微量成分采取逐级扩大法预混合后再与大宗饲料混匀,然后用F-75型双螺杆制粒机(华南理工大学)挤压成粒径分别为3.5和4.0 mm的2种颗粒饲料,风干备用。
表1 试验饲料组成及营养水平(风干基础)

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

项目
Items		 Ala-Gln添加量Ala-Gln addition amount/%
0 0.5 1.0 1.5
原料Ingredients
鱼粉Fish meal 40.00 40.00 40.00 40.00
玉米蛋白粉Corn gluten meal 4.00 4.00 4.00 4.00
小麦谷朊粉Wheat gluten 12.00 12.00 12.00 12.00
豆粕Soybean meal 12.05 12.05 12.05 12.05
面粉Wheat flour 17.00 17.00 17.00 17.00
鱼油Fish oil 4.00 4.00 4.00 4.00
豆油Soybean oil 2.00 2.00 2.00 2.00
大豆卵磷脂Soybean lecithin 2.00 2.00 2.00 2.00
项目
Items		 Ala-Gln添加量Ala-Gln addition amount/%
0 0.5 1.0 1.5
预混料Premix1) 2.00 2.00 2.00 2.00
氯化胆碱Choline chloride 0.50 0.50 0.50 0.50
磷酸二氢钙Ca(H2PO4)2 1.50 1.50 1.50 1.50
乙氧基喹啉Ethoxyquin 0.05 0.05 0.05 0.05
L-丙氨酰-L-谷氨酰胺Ala-Gln 0.50 1.00 1.50
微晶纤维素MCC 2.90 2.40 1.90 1.40
合计Total 100.00 100.00 100.00 100.00
营养水平Nutrient levels2)
粗蛋白质CP 48.68 49.37 50.08 48.28
粗脂肪EE 10.73 10.74 10.62 10.51
粗灰分Ash 1.56 1.57 1.60 1.55
水分Moisture 7.70 7.54 7.63 8.26

1)预混料为每千克饲料提供The premix provided the following per kg of diets:VA 5 000 IU,VD3 1 000 IU,VE 40 mg,VK3 1.0 mg,VB1 5.0 mg,VB2 1.0 mg,VB6 1.0 mg,VB12 2.0 mg,烟酸 nicotinic acid 40 mg,D-泛酸钙 D-calcium pantothenate 20 mg,叶酸 folic acid 3.0 mg,生物素 biotin 0.1 mg,VC 150 mg,Fe 100 mg,Cu 30 mg,Zn 50 mg,Mn 12 mg,I 0.8 mg,Se 0.3 mg,Co 0.2 mg。

2)营养水平为实测值。Nutrient levels were measured values.

1.2 试验用鱼、试验设计及饲养管理

1.2.1 试验用鱼

饲养试验在广东恒兴饲料实业股份有限公司863基地进行,养殖设施为室内24 h持续充气的流水养殖系统。试验用鱼为采自广东海洋大学海洋生物研究基地鱼类种子工程与健康养殖团队自繁自育的军曹鱼幼鱼,暂养于水槽(水体500 L)14 d后开始试验,暂养期间投喂不添加Ala-Gln的对照饲料进行饲料驯化。

1.2.2 试验设计

正式试验开始前,选取规格整齐、活力好、体格健壮的500尾军曹鱼幼鱼进行为期14 d的低氧胁迫,胁迫期间溶解氧浓度为(3.16±0.12) mg/L。以覆盖塑料薄膜、调整排气量、调控水流速度等为主要技术手段调节水体溶解氧浓度,具体调控方法参照Yang等[16]、王维政等[20]的研究。水体溶解氧浓度采用AZ8403型溶氧仪每4 h测定1次(台湾衡欣科技股份有限公司),辅以《水质 溶解氧的测定 碘量法》(GB 7489—1987)每8 h测定1次。胁迫结束后挑选360尾军曹鱼幼鱼[体重(105.16±6.01) g],随机分为4组,每组3个重复,每个重复30尾,用于正式试验。低氧胁迫期间试验鱼统一投喂对照饲料,胁迫后进入正式试验则4组试验鱼分别投喂添加0(对照组)、0.5%、1.0%、1.5% Ala-Gln的4种等氮等脂的试验饲料28 d。

1.2.3 饲养管理

试验过程中,每天08:00和17:00按照体质量的3%各投喂饲料1次,并在投喂1 h后用虹吸管移除残饵和粪便,投喂量依据天气情况和摄食状况做调整。
试验期间水质参数:溶解氧浓度在5~7 mg/L,水温(30±1) ℃,pH为7.8~8.2,盐度为28~30,氨氮浓度小于0.02 mg/L。

1.3 样品采集及分析方法

1.3.1 样品采集

在开始试验后的第7、14和28天进行采样。
采样前对每个水槽内的鱼进行体长、体重测定,用于生长指标计算。血样采集:每个水槽随机取鱼5尾,经MS-222麻醉后静脉抽血于2 mL离心管中,静置于4 ℃冰箱中过夜后,经3 700 r/min离心15 min,吸取上层血清置于1.5 mL离心管,放入-80 ℃超低温冰箱,用于血清生化与抗氧化指标的检测。

1.3.2 饲料营养成分的测定

饲料原料和试验饲料样品营养成分分析参照AOAC(1995)[21]的方法。索氏抽提法测定饲料粗脂肪含量,105 ℃烘干法测定饲料水分含量,凯氏定氮法测定饲料粗蛋白质含量,550 ℃马弗炉灰化法测定饲料粗灰分含量。

1.3.3 血清生化指标的测定

所测血清生化指标包括总蛋白(total protein,TP)、白蛋白(albumin,ALB)、葡萄糖(glucose,GLU)、甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,TC)含量和谷草转氨酶(glutamic oxaloacetic transaminase,AST)、谷丙转氨酶(glutamic pyruvic transaminase,ALT)活性,采用Chemray 240型全自动生化分析仪(深圳雷杜生命科学股份有限公司)进行测定,所用试剂购自南京建成海浩生物科技有限公司。

1.3.4 血清抗氧化指标的测定

所测血清抗氧化指标包括超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)活性和丙二醛(malonic dialdehyde,MDA)含量。SOD活性采用WST-1法测定甲臜染料的生成量后计算得出,CAT采用钼酸铵法测定,GPx通过采用微板法测定酶促反应中还原型谷胱甘肽(glutathione,GSH)的消耗量后计算得出,MDA含量采用硫代巴比妥酸(TBA)法测定,所用试剂盒购自南京建成生物工程研究所。

1.3.5 计算公式

增重率(weight gain rate,WGR,%)=[(W2-W1)/W1]×100;
特定生长率(specific growth rate,SGR,%/d)=[(lnW2-lnW1)/t]×100;
饲料系数(feed conversion ratio,FCR)=F/(W2-W1);
肝体比(hepatopancreas somatic index,HSI,%)=(Wh/W2)×100。
式中:W1为初始体重(g);W2为各采样时间点的体重(g);Wh为各采样时间点的肝脏重(g);t为饲喂时间(d);F为饲料消耗量(g)。

1.4 数据处理与分析

试验数据采用SPSS 26软件进行单因素方差分析(one-way ANOVA)和Duncan氏法多重比较检验,试验数据采用“平均值±标准差”表示,P<0.05为差异显著。

2 结果

2.1 Ala-Gln对低氧胁迫后军曹鱼幼鱼生长性能的影响

如表2所示,在饲喂7 d时,所有添加Ala-Gln组低氧胁迫后军曹鱼幼鱼的WGR、SGR和FCR与对照组相比均无显著差异(P>0.05);在饲喂14、28 d时,1.0%组与1.5%组低氧胁迫后军曹鱼幼鱼的WGR和SGR较对照组显著提高(P<0.05),FCR则较对照组显著降低(P<0.05);饲喂14、28 d时,0.5%组低氧胁迫后军曹鱼幼鱼的WGR和SGR均高于对照组,但无显著差异(P>0.05);低氧胁迫后军曹鱼幼鱼的HSI在任一饲喂时间时各组间均无显著差异(P>0.05)。
表2 Ala-Gln对低氧胁迫后军曹鱼幼鱼生长性能的影响

Table 2 Effects of Ala-Gln on growth performance of juvenile cobia after hypoxia stress (n=3)

项目
Items		 饲喂时间
Feeding time/d		 Ala-Gln添加量Ala-Gln addition amount/%
0 0.5 1.0 1.5
初始体重IBW/g 0 105.16±6.01 105.16±6.01 105.16±6.01 105.16±6.01
7 157.48±6.23 159.76±5.34 162.64±4.12 161.11±5.16
终末体重FBW/g 14 202.52±2.39a 204.40±2.85ab 220.17±4.01c 212.97±8.24bc
28 269.37±8.55a 277.65±5.78ab 307.51±4.07c 284.29±5.52b
7 49.75±5.92 51.92±5.08 54.66±3.92 53.20±4.91
增重率WGR/% 14 92.57±2.27a 94.37±2.71ab 109.36±3.81c 102.52±7.83bc
28 156.15±8.13a 164.03±5.50ab 192.42±3.88c 170.33±5.25bc
7 5.76±0.57 5.97±0.47 6.23±0.36 6.09±0.46
特定生长率SGR/(%/d) 14 4.68±0.09a 4.74±0.10ab 5.28±0.13c 5.04±0.27bc
28 3.36±0.11a 3.47±0.07bc 3.83±0.05c 3.55±0.07b
7 0.64±0.08 0.61±0.06 0.58±0.06 0.60±0.06
饲料系数FCR 14 0.73±0.02c 0.71±0.02bc 0.62±0.02a 0.66±0.05ab
项目
Items		 饲喂时间
Feeding time/d		 Ala-Gln添加量Ala-Gln addition amount/%
0 0.5 1.0 1.5
28 1.43±0.08c 1.36±0.04bc 1.16±0.02a 1.31±0.04b
7 1.82±0.14 1.86±0.25 2.24±0.27 1.91±0.26
肝体比HSI/% 14 1.87±0.06 1.97±0.16 2.00±0.14 1.95±0.22
28 2.06±0.32 2.01±0.19 2.11±0.29 2.02±0.09

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

In the same row, values with the same or no letter superscripts mean no significant difference (P>0.05), while with different letter superscripts mean significant difference (P<0.05). The same as below.

2.2 Ala-Gln对低氧胁迫后军曹鱼幼鱼血清生化指标的影响

如表3所示,在饲喂7 d时,0.5%组的血清TP含量与对照组相比显著上升(P<0.05);在饲喂28 d时,所有添加Ala-Gln组的血清TP均较对照组显著上升(P<0.05)。在饲喂14 d时,0.5%与1.5%组的血清ALB含量与对照组相比显著上升(P<0.05);在饲喂28 d时,所有添加Ala-Gln组的血清ALB含量均较对照组显著上升(P<0.05)。各添加Ala-Gln组的血清TP与ALB含量随饲喂时间的延长呈上升趋势,而对照组则随饲喂时间的延长呈先上升后下降的趋势。
表3 Ala-Gln对低氧胁迫后军曹鱼幼鱼血清生化指标的影响

Table 3 Effects of Ala-Gln on serum biochemical indexes of juvenile cobia after hypoxia stress (n=3)

项目
Items		 饲喂时间
Feeding time/d		 Ala-Gln添加量Ala-Gln addition amount/%
0 0.5 1.0 1.5
7 49.91±4.72ab 55.61±3.17b 45.99±3.40a 50.14±0.54ab
总蛋白TP/(g/L) 14 57.27±2.50 55.25±4.72 63.33±7.17 59.17±5.25
28 45.39±1.45a 66.06±5.46b 61.19±9.53b 60.48±6.38b
7 7.90±1.28 7.81±0.56 9.24±0.94 7.67±0.15
白蛋白ALB/(g/L) 14 8.72±0.39a 10.13±0.97b 9.37±0.28ab 10.40±0.54b
28 8.39±0.32a 9.97±0.41b 9.70±0.40b 9.63±0.46b
7 1.28±0.09a 2.26±0.27b 2.56±0.10b 2.60±0.21b
葡萄糖GLU/(mmol/L) 14 2.70±0.30a 3.07±0.28ab 3.42±0.30b 3.25±0.33ab
28 2.81±0.08a 4.28±0.24c 3.44±0.05b 3.54±0.45b
7 0.81±0.08 0.70±0.17 0.72±0.16 0.68±0.11
甘油三酯TG/(mmol/L) 14 0.99±0.13b 0.85±0.12ab 0.61±0.08a 0.68±0.16a
28 0.88±0.05b 0.64±0.07a 0.61±0.05a 0.65±0.13a
7 3.33±0.39 3.33±0.57 3.48±0.39 2.85±0.37
总胆固醇TC/(mmol/L) 14 4.76±0.61b 4.17±0.55ab 3.51±0.20a 3.56±0.39a
28 4.51±0.47b 3.67±0.57ab 3.31±0.49a 3.32±0.30a
7 22.86±1.53 24.90±3.45 25.16±3.83 25.28±4.47
谷草转氨酶AST/(U/L) 14 24.39±4.05 28.48±5.73 27.97±7.17 31.67±3.51
28 25.80±4.05a 25.16±3.83a 25.92±1.33a 33.71±2.89b
7 6.20±1.77 5.98±1.67 7.37±1.52 5.98±1.67
谷丙转氨酶ALT/(U/L) 14 7.59±2.25 7.48±0.93 7.80±1.45 7.90±1.67
28 6.30±1.11a 7.59±1.16ab 7.16±0.98ab 8.34±0.37b
如表3所示,所有添加Ala-Gln组的血清GLU含量均随饲喂时间的延长呈现上升趋势,在饲喂28 d时所有添加Ala-Gln组的血清GLU含量均较对照组显著上升(P<0.05)。各添加Ala-Gln组的血清TG与TC含量在饲喂7 d时均与对照组无显著差异(P>0.05),在饲喂14与28 d时1.0%组和1.5%组较对照组显著下降(P<0.05)。在饲喂28 d时,1.5%组的血清AST与ALT活性与对照组相比显著上升(P<0.05)。

2.3 Ala-Gln对低氧胁迫后军曹鱼幼鱼血清抗氧化指标的影响

如表4所示,随着饲喂时间的延长,各组的血清SOD活性先升高后降低,血清CAT和GPx活性则呈上升趋势,且随Ala-Gln添加量的增加上述指标均呈先上升后下降的趋势,且在饲喂14和28 d时1.0%组和1.5%组与对照组相比均显著上升(P<0.05)。各添加Ala-Gln组的血清MDA含量与对照组相比在饲喂14和28 d均显著下降(P<0.05)。
表4 Ala-Gln对低氧胁迫后军曹鱼幼鱼血清抗氧化指标的影响

Table 4 Effects of Ala-Gln on serum antioxidant indexes of juvenile cobia after hypoxia stress (n=3)

项目
Items		 饲喂时间
Feeding
time/d		 Ala-Gln添加量Ala-Gln addition amount/%
0 0.5 1.0 1.5
7 135.94±9.64 141.08±4.38 143.75±9.13 142.11±3.72
超氧化物歧化酶SOD/(U/mg) 14 139.23±6.79a 154.86±12.04ab 157.33±10.16b 159.80±2.14b
28 124.01±4.32a 132.03±5.89ab 143.75±3.27c 137.99±7.12bc
7 3.47±0.20 4.14±0.62 4.26±0.79 4.12±0.82
过氧化氢酶CAT/(U/mL) 14 3.73±0.11a 4.11±0.19ab 4.36±0.14b 4.47±0.44b
28 4.18±0.25a 4.62±0.14ab 4.98±0.31b 4.74±0.37b
7 197.87±6.47a 203.20±4.23a 214.93±8.05b 208.00±3.20ab
谷胱甘肽过氧化物酶GPx/(U/mL) 14 223.47±9.10a 233.07±6.47ab 259.73±5.14c 244.80±6.97bc
28 248.53±7.21a 282.67±6.47b 308.80±9.99c 301.33±10.89c
7 5.75±1.43 3.91±2.31 3.66±0.93 4.19±1.86
丙二醛MDA/(nmol/mL) 14 6.83±0.63b 5.06±0.35ab 4.69±1.74a 4.38±0.89a
28 7.23±0.60b 6.32±0.33ab 5.52±0.97a 5.80±0.52a

3 讨论

3.1 Ala-Gln对低氧胁迫后军曹鱼幼鱼生长性能的影响

虽然水产动物营养学中把Gln称为非必需氨基酸,在适宜环境中鱼类自身能自行合成以满足机体日常需求。但当鱼体处于快速生长、病理状态或应激状态时,内源合成的Gln已不能满足鱼体的需要量,甚至会导致体内其余氨基酸耗竭,此时Gln就变成了条件必需氨基酸。Liu等[22]使用添加0.5%~2.0% Gln的饲料投喂半滑舌鳎(Cynoglossus semilaevis)幼鱼30 d,结果表明Gln通过增强半滑舌鳎幼鱼的消化能力、抗氧化能力和耐缺氧应激能力来提高存活率和生长性能。在建鲤幼鱼上的研究发现,饲料中添加1.2% Gln可以起到提高生长性能,促进肠道发育的作用[23]。在草鱼(Ctenopharyngodon idella)上的研究发现,饲料中添加适当的Gln可促进草鱼的生长以及改善肌肉风味[24]。然而,在大黄鱼(Pseudosciaena crocea)[25]、斑点叉尾鮰(Ictalurus punctatus)[26]和侧带利齿脂鲤(Hoplias lacerdae)[27]上的研究显示,饲料中添加Gln并不能显著提高WGR和SGR。因此,Gln对不同物种的影响可能与试验鱼的种类、生长阶段、大小以及饲料中粗蛋白质含量和试验条件等有关,当鱼类自身自行合成的Gln可满足机体日常需求时,补充Gln或Ala-Gln并不能取得显著效果。
本研究中,随着饲料中Ala-Gln添加量的增加,军曹鱼幼鱼的生长指标呈先上升后下降的趋势,生长性能在添加量为1.0%时最优,且随着饲养时间的延长,效果逐渐显著。这说明适量添加Ala-Gln有助于低氧胁迫后军曹鱼幼鱼机体损伤恢复,促进其快速生长,并降低FCR,提高养殖经济效益。

3.2 Ala-Gln对低氧胁迫后军曹鱼幼鱼血清生化指标的影响

血清中TP和ALB含量可作为衡量鱼体蛋白质吸收与代谢状况的指标[28]。本研究结果显示,与对照组相比,饲喂添加Ala-Gln的饲粮28 d后军曹鱼幼鱼血清TP和ALB含量显著上升,这一试验结果与Pereira等[29]的报道一致,表明添加Ala-Gln可在一定程度上促进低氧胁迫后军曹鱼幼鱼体内蛋白质的合成与代谢。其原因可能是在饲料中适度的添加非必需氨基酸可替代必需氨基酸用作能量代谢的底物,这有助于提升鱼类对膳食蛋白质的利用效率以及增强鱼体内的蛋白质沉积,起到提高鱼类生长性能的效果[30-31]。
血清TC和TG含量可作为反映鱼体脂类代谢情况的指标[32]。本研究中,军曹鱼幼鱼血清TC和TG含量随Ala-Gln添加量的增加和饲喂时间的延长呈下降趋势,在饲喂7 d时,各添加Ala-Gln组血清GLU含量较对照组显著上升,与在金头鲷(Sparus aurata)[33]、草鱼[19]上的研究结果相一致。Gln在调节水产动物体内GLU和脂质的稳态中起重要作用[34]。血清中GLU含量的快速上升可能是因为Gln作为升糖氨基酸,参与了肝脏糖异生途径,促进了GLU的迅速生成;而血清中TC和TG含量的下降则表明Ala-Gln可调控脂肪的生成,有助于减少鱼体中脂肪的堆积。此外,血清中GLU含量的上升也可能是因为Gln可下调糖酵解途径中的关键限制酶——葡萄糖-6-磷酸脱氢酶(G6PDH)的mRNA表达量以及降低该酶的活性,导致糖酵解途径被抑制,鱼体内原本用于参与糖酵解的GLU得以保留,引起血清中GLU含量上升;而作为脂代谢底物的烟酰胺腺嘌呤二核苷酸(NADPH)、乙酰辅酶A、丙酮酸等糖酵解产物的含量减少,进一步导致血清中TG等脂代谢产物的含量下降[33]。本研究结果表明,低氧胁迫后在饲料中添加Ala-Gln可提高军曹鱼幼鱼血清中TP和ALB的含量,并降低TC和TG的堆积,这可能有助于改善低氧胁迫所带来的脂代谢与糖代谢紊乱的现象,但相关机理仍有待进一步的研究。
AST和ALT主要存在于肝细胞线粒体内,是水产动物医学上肝功能检查的重要指标,其在血清中的活性可用来判断水产动物的肝脏是否受到损害。当肝脏细胞受损时,会导致AST和ALT活性在血清中显著升高[14]。在草鱼的研究中发现,添加高剂量的Ala-Gln会加重肝脏的代谢负荷,造成鱼体肝脏损伤[19]。本研究中,1.5%组的血清AST与ALT活性随着饲喂时间的延长呈上升趋势,且在饲喂28 d时与对照组相比显著上升,表明1.5%的添加量已经给低氧胁迫后军曹鱼幼鱼的肝脏带来了显著性的损伤。

3.3 Ala-Gln对低氧胁迫后军曹鱼幼鱼血清抗氧化指标的影响

水产动物组织中活性氧自由基的含量与水产动物的健康有着密切的联系,当鱼类遭受到应激时,体内会产生大量的自由基,一旦不及时清除,会导致代谢异常,甚至对动物的机体和机能造成严重损伤。SOD、CAT和GPx是水产动物抗氧化机制中最重要的3种抗氧化酶[35],SOD可将超氧阴离子(O2-·)转化成过氧化氢(H2O2)和氧气(O2),CAT可进一步将H2O2转化成水(H2O),而GPx则通过催化GSH与H2O2反应生成H2O,保护细胞免受O2-·攻击,同时维护机体细胞的内环境稳定,三者在血清中的活性可用于衡量水产动物抗氧化能力。MDA作为脂质过氧化的产物,其含量的高低是衡量鱼体受氧化损伤轻重程度的指标[36]。
作为免疫增强剂,Gln在诸多水产动物的添加试验中取得了显著的效果。Harward等[37]的试验证明可通过补充Gln来维持鱼体中GSH的含量,在草鱼[38]、日本牙鲆[39]、杂交鲟[40]等水产动物的添加试验中也发现,饲喂Gln可引起鱼体血清中如SOD、CAT、GPx等抗氧化酶活性的上升。此外,饲喂由Gln作为合成前体物质的谷氨酸可保护鲤鱼肠细胞免受铜诱导的氧化损伤[41],Gln与其他氨基酸结合后可提高抗氧化能力,保护鲤鱼红细胞免受羟自由基(·OH)所诱导的凋亡氧化损伤并免于凋亡[42]。GSH在修复脂质氧化和蛋白质氧化的过程中起到关键作用[43],而Gln则是GSH合成的关键前体物质。低氧胁迫会导致血清中GSH含量下降以及MDA含量上升[14]。在本研究中,Ala-Gln的添加提高了军曹鱼幼鱼血清中GPx的活性,因为GPx活性通过采用微板法测定,通过测定酶促反应中GSH的消耗量后计算得出,因此GSH含量与GPx活性呈正相关,表明Ala-Gln降低脂质损伤可能部分归因于血清中GSH含量的增加。此外,SOD和GPx在大鼠肝脏修复中发挥重要作用,大鼠肝脏中SOD活性的增加有助于氧化损伤脂质的修复[44]。在哺乳动物中,GPx是抗氧化防御系统中的关键酶,其中的GPx4是唯一能够减少膜内氢过氧化物的GPx,可通过还原胸腺嘧啶氢过氧化物修复氧化损伤的DNA[45]。本试验结果显示,各添加Ala-Gln组血清SOD、CAT和GPx活性均随着添加量增加呈先上升后下降的趋势,在饲喂14和28 d时1.0%组和1.5%组较对照组显著上升,同时MDA含量显著下降,在添加量为1.0%时血清抗氧化酶活性达到最高,表明Ala-Gln通过增强军曹鱼幼鱼的抗氧化能力来降低低氧胁迫所带来的脂质氧化损伤。

4 结论

综上所述,在饲料中添加Ala-Gln可增强低氧胁迫后军曹鱼幼鱼的生长性能以及抗氧化能力,提高军曹鱼幼鱼血清中TP和ALB的含量以及减少血清中TC和TG的堆积,并在降低FCR的同时提高养殖经济效益。结合试验数据分析,低氧胁迫后军曹鱼幼鱼饲料中Ala-Gln的最适添加量为1.0%。

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