两种油脂水平下DHA添加对鲤生长及脂质代谢的影响

周继术 曹艳姿 吉 红 于海波

(西北农林科技大学动物科技学院水产科学系, 杨凌 712100)

二十二碳六烯酸(Docosahexaenoic acid, DHA)主要来源于海水鱼与藻类[1], 属于n-3系列的高不饱和脂肪酸(Highly unsaturated fatty acid, HUFA), 具有抗癌[2—4]、抗氧化[5—7]、防治心血管疾病[8]、提高免疫力[9,10]、调节脂代谢[11—13]等功能, 对大脑及神经系统发育[14,15]与视觉功能[16]密切相关。在水产动物上, DHA是海水鱼尤其是海水仔稚鱼正常生长发育的必需脂肪酸之一[17], 对黑鲷[18—20]、舌鳎仔鱼[21]等海水鱼脂质代谢也具有重要的调控作用, 而温水性鱼类如草鱼、鲤等具有将18:3n-3合成为DHA的能力, 一般认为DHA并非温水性鱼类的必需脂肪酸, 然而DHA对鱼类生长、脂代谢、细胞膜的流动性以及免疫功能显示出重要的调控作用[22—27]。但是高浓度HUFA的添加会给机体带来过高的过氧化压力[28—30], 从而影响草鱼组织脂质含量、抑制鲤及草鱼脂质沉积[31—33], 还可能造成生长减缓、饲料转化率降低等负面影响[34]。高浓度HUFA的添加对鱼类造成的负面效应是否和饲料中其他脂肪酸或脂肪总量水平相关, 尚未见报道。

为了进一步了解不同油脂水平下DHA的添加对淡水鱼类的营养生理作用, 本试验以鲤(Cyprinus carpioL.)为对象, 在6%与12%两个油脂水平下, 探讨了3%添加水平下DHA对鲤生长、生物学性状、体成份、血清理化性状及组织结构的影响, 旨在为研究淡水鱼类脂质营养及其代谢调控提供基本资料。

1 材料与方法

1.1 试验饲料

以酪蛋白、糊精、纤维素、大豆油以及复合鱼用预混料等为饲料原料, 采用Lovell[35]试验饲料配方配制鲤饲料。在6%与12%两个油脂水平下, 以DHA制品(无锡讯达化学品厂, 江苏, 无锡)分别替代3%的大豆油, 制成基础组、基础-DHA组、高脂组、高脂-DHA组4种等氮精制试验饲料。将各原料均粉碎过60目筛, 将原料按比例混匀后压制成直径为2 mm长3 mm颗粒, 于冰箱冷藏保存备用。DHA制品的脂肪酸组成见表 1, 试验饲料配方及营养成分见表 2。

1.2 试验鱼

试验鲤购自陕西省周至县某渔场。先挑选健康且个体相近的鲤驯养于容积为160 L的循环水养殖系统中, 经过7d驯养后, 选出体质健康的试验鱼360尾(14.81±0.13) g, 随机分成4个组, 每组3个重复,每个重复30尾鱼, 饲养于12个循环水养殖缸中。

1.3 饲养管理

将4组试验饲料分别投喂4组试验鱼, 投饲率3.5%, 每天4次(8:30、11:30、14:30和17:30), 每次持续投喂时间为1h, 每次投食后清除残饵和粪便。水源为经曝气的自来水, 每天记录水温及试验鱼摄食等情况。试验期间水温(27±2)℃, 水流速度为20—30 L/h, DO＞6 mg/L, pH 7.5—8.0。饲养中期称重一次以调整投饲量, 饲养试验为74d。在饲养试验结束后, 对所有试验鱼称重并测量体长和取样。

1.4 样品采集及分析

在饲养结束后, 将试验鱼用浓度为100 mg/kg MS-222麻醉后, 随机取9尾鱼采血。将所有鱼称其体重并测量其体长, 再将鱼进行解剖后, 分离其内脏、肝胰脏、脾脏、肠、腹腔脂肪、单侧肌肉并称重与量取鱼肠长。将分离的肌肉与肝胰脏于-20℃保存备用。

鲤生长指标的测定试验结束后, 称取所有试验鱼体重, 其生长指标测定如下:

存活率(%)=末尾数/始尾数×100;

绝对生长率(g/d)=(末尾均重-始尾均重)/饲养时间(d);

红琴平时很少叫风影的名字，开口闭口的总唤他和尚，林燕常常拿着风影敲过的木鱼，坐在门槛上敲。他的眼神郁郁的，在怀想出家时的生活。风影又体味着尘世的乐趣，特别是在红琴身上，他体验着那种女人特有的味道，阳光的温暖，雨丝的缠绵，兰花的馨香。对于林燕，村子里的好事者有各种猜测，他们用各自的缜密的逻辑推理得出了一个又一个结论，有人说是红琴落洞时山神在她身上播的种，也有人说是风影这个花和尚早就动了花心，和尚也是人，是男人都会想做那点事儿。嘴巴长在别人的脸上，人家要嚼舌头风影有什么办法。

相对生长率(%/d)=(末尾均重-始尾均重)/始尾均重×100;

饲料系数=饲料投喂量(g)/鱼体增重量(g)。

鲤生物学性状的测定分离鱼体内脏, 称取鱼体空壳、肝胰脏、脾脏、肠、腹腔脂肪、单侧肌肉等的重量并测量鱼体肠长。鲤生物学性状的测定指标如下:

肥满度(g/cm3%)=鱼体重(g)/体长(cm)3×100;

空壳比率(%)=空壳重(g)/体重(g)×100;

含肉率(%)=肌肉重(g)/体重(g)×100;

表 1 试验油脂脂肪酸组成Tab. 1 Fatty acids composition of dietary oils (%)

表 2 半纯化试验饲料配方及营养成分组成Tab. 2 Ingredient and proximate composition of the semi-purified experimental diet

肝胰脏指数(%)=肝胰脏重(g)/体重(g)×100;

腹脂指数(%)=腹腔脂肪重(g)/体重(g)×100;

肠重比(%)=肠重(g)/体重(g)×100;

脾脏指数(%)=脾脏重(g)/体重(g)×100。

饲料与肌肉及肝胰脏常规成分的测定饲料、肝胰脏与肌肉常规成分的测定根据AOAC[37]法, 分别用索氏抽提法测定肌肉及肝胰脏的粗脂肪含量, 用半微量凯氏定氮法测定粗蛋白含量。

血清样品制备和指标测定饲养试验结束后每箱随机取鲤9尾, 于其尾静脉处采血, 3000 r/min离心10min, 制备血清。血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、总蛋白(TP)、白蛋白(ALB)、球蛋白(GLO)、总胆固醇(Chol)、甘油三脂(TG)、葡萄糖(GLU)等指标由生化试剂盒(中生北控生物科技有限公司, 北京, 中国)及全自动生化分析仪(岛津ILAB600)条件下进行测定。

肝细胞及腹腔脂肪细胞观察在饲养及饥饿试验结束后, 每个处理随机取3尾鱼解剖, 分离肠道、肝胰脏、腹腔脂肪组织立即于波恩氏液中固定。24h后自来水洗涤, 不同浓度梯度的酒精脱水,二甲苯透明, 石蜡包埋, 连续切片, HE染色, 中性树胶封片。光学显微镜下进行组织学观察, 并利用显微镜的照相系统进行肝细胞直径及腹腔脂肪细胞的长轴与短轴距离的测量。将脂肪细胞长轴与短轴距离相乘, 计算脂肪细胞的面积。

1.5 数据分析

所得数据采用平均数±标准差(Mean±SD)表示。用SPSS17.0软件(Chicago, IL, USA)对试验数据进行单因素方差分析(One-way ANOVA)以及Duncan’s多重比较检验组间差异,P＜0.05为差异显著。

2 结果

2.1 DHA对鲤生长的影响

在6%油脂水平下, 基础-DHA组末尾均重(43.7±0.8) g、绝对生长率(0.40±0.01) g/d与相对生长率(2.67±0.03)%/d均显著低于基础组(P＜0.05)。在12%油脂水平下, 高脂-DHA组绝对生长率(0.45±0.01) g/d显著低于高脂组(0.49±0.01) g/d, (P＜0.05),而末尾均重与相对生长率在两高脂组间均无显著差异(P＞0.05)。基础-DHA组的饲料系数显著高于其他各组(P＜0.05), 而该饲料系数在其他三组间均无显著差异。饲料的摄食量(3972—4212 g)与鲤的存活率(93%—99%)在各组间均无显著差异(P＞0.05)。

2.2 DHA对鲤生物学性状的影响

由表 4可以看出: 基础-DHA组鱼体肥满度最低且显著低于高脂-DHA组(P＜0.05)。基础-DHA组腹脂指数为(0.37±0.05)%, 显著低于基础组(0.52±0.04)%、高脂组(0.70±0.05)%及高脂-DHA组(0.67±0.06)%的腹脂指数(P＜0.05)。同时, 12%水平下的高脂组与高脂-DHA组的腹脂指数均高于6%水平下的基础组与基础-DHA组(P＜0.05)。空壳比率、含肉率、比肠长与脾脏指数等在各组间均无显著差异(P＞0.05)。

2.3 DHA对鲤体成分的影响

从表 5可以看出, 在12%脂肪水平下鲤肌肉与肝胰及粗脂肪含量均分别显著高于6%脂肪水平组。在6%与12%脂肪水平下, DHA添加组的鲤肌肉粗脂肪含量[(1.82±0.07)%、(3.03±0.09)%]均分别显著低于无DHA组[(2.58±0.07)%、(3.51±0.11)%;P＜0.05)]。鲤肌肉粗蛋白含量在各组间均无显著差异(P＞0.05)。

表 3 DHA对鲤生长的影响Tab. 3 Effect of dietary DHA fortification on growth performance of common carp

表 4 DHA对鲤生物学性状的影响Tab. 4 Effect of dietary DHA fortification on biological parameters (n=13)

表 5 DHA对鲤肌肉及肝胰脏粗脂肪与粗蛋白含量的影响Tab. 5 Effect of dietary DHA fortification on crude lipid and crude protein of muscle and hepstosomatese (n=3)

2.4 DHA对鲤血清生化组成的影响

从表 6可以看出, 高脂-DHA组谷丙转氨酶为(126.9±31.2) U/L, 该值显著高于其他各组(58.9—70.2 U/L,P＜0.05), 同时该组的总蛋白与球蛋白等均达到各组中的最高值, 分别为(33.98±1.02) g/L与(23.66±0.89) g/L。分别在6%与12%油脂水平下,DHA的添加均显著升高了鲤血清胆固醇含量 (P＜0.05), 而DHA的添加有降低血清甘油三酯含量的趋势。血清谷草转氨酶、白蛋白、白球比与葡萄糖等指血清生化指标在各组间均无显著差异(P＞0.05)。

2.5 DHA对鲤肝胰脏与腹脂组织的细胞大小的影响

从表 7可以看出, 6%脂肪水平下, 基础-DHA组腹腔脂肪细胞面积为(67.5±7.0)×103μm2, 显著低于基础组; 当饲料脂肪水平上升至12%水平时, 两组间鱼体腹腔脂肪细胞面积无显著差异(P＜0.05)。

3 讨论

3.1 DHA对鲤生长的影响

在黑鲷这样海水鱼的研究中发现, 饲料中添加1.5%DHA, 黑鲷生长及生物学指标均没有显著变化, 但饲料效率显著增高[20]。DHA并非草鱼、鲤等温水性鱼类的必需脂肪酸, 本研究结果显示, 3%DHA制品的添加不但并未提高鲤的生长与饲料效率, 反而对其有明显的抑制作用(表 3), 这与Du等[30]在草鱼的研究中发现, 随饲料中鱼油添加量的增加, 草鱼摄食率及表观生长率下降相一致, 也与Takeuchi等[34]关于虹鳟饲料中添加过量n-3HUFA, 导致鱼体生长下降、饲料转化效率降低的研究结果相一致,也与鲤、草鱼[31—33]的研究结果相一致。

DHA的高不饱和性使其易受氧或其他自由基的攻击, 从而产生有害的过氧化产物, 这些过氧化产物的产生会损伤体抗氧化能力[25,37—39]。然而, 也有研究发现, DHA能够增强机体抗氧化酶活性以及促进自由基的清除[6,8,40—42]。因此, DHA对机体抗氧化能力的影响尚不明确。本研究结果显示, 3%DHA制品的添加不但未能提高鲤的生长与饲料效率, 反而对其有明显的抑制作用(表 3), 虽然本次试验没能检测谷胱甘肽过氧化物酶、超氧化物岐化酶等抗氧化指标, 然而结合本次试验结果, 笔者推测,DHA添加组的鲤较差的生长效果可能与DHA的添加与食入有关; 易于氧化的DHA在没有额外的抗氧化物质, 如维生素E的供给, 会给鲤机体带来过高的过氧化压力, 从而带给鲤较高的氧化应激, 进而影响鲤的生长, 降低鲤的饲料转化效率, 而这一结果在鲤的以往研究[34]及哺乳动物[43]中均有类似发现。

3.2 DHA对鲤脂质代谢的影响

在本试验的研究结果中发现, DHA的添加, 鱼体肥满度、腹脂指数、腹腔脂肪细胞面积与肌肉粗脂肪含量均低于同一脂肪水平下的无DHA的添加组(表 4—7)。本研究与Om等[18,19]的研究结果:饲料中DHA降低了黑鲷腹腔脂肪细胞脂质蓄积和细胞直径大小相一致。

表 6 DHA对鲤血清生理生化指标的影响Tab. 6 Effect of dietary DHA fortification on serum biochemical index (n=3)

表 7 DHA对鲤肝胰脏与腹脂组织的细胞大小的影响Tab. 7 Effect of dietary DHA fortification on cell area of hetopancreas and adipose tissues (n=3)

DHA等多不饱和脂肪酸(PUFA)是动物生命活动的重要能量来源, 也是细胞膜的重要组成成分,在细胞表面信号传导以及细胞生化代谢过程中起着重要作用[44]。对啮齿动物的研究表明, n-3PUFA能降低肥胖[45]。进一步的研究发现, PUFA能与鼠等哺乳动物中的肝脏过氧化物酶增殖物激活受体(PPARs)、固醇调节元件结合蛋白(SREBP)、X受体 (LXRs)等核受体特异性地结合, 使PUFA调节相关酶的转录基因, 从而减少脂肪合成和体脂沉积,促进了脂肪降解[44,46,47]。研究表明, 与陆生动物一样, PUFA能从分子水平上激活大西洋鲑PPAR受体, 调节相关酶的转录基因, 抑制脂合成酶的活性,促进脂肪降解, 最终减少体脂沉积[48—50]。因此, PUFA等长链脂肪酸是一种调控能量代谢的物质[51], 它通过作用于PPARs、SREBP等核受体实现对细胞脂质代谢的调控[52]。本研究室对水产动物尤其是对草鱼的长期研究也发现, DHA等PUFA对草鱼脂肪细胞发育及某些脂质氧化代谢基因, 如三酰甘油酯酶(ATGL)、过氧化物酶体增殖受体α激活因子1(PGC-1α)、过氧化物酶体增殖物激活受体(PPARs)等有显著影响[33,53—55], 从而促进了脂质的氧化。虽然本次试验并未检测以上脂质代谢基因, 但本研究结果显示, DHA的添加降低了鱼体肥满度、腹脂指数、腹腔脂肪细胞面积与肌肉粗脂肪含量, 为此,笔者推测, 这一结果可能与DHA促进了鲤脂质及脂肪酸的氧化代谢有关, 同时, 本试验结果与DHA的添加影响草鱼组织脂质含量、抑制鲤及草鱼脂质沉积的研究结果[31—33]相一致。

在塞内加尔舌鳎(Solea senegalensis)的研究发现, 提高DHA及饲料油脂水平, 将提高其生长率, 反之则降低其生长率[56]。本研究发现, 随饲料脂肪水平的提高, DHA的添加对鲤生长、肥满度、腹脂指数、腹腔脂肪细胞面积、肌肉粗脂肪含量与血清胆固醇含量等的抑制作用均有减轻的趋势(表3—7), 显示出脂肪水平对DHA添加所造成的损伤的补偿作用。

在本实验中添加DHA使鲤血清胆固醇含量并未显著降低(表 6), 这与饲料PUFA有降低哺乳动物血浆胆固醇含量的研究结果作用[57,58]不一致。HUFA对鱼类胆固醇代谢的影响与哺乳动物不同的原因还有待进一步研究。鲤血清甘油三酯含量随DHA添加有降低趋势(表 6), 这在鼠[59]上也有类似的研究结果。研究表明, DHA对鲤脂质代谢具有调控作用, DHA作为鲤的非必需脂肪酸, 其利用能力及其作用机制可能与哺乳动物不同, 其相关机制还需进一步研究。

4 总结

3%DHA制品的添加不但并未提高鲤的生长与饲料效率, 反而对其有明显的抑制作用。与此同时,DHA的添加对鱼体肥满度、腹脂指数、腹腔脂肪细胞面积与肌肉粗脂肪含量均有抑制作用, 这可能与DHA的添加量可能超过了鲤的营养需求量, 从而对鱼体形成了的过氧化压力, 进而影响了鱼机的生长与健康有关。随饲料脂肪水平的提高, DHA的添加对鲤生长、肥满度、腹脂指数、腹腔脂肪细胞面积、肌肉粗脂肪含量等所形成的抑制作用均有减轻趋势, 显示出提高饲料脂肪水平可在一定程度下补偿DHA添加所造成的机体损伤。

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