动物营养学报    2017, Vol. 29 Issue (10): 3761-3772    PDF    
长期饲喂不同蛋白质水平饲粮对猪脂肪代谢相关基因表达的影响
田志梅, 马现永, 王丽, 熊云霞, 邱月琴, 杨雪芬, 高开国, 温晓鹿, 胡友军, 蒋宗勇     
广东省农业科学院动物科学研究所, 畜禽育种国家重点实验室, 农业部华南动物营养与饲料重点实验室, 广东省动物育种与营养公共实验室, 广东省畜禽育种与营养研究重点实验室, 广州 510640
摘要: 本试验旨在研究长期饲喂不同蛋白质水平饲粮对猪脂肪代谢相关基因表达的影响。试验选用18头三元(杜×长×大)杂交28日龄断奶仔猪,随机分为3组,每组6个重复,每个重复1头。对照组[高粗蛋白质(HCP)组]采用符合NRC(2012)推荐营养需要的饲粮,试验组是根据NRC(2012)标准,在添加赖氨酸(Lys)、蛋氨酸(Met)、苏氨酸(Thr)、色氨酸(Try)4种必需氨基酸基础上,将饲粮蛋白质水平分别降低3%[中粗蛋白质(MCP)组]和6%[低粗蛋白质(LCP)组]。试验期125 d。结果表明:1)在肝脏中,与HCP组相比,LCP组显著降低脂肪酸合成相关基因乙酰辅酶A羧化酶(ACC)、脂肪酸合成酶(FAS)、苹果酸酶1(ME1)及锚蛋白1(ANK1)的基因表达量(P < 0.05);同时显著提高脂肪酸转运相关基因过氧化物酶体增殖物激活受体γ(PPARγ)及脂肪酸结合蛋白(FABP)的基因表达量(P < 0.05);而MCP组与HCP组相比,脂肪酸合成、转运相关基因表达量均无显著性差异(P>0.05);与HCP组相比,MCP及LCP组均显著降低脂肪酸氧化分解相关基因、过氧化物酶体增殖物激活受体α(PPARα)、肉毒碱棕榈酰转移酶(CPT)的基因表达量(P < 0.05)。2)在背最长肌(LDM)中,MCP组中脂肪酸合成相关基因胆固醇调节元件结合蛋白(SREBP)、FAS、硬脂酰辅酶A去饱和酶(SCD)的基因表达量显著高于其他2组(P < 0.05);ACCFABP在LCP组的基因表达量显著低于其他2组(P < 0.05);CPT基因表达量及LDM的肌内脂肪(IMF)含量在LCP及MCP组显著低于HCP组(P < 0.05)。3)肝脏和LDM中,各组甘油三酯脂酶(ATGL)及二烯酰辅酶A还原酶(DECR)基因表达量均无显著性差异(P>0.05)。由此可见,在NRC(2012)基础上,适当的降低饲粮蛋白质水平(3%)可促进LDM中脂肪酸合成、转运相关基因的表达,但对肝脏中脂肪酸合成相关基因的表达无显著影响;适当的降低饲粮蛋白质水平(3%)可降低LDM及肝脏中脂肪酸氧化分解相关基因的表达,但并不增加LDM肌内脂肪含量。
关键词: 饲粮     蛋白质     肥育猪     脂肪     代谢     基因    
Effects of Long-Term Feeding Diets with Different Protein Levels on Gene Expressions Related to Lipid Metabolism of Pigs
TIAN Zhimei, MA Xianyong, WANG Li, XIONG Yunxia, QIU Yueqin, YANG Xuefen, GAO Kaiguo, WEN Xiaolu, HU Youjun, JIANG Zongyong     
State Key Laboratory of Livestock and Poultry Breeding, Key Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture, Guangdong Public Laboratory of Animal Breeding and Nutrition, Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
Abstract: This experiment was conducted to study the effects of long-term feeding diets with different protein levels on gene expressions related to lipid metabolism of pigs. Eighteen cross-bred (Duroc×Landrace×Yorkshire) piglets weaned 28 days of age were randomly assigned to 3 groups with 6 replicates per group and 1 piglet per replicate. Piglets in control group[(high crude protein, HCP) group] were fed a diet based on the National Research Council (NRC) (2012) recommendation, those in experimental groups were fed diets with decreasing 3%[(medium crude protein, MCP) group] and 6% crude protein levels[(low crude protein, LCP) group], respectively, based on NRC (2012), and supplemented with 4 essential amino acids, which were lysine, methionine, threonine and tryptophane. The experiment lasted for 125 days. The results showed as follows:1) in liver, compared with HCP group, LCP group significantly decreased expressions of fatty acid synthesis genes including acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), malicenzyme 1 (ME1) and ankyrin 1 (ANK1) (P < 0.05), and significantly increased expressions of fatty acid transport genes like peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid binding proteins (FABP) (P < 0.05). However, there were no significant differences between MCP and HCP groups in hepatic expressions of fatty acid synthesis genes and fatty acid transport genes (P>0.05). Compared with HCP group, there were significant increases in hepatic expressions of fatty acid oxygenolysis genes like peroxisome proliferator-activated receptor alfa (PPARα) and carnitine palmitoyltransferase (CPT) in MCP and LCP groups (P < 0.05). 2) In the longissimus dorsis muscle (LDM), the expressions of fatty acid synthesis genes like sterol regulatory element binding protein (SREBP), FAS and stearoyl CoA desaturase (SCD) showed significantly higher in MCP group than those in the other two groups (P < 0.05), but ACC and FABP displayed significantly lower expressions in LCP group than those in the other two groups (P < 0.05). The expression of CPT gene and intramuscular fat (IMF) content of LDM were significantly lower in LCP and MCP groups compared to HCP group (P < 0.05). 3) In liver and LDM, there were no significant differences in expressions of adipose triglyceride lipase (ATGL) and 2, 4-dienoyl-CoA reductase (DECR) genes among three groups (P>0.05). In conclusion, decreasing dietary crude protein level (3%) properly can promote expressions of fatty acid synthesis and transport genes, but has no significant effects on hepatic expressions of fatty acid synthesis genes; decreasing dietary crude protein level (3%) properly can decrease hepatic expressions of fatty acid oxygenolysis genes, but does not affect IMF content of LDM.
Key words: diet     protein     finishing pigs     lipid     metabolism     gene    

猪肉是人类最主要的食物蛋白质源,我国是生猪生产及消费大国,饲粮蛋白质水平是影响养猪业的最主要的经济及环境因素之一。目前多数饲粮蛋白质水平远远超过动物生长发育需要量,不仅增加了生猪养殖业的成本,降低氮利用率,也增加养猪业的环境压力。饲粮蛋白质在动物生长发育中起到重要的营养及生理功能,目前有研究指出添加满足动物需求必需氨基酸的低蛋白质饲粮可达到相似的氮吸收利用率和体重(BW)[1-3];也有研究指出,降低饲粮蛋白质水平可降低猪的生长性能,并增加猪肉脂肪含量,改善肉品质[4-5]。Tian等[6]指出饲粮蛋白质水平从20%降到17%显著降低断奶仔猪的生长性能,提高仔猪肠道消化吸收。Madeira等[4]提出饲粮蛋白质水平从16%降为13%可增加肥育猪背部脂肪厚度以及猪肉肌内脂肪(intramuscular fat, IMF)含量、多汁性等肉品质,但降低肥育猪生长性能;同时也指出添加精氨酸的低蛋白质水平饲粮不影响肥育猪肌肉IMF含量,而添加亮氨酸的低蛋白质水平饲粮可增加肌肉IMF含量。然而也有研究指出,添加精氨酸影响猪肉品质,如降低猪总脂肪的沉积,增加肌肉IMF含量,提高肌肉的抗氧化能力等[7-8]。Tous等[3]指出62~97 kg猪饲粮蛋白质水平从12%降低到9.8%不影响猪的生长性能,但增加肌肉IMF含量;添加赖氨酸(Lys)可以提高猪肉品质,不影响猪的生长性能。

随着人们生活水平的提高,猪肉品质备受消费者的关注,是消费者首要衡量指标。脂肪及脂肪酸是决定猪肉食用品质的重要因素,尤其是IMF的含量在肉品质方面起到重要作用。目前通过降低饲粮蛋白质和添加氨基酸等饲养策略来提高猪体内的脂肪分配[9-11]。IMF含量是影响口感及风味等感官质量性状的重要因素之一,同时IMF含量也影响肌肉的滴水损失及多汁性[12-14]。近年来瘦肉型猪选育降低将近1%的IMF,这也是导致肉质变差的主要原因之一[15],因此人们对肉的IMF含量的关注也越来越高[16-18]。关于促进IMF沉积的研究也越来越多,通过降低饲粮中蛋白质水平来改变IMF的含量是典型的方法[19-22]。为此,本试验通过降低猪各生长阶段饲粮蛋白质水平,研究其对脂肪代谢相关基因表达的影响,为降低生猪养殖成本及氮排放量、提高猪肉品质提供理论依据。

1 材料与方法 1.1 试验动物及设计

选取18头三元(杜×长×大)杂交28日龄断奶仔猪[BW=(9.57±0.64) kg],随机分为3组,每组6个重复,每个重复1头。饲养在猪笼里, 每笼1头。对照组[高粗蛋白质(high crude protein, HCP)组]采用符合NRC(2012) 推荐营养需要的饲粮,试验组是根据NRC(2012) 标准,在添加Lys、蛋氨酸(Met)、苏氨酸(Thr)、色氨酸(Try)4种必需氨基酸基础上,将饲粮蛋白质水平分别降低3%[中粗蛋白质(medium crude protein, MCP)组]和6%[低粗蛋白质(low crude protein, LCP)组]。28日龄断奶仔猪经过7 d预试期后,进入正试期,正试期进行分阶段持续饲喂,至肥育结束。在仔猪阶段,LCP、MCP及HCP组分别饲喂粗蛋白质水平为14%、17%、20%的饲粮,共饲喂45 d;在生长猪阶段,LCP、MCP及HCP组分别饲喂粗蛋白质水平为12%、15%、18%的饲粮,共饲喂30 d;在肥育猪阶段,LCP、MCP及HCP组分别饲喂粗蛋白质水平为10%、13%、16%的饲粮,共饲喂50 d(表 1)。饲养试验结束后进行动物屠宰、样品采集。试验饲粮组成及营养水平见表 2。试验期间采取自由采食及饮水方式进行饲养。

表 1 饲粮粗蛋白质水平及饲喂时间 Table 1 Dietary crude protein levels and time for feeding
表 2 试验饲粮组成及营养水平(风干基础) Table 2 Composition and nutrient levels of experimental diets (air-dry basis)
1.2 屠宰及样品采集

屠宰前肥育猪进行电击处死,动物处死后,立即进行组织、脏器等分离并放于冰上。取80 g背最长肌(LDM)放于封口袋中置于-80 ℃保存,以备于检测IMF含量;分别取3 g LDM并分装到3个1.5 mL的EP管中,立即放于液氮中,最终转移到-80 ℃保存;取肝脏组织(每次切取保证部位一致性)大约3 g, 将肝脏组织用预冷磷酸盐缓冲液(PBS)冲洗,去除肝脏组织表面血液并用吸水纸吸去水分,分装到3个1.5 mL的EP管中,立即放于液氮中,最终转移到-80 ℃保存。

1.3 IMF含量测定

将LDM去筋膜后搅碎,取40 g肌肉样品放入培养皿中摊平后放入-80 ℃冰箱冷冻过夜,样品放入冷冻干燥器内冻干后称重。用滤筒称取冻干样品3 g,利用脂肪仪(FOSS-2055 SOXTEC)通过索氏萃取法检测IMF含量。计算公式如下:

IMF含量(%)=100×(油杯重-铝杯重)/上样量。

1.4 脂肪代谢相关基因表达量测定

与肉品质相关的脂肪代谢基因包括过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma, PPARγ)、过氧化物酶体增殖物激活受体α(peroxisome proliferator-activated receptor alfa, PPARα)、脂肪酸合成酶(fatty acid synthase, FAS)、胆固醇调节元件结合蛋白(sterol regulatory element binding protein, SREBP)、脂肪酸结合蛋白(fatty acid binding proteins, FABP)、肉毒碱棕榈酰转移酶(carnitine palmitoyltransferase, CPT)、硬脂酰辅酶A去饱和酶(stearoyl CoA desaturase, SCD)、甘油三酯脂酶(adipose triglyceride lipase, ATGL)、乙酰辅酶A羧化酶(acetyl CoA carboxylase, ACC)、苹果酸酶1(malicenzyme 1, ME1)、二烯酰辅酶A还原酶(2, 4-dienoyl-CoA reductase, DECR)、锚蛋白1(ankyrin 1, ANK1)基因。用TRIzol试剂盒(TaKaRa, 日本)提取100 mg肝脏组织或LDM样品总RNA并溶解在RNase-free水中。根据反转录试剂盒(TaKaRa,日本)说明书将总RNA(1 μg)反转录为cDNA。根据GenBank中猪的基因序列,使用Primer premier 5.0设计引物(表 2)。制作实时定量PCR标准曲线,确认最佳反应条件。反应体系(20 μL):2μL cDNA,10 μL SYBR Green 2×mix,上、下游引物各0.8 μL (100 nmol/L),6.4 μL的ddH2O。反应条件:95 ℃预变性3 min;95 ℃变性15 s,退火30 s,72 ℃延伸30 s,39个循环。通过对β肌动蛋白(β-actin)以及甘油醛-3-磷酸脱氢酶(GADPH)管家基因进行筛选,选择表达更稳定的β-actin作为实时定量PCR的内参基因。目的基因表达量计算采用2-ΔΔCt法。

表 3 实时定量PCR引物序列 Table 3 Primer sequences for real-time qPCR
1.5 统计分析

试验数据利用Prism 6软件的one-way ANOVA程序进行统计分析。数据均以平均值±标准误(mean±SE)表示,P < 0.05为差异显著。

2 结果 2.1 饲粮蛋白质水平对LDM中IMF含量的影响

图 1所示,在LDM中,HCP组中IMF的含量显著高于LCP及MCP组(P < 0.05),而LCP和MCP组中IMF的含量无显著性差异(P>0.05)。

数据柱形标注不同小写字母表示差异显著(P < 0.05)。 Data columns with different small letters mean significant difference (P < 0.05). 图 1 饲粮蛋白质水平对猪LDM中IMF含量的影响 Figure 1 Effects of dietary protein level on IMF content in LDM of pigs
2.2 饲粮蛋白质水平对猪肝脏组织脂肪代谢相关基因表达的影响

表 4可知,在肝脏中,SREBPACCME1、SCDANK1的基因表达量变化趋势具有一致性,而FABP在肝脏中的表达趋势则相反。与LCP组相比,MCP及HCP组中SREBPACCFASME1、SCD的基因表达量升高,其中FASACCME1基因表达量显著升高(P < 0.05);ANK1在HCP组的基因表达量显著高于LCP组(P < 0.05),而其在MCP组与其他2组相比无显著性差异(P>0.05)。在肝脏中,PPARγFABP的基因表达量变化趋势相同,LCP组显著高于MCP及HCP组(P < 0.05),但MCP组与HCP组无显著差异(P>0.05);PPARαCPT在LCP及MCP组的基因表达量无显著性差异(P>0.05),但均显著低于HCP组(P < 0.05);饲粮蛋白质水平对肝脏中SREBPSCDATGLDECR的基因表达量无显著差异(P>0.05)。

表 4 饲粮蛋白质水平对猪肝脏组织内脂肪代谢相关基因的影响 Table 4 Effects of dietary protein level on expressions of genes related in lipid metabolism in hepatic tissue of pigs
2.3 饲粮蛋白质水平对LDM脂肪代谢相关基因表达的影响

表 5可知,在LDM中,SREBPACCFASSCDFABPPPARα的基因表达量变化趋势具有相似性,MCP组高于LCP和HCP组,其中SREBPFASSCD在MCP组的基因表达量显著高于其他2组(P < 0.05);ACCFABP在LCP组的基因表达量显著低于MCP及HCP组(P < 0.05),但MCP组与HCP组差异不显著(P>0.05);ME1在HCP组的基因表达量显著高于LCP组(P < 0.05),而MCP组与其他2组无显著差异(P>0.05);ANK1在LCP和MCP组的基因表达量无显著差异(P>0.05),但均显著高于HCP组(P < 0.05);PPARαPPARγ在3组中的基因表达量无显著差异(P>0.05);CPT在HCP组的基因表达量显著高于其他2组(P < 0.05),但MCP组与LCP组无显著差异(P>0.05);饲粮蛋白质水平对LDM中ATGLDECR的基因表达量无显著影响(P>0.05)。

表 5 饲粮蛋白质水平对猪LDM脂肪代谢相关基因表达的影响 Table 5 Effects of dietary protein level on expressions of genes related in lipid metabolism in LDM of pigs
3 讨论 3.1 饲粮蛋白质水平对猪生长性能的影响

在目前养猪业的背景下,基于生态环境及经济压力,降低饲粮蛋白质水平既能降低养猪业成本,同时也能减少氮排放。有研究表示,低蛋白质饲粮可以在降低氮排放及氨释放的同时不影响猪的生长性能[23-25];也有研究指出,降低饲粮蛋白质水平将影响猪的生长性能[21, 26-27]。另有研究结果显示,在满足必需氨基酸Lys、Met、Thr、Try需要量的基础上,降低饲粮蛋白质水平影响仔猪及生长猪生长性能,但并不改变肥育猪生长性能[6, 28]。本课题组的另一项研究表明,仔猪断奶后随着饲粮蛋白质水平的降低,仔猪生长性能也出现不同程度的降低,而持续饲喂低蛋白质水平饲粮至生长猪阶段,饲粮蛋白质水平对生长猪生长性能与其对仔猪生长性能的影响具有一致性,但持续降低饲粮蛋白质水平并不影响肥育猪平均日增重、平均日采食量及料重比[29-30]。本课题组研究表明,在断奶初期降低饲粮蛋白质水平不利于仔猪克服断奶应激,影响断奶仔猪生长性能[6]。低蛋白质水平饲粮对仔猪生长性能的影响可能是由于仔猪处在断奶这一特殊时期引起的,仔猪断奶后发生应激反应,包括营养性应激、环境应激及心理应激。断奶后,能量来源的转变,肠道损伤及肠道微生物的改变,母源抗体的消失引起仔猪免疫力低下,影响仔猪生理生长状况。低蛋白质水平饲粮即使平衡了Lys、Met、Thr、Try,但可能由于其他必需氨基酸的不足,不能满足仔猪营养需求,从而导致其生长性能的降低。而随着饲喂时间延长,仔猪度过断奶应激时期后,生长猪生长性能依然随着饲粮蛋白质水平的降低而呈现显著下降,可能是由于低蛋白质饲粮降低仔猪的采食量,仔猪采食量的降低不利于其肠道等的消化道损伤的修复、断奶后变化的适应及断奶应激的忍受,从而影响后续生长猪的消化吸收功能;此外, 采食量是决定日增重的重要因素之一,Brillouet[31]证明仔猪日增重的降低会影响后期生长。但持续饲喂到肥育猪阶段,与对照组相比,低蛋白质水平饲粮对猪生长性能仍有持续性消极影响,但中蛋白质水平饲粮对肥育猪生长性能无显著影响,说明过低的蛋白质饲粮不利于猪的生长,而中蛋白质水平饲粮在后期肥育猪阶段并不影响猪的生长, 进一步证明持续地饲喂低蛋白质饲粮不利于猪的前期生长。

3.2 饲粮蛋白质水平对肝脏脂肪代谢相关基因表达的影响

肌肉脂肪含量及组成影响猪肉品质,动物体内脂肪的获得有2条途径,一是通过食物中摄取脂肪经小肠分解成甘油和脂肪酸消化吸收后,由肝脏合成脂肪酸后运输到身体各部位;另外肝脏中脂肪代谢酶催化糖及氨基酸代谢中间产物的内源性合成。肝脏内含有种类丰富的脂肪代谢相关酶,是动物体内脂肪酸合成的重要场所,在脂肪代谢中起重要作用,因此肝脏中脂肪代谢影响肌肉内脂肪代谢,进而影响肉品质。

过氧化物酶体增殖物激活受体(PPAR)及SERBP是调节脂质代谢的重要转录因子[10, 32]。PPARα和PPARγ属于PPAR超家族成员,通过线粒体及过氧化物酶体的β-氧化作用调控游离脂肪酸(free fatty acid,FFA)的氧化、吸收,参与调控脂肪代谢[33-34]SREBP作为脂类合成的候选基因调控FASME1、ACC等基因表达[33, 35-39]。本试验结果表明,低蛋白质水平饲粮降低肝脏中SREBPFASME1及ACC的基因表达量,其中FASME1及ACC的基因表达量显著低于其他2组,而其他2组间无显著差异。SREBPFASME1及ACC参与调控FFA的从头合成,因此低蛋白质水平饲粮通过降低FFA的从头合成,影响肝脏FFA的合成代谢[33, 40],而中蛋白质饲粮与高蛋白质饲粮无显著差异,说明适当降低饲粮蛋白质水平不影响肝脏脂肪酸合成相关基因的表达量。

PPARγ调控脂肪细胞分化、转运及脂肪沉积相关蛋白基因表达来调节脂肪代谢,而FABP通过细胞膜结构促进脂肪酸和其他脂类介质的转运[41-43],也可促进脂肪酸及亲脂类分子通过细胞质到核受体的细胞外转运[44-45]。本试验结果显示,PPARγFABP等参与脂肪酸转运基因的表达量变化与SREBPFASME1、ACC等脂肪酸合成基因相反,LCP组PPARγFABP的基因表达量显著高于其他2组,说明过度的降低饲粮蛋白质水平通过上调转录因子PPARγ的基因表达量、增加FABP的基因表达量,从而促进肝脏脂肪酸的转运[46]。以上结果说明,低蛋白质水平饲粮可降低肝脏脂肪酸合成,增加脂肪酸的转运;但饲喂中蛋白质水平饲粮与高蛋白质水平饲粮的猪肝脏中脂肪酸合成及转运基因表达量无显著差异,表明适当降低饲粮蛋白质水平并不影响猪肝脏中脂肪酸合成及转运过程。

PPARα通过调控脂类代谢基因的表达来调节线粒体及过氧化物酶体脂肪酸氧化、生酮及糖异生,影响脂肪代谢率[47-48],肝脏中PPARαCPT在HCP组的基因表达量显著高于LCP及MCP组,说明降低蛋白质水平饲粮不利于肝脏中脂肪酸的氧化分解及代谢。

因此,适当降低饲粮蛋白质水平不影响肝脏中脂肪酸合成及转运相关基因的表达,同时降低了肝脏内脂肪酸的氧化分解,说明适当降低饲粮蛋白质水平促进了肝脏脂肪酸合成代谢并降低其分解代谢,因此增加脂肪酸从肝脏向身体各组织的运输。

3.3 饲粮蛋白质水平对LDM脂肪代谢的影响

IMF影响肉的口感、肉色、风味等感官质量性状。肌肉IMF的含量与肌肉脂肪代谢密切相关,IMF含量取决于脂肪酸合成、分解、转运及沉积等[49]。因此研究肌肉脂肪代谢为改善猪肉品质奠定了理论依据。本试验结果显示,MCP组LDM SREBPACCFASSCDFABP的基因表达量最高,其中SREBPFASSCD的基因表达量显著高于LCP及HCP组,ACCFABP的基因表达量显著高于LCP组而与HCP组无显著差异,以上结果表明适当降低饲粮蛋白质水平促进LDM中脂肪酸合成及转运。LDM中ME1在HCP组的基因表达量显著高于LCP组,而其在MCP组表达量与LCP及HCP组无显著差异,说明低蛋白质水平饲粮不仅降低肝脏中脂肪酸的从头合成,同时也降低LDM中脂肪酸的从头合成,而中蛋白质饲粮对LDM中脂肪酸的从头合成无显著影响。有研究指出,生长猪及肥育猪阶段降低饲粮蛋白质水平可促进ACCFASME1等脂肪酸合成相关基因的表达[32, 40],说明长期过度降低饲粮蛋白质水平不利于肌肉中脂肪酸合成。Doran等[50]指出低蛋白质水平饲粮诱导肌肉中SCD基因的表达,从而促进肌肉中IMF的沉积。而本试验结果显示,适当的降低饲粮蛋白质水平虽然诱导LDM中SCD等脂肪酸合成基因的表达,同时也降低IMF的含量。

Teye等[51]指出低蛋白质饲粮可增加LDM的嫩度、多汁性及IMF等肉品质,同时低蛋白质饲粮增加脂肪酸的氧化分解。而本试验获得相反的结果,降低饲粮蛋白质水平不影响猪肝脏及肌肉组织中脂肪酸氧化分解相关基因ATGLDECR的表达,但降低LDM中CPT的基因表达量, 说明低蛋白质饲粮是通过降低了长链脂肪酸的氧化分解,而不是影响肝脏及肌肉组织中甘油三酯水解及不饱和脂肪酸氧化分解来降低肌肉内脂肪酸的氧化分解。而肌肉中PPARα基因表达量在3个蛋白质水平饲粮组中并没有表现出显著性差异,PPARγFABP的表达趋势不一致,说明PPAR在调控脂肪酸氧化分解、转运具有组织特异性,存在其他的通路与PPAR共同调控肌肉中脂肪酸的代谢。

Zhao等[49]指出IMF的含量取决于脂肪酸合成、转运及分解速度。本试验结果显示,MCP组LDM中脂肪酸合成相关基因SREBPACCFASSCD基因表达量高于其他2组,脂肪酸转运蛋白FABP的基因表达量也高于HCP组,同时脂肪酸氧化分解基因ATGLDECR基因表达量没有显著变化,脂肪酸氧化分解相关基因CPT的基因表达量也显著低于HCP组,但是IMF的含量却显著低于HCP组。降低饲粮蛋白质水平可降低肝脏中ANK1的基因表达量,而肌肉中ANK1的基因表达量变化则相反。锚蛋白(ANK)是重要的结构蛋白质家族基因,其中ANK1是与肉品质相关的功能性候选基因,尤其是IMF及系水力的影响[52-53]。降低饲粮蛋白质水平可增加肌肉中ANK1的基因表达量,可能是通过增加肌肉内细胞骨架蛋白质水解及重排降低IMF重要组成脂肪小球的聚集,从而降低了LDM的IMF含量,这也说明ANK1可能是决定IMF沉积的决定因素。上述研究结果提示,IMF的沉积可能不仅由脂肪酸合成、转运以及分解决定。研究指出,脂肪的沉积可能与三大营养物质蛋白质、糖类、脂类代谢过程紧密相关[54-56]。Hamill等[56]提出在IMF含量高的肌肉组织中,许多脂肪酸氧化分解途径相关基因表达下调,高IMF含量抑制肌肉中脂肪酸周转,从而影响肌肉中脂肪的沉积。

饲粮蛋白质水平降低影响仔猪及生长猪生长性能,即使在长期饲喂MCP饲粮并没有影响肥育猪的生长性能,但降低肥育猪的LDM中IMF含量。猪生长初期,尤其是断奶仔猪不适合降低饲粮蛋白质水平,断奶初期的饲喂低蛋白质水平饲粮也可能导致后期肥育猪肉品质下降。Gondret等[57-58]也提出长期降低饲粮蛋白质水平可导致IMF含量的降低,同时也指出IMF的聚集取决于脂肪细胞中脂肪酸合成与肌纤维细胞中脂肪酸氧化分解的平衡,也就是脂肪酸的周转。因此,长期持续适当降低饲粮蛋白质水平能够提高LDM中脂肪酸的合成、转运,并降低其分解,但没有提高LDM的IMF含量,说明提高IMF不只是与脂肪代谢相关,同时与其他营养物质代谢紧密相关。即使中蛋白质水平饲粮增加脂肪酸合成,由于从仔猪到生长肥育猪全程阶段降低蛋白质水平可能影响蛋白质、脂肪酸合成代谢,不能满足机体对蛋白质、脂肪酸的需求,导致脂肪酸及糖类物质向氨基酸的转换,从而影响了肌肉中脂肪的沉积。在肥育猪阶段合理降低饲粮蛋白质水平,不影响其生长性能前提下,可通过调节饲粮营养成分及比例,如添加某种氨基酸等方法增加IMF含量,提高肉品质。因此要通过降低饲粮蛋白质水平调节猪肉品质,除了调节饲粮中粗蛋白质水平还要考虑到降低蛋白质水平的幅度、添加氨基酸的种类、时间点的选择及期限,从而达到既能降低氮排放、饲养成本,又能提高猪肉品质的效果。

4 结论

① 适当降低饲粮蛋白质水平(6%)对肝脏中脂肪酸合成相关基因的表达无显著影响。

② 从仔猪到肥育猪阶段全程降低饲粮蛋白质水平可促进LDM中脂肪酸合成、转运相关基因的表达,但显著降低猪LDM中IMF的沉积,不利于肉品质的改善。

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