【作者】 罗杰；

【导师】 彭健；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 本试验的目的是研究在妊娠末期-泌乳期母猪日粮以及保育期仔猪日粮中添加长链n-3 PUFA对仔猪免疫成熟和肌肉生长的调控及其与仔猪生长的关系。选择18头妊娠末期(103d)长×大健康经产母猪,按胎次、体况和预产期相近的原则随机分成2组：对照组(C)(n=10),饲喂含7%猪油的日粮(下简称母猪对照组)；处理组(T)(n=8),饲喂含7%鱼油的日粮(下简称母猪处理组)。仔猪28d断奶。断奶后,来自同组母猪的仔猪分别合群,并以体重相近的原则分别选取28头仔猪(14头阉公猪和14头小母猪)随机分为两组,分别饲喂含7%猪油或鱼油的日粮。形成四个不同的处理组：CC、CT、TC和TT。每组14头仔猪,分为7个重复,每个重复2头仔猪(1头阉公猪和1头小母猪)。断奶仔猪试验期为35-70d。母猪分娩后21d采集乳样。在试验末,每组随机选取4头仔猪(2头阉公猪和2头小母猪)共16头仔猪屠宰。在仔猪屠宰时分别剥离了臂三头肌、臂二头肌、背阔肌、胸深肌、冈上肌、冈下肌、肩胛下肌、斜方肌、背最长肌、腰小肌、股二头肌、股四头肌、半膜肌、内收肌、臀中肌、腓肠肌和半腱肌共17块肌肉,分别称量肌肉块重量,同时采集脾脏、背最长肌、股二头肌和臂三头肌组织样品。仔猪于屠宰当天采集血样并分离血清。开展以下研究：第一部分：日粮中长链n-3 PUFA对仔猪生长性能、肌肉中脂肪酸组成和肌肉块重量的影响。采用气相色谱法分析饲料、猪乳以及背最长肌、股二头肌、臂三头肌中脂肪酸的组成；分析仔猪在哺乳期和保育期的生长性能；主要结果如下：添加7%猪油的日粮中脂肪酸主要以C16:0、C18:0、C18:1n-9和C18：2n-6为主,而添加7%鱼油的日粮中C16:1、C20:5n-3、C22:5n-3、C22:6n-3显著上升,其中n-3 PUFA含量是猪油日粮的8-9倍。饲喂猪油或鱼油日粮后猪乳中的乳脂率没有差异(P>0.05),但乳中脂肪酸组成差异显著。母猪处理组猪乳中C14:0、C16:1、C20:5n-3.C22:5n-3、C22:6n-3、n-3 PUFA和PUFA的含量极显著高于(P<0.01),但C18:1n-9、C18:2n-6、C20:4n-6、MUFA和n-6 PUFA的含量极显著低于(P<0.01)母猪对照组的猪乳。与添加7%的猪油相比,妊娠末期-泌乳期母猪日粮添加7%的鱼油显著提高了哺乳期仔猪ADG和保育期仔猪ADFI(P<0.05)、并有提高保育期仔猪ADG和仔猪试验末体重的趋势(P<0.10)。但是,在保育期仔猪日粮中添加7%鱼油却显著降低了保育期仔猪ADG(P<0.05)和仔猪试验末体重(P<0.05),并有降低FCR(P<0.10)的趋势。鱼油日粮饲喂母猪所产的仔猪腰肌重显著高于猪油日粮饲喂母猪所产的仔猪(P<0.01)。但保育期仔猪日粮添加7%的鱼油显著提高了试验期末背最长肌重(P<0.05),并有提高斜方肌(P<0.10)、股二头肌(P<0.10)和半膜肌重(P<0.10)的趋势。饲喂鱼油日粮显著提高了背最长肌、股二头肌和臂三头肌中n-3 PUFA的含量(P<0.01)。但不同的肌肉中n-3 PUFA的富集量不同,背最长肌中n-3 PUFA的富集量最高,臂三头肌中n-3 PUFA的富集量最低。这些结果表明,妊娠末期-泌乳期母猪日粮中添加7%的鱼油有利于所产仔猪的生长,而在断奶后仔猪日粮中添加7%鱼油对仔猪生长有不利影响；日粮中的长链n-3 PUFA能够提高试验末仔猪胴体中部分肌肉块的重量,并且长链n-3 PUFA对肌肉重量的影响与肌肉中的n-3 PUFA富集量有关。第二部分：研究日粮中长链n-3 PUFA对仔猪免疫成熟的调控。使用ELISA法分析试验末仔猪血清中干扰素γ(interferonγ,IFN-γ)和白介素10(interleukin 10,IL-10)的水平,并用RT-PCR法检测脾脏中IL-2、IL-12b、IL-12受体β2亚基(IL-12receptorβ2 subunit, IL-12Rβ2)、IFN-γ、GATA结合转录因子-3(GATA-binding transcription factor 3,GATA-3)、IL-4和IL-10 mRNA的表达丰度。结果如下：与添加7%的猪油相比,保育期仔猪日粮添加7%的鱼油有降低仔猪血清中IFN-γ/IL-10比值的趋势(P<0.10)。日粮中的长链n-3 PUFA显著或极显著提高了仔猪脾脏中IL-2(P<0.01)、IL-12b(P<0.05)和IL-12RP2(P<0.01) mRNA的表达丰度。母猪处理组仔猪脾脏中IFN-y mRNA的表达极显著上升(P<0.01),IL-4mRNA的表达极显著下降(P<0.01)。保育期仔猪日粮中添加7%的鱼油则显著提高了IL-4 mRNA的表达(P<0.05)。上述结果表明,日粮中的长链n-3 PUFA显著提高了仔猪脾脏中调控T淋巴细胞增殖的IL-2 mRNA的表达,提高了Thl特征基因的表达及Thl极化,促进仔猪的免疫成熟。第三部分：研究日粮中长链n-3 PUFA对仔猪体内促炎细胞因子表达的影响及其与生长的关系。使用RT-PCR法检测仔猪背最长肌和脾脏中IL-1β、IL-6和肿瘤坏死因子α(tumor necrosis factora,TNF-α),背最长肌中胰岛素样生长因子(insulin-like growth factor, IGF-I)、Ⅰ型IGF-Ⅰ受体(type I IGF-I receptor, IGFIR) mRNA的表达丰度。结果如下：母猪处理组仔猪背最长肌中IL-6和TNF-a mRNA的表达量显著降低(P<0.05),但背最长肌中IGF-I mRNA的表达以及脾脏中TNF-a mRNA的表达显著上升(P<0.05)。保育期仔猪日粮中添加7%的鱼油极显著提高了仔猪脾脏中促炎细胞因子(IL-1β、IL-6和TNF-α)mRNA的表达(P<0.01),但极显著降低了背最长肌中IGFIR mRNA的表达(P<0.01)。单因子相关分析显示,脾脏中IL-6 mRNA的表达与背最长肌中IGFIR mRNA的表达呈显著的负相关(r=-0.6580，P<0.05),背最长肌中TNF-a mRNA的表达与仔猪的ADG之间呈显著负相关(r=-0.6224,P<0.05)。上述结果表明,通过在妊娠末期-泌乳期母猪日粮中添加7%鱼油降低了仔猪肌肉中促炎细胞因子：mRNA的表达,减少了对肌肉IGF-I的抑制效应,促进了仔猪生长；而保育期仔猪日粮直接添加7%的鱼油则显著提高了脾脏中促炎细胞因子mRNA的表达,拮抗肌肉中IGF-I的功能,抑制仔猪生长。第四部分：日粮中长链n-3 PUFA对仔猪肌肉中MyHCs和MRFs mRNA表达及肌肉生长的影响。采用多重PCR法分析背最长肌、股二头肌和臂三头肌中MyHCs各亚型mRNA的比率,并用RT-PCR法分析背最长肌、股二头肌和臂三头肌中MRFs mRNA的表达丰度。结果如下：臂三头肌中MyHCs亚型以MyHC-Ⅰ和-Ⅱa为主,MyHC-Ⅱb比率较低,而背最长肌和股二头肌中各MyHCs亚型比率基本相似。母猪处理组仔猪背最长肌中MyHC-Ⅱx mRNA的比率极显著上升(P<0.01),MyHC-Ⅱa mRNA的比率显著降低(P<0.05)。保育期饲喂含7%鱼油日粮的仔猪股二头肌中的MyHC-Ⅱb mRNA的比率显著高于饲喂含7%猪油日粮的仔猪(P<0.05)。与饲喂7%的猪油日粮相比,保育期仔猪饲喂7%的鱼油日粮有提高背最长肌中MyoD mRNA表达的趋势(P=0.06)。以上试验结果表明,补饲长链n-3 PUFA能通过提高肌肉中MyoD基因的表达,促进肌肉中卫星细胞的增殖与分化并提高肌肉中酵解型肌纤维类型的比率,加快仔猪肌肉的生长。结论：1、长链n-3 PUFA的摄入,促进了仔猪的免疫成熟。妊娠末期-泌乳期通过母猪日粮添加7%鱼油经猪乳所提供的n-3 PUFA,降低了仔猪背最长肌中IL-6和TNF-αmRNA的表达,提高了仔猪背最长肌中IGF-ⅠmRNA的表达,有利于仔猪的生长：而在保育期日粮中直接添加7%的鱼油所提供的n-3 PUFA则促进仔猪脾脏中促炎细胞因子(IL-1β、IL-6和TNF-α)mRNA的过量表达,降低了背最长肌中IGFIR mRNA的表达,不利于仔猪的生长。2、日粮中补饲长链n-3 PUFA能通过在部分肌肉中富集一定含量的n-3 PUFA,提高了其中酵解型MyHC亚型mRNA的比率和MyoD mRNA的表达,提高了这些肌肉的生长潜力,加快了肌肉的生长。不同阶段日粮中的长链n-3 PUFA对仔猪肌肉生长的影响与仔猪整体生长的变化并不一致,其原因可能与不同阶段鱼油的饲喂在不同组织中富集的n-3 PUFA量不同有关。更多还原

【Abstract】 The aim of this study was to investigate the effect of long chain n-3 polyunsaturated fatty acids (PUFA) on immune maturation and muscle growth of piglets, and its potential mechanism involved in n-3 PUFA regulating piglets’growth. The diets of 18 gestating sows were supplemented with 7% lard (C) (n=10) or 7% fish oil (T) (n=8) from 10 d before parturition to weaning on d 28.At farrowing, litters were equalized within dietary treatments to the same number of piglets per litter(-10).At weaning,56 piglets,28 (half female and half castrated male) each from sows fed with fish oil diet or lard diet, were subdivided into four groups of 7 replicates (one female and one castrated male per replicate) based on both sow diet and post-weaning piglet diet (C had 7% lard and T had 7% fish oil):CC, CT, TC, TT, and were fed with the 7% fish oil or lard diet from d 35 to d 70. Milk samples were collected from lactating sows at 21d after farrowing. At d 70, four piglets per group (2 female and 2 castrated male) were sacrificed to collect spleen, longissimus dorsi muscle, biceps femoris muscle and triceps brachii muscle samples. Muscles of triceps brachii, latissimus dorsi, pectoralis profundus, biceps brachii, supraspinatus, infraspinatus, subscapularis, trapezius, longissimus dorsi, posas minor, biceps femoris, quadriceps femoris, adductor, gluteus medius, gastrocnemius, semitendinosus and semimembranosus were separated and weighted. Blood samples of piglets were collected and separated for sera at the end of the experiment.The first part:to investigate the effect of fish oil or lard supplementation to late gestation-lactating sows’and/or weaned piglets’diets on the growth performance, muscle mass of piglet and fatty acids composition in piglet muscle. The fatty acids composition of diets, milk, longissimus dorsi muscle, biceps femoris muscle and triceps brachii muscle were determined by gas chromatography method and growth performance of piglets were measured. C16:0, C18:0, C18:1n-9 and C18:2n-6 composed of the main content of fatty acids in the lard diets, while C16:1,C20:5n-3,C22:5n-3 and C22:6n-3 composed of the main content of fatty acids in the fish oil diets. The n-3 PUFA contents in fish oil diets were 8-9 folds higher than that in lard diets. The lipid contents in milk were not differ (P>0.05).However, C14:0, C16:1,C20:5n-3,C22:5n-3,C22:6n-3,n-3 PUFA and PUFA were extremely higher, while C18:1n-9, C18:2n-6, C20:4n-6, MUFA and n-6 PUFA were extremely lower in milk from fish oil diet fed sows than that from lard diet fed sows (P<0.01).In the lactation period, suckling piglets from fish oil diet fed sows grew more fast (P<0.05) than the controls, and in the post-weaning period these piglets also had higher average daily gain (ADG) (P<0.10), feed intake (P<0.05), and final body weight (P<0.10) than piglets from lard diet fed sows. However, piglets received fish oil diet had lower ADG (P<0.05), lower final body weight (P<0.05), and higher feed:gain ratio (P<0.10) than piglets received lard diet. Late gestation-lactating fish oil supplementation to sows’diet increased the muscle mass of posas minor (P<0.05) in piglets at the end of this experiment. However, longissimus dorsi muscle mass (P<0.05), trapezius muscle mass (P<0.10), biceps femoris muscle mass (P<0.10) and semimembranosus muscle mass (P<0.10) were higher in piglets fed with fish oil diet during post-weaning than that in lard diet fed piglets. Dietary fish oil significantly increased n-3 PUFA content in longissimus dorsi muscle, biceps femoris muscle and triceps brachii muscle. However, the n-3 PUFA content was higher in longissimus dorsi muscle than that in triceps brachii muscle after fish oil feeding. The results suggested that 7% fish oil addition to late gestation-lactating sows’diet was favorable to the growth of piglets. However, it was likely to have negative effect when 7% fish oil was supplemented to piglets’diet during post-weaning period. Dietary n-3 PUFA could increase masses of several muscles in piglet at the end of the experiment. And the effect of n-3 PUFA on muscles’ growth was dependent to the enrichment of n-3 PUFA in these muscles.The second part:to investigate the effect of dietary long chain n-3 PUFA on immune maturation. ELISA assay method was employeed to determine the interferon y (IFN-y) and interleukin 10 (IL-10) concentrations in sera and the genes expression of IL-2, IL-12b, IL-12 receptor O2 subunit (IL-12Rβ2), IFN-y, GATA-binding transcription factor 3 (GATA-3), IL-4 and IL-10 in spleen were determined by the method of relative quantitative RT-PCR. IFN-γ/IL-10 ratio of serum was tended to increase in piglets fed with fish oil diet during post-weaning. Dietary long chain n-3 PUFA supplementation increased splenic genes expression of IL-2 (P<0.01),IL-12b (P<0.05) and IL-12Rβ2 (P<0.01)in piglets.IFN-γgene expression was increased (P<0.01),while IL-4 gene expression was decreased (P<0.05) in spleen of piglets fed with fish oil diet in post-weaning period compared with that fed with lard diet. Splenic IL-4 gene expression in piglets from fish oil diet fed sows was higher (P<0.01)than that from lard diet fed sows. The results suggested that long chain n-3 PUFA could increase the mRNA expression of IL-2 and Thl genes in spleen, promote lymphocytes proliferation, Thl polarization and piglets’immune maturation.The third part:to investigate the effect of dietary long chain n-3 PUFA on pro-inflammatory cytokines gene expression and growth of piglets. IL-1β, IL-6 and tumor necrosis factora(TNF-α) expression in longissimus dorsi muscle and spleen and insulin-like growth factor (IGF-Ⅰ) and typeⅠIGF-Ⅰreceptor (IGFIR) expression in longissimus dorsi muscle of piglets were measured by the method of relative quantitative RT-PCR. Piglets from fish oil fed sows had lower genes expression of IL-6 (P<0.05) and TNF-a (P<0.01) but higher gene expression of IGF-I (P<0.05) in longissimus dorsi muscle and had higher TNF-a gene expressio (P<0.05) in spleen than piglets from lard diet fed sows.However, piglets fed with fish oil diet during post-weaning had higher splenic expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-a) (P<0.01)and lower expression of IGFIR (P<0.01) in the longissimus dorsi muscle than piglets fed with lard diet. Correlation analysis revealed that splenic IL-6 expression was negatively correlated with longissimus dorsi muscle IGFIR expression (r=-0.6580, P<0.05) and ADG was negatively correlated with longissimus dorsi muscle TNF-a expression (r=-0.6224, P<0.05).The results suggested that supplementation of 7% fish oil to sows’ diet during late gestation-lactating period down-regulated the genes expression of pro-inflammatory cytokines and attenuated the suppression of IGF-Ⅰgene expression in longissimus dorsi muscle of their progenies, promoted piglet growth. However,7% fish oil supplemented to piglets’diet during post-weaning period increased splenic genes expression of pro-inflammatory cytokines, decreased IGFIR gene expression in longissimus dorsi muscle, and suppressed piglest growth.The forth part:to investigate the effect of long chain n-3 PUFA on myosin heavey chain isotypes (MyHCs) and myogenic regulatory factors (MRFs) mRNA expression in muscle, and muscle growth of piglets. Multi-PCR was used to determine the ratio of MyHCs mRNA and relative quantitative RT-PCR was used to measure the genes expression of MRFs in longissimus dorsi muscle, biceps femoris muscle and triceps brachii muscle. The MyHC-Ⅰand-Ⅱa were the primary MyHCs in triceps brachii muscle. However, longissimus dorsi muscle and biceps femoris have equal ratios of MyHCs.Fish oil supplemented to sows’dets increased MyHC-Ⅱx mRNA ratio (P<0.01) but decreased MyHC-Ⅱa mRNA ratio (P<0.05) in their progenies’longissimus dorsi muscle at the end of this experiment. The MyHC-IIb mRNA ratio in biceps femoris muscle was also increased by post-weaning fish oil diet feeding in piglets (P<0.05).There was a tendency towards to increase the gene expression of MyoD (P<0.10) in longissimus dorsi muscle of piglets fed with fish oil diet after weaning compared with that fed with lard diet. The results indicated that dietary long chain n-3 PUFA could increase muscle growth via modulating MyoD gene expression, promoting satellite cells proliferation and differetiation and increase the ratio of glycolytic muscle fibre type.Conclusion:1.Dietary long chain n-3 PUFA could promote piglets’immune maturation. Seven percent of fish oil supplemented to sows’diets can decrease genes expression of pro-inflammatory cytokines and their unfavorable effects on IGF-I in longissimus dorsi muscle of their progenies, and in turn promote piglet growth. However, high levels of long chain n-3 PUFA supplementation to post-weaning piglets’diet may increase splenic genes expression of pro-inflammatory cytokines, holdback IGF-I function, and then negatively impact the growth of weaned piglets.2.The long chain n-3 PUFA in diets could deposit in skeletal muscles, increase the gene expression of MyoD and glycolytic muscle fibre type, promote postnatal muscle growth in piglets. However, the long chain n-3 PUFA supplementation in piglets’diets had different effects on muscle growth and growth performance of piglets and it was due to the different amounts of n-3 PUFA deposited in different tissues.更多还原