摘要
本课题以N-甲基-D-天冬氨酸(NMDA)为试验材料，以“杜洛克×长白×大白”肥育猪为试验对象，研究普通型、吸附型(纳米硅酸盐为载体)和钠米型(纳米级)NMDA对猪生长性能、胴体组成和内分泌水平的影响，探讨了NMDA对猪生长激素和生长抑素动态分泌模式、腺垂体生长激素(GH)mRNA丰度和下丘脑生长激素释放激素(GHRH)mRNA丰度的影响，并通过体外细胞培养试验，研究NMDA对腺垂体生长激素和下丘脑细胞信使物质分泌的影响。饲养试验中每种形式NMDA均以50，100，150mg／kg剂量添加于基础日粮。选择300头体重接近的肥育猪，随机分为10组，每组3个重复，每个重复10头，公母各半。其中一组设为对照，其余分别饲喂上述相应试验日粮，饲养试验为期6周。饲养试验结束前，从对照组及生长最佳组分别选取试验猪6头，喂食相应试验日粮，1h后，分别耳静脉采血，试验于13∶00至16∶00，15min间隔采样一次，并分别制备血清进行生长激素及生长抑素动态分泌模式研究；饲养试验结束后，分别从对照及不同形式NMDA处理生长最佳组中各选取体重接近的肥育猪(公母各半)12头，共48头，屠宰，进行胴体分割，并取血清及组织样品进行内分泌、腺垂体GH mRNA、下丘脑GHRH mRNA丰度及相关代谢指标分析。体外腺垂体和下丘脑细胞培养中NMDA设置10~(-8)，10~(-6)，10~(-4)mol／L 3个浓度梯度，每个浓度设6个重复，并设一个空白浓度，分别测定生长激素及细胞信使物质水平。试验获得以下主要结果：
1、饲养试验表明，各形式NMDA均不同程度改善了肥育猪日增重，并且在不同形式NMDA的三个剂量设置中，均呈现100mg／kg剂量效果优于同形式其他剂量组，但仅100mg／kg纳米型NMDA促生长达到显著水平，较对照组比较提高了9.15％(P＜0.05)。料重比的变化呈现与日增重变化相一致的趋势，100和150mg／kgNMDA均显著改善了料重比，分别较对照组降低了2.45％(P＜0.05)和2.75％(P＜0.01)。NMDA处理对猪采食量未产生显著影响。从试验可以看出，各剂量纳米NMDA的促生长效果优于普通型和吸附型NMDA。
2、屠宰试验显示，各形式NMDA改善了肥育猪胴体组成，并均以纳米型效果最为明显。与对照组比较，100mg／kg纳米NMDA处理组胴体瘦肉率提高了5.81％(P＜0.05)，脂肪率降低了18.93％(P＜0.05)，眼肌面积增加了8.78％(P＜0.05)。此外，100mg／kg吸附型NMDA亦显著提高了猪只胴体瘦肉率，较对照提高了
浙江大学博士学位论文:纳米型NMDA对肥育猪生长和胭体组成的影响及NMDA调控生长机理研究
4.81%(P<0 .05)。从背部不同位点背膘厚度可以看出，普通型和纳米型NMDA显
著降低了髻甲部背膘厚，较对照组分别下降了13.97%(尸<0 .05)和12.70%(尸<0 .05);
吸附型和纳米型NMDA显著降低了最后肋骨处背膘厚，分别较对照组降低了
26.20%(尸<0.05)和21.39%(尸<0.05);骨骼肌组成分析表明，在背最长肌和股二
头肌增重效果上，以100m妙g纳米型NMDA为最佳，分别较对照组提高了8.33%
(P<0.05)和10.53%(尸<0.05);而半健肌增重以100m留kg吸附型NMDA效果
最好，较对照组增加了17.50%(P<0.05)，纳米型组增加了12.50%(P>0.OS)。
3、生长激素和生长抑素动态分泌模式分析表明，NMDA处理显著提高了试验
猪只生长激素分泌的总体水平、基线水平和峰强度，分别较对照猪只提高了41.01%
(P<0.05)，44.70%(P<0.05)和29.44%(P< 0.05)，而NMDA对猪只生长激素分
泌频率和峰持续时间未产生显著影响。从性别角度而言，阉公猪生长激素分泌总体
水平增加36.91%(P<0.05)，基线水平提高了55.69%(尸<0.05)，而峰频率、峰持
续时间和峰强度无显著变化，母猪生长激素分泌总体水平提高了45.95%(尸<0.05)，
基线水平提高了50.10%(尸<0.05)，与阉公猪一致，峰频率、峰持续时间和峰强度
无显著变化。NMDA虽提高了阉公猪与母猪生长抑素的分泌水平，但差异不显著
(尸)0 .05)。
4、血清激素指标分析显示，各形式NMDA处理后，试验猪只血清胰岛素样
生长因子一I水平均较对照组有一定的提高，以100mg/k纳米型NMDA效果最为显
著，提高了21.91%(尸<0.05)。吸附型和纳米型NMDA均显著提高血清三碘甲腺
原氨酸(T3)和四碘甲腺原氨酸水平(T4)，其中T3较对照组分别提高了51.79%
(P<0.05)和60.71%(P<0.05)，T4分别提高了200.75%(P<0.05)和236.48%
(尸<0.05)。胰岛素略有增加，但差异不显著。
5、研究表明，吸附型和纳米型NMDA均显著降低了试验猪下丘脑cAMP水
平，分别较对照降低了10.12%(尸<0.01)和12‘73%(P<0.01)，而cGMP呈现增
加趋势。纳米型NMDA还显著提高了下丘脑一氧化氮合酶的活性。吸附型和纳米
型NMDA亦显著增加了腺垂体cAMP浓度，分别较对照组提高了23 .56%(P<0.05)
和52.67%(尸<0.05)。此外，纳米NMDA显著提高了肝脏cAMP水平，较对照组
增加了7.75%(p<0.05)。各试验组下丘脑天冬氨酸(ASP)含量较对照组均有提
高，但仅IOOm留kg纳米型NMDA组达显著水平，较对照组提高了14.56%(尸<0 .05)，
浙江大学博士学位论文:纳米型NMDA对肥育猪生长和胭体组成的影响及NMDA调控生长机理研究
腺垂体中ASP含量无显著差异。
6、血清相关代谢指标分析显示，不同形式NMDA对肥育猪血清
Different type NMDA were used to study the effect of NMDA ordinary NMDA(O-NMDA), adsorbed NMDA(A-NMDA) and nano NMDA(N-NMDA)on growth performance, carcass characteristics, incretion and approach to mechanism of NMDA affect growth by determining dynamic secretion model of growth hormone(GH) and somatostatin(SS) , as well as its influence to GH mRNA abundance in pituitary gland and growth hormone releasing hormone(GHRH) mRNA abundance in hypothalamus. GH secretion and cell messages were also investigated in vitro by pituitary and hypothalamus cell cultivation. 300 finishing pigs (DurocxLandracexYorkshire) which weighed about 52.5kg were selected in feeding experiment. Pigs were allotted to ten groups by weight and sex, each of which included 3 replicates of ten pigs. The group received the same corn- soybean based diet, supplemented with 0 (control group) and 50, 100 and 150 mg/kg of each type NMDA. All pigs were given adequate diets (four times per day) and free access to water. Before finishing the feeding e
xperiment, six pigs were chose from control and best growth group respectively and blood sample of each pig was collected to study dynamic secretion model of growth hormone (GH) and somatostatin (SS). On finishing the feeding trial, 12 pigs were picked out by similar body weight from control group and best growth group in each type NMDA treatment respectively to be slaughtered at the local abattoir to determine carcass characteristics. The skeletal muscle (Longissimus dorsi, Semi-membranosus, Semitendinosus, Quadriceps femoris and Biceps femoris ) were separated from the carcass of the pigs to investigate the response of different muscles to NMDA. The sample of liver, hypothalamus, pituitary gland and serum were
collected for laboratory analysis. In vitro trial. 10-8, 10-6 and 10-4 mol/L NMDA were set as concentration grads in cell culture and each concentration includes 6 replicates. The main results are as follows.
1. The results of feeding trial showed that all types NMDA improved average daily gain(ADG) of pigs, but only ADG in 100 mg/kg N-NMDA group increased significantly compared with control group (p<0.05). 100 and 150 mg/kg NMDA improved feed gain ratio obviously (p<0.05). There were no difference in feed intake between all the treatments (p>0.05).
2. Carcass composition was apparently changed by adding NMDA. lOOmg/kg N-NMDA group showed significant results, percentage of dissected lean of carcass (PDL) was increased by 5.81%(p<0.05); percentage of dissected fat (PDF) was decreased by 18.93% (p<0.05), and longissiums dorsi muscle area was increased by 8.78% (p<0.01). Backfat depth analysis showed O-NMDA and N-NMDA decreased scapular site backfat depth, A-NMDA and N-NMDA decreased which of the last rib site. The results also showed N-NMDA increased longissimus dorsi and Biceps femoris weight by 26.20% (P<0.05) 21.39% (P<0.05 ) respectively, and A-NMDA enhanced semitendinosus weight significantly by 17.50% (P<0.05) compared with the control.
3. Study of dynamic secretion model of GH and SS indicated that NMDA increased whole level, basal line and peak intensity of GH secretion significantly by 41.01%(P<0.05), 44.70% (P<0.05) and 29.44%(P<0.05)respectively, and the similar effect were showed in gilts and barrows. There were no obvious effects on GH peak frequency and peak lasting time. NMDA also have no effect on SS secretion in present experiment.
4. Assay of serum sample indicated that N-NMDA enhanced IGF-I concentration by 21.91%(P<0.05), A-NMDA and N-NMDA increased T3 by 51.79% (P<0.05) and 60.71% (P<0.05), and improved T4 by 200.75% (P<0.05) and236.48% (P<0.05) respectively. Adding NMDA has no obvious effect on insulin concentration.
5. A-NMDA and N-NMDA decreased cAMP level in hypothalamus by 10.12% and 12.73% (P<0.01). N-NMDA also increased NOS activity significantly. A-NMDA
and N-NMDA enhanced cAMP level in pituitary by 23.56% and 52.67% (P<0.05) . cAMP in liver was increased 7.75% (P<0.05) by N-NMDA added compared with control group. Furthermore,
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