N-氨甲酰谷氨酸对妊娠期湖羊母羊血液生化和繁殖性能的影响
本研究旨在分析N-氨甲酰谷氨酸(NCG)对初产湖羊母羊妊娠期血液生化、生殖激素及产羔性状的影响，为提高湖羊母羊繁殖效率奠定基础。选择300只年龄(8月龄)和体重(31.81 ± 1.20 kg)相近、健康的湖羊初产母羊，随机分为4组，每组75只，对照组母羊饲喂基础日粮，试验组母羊饲喂在基础日粮中每只每天分别添加1.0 (I组)、1.5 (II组)和2.0 g (III组)的NCG。配种采用本交方式，公羊放母羊圈中20 d，公羊出圈1个月后，通过B超检测每组选择40只妊娠母羊继续试验。整个试验从母羊配种前1个月开始至所有母羊产羔完成后结束。分别在妊娠前期(公羊配种结束后28 d)和妊娠后期(预产期前15 d)采集母羊血液，通过对母羊血液生化、生殖激素和产羔性状的测定，分析NCG对初产湖羊母羊的繁殖性能影响。结果表明：1)妊娠前期，Ⅲ组母羊血浆孕酮极显著高于对照组(P < 0.01)。Ⅰ组母羊血浆雌二醇极显著高于对照组(P < 0.01)；Ⅰ组母羊血浆精氨酸显著高于对照组(P < 0.05)；Ⅲ组母羊血浆总一氧化氮合酶显著高于对照组(P < 0.05)；Ⅲ组母羊血浆内皮型一氧化氮合酶显著高于对照组(P < 0.05)。2)妊娠后期，Ⅱ组母羊血浆葡萄糖极显著高于对照组(P < 0.01)；Ⅰ组母羊血浆催乳素(PRL)显著高于对照组(P < 0.05)。3)与对照组相比，添加NCG的各试验组对初产湖羊的妊娠率、胎产羔数和胎产活羔数均有不同程度的提高(P>0.05)，以II组母羊最高，分别提高了5.13%、0.15只、0.18只。综上所述，初产湖羊日粮中添加NCG可能通过增加血浆精氨酸和NO的含量，提高了初产湖羊的妊娠率、胎产羔数和胎产活羔数等繁殖性能，适宜剂量为每只每天1.5 g。
English
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精氨酸(Arg)是一种多功能的半必需或条件性必需的碱性氨基酸，是合成NO和多胺的前体物[1]。NO在胎盘血管生成和生长中发挥重要作用[2-3]。饲料中添加精氨酸可改善哺乳动物胚胎的着床，提高胚胎存活率和产仔数[4]。然而，由于精氨酸半衰期短、代谢快、使用成本高，机体补充精氨酸存在一定的局限性。N-乙酰基谷氨酸(N- acetyl glutamate,NAG)是一种内源性氨基酸衍生物，通过激活肝细胞和肠细胞线粒体中尿素循环途径的关键酶——磷酸氨基甲酰合成酶-1 (carbamoyl phosphate synthetase I,CPS-1)，促进精氨酸的内源性合成[5]。N-氨基甲酰谷氨酸(NCG)是N-乙酰谷氨酸(NAG)的结构类似物，可以代替NAG参与到动物体内尿素循环过程中，促进精氨酸的内源性合成[6]。NCG已被证明能够降低宫内生长受限胎儿的发生率[7]，促进新生羔羊的生长[8]，减轻胎儿氧化应激，抑制肠道炎症[9]。此外，NCG还可以通过调节哺乳动物胎盘血管生成[10]和子宫内膜蛋白组成[11]，抑制胚胎滋养细胞分化[12]来调节胎盘功能，从而促进哺乳动物的胎盘发育。NCG剂量的确定是基于以往在山羊[13]和绵羊[14-15]上的试验确定的。然而，关于NCG对初产湖羊繁殖性能影响的报道较少，特别是引入榆林地区的湖羊，其产羔性能并未达到理想水平。因此，本试验旨在研究妊娠期湖羊母羊日粮添加不同水平NCG对母羊血液生化、繁殖相关激素及产羔性状的影响，为提高榆林地区的湖羊母羊繁殖性能提供基础。
1. 材料与方法
1.1 试验地点与NCG厂家
试验地点为陕西省榆林市榆阳区某湖羊场，NCG购自福建旭牧联生物科技有限公司，纯度≥80%。
1.2 试验设计
随机选择300只8月龄初产湖羊母羊，体重(31.81 ± 1.20 kg)相近、健康状况良好的母羊进行试验，将300只母羊随机分为4组，每组75只，每15只母羊为1个重复，每组共5个重复。对照组母羊饲喂基础日粮，试验组母羊在基础日粮的基础上每只每天分别添加1.0 (I组)、1.5 (II组)和2.0 g (III组)的NCG。基础饲粮参照肉羊营养需要量标准NY/T816—2021体重40 kg的母绵羊妊娠前期的营养需要量配制，NCG按照I组、II组和III组分别对应0.5% (1 t配合饲料中含5 kg的NCG)、0.75% (1 t配合饲料中含7.5 kg的NCG)、1% (1 t配合饲料中含10 kg的NCG)的比例添加到精补颗粒料中，空怀期和妊娠前期每天每只母羊补饲精补颗粒料200 g (试验组饲喂对应浓度的NCG精补料200 g，对照组饲喂不含NCG精补料200 g)，妊娠后期每天每只母羊补饲精补颗粒料500 g (试验组饲喂200 g含NCG的精补料 + 300 g不含NCG的精补料，对照组饲喂500 g不含NCG的精补料)，母羊空怀期和妊娠前期日粮精粗比为26:74，妊娠后期日粮精粗比为40:60。除补饲的精补颗粒料外，其余精饲料和粗饲料按照饲粮配方比例搅拌均匀制成全混合日粮(TMR)后饲喂动物。基础日粮组成和总营养水平见表1。配种以本交的方式进行，公羊放母羊圈中20 d，公羊出圈1个月后，通过B超检测每组选择40只妊娠母羊继续试验，共选用160只，每10只母羊为1个重复，每组共4个重复。整个试验从母羊配种前1个月开始至所有母羊产羔完成后结束。母羊采血按不同生理阶段分为妊娠前期(公羊配种结束后28 d)和妊娠后期(预产期前15 d) 两个阶段，共两次，每组每次采样12只，采血时间为上午8:00颈静脉空腹采血。
表 1 基础日粮组成及营养水平(干物质基础)Table 1. Composition and nutrient levels of basal diets (dry matter basis)项目
Item原料
Ingredient含量 Content 营养水平
Nutrient levels含量 Content 妊娠前期
Early pregnancy妊娠后期
Late pregnancy妊娠前期
Early pregnancy妊娠后期
Late pregnancy粗饲料
Roughage/%玉米青贮
Corn silage51.80 42.00 代谢能
Metabolizable energy2)/(MJ·kg−1)7.83 17.49 玉米秸秆
Corn stalk11.10 9.00 干物质
Dry matter/%51.38 52.46 花生秧
Peanut seedling11.10 9.00 粗蛋白质
Crude protein/%11.94 14.54 精补料
Refined feed/%玉米
Corn15.16 23.32 中性洗涤纤维
Neutral detergent fibre/%42.36 44.85 麸皮
Wheat bran4.08 6.28 酸性洗涤纤维
Acid detergent fibre/%26.06 27.16 豆粕
Soybean meal3.90 6.00 粗脂肪
Crude fat/%2.9 3.6 小苏打
NaHCO30.39 0.60 粗纤维
Coarse fibre/%18.72 20.03 石粉
Limestone0.13 0.20 粗灰分
Crude ash/%8.18 8.89 碳酸氢钙
CaHPO40.13 0.20 钙
Calcium/%0.55 0.6 食盐
NaCl0.26 0.40 磷
Phosphorus/%0.45 0.48 预混料
Premix1)1.82 2.80 脱霉剂
Mildew remover0.13 0.20 合计
Total100.00 100.00 1)预混料为每千克饲粮提供：VA, 288～ 1152 KIU、VD2, 28.8～115.2 KIU、VE,1152 IU、Fe 0.8 g、Cu 0.16 g、Mn 0.8 g、Zn 0.8 g、I 8.0 mg、Co 1.6 mg、Se 1.6 mg。2)代谢能为计算值，其余为实测值。
1)The premix provided the following per kg of diets: VA, 288～1152 KIU, VD2, 28.8～115.2 KIU, VE,1152 IU, Fe 0.8 g, Cu 0.16 g, Mn 0.8 g, Zn 0.8 g, I 8.0 mg, Co 1.6 mg, Se 1.6 mg. 2)ME is a calculated value, whereas the others parameter values are measured values.1.3 饲养管理
试验开始前对圈舍进行彻底消毒，对所有试验动物按照羊场常规免疫程序进行疫苗免疫和驱虫等工作。试验羊每天分别于09:00和16:30进行两次饲喂，专人饲喂。试验期所有供试羊只在羊舍内设有饮水装置，自由饮水。其他饲养管理与羊舍环境条件一致。保证舍内清洁、干燥、通风良好、温度适宜，并进行常规清洁和定时消毒。
1.4 指标测定及方法
1.4.1 血液生化指标测定
用装有肝素钠抗凝剂的采血器分别对妊娠前期(公羊配种结束后28 d)和妊娠后期(预产期前15 d)的母羊采血5 mL，将采集好的血样置于冰盒中运输到实验室，进行3 000 rpm·min−1离心15 min，取上清液分装于1.5 mL的离心管中−80 ℃保存待测。用紫外/可见光分光光度计UV-6300B (上海美谱达仪器有限公司)，分别按试剂盒说明，对血浆样品进行总蛋白(TP)、氨基酸(AA)、甘油三酯(TG)、葡萄糖(GLU)、总胆固醇(TC)、谷丙转氨酶(ALT)、尿素氮(BUN)、一氧化氮(NO)的测量，试剂盒均购自上海酶联生物有限责任公司。
1.4.2 血浆精氨酸检测
用安捷伦Agilent
1200 HPLC液相色谱仪对母羊血浆样品进行精氨酸的测定。测定使用的检测器：VWD，C18； 色谱柱：250 mm × 4.6 mm，0.5 μm。 柱温：40 ℃； 波长：360 nm；流动相l mol·L−1乙酸钠溶液(pH 5.3)，甲醇：水 =1 ꞉ 1 (V ꞉ V)等度洗脱；流速1 mL·min−1 ； 进样量20 μL。1.4.3 血浆激素和一氧化氮合酶水平检测
将冻存的血浆样品置于冰盒中保存送往上海酶联生物有限责任公司，采用双抗体夹心法，用酶标仪Rayto (RT6100，深圳雷杜生命科学股份有限公司)进行雌二醇(E2)、孕酮(P4)、催乳素(PRl)、胰岛素(INS)、生长激素(GH)、总一氧化氮合酶(TNOS)、诱导型一氧化氮合酶(iNOS)和内皮型一氧化氮合酶(eNOS)的检测。
1.4.4 繁殖性能
记录母羊妊娠数、胎产羔羊数、胎产活羔数、死羔数、羔羊初生重等指标。妊娠率=妊娠母羊个数/配种母羊个数 × 100。
1.5 数据分析
试验数据采用Excel 2010进行初步整理，采用SPSS 26.0软件进行单因子方差(one-way ANOVA)分析，并采用LSD法进行多重比较。妊娠率采用卡方检验进行分析。结果均以“平均值 ± 标准差”表示。
2. 结果
2.1 NCG对妊娠期湖羊母羊血浆生化指标的影响
妊娠前期Ⅲ组母羊血浆尿素氮(BUN)显著低于Ⅰ组(P < 0.05)，各试验组与对照组之间无显著差异(P > 0.05) (表2)。Ⅰ组母羊血浆葡萄糖(GLU)较对照组有下降趋势(0.05 < P < 0.1)。母羊血浆中的总氨基酸(AA)、谷丙转氨酶(ALT)、总蛋白(TP)、甘油三脂(TG)和总胆固醇(TC)各试验组与对照组，以及各试验组之间均无显著差异(P > 0.05)。
表 2 NCG对妊娠期湖羊母羊血浆生化指标的影响Table 2. Effects of NCG on plasma biochemical indices in female Hu sheep during pregnancy项目
Item组别 Group P 对照组
Control groupⅠ组
Group ⅠⅡ组
Group ⅡⅢ组
Group Ⅲ妊娠前期
Early
pregnancy氨基酸 AA/ (µg·mL−1) 171.72 ± 13.26 170.73 ± 10.60 170.48 ± 11.02 167.89 ± 11.16 0.996 尿素氮 BUN/ (mg·mL−1) 0.21 ± 0.01ab 0.23 ± 0.02a 0.20 ± 0.01ab 0.18 ± 0.02b 0.195 谷丙转氨酶 ALT/(nmol·mL−1) 37.33 ± 4.59 36.93 ± 4.46 35.00 ± 4.41 32.48 ± 4.15 0.858 总蛋白 TP/(mg·mL−1) 29.03 ± 2.15 26.60 ± 4.15 29.70 ± 2.98 24.20 ± 2.20 0.437 甘油三酯 TG/(mg·mL−1) 0.83 ± 0.05 0.85 ± 0.04 0.71 ± 0.03 0.75 ± 0.03 0.103 葡萄糖 GLU/(µmol·mL−1) 0.41 ± 0.02 0.36 ± 0.01 0.36 ± 0.02 0.38 ± 0.02 0.236 总胆固醇 TC/(µmol·dL−1) 496.09 ± 33.30 466.15 ± 21.55 510.42 ± 38.65 493.82 ± 35.23 0.812 妊娠后期
Late
pregnancy氨基酸 AA/ (µg·mL−1) 208.63 ± 11.34 196.98 ± 9.37 215.92 ± 6.99 216.54 ± 12.54 0.557 尿素氮 BUN/ (mg·mL−1) 0.17 ± 0.01 0.21 ± 0.01 0.21 ± 0.01 0.15 ± 0.02 0.059 谷丙转氨酶 ALT/(nmol·mL−1) 37.64 ± 4.94 38.36 ± 2.96 43.48 ± 2.89 39.18 ± 3.69 0.692 总蛋白 TP/(mg·mL−1) 32.60 ± 2.04 28.40 ± 3.28 28.67 ± 2.58 28.43 ± 2.06 0.558 甘油三酯 TG/(mg·mL−1) 0.90 ± 0.06 0.91 ± 0.02 0.96 ± 0.05 0.96 ± 0.05 0.754 葡萄糖 GLU/(µmol·mL−1) 0.41 ± 0.01Bc 0.42 ± 0.02ABbc 0.49 ± 0.02Aa 0.47 ± 0.02ABab 0.124 总胆固醇 TC/(µmol·dL−1) 556.64 ± 22.08ab 546.88 ± 26.00b 619.47 ± 19.96a 558.92 ± 23.28ab 0.128 同行无字母或相同字母表示差异不显著(P > 0.05)，不同小写字母表示差异显著(P < 0.05)，不同大写字母表示差异极显著(P < 0.01)；Ⅰ组、Ⅱ组和Ⅲ组分别表示母羊在基础日粮的基础上每只每天分别添加1.0、1.5和2 g的N-氨甲酰谷氨酸。下表同。
In the same row, values with no letter or the same letter superscripts indicate no significant difference, while those with different small letter superscripts mean significant differences at the 0.05 level and those with different capital letter superscripts mean significant difference at the 0.01 level; Groups Ⅰ, II, and III indicate 1.0, 1.5, and 2 g of N-carbamylglutamic acid per ewe per day on the basis of the basal diet, respectively. This is applicable for the following tables as well. AA, amino acid; BUN, urea nitrogen; ALT, glutamic-pyruvic transaminase; TP, total protein; TG, triglyceride; GLU, glucose; TC, total cholesterol.妊娠后期Ⅱ组母羊血浆GLU极显著高于对照组(P < 0.01)，显著高于试验Ⅰ组(P < 0.05)；试验Ⅲ组母羊血浆GLU显著高于对照组(P < 0.05)，较试验Ⅰ组有上升趋势(0.05 < P < 0.1)。试验Ⅱ组母羊血浆TC显著高于试验Ⅰ组(P < 0.05)，各试验组与对照组之间无显著差异(P > 0.05)。母羊血浆中BUN、AA、ALT、TP和TG各试验组与对照组，以及各试验组之间均无显著性差异。
2.2 NCG对妊娠期湖羊母羊血浆Arg和NO以及NOS的影响
妊娠前期Ⅰ组母羊血浆精氨酸(Arg)显著高于对照组(P < 0.05)，较试验Ⅱ组和试验Ⅲ组有升高趋势(0.05 < P < 0.1) (表3)。试验Ⅲ组母羊血浆NO显著高于试验Ⅰ组(P < 0.05)。试验Ⅲ组母羊血浆总一氧化氮合酶(TNOS)显著高于试验Ⅰ组和对照组(P < 0.05)。试验Ⅲ组母羊血浆内皮型一氧化氮合酶(eNOS)显著高于试验Ⅰ组、试验Ⅱ组和对照组(P < 0.05)。试验Ⅲ组母羊血浆诱导型一氧化氮合酶(iNOS)较对照组有所提高，但无显著性差异。
表 3 NCG对妊娠期湖羊母羊血浆精氨酸和NO以及NOS的影响Table 3. Effects of NCG on plasma Arg and NO as well as NOS in pregnant female Hu Sheep项目
Item组别 Group P 对照组
Control groupⅠ组
Group ⅠⅡ组
Group ⅡⅢ组
Group Ⅲ妊娠前期
Early pregnancy精氨酸 Arg/(µg·mL−1) 12.23 ± 0.52b 14.94 ± 0.58a 12.79 ± 0.51ab 13.00 ± 1.14ab 0.128 一氧化氮 NO/(µmoL·L−1) 78.20 ± 4.97ab 72.12 ± 4.91b 79.78 ± 2.98ab 85.95 ± 3.34a 0.166 总一氧化氮合酶 TNOS/(ng·mL−1) 32.73 ± 1.66b 32.50 ± 1.64b 33.59 ± 1.77ab 38.68 ± 2.18a 0.085 诱导型一氧化氮合酶 iNOS/(ng·mL−1) 14.91 ± 0.90 14.84 ± 0.64 14.59 ± 0.51 15.76 ± 0.75 0.682 内皮型一氧化氮合酶 eNOS/(ng·mL−1) 82.28 ± 4.28b 82.03 ± 2.55b 83.12 ± 4.33b 94.43 ± 3.63a 0.086 妊娠后期
Late pregnancy精氨酸 Arg/(µg·mL−1) 12.85 ± 0.87 13.46 ± 1.33 13.91 ± 0.38 13.49 ± 1.10 0.898 一氧化氮 NO/(µmoL·L−1) 50.20 ± 2.49 51.93 ± 5.85 51.33 ± 5.20 56.49 ± 5.33 0.794 总一氧化氮合酶 TNOS/(ng·mL−1) 29.48 ± 1.82 25.29 ± 2.35 24.68 ± 2.34 26.68 ± 2.35 0.448 诱导型一氧化氮合酶 iNOS/(ng·mL−1) 11.48 ± 1.16 12.39 ± 0.98 11.86 ± 1.26 11.56 ± 0.93 0.933 内皮型一氧化氮合酶 eNOS/(ng·mL−1) 63.36 ± 5.30 65.93 ± 6.59 71.17 ± 3.62 59.62 ± 2.67 0.177 Arg, arginine; NO, nitric oxide; TNOS, total nitric oxide synthase; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase. 在妊娠后期，各NCG试验组母羊血浆Arg、NO和iNOS较对照组有所提高，而NCG试验组母羊血浆中的TNOS却低于对照组，以上指标在NCG各试验组之间以及NCG组与对照组之间均无显著性差异。
2.3 NCG对妊娠期湖羊母羊血浆激素浓度的影响
妊娠前期Ⅲ组母羊血浆孕酮(P4)极显著高于试验Ⅰ组、Ⅱ组和对照组(P < 0.01) (表4)。试验Ⅰ组母羊血浆雌二醇(E2)极显著高于试验Ⅰ组、Ⅱ组和对照组(P < 0.01)。母羊血浆生长激素(GH)和胰岛素(INS)各试验组与对照组，以及各试验组之间均无显著性差异(P > 0.05)。
表 4 NCG对妊娠期湖羊母羊血浆激素浓度的影响Table 4. Effects of NCG on plasma hormone concentration in pregnant female Hu Sheep项目
Item组别 Group P 对照组
Control groupⅠ组
Group ⅠⅡ组
Group ⅡⅢ组
Group Ⅲ妊娠前期
Early pregnancy孕酮 P4/(ng·mL−1) 26.15 ± 1.67B 23.85 ± 1.68B 27.62 ± 1.91B 34.46 ± 1.87A 0.001 雌二醇 E2/(pg·mL−1) 107.96 ± 5.32B 145.34 ± 5.83A 122.38 ± 6.16B 115.39 ± 6.79B 0.001 生长激素 GH/(ng·mL−1) 12.07 ± 0.79 12.12 ± 0.79 12.11 ± 0.69 12.12 ± 1.05 1.000 胰岛素 INS/(uIU·mL−1) 36.94 ± 2.73 35.19 ± 2.61 31.01 ± 3.40 30.67 ± 2.71 0.349 妊娠后期
Late pregnancy催乳素 PRL/(mIU·L−1) 438.41 ± 32.36b 537.42 ± 31.92a 488.9 ± 29.94ab 421.59 ± 22.92b 0.075 生长激素 GH/(ng·mL−1) 14.17 ± 1.37 14.58 ± 1.35 12.80 ± 0.40 15.77 ± 0.56 0.302 胰岛素 INS/(uIU·mL−1) 31.44 ± 3.01 36.05 ± 2.32 32.75 ± 2.06 34.70 ± 3.70 0.677 P4, progesterone 4; E2, estradiol 2; GH, growth hormone; INS, insulin; PRL, prolactin. 妊娠后期Ⅰ组母羊血浆催乳素(PRL)显著高于试验Ⅲ组和对照组(P < 0.05)。Ⅰ组和Ⅲ组的母羊血浆GH，以及3个试验组的母羊血浆INS较对照组有所提高，但均无显著性差异(P > 0.05)。
2.4 NCG对湖羊母羊妊娠与产羔性状的影响
与对照组相比，各试验组母羊妊娠率、母羊胎产羔数、胎产活羔数和活羔初生胎重均有所提高，试验Ⅰ组和试验Ⅱ组活羔初生个体重低于对照组，以上指标都无显著性差异(表5)；试验Ⅰ组和试验Ⅲ组均有1只母羊产两只死胎，对照组和试验Ⅲ组有两只母羊各产1只死胎，试验Ⅱ组有1只母羊产3只死胎。另外，试验Ⅰ组有俩只母羊发生产前瘫痪分别产2只和3只死胎。
表 5 NCG对湖羊母羊妊娠与产羔性状的影响Table 5. Effects of NCG on pregnancy and lambing traits of female Hu sheep项目
Item组别 Group P 对照组
Control groupⅠ组
Group ⅠⅡ组
Group ⅡⅢ组
Group Ⅲ妊娠率 Pregnancy rate of ewes/% 64.87 ± 0.09 66.67 ± 0.12 70.00 ± 0.04 70.00 ± 0.07 0.897 胎产羔羊总数 Total number of lambs born 1.75 ± 0.11 1.85 ± 0.11 1.90 ± 0.09 1.80 ± 0.09 0.742 胎产活羔数 Number of live lambs born 1.69 ± 0.10 1.81 ± 0.12 1.87 ± 0.09 1.74 ± 0.10 0.604 活羔初生胎重 Birth weight of live lambs/kg 5.45 ± 0.20 5.61 ± 0.23 5.63 ± 0.22 5.51 ± 0.20 0.925 活羔初生个体重 Birth weight of a live lambs/kg 3.04 ± 0.09 2.96 ± 0.08 2.91 ± 0.07 3.09 ± 0.08 0.379 死胎数 The number of dead fetus 4 7 3 4 — 3. 讨论
3.1 湖羊母羊妊娠期日粮中添加NCG对血浆生化指标的影响
胎儿内分泌系统作为动物重要的调节系统之一，会随着母体代谢状态的改变而发生适应性变化[16]。当母体营养不足时，内分泌系统会调动更多的身体储备来维持胎儿基本的生存需求。葡萄糖是为母体营养、胎儿生长和胎儿发育提供能量的主要底物[17]，胎儿葡萄糖代谢是胎儿氧化代谢的主要生理过程[16]。本研究中，在妊娠前期，试验Ⅲ组母羊血浆BUN显著低于试验Ⅰ组，且随着NCG添加剂量的增加而呈线性的降低，表明随着NCG剂量的增加，母羊利用饲料中的氨基酸的效率也在提高。可能原因是NCG改变了胃肠道微生物结构，进而改善了母羊胃肠道功能，提高了营养物质消化率[18-20]。在妊娠后期，与对照组相比，试验Ⅱ组和试验Ⅲ组母羊血浆葡萄糖水平均有显著提高。Zhang等[13]在内蒙古白绒山羊日粮中添加0.4 g·d−1的NCG，可以显著提高生成葡萄糖的氨基酸浓度，促进糖代谢。Li等[21]在体重为478.0 ± 13.7 kg的育肥荷斯坦公牛日粮中添加15或25 g·d−1的NCG，同样可显著提高公牛血液中GLU浓度，与本研究结果一致。表明一定浓度的NCG能够提高妊娠后期湖羊母体与胎儿之间的葡萄糖供应，增加胎儿的能量供给，可能的原因是随着胎儿的变大，机体能量需求也随之变大，NCG促进了生成GLU所需的更多的氨基酸的合成，加速了机体的糖代谢，从而促进胎儿生长发育。试验Ⅱ组母羊血浆TC显著高于试验Ⅰ组，与妊娠前期的结果相似，试验Ⅱ组母羊胎儿最多，能量需求最大，故而在糖代谢能量供应不足时，脂质代谢会加剧，来满足胎儿生长发育，因此增加了血浆中脂质代谢产物TC的含量。然而，对照组母羊血浆TP高于添加NCG的各试验组，可能的原因是NCG增加了胎儿的数量，增加了母体营养的掠夺，导致母羊营养水平欠佳，蛋白质合成减少，血浆TP下降。 此外，在妊娠前期，除了试验Ⅰ组母羊血浆TG外，对照组母羊血浆AA、ALT和TG浓度均高于各NCG试验组，而在妊娠后期，除了试验Ⅰ组母羊血液AA外，各试验组母羊血浆的AA、ALT和TG浓度均高于对照组，AA浓度的提高，表明NCG能够促进妊娠后期母羊氨基酸的合成，进而为胎儿提供更多的营养；ALT是肝脏功能检测的重要指标，其在一定限度内的升高，表明NCG改善了机体的肝脏功能，对机体是有益的。在限饲湖羊母羊妊娠110 d的胎儿血液中，TG和TC含量都明显升高，表明机体营养不足时，机体会加强脂质代谢来满足营养需求[22]。NCG各试验组TC和TG的升高，可能的原因是随着胎儿的变大，机体能量需求也随之变大，同时为了满足多胎胎儿的能量需求，除了糖代谢增加外，母羊的脂质代谢也随之增加，来满足胎儿的能量需求。
3.2 湖羊母羊妊娠期日粮中添加NCG对血浆精氨酸和NO以及NOS的影响
精氨酸作为胎儿必需的营养氨基酸，在胎儿发育中起着关键作用[23]。精氨酸是NO合成酶(NOS)合成NO和多胺的重要底物[24]。NO可以调节胎盘—胎儿血流，并在营养物质从母体向胎儿转移中发挥重要作用[3, 25]。本研究中，在妊娠前期，与对照组相比，试验Ⅰ组母羊血浆Arg显著提高，试验Ⅲ组母羊血浆TNOS和eNOS都显著提高，表明NCG能够促进Arg的内源性合成，进而促进NOS合成NO。这一结果与Zhang等[26]在宫内生长受限的哺乳羔羊中的研究结果一致。即NCG通过合成内源性精氨酸，在NOS参与下，代谢生成更多的NO来调控妊娠早期胎儿与胎盘以及母体之间的血流量，进而促进胎儿的生长发育。Wang等[27]在西藏患有高海拔肺动脉高压的荷斯坦小母牛日粮中添加20 g·d−1的NCG饲喂28 d，可显著提高母牛血液中L-Arg、eNOS和NO的浓度。NCG增强了L -Arg的合成，进而促进了NO合成。此外，Zhang等[28]在IUGR羔羊结肠的研究结果也证实了NCG能够促进NO依赖通路相关基因(iNOS和eNOS)的表达水平上调。
乳腺血流量和血液中的底物浓度是决定乳合成底物可用性和新生儿营养输送的主要因素[29]。本研究中，在妊娠后期，各NCG试验组母羊血浆中的精氨酸、NO和iNOS均高于对照组，而NCG各试验组母羊血浆中的TNOS却低于对照组，可能的原因是TNOS与精氨酸共同参与合成更多的NO所导致，NO在调节乳腺组织营养灌注中有着重要的潜在作用[30]，试验Ⅰ组和试验Ⅱ组的eNOS较对照组也有一定的提高，表明一定剂量的NCG能够提高妊娠后期母羊血浆中精氨酸和TNOS的含量，进一步合成更多的血浆NO，增加了血浆中NO的浓度，这可能会增加乳腺血流量和乳合成所需的营养物质的易得性，从而促进胎儿生长发育。
3.3 湖羊母羊妊娠期日粮中添加NCG对血浆激素浓度的影响
雌激素主要由卵巢发育中的卵泡颗粒细胞产生，并在发情周期早期对促黄体生成素的产生产生负反馈[31]。孕酮(P4)是由哺乳动物卵巢的卵泡排卵后形成的黄体以及胎盘分泌所得[32]。雌激素和孕激素作用于中枢神经系统、卵巢和子宫，对排卵、受精、胚胎着床、妊娠维持、乳腺发育和泌乳起着至关重要的作用[33]。本研究中，在妊娠前期，与对照组相比，试验Ⅰ组母羊血浆E2极显著提高；试验Ⅲ组母羊血浆孕酮(P4)极显著提高。蔡元等[15]在纯种初产湖羊母羊日粮中添加0.11%的NCG，添加时间为人工授精当天(第0天)至38 d，可显著提高第19天母羊血浆中孕酮的浓度，同时也可以提高第19天母羊血浆中E2的浓度和第38天母羊血浆中E2和P4的浓度，与本研究结果相吻合。表明一定剂量的NCG能够促进繁殖相关激素的分泌，有利于受精卵着床发育与胚胎附植。催乳素(PRL)具有促进乳腺生长发育、启动乳汁合成、维持泌乳和调节乳成分合成等功能[34]，本研究中，在妊娠后期，与对照组相比，试验Ⅰ组母羊血浆PRL显著提高，与张俊丽等[35]在经产滩羊上的研究结果类似。提示一定程度的NCG能够促进PRL的分泌。在哺乳动物中，精氨酸被证明是一种有效的刺激胰岛B细胞分泌胰岛素和垂体前叶分泌生长激素的物质[24]，胰岛素是葡萄糖和脂肪代谢、血管功能和生长促生长激素轴的主要调节激素[36]，本研究中，在妊娠前期，各NCG试验组母羊血浆生长激素基本相同，但对照组母羊血浆胰岛素高于各NCG试验组。有研究表明，NCG能提高机体血液中胰岛素的含量[37]，但也有NCG在滩羊中的研究结果表明，NCG并未使妊娠45 d滩羊血液中胰岛素含量升高，反而是降低了胰岛素含量[35]，与本研究结果相似，其具体机制还需进一步研究。在妊娠后期，除了试验Ⅱ组母羊血浆生长激素外，各NCG试验组母羊血浆中的生长激素和胰岛素均高于对照组，揭示NCG促进了妊娠后期母羊的体内的生长激素轴中的激素分泌，促进胎儿的生长。可能的原因是，NCG促进母羊体内合成更多精氨酸，精氨酸能够促进与生长相关激素的分泌来提高胎儿的生长。总体来说，NCG能够促进繁殖和生长相关激素的分泌，对母羊的繁殖和羔羊的生长是有益处的。
3.4 日粮中添加NCG对湖羊母羊妊娠与产羔性状的影响
NCG已被证明能够提高母羊的繁殖性能，主要表现在促进子宫内膜发育[13]、促进胎儿和胎盘发育[14]、提高妊娠早期胚胎和胎儿的存活，以及提高总胎儿数和活胎儿数等[15]，本研究中，与对照组相比，各试验组母羊妊娠率、母羊胎产羔数、胎产活羔数和活羔初生胎重均有所提高，以试验II组母羊最高，分别提高了5.13%、0.15只、0.18只和0.18 kg，与张俊丽等[35]在经产滩羊上的研究结果一致。试验Ⅰ组和试验Ⅱ组活羔初生个体重低于对照组，与蔡元等[38]在经产湖羊上的研究结果相类似。暗示一定程度的NCG能够提高初产湖羊母羊的妊娠率、产羔率和产活羔率，活羔初生重会因母羊产羔变多而下降，可能原因是受胎盘中多个胎儿之间的营养竞争所导致。总体而言，NCG对母羊的繁殖性能是有益的。
4. 结论
本研究条件下，初产湖羊日粮中添加NCG可能通过增加血浆精氨酸和NO的含量，提高了初产湖羊的妊娠率、胎产羔数和胎产活羔数等繁殖性能，适宜剂量为每天每只1.5 g。
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[22] ZHANG H, ZHANG Y, MA Y, ELSABAGH M, WANG H R, WANG M Z. Dietary rumen-protected L-arginine or N-carbamylglutamate attenuates fetal hepatic inflammation in undernourished ewes suffering from intrauterine growth restriction. Animal Nutrition, 2021, 7(4): 1095-1104. doi: 10.1016/j.aninu.2021.06.004
[23] ZHANG H, LIU X Y, ZHENG Y, ZHANG Y, LOOR J J, WANG H R, WANG M G. Dietary N-carbamylglutamate or L-arginine improves fetal intestinal amino acid profiles during intrauterine growth restriction in undernourished ewes. Animal Nutrition, 2022, 8(1): 341-349.
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[26] ZHANG H, PENG A, YU Y Q, GUO S, WANG M Z, COLEMAN D N, LOOR J J, WANG H R. N-carbamylglutamate and l-arginine promote intestinal absorption of amino acids by regulating the mTOR signaling pathway and amino acid and peptide transporters in suckling lambs with intrauterine growth restriction. The Journal of Nutrition, 2019, 149(6): 923-932. doi: 10.1093/jn/nxz016
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表 1 基础日粮组成及营养水平(干物质基础)
Table 1 Composition and nutrient levels of basal diets (dry matter basis)
项目
Item原料
Ingredient含量 Content 营养水平
Nutrient levels含量 Content 妊娠前期
Early pregnancy妊娠后期
Late pregnancy妊娠前期
Early pregnancy妊娠后期
Late pregnancy粗饲料
Roughage/%玉米青贮
Corn silage51.80 42.00 代谢能
Metabolizable energy2)/(MJ·kg−1)7.83 17.49 玉米秸秆
Corn stalk11.10 9.00 干物质
Dry matter/%51.38 52.46 花生秧
Peanut seedling11.10 9.00 粗蛋白质
Crude protein/%11.94 14.54 精补料
Refined feed/%玉米
Corn15.16 23.32 中性洗涤纤维
Neutral detergent fibre/%42.36 44.85 麸皮
Wheat bran4.08 6.28 酸性洗涤纤维
Acid detergent fibre/%26.06 27.16 豆粕
Soybean meal3.90 6.00 粗脂肪
Crude fat/%2.9 3.6 小苏打
NaHCO30.39 0.60 粗纤维
Coarse fibre/%18.72 20.03 石粉
Limestone0.13 0.20 粗灰分
Crude ash/%8.18 8.89 碳酸氢钙
CaHPO40.13 0.20 钙
Calcium/%0.55 0.6 食盐
NaCl0.26 0.40 磷
Phosphorus/%0.45 0.48 预混料
Premix1)1.82 2.80 脱霉剂
Mildew remover0.13 0.20 合计
Total100.00 100.00 1)预混料为每千克饲粮提供：VA, 288～ 1152 KIU、VD2, 28.8～115.2 KIU、VE,1152 IU、Fe 0.8 g、Cu 0.16 g、Mn 0.8 g、Zn 0.8 g、I 8.0 mg、Co 1.6 mg、Se 1.6 mg。2)代谢能为计算值，其余为实测值。
1)The premix provided the following per kg of diets: VA, 288～1152 KIU, VD2, 28.8～115.2 KIU, VE,1152 IU, Fe 0.8 g, Cu 0.16 g, Mn 0.8 g, Zn 0.8 g, I 8.0 mg, Co 1.6 mg, Se 1.6 mg. 2)ME is a calculated value, whereas the others parameter values are measured values.
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表 2 NCG对妊娠期湖羊母羊血浆生化指标的影响
Table 2 Effects of NCG on plasma biochemical indices in female Hu sheep during pregnancy
项目
Item组别 Group P 对照组
Control groupⅠ组
Group ⅠⅡ组
Group ⅡⅢ组
Group Ⅲ妊娠前期
Early
pregnancy氨基酸 AA/ (µg·mL−1) 171.72 ± 13.26 170.73 ± 10.60 170.48 ± 11.02 167.89 ± 11.16 0.996 尿素氮 BUN/ (mg·mL−1) 0.21 ± 0.01ab 0.23 ± 0.02a 0.20 ± 0.01ab 0.18 ± 0.02b 0.195 谷丙转氨酶 ALT/(nmol·mL−1) 37.33 ± 4.59 36.93 ± 4.46 35.00 ± 4.41 32.48 ± 4.15 0.858 总蛋白 TP/(mg·mL−1) 29.03 ± 2.15 26.60 ± 4.15 29.70 ± 2.98 24.20 ± 2.20 0.437 甘油三酯 TG/(mg·mL−1) 0.83 ± 0.05 0.85 ± 0.04 0.71 ± 0.03 0.75 ± 0.03 0.103 葡萄糖 GLU/(µmol·mL−1) 0.41 ± 0.02 0.36 ± 0.01 0.36 ± 0.02 0.38 ± 0.02 0.236 总胆固醇 TC/(µmol·dL−1) 496.09 ± 33.30 466.15 ± 21.55 510.42 ± 38.65 493.82 ± 35.23 0.812 妊娠后期
Late
pregnancy氨基酸 AA/ (µg·mL−1) 208.63 ± 11.34 196.98 ± 9.37 215.92 ± 6.99 216.54 ± 12.54 0.557 尿素氮 BUN/ (mg·mL−1) 0.17 ± 0.01 0.21 ± 0.01 0.21 ± 0.01 0.15 ± 0.02 0.059 谷丙转氨酶 ALT/(nmol·mL−1) 37.64 ± 4.94 38.36 ± 2.96 43.48 ± 2.89 39.18 ± 3.69 0.692 总蛋白 TP/(mg·mL−1) 32.60 ± 2.04 28.40 ± 3.28 28.67 ± 2.58 28.43 ± 2.06 0.558 甘油三酯 TG/(mg·mL−1) 0.90 ± 0.06 0.91 ± 0.02 0.96 ± 0.05 0.96 ± 0.05 0.754 葡萄糖 GLU/(µmol·mL−1) 0.41 ± 0.01Bc 0.42 ± 0.02ABbc 0.49 ± 0.02Aa 0.47 ± 0.02ABab 0.124 总胆固醇 TC/(µmol·dL−1) 556.64 ± 22.08ab 546.88 ± 26.00b 619.47 ± 19.96a 558.92 ± 23.28ab 0.128 同行无字母或相同字母表示差异不显著(P > 0.05)，不同小写字母表示差异显著(P < 0.05)，不同大写字母表示差异极显著(P < 0.01)；Ⅰ组、Ⅱ组和Ⅲ组分别表示母羊在基础日粮的基础上每只每天分别添加1.0、1.5和2 g的N-氨甲酰谷氨酸。下表同。
In the same row, values with no letter or the same letter superscripts indicate no significant difference, while those with different small letter superscripts mean significant differences at the 0.05 level and those with different capital letter superscripts mean significant difference at the 0.01 level; Groups Ⅰ, II, and III indicate 1.0, 1.5, and 2 g of N-carbamylglutamic acid per ewe per day on the basis of the basal diet, respectively. This is applicable for the following tables as well. AA, amino acid; BUN, urea nitrogen; ALT, glutamic-pyruvic transaminase; TP, total protein; TG, triglyceride; GLU, glucose; TC, total cholesterol.
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表 3 NCG对妊娠期湖羊母羊血浆精氨酸和NO以及NOS的影响
Table 3 Effects of NCG on plasma Arg and NO as well as NOS in pregnant female Hu Sheep
项目
Item组别 Group P 对照组
Control groupⅠ组
Group ⅠⅡ组
Group ⅡⅢ组
Group Ⅲ妊娠前期
Early pregnancy精氨酸 Arg/(µg·mL−1) 12.23 ± 0.52b 14.94 ± 0.58a 12.79 ± 0.51ab 13.00 ± 1.14ab 0.128 一氧化氮 NO/(µmoL·L−1) 78.20 ± 4.97ab 72.12 ± 4.91b 79.78 ± 2.98ab 85.95 ± 3.34a 0.166 总一氧化氮合酶 TNOS/(ng·mL−1) 32.73 ± 1.66b 32.50 ± 1.64b 33.59 ± 1.77ab 38.68 ± 2.18a 0.085 诱导型一氧化氮合酶 iNOS/(ng·mL−1) 14.91 ± 0.90 14.84 ± 0.64 14.59 ± 0.51 15.76 ± 0.75 0.682 内皮型一氧化氮合酶 eNOS/(ng·mL−1) 82.28 ± 4.28b 82.03 ± 2.55b 83.12 ± 4.33b 94.43 ± 3.63a 0.086 妊娠后期
Late pregnancy精氨酸 Arg/(µg·mL−1) 12.85 ± 0.87 13.46 ± 1.33 13.91 ± 0.38 13.49 ± 1.10 0.898 一氧化氮 NO/(µmoL·L−1) 50.20 ± 2.49 51.93 ± 5.85 51.33 ± 5.20 56.49 ± 5.33 0.794 总一氧化氮合酶 TNOS/(ng·mL−1) 29.48 ± 1.82 25.29 ± 2.35 24.68 ± 2.34 26.68 ± 2.35 0.448 诱导型一氧化氮合酶 iNOS/(ng·mL−1) 11.48 ± 1.16 12.39 ± 0.98 11.86 ± 1.26 11.56 ± 0.93 0.933 内皮型一氧化氮合酶 eNOS/(ng·mL−1) 63.36 ± 5.30 65.93 ± 6.59 71.17 ± 3.62 59.62 ± 2.67 0.177 Arg, arginine; NO, nitric oxide; TNOS, total nitric oxide synthase; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase.
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表 4 NCG对妊娠期湖羊母羊血浆激素浓度的影响
Table 4 Effects of NCG on plasma hormone concentration in pregnant female Hu Sheep
项目
Item组别 Group P 对照组
Control groupⅠ组
Group ⅠⅡ组
Group ⅡⅢ组
Group Ⅲ妊娠前期
Early pregnancy孕酮 P4/(ng·mL−1) 26.15 ± 1.67B 23.85 ± 1.68B 27.62 ± 1.91B 34.46 ± 1.87A 0.001 雌二醇 E2/(pg·mL−1) 107.96 ± 5.32B 145.34 ± 5.83A 122.38 ± 6.16B 115.39 ± 6.79B 0.001 生长激素 GH/(ng·mL−1) 12.07 ± 0.79 12.12 ± 0.79 12.11 ± 0.69 12.12 ± 1.05 1.000 胰岛素 INS/(uIU·mL−1) 36.94 ± 2.73 35.19 ± 2.61 31.01 ± 3.40 30.67 ± 2.71 0.349 妊娠后期
Late pregnancy催乳素 PRL/(mIU·L−1) 438.41 ± 32.36b 537.42 ± 31.92a 488.9 ± 29.94ab 421.59 ± 22.92b 0.075 生长激素 GH/(ng·mL−1) 14.17 ± 1.37 14.58 ± 1.35 12.80 ± 0.40 15.77 ± 0.56 0.302 胰岛素 INS/(uIU·mL−1) 31.44 ± 3.01 36.05 ± 2.32 32.75 ± 2.06 34.70 ± 3.70 0.677 P4, progesterone 4; E2, estradiol 2; GH, growth hormone; INS, insulin; PRL, prolactin.
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表 5 NCG对湖羊母羊妊娠与产羔性状的影响
Table 5 Effects of NCG on pregnancy and lambing traits of female Hu sheep
项目
Item组别 Group P 对照组
Control groupⅠ组
Group ⅠⅡ组
Group ⅡⅢ组
Group Ⅲ妊娠率 Pregnancy rate of ewes/% 64.87 ± 0.09 66.67 ± 0.12 70.00 ± 0.04 70.00 ± 0.07 0.897 胎产羔羊总数 Total number of lambs born 1.75 ± 0.11 1.85 ± 0.11 1.90 ± 0.09 1.80 ± 0.09 0.742 胎产活羔数 Number of live lambs born 1.69 ± 0.10 1.81 ± 0.12 1.87 ± 0.09 1.74 ± 0.10 0.604 活羔初生胎重 Birth weight of live lambs/kg 5.45 ± 0.20 5.61 ± 0.23 5.63 ± 0.22 5.51 ± 0.20 0.925 活羔初生个体重 Birth weight of a live lambs/kg 3.04 ± 0.09 2.96 ± 0.08 2.91 ± 0.07 3.09 ± 0.08 0.379 死胎数 The number of dead fetus 4 7 3 4 —
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