Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 8: 5433 - 5445

DOI: https://doi.org/10.12418/CJAN2025.441

RESEARCH PAPER

Effects of Diets with Different Energy and Protein Levels on Reproductive Performance and Gene Expression Related to Follicle Development in Female Rabbits

  • HAN Haodong ,
  • JIN Zhuoya ,
  • ZHAI Jiamin ,
  • ZHANG Yichao ,
  • LI Kexuan ,
  • DU Baobao ,
  • JIA Haofan ,
  • LI Youhao ,
  • DUAN Qianting ,
  • DONG Xianggui ,
  • SONG Bing ,
  • WANG Shuhui ,
  • CUI Wei ,
  • REN Zhanjun , *
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  • College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
*professor, E-mail:

Received date: 2024-12-19

  Online published: 2025-08-14

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Abstract

This study aimed to investigate the effects of feeding diets with different energy and protein levels during the lactation period on reproductive performance and gene expression related to follicle development in female rabbits, while maintaining the same energy and protein levels during the non-lactating period. One hundred Ira meat rabbits at 1 week before delivery were selected and randomly divided into 5 groups, with 5 replicates per group and 4 rabbits per replicate. The body weight [(5.21±0.58) kg] between replicates was similar. A 2-factor, 2-level and 1 control experimental design was adopted, and five diets with different energy and protein levels (Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ) were formulated. The trial was divided into three phases: phase 1 [from the start of the trial to the first delivery of the female rabbits (7 days)], all groups were fed diet Ⅴ; phase 2 [lactation period (35 days)], groups Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ were fed diets Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ, respectively; phase 3 [from the end of the lactation period to the second delivery of the female rabbits (7 days)], all groups were fed diet Ⅴ again. The group Ⅴ with unchanged nutritional levels in the three phases was used as the control group. The trial period was 49 days. The results showed as follows: 1) there were significant interactions between dietary energy and protein levels on the average daily feed intake (ADFI) during the 2nd, 3rd and 4th weeks of lactation and the body weight on day 35 of female rabbits (P<0.05). Dietary energy levels significantly affected the ADFI of female rabbits during the 1st and 5th weeks of lactation (P<0.05). Compared with group Ⅴ, the ADFI of female rabbits in groups Ⅰ, Ⅱ and Ⅲ decreased significantly during the 1st week of lactation (P<0.05); the ADFI in groups Ⅰ and Ⅳ decreased significantly during the 3rd and 4th weeks of lactation (P<0.05); the ADFI in groups Ⅰ and Ⅱ decreased significantly during the 5th week of lactation (P<0.05). The body weight of female rabbits in group Ⅳ before the first delivery was significantly lower than that in group Ⅴ (P<0.05). 2) There was a significant interaction between dietary energy and protein levels on the average milk yield of female rabbits during the 5th week of lactation (P<0.05). Dietary energy levels significantly affected colostrum yield, average milk yield of female rabbits during the 4th and 5th weeks of lactation (P<0.05), as well as the individual body weight of offspring during the 2nd and 5th weeks of lactation and the average daily gain (ADG) of offspring from 1 to 35 days of age (P<0.05). Dietary protein levels significantly affected the average milk yield during the 1st, 3rd and 5th weeks of lactation (P<0.05). Compared with group Ⅴ, the average milk yield of female rabbits in group Ⅱ decreased significantly during the 4th week of lactation (P<0.05); the individual body weight of offspring in group Ⅰ during the 5th week of lactation and the ADG from 1 to 35 days of age decreased significantly (P<0.05). 3) The proportion of tertiary follicles of female rabbits in groups Ⅲ and Ⅳ decreased significantly compared with group Ⅴ (P<0.05). 4) The mRNA relative expression levels of follicle-stimulating hormone receptor (FSHR) and anti-Müllerian hormone (AMH) in ovary of female rabbits in groups Ⅱ and Ⅳ increased significantly compared with group Ⅴ (P<0.05); the mRNA relative expression level of growth differentiation factor 9 (GDF9) in ovary of female rabbits in group Ⅰ increased significantly (P<0.05); the mRNA relative expression levels of forkhead box protein L2 (FOXL2), inhibin α (INHA) and estrogen receptor 1 (ESR1) in ovary of female rabbits in group Ⅱ increased significantly (P<0.05). In conclusion, under the conditions of this experiment, when female rabbits were fed a diet with a digestible energy level of 10.00 MJ/kg and a crude protein level of 16.93% during the non-lactating period, feeding a diet with a digestible energy level of 9.50 to 10.00 MJ/kg and a crude protein level of 16.93% to 18.00% during the lactation period was more beneficial for increasing feed intake and improving reproductive performance such as milk production and offspring growth. Conversely, feeding a diet with a digestible energy level of 10.50 MJ/kg and a crude protein level of 16.43% during the lactation period had a more significant effect on regulating genes related to follicle development in ovary of female rabbits, such as FSHR, AMH, FOXL2, INHA and ESR1.

Key words： energy; protein; female rabbits; reproductive performance; follicle development

Cite this article

HAN Haodong , JIN Zhuoya , ZHAI Jiamin , ZHANG Yichao , LI Kexuan , DU Baobao , JIA Haofan , LI Youhao , DUAN Qianting , DONG Xianggui , SONG Bing , WANG Shuhui , CUI Wei , REN Zhanjun . Effects of Diets with Different Energy and Protein Levels on Reproductive Performance and Gene Expression Related to Follicle Development in Female Rabbits[J]. Chinese Journal of Animal Nutrition, 2025 , 37(8) : 5433 -5445 . DOI: 10.12418/CJAN2025.441

兔肉作为一种高营养、低脂肪的健康肉类产品,近年来受到越来越多的关注[1]。在我国,工厂化集约养兔模式已成为主要的养殖方式,通过大规模化与机械化、自动化技术的有效结合,显著提升了生产效率[2-3]。目前,肉兔养殖常用42 d繁殖模式,即母兔产仔后第11天配种,妊娠24 d后仔兔断奶,断奶1周后母兔再次产仔[4-7]。在此过程中,母兔的营养需求会随着生理状态的变化而有所不同,特别是在泌乳期妊娠阶段,对能量和蛋白质的需求更为强烈。母兔营养需求的变化与其体重、生长速度和繁殖状态等因素密切相关[8],而饲粮中的能量和蛋白质水平可以从基因到内分泌层面影响母兔的生殖表现和生理功能[9]。研究表明,饲粮营养不适宜可能会对母兔繁殖性能和仔兔的生长发育造成负面影响[10-11]。
为更好地满足母兔在各生理阶段的营养需求,考虑到母兔在不同生理状态下对能量和蛋白质的分配与利用存在差异[12],本研究采用不同阶段饲喂不同饲粮的策略,以探究繁殖期母兔适宜的饲粮能量和蛋白质水平,具体策略为:在非泌乳期,各组均饲喂相同能量和蛋白质水平饲粮;而在泌乳期,各组则饲喂不同能量和蛋白质水平饲粮。该策略旨在通过精细化的饲养管理,优化母兔的繁殖性能,特别是在泌乳压力较大的阶段为其提供适宜的营养支持。

1 材料与方法

1.1 伦理声明

本试验经西北农林科技大学实验动物管理及伦理委员会审批批准,批准编号:NWAFU-DK2024043。

1.2 试验设计和饲养管理

动物试验于西安市临潼区三民循环有限公司养殖基地进行。选取产前1周的伊拉肉兔100只,随机分为5组,每组5个重复,每个重复4只兔,各重复之间体重[(5.21±0.58) kg]接近。采用2因素2水平+1对照的试验设计,配制5种不同能量和蛋白质水平的饲粮(Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ)。试验分为3个阶段:第1阶段[试验开始至母兔第1次产仔(7 d)]各组均饲喂饲粮Ⅴ;第2阶段[泌乳期(35 d)]Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ组对应饲喂饲粮Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ;第3阶段[泌乳期结束至母兔第2次产仔(7 d)]各组再次饲喂饲粮Ⅴ。以3个阶段营养水平保持不变的Ⅴ组作为对照组。试验期49 d。母兔采用单笼饲养,自由采食和饮水,保证良好的光照和通风条件,并按照正常的免疫程序进行免疫接种。

1.3 试验饲粮

以玉米、豆粕、玉米胚芽粕和小麦麸皮为主要原料,参照《Nutrition of the Rabbit》第2版[13]与《肉兔营养需要量》(NY/T 4049—2021)[14]推荐的营养需要配制试验饲粮,其组成及营养水平见表1。各组饲粮均制成直径4 mm、长度10 mm的颗粒料。饲粮粗蛋白质、粗脂肪、中性洗涤纤维、酸性洗涤纤维、酸性洗涤木质素、钙和总磷含量分别参照GB/T 6432—2018、GB/T 6433—2006、GB/T 20806—2022、NY/T 1459—2022、GB/T 20805—2006、GB/T 6436—2018和GB/T 6437—2018测定;消化能、氨基酸含量参照NY/T 4049—2021中各饲料原料营养价值数据结合原料使用量计算得出。
表1 试验饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of experimental diets (air-dry basis) %

项目
Items		 饲粮Diets
Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ
原料Ingredients
玉米Corn 18.00 18.20 18.00 15.00 15.00
次粉Wheat middling 5.00 5.00 8.76 13.30 9.85
豆粕Soybean meal 10.65 7.70 18.46 14.20 11.93
玉米胚芽粕Corn germ meal 29.94 32.00 19.93 20.40 24.04
小麦麸Wheat bran 15.27 15.00 5.00 8.80 15.54
稻壳粉Rice husk powder 8.97 9.60 17.85 16.30 11.64
白酒糟Distiller’s grain 5.00 5.00 5.00 5.00 5.00
豆油Soybean oil 1.17 1.50 1.00 1.00 1.00
预混料Premix1) 6.00 6.00 6.00 6.00 6.00
合计Total 100.00 100.00 100.00 100.00 100.00
营养水平Nutrient levels2)
消化能DE/(MJ/kg) 10.50 10.50 9.50 9.50 10.00
粗蛋白质CP 18.58 16.43 18.00 16.43 16.93
粗脂肪EE 3.70 3.24 3.32 3.49 3.06
中性洗涤纤维NDF 38.13 39.27 36.87 38.26 38.36
酸性洗涤纤维ADF 14.35 14.61 18.63 18.16 15.92
酸性洗涤木质素ADL 3.27 3.34 4.15 4.15 3.69
赖氨酸Lys 0.77 0.76 0.77 0.76 0.76
蛋氨酸+胱氨酸Met+Cys 0.62 0.61 0.62 0.61 0.62
钙Ca 1.73 1.68 1.68 1.65 1.67
总磷TP 0.74 0.71 0.72 0.70 0.70

1)预混料为每千克饲粮提供The premix provided the following per kg of diets:VA 1 000 IU,VD 900 IU,VE 50 mg,VK 2 mg,VB1 2 mg,VB2 6 mg,VB5 6.7 mg,VB12 0.02 mg,烟酸 nicotinic acid 50 mg,叶酸 folic acid 0.6 mg,生物素 biotin 0.2 mg,赖氨酸 Lys 1 500 mg,蛋氨酸 Met 1 500 mg,NaCl 5 g,Fe 90 mg,Cu 20 mg,Zn 70 mg,Mn 10 mg,Se 0.3 mg,I 0.2 mg,Co 0.15 mg。
2)消化能、氨基酸为计算值,其余为实测值。DE and amino acids were calculated values, while the others were measured values.

1.4 样品采集

试验结束后,每组选取4只试验兔,称重后进行屠宰。完整取出子宫,清除周围脂肪与结缔组织,进行称重。采集右侧卵巢,称重后立即于-80 ℃冻存,用于检测卵泡发育相关基因的表达;采集左侧卵巢,经生理盐水轻柔冲洗后,置于4%多聚甲醛溶液中固定,用于卵泡形态观察及卵泡计数。

1.5 指标测定

1.5.1 采食量和体重

每周测定各组母兔采食量,在产仔前、后和泌乳期第21天、第35天对母兔进行称重,计算母兔每周的平均日采食量以及泌乳期第21天、第35天和第2次产仔后的体重变化。计算公式为:

体重变化(kg)=母兔某阶段体重-母兔第1次产仔前体重。

1.5.2 繁殖性能指标

测定各组母兔初乳量以及每周的泌乳量,计算每周的平均泌乳量;测定仔兔出生时及每周的窝重和窝仔数,计算仔兔初生个体重、每周的个体重、平均日增重。计算公式为:

平均日增重(g/d)=(仔兔某日龄个体重-初生个体重)/仔兔日龄。

1.5.3 繁殖器官指数与卵泡形态观察、卵泡计数

根据下列公式计算子宫指数与卵巢指数:

子宫指数(mg/g)=子宫重(mg)/体重(g);

卵巢指数(mg/g)=卵巢重(mg)/体重(g)。

卵巢组织经固定后,依次进行脱水、修剪、包埋、切片、染色及封片处理。采用Pannoramic 250数字切片扫描仪和Panthera数码三目摄像显微系统进行图像采集。首先在40倍镜下观察全部组织,检查大体病变,随后分别在100倍和400倍镜下观察并记录原始卵泡、初级卵泡、次级卵泡、三级卵泡及闭锁卵泡的的数量并计算其比例(%)。各类卵泡依据卵母细胞体积、卵泡细胞形态与层数、透明带和卵泡腔的存在与否进行分型。原始卵泡位于卵巢皮质浅层,由一层扁平状卵泡细胞包围体积较小的卵母细胞构成,胞质淡染,核居中,周围无透明带;初级卵泡的卵母细胞体积明显增大,胞质均匀,核仁清晰,周围被一层或多层立方状卵泡细胞包绕,并形成透明带;次级卵泡的卵母细胞进一步增大,位于卵泡中央,周围由两层及以上颗粒细胞紧密排列,透明带清晰,但尚未形成卵泡腔;三级卵泡则具有明显卵泡腔,卵母细胞偏离中心,位于一侧,由卵丘细胞包裹,并被放射冠细胞环绕;闭锁卵泡表现为结构紊乱,卵母细胞和颗粒细胞变性或脱落,染色质浓缩或碎裂,卵泡腔消失或形态不规则。

1.5.4 卵泡发育相关基因的表达

采用Trizol法提取卵巢样品总RNA,用NanoDrop 2000微量分光光度计检测RNA浓度和纯度,检测合格后,用于实时荧光定量PCR(qRT-PCR)测定。根据GenBank的基因序列,利用Primer Premier 6软件设计引物,各引物序列见表2。以各组织样品总RNA为模板,使用反转录试剂盒将提取出的RNA反转录为cDNA。qRT-PCR采用SYBR Green Ⅰ法,参照TB Green Premix Ex Taq Ⅱ试剂盒说明书进行。以甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)作为内参基因进行数据的标准化处理。每次反应均设空白样品为阴性对照,每个样品设置3个重复,结果采用2-ΔΔCt法进行处理,分析组织样品目的基因的mRNA相对表达量。
表2 qRT-PCR引物序列

Table 2 qRT-PCR primer sequences

基因
Genes		 登录号
Accession number		 引物序列
Primer sequences (5'—3')		 产物长度
Product length/bp
甘油醛-3-磷酸脱氢酶
GAPDH		 NM_001082253.1 F:TGTTTGTGATGGGCGTGAA
R:CCTCCACAATGCCGAAGT		 150
促卵泡激素受体
FSHR		 XM_002709718.3 F:CCTTCCTCTATGCCATCTTCACCAA
R:GGAGTTGTGGACAGTGGATGAGAAT		 128
生长分化因子9
GDF9		 NM_001171350.1 F:AGTCGGTCCTGCTCTACACTCT
R:ATGGCTGCGTCTGGCTGGAA		 111
抗缪勒氏管激素
AMH		 NM_001082794.1 F:AGGATGCCGAGACAGTGACGA
R:ACCACAGGAGCAGGTGAAGAGA		 106
叉头框蛋白L2
FOXL2		 NM_001171115.1 F:TCGCCAAGTTCCCGTTCTAC
R:TCCAGTAGTTGCCCTTGCG		 245
抑制素α
INHA		 NM_001171115.1 F:CACATACGCAGCCGCAAGGT
R:GCAGCAGCAACACCAGGACA		 238
雌激素受体1
ESR1		 XM_008263696.3 F:TGGTCAGTGCCTTGTTGGAAGC
R:GGAACCCTCTTTGCCCAGTTGA		 151

1.6 数据统计与分析

采用Excel 2021对试验数据进行整理和初步计算,利用SPSS 27.0软件对各组间结果进行单因素方差分析(one-way ANOVA),并采用Duncan氏法进行多重比较。使用一般线性模型(GLM)进行双因素方差分析,以能量和蛋白质水平为主效应,并分析能量与蛋白质水平的交互作用。结果用平均值±标准差表示,P<0.05表示差异显著。

2 结果与分析

2.1 不同能量和蛋白质水平饲粮对母兔采食量和体重的影响

由表3可知,饲粮能量和蛋白质水平对母兔泌乳期第2、3、4周平均日采食量和第35天体重存在显著交互作用(P<0.05)。饲粮能量水平对母兔泌乳期第1、5周平均日采食量有显著影响(P<0.05)。
表3 不同能量和蛋白质水平饲粮对母兔采食量和体重的影响

Table 3 Effects of diets with different energy and protein levels on feed intake and body weight of female rabbits

项目
Items		 组别Groups P值P-value
Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ 能量水平
Energy
level		 蛋白质
水平
Protein
level		 能量水平×
蛋白质水平
Energy level×
protein level
平均日采食量ADFI/(g/d)
泌乳期第1周
Week 1 of lactation		 232.96
±51.59c		 267.53
±46.90bc		 302.35
±15.41b		 325.69
±80.04ab		 389.55
±40.26a		 <0.001 0.099 0.744
泌乳期第2周
Week 2 of lactation		 375.13
±68.64b		 492.65
±55.95a		 483.83
±48.78a		 390.52
±72.07b		 448.00
±45.90ab		 0.874 0.559 <0.001
泌乳期第3周
Week 3 of lactation		 303.82
±71.94b		 416.40
±55.03a		 405.72
±27.43a		 332.37
±78.72b		 390.85
±55.77a		 0.652 0.324 <0.001
泌乳期第4周
Week 4 of lactation		 326.57
±21.69b		 401.53
±16.56a		 405.22
±46.47a		 324.46
±63.51b		 436.35
±45.08a		 0.952 0.826 <0.001
泌乳期第5周
Week 5 of lactation		 397.68
±45.85b		 440.63
±43.95b		 583.73
±142.73ab		 518.31
±131.96ab		 638.35
±134.81a		 <0.001 0.727 0.115
第2次产仔前1周
1 week before 2nd
delivery		 377.46
±132.16		 363.56
±132.24		 400.19
±165.06		 378.25
±145.61		 408.56
±137.79		 0.762 0.752 0.946
体重BW/kg
第1次产仔前
Before 1st delivery		 4.98
±0.63ab		 5.37
±0.49a		 5.39
±0.63a		 4.80
±0.75b		 5.38
±0.53a		 0.711 0.619 0.019
第1次产仔后
After 1st delivery		 4.64
±0.46		 5.00
±0.50		 4.87
±0.31		 4.67
±0.42		 4.68
±0.47		 0.732 0.607 0.072
泌乳期第21天
Day 21 of lactation		 4.83
±0.64		 5.03
±0.60		 5.30
±0.42		 4.84
±0.35		 4.89
±0.39		 0.498 0.522 0.108
泌乳期第35天
Day 35 of lactation		 4.61
±0.81ab		 4.81
±0.40ab		 5.11
±0.47a		 4.46
±0.12b		 4.61
±0.48ab		 0.700 0.242 0.033
第2次产仔后
After 2nd delivery		 4.27
±0.71		 3.52
±0.90		 4.50
±0.59		 3.76
±0.40		 4.21
±0.46		 0.516 0.060 0.979
体重变化BW change/kg
泌乳期第21天
Day 21 of lactation		 -0.15
±0.64		 -0.33
±0.60		 -0.09
±0.42		 -0.54
±0.34		 -0.49
±0.37		 0.757 0.133 0.514
泌乳期第35天
Day 35 of lactation		 -0.37
±0.81		 -0.56
±0.40		 -0.29
±0.47		 -0.34
±0.12		 -0.77
±0.48		 0.318 0.906 0.719
第2次产仔后
After 2nd delivery		 -0.71
±0.71a		 -1.85
±0.90b		 -0.89
±0.59ab		 -1.04
±0.40ab		 -1.17
±0.46ab		 0.389 0.097 0.186

同行数据肩标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)。下表同。

In the same row, values with no letter or the same letter superscripts mean no significant difference (P>0.05), while with different small letter superscripts mean significant difference (P<0.05). The same as below.

在泌乳期第1周,与Ⅴ组相比,Ⅰ组、Ⅱ组和Ⅲ组母兔平均日采食量显著降低(P<0.05),Ⅳ组母兔平均日采食量无显著变化(P>0.05);在泌乳期第2周和第2次产仔前1周,与Ⅴ组相比,4组母兔平均日采食量均无显著变化(P>0.05);在泌乳期第3、4周,与Ⅴ组相比,Ⅰ组和Ⅳ组母兔平均日采食量显著降低(P<0.05),Ⅱ组和Ⅲ组母兔平均日采食量无显著变化(P>0.05);在泌乳期第5周,与Ⅴ组相比,Ⅰ组和Ⅱ组母兔平均日采食量显著降低(P<0.05),Ⅲ组和Ⅳ组母兔平均日采食量无显著变化(P>0.05)。
与Ⅴ组相比,Ⅳ组母兔第1次产仔前体重显著降低(P<0.05),其他3组母兔第1次产仔前体重无显著变化(P>0.05);4组母兔其他时期体重均无显著变化(P>0.05)。

2.2 不同能量和蛋白质水平饲粮对母兔繁殖性能的影响

由表4可知,饲粮能量和蛋白质水平对母兔泌乳期第5周平均泌乳量存在显著交互作用(P<0.05)。饲粮能量水平对母兔初乳量、泌乳期第4、5周平均泌乳量有显著影响(P<0.05),同时对泌乳期第2、5周仔兔个体重及1~35日龄仔兔平均日增重有显著影响(P<0.05)。饲粮蛋白质水平对泌乳期第1、3、5周母兔平均泌乳量有显著影响(P<0.05)。
表4 不同能量和蛋白质水平饲粮对母兔繁殖性能的影响

Table 4 Effects of diets with different energy and protein levels on reproductive performance of female rabbits

项目
Items		 组别Groups P值P-value
Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ 能量水平
Energy
level		 蛋白质
水平
Protein
level		 能量水平×
蛋白质水平
Energy level×
protein level
泌乳量Milk yield/(g/d)
初乳
Colostrum		 85.25
±17.88b		 89.75
±29.12b		 124.40
±30.80a		 93.60
±13.89ab		 112.20
±19.21ab		 0.031 0.175 0.072
泌乳期第1周
Week 1 of lactation		 179.40
±66.21		 142.50
±32.73		 186.17
±29.90		 132.00
±28.56		 155.50
±8.87		 0.917 0.019 0.072
泌乳期第2周
Week 2 of lactation		 161.00
±24.87		 172.80
±15.26		 197.00
±45.40		 177.00
±26.87		 183.80
±35.29		 0.765 0.155 0.255
泌乳期第3周
Week 3 of lactation		 164.40
±65.78		 120.00
±23.58		 172.00
±36.30		 115.00
±3.74		 159.00
±37.51		 0.952 0.033 0.771
泌乳期第4周
Week 4 of lactation		 187.78
±68.33ab		 135.00
±63.47b		 223.08
±63.95a		 208.63
±38.93ab		 227.58
±74.00a		 0.008 0.094 0.334
泌乳期第5周
Week 5 of lactation		 39.20
±23.72b		 34.29
±34.20b		 176.86
±121.30a		 50.50
±37.55b		 141.83
±80.63ab		 0.013 0.032 0.045
仔兔个体重BW of rabbit offspring/g
初生重
Birth weight		 56.22
±10.78		 57.28
±14.26		 62.22
±6.99		 58.14
±7.20		 63.26
±6.77		 0.243 0.605 0.380
泌乳期第1周
Week 1 of lactation		 109.08
±20.38		 109.36
±11.97		 124.73
±14.61		 107.71
±16.76		 124.18
±5.45		 0.318 0.236 0.211
泌乳期第2周
Week 2 of lactation		 186.38
±19.19		 173.88
±19.81		 209.61
±26.83		 221.46
±52.26		 208.35
±12.05		 0.022 0.982 0.399
泌乳期第3周
Week 3 of lactation		 271.49
±24.15		 255.94
±19.89		 295.86
±29.56		 279.35
±40.35		 283.71
±3.94		 0.129 0.297 0.975
泌乳期第4周
Week 4 of lactation		 395.16
±49.59		 368.82
±70.79		 426.48
±54.32		 398.74
±51.72		 427.69
±56.16		 0.078 0.118 0.967
泌乳期第5周
Week 5 of lactation		 468.56
±97.26b		 572.85
±100.62a		 659.75
±122.48a		 661.28
±78.85a		 664.95
±66.78a		 <0.001 0.168 0.180
平均日增重ADG/(g/d)
1~21日龄
1 to 21 days of age		 10.25
±1.15		 9.46
±0.95		 11.49
±1.32		 10.36
±1.71		 10.50
±0.19		 0.136 0.176 0.801
1~35日龄
1 to 35 days of age		 11.78
±2.78b		 14.73
±2.87ab		 17.07
±3.50a		 17.23
±2.25a		 17.19
±1.77a		 0.001 0.156 0.202
与Ⅴ组相比,Ⅱ组母兔泌乳期第4周平均泌乳量显著降低(P<0.05);其他泌乳时期4组母兔泌乳量均无显著变化(P>0.05)。与Ⅴ组相比,Ⅰ组泌乳期第5周仔兔个体重及1~35日龄平均日增重显著降低(P<0.05),其他组仔兔个体重无显著变化(P>0.05);其他时期各组之间仔兔个体重均无显著差异(P>0.05)。

2.3 不同能量和蛋白质水平饲粮对母兔卵巢和子宫发育的影响

图1展示了母兔卵巢中各级卵泡的形态。由表5可知,各组间母兔卵巢指数、子宫指数均无显著差异(P>0.05)。与Ⅴ组相比,Ⅲ组和Ⅳ组母兔三级卵泡比例均显著降低(P<0.05),Ⅰ组和Ⅱ组母兔三级卵泡比例无显著变化(P>0.05)。在其他卵泡发育阶段,各组间母兔卵泡比例均无显著差异(P>0.05)。
图1 卵泡形态观察

PrF:原始卵泡(400×);PF:初级卵泡(100×);SF:次级卵泡(100×);TF:三级卵泡(100×);AF:闭锁卵泡(100×)。

Fig.1 Follicle morphology observation

PrF: primordial follicles (400×); PF: primary follicles (100×); SF: secondary follicles (100×); TF: tertiary follicles (100×); AF: atretic follicles (100×).

表5 不同能量和蛋白质水平饲粮对母兔卵巢和子宫发育的影响

Table 5 Effects of diets with different energy and protein levels on ovary and uterus development of female rabbits

项目
Items		 组别Groups P值P-value
Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ 能量水平
Energy
level		 蛋白质
水平
Protein
level		 能量水平×
蛋白质水平
Energy level×
protein level
卵巢指数
Ovarian index/(mg/g)		 0.25
±0.08		 0.36
±0.06		 0.29
±0.09		 0.35
±0.10		 0.31
±0.13		 0.663 0.073 0.606
子宫指数
Uterine index/(mg/g)		 14.08
±12.12		 13.14
±3.15		 29.92
±24.64		 15.01
±1.02		 14.70
±8.79		 0.316 0.366 0.424
原始卵泡
Primordial follicle/%		 26.10
±17.66		 32.68
±12.02		 30.61
±6.21		 33.20
±10.73		 26.63
±5.87		 0.733 0.538 0.787
初级卵泡
Primary follicle/%		 19.41
±2.80		 18.32
±2.60		 26.30
±7.04		 20.75
±7.66		 19.14
±1.84		 0.183 0.330 0.506
次级卵泡
Secondary follicle/%		 24.97
±14.51		 15.31
±9.13		 18.47
±14.72		 21.52
±11.13		 16.78
±5.97		 0.985 0.662 0.408
三级卵泡
Tertiary follicle/%		 10.33
±6.42ab		 12.07
±0.67ab		 7.49
±3.58b		 8.59
±7.02b		 22.10
±11.99a		 0.313 0.642 0.915
闭锁卵泡
Atretic follicle/%		 19.19
±4.76		 21.61
±14.60		 17.12
±6.50		 15.94
±3.79		 15.36
±2.13		 0.455 0.903 0.724

2.4 不同能量和蛋白质水平饲粮对母兔卵巢卵泡发育相关基因表达的影响

如图2所示,与Ⅴ组相比,Ⅱ组和IV组母兔卵巢促卵泡激素受体(FSHR)和抗缪勒氏管激素(AMH)的mRNA相对表达量显著提高(P<0.05),Ⅰ组和Ⅲ组母兔卵巢FSHR和AMH的mRNA相对表达量无显著变化(P>0.05)。与Ⅴ组相比,Ⅰ组母兔卵巢生长分化因子9(GDF9)的mRNA相对表达量显著提高(P<0.05),其他3组母兔卵巢GDF9的mRNA相对表达量无显著变化(P>0.05)。与Ⅴ组相比,Ⅱ组母兔卵巢叉头框蛋白L2(FOXL2)、抑制素α(INHA)、雌激素受体1(ESR1)的mRNA相对表达量显著提高(P<0.05),其他3组母兔卵巢FOXL2、INHA和ESR1的mRNA相对表达量无显著变化(P>0.05)。
图2 不同能量和蛋白质水平饲粮对母兔卵巢卵泡发育相关基因表达的影响

数据柱形标注不同字母表示差异显著(P<0.05)。

Fig.2 Effects of diets with different energy and protein levels on expression of genes related to follicular development in ovary of female rabbits

Value columns with different letters mean significant difference (P<0.05).

3 讨论

3.1 不同能量和蛋白质水平饲粮对母兔采食量与体重的影响

本试验中,饲粮能量和蛋白质水平对母兔泌乳期第2、3、4周平均日采食量和第35天体重存在显著交互作用。饲粮能量水平显著影响母兔泌乳期第1、5周平均日采食量。相较于Ⅰ组,Ⅲ组和Ⅴ组(对照组)饲粮更有利于提高繁殖期母兔的采食量。程光民等[15]以消化能水平9.48 MJ/kg、粗蛋白质水平17.82%的饲粮饲喂泌乳母兔,获得了较好采食表现,这与本研究结果相似。Ⅰ组、Ⅱ组、Ⅲ组母兔第1次产仔前体重与Ⅴ组均无显著差异,而Ⅳ组母兔第1次产仔前体重显著低于Ⅴ组,由于此阶段各组均饲喂相同营养水平的饲粮,该差异可能是由其他因素导致的试验误差。有研究表明,当机体的基础需要被满足后,多余的营养会被用于生产和繁殖[16]。本试验中,Ⅱ组、Ⅲ组、Ⅴ组母兔泌乳期第3周的平均日采食量显著高于Ⅰ组和Ⅳ组,Ⅰ组和Ⅱ组母兔泌乳期第5周的平均日采食量显著低于Ⅴ组,但是各组母兔泌乳期第21天和第35天较产仔前的体重变化却无显著差异,这表明多余的营养主要用于胚胎发育和母兔泌乳。

3.2 不同能量和蛋白质水平饲粮对母兔繁殖性能的影响

母兔在繁殖期的营养摄取直接影响其泌乳量。妊娠期能量和蛋白质摄入过多或过低,均会对泌乳产生不利影响[17],并进一步影响断奶窝重[18]。泌乳期适宜的营养供给同样重要,此阶段较高的饲粮蛋白质水平可有效促进母兔泌乳[19],并能提升仔兔断奶成活率、断奶窝重和断奶体重[20]。同样,饲粮中适宜的能量水平也有助于改善泌乳性能[21]。
本试验中,饲粮能量和蛋白质水平对母兔泌乳期第5周平均泌乳量存在显著交互作用。饲粮能量水平显著影响母兔初乳量、泌乳期第4、5周平均泌乳量,这也间接影响了泌乳期第2、5周的仔兔个体重及1~35日龄的平均日增重。饲粮蛋白质水平对母兔泌乳期第1、3、5周平均泌乳量具有显著影响。从上述试验结果看,整个泌乳期Ⅲ组和Ⅴ组母兔的泌乳表现较好,表明较高的饲粮蛋白质水平有助于提高泌乳量;而饲粮能量水平较高的Ⅰ组和Ⅱ组母兔泌乳表现欠佳。有研究指出,当饲粮能量水平高于泌乳母兔营养需要时,母兔的采食量会随着饲粮能量水平的提高而降低[15],这可能导致母兔对蛋白质的摄入不足,从而影响泌乳。
仔兔在3周龄前主要依靠母乳摄取营养,3周龄后才开始大量采食固态饲粮[13],因此泌乳期前3周仔兔的生长主要受母乳因素影响。本试验结果显示,Ⅲ组、Ⅳ组和Ⅴ组仔兔体重的总体表现优于Ⅰ组和Ⅱ组,Ⅲ组和Ⅴ组仔兔的体重较高可能是由于这2组母兔的泌乳量较高,充足的母乳供应有利于仔兔的早期生长[22],而Ⅳ组母兔虽然泌乳量较低,但仔兔体重较高,可能是由于仔兔采食母兔饲粮所致,表明断奶前采食消化能水平为9.50 MJ/kg、粗蛋白质水平为16.43%的饲粮可能有益于仔兔生长。

3.3 不同能量和蛋白质水平饲粮对母兔卵巢和子宫发育的影响

家兔属于诱导发情动物,发情周期不规律。产后1~2 d,母兔会表现出较为活跃的性活动,通常在交配后10~13 h诱发排卵[23]。繁殖措施与营养供给策略均会对母兔卵泡发育及胚胎质量产生影响,最终决定其生殖能力[24]。本研究发现,Ⅴ组母兔产仔后卵巢中三级卵泡比例高于其他组,生殖状态更佳。

3.4 不同能量和蛋白质水平饲粮对母兔卵巢卵泡发育相关基因表达的影响

本试验结果显示,Ⅱ组母兔卵巢FSHR的mRNA相对表达量相较于其他组显著提高,FSHR作为卵泡刺激素受体[25],参与调控卵泡发育和成熟过程[26]。GDF9通过调控卵泡发育,影响雌二醇和黄体激素的合成[27]。Ⅰ组母兔卵巢GDF9的mRNA相对表达量显著高于其他组,表明充足的饲粮能量和蛋白质供应可通过调控GDF9的表达,促进卵泡发育和激素合成。AMH主要作用于卵巢中的颗粒细胞,是反映卵巢储备功能的重要指标之一[28]。Ⅱ组和Ⅳ组母兔卵巢AMH的mRNA相对表达量显著高于其他组,说明饲粮中较低的蛋白质水平可以调节卵巢储备功能。Ⅱ组母兔卵巢FOXL2的mRNA相对表达量显著高于其他组,提示该饲粮能量和蛋白质水平(消化能水平10.50 MJ/kg、粗蛋白质水平16.43%)可能有助于卵巢功能的维持[29]。INHA基因编码抑制素α亚基,α亚基与β亚基结合形成抑制素,通过负反馈抑制垂体促卵泡激素(FSH)的分泌,从而调节卵泡发育[30],Ⅱ组母兔卵巢INHA的mRNA相对表达量最高,表明饲粮能量水平较高、蛋白质水平较低,即能量和蛋白质水平比值较大时,能够通过影响抑制素分泌间接调控卵泡刺激素水平。ESR1在卵巢中的功能主要是调节雌激素分泌[31],缺失该基因会导致小鼠卵巢功能紊乱[32]。在本试验中,Ⅱ组母兔卵巢ESR1的mRNA相对表达量显著高于其他组,这表明相对较高的饲粮能量水平能促进ESR1的表达,对卵巢发育和成熟具有积极作用。综上所述,饲粮能量和蛋白质水平比值较大时,对母兔卵巢中FSHR、AMH、FOXL2、INHA、ESR1等卵泡发育相关基因具有显著调控作用,进而影响卵巢功能。

4 结论

本试验条件下,当非泌乳阶段饲喂消化能水平为10.00 MJ/kg、粗蛋白质水平为16.93%的饲粮时,泌乳阶段饲喂消化能水平为9.50~10.00 MJ/kg、粗蛋白质水平为16.93%~18.00%的饲粮对促进母兔采食和提高泌乳、子代生长等繁殖性能更为有利;而泌乳阶段饲喂消化能水平为10.50 MJ/kg、粗蛋白质水平为16.43%的饲粮对母兔卵巢中FSHR、AMH、FOXL2、INHA、ESR1等卵泡发育相关基因的调控作用更为显著。

感谢西北农林科技大学动物科技学院宫瑞光博士对文稿所提的宝贵意见。

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