Chinese Journal of Animal Nutrition >

2024 , Vol. 36 >Issue 1: 312 - 322

DOI: https://doi.org/10.12418/CJAN2024.028

RESEARCH PAPER

Effects of Epimedium Extract on Semen Quality, Reproductive Endocrine Hormone, Reproduction Performance of Aged Breeder Roosters

  • HAN Shuyu , 1 ,
  • SUN Haixia 1 ,
  • LIU Jing 1 ,
  • GUO Mengyu 1 ,
  • DAI Zhanhui 2 ,
  • AN Shengying , 1, * ,
  • LIU Guanzhong , 1, *
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  • 1 College of Animal Science and Technology, Hebei Agricultural University, Baoding 071000, China
  • 2 Baoding Xingrui Agriculture and Animal Husbandry Development Co., Ltd., Baoding 071000, China
* AN Shengying, associate professor, E-mail: ;
LIU Guanzhong, professor, E-mail:

Received date: 2023-07-23

  Online published: 2024-01-12

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Abstract

The experiment was to investigate the effects of different levels of epimedium extract on semen quality, reproductive endocrine hormone, reproduction performance of aged breeder roosters. Seventy-two healthy 65-week-old Dawu Jinfeng parent breeder roosters with similar body weight [(2.94±0.01) g] were randomly divided into 4 groups with 6 replicates per group and 3 breeder roosters per replicate, and the roosters fed separately. Breeder roosters in the control group were fed a basal diet, and breeder roosters in trial groups Ⅰ,Ⅱ and Ⅲ were fed diets supplemented with 0.05%, 0.10% and 0.15% epimedium extract, respectively. The feeding experiment lasted for 7 weeks. At the 4th (69-week-old) and 7th week (72-week-old) of experiment, semen was collected to measure semen quality, and blood was collected to measure reproductive endocrine hormone levels; qualified breeding eggs were collected and incubated them on a repeated basis at last two weeks of experiment to measure reproduction performance indexes. The results showed that, compared with the control group, 1) at the 4th week of the experiment, the sperm motility was significantly increased in all trial groups (P<0.05), the sperm abnormality was significantly decreased in trial groups (P<0.05), the number of available sperm in trial groups Ⅱ and Ⅲ was significantly increased (P<0.05), and the sperm density in trial group Ⅱ was significantly increased (P<0.05). At the 7th week of experiment, the ejaculation volume of trial groups Ⅱ and Ⅲ was significantly increased (P<0.05), and the sperm motility was significantly increased in trial group Ⅲ was significantly increased (P<0.05). 2) At the 4th week of the experiment, the seminal plasma glutamic oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities in trial groups Ⅱ and Ⅲ were significantly decreased (P<0.05); at the 7th week of the experiment, the seminal plasma GOT and GPT activities in trial group Ⅲ was significantly decreased (P<0.05). 3) At the 4th week of the experiment, the glutathione peroxidase (GSH-Px) activity was in all trial groups extremely significantly increased (P<0.01), and the seminal plasma total antioxidant capacity (T-AOC) in trial groups Ⅱ and Ⅲ was significantly increased (P<0.05); at the 7th week of the experiment, the seminal plasma total superoxide dismutase (T-SOD) activity in all trial groups was significantly increased (P<0.05), the seminal plasma malondialdehyde (MDA) content in trial groups Ⅱ and Ⅲ was significantly decreased (P<0.05), and the seminal plasma T-AOC in trial group Ⅱ was significantly increased (P<0.05). 4) At the 4th week of the experiment, the serum gonadotrophin releasing hormone (GnRH) level in all trial groups was significantly decreased (P<0.05), the serum luteinizing hormone (LH) and testosterone (T) levels in all trial groups was significantly increased (P<0.05), and the serum follicle-stimulating hormone (FSH) level in trial groups Ⅱ and Ⅲ was significantly increased (P<0.05); at the 7th week of the experiment, the serum GnRH level in all trial groups was significantly decreased (P<0.05), the serum FSH and LH levels in trial groups Ⅱ and Ⅲ were significantly increased (P<0.05), and the serum T level in trial group Ⅲ was significantly increased (P<0.05). 5) The fertility and hatchability of setting eggs in trial groups Ⅱ and Ⅲ were significantly increased (P<0.05), the hatchability of fertile eggs and healthy chick rate of setting eggs in trial group Ⅲ was significantly increased (P<0.05), and the embryonic mortality in trial group Ⅲ was significantly decreased (P<0.05). To sum up, epimedium extract can improve the semen quality of aged breeder roosters through modulating reproductive endocrine hormone, improving seminal plasma antioxidant capability, reducing sperm damage, and finally enhance the reproduction performance of aged breeder roosters, and the suitable supplemental level of epimedium extract in the diet is 0.15%.

Key words： epimedium extract; aged breeding roosters; semen quality; reproductive endocrine hormone; reproduction performance

Cite this article

HAN Shuyu , SUN Haixia , LIU Jing , GUO Mengyu , DAI Zhanhui , AN Shengying , LIU Guanzhong . Effects of Epimedium Extract on Semen Quality, Reproductive Endocrine Hormone, Reproduction Performance of Aged Breeder Roosters[J]. Chinese Journal of Animal Nutrition, 2024 , 36(1) : 312 -322 . DOI: 10.12418/CJAN2024.028

种公鸡在饲养后期精子密度、精子活力等下降,繁殖性能急剧减退,导致种蛋的受精率和孵化率降低,严重影响种鸡场经济效益,因此,提高老龄种公鸡的繁殖性能十分重要。现代药理学研究表明,淫羊藿具有提阳气[2]、抗氧化[3]、增强免疫[4]和提高动物精液品质[5-6]的作用。研究发现,淫羊藿复方中草药能够缓解由高频采精导致的种公鸡精液稀少的问题[7];淫羊藿复方中草药可以显著提高种公鸡血浆中生殖激素促卵泡素(FSH)、促黄体素(LH)和睾酮(T)水平[8]。淫羊藿提取物的主要成分是黄酮类和多糖[1]。研究显示淫羊藿多糖可显著提高雏鸡的抗氧化能力[3],但关于淫羊藿提取物在种公鸡繁殖性能方面的研究目前未见报道。本试验通过研究淫羊藿提取物对老龄种公鸡精液品质、血清生殖内分泌激素水平和繁殖性能的影响,考察淫羊藿提取物在种公鸡生产中的应用效果,并揭示其可能的作用机制,为淫羊藿提取物作为饲料添加剂在种公鸡上的应用提供科学依据。

1 材料与方法

1.1 试验材料

淫羊藿提取物由陕西某生物技术有限公司提供,规格为20∶1(20 g淫羊藿中草药提取出1 g淫羊藿提取物),提取方式为水提,简要步骤如下:水提、浓缩、醇沉、浓缩去醇、喷雾干燥、过筛,即得干浸膏粉。淫羊藿提取物为棕黄色粉末,主要活性物质为淫羊藿总黄酮(0.19%)、淫羊藿多糖(4.20%)。

1.2 试验设计

将72只65周龄、体重[(2.94±0.01) kg]相近、健康的种公鸡随机分成4组,每组6个重复,每个重复3只鸡,单笼饲养。对照组饲喂基础饲粮,试验Ⅰ、Ⅱ、Ⅲ组分别饲喂在基础饲粮中添加0.05%、0.10%和0.15%淫羊藿提取物的试验饲粮。饲养试验为期7周。

1.3 基础饲粮

参照《鸡饲养标准》(NY/T 33—2004)和《中国饲料成分及营养价值表(2019年第30版)》以及企业生产实际情况配制基础饲粮。基础饲粮组成及营养水平见表1。饲粮中粗蛋白质含量测定参照GB/T 6432—2018,钙含量测定参照GB/T 6436—2002,总磷含量测定参照GB/T 6437—2018。饲粮代谢能水平、非植酸磷和各氨基酸含量参考《中国饲料成分及营养价值表(2019年第30版)》中各原料成分的参考值计算得出。
表1 基础饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of the basal diet (air-dry basis)%

项目Items 含量Content
原料Ingredients
玉米Corn 63.67
豆粕Soybean meal 19.00
小麦麸Wheat bran 9.20
大豆油Soybean oil 0.50
沸石粉Zeolite powder 4.53
石粉Limestone 1.40
氯化钠NaCl 0.25
磷酸氢钙CaHPO4 1.10
DL-蛋氨酸DL-Met (99%) 0.12
维生素预混料Vitamin premix1) 0.03
矿物质预混料Mineral premix2) 0.20
合计Total 100.00
营养水平Nutrient levels3)
代谢能ME/(MJ/kg) 11.23
粗蛋白质CP 14.91
钙Ca 1.08
总磷TP 0.56
非植酸磷NPP 0.28
赖氨酸Lys 0.71
蛋氨酸Met 0.37
苏氨酸Thr 0.56
色氨酸Trp 0.17

1)维生素预混料为每千克饲粮提供 The vitamin premix provided the following per kg of the diet:VA 14 000 IU,VB1 4 mg,VB2 12 mg,VB6 12 mg,VB12 0.04 mg,VD3 5 200 IU,VE 64 IU,VK3 6 mg,烟酸 niacin 80 mg,泛酸钙 calcium pantothenate 24 mg,生物素 biotin 0.48 mg,叶酸 folic acid 2.4 mg。

2)矿物质预混料为每千克饲粮提供 The mineral premix provided the following per kg of the diet:Fe 60 mg,Cu 12 mg,Se 0.3 mg,Mn 120 mg,Zn 110 mg,I 1.5 mg。

3)粗蛋白质、钙和总磷为实测值,其余为计算值。CP, Ca and TP were measured values, while the others were calculated values.

1.4 饲养管理

饲养试验于保定兴芮农牧发展有限公司种禽五场进行,试验鸡采用单笼饲养,自由采食,自由饮水,每日喂料3次(06:30、11:30和15:30),其他饲养管理条件严格按照保定兴芮农牧发展有限公司的管理规定进行。

1.5 样品采集与指标测定

1.5.1 精液品质

采用背部按摩法对种公鸡进行采精,要求精液无粪便、血液等污染。供试鸡每3 d采精1次,试验第4周(69周龄)和第7周(73周龄)收集精液用于精液品质检测。试验第4周和第7周,每个重复随机抽取1只种公鸡采精,3 000 r/min离心10 min制备精浆,于-20 ℃保存待测。

1.5.1.1 精液常规参数

参照《家禽精液品质检测方法》(NY/T 4047—2021)测定精液量、精液pH、精子密度、精子活力、精子畸形率和有效精子数。

1.5.1.2 精子损伤

精浆中谷草转氨酶(GOT)和谷丙转氨酶(GPT)活性采用微板法测定,所用试剂盒购自南京建成生物工程研究所。

1.5.1.3 精浆抗氧化能力

精浆总抗氧化能力(T-AOC)采用ABTS法进行测定,总超氧化物歧化酶(T-SOD)活性采用黄嘌呤氧化酶法测定,谷胱甘肽过氧化物酶(GSH-Px)活性采用5,5'-二硫双(2-硝基苯甲酸)(DTNB)法测定,丙二醛(MDA)含量采用硫代巴比妥酸(TBA)法测定,所用试剂盒均购自南京建成生物工程研究所。

1.5.2 血清生殖内分泌激素水平

试验第4周和第7周,每个重复随机抽取1只种公鸡空腹翅下采血约5 mL,3 000 r/min离心10 min制备血清,于-20 ℃保存待测。血清中促性腺激素释放激素(GnRH)、FSH、LH和T水平的测定方法为酶联免疫法,测定仪器为华卫德朗DR-200BS酶标分析仪(北京华英生物技术研究所)。

1.5.3 繁殖性能

为了测定种公鸡的繁殖性能,在试验开始时选取同一环境饲养的288只母鸡,每个重复的种公鸡对应12只母鸡,进行人工授精,每6 d输精1次,试验最后2周收集合格种蛋以重复为单位入孵,每个重复42枚。在孵化第18天落盘照蛋,记录无精蛋数、受精蛋数、死胚蛋数,计算受精率、死胚率;在孵化第21天出雏,记录出雏数、健雏数,计算受精蛋孵化率、入孵蛋孵化率、健雏率以及入孵蛋健雏率。

受精率(%)=(受精蛋数/入孵蛋数)×100;

死胚率(%)=(死胚数/受精蛋数)×100;

受精蛋孵化率(%)=(出雏数/受精蛋数)×100;

入孵蛋孵化率(%)=(出雏数/入孵蛋数)×100;

健雏率(%)=(健雏数/出雏数)×100;

入孵蛋健雏率(%)=(健雏数/入孵蛋数)×100。

1.6 数据处理与分析

所用数据利用Excel 2021进行初始整理,再采用SPSS 19.0软件中的ANOVA过程进行单因素方差分析,并用Duncan氏法进行组间多重比较;此外,使用SPSS 19.0软件进行回归分析,并用曲线估计进行线性和二次分析。结果用平均值和均值标准误(SEM)来表示。以P<0.05表示差异显著,P<0.01表示差异极显著,P>0.05表示差异不显著。

2 结果与分析

2.1 淫羊藿提取物对老龄种公鸡精液品质的影响

2.1.1 淫羊藿提取物对老龄种公鸡精液常规参数的影响

由表2可知,随着淫羊藿提取物添加量的提高,69周龄种公鸡的精子密度呈线性(P<0.05)和二次变化(P<0.05),可建立回归方程y=-7 416.667x2+1 413.500x+290.300,由回归方程计算得出淫羊藿添加量为0.095%时种公鸡的精子密度最高;此外,精子活力和有效精子数呈线性升高(P<0.01),精子畸形率呈线性降低(P<0.01)。随着淫羊藿提取物添加量的提高,72周龄种公鸡的精液量、精子活力和有效精子数呈线性升高(P<0.01),精子畸形率呈线性降低(P<0.01)。试验第4周时,与对照组相比,试验Ⅱ、Ⅲ组有效精子数显著增加(P<0.05);各试验组精液量和精液pH无显著变化(P>0.05),精子活力显著提高(P<0.05),精子畸形率显著降低(P<0.05);试验Ⅱ组精子密度显著提高(P<0.05)。试验第7周时,各试验组精液pH、精子密度和精子畸形率无显著变化(P>0.05);试验Ⅲ组精子活力显著提高(P<0.05);试验Ⅱ、Ⅲ组精液量显著增加(P<0.05)。各试验组间相比,试验第7周时试验Ⅲ组精子活力显著高于试验Ⅰ组(P<0.05)。
表2 淫羊藿提取物对老龄种公鸡精液常规参数的影响

Table 2 Effects of epimedium extract on semen routine parameters of aged breeder roosters (n=6)

项目
Items		 对照组
Control
group		 试验Ⅰ组
Trial
group Ⅰ		 试验Ⅱ组
Trial
group Ⅱ		 试验Ⅲ组
Trial
group Ⅲ		 均值
标准误
SEM		 P值P-value
处理
Treatment		 线性
Linear		 二次
Quadratic
第4周The 4th week
精液量Ejaculation volume/mL 0.31 0.33 0.34 0.35 0.01 0.604 0.192 0.896
精液pH Semen pH 7.47 7.53 7.56 7.53 0.04 0.892 0.594 0.582
精子密度
Sperm density/(×109个/mL)		 29.43b 33.03ab 36.96a 33.14ab 0.93 0.029 0.048 0.031
精子活力Sperm motility/% 53.82b 65.31a 69.00a 69.33a 2.23 0.034 0.009 0.166
精子畸形率
Sperm abnormality/%		 6.94a 4.28b 3.74b 3.15b 0.49 0.020 0.004 0.225
有效精子数
Number of available sperm/
(×109个/mL)		 5.03b 7.06ab 8.57a 8.13a 0.46 0.019 0.005 0.125
第7周The 7th week
精液量Ejaculation volume/mL 0.41b 0.44ab 0.47a 0.48a 0.01 0.027 0.004 0.425
精液pH Semen pH 7.55 7.59 7.63 7.60 0.03 0.863 0.524 0.616
精子密度
Sperm density/(×109个/mL)		 28.05 28.52 29.68 27.30 1.34 0.946 0.933 0.623
精子活力Sperm motility/% 45.89b 47.56b 56.94ab 61.76a 2.20 0.018 0.002 0.673
精子畸形率
Sperm abnormality/%		 9.89 8.03 6.65 5.18 0.65 0.058 0.008 0.867
有效精子数
Number of available sperm/
(×109个/mL)		 5.46b 6.07ab 7.99a 8.05a 0.41 0.032 0.006 0.696

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),不同大写字母表示差异极显著(P<0.01)。下表同。

In the same row, values with no letter superscripts mean no significant difference (P>0.05), while with different small letter superscripts mean significant difference (P<0.05), and with different capital letter superscripts mean extremely significant difference (P<0.01). The same as below.

2.1.2 淫羊藿提取物对老龄种公鸡精子损伤的影响

由表3可知,随着淫羊藿提取物添加量的提高,69和72周龄种公鸡精浆GOT、GPT活性均呈线性降低(P<0.01)。试验第4周时,试验Ⅱ、Ⅲ组精浆GOT、GPT活性显著低于对照组(P<0.05)。试验第7周时,试验Ⅲ组精浆GOT、GPT活性显著低于对照组(P<0.05);同时,试验Ⅲ组精浆GOT活性显著低于试验Ⅰ组(P<0.05)。
表3 淫羊藿提取物对老龄种公鸡精子损伤的影响

Table 3 Effects of epimedium extract on sperm damage of aged breeder roosters (n=6)U/L

项目
Items		 对照组
Control
group		 试验Ⅰ组
Trial
group Ⅰ		 试验Ⅱ组
Trial
group Ⅱ		 试验Ⅲ组
Trial
group Ⅲ		 均值
标准误
SEM		 P值P-value
处理
Treatment		 线性
Linear		 二次
Quadratic
第4周The 4th week
谷草转氨酶GOT 406.67a 359.32ab 328.49b 303.91b 13.64 0.035 0.004 0.636
谷丙转氨酶GPT 14.61a 13.11ab 11.48b 10.99b 0.48 0.018 0.002 0.536
第7周The 7th week
谷草转氨酶GOT 327.07a 282.51a 205.76ab 145.46b 23.40 0.018 0.002 0.843
谷丙转氨酶GPT 15.64a 14.29ab 12.83ab 11.14b 0.62 0.048 0.006 0.878

2.1.3 淫羊藿提取物对老龄种公鸡精浆抗氧化能力的影响

由表4可知,随着淫羊藿提取物添加量的提高,69周龄种公鸡精浆GSH-Px活性呈线性(P<0.01)和二次变化(P<0.05),可建立回归方程y=-7 174.838x2+1 532.448x+94.664,由回归方程计算得出,淫羊藿提取物添加量为0.107%时种公鸡精浆GSH-Px活性最高;此外,种公鸡精浆T-AOC呈线性升高(P<0.05)。随着淫羊藿提取物添加量的提高,72周龄种公鸡精浆T-SOD活性呈线性(P<0.05)和二次变化(P<0.05),可建立回归方程y=-170.286x2+34.027x+11.113,由回归方程计算得出,淫羊藿提取物添加量为0.100%时种公鸡精浆T-SOD活性最高;此外,精浆GSH-Px活性呈线性升高(P<0.01),MDA含量呈线性降低(P<0.01),T-AOC呈二次变化(P<0.05)。试验第4周时,与对照组相比,各试验组精浆T-SOD活性和MDA含量无显著变化(P>0.05),GSH-Px活性极显著提高(P<0.01);试验Ⅱ、Ⅲ组精浆T-AOC显著提高(P<0.05)。试验第7周时,与对照组相比,各试验组精浆T-SOD活性显著提高(P<0.05);试验Ⅱ、Ⅲ组精浆MDA含量显著降低(P<0.05);试验Ⅱ组T-AOC显著提高(P<0.05);此外,试验Ⅱ、Ⅲ组精浆GSH-Px活性显著高于试验Ⅰ组(P<0.05);试验Ⅱ组精浆T-AOC显著高于试验Ⅰ、Ⅲ组(P<0.05);试验Ⅲ组精浆MDA含量显著低于试验Ⅰ组(P<0.05)。
表4 淫羊藿提取物对老龄种公鸡精浆抗氧化能力的影响

Table 4 Effects of epimedium extract on seminal plasma antioxidant capability of aged breeder roosters (n=6)

项目
Items		 对照组
Control
group		 试验Ⅰ组
Trial
group Ⅰ		 试验Ⅱ组
Trial
group Ⅱ		 试验Ⅲ组
Trial
group Ⅲ		 均值
标准误
SEM		 P值P-value
处理
Treatment		 线性
Linear		 二次
Quadratic
第4周The 4th week
谷胱甘肽过氧化物酶
GSH-Px/(U/mL)		 93.43Bb 157.06Aa 172.45Aa 164.34Aa 8.91 0.001 0.001 0.012
总超氧化物歧化酶
T-SOD/(U/mL)		 11.21 11.90 12.06 11.27 0.22 0.447 0.863 0.116
总抗氧化能力
T-AOC/(mmol/L)		 0.31b 0.38ab 0.54a 0.49a 0.03 0.043 0.014 0.268
丙二醛
MDA/(nmol/mL)		 4.46 4.31 3.87 3.58 0.31 0.756 0.295 0.912
第7周The 7th week
谷胱甘肽过氧化物酶
GSH-Px/(U/mL)		 143.50b 148.69b 177.90a 175.52a 4.97 0.011 0.003 0.647
总超氧化物歧化酶
T-SOD/(U/mL)		 11.10b 12.44a 12.76a 12.40a 0.22 0.029 0.022 0.038
总抗氧化能力
T-AOC/(mmol/L)		 0.37b 0.42b 0.68a 0.46b 0.04 0.019 0.081 0.049
丙二醛
MDA/(nmol/mL)		 4.80a 4.34ab 3.53bc 3.11c 0.22 0.020 0.002 0.953

2.2 淫羊藿提取物对老龄种公鸡血清生殖内分泌激素水平的影响

由表5可知,随着淫羊藿提取物添加量的提高,69和72周龄种公鸡血清GnRH水平呈线性降低(P<0.01),FSH、LH活性和T水平呈线性升高(P<0.01)。试验第4周时,与对照组相比,各试验组血清GnRH水平显著降低(P<0.05);试验Ⅱ、Ⅲ组血清FSH水平显著提高(P<0.05);各试验组血清LH、T水平显著提高(P<0.05)。试验第7周时,与对照组相比,各试验组血清GnRH水平极显著降低(P<0.01);试验Ⅱ、Ⅲ组血清FSH、LH水平显著提高(P<0.05);试验Ⅲ组血清T水平显著提高(P<0.05);此外,试验Ⅲ组血清T水平显著高于试验Ⅰ组(P<0.05)。
表5 淫羊藿提取物对老龄种公鸡血清生殖内分泌激素水平的影响

Table 5 Effects of epimedium extract on reproductive endocrine hormone levels of aged breeder roosters (n=6)

项目
Items		 对照组
Control
group		 试验Ⅰ组
Trial
group Ⅰ		 试验Ⅱ组
Trial
group Ⅱ		 试验Ⅲ组
Trial
group Ⅲ		 均值
标准误
SEM		 P值P-value
处理
Treatment		 线性
Linear		 二次
Quadratic
第4周The 4th week
促性腺激素释放激素
GnRH/(pg/mL)		 46.73a 35.70b 31.07b 30.26b 2.16 0.015 0.003 0.169
促卵泡素FSH/(mIU/mL) 4.32b 5.33ab 6.02a 6.64a 0.30 0.028 0.003 0.709
促黄体素LH/(mIU/mL) 5.74b 7.40a 7.76a 8.26a 0.32 0.023 0.004 0.301
睾酮T/(ng/mL) 0.22b 0.57a 0.68a 0.79a 0.07 0.017 0.003 0.315
第7周The 7th week
促性腺激素释放激素
GnRH/(pg/mL)		 86.58Aa 67.92Bb 63.85Bb 59.57Bb 3.03 0.002 <0.001 0.131
促卵泡素FSH/(mIU/mL) 3.75b 4.98ab 5.35a 5.96a 0.26 0.013 0.002 0.482
促黄体素LH/(mIU/mL) 5.11b 6.20ab 7.36a 7.56a 0.32 0.010 0.001 0.403
睾酮T/(ng/mL) 0.75b 0.86b 1.10ab 1.27a 0.07 0.015 0.002 0.759

2.3 淫羊藿提取物对老龄种公鸡繁殖性能的影响

由表6可知,随着淫羊藿提取物添加量的提高,种公鸡种蛋受精率、受精蛋孵化率、入孵蛋孵化率和入孵蛋健雏率呈线性升高(P<0.01或P<0.05),死胚率呈线性降低(P<0.01)。与对照组相比,各试验组健雏率无显著变化(P>0.05);试验Ⅱ、Ⅲ组受精率和入孵蛋孵化率显著提高(P<0.05);试验Ⅲ组受精蛋孵化率和入孵蛋健雏率显著提高(P<0.05),死胚率显著降低(P<0.05)。各试验组之间相比,试验Ⅲ组受精蛋孵化率显著高于试验Ⅰ组(P<0.05)。
表6 淫羊藿提取物对老龄种公鸡繁殖性能的影响

Table 6 Effects of epimedium extract on reproduction performance of aged breeder roosters (n=6)%

项目
Items		 对照组
Control
group		 试验Ⅰ组
Trial
group Ⅰ		 试验Ⅱ组
Trial
group Ⅱ		 试验Ⅲ组
Trial
group Ⅲ		 均值
标准误
SEM		 P值P-value
处理
Treatment		 线性
Linear		 二次
Quadratic
受精率Fertility 80.56b 84.92ab 89.42a 91.80a 1.58 0.046 0.006 0.725
死胚率Embryonic mortality 5.73a 3.83ab 3.46ab 1.82b 0.47 0.019 0.003 0.872
受精蛋孵化率
Hatchability of fertile eggs		 83.50b 86.71b 89.48ab 93.57a 1.20 0.012 0.001 0.825
入孵蛋孵化率
Hatchability of setting eggs		 69.84b 77.18ab 78.32a 81.12a 1.52 0.044 0.008 0.407
健雏率Healthy chick rate 97.56 98.38 98.54 98.96 0.45 0.762 0.312 0.835
入孵蛋健雏率
Healthy chick rate of setting eggs		 68.25b 76.16ab 76.19ab 79.40a 1.49 0.042 0.010 0.382

3 讨论

3.1 淫羊藿提取物对老龄种公鸡精液品质的影响

精子密度、精子活力、精子畸形率等直接影响精液品质。本试验中,饲粮中添加淫羊藿提取物可以明显提高种公鸡的精液量,与宫永胜等[7]用淫羊藿复合饲料添加剂饲喂种公鸡所得结果相似。本试验测定的公鸡精液pH为7.47~7.63,与王晓娟等[9]测定的黑凤鸡精液pH相似,接近弱碱性。本试验在炎热夏季进行,种禽场有完整的降温设施,但并不能完全避免热应激,试验中种公鸡的精子活力偏低,可能与季节有关,外界温度升高,精子活力相对较低。淫羊藿提取物显著提高老龄种公鸡的精子密度、精子活力,显著降低精子畸形率,说明淫羊藿提取物显著提高了老龄种公鸡的精液品质,与饶永超等[3]在种公鸡上应用淫羊藿复方中药所得结果相似。
精子破裂时渗漏出来的GOT和GPT可作为精子损伤程度的指示物[10-11]。当精子受损数较多时,精子释放到精清中的GOT和GPT活性就高[12]。GOT是催化谷氨酸与草肽乙酸在心肌内转变成α-酮戊二酸和天冬氨酸的关键酶。GPT是催化谷氨酸与丙氨酸之间氨基酸转化过程中的关键酶。研究表明,淫羊藿提取物(淫羊藿苷含量为10%)可以降低红螯螯虾血清中GPT、GOT活性[13]。本试验中,与对照组相比,各试验组种公鸡血清GOT、GPT活性均有不同程度降低,说明淫羊藿提取物能有效降低精子损伤。
活性氧通过使精子细胞膜产生脂质氧化反应,降低细胞膜的流动性,损伤线粒体,降低酶活性,从而降低精液品质。在老龄公鸡中,随着年龄增长,活性氧在睾丸中不断积累,进而使细胞产生氧化应激,精浆中的GSH-Px和SOD可以防止精子氧化应激[14]。童小峰等[15]研究表明,淫羊藿苷可以减少蛋鸡输卵管氧化应激。淫羊藿中含有的黄酮类具有良好的抗氧化活性[16-17],含有的多糖具有较强的羟基自由基清除能力[18⇓⇓⇓-22],具有开发为天然抗氧化剂的潜力。另有研究表明,淫羊藿、黄芪、当归等合理配伍构成的复方中药可以提高产蛋后期蛋鸡的抗氧化能力[23]。本试验中,淫羊藿提取物提高了老龄种公鸡精浆中GSH-Px、T-SOD活性,降低了MDA含量,总体上提高了整个机体的抗氧化性能,从而改善了老龄种公鸡的精液品质。

3.2 淫羊藿提取物对老龄种公鸡生殖内分泌激素的影响

种公鸡生殖内分泌激素对精液品质有重要的调节作用。下丘脑-垂体-睾丸轴(HPT轴)所分泌的激素调控精子的生成。下丘脑分泌抑制激素或GnRH,GnRH激活垂体上的特导性受体,刺激垂体促性腺激素细胞合成和分泌促性腺激素——FSH和LH。FSH作用于支持细胞,促进生精小管上皮细胞基因的表达,从而促进精子的生成[24]。LH与促黄体生成素受体结合激活腺苷酸环化酶,进而增加睾丸间质细胞内环磷腺苷(cAMP)水平,引发细胞内级联反应,最终导致睾丸间质细胞产生T[25-26],促进精子成熟以及睾丸的发育。淫羊藿中含有黄酮,Ding等[27]研究发现,淫羊藿苷(黄酮类)刺激下丘脑-垂体-性腺轴,提高雄性小鼠繁殖性能。秦绪光等[8]研究表明,淫羊藿复方添加剂可以提高种公鸡血浆FSH、LH、T水平;施金虎等[28]研究发现,淫羊藿可显著提高番鸭血清生殖激素FSH、LH和T水平。本试验中,淫羊藿提取物提高了老龄种公鸡血清中FSH、LH、T水平,说明淫羊藿提取物改善了老龄种公鸡的生殖内分泌功能。饶永超等[3]研究表明,淫羊藿复方中草药添加剂显著提高了种公鸡睾丸中T水平;高学勇等[29]研究表明,淫羊藿苷显著提高了环磷酰胺诱导的生精障碍大鼠血清中T水平。垂体和睾丸分泌的激素通过负反馈调节方式作用于下丘脑,与促性腺激素受体和性激素受体结合发挥作用[30],本试验中,淫羊藿提取物降低了老龄种公鸡血清中GnRH水平,原因可能是淫羊藿提取物提高了种公鸡体内T水平,干扰了下丘脑-垂体-性腺轴,抑制了GnRH的释放。

3.3 淫羊藿提取物对老龄种公鸡繁殖性能的影响

精液品质直接影响种公鸡繁殖性能,精子活力、精子密度可以影响受精率[31]。本试验中,淫羊藿提取物提高了老龄种公鸡的种蛋受精率,这与精子活力提高和精子密度增大有关。饶永超等[3]研究表明,淫羊藿复方中草药显著提高了贵州黄鸡的种蛋受精率、受精蛋孵化率、健雏率。入孵蛋孵化率既体现母鸡卵细胞与精子的结合能力,又反映胚胎和雏鸡的存活力。雷涛等[32]研究表明,中草药饲料添加剂淫羊藿散显著提高了安定鹅的受精率、孵化率。本试验中,淫羊藿提取物提高了老龄种公鸡的种蛋受精率、受精蛋孵化率和入孵蛋孵化率,与上述研究结果相似,说明淫羊藿提取物提高了老龄种公鸡的繁殖性能。

4 结论

综上所述,淫羊藿提取物可以通过调节生殖内分泌激素,提高精浆抗氧化能力,减少精子损伤,从而改善精液品质,最终提高老龄种公鸡的繁殖性能。淫羊藿提取物添加量为0.10%时,老龄种公鸡精浆中GSH-Px和T-SOD活性最高;淫羊藿提取物添加量为0.15%时,老龄种公鸡精浆中精浆MDA含量最低,并且精液品质和繁殖性能最佳。

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