动物营养学报 >

2026 , Vol. 38 >Issue 4: 2952 - 2962

DOI: https://doi.org/10.12418/CJAN2026.237

研究论文

饲粮中添加维生素A对长毛兔产毛性能及毛发发育相关基因表达的影响

  • 米巍巍 , 1, 2 ,
  • 孙海涛 2 ,
  • 杨丽萍 2 ,
  • 王威迤 2 ,
  • 刘公言 2 ,
  • 刘晓静 2 ,
  • 高淑霞 2 ,
  • 付冠华 , 1, * ,
  • 白莉雅 , 2, *
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  • 1 河北工程大学生命科学与食品工程学院, 邯郸 056038
  • 2 山东省农业科学院畜牧兽医研究所, 农业农村部畜禽生物组学重点实验室, 济南 250100
*付冠华,讲师,硕士生导师,E-mail: ;
白莉雅,副研究员,硕士生导师,E-mail:

米巍巍(2000—),女,河南滑县人,硕士研究生,从事动物营养与饲料研究。E-mail:

Office editor: 菅景颖

收稿日期: 2025-09-09

  网络出版日期: 2026-04-14

基金资助

山东省特种经济动物产业技术体系(SDARS-21)

国家兔产业技术体系济南综合试验站(CARS-43-G-7)

收起

Effects of Dietary Vitamin A Supplementation on Hair Production Performance and Hair Development-Related Gene Expression of Angora Rabbits

  • MI Weiwei , 1, 2 ,
  • SUN Haitao 2 ,
  • YANG Liping 2 ,
  • WANG Weiyi 2 ,
  • LIU Gongyan 2 ,
  • LIU Xiaojing 2 ,
  • GAO Shuxia 2 ,
  • FU Guanhua , 1, * ,
  • Bai Liya , 2, *
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  • 1 College of Life Science and Food Engineering, Hebei University of Engineering, Handan 056038, China
  • 2 Key Laboratory of Livestock and Poultry Multi-Omics of Ministry of Agriculture and Rural Areas, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Ji’nan 250100, China
*FU Guanhua, lecturer, E-mail: ;
BAI Liya, associate professor, E-mail:

Received date: 2025-09-09

  Online published: 2026-04-14

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摘要

本试验旨在探究饲粮中添加维生素A(VA)对长毛兔产毛性能及毛发发育相关基因表达的影响。选取5月龄体重相近的长毛兔96只,随机分为4组,每组6个重复,每个重复4只(公母各占1/2)。4组长毛兔分别饲喂在基础饲粮基础上额外添加0(Control组)、6 000(VA6组)、12 000(VA12组)、18 000 IU/kg(VA18组)VA的饲粮,试验期为73 d。结果表明:1)与Control组相比,各VA添加组的料毛比显著降低(P<0.05),兔毛的黄度与饱和度显著提升(P<0.05);VA12组兔毛产毛量、长度、亮度、断裂伸长率、弹性回复率及总毛囊密度显著增加(P<0.05),且毛囊细胞凋亡率显著降低(P<0.05)。2)在细胞水平显示,每3×103个毛乳头细胞(DPCs)24 h贴壁后在细胞培养液中添加0.1、0.3、0.5 μg/mL VA均可显著促进DPCs的增殖(P<0.05)。3)与Control组相比,VA12组、VA18组皮肤中VA代谢基因短链脱氢酶/还原酶家族16C成员5(SDR16C5)、醛脱氢酶1家族成员A2(ALDH1A2)的表达显著下调(P<0.05)。4)与Control组相比,VA12组、VA18组皮肤中成纤维细胞生长因子18(FGF18)的表达显著下调(P<0.05),而转化生长因子α(TGFα)的表达显著上调(P<0.05),同时血清中TGFα含量显著升高(P<0.05);此外,VA12组皮肤中角蛋白40(KRT40)及角蛋白相关蛋白16-1(KRTAP16-1)的表达显著上调(P<0.05)。5)与Control组相比,各VA添加组皮肤中谷胱甘肽代谢相关基因ChaC谷胱甘肽特异性γ-谷氨酰环转移酶1(CHAC1)的表达显著上调(P<0.05),以VA12组CHAC1的相对表达量最高,且该组皮肤中胱硫醚-γ裂解酶(CTH)的表达显著上调(P<0.05)。综上可知,VA可通过调控皮肤中VA代谢(SDR16C5、ALDH1A2)、生长因子(TGFα、FGF18)、角蛋白(KRT40、KRTAP16)及谷胱甘肽代谢(CTH、CHAC1)等相关基因的表达,对兔毛生长、毛囊密度及毛色亮度、断裂强度等产毛性能和毛品质指标产生影响。本试验条件下,长毛兔全价饲粮中额外添加VA的适宜水平为12 000 IU/kg。

关键词： 维生素A; 长毛兔; 产毛性能; 毛品质

本文引用格式

米巍巍 , 孙海涛 , 杨丽萍 , 王威迤 , 刘公言 , 刘晓静 , 高淑霞 , 付冠华 , 白莉雅 . 饲粮中添加维生素A对长毛兔产毛性能及毛发发育相关基因表达的影响[J]. 动物营养学报, 2026 , 38(4) : 2952 -2962 . DOI: 10.12418/CJAN2026.237

Abstract

This experiment aimed to investigate the effects of dietary vitamin A (VA) supplementation on hair production performance and the expression of hair development-related genes of Angora rabbits. Ninety-six 5-month-old Angora rabbits with similar body weight were selected and randomly divided into 4 groups, with 6 replicates per group and 4 rabbits per replicate (half male and half female). The four groups were fed a basal diet supplemented with 0 (Control group), 6 000 (VA6 group), 12 000 (VA12 group) and 18 000 IU/kg (VA18 group) of VA, respectively, for a 73-day experimental period. The results showed that: 1) compared with the Control group, the feed-to-wool ratio was significantly reduced (P<0.05) and the yellowness and saturation of rabbit wool were significantly increased in all VA-supplemented groups (P<0.05). The VA12 group showed significant increases in wool production, length, brightness, elongation at break, elastic recovery rate, and total hair follicle density (P<0.05), along with a significant decrease in hair follicle cell apoptosis rate (P<0.05). 2) At the cellular level, the addition of 0.1, 0.3 or 0.5 μg/mL VA in cell culture medium for 3×103 dermal papilla cells (DPCs) 24 hours after plating significantly promoted DPCs proliferation (P<0.05). 3) Compared with the Control group, the expression of VA metabolism genes short-chain dehydrogenase/reductase family 16C member 5 (SDR16C5) and aldehyde dehydrogenase 1 family member A2 (ALDH1A2) in the skin of the VA12 group and VA18 group was significantly down-regulated (P<0.05). 4) Compared with the Control group, the expression of fibroblast growth factor 18 (FGF18) in the skin of the VA12 group and VA18 group was significantly down-regulated (P<0.05), while the expression of transforming growth factor α (TGFα) was significantly up-regulated (P<0.05), accompanied by a significant increase in serum TGFα content (P<0.05). Furthermore, the expression of keratin 40 (KRT40) and keratin-associated protein 16-1 (KRTAP16-1) in the skin of the VA12 group was significantly up-regulated (P<0.05). 5) Compared with the Control group, the expression of the glutathione metabolism-related gene ChaC glutathione-specific γ-glutamylcyclotransferase 1 (CHAC1) in the skin of all VA-supplemented groups was significantly up-regulated (P<0.05), with the VA12 group showing the highest relative expression level of CHAC1. Additionally, the expression of cystathionine γ-lyase (CTH) in the skin of the VA12 group was significantly up-regulated (P<0.05). In conclusion, VA can affect hair production performance and quality indicators including hair growth, follicle density, hair brightness, and breaking strength-by regulating the expression of related genes involved in VA metabolism (SDR16C5, ALDH1A2), growth factors (TGFα, FGF18), keratin (KRT40, KRTAP16), and glutathione metabolism (CTH, CHAC1) in the skin. Under the conditions of this experiment, the recommended supplemental level of VA in complete feed for Angora rabbits is 12 000 IU/kg.

Key words： vitamin A; Angora rabbits; hair production performance; hair quality

我国长毛兔的养殖规模、兔毛产量及出口量均位居全球首位,是世界兔毛生产大国[1-2]。长毛兔所产兔毛具有轻、软、薄等优良特性,是纺织工业中重要的天然原料之一[3]。兔毛的经济价值主要取决于其产毛性能,具体体现在纤维细度、密度、长度、强度及毛色等关键指标[4]。毛发的生长与再生能力直接影响产毛性能,这一过程依赖于毛囊的持续周期性循环。位于毛囊底部毛球区的毛乳头细胞(DPCs)在此过程中发挥核心调控作用,是启动和维持毛发周期、调节毛发再生不可或缺的“指挥中心”[5]。因此,提升长毛兔的产毛性能,深入探究兔毛生长发育的调控机制,已成为该领域研究的重点方向。
维生素A(VA)在动物皮肤、毛发和皮脂腺的发育和维持中起着重要作用。研究表明,VA可显著促进小鼠皮肤损伤的修复[6],局部应用可增加胡须毛发密度[7]。Nan等[8]研究发现,VA通过转化生长因子β2(TGFβ2)通路调控DPCs的增殖和凋亡,从而影响毛囊的生长。此外,VA以剂量依赖方式调控毛囊干细胞(HFSCs),不仅参与调节毛发周期、伤口愈合与黑色素干细胞功能[9],还在协调皮肤损伤应答与毛发再生之间的平衡中发挥重要作用[10],并能通过激活HFSCs有效改善雄激素性脱发[11]。岳正凯[12]研究指出,在獭兔基础饲粮中额外添加12 000 IU/kg VA,可通过胰岛素样生长因子-1(IGF-1)信号通路调节热应激条件下DPCs的发育,显著提高兔毛长度与毛囊密度。综上所述,VA在促进动物毛发生长和提升毛发品质方面潜力显著,然而目前针对长毛兔的相关研究仍显不足,其具体作用机制亦有待进一步阐明。基于此,本试验在基础饲粮中额外添加不同水平的VA,拟通过探讨不同添加水平VA对长毛兔产毛性能及毛发发育相关基因表达的影响,为进一步阐释长毛兔产毛性能的调控机制提供依据,同时为优化长毛兔饲粮配方、提升其产毛性能提供理论参考。

1 材料与方法

1.1 试验设计

本试验经山东省农业科学院畜牧兽医所实验动物伦理委员会批准(IASM-2024-032)。以獭兔与长毛兔杂交选育的长毛兔F7代为研究对象,选取96只5月龄体重相近的个体,剪毛后称取体重,根据体重随机分为4组,分别为Control、VA6、VA12、VA18组,每组6个重复,每个重复4只,公母各占1/2。Control组饲喂基础饲粮,基础饲粮参考Lebas等[13]推荐的长毛兔营养需要配制,其组成及营养水平见表1。VA6、VA12、VA18组在基础饲粮中分别额外添加6 000、12 000和18 000 IU/kg的VA。饲养试验覆盖整个第3养毛期,共持续73 d。
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 10.0
豆粕Soybean meal 11.0
玉米胚芽粕Corn germ meal 8.0
次粉Wheat middlings 7.0
小麦麸皮Wheat bran 15.0
葵花粕Sunflower meal 7.5
豆油Soybean oil 0.5
羊草Chinese wildrye 10.0
黄蒿颗粒Artemisia granules 6.0
豆秸粉Bean straw powder 10.0
稻壳粉Rice hull powder 10.0
预混料Premix1) 5.0
合计Total 100.0
营养水平Nutrient levels2)
干物质DM 81.65
消化能DE/(MJ/kg) 9.01
粗蛋白质CP 15.79
粗脂肪EE 2.76
粗纤维CF 18.39
中性洗涤纤维NDF 34.07
酸性洗涤纤维ADF 20.95
酸性洗涤木质素ADL 3.88
粗灰分Ash 10.50
钙Ca 0.84
磷P 0.64
赖氨酸Lys 1.32
蛋氨酸Met 0.27
胱氨酸Cys 0.97

1)预混料为每千克饲粮提供 The premix provided the following per kg of the diet:VA 10 500 IU,VD3 1 800 IU,VE 18 IU,VK3 1.2 mg,VB1 2.7 mg,VB2 6 mg,VB6 3 mg,VB12 0.021 mg,叶酸 folic acid 1.2 mg,烟酰胺 niacinamide 24 mg,D-泛酸钙 D-calcium pantothenate 12 mg,D-生物素 D-biotin 0.06 mg。

2)消化能依据《肉兔营养需要量》(NY/T 4049—2021)中各原料对应数值计算,干物质(GB/T 6435—2014)、粗蛋白质(GB/T 6432—2018)、粗脂肪(GB/T 6433—2006)、粗纤维(GB/T 6434—2006)、中性洗涤纤维(GB/T 20806—2022)、酸性洗涤纤维(NY/T 1459—2022)、酸性洗涤木质素(GB/T 20805—2006)、粗灰分(GB/T 6438—2007)、钙(GB/T 6436—2018)、磷(GB/T 6437—2018)、氨基酸(GB/T 18246—2019)参照对应的国家标准进行测定。DE was calculated according to the corresponding value of each raw material in Nutrient Requirements of Meat Rabbis (NY/T 4049—2021), while DM (GB/T 6435—2014), CP (GB/T 6432—2018), EE (GB/T6433—2006), CF (GB/T 6434—2006), NDF (GB/T 20806—2022), ADF (NY/T 1459—2022), ADL (GB/T 20805—2006), Ash (GB/T 6438—2007), Ca (GB/T 6436—2018), P (GB/T 6437—2018) and amino acid (GB/T 18246—2019) were determined according to the corresponding national standards.

试验期间,试验兔饲养于开放式兔舍,采用单笼饲养模式,兔舍保持自然采光与通风,定期进行消毒。试验兔自由采食和饮水,并按常规程序完成免疫接种。整个养毛期内,每日记录环境温湿度(温度6.0~16.3 ℃,相对湿度45.2%~72.4%),并统计各重复的总采食量。

1.2 样本采集

73 d的养毛期结束时,禁食禁水12 h,统一剪毛,测定所有试验兔的体重、产毛量。从每组的每个重复中选取2只试验兔,在体测定背十字部兔毛的色差值。从每组每个重复中另选取1只试验兔,耳动脉采血,4 ℃下3 300×g离心10 min,取上清液,-80 ℃保存,用血清中生长因子含量的测定;采血后采集背十字部兔毛,用于毛品质指标测定;采集背十字部皮肤样本2份,一份固定在4%多聚甲醛溶液中,用于毛囊形态指标的测定,另一份-80 ℃保存,用于毛发发育相关基因表达的测定。

1.3 检测指标

1.3.1 生产性能

记录整个第3养毛期试验兔的初始体重及终末体重,统计产毛量和耗料量,计算平均日增重、平均日采食量和料毛比。

1.3.2 毛品质

参照刘策等[14]报道的方法,使用高密度手持色差仪测定兔毛的色差值。根据GB/T 13835-2009,采用手排法用游标卡尺测量兔毛的长度;使用元茂YM-2X纤维细度仪检测兔毛的细度;使用元茂YM-06E/PC电子式气动单纤维强力仪测定兔毛的断裂强度、断裂伸长率、弹性回复率。

1.3.3 毛囊形态及细胞凋亡率

将固定后的皮肤样本经脱水、浸蜡、包埋等步骤制成石蜡块,切片后依次进行贴片、脱蜡、染色及封片,最后进行显微镜观察。采用苏木精-伊红(HE)染色后测量毛囊密度与直径;采用TUNEL染色半定量分析细胞凋亡率,TUNEL阳性细胞在荧光显微镜下呈现绿色荧光,以同一视野下经DAPI蓝染的细胞总数作为总细胞数。细胞凋亡率计算公式为:
细胞凋亡率(%)=(TUNEL阳性细胞数/总细胞数)×100。

1.3.4 细胞增殖率

将实验室保存的长毛兔DPCs进行复苏后,放入含有1%双抗、10%胎牛血清(FBS)的DMEM/F-12细胞培养液,以每孔3×103个细胞铺于96孔板,37 ℃、5% CO2培养箱培养24 h贴壁后,分别更换为含不同浓度(0、0.1、0.3、0.5 μg/mL)VA的上述细胞培养液,每孔100 μL,继续培养48 h,每孔加10 μL CCK-8,3 h后用酶标仪测定450 nm处的吸光度(OD450)值。

1.3.5 皮肤中毛发发育相关基因

采用Trizol法提取皮肤总RNA,检测浓度和纯度合格后,试验TOYOBO FSQ-301反转录试剂盒合成cDNA。反应体系按照TOYOBO QPS-201荧光定量试剂盒进行操作。同一样品进行3个重复,以甘油醛-3-磷酸脱氢酶(GADPH)为内参基因,采用2-ΔΔCt法计算目的基因短链脱氢酶/还原酶家族16C成员5(SDR16C5)、醛脱氢酶1家族成员A2(ALDH1A2)、胱硫醚-γ裂解酶(CTH)、ChaC谷胱甘肽特异性γ-谷氨酰环转移酶1(CHAC1)、角蛋白40(KRT40)、角蛋白相关蛋白16-1(KRTAP16-1)、转化生长因子α(TGFα)、成纤维细胞生长因子18(FGF18)的mRNA相对表达量。内参基因和目的基因的实时荧光定量PCR引物信息见表2。
表2 实时荧光定量PCR引物信息

Table 2 Primer information of RT-qPCR

基因名称
Gene names		 引物序列
Primer sequence (5'—3')		 登录号
Accession
number		 产物大小
Product size/bp
甘油醛-3-磷酸脱氢酶
GADPH		 F:GCCGCTTCTTCTCGTGCAG
R:ATGGATCATTGATGGCGACAACAT		 NM_001082253.1 145
短链脱氢酶/还原酶家族16C成员5
SDR16C5		 F:CTTCTCGGCGGACAGCAA
R:GCGGCCTTGGGATTATGG		 XM_002710486.5 151
醛脱氢酶1家族成员A2
ALDH1A2		 F:ACCTCGAAATCAAGTACACC
R:ATCTGCCTTGTCTGCTTCT		 XM_002717704.5 134
胱硫醚-γ裂解酶
CTH		 F:TTCCAACATTTCGCCACG
R:GAGCCGCCACTGCTTTCT		 XM_008265065.4 190
ChaC谷胱甘肽特异性γ-谷氨
酰环转移酶1
CHAC1		 F:GGGAGACACCTTCCATCGG
R:TCTGTCACTTGCTCACCTCGTA		 XM_002717785.5 120
角蛋白40
KRT40		 F:GCAAGATCCGAGAACAGT
R:AAGTCCTCAGCAGCCAGT		 XM_002719141.5 166
角蛋白相关蛋白16-1
KRTAP16-1		 F:GCCACTCTGTCTGCTGTGAGC
R:CGGTAGGCAAAGGAGGGTCT		 XM_070060708.1 196
转化生长因子α
TGFα		 F:CTGCCTCGCCAGAGCCTTTT
R:TGGGAGTCTGGGCAGTCGTT		 XM_008254281.4 262
成纤维细胞生长因子18
FGF18		 F:CGCCCAGCGATGTATTCT
R:TGCGGAAGTCCACGTTCT		 XM_002710377.5 112

1.3.6 血清中生长因子含量

使用上海酶联生物科技有限公司生产的酶联免疫吸附试验(ELISA)试剂盒(货号:ml027304与ml028067),测定血清中TGFα和IGF-1含量。

1.4 数据统计与分析

使用Image Pro Plus 6.0软件对TUNEL染色切片进行半定量分析。试验数据采用R软件的agricolae包进行单因素方差分析,结合Duncan氏法对组间的差异显著性进行检验。数据以平均值±标准差表示,P<0.05为差异显著。采用GraphPad Prism 9.5软件进行数据及图形处理。

2 结果

2.1 VA对长毛兔生产性能的影响

由表3可知,与Control组相比,VA6、VA12组的ADFI显著降低(P<0.05);VA6、VA12、VA18组的ADG无显著变化(P>0.05),但料毛比显著降低(P<0.05),其中VA12组降低更为显著;此外,各VA添加组的产毛量都有所提高,且VA12组产毛量显著提高(P<0.05)。
表3 VA对长毛兔生产性能的影响

Table 3 Effects of VA on performance of Angora rabbits

项目
Items		 组别Groups P值
P-value
Control VA6 VA12 VA18
平均日增重ADG/g 7.27±2.52 6.52±3.49 6.21±3.44 7.53±3.60 0.538
平均日采食量ADFI/g 159.53±3.54a 150.66±6.27bc 148.01±8.46c 156.52±2.50ab 0.021
产毛量Wool yield/g 227.30±49.20b 248.87±45.07ab 268.56±58.12a 259.83±33.22ab 0.049
料毛比Feed/wool ratio 65.92±13.67a 56.02±9.11b 51.99±11.73b 55.10±7.41b 0.015

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.

2.2 VA对长毛兔毛品质的影响

由表4可知,与Control组相比,VA6、VA12和VA18组兔毛的细度无显著变化(P>0.05),但VA12组兔毛的长度显著增加(P<0.05);VA12、VA18组兔毛的亮度显著增加(P<0.05);VA6、VA12、VA18组兔毛的红度、断裂强度无显著变化(P>0.05),但黄度与饱和度显著增加(P<0.05);VA12组兔毛的断裂伸长率和弹性回复率显著增加(P<0.05)。
表4 VA对长毛兔毛品质的影响

Table 4 Effects of VA on hair quality of Angora rabbits

项目
Items		 组别Groups P值
P-value
Control VA6 VA12 VA18
长度Length/cm 6.18±0.51b 6.55±0.63ab 7.32±0.49a 6.85±0.87ab 0.040
细度Fineness/μm 15.42±1.07 15.21±1.10 15.29±1.25 15.56±1.51 0.964
亮度L* 89.68±3.41c 91.18±4.09bc 93.78±3.99ab 95.53±4.27a 0.016
红度a* -2.70±0.40 -3.07±0.30 -2.12±1.18 -2.48±1.01 0.286
黄度b* 10.43±1.07b 11.73±1.21a 11.69±1.41a 12.03±1.10a 0.016
饱和度C* 10.79±1.11b 12.13±1.25a 12.01±1.15a 12.32±1.19a 0.025
断裂强度
Breaking tenacity/(cN/dtex)		 0.20±0.06 0.23±0.06 0.27±0.04 0.24±0.60 0.220
断裂伸长率Elongation at break/% 33.65±3.68b 31.83±4.17b 37.90±2.92a 33.10±3.03b 0.037
弹性回复率Elastic recovery rate/% 40.55±5.41b 49.87±8.84ab 54.26±7.29a 52.25±10.80a 0.046

2.3 VA对长毛兔毛囊形态、细胞凋亡率及DPCs增殖的影响

由表5可知,与Control组相比,VA6、VA12和VA18组长毛兔初级毛囊直径、次级毛囊直径和次级毛囊密度无显著变化(P>0.05),但VA12组次级毛囊密度最高,且总毛囊密度与初级毛囊密度显著增加(P<0.05)。图1-A为背十字部皮肤HE染色切片图。
表5 VA对长毛兔毛囊形态的影响

Table 5 Effects of VA on hair follicles morphology of Angora rabbits

项目
Items		 组别Groups P值
P-value
Control VA6 VA12 VA18
初级毛囊直径
Primary follicle diameter/μm		 104.91±15.95 93.86±11.91 91.78±17.62 101.32±10.47 0.152
次级毛囊直径
Secondary follicle diameter/μm		 45.19±6.19 45.85±4.37 46.69±3.41 48.76±4.97 0.391
初级毛囊密度
Primary follicle density/(个/mm2)		 3.89±0.92b 3.98±1.04b 5.45±0.65a 4.62±1.10ab 0.034
次级毛囊密度
Secondary follicle density/(个/mm2)		 187.98±25.10 198.26±36.22 230.77±27.09 225.12±27.65 0.056
总毛囊密度
Total follicle density/(个/mm2)		 191.87±25.49b 202.24±35.74ab 236.21±27.68a 229.75±27.97a 0.048
图1 VA对长毛兔毛囊形态、细胞凋亡率及毛乳头细胞增殖的影响

A:HE染色分析毛囊形态(用于毛囊密度和直径测定);B:TUNEL染色分析毛囊细胞凋亡情况;C:毛囊细胞凋亡率(以阳性细胞占比表示);D:毛乳头细胞增殖(以OD450值表示)。Control、VA6、VA12和VA18分别指代Control、VA6、VA12和VA18组。数据柱形标注不同小写字母表示差异显著(P<0.05)。下图同。

Fig.1 Effects of VA on hair follicle morphology, cell apoptosis rate and DPCs proliferation of Angora rabbits

A: analysis of hair follicle morphology via HE staining (for determining hair follicle density and diameter); B: analysis of hair follicle cell apoptosis via TUNEL staining; C: apoptosis rate of hair follicle cells (expressed as the percentage of positive cells); D: proliferation of DPCs (expressed as OD450value). Control, VA6, VA12 and VA18 denoted the Control group, VA6 group, VA12 group and VA18 group, respectively. Data columns marked with different lowercase letters indicated significant difference (P<0.05). The same as below.

图1-B为背十字部皮肤TUNEL染色结果,TUNEL阳性细胞在荧光显微镜下呈现绿色荧光,阳性细胞可代表发生凋亡的细胞。图1-C可知,与Control组相比,VA12组细胞凋亡率显著降低(P<0.05),VA6和VA18组无显著变化(P>0.05)。由图1-D可知,在体外培养液中添加0.1、0.3、0.5 μg/mL VA可以显著促进DPCs的增殖(P<0.05)。

2.4 VA对长毛兔皮肤中毛发发育相关基因表达的影响

由图2可知,与Control组相比,VA6组皮肤中ALDH1A2、FGF18的相对表达量显著下调(P<0.05),SDR16C5、CHAC1、KRTAP16-1的相对表达量显著上调(P<0.05);VA12组皮肤中SDR16C5、ALDH1A2、FGF18的相对表达量显著下调(P<0.05),CTH、CHAC1、KRT40、KRTAP16-1、TGFα的相对表达量显著上调(P<0.05);VA18组皮肤中SDR16C5、ALDH1A2、FGF18的相对表达量显著下调(P<0.05),CHAC1、KRTAP16-1、TGFα的相对表达量显著上调(P<0.05)。
图2 VA对长毛兔皮肤中毛发发育相关基因表达的影响

SDR16C5:脱氢酶/还原酶家族16C成员5 short-chain dehydrogenase/reductase family 16C member 5;ALDH1A2:醛脱氢酶1家族成员A2 aldehyde dehydrogenase 1 family member A2;CTH:胱硫醚-γ裂解酶 cystathionine γ-lyase;CHAC1:ChaC谷胱甘肽特异性γ-谷氨酰环转移酶1 ChaC glutathione-specific γ-glutamylcyclotransferase 1;KRT40:角蛋白40 keratin 40;KRTAP16-1:角蛋白相关蛋白16-1 keratin-associated protein 16-1;TGFα:转化生长因子α transforming growth factor α;FGF18:成纤维细胞生长因子18 fibroblast growth factor 18。

Fig.2 Effects of VA on expression of hair development-related genes in skin of Angora rabbits

2.5 VA对长毛兔血清中生长因子含量的影响

由表6可知,各组长毛兔血清中IGF-1含量无显著差异(P>0.05),但VA12组在数值上相较Control组有明显增加;各VA添加组血清中TGFα含量与Control组相比均显著增加(P<0.05),但各VA添加组之间差异不显著(P>0.05)。
表6 VA对长毛兔血清中生长因子含量的影响

Table 6 Effects of VA on serum growth factor contents of Angora rabbits pg/mL

项目
Items		 组别Groups P值
P-value
Control VA6 VA12 VA18
胰岛素样生长因子-1 IGF-1 174.05±43.80 183.96±34.84 209.54±26.56 192.56±35.99 0.390
转化生长因子α TGFα 4.71±1.72b 8.87±3.30a 8.92±2.30a 9.34±2.13a 0.011

3 讨论

3.1 VA对长毛兔VA代谢的影响

VA在毛囊发育、毛发生长周期以及毛发质量的形成过程中,最主要活性形式是全反式视黄酸(ATRA)[11]。其生物合成依赖2步酶促反应:1)限速步骤——视黄醇脱氢酶催化全反式视黄醇(ROL)氧化为全反式视黄醛(RAL);2)终末转化——视黄醛脱氢酶将RAL氧化为ATRA。Goggans等[15]发现,SDR16C5是皮肤中主要的视黄醇脱氢酶,主导ROL向RAL转化,可调节毛囊周期、毛发成分和HFSCs标志物。SDR16C5/短链脱氢酶/还原酶家族16C成员6(SDR16C6)双敲除小鼠表现毛发生长加速[16]。此外,Adams等[17]研究发现,ATRA含量的升高会使SDR16C5基因显著下调。视黄醛脱氢酶ALDH1A2在毛囊的外根鞘、基底层及隆起区表达,是RAL向ATRA转化的关键酶[18]。Suo等[19]研究表明,SDR16C5和ALDH1A2定位于毛囊,通过抑制HFSCs功能维持休止期,参与毛囊的生长周期调节。本研究中,饲粮中添加12 000与18 000 IU/kg VA显著下调了长毛兔皮肤中SDR16C5和ALDH1A2的表达,与上述调控机制一致。这表明本试验中添加上述2个水平的VA影响了皮肤中VA的代谢,可能通过下调SDR16C5、ALDH1A2的表达减轻其对HFSCs的抑制作用,从而激活毛囊生长周期,最终提升毛发生长速率与产毛量。

3.2 VA对长毛兔产毛量的影响

产毛量是衡量毛兔经济效益的重要指标,而料毛比则直接反映其产毛效率[20-21]。在本试验条件下,各VA添加组均表现出产毛量增加与料毛比显著下降的趋势。其中,饲粮中添加12 000 IU/kg VA 时效果最为显著,该组不仅ADFI显著降低、料毛比最低,同时兔毛长度、产毛量及总毛囊密度均显著提高。细胞水平测定结果进一步显示,饲粮中添加12 000 IU/kg VA能够显著降低细胞凋亡率,且体外试验表明添加0.1、0.3、0.5 μg/mL VA可以显著促进DPCs的增殖,这与前人关于VA通过调节DPCs发育增加毛发长度与毛囊密度的研究结论[12]一致。因此,推断VA对毛发的调控作用可能与抑制毛囊细胞凋亡、促进DPCs增殖有关。IGF-1是毛发生长的关键正向调节因子[22],对促进长毛兔毛囊发育、提高产毛量具有重要作用[23]。本研究中,各VA添加组血清中IGF-1含量上升,皮肤中FGF18的表达则相应下调,其中以添加12 000 IU/kg VA的组变化最为明显。这表明VA可能通过提高皮肤中IGF-1含量并抑制FGF18表达,共同促进毛囊生长期延长与毛囊密度增加[24-25]。此外,各VA添加组长毛兔皮肤中TGFα的表达上调且血清中TGFα含量显著升高。TGFα属于表皮生长因子(EGF)家族,可通过结合表皮生长因子受体(EGFR)激活丝裂原活化蛋白激酶(MAPK)等信号通路,参与毛囊形态发生与细胞增殖[26],对维持毛发的正常发育与形态塑造具有关键作用[27-29]。综上所述,VA可能通过协同调控IGF-1、FGF18及TGFα等基因的表达,协同改善毛囊发育与毛发产量。本研究结果为进一步优化长毛兔产毛性能、开展精准营养调控提供了新的理论依据。

3.3 VA对长毛兔皮肤抗氧化能力及毛品质的影响

毛色是评价毛品质的重要指标,亮度与饱和度直接影响毛发的商业价值与视觉表现[14]。已有研究指出,VA缺乏会导致毛发稀疏、毛色暗淡等问题[30]。本试验结果显示,在饲粮中添加12 000 IU/kg VA可显著上调长毛兔皮肤中谷胱甘肽代谢相关基因CTH和CHAC1的表达。CTH催化同型半胱氨酸裂解为半胱氨酸,进而参与谷胱甘肽的合成,在机体内发挥抗氧化等关键作用[31]。CHAC1作为谷胱甘肽代谢中的重要调节酶,主要维持细胞内氧化还原平衡,从而间接影响毛发生长[32]。研究表明,提升幼龄绒山羊的抗氧化能力有助于减轻皮肤氧化应激,抑制毛囊细胞凋亡并促进毛囊发育,最终改善羊绒的产量与品质[33]。结合本试验结果,饲粮中添加12 000 IU/kg VA显著提高了兔毛的亮度与饱和度,同时伴随总毛囊密度显著上升与毛囊细胞凋亡率显著下降,说明VA可能通过上调CTH和CHAC1的表达,增强皮肤的抗氧化能力,改善毛色性状,并促进毛囊的发育。
南韦肖[34]研究发现,在水貂饲粮中添加20 000 IU/kg VA可改善其毛品质。本研究进一步显示,在长毛兔饲粮中添加12 000 IU/kg VA能显著提高兔毛的长度、断裂伸长率与弹性回复率,并使其断裂强度达到最佳水平;而添加18 000 IU/kg VA则导致兔毛的断裂伸长率下降,表明适宜剂量的VA具有促进毛发生长、增强毛发韧性的作用。角蛋白作为毛发的主要结构蛋白,与毛发的颜色[35]及力学性能[36]密切相关,是维持毛发强韧与光泽的关键物质。KRT40与KRTAP16-1均为与角蛋白结构和功能相关的基因,前者参与毛囊发育与角质形成细胞增殖[37-38],后者不仅在小鼠毛发生长中发挥作用[39],亦与KRT40等基因在长毛兔细毛中共同高表达[40],提示二者协同参与细毛的生长与分化。本试验中,饲粮中添加12 000 IU/kg VA显著上调了皮肤组织中KRT40与KRTAP16-1的表达,推测VA可能通过调控角蛋白相关基因的表达,促进毛发生长,维持其机械强度,进而提升兔毛品质。

4 结论

综上所述,VA可通过调控皮肤中SDR16C5、ALDH1A2、TGFα、FGF18、CTH、CHAC1、KRT40及KRTAP16等关键基因的表达,促进长毛兔兔毛生长、增加毛囊密度并最终改善毛品质。在本试验条件下,长毛兔全价饲粮中额外添加VA的适宜水平为12 000 IU/kg。

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