Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 8: 5475 - 5485

DOI: https://doi.org/10.12418/CJAN2025.444

RESEARCH PAPER

Response of Fibroblast Growth Factor 23/Klotho Axis to Dietary Vitamin D3 Deficiency or Excess and Its Effects on Egg Quality in Laying Hens

  • WANG Mengyao , 1 ,
  • CAO Linpeng 1 ,
  • QIN Lin 1 ,
  • ZHU Zijun 1 ,
  • LIU Fu 1 ,
  • XIE Yuesheng 1 ,
  • GUO Hui 2 ,
  • CAO Manhu , 1, *
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  • 1 College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China
  • 2 Guangzhou Yiru Biotechnology Co., Ltd., Guangzhou 510700, China
*professor, E-mail:

Received date: 2024-12-27

  Online published: 2025-08-14

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Abstract

This study was conducted to investigate the correlation between dietary vitamin D3 deficiency or excess and vitamin D metabolism, as well as its effects on egg quality of laying hens, from the perspective of the fibroblast growth factor 23/Klotho (FGF23/Klotho) axis. A single-factor completely randomized design was adopted. Ninety-six 42-week-old Roman pink laying hens were selected and randomly divided into 4 groups with 8 replicates per group and 3 hens per replicate. Hens in each group were fed basal diets supplemented with 0 (deficiency), 2 000 (low level), 4 000 (normal level) and 8 000 IU/kg (excess) vitamin D3, respectively. The experimental period lasted for 4 weeks. The results showed as follows: 1) in the 0 IU/kg group, eggshell strength and eggshell proportion of laying hens were lower than those in the 2 000 and 4 000 IU/kg groups, though the difference was not significant (P>0.05); tibia defatted weight was significantly lower than that in the 2 000 and 4 000 IU/kg groups (P<0.05); serum alkaline phosphatase activity was significantly higher than that in the other three groups (P<0.05), while serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and FGF23 levels were significantly lower than those in the other three groups (P<0.05), serum Klotho content was also lower than that in the other three groups, but the difference was not significant (P>0.05). The mRNA relative expression levels of nuclear vitamin D receptor (nVDR) in duodenum of laying hens in the 0 and 2 000 IU/kg groups were significantly lower than those in the 4 000 and 8 000 IU/kg groups (P<0.05), and the mRNA relative expression level of sodium-dependent type Ⅱb phosphate transporter (Na/Pi-Ⅱb) in duodenum was significantly lower than that in the 8 000 IU/kg group (P<0.05); the mRNA relative expression levels of FGF23 and cytochrome P450 family 27 subfamily B member 1 (CYP27B1) in kidney in the 0 IU/kg group were significantly higher than those in the other three groups (P<0.05), and the mRNA relative expression level of Klotho in kidney was significantly higher than that in the 2 000 and 8 000 IU/kg groups (P<0.05). 2) In the 8 000 IU/kg group, eggshell strength, eggshell proportion, and tibia ash weight of laying hens were lower than those in the 2 000 and 4 000 IU/kg groups, but the differences were not significant (P>0.05); tibia defatted weight was significantly lower than that in the 2 000 and 4 000 IU/kg groups (P<0.05); serum 1,25(OH)2D3 content was significantly lower than that in the 4 000 IU/kg group (P<0.05), and serum FGF23 and Klotho contents were also lower than those in the 4 000 IU/kg group, but the differences were not significant (P>0.05). The mRNA relative expression levels of membrane vitamin D receptor (mVDR), nVDR, and Na/Pi-Ⅱb in duodenum of laying hens in the 8 000 IU/kg group were significantly higher than those in the other three groups (P<0.05); there were no significant differences in the relative mRNA expression levels of FGF23, Klotho, and CYP27B1 in kidney between the 8 000 IU/kg group and the 2 000 and 4 000 IU/kg groups (P>0.05). In conclusion, both dietary vitamin D3 deficiency and excess can lead to decline in egg quality, which is related to the FGF23/Klotho axis and vitamin D metabolism. However, the response indices and regulatory mechanisms in laying hens differ between these two conditions. Under dietary vitamin D3 deficiency, FGF23,Klotho and CYP27B1 in kdiney of laying hens act as internal response indices to participate in the regulation of egg quality and calcium-phosphorus metabolism. In contrast, under dietary vitamin D3 excess, nVDR, mVDR and Na/Pi-Ⅱb in duodenum of laying hens serve as the internal response indices involved in regulating egg quality and calcium-phosphorus metabolism.

Key words： laying hens; vitamin D3; fibroblast growth factor 23/Klotho axis; egg quality

Cite this article

WANG Mengyao , CAO Linpeng , QIN Lin , ZHU Zijun , LIU Fu , XIE Yuesheng , GUO Hui , CAO Manhu . Response of Fibroblast Growth Factor 23/Klotho Axis to Dietary Vitamin D3 Deficiency or Excess and Its Effects on Egg Quality in Laying Hens[J]. Chinese Journal of Animal Nutrition, 2025 , 37(8) : 5475 -5485 . DOI: 10.12418/CJAN2025.444

维生素D是一种动物生长所必需的脂溶性维生素,在家禽体内具有重要的生理功能,尤其在维持钙磷代谢平衡、促进骨骼健康以及改善蛋品质等方面发挥着关键作用[1-2]。在蛋鸡饲养过程中,维生素D起着至关重要的作用,它不仅能够改善蛋鸡的生产性能和蛋壳质量,还能通过与维生素D受体(VDR)结合,调控下游基因的表达,从而对钙、磷的吸收及利用产生重要影响[3]。目前,关于维生素D影响和调控蛋品质的作用机制的研究,主要局限于其对钙、磷吸收的直接作用层面。近年来的研究表明,维生素D调节钙、磷吸收的作用与成纤维细胞生长因子23(fibroblast growth factor 23,FGF23)/克洛索蛋白(Klotho)轴存在密切关系[4-5]。FGF23被认为是继维生素D之后发现的又一调控钙、磷吸收的重要因子[6-7],医学上有研究发现,FGF23需要与Klotho形成协同受体发挥作用[8-9],因此Klotho也被共同认为是调控维生素D代谢和钙、磷吸收的重要因素,由于FGF23和Klotho之间的这种协同作用,它们被统称为FGF23/Klotho轴[10-11]。但是目前在蛋鸡生产领域关于FGF23/Klotho轴的研究相对较少。Yan等[12]和Ren等[13]研究发现,FGF23在蛋鸡中通过抑制肠道和肾脏对磷的吸收,维持体内的磷平衡。此外,FGF23/Klotho轴还可能与蛋鸡的骨骼健康和蛋壳形成有关,特别是在钙磷代谢失衡的情况下。因此,本研究以产蛋高峰期罗曼粉蛋鸡为试验对象,通过分析饲粮中添加不同水平维生素D3时血清中FGF23和Klotho含量的变化情况、对维生素D α-羟化过程的影响及其与1,25-二羟基维生素D3[1,25(OH)2D3]之间的关系,探究蛋鸡饲粮维生素D3缺乏或过量与维生素D代谢的相关性及其对蛋品质的影响,为生产中由于饲粮维生素D3缺乏或产蛋后期导致的蛋品质下降,提供调控因素和作用机制研究的切入点。

1 材料与方法

1.1 伦理声明

本研究采用的所有动物试验方案均符合《实验动物 福利伦理审查指南》(GB/T 35892—2018),并经湖南农业大学生物医学研究伦理委员会批准,批准编号为2024179。

1.2 试验材料

试验所用罗曼粉蛋鸡购自湖南某农牧有限责任公司,维生素D3(规格为50万IU/g)购自江苏某生物科技有限公司。

1.3 试验设计

采用单因素完全随机设计,选取96只42周龄罗曼粉蛋鸡,随机分为4组,每组8个重复,每个重复3只鸡。参照《罗曼粉商品蛋鸡饲养管理手册》并结合生产实际及维生素D3的相关特性,各组试验鸡分别饲喂在基础饲粮中添加0(缺乏)、2 000(低水平)、4 000(正常水平)和8 000 IU/kg(过量)维生素D3的饲粮。试验期4周。基础饲粮为参照NRC(1994)和我国《鸡饲养标准》(NY/T 33—2004)配制的粉料,其组成及营养水平见表1。
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 60.18
豆粕Soybean meal 24.50
鱼粉Fish meal 3.00
大豆油Soybean oil 0.50
DL-蛋氨酸DL-Met 0.07
磷酸氢钙CaHPO4 0.59
石粉Limestone 9.61
沸石粉Zeolite powder 0.62
食盐NaCl 0.30
氯化胆碱Choline chloride 0.10
植酸酶Phytase 0.03
矿物质预混料Mineral premix1) 0.20
维生素预混料Vitamin premix2) 0.30
合计Total 100.00
营养水平Nutrient levels3)
代谢能ME/(MJ/kg) 11.09
粗蛋白质CP 17.56
赖氨酸Lys 0.94
蛋氨酸Met 0.35
钙Ca 3.61
总磷TP 0.53
有效磷AP 0.36

1)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 25 mg,Cu 5 mg,Mn 100 mg,Zn 60 mg,Se 0.2 mg,I 0.5 mg,Co 0.1 mg。
2)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 10 000 IU,VE 20 mg,VK 3 mg,VB1 1 mg,VB2 4 mg,VB6 3 mg,VB12 15 mg,D-泛酸 D-pantothenic acid 8 mg,叶酸 folic acid 0.5 mg,烟酸 nicotinic acid 30 mg,生物素 biotin 25 mg。
3)粗蛋白质、钙和总磷为实测值,分别参考GB/T 6432—2018、GB/T 6436—2018和GB/T 6437—2018测定;其余为计算值,根据《中国饲料成分及营养价值表(2018年第29版)》计算得出。CP, Ca and TP were measured values, which were determined according to GB/T 6432—2018,GB/T 6436—2018 and GB/T 6437—2018, respectively; while the others were calculated values according to Tables of Feed Composition and Nutritive Values in China (29th edition, 2018).

1.4 饲养管理

试验在湖南农业大学实训楼进行。试验前对鸡舍、鸡笼、料筒和水管进行清洗和消毒,通风1星期后开始试验。采用密闭鸡舍3层阶梯式笼养,4组共32个重复随机分布于鸡舍各个位置,每天光照16 h,鸡舍温度控制在30~35 ℃,湿度控制在50%~80%,通风方式为自然通风和负压通风相结合。试验期间鸡只自由采食和饮水,定期清扫圈舍和消毒,每日观察圈舍情况(包括光照、温度、湿度、通风、饲粮和水消耗)及鸡只健康状况。

1.5 样品采集与指标测定

1.5.1 生产性能

以重复为单位记录每天的耗料量、产蛋数和蛋重,计算试验全期的平均日采食量、平均蛋重、料蛋比、产蛋率。相关计算公式如下:

平均日采食量(g)=总耗料量/(鸡只数×试验天数);

平均蛋重(g)=总蛋重/总产蛋数;

料蛋比=总耗料量/总蛋重;

产蛋率(%)=[总产蛋数/(鸡只数×试验天数)]×100。

1.5.2 蛋品质

试验第4周时,每个重复选取3枚接近平均蛋重的鸡蛋,进行蛋品质的测定。采用蛋壳强度测定仪(EFR-01,Ork公司,以色列)测定蛋壳强度;采用蛋壳厚度测定仪(NFN380,FHK公司,日本)测定蛋壳厚度;称量蛋重和蛋壳重,并计算蛋壳比重;使用塑料蛋液分离器分离蛋清与蛋黄,随后使用电子分析天平称量蛋黄重,并计算蛋黄比重;使用游标卡尺测定鸡蛋长径和短径值,并计算蛋形指数[14]。相关计算公式如下:

蛋壳比重(%)=(蛋壳重/蛋重)×100;

蛋黄比重(%)=(蛋黄重/蛋重)×100;

蛋形指数=长径值/短径值。

1.5.3 胫骨质量

饲养试验结束后,每个重复随机选取1只鸡进行屠宰,剥离左侧胫骨,放入密封袋中,于-20 ℃冰箱中保存待测。将胫骨在沸水中煮4 min左右,去除残余腓骨和肌肉组织,烘箱105 ℃烘干,冷却,用游标卡尺测量胫骨长度。石油醚浸泡去除脂肪,放入烘箱105 ℃烘干,称量胫骨脱脂重,随后将胫骨压碎,转入坩埚碳化完全后,于马弗炉550 ℃灰化,称量粗灰分重。胫骨中钙含量采用乙二胺四乙酸(EDTA)滴定法(GB/T 6436—2018)测定,磷含量采用钼黄比色法(GB/T 6437—2018)测定。

1.5.4 血清生化指标

饲养试验结束后,每个重复随机选取1只鸡进行颈静脉采血,860×g离心10 min,取血清于-80 ℃冰箱中保存待测。血清1,25(OH)2D3(货号:ml059823)、FGF23(货号:ml061098)和 Klotho(货号:ml541621)含量均采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)试剂盒测定。血清磷(货号:C006-1-1)含量和碱性磷酸酶(货号:A059-2)活性均采用南京建成生物工程研究所生产的试剂盒检测。具体操作步骤见试剂盒说明书。

1.5.5 肾脏和十二指肠磷吸收相关基因

将屠宰鸡只解剖后,分别收集肾脏和十二指肠黏膜组织样本各3份,包好投入液氮中暂存,随后将样品转移至-80 ℃冰箱保存待测。使用RNA提取试剂盒(货号:AG21017,艾科瑞生物工程有限公司)分别提取蛋鸡肾脏和十二指肠黏膜组织中的总RNA,采用反转录试剂盒(货号:AG11706,艾科瑞生物工程有限公司)进行反转录操作,然后采用荧光定量PCR试剂盒(货号:AG11701,艾科瑞生物工程有限公司)进行相关基因表达量的测定。PCR所用基因引物序列见表2。以β-肌动蛋白(β-actin)为内参基因,采用2-△△Ct方法计算目的基因的mRNA相对表达量。目的基因包括十二指肠膜维生素D受体(mVDR)、核维生素D受体(nVDR)、钠依赖Ⅱb型磷转运蛋白(Na/Pi-Ⅱb)、肾脏细胞色素P450家族27亚家族B成员1(CYP27B1)、FGF23和Klotho。
表2 基因引物序列

Table 2 Primer sequences of genes

基因
Genes		 引物序列
Primer sequences (5'—3')		 长度
Length/bp		 登录号
Accession number
膜维生素D受体
mVDR		 F:TCCCCACCATCTATTTCGCTC
R:CCTCCTTTGCCTTCTTCTTGG		 168 NM_204110.4
核维生素D受体
nVDR		 F:GGAGCAGCAGAAAGTCATCG
R:GCATCGGAGCCAAAGACATC		 192 NM_205098.2
钠依赖Ⅱb型磷转运蛋白
Na/Pi-Ⅱb		 F:CTTGGCTGGCTGGATACCTG
R:GGGTGAGGGGATAAGAACGC		 141 NM_204474.3
细胞色素P450家族27亚家族B成员1
CYP27B1		 F:GCTGTCACTGGGATTCTTTGC
R:CCAACCGAAAGGCACAAGTC		 160 AF470455
成纤维细胞生长因子23
FGF23		 F:CAGCCAAGAGGACTGTGTGT
R:ACTGGGAGTATGGTGGTGGA		 146 XM_425663.6
克洛索蛋白
Klotho		 F:TCACACTGGATAAAACCTCAACA
R:TACTTCTTGTCTTCTTCGCTGA		 179 XM_003641245.6
β-肌动蛋白
β-actin		 F:TGCGTGACATCAAGGAGAAG
R:TGCCAGGGTACATTGTGGTA		 300 NM_205518.2

1.6 数据统计与分析

利用SPSS 26.0软件对试验数据进行单因素方差分析(one-way ANOVA),并采用Duncan氏法进行多重比较检验。结果用平均值和均值标准误(SEM)表示,P<0.05为差异显著。使用GraphPad Prism 8.0软件绘制柱形图。

2 结果与分析

2.1 饲粮维生素D3添加水平对蛋鸡生产性能的影响

由表3可知,8 000 IU/kg组蛋鸡平均日采食量显著低于其他3组(P<0.05)。饲粮维生素D3添加水平对蛋鸡产蛋率、料蛋比和平均蛋重无显著影响(P>0.05),但0和8 000 IU/kg组蛋鸡产蛋率均低于2 000和4 000 IU/kg组。
表3 饲粮维生素D3添加水平对蛋鸡生产性能的影响

Table 3 Effects of dietary vitamin D3 supplemental levels on performance of laying hens

项目
Items		 维生素D3添加水平Vitamin D3 supplemental levels/(IU/kg) SEM P值
P-value
0 2 000 4 000 8 000
平均日采食量ADFI/g 114.67a 112.68b 114.93a 110.63c 0.434 <0.001
产蛋率Laying rate/% 90.00 93.13 94.38 88.75 1.816 0.687
料蛋比F/E 1.93 1.94 1.95 1.89 0.023 0.861
平均蛋重AEW/g 59.62 58.31 59.21 58.87 0.694 0.930

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 letter superscripts mean significant difference (P<0.05). The same as below.

2.2 饲粮维生素D3添加水平对蛋鸡蛋品质的影响

由表4可知,饲粮维生素D3添加水平虽未对蛋鸡蛋品质产生显著影响(P>0.05),但蛋壳强度和蛋壳比重均以0和8 000 IU/kg组较低,以4 000 IU/kg组为最高。
表4 饲粮维生素D3添加水平对蛋鸡蛋品质的影响

Table 4 Effects of dietary vitamin D3 supplemental levels on egg quality of laying hens

项目
Items		 维生素D3添加水平Vitamin D3 supplemental levels/(IU/kg) SEM P值
P-value
0 2 000 4 000 8 000
蛋壳强度Eggshell strength/N 45.22 47.87 48.17 41.30 1.232 0.164
蛋壳厚度Eggshell thickness/mm 0.42 0.41 0.42 0.42 0.006 0.966
蛋壳比重Eggshell proportion/% 12.76 12.80 12.88 12.61 0.139 0.927
蛋黄比重Yolk proportion/% 28.30 30.08 28.14 28.84 0.354 0.200
蛋形指数Egg shape index 1.34 1.32 1.32 1.32 0.006 0.740

2.3 饲粮维生素D3添加水平对蛋鸡胫骨质量的影响

由表5可知,饲粮维生素D3添加水平显著影响蛋鸡胫骨脱脂重(P<0.05),0和8 000 IU/kg组蛋鸡胫骨脱脂重均显著低于2 000和4 000 IU/kg组(P<0.05)。饲粮维生素D3添加水平对蛋鸡胫骨长度、粗灰分重、钙和磷含量均无显著影响(P>0.05)。
表5 饲粮维生素D3添加水平对蛋鸡胫骨质量的影响

Table 5 Effects of dietary supplemental levels of vitamin D3 on tibia quality of laying hens

项目
Items		 维生素D3添加水平Vitamin D3 supplemental levels/(IU/kg) SEM P值
P-value
0 2 000 4 000 8 000
胫骨长度Tibia length/mm 121.53 121.49 122.72 123.01 0.632 0.778
脱脂重Defatted weight/g 6.74b 8.31a 8.16a 6.40b 0.216 <0.001
粗灰分重Ash weight/g 2.89 3.36 3.21 2.88 0.092 0.165
钙含量Ca content/% 14.83 14.81 14.73 14.59 0.047 0.271
磷含量P content/% 4.39 4.52 4.36 4.36 0.039 0.400

2.4 饲粮维生素D3添加水平对蛋鸡血清生化指标的影响

由表6可知,饲粮维生素D3添加水平显著影响蛋鸡血清1,25(OH)2D3、FGF23、磷含量和碱性磷酸酶活性(P<0.05),且血清1,25(OH)2D3、FGF23、磷含量均以0 IU/kg组为最低,碱性磷酸酶活性以0 IU/kg组为最高。随着饲粮维生素D3添加水平的提高,血清1,25(OH)2D3、FGF23含量均呈先升高后降低的趋势,并在添加水平为4 000 IU/kg时达到峰值;血清Klotho含量也呈相同变化趋势,但并未达统计学上的显著水平(P>0.05)。
表6 饲粮维生素D3添加水平对蛋鸡血清生化指标的影响

Table 6 Effects of dietary vitamin D3 supplemental levels on serum biochemical indexes of laying hens

项目
Items		 维生素D3添加水平Vitamin D3 supplemental levels/(IU/kg) SEM P值
P-value
0 2 000 4 000 8 000
1,25-二羟基维生素D3
1,25(OH)2D3/(ng/mL)		 14.41c 22.61ab 25.16a 20.88b 0.966 <0.001
成纤维细胞生长因子23
FGF23/(pg/mL)		 174.15b 244.04a 257.12a 253.97a 8.659 <0.001
克洛索蛋白Klotho/(pg/mL) 514.00 602.59 604.86 581.82 16.286 0.163
磷P/(mmol/L) 0.73b 0.96a 0.88ab 0.94a 0.031 0.024
碱性磷酸酶AKP/(金氏单位/dL) 28.14a 13.40b 17.05b 20.04b 1.561 0.003

2.5 饲粮维生素D3添加水平对蛋鸡十二指肠和肾脏磷吸收相关基因表达的影响

由图1可知,0和2 000 IU/kg组蛋鸡十二指肠nVDR的mRNA相对表达量显著低于4 000和8 000 IU/kg组(P<0.05),十二指肠Na/Pi-Ⅱb的mRNA相对表达量显著低于8 000 IU/kg组(P<0.05)。8 000 IU/kg组蛋鸡十二指肠mVDR、nVDR和Na/Pi-Ⅱb的mRNA相对表达量显著高于其他3组(P<0.05),且Na/Pi-Ⅱb的mRNA相对表达量随饲粮维生素D3添加水平的提高呈阶梯式升高。
图1 饲粮维生素D3添加水平对蛋鸡十二指肠磷吸收相关基因表达的影响

数据柱形标注相同字母表示差异不显著(P>0.05),不同字母表示差异显著(P<0.05)。下图同。

Fig.1 Effects of dietary vitamin D3 supplemental levels on expression of genes related to phosphorus absorption in duodenum of laying hens

Value columns with the same letter superscripts mean no significant difference (P>0.05), while with different letters mean significant difference (P<0.05). The same as below.

由图2可知,0 IU/kg组蛋鸡肾脏FGF23和CYP27B1的mRNA相对表达量显著高于其他3组(P<0.05),肾脏Klotho的mRNA相对表达量显著高于2 000和8 000 IU/kg组(P<0.05),并且随着饲粮维生素D3添加水平的提高,FGF23和Klotho的mRNA相对表达量变化趋势基本一致,CYP27B1的mRNA相对表达量呈阶梯式降低。
图2 饲粮维生素D3添加水平对蛋鸡肾脏磷吸收相关基因表达的影响

Fig.2 Effects of dietary vitamin D3 supplemental levels on expression of genes related to phosphorus absorption in kidney of laying hens

3 讨论

维生素D是影响钙、磷代谢和吸收以及蛋品质的重要营养物质。然而,维生素D本身并不具备生物学活性,它是一类前体物质,需经过2次α-羟化过程才能转化为能够发挥生物学功能的活性物质[15]。具体羟化过程如下:在肝脏中,维生素D在25α-羟化酶和细胞色素P450家族24亚家族A成员1(CYP24A1)的作用下,转化为25-羟基维生素D3(25-OHD3),随后经血液运输至肾脏;在肾脏中,25-OHD3在1α-羟化酶和CYP27B1的作用下,形成具有生物活性的代谢产物1,25(OH)2 D 3 [16-17]。1,25(OH)2D3可通过调节mVDR和nVDR基因的表达[18-19],增加Na/Pi-Ⅱb与载体的结合位点数量,改变小肠刷状缘黏膜的组成和流动性,从而促进小肠中钙、磷的吸收,提高蛋品质。在生产实践中发现,蛋鸡体内维生素D的代谢过程受到多种因素的影响,如日龄、甲状旁腺激素(PTH)水平、FGF23含量等[20-22]。其中,FGF23是2001年在对低磷佝偻病、骨骼软化和骨纤维性结构不良等病症的医学研究中被发现的,并证实其是由骨组织中的成骨细胞和骨细胞分泌的内在激素蛋白,对磷的内稳态调控具有重要作用[23-24]。
杨涛[25]研究发现,饲粮中添加不同水平的维生素D3对蛋鸡蛋品质没有显著影响,但100 IU/kg组蛋鸡胫骨干重显著低于300、900和2 700 IU/kg组。康乐[26]研究发现,饲粮中添加不同水平(0、2 500和5 000 IU/kg)的维生素D3对蛋鸡平均蛋重并未产生显著影响;而0 IU/kg组蛋鸡的蛋壳强度、蛋壳比重和胫骨强度均显著低于其他维生素D3添加水平组。王荣梅[27]在饲粮中分别添加0和3 000 IU/kg的维生素D,结果显示,在4周的试验期内,2组蛋鸡采食量、产蛋率和产蛋量没有显著差异,且0 IU/kg组也未出现蛋壳强度和蛋壳厚度显著降低的情况;而0 IU/kg组蛋鸡血磷含量和肾脏VDR的mRNA相对表达量均低于3 000 IU/kg组。还有研究表明,在产蛋高峰期给蛋鸡饲喂不添加维生素D的饲粮,其产蛋率和蛋品质会下降,血清钙、磷和25-羟基维生素D(25-OHD)含量也会显著降低[28]。本研究发现,在短期(4周)试验条件下,饲粮维生素D3缺乏(0 IU/kg)对蛋鸡生产性能、蛋品质及部分胫骨质量指标均未产生显著影响。但是,与正常(4 000 IU/kg)组相比,维生素D3缺乏组蛋鸡胫骨脱脂重显著降低。值得注意的是,尽管蛋壳强度、蛋壳比重和胫骨粗灰分重等指标未达到统计学显著差异,但其数值在0 IU/kg组均低于4 000 IU/kg组。同时,0 IU/kg组蛋鸡血清1,25(OH)2D3、FGF23、Klotho、磷含量以及十二指肠nVDR、Na/Pi-Ⅱb的mRNA相对表达量均低于4 000 IU/kg组。这表明,当饲粮维生素D3缺乏时,蛋鸡体内钙、磷代谢及其稳态维持相关的重要因素平衡被打破,进而可能导致蛋壳强度、蛋壳比重、胫骨脱脂重及灰分重降低。然而,对基因指标的进一步检测显示,饲粮维生素D3添加水平为0 IU/kg时,与蛋鸡血清1,25(OH)2D3生成密切相关的编码1α-羟化酶的CYP27B1的mRNA相对表达量显著高于其他3种添加水平组。这与该添加水平下血清1,25(OH)2D3含量最低的结果相悖,其原因可能与动物机体本身存在的“营养饥饿”代偿机制有关,即当机体缺乏某种营养物质时,会通过激活相关基因的表达来代偿性弥补该营养物质的不足,以维持机体各代谢过程的正常运行和平衡。有研究也表明,在维生素D3短期缺乏的情况下,CYP27B1的表达可能上调,以维持体内活性维生素D的平衡[29],但维生素D3长期缺乏时,该代偿机制难以持续或失效。本研究中,饲粮维生素D3缺乏时,蛋鸡肾脏FGF23和CYP27B1的mRNA相对表达量较饲粮维生素D3正常(4 000 IU/kg)和过量(8 000 IU/kg)时均显著提高,而十二指肠nVDR和Na/Pi-Ⅱb的mRNA相对表达量却较饲粮维生素D3正常和过量时有所降低。这表明当饲粮维生素D3缺乏时,通过下调蛋鸡十二指肠中维生素D受体(nVDR)和磷吸收唯一载体蛋白(Na/Pi-Ⅱb)基因的表达,降低了磷的吸收,从而导致了胫骨脱脂重和粗灰分重下降,并最终导致蛋壳强度降低。同时,肾脏CYP27B1、FGF23、Klotho基因也对维生素D3的缺乏产生响应,但3种基因之间具体存在何种关系,还需进一步研究。
本研究结果还显示,当饲粮维生素D3过量时,蛋鸡蛋品质和血清生化指标也呈现出与维生素D3缺乏时类似的趋势,这表明饲粮维生素D3的添加水平并不是越高越好,过量时也同样会导致机体中钙、磷代谢紊乱和蛋品质下降。这与其他相关研究结果类似。研究表明,长期摄入过量维生素D3会导致动物体内钙、磷代谢失衡,并引发一系列生理变化,如因过量钙盐沉积导致的软组织矿化和肾功能受损等[30-31];游赣花等[32]用过量维生素D3灌胃Wistar大鼠,导致大鼠出现急性毒性反应,并造成多种组织器官钙化;另有研究表明,用维生素D3水平为68 348 IU/kg的饲粮饲喂蛋鸡,会使其生长速度减慢,生产性能下降[33]。与饲粮维生素D3缺乏时相反,饲粮维生素D3过量时蛋鸡十二指肠nVDR、mVDR和Na/Pi-Ⅱb的mRNA相对表达量均为4组中最高,这说明过量的维生素D3会促进维生素D受体的表达。本研究中,2 000、4 000和8 000 IU/kg组蛋鸡肾脏FGF23和CYP27B1的mRNA相对表达量无显著差异,但均显著低于0 IU/kg组,这表明肾脏FGF23和CYP27B1基因对饲粮维生素D3的过量添加并无响应;而饲粮维生素D3添加水平为8 000 IU/kg时,十二指肠nVDR、mVDR和Na/Pi-Ⅱb的mRNA相对表达量显著提高,可能是受到高水平维生素D3的直接影响。

4 结论

饲粮维生素D3缺乏或过量均会导致蛋品质下降,且与FGF23/Klotho轴及维生素D代谢有关,但这2种情况下蛋鸡体内的响应指标和调控机制存在差异。当饲粮维生素D3缺乏时,蛋鸡肾脏FGF23、Klotho和CYP27B1作为内在响应指标,参与蛋品质及钙磷代谢的调控;而当饲粮维生素D3过量时,蛋鸡十二指肠nVDR、mVDR和Na/Pi-Ⅱb为参与蛋品质和钙磷代谢调控的内在响应指标。

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