育成期能量限饲及转换为自由采食调控开产时蛋鸡生殖器官发育的关键基因和信号通路研究

doi:10.11843/j.issn.0366-6964.2025.02.024

摘要/Abstract

摘要：

本试验旨在基于转录组测序技术研究育成期(6~17周龄)能量限饲及转换为自由采食(18~20周龄)调控开产时(20周龄)蛋鸡生殖器官发育和激素水平的关键基因和信号通路。将720只6周龄海兰褐蛋鸡随机分为3组，每组6个重复，每个重复40只鸡。6~17周龄，试验鸡分别饲喂禽代谢能(ME)水平为12.34、11.11(90%)和9.87(80%)MJ·kg^-1，其它营养素水平相同的试验饲粮，12.34 MJ·kg^-1组试验鸡自由采食(对照组)，其它试验组蛋鸡按照对照组蛋鸡采食量定量饲喂，18~20周龄，各组试验鸡均饲喂相同营养水平试验饲粮自由采食。试验期6~20周龄。20周龄末，选取生殖器官发育和血液孕酮水平差异显著的对照组和80% ME摄入组试验鸡各4只，采集卵巢基质部进行RNA-seq分析，对差异表达基因进行GO功能富集分析和KEGG信号通路分析，并对测序结果进行qRT-PCR验证。结果表明：1)随育成期能量限饲强度增加，各试验组蛋鸡20周龄体重和体重变异系数(CV)均显著线性减少(P < 0.001)，6~20周龄平均日采食量(ADFI)和料重比(F/G)均显著线性增加(P < 0.001)，平均日代谢能摄入量(ADMEI)和平均日增重(ADG)显著线性减少(P < 0.001)。2)随育成期能量限饲强度增加，20周龄蛋鸡血清尿素氮(UN)含量显著线性增加(P=0.007)，血清甘油三酯(TG)和低密度脂蛋白(LDL)水平以及肝组织总胆固醇(TC)和游离脂肪酸(NEFA)含量均显著线性减小(P=0.045，P=0.029，P=0.024，P=0.003)。3)随育成期能量限饲强度增加，20周龄蛋鸡输卵管长度、长度体重比、重量和指数(P=0.012、0.016、0.042和0.045)、小黄卵泡的数量和指数(P=0.017和0.039)以及卵巢基质部重量和指数(P=0.046和0.047)均显著线性减少，血浆孕酮水平显著线性增加(P < 0.001)。4)对20周龄自由采食组(ALF20W)和80%能量限饲组(ERF20W)蛋鸡卵巢基质部进行RNA-Seq分析，纯净序列匹配到鸡参考基因组的比例均超过了93.77%，Q20和Q30的纯净序列含量分别高于97.03%和92.14%，两组共筛选出1 488个差异基因，ERF20W组600个下调，888个上调。GO功能分析发现细胞进程、发育和生殖等46个显著富集的GO条目，KEGG信号通路显著富集在28个显著富集的KEGG通路，其中类固醇激素生物合成通路、雌激素信号通路、卵巢类固醇生成通路和cAMP信号通路等是与能量代谢或生殖相关的通路，筛选到的cAMP反应元件结合蛋白(CREB)、类固醇生成急性调节蛋白(StAR)、细胞色素P450 1B1(CYP1B1)、胰岛素样生长因子I(IGF-I)、黑皮质素2受体(MC2R)、细胞骨架蛋白角蛋白18(KRT 18)和孕激素受体(PGR)等差异基因富集在以上信号通路，可能是育成期能量限饲及转换为自由采食调控开产时蛋鸡生殖器官发育和雌激素生成的潜在靶基因和通路。qRT-PCR结果显示10个差异表达基因的表达趋势与RNA-Seq结果一致。由此可见，育成期能量限饲及转换为自由采食显著影响了开产时(20周龄)蛋鸡脂质代谢、生殖器官发育和孕酮生成，随育成期能量限饲强度增加，开产时蛋鸡体重、体重CV、血清TG和LDL水平、肝TC和NEFA含量、输卵管长度、长度体重比、重量和指数、小黄卵泡的数量和指数以及卵巢基质部重量和指数均显著线性减少，而血浆孕酮水平显著线性增加。育成期能量限饲及转换为自由采食可能通过影响卵巢组织StAR、CREB1、CYP1B1、IGF-I、MC2R、KRT18和PGR等基因的表达，作用于类固醇激素生物合成通路、雌激素信号通路、卵巢类固醇生成通路和cAMP信号通路等通路，以调控开产时蛋鸡能量代谢、生殖器官发育和孕酮生成。

关键词: 蛋鸡, 育成期能量限饲, 生殖器官, RNA-seq, 卵巢

Abstract:

The aim of this experiment was to investigate the key genes and signaling pathways of regulation of energy-restricted feeding during rearing and conversion to ad libitum on the reproductive organ development and hormone level of laying hens at the initiation of laying period using transcriptome sequencing technology. A total of 720 6-week-old Hyline-Brown chicks were allocated equally to three groups with six replicates of 40 chicks each, and were fed one of three diets that were nutritionally equal with the exception of ME levels. From 6 to 17 weeks of age, the chicks in control group were given diet with 12.34 MJ·kg^-1 ME, and fed ad libitum. The levels of ME in diet of chicks in the experimental groups were 90% [11.11 (12.34×90%) MJ·kg^-1] and 80% [9.87 (12.34×80%) MJ·kg^-1] of that in control group, and the daily amount of feed was restricted to the absolute quantity of the diet consumed by chicks in control group. From 18 to 20 weeks of age, all laying pullets were fed a basal diet ad libitum. At 20 weeks of age, four chicks in each of the ad libitum feeding group and 80% energy-restricted feeding group with significant differences (P < 0.05) in reproductive organ development and plasma progesterone concentrations were selected to screen the novel mRNA implemented by the RNA-seq. The results showed as follows: 1) The body weight and body weight CV of chicks at 20 weeks of age decreased linearly with increasing energy restriction (P < 0.001), the ADFI and F/G (P < 0.001) from 6 to 20 weeks of age increased linearly, while the ADMEI and ADG (P < 0.001) decreased linearly with increasing the degree of energy restriction. 2) A gradual increase in the degree of energy restriction resulted in a gradual increase in the serum UN (P=0.007), but a gradual decrease in serum TG and LDL (P=0.045, 0.029), and in liver TC and NEFA (P=0.024, 0.003). 3) With the increase of energy restriction during rearing period, the length, ratio of length to body weight, weight and index of oviduct (P= 0.012, 0.016, 0.042, 0.045), the number and index of small yellow follicle (P=0.017, 0.039), and the weight and index of ovarian stroma (P=0.046, 0.047) decreased linearly, while the plasma progesterone level (P < 0.001) increased linearly. 4) The ovary stroma of chicks at 20 weeks of age in the ad libitum feeding group (ALF20W) and 80% energy-restricted feeding group (ERF20W) were used to screen the novel mRNA implemented by the RNA-seq. The proportion of pure reads matching to chicken reference genome was more than 93.77%, and the content of Q20 and Q30 was more than 97.03% and 92.14%, respectively. A total of 1 488 differential genes were screened in ALF20W and ERF20W, of which 600 were down-regulated and 888 were up-regulated in ERF20W. The GO functional enrichment analysis found that differentially expressed mRNAs were involved in biological processes such as biological regulation of cell proliferation, development, and reproduction. The KEGG pathway were significantly enriched in 28 pathways, among these pathways, the steroid hormone biosynthesis pathway, estrogen signaling pathway, ovarian steroidogenesis pathway, and cAMP signaling pathway were related to energy metabolism or reproduction. The differentially expressed genes including cAMP response element-binding protein (CREB), steroid-producing acute regulatory protein (StAR), cytochrome P450 1B1 (CYP1B1), insulin-like growth factor-1 (IGF-I), adrenocorticotropic hormone (MC2R), cytoskeletal keratin (KRT18), and progesterone receptor (PGR) were found to be enriched in the above signaling pathways, which maybe the potential target gene and pathway of energy restriction regulating reproductive organ development and estrogen production in laying chicks. The qRT-PCR results showed that the expression trends of 10 randomly selected differentially expressed genes were consistent with RNA-Seq results. The results showed that the body weight, body weight CV, serum TG and LDL, liver TC and NEFA, the length, ratio of length to body weight, weight and index of oviduct, the number and index of small yellow follicle, and the weight and index of ovarian stroma of chicks at 20 weeks of age decreased linearly with increasing the degree of energy restriction, while the plasma progesterone level increased linearly. It is suggested that energy restriction during the rearing period may regulate the expression of StAR, CREB1, CYP1B1, IGF-I, MC2R, KRT18 and PGR genes in ovarian tissues, and act on steroid hormone biosynthesis pathway, estrogen signaling pathway, ovarian steroidogenesis pathway, and cAMP signaling pathway, to regulate the energy metabolism, reproductive organ development and estrogen production of laying chicks at the initiation of laying period.

Key words: hens, energy restriction during rearing period, reproductive organs, RNA-seq, ovary

中图分类号:

S831.5

卢建, 马猛, 郭军, 王星果, 窦套存, 胡玉萍, 王强, 李永峰, 邵丹, 童海兵, 郭杰, 曲亮. 育成期能量限饲及转换为自由采食调控开产时蛋鸡生殖器官发育的关键基因和信号通路研究[J]. 畜牧兽医学报, 2025, 56(2): 737-754.

LU Jian, MA Meng, GUO Jun, WANG Xingguo, DOU Taocun, HU Yuping, WANG Qiang, LI Yongfeng, SHAO Dan, TONG Haibing, GUO Jie, QU Liang. Studies on Key Genes and Signaling Pathways of Regulation of Energy Restriction during Rearing and Conversion to Ad libitum on the Reproductive Organ Development of Hens at the Initiation of Laying Period[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 737-754.

图/表 16

表 1

试验饲粮组成及营养水平"

项目 Item	6~12周龄 6 to 12 weeks of age			13~17周龄 13 to 17 weeks of age			18~20周龄 18 to 20 weeks of age
	代谢能水平/(MJ·kg^-1) ME level			代谢能水平/(MJ·kg^-1) ME level
	12.34	11.11	9.87	12.34	11.11	9.87
原料Ingredient
玉米Corn	71.11	61.00	50.20	75.20	64.80	54.30	68.00
豆粕Soybean meal	25.67	27.60	29.40	20.80	22.70	24.50	24.00
石粉Limestone	1.40	1.40	1.40	2.00	2.00	2.00	6.00
沸石粉Zeolite powder	0.00	8.23	17.26	0.20	8.75	17.49	0.23
磷酸氢钙Dicalcium phosphate	0.105	0.105	0.105	0.105	0.105	0.105	0.105
磷酸二氢钙Monocalcium phosphate	0.595	0.595	0.595	0.595	0.595	0.595	0.595
食盐NaCl	0.25	0.25	0.25	0.25	0.25	0.25	0.25
50%胆碱50% Choline chloride	0.12	0.12	0.12	0.10	0.10	0.10	0.12
DL-蛋氨酸DL-methionine	0.15	0.16	0.17	0.15	0.15	0.16	0.20
赖氨酸Lysine	0.10	0.04	0.00	0.10	0.05	0.00	0.00
预混料Premix¹)	0.50	0.50	0.50	0.50	0.50	0.50	0.50
营养水平(计算值) Nutrient levels (calculated)
代谢能/(MJ·kg^-1) ME	12.34	11.11	9.87	12.34	11.11	9.87	11.72
粗蛋白质Crude protein	17.50	17.50	17.50	15.50	15.50	15.50	16.50
可消化氨基酸Digestible amino acid
赖氨酸Lysine	0.94	0.94	0.94	0.82	0.82	0.82	0.84
蛋氨酸Methionine	0.45	0.45	0.45	0.41	0.41	0.41	0.47
蛋氨酸+胱氨酸Met+Cys	0.73	0.73	0.73	0.67	0.67	0.67	0.74
色氨酸L-tryptophan	0.20	0.21	0.22	0.18	0.18	0.18	0.19
苏氨酸Threonine	0.66	0.66	0.66	0.59	0.59	0.59	0.62
钙Calcium	0.80	0.80	0.80	0.80	0.80	0.80	2.60
总磷Total phosphorus	0.50	0.50	0.50	0.50	0.50	0.50	0.50
非植酸磷Nonphytate phosphorus	0.27	0.27	0.27	0.27	0.27	0.27	0.27
营养水平(实测值) Nutrient levels (measured)
粗蛋白质Crude protein	17.75	17.59	17.63	15.61	15.58	15.58	16.59
赖氨酸Lysine	0.89	0.87	0.87	0.78	0.76	0.79	0.81
蛋氨酸Methionine	0.43	0.42	0.44	0.38	0.36	0.37	0.48
钙Calcium	0.84	0.86	0.79	0.87	0.88	0.83	2.68
总磷Total phosphorus	0.53	0.49	0.51	0.53	0.51	0.51	0.51

表 1

表 2

表 3

表 4

表 5

表 6

表 7

图 1

表 8

表 9

图 2

图 3

图 4

图 5

表 10

与能量代谢、性成熟相关的信号通路及差异表达基因"

信号通路Signaling pathway	基因Gene
ECM受体相互作用 ECM-receptor interaction	THBS2、COL1A1、FN1、ITGA7、GP1BB、COL6A3、COL6A1、COL6A2、ITGA11、COL4A1、COL4A2、THBS1、LAMA4、LAMC1、TNC、LAMB1、ITGA8、HSPG2、COL1A2
松弛素信号通路 Relaxin signaling pathway	COL1A1、CREB3L1、RXFP1、COL4A1、COL4A2、NOS3、H-RAS、MMP2、ACTA2、COL1A2、COL3A1、tctex1d1-b
脂肪细胞脂解的调节 Regulation of lipolysis in adipocyte	FABP4、PLIN1、MGLL、NPY1R、PTGS1、ABHD4
雌激素信号通路 Estrogen signaling pathway	KRT18、PGR、GRM1、CREB3L1、KRT40、KRT23、KRT13、KRT14、KRT1、NOS3、GNAQ、H-RAS、MMP2
催产素信号通路 Oxytocin signaling pathway	GUCY1A2、MYL6、CACNA1C、NOS3、PPP1R12A、MYL9、GNAQ、H-RAS、KCNJ4、CACNA2D1、CAMK2A、CACNB2、MYLK、CACNA1D
GnRH信号通路 GnRH signaling pathway	EGR1、CACNA1C、GNAQ、H-RAS、MMP2、CAMK2A、CACNA1D
类固醇激素生物合成 Steroid hormone biosynthesis	StAR、DHRS11、CYP3A4、HSD17B3、CYP1B1
醛固酮的合成和分泌 Aldosterone synthesis and secretion	ATP1B4、CREB3L1、CACNA1C、MC2R、GNAQ、CAMK2A、CACNA1D
促性腺激素释放激素分泌 GnRH secretion	CACNA1C、GNAQ、H-RAS、TRPC4、CACNA1D
卵巢类固醇生成 Ovarian steroidogenesis	StAR、IGF-I、CYP1B1
孕酮介导的卵母细胞成熟 Progesterone-mediated oocyte maturation	PGR、IGF-I、MOS、CCNA1、CCNB1
催乳素信号通路 Prolactin signaling pathway	JAK2、H-RAS、PRLR
脂肪的消化和吸收 Fat digestion and absorption	DGAT
卵母细胞减数分裂 Oocyte meiosis	CCNE1、PGR、IGF-I、MOS、CAMK2A、CCNB1
脂肪酸生物合成 Fatty acid biosynthesis	HTD2
脂肪酸代谢 Fatty acid metabolism	HTD2、SCD5、SCD
脂肪细胞因子信号通路 Adipocytokine signaling pathway	JAK2、LEPR
cAMP信号通路 cAMP signaling pathway	StAR、CREB1、MC2R等

表 10

图 6

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基因 Genes	序列号 Accession number	引物序列(5′→3′) Primer sequence(5′→3′)	产物长度/bp Product length
StAR	NM_204686	F：GTCCCTCGCAGACCAAGT R：TCCCTACTGTTAGCCCTGA	196
CREB1	NM_204450.3	F：GCACAGACCACAGATGGACA R：TTCAAGCACAGCCACTCGAT	285
ACTA2	NM_001031229.2	F：CGGAACGTGGCTACTCCTTT R：CCAGGGCCACATAACACAGT	84
CFTR	XM_015275972.3	F：CAACGACTGGAACTGTCGGA R：AAGTTGCCAGCTCTCTGTCC	103
COL3A1	XM_025152154.2	F：CCTTCGGAGAATGCTGTCCA R：CCCGGAAAGCCACTACCTC	217
CYP1B1	XM_040668785.1	F：GTGCCTGTTACCATCCCACA R：CACGGTGTCCTTGGGAATGA	75
ENTPD3	NM_001321564.2	F：TGTGATGTGAAAGGCCCTGG R：CAGGCACTCATCAAGGGGTT	78
HSPG2	XM_040688761.1	F：CGGAGAGCAGCACCTACATC R：AGTGCATCTCTCCCCCATCT	182
MC2R	NM_001031515.2	F：CGTCGTGGGGACTTTGAGAA R：CTGCAATAGCGAGCAAGCTG	97
NR4A3	XM_040665827.1	F：CACGTTTCTTGCTGAAGGGC R：GCTATACTGCGCTTGCACAC	85

项目 Item	代谢能水平/(MJ·kg^-1) ME level			SEM	P值P-value
项目 Item	12.34	11.11	9.87	SEM	代谢能 ME	线性 Linear	二次 Quadratic
体重/g Body weight	1 692.7^a	1 636.9^b	1 549.1^c	6.694	< 0.001	< 0.001	0.216
体重CV Body weight CV	8.45^a	8.15^a	6.93^b	0.254	0.023	0.010	0.320
体斜长/cm Body slope length	23.38	23.11	23.18	0.057	0.130	0.143	0.168
跖长/cm Shank length	8.62^b	8.73^a	8.62^b	0.018	0.029	0.970	0.008
跖围/cm Shank circumference	3.82	3.81	3.79	0.008	0.192	0.078	0.639

项目 Item	代谢能水平/(MJ·kg^-1) ME level			SEM	P值P-value
项目 Item	12.34	11.11	9.87	SEM	代谢能 ME	线性 Linear	二次 Quadratic
平均日采食量/g ADFI	71.0^b	71.5^b	72.9^a	0.255	0.001	< 0.001	0.220
平均日代谢能摄入量/kJ ADMEI	856.0^a	802.6^b	752.9^c	10.384	< 0.001	< 0.001	0.667
平均日增重/g ADG	13.45^a	12.80^b	11.95^c	0.172	< 0.001	< 0.001	0.615
料重比/(g·g^-1) F/G	5.29^c	5.58^b	6.11^a	0.088	< 0.001	< 0.001	0.115
代谢能转化比/(kJ·g^-1)MECR	63.77	62.63	63.09	0.374	0.478	0.474	0.331

项目 Item	代谢能水平/(MJ·kg^-1) ME level			SEM	P值P-value
项目 Item	12.34	11.11	9.87	SEM	代谢能 ME	线性 Linear	二次 Quadratic
葡萄糖GLU	11.58	10.68	11.11	0.211	0.224	0.354	0.143
尿素氮UN	1.00^b	1.18^ab	1.38^a	0.060	0.023	0.007	0.938
总胆固醇TC	3.19	2.70	3.02	0.118	0.252	0.570	0.122
甘油三酯TG	1.95^a	1.29^b	0.36^c	0.140	0.037	0.045	0.888
游离脂肪酸NEFA	0.41	0.38	0.28	0.036	0.305	0.143	0.667
高密度脂蛋白胆固醇HDL-C	1.74	1.94	2.27	0.105	0.111	0.041	0.744
低密度脂蛋白胆固醇LDL-C	1.18^a	0.70^ab	0.60^b	0.111	0.026	0.029	0.368

项目 Item	代谢能水平/(MJ·kg^-1) ME level			SEM	P值P-value
项目 Item	12.34	11.11	9.87	SEM	代谢能 ME	线性 Linear	二次 Quadratic
总胆固醇TC	1.64^a	1.57^ab	1.20^b	0.082	0.049	0.024	0.335
甘油三酯TG	0.33	0.27	0.19	0.035	0.251	0.102	0.928
游离脂肪酸NEFA	0.38^a	0.36^a	0.21^b	0.026	0.006	0.003	0.126
高密度脂蛋白胆固醇HDL-C	1.66	1.71	1.69	0.116	0.989	0.935	0.904
低密度脂蛋白胆固醇LDL-C	0.35	0.27	0.22	0.034	0.299	0.130	0.815