育成期代谢能摄入量对蛋鸡生殖器官发育、激素水平和卵巢基因表达的影响

doi:10.11843/j.issn.0366-6964.2024.11.025

Abstract

Abstract:

This experiment was conducted to evaluate the effects of metabolizable energy (ME) intake during rearing on the growth and development of reproductive organ, hormone level and gene expression in ovary of laying hens. A total of 720 6-week-old Hyline-Brown laying hens were allocated equally to three groups with six replicates of 40 hens 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 crude protein (CP) levels in diet were 17.5% (6-12 weeks) and 15.5% (12-17 weeks), respectively, and the ME levels in diet were 12.34, 11.11 (12.34×90%) and 9.87(12.34×80%) MJ ·kg^-1 (control group, also called ad libitum feeding group, 90% energy-restricted feeding group, and 80% energy-restricted feeding group), respectively. The hens in control group were fed ad libitum, and the daily amount of feed in experimental groups was restricted to the absolute quantity of the diet consumed by hens in control group. The results showed as follows: 1) Body weight, body slope length and shank circumference of hens at 17 weeks of age decreased linearly (P < 0.001) with increasing levels of energy restriction. 2) The serum UN and GLU content decreased linearly (P=0.040 and 0.044, respectively), while the plasma oestradiol concentrations increased linearly (P=0.026). 3)The ovary stroma of hens in the ad libitum feeding group (ALF17W) and 80% energy-restricted feeding group (ERF17W) with significant differences (P < 0.05) in plasma oestradiol concentrations 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 94.61%, and the content of Q20 and Q30 was more than 97.38% and 92.87%, respectively. A total of 1 299 differential genes were screened in ALF17W and ERF17W, of which 961 were down-regulated and 338 were up-regulated in ERF17W. The GO functional enrichment analysis found that differentially expressed mRNAs were involved in 48 GO terms such as cell proliferation, development, and reproduction. The KEGG pathway were significantly enriched in 25 pathways, among these pathways, the cAMP signaling pathway, estrogen signaling pathway, steroid hormone biosynthesis and ovarian steroidogenesis pathway were related to energy metabolism or reproduction. The screened cAMP response element-binding protein (CREB) and steroid-producing acute regulatory protein (StAR) are enriched in the cAMP signaling pathway, and progesterone receptor (PGR), metabotropic glutamate receptor 1 (GRM1) and cytoskeletal keratin (KRT18) are enriched in the estrogen signaling pathway, they may be the potential target genes of ME intake regulating estrogen production in laying hens. The qRT-PCR results showed that the expression trends of 10 randomly selected differentially expressed genes were consistent with the RNA-Seq results. The above results showed that the body weight, serum UN and GLU decreased linearly with the decrease of ME intake during the rearing period, but plasma oestradiol level increased linearly. It is suggested that ME intake during the rearing period may regulate the expression of StAR, CREB1, KRT18, PGR and GRM1 genes in ovarian tissues, and act on the cAMP and estrogen signaling pathways to regulate the production of estrogen in laying hens.

Key words: RNA-seq, rearing period, ME intake, estrogen, ovary, laying hens

CLC Number:

S831.5

Jian LU, Xiaojun JU, Xingguo WANG, Meng MA, Qiang WANG, Yongfeng LI, Taocun DOU, Yuping HU, Jun GUO, Dan SHAO, Haibing TONG, Liang QU. Effects of Metabolizable Energy Intake during Rearing on Development of Reproductive Organs, Hormone Level and Gene Expression in Ovary of Laying Hens[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5085-5100.

Figures/Tables 15

Table 1

Ingredients and nutrient levels of the experimental diet during rearing stage %"

项目Item	代谢能水平/(MJ·kg^-1) ME level
	6~12周龄 6 to 12 weeks of age			12~17周龄 12 to 17 weeks of age
	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
豆粕Soybean meal	25.67	27.60	29.40	20.80	22.70	24.50
石粉Limestone	1.40	1.40	1.40	2.00	2.00	2.00
沸石粉Zeolite powder		8.23	17.26	0.20	8.75	17.49
磷酸氢钙Dicalcium phosphate	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
食盐NaCl	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
DL-蛋氨酸DL-methionine	0.15	0.16	0.17	0.15	0.15	0.16
赖氨酸Lysine	0.10	0.04	0.00	0.10	0.05	0.00
预混料Premix¹⁾	0.50	0.50	0.50	0.50	0.50	0.50
营养水平(计算值)Nutrient levels (calculated value)
代谢能/(MJ·kg^-1) ME	12.34	11.11	9.87	12.34	11.11	9.87
粗蛋白质Crude protein	17.50	17.50	17.50	15.50	15.50	15.50
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
蛋氨酸Methionine	0.45	0.45	0.45	0.41	0.41	0.41
蛋氨酸+胱氨酸Met+Cys	0.73	0.73	0.73	0.67	0.67	0.67
色氨酸L-tryptophan	0.20	0.21	0.22	0.18	0.18	0.18
苏氨酸Threonine	0.66	0.66	0.66	0.59	0.59	0.59
钙Calcium	0.80	0.80	0.80	0.80	0.80	0.80
总磷Total phosphorus	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
营养水平(实测值)Nutrient levels (measured value)
粗蛋白质Crude protein	17.75	17.59	17.63	15.61	15.58	15.58
赖氨酸Lysine	0.89	0.87	0.87	0.78	0.76	0.79
蛋氨酸Methionine	0.43	0.42	0.44	0.38	0.36	0.37
钙Calcium	0.84	0.86	0.79	0.87	0.88	0.83
总磷Total phosphorus	0.53	0.49	0.51	0.53	0.51	0.51

Table 1

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Table 5

Table 6

Fig. 1

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Table 8

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Table 9

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基因名称 Gene name	登录号 GenBank accession number	引物序列(5′ $ \to $ 3′) Primer sequence(5′$ \to $3′)	产物长度/bp Product size
StAR	NM_204686	F: GTCCCTCGCAGACCAAGT R: TCCCTACTGTTAGCCCTGA	196
CREB1	NM_204450.3	F: GCACAGACCACAGATGGACA R: TTCAAGCACAGCCACTCGAT	285
ADIPOQ	NM_206991.2	F: CCCATCCGCTTCACCAAGAT R: GACGTCCGTCATGTAGACCG	129
PLTP	NM_001162406.2	F: CAGCCATGCACGCATACTTC R: AAGGTGGCTCTCAGCAGAAC	97
APOA1	XM_015297971.3	F: CTTGACCTGAAGCTGGCTGA R: CTCCTTCAGCTCCTCCTCCA	165
CD36	XM_040686380.1	F: GTTACTGGGTGTGGTGCAGA R: AGGAACCCATGAATGCGAGG	77
FABP5	NM_001006346.2	F: GGCCATCGACGCGTTTTTAG R: ACATCTGGTTTCGCCATGCT	123
COL3A1	XM_025152154.2	F: CCTTCGGAGAATGCTGTCCA R: CCCGGAAAGCCACTACCTC	217
MGST2	XM_040671687.1	F: AGTACTTCCACGGCTATGCG R: CCAACGTTGAAGCCCAGGTA	145
RAC2	NM_001201452.2	F: TGATGGTTGACAGCAAGCCT R: ACCACTTAGCACGGACGTTT	162

项目Items	代谢能水平/(MJ·kg^-1) ME level			SEM	P值 P-value
项目Items	12.34	11.11	9.87	SEM	代谢能 ME	线性 Linear	二次 Quadratic
6周龄体重/g Body weight at 6 weeks of age	374.5	379.9	376.1	1.953	0.504	0.731	0.266
6周龄体重CV Body weight CV at 6 weeks of age	10.77	12.38	11.20	0.345	0.140	0.591	0.059
17周龄体重/g Body weight at 17 weeks of age	1 474.3^a	1 404.9^b	1 249.8^c	8.460	< 0.001	< 0.001	0.001
17周龄体重CV Body weight CV at 17 weeks of age	6.50	6.77	7.33	0.376	0.713	0.439	0.869
17周龄体斜长/cm Body slope length at 17 weeks of age	21.45^a	21.07^b	20.68^c	0.052	< 0.001	< 0.001	0.976
17周龄跖长/cm Shank length at 17 weeks of age	8.57^b	8.68^a	8.64^ab	0.017	0.026	0.065	0.051
17周龄跖围/cm Shank circumference at 17 weeks of age	3.65^a	3.65^a	3.57^b	0.007	< 0.001	< 0.001	0.005

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	10.60^a	10.28^ab	9.82^b	0.175	0.019	0.044	0.098
尿素氮UN	1.22^a	0.73^b	0.68^b	0.116	0.049	0.040	0.355
总胆固醇TC	2.85	3.26	2.91	0.109	0.273	0.826	0.116
甘油三酯TG	0.33	0.27	0.31	0.013	0.136	0.585	0.057
游离脂肪酸UREA	0.41	0.38	0.28	0.036	0.305	0.667	0.667
高密度脂蛋白胆固醇HDL-C	1.88	2.04	1.82	0.065	0.367	0.689	0.180
低密度脂蛋白胆固醇LDL-C	0.78	0.81	0.74	0.039	0.791	0.694	0.583

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	1.70	1.41	1.23	0.096	0.125	0.046	0.792
甘油三酯TG	0.24	0.27	0.19	0.028	0.518	0.441	0.401
游离脂肪酸UREA	0.40	0.37	0.33	0.038	0.800	0.933	0.933
高密度脂蛋白胆固醇HDL-C	1.13	1.37	1.55	0.100	0.234	0.095	0.864
低密度脂蛋白胆固醇LDL-C	0.31	0.24	0.22	0.025	0.298	0.144	0.623

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
输卵管长度/cm Oviduct length	8.82	9.37	8.62	0.472	0.817	0.871	0.546
排卵前卵泡数/个 Preovulatory follicle amount	0.00	0.00	0.00	—	—	—	—
小黄卵泡数/个 Small yellow follicle amount	0.00	0.00	0.17	0.056	0.391	0.240	0.490
大白卵泡数/个 Big white follicle amount	2.83	2.00	1.83	0.475	0.682	0.421	0.754
输卵管重/g Oviduct weight	0.48	0.43	0.32	0.055	0.479	0.244	0.783
排卵前卵泡重/g Preovulatory follicle weight	0.00	0.00	0.00	—	—	—	—
小黄卵泡重/g Small yellow follicle weight	0.000	0.000	0.008	0.003	0.391	0.240	0.490
大白卵泡重/g Big white follicle weight	0.050	0.037	0.027	0.009	0.604	0.325	0.934
卵巢基质部重/g Ovary stroma weight	0.622^b	0.738^a	0.622^b	0.023	0.039	1.000	0.012
输卵管长度体重比/(cm·kg^－1) Oviduct length index	6.28	7.04	7.13	0.364	0.602	0.366	0.677
输卵管指数/(g·kg^－1) Oviduct index	0.347	0.317	0.262	0.039	0.693	0.406	0.886
排卵前卵泡指数/(g·kg^－1) Preovulatory follicle index	0.00	0.00	0.00	—	—	—	—
小黄卵泡指数/(g·kg^－1) Small yellow follicle index	0.000	0.000	0.007	0.002	0.391	0.240	0.490
大白卵泡指数/(g·kg^－1) Big white follicle index	0.035	0.027	0.022	0.007	0.748	0.458	0.914
卵巢基质部指数/(g·kg^－1) Ovary stroma index	0.442^c	0.555^a	0.512^b	0.018	0.021	0.071	0.024

Effects of Metabolizable Energy Intake during Rearing on Development of Reproductive Organs, Hormone Level and Gene Expression in Ovary of Laying Hens

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样本 Sample	原始序列数/条 Raw reads number	高质量序列数/条 Pure reads number	原始碱基数/Gb Raw data	高质量碱基数/Gb Pure data	Q20/%	Q30/%	GC/%	核糖体序列/% rRNA reads
ALF17W-1	50 962 344	50 799 300	7.64	7.59	97.54	93.18	48.36	0.20
ALF17W-2	53 402 274	53 214 066	8.01	7.94	97.38	92.87	48.38	0.22
ALF17W-3	43 229 032	43 075 756	6.48	6.43	97.42	92.98	48.95	0.18
ALF17W-4	75 650 346	75 361 666	11.35	11.25	97.56	93.30	48.49	0.20
ERF17W-1	99 725 586	99 386 002	14.96	14.85	97.54	93.19	47.96	0.15
ERF17W-2	91 464 834	91 146 292	13.72	13.61	97.70	93.58	48.12	0.24
ERF17W-3	98 418 144	98 067 256	14.76	14.65	97.75	93.70	48.62	0.16
ERF17W-4	94 560 800	94 224 928	14.18	14.07	97.52	93.16	48.68	0.11

样本 Sample	总序列数 Total reads number	未必对上序列数(占比) Unmapped reads number(ratio)	唯一比对上序列数(占比) Unique mapped reads number(ratio)	多基因组比对序列数(占比) Multiple mapped reads number(ratio)	总比对上序列数(占比) Mapped reads number(ratio)
ALF17W-1	50 699 230	2 646 173 (5.22%)	47 104 260 (92.91%)	948 797 (1.87%)	48 053 057 (94.78%)
ALF17W-2	53 098 502	2 759 806 (5.20%)	49 339 935 (92.92%)	998 761 (1.88%)	50 338 696 (94.80%)
ALF17W-3	42 997 384	2 292 500 (5.33%)	39 875 883 (92.74%)	829 001 (1.93%)	40 704 884 (94.67%)
ALF17W-4	75 208 912	4 051 145 (5.39%)	69 679 240 (92.65%)	1 478 527 (1.97%)	71 157 767 (94.61%)
ERF17W-1	99 236 998	4 736 818 (4.77%)	92 599 873 (93.31%)	1 900 307 (1.91%)	94 500 180 (95.23%)
ERF17W-2	90 928 890	4 404 556 (4.84%)	84 759 588 (93.22%)	1 764 746 (1.94%)	86 524 334 (95.16%)
ERF17W-3	97 907 900	4 817 719 (4.92%)	91 119 179 (93.07%)	1 971 002 (2.01%)	93 090 181 (95.08%)
ERF17W-4	94 120 564	4 674 565 (4.97%)	87 580 401 (93.05%)	1 865 598 (1.98%)	89 445 999 (95.03%)

信号通路Signaling pathway	基因Gene
ECM受体相互作用 ECM-receptor interaction	FN1、ITGA7、COL6A3、COL6A1、COL6A2、COL4A1、COL4A2、GP5、VWF、TNC、 ITGB6、LAMA5、FREM2、CD44、VTN、CD36、RELN、GP9、ITGA8、ITGB5、HSPG2、COL1A2、CEACAM20、COL1A2、CEACAM20
松弛素信号通路 Relaxin signaling pathway	COL1A1、MAPK11、MMP13、MMP9、COL4A1、RLN3、COL4A2、NOS3、TGFB1、SHC3、MMP2、ACTA2、COL1A2、COL3A1
雌激素信号通路 Estrogen signaling pathway	KRT18、PGR、GRM1、MMP9、KRT23、KRT13、KRT14、KRT17、CALML4、NOS3、GNAQ、SHC3、MMP2
醛固酮的合成和分泌 Aldosterone synthesis and secretion	CAMK2D、CALML4、AGTR1、CACNA1C、KCNK3、GNAQ、CYP21A1、CAMK1D、CAMK2A
胆固醇代谢 Cholesterol metabolism	PLTP、APOA1、LCAT、LRP1、CD36、ANGPTL4
催产素信号通路 Oxytocin signaling pathway	NOS3、MYL9、GNAQ、NFATC1、CAMK1D、NPR2、CACNA2D1、CAMK2A、MYLK
生长激素的合成、分泌和作用 Growth hormone synthesis, secretion and action	SOCS3、MAPK11、CACNA1C、SST、GNAQ、SSTR2、SSTR5、SHC3
cAMP信号通路 cAMP signaling pathway	StAR、CREB1
类固醇生物合成 Steroid biosynthesis	CEL
GnRH信号通路 GnRH signaling pathway	MAPK11、CAMK2D、CALML4、EGR1、CACNA1C、GNAQ、MMP2、CAMK2A

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