种鸽饲粮磷水平对乳鸽血清生化指标、非靶向代谢物及组织中磷转运载体基因表达的影响

doi:10.3864/j.issn.0578-1752.2023.23.017

Abstract

Abstract:

【Objective】The aim of this study was to investigate the effects of different levels of inorganic phosphorus (P) on serum biochemical parameters, non-targeted metabolites, and expression of phosphate transporter genes in the small intestine and kidney of pigeon, so as to provide a theoretical basis for the regulation of phosphorus nutrition in pigeon during the period of rapid growth.【Method】A total of 192 pairs of parental Silver King pigeons (40 weeks of age) were randomly allocated to four treatment groups, each consisting of eight replicates of 6 pairs per replicate. The study lasted for 46 days including the phases of egg-hatching and squab-rearing. Dietary treatments included the basal diet (containing 0.3% of P), the basal diet supplemented with 0.2%, 0.4%, or 0.8% inorganic P. The calcium (Ca) content in diets of parent birds were kept at 1.40% in every group. The levels of Ca, P, PTH and ALP activity in serum of 7, 14 and 21 d old squabs were determined, and the content of Ca and P in crop milk were analyzed. The gene expression of Pi transporters (NaPi-Ⅱb, PiT-1, and PiT-2) in small intestine (duodenum and jejunum), and Pi transporters (NaPi-Ⅱa, PiT-1, and PiT-2) in kidney of squabs were determined. Serum metabolome profiles of 14 and 21 d old squabs in the groups of control, 0.4% and 0.8% P-supplemented were analyzed. 【Result】(1) The P contents in crop milk sampled from squabs were very significantly (P<0.01) increased by P supplementation in diets of parent pigeons. (2) No significant effect of P supplementation in diet of parent birds on serum P level, ALP activity and PTH level of squabs were observed in this study. However, the serum Ca content in birds of 0.8% P supplemental group decreased very significantly (P<0.01). The serum ALP and PTH level of squabs significantly decreased with increased age (P<0.05). (3) There was a tendency of increased gene expression of NaPi-IIb in small intestine of squabs with dietary P levels of breeding birds, however, the difference was not significant (P>0.05). The expression profile of PiT-1 and PiT-2 in kidney of squabs at the age of 7 and 14 d were very significantly higher (P<0.01) than those in duodenum and jejunum. However, the difference of the mRNA expression level of PiT-1 and PiT-2 between kidney and intestine was not observed at the age of 21 d, and the PiT-1 value of kidney samples was even lower than that of jejunum. The mRNA expression levels of PiT-1 of 7 and 14 d old squabs were significantly increased by dietary P levels of breeding birds. The mRNA expression of PiT-2 in the group of dietary 0.4% P supplementation was significantly higher (P<0.05) than that of 0.8% group when the squabs were 7 and 21 d old. The interaction of tissue and dietary P levels was significant (P<0.05) on PiT-1 mRNA level of 7 and 14 d, and PiT-2 mRNA level of 21 d old birds. The mRNA expression level of NaPi-IIa in kidney of 14 d squabs significantly decreased in the group of 0.8% P supplementation, compared with the control and 0.4% group. (4) At the age of 14 and 21 d, about half of the significantly different metabolites among the control and P-added groups were included in the category of lipids and lipid-like molecules, organic acids and derivatives was the second, and benzenoids next. The biological pathways involved in the metabolism of differentially expressed metabolites were mainly carbohydrate metabolism, metabolism of protein and amino acids, translation and lipid metabolism etc. 【Conclusion】(1) There was a significant effect of P supplementation in the diet of parent pigeons on P content in crop milk fed to squabs. The values of serum P of young birds were stable. However, the serum Ca of squabs in the group of highest dietary P treatment decreased, which might indicate that pigeons were intolerant to high dietary P level. (2) The expression levels of P transporters (PiT-1 and PiT-2) mRNA in the kidney mostly increased with dietary P supplementation. The regulation mechanisms of P reabsorption in pigeons need to be studied further. (3)The serum metabolome profiles of squabs indicated that P was involved in many important metabolic processes, such as lipid metabolism and bone mineralization etc.

Key words: phosphorus, pigeon, biochemical index, metabolome, transporter

AN Yong, QIN ShiZhen, SHI ZhaoGuo, GONG LiYuan, ZHANG Shuai, JI Feng. Influences of Phosphorus Level in Diet of Parent Pigeons on Biochemical Index, Untargeted Metabolomics Profile of Serum, and Gene Expression of Phosphate Transporters in Squabs[J].Scientia Agricultura Sinica, 2023, 56(23): 4772-4788.

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Figures/Tables 12

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Table 6

Effects of dietary P levels on the relative expression of PiT-1 and PiT-2 mRNA in the small intestine and kidney of squabs at 7, 14 and 21 d old age"

项目 Item	7日龄 7 d old
	十二指肠Duodenum				空肠Jejunum				肾脏Kidney				SEM	组织Tissue section			SEM	磷添加水平P added level (%)				SEM	P值 P value
	0	0.2	0.4	0.8	0	0.2	0.4	0.8	0	0.2	0.4	0.8	SEM	十二指肠 Duodenum	空肠 Jejunum	肾脏 Kidney	SEM	0	0.2	0.4	0.8	SEM	组织 Tissue	P水平 P level	组织× P水平 Tissue×P level
PiT-1	1.02A	1.08A	1.17A	0.78A	1.53A	1.36A	1.93A	1.26A	3.76AB	5.11AB	7.35B	33.2C	1.24	1.01	1.52	12.34	0.62	2.10	2.52	3.48	11.7	0.72	0.00	0.00	0.00
PiT-2	1.03	0.95	1.14	0.94	0.71	0.66	0.98	0.51	2.10	1.98	2.19	2.05	0.12	1.01B	0.71A	2.08C	0.06	1.28ab	1.19ab	1.43b	1.17a	0.07	0.00	0.05	0.92
	14日龄 14 d old
	十二指肠Duodenum				空肠Jejunum				肾脏Kidney				SEM	组织Tissue section			SEM	磷添加水平 P added level (%)				SEM	P值 P value
	0	0.2	0.4	0.8	0	0.2	0.4	0.8	0	0.2	0.4	0.8	SEM	十二指肠 Duodenum	空肠 Jejunum	肾脏 Kidney	SEM	0	0.2	0.4	0.8	SEM	组织 Tissue	P水平 P level	组织× P水平 Tissue×P level
PiT-1	1.07A	0.89A	0.97A	1.09A	0.07A	0.59A	0.80A	1.24A	7.34B	7.89B	13.2C	12.7C	1.10	1.00	0.82	10.29	0.55	3.03	3.12	4.98	5.02	0.64	0.00	0.04	0.04
PiT-2	1.10	1.28	1.12	1.28	0.75	0.89	0.72	1.36	4.58	4.89	4.80	2.87	0.47	1.20A	0.93A	4.28B	0.24	2.15	2.35	2.21	1.84	0.27	0.00	0.60	0.10
	21日龄 21 d old
	十二指肠Duodenum				空肠Jejunum				肾脏Kidney				SEM	组织Tissue section			SEM	磷添加水平 P added level (%)				SEM	P值 P value
	0	0.2	0.4	0.8	0	0.2	0.4	0.8	0	0.2	0.4	0.8	SEM	十二指肠 Duodenum	空肠 Jejunum	肾脏 Kidney	SEM	0	0.2	0.4	0.8	SEM	组织 Tissue	P水平 P level	组织× P水平 Tissue×P level
PiT-1	1.18	1.17	1.35	1.50	2.06	3.02	1.63	2.11	1.18	1.17	1.35	1.59	0.33	1.3A	2.20B	1.32A	0.17	1.47	1.78	1.44	1.73	0.19	0.00	0.49	0.22
PiT-2	1.03A	1.14A	1.77B	1.28A	1.31AB	1.14A	0.93A	1.19A	1.03A	1.14A	1.77B	1.25AB	0.14	1.31	1.14	1.30	0.07	1.12	1.14	1.49	1.24	0.08	0.18	0.005	0.002

Table 6

Fig. 1

Fig. 2

Fig. 3

Table 7

Table 8

Table 9

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处理组 Treatment	1	2	3	4	处理组 Treatment	1	2	3	4
组成成分Ingredient (%)					营养水平 Nutrient Levels (%)
玉米 Corn	40.0	40.0	40.0	40.0	代谢能 ME (k cal·kg^-1)	2796.3	2796.3	2796.3	2796.3
豌豆 Pea	15.0	15.0	15.0	15.0	粗蛋白 CP ⁴⁾	15.5	15.0	15.2	15.2
小麦 Wheat	12.0	12.0	12.0	12.0	蛋氨酸 Methionine	0.25	0.25	0.25	0.25
高粱 Sorghum	10.0	10.1	10.1	10.1	赖氨酸 Lysine	0.82	0.82	0.82	0.82
豆粕 (46%) Soybean meal	15.0	15.0	15.0	15.0	Ca ⁴⁾	1.40	1.50	1.42	1.40
大豆油 Soybean oil	1.00	1.00	1.00	1.00	总磷 Total P (TP)⁴⁾	0.30	0.54	0.77	1.28
石粉 Stone powder	2.60	1.90	1.25	0.00
CaHPO₄·2H₂O	0.00	1.12	2.20	4.40
食盐 Salt	0.37	0.40	0.40	0.40
赖氨酸 Lysine	0.20	0.20	0.20	0.20
蛋氨酸 Methionine	0.12	0.12	0.12	0.12
多维 Vitamins¹⁾	0.02	0.02	0.02	0.02
复合微量元素 Minerals²⁾	0.15	0.15	0.15	0.15
氯化胆碱Choline chloride	0.04	0.04	0.04	0.04
砂砾 Sand³⁾	3.50	2.95	2.52	1.57

基因 Gene name	引物序列Primer sequence (5′ → 3′)		序列号Gene Bank accession number
NaPi-Ⅱa	Forward Reverse	ATCACGGGCATCGCAACAA GGGCTGTGCAGTTTGGAG	XM_021288850.1
NaPi-Ⅱb	Forward Reverse	CAGCATGGTGTCCTCCACAT GATTGTTGCCCCAGCAAAGG	XM_021286083.1
PiT-1	Forward Reverse	GAAGGCGTCAAGTGGTCTGA GAACGGGATCCGCCTTAGAG	XM_005505592.2
PiT-2	Forward Reverse	GAGGAAGGCGGCGTGGAGAT GGAAAAGCAGGTGGACCTGA	XM_021287576.1
β-actin	Forward Reverse	AGAGAGGCTACAGCTTCACCA CTGGCCATCAGGGAGTTCATAG	XM_005504502.2

项目 Item	磷添加水平 P supplemental level (%)	Ca (%)	P (%)
7日龄 7d	0	0.959	0.447B
	0.2	0.962	0.509C
	0.4	0.915	0.754E
	0.8	0.915	0.917G
14日龄 14d	0	1.018	0.304A
	0.2	1.019	0.453B
	0.4	1.027	0.649D
	0.8	0.904	0.911FG
21日龄 21d	0	1.063	0.303A
	0.2	1.092	0.452B
	0.4	1.108	0.624D
	0.8	1.023	0.863F
SEM		0.005	0.001
日龄 Day	7d	0.938A	0.657
	14d	0.992B	0.579
	21d	1.071C	0.560
SEM		0.012	0.007
磷添加水平 P added level (%)	0	1.014B	0.351
	0.2	1.024B	0.471
	0.4	1.017B	0.676
	0.8	0.947A	0.897
SEM		0.014	0.008
P值 P-Value	日龄 Day	0.000	0.000
	P水平P level	0.000	0.000
	日龄×P水平Day×P level	0.201	0.000

项目 Item	磷添加水平 P supplemental level (%)	Ca (mmol·L^-1)	P (mmol·L^-1)	ALP (U·L^-1)	PTH (ng·L^-1)
7日龄 7 d old	0	1.226	3.497abc	1328.0	221.9
	0.2	1.160	3.433ab	1267.3	196.7
	0.4	1.393	3.895bcd	1313.8	227.6
	0.8	1.211	4.171cd	1631.4	312.2
14日龄 14 d old	0	1.940	3.520abc	970.0	272.2
	0.2	2.060	3.768bcd	840.5	291.3
	0.4	1.966	3.526ab	1022.1	340.8
	0.8	1.663	3.049a	984.5	335.9
21日龄 21 d old	0	1.938	3.673abc	592.0	212.6
	0.2	2.256	3.666abc	781.0	134.9
	0.4	1.663	3.227ab	621.1	144.9
	0.8	1.740	4.340d	735.6	188.0
	SEM	0.074	0.424	163.0	39.7
日龄 Day	7 d	1.247A	3.749	1385.1C	239.6ABb
	14 d	1.907B	3.466	954.3B	310.0Bc
	21 d	1.937B	3.750	685.7A	170.1Aa
	SEM	0.052	0.119	73.632	28.1
磷添加水平 P supplemental level (%)	0	1.655AB	3.481	956.0	235.6
	0.2	1.773B	3.642	970.8	207.6
	0.4	1.827B	3.639	1017.4	237.8
	0.8	1.533A	3.857	1089.2	278.7
	SEM	0.060	0.137	84.3	22.9
P值 P-Value	日龄Day	0.000	0.151	0.000	0.000
	P水平P level	0.005	0.311	0.696	0.192
	日龄×P水平Day×P level	0.227	0.02	0.58	0.598

7日龄 7 d old
十二指肠 Duodenum				空肠 Jejunum				SEM	肠段 Intestinal segment		SEM	磷添加水平 P added level (%)				SEM	P值 P value
0	0.2	0.4	0.8	0	0.2	0.4	0.8	SEM	十二指肠 Duodenum	空肠 Jejunum	SEM	0	0.2	0.4	0.8	SEM	肠段 Segment	P水平 P level	肠段× P水平 Segment× P level
1.037	0.847	0.973	1.012	0.845	0.978	1.233	1.110	0.170	0.967	1.042	0.085	0.941	0.913	1.103	1.061	0.120	0.538	0.627	0.600
14日龄 14 d old
十二指肠 Duodenum				空肠 Jejunum				SEM	肠段 Intestinal segment		SEM	磷添加水平 P added level (%)				SEM	P值 P value
0	0.2	0.4	0.8	0	0.2	0.4	0.8	SEM	十二指肠 Duodenum	空肠 Jejunum	SEM	0	0.2	0.4	0.8	SEM	肠段 Segment	P水平 P level	肠段× P水平 Segment× P level
1.208	0.815	0.830	1.017	0.713	1.117	0.847	1.507	0.303	0.967	1.046	0.152	0.961	0.966	0.838	1.262	0.214	0.717	0.555	0.402
21日龄 21 d old
十二指肠 Duodenum				空肠 Jejunum				SEM	肠段 Intestinal segment		SEM	磷添加水平 P added level (%)				SEM	P值 P value
0	0.2	0.4	0.8	0	0.2	0.4	0.8	SEM	十二指肠 Duodenum	空肠 Jejunum	SEM	0	0.2	0.4	0.8		肠段 Segment	P水平 P level	肠段× P水平 Segment× P level
1.040	0.982	1.120	1.483	0.848	0.768	0.950	1.305	0.192	1.156	0.968	0.096	0.944	0.875	1.035	1.394	0.135	0.183	0.052	1.000

Influences of Phosphorus Level in Diet of Parent Pigeons on Biochemical Index, Untargeted Metabolomics Profile of Serum, and Gene Expression of Phosphate Transporters in Squabs

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序号 No.	差异代谢物 Differential metabolites	类别 Class	保留时间 RT (min)	质荷比 Ion (m/z)	Positive/ Negative	VIP		差异倍数FC		P值P value
序号 No.	差异代谢物 Differential metabolites	类别 Class	保留时间 RT (min)	质荷比 Ion (m/z)	Positive/ Negative	0.4% vs. Control	0.8% vs. Control	0.4% vs. Control	0.8% vs. Control	0.4% vs. Control	0.8% vs. Control
1	脱氢伏米酚 Dehydrovomifoliol	脂质和类脂质分子 Lipids and lipid-like molecules	5.69	267.1	neg	3.14	3.01	2.19	2.85	0.001	0.000
2	葫芦素B Cucurbitacin B	脂质和类脂质分子 Lipids and lipid-like molecules	10.20	579.3	neg	2.34	1.77	0.56	0.50	0.033	0.046
3	人参皂甙Rh6 Ginsenoside Rh6	脂质和类脂质分子 Lipids and lipid-like molecules	8.82	691.4	neg	2.13	1.39	0.74	0.75	0.011	0.026
4	3-羟基异庚酸 3-Hydroxyisoheptanoic acid	有机酸及其衍生物 Organic acids and derivatives	4.71	145.1	neg	1.50	1.57	0.91	0.87	0.007	0.001
5	3-甲氧基酪氨酸 3-Methoxytyrosine	有机酸及其衍生物 Organic acids and derivatives	5.30	194.1	pos	1.53	1.60	0.84	0.75	0.014	0.001
6	6-羟基氟伐他汀 6-Hydroxyfluvastatin	有机杂环化合物 Organoheterocyclic compounds	9.66	426.2	neg	1.27	1.18	1.07	1.11	0.027	0.006
7	Alhpa-tocopheronic acid		5.13	297.2	pos	1.91	1.74	0.77	0.72	0.006	0.001
8	1-（5-乙酰基-2-羟基苯基）-3-甲基-1-丁酮 1-(5-Acetyl-2-hydroxyphenyl)-3-methyl-1-butanone	有机氧化合物 Organic oxygen compounds	4.91	221.1	pos	1.46	1.43	1.17	1.27	0.046	0.001
9	2-[4,6-dihydroxy-3-(4-hydroxy-3-methylbut-2-en-1- yl)-2-methoxyphenyl]acetic acid	类苯Benzenoids	4.90	281.1	neg	1.79	1.61	1.14	1.19	0.009	0.001

日龄 Day (d)	比较组别 Groups	代谢通路 Metabolic pathways	代谢物个数 Number	P值 P value	校正P值 Corrected P value
14	0.4% vs. Control	胆汁分泌Bile secretion	1	0.023	0.023
	0.4% vs. Control	戊糖和葡萄糖醛酸的相互转化 Pentose and glucuronate interconversions	1	0.013	0.026
	0.8% vs. Control	维生素B6代谢Vitamin B6 metabolism	1	0.0262	0.0656
	0.8% vs. Control	氧化磷酸化Oxidative phosphorylation	1	0.0151	0.0753
21	0.4% vs. Control	胆酸生物合成Primary bile acid biosynthesis	1	0.0221	0.0221
	0.4% vs. Control	味觉传导Taste transduction	1	0.0151	0.0302
	0.8% vs. Control	多巴胺能突触Dopaminergic synapse	1	0.0085	0.0291
		催乳素信号通路Prolactin signaling pathway	1	0.0078	0.0312
		柠檬酸循环Citrate cycle (TCA cycle)	1	0.0141	0.0377
		苯丙氨酸、酪氨酸和色氨酸的生物合成 Phenylalanine, tyrosine and tryptophan biosynthesis	1	0.0239	0.0383
		单巴坦生物合成Monobactam biosynthesis	1	0.0274	0.0412
		β-丙氨酸代谢Beta-Alanine metabolism	1	0.0226	0.0416
		味觉传导Taste transduction	1	0.0226	0.0416
		丙氨酸、天冬氨酸和谷氨酸代谢Alanine, aspartate and glutamate metabolism	1	0.0198	0.0431
		硫胺素代谢Thiamine metabolism	1	0.0219	0.0437
		胰高血糖素信号通路Glucagon signaling pathway	1	0.0183	0.044
		氨基酰-tRNA生物合成Aminoacyl-tRNA biosynthesis	1	0.0365	0.0461
		组氨酸代谢Histidine metabolism	1	0.033	0.0466
		蛋白质消化和吸收Protein digestion and absorption	1	0.033	0.0466
		乙醛酸和二羧酸代谢Glyoxylate and dicarboxylate metabolism	1	0.0434	0.0496
		苯丙氨酸代谢Phenylalanine metabolism	1	0.042	0.0504
		黑色素生成Melanogenesis	1	0.0043	0.0511

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