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

摘要/Abstract

摘要：

【目的】通过探讨亲鸽饲粮中添加不同水平无机磷对乳鸽血清生化指标、非靶向代谢物和小肠及肾脏中磷转运载体基因表达量的影响，为快速生长期乳鸽的磷营养调控提供理论依据。【方法】选取192对健康成年银王鸽（40周龄），随机分为4组，每个处理组8个重复，每个重复6对种鸽，产蛋后由亲鸽自然孵化和哺育乳鸽。试验期包括孵化期和哺育期，共46d。对照组饲喂基础日粮（总磷含量0.3%），其余3个试验组分别在基础日粮中添加0.2%、0.4%和0.8%无机磷，各组饲粮中Ca含量均为1.40%。分别收集7、14和21日龄乳鸽血清分析Ca、P含量，碱性磷酸酶（ALP）活性和甲状旁腺素（PTH）水平；检测鸽乳中Ca和P含量；测定肠道（十二指肠和空肠）磷转运载体（NaPi-Ⅱb、PiT-1、PiT-2）及肾脏磷转运载体（NaPi-Ⅱa、PiT-1、PiT-2）的基因表达水平；检测14和21日龄对照组和磷添加（0.4%和0.8%）组乳鸽血清中的非靶向代谢物。【结果】（1）随着亲鸽日粮中磷添加水平的增加，鸽乳中磷含量极显著上升（P<0.01）。（2）饲粮添加磷对乳鸽血清磷、ALP活性和PTH水平均未产生影响，而0.8%磷添加组血清钙含量极显著下降（P<0.01）。随着日龄增长，乳鸽血清ALP和PTH水平均显著降低（P<0.05）。（3）各日龄乳鸽小肠NaPi-IIb表达水平随亲鸽饲粮磷含量增加均有上升趋势，但差异不显著（P>0.05）。7和14日龄乳鸽PiT-1和PiT-2 mRNA在肾脏的表达量均极显著高于肠道（P<0.01），但21日龄时肾脏和肠道的表达量相近，甚至PiT-1在肾脏的表达量低于空肠。7和14日龄乳鸽PiT-1 mRNA表达随饲粮磷水平增加而增加，7和21日龄时，0.4%加磷组PiT-2 mRNA表达水平高于0.8%组（P<0.05），采样部位和饲粮磷水平对7和14日龄PiT-1基因表达以及21日龄PiT-2基因表达有互作影响（P<0.05）。14日龄时，0.8%加磷组肾脏NaPi-Ⅱa mRNA表达水平低于对照组和0.4%处理组。（4）14和21日龄时，对照组和加磷组乳鸽之间的显著差异代谢物中约50%为脂质和类脂质分子，其次是有机酸及其衍生物，再次是苯系物等某些有机物，且差异代谢物涉及到的主要代谢途径包括碳水化合物代谢、蛋白质和氨基酸代谢、遗传物质合成及脂肪代谢等。【结论】（1）亲鸽饲粮中的磷水平显著影响其分泌的鸽乳含磷量，而乳鸽血磷含量较稳定，但是高磷组血钙含量下降，这有可能表明鸽对高磷耐受性较差。（2）乳鸽肾脏中PiT-1和PiT-2 mRNA表达大多随饲粮磷含量增加而增加，具体调控机制有待研究。（3）血液代谢组数据表明磷参与乳鸽体内多种重要的代谢过程，如脂肪代谢和骨骼矿化等。

关键词: 磷, 鸽, 生化指标, 代谢组, 转运载体

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

安勇, 秦士贞, 史兆国, 龚莉媛, 张帅, 计峰. 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[J]. 中国农业科学, 2023, 56(23): 4772-4788.

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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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