日粮锰源和水平对产蛋鸡蛋壳品质的影响及其机理
摘要
蛋壳问题常见于蛋鸡生产中，每年因蛋壳问题所造成的经济损失巨大，人们尝试通过各种营养调控手段改善蛋壳品质，已有的大多数研究集中于常量矿物质对蛋壳品质的影响，而关于微量元素的影响研究较少。锰是蛋鸡的必需微量元素之一，但关于锰对蛋壳品质的影响及其机理仍不清楚，有待深入研究。本文通过观测产蛋鸡生产性能、蛋壳品质、壳基质蛋白表达、蛋壳微观结构和材料力学分析系统研究了日粮锰来源和水平对产蛋鸡蛋壳品质的影响及其机理，并采用指数模型估计了有机锰在蛋壳品质上的相对生物学效价。
试验一日粮锰水平对蛋壳品质的影响及其机理
试验旨在研究日粮不同锰水平对蛋壳品质的影响及其调控机理。选取50周龄216只体重相近且健康的产蛋鸡，随机分为三个处理，每个处理6个重复，每个重复12只鸡。其中饲养试验分预饲期和正饲期，预饲期（50-58wk）内所有蛋鸡饲喂一种锰不足但其他营养素满足需要的基础日粮；正饲期（59wk-62wk）内三组组动物分别饲喂三种锰添加水平的日粮，分别是Mn0mg/kg，25mg/kg和100mg/kg。不同锰水平对蛋鸡生产性能影响不显著（P>0.05）。与对照组和25mg/kg水平组相比，添加100mg/kg锰显著提高了蛋壳厚度、强度和韧性（P<0.05）；扫描电镜图片显示，添加100mg/kg锰显著降低了乳突层厚度和乳突宽度（P<0.05），明显减少了蛋壳表面裂纹。与对照组相比，添加100mg/kg锰显著提高了蛋壳腺中糖基转移酶GlcAT-I mRNA表达（P<0.05），但基质蛋白OC116、OCX32、OCX36mRNA表达差异不显著（P>0.05）。添加100mg/kg锰显著提高了蛋壳膜中糖胺聚糖和糖醛酸的含量（P<0.05），提高了蛋壳中糖基转移酶表达。日粮中添加锰能通过改变蛋壳微观结构和提高蛋壳中糖胺聚糖和糖醛酸的含量以及参与钙化过程中的蛋白表达改善蛋壳品质。
试验二日粮锰水平对蛋壳基质蛋白表达的影响
试验旨在探讨日粮锰水平对蛋壳基质蛋白表达的影响。从试验一中0mg/kg锰对照组和100mg/kg锰处理组每个重复取鸡蛋蛋壳，采用同位素标记相对和绝对定量技术（isotope tags forrelative and absolute quantitation，iTRAQ）检测蛋壳中基质蛋白差异表达。与对照组相比，基础日粮添加100mg/kg锰显著影响蛋壳中69种基质蛋白表达，其中31种基质蛋白上调，37种蛋白下调。这些差异表达的蛋白或与蛋壳钙化有关，或与产蛋鸡免疫功能有关。日粮锰水平通过对蛋壳基质蛋白的影响而改变蛋壳钙化过程或产蛋鸡免疫功能。
试验三不同锰源和锰水平对蛋壳品质的影响及相对生物学效价比较
试验旨在研究不同锰源和锰水平对蛋壳品质的影响及相对生物学效价比较。648只50周龄体重相近且健康的产蛋鸡，随机分为9个处理，每个处理6个重复，每个重复12只鸡。其中饲养试验分预饲期和正饲期，预饲期（50-54wk）内所有蛋鸡饲喂一种锰不足但其他营养素满足需要的基础日粮；正饲期（55wk-62wk）内9组蛋鸡分别给饲9种不同锰源和锰添加水平日粮，分别是对照组：0mg/kg；无机锰：25mg/kg、50mg/kg、100mg/kg、200mg/kg；氨基酸锰：25mg/kg、50mg/kg、100mg/kg、200mg/kg。不同锰源和锰水平对产蛋鸡生产性能影响不显著（P>0.05）。与对照组相比，基础日粮中添加有机锰100mg/kg和200mg/kg显著降低了蛋重（P<0.05），但添加无机锰无显著影响（P>0.05）。与对照组相比，基础日粮中添加无机锰100mg/kg、200mg/kg和氨基酸锰100mg/kg、200mg/kg均显著提高了60wk和62wk产蛋鸡蛋壳破损力、厚度和韧性（P<0.05），对56wk和58wk产蛋鸡蛋壳品质各项指标影响不显著（P>0.05）。在等摩尔添加水平下，不同锰源对蛋壳品质各项指标影响差异不显著（P>0.05）。采用NLIN指数模型计算氨基酸锰在蛋壳品质指标上的相对生物学效价，结果表明：在等摩尔水平上，氨基酸锰相对于无机锰的生物学效价分别是蛋壳破损力159.1%、蛋壳弹性107.8%和蛋壳厚度189.2%。
试验四蛋壳微观材料力学模型的建立
试验旨在分析蛋壳微观结构改变引起蛋壳破损力变化的机理。根据试验一中扫描电镜所获蛋壳微观结构图像，建立了蛋壳的微观结构力学模型，用材料力学方法建立了蛋壳应力变化随着蛋壳微观结构变化的数学模型。乳突层排列越紧密，蛋壳应力越大，蛋壳破损力越大；乳突层越厚，栅栏层越薄，蛋壳应力越小，蛋壳破损力越小。这一结果与试验一的实际观测结果一致。通过建立蛋壳微观材料力学模型和分析，结果表明：日粮锰水平能通过影响蛋壳微观结构变化改变蛋壳稳定性。
Eggshell quality is of primary concern to the egg industry as the commercial processing andmarketing of eggs usually causes a high rate of cracked or broken eggshells. It is generally accepted thateggshell quality decreases with hen age, and the incidence of cracked eggs can exceed20%at the end ofthe laying period. Many nutritional approaches to improving eggshell quality have been explored.Numerous studies have concentrated on the effects of macro minerals (calcium and phosphorus) andvitamin D3on eggshell and bone quality. It is well known that macro minerals act as an eggshellstructural component and play an essential role in eggshell function. However, the relationship betweentrace elements and eggshell quality still awaits further study.Manganese (Mn) is an important traceelement in poultry nutrition, this study explored the mechanism of effect of Mn supplementation oneggshell quality, and investigated the bio-efficacy of organic Mn compare to the inorganic Mn oneggshell quality.
Experiment1.Effect of dietary Mn supplementation on eggshell quality
This study investigated the effect of dietary manganese (Mn) supplementation on eggshell quality,ultrastructure, glycosaminoglycan (GAG) and uronic acid content, and mRNA and protein expression ofGalβ1,3-glucuronosyltransferase (GlcAT-I). A total of216layers (Hy-Line Grey) at age50wk weredivided into three groups. In the first8wk of the12wk feeding trial, all groups were fed a basal dietthat met all layer nutrient requirements except for Mn. In the last4wk, each group was fed one of threediets supplemented with Mn levels at0,25, or100mg Mn/kg. Dietary Mn deficiency did not affect theegg performance of layers. Dietary Mn supplementation significantly improved the breaking strength,thickness and fracture toughness of eggshells (p<0.05). In photographs of eggshell ultrastructure, thesize of mammillary cones and cracks in the outer surface were decreased by dietary Mnsupplementation. The contents of glycosaminoglycans (GAGs) and uronic acids in eggshell membranewere significantly increased by dietary Mn addition (p<0.05). This result was further confirmed byincreased mRNA expression and protein expression of GlcAT-I when Mn was added to the diet. Thisstudy suggests that dietary Mn supplementation can improve eggshell quality by enhancing the GAGand uronic acid synthesis in the eggshell glands, which can affect the ultrastructure of eggshells.
Experiment2. Effect of dietary Mn supplementation on matrix protein in the eggshell
This study investigated the effect of dietary Mn supplementation on matrix protein expression inthe eggshell. The eggshell fragments were randomly sampled from the replicates of0mg/kg or100mg/kg Mn supplementation treatment. Isotope tags for relative and absolute quantitation technologywas applied to investigate the difference of matrix protein expression in the eggshell. Dietary Mnsupplementation significantly influence the68matrix proteins expression in the eggshell. There were31 matrix proteins up regulated and38matrix proteins down regulated by the dietary100mg/kg Mnsupplementation. These up or down regulated matrix proteins were related to eggshell calcification orlayer's immunity. In conclusion, dietary Mn supplementation influenced the process of eggshellcalcification and layer's physiological status.
Experiment3.Effect of different sources and levels dietary of Mn supplementation on eggshellquality and bio-efficacy comparison of organic and inorganic Mn on eggshell quality of layinghens.
This study investigated the effect of different Mn resources supplementation on eggshell qualityand compared the bio-efficacy of organic Mn to the inorganic Mn on eggshell quality. A total of648layers (Hy-Line brown) at age50wk were divided into nine groups. In the first4wk of the12wkfeeding trial, all groups were fed a basal diet that met all layer nutrient requirements except for Mn. Inthe left8wk, each group was fed one of nine diets supplemented with different Mn resources at:0mg,inorganic25,50,100,200mg; organic (Amino acid manganese, AA-Mn)25,50,100,200mg Mn/kg.Dietary Mn deficiency did not affect the egg performance of layers (p>0.05). Dietary Mnsupplementation significantly improved the breaking strength, thickness and fracture toughness ofeggshells at62wk (p<0.05). Dietary supplementation of AA-Mn100mg/kg or200mg/kg Mnsignificantly reduced the egg weight (p<0.05), but there were no difference between the inorganic Mntreatments(P>0.05). Compare to the control group, dietary supplementation of100mg or200mginorganic or organic Mn/kg significantly increased the breaking strength, thickness and fracturetoughness of eggshell at the age of60wk or62wk. On the equimolar basis, there were no difference ofeffect between AA-Mn and inorganic Mn treatments. Regression analysis revealed the AA-Mn was159.1%(breaking strength),107.8%(elastic modulus) and198.2%(thickness) as efficacious asinorganic Mn on the quimolar basis.
Experiment4.Modeling of mechanics of eggshell ultra-structure
This study discussed the mechanics of eggshell ultra-structure affected by dietary Mnsupplementation. A model of eggshell ultra-structure mechanic was set on the base of photographs ofscanning electronic microscope in experiment1. Formulas of material mechanics were applied to justifythe difference of ultra-structure lead to different stress in the eggshell. The eggshells with more compactand uniform mammilaries and thicker palisade layer were tested as with more compressive strength. Theconclusion justified from the eggshell model were in agreement with results from experiment1. Thisstudy revealed that dietary Mn supplementation can influence the mechanics of eggshell ultra-structure.
试验一日粮锰水平对蛋壳品质的影响及其机理
试验旨在研究日粮不同锰水平对蛋壳品质的影响及其调控机理。选取50周龄216只体重相近且健康的产蛋鸡，随机分为三个处理，每个处理6个重复，每个重复12只鸡。其中饲养试验分预饲期和正饲期，预饲期（50-58wk）内所有蛋鸡饲喂一种锰不足但其他营养素满足需要的基础日粮；正饲期（59wk-62wk）内三组组动物分别饲喂三种锰添加水平的日粮，分别是Mn0mg/kg，25mg/kg和100mg/kg。不同锰水平对蛋鸡生产性能影响不显著（P>0.05）。与对照组和25mg/kg水平组相比，添加100mg/kg锰显著提高了蛋壳厚度、强度和韧性（P<0.05）；扫描电镜图片显示，添加100mg/kg锰显著降低了乳突层厚度和乳突宽度（P<0.05），明显减少了蛋壳表面裂纹。与对照组相比，添加100mg/kg锰显著提高了蛋壳腺中糖基转移酶GlcAT-I mRNA表达（P<0.05），但基质蛋白OC116、OCX32、OCX36mRNA表达差异不显著（P>0.05）。添加100mg/kg锰显著提高了蛋壳膜中糖胺聚糖和糖醛酸的含量（P<0.05），提高了蛋壳中糖基转移酶表达。日粮中添加锰能通过改变蛋壳微观结构和提高蛋壳中糖胺聚糖和糖醛酸的含量以及参与钙化过程中的蛋白表达改善蛋壳品质。
试验二日粮锰水平对蛋壳基质蛋白表达的影响
试验旨在探讨日粮锰水平对蛋壳基质蛋白表达的影响。从试验一中0mg/kg锰对照组和100mg/kg锰处理组每个重复取鸡蛋蛋壳，采用同位素标记相对和绝对定量技术（isotope tags forrelative and absolute quantitation，iTRAQ）检测蛋壳中基质蛋白差异表达。与对照组相比，基础日粮添加100mg/kg锰显著影响蛋壳中69种基质蛋白表达，其中31种基质蛋白上调，37种蛋白下调。这些差异表达的蛋白或与蛋壳钙化有关，或与产蛋鸡免疫功能有关。日粮锰水平通过对蛋壳基质蛋白的影响而改变蛋壳钙化过程或产蛋鸡免疫功能。
试验三不同锰源和锰水平对蛋壳品质的影响及相对生物学效价比较
试验旨在研究不同锰源和锰水平对蛋壳品质的影响及相对生物学效价比较。648只50周龄体重相近且健康的产蛋鸡，随机分为9个处理，每个处理6个重复，每个重复12只鸡。其中饲养试验分预饲期和正饲期，预饲期（50-54wk）内所有蛋鸡饲喂一种锰不足但其他营养素满足需要的基础日粮；正饲期（55wk-62wk）内9组蛋鸡分别给饲9种不同锰源和锰添加水平日粮，分别是对照组：0mg/kg；无机锰：25mg/kg、50mg/kg、100mg/kg、200mg/kg；氨基酸锰：25mg/kg、50mg/kg、100mg/kg、200mg/kg。不同锰源和锰水平对产蛋鸡生产性能影响不显著（P>0.05）。与对照组相比，基础日粮中添加有机锰100mg/kg和200mg/kg显著降低了蛋重（P<0.05），但添加无机锰无显著影响（P>0.05）。与对照组相比，基础日粮中添加无机锰100mg/kg、200mg/kg和氨基酸锰100mg/kg、200mg/kg均显著提高了60wk和62wk产蛋鸡蛋壳破损力、厚度和韧性（P<0.05），对56wk和58wk产蛋鸡蛋壳品质各项指标影响不显著（P>0.05）。在等摩尔添加水平下，不同锰源对蛋壳品质各项指标影响差异不显著（P>0.05）。采用NLIN指数模型计算氨基酸锰在蛋壳品质指标上的相对生物学效价，结果表明：在等摩尔水平上，氨基酸锰相对于无机锰的生物学效价分别是蛋壳破损力159.1%、蛋壳弹性107.8%和蛋壳厚度189.2%。
试验四蛋壳微观材料力学模型的建立
试验旨在分析蛋壳微观结构改变引起蛋壳破损力变化的机理。根据试验一中扫描电镜所获蛋壳微观结构图像，建立了蛋壳的微观结构力学模型，用材料力学方法建立了蛋壳应力变化随着蛋壳微观结构变化的数学模型。乳突层排列越紧密，蛋壳应力越大，蛋壳破损力越大；乳突层越厚，栅栏层越薄，蛋壳应力越小，蛋壳破损力越小。这一结果与试验一的实际观测结果一致。通过建立蛋壳微观材料力学模型和分析，结果表明：日粮锰水平能通过影响蛋壳微观结构变化改变蛋壳稳定性。
Eggshell quality is of primary concern to the egg industry as the commercial processing andmarketing of eggs usually causes a high rate of cracked or broken eggshells. It is generally accepted thateggshell quality decreases with hen age, and the incidence of cracked eggs can exceed20%at the end ofthe laying period. Many nutritional approaches to improving eggshell quality have been explored.Numerous studies have concentrated on the effects of macro minerals (calcium and phosphorus) andvitamin D3on eggshell and bone quality. It is well known that macro minerals act as an eggshellstructural component and play an essential role in eggshell function. However, the relationship betweentrace elements and eggshell quality still awaits further study.Manganese (Mn) is an important traceelement in poultry nutrition, this study explored the mechanism of effect of Mn supplementation oneggshell quality, and investigated the bio-efficacy of organic Mn compare to the inorganic Mn oneggshell quality.
Experiment1.Effect of dietary Mn supplementation on eggshell quality
This study investigated the effect of dietary manganese (Mn) supplementation on eggshell quality,ultrastructure, glycosaminoglycan (GAG) and uronic acid content, and mRNA and protein expression ofGalβ1,3-glucuronosyltransferase (GlcAT-I). A total of216layers (Hy-Line Grey) at age50wk weredivided into three groups. In the first8wk of the12wk feeding trial, all groups were fed a basal dietthat met all layer nutrient requirements except for Mn. In the last4wk, each group was fed one of threediets supplemented with Mn levels at0,25, or100mg Mn/kg. Dietary Mn deficiency did not affect theegg performance of layers. Dietary Mn supplementation significantly improved the breaking strength,thickness and fracture toughness of eggshells (p<0.05). In photographs of eggshell ultrastructure, thesize of mammillary cones and cracks in the outer surface were decreased by dietary Mnsupplementation. The contents of glycosaminoglycans (GAGs) and uronic acids in eggshell membranewere significantly increased by dietary Mn addition (p<0.05). This result was further confirmed byincreased mRNA expression and protein expression of GlcAT-I when Mn was added to the diet. Thisstudy suggests that dietary Mn supplementation can improve eggshell quality by enhancing the GAGand uronic acid synthesis in the eggshell glands, which can affect the ultrastructure of eggshells.
Experiment2. Effect of dietary Mn supplementation on matrix protein in the eggshell
This study investigated the effect of dietary Mn supplementation on matrix protein expression inthe eggshell. The eggshell fragments were randomly sampled from the replicates of0mg/kg or100mg/kg Mn supplementation treatment. Isotope tags for relative and absolute quantitation technologywas applied to investigate the difference of matrix protein expression in the eggshell. Dietary Mnsupplementation significantly influence the68matrix proteins expression in the eggshell. There were31 matrix proteins up regulated and38matrix proteins down regulated by the dietary100mg/kg Mnsupplementation. These up or down regulated matrix proteins were related to eggshell calcification orlayer's immunity. In conclusion, dietary Mn supplementation influenced the process of eggshellcalcification and layer's physiological status.
Experiment3.Effect of different sources and levels dietary of Mn supplementation on eggshellquality and bio-efficacy comparison of organic and inorganic Mn on eggshell quality of layinghens.
This study investigated the effect of different Mn resources supplementation on eggshell qualityand compared the bio-efficacy of organic Mn to the inorganic Mn on eggshell quality. A total of648layers (Hy-Line brown) at age50wk were divided into nine groups. In the first4wk of the12wkfeeding trial, all groups were fed a basal diet that met all layer nutrient requirements except for Mn. Inthe left8wk, each group was fed one of nine diets supplemented with different Mn resources at:0mg,inorganic25,50,100,200mg; organic (Amino acid manganese, AA-Mn)25,50,100,200mg Mn/kg.Dietary Mn deficiency did not affect the egg performance of layers (p>0.05). Dietary Mnsupplementation significantly improved the breaking strength, thickness and fracture toughness ofeggshells at62wk (p<0.05). Dietary supplementation of AA-Mn100mg/kg or200mg/kg Mnsignificantly reduced the egg weight (p<0.05), but there were no difference between the inorganic Mntreatments(P>0.05). Compare to the control group, dietary supplementation of100mg or200mginorganic or organic Mn/kg significantly increased the breaking strength, thickness and fracturetoughness of eggshell at the age of60wk or62wk. On the equimolar basis, there were no difference ofeffect between AA-Mn and inorganic Mn treatments. Regression analysis revealed the AA-Mn was159.1%(breaking strength),107.8%(elastic modulus) and198.2%(thickness) as efficacious asinorganic Mn on the quimolar basis.
Experiment4.Modeling of mechanics of eggshell ultra-structure
This study discussed the mechanics of eggshell ultra-structure affected by dietary Mnsupplementation. A model of eggshell ultra-structure mechanic was set on the base of photographs ofscanning electronic microscope in experiment1. Formulas of material mechanics were applied to justifythe difference of ultra-structure lead to different stress in the eggshell. The eggshells with more compactand uniform mammilaries and thicker palisade layer were tested as with more compressive strength. Theconclusion justified from the eggshell model were in agreement with results from experiment1. Thisstudy revealed that dietary Mn supplementation can influence the mechanics of eggshell ultra-structure.
引文
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