Effects of Acid Protease Supplementation in Diets with Different Protein Levels on Growth Performance and Nutrient Apparent Digestibility of Broilers

LI Hongbin; LIU Yanjie; CHEN Ning; LI Fangfang; ZHU Yujing; ZHANG Ruiyang; YIN Dafei; ZHANG Yong

doi:10.12418/CJAN2023.275

2023 , Vol. 35 >Issue 5: 2943 - 2955

DOI: https://doi.org/10.12418/CJAN2023.275

RESEARCH PAPER

Effects of Acid Protease Supplementation in Diets with Different Protein Levels on Growth Performance and Nutrient Apparent Digestibility of Broilers

LI Hongbin ^,¹ ,
LIU Yanjie ² ,
CHEN Ning ³ ,
LI Fangfang ¹ ,
ZHU Yujing ¹ ,
ZHANG Ruiyang ¹ ,
YIN Dafei ¹ ,
ZHANG Yong ^,¹^,^**

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¹ College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China
² Jinan Bestzyme Bio-Engineering Co., Ltd., Jinan 251600, China
³ Modem Agricultural Production Base Construction Engineering Center of Liaoning Province, Shenyang 111000, China

**professor, E-mail: syndzhy@126.com

*Contributed equally

Received date: 2022-11-07

Online published: 2023-05-11

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Abstract

This experiment was conducted to investigate the effects of different dietary protein levels and different additional doses of acid protease on growth performance and nutrient apparent digestibility of broilers, and to provide reference for rational use of acid protease in the diet of broilers. A 4×2 factorial design was used in this experiment. Two factors were additional dose of acid protease and dietary protein level. The four additional doses of acid protease were 0, 125, 250 and 500 g/t, respectively. The two protein level were normal protein level (basal diet) and low protein level (low protein diet with 1% lower protein level on the basis of basal diet), respectively. A total of 1 440 healthy 7-day-old white feathered broilers were randomly divided into 8 groups with 6 replicates per group and 30 broilers per replicate. Broilers in the four groups were fed the basal diet (group Ⅰ), the basal diet+125 g/t acid protease (group Ⅱ), the basal diet+250 g/t acid protease (group Ⅲ), the basal diet+500 g/t acid protease (group Ⅳ), the low protein diet (group Ⅴ), the low protein diet+125 g/t acid protease (group Ⅵ), the low protein diet+250 g/t acid protease (group Ⅶ) and the low protein diet+500 g/t acid protease (group Ⅷ), respectively. the group Ⅰ which fed the basal diet and the group Ⅴ which fed low protein diet were the control group. The experiment lasted for 42 days. The results showed as follows: 1) under the condition of basal diet, compared with the control group (group Ⅰ), the average daily gain in the early stage of group Ⅱ was significantly increased (P<0.05), the average daily feed intake and average daily gain in the later stage of groups Ⅲ and Ⅳ were significantly increased (P<0.05), and the average daily gain in the whole stage of groups Ⅱ and Ⅳ was significantly increased (P<0.05); under the condition of low protein diet, compared with the control group (group Ⅴ), the average daily gain in the early stage of group Ⅶ was significantly increased (P<0.05), and the average daily gain in the whole stage of groups Ⅶ and Ⅷ was significantly increased (P<0.05). 2) Under the condition of basal diet, compared with the control group (group Ⅰ), the apparent digestibility of dry matter of broilers of groups Ⅱ, Ⅲ and Ⅳ was significantly increased (P<0.05); the apparent digestibility of crude protein, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine and proline of broilers of groups Ⅲ and Ⅳ was significantly increased (P<0.05); under the condition of low protein diet, compared with the control group (group Ⅴ), the apparent digestibility of dry matter, cysteine, valine and tyrosine of broilers of groups Ⅵ, Ⅶ and Ⅷ was significantly increased (P<0.05); the apparent digestibility of crude protein, serine, glycine and alanine of broilers of groups Ⅶ and Ⅷ was significantly increased (P<0.05); the apparent digestibility of aspartic acid, threonine, glutamic acid, methionine, isoleucine, leucine, phenylalanine, lysine, histidine, arginine and proline of broilers of group Ⅷ was significantly increased (P<0.05). 3) Under the condition of basal diet, the profit per chicken of groups Ⅱ, Ⅲ and Ⅳ was 0.64, 0.44 and 0.62 CNY higher than that of group Ⅰ, respectively; under the condition of low protein diet, the profit per chicken of groups Ⅵ, Ⅶ and Ⅷ was 0.32, 0.63 and 0.57 CNY higher than that of group Ⅴ, respectively. In conclusion, adding acid protease in diets with different protein levels can improve the growth performance and nutrient apparent digestibility, and reduce the mortality rate of broilers. In the basal diet, 125 and 500 g/t acid protease are recommended to be added to the diets of broilers in the early and later stages of the experiment, respectively. In the low protein diet, 250 and 500 g/t acid protease are recommended to be added to the diets of broilers in the early and later stages of the experiment, respectively.

Key words： broilers; acid protease; growth performance; amino acid apparent digestibility

Cite this article

LI Hongbin , LIU Yanjie , CHEN Ning , LI Fangfang , ZHU Yujing , ZHANG Ruiyang , YIN Dafei , ZHANG Yong . Effects of Acid Protease Supplementation in Diets with Different Protein Levels on Growth Performance and Nutrient Apparent Digestibility of Broilers[J]. Chinese Journal of Animal Nutrition, 2023 , 35(5) : 2943 -2955 . DOI: 10.12418/CJAN2023.275

蛋白质是肉鸡机体内所有生物代谢过程中的必需营养物质,而肉鸡饲粮中蛋白质的主要来源是豆粕。我国豆粕资源相对紧缺,大豆进口随着养殖行业的快速发展从2012年的5 838万t上升到了2021年的9 654万t。同时,我国肉鸡养殖主要采用玉米-豆粕型饲粮,虽然保证了肉鸡的快速生长,但也造成了蛋白质资源的浪费。因此,如何合理利用蛋白质和减少资源的浪费成为研究的热点话题。在肉鸡养殖中,肉鸡自身产生的内源性蛋白酶对饲粮中蛋白质的消化效率为60%~70%^[1-2]。而对于幼龄肉鸡来说,因机体消化系统未发育完全导致自身分泌的蛋白酶和采食量不协调,蛋白质更不容易消化。研究表明,在饲粮中通过添加外源性蛋白酶,可以有效补充内源性蛋白酶的不足,增强肠道中蛋白酶的活性,提高蛋白质的消化率,改善动物的生长性能^[3]。目前常见的蛋白酶一般为碱性蛋白酶,对酸性蛋白酶的研究较少,而苗华彪等^[4]通过体外法对饲用蛋白酶的研究发现酸性蛋白酶的水解效果优于中性、碱性蛋白酶。此外,目前对于酸性蛋白酶的研究多在同一饲粮蛋白质水平的基础上进行研究,对低蛋白质饲粮中酸性蛋白酶应用的研究较少。因此,本试验拟在不同蛋白质水平饲粮中添加不同剂量的酸性蛋白酶,以爱拔益加(AA)白羽肉鸡为研究对象,探讨低蛋白质饲粮中添加酸性蛋白酶对肉鸡生长性能、养分表观消化率的影响,为准确评价和使用酸性蛋白酶提供依据。

1 材料与方法

1.1 试验材料

本试验所使用的酸性蛋白酶购自济南某生物工程有限公司,酸性蛋白酶活性为100 000 U/g。

1.2 试验设计

采用4×2析因设计,2个因素分别为酸性蛋白酶添加剂量和饲粮蛋白质水平,4个酸性蛋白酶添加剂量分别为0(A)、125(B)、250(C)和500 g/t(D);2个饲粮蛋白质水平分别为正常蛋白质水平[基础饲粮,7~28日龄饲粮蛋白质水平为20.52%,29~49日龄饲粮蛋白质水平为19.98%]和低蛋白质水平(低蛋白质饲粮,蛋白质水平在正常蛋白质水平基础上降低约1%,7~28日龄饲粮蛋白质水平为19.49%,29~49日龄饲粮蛋白质水平为19.01%)。

挑选1 440只体况良好的7日龄AA白羽肉鸡,随机分为8个组,每组设6个重复,每个重复30只鸡,各组初始体重差异不显著(P>0.05)。各组分别饲喂基础饲粮(Ⅰ组)、基础饲粮+125 g/t酸性蛋白酶(Ⅱ组)、基础饲粮+250 g/t酸性蛋白酶(Ⅲ组)、基础饲粮+500 g/t酸性蛋白酶(Ⅳ组)、低蛋白质饲粮(Ⅴ组)、低蛋白质饲粮+125 g/t酸性蛋白酶(Ⅵ组)、低蛋白质饲粮+250 g/t酸性蛋白酶(Ⅶ组)和低蛋白质饲粮+500 g/t酸性蛋白酶(Ⅷ组),其中饲喂基础饲粮的Ⅰ组和饲喂低蛋白质饲粮的Ⅴ组为对照组。试验分为2个阶段,分别为试验前期(7~28日龄)和后期(29~49日龄),共42 d。

1.3 试验饲粮与饲养管理

试验饲粮为玉米-豆粕型饲粮,参照《鸡饲养标准》(NY/T 33—2004)中的营养需求配制,其组成及营养水平表1。

表1 试验饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of experimental diets (air-dry basis)%

项目 Items	7~28日龄 7 to 28 days of age		29~49日龄 29 to 49 days of age
项目 Items	基础饲粮 Basal diet	低蛋白质饲粮 Low protein diet	基础饲粮 Basal diet		低蛋白质饲粮 Low protein diet
原料 Ingredients
玉米 Corn	58.02	62.15		58.26		62.43
去皮豆粕 Dehulled soybean meal	26.00	23.27
豆粕(片,45%) Soybean meal (tablet, 45%)				25.00		22.30
玉米蛋白粉 Corn gluten meal	3.00	2.73		3.50		3.19
鸡油 Chicken oil	6.00	5.46		7.00		6.37
石粉 Limestone	1.10	1.00		1.00		0.91
磷酸氢钙 CaHPO₄	1.10	1.00		0.95		0.86
谷氨酸渣 Glutamic acid residue	2.00	1.82		2.00		1.82
羽毛粉 Feather meal	0.50	0.46
氯化钠 NaCl	0.28	0.25		0.28		0.25
L-赖氨酸盐酸盐 L-Lys·HCl	0.77	0.70		0.80		0.73
L-苏氨酸盐酸盐 L-Thr·HCl	0.10	0.09		0.10		0.09
DL-蛋氨酸盐酸盐 DL-Met·HCl	0.20	0.18		0.18		0.16
氯化胆碱 Choline chloride	0.10	0.09		0.10		0.09
甜菜碱 Betaine	0.03	0.03		0.03		0.03
腐植酸钠 Sodium humate	0.20	0.18		0.20		0.18
小苏打 NaHCO₃	0.10	0.09		0.10		0.09
预混料 Premix¹⁾	0.50	0.50		0.50		0.50
合计 Total	100.00	100.00		100.00		100.00
营养水平 Nutrient levels²⁾
粗蛋白质 CP	20.52	19.49		19.98		19.01
代谢能 ME/(MJ/kg)	13.50	13.50		13.81		13.81
钙 Ca	0.77	0.77		0.70		0.70
有效磷 AP	0.32	0.32		0.30		0.30
赖氨酸 Lys	1.34	1.34		1.32		1.32
苏氨酸 Thr	1.02	1.02		1.00		1.00
蛋氨酸 Met	0.54	0.54		0.51		0.51

1)预混料为每千克饲粮提供 The premix provided the following per kg of diets:Fe 76 mg,Cu 9.3 mg,Zn 72 mg,Mn 85 mg,Se 0.34 mg,I 0.68 mg,VA 11 000 IU,VE 14 IU,VD₃ 3 900 IU,VK 1.1 mg,烟酸 niacin 69 mg,VB₁ 1.4 mg,VB₂ 6.2 mg,VB₆ 3.1 mg,泛酸 pantothenic acid 15 mg,叶酸 folic acid 0.6 mg,生物素 biotin 0.13 mg,VB₁₂ 0.017 mg。

2)粗蛋白质水平为实测值,其他营养水平为计算值。CP was a measured value, while the other nutrient levels were calculated values.

试验开始前需要对鸡舍内进行清理和全面的消杀。肉鸡在试验期间自由采食和饮水,禁食期间保持水源的补给。鸡舍光照、温度、湿度、通风、卫生、免疫程序以及其他管理按照常规进行。

1.4 指标测定

1.4.1 生长性能

以每个重复为单位,在试验开始之前对试验肉鸡进行称重,在试验进行期间每7 d对肉鸡剩料量称重并记录每周的采食量;在试验第21天(28日龄)和第42天(49日龄)对试验肉鸡进行称重并记录肉鸡增重;在试验期间记录肉鸡的死亡和淘汰数量并计算出肉鸡的死淘率。通过肉鸡采食量和肉鸡增重统计数据计算出试验前期、后期和全期肉鸡的平均日采食量(ADFI)、平均日增重(ADG)与料重比(F/G)。

1.4.2 养分表观消化率

在肉鸡49日龄时,先饥饿4 h,采食1 h,再过3 h后从每组的每个重复中选取1只肉鸡进行屠宰,将回肠后1/2段内食糜轻轻挤于铝盒中,并迅速转入-20 ℃冰箱冷冻保存。将各组的饲粮和回肠食糜在冻干机内冻干,室温下回潮24 h后粉碎过40目筛并制备成分析试样。干物质(DM)含量采用直接烘干法(GB/T 6435—2014)测定,酸不溶灰分(AIA)含量采用灼烧法(GB/T 23742—2009)测定,粗蛋白质(CP)含量采用凯氏定氮法(GB/T 6432—2018)测定。

氨基酸含量测定参照GB/T 18246—2000。将分析试样送至沈阳农业大学分析测样中心,采用日立L-8900全自动氨基酸分析仪测定天门冬氨酸(Asp)、苏氨酸(Thr)、丝氨酸(Ser)、谷氨酸(Glu)、甘氨酸(Gly)、丙氨酸(Ala)、半胱氨酸(Cys)、缬氨酸(Val)、蛋氨酸(Met)、亮氨酸(Leu)、异亮氨酸(Ile)、酪氨酸(Tyr)、苯丙氨酸(Phe)、赖氨酸(Lys)、组氨酸(His)、精氨酸(Arg)、脯氨酸(Pro)的含量。

采用AIA法计算粗蛋白质、干物质和各种氨基酸的表观消化率^[5]。养分表观消化率计算公式如下:

某营养物质消化率(%)=[1-(B×C)/(A×D)]×100。

式中:A为饲粮中的某营养物质含量;B为回肠食糜中某营养物质含量;C为饲粮中AIA的含量;D为回肠食糜中AIA的含量。

1.5 数据处理与分析

试验采用SPSS 25.0软件进行单因素方差分析(one-way ANOVA),并采用Turkey法进行多重比较;采用SPSS 25.0软件对数据进行4×2析因分析。结果用“平均值±标准差”表示,P<0.05表示差异显著。

2 结果与分析

2.1 饲粮蛋白质水平和酸性蛋白酶添加剂量对肉鸡生长性能的影响

由表2可知,酸性蛋白酶添加剂量的主效应分析表明,添加B、C剂量时肉鸡前期ADG显著高于添加A、D剂量时(P<0.05);添加C、D剂量时肉鸡后期ADFI显著高于添加A、B剂量时(P<0.05);添加D剂量时肉鸡后期ADG显著高于添加A、B剂量时,添加C剂量时肉鸡后期ADG显著高于添加A剂量时(P<0.05);添加D剂量时肉鸡后期F/G显著高于添加A、B剂量时(P<0.05);添加C、D剂量时肉鸡全期ADFI显著高于添加A剂量时(P<0.05);添加B、C、D剂量时肉鸡全期ADG显著高于添加A剂量时(P<0.01);添加B、C、D剂量时肉鸡全期F/G显著高于添加A剂量时(P<0.05);添加B、C、D剂量时肉鸡全期死淘率高于添加A剂量时(P>0.05)。饲粮蛋白质水平的主效应分析表明,饲喂低蛋白质饲粮的肉鸡各阶段的ADG、ADFI和F/G与饲喂基础饲粮的肉鸡均差异不显著(P>0.05);饲喂低蛋白质饲粮的肉鸡全期死淘率高于饲喂基础饲粮的肉鸡(P>0.05)。酸性蛋白酶添加剂量和饲粮蛋白质水平的交互作用对肉鸡各阶段的ADG、ADFI和F/G以及全期死淘率均无显著影响(P>0.05)。

表2 饲粮蛋白质水平和酸性蛋白酶添加剂量对肉鸡生长性能的影响

Table 2 Effects of dietary protein level and acid protease additional dose on growth performance of broilers

项目 Items			7~28日龄 7 to 28 days of age						29~49日龄 29 to 49 days of age						7~49日龄 7 to 49 days of age
项目 Items			平均日采食量 ADFI/g		平均日增重 ADG/g		料重比 F/G		平均日采食量 ADFI/g		平均日增重 ADG/g		料重比 F/G		平均日采食量 ADFI/g		平均日增重 ADG/g		料重比 F/G		死淘率 Mortality rate/%
组别 Groups	Ⅰ		53.17±3.19		36.89±1.67^bc		1.44±0.04		97.68±2.33^c		41.14±2.98^c		2.38±0.18		75.43±2.34		39.02±1.77^bc		1.94±0.09		2.86±2.02
	Ⅱ		55.00±2.73		40.84±3.22^a		1.35±0.07		99.70±2.14^bc		43.27±1.79^bc		2.31±0.09		77.35±1.60		42.06±1.07^a		1.84±0.04		1.71±1.56
	Ⅲ		54.90±1.95		38.83±1.41^abc		1.41±0.04		108.62±3.73^a		48.17±2.36^ab		2.26±0.11		81.76±2.23		43.50±1.38^ab		1.88±0.06		1.71±2.56
	Ⅳ		52.56±2.64		36.49±1.59^bc		1.44±0.02		106.67±5.83^ab		50.32±6.66^a		2.14±0.26		79.61±4.01		43.40±3.78^a		1.84±0.13		3.43±3.73
	Ⅴ		52.06±2.90		35.83±2.25^c		1.45±0.05		97.54±3.16^c		40.21±3.86^c		2.44±0.17		74.80±1.89		38.02±1.82^c		1.97±0.05		4.57±3.83
	Ⅵ		54.44±3.42		38.52±2.18^abc		1.41±0.04		98.56±2.64^c		41.65±3.41^c		2.38±0.23		76.50±2.90		40.09±0.93^abc		1.91±0.10		2.86±2.86
	Ⅶ		55.41±2.71		39.38±1.68^ab		1.41±0.01		102.11±11.46^abc		44.67±7.17^abc		2.30±0.13		78.76±6.82		42.02±4.25^ab		1.88±0.04		3.43±3.73
	Ⅷ		53.21±4.88		36.97±2.01^bc		1.44±0.14		102.52±6.78^abc		46.44±4.42^abc		2.21±0.11		77.87±3.28		41.71±2.50^ab		1.87±0.08		1.14±1.56
剂量 Dose	A		52.62±2.93		36.36±2.87^b		1.45±0.06		97.61±2.35^b		40.67±2.71^c		2.41±0.17^a		75.12±2.25^b		38.52±1.07^b		1.95±0.04^a		3.71±3.03
	B		54.72±2.93		39.68±1.49^a		1.38±0.03		99.13±8.73^b		42.46±5.36^bc		2.34±0.19^a		76.92±5.04^ab		41.07±1.38^a		1.87±0.06^b		2.29±2.25
	C		55.16±2.24		39.10±1.72^a		1.41±0.09		105.37±6.35^a		46.42±5.71^ab		2.28±0.19^ab		80.26±3.57^a		42.76±3.78^a		1.88±0.13^b		2.57±3.14
	D		52.88±3.72		36.73±2.62^b		1.44±0.06		104.60±5.86^a		48.38±5.23^a		2.18±0.18^b		78.74±3.47^a		42.56±2.79^a		1.86±0.09^b		2.29±2.95
水平 Level	基础饲粮 Basal diet		53.91±2.68		38.26±2.34		1.41±0.07		103.17±6.77		45.73±5.18		2.27±0.18		78.54±4.11		41.99±2.94		1.87±0.08		2.43±2.50
水平 Level	低蛋白质饲粮 Low protein diet		53.78±1.95		37.67±0.04		1.43±2.62		100.18±3.29		43.24±0.17		2.33±2.03		76.98±1.77		40.46±2.93		1.91±0.08		3.00±3.14
P值 P-value	剂量 Dose		0.20		<0.01		0.11		<0.01		<0.01		0.03		0.02		<0.01		0.04		0.65
	水平 Level		0.90		0.38		0.40		0.10		0.09		0.27		0.17		0.06		0.21		0.53
	剂量×水平Dose×level	0.91		0.36		0.62		0.58		0.86		0.99		0.87		0.98		0.77		0.36

“剂量”表示酸性蛋白酶添加剂量,A、B、C和D对应的添加剂量分别为0、125、250和500 g/t;“水平”表示饲粮蛋白质水平,7~28日龄时基础饲粮和低蛋白质饲粮的蛋白质水平分别为20.52%和19.49%,29~49日龄时基础饲粮和低蛋白质饲粮的蛋白质水平分别为19.98%和19.01%。“剂量×水平”表示酸性蛋白酶添加剂量与饲粮蛋白质水平的交互作用。同列数据肩标相同或无小写字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05)。下表同。

“Dose” mean acid protease additional dose, and the corresponding additional doses of A, B, C and D were 0, 125, 250 and 500 g/t; “Level” mean dietary protein level, the protein levels of basal diet and low protein diet at 7 to 28 days of age and 29 to 49 days of age were 20.52% and 19.49%, 19.98% and 19.01%, respectively. “Dose×level” mean the interaction of acid protease additional dose and dietary protein level. In the same column, values with the same or no small letter superscripts indicated no significant difference (P>0.05), while with different small letter superscripts indicated significant difference (P<0.05). The same as below.

单因素方差分析表明,在饲喂基础饲粮时,与对照组(Ⅰ组)相比,Ⅱ组肉鸡前期ADG显著提高(P<0.01);Ⅲ、Ⅳ组肉鸡后期ADFI显著提高(P<0.05);Ⅲ、Ⅳ组后期肉鸡ADG显著提高(P<0.05);Ⅱ、Ⅳ组肉鸡全期ADG显著提高(P<0.05)。在饲喂低蛋白质饲粮中,与对照组(Ⅴ组)相比,Ⅵ组肉鸡前期ADG显著提高(P<0.01);Ⅶ、Ⅷ组肉鸡全期ADG显著提高(P<0.05)。在试验全期,无论是饲喂基础饲粮还是低蛋白质饲粮时,添加不同剂量酸性蛋白酶组肉鸡的死淘率与对照组均无显著差异(P>0.05)。

2.2 饲粮蛋白质水平和酸性蛋白酶添加剂量对肉鸡养分表观消化率的影响

由表3可知,酸性蛋白酶添加剂量的主效应分析表明,添加C、D剂量时肉鸡对CP、Met、Ile、Leu、Phe、His的表观消化率显著高于添加A、B剂量时(P<0.05);添加D剂量时肉鸡对Asp、Glu、Cys、Pro的表观消化率显著高于添加A、B剂量时(P<0.05);添加B、C、D剂量时肉鸡对DM、Thr、Ser、Gly、Ala、Cys、Val、Tyr的表观消化率显著高于添加A剂量时(P<0.05);添加C、D剂量时肉鸡对Asp、Glu、Gly、Cys、Val、Tyr、Lys、Arg、Pro的表观消化率显著高于添加A剂量时(P<0.05)。饲粮蛋白质水平的主效应分析表明,饲喂基础饲粮的肉鸡对CP、Cys、Tyr的表观消化率显著高于饲喂低蛋白质饲粮的肉鸡(P<0.05)。酸性蛋白酶添加剂量与饲粮蛋白质水平的交互作用对肉鸡对Met的表观消化率有显著影响(P<0.05)。

表3 饲粮蛋白质水平和酸性蛋白酶添加剂量对肉鸡养分表观消化率的影响

Table 3 Effects of dietary protein level and acid protease additional dose on apparent digestibility of nutrients of broilers%

项目 Items		粗蛋白质 CP	干物质 DM	天门冬氨酸 Asp	苏氨酸 Thr	丝氨酸 Ser	谷氨酸 Glu	甘氨酸 Gly
组别 Groups	Ⅰ	62.92±4.81^cd	94.67±0.59^cd	61.38±8.34^d	57.62±8.31^c	58.62±7.25^d	73.74±5.05^d	53.78±8.53^cd
	Ⅱ	70.47±0.74^bcd	96.48±1.25^ab	66.97±2.25^bcd	64.09±3.16^bc	64.46±2.93^cd	76.81±1.36^cd	59.97±3.34^bcd
	Ⅲ	75.06±7.14^ab	96.60±0.38^ab	76.11±7.75^ab	73.50±8.54^ab	74.28±8.50^abc	84.51±4.80^ab	70.14±8.88^ab
	Ⅳ	83.85±0.08^a	97.52±0.06^a	83.55±1.12^a	81.64±1.10^a	82.70±1.14^a	89.29±1.04^a	79.94±1.09^a
	Ⅴ	60.59±7.39^d	93.76±0.91^d	62.31±3.01^cd	57.73±0.62^c	57.92±0.39^d	75.09±2.71^cd	51.45±1.97^d
	Ⅵ	60.19±4.71^d	95.63±0.04^bc	70.51±2.39^bcd	65.86±1.68^bc	67.14±1.75^bcd	80.77±1.53^bcd	62.68±2.53^bcd
	Ⅶ	73.58±5.12^abc	96.94±0.35^ab	73.05±3.59^abc	66.51±0.65^bc	68.98±1.77^bc	81.43±1.58^bc	63.82±1.67^bc
	Ⅷ	73.58±5.12^abc	96.51±0.03^ab	77.67±0.48^ab	74.10±1.61^ab	75.51±0.63^ab	84.68±1.90^ab	71.66±0.65^ab
剂量 Dose	A	61.76±5.27^b	94.21±0.82^b	61.85±5.14^c	57.68±4.81^c	58.27±4.21^c	74.42±3.40^c	52.62±5.23^c
	B	65.33±6.54^b	96.06±0.87^a	68.74±2.79^bc	64.98±2.31^b	65.80±2.51^b	78.79±2.58^bc	61.33±2.88^b
	C	73.87±4.77^a	96.77±0.35^a	74.58±5.24^ab	70.01±6.38^b	71.63±5.87^b	82.97±3.41^ab	66.98±6.37^b
	D	78.72±6.63^a	97.01±0.58^a	80.61±3.47^a	77.87±4.49^a	79.11±4.22^a	86.99±2.94^a	75.80±4.84^a
水平 Level	基础饲粮 Basal diet	73.08±8.78^b	96.32±1.23	72.01±10.09	69.21±10.82	70.01±10.79	81.09±7.11	65.96±11.70
水平 Level	低蛋白质饲粮 Low protein diet	66.76±7.88^a	95.71±1.36	70.89±6.28	66.05±6.27	67.39±6.80	80.49±3.98	62.40±7.83
P值 P-value	剂量Dose	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
	水平Level	0.03	0.08	0.63	0.19	0.25	0.69	0.17
	剂量×水平 Dose×level	0.49	0.42	0.50	0.39	0.39	0.22	0.42
项目 Items		丙氨酸 Ala	半胱氨酸 Cys	缬氨酸 Val	蛋氨酸 Met	异亮氨酸 Ile	亮氨酸 Leu
组别 Groups	Ⅰ	67.44±4.79^e	66.30±6.88^cd	62.76±6.45^d	72.10±4.61^d	62.01±7.43^d	71.49±4.49^d
	Ⅱ	71.51±2.43^cde	71.02±0.07^bc	68.19±2.40^cd	72.68±1.57^d	66.80±3.25^cd	74.47±2.27^d
	Ⅲ	80.30±5.96^ab	77.74±1.42^ab	77.61±5.00^ab	84.16±5.15^ab	78.78±7.29^ab	84.28±5.43^ab
	Ⅳ	86.02±1.09^a	85.52±1.48^a	84.48±1.13^a	90.58±1.54^a	84.76±1.05^a	88.99±0.99^a
	Ⅴ	67.93±1.38^de	60.01±0.48^d	62.07±1.62^d	76.97±0.03^cd	65.41±2.04^cd	74.87±1.61^cd
	Ⅵ	74.96±0.77^bcd	72.32±4.42^bc	71.29±2.69^bc	80.78±1.99^bc	71.88±1.84^bcd	79.73±1.14^bc
	Ⅶ	75.97±0.81^bc	69.58±4.99^bc	71.92±0.42^bc	80.52±1.97^bc	74.25±2.75^abc	81.35±2.09^bc
	Ⅷ	81.42±0.44^ab	70.67±3.58^bc	78.41±3.44^ab	84.38±1.86^ab	82.65±4.37^ab	83.98±1.56^ab
剂量 Dose	A	67.68±2.89^d	63.15±5.39^c	62.41±3.86^c	74.53±3.87^c	63.71±4.86^c	73.18±3.37^b
	B	73.23±2.47^c	71.67±2.66^b	69.74±2.75^b	76.73±4.90^c	69.34±3.63^c	77.10±3.37^b
	C	78.13±4.28^b	73.66±5.58^ab	74.77±4.38^b	82.34±3.81^b	76.51±5.20^b	82.81±3.76^a
	D	83.72±2.74^a	78.10±8.86^a	81.44±4.08^a	87.48±3.84^a	83.70±2.87^a	86.49±3.08^a
水平 Level	基础饲粮 Basal diet	76.32±8.36	75.15±8.20^a	73.26±9.53	79.88±8.81	73.09±10.56	79.81±8.10
水平 Level	低蛋白质饲粮 Low protein diet	75.07±5.18	68.15±5.88^b	70.92±6.47	80.66±3.08	73.55±6.96	79.98±3.76
P值 P-value	剂量 Dose	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
	水平 Level	0.42	<0.01	0.21	0.60	0.84	0.91
	剂量×水平 Dose×level	0.23	0.08	0.26	0.02	0.42	0.10
项目 Items		酪氨酸 Tyr	苯丙氨酸 Phe	赖氨酸 Lys	组氨酸 His	精氨酸 Arg	脯氨酸 Pro
组别 Groups	Ⅰ	64.80±6.00^de	68.67±5.71^d	69.90±4.63^c	65.11±6.52^d	68.25±5.13^d	70.43±5.22^d
	Ⅱ	70.24±1.00^cd	71.84±1.89^cd	71.73±2.77^c	68.20±2.47^cd	70.51±1.99^cd	74.50±1.54^cd
	Ⅲ	77.61±4.45^b	81.55±6.09^ab	80.26±7.01^ab	79.56±7.79^ab	79.93±7.36^ab	82.39±5.36^ab
	Ⅳ	85.72±1.18^a	87.69±0.62^a	87.69±0.63^a	86.61±1.23^a	87.99±0.60^a	87.67±1.09^a
	Ⅴ	63.19±0.29^e	71.28±1.78^cd	69.41±1.94^c	67.28±1.18^cd	70.32±3.01^cd	71.89±2.99^cd
	Ⅵ	72.96±1.95^bc	77.09±2.22^bc	76.20±1.46^bc	74.07±2.73^bcd	78.07±3.09^bc	77.52±1.49^bcd
	Ⅶ	71.76±1.50^bcd	78.23±1.64^bc	77.18±1.88^bc	75.77±2.07^bc	78.04±1.83^bc	78.20±0.70^bc
	Ⅷ	75.87±1.81^bc	82.28±1.79^ab	83.48±0.65^ab	81.52±1.98^ab	83.78±0.37^ab	82.60±0.42^ab
剂量 Dose	A	64.00±3.59^c	69.98±3.76^b	69.66±2.91^c	66.20±4.03^b	69.28±3.63^c	71.16±3.57^c
	B	71.60±2.01^b	74.47±3.46^b	73.96±3.15^bc	71.14±4.00^b	74.29±4.85^bc	76.01±2.14^bc
	C	74.69±4.33^b	79.89±4.12^a	78.72±4.55^b	77.67±5.14^a	78.98±4.51^b	80.30±3.95^ab
	D	80.80±5.82^a	84.99±3.31^a	85.58±2.48^a	84.06±3.23^a	85.88±2.46^a	85.14±3.00^a
水平 Level	基础饲粮 Basal diet	74.59±8.89^b	77.44±8.73	77.39±8.31	74.87±10.07	76.67±9.10	78.75±7.74
水平 Level	低蛋白质饲粮 Low protein diet	70.95±5.18^a	77.22±4.44	76.57±5.46	74.66±5.65	77.55±5.42	77.55±4.27
P值 P-value	剂量 Dose	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
	水平 Level	0.04	0.90	0.63	0.92	0.64	0.45
	剂量×水平 Dose×level	0.07	0.16	0.33	0.25	0.20	0.22

单因素方差分析表明,在饲喂基础饲粮时,与对照组(Ⅰ组)相比,Ⅱ、Ⅲ、Ⅳ组肉鸡对DM的表观消化率显著提高(P<0.05);Ⅲ、Ⅳ组肉鸡对CP、Asp、Thr、Ser、Glu、Gly、Ala、Cys、Val、Met、Ile、Leu、Tyr、Phe、Lys、His、Arg、Pro的表观消化率显著提高(P<0.05)。在饲喂低蛋白质饲粮时,与对照组(Ⅴ组)相比,Ⅵ、Ⅶ、Ⅷ组肉鸡对DM、Cys、Val、Tyr的表观消化率显著提高(P<0.05);Ⅶ、Ⅷ组肉鸡对CP、Ser、Gly、Ala的表观消化率显著提高(P<0.05);Ⅷ组肉鸡对Asp、Thr、Glu、Met、Ile、Leu、Phe、Lys、His、Arg、Pro的表观消化率显著提高(P<0.05)。

2.3 酸性蛋白酶在实际应用中的经济效益

由表4可知,根据各组出栏体重、死淘率以及饲料消耗(疫苗和人工等成本不参与计算),分析各组的毛鸡利润(毛鸡利润=毛鸡收益-饲料成本)。在基础饲粮和低蛋白质饲粮中添加酸性蛋白酶的试验组所获得的经济收益均高于不添加酸性蛋白酶的对照组。在饲喂基础饲粮时,与不添

加酸性蛋白酶的Ⅰ组相比,添加125、250和500 g/t酸性蛋白酶的Ⅱ、Ⅲ、Ⅳ组每只鸡毛利润分别提高0.64、0.44、0.62元;在饲喂低蛋白质饲粮时,与不添加酸性蛋白酶的Ⅴ组相比,添加125、250和500 g/t酸性蛋白酶的Ⅵ、Ⅶ、Ⅷ组每只鸡毛利润分别提高0.32、0.63、0.57元。

表4 酸性蛋白酶在实际应用中的经济效益

Table 4 Economic benefit of acid protease in practical application

组别 Groups	总增重 Total weight gain/kg	毛鸡收益 Chicken income/ (元/只)	饲料价格 Feed price/ (元/t)	饲料成本 Feed cost/ (元/只)	毛鸡利润 Chicken profit (元/只)
Ⅰ	1.64±0.07^ab	11.48	3 200.00	10.14	1.34
Ⅱ	1.77±0.04^ab	12.39	3 205.85	10.41	1.98
Ⅲ	1.83±0.06^a	12.81	3 211.70	11.03	1.78
Ⅳ	1.82±0.16^a	12.74	3 223.40	10.78	1.96
Ⅴ	1.60±0.08^b	11.20	3 151.40	9.90	1.30
Ⅵ	1.68±0.04^ab	11.76	3 157.26	10.14	1.62
Ⅶ	1.77±0.18^ab	12.39	3 163.11	10.46	1.93
Ⅷ	1.75±0.10^ab	12.25	3 174.82	10.38	1.87

	酸性蛋白酶(100 000 U/g)价格为50元/kg,毛鸡市价为7元/kg。
	The price of acid protease (100 000 U/g) is 50 CNY/kg, and the market price of chicken is 7 CNY/kg.

3 讨论

3.1 不同蛋白质水平饲粮添加酸性蛋白酶对肉鸡生长性能的影响

已有研究显示,降低饲粮蛋白质水平会严重降低肉鸡的ADFI和ADG,并且显著降低肉鸡的F/G^[6⇓⇓-9],但可以通过添加外源蛋白酶来提高饲粮中蛋白质的利用率,以减少低蛋白质饲粮对肉鸡造成的负面影响,从而改善肉鸡的生长性能^[10],主要表现为提高肉鸡的ADFI和ADG,提高饲料的利用效率,有效改善肉鸡的生长性能,降低饲养成本^[11⇓-13]。Ding等^[14]将由地衣芽孢杆菌发酵生产出的蛋白酶(活性为100 000 U/g)添加到不同蛋白质水平的饲粮中发现,当降低饲粮蛋白质水平时,会导致肉鸡的ADFI和ADG降低,F/G增加。刘松柏等^[15]研究发现,饲粮蛋白质水平下调1.5%后添加125 g/t蛋白酶显著降低了肉仔鸡的F/G并提高了ADG。本试验中,在饲喂比基础饲粮蛋白质水平下调约1%的低蛋白质饲粮时,添加125或250 g/t酸性蛋白酶组肉鸡试验前期的ADG显著高于对照组,且高于添加500 g/t酸性蛋白酶组,这与刘松柏等^[15]的研究结果相符。在饲喂低蛋白质饲粮时,试验后期,随着酸性蛋白酶添加剂量的增加,肉鸡的ADFI和ADG逐渐增加,F/G逐渐降低,这与李东东等^[16]在蛋白质水平降低1%的玉米-豆粕型饲粮中添加300 g/t蛋白酶(活性100 000 U/g)后得出的22~24日龄肉鸡ADG显著提高且F/G显著降低的研究结果相符。在饲喂低蛋白质饲粮时,添加250或500 g/t酸性蛋白酶组肉鸡试验全期的ADG显著高于对照组,这与许程等^[17]在蛋白质水平降为基础饲粮95%的饲粮中添加0.125%蛋白酶后得出的肉鸭ADG显著高于对照组的结果相似。

不仅是在低蛋白质饲粮中,在基础饲粮中蛋白酶也能很好地促进饲粮中蛋白质的消化利用。大量研究表明,饲粮中添加蛋白酶可以改善畜禽的生长性能^[18⇓-20]。Mahmood等^[21]在饲粮中添加200 mg/kg组合蛋白酶,肉鸡试验前期的ADFI和ADG显著增加。宋增廷等^[22]在玉米-豆粕型饲粮中添加0.4%或0.8%的由蛋白酶和淀粉酶组成的复合酶,F/G显著降低,饲粮养分利用率极显著提高。侯振平等^[23]报道,饲粮中添加蛋白酶能够提高肉鸡的ADG,降低F/G,改善其生长性能。本试验中,在基础饲粮中添加125 g/t酸性蛋白酶组肉鸡试验前期的ADG显著高于对照组且显著高于添加500 g/t酸性蛋白酶组,ADG随酸性蛋白酶添加剂量的增加呈先升高后降低的变化趋势,与周梁^[24]的研究结果相吻合,即在生长前期随饲粮中酸性蛋白酶添加剂量的增加,肉鸡ADG呈先升高后降低的变化趋势。在基础饲粮中添加250或500 g/t酸性蛋白酶组肉鸡试验后期ADFI和ADG与对照组相比显著升高,且添加500 g/t酸性蛋白酶组还显著高于添加125 g/t酸性蛋白酶组,这与Glits等^[25]的研究结果相一致。在基础饲粮中添加125或500 g/t酸性蛋白酶组肉鸡试验全期ADG显著高于对照组,这与刘胜利等^[26]的研究结果基本一致。

在本试验中,酸性蛋白酶添加剂量和饲粮蛋白质水平的交互作用对肉鸡的生长性能无显著影响。但无论在基础饲粮还是在低蛋白质饲粮中添加酸性蛋白酶后,试验前期,随着酸性蛋白酶添加剂量的增加,肉鸡的ADFI和ADG均呈先升高后降低的变化趋势,尤其是肉鸡的ADG,有时变化达到显著水平;而试验后期,随着酸性蛋白酶添加剂量的增加,肉鸡的ADFI和ADG均持续升高。这可能是由于肉鸡早期消化道的发育尚未完成,消化道短且过于狭窄,对饲粮中营养物质消化不良,对病原微生物的抵抗力也较弱,易造成正常肠道菌群平衡紊乱及生长受阻,在饲粮中适量添加外源蛋白酶后可以补充内源蛋白酶的分泌不足^[27],但当添加超过一定剂量后,则可能反馈性地抑制内源酶分泌,从而影响了消化酶的活性。而在低蛋白质饲粮中添加外源蛋白酶可有效弥补饲粮中蛋白质的不足,但添加过量外源蛋白酶同样会抑制内源酶的分泌,故在本试验中高剂量的酸性蛋白酶均未起到提高养分消化率的作用^[24,28]。此外,食糜黏性过高,导致酸性蛋白酶作用底物有限,而且肉鸡对于营养物质的吸收也有限度,酸性蛋白酶添加剂量过高可能限制肉鸡对其他营养物质的利用,从而降低生长性能^[29]。而在肉鸡生长后期,随着肉鸡消化道发育成熟,内源酶分泌增多,限制肉鸡对酸性蛋白酶利用的因素减少,从而随着酸性蛋白酶添加剂量的增加生长性能将会逐渐提高。吕毅等^[30]研究表明,在肉鸡不同生长阶段,肉鸡需要的外源蛋白酶添加剂量会发生变化,本试验试验结果与此相符。

3.2 不同蛋白质水平饲粮添加酸性蛋白酶对肉鸡养分表观消化率的影响

外源蛋白酶制剂被广泛用于改善饲粮中养分的消化率。饲粮添加外源蛋白酶可降低肉仔鸡饲粮中不可消化氨基酸的比例,有效提高肉仔鸡对蛋白质和氨基酸的消化率^[31]。有研究表明,在肉仔鸡饲粮中添加蛋白酶可以提高饲粮中蛋白质和能量的利用率,进一步提高肉仔鸡的生长性能^[32]。此外,外源蛋白酶能通过补充家禽内源消化酶的不足来提高养分的利用率,并与之协同作用来提高营养物质及氨基酸的利用效率^[33]。Angel等^[34]研究表明,在7~22日龄饲粮中添加蛋白酶可提高肉仔鸡的CP、DM、能量和氨基酸的消化率。Stefanello等^[35]分别在豆粕型和玉米-豆粕型半纯合饲粮中添加蛋白酶,结果显示肉鸡的必需氨基酸和非必需氨基酸回肠末端表观消化率均显著提高,CP、DM、能量回肠末端表观消化率也显著提高。本试验中,在基础饲粮或低蛋白质饲粮中添加250或500 g/t酸性蛋白酶组的CP、DM和部分氨基酸的表观消化率与对照组相比显著提高,这与上述研究结果相符,表明酸性蛋白酶可以直接分解底物,在提高蛋白质、氨基酸等营养物质消化利用率的同时,还可以分解饲粮中的大分子物质,降解特异性多肽,补充肉鸡消化道内源蛋白酶分泌的不足,刺激内源酶的分泌,减轻饲粮中抗营养因子的作用,提高饲粮中其他营养物质的消化率^[36-37]。

随着肉鸡的集约化和规模化生产,人们不仅仅局限于肉鸡的生产效率,更多的是希望肉鸡能够更加健康、味道更加鲜美等,如何在保证肉鸡生产效率的前提下满足这些要求,氨基酸营养逐渐开始被人们所关注^[38]。研究表明,Lys可以提高肉鸡的日增重和饲料转化率并改善胴体品质^[39];Met可以改善肉鸡的生长性能和增强免疫力^[40];Thr具有调节肉鸡蛋白质合成、参与分化淋巴细胞增殖和免疫球蛋白合成、促进肠道发育等重要作用^[41];Leu和Val可以提高肉鸡胸肌率和腿肌率^[42];Gly和Ser复合物可以有效缓解肉鸡因接种球虫疫苗引发的免疫应激反应^[43];Arg能促进肉鸡肌肉蛋白质合成,增强机体氮存留,参与脂类代谢调控,刺激胰岛素和肾上腺素的生成^[44];鲜味氨基酸(包括Gly、Ala、Asp、Glu、Phe和Tyr)和甜味氨基酸(包括Thr、Ser、Gly、Ala、Lys、Pro)是鸡肉滋味的重要指标^[45]。本试验结果显示,与不添加酸性蛋白酶的对照组相比,添加250或500 g/t酸性蛋白酶组上述氨基酸的表观消化率得到了提高,尤其是Met的表观消化率,酸性蛋白酶添加剂量与饲粮蛋白质水平的交互作用对其具有显著影响。上述结果表明,在饲粮蛋白质充足时,酸性蛋白酶能增强对饲粮中氨基酸以及营养物质的消化吸收,从而增强机体免疫力;而在饲粮蛋白质不足时,酸性蛋白酶需在弥补饲粮蛋白质以及氨基酸不足之后再进行机体免疫的加强,导致低蛋白质饲粮中酸性蛋白酶的添加剂量要远远超过基础饲粮中酸性蛋白酶的添加剂量,这与Fru-Nji等^[46]的研究结果一致。

4 结论

① 饲粮中添加适量酸性蛋白酶可提高肉鸡的生长性能以及CP、DM和氨基酸表观消化率。

② 对于肉鸡的生长性能和养分表观消化率来说,在相同饲粮蛋白质水平下,肉鸡不同生长阶段酸性蛋白酶的最佳添加剂量不同,试验后期添加剂量要高于试验前期;在相同酸性蛋白酶添加剂量下,基础饲粮要优于低蛋白质饲粮。

③ 试验前期和后期基础饲粮中酸性蛋白酶的最佳添加剂量分别为125 和500 g/t,低蛋白质饲粮中酸性蛋白酶的最佳添加剂量分别为250和500 g/t。

④ 结合酸性蛋白酶在实际应用中的经济效益,选择试验前期和后期在低蛋白质饲粮中分别添加250和500 g/t酸性蛋白酶,此条件下的毛鸡利润不仅与基础饲粮中添加酸性蛋白酶时近乎一致,还可以减少饲粮中蛋白质的添加,从而减少豆粕的消耗。

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