Scholarly article on topic 'Nutrient density of prestarter diets from 1 to 10 days of age affects intestinal morphometry, enzyme activity, serum indices and performance of broiler chickens'

Nutrient density of prestarter diets from 1 to 10 days of age affects intestinal morphometry, enzyme activity, serum indices and performance of broiler chickens Academic research paper on "Animal and dairy science"

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{Chickens / Prestarter / "Intestinal histomorphology" / "Enzyme activity" / Energy / "Amino acids"}

Abstract of research paper on Animal and dairy science, author of scientific article — Ficinine V. Ivanovich, Ocmanyan A. Karlovich, Reza Mahdavi, Egorov I. Afanasyevich

Abstract A total of 480 day-old Cobb 500 broilers were used to investigate the effects of different levels of digestible amino acids (DAA; 100%, 107% and 114% of Cobb recommendations) and ME (3,000 or 2,900 kcal/kg) of prestarter diet on mixed sex broilers performance, enzyme activity, small intestine morphology, and serum metabolites. Broilers were randomly allotted to 6 treatments, where each treatment applied to 4 pens with 20 birds in each. The birds were subjected to their respective treatment diets from 1 to 10 days of age. This was followed by feeding common starter and finisher diets for the last 29 days. The enzyme activity of the pancreas was measured at 10 days of age. Morphometric indexes of jejunum were measured at 10 days of age and the end of the feeding period. Our results showed that the body weight (BW) increased as the DAA density of the prestarter diet increased from 100% to 114% over the first 10 days and the entire period of the study. Birds fed 114% DAA presented a better feed conversion ratio on day 10 (P < 0.05). At day 39, carcass weight and breast yield increased as the DAA levels increased from 100% to 114% (P < 0.05). The whole intestine length, small intestine length, and weights of the pancreas were lower in birds fed 100% DAA-diets at 10 days of age (P < 0.05). Increasing the dietary DAA and ME did not affect serum amylase, lipase, and protease concentrations and pancreatic amylase and lipase activity (P > 0.05); however, the activity of pancreatic protease increased as the DAA level increased from 100% to 114% (P < 0.05). The villus width and villus surface area (VSA) increased as the DAA level increased from 100% to 114% on day 10 (P < 0.05). At 10 days of age, crypt depth was the lowest in the birds fed plenty DAA prestarter diets (P < 0.05). It was found that dietary treatments at 39 days of age did not affect intestinal morphology. The results of the present work indicate that DAA level of 114% of Cobb recommendations and energy level of 2,900 kcal/kg diet may be recommended for starting broiler chicks.

Academic research paper on topic "Nutrient density of prestarter diets from 1 to 10 days of age affects intestinal morphometry, enzyme activity, serum indices and performance of broiler chickens"

Accepted Manuscript

Nutrient density of prestarter diets from 0 to 10 days of age affects intestinal morphometry, enzyme activity, serum indices and performance of broiler chickens

Ficinine V. Ivanovich, Ocmanyan A. Karlovich, Reza Mahdavi, Egorov I. Afanasyevich

S2405-6545(17)30042-2 10.1016/j.aninu.2017.06.005

Reference: ANINU 164

To appear in: Animal Nutrition Journal

Received Date: 20 February 2017 Revised Date: 7 May 2017 Accepted Date: 22 June 2017

Please cite this article as: Ivanovich FV, Karlovich OA, Mahdavi R, Afanasyevich EI, Nutrient density of prestarter diets from 0 to 10 days of age affects intestinal morphometry, enzyme activity, serum indices and performance of broiler chickens, Animal Nutrition Journal (2017), doi: 10.1016/j.aninu.2017.06.005.

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1 Nutrient density of prestarter diets from 0 to 10 days of age affects intestinal

2 morphometry, enzyme activity, serum indices and performance of broiler chickens

4 Ficinine V. Ivanovicha, Ocmanyan A. Karlovichb, Reza Mahdavib'*, Egorov I. Afanasyevicha

6 a All-Russian Research and Technological Institute of Poultry, Sergiev Posad, Moscow 141311,

7 Russia

8 b Special Animal Husbandry Department, Faculty of Animal Science and Biology, Moscow

9 Timiryazev Agricultural Academy, Moscow 127550, Russia 10

11 * Corresponding author.

12 E-mail address: re.mahdavi@gmail.com (R. Mahdavi).

20 21 22

25 Abstract

26 A total of 480 day-old Cobb 500 broilers were used to investigate the effects of different levels of

27 digestible amino acids (DAA; 100%, 107% and 114% of Cobb recommendations) and ME (3,000

28 or 2,900 kcal/kg) of prestarter diet on mixed sex broilers performance, enzyme activity, small

29 intestine morphology, and serum metabolites. Broilers were randomly allotted to 6 treatments,

30 where each treatment applied to 4 pens with 20 birds in each. The birds were subjected to their

31 respective treatment diets from 1 to 10 days of age. This was followed by feeding common starter

32 and finisher diets for the last 29 days. The enzyme activity of the pancreas was measured at 10

33 days of age. Morphometric indexes of jejunum were measured at 10 days of age and the end of

34 the feeding period. Our results showed that the body weight (BW) increased as the DAA density

35 of the prestarter diet increased from 100% to 114% over the first 10 days and the entire period of

36 the study. Birds fed 114% DAA presented a better feed conversion ratio on day 10 (P < 0.05). At

37 day 39, carcass weight and breast yield increased as the DAA levels increased from 100% to

38 114% (P < 0.05). The whole intestine length, small intestine length, and weights of the pancreas

39 were lower in birds fed 100% DAA-diets at 10 days of age (P < 0.05). Increasing the dietary

40 DAA and ME did not affect serum amylase, lipase, and protease concentrations and pancreatic

41 amylase and lipase activity (P > 0.05); however, the activity of pancreatic protease increased as

42 the DAA level increased from 100% to 114% (P < 0.05). The villus width and villus surface area

43 (VSA) increased as the DAA level increased from 100% to 114% on day 10 (P < 0.05). At 10

44 days of age, crypt depth was the lowest in the birds fed plenty DAA prestarter diets (P < 0.05). It

45 was found that dietary treatments at 39 days of age did not affect intestinal morphology. The

46 results of the present work indicate that DAA level of 114% of Cobb recommendations and

47 energy level of 2,900 kcal/kg diet may be recommended for starting broiler chicks.

48 Keywords: Chickens; Prestarter; Intestinal histomorphology; Enzyme activity

50 1. Introduction

51 The early life nutrition of broilers plays a crucial role in their productivity because of their

52 muscle cell proliferation and development of digestive tract during this period (Vieira and Moran,

53 1999). The transition from late embryogenesis to the early posthatch period is characterized by

54 major morphological and physiological changes allowing the bird to immediately consume

55 nutrients after hatching (Willemsen et al., 2010). This transition is fundamental as the bird shifts

56 from metabolism based on the lipid-rich yolk to a solid carbohydrate and protein based diet at

57 hatch. Diet is an important characteristic of poultry production, and different poultry species or

58 lines have different requirements depending on genetic, age, environment, and the health status of

59 the poultry. In the recent years, interest in early nutrition research has increased regarding the

60 strong positive correlation exists between early live weight and body weight (BW) at the end of

61 the production cycle (Tona et al., 2004; Willemsen et al., 2008). In this connection, an increase in

62 the average early weight will minimize the number of small birds that, for any reason, do not eat.

63 As a result, the overall lifetime performance of the chickens would be seriously affected by

64 posthatch nutrition (Geyra et al., 2001b). The ratio of macronutrients has a major influence on

65 posthatch performance and body composition of broiler chickens (Collin et al., 2003; Swennen et

66 al., 2007) and under- and over- formulation of macronutrients will decrease this performance

67 (Kidd et al., 2004). Therefore, the composition of prestarter diet (the content of crude protein,

68 amino acids, energy, macro, and microelements) can influence the subsequent growth and

69 development of broiler chickens. In general, diets with high ME or high energy-protein ratio

70 induces energy deposition as fat. Increasing the CP content of the diet beyond requirements will

71 result in leaner birds but with poorer efficiency, as the elimination of excess nitrogen is an energy

72 consuming process (Swennen et al., 2007). Amino acid balance and the ideal protein concept

73 (Emmert and Baker, 1997) can also affect broiler response to dietary energy. Amino acids in

74 excess of that required for protein synthesis and other aspects of body metabolism are

75 catabolized; which, as a result, incurs an energy cost and metabolic stress (Sklan and Plavnik,

76 2002). Wang et al. (2015) reported that high nutrient density diets (high amino acid and AME)

77 might improve broiler performance without affecting their intestinal structure during 8 to 21

78 days. In another study performed by Ullah et al. (2012), the performance of chicks in 35 days of

79 age was significantly higher in broilers fed 2,850 ME and 1.4% total lysine during the first 10

80 days posthatch than other treatments. Although the advantages of broiler performance of feeding

81 increased amino acid densities have been well documented, a limited number of works has been

82 reported on the effects of manipulation of nutrient densities in prestarter diets on the enzyme

83 activity and intestinal morphology.

84 Thus, the aim of the present study is to find the appropriate levels of ME and digestible amino

85 acids (DAA) in prestarter phase to the optimum production at the lowest cost and determine

86 whether providing dietary DAA higher and ME lower than Cobb recommendations affects the

87 performance, enzymes activity, intestinal morphology, and serum indices of broilers.

89 2. Material and methods

90 2.1. Experimental design

91 In this study, 480 day-old Cobb 500 broilers (mixed sex) were housed in cage pens (Battery)

92 according to a completely randomized design in factorial arrangement, consisting of 2 levels of

93 ME (2,900 and 3,000 kcal/kg) and 3 levels of DAA (100%, 107%, and 114% of Cobb

94 recommendations) during 0 to10 days of age. A fixed proportion of DAA relative to CP were

95 maintained in graded increments of CP from 21.4% to 24.6%. Water and feed were offered ad-

96 libitum. Birds were fed one of 6 experimental diets from 1 to 10 days of age, the same starter diet

97 from 11 to 21 days of age, and same finisher diet from 22 to 39 days of age. During the prestarter

98 phase, broilers were divided into six treatments as follows: 1) 3,000 kcal/kg ME and 100% of

99 DAA and CP (normal) of Cobb recommendations; 2) 3,000 kcal/kg ME and 107% of DAA and

100 CP (high) of Cobb recommendations; 3) 3,000 kcal/kg ME and 114% of DAA and CP (plenty) of

101 Cobb recommendations; 4) 2,900 kcal/kg ME and 100% of DAA and CP (normal) of Cobb

102 recommendations; 5) 2,900 kcal/kg ME and 107% of DAA and CP (high) of Cobb

103 recommendations; 6) 2,900 kcal/kg ME and 114% of DAA and CP (plenty) of Cobb

104 recommendations. Before formulation of the experimental diets, ingredients were analyzed for

105 nutrient concentration by NIRS DS 2500 FOSS. The analytical values obtained from these values

106 are shown in Table 1. The formula and chemical composition of the dietary treatments are shown

107 in Table 2.

109 2.2. Productive performance and serum indices

110 Body weight and feed consumption were obtained weekly followed by calculating body weight

111 gain (BWG), daily feed intake (FI), and feed conversion ratio (FCR) using these data. On day 10,

112 one chicken from each replicate of each treatment with BW close to the mean replicate was

113 selected and then sacrificed by neck dislocation and collected blood samples. The blood samples

114 were transferred into tubes and centrifuged, thereafter at 3,521 x g at 4 □ for 4 min using

115 HETTICH EBA 280S. The sera were removed and stored at -20 □ for further analysis.

116 Immediately after slaughter, the pancreas was removed, weighed, and stored in liquid nitrogen

117 for subsequent analyses. The length of intestine and small intestine was individually measured.

118 Serum protease was determined using BS 3000P spectrophotometer and Nabenzoyl-D,L-arginine

119 p-nitroanilide (BAPNA) according to the procedure described by Mikhailova et al. (2014).

120 Moreover, lipase and amylase in serum were determined with commercial kits (Human company,

121 Germany), using the Chem Well 2900 analyzer (Awareness Technology, Inc., USA). Pancreatic

122 protease activity was measured calorimetrically based on casein hydrolysis method as described

123 by Batoev (1971), amylase activity was determined based on starch hydrolysis by modified

124 Smith-Roy's method (Merina-Gluzkina, 1965). Pancreatic lipase was analyzed with commercial

125 kits (Diakon company, Russia) using semiautomatic spectrophotometer BS 3000P.

126 On day 39, 4 birds per treatment (had BW close to the mean replicate) were selected for carcass

127 traits evaluation. Birds were weighed, sacrificed by neck dislocation, bled, plucked, and

128 eviscerated. Carcass yield was calculated as hot eviscerated carcass weight (without feet, head,

129 and abdominal fat) relative to live body weight. Prime cuts yield included whole breast yield

130 (with skin and bones), legs yield (thighs and drumsticks with bones and skin), and abdominal fat

131 (fat located around the cloaca, cloacal bursa, gizzard, proventriculus, and adjacent abdominal

132 muscles).

134 2.3. Tissue sampling and Analysis of histological samples

135 At days 10 and 39, 4 birds per treatment were sacrificed by neck dislocation and their jejunum

136 (midpoint from the pancreatic duct to Meckel's diverticulum) was excised. The jejunum was of

137 particular interest because it is a major site of nutrient absorption in poultry (Horn et al., 2009)

138 and intestinal mass. In a study, it was observed that villus height and area increased several folds

139 in the jejunum and duodenum and less in the ileum to 10 days of age (Uni et al., 1999). Tissue

140 samples (5 cm) were removed and flushed with 0.9% NaCl and then fixed in10% neutral buffered

141 formalin solution for morphometric analysis. The tissues were processed by dehydration through

142 a series of graded alcohols, cleared with xylene. Paraffin-embedded tissue sections (5 p,m) were

143 prepared using an HM-325 universal automated microtome (Microm international GmbH,

144 Germany). Slides were stained by the Hematoxylin-Eosin method, as described by Uni et al.

145 (1995). Micrographs were taken with a Jenamed-2 light microscope (Carl Zeiss, Jena, Germany)

146 using Image Scope C (Systems for Microscopy and Analysis LLC, Russia) to calculate the

147 morphometric variables.

148 Morphometric parameters, including villus height from the tip of the villus to the crypt, midpoint

149 villus width, crypt depth from the base of the villi to the base of the crypt, and V/C ratio

150 (calculated by dividing villus height by crypt depth) were recorded in the next step. Villus surface

151 area was calculated as follows (Sakamoto et al., 2000):

152 Villus surface = 2n x (VW/2) x VH, where n = 3.14, VW = villus width, and VH = villus height.

154 2.4. Statistical Analysis

155 Data were analyzed in a 2 x 3 factorial arrangement of dietary treatments using analysis of

156 variance and General Linear Model (GLM) procedure of SAS (SAS/STAT Version 9.2, SAS

157 Institute Inc., Cary, NC) to determine the main effects of dietary ME, DAA and their interactions.

158 If a significant effect was detected, the differences between treatments were separated using

159 Duncan's multiple range test. Differences between mean values were considered significant at P

160 < 0.05. Also, all percentage data were subjected to angular transformation to stabilize variances

161 (arcsine square root percentage transformation) before statistical analysis.

163 3. Results

164 3.1. Performance parameters and carcass characteristics

165 Results showed that there was no significant effect of ME content of prestarter diet on productive

166 performance parameters (Table 3). The BW increased as the DAA density of the prestarter diet

167 increased from 100% to 114%, over the first 10 days (P < 0.0001) and the entire period of the

168 study (P < 0.01). Interaction of DAA and ME was not significant for the body weight, feed

169 consumption, and feed conversion ratio of the birds during the first 10 days of age and across the

170 39 days study (P > 0.05). When assessed for 39 days, birds fed plenty DAA prestarter diets

171 exhibited the highest body weight, regardless of diet ME content. Feed consumption was not

172 influenced by dietary treatments. Increasing DAA density of prestarter diet improved feed

173 conversion ratio only in the prestarter period (P < 0.0001).

174 As shown in Table 4, carcass weight, breast muscle weight, and legs weight were significantly

175 increased as the DAA density of the prestarter diet increased from 100% to 114% (P < 0.003).

176 There was not observed a significant effect of ME or interactions between ME and DAA density

177 of prestarter diets on carcass traits (P > 0.05). The main effects of ME, DAA, and their

178 interactions were not significant for the relative weight of carcass, legs, wings and abdominal fat

179 (P > 0.05).

181 3.2. Enzymes activity and serum metabolites

182 The results of the dietary treatments on the serum metabolites, pancreas weight, and enzymes

183 activity are shown in Table 5. No significant difference (P > 0.05) existed among treatments as

184 regard to serum amylase, lipase, and protease levels. Concerning pancreas weight, it was noticed

185 a significant increase (P < 0.01) in plenty DAA group (1.16 g) compared to normal and high

186 DAA groups (0.97 and 1.02 g). In addition, no significant effect was observed for ME or the

187 interactions between dietary ME and DAA content on the enzymes activities, serum metabolites,

188 and pancreas weight. Increasing dietary DAA from 100% to 114% significantly increased (P <

189 0.04) pancreatic protease from 139.50 to 159.63 mg/g per min. Besides, pancreatic amylase

190 (mg/g per min) and lipase (U/L) were not influenced by dietary treatments (P > 0.05).

192 3.3.Intestinal morphology

193 As shown in Table 6, the length of intestine was affected by DAA level of prestarter diets. As

194 compared to normal prestarter diets, the plenty inclusion of DAA increased the total intestine

195 length and small intestine length by 6.63 and 6.25 cm, respectively (P < 0.05). The main effect of

196 ME and interaction between DAA and ME was not significant for the whole intestinal length and

197 small intestinal length (P > 0.05). Our results showed that villus width and VSA at 10 days of age

198 increased as the DAA level of the prestarter diet increased from 100% to 114% (P < 0.05). At 10

199 days of age, VSA was increased from 121.01 to 146.56 and 138.73 mm as the DAA level

200 increased from 100% to 107% and 114%, respectively. At 10 days of age, crypt depth was the

201 lowest in birds fed plenty DAA diets (P < 0.09). Morphological examination showed that dietary

202 ME, DAA, and their interactions did not affect jejunum villus height, width, VSA, V/C ratio at

203 39 days of age.

205 4. Discussion

206 Although we observed that final performance of broiler is influenced by DAA and protein

207 concentration of prestarter diet, no effect of ME levels of the prestarter diet on performance

208 parameters at 10 days of age and across the 39-day study was observed. This observation may in

209 part be due to the fact that decreasing the dietary ME level of prestarter by 100 kcal/kg may not

210 be enough reduction to see differences in growth performance. A previous study (Vieira et al.,

211 2006) in broilers showed that the effect of the level of energy (from 2,870 to 3,100 kcal/kg) in

212 prestarter period was not significant on the BWG of broilers at 7 and 42 days of age whereas

213 3,000 kcal/kg energy diet improved feed conversion ratio at 7 days of age and 3,100 kcal/kg

214 energy diet significantly decreased feed consumption.

215 The BWG responses due to increased dietary DAA levels in the prestarter phase reported herein

216 agreed well with findings of Noy and Sklan (2002), Everaert et al. (2010), and Bahreiny et al.

217 (2013). Noy and Sklan (2002) showed that feed conversion ratio did not change significantly

218 with energy level (from 3,050 to 3,110 kcal/kg) of the diet but decreased with increasing protein

219 level (from 18% to 28%) during the 7 days posthatch. Previous studies showed that increasing

220 essential amino acids in a constant ratio to CP enhanced performance during the 7 days posthatch

221 (Sklan and Noy, 2003). Hargis and Creger (1980) reported that adequate protein availability in

222 the prestarter period seems to be essential to increase muscle development in later phases. The

223 strong effect of DAA and protein on performance of broilers at 10 days of age in the current

224 study can be explained by the high protein and amino acids requirements of newly hatched chicks

225 to meet the needs for rapid growth and the effects of additional supplementation of DAA and

226 protein on the better development of the gastrointestinal tract posthatch. It is well documented

227 that BW enhances fourfold to fivefold during the first 10 days of age, during which considerable

228 changes in gut weight and morphology are observed. The rapid growth of the intestine reaches a

229 maximum between 6 and 10 days and declines thereafter (Sklan et al., 2001). Uni et al. (1996)

230 reported that in broiler chicks the height and perimeter of villi in all segments of the small

231 intestine increased by 34% to 100% between 4 and 10 days after hatching. The crypt depth and

232 the number of enterocytes per longitudinal section of villi also increased with age. As shown in

233 Table 6, plenty DAA and protein concentration of prestarter diets increased the intestinal length

234 and VSA and decreased crypt depth. It is assumed that an increased villus height is paralleled by

235 an increased digestive and absorptive function of the intestine due to increased absorptive surface

236 area, expression of brush border enzymes, and nutrient transport systems (Amat et al., 1996).

237 Consequently, these changes might result in improvements in broiler performance. In addition,

238 the higher growth rate of chickens on a plenty DAA and protein diets is most likely the

239 consequence of their increased cumulative DAA and protein consumption. The lack of any

240 consistent interaction between dietary concentration of ME and DAA on BWG, FI, and FCR in

241 the current experiment suggests that the response in performance parameters to increasing

242 concentrations of DAA and CP from 21.4% to 24.6% CP in prestarter period was independent of

243 the dietary ME level over the range from 2,900 to 3,000 kcal/kg. A previous study found a

244 positive correlation between BW of 7 to10 days of age and final BW on 42 days of age (Vieira

245 and Moran 1999; Tona et al., 2004; Saki, 2005; Hooshmand, 2006). Li et al. (2009) reported that

246 nutritional deficiency during early posthatch development induced a permanent negative effect on

247 BW at slaughter in broilers.

248 As shown in Table 4, different levels of DAA in the prestarter period have a significant effect on

249 carcass traits. Total carcass, breast, and legs weight were significantly higher in birds fed the

250 plenty DAA prestarter diets, regardless of diet ME content. Zaboli and Miri (2013) showed that

251 prestarter diets with increased Lys densities resulted in increased breast meat and thigh yield.

252 However, treatments' effect on carcass yield was not significant. The relative weight of carcass,

253 legs, wings, and abdominal fat were not affected by dietary treatments. Thus, the improvement in

254 carcass, breast muscle, and legs weight at the end of experiment could be due to the higher BW

255 of birds fed plenty DAA and protein diets. However, Halevy et al. (2003) emphasized the effect

256 of posthatch feeding on the dynamics of satellite cells during the prestarter phase, which leads to

257 changes in the yield of meat cuts and in the composition of tissues of broilers at slaughter.

258 Digestion and absorption of nutrients early in life depend primarily on pancreatic enzyme activity

259 (Nitsan et al., 1991). As shown in Table 5, plenty DAA and protein concentration of prestarter

260 diets significantly increased protease activity. This finding was in agreement with Stringhini et al.

261 (2009) wherein the authors studied the effect of protein and amino acid supplementation levels

262 for broilers in the prestarter ration. The conclusion of the authors was that birds fed 20% CP and

263 non-supplemented with amino acids showed higher amylase activities and lower trypsin activities

264 than 22% CP supplemented with amino acids. Zhao et al. (2007) reported that the activities of

265 amylase and protease in the jejunal fluid of ducks are mainly dependent on dietary protein

266 content but not ME content. Dietary protein induces pancreatic proteases via secretion of

267 cholecystokinin, which is a potent pancreatic protease inducer (Green et al., 1992). Moreover,

268 dietary amino acids induce pancreatic protease activity by promoting translation, and transient

269 activation of translation initiation via mammalian target of rapamycin (mTOR ) pathway may be

270 associated with this induction (Hashimoto and Hara, 2003). In our experiment, pancreatic lipase

271 was not affected by different ME level. Differences in the effects of ME density on lipase activity

272 observed by other authors (Maiorka et al, 2004) may have been caused by differences in the

273 range of dietary ME used in the respective experiments.

274 In agreement with our findings, Swatson et al. (2002) and Abbasi et al. (2014) observed

275 significantly heavier pancreas in broilers fed increased levels protein diet. Maiorka et al. (2004)

276 reported that increasing the energy level of prestarter diet did not affect pancreas relative weight.

277 The increase in the pancreas weight in chicks fed plenty DAA and protein diets might be

278 attributed to their higher BW. Our statistical analysis showed a significant positive correlation

279 between BW and weight of pancreas.

280 In accordance with the present study, Maiorka et al. (2004) stated that dietary energy level (2,900

281 and 3,200 kcal/kg) had not significant effects on intestine length at 7 days posthatch. A previous

282 study in broilers showed that the increased intestinal length of broilers fed the higher level of

283 dietary Lys might be due to increased nutrient absorption (Jackson and Diamond, 1996). The

284 higher dietary levels of DAA and protein lead to increased nutrients in the small intestine. Such

285 an increase can result in an enhanced protein synthesis in the small intestine and promote the full

286 growth of small intestine, and subsequently, increased small intestinal length. The increase in

287 intestinal length, in turn, can improve digestion by increasing the exposure of nutrients to

288 brushborder hydrolytic enzymes as well as pancreatic and biliary secretions.

289 There is a lack of report linking the effects of amino acids and ME to the development of the

290 gastrointestinal tract of poultry during the prestarter period. In the current trial, small intestine

291 lengths, villus width, and VSA increased in broilers fed high or plenty inclusion of DAA diets on

292 day 10. A previous study showed that villus height increased as dietary threonine increased from

293 0.8% to 0.87% during the first 2 weeks posthatch. However, the threonine supplementation to

294 0.87% showed no significant effects on crypt depth and VSA (Moghaddam et al., 2011).

295 Laudadio et al. (2012) evaluated the morphometric indices of duodenum, jejunum, and ileum of

296 broilers at 49 days of age and the effect of protein level (from 18.5% to 22.5 %) in diet from 14

297 days of age until slaughter age (49 day) and determined that the VSA of all intestinal segments

298 did not change among groups. Instead, reducing the dietary protein level to 20.5% resulted in a

299 higher villus height and V/C ratio in the duodenum and ileum. The first 2 weeks posthatch

300 represent a period of rapid intestinal development in the broiler chickens (Geyra et al., 2001a).

301 During this period, dramatic morphological changes occur, including increases in the number and

302 proliferation rate of enterocytes, widening and lengthening of the villi, and deepening of crypts

303 (Sklan and Noy, 2000; Moran, 2007). Amino acids maintain intestinal viability and mass, in

304 addition to providing energy for normal intestinal function. As gastrointestinal tissues have

305 relatively high protein turnover rate, high amino acid and protein diets provide nutrients for basal

306 metabolism and cause a developed small intestine. In fact, wider villi in the birds that consumed

307 the high and plenty DAA and protein diets as compared with birds fed the normal DAA and

308 protein diets may increase total luminal villus absorptive area. Subsequently, a satisfactory

309 digestive enzyme action and higher transport of nutrients at the villus surface is reached. The

310 shallower crypt depth in chicks fed plenty DAA prestarter diets at 10 days of age may indicate

311 that more mature enterocytes inhabited the villi of these birds, with less cell recruitment and

312 hence less energy expenditure needed to maintain the absorptive function. More mature

313 enterocytes surrounding the villi may imply that there is a greater enterocyte functionality and,

314 hence, a greater absorptive surface. On the other hand, shorter crypt depths are indicative of a

315 longer time needed for cell regeneration (Xu, et al., 2003; Gao, et al., 2008; Markovicva, et al.,

316 2009). This result, combined with less energy needed to maintain cells on the villi potentially,

317 allows for more nutrients to be allocated to different regions of the body to support growth.

319 5. Conclusions

320 Data obtained in the current study demonstrated that increasing the dietary DAA and protein

321 concentration in the prestarter diet by up to 114% of Cobb recommendations significantly

322 improved final BW with a little change in feed intake. Also, increasing DAA density of prestarter

323 diet increased the intestinal length, pancreas weight, villus width, VSA, and pancreatic protease

324 while decreasing crypt depth in the prestarter period. Therefore, plenty DAA and protein levels in

325 the prestarter period can be financially attractive due to the relatively low contribution of the

326 prestarter diet to the total feed costs of a broiler up to market age. Moreover, decreasing the

327 dietary ME level of prestarter by 100 kcal/kg did not negatively affect the BWG and FCR of the

328 birds.

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450 Table 1. Nutrient analysis of ingredients used in experimental diets.

Analysis/Ingredient Corn Soybean meal Wheat

AME, kcal/kg 3,350 2,386 3,075

Crude protein, % 7.73 47.71 12.14

Dig-Lysine, % 0.20 2.66 0.28

Dig-Met + Cys, % 0.26 1.11 0.41

Dig-Thr, % 0.25 1.54 0.31

451 AME = apparent metabolizable energy.

471 Table 2. Composition of the diets (as-fed basis).

Prestarter

1 2 3 4 5 6 3 Lai ici r imsiiei

Ingredients, % /Treatment

Corn 44.33 39.44 34.56 46.78 41.90 37.01 23.45 22.14

Sunflower oil 2.59 3.36 4.14 0.55 1.32 2.10 3.97 4.54

Soybean meal 32.89 37.01 41.12 32.49 36.60 40.71 29.80 23.90

Wheat 15.00 15.00 15.00 15.00 15.00 15.00 38.00 45.00

DL-methionine 0.31 0.35 0.39 0.31 0.35 0.39 0.31 0.28

Lysine-HCl 0.26 0.25 0.25 0.27 0.26 0.25 0.28 0.30

L-threonine 0.14 0.15 0.16 0.14 0.15 0.16 0.14 0.13

Choline chloride 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

Limestone 2.39 2.37 2.34 2.39 2.37 2.34 2.17 1.98

NaCl (salt) 0.23 0.24 0.23 0.22 0.23 0.23 0.21 0.20

Vitamin premix1 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03

Mineral premix2 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10

Sodium sulfate 0.13 0.12 0.11 0.13 0.12 0.11 0.12 0.13

Ammonium phosphate 1.54 1.52 1.51 1.53 1.51 1.51 1.35 1.20

CelloLux3 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02

Calculated analyses

ME, kcal/kg 3,000 3,000 3,000 2,900 2,900 2,900 3,050 3,130

Crude protein, % 21.40 23.00 24.60 21.40 23.00 24.60 21.15 19.10

Dig-Lysine, % 1.19 1.28 1.37 1.19 1.28 1.37 1.15 1.03

Dig-Met + Cys, % 0.88 0.95 1.02 0.88 0.95 1.02 0.87 0.80

Dig-Thr 0.80 0.86 0.92 0.80 0.86 0.92 0.77 0.69

Calcium, % 0.96 0.96 0.96 0.96 0.96 0.96 0.88 0.80

Available P, % 0.48 0.48 0.48 0.48 0.48 0.48 0.44 0.40

ME = metabolizable energy

1 Vitamin premix provided 1 kg of diet with: vitamin A, 10,800 IU; vitamin D3, 2,160 IU; vitamin E, 15 IU; vitamin K3, 1.0 mg; vitamin B1, 4 mg; riboflavin, 5 mg; pantothenic acid 10 mg; niacin, 25 mg; vitamin B6, 8 mg; folic acid, 0.4 mg; vitamin B12, 0.08 mg; biotin, 0.15 mg.

476 2 Mineral premix provided 1 kg of diet with: I, 0.35 mg; Se, 0.15 mg; Zn, 40 mg; Cu, 8 mg; Fe, 80 mg; Mn, 100 mg.

477 3 CelloLux contains enzyme complex of cellulose, glucanase and xylanase.

478 Table3. Effect of dietary energy and DAA on the performance of broiler chicks (n = 4).

ME, kcal/ kg

DAA, BWG, % g

0 to10 11 to 21 22 to d d 39 d

0 to 39 d

0 to 10 d

11 to 21 d

22 to 39 d

0 to 39 d

0 to 10 d

1 to 21 d

22 to 39 d

0 to 39 d

3,00 100 0

150.06 ± 5.97

512.28 ± 30.89

1,313.5 5 ± 66.70

1,931.61 237.60 788.17 ± ± 102.34 ± 6.20 51.40

2,477.9

9±100.

3,503.76 ± 152.72

1.58 ± 0.04 a

3,00 107 0

164.57 ± 6.53

518.08 ± 17.27

1,335.8 9 ± 42.73

1,974.08 246.64 781.50 ± ±49.70 ± 15.24 28.50

2,522.8 7 ± 97.37

3,551.01 ± 100.60

1.50 ±0.09 a

3,00 114 0

191.72 ± 9.23

546.74 ± 12.02

1,397.7 7 ± 86.81

2,091.87 240.49 807.67 ± ± 102.83 ± 10.46 42.64

2,590.0 5 ± 53.36

3,638.21 ± 71.42

1.26 ± 0.10c

2,90 100 164.46 514.90± 1,346.0 0 ± 12.66 10.76 0 ±

1,981.10 253.39 780.12 ± ± 105.75 ± 18.69 49.92

2,496.6 6 ± 99.50

3,530.16 ± 161.43

1.54 ± 0.12a

2,90 107 0

173.01 ± 9.84

526.84 ± 26.04

1,341.6 5 ± 68.87

1,997.30 249.28 800.85 ± ± 90.18 ± 9.46 45.54

2,476.5 0 ±

127.95

3,526.62 ± 140.96

1.45 ±

0.13 ab

2,90 114 0

190.05 ± 9.71

556.06 ± 19.53

1,394.8 2 ± 47.70

2,096.18 247.173 813.68 ± ± 62.76 ± 8.50 23.91

2,563.4

132.62

3,624.34 ± 144.20

1.31 ± 0.06 bc

35.00 44.14

5.89 20.83

0.05 0.06 0.05

Factorial analysis ME, kcal/kg

3,000 168.78 525.70 1,349.0 1,999.18 241.57 792.45 ± 2,530.3 3,564.33 1.45 ± 1.51 1.88 1.79

± 18.97 ± 25.07 7 ± 71.77 ± 106.89 ± 10.91 39.65 0 ± 91.87 ± 117.87 0.16 ± 0.08 ± 0.10 ±0.0 9

2,900 175.84 532.60 1,360.8 2,024.86 249.94 798.22 ± 2,512.2 3,560.38 1.43 ± 1.50 1.85 1.76

± 14.80 ± 25.47 2 ± 69.89 ± 95.73 ± 11.44 40.12 2 ± 116.06 ± 142.94 0.14 ± 0.09 ± 0.13 ± 0.11

P-value 0.07 0.42 0.69 0.49 0.10 0.74 0.68 0.94 0.69 0.79 0.57 0.62

DAA, %

100 157.26 513.59± 1,329.7 1,956.35 245.49 784.14 ± 2,487.3 3,516.96 1.56 ± 1.53 1.88 1.80

± 11.97 21.46 b 7 ± ± 99.91 b ± 15.39 47.10 2 ± ± 146.16 0.08 a ±0.0 ± ±

c 76.68 93.21 8 0.10 0.10

107 168.79 522.46 1,338.7 1,985.69 247.96 791.17 ± 2,499.6 3,538.81 1.47±0. 1.52 1.87 1.79

± 8.03 b ± 20.99 b 7 ± 513.15 ± 68.55 b ± 11.83 36.66 9 ± 108.13 ± 114.10 11a ± 0.09 ± 0.12 ± 0.11

114 190.88 551.40 1,396.3 2,094.02 243.83 810.68 ± 2,576.7 3,631.28 1.28 ± 1.47 1.85 1.74

± 8.81 ± 15.82 0±64.8 ± 78.90 a ± 10.12 32.17 7 ± ± 105.61 0.08 b ± ± ±

a a 7 94.66 0.08 0.12 0.10

P-value <0.000 1 0.005 0.15 0.01 0.78 0.44 0.21 0.21 <0.000 1 0.45 0.91 0.48

479 DAA = digestible amino acids; ME = metabolizable energy.

480 a,b,c Within a column, means with different superscripts differ significantly (P < 0.05).

481 Table 4. Effect of dietary energy and DAA on the carcass traits (n = 4)._

Carcass parameter, g_ Carcass parameter, %

ME, DAA Body Carcass Breast Legs Wing Abdomi Carcass Breast Legs Wings Abdom kcal % weight s nal fat inal fat /kg_

3,00 100 1,983.00 ± 1,353.00 ± 454.50 ± 0 75.29 56.60 37.57

374.25

144.5 0 ± 11.59

29.50 ± 6.14

68.24 ± 1.88

33.55 ± 1.38

27.6 4 ± 0.62

10.68 ± 0.65

2.17 ± 0.37

3,00 107 2,033.00 ± 1,421.50 ± 509.00 ± 0 53.13 42.53 29.28

389.50

142.2 5 ± 10.66

33.25 ± 6.18

69.93 ± 1.58

35.79 ± 1.16

27.4 0 ± 1.65

10.02 ± 0.89

2.34 ± 0.41

3,00 114 2,145.00 ± 1,501.50 ± 538.00 ± 0 68.63 50.53 18.83

410.25

152.2 5 ± 7.72

33.25 ± 7.18

70.01 ± 1.67

35.83 ± 0.35

27.3 2 ± 0.93

10.14 ± 0.22

2.21 ± 0.43

2,90 100 2,044.00 ± 1,411.50 ± 495.75 ± 0 83.57 75.43 29.06

389.25

147.2 5 ± 14.57

32.50 ± 7.68

69.03 ± 1.04

35.13 ±1.27

27.6 1 ± 0.61

10.43 ± 0.73

2.31 ± 0.49

2,90 107 2,047.50 ± 1,393.75 ± 496.50 ± 0 82.44 71.14 25.36

376.75

140.5 0 ± 13.96

31.75 ± 4.19

68.05 ± 0.87

35.66 ± 1.88

27.0 2 ± 1.51

10.07 ± 0.77

2.29 ± 0.22

2,90 114 2,151.25 ± 1,502.00 ± 532.75 ± 0 53.86 38.40 25.43

410.00

149.7 5 ± 15.15

40.00 ± 6.73

69.82 ± 0.33

35.47 ± 1.19

27.2 8 ± 1.30

9.97 ± 0.85

2.66 ± 0.42

SEM 35.28

Factorial analysis

6.26 3.22

0.59 036

ME, kcal/kg

P-value DAA, % 100

P-value

2,053.67 ± 1,425.33 ± 500.50 ± 92.76 77.98 44.97

2,080.92 ± 1,435.75 ± 508.33 ± 85.15 76.06 30.12

64.67b

2,013.50 ± 1,382.25 ± 475.13 ±

2,040.25 ± 1,407.63 ± 502.75 ±

2,148.13 ± 1,501.75 ± 535.38 ±

391.33 146.3 32.00 ±

± 3 ± 6.18

26.68 10.19

392.00 145.8 34.75 ±

± 3 ± 6.96

27.07 13.80

0.95 0.92 0.31

381.75 145.8 31.00 ±

± 8 ± 6.63 19.37 b 12.28

383.13 141.3 32.50 ±

± 8 ± 4.96 28.36 b 11.54

410.13 151.0 36.63 ±

± 0 ± 7.39 22.48 a 11.21

0.06 0.33 0.22

69.40 ± 1.77

68.97 ± 1.05

68.64 ± 1.47

68.99 ± 1.55

69.91 ± 1.12

35.06 27.4 10.28 ± 2.24 ±

± 1.47 5 ± 0.66 0.37 1.05

35.42 27.3 10.16 ± 2.42 ±

± 1.36 0 ± 1.11

0.76 0.69

0.76 0.30

34.34 27.6 10.55 ± 2.24 ±

±1.49 2 ± 0.65 0.41 0.57

35.72 27.2 10.04 ± 2.31 ±

± 1.45 1 ± 0.77 0.30 1.47

35.65 27.3 10.05 ± 2.44 ±

± 0.84 0 ± 0.58 0.46 1.05

482 DAA = digestible amino acids; ME = metabolizable energy.

483 a,b Within a column, means with different superscripts differ significantly (P < 0.05).

485 Table 5. Effect of dietary energy and DAA on the enzyme activity (n = 4).

Pancreas Pancreatic enzymes__Serum enzymes_

weight,

ME, DAA% g Amylase, Lipase, U/L Protease, Amylase, U/L Lipase, Protease,

kcal/kg mg/g per min mg/g per U/L U/L

3,000 100 0.92 ± 0.09 14,656.67 ± 2,311.32 48,091.00 ± 5,413.00 134.25 ± 9.74 1,270.27 ± 109.84 19.18 ± 1.42 146.40 ± 8.34

3,000 107 1.05 ± 0.14 13,933.33 ± 2,217.11 45,257.33 ± 2,617.50 148.75 ± 15.78 ¿1,394.93 ± 191.26 19.11 ± 0.73 159.65 ± 12.92

3,000 114 1.14 ± 0.13 12,600.00 ± 2,078.46 48,694.00 ± 5,308.52 161.75 ± 9.50 1,354.43 ± 190.71 18.25 ± 2.22 170.40 ± 20.35

2,900 100 1.02 ± 0.11 13,933.33 ± 2,499.78 47,875.00 ± 4,624.91 144.75 ± 23.95 1,490.67 ± 304.06 18.63 ± 2.80 152.18 ± 2.17

2,900 107 0.99 ± 0.14 13,823.33 ± 2,086.73 47,920.50 ± 6,890.03 144.50 ± 11.36 1,347.00 ± 281.63 18.15 ± 0.82 152.80 ± 5.15

2,900 114 1.18 ± 0.08 14,085.00 ± 974.11 47,832.33 ± 3,975.58 157.50 ± 11.56 1,348.67 ± 222.66 19.28 ± 0.92 159.73 ± 47.62

SEM 0.06 1,043.43 2,491.65 7.28 112.98 0.75 11.08 ±

Factorial analysis

ME, kcal/kg

3,000 1.04 ± 13,733.33 ± 47,347.44 ± 148.25 ± 1,339.88 ± 18.84 ± 158.82 ±

0.14 2,184.98 4,471.48 16.00 161.60 1.49 16.81

2,900 1.06 ± 13,947.22± 47,875.94 ± 148.92 ± 1,395.45 ± 18.69 ± 154.90 ±

0.13 1,778.53 4805.47 16.38 255.56 1.35 25.29

P-value 0.63 0.80 0.80 0.91 0.55 0.80 0.67

DAA, %

100 0.97 ± 14,300.00 ± 47,983.00 ± 139.50 1,380.47 ± 18.90 149.29

0.11b 2263.02 4662.38 ±17.83b 242.22 ±1.68 ±6.43

107 1.02 ± 13,878.33 ± 46,588.92 ± 146.63 ± 1,370.97 ± 18.63 ± 156.23 ±

0.13b 1994.07 5030.71 12.93 ab 224.33 0.89 9.82

114 1.16 ± 13,342.50 ± 48,263.17 ± 159.63 ± 1,351.55 ± 18.76 ± 165.07 ±

0.10a 1699.46 4366.13 10.06 a 191.95 1.66 34.38

P-value 0.01 0.66 0.77 0.04 0.97 0.93 0.38

486 DAA = digestible amino acids; ME = metabolizable energy.

487 a,b Within a column, means with different superscripts differ significantly (P < 0.05).

488 Table 6. Effect of dietary energy and DAA on the intestinal histomorphology (n = 4).

10 days 10 days 39 days

ME, DAA, Intestin Small Villus Villu VSA, Crypt V/C Villus Villus VSA, Crypt V/C

kcal/k % al intestine height, s x10-3 depth, ratio, height, width, x10-3 depth, ratio,

g length, length, widt ^m2 ^m2

cm cm h, m

3,000 100

104.00 99.50 ± ± 2.83 3.19

504.25 ± 32.68

77.8 3 ± 8.66

123.79 196.00 ± 2.58 ± ±21.55 14.00 0.09

810.75 83.93± ± 14.21

213.5 203.5 4.00± 0± 0± 0.29 34.84 14.57

3,000 107

107.33 103.17 ± ± 3.30 3.92

537.50 ± 86.3 146.31 209.75 35.58 3 ± ± ±25.45

8.41 24.12

2.58 ± 839.00 96.00±

0.30 ± 11.61

251.8 216.7 3.87± 4± 5± 0.25 25.14 14.91

3,000 114 113.38 108.25 ± 501.75 ± ± 2.87 1.94 20.14

84.7 7 ±8.2 5

133.80 185.50 ± 2.71 ± ± 14.91 0.12

874.75 91.30± ± 11.70

249.7 206.7 4.27± 0± 5± 0.54 25.75 25.63

2,900 100

107.00 102.25 ± ± 5.72 5.51

497.00 ± 17.44

74.3 7 ± 5.90

115.86 194.00 ± 2.57 ± ± 5.75 11.27 0.14

868.00

83.77± 12.66

4.35±0 .45

2,900 107

107.25 103.13 ± ± 6.08 6.01

522.33 ± 88.9 145.79 205.00 ± 2.55 864.50 93.13± 251.0 213.7 4.07±

23.63 0 ± ± 3.61 ±0.11 ± 17.67 1± 5± 0.43

5.03 10.12 55.01 33.25 26.30

2,900 114 110.88 106.00 ± 522.75 ± ± 4.70 4.78 26.08

87.4 143.58 185.75 ± 2.83 ± 822.25 97.73± 249.7 220.5 3.77±

3 ± 16.69 0.18 ± 16.83 2± 0± 0.62

±9.2 17.28 74.39 21.12 30.77

4.46 9.94

37.87 8.26

15.91 13.61 0.26

Factorial analysis ME, kcal/kg

P-value DAA, % 100

108.24 103.64 ± 514.50± ± 4.83 4.69 31.36

108.38 103.79 ± 514.03± ± 5.33 5.21 23.42

0.94 0.93 0.97

105.50 100.88 ±4.47 b ±4.42b 3.76

82.9 134.63 197.08±

8.30 20.78

0.87 0.96

83.5 135.08 194.92± 7± ± 13.21

9.18 17.81

0± ±

6.91 14.76b

2.62± 0.18

2.65± 0.19

500.63±2 76.1 119.82 195.00±1 2.57±

841.50 90.41±

± 12.09 61.83

851.58 91.54±

± 15.06 62.37

839.38 83.85± ± 12.04

238.3 209.0 4.05±

5± 0± 0.37

31.23 17.57

242.5 211.7 4.06±

1± 5± 0.56

25.51 25.18

0.75 0.81

220.1 202.2 4.17± 5± 5± 0.39 27.15 18.12

107.29 103.15 ± 530.75± ± 4.53b 4.70ab 28.26

87.6 146.05 207.38± 2.57± 2± ± 16.46 a 0.20

6.36 16.55 a

851.75 94.57± ± 13.46

251.4 215.2 3.97± 2± 5± 0.33 26.37 19.20

112.13 107.13 ± 512.25± ± 3.84a 3.58a 23.80

86.1 0± 7.96

138.69 185.63±1 2.77±

848.50 94.52±1 ± 3.43

249.7 213.6 4.02± 1± 3± 0.58 21.06 26.42

P-value_0.02 0.04_0.20_0.05 0.06 0.09 0.11_0.94 0.36 0.13 0.60 0.73

489 DAA = digestible amino acids; ME = metabolizable energy; VSA = villus surface area; V/C ratio = villus /crypt ratio.

490 a,b Within a column, means with different superscripts differ significantly (P < 0.05).