Effects of Lysine Levels with Digestible Amino Acids on Growth Performance, Carcass Evaluation, Productive and Reproductive Traits of Aseel Chicken

EFFECTS OF LYSINE LEVELS WITH DIGESTIBLE AMINO ACIDS ON GROWTH PERFORMANCE, CARCASS EVALUATION, PRODUCTIVE AND REPRODUCTIVE TRAITS OF ASEEL CHICKEN

MUNAWAR HUSSAIN 2014-VA-509

A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN POULTRY PRODUCTION

UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES LAHORE, PAKISTAN

2018

To,

The Controller of Examinations, University of Veterinary and Animal Sciences, Lahore.

We, the Supervisory Committee, certify that the contents and form of the thesis,

submitted by Munawar Hussain, Reg. # 2014-VA-509 have been found satisfactory and

recommend that it be processed for the evaluation by the External Examiner (s) for

award of the Degree.

Supervisor ______

(Prof. Dr. Athar Mahmud)

Member ______

(Dr. Jibran Hussain)

Member ______

(Dr. Shafqat Nawaz Qaisrani)

DEDICATION This work is dedicated to

My Beloved

Parents& Family

My Teachers especially Prof. Dr. Muhammad Akram

ACKNOWLEDGEMENTS

All praises and thanks to ALMIGHTY ALLAH, the Lord of the worlds, the Omnipotent, the most Beneficent, the Merciful and the Gracious. Who is the creator of this world and heavens, the lord of the Day of Judgment, who is the entire source of knowledge and wisdom and endowed it to the mankind.

The work described in this thesis was made possible by contributions from various individuals and institutions to whom I am indebted. I thank all those who, in different ways, have walked beside me along the way; offering support and encouragement, challenging my thinking, and teaching me to consider alternative views.

The author would like to express his deep appreciation and thanks to my supervisor Prof. Dr. Athar Mahmud, Chairman, Department of Poultry Production and members, Dr. Jibran Hussain, Assistant Professor, Department of Poultry Production and Dr. Shafqat Nawaz Qaisrani, Assistant Professor, Department of Animal Nutrition, UVAS, Lahore, for their dynamic supervision, scholastic and constructive suggestions throughout the course of my studies, research and accomplishment of this manuscript.

I cannot find words to express my gratitude to Dr. Shahid Mehmood, Assistant Professor who guided me during my research work. All my prayers are due to Late Prof. Dr. Muhammad Akram, for his precious guidance. I would like to thank Mr. Sohail Ahmad, Mr. Faisal Hussnain, and Mr. Muhammad Usman, lecturers, Department of Poultry Production, UVAS, Lahore.

The authors gratefully acknowledge the support of anonymous readers and administration of SB Feeds Rawalpindi, Sindh Feeds Karachi, Islamabad Feeds Okara and ICGRC, Department of Poultry Production, University of Veterinary and Animal Sciences, Lahore. The expert guidance of Dr. Gulraiz Ahmed and Dr. Shahid Waheed Group Nutritionist and QA Manger (Feed) of SB Poultry, Rawalpindi is highly remarkable. They were so kind to me.

I am grateful to my dear friends Mr. Abdul Ghayas, Mr. Muhammad Tahir Khan, Mr. Abd ur Rehman Butt for his cooperation throughout the research. Thanks are also due to Dr. Aziz ur Rehman and Mr. Abd ur Rehman for the technical help and useful suggestions during writing of this manuscript.

I owe my special thanks to all of my classmates and friends for their company and moral support. Last but not least, warm thanks to my parents, brothers’ sister and wife; for support, moral strength and material welfare throughout my life and for being there for me during this time. Your love, encouragement and prayers made a big difference.

‘O’ Allah My Lord, I am in absolute need of the good You send me (Aamin).

Munawar Hussain

CONTENTS DEDICATION i

ACKNOWLEDGEMENTS ii

LIST OF TABLES iii

LIST OF FIGURES vi

ABSTRACT vii

SR. NO. CHAPTER PAGE NO.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 4

3 EXPERIMENT-I 36

4 EXPERIMENT-II 72

5 EXPERIMENT-III 91

6 SUMMARY 109

iii

LIST OF TABLES

TABLE NO. TITLE PAGE NO.

2.1 Body weight of Aseel at different ages 8

2.2 Egg weight in Aseel breed 10

2.3 Reproductive traits of Aseel 11

3.1.1 Feed formulation and nutrients calculations 48

Growth performance of indigenous chicken in response to 3.1.2 49 dietary amino acid regimens

Effect of dietary amino acid densities on immune 3.1.3 50 responses of birds

Effect of dietary amino acid densities on body 3.1.4 51 conformation traits of birds

3.2.1 Ingredient and nutrients composition of diets 69

Growth performance, lysine efficiency ratio and mortality 3.2.2 70 at six weeks of age

Effect of dietary amino acid regimens on meat composition 3.2.3 71 traits of Aseel varieties

4.1 Ingredient and nutrients composition of diets 87

Weight gain, final body weight, feed to gain ratio and 4.2 88 mortality at 18 weeks of age

Chemical composition of thigh and breast muscles of local 4.3 89 Aseel subsequent to dietary lysine regimens

4.4 Carcass characteristics of local Aseel at 18 weeks of age 90

5.1 Ingredient and nutrient composition of diets 104

Productive performance of local Aseel subsequent to 5.2 105 dietary lysine regimens

Egg geometry characteristics of local Aseel subsequent to 5.3 106 dietary lysine regimens

Egg quality characteristics of local Aseel subsequent to 5.4 107 dietary lysine regimens

Hatching traits of local Aseel subsequent to dietary lysine 5.5 108 regimens

LIST OF FIGURES

FIGURE NO. TITLE PAGE NO. Phase-wise weight gain and feed to gain ratio in varying 3.1.1 52 lysine diets

ABSTRACT This study evaluated the effect of varying dietary lysine (Lys) levels on growth, meat quality, productive and reproductive performance of Aseel chicken. Study was carried out in three phases, during first phase effect of dietary lysine (Lys) regimens was evaluated on growth performance and meat composition of Aseel chicken (Experiment 1). In total 540 birds, 180 from three Aseel varieties were studied. A Randomized Complete Block Design in factorial arrangement, with 9 treatments of 6 replicates with 10 birds each, was applied. Treatments consisted of 3 varieties [Mianwali (MW), Peshawari (PW), and Lakha (LK)] and 3 Lys levels

[1.35% (L1); 1.3% (L2); and 1.25% (L3)]. Growth performance (feed intake, weight gain, and feed: gain ratio) and meat composition (dry matter, ash, crude protein, and fat contents) were evaluated. In phase two, effect of dietary Lys regimens was evaluated on subsequent growth

(7-18 weeks) of Aseel chicken (Experiment 2). In total 378 birds, 126 from three Aseel varieties. A Randomized Complete Block Design under factorial arrangement, with 9 treatments of 6 replicates with 7 birds each, was employed. Treatments consisted of 3 varieties

[Mianwali (MW), Peshawari (PW), and Lakha (LK)] and 3 Lys levels [1.35% (L1); 1.3% (L2); and 1.25% (L3)]. Growth performance (feed intake, weight gain, and feed: gain ratio) were evaluated. In phase three, effect of dietary Lys regimens was evaluated on productive performance, egg characteristics and hatching traits of Aseel chickens (Experiment 3). In total

63 females and 9 males of 26 weeks were studied. Randomized Complete Block Design under factorial arrangement, with 9 treatments of 7 replicates with 7 females and 1 male each, was employed. Treatments consisted of 3 varieties [Mianwali (MW), Peshawari (PW), and Lakha

(LK)] and 3 Lys levels [1.35% (L1); 1.3% (L2); and 1.25% (L3)]. Productive performance (egg production, egg weight, egg mass, feed per dozen eggs and feed per Kg egg mass), egg characteristics (shape index, surface area and volume) and hatching traits (fertility and hatchability) were evaluated. Data were analyzed through factorial ANOVA using GLM

vii

procedures in SAS software, considering varieties and Lys levels as main effects and sex as block effect. Turkey’s HSD test was used to compare treatment means considering 5% probability level. There was significant influence of varieties and Lys levels on growth and meat quality. Improved WG (P=0.0002; 424.1±8.1) and F: G (P=0.0006; 2.84±0.05) was observed in MW variety as compared to PW (WG: 411.5±6.3; F: G=2.95±0.05) and LK (WG:

401.5±9.3; F: G=3.02±0.08). Among different (P<.0001) Lys regimens, higher and medium levels in the diet resulted in improved WG (423.3±8.2; 428.2±4.9), F: G (2.79±0.05; 2.80±0.03) and reduced FI (1175.8±3.7; 1198.0±5.4). Among dietary treatments medium dietary Lys regimen showed improved WG, F: G and final WG. Increased dry matter (P=0.0036;

73.80±0.17), lower ash contents (P<.0001; 1.23±0.03) and lower crude protein (P=0.0064;

21.97±0.17) contents were observed in thigh at medium Lys levels, whereas only difference

(P=0.0150; 1.30±0.04) in ash was found in breast with low Lys diet. While, the breast muscle ash % was lower for MW and PW variety (P<.0001; 1.30±0.03, 1.31±0.05). In subsequent phase results indicated higher WG (P<.0001; 1244.4±15.2) and improved F: G (P<.0001;

2.82±0.03) in MW variety compared to PW (WG: 1113.1±10.4; F: G: 3.05±0.02) and LK (WG:

1161.5±8.75; F: G: 2.94±0.03). For dietary treatments medium dietary Lys regimen showed improved WG, F: G and final WG. Increased dry matter (P=0.0176; 75.03±0.17), lower ash contents (P=0.026; 1.59±0.05) and lower crude protein (P=0.0175; 19.77±0.17) contents were observed in thigh under medium Lys levels, whereas the difference (P=0.0479) in CP was found only in breast, where L1 (22.30±0.17) and L2 (22.37±0.16). Carcass characteristics including slaughtering weight, dressed weight and dressing percentage showed higher (P<0.05) values in medium Lys dietary treatments. Among varieties MW variety showed overall enhanced carcass characteristics. In production and reproduction phase results showed differences in egg production, egg weight, egg mass, feed conversion per dozen and feed conversion per kg egg mass with in varieties, where PW variety showed higher egg production

viii

(P<0.0001; 40.9±0.54), MW and LK showed higher egg weight (P<0.0001;

46.52±0.55,45.19±0.65), PW and MW showed higher egg mass (P<0.0001, 1728.3±31.9,

1684.2± 39.1), PW showed lower feed conversion per dozen eggs (P<0.0001; 2.35±0.03) and

PW and MW indicated lower feed conversion per kg EM (P=<0.0001; 6.52±0.12, 6.72±0.15) and interaction of variety and dietary treatments. Similarly egg geometry and egg quality parameters showed differences with in varieties where MW variety showed higher egg length

(P<.0001; 54.57±0.50), lower shape index (P<.0001; 73.69±0.92), higher egg volume

(P<.0001; 42.5±0.50), higher egg surface area (P<.0001; 59.7±0.47) and interaction of variety and dietary treatments, where MW in interaction with medium Lys regimen. Improved hatching traits were found for fertility (P<.0001; 82.1±0.67) and hatchability (P<.0001;

59.9±0.65) in PW variety, moreover, PW variety in interaction with Lys regimens showed higher fertility (P<.0001) and hatchability (P<.0001). Fertility and hatchability did not show any significant variation in response to dietary treatments. It was concluded that 1.30% digestible Lys level regimen can be used to improve the early and subsequent growth rate of

Aseel chicken. Similarly, improved growth due to dietary Lys in juvenile phase has interaction with varieties in improving productive and reproductive performance of Aseel. Mianwali variety due to its higher growth may be exploited as a meat-type chicken.

Keywords: Aseel varieties, Lysine regimens, growth, meat quality, productive performance, egg characteristics, hatching traits

CHAPTER 1 INTRODUCTION

In Pakistan, poultry production contributes 1.3% to the national GDP, 6.1% towards agriculture and 10.8% in overall livestock sector. Total revenue involved in this sector is PKR 700/- billion.

Poultry accounts for 28% of total meat production of the country with a very encouraging growth rate of 8-10% per annum. This sector employs approximately 1.5 million people in

Pakistan (Hussain et al. 2015). Backyard poultry production is an important sub sector, contributing 11.12% in total poultry meat and 27.22% of the total egg production in the country

(Economic Survey, 2016-17). Despite having tremendous potential, very little attention has been given to indigenous poultry birds. These birds often possess better disease resistance and adaptability to the local environmental conditions as compared to imported commercial poultry strains. Local consumers also often express preference for meat from indigenous breeds (Barua and Howlider 1990).

Pakistan’s backyard poultry mainly consist of local breeds namely Aseel, Naked neck, Lyallpur

Silver Black, Desi and some other exotic breeds including Fayoumi, Black Australorp and

Rhode Island Red. Aseel is the oldest Asian game fowl having superior quality meat in terms of texture and taste, perhaps due to its parentage to the Cornish breed that is considered to be the main parent (male line) of today’s broilers (Dohner, 2001). Aseel can easily be reared in the tropical and sub-tropical regions due to its adaptability to extreme weather conditions.

Indigenous chicken has shown better growth through improved nutrition (Tadelle et al. 2003;

Kingori et al. 2007) and the performance of Aseel birds can also be improved through rearing in confinement (Chowdhury et al. 2006), selection, good management and improved nutrition

(Bhatti and Sohota 1994).

In poultry, early nutrition is very much important, due to the high rate of growth during the first weeks of life. Moreover, there is positive correlation present for early growth and

1 INTRODUCTION subsequent performance. Development of various physiological systems, especially digestive and immune systems, is faster at this young age and nutrition (e.g., amino acids, amino acid ratios and energy) plays a pivotal role in this development (Ullah et al. 2012).

Traditionally, crude protein was considered to be the base for the formulation of poultry diets.

But presently, specific amounts of individual amino acids are considered more essential for proper growth. It is now common to characterize ideal proteins or ideal amino acid balances and using tissue accretion methods to assess retention efficiency of amino acids (Tillman and

Dozier 2013). These methods can improve protein efficiency and decrease nitrogen excretion

(Chung and Baker 1992). In ideal amino acid formulation, lysine (Lys) is considered as base and all other essential amino acids are expressed as ratios to Lys (Baker and Han 1994).

Different studies were conducted on total amino acids and digestible amino acids of different ingredients and the differences in growth were observed between total and digestible amino acids. Currently, nutritionists formulate diets on the basis of digestible amino acids.

Interestingly, amino acids which were known as only building blocks for protein synthesis for years, are now also being considered to be involved directly or indirectly in the regulation of the protein synthesis. Feeding high density amino acid diets early in development improve subsequent performance and meat yield (Corzo et al. 2005), which is further supported by previous studies that demonstrated feeding broilers high Lys diets during the starting period increased subsequent breast meat yield (Holsheimer and Ruesink 1993; Kerr et al. 1999).

On the basis of available literature, it is very much clear that Aseel birds have significant potential as meat source and their productive potential can be enhanced through improved nutrition using modern nutritional tools. The present study aimed to investigate the effect of different Lys levels in diets formulated on the basis of ideal amino acid concept on: 1) growth performance; and 2) subsequent effects of this growth on rearing, egg production and reproductive (hatching) traits. The information gained can be used to enhance the

INTRODUCTION competitiveness and sustainability of small scale farmers who often use open-sided production systems with two thousand to ten thousand birds per farm and who are adversely affected by commercial environment controlled poultry farming. Moreover, the information can be used to improve free range production of indigenous birds in effort to meet consumer demand of organic poultry production.

CHAPTER 2 REVIEW OF LITERATURE

2.1. Aseel chicken; an important breed of Asian Sub-Continent, future prospects

(Submitted to World’s Poultry Science Journal)

Summary

Aseel is the most popular breed of chicken among the local breeds of the Asian subcontinent. The juvenile body weight of 250 to 300 g at 6 weeks of age indicates slow growth during the early period; however, the birds reach 5 Kg by 50 weeks of age indicating its potential to achieve higher weight. Slow early growth rate is a major problem of this breed and leads to higher time to reach market weight with poor Feed to gain ratio. In the past, genetic tools were applied to improve Aseel chicken performance without nutritional manipulations.

Available literature indicates that growth performance of the indigenous birds can be enhanced with nutritionally balanced diets. As such, along with genetic manipulations, improved nutritional strategies may further enhance early growth performance of Aseel chickens, and, consequently, later growth as well. The present review highlights the published literature about the growth performance of Aseel chicken and the challenges as well as the possible solutions to improve its performance.

Keywords: Aseel Chicken, Early Growth, Backyard Poultry, Subsequent Growth

Introduction

Backyard poultry production is an important sub sector of poultry in tropical and sub-tropical regions of the world providing a key source of valuable animal proteins (Roberts, 1999), and thus combating the nutritional deficiencies. In developing countries including Pakistan, these indigenous breeds are also a good source of income generation. Climate adaptability, greater disease resistance and lower nutritional demands of indigenous chickens are among the major factors driving the preference of rural community towards indigenous poultry breeds for meat as well as egg production (Romanov et al. 1996; Barua and Howlider 1990). Finally, the

REVIEW OF LITERATURE need to preserve (and enhance) the genetic base of indigenous birds is recognized throughout the world.

Central Avian Research Institute (CARI) India has worked on the genetic improvement of native breeds like Aseel, Malay, Kadaknath and Busra and has developed different meat

(Vanaraja, and CARI-Devendra) and egg type breeds (Grampriya, and CARIGold; Khan,

2002). Likewise, the Indigenous Chicken Genetic Resource Centre (ICGRC), Department of Poultry Production, University of Veterinary and Animal Sciences, Lahore and

University of Agriculture Faisalabad, Pakistan, are also working on the genetic improvement of local breeds including Aseel and Naked-Neck and development of new breeds and strains (LSB and Uni-Gold) to improve backyard poultry production.

Among the local poultry breeds, Aseel is the most popular and is known for its hardiness, larger body size (Mahmood et al. 2017), body length, upright posture, thick muscular thighs, strong legs, majestic gait, pea comb, small wattles, broad skull and good quality meat (Rajkumar et al. 2017). Its game-type behavior, vigor and aggressiveness are, furthermore, among the major factors related to its popularity in the Asian subcontinent

(Khan, 2004; Mahmood et al. 2017). Perhaps, due to its parentage in the Cornish breed that is considered to be the main parent (male line) of modern day broilers (Dohner, 2001), it is suggested that Aseel can be developed as a commercially suitable backyard meat-type bird.

Aseel Varieties and Characters

More than 16 varieties of Aseel chicken have been reported in Indo-Pak (Usman et al.

2014). In Pakistan, the most common varieties of Aseel are Lakha, Peshawari, Mianwali and, to a lesser extent, Mushki, Sindhi, Lassani and Java. Peela, Kulang, Yakub, Nuri,

Kagar, Chitta, Sabja, Teekar and Reza have been reported in India.

REVIEW OF LITERATURE

Aseel has multicolored (black, red, dark brown, white and golden) plumage with density feather coat (Sarker et al. 2012; Suganti, 2014; Rajkumar et al. 2017). Generally, red, white and black ear lobes are found in Aseel, and among them, red ear lobes are most prominent, whereas small size wattles are present in males (Sarker et al. 2012; Rajkumar et al. 2017). Pea comb is the breed characteristic of pure Aseel (Singh, 2001), whereas strawberry combs are also reported in Bangladeshi Aseel (Sarker et al. 2012). Similarly, white skin color is the breed character (Singh, 2001; Sarker et al. 2012; Rajkumar et al. 2017). Yellow beak coloration is found in most of the Aseel varieties except for Mushki Aseel that has black beak colour

(Rehman, 2017; Mahmood et al. 2017). Mostly yellow, black and white shanks are found in

Aseel (Sarker et al. 2012; Rajkumar et al. 2017). Spur is a sex linked characteristic in Aseel males (Iqbal et al. 2015; Rajkumar et al. 2017).

In Pakistan, Aseel varieties are distinct on the basis of their physical characteristics and geographical prevalence. Lakha (mottled) from the central Punjab has reddish brown plumage with brown egg shell color, Mushki from Khushab, Sargodha, Bhakkar and

Fatehjang districts have black plumage. Peshawari with wheaten colored plumage can be found in the regions of Peshawar, Nowshehra. Mardan and Mianwali varieties from

Mianwali, Sargodha and Kalabagh regions have dark brown plumage color (Babar et al.

2012; Mahmood et al. 2017). Similarly, the Sindhi variety, with wide shoulders, large and highly curved beak, muscular and compact body, pea comb and no wattles, droopy tail and hard feathers is found in Sindh province. In a microsatellite marker based genetic diversity study of Aseel varieties (Baber et al. 2012), more genetic variation was reported between

Peshawari and Mushki and less between Lakha and Mushki.

In India, different varieties of the Aseel are found in the area of Andhra Pradesh, Chhattisgarh,

Uttar Pradesh and Rsjasthan. Indian Aseel has various plumage coloring, including Peela with golden red plumage, Kulang with bluish colour, Yakub with black and red, Nuri with white,

REVIEW OF LITERATURE

Kagar has black, Chitta with black and white spotted, Java has black, Sabja with white and golden or black with yellow or silver, Teekar with brown and Reza has light red plumage colour

(Yadav et al. 2017). Aseel chickens are also found in Brahmanbaria district of Bangladesh

(Sarker et al. 2012).

Growth Performance of Aseel

In Pakistan, body weight in different varieties of Aseel ranges from 3-7 and 2-4.5 Kg for males and females respectively (Babar et al. 2012). In India, average body weight ranges from 3-5

Kg for cocks and 2-4 Kg for hens (Singh, 2001; Sharma and Chatterjee 2006; Rajkumar et al.

2017). In Bangladesh, Aseel adult male average weight is 3750 g and female is 2062g (Srker et al. 2012). Thus, it is clear that body weight in Aseel is a sex influenced trait (Haunshi et al.

2011; Rajkumar et al. 2017). Body weight also varies across different varieties of Aseel (Jatoi et al. 2014). Higher body weight is reported in Lakha and Mianwali compared to the Peshawari variety in Pakistan. Aseel body weights observed at different ages by various researchers are presented in Table1.

In Aseel chickens, juvenile body weight of 267.2 and 297.7g at 6 week of age indicates that growth in this early period is relatively slow. Aseel birds, however, reach 1853 g (females) and 2702.5g (males) at 40 weeks of age, indicating Aseel has potential to achieve sufficient body weight in later period (Haunshi et al. 2011; Rajkumar et al. 2017). Slow early growth rates translate increased time to reach market age. Coupled with poor Feed to gain ratio, there is a higher cost of production in Aseel birds. Different groups, however, have shown that Aseel growth performance improves when fed diets with higher levels of protein and energy (Haunshi et al. 2012).

REVIEW OF LITERATURE

Table. 2.1. Body weight of Aseel at different ages

Age (week) Body Weight (g) Reference 54.21 Valvani et al. 2016 88.51 Pathak et al. 2015 2nd 48.63 Sarker et al. 2012 72.88 Haunshi et al. 2011 65.1 Chatterjee et al. 2007 55.06 Gurung and Singh 1999 142.4 Rajkumar et al. 2017 86.08 Valvani et al. 2016 192.49 Pathak et al. 2015 4th 219.79 Jatoi et al. 2014 138.40 Sarker et al. 2012 150.62 Haunshi et al. 2011 154 Chatterjee et al. 2007 107.11 Gurung and Singh 1999 297.7 Rajkumar et al. 2017 203.48 Valvani et al. 2016 6th 346.95 Pathak et al. 2015 212.88 Sarker et al. 2012 267.19 Haunshi et al. 2011 161.19 Gurung and Singh 1999 610.08 Valvani et al. 2016 596.25 Jatoi et al. 2014 8th 360.0 Sarker et al. 2012 393 Chatterjee et al. 2007 234.14 Gurung, and Singh 1999 821.2 Rajkumar et al. 2017 1054.78 Valvani et al. 2016 12th 1074.2 Jatoi et al. 2014 743.75 Sarker et al. 2012 796 Chatterjee et al. 2007 408.22 Gurung, and Singh 1999 1122.5 Rajkumar et al. 2017 1455.28 Rehman, 2017 16th 1086.0 Sarker et al. 2012 1051.8 Haunshi et al. 2011 1216 Chatterjee et al. 2007 1381.4 (F) Rajkumar et al. 2017 20th 1840.9 (M) 1318.42 Haunshi et al. 2011 1704.4 (F) Rajkumar et al. 2017 40th 2702.5 (M) 1853 Haunshi et al. 2011 1755 Singh et al. 2007

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Rearing systems, additionally, have influence on body weight gain in Aseel and indigenous poultry breed with birds raised in confinement generally being more efficient (Chatterjee et al. 2002; Rehman, 2017).

Feed intake and feed conversion varies among different varieties of Aseel (Iqbal et al. 2012;

Jatoi et al. 2014). Usman et al. (2013) observed higher feed intake in Peshawari as compared with Lakha, Mianwali and Mushki Aseel (Usman et al. 2013). Similarly, higher feed intake has been recorded in Peshawari, Lakha and Mianwali varieties compared with the Mushki variety (Jatoi et al. 2014). This variation may be due to genetics; as similar variation has also been observed in different strains of commercial layers (Scheideler et al. 1998).

Productive Performance of Aseel

Most native chicken breeds, including Aseel, mature at a late age either in farm or field conditions compared to commercial hens (Haunshi et al. 2011; Rajkumar et al. 2017). Age of sexual maturity also varies among Aseel varieties. Various studies reported that Aseel hens reach sexual maturity at 154 days (Mohan et al. 2008), 213.25 (Haunshi et al. 2011), 174

(Haunshi et al. 2013) 144 (Valvani et al. 2016) or 214 days of age (Rajkumar et al. 2017).

Variation among the reports might be attributed to differences in rearing seasons as increasing day length at later growth stages may lead to earlier sexual maturity (Haunshi et al. 2013).

Egg production of Aseel hens has been reported as 160 eggs up to 78 weeks of age (Mohan et al. 2008a), 35.71 eggs/year (Sarker et al. 2012), 43.28 eggs (Valvan et al. 2016), 18 eggs at 40 weeks, 30 eggs at 52 weeks, 47 eggs at 64 weeks and 64 eggs up to 72 weeks (Rajkumar et al.

2017) and 35.90% in 31 to 46-week age (Rehman et al. 2017). Like growth rates and other performance measures, egg production may vary among different varieties of Aseel. Higher egg production (34.08%) was observed in the Mushki variety as compared to others (Usman et al. 2014). Ahmad et al. (2014) reported higher egg production in Peshawari variety in pre- and

REVIEW OF LITERATURE post-molt periods. Persistency of egg production in Aseel chickens, like other breeds, is inversely related to age. Production peaks during 24 to 36 weeks of age and then declines after

40 weeks of age (Haunshi et al. 2013; Rajkumar et al. 2017).

Egg weight, however, has positive correlation with age (Haunshi et al. 2011). Aseel egg weights reported by different studies are presented in Table 2.2.

Overall egg weight of Aseel hens is comparatively lower than eggs of commercial hens, which might be attributed to poor feeding and management systems in addition to genetics (Sarkar et al. 2006).

Table 2.2. Egg weight in Aseel breed

Age Egg Production Reference - 47.81 Singh et al. 2000a - 41 Singh et al. 2000b 40 47 Singh et al. 2007 26 40 Mohan et al. 2008 78 52 28 42.71 Haunshi et al. 2011 32 45.8 40 49.28 - 37-42 Sarker et al. 2012 - 41.2-45.9 Iqbal et al. 2012 - 45 Ahmad et al. 2013 - 51.62-55.65 Usman et al. 2014 40 38.8 Rajkumar et al. 2017 72 47.5 31 45.03 Rehman et al. 2017

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Egg geometry parameters have positive correlation with age (Usman et al. 2014). Haugh unit and yolk index was found to be 75.43 and 0.395 respectively as reported by Haunshi et al.

(2011). Usman et al. (2014), however, reported 81.95 Haugh unit score and 0.46 yolk index in

Aseel eggs. Egg shape index in Aseel chicken has been reported to be 75.46 (Singh et al. 2000a) and 77.36 (Haunshi et al. 2011).

Reproductive Performance of Aseel

Breed, nutrition, management and age of birds may affect reproductive performance of Aseel chickens. There is, however, a great deal of variation among reproductive traits (e.g., fertility, hatchability and hatch) in published literature (Table 2.3).

This variation in reproductive traits may be related to differences in birds’ rearing conditions, type of birds or random field data (Rajkumar et al. 2017).

Table 2.3. Reproductive traits of Aseel

Parameter Percentage Reference Fertility 84 Mohan et al. 2008a Hatchability 67 Hatch of fertile 88 Fertility 86.96 Haunshi et al. 2012 Hatchability 70.74 Hatch of fertile 81.21 Fertility 89.95 Ahmad et al. 2013a Hatchability 46.30 Hatch of fertile 49.85 Fertility 64.61 Ahmad et al. 2013b Hatchability 53.02 Hatch of fertile 84.20 Fertility 67.18 Rajkumar et al. 2017 Hatchability 44.71 Hatch of fertile 80.87 Fertility 79.0 Rehman, 2017 Hatchability 59.18 Hatch of fertile 74.86 embryonic mortality 19.69 A-grade chicks 52.23

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Meat Quality and Composition of Aseel Meat composition and quality in Aseel chickens is linked with different factors, including breed, nutrition, age, genetic makeup, sex, rearing systems, stress conditions, dressing and type of meat (Boscovik et al. 2010; Rajkumar et al. 2016). Normally, chicken meat crude protein ranges from 17.0 to 23.3%, fat from 1.0 to 8.9%, ash from 0.1 to 1.1% and dry mater from 60.4 to 76.14% (Karakok et al. 2010; Boscovik et al. 2010; Souza et al. 2011). Chemical composition of Aseel chicken meat is similar to other breeds with little variation. Aseel meat crude protein ranges from 24.94 to 26.85%, fat from 3.75 to 7.13% and dry matter from 83.35 to 88.28% in breast and thigh muscles (Haunshi et al. 2013). Rajkumar et al. (2017), however reported different results with Aseel chicken meat (breast) containing 21.5% crude protein,

3.4% fat, 2.0% ash and 73.3% moisture, 6.0 pH and 72.5 mg/100 gram cholesterol.

Meat quality is critical to consumer acceptance of meat products (Rajkumar et al. 2016) and pH is the major attributing factor for quality of meat (Dadgar et al. 2011). The pH values observed in Aseel is 6.0, which is comparable to pH range 5.8 to 6.3 of broiler meat (Sarsenbek et al. 2013).

Problems related to Aseel and Solutions

Aseel chicken has great potential for the improvement in growth performance (Yousaf et al.

2016). However, the poor early growth, late maturity, lower egg production with less persistency, high incidence of broodiness, low fertility and low hatchability, are the main challenges (Rajkumar et al. 2017). Most of the native chicken breeds mature at a late age

(Haunshi et al. 2011). However, early maturity and increased production (Mohan et al. 2008a) and loss of broodiness character due to improvement by long-term selection programs have also been reported. Moreover, low reproductive performance may be due to the fact that in field condition birds are reared under scarcity conditions leading to subsequent reduced fertility and hatchability (Rajkumar et al. 2017). Poor early growth under such conditions is also

REVIEW OF LITERATURE attributed to Aseel slow growing character which requires more time (20-24 Weeks) to achieve market weight (1.5 to 2 Kg) (Jatoi et al. 2014). Nutritional manipulation and managerial tools can be applied to improve early growth, which can accelerate subsequent performance and help in reducing time to market, while improving egg production, fertility and hatchability related issues. Birds need balanced feed for efficient performance and proper physiological functioning. Indigenous chicken has shown elevated growth through improved nutrition

(Tadelle et al. 2003; Kingori et al. 2007). In the near past, different studies, reported improved growth, productive and reproductive performance of Aseel through nutritional manipulations

(Khan et al. 2017; Zia et al. 2016, 2017).

Nutrition and Early Growth

Early period feeding of chicks has significant effects on intestinal tract development, muscular growth and immune system stimulation, resulting in ultimately faster weight gain (Saki, 2005;

Prabakar et al. 2016). Lysine is critical among all nutrients for early growth as it is directly linked to muscle development. Lysine comprises 7% of the protein in breast meat (Dozier et al. 2007; Mahdav et al. 2012). It has been reported that supplementation of Lys during early growth stages (Campestrini et al. 2010) is beneficial because Lys is involved in regulation of protein synthesis and increases muscle accretion (Dozier et al. 2007; Eits et al. 2003). Early nutrient supply is, furthermore, associated with the development of digestive system (Beski et al. 2015). Supplementation of 1.4% Lys in starter diets increases intestinal length and weight of digestive tract (Ullah et al. 2012), which is closely related to pancreatic enzyme activity and nutrients digestion and absorption (Sklan and Noy 2000).

During embryonic development, myoblasts develop from myogenic precursor cells, which are of mesodermal origin (Rehfeldt et al. 2000). Myoblasts, which stop cell division and exit from the cell cycle, come from the proliferation of myogenic precursors, which ultimately leads to the formation of multinucleated myotubes. After hatching, muscle

REVIEW OF LITERATURE growth occurs due to the difference in protein synthesis and protein degradation, which is termed as protein accretion or muscle accretion. This accretion occurs due to hypertrophy of muscle fibers that is complemented by proliferative activities of satellite cells as source of new nuclei (Stickland, 1983). The difference in development of muscles within breeds or varieties is due to genetics and physiology (Rehfeldt et al. 2000).

It has been found that, in addition to the effects on overall feed intake and energy balance, specific nutrients can significantly influence growth hormone receptor expression in muscles. Intake of a single amino acid, Lys, is extremely important for maintenance of normal circulating levels of plasma IGF-I and optimal growth. (Katsumata et al. 2002).

Early Growth Relation to Subsequent Growth

Early weight gain is positively correlated with time to reach market weight (Saki, 2005).

Provision of nutritionally balanced diet during early life may lead to increased satellite cell proliferation and activity in muscle cells leading to an elevated growth performance during later phases of life (Kadam et al. 2009; Prabakar et al. 2015). The supplementation of essential nutrients including Lys during birds’ early age has been shown to improve subsequent performance (Kidd et al. 1998). This subsequent improved growth performance may be due to increased satellite cells proliferation and activity in muscle cells (Kadam et al. 2009; Prabakar et al. 2015). Early nutrient supply is linked to the development of the digestive system, which is more rapid in early phase of life (Beski et al. 2015), and intestinal weight is closely related to pancreatic enzyme activity and ultimate nutrient digestion and absorption (Sklan and Noy

2000).

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Early Growth Relation to Production and Reproduction

The poorer production performance of the indigenous breeds may also be attributed to substandard flock management in traditional production systems. Body weights or feeding level play critical role in stimulating the birds to attain sexual maturity (Ciacciariello and Gous 2005). The relationships among live body measurement in egg-type poultry have been discussed by Adenowo et al. (2015). Breeders are thus interested in heritability of the traits and their genetic and phenotypic correlations. It is evident that larger hens within a bloodline lay larger eggs than those with smaller body weights (Du Plessis and

Erasmus 1972). Similarly, larger body size results in large egg length, width and mass, all factors affecting egg weight (Ricklefs and Marks 1983).

2.2. Amino Acids

Traditionally, crude protein was considered to be the base for the formulation of poultry diets.

It is now known that specific amounts of individual amino acids are more essential for proper growth, hence, common to characterize ideal proteins or ideal amino acid balances and using tissue accretion methods to assess retention efficiency of certain amino acids (Tillman and

Dozier 2013). These methods can improve protein efficiency and decrease nitrogen excretion

(Chung and Baker 1992). In ideal amino acid formulation, Lys is considered as base and all other essential amino acids are expressed as ratios to Lys (Baker and Han 1994). Different studies were conducted on total amino acids and digestible amino acids of different ingredients and the differences in growth were observed between total and digestible amino acids.

Currently, nutritionists formulate diets on the basis of digestible amino acids. Interestingly, amino acids which were known as only building blocks for protein synthesis for years, are now also considered to be involved directly or indirectly in the regulation of the protein synthesis.

Feeding amino acid density diets early in development improve subsequent performance and meat yield (Corzo et al. 2005), which supports previous studies that demonstrated feeding

REVIEW OF LITERATURE broilers high Lys diets during the starting period increased subsequent breast meat yield

(Holsheimer and Ruesink 1993; Kerr et al. 1999).

Amino acids and Growth performance

Amino Acids and Muscle Accretion

Dietary protein has been shown to regulate insulin-like growth factor 1 (IGF-I) concentration (Tesseraud et al. 2003) in avian species. IGF is an important positive modulator of body and muscle growth in chickens (Duclos et al. 1999). Doumit et al.

(1993) reported that IGF-I stimulates differentiation and proliferation of satellite cells.

The sensitivity and responsiveness of muscle to growth hormone and IGF-I may be regulated by the availability of nutrients such as amino acids. Although infusion of IGF-I alone does not alter muscle protein synthesis in rats, when IGF-I is given in combination with amino acids protein synthesis increases significantly in the gastrocnemius, oblique and soleus muscles (te Pas et al. 2004). It appears that IGF-II may be less involved than

IGF-I in controlling body and muscle growth of chickens. Tomas et al. (1998) suggested that IGF-I could induce fatness rather than lean tissue growth. Providing broilers, AA density diets increases breast meat yield, likely by increasing myofiber size via modulating protein synthesis and protein degradation. Lysine is involved in the release of the growth hormone, insulin like growth factor I (IGF-I) and modulation of bone growth by differentiation of osteoblasts and collagen synthesis (Nonis and Gous 2008). Depressed growth in Lys deficient diets may be due to decreased protein synthesis and accretion

(Tesseraud et al. 1996). Additionally, IGF-I may require the IGF binding protein, IGFBP-

3, in order to enhance protein synthesis in skeletal muscle (Svanberg et al. 2000). IGFs plays key roles in muscle development, the effects being dependent on stage of development. In general, prenatal effects are centered on proliferation and differentiation, whereas postnatal, the major effect is on hypertrophy. Increased amino acids and insulin

REVIEW OF LITERATURE results in hastening translation of messenger mRNA, which increases protein synthesis in muscles and other organs. The stimulation of protein synthesis by amino acids is attributed to stimulation of mRNA translation initiation (Suryawan et al. 2009).

Protein synthesis is the result of translation of mRNA, which is initiated by a high pool of amino acids. In muscle growth, size of myofibers increases, not the number as myonuclei have no further division ability, indicating the importance of IGF-I as it increases mRNA in skeletal muscles (Sacheck et al. 2004). In addition, methionine, by the action of S6K1 phosphorylation, activates mRNA translation (Métayer-Coustard et al. 2010). QM7 myoblasts S6K1 phosphorylation is carried out by a precursor of methionine keto- methylthio-butanoic acid. Furthermore, methionine is involved in DNA methylation and epigenetic gene regulation and ultimately gene expression (Waterland, 2006). Different interactions of amino acids, moreover, play vital roles in protein synthesis. As an example,

Lys, methionine and arginine interactions are involved in the synthesis of creatine

(Chamruspollert et al. 2002), which is further involved in the generation of creatine phosphate. Creatine phosphate has two functions in muscle cells: first, it can generate

ATP from ADP, which acts as energy to the muscle cells; secondly, it plays role in the transfer of high-energy phosphate into the myosin filaments from mitochondria (Smith et al. 2005). One of the most frequently explored signaling pathways is the mammalian target of rapamycin (mTOR) pathway. mTOR integrates signals from amino acids and hormones, e.g. insulin/insulin-like growth factors (Prod’homme et al. 2004). Amino acid and insulin/IGF signaling pathways share similarities or at least common kinases, such as mTOR and P70S6K, but are mediated by independent signals. Leucine (Kimball and

Jefferson 2006) is involved in activation of specific intracellular pathways involved in protein synthesis, mTOR activation, resulting in S6K1 phosphorylation, which leads to initiation of mRNA translation and protein synthesis.

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Amino acids and feed intake

Mild deficiencies of Lys and threonine in pigs (Ferguson and Gous 1997), and methionine in layers (Bunchasak, 2009) lead to small increase in feed intake. Excess dietary protein is often monitored through the plasma branched chain amino acid levels such as leucine, which are not metabolized in the liver. High levels of plasma leucine have been demonstrated to activate intracellular receptors (mTOR), which can lead to depressed food intake and enhanced energy expenditure (Black et al. 2009).

Tryptophan has largely been demonstrated to be involved in the regulation of feed intake and behavior, particularly in piglets (Le Floch et al. 2007). Interrelation of tryptophan and

Lys results in 17 % increased feed intake (Primot and Melchior 2008). Also, Jansman et al. (2000) showed increased feed intake and weight gain in response to decreasing protein content and higher tryptophan levels in pre-starter and starter diets. The effect of tryptophan on appetite regulation might be mediated through the control of the central production of the neuromediator: serotonin. Tryptophan being the precursor of serotonin and it has well been demonstrated that a decrease in brain tryptophan leads to decreased serotonin production (Pastuszewsaka et al. 2007). Additionally, tryptophan might have more drastic impact on bird behavior. Corzo et al. (2005) demonstrated an increased nervousness in broiler fed tryptophan deficient diets.

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Amino acids and immunity

Protein deficiency has long been known to impair immune function and increase the susceptibility of animals to disease. A dietary deficiency of protein reduces the availability of most AA in plasma, particularly Lys, TSSAs, Threonine, BCAAs, tryptophan arginine and glutamine (Li et al. 2007). Dietary deficiency of amino acids may depress immune system and lead to stress and ultimately disease (Bouyeh, 2012). Numerous groups have characterized the relationship between amino acid levels in diets and immune system function in term of lymphocyte proliferation and activation, cellular redox status, cytotoxic cell-mediated immunity, serum blocking activity, and hemagglutinin titer (Li et al. 2007;

Wu, 2009,2010). Deficiencies of essential amino acids, particularly during the growing phase, result in poor immune competence. The most important amino acid in relation to immunity is Lys as it is involved in lymphocyte proliferation and synthesis of cytokines (Konashi et al. 2000). Adequate Lys supply leads to enhanced cell-mediated immunity and antibody response to Newcastle Disease (ND) vaccination (Chen et al. 2003). Likewise, Bouyeh

(2013) reported improved immune response in birds against different diseases, especially

ND, with proper Lys supplementation.

After Lys, TSSA are the most important AAs for poultry, especially in corn/soy diets.

TSSA are important for immune responses as supplementation of methionine and cysteine results in T-cell proliferation, IgG secretion, leucocyte migration and increased antibody titre in birds infected with Newcastle Disease virus (Tsiagbe et al. 1987). Recent reviews

(Kidd, 2004; Li et al. 2007) concluded that higher methionine levels are required for optimized leukocyte migration inhibition, and taurine, which is a product of Sulphur containing amino acids, plays an important role in reducing pro inflammatory cytokines and prostaglandin E2 in lymphocytes by acting as an antioxidant. The studies also concluded that methionine is more important with respect to immune responses then

REVIEW OF LITERATURE cysteine, as it takes part in protein synthesis, protects cells from oxidative stress by reducing reactive oxygen species as a precursor of glutathione, takes part in cell division by the synthesis of spermine and spermidine (polyamines), and is involved in methylation reactions of DNA by donating methyl groups. Furthermore, methionine is required for thymus derived T-cell helper function (Grimble, 2006). Through the generation of decarboxylated S-adenosylmethionine, methionine is a donor of the methyl group that participates in the methylation of DNA and proteins, the synthesis of spermidine and spermine, and regulation of gene expression (Wu et al. 2006).

The third most important amino acid for proper growth of chicken is threonine, although it has a little impact on immune system of birds. Threonine is a major part of plasma y- globulin and its dietary supplementation results in increased serum and intestinal mucosal concentrations of IgA and IgG (Li et al. 2007). Similarly, branched chain amino acids (Val,

Ile, Leu), which are abundant in vegetable protein sources, especially in soybean meal, are important for development of immune system in terms of organ development (Kidd, 2004).

Like methionine, BCAA take part in protein synthesis, and, ultimately, immune cell functions

(Calder, 2006). Other amino acids that are relatively less important for growth, but have importance for immune system include arginine and glutamine. Arginine is important for

T lymphocytes development and multiple key biological processes, including proliferation, the expression of the T-cell receptor (TCR) complex and the (CD3zeta) z- chain peptide, the development of immune memory (Ochoa et al. 2001; Bronte et al. 2003), production of macrophages, increased heterophil to lymphocyte ratio, CD8+ cells and lymphoid organ weights (Kidd, 2004).

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Amino Acids and Meat Quality

Muscle growth can be modified by AA supply in the diet. Conde-Aguilera et al. (2010) showed that a deficient supply of methionine and cysteine in piglets affected the AA composition of body proteins. Among all tissues, the longissimus dorsi muscle responded most to methionine and cysteine deficiencies, with important reductions in weight, protein and methionine content of the muscle, but increased lipid contents. High and low rearing densities significantly alter the drip loss of the meat during storage. This effect was correlated with a higher ultimate pH value obtained with the high dietary Lys compared to low dietary supply (Berri et al. 2008). Free glutamate contributes to meat taste and is an important taste-active component of meat. It was found that among chicken, pork, and beef, chicken meat has more free glutamate then others (Kato et al. 1989). Thus, it is clear that feed plays a vital role in meat taste. Hence, it is conceivable that increasing free glutamate content in muscles can improve meat quality. It is further very much clear that free glutamate content in chicken meat can be regulated by dietary CP and BCAA. Dietary high CP level increases the free glutamate of meat, and a decrease in dietary Leucine induces an increase in free glutamate content and improved meat taste. Increases in Lys over and above NRC (1994) recommendations were reported to improve weight gain, feed efficiency and breast meat yield and reduced fat deposition in chicken carcass (Si et al.

2004).

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CHAPTER 3 EXPERIMENT-I

Title: Effect of Dietary Lysine Regimens on Growth Performance, Body Conformation,

Immune Responses and Meat Composition in Different Varieties of Aseel Chicken

3.1. Effect of dietary amino acid regimens on growth performance, body conformation

and immune responses in Aseel chicken (Published in Indian Journal of Animal

Research, DOI: 10.18805/ijar.B-767)

ABSTRACT

The present study was conducted to evaluate the effect of different levels of Lysine (Lys) on early growth, body conformation and immune response of three varieties of Aseel chicken.

Day-old chicks (n = 180) from each of the three Aseel varieties Mianwali [MW], Peshawari

[PW], and Lakha [LK], hence, a total of 540 chicks) were fed diets for four weeks containing three levels of Lys: 1.35% (L1); 1.3% (L2); and 1.25% (L3). Randomized Complete Block

Design (RCBD) under factorial arrangements with blocking on the basis of the sex of the chicks was applied. Each treatment was replicated six times with 10 chicks per replicate. At weekly intervals, feed intake (FI), weight gain (WG) and feed: gain ratio (F:G) were calculated.

Significantly higher (P<0.05; 610.8±6.0) FI was observed in PW variety followed by MW

(604.4±7.3) and LK (600.4±7.5). With respect to dietary treatments, lower FI (P<0.05;

578.1±2.2) was observed in the birds fed with L1 diets across all varieties. However, four-week body weight gain (P>0.05; 230.4±7.1, 230.1±4.6 and 240.6±7.3) did not show any significant variation and remained comparable in all varieties. Higher (P<0.05) BWG in birds fed L1 and

L2 diets (244.3±4.2 and 241.8±8.2) in comparison to L3 diets (214.9±3.6) were observed.

Interaction between varieties and AA densities displayed improved (P<0.05) BWG in MW with L1 diet (252.0±17.4), followed by MW with L2 (251.4±7.15) and LK with L1

(239.2±15.5) diets. The varieties showed non-significantly differences (P>0.05) in F:G.

EXPERIMENT-I

However, dietary treatment resulted in significant variations (P<0.05) for F:G, where improved

(P<0.05) F:G was observed as the diet density increased (2.99±0.05, 2.46±0.05, 2.44±0.08).

Birds fed high amino acid density diets depicted improved body conformation traits (length of body, drum stick, shank keel bone, circumference of drumstick and shank, wing spread and breast width), whereas immune response was not influenced by the dietary treatments. In conclusion, rearing the MW variety under 1.30% digestible Lys level with ideal amino acid ratio can be a good strategy to achieve higher early BWG and improved F:G in Aseel chicken.

Keywords: Amino Acids, Aseel Chicken, Body Conformation, Immune Responses

INTRODUCTION

Backyard poultry is an important sub-sector of poultry production in developing countries.

Indigenous breeds of poultry have disease resistance and adaptability to harsh environmental

(Khan et al. 2017). Moreover, local breeds have additional attraction to local folks as a meat source (Barua and Howlider 1990). Aseel is a prominent indigenous breed of the Indo-Pak subcontinent having higher final body weight compared to other local breeds (4 to 7 kg) (Babar et al. 2012; Usman et al. 2014) and superior quality of meat in term of taste and texture (Dohner,

2001). Problems related with Aseel birds are poor early growth and feed to gain ratio (F:G), higher cost of production and late maturity (Jatoi et al.2014).

Aseel can be used as a meat-type bird and its growth potential can be enhanced through improved nutrition using modern nutritional tools. Zia et al. (2017a, b) reported that organic selenium supplement improved the growth performance of Aseel birds. Similarly, increasing the level of energy and protein enhances the F:G of Aseel (Haunshi et al. 2012). Baker and Han

(1994) found that specific concentrations of individual dietary amino acids, termed as ideal amino acid ratio, are necessary for proper growth performance. Increasing the amount of amino acids (AA) improves growth and breast meat yield due to increases in protein synthesis (Dozier

EXPERIMENT-I et al. 2008). Lysine (Lys) is among the critical AAs contributing in increasing breast yield, and it is estimated that breast meat contains 7% Lys (Dozier et al.2007; Mahdav et al. 2012).

Therefore, increasing the level of Lys in the diet could increase breast yield and ultimately growth performance of the Aseel.

During last few years, ideal protein or ideal amino acid (IAA) and tissue accretion methods have been used to measure retention efficiency of AA in poultry (Tillman and Dozier 2013).

These methods can be used to improve protein efficiency and decrease N excretion. Numerous studies conducted on total and digestible AA of different ingredients revealed enhanced growth performance in birds with digestible AA (Lemme et al. 2004). With this background, the present study was executed to investigate the effect of including different levels of digestible amino acids in the diets formulated based on ideal amino acid concept on early growth performance, body conformation and immune response among three varieties of indigenous

Aseel chickens of Pakistan.

MATERIALS AND METHODS

The study was conducted at the Indigenous Chicken Genetic Resource Center (ICGRC),

Department of Poultry Production, University of Veterinary and Animal Sciences, Lahore-

Pakistan. Day-old chicks (n = 180) from each of the three Aseel varieties (Mianwali [MW],

Peshawari [PW], and Lakha [LK]; 540 chicks in total) were fed diets for four weeks containing three levels of Lys: 1.35% (L1); 1.3% (L2); and 1.25% (L3). Randomized Complete Block

Design (RCBD) with factorial arrangements was adopted with blocking on the bases of gender.

The experiment was replicated six times with 10 chicks (avg. wt.: 29 g) per treatment.

Three different diets for every two weeks phase with variable Lys levels were formulated. The experimental diets were formulated by using local grain data on the basis of ideal digestible amino acid ratios by Tillman and Dozier (2013). The control dietary treatment was formulated

EXPERIMENT-I in such a way that the allowance of 1.25% Lys was maintained throughout the experimental period (0 to 28 days). All the diets were iso-nitrogenous and iso-caloric (Table. 3.1.1). Before the start of trial, experimental diets were analyzed for dry matter (DM), crude protein (CP), crude fiber, fat, ash, calcium and phosphorus by following AOAC (2005). Amino acid analysis was executed using Biochrome 30+ Series amino acid analyzer resulting in ±2% from the calculated values and metabolizable energy was calculated through regression equation as described by NRC (1994). Linear formulation method was used for diets formulation.

Experimental diets were offered for the duration of 4week.

Animal Husbandry

All housing and equipment was cleaned prior to introducing birds. The equipment was rinsed with potassium permagnate and sun dried. Fumigation (35ml formalin + 17.5g potassium permagnate = 1X concentration) of house was completed one week prior to the chicks’ arrival.

Birds were kept in a multi deck cage house with 0.2-0.4 ft2of floor space allotted to each bird.

Management conditions like temperature, relative humidity and lighting schedule were maintained according to intensive broiler rearing.

Parameters Studied

The data were collected (weekly and pen-wise) to evaluate growth performance, immune response and body conformation at four weeks of age. The following parameters were studied;

Body weight gain (BWG) (g): Final weight – Day Old Chick Weight

Feed intake (FI) (g): Feed offered – Feed refusal

Feed to gain ratio (F:G): Feed Intake / Wight Gain

Protein efficiency ratio (PER): Weight Gain / Protein Intake

Lysine efficiency ratio (LER): Weight gain / Lysine Intake

EXPERIMENT-I

At the end of the experiment, blood samples were collected and serum was separated. The titres of Newcastle Disease (ND) using hemagglutination inhibition assay, Infectious Bronchitis (IB) and Infectious Bursal Disease (IBD) using ELISA test were performed according to the methods described by Chang el al. (2011). Body, drumstick, shank and keel bone length was measured with the help of measuring scale capable of measuring 0.05 mm. Drumstick, shank circumference and breast width were measured with the help of Vernier calliper capable of measuring up to 0.05 mm.

Statistical analysis

Before analysis, the data were checked for uniformity and homogeneity of variance and verified for the normality. Subsequently the data were analyzed in SAS 9.1 using two-way

ANOVA technique through GLM procedure assuming following mathematical model:

Yijk = μ + βi + Vj + Lk + (VL)jk + Ɛijk

Where, Yijk is dependent variables, µ is overall population mean, βi are blocks (sex), Vj is effect

th th of i treatment (i=3; varieties), Lk is effect of j treatment (j=3; Lys regimens), (VL)ij is interaction effect and Ԑijk is residual error. Aseel variety and Lys regimens were main effects.

Interactions were also tested. Treatment means were compared using Tukey’s HSD test at 5% probability level.

RESULTS AND DISCUSSION

Table 2 shows the growth performance of three Aseel varieties reared under different Lys regimens. The higher (P<0.05; 610.8±6.0) feed intake (FI) was observed in PW variety followed by MW (604.4±7.3) and LK (600.4±7.5) (Table 3.1.2). With respect to dietary treatments, lower (P<0.05; 578.1±2.2) FI was observed with L1 diet across all varieties. In interaction of varieties and different density diets, minimum FI (P<0.05; 569.8±4.4) was observed in LK variety fed L1 diet, whereas higher FI (640.7±3.6, 640.0±3.4 and 639.0±3.9)

EXPERIMENT-I was found in L3 diets fed birds of all the varieties(PW, MW and LK respectively). Among varieties higher FI in Peshawari variety could be attributed to the genetic variations among varieties. It was previously reported that birds of Peshawari variety had higher FI as compared to Lakha and Mianwali (Jatoi et al. 2014). Likewise, Corzo et al. (2005) reported that high density diets in birds reduced FI presumably due to satiety and gut fill. In the current study, decrease in FI might be attributed to satiated effect of high density diet on intake of birds. Similar finding of lower FI due to high density diets has also been reported in broilers

(Sklan and Plavnik 2002). Other possible reason of decreased feed intake might be branched chain AA resulting in decreased FI (Trottier and Easter 1995).

Body weight gain (P>0.05; 230.4±7.1, 230.1±4.6 and 240.6±7.3) was similar (P>0.05) in all varieties. Dietary treatments resulted in a higher (P<0.05) BWG in birds fed L1 and L2 diets

(244.3±4.2 and 241.8±8.2) in comparison to L3 Lys diets (214.9±3.6). Interaction between varieties and AA densities displayed improved (P<0.05) BWG in MW with L1 (252.0±17.4), followed by MW with L2 (251.4±7.15), LK with L2 (244.7±8.03) and L1 (239.2±15.5) diets.

Higher BWG (240.6±7.3) in MW Aseel reared under high density diets could be due to increased protein synthesis, which involves the role of dietary amino acids (Dozier et al.

2008). Interactions of amino acids plays a vital role in the protein synthesis like Lys, methionine and arginine interaction involves in the synthesis of creatine that improves the muscle growth (Chamruspollert et al. 2002). Similar to our findings, Ngambi et al. (2017) also reported that diets in higher density with threonine improved performance of slow- growing Venda chickens.

The varieties showed similar (P>0.05) F:G. The dietary treatment resulted in significant variation in (P<0.05) F:G, where linearly improved (P<0.05) F:G was observed by increasing diet densities from lower to higher side (2.99±0.05, 2.46±0.05, 2.44±0.08). Dietary treatments exhibited improved (P<0.05) F:G in all varieties under L1 and L2 diet and MW variety

EXPERIMENT-I

(2.35±0.06 and 2.37±0.17) in particular. Improved F:G of Mianwali Aseel might be attributed to the lower FI and higher weight gain, since birds’ performance improved with increasing AA densities (Zhai et al. 2013, 2014). Similar findings have also been reported in broilers fed high density AA supplemented diets (Quentin et al. 2003). Furthermore, Li et al. (2013) reported that increasing Lys level enhance F:G in local Chinese chicken. Phase wise WG and F:G also showed significant differences (P<0.05). Where for 0-14 day WG for all dietary treatments were 87.5±3.5, 87.5±2.3 and 64.9±4.2 and F:G were 2.04±0.04, 2.14±0.03 and 2.84±0.06 for

L1, L2 and L3 respectively. During 15-28 days, WG were 154.3±6.4, 156.8±4.9 and 150±4.8 and F:G was 2.67±0.09, 2.64±0.04, and 3.08±0.07 for L1, L2 and L3 Lys regimens, respectively (Figure-1).

The dietary treatment resulted in improved (P<0.05) protein efficiency ratio (PER), where linearly improved (P<0.05) PER was observed by increasing diet densities from low to higher side. Dietary treatments demonstrated improved (P<0.05) PER in all the varieties fed on high and medium density diets. Improved PER of Mianwali Aseel might be attributed to the lower

FI and higher weight gain, since birds perform well with increasing AA densities (Zhai et al.

2013, 2014). Similar findings have also been reported in broilers fed AA supplemented diets

(Quentin et al. 2003). Whereas varieties showed no differences (P>0.05) in PER. Lower

(P<0.05) Lysine efficiency ratio (LER) was observed in birds fed high density diets.

Dietary treatments had no significant effect (P>0.05) on immune status (Table 3.1.3). Higher growth rate in MW chicken did not compromise immune response, which might be attributed to the result of amino acids relation with immune system efficiency in terms of lymphocyte proliferation and activation (Tsiagbe et al. 1987), improved cellular redox status, cytotoxic cell-mediated immunity, serum blocking activity, and haemagglutinin titer (Konashi et al.

2000). Improved immune response against different diseases due to Lys and methionine supplementation especially against ND has already been highlighted (Bouyeh, 2012).

EXPERIMENT-I

Recently a study on Aseel chicken concluded that nutritional manipulation can increase glutathione peroxidase activity and ultimately can result in superior performance with improve blood biochemical profile (Zia et al. 2017d)

Dietary treatments had a significant effect (P<0.05) on all body conformation traits except keel length and shank circumference. Overall L2 diets showed higher (P<0.05) body conformation traits followed by L1 than L3 (Table 3.1.4). In an earlier study, length of feathers decreased in response to decreased dietary protein intake (Wylie and Hocking 1999). This effect on body conformation traits is expected to be due to differential nutrient partitioning from different nutrient densities, which possibly had resulted in improved body frame development (Sikur et al. 2004).

CONCLUSION

Based on the findings of the present study it can be concluded that diet containing 1.30 % digestible Lys level with ideal amino acid ratios, may improve early weight gain, F:G and body conformation in Mianwali Aseel chicken.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the support of anonymous readers and administration of

SB Feeds Rawalpindi, Sindh Feeds Karachi and ICGRC, Department of Poultry Production,

University of Veterinary and Animal Sciences, Lahore.

Conflict of interest: The authors declare that they have no conflict of interest.

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growth phases. Ind JAnimal Res. DOI:10.18805/ijar.v0iOF.6983

Zia MW, Khalique A, Naveed S, Hussain J. 2017b. Studies on growth pattern of different body

measurements in indigenous Aseel chicken fed with selenium supplemented diets. Ind

JAnimal Res. 51(4): 679-686.

Zia MW, Khalique A, Naveed S, Hussain J, Nadeem M, Ahmad S. 2017c. Comparative

evaluation of influence of dietary organic and inorganic selenium supplement on

growth performance of indigenous Aseel chickens. Ind JAnimal Res. 51(3):478-488

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Zia MW, Khalique A, Naveed S, Hussain J, Imran M. 2017d. Effect of selenium

supplementation on glutathione peroxidase (GPX), cholesterol, thyroxin (T4) and other

blood biochemicals in local Aseel. Ind JAnimal Res. DOI:10.18805/ijar.10765.

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Table 3.1.1. Feed formulation and nutrients calculations Ingredients Treatments 1 (%) 1 to 14 days 15 to 28 days L1 L2 L3 L1 L2 L3 Corn 61.97 62.44 62.66 65.28 64.03 65.78 Soybean Meal 23.05 19.94 21.97 20.93 23.72 20.20 Fish meal 8.00 11.13 9.04 6.06 5.46 6.09 Corn Gluten 3.00 3.00 3.00 3.00 2.00 3.00 60% CaCO3 0.97 0.97 0.99 0.95 0.80 0.95 DCP 0.20 0.20 - - 0.10 - Common Salt 0.30 0.30 0.30 0.30 0.30 0.30 NaHCO3 0.20 0.34 0.20 0.24 0.26 0.24 Vegetable Oil 0.20 - - 1.43 1.94 1.26 Vit-Min 0.50 0.50 0.50 0.50 0.50 0.50 Premix Lysine 0.61 0.46 0.40 0.54 0.37 0.67 Sulphate DL-Methionine 0.35 0.29 0.26 0.30 0.26 0.34 L-Threonine 0.25 0.21 0.18 0.19 0.14 0.23 L-Valine 0.09 0.03 0.38 0.06 0.01 0.11 L-Isoleucine 0.11 0.04 0.01 0.06 0.00 0.10 L-Tryptophan - - - 0.00 - 0.01 L-Arginine 0.19 0.16 0.10 0.16 0.11 0.23 Nutrients CP (%) 23 23 23 21 21 21 ME (Kcal/kg) 3000 3000 3000 3100 3100 3100 Calcium 1.00 1.00 1.00 0.80 0.80 0.80 Available 0.40 0.40 0.40 0.30 0.30 0.30 Phosphorus Digestible 1.35 1.30 1.25 1.20 1.15 1.25 Lysine Digestible 1.02 0.98 0.94 0.92 0.88 0.95 TSAA Digestible 0.95 0.91 0.88 0.83 0.79 0.86 Threonine Digestible 0.22 0.21 0.20 0.20 0.19 0.20 Tryptophan Digestible 1.42 1.37 1.32 1.28 1.22 1.33 Arginine Digestible 1.02 0.98 0.94 0.92 0.88 0.95 Valine Digestible 0.89 0.86 0.83 0.81 0.77 0.84 Isoleucine Digestible 0.85 0.82 0.79 0.76 0.72 0.79 Phenylalanine 1L1=high d-lysine L2=Medium d-lysine L3= control diet

Table 3.1.2. Growth performance of indigenous chicken in response to dietary lysine regimens

Variables1 Parameters2 FI BW F:G LER PER Varieties LK 600.4±7.5b 230.4±7.1 2.66±0.10 28.61±1.2 1.57±0.06 MW 604.4±7.3b 240.6±7.3 2.56±0.09 29.59±1.3 1.63±0.07 PW 610.8±6.0a 230.1±4.6 2.68±0.07 27.59±1.2 1.53±0.04 AA Densities L1 578.1±2.2c 241.8±8.2a 2.44±0.08b 22.94±0.9b 1.71±0.06a L2 597.6±2.9b 244.3±4.2a 2.46±0.05b 31.96±0.6a 1.67±0.04a L3 639.9±2.0a 214.9±3.6b 2.99±0.05a 30.88±0.6a 1.35±0.03b Varieties × AA Densities LK L1 569.8±4.4e 239.2±15.5abcd 2.43±0.16b 23.01±1.5b 1.71±0.12a L2 590.5±1.8c 244.7±8.03abc 2.43±0.08b 32.61±1.0a 1.71±0.06a L3 640.7±3.6a 207.4±6.42d 3.11±0.10a 30.20±1.3a 1.30±0.05c MW L1 583.7±2.6cd 252.0±17.4a 2.37±0.17b 23.86±1.93b 1.77±0.14a L2 589.4±2.5cd 251.4±7.15ab 2.35±0.06b 33.57±0.61a 1.75±0.05a L3 640.0±3.4a 218.4±6.49cd 2.95±0.09a 31.34±1.15a 1.38±0.05bc PW L1 580.7±1.0d 234.4±9.95abcd 2.50±0.12b 21.95±1.06b 1.64±0.08a L2 612.8±2.3b 236.7±6.44abcd 2.60±0.08b 29.72±1.08a 1.57±0.05ab L3 639.0±3.9a 219.2±5.73bcd 2.92±0.07a 31.11±0.96a 1.38±0.04bc

Linear and Quadratic Responses Due to Dietary Treatments

EXPER L * NS NS NS NS Q NS * * * * 1 2 LK=Lakha, MW=Mianwali, PW=Peshawari, L1=high d-lysine, L2=Medium d-lysine, L3= control diet FI=feed intake, BW=body I

MENT weight, F:G= Feed to gain ratio, PER= protein efficiency ratio, LER= lysine efficiency ratio. Value with different superscript within column differ significantly (P≤0.05)

Table 3.1.3. Effect of dietary amino acid densities on immune responses of birds

Treatments1 Parameters SE L1 L2 L3

NDV 4.67 4.67 5.0 0.69

IB 4491.7 4459.1 4790 690.7

IBD 1409.5 1339.7 1420.9 143.8

SP N/D2 N/D N/D -

MG N/D N/D N/D -

1L1=high d-lysine L2=Medium d-lysine L3= control diet 2 Not Detected

Value with different superscript within row differ significantly (P≤0.05) EXPER

MENT

Table 3.1.4. Effect of dietary amino acid densities on body conformation traits of birds

Traits2

Variables1

BL DSL DSC SL SC WS WSPN KBL BW

ab b a a ab a b ab a L1 12.8±0.13 2.52±0.06 2.21±0.09 1.83±0.05 1.00±0.03 7.58±0.08 15.9±0.15 2.09±0.03 24.4±0.53

a a a a a a a a a L2 13.08±0.17 2.66±0.06 2.09±0.06 1.91±0.03 1.04±0.03 7.61±0.10 16.3±0.10 2.15±0.03 24.9±0.42

b b b b b b b b b L3 12.5±0.07 2.52±0.01 1.84±0.05 1.67±0.03 0.94±0.02 7.22±0.05 15.7±0.16 2.03±0.06 21.9±0.49

1L1=high d-lysine L2=Medium d-lysine L3= control diet 2 BL=body length, DSL=drum stick length, DSC=drum stick circumference, SL=shank length, SC=shank circumference, WS=wing spread, WSPN=wing span, KBL=keel bone length and BW=breast width

Value with different superscript within column differ significantly (P≤0.05) EXPER

MENT

Figure 3.1.1. Phase wise weight gain and Feed to gain ratio in varying lysine diets

300 3.5 2.99 3.08 241.8 244.3 3 2.84 250 2.67 2.64 214.9 2.44 2.46 2.5 2.14 200 2.04 154.3 156.8 150 2 150 FCR 1.5 100 87.5 87.5 Weight Weight Gain (g) 64.9 1

50 0.5

0 0 L1 L2 L3 L1 L2 L3 Dietery Treatments Dietery Treatments 0-14 0-28 15-28 0-14 0-28 15-28 Weeks Weeks

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3.2. Effect of Dietary Lysine Regimens on Growth Performance and Meat Composition in

Aseel Chicken (Accepted for Publication in Brazilian Journal of Poultry Science ID: RBCA-

2017-0584; Vol. 2, 2018)

Abstract

The present study was conducted with objective to evaluate effect of dietary lysine (Lys) regimens on growth performance and meat composition of Aseel chicken. Day-old chicks (n = 180) from each of the three Aseel varieties (Mianwali [MW], Peshawari [PW], and Lakha [LK]; hence 540 chicks in total) were fed diets containing three different levels of Lys: 1.35% (L1); 1.3% (L2); and

1.25% (L3). Randomized Complete Block Design (RCBD) with factorial arrangements was adopted with blocking on the bases of gender. The experiment was replicated six times with 10 chicks (avg. wt.: 29 g) per treatment. At weekly intervals, feed intake (FI), weight gain (WG) and feed: gain ratio (F:G) were measured. At 42nd day of age, three chicks per replicate were euthanized and carcasses evaluated for dry matter (DM), ash, crude protein (CP) and fat contents.

Results showed higher WG (P=0.0002; 424.1±8.1) and improved F:G (P=0.0006; 2.84±0.05) in

MW variety compared to PW (WG:411.5±6.3; F:G=2.95±0.05) and LK (WG: 401.5±9.3;

F:G=3.02±0.08). Among different (P<.0001) Lys regimens, L1 and L2 resulted in enhanced WG

(423.3±8.2; 428.2±4.9), F:G (2.79±0.05; 2.80±0.03) and reduced FI (1175.8±3.7; 1198.0±5.4).

Reduced FI was observed in higher and medium Lys regimens across all varieties. Similarly, increased (P<.0001; 0.0150) ash content in thigh and breast with increased level of Lys in the early life period (0-6 weeks) was also observed. Increased dry matter (P=0.0036; 73.80±0.17), lower ash contents (P<.0001; 1.23±0.03) and lower crude protein (P=0.0064; 21.97±0.17) contents was observed in thigh of the birds fed diets with medium Lys levels, whereas significantly higher

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(P=0.0150; 1.30±0.04) ash content was found in breast with low Lys diet. It may be concluded that, 1.30% digestible Lys level regimen may improve early growth rate of Aseel chicken.

Keywords: Amino Acid, Aseel Chicken, Growth Performance, Meat Composition

Introduction

Aseel is the oldest Asian game fowl having superior meat quality in term of taste and texture compared to other breeds (Dohner, 2001). The pugnacity, upright posture, firm muscular thighs with strong legs (Usman et al. 2014) and heavy body weight (Jatoi et al. 2014) are well-known characteristics of the Aseel. With a large body size, Aseel mature body weight (BW) ranges from three to five kg. These BW characteristics may be due to the incorporation of Cornish breeds, the parent of modern day broilers, in early breeding of Aseel (Usman et al. 2014). On the basis of its body weight, sturdiness, disease resistance, adaptability, survival in harsh environment and superior quality meat, Aseel can be a good option as a meat-type chicken (Khan et al. 2016). The main hurdle in the use of Aseel as a meat-type bird, however, is its poor early growth rate and feed conversion. Different techniques, however, including genetic selection, management and nutritional manipulations can be applied to improve the early poor growth of Aseel chicken (Khan et al. 2017).

The concept of providing individual amino acids for proper growth is not new. Certain levels of individual amino acids are necessary for proper growth performance across species (Baker and

Han 1994; Ayasan and Okan 2014). Lysine, a basic building block for protein synthesis, is involved both directly and indirectly in the regulation of the protein synthesis (Dozier et al. 2007). In chickens, Lys accounts for 7% of the protein in breast meat (Eits et al. 2003). Dietary Lys is important for maintenance and skeletal muscle accretion (Dozier et al. 2007; Ayasan and Okan

2010). Supplementation of Lys during early growth stages (Ayasan and Okan 2006; Campestrini et

EXPERIMENT-I al. 2010) is beneficial and increases muscle growth (Eits et al. 2003) and improves weight gain and feed efficiency (Quentin et al. 2003; Si et al. 2004). Chickens grow faster with relatively high dietary Lys concentrations in the diet compared with diet deficient in Lys (Li et al. 2013).

Deficiency of Lys in the diet results in decreased protein synthesis and accretion (Liao et al.

2015). As such, it was hypothesized that balanced diets supplemented with Lys may ameliorate the early poor growth rate of indigenous Aseel chicken. The present study was, therefore, planned to evaluate the effect of different Lys regimens on growth performance and meat composition in different varieties of indigenous Aseel chicken.

MATERIALS AND METHODS

Ethical note

Experimental procedures were in accordance with the guidelines and code of practice of University of Veterinary and Animal Sciences Lahore. Ethical approval was obtained before the conduct of the study (Protocol Number UVAS-DAS-4901).

Housing, Experimental Birds and Diets

The study was conducted at Indigenous Chicken Genetic Resource Center, Ravi Campus,

University of Veterinary and Animal Sciences, Lahore. In total, 540 day-old chicks, 180 from each variety (90 male and 90 female), were randomly assigned to 9 experimental groups in a 3

(Varieties: Mianwali (MW), Peshawari (PW), and Lakha (LK)) × 3 (Lys regimens: L1, L2 and

L3:1.35, 1.30 and 1.25%) factorial arrangement under randomized complete block design (RCBD) with sex as block. Each experimental group was replicated 6 times (three for male and female each) times with 10 birds in each. The experiment was conducted in multi deck cage house with stocking density of 0.2-0.4 ft2 /bird. House preparation for the experiment was done by cleaning

EXPERIMENT-I and washing of equipment’s rinsing with potassium permagnate and sun drying. Fumigation (35ml formalin + 17.5g potassium permagnate = 1X concentration) of house was completed before one week of chicks’ arrival. Management conditions like temperature, relative humidity and lighting schedule were maintained according to intensive broiler rearing. Water and feed were provided ad-libitum through trough feeders and nipple drinking system.

Three diets with variable Lys levels were formulated for every two-week phase. All the diets were iso-nitrogenous and iso-caloric (Table. 3.2.1). Before the start of trial, chemical analysis for crude protein (CP), dry matter (DM), fat, crude fiber CF), ash, calcium and phosphorus of experimental diets were performed following AOAC (2005) while, amino acid analysis was executed using

Biochrome 30+ Series amino acid analyzer resulting in ±2% from the calculated values and metabolizable energy was calculated through regression equation as described by NRC (1994).

Linear formulation method was used for diets formulation. Experimental diets were offered for the duration of 6 weeks.

Parameters

Cumulative feed intake was determined by combining each week feed intake (FI). Initial and final body weights (FBW) were recorded to calculate the WG, feed:gain ratio (F:G) and Lys efficiency ratio (LER). In total, 162 birds, three birds with average weights from individual replicates were picked up and kept off feed for six hours. Afterwards, birds were euthanized to collect meat samples from the thigh and breast portion of the carcass. Proximate analysis (DM, Ash, CP and

EE) of the meat samples was performed using standard methods, DM was determined through hot air oven at 80 Cº for 48 hours (Haunshi et al. 2012), CP by Kjeldahl method (AOAC 1999). Fat

EXPERIMENT-I was determined by the Soxhlet apparatus method (AOAC 1999) and ash was determined by complete burning of sample using a furnace with a heating temperature of 600°C (AOAC 1999).

Statistical Analysis

Before analysis, the data were checked for uniformity and homogeneity of variance and verified for the normality. Subsequently, the data were analyzed through ANOVA technique in factorial arrangement under RCBD by using GLM procedure of SAS assuming following mathematical model:

Yijk = μ + βi + Vj + Lk + (VL)jk + Ɛijk

Where, Yijk is Dependent variables, µ is overall population mean, βi are the blocks that is sex, Vj

th th is effect of i treatment (i=3; Varieties), Lk is effect of j treatment (j=3; Lys Regimens), (VL)ij is interaction effect and Ԑijk is residual error. Aseel variety and Lys regimens were taken as main effects. Interaction of these was also tested. The treatment means were compared through Tukey’s

HSD test at 5% probability level. Each replicate was considered as an experimental unit.

Results and Discussions

Feed Intake, Weight Gain and Feed:Gain Ratio

Results showed improved WG (P=0.0002; 424.1±8.1) and F:G (P=0.0006; 2.84±0.05) in MW variety compared to PW (WG:411.5±6.3; F:G=2.95±0.05) and LK (WG: 401.5±9.3;

F:G=3.02±0.08). Among different (P<.0001) Lys regimens, higher and medium levels in the diet resulted in enhanced WG (423.3±8.2; 428.2±4.9), F:G (2.79±0.05; 2.80±0.03) and reduced FI

(1175.8±3.7; 1198.0±5.4. Among different Lys regimens linearly increasing (P = 0.0001) feed intake was observed from higher to lower Lys density diets (1175.8±3.7, 1198.0±5.4, 1232.9±4.5)

EXPERIMENT-I and the interaction with different Aseel varieties (P= 0.0496) showed variations in feed intake

(highest in low Lys diets across all varieties and low with high and medium Lys across all varieties), whereas, Aseel varieties independently (P= 0.1005) did not differ in feed intake (Table

3.2.2). Reduced FI in higher and medium Lys levels may be attributed to the fact that high amino acid density diets in less quantity might have fulfilled nutrients requirement of the birds and made them satiated. Similar results were reported in other studies also, where lower FI due to high amino acid density diets were observed in broilers (Sklan and Plavnik 2002; Corzo et al. 2005). Likewise, supplementation of branched chain amino acids (valine, leucine and isoleucine) resulted in decreased FI in animals (Trottier and Easter 1995).

High and medium level of Lys demonstrated higher weight gain compared to low level. Among different varieties, (P= 0.0006) birds of MW variety showed enhanced weight gain followed by

PW and LK. Feed to gain ratio showed marked variations (P<.0001) with respect to Lys regimens,

Aseel varieties (P=0.0006), and their interaction (P= 0.0026). Higher and medium Lys regimens resulted in improved F:G than low level of Lys. Birds of MW variety exhibited lower F:G than those of LK and PW. Lysine efficiency showed similar trend to that of F:G in dietary treatments

(P<.0001) and varieties (P=0.0007). Interaction of dietary treatments and varieties showed differences (P=0.0496, 0.0034, 0.0026 and 0.009) in FI, WG, F:G and LER, where higher FI was found in birds fed low Lys diets. Weight gain was higher in medium Lys diet fed birds of MW variety, F:G and LER were lower in the all varieties fed high and medium Lys diet.

Higher and medium level Lys regimens in the diets of Aseel chicken improved WG and F:G. It is quite possible that supplementation of higher levels of Lys might have promoted muscle accretion in early two weeks of growth, resulting in increased final weight gain. It is reported that supplementation of Lys during early growth stages (Campestrini et al. 2010) is beneficial. It

EXPERIMENT-I involves the regulation of the protein synthesis and increases muscle accretion (Dozier et al. 2007;

Eits et al. 2003). Hence, chickens grow faster at a relatively high dietary Lys concentration in the diets compared with diet having less Lys (Li et al. 2013). Moreover, increased weight gain in higher and medium Lys diets may also be attributed to its distinct characteristic of increasing villous height in jejunum and ileum with increasing Lys levels (Vaezi et al. 2011; Wang et al.

2009) and increasing crypt depth in duodenum and jejunum

(Franco et al. 2006), ultimately accelerating early growth through improved nutrient digestion and uptake.

Similar to these findings, increment in Lys over and above NRC (1994) recommendations was reported to improve WG and F:G (Si et al. 2004).It was reported that meat-type birds performed well with increasing amino acids densities (Zhai et al. 2013, 2014). Panda et al. (2011) fed broilers

1.3% Lys with a specific ratio to other amino acids and observed improved results, indicating that concentrated diets can improve F:G in broiler (Quentin et al. 2003). Moreover, improved F:G in high and medium Lys groups may be attributed to the higher WG and lower FI in the same group.

On the other hand, decreased WG in low Lys density diets may be explained by the fact that inadequate Lys supply depresses immune system of the birds (Geraert and Mercier2010) showing adverse effects on livability and growth performance (Liao et al. 2015). Similarly, it was reported that depressed growth in Lys deficient diets may be due to decreased protein synthesis and accretion (Tesseraud et al. 1996).

The observed difference in growth performance between varieties may be the result of genetic diversity between the breeds and varieties (Leeson et al. 1997). Similarly, differences in WG and

F:G were observed in different varieties of Aseel, where MW variety showed higher WG and F:G

(Jatoi et al. 2014).

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Mortality percentage also differed significantly among different (P= 0.028) dietary treatments, where, higher mortality was found in groups fed low Lys diets (Table. 2). Higher and medium Lys diets showed reduced mortality. Higher mortality in low Lys diet may be attributed to the fact that inadequate Lys supply may depress immune system of the birds (Geraert and Mercier 2010), as deficiency of dietary Lys leads to increased susceptibility of birds to diseases (Datta et al. 2001;

Li et al. 2007). The reason behind is because Lys is an integral component for protein synthesis, any deficiency of Lys can limit the immune related protein synthesis including antibodies and cytokines. Chen et al. (2003) reported that deficiency of Lys negatively affected antibody response and cell mediated immunity, also showing adverse effects on growth performance (Liao et al.

2015). Similar to the finding of the current study, Mushtaq et al. (2015) fed higher Lys level to the birds and observed reduced mortality, concluding that higher Lys level in the diets improved livability.

Meat Composition

Increased dry matter (P=0.0036; 73.80±0.17), lower ash contents (P<.0001; 1.23±0.03) and lower crude protein (P=0.0064; 21.97±0.17) contents was observed in thigh of the birds fed diets with medium Lys levels, whereas significantly higher (P=0.0150; 1.30±0.04) ash content was found in breast with low Lys diet. Breast muscle ash % was lower in MW and PW variety (P<.0001;

1.30±0.03, 1.31±0.05). Whereas, DM content in breast and thigh remained unchanged (P= 0.1728) among all varieties. Among different varieties, birds of PW variety indicated higher (P= 0.0002;

1.37±0.04) ash content in thigh, whereas ash content of breast was found to be higher (P<.0001;

1.47±0.03) in LK variety of Aseel. Birds fed with lower Lys indicated higher (P= 0.0064;

22.57±0.17) CP content in thigh, whereas, CP remained unchanged (P= 0.7139) in breast.

Moreover, Aseel varieties remained unresponsive (P= 0.7447; 0.3339) regarding CP in both thigh

EXPERIMENT-I and breast. All treatments separately and in interaction did not show any pronounced effect on EE in both thigh and breast meat samples (Table. 3.2.3). The findings of the current study confirm that different dietary Lys regimens significantly affected meat composition (DM, Ash, CP and EE) of indigenous Aseel chicken that might be due to incremental levels of amino acids modifying the muscle growth by increasing myofiber size (Tesseraud et al. 1996). Lower Lys level during early growth stages in the present study did not show any adverse effect on proximate meat composition.

However, contrary to the finding of the present study, it was observed that amino acid deficiency can lead to protein decrease and crude fat increase in meat (Corzo et al. 2005; Lilly et al. 2011).

Among different varieties, ash content in thigh was found to be higher in birds of PW variety, whereas, in breast it was found to be higher in LK variety of Aseel. However, DM, CP and EE contents in both thigh and breast remained unchanged among all the varieties. Higher ash content in PW and LK might be due to their genetic effect. Similarly, differences in ash content among different genotypes of poultry have already been reported as Wattanachant et al. (2004) observed that Thai Indigenous chicken meat contained lower percentages of ash contents than those of commercial broiler, indicating that ash content varies with genotype (Wattanachant 2008; Sogunle et al. 2010) and variety (Okarini et al. 2013). Samooel et al. (2015) observed significantly higher ash content in meat proximate composition of yellow-brown plumage color birds.

In the current study, no effect of variety on DM, CP and EE was observed. In contrast, difference in DM (Tougan et al. 2013), CP (Wattanachant, 2008) and EE (Samooel, 2015) was reported among different genotypes of poultry, indicating that these parameters of meat composition may vary breed to breed (Mohan et al. 2008; Okarini et al.2013), strain to strain (Sogunle et al. 2010) and genotypes to genotypes (Liu et al. 2012). Higher ash contents than the present study was reported in indigenous chicken meat (2%) (Ogunmola et al. 2013), while, lower ash contents

EXPERIMENT-I

(0.95±0.5%) has also been reported (Tougan et al.2013; Chepkemoi et al. 2017). Whereas, protein content is higher in the present study than those reported in the literature for indigenous chicken meat 18.15% by Chepkemoi et al. (2017), and 20.5±0.5 Tougan et al. (2013). These variations in nutritional contents may be attributed to variation in the breed, feed, sex, production system, processing, age at slaughter, and the part of the cut (Haunshi et al. 2010).

Based on the findings of the present study it can be concluded that 1.30% digestible Lys level with ideal amino acid ratio during early weeks of growth can improve early growth of native Aseel chicken. Furthermore, Mianwali variety due to its higher early growth performance can be used in future breeding plans for the development of a modern slow growing meat-type breed.

Acknowledgements

The authors gratefully acknowledge the support of Quality Control Department, Islamabad Feeds and ICGRC, Department of Poultry Production, University of Veterinary and Animal Sciences,

Lahore.

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Table 3.2.1. Ingredient and nutrients composition of diets*

Ingredients (%) 1 to 14 days 15 to 28 days 29 to 42 days L1 L2 L3 L1 L2 L3 L1 L2 L3 Corn 61.97 62.44 62.66 65.78 64.03 65.78 71.9 61.98 61.45 Soybean Meal 23.05 19.94 21.97 20.20 23.72 20.20 13.7 25.82 26.59 Fish meal 8.00 11.13 9.04 6.09 5.46 6.09 6.34 0.69 0.66 Corn Gluten 60% 3.00 3.00 3.00 3.00 2.00 3.00 3.00 3.00 3.00 CaCO3 0.97 0.97 0.99 0.95 0.80 0.95 0.96 0.80 0.80 DCP 0.20 0.20 - - 0.10 - - 1.00 1.00 Common Salt 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 NaHCO3 0.20 0.34 0.20 0.24 0.26 0.24 0.24 0.34 0.34 Vegetable Oil 0.20 - - 1.26 1.94 1.26 2.3 4.29 4.47 Vit-Min Premix 0.50 0.50 0.50 0.50 0.50 0.50 0.5 0.50 0.50 Lysine Sulphate 0.61 0.46 0.40 0.67 0.37 0.67 0.47 0.58 0.45 DL-Methionine 0.35 0.29 0.26 0.34 0.26 0.34 0.21 0.32 0.27 L-Threonine 0.25 0.21 0.18 0.23 0.14 0.23 0.13 0.17 0.13 L-Valine 0.09 0.03 0.38 0.11 0.01 0.11 0.03 0.08 0.03 L-Isoleucine 0.11 0.04 0.01 0.10 0.00 0.10 0.05 0.05 0.01 L-Tryptophan - - - 0.01 - 0.01 - - - L-Arginine 0.19 0.16 0.10 0.23 0.11 0.23 0.14 0.11 0.03 Nutrients1 CP (%) 23 23 23 21 21 21 20 20 20 Metabolizable 3000 3000 3000 3100 3100 3100 3200 3200 3200 Ca 1.00 1.00 1.00 0.80 0.80 0.80 0.80 0.80 0.80 Ava.Energy P (Kcal/Kg) 0.40 0.40 0.40 0.30 0.30 0.30 0.30 0.30 0.30 Dig. Lysine 1.35 1.30 1.25 1.20 1.15 1.25 1.05 1.15 1.10 Dig. TSAA 1.02 0.98 0.94 0.95 0.88 0.95 0.77 0.89 0.85 Dig. Threonine 0.95 0.91 0.88 0.86 0.79 0.86 0.68 0.78 0.75 Dig. Tryptophan 0.22 0.21 0.20 0.20 0.19 0.20 0.16 0.18 0.18 Dig. Arginine 1.42 1.37 1.32 1.33 1.22 1.33 1.07 1.23 1.18 Dig. Valine 1.02 0.98 0.94 0.95 0.88 0.95 0.77 0.89 0.85 Dig. Isoleucine 0.89 0.86 0.83 0.84 0.77 0.84 0.68 0.78 0.75 Dig. 0.85 0.82 0.79 0.79 0.72 0.79 0.63 0.72 0.69 *L1=High lysine regimen, L2=Medium lysine regimen and L3=Low lysine regimen 1 Analyzed aminoPhenylalanine acids is in the range of ±2% of the calculated values

Table 3.2.2. Growth performance, lysine efficiency ratio and mortality at 6 weeks of age

§ Effects Parameters FI WG F:G LER Mortality Dietary Treatments¶ L1 1175.8±3.7c 423.3±8.2a 2.79±0.05b 27.97±0.58a 0.56± 0.56b L2 1198.0±5.4b 428.2±4.9a 2.80±0.03b 27.80±0.30a 1.12± 0.76b L3 1232.9±4.5a 385.6±7.2b 3.22±0.06a 24.15±0.46b 3.89± 1.18a Varieties‡ LK 1199.3±7.2 401.5±9.3c 3.02±0.08a 25.99±0.74b 1.12± 0.76 MW 1198.5±7.5 424.1±8.1a 2.84±0.05b 27.51±0.56a 1.67± 0.91 PW 1208.9±7.1 411.5±6.3b 2.95±0.05a 26.43±0.49b 2.78± 1.09 Dietary Treatments × Varieties L1 LK 1174.0±5.62b 394.7±15.3ab 3.0±0.12 d 28.02±1.12a 1.12 MW 1176.7±5.43b 396.0±18.7ab 3.0±0.14 d 28.04±1.28a 0.56 PW 1177.0±8.67b 393.4±9.34ab 3.0±0.08 d 27.86±0.69a 2.78 L2 LK 1191.6±6.09b 393.0±4.04ab 3.03±0.03 d 27.49±0.25a 1.12 MW 1184.4±9.86b 406.2±12.5a 2.93±0.07 d 28.56±0.71a 2.78 PW 1218.0±6.21a 400.0±7.12ab 3.05±0.05d 27.36±0.42a 3.89 L3 LK 1232.4±9.45a 332.2±10.5d 3.73±0.11a 22.47±0.72d 2.78 MW 1234.4±6.62a 384.2±10.0b 3.22±0.07c 25.39±0.57b 2.78 PW 1232.0±8.14a 355.8±6.58c 3.47±0.05b 24.06±0.34c 3.89 P-value

DT* <.0001 <.0001 <.0001 <.0001 0.028 EXPERIMENT VA† 0.1005 0.0002 0.0006 0.0007 0.4238 DT×VA 0.0496 0.0034 0.0026 0.009 0.9727 *DT, Dietary Treatments †VA= Varieties ‡LK=Lakha, MW=Mianwali, PW=Peshawari ¶L1=High lysine regimen, L2=Medium lysine regimen and L3=Low lysine regimen §FI=feed intake, BWG=body weight gain, F:G= feed:gain ratio, LER= lysine efficiency ratio

Value with different superscript within column differ significantly (P>0.05)

Table 3.2.3. Effect of dietary amino acid regimens on meat composition traits of Aseel varieties

Variables* Thigh Breast DM Ash CP EE DM Ash CP Fat Dietary Treatments L1 73.21±0.34ab 1.40±0.04a 22.32±0.22ab 0.63±0.02 71.62±0.21 1.42±0.04a 24.26±0.15 0.51±0.02 L2 73.80±0.17a 1.23±0.03b 21.97±0.17b 0.62±0.04 71.60±0.25 1.36±0.04ab 24.49±0.19 0.55±0.04 L3 72.72±0.19b 1.23±0.04b 22.57±0.17a 0.66±0.04 71.22±0.26 1.30±0.04b 23.66±1.26 0.52±0.03 Varieties LK 73.15±0.30 1.26±0.04b 22.33±0.20 0.63±0.03 71.79±0.25 1.47±0.03a 24.15±0.19 0.49±0.02 MW 73.19±0.29 1.23±0.03b 22.32±0.22 0.64±0.04 71.38±0.23 1.30±0.03b 23.35±1.24 0.52±0.04 PW 73.40±0.21 1.37±0.04a 22.21±0.16 0.63±0.04 71.27±0.23 1.31±0.05b 24.90±0.18 0.56±0.03 P-value DT 0.0036 <.0001 0.0064 0.6340 0.1728 0.0150 0.7139 0.3272 VA 0.6635 0.0002 0.7447 0.9806 0.0786 <.0001 0.3339 0.1159 VA x DT 0.3528 <.0001 0.0005 0.4449 0.0025 <.0001 0.6189 0.1190

*VA= Varieties, DT, Dietary Treatments, LK=Lakha, MW=Mianwali, PW=Peshawari, L1=High lysine regimen, L2=Medium lysine regimen and L3=Low lysine regimen, DM= Dry Matter CP= Crude Protein

Value with different superscript within column differ significantly (P>0.05) EXPERIMENT

CHAPTER 4 EXPERIMENT-II

Title: Carryover effect of dietary lysine regimens on growth performance, carcass

characteristics and meat chemical composition in growing phase of Aseel chicken

(Accepted for Publication in Brazilian Journal of Poultry Science ID: RBCA-2017-0681;

Vol. 3, 2018)

ABSTRACT

The aim of present study was to evaluate the subsequent growth, carcass and meat proximate analysis of Aseel chickens fed different levels of dietary lysine (Lys) in early growth phases. In total, 378 day-old chicks [126 from three Aseel varieties i.e., Mianwali (MW), Peshawari (PW), and Lakha (LK)] were reared under three levels of Lys: 1.35% (L1); 1.3% (L2); and 1.25% (L3) during juvenile period (0-6 weeks). A 3×3 factorial arrangement of treatments were employed under Randomized Complete Block Design (RCBD). Treatments were divided into 6 replicates having 7 chicks in each. Feed intake (FI), weight gain (WG) and feed: gain ratio (F:G) were measured on weekly basis. At the end of 18th week, all birds were euthanized and slaughtered to evaluate carcass traits and meat proximate analysis [DM, ash, crude protein (CP) and fat%]. MW variety had improved WG (P<.0001; 1244.4±15.2), F:G (P<.0001; 2.82±0.03) and carcass traits compared to PW (WG: 1113.1±10.4; F:G: 3.05±0.02) and LK(WG: 1161.5±8.75; F:G: 2.94±0.03).

Regarding dietary treatments, bird fed with medium dietary Lys regimen in early phase showed improved WG, F:G, and carcass traits (slaughtering weight, dressed weight and dressing percentage) compared to the birds fed with high or low levels of Lys. In terms of meat composition,

CP (P=0.0175, 20.23±0.19) and Ash (P=0.026, 1.76±0.06) were higher in bird’s thigh fed with higher Lys regimen as compared to the birds fed with medium and lower Lys. It may be concluded that, feeding Aseel chickens diets containing medium levels of Lys during early growth periods

EXPERIMENT-II may performance improving effects later in the chicken’s life compared to similar chickens fed higher or lower levels of Lys. Similarly, across treatments, MW Aseel chickens scored higher carcass characteristics compared to PW and LK Aseel varieties. Taken together, MW Aseel chickens fed medium levels of Lys have potential for use as a native meat-type chicken in tropics.

Keywords: Lysine, performance, carcass characteristics, Aseel Chicken

INTRODUCTION

Importance of backyard poultry is well understood in tropical and sub-tropical regions where it is described as the major source of income especially for women. Indigenous poultry breeds are of the most significant component for these poultry producers due to well adaptability under harsh climate and more resistance to cope with local pathogens. Aseel is the oldest indigenous breed of

Indo-Pak subcontinent with five common varieties i.e. Lakha (LK) of reddish brown color with white mottling, Mianwali (MW) with dark brown color, Peshawari (PW) with wheaten color,

Mushki of black plumages (Babar et al. 2012), Sindhi variety having reddish brown plumage

(Rehman et al. 2017). Growth potential of Aseel can be well assessed with its average adult body weight of 4 to 7 kg which is the major reason behind public preference for Aseel farming rather than other native breeds (Babar et al. 2012; Usman et al. 2014). Aseel is resilient to survive in harsh climatic conditions and has execellent meat producing qualities (Rajkumar et al. 2016;

2017). On the basis of these facts, it can be used as slow growing organic chicken in open-sided poultry houses for production and marketing of organic poultry. However, the main hurdle in its propagation is its poor early growth rate (20-24 weeks) to achieve market weight (1.5 to 2 kg)

(Jatoi et al. 2014). Unfortunately, this unique bird has been neglected and no serious efforts were made for optimum nutritional and managemental recommendations. Indigenous chicken has shown enhanced growth through improved nutrition (Tadelle et al. 2003; Kingori et al. 2007).

EXPERIMENT-II

Nutrient optimization and choice of suitable variety may improve early growth, which might have acceleratory effect on subsequent performance and may help in reducing the market age.

Early nutrient supply is closely associated with the development of digestive system during early phase of life (Beski et al. 2015). Lysine (Lys) among all amino acids is critical for early growth

(Campestrini et al. 2010) and its supplementation during early growth stages is required for regulation of the protein synthesis and muscle growth (Eits et al. 2003). Supplementation of Lys during early phase of life improves the subsequent performance of birds (Kidd et al. 1998) and inclusion of 1.4% Lys in starter diets increases intestinal length and weight of digestive tract (Ullah et al. 2012) which leads to higher pancreatic enzyme activity and nutrients digestion and absorption

(Sklan and Noy 2000). The information regarding Lys supplementation in early phase of life in native Aseel chicken is scarce and need to be exploited. It was hypothesized that supplementation of Lys in diets of Aseel chicks during early phase may bring improvement in subsequent performance and help in reducing market age. Therefore, the present study was planned to examine the subsequent effects of Lys on growth performance, carcass characteristics and meat proximate composition in Aseel chicken during growing phase.

MATERIALS AND METHODS

The study was conducted at Indigenous Chicken Genetic Resource Center, Ravi Campus,

University of Veterinary and Animal Sciences, (UVAS), Lahore. Experimental procedures were in accordance with the guidelines and code of practice of UVAS, Lahore. Ethical approval was obtained before the conduct of the study (Protocol Number UVAS-DAS-4901). In total, 378 day- old chicks were kept for the period of 12 weeks (7 to 18 weeks), 126 bird from each variety (63 male and 63 female), were allotted to 9 experimental groups in a 3 (varieties) × 3 (Lys regimens)

EXPERIMENT-II factorial arrangement, using Randomized Complete Block Design (RCBD). Experimental groups were replicated 6 (three for male and three for female) times with 7 birds in each.

Birds Husbandry

Experiment was conducted in three tier multi deck battery cage house with stocking density of 1 ft2 /bird. Cleaning and washing of equipments were performed with potassium permagnate and sun drying. Fumigation (35ml formalin + 17.5g potassium permagnate = 1X concentration) of house was completed before one week of birds’ arrival. Management conditions like temperature, relative humidity and lighting schedule were maintained according to intensive broiler rearing.

Water and feed were provided ad-libitum through trough nipple drinking system and feeders, respectively.

The diets were formulated (Table 4.1) on the bases of ideal amino acid concept. Birds were fed with nine diets formulated for juvenile phase 0-6 weeks (Hussain et al. 2017). After 6th weeks of age, all the experimental units were switched to this diet offered up to 18 week of age. Chemical analysis for crude protein (CP), dry matter (DM), fat, crude fiber CF), ash, calcium and phosphorus of experimental diets were performed following AOAC (2005), while, amino acid analysis was executed using Biochrome 30+ Series Amino acid analyzer.

Parameters

Cumulative feed intake was determined by combining the weekly feed intake. Initial and final body weights (FBW) were recorded to calculate the cumulative weight gain. Feed to gain ratio

(F:G) and final weight gain and final feed to gain ratio (FF:G) (0 to18 weeks) were also recorded from day 1 to 18 weeks of age. In total, 108 birds, two from each replicate with average weights, were picked up for slaughtering parameters. Birds were given a withdrawal period of six hours

EXPERIMENT-II before slaughtering. Thereafter, birds were slaughtered to collect meat samples from the thigh and breast. Proximate analysis (DM, Ash, CP and EE) of the meat samples was performed using standard methods, dry matter was determined through hot air oven at 800C for 48 hours (Haunshi et al. 2012), crude protein by Kjeldahl method (AOAC, 1999). Fat was determined by the Soxhlet apparatus method (AOAC, 1999); and ash was determined with a furnace at 600°C (AOAC, 1999).

Carcass characteristics including slaughter weight, breast weight, breast yield, thigh weight, thigh yield, dressed weight, dressing percentage and abdominal fat were recorded.

Statistical Analysis

Before analysis, the data were tested for uniformity and homogeneity of variance and were verified for the normality. After that, the data were analyzed through ANOVA technique in factorial arrangements under RCBD by using the GLM procedure of SAS assuming following mathematical model:

Yijk = μ + βi + Vj + Lk + (VL)jk + Ɛijk

Where, Yijk is Dependent variables, µ is overall population mean, βi are the blocks that is sex, Vj

th th is effect of i treatment (i=3; Varieties), Lk is effect of j treatment (j=3; Lys regimens), (VL)ij is interaction effect and Ԑijk is residual error. Aseel variety and Lys regimens were taken as main effects. Interactions of these were also tested. Treatment means were compared through Tukey’s

HSD test at 5% probability level. Each replicate was considered as experimental unit.

EXPERIMENT-II

RESULTS

Growth Performance

MW variety had improved WG (P<.0001; 1244.4±15.2) and F:G (P<.0001; 2.82±0.03) as comparative to the PW (WG: 1113.1±10.4; F:G: 3.05±0.02) and LK(WG: 1161.5±8.75; F:G:

2.94±0.03). For dietary treatments, birds fed with medium dietary Lys regimen showed improved

WG (P=0.0201; 1195.5±18.5), F:G (P=0.0058; 2.87±0.03) and final WG(P<.0001; 1651.8±18.1) after juvenile period. MW variety when fed with medium Lys regimes showed improved weight gain and feed to gain ratio (Table. 4.2).

Meat Composition

CP (P=0.0175, 20.23±0.19) and Ash (P=0.026, 1.76±0.06) were higher in bird’s thigh fed with higher Lys regimen as compared to the birds fed with medium and lower Lys (Table 4.3). Higher ash contents in thigh (P<.0001; 1.80±0.05) and breast (P=0.0052; 2.02±0.04) were observed in

PW and LK variety, respectively. Regarding dry matter content in thigh and breast, neither Aseel varieties nor its interaction with Lys regimens show any significant differences (P>0.05).

Regarding breast meat, higher CP content (P=0.0479; 0.0055) was found in PW varieties compared to the rest of the varieties. Regarding dietary regimens, lower Lys regimens fed bird showed higher

CP contents as compared to the birds fed with medium and high Lys regimens. LK variety fed with high Lys regimen (P=0.0084, 20.72±0.26) had the higher CP in breast. Varieties, Lys regimens and their interaction did not show any effect on EE of Thigh (P= 0.5744; 0.9179; 0.6995) and breast meat (0.6329; 0.2314; 0.6397).

EXPERIMENT-II

Carcass Characteristics

Dietary Lys regimens showed significant variations regarding pre-slaughter weight (P=0.0001), dressed weight (P=0.0042) and dressing percentage (P<.0001), while, all the other characteristics, such as, breast weight, breast yield, thigh weight, thigh yield and abdominal fat remained unchanged. Birds fed medium Lys regimens showed higher pre-slaughter weight (1696.5±14.8), and dressed weight (927.6±11.2) comparative to high and lower Lys dietary treatments.

Among different varieties, significant variations were noted in terms of carcass traits. Higher pre- slaughtering weight (P=0.0001, 1651.8±18.1, 1624.3±17.9), breast weight (214.5±2.5, 210.1±2.7) and thigh weight (183.5±2.2, 180.6±1.8) were observed in MW and LK varieties. Dressed weight

(961.7±7.8) and dressing percentage (58.3±0.6) was higher was higher in MW. Moreover, abdominal fat was higher in LK (1.48±0.04) compared to MW and PW. All interactions of dietary treatments and varieties remained non-significant (Table 4.4).

DISCUSSIONS

Feed intake, Weight Gain and Feed to gain ratio

Medium Lys regimen in the diets of Aseel chicken improved weight gain and Feed to gain ratio.

It is quite possible that supplementation of medium level of Lys as compared to lower level might have promoted muscle accretion in the birds in early weeks of growth, resulting in increased final weight gain. Earlier, its role in regulation of protein synthesis and muscle mass accretion during early growth stages has been reported (Campestrini et al. 2010; Dozier et al. 2007; Eits et al. 2003).

Good feed management in early life of chicks induce significant influence on intestinal tract development, muscular growth and immune system stimulation, resulting in ultimately faster weight gain (Saki, 2005; Prabakaret al. 2016) leading to higher early weight gain and weight at market (Saki, 2005; Hooshmand, 2006). Higher growth rate in later life has been attributed to

EXPERIMENT-II access to feeding essential nutrients like carbohydrates, proteins and amino acids in early life which possibly increased satellite cells proliferation and activity in muscle cell (Kadam et al.

2009). Early nutrient supply is linked to development of digestive system as it is more rapid in early phase of life (Beski et al. 2015) and intestinal weight is closely related with pancreatic enzyme activity and ultimate nutrients digestion and absorption (Sklan and Noy, 2000). Hence, chickens had faster growth at a relatively higher dietary Lys concentration in diet compared with diets having less Lys (Li et al. 2013). Similarly, 14% increase in dietary digestible Lys than the commercial recommendations resulted in greater weight gain (Ivanovich et al. 2017). Similarly, for varieties, difference in weight gain and Feed to gain ratio was observed in different varieties of Aseel by Jatoi et al. (2014). Moreover, feed intake and feed conversions were also different in different varieties of Aseel (Iqbal et al. 2012; Jatoi et al. 2014), Peshawari variety showed significantly higher feed intake compared with Lakha, Mianwali and Mushki Aseel (Usman et al.

2013). Similarly, higher feed intake, has been recorded in Peshawari, Lakha and Mianwali varieties

(Jatoi et al. 2014). The observed difference in growth performance between varieties may be the result of genetic diversity (Leeson et al. 1997), as similar kind of variation also observed in different strains of commercial layers (Scheideler et al. 1998) and Japanese quail (Jatoi et al. 2013).

Meat Composition

The findings of the current study confirm that different dietary Lys regimens had significant effect on dry matter, ash and crude protein of meat from indigenous Aseel chicken that might be the result of incremental levels of amino acids, modifying the muscle growth by increasing myofiber size (Dozier et al. 2008). Lower protein level during early growth stages in the present study did not show adverse effect on proximate meat composition. However, contrary to that the finding of

EXPERIMENT-II the previous studies have indicated that amino acid deficiency can lead to protein decrease and crude fat increase in meat (Corzo et al. 2005; Lilly et al. 2011).

Differences in ash content of thigh and breast as well as crude protein content of thigh in different varieties might be due to their inherent potential. Similar to these finding, difference in ash content among different genotypes of poultry has been reported by Wattanachant et al. (2004) who observed lower percentages of ash contents in Thai chicken than those of commercial broiler

(Wattanachant, 2008; Sogunle et al. 2010). Difference among the varieties has also been indicated in some instances (Okarini et al. 2013). Samooel et al. (2015) observed significantly higher ash content in meat proximate composition of yellow-brown plumage color birds. Chemical composition of Aseel chicken meat is akin to broiler meat with little differences. Previous studies witnessed difference in meat composition which shows that, Aseel meat contain crude protein from

26.85 to 24.94, fat from 3.75, 7.13 and dry matter from 88.28 to 83.35% in breast and thigh muscles

(Haunshi et al. 2013). Moreover, Rajkumar et al. (2017) reported 21.5 crude protein, 3.4 fat, 2.0 ash and 73.3% moisture in Aseel breast meat.

Carcass Characteristics

Carcass characteristics showed mixed variation to dietary treatments, which is in line with the previous results where, carcass yield was not affected by different pre-starter diets (Ullah et al.2012). Contrary to that, high Lys level in the starter period resulted in more fat pad weight and carcass yield than did the birds fed the lower level of Lys in the starter period (Kidd et al 1998).

Variations in carcass traits due to varieties maybe due to the fact that these traits can vary breed to breed (Okarini et al. 2013), strain to strain (Sogunle et al. 2010) and genotypes to genotypes (Liu et al. 2012).

EXPERIMENT-II

CONCLUSIONS

Based on the findings of the present study, it can be stated that medium Lys regimen (1.3%) during early age periods may improve growth performance and carcass characteristics in Aseel varieties at market age. Moreover, Mianwali variety of Aseel possesses a good growth potential as a meat- type chicken.

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EXPERIMENT-II

Table 4.1. Ingredients and nutrients composition of diets*

Ingredients (%) 7-18 weeks

Corn 44 Soybean Meal 12.8 Soybean Full-Fat 16.5 Rice Broken 10 Canola Meal 8 Sunflower Meal 5

DCP 0.7 Common Salt 0.25 NaHCO3 0.29 CaCO3 1.2 Vit-Min Premix 0.5 Lysine Sulphate 0.38 DL-Methionine 0.28 L-Threonine 0.1 Nutrients Analysis1 CP (%) 20.5 Metabolizable Energy (Kcal/Kg) 3000 Ca 0.8 Ava. P 0.4 Dig. Lysine 1.1 Dig. TSAA 0.85 Dig. Threonine 0.75 Dig. Tryptophan 0.22 Dig. Arginine 1.2 Dig. Valine 0.85 Dig. Isoleucine 0.75 1 Analyzed amino acids is in the range of ±2% of the calculated values

Table 4.2. Weight gain, final body weight, feed to gain ratio and mortality at 18 weeks of age Parameters Variables* FI WG F:G FBW FF:G Mortality (g) (g) (g) (%) Dietary Treatments L1 3431.4±44.9 1172.9±19.1ab 2.94±0.03ab 1624.3±17.9a 2.84±0.04b 0.38± 0.12 L2 3432.8±47.3 1195.5±18.5a 2.87±0.03b 1651.8±18.1a 2.81±0.03b 0.33± 0.11 L3 3434.5±33.9 1150.5±15.3b 2.98±0.03a 1564.5±15.3b 2.99±0.03a 0.50± 0.12 P-value 0.9987 0.0201 0.0058 <.0001 <.0001 0.6262 Varieties LK 3405.3±30.7 1161.5±8.75c 2.94±0.03a 1591.2±10.5b 2.89±0.03b 0.45± 0.12 MW 3500.6±47.6 1244.4±15.2a 2.82±0.03b 1696.5±14.8a 2.77±0.03c 0.45± 0.12 PW 3392.8±42.3 1113.1±10.4b 3.05±0.02a 1552.8±11.6b 2.97±0.02a 0.34± 0.11 P-value 0.1533 <.0001 <.0001 <.0001 <.0001 0.7644 Varieties × Dietary Treatments L1 3421.7±63.9 1147.2±18.1bcde 2.98±0.04bc 1598.4±11.9bc 2.88±0.03cd 0.50± 0.22 LK L2 3343.4±53.3 1174.5±15.1bc 2.85±0.03de 1623.0±13.5b 2.79±0.03de 0.34± 0.21 L3 3450.8±37.8 1162.7±12.4bcd 2.97±0.04bcd 1552.2±17.1c 3.02±0.04ab 0.50± 0.23 L1 3486.7±93 1260.8±23.9a 2.77±0.07e 1713.5±20.5a 2.73±0.07e 0.34± 0.21 MW L2 3570.8±95.1 1278.0±27.4a 2.79±0.03e 1741.4±20.7a 2.73±0.03e 0.50± 0.23

L3 3444.2±61.3 1194.2±15.2b 2.88±0.04cde 1634.5±13.2b 2.86±0.04cd 0.50± 0.23 L1 3385.8±82.7 1110.8±18.9de 3.04±0.03ab 1560.8±13.7c 2.93±0.03bc 0.34± 0.21 cde bc bc cd

PW L2 3384.2±70.9 1133.8±17.4 2.98±0.04 1591.0±14.1 2.89±0.04 0.17± 0.17 EXPERIMENT L3 3408.4±79.6 1094.5±16.9e 3.12±0.04a 1506.7±15.7d 3.08±0.03a 0.50± 0.23 P-value 0.5748 <.0001 <.0001 <.0001 <.0001 0.9539 *LK=Lakha, MW=Mianwali, PW=Peshawari L1=High lysine regimen, L2=Medium lysine regimen and L3=Low lysine regimen §FI=feed intake, BWG=body weight gain, F:G= feed to gain ratio,

FBW= final body weight, FF:G= final feed to gain ratio

- Value with different superscript within column differ significantly (P>0.05) II

Table 4.3. Chemical composition of thigh and breast muscles of local Aseel subsequent to dietary lysine regimens

Variables* Thigh Breast DM Ash CP EE DM Ash CP Fat Dietary Treatments L1 74.45±0.30ab 1.76±0.06a 20.23±0.19a 1.98±0.02 73.35±0.23 1.96±0.04 22.30±0.17 b 1.17±0.03 L2 75.03±0.17a 1.59±0.05b 19.77±0.17b 2.00±0.05 73.28±0.23 1.91±0.04 22.37±0.16 b 1.21±0.04 L3 74.07±0.18b 1.69±0.06ab 20.39±0.14a 2.06±0.07 73.07±0.24 1.91±0.04 22.81±0.18 a 1.21±0.03 P-value 0.0176 0.026 0.0175 0.5744 0.6399 0.5042 0.0479 0.6329 Varieties LK 74.44±0.26 1.51±0.04b 20.24±0.18 2.03±0.05 73.41±0.26 2.02±0.04a 22.12±0.12b 1.17±0.03 MW 74.41±0.26 1.73±0.06a 20.15±0.21 2.01±0.06 73.14±0.21 1.88±0.04b 22.51±0.17 ab 1.18±0.03 PW 74.70±0.19 1.80±0.05a 20.00±0.15 2.00±0.05 73.16±0.23 1.87±0.04b 22.85±0.19 b 1.24±0.03 P-value 0.618 <.0001 0.5476 0.9179 0.6134 0.0052 0.0055 0.2314 Varieties × Dietary Treatments LK L1 74.0±0.58 1.58±0.07bc 20.72±0.26a 1.99±0.05 72.71±0.45 2.08±0.06 22.22±0.12ab 1.11±0.03 L2 75.19±0.24 1.42±0.05c 19.46±0.19b 1.97±0.08 73.76±0.30 2.08±0.06 21.89±0.15b 1.17±0.04 L3 74.11±0.38 1.52±0.05c 20.54±0.24ab 2.12±0.06 73.76±0.46 1.89±0.04 22.24±0.31ab 1.21±0.04 MW L1 74.7±0.62 1.71±0.06abc 19.60±0.08ab 1.97±0.08 73.69±0.38 1.81±0.05 21.95±0.24b 1.14±0.04 L2 74.75±0.38 1.63±0.09bc 20.15±0.41ab 1.97±0.12 72.91±0.42 1.84±0.05 22.39±0.16ab 1.20±0.07 L3 73.76±0.21 1.87±0.12ab 20.69±0.17ab 2.10±0.1 72.81±0.20 1.99±0.05 23.19±0.24a 1.21±0.04 PW L1 74.63±0.35 2.00±0.09a 20.36±0.29ab 1.98±0.10 73.66±0.21 1.97±0.06 22.73±0.42ab 1.26±0.05 L2 75.14±0.28 1.72±0.04abc 19.69±0.19ab 2.06±0.05 73.16±0.23 1.82±0.06 22.84±0.32ab 1.23±0.07

abc ab ab

L3 74.33±0.33 1.68±0.07 19.95±0.24 1.96±0.12 72.64±0.33 1.82±0.08 23.00±0.29 1.21±0.05 EXPERIMENT P-value 0.6245 0.0391 0.0084 0.6995 0.1410 0.1938 0.0076 0.6397 *LK=Lakha, MW=Mianwali, PW=Peshawari, L1=High lysine regimen, L2=Medium lysine regimen and L3=Low lysine regimen, DM= Dry Matter CP= Crude Protein

Value with different superscript within column differ significantly (P>0.05)

Table 4.4. Carcass characteristics of local Aseel at 18 weeks of age

SW BW BY TW TY DW DP AF Dietary Treatments Variables* L1 1591.2±10.5b 199.2±5.1 21.8±0.38 171.5±3.5 18.8±0.26 912.0±10.3b 57.4±0.6a 1.36±0.03 L2 1696.5±14.8a 200.1±5.4 21.5±0.39 175.0±4.0 18.8±0.28 927.6±11.2a 54.7±0.4b 1.42±0.04 L3 1552.8±11.6c 198.7±4.9 22.1±0.41 170.9±3.8 19.0±0.32 900.8±9.2b 58.1±0.4a 1.35±0.04 Varieties LK 1624.3±17.9a 210.1±2.7a 23.2±0.26a 180.6±1.8a 20.1±0.18a 904.7±4.8b 55.8±0.5b 1.48±0.04a MW 1651.8±18.1a 214.5±2.5a 22.3±0.12b 183.5±2.2a 19.1±0.14b 961.7±7.8a 58.3±0.6a 1.35±0.03b PW 1564.5±15.3b 173.5±2.2b 19.9±0.20c 153.3±1.6b 17.6±0.16c 874.1±4.4c 55.9±0.5b 1.29±0.02b P-value DT 0.0001 0.9261 0.1924 0.2255 0.7424 0.0042 <.0001 0.2312 VA 0.0001 <.0001 <.0001 <.0001 <.0001 <.0001 0.0002 0.0002 VA x DT 0.7944 0.2769 0.2143 0.2783 0.4050 0.6726 0.9749 0.6781

*VA= Varieties, DT, Dietary Treatments, LK=Lakha, MW=Mianwali, PW=Peshawari, L1=High lysine regimen, L2=Medium lysine regimen and L3=Low lysine regimen, SW=Slaughter weight,

BW=Breast Weight, BY=Breast Yield, TW=Thigh Weight, TY=Thigh Yield, DW= Dressed Weight, DP=Dressing Percentage, AF=Abdominal Fat

EXPERIMENT

Value with different superscript within column differ significantly (P>0.05)

CHAPTER 5 EXPERIMENT-III

Title: Subsequent effect of dietary lysine/amino acid regimens on productive performance,

egg characteristics, and hatching traits of Aseel chickens (Submitted to Indian

Journal of Animal Research)

ABSTRACT

This study was conducted to examine subsequent effect of dietary lysine (Lys) regimens on productive performance, egg characteristics, and hatching traits of Aseel chickens. In total, 72 (63 females and 9 male) birds of 26 weeks of age were fed diets containing three Lys levels (L1, L2,

L3; 8 birds per treatment). A 3×3 factorial arrangement of treatments were employed in

Randomized Complete Block Design (RCBD). Data were recorded regarding productive performance, egg quality and hatching traits for the duration of 20 weeks (26-46 weeks).

Significant differences (P≤0.05) were observed regarding varieties and their interaction with dietary regimens regarding egg production, egg weight, egg mass, feed conversion per dozen and feed conversion per kg egg mass, where PW variety showed higher egg production (P<0.0001;

40.9±0.54), MW and LK showed higher egg weight (P<0.0001; 46.52±0.55,45.19±0.65), PW and

MW showed higher egg mass (P<0.0001, 1728.3±31.9, 1684.2± 39.1), PW and MW depicted improved feed conversion per dozen eggs (P<0.0001; 2.35±0.03) and feed conversion per kg EM

(P=<0.0001; 6.52±0.12, 6.72±0.15). In terms of egg morphometry significantly higher egg length

(P<.0001; 54.57±0.50), egg volume (P<.0001; 42.5±0.50) and egg surface area (P<.0001;

59.7±0.47) were observed in MW variety. PW variety showed better fertility (P<.0001; 82.1±0.67) and hatchability (P<.0001; 59.9±0.65). It was concluded that, dietary Lys in juvenile phase or improved growth has interaction with varieties in elevating productive and reproductive performance of Aseel, especially MW variety.

EXPERIMENT-III

Keywords: juvenile growth, egg production, egg quality, hatching traits, Aseel chicken

INTRODUCTION

Aseel is the popular indigenous chicken for Indo-Pak region and well known for pugnacity, upright posture, agility, firm and well-developed thighs and legs (Dohner, 2001; Singh, 2001) and density body weight (Jatoi et al. 2014). Aseel chickens have potential for higher productivity in terms of growth and production. However, there are some hurdles related to the propagation of Aseel breed, e.g., poor early growth, late maturity, less persistence and number of egg production, broodiness and low fertility and hatchability rates. Low reproductive performance is due to the fact that under field conditions birds are reared under scarcity conditions, with depressed growth it leads to subsequent reduced fertility and hatchability in Aseel birds (Rajkumar et al.2017). The low productivity of the indigenous stock could also partially be attributed to low management standards often seen in traditional production systems. The production performance of the indigenous chickens can be enhanced by improved nutrition (Solomon, 2007). Body weight or feeding level plays a critical role in stimulating the birds to attain sexual maturity

(Ciacciariello and Gous 2005). The relationship among live body measurement in egg-type poultry is well known e.g. body structure and egg production and size (Adenowo et al. 2015).

Breeders are thus interested in heritability of egg production traits and their genetic and phenotypic correlations.

Early chick performance impacts subsequent performance in later stages of the chicken’s life. In poultry, development during the early post-hatch is critical as different body systems like digestive, muscular, and immune systems are developing fast and any deficiency in nutrition at this stage may affect subsequent performance (Prabakaret al. 2016). Feeding essential nutrients like carbohydrates, proteins and amino acids in early life increases satellite cell proliferation and

EXPERIMENT-III activity in muscle cell ultimately elevated final productivity of birds (Kadam et al. 2009; Prabakar et al. 2016). Similarly, early nutrient supply is linked to development of digestive system as it is more rapid in early phase of life (Beski et al. 2015), and intestinal weight is closely related pancreatic enzyme activity and ultimate nutrient digestion and absorption (Sklan and Noy 2000).

More specific in this regard Lys supplementation in early weeks of life results in significantly enhanced performance in later life span of poultry birds (Kidd et al. 1998). Furthermore, 1.4% Lys starter feeds results in increased intestinal length and weight of digestive tract (Ullah et al. 2012).

Muscle development is extremely important to broiler and turkey production and for developing layer pullets, as one of the major issues in the layer industry today is reaching an optimal pullet body weight to start the laying cycle (Prabakar et al. 2016).

On the basis of available literature, Aseel birds have significant potential as meat-type birds and their productive potential can be enhanced through improved nutrition using modern nutritional tools. The present study aimed to investigate the subsequent effect of dietary Lys regimens on productive performance, egg characteristics, and hatching traits of Aseel chickens.

MATERIALS AND METHODS

Ethical note

Experimental procedures were in accordance with the guidelines and code of practice of UVAS,

Lahore. Ethical approval was granted before the conduct of the study (Protocol Number: UVAS-

DAS-4901).

Housing, Experimental Birds and Diets

The study was conducted at Indigenous Chicken Genetic Resource Center, Ravi Campus,

University of Veterinary and Animal Sciences Lahore (UVAS). In total 63, females and 9 males with 21 females and 3 males from each variety were randomly selected at 26 weeks age from

EXPERIMENT-III already reared flock. Birds were maintained as per their brooding phase experimental arrangements with three dietary Lys regimens. The male birds were offered male breeder diets while the females were offered female breeder diets (Table.1). The birds were kept in individual cage units to have complete records of every single bird with the floor space of 1.5 ft2 in three-tiered laying cages with steep wire floor to facilitate egg collection. The removable dropping trays were placed under the floor of cage to collect fecal material.

Parameters studied

Productive performance

Productive performance of selected birds was evaluated till the age of 46 weeks. Average body weight of female, age at first egg, eggs production, egg weight, feed conversion per dozen and feed conversion per kg egg mass were studied in this context.

Egg geometry and quality

Egg quality during production cycle was also studied. Three eggs from each female were used.

Parameter for quality includes Egg weight, Yolk index, Egg shell thickness, Haugh Unit score,

Meat spot, Blood spot and Yolk color. The parameters for geometry also included Shape index,

Surface area and volume of eggs.

Hatching results

Four different hatches were set at the age of 30 to 42 weeks. After hatching parameters includes fertility, hatchability, dead germs and dead in shell were studied for each female.

Statistical Analysis

Before analysis, the data were checked for uniformity and homogeneity of variance and were verified for the normality. After that, the data were analyzed through ANOVA technique in

EXPERIMENT-III factorial arrangement under CRD by using the GLM procedure of SAS (SAS Institute Inc., 2002–

03) assuming following mathematical model:

Yijk = µ + Vi + AAj + (VAA)ij + Ԑijk

th Where, Yijk is Dependent variables, µ is overall population mean, Vi is effect of i treatment (i=3;

th Varieties), AAj is effect of j treatment (j=3; AA Levels), (VAA)ij is interaction effect and Ԑijk is residual error. Aseel variety and Lys level were taken as main effects. Interaction of these was also tested. Treatment means were compared through Tukey’s HSD test at 5% probability level. Each bird was considered as replicate and experimental unit.

RESULTS

Production performance

Statistical analysis of production performance showed differences in BW due to dietary treatment, varieties and their interaction (P=0.0105, <0.0001, <0.0001). Significant differences were observed regarding egg production, egg weight, egg mass, feed conversion per dozen and feed conversion per kg egg mass among different varieties, where PW variety showed higher egg production (P<0.0001; 40.9±0.54), MW and LK showed higher egg weight (P<0.0001;

46.52±0.55,45.19±0.65), PW and MW showed higher egg mass (P<0.0001, 1728.3±31.9, 1684.2±

39.1), PW and MW showed improved feed conversion per dozen eggs (P<0.0001; 2.35±0.03) and feed conversion per kg EM (P<0.0001; 6.52±0.12, 6.72±0.15). Regarding dietary treatment, Egg production (P=0.1715), AEW (P=0.3077), EM (P=0.0856), FCR per dozen (P=0.2108) and FCR per kg EM (P=0.0727) remained unchanged throughout the experimental period. In the interaction between varieties and dietary treatment significant differences were observed regarding egg production (P<0.0001), AEW (P=0.0009), EM (P=0.0006), FCR per dozen (P<0.0001) and FCR

EXPERIMENT-III per kg EM (P=0.0003). on overall basis, PW variety showed better productive performance compared to the rest of the varieties. PW variety when fed with high or medium Lys regimes during early ages showed better subsequent performance (Table 2).

Egg geometry and quality

MW variety had higher EL (P<.0001; 54.57±0.50), higher EV (P<.0001; 42.5±0.50) and ESA

(P<.0001; 59.7±0.47) as compared to PW and LK. The interaction between dietary treatments and varieties showed significant (P<0.0001, 0.0007, 0.0009 and 0.0008) differences in EL, SI, EV and

ESA, where, MW variety with medium amino acid during early period showed improved egg quality (Table 3). MW variety showed improvement in egg quality traits followed by LK and PW.

LK variety when fed with higher and medium Lys regimens during their early life revealed better egg quality traits (Table 4). However, incidence of blood or meat spots were found in eggs of all treatments.

Hatching traits

PW variety were better in terms of fertility (P<.0001; 82.1±0.67) and hatchability (P<.0001;

59.9±0.65). Furthermore, the interaction of variety with Lys regimens showed significant effect on fertility (P<.0001) and hatchability (P<.0001). However, other parameters (SE, DG, DIS, HOF and AGC) remained unchanged throughout the experimental period (Table 5).

DISCUSSION

Production performance

Most of the native chicken breeds including Aseel mature at a late age either in farm or field conditions (Haunshi et al. 2011; Rajkumar et al. 2017). Age at first egg in this study was 195 days which is different from those of the other studies indicating that sexual maturity varies among

EXPERIMENT-III

Aseel chickens. Various studies reported that Aseel mature at 154 (Mohanet al. 2008), 213.25

(Haunshiet al. 2011), 174(Haunshiet al. 2013) 144 (Valvani et al. 2016) or 214 days of age

(Rajkumar et al.2017). Variation among the reports might be attributed to differences in rearing seasons because increasing day length at later growing stage may lead to earlier sexual maturity

(Haunshiet al. 2013). Differences in age at first egg under dietary treatments and interactions might be the due to the fact that body weight or feeding level play a critical role in stimulating the birds to attain sexual maturity (Ciacciariello and Gous 2005). The relationships among live body measurement in egg-type poultry have been discussed by Adenowo et al. (2015).

Egg production results of this study are in line with the results of previous studies with Aseel, where, 35.90% (Rehman et al. 2017), 34.08% (Usman et al. 2014) egg production was observed.

Moreover, Ahmad et al. (2014) reported higher egg production in Peshawari variety in pre- and post-molt periods. Some other studies showed different egg productions in Aseel, where 160 eggs up to 78 week of age (Mohan et al.2008), 35.71 eggs/year (Sarker et al. 2012), 43.28 eggs (Valvan et al. 2016), 18 eggs at 40 weeks, 30 eggs at 52 weeks, 47 eggs at 64 weeks and 64 eggs up to 72 weeks (Rajkumar et al. 2017) were recorded. Persistency of egg production in Aseel chicken like other breeds is inversely related to age. Enhanced persistency occurs during 24 to 36 weeks of age and then declining trend may be observed after 40 weeks of age (Haunshi et al. 2013; Rajkumar et al. 2017).

Overall egg weight is less comparative to other breeds of chicken which might be attributed, somehow to poor feeding and management systems in addition to genetics (Sarkar et al. 2006).

Egg geometry and quality

The results of egg geometry with respect to varieties of Aseel in this study are in line with the previously reported results where Haugh unit and yolk index were found to be 75.43 and 0.395,

EXPERIMENT-III respectively (Haunshi et al. 2011). However, Usman et al. (2014) observed 81.95 Haugh unit score and 0.46 yolk index in Aseel eggs. Egg shape index in Aseel chicken has been reported to be 75.46

(Singh et al. 2000a) and 77.36 (Haunshi et al. 2011). Egg geometry parameters have positive correlation with age (Usman et al. 2014).

Hatching traits

Hatching traits in this study are similar to those reported ina previous study indicating where fertility 79.0%, hatchability 59.18%, hatch of fertile 74.86%, embryonic mortality 19.69% and A- grade chicks 52.23% of total in different varieties of Aseel (Rehman, 2017). Meanwhile, differences in reproductive traits have been reported in many previous studies showing fertility

84%, hatchability 67% and hatch of fertile 88% (Mohan et al. 2008), fertility 86.96%, hatchability

70.74% and hatch of fertile 81.21% (Haunshi et al. 2012), fertility 89.95%, hatchability 46.30% and hatch of fertile 49.85% in Mushki Aseel, similarly, fertility 64.61%, hatchability 53.02% and hatch of fertile 84.20% in Peshawari Aseel (Ahmad et al. 2013a,b), fertility 67.18%, hatchability

44.71% and hatch of fertile 80.87% (Rajkumar et al. 2017). This variation in reproductive traits could be due to the difference in birds’ condition, selected birds or random field collected birds

(Rajkumar et al. 2017).

CONCLUSIONS

It was concluded that, dietary Lysin juvenile phase or improved growth has interaction with varieties in elevating productive and reproductive performance of Aseel, especially MW variety.

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Table 5.1. Ingredient and nutrients composition of diets*

Ingredients (%) 26-46 weeks Corn 47.4 Rice Polish 15 Soybean Meal 11.3 Soybean Full-Fat 4.2 Sunflower Meal 2 Canola Meal 1 Fish meal 4 DDGS 5 CaCO3 8.7 DCP 0.16 Common Salt 0.16 NaHCO3 0.24 Vit-Min Premix 0.5 Lysine Sulphate 0.08 DL-Methionine 0.22 L-Threonine 0.03 L-Tryptophan 0.01 Nutrients1 CP (%) 17.25 Metabolizable Energy (Kcal/Kg) 2750 Ca 3.4 Ava. P 0.4 Dig. Lysine 0.72 Dig. TSAA 0.65 Dig. Threonine 0.55 Dig. Tryptophan 0.17 Dig. Valine 0.66 Dig. Isoleucine 0.56 1 Analyzed amino acids is in the range of ±2% of the calculated values

102

Table 5.2. Productive performance of local Aseel subsequent to dietary lysine regimens

Parameters * Variables AFE BW EP AEW EM FCR/Dozen FCR/Kg Dietary Treatments EM L1 195.5±1.36b 2404.5±24.1 ab 36.9±0.88 44.95±0.71 1657.9±42.5 2.63±0.06 6.85±0.18 L2 196.4±2.03ab 2413.8±22.7 a 37.4±0.95 45.10±0.80 1676.4±38.1 2.60±0.07 6.75±0.15 L3 201.1±1.34a 2366.7±20.5 b 35.9±0.82 43.90±0.56 1575.2±37.4 2.70±0.06 7.19±0.18 P-value 0.0373 0.0105 0.1715 0.3077 0.0856 0.2108 0.0727 Varieties LK 203±1.66 ]a 23.92.2±11.6 b 33.2±0.49c 45.19±0.65a 1496.9±29.8b 2.91±0.04a 7.54±0.15a MW 198±1.08b 2504.1±12.7 a 36.2±0.59b 46.52±0.55a 1684.2± 39.1a 2.67±0.04b 6.72±0.15b PW 192±1.32c 2288.8±11.2 c 40.9±0.54a 42.24±0.56b 1728.3±31.9a 2.35±0.03c 6.52±0.12b P-value <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 Varieties × Dietary Treatments L1 197.7±2.2abcd 2415.7±20.5bc 33.6±0.79ed 45.43±1.11abc 1525.8±55.5ab 2.87±0.07ab 7.40±0.27ab LK L2 204.7±3.6ab 2401.5±17.8bc 33.3±1.04cd 46.29±1.15abe 1536.1±42.9ab 2.90±0.09a 7.33±0.21ab L3 206.6±1.7a 2359.3±17.9cd 32.6±075d 43.86±1.06abc 1428.8±53.1b 2.96±0.07a 7.90±0.29a L1 197.6±2.2abcd 2515.8±19.1a 36.3±0.98bcd 46.6±1.07ab 1690.3±73.8ab 2.66±0.07abc 6.70±0.30b MW L2 195.4±1.7bcd 2532.2±19.3a 37.4±1.20abc 47.3±1.17a 1766.7±75.4a 2.59±0.08bcd 6.41±0.27b L3 201.0±1.3abc 2464.3±21.2ab 34.9±0.75cd 45.7±0.57abc 1595.5±41.2ab 2.76±0.06ab 7.05±0.18ab L1 191.3±2.3cd 2282.2±21.2d 41.02±1.22a 42.86±1.20abc 1757.5±69.6a 2.35±0.07cd 6.43±0.26b d d a c a d b

PW L2 189.0±2.2 2307.9±15.2 41.5±0.95 41.71±0.92 1726.4±43.4 2.32±0.05 6.51±0.17

EXPERIMENT L3 195.7±1.9bcd 2276.5±21.9d 40.3±0.62ab 42.14±0.83bc 1701.2±56.7a 2.39±0.04cd 6.63±0.22b P-value <.0001 <.0001 <.0001 0.0009 0.0006 <.0001 0.0003 *LK=Lakha, MW=Mianwali, PW=Peshawari L1=high d-lysine L2=Medium d-lysine L3= Low d-lysine, AFE= Age at Frist Egg BW= Body Weight, EP= Egg Production, AEW= Average Egg Weight, EM= Egg Mass, FCR= Feed to gain ratio

Value with different superscript within column differ significantly (P>0.05)

III

103

Table 5.3. Egg Geometry Characteristics of local Aseel subsequent to dietary lysine regimens

Parameters Variables* EL EB SI EV ESA Dietary Treatments L1 51.00±0.79 39.81±0.41 78.29±0.98 41.1±0.65 58.4±0.62 L2 51.81±0.82 40.33±0.35 78.23±1.41 41.2±0.73 58.5±0.70 L3 51.43±0.82 39.57±0.39 77.40±1.61 40.1±0.52 57.42±0.50 P-value 0.5577 0.3517 0.8259 0.3077 0.3187 Varieties LK 51.95±0.50 b 40.3±0.41 77.69±1.09 b 41.3±0.59a 58.6±0.56a MW 54.57±0.50 a 40.2±0.33 73.69±0.92 c 42.5±0.50a 59.7±0.47a PW 47.71±0.54 c 39.3±0.39 82.54±1.25 a 38.7±0.51b 55.9±0.49b P-value <.0001 0.1385 <.0001 <.0001 <.0001 Varieties × Dietary Treatments L1 51.14±0.80abc 40.29±0.68 78.84±1.44abc 41.48±1.01abc 58.75±0.96abc LK L2 52.86±0.77a 41.14±0.59 77.96±1.74abc 42.26±1.05abc 59.49±0.99abc L3 51.86±0.99ab 39.43±0.78 76.28±2.48abc 40.04±0.96abc 57.38±0.93abc L1 54.43±0.84a 40.43±0.84 74.37±1.52bc 42.52±0.97bc 59.74±0.92ab MW L2 54.43±1.13a 40.43±0.48 74.45±1.66bc 43.17±1.07a 60.35±1.01a L3 54.86±0.67a 39.57±0.57 72.24±1.71c 41.74±0.52abc 59.02±0.49abc

L1 47.43±0.87c 38.71±0.68 81.66±0.84ab 39.13±1.10abc 56.49±1.07abc PW L2 48.14±1.12bc 39.43±0.65 82.28±0.65ab 38.09±0.84c 55.49±0.82c EXPERIMENT L3 47.57±0.92c 39.71±0.75 83.69±2.34a 38.48±0.76bc 55.88±0.73bc P-value <.0001 0.2942 0.0007 0.0009 0.0008 *LK=Lakha, MW=Mianwali, PW=Peshawari, L1=high d-lysine L2=Medium d-lysine L3= Low d-lysine, EL=Egg Length, EB= Egg Breath SI= Shape Index, EV= Egg Volume, ESA= Egg Surface Area

Value with different superscript within column differ significantly (P>0.05) III

104

Table 5.4. Egg Quality Characteristics of local Aseel subsequent to dietary lysine regimens

Parameters Variables* EST YI HUS YC Dietary Treatments L1 0.36 0.51 71.28±0.7 11.86±0.26 L2 0.36 0.52 70.57±0.6 11.81±0.28 L3 0.35 0.51 69.91±0.6 11.76±0.25 P-value 0.4386 0.0933 0.5502 0.894 Varieties LK 0.35 0.53a 72.05±0.58a 11.34±0.24 MW 0.37 0.51b 68.67±0.51b 12.19±0.24 PW 0.36 0.50b 71.05±0.58a 11.91±0.28 P-value 0.1491 <.0001 0.0002 0.0607 Varieties × Dietary Treatments L1 0.35 0.54ab 73.72±0.81a 11.14±0.41 LK L2 0.36 0.55a 71.43±1.02ab 11.28±0.42 L3 0.35 0.52abc 71.00±0.98ab 11.57±0.48 L1 0.37 0.51bc 68.72±0.99b 12.42±0.37 MW L2 0.37 0.52abc 69.43±0.78ab 12.14±0.51 L3 0.36 0.5c 67.85±0.88b 12.00±0.38

c ab L1 0.36 0.5 71.43±1.02 12.00±0.49 EXPERIMENT PW L2 0.36 0.51bc 70.86±1.26ab 12.00±0.58 L3 0.36 0.51abc 70.85±0.83ab 11.72±0.47 P-value 0.6345 0.0001 0.0047 0.5887 *LK=Lakha, MW=Mianwali, PW=Peshawari, L1=high d-lysine L2=Medium d-lysine L3= Low d-lysine, EST= Egg Shell Thickness, YI= Yolk Index, HUS= Hough Unit Score, YC= Yolk Color. Value with different superscript within column differ significantly (P>0.05)

III

105

Table 5.5. Hatching Traits of local Aseel subsequent to dietary lysine regimens

Parameters Variables* SE FR DG DIS HA HOF AGC Dietary Treatments x Varieties L1 96.14±0.5 73.29±1.3c 10.71±1.2 19.33±1.2 51.14±0.9b 69.96±2.2 59.29±2.9 LK L2 95.86±0.7 75.00±0.8bc 11.43±1.3 19.9±1.35 51.43±1.5b 68.67±2.4 58.86±2.5 L3 95.14±0.5 72.71±1.4c 10.71±1.6 18.08±1.1 51.71±1.7b 71.21±2.5 56.71±1.8 L1 96.0±0.5 79.57±1.3ab 8.86±1.2 19.93±1.1 56.57±1.1ab 71.22±1.8 61.71±2.7 MW L2 95.86±1.2 80.43±1.3ab 11.43±1.4 18.15±2.2 56.43±1.5ab 70.42±2.9 62.43±1.6 L3 94.86±0.9 80.86±1.6a 10.43±1.1 17.62±2.2 58.00±1.1a 71.95±2.2 60.0±2.8 L1 95.86±0.8 81.86±1.3a 8.86±0.8 16.09±1.9 61.29±1.1a 75.06±2.3 64.86±2.6 PW L2 95.86±0.8 81.43±0.8a 10.14±1.0 16.79±1.2 59.43±1.4a 73.07±2.1 58.86±2.8 L3 95.57±0.7 83.00±1.4a 10.71±0.8 18.09±1.1 59.00±0.9a 71.19±1.5 60.86±3.1 P-value 0.9598 <.0001 0.7265 0.6539 <.0001 0.6998 0.5477 Dietary Treatments L1 96.00±0.35 78.24±1.08 9.48±0.60 18.45±0.86 56.33±1.09 72.08±1.22 61.95±1.59 L2 95.86±0.49 78.95±0.84 11.0±0.69 18.28±0.94 55.76±1.09 70.72±1.42 60.05±1.32 L3 95.19±0.39 78.86±1.27 10.6±0.67 17.93±0.85 56.24±1.00 71.45±1.15 59.19±1.48 P-value 0.3847 0.7564 0.2624 0.9154 0.8407 0.7558 0.4073 Varieties

LK 95.7±0.34 73.7±0.69b 10.9±0.75 19.1±0.67 51.5±0.79a 69.95±1.29 58.3±1.35 EXPERIMENT MW 95.6±0.5 80.3±0.78a 10.3±0.71 18.6±1.06 57.0±0.70b 71.19±1.29 61.4±1.34 PW 95.8±0.41 82.1±0.67a 9.90±0.51 17.0±0.80 59.9±0.65c 73.11±1.13 61.5±1.65 P-value 0.9502 <.0001 0.5392 0.224 <.0001 0.2223 0.2258 *LK=Lakha, MW=Mianwali, PW=Peshawari, L1=high d-lysine L2=Medium d-lysine L3= Low d-lysine, SE= Settable Eggs, FR=Fertility, DG=Dead Germ, DIS= Dead in Shell, HA= Hatchability, HOF= Hatch of Fertile, AGC= A Grade Chicks

III

Value with different superscript within column differ significantly (P>0.05)

106

CHAPTER 6 SUMMARY

Backyard poultry production is an important sub sector of poultry in tropical and sub-tropical countries of the world. Aseel is the most popular breed among the local breeds of Asian subcontinent. Aseel chickens has juvenile body weight of 250 to 300g at 6 week of age showing slow growth during the early period, however, may grow up to 5 Kg at adult stage, indicating that

Aseel has the potential to gain good body weight. But the main problem is its’ poor early growth, taking more time to achieve market weight resulting in poor feed conversion and higher cost of production. Aseel chicken has great potential to be improved for growth related characters.

Genetic tools are being used to improve Aseel performance but in continuation to that nutritional manipulations and managerial tools can also be applied to improve early growth, which can further improve subsequent performance. Indigenous chicken has shown elevated growth by improved nutrition, so nutritional manipulations need to be focused to improve early growth in Aseel chicken. After hatching, early few weeks are much important because feeding of chicks during early period produces significant effect on intestinal tract development, muscular growth and immune system stimulation, resulting in ultimately faster weight gain. Lysine (Lys) is critical among all nutrients for early growth as it is directly linked to the muscle development. Lysine contents also own major share of the protein in breast meat. It has been reported that supplementation of Lys during early growth stages is beneficial because it involves in regulation of protein synthesis and increases muscle accretion. Moreover, early nutrient supply is closely associated with the development of digestive system during early phase of life. Supplementation of Lys in starter diets increases intestinal length and weight of digestive tract, which is closely related to pancreatic enzyme activity and nutrients digestion and absorption.

107

SUMMARY

Keeping above in view a study was conducted to investigate the effects of different dietary Lys levels with digestible amino acids on growth performance, carcass evaluation, productive and reproductive traits of Aseel chicken. This study consisted of three different experimental phases.

The experimental Phase-I was conducted with the objective, to evaluate the effect of dietary Lys regimens on growth performance and meat composition of Aseel chicken. In total, 540 day-old chicks, 180 from individual variety, were randomly assigned to 9 experimental groups in a 3

(Varieties: Mianwali (MW), Peshawari (PW), and Lakha (LK)) × 3 (Lys regimens: L1, L2 and

L3:1.35, 1.30 and 1.25%) factorial arrangement under Randomized Complete Block Design

(RCBD) with sex as block. Experimental groups were replicated 6 times with 10 birds in each with average weight of 29 grams. Feed intake (FI), weight gain (WG) and feed to gain ratio (F:G) parameters of growth performance and Dry Matter (DM), Ash, Crude Protein (CP) and Fat parameters of meat composition were evaluated. The second phase was designed to examine growth performance of Aseel chicken subsequent to juvenile dietary Lys regimes. In total 378 birds were randomly picked up (126 from each variety) and distributed into 9 experimental groups in a 3×3

{three varieties of Aseel chicken MW, PW and LK reared under three dietary Lys regimes (L1, L2 and L3) during juvenile period (0-6 weeks)} factorial arrangement under RCBD. Each experimental group was replicated 6 times with 7 birds in each and reared on the similar diet. The third phase was undertaken to examine the subsequent effect of dietary Lys regimens on productive performance, egg characteristics, and hatching traits of Aseel chickens. In total, 72 (63 female and

9 male) birds of 26 weeks of age were kept for a period of 20 weeks (26 to 46 weeks), 24 from each variety, were randomly assigned to 9 experimental groups in a 3 {(varieties) MW, PW, and

LK} × 3 (juvenile Lys regimen groups L1, L2 and L3) factorial arrangement, using Completely

Randomized Design (CRD). Each bird in experimental group was considered as a replicate. The

108

SUMMARY data were analyzed by using 2-way ANOVA technique under factorial arrangement using GLM procedure of SAS 9.1 software (SAS Institute Inc., 2002-03). Means were compared through

Tukey’s HSD test at 5% probability level. Results indicated higher WG and lower F:G in MW verities. Among different Lys regimens, higher and medium levels in the diet showed improved

WG, F:G and reduced FI. Similarly, increased ash, dry matter and crude protein content in thigh and breast due to increased level of Lys in the early life period was also observed. In subsequent phase results indicated that MW variety exhibited higher weight gain and lower feed to gain ratio.

Medium dietary Lys regimen, improved weight gain, feed to gain ratio, final weight gain.

Similarly, medium dietary Lys regimen also showed higher DM in thigh while CP was found to be significantly higher in low Lys regimen. Carcass characteristics including slaughtering weight, dressed weight and dressing percentage showed higher values under medium dietary treatments during juvenile period, whereas MW variety showed overall improved carcass characteristics. In production and reproduction phase results showed differences among verities in egg production, egg weight, egg mass, feed conversion per dozen and feed conversion per kg egg mass, while non- significant differences on the basis of dietary treatments. Egg geometry and egg quality parameters showed differences in most of the results on the basis of varieties and their interactions. Variation in hatching traits were found for fertility and hatchability due to varieties and interaction, PW variety in interaction with Lys regimens showed higher fertility and hatchability.

109

CONCLUSIONS

 It was concluded that, 1.30% digestible Lys level regimen can be used to improve the early

and subsequent growth rate of Aseel chicken.

 Mianwali variety of Aseel compared to PW and LK showed improved growth and meat

composition.

 Different Lysine regimens did not show any particular effect on immune status of birds as

measured here.

 In production performance, high lysine regimens in interaction with PW variety exhibited

best results for egg production and MW for egg geometry.

 Hatching traits were higher in PW and MW varieties than LK.

SUGGESTIONS AND RECOMMENDATIONS

 More studies in this direction can help explore more ways to propagate Aseel and develop

this bird as a meat-type chicken.

 In native chickens, especially Aseel, improved productive performance via improved

nutrition with higher economic returns may attract rural farmers for improvement of their

household economy.

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Supplementary Data

Table I. Block comparisons of Feed Intake, Weight gain and Feed to gain ratio at 6 and 18 weeks of age

Variables1 Parameters2 FI WG F:G

6th Week

M 1211.6±5.7a 403.0±6.4a 3.03±0.06b

F 1192.8±5.6b 364.9±4.5b 3.28±0.05a

P-value 0.0218 <.0001 0.0030

18th weeks

M 3366.5±28.7b 1150.8±12.7a 2.93±0.03

F 3499.3±34.5a 1195.1±13.6a 2.94±0.02

P-value 0.0047 0.0209 0.9814 1 M= Male, F=Female, 2FI= Feed Intake WG= Weight Gain F:G= Feed to Gain Ratio

Value with different superscript within column differ significantly (P>0.05)

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Table II. Block comparisons of thigh and breast muscles composition at 6th and 18th week in of local Aseel.

Parameters

Variables* Thigh Breast

DM Ash CP Fat DM Ash CP Fat

6th week

M 72.82±1.11b 1.32±0.16 22.45±0.92 0.72±0.15a 71.27±0.97 1.36±0.16 23.97±4.34 0.52±0.16

F 73.66±0.97a 1.26±0.18 22.12±0.66 0.56±0.12b 71.70±1.04 1.37±0.19 24.31±0.91 0.53±0.11

P-value 0.0046 2470 1422 <.0001 1246 8419 6925 7262

18th week

M 74.14±0.99b 1.75±0.23a 20.24±0.81 2.10±0.23a 73.07±0.97 1.91±0.17 22.67±0.77 1.19±0.14

F 74.88±0.89a 1.62±0.24b 20.02±0.69 1.92±0.18b 73.40±0.96 1.94±0.17 22.32±0.68 1.20±0.11

P-value 0.0056 0.0389 2644 0.0031 2062 5722 0.0778 0.6485

* M= Male, F=Female, DM= Dry Matter CP= Crude Protein

Value with different superscript within column differ significantly (P>0.05)

112

Table III. Meat Quality Traits at 18 weeks of age Parameters Variables* IPH FPH DL WHC Gender (Block) M 6.32±0.13 6.14±0.05 2.72±0.09 18.99±0.14 F 6.32±0.08 6.15±0.04 2.68±0.12 18.98±0.16 P-value 0.9712 0.4794 0.2129 0.7909 Dietary Treatments L1 6.33±0.12 6.15±0.04 2.68±0.12 19.04±0.10 L2 6.30±0.12 6.14±0.04 2.72±0.09 18.99±0.18 L3 6.32±0.09 6.16±0.05 2.70±0.12 18.94±0.16 Varieties LK 6.35±0.11 6.16±0.04 2.73±0.07 19.04±0.14 MW 6.31±0.10 6.15±0.04 2.70±0.12 18.97±0.15 PW 6.29±0.13 6.14±0.05 2.67±0.13 18.95±0.15 Varieties × Dietary Treatments L1 6.39±0.12 6.17±0.04 2.74±0.07 19.06±0.08 LK L2 6.32±0.11 6.15±0.04 2.69±0.07 19.12±0.14 L3 6.35±0.12 6.17±0.04 2.77±0.05 18.96±0.18 L1 6.43±0.12 6.15±0.04 2.70±0.08 19.02±0.09 MW L2 6.30±0.09 6.15±0.03 2.76±0.07 18.89±0.19

L3 6.29±0.08 6.14±0.06 2.63±0.15 19.01±0.11 L1 6.25±0.08 6.13±0.04 2.61±0.15 19.03±0.09 PW L2 6.29±0.17 6.14±0.07 2.70±0.13 18.96±0.16 L3 6.35±0.10 6.16±0.05 2.70±0.11 18.86±0.16 P-value 0.4466 0.5057 0.0914 0.1615 *LK=Lakha, MW=Mianwali, PW=Peshawari L1=High lysine regimen, L2=Medium lysine regimen and L3=Low lysine regimen §FI=feed intake, IPH=Initial pH, FPH= Final pH, DL= Drip Loss,

WHC= Water Holding Capacity

Value with different superscript within column differ significantly (P>0.05)

113

Table IV. Economic at 6 weeks of age (Pakistani Rupee)

Parameters

Variables* Feed Cost/ Feed Intake Cost of Feed Body Weight Cost Per Kg Kg

L1 1175.8 45.90 53.97 452.3 119.3

L2 1198.0 44.96 53.86 457.2 117.8

L3 1232.9 44.50 54.83 414.6 132.3

*L1=High lysine regimen, L2=Medium lysine regimen and L3=Low lysine regimen

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Table V. Organ Development at 6 weeks of age Parameters Variables* Liver Gizzard Heart Spleen Intestine (%BW) (%BW) (%BW) (%BW) (%BW) Gender

F 3.04±0.11b 5.85±0.10 0.63±0.04b 0.39±0.04 1.67±0.11b

M 3.13±0.06a 5.91±0.13 0.69±0.03a 0.42±0.05 1.80±0.17a

Dietary Treatments

L1 3.08±0.10 5.86±0.12 0.66±0.05 0.41±0.05 1.73±0.17

L2 3.09±0.11 5.87±0.11 0.65±0.05 0.40±0.04 1.76±0.17

L3 3.09±0.10 5.92±0.10 0.65±0.04 0.42±0.04 1.73±0.14

Varieties

LK 3.01±0.11 5.89±0.13 0.65±0.045 0.41±0.044 1.71±0.14

MW 3.14±0.07 5.87±0.11 0.66±0.048 0.40±0.043 1.76±0.17

PW 3.12±0.06 5.89±0.12 0.66±0.045 0.42±0.041 1.76±0.15

*M= Male, F=Female, LK=Lakha, MW=Mianwali, PW=Peshawari L1=High lysine regimen, L2=Medium lysine regimen and L3=Low lysine regimen §FI=feed intake, BW=body weight

Value with different superscript within column differ significantly (P>0.05)

115

Table VI. Blood Biochemistry at 6 weeks of age

GLU TP ALB GLB CRT UA TRIG CHOL ALAST ASAT LDL HDL Variables* (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) U/L U/L (mg/dl) (mg/dl)

Gender

F 210.7±11.4 4.69±0.19 3.03±0.08 1.84±0.14 0.67±0.06 9.19±0.58 105.3±4.43 139.2±6.62 7.54±0.08 55.35±1.75 44.71±1.22 69.2±1.89

M 208.2±13.4 4.65±0.12 3.04±0.07 1.83±0.10 0.65±0.06 9.07±0.62 105.8±4.52 138.6±6.25 7.53±0.08 55.23±1.58 44.69±1.37 68.7±1.88

Varieties

LK 211.4±13.1 4.66±0.11 3.05±0.10 1.74±0.09c 0.66±0.07 8.99±0.64b 106.5±5.01a 139.6±5.94 7.56±0.08 55.16±1.55 44.63±1.52 69.1±1.94

MW 208.2±10.3 4.65±0.11 3.02±0.05 1.85±0.07b 0.65±0.05 9.05±0.39ab 106.4±4.76ab 135±5.64 7.53±0.07 55.74±1.61 44.85±1.23 68.6±2.01

PW 212.4±10.4 4.72±0.23 3.05±0.07 1.93±0.11a 0.67±0.06 9.34±0.69a 103.7±2.91b 141.2±6.63 7.51±0.08 54.96±1.78 44.63±1.15 69.3±1.71

Treatments

L1 212.2±10.7 4.63±0.07 3.06±0.07a 1.84±0.13 0.64±0.06b 8.97±0.61b 106.8±4.56a 137.1±5.03 7.54±0.08 55.16±1.69 44.55±1.41 68.9±1.89

L2 207.1±9.71 4.70±0.12 3.05±0.82a 1.84±0.12 0.68±0.05a 9.11±0.54ab 102.8±2.88b 139.3±6.57 7.53±0.08 55.57±1.57 44.86±1.34 68.9±2.00

L3 212.7±12.8 4.69±0.24 3.00±0.06b 1.84±0.13 0.66±0.07ab 9.31±0.64a 107.0±4.53a 140.3±7.28 7.54±0.08 55.14±1.73 44.69±1.16 68.9±1.84

*LK=Lakha, MW=Mianwali, PW=Peshawari L1=High lysine regimen, L2=Medium lysine regimen and L3=Low lysine regimen §GLU=Glucose, TP=Total Protein, ALB=Albumin, GLU= Globulin, CRT= Creatinine, UA= Uric acid, TRIG= Triglyceride, CHOL= Cholesterol, ALAST= Alanine amino transferase, ASAT= Aspartate amino transferase LDL= Low density lip oprotein HDL= High density lipoprotein

Value with different superscript within column differ significantly (P>0.05)

116