Poultry Nutrition Printed Edition of the Special Issue Published in Animals www.mdpi.com/journal/animals Vincenzo Tufarelli Edited by Poultry Nutrition Poultry Nutrition Editor Vincenzo Tufarelli MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editor Vincenzo Tufarelli University of Bari Aldo Moro Italy Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Animals (ISSN 2076-2615) (available at: https://www.mdpi.com/journal/animals/special issues/ Poultry Nutrition). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Volume Number , Page Range. ISBN 978-3-03943-853-2 (Hbk) ISBN 978-3-03943-854-9 (PDF) Cover image courtesy of Vincenzo Tufarelli. c © 2020 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Preface to ”Poultry Nutrition” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii Nimra Azeem, Muhammad Nawaz, Aftab Ahmad Anjum, Shagufta Saeed, Saba Sana, Amina Mustafa and Muhammad Rizwan Yousuf Activity and Anti-Aflatoxigenic Effect of Indigenously Characterized Probiotic Lactobacilli against Aspergillus flavus —A Common Poultry Feed Contaminant Reprinted from: Animals 2019 , 9 , 166, doi:10.3390/ani9040166 . . . . . . . . . . . . . . . . . . . . 1 Daniel Hernandez-Patlan, Bruno Sol ́ ıs-Cruz, Karine Patrin Pontin, Juan D. Latorre, Mikayla F. A. Baxter, Xochitl Hernandez-Velasco, Ruben Merino-Guzman, Abraham M ́ endez-Albores, Billy M. Hargis, Raquel Lopez-Arellano and Guillermo Tellez-Isaias Evaluation of the Dietary Supplementation of a Formulation Containing Ascorbic Acid and a Solid Dispersion of Curcumin with Boric Acid against Salmonella Enteritidis and Necrotic Enteritis in Broiler Chickens Reprinted from: Animals 2019 , 9 , 184, doi:10.3390/ani9040184 . . . . . . . . . . . . . . . . . . . . 11 Alaeldein M. Abudabos, Muttahar H. Ali, Mohammed A. Nassan and Ahmad A. Saleh Ameliorative Effect of Bacillus subtilis on Growth Performance and Intestinal Architecture in Broiler Infected with Salmonella Reprinted from: Animals 2019 , 9 , 190, doi:10.3390/ani9040190 . . . . . . . . . . . . . . . . . . . . 25 Mahmoud Mostafa Azzam, Shou-qun JIANG, Jia-li CHEN, Xia-jing LIN, Zhong-yong GOU, Qiu-li FAN, Yi-bing WANG, Long LI and Zong-yong JIANG Effect of Soybean Isoflavones on Growth Performance, Immune Function, and Viral Protein 5 mRNA Expression in Broiler Chickens Challenged with Infectious Bursal Disease Virus Reprinted from: Animals 2019 , 9 , 247, doi:10.3390/ani9050247 . . . . . . . . . . . . . . . . . . . . 31 Youssef A. Attia, Nicola F. Addeo, Abd Al-Hamid E. Abd Al-Hamid and Fulvia Bovera Effects of Phytase Supplementation to Diets with or without Zinc Addition on Growth Performance and Zinc Utilization of White Pekin Ducks Reprinted from: Animals 2019 , 9 , 280, doi:10.3390/ani9050280 . . . . . . . . . . . . . . . . . . . . 43 Moataz M. Fathi, Ibrahim Al-Homidan, Tarek A. Ebeid, Ahmed Galal and Osama K. Abou-Emera Assessment of Residual Feed Intake and Its Relevant Measurements in Two Varieties of Japanese Quails ( Coturnixcoturnix japonica ) under High Environmental Temperature Reprinted from: Animals 2019 , 9 , 299, doi:10.3390/ani9060299 . . . . . . . . . . . . . . . . . . . . 55 Ivana Prakatur, Maja Miskulin, Mirela Pavic, Ksenija Marjanovic, Valerija Blazicevic, Ivan Miskulin and Matija Domacinovic Intestinal Morphology in Broiler Chickens Supplemented with Propolis and Bee Pollen Reprinted from: Animals 2019 , 9 , 301, doi:10.3390/ani9060301 . . . . . . . . . . . . . . . . . . . . 65 Jason D. Keegan, Giorgio Fusconi, Mauro Morlacchini and Colm A. Moran Whole-Life or Fattening Period Only Broiler Feeding Strategies Achieve Similar Levels of Omega-3 Fatty Acid Enrichment Using the DHA-Rich Protist, Aurantiochytrium limacinum Reprinted from: Animals 2019 , 9 , 327, doi:10.3390/ani9060327 . . . . . . . . . . . . . . . . . . . . 77 v Doaa Ibrahim, Asmaa T.Y. Kishawy, Safaa I. Khater, Ahmed Hamed Arisha, Haiam A. Mohammed, Ahmed Shaban Abdelaziz, Ghada I. Abd El-Rahman and Mohamed Tharwat Elabbasy Effect of Dietary Modulation of Selenium Form and Level on Performance, Tissue Retention, Quality of Frozen Stored Meat and Gene Expression of Antioxidant Status in Ross Broiler Chickens Reprinted from: Animals 2019 , 9 , 342, doi:10.3390/ani9060342 . . . . . . . . . . . . . . . . . . . . 91 Sivakumar Allur Subramaniyan, Da Rae Kang, Jin Ryong Park, Sharif Hasan Siddiqui, Palanisamy Ravichandiran, Dong Jin Yoo, Chong Sam Na and Kwan Seob Shim Effect of In Ovo Injection of L-Arginine in Different Chicken Embryonic Development Stages on Post-Hatchability, Immune Response, and Myo-D and Myogenin Proteins Reprinted from: Animals 2019 , 9 , 357, doi:10.3390/ani9060357 . . . . . . . . . . . . . . . . . . . . 111 Seyed Mohammad Ghoreyshi, Besma Omri, Raja Chalghoumi, Mehrdad Bouyeh, Alireza Seidavi, Mohammad Dadashbeiki, Massimo Lucarini, Alessandra Durazzo, Rene van den Hoven and Antonello Santini Effects of Dietary Supplementation of L-Carnitine and Excess Lysine-Methionine on Growth Performance, Carcass Characteristics, and Immunity Markers of Broiler Chicken Reprinted from: Animals 2019 , 9 , 362, doi:10.3390/ani9060362 . . . . . . . . . . . . . . . . . . . . 129 Shad Mahfuz and Xiang Shu Piao Application of Moringa ( Moringa oleifera ) as Natural Feed Supplement in Poultry Diets Reprinted from: Animals 2019 , 9 , 431, doi:10.3390/ani9070431 . . . . . . . . . . . . . . . . . . . . 147 K. F. M. Abouelezz, Y. Wang, W. Wang, X. Lin, L. Li, Z. Gou, Q. Fan and S. Jiang Impacts of Graded Levels of Metabolizable Energy on Growth Performance and Carcass Characteristics of Slow-Growing Yellow-Feathered Male Chickens Reprinted from: Animals 2019 , 9 , 461, doi:10.3390/ani9070461 . . . . . . . . . . . . . . . . . . . . 167 Ahmed A. Saleh, Abeer A. Kirrella, Safaa E. Abdo, Mahmoud M. Mousa, Nemat A. Badwi, Tarek A. Ebeid, Ahmed L. Nada and Mahmoud A. Mohamed Effects of Dietary Xylanase and Arabinofuranosidase Combination on the Growth Performance, Lipid Peroxidation, Blood Constituents, and Immune Response of Broilers Fed Low-Energy Diets Reprinted from: Animals 2019 , 9 , 467, doi:10.3390/ani9070467 . . . . . . . . . . . . . . . . . . . . 181 Ayman E. Taha, Osama A. AbdAllah, Khalil M. Attia, Ragaa E. Abd El-Karim, Mohamed E. Abd El-Hack, Mohamed A. El-Edel, Islam M. Saadeldin, Elsayed O. S. Hussein and Ayman A. Swelum Does in Ovo Injection of Two Chicken Strains with Royal Jelly Impact Hatchability, Post-Hatch Growth Performance and Haematological and Immunological Parameters in Hatched Chicks? Reprinted from: Animals 2019 , 9 , 486, doi:10.3390/ani9080486 . . . . . . . . . . . . . . . . . . . . 193 Jun Li, Yefei Cheng, Yueping Chen, Hengman Qu, Yurui Zhao, Chao Wen and Yanmin Zhou Dietary Chitooligosaccharide Inclusion as an Alternative to Antibiotics Improves Intestinal Morphology, Barrier Function, Antioxidant Capacity, and Immunity of Broilers at Early Age Reprinted from: Animals 2019 , 9 , 493, doi:10.3390/ani9080493 . . . . . . . . . . . . . . . . . . . . 207 Wenchao Liu, Yilin Yuan, Chenyu Sun, Balamuralikrishnan Balasubramanian, Zhihui Zhao and Lilong An Effects of Dietary Betaine on Growth Performance, Digestive Function, Carcass Traits, and Meat Quality in Indigenous Yellow-Feathered Broilers under Long-Term Heat Stress Reprinted from: Animals 2019 , 9 , 506, doi:10.3390/ani9080506 . . . . . . . . . . . . . . . . . . . . 219 vi Enayatullah Hamdard, Zengpeng Lv, Jingle Jiang, Quanwei Wei, Zhicheng Shi, Rahmani Mohammad Malyar, Debing Yu and Fangxiong Shi Responsiveness Expressions of Bitter Taste Receptors Against Denatonium Benzoate and Genistein in the Heart, Spleen, Lung, Kidney, and Bursa Fabricius of Chinese Fast Yellow Chicken Reprinted from: Animals 2019 , 9 , 532, doi:10.3390/ani9080532 . . . . . . . . . . . . . . . . . . . . 233 Mahmoud Alagawany, Shaaban S. Elnesr, Mayada R. Farag, Mohamed E. Abd El-Hack, Asmaa F. Khafaga, Ayman E. Taha, Ruchi Tiwari, Mohd. Iqbal Yatoo, Prakash Bhatt, Gopi Marappan and Kuldeep Dhama Use of Licorice ( Glycyrrhiza glabra ) Herb as a Feed Additive in Poultry: Current Knowledge and Prospects Reprinted from: Animals 2019 , 9 , 536, doi:10.3390/ani9080536 . . . . . . . . . . . . . . . . . . . . 251 Mahmoud Alagawany, Shaaban S. Elnesr, Mayada R. Farag, Mohamed E. Abd El-Hack, Asmaa F. Khafaga, Ayman E. Taha, Ruchi Tiwari, Mohd. Iqbal Yatoo, Prakash Bhatt, Sandip Kumar Khurana and Kuldeep Dhama Omega-3 and Omega-6 Fatty Acids in Poultry Nutrition: Effect on Production Performance and Health Reprinted from: Animals 2019 , 9 , 573, doi:10.3390/ani9080573 . . . . . . . . . . . . . . . . . . . . 263 Mahmoud Mostafa Azzam, Rashed Alhotan, Abdulaziz Al-Abdullatif, Saud Al-Mufarrej, Mohammed Mabkhot, Ibrahim Abdullah Alhidary and Chuntian Zheng Threonine Requirements in Dietary Low Crude Protein for Laying Hens under High-Temperature Environmental Climate Reprinted from: Animals 2019 , 9 , 586, doi:10.3390/ani9090586 . . . . . . . . . . . . . . . . . . . . 283 Zaheer Ahmad, Ming Xie, Yongbao Wu and Shuisheng Hou Effect of Supplemental Cyanocobalamin on the Growth Performance and Hematological Indicators of the White Pekin Ducks from Hatch to Day 21 Reprinted from: Animals 2019 , 9 , 633, doi:10.3390/ani9090633 . . . . . . . . . . . . . . . . . . . . 295 Federica Mannelli, Sara Minieri, Giovanni Tosi, Giulia Secci, Matteo Daghio, Paola Massi, Laura Fiorentini, Ilaria Galigani, Silvano Lancini, Stefano Rapaccini, Mauro Antongiovanni, Simone Mancini and Arianna Buccioni Effect of Chestnut Tannins and Short Chain Fatty Acids as Anti-Microbials and as Feeding Supplements in Broilers Rearing and Meat Quality Reprinted from: Animals 2019 , 9 , 659, doi:10.3390/ani9090659 . . . . . . . . . . . . . . . . . . . . 305 Mingming Shen, Zechen Xie, Minghui Jia, Anqi Li, Hongli Han, Tian Wang and Lili Zhang Effect of Bamboo Leaf Extract on Antioxidant Status and Cholesterol Metabolism in Broiler Chickens Reprinted from: Animals 2019 , 9 , 699, doi:10.3390/ani9090699 . . . . . . . . . . . . . . . . . . . . 321 Enayatullah Hamdard, Zhicheng Shi, Zengpeng Lv, Ahmadullah Zahir, Quanwei Wei, Mohammad Malyar Rahmani and Fangxiong Shi Denatonium Benzoate-Induces Oxidative Stress in the Heart and Kidney of Chinese Fast Yellow Chickens by Regulating Apoptosis, Autophagy, Antioxidative Activities and Bitter Taste Receptor Gene Expressions Reprinted from: Animals 2019 , 9 , 701, doi:10.3390/ani9090701 . . . . . . . . . . . . . . . . . . . . 335 vii Sylwia Orczewska-Dudek and Mariusz Pietras The Effect of Dietary Camelina sativa Oil or Cake in the Diets of Broiler Chickens on Growth Performance, Fatty Acid Profile, and Sensory Quality of Meat Reprinted from: Animals 2019 , 9 , 734, doi:10.3390/ani9100734 . . . . . . . . . . . . . . . . . . . . 365 Shuyun Ji, Xi Qi, Shuxue Ma, Xing Liu and Yuna Min Effects of Dietary Threonine Levels on Intestinal Immunity and Antioxidant Capacity Based on Cecal Metabolites and Transcription Sequencing of Broiler Reprinted from: Animals 2019 , 9 , 739, doi:10.3390/ani9100739 . . . . . . . . . . . . . . . . . . . . 381 Cebisa Kumanda, Victor Mlambo and Caven Mguvane Mnisi Valorization of Red Grape Pomace Waste Using Polyethylene Glycol and Fibrolytic Enzymes: Physiological and Meat Quality Responses in Broilers Reprinted from: Animals 2019 , 9 , 779, doi:10.3390/ani9100779 . . . . . . . . . . . . . . . . . . . . 395 Alberto Vi ̃ nado, Lorena Castillejos and Ana Cristina Barroeta Soybean Lecithin High in Free Fatty Acids for Broiler Chicken Diets: Impact on Performance, Fatty Acid Digestibility and Saturation Degree of Adipose Tissue Reprinted from: Animals 2019 , 9 , 802, doi:10.3390/ani9100802 . . . . . . . . . . . . . . . . . . . . 407 Minyu Zhou, Yuheng Tao, Chenhuan Lai, Caoxing Huang, Yanmin Zhou and Qiang Yong Effects of Mannanoligosaccharide Supplementation on the Growth Performance, Immunity, and Oxidative Status of Partridge Shank Chickens Reprinted from: Animals 2019 , 9 , 817, doi:10.3390/ani9100817 . . . . . . . . . . . . . . . . . . . . 421 Sajid Khan Tahir, Muhammad Shahbaz Yousaf, Sohrab Ahmad, Muhammad Khurram Shahzad, Ather Farooq Khan, Mohsin Raza, Khalid Abdul Majeed, Abia Khalid, Hafsa Zaneb, Imtiaz Rabbani and Habib Rehman Effects of Chromium-Loaded Chitosan Nanoparticles on the Intestinal Electrophysiological Indices and Glucose Transporters in Broilers Reprinted from: Animals 2019 , 9 , 819, doi:10.3390/ani9100819 . . . . . . . . . . . . . . . . . . . . 433 Tengfei He, Shenfei Long, Shad Mahfuz, Di Wu, Xi Wang, Xiaoman Wei and Xiangshu Piao Effects of Probiotics as Antibiotics Substitutes on Growth Performance, Serum Biochemical Parameters, Intestinal Morphology, and Barrier Function of Broilers Reprinted from: Animals 2019 , 9 , 985, doi:10.3390/ani9110985 . . . . . . . . . . . . . . . . . . . . 445 Shad Mahfuz and Xiangshu Piao Use of Medicinal Mushrooms in Layer Ration Reprinted from: Animals 2019 , 9 , 1014, doi:10.3390/ani9121014 . . . . . . . . . . . . . . . . . . . . 455 Damini Kothari, Woo-Do Lee, Kai-Min Niu and Soo-Ki Kim The Genus Allium as Poultry Feed Additive: A Review Reprinted from: Animals 2019 , 9 , 1032, doi:10.3390/ani9121032 . . . . . . . . . . . . . . . . . . . 469 Abd Ur Rehman, Muhammad Arif, Muhammad M. Husnain, Mahmoud Alagawany, Mohamed E. Abd El-Hack, Ayman E. Taha, Shaaban S. Elnesr, Mervat A. Abdel-Latif, Sarah I. Othman and Ahmed A. Allam Growth Performance of Broilers as Influenced by Different Levels and Sources of Methionine Plus Cysteine Reprinted from: Animals 2019 , 9 , 1056, doi:10.3390/ani9121056 . . . . . . . . . . . . . . . . . . . . 491 viii Majid Shakeri, Jeremy James Cottrell, Stuart Wilkinson, Weicheng Zhao, Hieu Huu Le, Rachel McQuade, John Barton Furness and Frank Rowland Dunshea Dietary Betaine Improves Intestinal Barrier Function and Ameliorates the Impact of Heat Stress in Multiple Vital Organs as Measured by Evans Blue Dye in Broiler Chickens Reprinted from: Animals 2020 , 10 , 38, doi:10.3390/ani10010038 . . . . . . . . . . . . . . . . . . . . 503 Silje Granstad, Anja B. Kristoffersen, Sylvie L. Benestad, Siri K. Sjurseth, Bruce David, Line Sørensen, Arnulf Fjermedal, Dag H. Edvardsen, Gorm Sanson, Atle Løvland and Magne Kaldhusdal Effect of Feed Additives as Alternatives to In-feed Antimicrobials on Production Performance and Intestinal Clostridium perfringens Counts in Broiler Chickens Reprinted from: Animals 2020 , 10 , 240, doi:10.3390/ani10020240 . . . . . . . . . . . . . . . . . . . 517 Mahmoud M. Abo Ghanima, Mohamed F. Elsadek, Ayman E. Taha, Mohamed E. Abd El-Hack, Mahmoud Alagawany, Badreldin M. Ahmed, Mona M. Elshafie and Karim El-Sabrout Effect of Housing System and Rosemary and Cinnamon Essential Oils on Layers Performance, Egg Quality, Haematological Traits, Blood Chemistry, Immunity, and Antioxidant Reprinted from: Animals 2020 , 10 , 245, doi:10.3390/ani10020245 . . . . . . . . . . . . . . . . . . . 537 Xingyong Chen, Kaiqin He, Congcong Wei, Wanli Yang and Zhaoyu Geng Green Tea Powder Decreased Egg Weight Through Increased Liver Lipoprotein Lipase and Decreased Plasma Total Cholesterol in an Indigenous Chicken Breed Reprinted from: Animals 2020 , 10 , 370, doi:10.3390/ani10030370 . . . . . . . . . . . . . . . . . . . 553 Diaa E. Abou-Kassem, Mohamed E. Abd El-Hack, Ayman E. Taha, Jamaan S. Ajarem, Saleh N. Maodaa and Ahmed A. Allam Detoxification Impacts of Ascorbic Acid and Clay on Laying Japanese Quail Fed Diets Polluted by Various Levels of Cadmium Reprinted from: Animals 2020 , 10 , 372, doi:10.3390/ani10030372 . . . . . . . . . . . . . . . . . . . 563 Youssef Attia, Mahmoud El-kelawy, Mohammed Al-Harthi and Ali El-Shafey Impact of Multienzymes Dose Supplemented Continuously or Intermittently in Drinking Water on Growth Performance, Nutrient Digestibility, and Blood Constituents of Broiler Chickens Reprinted from: Animals 2020 , 10 , 375, doi:10.3390/ani10030375 . . . . . . . . . . . . . . . . . . . 581 Ahmed A. Saleh, Khairy A. Amber, Mahmoud M. Mousa, Ahmed L. Nada, Wael Awad, Mahmoud A.O. Dawood, Abd El-Moneim E. Abd El-Moneim, Tarek A. Ebeid and Mohamed M. Abdel-Daim A Mixture of Exogenous Emulsifiers Increased the Acceptance of Broilers to Low Energy Diets: Growth Performance, Blood Chemistry, and Fatty Acids Traits Reprinted from: Animals 2020 , 10 , 437, doi:10.3390/ani10030437 . . . . . . . . . . . . . . . . . . . 595 Long Li, K.F.M. Abouelezz, Zhonggang Cheng, A.E.G. Gad-Elkareem, Qiuli Fan, Fayuan Ding, Jun Gao, Shouqun Jiang and Zongyong Jiang Modelling Methionine Requirements of Fast- and Slow-Growing Chinese Yellow-Feathered Chickens during the Starter Phase Reprinted from: Animals 2020 , 10 , 443, doi:10.3390/ani10030443 . . . . . . . . . . . . . . . . . . . 605 ix Elsayed O.S. Hussein, Shamseldein H. Ahmed, Alaeldein M. Abudabos, Gamaleldin M. Suliman, Mohamed E. Abd El-Hack, Ayman A. Swelum and Abdullah N. Alowaimer Ameliorative Effects of Antibiotic-, Probiotic- and Phytobiotic-Supplemented Diets on the Performance, Intestinal Health, Carcass Traits, and Meat Quality of Clostridium perfringens - Infected Broilers Reprinted from: Animals 2020 , 10 , 669, doi:10.3390/ani10040669 . . . . . . . . . . . . . . . . . . . 623 Ahmed A. Saleh, Bilal Ahamad Paray and Mahmoud A.O. Dawood Olive Cake Meal and Bacillus licheniformis Impacted the Growth Performance, Muscle Fatty Acid Content, and Health Status of Broiler Chickens Reprinted from: Animals 2020 , 10 , 695, doi:10.3390/ani10040695 . . . . . . . . . . . . . . . . . . . 637 Fayiz M. Reda, Mohamed T. El-Saadony, Shaaban S. Elnesr, Mahmoud Alagawany and Vincenzo Tufarelli Effect of Dietary Supplementation of Biological Curcumin Nanoparticles on Growth and Carcass Traits, Antioxidant Status, Immunity and Caecal Microbiota of Japanese Quails Reprinted from: Animals 2020 , 10 , 754, doi:10.3390/ani10050754 . . . . . . . . . . . . . . . . . . . 653 Vera Perricone, Marcello Comi, Carlotta Giromini, Raffaella Rebucci, Alessandro Agazzi, Giovanni Savoini and Valentino Bontempo Green Tea and Pomegranate Extract Administered During Critical Moments of the Production Cycle Improves Blood Antiradical Activity and Alters Cecal Microbial Ecology of Broiler Chickens Reprinted from: Animals 2020 , 10 , 785, doi:10.3390/ani10050785 . . . . . . . . . . . . . . . . . . . 667 Bozena Hosnedlova, Katerina Vernerova, Rene Kizek, Riccardo Bozzi, Jaromir Kadlec, Vladislav Curn, Frantisek Kouba, Carlos Fernandez, Vlastislav Machander and Hana Horna Associations Between IGF1, IGFBP2 and TGFß3 Genes Polymorphisms and Growth Performance of Broiler Chicken Lines Reprinted from: Animals 2020 , 10 , 800, doi:10.3390/ani10050800 . . . . . . . . . . . . . . . . . . . 681 Roua Gabriela Popescu, Sorina Nicoleta Voicu, Gratiela Gradisteanu Pircalabioru, Alina Ciceu, Sami Gharbia, Anca Hermenean, Sergiu Emil Georgescu, Tatiana Dumitra Panaite and Anca Dinischiotu Effects of Dietary Inclusion of Bilberry and Walnut Leaves Powder on the Digestive Performances and Health of Tetra SL Laying Hens Reprinted from: Animals 2020 , 10 , 823, doi:10.3390/ani10050823 . . . . . . . . . . . . . . . . . . . 705 Muhammad Abdul Basit, Arifah Abdul Kadir, Teck Chwen Loh, Saleha Abdul Aziz, Annas Salleh, Ubedullah Kaka and Sherifat Banke Idris Effects of Inclusion of Different Doses of Persicaria odorata Leaf Meal (POLM) in Broiler Chicken Feed on Biochemical and Haematological Blood Indicators and Liver Histomorphological Changes Reprinted from: Animals 2020 , 10 , 1209, doi:10.3390/ani10071209 . . . . . . . . . . . . . . . . . . 721 x About the Editor Vincenzo Tufarelli is an Associate Professor in Animal Nutrition at the Department of Emergency and Organ Transplants (DETO), Section of Veterinary Science and Animal Production of the University of Bari Aldo Moro, Italy. He has considerable experience in animal and poultry science, with a particular interest in nutrition and feed technology. He is involved in many research collaborations, even with international institutions, in the field of animal science and feed quality. He serves as an editorial board member and peer reviewer for many indexed journals and he is the author of more than 180 scientific papers published in international journals and proceedings of national and international conferences. xi Preface to ”Poultry Nutrition” Nutrition is defined in a range of ways, but is frequently inadequately understood. It is a simple concept, yet encompasses much complexity. In recent years, advances in poultry production, introduction of new and alternative products, and the development of new dietary management approaches have made it possible to increase poultry performance. However, to realize this, there must be further focus on diet quality. Producing suitable poultry products requires knowing the factors affecting quality, then exercising management accordingly. This book presents cross-discipline studies covering many aspects, ranging from poultry production and nutrition to alternative feeding systems, with the aim of disseminating information suitable for improving poultry health and products quality. Moreover, the purpose of this book is also to provide information about feed quality and alternative ingredients testing that can be used to improve poultry performance and producers’ returns. Vincenzo Tufarelli Editor xiii animals Article Activity and Anti-Aflatoxigenic E ff ect of Indigenously Characterized Probiotic Lactobacilli against Aspergillus flavus —A Common Poultry Feed Contaminant Nimra Azeem 1 , Muhammad Nawaz 1, *, Aftab Ahmad Anjum 1 , Shagufta Saeed 2 , Saba Sana 1 , Amina Mustafa 1 and Muhammad Rizwan Yousuf 3 1 Department of Microbiology, University of Veterinary and Animal Sciences, Lahore 54000, Punjab, Pakistan; nimra_azeem2011@hotmail.com (N.A.); aftab.anjum@uvas.edu.pk (A.A.A.); saba.sana@uvas.edu.pk (S.S.); aminamustafa046@gmail.com (A.M.) 2 Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore 54000, Punjab, Pakistan; shagufta.saeed@uvas.edu.pk 3 Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore 54000, Punjab, Pakistan; mryousuf@uvas.edu.pk * Correspondence: muhammad.nawaz@uvas.edu.pk; Tel.: + 92-03337773240; Fax: + 92-9211449-178 Received: 14 March 2019; Accepted: 11 April 2019; Published: 15 April 2019 Simple Summary: Mycotoxicosis in poultry has been seriously damaging the poultry production in Pakistan, resulting in economic losses to the country. The present study may act as a preliminary step for exploring the e ff ect of indigenously characterized potential probiotic lactobacilli on aflatoxin production by Aspergillus flavus . The present study explored anti-fungal Lactobacillus strains. Further investigations revealed their in vitro aflatoxin binding and anti-aflatoxigenic capabilities. These findings demonstrated L. gallinarum PL 149 to be an e ff ective binder of aflatoxin B1 which may be used as a biocontrol agent against A. flavus and aflatoxin B1 production. It may be further employed for aflatoxin binding in poultry gut after in vivo evaluations. Abstract: Aflatoxin contamination in human food and animal feed is a threat to public safety. Aflatoxin B1 (AFB1) can be especially damaging to poultry production and consequently economic development of Pakistan. The present study assessed the in vitro binding of AFB1 by indigenously characterized probiotic lactobacilli. Six isolates ( Lactobacillus gallinarum PDP 10, Lactobacillus reuetri FYP 38, Lactobacillus fermentum PDP 24, Lactobacillus gallinarum PL 53, Lactobacillus paracasei PL 120, and Lactobacillus gallinarum PL 149) were tested for activity against toxigenic Aspergillus flavus W-7.1 (AFB1 producer) by well di ff usion assay. Only three isolates (PL 53, PL 120, and PL 149) had activity against A. flavus W-7.1. The ameliorative e ff ect of these probiotic isolates on AFB1 production was determined by co-culturing fungus with lactobacilli for 12 days, followed by aflatoxin quantification by high-performance liquid chromatography. In vitro AFB1 binding capacities of lactobacilli were determined by their incubation with a standard amount of AFB1 in phosphate bu ff er saline at 37 ◦ C for 2 h. AFB1 binding capacities of isolates ranged from 28–65%. Four isolates (PDP 10, PDP 24, PL 120, and PL 149) also ceased aflatoxin production completely, whereas PL 53 showed 55% reduction in AFB1 production as compared to control. The present study demonstrated Lactobacillus gallinarum PL 149 to be an e ff ective candidate AFB1 binding agent against Aspergillus flavus . These findings further support the binding ability of lactic acid bacteria for dietary contaminants. Keywords: Aflatoxin B1; Lactobacillus ; anti-fungal; Aspergillus flavus ; in vitro; poultry Animals 2019 , 9 , 166; doi:10.3390 / ani9040166 www.mdpi.com / journal / animals 1 Animals 2019 , 9 , 166 1. Introduction Poultry is one of the major sectors playing a role in the enhanced economic activity of Pakistan but still it faces a lot of problems, including mycotoxicosis. Mycotoxins are toxic secondary metabolites of fungal origin, which can cause various diseases and death in animals and humans. Ergot alkaloids, fumonisins, patulin, aflatoxin, citrinin, trichothecenes, ochratoxin A, and zearalenone are all examples of some di ff erent mycotoxins. Aflatoxins, produced by Aspergillus parasiticus , Aspergillus flavus , and Aspergillus nomius , are of great importance because of their biological and biochemical e ff ects on living systems [ 1 ]. Aflatoxin-producing molds are globally and can flourish on a variety of food and feed commodities during production, processing, storage, and transportation procedures [ 1 – 3 ]. These molds can infect crops, especially in hot and humid conditions, resulting in economic loss and adverse e ff ects on consumers’ health. Aflatoxin is a potent carcinogen, mutagen, contains hepatotoxic and immunosuppressant e ff ects and inhibit several metabolic systems resulting in liver and kidney damage [ 1 , 4 ]. Aflatoxin and citrinin cause increased fragility of the vascular system and produce hemorrhages in body tissues. Among aflatoxins, aflatoxin B1 is the most potent, and it is categorized among class 1 human carcinogens. Di ff erent factors including pH, temperature, water activity, available nutrients, and competitive inhibition by other microorganisms can a ff ect aflatoxin production in feed [ 3 ]. Appropriate harvesting and storage conditions of crops and feed play important roles in aflatoxin reduction. Various methods have been employed for the removal or inactivation of aflatoxins, including physical, biological, and chemical methods. Chemical treatments may include roasting, ammoniation, and other solvent extraction techniques. Many aflatoxin binders, like activated carbon and various mineral clays, are commercially available and act as sequestering agents and tightly bind aflatoxin; the resulting binding complex is then excreted from the animal’s body [ 5 ]. These toxin binders can restore the nutritional value of the feed, but these chemical methods are unsafe, unhealthy, and expensive [ 6 ]. Toxin removal by microorganisms is a promising and economical method for decontaminating raw materials and food [ 7 ]. Numerous investigations have reported the inhibitory e ff ects of microbes including actinomycetes, yeast, mold, and bacteria on mold growth and aflatoxin production [ 3 ]. Thus, beneficial microorganisms may serve as an alternative therapy for mycotoxicosis. Anti-mutagenic lactic acid bacteria can remove mutagens from food by physical means [ 8 ]. Toxin binding by bacteria occurs through cell wall components, namely polysaccharides or polypeptides. Many researchers have studied this binding mechanism, but the exact mechanism of binding is still unknown [9]. Researchers are paying more attention towards preventing the absorption of aflatoxins in the gastrointestinal tracts of users by the aid of probiotic bacterial supplements in food and feed [ 10 ]. According to the World Health Organization (WHO), probiotics are defined as live microorganisms which when administered in adequate amounts exert healthy e ff ects to host [ 11 ]. Lactobacillus , Bifidobacterium , Enterococcus , Saccharomyces , and Bacillus may serve as probiotics. Lactobacilli can e ffi ciently remove aflatoxins from contaminated broth. The toxin removal mechanism involves sequestration by binding the toxin to the cell wall instead of metabolic degradation [ 12 ]. The present study may act as a preliminary step for studying the e ff ect of indigenously characterized potential probiotic lactobacilli on aflatoxin production by Aspergillus flavus , so that lactobacilli can be used as biocontrol agents. The present study also assessed the in vitro AFB1 binding capacity of Lactobacillus spp., so that these probiotic strains can be employed as toxin binders in place of chemicals in animal feed and thereby the harmful e ff ects of chemical toxin binders can be avoided. 2 Animals 2019 , 9 , 166 2. Materials and Methods 2.1. Identification of Isolates Previously characterized probiotic lactobacilli ( n = 6) of poultry and fermented food origin [ 13 ] and toxigenic Aspergillus flavus W-7.1 were procured from the Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, as listed in Table 1. Lactobacilli were revived using De Man, Rogosa, and Sharpe (MRS) agar and identified as describe previously [ 14 ]. Fungal strain was cultured on Sabouraud Dextrose Agar (SDA) medium incubated at 37 ◦ C for 5–6 days. Culture and microscopic characters were observed for identification as described previously [15]. Table 1. Antifungal activity of cell free supernatants of lactobacilli. Isolates GenBank Accession # Zones of Inhibition (mm) pH 4 pH 7 L. gallinarum PDP 10 MF980924 NZ NZ L. reuteri PDP 24 MF980925 NZ NZ L. fermentum FYP 38 MF980923 NZ NZ L. gallinarum PL 53 MK182967 13 12 L. paracasei PL 120 MK182968 16 14 L. gallinarum PL 149 MK182969 17 15 NZ: No zone of inhibition. 2.2. Antifungal Activity of Lactobacilli Antifungal activity of lactobacilli ( n = 6) was determined by well di ff usion assay as described elsewhere [ 16 ]. Briefly, SDA medium seeded with fungal spores (10 7 spores / mL) was poured into sterile Petri dishes and allowed to solidify. Wells were punctured in the medium which were then sealed with sterile molten agar. Cell free supernatant (100 μ L) of each lactobacilli strain was added into the respective wells. After 3–4 days incubation at 28 ◦ C aerobically, the diameter of zones of inhibition (mm) was measured. 2.3. E ff ect of Lactobacilli on Aflatoxin Production The effect of lactobacilli on aflatoxin production by Aspergillus flavus was observed by inoculating 1 mL bacterial suspension (1 McFarland) in yeast extract sucrose broth (YESB) supplemented with a standard amount of fungal spores (10 7 spores / mL), followed by incubation at 28 ◦ C and 100 rpm for 10 days. YESB media supplemented with known fungal spores and plain YESB media without any inoculation were also incubated as positive and negative controls, respectively. After incubation, medium containing lactobacilli and fungus was filtered through Whatman filter paper no 1 and aflatoxin B1 quantity in filtrate was measured by high-performance liquid chromatography (HPLC) and compared with controls [6]. Aflatoxin B1 was detected by HPLC and quantified using the following formulae: Quantity of Aflatoxins ( ng mL ) = peak area of sample peak area of standard × 100 (1) % age reduction = 1 − ( Peak area of AFB1 in treatment ) ( Peak area of AFB1 in control ) (2) 2.4. Aflatoxin B1 Extraction For toxin extraction, a previously established protocol was used with modifications [ 17 ]. Briefly, broth culture of Aspergillus flavus was autoclaved at 121 ◦ C and 15 psi and then homogenized using homogenizer. Twenty-five grams of homogenate was treated with chloroform (90 mL), methanol (10 mL), NaCl (5 g), and distilled water (10 mL) and incubated at 37 ◦ C with continuous shaking 3 Animals 2019 , 9 , 166 (150–160 rpm) for 30 min. Filtration was carried out using Whatman filter paper #4 and filtrate was concentrated in a water bath at 50 ◦ C. Concentrate was ground to fine powder and reconstituted in 3 mL chloroform volume and stored at 4 ◦ C. 2.5. Toxin Binding Assay Standard aflatoxin B1 solution was prepared by the method described elsewhere [ 18 ]. Prepared standard aflatoxin solution was then added to sterile phosphate bu ff er saline (PBS) containing lactobacilli culture (1 McFarland). After 2 h of incubation, cells with bound toxin were separated by centrifugation at 10,000 rpm for 5 min and unbound aflatoxin in supernatant was quantified by HPLC. 2.6. High Performance Liquid Chromatography (HPLC) Aflatoxins were quantified by Agilent HPLC system, 1100 series (Agilent, Santa Clara, CA, USA) as described previously [ 19 ]. A mixture of acetonitrile, water, and methanol was used as mobile phase at a flow rate of 1 mL per minute. Mobile phase was firstly purified using a filtration assembly and then sonicated for 10 min at 20 ◦ C in order to avoid gas bubbles. Next, 20 μ L samples were injected using a micro-syringe. After 15 min, ultra violet (UV) absorbance was recorded at 254 nm. Sample peaks were analyzed and compared with standard UV absorption data of secondary metabolites at various retention times. Limit of detection (LOD) and limit of quantification (LOQ) of standard aflatoxin were 0.01 ng / mL–100 μ g / mL and 0.1 ng / mL–100 μ g / mL, respectively. 2.7. Statistical Analysis Mitigation of aflatoxin production and toxin binding capacity of lactobacilli was compared by one-way ANOVA (analysis of variance) followed by Turkey’s multiple comparison test using Graph pad prism 5.0 software (GraphPad Software, San Diego, CA, USA). 3. Results A total of six potential probiotic lactobacilli, including Lactobacillus gallinarum PDP 10, Lactobacillus reuteri PDP 24, Lactobacillus fermentum FYP 38, Lactobacillus gallinarum PL 53, Lactobacillus paracasei PL 120, and Lactobacillus gallinarum PL 149, were procured from the Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan. All isolates were Gram-positive rods and catalase negative. Only three isolates (PL 53, PL 120, and PL 149) had antifungal activity observed by well di ff usion assay, as illustrated in Table 1 and Figure 1. Figure 1. Activity of cell free supernatant of Lactobacillus gallinarum PL 149 against Aspergillus flavus. 4 Animals 2019 , 9 , 166 Four isolates (PDP 10, PDP 24, PL 120, and PL 149) showed 100% removal of AFB1, PL 53 caused 55.2% reduction, while FYP 38 showed an enhancing e ff ect on aflatoxin B1 production, as described in Table 2. All isolates showed a varied degree of toxin binding capacities, as described in Table 3 and Figure 2. PL 149 was the most e ff ective binder of aflatoxin B1, with 65% capacity. Table 2. E ff ect of lactobacilli on aflatoxin B1 production. Isolates Peak Areas Quantity of AFB1 (ng / mL) % Age Reduction Standard 120.205 100 - Control 0.58439 0.4 - L. gallinarum PDP 10 ND ND 100% L. fermentum FYP 38 0.815847 0.6 − 39.6% L. reuteri PDP 24 ND ND 100% L. gallinarum PL 53 0.26124 0.2 55.2% L. paracasei PL 120 ND ND 100% L. gallinarum PL 149 ND ND 100% AFB1: Aflatoxin B1; ND: Not detected. Table 3. Aflatoxin B1 binding capacity of probiotic lactobacilli. Isolates Peak Areas Quantity of AFB1 Bound (ng / mL) % Age Reduction (Binding Capacity) Standard 108.246 100 - Control 927.763 857 - L. gallinarum PDP 10 451.63 417.2 51.3% L. fermentum FYP 38 407.553 376.5 56% L. reuteri PDP 24 909.624 840 2% L. gallinarum PL 53 546.523 504.8 42% L. paracasei PL 120 676.472 624.9 28% L. gallinarum PL 149 326.775 301.8 65% AFB1: Aflatoxin B1. Figure 2. High-performance liquid chromatography chromatograms of aflatoxin B1 present in control and suspension after treatment with lactobacilli: ( a ) Control; ( b ) PDP 10; ( c ) FYP 38; ( d ) PL 149. 5