The Journal of Applied Poultry Research

VOLUME 23 NUMBER 1 MARCH 2014 THE JOURNAL OF APPLIED POULTRY RESEARCH

March 2014 Volume 23, Number 1

CONTENTS Research Reports Effect of a synbiotic on the intestinal microflora of chickens Seyed Morteza Dibaji, Alireza Seidavi, Leila Asadpour, and Fernando Moreira da Silva 1 Exposure of workers to dust and bioaerosol on a poultry farm S. B. Jerez, Y. Cheng, and J. Bray 7 Effect of different sources and levels of selenium on performance, meat quality, and tissue characteristics of broilers T. F. B. Oliveira, D. F. R. Rivera, F. R. Mesquita, H. Braga, E. M. Ramos, and A. G. Bertechini 15 The effects of an enzyme complex in moderate and low nutrient-dense diets with dried distillers grains with solubles in laying hens Dana Hahn-Didde and Sheila E. Purdum 23 Relationships of incubational hatching egg characteristics to posthatch body weight and processing yield in Ross × Ross 708 broilers E. D. Peebles, R. Pulikanti, W. Zhai, and P. D. Gerard 34 Effects of distillers dried grains with solubles and mineral sources on gaseous emissions W. Li, Q.-F. Li, W. Powers, D. Karcher, R. Angel, and T. J. Applegate 41 Wet litter not only induces footpad dermatitis but also reduces overall welfare, technical performance, and carcass yield in broiler chickens Ingrid C. de Jong, H. Gunnink, and J. van Harn 51 Effect of production system and flock age on egg quality and total bacterial load in commercial laying hens Samiullah, J. R. Roberts, and K. K. Chousalkar 59 The effect of raw and roasted soybeans on intestinal health, diet digestibility, and pancreas weight of broilers C. Rocha, J. F. Durau, L. N. E. Barrilli, F. Dahlke, P. Maiorka, and A. Maiorka 71 Economic effects of proposed changes in living conditions for laying hens under the National Organic Program Tomislav Vukina, Kenneth Anderson, and Mary K. Muth 80 Suitability of poultry litter ash as a feed supplement for broiler chickens J. P. Blake and J. B. Hess 94 Poultry litter ash as a replacement for dicalcium phosphate in broiler diets J. P. Blake and J. B. Hess 101 Time study examining the effect of range, cage-free, and cage environments on man-hours committed to bird care in 3 brown egg layer strains Kenneth E. Anderson 108 Salmonella Typhimurium in chicken manure reduced or eliminated by addition of LT1000 C. L. Sheffield, T. L. Crippen, R. C. Beier, and J. A. Byrd 116 The inner perivitelline layer sperm hole assay: Use of filter paper rings for the isolation of the perivitelline layer overlying the germinal disc and new observations on its morphology Murray R. Bakst, Janet Eastridge, and Irek A. Malecki 121 Field Reports The effect of feeding Hydrogel-95 to emu chicks at hatch Zachary Lowman and Carmen Parkhurst 129 Evaluation of recovery of Salmonella from trachea and ceca in commercial poultry G. Kallapura, A. Botero, S. Layton, L. R. Bielke, J. D. Latorre, A. Menconi, X. Hernández-Velasco, D. J. Bueno, B. M. Hargis, and G. Téllez 132

Please note that the Instructions to Authors can now be found online (http://japr.oxfordjournals.org) and do not appear in the print issue. © 2014 Poultry Science Association, Inc. Effect of a synbiotic on the intestinal microflora of chickens 1

Seyed Morteza Dibaji ,* Alireza Seidavi ,*2 Leila Asadpour ,† and Fernando Moreira da Silva ‡2

* Department of Animal Science, and † Department of Veterinary Science, Rasht Branch, Islamic Azad University, Rasht 41335-3516, Iran; and ‡ University of the Azores, Faculty of Agrarian Sciences, Department of Animal Reproduction, Research Centre for Agricultural and Environmental Science and Technology of the Azores, Angra do Heroísmo 9700, Portugal Downloaded from

Primary Audience: Agricultural Faculty, Poultry Farmers, Poultry Microbiologists

SUMMARY http://japr.oxfordjournals.org/ The current experiment was conducted to evaluate the effect of a synbiotic (Biomin Imbo) on intestinal microflora of Ross broiler chickens. A total of 200 male chickens were randomly divided into 20 groups of 10 birds each, kept in 20 pens (1.5 × 1 m each), and fed for 42 d at different synbiotic levels. Treatments included (1) a basal diet without synbiotics (control), (2)

a basal diet with synbiotic levels proposed by the manufacturer, (3) a basal diet with synbiotic levels 25% lower than those proposed by the manufacturer, (4) a basal diet with synbiotic levels

50% higher than those proposed by the manufacturer, and (5) a basal diet with synbiotic levels by guest on February 24, 2014 25% higher than those proposed by the manufacturer. At the end of the 42-d feeding period, 1 bird was randomly selected from each experimental unit, humanely euthanized, and the cecum was removed to measure the microbial population. The cecal contents were collected in discharge containers for microbial cultures, and counts were conducted after microbial culture. The addition of the synbiotic reduced Escherichia coli and total coliform populations in the intestines of broiler chickens. Conversely, different levels of probiotic increased the numbers of lactobacilli in the intestine of broiler chickens. Concentrations of the synbiotic higher than the recommended levels in the diet increased the lactic acid bacteria population in the gut of broiler chickens.

Key words: synbiotic, intestinal microflora, broiler chicken 2014 J. Appl. Poult. Res. 23 :1–6 http://dx.doi.org/ 10.3382/japr.2012-00709

DESCRIPTION OF PROBLEM hunger persists in many locations and almost 1 billion people are reported to be malnourished The population of the world has increased [1]. By 2050, farmers will need to double crop from 3 billion in 1959 to more than 7 billion in production to meet the demand. In this con- March 2012. As the world’s population grows, text, the world needs food products with annual

1This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 Corresponding authors: [email protected] and [email protected] 2 JAPR: Research Report growth of 2.5% for the next 10 yr [2]. Consider- thereby increasing the resistance to disease [9]. ing these statistics, many researchers and nutri- This study was designed to evaluate the effect of tion experts believe that poultry production can synbiotic Biomin Imbo on intestinal microflora play a role in increasing food production. Poul- of broiler chickens. try can make positive contributions to the diet of those with low incomes, as it is consistently high MATERIALS AND METHODS quality, low in saturated fats, as well as more At the Faculty of Agriculture, Islamic Azad healthy and affordable when compared with University, 200 one-day-old male Ross 308 other meats. Thus, it is a source of essential nu- chicks were randomly divided into 20 groups of trients and sought after worldwide. In addition, 10 birds each and kept in 20 pens (1.5 × 1 m as chicken has an FCR of less than 2.0, which each) in the same room separated from each oth- is much lower than other livestock, poultry can er by a metal mesh wall. Birds received 22L:2D play an important role providing an inexpensive throughout the study period. Room temperature protein source and alleviating world hunger. was maintained at 30 to 33°C for the first week Downloaded from To protect health and enhance the perfor- and then reduced gradually 2.8°C every week mance of chickens, such as immune response, to 20°C; this temperature was then maintained intestinal health status, reducing morbidity and for the duration of the study. Each treatment mortality, improving digestion, and feed conver- included 4 repetitions (10 birds per repetition), sion, additives (e.g., antibiotics) are sometimes where birds had ad libitum access to feed and http://japr.oxfordjournals.org/ used, possibly causing gastrointestinal distur- water provided in chute feeders for 42 d, with bances and adding to concern over antibiotic different synbiotic levels (Biomin Imbo [10]). resistance. Probiotic bacteria are a sustainable approach to modulate the gut microflora toward • Treatment 1: Basal diet without synbiotic a more favorable composition. Probiotics should (control). contribute to efficient production in a sustain- • Treatment 2: Standard Ross diet based on able way, promoting healthy and robust animals catalog (base) with the levels of Biomin [3]. Probiotics are considered live microorgan- Imbo recommended by the manufacturer by guest on February 24, 2014 isms that are thought to be beneficial to the host (0.1% for the starter period, 0.05% for the organism. The concept behind probiotics was growth period, and 0.025% for the finish- introduced in the early 20th century by Elie er period). Metchnikoff, known as the father of probiotics. • Treatment 3: Standard Ross diet based on Probiotics were thought to benefit the host by catalog (base) with a 25% reduction in the improving its intestinal microbial balance, thus level of the synbiotic recommended by the inhibiting pathogens and toxin-producing bacte- manufacturer (0.075% for the starter pe- ria [4]. Today, specific health effects are being riod, 0.0375% for the growth period, and investigated and documented, including allevia- 0.01875% for the finisher period). tion of chronic intestinal inflammatory diseases • Treatment 4: Standard Ross diet based on [5], and prevention and treatment of pathogen- catalog (base) with a 50% increase in the induced diarrhea [6], urogenital infections [7], level of the synbiotic recommended by and atopic diseases [8]. the manufacturer (0.15% for the starter The probiotic part of Biomin Imbo is Entero- period, 0.075% for the growth period, and coccus faecium (5 × 1110 cfu/kg), which helps 0.0375% for the finisher period). to prevent the growth of pathogenic microorgan- • Treatment 5: Standard Ross diet based on isms such as Salmonella [9]. This probiotic also catalog (base) with a 25% increase in the contains fructo-oligosaccharides that stimulate level of the synbiotic recommended by the the growth of bifidobacteria, which, along with manufacturer (0.125% for the starter pe- Enterococcus faecum, cause intestinal microflo- riod, 0.0625% for the growth period, and ra stability and act as a barrier against pathogens. 0.03125% for the finisher period). Other components of this probiotic (extracted Composition of the basal diet and its nutritive from seaweed) stimulate the activity of macro- characteristics are represented in Table 1 and phages and lymphocytes of the immune system, Table 2, respectively. Dibaji et al.: SYNBIOTIC IN CHICKEN 3

Table 1. Composition of the basal diet used in the experiment

Ingredient (%) Starter Grower Finisher

Corn 46.09 50.91 48.88 Soybean meal 40.00 35.00 39.97 Fish meal 3.00 3.00 — Meat meal 3.00 3.00 — Oil 4.56 5.45 7.38 dl-Met 0.29 0.23 0.17 l-Lys 0.04 — — l-Thr 0.03 — — Ca 22%, P 18% 0.99 0.75 1.64

CaCO3 0.98 0.76 1.00 KHCO3 0.05 0.03 — NaCl 0.37 0.37 0.45 Vitamin and mineral mixture1 0.60 0.50 0.50 Total 100 100 100 Downloaded from 1 Vitamin A: 5,000 IU/g; vitamin D3: 500 IU/g; vitamin E: 3 mg/g; vitamin K3: 1.5 mg/g; vitamin B2: 1 mg/g; calcium pantothenate: 4 mg/g; niacin: 15 mg/g; vitamin B6: 13 mg/g; Cu: 3 mg/g; Zn: 15 mg/g; Mn: 20 mg/g; Fe: 10 mg/g; K: 0.3 mg/g.

Birds were in pens for the entire 42 d. Seed in this study was approved by the Islamic Azad http://japr.oxfordjournals.org/ containers and water bottles were cleaned us- University Ethics Committee. The experimental ing a disinfectant solution of Despadak (ratio of procedures described herein were also approved 1:100 [11]). In addition, facilities (walls, ceil- by this committee, and care was taken to mini- ings, and shelves) were sprayed with the same mize the number of birds used. disinfectant. Manure produced during the exper- Agar plates were streaked with cecal con- iment was collected after the study ended. tents and sent to the laboratory of nutrition and At the end of the trial, a bird from each rep- dairy industry from the Islamic Azad University,

etition was euthanized and the cecum was re- about 100 m from bird housing. To determine by guest on February 24, 2014 moved for further culture. Use and care of birds bacterial growth and colony counts, the agar

Table 2. Nutritional composition (calculated) of basal diets used in the experiment

Ingredient (%, unless otherwise noted) Starter Grower Finisher

ME (kcal/kg) 3,025 3,150 3,200 CP 24.9 23 22 Lys 1.41 1.26 1.22 Met 0.67 0.59 0.50 Met + Cys 1.05 0.94 0.85 Thr 1.98 0.87 0.85 Trp 0.30 0.27 0.28 Arg 1.68 1.54 1.51 Ile 1.04 0.95 0.94 Val 1.60 1.07 1.03 Leu 1.99 1.87 1.82 Ca (%) 1.05 0.90 0.85 Available P 0.50 0.45 0.42 Na 0.23 0.23 0.20 K 1.00 0.90 0.93 Cl 0.30 0.30 0.30 Dietary cation-anion balance (mEq/kg) 272.12 244.55 242.77 Choline (g/kg) 1.48 1.37 1.37 Linoleic acid 1.21 1.27 1.24 CF 3.78 3.52 3.73 EE 6.84 7.87 9.22 4 JAPR: Research Report plates streaked on the site were used. Collect- amount of the total bacteria in the gut of broil- ing tubes were weighed, wrapped in an alu- ers (P < 0.05). Comparison of averages obtained minum sheet, and autoclaved for 10 min. The from the experiments showed that treatment 3, culture media were prepared 24 h before col- the recommended dosage, had the most influ- lected samples were poured into petri dishes. de ence on the total bacteria (Table 3). Concerning Man, Rogosa, Sharpe agar was used to culture the effect of different synbiotic levels on E. coli lactobacilli, eosin methylene blue was used to and coliform populations, results showed that culture Escherichia coli, MacConkey agar was additions of this probiotic reduced E. coli bac- used to culture coliforms, and nutrient agar was teria and total coliform populations in the intes- used to culture total aerobic bacteria. Samples tines of broiler chickens (P < 0.05). were transferred to the laboratory in the listed Different levels of probiotics increased the tubes and weighed again. The amount of sample numbers of lactobacilli in the intestine of broiler in each tube was calculated from the difference chickens (P < 0.05). Adding a higher concentra- between these 2 values. Tubes were shaken for tion than the recommended levels increased the Downloaded from approximately 30 min; the action was performed lactic acid bacteria population in gut of broiler for bacteria isolated from gastrointestinal con- chickens (P < 0.01). In contrast, adding recom- tents and preparation of suspension. One milli- mended or levels below recommendation did not liter was removed from the prepared suspension change the populations of lactic acid bacteria. and added to 9 mL of PBS in the other tube. The Based on our experiments, we found that the http://japr.oxfordjournals.org/ suspension was prepared from 10−1 dilutions supplement used had statistically significant ef- and serial dilutions were done (10−2, 10−3, 10−4, fects. Total bacteria and lactobacilli increased 10−5, and 10−6). Next, 100 μL was removed from in all treatments. Escherichia coli and coliform dilutions (10−4, 10−5, and 10−6) and poured into populations decreased in all treatments. Lac- the petri dish previously prepared containing the tic acid bacteria increased when the probiotic medium and completely distributed to all parts was added in excess of recommended dosages of the medium. Lactobacilli were incubated at (P < 0.05). Razavi et al. [13] studied the effect 37°C in anaerobic conditions for 72 h. An an- of probiotics and virginiamycin on the lactoba- by guest on February 24, 2014 aerobic jar was used to create anaerobic condi- cilli population in the ileum of broiler chickens tions. Total aerobic bacteria were incubated at and reported that the highest population was 37°C under aerobic conditions for 48 h. Count- observed in chickens that consume probiotics ing of bacteria in the petri dishes was done by a and antibiotics. Probiotics and can improve per- colony counter. Bacterial counts were reported formance in the ileum; however, organic acids as logarithm number of bacteria per 1-g sample. and virginiamycin did not improve performance Data were analyzed by SPSS [12] statistical compared with control treatment. Abrishami et software, and averages were compared by the al. [14] studied the effect of different prebiot- Tukey test. The statistical design was Xij = μ + Tj ics (Fermecto) and probiotics (cultured intesti- + eij, where Xij is the number of each control in nal contents) on the performance and microbial the experiment; μ represents the overall mean; population of the digestive system. Supplement- Tj is the effect of each group of the experimental ing with Fermecto, the number of lactobacilli in- diet; and eij is the effect of the error. The total creased aerobic bacteria up to 42 d in the diges- value of any observed treatment effects and the tive system. Adding cultured intestinal contents average test error was the result of the whole increased the number of lactobacilli and aero- population. Before performing statistical analy- bic bacteria up to 42 d in the digestive system. sis of data, all data were tested by a normality Those authors reported a significant effect in in- test. creasing the number of lactobacilli and aerobic bacteria. Adding Fermecto as a prebiotic (at the RESULTS AND DISCUSSION suggested level based on the source) can cause Microflora identified in each treatment (total performance improvement and partial change in bacteria, E. coli, coliform bacteria, lactobacilli, bacterial population. and lactic acid bacteria) are summarized in Ta- Other related studies included projects by ble 3. Additions of Biomin Imbo increased the Sabouni et al. [15], who demonstrated that pro- Dibaji et al.: SYNBIOTIC IN CHICKEN 5

biotics and prebiotics improve the number of lactobacilli and total bacteria in the ileum. Kabir et al. [16] demonstrated that probiotics are able ± 7.331 ± 7.065 ± 7.390 ± 7.293 ± 3.983 a b b b ab to eliminate harmful pathogens through compe- bacteria bacteria (log cfu/g) Lactic acid Lactic acid tition for joining to small gut. Broiler chickens fed probiotics had histological changes in the

intestine, including increased villi length and cellular levels. In addition, chickens fed with

± 7.106 ± 7.250 ± 7.217 8.448 ± 7.271 8.441 ± 6.757 8.462 8.542 8.591 lactobacilli have fewer coliforms in their feces. a b ab ab ab

bacteria bacteria Intestinal microflora can also be improved by (log cfu/g) Lactobacillus changing intestinal acidity by increasing lactic acid concentration in the intestine, reducing the activities of harmful bacteria (Salmonella and E. coli), and increasing lactobacilli [17]. Downloaded from ± 6.662 ± 2.893 ± 7.000 7.715 7.855 ± 7.039 7.822 ± 7.021 8.112 8.051 a b b Chichlowski et al. [18] reported that use of pro- ab ab bacteria bacteria Coliforms (log cfu/g) biotic and prebiotic products can improve intestine bacteria balance. Overall, the results of the above experiments [13–22] agree with our results. The use of the http://japr.oxfordjournals.org/ present probiotic resulted in an increase in to- ± 5.509 ± 6.487 ± 6.706 7.544 ± 6.149 6.753 7.829 6.761 ± 6.815 8.035 tal bacteria and gram-positive bacteria, such as a b b b b Escherichia

coli (log cfu/g) lactobacilli and lactic acid bacteria, but reduced coliform and E. coli. Our results do not agree with Khalaji et al. [21] and Rakhshan et al. [23], possibly because those researchers used differ- P < 0.05). ± 7.212 ± 0.222 8.037 ± 0.179 7.647 ± 7.937 7.554 ± 6.991 7.420 7.459 a a a a b ent additives. Khalaji et al. [21] studied the ef-

(log cfu/g) fect of the prebiotic Technomoss on the diges- by guest on February 24, 2014 Total bacteria bacteria Total 8.725 8.726 8.689 8.621 8.004 tive health of broiler chickens. According to the poultry industry, gastrointestinal disease is one of the most important problems, and concerns of resistance to antibiotics also exist. There- fore, a new trend has emerged toward the use of probiotics. The effect of a moss prebiotic on gut morphology and microbial intestine population in broiler chickens was studied by Rakh- shan et al. [23]. No significant difference was observed in the microbial number of lactobacilli and coliforms of intestinal contents among the different groups [23]. According to available reports, increasing congenital malformations, the occurrence of chronic diseases, and some other complications in humans could be avoided or reduced if the use of antibiotics was reduced and if probiotics and synbiotics, as alternatives, were more accepted [24]. Control Recommended amount by company 25% lower than the recommended amount by company 50% higher than the recommended amount by company 25% higher than the recommended amount by company Description CONCLUSIONS AND APPLICATIONS

1. In the present study, positive results were obtained when male Ross 308 chicks Treatment means that are not followed by the same letter differ significantly from one another ( means that are not followed by the same letter differ Treatment 1 2 3 4 5 Table 3. Mean comparison (± SEM) of cecum microflora among treatments Table Treatment a,b were fed a standard diet with a probiotic 6 JAPR: Research Report

added at 50% of the level recommended Feremco and perobiotic levels of cultured intestinal con- by the manufacturer, increasing benefi- tents on performance and bacterial population of digestive system. Pages 699–702 in 4th Iranian Animal Science Con- cial bacteria such as lactobacilli and lac- gress, Tehran University, Tehran-Karaj, Iran. tic acid. 15. Sabouni, S., N. Eila, M. Aalehi, and V. B. Gholam- 2. Because good microbes help develop a hossein. 2010. The effect of probiotics and prebiotic diet on performance and bacterial population and morphology of strong immune system, reduce morbidity gut ileum of broiler chicken. Pages 1070–1077 in 4th Iranian and mortality, and improve digestion and Animal Science Congress, Tehran University, Tehran-Karaj, feed conversion, the use of this probi- Iran. otic ameliorates the bird’s performance, 16. Kabir, S., M. M. Rahman, M. B. Rahman, and S. U. Ahmad. 2004. The dynamics of probiotics on growth perfor- thereby reducing production costs. mance and immune in broiler. Jpn. Poult. Sci. 3:61–64. 17. Vegas, J. L. 2004. Prebiotics, probiotic, acid fires and antibiotic growth promoters. Pages 339–346 in Poultry Dis- REFERENCES AND NOTES eases: A Guide for Farmers and Poultry Professionals. Inter- national Book Distributing Co., Charbagh, Lucknow, India. 1. Alexander, R. 2013. Does a child die of hunger ev- 18. Chichlowski, M., W. J. Croom, F. W. Edens, B. W. Downloaded from ery 10 seconds? BBC NEWS Magazine. Accessed Oct. 15, McBride, R. Qiu, C. C. Chiang, L. R. Daniel, G. B. Haven- 2013. http://www.bbc.co.uk/news/magazine-22935692. stein, and M. D. Koci. 2007. Microarchitecture and spatial 2. Mack, M. 2009. Role of technology is crucial in im- relationship between bacteria and ileal, cecal, and colonic proving food security. Syngenta CEO in USDA Outlook epithelium in chicks fed a direct-fed microbial, PrimaLac, Forum, Washington, DC. Syngenta International AG, Basel, and salinomycin. Poult. Sci. 86:1121–1132. Switzerland. 19. Willis, W. L., and L. Reid. 2008. Investigating the ef- 3. Brittain, A., C. S. Lee, and P. J. O’Brien. 2002. Mi- fects of dietary probiotic feeding regimens on broiler chick- http://japr.oxfordjournals.org/ crobial contributions to aquatic nutrition. Page 220 in Mi- en production and Campylobacter jejuni presence. Poult. crobial Approaches to Aquatic Nutrition in Environmentally Sci. 87:606–611. Sound Aquaculture Production Systems. C. S. Lee and P. 20. Lee, K. W., S. H. Lee, H. S. Lillehoj, G. X. Li, S. O’Bryen, ed. World Aquaculture Society, Baton Rouge, LA. I. Jang, U. S. Babu, M. S. Park, D. K. Kim, E. P. Lillehoj, 4. Metchnikoff, E. 1907. Essais optimistes. Paris. The A. P. Neumann, T. G. Rehberger, and G. R. Siragusa. 2010. prolongation of life. Optimistic studies. Chalmers. P. Mitch- Effects of direct-fed microbials on growth performance, gut el, ed. Springer Publishing Co. Inc., New York, NY. morphometry, and immune characteristics in broiler chick- 5. Mach, T. 2006. Clinical usefulness of probiotics in in- ens. Poult. Sci. 89:203–216. flammatory bowel diseases. J. Physiol. Pharmacol. 57(Sup- 21. Kannan, M., R. Karunakaran, V. Balakirishnan, and pl. 9):23–33. T. G. Parabhaken. 2005. Influence of probiotics supplemen- by guest on February 24, 2014 6. Yan, F., and D. B. Polk. 2006. Probiotics as functional tation on lipid profile of broilers. Int. J. Poult. Sci. 4:994– food in the treatment of diarrhea. Curr. Opin. Clin. Nutr. 997. Metab. Care 9:717–721. 22. Khalaji, S., M. Zaghari, and V. S. Nezafati. 2010. The 7. Reid, G. 2008. Probiotic lactobacilli for urogenital effect of probiotics Tecnomoss on digestive health, safety health in women. J. Clin. Gastroenterol. 42(Suppl 3 Pt and performance of broiler chicken. Pages 207–209 in 4th 2):S234–S236. Iranian Animal Science Congress, Tehran University, Teh- 8. Vanderhoof, J. A. 2008. Probiotics in allergy manage- ran-Karaj, Iran. ment. J. Pediatr. Gastroenterol. Nutr. 47(Suppl 2):S38–S40. 23. Rakhshan, M., S. Shivazad, M. Mousavi, and M. 9. Guarnera, F., and G. J. Schaafsmab. 1988. Probiotics. Zaghari. 2010. The effect of probiotics teknomoss on gut Int. J. Food Microbiol. 39:237–238. morphology and intestinal bacterial population in broiler chicken. Pages 703–706 in 4th Iranian Animal Science Con- 10. Biomin Holding GmbH, Herzogenburg, Austria. gress, Tehran University, Tehran-Karaj, Iran. 11. Lab. Calier S.A., Barcelona, Spain. 24. Azimi, J. 2008. Study of the effect of probiotics on 12. SPSS Inc., Chicago, IL. poultry feed. MSc Diss. Tehran University, Tehran, Iran. 13. Razavi, S. H., H. A. Shahriar, Y. Ebrahimnejad, A. Ahmadzadeh, M. Ghaderi, S. Goli, and V. N. Rezaee. 2010. The effect of probiotic, organic acid, prebiotic and antibiotic Acknowledgments on performance and Lactobacillus population in ileum of This manuscript is obtained from the MS thesis of Seyed broiler chicken. Pages 994–997 in 4th Iranian Animal Sci- Morteza Dibaji at Islamic Azad University. We are grateful ence Congress, Tehran University, Tehran-Karaj, Iran. to Islamic Azad University for support. The Research Centre 14. Abrishami, M. H., V. K. Zareaha, H. Kermanshahi, for Agricultural and Environmental Science and Technology and V. M. Pilehvar. 2010. The effect of different prebiotic of the Azores is also fully acknowledged. © 2014 Poultry Science Association, Inc. Exposure of workers to dust and bioaerosol on a poultry farm

S. B. Jerez ,*1 Y. Cheng ,* and J. Bray †

* Division of Environmental Science, and † Department of Agriculture, Arthur Temple College of Forestry and Agriculture, Stephen F. Austin State University, Nacogdoches, TX 75962

Primary Audience: Researchers, Flock Supervisors, Poultry Workers, Production Managers Downloaded from

SUMMARY

Poultry houses are known for generating excessive dust, which originates from bedding http://japr.oxfordjournals.org/ materials, fiberglass insulations, feed, dried fecal materials, and feather particles. Dust may contain microorganisms, including endotoxins, fungi, and bacteria, that may affect living things when inhaled. Dust that contains living organisms is referred to as bioaerosol, and its particle size may range from 0.5 to 100 µm. Respirable dust, which has an aerodynamic diameter of less than or equal to 4 µm, can travel to and be deposited in the gas-exchange region of the hu-

man respiratory system. This is of particular concern because of the greater health hazard that it poses. The concentrations of respirable dust and bioaerosol measured with samplers attached 3 to the workers (worker-exposure concentrations) were more than 3 (0.82 vs. 0.26 mg/m ) and by guest on February 24, 2014 one-and-a-half times (58.46 vs. 33.79 cfu/m3) higher, respectively, than the concentrations measured with stationary samplers indoors. The respirable dust is still below the permissible exposure limit (5 mg/m3) set by the Occupational Safety and Health Administration, but beyond the limit for animal buildings suggested by other researchers.

Key words: bioaerosol, dust , worker exposure 2014 J. Appl. Poult. Res. 23 :7–14 http://dx.doi.org/ 10.3382/japr.2012-00710

DESCRIPTION OF PROBLEM sues in and around these facilities have become significant. Concentrated animal feeding operations Malmberg and Larson [3] reported that in- greatly contribute to the ability of US producers halation of organic dust may cause an acute to meet mounting demands for the production of inflammatory reaction in the airways and fever meat, milk, poultry, and eggs [1]. Approximate- in nonsensitized subjects, which is called toxic ly 2,204,792 farm workers exist in the United pneumonities or organic dust syndrome. In addi- States, with an estimated 260,000 persons work- tion, a person exposed to a high level of dust may ing in livestock, dairy, and poultry farm facili- experience increased phlegm production and ties [2]. As livestock and poultry facilities have pulmonary inflammation 4 to 10 h after expo- evolved from small backyard farms to large con- sure that can last up to 24 h; conversely, chronic fined structures, health and environmental is- exposure may result in bronchitis and asthma

1 Corresponding author: [email protected] 8 JAPR: Research Report

[4]. The severity of dust damage to health not Broiler Research Center (BRC) at the Walter C. only depends on the inhaled concentrations but Todd Agricultural Research Center of Stephen on the size of the dust as well. Fine (≤2.5 µm F. Austin State University. One of the 4 tunnel- in aerodynamic diameter) and coarse particles ventilated buildings at BRC was used for indoor (between 2.5–10 µm in aerodynamic diameter) measurements. This farm produces 110,400 have been linked to higher rates of total mortal- commercial broiler chickens in 7 wk for one ity, mortality from major cardiovascular diseas- flock. Including the preliminary tests, the study es, and increased rates of morbidity expressed covered 2 flocks: flock 37 and 38. The center primarily as hospital admission for those popu- raises about 5.5 flocks each year, with 14 to 21 lations with long-term exposure to heavier loads d of down time between flocks. Wood shavings of dust. The smaller the particles are, the more were used as bedding used at BRC, as wood is intense the damage is, as small particles may be an abundant resource in east Texas. composed of adsorbed organic molecules, bioaerosols, and other materials. Area Sampling for Respirable Downloaded from The exposure to and effects of pollutants on Dust and Bioaerosol the health of workers in animal buildings have not been fully studied. Previous exposure stud- The building was tunnel ventilated with ten ies in animal buildings primarily dealt with the 52-in fans and one 48-in fan (Figure 1). It had 29 concentrations of dust measured at stationary lo- adjustable drop-down inlets and 2 cooling pads http://japr.oxfordjournals.org/ cations indoors. Results of stationary sampling on opposite ends to cool the air that was drawn with short sampling time correlates poorly with into the house during warm weather. Three ven- health effects and probably is not a surrogate tilation schemes were used in this building— measure of worker’s exposure [5, 6]. In addi- minimum, tunnel, and transitional—to main- tion, Riegel et al. [7] found that the measured tain a temperature range of 70 to 88°F and RH concentrations of endotoxins and bacteria in between 40 and 60%, depending on the growth dust collected using samplers attached to the stage of the chickens. Three forced-fan heaters workers are higher than those measured from [8] were located on one side wall in the house by guest on February 24, 2014 stationary samplers indoors. However, the expo- that put out 250,000 BTU/heater and 16 infra- sure of workers to higher concentrations of dust conic radiant heater brooders [9] that generate could be attributed to activities that they do out- 16,000 BTU/brooder. side of the buildings, such as loading litters into Area sampling was when stationary samplers trucks for disposal, unloading new shavings, consisting of respirable cyclones [10] connected mowing, cleaning the barns between flocks, and to personal sampling pumps [11] were used to so on. Therefore, relying only on measurements measure the concentrations of respirable dust indoors may not be adequate to quantify the real (particles with diameter of ≤4 µm) at a height exposure of workers to dust during their entire of about 1.5 m at 6 sampling locations in the work hours. The objectives of this study were to building (Figure 1). The cyclone has a cut-point quantify the worker-exposure of poultry work- of 4 µm at a flow rate of 2.5 L/min. Cut-point ers to respirable dust and bioaerosols, and com- diameter is the aerodynamic diameter of the pare those with the measured concentrations at particles collected at 50% efficiency or where stationary locations indoors. With representa- half of these particles are captured on the filter tiveness of samples being a critical component and the other half are not. Based on results of of exposure assessment studies, this research ad- preliminary experiments in which 3 types of fil- dresses the importance of adopting the method ters (gelatin, glass fiber, and Teflon) were tested that will more adequately represent the condi- side-by-side for dust loading and growth of mi- tion to which poultry workers are exposed. croorganism colonies, Teflon filters [12] were determined to be best suited for mass concentra- MATERIALS AND METHODS tion and microorganism colony quantifications (data not shown); thus, Teflon filters were used This study was conducted during the spring in the measurements. Filters were conditioned and summer of 2009 (April to July) in the in a desiccator (RH = 20 to 30%; temperature Jerez et al.: POULTRY WORKER EXPOSURE 9

Figure 1. Location of stationary samplers inside one of the poultry buildings at the Walter C. Todd Broiler Research Center at Stephen F. Austin State University. The building had 11 fans: 6 on one end wall, 4 on the adjoining side- walls, and 1 on the opposite endwall. Not drawn to scale.

= 25 ± 2°C) 24 h before and after sampling. All buildings but within the farm. Samplers were pumps were calibrated before use using a pri- worn while they were in the farm so the respi- mary flow calibrator [13]. Sampling indoors rable dust the workers collected came from a va- Downloaded from lasted for about 20 h to ensure that a measur- riety of sources indoors and outdoors. able amount (≥1 mg) of dust was collected on The workers were trained on how to use the the filters. The total number of sampling events samplers before the start of the study. The sam- was 16 d. Every sample was analyzed for dust plers were placed in secured, clean, and sani- mass concentration and colony growth. An ana- tized containers by the workers after completing http://japr.oxfordjournals.org/ lytical balance [14] with a resolution of 0.1 mg their measurements and were collected at the was used in weighing the filters before and after end of the day by the investigators for analysis sampling to get the weight of respirable dust. in the laboratory. The workers were not required The bioaerosol component of dust was quan- to record the start and end times of the measure- tified following the procedure outlined in Predi- ments, as the actual run time of the pumps were cala et al. [15]. The filters were loaded onto R2A automatically recorded. The collected filters agar plates after the mass concentration of dust were analyzed for dust mass concentration and has been determined. The samples were then in- colony growth following the same procedures by guest on February 24, 2014 cubated at 30°C for 3 d. After incubation, the used for filters collected from the stationary colony-forming units of microorganisms were samplers indoors. counted with a hand-held electronic colony counter [16].

Worker-Exposure Sampling for Dust and Bioaerosol

In worker-exposure sampling, the samplers were attached to the workers’ lapels near their breathing zones during the entire sampling period, as shown in Figure 2. Four workers were present at the farm. All 4 volunteered to par- ticipate in this study; however, only 2 wore 2 samplers each during each sampling event. The cyclone was connected to a sampling pump that was enclosed in a belted noise-reducing cover to minimize noise. Each pump weighed about 450 g, whereas the cyclone was about 40 g. The working hours spent on the farm varied from 170 to 520 min. Workers spent about 40 to 90 min in all 4 poultry buildings at the BRC to pick Figure 2. Two respirable cyclones were worn by each worker. Each cyclone was connected to a pump en- up dead chicken and check the equipment. The closed in a noise-reducing cover. Color version avail- rest of their time was spent working outside the able in the online PDF. 10 JAPR: Research Report

Data Analysis on) were applied throughout, the assumption was that the environmental conditions inside The mass concentration of respirable dust were also similar and all 4 buildings were es- was the mass of dust divided by the volume of sentially the same. To confirm this assumption, air sampled. The mass of dust was the difference environmental conditions (temperature and between the weights of the filter before and after RH), and the weight and mortality of birds in sampling. The volume of air was the product of all 4 buildings during 2 flocks (flocks 37 and the sampling airflow rate and the sampling time. 38) were collected and compared. In the com- As for the bioaerosol concentration, it was cal- parisons, the one-way ANOVA was used. All culated as the number of colony-forming units chickens in the 4 buildings had the same growth of the microorganism divided by the volume of level. air sampled. The air temperatures in all 4 buildings dur- The randomized complete block ANOVA ing both flocks did not vary significantly (P > was used to determine if a significant difference 0.05). The average temperature in all 4 build- Downloaded from existed between the means of the concentrations ings during flock 38, however, was about 4°F of respirable dust and bioaerosols indoors and higher (84.3 vs. 80.3°F) than during flock 37, those collected by the workers. The sampling as shown in Table 1. The mean temperature lev- method was considered as a fixed factor and each els in all 4 buildings varied from 72.3 to 88.3°F sampling event (day) was considered a random during flock 37, whereas they varied from 78.3 http://japr.oxfordjournals.org/ factor, which was used as a block. To determine to 90.2°F during flock 38. In the building that whether differences existed among each sam- was tested, the temperature varied from 73.1 to pling event for indoors and worker exposure of 86.8°F during flock 37 and from 78.3 to 88.4°F dust and bioaerosol concentrations, the repeated during flock 38. The temperature setting in the measures design was used. Repeated measures building was varied from d 1 to 49 to provide provided information on how the concentration proper temperature for different growth levels varied with time. Data analyses were completed of chickens. When the chickens were younger, using the statistical software SAS [17]. a higher temperature was needed to keep them by guest on February 24, 2014 warm. The air temperature in the building was RESULTS AND DISCUSSION reduced as the chickens became bigger. Comparisons of the Environmental Relative humidity plays an important role in Conditions and Poultry Data in All Buildings dust and bioaerosol concentrations. Lower humidity and higher temperature in the house re- Due to the limited number of samplers avail- sult in higher concentrations of microorganisms able for indoor measurements, area samplings in the air [18]. Significant differences (P < 0.05) were conducted in just one building. However, were observed in RH among the 4 buildings dur- personal samplers were carried by the workers ing flocks 37 and 38. Relative humidity fluctu- in all 4 buildings. Because all 4 buildings were ated from d 1 to 49, varying from 45 to 84% for located side-by-side and the same management flock 37 and from 52 to 87% for flock 38. The practices (manure, feeding, ventilation, and so average RH was about 64 and 68% for flocks

Table 1. Comparison of average environmental conditions in all 4 buildings at the Broiler Research Center of Stephen F. Austin State University for flocks 37 and 38

Flock 37 Flock 38

Parameter P-value Mean P-value Mean

Temperature, °F 0.59 80.3 0.71 84.3 Humidity, % <0.001 63.6 <0.001 68.0 Water consumption, gal 0.61 1,194.1 0.96 1,235.4 Weight of birds, lb 0.98 2.13 0.93 2.21 Mortality 0.13 15 0.39 18 Jerez et al.: POULTRY WORKER EXPOSURE 11

37 and 38, respectively, close to the desired RH m3 indoors and from 0.07 to 4.07 mg/m3 with a of 60%. mean of 0.82 mg/m3 for worker exposure. The Chicken activity has a significant effect on average worker-exposure dust concentration dust concentration, and their level of activity was 3 times higher than the dust concentration could be represented by their water consump- indoors. Ellen et al. [19] obtained higher respi- tion. No significant differences (P > 0.05) were rable dust concentrations in the poultry houses observed among the 4 houses in terms of the that they monitored, ranging from 1.4 to 6.5 mg/ water consumption for both flocks 37 and 38. m3. In addition, their measured maximum dust The average water consumption in the 4 build- concentrations were more than 6 times higher ings for flock 37 ranged from 8 to 2,529 gallons. than the measurements in the current study. For flock 38, the average water consumptions This large discrepancy in the maximum value in the 4 buildings ranged from 5 to 2,410 gal- can be attributed to the fact that their samplings lons. Based on no significant differences be- were conducted mostly during the day, when ing observed among water consumption in the the animals were more active, and also during Downloaded from buildings during the 2 flocks, the chicken ac- winter, when the ventilation rates were low. In tivity may have been similar. Based on the re- the current study, none of the average indoor or sults of the comparisons of the environmental worker-exposure measurements exceeded the conditions, water consumption, mortality, and threshold value for respirable dust of 3 mg/m3 chickens weight, it could be concluded that all 4 recommended by the American Conference of http://japr.oxfordjournals.org/ buildings were similar. Also, due to the limited Governmental Industrial Hygienists [20]; how- number of samplers available, using one build- ever, the values did exceed the recommended ing in the data collection was deemed to be suf- exposure limit of 0.16 mg/m3 recommended by ficient. Donham et al. [21]. The area dust concentrations fluctuated from Comparisons of the Area and Worker- d 1 to 49 for both flocks. As shown in Figure Exposure Sampling for Respirable Dust 3, the initial dust concentration was high due to the resuspended dust brought about by intense by guest on February 24, 2014 The random block design was used to test if activity in the building with new chicks being significant differences existed between the con- brought in. Conventional wisdom was that the centrations of dust and bioaerosols measured at dust concentration will continue to increase as stationary locations indoors (area sampling) and the birds become bigger, as they tend to generate at the samplers attached to the workers (work- more particles emanating from their feathers and er-exposure sampling). Sampling type was the resuspend more dust from their disturbance of fixed-effect factor and each sampling event was the litter. Because the mass concentration fluc- a block. The area dust concentrations were sig- tuated throughout the growing period, results nificantly lower than the worker-exposure dust may indicate that majority of the resuspended concentrations (P < 0.05). As shown in Table 2, dust was not of a respirable fraction. Similarly, respirable dust concentrations varied from 0.03 no uniform pattern emerged for the measured to 1.03 mg/m3 with an average value of 0.23 mg/ worker-exposure concentrations (Figure 3). The

Table 2. Worker-exposure and area concentrations of respirable dust and bioaerosols

Lower Upper 95% confidence 95% confi- Item limit dence limit Mean Minimum Maximum SD

Worker exposure Respirable dust 0.56 1.08 0.82 0.07 4.07 0.87 Respirable bioaerosols 41.7 75.2 58.5 0 259.3 57.1 Indoors Respirable dust 0.19 0.26 0.23 0.03 1.03 0.18 Respirable bioaerosols 28.0 39.6 33.8 2.3 128.0 27.4 12 JAPR: Research Report Downloaded from http://japr.oxfordjournals.org/

Figure 3. Variation in respirable dust concentrations measured indoors and for worker exposure from April to July 2009. Error bars represent SEM. Color version available in the online PDF. measured worker-exposure concentrations were varied from 17.5 to 176.8 cfu/m3. The worker- generally higher than those of the area measure- exposure bioaerosol concentrations increased by guest on February 24, 2014 ments, suggesting that measuring the concentra- from d 1 to 49 for both flocks, which was cor- tions at stationary locations indoors may under- related with the increase in weight of the birds. estimate workers exposure level to contaminants According to Scheff et al. [22], the acceptable such as respirable dust. range of values for total bacteria in most indoor environments is from 100 to 1,000 cfu/m3. The Comparisons of the Area and Worker- measured respirable bioaerosol concentrations 3 Exposure Bioaerosol Concentrations in this study never exceeded 300 cfu/m . Studies on bioaerosol measurements in poultry build- The bioaerosol concentration indoors was ings are limited. Hinz and Linke [23] reported a significantly different from the worker-exposure total bioerosol concentration of 7.7 × 106 cfu/m3 concentration (P < 0.05). However, no signifi- in measurements done in poultry-caged layers. cant differences in bioaerosol concentrations Based on linear correlation of the bioaerosol were observed among the sampling events. As concentrations and RH, a weak correlation (r = shown in Figure 4, the average area bioaerosol 0.24 for worker exposure and 0.28 for indoors) concentrations indoors for flock 37 were higher was observed between the parameters. In gen- than for flock 38 and fluctuated throughout the eral, higher RH in the building is associated whole flock season. During flock 37, the area with higher bioaerosol concentration. Similar bioaerosol concentrations ranged from 5 to 128 to the respirable dust fraction comparisons, cfu/m3, whereas the worker-exposure concentra- based on Figure 4, worker exposure of bioaero- tion ranged from 2 to 259 cfu/m3. During flock sol was higher than the area concentrations, 38, area bioaerosol concentrations were some- suggesting that measuring exposure by attach- what steady from day to day. The indoor con- ing personal samplers to workers will yield centrations in flock 38 ranged from 6 to 103 cfu/ more representative results compared with area m3, whereas the worker-exposure concentrations measurements. Jerez et al.: POULTRY WORKER EXPOSURE 13 Downloaded from http://japr.oxfordjournals.org/

Figure 4. Variation in respirable bioaerosol concentrations measured indoors and for worker exposure from April to July 2009. The average RH during days of sampling is also shown. Error bars for the dust and bioaerosol concentrations represent SEM. Color version available in the online PDF.

CONCLUSIONS AND APPLICATIONS REFERENCES AND NOTES

1. Sweeten, J. M., L. Erickson, P. Woodford, C. B. 1. The concentrations of respirable dust Parnell, K. Thu, T. Coleman, R. Flocchini, C. Reeder, J. by guest on February 24, 2014 and bioaerosols obtained using personal R. Master, W. Hambleton, G. Bluhm, and D. Tristao. 2000. samplers were usually higher than those Air quality research and technology transfer White paper and recommendations for concentrated animal feeding op- measured using stationary samplers in- erations. Accessed October 2012. http://www.nrcs.usda.gov/ doors. The higher measurements in per- Internet/FSE_DOCUMENTS/stelprdb1046310.pdf. sonal samplers could be attributed in part 2. NASS. 2007. Census of agriculture shows growing to dust resuspension due to increased diversity in U.S. Farming. USDA–National Agricultural Statistics Service. Accessed October 2012. http://www.usda. bird activities when disturbed and to gov/wps/portal/usda/usdahome?contentid=2009/02/0036. their exposure to dust outside the build- xml&contentidonly=true. ings. This confirms the results of similar 3. Malmberg, P., and A. Rask-Andersen. 1993. Organic studies done in an indoor environment. dust toxic syndrome. Semin. Resp. Med. 14: 34–38. 4. Cole, D., L. Todd, and S. Wing. 2000. Concentrated Therefore, to determine the true expo- animal feeding operations and public health: A review of oc- sure of poultry workers to dust and other cupational and community health effects. Environ. Health pollutants, personal samplers may yield Perspect. 108:685–699. more representative measure. 5. Su, H. J., P. C. Wu, H. L. Chen, F. C. Lee, and L. L. Lin. 2001. Exposure assessment of indoor allergens, endo- 2. Respirable dust fractions in a poultry toxins, and airborne fungi for homes in Southern Taiwan. house can exceed the more stringent Environ. Res. 8:241–252. limit proposed by other researchers, 6. Verhoeff, A. P., and H. A. Burge. 1997. Health risk but not the recommended threshold by assessment of fungi in home environments. Ann. Allergy Asthma Immunol. 78:544–554. American Conference of Governmental 7. Rieger, M. A., M. Lohmeyer, M. Nubling, S. Neu- Industrial Hygienists. haus, H. Diefenbach, and F. Hofmann. 2005. A description 3. The measured respirable bioaerosol con- of the standardized measurement procedures and recommended threshold limit values for biological hazards in Ger- centrations in this poultry house were many. J. Agric. Saf. Health 11:185–191. lower than the published results by other 8. Forced-fan heaters, Model Guardian 250, L.B. White researchers. Co., Onalaska, WI. 14 JAPR: Research Report

9. Infraconic radiant heater brooders, Model I-34, L.B. 19. Ellen, H. H., R. W. Bottcher, E. V. Wachenfelt, and White Co., Onalaska, WI. H. Takai. 2000. Dust levels and control methods in poultry 10. Respirable dust aluminum cyclone, Model 225–01– houses. J. Agric. Saf. Health 6:275–282. 02, SKC Inc., Eighty Four, PA. 20. ACGIH. 2010. Threshold Limit Values for Chemi- 11. Personal sample pump, Model AirChek XR5000, cal Substances and Physical Agents & Biological Exposure SKC Inc., Eighty Four, PA. Indices. American Conference of Governmental Industrial 12. Teflon filters, pore size of 2 µm, Model 225–1709, Hygienists, Cincinnati, OH. SKC Inc., Eighty Four, PA. 21. Donham, K. J., D. Cumro, S. Reynolds, and J. Mer- 13. Primary flow calibrator, Model Gilibrator 2, Sensi- chant. 2000. Dose-response relationship between occupa- dyne, Clearwater, FL. tional exposures and cross-shift declines of lung function in poultry workers: Recommendations for exposure limit. J. 14. Analytical balance, Model AB104-S, Mettler Toledo, Occup. Environ. Med. 42:260–269. Columbus, OH. 22. Scheff, P. A., V. K. Paulius, L. Curtis, and L. M. 15. Predicala, B. Z., J. E. Urban, R. G. Maghirang, S. B. Conroy. 2000. Indoor air quality in a middle school, part II: Jerez, and R. D. Goodband. 2002. Assessment of bioaerosols Development of emission factors for particulate matter and in swine barns by filtration and impaction. Curr. Microbiol. bioaerosols. Appl. Occup. Environ. Hyg. 15:835–842. 44:136–140. 23. Hinz, T., and S. Linke. 1998. A comprehensive ex- 16. Electronic colony counter, Bel-Art Products, Pequan- perimental study of aerial pollutants in and emissions from Downloaded from nock, NJ. livestock buildings. Part 2: Results. J. Agric. Eng. Res. 17. SAS User’s Guide. 2008. Version 9 ed. SAS Inst. 70:119–129. Inc., Cary, NC. 18. Rautiala, S., J. Kangas, K. Louhelainen, and M. Rei- Acknowledgments man. 2003. Farmers’ exposure to airborne microorganisms The financial support provided by the Office of Research in composting swine confinement buildings. AIHA J. (Fari- and Sponsored Programs of Stephen F. Austin State Univer- fax, VA) 64:673–677. sity (FRG 2008-105) is acknowledged. http://japr.oxfordjournals.org/ by guest on February 24, 2014 © 2014 Poultry Science Association, Inc. Effect of different sources and levels of selenium on performance, meat quality, and tissue characteristics of broilers

T. F. B. Oliveira,* 1 D. F. R. Rivera ,* F. R. Mesquita ,* H. Braga ,* E. M. Ramos ,† and A. G. Bertechini* 1

* Department of Animal Science, and † Department of Food Science, Universidade Federal de Lavras, Lavras, MG, Brasil CEP 37200-000 Downloaded from Primary Audience: Nutritionists, Researchers, Meat Scientists, Consumers

SUMMARY http://japr.oxfordjournals.org/ The objective of this study was to evaluate the effects of dietary levels of inorganic and organic selenium (Se) on performance, meat physicochemical characteristics, and Se deposition in the tissues of broilers chickens. A total of 2,880 one-day-old broilers (Cobb 500 strain) were divided into 96 experimental pens of 30 birds each. The 12 experimental treatments were arranged in a factorial design of 4 × 3 [selenium levels of 0.15, 0.30, 0.45, and 0.60 ppm, and

organic (selenium yeast, SY) or inorganic (sodium selenite, SS) sources of selenium and their association (SY + SS)], with 8 replicates in a completely randomized design. No differences (P

> 0.05) among Se levels or sources were detected with regard to any performance parameters. by guest on February 24, 2014 The average values of ADFI, ADG, and FCR were 106 g/bird per day, 63 g/bird per day, and 1.684 kg/kg, respectively. No differences (P > 0.05) were found between treatments regarding pH (5.79) and shear force (30.08 kgf). Those birds receiving 0.15 ppm of Se showed a significantly higher (21.92%) cooking loss of breast meat (P < 0.05). The organic form of Se decreased cooking loss, and the smallest loss was found when the diet was supplemented with 0.60 ppm of Se (15.87%). Deposition of Se in the liver increased from 0.97 (0.15 ppm of Se) to 2.43 mg/kg (0.60 ppm of Se) when using SY. The concentration of Se in the breast meat increased linearly from 0.23 to 1.42 mg/kg with increasing dietary levels of SY. Therefore, dietary supplementation with 0.15 ppm of Se, independent of source, can maintain normal bird performance. The SY was more efficient in depositing Se to the liver and breast muscle than the SS.

Key words: antioxidant, mineral, organic, absorption , meat 2014 J. Appl. Poult. Res. 23 :15–22 http://dx.doi.org/ 10.3382/japr.2013-00761

DESCRIPTION OF PROBLEM tant part of its structure [1]. The poultry industry has commonly supplemented diets with Se Selenium (Se) is an essential mineral that has because of the wide variation of this element’s antioxidant properties in animals. The enzyme concentration in feed grains around the world glutathione peroxidase protects cells and cell [2]. In Brazil, the soil is, on average, very defi- membranes from oxidation, and Se is an impor- cient in Se (0.02–0.05 mg/kg), making supple-

1 Corresponding author: [email protected] or [email protected] 16 JAPR: Research Report mentation necessary for maintaining the high information on the effects of Se source on the performance of modern broiler strains [3]. The performance and meat quality characteristics feed industry has historically used sodium sel- of broilers, little information is available on de- enite as an inorganic form of supplementation, position of Se in the breast, the combination of but in 2000, the use of organic Se to supple- different sources of Se on the development of ment poultry diets was allowed [4]. There has broilers, and the absorption sites of Se in the in- been a subsequent interest in either partly or testine. completely replacing the inorganic Se with an The present study was thus conducted to de- organic form, the latter being deposited in the termine the optimum level and source of Se di- body tissues more efficiently than the inorganic etary supplementation on broiler performance, form [5]. Selenomethionine is an organic form physicochemical breast meat characteristics, commonly used by the poultry industry, and it is and Se concentration in different tissues. readily absorbed by the erythrocytes through an active mechanism similar to that of methionine MATERIALS AND METHODS Downloaded from itself. In contrast, inorganic Se, similar to sodium selenite, is absorbed by simple diffusion [6]. Experimental Chickens and Diets For this reason, inorganic Se is retained in lesser This project was approved and conducted un- concentrations in the muscles and is excreted der the supervision of the Federal University of in higher concentrations than organic Se. The Lavras Animal Care and Use Committee. A total http://japr.oxfordjournals.org/ absorbed Se, which is not immediately metabo- of 2,880 one-day-old Cobb 500 chicks from a lized, is incorporated into certain tissues with local commercial hatchery were assigned to 12 high levels of protein syntheses, such as skeletal treatments with 8 replicates of 30 male birds muscles, erythrocytes, pancreas, liver, kidney, each. The broilers were randomly placed in floor stomach, and gastrointestinal epithelium [7]. pens (2.0 m × 1.1 m) with continuous lighting The availability of Se for tissue assimilation is for 24 h. Fresh water and feed were offered ad affected by its form and concentration in the diet libitum throughout the trial (Table 1). The birds [5, 8, 9]. Consumer demands for meat quality were housed in an environmentally controlled by guest on February 24, 2014 have increased in recent years; the industry has room at 32°C. The temperature of the room was thus been challenged to improve the nutritional reduced by 3°C each week until the birds were value, quality, and shelf life of meat, and Se 21 d of age. supplementation might improve these character- The 12 treatments were as follows: a corn- istics. The concentration of certain nutrients is soybean meal basal diet (Table 1) supplemented an important market issue because today’s con- with 3 sources of Se: (1) 100% selenium yeast sumers are more interested in enriched products (SY); (2) 50% SY + 50% sodium selenite (SS; that are considered healthier [10]; therefore, Se SY+SS); and (3) 100% SS, and 4 supplemen- is considered a nutrient of consumer interest. tation levels (0.15, 0.30, 0.45, and 0.60 ppm) The loss of water during handling and cooking of each selenium source in a 4 × 3 factorial ar- is also considered an indicator of meat quality rangement. The analyzed dietary Se content of by consumers. One of the most important qual- each treatment is shown in Table 2. Sodium sel- ity characteristics is the water-holding capacity enite (45.6%) was used as the inorganic Se, and of breast meat. The water retention of the mus- Se yeast (Selemax 2,000 ppm) [13] was used as cle and the oxidative stability of chicken meat the organic Se. The feeding program was divid- during refrigerated storage can be improved by ed into 4 phases (0–7 d, 8–21 d, 22–35 d, and increasing dietary levels of Se [11]. Oxidation 36–42 d), and the diets in each phase were for- can damage cell membranes, thus reducing their mulated to be commercial grade, exceeding the integrity and allowing the seepage of intracel- NRC [2] requirements for essential amino acids. lular fluids [12]. The amount of water that seeps from the tissue, which reduces the appearance of Performance packaged meat and the juiciness of the cooked product, can be measured by drip loss and cook- Body weight and feed intake were recorded ing loss, respectively. Despite the extensive after each feeding phase to determine feed in- Oliveira et al.: SELENIUM IN BROILERS 17

Table 1. Composition (%) of the experimental diets

Prestarter Starter Grower Finisher Item (1–7 d) (8–21 d) (22–34 d) (35–42 d)

Ingredient Yellow maize 56.274 58.700 60.608 54.950 Soybean meal 37.000 34.080 31.300 27.170 Vegetable oil 2.162 3.072 4.240 4.158 Dicalcium phosphate 1.924 1.875 1.720 1.570 Limestone 0.875 0.816 0.774 0.740 Salt (NaCl) 0.470 0.460 0.440 0.420 dl-Methionine 0.373 0.273 0.245 0.241 l-Lysine HCl 0.395 0.260 0.230 0.287 l-Threonine 0.158 0.082 0.061 0.082 Vitamin¹ 0.100 0.100 1.000 0.100 Mineral² 0.100 0.100 0.100 0.100 Choline chloride 0.057 0.570 0.057 0.057 Downloaded from Nutrient and energy level (calculated) ME kcal/kg 2,956 3,050 3,150 3,200 CP 22.35 20.98 19.80 18.32 Met + Cys 0.97 0.84 0.79 0.76 Lysine 1.36 1.19 1.10 1.05 Threonine 0.89 0.77 0.71 0.68 http://japr.oxfordjournals.org/ Tryptophan 0.25 0.24 0.22 0.20 Arginine 1.39 1.30 1.22 1.10 Isoleucine 0.86 0.81 0.76 0.69 Sodium 0.22 0.22 0.21 0.20 Calcium 0.94 0.90 0.84 0.78 Available phosphorus 0.47 0.45 0.42 0.39 1 The vitamin premix provided (per kilogram of diet): 12,000 IU of vitamin A, 2,400 IU of vitamin D3, 40 mg of vitamin E, 31.8 mg of vitamin K, 2.5 mg of vitamin B1, 4.0 mg of vitamin B2, 2.0 mg of vitamin B6, 15 µg of vitamin B12, 60 µg of biotin, 30

mg of niacin, and 1.8 mg of folic acid. by guest on February 24, 2014 2The mineral premix provided (per kilogram of diet): 80 mg of Fe, 70 mg of Zn, 70 mg of Mn, 1 mg of I, and 10 mg of Cu. take (FI), BW gain (BWG), and FCR. Based on Science Department processing facility located these data, the ADFI and ADG were calculated. approximately 300 m from the broiler house. The birds that died were weighed daily to adjust After exsanguination, the birds were scalded the FI accordingly. in a set of 3 triple-pass scalding tanks [14] at 52°C for 220 s (the shackle speed was set so that Physicochemical Characteristics the carcasses spent 30 s in each scalding tank with 30 s in between), picked for 180 s using Feed was withdrawn from the birds approxi- a rotary drum picker [15], and mechanically mately 6 h before slaughter, and the birds were eviscerated [16] in an automated line system. At transported to the processing facility in plastic this stage, the carcasses were removed from the crates. The birds were slaughtered at the Feed shackles before chilling for the sample collec-

Table 2. Selenium analyzed (mg/kg) according to dietary source and supplementation level1

Se supplementation level (ppm)

Se source 0.15 0.30 0.45 0.60

Organic Se 0.190 0.335 0.485 0.630 Inorganic Se 0.195 0.340 0.490 0.645 Organic + inorganic Se 0.195 0.335 0.485 0.635 1The difference between supplemented Se and analyzed Se is the Se from the ingredients. 18 JAPR: Research Report tion. After slaughter, the breast (including bone Statistical Analysis and skin) and thigh (bone and skin) muscles The data were subject to an ANOVA proce- were removed from the carcass. For each of dure using SAS software [23] to measure the ef- these muscle types, 2 carcasses from each rep- fects of the levels and the sources, as well as their licate (n = 16 per treatment) were selected for interaction. Those response variables resulting meat quality evaluation. in a significant F-test were further analyzed us- After 15 min, postmortem pH was measured ing the Tukey test. Significance was accepted at in the selected breast muscles. The breast and P < 0.05. A regression analysis was applied for thigh samples were then placed into polyethyl- the dietary Se levels within each source. ene bags and transported in a cool box at 4°C to the laboratory. At 24 h postmortem, the whole breasts were separated into right and left halves. RESULTS AND DISCUSSION The breasts and thighs from the left side of the No significant differences among the treat- carcass were bagged individually according to ments (P > 0.05) were observed for any per- Downloaded from treatment groups, stored at 4°C for 5 d, and used formance parameter or mortality measurement for the pH measurements. The breasts from the made over the entire trial period. No adverse right side of the carcass were vacuum-packaged effect of Se on the growth of the chickens was [17]. All of the meat quality analyses were con- observed during the trial. The averages of ADFI, ducted in the Meat Laboratory of the Food Sci-

ADG, FCR, and mortality were 106 g/bird per http://japr.oxfordjournals.org/ ence Department. day, 63 g/bird per day, 1.684 kg/kg, and 4.68%, To measure cooking loss, the breast meat respectively. was refrigerated overnight at 4°C. The meat Similarly, Peric et al. [12] observed no effects was removed from the refrigerator until it on the performance of the broilers when using reached room temperature. The samples were different concentrations (0.0 and 0.3 ppm) of SS weighed and packed in aluminum paper. and SY, and a combination of both. Edens et al. The samples were constantly monitored dur- [24] and Spears et al. [25] also reported no dif- ing cooking. After the external temperature ferences in BW or FE when broilers were fed by guest on February 24, 2014 reached 85°C, the samples were constantly diets containing SS or SY. turned until the internal temperature reached The supplemented Se levels appeared to 72 ± 2°C [18]. The cooked muscle was cooled be sufficient to maintain normal growth of the to room temperature and weighed to determine birds. Because of the low level of Se in the lo- the cooking loss. The cooked meat was used cal maize (0.03 mg/kg), a control diet without to measure the shear force. The samples were supplementation was not used. In our previous cut into 1 × 1 × 2 cm slices perpendicular to study, mortality was controlled when only 0.05 the fiber orientation of the muscle [19]. Tex- mg/kg Se was supplemented (data not pub- ture analysis was performed using a Warner- lished). According to the NRC [26], chickens Bratzler shear tool fitted to a TA.XT2i Texture receiving diets with a level of Se <0.1 mg/kg Analysis System [20] connected to a computer can present symptoms of exudative diathesis at equipped with the Texture Expert software 2 to 3 wk of age and can die by 3 to 4 wk of age. [20]. The tests were performed at a cross-head In this study, the yields of cut-up carcasses speed of 60 mm/min. were not influenced by the source or level of Se, in agreement with the results of Deniz et al. [27]. Tissue Concentration Analysis The average carcass, breast, and leg yields were 69.50, 25.86, and 22.57%, respectively. Choct et The liver, duodenum, jejunum, ileum, and al. [8] and Payne and Southern [5] reported sig- breast meat samples were finely ground before nificant improvements in breast and leg yields analysis. The Se concentration of the tissues was when supplementing with SY. determined according to the method described Supplementation with SY produced the low- [21] using a hydrate generation atomic absorp- est cooking loss among all sources for supple- tion spectrophotometer [22]. mentation levels >0.30 ppm (Figure 1). The Oliveira et al.: SELENIUM IN BROILERS 19 Downloaded from

Figure 1. Effect of Se source [selenium yeast (SY, Figure 2. Effect of Se source [selenium yeast (SY, organic) or sodium selenite (SS, inorganic)] and level organic) or sodium selenite (SS, inorganic)] and level on the cooking loss (%) in the breast meat of 42-d- on the deposition of Se (mg/kg) in the liver of 42-d- http://japr.oxfordjournals.org/ old broiler chickens. SY: y = 25.276803 – 29.138442x old broiler chickens. SY: y = −0.089000 + 8.071167x + 22.440097x2, R2 = 99.53%; SY+SS: y = 25.997792 − 6.450000x2, R2 = 97.65%; SY+SS: y = 0.178875 – 33.941205x + 38.274514x2, R2 = 84.88%; SS: y = − 7.331833x − 7.061111x2, R2 = 92.04%; SS: y = 26.105563 – 34.520585x + 44.333319x2, R2 = 91.86%. −0.050063 + 7.916583x −8.502778x2, R2 = 98.12%. combination of the two sources (SY+SS) pro- selenite is retained less efficiently, and the reduced intermediate results, as expected. In this maining Se is excreted in the urine [31]. experiment, we demonstrated that increasing These observations are consistent with those by guest on February 24, 2014 the dietary level of Se and using mainly SY as a reported by Kuricová et al. [32] and Wang and source may improve some of the physicochemi- Xu [33], who found similar improvements in the cal characteristics of the meat. The pH and shear deposition of Se in the liver in broilers fed SY. force were not affected by treatment, and their Baowei et al. [34], evaluating similar Se levels averages were 5.79 and 30.08 kgf, respectively. in geese (0.0, 0.1, 0.3, and 0.5 ppm), found that Similarly, Naylor et al. [28] found an im- the Se deposition increased with increasing di- provement in cooking loss when supplementing etary concentrations of SY. Deposition of Se in with 0.30 and 0.40 ppm of SY. However, Mieze- the liver is interesting in cases of Se deficiency, liene et al. [29] and Mikulski et al. [30] observed as the bird could mobilize Se from the liver to no significant differences in cooking loss in the meet its vital functions without sacrificing per- breast meat of broiler chickens and turkeys, re- formance. spectively. Regardless of the sources and levels of Se The concentrations of Se in the liver tissue studied, the duodenum was the segment of the of chickens fed varying amounts and forms of small intestine where higher concentrations of this microelement are shown in Figure 2. The Se were found, followed by the jejunum and ile- highest Se levels were found in the birds fed the um. Therefore, absorption decreased in the order largest amounts of Se, except in those fed SS. of duodenum, jejunum, and ileum, which agrees Significant differences among the effects of SY, with the results of studies conducted using rats SY+SS, and SS were apparent after 6 wk. [35] and cocks [36]. The higher Se content in the liver of the birds Assessing the apparent retention of Se in that were fed SY may be explained by the abil- broilers, Yoon et al. [37] and Gomes et al. [3] ity of selenomethionine, which is not used in found higher retention levels with SY. The re- the synthesis of selenoproteins, to be retained at sults observed by those authors agree with our high rates in tissues, including the liver. Sodium results, confirming that absorption of SY is 20 JAPR: Research Report Downloaded from

Figure 3. Effect of Se source [selenium yeast (SY, organic) or sodium selenite (SS, inorganic)] and level on Figure 4. Effect of Se source [selenium yeast (SY, the deposition of Se (mg/kg) in the jejunum of 42-d- organic) or sodium selenite (SS, inorganic)] and http://japr.oxfordjournals.org/ old broiler chickens. SY: y = −0.3250 + 6.5749 x − level on the deposition of Se (mg/kg) in the ileum of 8.8694x2, R2 = 99.18%; SY+SS: y = 0.3285 + 3.1188x 42-d-old broiler chickens. SY: y =0.0905 + 1.2823x – − 2.9611x2, R2 = 83.66%; SS: y = 0.3182 + 6.6628x 2.2555x2, R2 = 73.13%; SY+SS: y = −0.6923 + 7.9275x − 9.0944 x2, R2 = 94.99%. − 8.2944x2, R2 = 94.91%; SS: y = 0.9225 + 0.1462 + 2.5305x2, R2 = 73.20%. greater than that of SS. Sodium selenite is re- the FAO [38] is 0.065 and 0.055 mg for men and tained at higher concentrations in the jejunum women aged 19 to 65 yr old, respectively. and ileum (Figures 3 and 4) due to its absorp- Except for the birds fed SS, dietary supple- by guest on February 24, 2014 tion mechanism of simple diffusion compared mentation favored the deposition of Se in the to the active mechanism of SY. That the highest breast, perhaps due to the high capacity of Se concentration of Se was observed in the duode- to be incorporated into muscle. Selenium yeast, nums of birds fed SS cannot be physiologically predominantly composed of selenomethionine, explained. incorporates into muscle at a rate similar to the The Se concentration in the breast remained amino acid methionine. Thus, SY is deposited constant with increasing dietary Se levels for the more efficiently than SS in tissues with slow birds that were fed SS. Supplementation with protein turnover, such as muscles. SY and with SY+SS increased the Se concentration in the breast, as shown in Figure 5. The Se CONCLUSIONS AND APPLICATIONS deposited in the breast of those birds fed SY was 68.15% and 246.86% higher than those for the 1. Based on these results, the Se-deficient birds fed SY+SS and SS, respectively. basal diet supplemented with the mini- A daily intake of 150 g of breast meat from mum level specified by the NRC [38] broilers receiving diets supplemented with 0.30 (0.15 ppm of Se), independent of the ppm of SY, SY+SS, or SS would be sufficient source, maintained normal performance to provide 117.00 and 138.27%, 114.92 and of the birds. 135.81%, and 38.31 and 45.27% of the daily 2. Selenium yeast was more efficient in de- Se recommendations for men and women, re- positing Se into the liver, which could be spectively. Consumption of the same amount useful during Se deficiency. of breast meat from broilers receiving 0.45 and 3. The Se concentration in breast meat 0.60 ppm of SY or SY+SS would provide more improved when the birds were fed SY, Se than is recommended by the FAO [38]. The but supplementation with 0.30 ppm of recommendation for the daily intake of Se by SY+SS provided the Se required for a Oliveira et al.: SELENIUM IN BROILERS 21

meat yield and feather coverage. Br. Poult. Sci. 45:677– 683. 9. Skrivan, M., J. Simáne, and G. Dlouhá. 2006. Ef- fect of dietary sodium selenite, Se-enriched yeast and Se- enriched Chlorella on egg Se concentration, physical parameters of eggs and laying hens production. Czech J. Anim. Sci. 51:163–167. 10. Grashorn, M. A. 2007. Functionality of poultry meat. J. Appl. Poult. Res. 16:99–106. 11. Smet, K., K. Raes, G. Huyghebaert, L. Haak, S. Ar- nouts, and S. De Smet. 2008. Lipid and protein oxidation of broiler meat as influenced by dietary natural antioxidant supplementation. Poult. Sci. 87:1682–1688. 12. Perić, L., N. Milošević, D. Žikić, Z. Kanački, N. Džinić, L. Nollet, and P. Spring. 2009. Effect of selenium sources on performance and meat characteristics of broiler chickens. J. Appl. Poult. Res. 18:403–409.

13. Selemax, Biorigin, Brazil. Downloaded from 14. Scalding Tank model SGS-3CA, Stork Gamco, Gainesville, GA. 15. Model d-8, Stork Gamco, Gainesville, GA. Figure 5. Effect of Se source [selenium yeast (SY, or- 16. Model PNT-24, Stork Gamco, Gainesville, GA. ganic) or sodium selenite (SS, inorganic)] and level on 17. Model VM-18, Orved, Venezia, Italy. http://japr.oxfordjournals.org/ the deposition of Se (mg/kg) in the breast meat of 42-d- 18. Rodrigues, E. C., E. M. Ramos, L. V. Rossato, A. G. old broiler chickens. SY: y = −0.156750 + 2.629667x, Beterchini, L. M. G. Botega, and F. A. Gomes. 2007. Quali- R2 = 97.13%; SY+SS: y = 0.247625 + 0.653833x, R2 = dade da carne do peito de frangos suplementados com dife- 91.81; SS: y = 0.239. rentes fontes e concentrações de selênio. Pages 327–329 in Proc. IV Congr. Bras. Ciênc. Tecn. Carnes, Campinas, Bra- zil. Colégio Brasileiro de Nutrição Animal, Campinas, SP, human adult when consuming 150 g of Brazil. breast meat daily. 19. Froning, G. W., and T. G. Uijttenboogart. 1988. Ef- fect of postmortem electrical stimulation on color, texture, pH, and cooking losses of hot and cold deboned chicken broiler breast meat. Poult. Sci. 67:1536–1544. by guest on February 24, 2014 REFERENCES AND NOTES 20. Stable Micro Systems Ltd., Godalming, UK. 21. Tinggi, U. 2003. Essentiality and toxicity of selenium 1. Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swan- and its status in Australia: A review. Toxicol. Lett. 137:103– son, D. G. Hafeman, and W. G. Hoekstra. 1973. Selenium: 110. Biochemical role as a component of glutathione peroxidase. 22. SpectrAA-2000, Varian, Palo Alto, CA. Science 179:588–590. 23. SAS Institute. 2001. SAS User’s Guide: Statistics. 2. NRC. 1994. Nutrient Requirements of Poultry. 9th Version 8.02 ed. SAS Institute Inc., Cary, NC. ed. National Academy Press, Washington, DC. 24. Edens, F. W. 1996. Organic selenium: From feathers 3. Gomes, F. A., A. G. Bertechini, R. L. Dari, J. A. G. to muscle integrity to drip loss. Five years onward: No more Brito, E. J. Fassani, P. B. Rodrigues, and L. A. Silva. 2011. selenite! Pages 165–185 in Proc. Alltech’s 12th Ann. Symp., Effect of sources and levels of selenium on physiological Nottingham, UK. traits in broilers. Arq. Bras. Med. Vet. Zool. 63:633–640. 25. Spears, J. W., J. Grimes, K. Lloyd, and T. L. Ward. 4. Food and Drug Administration. 2000. FDA approves 2003. Efficacy of a novel organic selenium compound (zinc- food additive petition for selenium yeast. Page 10 in FDA 1.selenomethionine, available Se) in broiler chicks. Pages Veterinarian Newsletter (July/August). Food and Drug Ad- 197–198 in Proc. 1st Latin Am. Congr. Anim. Nutr., Cancun, ministration, Washington, DC. Mexico. 5. Payne, R. L., and L. L Southern.. 2005. Compari- 26. NRC. 1983. Selenium in Nutrition. National Acad- son of inorganic and organic selenium sources for broilers. emy Press, Washington, DC. Poult. Sci. 84:898–902. 27. Deniz, G., S. S. Genzen, and I. I. Turkmen. 2005. Ef- 6. Surai, P. F., and N. H. C. Sparks. 2000. Tissue-specif- fects of two supplemental dietary selenium sources (mineral ic fatty acid and α-tocopherol profiles in male chickens de- and organic) on broiler performance and drip-loss. Rev. pending on dietary tuna oil and vitamin E provision. Poult. Med. Vet. (Toulouse) 156:423–426. Sci. 79:1132–1142. 28. Naylor, A. J., M. Choct, and K. A. Jacques. 2000. 7. Schrauzer, G. N. 2001. Commentary: Nutrition sele- Effect of feeding Sel-Plex TM organic selenium in diets of nium supplements: Product types, quality, and safety. J. Am. broiler chickens on liver selenium. In Proc. Concent. South- Coll. Nutr. 20:1–4. ern Poult. Sci., Atlanta, GA. 8. Choct, M., A. J. Naylor, and N. Reinke. 2004. Sele- 29. Miezeliene, A., G. Alencikiene, R. Gruzauskas, and nium supplementation affects broiler growth performance, T. Barstys. 2011. The effect of dietary selenium supple- 22 JAPR: Research Report mentation on meat quality of broiler chickens. Biotechnol. dant capacity of goose. J. Anim. Physiol. Anim. Nutr. (Berl.) Agron. Soc. Environ. 15:61–69. 95:440–448. 30. Mikulski, D., J. Jankowski, Z. Zdunczyk, M. Wro- 35. Whanger, P. D., P. H. Weswig, J. A. Schmitz, and J. blewska, K. Sartowska, and T. Majewska. 2009. The effect E. Oldfield. 1976. Effects of selenium, cadmium, mercury, of selenium source on performance, carcass traits, oxidative tellurium, arsenic, silver and cobalt on white muscle disease status of the organism, and meat quality of turkeys. J. Anim. in lambs and effect of dietary forms of arsenic on its accu- Feed Sci. 18:518–530. mulation in tissues. Nutr. Rep. Int. 14:63–72. 31. Kim, Y. Y., and D. C. Mahan. 2003. Biological as- 36. Humaloja, T., and H. M. Mykkanen. 1986. Intesti- pects of selenium in farm animals. Asian-australas. J. Anim. nal-absorption of Se-75 labeled sodium selenite and sele- Sci. 16:435–444. nomethionine in chicks—Effects of time, segment, sele- 32. Kuricova, S., K. Boldizarova, L. Gresakova, R. Bob- nium concentration and method of measurement. J. Nutr. cek, M. Levkut, and L. Leng. 2003. Chicken selenium sta- 116:142–148. tus when fed a diet supplemented with Se-yeast. Acta Vet. 37. Yoon, I., T. M. Werner, and J. M. Butler. 2007. Effect (Brno) 72:339–346. of source and concentration of selenium on growth perfor- 33. Wang, Y. B., and B. H. Xu. 2008. Effect of differ- mance and selenium retention in broiler chickens. Poult. ent selenium source (sodium selenite and selenium yeast) on Sci. 86:727–730. broiler chickens. Anim. Feed Sci. Technol. 144:306–314. 38. FAO. 2002. Human Vitamin and Mineral Require-

34. Baowei, W., H. Guoqing, W. Qiaoli, and Y. Bin. ments. Food and Agriculture Organization of the United Na- Downloaded from 2011. Effects of yeast selenium supplementation on the tions (FAO), Rome, Italy. growth performance, meat quality, immunity, and antioxi- http://japr.oxfordjournals.org/ by guest on February 24, 2014 © 2014 Poultry Science Association, Inc. The effects of an enzyme complex in moderate and low nutrient-dense diets with dried distillers grains with solubles in laying hens

Dana Hahn-Didde and Sheila E. Purdum 1

Animal Science Department, University of Nebraska, Lincoln 68583

Primary Audience: Nutritionists, Egg Producers, Distillers Dried Grains with Solubles

Suppliers, Laying Hen Managers Downloaded from

SUMMARY A study was conducted to examine the effects of an enzyme complex containing xylanase, http://japr.oxfordjournals.org/ amylase, and protease in moderate- and low-ME diets with dried distillers grains with solubles (DDGS) in laying hen diets. A total of 192 hens were fed 1 of 4 dietary treatments across phase 1 and 2 of the production cycle. Dietary treatments were arranged in a 2 × 2 factorial design, factors consisted of moderate ME (2,900 kcal/kg, phase 1; 2,880 kcal/kg, phase 2) or low ME (2,860 kcal/kg, phase 1; 2,800 kcal/kg, phase 2) and enzyme complex inclusion (0 or 0.0375%).

All diets contained 15% DDGS and phytase included at 300 phytase units per kilogram and were formulated to contain 0.30% available P and a 10% reduction of Ca. Significant ME by time interactions were noted for egg production and feed intake at the conclusion of phase 1, by guest on February 24, 2014 showing diets containing low ME to be higher performing during a heat stress period. Signifi- cant effects on nutrient retention were also observed. Diets containing the enzyme complex had higher retention values for Ca, P, and CP across both phases and also had higher AME retention values for phase 1. Overall, no negative effects on lowering ME, Ca, and P were observed while supplementing with an enzyme complex.

Key words: phytase , avizyme 1502 , metabolizable energy , laying hen 2014 J. Appl. Poult. Res. 23 :23–33 http://dx.doi.org/ 10.3382/japr.2013-00764

DESCRIPTION OF PROBLEM dition, alternative ingredients, such as dried distillers grains with solubles (DDGS), have been Finding ways to reduce feed costs of produc- shown to be an acceptable ingredient in poultry tion is not a new concept. As grain prices have rations [2, 3]. Masa’deh et al. [3] showed that continued on an upward trend, the animal feed DDGS has the potential to replace corn, soybean industry as a whole has been working toward meal, dicalcium P, and salt in layer rations, all lowering nutritional costs. The addition of ex- of which have the potential for substantial cost ogenous enzymes has proven to be an effective savings. cost-saving mechanism. Exogenous enzymes Exogenous enzyme supplementation in the have increased the availability of phosphorous, diet improves production efficiency of poultry calcium, AME, and nitrogen retention [1]. In ad- by increasing digestion of low-quality products

1 Corresponding author: [email protected] 24 JAPR: Research Report and reducing nutrient loss through excreta [4]. Nebraska-Lincoln Poultry Research Facility. Exogenous enzymes can offer nutritional ben- The hens were housed 4 per cage (600 cm2/hen) efits in a variety of ways, including hydrolyzing in a 4-tiered manure belt cage system [13]. The nonstarch polysaccharides (NSP) that could be experiment was conducted late spring through used by the bird [4, 5]. This leads not only to mid fall in a tunnel-ventilated building. Hens more dietary energy being used by the bird, but were maintained on a 16L:8D photoperiod. The gut viscosity is also decreased for some ingredi- hens were fed experimental diets from 25 to 51 ents. It is well known that NSP is a factor in diets wk of age, consisting of 2 phases; phase 1 was containing wheat and barley; however, corn and from 25 to 36 wk of age and phase 2 was from soybean meal-based diets also contain varying 37 to 51 wk of age. Feed intake was recorded levels of NSP [6–8]. Although an increase in weekly and calculated on a per hen per day ba- gut viscosity is not usually an issue when uti- sis. Egg production was recorded daily and cal- lizing corn-soy diets, the NSP compounds that culated as percent egg production per hen per are present do have the ability to encapsulate day. The University of Nebraska Institutional Downloaded from nutrients, decreasing the accessibility to the bird Animal Care and Use Committee approved all [8–10]. procedures. The hens underwent a significant Dried distillers grains with solubles is a by- heat stress at the conclusion of phase 1, when product of ethanol production and contains a the hens were 34 to 36 wk of age; during this mixture of solid and liquid fermentation resi- time barn temperatures exceeded 32°C. http://japr.oxfordjournals.org/ dues [11, 12]. Due to processing, DDGS contain higher amounts of protein, fat, minerals, Diets and starch [12]. The increase in protein, fat, and A phase feeding program was used during available P makes DDGS an attractive ingredi- the experiment, phase 1 was fed from 25 to 36 ent for least-cost formulations [12]. However, wk of age and phase 2 was fed from 37 to 51 the main limiting factor of DDGS in poultry ra- wk of age. Diets were formulated based on ex- tions is the increased NSP in the diet, leading pected feed intake and age of the hens. Recom- to a decrease in nutrient availability [12]. As a mendations for dietary nutrients were based on by guest on February 24, 2014 result of the high amount of NSP, an inclusion Hy-Line W-36 commercial management guide rate of 15% has been shown to be the maximum recommendations [14] and NRC recommenda- in poultry diets [3, 12]. As a result of the higher tions [15]. The diets were arranged in a 2 × 2 NSP found in DDGS, it is important to exam- factorial design, with the variables being en- ine how enzyme complexes that hydrolyze NSP zyme complex level (0 or 0.0375% Avizyme perform with DDGS. The objective of this study 1502 [16]) and ME level (moderate vs. low). was to examine if lowering the amount of ME, All diets contained Phyzyme XP 5000 G [16] P, and Ca with and without the addition of an calculated to contain approximately 300 FTU/ enzyme complex would affect production pa- kg (phytase). For phase 1, ME levels were either rameters and nutrient retention. 2,900 or 2,860 kcal/kg for moderate- or low-ME diets, respectively. For phase 2, ME levels were MATERIALS AND METHODS either 2,880 or 2,800 kcal/kg for moderate- or Birds and Housing low-ME diets, respectively. Therefore, dietary arrangements were moderate ME, moderate ME One hundred ninety-two Hy-Line W-36 with 0.0375% Avizyme 1502, low ME, and low white leghorn laying hens were used in this re- ME with 0.0375% Avizyme 1502. Analysis of search. The hens were provided access to 100 g phytase activity received at the conclusion of of feed per hen per day, water was also provided phase 1 indicated a high level of phytase activity ad libitum via a water nipple drinker system for (1,100–2,300 FTU/kg). As a result, a reduction every cage. The current study used 48 cages in was noted in the inclusion rate of phytase for total and each cage was randomly assigned to phase 2, leading to an analyzed values ranging 1 of 4 dietary treatments, resulting in 12 rep- from 190 to 400 FTU/kg. licate cages per treatment. Hens were housed Avizyme 1502 was calculated to contain 225 in the F-research building at the University of U/g of xylanase, 300 U/g of amylase, and 3,000 Hahn-Didde and Purdum: ENZYME COMPLEX IN LAYERS 25

Table 1. Composition of diets for phase 1

Item Diet 1 Diet 2 Diet 3 Diet 4

Ingredient (%) Corn 55.61 55.55 56.62 56.56 Soybean meal1 16.14 16.51 15.95 15.96 Distillers dried grains with solubles 15.00 15.00 15.00 15.00 Animal-vegetable fat 2.51 2.52 1.69 1.70 Dicalcium P 0.45 0.45 0.45 0.45 Limestone 9.56 9.56 9.56 9.56 NaCl 0.29 0.30 0.30 0.30 dl-Met 0.14 0.15 0.15 0.15 Vitamin premix2 0.10 0.10 0.10 0.10 l-Lys HCl 0.15 0.15 0.15 0.15 Phyzyme 0.02 0.02 0.02 0.02 Avizyme 0.00 0.0375 0.00 0.0375 Calculated nutrient analysis (%, unless otherwise noted) Downloaded from ME (kcal/kg) 2,900 2,900 2,860 2,860 Ca 3.79 3.79 3.79 3.79 Total P 0.45 0.45 0.45 0.45 Available P 0.33 0.33 0.33 0.33 Lys 0.88 0.88 0.88 0.88 Met 0.43 0.43 0.43 0.43 http://japr.oxfordjournals.org/ Protein 17.00 17.00 17.00 17.00 Met + Cys 0.72 0.72 0.72 0.72 Analyzed nutrient analysis (%, unless otherwise noted) ME 3,700 3,720 3,670 3,700 Ca 4.35 4.53 4.02 4.31 Total P 0.53 0.52 0.49 0.47 Protein 16.70 15.60 16.60 15.67 Phytase3 (phytase units/kg) 1,200.00 1,100.00 2,300.00 1,100.00 3

Amylase (U/kg) <100 1,400.00 220.00 1,800.00 by guest on February 24, 2014 1Soybean meal contained 48% CP. 2 Vitamin and trace mineral premix provided the following per kilogram: vitamin A (retinyl acetate), 6,600 IU; vitamin D3, 2,805 IU; vitamin E (dl-α-tocopheryl acetate), 10 IU; vitamin K3 (menadione dimethpyrimidinol), 2.0 mg; riboflavin, 4.4 mg; −1 −1 pantothenic acid, 6.6 mg; niacin, 24.2 mg; choline, 110 mg ; vitamin B7 (biotin), 8.8 mg ; ethoxyquin, 1.1 mg; Mn (MnO), 88 mg; Cu (CuSO4H2O), 6.6 mg; Fe (FeSO4H2O), 8.5 mg; Zn (ZnO), 88 mg; and Se (Na2SeO3), 0.30 mg. 3Eurofins Nutrition Analysis Center [22].

U/g of protease, formulated to contribute 87 biweekly egg weight, and monthly hen weight. kcal/kg to the diet (referred to as enzyme com- Hen CV was determined by using individual hen plex). The DDGS was added to the diets at 15 weights from each cage; weights from each cage % during both phases. The tabulated nutrient were averaged and SD was determined for each composition of the DDGS [17] used in the cur- cage. Hen CV was determined using rent study were 27% CP, 10.3% fat, 2,800 kcal/ kg, and 0.42% available P. Available P was for- CV (%) = (cage SD/cage average) × 100. mulated at 0.33% and Ca was lowered approximately 10%, as recommended by Phycheck At the conclusion of phase 1 and phase 2, software [18]. Phytase contributed 0.12% avail- chromic oxide was added to the diets as (1%) able P and 0.108% Ca. The diet formulations for an indigestible marker and the diets were fed phase 1 and phase 2 are shown in Tables 1 and for a week. Following the week of feeding 2, respectively. chromic oxide, fecal samples were collected from each pen and frozen (−20°C). The fecal Measurements and Analysis samples were then freeze-dried in FTS Systems Measurements during both phase 1 and 2 in- Drua-Dry (model FD-20–54) [19], ground in a cluded daily egg production, weekly feed intake, Cyclotec-1903 sample mill [20], and then sifted 26 JAPR: Research Report

Table 2. Diet composition for phase 2

Item Diet 1 Diet 2 Diet 3 Diet 4

Ingredient (%) Corn 58.70 56.56 58.02 58.57 Distillers dried grains with solubles 15.00 15.00 15.00 15.00 Soybean meal1 13.14 15.20 15.18 14.65 Limestone 10.12 10.11 10.11 10.11 Animal-vegetable fat 1.90 2.23 0.680 0.600 Dicalcium P 0.39 0.38 0.37 0.38 NaCl 0.30 0.30 0.30 0.30 dl-Met 0.17 0.14 0.13 0.14 l-Lys HCl 0.18 0.11 0.10 0.12 Vitamin premix2 0.10 0.10 0.10 0.10 Phyzyme 0.006 0.006 0.006 0.006 Avizyme 0.00 0.0375 0.00 0.0375 Calculated nutrient analysis (%, unless otherwise noted) Downloaded from ME (kcal/kg) 2,880 2,880 2,800 2,800 Ca 3.98 3.98 3.98 3.98 Total P 0.43 0.43 0.43 0.43 Available P 0.30 0.30 0.30 0.30 Lys 0.82 0.82 0.82 0.82 Met 0.45 0.42 0.41 0.42 http://japr.oxfordjournals.org/ Protein 15.86 16.50 16.69 16.50 Met + Cys 0.72 0.70 0.70 0.70 Analyzed nutrient analysis (%, unless otherwise noted) ME 3,610 3,630 3,540 3,560 Ca 4.94 4.84 4.75 4.98 Total P 0.51 0.50 0.47 0.53 Protein 15.34 15.03 15.08 15.22 Phytase3 (phytase units/kg) 260.00 400.00 350.00 190.00 3

Amylase (U/kg) <100.00 1,000.00 <100.00 1,300.00 by guest on February 24, 2014 1Soybean meal contained 48% CP. 2 Vitamin and trace minerals provided the following per kilogram: vitamin A (retinyl acetate), 6,600 IU; vitamin D3, 2,805 IU; vitamin E (dl-α-tocopheryl acetate), 10 IU; vitamin K3 (menadione dimethpyrimidinol), 2.0 mg; riboflavin, 4.4 mg; panto- −1 −1 thenic acid, 6.6 mg; niacin, 24.2 mg; choline, 110 mg ; vitamin B7 (biotin), 8.8 mg ; ethoxyquin, 1.1 mg; Mn (MnO), 88 mg; Cu (CuSO4H2O), 6.6 mg; Fe (FeSO4H2O), 8.5 mg; Zn (ZnO), 88 mg; and Se (Na2SeO3), 0.30 mg. 3Eurofins Nutrition Analysis Center [22]. through a 1-mm screen. Retention calculations AME = 100 − [(dietary Cr/fecal Cr) were [21] × (fecal GE/dietary GE) × 100].

CP retention (%) = {[dietary Cr (%)/excreta Diet samples were taken after each mixing event for analysis. The samples were ground in Cr (%)] × [excreta CP/dietary CP]} × 100; a Cyclotec 1903 sample mill and sifted through a 1-mm screen. Samples of the diets were taken Ca retention (%) = {[dietary Cr (%)/excreta at each mixing (every 2 wk), samples were then analyzed and the results for the feed analysis Cr (%)] × [excreta Ca/dietary Ca]} × 100; are an average of the analysis performed across all samples for each phase. At the end of each P retention (%) = {[dietary Cr (%)/excreta phase, the diets were pooled and analyzed at the Eurofins Nutrition Analysis Center [22] for CP Cr (%)] × [excreta P/dietary P]} × 100; [23], moisture, crude fat, crude fiber, ash, Ca [24], P [25], chromium [26], and gross ME by P retention (mg/hen per d) = [feed consumed bomb calorimetry; AME for phase 1 was then calculated. Fecal samples were analyzed for (mg) × available P] × P retention (%); and CP [23], Ca [24], P [25], chromium [26], and Hahn-Didde and Purdum: ENZYME COMPLEX IN LAYERS 27 gross ME by bomb calorimetry. Fecal analysis data were analyzed separately (Table 4). A sig- for chromium, Ca, P, and CP were analyzed at nificant effect of ME level (P ≤ 0.015) on egg the University of Nebraska-Lincoln; gross ME production (Table 4) was observed. Diets con- values for the feces were determined by Euro- taining low ME (2,860 kcal/kg) had an overall fins [22]. egg production of 89.25%, which is substantially higher than their moderate ME counterparts Data Analysis (2,900 kcal/kg; 80.88%; Table 4). Therefore, during the heat stress, egg production was more Data were analyzed for repeated measures us- dependent on ME level of the diet rather than ing the mixed models (Proc Mixed) procedures enzyme combination. Furthermore, during this in SAS [27] for a randomized complete block time period, hens fed diets containing low ME design with a 2 × 2 factorial arrangement. The (2,860 kcal/kg) consumed significantly more data were tested for the main effects of enzyme kilocalories per hen per day (P ≤ 0.0291) than combination and ME level and their interaction. hens fed moderate-ME diets (2,900 kcal/kg; Ta- Downloaded from Cages were blocked by side (north and south) ble 4). We suggest that hens fed a low-ME diet and by tier level (1–6). Each block consisted of 8 during times of heat stress will maintain higher cages. Blocking was done to reduce the amount egg production. of variation in temperature between the tiers. No significant interaction was observed be- The repeated measures procedure was used to tween enzyme, ME, and time at the conclusion http://japr.oxfordjournals.org/ examine the treatment effect over a specific time of phase 1 (P ≤ 0.242; Table 3) for feed intake. period and possible treatment by time interac- However, a significant ME by time interaction (P tions. The data analyzed via repeated measures ≤ 0.029; Table 3) was observed. Based on calcu- included egg production, feed intake, feed con- lated kilocalories per hen per day consumption, version, hen weight, and hen CV. Separation of hens had similar energy consumption across all the means was done using a least squares means diets (265.83, 268.43, 267.78, and 267.37 kcal/ statement with the pdiff option. Nutrient reten- hen per day; Table 3). These findings support tion data were analyzed via Proc Glimmix in the theory that the laying hen consumes feed to by guest on February 24, 2014 SAS [27], along with tibia measurements for meet energy requirements [29]. These results are length, diameter, and bone ash. Means were consistent with the findings of Valkonen et al. separated using a least squares means statement [30], that low-ME diets had a higher feed intake with the slicediff option. than high-ME diets through phase 1. The effect ME level has on feed intake is further confound- RESULTS AND DISCUSSION ed when examining the heat stress that occurred at the conclusion of phase 1. Within this 2-wk Production Parameters period, all diets observed approximately a 10% Overall, no significant interaction was ob- drop in feed intake. Although no significant served between enzyme supplementation, ME ME by enzyme supplementation interaction or level, and time through phase 1 or phase 2 (Table significant enzyme supplementation effect was 3). During phase 1, a significant interaction was observed, a significant effect of ME level (P ≤ noted between ME and time (P ≤ 0.005; Table 3; 0.027; Table 4) was noted. This effect is mir- Figure 1). During the final 2 wk of phase 1 (P ≤ rored when examining calculated ME consump- 0.015; Table 4), diets with low ME had higher tion, as diets containing low levels of ME had egg production than diets containing moderate higher kilocalories per hen per day consumption ME. This complements the findings of Peguri than their moderate ME counterparts (Table 4). and Coon [28], who reported that an increase This finding corresponds to previous work that in ME leads to a reduction in egg production. reported a reduction of ME level in the diet dur- This effect is most likely confounded by the ing heat stress leads to an increase in feed intake heat stress hens experienced during the final 2 [31–33]. These differences between ME levels wk of phase 1. At this time, house temperatures over time did not continue into phase 2 (P ≤ exceeded 35°C. To further analyze the effect of 0.833). Enzyme supplementation, ME level, and heat stress, the final 2 wk of phase 1 production time interaction effect was near significance (P 28 JAPR: Research Report egg) (g of feed:g Feed conversion Feed conversion day) 1 ME intake ME intake (kcal/hen per Phase 2 Feed intake Feed intake (g/hen per day) Downloaded from (%) 1.657 4.2940.855 12.288 0.5500.107 0.025 0.558 0.815 0.273 0.734 0.319 1.172 3.036 8.689 0.018 0.532 0.721 0.716 0.769 0.045 0.650 0.642 0.574 0.130 0.833 0.812 0.875 0.668 0.065 0.065 0.168 84.8783.5386.5687.29 85.98 90.06 88.54 89.53 247.6385.41 259.3885.71 247.91 250.68 89.80 1.57 84.20 87.26 1.60 86.93 1.59 1.63 255.03 88.02 247.77 89.03 1.61 253.50 1.59 249.30 1.58 1.61 Egg production Egg production http://japr.oxfordjournals.org/ egg) 0.020 (g of feed:g Feed conversion Feed conversion 0.723 0.918 0.885 0.140 2.639 0.014 day) by guest on February 24, 2014 ME intake ME intake (kcal/hen per 1 Phase 1 Feed intake Feed intake (g/hen per day) (%) 0.015 1.8340.669 3.418 0.725 0.095 0.183 0.011 0.914 0.130 0.628 0.628 0.197 0.499 0.077 0.074 0.686 0.005 0.029 0.024 0.358 0.321 0.242 0.238 0.812 91.7588.8291.9993.65 91.66 92.56 93.63 93.49 265.8391.23 268.4391.87 267.78 267.37 1.66 93.02 1.68 90.28 92.65 1.72 92.82 1.69 267.90 92.11 266.80 93.56 1.67 267.13 1.71 267.57 1.69 1.69 Egg production Egg production 3 3 5 5 ME level Moderate Moderate Low Low 3 4 4 4 5 2 2 2 0 P -value Low P -value 0.0375 Moderate SEM 0 0.0375 0 0.0375 SEM Enzyme ME Enzyme × ME Enzyme × time ME × time Enzyme × ME time Phase 1 = 25–36 wk of age; phase 2 37–51 age. Animal Nutrition [16]. Phyzyme 5000G XP; Danisco Moderate ME level, phase 1 = 2,900 kcal/kg; 2 2,860 kcal/kg. Animal Nutrition [16]. 1502 and Phyzyme 5000G XP; Danisco Avizyme Low ME level, phase 1 = 2,880 kcal/kg; 2 2,800 kcal/kg. Table 3. Egg production, intake, and feed conversion Table Diet 1 2 3 4 5 Enzyme supplementation Enzyme supplementation (% enzyme complex)

Main effect

Interaction

Hahn-Didde and Purdum: ENZYME COMPLEX IN LAYERS 29

could possibly be due to the enzyme effect, as the enzyme complex and phytase have been shown to recoup energy within the diet [1, 6, 34]. This most likely resulted in a less dramatic effect on feed intake during phase 2. No significant differences were observed between ME level and enzyme combination throughout phase 1 or phase 2 on feed conversion (Table 3). However, at the conclusion of phase 1, during the period of heat stress, a significant difference (P ≤ 0.035) was noted due to ME level, with low-ME diets having a higher feed conversion (1.59 g of feed:g of egg) than diets containing moderate levels of ME (1.52 g Downloaded from Figure 1. Egg production through phase 1 (25–36 wk of feed:g of egg; Table 4). This correlates with of age; P ≤ 0.005), showing that from 32 to 36 wk of the feed intake data during this time period. No age egg production was higher for birds fed diets con- significant differences were observed between taining low ME than for birds fed moderate ME. SEM = 0.015. phases 1 and 2 for hen weight, hen CV, or egg weight (Table 5). http://japr.oxfordjournals.org/ ≤ 0.065), trending toward diets containing en- Apparent Nutrient and Energy Retention zyme complex, phytase, and low ME to have a higher feed intake than their counterparts (Table No significant interaction was observed for 3). Overall, the lack of differences in this study calcium retention between enzyme combination

Table 4. Final 2 wk of phase 1 (34–36 wk of age)1

Measurement by guest on February 24, 2014

Egg Feed ME production Feed intake ME consumption conversion Diet level (%) (g/hen per day) (kcal/hen per day) (g of feed:g of egg)

Enzyme supplementation (% enzyme complex) 02 Moderate3 84.92 83.27 241.48 1.49 0.03754 Moderate3 76.83 82.42 239.03 1.54 02 Low5 88.00 87.78 251.05 1.61 0.03754 Low5 90.50 87.04 248.94 1.56 SEM 0.033 1.422 4.812 0.033 Main effect Enzyme 0.03754 83.67 84.73 243.99 1.55 02 86.46 85.52 246.26 1.55 P-value 0.402 0.690 0.605 0.948 ME Moderate3 80.88b 82.85b 240.26b 1.52b Low5 89.25a 87.41a 249.99a 1.59a P-value 0.015 0.027 0.0291 0.035 SEM 0.236 1.995 3.679 0.023 Enzyme × ME 0.116 0.979 0.699 0.124 a,bMeans within a column lacking a common superscript differ significantly (P ˂ 0.05). 1Temperatures exceeded 35°C at 36 wk of age and 37.7°C at 37 wk of age. 2Phyzyme 5000G XP; Danisco Animal Nutrition [16]. 3Moderate ME level, phase 1 ME = 2,900 kcal/kg; phase 2 ME = 2,860 kcal/kg. 4Avizyme 1502 and Phyzyme 5000G XP; Danisco Animal Nutrition [16]. 5Low ME level, phase 1 ME = 2,880 kcal/kg; phase 2 ME = 2,800 kcal/kg. 30 JAPR: Research Report

Table 5. Hen weight, hen CV, and egg weight

Phase 11 Phase 21

ME Hen weight Hen Egg weight Hen weight Hen Egg weight Diet level (g) CV (%) (g) (g) CV (%) (g)

Enzyme supplementation (% enzyme complex) 02 Moderate3 1,415.05 7.60 54.47 1,420.42 8.60 58.09 0.03754 Moderate3 1,439.94 8.20 54.55 1,416.73 7.60 57.38 02 Low5 1,418.64 7.90 53.97 1,411.18 8.60 58.01 0.03754 Low5 1,436.60 6.90 54.69 1,451.13 9.30 57.63 SEM 18.190 0.010 0.903 28.633 0.011 0.578 Main effect Enzyme 0.03754 1,438.27 7.60 54.62 1,433.94 8.50 57.51 02 1,416.85 7.70 54.22 1,415.81 8.60 58.05 Downloaded from P-value 0.251 0.823 0.812 0.530 0.889 0.296 ME Moderate3 1,427.50 7.90 54.51 1,418.58 8.10 57.74 Low5 1,427.62 7.50 54.33 1,431.16 8.90 57.82 P-value 0.995 0.603 0.313 0.663 0.479 0.871 SEM 13.004 0.008 0.831 20.249 0.008 0.457 http://japr.oxfordjournals.org/ Enzyme × ME 0.851 0.367 0.796 0.450 0.468 0.742 Enzyme × time 0.271 0.340 0.730 0.523 0.531 0.552 ME × time 0.730 0.688 0.314 0.261 0.543 0.117 Enzyme × ME × time 0.636 0.369 0.848 0.343 0.978 0.163 1Phase 1: 25–36 wk of age; phase 2: 37–51 wk of age. 2Phyzyme 5000G XP; Danisco Animal Nutrition [16]. 3Moderate ME level, phase 1 ME = 2,900 kcal/kg; phase 2 ME = 2,860 kcal/kg. 4Avizyme 1502 and Phyzyme 5000G XP; Danisco Animal Nutrition [16]. 5Low ME level, phase 1 ME = 2,880 kcal/kg; phase 2 ME = 2,800 kcal/kg. by guest on February 24, 2014 and ME level during phase 1. Examining the respectively; Table 6). Similar to phase 1, diets main effects, significant differences were noted containing enzyme complex had higher Ca re- between enzyme combination (P ≤ 0.013) and tention (65.51%) than the control diets (61.07%; ME level (P ≤ 0.0014; Table 6). Diets contain- Table 6). Diets containing low ME had higher ing the enzyme complex had higher levels of Ca retention (66.53 %) than their moderate ME Ca retention than the control diets. This finding counterparts (60.06%; Table 6). contradicts previous results, refuting the effects Overall, a significant difference was noted of carbohydrases and protease mixtures on min- between diets in P retention (Table 6) during eral retention [6]. However, our report and oth- phase 1 (P ≤ 0.002), with average retention val- ers have shown that the addition of the enzyme ues quite low. This was likely due to the fact complex can have a positive effect on mineral that the hens were exposed to heat stress con- retention, especially during times of heat stress ditions during the final 2 wk of phase 1 when [35, 36]. Examining ME level, diets containing fecal samples were collected. The diet that had moderate ME had higher Ca retention than low- the greatest P retention (54.91 mg/hen per day) ME diets. This also could have been due to the was the low-ME control diet (Table 6). Both di- heat stress undergone by the hens at the conclu- ets without enzyme complex exhibited higher sion of phase 1 and during the retention trial. P retention values than the diets supplemented Phase 2 was similar to phase 1, as no signifi- with the enzyme complex. It has been reported cant interaction was observed between enzyme that, with increasing ambient temperature, an supplementation and ME level (Table 6). Main increase of mineral excretion by the hen will effects of enzyme supplementation and ME lev- occur, especially P [37, 38]. In addition to the el were significant (P ≤ 0.025 and P ≤ 0.002, previous findings, it is known that during times Hahn-Didde and Purdum: ENZYME COMPLEX IN LAYERS 31

Table 6. Nutrient retention analysis of Ca, P, CP, and AME

Phase 11 Phase 21

P P ME Ca (mg/hen CP AME Ca (mg/hen per CP Diet level (%) per day) (%) (kcal/kg) (%) day) (%)

Enzyme supplementation (% enzyme complex) 02 Moderate3 58.33 14.44c 46.97c 2,630.00b 56.93 101.15 63.56 0.03754 Moderate3 60.53 39.71ab 51.81ab 2,740.00a 63.18 132.62 70.83 02 Low5 49.33 57.16a 54.91a 2,710.00a 65.21 138.76 64.94 0.03754 Low5 56.77 24.70bc 50.92b 2,710.00a 67.84 111.85 70.60 SEM 2.091 9.65 1.335 0.022 2.132 7.735 2.095 Main effect Enzyme 0.03754 58.65a 32.21 51.36 2,723.00a 65.51a 135.69a 70.72a Downloaded from 02 53.83b 35.80 50.94 2,670.00b 61.07b 106.50b 64.25b P-value 0.013 0.699 0.726 0.006 0.025 0.0005 <0.0001 ME Moderate3 59.43a 27.08 49.39b 2,685.00 60.058b 116.88 67.19 Low5 53.05b 40.93 52.91a 2,708.00 66.525a 125.30 67.77 P-value 0.0014 0.141 0.006 0.197 0.002 0.283 0.601 http://japr.oxfordjournals.org/ SEM 1.634 7.11 1.033 0.018 1.665 5.469 1.950 Enzyme × ME 0.166 0.003 0.001 0.004 0.349 0.769 0.471 a–cMeans within a column lacking a common superscript differ significantly (P ˂ 0.05). 1Phase 1: 25–36 wk of age; phase 2: 37–51 wk of age. 2Phyzyme 5000G XP; Danisco Animal Nutrition [16]. 3Moderate ME level, phase 1 ME = 2,900 kcal/kg; phase 2 ME = 2,860 kcal/kg. 4Avizyme 1502 and Phyzyme 5000G XP; Danisco Animal Nutrition [16]. 5Low ME level, phase 1 ME = 2,880 kcal/kg; phase 2 ME = 2,800 kcal/kg. by guest on February 24, 2014 of heat stress and decreased feed intake an in- for moderate and low ME, respectively). How- flammatory response takes place, which can ever, both control diets had different CP reten- also negatively affect P retention [39]. Both of tion values; diets containing moderate ME had these factors most likely contributed to the low a CP retention value of 46.97 %, whereas diets P retention at the conclusion of phase 1. In phase containing low ME had a CP retention value of 2, a main effect difference between enzyme sup- 54.91 % (Table 5). These differences in CP re- plementation (P ≤ 0.0001) on P retention was tention depending on ME are most likely due observed. Diets containing the enzyme complex to the hen consuming more when fed diets with had higher P retention (135.69 mg/hen per day) low ME. This was especially pronounced at the than the control diets (106.50 mg/hen per day). conclusion of phase 1, as with the heat stress The effect of the enzyme complex observed the hens with low-ME diets had a higher feed again illustrates that enzyme complex supple- intake (Table 4). At the conclusion of phase 2, mentation has a positive effect on mineral reten- no significant interaction between ME level and tion. It has been previously reported that the ad- enzyme combination (Table 6) was observed. dition of enzyme complex to fibrous ingredients Upon examination of the main effects, the en- assists in release of minerals, such as Ca and P zyme combination had a significant effect (P [35, 36]. ≤ 0.0001), resulting in diets with the enzyme When examining CP retention values, a sig- combination having higher CP retention than nificant interaction was observed between en- the control diets. These results are consistent zyme supplementation and ME at the conclu- with results found in previous studies, in which sion of phase 1 (P ≤ 0.001). Diets containing the the addition of a protease, such as that found in enzyme complex had CP values that were simi- enzyme complex, aids in protein and nitrogen lar regardless of ME level (51.08 and 50.91% retention [9]. 32 JAPR: Research Report

Apparent ME values were measured at the tion of energy, minerals and amino acids. Br. Poult. Sci. conclusion of phase 1, and we observed a sig- 49:37–44. 2. Lumpkins, B., A. Batal, and N. Dale. 2005. Use of nificant enzyme combination by ME effect (P distillers dried grains plus soluble in laying hen diets. J. ≤ 0.004; Table 6). The AME value of the mod- Appl. Poult. Res. 14:25–31. erate-ME control diet was significantly lower 3. Masa’deh, M. K., S. E. Purdum, and K. J. Hanford. than the other 3 diets (2,630.00 kcal/kg; Table 2011. Dried distillers grains with soluble in laying hen diets. Poult. Sci. 90:1960–1966. 6). This is possibly the result of the heat stress 4. Costa, F. G. P., C. C. Goulart, D. F. Figueiredo, C. F. that occurred during this collection period. S. Oliveria, and J. H. V. Silva. 2008. Economic and envi- Hens consuming moderate-ME diets had lower ronmental impact of using exogenous enzymes on poultry kilocalories per hen per day consumption dur- feeding. Int. J. Poult. Sci. 7:311–314. 5. Buchanan, N. P., L. B. Kimbler, A. S. Parsons, G. E. ing this time (Table 4), which could contribute Seidel, W. B. Bryan, E. E. D. Felton, and J. S. Moritz. 2007. to the significantly lower AME value for the The effects of nonstarch polysaccharide enzyme addition moderate-ME control diet. Diets containing low and dietary energy restriction on performance and carcass ME showed stable AME values, regardless of quality of organic broiler chickens. J. Appl. Poult. Res. 16:1–12. Downloaded from enzyme supplementation (Table 6). However, 6. Cowieson, A. J., and O. Adeola. 2005. Carbohy- in moderate-ME diets, supplementation of the drases, protease, and phytase have an additive beneficial ef- enzyme complex increased AME retention. Di- fect in nutriationally marginal diets for broiler chicks. Poult. Sci. 84:180–186. ets containing the enzyme complex had higher 7. Meng, X., and B. A. Slominski. 2005. Nutritive val- AME values than the control diets (Table 6).

ues of corn, soybean meal, canola meal and peas for broiler http://japr.oxfordjournals.org/ In addition, more consistent AME values were chickens as affected by a multicarbohydrase preparation of observed with the addition of the enzyme com- cell wall degrading enzymes. Poult. Sci. 84:1242–1251. 8. Yegani, M., and D. R. Krover. 2013. Effects of corn plex, moving from moderate-ME to low-ME di- source and exogenous enzymes on growth performance ets. Therefore, the addition of enzyme complex and nutrient digestibility in broiler chickens. Poult. Sci. helping to recoup the difference in the low-ME 92:1208–1220. diets when compared with moderate-ME diets. 9. Cowieson, A. 2005. Factors that affect the nutritional value of maize for broilers. Anim. Feed Sci. Technol. It has been well documented that the addition 119:293–305. of an enzyme complex to the diet assists with 10. Choct, M. 2006. Enzymes for the feed industry: Past, by guest on February 24, 2014 increasing ME [30, 35, 36]. Therefore, it was no present and future. World’s Poult. Sci. J. 62:5–16. surprise that diets containing the enzyme com- 11. Rausch, K. D., and R. L. Belayea. 2006. The future of co-products from corn processing. Appl. Biochem. Biotech- plex had higher AME values than the control nol. 128:47–86. diets. 12. Oryschak, M., D. Krover, M. Zuidhof, X. Meng, and E. Beltranena. 2010. Comparative feeding value of extruded and nonextruded wheat and corn distillers dried grains with CONCLUSIONS AND APPLICATIONS soluble for broilers. Poult. Sci. 89:2183–2196. 13. Farmer Automatic of America, Statesboro, GA. 1. In the current research, positive results 14. Hy-Line International. 2008. Hy-Line W36 Commer- were observed when limiting Ca, P, and cial Management Guide 2007–2008. Hy-Line International, ME and supplementing with an enzyme West Des Moines, IA. 15. NRC. 1994. Nutrient Requirements of Poultry. 9th complex. ed. Natl. Acad. Press, Washington, DC. 2. The addition of an enzyme complex re- 16. Danisco Animal Nutrition, St. Louis, MO. stored ME along with increased levels of 17. Poet Nutrition, Sioux Falls, SD. absorption of Ca, P, CP, and AME. 18. Phycheck software, Danisco Animal Nutrition, 3. During heat stress, birds fed diets con- Wilmington, Denmark. taining low ME outperformed birds fed 19. FTS Systems Inc., Stony Ridge, NY. moderate-ME diets, regardless of en- 20. Tecator, Huganas, Sweden. 21. Leeson, S., and J. D. Summers. 2001. Scott’s Nutri- zyme supplementation. tion of the Chicken. 4th ed. University Books, Guelph, Can- ada. REFERENCES AND NOTES 22. Eurofins Nutrition Analysis Center, Des Moines, IA. 23. AOAC. 1995. Kjeldahl method, 954.06. Official 1. Cowieson, A. J., and V. Ravindran. 2008. Effect of Methods of Analysis. AOAC, Gaithersburg, MD. exogenous enzymes in maize-based diets varying in nutrient 24. AOAC. 1995. Calcium analysis 968.08. Official density for young broilers: Growth performance and reten- Methods of Analysis. AOAC, Gaithersburg, MD. Hahn-Didde and Purdum: ENZYME COMPLEX IN LAYERS 33

25. AOAC. 1995. Phosphorus analysis 964.06. Official 35. Olukosi, O. A., A. J. Cowieson, and O. Adeola. 2007. Methods of Analysis. AOAC, Gaithersburg, MD. Age-related influence of a cocktail of xylanase, amylase and 26. Williams, C. H., D. J. David, and O. Iismaa. 1962. protease or phytase individually or in combination in broil- The determination of chromic oxide in fecal samples by ers. Poult. Sci. 86:77–86. atomic absorption spectrometry. J. Agric. Sci. 59:381–385. 36. Francesch, M., and P. A. Geraert. 2009. Enzyme 27. SAS Institute. 2008. SAS User’s Guide. Statistics. complex containing carbohydrases and phytase improves SAS Institute Inc., Cary, NC. growth performance and bone mineralization of broilers 28. Peguri, A., and C. Coon. 1991. Effect of temperature fed reduced nutrient corn-soybean-based diets. Poult. Sci. and dietary energy level on layer performance. Poult. Sci. 88:1915–1924. 70:126–138. 37. Belay, T., C. J. Wiernusz, and R. G. Teeter. 1992. 29. Hill, F. W., D. L. Anderson, and L. M. Dansky. 1956. Mineral balance and urinary and fecal mineral excretion Studies of the energy requirements of chickens 3. The effect profile housed in thermoneutral and heat-distressed environ- of dietary energy level on the rate and gross efficiency of egg ments. Poult. Sci. 71:1043–1047. production. Poult. Sci. 35:54–59. 38. Belay, T., and R. G. Teeter. 1996. Effects of ambient 30. Valkonen, E., E. Venäläinen, L. Rossow, and J. Va- temperature on broiler mineral balance partitioned into uri- laja. 2008. Effects of dietary energy content on the perfor- nary and fecal loss. Poult. Sci. 37:423–433. mance of laying hens in furnished and conventional cages. 39. Quinteiro-Filho, W. M., A. Ribeiro, V. Ferraz-de-Pau- Poult. Sci. 87:844–852. la, M. L. Pinheiro, M. Sakai, L. R. M. Sá, A. J. P. Ferreira, Downloaded from 31. Scott, T. A., and D. Balnave. 1989. Responses of and J. Palermo-Neto. 2010. Heat stress impairs performance sexually-maturing pullets to self-selection feeding under parameters, induces intestinal injury, and decreases mac- different temperature and lighting regimens. Br. Poult. Sci. rophage activity in broiler chickens. Poult. Sci. 89:1905– 30:135–150. 1914. 32. Balnave, D., and T. M. Murtisari Abdoellah. 1990. Acknowledgments Self-select feeding of commercial pullets using a complete http://japr.oxfordjournals.org/ layer diet and a separate protein concentrate at cool and hot Thanks go to Curtis Novak with Land O’Lakes (Kansas temperatures. Aust. J. Agric. Res. 41:102–107. City, MO) and Ernie Pearson of DuPont (Danisco, St. Louis, MO) for their assistance in diet formulation. Thanks also 33. Balnave, D., and J. Brake. 2005. Nutrition and man- go to Patricia Weber and Ruth Deidrichsen (University of agement of heat-stressed pullets and laying hens. World’s Nebraska-Lincoln) for their assistance with sample collec- Poult. Sci. J. 61:399–406. tion and laboratory analysis. 34. Novak, C. L., H. M. Yakout, and J. Remus. 2007. Re- sponse to varying dietary energy and protein with or without enzyme supplementation on growth and performance of leg- horns: Growing period. J. Appl. Poult. Res. 16:481–493. by guest on February 24, 2014 © 2014 Poultry Science Association, Inc. Relationships of incubational hatching egg characteristics to posthatch body weight and processing yield in Ross × Ross 708 broilers 1 , 2

E. D. Peebles,* 3 R. Pulikanti ,* W. Zhai ,* and P. D. Gerard †

* Department of Poultry Science, Mississippi State University, Mississippi State 39762; and † Department of Mathematical Sciences, Clemson University, Clemson, SC 29634

Primary Audience: Primary Breeders, Hatchery Personnel, Avian Reproductive Downloaded from Physiologists

SUMMARY http://japr.oxfordjournals.org/ The characteristics of broiler hatching eggs have the potential to effect broiler posthatch growth and processing yield. The association of set egg weight (SEW), length of incubation (LI), and mean daily percentage of incubational weight loss (MDPEWL) of embryonated Ross × Ross 708 broiler hatching eggs with subsequent posthatch BW and processing yield was investigated. Sixty Ross × Ross 708 broiler hatching eggs were randomly set on each of 8

replicate tray levels of an incubator. Weight loss of individual embryonated eggs between 0 and 10.5, 10.5 and 18.5, and 0 and 18.5 d of incubation was determined for the calculation

of MDPEWL. Furthermore, on 18.5 d of incubation, embryonated eggs were transferred to a by guest on February 24, 2014 hatcher unit where they were individually monitored for hatch every 12 h for determination of LI. Chicks were placed in corresponding replicate floor pens and were grown out from 0 (21.5 d of incubation) to 49 d posthatch. Live bird BW as a percentage of SEW on 21.0 d of incubation and d 0 and 49 posthatch, and bird sex on d 49 posthatch were determined. After commercial processing, carcass weight as a percentage of SEW, and carcass, abdominal fat pad, wings, breast muscle, tenders, drumsticks, and thighs weights as percentages of live BW were determined. Bird BW on 21.0 d of incubation and on d 0 and 49 posthatch were positively correlated or interrelated with SEW. Between 0 and 10.5 d of incubation, MDPEWL was negatively correlated with absolute and relative BW on 21.0 d of incubation, absolute BW on d 0 posthatch, and relative tenders weight. Further, LI was positively correlated with absolute and relative BW on 21.0 d of incubation and d 0 posthatch, but was negatively correlated with relative (percentage of live BW) carcass weight on d 49 posthatch. The MDPEWL of modern strain broiler hatching eggs should be closely monitored, particularly during the first half of incubation, for the regulation of LI and hatchling BW and for their potential effects on processing yield characteristics.

Key words: body weight , broiler , egg weight loss , incubation length, processing yield 2014 J. Appl. Poult. Res. 23 :34–40 http://dx.doi.org/ 10.3382/japr.2013-00784

1 This is Journal Article Number J-12335 from the Mississippi Agricultural and Forestry Experiment Station supported by MIS- 322270. 2 Use of trade names in this publication does not imply endorsement by Mississippi Agricultural and Forestry Experiment Sta- tion of these products, nor similar ones not mentioned. 3 Corresponding author: [email protected] Peebles et al.: INCUBATIONAL AND YIELD RELATIONSHIPS 35

DESCRIPTION OF PROBLEM daily percentage of incubational weight loss of embryonated eggs [MDPEWL; percentage of A better understanding of the relationships set egg weight (SEW)] between 10.5 and 18.5 between pre- and posthatch physiological vari- d of incubation and relative chick BW on d 0 ables of modern strain broilers is important, as and 0.5 posthatch. More recently, Pulikanti et the embryonic growth period in those strains al. [6] reported that on d 48 posthatch, the rela- spans over almost one-third of the broiler’s en- tive body and breast muscle weights of broilers tire growth period. Previous researchers [1, 2] were positively correlated with specific egg- have suggested the importance of understanding shell water vapor conductance, and that rela- and monitoring prehatch physiological variables tive BW was positively correlated with length of broiler hatching eggs and embryos for the im- of incubation (LI). Conversely, relative breast provement of hatchery and grow-out manage- muscle weight was negatively correlated with ment practices essential for the realization of internal egg temperature. increased slaughter yield. Eggshell conductance and MDPEWL are Downloaded from Manipulation of incubational temperature has highly interrelated variables, as MDPEWL is been shown to affect subsequent broiler breast used in the calculation of conductance [7, 8]. muscle yield at 35 [3] and 70 [4] d posthatch. The variable MDPEWL may also serve as an in- The relationships of various physiological char- dex of the functional porosity of the eggshell to acteristics of broiler hatching eggs with those of oxygen, carbon dioxide, and water [9]. Hocking http://japr.oxfordjournals.org/ corresponding broiler hatchlings during the ear- [10] has suggested that an increase in eggshell ly [5] and middle to late [6] periods of grow-out conductance facilitates increased oxygen con- have also been examined in other studies. Inter- sumption by the embryo, which subsequently nal egg temperature and specific eggshell water increases embryonic metabolic rate. However, vapor conductance (eggshell water conductance no other previous studies have examined rela- adjusted to a 100-g egg weight basis) were the tionships of various physiological parameters of prehatch variables that were of primary focus in hatching eggs, including MDPEWL, with sub- the reports by Pulikanti et al. [5, 6]. The variables sequent broiler processing yield characteristics. by guest on February 24, 2014 in chicks that were examined by Pulikanti et al. Therefore, the current study was conducted to [5] through 10 d posthatch included total BW, examine the relationships of various hatching and the weights, moisture concentrations, and egg physiological parameters, including MD- nutrient compositions (glucose, glycogen, fat, PEWL, with the BW, and relative weights of and protein) of their carcasses, pipping muscles, the carcass, abdominal fat pad, wings, breast livers, and left gastrocnemius muscles; whereas muscle, tenders, drumsticks, and thighs of the posthatch variables examined by Pulikanti et 49-d-old broilers. The selected prehatch physi- al. [6] included total BW, carcass weight, and ological variables used in this study included the weights, moisture concentrations, and nutri- MDPEWL and LI because they are noninvasive ent compositions (glucose, glycogen, fat, and procedures that can be pragmatically assessed in protein) of the livers, breast muscles, and left commercial hatcheries and because they serve gastrocnemius muscles of middle (28-d-old) and as accurate estimations of the physiological late (48-d-old) posthatch broilers. functionality of the eggshell and of the rate of Among the various relationships established embryogenesis [11–13]. by Pulikanti et al. [5], it was shown that positive correlations or interrelationships existed MATERIALS AND METHODS between internal egg temperature and percentage yolk sac weight as well as between spe- General cific eggshell water vapor conductance and percentage chick carcass weight. Conversely, The current experimental protocol was ap- in the same study, specific eggshell water va- proved by the Institutional Animal Care and por conductance was negatively correlated Use Committee of Mississippi State University. with percentage yolk sac weight. Furthermore, Seven hundred twenty Ross × Ross 708 (Avia- a negative correlation existed between mean gen Inc., Huntsville, AL) broiler hatching eggs 36 JAPR: Research Report were collected from a 30-wk-old breeder flock. Posthatch Grow Out The eggs were held under standard storage conditions for 3 d before setting. The hatchlings used for correlation analysis included only those that had hatched after 20.0 d of incubation (were observed as new hatches Incubation on 20.5 and 21.0 d of incubation). However, on On d 0 of incubation, the eggs that weighed d 21.5 of incubation (d 0 posthatch), all hatch- within ±10% of the mean weight of all eggs col- lings were pulled from the hatcher unit for post- lected and those that were not contaminated or hatch grow-out. Therefore, all hatchlings that visibly abnormal were selected for incubation. were subsequently grown-out had remained Four hundred and eighty selected eggs were ran- in the hatcher for a maximum of 36 h before domly labeled and weighed to record their indi- placement. From each tray level in the hatcher, vidual SEW (g) and were then set on each of 8 18 chicks were randomly selected for grow-out replicate tray levels (60 eggs/level) of a James- at a commercial density between 0 and 48 d Downloaded from way Model 500 single stage incubator [14]. The posthatch on corresponding climate controlled eggs were incubated for 18.5 d under standard floor pens (8 replicate floor pens) containing commercial conditions at approximately 37.5°C wood shavings. They were provided ad libitum dry bulb and 29.3°C wet bulb temperatures. The feed and water and heated brooding, and were 480 eggs were evenly distributed among the 8 maintained under standard industry wet and dry http://japr.oxfordjournals.org/ central trays to ensure an even air flow among bulb temperature settings. The birds were pro- all the eggs [5]. On d 10.5 of incubation, all eggs vided Mississippi State University basal starter, were weighed (g) and then candled to determine grower, and finisher broiler diets that met or ex- egg fertility and embryo viability. Eggs that ceeded NRC [15] recommendations, and were were infertile or that contained nonviable em- allotted according to standard industry feeding bryos were discarded. On d 18.5 of incubation, regimens. The ingredient percentages and calcu- each egg was again candled to determine em- lated analysis of the diets were as described by bryo viability. Eggs containing viable embryos Peebles et al. [16]. by guest on February 24, 2014 were then weighed (g), and 34 eggs per replicate level that contained live embryos were placed Processing and Data Collection in individual hatching baskets and subsequently transferred to their corresponding tray levels in For approximately 16 birds per replicate pen, a Jamesway Model 500 hatcher unit [14]. The live bird BW (g) was determined and was fur- eggs in the hatcher were incubated at approxi- ther calculated as a percentage of SEW on d 21.0 mately 37.4°C dry bulb and 29.9°C wet bulb of incubation, and on d 0 and 49 posthatch. In temperatures. Three precalibrated wireless data addition, on d 49 posthatch, the sex of the birds loggers in different locations were used in the was determined. The birds were then routinely setter and hatcher to more accurately monitor processed; the fat pad, carcass, wings, breast actual wet and dry bulb temperatures [6, 8]. muscle, tenders, drumsticks, and thighs of the birds were collected and weighed (g), and the Determination of Length of Incubation and relative weights (percentages of live bird BW) Percentage Incubational Egg Weight Loss of each were determined. In addition, carcass weight as a percentage of SEW was also deter- Beginning on d 18.5 of incubation, eggs were mined. individually monitored for hatch every 12 h (0.5 d) through d 21.5 of incubation for determina- Statistical Analysis tion of LI (d) [8]. Incubational weight loss values (g) of individual embryonated eggs between Using the GLM procedure of SAS [17], par- 0 and 10.5, 10.5 and 18.5, and 0 and 18.5 d of tial correlations between the pre- and posthatch incubation were determined for the subsequent variables were generated with data for each calculation of MDPEWL in each of the 3 respec- individual egg. Only those eggs that produced tive time intervals. chicks that were ultimately sampled at process- Peebles et al.: INCUBATIONAL AND YIELD RELATIONSHIPS 37 ing age were used for correlation analysis. In the to 10.5, 10.5 to 18.5, and 0 to 18.5 d of incuba- correlation analyses for each of the individual tion MDPEWL were negatively correlated with eggs and their corresponding hatchlings (ap- BWI21, BWI21 as a percentage of SEW, and proximately 16 samples × 8 replicate units = tenders weight as a percentage of live bird BW. approximately 128 total samples), the prehatch Further, 0 to 10.5 d of incubation MDPEWL was variables were SEW, 0 to 10.5 d of incubation negatively correlated with BW0. In addition, LI MDPEWL, 10.5 to 18.5 d of incubation MD- was positively correlated with BWI21, BWI21 PEWL, 0 to 18.5 d of incubation MDPEWL, and as a percentage of SEW, BW0, and BW0 as a LI. The posthatch variables were 21.0 d of incu- percentage of SEW, but was negatively corre- bation BW (BWI21); BWI21 as a percentage of lated with CWBW49. The numbers of samples, SEW; d 0 BW (BW0); BW0 as a percentage of mean, and SEM for each of the significantly cor- SEW; d 49 BW (BW49); BW49 as a percent- related variables are provided in Table 2. age of SEW; d 49 carcass weight as a percent- The loss of mass in eggs during incubation age of SEW; d 49 carcass weight as a percent- is primarily a function of the loss of water via Downloaded from age of live bird BW (CWBW49); and d 49 fat transpiration through pores in the eggshell [18]. pad, wings, breast muscle, tenders, drumsticks, It is also known that embryonic metabolism and thighs weights as percentages of live bird [10] and the subsequent posthatch growth and BW. Each of the 8 tray levels and correspond- performance of broilers [5, 6] are influenced by ing pens were considered as replicate units. The the rate at which water is lost through the pores http://japr.oxfordjournals.org/ GLM procedure was used to compute the par- of the eggshell. Nevertheless, because the diffu- tial correlations in a model that simultaneously sion rate of water through pores in the eggshell fit the variables to be correlated with sex and is also associated with the rates of exchange of replicate tray level or floor pen as fixed effects. the vital gases, oxygen and carbon dioxide [9], The MANOVA statement with the printe option changes in the diffusion rates of the vital gases produced the appropriate partial correlations. in conjunction with changes in the rate of water Partial correlation coefficients were considered loss can, likewise, effect embryonic metabo- significant at P ≤ 0.05. lism and the subsequent posthatch growth and by guest on February 24, 2014 performance of broilers. Hatching success is in RESULTS AND DISCUSSION part dependent on the RH within commercial in- cubators [18], and changes in incubational RH Mean embryonic mortality between 0 and affect the rates of vital gas exchange and water 10.5 and 10.5 and 18.5 d of incubation were loss through pores in the shells of eggs during 1.47 and 0.701%, respectively. Only eggs con- incubation [7–9, 19]. Because modern strain taining viable embryos were transferred to the commercial broilers spend a larger proportion of hatcher; all hatchlings used for grow-out had their lives in the egg while undergoing a more remained in the hatcher for a maximum of 36 rapid developmental process, the physiological h before pen placement, and only broilers that properties of the eggshell and their subsequent were processed were used for pre- and posthatch influence on embryogenesis are more likely to correlation analyses. Furthermore, only signifi- exert an ultimate influence on the processing cant (P ≤ 0.05) correlations that existed between yield of modern strain commercial broilers at pre- and posthatch variables were reported, and slaughter age. Therefore, because MDPEWL when a particular variable was used in the cal- can be altered by incubational RH, the adjust- culation of a dependent variable, the correlation ment of RH in the incubator has the potential to between those 2 variables was not reported (e.g., affect subsequent processing yield. SEW and d 49 carcass weight as a percentage Pinchasov [20] reported that a close correla- of SEW). tion or association exists between the weight of Significant correlations or levels of interde- eggs from commercial broiler breeder hens and pendence between variables in accordance with hatching chick weight, but that the high correla- the aforementioned criteria are provided in Ta- tion diminished and became insignificant by 5 d ble 1. Set egg weight was positively correlated posthatch. However, broiler BW was observed with BWI21, BW0, and BW49. Conversely, 0 by Morris et al. [21] to have a strong linear rela- 38 JAPR: Research Report

Table 1. Coefficients and P-values for statistically significant (P ≤ 0.05) partial correlations (interrelationships) of prehatch variables with respective posthatch variables1,2

Item, r 0 to 10.5 10.5 to 18.5 0 to 18.5 (P-value) SEW MDPEWL MDPEWL MDPEWL LI

BWI21 0.90 (0.0001) −0.27 (0.004) −0.23 (0.01) −0.26 (0.006) 0.29 (0.002) BWSEWI21 ─ −0.37 (0.0001) −0.38 (0.0001) −0.38 (0.0001) 0.51 (0.0001) BW0 0.85 (0.0001) −0.19 (0.04) ─ ─ 0.22 (0.02) BWSEW0 ─ ─ ─ ─ 0.27 (0.004) BW49 0.25 (0.007) ─ ─ ─ ─ CWBW49 ─ ─ ─ ─ −0.20 (0.03) TWBW49 ─ −0.24 (0.01) −0.21 (0.03) −0.23 (0.01) ─ 1Approximately 16 eggs within each of 8 replicate groups and their corresponding hatchlings were used for correlations. 2Prehatch variables of embryonated broiler hatching eggs were set egg weight (SEW; g); mean daily percentage of egg weight loss (MDPEWL) between 0 and 10.5 d of incubation (0 to 10.5 MDPEWL); 10.5 and 18.5 d of incubation (10.5 to 18.5 MDPEWL); 0 and 18.5 d of incubation (0 to 18.5 MDPEWL); and length of incubation (LI; d). The posthatch variables were BW on 21.0 d of incubation (BWI21; g); BWI21 as a percentage of SEW (BWSEWI21); BW on d 0 (BW0; g); BW0 as a Downloaded from percentage of SEW (BWSEW0); BW on d 49 (BW49; g); carcass weight as a percentage of live bird BW on d 49 (CWBW49); and tenders weight as a percentage of live bird BW on d 49 (TWBW49). tionship with hatching egg weight through 84 d MDPEWL and LI has been established. A de- posthatch, although the influence of egg weight crease in MDPEWL, as a result of an increase http://japr.oxfordjournals.org/ on BW declined with posthatch age. Likewise, in RH in the incubator, is expected to increase Rahn et al. [22] demonstrated mathematically LI in accordance with the established inverse that the posthatch BW of birds is positively re- relationship between MDPEWL and LI [7, 11, lated to the weight of the egg from which they 12, 19]. However, although MDPEWL through- hatch. In reports by Rahn and Ar [11] and Rahn out the setter period was negatively correlated et al. [22], a positive mathematical relationship with tenders weight as a percentage of live bird between LI and posthatch BW has been illustrat- ed. As would be expected upon consideration of by guest on February 24, 2014 the results of the aforementioned reports, a posi- Table 2. Number of samples (N), mean, and SEM of tive relationship has also been demonstrated to the significantly correlated (interrelated) prehatch and 1 exist between SEW and LI [11]. The current data posthatch variables presented in Table 1 confirm these earlier established relationships. Item N Mean SEM Nevertheless, the current study is unique from SEW, g 126 57.1 0.27 previous investigations in that changes in SEW, 0 to 10.5 MDPEWL, % 126 0.56 0.007 LI, and MDPEWL were associated with those 10.5 to 18.5 MDPEWL, % 126 0.51 0.007 of various slaughter yield variables in addition 0 to 18.5 MDPEWL, % 126 0.54 0.006 to posthatch BW. Furthermore, the responses of LI, d 126 20.6 0.02 corresponding slaughter yield variables to MD- BWI21, g 126 42.3 0.24 BWSEWI21, % 126 74.0 0.18 PEWL and LI were traced in individual Ross × BW0, g 126 40.0 0.23 Ross 708 broilers that were hatched from eggs BWSEW0, % 126 70.0 0.20 incubated under an RH (approximately 54%) BW49, g 126 3,278 43 that was close to optimal for hatchability [23]. CWBW49, % 122 70.1 0.13 The current results, that absolute and rela- TWBW49, % 125 4.35 0.039 1 tive BW on 21 d of incubation and on 0 d post- Prehatch variables of embryonated broiler hatching eggs were set egg weight (SEW); mean daily percentage egg hatch were positively correlated with LI, that weight loss (MDPEWL) between 0 and 10.5 d of incubation absolute and relative BW on 21.0 d of incuba- (0 to 10.5 MDPEWL); 10.5 and 18.5 d of incubation (10.5 tion were negatively correlated with MDPEWL to 18.5 MDPEWL), and 0 and 18.5 d of incubation (0 to 18.5 MDPEWL); and length of incubation (LI). The post- throughout the setter period, and that BW0 was hatch variables were BW on 21.0 d of incubation (BWI21); negatively correlated with MDPEWL during the BWI21 as a percentage of SEW (BWSEWI21); BW on d 0 first half of incubation, are in agreement with (BW0); BW0 as a percentage of SEW (BWSEW0); BW on d 49 (BW49); carcass weight as a percentage of live bird BW reports by Rahn and Ar [11] and Ar and Rahn on d 49 (CWBW49); and tenders weight as a percentage of [12], in which an inverse relationship between live bird BW on d 49 (TWBW49). Peebles et al.: INCUBATIONAL AND YIELD RELATIONSHIPS 39

BW on d 49, LI was negatively correlated with possibly affect various processing yield CWBW49. The negative correlation that LI had characteristics through d 49 posthatch. with CWBW49 was, therefore, not expected, but 3. Because MDPEWL can be altered by does suggest that a shorter LI may be favorable adjusting RH in the incubator, desired to a specific increase in carcass yield on d 49. decreases in MDPEWL (as suggested in Nevertheless, based on these results, we sug- the previous statement) may be achieved gest that LI and MDPEWL (particularly during through fine incremental increases in the first half of incubation), influence hatchling incubator RH. This can subsequently BW and may have subsequent finite effects on influence embryonic metabolism by al- slaughter yield in modern strain broilers. Adjust- tering water loss and vital gas exchange ments in incubator RH should be considered as across the eggshell. potential means by which to control hatchling BW and possibly influence processing yield in Ross × Ross 708 broilers through d 49 posthatch. REFERENCES AND NOTES Downloaded from Because the total life span of the modern commercial broiler has been substantially short- 1. Bamelis, F., B. Kemps, K. Mertens, B. De Ketelaere, E. Decuypere, and J. DeBaerdemaeker. 2005. An automatic ened, broiler development during the embry- monitoring of the hatching process based on the noise of the onic period has become particularly important. hatching chicks. Poult. Sci. 84:1101–1107. A slight increase in LI may positively influence 2. Peebles, E. D., R. W. Keirs, L. W. Bennett, T. S. Cum- http://japr.oxfordjournals.org/ hatchling BW with limited effects on subse- mings, S. K. Whitmarsh, and P. D. Gerard. 2005. Relation- ships among prehatch and posthatch physiological param- quent processing yield in modern strain broilers. eters in early nutrient restricted broilers hatched from eggs A fine change in incubator RH may further ben- laid by young breeder hens. Poult. Sci. 84:454–461. efit embryogenesis by modulating the embryo’s 3. Piestun, Y., O. Halevy, D. Shinder, M. Ruzal, S. Druyan, and S. Yahav. 2011. Thermal manipulations during rate of metabolism. However, the actual physi- broiler embryogenesis improve post hatch performance un- ological bases for these relationships can only der hot conditions. Therm. Biol. 36:469–474. be speculated upon at present, as the eggshell 4. Piestun, Y., S. Druyan, J. Brake, and S. Yahav. 2013. diffusion rates of the vital gases (i.e., oxygen Thermal manipulations during broiler incubation alter performance of broilers at 70 days of age. Poult. Sci. 92:1155– by guest on February 24, 2014 and carbon dioxide) as well as that of water can 1163. be affected by the incubational environment. It 5. Pulikanti, R., E. D. Peebles, W. Zhai, L. W. Bennett, is suggested that further research be conducted and P. D. Gerard. 2012. Physiological relationships of the to delineate the relative influences of alterations early post-hatch performance of broilers to their embryo and eggshell characteristics. Poult. Sci. 91:1552–1557. in water loss and vital gas exchange, in response 6. Pulikanti, R., E. D. Peebles, L. W. Bennett, W. Zhai, to changes in incubator RH, on embryonic me- and P. D. Gerard. 2013. Physiological relationships of the tabolism. middle and late post-hatch performance of broilers to their embryo and eggshell characteristics. J. Poult. Sci. 50:375– 380. CONCLUSIONS AND APPLICATIONS 7. Ar, A., C. V. Paganelli, R. B. Reeves, D. G. Greene, and H. Rahn. 1974. The avian egg: Water vapor conductance, shell thickness, and functional pore area. Condor 1. The MDPEWL of modern strain broiler 76:153–158. hatching eggs should be closely moni- 8. Pulikanti, R., E. D. Peebles, and P. D. Gerard. 2011. tored, particularly during the first half of Use of implantable temperature transponders for the determination of air cell temperature, eggshell water vapor con- incubation, for the regulation of LI and ductance, and their functional relationships in embryonated hatchling BW, and for their potential ef- broiler hatching eggs. Poult. Sci. 90:1191–1196. fects on processing yield characteristics. 9. Deeming, D. C. 2002. Functional characteristics of 2. Small incremental decreases in MD- eggs. Pages 28–42 in Avian Incubation-Behaviour, Environ- ment, and Evolution, D. C. Deeming, ed. Oxford University PEWL between 10.5 and 18.5 d of in- Press, New York, NY. cubation should be tested as a means by 10. Hocking, P. M., ed. 2009. Biology of Breeding Poul- which to promote an increase in broiler try, Poultry Science Symposium Series. Vol. 29. CABI, BW on 21.0 d of incubation. Similar Wallingford, UK. 11. Rahn, H., and A. Ar. 1974. The avian egg: Incubation changes in MDPEWL between 0 and time and water loss. Condor 76:147–152. 10.5 d of incubation might also be used 12. Ar, A., and H. Rahn. 1978. Interdependence of gas to promote an increase in bird BW and to conductance, incubational length, and weight of the avian 40 JAPR: Research Report egg. Pages 227–236 in Respiratory Function in Birds, Adult 20. Pinchasov, Y. 1991. Relationship between the weight and Embryonic, J. Piiper, ed. Springer-Verlag, Berlin, Ger- of hatching eggs and subsequent early performance of broil- many. er chicks. Br. Poult. Sci. 32:109–115. 13. Tullett, S. C., and F. G. Burton. 1982. Factors affect- 21. Morris, R. H., D. F. Hessels, and R. J. Bishop. 1968. ing the weight and water status of the chick at hatch. Br. The relationship between hatching egg weight and sub- Poult. Sci. 23:361–369. sequent performance of broiler chickens. Br. Poult. Sci. 14. Jamesway Incubator Company, Inc., Cambridge, ON, 9:305–315. Canada. 22. Rahn, H., C. V. Paganelli, and A. Ar. 1975. Relation 15. NRC. 1994. Nutrient Requirements of Poultry. 9th of avian egg weight to body weight. Auk 92:750–765. rev. ed. Natl. Acad. Press, Washington, DC. 23. Robertson, I. S. 1961. Studies in the effect of humid- 16. Peebles, E. D., C. D. Zumwalt, P. D. Gerard, M. A. ity on the hatchability of hen’s eggs. I. The determination of Latour, and T. W. Smith. 2002. Market age live weight, car- optimum humidity for incubation. J. Agric. Sci. 57:185– cass yield, and liver characteristics of broiler offspring from 194. breeder hens fed diets differing in fat and energy contents. Poult. Sci. 81:23–29. Acknowledgments 17. SAS Institute. 2003. SAS Proprietary Software. Re- The authors express their appreciation for the expert lease 9.1. SAS Inst. Inc., Cary, NC. technical assistance of Alejandro Corzo and Sharon K. 18. Ar, A., and H. Rahn. 1980. Water in the avian egg: Womack of the Mississippi State University Poultry Science Downloaded from Overall budget of incubation. Am. Zool. 20:373–384. Department, and for the assistance of graduate students and 19. Rahn, H., R. A. Ackerman, and C. V. Paganelli. 1977. undergraduate student workers of the Mississippi State Uni- Humidity in the avian nest and egg water loss during incuba- versity Poultry Science Department. tion. Physiol. Zool. 50:269–283. http://japr.oxfordjournals.org/ by guest on February 24, 2014 © 2014 Poultry Science Association, Inc. Effects of distillers dried grains with solubles and mineral sources on gaseous emissions

W. Li,* Q.-F. Li,* W. Powers ,*1 D. Karcher ,* R. Angel ,† and T. J. Applegate ‡

* Department of Animal Science, Michigan State University, East Lansing 48824; † Department of Animal and Avian Sciences, University of Maryland, College Park 20742; and ‡ Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-2054

Primary Audience: Laying Hen Producers, Nutritionists, Environmentalists, and Researchers Downloaded from

SUMMARY

Distillers dried grains with solubles (DDGS), an important ethanol industry co-product, has http://japr.oxfordjournals.org/ been used as a high-protein feed in poultry production. Limited studies exist on their effect on air emissions, however. In the current study, 4 diets (2 × 2 factorial design: 0 or 20% DDGS + inorganic or organic mineral sources) were fed to Hy-line W36 hens from 50 to 53 wk of age and the effects of DDGS level and mineral sources on air emissions were monitored continu- ously for a 23-d period in environmentally controlled chambers. The NH3, H2S, CH4, nonmeth-

ane hydrocarbons, N2O, CO2, and CO2-equivalent emissions ranged from 0.51 to 0.64 g/day- hen, 0.71 to 0.84 mg/day-hen, 33.9 to 46.0 mg/day-hen, 54.1 to 60.0 mg/day-hen, 66.0 g to 72.2

g/day-hen, and 83.1 to 92.1 g/day-hen, respectively. Feeding DDGS to laying hens resulted in by guest on February 24, 2014 14% decreased NH3 emissions but a 19% increase in CH4 emissions without affecting other gaseous emissions. More than 30% of N, 80% of P, 80% of K, and 50% of Ca was retained in the manure. In conclusion, feeding laying hens a diet containing 20% DDGS may be beneficial for the environment. Substitution for organic trace minerals did not affect hen performance or air emissions.

Key words: air emissions, distillers dried grains with solubles, laying hen, mineral source 2014 J. Appl. Poult. Res. 23 :41–50 http://dx.doi.org/ 10.3382/japr.2013-00802

DESCRIPTION OF PROBLEM grain sources [1, 2]. Feeding increased dietary concentrations of DDGS could result in exces- As a result of the ethanol industry’s continu- sive protein intake, which may alter air emis- ous growth, co-products such as distillers dried sions [3]. grains with solubles (DDGS) are available as Typically, DDGS is included in laying hen an energy source in livestock and poultry feeds. diets at 5 to 15% to avoid negative effects on Corn is the primary grain used in US ethanol animal performance including egg production production due to its consistent availability. Dis- and egg weight. Lumpkins et al. [4] reported tillers dried grains with solubles are higher in a significant reduction in hen-day egg produc- energy, mineral, and protein content, but lower tion when feeding 15% DDGS and suggested a in starch content compared with the original maximum inclusion level of 10 to 12% DDGS.

1 Corresponding author: [email protected] 42 JAPR: Research Report

Roberson et al. [5] found linear decreases in egg ganic minerals (20_Org). These 4 different diets production, egg weight, and egg mass as DDGS provided very similar energy (ME ranged from increased. In recent reports, researchers have 2,800 to 2,847 kcal/kg) and similar mineral lev- suggested that 20% DDGS can be fed with no els (Table 1). All diets were formulated to meet negative effects on ADFI and egg production [6] or exceed NRC nutrient recommendations for and only slightly reduced egg weight [7]. laying hens [1]. Research involving organic trace mineral Hens were provided ad libitum access to feed sources is increasing [3, 8]. Huang et al. [8] con- and water using a feed trough and nipple water- ducted a study to estimate relative bioavailabil- ers. Daily amount of feed was added into feed ity of Zn in 3 organic zinc sources with different troughs every morning between 0600 and 0730 chelation strength compared with ZnSO4, and h. Average daily feed intake for each room was found organic minerals may be better used by calculated weekly based on total amount of feed laying hens. It has been demonstrated that in- added during the week and the amount of feed cluding organic mineral sources in swine diets left at the end of the week. Feed was sampled Downloaded from containing 20% DDGS reduced H2S emissions weekly and pooled by treatment at the end of the by 25% compared with a DDGS diet containing study. Eggs were collected daily from each room inorganic trace minerals [3]. Therefore, the pos- and egg weight and number was recorded. On sibility exists of applying organic trace minerals the last day of the study, excreta was removed, in poultry to alleviate H S emissions resulting weighed, mixed, and subsampled for each room.

2 http://japr.oxfordjournals.org/ from feeding diets with higher levels of DDGS. Temperature was maintained at 22°C ± 0.4 for The objective of the current study was to inves- the entire study. Humidity ranged from 36 to tigate the effects of a combination of feed in- 70% throughout the 3-wk period, and light (20 gredients, mainly DDGS (0% or 20%), and or- lx) was provided from 0600 to 2000 h. ganic versus inorganic diet mineral courses on gaseous emissions. Measurements of Gaseous Concentrations MATERIALS AND METHODS Twelve rooms (height = 2.60 m, width = 2.37 by guest on February 24, 2014 All animal procedures were approved by the m, length = 4.11 m) were designed to continu- Michigan State University Institutional Animal ously monitor incoming and exhaust concen- Care and Use Committee. A total of 672 Hy- trations of gases [3, 10]. Ammonia (NH3) was Line W36 hens at 50 wk of age were randomly measured using a chemiluminescence NH3 ana- assigned to 1 of 12 environmentally controlled lyzer with a detection limit of 0.001 ppm [11], 2 rooms (7 hens/cage; 355 cm of cage space/hen; which is a combination NH3 converter and NO- 8 cages/room) for a 23-d experimental period. NO2-NOx analyzer. Hydrogen sulfide (H2S) was The experimental unit was the room (group of analyzed using pulsed fluorescence SO2-H2S hens) to which the diet treatment was randomly analyzer with a detection limit of 0.003 ppm (er- assigned. The measurement unit was the same ror = 1% of full-scale at 1 ppm [12]). Methane as the experimental unit and the measurements (range: 0~100 ppm; detection limit: 0.05 ppm) (such as total egg number, feed consumption) and nonmethane total hydrocarbon (NMTHC; were collected from each room (group of hens). range: 0~10 ppm; detection limit: 0.02 ppm) was determined by a back-flush gas chromatog- Diets and Management raphy system [13]. Concentrations of CO2 (5.1 ppm detection limit at 1,000 ppm range) and Diets were arranged in a 2 × 2 factorial de- N2O (0.03 ppm detection limit at 50,000 ppm sign based on level of DDGS (0 and 20%) and range) were measured using an INNOVA 1412 source of trace minerals (inorganic and organic). photoacoustic analyzer [14]. Through software Organic minerals were supplied by Pancosma control [15], monitoring of gas concentrations in [9]. The 4 diets were 0% DDGS with inorganic each room occurred in a sequential manner. Gas minerals (0_Inorg), 0% DDGS with organic emission rates were calculated as the product of minerals (0_Org), 20% DDGS with inorganic ventilation rates and concentration differences minerals (20_Inorg), and 20% DDGS with or- between exhaust and incoming air. Li et al.: GASEOUS EMISSIONS IN HENS 43

Table 1. Diet and nutrient composition of a control diet or diets containing distillers dried grains with soluble (DDGS) with inorganic or organic trace minerals sources1

Item 0_Inorg 0_Org 20_Inorg 20_Org

Ingredient (% of mix, prepared from a basal mix) Corn 54.4 54.48 41.7 41.7 DDGS — — 20.0 20.0 Soybean meal (48%) 29.6 29.6 20.3 20.3 Soy oil 2.61 2.61 4.60 4.60 Iodized sodium chloride 0.41 0.41 0.33 0.33 dl-Met 0.19 0.19 0.18 0.18 l-Lys, HCl — — 0.15 0.15 Limestone 9.72 9.72 9.92 9.92 Dicalcium phosphate 1.78 1.78 1.47 1.47 Diatoamaceous earth 1.00 1.00 1.00 1.00 Trace mineral and vitamin premix2 0.35 0.35 0.35 0.35 Total 100 100 100 100 Downloaded from Analyzed (calculated) dietary composition (as-is basis) CP (%) 18.1 17.9 19.0 18.0 NDF (%) 8.40 6.20 14.7 14.9 Total P (mg/kg) 6,762 7,219 6,597 6,697 K (mg/kg) 9,577 9,788 9,317 9,065 Ca (%) 4.77 4.86 4.20 4.80 http://japr.oxfordjournals.org/ Mg (mg/kg) 1,770 1,831 2,056 1,975 Na (mg/kg) 1,398 1,519 1,218 1,514 Fe (mg/kg) 348 409 310 341 Cu (mg/kg) 10.7 18.9 11.3 12.8 S (mg/kg) 2,506 2,600 3,018 3,172 ME (cal/kg) 2,847 2,847 2,800 2,800 10_Inorg = 0% DDGS + inorganic minerals; 0_Org = 0% DDGS + organic minerals; 20_Inorg = 20% DDGS + inorganic minerals; and 20_Org = 20% DDGS + organic minerals. 2 Inorganic trace mineral mix used in the 0_Inorg and 20_Inorg treatment. The mix provided per kilogram of diet: copper from by guest on February 24, 2014 copper sulfate, 8.34 mg; iron from ferrous sulfate, 83.75 mg; zinc from zinc sulfate, 100.06 mg; manganese from 50% manganese oxide and 50% manganese sulfate, 33.34 mg; iodine from calcium iodate, 0.86 mg; selenium from sodium selenite, 0.3 mg. Organic trace mineral mix used in the 0_Org and 20_Org treatment. The mix provided per kilogram of diet: copper from copper glycinate, 8.34 mg; iron from ferrous glycinate, 83.75 mg; zinc from zinc glycinate, 100.06 mg; manganese from manganese glycinate, 33.34 mg; iodine from ethylenediamine dihyrdroiodide, 0.86 mg; selenium proteinate from B-Traxim Selenium (Pancosma S.A., Switzerland), 0.3 mg. Luprosil Salt, calcium salt of propionic acid containing a minimum of 98% calcium propionate; BASF Corporation, Florham Park, New Jersey.

Chemical Analyses Global Warming Potential and N Excretion Calculation Feed and excreta N content were determined using the Kjeldahl method [16]. Feed amino Predicted daily global warming potential acid content was analyzed by the University of (GWP) was calculated based on sum of the Missouri Agriculture Experiment Station Labo- 100-year GWP of CH4, N2O, and CO2. All emis- ratory [17] using HPLC methods [18]. Feed sion masses were converted to a CO2-equivalent energy and mineral content (Ca, P, Mg, K, Na, (CO2e) basis. The 100-yr GWP of CH4 is 21 S, Cu, Zn, Fe, Mn, Mo) were analyzed by the times that of CO2 and the GWP of N2O is 310 University of Arkansas Center for Excellence in times that of CO2 [23]. Poultry Science [19] laboratory using bomb calorimetry and microwave digestion followed by Nutrient and Mineral Balance inductively coupled plasma mass spectrometry, respectively. Manure P content was analyzed by Nutrient balance was calculated using the Dairy One [20] using a Foss NIR System Mod- amount of each nutrient in consumed feed and el 6500 with Win ISI II v1.5 [21, 22]. Manure the amount of each nutrient that was deposited NH4-N content was measured by distillation in a in the egg, excreted in the manure, and emitted Michigan State University laboratory [16]. to air. The nutrient and minerals data for whole 44 JAPR: Research Report fresh egg were from literature (Table 2), and the Table 2. Nutrient composition for whole raw and fresh average N content of protein was estimated at eggs [24] 16% [24]. Nutrient or mineral Value per 100 g

Water (g) 76.15 Statistical Analyses Energy (kcal) 143 Protein (g) 12.56 Emissions data and hen performance data, in- Phosphorus (mg) 198 cluding egg weight, egg production, and ADFI, Calcium (mg) 56 were analyzed using a mixed model procedure Sodium (mg) 142 [25]. Significant differences were declared at P Iron (mg) 1.75 Copper (mg) 0.072 < 0.05. All data were analyzed as repeated mea- Magnesium (mg) 12 surements (the response variable is measured Zinc (mg) 1.29 multiple times on the same experimental unit), Manganese (mg) 0.028 and the model consisted of a DDGS factor, a Potassium (mg) 138 1 Downloaded from mineral source factor, and their interaction. Sulfur (mg) 144 1 A modification of Akaike’s Information Stadelman and Pratt [28]. Criterion (AICc), which is more appropriate for small sample sizes, was applied to choose 4 different treatments ranged from 0.51 to 0.64 among correlation structures after an interaction g/day-hen (Table 5), similar to the results (0.60 http://japr.oxfordjournals.org/ was tested. If the interactions were not signifi- and 0.62 g/day-hen) of Wang-Li et al. [26] and cant, then main effects would be analyzed. If the means reported (0.95 g/day-hen) by Lin et al. first-order interactions were significant, then the [27]. The H2S emission rates from the 4 different structure would be treated as a one-way ANOVA treatments ranged from 0.71 to 0.86 g/day-hen, where each unique treatment combination rep- which were lower than the means (1.27 g/day- resented a separate treatment group. Manure hen) reported by Lin et al. [27]. characteristics data were analyzed using GLM Based on AICc, compound symmetry cor- procedure [25]. Tukey’s studentized range test relation structure was applied and the results of by guest on February 24, 2014 was performed to assess all diet combination ef- interactions, main effect of DDGS level, source fects at P < 0.05 level. of mineral, and hen age were listed in Table 6. No significant interactions of DDGS levels with RESULTS AND DISCUSSION mineral sources were observed. For NH3 and Hen Performance and Diet Effect CH4, DDGS level had a significant effect and mineral source had no significant effect. Fol- Daily egg production profile from rooms 1 to lowing feeding of 0% DDGS, NH3 had 14% 12 with different diet treatments showed simi- higher emission rate than feeding 20% DDGS, lar egg production rates. The overall egg pro- although the N intakes were not significantly duction ranged from 86.4 to 90.3%, egg mass different (0%: 2.92 g of N/hen-day vs. 20%: ranged from 55.5 to 57.9 g of eggs/hen-day, and 2.89 g of N/hen-day; P = 0.40). For CH4 emis- ADFI ranged from 99.8 to 102.7 g of feed/hen- sions, feeding the 20% DDGS diet resulted in day (Table 3). Neither the interaction nor the 19% higher emissions than feeding the 0% main effect of DDGS level nor the main effect DDGS diet. Neither DDGS levels nor mineral of mineral source was significant on egg pro- source had a significant effect on H2S, NMTHC, duction, egg weight, or ADFI (Table 3). N2O, CO2, or CO2e emissions.

Gaseous Emissions and Diet Effect Nutrient and Mineral Balance: Retention, Emission, and Excretion Emission rates of NH3, H2S, CH4, NMTHC, N2O, CO2, and CO2e were calculated based on Daily manure excretion ranged from 62.7 to g/day-hen (hen specific), g/kg of feed (feed 73.9 g/day-hen, and the excretion mass from specific), and g/dozen egg (egg production spe- hens fed 20_Inorg was significantly higher than cific; Table 4). The NH3 emission rates from the excretion mass from hens fed 0_Inorg (Table 6). Li et al.: GASEOUS EMISSIONS IN HENS 45

2 ADFI 0.40 0.54 0.18 (g of feed/hen-day) 101.4 100.3 100.5 101.2 99.8 ± 6.16 (75, 102.8) 100.1 ± 6.63 (75, 102.6) 102.7 ± 6.72 (75, 105.7) 100.8 ± 6.41 (75, 103.0) Downloaded from Egg mass 0.74 0.32 0.54 56.4 55.9 55.3 57.1 (g of eggs/hen-day) 55.0 ± 7.53 (72, 54.0) 57.9 ± 7.04 (72, 57.4) 55.5 ± 8.62 (72, 54.8) 56.2 ± 6.24 (72, 56.3) http://japr.oxfordjournals.org/ (%) by guest on February 24, 2014 0.61 0.31 0.48 Egg production Egg production 88.3 87.2 86.6 88.9 86.4 ± 10.9 (72, 85.6) 90.3 ± 10.3 (72, 89.3) 86.8 ± 12.6 (72, 86.4) 87.6 ± 9.19 (72, 87.4) 8 8 8 df -value) 1 0 20 Inorganic Organic DDGS Minerals DDGS × minerals DDGS Mineral source Mineral Main effect least squares means Main effect Type 3 tests of fixed effects ( P 3 tests of fixed effects Type 0_Inorg = 0% distillers dried grains with solubles (DDGS) + inorganic minerals; 0_Org = 0% DDGS + organic mineral; 20_Inorg = 20% DDGS + inorganic minerals; and 20_Org = 20% 20_Org and minerals; inorganic 20% DDGS + = 20_Inorg mineral; organic 0% DDGS + = 0_Org minerals; inorganic (DDGS) + solubles with grains dried 0% distillers = 0_Inorg Daily average feed consumption calculated using total period data. 0_Inorg Egg production, egg mass, and ADFI for hens fed different dietary treatments (mean ± SD; n, median) ADFI for hens fed different 3. Egg production, egg mass, and Table Treatment 1 minerals. DDGS + organic 2 0_Org 20_Inorg 20_Org

Effect

46 JAPR: Research Report (g) 3 e 2 CO (g) 2 CO O (mg) Downloaded from 2 N (mg) 2 http://japr.oxfordjournals.org/ NMTHC (mg) ± 29.3 ± 27.3 ± 21.9 ± 23.3 3.56 ± 5.70 2.89 ± 5.51 ± 310 2.66 ± 4.36 ± 279 3.04 ± 4.11 ± 223 55.5 ± 34.0 ± 240 59.6 ± 28.5 36.6 ± 60.5 54.1 ± 44.2 28.1 ± 56.0 ± 414 60.0 ± 27.0 66.0 ± 18.9 26.0 ± 45.2 ± 375 73.4 ± 17.8 30.4 ± 42.5 ± 317 72.2 ± 13.6 558 ± 351 83.1 ± 26.7 ± 331 72.1 ± 15.9 582 ± 283 50.5 ± 85.9 92.1 ± 25.2 539 ± 422 37.0 ± 73.4 89.6 ± 23.2 599 ± 267 37.3 ± 61.0 91.3 ± 23.3 663 ± 210 42.3 ± 58.4 718 ± 188 782 ± 512 716 ± 188 798 ± 387 838 ± 298 723 ± 164 773 ± 547 903 ± 264 828 ± 381 891 ± 226 927 ± 294 917 ± 23.3 987 ± 275 1,009 ± 237 1,165 ± 411 997 ± 250 1,227 ± 358 1,254 ± 367 1,258 ± 352 4 a a a a a a b b b b b b CH by guest on February 24, 2014 P < 0.05). S (mg) 2 H (g) 3 ± 0.24 ± 0.25 ± 0.23 0.71 ± 0.24 ± 0.23 0.81 ± 0.26 0.86 ± 0.29 ± 2.23 0.76 ± 0.24 33.9 ± 2.24 40.4 ± 2.15 7.17 ± 2.65 46.0 ± 2.14 7.93 ± 2.83 45.0 8.64 ± 3.30 ± 3.39 7.70 ± 2.77 ± 3.65 349 ± 3.34 401 10.0 ± 3.57 ± 3.37 458 10.8 ± 3.70 454 12.1 ± 4.88 10.5 ± 3.27 471 537 642 621 a a a a a a b b b b b b NH n 69 0.54 6969 0.64 69 0.51 0.51 6969 5.28 69 6.18 69 5.01 5.02 6969 7.55 69 8.70 69 7.13 7.07 equivalent. 2 1 e = CO 2 Means within a column with no common superscript differ significantly ( Means within a column with no common superscript differ 0_Inorg 0_Org 20_Inorg 20_Org 0_Inorg 0_Org 20_Inorg 20_Org 0_Inorg 0_Org 20_Inorg 20_Org 0_Inorg = 0% distillers dried grains with solubles (DDGS) + inorganic minerals, 0_Org = 0% DDGS + organic minerals, 20_Inorg = 20% DDGS + inorganic minerals, and 20_Org = 20% and 20_Org minerals, = 20% DDGS + inorganic 20_Inorg minerals, = 0% DDGS + organic 0_Org minerals, dried grains with solubles (DDGS) = 0% distillers + inorganic 0_Inorg NMTHC = nonmethane hydrocarbons. CO Gas emissions from hens fed different dietary treatments (mean ± SD) 4. Gas emissions from hens fed different Table Treatment a,b 1 minerals. DDGS + organic 2 3

Day-hen Kilogram of feed Dozen egg

Li et al.: GASEOUS EMISSIONS IN HENS 47 egg 3 e g/dozen 2 CO hen g/day- egg 2 g/dozen CO hen g/day- egg Downloaded from O mg/dozen mg/dozen 2 N hen mg/day- http://japr.oxfordjournals.org/ 2 egg mg/dozen mg/dozen NMTHC hen 3.222.85 43.7 39.8 57.6 57.0 790 800 69.7 72.1 957 1,003 87.6 90.5 1,196 1,256 mg/day- a b egg 504 631 4 by guest on February 24, 2014 mg/dozen mg/dozen CH a b hen mg/day- P < 0.05). 0.33 <0.01 <0.01 0.62 0.74 0.87 0.84 0.31 0.29 0.33 0.25 egg S mg/dozen mg/dozen 2 H hen 0.790.78 11.1 10.6 40.0 42.7 556.5 578.6 3.11 2.96 43.9 39.6 54.8 59.8 778 813 69.1 72.8 968 992 86.3 91.7 1,210 1,243 0.760.81 10.4 11.3 37.1 45.5 mg/day- b a 1 0.96 0.62 0.18 0.47 0.85 0.72 0.14 0.48 0.15 0.57 0.09 0.51 egg 8.12 7.10 3 g/dozen NH b a hen 0.51 0.530.58 7.34 7.86 0.020.11 0.010.10 0.1 0.08 0.42 0.59 0.14 0.18 0.08 0.17 0.49 0.45 0.78 0.69 0.14 0.40 0.23 0.57 g/day- -value lower than the significance level of 0.05, the null hypothesis is rejected. Values within a column were not significantly different at 95% CI. within a column were not significantly different Values the null hypothesis is rejected. P -value lower than the significance level of 0.05, equivalent. 2 1 e = CO 2 20% Inorganic Organic 0% Means within a column with no common superscript differ significantly ( Means within a column with no common superscript differ Mineral source DDGS Minerals DDGS × minerals DDGS If the test of significance gives a NMTHC = nonmethane hydrocarbons. CO

least squares means Main effect Type 3 tests of fixed effects 3 tests of fixed effects Type ( P -value) The effect of distillers dried grains with solubles (DDGS) and mineral source on gas emission rates The effect 5. Table Effect a,b 1 2 3 48 JAPR: Research Report

Table 6. The effect of distillers dried grains with solubles (DDGS) and mineral source on excretion characteristics (mean ± SD with n in parentheses)1

Item 0_Inorg 0_Org 20_Inorg 20_Org

Manure (g/day-hen) 62.7b ± 3.97 (3) 66.3ab ± 3.71 (3) 73.9a ± 4.45 (3) 69.8ab ± 4.42 (3) DM (%) 34.1a ± 1.17 (3) 32.9a ± 1.55 (3) 34.6a ± 1.40 (3) 34.8a ± 1.85 (3) + b b a a NH4 -N (g/day-hen) 0.39 ± 0.06 (3) 0.41 ± 0.02 (3) 0.59 ± 0.05 (3) 0.59 ± 0.01 (3) TKN2 (g/day-hen) 0.98b ± 0.07 (3) 0.99b ± 0.10 (3) 1.24a ± 0.06 (3) 1.17ab ± 0.05 (3) pH 7.18a ± 0.63 (21) 7.29a ± 0.64 (21) 7.07a ± 0.54 (21) 7.13a ± 0.65 (21) P (g/day-hen) 0.57a ± 0.03 (3) 0.65a ± 0.09 (3) 0.61a ± 0.06 (3) 0.59a ± 0.05 (3) Ca (g/day-hen) 2.60b ± 0.05 (3) 2.96ab ± 0.12 (3) 3.01a ± 0.12 (3) 2.82ab ± 0.21 (3) Na (g/day-hen) 0.14a ± 0.00 (3) 0.15a ± 0.02 (3) 0.16a ± 0.00 (3) 0.16a ± 0.01 (3) Fe (mg/day-hen) 35.1a ± 1.22 (3) 35.2a ± 8.98 (3) 36.5a ± 1.95 (3) 35.1a ± 3.22 (3) Cu (mg/day-hen) 1.91a ± 0.17 (3) 1.88a ± 0.24 (3) 1.81a ± 0.04 (3) 1.62a ± 0.08 (3) Mg (g/day-hen) 0.16a ± 0.01 (3) 0.17a ± 0.10 (3) 0.18a ± 0.01 (3) 0.17a ± 0.02 (3) Zn (mg/day-hen) 13.6a ± 0.55 (3) 11.7b ± 0.45 (3) 13.9a ± 0.38 (3) 11.8b ± 0.82 (3) Mn (mg/day-hen) 11.3a ± 0.63 (3) 13.0a ± 0.60 (3) 12.0a ± 0.48 (3) 12.0a ± 1.33 (3) Downloaded from K (g/day-hen) 0.83a ± 0.01 (3) 0.85a ± 0.08 (3) 0.81a ± 0.04 (3) 0.77a ± 0.07 (3) a,bMeans within a column with no common superscript differ significantly (P < 0.05). 10_Inorg = 0% DDGS + inorganic minerals, 0_Org = 0% DDGS +organic minerals, 20_Inorg = 20% DDGS + inorganic minerals, and 20_Org = 20% DDGS + organic minerals. 2

TKN = total Kjeldahl nitrogen. http://japr.oxfordjournals.org/

Table 7. Mineral intake and retention, emission, and excretion from hens fed different dietary treatments

Item Feed intake2 Excreted in Retained Excreted to Unaccounted (g/day-hen) Diet1 (mean ± SD) eggs (%) (%) air (%) (%)

N 0_Inorg 2.90 ± 0.19 38.0 33.9 16.5 11.6 by guest on February 24, 2014 0_Org 2.94 ± 0.19 39.4 33.8 19.3 7.49 20_Inorg 2.90 ± 0.19 38.2 42.5 15.8 3.46 20_Org 2.87 ± 0.18 39.1 40.6 15.9 4.33 Average 2.90 38.7 37.7 16.9 6.72 S 0_Inorg 0.25a ± 0.17 31.6 58.7 0.27 9.5 0_Org 0.27b ± 0.18 31.2 57.4 0.28 11.1 20_Inorg 0.30c ± 0.19 26.3 79.5 0.27 −5.99 20_Org 0.32d ± 0.20 25.6 73.1 0.23 1.13 Average 0.29 28.7 67.1 0.26 3.94 P 0_Inorg 0.68a ± 0.04 16.1 84.5 — −0.57 0_Org 0.74b ± 0.05 15.5 88.3 — −3.79 20_Inorg 0.67a ± 0.04 16.5 92.1 — −8.61 20_Org 0.67a ± 0.04 16.7 88.0 — −4.61 Average 0.69 16.2 88.2 −4.40 K 0_Inorg 0.96b ± 0.06 7.92 86.2 — 5.88 0_Org 1.00c ± 0.07 7.95 84.3 — 7.71 20_Inorg 0.94b ± 0.06 8.15 86.2 — 5.68 20_Org 0.90a ± 0.06 8.58 85.6 — 5.81 Average 0.95 8.15 85.6 6.27 Ca 0_Inorg 4.77b ± 0.32 0.65 54.5 — 44.8 0_Org 4.99c ± 0.33 0.65 59.4 — 40.0 20_Inorg 4.23a ± 0.27 0.73 71.2 — 28.1 20_Org 4.79b ± 0.30 0.66 58.9 — 40.5 Average 4.70 0.67 61.0 38.4 a–dMeans within a column with no common superscript differ significantly (P < 0.05). 10_Inorg = 0% DDGS + inorganic minerals, 0_Org = 0% DDGS + organic minerals, 20_Inorg = 20% DDGS + inorganic minerals, and 20_Org = 20% DDGS + organic minerals. 2Water nutrient input not included. Li et al.: GASEOUS EMISSIONS IN HENS 49 Downloaded from http://japr.oxfordjournals.org/ by guest on February 24, 2014

Figure 1. Average excretion characteristics for (I) N balance, (II) S balance, (III) P balance, and (IV) K balance. TKN = total Kjeldahl nitrogen.

Manure generated from the different treatments tion was not correlated with Ca intake rates. The + had similar DM content. The daily NH4 -N ex- Zn excretion from hens fed inorganic mineral cretion from hens fed 20_Inorg and 20_Org was sources (0_Inorg and 20_Inorg) was higher than significantly higher than from hens fed 0_Inorg hens fed organic mineral sources (0_Org and + and 0_Org. However, the higher NH4 -N ex- 20_Org). cretion did not result in higher NH3 emissions. Excretion characteristics are listed in Table The manure pH was similar across all diet treat- 7 and depicted in Figure 1. Approximately 40% ments. Excreted P, Na, Fe, Cu, Mg, Mn, and K of N consumed remained in excreta, 40% was masses were not different by diet treatment. The retained in eggs, and 15% was emitted to air. Ca excretion was higher from hens fed 20_Inorg Approximately 70% of S consumed remained than from hens fed 0_Inorg; however, the excre- in excreta, 30% was retained in eggs, and less 50 JAPR: Research Report than 1% was emitted to air. More than 80% of as a feed ingredient on air emissions and performance from consumed P, 80% of consumed K, and 50% of laying hens. Poult. Sci. 89:1355–1359. 7. Masa’deh, M. K., S. E. Purdum, and K. J. Hanford. consumed Ca were excreted in manure. Quanti- 2011. Dried distillers grain with solubles in laying hen diets. fied excreta, eggs, and air emissions accounted Poult. Sci. 90:1960–1966. for 88% or more of the feed inputs except for 8. Huang, Y. L., L. Lu, S. F. Li, X. G. Luo, and B. Liu. Ca. The remaining 12% likely resulted from par- 2009. Relative bioavailabilities of organic zinc sources with different chelation strengths for broilers fed a conventional ticulate matter loss, systematic errors in chemi- corn-soybean meal diet. J. Anim. Sci. 87:2038–2046. cal quantification, and uncertainties in reference 9. Pancosma, Geneva, Switzerland. values. 10. Powers, W. J., S. B. Zamzow, and B. J. Kerr. 2007. Reduced crude protein effects on aerial emissions from swine. Appl. Eng. Agric. 23:539–546. CONCLUSIONS AND APPLICATIONS 11. TEI Model 17i, Thermo Fisher, Franklin, MA. 12. TEI Model 450i, Thermo Fisher, Franklin, MA. 1. In the current study, neither DDGS nor 13. TEI Model 55i, Thermo Fisher, Franklin, MA.

the source of mineral had a significant 14. Lumasense Technologies, Ballerup, Denmark. Downloaded from effect on laying hen performance or 15. LabVIEW Version 8.2; National Instruments Corp., large effects on air emissions. Austin, TX. 2. Feeding 20% DDGS to laying hens re- 16. AOAC. 2000. Method 928.08, Official Methods of Analysis. AOAC International, Gaithersburg, MD. sulted in a slight decrease in NH3 emis- 17. University of Missouri Agriculture Experiment Sta-

sions but an increase in CH4 emissions tion Laboratory, Columbia, MO. http://japr.oxfordjournals.org/ without affecting other gaseous emis- 18. AOAC. 2006. Method 982.30, Official Methods of sions. More than 30% of consumed N, Analysis. AOAC International, Gaithersburg, MD. 80% of consumed P, 80% of consumed 19. University of Arkansas Center for Excellence in Poultry Science, Fayetteville, AR. K, and 50% of consumed Ca were ex- 20. Dairy One Inc., Ithaca, NY. creted in manure. 21. AOAC. 1984. Method 941.04, Official Methods of 3. Based on laying hen performance and Analysis. AOAC, Gaithersburg, MD. the measured emissions, DDGS (20%) 22. AOAC. 2005. Method 2001.11, Official Methods of Analysis. AOAC International, Gaithersburg, MD.

can be fed to laying hens without ad- by guest on February 24, 2014 23. Grubb, M., C. Vrolijk, and D. Brack. 1999. The verse environmental effects. Substitu- Kyoto protocol: A guide and assessment. Royal Institute of tion with organic trace minerals did not International Affairs, London, UK. have an effect on hen performance or air 24. USDA. 2012. National nutrient database for standard emissions. reference: Release 24. Accessed August 31, 2012. http://ndb. nal.usda.gov/ndb/foods/show/111?fg=&man=&lfacet=&co unt=&max=25&sort=&qlookup=egg&offset=&format=Stat s&new=. REFERENCES AND NOTES 25. SAS 9.3, SAS Institute Inc., Cary, NC. 26. Wang-Li, L., Q.-F. Li, K. Wang, B. W. Bogan, J.-Q. 1. NRC. 1994. Nutrient Requirements of Poultry. 9th Ni, E. L. Cortus, and A. J. Heber. 2012. National air emis- rev. ed. Natl. Acad. Press, Washington, DC. sions monitoring study – Southeast layer site: Part II. Am- 2. Pedersen, C., M. G. Boersma, and H. H. Stein. 2007. monia concentrations and emission from high-rise layer Digestibility of energy and phosphorus in ten samples of houses. Trans. ASABE (In press). distillers dried grains with solubles fed to growing pigs. J. 27. Lin, X., E. L. Cortus, R. Zhang, S. Jiang, and A. J. Anim. Sci. 85:1168–1176. Heber. 2012. Ammonia, hydrogen sulfide, carbon dioxide 3. Li, W., W. Powers, and G. M. Hill. 2011. Feeding dis- and particulate matter emissions from California high-rise tillers dried grains with solubles and organic trace mineral layer houses. Atmos. Environ. 46:81–91. sources to swine and the resulting effect on gaseous emis- 28. Stadelman, W. J., and D. E. Pratt. 1989. Factors influ- sions. J. Anim. Sci. 89:3286–3299. encing composition of the hen’s egg. World’s Poult. Sci. J. 4. Lumpkins, B., A. Batal, and N. Dale. 2005. Use of 45:247–266. distillers dried grains plus solubles in laying hen diets. J. Appl. Poult. Res. 14:25–31. Acknowledgments 5. Roberson, K. D., J. L. Kalbfleisch, W. Pan, and R. A. The authors thank Stéphane Durosoy (Anamine, Sillingy, Charbeneau. 2005. Effect of corn distillers dried grains with France) for product donation and Jolene Roth (Michigan solubles at various levels on performance of laying hens and State University) for laboratory oversight. egg yolk color. Int. J. Poult. Sci. 4:44–51. 6. Wu-Haan, W., W. Powers, R. Angel, and T. J. Apple- gate. 2010. The use of distillers dried grains plus solubles © 2014 Poultry Science Association, Inc. Wet litter not only induces footpad dermatitis but also reduces overall welfare, technical performance, and carcass yield in broiler chickens

Ingrid C. de Jong ,1 H. Gunnink , and J. van Harn

Wageningen UR Livestock Research, PO Box 65, 8200 AB Lelystad, the Netherlands

Primary Audience: Flock Supervisors, Researchers, Extension Services, Veterinarians Downloaded from

SUMMARY This study investigated whether a high level of footpad dermatitis (FPD) in broiler chickens induced by increased litter moisture content is accompanied by negative effects on technical performance, carcass yield, and other welfare aspects. Litter moisture content was increased http://japr.oxfordjournals.org/ by systematically spraying water over the litter from 6 d of age onward (lesion-induction treatment). Results were compared with a control group kept on relatively good quality litter and having a very low prevalence of footpad lesions. Litter quality significantly decreased from 7 d of age onward in the pens with the lesion-induction treatment compared with the control pens.

At 21 and 36 d of age, significantly more FPD was observed in the lesion-induction groups compared with the control groups. Technical performance of the broilers was negatively affected for the lesion-induction groups from 28 d of age onward; lesion-induction groups had by guest on February 24, 2014 significantly lower BW gain, feed intake, and water intake and significantly higher FCR compared with control groups. As a result of the lower BW gain, carcass weight was less for the lesion-induction group and significantly more rejections for commercial parts were found. The lesion-induction groups also had significantly more hock burns and breast irritations at d 21 and 36, and were dirtier but had fewer thigh scratches at d 35 compared with the control groups. In addition, locomotion was negatively affected among the lesion-induction group, as they had a higher gait score at d 36 compared with the control group. We concluded that increased litter moisture content not only caused severe FPD but also reduced broiler performance and carcass yield and had a negative effect on other welfare aspects.

Key words: animal welfare , broiler , carcass yield, footpad dermatitis, performance 2014 J. Appl. Poult. Res. 23 :51–58 http://dx.doi.org/ 10.3382/japr.2013-00803

DESCRIPTION OF PROBLEM in different countries have shown that the prevalence of FPD in broiler chicken varies consid- Footpad dermatitis (FPD), also called foot- erably. For example, in a recent study in the pad lesions or pododermatitis, is a welfare con- Netherlands it was reported that the prevalence cern in broiler chickens. Severe FPD is in gen- of severe FPD in regular, fast-growing broiler eral considered to be painful for the birds [1], chicken flocks in indoor housing systems was and because of its association with litter quality 38.4% [3]. Researchers in France in 2009 re- it also affects other welfare aspects [2]. Studies ported levels of about 70% of severe FPD in a

1 Corresponding author: [email protected] 52 JAPR: Research Report wide range of production systems [4], and simi- the Netherlands. One-day-old broiler chickens lar levels were found in a study in Portugal in (7,200 Ross 308, as hatched) were housed in a wide range of production systems [5]. In an 8 identical pens of 47.5 m2 in 2 identical, cli- older study in the United Kingdom, researchers mate-controlled rooms until slaughter at 37 d reported a prevalence of 14.8% severe FPD in of age. Per pen, 900 broilers were housed (19 different production systems [6]. chicks/m2 at placement). Wood shavings (1 kg/ Wet litter is the most important FPD-causing m2) were used as bedding material. Each pen factor in broiler chickens [7]. It can be expected had 11 feeder pans and 2 drinking lines with 72 that the prevalence of severe FPD is accompa- nipples with drip cups. Environmental tempera- nied by other negative effects on welfare and ture was gradually reduced from 33 to 19°C at productivity caused by deteriorated litter qual- d 1 and 37, respectively. Lights were continu- ity. Fast-growing broiler chickens from 3 wk ously on at d 1, 2, and 37; from d 3 to 36, an of age onward spend a considerable amount of intermittent lighting schedule was applied of time sitting [8]. When sitting, the hocks, as well 4L:4D:3L:1D:3L:1D:3L:1D:3L:1D, with dark Downloaded from as the chest, are in contact with litter. Therefore, periods between 2300 to 0300, 0600 to 0700, it can be expected that increased litter moisture 1000 to 1100, 1400 to 1500, and 1800 to 1900 not only increases the risk for FPD, but also for h. Light intensity was 20 lx at bird height. Birds hock burns (hock dermatitis) and dermatitis of were vaccinated according to commercial prac- the skin on the chest [4, 9, 10]. This may, in turn, tice (i.e., IB primer at d 1 at the hatchery, NCD http://japr.oxfordjournals.org/ lead to reduced carcass quality of the broilers at d 14, and Gumboro at d 22). and an increased number of downgraded car- Birds received a commercial multiphase diet casses at the slaughter house. In addition, broil- (i.e., starter from d 0–10, grower I from d 10– ers suffering from pain caused by FPD [10, 11], 18, grower II from d 18–29, and finisher from and possibly also from dermatitis on the hocks d 29–37). Diets were produced and supplied by and breast, may be less eager to go to the feed- a commercial compound feed company in The ers and drinkers, and thus may show less weight Netherlands. gain compared with broilers with no FPD. by guest on February 24, 2014 In commercial flocks, in some studies, posi- Treatments tive relationships were observed between different types of contact dermatitis [4], whereas, in The experiment aimed to have 2 contrasting others studies [2, 12], no such relationships were treatments with respect to litter moisture content observed; but relationships between FPD and and FPD. The control groups of broilers had dry technical performance and carcass yield were litter and a very low percentage of FPD (n = 4 often not determined. Therefore, in the current pens), whereas the lesion-induction groups of study, we compared technical performance, broilers had wet litter and a very high percent- carcass yield, and additional welfare indicators age of severe FPD (n = 4 pens); that is, for the in an experimental setting in broiler chickens control group, an FPD score of around 20 versus where FPD was induced by increasing litter more than 180 points (on a scale from 0–200) moisture content compared with control groups at the day of slaughter for the lesion-induction having low incidences of FPD. Negative effects group. The FPD score was calculated as of wet litter on production and carcass yield may function as an additional incentive for farmers to reduce the risk for FPD and thus improve wel- FPD score = [(number of feet with lesion score fare in their flocks. of 0 × 0) + (number of feet with lesion score MATERIALS AND METHODS 1 × 0.5) + (number feet with lesions score Birds and Housing 2 × 2)] × 100/total number of scored feet, [3]

The experiment was carried out between where feet with no lesions were assigned a score October and November 2012 at the research fa- of 0, mild lesions a score of 1, and severe lesions cilities of Schothorst Feed Research, Lelystad, a score of 2 according to Berg [13]. Both feet de Jong et al.: BROILER FOOTPAD LESIONS 53 were assessed and the most severe score per bird or very small discoloration; score 1 = discolor- was used for the calculation. ation but no deep lesion; score 2 = deep lesion Footpad dermatitis was induced by system- with ulcers or scabs, bumble foot [13]). Hock atically spraying water over the litter (300 mL/ burns were scored for both hocks according to m2 per day) according to McKeegan [14], start- The Welfare Quality Consortium [15], with a ing at d 6 of age and during 5 subsequent days score ranging from 0 (no hock burn) to 4 (large per week until the end of the experiment. As black spot). Cleanliness was scored by assess- the progress in development of FPD was less ing the breast and assigning a cleanliness score than expected, the amount of sprayed water was between 0 and 3 [15], with a score of 0 for clean substantially increased in the final week to 1.5 and a score of 3 for being very dirty. Breast irri- L/m2 per day for 5 d until the desired contrast tation was scored as 0 for no irritation or redness between the treatment groups was observed. of the breast; 1 for slight discoloration (redness) In the control groups, the development of FPD of the breast; 2 for large discoloration (redness) was prevented by keeping the litter as dry as and presence of small brown spots; and 3 for Downloaded from possible. This was done by replacing wet spots large brown spots or blister(s). Scratches were underneath the drinking lines by fresh litter, scored by inspecting the thigh area at both sides adding fresh litter on top, and by daily raking and assigning a score for the number and sever- the litter underneath the drinking lines from ity of scratches: 0 for no scratches; 1 for small, 2 wk of age onward. Because the experiment superficial scratch or scratches; 2 for scratch or http://japr.oxfordjournals.org/ was carried out in the autumn and winter pe- wound (<1.5 cm) or healed scratch; and 3 for riod (usually the wet season) and litter quality large scratch or wound (>1.5 cm). decreased during the experiment in the control Technical Performance. Body weight was groups, a complete replacement of the litter of determined at d 1 (sample of 180 randomly the control groups was conducted at the end of chosen chicks per pen) and 37 (by weighing all the second week of age. remaining birds per pen). Body weight at d 10, In total, 8 pens (4 pens/room) were randomly 18, and 28 was determined using an automated assigned to the present experiment and treat- weighing plateau in the pen [16]. Feed intake by guest on February 24, 2014 ments were equally divided over both rooms and and water intake were determined per pen per over rows. Each room was divided by a central feeding phase (d 0–10, 10–18, 18–28, 28–37). corridor with 8 pens on each side. Pens not as- Mortality was recorded daily. signed to the current experiment were used for Gait Score. The gait score was used to de- another study (using broilers of the same batch). termine the quality of locomotion of the birds The experiment was approved by the Animal [17]. A random sample of at least 40 birds was Care and Use Committee of Wageningen Uni- released one-by-one from a catching pen and versity and Research Centre. assigned a gait score between 0 and 5 (normal, dextrous and agile to incapable of walking [15]). Measures Slaughter Yield and Rejections. On d 37, 10 male and 10 female broilers were randomly se- Litter Quality. Litter quality was scored per lected from each pen, individually marked and pen by visual inspection at d 7, 14, 21, 28, and weighed, and transported to a commercial pro- 36 as a standard procedure at the experimental cessing plant. The birds were slaughtered and farm. Litter of each pen received a score be- hand eviscerated by trained slaughter plant per- tween 0 (very wet) to 10 (completely dry) from sonnel on a small, partially automated poultry 2 independent observers. Scores of both observ- processing line. Yields of carcass weight, car- ers were averaged per pen. cass yield (as a percentage of the live weight), Footpad Dermatitis, Hock Burns, Cleanli- and processing yields (as a percentage of the ness, Breast Irritation, and Scratches. Per pen, carcass weight) of the different commercial parts 20 males and 20 females were randomly selected (wings, legs, breast fillet) were determined. In at 21 and 36 d of age and inspected. Footpad der- addition, for each pen, the percentage of rejec- matitis was scored for both feet according to the tions for whole birds and commercial parts were Swedish classification (i.e., score 0 = no lesions determined. 54 JAPR: Research Report

Statistical Analysis Table 1. Average litter quality scores1 for the control and lesion-induction groups The experiment was carried out as a random- Lesion ized block design with treatment (percentage of Age (d) Control induction LSD P-value FPD: low and high) as factor and room and row a b within the room as blocking factors. Data were 7 8.0 7.3 0.69 <0.05 14 10.0a 5.8b 0.35 <0.001 first analyzed for outliers. Significant outliers 21 8.0a 4.1b 0.35 <0.001 (i.e., observed value with residual outside the 28 7.0 4.0 —2 range of 2.5 × SE of residuals) were excluded 36 7.8a 3.0b 0.69 <0.001 from the data set before statistical analysis. Data a,bDifferent superscripts within a row indicate a significant were statistically analyzed by ANOVA using treatment effect (P < 0.05). 1Litter scores ranging from 1 (very wet) to 10 (completely Y = µ + Room + Block + Treatment + error, dry). ijk i j k 2As there was no variation in litter scores within treatments at this particular age, no statistical analysis could be per- where Y = the response parameter; μ = the gen- formed. Downloaded from eral mean; Room = the effect of climate room (i = 1,2); Block = the effect of row within room (j = 1,2); Treatment = the effect of treatment (k = creasing the litter moisture started from d 6 of 1,2); and error = the error term. Effects with P ≤ age onwards, but the effects on technical per- 0.05 were considered to be statistically signifi- formance of the birds were observed from 28 d http://japr.oxfordjournals.org/ cant, whereas 0.05 < P ≤ 0.10 were considered of age onwards. The lesion-induction group had trends. All analyses were performed using Gen- stat Release 14.2 [22].

RESULTS AND DISCUSSION As a result of water spraying in the lesion- induction groups and the active litter manage- by guest on February 24, 2014 ment (raking, litter adjustment and litter replacement) to prevent the development of FPD in the control groups, litter quality was significantly worse in the lesion-induction groups as compared with the control groups at all ages (Table 1). The increased litter moisture content induced the development of FPD in the birds as expected according to the literature [14, 18]. Figure 1 shows the prevalence of FPD in both treatment groups at 21 and 36 d of age. A significant treatment effect was observed on the average FPD score at both ages [d 21 = 1.4 vs. 0.3 (P < 0.001) and d 36 = 2.0 vs. 0.5 (P < 0.001) for lesion- induced vs. control, respectively]. Average FPD flock scores at 21 d of age were 110 versus 14 points for lesion-induced and control groups, respectively; at 36 d of age, scores were 198 versus 28 points for lesion-induced and control groups, respectively. At d 36, 99% of the birds in the lesion-induction groups had severe FPD compared with only 2% in the control groups. Table 2 shows the technical performance of Figure 1. Average percentage of birds with different both treatment groups for the different feeding footpad dermatitis (FPD) lesion scores for the control phases and the overall production period. In- and lesion-induction groups. de Jong et al.: BROILER FOOTPAD LESIONS 55

Table 2. Technical performance for the control and lesion-induction groups per production phase and for the overall production period

Lesion Item Control induction LSD P-value

Day 0–10 BW gain (g) 247 247 33.7 0.99 Feed intake (g) 313 309 7.7 0.18 FCR 1.27 1.25 0.17 0.77 Water intake (mL) 540 540 32.0 0.95 Water-to-feed ratio 1.71 1.75 0.10 0.28 Mortality (%) 0.9c 1.3d 0.49 0.06 Day 10–18 BW gain (g) 406 410 25.1 0.72 Feed intake (g) 568 574 18.1 0.39 FCR 1.40 1.40 0.11 0.93 Water intake (mL) 940 940 53.8 0.99 Downloaded from Water-to-feed ratio 1.65 1.64 0.09 0.61 Mortality (%) 0.4 0.3 0.37 0.36 Day 18–28 BW gain (g) 714 716 101.8 0.97 Feed intake (g) 1,142 1,158 55.6 0.47 FCR 1.60 1.62 0.15 0.77 http://japr.oxfordjournals.org/ Water intake (mL) 1,726 1,717 147.4 0.87 Water-to-feed ratio 1.51 1.48 0.10 0.49 Mortality (%) 0.4 0.4 0.25 0.58 Day 28–37 BW gain (g) 678a 531b 129.3 <0.05 Feed intake (g) 1,279a 1,159b 89.3 <0.05 FCR 1.89B 2.22A 0.41 0.08 Water intake (mL) 2,289a 1,814b 227.5 <0.01 a b

Water-to-feed ratio 1.82 1.56 0.10 <0.01 by guest on February 24, 2014 Mortality (%) 0.3 0.7 0.80 0.24 Day 0–37 BW at d 37 (g) 2,090a 1,948b 83.2 <0.01 BW gain (g) 2,064a 1,904b 84.7 <0.01 Feed intake (g) 3,331a 3,199b 65.2 <0.01 FCR 1.61b 1.68a 0.06 <0.05 Water intake (mL) 5,490a 5,010b 309.1 <0.05 Water-to-feed ratio 1.66a 1.57b 0.09 <0.05 Mortality (%) 2.0 2.6 0.99 0.13 EPEF1 336a 298b 23 ≤0.05 a–dDifferent superscripts within a row indicate a significant treatment effect (P ≤ 0.05). A,BDifferent superscripts within a row indicate a tendency (0.05 ≤ P ≤ 0.10). 1European Production Efficiency Factor = {BW gain (g/d) × [100 − mortality (%)]}/(feed conversion × 10). significantly decreased growth, feed intake, and bird cleanliness, increased incidence of hock water intake and an increased FE as compared burn and breast irritation at 21 and 36 d of age, with the control group in the finisher phase of and worse locomotion at 36 d of age compared the production period. This resulted in a worse with the control groups (Table 3). At 36 but technical performance over the overall produc- not at 21 d of age, more thigh scratches were tion period for the lesion induction group com- found in the control group as compared with the pared with the control group. No differences lesion-induction group (Table 3). were observed in mortality between the treat- Table 4 shows the results of the carcass yields ments. and the percentages of rejected birds or com- The induction of FPD by increased litter mercial parts. A higher BW and carcass weight moisture was accompanied by a reduction in were found for the control group as compared 56 JAPR: Research Report

Table 3. Average scores for breast cleanliness, breast irritation, thigh scratches, hock burn, and gait score1 for the control and lesion-induction groups at 21 and 36 d of age

d 21 d 36

Lesion Lesion Indicator Control induction LSD P-value Control induction LSD P-value

Breast cleanliness 0.4b 1.4a 0.3 <0.001 1.2b 2.6a 0.1 <0.001 Breast irritation score 0.6b 1.1a 0.1 <0.001 1.0b 2.1a 0.5 <0.01 Thigh scratches 0.0 0.0 0.0 0.36 0.2a 0.0b 0.06 <0.001 Hock burn score 0.6b 1.2a 0.2 0.001 1.0b 3.5a 0.3 <0.001 Gait score ———— 2.1b 2.5a 0.3 <0.05 a,bDifferent superscripts within a row indicate a significant treatment effect (P < 0.05). 1Breast cleanliness, breast irritation, and thigh scratch scores ranging from 0 (none) to 3 (severe); hock burn scores ranging from 0 (none) to 4 (severe); gait scores ranging from 0 (excellent gait) to 5 (unable to walk). Downloaded from with the lesion-induction group at 37 d of age wetted litter showed lower BW gain, which is in (Table 4). Conversely, for processing yields, a line with the results of the present study. Lower lower wing percentage was found for the control BW gain might be due to a pain-induced reduc- group compared with the lesion-induction group tion in feed intake in broilers with severe foot- (Table 4). No differences were found in leg and pad lesions [10, 11]. The deteriorated litter qual- http://japr.oxfordjournals.org/ breast fillet percentage between control and le- ity in the lesion-induction group also seemed sion-induced group. No differences in percent- to lead to thermal discomfort in chicks, which ages of downgraded carcasses were observed, may also have affected BW gain. More huddling but the percentage of downgraded commercial was observed in these groups, although we did parts was significantly higher in the lesion- not quantify these data. We also observed worse induction group as compared with the control locomotion in birds from the lesion-induction group (Table 4). groups compared with the control groups, which In the current experiment, we experimentally is likely to have a negative effect on the feed in- by guest on February 24, 2014 induced FPD by increasing the litter moisture take. It has been suggested in turkeys that foot- content. However, deteriorated litter quality pad lesions may provide a route for systematic also has negative effects on technical perfor- invasion of microorganisms that could settle in mance and other welfare indicators, such as the leg joints causing leg weakness [7], which hock burns, breast irritation, gait score, cleanli- might also explain the worse gait score in the ness, and rejections at the slaughter plant. Thus, lesion-induced group as compared with the broiler farms having problems with litter quality control group. Conversely, broilers with severe not only have a risk for birds developing FPD, footpad dermatitis may prefer to rest on their but also have a greater risk for a reduced perfor- hocks instead of standing or walking, and this mance as compared with broiler farms that are decreased activity might have caused the worse able to keep the litter dry. locomotion in the lesion-induction groups [19]. A large contrast in FPD was created between The difference in activity may also explain the the control and lesion-induction groups by sig- higher number of thigh scratches in the con- nificantly deteriorating the litter quality through trol groups compared with the lesion-induction increasing litter moisture content. This is in line groups and is in line with a study of commer- with earlier studies showing that increasing lit- cial flocks [4]. Thigh scratches negatively affect ter moisture content significantly increases the bird welfare, as they might be painful; however, prevalence of FPD in broilers [14, 18]. Although on average, only small, single, and superficial many studies have focused on litter quality as scratches were observed at a low prevalence in the cause of FPD in broilers, only a few stud- the control group. ies report that wet litter also has negative effects The reduced technical performance in the on technical performance. A recent study [18] lesion-induced group compared with the control reported that broilers placed on experimentally group is in line with reduced cold carcass weight. de Jong et al.: BROILER FOOTPAD LESIONS 57

Table 4. Carcass yield and percentage of downgrade for the control and lesion-induction groups

Lesion Item Control induction LSD P-value

Carcass yield Live weight (g) 2,174a 1,976b 91 <0.01 Carcass weight (g) 1,430a 1,307b 69 <0.01 Carcass (% of live weight) 65.7 66.1 1.0 0.35 Wing (% of carcass weight) 10.8b 11.0a 0.2 <0.01 Leg (% of carcass weight) 34.5 34.3 0.4 0.26 Back (% of carcass weight) 16.7 16.5 0.5 0.39 Fillet (% of carcass weight) 30.3 30.3 1.0 0.95 Skin (% of carcass weight) 3.1 3.2 0.2 0.28 Downgrade (%) Whole chicken 0.39 0.83 0.69 0.16 Commercial parts 0.14b 0.38a 0.23 <0.05 Total 0.53 1.22 0.94 0.11 Downloaded from a,bDifferent superscripts within a row indicate a significant treatment effect (P < 0.05).

The higher rejection percentage for commercial was found [4]; this seems to be in contrast with parts is in line with the higher incidence of breast the current experiment, where we found that wet http://japr.oxfordjournals.org/ irritation, hock burn, and dirtier breasts of the litter resulted not only in more FPD, but also broilers of the lesion-induced group compared more severe hock burn, breast irritation, and with the control group. An economic calculation dirtier birds. An explanation for these different was performed based on the technical results of findings might be that lesions initially appear on the current experiment (Table 5). The lesion- the feet, subsequently on the hocks, and finally induction group had a lower margin of €0.089/ on the breast [3, 8], because hocks and breasts broiler placed than the control group, which is a are increasingly in contact with litter as activity considerable difference. decreases. If broilers are slaughtered before or at by guest on February 24, 2014 Hock burn is a type of contact dermatitis, the first stages of development of hock burn, for such as FPD and breast irritation and is clearly example, no correlation will be found. associated with high litter moisture content [2], although a correlation between hock burn and CONCLUSIONS AND APPLICATIONS FPD has been found in some [4, 20], but not all studies [2, 12]. A negative correlation between 1. Not only did FPD in broiler chickens breast blisters and hock burns, and breast blis- increase with high litter moisture con- ters and FPD measured in commercial flocks tent, but broiler performance was also

Table 5. Calculated economic results for the control and lesion-induction groups1

Lesion Parameter Control induction

Live weight (g) 2,090 1,948 FCR 1.614 1.681 Economic results (€/broiler placed) Gross income2 (sale of birds) 1.5464 1.4230 1-d-old chickens 0.2950 0.2950 Feed costs 0.8450 0.8109 Total variable expenses 0.1852 0.1851 Gross margin3 0.2212 0.1320 1Calculations were based on reference values for Dutch broiler farms according to [21]; a return price of €0.755/kg per broiler was delivered at slaughter along with a feed price of €0.26/kg. 2Because of a difference of 0.7% in total rejections, the return price has been lowered by €0.005/kg according to commercial practice. 3Gross margin = [gross income − (1-d-old chickens + feed costs + total variable expenses)]. 58 JAPR: Research Report

reduced and more rejections of com- 9. Greene, J. A., R. M. McCracken, and R. T. Evans. mercial parts at the slaughter house were 1985. A contact-dermatitis of broilers – Clinical and patho- logical findings. Avian Pathol. 14:23–38. also found. 10. Martland, M. F. 1985. Ulcerative dermatitis in 2. Other welfare indicators, such as breast broiler-chickens – The effects of wet litter. Avian Pathol. cleanliness, breast irritation, hock burn, 14:353–364. and gait, were negatively affected in 11. Martland, M. F. 1984. Wet litter as a cause of plantar pododermatitis, leading to foot ulceration and lameness in broilers kept on litter with high moisture fattening turkeys. Avian Pathol. 13:241–252. content as compared control groups of 12. Kristensen, H. H., G. C. Perry, N. B. Prescott, J. broilers kept on dry litter. Ladewig, A. K. Ersboll, and C. M. Wathes. 2006. Leg health 3. Therefore, keeping litter moisture levels and performance of broiler chickens reared in different light environments. Br. Poult. Sci. 47:257–263. low is preferred from a welfare point of 13. Berg, C. C. 1998. Foot-pad dermatitis in broilers and view and also from an economic point of turkeys – Prevalence, risk factors and prevention. PhD Diss. view; not only because footpad lesions Swedish University of Agricultural Sciences, Uppsala, Swe- have an economic value in itself, but den. 14. McKeegan, D. 2010. Foot pad dermatitis and hock Downloaded from also because of the negative associations burn in broilers: Risk factors, aetiology and welfare con- with broiler performance and downgrad- sequences. Accessed April 2013. http://randd.defra.gov.uk/ ed carcass quality. Default.aspx?Menu=Menu&Module=More&Location=Non e&Completed=0&ProjectID=14546. 15. The Welfare Quality Consortium. 2009. The Welfare

Quality Assessment Protocol for Poultry (Broilers, Laying http://japr.oxfordjournals.org/ REFERENCES AND NOTES Hens). The Welfare Quality Consortium, Lelystad, the Neth- erlands. 1. Michel, V., E. Prampart, L. Mirabito, V. Allain, C. Arnould, D. Huonnic, S. LeBouquin, and O. Albaric. 2012. 16. Opticon DWS-20, Opticon Agricultural Systems, Histologically-validated footpad dermatitis scoring system Venlo, the Netherlands. for use in chicken processing plants. Br. Poult. Sci. 53:275– 17. Kestin, S. C., T. G. Knowles, A. E. Tinch, and N. G. 281. . Gregory. 1992. Prevalence of leg weakness in broiler chick- 2. Haslam, S. M., T. G. Knowles, S. N. Brown, L. J. ens and its relationship with genotype. Vet. Rec. 131:190– Wilkins, S. C. Kestin, P. D. Warriss, and C. J. Nicol. 2007. 194. Factors affecting the prevalence of foot pad dermatitis, hock 18. Cengiz, O., J. B. Hess, and S. F. Bilgili. 2011. Effect burn and breast burn in broiler chicken. Br. Poult. Sci. of bedding type and transient wetness on footpad dermatitis by guest on February 24, 2014 48:264–275. in broiler chickens. J. Appl. Poult. Res. 20:554–560. 3. de Jong, I. C., J. van Harn, H. Gunnink, V. A. Hindle, 19. Reiter, K., and W. Bessei. 2009. Effect of locomotor and A. Lourens. 2012. Footpad dermatitis in Dutch broil- activity on leg disorder in fattening chicken. Berl. Munch. er flocks: Prevalence and factors of influence. Poult. Sci. Tierarztl. Wochenschr. 122:264–270. 91:1569–1574. 20. Sørensen, P., G. Su, and S. C. Kestin. 2000. Effects of 4. Allain, V., L. Mirabito, C. Arnould, M. Colas, S. Le age and stocking density on leg weakness in broiler chick- Bouquin, C. Lupo, and V. Michel. 2009. Skin lesions in ens. Poult. Sci. 79:864–870. broiler chickens measured at the slaughterhouse: relation- 21. Wageningen UR Livestock Research. 2012. KWIN ships between lesions and between their prevalence and Veehouderij 2012–2013. Wageningen UR Livestock Re- rearing factors. Br. Poult. Sci. 50:407–417. search, Lelystad, the Netherlands. 5. Gouveia, K. G., P. Vaz-Pires, and P. M. da Costa. 22. Genstat Release 14.2, VSN International Ltd., Hemel 2009. Welfare assessment of broilers through examination Hempstead, UK. of haematomas, foot-pad dermatitis, scratches and breast blisters at processing. Anim. Welf. 18:43–48. Acknowledgments 6. Pagazaurtundua, A., and P. D. Warriss. 2006. Levels This experiment was financially supported by the Minis- of foot pad dermatitis in broiler chickens reared in 5 differ- try of Economic Affairs, project number BO12.02-002.40.07 ent systems. Br. Poult. Sci. 47:529–532. and subsidized by the Product Board of Poultry and Eggs. 7. Shepherd, E. M., and B. D. Fairchild. 2010. Footpad Teun Veldkamp and Thea van Niekerk are acknowledged for dermatitis in poultry. Poult. Sci. 89:2043–2051. their comments on the manuscript. We thank the personnel 8. Alvino, G. M., G. S. Archer, and J. A. Mench. 2009. of Schothorst Feed Research (Lelystad, the Netherlands) for Behavioural time budgets of broiler chickens reared in vary- assistance in the experiment. ing light intensities. Appl. Anim. Behav. Sci. 118:54–61. © 2014 Poultry Science Association, Inc. Effect of production system and flock age on egg quality and total bacterial load in commercial laying hens

Samiullah ,* J. R. Roberts ,*1 and K. K. Chousalkar †

* School of Environmental and Rural Science, University of New England, Armidale, Australia 2351; and † School of Animal and Veterinary Science, University of Adelaide, Roseworthy, Australia 5371

Primary Audience: Egg Producers, Food Safety Authorities, Researchers Downloaded from

SUMMARY

The shell of the egg is essential in providing shape to the egg and ensuring safe packaging of http://japr.oxfordjournals.org/ the internal contents; however, shell defects have been shown to increase the risk of microbial contamination of eggs. Eggs were collected from commercial cage and free-range flocks at the ages of 25, 35, 45, 55, 65, and 75 wk. From each collection per flock, 30 eggs were processed for the eggshell and egg internal quality determination, 30 eggs for cuticle estimation, 30 eggs for mammillary layer ultrastructural features scoring, and 60 eggs for egg microbial enumeration. Translucency score and shell reflectivity were significantly higher in free-range eggs and increased with flock age in both production systems. Egg weight, shell weight, percentage

shell, shell thickness, albumen height, Haugh unit, and yolk color were higher for cage eggs. by guest on February 24, 2014 The amount of cuticle was higher in cage eggs and fluctuated with flock age in both production systems. For the mammillary layer ultrastructural variables, a significant effect of production system and flock age was observed for early fusion, Type A bodies, and Type B bodies, whereas aragonite, depression, erosion, and hole were rarely observed. Variability of mammillary cap size, the incidence of poor mammillary cap quality, incidence of late fusion, alignment, Type A bodies, Type B bodies, and cubic cone formation were greater in the free-range versus cage system and increased with flock age in both production systems. The incidence of confluence and early fusion were greater in cage eggs and decreased with age in both production systems. Significantly lower total microbial load was observed for cage compared with free-range eggs, but the overall bacterial load recorded in this study was low. It can be concluded that cage eggs were better in overall quality when directly compared with free-range eggs.

Key words: conventional cage, free range , cuticle, shell mammillary layer , bacterial load , Enterobacteriaceae 2014 J. Appl. Poult. Res. 23 :59–70 http://dx.doi.org/ 10.3382/japr.2013-00805

DESCRIPTION OF PROBLEM tralian egg production market in 2011 of 55 and 34%, respectively [1]. Free-range production is In Australia, conventional cage and free range increasing, as its market share value in the year are the main commercial egg production sys- 2011 (34%) was higher than for 2010 (28%) [2]. tems, with an estimated retail share of the Aus- Eggs obtained from conventional cage systems

1 Corresponding author: [email protected] 60 JAPR: Research Report have been reported as being cleaner and having in an egg-candling box and observing the ex- better overall egg quality compared with free tent of light penetration through the shell. Eggs range [3]. Consumers prefer an egg in which the were scored for the incidence of translucency albumen is firm, the yolk has a dense color, the on the scale of 0 being least translucent and egg is of an appropriate size with intact shell, 5 being the highest incidence of translucency. and is free from pathogens. In the present study, Eggshell and egg internal quality parameters the effect of 2 production systems (cage vs. free were measured by TSS [4] equipment. Egg- range) and of increasing flock age were evaluat- shell parameters measured were shell reflectiv- ed for overall egg quality, the amount of cuticle ity (TSS-QCR), egg weight and shell weight cover on the eggshell, the incidence of various (g; TSS QCBi), shell breaking strength (N) by mammillary layer ultrastructural features, and quasi-static compression and shell deforma- the total bacterial load on the eggshell surface, tion (µm; TSS QC-SPA – 50 N load cell), shell in shell crush, and egg internal contents. thickness (µm; custom built gauge based on a Mitutoyo dial comparator gauge [5]), and per- Downloaded from MATERIALS AND METHODS cent shell (%; calculated from shell weight and egg weight). For determination of shell break- Eggs were collected, on farm, at 25, 35, 45, ing strength, eggs were presented horizontally 55, 65, and 75 wk of age from one conventional in the TSS QC-SPA equipment and force was cage (CC) flock and one free-range (FR) flock applied directly to the egg equator. Internal of Hy-Line Brown laying hens. Both were flocks http://japr.oxfordjournals.org/ quality was measured as albumen height (mm) in commercial production and the 2 flocks were and Haugh unit (TSS QCH) and yolk color 4 wk apart in age. Although the feed was not (TSS QCC). identical for both flocks, diets were formulated according to Hy-Line Australia specifications and contained wheat (or wheat plus sorghum), Estimation of the Amount of Cuticle soybean meal, meat meal, vegetable oil, limestone, and yolk color pigment as major compo- For estimation of the amount of cuticle, nents. The CC flock was in an environmentally eggs were soaked in MST cuticle blue stain by guest on February 24, 2014 controlled conventional cage system with 6 hens [6] for 1 min and rinsed in tap water 3 times in each cage (550 cm2/bird). The FR flock size to remove excess stain. Shell color (L*a*b*) was 16,000 hens with free access to an outside was measured with a hand-held Konica Mi- confined range (10 m2/bird in the poultry house, nolta spectrophotometer [7]. The reading was 4 m2/bird on the range). taken 3 times per location at 3 locations around During the study period, a total of 900 eggs the equator of each egg and an average was were processed from each production system recorded. The Konica Minolta spectrophotom- (150 eggs per collection per flock). Out of 900 eter functions on the L*a*b* space system, eggs from each flock, 540 eggs (90 per collection where L* represents the grading between white per flock) were processed for the determination (100) and black (0). The higher the value for of traditional egg external and internal quality L*, the lighter the shell color and vice versa. parameters, amount of cuticle estimation, and The value for a* represents the color grading the scoring of eggshell ultrastructural features. between green and red, where green is toward The remaining 360 eggs (60 per collection per the negative end of the scale and red toward flock) were processed for egg microbiology in the positive end. More negative values for a* which the total bacterial load on eggshells, in mean the eggs acquired more stain and thus eggshell crush, and internal contents was recov- the amount of cuticle is greater and vice versa. ered and enumerated. The b* component is the grading between yellow and blue. For b*, blue is toward the nega- Eggshell and Egg Internal tive end and yellow toward the positive end of Quality Measurements the scale. In the present study, among all the 3 components of the L*a*b* color space system, The incidence of translucency was deter- a* was the most important, as it indicated the mined by placing intact eggs over a light source amount of cuticle present on the stained eggs. Samiullah et al.: EGG QUALITY AND BACTERIA 61

Ultrastructural Scoring of Shell BGA plates showed TEC, whereas MacCo- Mammillary Layer nkey agar plates showed TBC. The eggs used for shell wash were dipped for approximately The ultrastructural features of the mammil- 30 s in 70% ethanol (C2H5OH) to kill shell sur- lary layer were scored using a scanning electron face bacteria and remove any possible external microscope [8]. Pieces of shell, approximate- contamination. Eggs were allowed to dry in a ly 1 cm square, were cut out from around the biosafety cabinet. Egg internal contents were equator of the eggshell using a Dremel tool [9] removed into a sterile container (200 mL) by and soaked overnight in tap water. Shell mem- breaking the egg at the edge of sterile container branes were peeled off manually and pieces of into 2 equal halves. Eggshell internal walls were shell were allowed to dry thoroughly. The dried washed with PBS to remove all the adherent al- pieces were then plasma etched in a BioRAD bumen. Eggshells (n = 6) with intact shell mem- RF Plasma Barrel Etcher PT 7150 [10] for 4 h branes were transferred into a sterile bag [15] following the method of Reid [11] to remove and crushed to expose all the shell pores; 60 mL Downloaded from the outer shell membrane. The specimens were of PBS was added into each bag. The shell crush mounted on a 9-mm diameter aluminum stub was processed for bacterial enumeration in the using aqueous conductive silver liquid scanning same way used for shell wash. For the process- electron microscope adhesive [12], sputter coat- ing of egg internal contents, 1 mL of the homo- ed in a Neocoater [13] for 5 min, and viewed un- geneously mixed pooled sample (n = 3) in a ster- http://japr.oxfordjournals.org/ der the scanning electron microscope at various ile container was added to 4 mL of BPW, mixed magnifications. Eggshell ultrastructural features thoroughly, and incubated at 37°C. A 0.1-mL of the mammillary layer were scored after the sample of the incubated BPW enrichment was method of Solomon [14]. inoculated into each of the MacConkey and VR- Mammillary cap size was scored as 1 (simi- BGA plates. The plates were incubated at 37°C lar), 2 (variable), or 3 (highly variable). Mam- overnight. After incubation, all the plates were millary caps were scored according to their examined for the growth of bacterial colonies. quality, which was assessed as both the size of the cap in relation to its cone and the degree of by guest on February 24, 2014 membrane attachment, from 1 (best) to 5 (worst). Data Analysis Confluence, early fusion, late fusion, alignment, Data were analyzed using Statview Software Type A bodies, Type B bodies, aragonite, cu- [16]. A 2-way ANOVA was conducted taking bics, cubic cone formations, changed membrane production system and flock age as independent (membrane not removed by plasma ashing), and variables and all other variables as dependent. cuffing were each ranked for incidence from 1 Level of significance was indicated by prob- (none) to 4 (extensive). The same was done for ability of less than 5%. The Fisher’s LSD test the incidence of depressions, erosion, and holes, was used to differentiate levels of significance although these were rarely observed. between mean values. Egg Microbial Enumeration RESULTS AND DISCUSSION To enumerate the total bacterial count (TBC) and total Enterobacteriaceae count (TEC) on Eggshell and Egg Internal the surface of the eggshell, pooled samples (n = Quality Measurements 6) were washed in 60 mL of PBS (pH 7.4) for 1 min. From the shell rinsate, 0.1 mL was plated For the eggshell quality parameters, a sig- onto MacConkey and Violet Red Bile Glucose nificant main effect was observed for both pro- Agar (VRBGA). From the same rinsate, 1 mL duction system and flock age and interaction was inoculated into 4 mL of buffered peptone between the 2 for translucency score, shell re- water (BPW). Inoculated plates and BPW vials flectivity (%; a measure of shell color lightness), were placed in the incubator overnight at 37°C. egg weight (g), shell weight (g; P < 0.0001), The plates were examined for colony-forming and shell thickness (μm; P = 0.0088; Table 1). units and the colonies were counted. The VR- A statistically significant interaction between 62 JAPR: Research Report production system and flock age indicates that Egg weight in the CC flock was significantly the pattern of change with increasing flock age higher than for the FR birds at all flock ages. In was different between the 2 production systems. the FR flock, egg weight (g) increased between Shell breaking strength (N), shell deformation 25 and 35 wk and then remained relatively con- (μm), and percentage shell were not significant- stant. In contrast, in the CC system, a consistent ly affected by production system, although a sig- slight increase in egg weight was observed with nificant interaction was noted between produc- increasing flock age. These observations are in tion system and flock age. For the egg internal contrast to the findings of previous studies [18, quality, a significant main effect was observed 19] in which a greater increase in egg weight for both production system and flock age and a was observed in free-range eggs. A slightly significant interaction between the 2 was noted higher egg weight in a cage flock compared with for albumen height (mm), Haugh unit, and yolk a free-range flock has been reported previously color. Translucency score (P < 0.0001) and shell [20, 21], which is similar to the current findings. reflectivity (P = 0.0001) were higher in the FR Shell weight and shell thickness were higher for Downloaded from flock and increased with flock age in both sys- CC versus FR eggs in the present study. No sig- tems, whereas egg weight, shell weight, percent- nificant difference was recorded for shell weight age shell, and shell thickness were higher in CC between cage and floor pen and litter system (P < 0.0001) and generally increased with flock eggs in previous studies [22–24]. In the studies age, irrespective of production system. The egg of Abrahamsson and Tauson [25], higher shell http://japr.oxfordjournals.org/ internal quality parameters, albumen height, weight was recorded for cage eggs compared Haugh unit, and yolk color, were higher in the with an aviary system. CC production system, as compared with FR (P Shell thickness was not significantly different < 0.0001), and decreased with flock age, except between free-range and cage systems in several for the yolk color. previous studies [18, 19, 22], contrary to the Translucency develops when moisture es- findings of the present study. The current results capes from the egg albumen through the shell for higher shell thickness in CC eggs cannot be membranes into the ultrastructure of the mam- compared directly with previous research find- by guest on February 24, 2014 millary layer. Most freshly laid eggs show rela- ings, as most of the authors compared cage eggs tively few translucent spots, and translucency with production systems that were different from develops within the first 24 h after laying. The the free-range production system of the present incidence of translucency increases with the study. Housing system had little effect on shell passage of time until 6 to 7 d after the egg is breaking strength in the present study. Break- laid. The incidence of translucency varies from ing strength was higher for FR than for CC at egg to egg and is affected by both storage time 45 and 55 wk, but the reverse was true at 65 wk. and factors, such as flock age; it ranges from Higher shell strength in conventional cage eggs small pin points to hair-like lines and circular compared with all other systems has been re- spots of approximately 1 mm2. The higher shell ported [26], and a higher value for shell breaking reflectivity in FR eggs might be due to the time strength was recorded in cage eggs than for a lit- lapse between collection and processing. The ter system [22]. An increase in shell strength to- lower shell reflectivity (darker shell color) in ward peak production, followed by a subsequent CC versus FR eggs and increasing shell reflec- decrease with flock age in cage and outdoor tivity with flock age in both systems has been flocks, has been reported [27]. Shell deformation reported previously [17]. In the current study, (μm) showed a similar pattern to shell breaking shell reflectivity generally increased to a greater strength, with no overall difference between pro- extent in the FR flock as flock age increased. duction systems, indicating that housing system Shell reflectivity varies with the amount of pig- had little effect on shell elasticity or fragility. A ment (protoporphyrin IX) deposited in the egg- significant interaction (P < 0.0001) between the shell. The lighter the shell color, the higher the production system and flock age for shell defor- shell reflectivity and vice versa. Shell reflectiv- mation was observed, which was higher for CC ity can be used to measure stress conditions in than FR at 45 and 55 wk. Shell deformation was brown shelled laying hens. greater in conventional cage eggs versus all other Samiullah et al.: EGG QUALITY AND BACTERIA 63 P × A 1 A P -value P 75 Downloaded from 65 55 http://japr.oxfordjournals.org/ Free range 45 35 by guest on February 24, 2014 25 Flock age (wk) 75 65 299.0 ± 5.04 294.0 ± 7.62 320.0 ± 15.8 334.6 ± 7.55 316.6 ± 6.58376.9 ± 5.13 322.6 ± 8.01* 392.4 ± 6.02 321.3 ± 6.57* 389.0 ± 5.86 294.0 ± 12.0 354.8 ± 5.41* 261.6 ± 8.04 352.5 ± 5.74* 385.3 ± 3.55 381.5 ± 6.01 0.8885 387.3 ± 4.60 389.3 ± 4.72 <0.0001 <0.0001 0.0088 <0.0001 <0.0001 55 45 Conventional cage 35 60.35 ± 0.87 63.90 ± 0.75 63.27 ± 0.89328.3 ± 12.0 62.97 ± 0.78 64.47 ± 0.82 307.3 ± 5.45 68.60 ± 0.82 298.0 ± 5.37 51.68 ± 0.59* 62.57 ± 0.85 64.30 ± 0.58370.1 ± 3.49 61.73 ± 0.84 389.4 ± 3.82 61.97 ± 0.85* 378.0 ± 3.85 63.84 ± 1.0*103.5 ± 0.62 101.0 ± 0.7710.07 ± 0.09 99.23 ± 0.92 <0.0001 10.90 ± 0.16 95.40 ± 0.96 98.60 ± 1.03 ± 0.13 11.03 <0.0001 96.67 ± 1.57 10.50 ± 0.15 ± 0.13 11.70 96.50 ± 0.89* <0.0001 ± 0.16 11.40 95.17 ± 1.14* 90.77 ± 1.11* 7.73 ± 0.31* 80.13 ± 1.98* 84.20 ± 1.38* 10.03 ± 0.15* 63.33 ± 2.31* 9.80 ± 0.11* 10.07 ± 0.19 9.37 ± 0.19* <0.0001 ± 0.21 11.80 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 25 (μm)

(μm)

unit

thickness

P = production system; A = age; P × A = production system and flock age interaction. A × = age; P A = production system; P Translucency scoreTranslucency Shell reflectivity (%)Egg weight (g) 2.23 ± 0.20 27.10 ± 0.67Shell breaking strength (N) 40.07 ± 1.4 1.17 ± 0.18 30.33 ± 0.41Deformation 28.87 ± 0.59 1.03 ± 0.19 41.83 ± 0.96 30.17 ± 0.82Shell weight (g) 2.40 ± 0.21 29.70 ± 0.67 39.55 ± 0.90 38.19 ± 0.99 29.40 ± 0.57 1.63 ± 0.26Percentage shell 40.22 ± 1.60 29.67 ± 0.79* 36.67 ± 1.70 1.43 ± 0.21 32.57 ± 0.92*Shell 41.31 ± 1.60 33.37 ± 0.52* 2.77 ± 0.27 37.63 ± 1.30* 5.40 ± 0.07Albumen height (mm) 38.45 ± 1.50 38.70 ± 0.98* 33.97 ± 1.2* 2.40 ± 0.25* 42.74 ± 1.30* 9.02 ± 0.09Haugh unit 5.88 ± 0.07 41.50 ± 1.30* ± 0.14 11.04 3.97 ± 0.17* 35.52 ± 1.50* color Yolk 9.21 ± 0.09 37.49 ± 1.50 3.13 ± 0.19* 5.85 ± 0.08 10.65 ± 0.18 <0.0001 5.67 ± 0.08 2.97 ± 0.18*1 10.24 ± 0.21 9.26 ± 0.11 <0.0001 9.39 ± 0.20 5.99 ± 0.12 3.10 ± 0.17* 9.03 ± 0.14 10.07 ± 0.20 0.9182 6.14 ± 0.13 9.29 ± 0.13 9.95 ± 0.28 0.0001 8.96 ± 0.17 4.73 ± 0.09* <0.0001 0.0173 9.04 ± 0.18* 5.20 ± 0.11* 9.15 ± 0.15 9.33 ± 0.23* 0.0002 5.81 ± 0.06 0.0112 8.53 ± 0.20* 8.33 ± 0.15* 5.51 ± 0.12 6.76 ± 0.29* <0.0001 9.05 ± 0.09 7.32 ± 0.23* 5.70 ± 0.09 8.92 ± 0.13 4.81 ± 0.24* 5.94 ± 0.12 9.22 ± 0.14 9.33 ± 0.15 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0853 0.0009 0.0048 0.0002 *Significant difference from cage system at the same age. *Significant difference Table 1. Eggshell and egg internal quality measurements (mean ± SE) Table Variable 64 JAPR: Research Report systems in a previous study [26], and higher shell production cycle which, in turn, decreases albu- deformation was recorded for cage eggs com- men height. pared with aviary system [25]. However, another Yolk color was generally more consistent for study reported no significant effect of production the CC system. A significantly higher yolk color system on shell deformation [28]. in free-range compared with cage eggs was re- In the current study, percentage shell was corded in some previous studies [20, 24, 33]. In not significantly affected by production system, other studies [17], no significant difference was which is similar to other published data [17]. observed in yolk color between free-range and A higher percentage shell has been recorded in cage eggs. A higher yolk color for floor flock cage system eggs versus other systems by Hi- versus cage system eggs was recorded [34]. It dalgo et al. [26]. A slightly higher percentage can be concluded that yolk color varies more shell was recorded for free-range eggs versus a with flock nutrition than age or production sys- cage system in other studies [18, 19], which is in tem and will be dependent on both the amount contradiction to the current study. of pigment added to the feed and, in the case of Downloaded from Albumen height (mm) was statistically sig- free-range production, how much vegetation is nificantly different (P ≤ 0.05) between the 2 consumed by the birds. production systems at different ages. However, a longer time interval was noted between egg Estimation of the Amount of Cuticle collection and egg analysis of the free-range http://japr.oxfordjournals.org/ eggs, which would explain much of this differ- Spectrophotometry (L*a*b*) Measurements ence. A variable albumen height in cage eggs of Stained Eggshell. A significant main effect with increasing hen age, with a linear decrease was observed for production system and flock in outdoor hen eggs, has been recorded previ- age and a significant interaction was observed ously [18]. A relatively higher albumen height between production system and flock age for was recorded in free-range eggs compared with the L* and b* components of the L*a*b* space cages, with an overall decrease with flock age system, as shown in Table 2. A significant main [19], although no significant effect of produc- effect of production system and flock age was by guest on February 24, 2014 tion system on albumen height was observed noted for a* components of the L*a*b* space [19, 20]. system, but no significant interaction was ob- A statistically significant effect (P ≤ 0.05) of served between the two. production system and the interaction between The higher values of L* for FR compared production system and flock age was found for with CC eggs indicated less pigment in FR com- Haugh unit; this followed a similar pattern to al- pared with CC system eggs. From the current bumen height. Haugh unit decreased with flock spectrophotometric measurements of stained age more in FR, whereas it followed a consistent eggs, it can be concluded L* values are less sig- decline in CC. A higher Haugh unit in cage eggs nificantly affected by MST blue stain. The a* versus other systems was reported previously is the most important component in the L*a*b* [22, 29–31]. A slight decrease and then increase space system, as it shows the amount of the in Haugh unit occurred in an outdoor production MST stain acquired by the cuticle. The more system at the same time as a linear decrease oc- negative values in CC versus FR indicated more curred in a cage production system (26–50 wk of cuticle present in the cage eggs. The lack of sig- flock age) [18]. Free-range eggs had the lowest nificant interaction between production system Haugh unit scores compared with other systems and flock age indicated that flock age had simi- [26]. In other studies [17, 24, 32], no significant lar effects on the amount of cuticle deposition in effect of production system on Haugh unit was both production systems. The b* component of reported, and one study reported a higher Haugh the L*a*b* color space system is the grading be- unit in free-range compared with cage and deep tween blue and yellow, where yellow is toward litter systems [21]. Higher albumen height in the positive end of the scale and blue is toward CC eggs showed that these eggs were better in the negative end. Significant main differences internal quality compared with FR eggs. Viscos- were observed between production systems, ity of albumen decreases toward the end of the with FR flocks being higher. Samiullah et al.: EGG QUALITY AND BACTERIA 65

Ultrastructural Scoring of Shell Mammilla- ry Layer. Scanning electron microscopic obser- P × A vations indicated that no 2 eggs from the same

2 flock at the same age possess the same ultrastructural characteristics, which, in turn, reflects A a high rate of variation in the secretion of egg- P -value shell precursors in the shell gland of the hen’s reproductive system. Thus, oviducal malfunc- P

<0.0001 <0.0001 0.0699 tion may result in the breaching of the shell’s inherent defense mechanisms [35]. Analysis of the uterine fluid proteins [36] at various stages 1.24 of shell formation and in vitro crystallization ex- ±

periments [37] have revealed egg-specific pro- 75 teins that influence the process of calcification Downloaded from

0.97 −2.58 by modifying and modulating crystal growth

± [38]. Shell quality decreases with increased

65 flock age, and old hen eggs are reported to be more prone to microbial penetration as com- 1.02 −1.35

pared with eggs from younger flocks [39]. http://japr.oxfordjournals.org/ For the ultrastructural variables of the mam- 55 millary layer, a significant main effect of pro- Free range 0.92 −2.15

± duction system was recorded for mammillary

cap size, mammillary cap quality, early fusion, −0.85 45 Type A bodies, Type B bodies, aragonite, cuffing, and erosion (Table 3). A significant interaction between production system and flock

35 age (P < 0.05) was found for mammillary cap by guest on February 24, 2014 size, late fusion, alignment, Type A bodies, Type B bodies, cubic cone formation, cuffing, 3.15 ± 0.98* 2.52 ± 0.83 25 and changed membrane. No significant effect of production system or interaction between pro- Flock age (wk) 1.01

± duction system and flock age was observed for

the incidence of confluence, cubics, depression, 75 or hole. Variability of mammillary cap size, the 0.66 −4.61

± incidence of poor mammillary cap quality, inci-

dence of late fusion, alignment, Type A bodies, 65 Type B bodies, and cubic cone formation were 1.14 −1.05 greater in FR versus CC and increased with flock ±

age in both production systems. The incidence 55 of confluence and early fusion were greater in 1.20 −4.83 CC and decreased with age in both production ±

Conventional cage systems. More variable mammillary cap size −3.84 45 leads to poor membrane attachment and poor cap quality, which can affect the overall quality of the shell ultrastructure. Overall, cap qual- 0.26 ± 0.78 35 ity was poorer in FR compared with cage eggs.

0.75 Good cap quality cones show higher affinity for

± membrane attachment, thus making the shell −2.67 32.31 ± 0.30 32.4 ± 0.41 28.46 ± 0.64 29.44 ± 0.29 31.92 ± 0.43 28.97 ± 0.42 34.47 ± 0.42* 34.67 ± 0.50* 32.42 ± 0.45* 33.65 ± 0.88* 30.76 ± 0.58 31.29 ± 0.54* <0.0001 <0.0001 <0.0001 25

1 stronger. A higher incidence of early fusion has a positive effect on shell strength and vice versa

L* = between white (100) and black (0); a* = between green (negative) and red (positive); b* = between blue (negative) and yellow (positive). L* = between white (100) and black (0); a* green (negative) red = production system and flock age interaction. A × = age; P A = production system; P [14]. The overall incidence of early fusion was b* Table 2. Spectrophotometry values of stained eggshells (mean ± SE) Table Variable L*a* 51.06 ± 0.491 54.76 ± 0.61 56.47 ± 1.232 54.28 ± 0.86 55.09 ± 0.53 from cage system at the same age. *Significant difference 53.09 ± 0.86 56.12 ± 0.75* 57.09 ± 0.53* 59.79 ± 0.65* 60.44 ± 0.93* 62.08 ± 0.96* 60.96 ± 0.73* <0.0001 <0.0001 0.0029 66 JAPR: Research Report P × A 2 A P -value P 75 Downloaded from 65 55 http://japr.oxfordjournals.org/ Free range 45 35 by guest on February 24, 2014 25 Flock age (wk) 75 65 55 Conventional cage 45 35 2.17 ± 0.17 2.00 ± 0.14 2.30 ± 0.11 2.87 ± 0.13 2.77 ± 0.12 2.47 ± 0.12 1.97 ± 0.12 2.33 ± 0.10 *2.77 ± 0.09 2.77 ± 0.10 2.67 ± 0.11 *2.87 ± 0.12 0.0600 <0.0001 0.0111 25 1.17 ± 0.07 1.37 ± 0.12 1.23 ± 0.081.00 ± 0.00 1.50 ± 0.09 1.10 ± 0.06 1.57 ± 0.09 1.03 ± 0.03 1.47 ± 0.091.10 ± 0.06 1.13 ± 0.09 *1.00 ± 0.00 1.13 ± 0.06 1.07 ± 0.05 1.27 ± 0.08 1.00 ± 0.00 1.17 ± 0.082.86 ± 0.12 1.30 ± 0.08 1.13 ± 0.08 1.00 ± 0.00 2.83 ± 0.09 1.13 ± 0.06 1.77 ± 0.10 1.20 ± 0.11 1.93 ± 0.11 1.07 ± 0.05 1.70 ± 0.11 1.47 ± 0.12 1.17 ± 0.09 *1.90 ± 0.091.00 ± 0.00 1.03 ± 0.03 1.37 ± 0.09 1.33 ± 0.10 1.00 ± 0.00 1.30 ± 0.091.00 ± 0.00 1.03 ± 0.03 1.07 ± 0.05 0.0440 1.07 ± 0.05 1.00 ± 0.00 1.20 ± 0.07 2.90 ± 0.14 1.03 ± 0.03 1.07 ± 0.05 <0.0001 1.00 ± 0.00 1.07 ± 0.05 *2.13 ± 0.14 1.10 ± 0.06 1.97 ± 0.11 1.03 ± 0.03 1.00 ± 0.03 1.03 ± 0.03 0.0381 1.00 ± 0.00 1.10 ± 0.07 0.0082 1.27 ± 0.08 1.00 ± 0.00 1.00 ± 0.00 1.17 ± 0.07 0.0032 1.20 ± 0.07 1.00 ± 0.00 1.03 ± 0.03 0.4327 1.07 ± 0.05 1.03 ± 0.00 0.5660 <0.0001 1.23 ± 0.08 1.03 ± 0.00 0.1947 1.10 ± 0.06 1.00 ± 0.03 0.0024 1.17 ± 0.07 1.00 ± 0.00 0.7456 1.00 ± 0.00 0.0069 0.0393 0.5527 0.0131 0.9999 0.2626 0.3104 1 Mammillary cap size = 1 (similar), 2 (variable), 3 (highly variable); mammillary cap quality = 1 (best) to 5 (worst); confluence, early fusion, late fusion, alignment, Type A bodies, Type B Type bodies, A Type fusion, alignment, early fusion, late = 1 (best) to 5 (worst); confluence, cap quality mammillary 3 (highly variable); 2 (variable), cap size = 1 (similar), Mammillary = production system and flock age interaction. A × = age; P A = production system; P Table 3. Scores for ultrastructural features of shell mammillary layer (mean ± SE) Table Variable Mammillary cap size 1.48 ± 0.11Confluence 1.70 ± 0.11Cap quality 2.33 ± 0.12Early fusion 2.13 ± 0.18Late fusion 2.00 ± 0.09 3.00 ± 0.11 1.90 ± 0.10Alignment 2.83 ± 0.11 1.48 ± 0.11 *1.83 ± 0.09 3.21 ± 0.11 2.37 ± 0.13 1.67 ± 0.15 *2.10 ± 0.12 2.03 ± 0.14 2.23 ± 0.14 2.80 ± 0.10 2.07 ± 0.14 2.45 ± 0.14 1.77 ± 0.14 2.13 ± 0.12 2.57 ± 0.11 2.77 ± 0.08 1.83 ± 0.16 3.40 ± 0.12 2.27 ± 0.11 2.37 ± 0.18 2.20 ± 0.13 *2.20 ± 0.09 2.23 ± 0.11 2.67 ± 0.09 2.70 ± 0.14 2.37 ± 0.09 2.40 ± 0.09 3.00 ± 0.07 1.60 ± 0.12 2.73 ± 0.11 3.07 ± 0.08 0.0067 3.00 ± 0.09 2.33 ± 0.14 3.07 ± 0.07 2.03 ± 0.18 <0.0001 *2.37 ± 0.10 *2.57 ± 0.18 2.30 ± 0.17 2.73 ± 0.13 *2.37 ± 0.11 1.87 ± 0.16 2.73 ± 0.16 2.13 ± 0.12 3.13 ± 0.08 2.43 ± 0.16 *1.40 ± 0.12 0.0090 2.20 ± 0.12 2.40 ± 0.12 *2.97 ± 0.09 2.33 ± 0.09 3.03 ± 0.03 0.1401 2.97 ± 0.03 3.00 ± 0.05 0.0070 <0.0001 0.00031 <0.0001 <0.0001 0.3149 0.5407 depression, erosion, and hole = 1 (none) to 4 (extensive). bodies, aragonite, cubics, cubic cone formations, changed membrane, cuffing, 2 0.6687 <0.0001 0.3285 from cage system at the same age. *Significant difference 0.0062 Type A A body Type Type B bodyType Aragonite 1.52 ± 0.12Cubic 1.83 ± 0.14Cubic cone formation 1.00 ± 0.00 2.00 ± 0.14Cuffing 1.37 ± 0.09 2.13 ± 0.12Changed membrane 1.87 ± 0.09 2.90 ± 0.12 1.90 ± 0.11Depression 1.86 ± 0.22 1.90 ± 0.12 2.87 ± 0.12 1.40 ± 0.12Erosion 1.20 ± 0.07 1.93 ± 0.13 1.97 ± 0.11 1.23 ± 0.10Hole 1.00 ± 0.00 1.40 ± 0.10 2.20 ± 0.16 1.10 ± 0.06 1.07 ± 0.05 *2.10 ± 0.15 *2.53 ± 0.10 1.10 ± 0.06 *2.63 ± 0.14 *2.57 ± 0.11 *2.80 ± 0.18 1.03 ± 0.03 2.13 ± 0.11 *2.63 ± 0.17 *2.40 ± 0.13 1.00 ± 0.00 *1.53 ± 0.10 2.13 ± 0.13 <0.0001 1.10 ± 0.07 1.10 ± 0.06 1.13 ± 0.06 <0.0001 *1.00 ± 0.00 1.00 ± 0.00 1.17 ± 0.08 0.3520 1.00 ± 0.00 1.00 ± 0.00 0.0006 <0.0001 1.20 ± 0.09 0.1350 <0.0001 1.10 ± 0.06 1.07 ± 0.05 <0.0001 1.10 ± 0.05 1.20 ± 0.10 <0.0001 0.0854 0.5658 0.0577 Samiullah et al.: EGG QUALITY AND BACTERIA 67

slightly higher in CC versus FR eggs. A higher incidence of early fusion increases the effective P × A thickness of palisade columns [14] and is a posi- 2 tive feature of the mammillary layer. Increased

A incidence of cuffing has a positive effect on the

P -value mammillary layer, and its higher incidence in CC eggs suggests better ultrastructural quality. P Late fusion, alignment, cubics, cubic cone formation, changed membrane, depression, and hole negatively affect mammillary layer quality. Their incidence was not significantly different between the 2 production systems. The higher 75 incidence of mammillary layer variables, such as Type A bodies, Type B bodies, and aragonite, Downloaded from in FR compared with CC eggs indicates better 65 quality of CC eggs.

Egg Microbial Enumeration 55 http://japr.oxfordjournals.org/

Free range A significant main effect (P < 0.05) of production system and flock age and the interaction

45 between the 2 was recorded only for TBC on the eggshell surface. A significant main effect of flock age was observed on TBC in shell crush,

35 but no significant effect was noted for production system or interaction between production system and flock age, as shown in Table 4. The by guest on February 24, 2014 TEC on shell was significantly affected by flock 25 age and interaction between production system

Flock age (wk) and flock age, whereas TEC in shell crush was only significantly affected by flock age. None of

75 the egg internal contents were positive for bacteria. Understanding external contamination of 65

count. shells is important to evaluate the shelf life and food safety of commercial eggs. In most cases, egg internal contamination results from penetra- 55 tion of bacteria deposited on the shell surface as it is being laid or after it has been laid [40]. Sal- Conventional cage

Enterobacteriaceae monella poisoning related to egg products has 45 attracted the attention of food safety authorities in Australia and all over the world. The shell wash method used in the current study has been 35 used successfully by other workers for microbial enumeration from the eggshell surface [41]. In the current study, the TBC on shell surface 25 was significantly higher in FR eggs compared with CC eggs. On average, 20 to 30 times more

1 bacteria have been isolated previously from the surface of litter floor eggs compared with

TBC = total bacterial count; TEC = total TBC = total bacterial count; = production system and flock age interaction. A × = age; P A = production system; P a wire floor [42]. A greater chance of eggshell Table 4. Bacterial load in colony-forming units per eggshell of whole shell and crush (mean ± SE) Table Variable TBC on eggshell 3.42 ± 0.08 1.55 ± 0.52 3.09 ± 0.35 3.48 ± 0.59 3.29 ± 0.22 4.07 ± 0.11 3.81 ± 0.12*1 2.50 ± 0.33* 2.89 ± 0.20 3.47 ± 0.292 3.79 ± 0.15* 4.39 ± 0.09* from cage system at the same age. *Significant difference <0.0001 <0.0001 0.0012 TBC in shell crush 0.35 ± 1.56 ± 0.53TEC on eggshell 1.31 ± 0.45 1.19 ± 0.41 1.96 ± 0.55TEC in shell crush 1.63 ± 0.45 0.86 ± 0.48 0.00 ± 0.58 ± 0.39 1.79 ± 0.52 1.51 ± 0.41 3.08 ± 0.36 0.34 ± 0.22 1.71 ± 0.38 1.57 ± 0.44 0.20 ± 1.68 ± 0.47 0.20 ± 1.46 ± 0.37 0.43 ± 0.29 2.10 ± 0.38 1.20 ± 0.28 1.26 ± 0.26 0.05 ± 0.03 0.84 ± 0.27 1.88 ± 0.33 1.74 ± 0.38 1.42 ± 0.31 1.47 ± 0.40 2.06 ± 0.35* 1.23 ± 0.26 1.26 ± 0.29* 0.20 ± 0.13 1.32 ± 0.29 0.64 ± 0.22 0.6112 0.91 ± 0.29 0.0671 0.0332 0.2111 0.0265 0.5984 <0.0001 0.0133 0.0709 68 JAPR: Research Report contamination exists in litter and free-range sys- nesses, increased with flock age and tems compared with cages, as freshly laid eggs were higher in the CC versus the FR can be contaminated when coming in contact flock. with contaminated surfaces [43, 44]. A 15 times 2. The egg internal quality variables, albu- greater bacterial load on the eggshell surfaces men height and Haugh units, decreased of deep litter eggs compared with cage eggs with flock age and were higher in the CC has been reported [44]. Total aerobic flora were compared with the FR flock, whereas higher (more than 1.0 log) on eggs from an avi- yolk color was higher in cage eggs and ary housing system compared with conventional fluctuated with flock age in both produc- and furnished cage systems [41]. In another tion systems. study [45], the total bacterial load and Entero- 3. The amount of cuticle present on the bacteriaceae on the eggshell were significantly eggshell varied significantly with flock higher for furnished cage eggs compared with age and was significantly higher in CC conventional cage eggs. In the present study, the versus FR eggs. Downloaded from TBC in shell crush was not significantly differ- 4. The increased incidence of late fusion, ent between production systems and remained alignment, Type A bodies, and Type below 2 log cfu/mL of rinsate throughout pro- B bodies with increasing flock age induction in both systems. The TEC on the shell dicates that shell quality decreases as and shell crush was not affected by the produc- hen age increases. In the present study, http://japr.oxfordjournals.org/ tion system and, compared with TBC, was quite a significantly higher incidence of poor low. Enterobacteriaceae are the main bacteria mammillary cap quality, incidence of that cause food poisoning. Bacterial contamina- late fusion, alignment, Type A bodies, tion of the eggshell is greatly affected by factors Type B bodies, and cubic cone formation such as diet [46] and poultry house environment was recorded in FR versus CC eggs. [41]. A significantly higher Enterobacteriaceae 5. The total bacterial and Enterobacteria- count on eggshells from furnished cage produc- ceae load was relatively low and was tion (12.3%) compared with conventional cages significantly higher in FR compared by guest on February 24, 2014 (5.8%) was recorded, but the egg internal con- with CC eggs. tents from both production systems were free from bacteria [45]. In the present study, egg internal contents from both production systems REFERENCES AND NOTES were free from bacteria. Salmonella was not isolated from egg internal contents collected from 1. Australian Egg Corporation Limited. 2012. AECL annual report 2011/2012. http://www.aecl.org/about-us/ different production systems in Australia [35]. annual-reports/. Comparing different housing systems, previous 2. Australian Egg Corporation Limited. 2011. AECL an- studies [41, 42, 45, 47, 48] have shown that eggs nual report 2010/2011. 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50. Mertens, K., F. Bamelis, B. Kemps, B. Kamers, E. Acknowledgments Verhoelst, B. De Ketelaere, M. Bain, E. Decuypere, and J. This research was conducted within the Poultry CRC, es- De Baerdemaeker. 2006. Monitoring of eggshell breakage tablished and supported under the Australian Government’s and eggshell strength in different production chains of con- Cooperative Research Centres Program. The participation of sumption eggs. Poult. Sci. 85:1670–1677. Rowly Horn and Australian egg industry producers is very much appreciated. Downloaded from http://japr.oxfordjournals.org/ by guest on February 24, 2014 © 2014 Poultry Science Association, Inc. The effect of raw and roasted soybeans on intestinal health, diet digestibility, and pancreas weight of broilers

C. Rocha ,*1 J. F. Durau ,* L. N. E. Barrilli ,* F. Dahlke ,† P. Maiorka ,‡ and A. Maiorka *

* Department of Animal Science, Universidade Federal do Paraná, Curitiba, Brazil 80035-050; † Department of Animal Science, Universidade Federal de Santa Catarina, Florianópolis, Brazil 88040-900; and ‡ Department of Pathology, Universidade de São Paulo—Faculdade de Medicina Veterinária e Zootecnia, São Paulo, Brazil 05508 270 Downloaded from

Primary Audience: Nutritionists, Researchers

SUMMARY http://japr.oxfordjournals.org/ Soybeans have high protein and energy content; however, they also contain antinutritional factors that limit inclusion in feeds for nonruminant animals. Therefore, the aim of the present study was to evaluate the influence of feeding raw (RFFS), deactivated (DFFS), or over- heated (OFFS) full-fat soybeans on the intestinal health, diet digestibility, and pancreas weight of broilers. One hundred twenty broilers were fed diets containing 15% DFFS, 10% DFFS + 5% RFFS, 15% RFFS, or 10% DFFS + 5% OFFS; data were submitted to ANOVA and means

were compared by Tukey’s test. Broilers fed 15% RFFS presented higher goblet cell counts by guest on February 24, 2014 and sialic acid levels compared with those fed diets containing DFFS and OFFS. Intestinal villi were shorter in birds fed 15% RFFS than those fed 10% DFFS + 5% RFFS. The coefficient of apparent digestibility of nutrients was affected by the dietary treatments. The pancreas of the broilers fed the diets containing RFFS was significantly heavier of those fed DFFS or OFFS. The intestinal integrity, as well as pancreas structure, is impaired by the inclusion of RFFS in broiler diets. Conversely, soybean over-heating hinders nutrient utilization by broilers.

Key words: digestibility , Glycine max , intestinal health, sialic acid, soybean processing 2014 J. Appl. Poult. Res. 23 :71–79 http://dx.doi.org/ 10.3382/japr.2013-00829

DESCRIPTION OF PROBLEM ing their use as both a protein and energy source [1, 2]. however, soybeans must be treated with Corn and soybean products are the main feed- heat to eliminate part of their antinutritional fac- stuffs in feed formulation. The combination of tors, which negatively affect the metabolism and soybean meal with oil or fat sources in feeds is health of nonruminant animals. often used to supply the high protein and energy Raw soybeans contain several antinutri- requirements of modern broiler strains. Full-fat tional factors, such as protease inhibitors, lec- soybeans may be used as an alternative to that tins (hemagglutinins), and allergens (glycinin combination because they are rich in protein and β-conglycinin), which are heat unstable (370–420 g/kg) and oil (180–220 g/kg), allow- and therefore can be inactivated by heat treat-

1 Corresponding author: [email protected] 72 JAPR: Research Report ments. Once inactivated, the antinutritional fac- fat soybeans (DFFS), or over-heated full-fat tors in full-fat soybeans will no longer cause any soybeans (OFFS), as shown in Table 1. Full-fat damage to the animals. In addition to the heat- soybeans were deactivated in wet heat (150°C unstable antinutritional factors, soybeans also for 15 min), and OFFS were submitted to 180°C contain heat-stable antinutritional factors, such for 50 min. The chemical composition of the dif- as oligosaccharides, nonstarch polysaccharides, ferent soybean products and their protein quality and phytates, which can be broken down by ex- parameters are shown in Table 2. Birds were of- ogenous enzymes that can be added to the diet. fered feed and water ad libitum during the entire Antinutritional factors may render feedstuff nu- experimental period. trients unavailable, including calcium and phos- On d 21, six birds per treatment were random- phorus, cause pancreas hypertrophy, and destroy ly chosen, individually weighed, and euthana- intestinal microvilli, increasing the loss of en- tized by cervical dislocation for the evaluation dogenous essential amino acids, thereby reduc- of villus height, crypt depth, goblet cell count, ing growth rate and FE [3–7]. pancreas weight, and N-acetylneuraminic acid Downloaded from Soybean heat treatment needs to be opti- (sialic acid) concentration. Each bird was con- mized to reduce the presence of antinutritional sidered one experimental unit. Ileum samples factors and, at the same time, over-heating must (2 cm) were collected 2 cm below Meckel’s di- be prevented. Considering the nutritional impor- verticulum for intestinal morphometric analysis tance of soybeans in the diets of nonruminant (villus height and crypt depth) [8]. Images of 20 http://japr.oxfordjournals.org/ animals, the objective of this study was to evalu- villi and 20 crypts per bird at 40× magnification ate the influence of the dietary inclusion of raw, were collected using an image-analyzing system deactivated, and over-roasted full-fat soybeans [9]. Villus height was measured from the base, on the intestinal health, nutrient digestibility, starting immediately after the higher section of and pancreas weight of 21-d-old broilers. the crypt, to the top. Crypt depth was measured from the base of crypt to the crypt:villus transi- MATERIALS AND METHODS tion region. The number of goblet cells was enumerated along 200 µm of the intermediate sec- by guest on February 24, 2014 The current experiment was approved by tion of 20 villi at 200× magnification using the the Committee of Ethics on Animal Use of the previously mentioned image-analyzing system sector of Agricultural Sciences of the Federal [9]. Pancreas was collected and its wet weight University of Paraná, under protocol number was recorded. 057/2011. Sialic acid concentration was determined in In this trial, 120 one-day-old broiler chicks the ileal content of the same birds used for in- with an average initial weight 46.59 ± 0.56 g testinal morphometric evaluation. The content were distributed according to a completely ran- present from 6 cm after Meckel’s diverticulum domized experimental design into 4 treatments until 4 cm before the ileocecal valve was col- with 6 replicates of 5 birds each. During the lected. Samples were vacuum freeze-dried for 21-d experimental period, birds were housed in 72 h, manually ground in a crucifer, and ana- 2.25-m2 pens with reused wood shavings litter lyzed according to the method described by equipped with bell drinkers and tube feeders. Jourdian et al. [10]. Chicks were brooded until 14 d of age with one The four remaining 21-d-old birds per rep- electrical lamp with an incandescent bulb per licate were slaughtered by neck dislocation for pen. Thereafter, environmental temperature was the nutrient digestibility assay. The ileal con- controlled by side-curtain management. tent was collected, homogenized, placed in The experimental diets were based on corn plastic bags, and stored in a freezer at −18°C. and soybean meal and were formulated to con- Samples were subsequently thawed to room tain the nutritional levels recommended by temperature and dried in a forced-ventilation Rostagno et al. [2]. Feeds were fed in the mash oven at 55°C until a constant weight was at- form. The experimental treatments consisted of tained. The experimental diets and the ileal the dietary inclusion of different proportions of content were ground to a 1-mm mesh size and raw full-fat soybeans (RFFS), deactivated full- analyzed for DM content, CP, EE, and ash at Rocha et al.: SOYBEAN-FED BROILERS 73

Table 1. Composition and nutritional levels of experimental diets1

10% DFFS 10% DFFS Item 15% DFFS + 5% RFFS 15% RFFS + 5% OFFS

Ingredient (g/kg) Corn 552.4 552.4 552.4 552.4 Soybean meal (45.6%) 236.9 236.9 236.9 236.9 RFFS — 50.0 150.0 — DFFS 150.0 100.0 100.0 OFFS — — — 50.0 Soybean oil 13.6 13.6 13.6 13.6 Limestone 9.6 9.6 9.6 9.6 Dicalcium phosphate 16.0 16.0 16.0 16.0 Salt 4.4 4.4 4.4 4.4 l-Lys 1.0 1.0 1.0 1.0 dl-Met 2.7 2.7 2.7 2.7 Downloaded from l-Thr 0.5 0.5 0.5 0.5 Choline chloride 1.3 1.3 1.3 1.3 Celite2 10.0 10.0 10.0 10.0 Vitamin premix3 1.0 1.0 1.0 1.0 Mineral premix4 0.5 0.5 0.5 0.5 Determined chemical composition (g/kg) Moisture 107.3 108.3 112.0 105.1 http://japr.oxfordjournals.org/ CP 227.9 227.0 212.7 227.3 EE 69.9 68.6 71.5 67.6 Ash 62.1 61.9 62.9 59.4 Calculated chemical composition (g/kg, unless otherwise noted) AME (kcal/kg) 3,022 3,022 3,022 3,022 Calcium 10.1 10.1 10.1 10.1 Available P 4.1 4.1 4.1 4.1

Digestible Lys 10.9 10.9 10.9 10.9 by guest on February 24, 2014 Digestible Met + Cys 5.8 5.8 5.8 5.8 Digestible Met 2.9 2.9 2.9 2.9 Digestible Thr 7.0 7.0 7.0 7.0 Digestible Trp 2.2 2.2 2.2 2.2 1RFFS = raw full-fat soybean; DFFS = deactivated full-fat soybean; OFFS = over-heated full-fat soybean. 2Celite Corp., Lompoc, CA. 3 Multivitamin premix composition (per kilogram): vitamin A = 8,000 IU; vitamin D3 = 2,400 IU; vitamin E = 16.65 mg; vitamin K3 = 1.5 mg; vitamin B1 = 0.6 mg; vitamin B2 = 2.36 mg; vitamin B6 = 0.6 mg; vitamin B12 = 1,320 μg; biotin = 0.15 mg; pantothenic acid = 9.32 mg; niacin = 30.12 mg; folic acid = 1.42 mg. 4Multimineral premix composition (per kilogram): selenium = 0.65 mg; iodine = 0.35 mg; iron = 57.72 mg; copper = 12.30 mg; zinc = 141.48 mg; manganese = 173.0 mg.

105°C according to the methodology described in the feedstuff, MCf is marker content in the by AOAC [11]. Gross energy (GE) was deter- feed, and MCi is marker content in the ileal di- mined in a bomb calorimeter [12]. Acid-insol- gesta [14]. Ileal digestible energy (DE) content uble ash (Celite) was added at 1% in the diet as was also calculated (in kcal/kg). indigestible marker [13]. Data were submitted to Bartlett’s test to Based on those results, the coefficients of ap- evaluate the homogeneity of variances and the parent ileal digestibility of DM, CP, EE, ash, and Shapiro-Wilk test to verify the normality of GE were calculated according to the equation residues. After data normal distribution and the CAID = 100 − [100 × (Ni/Nf) × (MCf/MCi)], absence of outliers were determined, data were where CAID is the coefficient of apparent ileal submitted to ANOVA and mean were compared digestibility, Ni is the nutrient concentration in by Tukey’s test at the 5% probability level using the ileal digesta, Nf is the nutrient concentration the software program Statistix [15]. 74 JAPR: Research Report

Table 2. Chemical composition and protein quality heat treatments than that required for the dena- variables of raw (RFFS), deactivated (DFFS), and turation of protease inhibitors. over-heated full-fat soybean (OFFS) The index of protein solubility in KOH at Item 0.2% of soybeans submitted to increasing (0–21 (g/kg of DM, min) times of steam heating was evaluated by unless otherwise noted) RFFS DFFS OFFS Anderson-Hafermann et al. [19]. Although pro- Moisture 60.9 41.3 7.9 tein solubility did not significantly change as CP 405.5 382.2 416.4 autoclaving times increased, the performance of EE 211.3 229.7 165.7 broilers fed full-fat soybeans steam-heated for Ash 46.3 46.9 51.6 12 to 18 min was better compared with those fed CF 25.6 27.3 122.1 KOHSP1 86.16 79.42 10.37 RFFS. In a second experiment, those authors Urease (ΔpH) 0.84 0.15 0.00 submitted RFFS and raw, full-fat Kunitz trypsin 1Soluble protein 0.2% potassium hydroxide. inhibitor-free soybeans (KFSB) to steaming at 121°C for 0 to 21 min and concluded that KFSB Downloaded from RESULTS AND DISCUSSION presented better nutritional value than RFFS. However, it also demonstrated that heating The results of protein solubility in potassium significantly affects soybean nutritional values hydroxide (KOH) at 0.2% were 86.16, 79.42, for broilers because, although trypsin inhibitor and 10.37% for RFFS, DFFS, and OFFS, re- activity in KFSB is 40 to 50% lower compared http://japr.oxfordjournals.org/ spectively (Table 2). According to Butolo [16], with RFFS, the level of this inhibitor is 16 times protein solubility values greater than 85% indi- higher in KFSB compared with soybean meal. cate under-heating of soybeans. Urease activity Table 3 presents sialic acid concentration in obtained for RFFS was 0.84 (∆pH), which is the ileal content, villus height, crypt depth, and lower than those reported in literature, ranging goblet cell count results. Ileal villi of broilers between 2.0 and 2.5 for this feedstuff [16, 17]. fed 15% RFFS were shorter than of those fed Methods such as protein solubility in KOH 10% DFFS + 5% RFFS (P < 0.05), but were not 0.2%, urease activity, trypsin inhibitory activ- different from the other treatments. Ileal crypt by guest on February 24, 2014 ity, and dispersible protein index are extremely depth was not significantly different (P > 0.05) important to evaluate the effectiveness of the among treatments. processing of full-fat soybeans and its coprod- Broilers fed 15% RFFS had a higher number ucts [18]. Whereas urease and trypsin inhibitory of goblet cells (P < 0.05) compared with those activities are commonly used to determine soy- fed diets containing only DFFS or DFFS in com- bean under-heating, protein solubility in KOH bination with OFFS. These results are consistent at 0.2% and dispersible protein index are good with those of Oliveira et al. [22], who also veri- indicators of soybean over-heating. Therefore, fied an increase in goblet cell counts in starter it is recommended that a combination of these broilers fed lectin-rich feedstuffs. According techniques be applied (e.g., the use of the meth- to Bedford [23], the gut responds to changes odology of protein solubility in KOH at 0.2% and challenges posed by the diet changing its together with urease activity tests) [16, 19, 20]. weight, length, absorption surface area, and cell Hemagglutinin and trypsin inhibitor levels turnover rate. in soybeans depend on previous soybean heat Lectins or hemagglutinin are glycoproteins treatment. Trypsin inhibitor activity in raw soy- that have the capacity of binding to specific mol- beans ranges between 20 and 35 mg/g, but after ecules on the surface of the brush border of the heat treatment it is reduced to a residual level of intestinal mucosa [6, 24]. This disrupts the intes- 4 mg/g [7]. Barca et al. [21] analyzed hemagglu- tinal epithelium, reduces villus height, changes tinin content in raw and heat-treated soybeans the activity of brush border enzymes, causes en- and found higher levels in raw soybeans (3,600 dogenous protein hypersecretion, increases the μg/g) compared with heat-treated soybean prod- number of the mucin-secreting goblet cells, and ucts (1,692 μg/g for whole soybean flour and damages microvilli [16, 25, 26]. 155 μg/g for defatted soybean flour). According The broilers fed the diets containing RFFS to those authors, lectins are destroyed by milder (15% RFFS and 10% DFFS + 5% RFFS) pre- Rocha et al.: SOYBEAN-FED BROILERS 75

Table 3. Effect of raw or roasted full-fat soybean on villus height (VH), crypt depth (CD), number of goblet cells (GC), and sialic acid concentration (SA) of the ileum in broilers at 21 d of age

VH CD GC SA Item1 (µm) (µm) (no. in 200 µm) (mg/kg per day)

15% DFFS 995.0ab 115.2 11b 8.72b 10% DFFS + 5% RFFS 1,052.2ª 116.9 14ab 11.66a 15% RFFS 920.7b 114.0 15a 10.68ª 10% DFFS + 5% OFFS 1,011.1ab 112.2 11b 8.84b P-value 0.0274 0.7370 0.0091 0.0003 SEM 14.14 1.46 0.45 0.34 a,bMeans in a column with no common superscript differ significantly by Tukey’s test (P < 0.05). 1RFFS = raw full-fat soybean; DFFS = deactivated full-fat soybean; OFFS = over-heated full-fat soybean.

sented higher sialic acid concentrations (P < well DE are shown in Table 4. Dry matter and Downloaded from 0.05) relative to those fed only DFFS or in com- CP digestibility was higher in broilers fed 15% bination with OFFS. According to Larsen et al. DFFS and 10% DFFS + 5% RFFS relative to [27] and Pirgozliev et al. [28], sialic acid is asso- the diets with 15% RFFS and 10% DFFS + 5% ciated with mucin production by the goblet cells OFFS. The CP digestibility results obtained for

and its level in biological samples can be used as the treatment containing 5% OFFS may be ex- http://japr.oxfordjournals.org/ an indication of mucin loss by the gastrointesti- plained by the fact that over-heating damages nal tract; likewise, it is considered a useful sci- heat-sensitive amino acids, such as lysine, me- entific tool to evaluate metabolism and intestinal thionine, and cystine, and therefore reduces their health status in animals. digestibility [3, 34]. Sialic acids are a family of sugars with 9 car- Parsons et al. [35] evaluated the effects of bon atoms. They derive from neuraminic acid over-heating on the availability of soybean meal and are widely distributed as components of amino acids in broilers, and found that the true

mucin, glycoproteins, gangliosides, milk oligo- digestibility of several amino acids decreased by guest on February 24, 2014 saccharides, and so on [10]. The acid N-acetyl- as autoclaving time increased from 0 to 40 min. neuraminic acid, measured in the present study, Adequate heat treatment, conversely, increases is the most widely distributed of the sialic acids protein availability, rendering more susceptible [29]. Sialic acid concentration usually increases to digestive enzymes [36], which was confirmed in conditions involving cell senescence, bacte- by the results obtained with the diets containing rial infections, and osmotic fragility [30]. 15 and 10% DFFS. The increase in sialic acid secretion, as ob- Relative to the diet with 15% RFFS, its low served in the present study, was probably due CP digestibility may be attributed to the pres- to the higher number of goblet cells determined ence of protease inhibitors and lectins in raw in the broilers fed diets containing raw full-fat soybeans. Moreover, this result may also be ex- soybeans. The main physiological functions of plained by the presence of lectins that, in addi- mucin in the gastrointestinal tract are lubrication tion to stimulating intestinal tissue proliferation, of the epithelial surface and mucosa protection, also stimulate the glycoprotein synthesis by the acting as a barrier against the diffusion of nutri- goblet cells, thereby creating a mucin layer on ents and toxins [31]. Therefore, mucin produc- the intestinal mucosa [22], preventing nutrient tion increases when the mucosa needs to be pro- digestion by the brush border enzymes, and con- tected [32]. Nevertheless, mucins secreted in the sequently their utilization by the birds. intestinal lumen are rich in amino acids and in- Ether extract and ash coefficients of digest- creasing its production for intestinal protection ibility were lower (P < 0.05) when broilers were is nutritionally costly because there is inevitable fed 10% DFFS + 5% OFFS compared with the endogenous amino acid loss and maintenance other treatments. According to Hurrell [3], the energy requirements increase [33]. Maillard reaction, in addition to causing the The coefficients of apparent ileal digestibil- formation of amino acid complexes, may also ity of DM, CP, EE, ash, and GE fractions, as reduce the bioavailability of some minerals and 76 JAPR: Research Report

Table 4. Effect of raw or roasted full-fat soybean on ileal digestibility of DM, CP, EE, ash, gross energy (GE), and digestible energy (DE) of broilers at 21 d of age

Item1 DM (%) CP (%) EE (%) Ash (%) GE (%) DE (kcal/kg)

15% DFFS 68.2a 76.8a 81.0a 37.74a 69.8ª 3,279ª 10% DFFS + 5% RFFS 68.1a 75.6a 77.9a 37.17ª 68.1ab 2,994b 15% RFFS 63.0b 65.3b 78.8a 31.8a 63.9bc 2,892bc 10% DFFS + 5% OFFS 59.9b 65.7b 72.2b 19.4b 60.6c 2,655c P-value <0.0001 <0.0001 0.0071 <0.0001 <0.0001 <0.0001 SEM 0.91 1.36 1.02 1.76 0.96 49.72 a–cMeans in a column with no common superscript differ significantly by Tukey’s test (P < 0.05). 1RFFS = raw full-fat soybean; DFFS = deactivated full-fat soybean; OFFS = over-heated full-fat soybean.

the concentration of some vitamins (B6, A, and birds fed diets containing over-heated soybeans

C). Andrieux and Sacque [37] heated rat diets in their experiment. Downloaded from to obtain the Maillard reaction and observed re- Nonstarch polysaccharides are also consid- duced calcium and magnesium absorption in the ered major antinutritional factors in the diet of small intestine. Those authors suggested that the monogastric animals; they make up 20 to 30% absorption of minerals such as magnesium may of soybean content. However, unlike lectins and be compensated during feed passage along the protease inhibitors, nonstarch polysaccharides http://japr.oxfordjournals.org/ large intestine, suggesting that chelated form or are heat stable and can only be broken down the complexes formed could be destroyed by in- when exogenous enzymes are added to the feed. testinal microflora, with the mineral being liber- Among these compounds, those classified as ated and subsequently absorbed in the colon. In noncellulose polymers and pectic polysaccha- the present study, however, only the ileal content rides are water soluble because they contain was collected; therefore, it was not possible to many hydroxyl groups, and may significantly verify any possible action of cecal microorgan- interfere in gastrointestinal tract physiological isms on mineral absorption. processes [39]. These molecules are highly hy- by guest on February 24, 2014 The absence of significant differences in EE groscopic and thereby increase digesta viscosity, digestibility among diets containing raw or de- hindering the interaction between endogenous activated full-fat soybeans verified in the present enzymes and nutrients, as well as nutrient diffu- study is consistent with the findings of Wood et sion through the intestinal mucosa, and, conse- al. [38]. The GE digestibility and DE content of quently, reducing diet digestibility [40]. the ileal digesta of broilers fed 15% DFFS were Live weight and pancreas weight results are higher than those fed 15% RFFS and 10% DFFS shown in Table 5. Live weight was not different + 5% OFFS. According to Brito et al. [18], the among treatments (P > 0.05). Conversely, pan- heating of feedstuffs may affect their energy creas absolute weight and pancreas weight rela- values, as shown by the reduced ME values in tive to BW were statistically different among

Table 5. Effect of raw or roasted full-fat soybean on live weight (LW), absolute weight (AW), and relative weight (RW) of pancreas of broilers at 21 d of age

RW Item1 LW (g) AW (g) (g/100 g of LW)

15% DFFS 989.2 3.32c 0.334c 10% DFFS + 5% RFFS 925.0 4.05b 0.439b 15% RFFS 900.0 5.00a 0.556a 10% DFFS + 5% OFFS 953.3 3.19c 0.335c P-value 0.1699 <0.0001 <0.0001 SEM 14.83 0.17 0.02 a–cMeans in a column with no common superscript differ significantly by Tukey’s test (P < 0.05). 1RFFS = raw full-fat soybean; DFFS = deactivated full-fat soybean; OFFS = over-heated full-fat soybean. Rocha et al.: SOYBEAN-FED BROILERS 77 treatments (P < 0.05). The broilers fed the 15% DFFS and 10% DFFS + 5% OFFS diets presented lower absolute and relative pancreas weights compared with those fed the diets containing RFFS. The highest pancreas weights were obtained in broilers fed 15% RFFS relative to the other treatments. These results are in agreement with the findings of Perez-Maldonado et al. [41] and Brito et al. [18], who found that broilers fed raw and under-heated full-fat soybeans had heavier pancreas than those fed diets containing properly-treated full-fat soybeans or soybean meal. This increase in pancreas weight in broil- Downloaded from ers fed raw or under-heated full-fat soybeans may be explained by the presence of protease inhibitors. According to Han et al. [4] and Lee- son and Summers [42], the presence of protease inhibitors causes pancreatic hypertrophy and http://japr.oxfordjournals.org/ hyperplasia due to the stimulation of pancreatic secretion, as well as depresses small intestine proteolytic activity, resulting in reduced release of free amino acids. The histological examination of the pancreatic tissue showed that broilers fed RFFS presented an increase in the size and number of exocrine cells, explaining the increase in size by guest on February 24, 2014 and weight of this organ (Figure 1). This result can be interpreted as a physiological response to antinutritional factors found in the intestinal content that stimulate exocrine pancreas to enhance production of enzymes in a compensatory way tentatively to digest these factors. Protease inhibitors act by binding and blocking the activity of the proteolytic enzymes secreted by the pancreas (e.g., trypsin and chymotrypsin). Tryp- Figure 1. (A) Chickens fed the control diet where nor- sin pancreatic secretion is controlled by nega- mal pancreas parenchyma was observed. (B) Chick- tive feedback, that is, when trypsin levels in the ens fed 15% raw full-fat soybean showed hypertrophy small intestine are low—such as the case of the and hyperplasia of the exocrine component (as evidenced by the increase in organ size in a macroscopic formation of a complex between trypsin and its view). inhibitor—the pancreas is stimulated by the hor- mone cholecystokinin to produce more trypsin, leading to pancreas hypertrophy. The increase with increasing RFFS levels (0–20%) for layers in the synthesis of trypsin and chymotrypsin in- for 28 d and observed that pancreas weight increase the utilization of sulfur amino acids, on creased (P < 0.05) with increasing raw full-fat which these enzymes are based [43]. soybeans dietary levels. The heaviest pancreas The effects of raw soybeans on the pancreas was obtained in hens fed 15 and 20% raw full- have been studied for some time [38, 44], and, fat soybeans. according to Grant et al. [26], both lectins and Zhang et al. [46] compared diets with partial trypsin inhibitors may affect pancreatic func- or total replacement of raw full-fat soybeans by tion. Latshaw and Clayton [45] supplied diets KFSB and one soybean meal diet and found that 78 JAPR: Research Report laying hens fed 30% raw full-fat soybeans had 3. hurrell, R. F. 1990. Influence of the Maillard reac- heavier pancreas than those fed diets containing tion on the nutritional value of foods. Pages 245–259 in The Maillard Reaction in Food Processing, Human Nutrition and trypsin inhibitor-free full-fat soybeans or soy- Physiology. Birkhauser Verlag, Basel, Switzerland. bean meal. Those authors also observed heavier 4. han, Y., C. M. Parsons, and T. Hymowitz. 1991. Nu- pancreases in layers fed trypsin inhibitor-free tritional evaluation of soybeans in trypsin inhibitor content. full-fat soybeans compared with those fed soy- Poult. Sci. 70:896–906. 5. Palacios, M. F., R. A. Easter, K. T. Soltwedel, C. bean meal. It was concluded that the nutritional M. Parsons, M. W. Douglas, T. Hymowitz, and J. E. Pet- value of trypsin inhibitor-free full-fat soybeans tigrew. 2004. Effect of soybean variety and processing on is better compared with raw full-fat soybeans, growth performance of young chicks and pigs. J. Anim. Sci. 82:1108–1114. but worse than soybean meal. The authors at- 6. Fasina, Y. O., H. L. Classen, J. D. Garlich, B. L. tributed this finding to the fact the Kunitz tryp- Black, P. R. Ferket, Z. Uni, and A. A. Olkowski. 2006. Re- sin inhibitor is not the only protease inhibitor in sponse of turkey poults to soybean lectin levels typically soybeans. According to Douglas et al. [47], tryp- encountered in commercial diets. 2. Effects on intestinal development and lymphoid organs. Poult. Sci. 85:870–877. sin inhibitors are more relevant as antinutritional 7. Clark, J. A., and C. M. Coopersmith. 2007. Intestinal Downloaded from factors for young broilers than lectins; however, crosstalk: A new paradigm for understanding the gut as the both these factors have complementary effects “motor” of critical illness. Shock 28:384–393. on growth rate reduction [5]. 8. The collected segments were longitudinally cut, extended on cardboard and pinned at the ends, carefully rinsed In the present study, we have shown that both with saline solution at 0.9%, and fixed in buffered formal- under- and over-heating of soybeans, despite dehyde at 10% for 48 h. Segments were then dehydrated in http://japr.oxfordjournals.org/ their different modes of action, impair broiler graded alcohol series (70–100%), cleared in xylol, and em- bedded in paraffin. Villus height and crypt depth measure- performance. Over-heating reduces the nutri- ments and goblet cell counts were carried out in 5-µm tissue tional value of full-fat soybeans, and this effect sections stained with hematoxylin-eosin and alcian blue. is often irreversible, reducing dietary nutrient 9. Motic Images Plus 2.0, optical microscope Olympus digestibility. Conversely, raw full-fat soybeans BH2, Olympus America Inc., Melville, NY. 10. Joudian, G., L. Dean, and S. Roseman. 1971. A damage body cell structures, preventing homeo- periodate-resorcinol method for the quantitative estimation stasis in the gastrointestinal tract of broilers. of free siálico acids and their glycosides. J. Biol. Chem. 246:430–435. 11. AOAC International. 1995. Official and Tentative by guest on February 24, 2014 CONCLUSIONS AND APPLICATIONS Methods of Analysis of AOAC. 16th ed. AOAC Int., Wash- ington, DC. 1. The antinutritional factors present in raw 12. Model 1261, Parr Instrument Co., Moline, IL. full-fat soybeans damage the integrity of 13. Scott, T. A., and F. Boldaji. 1997. Comparison of in- ert markers [chromic oxide or insoluble ash (CeliteTM)] for the intestinal epithelium and negatively determining apparent metabolizable energy of wheat- or bar- affect both gross and microscopic pan- ley- based broiler diets with or without enzymes. Poult. Sci. creatic structure. 76:594–598. 2. Over-heating affects soybean nutritional 14. Sakomura, N. K., and H. R. Rostagno. 2007. Métodos de Pesquisa em Nutrição de Monogástricos. Funep, Jaboti- profile, reducing nutrient utilization in cabal, Brazil. broilers. 15. Analytical Software. 2008. Statistix for Windows. 3. Proper full-fat soybean processing is Analytical Software, Tallahassee, FL. critical, because the inclusion of raw or 16. Butolo, J. E. 2002. Ingredientes de origem vegetal. Pages 93–238 in Qualidade de Ingredientes na Alimentação over-heated full-fat soybeans in broiler Animal. Colégio Brasileiro de Nutrição Animal, Campinas, diets is not recommended. Brazil. 17. Purushotham, B., P. M. Radhakrishna, and B. S. Sherigara. 2007. Effects of steam conditioning and extrusion temperature on some anti-nutritional factors of soybean REFERENCES AND NOTES (Glycine max) for pet food applications. Am. J. Anim. Vet. Sci. 2:1–5. 1. Waldroup, P. W. 1982. Whole soybeans for poultry 18. Brito, C. O., L. F. T. Albino, H. S. Rostagno, P. C. feeds. World’s Poult. Sci. J. 38:28–35. Gomes, D. C. O. Carvalho, and A. 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5:86–95. ed full-fat soybeans by the chick. Poult. Sci. 50:1392–1399. Downloaded from 24. Liener, I. E. 1981. Factors affecting the nutritional 39. Choct, M., Y. Dersjant-Li, J. McLeish, and M. quality of soya products. J. Am. Oil Chem. Soc. 58:406– Peisker. 2010. Soy oligosaccharides and soluble non-starch 415. polysaccharides: A review of digestion, nutritive and anti- 25. King, T. P., A. Pusztai, and E. M. W. Clarke. 1980. nutritive effects in pigs and poultry. Asian-australas. J. Kidney bean (Phaseolus vulgharis) lectin-induced lesions in Anim. Sci. 23:1386–1393.

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Eur. Symp. Poult. Nutr. Balatonfüred, Hungary. World’s by guest on February 24, 2014 Poult. Sci. Assoc., Beekbergen, the Netherlands. Books, Guelph, Canada. 29. Varki, A. 1992. Diversity in the siálico acids. Glyco- 43. Sgarbieri, V. C. 1996. Proteínas em Alimentos Pro- biology 2:25–40. teicos: Propriedades, Degradações, Modificações. Livraria Varela, São Paulo, Brazil. 30. Pirgozliev, V., O. Oduguwa, T. Acamovic, and M. R. Bedford. 2007. Diets containing Escherichia coli-derived 44. Rogler, J. C., and C. W. Carrick. 1964. Studies on phytase on young chickens and turkeys: Effects on perfor- raw and heated unextracted soybeans for layers. Poult. Sci. mance, metabolizable energy, endogenous secretions, and 43:605–612. intestinal morphology. Poult. Sci. 86:705–713. 45. Latshaw, J. D., and P. C. Clayton. 1976. Raw and 31. Forstner, J. F., and G. G. Forstner. 1994. Gastrointes- heated full fat soybeans in laying diets. Poult. Sci. 55:1268– tinal mucus. Pages 1255–1283 in Physiology of the Gastro- 1272. intestinal Tract. Raven Press, New York, NY. 46. Zhang, Y., C. M. Parsons, and T. Hymowitz. 1991. 32. Cowieson, A. J., T. Acamovic, and M. R. Bedford. Effect of soybeans varying in trypsin inhibitor content on 2004. The effect of phytase and phytic acid on the loss of en- performance of laying hens. Poult. Sci. 70:2210–2213. dogenous amino acids and minerals from broilers chickens. 47. Douglas, M. W., C. M. Parsons, and T. Hymowitz., Br. Poult. Sci. 45:101–108. T. 1999. Nutritional evaluation of lectin-free soybeans for 33. Nyachoti, C. M., C. F. M. De Lange, B. W. Mcbride, poultry. Poult. Sci. 78:91–95. and H. Schulze. 1997. Significance of endogenous gut nitrogen losses in the nutrition of growing pigs: A review. Can. J. Acknowledgments Anim. Sci. 77:149–163. The authors thank Coordenação de Aperfeiçoamento de 34. Kouzeh-Kanani, M., D. J. Van Zuilichem, J. P. Pessoal de Nível Superior (Brasília, Brazil) for ﬁnancial Roozen, and W. Pilnik. 1981. A modified procedure for low support. © 2014 Poultry Science Association, Inc. Economic effects of proposed changes in living conditions for laying hens under the National Organic Program

Tomislav Vukina ,*1 Kenneth Anderson ,† and Mary K. Muth ‡

* Department of Agricultural and Resource Economics, North Carolina State University, Raleigh 27695-8109; † Prestage Department of Poultry Science, North Carolina State University, Raleigh 27695; and ‡ RTI International Research, Triangle Park, NC 27709

Primary Audience: Researchers, Policy Analysts, Organic Producers Downloaded from

SUMMARY In this paper, we estimate the costs and benefits of implementing the proposed National Or-

ganic Program for laying hens compared with alternatives. For the regulatory proposals under http://japr.oxfordjournals.org/ option 2, the regulatory cost will be zero because most producers are already in compliance with the proposed regulation. The anticipated benefits of this regulation will be zero as well, because the current market prices already reflect consumers’ willingness to pay for the existing animal welfare conditions. For the regulatory proposals under option 3, before market adjustments, the average regulatory burden for the entire organic egg industry will amount to $0.09

per dozen eggs, with extreme variations between $0 for small operations and $2.30 per dozen for large operations. If we rely on the average price of organic eggs, $2.69 per dozen, and as- sume a maximum estimated benefit associated with improved animal welfare conditions, that by guest on February 24, 2014 consumers would be willing to pay of about 30% above the current market price, the estimated benefit of regulation amounts to $0.81 per dozen eggs. Based on the findings, we conclude that option 2 is welfare neutral and could be easily adopted because it already has been adopted by representative producers. For option 3, the benefit-cost ratio is larger than 1, which indicates that the proposal passes the benefit-cost ratio test. The obtained result, however, has to be interpreted with serious reservation because of the differential effect that the proposed regulation would have on different industry participants. Under option 3, the effect of the proposed changes on small organic egg producers is negligible because most small producers are operating under conditions similar to the proposed living standards. However, costs will increase substantially for large organic egg producers and likely cause a substantial number of producers to exit organic production and switch to conventional production, which would cause a substantial decline in the prices of conventional eggs and organic feed in the short run.

Key words: organic, poultry , egg , living conditions, benefit-cost analysis 2014 J. Appl. Poult. Res. 23 :80–93 http://dx.doi.org/ 10.3382/japr.2013-00834

DESCRIPTION OF PROBLEM Program (NOP). As part of the rulemaking process, AMS may conduct economic effect analy- The USDA-Agriculture Marketing Service ses of amendments to the national standards for (AMS) oversees the USDA National Organic production and handling of organic agricultural

1 Corresponding author: [email protected] Vukina et al.: ORGANIC LAYERS’ LIVING CONDITIONS 81 products. With potential changes in the require- Option 1 provides for no substantial changes ments for living conditions for organic poultry, to existing regulations. Living conditions under the NOP must consider the economic effects of §205.239 do not specify indoor or outdoor stock- these changes on the regulated industry. The ing rates but require maintaining year-round liv- USDA NOP regulations at 7 Code of Federal ing conditions that accommodate the health and Regulations Part 205 set forth the national stan- natural behavior of animals. All animals must dards for production and handling of organic ag- have year-round access to the outdoors, shade, ricultural products. The NOP regulations were shelter exercise areas, fresh air, clean water, and first published in 2000 and were updated in direct sunlight suitable to the species, stage of February 2010 to include a substantial practice life, and climate. Use of covered porches or runs standard amendment regarding access to pas- is acceptable and soil contact is not required. ture for livestock. Livestock living conditions as Pullets may be confined until 20 wk of age if they apply to poultry are regulated by §205.238 necessary [per §205.239(b)]. (Livestock Health Care Practice Standard) and Option 2 is similar to existing animal welfare Downloaded from §205.239 (Livestock Living Conditions). standards. In indoor housing, birds must be able The NOP regulations do not set specific to move freely and engage in natural behav- stocking rates for either inside housing or out- iors (turn around, flap wings, scratch, and dust side access areas. The NOP issued a general pol- bathe). Scratch areas and dust baths must be pro- icy memo in October 2002 affirming that outside vided. Houses with slatted floors must have a http://japr.oxfordjournals.org/ access areas are required, but it did not specify minimum of 15% of available floor space as dust size or other details. The NOP subsequently pro- bathing areas. For layers, perches are required vided a memo regarding exemption to outside with a minimum of 15.3 cm (6 in.) per bird, rails access for purposes of biosecurity, as well as a may be included in front of nest boxes, and the decision that outside access could be provided floor may be slatted or mesh. Layers in single- in a fenced, roofed, and floored outside area (a level houses must have 1,394 cm2 (1.5 ft2), lay- “porch or veranda” attached to a poultry house). ers in raised roost-type houses must have 1,115 To obtain organic certification, poultry produc- cm2 (1.2 ft2), and layers in multi-tier houses by guest on February 24, 2014 ers must submit to NOP an organic system plan must have 929 cm2 (1.0 ft2), provided that over- describing outside access. The organic system head perches and platforms provide for at least plan is subsequently reviewed by USDA-ac- 55% of hens to perch. Natural light is required credited certification agents, who then interpret such that reading is possible on a sunny day with the regulations, review the organic system plan the lights turned off. With artificial lighting, a for sufficiency, and conduct on-site inspections dark period of at least 8 h must be provided each to verify compliance by organic operations. day. Ventilation must be sufficient to ensure less The National Organic Standards Board than 25 ppm ammonia. (NOSB), the NOP citizen advisory panel, made Under option 2, exit doors must be distrib- recommendations in April 2002, November uted around the building and provide ready ac- 2009, and December 2011 on animal welfare cess to the outdoors such that more than 1 bird issues concerning appropriate living conditions can exit at a time. For layers, exit doors must for poultry. On December 2, 2011, based on the be at least 41 by 36 cm (16 in wide by 14 in NOSB recommendation and independent animal high). For outdoor access, pullets must be out- welfare standards, NOP submitted 3 options for side by 16 wk. Outdoor access must be available regulations regarding outdoor access for poultry. when temperatures are over 50°F and provide The specific regulatory options that were con- direct sunlight, although solid roofs are allowed. sidered are (1) make no substantial changes to The surface of the run can be concrete but must the existing regulation, (2) adopt modified ani- have a well-maintained substrate of sawdust and mal welfare standards similar to existing stan- wood chips, and scratch areas and dust baths in dards, and (3) adopt animal welfare standards soil or suitable substrate must be available. Lay- that differ substantially from existing standards. ers must have a minimum of 1,858 cm2 (2.0 ft2) The implementation period for options 2 and 3 per bird for a minimum of 5% of the total flock is 5 yr. population. Mobile outdoor pen units must pro- 82 JAPR: Research Report vide a minimum of 1,858 cm2 (2 ft2) per bird sis. The approach relies on measuring benefits and be moved to provide vegetative cover at all and costs associated with the proposed project times. or policy; if the benefits are larger than the costs, Option 3 modifies the indoor living condi- or if the so-called benefit-cost ratio is greater tions (§205.239) under option 2 to provide more than 1, the project or policy passes the test and indoor space, increases minimum requirements is potentially approved. for scratch areas and dust baths, increases the exit door area, modifies outdoor living condi- Benefits of Regulation tions to eliminate solid roofs, specifies stocking rates, requires soil scratching areas, and requires Estimation of the monetary benefits of the year-round vegetative cover. Under option 3, regulatory options in this study relies on the ben- stocking rates, which are calculated by the floor efits transfer approach, which consists of a sys- perimeter of the building not including nest box- tematic review of the economics literature to de- es or perch areas, must provide a minimum of termine if benefits estimates can be transferred Downloaded from 1,858 cm2 (2 ft2) per laying hen and 1.4 kg (3 from other similar studies and adjusted to reflect lb) of live weight for pullets. For layers, perches the regulatory proposals. The most important are required with 15.2 cm (6 in) per bird with at part of the proposed regulations for living con- least 35 cm (14 in) elevation. Pullets must have ditions for organic poultry relates to reducing perches at 4 wk of age. Scratch areas and dust

stocking densities, both indoors and outdoors; http://japr.oxfordjournals.org/ baths must be available for at least 30% of avail- thus, studies addressing this particular aspect of able floor space. Houses with slatted floors are animal welfare improvement are most relevant. permitted if scratch areas are provided. The economics literature shows that consumers For option 3, exit doors must provide ready value improvements in animal welfare, and the access to the outdoors with a minimum of 1.8 hypothetical willingness to pay for increased m (6 ft) per 1,000 birds and a minimum height animal space could be quite substantial. of 35 cm (14 in). For the outdoor area, no solid Specifically for the organic egg industry roofs are permitted except for shade structures, a segment, the literature does not contain any by guest on February 24, 2014 shaded area must be provided in warm weather, consumer preference studies of animal welfare and birds must have soil contact. In addition, (living conditions) that are similar to the regu- 50% vegetative cover must be provided year- latory options. Therefore, we based our esti- round. Layers must have a minimum of 1,858 mates on the assumption that benefits associated cm2 (2 ft2) per bird. with improved animal welfare are going to be The objective of this project was to provide similar across poultry species, which allows us an independent economic effect analysis of pro- to use broiler studies to make comparable esti- posed regulatory changes for the living condi- mates about organic eggs. As we found out via tions for organic poultry. In the current paper, industry interviews, the representative organic we estimate the costs and benefits of implement- egg producer already satisfies the regulatory re- ing the proposed rule for laying hens, compared quirements related to stocking rates proposed in with alternatives (as per Executive Order 12866 option 2. Therefore, consumers’ willingness to [1]). The intention is for these results to help pay for the reduction in animal density has been guide the decisions of the NOSB when contem- already incorporated into the price of organic plating the adoption of any of the proposed reg- eggs, so no additional benefits are associated ulatory proposals. Given other urgent priorities with option 2. For option 3, we concluded that at this time, NOP does not anticipate addressing the benefits could be at most valued at a 30% the NOSB proposals on animal welfare in the increase in willingness to pay over and above near future. the current market prices [2].

MATERIALS AND METHODS Cost of Regulation The methodological approach frequently used in economics to evaluate projects or policy We turn now to the methodology for esti- proposals is referred to as a benefit-cost analy- mating the increased costs associated with the Vukina et al.: ORGANIC LAYERS’ LIVING CONDITIONS 83 proposed regulations. The employed methodol- quiring prior approval from the Office of Man- ogy relies on the standard enterprise budgeting agement and Budget. Under the Paperwork Re- techniques [3]. In constructing the budgets, we duction Act, Office of Management and Budget focused primarily on the cost aspects, because approval is required before collecting data from these are most relevant in analyzing the eco- more than 9 entities under government-spon- nomic effects of various regulatory scenarios. sored studies [6]. The imputed values for total revenue were calculated based on the break-even price, which Baseline Cost Estimation implicitly assumes the zero-profit condition [4]. The cost estimation methodology involved 2 The baseline cost estimates presented in this steps. In the first step, we established the base- section reflect stylized approximations of highly line cost structure and the break-even price. In idiosyncratic individual real-life cases and are the second step, we analyzed whether any of not intended to be used to assess an individual the specific regulatory requirements in options producer’s profitability or cash flow. The base- Downloaded from 2 and 3 will have an effect on the established line scenarios reflect the average situations for baseline cost structure. All regulatory proposal the most frequently observed configurations of items that could have an effect on the represen- production space. In presenting the budgets, tative operation’s baseline costs were quantified we focus primarily on the cost aspects, because to obtain the new (postregulation) cost structure these are most relevant in analyzing the eco- http://japr.oxfordjournals.org/ and the new break-even price. The comparison nomic effects of various regulatory scenarios. of the new (postregulation) and old (baseline) The hypothetical values for total revenue are break-even prices is finally expressed as a per- calculated based on the break-even price, which centage increase in the break-even price relative implicitly assumes the zero-profit condition. to the baseline and represents the cost increase The basic assumptions employed throughout due to regulation. can be summarized as We simplified the analysis by developing a set of representative operations defined by the • simple linear (straight-line) depreciation by guest on February 24, 2014 size of organic egg operations. Development of of assets with zero salvage value; representative operations is a method frequently • annual opportunity cost of capital of 3%; used in conducting economic effect analyses be- • homogenous labor hired at $13.25 per h; cause it facilitates estimation of industry costs • property tax rate of 0.8% of the value of with relatively limited data. This method avoids the assets; the need to develop a specific cost estimate for • annual insurance costs of 0.5% of the val- each potentially affected entity, which would be ue of the assets; and a time-intensive and costly process and likely • price variability for inputs according to require an extensive industry survey beyond the the size of the flock. scope of this project. For the purposes of study- ing the effects of proposed regulations on the In addition to the above assumptions, land cost of producing organic eggs, we focused our prices were constructed based on average real analysis on 3 sizes of organic layer operations: estate values for farm land per acre in 2011 [7]. small, midsize, and large flocks. Land prices were calculated as the average of In line with this approach, we developed the published land prices in the top organic-egg- structured interview guides [5], where the first producing states. Prices for land in New York, group of questions was set up to uncover the ba- Massachusetts, Michigan, North Carolina, and sic cost structure of the enterprise and the sec- California were averaged to obtain a land price ond group of questions focused on the typical of $5,675 per acre. The annual rental rate was costs involved in complying with the proposed obtained by multiplying the value of land with regulations. When conducting industry inter- the 3% interest rate, resulting in annual rates of views, we employed a method consistent with $170 per acre. the limitations on the number of establishments Labor costs were estimated using data ob- that may be contacted (fewer than 9) without retained on hourly wages for farming, fishing, 84 JAPR: Research Report and forestry occupations published by the Bu- bined effect of the proposed regulations on the reau of Labor Statistics for states with high organic egg industry as a whole will depend on concentrations of organic egg production. We the market shares that different size producers calculated an average hourly wage rate using have in the total national production. wage rates from 8 states—California, Iowa, To solve this problem, we relied on the data Massachusetts, Michigan, New York, North on the number of certified organic egg producers Carolina, Oregon, and Pennsylvania—result- and operations in 2011 obtained by the USDA- ing in an average hourly wage rate of $13.25. AMS survey of 36 USDA-accredited state and Organic certification costs were calculated as private organic certifiers (for details see [5]). the average of California Certified Organic Corresponding to our baseline enterprise bud- Farmers and Iowa Organic Certification Pro- get scenarios, we divided the egg industry into gram posted fees for each organic production 3 segments: small (fewer than 16,000 layers), sales range category. midsize (between 16,000 and 100,000 layers), All budgets were prepared based on the ex- and large producers (more than 100,000 layers). Downloaded from isting literature [3, 8–19], personal communica- To obtain the distribution by the defined size tions with extensions specialists and industry categories, we calculated the average producer’s leaders, and the authors’ expert opinions and size for each certifying agency by dividing the insights based on their research on the poultry number of birds by the number of producers that industry [20]. The budgeting analyses show each individual certifying agency certified in http://japr.oxfordjournals.org/ that the baseline break-even organic price for 2011. Next, we calculated the percentage share a representative small organic layer operation of each certifying agency in the industry total is $3.944 per dozen eggs, for a midsize organic (for each of the poultry industries separately), layer operation it is $2.475, and for a large or- and we multiplied this percentage share with the ganic operation it is $2.396. average producer size for this certifying agency. Finally, we summed these numbers in each Regulatory Cost Estimation of the individual size categories to obtain the percentage shares of each size category in the by guest on February 24, 2014 Using the baseline enterprise budgets de- industry total. As shown in Table 1, 30% of pro- veloped in the first step of the cost estimation duction is produced by small producers, 54% by methodological approach, in the second step, midsize producers, and 16% by large producers. we analyzed the effect of the regulation on the Likewise, we summed the number of producers baseline cost structure. We present the estimated in each size category based on the average pro- costs of compliance for each regulatory option ducer size of the certifying agency. The results separately. In each case, we present costs for show that 74% of producers are estimated to be representative farms of different sizes. In some small, 25% are estimated to be midsize, and 1% cases, the representative organic producers are are estimated to be large. in compliance with the regulatory options; thus, Given the lack of better data, this approach no incremental costs exist due to the proposed only depends on an assumption that the distri- regulation. In other cases, the effect of the regu- bution of producers by size within one certify- lation on costs can be substantial. ing agency is not too wide such that the mean size is a good representation of the observed Sizes of Organic Egg Operations size. Practically, this means that a typical certi- fier does not certify very small and very large One of the important difficulties encountered producers at the same time. To the extent that in this study is the lack of precise data on the some specialization of certifying agencies ex- distribution of producers by farm size. Having ists, such that, for any particular commodity, this information is very important because the some of them specialize in certifying small pro- regulatory proposals we analyzed clearly have ducers and others specialize in certifying large significantly different effects on producers de- producers, the obtained size distribution should pending on their size. Consequently, the com- be fairly reliable. Vukina et al.: ORGANIC LAYERS’ LIVING CONDITIONS 85

Table 1. Estimated number of Certified Organic Poultry and Egg Producers and Operations by size in 20111

Estimated Estimated Number percentage number Percentage Stock or species of birds of production of producers2 of producers

Layer hens (inventory) 7,673,085 100 580 100 Small (1,000 to 16,000 hens) 2,301,925 30 430 74 Midsize (16,000 to 100,000 hens) 4,143,466 54 145 25 Large (more than 100,000 hens) 1,227,694 16 5 1 1Based on information collected by USDA-Agricultural Marketing Service [21]. 2The number of producers for each size category is estimated by assigning all producers of each certifying agency to a size category based on the average production of operations under the certifying agency.

RESULTS AND DISCUSSION • an increased requirement for more outdoor access, which will be reflected in The analysis of the proposed rules on the cost Downloaded from fencing costs and the increased cost of of production of organic eggs starts with the pro- land; increased mortality and reduced feed duction of layer pullets as the first stage in the conversion associated with a substantially egg production cycle. Small pullet operations increased outdoor area; and additional will typically not be affected by the proposed heating costs to maintain the indoor envi- regulations in either regulatory scenario. In con- http://japr.oxfordjournals.org/ ronment within the thermal neutral zone trast, large pullet producers will be affected in of the chickens. both regulatory scenarios related to the outdoor access after 16 wk of age. To mitigate the effect of these proposed rules, the pullet growers However, when it comes to large producers, indicated in interviews that they would shift the the most significant effect of option 3 will be re- growing cycle so that the pullets would be moved flected in the requirement to significantly reduce into the laying facility by wk 16. Subsequently, the population density on the established farms this would shift the costs of raising the pullets in response to the proposed regulation regarding by guest on February 24, 2014 from 16 to 18 wk, where there is no egg pro- the indoor density, with an enormous effect on duction to the egg producer. This would result in the revenue reduction that could cause some of the suppression of feed conversion as well as the the large producers to exit the organic industry increase in some utility costs during those 2 wk. and convert their operations into conventional However, the price of pullets, if transferred 2 egg production. The combined effect of the pro- wk earlier, will have to drop, thereby offsetting posed regulation in option 3 is estimated to be a an increase in cost incurred by layer operations. 6.8% increase in the break-even price for mid- Therefore, the net effect of regulation on organic size producers and a 96% increase in the break- egg production through the pullets segment of even price relative to the baseline cost scenario the market is likely to be zero. for large producers. We describe the derivation Next, we turn to eggs. Based on our informa- of these estimates in subsequent sections. tion gathering, the representative typical organic egg producers, regardless of size, currently op- Small Operations (Fewer than 16,000 Hens) erate under the requirements proposed under option 2; hence, the effect of proposed regula- The summary of the regulatory effects of dif- tion on the break-even price is zero. In contrast, ferent regulatory options vis-à-vis the baseline the regulatory proposal summarized in option 3 for small egg producers is represented in Table 2 will have multiple effects on the cost structure of [22]. As far as indoor housing requirements are representative midsize- and large-scale organic concerned, a typical small organic egg producer egg producers through should automatically satisfy all of the regulatory option 2 requirements. The same is true for the • a one-time (fixed) cost associated with outdoor access requirement. As a result, the per- retrofitting the house to install more exit centage increase in the break-even organic price holes, and relative to the baseline is 0%. Similar to option 86 JAPR: Research Report

Table 2. Estimated costs of producing organic eggs under different scenarios for small operations in 2011

Item Baseline Option 2 Option 3

Production volume Birds per operation (n) 1,000 1,000 1,000 Organic eggs (dozen) 17,904 17,904 17,904 Breaker market eggs (dozen) 3,160 3,160 3,160 Costs per farm ($) Total fixed costs 28,892 28,892 28,892 Annualized fixed costs 4,113 4,113 4,113 Variable costs 68,830 68,830 68,830 Total annual costs 72,944 72,944 72,944 Breaker market eggs revenue adjustment1 2,338 2,338 2,338 Costs per dozen eggs Break-even revenue per bird ($) 70.61 70.61 70.61 Break-even price per dozen organic eggs ($) 3.94 3.94 3.94 Percentage increase over baseline — 0.0 0.0 Downloaded from 1Breaker market egg price assumes $0.74 per dozen.

2, when indoor housing requirements are con- ing type and equipment configuration. For a sin- cerned, the typical small organic egg producer gle-level house, the reduction in the flock size http://japr.oxfordjournals.org/ should satisfy all of the regulatory option 3 re- would be 12.5%. The reduction in flock size quirements as well. In addition, small producers would cause a heat loss inside the building that typically already provide outdoor access, which will need to be replaced by an additional heating would meet option 3 conditions. Hence, the per- requirement in winter months (120 d) in colder centage increase in the break-even organic price climates. Assuming laying hens generate heat of relative to the baseline is also 0%. 40 BTU per hen per hour [12] and 91,600 BTU per gallon of propane, valued at a price of $1.51

Midsize Operations per gallon, the heat replacement cost amounts by guest on February 24, 2014 (Between 16,000 and 100,000 Hens) to $3,798 annually. In addition to the reduction in flock size, some modifications to the hous- To satisfy the organic certification require- ing structures will be required. In particular, ments, most typical midsize producers are al- the number and size of exit doors are typically ready operating under the indoor stocking rates inadequate. Our estimates are based on the in- required by option 2 and, in some cases, are stallation of 14 exit doors at a one-time expense exceeding these stocking rates. Currently, they of $400 per door. On an annual basis, this cost are operating using a combination of natural and translates into a $644 increment plus the cor- artificial lighting to achieve the 16 h of daylight responding increases in insurance and property for optimal performance in the older single-level taxes. houses. Outdoor access requirements under op- In our interviews with producers, we found tion 2 are also being met. A typical organic egg out that midsize organic egg producers would be producer is providing access at 2 ft2 per hen able to expand outdoor access to meet option 3 based on approximately 10% of the hens us- requirements of 1,858 cm2 (2 ft2) per hen housed. ing the verandas. Some producers have allowed However, this would come at a cost of adding for the outdoor space at the 2 ft2 for 33% of the approximately 1 acre of land with its rental rate flock. As the result, the percentage increase in of $170 per year. The cost of additional fencing the break-even organic price relative to the base- is assumed trivial and is not explicitly accounted line is 0%. for. Even though the hens would consume some To satisfy option 3 organic certification re- nutrients on a more extensive system, typically quirements, most of these companies would what is seen is an increased feed consumption have to alter their indoor stocking rates. This from 1.7 kg (3.8 lb) per dozen to 1.8 kg (4.0 lb) would require a reduction in flock size to meet per dozen, or higher, due to the repartitioning of the indoor stocking rate depending on the hous- the nutrients consumed to support the foraging Vukina et al.: ORGANIC LAYERS’ LIVING CONDITIONS 87

Table 3. Estimated costs of producing organic eggs under different scenarios for midsize operations in 2011

Item Baseline Option 2 Option 3

Production volume Birds per operation (n) 16,000 16,000 14,000 Organic eggs (dozen) 314,899 314,899 261,595 Breaker market eggs (dozen) 78,725 78,725 65,399 Costs per farm ($) Total fixed costs 518,225 518,225 523,900 Annualized fixed costs 58,210 58,210 58,454 Variable costs 779,345 779,345 680,717 Total annual costs 837,555 837,555 739,172 Breaker market eggs revenue adjustment1 58,256 58,256 48,395 Costs per dozen eggs Break-even revenue per bird ($) 48.71 48.71 49.34 Break-even price per dozen organic eggs ($) 2.47 2.47 2.64 Percentage increase over baseline — 0.0 6.7 Downloaded from 1Breaker market egg price assumes $0.74 per dozen. activity and the increased movement to reach Several interesting results are worth high- resources. With current feed costs, this would lighting with respect to the new cost structure http://japr.oxfordjournals.org/ mean an added increase in per-dozen egg costs. under regulatory option 3. First, the new indoor Finally, larger outdoor access is likely to cause stocking rate requirement will force producers to mortality to increase from 8.3 to 18% [15]. The reduce the number of hens, which will, in turn, effect on reduced egg production was calculat- reduce some of the variable cost components ed under the assumption that mortality will be (e.g., feed) and increase others (e.g., energy). evenly distributed throughout the entire produc- Second, because of required investments in land tion cycle. Taking all these effects jointly into and equipment, the annualized fixed costs go up, consideration, we find the projected increase in but only moderately. Finally, the reduction in the by guest on February 24, 2014 the break-even organic eggs price amounts to number of birds placed reduces the quantity of 6.7%. eggs produced with the negative effect on both The summary of the estimated costs of regu- the average cost per dozen eggs and on the to- lation and their relationship to the baseline cost tal revenue. All effects combined result in a re- scenario is presented in Table 3. As mentioned quired increase in price necessary to break even. before, the typical midsize organic egg producer appears to be automatically in compliance with Large Operations (More than 100,000 Hens) the regulatory proposal contained in option 2; hence, the increase in the average total cost, and As it relates to option 2, the interviews with thus the break-even price relative to the base- industry participants revealed that large pro- line, amounts to 0%. A negligible effect on mid- ducers are already operating under the required size producers is definitely associated with the indoor stocking rates. Their production facili- types of production operations they are integrat- ties are operated using predominantly artificial ing into organic egg production. These are typi- lighting; natural sunlight is provided by out- cally modified older facilities previously used door access. Outdoor access requirements un- for broiler breeder fertile egg production, which der option 2 are also being met; large organic is typically smaller in size and has a single pro- egg producers are providing outdoor access us- duction unit on a farm. This allows for greater ing verandas at 2 ft2 per hen based on the flock flexibility associated with indoor hen density utilization ranging from 10 to 33%. Most of the and outdoor access. If they were to build new veranda areas are wire pens with covers to pre- facilities, the effect of the new regulations, even vent wild birds and predators from having direct under option 2, would be greater and may be contact with the flock. This does not prevent in- comparable to the effects on large producers. direct contact from occurring nor small rodents 88 JAPR: Research Report from entering the verandas. The construction of to 1.8 kg (3.8 to 4.0 lbs) per dozen eggs. Accord- verandas ranges from concrete covered in a litter ing to our estimates, all these effects combined to soil with no vegetation. From the perspective produce a stark increase in the break-even price of a typical large organic egg producer, compli- of 96%. ance with the proposed option 2 requirements The summary of the estimated costs of regu- will have no appreciable additional costs; hence, lation and their relationship to the baseline cost the percentage increase in the break-even organ- scenario for the representative large organic ic price relative to the baseline is 0%. eggs producer is presented in Table 4. As seen For option 3, the most significant effect of the from the table, the typical large organic eggs proposed regulation will be felt by large-scale producer is automatically in compliance with egg producers. Based on our analyses, the repre- the regulatory proposal contained in option 2; sentative large-scale egg producer could satisfy hence, the increase in the average total cost, and the proposed requirements only through a rather thus the break-even price relative to the base- dramatic increase in costs. The typical produc- line, amounts to 0%. However, this is most defi- Downloaded from tion system is some type of multi-level housing, nitely not the case for option 3. The reduction in either an aviary or an integrated multi-level slat the number of birds and, consequently, the re- system. In addition, many of the large produc- duction in the number of eggs and total revenue ers have integrated their production into com- are staggering. plexes that contain multiple houses along with At this point, it is important to emphasize http://japr.oxfordjournals.org/ feed milling and waste disposal facilities. Their that the time horizon of 5 yr that we implicitly ability to provide increased indoor and outdoor used in this analysis eliminates the need to con- space, for which they are approved under the sider the possibility of constructing an entirely current standards, is limited. The limiting space new production complex that would satisfy the requirements are related to both indoor and out- stringent stocking rates requirement envisioned door stocking rates. Even after a dramatic reduc- in option 3. Moreover, based on our interviews tion in indoor population density, the outdoor with producers, they unanimously ruled out the space requirement under option 3 still remains a possibility of investing in the construction of by guest on February 24, 2014 binding constraint. In the example provided by new houses, even in the long run, and claimed our baseline scenario, the indoor stocking rate that they would exit the organic industry instead. of 2 ft2 per hen, calculated by floor perimeter A few other points are worth clarifying. First, a of the building, would require a reduction in the small reduction in annualized fixed costs exists number of hens from 100,000 to 13,500 hens due to the reduction in annual organic certifica- because the typical house (60 × 450 ft) has the tion fees resulting from the reduction in revenue. floor surface of 27,000 ft2. This dramatic reduc- Second, a dramatic reduction in total variable tion in flock size has consequences over the en- cost was noted due to the reduction in the vol- tire cost structure. A particularly important cost ume of output. This reduction in total variable item becomes the cost of providing additional cost is somewhat dampened by the need to use heat in a significantly depopulated house. After more energy to offset the heat loss inside the this 86.5% reduction in flock size, it is interest- building resulting from lower population den- ing to note that the outdoor space requirement of sity, the phenomenon that was well explained 1,858 cm2 (2 ft2) per bird proposed by option 3 is in the section regarding midsize producers. All still not met. To satisfy this requirement, outdoor these effects combined produce a dramatic in- space has to be increased from the current 1,855 crease in break-even price of 96% over the base- m2 [20,000 ft2; 1,858 cm2 (2 ft2)/bird for 10% of line. 100,000 birds] to 2,505 m2 (27,000 ft2). From our interviews, this 35% increment in outdoor Estimated Total Industry Costs space could be accommodated by most large producers at the proportionate 35% increase in Using the per-farm estimated regulatory the annual veranda-related cost. The only other costs above and the estimates of production vol- cost related to increased outdoor access is re- umes and actual prices, we calculated the total flected in the reduced feed conversion from 1.7 estimated industry costs due to regulation under Vukina et al.: ORGANIC LAYERS’ LIVING CONDITIONS 89

Table 4. Estimated costs of producing organic eggs under different scenarios for large operations in 2011

Item Baseline Option 2 Option 3

Production volume Birds per operation (n) 100,000 100,000 13,500 Organic eggs (dozen) 1,968,120 1,968,120 265,696 Breaker market eggs (dozen) 492,030 492,030 66,424 Costs per farm ($) Total fixed costs 3,986,200 3,986,200 3,986,200 Annualized fixed costs 418,234 418,234 414,184 Variable costs 4,661,742 4,661,742 882,758 Total annual costs 5,079,975 5,079,975 1,296,943 Breaker market eggs revenue adjustment1 364,102 364,102 49,154 Costs per dozen eggs Break-even revenue per bird ($) 47.16 47.16 92.43 Break-even price per dozen organic eggs ($) 2.40 2.40 4.70 Percentage increase over baseline — 0.0 96.0 Downloaded from 1Breaker market egg price assumes $0.74 per dozen. each of the regulatory options and contrasted In light of this information, the proposed these numbers with the industry total revenue. regulation regarding indoor and outdoor stock- http://japr.oxfordjournals.org/ Under some scenarios, the estimated total indus- ing rates was analyzed by first adjusting the in- try costs are zero because the representative op- door stocking rates by reducing the number of erations are in compliance with the regulation. animals until the condition is satisfied. In other Table 5 shows that the estimated total organic words, we ignored an unlikely possibility that eggs industry costs due to the proposed regula- a producer would opt to construct a brand new tion under option 2 are $0. Table 6 presents the housing facility to satisfy the indoor stocking estimated total industry costs under option 3, for rate constraint to keep the production at the which the total annual regulatory costs are esti- original preregulation level. If and when, after by guest on February 24, 2014 mated to be $68.1 million. These estimates rep- this adjustment took place, the new proposed resent 17% of estimated total industry revenue. outdoor stocking rate is still binding, the pro- All baseline and cost-shifting scenarios are ducer was allowed to purchase additional land based on the assumption of a representative pro- at the prevailing market land prices. In some ducer. To the extent that the entire egg industry is cases, the stocking rate regulation requirements fairly homogenous with respect to its cost struc- are so severe, based on the interview responses, ture within each size category, the representative we found out that the reduction in revenue asso- agent approach is adequate. However, if the in- ciated with the required reduction in the number dustry is technologically highly heterogeneous, of animals and the corresponding increase in av- then the representative agent approach is not go- erage total cost would force some firms to exit. ing to capture all specific nuances and idiosyn- crasies of different production processes, and a complete industry survey would be required. Regulatory Feasibility of Organic Egg All cost-shift scenarios are based on the Production Under Option 3 intermediate length of the run (5-yr horizon), where changes in variable cost through input In conducting data collection and analy- and output adjustments are possible together ses for the regulatory options, we identified with some changes in fixed cost through smaller several concerns regarding the feasibility of adjustments in land, buildings, and equipment. complying with the requirements under option However, potential entry and exit of firms, as 3 for egg production. The interviews with or- well as the new construction of large-scale pro- ganic egg industry participants and other ex- duction facilities by existing firms as the result perts revealed important reservations about the of regulation, is not considered in the current proposed regulations as presented in the option analysis. 3 scenario. 90 JAPR: Research Report 0 0 0 0 0 13,334 54,784 cost ($) cost ($) 68,118,000 Total industry industry Total Total industry industry Total 0.00 0.00 0.00 0.00 0.09 0.00 0.17 2.30 Regulatory Regulatory Regulatory Regulatory cost per unit ($) cost per unit ($) Downloaded from ($) 2 ($) 2 revenue in 2011 revenue Total industry industry Total in 2011 Total industry industry Total http://japr.oxfordjournals.org/ Unit Unit by guest on February 24, 2014 1 1 number of units Baseline Baseline 44,657,000 Dozen eggs 120,110,000 80,383,000 Dozen eggs 216,197,000 23,817,000 Dozen eggs 64,058,000 148,858,000 Dozen eggs 400,366,000 number of units Baseline Baseline 80,383,00023,817,000 Dozen eggs Dozen eggs 216,197,000 64,058,000 44,657,000 Dozen eggs 120,110,000 148,858,000 Dozen eggs 400,366,000 30 54 16 100 % of 54 16 30 Production 100 % of Production Total estimated dozens of organic eggs are based on laying hen counts published by USDA-National Agricultural Statistics Service [23, 24] assuming 19.4 dozens of eggs per laying hen. Note Agricultural eggs are based on laying hen counts published by USDA-National dozens of organic estimated Total Chief Haller, by USDA-Agricultural Marketing Service [21] and prices based on simple averages of monthly provided Lawrence obtained Revenue derived from production estimates Agricultural Statistics Service [23, 24] assuming 19.4 dozens of eggs per laying hen. by USDA-National eggs are based on laying hen counts published estimated dozens of organic Total Chief Haller, by USDA-Agricultural Marketing Service [21] and prices based on simple averages of monthly provided Lawrence obtained Revenue derived from production estimates Total estimated annual industry costs of regulations under option 2 Total 5. Table Item 1 budgets with 20% loss to the breaker market producers’ between this assumption and the of 308 eggs per hen that we use in our midsize large that there is a small difference eggs per hen) and 284 hen with 15% loss to the breaker market in small producer budget (21.1 dozens hen). (20.5 dozens of organic 2 Economist, USDA-Agricultural Marketing Service, Poultry Programs. estimated annual industry costs of regulations under option 3 Total 6. Table Item 1 2 Economist, USDA-Agricultural Marketing Service, Poultry Programs. Total organic egg production organic Total Total organic egg production organic Total Eggs, midsize operations operations Eggs, large Eggs, small operations Eggs, small operations Eggs, midsize operations Eggs, large operations Eggs, large Vukina et al.: ORGANIC LAYERS’ LIVING CONDITIONS 91

• The number and size of exit doors required of midsize and large producers to restrict per 1,000 hens appears to be excessive be- the access of wild animals and rodents to cause their installation could sometimes the flock of laying hens and, ultimately, jeopardize the physical integrity of the increases access to the poultry by wild housing structure, rendering it unusable birds and aerial predators. Under the pro- for continued production. The number of posed extensive outdoor access systems, exit doors added depends on the ultimate it would be impossible for the producers determination of indoor stocking rates. to meet the intent of the Food and Drug The proposed regulation for increased out- Administration Egg Safety Plan. door space was indicated as excessive by • Most of the large producers indicated that all producers interviewed. The numbers of the option 3 regulatory levels for both layers that actually go outside decreases indoor and outdoor space would induce as the flock size increases. Typically, most them to exit the organic industry and of the midsize and large producers indi- convert their operations to conventional Downloaded from cated that less than 10% of the flock was production practices. In the interviews, outdoors at any point in time. Granted that they indicated that because of the capital these are anecdotal observations, they are investment in their operations they would consistent across regions and producer not be able to produce organic eggs. sizes. Also chickens are prey animals and • Within the Clean Water Act [25, 26], op- http://japr.oxfordjournals.org/ are unlikely to venture very far away from erations that confine poultry to a specific the chicken house; hence, significant areas paddock for more than 45 d out of 12 mo of added space could be left unused. If a are defined as animal feeding operations. requirement for 50% forage cover is add- Many of the midsize organic produced, the costs for paddock expansion would ers fall into the medium contained area need to more than double to provide suffi- feeding operation (CAFO) definition. As cient space. None of the mid- to large-size with all free-range poultry operations, a producers indicated that they would be significant portion of the paddock is bare by guest on February 24, 2014 capable of maintaining this type of forage soil throughout the year regardless of the cover within the outdoor access areas. season. In our interviews with producers, • Organic producers that have more than we learned that several producers with 50,000 hens are currently subject to the operations in sensitive watersheds have Food and Drug Administration’s Egg been informed [27, 28] that they would be Safety Plan, and, as of July 9, 2012, pro- prohibited from providing free-range ac- ducers having 3,000 to 49,999 hens were cess to their laying flocks without adding under the same regulation. Regulation 21 600 acres to their operation, constituting CFR Part 118.4(b) [4] states that, as part a $3.4 million investment in land that is of an effective biosecurity program, pro- not available in their area. They indicated ducers must “prevent stray poultry, wild that they would have to close their op- birds, cats, and other animals from enter- erations and move to another location to ing poultry houses”; based on the inter- continue production. The current CAFO pretation of the rule and the need for a Rule [25] prohibits the discharge of fecal rodent and pest control program [21 CFR coliforms from manure into surface wa- Part 118.4(c)], a prevention program must ters from CAFO. All of the producers in be instituted to limit rodents in the build- this case indicated they would be forced ing as well. Currently, large producers use to abandon organic production because verandas and covered porches, which in of the inability to acquire land, or they essence does limit other animals’ access would have to construct a new facility to to poultry facilities. Introduction of rules accommodate potential changes in current that mandate greater outdoor access areas and future regulations. In the future, the eliminates the use of covered verandas US Environmental Protection Agency is and porches. This eliminates the ability examining the regulations pertaining to all 92 JAPR: Research Report

of the sensitive watersheds and is looking most small producers are operating un- into establishing new runoff rules for the der conditions similar to the proposed Mississippi watershed, which will affect living standards. some of the larger organic operations in 5. However, under option 3, costs will in- the United States. crease substantially for large organic egg producers and likely cause a substantial number of producers to exit organic CONCLUSIONS AND APPLICATIONS production and switch to conventional production. The switch from organic to 1. For the regulatory proposals under op- conventional production for large organ- tion 2, we found that the regulatory cost ic egg producers could substantially dis- will be zero because most of the produc- turb the conventional egg market caus- ers are already in compliance with the ing substantial price declines, at least in the short term. This change would also proposed regulation. We also found that Downloaded from the anticipated benefits of this regula- affect markets for organic corn and soy- tion are going to be zero as well because beans used as feed and cause a substan- the current market prices already reflect tial decline in the prices of organic feed. consumers’ willingness to pay for the ex-

isting animal welfare conditions. REFERENCES AND NOTES http://japr.oxfordjournals.org/ 2. For the regulatory proposals under op- 1. Office of Information and Regulatory Affairs. 2009. tion 3, we found that, before market ad- Executive Order 12866 Regulatory Planning and Review. justments, the average regulatory burden Office of Management and Budget, Executive Office of the for the entire organic egg industry will President. Accessed August 2012. http://www.reginfo.gov/ public/jsp/Utilities/EO_Redirect.jsp. amount to $0.09 per dozen eggs, with 2. See detailed list of literature references that leads to extreme variations between $0 for small this conclusion in the Phase 2 Report in Economic Impact operations and $2.30 per dozen for large Analysis of Proposed Regulations for Living Conditions for Organic Poultry (USDA-Agricultural Marketing Service, by guest on February 24, 2014 operations. If we take the average price 2012). of organic eggs, $2.69 per dozen, and as- 3. Boehlje, M., and V. Eidman, ed. 1984. Farm Manage- sume the maximum estimated benefits ment. Wiley, New York, NY. associated with improved animal wel- 4. The break-even price represents the sale price that the fare conditions, that consumers would producer must charge for a product in order for the revenues to just cover the expenses. be willing to pay of about 30% above the 5. USDA-Agricultural Marketing Service. 2012. Eco- current market price, we end up with an nomic Impact Analysis of Proposed Regulations for Liv- estimated benefit of regulation of about ing Conditions for Organic Poultry. National Organic Pro- gram. Accessed August 2012. http://www.ams.usda.gov/ $0.81 per dozen eggs. AMSv1.0/getfile?dDocName=STELPRDC5103929. 3. Based on our findings, we conclude that 6. Office of Management and Budget. 1980. Paperwork option 2 is welfare neutral and could be Reduction Act. (44 U.S.C. Chapter 35). Reviewed/updated adopted because it has already been ad- January 17, 2013. Accessed August 2012. http://www. howto.gov/web-content/requirements-and-best-practices/ opted by representative producers. As laws-and-regulations/paperwork-reduction-act. for option 3, we found that, on average, 7. USDA-National Agricultural Statistics Service. the benefit-cost ratio is larger than 1, 2011. Land Values 2011 Summary: August 2011. Accessed which indicates that the proposal would January 2013. http://usda01.library.cornell.edu/usda/nass/ AgriLandVa/2010s/2011/AgriLandVa-08-04-2011.pdf. pass the benefit-cost ratio test. 8. Anderson, K. E. 2009. Single Cycle Report of the 37th 4. However, that option 3 passed the ben- North Carolina Layer Performance and Management Test. efit-cost ratio test has to be interpreted Vol. 37, No. 4. North Carolina State University, Coopera- tive Extension Service, Department of Poultry Science, Ra- with serious reservation because of the leigh, NC. Accessed August 2012. http://www.ces.ncsu.edu/ differential effect that the proposed regu- depts/poulsci/tech_manuals/layer_reports/37_single_cycle_ lation would have on different industry report.pdf. participants. Under option 3, the effect 9. Anderson, K. E. March 3, 2010. Small farm egg production. Small Farm Conference, West Virginia University, of the proposed changes on small organ- Extension Service, Small Farm Center, Morgantown, WV. ic egg producers is negligible because North Carolina Cooperative Extension, Raleigh. Vukina et al.: ORGANIC LAYERS’ LIVING CONDITIONS 93

10. Anderson, K. E. 2011. Single Production Cycle Re- 20. The detailed presentation of the baseline cost esti- port of the 38th North Carolina Layer Performance and mates can be found in Appendix C of the Phase 2 Report Management Test (NCLP&MT). Vol. 38, No. 4. North Caro- in Economic Impact Analysis of Proposed Regulations for lina State University, Cooperative Extension Service, De- Living Conditions for Organic Poultry (USDA-Agricultural partment of Poultry Science, Raleigh, NC. Accessed August Marketing Service, 2012). 2012. http://www.ces.ncsu.edu/depts/poulsci/tech_manuals/ 21. USDA-Agricultural Marketing Service. 2011. Poul- layer_reports/38_single_cycle_report.pdf. try Market News and Analysis Reports. USDA, Washington, 11. Anderson, K. E. 2014. Time study examining the DC. Accessed August 2012. http://www.marketnews.usda. effect of range, cage-free, and cage environments on man- gov/portal/py. hours committed to bird care in 3 brown egg layer strains. J. 22. The detailed estimates for the regulatory impacts of Appl. Poult. Res. 23:108–115. options 2 and 3 for each of the size categories are available 12. Bell, D., and W. Weaver, ed. 2002. Commercial in the USDA-Agricultural Marketing Service, National Or- Chicken Meat and Egg Production. Kluwer Academic Pub- ganic Program, National Organic Program Insider (USDA- lishers, Norwell, MA. Agricultural Marketing Service, 2012). 13. Conner, B. 2010. Pastured Poultry Budgets: Slow 23. USDA-National Agricultural Statistics Service. Growing Broiler and Organic Comparisons. National Sus- 2012a. 2010 Agricultural Statistics Annual. USDA, Wash- tainable Agriculture Information Service, National Center ington, DC. Accessed August 2012. http://www.nass.usda.

for Appropriate Technology, Butte, MT. gov/Publications/Ag_Statistics/2010. Downloaded from 14. Cunningham, D. L. 2011. Cash flow estimates for 24. USDA-National Agricultural Statistics Service. contract broiler production in Georgia: A 30-year analysis. 2012b. Chickens and Eggs. USDA, Washington, DC. Ac- Cooperative Extension Bulletin 1228. University of Geor- cessed August 2012. http://www.nass.usda.gov/Publica- gia, Athens. tions/Todays_Reports/reports/ckeg0112.pdf. 15. Golden, J. B., D. V. Arbona, and K. E. Anderson. 25. US Environmental Protection Agency. 2008. Concen- 2012. A comparative examination of rearing parameters trated Animal Feeding Operations Final Rulemaking–Q & and layer production performance for brown egg-type pul- A. US EPA, Washington, DC. Accessed August 2012. http:// http://japr.oxfordjournals.org/ lets grown for either free-range or cage production. J. Appl. www.epa.gov/npdes/pubs/cafo_final_rule2008_qa.pdf. Poult. Res. 21:95–102. 26. US Environmental Protection Agency. 1980. Clean 16. Jones, D. R., K. E. Anderson, and G. S. Davis. 2001. Water Act of 1977, 33 U.S.C. § 1251 et seq. Accessed Au- The effects of genetic selection on production parameters of gust 2012. http://www.law.cornell.edu/uscode/text/33/1251. single comb white leghorn hens. Poult. Sci. 80:1139–1143. 27. Pula, W. E. 2001. Letter to Mr. George Bass, “The 17. Kuney, D. R., S. Bokhari, D. Bell, and G. Zeider. country hen,” organic egg producer, from MA-MDC, Divi- 1995. Labor costs and the packaging of table eggs 1962 to sion of Watershed Management, Quabbin Section. Belcher- 1991. J. Appl. Poult. Res. 4:94–99. town, MA. 18. Pennsylvania State University. 1999. Agricultural Al- 28. Pula, W. E. 2010. Letter to Mr. George Bass, “The ternatives: Small Scale Egg Production (Organic and Non- country hen,” organic egg producer, from MA-EQS-DCR, by guest on February 24, 2014 organic). Pennsylvania State University College of Agricul- Office of Watershed Management, Quabbin and Ware Sec- tural Sciences Extension, University Park. tion. Belchertown, MA. 19. Rhodes, J. L., J. Timmons, J. R. Nottingham, and W. Musser. 2011. Broiler production management for po- Acknowledgments tential and existing growers. University of Maryland Ex- This study was funded by the USDA-Agricultural tension, College Park, MD. Accessed August 2012. https:// Marketing Service (Washington, DC), Contract AG- extension.umd.edu/sites/default/files/_docs/POULTRY_ 6395-P-11-0638. All opinions stated are those of the authors BroilerProductionManagement_final1.pdf. and not of the USDA. © 2014 Poultry Science Association, Inc. Suitability of poultry litter ash as a feed supplement for broiler chickens

J. P. Blake1 and J. B. Hess

Department of Poultry Science, Auburn University, Auburn, AL 36849

Primary Audience: Nutritionists, Researchers, Producers

SUMMARY Downloaded from An experiment was conducted to evaluate poultry litter ash (PLA) at graded levels (0, 25, 50, 75, and 100%) as a dietary substitute for dicalcium phosphate (dicalP) for broiler chickens, where PLA was substituted for dicalP on a wt/wt basis. A decreased BW gain was observed at 21 d from PLA at the 100% substitution rate when compared with the 25% substitution rate. No effects on BW gain, feed consumption, or FCR were incurred by study termination at 41 http://japr.oxfordjournals.org/ d, and the 100% substitution rate of dicalP did not cause any detrimental effects on the live performance in market age broilers. Although femur ash percentages decreased with increasing levels of PLA, bone integrity was not compromised by the addition of PLA to the diet. Dry matter digestibility of dietary Ca (26.32–58.65%) and P (30.23–42.68%) increased with increasing

levels of PLA (0–100%), respectively, inferring that the Ca and P component of the diet was more efficiently used as the level of PLA increased. In conclusion, PLA resulting from the combustion of poultry litter can be used as a P and Ca source for broilers. by guest on February 24, 2014

Key words: poultry litter ash , dicalcium phosphate , digestibility , broiler 2014 J. Appl. Poult. Res. 23 :94–100 http://dx.doi.org/ 10.3382/japr.2013-00836

DESCRIPTION OF PROBLEM Feed phosphates are a costly component of the poultry diet and are mirrored by rising costs Recycling nutrients contained in poultry main the extraction and production of animal feed nure is an ongoing issue, and addressing associ- phosphates [3]. The potential for recycling P ated financial and societal effects must always from poultry manures exists, especially where be considered. The traditional practice of fertil- poultry litter as a feedstock for energy genera- izing crops with poultry manure is and will con- tion has been demonstrated [4]. The primary tinue to be a good agronomic practice. However, product remaining after combustion is poultry practical alternatives to land application of poul- litter ash (PLA), which has potential for use as try manure are needed because, in some areas, a P supplement for use in poultry diets and has the application of poultry manure to agricultural greater value in this respect as compared with its lands has resulted in soil phosphorus levels that fertilizer value [5]. Local sources of products, exceed the amount that can be removed in the such as PLA, provide a distinct economic advan- harvested crop [1]. Phosphorus runoff into sur- tage in reducing feed costs and maintaining bird face waters where manure is applied at exces- performance. An experiment was conducted to sive rates or rates outside established require- evaluate graded levels of PLA in the diet and the ments for crop production is a concern [2]. digestibility of P and Ca for the broiler chicken.

1 Corresponding author: [email protected] Blake and Hess: POULTRY LITTER ASH FOR BROILERS 95

MATERIALS AND METHODS hundred-sixty 1-d-old mixed-sex broiler chicks (Cobb × Cobb) were randomly assigned to 5 di- Evaluation of PLA etary treatments with 9 replicates of 8 birds/pen. Samples of PLA were obtained from a pilot In this study, birds were housed in Petersime plant test of an Energy Products of Idaho flu- batteries [11] within a temperature-controlled idized bed system designed to combust poultry room having an L:D cycle of 23:1 h. Feed and litter with the intent to produce process steam water were provided for ad libitum consumption [6]. The broiler litter was wood shavings based through termination of the experiment on d 41. and supported 6 continuous flocks reared in Birds and feed were weighed on d 1, 21, and 41 Alabama over the course of approximately 1 for the determination of BW gain (BWG), feed y. Initial analyses were completed to determine consumption, and FCR for the 0- to 21-, 21- to mineral composition of the PLA (Table 1) along 41-, and 0- to 41-d periods. Feed consumption with values used in diet formulation [7]. It must (FC) was calculated by the difference in feed offered and feed remaining on a pen basis at 21 be noted that a high level of Al in the PLA may Downloaded from be attributed to base-soil contamination of the and 41 d. Feed conversion ratio was expressed litter during the clean-out process. The PLA was as FC divided by BWG for each period. Mor- analyzed for dioxins where a toxic equivalency tality was recorded daily. On d 41, femur bones factor (TEF) was assigned when compared with were randomly obtained from 3 birds per pen and pooled (by pen) for ashing. Femur samples the toxicity of 2,3,7,8 tetrachlorodibenzo-p-di- http://japr.oxfordjournals.org/ oxin [7]. In 2005, the World Health Organiza- were cleaned and oven-dried at 100°C for 24 h tion derived the TEF for dioxin-like compounds to obtain a dry weight, and ashed in a muffle that most of the world uses now [8]. Currently, furnace at 500°C for 24 h in porcelain crucibles the maximum limit for dioxin in a feed ingredi- to obtain femur ash weight, which was divided ent that falls into the category for PLA is 1.0 ng by dry weight and multiplied by 100 to calcu- of World Health Organization-polychlorinated late percentage of femur ash. Animal transport, dibenzo-p-dioxins/polychlorinated dibenzofu- rans-toxic equivalents per kilogram [9]. Based by guest on February 24, 2014 on the PLA analysis and calculation of TEF, the Table 1. Composition of poultry litter ash dioxin level was 0.63 ng/kg, whereas the level Analyzed Computer of nonquantified polychlorinated biphenyls was Nutrient value1 value2 found to be 115 ng/kg [7]. Therefore, PLA was deemed safe for use as a feed supplement in Calcium (%) 16.68 16.70 poultry diets, whereby no related compounds Phosphorus (%) 10.08 10.00 Copper (%) 0.17 1,500.00 ppm appear to pose a potential threat to bird health or Iron (%) 0.59 5,000.00 ppm meat quality [9]. Magnesium (%) 2.65 2.70 Manganese (%) 0.21 1,900.00 ppm Potassium (%) 7.64 7.50 Experimental Procedures Sodium (%) 4.34 4.20 Chloride (%) 0.99 1.00 Direct substitution of PLA for dicalcium Zinc (%) 0.14 1,300.00 ppm phosphate (dicalP) on a wt/wt basis was ac- Selenium (ppm) 2.40 2.40 complished at dietary levels of 0, 25, 50, 75, Fluoride (ppm) 436.00 and 100% (Tables 2 and 3). All diets were com- Aluminum (ppm) 6,258.00 puter formulated based on commercial modern- Antimony (ppm) <5.00 Arsenic (ppm) 52.00 type broiler diets and do not necessarily adhere Cadmium (ppm) 0.80 to NRC requirements for the broiler chicken, Chromium (ppm) 34.00 which are somewhat dated [10]. As a result, the Lead (ppm) 4.40 starter and grower diets were formulated with Mercury (ppm) <0.10 lower levels (ca. 95%) of CP, ME, Ca, and P as Vanadium (ppm) 26.00 1 compared with NRC requirements. A starter diet Values obtained from analysis of sample submitted to Eu- rofins [7]. was fed from 0 to 21 d (Table 2) followed by a 2Values assigned to poultry litter ash when used as an ingre- grower diet from 21 to 41 d (Table 3). Three- dient in the computer feed formulation matrix. 96 JAPR: Research Report housing, and handling procedures were in ac- percentage of the difference in wet weight and cordance with the guidelines of Auburn Univer- freeze-dried weight before grinding and analysis sity’s Institutional Animal Care and Use Com- for Ca and P by ICP at the Auburn University mittee. Soil Testing Laboratory. Apparent digestibility The digestibility of Ca and P from each di- of Ca and P was determined as a percentage of etary treatment was determined. For this evalu- the difference between amounts consumed ver- ation, the 45 pens of birds were used for a 24-h sus amount excreted. Statistical analysis was total excreta collection to calculate mineral di- conducted using JMP software [12]. gestibility. Birds were introduced to the grower diet (Table 3) on d 21 of the experiment and al- RESULTS AND DISCUSSION lowed a 2-d orientation period. On d 23, feed weights were obtained and excreta collection Poultry litter ash was fed at graded levels (0, trays were lined with aluminum foil. Follow- 25, 50, 75, and 100%) as a direct substitute for ing a 24-h period, feed weights were obtained dicalP on a wt/wt basis. Phosphorus is the sec- Downloaded from and excreta were quantitatively collected and ond most expensive dietary ingredient after pro- weighed. Excreta moisture was determined as a tein, and PLA may offer exceptional advantage

Table 2. Composition of starter diets http://japr.oxfordjournals.org/

Level of poultry litter ash (%)

Item (%, unless otherwise noted) 0 25 50 75 100

Ingredient Ground yellow corn 58.52 58.52 58.52 58.52 58.52 Soybean meal (48% CP) 30.80 30.80 30.80 30.80 30.80 Poultry by-product meal (50% CP) 4.00 4.00 4.00 4.00 4.00

Poultry oil 2.80 2.80 2.80 2.80 2.80 by guest on February 24, 2014 Dicalcium phosphate1 1.40 1.05 0.70 0.35 0.00 Limestone (38% Ca) 1.12 1.12 1.12 1.12 1.12 Poultry litter ash2 0.00 0.35 0.70 1.05 1.40 Salt 0.40 0.40 0.40 0.40 0.40 dl-Methionine 0.26 0.26 0.26 0.26 0.26 l-Lysine 0.12 0.12 0.12 0.12 0.12 Vitamin premix3 0.25 0.25 0.25 0.25 0.25 Trace mineral premix4 0.25 0.25 0.25 0.25 0.25 Coban-605 0.08 0.08 0.08 0.08 0.08 Total 100.00 100.00 100.00 100.00 100.00 Calculated composition CP 22.00 22.00 22.00 22.00 22.00 ME (kcal/kg) 3,087.00 3,087.00 3,087.00 3,087.00 3,087.00 Ca 0.93 0.90 0.88 0.85 0.83 Nonphytate P 0.45 0.44 0.39 0.36 0.33 Met 0.62 0.62 0.62 0.62 0.62 Met + Cys 0.95 0.95 0.95 0.95 0.95 Lys 1.27 1.27 1.27 1.27 1.27 1Contains 18.5% P and 24.1% Ca. 2Poultry litter ash was added to the diet at the expense of dicalcium phosphate on a wt/wt basis. 3Supplied the following per kilogram of complete feed: vitamin A, 8,000 IU (vitamin A acetate); vitamin D, 2,000 IU (cho- lecalciferol); vitamin E, 8 IU (dl-α tocopheryl acetate); menadione, 2 mg (menadione sodium bisulfite complex); riboflavin, 5.5 mg (riboflavin); pantothenic acid, 13 mg (d-calcium pantothenate); niacin, 36 mg (niacinamide); choline, 500 mg (choline chloride); vitamin B12, 0.02 mg (cyanocobalamin); folacin, 5 mg (folic acid); thiamine, 1 mg (thiamine mononitrate); pyridoxine, 2.2 mg (pyridoxine hydrochloride); biotin, 0.05 mg (d-biotin); ethoxyquin, 125 mg. 4Supplied the following per kilogram of complete feed: manganese, 125 mg (manganous oxide); iodine, 1 mg (ethylene di- amine dihydriodide); iron, 55 mg (iron carbonate); copper, 6 mg (copper oxide); zinc, 55 mg (zinc oxide), selenium, 0.3 mg (sodium selenite). 5Monensin sodium [16]. Blake and Hess: POULTRY LITTER ASH FOR BROILERS 97

Table 3. Composition of grower diets

Level of poultry litter ash (%)

Item 0 25 50 75 100

Ingredient (%) Ground yellow corn 64.00 64.00 64.00 64.00 64.00 Soybean meal (48% CP) 25.67 25.67 25.67 25.67 25.67 Poultry by-product meal (50% CP) 4.00 4.00 4.00 4.00 4.00 Poultry oil 2.77 2.77 2.77 2.77 2.77 Dicalcium phosphate1 1.28 0.96 0.64 0.32 0.00 Limestone (38% Ca) 0.98 0.98 0.98 0.98 0.98 Poultry litter ash2 0.00 0.32 0.64 0.96 1.28 Salt 0.42 0.42 0.42 0.42 0.42 dl-Methionine 0.22 0.22 0.22 0.22 0.22 l-Lysine 0.08 0.08 0.08 0.08 0.08 Vitamin premix3 0.25 0.25 0.25 0.25 0.25 Downloaded from Trace mineral premix4 0.25 0.25 0.25 0.25 0.25 Coban-605 0.08 0.08 0.08 0.08 0.08 Total 100.00 100.00 100.00 100.00 100.00 Calculated composition CP (%) 20.00 20.00 20.00 20.00 20.00 ME (kcal/kg) 3,153.00 3,153.00 3,153.00 3,153.00 3,153.00 http://japr.oxfordjournals.org/ Ca (%) 0.84 0.82 0.79 0.77 0.75 Nonphytate P (%) 0.42 0.39 0.37 0.34 0.31 Met (%) 0.56 0.56 0.56 0.56 0.56 Met + Cys (%) 0.86 0.86 0.86 0.86 0.86 Lys (%) 1.10 1.10 1.10 1.10 1.10 1Contains 18.5% P and 24.1% Ca. 2Poultry litter ash was added to the diet at the expense of dicalcium phosphate on a wt/wt basis. 3Supplied the following per kilogram of complete feed: vitamin A, 8,000 IU (vitamin A acetate); vitamin D, 2,000 IU (cho- lecalciferol); vitamin E, 8 IU (dl-α tocopheryl acetate); menadione, 2 mg (menadione sodium bisulfite complex); riboflavin, by guest on February 24, 2014 5.5 mg (riboflavin); pantothenic acid, 13 mg (d-calcium pantothenate); niacin, 36 mg (niacinamide); choline, 500 mg (choline chloride); vitamin B12, 0.02 mg (cyanocobalamin); folacin, 5 mg (folic acid); thiamine, 1 mg (thiamine mononitrate); pyridoxine, 2.2 mg (pyridoxine hydrochloride); biotin, 0.05 mg (d-biotin); ethoxyquin, 125 mg. 4Supplied the following per kilogram of complete feed: manganese, 125 mg (manganous oxide); iodine, 1 mg (ethylene di- amine dihydriodide); iron, 55 mg (iron carbonate); copper, 6 mg (copper oxide); zinc, 55 mg (zinc oxide), selenium, 0.3 mg (sodium selenite). 5Monensin sodium [16]. as a source of P in meeting the bird’s require- minerals as compared with the control diet [13]. ment. Although PLA contains micro minerals Such decreases may also present a scenario that such as Fe, Zn, Cu, Mn, and Se, changes in the yields a marginal deficiency, whereby the bird rate of micro mineral addition were not altered responds with self-conservation and improved via the trace mineral premix in this experiment. capability to meet its nutrient requirements for This approach to diet formulation resulted in P and Ca. decreasing levels of Ca and P with increasing An increased BWG (P = 0.010) was observed levels of dietary PLA (Tables 2 and 3). It can during the 0- to 21-d period in birds receiving be recognized that this approach may produce the 25% PLA diet versus the 100% PLA diet adverse effects due to inadequacies in meeting (Table 4). Factors contributing to this increased nutrient requirements, but may also challenge performance could not be identified. A linear the availability of Ca and P in the test ingredi- decrease in BWG (P = 0.001) was observed in ent. When faced with a decrease in dietary P, and birds fed increasing levels of PLA in their diet possibly Ca, the bird will adapt by increasing P versus those fed the control diet during the 0- to utilization capacity to maintain P homeostasis, 21-d period, but this effect on growth rate dis- resulting in more efficient utilization of these appeared by d 41. This linear decrease in BWG 98 JAPR: Research Report coincided with a decrease in P and Ca level as P and Ca levels decreased as the level of PLA PLA inclusion increased (Tables 2 and 3). As the increased, no negative effect on bone mineral- resulting diets were formulated based on a wt/wt ization was observed. substitution of dicalP with PLA, the concomi- The digestibility of Ca and P increased lin- tant decrease in P and Ca levels in the diet may early (P = 0.0001) with increasing levels of PLA have contributed to a decreased BWG, as the (Table 5). Such a relationship infers that the Ca substitution rate increased during the 0- to 21-d and P component of the diet was more efficient- growth phase. Mortality averaged 2.1% during ly used, as the level of PLA increased in combi- the 41-d experimental period with no significant nation with a decrease in dietary Ca and P levels. differences (P > 0.05) among treatments (data It is plausible that the decreasing Ca and P lev- not shown). By d 41, the substitution of dicalP els promoted more efficient utilization of these with PLA failed to significantly affect (P > 0.05) minerals as compared with the control diet. Yan BWG, FC, or FCR in market age broilers. et al. [13] have reported that chicks exposed to With increasing levels of PLA, a significant low-Ca or -P diets will adapt by increasing their Downloaded from (P < 0.0001) linear increase in excreta mois- P utilization capacity to maintain P homeostasis. ture was noted (Table 5). It was not determined The bioavailability of PLA derived from tur- whether this increase was related to an increased key [14] and broiler [15] litter has been investi- water intake by birds fed increasing levels of gated. The analyzed values for Ca and P for the PLA. However, dietary sodium increased lin- PLA used in the current experiment was 16.68 http://japr.oxfordjournals.org/ early, from 1,000 to 3,000 ppm, with increasing and 10.08%, respectively, which compared well level of PLA, which supports a nonvalidated in- to that of other investigators indicating levels of crease in water consumption concomitant with Ca and P of 17.50 and 9.86 [13] and 15.45 and increased excreta moisture. Although femur ash 9.40% [14], respectively. Previous research was percentages did not vary greatly among treat- focused on determining the biological value of ments, a slight linear decrease was observed in the P content of the PLA [13, 14], whereas in which birds receiving the 100% PLA exhibited the current experiment we took a more practi- the lowest value (P = 0.006), whereas the 25, 50, cal approach. For turkeys [13] and broilers by guest on February 24, 2014 and 75% PLA additions yielded adequate or de- [14], the bioavailability values were reported clining [but not significant (P ≥ 0.05)] femur ash as 72.4 to 87.5 and 77.6 to 82.9%, respectively, values (Table 5). As compared with the control in comparison to calcium phosphate monobasic (0%), femur ash was not compromised by the monohydrate. Nonetheless, their conclusions addition of PLA to the diet. Even though dietary also confirmed that the PLA resulting from the

Table 4. Performance of mixed sex broilers fed graded levels of poultry litter ash (PLA)1,2

BWG (g) FC (g/bird) FCR (FC:BWG)

Item 0–21 d 21–41 d 0–41 d 0–21 d 21–41 d 0–41 d 0–21 d 21–41 d 0–41 d

PLA3 (%) 0 820ab 1,493 2,312 1,146 3,043 4,189 1.40 2.04 1.81 25 832a 1,420 2,252 1,170 2,965 4,135 1.41 2.09 1.84 50 802ab 1,430 2,232 1,120 2,957 4,077 1.40 2.07 1.83 75 793ab 1,398 2,191 1,110 2,889 4,000 1.40 2.07 1.83 100 778b 1,440 2,218 1,125 2,968 4,093 1.45 2.07 1.85 Pooled SEM 11.1 35.4 37.8 20.4 52.0 65.0 0.02 0.03 0.01 P-value Linear response 0.001 0.258 0.039 0.120 0.175 0.115 0.178 0.765 0.233 Quadratic response 0.004 0.194 0.066 0.778 0.193 0.153 0.201 0.658 0.496 r2 0.22 0.03 0.10 0.06 0.04 0.06 0.04 0.01 0.04 a,bMeans in a column with different superscripts are significantly different (P < 0.05). 1All values represent least squares means of measurements on 9 battery cages each having 8 birds. 2BWG = BW gain; FC = feed consumption; FCR = calculated as the ratio of unit of FC per unit of BWG. 3Percentage of PLA used as a replacement for dicalcium phosphate on a wt/wt basis. Blake and Hess: POULTRY LITTER ASH FOR BROILERS 99

Table 5. Percentage of excreta moisture, femur ash, and digestibility of Ca and P in broiler diets utilizing graded levels of poultry litter ash (PLA)1

Excreta Femur Calcium Phosphorus Item moisture ash digestibility digestibility

PLA2 (%) 0 72.93c 30.61ab 26.32d 30.23b 25 73.63c 31.29a 37.71c 35.21b 50 76.45b 30.51ab 56.37ab 35.67b 75 79.49a 30.12ab 50.42b 42.29a 100 81.72a 29.40b 58.65a 42.68a Pooled SEM 0.641 0.382 1.823 1.532 P-value Linear response 0.0001 0.006 0.0001 0.0001 Quadratic response 0.0001 0.006 0.0001 0.0001 r2 0.75 0.17 0.68 0.50 Downloaded from a–dMeans in a column with different superscripts are significantly different (P < 0.05). 1All values represent the least squares means of 9 pens per treatment. Excreta moisture, calcium digestibility, and phosphorus digestibility were obtained on a pen basis (8 birds per pen), whereas femur ash values represent the means of 1 pooled sample from 3 birds per pen per treatment. 2Percentage of PLA used as a replacement for dicalcium phosphate on a wt/wt basis. http://japr.oxfordjournals.org/ combustion of poultry litter could be used as a P 4. Power Plant Research Program. 1998. Engineering source in poultry diets. and economic feasibility of using poultry litter as a fuel to generate electric power at Maryland’s Eastern Correctional Institute. Department of Natural Resources, Annapolis, MD. Accessed June 6, 2013. http://esm.versar.com/pprp/eci/eci. CONCLUSIONS AND APPLICATIONS htm. 5. USDA. 2009. Manure use for Fertilizer and for En- 1. The substitution of dicalP with PLA on a ergy: Report to Congress. USDA Economic Research Ser- vice, Washington, DC. Accessed June 6, 2013. http://www.

wt/wt basis failed to compromise BWG, by guest on February 24, 2014 ers.usda.gov/media/156155/ap037_1_.pdf. FC, FCR, or bone development in mar- 6. Energy Products of Idaho, Coeur d’Alene, ID. ket age broilers. 7. Eurofins, Memphis, TN. 2. The digestibility of dietary Ca and P in- 8. Van den Berg, M., L. S. Birnbaum, M. Denison, M. creased with increasing levels of PLA, De Vito, W. Farland, M. Feeley, H. Fiedler, H. Hakansson, where decreasing Ca and P levels pro- A. Hanberg, L. Haws, M. Rose, S. Safe, D. Schrenk, C. To- hyama, A. Tritscher, J. Tuomisto, M. Tysklind, N. Walker, moted more efficient utilization of these and R. E. Peterson. 2006. The 2005 World Health Organiza- minerals as compared with the control tion reevaluation of human and mammalian toxic equivalen- diet. cy factors for dioxins and dioxin-like compounds. Toxicol. Sci. 93:223–241. 3. Poultry litter ash resulting from the com- 9. US Environmental Protection Agency. 2010. Recom- bustion of poultry litter can be used as a mended Toxicity Equivalence Factors (TEFs) for Human P and Ca source for broilers. Health Risk Assessments of 2,3,7,8-Tetrachlorodibenzo-p- dioxin and Dioxin-Like Compounds. Risk Assessment Fo- rum, Washington, DC. 10. NRC. 1994. Nutrients Requirements of Poultry. 9th REFERENCES AND NOTES rev. ed. Natl. Acad. Press, Washington, DC. 11. Petersime Incubator Co., Gettysburg, OH. 1. Codling, E. E., R. L. Chaney, and J. Sherwell. 2002. 12. Data were subjected to one-way ANOVA and, where Poultry litter ash as a potential phosphorus source for agri- significant, means were separated by Tukey’s Honestly cultural crops. J. Environ. Qual. 31:954–961. Significant Difference test. Data were further analyzed by 2. Sharpley, A. N., S. H. Chapra, R. Wedepohl, J. T. regressing the measured variable on level of PLA. All per- Sims, T. C. Daniel, and K. R. Reddy. 1994. Managing ag- centage data were subjected to arcsine square root trans- ricultural phosphorus for protection of surface waters: Issue formation prior to analysis. The experimental unit for each and options. J. Environ. Qual. 23:437–451. analysis was pen of birds. Statistical significance was con- 3. Auman, S. 2010. Phosphorus availability—When sidered as P ≤ 0.05 for dietary treatment differences. Data will we run out of phosphorus? Pages 1–12 in Proc. 2010 were analyzed using JMP software (JMP 11.0 Statistical Natl. Poult. Anim. Waste Manag. Sym. J. B. Hess, J. P. Discovery Software, 2013, SAS Institute Inc., Cary, NC). Blake, and K. S. Macklin, ed. National Poultry Waste Man- 13. Yan, F., R. Angel, C. Ashwell, A. Mitchell, and M. agement Symposium Committee, Auburn, AL. Christman. 2005. Evaluation of the broiler’s ability to adapt 100 JAPR: Research Report to an early moderate deficiency of phosphorus and calcium. 15. Muir, F., R. M. Leach Jr., and B. S. Heinrichs. 1990. Poult. Sci. 84:1232–1241. Bioavailability of phosphorus from broiler litter ash for 14. Apke, M. P., P. E. Waibel, and R. V. Morey. 1984. chicks. Poult. Sci. 69:1845–1850. Bioavailability of phosphorus in poultry litter biomass ash 16. Elanco Animal Health Inc., Indianapolis, IN. residues for turkeys. Poult. Sci. 63:2100–2102. Downloaded from http://japr.oxfordjournals.org/ by guest on February 24, 2014 © 2014 Poultry Science Association, Inc. Poultry litter ash as a replacement for dicalcium phosphate in broiler diets

J. P. Blake1 and J. B. Hess

Department of Poultry Science, Auburn University, Auburn, AL 36849

Primary Audience: Nutritionists, Researchers, Producers

SUMMARY Downloaded from An experiment was designed to evaluate the nutritional value of poultry litter ash (PLA) under commercial-type conditions. Diets were formulated to meet the nutrient requirements of the broiler utilizing PLA at combinations of 0, 25, 50, 75, or 100% in starter, grower, and finisher

diets as a replacement for dicalcium phosphate (DP). No significant effects were observed on http://japr.oxfordjournals.org/ BW, feed consumption, FCR, or mortality when broilers were fed PLA to 100% replacement for DP to 41 d of age. Processing performance, as measured by carcass and meat yield, of broilers at 42 d of age was also unaffected (P > 0.05). The complete substitution of DP with PLA failed to compromise growth and processing performance in market age broilers and the PLA produced via the combustion of poultry litter can be used as a phosphorus source in poultry diets.

Key words: poultry litter ash, dicalcium phosphate, broiler

2014 J. Appl. Poult. Res. 23 :101–107 by guest on February 24, 2014 http://dx.doi.org/ 10.3382/japr.2013-00838

DESCRIPTION OF PROBLEM supplement for use in poultry diets as compared with its fertilizer value [4]. Feed phosphates are The fertilization of crops with poultry ma- a costly component of the poultry diet and are nure is and will continue to be a good agronomic mirrored by rising costs in the extraction and practice. However, alternatives to land applica- production of animal feed phosphates [5]. Local tion of poultry manure are mandated because, in sources of products, such as PLA, provide a dis- some areas, the application of poultry manure tinct economic advantage in reducing feed costs. to agricultural lands has resulted in soil phos- An experiment was designed to evaluate PLA phorus levels that exceed the amount that can as a substitute for dicalcium phosphate (DP) in be removed in the harvested crop [1]. Concerns commercial-type diets for the broiler chicken. about P runoff into surface waters where manure is applied at excessive rates or not according to MATERIALS AND METHODS established requirements for crop production also exist [2]. General Considerations Potential exists for recycling P from poultry manures, especially where poultry litter can be A total of 1,600 broiler chicks were obtained obtained as a feedstock for energy generation from a commercial hatchery (Cobb × Ross) and [3]. After combustion, the remaining poultry lit- 25 birds were randomly assigned to each of 64 ter ash (PLA) has greater value for use as a P floor pens, each being 1.98 × 2.29 m. The cur-

1 Corresponding author: [email protected] 102 JAPR: Research Report

Table 1. Composition of poultry litter ash Table 2. Assignment of dietary treatments

Nutrient Poultry litter ash (%) (%, unless Analyzed Computer otherwise noted) value1 value2 Treatment1 Starter Grower Finisher

Calcium 16.68 16.70 0/0/0 0 0 0 Phosphorus 10.08 10.00 25/25/25 25 25 25 Copper 0.165 1,500.00 ppm 50/50/50 50 50 50 Iron 0.593 5,000.00 ppm 75/75/75 75 75 75 Magnesium 2.650 2.70 100/100/100 100 100 100 Manganese 0.209 1,900.00 ppm 25/100/100 25 100 100 Potassium 7.640 7.5 50/100/100 50 100 100 Sodium 4.340 4.2 75/100/100 75 100 100 Chloride 0.990 1.0 1Percentage of poultry litter ash used as a replacement for Zinc 0.136 1,300.00 ppm dicalcium phosphate in the starter, grower, and finisher diets. Selenium (ppm) 2.40 2.40 Downloaded from 1Values obtained from analysis of sample submitted to Eu- rofins [8]. 2Values assigned to poultry litter ash when used as an ingredient in the computer feed formulation matrix. http://japr.oxfordjournals.org/ Table 3. Composition of starter diets

Level of poultry litter ash (%) Item (%, unless otherwise noted) 0 25 50 75 100

Ingredient Ground yellow corn 55.75 55.44 55.14 54.79 54.47 Soybean meal (48% CP) 35.11 35.12 35.14 35.17 35.19

Poultry oil 4.53 4.65 4.77 4.90 5.02 by guest on February 24, 2014 Dicalcium phosphate1 1.73 1.30 0.86 0.43 0.00 Limestone (38% Ca) 1.23 1.12 1.01 0.90 0.79 Poultry litter ash2 0.00 0.80 1.60 2.41 3.22 Salt 0.45 0.37 0.28 0.20 0.11 dl-Methionine 0.27 0.27 0.27 0.27 0.27 l-Lysine 0.10 0.10 0.10 0.10 0.10 Vitamin premix3 0.50 0.50 0.50 0.50 0.50 Trace mineral premix4 0.25 0.25 0.25 0.25 0.25 Coban-605 0.08 0.08 0.08 0.08 0.08 Total 100.00 100.00 100.00 100.00 100.00 Calculated composition CP 21.50 21.50 21.50 21.50 21.50 ME (kcal/kg) 3,142.00 3,142.00 3,142.00 3,142.00 3,142.00 Ca 0.93 0.93 0.93 0.93 0.93 Nonphytate phosphorus 0.45 0.45 0.45 0.45 0.45 Met 0.62 0.62 0.62 0.62 0.62 Met + Cys 0.95 0.95 0.95 0.95 0.95 Lys 1.27 1.27 1.27 1.27 1.27 1Contains 18.5% P and 24.1% Ca. 2Poultry litter ash was added to the diet at the expense of dicalcium phosphate. 3Supplied the following per kilogram of complete feed: vitamin A, 8,000 IU (vitamin A acetate); vitamin D, 2,000 IU (cho- lecalciferol); vitamin E, 8 IU (dl-α tocopheryl acetate); menadione, 2 mg (menadione sodium bisulfite complex); riboflavin, 5.5 mg (riboflavin); pantothenic acid, 13 mg (d-calcium pantothenate); niacin, 36 mg (niacinamide); choline, 500 mg (choline chloride); vitamin B12, 0.02 mg (cyanocobalamin); folacin, 5 mg (folic acid); thiamine, 1 mg (thiamine mononitrate); pyridoxine, 2.2 mg (pyridoxine hydrochloride); biotin, 0.05 mg (d-biotin); ethoxyquin, 125 mg. 4Supplied the following per kilogram of complete feed: manganese, 125 mg (manganous oxide); iodine, 1 mg (ethylene di- amine dihydriodide); iron, 55 mg (iron carbonate); copper, 6 mg (copper oxide); zinc, 55 mg (zinc oxide), selenium, 0.3 mg (sodium selenite). 5Monensin sodium [12]. Blake and Hess: ASH IN BROILER DIETS 103

Table 4. Composition of grower diets

Level of poultry litter ash (%) Item (%, unless otherwise noted) 0 25 50 75 100

Ingredient Ground yellow corn 63.00 62.70 62.31 62.01 61.62 Soybean meal (48% CP) 29.70 29.71 29.81 29.82 29.93 Poultry oil 3.28 3.41 3.53 3.65 3.77 Dicalcium phosphate1 1.60 1.20 0.80 0.40 0.00 Limestone (38% Ca) 1.09 0.99 0.89 0.80 0.70 Poultry litter ash2 0.00 0.74 1.48 2.22 2.96 Salt 0.45 0.37 0.30 0.22 0.14 dl-Methionine 0.23 0.23 0.23 0.23 0.23 l-Lysine 0.07 0.07 0.07 0.07 0.07 Vitamin premix3 0.25 0.25 0.25 0.25 0.25 Trace mineral premix4 0.25 0.25 0.25 0.25 0.25 Downloaded from Coban-605 0.08 0.08 0.08 0.08 0.08 Total 100.00 100.00 100.00 100.00 100.00 Calculated composition CP 19.50 19.50 19.50 19.50 19.50 ME (kcal/kg) 3,153.00 3,153.00 3,153.00 3,153.00 3,153.00 Ca 0.84 0.84 0.84 0.84 0.84 http://japr.oxfordjournals.org/ Nonphytate phosphorus 0.42 0.42 0.42 0.42 0.42 Met 0.56 0.56 0.56 0.56 0.56 Met + Cys 0.86 0.86 0.86 0.86 0.86 Lys 1.10 1.10 1.10 1.10 1.10 1Contains 18.5% P and 24.1% Ca. 2Poultry litter ash was added to the diet at the expense of dicalcium phosphate. 3Supplied the following per kilogram of complete feed: vitamin A, 8,000 IU (vitamin A acetate); vitamin D, 2,000 IU (cho- lecalciferol); vitamin E, 8 IU (dl-α tocopheryl acetate); menadione, 2 mg (menadione sodium bisulfite complex); riboflavin,

5.5 mg (riboflavin); pantothenic acid, 13 mg (d-calcium pantothenate); niacin, 36 mg (niacinamide); choline, 500 mg (choline by guest on February 24, 2014 chloride); vitamin B12, 0.02 mg (cyanocobalamin); folacin, 5 mg (folic acid); thiamine, 1 mg (thiamine mononitrate); pyridoxine, 2.2 mg (pyridoxine hydrochloride); biotin, 0.05 mg (d-biotin); ethoxyquin, 125 mg. 4Supplied the following per kilogram of complete feed: manganese, 125 mg (manganous oxide); iodine, 1 mg (ethylene di- amine dihydriodide); iron, 55 mg (iron carbonate); copper, 6 mg (copper oxide); zinc, 55 mg (zinc oxide), selenium, 0.3 mg (sodium selenite). 5Monensin sodium [12]. tain-sided house had thermostatically controlled Mortalities were weighed daily and recorded heating and cross ventilation. Pens were sepa- before disposal. Feed consumption (FC) was rated by wire partitions and the floor and isles calculated by the difference in feed offered and were concrete. Each pen was equipped with an feed remaining on a pen basis at 14, 28, and 41 electric brooder and ceiling-mounted forced- d. Mortality-corrected FCR (FC:BW) was de- air furnaces were used to maintain a brooding termined as the ratio of the FC divided by the temperature of 95°F (35°C) for the first week; pen weight plus BW of mortality during the thereafter, brooding temperature was reduced 14-, 28-, and 41-d periods. Statistical analysis 5°F (2.8°C) weekly through 4 wk of age. Each was conducted using JMP software [6]. Animal pen had fresh pine shavings (5 cm) and was transport, housing, and handling procedures equipped with 1 hanging feeder (22.5-kg capac- were in accordance with guidelines of Auburn ity) and nipple water line system. Chicks were University’s Institutional Animal Care and Use vaccinated for Marek’s disease at the hatchery. Committee. Feed and water were provided ad libitum, accompanied by an L:D cycle of 23:1 for the 41-d Experimental Diets experimental period. Body weight was recorded initially and at The PLA was obtained from a pilot plant test 14, 28, and 41 d of age to determine BW gain. of an Energy Products of Idaho [7] fluidized 104 JAPR: Research Report

Table 5. Composition of finisher diets

Level of poultry litter ash (%) Item (%, unless otherwise noted) 0 25 50 75 100

Ingredient Ground yellow corn 72.55 72.24 71.95 71.63 71.32 Soybean meal (48% CP) 21.76 21.82 21.87 21.92 21.97 Poultry oil 1.98 2.08 2.18 2.30 2.41 Dicalcium phosphate1 1.38 1.03 0.69 0.35 0.00 Limestone (38% Ca) 1.02 0.94 0.85 0.76 0.68 Poultry litter ash2 0.00 0.64 1.28 1.92 2.56 Salt 0.45 0.39 0.32 0.26 0.20 dl-Methionine 0.24 0.24 0.24 0.24 0.24 l-Lysine 0.12 0.12 0.12 0.12 0.12 Vitamin premix3 0.25 0.25 0.25 0.25 0.25 Trace mineral premix4 0.25 0.25 0.25 0.25 0.25 Downloaded from Total 100.00 100.00 100.00 100.00 100.00 Calculated composition CP 16.50 16.50 16.50 16.50 16.50 ME (kcal/kg) 3,175.00 3,175.00 3,175.00 3,175.00 3,175.00 Ca 0.75 0.75 0.75 0.75 0.75 Nonphytate phosphorus 0.37 0.37 0.37 0.37 0.37 http://japr.oxfordjournals.org/ Met 0.54 0.54 0.54 0.54 0.54 Met + Cys 0.79 0.79 0.79 0.79 0.79 Lys 0.92 0.92 0.92 0.92 0.92 1Contains 18.5% P and 24.1% Ca. 2Poultry litter ash was added to the diet at the expense of dicalcium phosphate. 3Supplied the following per kilogram of complete feed: vitamin A, 8,000 IU (vitamin A acetate); vitamin D, 2,000 IU (cho- lecalciferol); vitamin E, 8 IU (dl-α tocopheryl acetate); menadione, 2 mg (menadione sodium bisulfite complex); riboflavin, 5.5 mg (riboflavin); pantothenic acid, 13 mg (d-calcium pantothenate); niacin, 36 mg (niacinamide); choline, 500 mg (choline chloride); vitamin B12, 0.02 mg (cyanocobalamin); folacin, 5 mg (folic acid); thiamine, 1 mg (thiamine mononitrate); pyridox- by guest on February 24, 2014 ine, 2.2 mg (pyridoxine hydrochloride); biotin, 0.05 mg (d-biotin); ethoxyquin, 125 mg. 4Supplied the following per kilogram of complete feed: manganese, 125 mg (manganous oxide); iodine, 1 mg (ethylene di- amine dihydriodide); iron, 55 mg (iron carbonate); copper, 6 mg (copper oxide); zinc, 55 mg (zinc oxide), selenium, 0.3 mg (sodium selenite). bed system designed to combust poultry litter finisher (ca. 6.7 lb/bird) on a pen basis during with the intent to produce process steam. Initial the 41-d experimental period (Tables 3, 4 and 5). analyses [8] were completed to determine its mineral composition and establish feed formula- Processing and Yield Determination tion values (Table 1). For diet formulation, PLA (16.70% Ca, 10.00% P) was substituted for DP Carcass yield was evaluated at 42 d of age (24.1% Ca, 18.5% P) where the mineral contri- for 10 broilers randomly selected, wing-banded, bution of Ca, P, and Na were used in the feed for- and placed back in the pens at terminal weighing mulation matrix. The 8 dietary treatments were (d 41) with their pen mates and maintained on 0, 25, 50, 75, or 100% PLA as a replacement full feed. Feed and water withdrawal was intro- for DP in the starter, grower, and finisher diets duced 8 h (2300 h) before processing. Marked or 25, 50, or 75% in the starter diet followed birds were weighed before processing following by 100% PLA in the grower and finisher diets the 8-h feed withdrawal period. Carcass and ab- (Table 2). Each diet was fed to 8 pens with 25 dominal fat weight were determined after a 2-h birds/pen. The corn-soybean meal pelleted diets ice chilling to slightly less than 4.4°C (40°F). were formulated based on available informa- Following chilling, the front half and rear half tion to meet or exceed NRC recommendations were separated, weighed, and the respective [9] and birds were allocated a specified amount yield of each component calculated as a percent of starter (1.8 lb/bird), grower (3.5 lb/bird), and of preslaughter live weight. Blake and Hess: ASH IN BROILER DIETS 105 3 — 2.50 1.50 2.50 2.50 2.50 2.50 2.50 2.50 0.136 0–41 d Mortality (%) 1.906 0–41 d 2.111 28–41 d Downloaded from FCR (FC:BW) 14–28 d 0–14 d http://japr.oxfordjournals.org/ 0–41 d 28–41 d by guest on February 24, 2014 14–28 d Feed consumption (kg/bird) 0–14 d 41 d BW (kg) BW 28 d 14 d (g) 2 BW 0 d 0.130 0.258 0.076 0.894 0.100 0.771 0.770 0.315 0.092 0.281 0.054 0.794 46.73 452.2 1.476 2.557 0.516 2.041 2.295 4.852 1.141 1.993 2.123 1.898 47.05 469.6 1.517 2.567 0.511 2.081 2.294 4.886 1.088 1.988 2.185 1.896 46.88 465.6 1.500 2.603 0.514 2.085 2.334 4.933 1.104 2.016 2.116 1.895 47.60 464.0 1.495 2.562 0.521 2.059 2.240 4.820 1.123 1.997 2.099 1.881 1 Percentage of poultry litter ash used as a replacement for dicalcium phosphate in the starter, grower, and finisher diets fed as an allocation of 1.8, 3.5, and ca. 6.7 lb/bird during the 41-d ex - and finisher diets fed as an allocation grower, phosphate in the starter, for dicalcium ash used as a replacement of poultry litter Percentage are grand means involving 64 pens each with 25 chicks at placement. Values exists, as data were transformed and subject to analysis. mortality percentages were transformed to the arcsine, whereas no valid SEM Total 0/0/0 Table 6. Performance of mixed sex broilers fed graded levels poultry litter ash Table Item 1 perimental period. 2 3 25/25/25 50/50/50 75/75/75 100/100/100 47.50 461.2 1.452 2.517 0.486 2.037 2.261 4.784 1.054 2.055 2.123 1.901 25/100/100 46.18 473.5 1.452 2.537 0.521 2.065 2.213 4.799 1.100 2.109 2.040 1.892 50/100/100 46.80 466.9 1.462 2.555 0.512 2.051 2.307 4.870 1.097 2.061 75/100/100 46.75 464.9 1.477 2.596 0.519 2.087 2.394 5.000 1.117 2.062 2.139 1.926 Pooled SEM 0.35 5.47 0.017 0.044 0.009 0.026 0.074 0.056 0.019 0.044 0.034 0.014 P -value 106 JAPR: Research Report (%) Yield Yield Rear half (g) Weight Weight (%) Yield Yield Front half 0.897 0.080 0.721 0.446 Downloaded from (g) Weight Weight 5 (%) http://japr.oxfordjournals.org/ Yield Yield (g) Abdominal fat Weight Weight

6 4 (%) Yield by guest on February 24, 2014 0.862 0.265 0.487 0.263 (g) Chilled carcass 61.29 1.16 1.78 0.051 32.56 0.419 33.55 0.978 Weight Weight

3 (%) 1.16 0.251 71.39 1,938 73.20 48.00 1.81 1,072 40.49 818 30.90 74.19 2,045 76.00 48.50 1.81 1,109 41.25 887 32.94 70.07 1,945 71.92 50.00 1.86 1,072 39.60 823 30.46 71.36 1,944 73.23 49.63 1.87 1,048 39.45 847 31.90 72.85 1,983 74.77 51.13 1.93 1,075 40.47 857 32.37 71.69 1,993 73.66 53.25 1.97 1,100 40.62 840 31.07 70.45 1,912 72.38 51.13 1.94 1,057 39.99 804 30.46 70.83 1,955 72.75 51.63 1.92 1,087 40.43 817 30.40 Carcass yield (g) 2 0.993 66.74 2,648 2,686 2,701 2,649 2,654 2,707 2,641 2,688 Preslaughter live weight 1 Percentage of poultry litter ash used as a replacement for dicalcium phosphate in the starter, grower, and finisher diets fed as an allocation of 1.8, 3.5, and ca. 6.7 lb/bird during the 41-d ex - and finisher diets fed as an allocation grower, phosphate in the starter, for dicalcium ash used as a replacement of poultry litter Percentage carcasses. All values represent least squares means of 8 pens, each providing data from 10 SEM values were estimates derived from actual percentages. Statistical analysis employed transformed values (arcsine), whereas the respective the from removed fat Depot bird. live full-fed the to relative and basis absolute an on expressed fat abdominal of removal and chilling slush ice of h 2 after giblets and neck without Carcass Abdominal fat expressed on an absolute basis and relative to the chilled carcass. As a percent of preslaughter live weight. Table 7. Processing performance of broilers at 42 d age fed graded levels poultry litter ash Table Start/Grow/Finish 0/0/0 1 2 3 4 5 6 perimental period. abdominal cavity of carcasses without neck and giblets after 2 h slush ice chilling expressed on an absolute basis relative to the full-fed live weight. 25/25/25 50/50/50 75/75/75 100/100/100 25/100/100 50/100/100 75/100/100 Pooled SEM P -value Blake and Hess: ASH IN BROILER DIETS 107

RESULTS AND DISCUSSION REFERENCES AND NOTES

This experiment was designed to evaluate the 1. Codling, E. E., R. L. Chaney, and J. Sherwell. 2002. use of PLA as a substitute for DP under com- Poultry litter ash as a potential phosphorus source for agricultural crops. J. Environ. Qual. 31:954–961. mercial-type conditions. No significant effects 2. Sharpley, A. N., S. H. Chapra, R. Wedepohl, J. T. (P > 0.05) were observed on BW, FC, FCR, or Sims, T. C. Daniel, and K. R. Reddy. 1994. Managing ag- mortality when broilers were fed graded levels ricultural phosphorus for protection of surface waters: Issue of PLA to 100% replacement for DP, as repre- and options. J. Environ. Qual. 23:437–451. 3. Power Plant Research Program. 1998. Engineering sented by the 100/100/100 treatment (Table 6). and economic feasibility of using poultry litter as a fuel to Also, no significant differences (P > 0.05) were generate electric power at Maryland’s Eastern Correctional observed in the processing performance of broil- Institute. PPES-96–1. Dept. of Natural Resources, Annapo- lis, MD. Accessed June 6, 2013. http://esm.versar.com/pprp/ ers at 42 d of age due to the substitution of DP eci/poultry.htm. with PLA (Table 7). 4. USDA. 2009. Manure use for Fertilizer and for En- The use of PLA as a replacement for DP has ergy: Report to Congress. USDA, Economic Research Ser- been met with limited investigation in the past vice, Washington, DC. Accessed June 6, 2013. http://www. Downloaded from ers.usda.gov/media/156155/ap037_1_.pdf. with turkeys [10] and broilers [11]. Both inves- 5. Auman, S. 2010. Phosphorus Availability—When tigators concluded that PLA obtained from the will we run out of phosphorus? In Proc. 2010 Natl. Poult. combustion of turkey or broiler litter could be Anim. Waste Manag. Sym. J. B. Hess, J. P. Blake, and K. S. Macklin, ed. Natl. Poult. Waste Manag. Sym. Comm., Au- used as a P supplement for poultry. In the current burn, AL. http://japr.oxfordjournals.org/ study, the complete substitution of DP with PLA 6. Data were subjected to or one-way analysis of vari- failed to compromise growth and processing ance and, where significant, means were separated by performance in market age broilers. Therefore, Tukey’s honestly significant difference test at the probability level of 0.05 using JMP software (JMP The Statistical the PLA evaluated in this and previous experi- Discovery Software, 2005, SAS Institute Inc., Cary, NC). ments confirms that the PLA produced via the All percentage data were subjected to arcsine square root combustion of poultry litter can be used as a transformation prior to analysis; however, actual data are reported. phosphorus source in poultry diets. 7. Energy Products of Idaho, Coeur d’Alene, ID. 8. Eurofins, Memphis, TN. by guest on February 24, 2014 CONCLUSIONS AND APPLICATIONS 9. NRC. 1994. Nutrients Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. 1. The use of PLA as a replacement for DP 10. Apke, M. P., P. E. Waibel, and R. V. Morey. 1984. in commercial-type broiler diets had no Bioavailability of phosphorus in poultry litter biomass ash residues for turkeys. Poult. Sci. 63:2100–2102. negative effects on growth or processing 11. Muir, F., R. M. Leach Jr., and B. S. Heinrichs. 1990. characteristics of market age broilers. Bioavailability of phosphorus from broiler litter ash for 2. The PLA produced by the combustion chicks. Poult. Sci. 69:1845–1850. of poultry litter and used in this evalua- 12. Elanco Animal Health Inc., Indianapolis, IN. tion can be used as a substitute for DP in broiler diets without consequence. © 2014 Poultry Science Association, Inc. Time study examining the effect of range, cage-free, and cage environments on man-hours committed to bird care in 3 brown egg layer strains

Kenneth E. Anderson 1

Department of Poultry Science, North Carolina State University, Raleigh 27695-7608

Primary Audience: Egg Producers, Complex Managers, Flock Supervisors, Researchers Downloaded from

SUMMARY Growing consumer awareness concerning layer hen welfare has caused the egg industry to consider alternative environments for egg-laying hens. However, the time commitment for alternative care has not been researched in detail since the 1950s. In 2 experiments, we evalu- http://japr.oxfordjournals.org/ ated the man-hour commitment associated with 3 different environments, including range (R), cage-free (CF), and cage systems (C). Concurrently within the R and CF systems, 3 strains of birds, Hy-Line Silver Brown, Hy-Line Brown, and Barred Plymouth Rock, were examined to see if strain differences were associated with man-hours associated for care. All birds were

raised within the environment they were going to be laying in at 17 wk of age, the time data collection started. The system study was a 3-factor randomized design that commenced at 17 wk, when the laying phase commenced with 8 R replicates, 24 CF replicates, and 4 C replicates. The strain study consisted of R and CF systems and 3 strains of Hy-Line Brown, Hy-Line by guest on February 24, 2014 Silver Brown, and Barred Plymouth Rock. All husbandry was provided as appropriate to the production system and in accordance with the approved animal care procedures. Time was recorded for all of the procedures done within the replicates (i.e., egg collection, feeding, and so on). The data were transformed first by man-hours per bird housed and second by man-hours per bird surviving. All time data were analyzed using PROC GLM in SAS. Man-hours per hen decreased from 17 to 37 wk in all production systems. The production environment C required only 0.334 h/hen housed, which is lower as compared with either the CF at 0.486 h/hen housed or R at 1.268 h/hen housed. Strain alone did not influence man-hours; however, the strain with the poorest livability had the greatest man-hour requirement for hens surviving. This study substantiates previous findings that moving from intensive to extensive production systems will increase man-hours per hen by 3 or 4 times.

Key words: brown egg layer , heritage layer , range , cage-free, cage, labor 2014 J. Appl. Poult. Res. 23 :108–115 http://dx.doi.org/ 10.3382/japr.2013-00852

DESCRIPTION OF PROBLEM constituents forcing this growth are creating great change, and with these changes come un- Alternative production systems of cage-free certainty in relation to the future for intensive or range egg production are increasing in num- egg production. Concerns still exist regarding ber due to outside influences [1]. The outside the effect of the cage environment on laying hen

1 Corresponding author: [email protected] Anderson: CAGE ENVIRONMENTS 109 well-being, but along with this are concerns re- 2 studies were conducted concurrently looking lated to the cage-free and range systems as they at the production environments of caged, cage- relate to the time commitment to actually care free, and range environments, with the second for the birds. Labor inputs for egg production study examining the influence of breed on man- have always been of concern, and means of hours in the cage-free and free-range systems. minimizing labor has been discussed in early The birds were hatched and grown at the Pied- works [2–4]. Those authors indicated that labor mont Research Station [6] in accordance with varied across season and unit size, but indicated the methods used for the North Carolina Layer that man-hours per bird ranged from about 2.2 to Performance and Management Test [7] and the 2.6 h/hen. To examine labor with modern com- North Carolina State University Institutional mercial layers, a preliminary study by Anderson Animal Care and Use Committee approval in- [5] tracked the man-hours to care for free-range cluding that for range production. The hens were hens and hens in cage production facilities. In reared in a manner that facilitated their learning this preliminary study it was found that within the function of the many aspects of the laying Downloaded from the North Carolina Layer Performance and environment for cage-free and free-range sys- Management Test, through an 85-wk production tems. Within the rearing environment it is im- cycle, hens on the range required 2.663 man- perative that pullets learn to properly use the hours/hen, whereas the caged hens only required facilities in the production environments, such 0.173 man-hours/hen. The man-hours for the as range, roosts, and nests for egg production, http://japr.oxfordjournals.org/ range hens were consistent with previous works and foraging behaviors to ensure productivity of [2–4]. Whereas large producers are dealing with the flock. All of the brown layers were reared in cage versus cage-free systems, small producers, the cage-free and free-range systems depending with flocks ranging in size from 100 to 3,000 on the production for which they were intended hens using commercial and heritage strains (i.e., free range or cage free). The chicks were of chickens, are increasing egg production in hatched concurrently then identified as to the both cage-free and range settings. Still, one of production environment, range and cage free, the current issues is that our knowledge base they were destined for. The birds were brooded by guest on February 24, 2014 is small as to how these alternative production in comingled floor pens on litter at 689 cm2/bird. methods influence man-hours. Current informa- The range pullets were moved to the range after tion was collected with specific pure breeds and brooding to complete their rearing. The birds modern lines of poultry selected for very high were maintained on the same rearing dietary rates of egg production [1]. Despite the degree regimen, vaccination, and supplemental lighting of environmental protection and economic fea- program throughout the rearing program; how- sibility offered by traditional poultry houses, the ever, at 12 wk, the range birds went on natural practice of housing laying hens in cages has the light supplemented with controlled day lengths. public perception of providing poor welfare for The following procedures were conducted in ac- laying hens with little or no perspective of the cordance with the research protocol. The typi- effect on hens in cage-free or range settings. As cal procedures included in the time study were a result of increasing public interest in laying production data recorded daily: eggs, mortal- hen welfare, alternative management systems, ity, feed distribution by weight as needed with including cage-free and free-range environ- weigh backs taken at the close of each 28-d ments, have been developed. The objective of period, egg quality data collected and recorded this study was to examine the man-hour require- once every period, manure removal as needed, ments between 3 housing systems using 2 com- maintenance, and rotating paddocks mercial strains and 1 heritage strain throughout a single production cycle. Experiment 1

MATERIALS AND METHODS The time study examining production environments used range, cage-free, and cage pro- To examine differences in man-hours for production, described in Table 1, as follows using duction systems and breed influences on labor, commercial brown egg strains. The strains were 110 JAPR: Research Report

Table 1. Replicate and hen numbers associated with experiments 1 and 2

Time study Cage sets/ Hens/ Total hens Item replicate replicate replicate housed

Experiment 1 House 4, cage 2 36 738 1,476 House 5, cage 2 36 756 1,476 House 2, cage free 20 1 216 4,320 Hut 1 and 2, free range 8 1 75 600 Experiment 2 House 2, cage free 8 1 216 1,728 Hut 1 and 2, free range 6 1 75 450 the same for the North Carolina Layer Perfor- freezing). Tube feeders were available in each mance and Management Test [8]. pen: 1 inside and a covered feeder outside pro- Downloaded from Range Layout Description. The range layout viding 5.1 cm of feeder space per hen. consisted of 2 range huts, each consisting of 4 Cage-Free Layout. The cage-free layout pens with associated paddocks. Hens were pro- consisted of 24 cage-free pens that were a slat- vided a minimum of 929 cm2/hen in the range litter floor combination. The slat-litter facility hut in pens that were all slats. Hens were pro- pens were 3.7 × 5.5 m (12 × 18 ft) with two- http://japr.oxfordjournals.org/ vided 13 cm of roosting space per hen and 1 nest thirds slats and one-third litter for a total of 20.4 per 8 hens. The range hut had a timer and light m2 (216 ft2). The hens were housed at approxi- powered via battery and solar panels and a sup- mately 929 cm2 (144 in2) per hen for a total hen plemental propane heater for winter conditions population of 216 hens/pen. Roosts were pro- to maintain an interior temperature above 7.2°C vided at 13 cm/hen and 1 nest per 8 hens, which (45°F), which is the lower level of the chickens’ provides adequate use of nests by the hens. Heat effective thermal neutral zone where body tem- for winter conditions was provided by hen body perature will be maintained via a feed intake in- heat, which maintained an interior temperature by guest on February 24, 2014 crease. The hens had free access to the outdoors above 12.7°C (55°F), which is the lower level of throughout the day and night, but appeared to re- the chickens’ thermal neutral zone where body turn to the range hut during the dark for roosting temperature was maintained without a feed in- and protection. Husbandry, lighting, and supple- take increase. mental feed were allocated on the same basis as Cage Layout. The cage layout consisted flock mates in cages to minimize the variables of 2 cage production houses, with each house between flock mates as much as possible. Range containing 2 rows of commercial layer cages density was based upon a 500 hen/acre static used in this study. In both houses, each side of equivalency of 8.04 m2/hen (86 ft2/hen). The a bank was designated as a row and each row range pens were 21.3 × 21.3 m (70 × 70 ft) and was divided into 36 eight-foot cage groups with were enclosed by a 1.8-m (6-ft) fence with the 9 groups on each of 4 levels. The replicates lower chain link section being 1.2 m (4 ft) high. were equipped with feed hoppers to supply and To facilitate range forage replenishment, each of monitor feed consumption for each individual the paddocks were divided in half with a diago- replicate and the feed was distributed by an nal fence providing 4.04 m2/hen (43 ft2/hen) and automatic feeding system. House 4 was a high rotated every 4 wk. One week before rotation rise, environmentally controlled laying facility the paddocks were mowed to an approximate with 2 rows of a quad-deck (4-tier) cage system. height of 15 cm (6 in). Hen movement was con- The replicate blocks contained cages that were trolled by an access a gate that allowed access either 61 or 81 cm wide. Two replicate banks to one-half of the paddock at any point in time. of cages were used with a total of 1,476 hens. The veranda area was a 3.04- × 4.6-m (10- × 15- House 5 was a standard height, totally enclosed, ft) shaded area of bare dirt. Each range hut had force-ventilated laying house with a scraper 8 nipple drinkers inside each pen and 8 nipple pit manure-handling system. It had 2 rows of drinkers outside (turned off in winter to prevent a quad-deck (4-tier) cage system. The replicate Anderson: CAGE ENVIRONMENTS 111 blocks contained cages that were either 61 or the same procedure for the individual replicates 81 cm wide. Two replicate banks of cages were to capture all aspects of bird care. used with a total of 1,476 hens. Heat for winter conditions was provided by hen body heat, Statistical Analysis which maintained an interior temperature above 12.7°C (55°F), which is the lower level of the The raw data was summarized by calculating chickens’ thermal neutral zone where body tem- the total time (number of caretakers × minutes) perature was maintained without a feed intake for each replicate. The data were transformed increase. in 2 ways to capture the man-hours per bird. By analyzing the time data based upon the number of hens housed and then by hens surviving, we Experiment 2 were able to capture the increase in time due to In this experiment, the evaluated man-hour mortality of the flock. For experiments 1 and 2, all time data was analyzed using PROC GLM commitment associated with 2 commercial Downloaded from strains of brown egg layers [Hy-Line Silver in SAS [9]. Means determined to be signifi- Brown (HBS) and Hy-Line Brown (HB)] and a cantly different were separated using LSMeans/ heritage strain [Barred Plymouth Rock (BPR)] PDIFF. were examined. Man-hour commitments were RESULTS AND DISCUSSION collected such that strain and production sys- http://japr.oxfordjournals.org/ tem environments differences were elucidated. Experiment 1 Environments included the range and cage-free systems. Within the range system, 6 replicates of Hen Age and Mortality on Man-Hours. 75 hens (2 reps for each strain/breed) were used; Figure 1 illustrates that care for the range hens the cage-free system consisted of 8 replicates of housed was higher (P < 0.0001) throughout the 216 hens (3 for each commercial strain and 2 study. The hens in the range paddocks required for BPR), as shown in Table 1. The production more man-hours early in the production phase cycle was monitored from 17 to 85 wk of age on when the hens were young; man-hours stabilized by guest on February 24, 2014 a 28-d period basis [8]. somewhat by 29 wk of age. This may be indica- tive of the animal caretakers becoming accus- Collection of Man-Hour Data tomed to the pen and becoming more efficient in their work patterns. The man-hours from 21 To provide producers with a more conclusive to 61 wk of age were greater (P < 0.0001) in the comparison of husbandry practices associated cage-free than in the cage system. This appears with the production environment, the man-hour to be related to the time where egg production study consisted of range, cage-free, and cage would have been greatest. The time associated row replicates as described for experiments 1 with egg collection may have been a contrib- and 2. The time spent working within each of uting factor, as workers had to collect eggs 2 the replicates was recorded by personnel to times daily in the cage system during high egg within 1 min. This was converted to man-hours production, whereas once a day the eggs rolled per hen for each of the production periods. The out into egg trays in the nest boxes. Figure 2 il- time study records commenced with the first lustrates the effect of hen mortality on the man- period and consisted of feeding, collection of hours needed in the different production envi- production records, and all associated hen care ronments. As hens age the man-hours per hen procedures were collected each period. The ani- decreases on a hen-housed basis from 0.06 to mal caretaker staff used a sign in-sign out record 0.04 h/bird, but the man-hours per hen surviving sheet for individual pens, paddocks, or rows; increased by 25%. The hours required for hen they recorded the actual time going in, complet- care were very similar to hen housed shown in ed the required tasks, and then recorded the time Figure 3, except for the range production start- out. The number of people required to complete ing at 53 wk of age, when the man-hours per hen the tasks were documented and time was then began to reflect the cumulative mortality with multiplied accordingly. Maintenance staff used significantly increased (P < 0.01) man-hours 112 JAPR: Research Report Downloaded from http://japr.oxfordjournals.org/ Figure 1. Effect of conventional cage, cage-free, and free-range production environments on man hours per hen housed for hen care as the flock ages in experiment 1. The free-range hens required significantly (P < 0.0001) more man hours throughout the study. Symbols (†P < 0.0001) indicate significant differences between cage and cage-free means. over the man-hours based on hen housed in sub- fewer hens were in the paddocks, the amount of sequent periods through the end of the study. time needed to care for the paddock remained This was due to the cumulative mortality in the the same, resulting in an increase in man-hours

later stages of the production cycle. Even though per hen increasing. by guest on February 24, 2014

Figure 2. The interaction of mortality and production environment on man hours per hen as the flock ages in experiment 1. The free-range hens required significantly (P < 0.0001) more man hours throughout the study. Symbols (†P < 0.0001) indicate significant differences between cage and cage-free means. Anderson: CAGE ENVIRONMENTS 113 Downloaded from http://japr.oxfordjournals.org/ Figure 3. Comparison of hen-housed man hours per hen as compared with surviving hens associated with cumulative mortality in the flock from 17 through 81 wk of age in experiment 1. Symbols (**P < 0.01) indicate significant differences between means at the hen ages.

Over the entire production cycle, a signifi- range hut and the protective fencing. This type cant (P < 0.0001) increase of 0.334, 0.486, and of paddock rotation, though effective, was very 1.268 h/hen was observed in man-hours per hen labor intensive. These man-hours per hen were (Table 2) between the intensive cage system, the slightly higher but seem appropriate, as this was less intensive cage-free system, and the exten- a research setting with additional labor inputs. by guest on February 24, 2014 sive range system, respectively. If mortality is This correlated well with early research from the taken into account, the rate increases to 0.351, 1940s and 1950s, which were consistent at ap- 0.520, and 1.512 h/hen. Initial range research us- proximately 2.6 h per hen for the life of the flock ing commercial brown egg strains indicated that [2, 4]. The reason for the reduction of more than man-hours per bird were 0.173 h/bird for cage 1 h/hen was the result of the paddock rotation production and 2.663 h/bird for range produc- method. In this study we developed a rotation tion [5]. In this initial time study, the hens were program where we merely had to open and close maintained on a static paddock with a single a gate to switch paddocks every 28-d period. Us- move of the range paddock halfway through the ing this method we were able to maintain good production period; as in the early work [2, 4], the forage cover and allowed for paddock cleansing paddock was changed by physically moving the by rain and sunlight, which helped minimize parasites. This study highlights why complexes gained popularity: the labor input on a per-hen Table 2. Effect of the production environment during basis continued to decline to about 0.03 h/hen in the laying period on total man-hours per hen in a 1 million hen complex [10]. experiment 1

Production Hen housed Hen surviving Experiment 2 environment (h/hen) (h/hen) Effect of Strain on Man-Hours per Hen. C C Cage 0.334 0.351 The different strains or breeds required similar Cage free 0.486B 0.520B Range 1.268A 1.512A time commitments when evaluated on a hen- SEM ±0.016 ±0.033 housed basis (Table 3). The HB, HSB, and BPR A–CValues with different superscripts within columns are sig- required similar time commitments of 0.052, nificantly different (P < 0.0001). 0.051, and 0.049 h/hen per period, respectively. 114 JAPR: Research Report

Table 3. Effect of strain of chicken and mortality on Table 4. Effect of the cage-free and range production average man-hours per hen within each period environment during the laying period on total man- hours per hen in experiment 2 Hen housed Hen surviving Strain (h/hen) (h/hen) Production Hen housed Hen surviving environment (h/hen) (h/hen) Hy-Line Brown 0.052 0.058b Hy-Line Silver Brown 0.051 0.063a Cage free 0.459B 0.484B Barred Plymouth Rock 0.049 0.057b Range 1.266A 1.530A SEM ±0.001 ±0.001 SEM ±0.014 ±0.033 a,bValues with different superscripts within columns are sig- A,BValues with different superscripts within columns are significantly different (P < 0.001). nificantly different (P < 0.0001).

However, when the hour per hen rate is exam- An interaction (P < 0.01) was observed be- ined for the hens surviving each period, HSB tween the production environment and the strain required the greatest (P < 0.001) time input over of hen used on a hen-housed basis (Figure 4). Downloaded from the HB or BPR hens. The HSB required the This appears to be related to the response of the greatest time associated with hen survival due to HSB hens to the production environments. In the higher mortality rate in the cage-free setting the cage-free environment, HB and HSB hens than the other strains [5]. had the highest man-hour needs; however, in the

Effect of Management System on Man- range environment, BPR hens had the highest (P http://japr.oxfordjournals.org/ Hours per Hen. As in experiment 1, the range < 0.01) man-hour needs. The HB was intermedi- hens required the greatest number of man-hours ate in man-hour use; this seems to indicate that per hen over the course of the production cycle the strains used in a particular production system than did the cage-free hens. In both cases, the would influence the time needed for hen care. shift from cage-free to range required approximately 3 times the man-hour input per hen (Ta- CONCLUSIONS AND APPLICATIONS ble 4). Surprisingly, the difference in egg production [5] between the HB, HSB, and BPR did 1. Moving from intensive (cage) to exten- by guest on February 24, 2014 not result in the man-hour requirement for bird sive production systems requires signifi- care. cant increases in time commitments: a

Figure 4. Interaction of production environment and strain on man hours per hen during the entire laying period on a hen-housed basis. Different letters (a–d) indicate significant differences between means (P < 0.001). HB = Hy- Line Brown; HSB = Hy-Line Silver Brown; BPR = Barred Plymouth Rock. Anderson: CAGE ENVIRONMENTS 115

45% increase in man-hours from cage to 2. Lee, C. L. 1948. Profitable Poultry Management. The cage free; a 279% increase in man-hours Beacon Milling Company Inc., Cayuga, NY. 3. Winter, A. R., and E. M. Funk. 1949. Poultry Farm from cage to range; and a 161% increase Management. Pages 540–551 in Poultry Science and Prac- in man-hours from cage free to range. tice. 3rd ed. R. W. Gregory, ed. J. B. Lippincott Company, 2. The man-hours needed for the care of New York, NY. hens in all of the production environ- 4. Juhl, M. A. 1951. Housing and Yarding Principles and Practice. Pages 253–254 in Poultry Husbandry, 3rd ed. ments decrease as the hens age. McGraw-Hill Book Company Inc., New York, NY. 3. Flock mortality will result in increased 5. Anderson, K. E. 2009b. Single Production Cycle man-hours per hen in the later periods of Report of the 37th North Carolina Layer Performance and the production cycle Management Test. Vol. 37, No.4. February 2009. 4. The strain of commercial laying hen or 6. Piedmont Research Station, Salisbury, NC. 7. Anderson, K. E. 2010. Report on Pullet Rearing the use of a heritage breed may affect the Period of the 38th North Carolina Layer Performance and man-hours per hen needed to care for the Management Test. Vol. 38, No. 2, July 2010. flock. This may be associated with the 8. Anderson, K. E. 2011. Single Production Cycle Re- Downloaded from productivity of the hens, mortality of the port of the 39th North Carolina Layer Performance and Management Test. Vol. 38, No.4. November 2011. flock, or other production influences of 9. SAS Institute Inc., Cary, NC. the strain. 10. Bell, D. D., and W. D. Weaver. 2002. Commercial Chickens Meat and Egg Production. 5th ed. Springer Sci- ence and Business Media, New York, NY.

REFERENCES AND NOTES http://japr.oxfordjournals.org/

1. Anderson, K. E. 2009a. Overview of natural and organic production: Looking back to the future. J. Appl. Poult. Res. 18:348–354. by guest on February 24, 2014 © 2014 Poultry Science Association, Inc. Salmonella Typhimurium in chicken manure reduced or eliminated by addition of LT1000

C. L. Sheffield ,1 T. L. Crippen , R. C. Beier , and J. A. Byrd

Food and Feed Safety Research Unit, Southern Plains, Agricultural Research Center, College Station, TX 77845

Primary Audience: Food Safety Researchers and Regulators, The Poultry Industry (worldwide) Downloaded from

SUMMARY Poultry are normally reared on bedding materials such as wood shavings or rice hulls. Poul-

try litter reuse for multiple flocks has become economically important in modern broiler pro- http://japr.oxfordjournals.org/ duction. However, this practice results in the litter serving as a reservoir of numerous microbial organisms, including, yeasts, molds, multiple types of viruses, and bacterial pathogens such as Salmonella, Escherichia, Campylobacter, Clostridium, Staphylococcus, and Pseudomonas. The foodborne pathogens are of particular importance for poultry producers. During the preharvest feed withdrawal period, consumption of contaminated litter and feces by the birds can lead to infection of the upper gastrointestinal tract with Salmonella, which presents substantial problems at processing. The current study was conducted to determine whether the use of a liquid

bacterial product (LBP), such as LT1000, could reduce the load of Salmonella Typhimurium by guest on February 24, 2014 in poultry manure. The LBP was added to sterile poultry manure then challenged with 108 cfu/ mL of Salmonella Typhimurium. The concentration of Salmonella Typhimurium was measured over 9 d or until the Salmonella Typhimurium was no longer detected. In 91% of the trials, Sal- monella Typhimurium was completely eliminated within 9 d. This demonstrates that the LBP used in the current study has the potential to substantially improve the overall microbiological safety of used poultry litter.

Key words: Salmonella Typhimurium, poultry , manure , litter , effective microorganism 2014 J. Appl. Poult. Res. 23 :116–120 http://dx.doi.org/ 10.3382/japr.2013-00867

DESCRIPTION OF PROBLEM monella bacterium is commonly found within the gastrointestinal tract of chickens and on fin- Salmonella is one of the most frequently iso- ished retail poultry products [6–8]. lated foodborne pathogens associated with hu- Additionally, animal manure has been effec- man illness and has been estimated to cause over tively used as fertilizer for centuries, and poultry a million illnesses each year in the United States waste is the most desirable of the organic fertiliz- [1], costing over $14 billion [2]. Approximately ers because of its high nitrogen content [9]. How- 95% of human cases of salmonellosis are food- ever, it is also source of some major human patho- borne in origin [3] and frequently linked to the gens, such as Salmonella, Staphylococcus, and consumption of poultry products [4, 5]. The Sal- Campylobacter, all of which have the potential

1 Corresponding author: [email protected] Sheffield et al.: LT1000 REDUCES SALMONELLA 117 to cause food safety issues [10]. Chinivasagam et cooler litter from the outside of the pile to the al. [11] detected Salmonella in 83% of farms that higher temperatures generated internally for ef- reuse litter and 68% of farms that dispose of litter fective composting. Zakia et al. [19] found that after utilization by a single flock of broilers. composting reduced the Salmonella spp. count Due to rising costs and the difficulty of in poultry litter by 70.59%; however, to achieve procuring bedding material, especially wood this result, the compost required daily turning shavings, it has become a common practice for and was composted for a total of 35 d. Despite broiler producers to grow-out multiple flocks of its increased utilization, the process of in-house broilers on the same litter. Using the same litter windrowing is still far from an ideal alternative for multiple grow-outs can cause many prob- for poultry producers. lems for poultry producers, including disease A liquid bacterial product (LBP; LT1000) outbreaks, higher litter moisture, and increased composed of 3 groups of microbes—yeast (Sac- NH3 production. One tactic for dealing with charomyces cerevisae), photosynthetic bacteria these issues is to leave the poultry house free of (Rhodopseudomonas palustris), and lactic acid Downloaded from birds for 2 or more months, as this will allow bacteria (Lactobacillus casei)—is purported to for the reduction of bacteria due to desiccation work synergistically to modify the surrounding within the litter [12]. However, leaving poultry microbial environment, encouraging the break- houses empty for an extended time is not a re- down of ammonia and enhancing the efficacy alistic option due to the economic losses for the of composting [20]. A slightly different formu- http://japr.oxfordjournals.org/ producer. Another tactic is for poultry produc- lation of this material, EM•1, has been shown ers is to use litter amendments, such as Poultry to be safe for consumption and to enhance the Litter Treatment [13], which has been shown to immune response in chickens [21]. The object of reduce pH, NH3, and bacterial load [14]. the current study was to evaluate the efficacy of Approximately 44 million tons of poultry this LBP to reduce the concentration of Salmo- manure was produced in the United States in nella Typhimurium in poultry manure. 2008 [14]; in addition, the US poultry industry must meet stringent new performance standards MATERIALS AND METHODS by guest on February 24, 2014 proposed by the USDA-Food Safety Inspection Service aimed at reducing Salmonella in poultry All procedures in this study were approved [15]. Preharvest Salmonella-reduction strate- by the USDA-Agricultural Research Service- gies, such as prebiotics, probiotics, competitive Southern Plains Agricultural Research Center exclusion, and bacteriophage treatment, have Institutional Animal Care and Use Committee all been attempted with varying degrees of suc- (IACUC protocol # 09–12). The poultry ma- cess [16, 17]. In-house windrowing and partial nure used in this study was collected from ma- house cleanout are 2 approaches designed to aid ture Single Comb White Leghorn hens obtained in the reuse of litter for an extended period of from the Texas A&M Poultry Research facility. time [18]. On-farm composting and in-house They were housed individually in commercial windrowing has been underutilized in the past; layer cages and provided free access to water but, with the lack of viable alternatives to ac- and balanced, unmedicated corn-soybean-based commodate the increased practice of reutiliza- mash layer diet that met or exceeded the NRC tion of litter for several flock rotations, in-house recommendations for nutrients [22]. The ma- windrowing is becoming the method of choice nure was collected and stored in sealed contain- for making organic wastes safe before applica- ers at 4°C. All manure collected over a period tion to land. In-house windrowing is a compost- of 3 wk was combined, aliquoted into 500-mL ing technique that uses grade blades on tractors, polypropylene containers, autoclaved at 121°C skid-steer loaders, or specially designed aera- for 20 min, and stored at 4°C until use. Manure tion equipment to pile litter into one or multiple was used in these tests to ensure a very consis- conical piles (windrows) that extend the length tent test matrix. The LBP (LT1000) material was of a poultry house and incubate for a period of provided by TeraGanix Inc. [23] and maintained 10 d or more. The technique requires the piles at room temperature per the manufacturer’s di- be turned during the incubation period to rotate rections. The colony-forming units per milliliter 118 JAPR: Research Report

Table 1. Alteration in Salmonella Typhimurium concentration over time

Days postinoculation Trial Positive (cfu/mL) control 5 7 9

1 8.701 5.18 0.00 0.00 2 8.70 8.16 6.64 5.00 3 8.60 7.10 6.50 0.00 4 8.62 7.43 6.50 0.00 Mean ± SD 8.66 ± 0.05a 6.97 ± 1.27b 4.91 ± 3.27b 1.25 ± 2.50b a,bValues with different superscripts differ significantly as analyzed by ANOVA and Bonferroni’s test (P < 0.05). 1 Log10 transformed mean (cfu/mL) of 3 replicates, except trial 1, which had only 2 replicates. of the indicator bacteria, L. casei, within the LBP Data were analyzed using commercially was determined for each sample experiment by available statistical software [25]. Descriptive Downloaded from spread-plating a serial dilution of the stock ma- statistics were generated using the mean and terial onto de Man, Rogosa, Sharpe agar [24] standard deviation and presented in table for- and maintained in a high-CO2 environment at mats. Comparison of treatment effect was ana- 37°C for 48 h before counting colonies. The lyzed by ANOVA followed by a Bonferroni’s

Salmonella Typhimurium was obtained from the Multiple Comparison Test. http://japr.oxfordjournals.org/ USDA-Agricultural Research Service-Southern Plains Agricultural Research Center microbial RESULTS AND DISCUSSION collection after confirmation by agglutination testing and 16s rRNA sequencing. We report here on the efficacy of a LBP The Salmonella Typhimurium was cultured (LT1000) to reduce Salmonella Typhimurium on tryptic soy agar at 37°C for 24 h; harvested levels within poultry manure. Salmonella Ty- and resuspended for use in experimentation in phimurium was eliminated from 91% of the

PBS to an optical density of ~0.7 at 620 nm. The manure samples within 9 d after the addition of by guest on February 24, 2014 final inoculum concentration was determined by LBP. In trial 1, the treatment marker bacteria (L. serial dilution onto tryptic soy agar plates. An casei) could not be detected in 1 of the samples aliquot of 42 µL of LBP (1 gal/1,000 ft2) mixed at d 9; therefore, this sample was not included with 1 mL of tryptic soy broth (TSB) was added in the final data analysis. Both of the remaining to 10 g of autoclaved poultry manure in each of treated samples in trial 1 demonstrated elimina- 3 sterile 300-mL plastic tubs (treated). A tub of tion of Salmonella Typhimurium at d 5 and 7, poultry manure without LBP served as a positive respectively. The treated samples in trials 2 to control for Salmonella Typhimurium growth 4 showed between 1- to 3-log reductions by d 5 (control). The 4 samples tubs were placed in an and 7, respectively. By d 9, 10 of the 11 samples incubator at 37°C with normal atmospheric air. had eliminated Salmonella Typhimurium; the Every day for the duration of the experiment, 1 remaining sample did, however, show a 3-log mL of TSB only was added to the manure and reduction in Salmonella Typhimurium. gently mixed to maintain moisture levels within The concentration of Salmonella Typhimuri- the manure similar to those found in commercial um significantly (P < 0.05) decreased over time poultry facilities (i.e., at or below 30%). On d 3, (Table 1) in the samples where LBP was present. 100 µL of Salmonella Typhimurium inoculum In 3 of the 4 trials, no Salmonella Typhimurium with a mean concentration of 4.42 × 108 cfu/mL could be detected in the manure by either d 7 (±0.52 × 108) diluted in 1 mL of TSB was added or 9 in the study. In trial 2, however, the load to each of the 4 tubs. The manure was sampled of Salmonella Typhimurium in the tub was only for Salmonella Typhimurium and L. casei ev- reduced, not eliminated. The concentration level ery other day for 9 d or until no Salmonella Ty- of the marker bacteria (L. casei) was also dy- phimurium was detected, following the culture namic over the course of the trials. The L. casei methods described above. The experiment was levels consistently declined over 9 d in 3 of the replicated 4 times. 4 trials (Table 2). However, in trial 3, the L. ca- Sheffield et al.: LT1000 REDUCES SALMONELLA 119

Table 2. Alteration in Lactobacillus casei concentration over time

Days postinoculation Trial Positive (cfu/mL) control 5 7 9

1 8.001 7.00 6.00 5.85 2 8.00 7.39 7.22 6.85 3 7.78 7.00 8.12 6.00 4 7.78 7.15 7.15 6.70 Mean ± SD 7.89 ± 0.13a 7.14 ± 0.18a 7.12 ± 0.87a 6.35 ± 0.50b a,bValues with different superscripts differ significantly as analyzed by ANOVA and Bonferroni’s test (P < 0.05). 1 Log10 transformed mean (cfu/mL) of 3 replicates, except trial 1, which had only 2 replicates. sei levels fluctuated over the course of the trial. was a decrease in its concentration over that

The cause of the difference in growth patterns time. Future studies should evaluate the longev- Downloaded from is unclear. Statistical analyses indicated that no ity of LBP constituents and effectiveness in the significant differences (P < 0.05) could be found litter. Based on a comparison of the efficacy to between the mean L. casei levels in the control control of Salmonella Typhimurium in poultry and treated samples until d 9, where a reduction manure or litter of LBP to the other approaches in L. casei concentration was detected in all 4 from the literature, LBP offers promise to pro- http://japr.oxfordjournals.org/ trials. vide an effective, easy, and safe means of con- Other approaches to reducing Salmonella Ty- trolling Salmonella Typhimurium in the boiler phimurium levels within poultry manure or lit- production arena. ter have had mixed results. Williams et al. [26] reported that the addition of sodium bisulfate CONCLUSIONS AND APPLICATIONS actually led to an increase in survivability of Salmonella Typhimurium. Larrison et al. [27] examined 2 litter treatments, one with an acidi- 1. An LBP administered at a level of 1 by guest on February 24, 2014 2 fier and one without, and reported that neither gal/1,000 ft significantly (P < 0.05) re- treatment was effective in reducing Salmonella duced Salmonella Typhimurium in poul- colonization. Stringfellow et al. [28] found that try manure over 9 d in a laboratory study. quick lime and steam pasteurization were effec- This material is easy to incorporate into tive at controlling Salmonella Typhimurium in litter, safe for poultry and humans, and poultry litter; however, steam pasteurization is requires no specialized equipment. time consuming and requires specialized equip- 2. Further research should to be conducted ment. Furthermore, to enhance the performance on the usefulness, efficacy, and the per- of quick lime, water must be added to the lit- sistence of this LBP under commercial ter. This increased moisture can lead to excess broiler production conditions. production of ammonia and other associated problems. Additionally, studies by Bennett et al. [29, 30] with day-of-hatch chicks showed REFERENCES AND NOTES that lime levels in excess of 5% (wt/vol) caused 1. Scallan, E., R. M. Hoekstra, F. J. Angulo, R. V. Tauxe, mild but obvious ocular and respiratory irrita- M.-A. Widdowson, S. L. Roy, J. L. Jones, and P. M. Griffin. tion. Vicente et al. [31] found that Poultry Guard 2011. Foodborne illness acquired in the United States—Ma- litter amendment, a litter acidifier, significantly jor pathogens. Emerg. Infect. Dis. 17:7–15. reduced Salmonella enteritidis levels in broiler 2. Scharff, R. L., J. Mcdowell, and L. Medeiros. 2009. Economic cost of foodborne illness in Ohio. J. Food Prot. chicks at 11 d post-treatment; however, this ap- 72:128–136. parent reduction did not hold up over time, as 3. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. no significant difference was noted between the S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. treated and control chicks at 21 d post-treatment. Infect. Dis. 5:607–625. Our study did not measure the persistence of the 4. Kimura, A. C., V. Reddy, R. Marcus, P. R. Cieslak, L. casei present in the manure past 9 d, but there J. C. Mohle-Boetani, H. D. Kassenborg, S. D. Segler, F. P. 120 JAPR: Research Report

Hardnett, T. Barrett, and D. L. Swerdlow. 2004. Chicken rent and developing strategies to reduce contamination at consumption is a newly identified risk factor for sporadic farm level. J. Food Prot. 73:774–785. Salmonella enterica serotype Enteritidis infections in the 18. Tabler, T., and J. Wells. 2012. Poultry litter manage- United States: A case-control study in FoodNet sites. Clin. ment. The Poultry Site. Accessed March 2013. http://www. Infect. Dis. 38:S244–S252. thepoultrysite.com/articles/2700/poultry-litter-managment. 5. Guo, C., R. M. Hoekstra, C. M. Schroeder, S. M. 19. Zakia, A. M. Ahmed, Z. M Sedik, M. D. Alharery, M. Pires, K. L. Ong, E. Hartnett, A. Naugle, J. Harman, P. Ben- A. Khalaf, S. A. Nasr, and H. A. Abdelrahman. 2012. Micro- nett, P. Cieslak, E. Scallan, B. Rose, K. G. Holt, B. Kissler, bial ecology of composting dead poultry and their wastes. E. Mbandi, R. Roodsari, F. J. Angulo, and D. Cole. 2011. Glob. Vet. 9:683–690. Application of Bayesian techniques to model the burden of 20. EM Research Organization. 2013. Livestock. Ac- human salmonellosis attributable to US food commodities cessed March 2013. http://www.emrojapan.com/applica- at the point of processing: Adaptation of a Danish model. tion/agriculture/livestock.html. Foodborne Pathog. Dis. 8:509–516. 21. Esatu, W., G. Terefe, and T. Dessie. 2012. Immuno- 6. Bailey, J. S., and D. E. Cosby. 2005. Salmonella prev- modulatory effect of effective microorganisms (EM®) in alence in free-range and certified organic chickens. J. Food chickens. Research Journal of Immunology 5:17–23. Prot. 68:2451–2453. 22. NRC. 1984. Nutritional Requirements of Poultry. 8th Braden, C. R. 2006. Salmonella enterica serotype En 7. - rev. ed. Natl. Acad. Press, Washington, DC.

teritidis and eggs: A national epidemic in the United States. Downloaded from Clin. Infect. Dis. 43:512–517. 23. TeraGanix Inc., Alto, TX. 8. Zhao, C., B. Ge, J. DeVillena, R. Sudler, E. Yeh, S. 24. Becton, Dickinson, Co., Franklin Lakes, NJ. Zhao, D. G. White, D. Wagner, and J. Meng. 2001. Preva- 25. Prism v. 5.01, GraphPad Software Inc., La Jolla, CA. lence of Campylobacter spp., Escherichia coli, and Salmo- 26. Williams, Z. T., J. P. Blake, and K. S. Macklin. 2012. nella serovars in retail chicken, turkey, pork, and beef from The effect of sodium bisulfate on Salmonella viability in the greater Washington, DC, area. Appl. Environ. Microb. broiler litter. Poult. Sci. 91:2083–2088. http://japr.oxfordjournals.org/ 67:5431–5436. 27. Larrison, E. L., J. A. Byrd, and M. A. Davis. 2010. Ef- 9. Moore, P. A. Jr., T. C. Daniel, A. N. Sharpley, and C. fects of litter amendments on broiler growth characteristics W. Wood. 1995. Poultry manure management: Environmen- and Salmonella colonization in the crop and cecum. J. Appl. tally sound options. J. Soil Water Conserv. 50:321–327. Poult. Res. 19:132–136. 10. Terzich, M., M. J. Pope, T. E. Cherry, and J. Hol- 28. Stringfellow, K., D. Caldwell, J. Lee, A. Byr, J. Care, linger. 2000. Survey of pathogens in poultry litter in the K. Kessler, J. McReynolds, A. Bell, R. Stipanovic, and M. United States. J. Appl. Poult. Res. 9:287–291. Farnell. 2010. Pasteurization of chicken litter with steam 11. Chinivasagam, H. N., T. Tran, and P. J. Blackall. and quicklime to reduce Salmonella Typhimurium. J. Appl. 2012. Impact of the Australian litter re-use practice on Sal- Poult. Res. 19:380–386. monella in the broiler farming environment. Food Res. Int. 29. Bennett, D. D., S. E. Higgins, R. W. Moore, R. Bel- 45:891–896. tran, D. J. Caldwell, J. A. Byrd, and B. M. Hargis. 2003. by guest on February 24, 2014 12. Lovanh, N., K. L. Cook, M. J. Rothrock, D. M. Miles, Effects of lime on Salmonella enteritidis survival in vitro. J. and K. Sistani. 2007. Spatial shifts in microbial population Appl. Poult. Res. 12:65–68. structure within poultry litter associated with physicochemi- 30. Bennett, D. S., S. E. Higgins, R. Moore, J. A. Byrd, R. cal properties. Poult. Sci. 86:1840–1849. Beltran, C. Corsiglia, D. Caldwell, and B. M. Hargis. 2005. 13. Jones-Hamilton Co., Walbridge, OH. Effect of addition of hydrated lime to litter on recovery of 14. Bolan, N. S., A. A. Szogi, T. Chuasavathi, B. Se- selected bacteria and poult performance. J. Appl. Poult. Res. shadri, M. J. Rothrock, and P. Panneerselvam. 2010. Uses 14:721–727. and management of poultry litter. World’s Poult. Sci. J. 31. Vicente, J. J., S. E. Higgins, B. M. Hargis, and G. 66:673–698. Tellez. 2007. Effect of Poultry Guard litter amendment on 15. USDA-Food Safety and Inspection Service. 2011. horizontal transmission of Salmonella enteritidis in broiler New performance standards for Salmonella and Campy- chicks. Int. J. Poult. Sci. 6:314–317. lobacter in chilled carcasses at young chicken and turkey slaughter establishments. FSIS Notice. 31–11:1–16. Acknowledgments 16. Bucher, O., A. Fazil, A. Rajić, A. Farrar, R. Wills, Mention of trade name, proprietary product, or specific and S. A. McEwen. 2012. Evaluating interventions against equipment does not constitute a guarantee or warranty by Salmonella in broiler chickens: Applying synthesis research the USDA and does not imply its approval to the exclusion in support of quantitative exposure assessment. Epidemiol. of other products that may be suitable. Infect. 140:925–945. 17. Vandeplas, S., R. Dauphin Dubois, Y. Beckers, P. Thonart, and A. Théwis. 2010. Salmonella in chicken: Cur- © 2014 Poultry Science Association, Inc. The inner perivitelline layer sperm hole assay: Use of filter paper rings for the isolation of the perivitelline layer overlying the germinal disc and new observations on its morphology

Murray R. Bakst ,*1 Janet Eastridge ,* and Irek A. Malecki †‡

* Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, USDA Agricultural Research Service, Beltsville, MD 20705; † School of Animal Biology, University of Western Australia, Crawley 6009, WA, Australia; and ‡ Department of Animal Sciences, University of Stellenbosch, Matieland 7600, South Africa Downloaded from

Primary Audience: Researchers, Breeder-Hatchery Personnel http://japr.oxfordjournals.org/

SUMMARY The inner perivitelline layer (IPL) sperm hole assay provides an objective assessment of the number of sperm penetrating the hen’s ovum at the germinal disc. That number will provide insight into numerous parameters concerning fertility, including, but not limited to, the prob-

ability of the ovum being fertilized (true fertility), the duration of fertility, an assessment of mating activity, the effect of inseminating in vitro-stored semen, and the influence of spiking a breeder flock. In the current paper, we discuss the use of a filter paper ring to isolate and process by guest on February 24, 2014 the IPL overlying the germinal disc (PL disc) in 5 species of domestic birds. The morphology of the PL disc is also described to assist those using the sperm hole assay during sperm hole counting. The filter ring approach was successfully used with chicken, turkey, duck, goose, and Japanese quail eggs; the PL discs remained intact and stretched across the filter ring and sperm holes, when present, were clearly discernible. Morphologically, the PL disc appeared to be a silhouette of the germinal disc, including the germinal vesicle observed in follicular oocytes. The fibers forming the IPL had a honeycomb-like arrangement that could be mistaken for actual sperm holes in faintly stained slides. Given its diversity of applications and relative simplicity using the filter ring, it is recommended that the sperm hole assay be part of a quality control program at the hatchery. Already used in the scientific community, this filter ring method has the additional advantage that the blastoderm may be collected before the staining process.

Key words: inner perivitelline layer , fertility assessment, poultry 2014 J. Appl. Poult. Res. 23 :121–128 http://dx.doi.org/ 10.3382/japr.2013-00873

DESCRIPTION OF PROBLEM fertility determination procedures exist [1] that may provide some insight into the possible basis The biological basis for flock fertility or of impaired fertility, the single strongest quanti- hatchability problems is often difficult to diag- tative assay is the inner perivitelline layer (IPL) nose. Whereas numerous semen evaluation and sperm hole assay [2, 3]. This assay reveals the

1 Corresponding author: [email protected] 122 JAPR: Research Report number of sperm interacting with the IPL over- of the PL preparation and reduced PL wrinkling lying the germinal disc (GD) at the time of fer- and was subsequently adapted as a quality con- tilization, thus providing insight regarding both trol measure by a commercial turkey breeder the overall semen quality at mating or artificial [21]. A third method, introduced by Malecki and insemination and the approximate duration of the Martin [20] and Malecki and Fraser [16, 17] in fertility of a given hen (sperm storage capacity) their work with emus and ducks, respectively, from the time the egg examined was laid [4–6]. involves the use of a filter ring to isolate, clean, The inner IPL is an acellular, fibrous invest- and stain the PL disc used for the sperm hole ment surrounding the ovum (yolk) at ovulation assay. The technique used by these authors was and represents a barrier sperm must hydrolyze to similar to that described by Gupta and Bakst reach the female haploid pronucleus within the [22] for the isolation of the intact blastoderm, GD. Sperm preferentially bind to the IPL over- except yolk and cells were removed from the PL lying the GD (henceforth referred to as the PL adhering to the filter ring. disc) and then release a trypsin-like enzyme (ac- Given its diversity of applications, the sperm Downloaded from rosin) that hydrolyzes the IPL fibers, thus form- hole assay has become an important technique ing a sperm hole [see 7 for details]. Bramwell et used by industry personnel, poultry scientists, al. [2] introduced a staining procedure that pro- and scientists working with nondomestic birds. duced the visualization of the sperm holes and The purpose of the current paper is to provide a enabled their subsequent quantification. Their detailed description of the use of a filter ring to http://japr.oxfordjournals.org/ original publication on the sperm hole assay process the PL disc when performing the sperm has subsequently been both modified and used hole assay. Examples of its application and ex- by numerous scientists examining the dynam- planations of our observations were based on re- ics of sperm:ovum interactions under a variety sults from chicken, turkey, Japanese quail, duck, of treatment conditions in both domestic [4–6, and goose eggs. In addition, the morphologi- 8–17] and nondomestic [18–20] birds. cal characteristics of the stained PL discs were One downside of the sperm hole assay is that described to assist those performing the sperm it is time consuming to perform. Currently, the hole assay when confronted with over- or under- by guest on February 24, 2014 most commonly used procedure for the sperm stained or dirty slide preparations. hole assay is probably that described by Bram- well and Donoghue [3]. Briefly, the PL disc is MATERIALS AND METHODS isolated, washed clean of yolk and blastoderm cells (if present), and placed on a slide for fixa- The eggs used in this study were obtained tion and staining. The placement of the excised from commercial breeders (chickens, ducks, and perivitelline layer (PL), which in the laid egg geese) raised under standard husbandry condi- consists of an inner and outer layer, without tions and a Japanese quail breeder colony locat- wrinkles and folds could be challenging and ed at the Patuxent Wildlife Research Center [23]. time consuming. An alternative, more time ef- Turkeys were artificially inseminated weekly, ficient method of preparing wrinkle-free PL whereas chickens, ducks, Japanese quail, and slide preparations was described by Spasojevic geese mated naturally. All eggs used were stored [21]. Briefly, super glue was applied to a slide to no longer than 7 d postlaying. The application outline a square (about 20 mm per side). Excess of the filter ring for the sperm hole assay was albumen was removed from the ovum around initially described by Malecki and Martin [20] the GD and the slide with the super glue was and Malecki and Fraser [16, 17]. Isolating the firmly placed over the GD region. After the glue PL using a filter ring was performed by remov- was allowed to set, the PL was cut around the ing the upper third of the egg shell while holding outside border of the super glue, gently lifted off the egg with its blunt end up and decanting the the yolk, and the slide was washed to remove thick and thin albumen out of the remaining egg the yolk and blastoderm, if present. This prepa- shell. The yolk was then carefully poured into ration was then stained as described by Bram- either a small bowl or one’s hand and rotated well and Donoghue [3]. With commercial turkey manually until the GD was on the upper surface eggs, this modification improved both the speed of the yolk. Bakst et al.: PERIVITELLINE LAYER ISOLATION 123

Before the next step (placement of the filter the PL disc adhering to the slide, and then the ring over the GD), the PL should have a flat, slides should be stored for future use. nonglossy appearance. If glossy, layers of albu- The stained PL discs were observed with men remain that should be removed either by a Nikon stereoscopic zoom microscope brushing a finger gently across the surface of (SMZ1500) with NIS Elements Software and a the yolk in the vicinity of the GD or by plac- Nikon DS-2Mv camera for capture of digital im- ing a single unfolded sheet of tissue paper over ages. Differential interference contrast micros- the GD region and slowly lifting the tissue with copy was performed with a Zeiss Axioskop [30] the adhering albumen [24]. Using forceps, the with QImaging QICam digital camera using the filter ring [25] was placed over the GD such that NIS Elements Software package [31]. the blastoderm or GD was centered in the ring. Iris scissors were used to cut the PL around the RESULTS AND DISCUSSION outside diameter of the filter ring. The edges of the filter ring and PL were grasped with forceps, The use of a filter ring to support the PL in Downloaded from lifted so that the filter ring, PL, and adherent performing the sperm hole assay was successful yolk were perpendicular to the surface of the in chicken, turkey, Japanese quail, goose, and yolk, and then quickly transferred to and totally duck eggs. Wrinkling of the PL was minimal immersed into a Petri dish containing PBS [26]. as long as the PL adhered to the filter ring, thus The Petri dish with the filter ring supporting the the need to grasp both the edge of the PL and http://japr.oxfordjournals.org/ PL was transferred to a stereomicroscope; while the filter ring with forceps during the isolation viewing at low magnification (0.75–1.5×), the and transfer steps. If the PL partially separated yolk and blastodermal cells were removed with from the filter ring during manipulation it was a hair loop [27] and by flushing a gentle stream straightened with a hair loop after placement on of PBS over the entire filter ring until all yolk the slide. If the PL separated completely from and cells were removed from the PL. Any re- the filter paper ring during manipulation, the maining yolk on the periphery of the filter ring isolated PL was retrieved, placed on a slide, and may dislodge during processing and interfere prepared as described by Bramwell and Dono- by guest on February 24, 2014 with visualization of the sperm holes. ghue [3]. The filter ring with the cleaned, nearly trans- parent PL was placed on a glass slide and fixed Appearance of the PL Overlying the GD by pipetting about 30 μL of 10% neutral buffered formalin [28] over the filter ring to cover In 70 to 80% of the PL slide preparations the PL. After 15 to 20 s, the neutral buffered from all species examined, the PL disc was formalin was poured off and about 30 to 40 μL clearly discernible visually as a lighter stained of the Schiff’s reagent [28] was added in the disc surrounded by the more densely stained PL same manner. The slide was manually rocked of the non-GD region. The PL disc was further back and forth to evenly distribute the Schiff’s characterized by a central dark spot (Figures reagent across the filter ring [29]. To avoid pos- 1A, 2A). After examining the GD in follicular sible precipitates on the PL, the Schiff’s reagent oocytes, Perry et al. [32] described a central was applied to the slide using a 5-mL syringe zone characterized by the germinal vesicle and wrapped in aluminum foil (to minimize light an outer zone consisting of the remainder of the reaching the Schiff’s reagent) fitted with a 0.45- GD. These regional differences clearly corre- µm syringe filter [29]. Once the PL developed a spond to the 2 zones observed on the PL disc in magenta coloration (~30 s), the excess Schiff’s stained PL preparations (Figures 1A, 1B), which reagent was poured off, the slide dipped in dis- can be seen in yellow yolk follicular oocytes in tilled water, and then carefully blotted, taking situ (Figure 2A). Using stereomicroscopy, the care not to touch the filter ring. The PL was al- same zones were more clearly observed; the lowed to air dry for a minimum of 1 min before PL disc possessed a lighter stained zone where microscopic examination. For storage, the prep- sperm holes, if present, were concentrated, as aration should be allowed to completely dry, the well as a more densely stained central area in filter ring should be carefully removed, leaving which sperm holes were rarely observed (Fig- 124 JAPR: Research Report Downloaded from http://japr.oxfordjournals.org/

Figure 1. The perivitelline layer (PL) overlying the germinal disc (GD; PL disc) was disc-shaped, lighter stained than the non-GD region disc, and was characterized by a densely stained central region (A; the black in the periphery is the filter-ring). Sperm holes (A, B) were concentrated in the PL disc, except in the dense center region where sperm holes were rarely observed. The lighter stained region of the PL disc could not be clearly discerned when albumen was not sufficiently removed (B) during preparation. Sperm holes, which varied in diameter, possessed a fibrillar ground substance (C). In all species examined, the PL disc had a honeycomb appearance (C, D, E), which may or may not (D) be observed in its dense central region (DC). The honeycomb appearance of the PL (E) overlying the by guest on February 24, 2014 PL disc became less apparent as it merged with the non-PL disc region (NPD). [Specimens shown in A (bar = 1,000 μm), B (bar = 1,000 μm), C (bar = 100 μm), and D (bar = 100 μm) were from chicken eggs; E (bar = 100 μm) was from a turkey egg; C, D, and E were examined using differential interference contrast.] ures 1A, 1B). The PL disc, despite being an acel- 1E, 2B, and 2C). This pattern is similar to that lular, fibrous reticulum, clearly maintains the observed in a top view of columnar epithelial impression of the follicular oocyte’s GD with an cells [33; see Figures 10 and 12], and appears intact germinal vesicle. This is not due to dif- to represent impressions of the cells compris- ferential staining with Schiff’s reagent, as the ing the granulosa cell layer that was adjacent morphology of the PL disc is unchanged when to the IPL during follicular maturation. In uni- unstained specimens were viewed by dark field formly, densely stained preparations where the microscopy [6, 18]. Stepinska and Bakst [7] pro- PL disc cannot be clearly differentiated from vided a comprehensive review of the possible the surrounding PL, sperm holes (Figure 1B) or basis of the preferential attraction to and bind- the honeycomb appearance of the PL disc may ing of sperm to the PL disc. As those authors be discernible. However, when the albumen is noted, whatever factor or factors responsible for too thick, all evidence of the PL disc may be the preferential binding of sperm to and hydro- masked. With lightly stained PL disc prepara- lysis of the PL disc originates from the GD and, tions, care must be taken to correctly differenti- in addition, is a constitutive component of the ate sperm holes from the honeycomb-like spac- PL disc. es, as the width of their diameters may overlap Another discerning characteristic of the PL (Figure 2C). disc was the honeycomb-like configuration of As suggested previously, the morphological the fibers forming the PL disc (Figures 1C, 1D, characteristics of the stained PL disc relates to Bakst et al.: PERIVITELLINE LAYER ISOLATION 125 Downloaded from http://japr.oxfordjournals.org/

Figure 2. The lighter stained perivitelline layer overlying the germinal disc (GD; PL disc) and its densely stained central region (arrow) from a Japanese quail egg are observed under low magnification (A). That the PL disc is by guest on February 24, 2014 an impression derived from the germinal disc with an intact germinal vesicle (arrow) during follicular maturation is evident in the insert (A). The insert is the germinal disc region of an intact 20.8-mm follicular oocyte (turkey). The honeycomb appearance of the Japanese quail PL disc (B) is not clearly evident in its dense center region (DC) and is further obscured by a partial layer of stained albumen. In duck preparations (C and D), PL sperm holes may vary in shape and size (C). Of the 5 sperm holes observed in the black circle (C), 4 are about the same diameter as the honeycomb compartments in the PL and 1 is considerably smaller. In the lighter stained preparations, these sperm holes may be overlooked during counting. Rarely, and only in duck preparations, relatively large areas of the PL disc appeared to be hydrolyzed by sperm (D). [Specimens shown in A (bar = 1,000 μm) and B (bar = 100 μm) were from Japanese quail eggs; C (bar = 220 μm) and D (bar = 40 μm) were from duck eggs.] its location over the GD during follicular oocyte were irregularly shaped, less regularly spaced, maturation. In the chicken, the IPL was formed and had a mitotic index greater than the granu- during the last 7 to 10 d of follicular oocyte mat- losa cells overlying the non-GD region. These uration and at ovulation had an average width of observations led them to suggest that the granu- 2.4 μm [34]. Perry et al. [35] indicated that the losa cells overlying the GD were relatively less chicken IPL overlying the non-GD region in 30- mature and represented a growth center for the mm diameter follicular oocytes averaged 2 μm lateral expansion of the granulosa layer during in width. In contrast, Perry et al. [32] also noted follicular oocyte maturation. Whether the hon- that, in 25-mm diameter follicular oocytes, the eycomb-shaped spaces in the PL overlying the IPL overlying the GD was 1.5 μm in width and GD were a result of the PL being thinner than its fibers were thinner and more numerous than the surrounding PL or represent a structural the IPL fibers over the non-GD region. This dif- adaption of the PL to facilitate granulosa cell ferential in IPL thickness may account for the proliferation and lateral expansion is not known. lighter staining of the PL disc when compared For yet unknown reasons, the densely stained with the non-GD region. Perry et al. [32] also center of the PL disc rarely contained sperm noted that the granulosa cells overlying the GD holes (Figures 1A, 2A). Its size and location 126 JAPR: Research Report indicates that the densely stained center of the holes in the chicken and turkey IPL can exceed PL disc was directly over the germinal vesicle several hundred, this was generally limited to during follicular oocyte maturation (Figure 2A). less than 20% of the PL examined [37]. How- In describing the ultrastructural changes of the ever, of the duck IPL examined (n = 232), 64% GD 6 h before ovulation, Yoshimura et al. [36] had greater than 200 sperm holes (Figure 2C), observed a fluid-filled space over the germinal the maximum number of sperm holes counted vesicle between the vitelline membrane (the true [37]. Two or 3 sperm holes were occasionally plasmalemma of the oocyte, also referred to as merged in PL discs isolated from chicken, tur- the oolemma) and the ventral face of the IPL, key, goose, and Japanese quail eggs. However, which was not observed in follicular oocytes 25 this was much more common in the duck PL h before ovulation. Given this location, the fluid disc, as large, pleomorphic areas of the hydro- observed by Yoshimura et al. [36] may have in- lyzed PL were observed (Figures 2C, 2D). The fluenced both the staining and sperm penetration central dark staining area in the duck PL disc properties of the densely stained center of the lacked sperm holes. Downloaded from PL disc. Japanese Quail. Although the yolk was Individual sperm holes in the PL disc varied considerably smaller in diameter than the other in shape (round to oval) and size within and be- species examined, the same size filter ring was tween the species examined (Figure 1C, 2C), but successfully used to isolate the PL disc (Figure tended to be larger within the PL disc compared 2A). The PL disc had the same staining char- http://japr.oxfordjournals.org/ with the sperm holes outside the PL disc. When acteristics as described above. However, unlike observed with differential interference contrast, the other species examined, the distribution of sperm holes appeared to possess a ground sub- sperm holes was not primarily limited to the stance (Figure 1C) that by transmission electron PL disc but extended to the non-PL disc region. microscopy consisted of a fibrillar matrix [7]. The honeycomb pattern of the IPL was observed In PL preparations with greater than 300 sperm throughout the PL disc (Figure 2B). holes, overlapping hydrolyzed areas formed ir- Goose. The albumen surrounding the yolk regularly shaped areas consisting of this fibrillar was similar to that of the duck but required more by guest on February 24, 2014 matrix (Figure 2C, 2D). time to remove than that of the duck, chicken, turkey, and Japanese quail. Notwithstanding removal time, the morphology of the PL disc Observations from the resembled that of the other species examined, Different Species Examined including the honeycomb pattern of the IPL. As Chicken and Turkey. The sperm hole assay observed in other species, sperm holes tended to had been used extensively in chicken and tur- be larger in the PL disc than in the non-PL disc keys eggs to evaluate sperm numbers penetrat- region and some sperm holes merged with one ing the PL in the PL disc region under various another. treatment conditions, such as reproductive efficiency [2, 4–6, 9, 21], effect of in vitro semen CONCLUSIONS AND APPLICATIONS storage on sperm:egg interaction in turkeys [8], and effect of diet on production traits and hatch- 1. Isolating and processing the PL disc re- ability [10]. Morphological differences between gion for the sperm hole assay using the the chicken and turkey PL discs were limited to filter ring technique is relatively quick the central dense region of the PL disc: in the and consistently produces preparations turkey it was more uniformly dense and circular with minimal wrinkling or folding of the than that of the chicken. PL. Duck. Using a filter ring to isolate the PL, 2. Though subtle differences exist in Malecki and Fraser [16] determined the ratio of the morphological appearances of the sperm holes in the IPL to sperm trapped in the stained PL disc within and between the outer PL in Pekin ducks, whereas Malecki and species examined, these differences Fraser [17] reported about 20 sperm hole num- should not influence the results when us- bers/mm2 of IPL. Though numbers of sperm ing the sperm hole assay unless excess Bakst et al.: PERIVITELLINE LAYER ISOLATION 127

albumen obliterates visualization of the 17β-trenbolone on male and female reproduction in Japa- sperm holes. nese quail (Coturnix japonica) . Avian Biol. Res. 5:61–68. 12. Froman, D. P., J. C. Wardell, and A. J. Feltmann. 3. Given its diversity of application and its 2006. Sperm mobility: Deduction of a model explaining relative simplicity when using the filter phenotypic variation in roosters (Gallus domesticus). Biol. ring, it is recommended that the sperm Reprod. 74:487–491. hole assay be part of both a quality con- 13. Gumułka, M., and E. Kapkowska. 2005. Age effect of broiler breeders on fertility and sperm penetration of the trol program at the hatchery and used in perivitelline layer of the ovum. Anim. Reprod. Sci. 90:135– the laboratory when addressing the ef- 148. fect of different condition or treatments 14. Santos, T. C., A. E. Murakami, J. C. Fanhani, and C. on reproductive efficiency in domestic A. L. Oliveira. 2011. Production and reproduction of egg- and meat-type quails reared in different group sizes. Rev. and nondomestic birds. Bras. Ciênc. Avíc. 13:1–12. 15. Farooq, U., I. A. Malecki, and A. Etherington. 2012. Effect of age on fertility in the Japanese quail (Coturnix japonica). Proc. Austr. Poult. Sci. Symp. 23:194–197.

REFERENCES AND NOTES 16. Malecki, I. A., and G. Fraser. 2010a. The rate of egg Downloaded from fertilization in the Pekin duck is higher in between- than 1. Bakst, M. R., and J. A. Long. 2010. Techniques for within-strain matings. Avian Biol. Res. 3:131–132. Semen Evaluation, Semen Storage, and Fertility Determina- tion. 2nd ed. Published as a CD by The Midwest Poultry 17. Malecki, I. A., and G. Fraser. 2010b. The effect of Federation, Buffalo, MN. duck body weight on sperm hole numbers in the vitelline membrane. Proc. XIII Euro. Poult. Conf. August 23–27, 2. Bramwell, R. K., H. L. Marks, and B. Howarth Jr..

2010, Tours, France. CAB Abstracts, CABI, Wallingford, http://japr.oxfordjournals.org/ 1995. Quantitative determination of spermatozoa penetra- UK. tion of the perivitelline layer of the hen’s ovum as assessed on oviposited eggs. Poult. Sci. 74:1875–1883. 18. Birkhead, T. R., B. C. Sheldon, and F. Fletcher. 1994. A comparative study of sperm-egg interactions in birds. J. 3. Bramwell, R. K., and A. M. Donoghue. 2010. Pre- Reprod. Fertil. 101:353–361. dicting fertility: Section 4. Determination of holes made by sperm in the perivitelline layer of laid eggs: The sperm pen- 19. Birkhead, T. R., J. Hall, E. Schut, and N. Hemmings. etration assay. Pages 90–94 in Techniques for Semen Evalu- 2008. Unhatched eggs: Methods for discriminating between ation, Semen Storage, and Fertility Determination. 2nd ed. infertility and early embryo mortality. Ibis 150:508–517. M. R. Bakst and J. A. Long, ed. Published as a CD by The 20. Malecki, I. A., and G. B. Martin. 2005. Reproductive Midwest Poultry Federation, Buffalo, MN. Technologies for Ratite Farming. Publication No 05/200.

4. Wishart, G. J. 1997. Quantitative aspects of Rural Industries Research and Development Corporation, by guest on February 24, 2014 sperm:egg interaction in chickens and turkeys. Anim. Re- Barton, ACT, Australia. prod. Sci. 48:81–92. 21. Spasojevic, R. 2010. Two hundred million sperm 5. Wishart, G. J., and H. J. Staines. 1999. Measuring cells per hen? No way! In 2010 Proc. Midwest Poult. Fed. sperm:egg interaction to assess breeding efficiency in chick- Turkey Breeder Workshop. Published as a CD by The Mid- ens and turkeys. Poult. Sci. 78:428–436. west Poultry Federation, Buffalo, MN. 6. Hazary, R. C., H. J. Staines, and G. J. Wishart. 2000. 22. Gupta, S. K., and M. R. Bakst. 1993. Turkey embryo Assessing the efficiency of mating in broiler breeder flocks staging from cleavage through hypoblast formation. J. Mor- by enumerating the spermatozoa which penetrate the inner phol. 217:313–325. perivitelline layer over the germinal disc. Br. Poult. Sci. 23. Patuxent Wildlife Research Center, Laurel, MD. 41:395–400. 24. If removal of the albumen proved difficult, tissue pa- 7. Stepinska, U., and M. R. Bakst. 2007. Fertilization. per was placed over the exposed GD, dampened with PBS, Pages 553–587 in Reproductive Biology and Phylogeny of and allowed to sit for 5 min to hydrate the albumen, at which Aves (Birds). B.G.M. Jamieson, ed. Science Publishers, En- point it can be easily removed (Malecki, I. 2013. University field, NH. of Western Australia, Crawley 6009 WA, Australia. Personal 8. Donoghue, A. M. 1996. The effect of 24 hour in vitro observation). storage on sperm hydrolysis through the perivitelline mem- 25. Filter-rings were cut from Whatman #1 or #5 filter brane of ovipositioned turkey eggs. Poult. Sci. 75:1035– paper (GE Healthcare Life Sciences, Piscataway, NJ). Each 1038. filter-ring had an outside diameter of approximately 15 mm 9. Fairchild, B. D., and V. L. Christensen. 2005. Influ- and an inside diameter of 6 mm, the later made with a paper ence of hen age and number of inseminated sperm on the hole puncher. number of holes hydrolyzed in the inner perivitelline layer 26. If the GD failed to adhere to the filter-ring, most like- of turkey eggs. J. Appl. Poult. Res. 14:576–581. ly there was too much albumen left associated with PL or 10. Renema, R. A. 2004. Reproductive responses to Sel- the filter-ring did not make sufficient contact with the yolk’s Plex organic selenium in male and female broiler breeders: surface. impact on production traits and hatchability. Pages 81–91 in 27. To make a hair loop, a strand of hair is looped (about Nutritional Biotechnology in the Feed and Food Industries, 3 mm in diameter) over the tip of a wooden applicator stick T. P. Lyons and K. A. Jacques, ed. Nottingham University and glued in place. Press, Nottingham, UK. 28. Electron Microscopy Sciences, Hatfield, PA. 11. Henry, P. F. P., V. G. Akuffo, Y. Chen, N. K. Karou- 29. To avoid possible precipitates on the PL, the Schiff’s na-Renier, D. T. Sprague, and M. R. Bakst. 2012. Effect of reagent was applied to the slide using a 5-mL syringe 128 JAPR: Research Report wrapped in aluminum foil (to minimize light reaching the 35. Perry, M. M., A. B. Gilbert, and A. J. Evans. 1978b. Schiff’s reagent) fitted with a 0.45-µm syringe filter (Ther- Electron microscope observations on the ovarian follicle of mo-Fisher Scientific, Pittsburg, PA). Schiff’s reagent should the domestic fowl during the rapid growth stage. J. Anat. be colorless at the time of use. If it has a magenta or purple 125:481–497. color as a result of light exposure, expect longer staining 37. Yoshimura, Y., T. Okamoto, and T. Tamura. 1993. times or replace with fresh, clear stain. Ultrastructural changes of oocyte and follicular wall during 30. Zeiss, Thornwood, NY. oocyte maturation in the Japanese quail (Coturnix coturnix 31. Nikon Instruments, Melville, NY. japonica). J. Reprod. Fertil. 97:189–196. 32. Perry, M. M., A. B. Gilbert, and A. J. Evans. 1978a. 37. Bakst, M. 2013. Beltsville Agricultural Research The structure of the germinal disc region of the hen’s ovarian Center, USDA, Beltsville, MD. Unpublished observations. follicle during the rapid growth phase. J. Anat. 127:379– 392. Acknowledgments 33. Bakst, M. R. 1993. The anatomy of reproduction in The authors thank Aviagen Inc. (Huntsville, AL) for the birds with emphasis on poultry. Pages 15–28 in Manipula- chicken and turkey eggs, Maple Leaf Farms (Leesburg, IN) tion of the Avian Genome. R. J. Etches and A. M. Verrander for the duck eggs, Metzer Farms (Gonzales, CA) for the Gibbins, ed. CRC Press, Ann Arbor, MI. goose eggs, and P. Henry (Patuxent Wildlife Research Cen- 34. Bellairs, R., M. Harkness, and R. D. Harkness. 1963. ter, Laurel, MD) for the Japanese quail eggs. The vitelline membrane of the hen’s egg: A chemical and Downloaded from electron microscopical study. J. Ultrastruct. Res. 8:339– 359. http://japr.oxfordjournals.org/ by guest on February 24, 2014 © 2014 Poultry Science Association, Inc. The effect of feeding Hydrogel-95 to emu chicks at hatch

Zachary Lowman 1 and Carmen Parkhurst

Prestage Department of Poultry Science, North Carolina State University, Raleigh 27606

Primary Audience: Researchers, Emu Producers

SUMMARY Downloaded from Very little research has been conducted on emu (Dromaius novaehollandiae) in comparison to other types of poultry. Much of the information that is available to producers is very conflicting. Feed restriction for the emu chick for the first 4 d of life is a common practice among

the emu industry; however, scientific research has not supported this practice. To the best of http://japr.oxfordjournals.org/ our knowledge, the effects of hydration supplements have not been investigated in the emu. Therefore, weights were recorded on emus fed Hydrogel-95 and were compared with emus that were not fed any supplements to determine if the supplement had any effect on the growth parameters of emu chicks. From these 2 field trials, we conclude that feeding Hydrogel-95 significantly decreases weight loss in emu chicks during the first week of life.

Key words: emu , body weight , hydration supplements, hydrogel-95

2014 J. Appl. Poult. Res. 23 :129–131 by guest on February 24, 2014 http://dx.doi.org/ 10.3382/japr.2013-00835

DESCRIPTION OF PROBLEM are 4 d old [5]. However, others recommend feeding chicks as soon as they are removed from Emu (Dromaius novaehollandiae) are a the hatcher [1]. Currently, no published studies flightless bird from the ratite family. Emu are exist with analysis of the effects of immedi- typically raised for either meat or oil [1]. Emu ate feeding compared with fasting. Prefeeding oil has been shown to possess anti-inflammato- hatching supplements, such as Oasis and similar ry, antiviral, and antibacterial, as well as burn products, have been used in both chicken and wound-healing properties [1, 2]. The literature turkey hatcheries. Many studies have reported concerning emu is very limited compared with that these products provided to chicks and poults other types of commercial poultry and even increased weight gain and other growth param- other ratite groups. Much debate and conflicting eters [6, 7]. Hydrogel-95 is a novel green hatch- data exists on management practices for emu, ing gel supplement (HGS). It is 95% water and ranging from incubation temperatures, humid- provides chicks with crucial hydration and extra ity, egg storage time and temperature, and even nutrients to allow for an optimal start to life. It what day chicks should be allowed first access has been successfully used in chick and poult to feed [3, 4]. The data pertaining to feeding boxes during shipment to farms. The objective emu chicks is quite contradictory; many sources of this field study was to determine the effect of recommend not providing chicks feed until they an HGS fed to emu posthatch.

1 Corresponding author: [email protected] 130 JAPR: Field Report

MATERIALS AND METHODS Table 1. Mean weight loss (g) of emu chicks in 7 d by treatment This trial was conducted with a local emu pro- Treatment n Mean SE ducer in North Carolina. The emu chicks were handled and reared in accordance with proceed- Control 24 −39.45a 4.5196 1 b ings described in the Guide for the Care and Use Hydration supplement 38 −27.50 3.5918 of Agricultural Animals in Research and Teach- a,bDenotes significant difference at P < 0.0427. 1 ing [8]. The eggs were obtained from several Hydrogel-95 [9]. different emu farms and 2 trials were conducted. In trial 1, we used a temperature of 97.5°F and 30% RH for 53 d during incubation. The hatchery manager changed the incubation parameters In trial 2, the HGS group performed better slightly in the incubator for the second trial due than control birds. However, unlike the first to a decision by the farm owners after reading trial, the birds on the HGS diet actually gained a literature pertaining to ratite incubation. In trial significantly different amount of weight, on av- Downloaded from 2, eggs were incubated at a temperature of 97°F erage 38.76 g, whereas control birds lost 20.19 g and 33% RH. The eggs from each trial were on average (P < 0.0001; Table 2). It is worth not- separated into 2 groups: one group was given ing that birds on HGS appeared to be more ac- an HGS [9] in the hatcher for immediate access tive and produced noticeably more manure than and the other group was not provided with HGS the control group. This improvement in growth http://japr.oxfordjournals.org/ (control). At hatch, chicks were weighed as soon is thought to be due to the increased availability as they had dried and were leg banded for iden- of nutrients and hydration that the supplement tification. Chicks were left in the hatcher for 24 provided to the newly hatched chicks. The ben- h and then transferred to brooding pens (3 × 6 eficial effects of providing extra nutrients to ft) with 10 to 12 birds per pen. The temperature chicks immediately posthatch have been recog- was kept at 29°C for all 7 d of the trial. Thirty- nized for almost 40 yr [11]. Several observations eight birds hatched from the HGS group and 24 have been made as to why these birds perform birds hatched from the control group for a total more efficiently. Birds that are not allowed ac- by guest on February 24, 2014 of 62 emu chicks for trial 1. For trial 2, 18 chicks cess to feed have been shown to have decreased hatched from the control group and 23 hatched rates of yolk utilization [12]. This lack of feed from the HGS group for a total of 41 emu. The causes slower maturation of the gastrointestinal experimental group was provided HGS in addi- tract as demonstrated by decreased height and tion to common starter feed for 7 d after place- area of villi in the small intestine [13]. Thus it ment. The control group was provided with only has been suggested that hydration supplements the starter feed. Birds were weighed at 7 d of age. may stimulate the gastrointestinal tract, or even stimulate energy metabolism and utilization RESULTS AND DISCUSSION leading to the beneficial effects that result from the use of these supplemental products [6]. Fur- The weights were analyzed using JMP Pro 10 ther research will be required to determine the [10] one-way ANOVA. An overall decrease was actual mechanism by which Hydrogel-95 facili- observed in chick BW for the 7-d period for both tates increased growth and performance. groups in trial 1. Majewska (2001) and Nelson (1992) reported similar results, in that emu have very low growth rates and a reduction in weight may commonly occur during the first week of Table 2. Mean weight loss-to-gain (g) of emu chicks in life [3, 4]. Those authors attributed this decrease 7 d by treatment to chicks mainly relying on nutrients from the yolk [3, 4]. In the current study, both the con- Treatment n Mean SE trol and experimental groups had decreases in Control 18 −20.198b 7.864 BW; however, birds fed HGS had significantly Hydration supplement1 24 38.757a 6.957 less (P < 0.0427) weight loss than birds fed the a,bDenotes significant difference at P < 0.05. starter ration (Table 1). 1Hydrogel-95 [9]. Lowman and Parkhurst: HYDROGEL-95 FOR EMU 131

CONCLUSIONS AND APPLICATIONS 6. Batal, A. B., and C. M. Parsons. 2002. Effects of fasting versus feeding oasis after hatching on nutrient utilization in chicks. Poult. Sci. 81:853–859. 1. Providing Hydrogel-95 may serve as a 7. Knight, C. D., and J. J. Dibner. 1998. Nutritional pro- beneficial practice when hatching and graming in hatchling poultry: Why a good start is important. brooding emu. Poult. Dig. Aug-Sept:20–26. 8. Federation of Animal Science Societies. 2010. Guide 2. Feeding Hydrogel-95 to emu chicks has for the Care and Use of Agricultural Animals in Research beneficial effects on growth performance and Teaching. 3rd ed. FASS, Champaign, IL. as similar products in other poultry. 9. Hydrogel-95, Clear H2O, Portland, OR. 10. SAS Institute Inc., Cary, NC. 11. Thaxton, J. P., and C. R. Parkhurst. 1976. Growth, REFERENCES AND NOTES efficiency, and livability of newly hatched broilers as influenced by hydration and intake of sucrose. Poult. Sci. 1. Beckerbauer, L. M., R. Thiel-Cooper, D. U. Ahn, J. L. 55:2275–2279. Sell, F. C. Parrish, and D. C. Beitz. 2001. Influence of two 12. Noy, Y., and D. Sklan. 1999. Different types of ear- dietary fats on the composition of emu oil and meat. Poult. ly feeding and performance in chicks and poults. J. Appl. Sci. 80:187–194. Poult. Res. 8:16–24. Downloaded from 2. O’Banion, S., and J. Griswold. 1998. Evaluation of 13. Uni, Z., S. Ganot, and D. Sklan. 1998. Posthatch de- emu oil in lubrication and treatment of healed burn wounds. velopment of mucosal function in the broiler small intestine. Proc. Am. Burn Assoc. March 18, 1998. American Burn As- Poult. Sci. 77:75–82. soc. Meeting, Chicago, IL. 3. Majewska, D. 2001. The influence of emu (Dromaius Acknowledgments Novaehollandiae) egg storage time on hatchability and chick The authors thank RDM Products (Fayetteville, NC) and http://japr.oxfordjournals.org/ survival. Electron. J. Pol. Agric. Univ. 4:123–136. the Charles Lee Guy Fund (Fayetteville, NC) for their fund- 4. Nelson, A. H. 1992. Emu Farming, Emu Today and ing and contributions to this trial. Tomorrow. Annual Edition 1991–1992. Schatz Publishing Group, Nardin, OK. 5. Jodoin, P.1995. Emu chick rearing, The Ratite Ency- clopedia, Ostrich, Emu, Rhea. Ratite Records Inc., San An- tonio. by guest on February 24, 2014 © 2014 Poultry Science Association, Inc. Evaluation of recovery of Salmonella from trachea and ceca in commercial poultry

G. Kallapura ,* A. Botero ,† S. Layton ,‡ L. R. Bielke,* J. D. Latorre ,* A. Menconi ,* X. Hernández-Velasco ,§ D. J. Bueno ,# B. M. Hargis ,* and G. Téllez* 1

* Department of Poultry Science, University of Arkansas, Fayetteville 72701; † Private Consultant, Bucaramanga, Colombia 68001000; ‡ Vetanco Argentina S.A., Vicente López, Buenos Aires, Argentina B1603CMA; § Departamento de Medicina y Zootecnia de Aves, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, México City 04510, México; and # Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Downloaded from Agropecuaria Concepción del Uruguay, Casilla de Correo 3260, Entre Ríos, Argentina

Primary Audience: Flock Supervisors, Quality Assurance and Laboratory Personnel, http://japr.oxfordjournals.org/ Researchers, Veterinarians

SUMMARY

Unpublished data from our laboratory suggest that the respiratory tract may be a viable portal of entry for Salmonella infection. Further, field reports have indicated that tracheal sampling can be a sensitive tool for monitoring for Salmonella incidence in commercial flocks. In the by guest on February 24, 2014 present study we conducted a series of field trials in North and South America to evaluate the association between cecal and tracheal recovery of Salmonella in chickens and turkeys from commercial flocks. Environmental humidity and temperature were measured to evaluate their effects on frequency of isolation from the organs. Salmonella was recovered from tracheal samples in all trials. In 3 of the 4 trials in which both trachea and ceca were sampled, the incidence of Salmonella recovery was higher in tracheal samples. Though Salmonella was not recovered from ceca in trial 2, 5% of liver and spleen samples indicated infection. Environmen- tal conditions were not associated with incidence of Salmonella recovery. These data suggest that tracheal contamination can be a good indicator of Salmonella infection under commercial poultry conditions.

Key words: Salmonella infection, Salmonella recovery , trachea, ceca, chicken 2014 J. Appl. Poult. Res. 23 :132–136 http://dx.doi.org/ 10.3382/japr.2013-00854

DESCRIPTION OF PROBLEM tracheal inoculation is indeed possible, and that low doses of Salmonella administered directly Our laboratory hypothesized that tracheal into the trachea can cause systemic infection [1, sampling may be a viable method for detecting 2]. Thus, if infection does occur through respira- Salmonella contamination in poultry. Very re- tory inoculation, Salmonella should be able to cent research from our laboratory suggested that be isolated from the trachea and may be a reli-

1 Corresponding author: [email protected] Kallapura et al.: TRACHEAL RECOVERY OF SALMONELLA 133 able organ for detection purposes. The purpose Field Trial 2 of this study was to compare the frequency of isolation of Salmonella between tracheal, cecal, A commercial poultry company from Bue- and liver and spleen samples under a variety of nos Aires, Argentina, participated in this study environmental and commercial conditions in during December 2012, and involved 4 conven- several countries in North and South America. tional commercial broiler farms housing 80,000 Environmental humidity and temperature were broiler chickens in each farm. Average maxi- measured to evaluate their effects on frequency mum and minimum temperature for the area was of isolation from the organs. recorded to be in the range of 21 to 28°C and the average air humidity was recorded to be 63% for MATERIALS AND METHODS the monitored period. Chickens were screened via meconium sampling in the hatchery for Sal- Culture Methodologies monella and were deemed negative. On d 3 and 13, all farms were treated with a proprietary wa-

Conventional methodologies were used for Downloaded from ter applied nutritional supplement with potential the isolation and enrichment of salmonellae in to reduce Salmonella, increase flock health, and the laboratories from each of these trials. Tra- increase production parameters. At 28 d of age, chea, ceca, and liver and spleen samples were 5 chicks from each farm were cultured for Sal- aseptically collected for determination of Sal- monella recovery in trachea, ceca, and liver and monella incidence. In trials 2, 3, and 5, samples http://japr.oxfordjournals.org/ spleen. were enriched with tetrathionate enrichment broth [3]. Samples were pre-enriched in peptone water [4] for 8 h and then enriched with double- Field Trial 3 strength tetrathionate enrichment broth in trials 1 and 4. Detection agars were McConkey agar A commercial broiler farm from Guana- [5] in trial 1, brilliant green agar [6] with 25 µg/ juato, Mexico, participated in this trial, which mL of novobiocin [7] in trial 2, xylose lysine de- was performed in the month of July 2012. Av- erage maximum and minimum temperatures oxycholate agar [8] with novobiocin in trials 3 by guest on February 24, 2014 and 4, or xylose lysine tergitol-4 agar [9] in trial for the area were recorded at 18 and 28°C and 5. The presence or absence of typical lactose- the average air humidity was recorded at 65%. negative colonies of Salmonella was determined One hundred 49-d-old broiler chicks from the after enrichment incubation at 37°C for all trials, farm under study were cultured for enumeration followed by selective plating on agar. In trials 1, of Salmonella incidence in ceca and trachea. 3 and 5, Salmonella serogroup was confirmed Standard drag swab technique was employed with poly-O Salmonella-speciﬁc antiserum [10]. for the house, with subsequent enrichment and selective plating as previously described. Drag Field Trial 1 swabs were assembled as previously described by Caldwell et al. [11]. Standard sterile cotton A diagnostic laboratory for a poultry compa- swabs were used for taking drag swabs of each ny in Bucaramanga, Santander, Colombia, par- fan duct in the house, with subsequent enrich- ticipated in this study. Tracheas were sampled ment and selective plating as described above. from broiler chickens of various ages over a period of 16 mo, ranging from January 2012 to April 2013, involving conventional commercial Field Trial 4 broiler farms associated with the company. A total of 1,061 samples were collected over this A commercial turkey farm from Arkansas period of 13 mo, and the previously mentioned was sampled in December 2011. Average mini- culture methodologies were employed for Sal- mum and maximum temperature for the area monella isolation. Average maximum and mini- was recorded to be in the range of −2.9 and mum temperature for the area was recorded to 4.9°C and the average air humidity was record- be in the range of 25.3 to 18.9°C and the aver- ed at 67%. One hundred 16-wk-old female tur- age air humidity was recorded to be 82% for the keys were cultured for Salmonella incidence in monitored period. trachea and ceca. 134 JAPR: Field Report

Field Trial 5 under commercial conditions. All data for this report are represented in Table 1. In trial 1, con- A commercial broiler company from Ar- ducted with a diagnostic laboratory of a poultry kansas participated in this study. The trial was company from Bucaramanga, Colombia, a total performed during the months of January and of 1,061 cases of broiler chickens were tested February 2013 and involved 6 conventional for presence of Salmonella, of which 96 (9.04%) commercial broiler farms. Average minimum enriched tracheal samples were found to be pos- and maximum temperatures for the area were itive. Serogroup determination showed that 89 recorded at −2.7 and 7.7°C and the average air (92.7%) of the samples belonged to group B, humidity was recorded at 69%. Chickens were confirmed to be Salmonella Heidelberg, and 7 screened in the hatchery for Salmonella and (7.3%) belonged to group A (data not shown). were deemed negative. At approximately 24 d of Trials 2 and 3 evaluated the presence of Salmo- age, 25 chicks from each farm were cultured for nella in environmental samples, with enumera- Salmonella recovery in trachea, ceca, and liver tion of colony-forming units per gram of litter and spleen. Downloaded from (Trial 2) and incidence of Salmonella in litter RESULTS AND DISCUSSION through drag swabs (Trial 3), and approximately 104 cfu of Salmonella was estimated to be pres- Aerosolization is a traumatic process for ent per gram of litter in trial 2, along with 7 of most microorganisms, and survival can be de- 8 (87.5%) litter drag swabs and 5 of 10 (50%) http://japr.oxfordjournals.org/ pendent on the mechanisms of aerosolization, fan duct swab samples being positive for Sal- the climate into which these organisms are monella in trail 3 (data not shown). These re- launched, the distance they are traveling, and sults correlated with the incidence of recovery time involved in the whole process. Salmonella of Salmonella from the trachea of birds at 8 of has proven to be viable in laboratory-generated 20 (40%) in trial 2 and 28 of 100 (28%) in trial aerosols for more than 2 h [12]. Likewise, it has 3. These data support previously described stud- been shown that the death rate of Salmonella ies demonstrating the presence of Salmonella in was influenced by the protective nature of the aerosols and dust inhaled by poultry in grow-out by guest on February 24, 2014 media during aerosolization, along with overall barns [11, 13, 15–19]. prevailing RH and temperature of the air [13]. Despite a low sample number in trial 2, a rel- Environmental temperature, humidity, and tem- atively high incidence of tracheal samples (8/20; peratures within litter may have a role in sup- 40%) were positive for Salmonella, which was porting the continued survival of the organism similar to levels reported in trials 3 and 4 [28/100 in dust and aerosol generated in the poultry fa- (28%) and 34/100 (34%), respectively]. Trache- cility. In fact, humidity is known to play a major al recovery was low in trial 5 (3/150; 2%) de- role in survivability of Salmonella in aerosols, spite ceca and liver and spleen incidence similar and dependence on humidity is known to be the to other trials, suggesting that even low levels of characteristic of many gram-negative organisms tracheal contamination can be an indicator of in- [14]. The average minimum and maximum tem- fection, with respect to our hypothesis; however, peratures and average humidity were recorded this might not be the sole explanation. Addition- at the locations in which the field studies herein ally, despite previous reports of an association were conducted. between high environmental temperature and In the present study, 5 field trails were con- humidity and increased Salmonella incidence ducted to determine the recovery rate of Salmo- [13, 20–22], such a correlation was not noted nella from the trachea of commercial poultry. in the present study. The highest temperatures Ceca and liver and spleen samples were also and humidity, 18.9 to 25.3°C and 82%, were re- collected for comparison to traditionally ac- corded in trial 1, which had only 9.04% tracheal cepted sampling methods of detection (except recovery of Salmonella. However trials 2, 3, and for field trial 1); temperature and humidity mea- 4 each reported similar or relatively higher lev- surements were recorded to determine the ef- els of Salmonella recovery, despite wide varia- fects these parameters may have on the ability tions in temperature and humidity. Therefore, a to detect Salmonella contamination in poultry previously reported association between high Kallapura et al.: TRACHEAL RECOVERY OF SALMONELLA 135

environmental temperature and humidity and increased Salmonella incidence was nonexis- tent, as far as the present study is concerned. In ND

Liver summary, Salmonella was recovered from tra-

and spleen cheal samples in all trials evaluated in the present study. These data support the hypothesis that tracheal samples can be an indicator of Salmo- nella contamination, and such contamination is 1 likely an indication of infection, as evidenced Ceca by positive recovery in gastrointestinal and liver and spleen sampling.

recovery, n (%) Salmonella recovery, CONCLUSIONS AND APPLICATIONS Downloaded from 1. These data offer further evidence that Trachea 3/150 (2%) 27/150 (18%) (7.3%) 11/150 8/20 (40%) 0/20 (0%) 2/20 (10%) airborne transmission may be important 34/100 (34%) 17/100 (17%) ND 28/100 (28%) 10/100 (10%) ND 96/1,061 (9.0%) ND for Salmonella transmission within and between poultry flocks.

2. The results of these studies suggest tra- http://japr.oxfordjournals.org/ cheal recovery of Salmonella is a viable 69 67 65 82 63 and sensitive site for detection of Salmo- Average Average

humidity, % humidity, nella infection. 3. Tracheal samples can be used as a complementary tissue to ceca and liver and spleen to increase Salmonella recovery 7.7 4.9 28 28 in positive poultry. Maximum

4. Tracheal Salmonella recovery was effec- by guest on February 24, 2014 tive even with the variations in temperature and humidity in the trials. 18 18.9 25.3 21 −2.7 −2.9 Average temperature, °C Average Minimum REFERENCES AND NOTES

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Liu H. A. Olanrewaju D. Skinner-Noble North Carolina State University USDA St. Gregory’s University J. Lu A. Olkowski B. A. Slominski College of Animal Science University of Saskatchewan University of Manitoba and Technology C. M. Owens University of Arkansas M. U. Sohail B. A. Ventura M. Wineland GC University Faisalabad University of Maryland North Carolina State University P. Sørensen F. M. Vieira J. W. Worley Danish Institute of Federal University of Technology - University of Georgia Agricultural Sciences Paraná (UTFPR) J. Wu R. S. Spurio S. L. Vieira University of Alberta Londrina State University UFRGS S. Yalçin C. Stark A. J. Wade Ankara University North Carolina State University EW-GROUP F. Yan H. Stilborn P. Waldroup Novus International Stilborn Consulting University of Arkansas X. Yang H. Su K. G. Wamsley College of Animal Science Texas A&M University Mississippi State University and Technology, J. B. Sutherland J. Wang Northwest A&F University National Center for Toxicol. Res. Dankook University S. Yasar B. Svihus N. E. Ward Suleyman Demirel University Norwegian University of Life Sciences DSM Nutritional Products Ltd. M. Yegani N. Tablante S. Watkins University of Alberta University of Maryland University of Arkansas G. R. Zaboli G. T. Tabler G. Weber University of Zabol Mississippi State University DSM Nutritional Products Ltd. W. Zhai A. M. Thabet A. B. Webster Mississippi State University University of Science and Technology University of Georgia F. Zhao A. J. Thachil M. Wick Carver Research Foundation University of Minnesota The Ohio State University H. Zhuang P. B. Tillman R. Wideman USDA, ARS Ajinomoto Heartland University of Arkansas N. G. Zimmermann J. R. Timmons H. R. Wilson University of Maryland-College Park University of Maryland University of Florida LESREC G. Van Wicklen Private Consultant ABBREVIATIONS

The following abbreviations may be used without definition in Journal of Applied Poultry Research. Plurals do not require “s”. Chemical symbols and three-letter abbreviations for amino acids do not need definition. Other abbreviations should be defined at first use in the summary and main text, as well as in each table or figure in which they appear. Updated December 2011 ADF acid detergent fiber LSD least significant differences ADFI average daily feed intake m meter ADG average daily gain µ micro AME apparent metabolizable energy M molar AMEn nitrogen-corrected apparent metabo- ME metabolizable energy lizable energy MEn nitrogen-corrected metabolizable en- ANOVA analysis of variance ergy AOAC Association of Official Analytical MHC major histocompatibility complex Chemists mRNA messenger ribonucleic acid BSA bovine serum albumin min minute BW body weight mo month °C Celsius MS mean squares cDNA complementary DNA n number of observations CF crude fiber N normal cfu colony-forming units NAD nicotinamide adenine dinucleotide CI confidence interval NADH reduced form of NAD CP crude protein NDF neutral detergent fiber cpm counts per minute NRC National Research Council CV coefficient of variation NS not significant d day PBS phosphate-buffered saline df degrees of freedom PCR polymerase chain reaction DM dry matter ppm parts per million DNA deoxyribonucleic acid r correlation coefficient EDTA ethylenediaminetetraacetate r2 coefficient of determination, simple EE ether extract R2 coefficient of determination, multiple ELISA enzyme-linked immunosorbent assay RH relative humidity °F Fahrenheit RIA radioimmunoassay FCR feed conversion ratio RNA ribonucleic acid ft foot rpm revolutions per minute FE feed efficiency s second g gram SAS Statistical Analysis System gal gallon s.c. subcutaneous G:F gain-to-feed ratio SD standard deviation GLM general linear model SE standard error h hour SEM standard error of the mean HEPES N-(2-hydroxyethyl)piperazine-N′-2- SNP single nucleotide polymorphism ethanesulfonic acid SRBC sheep red blood cells HPLC high-performance (high-pressure) TBA thiobarbituric acid liquid chromatography T cell thymic-derived cell ICU international chick units TME true metabolizable energy Ig immunoglobulin TMEn nitrogen-corrected true metabolizable IL interleukin energy i.m. intramuscular TSAA total sulfur amino acids in. inch USDA United States Department i.p. intraperitoneal of Agriculture IU international units UV ultraviolet i.v. intravenous vol/vol volume to volume kcal kilocalorie vs. versus L liter* wt/vol weight to volume lb pound wt/wt weight to weight L:D hours of light:hours of darkness in a wk week photoperiod yr year

*Also capitalized with any combination, e.g., mL. Patrons

Gold Alpharma Cobb-Vantress, Inc. Evonik-Degussa Corporation Novus International, Inc.

Silver AB Vista Feed Ingredients ChemGen Corp. Foster Farms Griffin Industries, Inc. Hendrix Genetics Hy-Line International Perdue Farms, Inc. Zinpro Corporation