Evaluation of Sodium Metabisulphite in Sorghum-based Meat Chicken Diets

Transcription

1 Evaluation of Sodium Metabisulphite in Sorghum-based Meat Chicken Diets January 2015 RIRDC Publication No. 15/001

2 Evaluation of Sodium Metabisulphite in Sorghum-based Meat Chicken Diets by Peter H Selle January 2015 RIRDC Publication No 15/001 RIRDC Project No PRJ

3 2015 Rural Industries Research and Development Corporation. All rights reserved. ISBN ISSN Evaluation of sodium metabisulphite in sorghum-based meat chicken diets Publication No. 15/001 Project No. PRJ The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone Researcher Contact Details Peter H Selle Poultry Research Foundation 425 Werombi Road Camden NSW 2570 Phone: peter.selle@sydney.edu.au In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: rirdc@rirdc.gov.au. Web: Electronically published by RIRDC in January 2015 Print-on-demand by Union Offset Printing, Canberra at or phone ii

4 Foreword Grain sorghum is an important feedstuff for chicken meat production in Australia; however, there is a consensus that the performance of chickens offered sorghum-based diets is inferior to their counterparts on wheat. Wheat-based chicken diets invariably contain non-starch polysaccharidedegrading enzymes, which is advantageous, but this is not equally the case with sorghum as it is a non-viscous grain. Nevertheless, the possibility remains that alternative feed additives may advantage meat chickens offered sorghum-based diets. Several reducing agents, including sodium bisulphite/sodium metabisulphite, have been shown to enhance in vitro pepsin digestibility of sorghum on repeated occasions. Consequently, the inclusion of sodium metabisulphite in sorghum based chicken diets may have the potential to enhance chicken performance; if so, this would clearly benefit the chicken meat industry. This project was therefore conducted to investigate whether sodium metabisulphite addition to sorghum-based chicken diets advantages chicken performance. This project was funded by the RIRDC chicken meat program from producer levies, which is matched by Australian government funds. This report is an addition to RIRDC s diverse range of over 2000 research publications and it forms part of our Chicken Meat R&D program, which aims to support increased sustainability and profitability in the chicken meat industry through focussed research and development. Most of RIRDC s publications are available for viewing, free downloading or purchasing online at Purchases can also be made by phoning Craig Burns Managing Director Rural Industries Research and Development Corporation iii

5 Acknowledgments The inputs of a number of colleagues into the completion of this project should be acknowledged. The Principal Investigator would especially like to thank Ms Sonia Yun Liu (Poultry Research Foundation) and Dr Jingwen Cae, Dr Robert Caldwell, Mr Ali Khoddami and Dr Tom Roberts (Faculty of Agriculture and Environment of the University of Sydney) for their contributions. The Principal Investigator would also like to thank all his fellow members of the Poultry Research Foundation, including its Director at the time that the research was conducted, Dr Aaron Cowieson, and Ms Joy Gill, in particular, for their indispensable support. Dr Vivien Kite and the RIRDC Chicken Meat Advisory Committee are to be acknowledged for their tangible support and interest in the project and I would like to thank Mr Greg Hargreave and Mr Greg McDonald for their recommendations and suggestions. Many thanks for all your contributions. Peter H Selle Poultry Research Foundation iv

6 Abbreviations ADF AIA AME AMEn AUC Ca Cps DI DJ GE LSD MRT N Na NBD-Cl NIR/NIRS NOAEL NSP PDN PDS PI PJ PSI RVA S SEM -SH SMBS -S-S- Acid detergent fibre Acid insoluble ash Apparent metabolisable energy Nitrogen-corrected apparent metabolisable energy Area under curve Calcium Centipoise Distal ileum Distal jejunum Gross energy Least significant difference Mean retention time Nitrogen Sodium 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole Near infra-red spectroscopy No-observed-adverse-effect level Non-starch polysaccharides Potential digestible nitrogen Potential digestible starch Proximal ileum Proximal jejunum Particle size index Rapid visco analyser Sulphur Standard error of the mean (pooled) Free sulphydryl group Sodium metabisulphite Disulphide bond (bridge, linkage) v

7 Contents Foreword... iii Acknowledgments... iv Abbreviations... v Executive Summary... x Introduction... 1 Objectives... 5 Methodology... 6 Experiment One: The effects of graded sodium metabisulphite inclusion levels in allsorghum diets... 9 Outline... 9 Results Discussion Summary Experiment Two: The effects of graded sodium metabisulphite inclusion levels in sorghum-casein diets Outline Results Discussion Summary Experiment Three: The effects of graded sodium metabisulphite inclusion levels in complete sorghum-based chicken diets Outline Materials and Methods Results Discussion Summary Summary and Implications Oxidative reductive depolymerisation of starch polysaccharides Tolerance of meat chickens to sodium metabisulphite Conclusions and Recommendations References Publications Arising from this Project vi

8 Tables Table 1. Nutritive characteristics of red sorghum # Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. Table 8. Composition of eight all-sorghum diets with graded sodium metabisulphite inclusion levels, dietary electrolyte balance (DEB) and additional levels of sulphur (S) and sodium (Na) derived from sodium metabisulphite Effect of sodium metabisulphite inclusion levels on concentrations of free sulphydryl groups and disulphide bonds expressed as μmol/g protein (analyses completed in triplicate) Effect of sodium metabisulphite inclusion levels in all-sorghum diets on growth performance and mortality rates of chickens from 14 to 21 days post-hatch Effect of sodium metabisulphite inclusion levels in all-sorghum diets on nutrient utilisation [apparent metabolisable energy (AME), nitrogen (N) retention and N- corrected AME (AMEn)] in chickens at 19 to 21 days post-hatch Effect of sodium metabisulphite inclusion levels on apparent digestibility coefficients of nitrogen in four small intestinal sites at 21 days post-hatch Effect of 5.0 g/kg sodium metabisulphite on apparent digestibility coefficients of start and nitrogen in four small intestinal sites at 21 days post-hatch Effects of 5.0 g/kg sodium metabisulphite on small intestinal site of sorghum starch disappearance in chickens from 14 to 21 days post-hatch and Pearson correlations with nutrient utilisation parameters Table 9. Nutritive characteristics of red sorghum # Table 10. Composition of sorghum-casein diets Table 11. Trial design of Experiment Two Table 12. Table 13. Table 14. Table 15. Table 16. Table 17. The effect of sodium metabisulphite (SMBS) on concentrations of free sulphydryl groups and disulphide bonds in steam-pelleted sorghum (analyses completed in triplicate) The effect of sodium metabisulphite (SMBS) on growth performance of birds offered cold-pelleted, sorghum-casein diets (treatments 4 to 7 inclusive) from 18 to 26 days posthatch The effect of sodium metabisulphite (SMBS) on nutrient utilisation of birds offered coldpelleted, sorghum-casein diets (treatments 4 to 7 inclusive) Dietary composition and (common) nutrient specifications of seven sorghum-based diets with graded inclusion levels of sodium metabisulphite Effects of graded sodium metabisulphite inclusion levels on Promatest protein solubility (%) and concentrations (μmol/g protein) of free sulphydryl groups and disulphide bonds in sorghum-based diets Pearson correlations between sodium metabisulphite inclusion levels (g/kg), Promatest protein solubility (%), and concentrations (μmol/g protein) of free sulphydryl groups and disulphide bonds in sorghum-based diets vii

9 Table 18. Table 19. Table 20. Table 21. Effects of graded sodium metabisulphite inclusion levels on RVA properties of starch contained in sorghum-based diets (analyses completed in duplicate) Effects of graded sodium metabisulphite inclusion levels on RVA properties of starch extracted from sorghum-based diets Pearson correlations between sodium metabisulphite inclusion levels (g/kg) and RVA starch properties of sorghum-based diets including peak viscosity (cp), holding viscosity (cp), break-down viscosity (cp), final viscosity (cp), setback viscosity (cp), peak time (minutes) and pasting temperature ( C) Effects of graded sodium metabisulphite dietary inclusion levels on growth performance of meat chickens from days post-hatch Table 22. Effects of graded sodium metabisulphite dietary inclusion levels on nutrient utilisation 35 Table 23. Table 24. Table 25. Table 26. Table 27. Table 28. Table 29. Table 30. Table 31. Table 32. Pearson correlations between sodium metabisulphite inclusion levels (g/kg) and weight gain (g/bird), feed intake (g/bird), feed conversion ratios, AME (MJ/kg and MJ/day), N retention (%) and N-corrected AME (AMEn; MJ/kg) Significant Pearson correlations between RVA starch properties of sorghum-based diets containing graded levels of sodium metabisulphite and parameters of growth performance and nutrient utilisation in meat chickens Significant Pearson correlations between Promatest protein solubilities and concentrations of free sulphydryl groups and disulphide bonds of sorghum-based diets containing graded levels of sodium metabisulphite and parameters of growth performance and nutrient utilisation in meatchickens Effects of graded sodium metabisulphite dietary inclusion levels on apparent digestibility coefficients of nitrogen in four small intestinal segments Effects of graded sodium metabisulphite dietary inclusion levels on apparent digestibility coefficients of starch in four small intestinal segments Effects of graded sodium metabisulphite dietary inclusion levels on mean retention times of digesta in four small intestinal segments Effects of graded sodium metabisulphite dietary inclusion levels on potential digestible nitrogen coefficient (PDN), nitrogen digestion rate (K nitrogen ), potential digestible starch coefficient (PDS), starch digestion rate (K starch ) and area under starch digestion curve (AUC) Effects of graded sodium metabisulphite dietary inclusion levels on small intestinal site of sorghum starch disappearance in chickens from 10 to 24 days post-hatch Effects of graded sodium metabisulphite dietary inclusion levels on small intestinal site of dietary nitrogen disappearance in meat chickens from 10 to 24 days post-hatch Pearson correlations between small intestinal site of sorghum starch disappearance in meat chickens and selected parameters of growth performance, nutrient utilisation, kinetics of starch digestion and RVA starch properties viii

10 Figures Figure 1. Figure 2. Figure 3. Figure 4. Effect of sodium metabisulphite inclusion levels on concentrations of free sulphydryl groups and disulphide bonds expressed as μmol/g protein Linear relationship (r = ; P < 0.001) between dietary concentrations of sodium metabisulphite and voluntary feed intakes of meat chickens The proportion of total starch disappearance from four small intestinal segments and the sum of the posterior three segments in negative control diet (green) and mean values for six sodium metabisulphite-supplemented diets (red) The negative linear relationship (r = ; P < 0.015) between final starch viscosity and starch disapperance from the three posterior small intestinal segments (mean values only shown for purposes of illustration) ix

11 Executive Summary What the report is about According to the Australian Chicken Meat Federation, approximately 596 million birds will be processed to generate million tonnes of chicken-meat in (ACMF, 2014). Thus the chicken meat industry has an annual requirement of approximately 3 million tonnes of feedstuffs to meet this demand. Approximately 600,000 tonnes of this total comprises grain sorghum, which provides both energy and protein in chicken diets. However, in comparison to wheat-based diets, the performance of meat chickens on sorghum-based diets is often sub-standard. This report describes an investigation into the dietary inclusion of sodium metabisulphite in sorghumbased diets as a means to enhance chicken performance. Sulphite reducing agents, including sodium metabisulphite, have the capacity to reduce disulphide cross-linkages in proteins. Disulphide crosslinkages in kafirin, the dominant sorghum protein fraction, are believed to be important limiting factors for the utilisation of sorghum-derived protein and starch utilisation by meat chickens. This tenet formed the premise of the present investigation. Who is the report targeted at? This report is targeted primarily at poultry nutritionists within integrated operations and independent feed-mills and nutritional consultants to the chicken meat industry. The secondary target is poultry nutritionists within tertiary institutions and government departments engaged in research, teaching and extension. Where are the relevant industries located in Australia? The Chicken Meat industry operates in all states of Australia, generally in close proximity to capital cities and major areas of population. However, sorghum use in meat chicken diets is generally restricted to regions close to where sorghum is produced, and in particular to chicken meat operations in Queensland and NSW, from which more than 50% of chicken meat production is derived. Background Under Australian conditions, the performance of meat chickens offered sorghum-based diets is considered to be inferior in comparison to wheat-based diets, which are invariably advantaged by the inclusion of non-starch polysaccharide (NSP)-degrading enzymes. In relation to this inferior performance, Taylor (2005) proposed that the evidence points clearly to disulphide bond crosslinking of the β- and γ-kafirin proteins being a major factor in reducing protein and starch digestibility. Kafirin is the dominant protein fraction in sorghum and is found in protein bodies in sorghum endosperm where both protein bodies and starch granules are located in close proximity, embedded in a glutelin protein matrix. Disulphide cross-linkages in the relatively cystine-rich γ- and β-kafirin fractions located in the periphery of protein bodies are believed to be of particular importance. They are thought to impede digestion of α-kafirin directly, which is located in the central core of protein bodies, and to impede digestion of starch indirectly via biophysical and biochemical interactions. If this is the case, it follows that the inclusion of a sulphite reducing agent, with the capacity to cleave disulphide cross-linkages, in sorghum-based chicken diets would be advantageous. Sulphite reducing agents include sodium sulphite (Na 2 SO 3 ), sodium bisulphite (NaHSO 3 ) and sodium metabisulphite (Na 2 S 2 O 5 ). Sodium sulphite is commonly used in the dry-extrusion of whole ( full-fat ) soybeans for pigs and poultry so there is precedence for its inclusion in animal diets. For this project it was decided to evaluate sodium metabisulphite because it contains more sulphur (S) and less sodium (Na) than sodium sulphite. Sodium metabisulphite and sodium bisulphite are effectively x

12 interchangeable as it is understood that sodium metabisulphite, a powder, is converted to sodium bisulphite in liquid phases. Aims/objectives The fundamental objective of this research was to ascertain if sodium metabisulphite inclusions in sorghum-based chicken diets would enhance growth performance and nutrient utilisation. Methods used On the basis of in vitro methods, the effects of sodium metabisulphite on concentrations of disulphide bonds and free sulphydryl groups in sorghum and sorghum-based diets were determined and related to dietary protein solubility assessed by the Promatest method. In addition, the effects of sodium metabisulphite on starch pasting properties of sorghum-based diets, and starch extracted from those diets, were determined by rapid visco analysis (RVA). Three chicken feeding studies were conducted within this project. Two preliminary feeding studies were completed with all-sorghum and sorghum-casein mash diets with graded sodium metabisulphite inclusions to assess appropriate inclusion rates from the standpoint of efficacy and toxicity. In a third feeding study, graded levels of sodium metabisulphite were included in conventional sorghum-based meat chicken diets, which were steam-pelleted at a conditioning temperature of 84 C. The parameters assessed in these studies included growth performance (weight gain, feed intake, feed conversion ratio), nutrient utilisation (apparent metabolisable energy expressed as MJ/kg and MJ/day, N retention, N-corrected apparent metabolisable energy) and apparent digestibility coefficients of starch and nitrogen in four small intestinal sites. Also, the effects of sodium metabisulphite on the dynamics of nitrogen and starch digestion (extent, rate and site of digestion/absorption in the small intestine) were determined. Results/key findings The inclusion of sodium metabisulphite in steam-pelleted, sorghum-based chicken diets enhanced energy utilisation and efficiency of feed conversion. Sodium metabisulphite dietary inclusions ranging from 0 to 5.25 g/kg significantly influenced apparent metabolisable energy (r = 0.462; P < 0.005), N- corrected apparent metabolisable energy (r = 0.437; P < 0.01) and efficiency of feed conversion (r = 0.416; P < 0.02) in a quadratic manner. Moreover, optimum sodium metabisulphite inclusion levels may be calculated from the relevant regression equations. It can be deduced that 3.76 g/kg sodium metabisulphite improved AME by 0.35 MJ (13.84 versus MJ/kg), 4.17 g/kg sodium metabisulphite improved AMEn by 0.37 MJ (12.29 versus MJ/kg) and 4.50 g/kg sodium metabisulphite improved FCR by 2.75% (1.416 versus 1.456). In the final feeding study, sodium metabisulphite significantly increased both AME and AMEn at all six inclusion levels ranging from 1.5 to 5.25 g/kg and significantly improved FCR at five inclusion levels. On average, sodium metabisulphite increased AME by 0.35 MJ (13.80 versus MJ/kg), AMEn by 0.40 MJ (12.25 versus MJ/kg) and improved FCR by 2.33% (1.424 versus 1.458). Thus the inclusion of sodium metabisulphite in sorghum-based chicken diets holds promise. This is only further emphasised when consideration is given to the lowest inclusion rate evaluated. At 1.50 g/kg, sodium metabisulphite significantly improved AME by 0.36 MJ (13.81 versus MJ/kg; P = 0.005), AMEn by 0.45 MJ (12.30 versus MJ/kg; P = 0.001) and FCR by 2.47% (1.422 versus 1.458; P = 0.025) on the basis of pair-wise comparisons. Responses of this magnitude at the 1.50 g/kg level suggest that it would be economically feasible to include sodium metabisulphite in sorghumbased chicken diets. xi

13 Sodium metabisulphite was evaluated because of its potential to reduce disulphide bonds, especially in the β- and γ-kafirin fractions of sorghum protein. Moreover, it was established in vitro that sodium metabisulphite decreased disulphide bonds, increased free sulphydryl groups and increased protein solubility of sorghum-based diets. However, the enhancements in energy utilisation and feed conversion efficiency observed in the studies reported here do not appear to be related to reductions of disulphide cross-linkages because essentially sodium metabisulphite did not influence N digestibility. This may have been because sodium metabisulphite exacerbated endogenous amino acid flows. Alternatively, sodium metabisulphite profoundly influenced RVA starch pasting properties in vitro, almost certainly via the oxidative-reductive depolymerisation of starch polysaccharides. Another key finding was that sodium metabisulphite altered the sites of small intestinal starch digestion. Abruptly digestible starch is defined as the fraction of starch that is absorbed from the proximal jejunum, while gradually digestible starch is the starch absorbed from the distal jejunum, proximal and distal ileum. On this basis, sodium metabisulphite generated more gradually digestible starch coupled with less abruptly digestible starch and this change in the sites of starch digestion appears to have driven the enhancements energy utilisation and feed conversion efficiency. The advantages and underlying mechanisms of slowly digestible starch in relation to feed conversion efficiency in meat chickens have been extensively investigated by Weurding et al. (2001a,b; 2003a,b). There is good evidence that slowly digestible starch has a protein-sparing effect because its presence in the lower small intestine causes a reduction in the oxidation of amino acids, especially glutamic acid, to provide energy for gut function. The explanation as to how sodium metabisulphite generated more gradually digestible starch, presumably via the oxidative-reductive depolymerisation of starch polysaccharides, is not straightforward. However, it may be related to the fact that chemically modified, including oxidised, starch has been shown to be less readily digestible. It is also possible that depolymerised starch had a more rapid transit through the proximal jejunum, which is consistent with the sodium metabisulphite induced reductions in starch digestibility observed in this small intestinal segment. Potentially, sodium metabisulphite is toxic; however, the maximum inclusion that can be tolerated in meat chickens on steam-pelleted diets appears to be in excess of 5.0 g/kg. The emission of sulphur dioxide from sodium metabisulphite, particularly in mash diets, appears to depress feed intakes. Other potential toxic effects could include exacerbated endogenous amino acid flows, increased uric acid excretion and a deficiency of thiamin. If these occur, they would be expected to compromise apparent digestibility of protein/amino acids, nitrogen retention and voluntary feed intakes, respectively. Nevertheless, if dietary inclusions in the order of 1.50 g/kg sodium metabisulphite are efficacious then toxicity should not present problems. Implications for relevant stakeholders The majority of relevant stakeholders believe that the performance of meat chickens offered sorghumbased diets is either occasionally or consistently inferior to wheat. However, poor quality ( low ME ) wheat is advantaged by the dietary inclusion of NSP-degrading enzymes. However, there is now the real possibility that the inclusion of sodium metabisulphite in sorghum-based diets will enhance chicken performance and will be economically feasible. Moreover, the advantages of including sodium metabisulphite in chicken diets may not be necessarily limited to those that are sorghum-based and may extend to other cereals. Recommendations The conclusions from this project are effectively based on one feeding study and ideally should be confirmed in one or more additional feeding studies which should have two objectives. From an applied standpoint, attention should be paid to the optimum sodium metabisulphite inclusion level. In this context it is noteworthy that sodium bicarbonate contains 274 g/kg Na and the Na concentration xii

14 in sodium metabisulphite is 242 g/kg. Thus the inclusion of sodium metabisulphite in a formulation should displace a similar quantity of sodium bicarbonate to preserve dietary sodium concentrations and dietary electrolyte balances. Importantly, however, this would also offset the inclusion cost of sodium metabisulphite. From a research standpoint, the mechanisms whereby sodium metabisulphite generates more gradually digestible starch and this in turn positively impacts on energy utilisation and feed efficiency are an obvious area of investigation. Finally, if sodium metabisulphite advantageously modifies the extent, rate and site of starch digestion along the small intestine in sorghum-based diets, which appears to be the case, then these advantages may be equally applicable to wheat- and maize-based chicken diets. Thus, feeding studies to confirm the efficacy of sodium metabisulphite need not be confined to sorghum and should also include other cereals. The impact of sodium metabisulphite on diets consisting of whole grain and a pelleted balancing concentrate is also relevant. xiii

15 Introduction From 2008 to 2012 inclusive, the Australian sorghum harvest averaged million tonnes and the entire crop could be utilised as an animal feedstuff for pigs, poultry and beef feedlot cattle. However, up to 500,000 tonnes of sorghum has been exported annually, which may indicate that these primary industries are reluctant to incorporate sorghum into their diets, despite the fact that sorghum is usually available at a discount price relative to wheat. An informal survey of 15 practicing nutritionists was completed by the Poultry Research Foundation to seek their opinions of the performance of pigs and poultry on sorghum-based diets. A clear majority (71%) believed that the performance of pigs and poultry on sorghum-based diets was either routinely or occasionally inferior to wheat-based diets. The stigma of bird-proof sorghums with high levels of condensed tannin, a potent anti-nutritive factor, may be contributing to this negative assessment. This is because 19% of respondents in the survey considered that grain sorghum contains sufficient concentrations of condensed tannin to impair animal performance. However, it is highly improbable that this is the case. The Poultry Research Foundation detected the presence or absence of pigmented testas in eleven sorghum varieties harvested since All eleven varieties did not contain a pigmented testa, indicating they are Type I sorghums that, by definition, do not contain condensed tannin as they do not have the requisite genotype. Therefore, it is highly unlikely that condensed tannin is contributing to the inconsistent or sub-optimal performance of meat chickens offered sorghum-based diets. However, real attention has been paid to kafirin, the dominant protein fraction in sorghum, as the prime cause of the nutritive shortcomings of grain sorghum both generally and in poultry specifically (Bryden et al., 2009; Selle et al., 2010; Selle, 2011). Kafirin is located in protein bodies within sorghum endosperm; the central core is composed of α-kafirin while the peripheral layers consist of β- kafirin and γ-kafirin. Moreover, there has been a very real focus on disulphide cross-linkages in β- kafirin and γ-kafirin and hydrothermal processes (eg wet-cooking, which usually consists of boiling sorghum in water for ten minutes) induce their formation and steam-pelleting of sorghum-based chicken diets constitutes a hydrothermal process. The rationale for this considerable interest in disulphide cross-linkages in kafirin is reflected in the following conclusion in a sorghum review drawn by Taylor (2005): the evidence points clearly to disulphide bond cross-linking of the β- and γ- kafirin proteins being a major factor in reducing protein and starch digestibility. If this is the case, the evaluation of reducing agents with the capacity to cleave disulphide bonds in sorghum-based chicken diets is a logical progression. The sulphite reducing agent, sodium metabisulphite, was selected partially because of the principal researcher s background in using sodium sulphite in the dry extrusion of whole ( full-fat ) soybeans to facilitate trypsin inhibitor destruction and enhance protein solubility. Extruded full-fat soy was included in diets for weaner pigs and chickens and also as the basis of a range of equine supplements. In relation to disulphide cross-linkages in kafirin depressing protein digestibility, the pivotal paper is that of Hamaker et al. (1987) entitled Improving the in vitro protein digestibility of sorghum with reducing agents. Wet cooking (boiling in a water-bath for 20 minutes) sorghum reduced its pepsin digestibility by 30.3% (0.563 versus 0.808). In contrast, wet cooking maize, barley, rice and wheat reduced their average pepsin digestibility by only 8.8% (0.819 versus 0.898). The reducing agent, 2- mercaptoethanol, increased the protein digestibility of raw and wet-cooked sorghum by an average of 25.9% (0.881 versus 0.700) following exposure to pepsin. Similarly, 2-mercaptoethanol increased protein digestibility by 31.4% when sorghum was exposed to trypsin and chymotrypsin. The pepsin digestibility of wet-cooked sorghum was enhanced by four reducing agents where sodium bisulphite was more effective than 2-mercaptoethanol (dithioreitol > sodium bisulphite > 2-mercaptoethanol > L-cysteine). In the periphery of protein bodies, both β- and γ-kafirin contain relatively high levels of cystine. The induction of disulphide cross-linkages by wet-cooking in the periphery of protein bodies has been likened to case-hardening of these structures (Belton, 2004) so that the central α-kafirin core is protected from enzymatic proteolysis by an encompassing layer of relatively insoluble 1

16 protein. Given α-kafirin comprises from 66 to 84% of total kafirin (De Mesa-Stonestreet et al., 2010), a 110 g/kg protein sorghum with a 55% kafirin content would contain in the order of 45 g/kg α-kafirin or 40% of total sorghum protein. Hence disulphide cross-linkages, especially in the β- and γ-kafirin fractions, which are amplified by hydrothermal processing of sorghum, depress the protein digestibility of β- and γ-kafirin and, in turn, α-kafirin. In relation to disulphide cross-linkages in kafirin compromising starch digestibility, the situation is not equally straightforward. However, kafirin protein bodies may physically limit the gelatinisation of starch in sorghum endosperm and the possibility remains that kafirin may interact with starch granuleassociated proteins via inter-molecular disulphide cross-linkages. While there is a general consensus that this is broadly the case, Gidley et al. (2011) have expressed reservations regarding the contention that disulphide cross-linkages in kafirin are not a key factor in relation to the digestibility of sorghum starch. Nevertheless, it would be a highly valuable outcome if disulphide cross-linkages in either kafirin protein bodies and/or the glutelin protein matrix are compromising starch digestibility of starch granules in sorghum endosperm and it can be demonstrated that this is countered by the dietary inclusion of reducing agents. Thus reducing agents, including sodium metabisulphite, appear to have the potential to enhance sorghum protein and perhaps starch digestibility in chickens on the basis of in vitro data. However, despite this apparent promise, there do not appear to be any published reports where the inclusions of reducing agents in sorghum-based chicken diets have been evaluated. There are several sulphite reducing agents with the potential to cleave disulphide bonds, including sodium bisulphite (NaHSO 3 ) and sodium metabisulphite (Na 2 S 2 O 5 ), with sulphur contents of 30.8% and 33.7%, respectively. However, sodium bisulphate and sodium metabisulphite are effectively interchangeable. Throughout this project, sodium metabisulphite was added to the diets in powder form but it is insoluble and is converted to sodium bisulphite in liquid phases, which presumably takes place in the avian gut. In addition to the Hamaker et al. (1987) study, Rom et al. (1992), Oria et al. (1995), Arbab and El Tinay (1997) and Elkhalifa et al. (1999) have shown that sulphite reducing agents ameliorate the negative impact of wet-cooking on sorghum pepsin digestibility. Rom et al. (1992) found that sodium bisulphite increased uncooked sorghum pepsin digestibility by 21.5% (0.96 versus 0.79) and wetcooked sorghum by 36.2% (0.58 versus 0.79) following 120 minutes incubation. Oria et al. (1995) found that sodium bisulphite increased uncooked sorghum pepsin digestibility by 34.4% (0.930 versus 0.692) and wet-cooked sorghum by 28.9% (0.562 versus 0.436). Arbab and El Tinay (1997) reported that two reducing agents, sodium bisulphite and ascorbic acid, improved the in vitro pepsin digestibility of uncooked and cooked sorghum of two cultivars that were either high (31 g/kg) or low (3.5 g/kg) in tannin content. Finally, Elkhalifa et al. (1999) showed that increasing concentrations of sodium metabisulphite enhanced the pepsin digestibility of wet-cooked sorghum. Thus sulphite reducing agents tangibly increased pepsin digestibility of both raw and wet-cooked sorghums in these reports. As discussed, this is usually attributed to the reduction of disulphide bonds mainly in the relatively cystine-rich β- and γ-kafirin fractions in the periphery of protein bodies, which facilitates the enzymatic digestion of the central α-kafirin core. The effects of a protease and potassium metabisulphite on the proteolysis of sorghum endosperm proteins while mashing raw grain were investigated by Ng andwe (2008). Protease increased free amino nitrogen production from approximately 36 to 75 mg/100 g sorghum, but the combination of protease and potassium metabisulphite generated a further increase in free amino nitrogen to 91 mg/100 g sorghum. However, transmission electron microscopy showed that protease predominantly hydrolysed the glutelin matrix protein surrounding the kafirin protein bodies, but in the presence of potassium metabisulphite there was also a substantial breakdown of the protein bodies. Potassium metabisulphite had the effect of reducing kafirin polymers and oligomers into monomers as revealed by gel electrophoresis. Ng andwe et al. (2008) concluded that the addition of potassium 2

17 metabisulphite in a sorghum grain mashing system significantly improved the rate of sorghum protein hydrolysis because of the reduction of intermolecular disulphide bonds in kafirin protein, which afforded protease better access to the substrate. Disulphide bonds link two cysteine moieties to form sulphur containing amino acid cystine. The reduction of disulphide bonds in cystine by sulphite (SO 3 2- ) reducing agents is represented by the following equation of Cecil and Wake (1962). The reaction is strictly reversible and will only proceed to completion with low RS - (cysteine) concentrations: 2- RS*SR + SO 3 RS RS*SO 3 where RS*SR represents cysteine and RS - represents cysteine. While several in vitro reports have demonstrated the positive effects of reducing agents on the pepsin digestibility of sorghum, very few studies have determined concentrations of disulphide bonds and free sulphydryl groups. However, Ezeogu et al. (2008) used the method of Chan and Wasserman (1993) to analyse concentrations of free sulphydryl groups. In this study the average concentration of free sulphydryl groups in floury and vitreous sorghum endosperm was 23.3 nmol/g protein which was decreased by 89% to 2.6 nmol/g protein by wet-cooking (96 C for 10 minutes). However, wet-cooking in the presence of a reducing agent (2-mercaptoethanol) increased free sulphydryl groups by 73% from 23.3 to 40.2 nmol/g protein. Thus, the overall effect of 2-mercaptoethanol on free sulphydryl group concentrations in floury and vitreous endosperm of wet-cooked sorghum was a profound, fifteen-fold increase from 2.6 to 40.2 nmol/g protein. Interestingly, in an earlier study, Ezeogu et al. (2005) found that wet-cooking vitreous endosperm sorghum flour with 2-mercaptoethanol increased in vitro starch digestibility by 14.4% (0.995 versus 0.870) and by 5.74% (0.995 versus 0.941) in the floury endosperm. Similarly, Zhang and Hamaker (1998) reported that sodium metabisulphite increased starch digestibility of wet-cooked sorghum flour by 12.6%. Also, Choi et al. (2008), using laser scanning microscopy, reported that sodium bisulphite reduced the scale of the protein matrix surrounding starch granules in sorghum endosperm and this reduction may facilitate starch digestion. These findings suggest that the extent of disulphide cross-linkages in kafirin and glutelin has a negative influence on starch digestibility of sorghum under in vitro conditions. Evaluations of reducing agents in sorghum-based diets offered to pigs and poultry are limited. Sodium sulphite is used commercially in the extrusion of whole ( full-fat ) soybeans because it enhances protein solubility and facilitates trypsin inhibitor degradation. Thus the majority of in vivo evaluations of sulphite reducing agents are related to soy protein and responses could stem from improvements in either the destruction of trypsin inhibitors and/or the reduction of disulphide bonds and enhanced protein solubility. Burnham et al. (2000) reported that sodium sulphite improved weaner pig performance on maize-based diets containing soybean meal, extruded soybean meal or dry-extruded soybeans. As a main effect, sodium sulphite significantly improved weight gain by 14.9%, feed intake by 7.2% and feed efficiency by 7.3% to 28-days post-weaning. The average dietary inclusion rate of sodium bisulphite was g/kg in the Burnham et al. (2000) feeding studies. Kim and Kim (1997) found that dry-extrusion of full fat soy with sodium sulphite numerically increased weight gain and feed intake but significantly improved feed efficiency by 11.7% in young pigs to 35 days postweaning. Also, Kim et al. (2000) reported that the addition of sodium sulphite to dry-extruded full-fat soy numerically improved ileal N digestibility, N retention and apparent ileal digestibility coefficients of amino acids in growing pigs. In poultry, Herkelman et al. (1991) found the addition of 10 and 20 g/kg sodium metabisulphite to whole soybeans heated for 20 minutes at 121ºC improved weigh gain by 12.6% and feed efficiency by 5.0% in meat chickens. These outcomes indicate that the benefits of sodium metabisulphite inclusion in sorghum-based chicken diets need not be confined to the sorghum component. It is relevant that cystine was the least digestible amino acid in 19 meat-and-bone meal samples with apparent ileal digestibility coefficients ranging from to in meat-strain chicks as reported by Ravindran et al. (2002). Wang and Parsons (1998) recorded similar results and suggested that the formation of lanthionine during the 3

18 rendering process may have been responsible because of the limited availability of the cystine moiety in lanthionine in poultry (Robbins et al., 1980). However, it is likely that the rendering process induced disulphide bond formation in meat-and-bone meal, thereby reducing cystine digestibility. If so, it follows that sodium metabisulphite would attenuate the formation disulphide linkages and enhance cystine digestibility. This is important given that Wang et al. (1997) considered that cystine is the first limiting amino acid in meat-and-bone for meat chickens. Because of this background, meatand-bone meal was included in the diets of the third feeding study undertaken in the current project and in which where complete, standard sorghum-based diets were offered to meat chickens. 4

19 Objectives The primary objective of this project was to ascertain if the inclusion of a sulphite reducing agent (sodium metabisulphite) in sorghum-based diets for chickens is advantageous. The rationale for this approach is encapsulated in the following statement of Taylor (2005): the evidence points clearly to disulphide bond cross-linking of the β- and γ-kafirin proteins being a major factor in reducing protein and starch digestibility (in grain sorghum). Given that this is the case it follows that a sulphite reducing agent would be advantageous and there are precedents for their inclusion in pig and poultry diets. In undertaking this research, one outcome considered likely was that sodium metabisulphite would be verified to be an economically viable feed additive for sorghum-based poultry diets. However, it was also considered likely that sodium metabisulphite could act as a vehicle to assess the nutritional importance of di-sulphide cross-linkages in grain sorghum and other relevant protein sources. 5

20 Methodology Summary of studies undertaken Two preliminary feeding studies were completed with all-sorghum and sorghum-casein diets with graded sodium metabisulphite inclusion levels to assess appropriate inclusion rates from the standpoint of efficacy and toxicity. In the third feeding study graded levels of sodium metabisulphite were included in a conventional sorghum-based chicken diet, which was steam-pelleted at a conditioning temperature of 84 C. In this study, the effects of the reducing agent on growth performance, nutrient utilisation, extent, rate and site of small intestinal digestion of nitrogen and starch were determined. Concentrations of disulphide bonds and free sulphydryl groups Milled samples of sorghum and complete diets were analysed for free sulphydryl (-SH) concentrations using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) according to the proteolytic extraction method of Andrews et al. (1995). Total sulfhydryl concentrations were determined after reduction of the extract with sodium borohydride and derivatisation with NBD-Cl; disulphide (-S-S-) concentrations were then determined by difference. Concentrations are expressed as μmoles of -SH or S-S- per gram of extracted protein. Promatest protein solubility The protein solubilities of sorghum and complete diets were determined by the Promatest method (Germ Services; Montardon, France). Essentially, this method compares the solubility of the target protein with that of albumin as the 100% soluble standard. This methodology has been described in detail by Odjo et al. (2012) where the solubility of extracted protein is compared to albumin via a colorimetric reaction with Coomassie Blue. Starch pasting properties The RVA pasting properties of sorghum-based diets and extracted strach were determined using a Rapid-Visco-Analyzer as outlined by Hernandez et al. (2008). Starch was isolated from the sorghumbased diets by sonication using the method described by Park et al. (2006). Within 13 minute intervals, a 28g mixture of sorghum or diet and water (15:85 w/w) were prepared and held at 50 C temperature for 1 minute and then heated from 50 C to 95 C. After holding the hot paste at 95 C for 2.5 minutes, the slurry was again cooled to 50 C, and then held at that temperature for 2 minutes. Growth performance The growth performance parameters determined included weight gain (g/bird), feed intake (g/bird), feed conversion ratio (g/g) and mortality/cull rates (%). Male Ross 308 chicks were used in the three feeding studies and were offered dietary treatments from 14 to 21, 18 to 26 and 10 to 24 days posthatch having been fed a propriety starter ration initially. On days 14, 18 and 10, chicks were individually identified (by wing-band), weighed and allocated into bioassay cages on the basis of body weight in an environmentally-controlled facility. Birds had unlimited access to feed and water under a 23 hours on - 1 hour off lighting regimen. Feed intakes were recorded and the birds were reweighed at days 21, 26 and 24, to determine growth rates and feed per gain. The incidence of dead or culled birds was recorded daily and their body weights used to adjust feed conversion ratio calculations. 6

21 Nutrient utilisation The total excreta collection method over a 72 hour period was used to determine apparent metabolisable energy on a dry matter basis (AME; expressed as MJ/kg and MJ/day), nitrogen (N) retention and N-corrected apparent metabolisable energy (AMEn). Total excreta were quantitatively collected from each cage and feed intakes recorded for 72 hours usually from 18 to 20 days post-hatch in order to determine AME. Excreta were dried in a forced-air oven at 80ºC for 24 hours and the gross energy (GE) of excreta and diets were determined using an adiabatic bomb calorimeter. The AME values of the diets on a dry matter basis were calculated from the following equation: AME diet (MJ/kg DM) = (feed intake x GE diet ) (excreta output x GE excreta ) feed intake AME intakes (MJ/day DM) were calculated from dietary energy densities and average daily feed intakes over the entire feeding period. N contents of diets and excreta were determined using a nitrogen determinator (Leco Corporation, St Joseph, MI.) and N retentions calculated from the following equation: N retention (%) = (feed intake x N diet ) (excreta output x N excreta ) x 100 (feed intake x N diet ) N-corrected AME (AMEn MJ/kg DM) values were calculated by correcting to zero N retention using the factor of kj/g N retained in the body (Hill and Anderson, 1958). Apparent starch and crude protein (N) digestibility coefficients Acid insoluble ash (Celite ) was included in diets at 2% as an inert marker to determine starch and N digestibility. The small intestines were removed from euthanised birds and samples of digesta were gently expressed from the proximal jejunum, distal jejunum, proximal ileum and distal ileum in their entirety and pooled for each cage. Where relevant, proximal jejunal samples were taken from the end of the duodenal loop to the mid-point with Meckel s diverticulum and distal jejunal samples from the mid-point to the diverticulum. Proximal ileal samples were taken from Meckel s diverticulum to the mid-point with the ileo-caecal junction and distal ileal samples were taken from below this mid-point. The digesta samples were freeze-dried to determine apparent digestibilities of starch and crude protein (N) using acid insoluble ash (AIA) as the inert dietary marker. Starch concentrations in diets and digesta were determined by a procedure based on dimethyl sulphoxide, α-amylase and amyloglucosidase as described by Mahasukhonthachat et al. (2010). N concentrations were determined as already stated and AIA concentrations were determined by the method of Siriwan et al. (1993). The apparent digestibility coefficients for starch and protein (N) at up to four small intestinal sites were calculated from the following equation: Apparent digestibility coefficient = (nutrient/aia) diet (nutrient/aia) digesta (nutrient/aia) diet Dynamics of starch and crude protein (N) digestion Starch and nitrogen digested in proximal jejunum (abruptly digestible) and the three posterior segments including distal jejunum, proximal ileum and distal ileum (gradually digestible) were calculated by the following equations: 7

22 Abruptly digestible nutrient = feed intake dietary concentration PJ digestibility coefficient Gradually digestible nutrient = feed intake dietary concentration (DI PJ digestibility coefficient) Mean retention time (MRT) was calculated using the following equation: MRT (min) = (1440 AIA digesta W) / FI 24hr AIA feed Where AIA digesta is the AIA concentration in the digesta (mg/g), W is the weight of dry gut content (g), FI 24hr is the feed intake over 24 h before sampling (g), AIA feed is the AIA concentration in the feed (mg/g) and 1440 equals minutes per day. The mean retention time in the duodenum was estimated to be five minutes (Weurding et al., 2001a). The pattern of fractional digestibility coefficients was described by relating the digestion coefficient at each site with the digestion time (t). The digestion time (t) was calculated from the sum of MRT determined in each intestinal segment. The curve of digestion was described by exponential model developed by Orskov and McDonald (1979): Equation 1 Where (g/g starch or nitrogen) is the proportion of starch or nitrogen that digested at time t (min), the fraction is the amount of potential digestible starch or nitrogen (asymptote) (g/g starch or nitrogen), digestion rate constant k (per unit time, min -1 ) would indicate how rapidly starch or nitrogen was digested. Starch absorption was assumed not to take place proximal to the small intestine. N digestion in this study was determined as apparent N digestibility and it is impacted by endogenous N flows, which is not the case with starch. The glycaemic indices are predicted by the area under the digestogram (AUC) (Liu et al., 2013), which is obtained by integrating Equation (1) between digestion times t 1 (usually t = 0) and t 2 (Equation 2). Equation 2. The Microsoft Excel Solver was used to compute parameters of the modified first-order kinetic (Equation 1) by minimising the sums of squares of residuals with the constraint that 1 g/g. Statistical analyses Experimental data was analysed using the IBM SPSS Statistics 20 program (IBM Corporation. Somers, NY USA) and the JMP program (SAS Institute Inc. Cary, NC USA). The experimental units were cage means and statistical procedures included univariate analyses of variance using the general linear models procedure, Pearson correlations and single and multiple linear regressions and quadratic regressions. A probability level of less than 5% was considered to be statistically significant. 8