Safe and Effective Grain Feeding for Horses

Safe and Effective Grain Feeding for Horses

A report for the Rural Industries Research and Development Corporation by James Rowe, Wendy Brown, and Simon Bird

November 2001 RIRDC Publication No 01/148 RIRDC Project No UNE-62A

© 2001 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0 642 58368 4 ISSN 1440-6845 Safe and effective grain feeding for horses Publication No. 01/148 Project No. UNE-62A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.

Researcher Contact Details Prof James Rowe University of New England Armidale, NSW 2351 Phone: (02) 6773 2225 Fax: (02) 6773 3275 Email: [email protected]

RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4539 Fax: 02 6272 5877 Email: [email protected]. Website: http://www.rirdc.gov.au

Published in November 2001 Printed on environmentally friendly paper by Canprint

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Foreword The aim of this research program was to develop safer and more effective ways of feeding grain to horses. Horses have a limited ability to digest starch in the small intestine and the rapid fermentation of undigested starch in the caecum and colon lead to an accumulation of acidic end products that initiate a cascade of adverse effects – the best known being laminitis or founder. The focus of the project was to develop and calibrate an in vitro method for predicting pre-caecal starch digestion and then use this to identify the safest grains and best processing methods to reduce the amount of starch passing undigested to the caecum and colon. This report covers details of two in vitro assays, one for estimating enzyme starch digestion and the second for measuring rate of fermentation. These assays were then used to examine differences between a wide range of feed grains and processing methods. There are descriptions of two in vivo experiments designed to confirm the in vitro predictions in relation to pre-caecal starch digestion and investigate the role of exogenous enzyme preparations to improve digestion of wheat grain. There is a review of the characteristics of oat grain that make its digestibility so variable and a report on the potential value of triticale as a suitable grain for horses. This project was funded from industry revenue which is matched by funds provided by the Federal Government. This report, a new addition to RIRDC’s diverse range of over 700 research publications, forms part of our Equine R&D program, which aims to develop better feeds for growth and development and reduce the incidence of grain related problems of hoof care. Most of our publications are available for viewing, downloading or purchasing online through our website: • •

downloads at www.rirdc.gov.au/reports/Index.htm purchases at www.rirdc.gov.au/eshop

Peter Core Managing Director Rural Industries Research and Development Corporation

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Acknowledgements Parts of the project described in this final report were conducted in parallel with the Project: “Premium Feed Grains for Livestock” coordinated by the GRDC and supported by MLA, RIRDC (Chicken Meat and Eggs), DRDC and PRDC. Development of the in vitro assays was conducted as joint activity between UNE62A and the GRDC-coordinated feed grains project. Ben Barwick and Karen Roberts made valuable contributions to the experiments measuring in vivo starch digestion as part of their Bachelor of Rural Science honours projects. The section on oat digestibility is based on a recent paper by Rowe, J.B., May, P.J. and Crosbie, G. (2001) ‘Knowing your oats’ Recent Advances in Animal Nutrition in Australia, 13 (in press). Measurement of oat digestibility in the horse form part of a research thesis by Bianca Wilson and access to the unpublished results is gratefully acknowledged.

Abbreviations NSP

Non-starch polysaccharide. Insoluble NSPs are mainly the structural carbohydrates that are an important part of the plant cell walls. There are also soluble NSPs and these comprise sugars, fructans and other soluble carbohydrates.

VFA

Volatile fatty acids are produced as the main end-products of fermentation that are available to the animal for energy metabolism. Quantitatively the most important VFA is acetate, followed by propionate and butyrate.

About the authors JAMES B. ROWE is Professor of Animal Science at the University of New England and has been the coordinator for this project. His area of special interest is animal nutrition and he has focussed much of his research in recent years on problems related to fermentative acidosis. His research on the management of acid accumulation during fermentation of starch in the digestive tract of horses and ruminant animals was instrumental in the development of the product Founderguard based on the antibiotic virginiamycin. More recently he has studied characteristics of feed grains and feed grain processing as complementary strategies to reduce the risk of fermentative acidosis and the subsequent problems of laminitis and behavioural changes. WENDY BROWN has been the technical coordinator for this project and managed all aspects of the in vivo studies including compilation of data and reporting. The wide range of technical skills and her expert knowledge of horse management have been essential for success of the project. SIMON H. BIRD completed his doctoral studies in the field of ruminant nutrition and has developed an impressive range of research achievements on the role of protozoa in the fermentation process and on the relative importance of dietary nutrients such as protein and lipids. For the last four years his research has focused on the nutritive value of feed grains and he has been the principal post-doctoral Research Fellow working on this project. He has been directly responsible for development of the in vitro assays described in this report and has made a major contribution to the analysis of the in vivo studies.

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Contents Foreword.................................................................................................................................................................iii Acknowledgements.................................................................................................................................................iv Abbreviations..........................................................................................................................................................iv About the authors....................................................................................................................................................iv Executive Summary................................................................................................................................................vi Implications .......................................................................................................................................................vii 1. Introduction..........................................................................................................................................................1 2. Starch digestion in the horse ................................................................................................................................2 Processing to improve small intestinal starch digestibility ..................................................................................2 Measuring starch digestion in the horse...............................................................................................................2 3. New in vitro assays for measuring starch digestion.............................................................................................4 An in vitro assay to simulate digestion in the small intestine ..............................................................................4 Protocol for in vitro enzyme digestion of starch ..............................................................................................5 An in vitro assay to simulate fermentation in the caecum and colon...................................................................5 Protocol for in vitro fermentation assay...........................................................................................................6 Results from in vitro assays – enzyme digestion and fermentation .....................................................................6 Effects of processing............................................................................................................................................8 Conclusions on in vitro assays.............................................................................................................................9 4. Measuring starch digestibility in horses.............................................................................................................10 In vivo study 1 Barley, oats, triticale and sorghum............................................................................................10 Experimental Design......................................................................................................................................10 Feeding and management ..............................................................................................................................10 Measurements ................................................................................................................................................11 Results............................................................................................................................................................11 Conclusion In vivo study 1 ............................................................................................................................12 In vivo study 2 - Triticale (two cultivars) and wheat (+/-enzymes)..................................................................13 Experimental Design......................................................................................................................................13 Feeding and management ..............................................................................................................................14 Enzymes.........................................................................................................................................................14 Measurements ................................................................................................................................................14 Results............................................................................................................................................................14 Discussion......................................................................................................................................................15 Ratio of groats to non-groat material .................................................................................................................17 Hull lignin as a factor influencing digestibility..............................................................................................18 Level of feeding .............................................................................................................................................19 Combining hull lignin and level of intake with ADF to predict DE ..............................................................20 Processing oat grain for animal feeding.............................................................................................................21 Measuring lignin content of oat hulls ................................................................................................................22 Visual appearance of low- and high-lignin oat grain .........................................................................................22 Digestibility of low- and high-lignin oat grain by horses ..................................................................................22 In vitro assays ....................................................................................................................................................24 Triticale as a new feed grain for horses .............................................................................................................24 Basis for selecting more digestible cultivars of oat grain ..................................................................................24 Exogenous feed enzymes...................................................................................................................................24 8. References..........................................................................................................................................................25

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Executive Summary Cereal grain is an important feed ingredient for most intensively managed horses and although cereals provide a valuable source of digestible energy their feeding to horses is always associated with some risk. The danger in feeding cereal grain to horses lies in the risk of incomplete digestion of starch in the small intestine and the possibility that significant amounts of starch can pass through to the caecum and colon (large intestine, or hind gut). Starch entering the hind gut is fermented very much more quickly than roughage and this rapid fermentation leads to accumulation of acidic end products and low pH. Just how the acid build up in the hind gut affects the horse is not clear but there is no doubt that acid accumulation in the hind gut is the primary cause of laminitis as well as many of the behavioural changes commonly associated with feeding grain to horses. At the start of this project there was no simple method for determining the intestinal or pre-caecal starch digestibility of different grains and/or the effectiveness of various processing methods designed to make cereal grains more digestible and therefore safer for horses. Several researchers have made measurements of pre-caecal starch digestion using inert markers as references or mobile nylon bags removed from the caecum but these methods are time consuming and subject to variability between animals. A simple and repeatable estimate of pre-caecal digestibility is essential if we are to understand and improve the safety of grain feeding and the development of such an assay was a primary focus for this project. An objective measurement of the starch digestibility characteristics of a raw or processed grain sample is a valuable tool in that it is not complicated by animal to animal variation or the level or method of feeding. Such a tool allows us to quickly build a data base on different feeds and the consequences of different processing techniques. Two in vitro assays were developed during the project. The first is an enzyme-based assay designed to predict pre-caecal starch digestibility and the second was established to measure the rate of fermentation. The theory behind these two assays is that we need to understand the risk of starch entering the hind gut as well as the rate at which that starch is likely to ferment when it comes into contact with the gut microbes. Once the two assays were developed we analysed a set of 55 samples selected to represent different grains, a range of different cultivars of each grain and, with selected cultivars, samples taken from geographically distinct locations. The results were analysed by comparing the enzyme digestibility of each grain with its potential rate of fermentation. What we were looking for was grains that were highly digestible in the enzyme assay (low risk of starch reaching the hind gut) or grains that had a reasonable level of enzyme digestibility and low rate of fermentation. By and large the grains with a high level of intestinal (enzyme) digestibility were also those that fermented most rapidly but there were a few exceptions. Two cultivars of triticale stood out as having the highest enzyme digestibility of all grains tested and far higher than would be expected from their rate of fermentation. On the basis of this result we went on to examine the digestibility of triticale in horses to confirm that the cultivar difference observed in vitro was also reflected in actual digestibility in the horse. We were very pleased that this was the case. Further in vitro testing of triticale has confirmed that the cultivars of Madonna and Abacus are significantly more digestible than Tahara. Studies of starch digestibility of the two triticale parents, Durum wheat and rye show that the digestibility of triticale lies in between that of wheat and rye with the higher digestibility cultivars such as Abacus being far closer to rye than Durum Wheat. This finding is one that will be of interest to plant breeders, feed manufacturers and to horse owners purchasing grain for supplementary feeding. The in vitro assays showed that oat grain starch was very highly digestible and also very rapidly fermented. This suggests that while oats is probably one of the safest grains in terms of a low risk of starch entering the hind gut, feeding strategies should still be employed that minimise this risk as any oat starch finding its way into the hind gut will ferment extremely rapidly. vi

Grains were ranked by the in vitro assay for enzyme digestion as follows: Highly digestible Oats and Cultivars of triticale

Cultivars of triticale, barley

Wheat

Poorly digestible Sorghum and maize

A second objective of the project was to examine the potential importance of oat hull digestibility in the utilisation of oat grain by horses. Studies in sheep and cattle had indicated that lignin content of the oat hull is genetically controlled and can have a significant effect (approximately 10 percentage units) on apparent digestibility. This research has been extensively reviewed as part of this final report and it is suggested that lignin content is likely to have an important effect on digestibility as well as gut fill and body weight. Unfortunately the results of the experiments on digestibility of oat grain conducted as part of this project were not clear cut and only preliminary results are reported. These results suggest that lignin could influence digestibility in the horse and that processing the oats by rolling could also be important in improving its digestibility. Exogenous enzymes that target the non-starch polysaccharides that make up the cell walls in the cereal grain have been found to be most effective in increasing starch digestion in poultry. Wheat is consistently improved by the use of exogenous enzymes and we therefore conducted an experiment to measure the response of a commercial exogenous enzyme mix on pre-caecal starch digestion in the horse. The enzymes appeared to work very well and significantly improved pre-caecal starch digestibility.

Implications This project has provided a new tool in the form of powerful in vitro assays. These assays will be useful for plant breeders, feed manufacturers and horse owners. It is recommended that the in vitro assays developed as part of this project be made available on a commercial basis for these user groups to test samples prior to use for horse feeding. It is expected that the in vitro enzyme assay will cost around $85/sample at this stage but could become cheaper (around $45/sample) if an NIR calibration can be developed. The identification of high-digestibility triticale for horses is an exciting new development for the horse industry as it was previously not known that this grain was potentially suitable for horses. Further work in collaboration with feed manufacturers is indicated to explore the effect of processing methods such as steam flaking in combination with selection of the more digestible cultivars of triticale. Unfortunately it is not possible to make a clear statement about lignin content of oat hulls and its nutritional value in horses. However, because of the importance of oat grain in the diet of horses, and the very good digestibility of oat starch, it is recommended that further work be undertaken to provide unequivocal data on this factor. The use of exogenous enzymes to improve pre-caecal digestion of wheat in the horse was clearly shown in this project. Exogenous feed enzymes are commercially available and it should be possible for feed manufacturers to take immediate advantage of this information.

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1. Introduction Cereal grains are commonly used to supplement the diet of horses when an increase in digestible energy intake is required. Cereal grains contain high levels of starch (roughly 60-80% DM) and relatively low levels of fibrous material and provide approximately 50% more digestible energy (on a weight/weight basis) than forages. While it is recognised that provision of proteins and minerals from grains is important, it is the efficiency with which the carbohydrate component of grain is utilised that is considered to be the major determinant of nutritive value. Apart from nutritive value, the risk of metabolic disorders is the other major consideration when feeding grain to horses. To understand why grain may cause metabolic problems it is necessary to understand how food is digested in the horse. Two major digestive processes occur in the horse. Food consumed by the horse is first exposed to acid digestion in the stomach and enzymatic digestion in the small intestine. Undigested material passing from the small intestine is then subjected to microbial activity resulting in fermentation of feed material in the caecum and colon. It is important to note that the end products of enzyme digestion and microbial fermentation are different. Enzyme digestion of starch in the small intestine gives rise to glucose that is absorbed across the intestine wall. In contrast fermentation of feed material in the hindgut gives rise to organic acids that can be absorbed across the gut wall. While both these digestive processes are normal, excessive or rapid production of acid in the hindgut (acidosis) may cause metabolic disorders. Although the enzymes secreted into the small intestine are capable of digesting all dietary starch, digestion in the small intestine may be incomplete because either a high intake of starch has exceeded the digestive capacity of the small intestine or the enzymes cannot readily access the starch in the grain. In this latter situation the microstructure of some grains is believed to restrict the access of enzymes to the starch. If significant amounts of starch pass undigested into the hindgut extensive fermentation may lead to the accumulation of acid within the gut. The resulting acidosis is the primary cause of a wide range of problems for the horse, the most devastating of which is laminitis. While acute laminitis is widely recognised as a major problem in the horse industry, it is likely that chronic acidosis is also responsible for many secondary conditions of lameness and poor performance. Fermentative acidosis, as a consequence of grain feeding, has also been associated with adverse behaviour in horses. The major problem associated with feeding grain to horses is the passage of large quantities of undigested starch to the caecum and hindgut. For this reason, the primary objective of safer grain feeding strategies is to maximise pre-caecal digestion of the starch component of the grain. This objective can only be achieved when the factors limiting starch digestion in the small intestine are clearly identified. The feeding of grain to horses can only be made safe when the intestinal digestibility of the starch in raw or processed grain can accurately be predicted for any given feeding regime.

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2. Starch digestion in the horse Cereal grains are fed to all classes of domestic livestock and the digestibility of starch in pigs, poultry, sheep and cattle has been intensively studied. Much is known about the site of starch digestion in these animals and published results indicate that starch digestibility varies considerably both between and within grain type. Published results also indicate that starch digestibility varies between animal species, for example poultry can completely digest starch in sorghum whereas digestibility of sorghum starch in cattle may be as low as 70%. This variability is believed to be due to the microstructure of the grain and the composition of the starch itself. Unfortunately there is a paucity of information available on the relative digestibility of the cereal grains in horses. The traditional measurement of in vivo starch digestibility involves measurements of intake and faecal output. However these measurements do not tell us anything about the site of starch digestion in the horse and do not differentiate between enzymatic digestion in the small intestine and fermentative digestion in the hindgut. Therefore most of the current digestibility data does not provide sufficient information on which to select and design more effective grain-feeding systems for horses. One of the primary aims of this project was to develop a reliable and accurate assay that will allow us to rank raw and processed grains for small intestinal digestibility in the horse.

Processing to improve small intestinal starch digestibility The selection of grains that have an inherently high intestinal digestibility of starch is one way of making grain feeding safer for horses. However these grains may not always be available or there may be situations where it is not economic to feed these grains. Grain processing can be used to increase the digestibility of starch and offers an alternative strategy to grain selection. Grain processing also increases the range of grains that can be considered for horse diets. While the benefits of grain processing are well documented for some livestock animals, for example steam flaking of sorghum increases the digestibility of starch in cattle from 70% to approximately 95%, the benefits of processing grains for horses have not received the same degree of attention. Physical treatments (hammer milling or rolling) are commonly used to process grains for horses. These treatments increase digestibility through a reduction in particle size of the feed and a subsequent increase the surface area exposed to enzyme attack. Whilst the established practices for processing grains are recognised, the efficacy of adding exogenous enzymes with the grain was also examined in this project. Enzymes are routinely used in the pig and poultry industry, and we believe that their use in horses warrants investigation.

Measuring starch digestion in the horse Measurement of the actual amount of starch digested in the small intestine (pre-caecal) is very difficult. In vivo methods rely either on surgical modification and the placement of a cannula in the ileum or caecum, or alternatively to slaughter horses at various times after feeding test diets to measure the amount of starch remaining in the different parts of the digestive tract. Although these in vivo methods have produced some valuable information the cost and logistical difficulties have severely limited the number of feeds that can be tested. In addition there is generally poor agreement between the results of different studies even when the grains are apparently the same (See Figure 1) More recent studies by Cuddeford (1999), and Pagan (1999) have moved towards techniques for ranking grains and processing methods using indirect measurements. The results of Cuddeford and his colleagues are based on caecal cannulation and the recovery of mobile bags containing grain samples that had been administered directly into the stomach using a naso-gastric tube. Starch digestibility was estimated by measuring the disappearance of starch from the bag. Results from these studies have shown quite clearly the benefits of micronisation in increasing small intestine digestibility of barley starch compared to dry rolled grain. The methods used by Pagan and colleagues are based on the glycaemic response, which is used extensively in human nutrition as a method of determining the effect of starch rich foods on postprandial blood glucose concentrations. Following the consumption of a test diet, blood samples are taken at regular intervals from horses to monitor the rise and fall of 2

blood glucose concentration. The peak glucose concentration and the area under the curve are related to starch digestion and glucose absorption. This technique has been useful in examining differences between grains (Pagan, 1999) as well as investigating the efficacy of grain processing. While the glycaemic index measurements are non-invasive and less expensive than measurements involving cannulation and the use of markers to measure digesta flow it is still a relatively expensive and timeconsuming measurement. A major disadvantage in all of the in vivo methods used to determine the intestinal starch digestion of different grains is the large variation that occurs between animals in the efficiency of starch digestion and glucose absorption. This variation in starch digestion is not unexpected given that the rate of eating, extent of chewing and also the amount of amylase produced in the intestines of horses has been reported to vary between animals (Meyer, 1995). Comparison of results reported from different studies is difficult because the amount and nature of the roughage component of the diet, the level of feeding and the number of meals fed per day varies between studies. Although it is important to account for differences between animals when designing feeding programs it is a source of variation that makes characterization of the diet extremely difficult. In vitro studies offer an alternative to in vivo studies with the advantages of being cheaper and allowing a large number of samples to be compared under a standard set of conditions.

Corn

Barley

Sorghum

100 90 80 70

Whole

60

Rolled Ground

50

Micronised

40 30 20 10 Arnold et al. 82

Householder et al. 77

Arnold et al. 81

Meyer et al. 93

Arnold et al. 82

Hintz et al 71

Hinkle et al. 83

Arnold et al. 82

Kienzle 92

Meyer et al. 93

Arnold et al. 81

Arnold et al. 82

Householder et al. 77

Meyer et al. 95

Kienzle 92

0 Meyer et al. 93

Small intestine starch digestion (% of intake)

Oats

Figure 1 Intestinal digestibility of starch in the small intestine of horses fed different grains processed in different ways showing the variability between studies in estimated digestibility. From Rowe et al. (2001). In our studies we have devoted considerable effort to the development of in-vitro assays that simulate the digestion of starch in the small intestine of the horse. In this project two separate in vitro assays were developed. The first was designed to predict the digestion of starch in the small intestine. The second assay was designed to emulate the fermentation of grain in the caecum and colon. The methods for both of these assays are described in the sections that follow. These two assays were then used to test a variety of grains and their cultivars. From the results of these assays, specific grains were selected for testing in vivo to establish the validity of the assays for use in horses.

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3. New in vitro assays for measuring starch digestion An in vitro assay to simulate digestion in the small intestine Starch occurs in two forms: α-amylose and amylopectin. Amylose is a linear polymer of α-1,4-linked glucose units. Amylopectin is a much larger polymer consisting of linear chains of α-1,4-linked glucose units with α-1, 6 branch points occurring, on average, every 12 glucose units (Lehninger 1970). Amylose and amylopectin are hydrolysed into small oligosaccharides of two or three glucose units by α-amylase secreted into the duodenum by the pancreas. The oligosaccharides are then hydrolysed to produce glucose by maltase (amyloglucosidase). The enzyme assay developed for these studies is based on these two major enzymes responsible for the digestion of starch in the duodenum, and is conducted under similar physiological conditions of pH (7) and temperature (390C) found in the intestines. Therefore ranking grains according to the amount of starch hydrolysed to glucose in this assay can be expected to provide a reliable in vivo index of intestinal starch digestibility for the test grains. The in vitro enzyme assay developed for these studies was adapted from the established method for starch determination (McCleary et al., 1997). The method of McCleary et al. (1997) is based on two enzymes: amyloglucosidase (AMG) and α-amylase (AA) and high temperature treatment to gelatinise the starch. Starch digestion was determined from the amount of starch hydrolysed to glucose. Various combinations of incubation times, pH conditions and temperature were examined. There were clear differences between the enzyme digestibility of sorghum, oats and barley and these differences were evident for all combinations of pH, temperature and incubation time. The conditions selected for standardisation of our assay were: an incubation time of 1 h; pH of 7; and a temperature of 39o C. The concept of this assay is summarised in Figure 2 below. Barrier between enzymes and starch Cell walls Protein matrix Protein bodies

Amylose Amyloglucosidase

Starch

Assay conditions set to limit enzyme activity

Factors affecting availability of starch

Temp 39 C Time 1 hr pH 7 Grinding 0.5 mm

Figure 2

Glucose

Particle size Gelatinisation Starch structure

Summary of the in-vitro test to measure intestinal starch digestion. The extent of glucose released is an indicator of the accessibility of starch to the amylolytic enzymes. 4

Protocol for in vitro enzyme digestion of starch Mill sample grain to pass through a 0.5mm sieve. Accurately weigh 100±2mg of sample (in duplicate) into the bottom of pre-weighed 20ml culture tubes (+ magnetic stirrer bar). Add duplicates of wheat starch standard. Add 200µl of 80% ethanol to each tube to whet sample. Add 3.0ml of MOPS/Enzyme solution to each tube*. Add 100µl of AMG to the bottom of each tube. Place tubes in heating/stirring block at 40oC for 1hr (1.5 on the stir speed setting). Remove tubes from block and adjust volume to 10ml (10g) with QH2O. Vortex, and centrifuge samples at 3000rpm for 10mins. Dilute sample supernatant 1 in 10 with QH2O (0.5ml sample + 4.5ml QH2O). Vortex. Transfer 100µl of diluted sample (in duplicate) to the bottom of 10ml culture tubes. Include triplicates of wheat starch control and glucose standards (0, 50, 100µg). Add 3.0ml of GOPOD reagent to each tube. Vortex, and place in shaking water bath at 50oC for 20mins. Remove from water bath and allow to cool for 20mins. Vortex. Read absorbance at 510nm against QH2O. *MOPS/Enzyme solution is made by adding 3.0ml of α-Amylase (B.licheniformis 3000 U/ml) to MOPS Buffer to a final volume of 90ml. For MOPS buffer preparation refer to Megazyme Total Starch Assay procedural booklet. Calculations: All results are expressed as %starch of ‘as is’ weight of sample. % Starch =

A x F x 1000 x 1/1000 x 100/W x 162/180

A = Absorbance of Sample – Absorbance of Reagent Blank F = 100 / (Absorbance for 100 g glucose) or 100 / (Slope of glucose standards curve) 1000 = Volume correction (0.1ml taken from 10ml) 1/1000 = Conversion from micrograms to milligrams 100/W = Factor to express “starch” as a percentage of sample weight W = Weight of ‘as is’ sample in milligrams 162/180 = Adjustment from free glucose to anhydro-glucose (as occurs in starch)

An in vitro assay to simulate fermentation in the caecum and colon Initial work on the development of an in vitro fermentation assay concentrated on the published method of Opatpatanakit (1994). This method was based on the disappearance of starch and some of the end products of fermentation (gas and volatile fatty acids). Incubations were conducted with small amounts of feed (0.1 g) in a sealed tube containing buffered fluid. Several adaptations to these methods were made to meet our requirements for this assay. The size of the fermentation vessel was increased to one litre and the sample size to 30 g to allow the system to handle whole processed grain samples. The second important change was the removal of glucose from the incubation mixture. Published methods generally included glucose, but in our studies it was found that the fermentation of glucose had a confounding effect on the fermentation of grain. During the development of this assay it became apparent that the measurement of a single end product of fermentation was unlikely to provide an adequate description of the fermentation of grain. Therefore the in vitro fermentation assay was extended to include the measurements of gas production, change in pH, volatile fatty acid and lactic acid production and the disappearance of starch during 5 hours of incubation at 39o C.

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Protocol for in vitro fermentation assay Dry unprocessed grain samples were first hammer-milled and sieved through a 0.5 mm screen. A sample of milled grain (30 g) was added to a culture jar (1 L). One hour prior to the commencement of the assay 375 ml of McDougalls buffer and urea (1.2 g/l) were added to the jar and stirred with a rod until the grain was completely wet. The jar was sealed and flushed with CO2 (via a three-way gas tap) and placed in a shaking water bath at 39o C. Rumen fluid* (2 L/animal) was collected from two steers (fitted with permanent rumen cannula) prior to feeding. The diet of the cattle (fed once/day am) contained approximately 50% mixed grain (oats, barley, wheat maize and sorghum) and 50% chaffed hay. The assay was initiated with the addition of 125 ml of rumen fluid to the culture jar. The assays were started approximately 30 min after the collection of rumen fluid from the cattle. Measurements of gas production and pH were made every hour. The incubation was stopped after 5 h with the addition of H2SO4 (15 ml 20% w/w) and a liquid sample was collected for the determination of volatile fatty acids and lactic acid. The remainder of incubation contents were dried and assayed for starch content. * Note the bacterial inoculum was obtained from fistulated steers rather than horses. The maintenance of fistulated horses was considered to be impractical. It is believed that the activity of the microbes in the rumen of cattle is comparable to the microbial activity in the caecum and colon of horses.

Results from in vitro assays – enzyme digestion and fermentation Initially a total of 55 grains were tested in both assays. All the major cereals grown in Australia were represented in this collection and the individual cultivars were sourced from a range of geographical locations and growing conditions. The results of the assays are presented in Table 1.The most obvious feature of these results is the variation both between and within grain types for starch fermentation and enzyme digestion. Some of the differences between grain types were expected, as results from in vivo studies reported in the literature indicate that starch digestibility is influenced by grain type. For example barley and sorghum are commonly fed to feedlot cattle and it is well known that dry-rolled barley is used more efficiently than dry-rolled sorghum (Saba et al., 1964). Incomplete whole-tract digestion of sorghum starch is the primary reason for the poor utilisation of this grain by cattle. Starch content of faeces collected from steers fed either dry-rolled sorghum or barley was 25% and 4% respectively (Watts and Tucker, 1993). Encouragingly the assay data provide an explanation for these in vivo results. The average in vitro fermentability of barley starch (67%) was clearly superior to the average fermentability of sorghum starch (44%). A similar difference in enzyme digestibility of starch between these two grains (barley 45% vs sorghum 28%) was also observed. Therefore considering that both enzyme digestibility and fermentability of sorghum starch is low it is not surprising that a significant amount of sorghum starch passes through the digestive tract of cattle undigested. The assay results for sorghum also suggest that this grain would not be suitable for horses. Oat grain is regularly included in the diet of horses and is believed to be the safest of all the cereal grains. This belief appears to be well founded, as the in vitro enzyme digestibility of starch in oats was the highest of the grains tested, with the exception of triticale. Therefore it is unlikely that sufficient oat starch would escape digestion in the intestine and reach the hindgut where it may cause metabolic problems. The data presented in Table 1 indicates that there is a considerable range in the fermentability and enzyme digestibility of starch within each grain type. These differences, (up to 16 percentage points for barley), may provide an explanation for some of the metabolic problems that are occasionally encountered when switching from one batch of feed to another, even though the grain type in each batch is the same. These results highlight the importance of developing a test that will allow us to predict the digestibility of cereal starch in horses.

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Table 1

Results from in vitro fermentation and enzyme digestion of starch in 55 finely milled (0.5 mm screen) samples of grain. Starch digestibility (%) (expressed as a percent of original starch content in grain)*

Number of cultivars

Grain type Barley Wheat Oat Sorghum Triticale Maize

Fermentation Mean 67 48 72 44 60 42

20 7 4 20 3 1

Enzyme digestion

Range 52-76 35-63 70-77 35-51 52-78

Mean 45 43 61 28 70 29

Range 37-53 37-47 57-66 23-33 65-76

Enzyme digestion (% starch present)

There was a weak positive correlation (r2=0.46) between the enzyme digestibility of starch and starch fermentation (Figure 3). The finding that this correlation is not very strong suggests that the grain characteristics that influence enzyme digestibility of starch are not necessarily the same characteristics that influence microbial fermentation. Therefore the results from the enzyme assay cannot be used to predict fermentability and vice versa. Given the importance of intestinal starch digestion in the horse we believe that the results from the enzyme assay will be more relevant than the fermentation assay in the development of successful grain-feeding systems for the horse.

80 70 Wheat

60

Barley Triticale

50

Oats Sorghum

40

maize

30 20 30

40

50

60

70

80

Fermentation (% starch present)

Figure 3

Relationship between fermentation and enzyme digestibility of starch in finely milled samples of grain.

In comparison with barley, the in vitro fermentability and enzyme digestibility of starch in triticale varieties was clearly superior (Table 1). The results for triticale were unexpected. This grain is not commonly fed to domestic livestock, yet the in vitro starch fermentation and enzyme digestion characteristics suggest that triticale may have superior qualities to either wheat or barley. Of particular significance is the high enzyme digestibility of starch in triticale (70%) which is clearly superior to 7

that of barley (45%) or wheat (43%). This result implies that the digestion of starch in the small intestine will be more efficient for triticale than for either barley or wheat. The assay results have highlighted the potential value of triticale as a suitable grain for inclusion in horse diets. Triticale may prove to be an even better grain than oats because it contains approximately 40% more starch than oats.

Effects of processing Rolling and hammer milling are the most common methods used to process grains for livestock and the benefits have been well documented. Physical processing increases digestibility because the grain is broken into smaller pieces increasing the area of starchy endosperm that is exposed to intestinal and microbial enzymes. Therefore it was important to determine whether our assays were responsive to changes in particle size. The relationship between particle size (four screen sizes) and grain type (three grains) was tested with the assays. Particle size had a significant effect on starch digestibility in the three grain types tested (Figure 4). As particle size decreased starch digestibility increased, which is consistent with results reported from in vivo studies.

Starch digestibility (%)

80

Oats

60

Barley

40

20 Sorghum

0 0.0

1.0

2.0

3.0

4.0

5.0

Screen mesh size (mm) Figure 4

Effect of processing (particle size) on the in vitro digestibility of starch in oat, barley and sorghum grain

8

Conclusions on in vitro assays The in vitro assays developed in this project have identified large differences between and within grains with respect to both susceptibility of grain to microbial fermentation and enzyme digestibility of starch. Encouragingly the results obtained with the assays were consistent with results reported from previous in vivo studies suggesting that these assays may provide the basis for developing a reliable method for evaluating the suitability of grain for horses. Given the importance of intestinal starch digestion in the horse we believe that the results from the enzyme assay will be more relevant than the fermentation assay in the development of successful grain-feeding systems for the horse. However because there is limited information currently available for grain-feeding studies with horses further in vivo calibration of the assays was required. Also, due to the wide variations found within grains, it was necessary to conduct these in vivo studies using the exact grains that we had tested in our assays for this calibration to be meaningful. The next section describes the first of two in vivo studies in which horses were slaughtered after being fed test grains for 14 days. Samples of digesta were taken from the digestive tract to measure starch digestibility.

9

4. Measuring starch digestibility in horses In vivo study 1 Barley, oats, triticale and sorghum The aim of the experiment was to investigate both the site and extent of starch digestion in the gastrointestinal tract of the horse. Oats, barley, sorghum and triticale were the test grains selected for our initial in vivo measurements of starch digestion. Results from the in vitro enzyme assay indicated that intestinal starch digestibility in order from highest to lowest should be oats and triticale high, barley intermediate and sorghum low. If this hypothesis is correct the amount of starch measured in caecal digesta of the slaughtered horses will be highest for the sorghum-fed horses and lowest for the horses fed oats and triticale.

Experimental Design Fourteen mares and geldings of mixed breed were purchased for this experiment. On their arrival at the stables all horses were weighed, checked for any signs of abnormality, identifying features recorded, identification collars attached and a broad-spectrum anthelmintic paste administered orally. Any animals that were considered to be unsuitable on the basis of health or temperament were excluded from entering the trial. Horses were allowed a seven-day adaptation period for acclimatisation to surroundings and adaptation to basal diets and feeding regimes. During this adaptation period, 2 horses were excluded from the trial on the basis of poor feed intake. The remaining 12 horses, ranging in size from 275 kg to 445 kg, were stratified according to body weight and within each stratum were allocated at random to one of four dietary treatments: barley, oats, sorghum or triticale (Table 2). All grains were dry-rolled. Horses were fed these diets for the 14-day experimental period prior to slaughter.

Table 2 Summary of Study Design Group Number of Test Grain Animals 1 3 Barley 2 3 Oats 3 3 Sorghum 4 3 Triticale

Variety Tantangara Yarran Western Red Tahara

Starch content of grain (%) 53 35 64 59

Feeding and management During the 7-day adaptation period all horses were fed lucerne chaff (2% of body weight per day) and increasing levels of a mixed grain diet starting at 500 grams per day and increasing to 1 % body weight per day. The mixed grains consisted of equal quantities of each of the grains to be used in the experimental diets (oats, barley, triticale, and sorghum). For the 14-day experimental period that followed, all horses were fed lucerne chaff (1.5% of bodyweight per day) and the grain to be tested at 1% bodyweight per day. The amount fed each day was divided between two equal feeds offered at 8 AM and 4 PM. Horses were fed individually in separate stables, and any refusals were weighed and recorded. Fresh drinking water was available at all times. Horses were exercised daily in a round yard while the stables were cleaned. During the final 5 days of the experiment, ytterbium acetate was added to the grain component of the diets as an inert marker for digesta flow. Ytterbium is a rare earth metal that is not absorbed from the digestive tract so the ratio of ytterbium to starch in digesta can be used to estimate starch digestion.

10

Measurements Horses were weighed on arrival at the stables and at weekly intervals thereafter. Feed intake was measured daily, and fresh faecal samples were collected on days 4, 6 and 8 of the experimental period. Faecal samples were mixed 50/50 by weight with distilled water and the pH of each sample was measured and recorded. At the conclusion of the experimental feeding period, horses were transported to the local pet food abattoir. Horses were slaughtered approximately 16 hours after their last feed and immediately after slaughter the digestive tract was removed and ties placed at the following sites: duodenum, ileum, junction between caecum and colon and between the proximal and distal colon. Each section of the gut was weighed before removing sub-samples for analysis of starch, dry matter, pH, VFA and ytterbium. The digestibility of starch in different parts of the digestive tract was estimated from the ratio of ytterbium to starch in digesta samples.

Results Table 3 The apparent digestibility of starch prior to the caecum in horses fed supplements of cereal grain. Measurements

Treatment diets Oats Sorghum 1223 2498

Barley Triticale Starch intake g/d 2041 2449 Apparent digestibility of 96 97 64 97 starch prior to caecum (%)* Apparent digestion of starch 1959 1186 1598 2375 prior to caecum (g/d) Starch entering the caecum 82 37 900 74 (g/d) *Digestibility of starch was estimated from the amount of starch that had disappeared between the mouth and the caecum, and expressed as a % of starch intake. The results from the slaughter study (Table 3) indicate that the pre-caecal digestibility of sorghum starch was considerably lower than the pre-caecal digestibility of starch in the other grains tested. The low digestibility of sorghum starch was consistent with the in vitro assay results (Table 5). However the in vitro assay results also predicted that the pre-caecal digestibility of barley starch should be lower than the digestibility of starch in either triticale or oats but this difference was not obvious in the slaughter data. The lack of agreement between the enzyme assay and the slaughter results may have been due to the length of time that elapsed between the final feed and time of slaughter (16h). During this time, starch reaching the caecum will have been exposed to microbial enzyme attack and so the starch in the more fermentable grains may have been almost completely fermented. This problem was anticipated and measurements of acidity (pH) and VFA concentration in caecal digesta were made as it was expected these results would provide an indirect indication of the amount of starch reaching the caecum. The results for caecal pH and VFA concentration are presented in Table 4 and clearly indicate that compared with oats, triticale and barley, more starch was reaching the caecum of horses fed sorghum. Total VFA concentration in the caecum of the sorghum-fed horses was 35% higher than VFA concentration in the caecum of horses fed the other grains. The pH and concentration of VFA in caecal digesta were comparable for horses fed oats, sorghum and triticale diets but again too much time may have elapsed after the final meal for differences to be detected. Further in vivo studies are required to determine whether the in vitro enzyme assay can accurately predict the intestinal digestibility of starch in the more digestible grains.

11

Grain supplements were fed as a fixed percent (1%) of bodyweight so the large variation in starch intake between the dietary treatments (Table 3) was due mainly to the different starch contents of the grains fed in this study (Table 2). The most significant feature of the results reported in Table 3 is the large amount of starch that was apparently digested prior to the caecum in horses fed triticale. This is an important result for several reasons. Firstly, the enzyme assay predicted that triticale starch should be highly digestible in the intestine of the horse and the slaughter data has confirmed this prediction. Secondly, triticale is not currently used in horse diets so this finding has important implications for the horse industry. Finally, the results indicate that provided starch is readily accessible to enzyme attack, the intestines in the horse have a high capacity to digest starch. The pH of faeces provides an indirect indication of pH conditions in the caecum and so this parameter was monitored during the grain-feeding period. The results presented in Table 4 show that faecal pH was highest for the triticale-fed horses which is a further indication that most of the triticale starch was digested prior to the caecum.

Table 4

Acidity measurements in caecal and faecal samples collected from horses fed supplements of cereal grain.

Measurements Barley 6.63 6.33 6.53

Faecal pH - Day 4 Faecal pH – Day 6 Faecal pH – Day 8 Caecal pH – at slaughter Caecal VFA (mmol/L)

Table 5 Grain Type

Barley Oats Sorghum Triticale

6.60 67

Oats

Treatment diets Sorghum 6.43 5.63 6.15 5.90 6.70 6.75 6.70 67

Triticale 6.63 6.78 7.15

6.40 93

6.70 69

Comparison of in vitro assay estimates of starch digestion and estimates of precaecal starch digestion in slaughtered horses. Cultivar

Tantangara Yarran Western Red Tahara

Starch digestion (%) In vitro assay Enzyme Fermentation

In vivo Pre-caecal

48.1 66.5 32.6 65.0

95.7 97.3 63.7 97.1

69.0 73.9 44.8 76.9

Conclusion In vivo study 1 The predictions from the in vitro assay were largely confirmed by results from the slaughter study. Therefore we believe that the enzyme assay developed in this project will prove to be very important in the development of successful grain-feeding systems for the horse. Of particular note was the confirmation that most of the starch in triticale was digested in the intestine. This finding has important implications for the horse industry. More sensitive in vivo studies are required to determine whether the in vitro enzyme assay can accurately predict the intestinal digestibility of starch in the horse for more digestible grains.

12

In vivo study 2 - Triticale (two cultivars) and wheat (+/-enzymes) Results from our in vitro assay indicated a large variation in the digestibility of different cultivars of triticale. For this reason, a second experiment was conducted to investigate the starch digestibility in vivo of two cultivars of triticale determined by our in vitro assay as being of high and low digestibility relative to each other. If this difference between samples of triticale turns out to be a geneticallylinked then it provides a very important lead for plant breeders as well as for-end users keen to purchase grains with high starch digestibility. The second aspect of this study was to investigate the potential application of exogenous enzymes for improvement of starch digestion of grains not often used in horses. Wheat is a grain with a relatively low digestibility even in poultry and the risks associated with feeding this grain to ruminants are well known. In poultry the poor digestibility has been linked to the presence of non-starch polysaccharide components of the cell walls increasing viscosity. The digestibility of these “low ME” wheats can be considerably improved by the use of exogenous enzymes that break down the NSPs. This method of adding exogenous enzymes to the diet of poultry to improve starch digestibility is very widespread and it is important to know whether this approach will be effective as a method of improving starch digestibility in the horse. The effect of exogenous enzymes active against the NSP fraction is partly through modified physical characteristics of the digesta and it is therefore unlikely that efficacy of exogenous enzyme use can be examined using the in vitro assay described above. For this reason we included the treatment in the current in vivo experiment.

Experimental Design Sixteen mares and geldings of mixed breed and aged between 3 and 26 years were purchased for this experiment. On their arrival at the stables all horses were weighed, checked for any signs of abnormality, identifying features recorded, identification collars attached and a broad-spectrum anthelmintic paste administered orally. Two animals that were considered by the examining veterinarian to be unsuitable on the basis of health were excluded from entering the trial. Horses were allowed a seven-day adaptation period for acclimatisation to surroundings and adaptation to diets and feeding regimes. During this adaptation period, 1 horse was excluded from the trial on the basis of poor health. The remaining 13 horses, ranging in size from 332 kg to 452 kg, were stratified according to body weight and within each stratum were allocated at random to one of four dietary treatments: Triticale Madonna, Triticale Tahara, wheat or wheat with enzyme. All grains were hammer milled, and this was to reduce the risk of feeding wheat grain, to decrease variability of mechanical chewing between horses due to age differences (several old horses had worn teeth) and to allow maximum opportunity for the enzyme included to have access to the starch content of the grain. Horses were fed these diets for the 14-day experimental period prior to slaughter. Table 6

Summary of sudy deign, starch content of the different grains, enzyme starch digestibility measured in vitro and rate of fermentation (% starch fermented in 3 hours).

Group

Number of Animals

1 2 3 4

3 3 4 3

Test Grain

Triticale Triticale Wheat Wheat + enzyme

Variety

Starch content of grain (%)

Madonna Tahara Janz Janz

55.5 57.2 56.3 56.3

13

In vitro enzyme assay (starch digested) 65 76 43

In vitro fermentation (% starch) 52 78 58

Feeding and management During the 7-day adaptation period all horses were fed lucerne chaff mixed with increasing levels of test grain starting at 500 grams per day and increasing to 1 % body weight per day. For the 14-day experimental period that followed, all horses were fed lucerne chaff (0.65 % of bodyweight per day) and the grain to be tested at 1% bodyweight per day. The amount fed each day was divided between two equal feeds offered at 8 AM and 4 PM. Horses were fed individually in separate stables, and any refusals were weighed and recorded. Fresh drinking water was available at all times. Horses were exercised daily in a round yard while the stables were cleaned. During the final 5 days on the experimental diets ytterbium was added to the grain component of the diets as an inert marker for digesta flow.

Enzymes Water or enzyme solution (0.4% of bodyweight per day) was added to all feeds 30 minutes prior to feeding. Group 4 horses had enzymes included in their diets whilst horses in groups 1, 2, and 3 received water only. The enzyme solution contained a mixture of Bio-Feed Alpha (500 U/g βglucanase and 150 U/g α-amylase) and Bio-Feed Wheat (1000 U/g xylanase) and was included at a rate of 800ml and 600ml per tonne of feed respectively (Novozymes Pty Ltd, North Rocks, NSW, Australia).

Measurements Horses were weighed on arrival at the stables and at weekly intervals thereafter. Feed intake was measured daily, and fresh faecal samples were collected on days 5, 6 and 9 of the experimental period. Faecal samples were mixed 50/50 by weight with distilled water and the pH of each sample was measured and recorded. At the conclusion of the experimental feeding period, horses were transported to the local pet food abattoir. Horses were slaughtered approximately 16 hours after their last feed and immediately after slaughter the digestive tract was removed and ties placed at the duodenum, ileum, the junction between caecum and colon and between the proximal and distal colon. Each section of the gut was weighed before removing sub-samples for analysis of starch, dry matter, pH, VFA and ytterbium. The digestibility of starch in different parts of the digestive tract was estimated relative to ytterbium concentration.

Results The key results are summarised in Tables 7 and 8. Table 7 shows differences between treatment diets in the amount of starch remaining in the caecum relative to the inert marker 16 hours after the last feed of grain. The intake of starch and rate of fermentation was similar for all grains. There was more starch entering the caecum in horses fed the triticale Tahara diet and the wheat diet than in horses fed triticale Madonna or the wheat with the exogenous enzymes added. Table 7

The apparent digestibility of starch prior to the caecum in horses fed supplements of cereal grain.

Measurements

Treatment diets Tahara Wheat

Madonna Starch intake g/d Apparent digestibility of starch prior to caecum (%)* Apparent flow of starch entering caecum (g/d)

2317

2260

2533

Wheat + Enzyme 2427

99.5

96.5

95.3

99.1

12

80

116

22

14

*Digestibility of starch was estimated from the amount of starch that had disappeared between the mouth and the caecum, and expressed as a % of starch intake. The faecal pH, caecal pH at slaughter and caecal VFA concentrations all showed differences consistent with measured differences of starch flow to the caecum. In the Tahara and wheat treatments there was lower pH in both faeces and caecal digesta than measured in horses fed Madonna and the wheat with enzymes. In those horses and treatments with low pH (Tahara and wheat) there were the highest concentrations of VFA. There were also higher concentrations of VFA in the caecal digesta of horses fed wheat plus enzymes. Table 8

Faecal and caecal pH measured in horses fed two cultivars of triticale and wheat grain with or without a mixture of exogenous enzymes active against NSP and starch.

Measurements

Treatment diets Tahara Wheat

Madonna Faecal pH - Day 5 Faecal pH – Day 6 Faecal pH – Day 9

6.95 6.82 6.72

6.64 6.58 6.57

6.57 6.46 6.36

Wheat + Enzyme 6.68 6.59 6.85

Caecal pH – at slaughter Caecal VFA (mmol/L)

6.93 38.2

6.38 67.6

6.61 70.4

6.75 62.4

Discussion The in vivo measurement of pre-caecal starch digestion and the fermentation characteristics in the caecal and faecal samples are consistent with the in vitro predictions for Madonna, Tahara and for wheat. That is the in vitro enzyme assay ranked the pre-caecal digestibility in the exact order in which it was measured for these three grains. In addition there was a close regression relationship between the predicted pre-caecal starch digestibility and measured flow of starch to the caecum. It is encouraging that the in vitro assay accurately predicted differences between the two cultivars of triticale as well as between the triticale and wheat. 140

Starch passing to caecum (g/d)

120

Wheat y = -2.55x + 221 R2 = 0.74

100

Barley

80

Triticale (Tahara)

60

40

Oats

20 Triticale (Maddona)

0 40

45

50

55

60

65

70

75

80

In vitro enzyme assay (% starch digested)

Figure 5

In vivo enzyme starch digestion assay and the amount of starch flowing to the caecum measured in vivo. 15

The in vitro assay is not able to measure likely changes in digestibility due to the action of exogenous enzymes. The effects of exogenous enzymes need to be measured in vivo. It is clear that a similar improvement in starch digestion is possible in the horse as is now well documented in poultry. The only concern about the action of the enzymes is the high VFA concentration. This high VFA concentration is similar to findings in poultry where enzymes have increased both starch digestion and also the rate of fermentation in the caecum. The increased fermentation could be due to the effect of enzymes on the cell wall material increasing starch exposure to microbial fermentation. When the results of the two in vivo studies are considered together (with the exception of sorghum) there is a very good relationship (R2 = 0.74) between the in vitro prediction of enzyme starch digestion and the amount of starch passing to the caecum measured in vivo. It is clear that sorghum is very much less digestible than the other grains with approximately 900 g/d starch passing to the caecum compared with around 100 g/d for the next highest (wheat). While the in vitro assay correctly shows sorghum as a feed of low potential digestibility in the small intestine of the horse the actual digestibility appears not to be on a linear scale with other grains.

16

5. Digestibility of oat grain Oat grain has long been recognised as an excellent ingredient in the diet of humans and animals. Shaw (1907) declares that “viewed from the standpoint of adaptation for feeding live stock no cereal grain grown in [the USA] compares with the oat”. However, while oats is widely used as a feed grain for ruminants and horses its value is often discounted because of the variability in its nutritional value. Variability in the nutritional value of oat grain has been the subject of numerous studies (e.g. Pickering et al. 1982; Crosbie et al. 1985; Margan et al. 1987; Rowe and Crosbie 1987; Oddy et al. 1990) which show clearly that there are a number of factors to be considered if we are to accurately predict the performance of animals given this feed. This section of the report includes a review of literature and some experiments covering utilisation of oats in ruminant animals and also discusses an experiment conducted as part of the current project. It is recognised that ruminants and equids have different digestive anatomy and physiology but we believe that the common element of fermentative digestion of NSP ‘fibre’ components are likely to be similar in both animal species.

Ratio of groats to non-groat material The dominant factor determining nutritional value of any particular sample of oat grain is the ratio of groats to non-groat material. The non-groat fraction consists mainly of the fibrous hull but can also include variable amounts of other material such as weeds and seeds from other plants as well as chaffed head, leaf and stem material not separated from the grain in the harvesting process. Samples of oat grain can also contain significant quantities of free groats if the hulls and groats are separated during harvesting and when this occurs the nutritive value of the sample can be improved considerably. The amount of non-groat material and the presence of free groats in an oat sample can be seen easily, but unfortunately it is not always accounted for in ascribing variability to oat grain quality. Increasing amounts of non-groat material tend to decrease the density of an oat sample and is almost certain to decrease its nutritional value (Pickering et al. 1982). For this reason oat grain is sometimes traded on the basis of hectolitre or bushel weight. Oddy et al. (1990) reported that bulk density measured as kg/hL or as weight per hundred grains was poorly correlated with acid detergent fibre (ADF) and these authors suggested bulk density is of little or no value in predicting oat grain quality. This conclusion regarding hectolitre weight, and therefore groat to hull ratio, is based on a range of diets for sheep constructed from oat fractions to match the extreme range of chemical compositions of oats grain found ‘in the field’ (Oddy et al. 1990) and so overlooks the value of grain density as a predictor of groat to hull ratio in clean samples of oats. The study of Oddy et al. (1990) with the constructed diets shows the importance of accounting for the variable hull to groat ratio. Although the relationship with digestible energy is very good (Figure 6) it is important to realise that the most digestible ‘oat’ diet in this study was in fact pure groats and that the least digestible ‘oat ‘ contained approximately 75% oats and 25% additional oat hulls. Therefore while this is a very important predictive relationship when buying or selling oats with either a lot of free groats or an unusually high proportion of non-groat material, it does not necessarily provide an accurate description of differences among ‘normal’ oats with only small contents of free groats or nongroat material. The problem of using ADF to predict the digestibility of ‘normal’ oats is shown in Figure 7. The data have been taken from three experiments with sheep in which the digestibility of different oat samples were measured; two samples were the true oat grain samples described by Oddy et al. (1990), eight values come from the data of (Margan et al. 1987) and two come from Rowe and Crosbie (1988). The challenge is to understand the causes of the variability shown.

17

Digestible energy (MJ/kg DM)

18

17

16

15 y = -0.021x + 18.9

14

R2 = 0.94

13

12 0

50

100

150

200

250

300

Acid detergent fibre (g/kg)

Figure 6

Relationship between ADF and digestible energy of oat diets measured in sheep feeding experiments as reported by Oddy et al. (1990).

Digestible energy (MJ/kg DM)

18 17 16 15 14 y = -0.012x + 16.7 R2 = 0.18

13 12 50

100

150

200

250

Acid detergent fibre (g/kg)

Figure 7

Relationship between ADF and digestible energy of oat grain without added groats or hulls. Two values are the grain samples with 180 g ADF/kg and 25 g ADF/kg of Oddy et al. (1990), eight are taken from the data of Margan et al. (1987) and two from Rowe and Crosbie (1988).

Hull lignin as a factor influencing digestibility Crosbie et al. (1985) reported that there was considerable variation in hull lignin content between different cultivars of oat grain and that these differences are predominantly genetically controlled. Most cultivars were found to be either ‘high’ lignin with around 3% lignin in the whole grain and around 6 – 10 % in the hull fraction, or ‘low’ lignin with around 1% lignin in the whole grain and 1 – 3% in the hull. This discovery provided a possible explanation for the experience of many livestock managers that certain cultivars of oat grain were better for livestock production than others. Rowe and Crosbie (1988) then showed that the differences in hull lignin content in high- and low-lignin oat cultivars had a very significant effect not only on the digestibility of the hulls but of the whole grain. With ADF, the ratio of hulls to groats, and the concentrations of protein and ash all held constant 18

between two cultivars, Rowe and Crosbie (1988) found that a difference of approximately 20 g lignin per kg grain resulted in an improved digestibility of the order of 15%. An independent study by Margan et al. (1987) reported significant differences in digestibility between the grains from two cultivars, Coolabah and Cooba. They also differed in lignin content, though that was not the reason they were chosen for the study, and so their results add considerably to appreciation of the importance of hull lignin content. Table 8 summarises the effect of lignin content on digestibility of oat grain in sheep fed principally oats at a rate of approximately 1.6% of body weight per day. While the ADF content of grains of the high-lignin cultivars is slightly higher, this difference would only explain approximately one third of the change in DE with ADF indicated by the predictive equation shown in Figure 6. Table 8

Analysis of the major components (g/kg dry matter) of four samples of oat grain and the digestible energy (DE, MJ/kg dry matter) obtained with sheep fed these diets at rates equivalent to approximately 1.6% of body weight per day. Data for ‘Cooba’ and ‘Coolabah’ were derived from the study of Margan et al. (1987) and for ‘Murray’ and ‘Mortlock’ from Rowe and Crosbie (1988).

Cultivar Low-lignin Murray Cooba High-lignin Mortlock Coolabah Average difference between high- and low-lignin cultivars

Lignin

ADF

DE

8 10

133 110

15.6 15.4

23 30 17.5

144 150 25.5

14.0 13.5 1.8

Level of feeding With all feeds it is accepted that as the level of feeding increases there is a slight decrease in the efficiency of digestion, and DE/kg of feed consumed is reduced. For most feeds the decrease in digestibility with increasing level of feed intake is relatively minor. However, in the case of whole oats, level of intake appears to be an important factor in sheep and cattle as shown in Figure 8 which uses data from Margan et al. (1987). Level of feed intake is also likely to be a factor in determining the digestibility of oat grain in horses.

Dige s tible e ne rgy (k g/M J DM )

16

15

y = -0.85x + 15.6 R2 = 0.81

14

13

12 0

0.5

1

1.5

2

2.5

3

3.5

Level of intake (g/100 g body w eight)

Figure 8

Decrease in digestible energy value of oats grain with increasing intakes by sheep. Data are for Coolabah oats as reported by Margan et al. (1987)

19

Combining hull lignin and level of intake with ADF to predict DE When we account for both level of feed intake and lignin content of oat hulls in the prediction of digestible energy of oats grain we are able to explain much of the variability shown in Figure 7. Figure 9 illustrates the same data as those used in Figure 7, but level of feed intake has replaced ADF as the independent variable and grains have been separated on the basis of their lignin content. The two data points representing 18% ADF and 25% ADF oats are shown as the filled triangles each with a single line through them; that they fall either side of the line of best fit suggests that the prediction of DE based on feed intake and lignin could be further improved by considering ADF as a measure of groat to non-groat material. A robust method of predicting DE would be to use the predictive equation based on the results of Oddy et al. (1990), add 1.5 MJ/kg if the grain is low lignin, and then adjust DE for level of intake by 0.86 MJ/kg for each 1% of body weight consumed above or below the 1% base level used by Oddy et al. (1990).

Digestible energy (MJ/kg dry matter)

17 y = -0.85x + 16.9 R2 = 0.95

16

15

14

y = -0.87x + 15.6 R2 = 0.67

13

12 0

0.5

1

1.5

2

2.5

3

3.5

Intake (g oats/100 g bodyw eight)

Figure 9

Prediction of digestible energy of oat grain taking into account level of feeding and grains as high-lignin (▲) or low-lignin (○). As for Figure 2, eight values (plain triangles and circles) are taken from the data of Margan et al. (1987), two (closed triangles with a single horizontal line) are from Oddy et al (1990), and two (triangles and circles with an X) are from Rowe and Crosbie (1988).

Hull lignin content and gut fill in sheep and cattle A feeding experiment was conducted in cattle by May et al. (1989) using low- and high-lignin oat cultivars, Murray and Mortlock respectively, and demonstrated a significant effect of high-lignin oats on gut fill and dressing percentage. There was accumulation of low digestibility roughage in the rumen and a much larger rumen in cattle fed high-lignin Mortlock grain. Similarly in sheep fed supplements of high or low-lignin grain there was significantly higher weight of digesta in animals fed the lower digestibility high-lignin grain (see Table 9). Both sets of data in Table 3 suggest that lignin content of the oats could make a difference of around 10 kg for a 500kg animal.

20

Table 9

Differences in gut fill in cattle and sheep fed high-lignin (Mortlock cultivar) or lowlignin (Murray cultivar). Data taken from May et al. (1989a) and Rowe and Coss (1992) Mortlock

Lignin (g/kg) In vivo dry matter digestibility (%) Measured in sheep (maintenance) Measured in cattle (intake approx. 2.2% of body weight) Dressing % measured in cattle (Carcase weight as % of body weight) Weight of reticulo-rumen (kg) measured in sheep

Murray

Signif. of diff (P)

24

11

70.2 64.1

82.4 71.6

49.8

51.8

0.003

4.70

3.71

0.01

The results of Johnson et al., (1998) and Pagan (2001), show that the gut contents and weight of the horse, like sheep and cattle, increases in response to consumption of lower digestibility feeds. It is therefore almost certain that horses fed lower lignin oat grain will have less gut fill and be lighter than horses fed an equivalent amount of high-lignin grain.

Processing oat grain for animal feeding The question of whether or not there is any advantage to be gained from processing oat grain before feeding it to cattle and horses is a very important one in determining its suitability for on-farm use in situations where no grinding and mixing equipment are available. We are aware of three studies in cattle examining this. The consistent finding in all three studies was that there is little or no improvement in digestibility or animal performance in response to processing oat grain by rolling or hammer milling. Toland (1976) reported improvements of between 60 and 100% in the digestibility of wheat and barley starch as a result of rolling compared to feeding whole grain, but found no significant benefits in the case of oats. It is therefore safe to conclude that oat grain can be fed whole to cattle without any risk that it will be digested or utilized inefficiently for production. The data of May et al. (1989b) even suggest possible benefits in whole grain feeding to achieve slightly higher consumption. As is the case with all cereal grains, there is no benefit in rolling or grinding oats before feeding it to sheep. A study on the effect of rolling on oats on their subsequent digestibility by horses (Wilson 2001) suggests that there are significant improvements in digestibility in response to this form of processing - 50.5% for rolled oats vs 44.6 % in the case of unprocessed whole grain. Table 10

Categorisation of oat cultivars tested with a combination of wet chemistry and the Crosbie colour test for lignin content. Information for this table was obtained from Crosbie et al. (1985), G.B. Crosbie (pers. comm.) and A.G. Kaiser (pers. comm.). High lignin Dalyup Echidan Kalgan Moore Mortlock Coolabah Graza-50 Pallinup Dumont Bettong Cleanleaf Panorama-5

Low lignin Irwin Murray Swan Yilgarn Cooba Yarran Graza-70 Marloo Culgoa Amby Bimbil Carbeen Nile 21

Medium West

Measuring lignin content of oat hulls Measurement of lignin content by wet chemistry is time consuming and expensive, and if the analysis is not made by an experienced technician it can yield uncertain results. Probably due to the painstaking task of measuring large numbers of lignin concentrations, combined with the difficult process of hand separation of hulls from groats, there are as yet no NIR prediction equations for hull lignin. Although not quantitative, a test developed by Crosbie and his co-workers (Rowe et al., 2001) provides a quick and accurate indication of whether samples of oat grain fall into the category of ‘high-lignin’ or of ‘low-lignin’. The Crosbie oat hull assay is based on the pink colour that develops when a solution of phloroglucinol stains the lignin component of the hulls, and is most easily made by adding approximately 2 ml of a phloroglucinol solution to about 10 whole oat grains or to separated hulls from 3 to 5 grains. The solution is prepared by dissolving 1 g of phloroglucinol in 80 ml 2 M HCl plus 20 ml ethanol, and then filtering. The phloroglucinol solution should be kept in a dark bottle in a refrigerator (4 oC) and can be used for about 1 month if properly stored. Sufficient solution is added to cover the grains and/or hulls placed in a white ice-block tray; the volume of each well easily accommodates the sample and solution. The multiple compartments facilitate inclusion of known high- and low-lignin samples, and the white background enables ready detection of differences in colour. A list of major cultivars of oat grains categorised by hull lignin content by the colorimetric method described above is summarized in Table 10. About half of samples of the West cultivar, categorised as ‘medium’ had high concentrations of lignin and half had low concentrations. When individual grains were grown to produce seed the low-lignin seeds produced grains with low hull lignin, and the highlignin seeds produced high-lignin grain.

Visual appearance of low- and high-lignin oat grain The visual assessment of all grains for evidence of mould or moisture damage is important because fungal contamination can have adverse effects on feed intake as well as a range of toxic effects. The presence of fungus is most easily identified as darker colorations because mycelium and spores are often black in colour. There has therefore been a preference amongst buyers for bright, light yellow colours in oat hulls. It is important to note that the hulls of low-lignin oat grain are often slightly darker than that of high-lignin grains, possibly due to accumulation of pigmented phenolics in the hull of low-lignin cultivars. This darker coloration of low-lignin hulls is natural and does not signify fungal contamination. In order for buyers to have complete confidence when purchasing low-lignin grains of slightly darker colour it may be necessary to develop a quick assay for fungal contamination to complement the rapid colour method for differentiating between high- and low-lignin cultivars.

Digestibility of low- and high-lignin oat grain by horses An objective of this project was to investigate the role of lignin in determining the digestibility of oat grain in horses. There have been some problems in interpreting the results of this study. A partial analysis of the data using data from 4 horses suggests that there was a slightly higher digestibility of the low-lignin Yarran cultivar than the lower lignin mortlock cultivar (50.6% vs 44.4%).

Conclusions - oats We consider that breeding and cultivation of low-lignin oat cultivars should be encouraged by grain users being prepared to pay more for the higher digestibility grain than for the high-lignin low digestibility cultivars. There are currently a number of low-lignin oat cultivars grown commercially and we are not aware of problems, such as lodging or rust resistance, being linked to lower levels of lignin in the hull. It is essential that we obtain unequivocal data for horses on the importance of hulllignin, processing and level of intake. 22

6. Triticale In addition to the in vitro and in vivo studies reported above on the digestibility of triticale we have conducted further tests on low- and high-digestibility cultivars to determine whether the differences are consistent between different samples of each cultivar. These results are summarised in Table 11 below. Table 11

In vitro digestibility measured in different samples of three cultivars of triticale.

Average in vitro digestibility (%)

Tahara Abacus Madonna 52 65 57

Standard error of mean

Number of assays

4.8

6.3

4.6

5

3

4

Ttriticale is a cross between durum wheat and rye. It is therefore reassuring to find that the in vitro digestibility of triticale lies between that of durum wheat and rye. The cultivars of Madonna and Abacus have digestibilities closer to that of rye while the digestibility of Tahara is closer to durum wheat (see Figure 10). These results suggest that it will be possible to select for cultivars of triticale that are highly digestible by the horse. Even the use of cultivars such as Abacus and, to a lesser extent Madonna, offer some immediate opportunities for the horse industry. In the same way as variability has reduced the value of oats it is possible that variability between triticale cultivars may diminish the perceived value of triticale – particularly if the reasons for the variability of performance of horses fed triticale are not understood by processors and feeders.

In vitro e nz ym e a ssa y (% sta rch dige ste d)

80 70 60 50 40 30 20 10 0 Durum

Figure 10

Tahara

M adonnah

Abacus

Rye

In vitro digestibility of the parents of triticale (rye and durum wheat) and of three cultivars of triticale.

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7. Implications In vitro assays Two new assays for the horse feed industry and, particularly the enzyme assay to predict pre-caecal starch digestion will be useful in helping to ensure that any person feeding grain to a horse will be able to do so knowing the relative safety of the grain for the feeding purpose. In its simplest form the assay has helped to confirm differences between grains with respect to starch digestibility and we suggest the following ranking: Highly digestible Oats and Cultivars of triticale

Cultivars of triticale, barley

Wheat

Poorly digestible Sorghum and maize

The in vitro assay will also find a place in quantifying the effects due to processing of horse feeds and providing a comparison of ‘before and after’. As illustrated by Channon and Rowe (2001) processing using the steam flaking can produce variable results due to differences in the extent of cooking and fineness of the flake.

Triticale as a new feed grain for horses The results of the in vitro and in vivo tests have identified triticale as a grain highly digestible by the horse. There appear to be certain cultivars of triticale that are more digestible than others and this information will be of value to plant breeders, horse owners and feed manufacturers to produce and use the most appropriate grains for horses.

Basis for selecting more digestible cultivars of oat grain The review of oat digestion and the preliminary study in the horse has identified the possible role of hull lignin altering digestibility and utilisation of the grain by horses. The question of relative importance of hull lignin, processing (rolling and/or steam rolling) and the level of feed intake in determining gut fill and digestible energy deserves further experimentation. Since oat grain is so widely used as a feed for horses it is important to have a clear understanding of the factors influencing its utilisation. If hull lignin content emerges as an important factor influencing its digestibility then the rapid test for hull lignin and the fact that many cultivars of oats are known to have low levels of lignin will assist breeders, growers and users to select and use the most appropriate oat grain for feeding to horses.

Exogenous feed enzymes The inclusion of exogenous enzyme preparation active against NSP and starch were shown to improve pre-caecal wheat starch digestion. This demonstrates the potential for using exogenous enzymes to increase the usefulness of a wider range of grains than are currently used in preparation of diets for horses. In other species such as poultry exogenous enzymes have been a cost-effective and efficacious way to improve nutritive value of wheat and barley. In situations where the choice of grain is limited and it is necessary to feed wheat then it is likely that exogenous enzymes may be an excellent way of improving the safety and effectiveness of feeding grain to horses.

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8. References Arnold, F. F. (1982). Precaecal, post ileal and total tract starch digestion in ponies fed corn, oats, barley or sorghum grain. M.S. Thesis, Texas A&M. Bird, S. H., Rowe, J. B., Choct, M., Stachiw, S., Tyler, P., and Thompson, R. D. (1999). In vitro fermentation of grain and enzymatic digestion of cereal starch. Recent Advances in Animal Nutrition in Australia 12. Crosbie, G. B., Tarr, A. W., Portmann, P. A. and Rowe, J. B. (1985). Variation in hull composition and digestibility among oat genotypes. Crop Science 25, 678-680. Cuddeford, D. (1999). Recent advances in equine nutrition. Recent Advances in Animal Nutrition in Australia 12, 99-105. Diez-Gonzalez, F., Callaway, T. R., Kizoulis, M. G., and Russell, J. B. (1998). Grain feeding and the dissemination of acid-resistant Escherichia coli from cattle. Science 281, 1666-1668. Hintz, H. F., Hogue, D. E., Walker, E. F., Lowe, J. E., and Schryver, H. F. (1971). Apparent digestion in various segments of the digestive tract of ponies fed diets with varying roughage-grain ratios. Journal of Animal Science 32, 245-248. Householder, D. D., Potter, G. D., and Lichtenwalner, R. E. (1977). Nutrient utilization in different segments of the equine digestive tract. In "Proceedings of the 5th Equine Nutrition and Physiology Symposium", pp. 44-45. ENPS, Missouri. Johnson, K., G., Tyrrell, J., Rowe, J. B., and Pethick, D. W. (1998). Behavioural changes in stabled horses given nontherapeutic levels of virginiamycin. Equine Veterinary Journal 30, 139-143. Kienzle, E., Radicke, S., Wilke, S., Landes, E., and Meyer, H. (1992). Praeileale Staerkeverdauung in Abhaengigkeit von Staerkeart und -zubereitung. In "Europaische Konfernz uber die Ernahrung des Pferdes", pp. 103-106, Hannover. Krueger, A., Kinden, D., Garner, H., and Sprouse, R. (1986). Ultrastructural study of the equine cecum during onset of laminitis. Am J Vet Res 47, 1804. Margan, D. E., Graham, N. M. and Searle, T. W. (1987). The energy value of whole oats grain in adult wether sheep. Australian Journal of Experimental Agriculture 27, 223-230. May, P. J., Barker, D. J., Jones, W. M., Snowdon, J. M. and McMullen, G. R. (1989b). Effect of processing oats in finishing diets for young cattle. Division of Animal Production, Department of Agriculture WA, Annual Report 1988/89, pp. 106-108. May, P.J.., Barker, D. J., Jones, W. M., Snowdon, J. M. and McMullen, G. R. (1989a). Oat grain of high or low lignin content in diets for finishing beef cattle. Division of Animal Production, Department of Agriculture WA, Annual Report 1988/89, pp. 103-105. Meyer, H. (1993). Investigation on preileal digestion of oats, corn and barley, starch in relation to grain processing. In "Proceedings 13th Equine Nutrition and Physiology Symposium", pp. 92-97. ENPS, Florida. Meyer, H., Radicke, S., Kienzle, E., Wilke, S., Kleffken, D., and Illenseer, M. (1995a). Investigations on Preileal Digestion of Starch from Grain, Potato and Manioc in Horses. J. Vet. Med. A. 42, 371-381. Oddy, V. H., Ewoldt, C.L., Jones,A.W. and Warren,H.M. (1990). Metabolisable energy content of diets based on oat grain. Australian Journal of Experimental Agriculture 30, 503-506. Pagan, J. D. (1999). Recent developments in equine nutrition. In "Proceedings 1999 Cornell Nutrition Conference for FeedManufacturers", pp. 160-167, Rochester, NY. Pickering, F. S., Barrett, N. C., Farrell, D. J. and Corbett, J. L. (1982). Physical and chemical attributes of oats grain and nutritional value. Animal Production in Australia 14, 675. Potter, G., Arnold, F., Householder, D., Hansen, D., and Brown, K. Digestion of starch in the small or large intestine of the equine. Rowe, J. B. and Coss, L. (1992). The importance of lignin content of oat hulls in ruminant nutrition. Proceedings of the Nutrition Society of Australia 17, 218. Rowe, J. B. and Coss, L. (1994). Lignin content of oats - does it affect animal performance when sheep are given oats as a supplementary feed? Animal Production in Australia 20, 421. Rowe, J. B. and Crosbie, G. B. (1988). The digestibility of grain of two cultivars of oats differing in lignin content. Australian Journal of Agricultural Research 39, 639-644.. Shaw, T. (1907). Feeding Farm Animals. Orange Judd Company, London, UK. Toland, P. C. (1976). The digestibility of wheat, barley, or oat grain fed either whole or rolled at restricted levels with hay to steers. Australian Journal of Experimental Agriculture and Animal Husbandry 16, 71-75. Welch, W., Hayward, M. V., Iorwerth, D. and Jones, H. (1983). The composition of oat husk and its variation due to genetic and other factors. Journal of the Science of Food and Agriculture 34, 417-426.

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