Rice-based Diets
in Pigs–for protection
against intestinal bacterial
infections
A report for the Rural Industries
Research and Development
Corporation
by Associate Professor John Pluske and
Professor David Hampson
September 2005
RIRDC Publication No 05/143
RIRDC Project No. UMU-30A
© 2005 Rural Industries Research and Development Corporation
All rights reserved.
ISBN1 74151 206 9
ISSN 1440-6845
Rice-based Diets in Pigs – for protection against intestinal bacterial infections
Publication No. 05/143
Project No. UMU-30A
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Researcher Contact Details
Associate Professor J.R. Pluske
School of Veterinary and Biomedical Sciences
Murdoch University
Murdoch WA 6150
Phone: (08) 9360 2012
Fax:
(08) 9360 2487
Email: [email protected]
In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.
RIRDC Contact Details
Rural Industries Research and Development Corporation
Level 1, AMA House
42 Macquarie Street
BARTON ACT 2600
PO Box 4776
KINGSTON ACT 2604
Phone: 02 6272 4819
Fax:
02 6272 5877
Email: [email protected].
Website: http://www.rirdc.gov.au
Published in September 2005
Printed on environmentally friendly paper by Canprint
ii
Foreword
In Australia, post–weaning diarrhoea (PWD) represents a major constraint to efficient and profitable
pig production. Antibiotic resistance caused by the routine inclusion of antibiotics in diets is becoming
a major reason for the perseverance of this disease. The European Union has banned the use of growth
promoting antibiotics in diets for pigs from 1st January 2006, although in some EU countries such as
Sweden and Denmark, a voluntary (industry) ban on the use of dietary growth promoting antibiotics
has been enforced for some years. Growth promoting antibiotics in diets for pigs are still permitted in
Australia, however in countries where growth-promoting antibiotics are banned, the incidence of PWD
has increased dramatically, concomitant with an increase in mortality, compromised welfare and
deterioration in feed conversion efficiency. The increased use of antibiotics for therapeutic use
together with the persistent occurrence of PWD has increased costs, contributes further to antibiotic
resistance, and has highlighted the need for greater understanding of the aetiology of PWD.
This research package was designed to identify the effects of cooked white rice in diets for weanling
pigs as a nutritional strategy for controlling PWD without reliance on growth promoting antibiotics.
The general hypothesis tested was that the incidence of PWD could be reduced by strategic nutritional
interventions using cooked white rice in the post-weaning period. The overall aim of this research
project was to investigate the scientific bases of interactions between diet composition and the
occurrence of PWD, focusing specifically on rice processing and ingredient interactions to ultimately
develop specialised rice-based diets that can be fed to young pigs to control PWD in Australia. This
would have potential value-adding benefits for the Australian rice industry.
This publication summarises a series of experiments conducted in vitro and in vivo to ascertain the
potential beneficial properties of Australian-grown rice in ameliorating PWD in piglets and
maintaining/enhancing production in the post-weaning period. The experiments investigated the
physico-chemical properties of rice, again both in vitro and in vivo, to determine the most suitable
type(s) of rice for potential use in the pig industry to overcome PWD without the use of growth
promoting antibiotics.
This project was funded from industry revenue that is matched by funds provided by the Australian
Government.
This report is an addition to RIRDC’s diverse range of over 1200 research publications. It forms part
of the Rice R&D Sub-program which aims to improve the profitability and sustainability of the
Australian rice industry.
Most of our publications are available for viewing, downloading or purchasing online through our
website:
•
•
downloads at www.rirdc.gov.au/fullreports/index.html
purchases at www.rirdc.gov.au/eshop
Peter O’Brien
Managing Director
Rural Industries Research and Development Corporation
iii
Acknowledgments
The technical assistance of Ms Fiona Cavaney and Dr Jae-Cheol Kim from Murdoch University is
thanked throughout the course of this research project. Associate Professor David Pethick, School of
Veterinary and Biomedical Sciences, Murdoch University, is also thanked for critical input and
discussion. Dr Melissa Fitzgerald, NSW Agriculture, Yanco, NSW, is thanked for early helpful
discussions and chemical analysis in the screening component of the project. Dr Bruce Mullan, WA
Department of Agriculture, is thanked for assistance with diet formulation and diet preparation. The
staff at the Medina Research Station, particularly Messrs Bob Davis and Richard Seaward, are thanked
also for assistance with running of pig feeding trials. Dr Robert van Barneveld, Barneveld Nutrition, is
thanked for arranging the extrusion of rice used in the feeding trials in this project. Dr Lucile
Montagne, a post-doctoral fellow from INRA in Rennes, France, is also thanked for her involvement
in the work during her visit to Murdoch University.
Abbreviations
ANOVA:
AOAC:
CP:
CTTAD:
DE:
DF:
DM:
FCR:
FDS:
GE:
PWC:
PWD:
N:
NE:
NH3:
NSP:
P:
RS:
SED:
SEM:
VFA:
analysis of variance.
Association of Official Agricultural Chemists.
crude protein.
coefficient of total tract apparent digestibility.
digestible energy.
dietary fibre.
dry matter.
feed conversion ratio (grams of feed per gram of daily bodyweight gain).
fast digestible starch.
gross energy.
post-weaning colibacillosis.
post-weaning diarrhoea.
nitrogen.
net energy.
ammonia.
non-starch polysaccharides.
phosphorus.
resistant starch.
standard error of difference.
standard error of the mean.
volatile fatty acids.
iv
Contents
Foreword ................................................................................................................................iii
Acknowledgments.................................................................................................................iv
Abbreviations ........................................................................................................................iv
Executive Summary .............................................................................................................vii
1. Introduction .....................................................................................................................1
1.1 Background ........................................................................................................................... 1
1.2 Post-weaning diarrhoea (PWD)............................................................................................. 2
1.3 Associations between diet and PWD..................................................................................... 3
1.4 Digestibility of rice and effects of processing....................................................................... 4
1.5 Interactions with other feed ingredients ................................................................................ 4
2. Objectives ........................................................................................................................6
3. Methodology ....................................................................................................................7
3.1 Database of physical and chemical characteristics of Australian rice for pigs...................... 7
3.2 Selection of rice varieties and processing methods on physico-chemical effects in the
weaned pig............................................................................................................................. 7
3.3 Interactive effects of cooked white rice with vegetable and animal protein sources on
digesta and fermentation characteristics and the faecal shedding of haemolytic E. coli....... 8
3.4 Effects of extrusion of rice and dietary protein sources on production, digestibility and
PWD ...................................................................................................................................... 8
3.5 Effects of added oat hulls to extruded rice-based diets on production, digestibility and the
incidence of PWD ................................................................................................................. 9
3.6 The nutritive value of extruded rice and cooked (autoclaved) rice for weaner and grower
pigs ........................................................................................................................................ 9
3.7 On-farm testing of processed rice-based diets..................................................................... 10
4. Screening and selection of rice varieties for in vitro and feeding trials ..................11
4.1 Summary ............................................................................................................................. 11
4.2 Introduction ......................................................................................................................... 11
4.3 Materials and Methods ........................................................................................................ 11
4.4 Results and Discussion........................................................................................................ 12
5. Selection of rice varieties and processing methods on physico-chemical effects in
the weaned pig ..............................................................................................................14
5.1 Summary ............................................................................................................................. 14
5.2 Introduction ......................................................................................................................... 14
5.3 Materials and Methods ........................................................................................................ 15
5.4 Results ................................................................................................................................. 16
5.5 Discussion ........................................................................................................................... 20
6. Selection of rice varieties and processing methods on physico-chemical effects in
the weaned pig ..............................................................................................................22
6.1 Summary ............................................................................................................................. 22
6.2 Introduction ......................................................................................................................... 22
6.3 Materials and Methods ........................................................................................................ 23
6.4 Results ................................................................................................................................. 27
6.5 Discussion ........................................................................................................................... 34
v
7.
Interactive effects of cooked white rice with vegetable and animal protein sources
on digesta and fermentation characteristics and the faecal shedding of haemolytic
E. coli..............................................................................................................................37
7.1 Summary ............................................................................................................................. 37
7.2 Introduction ......................................................................................................................... 37
7.3 Materials and Methods ........................................................................................................ 38
7.4 Results ................................................................................................................................. 40
7.5 Discussion ........................................................................................................................... 43
8. Effect of extrusion of rice and dietary protein sources on production, digestibility
and faecal shedding of E. coli......................................................................................46
8.1 Summary ............................................................................................................................. 46
8.2 Introduction ......................................................................................................................... 46
8.3 Materials and Methods ........................................................................................................ 47
8.4 Results ................................................................................................................................. 50
8.5 Discussion ........................................................................................................................... 54
9. Effect of added oat hulls to extruded rice- and wheat-based diets on production,
digestibility and the incidence of PWD .......................................................................56
9.1 Summary ............................................................................................................................. 56
9.2 Introduction ......................................................................................................................... 56
9.3 Materials and Methods ........................................................................................................ 57
9.4 Results ................................................................................................................................. 60
9.5 Discussion ........................................................................................................................... 67
10. The nutritive value of extruded rice and cooked (autoclaved) rice for weaner and
grower pigs ....................................................................................................................70
10.1 Summary ............................................................................................................................. 70
10.2 Introduction ......................................................................................................................... 70
10.3 Materials and Methods ........................................................................................................ 71
10.4 Results ................................................................................................................................. 72
10.5 Discussion ........................................................................................................................... 75
11. Implications and Recommendations...........................................................................79
12. References .....................................................................................................................81
vi
Executive Summary
Seven experiments were conducted in this research programme to test the general hypothesis that the
incidence of post-weaning diarrhoea (PWD) could be reduced by strategic nutritional interventions in
the post-weaning period using Australian-grown rice. An associated purpose of the study was to
examine the use of rice-based diets on production indices in pigs and the physico-chemical properties
of rice in the gastrointestinal tract. The major objectives of this research project were the development
of a database describing the physical and chemical characteristics of Australian rice and their
suitability in diets for pigs, the commercial development and uptake by the Australian pig Industry of
specialty processed rice-based diets for protection against PWD caused by enterotoxigenic
Escherichia coli (E. coli) and increased production, an increased understanding of the mechanisms
whereby such diets afford protection, and potentially, although outside the scope of this project, a
biomedical avenue into the use of such diets for control of enteric conditions in man. The potential
benefits of this research programme were an increased utilisation of Australian rice particularly in
value-added markets, the development of high-value specialty or ‘boutique’ diets based on processed
rice for feeding the young pig and (or) pigs in states of enteric disease, and a possible avenue for rice
into the biomedical industries, i.e. “functional foods”.
Rice is a staple food for much of the world’s human population but has received relatively little
attention as a possible feedstuff for the animal industries, in this case the Australian pig industry. Rice,
once gelatinised, is highly digestible within the gastrointestinal tract of the pig that, in turn, could
enhance performance over pigs fed wheat, the most commonly used grain used in Australia.
Furthermore, and based on previous studies conducted in Australia and overseas, rice has potential to
ameliorate PWD, a disease which costs the Australian pig industry millions of dollars annually. The
increasing pressure that governments are facing worldwide to ban the use of dietary growth promoting
antibiotics and dietary heavy metals such as zinc and copper in pig diets has meant that alternative
strategies for the control of PWD, and indeed other enteric diseases, need to be found. This has already
occurred in the European Union, for example. Based on these previous studies, cooked white rice
appears to offer promise as an alternative nutritional strategy to the current use of antimicrobial
compounds for mitigating PWD. However, more research was needed in order to refine further dietary
recommendations that could be made to the rice and pig industries regarding the feeding of rice to pigs
to reduce PWD without recourse to antimicrobials in the diet.
The seven experiments conducted in this programme are presented as chapters, as follows:
1.
2.
3.
4.
5.
6.
7.
Screening and selection of rice varieties for feeding and in vitro trials.
In vitro assessments of starch-related properties in response to rice type, cooking methods and
cooling after cooking.
Effect of rice type fed to piglets after weaning on starch digestion, digesta and fermentation
characteristics and the faecal shedding of haemolytic E. coli.
Interactive effects of cooked rice products with vegetable and animal protein sources on digesta
and fermentation characteristics and the faecal shedding of haemolytic E. coli.
Effect of extrusion of rice and dietary protein sources on production, digestibility and faecal
shedding of E. coli.
Effect of added oat hulls to extruded rice- and wheat-based diets on performance and diarrhoea
after weaning.
The energy value of extruded rice and cooked (autoclaved) rice for weaner and grower pigs.
Results obtained in this research project have demonstrated that cooked (processed) white rice, either
in medium-grain or long-grain form, included in diets for weanling pigs can be used as a replacement
for wheat without a loss of production in the immediate post—weaning period. The decision to replace
a cereal such as wheat in diets for weanling pigs, therefore, is likely to be one of price differential.
Cooking broken white rice, particularly in medium-grain and waxy rice that have lower amylose levels
than long-grain rice, increases starch digestibility when measured at the end of the small intestine. This
vii
could be predicted with accuracy in vitro using a “fast digestible starch” assay modified for rice in our
laboratory. Regardless of the type and variety of rice used, however, pigs fed cooked white rice
partition more digested nutrients into carcass gain than pigs fed other cereals such as wheat and barley,
although the type of proteins fed to pigs will also influence this. In this regard, the use of extruded rice
plus sources of animal protein (eg, milk powders, fishmeal, meat and bone meal) appear the best
dietary combination for production purposes. Feeding vegetable (plant) proteins typically increased
the weight of the gastrointestinal tract as a consequence of increased fermentative activity in the large
intestine, and reduced bodyweight gain and FCR. Determination of the energy (DE and NE) values of
extruded medium-grain (Amaroo) and long-grain (Doongara) rice confirmed the superior energy value
of these two rice types over existing cereals used in Australian feeding of pigs, such as wheat.
The effects of feeding cooked white rice on reducing faecal shedding of the bacterium (E. coli)
responsible for causing PWD were generally unchanged, or even exacerbated, when the rice plus
animal protein diets were fed compared to commercially-based diets that were considered a
contributing factor to the incidence of PWD. The extent and duration of faecal shedding of
enterotoxigenic E. coli found in the studies conducted was generally low, and this might have
influenced the capacity of the rice-based diets to exhibit protective effects. It was hypothesised also
that an imbalance in the amounts of carbohydrate versus protein entering the large intestine might
have predisposed the pig to PWD, due to a change in the types of microbiota and subsequent
production of compounds implicated in non-infectious diarrhoea. The results of Chapter 9 advocate
the inclusion of a quantity of slowly or moderately fermentable dietary fibre to extruded rice-based
diets consisting of animal protein to ameliorate the diarrhoea that is sometimes observed when feeding
this diet, although in this instance 20 g kg- oat hulls depressed digestibility and production after
weaning. Nevertheless, this proposition is consistent with European experiences of feeding processed
rice to piglets after weaning. In this respect, it is feasible that the addition of rice bran and (or) rice
hulls, or possibly the use of brown rice, in diets for piglets after weaning could achieve similar results.
An unfortunate consequence of the drought in the rice-growing regions of NSW for this particular
project, however, was the inability to perform an on-farm trial implementing some of the findings and
conclusions arising from this research project.
The major recommendations arising from this project are as follows:
1.
2.
3.
4.
5.
Medium-grain rice (variety Amaroo) or long-grain rice (variety Doongara) was identified as being
the most suitable rice cultivars for utilisation in piglet feeds in Australia. Waxy rice, but not
parboiled rice, would also be suitable, but its lower production tonnage in Australia at present
would increase its price relative to other rice types and other cereals and hence limit its
usefulness.
Processed (extruded) medium-grain rice (variety Amaroo) or long-grain rice (variety Doongara) is
a suitable replacement for cereals currently fed to weanling pigs in Australia such as wheat and
barley. Adoption of processed rice by the pig industry will be predominately driven by the price
differential between processed rice and these alternative cereals.
Starch digestion at the end of the small intestine, as well as the colon, can be predicted accurately
with a “fast digestible starch” assay modified for use in our laboratory. This test could be used by
the rice industry as part of a broader screening process for potentially new varieties of rice
suitable for the pig industry, however the assay is capable of being tailored for use in other
species, including man.
Sources of animal protein in diets containing processed (extruded) rice generally cause superior
production after weaning compared to vegetable (plant) sources of protein, although vegetable
proteins showed reduced faecal shedding of haemolytic E. coli compared to animal sources of
protein.
Producers feeding extruded rice-based diets with animal protein sources are encouraged to include
some slowly or moderately fermentable dietary fibre, such as oat hulls, wheat bran and (or) beet
pulp, to ameliorate the diarrhoea that is sometimes observed when feeding this diet. Future studies
should investigate the addition of rice bran and (or) rice hulls, or possibly the use of brown rice, in
diets for piglets after weaning that could accomplish similar results.
viii
6.
7.
The average (mean) digestible energy (DE) content (MJ/kg as-fed) of extruded rice is 15.3 MJ/kg
as-fed. Medium-grain (Amaroo) rice has a 0.4 MJ/kg higher DE content than the long-grain rice
(Doongara).
Pig producers should use different DE values for pigs of different ages/weights. Weanling pigs (8
kg) extracted less energy from both extruded rices than grower (55 kg) pigs (up to 0.5 MJ/kg
difference). Producers using a net energy (NE) system should use a common value of 11.5 MJ/kg
as-fed.
ix
x
1. Introduction
1.1
Background
Rice is the staple cereal consumed by much of the world’s population, and a plethora of studies exist
investigating the physical and chemical properties of cooked rice for man. The vast majority of these
studies relate to the starch properties of rice, presumably because starch constitutes in excess of 75%
of rice’s composition (Marsono and Topping, 1993), and hence forms the major carbohydrate
consumed. The high starch content of cooked rice coupled with a very low non-starch polysaccharide
(NSP) level makes cooked rice a ready source of absorbable glucose, and hence energy, for the human
population. More recently, there is interest in the use of rice-based oral rehydration formulas for
controlling enteric diseases in children (eg, Iyngkaran and Yadav, 1998; Ramakrishna et al., 2000) and
animals (eg, Wingertzahn et al., 1999; Hampson et al., 2001).
In contrast, there is less information pertaining to the feeding of rice to animals, especially the pig,
with respect to effects on production and intestinal “health”, which incorporates enteric disease. This
is predominately because other cereal sources, such as wheat, barley, corn and sorghum, are used in
pig production and can be fed to pigs cheaper than rice. Nevertheless, and given the information
available from the human literature with respect to the cooking and milling properties of rice, potential
exists for the use of processed (cooked) rice in certain diets for pigs, especially the young pig. This is
particularly when the intestine is compromised by enteric pathogens such as Escherichia coli, the
agent of post-weaning colibacillosis (PWC) or, as it is more commonly recognised, post-weaning
diarrhoea (PWD). Incorporation of processed rice into such diets has potential to add value to the
Australian rice industry and reduce the pig Industry’s reliance on the use of growth promoting
antibiotics. Furthermore, spin-offs into the biomedical field in the control of human enteric pathogens
may be possible.
The Australian pig industry currently has approximately 300,000 sows producing around 6 million
pigs per year. The gross value of pig meat production is estimated at $900 million. Yearly feed
consumption for pigs in Australia is estimated at 1.5 million tonnes, with weaner pigs (i.e., those from
weaning to around 20 kg) consuming around 12%, or 180,000 tonnes, of this amount. Of this, around
20% is fed in the first two weeks after weaning, or 36,000 tonnes, which at present-day diet prices is
worth between $25 and $30 million annually. Currently young pigs are fed predominantly wheatbased diets, however wheat is associated with increased incidence of PWD (McDonald et al., 1999).
Protective diets based on processed rice, therefore, are ideally positioned to capture a considerable
proportion of this market, particularly if antimicrobials such as growth promoting antibiotics are
banned.
Enteric bacterial infections such as PWD cause extensive morbidity and loss of production in the pig
industry, and losses are currently valued in excess of $60 million annually (Cutler, 1992). Postweaning diarrhoea (PWD), which is the locus of this research proposal, costs the Australian pig
Industry between $22 and $26 million annually (Cutler, 1992). More recently, Cutler (2001) estimated
that a 1% increase in post-weaning mortality, which incidentally occurred in Denmark following their
ban on antimicrobials in weaner diets (Larsen, 2004), would reduce profit by $18 per sow per year;
this equates to a total Australian industry cost of approximately $6 million.
Antimicrobial agents are presently the main tool used for control of PWD, and are provided to pigs to
treat overt disease, to provide prophylaxis in situations where disease is liable to occur, and to improve
growth rates in the absence of disease. However, problems are arising over the use of antimicrobials in
the pig industry. Their long-term use eventually selects for the survival of resistant bacterial species or
strains, and genes encoding this resistance also can be transferred to other formerly susceptible
bacteria. Currently a variety of bacterial pathogens of pigs are showing resistance to a range of
antimicrobial drugs. Not only is this reducing the number of antimicrobials available to control
1
bacterial diseases in pigs, but this resistance also poses risks to human health. Risks include the
transfer of multidrug resistant zoonotic pathogens (eg, Salmonella spp. and Campylobacter spp.) from
pigs to humans, the direct or indirect transfer of resistance genes from the porcine intestinal microflora
to human bacterial strains, and the presence of antimicrobial drug residues in pig meat (Hampson et
al., 2001). Public concern about these issues is leading to reduced availability or the complete banning
of certain antimicrobial agents for use in pig production, as has occurred in certain parts of Europe.
Consequently it is import to develop alternative means, such as the use of nutrition, both of controlling
bacterial infections and promoting growth in pigs without recourse to the use of antimicrobials.
In countries where growth-promoting antibiotics are banned, such as Sweden and Denmark, the
incidence of PWD has increased dramatically, concomitant with an increase in mortality,
compromised welfare and deterioration in feed conversion efficiency in the period after weaning
(Larsen, 2004). Presently, where growth-promoting antibiotics cannot be included in diets, producers
therapeutically treat pigs displaying overt signs of diarrhoea. However, the increased use of antibiotics
for therapeutic use together with the persistent occurrence of PWD has increased costs, contributes
further to antibiotic resistance, and has highlighted the need for greater understanding of the aetiology
of PWD.
There are currently no bans on the use of growth promoting antibiotics in the Australian pig Industry.
However, Australian Pork Limited (APL), which represents the interests of Australian pork producers
and is responsible for formulating policies on their behalf, has outlined strategies for the use of
antibiotics. In this document (available at www.australianpork.com.au), APL stated: "The elimination
or prudent use of antibiotics……is vital for success in this sophisticated, exacting and globalised
marketplace, and should be embraced". This concern has arisen, at least partly, in recognition of the
growing problem of antibiotic resistance in Australia (Barton, 1999, 2000) of several economically
important enteric pathogens, such as E. coli, the causative agent of PWD. Barton (1999), for example,
found that approximately 90% of the porcine E. coli isolates tested in Australia were resistant to three
or more classes of antibiotics, with around 20% of isolates resistant to six or more classes.
Collectively, there is a need to investigate alternative ways to ameliorate enteric conditions such as
PWD without antibiotics. The use of nutritional intervention is one important means by which this
could be achieved.
1.2
Post-weaning diarrhoea (PWD)
PWD is a diarrhoeal disease that typically starts 3-4 days after weaning and continues until 9-12 days
post-weaning. Piglets usually develop watery diarrhoea and show a rapid loss of condition, with most
members of a litter being affected. PWD is endemic on some farms, being present in many litters, and
repeatedly occurring in successive litters over many years. Besides potential mortalities, and the cost
of treatment, piglets fail to gain weight immediately after weaning, and this could extend the total time
to reach slaughter weight (Williams, 2003). PWD is a complex and multifactorial disease,
incorporating many aspects of management (Madec et al., 1998), but important aetiological agents
include E. coli, and sometimes rotaviruses (Lecce, 1983).
The main aetiological agent(s) associated with PWD is (are) specialised strains of E. coli, which differ
from common non-pathogenic types that occur in the intestinal tracts of healthy pigs. Unlike nonpathogenic strains, these pathogenic strains can adhere to the luminal surface of small intestinal
enterocytes or the mucus covering the villi, particularly in the anterior small intestine, preventing them
being flushed away to the more distal parts of the tract by normal peristaltic movement of the luminal
contents. Attachment is through bacterial rod-like surface structures called fimbriae or pili. In
pathogenic E. coli strains, adhesins K88 (also known as F4), and F18 (formerly F107) are most
commonly associated with PWD, and these both exhibit several antigenic variants (Francis, 2002). At
this site adjacent to the enterocyte surface they deliver powerful toxins that disrupt the normal
functionality of the enterocytes. As the anterior small intestine has a critical function in both digestion
and absorption, disruption of function here is particularly harmful.
2
The most common and significant pathogenic types associated with PWD are enterotoxigenic E. coli
(ETEC). Different ETEC strains release different combinations of two toxin types, heat labile toxin
(LT) and heat stable toxin (ST; variants STa and STb), both of which provoke hypersecretory
diarrhoea as a result of loss of water and electrolyte into the intestinal lumen. These processes result in
an excess volume of fluid and electrolyte in the gut lumen of infected pigs. This volume can only be
fully reabsorbed if the colon is healthy, has a stable, well-balanced microflora, and is not physically
overloaded (Argenzio, 1992). In addition, the E. coli strains that cause PWD are usually able to lyse
red blood cells present in the blood agar plates that are used for their isolation, and consequently these
bacteria are known as β-haemolytic E. coli. The haemolytic activity is a useful marker of strains that
are liable to be involved in PWD. There is a small number of O-serotypes that are repeatedly observed
in association with PWD, of which the most common are: O149, O138, O139, O141 and O8
(Hampson, 1994).
Haemolytic E. coli are uncommon in the intestinal tract of healthy unweaned pigs, although
occasionally these strains are present in unweaned diarrhoeic pigs. Following weaning, these
organisms frequently proliferate in the gastrointestinal tract of both healthy pigs and pigs that go on to
develop diarrhoea. The key difference is that the number and proportion of potentially pathogenic
strains of E. coli in the gastrointestinal tract and faeces is higher in pigs with PWD, compared against
those that remain healthy (Hampson, 1994). Pigs with PWD have up to 109 colony forming units of
such haemolytic E. coli in the small intestine, whilst there is minimal change in other resident bacterial
populations at this time (Smith and Jones, 1963). Richards and Fraser (1961) first reported the link
between excessive multiplication of haemolytic E. coli in the small intestine and the development of
diarrhoea in weaner pigs. The associated disease, which has been called post-weaning colibacillosis
(PWC), is characterised by diarrhoea, dehydration, rapid loss of weight, metabolic acidosis, poor
condition and shivering (Bertschinger, 1999). The terms PWC and PWD tend to be used
interchangeably, but PWC is a more specific term where the disease is completely or predominantly
attributable to the E. coli infection. On the other hand, the term PWD acknowledges that the diarrhoea
that often occurs in piglets after weaning may have other aetiologies and (or) complex interactions
superimposed on the E. coli infection (Hampson, 1994). Faecal-oral spread between animals is the
primary means of transmission (Bertschinger, 1999).
1.3
Associations between diet and PWD
Research conducted previously at Murdoch University has shown that feeding cooked (ie, 121° C in
an autoclave) white rice to pigs experimentally-infected with a number of economically-significant
enteric pathogens, including the causative agent of PWD, E. coli, offers protection against the
proliferation of such bacteria in the intestines with a subsequent reduction in the clinical expression of
diarrhoea (see papers by Siba et al., 1996; Pluske et al., 1996; Pluske et al., 1998; McDonald et al.,
1999; Hampson et al., 2000; McDonald et al., 2001). This is believed to be related to the type of diet
fed after weaning.
The main source of growth substrate for the gastrointestinal microflora comes from the diet. Simple
sugars tend to act as the main growth substrate in the upper part of the gastrointestinal tract, whilst in
the large intestine, where the main bacterial biomass is located, dietary fibre (DF) serves as the major
bacterial substrate. Our data at present suggests that it is the soluble non-starch polysaccharide (NSP)
component of feedstuffs that most likely promotes proliferation of E. coli in the gut and causes
diarrhoea, although the role of resistant starch (RS) cannot be dismissed without further studies. This
may be mediated by enhanced viscosity of the intestinal contents. As such, cooked rice with its low
soluble NSP content, high concentration of readily digestible starch and low viscosity-inducing
properties, offers promise as a grain that may be used to control PWD.
3
While it is recognised that different types of fibre in the diet can broadly influence the composition
and metabolic activity of the large intestinal microflora in pigs (Varel et al., 1982; Varel and Pond,
1985; Bach Knudsen et al., 1991; Jensen and Jorgensen, 1994; Reid and Hillman, 1999), little is
known about the way in which these bacteria interact with pathogenic species of bacteria. This lack of
information makes it difficult to predict how a given dietary component could be used to indirectly
influence a given enteric pathogen. Besides influencing the normal gastrointestinal microflora, diet
could also influence colonization by pathogens through other routes. For example it could act by
modulating the amount of specific substrate available for the pathogen at a given site, by influencing
viscosity of the intestinal contents and hence altering accessibility of receptor sites and (or) affecting
intestinal motility, and by direct or indirect effects on the intestinal mucosa. As an example of the
latter effect, different cereal types and particle size have been shown to alter epithelial cell
proliferation and lectin binding patterns of the epithelium in the large intestine of pigs (Brunsgaard,
1998). Similar changes may occur in specific colonization sites or bacterial receptors on the
enterocytes. The diet also might influence intestinal function; for example, components in boiled rice
inhibit secretion in the small intestine, and hence reduce the magnitude of secretory diarrhoea due to
pathogens such as enterotoxigenic E. coli (Mathews et al., 1999). Many of these questions remain
unanswered.
1.4
Digestibility of rice and effects of processing
Despite the plethora of information pertaining to the physical and processing characteristics of rice,
information concerning the pattern of digestibility in vivo along the gastrointestinal (GI) tract in the
pig is scarce. It is important to assess the digestibility of rice along the GI tract because proliferation of
E. coli generally occurs more anteriorly in the small intestine. Hence it is necessary to maximise
digestion of carbohydrate as anteriorly (ie, towards the mouth) as possible so that little or no substrate
is available for the bacteria.
Marsono and Topping (1993) demonstrated that the choice of cultivar as well as milling and further
processing influences the level and types of DF (particularly NSP) in Australian rice. However, and as
highlighted by these authors, it remains to be seen whether these chemical changes modify the
physiological effects in vivo of processed rice products, although Devi and Geervani (2000) reported
that in vitro starch digestibility values in puffed, boiled and parboiled-boiled rice samples were higher
than in raw and parboiled rice samples. In addition, Sagum and Arcot (2000) reported some
redistribution from insoluble to soluble NSP after heat processing, while Tetens et al. (1997) reported
that the rate of starch digestion was influenced by both variety and method of cooking when assessed
using an in vitro starch digestibility assay. In this particular study it was parboiling, however other
forms of processing such as extrusion are also likely to influence the rate of starch digestion.
Again, and in the pig, this type of information is unknown but is crucial if rice-based diets are to be
used in the control of PWD. This is especially the case given our previous data implicating soluble
NSP in the aetiology of PWD (McDonald et al., 1999). It is imperative, therefore, that these possible
effects are investigated in vivo and that there is no increase in the proliferation of E. coli and increased
incidence of PWD in (processed) rice-fed pigs. A pivotal study to answer this question, therefore, is an
investigation of the sites and rates of starch digestion along the GI tract study in several rice cultivars
in association with the processing method.
1.5
Interactions with other feed ingredients
An important consideration in this work will be the interaction between rice and other dietary
ingredients. These ingredients will predominantly be protein-based feedstuffs of either animal or plant
origin, and generally comprise 25-30% of a weaner diet. There is some evidence that PWD is
influenced by the protein content and quality of the diet (Hampson et al., 2001), and we suspect that
this is attributable to vegetable protein sources that can contain up to 300 g kg-1 NSP. It is crucial,
4
therefore, that any likely interactions between rice type, rice processing and different protein
feedstuffs are studies to minimise the risk of pigs succumbing to PWD with processed rice diets.
The general hypothesis tested in this research programme was that the incidence of PWD could be
reduced by strategic nutritional interventions based on the incorporation of Australian-grown, cooked
white rice in diets in the post-weaning period. The overall aim of this research project was to
investigate the scientific bases of interactions between diet composition, the use of cooked white rice
and the occurrence of PWD, focusing specifically on rice processing and ingredient interactions to
ultimately develop specialised rice-based diets that can be fed to young pigs to control PWD in
Australia. This would have potential value-adding benefits for the Australian rice industry.
5
2. Objectives
The major objectives of this research project were as follows:
•
•
•
•
development of an Australian database describing the physical and chemical characteristics of
processed rice, and their suitability in diets for pigs
commercial development and uptake by the pig Industry of specialty processed rice-based diets for
protection against post-weaning diarrhoea caused by enterotoxigenic Escherichia coli and
increased production
understanding of the mechanisms whereby such diets afford protection
a biomedical avenue into the use of such diets for control of enteric conditions in man.
The potential benefits of this research programme can be summarised as follows:
• increased utilisation of rice in added-value markets
• development of high-value specialty or ‘boutique’ diets based on processed rice for feeding the
young pig and (or) pigs in states of enteric disease
• a possible avenue for rice into the biomedical industries, ie “functional foods”. An important, and
potentially lucrative, spin-off from this research proposal is the development of specialty
products/diets for use in the treatment of enteric infections in the human population.
6
3. Methodology
This research programme comprised several distinct but interrelated approaches to assess the
utilisation of cooked white rice in pig diets against intestinal bacterial infections, specifically
enterotoxigenic E. coli, the agent of post-weaning colibacillosis (PWC). This research plan was
developed following discussions with Dr Keith Hutton, General Manager of Coprice Feeds and ByProducts Group, Dr Melissa Fitzgerald, NSW Agriculture Yanco, feed manufacturers and
representatives from the Australian pig industry. Discussions were also held with Dr Jeff Davis,
RIRDC Rice R&D Program Manager, throughout the project.
This summary provides an overview of the major methodological approach to the experiments
conducted. Methodologies and techniques relevant to specific experiments/procedures are detailed in
the experimental chapters that follow this section.
3.1
Database of physical and chemical characteristics of
Australian rice for pigs
The major aim of this part of the programme was to characterise the starch-related properties of a
number of different rice types and rice varieties grown by NSW Agriculture. This process acted as a
way of ‘screening’ varieties/types of rice for their suitability in diets for piglets, as well as acting as a
valuable composition database for the Australian rice industry. A monitoring process such as this will
also aid in selection of rice for subsequent trials in this project.
Unfortunately the persistent drought that affected the rice-growing regions in NSW over the duration
of this particular project restricted this study to samples harvested only in 2001, because it was not
possible to continue with such work given the lack of rice available and the time-scale of the project.
Nevertheless, the wide range of samples examined provided a valuable screening database and allowed
progression of the project onto its next stage. It met its aim in allowing the selection of rice types
deemed suitable for use in piglet feeding trials and associated in vitro studies.
The results from this experiment are outlined in Chapter 4.
3.2
Selection of rice varieties and processing methods on
physico-chemical effects in the weaned pig
On the basis of 3.1 (above), and considering the relative contribution of the different rice types
produced in Australia to the total tonnage produced annually, a common medium-grain variety (cv.
Amaroo) and a common long-grain variety (cv. Doongara) were selected as ‘candidate’ rice types for
use in the pig industry. Both in vitro (Experiment 3.2a) and in vivo (Experiment 3.2a) studies were
examined.
3.2a
In vitro assessments of starch-related properties in response to rice
type, cooking methods and cooling after cooking
The first part of this experiment examined in vitro the starch-related properties of two rice varieties
both before and after cooking. This experiment permitted the establishment of a number of chemical
techniques in our laboratory, such as the analysis of total starch, resistant starch, amylose:amylopectin
ratio and fast digestible starch (FDS), and provided a foundation for 3.2b (below) with respect to
determining the optimum rice:water ratio and method of cooling after cooking that would allow for
maximum digestibility of starch in the GI tract of weanling pigs. Furthermore, a sample of extruded
rice allowed a preliminary examination of its potential physico-chemical properties in vivo in the
newly-weaned piglet.
7
The results from this experiment are outlined in Chapter 5.
3.2b
Effect of rice type fed to piglets after weaning on starch digestion,
digesta characteristics and the incidence of PWD
The aim of the second part of this study was to ascertain in vivo the feeding of rice of different
chemical composition to pigs on aspects of production and PWD. The rice types selected were (i) an
Australian-grown medium-grain variety (cv. Amaroo), (ii) an Australian-grown long-grain variety (cv.
Doongara), and (iii) waxy rice sourced from Thailand. The waxy rice was chosen because of its high
amylopectin content, which is thought to have a higher starch digestibility in the GI tract than rices of
lower amylopectin (higher amylose) content. It was necessary to source the imported waxy rice
because sufficient quantity of an Australian-grown waxy rice was unavailable.
In combination with this study was the development of a technique (based on that of Tetens et al.,
1997) to measure starch digestion in vitro; this can then be verified against the in vivo data that will be
gathered.
The results from this experiment are outlined in Chapter 6.
3.3
Interactive effects of cooked white rice with vegetable and
animal protein sources on digesta and fermentation
characteristics and the faecal shedding of haemolytic E. coli
Previous studies using cooked white rice in diets for young pigs have always included other dietary
ingredients that are low in NSP and (or) RS. These ingredients are primarily animal-based, such as
meat and bone meal, fishmeal, blood meal, and milk powders. Vegetable protein sources, such as
lupins, and peas, or oilseed meals such as canola meal and soybean meal, were specifically excluded
from diets since the hypothesis has been that diets low in soluble NSP prevent PWD. Furthermore,
German data derived in the 1970’s shows that ‘resistant protein’ (protein escaping digestion in the
small intestine into the large intestine) might be a contributor to PWD in its own right. However, the
inclusion of vegetable proteins in diets for young pigs is commonplace due to their cost effectiveness,
but these tend to be ingredients containing high quantities of soluble NSP such as lupins and soybean
meal. In addition, future pressure on the use of animal protein products such as meat and bone meal
will place greater reliance on the use of vegetable proteins in young pig diets It was necessary,
therefore, to examine other sources of vegetable proteins such that the benefits of cooked rice are not
diminished by other ‘non-beneficial’ dietary ingredients.
This study examined the effects of the sources of protein, that is, plant (vegetable) protein sources
versus animal protein sources. This comparison was made because the animal protein sources (eg,
fishmeal, meat and bone meal) are believed to be less likely to cause PWD since they do not contain
NSP. Part of this study necessitated the oral infection of pigs with a virulent strain of E. coli and
subsequent monitoring of bacterial shedding and diarrhoea after weaning.
The results from this experiment are outlined in Chapter 7.
3.4
Effects of extrusion of rice and dietary protein sources on
production, digestibility and PWD
The intention in this experiment was to use information acquired from the previous experiments to
assist further in the commercial development of specialty (processed) rice-based diets against PWD
and to enhance production. A number of different processing options for rice are available to the
Australian pig industry, such as micronisation, expansion and extrusion. Some form of heat and
8
moisture processing of rice is required before the grain can be fed to pigs to gelatinise the starch and
improve its feeding and nutritive value. Discussions were held with Australian feed manufacturers and
the pig industry regarding the relative merits of these processing techniques, and it was considered that
extrusion was the most viable processing option for rice and its subsequent incorporation into a diet.
Discussions were held with Dr Robert van Barneveld, Barneveld Nutrition, Queensland, who has
previously extruded rice at the Roseworthy Campus of the University of Adelaide. Two-tonne batches
each of a medium-grain rice (predominately Amaroo) and a long-grain rice (Doongara) were bought
from a trading company in Melbourne, because Coprice Feeds Pty Ltd was unable to supply us with
the rice. The rice was then transported to Roseworthy, extruded, and then transported to the Medina
Research Station in Western Australia, where it was stored in sealed bulka-bags ready for use in
animal experiments.
Based on results achieved in Chapters 4 and 5, it was decided to compare the two extruded rice types
against wheat, the most common cereal included in diets for young pigs in Australia. Further, the
experiment allowed an opportunity to test each rice type with either plant (vegetable) or animal
sources of protein. Production indices, digestibility and diarrhoea were monitored in this three-week
feeding experiment, as were the number of antibiotic treatments given to pigs for post-weaning
diarrhoea.
The results from this experiment are outlined in Chapter 8.
3.5
Effects of added oat hulls to extruded rice-based diets on
production, digestibility and the incidence of PWD
Results described in Chapters 7 and 8 indicated that pigs fed a diet based on cooked white rice and
animal protein sources generally resulted in better performance, particularly in the first week after
weaning, but unfortunately the incidence of diarrhoea appeared to be exacerbated. Recent work
originating from Europe, where many pig-producing countries are not permitted to use growthpromoting antibiotics, demonstrates that modulation of the diet is a key tool in the amelioration of
PWD. Researchers have been investigating the influence of added insoluble/moderately fermentable
sources of DF to reduce PWD. On the basis of this work, and after discussions with Dr Jeff Davis, it
was decided to conduct an additional experiment using oat hulls, a rich source of insoluble DF, to
establish its influence on post-weaning performance and PWD.
The results from this experiment are outlined in Chapter 9.
3.6
The nutritive value of extruded rice and cooked (autoclaved)
rice for weaner and grower pigs
The supply of extruded rice (Chapter 8) provided an ideal opportunity to examine the energy value of
extruded medium-grain and extruded long-grain rice in pigs of two different ages – weaner and grower
pigs. An accurate estimate of the energy value of rice was deemed important for the Australian feed
manufacturing industry because accurate diet formulation for piglet diets depends, in part, on the
accuracy of the estimates manufactures have for energy value. To our knowledge, there are no current
estimates of the energy values of extruded rice for pigs; establishment of these estimates would be of
value to the pig industry and potentially mean fewer barriers to the use of rice in pig diets.
The results from this experiment are outlined in Chapter 10.
9
3.7
On-farm testing of processed rice-based diets
The final part of the research project was the conduct of some on-farm trials to test the efficacy of
specialty rice-based diets against PWD and their effects on production in a practical setting. The
formulation of the diets was to be based on the information derived from the aforementioned trials.
The studies were planned for Australia’s largest producer of pigs, QAF Meat Industries (ex Bunge
Meat Industries) in Corowa, NSW, and an experiment was also planned for Wandalup Farms in
Western Australia.
After discussions with Dr Jeff Davis, RIRDC Rice R&D Programme manager, these studies could
unfortunately not be performed. The persistent drought in NSW over the past few years severely
reduced the total quantity of rice harvested and therefore available for use in such trial work. The
industry (in-kind) partner in the project, Coprice Feeds Pty Ltd in Leeton, advised that they could not
supply sufficient rice for use in such trials.
10
4. Screening and selection of rice
varieties for in vitro and feeding trials
4.1
Summary
A number of different rice varieties representing waxy, medium-grain and long-grain types were
obtained from the 2001 harvest at NSW Agriculture, Yanco, for assessment of potential suitability for
use in specialty diets for young pigs after weaning, based on in vitro chemical characteristics. The
waxy and medium-grain rice varieties were judged to be the most suitable for use in piglet diets due to
their lower amylose contents, which should result in higher starch digestibility in vivo. However, and
based on quantities of the different rice varieties produced in Australia at the present time, the
medium-grain rice Amaroo was considered the most promising to pursue for feeding to piglets. Waxy
rice varieties, although most probably affording high starch digestibility coefficients, are produced in
lesser quantities and the ‘waxy’ nature of the starch would most likely cause feed manufacturing
problems on a commercial basis.
4.2
Introduction
Rice is used mainly as a human foodstuff around the world, and only rice by-products (eg, broken
rice) are generally used in the animal industries. A plethora of studies exist investigating the physical
and chemical properties of cooked rice for man. The high starch content of cooked rice and its low DF
level makes cooked rice a ready source of absorbable glucose, and hence energy. To the converse, less
information exists regarding rice fed to pigs regarding influences on production and the prevention of
enteric disease. However, and given information that exists in the human literature about the cooking
and milling properties of rice, potential exists for the use of cooked rice in diets for young pigs. This is
because increasing pressure is being placed on governments to restrict/ban the use of growthpromoting antibiotics in the intensive livestock industries for the control of enteric pathogens such as
E. coli, the agent of PWD (Pluske et al., 2002). Use of processed rice in ‘specialty’ diets has potential
to add value to the Australian rice industry and reduce the pig Industry’s reliance on the use of
antibiotics.
The major aim of this particular study was to characterise and ‘screen’ the starch-related properties of
different rice varieties grown in NSW for potential use in diets for pigs. The three major types of rices
considered most suitable for inclusion in Australian pig diets were waxy, medium-grain and longgrain.
4.3
Materials and Methods
Dr Melissa Fitzgerald sourced a variety of different rice samples from the 2001 rice harvest at NSW
Agriculture, Yanco. The waxy rice varieties were Shimuzi and Tarra 140, the medium-grain rice
varieties were Amaroo, Millin and Langi, and the long-grain rice varieties were Doongara and L203.
4.3.1 Chemical analyses
The amylose content was determined at NSW Agriculture, Yanco, according to a PCR-based method
established in Dr Fitzgerald’s laboratory at Yanco, NSW. The amylopectin content of rice was
determined by difference from total starch. Total Starch and Megazyme Resistant Starch kits
(Megazyme International Ireland, Ltd., Wicklow, Ireland) were used for analysis of total starch and
RS, respectively. For total starch analysis, 100-mg samples were pre-incubated with 2 mL DMSO in a
boiling water bath for 5 min prior to incubation with the thermostable α-amylase and then with
amyloglucosidase. The starch content was determined at 510 nm using a spectrophotometer (UVmini-
11
1240, Shimadzu, Kyoto, Japan). For RS determination, 100-mg samples were incubated at 37 °C for
16 hrs with pancreatic α-amylase and amyloglucosidase. The hydrolysed sugars were washed several
times with ethanol. The remainder was then hydrolysed with 2M KOH and then with
amyloglucosidase. The RS content was determined at 510 nm. The fast digestible starch (FDS) content
of the raw rices was determined using the method of Zarrinkalam et al. (2001), which is a modification
of the AOAC official method 996.11 (AOAC 1997).
4.4
Results and Discussion
The starch-related characteristics of the rice samples are presented in Table 4.1. The amylose content
varied five-fold, ranging from 5.1% in the waxy Shimuzi variety to 25.7% in the long-grain variety
L203. The amylose:amylopectin ratio in these two varieties similarly differed, ranging between 0.05 to
0.35. Gelatinisation temperature varied between 61.6 ºC for Shimuzi to 78.8 ºC for the long-grain
variety Langi.
The amylose content of rice provides an indication of the texture of cooked rice, and the higher the
amylose content, then in general the firmer the rice but the less digestible the rice will be. During
starch digestion in the pig, the α–amylase produced in the pancreas breaks down amylose to maltose
and maltotriose and amylopectin is broken down to maltose, maltotriose and α–limit dextrins (Li et
al., 2004). Amylopectin is regarded as being more digestible than amylose in starch because its
branched structure allows more complete digestion by α–amylase, so in this regard, and for the young
pig that has a lower capacity to digest amylose (Black, 2001), it is suggested that a waxy rice or
medium-grain rice would be most suitable for incorporation into diets for young pigs, at least for
production purposes. The digestibility of starch in the long-grain varieties, ie, Doongara and L203,
might be reduced given the higher amylose content. This, in turn, could cause poorer performance and
(or) result in increased susceptibility to enteric pathogens such as E. coli because of a higher bypass of
RS entering the distal regions of the GI tract.
Table 4.1 Starch-related characteristics of selected rice varietiesA.
Type
rice
of Variety
Total
starch,
g/kg
776
774
Amylose, %
Amylopectin, %
Am:ApB
Gelatinisation
temperature, ºC
5.1
8.6
94.9
91.4
0.05
0.09
61.6
78.7
Waxy
Shimuzi
Tarra 140
Medium
grain
Amaroo
799
18.2
81.8
0.22
67.9
Millin
Langi
791
772
16.4
17.2
83.6
82.8
0.20
0.21
69.3
78.8
Doongara
L203
775
801
23.8
25.7
76.2
74.3
0.31
0.35
75.7
73.8
Long grain
A
All analyses conducted by Dr Melissa Fitzgerald, NSW Agriculture, Yanco (except total starch).
Am:Ap: amylose to amylopectin ratio.
B
Gelatinisation temperature is the temperature required to melt the amylopectin crystals within starch
(Fitzgerald et al., 2003). This is important information to know for the pig feed industry given that rice
used in diets will need to undergo some form of heat processing. In this regard, it would appear that in
all varieties examined, the temperature at which all starch crystals ‘melt’ (are gelatinised) will present
no difficulties to pig feed manufacturers. This is because pelleting of feeds generally occurs at
temperatures ranging between 75 ºC and 90 ºC. Extrusion temperatures commonly exceed 120 ºC.
12
In addition to these measurements, an in vitro ‘fast digestible starch’ (FDS) technique adapted in our
laboratory was used as a rapid screening method for the suitability of rice in diets for young pigs.
Analysis performed using this technique showed that the amount of glucose released in vitro
correlated to the amylose content in the rice, suggesting that this method has possible merit as a means
of screening rices for potential inclusion in diets. Further implementation and validation of this
technique is described in Chapter 5.
It was the intention of this study to track these particular varieties over three successive growing
seasons to examine the variation that occurred in the selected measured parameters, and hence
establish a nutrient database for the Australian rice and pig industries. Unfortunately the drought that
affected the rice-growing regions in NSW over the duration of this particular project restricted this
study to samples harvested only in 2001, because it was not possible to continue with such work given
the lack of rice available and the time-scale of the project.
In conclusion, the wide range of samples examined in the harvest from 2001 provided a valuable
screening database and allowed progression of the project onto its next stage. This study met its aim in
allowing the selection of rice types deemed suitable for use in piglet feeding trials and associated in
vitro studies, considering the tonnage of rice produced in Australia, the quantities of ‘brokens’
available for use in the Australian pig industry, and the requirements for processing of rice by the feed
manufacturing industry.
13
5. Selection of rice varieties and
processing methods on physicochemical effects in the weaned pig
Experiment A: In vitro assessments of starch-related properties in
response to rice type, cooking methods and cooling after cooking
5.1
Summary
Three in vitro experiments were conducted to examine the effect of rice type, rice:water ratio and
cooling on the resistant starch (RS) content of autoclaved rice. The three types of rice used were
Amaroo (lower amylose, medium-grain variety), Doongara (higher amylose, long-grain variety) and
parboiled rice. In Experiment 1, total starch, amylose, fast digestible starch (FDS) and RS contents in
three uncooked rice samples were examined. The variety Amaroo contained less amylose (18.8 g
100g-1, P=0.001), a higher FDS content (21.7 g 100g-1, P<0.001) and less RS (0.1 g 100g-1, P<0.001)
than Doongara (25.6, 15.9, 0.4, respectively). Parboiled rice contained the highest FDS (33.9 g 100g-1)
and RS (0.72 g 100g-1) contents with an amylose content of 25.4 g 100g-1. In Experiment 2, the effects
of rice type, rice:water ratio (1:1 or 1:2 w/w) and cooling (freshly dried or dried after refrigeration at 4
°C for 24 hrs) on the RS content of autoclaved rice were examined. The RS contents differed
according to rice type (0.6, 1.4, 3.7 g 100g-1 for Amaroo, Doongara and parboiled, respectively,
P<0.001). Decreasing the rice:water ratio (1:2) and cooling (24 hours at 4oC) after autoclaving
increased the RS content (P<0.001). In Experiment 3, extrusion of rice using a single-screw extruder
decreased the RS content only in Doongara, which had the highest RS content in its unextruded (raw)
form (0.42 to 0.16 g 100g-1, P=0.02). The results indicate that more amylose, a lower rice:water ratio
and cooling after autoclaving increase the RS content of rice, while extrusion decreases the RS
content.
5.2
Introduction
Resistant starch (RS) has been defined as starch that is resistant to enzymatic digestion in the small
intestine but is fermented by the microbiota in the large intestine of non-ruminant animals (Englyst et
al., 1992). Debate still exists as to whether RS should be included as part of dietary fibre (DF) because
while it resists enzymatic digestion in the small intestine and behaves similarly to DF in the gut, it has
the chemical structure of hydrolysable alpha-linked hexoses (Trowell et al., 1976; Englyst and
Cummings, 1987). It is evident that some feed ingredients containing RS are fermentable in the small
intestine (eg, Berggren et al., 1995; Heijnen and Beynen, 1997; Govers et al., 1999). Resistant starch
is generally classified as RS1, physically inaccessible starch due to structural encapsulation which is
mainly found in raw forms of grain; RS2, enzyme-resistant, B-type starch granules found mainly in
unripe bananas and raw potato; and RS3, retrograde starch that is found mainly in processed grains
and also occurs after cooking and cooling (Englyst et al., 1992).
Rice is characterised by its high starch content, low non-starch polysaccharide (NSP) content and
lower protein content in comparison to other cereals (Juliano, 1992). Rice has been investigated in
diets for young pigs because of its positive effects on production relative to cereals such as maize,
wheat and sorghum (Alcantara et al., 1989; Mateos et al., 2001, 2002; Martin et al., 2003; Vicente et
al., 2004). Furthermore, the use of cooked rice has been associated with reductions in PWC and swine
dysentery (see review by Pluske et al., 2002; Pluske et al., 2003; Hopwood et al., 2004). Moreover,
Reid and Hillman (1999) reported lower coliform populations and higher Lactobacillus populations
when retrograde RS was fed to pigs in the form of raw potato starch, suggesting that RS might play a
role in altering the microbial ecology of the lower gut. Resistant starch is also known to influence ileal
14
starch and faecal crude fat and energy digestibility, and the proliferation of colonic microbes (De
Schrijver et al., 1999).
It is most likely that rice requires some form of cooking and drying, such as extrusion, to enable
gelatinisation of the starch and its incorporation into a commercial piglet diet. Generally, the RS
content of rice increases with cooking and the amylose content is positively correlated with the
increase in RS content in cooked rice (Juliano, 1992). Quantifying the RS contents of rice having
different amylose contents and with various cooking and cooling combinations, therefore, should
assist in the selection of rice types and processing conditions for the formulation of diets for piglets.
A number of in vitro studies were conducted to examine whether: 1) rice with a higher amylose
content contains a higher RS content, 2) autoclaving (cooking) will increase the RS content, 3) cooling
after autoclaving will increase the RS content of rice, and 4) extrusion reduces the RS content of rice.
5.3
Materials and Methods
5.3.1 Rice varieties
Three varieties of commercially available rice were obtained: (i) Amaroo (a lower amylose, mediumgrain rice; Australian Ricegrowers’ Cooperative, Leeton, NSW, Australia), (ii) Doongara (a higher
amylose, long-grain rice; Australian Ricegrowers’ Cooperative, Leeton, NSW, Australia), and (iii)
parboiled rice (‘Tastic’ premium parboiled rice; Riviana Foods Pty Ltd, Australia).
5.3.2 Experimental design, cooking and cooling
Three in vitro experiments were conducted. In Experiment 1, the total starch and RS contents were
compared in the three uncooked rice samples. In Experiment 2, the effects of rice type, rice:water ratio
and cooling combinations on the RS content of autoclaved rice were tested using a 3 x 2 x 2 factorial
design. The respective factors in the study were the three rice types (Amaroo, Doongara, parboiled),
two rice:water ratios (1:1 or 1:2 w/w) and two cooling treatments (non-refrigerated, or refrigerated at 4
°C for 24 hrs). All samples of rice were cooked in an autoclave (121 °C, 20 min steaming plus 20 min
drying) with rice: water ratios of either 1:1 or 1:2 (w/w). After cooking, the rice was divided into two
portions. The first portion was sampled immediately after cooking (‘Fresh’) whilst another sample was
taken following refrigeration at 4 °C for 24 hours (‘Cooled’). These samples of rice were then dried at
70 °C for 48 hours to enable chemical analyses. After drying, the samples were milled to pass through
a 1 mm screen twice. The milled samples were later analysed for total starch and RS contents. In
Experiment 3, the effect of extrusion was examined by comparing the RS contents in raw and extruded
forms of Amaroo and Doongara. The extruded rice samples were obtained from the Roseworthy
Campus of the University of Adelaide.
5.3.3 Chemical analyses
Dry matter (DM) was measured using the AOAC official method 930.15 (AOAC, 1997). Total starch
and RS were determined as described previously (Chapter 4.2.1). The fast digestible starch (FDS)
content of the raw rices was determined using the method of Zarrinkalam et al. (2001), which is a
modification of the AOAC official method 996.11 (AOAC 1997). Ten sub-samples of each treatment
combination were analysed in duplicate for the analysis of total starch, RS and the FDS contents. The
amylose content of raw rice was determined by the calorimetric measurement of the iodine binding
capacity of the amylose using a Megazyme Amylose/Amylopectin Assay Kit (Megazyme International
Ireland, Ltd., Wicklow, Ireland). Amylopectin content was determined as the difference between the
measured amylose and total starch contents.
15
5.3.4 Statistical analysis
For Experiment 1, the effect of rice type on the total starch, amylose, FDS and RS contents were
assessed by one-way ANOVA. In Experiment 2, treatment effects were assessed by ANOVA for a
factorial arrangement with the main effects being rice type, rice:water ratio and refrigeration. The
effects were considered as fixed effects in the model. For Experiment 3, the effect of extrusion
cooking on the RS content was assessed by ANOVA for a factorial arrangement with the main effects
being rice type and extrusion cooking. All statistical analyses were conducted using the statistical
package StatView 5.0 for Windows, SAS Inc. (AddSoft Pty. Ltd., Woodend, Vic., Australia).
Statistical significance was accepted at P < 0.05.
5.4
Results
5.4.1 Experiment 1
Total starch, amylose, FDS and RS content in three uncooked rice samples are presented in Table 5.1.
Total starch contents of uncooked rice were similar in Amaroo and Doongara, but were higher in
parboiled rice (P<0.001). Amylose content was lower in Amaroo than in Doongara and parboiled rice
(P<0.001). The FDS content decreased in the order of parboiled, Amaroo and Doongara (P<0.001).
The RS content decreased in the order of parboiled, Doongara and Amaroo (P<0.001). The RS
contents of raw rice ranged from 0.1 to 0.7 g 100-1.
5.4.2 Experiment 2
The main effects of rice type, rice:water ratio, refrigeration on RS content of autoclaved rice are
presented in Table 5.2. The total starch contents in autoclaved rice were not influenced by any
treatment. However, the RS content was significantly different in the three autoclaved rice types
(P<0.001). The range in RS content was 0.6 g to 5.1 g 100g-1 DM. Decreasing the rice:water ratio and
refrigeration (‘Cooled’, ie, 24 hours at 4oC) after autoclaving significantly increased the RS content
(P<0.001). There were significant three-way interactions between rice, rice:water ratio and
refrigeration (P<0.002) (Figure 5.1).
5.4.3 Experiment 3
The effect of extrusion on RS content is presented in Table 5.3. Extrusion decreased the RS content in
Doongara to the level of the lower amylose, medium-grain variety Amaroo. However, the RS content
was not influenced by extrusion in variety Amaroo as evidenced by a significant rice by extrusion
interaction (P=0.005).
16
1
Table 5.1 Starch-related properties (DM) in three types of uncooked (raw) rice (Experiment 1)A.
Amaroo
Doongara
Parboiled
Pooled mean
SEMB
Probability, P=
Total starch (g 100g-1)
86.1a
85.6a
89.4b
87.0
0.33
<0.001
Amylose (g 100g-1)
18.8a
25.6b
25.4b
22.8
1.15
<0.001
Amylopectin (g 100g-1)
67.3a
60.0c
64.0b
64.5
0.98
0.002
Amylose:amylopectin ratio (%)
0.28a
0.43b
0.40b
0.36
0.022
<0.001
FDS (g 100g-1)C
21.7b
15.9a
33.9c
20.5
1.37
<0.001
Resistant starch (g 100g –1)C
0.10a
0.42b
0.72c
0.41
0.058
<0.001
Prop. RS of total starch (%)
0.11a
0.49b
0.81c
0.47
0.066
<0.001
Rice
2
3
4
5
A
Values are mean of 10 observations, except amylose (3) and FDS (8).
SEM: standard error of the mean.
C
FDS: fast digestible starch; RS: resistant starch.
abc
Values within a row with different superscripts are significantly different (P<0.05).
B
17
1
Table 5.2 Main effects of rice type, rice:water ratio and refrigeration on starch-related properties (DM; Experiment 2)A.
Total starch (g 100g-1)
Treatment
Resistant starch (RS)
Prop. RS of total starch (%)
–1
(g 100g )
Amaroo
90.2
0.60a
0.66a
Doongara
90.4
1.42b
1.57b
Parboiled
90.1
3.74d
4.15d
1:1
90.3
1.52b
1.69b
1:2
90.1
2.31c
2.56c
Fresh
90.2
1.54b
1.71b
Cooled
90.2
2.29c
2.54c
Pooled mean
90.2
1.92
2.13
SEMC
0.07
0.149
0.157
Rice
Rice:water ratio
RefrigerationB
StatisticsD
2
3
4
5
6
7
Probability, P=
Rice
0.327
<0.001
<0.001
Rice:water ratio
0.245
<0.001
<0.001
Refrigeration
0.669
<0.001
<0.001
A
Values from mean of 10 observations for each treatment combination.
B
Fresh’: autoclaved and dried at 70 °C, 48 hrs; ‘Cooled’: autoclaved and stored at 4 °C, 24 hrs and dried at 70 °C, 48 hrs.
C
SEM: Standard error of the mean.
D
Three way interactions were significant for RS (P=0.002) and prop. RS of total starch (P=0.005); abcValues within a column with different superscripts are
significantly different (P<0.05).
.
18
Table 5.3 Effect of extrusion on the RS content (g 100g-1 DM) of rice (Experiment 3)A.
Resistant starch (g 100g-1) DM
Raw
Extruded
Amaroo
0.10
0.13
Doongara
0.42
0.16
Pooled mean
0.26
0.14
SEMB
0.051
0.028
Statistics
Probability, P=
Rice (R)
<0.001
Extrusion (E)
0.020
R x E interaction
0.005
A
Values are mean of 10 observations for raw rice and mean of 5 observations for extruded rice.
SEM: standard error of the mean.
B
Resistant starch (g 100g-1 DM)
6
5
4
3
2
1
0
Refrigeration
Rice:water
Rice
UC
F
R
1:1
F
R
UC
F
1:2
Amaroo
R
1:1
Doongara
F
1:2
R
UC
F
R
F
1:1
R
1:2
Parboiled
Figure 5.1 Interaction plot for the RS content (g 100g-1 DM) against rice type (Amaroo, Doongara and
parboiled), rice:water ratio (1:1 versus 1:2) and cooking (‘Fresh’ versus ‘Cooled’) combinations in
autoclaved rice (Experiment 2). The UC, F and R represents uncooked, fresh and refrigerated
(‘Cooled’) rice, respectively.
19
5.5
Discussion
Experiment 1 examined the content of RS1 in three types of rice, although parboiled rice presumably
contains both RS1 and RS3. As hypothesised, the higher amylose rice variety Doongara had a higher
RS content than the lower amylose variety Amaroo, which is in agreement with previous reports
(Marsono and Topping, 1993; Sagum and Arcot, 2000). The RS contents in the three types of raw rice
were within the range reported previously (Muir and O’Dea, 1993; Tetens et al., 1997), with the
parboiled rice having the highest RS content. Parboiling leads to the formation of very heat resistant
amylose-lipid complexes (Juliano, 1992), which have been shown to reduce the rate of starch
digestion in rats (Holm et al., 1983). Tetens et al. (1997) reported that the amylose content of rice was
negatively correlated to the degree in vitro of starch digestion, and that parboiling caused significantly
lower rates of starch digestion compared to non-parboiled rice varieties. Data from our study only
partly supports these findings because although Amaroo had lower amylose and RS contents than the
parboiled rice, Doongara had a similar amylose content to the parboiled rice but had significantly less
RS and a lower FDS content. Tetens et al. (1997) commented, however, that the extent to which rice is
parboiled varies considerably and hence this could be an explanation for this discrepancy.
Starch with a high amylose:amylopectin ratio has a dense and rigid structure due to the extensive
hydrogen bonding between amylose molecules and possible amylose-lipid complexes, which results in
hard starch gels in cooked rice (Tetens et al., 1997). The tight helical twists of amylose chains
encapsulate some starch that makes it less accessible to pancreatic α-amylase in the small intestine
(Zobel and Stephen, 1995; Black 2001). Therefore, the starch in waxy or lower amylose rice is
generally hydrolysed more rapidly and more completely than that in a higher amylose rice variety (Hu
et al., 2004). The highest RS content found in uncooked parboiled rice is due mainly to the formation
of RS3 during the parboiling process (Marsono and Topping, 1994; Devi and Geervani, 2000).
Experiment 2 demonstrated that the type of rice, rice:water ratio and refrigeration influenced the
formation of RS. The higher amylose variety Doongara had a higher RS content after autoclaving,
which is in agreement with previous reports by Juliano (1992), Marsono and Topping (1993), Mangala
et al. (1999b) and Sagum and Arcot (2000). Autoclaving causes gelatinisation of the starch, but then
retrogradation to RS3 occurs if the rice is cooled after the cooking process. Gelatinisation disorders
the molecular structure of starch and the dispersed starch polymers reform semi-crystalline structures
(retrogradation) when cooled (Garcia-Alonso et al., 1998). The presence of higher amounts of linear
amylose increased RS formation upon cooking and cooling because the amylose molecules align
themselves or associate with each other during retrogradation (Sagum and Arcot, 2000), and
retrograded amylose is particularly resistant to hydrolysis by pancreatic α-amylase (Gee et al., 1991).
Although long unbranched chains of amylopectin are possibly involved in the formation of RS when
cooked starch was stored for up to 20 days (Mangala et al., 1999a), structural studies revealed that the
majority of RS formed upon cooking and cooling was a linear (1Æ4)-a-D-glucan, originating from
amylose (Mangala and Tharanathan, 1999). Also, amylose chain length was positively related to the
RS formation in a study with potato starch (Eerlingen et al., 1993). Therefore, RS formation in
autoclaved rice most likely increases with the increasing amount of amylose in rice, as espoused by
Juliano (1992). Highest RS contents have been observed when parboiled rice was autoclaved since
repeated cooking further increases the RS content in rice (Mangala et al., 1999a). In addition, a
decreased rice:water ratio increased RS formation after cooking. With excess water, the amorphous
phase of the starch granule swells and the crystalline regions become even more disrupted. This higher
disruption contributes to the higher RS formation when the starch retrogrades (Sagum and Arcot,
2000).
In the current study, refrigeration of cooked rice after autoclaving (‘Cooled’) increased RS formation
by promoting retrogradation of starch polymers. Temperature plays an important role during the
retrogradation process. Marsono and Topping (1993) found significantly increased RS contents in rice
cooked in a rice-cooker and in microwave-cooked rice when they were stored at 0-4 °C compared to
20
freshly cooked rice. Mitsuda (1993) found that rice stored at –20 °C retrogrades more than one stored
in a refrigerator at approximately 4 °C. In a more detailed study, Kavita et al. (1998) found that the RS
content of cooked rice was 1.96 g 100g-1 DM. When samples were stored for 24 hrs or 48 hrs at 4 °C,
the RS contents increased to 3.37 g and 4.38 g 100g-1 DM, respectively. In their next experiment,
Kavita et al. (1988) reported that the RS content increased to 14%, 20% and 27%, when the cooked
rice (RS content of 1.14 g 100g-1 DM) was stored for 12 hrs at 27 °C, for 12 hrs at 4 °C and for 24 hrs
at 4 °C, respectively. In our study, the significant three-way interaction between rice, the rice:water
ratio and refrigeration (Figure 5.1) was due to disparity in the RS values with different combinations
of cooking and refrigeration. For example, the increase in RS content in Amaroo after cooking
(autoclaving) and refrigeration attained the same level as that in the ‘Fresh’ high amylose rice.
Extrusion decreased the RS content in Doongara but not in the lower amylose rice Amaroo, which
caused a significant statistical interaction (P=0.005; Experiment 3). Parchure and Kulkarni (1997)
reported that extruded rice had a lower RS content compared to that of raw and cooked rice, a finding
found also by Pluske et al. (1996) but in a comparison of whole (ground) wheat versus extruded
wheat.
In conclusion, the range in RS formation as a consequence of the various cooking and cooling
combinations under test was from 0.6 g to 5.1 g 100g-1 DM. The extent of RS formation was
significantly influenced by the amylose content of rice, the volume of water used for cooking, and
cooling after cooking. Further work is required using pigs to ascertain the importance, or otherwise, of
these treatments on pig production and resistance/susceptibility to PWC. This was investigated in part
in the next experiment.
21
6. Selection of rice varieties and
processing methods on physicochemical effects in the weaned pig
Experiment B: Effect of rice type fed to piglets after weaning on
starch digestion, digesta and fermentation characteristics and the
faecal shedding of haemolytic E. coli
6.1
Summary
An experiment was conducted in male pigs weaned at approximately 21 days of age to examine the
effects of different types of cooked white rice mixed with animal protein sources on starch digestion,
fermentation characteristics, shedding of E. coli and performance in the 14 days following weaning.
Pigs were allocated in a completely randomised block design having four dietary treatments, with 12
pigs allocated to each diet. The three rice types used were a medium-grain rice (Amaroo; AM), a longgrain rice (Doongara; DOON) and a waxy rice (WAXY), which were included in a diet at an inclusion
level of 703 g kg-1. These three diets were compared against a diet comprising predominately wheat,
barley and lupins (WBL). All diets contained the marker titanium dioxide (TiO2; 1 g/kg of diet) for
subsequent determination of apparent starch digestibility. On days 1, 3, 7 and 8/9 after weaning, a
faecal swab was taken for assessment of β–haemolytic E. coli and faecal consistency. Pigs were
euthanased for sample collection after 14 days. Apparent digestibility of starch measured at the
terminal ileum was highest (P=0.004) in AM (≈ 96%) and WAXY (≈ 99%) and lowest, but the same,
in diets DOON and WBL (≈ 88%). Starch digestibility measured in the distal colon was highest
(P<0.001) in all three rice-based diets. Pigs fed cooked rice generally consumed more dry matter and
grew favourably relative to pigs fed diet WBL in the 14 days after weaning, and diverted more
absorbed nutrients to carcass gain (P<0.001) than to growth of the visceral organs than pigs fed diet
WBL. Pigs fed diet WBL produced more VFA in the gastrointestinal tract than pigs fed rice (P<0.05).
Relatively low levels of infection with haemolytic E. coli were observed in this experiment making
interpretation of the dietary effects on PWD difficult to interpret, however pigs fed diets AM and
WBL appeared to shed less haemolytic E. coli than pigs fed diets DOON and WAXY. It is thought
that the lack of a slowly/moderately fermentable source of DF in the rice-based diets could have
shifted the balance of the bacteria to proteolytic rather than saccharolytic and predisposed the pigs to
PWD.
6.2
Introduction
Restrictions in some countries on the use of growth promoting antibiotics and heavy minerals such as
zinc and copper in diets for young pigs has seen the development of alternative feed- and
management-related strategies to maintain production and prevent enteric disorders, such as PWC, in
the absence of antimicrobials (Pluske et al., 2002). A large number of products are marketed to the pig
industry as alternatives/replacements to growth promoting antibiotics and heavy minerals, but these
sometimes show inconsistent and variable effects.
Another, or complementary, approach to feeding the weanling pig without dietary antimicrobials is to
modify the ingredients used. Most pig starter diets are based on cereals, such as wheat, barley, oats
and (or) maize, and a combination of animal and vegetable proteins. Some of these feedstuffs are high
in certain NSP, which have been shown to reduce live weight gain (eg, McDonald et al., 1999),
increase growth of the gastrointestinal organs at the expense of body gain (eg, McDonald et al., 1999;
McDonald et al., 2001; Hopwood et al., 2003; Pluske et al., 2003) and predispose pigs to PWC
(McDonald et al., 1999; McDonald et al., 2001; Hopwood et al., 2002). For example, Hopwood et al.
22
(2004) demonstrated that pearl (dehulled) barley fed to weanling pigs increased digesta viscosity and
reduced starch digestion concomitant with greater proliferation of β–haemolytic Escherichia coli, in
comparison to feeding cooked white rice. Furthermore, Pluske et al. (2003) reported beneficial effects
of feeding a diet based on cooked white rice to weanling pigs in terms of reduced antibiotic treatments
and empty bodyweight gain.
Rice would appear to offer, therefore, a promising alternative to other cereals because of its higher
starch and lower NSP contents, and its association with reduced shedding of enterotoxigenic E. coli.
Unfortunately the higher price of rice relative to other cereals such as wheat may preclude its
widespread use. Nevertheless, work from Spain (eg, Mateos et al., 2001, 2002) has shown that cooked
white rice fed to weanling pigs, either alone or in combination with oats, enhances performance after
weaning and reduces diarrhoea.
Many types of rice are grown and, as would be expected, they differ in chemical characteristics such
as amylose:amylopectin, starch and RS levels and gelatinisation temperature (Marsono and Topping,
1993; Fitzgerald et al., 2003). These differences, in turn, are likely to alter their physico-chemical
characteristics in the gastrointestinal tract of pigs. If rice is to be considered for use in young pig diets,
then it is important to examine different rice types that could be available to the feed manufacturing
industry. This experiment investigated a number of cooked rice-based diets on apparent starch
digestibility, fermentation and digesta characteristics, shedding of β–haemolytic E. coli and
performance after weaning. The general hypothesis tested was that rice containing a lower
amylose:amylopectin ratio would cause a higher rate of starch digestion concomitant with decreased
shedding of β–haemolytic E. coli from the gastrointestinal tract.
6.3
Materials and Methods
6.3.1 Animals and housing
Forty-eight entire male pigs (Large White x Landrace) aged 19-24 days of age and weighing 6.7 ±
0.24 kg (mean ± SE) were used in the experiment. Pigs were obtained from a commercial farm on the
day of weaning and transported in an insulated horse float to the experimental facility at Murdoch
University. Upon arrival, the pigs were ear-tagged, weighed, and stratified into pens of four pigs each
according to treatment and liveweight. Pens were of wire-mesh construction with slatted metal floors,
and measured 2.5 m2 in floor area. Each pen had an enclosed wooden box containing a heat lamp, and
was equipped with a nipple water drinker and a feed trough. The ambient temperature varied between
19 and 26°C throughout the study. The Murdoch University Animal Ethics Committee and the Animal
Ethics and Experimentation Committee of the WA Department of Agriculture approved this
experiment.
6.3.2 Experimental design, diets and feeding
Pigs were allocated in a completely randomised block design having four experimental (dietary)
treatments, with 12 pigs allocated to each treatment. The experiment was conducted in three replicates,
with 16 pigs (ie, one pen of four pigs per treatment combination) constituting a single replicate. The
three rice-based diets in the experiment comprised: (i) medium-grain rice (cultivar Amaroo) plus an
animal protein supplement (AM), (ii) long-grain rice (cultivar Doongara) plus an animal protein
supplement (DOON), (iii) waxy rice plus an animal protein supplement (WAXY), and (iv)
commercially based weaner diet (WBL) (Table 6.1). The rice types were chosen on the basis of results
in Chapter 4 because they each differed in their chemical characteristics (eg, amylose:amylopectin)
and would therefore be expected to cause different physico-chemical effects in the gastrointestinal
tract.
The three diets based on rice were fed daily by mixing the cooked rice with the remainder of the diet
(on an as-fed basis) immediately before feeding. A small amount (150 to 200 mL) of warm water was
23
used to assist with mixing. Each rice type was cooked in an autoclave at 121°C for 20 minutes using a
ratio of 2:1 water: dry rice, and was left to cool overnight in a cool room (4° C) prior to feeding the
following day. Diet WBL was fed as a meal. Pigs were fed between 0900 and 1030 daily, and any
residue was recorded the following day. Pigs were fed the experimental diets on an ad libitum basis
for 14 days, after which they were euthanased for sample collection (see below). Antibiotics, zinc
oxide or copper sulphate were not included in the diets.
6.3.3 Faecal shedding of β-haemolytic E. coli
All pigs were swabbed upon arrival at Murdoch University by inserting a soft cotton bud into the anus
of the pig. Swabs were then cultured for the presence of β-hemolytic E. coli on sheep blood agar
plates (after McDonald et al., 2001). Swabs from all pigs were also collected and subsequently
cultured on day 1, day 3, day 6, day 7 and day 8 of the experiment. Plates were scored and the faecal
consistency determined according to the methods of Montagne et al. (2004).
6.3.4 Post-mortem procedures
Pigs in each pen were fed their morning meal on the day of sampling in a staggered fashion across
each dietary treatment, such that the period of time between feeding and euthanasia was 3-6 hours.
Pigs were transported a distance of approximately 200 metres from the experimental facility to a
necropsy room. Pigs were euthanased with a lethal dose of sodium pentobarbitone solution
administered intravenously (Lethobarb; May and Baker Pty. Ltd., Australia). Cervical dislocation and
exsanguinations followed, reducing the amount of blood present as a potential contaminant during
sample collection and weighing. A faecal sample was taken at this time. The abdomen was then
opened from the sternum to the pubis, and the gastrointestinal tract was removed. It was then divided
into four sections (stomach, small intestine, caecum and colon) that were tied off with light twine
before being separated. The small intestine was stripped free of its mesentery and laid on a table into
sections of equal length.
The full and empty weights of the small intestine, caecum and colon were determined by first
weighing the organ containing its contents and then reweighing the organ after the contents were
removed and the organ was blotted dry. Samples of digesta from the ileum, caecum, and proximal and
distal colon were collected into plastic jars, placed immediately on ice, and later frozen at –20° C for
subsequent chemical analyses (see below). The pH of digesta was measured by inserting the electrode
of a calibrated portable pH meter (Schindengen pH Boy-2; Schindengen Electric MFG, Tokyo, Japan)
into the collected sample. A sub-sample of digesta from the ileum and caecum was collected for
determination of viscosity. Finally, a segment of adipose tissue was collected adjacent to the
longissumus dorsi, placed in a sterile container, and immediately placed in liquid nitrogen for
subsequent determination of ATP-citrate lyase (see below).
6.3.5 Analytical methods
The DM content of the digesta and faecal samples and the starch, FDS and amylose contents were
measured as described previously (Chapter 4.2.1). Titanium dioxide (TiO2) was measured according to
the methods described by Short et al. (1996).
24
Table 6.1 Composition of experimental diets (air-dry basis, g kg-1).
DietA,B
Ingredient
Rice
Wheat
Barley
Australian sweet
lupins
Skim milk powder
Soybean meal
Whey powder
Blood meal
Fish meal
Meat and bone
meal
Canola oil
L-lysine
DL-methionine
L-threonine
L-tryptophan
Choline chloride
(60%)
Dicalcium
phosphate
Limestone
Salt
Vitamin and
mineral premixC
Calculated
analysis:
DE (MJ/kg)
Crude protein, g
kg-1
Total lysine, g kg-1
A
AM
702.7
-
DOON
702.7
-
WAXY
702.7
-
WBL
508.5
100
100
100
30
100.5
51.5
100
30
100.5
51.5
100
30
100.5
51.5
50
42.8
50
70
14.4
5
2.8
0.4
1.4
0.3
4
5
2.8
0.4
1.4
0.3
4
5
2.8
0.4
1.4
0.3
4
40
5.1
0.8
1.9
10.9
0.4
-
-
-
2.1
1
0.7
1
0.7
1
0.7
1.6
1.5
15.3
200
15.3
200
15.3
200
15.2
220
13.0
13.0
13.0
12.9
Diets were formulated to contain 8 g Ca/kg and 4.5 g available P/kg.
B
Refer to the text for details of diets.
C
Premix provided (mg/kg air-dry diet): retinyl acetate 3.44, cholecalciferol 0.065, α-tocopheryl acetate 20,
menadione 4.4, riboflavin 4, pyridoxine 1.6, cyanocobalamin 0.02, pantothenic acid 14, nicotinic acid 20, Co
0.2, I 0.6, Fe 120, Mn 60, Zn 100, Cu 10, Se 0.13.
25
To determine volatile fatty acid (VFA) concentration (C2: C6), thawed digesta samples from the ileum,
caecum, proximal colon and distal colon were diluted either 1:1 (w:v) (ileal digesta) or 1:2 (w:v)
(caecal and colonic digesta) with distilled water, mixed, centrifuged and the supernatant fraction
analysed chromatographically. The supernatant fraction (0.1 mL) was added to 1 mL internal standard
solution containing valeric acid before processing on a capillary GC. A working standard and a control
(distilled water) were included in each run of the analysis, with the working standard containing acetic
acid (60 mM), propionic acid (20 mM), isobutyric acid (6.67 mM), butyric acid (20 mM), isovaleric
acid (10 mM), valeric acid (10 mM) and caproic acid (4 mM). The Hewlett Packard 5890A capillary
GC (Agilent Technologies, Forrest Hill, Victoria, Australia) was maintained at injector and detector
FID settings of 260 °C and 265 °C respectively, and an initial and final oven temperature of 120 °C
and 240 °C, respectively. The carrier gas flow rate was 5 mL/min and the split-flow rate was 70
mL/min. The Hewlett Packard Chemstation integration system was used to calculate the VFA
concentrations from the area of the peaks.
For viscosity, an aliquot of the digesta from the ileum was placed in an Eppindorf tube, mixed on a
vortex, and centrifuged at 12000 g for 8 min (Quantum Scientific Pty. Ltd., Milton, QLD, Australia).
The supernatant fraction (0.5 mL) was placed in a Brookfield LVDV-II+ cone-plate rotational
viscometer (CP40; Brookfield Engineering Laboratories Inc., Stoughton, MA, USA), and the viscosity
of all samples was measured. The viscosity was measured in mPas.
The specific activity of ATP-citrate lyase in adipose tissue was determined according to the
methodology established in the Biochemistry Laboratory at Murdoch University. ATP-citrate lyase
was used in this study as a biomarker of starch digestion and hence glucose availability, since this
enzyme is the rate-limiting step in the conversion of glucose to adipose tissue. Briefly, stock premix
buffer was prepared just prior to the assay. Assay ingredients were added to one blank and two
duplicate, 1-ml cuvettes (path length = 1 cm). The assay protocol is summarised in Table 5.2. The
assay was started after 10 minutes pre-incubation at 37 ºC by adding the assay mix, which contained
3.025 mg ATP and 380 μl NaOH (1M) made up to 2 ml with deionised water. The rate of NADH
disappearance was measured over 5 minutes.
Table 6.2 Conditions used in the ATP-citrate lyase assay.
Reagent
Blank cuvette (μl)
Duplicate sample cuvettes (μl)
A
Premix buffer
750
750
Cytosol extractB
200
200
Water
50
Assay mix
50
A
0.1 ml of 0.15 M Tris buffer (pH 7.4), 0.1 ml of MgCl2 (0.1 M), 0.1 ml of K3citrate (0.2 M), 0.4 mg
of CoA, 0.21 mg of NADH, 0.7 μl of mercaptoethanol, 2 μl of malic dehydrogenase, made up to 0.95
ml using deionised water.
B
Prepared by pulverising frozen sample of adipose tissue and then sonicating to release cytosolic
contents.
6.3.6 Calculations and statistical analyses
Apparent starch digestibility at the terminal ileum and in the colon was calculated using ratios of the
TiO2 concentration in the feed and digesta according to the following calculation:
Apparent starch digestibility = 1-[(ID x AF) / (IF x AD),
where ID is the concentration of marker (TiO2) in the diet, AF is the starch concentration in the digesta,
IF is the marker concentration (TiO2) in the digesta, and AD is the starch concentration in the diet. All
digestibilities are presented as percentages.
26
Empty body weight (EBW) was determined as the liveweight of the pig minus the contents (digesta)
contained in the stomach, small intestine, caecum, colon and bladder (if applicable). The percentage of
the pig that was carcass was calculated as:
[(Liveweight of pig minus weight of gastrointestinal tract)/liveweight] x 100.
Statistical analyses were performed using Statview for Macintosh (version 5.0; SAS Institute Inc.,
Cary, NC, USA). Replicate was included as an independent variable for all dependent variables
analysed, but was removed whenever found to be non-significant (P>0.05). Following removal of this
term, one-way ANOVA determined significant differences between treatment groups, and the mean
values were compared using Fisher’s-protected least significant difference test. Statistical significance
was accepted at P<0.05. The unit of replication was the individual pig for all measurements, except in
the case of dry matter intake where the pen was used as the experimental unit.
6.4
Results
6.4.1 Chemical characteristics of rice
Amylose:amylopectin ratios for rice types AM, DOON and WAXY were 0.22, 0.31 and 0.03,
respectively. The low ratio for WAXY indicates that this rice type is almost exclusively amylopectin,
with AM also having a higher level of amylopectin than DOON. The FDS content, expressed as a
percentage of total starch, mirrors the amylose content of the rice types in both raw and cooked forms.
Gelatinisation temperature was highest in DOON (75.8 °C) and lowest in WAXY (69.6 °C). The level
of RS varied from 0.6 g kg-1 in Amaroo to 1.4 g kg-1 in Doongara (Table 6.3).
Table 6.3 Selected chemical characteristics of the three rice types used.
Characteristic
Total starch, g kg-1
DMA
Raw
Cooked
Resistant starch, g kg-1
DM
FDSB, % total starch
Raw
Cooked
Amylose content, %
Gelatinisation
temperature, °C
Medium-grain
(Amaroo; AM)
Type of rice
Long-grain (Doongara;
DOON)
Waxy (WAXY)
878
913
0.60
860
903
1.42
883
914
0.75
23.2
86.7
18.2
69.8
18.3
84.0
23.8
75.8
26.5
90.0
6.1
69.6
A
DM: dry matter.
FDS: fast digestible starch.
B
27
6.4.2 Apparent digestibility of starch and viscosity
Apparent digestibility of starch measured at the terminal ileum was lowest in pigs fed DOON and diet
WBL and highest in pigs fed AM and WAXY (P=0.004). Starch had virtually been fully digested in
the distal region of the colon but was still about 2% digestibility units lower in pigs fed diet WBL
compared to pigs fed any of the rice types (P<0.001). Residual starch content reflected apparent starch
digestibility, although pigs fed diet WBL had between 6 and 12 times the level of starch in their distal
colons compared to pigs fed the three rice-based diets (P<0.001) (Figure 6.1). Digesta viscosity was
highest in pigs fed diet WBL in both the ileum (P<0.001) and the caecum (P=0.027). The specific
activity of ATP-citrate lyase varied between 37 μmol/mg protein for pigs fed diet WBL to 148
μmol/mg protein for pigs fed WAXY. Pigs fed WAXY had activities higher than pigs fed AM and
WBL (P=0.005) but were similar (P>0.05) to pigs fed DOON. Pigs fed AM had similar ATP-citrate
lyase activities to pigs fed DOON and WBL (P>0.05) (Table 6.4).
Starch content
(g/100 g digesta)
12
10
8
Ileum
6
4
2
Colon
0
Calrose
Doongara
Double
Elephant
Commercial
Diet Type
Figure 6.1 The starch content in the terminal ileum (end of small intestine) and colon (large intestine)
of pigs fed different rice types (medium-grain - AM; Long-grain – DOON; WAXY – Double
Elephant) and WBL for 14 days after weaning.
6.4.3 Performance data
There were no statistically significant differences in the liveweight of pigs or rate of daily gain
between diets after weaning, although there was a trend (P=0.085) for pigs in the first week eating rice
types AM and WAXY to grow faster than pigs eating DOON and WBL. There was a significant
replicate effect on daily gain, with pigs in replicate 1 performing better (P=0.032) than pigs in
replicates 2 and 3 (204 g/day versus 139 and 180 g/day, respectively). Similarly, replicate was
significant (P<0.001) for the liveweight of pigs after 14 days, with pigs in replicate 1 weighing more
(10.4 kg) than pigs in replicate 3 (9.4 kg), with pigs in replicate 2 being lightest (7.5 kg). These
weights reflected differences in starting weight (7.6, 6.9 and 5.5 kg for replicates 1, 3 and 2,
respectively). The EBW of pigs was greatest (94.6-95.4%) in pigs fed the three rice-based diets and
lowest (90.1%; P<0.001) for pigs fed diet WBL (Table 6.5).
28
Table 6.4 Apparent digestibility of starch, residual starch content, viscosity and the ATP-citrate lyase
activity of the digesta in pigs fed different diets after weaning.
DietA
Item
Starch
digestibility, %
Ileum
Distal colon
Residual starch,
mg/g DMC
Ileum
Distal colon
Viscosity, mPas
Ileum
Caecum
ATP-citrate lyase,
μmol/mg protein
AM
DOON
WAXY
WBL
SEDB
P=
96.2 a
99.8 a
88.6 b
99.8 a
99.1 a
99.9 a
88.5 b
97.6 b
5.78
0.55
0.004
<0.001
44 a
6a
110 b
7a
20 a
3a
98 b
36 b
26.4
7.9
<0.001
<0.001
2.9 a
2.8 a
79 ac
2.9 a
2.9 a
105 ab
3.8 a
2.6 a
148 b
8.2 b
4.7 b
37 c
1.77
1.19
24.5
<0.001
0.027
0.005
A
Refer to the text for details of diets.
SED: standard error of difference between treatment means.
C
DM: dry matter.
abc
Means in the same row lacking a common superscript are significantly different.
B
Table 6.5 The performance of pigs fed different diets after weaning.
DietA
Item
Body weight, kg
Weaning
Day 7
Day 14
EBWC at
slaughter, kg
Pig weight
expressed as % of
EBWC
Average daily gain,
g
Day 1-7
Day 8-14
Day 1-14
Dry matter
disappearance,
g/pen (n=3)
Day 1-7
Day 8-14
Day 1-14
A
AM
DOON
WAXY
WBL
SEDB
P=
6.8
7.6
9.4
8.9
6.6
7.0
8.6
8.2
6.7
7.6
9.5
9.1
6.7
7.3
9.2
8.4
0.75
0.79
1.24
1.12
0.979
0.785
0.621
0.465
95.4 a
94.6 a
95.3 a
90.1 b
1.08
<0.001
117
253
185
54
228
141
123
270
196
84
280
182
49.8
90.0
57.2
0.085
0.770
0.865
977
1812
1395
709
1608
1159
979
1816
1397
481
1347
913
291.1
440.7
358.3
0.434
0.770
0.621
Refer to the text for details of diets.
SED: standard error of difference between treatment means.
C
EBW: empty body weight; calculated as bodyweight of pig minus weight of gastrointestinal tract contents.
abc
Means in the same row lacking a common superscript are significantly different.
B
29
6.4.4 PWD and faecal shedding of E. coli
Only sporadic diarrhoea was observed in this particular study, with only a few pigs developing serious
PWD. There were no significant differences between diets on days 1, 3, and 7 regarding the faecal E.
coli score (P>0.05), although on days 8/9, pigs fed diets AM, WAXY and WBL shed less haemolytic
E. coli in the faeces than pigs fed diet DOON (P=0.011). The consistency of faeces varied
considerably in pigs fed different diets after weaning, which prevented statistical differences between
diets. Nevertheless, it was evident that pigs fed WBL had more moist faeces on days 7 and 8/9 after
weaning than pigs fed any of the rice-based diets (Table 6.6).
Table 6.6 The haemolytic E. coli score in faeces and the faecal consistency in pigs assessed at various
time points after weaning.
DietA
Item
E. coli score in
faecesC
Day 1
Day 3
Day 7
Day 8/9
Faecal
consistency, %D
Day 3
Day 7
Day 8/9
A
AM
DOON
WAXY
WBL
SEDB
P=
0.58
0.42
0.50
0.33 a
0.25
0.50
1.67
1.75 b
0.45
0.27
2.00
0.91 ab
0.33
0.25
0.50 a
0.121
0.107
0.204
0.169
0.767
0.309
0.069
0.011
42
25
33
38
36
44
29
30
31
33
39
50
6.0
4.6
5.3
> 0.05
> 0.05
> 0.05
Refer to the text for details of diets.
SED: standard error of difference between treatment means.
C
Faecal score: agar plates were scored from 0-5 acording to the number of streaked sections that had viable
growth of haemolytic E. coli, where 0 = no growth, 1 = E. coli in first section, and so on (5 = heaviest growth of
haemolytic E. coli).
D
Faecal consistency: expressed as % cumulative score per day of pigs having more liquid faeces; higher values
are associated with more liquid faeces.
abc
Means in the same row lacking a common superscript are significantly different.
B
6.4.5 Fermentation characteristics
The acidity of digesta in the ileum (P=0.055), and the caecum, proximal colon and distal colon (all
P<0.001), was highest for pigs fed diet WBL and not statistically significant between any of the three
rice diets. Faecal pH was similar (P>0.05) in pigs fed all diets. The total VFA concentration was
similar for pigs fed all diets in the ileum and distal colon. In the caecum, pigs fed the three rice diets
had VFA concentrations ranging from 140-175 mM, but only pigs fed AM and WAXY had less VFA
than pigs fed diet WBL (196 mM; P=0.026). In the proximal colon, pigs fed diet WBL had a
significantly higher concentration of VFA than pigs fed the three rice-based diets (P<0.001). The
concentration of acetate was similar (P>0.05) at all sites of the gastrointestinal tract in the four diets,
but the concentration of propionate was highest in the caecum of pigs fed diets DOON and WBL
(P=0.044) and in the proximal colon of pigs only fed WBL (76 mM; P=0.010). Pigs fed WBL had a 23-fold higher concentration of butyrate at all gastrointestinal sites than pigs fed the three rice-based
diets (0.017>P<0.001). Conversely, pigs fed any of the three rice-based diets consistently had higher
concentrations (0.030>P<0.001) of isovalerate and isobutyrate in their digesta than pigs fed WBL
(Table 6.7).
30
6.4.6 Organ weights
The percentage of pig that was carcass was 4-6 points higher in pigs fed the three rice-based diets than
in pigs fed WBL (P<0.001). This difference reflected lower weights of the stomach (P=0.002), caecum
(P=0.024) and colon (P=0.032) commensurate with significantly less digesta (0.025>P<0.001) being
present in these organs. There was also significantly less digesta found in the small intestine (P=0.010)
of pigs fed the rice diets compared to pigs fed diet WBL. When expressed on a relative basis (%
EBW), pigs fed AM had a significantly lighter caecum and colon than pigs fed either DOON or WBL,
whereas the relative weights of the caecum and colon of pigs fed WAXY was comparable to those in
pigs fed DOON. The relative weight of the colon was heaviest (1.63%; P<0.001) in pigs fed WBL.
Pigs fed diet WBL had the heaviest pancreas (P=0.002; Table 6.8).
6.4.7 Prediction of residual starch content using fast digestible starch (FDS)
The data in Figure 6.2 show that more starch remained at the end of the small intestine (ileum) in the
digesta of pigs fed DOON and diet WBL compared to pigs fed AM and WAXY. These data concur
with the apparent starch digestibility figures expressed in Table 6.4. In the colon, where significant
microbial fermentation of starch can occur, the amount of starch remaining in pigs fed all rice types
was not different, but remained elevated in pigs fed WBL. The digestibility of starch in the small
intestine could be predicted from the ‘fast digestible starch’ in vitro test (Figure 6.2).
31
Table 6.7 Fermentation characteristics of digesta in pigs fed different diets after weaning.
DietA
Item
pH
Ileum
Caecum
Proximal
colon
Distal colon
Faeces
Total VFAC,
mM
Ileum
Caecum
Proximal
colon
Distal colon
Acetate, mM
Ileum
Caecum
Proximal
colon
Distal colon
Propionate, mM
Ileum
Caecum
Proximal
colon
Distal colon
Butyrate, mM
Ileum
Caecum
Proximal
colon
Distal colon
Isobutyrate,
mM
Caecum
Proximal
colon
Distal colon
Isovalerate, mM
Caecum
Proximal
colon
Distal colon
A
AM
DOON
WAXY
WBL
SEDB
P=
6.7 a
6.3 a
6.5 a
6.7 a
6.1 a
6.4 a
6.7 a
6.3 a
6.5 a
6.2 b
5.4 b
5.6 b
0.36
0.30
0.30
0.055
<0.001
<0.001
6.8 a
6.9
6.7 a
7.0
6.9 a
7.0
6.3 b
6.9
0.30
0.29
0.006
0.730
19
140 a
121 a
27
175 ab
137 a
25
145 a
136 a
30
196 b
213 b
10.9
34.2
39.4
0.372
0.026
<0.001
103
110
96
150
35.7
0.071
12
75
61
16
79
61
16
69
62
10
58
64
7.8
13.3
15.3
0.480
0.061
0.984
42
43
34
52
12.4
0.126
0
42 a
37 a
2
64 bc
47 a
0.4
48 ab
46 a
2
75 c
76 b
2.88
20.8
20.5
0.582
0.044
0.010
29
34
30
49
15.9
0.142
7a
16 a
16 a
7a
22 a
19 a
8a
17 a
18 a
18 b
52 b
57 b
6.4
10.3
12.1
0.017
<0.001
<0.001
16 a
17 a
17 a
37 b
10.9
0.008
1a
1a
1a
1a
2a
2a
0.1 b
0.1 b
1.2
1.04
0.014
0.008
5a
4a
5a
0.9 b
2.04
0.010
3a
4 ab
3a
3a
4a
5b
0.6 b
1c
1.28
1.2
<0.001
<0.001
7a
6a
7a
3b
2.4
0.030
Refer to the text for details of diets.
SED: standard error of difference between treatment means.
C
VFA: volatile fatty acids (expressed as mM, or mmol/kg wet digesta).
abc
Means in the same row lacking a common superscript are significantly different.
B
32
Table 6.8 Weight of the carcass at euthanasia, organ weight (empty, and as a % of empty bodyweight)
and the weight of organ contents in pigs fed different diets after weaning.
DietA
Item
Carcass, %C
Empty organ
weight, g
Stomach
Small intestine
Caecum
Colon
Pancreas, g
% EBWD
Stomach
Small intestine
Caecum
Colon
Contents, g
Stomach
Small intestine
Caecum
Colon
A
AM
88.0 a
DOON
86.8 a
WAXY
87.7 a
WBL
82.5 b
SEDB
1.41
P=
<0.001
71 a
487
22 a
104 a
17 a
64 a
460
24 a
108 a
17 a
71 a
499
23 a
108 a
16 a
92 b
490
28 b
137 b
24 b
12.2
69.3
3.8
21.0
3.0
0.002
0.779
0.024
0.032
0.002
0.78 a
5.44
0.24 a
1.15 a
0.80a
5.68
0.30 bc
1.35 b
0.78 a
5.57
0.26 ab
1.20 ab
1.10 b
5.90
0.34 c
1.63 c
0.096
0.559
0.044
0.177
< 0.001
0.542
0.003
<0.001
216 a
122 a
26 a
83 a
189 a
134 a
40 ab
96 a
205 a
127 a
41 b
82 a
341 b
218 b
77 c
217 b
91.7
53.7
14.3
27.9
0.025
0.010
<0.001
<0.001
Refer to the text for details of diets.
SED: standard error of difference between treatment means.
C
Carcass, %: Calculated as [(Liveweight of pig minus weight of gastrointestinal tract)/liveweight] x 100.
D
EBW: empty body weight (calculated as liveweight of pig minus total weight of gastrointestinal tract contents.
abc
Means in the same row lacking a common superscript are significantly different.
B
33
Starch content
g/100 g digesta
10
Doongara
7.5
Ileum
5
Colon
2.5
Doongara
Calrose
Elephant
0
74
76
78
80
82
84
Fast digestible
starch %
Figure 6.2 The relationship between fast digestible starch content (%) and the amount of starch
remaining in the terminal ileum and colon of pigs killed 14 d after weaning.
6.5
Discussion
These data clearly demonstrate that there are differences between different types of cooked rice in the
digestibility of starch. The major findings of this study were that (i) pigs fed Amaroo (AM) and
Double Elephant (WAXY, ie, ≈ 97% amylopectin) had a higher apparent starch digestibility at the end
of the small intestine (ileum) and performed best (growth rate) in the study; (ii) pigs fed Doongara
(DOON) and the commercial wheat-and barley-based diet (WBL) had inferior starch digestion in the
small intestine, presumably because of the higher amylose content in DOON and the anti-nutritional
effects of NSP in the wheat and barley in diet WBL; (iii) pigs fed all rice types ate more energy and
were more efficient at converting energy from the feed into carcass tissue than pigs fed the
commercial diet, indicating a strong, positive effect for feeding a cooked white-rice-based diet after
weaning; and (iv) the faecal score of E. coli (incidence of diarrhoea) was reduced in pigs fed AM and
the commercial diet WBL, but was higher in pigs fed DOON and WAXY. Collectively, these data
suggest that piglet diets in Australia should preferably be based on the use of medium-grain rather
than long-grain rice, although a mixture of the two cannot be precluded from possible use. In addition,
increased digestion of starch in the medium- (AM) and waxy-rice (WAXY) types suggests that any
diets based on these rices will cause better performance than those currently used.
Few reports have investigated the effect of cooked white rice fed to piglets on apparent starch
digestion. Hopwood et al. (2004) reported an apparent starch digestibility of 96% and 100%,
respectively, in the ileum and faeces of piglets killed 10 days post-weaning after being fed a diet based
on cooked white medium-grain rice and animal protein sources. Incorporation of increasing amounts
of pearl barley at the expense of cooked white rice by Hopwood et al. (2004) significantly reduced
starch digestibility in the ileum, but faecal digestibility of starch was unaffected (P=0.096) by the
34
incorporation of pearl barley. Feeding pigs diet WBL in the current study significantly depressed
small intestinal starch digestion to approximately 88% but only to the level of pigs fed the diet using
DOON (Table 6.4). Presumably the higher amylose content of DOON, which is regarded as being less
digestible by pancreatic amylase in the small intestine (Black, 2001), accounted for the lower
disappearance of starch than in pigs fed AM and WAXY, which have more amylopectin. The resultant
excess starch present within the lumen of pigs fed diets DOON and WBL would pass directly into the
caecum and colon and potentially cause considerable microbial fermentation, resulting in a lowering
of pH and higher VFA production, and organ hypertrophy, although this was only evident in pigs fed
diet WBL (Tables 6.7 and 6.8). The greater NSP content of diet WBL together with greater viscosity
of the digesta and the undigested starch undoubtedly caused the significantly higher level of VFA
production, greater gut weights and lower carcass percentage compared to pigs fed all three rice-based
diets, which concurs with previous work (eg, McDonald et al., 1999, 2001; Pluske et al., 2003).
The lack of any differences in growth performance between diets, except for a trend (P=0.085; Table
6.5) for improved daily gain in pigs fed diets AM and WAXY in the first seven days of the study,
suggests that cooked rice-based diets can be used successfully to maintain weight gain in the
immediate post-weaning period. Piglets generally suffer a period of sub-optimal growth caused by low
feed intake in the period after weaning (Pluske et al., 1997), and feeding diets with AM and WAXY
assisted in alleviating the period of temporary anorexia that generally accompanies weaning under
commercial conditions. The compensation seen in pigs fed diet WBL and the failure of pigs eating
diets AM and WAXY to continue their weight gain advantage could be due to a lower energy value
ascribed to rice during feed formulation, which would reduce growth rate. The final study in this
project (Chapter 10) showed a DE value of ≈ 15.3 MJ/kg, whereas the DE value used to calculate the
DE content of these particular diets was ≈ 14.6 MJ/kg (Dr B. Mullan, personal communication). The
lower DE value ascribed to rice limited energy supply to the pig and restricted growth to below
genetic potential. Commercial use of the determined DE value of rice described in Chapter 10 will
improve the accuracy of diet formulation and provide feed manufacturers with more confidence to use
rice in piglet diets to attain maximum growth. The in vitro test developed herein using fast digestible
starch (FDS) as a predictor of starch digestion at the ileum (Figure 6.2) offers promise as a screening
tool also to the feed manufacturing industry.
Nevertheless, pigs consuming all three rice-based diets ate more dry matter (albeit not significantly)
and converted absorbed nutrients more efficiently to body gain (ie, in the carcass) than pigs fed diet
WBL, as evidenced by the higher percentage of carcass weight. This reflects, in part, lower visceral
weights recorded for pigs eating rice (Table 6.8). These data agree with previously published work
from this University (eg, McDonald et al., 1999, 2001; Pluske et al., 2003). These data are further
supported, again in part, by the measured ATP-citrate lyase activities (Table 6.4) that show a lower
level in adipose tissue in pigs fed the commercial WBL diet. This most probably indicates a
slower/reduced incorporation of the absorbed glucose into adipose tissue that, in turn, mirrors the
reduced digestion of starch seen in the small intestine. The higher ATP-citrate lyase activity for
DOON, which had a similar ileal starch digestibility value to pigs fed diet WBL, might be explained
by the higher level of dry matter intake observed in these pigs, and hence an overall greater absorption
of glucose.
Only sporadic instances of spontaneous diarrhoea were observed in this study, and in general the
health of all pigs remained high. It is difficult to draw conclusions from a study such as this where a
natural infection is relied upon to examine dietary effects on PWD, and hence the results obtained are
ambiguous compared to a study where pigs are experimentally infected. However, pigs fed all three
rice-based diets, particularly diets DOON and WAXY, shed haemolytic E. coli in their faeces, and in
general at higher levels than pigs fed diet WBL. For example, on day 8/9 of the study, pigs fed DOON
and WAXY had E. coli scores of 1.75 and 0.91 respectively compared to 0.33 and 0.50 for pigs fed
AM and WBL, respectively. These data compare favourably to those presented by Hopwood et al.
(2004), who also reported no differences in faecal E. coli swab score in experimentally-infected pigs
fed cooked white rice or pearl barely; however, these authors used an infection model that causes a
greater infection pressure in the gastrointestinal tract, and subsequently detected a significant
35
amelioration of haemolytic E. coli in the small intestine in pigs fed cooked white rice. It was not
possible to do this in the current study because pigs were not experimentally infected. However, the
consistency of faeces was firmer (lower value) in pigs fed rice than in pigs fed WBL, albeit nonstatistically (Table 6.6).
The production of iso (branched chain) VFA and other nitrogenous compounds such as NH3 and
biogenic amines have been implicated in the aetiology of PWD (eg, Bolduan et al., 1988; Aumaitre et
al., 1995; Awati, unpublished PhD Thesis, Wageningen University, The Netherlands). Bolduan et al.
(1988) hypothesised that DF added to piglet diets, particularly insoluble DF, was beneficial in
reducing PWD because the production of such nitrogenous compounds was reduced. There is some
recent support for this proposition, and it was noticeable in the current study that pigs fed all three
rice-based diets produced significantly greater quantities of isobutyrate and isovalerate than pigs fed
diet WBL (Table 6.7). It is difficult to implicate cause and effect with respect to the greater shedding
of haemolytic E. coli observed, however the lack of a slowly/moderately fermentable source of DF in
the rice-based diets could have shifted the balance of the bacteria to proteolytic rather than
saccharolytic and predisposed the pigs to PWD.
In conclusion, data in this experiment showed that pigs fed either the medium-grain rice (AM) or the
waxy rice (WAXY) had a superior starch digestibility in the small intestine than pigs fed the longgrain rice (DOON) or the commercial WBL diet. Pigs fed rice consumed more dry matter and grew
favourably relative to pigs fed diet WBL in the 14 days after weaning, and diverted more absorbed
nutrients to carcass gain than to growth of the visceral organs than pigs fed diet WBL. Relatively low
levels of infection with haemolytic E. coli were observed in this experiment and hence dietary effects
on PWD were difficult to interpret, however pigs fed diets AM and WBL appeared to shed less
haemolytic E. coli than pigs fed diets DOON and WAXY. It is postulated that the lack of a a
slowly/moderately fermentable source of DF in the rice-based diets could have shifted the balance of
the bacteria to proteolytic rather than saccharolytic and predisposed the pigs to PWD.
36
7. Interactive effects of cooked white rice
with vegetable and animal protein
sources on digesta and fermentation
characteristics and the faecal shedding
of haemolytic E. coli
7.1
Summary
Sixty-four piglets weaned at approximately 21 days of age and fed different diets for a period of 10
days after weaning. Diets were based on either cooked white rice or wheat as the two cereal types, and
these were supplemented with protein derived from either animal sources or plant sources. Pigs were
inoculated with an enterotoxigenic serotype of E. coli (O149;K91;K88) at 48, 72, 96 and 120 h after
weaning. At 10 days after weaning, all pigs were euthanased, and samples were taken for
bacteriological assessment and measures of fermentation and digestive physiology. Pigs fed cooked
white rice consumed more feed than pigs fed wheat (P<0.001). At euthanasia, pigs fed wheat were
found to have a higher viscosity in their ileal digesta than pigs fed rice (P<0.001). A higher viscosity
did not correlate to a greater colonisation by E. coli of the wheat-fed pigs and, overall, levels of
colonisation in pigs fed all diets were low. A number of possible reasons for the results obtained are
discussed.
7.2
Introduction
Different diets appear to influence the colonisation of enterotoxigenic E. coli in the gastrointestinal
tract of weaner pigs (Pluske et al., 2002). Colonisation and proliferation of, in particular haemolytic
strains, of enterotoxigenic E. coli in the small intestine characterises PWD, which generally results in
diarrhoea, dehydration, weight loss, and sometimes death in the first two weeks following weaning.
Some authors have viewed diarrhoea, an excretion of cellular solutes, as a protective defence
mechanism in response to this infective agent in the gut (Stewart et al., 2001). Regardless, PWC/PWD
is a significant disease causing major economical loss to the pig industry.
Diet influences pH, nutrient composition, and flow of digesta, which in turn influences distribution,
composition and metabolic activity of gut flora (Stewart et al., 2001; Pluske et al., 2002). It is
important to find effective methods of control of specific enteric bacterial pathogens that are cost
effective and plausible, and diet provides an excellent medium. This is especially relevant given the
ban placed on a number of antibiotics in Europe recently, and reported cases in Australia of antibiotic
resistance to strains of E. coli causing PWC (Barton, 1999).
One of the components of dry matter digested mainly in the large intestine is known as dietary fibre
(DF). Key components of DF are soluble NSP that are found in plant cell walls, and are broken down
into metabolites, gases, and microbial biomass in the large intestine and terminal ileum (Pluske et al.,
1999). Pigs are unable to release the energy portion of the complex carbohydrate, as it is resistant to
the pigs’ own endogenous enzymes. The volatile fatty acids (VFA) that are produced from the
breakdown of NSP, mainly acetate, propionate and butyrate (Mosenthin et al., 2001), decrease the pH
of digesta. In addition, a diet that has high levels of soluble NSP takes up more water in the intestine,
making it more viscous. It is likely that diets containing plant protein are more viscous than those with
animal protein sources, due to the soluble NSP in plant proteins.
Previous studies conducted at Murdoch University (eg, McDonald et al., 1999) have used cooked
white rice in diets for young pigs with other dietary components that are low in NSP and (or) resistant
37
starch (RS). These supplements are predominantly animal-based, for example fishmeal, meatmeal,
bloodmeal, bonemeal and skimmed milk powder. Vegetable protein sources had deliberately been left
out as these contain soluble NSP, and the hypothesis tested was that diets low in soluble NSP prevent
PWC. However, because of their availability and cost effectiveness, vegetable proteins, derived from
lupins and peas for example, are often used in diets. Also, the continued use of animal proteins
worldwide in diets is under recurrent scrutiny and their use is actually banned in the European Union.
There is likely to be a greater reliance on vegetable proteins in young pig diets in the future, so
potential benefits of cooked rice combined with this needs to be investigated.
Wheat contains about 10 g kg-1 viscous soluble NSP (Kim et al., 2003). In comparison, rice contains
very little soluble NSP (Marsono and Topping, 1993). Increased intestinal viscosity is believed to be
associated with increased microbial fermentation and microbial numbers, and greater microbial
numbers may cause more microbial fermentation (McDonald et al., 1999). In addition, increased
viscosity slows transit time of digesta through the upper small intestine, which allows more time for
microbial pathogens to proliferate. McDonald et al. (2001) have demonstrated increased numbers of
enterotoxigenic E. coli in the small intestine by using carboxymethylcellulose to increase viscosity in
diets. Increasing intestinal viscosity also decreases absorption of glucose (Rainbird et al., 1984) and
other nutrients (Ehrlein and Stockmann, 1998). In other studies performed by Pluske et al. (1996), it
was found that a diet higher in NSP content (based on wheat) caused swine dysentery in 75% of pigs
after infecting with S. hyodysenteriae, whereas there were no cases of swine dysentery in pigs fed
cooked white rice. Collectively, these data suggests that diets based on wheat might predispose pigs to
a number of enteric bacterial diseases, with viscosity being a causative or associative factor in the
aetiology of the diseases.
In regard to PWC, it is possible that newly-weaned pigs fed a wheat diet will have a higher
colonisation of E. coli in the small intestine than pigs fed a rice based diet, as a result of greater
viscosity in the small intestine. In addition, weaner pigs fed a diet supplemented by plant protein could
have a higher number of E. coli colonised in the small intestine than a diet supplemented with animal
protein, due to higher levels of soluble NSP. Therefore, the hypotheses tested in this experiment were
as follows: a) diets based on wheat, irrespective of the mixture of supplementary protein sources, will
cause a higher colonisation of E. coli in the small intestine than pigs fed rice-based diets, and b) a rice
diet supplemented with a plant protein mixture will cause a higher colonisation of E. coli in the small
intestine, when compared to a rice-animal protein diet.
7.3
Materials and Methods
7.3.1 Animals and procedures
A total of 64 mixed-sex pigs (Large White x Landrace) were used in the trial. The trial was run in two
replicates of 32 pigs. Pigs were weaned at approximately 21 days of age and transported to Murdoch
University from Wandalup Farms, Western Australia. Pigs were then randomly divided into four
groups on the basis of gender and liveweight, so that average weights in each pen were similar. Each
of these groups (n=4 per treatment) was fed a different diet. Daily records were kept of voluntary food
intake in each pen. Pigs were housed in wire-mesh pens with slatted raised pens, four per pen, in an
isolation animal house at Murdoch University. Heating was provided by the use of electric heaters.
The Animal Ethics Committee of Murdoch University approved this experiment.
On arrival at Murdoch University, pigs were weighed and faecal swabs were taken to record initial E.
coli presence. Faecal swabs were cultured for the presence of haemolytic E. coli on day 2 of the
experiment, which was the first day of infection, and then on days 6 to 9 after weaning. Pigs were
infected orally with 107 CFU/mL of E. coli serotype O149;K91;K88 at 48, 72, 96 and 120 hours after
arrival.
38
7.3.2 Experimental design, diets and feeding
The experiment was arranged as a 2 x 2 factorial arrangement of treatments with the respective factors
tested being cereal type (cooked white rice vs. wheat) and type of protein source (plant, or vegetable,
protein sources vs. animal protein sources). The wheat-plant protein (WPP) diet consisted of wheat
and was made up to a complete diet only with plant protein sources. The wheat-animal protein (WAP)
diet contained wheat mixed with animal protein sources. The rice-based diets used cooked, mediumgrain, white rice (Sunwhite Calrose®; Australian Rice Growers Co-operative, Australia; variety
Amaroo), mixed with a plant protein RPP) or animal protein (RAP) supplement (Table 7.1). The rice
was cooked with water (1:2 rice:water ratio) in an autoclave for 20 minutes at 121 ºC, and was then
allowed to cool overnight at 4 ºC before incorporation into the diet with either the plant or animal
protein supplement. A small amount of warm water was added to facilitate mixing the rice and either
the plant or animal protein mixture.
Pigs were fed one of the four diets (Table 7.1) on an ad libitum basis for 10 days after weaning, by
placing the mixed feed in open troughs once daily. Water was available at all times through a nipple
drinker located in each pen.
Table 7.1 Composition of the experimental diets (g kg-1 on as-fed basis).
Ingredient
Wheat
Rice
Canola meal
Lupins
Full fat soybean
Meatmeal (50% CP)
Whey powder
Blood meal (85% CP)
Fishmeal (65% CP)
Canola oil
Salt
Vitamin/mineral premixA
L-Lysine
DL-Methionine
L-Threonine
Tryptosine
Dicalcium phosphate
Choline chloride
Calculated analysis:
DE (MJ/kg)
Available Lysine (g/MJ)
Crude protein (%)
Wheat-based diets
Plant Protein
Animal
Protein
750
780
50
50
74.9
50
50
25
50
30
28.3
1
1
1.5
1.5
5.2
2.9
2.2
1.2
4.6
2.7
6.6
4.2
18.5
0.7
0.4
0.4
15.00
0.85
16.7
15.00
0.85
19.7
A
Rice-based diets
Plant Protein
Animal
Protein
660
750
100
100
87.2
50
95.4
26.4
50
5
5
1
1
1.5
1.5
3.3
1.1
2
1.5
4.4
2.9
7.5
6.3
20.3
8.4
0.4
0.4
15.15
0.85
16.0
15.20
0.85
17.1
Premix provided (mg/kg air-dry diet): retinyl acetate 3.44, cholecalciferol 0.065, α-tocopheryl acetate 20,
menadione 4.4, riboflavin 4, pyridoxine 1.6, cyanocobalamin 0.02, pantothenic acid 14, nicotinic acid 20, Co
0.2, I 0.6, Fe 120, Mn 60, Zn 100, Cu 10, Se 0.13.
39
7.3.3 Post-mortem procedures
Refer to the previous experiment (Chapter 6.3.4) for details of procedures used. In addition, and after
the small intestine was stripped of its mesentery, it was laid out in four sections and keyhole incisions
were made. Individual sterile swabs were wiped along the mucosal surface at distances approximately
25% and 75% along the small intestine. Swabs were also applied to the digesta in the caecum,
proximal colon and the faeces. Swabs were then rolled onto Sheep Blood Agar (SBA) plates. Plates
remained at room temperature until they were streaked using standard procedures, after which they
were incubated overnight at 37 °C and scored the following day for the presence of haemolytic E. coli
colonies (see 7.3.4 below).
Furthermore, a 10-cm section was cut out halfway along the small intestine (mid jejunum), and 1g of
epithelium were stripped from its mucosal surface. This was added to 9mL of sterile PBS. Further 1/10
serial dilutions were performed, and 100 µL was dropped onto SBA plates. These were also incubated
overnight in air at 37 °C before being counted for haemolytic E. coli colonies the following day (see
7.3.4 below).
Samples of digesta were taken from the stomach, ileum, caecum, proximal colon and faeces, and pH
was measured immediately (Boy-2 pH meter). The viscosity of the digesta was determined according
to methods described in Chapter 6.3.5.
7.3.4 Microbial assessment
Plates were assessed for β-haemolytic colonies displaying morphology characteristic of E. coli, after
overnight incubation. Some representative colonies of E. coli were selected and streaked onto sterile
nutrient agar slopes to be serotyped by the National E. coli Reference Laboratory at the Department of
Natural Resources and Environment Agriculture, Bendigo, Victoria, Australia, by slide coagglutination.
7.3.5 Statistical analyses
Statistical analyses were calculated using SuperANOVA (version 1.11; Abacus Concepts, 1989-1991)
according to a two-way ANOVA. The unit of replication for all measurements of body weight,
internal measurements, and microbiological data was the individual pig. The unit of replication for
food intake was each pen of four pigs. Statistical significance was accepted at P<0.05. Differences
between treatments for significant main effect means were examined using Fisher’s-protected least
significant difference test.
7.4
Results
7.4.1 Microbial assessments
There was no significant difference between faecal swabs taken from pigs on any day, except day 8
after weaning. It was expected that there would be low counts from faecal swabs for days 1 and 3, as
these were taken before infection of pigs with E. coli. All counts were low, except day 6, which is also
the 4th and final day of infection. Generally, pigs fed rice had a higher colonisation of E. coli than
those fed wheat. Also, the organs of pigs fed rice had higher averages of swab counts than organs of
pigs fed wheat. Most averages between the two different protein types were quite similar.
Significantly more bacteria were found in pigs fed rice than those fed wheat, in the digesta 25% along
the length of the small intestine. There was no significant difference between numbers of bacteria
detected at any of the other sites measured along the digestive tract (Table 7.2).
40
Table 7.2 β–haemolytic E. coli swab score assessed from faecal swabs taken at different times after
weaning, the swab score assessed from different sites along the gastrointestinal tract at euthanasia on
day 10, and the number of colony forming units (CFU) found in the small intestine at euthanasia.
(i) Swab scoreC
Day 1
Day 3
Day 6
Day 7
Day 8
Day 9
A
(ii) Swab score at
day 10 collected
from:
Caecum
Colon
Small intestine 25% along tract
Small intestine 75% along tract
Faeces
(iii) CFU (x 106)
Cereal Source
Rice
Wheat
Protein Source
Animal
Plant
Probability, P=A,B
C
P
C*P
0.31
0.28
2.40
1.90
1.90
0.78
0.30
0.20
1.70
1.60
1.20
0.93
0.45
0.32
2.00
1.80
1.50
1.00
0.16
0.16
2.00
1.80
1.60
0.67
NS
NS
NS
NS
*
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
**
NS
1.00
1.00
0.34
0.60
0.70
0.00
0.81
0.84
0.12
0.82
0.86
0.22
NS
NS
**
NS
NS
NS
NS
NS
NS
0.47
0.20
0.29
0.38
NS
NS
NS
0.69
1.9
0.40
0.16
0.51
1.0
0.57
1.9
NS
NS
NS
NS
NS
NS
C- Cereal; P- Protein; C*P- Cereal by protein interaction.
NS- Not Significant; *- P≤0.05 to P<0.01; **- P≤0.01 to P<0.001; and ***- P≤0.001
C
Method of scoring swabs:
0= no colonies grown on the SBA plate exhibit morphology characteristic of haemolytic E. coli;
1= the first swab of primary inoculum displays colonies that are characteristic of haemolytic E. coli;
2= the second streak with a flamed wire loop, diluting the primary inoculum, displays colonies' characteristic of
haemolytic E. coli;
3= the third streak with a flamed wire loop, diluting the primary inoculum, displays colonies' characteristic of
haemolytic E. coli;
4= the fourth streak with a flamed wire loop, diluting the primary inoculum, displays colonies' characteristic of
haemolytic E. coli;
5= the fifth streak with a flamed wire loop, diluting the primary inoculum, displays colonies' characteristic of
haemolytic E. coli.
B
7.4.2 Performance data
Pigs fed the rice diets ate, on average, more than double the amount of DM in the 10 days after
weaning than their counterparts offered the wheat-based diets (310 vs. 150 g DM per pig per day,
P<0.01). However this increase in DM intake failed to translate into an improvement in liveweight and
average daily liveweight gain after weaning, as there were no significant differences in bodyweight
between experimental treatment diets before and after the experiment. Growth rates were generally
very poor, with newly-weaned pigs only growing between 26 and 44 g per day (Table 7.3).
41
Table 7.3 The performance of pigs fed different diets for the first 10 days after weaning.
Item
DMIC,
kg/pig/day
Bodyweight,
kg
Start
Finish
Daily
liveweight
gain, grams
Cereal Source
Rice
Wheat
0.31
0.15
Protein Source
Animal
Plant
0.24
0.22
Probability, P=A,B
C
P
C*P
***
NS
NS
6.1
6.4
6.3
6.6
6.2
6.6
6.2
6.4
NS
NS
NS
NS
NS
NS
36
34
44
26
NS
NS
NS
A
C- Cereal; P- Protein; C*P- Cereal by protein interaction.
NS- Not Significant; *- P≤0.1 to P<0.05; **- P≤0.05 to P<0.01; ***- P≤0.01 to P<0.001.
B
C
DMI: dry matter intake.
7.4.3 Fermentation characteristics
Pigs fed cooked whole rice had a higher pH than pigs fed wheat-based diets in the ileum, caecum and
proximal colon (P<0.01). A significant difference in the pH of the caecum contents and faeces was
also found in pigs fed different protein diets. Pigs fed animal protein had a higher pH (P<0.05) in the
caecum, but a lower pH in the faeces (P<0.05). A significant interaction occurred for digesta pH in the
faeces, with pigs fed diet RPP having the highest pH and pigs fed diet WAP had the most acidic pH
(P<0.1) (Table 7.4).
Table 7.4 The pH of digesta in the gastrointestinal tract of pigs fed different diets.
Cereal Source
Rice
Wheat
A
pH
Stomach
Ileum
Caecum
Proximal colon
Faeces
2.2
7.1
6.1
6.2
7.1
2.4
6.4
5.8
5.9
6.8
Protein Source
Animal
Plant
2.4
6.7
6.0
6.1
6.7
2.3
6.9
5.8
5.9
7.2
Probability, P=A,B
C
P
C*P
NS
***
**
**
NS
NS
NS
**
NS
**
NS
NS
NS
NS
*
C- Cereal; P- Protein; C*P: Cereal by protein interaction.
NS- Not Significant; *- P≤0.1 to P<0.05; **- P≤0.05 to P<0.01; ***- P≤0.01 to P<0.001.
B
Differences between full and empty organ weights were significant when expressed as a percentage of
(live) bodyweight. Most of the statistical differences in organ weights were between pigs fed cooked
white rice or wheat. The weights of the full stomach, caecum and colon were heavier (P<0.05) in pigs
fed wheat when compared to those that ate rice, however only the empty stomach weight was heavier
in pigs fed wheat (P<0.01). Weights of the full caecum and colon were heavier (P<0.01) in pigs fed
plant protein sources compared to animal protein sources, however on an empty bodyweight basis,
only the colon was heavier in pigs fed plant protein. Pigs fed a wheat-based diet had more than a
twofold higher digesta viscosity in the ileum (4.5 vs. 2.1 cP, P<0.01) than pigs fed rice (Table 7.5).
42
Table 7.5 Organ weights of pigs fed different diets and digesta viscosity in the ileum of pigs fed
different diets.
A
FOW3, g
Stomach
Caecum
Colon
Small
intestine
EBWC, g
Stomach
Caecum
Colon
Small
intestine
Viscosity, cPD
Cereal Source
Rice
Wheat
Protein Source
Animal
Plant
Probability, P=A,B
C
P
C*P
2.1
0.83
2.4
6.0
4.1
1.0
2.9
6.6
3.1
0.73
2.3
6.5
3.1
1.1
3.0
6.1
***
*
**
NS
NS
***
***
NS
NS
NS
NS
NS
0.75
0.31
1.3
5.1
0.87
0.31
1.4
5.2
0.77
0.30
1.2
5.2
0.85
0.32
1.4
5.1
***
NS
NS
NS
**
NS
*
NS
NS
NS
NS
NS
2.1
4.5
3.0
3.6
***
NS
NS
C- Cereal; P- Protein; and C*P- Cereal * Protein
B
NS- Not Significant; *- P≤0.1 to P<0.05; **- P≤0.05 to P<0.01; ***- P≤0.01 to P<0.001.
C
FOW- Full organ weight expressed as a percentage of pig liveweight; EBW: Empty organ weight expressed as
a percentage of liveweight.
D
CP: centipoise (mPas).
7.5
Discussion
The low faecal swab scores and low jejunal counts of haemolytic E. coli contributed to a general lack
of any significant differences between diets in this study, illustrating the low degree of colonisation
that occurred in these pigs despite experimental infection. More bacteria were found in the digesta
25% of the way along the small intestine in pigs fed rice when compared to wheat (Table 7.2), and the
general trend was that pigs fed a rice-based diet had higher counts of haemolytic E. coli than wheatfed pigs. This occurred despite pigs fed wheat having a significantly higher viscosity than pigs fed
cooked rice, and does not support the first hypothesis proposed in this study that increased intestinal
viscosity will increase susceptibility of pigs to E. coli colonisation, and hence a higher incidence of
PWD. In addition, there were no significant differences in colonisation or scores of E. coli found
between pigs fed different protein sources, which does not support the second hypothesis that pigs fed
plant protein sources containing more NSP will also show greater colonisation of the gastrointestinal
tract with haemolytic E. coli.
Data from this study showed clearly that wheat is a more viscous cereal than rice when assessed at the
terminal ileum (2.1 vs. 4.5 cP; Table 7.5). Dietary composition, the time of measurement after feeding
and the extent of hydration of the dietary NSP all influence the viscosity value (McDonald et al.,
2001). However, and based upon previous work done at Murdoch University by McDonald et al.
(2001) and Hopwood et al. (2003), the expectation in this experiment was that weaner pigs fed wheat
would be more susceptible to PWC since their digesta was more viscous. There was little difference
between feacal swabs from day to day, apart from day 8, in the colonisation of the gastrointestinal
tract by the inoculated E. coli, with diet RPP having the highest faecal swab score and diet WAP the
lowest faecal swab score. Nevertheless pigs fed the plant protein sources had numerically higher
numbers of haemolytic E. coli than pigs offered the animal protein sources, but the large variation
between pigs meant that this difference was not significant either.
43
The results of this experiment, therefore, are contrary to a previous study performed by McDonald et
al. (2001), where effects of viscosity were given as a possible reason for a marked increase in the
numbers of colonisation of E. coli and incidence of PWC. A possible reason for this is that these
piglets may have developed some prior immunity to the serotype that the pigs were inoculated with.
Upon entry to Murdoch University from the donor piggery all pigs were swabbed, and it was found
that just over 15% of the weaner piglets were positively identified as having colonies displaying
characteristics of haemolytic E. coli. One of these pigs was positively identified as carrying serotype
0149;K91;K88. Pigs may also be immune through a genetic trait, that is, a lack of enterocyte receptors
for enterotoxigenic E. coli, to match the K88 fimbrial attachment. It is highly unlikely, however, that
the pigs sourced from Wandalup Farms for this study are genetically immune to this particular E. coli
because piglets have successfully been inoculated and succumbed to PWD in previous experiments at
Murdoch University.
Another reason for the poor colonisation in this study might have been low levels of voluntary feed
intake, such that the amount of diet consumed by the pigs may have been insufficient to predispose
pigs to the enterotoxigenic effects of the bacterium. Kelly et al. (1984) noted that pigs eat insufficient
food to provide energy for maintenance shortly after weaning, which may contribute to changes in
intestinal morphology. Hampson (1987), Rantzer et al. (1995) and numerous other workers have
reported previously that pigs eating less feed after weaning were less likely to develop PWC than pigs
that consumed more feed, although Madec et al. (1998) reported the converse in a French study.
Furthermore, diets used in this study were isoenergetic and contained the same lysine:DE ratio,
however they contained less protein (≈ 170 g/kg) than would typically be found in a starter diet in
Australia (210-230 g/kg). There is a growing body of evidence (eg, see review by Pluske et al, 2002)
that pigs fed diets lower in crude protein (170-200 g/kg) shed less enterotoxigenic E. coli and are less
likely to develop PWC than pigs fed commercial levels of protein (> 230 g/kg). Some authors have
associated the production of products from bacterial protein digestion, such as biogenic amines and
phenolics, with a higher incidence of PWC (Bolduan et al., 1988; Gaskins, 2001). A greater CP
content of the diet might cause more of these products to be formed, even at low levels of feed intake.
The low dietary CP levels coupled with low levels of feed consumption seen in this study might have
contributed to the lack of E. coli colonisation observed.
An increase in the DF, or NSP, level of the diet usually increases the amount of VFA produced in the
large intestine. Acetate, propionate and butyrate are the main VFA resulting from the breakdown of
NSP by bacteria (Mosenthin et al., 2001), hence an increase in VFA in the caecum and colon increases
acidity of the gastrointestinal contents (McDonald et al. 2001). It was evident that pigs fed the wheatbased diets had greater levels of microbial fermentation in their caecum and proximal colon, and most
likely higher VFA levels, than rice-fed piglets, which in turn caused lower pH values. The higher
soluble NSP content in wheat would have allowed more substrate for fermentation by the microbes
(Pluske et al., 1999). A lower pH in the ileum may also be due to a change in the relative populations
of microbes, as is noted in chickens fed the viscous compound sodium carboxymethylcellulose (van
der Klis et al., 1993; Smits et al., 1998).
Concomitant with changes in acid production were changes in the weights of organs associated with
feeding different cereal diets. Both full and empty organs from pigs fed wheat diets all weighed equal
to, or more than, organs from rice-fed pigs (see Table 7.5). However, pigs fed the rice diets ate
significantly more DM than those on the wheat diets, which might be attributable to the increased
viscosity of the wheat-based diets and negative feedback effects on transit time through the
gastrointestinal tract. Wheat is the more viscous of the two cereals, and requires more frequent and
forceful peristaltic contractions to move digesta posteriorly (Cherbut et al., 1990). This leads to more
muscle accretion and hence heavier organ weights. The plant (vegetable) proteins used also had high
levels of soluble NSP, however the sources used also contained appreciable levels of oligosaccharides
(eg, verbascose, stachyose and raffinose in lupins) that also contribute to fermentation. Microbial
fermentation was the most likely reason for the increase in empty stomach and colon weights seen.
Excepting the small intestine, organs, both full and empty, from pigs fed plant protein sources were
heavier than those obtained from pigs fed animal protein organs. This is attributable to both the higher
44
viscosity of plant protein, which most likely causes the development of smooth muscle in these
organs, and the greater apparent digestibility of protein from the animal protein sources. A higher
digestibility leaves fewer residues available for the proteolytic bacteria in the distal part of the small
intestine and the large intestine.
45
8. Effect of extrusion of rice and dietary
protein sources on production,
digestibility and faecal shedding of E.
coli
8.1
Summary
A 3 x 2 factorially arranged group of dietary treatments using 84 male weaned piglets aged
approximately 21 days of age and weighing 6.7 ± 0.13 kg (mean ± SEM) was used to investigate the
effects of diet on performance and PWD. The experimental factors were (a) three cereal types, ie, the
medium-grain, lower-amylose rice Amaroo, the long-grain, higher-amylose rice Doongara, and wheat
and (b) two protein sources, namely plant (vegetable) and animal protein types. Diets are subsequently
referred to as: MGAP: medium-grain rice plus animal protein, MGPP: medium-grain rice plus plant
protein, LGAP: long-grain rice plus animal protein, LGPP: long-grain rice plus plant protein, WAP:
wheat plus animal protein, and WPP: wheat plus plant protein. The experiment lasted for 21 days,
during which time performance indices were monitored, piglets were swabbed for the presence or
absence of haemolytic E. coli, piglets were injected with antibiotics if deemed to require treatment by
the stockperson, and the coefficient of total tract apparent digestibility (CTTAD) of selected dietary
components was assessed. Pigs fed extruded medium-grain or long-grain rice performed equally
(P>0.05) to pigs fed wheat as the sole cereal in diets in the 21 days after weaning. The inclusion of
plant (vegetable) protein sources in the diet decreased growth rate (P<0.001) and feed intake
(P=0.007) and increased FCR (P=0.028) in weeks two and three of the trial compared to the use of
animal protein sources, indicating that potential users of extruded rice for specialty piglet diets need to
select dietary protein sources judiciously in order to maximise the efficiency of rice. Significant
interactions occurred for the CTTAD of dietary components when assessed in week three of the study.
In general, pigs fed diets MGAP and LGAP had higher (P<0.001) faecal digestibilities than pigs fed
all other diets, and plant proteins depressed (P<0.001) digestibility compared to animal proteins.
Faecal shedding of haemolytic E. coli was low and similar (P>0.05) across all dietary treatments The
number of antibiotic injections given by the stockperson for PWD was highest for pigs fed animal
protein sources than vegetable protein sources (main effect of protein: P=0.057), although there was a
tendency for an interaction (P=0.069) because pigs fed diet MGPP had a higher swab score than their
counterparts fed MGAP (0.7 vs. 0.5). In summary, pigs tolerated the extruded-rice based diets well
and performed equivalently to pigs fed wheat as the sole cereal.
8.2
Introduction
Results obtained in Chapters 6 and 7 showed that pigs fed a diet after weaning based on cooked white
rice and animal protein generally showed better performance, particularly in the first week after
weaning, but effects on ameliorating the shedding of E. coli and incidence of PWD were equivocal.
Furthermore, the results seen in Chapter 6 showed that pigs fed plant protein sources seemingly
succumbed to more diarrhoea after weaning and grew slower than pigs fed animal protein sources.
Whilst the latter finding is not unexpected, our findings that the cooked rice-based diet did not
ameliorate the incidence of scouring after weaning is in contrast to earlier work reported from this
laboratory (eg, McDonald et al., 1999, 2001). The reason(s) for these ambiguous findings are not
known but warranted further investigation.
Recent work from Spain (eg, Mateos et al., 2002; Solà-Oriol et al., 2004) has demonstrated that rice
processed in a similar way to extrusion outperforms other cereals such as maize (corn), wheat and
sorghum in terms of piglet performance and reduced mortality when growth promoting antibiotics and
zinc oxide are not added to the diet. A recent Chinese study (Li et al., 2004), however, demonstrated
46
that extrusion of Chinese stored brown rice did not influence performance after weaning compared to
non-extruded Chinese stored brown rice, and in fact caused inferior feed conversion efficiency.
Another Chinese study using whole brown rice fed to growing pigs (44 kg) reported improved nutrient
digestibility (Piao et al., 2002). For Australian-grown rice to be used commercially in piglet diets in
Australia, it was imperative that rice types identified in Chapter 4 as potentially being of benefit to
young pigs be processed in such a way to simulate commercial reality. Furthermore, it was important
to assess any possible interactions between extruded rice and different protein sources.
The hypotheses tested in this experiment, therefore, were that (a) diets based on extruded rice will
cause faster growth after weaning and less faecal excretion of haemolytic E. coli than a traditional,
pelleted weaner diet based on wheat, (b) diets based on extruded Amaroo (medium-grain rice) will
cause better performance, irrespective of the protein source, than diets based on Doongara (long-grain)
rice, and (c) rice-based diets using a combination of animal protein sources will cause less excretion of
haemolytic E. coli than diets based on vegetable proteins.
8.3
Materials and Methods
8.3.1 Animals, procedures and housing
A total of 84 entire male piglets (Large White x Landrace) aged approximately 21 days of age and
weighing 6.7 ± 0.13 kg (mean ± SEM) was used in this experiment. The piglets were obtained from
Wandalup Farms, Mandurah, WA. On arrival at the Medina Research Station, the pigs were eartagged, weighed, and stratified into pens of three or four pigs each according to treatment and
liveweight. Pigs were offered their respective diets (see below) in groups of four for the first seven
days after weaning, to accustom them to their new surroundings. For the final two weeks of the study,
pigs were housed individually. Pens were of wire-mesh construction with slatted metal floors, and
measured 1.68 m2 in floor area (0.42 m2 per pig). Each pen was equipped with a nipple water drinker
and a stainless steel feed trough. The ambient temperature was maintained between 26 and 28° C
throughout the study using two reverse-cycle air conditioning units. The room containing the pens was
cleaned daily. The Murdoch University Animal Ethics Committee and the Animal Ethics and
Experimentation Committee of the WA Department of Agriculture approved this experiment. The
experiment was conducted in two replicates.
8.3.2 Experimental design, diets, feeding and sample collection
The experiment was designed as a 3 x 2 factorial arrangement of treatments with the respective factors
being (a) three cereal types, ie, the medium-grain, lower-amylose rice Amaroo, the long-grain, higheramylose rice Doongara, and wheat and (b) two protein sources, namely plant (vegetable) and animal
protein types. Diets are subsequently referred to as: MGAP: medium-grain rice plus animal protein,
MGPP: medium-grain rice plus plant protein, LGAP: long-grain rice plus animal protein, LGPP: longgrain rice plus plant protein, WAP: wheat plus animal protein, and WPP: wheat plus plant protein.
Diets were formulated to contain adequate levels of energy and nutrients for pigs of this genotype and
age. The extruded rice sourced from the University of Adelaide had to be passed through a hammer
mill to reduce particle size before incorporation into the meal-based diets. The diet was offered to
piglets in mash form, and the diet composition is presented in Table 8.1. Titanium dioxide (TiO2) was
added as an inert marker for CTTAD estimation. Faecal samples were collected from the wire-mesh
floor for weaner pigs at 0800, 1000, 1200, 1400, 1600 h on days 18-21 of the experiment. Samples
collected over the three-day period were pooled, kept at –20 ºC and later thawed, mixed, freeze-dried
and ground through a laboratory hammer mill (1 mm screen) prior to chemical analysis.
47
8.3.3 Microbial assessment
Faecal swabs were taken to record initial E. coli presence upon arrival, and then on days 2, 5, 6 and 8
after weaning. Faecal swabs were cultured for the presence of haemolytic E. coli, and plates were
assessed for β-haemolytic colonies displaying morphology characteristic of E. coli, after overnight
incubation. The presence of haemolytic E. Coli was then scored from no growth (0) to heavy
colonisation (5), as has been described previously (Chapter 6.3.3).
Piglets were monitored daily by the stockperson for clinical signs of diarrhoea. Affected pigs (as
assessed by the stockperson, who was unaware of the treatment allocation of pigs) were treated for
diarrhoea by intramuscular injection with Trisoprim-480 [(trimethropin 80 mg/mL, sulfadiazine, 400
mg/mL), 1.5 mL/30 kg body weight; Troy Laboratories, Smithfield, NSW, Australia]; treatment
continued until the diarrhoea ceased. Records were kept of the duration of treatment required for each
treated piglet.
8.3.4 Chemical analyses
The dry matter (DM), nitrogen (N), gross energy (GE), total starch, RS, amylose and amylopectin
contents of extruded rice were determined as described previously (Chapter 4.2.1). Crude protein (CP)
content was calculated as N x 6.25. The DM, GE and TiO2 content of diets and faecal samples were
determined for estimation of apparent digestibilities of GE and the DE content of the experimental
diets. The GE content of the rice, diet, and faecal samples was determined using a Ballistic Bomb
Calorimeter (SANYO Gallenkamp, Loughborough, UK). The TiO2 contents of diet and feacal samples
were determined using the method described by Short et al. (1996).
8.3.5 Statistical analyses
Treatment effects were assessed by two-way ANOVA for a factorial design with the main effects
being cereal (medium-grain rice, long-grain rice and wheat) and protein type (plant and animal).
Average daily gain in the first week after weaning was assessed using the pen as the unit of
replication. Daily gain, voluntary feed intake and FCR in weeks two and three of the study used the
individual pig as the experimental unit, because pigs were housed individually. For the CTTAD of
DM, starch, GE and CP, the individual pig was considered the unit of replication. All effects were
considered as fixed effects in the model. Fisher’s-protected least significant difference test were used
(at 5% significance level) for comparison between mean values of different variables. All statistical
analyses were conducted using the statistical package StatView 5.0 for Windows (AddSoft Pty. Ltd.,
Woodend, Vic., Australia).
48
Table 8.1 Composition of the experimental diets used in the experiment (g kg-1 as-fed basis).
Ingredient
Rice
Wheat
Meat
and
bone meal
Whey
powder
Bloodmeal
(85% CP)
Fishmeal
(65% CP)
Lupins
Canola meal
Full-fat
soybean meal
Canola oil
L-lysine
DLmethionine
L-threonine
L-tryptophan
Choline
chloride
Dicalcium
phosphate
Limestone
Salt
Vitamin and
mineral
premixA
Titanium
dioxideB
Calculated
analysis:
DE (MJ/kg)
CP, g/kg
Available
lysine, %
Calcium %
Available P,
%
Medium-grain rice
Animal
Plant
protein
protein
705.6
528.4
51.6
-
Long-grain rice
Animal
Plant
protein
protein
705.6
528.4
51.6
-
Wheat
Animal
protein
780
50
Plant
protein
532.8
-
100
-
100
-
50
-
30
-
30
-
25
-
100.4
-
100.4
-
50
-
-
100
150
185.2
-
100
150
185.2
-
100
150
151.6
5
2.78
0.36
6.4
1.12
5
2.78
0.36
6.4
1.12
28.3
6.04
1.2
30
6.84
1.47
1.43
0.28
0.4
2.68
0.34
0.4
1.43
0.28
0.4
2.68
0.34
0.4
2.7
0.42
0.4
2.54
0.23
0.4
-
18.7
-
18.7
0.7
17
1
0.7
4.4
1
0.7
1
0.7
4.4
1
0.7
1
0.7
5.2
1
0.7
1
1
1
1
1
1
15.3
200
1.30
15.4
200
1.31
15.3
200
1.30
15.4
200
1.31
15.0
197
1.28
15.3
215
1.30
1.2
0.6
0.8
0.45
1.2
0.6
0.8
0.45
0.91
0.49
0.8
0.45
A
Provided the following nutrients (per kg of air-dry diet): Vitamins: A 1500 IU, D3 300 IU, E 37.5 mg, K 2.5
mg, B1 1.5 mg, B2 6.25 mg, B6 3 mg, B12 37.5 μg, Calcium pantothenate 25 mg, Folic acid 0.5 mg, Niacin 30
mg, Biotin 75 μg; Minerals: Co 0.5 mg (as cobalt sulphate), Cu 25 mg (as copper sulphate), Iodine 1.25 mg (as
potassium iodine), Iron 150 mg (as Ferrous sulphate), Mn 100 mg (as Manganous oxide), Se 0.5 mg (as Sodium
Selenite), Zn 0.25 mg (as zinc oxide). (Hogro Bronze Weaner and Grower, Rhone-Poulenc Animal Nutrition Pty
Ltd., Queensland, Australia).
B
Titanium dioxide (TiO2; Sigma Chemical Company, St. Louis, MO, USA).
49
8.4
Results
8.4.1 Faecal shedding of haemolytic E. coli and antibiotic treatments
The mean number of antibiotic treatments and the mean faecal swab score are shown in Table 8.2. The
number of antibiotic treatments given by the stockperson for clinical PWD was similar (P>0.05)
across all dietary treatments. However, shedding of haemolytic E. coli ascertained via faecal swabs
showed a significant main effect of protein source on the faecal swab score, with pigs fed animal
protein demonstrating a higher score than pigs fed vegetable protein (0.6 vs. 0.4, P=0.057).
Furthermore there was a tendency for an interaction between cereal type and protein source (P=0.069),
with pigs fed diet MGPP having a higher swab score than pigs fed diet MGAP (Table 8.2).
Table 8.2 Interaction means for average number of antibiotic treatments and the average faecal swab
score for piglets after weaning.
Dietary treatment
Cereal type
Protein source
Medium-grain
rice
Long-grain rice
Wheat
Animal
Plant
Animal
Plant
Animal
Plant
Pooled mean
SEMA
Number of
antibiotic
treatments
1.4
0.7
1.0
0.6
1
1
Faecal swab
scoreB
0.9
0.0.72
0.5
0.30
0.5
0.7
0.6
0.3
0.8
0.3
Probability, P=
Cereal type
0.792
0.461
Protein source
0.230
0.057
Cereal * Protein
0.638
0.069
A
SEM: standard error of the mean.
B
Faecal swab score is the mean score per pig determined from swabs taken on days 2, 5, 6 and 8 after
weaning.
8.4.2 Performance data
Pigs generally adapted well to their new environment. There were no statistically significant
differences between treatment groups for average daily gain in the first week after weaning, although
there was a tendency for pigs offered animal protein to outperform pigs offered plant protein (78 vs.
43 g/day, P=0.152). Pigs fed rice numerically performed better than pigs fed wheat (an average of 69
vs. 44 g/day), however large variation between pens of pigs precluded statistical differences (Table
8.3).
In weeks two and three, there was no significant main effect of cereal type on any of the indices
measured, although there was suggestion of an improved FCR with wheat compared to rice (P=0.191).
There was a significant main effect, however, of protein source. Pigs fed animal protein rather than
plant (vegetable) protein were heavier at the end of the experiment (11.80 vs. 10.36) because they
grew faster (317 vs. 242 g/day). This was a consequence of a higher voluntary feed intake (580 vs. 500
g/day) and improved FCR (1.87 vs. 2.31). No interactions occurred for any of the production indices
(Table 8.4).
50
Table 8.3 Interaction means for average daily gain of pigs kept in groups in the first week after
weaning.
Dietary treatment
Cereal type
Protein source
Medium-grain
Animal
rice
Plant
Animal
Long-grain rice
Plant
Animal
Wheat
Plant
Pooled mean
SEMA
Cereal type
Protein source
Cereal * Protein
A
SEM: standard error of the mean.
Start Liveweight,
kg
6.71
6.66
6.68
6.69
6.97
6.81
Liveweight after 7
days, kg
7.32
7.04
7.31
6.98
7.46
7.03
Daily gain, g
6.73
0.131
7.15
0.177
60
110.2
Probability, P=
0.900
0.991
0.952
0.465
0.996
0.984
0.625
0.152
0.903
87
54
91
42
55
32
8.4.3 Coefficient of total tract apparent digestibility
The CTTAD of DM, starch, energy and CP are depicted in Table 8.5. Significant interactions between
the cereal type and protein source occurred for DM, starch, energy and CP. The CTTAD for DM was
higher in diets MGAP and LGAP than in the corresponding wheat-based diet (WAP) (0.92 and 0.92
vs. 0.85, P<0.001), however DM digestibility was similar between all three diets when plant proteins
were fed to pigs instead of animal proteins (0.83, 0.82 and 0.80 for diets MGPP, LGPP and WPP,
respectively). A similar interaction occurred between cereal type and protein source for the CTTAD of
energy, with diets MGAP and LGAP having the highest coefficients of energy digestibility compared
to WAP (0.92 and 0.91 vs. 0.83, P<0.001).
Total-tract starch digestibility was very high in all diets (range 0.989 to 0.999). The CTTAD for starch
was higher in diet WPP than in diet WAP (0.993 vs. 0.989), which resulted in a significant interaction
(P<0.001). Surprisingly, the CTTAD for CP was higher in pigs fed diet WPP compared to those fed
diets MGPP and LGPP (0.76 vs. 0.67 and 0.66 respectively, P=0.016) (Table 8.5).
51
Table 8.4 Interaction means for performance of pigs fed different diets in weeks two and three of the experiment.
Dietary treatment
Cereal type
Protein source
Animal
Medium-grain rice
Plant
Animal
Long-grain rice
Plant
Animal
Wheat
Plant
Pooled mean
SEMA
Liveweight at start
of Week 2, kg
7.31
6.98
7.32
7.04
7.46
7.03
Liveweight at end
of Week 3, kg
11.66
10.39
11.69
9.97
12.06
10.71
7.15
0.170
11.07
0.284
0.981
0.322
0.987
0.723
0.010
0.942
311
243
312
219
329
264
Daily feed intake, g
day-1
586
527
586
488
569
485
278
10.4
540
15.1
2.13
0.122
0.330
<0.001
0.803
0.712
0.007
0.874
0.191
0.028
0.545
Daily gain, g
FCR (g feed:g gain)
1.92
2.69
1.98
2.32
1.71
1.92
Probability, P=
Cereal type
Protein source
Cereal * Protein
A
SEM: standard error of the mean.
52
Table 8.5 The coefficient of total tract apparent digestibility (CTTAD) of dry matter (DM), starch, energy and crude protein (CP) in pigs fed different diets
after weaning.
Dietary treatment
Protein source
Animal protein
Medium-grain rice
Plant protein
Animal protein
Long-grain rice
Plant protein
Animal protein
Wheat
Plant protein
Cereal type
Pooled mean
SEMB
Cereal type
Protein source
Cereal * Protein
A
DM: dry matter; CP: crude protein.
B
SEM: standard error of the mean.
CTTAD of:
DMA
0.92
0.83
0.92
0.82
0.85
0.80
Starch
0.999
0.998
0.999
0.997
0.993
0.989
Energy
0.92
0.82
0.91
0.81
0.83
0.80
CPA
0.79
0.67
0.78
0.66
0.78
0.76
0.86
0.006
0.996
0.0012
0.85
0.006
0.74
0.009
<0.001
<0.001
<0.001
0.022
<0.001
0.016
Probability, P=
<0.001
0.837
0.016
<0.001
<0.001
<0.001
53
8.5
Discussion
Weaned piglets fed extruded medium-grain rice or long-grain rice performed equally to pigs fed
wheat in the first three weeks after weaning, indicating that extruded rice can replace wheat as the
sole cereal in piglet diets after weaning. There was a suggestion, however, that pigs fed wheat as
the cereal converted feed to daily gain more efficiently than pigs fed the two extruded rice-based
diets (P=0.191; Table 8.3). As alluded to in Section 7.5 and highlighted further in Chapter 10,
however, it seems that the energy value of extruded rice might have been underestimated, which
would account for the inferior feed conversion of the rice-fed piglets. This is further supported by
the findings that pigs fed the extruded medium-grain rice or long-grain rice diets had higher
CTTAD for starch, energy and CP that would have yielded more nutrients in the digestive tract for
absorption and body growth, although the response of the cereal depended on the type of protein
sources fed to the pigs.
The inclusion of vegetable (plant) protein sources in the diet depressed growth rate (P<0.001), feed
intake (P=0.007) and FCR (P=0.028) in weeks two and three of the trial compared to the use of
animal protein sources. This is an important finding because it provides clear direction to potential
users of extruded rice regarding the selection of dietary protein sources to maximise the efficiency
of rice. Vegetable proteins such as the lupins, soybean meal and canola meal used in this study
contain appreciable levels of NSP and oligosaccharides that are anti-nutritive in the gastrointestinal
tract of the pig, reducing the extent of digestion and absorption of nutrients available for body
growth (Pluske et al., 1999). Animal protein sources are more digestible than plant protein sources
and hence more nutrients became available for growth and development. However, the presence of
significant interactions between cereal and protein sources for CTTAD indicates that the dietary
component responded differently to both dietary variables. For example, CTTAD for energy was
significantly higher in both extruded rice-based diets irrespective of whether animal or plant
protein was added compared to diets WAP and WPP, whereas for CP digestion, the significant
interaction (P=0.016) was caused by an apparently higher digestibility in pigs fed diet WPP
compared to pigs fed diets MGPP and LGPP. It is difficult to explain the higher CP digestibility in
pigs fed diet WPP compared to pigs fed diets MGPP and LGPP given the higher DM and energy
digestibilities observed in the extruded rice-based diets when plant protein sources were added, but
might be attributable to a higher formation of microbial protein causing an overall depressed total
tract digestibility (Pluske et al., 2003). Interpretation of total tract digestibility coefficients for CP
is fraught, therefore, because the formation of protein by the microbiota provides no real indication
of ileal digestibility of CP and absorption of amino acids, which cannot be absorbed by the pig in
the large intestine.
Seemingly in contrast to these findings, Montagne et al. (2004) reported no differences in average
daily gain or FCR when pigs were fed diets based on cooked (autoclaved) white rice with either an
animal or plant protein supplement. The reason(s) for this difference is (are) hard to explain, but
could be attributable to the fact that Montagne et al. (2004) infected pigs experimentally with
enterotoxigenic E. coli that could have disturbed the intestinal milieu associated with digestion and
absorption, and (or) there was an effect of cooking form on digestibility and subsequent growth
rate. Extruded rice contains very low levels of RS whereas cooked (autoclaved) white rice that is
then cooled prior to feeding contains approximately 20 times the RS content of extruded rice
(Chapter 5). The RS level of the extruded products were not considered in the derivation of the
energy value of extruded rice used in the formulation of the diets, and hence there could have been
a misbalance in energy contributions between the protein sources and the extruded rice that
contributed to the inferior performance of the pigs fed plant protein.
The number of antibiotic treatments given by the stockperson for clinical PWD in the 21 days after
weaning was similar (P>0.05) in pigs across all treatment groups. In contrast, the faecal shedding
of E. coli in the first eight days after weaning showed that vegetable proteins (as a main effect in
the statistical analysis) reduced (P=0.059) the mean faecal swab score recorded in the first eight
54
days after weaning compared to pigs fed animal protein sources. This difference was caused
predominately by the greater swab score recorded in pigs fed diets LGAP and WAP, suggesting
that in these two cereal sources the presence of vegetable proteins reduced colonisation and
subsequent shedding of the haemolytic E. coli. Vegetable proteins contain considerable levels of
DF, which are possibly thought to influence PWD and shedding of E. coli (Bolduan et al., 1988).
These data contrast to the work of Pluske et al. (2003) where the addition of various sources of DF,
which are present also in vegetable proteins, increased the number of antibiotic injections required
in pigs that displayed PWD. However, the quantity and chemical composition of the DF sources
used Pluske et al. (2003) contrasted greatly with the predominately uronic acid-based DF present in
vegetable proteins (Pluske et al., 1999), which could help explain the difference. Moreover, the use
of extruded rice instead of cooked (autoclaved) rice could also have contributed to the differences
observed because of changes to the physico-chemical properties (Marsono and Topping, 1993).
Montagne et al. (2004) noted a lack of difference in pigs fed diet WPP compared to pigs fed diets
MGAP and MGPP for the faecal shedding of haemolytic E. coli, which again could reflect the
difference in the form (ie, extrusion vs. autoclaving) of rice used in the experiments.
Nevertheless, and as depicted in Table 8.4, pigs fed diets MGAP or LGAP performed equivalently
to their counterparts fed wheat-based diets, and mortality on the rice-based diets was zero.
Professor Gonzales Mateos (personal communication), who has conducted similar studies with
processed rice in Spain, has sometimes observed a higher incidence of PWD in piglets fed a cooked
rice-based diet, although this did not cause detrimental performance. Professor Mateos believes that
the (transient) diarrhoea after weaning caused by feeding rice is a consequence of higher feed
intake in the first week. It was not possible to measure individual feed intake in the first week postweaning in our experiment because pigs were housed in groups of four, however there was a clear
trend for pigs fed the extruded rice and animal protein-based diets (MGAP and LGAP) to
numerically perform better than pigs fed wheat (an average of 69 vs. 44 g/day), a difference that
undoubtedly reflects a higher voluntary feed intake.
In conclusion, pigs fed the extruded rice-based diets with either animal protein sources or plant
protein sources performed equivalently to pigs fed either of the wheat-based diets in the first three
weeks following weaning. The digestibility of DM and the dietary components starch and energy
was generally improved by the use of extruded rice compared to wheat, and was increased by the
use of animal rather than vegetable proteins. The lack of difference in antibiotic treatments and
faecal shedding of haemolytic E. coli might have been a general reflection of the low levels of
PWD seen in this study, although the reduction in shedding seen in pigs fed vegetable proteins
implicated a role for DF in the expression of PWD. This feature will be pursued in the next chapter.
55
9. Effect of added oat hulls to extruded
rice- and wheat-based diets on
production, digestibility and the
incidence of PWD
9.1
Summary
This experiment was conducted in male weaner pigs (96 pigs with initial weight of 5.16 ± 0.08 kg)
to examine the effects of added oat hulls (20 g kg-1) in either an extruded rice- or wheat-based diet
on performance, the incidence and treatment of PWD, coefficients of total tract apparent
digestibility (CTTAD) of dietary components and the levels of blood and faecal metabolic
indicators of hindgut digestion in the post-weaning period. The diets used were: (i) extruded
medium-grain rice (R; variety Amaroo) plus animal protein (RAP); (ii) diet (i) with added oat hulls
(20 g kg-1; RAPOH); (iii) wheat (W) plus animal protein (WAP); and (iv) diet (iii) with added oat
hulls (20 g kg-1; WAPOH). All diets were formulated to contain 14.4 MJ DE kg-1 and 0.80 g lysine
MJ DE-1. Pigs (24 pigs per treatment combination; 4 pigs per pen and 6 pens per treatment) were
randomly allocated based on initial live weight and fed their respective diets ad libitum. Blood and
faecal samples were collected on days 7 and 14 after weaning. Pigs fed wheat diets retained more
moisture in their faeces than pigs fed R diets (P<0.01). Pigs fed diet RAP had more diarrhoea and
hence received more antibiotic treatment than pigs fed diet WAP. Addition of oat hulls to diet RAP
decreased the incidence of PWD and the number of antibiotic treatments. The number of pigs
shedding haemolytic E. coli measured five days after weaning was lower (P<0.05) in pigs fed oat
hull-supplemented diets. Mean E. coli scores were lower in pigs fed wheat-based diets than in pigs
fed extruded rice-based diets regardless of oat hull supplementation. The CTTAD of all dietary
components (DM, starch and energy) were higher in extruded rice-based diets than in wheat-based
diets (P<0.001). The addition of oat hulls decreased the CTTAD (P<0.001). Pigs fed extruded ricebased diets had higher plasma urea concentrations and lower faecal biogenic amine concentrations
compared to pigs fed wheat-based diets. Plasma creatinine concentration was positively correlated
to haemolytic E. coli scores after weaning (P<0.015). Supplementation of oat hulls tended to
decrease biogenic amine concentrations (P=0.103). In conclusion, insoluble NSP added in the form
of oat hulls in extruded rice and animal protein-based diets for weaner pigs reduced the incidence
of PWD in the first three weeks after weaning.
9.2
Introduction
Numerous recent studies in pigs have demonstrated that cooked white rice shows excellent
potential for inclusion in diets as a replacement for more traditional cereals such as maize and
sorghum (Alcantara et al., 1989; Mateos et al., 2001; 2002; Solà-Oriol et al., 2004; Vicente et al.,
2004). Furthermore, the use of cooked white rice has been associated with reductions in PWC and
swine dysentery (Pluske et al., 2002; 2003; Hopwood et al., 2004). However, results found
previously in Chapters 7 and 8 showed an increased incidence of PWD, or certainly no reduction in
PWD, when pigs were fed either extruded rice-based diets or a cooked rice-based diet with animal
protein compared to pigs fed a WAP or WPP diet. It has been postulated that an imbalance between
the reduced amount of (fermentable) carbohydrates entering the hindgut from rice, as it is all
starch, and an excess of undigested nitrogen (N) relative to carbohydrate entering the large intestine
in pigs fed diet RAP, may have contributed to PWD because the microbiota decarboxylate the N
into diamines which are thought to be implicated in the aetiology of PWD (Bolduan et al., 1988;
Gaskins, 2001; Pluske et al., 2002).
56
Substrates such as carbohydrate and N entering the caecum most likely change the ratio of
saccharolytic and proteolytic microbiota in the large intestine. For example, a diet containing a
limited amount of fermentable carbohydrates (NSP and RS) and (or) is high in N will cause the
proteolytic microbiota to predominate over saccharolytic bacteria (Piva et al., 1995; Reid and
Hillman, 1999). The proliferation of proteolytic bacteria in the large intestine of pigs can produce
many potentially toxic metabolic by-products including branched-chain VFA, NH3, volatile
phenols, indoles and biogenic amines (Williams et al., 2001). When undigested N enters the
caecum, deamination or decarboxylation processes produce NH3 and amines, respectively.
Putrescine, cadaverine, histamine and β-phenylethylamine are produced in the pig’s large intestine
from arginine, lysine, histidine and phenylalanine, respectively, by a number of bacterial groups
including Bacteroides, Clostridium, Enterobacterium, Lactobacillus and Streptococcus (Gaskins,
2001). These microbially generated amines (biogenic amines) could contribute to the occurrence of
diarrhoea after weaning. However, the addition of insoluble and (or) slowly fermentable NSP
sources in weaner pig diets showed a linear reduction in biogenic amines and less PWD (Bolduan
et al., 1988; Aumaitre et al., 1995).
Given the unexpected results found in Chapters 7 and 8, the purpose of the current study was to (1)
determine whether the type of cereal (low fermentable carbohydrate diet RAP vs. high fermentable
carbohydrate diet WAP) influences the incidence of diarrhoea and excretion of haemolytic E. coli,
(2) see whether the addition of insoluble NSP (20 g oat hulls kg-1) reduced the deleterious effects
feeding an extruded rice plus animal protein diet appeared to have on PWD, and (3) examine
further the digestibility of selected dietary components and performance of weaner pigs in response
to cereal type and added oat hulls.
9.3
Materials and Methods
9.3.1 Experimental design
A 2 x 2 factorially designed experiment was conducted, with the respective factors being cereal
type (extruded medium-grain rice vs. wheat) and oat hull addition (with or without 20 g oat hulls
kg-1), to test the effect of cereal source and insoluble NSP supplementation on faecal moisture
content, the incidence of PWD, the number of antibiotic treatments, faecal scores of haemolytic E.
coli, blood urea and creatinine, the formation of biogenic amines, CTTAD of dietary components
and performance after weaning. The Murdoch University Animal Ethics Committee and the
Animal Ethics and Experimentation Committee of the WA Department of Agriculture approved the
experimental protocol.
9.3.2 Animals, diets, feeding, recording and sample collection
Ninety-six male pigs (Large White x Landrace) weaned at approximately 18-22 days of age were
obtained from a commercial supplier (Wandalup Farms, Mandurah, WA). The pigs were
transported to an isolated animal house at Murdoch University where they housed in metal wiremashed pens with a floor space of 2.5 m2. The experiment was conducted in two replicates with 48
pigs each. The average live weights (mean ± SEM) of pigs at arrival were 5.5 ± 0.05 kg for
replicate 1 and 4.9 ± 0.08 kg for replicate 2. Pigs were randomly accommodated to a pen (4
pigs/pen, 6 pens/treatment = 24 pigs per treatment) based on their live weight at arrival. The
ambient temperature was maintained at 29 ± 1°C. Water and feed were freely accessible during the
whole experiment through a nipple drinker and feed trough, respectively, set in each pen.
The pigs were offered their respective experimental diet ad libitum for three weeks. The diets used
were: (i) extruded medium-grain rice (R; variety Amaroo) plus animal protein (RAP); (ii) diet (i)
with added oat hulls (20 g kg-1; RAPOH); (iii) wheat (W) plus animal protein (WAP); and (iv) diet
(iii) with added oat hulls (20 g kg-1; WAPOH). All diets were formulated to contain 14.4 MJ DE
kg-1 and 0.80 g lysine MJ DE-1. Oat hulls (Glen Forrest Stockfeeds, Glen Forest, WA) were passed
57
twice through a hammermill fitted with a 4.5 mm screen prior to incorporation into diets. The
extruded rice was that used previously (Chapter 8.3.2), and was passed through a hammermill
without the screen to achieve a more uniform particle size. The wheat was passed through the same
hammermill but with a 4.5 mm screen, to reduce particle size. Titanium dioxide (TiO2) was added
as an inert marker for estimation of the CTTAD. The composition and analysed chemical
composition of experimental diets are presented in Table 9.1.
Pigs were weighed every week and feed intake was recorded on a weekly basis as feed
disappearance from the feeder. Pigs were monitored for the presence of diarrhoea at least twice
daily. Pigs with diarrhoea were treated immediately with an intramuscular injection of Trisprim480 (trimethropin 80 mg/mL, sulfadiazine, 400 mg/mL; Troy Laboratories, Smithfield, NSW,
Australia) until the diarrhoea ceased. Faecal swabs were taken for culture on days 0, 2, 5 and 6
after weaning. Blood samples (10 ml) from two randomly selected pigs per pen were taken from
the anterior vena cava into heparinised EDTA-vacutainer tubes on days 7 and 14. The samples
were immediately transported and analysed for plasma urea and creatinine contents. Faecal ‘grab’
samples were collected from each pen at 0800, 1000, 1200, 1400, 1600 h on day 7 and 14 of the
trial. The samples were then kept at –20 ºC and later thawed, mixed, freeze-dried and ground
through a laboratory hammer mill (1 mm screen) prior to chemical analysis.
9.3.3 Chemical and microbial analyses
All analyses were made in duplicate. Dry matter (DM) was measured using AOAC official method
930.15 (AOAC, 1997). Total starch was determined as described previously (Chapter 4.2.1). The N
content was determined with a LECO FP-428 Nitrogen Analyser using a combustion method
(AOAC official method 990.03; AOAC, 1997). The gross energy (GE) content was determined
using a Ballistic Bomb Calorimeter (SANYO Gallenkamp, Loughborough, UL). NSP and their
constituent sugar contents were determined as alditol acetates by gas-liquid chromatography at The
University of New England, using the method of Theander and Westerlund (1993). Water holding
capacity (WHC) of diets was determined using a method by Kyriazakis and Emmans (1995).
Titanium dioxide (TiO2) was determined as described by Short et al. (1996).
The E. coli score was determined by scraping faecal swabs on a sheep blood agar plate and grown
overnight at 37 °C in air. The presence of haemolytic E. coli was then scored from no growth (0) to
heavy colonisation (5.) Faecal consistency was measured and scored as normal, moist, wet and
diarrhoea. Plasma urea content was determined using an enzymatic (urease) kinetic method
(Randox). Plasma creatinine assay was performed on an automated analyser (Daytone RX, Randox,
Northern Ireland) using alkaline picrate without deproteinisation. Both metabolites were
determined in the Clinical Pathology laboratory at Murdoch University. The concentration of
biogenic amines in the feed and faeces of pigs was determined by the State Chemistry Laboratories
of the Department of Primary Industries, Victoria, Werribee.
9.3.4 Statistics
The pen was considered as an experimental unit for all statistical analyses. For pig performance, the
treatment effects were assessed by ANOVA for a factorial arrangement with the main effects being
cereal type and oat hull supplementation. For CTTAD, blood metabolites and biogenic amine
levels, treatment effects were assessed by repeated measures ANOVA. Pearson’s correlation study
was performed to examine relationships between the plasma creatinine concentration and
haemolytic E.coli scores. The effects were considered as fixed effects in the model. Fisher’sProtected least significant difference test were used (at 5% significance level) for comparison of
treatment differences. The digestibility at week one was compared with the results repeated after
two weeks using one-way ANOVA. All statistical analyses were conducted using the statistical
package StatView 5.0 for Windows, SAS Inc. (AddSoft Pty. Ltd., Woodend, Vic., Australia).
58
Table 9.1 DietA composition (g kg-1, as-fed basis) and analysed nutrient content (g kg-1 DM).
Ingredient
RAP
Extruded rice
702
Wheat
Meat and bone meal
52
Whey powder
100
Bloodmeal
30
Fishmeal
100
Oat hulls
Canola oil
5
L lysine-HCl
2.8
DL Methionine
0.4
L Threonine
1.5
Tryptophan
2.8
Choline chloride
0.4
Dicalcium phosphate
Salt
0.1
B
0.7
Vit/Min premix
Titanium dioxide
0.1
-1
Calculated analysis (g kg DM):
Crude protein
195
Starch
569
GE (MJ/kg DM)
19.03
Total NSP
11.94
Insoluble NSP
8.95
Soluble NSP
3.00
Free sugars
31.66
Biogenic amines (mg kg-1 DM)
β-Phenylethylamine
Putrescine
Cadaverine
Histamine
RAPOH
682
52
100
30
100
20
5
2.8
0.4
1.5
2.8
0.4
0.1
0.7
0.1
WAP
782
50
50
25
50
28
6
1.2
2.7
0.4
0.4
0.7
0.1
0.7
0.1
WAPOH
762
50
50
25
50
20
28
6
1.2
2.7
0.4
0.4
0.7
0.1
0.7
0.1
188
568
19.03
23.02
20.19
2.82
31.20
204
543
19.32
76.99
65.74
11.24
32.37
190
563
19.35
94.07
83.36
10.71
28.34
19
24
43
4
22
17
26
3
22
17
24
3
15
24
41
4
Water holding capacity
3.67
3.71
1.69
1.65
(g g-1 dried diet)
A
RAP: Rice animal protein; RAPOH: Rice animal protein and oat hulls; WAP: wheat animal
protein; WAPOH: wheat animal protein and oat hulls
B
Provided the following nutrients (per kg of air-dry diet): Vitamins: A 1500 IU, D3 300 IU, E 37.5
mg, K 2.5 mg, B1 1.5 mg, B2 6.25 mg, B6 3 mg, B12 37.5 g, Calcium pantothenate 25 mg, Folic
acid 0.5 mg, Niacin 30 mg, Biotin 75 g; Minerals: Co 0.5 mg (as cobalt sulphate), Cu 25 mg (as
copper sulphate), Iodine 1.25 mg (as potassium iodine), Iron 150 mg (as Ferrous sulphate), Mn 100
mg (as Manganous oxide), Se 0.5 mg (as Sodium Selenite), Zn 0.25 mg (as zinc oxide). (Hogro
Bronze Weaner and Grower, Rhone-Poulenc Animal Nutrition Pty Ltd., Queensland, Australia).
59
9.4
Results
Faecal consistency scores on 2, 5 and 6 days after weaning are presented in Table 9.2. Pigs fed the
extruded rice-based diet had firmer faeces than pigs fed the wheat-based diet on day 2 (P=0.011), but
the difference was not significant on day 5 and 6 after weaning. Follow-up determination of faecal
moisture content one and two weeks after weaning showed significantly higher moisture content in
pigs fed wheat diets compared to pigs fed extruded rice diets (see Table 9.5, below).
Table 9.2 Faecal consistency scoreA of pigs fed different diets after weaning.
DietsB
RAP
RAPOH
WAP
WAPOH
Pooled means
SEM
Probability,
P=C
Day 2
1.2
1.6
2.0
1.7
1.6
0.087
0.011
Day 5
1.6
1.6
2.0
1.7
1.7
0.090
0.340
Day 6
1.7
1.9
2.0
1.7
1.8
0.094
0.429
A
Faecal consistency scored defined as; 1: normal, 2: moist, 3: wet and 4: diarrhoea.
Refer text for details of diets.
C
From one-way ANOVA.
B
The incidence of scouring and the number of pigs injected with antibiotics are presented in Table 9.3.
Pigs in replicate 1 had no diarrhoea, so the data presented in Table 9.3 and Figure 9.1 refer only to
observations in replicate 2. Pigs fed diet RAP diet showed a higher incidence of scouring and received
more antibiotic injections than pigs fed diet WAP. Addition of oat hulls in the RAP diet significantly
decreased the incidence of scouring and the number of antibiotic treatments. The presence of
haemolytic E. coli during the first week after weaning is illustrated in Figure 9.1. The number of pigs
shedding haemolytic E. coli on day 5 was significantly lower in oat hull-supplemented groups. Mean
E. coli scores were lower in pigs fed wheat diets than in pigs fed extruded rice diets regardless oat hull
supplementation.
Table 9.3 Incidence of scouring and number of antibiotic treatments of pigs fed different diets after
weaning.
DietA
RAP
RAPOH
WAP
WAPOH
No. of pigs injected
4
2
0
1
Incidence of scouring
9
2
0
1
No. of antibiotic treatmentsB
21
6
0
3
A
Refer text for details of diets.
Intramuscular injection of Trisoprim-480 (refer text for details).
B
60
2.5
7
Mean heamolytic E. coli score
No. of pigs shedding haemolytic E. coli
8
6
5
4
3
2
2
1.5
1
1
0
Day 0
Day 2
Day 5
0.5
Day 0
Day 6
Day 2
Day after weaning
RAP
RAPOH
WAP
Day 5
Day 6
Day after weaning
WAPOH
RAP
RAPOH
WAP
WAPOH
Figure 9.1 The number of pigs shedding haemolytic E. coli (left) and mean E. coli score of pigs fed different diets after weaning (right). Mean E. coli score
calculated only from the pigs shedding E. coli on the measurement day
61
Performance indices are summarised in Table 9.4. Daily gain (P<0.05) and FCR (P<0.01) were
affected by the cereal source, while feed intake remained unchanged. Pigs fed wheat diets grew faster
than pigs fed extruded rice-based diets. Supplementation of 20 g kg-1 oat hulls did not affect the
performance of pigs. There were significant replicate effects for daily gain and intake (P<0.001) but
not FCR because of the unavoidable difference in average starting weights between the two replicates
(mean live weight of 5.5 ± 0.05 kg for replicate 1 and 4.9 ± 0.08 kg for replicate 2). The significant
effect of replicate is not shown in the table because interactions between replicates and independent
variables were not significant.
Effects of cereal source and oat hull supplementation on the CTTAD are presented in Table 9.5.
Cereal source significantly influenced the CTTAD of DM, starch, GE and the DE content of the total
diet (P<0.001). Generally, the extruded rice-based diets were more digestible than the wheat-based
diets. Supplementation with 20 g oat hulls kg-1 reduced the CTTAD of DM and GE (P<0.001).
However, the CTTAD of starch was not affected by oat hull supplementation. The interaction between
cereal source and oat hull supplementation for all measurements was not significant. Faecal moisture
contents were higher in the wheat diets than in the extruded rice diets (P<0.01), and were not
influenced by oat hull supplementation.
Table 9.6 shows the difference in CTTAD when measured one or two week(s) after weaning. The
CTTAD of DM (P=0.108) and energy (P=0.084) but not starch tended to increase as the pigs aged.
The DE content of diet (P<0.05) was significantly improved as the pigs aged.
The effects of cereal source and oat hull supplementation on blood metabolites and formation of
biogenic amines are presented in Table 9.7. Plasma urea content (mmol L-1) tended to be higher in the
extruded rice-based diets than in the wheat-based diets (P=0.064). There was no significant main
effect (P=0.160) of oat hulls in lowering plasma urea levels, however a significant cereal source by oat
hull interaction was observed, such that supplementation of oat hulls significantly reduced plasma urea
concentration in the extruded rice-based diet (P<0.01) while no significant difference was observed in
the wheat-based diet. Plasma creatinine content (mmol L-1) was significantly higher in the extruded
rice-based diets than in the wheat-based diets (P<0.01). However, oat hull supplementation did not
alter the plasma creatinine concentration. Plasma creatinine concentration was positively associated
with mean haemolytic E. coli scores (Figure 9.2, P<0.015).
There were significant increases in faecal contents of putrescine, cadaverine and histamine (P<0.001)
in the wheat-based diets compared to the extruded rice-based diets. Also, the content of βphenylethylamine tended to be higher in the wheat-based diets (P=0.066). Adding oat hulls tended to
decrease the contents of β-phenylethylamine (P=0.066) and cadaverine (P=0.104) but not the contents
of putrescine and histamine. There was a tendency for an interaction between cereal source and oat
hull supplementation for the content of β-phenylethylamine (P=0.077), such that oat hull
supplementation significantly decreased β-phenylethylamine content in the wheat-based diet (P<0.05)
but did not alter β-phenylethylamine content in the extruded rice diet. Overall, total amine content was
significantly higher in wheat-based diets (P<0.001), and oat hull supplementation tended to decrease
total amine production in the large intestine (P=0.103).
62
Table 9.4 Effect of cereal source and oat hull supplementation on performance indicesA of piglets for three weeks after weaning.
Cereal
Extruded rice
Wheat
-1
-1
Probability, P=B
Pooled
mean
SEM
Cereal source
Oat hull
Interaction
Oat hulls
No
+20 g oat hulls kg
No
+20 g oat hulls kg
No. of pigs
24
24
24
24
Start wt. (kg)
5.20
5.21
5.06
5.17
5.16
0.08
NS
NS
NS
Finish wt. (kg)
9.88
9.81
10.77
10.99
10.36
0.30
*
NS
NS
Gain (g day-1)
223
219
271
277
248
11.6
*
NS
NS
Intake (g day-1)
443
437
423
440
436
13.0
NS
NS
NS
FCR (g g-1)
2.05
2.01
1.58
1.60
1.81
0.06
**
NS
NS
A
Values are LS means from 24 observations.
Repeated measures ANOVA; NS: non-significant, *P>0.05; **P<0.01.
B
63
Table 9.5 Effects of cereal source and oat hull supplementation on the CTTAD measured at one and two weeks after weaningA
Cereal
Oat hull
Extruded rice
No
Wheat
-1
+20 g oat hulls kg
No
Probability, P=B
+20 g oat hulls kg
Pooled
mean
SEM
Cereal source
Oat hull
Interaction
-1
CTTAD of
DM
0.90a
0.87b
0.86c
0.84d
0.89
0.004
***
***
NS
Starch
0.999a
0.999a
0.994b
0.995b
0.997
0.004
***
NS
NS
Gross energy
0.89a
0.87b
0.84c
0.83d
0.86
0.004
***
***
NS
DE (MJ/kg DM)
17.0a
16.5b
16.3b
16.0c
16.4
0.07
***
***
NS
64a
66ab
68ab
71b
67
0.8
**
NS
NS
Faecal moisture
content (g per 100g)
A
Values are least-squares means from 24 observations (CTTAD was measured at week one and two from 12 pens). abcValues in row without common
superscripts are significantly different at 5% significance level.
B
Repeated measures ANOVA; NS: non-significant, **P<0.01; ***P<0.001.
64
Table 9.6 Effect of collection time after weaning on the CTTADA of selected dietary components.
Week after weaning
2
Pooled
mean
SEM
Probability,
P=B
Treatment
1
CTTAD of
DM
0.86
0.87
0.87
0.004
0.108
Starch
0.996
0.997
0.997
0.004
NS
Gross energy
0.85
0.86
0.86
0.004
0.084
DE (MJ/kg DM)
16.3
16.6
16.4
0.07
*
Faecal moisture
content (g g-1)
67
68
67
0.8
NS
A
Values are LS means from 12 observations.
ANOVA; NS: non-significant, *P>0.05; ***P<0.001.
B
Mean plasma creatinine μ
( mol/L)
90
80
70
60
0
1
2
3
4
Total haemolytic E. coli score
Figure 9.2 Relationship between plasma creatinine concentration and rectal haemolytic E. coli
score of pigs during 14 days post-weaning (P<0.015).
65
Table 9.7 Main effects of cereal source and oat hull supplementation on blood metabolitesA and formation of biogenic aminesB in weanling pigs.
Cereal
Oat hull
Extruded rice
No
Wheat
+20 g oat hulls kg-1
No
+20 g oat hulls kg-1
Probability, P=C
Pooled
mean
SEM
Cereal source
Oat hull
Interaction
Blood metabolites
Urea (mmol L-1)
Creatinine ( mol L-1)
3.5a
2.6b
2.5b
2.7b
2.8
0.10
0.064
0.160
*
74.1ab
76.4a
69.2b
68.4b
72.1
1.00
**
NS
NS
Biogenic amines (mg kg-1)
β-Phenylethylamine
28a
28a
41b
28a
31
1.93
0.066
0.066
0.077
Putrescine
43a
49a
177b
153b
106
11.19
***
NS
NS
Cadaverine
353a
211a
868b
730b
504
59.54
***
0.104
NS
Histamine
7a
6a
64b
63b
35
4.70
***
NS
NS
431a
292a
1150b
974b
712
72.42
***
0.103
NS
Total amine
A
abc
Values are least-squares means means from 12 observations. Values in row without common superscripts are significantly different at 5% significance
level.
B
Values are least-squares means from 24 observations (biogenic amines were measured at week one and two from 6 pens).
C
Repeated measures ANOVA; NS: non-significant, P>0.05; **P<0.01; ***P<0.001.
66
9.5
Discussion
9.5.1 Incidence of diarrhoea and antibiotic treatment after weaning
As was observed in the previous trial (Chapter 8), pigs fed an extruded rice diet with animal protein
supplements (diet RAP) showed a higher incidence of diarrhoea after weaning. These two experiments
are seemingly in contrast to previous reports from this laboratory showing that cooked white rice is
associated with reductions in PWC. Previous experiments at this laboratory have always used
autoclaving as the method for cooking rice. For example, Pluske et al. (2003) and Hopwood et al.
(2004) autoclaved the rice and then cooled it at 4 ºC overnight before feeding it to pigs. It is well
documented that cooking and cooling of rice can significantly increase the RS content by promoting
retrogradation of starch polymers (Marsono and Topping, 1993). Studies conducted as part of this
research project (Chapter 5) have shown that autoclave cooking and then cooling in a refrigerator
increases the RS content by up to 12 times in the medium-grain (Amaroo) rice. Extrusion of the
medium- and long-grain rice, in contrast, showed decreases in the RS content, due most likely to the
solubilisation of starch by degradation of its macromolecular structure (Parchure and Kulkarni, 1997).
Therefore, it is possible that the (readily fermentable) RS formed during the (autoclave) cooking and
cooling of rice somehow prevented adhesion of haemolytic E. coli and (or) manipulated the hindgut
microbiota to reduce coliform shedding in the faeces, as was observed by, for example, Pluske et al.
(2003) and Hopwood et al. (2004).
Reid and Hillman (1999) reported that feeding retrograded waxy maize to weaner pigs increased the
Lactobacilli:coliform ratio in the large intestine, which the authors’ claim is beneficial to gut “health”
and reduces infection by intestinal pathogens. These studies used diets containing very low amounts of
RS and NSP, which may have limited the proliferation of health-promoting saccharolytic microbes
and showed that fermentable RS could depress expression of pathogens (Pluske et al., 2003, Hopwood
et al., 2004) and potentially harmful proteolytic microbes such as Bacteroides spp (Reid and Hilman,
1999). In this current study, it was evident that dietary supplementation with 20 g kg-1
insoluble/slowly digestible NSP as oat hulls to a low-fibre diet containing highly digestible
ingredients, such as diet RAP, positively influenced piglet health after weaning because there was less
diarrhoea and less antibiotic treatments. Martin et al. (2003) similarly reported that addition of 20 g
oat hulls kg-1 in a rice-based diet reduced the incidence of PWD between 21 and 41 days of age.
Surprisingly, the wheat-based diets used in this study were protective against PWD and pigs remained
healthy throughout the study. We have no obvious explanation for this finding because it contrasts to
previous work in our laboratory. However, it is well recognised that PWD is a very complex disease
and its aetiology still remains relatively misunderstood. It is possible that the overall level of infection
in this particular batch of pigs was generally low (as evidenced by the lack of disease in replicate 1)
and (or) the level of immunity was high, and feeding diet RAP was sufficient to cause mild diarrhoea.
The NSP in the wheat, under these infection conditions, might have been of just the correct
quantity/fermentability to limit shedding of haemolytic E. coli. Nevertheless, when feeding extruded
rice-based, low-fibre diets to weaner pigs, the addition of an appropriate insoluble / slowly digestible
fibre source is recommended. Further work is required in this area.
9.5.2 Digestibility and performance
The RAP diet showed significantly higher total-tract digestibility of all measured dietary components
than the WAP diet. However, pigs fed diet WAP grew faster and had a better feed efficiency (FCR)
than their counterparts fed diet RAP. Several reports showed higher digestibility of dietary
components and better performance indices in pigs fed a cooked rice-based diet compared to corn-,
wheat- and sorghum-base diets (Bonet et al., 2003; Lopez et al., 2003; Martin et al., 2003; Solà-Oriol
et al., 2004; Vicente et al., 2004). In the current study, we suspect that the inferior performance shown
in extruded rice-fed piglets was attributable to the use of a lower DE value and a higher CP value in
the formulation of the experimental diets despite the higher CTTAD of dietary components observed.
67
The DE value determined with weaner pigs using the same extruded rice samples (Chapter 10) showed
a higher value than the tabulated DE value that was used in the formulation, and hence the dietary DE
content of diet RAP was higher than the WAP diet (see Table 9.4). Also, chemical analysis showed
that the extruded rice had only 64 g CP kg-1 (DM), which is a much lower value compared to raw rice
(73 g CP kg-1). Therefore, an unbalanced energy: protein ratio was the most likely cause of the
disparity between CTTAD and performance figures. Supplementation of 20 g oat hulls kg-1 did not
influence performance of piglets but significantly decreased CTTAD of DM, GE and dietary DE
content, which is in agreement with Lopez et al. (2003) and was most likely due to the dilution effects
of the insoluble fibre. Increasing age after weaning tends to increase the CTTAD of dietary
components, as other studies have reported age-related developments in the small intestinal enzyme
systems and enhanced colonisation of the microbiota in the large intestine (eg, Martin et al., 2003;
Kim et al., 2005a).
9.5.3 Blood metabolites and biogenic amines
Urea is synthesised in the liver through the catabolism of amino acids when either (1) excess dietary
amino acids enter into the portal blood system, (2) the excess amino acids are not used for
gluconeogenesis, or (3) dietary amino acids are not ideally balanced (Eggum, 1970). Plasma urea
concentration can also be influenced when microbial fermentation of nitrogenous compounds are
increased in the large intestine, either by increased undigested or endogenous nitrogen entering the
large intestine and (or) by overwhelming proliferation of proteolytic bacteria over saccharolytic
microbes (Younes et al., 1996, 1998). Catabolism of amino acids by microbes produces NH3 that
crosses the colonic epithelium and diffuses into the portal blood system, where it is converted to urea
in the liver. The urea synthesised in the liver is either excreted as urine or diffuses back into the
caecum and is incorporated into microbial N (Younes et al., 1996, 1998). Therefore, the higher plasma
urea content in pigs fed diet RAP compared to pigs fed diet WAP could be a reflection of either
inefficient utilisation of dietary protein or increased protein fermentation due to the proliferation of
proteolytic microbes in the pigs fed diet RAP. However, the latter is the most likely explanation
because supplementation of oat hulls decreased the plasma urea level in the low-fibre RAP diet,
possibly through the modification of the large intestinal microbiota. These data concur with the
findings of Bolduan et al. (1998), who reported a linear decline in plasma urea content with increasing
crude fibre levels in diets of weaner pigs. Furthermore, pigs fed diet WAP with oat hulls did not show
a modified plasma urea concentration, presumably because there was already sufficient NSP from
wheat to maintain saccharolytic bacterial activity in the large intestine.
Creatine is synthesised from arginine, glycine and methionine, and is used as a high-energy phosphate
reserve in muscle. Degradation from creatine and creatine phosphate in muscle is the main route of the
creatinine fluxed into the portal blood system, although some originates from diets. Therefore a
determinant of plasma creatinine levels is muscle mass (about 0.3 to 0.5% of muscle weight; Braun et
al., 2003). However, when animals are dehydrated such as can occur with diarrhoea after weaning,
plasma creatinine levels can be increased due either to simple dehydration of plasma contents or to
increased mobilisation of protein reservoirs from muscle (eg, the viscera) to compensate for decreased
nutrient intake and (or) absorption (Wannemacher, 1977; Segales et al., 1998). The association
between plasma creatinine levels and shedding of haemolytic E. coli in the post-weaning period found
in our study (Figure 9.2) adds testament to this theory, albeit that no assessment of total body water
content was made in the work.
Biogenic amines produced by the decarboxylation of amino acids in the large intestine have been
considered by some authors to increase the incidence of PWD, particularly in pigs fed diets containing
low DF and high CP levels (Aumaitre et al., 1995; Bolduan et al., 1988; Pluske et al., 2002). The
biogenic amines are thought to produce proteolytic and osmotic diarrhoea due to their ability to irritate
the colonic mucosa and to its high osmotic pressure (Nollet et al., 1999). However, our finding that
the levels of biogenic amines were markedly lower in the RAP diet suggests that (a) the
amount/composition of the protein entering the hindgut was different to that in the wheat-based diet
68
and caused lower production of amines, and (or) (b) that the diarrhoea seen in diet RAP was unrelated
to amine production. Hence it appears that the type and concentration of amines produced in the large
intestine depends on the type of cereal that pigs received and the amino acid flow into the hindgut.
Further, the significant interaction between oat hull supplementation and different responses of the
individual amines (eg, decreased β-phenylethylamine and cadaverine but not putrescine and
histamine) suggests that oat hulls depressed the microbiota metabolising phenylalanine and lysine but
not the microbiota metabolising arginine and histidine. Higher biogenic amines in wheat-based diets
could be the result of (a) increased endogenous secretions in the WAP diets due to its higher NSP
content (Low, 1989), (b) increased entry of dietary sources of N (amino acids) into the large intestine
because the higher NSP content in the wheat decreases N digestibility at the terminal ileum (Bedford
et al., 1992; Baidoo et al., 1998) and (or) reduces digesta retention time in the small intestine (Freire et
al., 2000), and (or) (c) increased decarboxylation of N rather than catabolism of amino acids in WAP
diet due to the favourable environment for microbiota such as E. coli, Proteus and Clostridia (Nollet
et al., 1999). Although it was not statistically significant, supplementation of oat hulls tended to
decrease amine production, which is in general agreement with reports by Aumaitre et al. (1995) and
Bolduan et al. (1988). This finding implies that supplemental oat hulls may have modified the activity
of proteolytic microbes in the large intestine.
In conclusion, and as indicated in the digestibility indices, extruded rice is an excellent replacement
for the traditional cereals such as wheat fed to pigs. The next study was conducted with the aim of
refining the nutritive value of extruded rice for the Australian pig industry, Furthermore, it appears
that under certain (and not yet properly defined) conditions, feeding a highly-digestible, extruded ricebased diet with animal protein in the absence of a source (or sources) of insoluble/slightly fermentable
DF could predispose weanling pigs to an increased risk of infection by intestinal pathogens such as E.
coli.
69
10. The nutritive value of extruded rice and
cooked (autoclaved) rice for weaner
and grower pigs
10.1 Summary
An experiment was conducted to examine the digestible energy (DE) and calculated net energy (NE)
contents of two varieties of extruded rice (medium-grain variety Amaroo and long-grain variety
Doongara) in pigs of two body weight groups (8 and 55 kg). Diets contained 857 g rice kg-1, 50 g meat
and bone meal kg-1, 82 g fish meal kg-1 and other trace ingredients. Diets were fed at 5% of body
weight for 8 kg pigs and at 3.75% of body weight for 58 kg pigs. Digestibility of GE was determined
using an indigestible marker (TiO2) and the DE content of rice was calculated by subtracting the DE
content of ingredients other than rice. The mean (± SEM) CTTAD of gross energy and DE content
(MJ/kg as-fed) of rice were 0.917 (0.003) and 15.26 (0.08), respectively. Variety significantly
influenced the DE content of rice such that the medium-grain rice had a higher DE content than the
long-grain rice (0.3 MJ difference, P<0.001). Body weight of the pig also significantly influenced the
DE content of rice such that weaner pigs extracted less energy from rice than grower pigs (up to 0.5
MJ, P<0.001). In a separate trial, the CTTAD of GE and DE content (MJ/kg) of cooked medium-grain
rice Amaroo (autoclaved for 20 min at 120 °C; rice:water ratio of 1:2 w/w) were examined with 10-kg
pigs. The mean values (± SEM) were 0.918 (0.001) and 15.1 (0.031), respectively. Estimation of the
net energy (NE) content of rice using CVB (Dutch) and INRA (French) prediction equations showed a
mean NE content for both rice types of 11.5 MJ/kg (air-dry basis). The DE and NE values found for
rice are higher than for other cereals used in weaner pig diets such as wheat.
10.2 Introduction
Approximately 600 million tonnes of rice are produced annually in the world (FAO, 2003), with the
overwhelming majority of this entering the human food market. Rice is characterised by its high starch
content, low NSP content and lower CP content in comparison to other cereals (Juliano, 1992).
Several recent studies in pigs, however, have demonstrated that cooked white rice shows excellent
potential for inclusion in diets as a replacement for more traditional cereals such as maize and
sorghum. For example, Alcantara et al. (1989), Mateos et al. (2001, 2002), Solà-Oriol et al. (2004)
and Vicente et al. (2004) all reported positive effects on growth when rice was included in diets for
pigs. Furthermore, the use of cooked rice has been associated with reductions in post-weaning
colibacillosis and swine dysentery (Pluske et al., 2002, 2003; Hopwood et al., 2004).
Aside from the NRC (1998) estimate of energy value for rice of 14.9 MJ DE/kg, further published data
pertaining to the energy content of cooked rice for pigs is scarce. This information is necessary for
nutritionists to more accurately formulate diets. The purpose of this study was to examine the DE
content of two types of extruded rice (lower or higher amylose:amylopectin ratio) in weaner pigs and
grower pigs. It is recognised though that body weight (Bell and Keith, 1989; Kim et al., 2005a) and
starch structure influence starch digestibility in pigs (Black, 2001; Lindberg et al., 2003; Kim et al.,
2005b), so in this experiment the hypotheses tested were that (i) rice with a lower
amylose:amylopectin ratio will have a higher DE content compared to a variety with a higher
amylose:amylopectin ratio, and (ii) heavier pigs will extract more energy than lighter pigs from rice.
70
10.3 Materials and Methods
10.3.1 Animals and experimental design
Thirty-two male pigs (Large White x Landrace, 16 pigs per each body weight groups) were used in
this experiment. The experiment was designed as a 2 x 2 factorial arrangement of treatments with the
respective factors being (a) two extruded rice varieties (medium-grain, lower amylose:amylopectin
Amaroo vs. long-grain, higher amylose:amylopectin Doongara) and (b) two body weight groups
(weaner and grower). The rice was extruded at described previously (Chapter 8.3.2). The average
body weights for each group were mean (± SEM) of 7.9 (± 0.16) kg and 55.4 (±3.10) kg, respectively.
Pigs in each body weight group were weighed and sub-divided randomly into two groups of eight pigs
according to their body weight. The Murdoch University Animal Ethics Committee and the Animal
Ethics and Experimentation Committee of the WA Department of Agriculture approved this
experiment.
10.3.2 Housing, diet preparation, feeding and sample collection
The weaner pigs were kept in individual wire-mesh floored metabolism crates in a room where the
temperature was maintained as constant as possible (27 ± 1°C). The grower and finisher pigs were
kept in individual concrete floor pens in a conventional room without heating. Water was freely
accessible during the whole experiment through a nipple drinker set in each crate and pen. From the
time of arrival, the pigs were offered their respective experimental diet at a rate of 5% of body weight
for weaner pigs and 3.75% of body weight for grower pigs, for a period of 10 days. The diet was
offered in mash form, and the diet composition is presented in Table 10.1. Titanium dioxide (TiO2)
was added as an inert marker for CTTAD estimation. Pigs were adapted to experimental diets for 7
days and the faecal samples were collected from the wire-mesh floor for weaner pigs and from the
concrete pan for grower pigs at 0800, 1000, 1200, 1400, 1600 h for the final 3 days. Samples collected
over the three-day period were pooled, kept at –20 ºC and later thawed, mixed, freeze-dried and
ground through a laboratory hammer mill (1 mm screen) prior to chemical analysis.
An additional experiment was conducted to determine the DE content of cooked rice (medium-grain
variety Amaroo) with 8 pigs weighing 10.3 kg (± 0.16). Rice was autoclaved for 20 min at 120 ºC
with a rice:water ratio of 1:2 (w/w) and kept at 4 ºC overnight. The cooked rice was then mixed with a
mixture of other ingredients (see Table 10.1) before feeding. The experimental conditions were the
same with the weaner pigs in the first trial.
10.3.3 Chemical analyses
The dry matter (DM), nitrogen (N), gross energy (GE), total starch, amylose and amylopectin contents
of extruded rice were determined as described previously (Chapter 4.2.1). The RS contents of extruded
rice were determined using a Megazyme Resistant Starch kit (Megazyme International Ireland, Ltd.,
Wicklow, Ireland). Crude protein (CP) content was calculated as N x 6.25. The DM, GE and TiO2
content of diets and faecal samples were determined for estimation of apparent digestibilities of GE
and the DE content of the experimental diets. The GE content of the rice, diet, and faecal samples was
determined using a Ballistic Bomb Calorimeter (SANYO Gallenkamp, Loughborough, UK). The TiO2
contents of diet and feacal samples were determined using the method described by Short et al.
(1996). The DE content of rice was calculated by subtracting the DE content of ingredients other than
rice from the DE content of the complete diet and expressed on a DM basis.
71
Table 10.1 The composition of the experimental diets (g kg-1).
Amount (g kg-1)
857
50.5
82.3
1
3.7
1.8
0.5
0.4
1
0.7
1
Ingredient
Test riceA
Meat and bone meal
Fish meal
Canola oil
Lysine
Threonine
Tryptophan
Choline
Salt
Vitamin/Mineral mixB
Titanium dioxideC
Calculated analysis:
DE (MJ/kg)
Available Lysine (g/MJ DE)
Crude protein (g/kg)
Crude fat (g/kg)
Neutral Detergent Fibre (NDF, g/kg)
Available phosphorus (g/kg)
Calcium (g/kg)
15.29
0.60
144
20.5
10.9
4.5
8.8
A
Either extruded Amaroo or Doongara.
Provided the following nutrients (per kg of air-dry diet):Vitamins: A 1500 IU, D3 300 IU, E 37.5 mg, K 2.5
mg, B1 1.5 mg, B2 6.25 mg, B6 3 mg, B12 37.5 μg, Calcium pantothenate 25 mg, Folic acid 0.5 mg, Niacin 30
mg, Biotin 75 μg; Minerals: Co 0.5 mg (as cobalt sulphate), Cu 25 mg (as copper sulphate), Iodine 1.25 mg (as
potassium iodine), Iron 150 mg (as Ferrous sulphate), Mn 100 mg (as Manganous oxide), Se 0.5 mg (as Sodium
Selenite), Zn 0.25 mg (as zinc oxide). (Hogro Bronze Weaner and Grower, Rhone-Poulenc Animal Nutrition Pty
Ltd., Queensland, Australia).
C
Titanium dioxide (TiO2; Sigma Chemical Company, St. Louis, MO, USA).
B
10.3.4 Statistical analyses
The pig was considered as the experimental unit. For CTTAD of GE and DE content of rice, the
treatment effects were assessed by two-way ANOVA for a factorial design with the main effects being
rice variety and body weight. The effects were considered as fixed effects in the model. Fisher’sprotected least significant difference test were used (at 5% significance level) for comparison of the
CTTAD of GE and DE content between mean values of different variables. All statistical analyses
were conducted using the statistical package StatView 5.0 for Windows (AddSoft Pty. Ltd., Woodend,
Vic., Australia).
10.4 Results
Pigs generally adapted well to the experimental diets by the end of the adaptation period. One pig
from the weaner group was removed from the trial due to persistent low feed intake.
The chemical composition of the two extruded rice varieties is presented in Table 10.2. Although total
starch contents of the two rice varieties were the same, Doongara had a higher amylose, CP and lower
crude fat and crude fibre content than the variety Amaroo. The RS contents of extruded Amaroo and
Dongara were similar and lower than the RS contents in raw rice (0.1 and 0.4 g/100g, respectively).
72
Table 10.2 Chemical composition of the extruded rice (g kg-1).
Composition, g kg-1 (DM)
DM
Total starch
Amylose
Amylopectin
Amylose:amylopectin
Resistant starch
Crude protein
Crude fatB
Crude fibre
Gross energy (MJ/kg DM)
A
Medium grain riceA
859
879
193
686
0.28
1.3
64.0
32.0
5.0
18.7
Long grain riceA
863
880
253
627
0.40
1.6
76.0
28.5
3.8
18.9
Variety: Amaroo and Doongara.
Canola oil was sprayed in the process of extrusion; the crude fat contents of corresponding raw rice were 10.9
g and 10.8 g/kg DM, respectively. The energy value of added fat was adjusted when estimating rice DE.
B
The effects of variety and body weight of pigs on the DE content of extruded rice are presented in
Table 10.3. The CTTAD of GE and the DE content of rice were 0.917 (range from 0.899 to 0.930) and
15.3 MJ/kg (range from 14.8 to 15.6), respectively. Variety influenced the DE content of rice
(P<0.001) with it being higher in the lower amylose:amylopectin variety Amaroo than the higher
amylose:amylopectin variety Doongara. Also, body weight of the pig significantly influenced the
CTTAD of GE and the DE content of rice (P<0.001). Weaner pigs extracted less energy from a given
rice than grower pigs. The interactions between variety and body weight of pigs were not significant
for the CTTAD and DE. The DE content of (autoclaved) cooked Amaroo rice was within the range of
extruded rice with a mean of 15.1 MJ/kg (Table 10.4).
The net energy (NE) contents of the two extruded rice types, from the determined DE values and
chemical compositions of the rices, was made using CVB (Dutch) and INRA (French) prediction
equations. The results are presented in Table 10.5. The mean NE value of extruded Amaroo and
Dongara was 11.5 MJ/kg as-fed.
73
Table 10.3 Main effects of variety and body weight of pigs on the digestible energy (DE) content of extruded rice.
n1
Treatment
Initial wt. (kg)
Final wt. (kg)
Growth (g/day)
CTTAD of GE
DE (MJ/kg as is)
DE (MJ/kg DM)
Medium rice
16
0.92ab
15.4b
17.9b
Long rice
15
0.92ab
15.1a
17.5a
Weaner
15
7.9
8.7
80
0.90a
15.0a
17.4a
Grower
16
55.4
61.4
301
0.93b
15.5b
18.0b
Pooled mean
33.2
35.1
190
0.92
15.3
17.7
SEM
4.6
4.8
34
0.003
0.07
0.08
Rice variety
2
Body weight
Statistics
Probability, P
=
Rice variety (V)
0.336
0.001
0.001
Body weight (BW)
0.001
0.001
0.001
V x BW
0.350
0.273
0.281
1
One pig from weaner group was removed from the trial.
Medium grain rice (lower amylose:amylopectin; variety Amaroo) and long grain rice (higher amylose:amylopectin; variety Doongara)
Values within a column without common superscript are significantly different at P<0.05.
2
74
Table 10.4 The DE content (MJ/kg) of cooked Amaroo rice in piglets.
Rice
n=
Cooked rice (Amaroo)
SEM
8
Initial wt. (kg)
10.3
0.16
Final wt. (kg)
12.6
0.17
Growth (g/day)
236
7.34
CTTAD of GE
0.92
0.001
DE (MJ/kg DM)
17.3
0.03
DE (MJ/kg as is)
15.1
0.03
10.5 Discussion
The DE content of extruded rice reported in this paper (15.3 MJ/kg; range from 14.8 to 15.6) showed
much higher values than the NRC value (14.9 MJ/kg). Eggum (1977) reported that the mean DE
content of raw and cooked rice in the rat was 15.6 MJ/kg DM (range from 15.4 to 15.8 MJ/kg), which
is also higher than the current NRC value for pigs. Extrusion most likely improves the energy
digestibility and the DE content of rice, since the RS content significantly decreased with extrusion
compared to polished raw rice (Parchure and Kulkarni, 1997; Faraj et al., 2004). However, the
extrusion effect alone does not explain the higher DE value reported in this paper, because the energy
contribution from the reduced RS by extrusion (0.3 g 100g-1) was less than 0.05 MJ/kg DM
(MacDonald et al., 1995). Therefore, the current DE value of rice used for formulation of pig diet may
be underestimated by as much as 0.4 MJ/kg.
The lower amylose, medium-grain variety Amaroo had a 0.3 MJ/kg higher DE content than the higher
amylose, long-grain variety Doongara. The amylose:amylopectin ratio is known to determine many
physical and chemical properties of processed rice. Generally, increased amylose content is associated
with the increased water holding capacity of the starch granule and increased capacity of
retrogradation through increasing capacity of hydrogen bonding (Juliano, 1992). A higher amylose
content in rice is known to inversely correlate to the in vitro starch digestion index (SDI; Tetens et al.,
1997; Rashmi and Urooj, 2003) and to increase the RS content (Sagum and Arcot, 2000). Retrograded
RS has been shown to decrease ileal starch digestibility and faecal fat and energy digestibility in pigs
(De Schrijver et al, 1999).
In the current study, the varietal difference in the DE content was higher in weaner pigs (0.4 MJ/kg)
than in grower pigs (0.2 MJ/kg), suggesting that weaner pigs were less able to extract energy in
response to differences in the chemical structure of starch, namely the higher amylose content and (or)
the higher RS content. The lower DE content of the higher amylose rice used in the present study was
most likely due to 1) the higher amount of undigested starch from RS reaching to the hindgut, and (or)
2) less fermentation capacity in the hindgut of weanling pigs. A similar observation was reported in a
rat study where digestibility values were compared in five rice cultivars (raw and cooked) differing in
amylose content (22–284 g/kg DM) (Eggum et al., 1993). In this particular study, rice with more
amylose showed a lower energy digestibility coefficient compared to rice with less amylose when
hindgut fermentation was suppressed by addition of the antibiotic Nabacitin. However, rats fed a nonantibiotic diet showed no differences in energy content. These data implicate the extent of hindgut
75
fermentation by the microbiota in different aged pigs as a possible reason for differences between
varieties in different aged pigs.
Nevertheless, it is unlikely that the type of starch alone influenced the DE content of rice, because
even retrograded RS is almost completely fermented by microbes in the large intestine of adult rats
(CTTAD of rice starch >0.99 in rat, Eggum, 1977; Eggum et al., 1993). Instead the difference in
starch, lipid and protein digestibility and in NSP content may be attributed to the overall slightly lower
DE content in the higher amylose variety Doongara. The NSP content of rice was reported to be higher
in a higher amylose rice variety. For example, variety Doongara (311 g amylose kg-1 DM) had 18 g
NSP kg-1 DM, while the medium- and lower-amylose rice varieties Inga (202 g amylose kg-1 DM) and
Japonica (115 g amylose kg-1 DM) had 16 g and 10 g NSP kg-1 DM, respectively (Sagum and Arcot,
2000).
The DE content of the cooked (autoclaved) rice was similar to the value determined with extruded
rice. Generally, autoclaving of rice is known to increase the RS content during cooling (Mangala et al.,
1999), and cooked rice had a lower digestibility of energy and protein compared to raw rice in the rat
(Eggum et al., 1993). However in the current study, the DE content of the cooked rice was within the
range of the extruded rices.
Body weight of pigs significantly influenced the DE content of extruded rice, which is in agreement
with earlier studies investigating other grains such as wheat (Bell and Keith, 1989; Kim et al., 2004).
The DE content determined with weaner pigs was significantly lower than that determined with
grower pigs, which was most likely due to a less developed hindgut and hence a lower level of
fermentation. However, the between-animal variation for the DE content (DM) was high in weaner
pigs (standard deviation, SD, of 0.250) compared to grower pigs (SD 0.156). Since the weaner pigs
were used only one week after weaning (aged approximately 35 ± 2 days), it is possible that variation
in recovery rate from the damaged gut integrity may cause the variation in DE content (Pluske et al.,
1997). The cooked rice experiment further supports this because pigs fed cooked rice (aged 49 ± 2
days) had a lower variation in the DE content (SD 0.09).
An early report on the DE and NE values of rice by Robles and Ewan (1982) indicated figures of 13.9
MJ and 9.5 MJ/kg, respectively. Furthermore, the NRC (1998) has shown the NE value of rice to be
9.61 MJ/kg. However, the DE and NE values determined in our study showed higher values than in
the abovementioned reports. The mean DE and NE values reported herein are in agreement with the
values reported in the Dutch tables of composition and nutritional value of feed materials (Sauvant et
al., 2004; 14.8 MJ DE and 11.8 MJ NE/kg) and in Valdivié (2004; 14.9 MJ ME/kg). The NE
estimation using CVB (Dutch) and INRA (French) prediction equations showed that the INRA
formulas gave a higher NE value than CVB formula (see Table 10.5). Similar results showing slightly
higher NE values by using INRA formula compared to CVB formula for corn, wheat and barley were
reported (Gowans, 2004). Nevertheless, both the CVB and INRA estimations of NE are widely
accepted as valid equation as they correlated well, and values were 96% equivalent (Gowans, 2004).
76
Table 10.5 Estimated net energy (NE) values of rice (MJ/kg as-fed).
NRC
CVBA
B
INRA mean
Medium grain
Long grain
Remarks
9.61
-
NRC 1998
11.10
11.17
Sauvant et al., 2004
Noblet et al., 2004
11.72
11.83
C
11.74
11.83
INRA-2D
11.79
11.91
11.63
11.76
11.88
11.70
INRA-2 WeanerG
12.03
11.71
H
11.86
11.55
12.06
11.98
INRA-2 GrowerJ
12.31
12.14
INRA-3 GrowerK
12.14
11.99
11.89
11.84
11.50
11.51
INRA-1
INRA-3
E
F
INRA Weaner mean
INRA-3 Weaner
I
INRA Grower mean
L
Overall INRA mean
M
Overall mean
Using calculated DE
Using determined DE value
Using determined DE value
Formulas used to calculate NE are:
A
CVB= 0.108 x DCP + 0.361 x DEE + 0.137 x (Starch – fermentable starch) x Dstarch + 0.124 x Dsugars +
0.96 x (NSP + fermentable starch) x DNSP; 0.96 is the correction factor for disaccharides for rice. Digestible
nutrient contents are expressed as g/100g as-fed and NE is expressed as MJ/kg as-fed.
B
Mean of 3, 4, and 5.
C
INRA-1= 0.121 x DCP + 0.350 x DEE + 0.143 x starch + 0.119 x sugars + 0.086 x Dresidue; digestible
residue= DOM – (DCP + DEE + starch + sugars). Digestible nutrient contents are expressed as g/100g DM and
NE is expressed as MJ/kg DM.
D
INRA-2= 0.703 x DE + 0.066 x EE + 0.020 x starch – 0.041 x CP – 0.041 x CF.
E
INRA-3= 0.73 x ME + 0.005 x EE + 0.015 x starch – 0.028 x CP – 0.041 x CF; Crude nutrients are expressed
as kg/kg DM and NE is expressed as MJ/kg DM.
Abbreviations: D: digestible, CP: crude protein, EE: ether extract, NSP: non-starch polysaccharides, OM:
organic matter, CF: crude fibre.
Digestibility coefficients applied for CVB formula: OM: 0.94, CP: 0.67, EE: 0.34, CF: 0.96, NSP: 0.60, Starch:
1.0, sugars: 1.0.
Digestibility coefficients applied for INRA formula: OM: 0.98, CP: 0.89, EE: 0.24, NSP: 1.0, Starch: 1.0,
sugars: 1.0.
F
Mean of 3, 7 and 8.
G
Calculated using INRA-2 formula and determined DE content with weaner pigs.
H
Calculated using INRA-3 formula and the ME value used in the formula was calculated from the determined
DE content with weaner pigs.
I
Mean of 3, 10 and 11.
J
Calculated using INRA-2 formula and determined DE content with grower pigs.
K
Calculated using INRA-3 formula and the ME value used in the formula was calculated from the determined
DE content with grower pigs.
L
Mean of 2, 6 and 9.
M
Mean of 1 and 12.
In conclusion, this study suggests that the DE and NE content of rice might be higher than originally
thought, and this should be considered for practical diet formulation if rice is being used. The DE and
NE values for rice are also higher than for other cereals used in weaner pig diets such as wheat. In
addition, weaner pigs could extract approximately 0.5 MJ/kg less DE (as-fed) from a given rice
77
compared to grower pigs. These data will be invaluable to nutritionists and feed formulators in the
Australian pig industry who wish to incorporate rice into diets for piglets.
78
11. Implications and Recommendations
Results obtained in this research project have demonstrated that cooked (processed) white rice, either
in medium-grain or long-grain form, included in diets for weanling pigs can be used as a replacement
for wheat without a loss of production in the immediate post—weaning period. The decision to replace
a cereal such as wheat in diets for weanling pigs, therefore, is likely to be one of price differential.
Cooking broken white rice, particularly in medium-grain and waxy rice that have lower amylose levels
than long-grain rice, increases starch digestibility when measured at the end of the small intestine and
reduces shedding of haemolytic E. coli. This could be predicted with accuracy in vitro using a “fast
digestible starch” assay modified for rice in our laboratory. Regardless of the type and variety of rice
used, however, pigs fed cooked white rice partition more digested nutrients into carcass gain than pigs
fed other cereals such as wheat and barley, although the type of proteins fed to pigs will also influence
this. In this regard, the use of commercially manufactured extruded rice plus sources of animal protein
(eg, milk powders, fishmeal, meat and bone meal) appear the best dietary combination for production
purposes. Feeding vegetable (plant) proteins typically increased the weight of the gastrointestinal tract
as a consequence of increased fermentative activity in the large intestine, and reduced bodyweight gain
and FCR. Determination of the energy (DE and NE) values of extruded medium-grain (Amaroo) and
long-grain (Doongara) rice confirmed the superior energy value of these two rice types over existing
cereals used in Australian feeding of pigs, such as wheat.
The effects of feeding cooked white rice on reducing faecal shedding of the bacterium (E. coli)
responsible for causing PWD were generally unchanged, or even exacerbated, when the rice plus
animal protein diets were fed compared to commercially-based diets that were considered a
contributing factor to the incidence of PWD. The extent and duration of faecal shedding of
enterotoxigenic E. coli found in the studies conducted was generally low, and this might have
influenced the capacity of the rice-based diets to exhibit protective effects. It was hypothesised also
that an imbalance in the amounts of carbohydrate versus protein entering the large intestine might have
predisposed the pig to diarrhoea after weaning, due to a change in the types of microbiota and
subsequent production of compounds implicated in non-infectious diarrhoea. The results of Chapter 9
advocate the inclusion of a quantity of slowly or moderately fermentable dietary fibre to extruded ricebased diets consisting of animal protein to ameliorate the diarrhoea that is sometimes observed when
feeding this diet, although in this instance 20 g kg- oat hulls impacted upon digestibility and
production after weaning. Nevertheless, this proposition is consistent with European experiences of
feeding processed rice to piglets after weaning. In this respect, it is feasible that the addition of rice
bran and (or) rice hulls, or possibly the use of brown rice, in diets for piglets after weaning could
achieve similar results. An unfortunate casualty of the drought for this particular project, however, was
the inability to perform an on-farm trial implementing some of the findings and conclusions arising
from this research project.
The major recommendations arising from this project are as follows:
1. Medium-grain rice (variety Amaroo) or long-grain rice (variety Doongara) was identified as
being the most suitable rice cultivars for utilisation in piglet feeds in Australia. Waxy rice, but
not parboiled rice, would also be suitable, but its lower production in Australia at present
would increase its price relative to other rice types and other cereals and hence limit its
usefulness.
2. Processed (extruded) medium-grain rice (variety Amaroo) or long-grain rice (variety
Doongara) are a suitable replacement for cereals currently fed to weanling pigs in Australia
such as wheat and barley. Adoption of processed rice by the pig industry will be
predominately driven by the price differential between processed rice and these alternative
cereals.
3. Starch digestion at the end of the small intestine, as well as the colon, can be predicted
accurately with a “fast digestible starch” assay modified for use in our laboratory. This test
could be used by the rice industry as part of a broader screening process for potentially new
79
4.
5.
6.
7.
varieties of rice suitable for the pig industry, however the assay is capable of being tailored for
use in other species, including man.
Sources of animal protein in diets containing processed (extruded) rice generally cause
superior production after weaning compared to vegetable (plant) sources of protein, although
vegetable proteins showed reduced faecal shedding of haemolytic E. coli compared to animal
sources of protein.
Producers feeding extruded rice-based diets with animal protein sources are encouraged to
include some slowly or moderately fermentable dietary fibre, such as oat hulls, wheat bran and
(or) beet pulp, to ameliorate the diarrhoea that is sometimes observed when feeding this diet.
Future studies should investigate the addition of rice bran and (or) rice hulls, or possibly the
use of brown rice, in diets for piglets after weaning that could accomplish similar results.
The mean digestible energy (DE) content (MJ/kg as-fed) of extruded rice is 15.26 MJ/kg asfed. Medium-grain (Amaroo) rice has a 0.4 MJ/kg higher DE content than the long-grain rice
(Doongara).
Pig producers should use different DE values for pigs of different ages/weights. Weanling pigs
(8 kg) pigs extracted less energy from both extruded rices than grower (55 kg) pigs (up to 0.5
MJ/kg difference). Producers using a net energy (NE) system should use a common value of
11.5 MJ/kg as-fed.
80
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