lactose in weaning pigs

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Please cite this article in press as: ODoherty, J.V., et al., The effects of lactose inclusion and seaweed extract derived from

Laminaria spp. on performance, digestibility of diet components and microbial populations in newly weaned pigs. Anim.

Feed Sci. Technol. (2010), doi:10.1016/j.anifeedsci.2010.03.004

ARTICLE IN PRESSG Model

ANIFEE-12232; No.of Pages 8

Animal Feed Science and Technology xxx (2010) xxxxxx

Contents lists available at ScienceDirect

Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

The effects of lactose inclusion and seaweed extract derived from

Laminaria spp. on performance, digestibility of diet components

and microbial populations in newly weaned pigs

J.V. ODoherty, S. Dillon, S. Figat, J.J. Callan, T. Sweeney

School of Agriculture, Food Science & Veterinary Medicine, College of Life Sciences, Lyons Research Farm,

Newcastle, Co. Dublin, Ireland

a r t i c l e i n f o

Article history:

Received 21 January 2009

Received in revised form 1 March 2010

Accepted 4 March 2010

Available online xxx

Keywords:

Lactose

Laminarin

Fucoidan

Seaweed

Pig

a b s t r a c t

A 22 factorial experiment was conducted to investigate the interactions between two

different lactose (L) levels (150 g/kg vs. 250 g/kg) and seaweed extract (SWE): (0 g/kg vs.

2.8 g/kg; containing laminarin and fucoidan) derived from Laminaria spp. on growth per-

formance, coefficient of total tract apparent digestibility (CTTAD) and faecal microbial

populations in the weanling pig. Two hundred and forty pigs (120 male and 120 female)

were selected after weaning (24 days of age, 7.60.9 kg live weight) and blocked on the

basisof live weightand withineachblock assignedto oneof thefourdietarytreatments.The

pigs were offered the followingdietson an ad libitum basis for 25days: (T1) 150 g L/kg; (T2)

150g L/kg + SWE; (T3) 250g L/kg; (T4) 250 g L/kg + SWE. Pigs offered diets supplemented

with SWE hada higheraveragedailygain (ADG):(322g vs. 281 g,s.e.9.0; P


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ANIFEE-12232; No.of Pages8

2 J.V. ODoherty et al. / Animal Feed Science and Technology xxx (2010) xxxxxx

susceptibility to scouring and disease (Hampson, 1986). In the past, these problems have been managed in a controlled

fashion by the use of prophylactic antibiotics (Williams et al., 2001). Also high concentrations of dietary minerals, e.g. zinc

in the form of zinc oxide, were shown to decrease the incidence of nonspecific post-weaning scours ( Poulsen, 1995) and

to improve growth performance of newly weaned pigs (ODoherty et al., 2005). However, the use of high concentrations of

inorganic minerals has raised some environmental concerns.

Much research exists regarding the benefits of lactose (L) supplementation in terms of improving pig performance

(ODoherty et al., 2005; Pierce et al., 2006). The inclusion of high levels of L in weaning pig diets resulted in a reduction

in intestinal pH and increases in Lactobacilli spp. and short-chain fatty acids and reductions in coliforms (Pierce et al., 2006).

Recently seaweeds and sea weed extracts (SWE) have been researched as an alternative to in-feed antibiotics in pig diets

(Gardiner et al., 2008; Reilly et al., 2008; Gahan et al., 2009). Seaweeds are rich in undigestible polysaccharides and are a

potential source of soluble dietary fibres. The most abundant polysaccharides in brown seaweeds are laminarin, fucoidan

and alginic acid (Zvyagintseva et al., 2003). Laminarin has a chemical structure consisting mainly of a linear-(13)-linkedglucose backbone with some random -(16)-linked glucose side-chains depending on the variety of seaweed ( Brownand Gordon, 2005). Recently, Lynch et al. (2010) showed that laminarin had antimicrobial properties. Deville et al. (2007)

noted that laminarin can influence the adherence and the translocation of bacteria across the epithelial wall and seems to

be a modulator of the intestinal metabolism by its effects on mucus composition, intestinal pH and short-chain fatty acid

production, especially butyrate. Fucoidans are sulfated polysaccharides extracted from the cell wall of various species of

brown seaweeds (Berteau and Mulloy, 2003). Fucoidans have been shown to have antitumor (Zhuang et al., 1995), antiviral

(Lee et al., 2004) and antibacterial (McClure et al., 1992) properties. Thus, fucoidan and laminarin have a wide spectrum of

biological actions which may alleviate the need for high concentrations of L (ODoherty et al., 2004) in antibiotic growth

promoter (AGP)-free diets.

The hypothesis of this study is that the inclusion of a seaweed extract containing laminarin and fucoidan from Lami-

naria spp. will reduce the counts of Escherichia coli, allowing levels of L to be reduced while maintaining post-weaning pig

performance in AGP-free diets.

2. Materials and methods

All procedures described in this experiment were conducted under experimental licence from the Irish Department of

Health in accordance with the Cruelty to Animals Act 1876 and the European Communities (Amendments of the Cruelty to

Animals Act 1976) Regulations, 1994.

2.1. Experimental design and dietary treatments

This experiment was designed as a 22 factorial arrangement of dietary treatments with the respective factors beingtwo different levels of L: low (150g/kg) and high (250g/kg); and the presence (2.8g/kg) or absence of SWE (AGP-free diets).

This experiment was carried out over two periods of 25 days. Two hundred and forty pigs (120 male and 120 female) were

selected after weaning at 24 days of age and blocked on the basis of weaning weight and within each block assigned to one

of four dietary treatments (equal numbers of males and females were allocated to each pen). The pigs in periods 1 and 2

had an initial live weight of 7.2 and 7.8 kg (s.d. 0.9 kg) respectively. During the experiment (d 025) the pigs were offered

the following diets: (T1) 150g L/kg; (T2) 150g L/kg + SWE; (T3) 250 g L/kg; (T4) 250g L/kg + SWE. The SWE was included

at 2.8g/kg and derived from Laminaria digitata. The extract contained both laminarin (112 g/kg), fucoidan (89 g/kg) and

ash (799 g/kg) and was sourced from Bioatlantis Ltd. (Tralee, County Kerry, Ireland). The concentration of SWE used was

deemed as the optimum dietary inclusion level based on results of work from Gahan et al. (2009) and Reilly et al. (2008). The

composition and chemical analysis of the experimental diets are presented in Table 1. Diets were formulated to have similar

concentrations of digestible energy (16 MJ/kg) and standardised ileal digestible lysine (14 g/kg). All amino acid requirements

were met relative to lysine (Close, 1994).

2.2. Animals and management

Pigs were housed in groups of four (15 replicates per treatment, 9 replicates in period 1 and 6 replicates in period 2)

on fully slatted pens (1.68 m1.22 m). House temperature was maintained at 30 C in the first week and then reduced by

2 C per week. Pigs were weighed at the beginning of the experiment (day of weaning= day 0), day 7, day 14 and day 25.

The pigs were fed on an ad libitum basis from a four space feeder with precautions taken to avoid wastage of feed. Water

was available on an ad libitum basis from nipple drinkers. Food remained in the feeders until the time pigs were weighed,

and then was weighed back for the purpose of calculating G:F ratio. During the experiment, feed samples were collected at

time of feeding and were retained for chemical analysis. Multiple fresh faecal samples were collected daily from all pens on

days 1015 and mixed with sodium benzoate and phenylmethylsulfonyl fluoride, in order to stop any bacterial activity and

minimize the effects of post-thawing fermentation on resulting volatile fatty acid (VFA) concentrations. The samples were

thenstoredat20 C untilanalysis forVFA andacid insolubleash (AIA)contents. Chromic oxide (150 mg/kg) wasadded to the

feed at manufacture. Multiple fresh faecal samples were collected from all pens on day 10 and stored in sterile containers
http://dx.doi.org/10.1016/j.anifeedsci.2010.03.004http://dx.doi.org/10.1016/j.anifeedsci.2010.03.004


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J.V. ODoherty et al. / Animal Feed Science and Technology xxx (2010) xxxxxx 3

Table 1

Composition and chemical analysis of experimental starter diets (g/kg as fed).

Treatment

T1 T2 T3 T4

Lactose 150 (g/kg) 150 (g/kg) 250 (g/kg) 250 (g/kg)

Seaweed extract + +

Ingredients (g/kg)

Whey permeate 107 107 215 215Wheat 445 445 349 349

Soya bean meal 160 160 160 160

Whey protein isolate 130 130 130 130

Full fat soya bean 80 80 80 80

Soya oil 65 65 55 55

Lysine HCL 4.5 4.5 3.7 3.7

dl-Methionine 1.6 1.6 1.6 1.6

l-Threonine 2.2 2.2 1.8 1.8

Vitamin and mineral premixa 4 4 4 4

Analysed composition (g/kg)

Dry matter 902 899 913 903

Lactose 148 153 247 249

Crude protein (N*6.25) 204 201 198 196

Neutral detergent fibre 85.9 96.2 85.6 78.5

Ash 43 45 50 50

Gross energy (MJ/kg) 17.7 17.8 17.5 17.3

Lysineb 16 16 16 16

Methionine and cysteineb 8.8 8.8 8.8 8.8

Threonineb 10.4 10.4 10.4 10.4

Laminarin (mg/kg) 0 300 0 300

Fucoidan (mg/kg) 0 236 0 236

Lactofeed 70: Volac International Ltd., Orwel, Royston, SG8 5QX, UK. The chemical analysis is as follows (g/kg unless otherwise stated): DM 955, crude

protein 125, oil 50, ash 90, fibre 10, GE content of 15.5 MJ/kg and a pH of 6.57.a Provided (mg/kgcompletediet): Cu 100, Fe 140, Mn 47,Zn 120, I .6,Se 0.3, retinol 1.8, cholecalciferol 0.025,alpha-tocopherol 67,phytylmenaquinone4,

cyanocobalamin 0.01, riboflavin 2, nicotinic acid 12, pantothenic acid 10, choline chloride 250, thiamine 2, pyridoxine 0.015. Chromium oxide included

at 150 mg/kg complete diet.b Calculated from total amino acid analysis (Sauvant et al., 2004).

(Sarstedt, Wexford, Ireland) on dry ice and transported to the laboratory within 3 h, for the enumeration of E. coli andLactobacilli.

2.3. Faeces scoring and morbidity

Faecal scores (FS), cleanliness scores (CS) and health scores (HS) were recorded daily (09.00 h) in the individual pens,

by the same operator throughout the experiment using a subjective score on a one-point scale ranging from 1 to 4. FS:

1 = firm, 2 = soft, spreads slightly, 3 = very soft, spreads readily, 4 = watery liquid consistency. CS: 1 = clean, free of faecal

contamination, 2 = some indication of faecal contamination, 3 = contaminated with faecal material, 4 = heavily contaminated

with faecal material. Scores were given daily for individual pens and the average faecal score value per pen was given. HS:

1 = no indication of impaired health, lameness or scours, 2 = some indication of impaired health (e.g. slow to respond to

human presence), lameness or scours, 3 = clear indications of impaired health (e.g. shivering), lameness or scours, 4 = serious

signs of impaired health (e.g. showing pain), lameness or scours.

2.4. Laboratory analysis

Both concentrates and faeces were analysed for N, dry matter (DM), ash, GE, neutral detergent fibre (NDF) and

AIA. Following collection, faeces were dried at 100 C for 48 h. The concentrates and dried faeces samples were milled

through a 1mm screen (Christy and Norris hammer mill). The DM of faeces and feed were determined after drying

over night at 103 C. Ash was determined after ignition of a known weight of concentrates or faeces in a muffle fur-

nace (Nabertherm, Bremen, Germany) at 500 C. The N content of both feed and faeces was determined using the LECO

FP 528 instrument (Leco Instruments, U.K Ltd., Newby Road, Hazel Grove, Stockport, SK7 5DA, Cheshire, UK). The NDF

was determined using a Fibertec extraction unit (Tecator, Hoganans, Sweden). The GE of the feed and faeces was deter-

mined using a Parr 1201 oxygen bomb calorimeter (Parr, Moline, IL, USA). The AIA analysis was determined according

to the method of McCarthy et al. (1974). The total laminarin content in the diets was determined using a Megazyme

kit (Megazyme, Bray, Ireland). The fucoidan level in the diets was determined according to the method described

by Usov et al. (2001). Thawed faecal samples were analysed for VFA concentration and profile using the method of


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OConnell et al. (2006). The L concentration of the diets was determined using the method of Birch and Mwangelwa

(1974).

2.5. Microbiology

A 1.0g sample wasremoved from thefaecal sample, serially diluted (1:10)in 9.0ml aliquotsof maximum recovery diluent

(MRD, Oxoid, Basingstoke, UK), and spread plated (0.1 ml aliquots) onto selective agars, as follows. Lactobacillus spp. were

isolated on de Man, Rogosa and Sharp (MRS) agar (Oxoid) with overnight (1824 h) incubation at 37

C in an atmosphereenriched with CO2 5%, as recommended by the manufacturers (Oxoid). The API 50 CHL (BioMerieux, France) kit was used to

confirm suspect Lactobacilli spp. The E. coli species were isolated on MacConkey agar (Oxoid), following aerobic incubation

at 37 C for 1824 h. Suspect colonies were confirmed with API 20E (BioMerieux, France). This API system identifies the

suspect colonies by measuring their ability of produce cytochrome oxidase. Typical colonies of each bacteria on each agar

were counted and the numbers of bacteria expressed per gram of faeces after being serially diluted.

2.6. Statistical analysis

The experimental data was analysed as a 2 2 factorial using the General Linear Model procedure of Statistical Analysis

Systems Institute (1985). Themodels for both performance anddigestibility analysis included themain effects of L level,SWE

inclusion, period and the interaction between L and SWE. Both the performance and CTTAD data were adjusted for initial

live weight by covariance analysis. All the data were checked initially for outliers and normality using the PROC Univariate

procedure of Statistical Analysis Systems Institute (1985). The microbial counts were log transformed prior to statisticalanalysis. The least significant difference test was used to separate main effect means. The probability level that denotes

significance is P


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Table 3

The effect of dietary treatment on the coefficient of total tract apparent digestibility (CTTAD) of organic matter (OM), nitrogen (N), gross energy (GE) and

neutral detergent fibre (NDF) (least square interaction means standard error of mean).

Treatment SEM Significance

T1 T2 T3 T4 L SWE LSWE

150 (g/kg)a 150 (g/kg)a 250 (g/kg)a 250 (g/kg)a

b +b b +b

Digestibility coefficients

c

OM 0.862 0.901 0.898 0.937 0.005 *** *** ns

N 0.821 0.880 0.853 0.907 0.011 * *** ns

GE 0.839 0.889 0.882 0.925 0.007 *** *** ns

NDF 0.375 0.650 0.619 0.746 0.027 *** *** *

Probability of significance: *P


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between L level and SWE on pig health between d 714 (P


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fucoidan could substitute for L in the absence of in-feed antibiotics. However, there was still a synergistic response to the

SWE at high L inclusion levels.

High L levels were found to improve the CTTAD of diet components in the current study. The improved N digestibility

observed indicates that the high L level determines conditions in the gut conducive to proteolytic activity. The microbial

fermentation of L to lactic acid creates an environment ideal for the growth of lactic acid bacteria (Pollmann et al., 1980). The

fermentation of the feed may affect the digestibility of the feed components. The increased NDF disappearance observed in

the current study may be the result of increased bacterial fermentation due to the increased L inclusion.

Despite the increase in the Lactobacilli population with high L diets, there was no dietary effect on VFA concentration or

profiles. Faecal VFA concentrations are the outcome of production, absorption and degradation within the gut. Faecal VFA

concentrations may not be a totally accurate way to demonstrate fermentation intensity in the large intestine.

5. Conclusion

Theinclusion of either a highdietary concentration of L or a laminarin-fucoidanextractimproved performance parameters

of weaner pigs, increased the CTTAD of diet components and reduced the counts of E. coli in the faeces. Further studies will

explore the potential of these diets to prevent the negative impact of pathogen challenges.

Acknowledgements

Funding for this research was provided under the National Development Plan through the Research Stimulus Fund

administered by the Irish Department of Agriculture, Fisheries and Food.

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