Animal Science 1996, 62: 131-144C 1996 British Society of Animal Science

1357-7298/96/56250131S20-00

Maintenance of villous height and crypt depth in piglets byproviding continuous nutrition after weaning

J. R. Piuske't, I. H. Williams1 and F. X. Aherne2

1 Animal Science, Faculty of Agriculture, University of Western Australia, Nedlands, WA 6009, Australia2Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton AB T6G 2P5, Canada

Abstract

Thirty-two piglets weaned at 28 days of age were used to test the hypothesis that maintenance of nutrition afterweaning would prevent the normal decline in villous height and increase in crypt depth and hence preserve thestructure and function of the small intestine. Piglets were allocated to one of four treatments at weaning: (1)control group killed at weaning; (2) piglets offered a dry starter diet ad libitum; (3) piglets offered ewes' fresh milk;and (4) piglets offered ewes' fresh milk plus 20 g t-glutamine per I. Piglets in treatments (3) and (4) were offeredewes' fresh milk every 2 h in a feeding schedule that increased from 1-2 I per piglet on the 1st day after weaning to2-4 I on days 4 and 5. On the 5th day all piglets were killed and samples of small intestine were taken forhistological and biochemical examination. Feeding ewes' milk or ewes' milk plus 20 g L-glutamine per I maintained(P > 0-05) villous height and crypt depth compared with piglets killed at weaning. In contrast, piglets given a drystarter diet had shorter villi (P < 0-001), deeper crypts (P < 0-001), and proportionately 0-21 to 0-28 less protein(P>0-05) in their intestinal mucosa. Piglets given the starter diet proportionately grew from 0-49 to 0-62 moreslowly (P < 0-01), ate the same amount of dry matter (DM; P > 0-05), but consumed proportionately 0-30 lessenergy (P < 0-001) than their counterparts given the milk diets. No treatment differences in the specific activity oflactase and sucrase were observed (P>0-05). Significant correlations existed between voluntary food intake andvillous height at the proximal jejunum for piglets given the starter diet and ewes' milk (P < 0-05 and P = 0-073,respectively). In turn, villous height was significantly correlated (r = 0-78 to 0-87, P < 0-05) with the rate of body-weight gain after weaning in these two groups. For piglets offered ewes' milk plus glutamine, an increase in DMintake was associated only with increases in crypt depth (P < 0-01). These data show that the structure andfunction of the small intestine can be preserved when a milk diet is given after weaning, and suggest an associationbetween food intake and villous height in determining post-weaning weight gain.

Keywords: continuous nutrition, crypt depth, glutamine, piglets, villous height.

IntroductionThe small intestine of the newly weaned pigletgenerally undergoes a reduction in villous heightand an increase in crypt depth (Gay, 1976; Gay,Barker and Moore, 1976; Hampson, 1986a and b;Miller, James, Smith and Bourne, 1986; Cera, Mahan,Cross, Reinhart and Whitmoyer, 1988; Kelly, Smythand McCracken, 1991b and c). These changes havegenerally been associated with reductions in the

t Present address: School of Veterinary Studies, MurdochUniversity, Murdoch WA 6150, Australia.

specific activity of disaccharidase enzymes (Gay etal., 1976; Hampson and Kidder, 1986; Miller et al.,1986) and a reduced capacity of the gut to absorbxylose (Miller, Newby, Stokes and Bourne, 1984;Hampson and Kidder, 1986; Hampson and Smith,1986) and alanine (Smith, 1984; Miller et al., 1986).The net effect of these alterations is thought toreduce the digestive and absorptive function of thegut (Gay et al., 1976; Hampson, 1983). From aproduction perspective these changes are importantbecause they may reduce growth and extend thetime pigs take to reach market weight.

131

132 Pluske, Williams and Aherne

Numerous factors have been proposed to account forthe apparent decrease in digestive and absorptivecapacity of the small intestine after weaning (seeHampson, 1987; McCracken and Kelly, 1993). Inmany studies the possible contribution that foodintake per se may play in the maintenance of gutintegrity has largely been ignored, or considered notto be of major aetiological importance (e.g.Hampson, 1983), and only Kelly et al. (1991c) haveexamined effects of a controlled level of food intakeon gut structure and function. Some workers(Robertson, Clark and Bruce, 1985; Bark, Crenshawand Leibbrandt, 1986) have shown that after weaningthere is a period of temporary starvation duringwhich piglets fail to eat sufficient food to cover theirmaintenance energy requirement. McCracken (1984)and McCracken and Kelly (1984) suggested thatvillous atrophy and reductions in digestive enzymeactivity after weaning may be related more to thelack of a continuous supply of substrate than to anyantigenicity of the diet or inherently low levels ofdisaccharidase activity. In rodents and miniaturepiglets a period of food restriction (Steiner, Bourges,Freedman and Gray, 1968; McManus andIsselbacher, 1970; Altmann, 1972), or exclusion ofnutrients from the gut when animals were fedparenterally (McNeill and Hamilton, 1971; Shulman,Fiorotto, Sheng and Garza, 1984; Goldstein,Hebiguchi, Luk, Taqi, Guilarte, Franklin, Niemiecand Dudgeon, 1985; Castillo, Feng, Stevenson,Kerner and Kwong, 1990), resulted in villous atrophyand decreases in mucosal protein content anddigestive enzyme activity. These findings suggest adirect role for the presence of nutrients in the lumenfor the maintenance of gut structure and functionafter weaning.

Gut structure and function after weaning may alsobe limited by availability of the amino acid L-glutamine. Glutamine is the principal fuel used bythe gut epithelium (Windmueller, 1982), is presentin high concentrations in sow's milk around thetime of weaning (Wu and Knabe, 1994), andnumerous studies have demonstrated thatglutamine is required by villous enterocytes tosupport metabolism, and the structure and functionof the gut (Souba, 1991, 1992 and 1993). Provision oforal glutamine stimulates net uptake of glutamineby enhancing brush-border transport rates (Salloum,Souba, Fernandez and Stevens, 1990), and supportsmucosal growth by stimulating the activity ofglutaminase (Klimberg, Salloum, Kasper, Plumley,Dolson, Hautamaki, Mendenhall, Bova, Bland,Copeland and Souba, 1990; Salloum, Souba,Klimberg, Plumley, Dolson, Bland and Copeland,1989). At a time when the piglet's supply ofmaternal glutamine disappears, supplementation ofdiets with synthetic glutamine offers a means of

enhancing the structure and function of the gut afterweaning.

The hypothesis tested in this study was thatmaintenance of voluntary food intake after weaningwould preserve the structure and function, andtherefore maintain the digestive and absorptivecapacity of the small intestine when piglets werekilled 5 days later. Furthermore, we proposed thatsupplementation of L-glutamine in the milk dietwould augment the digestive and absorptivecapacity of the gut following weaning. An abstract ofthis work has been presented previously (Pluske,Williams and Aherne, 1991).

Material and methodsAnimals and housingThirty-two piglets from four primiparous gilts (LargeWhite X Landrace) bred at Medina Research Centre,Medina, Western Australia, were used in the study.Gilts were transported to the University of WesternAustralia on about day 75 of gestation and werehoused in environmentally controlled roomsmaintained at 19°C. After farrowing, litter size wasstandardized to eight piglets per litter. Routinemanagement of piglets included teeth clipping, taildocking and an injection (200 mg i.m.) of irondextran. Piglets were housed with their mothersduring lactation in standard farrowingaccommodation.

At an average age of 28 (s.e. 0-4) days when theyweighed 8-9 (s.e. 0-34) kg, piglets were transported300 m to an air-conditioned experimental room thatcontained 12 individual stainless-steel pensarranged in four banks of three pens each. Eachpen had a measured floor area of 0-45 m2. Ambienttemperature was maintained at 25-6 (s.e. 0-44)°Cwith relative humidity of 0-37 (s.e. 0-07) for the 5-day duration of the study. Galvanized steel trayswere placed under each pen for the collection ofspilt food, faeces and urine. Two galvanisedtroughs were placed in each pen, one for food andthe other for fresh water.

Experimental treatmentsThe experiment was a completely randomizedblock design. Piglets were allocated randomly onthe basis of litter, sex and live weight to one offour treatment groups as follows: (1) sow-reared,control group killed on the day of weaning (SR)(no. = 8); (2) piglets offered a pelleted starter diet(starter) (no. = 8); (3) piglets offered ewes' freshmilk (EM) (no. = 8); (4) piglets offered ewes'fresh milk plus 20 g L-glutamine per 1 (EM+Gln)(no. = 8).

Gut morphology and nutrition in piglets 133

Table 1 Composition (g/kg) and analysis of the starter diet

ingredientsWheat 276Barley 200Oat groats 150Skim-milk powder 250Fish meal 70Vegetable oil 30Limestone 7Dibasic calcium phosphate 8L-lysine HC1 2DL-methionine 1i.-threonine 1Vitamin and mineral pre-mixt 5

CompositionDry matter} 889Crude protein (g N X 6-25)} 213Ether extract} 128Ash} 76Digestible energy (MJ/kg)§ 15-0Total lysine§ ' 14Calcium§ 12Phosphorus§ 8

t Provided the following nutrients (per kg of air-dry diet):vitamins: retinol 1500 ug, cholecalciferol 20 ug, a-tocopherol10 mg, riboflavin2-5 mg,niacinl6 mg,pantothenicacidlO mg,pyridoxine 2 mg, choline chloride 140 mg, cyanocobalamin10 ug; minerals: NaC13-5 g, Cu 10 mg, Zn 100 mg, Mn20 mg,Fe 60 mg, Co 0-2 mg.} Determined by proximate analysis of diet.§ Calculated from ingredients.

Diets and feeding regimenPiglets were offered ewes' fresh milk every 2 h for 5days in a feeding schedule that increased from 1-2 1per pig on the 1st day after weaning (i.e. 100 ml perfeed) to 1-4 1 on day 2, 1-81 on day 3, and to 24 1 ofewes' milk on days 4 and 5. In a previous studyusing the same genotype, Pluske and Williams (1988)determined that 25-day-old sucking piglets drankabout 50 ml milk per suckling. Since the sow suckles21 to 24 time per day (Pluske and Williams, 1988), itwas considered that 1-2 1 offered on the 1st day afterweaning equated to the milk intake of these pigletsprior to weaning and would ensure a reasonable rateof live-weight gain. For piglets offered ewes' milkplus glutamine, L-glutamine was added to the milkat a rate of 20 g/1 milk just prior to feeding, andshaken thoroughly to ensure that the glutaminedissolved fully.

The pelleted starter diet (Table 1) was fed ad libitumso that proportionately at least 0-2 of the previousday's intake was always in front of the piglet. Onsome occasions a pig was observed to urinate ordefecate in its food trough. When this occurred thefood was removed, weighed and an equivalentamount of fresh pellets was replaced in the feeder.

Experimental procedureEach day at 14.00 h all piglets were weighed and, forpiglets given the starter diet, any food that had spiltinto the collection trays was collected and the weightrecorded. For piglets given the milk diets every 2 h,the amount offered was recorded and, after 10 to15 min, troughs were removed from each pen andany residual milk was weighed in tared containersand recorded. Between feedings all troughs werewashed and scrubbed thoroughly with hot water.

Post-mortem procedureFood was withdrawn from piglets 2 h prior toslaughter on the afternoon of the 5th day followingweaning. Sow-reared piglets were removed from thesow about 1 h prior to slaughter on the day ofweaning. Weaned piglets were removed from theirpens and restrained on a surgical table in a dorsalposition. Following the withdrawal of 5 ml bloodfrom the heart, piglets were killed by an injection ofsodium pentobarbitone. The abdomen was openedimmediately, from the sternum to the pubis, and theentire gastro-intestinal tract was removed. Twosegments of small intestine (= 10 cm in length), eachat distances of proportionately 0-25, 0-5 and 0-75along the gut from the gastric pylorus to the ileo-caecal valve, were clamped with haemostats. Onesegment at each site was filled by syringe with 5 to10 ml ice-cold, 10% phosphate-buffered formalin (pH7-4) for subsequent histological examination. Theadjacent segment at each site was filled with anequivalent volume of ice-cold, 0-01 mol/1 phosphate-buffered saline (pH 7-4), excised, wrappedimmediately in aluminium foil, and then immersedin liquid nitrogen for subsequent disaccharidase andmucosal protein determinations. Total processingtime from killing to obtaining gut samples was about20 min. In the text, sites at 0-25, 0-5 and 0-75 are usedinterchangeably with their approximate positionalong the small intestine, i.e. proximal jejunum, midjejunum and distal ileum, respectively, or arereferred to as sites 1 to 3.

HistologyAfter fixation for several days, ring-shaped lengthsof small intestine from all three sites were excised,dehydrated, and embedded in paraffin wax. Fromeach of these, six transverse sections (4 to 6 urn) werecut, stained with haematoxylin and eosin, andexamined under a light microscope. Measurementsof villous height and crypt depth were taken onlyfrom sections where the plane of section ranvertically from the tip of the villus to the base of anadjacent crypt. From each section a calibratedeyepiece graticule was used to measure 10 of thetallest well oriented villi from tip to crypt mouth,and 10 associated crypts from crypt mouth to base(after Hampson, Fu and Smith, 1988).

134 Pluske, Williams and Aherne

Digestive enzyme determinationsMethods used for the determination of lactase ((3-galactosidase; EC 3.2.1.23) and sucrase (sucrose-a-glucosidase; EC 3.2.1.48) activity were adaptedfrom techniques described by Kidder and Manners(1980) and Hampson (1983). Approximately 200 mgof mucosa was gently scraped from partiallythawed lengths of small intestine using a spatula.The mucosa was then placed into a plastic vialcontaining 40 ml of distilled water to make a 1/200(w/v) mucosal homogenate. Followinghomogenization for 30 s in a polytron (Model CH-6010, Kinematica, Kriens-Luzern, Switzerland) atsetting no. 10, the contents were transferred to aplastic centrifuge tube and spun at 3000 r.p.m. for15 min. The supernatant was decanted, and 5 mlwas kept in a plastic tube at 4°C for analysis oflactase and sucrase activity. A further 5 ml wasretained for determination of mucosal proteinconcentration.

Substrate concentrations and incubation conditionsused for lactase and sucrase determinations werethe same as those used by Kidder and Manners(1980) and Kelly (1985). Briefly, equal quantities ofsubstrate and 0-2 mol/1 phosphate buffer weremixed thoroughly in a 50 ml volumetric flask.From this solution 200 j_il was dispensed into cleanglass test tubes, a further 100 JJ.1 supernatant ordistilled water (blank) was added, and then alltubes were incubated in a water bath set at 37°Cfor exactly 30 min. After 2 min the reaction wasterminated by submerging the tubes in boilingwater for 2 min. The free glucose liberated by theaction of lactase and sucrase was then determinedusing the glucose-6-phosphate dehydrogenase (EC1.1.1.49)-hexokinase (EC 2.7.1.1) assay (Boehringer-Mannheim Biochemica, Germany) for the UVdetermination of glucose, as adapted by Kelly(1985).

Mucosal protein concentrationThe protein content of the mucosal homogenate wasdetermined using the method described by Gornall,Bardawill and David (1949), as adapted by Kelly(1985).

AnalysisSamples of ewes' fresh milk were collected in plasticvials and frozen at -20°C for analysis of fat, proteinand total solids. Milk was analysed using aMilkoScan® (Foss Electric, Denmark) automatedanalyser. The proportion of fat, protein and totalsolids contained in ewes' milk (no. = 36) was 73 (s.e.4-0), 48 (s.e. 1-6) and 185 (s.e. 3-5) g/kg respectively.The calculated gross energy (GE) content of the milkwas 4-65MJ/kg, or 25-14 MJ/kg dry matter (DM),according to the equation of Perrin (1958).

Levels of free plasma glutamine were determinedaccording to the method of Jones and Gilligan (1983)using a Varian 5000 high-performance liquidchromatograph and a Varian Flourichom detector.The internal standard for glutamine was 0-0913 gcx-amino-guanidino-propionic acid dissolved in100 ml double-deioinized water to yield aconcentration of 5 mmol/1. Plasma urea and alkalinephosphatase were measured using a biochemicalanalyser (COBAS MIRA, Roche Diagnostica,Switzerland).

Statistical analysis and presentation of resultsAll data were subjected to one-way analysis ofvariance for treatment effects using SYSTAT®(Wilkinson, 1990). As this experiment wasconducted over two time periods owing to theavailability of piglets, time was included as anindependent variable in the initial analysis ofvariance. The effect of time was not significant forany of the variables measured, so the data were re-analysed with treatment group being the onlyindependent variable. Pairwise comparisonsbetween treatment means were made usingFisher's-protected least significant difference (LSD)procedure (Maindonald, 1992). Where appropriate,simple linear regressions were performed usingSYSTAT® (Wilkinson, 1990).

The weight of the empty body was determined bymultiplying the starting live weight of each piglet bythe proportion of the carcass that was empty body inthe piglets killed at weaning (Noblet and Etienne,1987). This was 981 (s.e. 6-8) g/kg (CV = 0-0069).Empty body-weight gain was calculated, therefore,as the difference beween the recorded weight of theempty body at slaughter 5 days after weaning andthe estimated weight of the empty body at weaning.The protein content of the mucosa and the specificenzyme activity are presented as the mean of allthree sites (0-25, 0-5 and 0-75) along the smallintestine.

ResultsAll piglets offered the milk diets began drinkingwithin 8 h of weaning, and became accustomed tothe 2-hourly feeding schedule very quickly. Onceconditioned to the procedure, piglets eagerlyawaited the arrival of their food. When the troughwas placed in the pen, piglets drank their milkvoraciously. Most animals consumed all their milkwithin 1 to 3 min and there was no spillage. Thehealth of all piglets during the experiment wasexcellent, and no diarrhoea was observed excepton the morning of the final day when severalpiglets given the milk diets developed a very mild

Gut morphology and nutrition in piglets

Table 2 Villous height and crypt depth of piglets killed at weaning or 5 days later

135

Villous height (|im)

Mean:];Crvpt depth (|jm)

Mean}:

Proportion ofintestine

0-250-500-75

0-250-500-75

SR

550''464''264426b

116a

124-'97''

112b

Treatmentt

Starter

330b

316b

261299"

182C

179b

172C

178"

EM

569"428a

258418b

137b

137"123b

132b

EM + Gin

561"444"308438b

139b

134"113"b

129b

s.e.d.

53-745-930-133-2

11-812112-510-7

Significance

***

***

************

1 b c Within rows, means not followed by a common superscript differ significantly.t SR: piglets killed at weaning; Starter: piglets given dry starter diet ad libitum; EM: piglets given ewes ' fresh milk; EM + Gin:piglets given ewes' fresh milk plus 20 g/1 L-glufamine.X Mean of all three sites along the small intestine.

Villous height and crypt deptliFeeding ewes' milk or ewes' milk plus glutaminemaintained (P > 0-05) villous height at sites 0-25 and0-5 along the small intestine compared with pigletskilled at weaning. In contrast, piglets given thestarter diet had villi that were proportionately from0-26 to 0-72 shorter (P < 0-01) at both sites than allother treatment groups. Villous height at the distalileum was similar (P > 0-05) between treatments.Mean villous height along the length of the smallintestine was proportionately 0-34 to 0-45 lower(P < 0-001) in piglets given the starter diet than it wasin piglets killed either at weaning or given both milkdiets. Feeding ewes' milk or ewes' milk plusglutamine did not reduce mean villous height incomparison with piglets killed at weaning. Villousheight decreased (P < 0-001) from the proximal to thedistal part of the small intestine (Table 2).

Crypt depth at the proximal jejunum had increasedin all treatments by the time piglets were killed 5days after weaning. This increase was less for pigletsgiven ewes' milk or ewes' milk plus glutamine(P < 0-001) and was greatest for piglets offered thestarter diet (P < 0-001). Piglets given the starter dietshowed a 0-32 proportional increase in crypt depth atthe proximal jejunum in comparison with bothgroups of piglets given milk (182 um v. an average of138 urn, P< 0-001). At the mid jejunum and distalileum, crypt depth was similar in piglets killed atweaning and those given ewes' milk or ewes' milkplus glutamine (P > 0-05), but was always greater inpiglets given the starter diet (P < 0-001). Mean cryptdepth in piglets given ewes' milk or ewes' milk plusglutamine was, on average, 16 urn deeper (P > 0-05)than in piglets killed at weaning whereas, in pigletsgiven the starter diet, mean crypt depth was

proportionately 0-35 to 0-59 deeper than in othergroups (P < 0-001). There was a trend (P = 0-128) forcrypt depth to decrease from the proximal to thedistal part of the small intestine (Table 2).

Piglet performanceEmpty body-weight gain of piglets given the starterdiet after weaning was 252 g/day. This wasproportionately 0-49 less (P = 0-023) than pigletsoffered ewes' milk and 0-62 less (P = 0-006) thanpiglets given ewes' milk plus glutamine. Voluntaryfood intake between treatments was similar(P > 0-05), however piglets given both milk dietsconsumed proportionately 0-30 more energy (7-45 v.5-7 MJ GE per day, P = 0-006) than those given thestarter diet (Table 3). The variation in voluntary foodintake of piglets given the starter diet was twice thatof animals given the milk diets (CV: 0-25 v. 0-12).

Piglets offered ewes' milk or ewes' milk plusglutamine were more efficient in converting DM intoempty body gain than piglets given the starter diet(0-8 and 0-7 v. 1-3, P < 0-001). However the inclusionof one piglet in the initial data set for starter-fedpiglets, whose stomach contained = 750 g of food atslaughter but grew only 280 g after weaning,resulted in an increase in food conversion ratio (FCR)from 1-3 to 1-8. This pig was considered aberrant andwas therefore removed from the calculations for FCRand energetic efficiency of body gain. The energeticcost of 1 g of empty body-weight gain was around19 kj digestible energy, and was similar (P > 0-05) forall treatments (Table 3).

Pattern of voluntary food intake after weaningOn the 1st day after weaning piglets eating thestarter diet consumed less DM (129 « 184 av. 184 g,

136 Pluske, Williams and Aherne

Table 3 Performance of piglets after weaning

Live weight (kg)weaningafter 5 days

Empty body weight (kg)weaningafter 5 days

Daily gain (g/day)live weightempty body weight

Voluntary food intake(g dry matter per day)

Energy intake (MJ GE per day)Food conversion ratio(g dry matter : g EBWG)$

Energy cost/g EBWGt (kj DE per g)

SR

8-6

8-5

Treatmentt

Starter

8-9104

8-710-0

307a

252a

3205-7a

1-318-0

EM

9-111-3

8-910-8

435b

375b

2957.4b

0-820-2

EM + Gin

8-9111

8-810-8

443b

407b

2977-5b

0-718-2

s.e.d.

0-870-85

0-850-83

47-650-6

2740-55

0450-96

Significance

***

***

a b Within rows, means not followed by a common superscript differ significantly.t See Table 2.^ EBWG: empty body-weight gain.

P = 0-306) than piglets given the milk diets. DMintake between treatment groups did not differ(P > 0-05) on any day after weaning. However on the5th day piglets offered both milk diets consumed, onaverage, 82 g DM less (P = 0-142) than piglets giventhe pelleted diet (Figure 1).

Relationships beween gut morphology, voluntary foodintake and the rate of gainSignificant within-treatment relationships existedbetween total DM intake and villous height at the

500 1

60400 -

.S 300 "

D

200 -

100 J

1 2 3 4 5Day after weaning

Figure 1 The daily pattern of dry matter intake (g) in pigletsoffered either a pelleted starter diet (O), ewes' liquid milk (A),or ewes' liquid milk fortified with 20 g/1 L-glutamine (•) for5 days after weaning. Values are mean ± s.e. for eight pigletsper treatment.

proximal jejunum in piglets given the starter diet (r =0-78, P = 0-039) and ewes' milk (r = 0-65, P = 0-073),but were not found in animals given ewes' milksupplemented with 20 g/1 glutamine. Crypt depthalong the small intestine of piglets given the starterdiet and ewes' milk showed no relationship withtotal DM intake (P > 0-05). However for piglets givenewes' milk plus glutamine, an increase in glutamineintake explained a significant proportion of thevariation in crypt depth at the proximal (r = 0-71, P =0-051) and mid (r = 0-92, P < 0-01) jejunum. As aconsequence, DM intake was highly correlated tomean crypt depth (r = 0-88, P = 0-004) (Table 4).

Villous height at the proximal jejunum wassignificantly correlated with total empty body-weight gain in piglets given the starter diet (r = 0-78,P = 0-024) and ewes' milk (r = 0-87, P = 0-005) afterweaning. In contrast, no relationships existedbetween these parameters in piglets offered ewes'milk plus glutamine. No significant correlations(P > 0-05) were recorded between crypt depth andrate of gain in any treatment groups. However atsites proportionately 0-5 (P = 0-058) and 0-75 (P =0-073) along the gut, an increase in empty body-weight gain was associated with an increase in cryptdepth for piglets given ewes' milk plus glutamine(Table 5).

Digestive enzyme activityThe mean protein content of the mucosa of the smallintestine did not differ between treatments (P > 005)nor show any difference along the small intestine(P > 0-05) at the sites sampled. However the

Gut morphology and nutrition in piglets 137

concentration of mucosal protein in piglets given thestarter diet was from 0-21 to 0-28 lower (P = 0-574)than in the other groups. Similarly, the specificactivity of lactase arid sucrase did not differ betweentreatments (P > 0-05; Table 6). The lack of statisticalsignificance between treatment groups was due to

Table 4 Correlation coefficients between total dry matter in take (g)and villous height and crypt depth (\xm) when measured 5 days afterzveaning

Villous height

Mean}:Crvpt depth

Meant

Proportionof

intestine

0-250-500-75

0-250-500-75

Starter

0-78*0-360140-420-280-560-370-35

Treatmentt

EM

0-65§0-480-200-65§0-580-490-550-56

EM + Gin

0-100-200-510-140-71*0-92**0-530-88**

t See Table 2.X Mean of all three sites along the small intestine.§ P= 0-073.

Table 5 Correlation coefficients between total empty body-weightgain (g) and villous height and crypt depth (\xm) when measured 5days after weaning

Variable

Villous height

Mean:}Crvpt depth

Mean}

Proportionnf

intestine

0-250-500-75

0-250-500-75

Starter

0-78*0-68§0-420-6910-280-510-490-44

Treatmentt

EM

0-87**0-370-140-71*0-140-300-450-39

EM + Gin

0-100-530-660-360-410-69§0-660-63

t See Table 2.% Mean of all three sites along the small intestine.§ P = 0-062.I P = 0-058.

the large variation in protein content and digestiveenzyme activity that occurred. For example, the CVfor the mean specific activity of lactase in pigletsgiven ewes' fresh milk ranged from 0-36 to 1-09,whilst that of sucrase for piglets offered the starterdiet varied from 0-49 to 1-23.

The specific activity of lactase declined (P = 0-060)from the proximal to the distal part of the smallintestine. Values decreased from 95 to 68 to 38 umol/min per g mucosal protein at sites 1 to 3 along thesmall intestine. Sucrase activity, however, showed noproximal to distal decline along the gut (P = 0-371).

Plasma metabolitesLevels of plasma glutamine were similar (P > 005)between dietary treatments when measured 5 daysafter weaning, although the level of circulatingglutamine was from 0-84 to 1-15 higher (P < 0-01) inthese groups than when piglets were killed on theday of weaning. Plasma urea was three times higher(P < 0-001) in piglets offered ewes' milk plusglutamine than that recorded in piglets killed eitherat weaning or those given a solid diet after weaning.A difference of 3-8 mmol/1 urea (P < 0-001) existedbetween piglets given ewes' milk plus glutamine and

Table 6 Protein content of the mucosa and the specific activity oflactase (EC 3.2.1.23) and sucrase (EC 3.2.1.48) of piglets killed atweaning or 5 days later

Treatmentt

SR Starter EM EM + Gin s.e.d.

Mucosal proteincontent

(mg/g mucosa)}: 134 106 150 148 29-4Lactase activity

(Hmol/min per gprotein)}: 67 64 76 60 25-6

Sucrase activity((imol/min per g

protein)}: 63 78 58 59 24-0

t See Table 2.X Mean of all three sites along the small intestine.

Table 7 The concentration of me

Glutamine (nmol/1)Urea (mmol/1)Alkaline phosphatase (U/l)

tabolites in the plasma

SR

382a

2-2a

428

of piglets killed at weaning

Treatmentt

Starter

705b

2-0a

326

EM

822b

2-6b

310

or 5 days later

EM + Gin

803b

6-4c

335

s.e.d.

95-70-48

26-0

Significance

a'b'c Within rows, means not followed by a common superscript differ significantly,t See Table 2.

138 Pluske, Williams and Aherne

those given ewes' milk alone. Urea levels in pigletsgiven ewes' milk only was elevated, proportionatelyon average, 0-24 (P < 0-001) above that found ineither piglets killed at weaning or those given thestarter diet. There was no treatment difference(P > 0-05) in the plasma concentration of alkalinephosphatase (Table 7).

DiscussionMaintenance of villous height and crypt depth afterweaningFeeding ewes' milk or ewes' milk plus 20 g/1 L-glutamine to piglets every 2 h for 5 days afterweaning preserved villous height and crypt depthalong the entire length of the small intestinecompared with piglets killed at weaning. Thissupports our hypothesis that maintaining nutritionafter weaning at levels approximating pre-weaningintake prevents villous atrophy and an increase incrypt depth. Supplementation of ewes' fresh milkwith glutamine did not enhance the structure andfunction of the small intestine as it was similar to thatin piglets receiving ewes' milk alone. This failed tosupport our proposition that addition of glutamineto the diet would augment the digestive andabsorptive function of the gut after weaning.However, the significant correlation observedbetween DM intake and crypt depth (Table 5)suggests that glutamine may be an importantsubstrate for cell renewal in the small intestine, andwill be discussed later.

The preservation of villous height and crypt depththat occurred when piglets were given milk or milkplus glutamine suggests that the balance betweencell loss from the villi and cell production in thecrypts was not altered after weaning. In contrast,piglets given the starter diet ad libitum had villi thatwere shorter and crypts deeper by the 5th day afterweaning compared with piglets given both of themilk diets. An increase in crypt depth is compatiblewith an increase in crypt-cell production rate and anoverall stimulation of cell turn-over in the smallintestine (Al-Mukhtar, Polak, Bloom and Wright,1982) that has generally been associated with areduced digestive and absorptive capacity (Gay et ah,1976; Hampson, 1983). In the present experimentthere was a marked difference in the rate of emptybody-weight gain of piglets given the starter diet andthose given both milk diets (252 v. an average of391 g/day).

Despite consuming an equivalent amount of DM,piglets given both milk diets ingested significantlymore energy than those offered the solid diet. Adecrease in energy intake decreases both fasting heatproduction (Koong, Nienaber, Pekas and Yen, 1982)

and the quantity of mucosa in the small intestine(Pekas, 1986a and b). The amount of mucosa in thesmall intestine is highly correlated with fasting heatproduction of the growing pig (Pekas, 1986b). Sinceheat production is associated with protein synthesis(Webster, 1980 and 1981), and this is most active inthe gut epithelium due to the high rate of cell turn-over (Cheng and Leblond, 1974; Pekas and Wray,1991), it is likely that an increase in energy intakewill supply more substrate for mucosal growth(Pekas, 1986b). In this regard the likely contributionof the 0-3 proportional difference in energy intake toboth the maintenance of gut structure and functionand the disparity in growth between treatmentsremains unknown. This will be examined in our nextexperiment (Pluske, Williams and Aherne, 1996).

Although we have clearly demonstrated thatmucosal integrity can be maintained after weaningwe do not know the importance of milk per se. In thisstudy we used ewes' fresh milk because of (a) itssimilarity in gross composition to sows' milk, and (b)we reasoned that offering weaned piglets a familiarnutritional source (i.e. milk) would help overcomethe interruption to food intake that normally occursimmediately after weaning. We offered ewes' milkthat was collected fresh each day and offered it topiglets in an unhomogenized and unpasteurizedform. Piglets adapted readily to this diet andconsumed large quantities within hours of weaningwhereas piglets often take a number of days toconsume similar quantities of a dry diet. However,milk is known to contain a variety of biologicallyactive compounds (Cera, Mahan and Simmen, 1987;Jaeger, Lamar, Bottoms and Cline, 1987; Koldovsky,1989; King and Kelly, 1990; Kelly, King, Brown andMcFadyen, 1991d; Kelly, King, McFadyen andTravis, 1991a; Kelly, McFadyen, King and Morgan,1992) that are likely to influence intestinaldifferentiation and cell turn-over. Some workers (e.g.Hampson, 1986b) have also commented that thewithdrawal of specific factor(s) associated with theconsumption of sows' milk might cause decreases invillous height after weaning. Therefore it is notpossible to discern whether villous height and cryptdepth were attenuated by the presence of thesecompounds in milk or if it was attributable to theactual amount of food the piglets' consumed.Delineation of these factors forms the basis of ournext experiment (Pluske et ah, 1996).

Piglet peformance and relationship to gut structureDM intakes and growth rates of piglets given allthree diets were excellent. Piglets offered ewes' milkor ewes' milk plus glutamine averaged between 375and 407 g empty body-weight gain per day, andconverted milk DM to empty body-weight gain withan efficiency similar to piglets sucking the sow

Gut morphology and nutrition in piglets 139

(Lucas and Lodge, 1961; Noblet and Etienne, 1987).These data exemplify the rapid growth potential ofthe young piglet, and support impressive growthrates reported by other workers (e.g. Hodge, 1974;Williams, 1976; Harrell, Thomas and Boyd, 1993)when piglets were offered milk diets. It wasapparent, however, that piglets given the starter dietwere limited in their ability to ingest an equivalentamount of energy as those piglets given both milkdiets despite being offered ad libitum. Some authorshave attributed this phenomenon to the weanedpiglet having a limited 'gut capacity' (Campbell,1989; Cranwell and Moughan, 1989; Tybirk, 1989),although the factors influencing this are not welldefined. Nevertheless, we and others (Kelly et al,1991b and c; Pluske et al, 1996) have been unable toconfirm associations between a 'reduced' digestiveand absorptive capacity of the small intestine withsignificant decreases in the activities of lactase andsucrase or the amount of xylose absorbed, suggestingthat measurement of these disaccharidases may bean inappropriate measure of digestive andabsorptive function of the gut.

The reduced growth rate of piglets offered thepelleted starter diet was associated with villousshortening and an increase in crypt depth but whatcannot be deduced from our data is whether or notthese changes were caused by the type of food per seor the fact that piglets consumed less energy.However, a positive linear relationship between foodintake and villous height was observed and, in turn,between villous height and empty body-weight gainafter weaning. These relationships suggest thatpiglets consuming more dry food after weaninggrew faster because villous height was preserved.The presence of more nutrients in the lumen of thegut is likely to influence a multitude of growthfactors, biologically active peptides and guthormones that are responsible for attenuating thestructure and function of the gut.

The findings in our study provide support for thetheory of 'luminal nutrition' (Gleeson, Cullen andDowling, 1972; Williamson, 1978; Diamond andKarasov, 1983) as an explanation for both intestinalstructure and the variation in intestinal morphologyseen along the small intestine. For piglets offered thestarter diet or ewes' milk only, villous height at theproximal jejunum responded linearly to an increasein DM (Table 4). This confirms the existence of astrong relationship between nutrition and gutstructure and, because villous height at the proximaljejunum can be used as a predictor of body gain(Table 5), emphasizes the importance of food intakeper se in the performance of piglets after weaning.This result is not surprising since the proximal smallintestine is the major site for digestion and

absorption in the gut (Kidder and Manners, 1980;Diamond and Karasov, 1983; Hampson, 1983; Puchaland Buddington, 1992). The decrease in villousheight and lactase activity from the proximal to thedistal part of the small intestine seen in the presentstudy most likely parallels the decreasing gradient innutrient concentration that occurs along the gut.

Mucosal protein content and digestive enzyme activitiesThe mean protein content of the mucosa in pigletskilled at weaning and those given ewes' milk orewes' milk plus glutamine is in excellent agreementwith results for sow-suckled piglets reported byKelly et al. (1991b). These workers reported a proteinconcentration of 149 mg/g mucosa in unweanedpiglets slaughtered at 22 days of age and the averageprotein content of both groups given milk in ourexperiment was identical. Similarly, theconcentration of protein in the mucosa in pigletsgiven the solid diet was similar to that recorded bythese authors on the 5th day after weaning (106 v.113 mg protein per g mucosa), and was in the range(52 to 164 mg/g fresh mucosal scraping) reported byKidder and Manners (1980) for weaned piglets. Nostatistical difference for mucosal protein contentbetween piglets given the starter diet and milk dietswas found, presumably because of the small amountof mucosa scraped (200 mg) and the large CV (0-27 to0-86) noted between samples. It was of interest,however, that the proportional reduction in mucosalprotein concentration in piglets given the pelleteddiet was 0-29. This is identical to the 0-3 proportionaldifference in energy intake between piglets given thestarter diet and those offered ewes' milk and ewes'milk plus glutamine.

The mean values for specific lactase and sucraseactivity observed in this experiment are lower thanthose recorded by Kelly et al. (1991b and c). Forexample, mean specific lactase activity in pigletsgiven the starter diet on the 5th day after weaningwas 64 umol/min per g mucosal protein whereasKelly et al. (1991b) recorded a value of 109-5 (imol/min per g mucosal protein. Similarly, the meanspecific activity of sucrase was 78 as opposed to96-2 umol/min per g mucosal protein found by Kellyet al. (1991b). The higher activities recorded by Kellyet al. (1991b) are best explained by the youngerpiglets used in that experiment. Kelly et al. (1991b)weaned piglets at 14 days of age whereas we weanedanimals at 28 days of age. The mean villous height ofpiglets used in this experiment was lower by the 5thday after weaning (302 v. 424 urn) and, becauselactase is located more apically on the villus(Dahlqvist and Nordstrom, 1966; Nordstrom andDahlqvist, 1973; Kelly et al, 1991a) and lactaseactivity declines with age (Bailey, Kitts and Wood,1956; Kidder and Manners, 1980; James, Smith, Tivey

140 Pluske, Williams and Aherne

and Wilson, 1987; Sangild, Cranwell, S0rensen,Mortensen, Noren, Wetteberg and Sjostrom, 1991),piglets of a younger age having longer villi would beexpected to show greater lactase activity.

A salient aspect of this experiment was the poorassociation between in vitro estimates of lactase andsucrase activity and the preservation of villousheight and crypt depth, and the high rate of bodygain observed, in piglets given the milk diets. Forinstance, piglets consuming the dry starter diet afterweaning suffered a 041 proportional decrease inmean villous height and a 0-36 proportional increasein mean crypt depth compared with piglets givenmilk, yet showed no reduction in specific activities oflactase and sucrase. A reduction in villous height andincrease in crypt depth is indicative of increasedrates of cell production in the crypts and enterocytemigration, thereby leaving fewer mature enterocyteson the villus available for digestion and absorption(Rey, Schmitz, Rey and Jos, 1971; Smith, 1984;Hampson, 1986a). Under these circumstances wewould have predicted a decrease in the activity ofthese enzymes. However, we caution that only thespecific activity of these enzymes was measured.Expression of enzyme activity on a total basis mayhave yielded more meaningful data, although datapresented by Kelly et al. (1991b) suggests that eventhis in vitro measure does not adequately reflect thelarge differences seen in intestinal architecture.

Structure and function of the small intestine in pigletsoffered L-glutamineThe digestive and absorptive capacity of the smallintestine, as assessed by villous height and cryptdepth, the specific activities of lactase and sucrase,and the rate of empty body-weight gain, was similarbetween piglets offered ewes' milk and those givenewes' milk supplemented with 20 g/1 L-glutamine.These findings failed to support our hypothesis thatglutamine would augment the structure and functionof the small intestine after weaning. The mostprobable reason for the lack of an effect of glutaminewas that piglets did not undergo starvation and /orthere was no stimulation of the adrenal cortex.During these states glutamine may become a'conditionally essential nutrient' (Souba, 1991 and1992) for the mucosa because high rates of glutaminemetabolism provide adequate amounts of keyintermediates for nucleic acid biosynthesis (Klimberget al, 1990) and mucosal renewal (Souba, 1993).Under these circumstances enterocytes would beexpected to increase their demand for glutaminebecause it is more important than glucose as anoxidative fuel (Windmueller and Spaeth, 1980).

In the present experiment there was no period offasting after weaning. In view of the high level of

milk (and glutamine) intake achieved, the exogenoussupply of glutamine was most likely in excess of thatrequired by the epithelium. Support for this notioncomes from the 1-46 proportional increase in theconcentration of plasma urea (Table 7) in pigletsgiven ewes' milk plus glutamine compared withpiglets offered ewes' milk only. An increase in urealevels following glutamine supplementation was alsoreported by Dechelotte, Darmaun, Rongier,Hecketsweiler, Rigal and Desjeux (1991). An increasein urea concentration is consistent with thehydrolysis of glutamine to glutamate by glutaminasewith its subsequent deamination to ammonia andrelease into the portal system. Ammonia, in turn, isdetoxified in the urea cycle in the liver and releasedas urea into the circulation.

Despite glutamine having no apparent effect on thestructure and function of the small intestine afterweaning, there are two aspects of this experimentthat warrant further examination. First, the lack ofany correlation between villous height and foodintake in piglets given ewes' milk plus glutamine(Table 4) suggests that this amino acid might havehad an ameliorative role on intestinal structure thatwas not entirely related to the level of food intake perse. This may occur by stimulating the activity ofmitochondrial glutaminase and/or the rate oftransport across enterocytes already present on thevillus. Glutamine has been shown to expedite both ofthese processes (Klimberg et al., 1990). Secondly, thelinear increase in crypt depth as glutamine intakeincreased is indicative of an increase in glutaminemetabolism in the mucosa. An increase in glutaminetransport across the enterocytes increases the activityof mitochondrial glutaminase (Klimberg et al., 1990);Salloum et al., 1990) that would, in turn, acceleratethe hydrolysis of glutamine into products that candirectly enter the tricarboxylic acid cycle andgenerate adenosine triphosphate. This energy couldbe used to support cell division in the proliferativezone in the crypts of Lieberkiihn (Newsholme,Crabtree and Ardawi, 1985; Klimberg et al., 1990)since glutamine is a requisite substrate for de novoDNA biosynthesis.

Several recent studies in the weaned piglet providesome support to these speculations. Wu and Knabe(1993 and 1994) have highlighted the possibleimportance of glutamine as an energy substrate forthe small intestine of the pig both during sucklingand after weaning. Wu and Knabe (1994) reportedthat glutamine was the most abundant free andprotein-bound amino acid in sows' milk at days 22and 29 of lactation. After weaning, Wu and Knabe(1993) measured glutamine metabolism in isolatedenterocytes and found two-and 10-fold increases inthe rate of oxidation of glutamine to CO2 in

Gut morphology and nutrition in piglets 141

enterocytes from 29-day-old weaned pigletscompared with 21-day-old sucking piglets. Thesedata suggest that glutamine may serve as anincreasingly important energy substrate for theenterocytes of weaned piglets. Rates of glutamineoxidation were not measured in the present study,although tentative support for the findings of Wuand Knabe (1993) comes from the significant linearrelationship observed between glutamine intake andcrypt depth (Table 4). However to observe abeneficial effect of supplemental glutamine on thedigestive and absorptive capacity of the smallintestine and therefore on growth rate, it is likely thatan experiment lasting longer than 5 days is required.This would give cells that are formed in the cryptbase time to migrate up the villus and develop fulldigestive and absorptive capacity. In this regard,Meier, Knabe, Wu and Borbolla (1993) reported thatthe addition of 10 g/kg glutamine to a maize-soya-bean diet prevented villous atrophy in the jejunumon the 7th day after weaning.

We have demonstrated that villous height andcrypt depth can be maintained if piglets do notsuffer the nutritional stress of interrupted intakeimmediately after weaning. In contrast pigletsoffered a solid, pelleted diet at the same level ofvoluntary food intake suffered a decrease in villousheight and an increase in crypt depth afterweaning. Piglets drinking ewes' milk and ewes'milk plus glutamine, however, ingestedproportionately 0-30 more energy than animalseating the solid diet. The possible contribution ofthis increase to the preservation of gut structureand function and the disparity in body-weight gaincould not be resolved from this study, but will beexamined in the next experiment (Pluske et ah,1996).

AcknowledgementsJRP was in receipt of a Junior Research Fellowship from thePig Research and Development Corporation (PRDC) ofAustralia during this study. We are grateful to ProfessorD. J. Hampson for assistance with preparation ofhistological slides, and to Dr D. Kelly for advice on enzymeassays. Thanks is also expressed to (the late) RosemaryHead, John Beesley, Rob Smits, Andrew Williams, JamesFisher, Dave Miller and Janet Paterson for their help duringthe experiment. Financial support from PRDC isacknowledged.

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