Villous height and crypt depth in piglets in response to ... · 2-5 Ma; and (5) piglets given cow's fresh milk a Ond libitum the 5th day. all piglets were killed and samples of gut

Animal Science 19%, 62: 145-158 1357-7298/96/56260145$20-00© 1946 British Society of Animal Science

Villous height and crypt depth in piglets in response to increases inthe intake of cows' milk after weaning

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

1 Animal Science, Faculty of Agriculture, University of Western Australia, Nedlands, WA 6907, Australia2Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton AB T6G 2P5, Canada

Abstract

The hypothesis tested in this experiment was that the structure and function of the small intestine of piglets given amilk liquid diet after weaning depends on their level of energy intake. At weaning (28 days), 42 piglets wereallocated to one of five treatments: (1) control group killed at weaning; (2) piglets offered a dry starter diet adlibitum; (3) piglets given cow's fresh milk at maintenance energy intake (Ma); (4) piglets given cow's fresh milk at2-5 Ma; and (5) piglets given cow's fresh milk ad libitum. On the 5th day all piglets were killed and samples of gutwere taken for histological and biochemical examination. Piglets given milk ad libitum grew faster (P < 0-001)than piglets on all other treatments. Piglets offered the dry starter diet ingested similar quantities of dry matter andenergy, and grew at the same rate as piglets given cows' milk at 2-5Ma. As predicted, piglets given milk atmaintenance energy intake grew slower fP < 0-001) and consumed less food (P < 0-001) than piglets in all othertreatments. For piglets given both cows' fresh milk and the dry starter diet, there were significant linearrelationships (r = 0-72 to 0-82, P <0-05) between villous height and crypt depth with dry matter (energy) intakeafter weaning. In turn, mean villous height in both milk-fed and starter-fed piglets was significantly correlated (x =0-68 to 0-79, P < 0-05) to empty body-weight gain in the first 5 days after weaning. Estimates of digestive enzymeactivity and absorptive capacity of the gut did not corroborate the large differences seen between treatments invillous height and crypt depth, and possible reasons are discussed. These data illustrate the interdependencebetween voluntary food intake and mucosal architecture in determining piglet performance after weaning.

Keywords: crypt depth, energy intake, piglets, villous height.

Introduction of milk intake or to some protective and/orIn our previous experiment (Pluske, Williams and stimulatory effect on the gut mucosa afforded byAherne, 1996) we demonstrated that the regular milk perse.feeding of a milk diet prevented the decrease invillous height and increase in crypt depth generally Prolonged lactation does not prevent the normal lossassociated with weaning. In contrast, piglets of lactase activity that occurs with increasing ageconsuming a dry starter diet displayed villous (Lebenthal, Sunshine and Kretchmer, 1973; Kelly,atrophy, an increase in crypt depth, and a reduced King, McFadyen and Travis, 1991a). In addition,concentration of mucosal protein. A dry-matter (DM) several authors (Hampson, 1986b; Kelly, O'Brien andintake of 296 g/day supplying 74 MJ gross energy McCracken, 1990b; Kelly, Smyth and McCracken,(GE) per day was sufficient to maintain the structure 1990a) have proposed that villous atrophy and cryptand function of the gut in piglets given ewes' fresh hyperplasia may be related to the withdrawal ofmilk. However what could not be resolved was 'intrinsic factors', such as IgA, in sows' milk,whether the maintenance of villous height and crypt Mammalian milks, including that of the sow, alsodepth after weaning was due to this particular level contain an array of biologically active peptides (e.g.

epidermal growth factor) and compounds (e.g.t Present address: School of Veterinary Studies, Murdoch polyamines) that may stimulate protein and DNAUniversity, Murdoch WA 6150, Australia. synthesis and have functional roles in intestinal

145

146 Pluske, Williams and Aherne

differentiation. Consumption of ewes' milk afterweaning in our previous study (Pluske et al., 1996),especially as it was offered in an unhomogenizedand unpasteurized state, may have attenuated theactivity of enzymes, peptides and growth factors,and hence contributed to the integrity of the smallintestine.

Studies with rodents (Stevens Hooper and Blair,1958; Steiner, Bourges, Freedman and Gray, 1968;McManus and Isselbacher, 1970; Altmann, 1972;Hopper, Rose and Wannemacher, 1972; Al-Dewachi,Wright, Appleton and Watson, 1975; Rudo,Rosenberg and Wissler, 1976; Goodlad and Wright,1984; Goodlad, Plumb and Wright, 1988), however,have demonstrated a hypoproliferative effect offasting on the intestinal mucosa that causes adecrease in villous height, crypt depth and/or theprotein content of the mucosa. Our intention in thisexperiment was to feed milk to piglets at differentlevels of energy intake after weaning, ranging frommaintenance through to ad libitiiin-intake, so that agraded response between energy intake and thestructure and function of the small intestine could beestablished. This would allow an effect of voluntaryfood intake to be demonstrated that was notconfounded with feeding a milk diet per se.Therefore, the hypothesis tested in this experimentwas that the structure and function of the smallintestine of piglets fed a milk diet after weaningdepends on their level of energy intake. Pigletsconsuming a milk diet at their maintenance energyrequirement would be expected to have shorter villiand shorter crypts than those on energy intakesabove maintenance.

Material and methodsAnimals and housingForty-two piglets from six primiparous gilts (LargeWhite X Landrace) were used in the study. Thegenotype, management, feeding and housing of giltsand piglets have been described previously (Pluskec/ al., 1996). Creep food was not offered duringsuckling. Piglets were weaned at 29 (s.e. 0-2) days ofage when they weighed 9-1 (s.e. 0-29) kg, and werehoused in individual accommodation as alreadydescribed (Pluske et al, 1996).

Experimental treatmentsThe experiment was a completely randomized blockdesign. Piglets were allocated randomly on the basisof litter, sex and live weight to one of five treatmentgroups as follows: (1) sow-reared, control groupkilled on the day of weaning (SR; no. = 10); (2)piglets offered a pelleted starter diet (starter; no. = 8);(3) piglets offered cows' fresh milk at maintenanceenergy intake (Ma; no. = 8); (4) piglets offered cows'

fresh milk at 2-5 times maintenance energy intake(2-5 Ma; no. = 8); (5) piglets offered cows' fresh milkad libitum (AL; no. = 8).

Diets, feeding regimen and experimental proceduresPiglets were offered cows' fresh milk every 2 haccording to the general feeding pattern outlinedpreviously (Pluske et al., 1996). Cows' milk was usedin this study because it was readily available in largequantities. Food intake in treaments Ma and 2-5 Mawas determined on a metabolic live-weight basis forcalculating the daily maintenance requirement ofyoung piglets: MEm = 0-485 M07S MJ ME per day,where MEm is the amount of metabolizable energyrequired by the pig for daily maintenance, and M isthe live weight (kg) of the pig (after Close andFowler, 1985)"

At 14.00 h each day all piglets were weighed and, forpiglets given food at Ma and 2-5 Ma, their energyrequirement for the next 24 h was calculated. Thiswas then divided into 12 equal feeds and offeredevery 2 h. For piglets that were given milk 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. For piglets offered milk ad libitum,troughs were left in the pens for longer (30 to 45 min)because some animals drank their milk in twoepisodes. Between feedings all troughs were washedand scrubbed thoroughly with hot water. For pigletsgiven milk ad libitiuiu, the amount offered wasincreased proportionately by 01 at each feed if thepiglet consumed all the milk offered to it in theprevious meal. This ensured that more milk wasoffered than could be drunk.

The dry starter diet used in this experiment has beendescribed previously (Pluske et al., 1996). For pigletsgiven this pelleted diet, any food that had spilledinto the collection trays was collected and the weightrecorded. Piglets sometimes urinated or defecated intheir troughs and, when this happened, the food wasremoved, weighed, and an equivalent amount offresh pellets replaced in the feeder.

One hour before slaughter on the 5th day afterweaning the absorptive function of the smallintestine was assessed using o-xylose. A xylosesolution (equivalent to 2 ml of 5'-! (w/v) per kglive weight) was either added to the troughs ofpiglets receiving milk or, in the case of pigletsreceiving solid food, was administered using astomach tube.

Post-mortem procedureThe slaughter and post-mortem procedure for theprocessing of all piglets has been described in our

Gut morphology and energy intake 147

previous publication (Pluske et al., 1996). The pH ofcaecal contents was measured directly using a digitalpH meter (Suntex SP-32, Stansens, Taiwan).

AnalysisSamples of cows' liquid milk were collected in plasticvials and frozen at -20°C for analysis of fat, protein,and total solids. Milk was analysed using aMilkoSean* (Foss Electric, Denmark). The proportionof fat, protein and total solids contained in cows'whole milk (no. = 64) was 33-4 (s.e. 1-3), 33-2 (s.e. 0-4)and 121-4 (s.e. 2-6) g/kg. The calculated GE contentof the milk was 2-69 MJ GE per kg, or 22-16 MJ GEper kg DM, according to the equation of Perrin(1958)!

Plasma glycerol was determined by capillary gaschromatography according to the techniquedeveloped by Fenton and Aherne (1987). Plasmaalkaline phosphatase and urea were determined (induplicate) using a biochemical analyser (COBASM1RA, Roche Diagnostica, Switzerland). The level offree thyroxine (T4) in plasma was determined usingan Amerlex-MAB FT4 kit (Amersham International,UK). This was a direct, competitive assay for free T4using a high specific-activity, |I2SI [-labelledmonoclonal antibody raised to T4. Plasma xylose wasanalysed according to the method of Merritt andDuelly (1983).

Histology and brush-border lactase and sucrase specificactivityVillous height, crypt depth and the specific activitiesof lactase and sucrase were measured at three sitesalong the small intestine, as described previously(Pluske ctai, 1996).

Statistical analysis and presentation of resultsAll data were subjected to least-squares, one-wayanalysis of variance for treatment effects usingSYSTAT* (Wilkinson, 1990). Since this experiment wasconducted in three parts, time was included as anindependent variable in the initial analysis ofvariance. The effect of time was not significant forany variables analysed and the data were re-analysed with treatment group being the onlyindependent variable. Pairwise comparisonsbetween treatment means were made using Fisher's-protecled least significant difference (LSD)procedure (Maindonald, 1992).

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 between the recorded weight of theempty body at slaughter 5 days after weaning andthe estimated weight of the empty body at weaning.

Data were combined from the three milk treatmentsto test the hypothesis that there is a linear increase invillous height with increasing levels of voluntaryfood intake. Simple linear regression analysis wasconducted using SYSTAT® (Wilkinson, 1990). Toanalyse the response of intestinal structure to foodintake, both linear (i/ = (7 + bx) and quadraticregressions (y = a + bx + ex2) were calculated.

The protein content of the mucosa and specificenzyme activity are presented as the means of allthree sites sampled proportionately 0-25, 0-50 and0-75 along the small intestine.

ResultsAll piglets offered cows' fresh milk began drinkingwithin 8 h of weaning. Piglets receiving milk at Maor 2-5 Ma finished their meal within 4 min of placingthe trough in the pen, whereas those given food adlibitum took longer. The health of all piglets wasexcellent and no animals developed diarrhoeaduring the 5-day period.

Villous height and crypt depth after weaningFeeding cows' milk ad libitum after weaningmaintained villous height at the proximal jejunumat a level similar to that of piglets killed atweaning. At the mid jejunum and distal ileum,villous height in piglets given food ad libitum wasproportionally 0-23" (P < 0-05) and 0-33 (P<0-01)higher relative to piglets at weaning. Piglets givencows' milk at Ma had shorter villi (P < 0-001) at allsites along the small intestine than did pigletsgiven milk ad libitum. Differences in villous heightbetween piglets given milk at Ma and 2-5 Ma wereless distinct, with only values at the mid jejunumbeing statistically significant (P < 0-05). Villousheight in animals given milk at 2-5 Ma and adlibitum was similar at all sites except at the distalileum. For piglets offered the dry starter diet,villous height was similar (P < 0-05) to those givenmilk at Ma but lower (P<0-01) at all sites alongthe gut than in piglets given milk ad libitum.Relative to animals given milk at 2-5 Ma, villousheight differed only at the mid jejunum (P< 0-051;Table 1).

Mean villous height was similar in piglets given thestarter diet and those offered cows' milk at Ma,despite there being nearly a three-fold difference inDM intake (Table 2). Mean villous height in pigletsgiven cows' milk at 2-5 Ma was proportionately 013lower (P < 0-05) than those offered milk ad libitum,


Table 1 Villous height and crypt depth of piglets killed either at weaning or 5 days later

Villous height (um)

Mean:):Crypt depth (urn)

Mean:):

Proportion

ofintestine

0-250-500-75

0-250-500-75

SR

569a

445"b

333"449"

122"116"102"114"

Starter

413b

384"300"b

366b

161b

169b

141b

157C

Treatmentt

Ma

379b

363"249b

330b

122"128ac

107"119a

2-5 Ma

458bc

494bc

342"432"

151b

144bc

126"b

141b

Ad libitum

508"c

547C

442C

499c

148b

157b

148b

151bc

s.e.d.

47-750-637-927-0

13-514-416-48-7

Significance

***

******

a.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; Ma: piglets given cows' fresh milk at maintenance;2-5 Ma: piglets given cows' fresh milk at 2-5 times maintenance; ad libitum: piglets given cows' fresh milk ad libitum.% Mean of all three sites.

was proportionately 0-18 higher (P < 0-05) than thoseoffered the starter diet, but was comparable withthose killed at weaning. Mean villous height inpiglets given milk ad libitum was higher (P < 0-001)than in all other treatment groups, including thosekilled at weaning (Table 1).

Feeding the starter diet or cows' milk at either 2-5 Maor ad libitum after weaning increased crypt depth(P < 0-05) at all sites along the gut compared withpiglets killed at weaning, or piglets offered milk atMa. Crypt depth at all sites was similar for pigletsoffered either the starter diet or cows' milk at the two

higher levels of intake. Feeding milk to piglets at Madid not alter mean crypt depth compared withpiglets killed at weaning, whereas mean crypt depthin the remaining three groups (starter, 2-5 Ma and adlibitum) was proportionately from 0-21 to 0-35 deeper(P < 0-001) than in piglets given milk at Ma. Meancrypt depth in piglets given milk at 2-5 Ma was lower(P < 0-05) than in piglets offered the starter diet(Table 1).

Performance of piglets after weaningPiglets offered cows' milk ad libitum gained emptybody weight at twice the rate (463 v. 231 g/day,

Table 2 Performance of piglets after weaning

Treatmentt

SR Starter Ma 2-5 Ma Ad libitum s.e.d. Significance

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 DM per day)Energy intake (MG GE per day)Food conversion ratio (g DM : g EBWGpEnergy cos t /g EBWGf (kj DE per g)

8-9

8-7

9010-5

8-8100

288"231"286"

5-1"1-0

21-1

9-19-4

8-99-2

58b

49b102b

2-3b

9-210-5

9010-3

272"253a

234"5-2a

1-123-8

9-211-7

9-011-3

514C

463C

400c

8-9c

0-918-9

0-860-86

0-850-84

80-174-241-0

0-760-071-61

a,b,c vvithin rows, means not followed by a common superscript differ significantly.t See Table 1.% EBWG: empty body-weight gain.


500 - i

"sc 4 0 0

c 300 -

E 200 -

100 -

0 J

1 2 3 4 5Day after weaning

Figure 1 The daily pattern of dry-matter intake (g) in pigsweaned at 29 (s.e. 0-2) days of age and fed for 5 days afterweaning with either a pelleted starter diet (•) or cows' liquidmilk at three levels of energy intake: maintenance (•), 2-5maintenance (•), and ad libitum (O). Values are mean + s.e. foreight pigs per treatment.

P < 001) of piglets offered the dry starter diet adlibitum in the 5 days after weaning. During this timepiglets given milk ad libitum consumedproportionately 040 more DM (400 v. 286 g/day,P = 0-010) and 0-74 more energy (8-9 v. 5-1 MJ GE perday, P < 0-010) than piglets given dry pellets. Pigletsoffered the starter diet ingested proportionately 0-20more DM (P > 0-05) but similar amounts of energy,and grew at the same rate, as piglets given cows'milk at 2-5 Ma. Animals given milk at Ma grewslower (P < 0-001) and were given less food(P < 0-001) than piglets in all other treatment groups(Table 2). The CV in daily empty body-weight gainranged from 1-10 for piglets offered the starter diet to0-15 for piglets given cows' milk ad libitum. Pigletsdrinking cows' milk ad libitum consumed around 3-5times their maintenance level of energy intake overthe duration of the experiment. In comparison,piglets consuming the starter diet ate the equivalentof 1-6 times their estimated maintenance requirementfor energy.

Food conversion ratio was similar (P = 0-098) for alltreatment groups except those given milk at Ma.Similarly, the energetic cost of 1 g of empty body-weight gain did not differ (P = 0-098) between pigletsgiven milk at 2-5 Ma or ad libitum and piglets giventhe pelleted diet (Table 2).

Pattern of voluntary food intake after weaningDM intake was highest (P < 0-001) on all days afterweaning for piglets offered cows' milk ad libitum.Piglets given the starter diet consumed only 110 g onthe 1st day after weaning but increased their intake

rapidly and consumed 365 g by day 5. This was stillproportionately 0-20 less DM than for piglets offeredcows' milk ad libitum (Figure 1). DM intake in pigletsoffered the starter diet and those offered cows' milkat 2-5 Ma was similar over the duration of the study.The CV of DM intake was greatest in piglets offeredthe starter diet, decreasing from 0-96 on the 1st dayafter weaning to 0-54 on the final day of theexperiment. In contrast, the CV for piglets offeredcows' milk ad libitum decreased from 0-21 on day 1 to0-14 by day 5.

Relationships between intestinal structure and-performance after weaningData from the three groups given cows' milk werecombined and the following relationships betweenfood intake, intestinal structure, and the rate ofempty body-weight gain were established. For allrelationships, the inclusion of the quadratic term (x2)

700 - i

600 -

"3 500 -

| 400 -

cS 300 -

200 ->

(a)

0 1000 2000Total dry-matter intake (g)

3000

200 -

"g 180 -

a, 1 4 0 ~bc 120 -

s5 100 -

(b)

1000 2000 3000

Total dry-matter intake (g)

Figure 2 Relationship between total dry-matter intake and(a) mean villous height (y = 279-7 + 0-12x, r = 0-82, P < 0-001; no.= 22] and (b) mean crypt depth [y = 114-3 + 0-02x, r = 0-44,P < 0-05; no. = 22] along the entire length of the small intestinefor pigs receiving cows' liquid milk.


3000 - i

^ 2000 -be

"31000 -

<u0 -

' - 1 0 0 0 ->

(a)

300 400 500

Villous height (u.m)

i I r600 700

3000 - i

6C

3

I

u em

pty

1

o

2000 -

1000 -

0 -

-1000 -

80 100 120 140 160

Crypt depth

180 200

Figure 3 Relationship between (a) mean villous height andtotal empty body-weight gain [y = -1788-3 + 7-24x, r = 0-68, P =0-002; no. = 22] and (b) mean crypt depth and total emptybody-weight gain [y = -919-3 + 15-86*, r = 0-45, P < 0-05; no. =22] for pigs receiving cows' liquid milk.

failed to explain any more variation than the linearregression, so the response was assumed to be linearrather than quadratic.

Total DM intake in the first 5 days after weaning washighly correlated to mean villous height along thesmall intestine for all piglets fed cows' liquid milk(r = 0-82, P< 0-001) (Figure 2a). Significantrelationships were also found between villous heightand energy intake for all piglets given milk (r = 0-47(P < 0-05), r = 0-53 (P < 0-01) and r = 0-73 (P < 0-001),for sites 0-25, 0-50 and 0-75 along the gut,respectively). For crypt depth, only proportionately0-19 of the total variation (P < 0-05) along the lengthof the small intestine was related to milk energyintake after weaning (Figure 2b). For individual sitesalong the gut, only crypt depth at the distal ileumshowed any correlation (r = 0-47, P < 0-010) with DMintake in the first 5 days after weaning. For pigletsgiven the starter diet, total DM intake wassignificantly correlated to villous height at sites 0-50(r = 0-72, P = 0-069) and 0-75 (r = 0-76, P < 0-05) alongthe small intestine. Crypt depth at these two siteswas also associated with the amount of food pigletsconsumed in the 5 days after weaning, althoughcorrelations were weaker (r = 0-71, P = 0-078 for midjejunum, and r = 0-62, P = 0-096, for distal ileum). DMintake was correlated to mean crypt depth (r = 0-69,P = 0-058), but was not related to mean villousheight.

When used as a predictor of growth after weaning,mean villous height along the length of the smallintestine was highly correlated to empty body-weight gain for milk-fed piglets (r = 68, P<0-01)(Figure 3a). Significant relationships were also foundbetween villous height and total body gain for all

Table 3 Protein content of the mucosa, the specific activity of lactase (EC 3.2.1.23) and sucrase (EC 3.2.1.48), and plasma xyloseconcentration of piglets killed at weaning or 5 days later

Treatmentt

SR

129-1

' 77ab

65ab

22

Starter

98b

105a

86b

16

Ma

106b

80ab

51a

20

2-5 Ma

133a

7 2 b

57a

18

Ad libitum

134a

52b

45a

16

s.e.d

10-4

17-4

12-6

2-6

Significance

Mucosal protein content (mg/g mucosa):):

Lactase activity (nmol/min per g protein):):

Sucrase activity (umol/min per g protein):):

Xylose (mg/100 ml)

•lbc Within rows, means not followed by a common superscript differ significantly.t See Table 1.X Mean of all three sites.


5 0 0 -

wei

ghbo

dy-

3 0 0 -

1 0 0 -

- 1 0 0 -

(a)

- 3 0 0 •

200 300 400Villous height (|im)

500

£sb

500 -

-

3 0 0 -

100 H

1 -100 H

a.£ -300 -J

(b)

O

I50

I100 150 200

Crypt depth (p.m)250

Figure 4 Relationship between (a) mean villous height andtotal empty body-weight gain [y = -6120 + 2-33.r, r = 0-79, P =0020; no. = 8] and (b) mean crypt depth and total empty body-weight gain [y = -536-2 + 5-02J, r = 0-75, P < 0-05; no. = 8] forpigs given the pelleted starter diet.

piglets given cows' milk (r = 040 (P = 0-055), r = 0-55(P < 0-01) and r = 0-71 (P < 0-001), for sites 0-25, 0-50and 0-75 along the gut respectively). The relationshipbetween mean crypt depth and empty body-weightgain after weaning was weaker (r = 0-45, P < 0-05)(Figure 3b), and was better correlated to crypt depthat the distal ileum that accounted for proportionately0-29 (P < 0-01) of the total variation. For piglets giventhe starter diet, villous height was significantlycorrelated to empty body-weight gain after weaningat sites 0-25 (r = 74, P < 0-05), 0-50 (r = 0-86, P = 0-012),and 0-75 (r = 0-79, P < 0-05) along the gut. As aconsequence, mean villous height was highlycorrelated (r = 0-79, P < 0-05) to empty body-weightgain (Figure 4a). Crypt depth at sites 0-25 (r = 0-75,P < 0-05) and 0-50 (r = 0-69, P = 0-086) along the gut,and mean crypt depth (r = 0-76, P < 0-05) (Figure 4b),were correlated to empty body-weight gain afterweaning.

Mucosal protein and digestive enzyme activityMean protein content of the mucosa of the smallintestine was proportionately from 0-27 to 0-36higher (P < 0-001) in piglets that were killed either atweaning or received milk at 2-5 Ma or ad libitumcompared with piglets receiving milk at Ma or thestarter diet. Specific lactase activity was greatest(P < 0-05) for piglets offered the pelleted starter diet,and this was higher (P < 0-05) than for piglets givenmilk at 2-5 Ma or ad libitum. Lactase activity wassimilar in the three groups of piglets given cows'milk (P > 0-05) and piglets killed at weaning. Thespecific activity of sucrase was highest for pigletsgiven the pelleted diet and for those animals killed atweaning (P = 0-012). All groups given milk recordeda similar activity for sucrase (range: 45 to 57 (imol/min per g protein; Table 3). Mean lactase activityaveraged over all treatments was highest (P < 0-05) atthe proximal jejunum and declined (P > 0-05) to thedistal ileum. In contrast, sucrase activity was highest(P = 0-09) towards the distal part of the smallintestine. The amount of xylose absorbed by pigletson the 5th day after weaning was similar in alltreatment groups (Table 3).

Organ weights and pH valuesSignificant differences between treatments werefound for all organ weights when expressed asgrams or g/kg empty body weight (EBW). Pigletsgiven milk at Ma had similar values (P > 0-05) tothose killed at weaning, but values were generallylower (P < 0-001) than for other treatments for allorgans when expressed on an absolute weight basis.When corrected for empty body weight (g/kg),piglets given milk at Ma had values similar to theother groups given milk, but these were lower(P < 0-001) than in piglets given the starter diet. Thelength of the small intestine was similar in groups ofpiglets given food after weaning but was shorter inpiglets killed at weaning (P < 0-05). Expressed asg/kg, the small intestine was identical (0-9 m/kgEBW) between groups.

The large intestine was heaviest (P < 0-001) in pigletsgiven the starter diet but similar to that of pigletsgiven milk ad libitum (172 v. 159 g, P < 0-068). Whenexpressed on a g/kg basis, the large intestine wasproportionately from 0-18 to 0-41 heavier (P < 0-01)in piglets given the starter diet than in piglets fromother treatments. Piglets given the starter diet afterweaning had more acidic contents in their caeca (pH= 6-0; P = 0-056) (Table 4).

Plasma metabolitesThe concentration of glycerol in plasma did notdiffer between treatment groups. Blood ureaconcentration was highest (P < 0-01) in piglets givencows' milk ad libitum and lowest in piglets given milk


Table 4 Organ weights and caecal pH of piglets killed at weaning or 5 days later

Length of small intestinemm/kg EBWJ

Stomachgg/kgEBWt

Small intestinegg/kgEBWt

Large intestinegg / k g EBW:):

pH caecum

SR

7-7*0-9

40"4-6"

218"25-3"

111"12-8"

6-5"

Starter

8-8b

0-9

60"6-0b

305b

304 b

172b

17-3b

6-0b

Treatmentt

Ma

8-3"b

0-9

46"c

5-1"

221"25-6"c

132"c

13-2"6-9"

2-5 Ma

8-7b

0-9

53bc

5-3"b

294b

29-2"b

124"12-3"6-6"

Ad libitum

8-7b

0-8

58b

5-2ab

328"29-9bc

159bc

14-6"6-6"

s.e.d.

0400-07

3-8041

2502-23

1441-300-28

Significance

***

*

***

";b'c Within rows, means not followed by a common superscript differ significantly.t See Table 1.% EBW: empty body weight.

at Ma. The level of free thyroxine was similar inpiglets receiving cows' fresh milk at 2-5 Ma and adlibitum, but was higher (P = 0-012) in piglets givenmilk ad libitum than in animals killed at weaning,offered milk at Ma, or given the starter diet (Table 5).

DiscussionVillous height and crypt depth after weaningIn this experiment we tested the hypothesis that thestructure and function of the small intestine ofpiglets given a milk diet after weaning would vary indirect response to increasing levels of milk energyintake. Positive correlations between voluntary foodintake and both villous height and crypt depthclearly demonstrated that a component of themucosal response of the small intestine afterweaning is driven by the amount of food piglets eat.Similar relationships were recorded for piglets eatinga dry starter diet. Furthermore, both villous height

and crypt depth explained a significant proportion ofthe total variation in EBW gain in piglets given bothcows' fresh milk and the starter diet. Whilst wecannot state definitively that cause and effectrelationships exist between gut structure and weightgain, these data illustrate the apparentinterdependence between voluntary food intake andmucosal structure in determining the performance ofpiglets after weaning, and concur with similarrelationships found in our previous study (Pluske etah, 1996).

The dramatic effect of a reduction in voluntary foodintake on mucosal growth was seen in piglets offeredmilk at maintenance. These piglets had a shortersmall intestine, a reduced concentration of protein inthe mucosa, and possessed shorter villi and cryptsthan piglets offered milk at the two higher levels ofintake. These data also demonstrate that whenpiglets consumed a conventional starter diet at the

Table 5 The concentration of metabolites in

Glycerol (mg/1)Urea (mmol/1)Insulin (|iU/l)Free thyroxine (T4) (pmol/1)

the plasma of piglets

SR

104l-9b

6-611-5"

Starter

842-lb

12412-2"b

killed at weaning or 5 days

Treatmentt

Ma

12-31.2"84

12-6"b

2-5 Ma

8-6l-9b

94154bc

later

Ad libitum

11-33-0c

9-116-8C

s.e.d.

2-790-661-211-04

Significance

a.b,c vVithin rows, means not followed by a common superscript differ significantly,t See Table 1.


same level of energy intake as piglets drinking aliquid milk diet (i.e. = 2-5 Ma), villous atrophy, anincrease in crypt depth, and a decrease in mucosalprotein content occurred. Despite these markedchanges in gut morphology, piglets given the starterdiet grew at the same rate after weaning as pigletsgiven milk.

The absence of nutrients from the lumen caused, forexample, by starvation (Altmann, 1972; Hampson,1983; Goodlad et al., 1988) or parenteraladministration of the diet (Feldman, Dowling,McNaughton and Peters, 1976; Goldstein, Hebiguchi,Luk, Taqi, Guilarte, Franklin, Niemiec and Dudgeon,1985; Castillo, Feng, Stevenson, Kerner and Kwong,1990), causes villous atrophy and a decrease in therate of cell production in the crypts of Lieberkiihn(Goodlad and Wright, 1984). This is mediated by anincrease in cell-cycle time (Al-Dewachi et al., 1975;Goodlad et al., 1988) that respectively increasesduring starvation and decreases after refeeding. Atemporary reduction in crypt-cell production ratewas reported by Hampson (1986a) and Hall andByrne (1989) following weaning onto a solid diet inboth conventional and gnotobiotic piglets. Hampson(1986a) considered that this brief respite in crypt-cellproduction may be a possible mediator of thereduction in enterocyte number on the villus afterweaning. However the decrease in both villousheight and crypt-cell production rate reported inthese studies may also be explained by the period oftemporary starvation that occurs after weaning.

Support for this notion comes from the studies ofKoga and Kimura (1978, 1979 and 1980). Restrictingadult mice to proportionately 0-60 of ad libitum intakecaused a decrease in cell migration rate from thecrypt onto the villus and a reduction in villous height(Koga and Kimura, 1978), a lower mitotic activity inthe crypts (Koga and Kimura, 1979) and, in their finalstudy, Koga and Kimura (1980) found that the cell-cycle time in the crypts of duodenal and jejunal cells,in particular the duration of the Gj phase, wasprolonged under dietary restriction. The shortercrypt depth seen in this experiment in piglets givencows' milk at Ma is consistent with thesemechanisms.

It is not possible to discern the precise aetiologywhereby low food intake effects these changes to themitotic cycle of undifferentiated cells in the crypts.Feldman et al. (1976) listed four ways in which theabsence of nutrients from the lumen of the smallintestine causes mucosal hypoplasia, and it is likelythat some, if not all, of these mechanisms wereimplicated in the results observed in this experiment:(1) the absence of food may have changed thebalance or numbers of bacterial flora in the gut,

leading to secondary changes in the small intestinemucosa; (2) ingested food may have been useddirectly by the intestinal mucosa during transport,either as an energy source or as substrates for cellsynthesis; (3) the absence of luminal contents couldhave led to smaller than normal release of gastro-intestinal polypeptide hormones which, themselves,may be trophic to the intestine (e.g. enteroglucagon)(Williamson, 1978); and (4) the absence of food in thegut may have failed to trigger cholecystokinin(Kanayama and Liddle, 1991) and secretin releasefrom the intestinal mucosa and, in turn, the resultantexocrine pancreatic hyposecretion may have led tohypoplasia (Altmann and Leblond, 1970; Altmann,1971; Tivey and Shulman, 1991).

Hampson (1986b) also conducted a study in whichthe amount of a liquid milk diet offered afterweaning was quantified, and then the structure andfunction of li;-.1 small intestine assessed. In his studypiglets achieved an average intake in the first 5 daysafter weaning of 400 g/day. The diet offered was asow-milk replacer made up in a ratio of 1: 3 in water(i.e. 330 g/kg DM), and this equated to an averageDM intake of 133 g per pig per day, or 2-9 MJ GE perpig per day. Since piglets still displayed villousatrophy and reductions in brush-border enzymeactivity, Hampson (1986b) concluded that 'neitherpoor and irregular intake of food after weaning norphysical trauma from the weaning diet are normallyinvolved in these changes'. In the presentexperiment, piglets given cows' milk at Maconsumed 102 g DM, or 2-3 MJ GE, per day. Thesepiglets displayed villous atrophy and no increase incrypt depth but, in contrast to the study of Hampson(1986b), showed no decline in the specific activity oflactase and sucrase. Since the amount of milkconsumed by piglets at maintenance was only »30 gDM per day less than the intakes reported byHampson (1986b), it is likely that the villous atrophyreported by Hampson (1986b) was due to nutrientdeprivation in the small intestine. The similarity inconsumption between the two studies providesfurther support for our hypothesis, and embraces thenotion that low voluntary food intake after weaningis most likely limiting the development of digestiveand absorptive processes in the small intestine. Thiscomparison also reinforces the likelihood thatchanges to the structure and function of the gut that,in the past, have been attributed to factors such aschronic stress (Bjork, 1989), dietary antigens (Miller,Newby, Stokes and Bourne, 1984), and a decrease indigestive and absorptive capacity (Gay, Barker andMoore, 1976; Hampson, 1983), are most likelyconfounded with the amount of food piglets eat.

Only Kelly, Smyth and McCracken (1991c) havestudied the effects of different levels of voluntary


food intake on the digestive and absorptivedevelopment of the weaned piglet. These authorsoffered (by stomach tube) a restricted (0, 25, 50, 75,100 g/day) or continuous (150, 175, 200, 225, 250 g/day, on average) amount of cereal-based diet topiglets weaned at 14 days of age for the first 5 daysafter weaning. Piglets given the restricted dietsuffered a 0-19 proportional reduction in meanvillous height and 013 decrease in mean crypt depth.Data from this and our previous study (Pluske et al.,1996) corroborate the work of Kelly et al. (1991c), andhighlight the marked effect luminal nutrition has ongut morphology.

Digestive enzyme activity after weaningIn vitro estimates of specific lactase and sucraseactivity failed to reflect large differences in villousheight and empty body-weight gain recorded inthese studies. Lactase and sucrase activities weregenerally higher in piglets given the starter diet andthose given milk at maintenance, although statisticaldifference was not always achieved because of thelarge variation observed between values. Thefindings from this and the previous experiment(Pluske et al., 1996) show an anomaly between thehistological structure and biochemical function of thesmall intestine that is not related to the observedgrowth rate. For example, piglets given ewes' milk orewes' milk plus glutamine in our previous reportconverted DM to body gain (food conversion ratio of0-7 to 0-8) with an efficiency analogous to that ofsucking piglets (Lucas and Lodge, 1961; Noblet andEtienne, 1987). In this study piglets given cows' milkad libitum grew almost 10 times faster and had villiproportionately 0-51 higher than piglets given Mabut had similar levels of lactase and sucrase.

The disaccharidase enzymes lactase and sucrasewere chosen as 'markers' of enterocyte maturity andfunctional capacity since Berg, Dahlqvist, Lindbergand Norden (1973) reported significant correlationsbetween the activity of these two enzymes andvillous architecture in biopsies of human smallintestine. Gay et al. (1976), Hampson and Kidder(1986) and Miller, James, Smith and Bourne, (1986)reported large reductions in the specific activity oflactase and sucrase with minimum values at 4 to 5days after weaning. These reductions may in fact beunderestimates of the real loss of disaccharidaseactivity since Miller et al. (1986), using cytochemicaltechniques that can measure enzyme activity amonga single villus, reported an additional three- to five-fold fall in the ability of individual enterocytes toexpress lactase activity, and a two-fold reduction insucrase expression. Since these changes werecoincidental with villous atrophy and crypthyperplasia, Hampson and Kidder (1986) postulatedthat an increased rate of enterocyte production in the

crypts, coupled with an increased rate of cellularmigration up the villus (Smith, 1984), resulted inenterocytes having insufficient time to differentiatefully and express maximum digestive and absorptivecapacity before being extruded from the villous apexinto the lumen (Rey, Schmitz, Rey and Jos, 1971).This is generally thought to result in cell loss beforemaximum digestive and absorptive capacity can beexpressed (Smith, 1984). The greater reduction inlactase activity than sucrase activity found in thesestudies was most likely due to the more apicaldistribution of lactase on the villus (Nordstrom andDahlqvist, 1973; Nichols and Nichols, 1988; Kelly etal., 1991a). An increase in crypt depth, therefore,would be expected to reduce the activity of lactasemore than sucrase after weaning.

Our data agree with those found by Kelly, Smythand McCracken (1991b and c). Kelly et al. (1991b)found a 0-55 proportional reduction in villous heightand a 0-23 increase in crypt depth by the 5th dayafter weaning, but found no statistical decrease ineither the specific or total activity of lactase or thespecific activity of sucrase, that might have beenpredicted from such large changes in gut structure.However on the 7th day after weaning, lactaseactivity was less than half that of unweaned pigletskilled at 22 days of age, and this coincided with asignficiant increase in crypt depth. As suggested byKelly et al (1991b), the delay in the loss of lactaseactivity may have been attributable to a highersupply of nutrients to the gut and /or be an artefactof the clean environment that the piglets werehoused, since it is known that exposure of weanedpiglets to a 'dirty' as opposed to a 'clean'environment causes a significant increase in cryptdepth (Miller et al., 1986). In the present experiment,crypt depth in piglets offered the starter diet hadincreased significantly by the 5th day after weaning,but the specific activity of lactase and sucrase werenot different from those given either of the milkdiets. Furthermore, Kelly et al. (1991c) reported a 019proportional decrease in mean villous height whenpiglets were offered a reduced quantity of food afterweaning, yet the specific activity of lactase andsucrase increased compared with piglets given fourtimes as much diet over the 5-day period. Pigletsgiven a restricted basis had shorter cryptscharacteristic of a reduction in crypt-cell productionrate (Al-Dewachi et al., 1975; Goodlad and Wright,1984; Goodlad et al, 1988) that, according to theliterature, would be expected to reduce the loss ofenzyme activity from the villous tip. Expression ofenzyme activity, either as specific or total activity,appears to provide only a crude appraisement of invivo digestive activity (Kelly et al, 1991c). The mostprobable reasons for differences between the variousreports are in the level of food intake achieved by


piglets between experiments and variations in themethodology for determining activity.

Absorptive capacity of the small intestine after weaningVillous atrophy at 3 to 4 weeks of age not only leavesfewer absorptive cells on the villi after weaning butis also associated with a reduction in the relativematurity of enterocytes because of faster rates ofproduction in the crypts (Hampson, 1986a). In thisinstance enterocytes may be extruded from thevillous apex before they express full absorptivecapacity (Rey et al., 1971; Smith, 1984; Miller et al,1986). However evidence to support this notion inthe weaner is equivocal. Numerous workers haveshown a reduced ability of piglets to absorb xylose(Miller et al., 1984; Hampson and Kidder, 1986;Hampson and Smith, 1986) and alanine (Smith, 1984;Miller et al., 1986) after weaning. In contrast, Kelly etal. (1991c) showed no decrease in the ability ofpiglets to absorb a standard xylose dose 5 days afterweaning.

In the present experiment there was no statisticaldifference between treatment groups in theircapacity to absorb xylose after weaning despiteappreciable differences in villous height. Lowestvalues were recorded in piglets offered cows' milkat 2-5 Ma and ad libitum, so perhaps animals in thesetreatments metabolized xylose to a greater extentand /or excreted more xylose in the urine since theywere growing faster. This would have resulted in alower level of xylose in the blood. Alternatively, theenterocytes in piglets given milk at maintenancemay have exhibited an adaptive increase inabsorptive capacity, since there are numerousreports, mainly in the adult rat, where starvationcauses a transient increase in absorption (Kershaw,Neame and Wiseman, 1960; Dowling, 1967; Lifshitz,Hawkins, Diaz-Bensussen and Wapnir, 1972;Wapnir and Lifshitz, 1974; Esposito, 1967; Bardocz,Grant, Brown, Ewen, Stewart and Pusztai, 1991).Gupta and Waheed (1992) reported that the surfacearea of the microvilli, the fluidity of the brush-border membrane and D-glucose transport throughintestinal epithelial membranes all showed anincrease during starvation compared with well fedcontrols. The increase in xylose absorption seen inthis study in piglets given cows' milk atmaintenance provides some support for this finding,but more precise techniques than the 'xyloseabsorption test' would have to be implemented toconfirm this possibility.

Compensatory mechanisms of growth for piglets fed apelleted starter dietPiglets given the starter diet grew at the same rate asthose given cows' milk at 2-5 Ma in the 5 days afterweaning despite suffering a 0-18 proportional

decrease in villous height and an 0-11 increase incrypt depth. The decrease in villous height suggestsa reduction in the surface area available for thedigestion and absorption of nutrients in the gut(Miller et al., 1986). It is likely that piglets given thepelleted diet compensated for this apparent loss ofdigestive and absorptive capacity by one of twomechanisms, or both: (i) an increase in the activity ofstarch-reducing enzymes, such as maltases andglucoamylase, located in the brush-bordermembrane, and (ii) production of short-chain fattyacids in the caecum.

Results from Kelly et al. (1990a and b) and Kelly et al.(1991b and c) showed increases in brush-bordermaltase and glucoamylase activity for piglets given asolid diet, even by 3 days after weaning, and supportMcCracken's (1984) notion of rapid substrateinduction of brush-border enzymes. Kelly et al.(1991b) found maximum activity of these enzymeson the 5th day after weaning, yet activity did notdiffer between piglets that differed in their level offood intake (Kelly et al., 1991c). This suggests that byday 5 after weaning the limit to growth may be theabsorption of these hydrolytic products. Assuggested by Kelly et al. (1991b), it is probable thatthe levels of these two enzymes may be morerelevant in limiting absorption of a weaner diet thaneither lactase of sucrase.

The increase in both the absolute and relative weight(g/kg EBW) of the large intestine followingconsumption of the pelleted solid diet after weaningconcurs with the findings of Kelly et al. (1991b), andsuggests a trophic effect of the starter diet onmucosal growth in the caecum and colon. The largeintestine is the major site of water and electrolyteabsorption in the weaned piglet (Hamilton and Roe,1977; Buddie and Bolton, 1992) and has considerablepropensity to produce volatile fatty acids. Theacidification of the caecum (pH = 6-0) and increasedgrowth of the large intestine of piglets given thestarter diet (Table 5) is consistent with thisproposition, and suggests a role for these acids in thesupply of energy to the newly weaned piglet. In abiological system where renewal of villousenterocytes may take 3 to 4 days following villousatrophy (Moon, 1971), the production of volatile fattyacids in the hind gut may be an important 'reservoir'of energy for the young pig. However a differentconclusion was reached by Hampson (1987), whoreported that in 26-day-old piglets weaned for 5days, microbial activity in the large intestine is notlikely to be developed sufficiently to have a majorinfluence on absorptive processes and maypredispose the weaned pig to diarrhoea. Ultimately,the extent of diarrhoea will depend on digestive andabsorptive processes in the small intestine and the


osmolality of active particles in the lumen of thelarge intestine.

Plasma metabolitesMetabolites were measured in this experiment togive an indication of the metabolic status of pigletsunder controlled conditions of voluntary food intake.Despite large differences in intake and the rate ofempty body-weight gain, only the plasma levels ofurea and thyroxine differed between treatmentgroups. In piglets given cows' milk from Ma throughto ad libitum intake, blood urea increased from 1-2 to1-9 to 3-0 mmol/1 (P < 0-01) in line with an increase inmilk protein intake. In the DM, nitrogen intake ofpiglets on a daily basis was 4-5 g, 10-2 g and 17-5 gfor piglets given milk at Ma, 2-5 Ma and ad libitumrespectively. The higher levels of urea found inpiglets consuming more energy may reflect an intakeof dietary protein in excess of that required bypiglets for protein deposition. The higher level offree thyroxine present in piglets given milk at 2-5 Maand ad libitum is most likely a reflexion of the higheranabolic state of these animals. Support for thisnotion comes from a review by Aherne, Williamsand Head (1991) showing that tissue anabolism inthe lactating sow is associated with an increase in thelevel of circulating thyroxine.

ConclusionsThis experiment showed that, when piglets are givenmilk above maintenance and the period of temporarystarvation after weaning is avoided, the balancebetween cell loss from the villous tip and celldivision in the crypts can be maintained comparedwith piglets killed at weaning. The linear response invillous height and crypt depth with an increase inDM intake in piglets given cows' fresh milk supportsthe theory of 'luminal nutrition' and its effects on gutmorphology after weaning. The biologicalsignificance of these relationships was strengthenedby the highly significant correlations found betweenvillous height, crypt depth and the rate of emptybody-weight gain. These relationships support thedirect biological link between voluntary food intakeand piglet growth after weaning. The dramatic effectof a reduction in voluntary food intake on mucosalmorphology was observed in piglets given food atMa. Since piglets suffer a period of 'temporarystarvation' immediately after weaning, these resultsprovide evidence that the villous atrophy observedin many previous studies, and thought attributableto a variety of causes, is likely to be confounded witha failure to control and quantify the amount of foodconsumed after weaning

Villous height and crypt depth in piglets given thestarter diet after weaning were also correlated to thelevel of voluntary food intake. However piglets in

this group suffered a decrease in villous height andan increase in crypt depth compared with pigletsgiven milk at similar levels of energy intake. Despitethese changes to the histological appearance of thegut, piglets given the starter diet grew at the samerate as those given milk (at 2-5 Ma). In the absence ofsupporting data, we can only speculate thatcompensatory mechanisms, including an increase inthe hydrolytic activity of maltase and glucoamylaseand the production of volatile fatty acids in thecaecum, must have provided more substrate forgrowth and been responsible for the similar rate ofgrowth observed. Since there was no disparity infood intake, the exact aetiology of alterations to gutmorphology cannot be resolved from this study.

AcknowledgementsJRP was in receipt of a Junior Research Fellowship from thePig Research and Development Corporation (PRDC) ofAustralia during this study. We are grateful to Professor D.J. Hampson from Murdoch University, Murdoch, WesternAustralia, for assistance with preparation of histologicalslides. 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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(Received 6 February ^995—Accepted 16 August 1995)

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Villous height and crypt depth in piglets in response to ... · 2-5 Ma; and (5) piglets given cow's fresh milk a Ond libitum the 5th day. all piglets were killed and samples of gut