J. Phy&iol. (1966) 183, pp. 570-575 With 1 plate Printed in Great Britain
THE SURFACE AREA OF THE INTESTINAL MUCOSA IN THE LACTATING RAT
BY R. BOYNE, B. F. FELL AND I. ROBB From the Rowett Research Institute, Buccksburn, Aberdeen

(Received 24 May 1965)

SUMMARY
1. The mucosal and serosal surface dimensions of the rat duodenum, jejunum and ileum were obtained by measurement of photographs of histological sections. The surface areas were calculated from these data. 2. Measurements were made on the intestines from virgin rats and from rats on the sixteenth day of lactation. The mucosal area/unit length of serosa was appreciably increased in all parts of the intestine during lactation. The mucosal area/serosal area (MA/SA) ratio was also markedly increased, but to a lesser extent distally, where an important component of the intestinal hypertrophy was dilatation of the intestine.

INTRODUCTION

Recent work has shown that there is a considerable increase in the weight and total nitrogen content of the wall of the alimentary canal in the lactating animal, and that this is associated with a general hypertrophy of the alimentary tissues. These changes, which progress throughout the suckling period, have been described in some detail in the rat (Fell, Smith & Campbell, 1963; Campbell & Fell, 1964), in grazing hill sheep (Fell, Campbell & Boyne, 1964), and have been noted also in the mouse (Campbell & Fell, 1964). Campbell & Fell were able to reduce or abolish the hypertrophy in rats by controlled feeding and they suggested that the hypertrophy is caused by the increased food intake normally associated with lactation. They found that lactating rats with both pronounced and partial hypertrophy, and control rats with normal intestines, all showed the same apparent coefficient of digestibility of crude protein, suggesting that the hypertrophy is an adaptive response that enables the animals to maintain a constant coefficient of digestibility at increased levels of food consumption. In histological preparations, the intestinal hypertrophy as assessed by the height of the villi appeared to be relatively greater in the jejunum and ileum than in the duodenum (Fell et at. 1963).
571 INTESTINAL SURFACE AREA In this paper we have tried to present a more objective description of the changes in the surface area of the intestinal mucosa, by the measurement of enlarged photographs of histological sections.
METHODS The rats used were eight virgin Hooded Lister fomales, and eight parous rats of the same strain and of similar body weight, at the sixteenth day of lactation. The food was Diet 86 rat cubes (North-Eastern Agricultural Co-op. Soc. Ltd., Aberdeen), fed ad libitum. Laparotomy was performed under pentobarbitone sodium anaesthesia (Nembutal: Abbott Laboratories Ltd.) and the intestines were prepared by Warren's (1939) method as modified by Wood (1944). The modified technique results in a preparation of intestine that has been rapidly fixed under standard conditions of distension. After fixation in 10 % formol-saline, single pieces of intestine approximately 8 cm long were cut from the duodenum (5 cm from pylorus), jejunum (middle of the small intestine) and ileum (5 cm from ileo-caecal valve). After a further period of fixation, two segments of 2 cm, and two segments of 1 cm were cut from each piece. The selected tissues were processed in the normal way for the preparation of paraffin-wax sections. Transverse sections were prepared from the short segments, and longitudinal sections from the 2 cm lengths, after these had been trimmed until the greatest diameter was reached. The sections were stained by haematoxylin and eosin. Photographic technique. The microscope slide on which the section was mounted was placed in the negative holder of a Leitz Focomat II C enlarger and the image was projected on to an Ilford 'Special Contrasty' 3% x 31 in. (8% x 8% cm) bromide lantern plate. After processing, the enlarger was used to obtain from the plate a greatly enlarged image on Kodak Normal Bromide WSG 2S paper cut from a '20 in. x 10 m.' (50-8 cm x 10 m) roll. This was developed and fixed. In this way, 192 photographs were obtained at magnifications of 60 times or more (P1. 1, figs. 1 and 2). Two of the photographs were wholly unsuitable, and many others had short lengths that were unsuitable for measurement owing to tangential sectioning or other defects. Measurement and calculation of surface areas. The serosal and mucosal outlines of the photographs were measured with a rotameter, except for the unsuitable areas of mucosa and the lengths of serosa related to them. Magnifications were obtained by direct measurement of the photographs, and of the original sections by the combined use of calibrated microscope ocular lens and mechanical stage. The actual measurements of the histological preparations were calculated from these data. Average results were calculated for the duodenum, jejunum and ileum of each rat from the two transverse and the two longitudinal preparations of each part, to obtain representative mucosal and serosal circumferences, and mucosal and serosal lengths. The area of the mucous membrane was calculated using the formula recommended by Warren (1939). If SC = serosal circumference, MC = mucosal circumference, SL = serosal length, ML = mucosal length, SA = serosal area, MA = mucosal area, then an approximation for mucosal area is an estimated area, EA, and EA MC ML SA SC+SLRESULTS

When the photographs were measured by different workers, the results differed by not more than 6 % and usually by considerably less than this. Determination of the magnification factors was done twice, by two of us,
572 B. BOYNE, B. F. FELL AND I. ROBB and where discrepancies occurred the measurements were repeated until full agreement was reached. The estimated mucosal surface areas of the different parts of the intestines of normal and lactating rats are given in Tables 1 and 2. The data show that in the control virgin rats the jejunum has the greatest, and the ileum the least mucosal area/unit area of serosa. There is a considerable increase in the mucosal surface area/unit area serosa in all parts of the small intestine during lactation.
TABLE 1. The surface area of the intestinal mucosa in virgin rats
Duodenum Estimated Body mucosal area/ weight unit length of MA/SA Rat (g) serosa (cm2) ratio 1 5-62 7-5-240 6-8-77 7*4 5*6-5*6-4-38 5*7 5*7*4-54 5-Average values 6-76

Jejunum

Estimated Estimated mucosal area/ mucosal area/ unit length of MA/SA unit length of MA/SA serosa (cm2) ratio serosa (cm2) ratio 7-52 6-66 4-41 4*41 6-24 6-65 5-23 5-94 6-61 6-5*86 8-21 6-83 5-80 7-06 5-85 6.35 5*90 6-03 6-79 6-41 6-59 7*24 7*29 8-91 6-9-45 10-70 6*07 7*46 5*69 6-20
TABLE 2. The surface area of the intestinal mucosa in rats on the sixteenth day of lactation

Duodenum

Body weight Rat (g) Average values
Estimated mucosal area/ unit length of MA/SA serosa (cm2) ratio 12-91 12*91 10-06 9-98 8-89 10-72 9-16 9-53

9-87 13-17

11-43 10-75
10-41 12-54 11-56 12-44 11-26
Estirnated Estimated mucosal area/ mucosal area/ unit length of MA/SA unit length of MA/SA serosa (cm2) ratio serosa (cm2) ratio 11-19 11.08 10-82 8-38 10-58 7-89 9-83 7*28 13-79 9*18 -8*95 7-64 13-36 10*77 9-22 7-20 12-50 10.08 11.05 8-64 15-72 11-39 9.09 6-74 10-15 7-64 8-77 7-10-28 9-64 8-68 12-32 9.79 9-67 7.73

DISCUSSION

The validity of the method used to estimate the mucosal area of the intestine is discussed by Warren (1939) and by Fisher & Parsons (1950). The values obtained by this method are approximations and Fisher & Parsons have derived a new and more accurate expression that takes into account the fractions of mucosal length and circumference not occupied by vili. This requires additional separate measurements of serosal projections of the lengths of flat mucosa between the villi.
573 INTESTINAL SURFACE AREA Our results differ from those of Fisher & Parsons (1950) in that we obtained the maximum average values for mucosal area/unit length of serosa at the middle of the intestine. This would be about 50 cm from the ileo-caecal valve. Fisher & Parsons, using fewer rats but a more refined method, concluded that the mucosal area/unit length of small intestine increased linearly with distance from the ileo-caecal valve. It seemed possible that the discrepancy could be due to the different expressions used to calculate the mucosal areas; we therefore made the extra measurements and applied the modified formula to rats four and five from each of our two groups.

TABLE 3. The surface area of the intestinal mucosa of the rat calculated by the method of Fisher & Parsons (1950)
MA/1 cm length (cm2) 4-41 4-25 4-33 7-01 8-25 7-63

MA/SA ratio 5-66 5.0

MA/i cm

length

MA/I cm
MA/SA ratio 6-03 5-82 8-89
Rat Virgin no. 4 Virgin no. 5 Average values Lactating no. 4 Lactating no. 5 Average values

7-29 8-70 8*0

(cm2) 5.49 4-63 5-06 11-03 9-96 10*50

MA/SA ratio 5-65 4-99

5-88 7X52 6-70

9-62 8-58

8503 8546
These results, which are set out in Table 3, are lower than before, but the situation remains essentially unchanged, with the highest values for mucosal area occurring in the jejunum. The results for jejunum and ileum in the control rats are comparable with those reported by Fisher & Parsons (1950). The differences we found between the results from virgin and lactating rats using either expression appear to be substantial enough to show that the mucosal surface area of the intestine is greatly increased in lactating rats feeding ad libitum. The components of the hypertrophy are dilatation of the intestine and villus enlargement. An increase in length of the intestine in lactating rats was noted earlier (Fell et al. 1963). When the microvilli of the epithelial cells are taken into account the intestinal surface area in both control and lactating rats is further increased, probably by a factor of 24 (Palay & Karlin, 1959). We have no data on possible changes in the form of the microvilli during lactation. It was suggested earlier (Fell et al. 1963) that the greatest villus enlargement during lactation occurs in the more distal parts of the small intestine, but it now appears that the duodenum shows the greatest increase in estimated mucosal surface area/unit area of serosa. The comparison by counting of villi in specimens photographed at the same magnification, showed that there was not a significant increase in the number of villi/unit length of section of duodenum in lactating rats. Dilatation of
B. BOYNE, B. F. FELL AND I. ROBB the intestine would obviously have an important effect on the results given in the Tables. When the measurements of serosal circumference of the organs in control and lactating rats were compared it was found that the average for the duodenum increased from 0-81 to cm, for the jejunum from to cm and for the ileum from 0.92 to cm. These increases doubtless reflect a physiological enlargement but it is possible also that the duodenum might have had more resistance to distension by fixative than did the other organs. Fisher & Parsons (1950) concluded that there is negligible stretching of the mucosa except in extreme distension of the organ; it seems probable, however, that the MA/SA ratio would tend to be reduced by distension. In the duodenum the villi were appreciably taller than in the controls: they were also thicker and presented a complex outline due to branching or fusion. In the jejunum and ileum, relatively simple elongation of villi was the main change noted. It appears that rats that are lactating must be excluded from the generalization, made for growing rats by Cori (1925), and extended to adults by Wood (1944), that there is a constant ratio between intestinal absorbing surface area and body weight. Cori arrived at his generalization by studying the rate of absorption of sugars from the alimentary canal in rats. He showed that there is a proportionality between the amount absorbed and body weight, and hence between intestinal surface area and body weight. The matter is discussed by Wiseman (1964) who notes that later workers have found that absorption coefficients based on body weight are inadequate.

We thank Professor R. C. Garry of the Institute of Physiology, University of Glasgow, for his suggestion that we should make these measurements.

REFERENCES

CAMPBELL, R. M. & FELL, B. F. (1964). Gastro-intestinal hypertrophy in the lactating rat and its relation to food intake. J. Phy8iol. 171, 90-97. CoRI, C. F. (1925). The fate of sugar in the animal body. I. The rate of absorption of hexoses and pentoses from the intestinal tract. J. biol. Chem. 66, 691-715. FELL, B. F., CAMPBELL, R. M. & BOYNE, R. (1964). Observations on the morphology and nitrogen content of the alimentary canal in breeding hill sheep. Res. vet. Sci. 5, 175-185. FELL, B. F., SMITH, K. A. & CAMPBELL, R. M. (1963). Hypertrophic and hyperplastic changes in the alimentary canal of the lactating rat. J. Path. Bact. 85, 179-188. FISHER, R. B. & PARSONS, D. S. (1950). The gradient of mucosal surface area in the small intestine of the rat. J. Anat. 84, 272-282. PALAY, S. L. & KARLIN, L. J. (1959). An electron microscopic study of the intestinal villus. I. The fasting animal. J. biophy8. biochem. Cytol. 5, 363-372. WARREN, R. (1939). Serosal and mucosal dimensions at different levels of the dog's small intestine. Anat. Rec. 75, 427-437. WISEMAN, G. (1964). Ab8orption from the Intestine, pp. 22-23. London: Academic Press. WOOD, H. 0. (1944). The surface area of the intestinal mucosa in the rat and in the cat. J. Anat. 78, 103-105.
The Journal of Physiology, Vol. 183, No. 3

Fig. 1

Fig. 2
R. BOYNE, B. I. FELL AND I. ROBB

(Facing p. 575)

INTESTINAL SURFACE AREA
EXPLANATION OF PLATE Fig. 1. Histological preparation of rat jejunum photographed against a 1 m rule and against the enlarged photograph of the section, as used for measuring. Fig. 2. Part of a photographic print of rat jejunum obtained from the original tissue section by projection through the Focomat IIC enlarger.