Effect of Dietary Copper on Intestinal Mucosa Enzyme Activity, Morphology, and Turnover Rates in Weanling Pigs' S. V. Radecki2, P. K. Ku*,M. R. Benninkt, M. T. Yokoyama", and E. R. Miller*J Departments of *Animal Science and +Food Science and Human Nutrition, Michigan State University, East Lansing 48824
ABSTRACT: Twenty-four pigs from four litters weaned a t 21 d of age (6.6 kg of BWI were used to evaluate the influence of 250 ppm of dietary Cu on
intestinal mucosa glucose-6-phosphatase (GPI, alkaline phosphatase (AP), and adenosine triphosphatase (ATPase) activity; mucosal morphology; and the turnover rate of the intestinal mucosa throughout the gastrointestinal tract. Pigs were allotted into four pens of six pigs each based on sex, litter, and weight. Pens were then assigned to one of two treatments: 1) corn-soybean meal-whey diet with no antimicrobials (CO), or 2) CO + 250 ppm of Cu. Pigs were fed twice daily an amount approximately equal to a d libitum intake for 14 d. On d 14, pigs were injected i.p. with i3Hlthymidine (50 @/kg of BW) 10 h after the morning meal. One pig from each pen was euthanatized at 1, 6, 12, 20, 32, and 44 h postinjection, and intestinal tissue was collected from the duodenum, two
jejunum sites (upper and lower), ileum, cecum, and colon. The activity of GP and AP in the lower jejunum tended to decrease in pigs fed Cu (P c .11, P e .08,respectively). The ATPase activity was not affected by treatment (P > .lo). Crypt death, villus height, or epithelial cell size (P > .lo) were not affected by feeding Cu. Migration rate of epithelial cells up the villus was also not affected by treatment (P > .lo). Turnover rate of the intestinal mucosa of the upper and lower jejunum was slower (P c .lo; P c .05, respectively) and the lower jejunal cell generation interval was longer (P e .05)in pigs fed the Cu diet. From these data we conclude that the addition of 250 ppm of Cu to the diet of weanling pigs slows the turnover rate of the jejunal intestinal mucosa, which may result in a lower energy requirement for the maintenance of this tissue.
Key Words: Pigs, Antimicrobials, Copper, Intestinal Mucosa
J. h i m . Sci. 1992. 70:1424-1431
Introduction The addition of "supranutritive" levels of Cu to the diets of young growing pigs increases their average daily gain and gain efficiency (Zimmerman, 1986).However, the mechanism by which this feed additive functions is not well understood. It is
'Acknowledgement is made to the Michigan Agricultural Experiment Station for their support of this research. 2Present address: Dept. of h i m . Sci., Rutgers Univ., New Brunswick, NJ 08903. 3To whom correspondence should be addressed. Received June 17, 1991. Accepted December 2, 1981.
likely that toxic compounds produced by the microflora in the gastrointestinal tract (GIT) of the pig are involved, because little improvement in growth performance is seen when germ-free animals are fed high Cu or other growth-enhancing, antimicrobially supplemented diets (Whitehair and Thompson, 1956; Coates et al., 1963; Shurson et al., 19901. Reducing the production of these toxic compounds through the use of Cu may be one mechanism by which this feed additive improves growth performance. Various toxic compounds, especially ammonia, can alter cell metabolism and cell life span in a manner that may be detrimental to the animal (Visek, 1972). In rats, the presence of bacteria in the GIT decreases the cell
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COPPER AND THE INTESTINAL MUCOSA IN PIGS
generation interval of intestinal epithelial cells (Lesher et al., 1964). Also, the activity of enzymes important in the transport of nutrients across the intestinal wall is decreased when bacteria are present in the GIT (Kawai and Morotomi, 1978). Thus, decreasing the concentration of toxic bacterial metabolites in the GIT may slow the turnover rate of the mucosa and consequently decrease the energy requirements of the intestinal epithelial cells. The energy spared by these processes may be available to the pig for lean tissue gain. In this study, the influence of 250 ppm of dietary Cu on these metabolic processes of the intestinal mucosa was investigated. In addition to these objectives, measures of intestinal epithelial cell turnover rates were determined, because these measures have not been reported for swine.
Materials and Methods Animals and Diet. Twenty-four crossbred pigs (Yorkshire x Landrace) were weaned a t 21 d of age (6.6 kg of BW) and assigned to one of two treatments based on litter, sex, and weight. Pigs were housed in 1-m x 2-m stainless steel pens (six pigs per pen) in a n environmentally controlled (27’C) nursery. Treatments included a control diet (CO,Table 1) devoid of antimicrobials, and CO + 250 ppm of Cu as CuS04.5H20 (CUI. All pens of pigs were fed twice daily (0800 and 1700) a n amount approximately equal to a d libitum intake (6to 8% of BW) for 14 d. Water was available at all times. Average daily gain was calculated for the 14-d treatment period. On d 14, the morning meal was fed to each pen a t 1-h intervals starting at 0600, as compared to feeding all pens at 0800. This change in feeding regimen on d 14 helped to facilitate subsequent tissue collection and likely had little effect on pig performance. Ten hours postprandial, I3Hlthymidine (specific activity 43 Ci/mmol, Amersham, Arlington Heights, IL) was injected (50 pCi/ kg of BWI i.p. The 13Hlthymidinewas diluted with sterile saline to 100 pCi/mL before injection. Tissue Collection. One pig from each pen (two per treatment) was euthanatized at 1, 6, 12, 20, 32, and 44 h after the t3Hlthymidine injection. Animals were euthanatized by asphyxiation with C02 gas in a gassing chamber. The GIT was quickly removed, and intestinal tissue samples were collected from the duodenum, jejunum (two sites], ileum, cecum, and colon. The duodenum was defined as the region of the small intestine between the stomach and the point at which the bile duct entered the intestinal tract (approximately 10 cm). The ileal-cecal valve and the Downloaded from https://academic.oup.com/jas/article-abstract/70/5/1424/4705245 by University of California, Berkeley/LBL user on 04 August 2018
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Table 1. Composition of the basal dieta Inmedient Ground, shelled corn Soybean meal, 4 4 % CP Dried whey Ground limestone Mono-dicalcium phosphate Selenium-vitamin E premixb Vitamin-mineral premixC L-lysine.HCl(78%) Salt Calculated analysis Lysine, % Ca, % p. %
0%
55.35 25.0 15.0 1 .o 1.5 1 .o .75 .15 .25 1.12 .88 .75
*The treatment diet was formulated by adding 250 ppm of Cu (as copper sulfate1 at the expense of ground, shelled corn. bF’rovided .1 mg of Se and 17 IU of vitamin E per kilogram of diet. CProvided the following amounts of vitamins and minerals per kilogram of diet: vitamin A, 3,300 IU;vitamin D, 000 IU; menadione sodium bisulfite, 2.2mg; riboflavin, 3.3 mg; niacin, 18 mg; D-pantothenic acid, 13 mg; choline, 110 mg; vitamin Bl2, 20 pg; Zn, 75 mg; Mn, 34 mg; Fe, 60 mg;I, .5 mg.
ileocolic artery were used to demarcate the end points of the ileum (approximately 10 cml. Jejunum samples were taken from the upper (jejunum-A) and lower (jejunum-B) halves of the section of intestine between the duodenum and ileum. Samples collected from the cecum were obtained from the end of the cecum, and the colon sample was taken from the spiral colon. Tissue samples (1 to 2 gl were washed with saline and quickly frozen in a dry ice/acetone bath for enzyme analysis. For morphology and autoradiography, lengths of GIT (2 to 4 cm) were removed, gently flushed with saline, ligated at both ends with string, and filled with 10% buffered formalin using a syringe. These tissue samples were then placed in 10% buffered formalin. All samples were collected within 15 min after death. The use and handling of animals was approved by the Michigan State University Committee on Animal Use. Enzyme Assays. Approximately .5 g of tissue from each GIT site was minced with scissors and placed in 18-mm x 150-mm test tubes. Tissues were homogenized in Tris buffer (50 mM) with a Polytron Homogenizer (Brinkman Instruments, Westbury, NY) for 15 to 30 s. The homogenate was centrifuged (850 x g) for 10 min at 4°C. The supernatant was used for enzyme and protein analysis. Alkaline phosphatase activity was assayed colorimetrically by monitoring the appearance of p-nitrophenol using Sigma Kit 245 (Sigma Chemical, St. Louis, MO). Enzyme activity was expressed as micromoles of inorganic phosphorus (Pi) liberated per minute per milligram of protein.
RADECKI ET AL.
1426 100
pen 1
d-
+ -0-
.-u)
.-c E
-0
pen2 pen3
4 3 pen4
70
60
20 10 1
01
.
1
1
1
/
6
1
1
1
/
1
1
1
1
1
l
1
12
l
1
/
1
1
1
1
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32
20
I
l
l
l
l
l
/
l
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~
l
l
l
l
I
44
Hours post-injection Figure 1. Percentage of labeled mitoses in the ileum. Each point represents one pig. Pens 1 and 2, copper-fed pigs; pens 3 and 4, control-fed pigs.
The activity of adenosine triphosphatase (ATPase) was assayed by the differential method of Hirschhorn and Rosenberg (1968).Briefly, .1 mL of the supernatant was added to 2.4 mL of a medium that contained 140 mM NaCl and 16 mM KC1. A second .l-mL aliquot was added to 2.4 mL of a second medium containing no added NaCl or KC1. Both media contained 2.5 mM ATP (dipotassium salt) and 30 mM Tris buffer. The final pH of both media was 7.4. Reactions were carried out at 37°C for 60 min. The reaction was stopped with 1.5 mL of 6 M perchloric acid. The amount of Pi in the media (indicating the activity of the phosphatase) was determined colorimetrically (Gomori, 1942). The activity of Na/K dependent ATPase was calculated by subtracting the enzyme activity in the medium with no added Na or K (Na/K independent ATPase) from the activity detected in the medium with added Na and K (total ATPase activity). Enzyme activity was expressed as micrograms of Pi liberated per hour per milligram of protein. The activity of glucose-6-phosphatase was determined by monitoring the release of Pi from glucose-6-phosphate in 30 min (modified from Cori and Cori, 19521. The media contained .5 mL of .01 M glucose-6-phosphate, .3 mL of .1 M potassium citrate, and .2 mL of the supernatant. The reaction was incubated for 30 min at 37°C then stopped with 10% trichloroacetic acid. Supernatant was Downloaded from https://academic.oup.com/jas/article-abstract/70/5/1424/4705245 by University of California, Berkeley/LBL user on 04 August 2018
also added to a second set of media and acidified immediately. Acidified media were centrifuged (2,000 x gl for 5 min, and the concentration of Pi liberated by the reaction was determined (Gomori, 19421. Glucose-6-phosphatase activity was then calculated as the difference between the amount of Pi detected in these two media for each GIT site and expressed as micrograms of Pi liberated per minute per 100 milligrams of protein. Total soluble protein was determined by the procedure of Lowry et al. (1951). Morphology and Autoradiography. Formalin-fixed tissue was embedded in paraffin, sliced into 6 - p sections, and mounted on glass slides. Paraffin was removed with xylene, and the tissue sections were hydrated with increasing proportions of water in ethanol. After hydration, the slides were dipped in Kodak NBT-29 (Eastman Kodak, Rochester, NY) emulsion in a darkroom and placed in light-proof boxes. Slides were stored at 4°C during a 14- to 38-d exposure period and then developed. Slides were then stained with hematoxylin and eosin. The number of cells in crypt and villus columns were counted, and the height of these columns was measured with a n ocular micrometer. Columns were defined as a single row of epithelial cells extending from the base of the crypt to the tip of the villus. The position of the 13Hlthymidinelabeled cells along the columns was also noted.
1427
COPPER AND THE INTESTINAL MUCOSA IN PIGS
Ten columns per sample were counted when possible, and these counts were averaged. For each GIT site, the percentage of labeled mitosis (number of labeled mitotic cells divided by the total number of cells in mitosis1 was plotted against time between 13Hlthymidine injection and euthanasia of the pig, for each pen. Figure 1 is a n example of this plot for the ileum. From these plots, the DNA synthesis phase can be estimated as the time interval between one-half of the maximum percentage of labeling on the ascending and descending slopes of the first peak (Cleaver, 1967). The percentage of cells labeled at 1 h postdose was also determined, by counting the total number of cells labeled in the crypt and dividing by the total number of cells in the crypt. This population of cells represents those cells that have incorporated the [3Hlthymidine but have yet to divide again. The cell generation interval can then be calculated as the length of the DNA synthesis phase divided by the percentage of cells labeled 1 h after the 13Hlthymidine administration (Cleaver, 19671. Therefore, because each point represents one pig, and each line in Figure 1 represents one pen, the experimental unit is the pen.
100
80
The turnover rate of the mucosa was calculated as the amount of time necessary for the leading labeled epithelial cell in the crypt or villus to reach the tip of the crypt or villus, depending on the site. For those sites with villi (duodenum, jejunum, and ileum), migration starts a t the crypt/villus junction. In the colon and cecum, migration rates were calculated starting from the base of the crypt. These turnover rates were estimated from the migration rate (slope of the regression line) of the labeled cells up the crypt or villus for each pen. Figure 2 is a n example of this plot in the lower jejunum. Statistical Analysis. The influence of Cu and(or1 GIT site on mucosal morphology and epithelial cell enzyme activity was detected using a repeated measure analysis (Gill, 1978). In this analysis, the main effects of treatment and GIT site, as well as the interaction of treatment and GIT site, could be evaluated. The error term used to test the effect of treatment was animals (or pens1 within treatment, and the error term to test GIT site or the interaction of GIT site and treatment was the residual error term. When the variation among pens was trivial (P r .251, pen variation was pooled with animal within treatment error, and
1
70 60
50 40 c
0
30 / *
, --e-
- /
C
.-2 0
v)
20
-8
0
a
10 c
+
pen2
-0-
pen3
-3 p e n 4
Figure 2. Jejunum-B epithelial cell migration along the villus, expressed as a function of time after the injection of [3H]thymidine. Regression equations for each pen of pigs were as follows: Pen 1, Y = 1 . 3 1 -~ 32.55, r = .91; Pen 2, 1.26~- 32.07, r = .84. Each point Y = 1 . 2 3 ~- 34.7, r = 3 4 ; Pen 3, Y = 1 . 5 7 ~- 27.25, r = .93; Pen 4, Y represents one pig. Data points at 6 and 32 h for Pen 2 and at 6 and 44 h for Pen 3 were not determined due to a lack of well-positioned villi for the pigs euthanatized at these time points. Pens 1 and 2, copper-fed pigs; Pens 3 and 4, control-fed pigs. Figure is representative of other GIT sites.
-
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1428
RADECKI ET AL.
Table 2. Influence of dietary copper on weight gain in weanling pigs Treatment Item n Day 0, kg Day 14, kg ADG, gb
Control 12 6.6 6.3
-24
Copper
SEM
P
12 6.6
.19 .22 9.2
7.4 56
NSa ,003 ,001
aNot significant, P > ,113. bAverage daily gain for the 14-d treatment period.
animal served as the experimental unit. Otherwise, pen served as the experimental unit. The influence of time after the [3Hlthymidine injection was not included in the statistical analysis of these measurements. Treatment means were separated using a Bonferroni t-test (Gill, 1978). 'The influence of Cu and(or1 GIT site on the generation interval was determined similarly; however, pen served as the experimental unit, because the determination of these values was based on the values obtained over time for each pen of pigs. Mucosal turnover rates were estimated using linear regression equations determined for each pen at each GIT site, regressing the location of the labeled cell on the cell column over time. Correlation coefficients (r) ranged from .57 to 98, with a
Table 3. Influence of dietary copper on the specific activity of glucose-6-phosphatase and alkaline phosphatase in the gastrointestinal tract of weanling pigsa Treatment Intestinal site Glucose-6-phosphataseb Duodenum Jejunum-A Jejunum-B Ileum Cecum Colon Alkaline phosphatased Duodenum Jejunum-A Jejunum-B Ileum Cecum Colon
Control
Copper
7.1 7.6 9.4 6.4 2.2 2.4
6.1 6.7 6.5 5.0 3.2 2.4
50.1 146.6 168.1 112.5 72.6 68.7
35.2 136.6 109.8 101.4 81.4 74.8
P
NSC NS .ll
NS NS NS NS NS .08
NS NS NS
*Values represent the average of 12 pigs. bMicrograms of inorganic P released per minute per 100 mg of protein. SEM, treatment = 1.01;gastrointestinal site = 1.19. Main effect of gastrointestinal site, P < ,001. CNot significant, P > .25. dMicromole of inorganic P released per minute per milligram of protein. SEM, treatment = 13.35;gastrointestinal site = 17.65.Main effect of gastrointestinal site, P < ,001. Downloaded from https://academic.oup.com/jas/article-abstract/70/5/1424/4705245 by University of California, Berkeley/LBL user on 04 August 2018
mean of .84 across all sites and pens. The slope of these regression equations served as a measure of the rate of migration of the epithelial cell up the villus or crypt. An inverse prediction equation was then formulated to calculate the turnover rate, using the migration rate and the length in cells of the villus (duodenum, jejunum-A, jejunum-B, and ileum) or crypt (cecum and colon). The influence of treatment on the predicted turnover rate of the intestinal mucosa was tested by calculating confidence intervals, which were estimated using error associated with the epithelial cell column length, error associated with the slope of the regression equation, and the error associated with time (Gill, 1978).
Initial and final weight, as well as average daily gain data, were analyzed as a one-way ANOVA, with pigs serving as the experimental unit.
Results Pigs fed Cu were heavier on d 14 and thus grew faster than CO-fed pigs (Table 2). There were no GIT site x treatment interactions (P > .25). Therefore, only the main effects of GIT site and treatment will be discussed. Glucose-6-phosphatase activity tended to be lower (P c .11) in the mucosa of the lower jejunum of pigs fed the Cu diet than in the mucosa of the lower jejunum of pigs fed the CO diet (Table 3). When the activity of this enzyme was pooled across small intestine sites, the decrease in activity due to Cu was more evident ( P e .011. There was a main effect ( P c .001) of GIT site on the activity of glucose-6-phosphatase. The activity of alkaline phosphatase also tended to be lower ( P c .08) in the mucosa of the lower jejunum when pigs were fed the Cu diet rather than the CO diet (Table 31. Again, when small intestine sites were pooled, the decrease in activity due to Cu was more evident ( P c .02). The activity of this enzyme was also affected by GIT site ( P c
.oo I). Total ATPase, Na/K dependent ATPase, and Na/K independent ATPase activity were unaffected ( P > .lo) by treatment (data not shownl. Total ATPase, Na/K dependent ATPase, and Na/ K independent ATPase activity averaged 128 G E M = 8.61, 27 G E M = 3.51, and 101 (SEM = 7.5) micrograms of phosphate released per hour per milligram of protein, respectively. The addition of 250 ppm of Cu to the diet had no effect on crypt depth (number of cells or micrometers), villus height (number of cells or micrometers), total height of the villus/crypt structure (number of cells or micrometers), or cell size (micrometers; Tables 4 and 5).
1429
COPPER AND THE INTESTINAL MUCOSA IN PIGS
Duodenal estimates of these measures of the mucosal morphology could not be determined due to a lack of well-positioned crypts and villi. Copper had little influence on the length of the DNA synthesis phase (Table 61. However, the cell generation interval (length of the cell cycle) of animals fed the Cu diet was longer (P c .05) in the lower jejunum than in pigs fed the CO diet (Table 6). Generation interval was not affected by treatment at other GIT sites (P > .lo). The rate of cell migration (number of cell positions/hourl is shown in Table 7. Copper tended to decrease the rate of cell migration up the crypt (P < .08) in the cecum. The turnover rate of the intestinal mucosa of the upper jejunum tended to be slower (P e .lo) when pigs were fed the Cu diet than when they consumed the CO diet (Table 7). The turnover rate of the mucosa of the lower jejunum was slower (P c .05) when pigs were fed Cu (Table 7).
Discussion
similar to that in the germ-free animal, antimicrobials may similarly increase the activities of these enzymes. However, the addition of Cu to the diet did not increase the activity of these enzymes. The addition of Cu to the diet tended to decrease the activity of glucose-6-phosphatase(lower jejunum; P < .11) and alkaline phosphatase (lower jejunum; P c .081. The activity of these enzymes at the other GIT sites, together with the activity of ATPase, were not affected by the addition of Cu to the diet (P > .lo). The influence of GIT site on the activity of glucose-6-phosphatase, alkaline phosphatase, and ATPase is not surprising, because the digestive and absorptive capacity of the small intestine is known to be greater than that of the cecum and colon. In previous work with high-Cu diets fed to weanling pigs, Shurson et al. (1990) observed a n increase in villus height and crypt depth. Changes in morphology due to the addition of 250 ppm of Cu to the diet were not significant in our study. However, tendencies did exist that were similar to the observations of Shurson et al. (19901. Values
The activities of glucose-6-phosphatase, alkaline phosphatase, and adenosine triphosphatase have been shown to be greater throughout the GIT of germ-free mice than in the GIT of conventionally reared mice (Kawai and Morotomi, 19781. If antimicrobials function to produce a situation in the GIT
Table 5. Influence of dietary copper on the morphology of the gastrointestinal tract of weanling pigs (micrometers per crypt-villus column)a Treatment Intestinal site
Table 4. Influence of dietary copper on the morphology of the gastrointestinal tract of weanling pigs (number of epithelial cells per crypt-villus column)a Treatment Intestinal site Crypt dept hb Jejunum-A Jejunum-B Ileum Cecum Colon Villus heightd Jejunum-A Jejunum-B Ileum Total heighte Jejunum-A Jejunum-B Ileum
Control
Copper
P NSC
32.2 28.8 29.1 54.9 53.8
32.6 32.5 30.0 49.0 58.3
52.7 49.2 47.6
66.4 52.1 63.3
NS
84.9 78.1 76.6
98.2 85.1 93.0
NS
NS NS .20 .25 .20 .15 .20 .15
aEach mean represents 12 pigs. bSEM, treatment = 2.67; site = 3.15. Main effect of site, P e
.oo 1.
'Not significant, P =. .25. dSEM, treatment = 6.40, site = 6.94. eVillus t crypt height. SEM, treatment Main effect of site, P c .07.
Crypt depthb Jejunum-A Jejunum-B Ileum Cecum Colon Villus heightd Jejunum-A Jejunum-B Ileum Total heighte Jejunum-A Jejunum-B Ileum Cell sizef Jejunum-A Jejunum-B Ileum Cecum Colon
P
Control
Copper
189.6 171.5 184.9 376.6 387.3
181.7 209.9 195.8 345.3 412.4
368.5 327.0 290.0
44 1.3 371.3 386.3
NS
558.1 498.5 474.9
620.5 587.2 582.1
.25 .20 .15
6.6 6.4 6.2 6.9 7.2
6.4 6.7 6.4 7.1 7.2
NS
~~
NSC .20
NS .25
NS .25 .15
NS NS NS NS
~_________
*Each mean represents 12 pigs. bSEM, treatment = 18.40; site = 21.99. Main effect of site, P < ,001.
CNot significant, P > .25. dSEM, treatment = 44.04; site
= 42.07.
Main effect of site, P
= 41.75.
Main effect of site, P
e .03.
%EM, treatment
= 43.45;
site
< .04. =
6.19;
site
= 6.45.
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fMicrometers divided by number of cells (from Table 41. SEM, treatment = .34; site = .40. Main effect of site, P e .004.
RADECKI ET AL
1430
Table 6. Influence of dietary copper on the DNA synthesis phase of the cell cycle and the cell generation interval of epithelial cells of the gastrointestinal tract of weanling pigsa Treatment Intestinal site DNA synthesis phaseb Duodenum Jejunum-A Jejunum-B Ileum Cecum Colon Cell generation i n t e n d Duodenum Jejunum-A Jejunum-B Ileum Cecum Colon
Control
Copper
8.6 8.2 9.7 6.9 10.4 8.5
8.5 9.3 7.2 7.5 12.6 9.2
41.2 26.5 20.4 19.9 38.2 35.1
27.4 33.0 44.6 22.4 37.5 30.6 ~
P NSC .20 .10
NS .10
NS .15
NS .05
NS NS NS ~~
&Two pens per treatment. bHours. SEM, treatment = .43; site = .77. CNot significant, P > .25. dHours. SEM, treatment = 5.65; site = 5.71.
for the different morphological features were within the range of previously reported values (Moon, 1970; Shurson et al., 1990). There are no published data on the number of cells in the crypt or villus columns in the pig GIT. The main effect of GIT site on the various morphological measures was expected, because this result has also been noted in the chick (Imondi and Bird, 1966) and mouse (Cooper et al., 1974). Length of the epithelial cell generation interval, D N A synthesis phase, and the migration rate of epithelial cells up the villus or crypt have not been reported previously for swine. In this study, the D N A synthesis phase ranged from 6.85 h in the ileum to 10.4 h in the cecum (control pigs). Cell generation interval ranged from 19.93 h in the ileum to 41.18 h in the duodenum (control pigs). The migration rate of the epithelial cells in the small intestine varied from .77 cell positiondh in the ileum to 1.42 cell positiondh in the lower jejunum (control pigs). In the cecum, the migration rate averaged .52 cell positions/h, whereas in the colon, migration of the epithelial cells up the crypt averaged .57 cell positions/h in the control pigs. The migration rate is apparently much greater in the jejunum, but even with longer villi at this GIT site, turnover rates seem to be faster. Thus, the turnover rate of the mucosal tissue of the jejunum is quite dynamic compared with other GIT sites. The values reported in this study for the D N A synthesis phase and the migration rates are in the range reported for other species. However, the length of the cell generation interval seems to be much longer in the pig than in other species. In the rat, the D N A synthesis phase of the epithelial cells Downloaded from https://academic.oup.com/jas/article-abstract/70/5/1424/4705245 by University of California, Berkeley/LBL user on 04 August 2018
in the duodenum, jejunum, and ileum averaged 7.5 h, whereas the generation interval varied from 10 to 14 h (Cairnie et al., 1965). The chicken duodenum epithelial cell D N A synthesis phase is approximately 5 to 6 h and the generation interval 11.5 h (Cameron, 1964). Using the methods described in this study, Tsubouchi (1983) found that the migration rate of jejunal epithelial cells up the villus was 2.04 cell positiondh in the mouse. The apparent difference in the generation intervals may be attributed in part to species differences and(or1 to the age of the animal. The energy required to maintain the GIT has been estimated to be approximately 24% of the animal’s daily maintenance energy requirements (Yen et al., 1988). Decreasing the turnover rate of this tissue may spare some of this energy and make it available for body weight gain. The addition of Cu to the diet of pigs slowed the time in which the mucosa of the jejunum (A and B1 was replaced approximately 30% (Cu, 53.84 h; CO, 41.18 h). The turnover rate of this tissue can be affected by the length of the cell generation interval, as well as the migration rate of the epithelial cells up the crypt/villus structure. From this study, it seems that the key factor involved in the slower turnover rate of the jejunal mucosa is a lengthening of the cell generation interval (Cu, 38.78 h; CO, 23.42 hl, because the addition of Cu to the diet had little effect on the migration rate of epithelial cells (Cu, 1.19; CO, 1.30 cell positions/h). This observation lends support to the hypothesis that 250 ppm
Table 7. Influence of dietary copper on the turnover rate of the gastrointestinal tract mucosa in weanling pigsa Treatment Intestinal site
Control
Migration rate, cells/hb 1.18 Jejunum-A Jejunum-B 1.42 Ileum .77 Cecum .52 Colon .57 Turnover rate, he Jejunum-A 48.56 J ejunum-B 33.80 Ileum 60.83 Cecum 83.84 Colon 47.61
(.35F (.301 (.241 L131 (.151 (7.11P (4.571 (48.101 (16.391 (.a21
Copper 1.11 1.27 .77 .21 .66
(.371 (.301 (.381 (.091 LO51
64.33 (8.081 43.34 (4.571 101.59 (48.101 61.49 (18.23) 49.07 (.E21
P
NSd NS NS .08
NS .10 .05
NS NS NS
*Estimated from two pens per treatment. bRate at which intestinal epithelial cells migrate up the villus. CStandard error of the average slope of two pens. dNot significant, P > .25. eEstimated for each pen from regression equations from each pen. fConfidence interval.
COPPER AND THE INTESTINAL MUCOSA IN PIGS
of Cu may decrease the energy requirements of the GIT, and consequently decrease the maintenance energy requirements of the pig. A reduction in the turnover rate observed in the jejunum of pigs fed Cu may allow for more dietary energy to be used for body weight gain, instead of the maintenance of the jejunal epithelium. Estimates of turnover rates reported here are similar to those reported by Moon (1970) in 1-wk-old pigs. Other studies in pigs have also demonstrated changes in GIT epithelial cell activity when antimicrobials are added to the diet. A decrease in the mitotic index (an estimate of the rate of cell division) has been observed in the intestinal mucosa of pigs fed a diet containing 250 ppm of Cu (Menten, 1988). Carbadox has been shown to decrease the amount of oxygen used by the portaldrained viscera, which includes the GIT (Yen et al., 1989). Because oxygen consumption can be used to indicate energy use by the tissue, this finding suggests that this antimicrobial may change the energy requirements of the GIT. Radecki (19901 indicated that the addition of carbadox, chlortetracycline, or Cu decreased urea hydrolysis in the jejunum, which may lead to a reduction in the exposure of the epithelial cells to ammonia, a compound known to be toxic to mammalian cells (Visek, 1972). This reduction in ammonia may be linked to the reduction in the replacement rate of the mucosal tissue of the GIT and may be one factor involved in the promotion of growth by antimicrobial compounds.
Implications From this study, it seems that 250 ppm of dietary Cu can slow the replacement rate of intestinal epithelial cells, especially in the jejunum. If this change in turnover rate occurs, it may lead to a reduction in the amount of energy required to maintain the gastrointestinal tract, subsequently increasing the amount of energy available for body weight gain. Further investigations of the influence of Cu on the turnover rate of the intestinal epithelial mucosa need to be conducted to provide more definitive evidence for the changes in turnover reported in this study.
Literature Cited Cairnie, A. B., L. F. Lamerton, and G. C. Steel. 1985. Cell proliferation studies in the intestinal epithelium of the rat. Exp. Cell Res. 39:528. Cameron, I. L. 1984.Is the duration of DNA synthesis in somatic cells of mammals and birds a constant? J. Cell Biol. 20:185.
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143 1
Cleaver, J. E. 1907.Thymidine metabolism and cell kinetics. In: A. Neuberger and E. L. Tatum [Ed.) Frontiers of Biology. Vol 8. North-Holland Publishing Co., Amsterdam. Coates, M. E., R.Fuller, G. F. Harrison, M. Lev, and S.F. Suffolk. 1983.A comparison of the growth of chicks in the Gustafsson germ-free apparatus and in conventional environment, with and without dietary supplements of penicillin. Br. J. Nutr. 17:141. Cooper, J. W., R. F. Hagemann, and G. J. Brodmerkel, Jr. 1974. On the determination of intestinal epithelial cell generation time from labeling index and DNA synthesis duration. Experientia 30:188. Cori, G. T. and C. F. Cori. 1952. Glucose-8-phosphatase of the liver in glycogen storage disease. J. Biol. Chem. 199:861. Gill, J. L. 1978. Design and Analysis of Experiments. The Iowa State University Press, Ames. Gomori, G. 1942.A modification of the colorimetric phosphorus determination for use with the photoelectric colorimeter. J. Lab. Clin. Med. 27:955. Hirschhorn, N., and I. H. Rosenberg. 1988. Sodium-potassium adenosine triphosphatase of the small intestine of man: Studies in cholera and diarrheal disease. J. Lab. Clin. Med. 72:28.
Imondi, A. R.,and F. H. Bird. 1965. The turnover of intestinal epithelium in the chick. Poult. Sci. 45:142. Kawai, Y., and M. Morotomi. 1978. Intestinal enzyme activities in germfree, conventional, and gnotobiotic rats associated with indigenous microorganisms. Infect. Immunol. 19:i'i'l. Lesher, S.,H. E. Walburg, and G. A. Sacher. 1984. Generation cycle in the duodenal crypt cells of germ-free and conventional mice. Nature 202:884. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265. Menten, J.F.M. 1988. Effects of high dietary copper on the utilization of nutrients and on blood and intestinal variables of starter pigs. Ph.D. Dissertation. Michigan State Univ., East Lansing. Moon, H. W. 1970. Epithelial cell migration in the alimentary mucosa of the suckling pig. R o c . SOC.Exp. Biol. Med. 137: 151.
Radecki, S.V. 1990.The influence of dietary antimicrobials on intestinal fermentation and mucosal morphology, enzyme activity, and the turnover rate of the intestinal mucosa in weanling pigs. Ph.D. Dissertation. Michigan State Univ., East Lansing. Shurson, G. C., P. K. Ku, G. L. Waxler, M. T. Yokoyama, and E. R. Miller. 1990. Physiological relationships between microbiological status and dietary copper levels in the pig. J. Anim. Sci. 68:1061. Tsubouchi, S. 1983. Theoretical implications for cell migration through the crypt and the villus in labelling studies conducted a t each position within the crypt. Cell Tissue Kinet. 10:441.
Visek, W. J. 1972.Effects of urea hydrolysis on cell lifespan and metabolism. Fed. Proc. 31:1178. Whitehair, C. K., and C.M. Thompson. 1956.Observations on rearing disease-free swine. J. Am. Vet. Med. Assoc. 128:94. Yen, J. T., J. A. Nienaber, D. A. Hill, and W. G. Pond. 1988. Oxygen consumption by whole body and by portal drained organs in swine. J. Anim. Sci. 66(Suppl. 1):331(Abstr.). Yen, J. T., J. A. Nienaber, D. A. Hill, and W. G. Pond. 1988.Effect of carbadox on fasting oxygen consumption by whole body and by portal vein drained organs in swine. J. Anim. Sci. 67(suppl. 2):127 (Abstr.). Zimmerman, D. R. 1988. Role of subtherapeutic levels of antimicrobials in pig production. J. Anim. Sci. 62[Suppl. 3):0.
ABSTRACT: Twenty-four pigs from four litters weaned a t 21 d of age (6.6 kg of BWI were used to evaluate the influence of 250 ppm of dietary Cu on
intestinal mucosa glucose-6-phosphatase (GPI, alkaline phosphatase (AP), and adenosine triphosphatase (ATPase) activity; mucosal morphology; and the turnover rate of the intestinal mucosa throughout the gastrointestinal tract. Pigs were allotted into four pens of six pigs each based on sex, litter, and weight. Pens were then assigned to one of two treatments: 1) corn-soybean meal-whey diet with no antimicrobials (CO), or 2) CO + 250 ppm of Cu. Pigs were fed twice daily an amount approximately equal to a d libitum intake for 14 d. On d 14, pigs were injected i.p. with i3Hlthymidine (50 @/kg of BW) 10 h after the morning meal. One pig from each pen was euthanatized at 1, 6, 12, 20, 32, and 44 h postinjection, and intestinal tissue was collected from the duodenum, two
jejunum sites (upper and lower), ileum, cecum, and colon. The activity of GP and AP in the lower jejunum tended to decrease in pigs fed Cu (P c .11, P e .08,respectively). The ATPase activity was not affected by treatment (P > .lo). Crypt death, villus height, or epithelial cell size (P > .lo) were not affected by feeding Cu. Migration rate of epithelial cells up the villus was also not affected by treatment (P > .lo). Turnover rate of the intestinal mucosa of the upper and lower jejunum was slower (P c .lo; P c .05, respectively) and the lower jejunal cell generation interval was longer (P e .05)in pigs fed the Cu diet. From these data we conclude that the addition of 250 ppm of Cu to the diet of weanling pigs slows the turnover rate of the jejunal intestinal mucosa, which may result in a lower energy requirement for the maintenance of this tissue.
Key Words: Pigs, Antimicrobials, Copper, Intestinal Mucosa
J. h i m . Sci. 1992. 70:1424-1431
Introduction The addition of "supranutritive" levels of Cu to the diets of young growing pigs increases their average daily gain and gain efficiency (Zimmerman, 1986).However, the mechanism by which this feed additive functions is not well understood. It is
'Acknowledgement is made to the Michigan Agricultural Experiment Station for their support of this research. 2Present address: Dept. of h i m . Sci., Rutgers Univ., New Brunswick, NJ 08903. 3To whom correspondence should be addressed. Received June 17, 1991. Accepted December 2, 1981.
likely that toxic compounds produced by the microflora in the gastrointestinal tract (GIT) of the pig are involved, because little improvement in growth performance is seen when germ-free animals are fed high Cu or other growth-enhancing, antimicrobially supplemented diets (Whitehair and Thompson, 1956; Coates et al., 1963; Shurson et al., 19901. Reducing the production of these toxic compounds through the use of Cu may be one mechanism by which this feed additive improves growth performance. Various toxic compounds, especially ammonia, can alter cell metabolism and cell life span in a manner that may be detrimental to the animal (Visek, 1972). In rats, the presence of bacteria in the GIT decreases the cell
1424
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COPPER AND THE INTESTINAL MUCOSA IN PIGS
generation interval of intestinal epithelial cells (Lesher et al., 1964). Also, the activity of enzymes important in the transport of nutrients across the intestinal wall is decreased when bacteria are present in the GIT (Kawai and Morotomi, 1978). Thus, decreasing the concentration of toxic bacterial metabolites in the GIT may slow the turnover rate of the mucosa and consequently decrease the energy requirements of the intestinal epithelial cells. The energy spared by these processes may be available to the pig for lean tissue gain. In this study, the influence of 250 ppm of dietary Cu on these metabolic processes of the intestinal mucosa was investigated. In addition to these objectives, measures of intestinal epithelial cell turnover rates were determined, because these measures have not been reported for swine.
Materials and Methods Animals and Diet. Twenty-four crossbred pigs (Yorkshire x Landrace) were weaned a t 21 d of age (6.6 kg of BW) and assigned to one of two treatments based on litter, sex, and weight. Pigs were housed in 1-m x 2-m stainless steel pens (six pigs per pen) in a n environmentally controlled (27’C) nursery. Treatments included a control diet (CO,Table 1) devoid of antimicrobials, and CO + 250 ppm of Cu as CuS04.5H20 (CUI. All pens of pigs were fed twice daily (0800 and 1700) a n amount approximately equal to a d libitum intake (6to 8% of BW) for 14 d. Water was available at all times. Average daily gain was calculated for the 14-d treatment period. On d 14, the morning meal was fed to each pen a t 1-h intervals starting at 0600, as compared to feeding all pens at 0800. This change in feeding regimen on d 14 helped to facilitate subsequent tissue collection and likely had little effect on pig performance. Ten hours postprandial, I3Hlthymidine (specific activity 43 Ci/mmol, Amersham, Arlington Heights, IL) was injected (50 pCi/ kg of BWI i.p. The 13Hlthymidinewas diluted with sterile saline to 100 pCi/mL before injection. Tissue Collection. One pig from each pen (two per treatment) was euthanatized at 1, 6, 12, 20, 32, and 44 h after the t3Hlthymidine injection. Animals were euthanatized by asphyxiation with C02 gas in a gassing chamber. The GIT was quickly removed, and intestinal tissue samples were collected from the duodenum, jejunum (two sites], ileum, cecum, and colon. The duodenum was defined as the region of the small intestine between the stomach and the point at which the bile duct entered the intestinal tract (approximately 10 cm). The ileal-cecal valve and the Downloaded from https://academic.oup.com/jas/article-abstract/70/5/1424/4705245 by University of California, Berkeley/LBL user on 04 August 2018
1425
Table 1. Composition of the basal dieta Inmedient Ground, shelled corn Soybean meal, 4 4 % CP Dried whey Ground limestone Mono-dicalcium phosphate Selenium-vitamin E premixb Vitamin-mineral premixC L-lysine.HCl(78%) Salt Calculated analysis Lysine, % Ca, % p. %
0%
55.35 25.0 15.0 1 .o 1.5 1 .o .75 .15 .25 1.12 .88 .75
*The treatment diet was formulated by adding 250 ppm of Cu (as copper sulfate1 at the expense of ground, shelled corn. bF’rovided .1 mg of Se and 17 IU of vitamin E per kilogram of diet. CProvided the following amounts of vitamins and minerals per kilogram of diet: vitamin A, 3,300 IU;vitamin D, 000 IU; menadione sodium bisulfite, 2.2mg; riboflavin, 3.3 mg; niacin, 18 mg; D-pantothenic acid, 13 mg; choline, 110 mg; vitamin Bl2, 20 pg; Zn, 75 mg; Mn, 34 mg; Fe, 60 mg;I, .5 mg.
ileocolic artery were used to demarcate the end points of the ileum (approximately 10 cml. Jejunum samples were taken from the upper (jejunum-A) and lower (jejunum-B) halves of the section of intestine between the duodenum and ileum. Samples collected from the cecum were obtained from the end of the cecum, and the colon sample was taken from the spiral colon. Tissue samples (1 to 2 gl were washed with saline and quickly frozen in a dry ice/acetone bath for enzyme analysis. For morphology and autoradiography, lengths of GIT (2 to 4 cm) were removed, gently flushed with saline, ligated at both ends with string, and filled with 10% buffered formalin using a syringe. These tissue samples were then placed in 10% buffered formalin. All samples were collected within 15 min after death. The use and handling of animals was approved by the Michigan State University Committee on Animal Use. Enzyme Assays. Approximately .5 g of tissue from each GIT site was minced with scissors and placed in 18-mm x 150-mm test tubes. Tissues were homogenized in Tris buffer (50 mM) with a Polytron Homogenizer (Brinkman Instruments, Westbury, NY) for 15 to 30 s. The homogenate was centrifuged (850 x g) for 10 min at 4°C. The supernatant was used for enzyme and protein analysis. Alkaline phosphatase activity was assayed colorimetrically by monitoring the appearance of p-nitrophenol using Sigma Kit 245 (Sigma Chemical, St. Louis, MO). Enzyme activity was expressed as micromoles of inorganic phosphorus (Pi) liberated per minute per milligram of protein.
RADECKI ET AL.
1426 100
pen 1
d-
+ -0-
.-u)
.-c E
-0
pen2 pen3
4 3 pen4
70
60
20 10 1
01
.
1
1
1
/
6
1
1
1
/
1
1
1
1
1
l
1
12
l
1
/
1
1
1
1
1
1
/
1
l
32
20
I
l
l
l
l
l
/
l
l
~
l
l
l
l
I
44
Hours post-injection Figure 1. Percentage of labeled mitoses in the ileum. Each point represents one pig. Pens 1 and 2, copper-fed pigs; pens 3 and 4, control-fed pigs.
The activity of adenosine triphosphatase (ATPase) was assayed by the differential method of Hirschhorn and Rosenberg (1968).Briefly, .1 mL of the supernatant was added to 2.4 mL of a medium that contained 140 mM NaCl and 16 mM KC1. A second .l-mL aliquot was added to 2.4 mL of a second medium containing no added NaCl or KC1. Both media contained 2.5 mM ATP (dipotassium salt) and 30 mM Tris buffer. The final pH of both media was 7.4. Reactions were carried out at 37°C for 60 min. The reaction was stopped with 1.5 mL of 6 M perchloric acid. The amount of Pi in the media (indicating the activity of the phosphatase) was determined colorimetrically (Gomori, 1942). The activity of Na/K dependent ATPase was calculated by subtracting the enzyme activity in the medium with no added Na or K (Na/K independent ATPase) from the activity detected in the medium with added Na and K (total ATPase activity). Enzyme activity was expressed as micrograms of Pi liberated per hour per milligram of protein. The activity of glucose-6-phosphatase was determined by monitoring the release of Pi from glucose-6-phosphate in 30 min (modified from Cori and Cori, 19521. The media contained .5 mL of .01 M glucose-6-phosphate, .3 mL of .1 M potassium citrate, and .2 mL of the supernatant. The reaction was incubated for 30 min at 37°C then stopped with 10% trichloroacetic acid. Supernatant was Downloaded from https://academic.oup.com/jas/article-abstract/70/5/1424/4705245 by University of California, Berkeley/LBL user on 04 August 2018
also added to a second set of media and acidified immediately. Acidified media were centrifuged (2,000 x gl for 5 min, and the concentration of Pi liberated by the reaction was determined (Gomori, 19421. Glucose-6-phosphatase activity was then calculated as the difference between the amount of Pi detected in these two media for each GIT site and expressed as micrograms of Pi liberated per minute per 100 milligrams of protein. Total soluble protein was determined by the procedure of Lowry et al. (1951). Morphology and Autoradiography. Formalin-fixed tissue was embedded in paraffin, sliced into 6 - p sections, and mounted on glass slides. Paraffin was removed with xylene, and the tissue sections were hydrated with increasing proportions of water in ethanol. After hydration, the slides were dipped in Kodak NBT-29 (Eastman Kodak, Rochester, NY) emulsion in a darkroom and placed in light-proof boxes. Slides were stored at 4°C during a 14- to 38-d exposure period and then developed. Slides were then stained with hematoxylin and eosin. The number of cells in crypt and villus columns were counted, and the height of these columns was measured with a n ocular micrometer. Columns were defined as a single row of epithelial cells extending from the base of the crypt to the tip of the villus. The position of the 13Hlthymidinelabeled cells along the columns was also noted.
1427
COPPER AND THE INTESTINAL MUCOSA IN PIGS
Ten columns per sample were counted when possible, and these counts were averaged. For each GIT site, the percentage of labeled mitosis (number of labeled mitotic cells divided by the total number of cells in mitosis1 was plotted against time between 13Hlthymidine injection and euthanasia of the pig, for each pen. Figure 1 is a n example of this plot for the ileum. From these plots, the DNA synthesis phase can be estimated as the time interval between one-half of the maximum percentage of labeling on the ascending and descending slopes of the first peak (Cleaver, 1967). The percentage of cells labeled at 1 h postdose was also determined, by counting the total number of cells labeled in the crypt and dividing by the total number of cells in the crypt. This population of cells represents those cells that have incorporated the [3Hlthymidine but have yet to divide again. The cell generation interval can then be calculated as the length of the DNA synthesis phase divided by the percentage of cells labeled 1 h after the 13Hlthymidine administration (Cleaver, 19671. Therefore, because each point represents one pig, and each line in Figure 1 represents one pen, the experimental unit is the pen.
100
80
The turnover rate of the mucosa was calculated as the amount of time necessary for the leading labeled epithelial cell in the crypt or villus to reach the tip of the crypt or villus, depending on the site. For those sites with villi (duodenum, jejunum, and ileum), migration starts a t the crypt/villus junction. In the colon and cecum, migration rates were calculated starting from the base of the crypt. These turnover rates were estimated from the migration rate (slope of the regression line) of the labeled cells up the crypt or villus for each pen. Figure 2 is a n example of this plot in the lower jejunum. Statistical Analysis. The influence of Cu and(or1 GIT site on mucosal morphology and epithelial cell enzyme activity was detected using a repeated measure analysis (Gill, 1978). In this analysis, the main effects of treatment and GIT site, as well as the interaction of treatment and GIT site, could be evaluated. The error term used to test the effect of treatment was animals (or pens1 within treatment, and the error term to test GIT site or the interaction of GIT site and treatment was the residual error term. When the variation among pens was trivial (P r .251, pen variation was pooled with animal within treatment error, and
1
70 60
50 40 c
0
30 / *
, --e-
- /
C
.-2 0
v)
20
-8
0
a
10 c
+
pen2
-0-
pen3
-3 p e n 4
Figure 2. Jejunum-B epithelial cell migration along the villus, expressed as a function of time after the injection of [3H]thymidine. Regression equations for each pen of pigs were as follows: Pen 1, Y = 1 . 3 1 -~ 32.55, r = .91; Pen 2, 1.26~- 32.07, r = .84. Each point Y = 1 . 2 3 ~- 34.7, r = 3 4 ; Pen 3, Y = 1 . 5 7 ~- 27.25, r = .93; Pen 4, Y represents one pig. Data points at 6 and 32 h for Pen 2 and at 6 and 44 h for Pen 3 were not determined due to a lack of well-positioned villi for the pigs euthanatized at these time points. Pens 1 and 2, copper-fed pigs; Pens 3 and 4, control-fed pigs. Figure is representative of other GIT sites.
-
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1428
RADECKI ET AL.
Table 2. Influence of dietary copper on weight gain in weanling pigs Treatment Item n Day 0, kg Day 14, kg ADG, gb
Control 12 6.6 6.3
-24
Copper
SEM
P
12 6.6
.19 .22 9.2
7.4 56
NSa ,003 ,001
aNot significant, P > ,113. bAverage daily gain for the 14-d treatment period.
animal served as the experimental unit. Otherwise, pen served as the experimental unit. The influence of time after the [3Hlthymidine injection was not included in the statistical analysis of these measurements. Treatment means were separated using a Bonferroni t-test (Gill, 1978). 'The influence of Cu and(or1 GIT site on the generation interval was determined similarly; however, pen served as the experimental unit, because the determination of these values was based on the values obtained over time for each pen of pigs. Mucosal turnover rates were estimated using linear regression equations determined for each pen at each GIT site, regressing the location of the labeled cell on the cell column over time. Correlation coefficients (r) ranged from .57 to 98, with a
Table 3. Influence of dietary copper on the specific activity of glucose-6-phosphatase and alkaline phosphatase in the gastrointestinal tract of weanling pigsa Treatment Intestinal site Glucose-6-phosphataseb Duodenum Jejunum-A Jejunum-B Ileum Cecum Colon Alkaline phosphatased Duodenum Jejunum-A Jejunum-B Ileum Cecum Colon
Control
Copper
7.1 7.6 9.4 6.4 2.2 2.4
6.1 6.7 6.5 5.0 3.2 2.4
50.1 146.6 168.1 112.5 72.6 68.7
35.2 136.6 109.8 101.4 81.4 74.8
P
NSC NS .ll
NS NS NS NS NS .08
NS NS NS
*Values represent the average of 12 pigs. bMicrograms of inorganic P released per minute per 100 mg of protein. SEM, treatment = 1.01;gastrointestinal site = 1.19. Main effect of gastrointestinal site, P < ,001. CNot significant, P > .25. dMicromole of inorganic P released per minute per milligram of protein. SEM, treatment = 13.35;gastrointestinal site = 17.65.Main effect of gastrointestinal site, P < ,001. Downloaded from https://academic.oup.com/jas/article-abstract/70/5/1424/4705245 by University of California, Berkeley/LBL user on 04 August 2018
mean of .84 across all sites and pens. The slope of these regression equations served as a measure of the rate of migration of the epithelial cell up the villus or crypt. An inverse prediction equation was then formulated to calculate the turnover rate, using the migration rate and the length in cells of the villus (duodenum, jejunum-A, jejunum-B, and ileum) or crypt (cecum and colon). The influence of treatment on the predicted turnover rate of the intestinal mucosa was tested by calculating confidence intervals, which were estimated using error associated with the epithelial cell column length, error associated with the slope of the regression equation, and the error associated with time (Gill, 1978).
Initial and final weight, as well as average daily gain data, were analyzed as a one-way ANOVA, with pigs serving as the experimental unit.
Results Pigs fed Cu were heavier on d 14 and thus grew faster than CO-fed pigs (Table 2). There were no GIT site x treatment interactions (P > .25). Therefore, only the main effects of GIT site and treatment will be discussed. Glucose-6-phosphatase activity tended to be lower (P c .11) in the mucosa of the lower jejunum of pigs fed the Cu diet than in the mucosa of the lower jejunum of pigs fed the CO diet (Table 3). When the activity of this enzyme was pooled across small intestine sites, the decrease in activity due to Cu was more evident ( P e .011. There was a main effect ( P c .001) of GIT site on the activity of glucose-6-phosphatase. The activity of alkaline phosphatase also tended to be lower ( P c .08) in the mucosa of the lower jejunum when pigs were fed the Cu diet rather than the CO diet (Table 31. Again, when small intestine sites were pooled, the decrease in activity due to Cu was more evident ( P c .02). The activity of this enzyme was also affected by GIT site ( P c
.oo I). Total ATPase, Na/K dependent ATPase, and Na/K independent ATPase activity were unaffected ( P > .lo) by treatment (data not shownl. Total ATPase, Na/K dependent ATPase, and Na/ K independent ATPase activity averaged 128 G E M = 8.61, 27 G E M = 3.51, and 101 (SEM = 7.5) micrograms of phosphate released per hour per milligram of protein, respectively. The addition of 250 ppm of Cu to the diet had no effect on crypt depth (number of cells or micrometers), villus height (number of cells or micrometers), total height of the villus/crypt structure (number of cells or micrometers), or cell size (micrometers; Tables 4 and 5).
1429
COPPER AND THE INTESTINAL MUCOSA IN PIGS
Duodenal estimates of these measures of the mucosal morphology could not be determined due to a lack of well-positioned crypts and villi. Copper had little influence on the length of the DNA synthesis phase (Table 61. However, the cell generation interval (length of the cell cycle) of animals fed the Cu diet was longer (P c .05) in the lower jejunum than in pigs fed the CO diet (Table 6). Generation interval was not affected by treatment at other GIT sites (P > .lo). The rate of cell migration (number of cell positions/hourl is shown in Table 7. Copper tended to decrease the rate of cell migration up the crypt (P < .08) in the cecum. The turnover rate of the intestinal mucosa of the upper jejunum tended to be slower (P e .lo) when pigs were fed the Cu diet than when they consumed the CO diet (Table 7). The turnover rate of the mucosa of the lower jejunum was slower (P c .05) when pigs were fed Cu (Table 7).
Discussion
similar to that in the germ-free animal, antimicrobials may similarly increase the activities of these enzymes. However, the addition of Cu to the diet did not increase the activity of these enzymes. The addition of Cu to the diet tended to decrease the activity of glucose-6-phosphatase(lower jejunum; P < .11) and alkaline phosphatase (lower jejunum; P c .081. The activity of these enzymes at the other GIT sites, together with the activity of ATPase, were not affected by the addition of Cu to the diet (P > .lo). The influence of GIT site on the activity of glucose-6-phosphatase, alkaline phosphatase, and ATPase is not surprising, because the digestive and absorptive capacity of the small intestine is known to be greater than that of the cecum and colon. In previous work with high-Cu diets fed to weanling pigs, Shurson et al. (1990) observed a n increase in villus height and crypt depth. Changes in morphology due to the addition of 250 ppm of Cu to the diet were not significant in our study. However, tendencies did exist that were similar to the observations of Shurson et al. (19901. Values
The activities of glucose-6-phosphatase, alkaline phosphatase, and adenosine triphosphatase have been shown to be greater throughout the GIT of germ-free mice than in the GIT of conventionally reared mice (Kawai and Morotomi, 19781. If antimicrobials function to produce a situation in the GIT
Table 5. Influence of dietary copper on the morphology of the gastrointestinal tract of weanling pigs (micrometers per crypt-villus column)a Treatment Intestinal site
Table 4. Influence of dietary copper on the morphology of the gastrointestinal tract of weanling pigs (number of epithelial cells per crypt-villus column)a Treatment Intestinal site Crypt dept hb Jejunum-A Jejunum-B Ileum Cecum Colon Villus heightd Jejunum-A Jejunum-B Ileum Total heighte Jejunum-A Jejunum-B Ileum
Control
Copper
P NSC
32.2 28.8 29.1 54.9 53.8
32.6 32.5 30.0 49.0 58.3
52.7 49.2 47.6
66.4 52.1 63.3
NS
84.9 78.1 76.6
98.2 85.1 93.0
NS
NS NS .20 .25 .20 .15 .20 .15
aEach mean represents 12 pigs. bSEM, treatment = 2.67; site = 3.15. Main effect of site, P e
.oo 1.
'Not significant, P =. .25. dSEM, treatment = 6.40, site = 6.94. eVillus t crypt height. SEM, treatment Main effect of site, P c .07.
Crypt depthb Jejunum-A Jejunum-B Ileum Cecum Colon Villus heightd Jejunum-A Jejunum-B Ileum Total heighte Jejunum-A Jejunum-B Ileum Cell sizef Jejunum-A Jejunum-B Ileum Cecum Colon
P
Control
Copper
189.6 171.5 184.9 376.6 387.3
181.7 209.9 195.8 345.3 412.4
368.5 327.0 290.0
44 1.3 371.3 386.3
NS
558.1 498.5 474.9
620.5 587.2 582.1
.25 .20 .15
6.6 6.4 6.2 6.9 7.2
6.4 6.7 6.4 7.1 7.2
NS
~~
NSC .20
NS .25
NS .25 .15
NS NS NS NS
~_________
*Each mean represents 12 pigs. bSEM, treatment = 18.40; site = 21.99. Main effect of site, P < ,001.
CNot significant, P > .25. dSEM, treatment = 44.04; site
= 42.07.
Main effect of site, P
= 41.75.
Main effect of site, P
e .03.
%EM, treatment
= 43.45;
site
< .04. =
6.19;
site
= 6.45.
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fMicrometers divided by number of cells (from Table 41. SEM, treatment = .34; site = .40. Main effect of site, P e .004.
RADECKI ET AL
1430
Table 6. Influence of dietary copper on the DNA synthesis phase of the cell cycle and the cell generation interval of epithelial cells of the gastrointestinal tract of weanling pigsa Treatment Intestinal site DNA synthesis phaseb Duodenum Jejunum-A Jejunum-B Ileum Cecum Colon Cell generation i n t e n d Duodenum Jejunum-A Jejunum-B Ileum Cecum Colon
Control
Copper
8.6 8.2 9.7 6.9 10.4 8.5
8.5 9.3 7.2 7.5 12.6 9.2
41.2 26.5 20.4 19.9 38.2 35.1
27.4 33.0 44.6 22.4 37.5 30.6 ~
P NSC .20 .10
NS .10
NS .15
NS .05
NS NS NS ~~
&Two pens per treatment. bHours. SEM, treatment = .43; site = .77. CNot significant, P > .25. dHours. SEM, treatment = 5.65; site = 5.71.
for the different morphological features were within the range of previously reported values (Moon, 1970; Shurson et al., 1990). There are no published data on the number of cells in the crypt or villus columns in the pig GIT. The main effect of GIT site on the various morphological measures was expected, because this result has also been noted in the chick (Imondi and Bird, 1966) and mouse (Cooper et al., 1974). Length of the epithelial cell generation interval, D N A synthesis phase, and the migration rate of epithelial cells up the villus or crypt have not been reported previously for swine. In this study, the D N A synthesis phase ranged from 6.85 h in the ileum to 10.4 h in the cecum (control pigs). Cell generation interval ranged from 19.93 h in the ileum to 41.18 h in the duodenum (control pigs). The migration rate of the epithelial cells in the small intestine varied from .77 cell positiondh in the ileum to 1.42 cell positiondh in the lower jejunum (control pigs). In the cecum, the migration rate averaged .52 cell positions/h, whereas in the colon, migration of the epithelial cells up the crypt averaged .57 cell positions/h in the control pigs. The migration rate is apparently much greater in the jejunum, but even with longer villi at this GIT site, turnover rates seem to be faster. Thus, the turnover rate of the mucosal tissue of the jejunum is quite dynamic compared with other GIT sites. The values reported in this study for the D N A synthesis phase and the migration rates are in the range reported for other species. However, the length of the cell generation interval seems to be much longer in the pig than in other species. In the rat, the D N A synthesis phase of the epithelial cells Downloaded from https://academic.oup.com/jas/article-abstract/70/5/1424/4705245 by University of California, Berkeley/LBL user on 04 August 2018
in the duodenum, jejunum, and ileum averaged 7.5 h, whereas the generation interval varied from 10 to 14 h (Cairnie et al., 1965). The chicken duodenum epithelial cell D N A synthesis phase is approximately 5 to 6 h and the generation interval 11.5 h (Cameron, 1964). Using the methods described in this study, Tsubouchi (1983) found that the migration rate of jejunal epithelial cells up the villus was 2.04 cell positiondh in the mouse. The apparent difference in the generation intervals may be attributed in part to species differences and(or1 to the age of the animal. The energy required to maintain the GIT has been estimated to be approximately 24% of the animal’s daily maintenance energy requirements (Yen et al., 1988). Decreasing the turnover rate of this tissue may spare some of this energy and make it available for body weight gain. The addition of Cu to the diet of pigs slowed the time in which the mucosa of the jejunum (A and B1 was replaced approximately 30% (Cu, 53.84 h; CO, 41.18 h). The turnover rate of this tissue can be affected by the length of the cell generation interval, as well as the migration rate of the epithelial cells up the crypt/villus structure. From this study, it seems that the key factor involved in the slower turnover rate of the jejunal mucosa is a lengthening of the cell generation interval (Cu, 38.78 h; CO, 23.42 hl, because the addition of Cu to the diet had little effect on the migration rate of epithelial cells (Cu, 1.19; CO, 1.30 cell positions/h). This observation lends support to the hypothesis that 250 ppm
Table 7. Influence of dietary copper on the turnover rate of the gastrointestinal tract mucosa in weanling pigsa Treatment Intestinal site
Control
Migration rate, cells/hb 1.18 Jejunum-A Jejunum-B 1.42 Ileum .77 Cecum .52 Colon .57 Turnover rate, he Jejunum-A 48.56 J ejunum-B 33.80 Ileum 60.83 Cecum 83.84 Colon 47.61
(.35F (.301 (.241 L131 (.151 (7.11P (4.571 (48.101 (16.391 (.a21
Copper 1.11 1.27 .77 .21 .66
(.371 (.301 (.381 (.091 LO51
64.33 (8.081 43.34 (4.571 101.59 (48.101 61.49 (18.23) 49.07 (.E21
P
NSd NS NS .08
NS .10 .05
NS NS NS
*Estimated from two pens per treatment. bRate at which intestinal epithelial cells migrate up the villus. CStandard error of the average slope of two pens. dNot significant, P > .25. eEstimated for each pen from regression equations from each pen. fConfidence interval.
COPPER AND THE INTESTINAL MUCOSA IN PIGS
of Cu may decrease the energy requirements of the GIT, and consequently decrease the maintenance energy requirements of the pig. A reduction in the turnover rate observed in the jejunum of pigs fed Cu may allow for more dietary energy to be used for body weight gain, instead of the maintenance of the jejunal epithelium. Estimates of turnover rates reported here are similar to those reported by Moon (1970) in 1-wk-old pigs. Other studies in pigs have also demonstrated changes in GIT epithelial cell activity when antimicrobials are added to the diet. A decrease in the mitotic index (an estimate of the rate of cell division) has been observed in the intestinal mucosa of pigs fed a diet containing 250 ppm of Cu (Menten, 1988). Carbadox has been shown to decrease the amount of oxygen used by the portaldrained viscera, which includes the GIT (Yen et al., 1989). Because oxygen consumption can be used to indicate energy use by the tissue, this finding suggests that this antimicrobial may change the energy requirements of the GIT. Radecki (19901 indicated that the addition of carbadox, chlortetracycline, or Cu decreased urea hydrolysis in the jejunum, which may lead to a reduction in the exposure of the epithelial cells to ammonia, a compound known to be toxic to mammalian cells (Visek, 1972). This reduction in ammonia may be linked to the reduction in the replacement rate of the mucosal tissue of the GIT and may be one factor involved in the promotion of growth by antimicrobial compounds.
Implications From this study, it seems that 250 ppm of dietary Cu can slow the replacement rate of intestinal epithelial cells, especially in the jejunum. If this change in turnover rate occurs, it may lead to a reduction in the amount of energy required to maintain the gastrointestinal tract, subsequently increasing the amount of energy available for body weight gain. Further investigations of the influence of Cu on the turnover rate of the intestinal epithelial mucosa need to be conducted to provide more definitive evidence for the changes in turnover reported in this study.
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