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Department of Animal and Range Sciences, North Dakota State University, Fargo 58105
2 Correspondence—phone:
701-231-7653; fax: 701-231-7590: E-mail:
Joel.Caton{at}ndsu.nodak.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Key Words: Cattle • Cell Growth • Growth • Oxygen Consumption • Pregnancy • Small Intestine
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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The small intestinal epithelium is a diverse population of cells requiring integration of cellular proliferation, differentiation, and senescence (Podolsky, 1993). These integrations, coupled with the observation that the portal-drained viscera accounts for 20 to 35% of cardiac output, whole-body protein turnover, and energy expenditure while only contributing 3 to 6% of BW (Reeds et al., 1999), highlight the pivotal role of intestinal tissues in both whole-body nutrient demand and systemic tissue supply. In addition, the portal-drained viscera are also adaptive to various conditions such as dietary carbohydrate, fiber, caloric density, starvation, pregnancy, and lactation (Ferraris and Diamond, 1997). We hypothesize that visceral tissues will modulate metabolic processes in response to increased nutrient demand associated with advancing gestation. Our specific objectives were to evaluate adjustments in maternal metabolism as reflected by energy use and gastrointestinal tissue growth due to pregnancy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Beef heifers, 2 yr of age and with an initial BW of 378 ± 32.1 kg, were used in a serial slaughter experiment as described by Scheaffer et al. (2001). Briefly, heifers were grouped in common pens and fed corn silage- and hay-based diets formulated to provide 0.45 kg of ADG in nonpregnant (NP) cows (Scheaffer et al., 2001). Twenty-two heifers were naturally serviced and resulted in the pregnant (PR) treatment, whereas 17 NP heifers served as controls. Pregnant heifers were slaughtered on d 40 (n = 6), 120 (n = 5), 200 (n = 7), and 270 (n = 4) of gestation, and NP heifers were slaughtered on d 40 (n = 5), 120 (n = 5), 200 (n = 5), and 270 (n = 2). Heifers were stunned, exsanguinated, and eviscerated. Experimental protocol followed the local institutional care and use committee guidelines.
Upon evisceration, intestinal and hepatic tissue samples were collected (Jin et al., 1994; Scheaffer et al., 2001). Small intestinal samples were collected at three sites. Duodenal samples were taken 20 cm distal to the entry of the pancreatic duct into the intestinal lumen. Jejunal samples were collected from the center third of the small intestine dissected from the mesentery. Ileal samples were collected 40 cm proximal from the ileo-cecal junction. Hepatic tissue samples were collected from a random point of the liver.
Small Intestinal Cellularity
Samples (approximately 2 g) were collected, placed on dry ice, and stored at -80°C for analysis of RNA, DNA, and protein content (Jin et al., 1994). Tissue was minced and 0.5 g was homogenized in TNE buffer (0.05 M Tris, 2.0 M NaCl, 2 mM EDTA, pH 7.4) using a Polytron with a PT-10s probe (Brinkmann, Westbury, NY). Homogenization was done on ice to a consistent dispersal of tissue. Tissue homogenates were analyzed for concentrations of DNA and RNA by using diphenylamine (Johnson et al., 1997) and orcinol procedures (Reynolds et al., 1990). Standards were DNA type-I from calf thymus and RNA type-IV from calf liver (Sigma, St. Louis, MO). Protein in tissue homogenates was determined with Coomassie brilliant blue G (Sigma; Bradford, 1976) with BSA (Fraction V, Sigma) as the standard (Johnson et al., 1997). The concentration of DNA was used as an index of hyperplasia and RNA:DNA and protein:DNA ratios were used as indices of hypertrophy (Swanson et al., 2000). Intestinal section DNA, RNA, and protein content were calculated by multiplying DNA concentration by fresh sample weight.
Small Intestinal Oxygen Consumption and Energy Use
Tissue samples were collected and placed into Krebs-Ringer bicarbonate (20 mL) fortified with sodium pyruvate (5 mM), sodium glutamate (5.0 mM), sodium acetate (4.5 mM), glucose (12.8 mM), and malic acid (4.5 mM) buffer at room temperature and pressure. Tissue samples were immediately transported to the laboratory in 10 mL of buffer. Tissue samples were sliced 0.5 mm thick with a Stadie-Riggs microtome (Thomas, Philadelphia, PA), transferred into Petri dishes containing buffer, and maintained at 37°C. Sliced tissue samples were subsampled (200 ± 10 mg; Reynolds et al., 1990), placed into 3 mL of buffer, and analyzed for in vitro oxygen consumption, using a Clarke polariographic electrode (model 5300, Yellow Springs Instruments, Yellow Springs, OH). All tissues were analyzed within 4 h of collection. Oxygen consumption estimates were converted to organ energy use per day (kcal/d), similar to the calculations of Huntington et al. (1988).
Jejunum Morphometry and Cell Proliferation
Jejunal samples of 5 to 10 g were collected and immersed into 10% formalin and Carnoy’s fixative, placed into paraffin blocks, sectioned to 5 µm, and mounted on glass slides. Tissue sections were treated with blocking buffer consisting of PBS and 1.5% (vol/vol) normal horse serum (Vector Lab, Burlingame, CA) for 20 min. Sections of fixed tissue were incubated with mouse anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (Clone PC-10; Roche Diagnostics Corp., Indianapolis, IN) at 1 µg/mL in blocking buffer (Fricke et al., 1997). Primary antibody was detected by using a biotinylated secondary antibody (horse anti-mouse immunoglobulin G, Vectastain, Vector Lab) and the Avidin-Biotin complex system (Vetcatstain; Vector Lab). Negative control staining consisted of omission of the primary antibody and positive control staining was done in the early cycle corpus luteum of sheep. Tissue sections were counterstained (1 min) with Nuclear Fast Red to visualize nonlabeled nuclei.
An image analysis system (Roche Image Analysis Systems, Elon College, NC) was used to evaluate PCNA labeling (Jin et al., 1994; Zheng et al., 1994). The total area of PCNA-labeled crypt cell nuclei was determined within 10 random fields per tissue for each heifer (Jin et al., 1994; Swanson et al., 2000).
Analysis and Design
Data were analyzed as a completely randomized design with a 2 x 4 factorial arrangement of treatments using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). The model (with treatments and slaughter period arranged as a 2 x 4 factorial) contained treatment, slaughter period, and treatment (slaughter period interactions. In the absence of interactions (P > 0.10), main effects of pregnancy and slaughter period were evaluated. Linear, quadratic, and cubic contrasts were used to assess slaughter period responses and were protected by a significant F-test. In the presence of interactions (P < 0.10), interactive means were separated by least significant difference (Snedecor and Cochran, 1989). Slaughter period effects for variables with interactions were also evaluated with separate analysis using contrasts, which were constructed for linear, quadratic, and cubic effects within either NP or PR heifers.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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). This response to slaughter period is likely explained by changing environmental and dietary composition as slaughter period advanced (Scheaffer et al., 2001). Duodenal protein (mg/g) was unresponsive to pregnancy and slaughter period.
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Jejunal tissues were thought to be the most likely tissue to respond to physiological demands of gestation. The jejunum has been shown to be responsive to diet quality (Jin et al., 1994) and quantity (Burrin et al., 1992) when other sections of the small intestine have been unaffected. Jejunal protein (mg/g) was unaltered, whereas cell number (DNA, mg/g; Table 1
) was increased (P = 0.10) by pregnancy, with PR heifers having greater cell numbers compared with NP. Burrin et al. (1992) also demonstrated an increase in jejunal DNA (mg/g) concentration in sheep fed at maintenance compared with those provided ad libitum access to diet. Jin et al. (1994) observed an increase in jejunal cell numbers in response to increasing dietary fiber. It appears as though the jejunal mucosa is responsive to nutrient demand of the animal, as well as being responsive to luminal nutrient supply.
The cell size estimate (protein:DNA; Table 1) in the jejunum revealed an interaction (P = 0.05) between pregnancy and slaughter period. This interaction resulted from an increase in jejunal protein:DNA of NP heifers at the d 200 slaughter period. Across slaughter periods, NP heifers responded quadratically (P < 0.10), whereas the PR heifers remained similar. No effects were observed for jejunal RNA:DNA (P > 0.19).
Ileal protein concentrations (mg/g) were not influenced by pregnancy or slaughter day. Cell number estimates, within the ileum, interacted between treatment and slaughter period (P = 0.06; Table 1). The ileum of pregnant heifers had decreased DNA concentration at d 200 (NP = 3.52 vs. PR = 2.01 mg/g; P = 0.07) and 270 (NP = 5.11 vs. PR = 3.35; P = 0.10). The NP heifers’ response was quadratic as slaughter period advanced, decreasing at d 120 and 200 and then increasing at d 270, whereas the PR heifers responded to all contrasts tested. This response in the ileum may be an energetic conservation mechanism in maternal systems. This section of the small intestine responded similarly in its mass (Scheaffer et al., 2001).
The duodenum may be less responsive to changing luminal nutrient content, partially due to reduced enzymatic hydrolysis. The concept that distal portions of the small intestine may be more responsive to diet than proximal sections has been reported (Erickson et al., 1995). In rats fed isocaloric low- or high-protein diets, the mid- and distal portions of the small intestine responded by increasing the concentration of amino acid transporter messenger RNA expression relative to the proximal small intestine. This indicates that the mid- and distal small intestine is responsive to luminal concentrations of nutrients. This organization of function in the intestine has been reviewed by Ganapathy et al. (1994), who reported that the intracellular peptidases have the highest activity in the distal and/or middle segment of the small intestine. Thus, a greater proportion of peptides are hydrolyzed and transported in the jejunum and proximal ileum.
Altmann and LeBlond (1969) described a proximal-distal gradient in the intestine, with the greatest villus length in the duodenum and decreasing villus length and crypt depth as sampling site approaches the terminal ileum. Observations that essential AA absorption by duodenal tissues was unaffected by intake (MacRae et al. 1997) provide the functional support to the histological observations of Altmann and Leblond (1969) and partially explain the lack of response of duodenal tissue to pregnancy in this experiment; luminal nutrient concentration would be expected to be similar in animals offered the same diet. In addition, the gut demonstrates variable functionality, with the greatest glucose absorption occurring in the proximal intestine (Freeman, 1995; Bauer et al., 2001) and the majority of AA absorption occurring in the distal intestine (MacRae et al., 1997).
Due to its position within the gastrointestinal tract, the duodenum has the opportunity to be the most responsive of intestinal segments to luminal nutrient concentration. The epithelial layer of the duodenum may be exposed to the highest concentrations (depending on degree of hydrolysis) of luminal nutrients and respond maximally to the levels of nutrient intake relative to the distal portions of the small intestine. After 3 d of fasting, cellular proliferation in the duodenum of rats was reduced, whereas a reduction in proliferation did not occur in the ileum (Holt et al., 1986). Conversely, the data of Jin et al. (1994) showed that diets with similar total caloric and protein concentrations, but differing fiber levels did not elicit a response in duodenal tissue; this phenomenon is also seen in the current data set (Table 1). The data of Jin et al. (1994) demonstrate that when luminal nutrient supply is similar in its total concentration, duodenal tissue is unresponsive; this should also be the case with differing physiological states, as in the present report. Thus, the duodenum appears to be unresponsive to the increased nutrient demand of pregnancy (Table 1) due to the expected similarity of luminal nutrient content in NP and PR heifers.
The proximal-distal gradient of villus length allows for differing responses to dietary variations among the duodenum, jejunum, and ileum. This flexibility allows the small intestine to respond to physiological signals that communicate a greater whole-animal nutrient demand for either glucose (Kojima et al., 1999) or AA (MacRae et al., 1997; Howell et al., 2001).
Liver protein (mg/g, Table 1) was reduced due to pregnancy (P = 0.07); however, other liver cellularity variables were unaffected by pregnancy or advancing slaughter period. This reduction in protein concentration observed in the liver may indicate a decrease in export proteins from the liver of pregnant heifers. However, this does not seem to be a plausible explanation because hepatic oxygen uptake has been shown to increase due to fetal number in ewes (Freetly and Ferrell, 1998).
In vitro O2 consumption data (Table 2) indicate that metabolic rates of the duodenum and jejunum did not respond to pregnancy per unit of tissue mass. Although, the jejunum tended to increase in cell number (P = 0.10) in pregnant animals (Table 1), this response in cell number was not reflected in in vitro O2 consumption. The concentration of RNA and the ratio of protein:DNA in the jejunum tended to interact between treatment and slaughter period (Table 1). Jejunal RNA concentration, an indication of increased metabolic rate, was greater in the PR heifers at d 200 compared with NP heifers (P = 0.07); however, an increase in metabolic rate was not supported by O2 consumption data (Table 2). Variable responses of measures within the jejunum were observed by Burrin et al. (1990; 1992) in studies where level of intake, ad libitum or maintenance, did not affect in vitro O2 consumption, whereas the maintenance intake increased the estimate of jejunal cell number. A similar response has also been reported by Nyachoti et al. (2000), who demonstrated that in vitro O2 consumption did not respond when expressed per unit of tissue mass in growing pigs fed diets of differing crude fiber content.
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; P = 0.05). These data suggest that ileal tissues were functioning at a lower metabolic rate and possibly conserving energy during pregnancy. This concept is supported by the cellularity data in Table 1, in which the PR heifers had fewer cells per unit tissue in the ileum. Hepatic in vitro O2 consumption responded in a cubic pattern to slaughter period and was higher at d 40 and 200 (Table 2; P = 0.04). Likewise, O2 consumption within specific small intestine segments also responded to slaughter day. These responses, because of the absence of pregnancy x slaughter date interactions, were likely due to changes in diet composition or environment.
Calculated daily energy consumption is the combined response of in vitro O2 consumption and tissue mass (Table 3). The three small intestinal sections each responded to slaughter period, and an interaction tended (P = 0.11) to be present for the ileum. The interaction was a result of magnitude since NP heifers used more energy at the d 40 and 200 slaughter periods (P ≤ 0.06). Total small intestinal energy use tended (P = 0.15) to be reduced in PR vs. NP heifers. Moreover, this difference was significant at d 40 and 270. Total liver energy use also responded to slaughter period (Table 3). An interesting point from the data in this table is that in all the variables, the PR heifers had numerically less total tissue energy use.
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The results presented here describe the response of the intestine to increased nutrient demand of gestation. However, it is interesting to consider the impact that increased systemic blood flow and cardiac output due to pregnancy might have on intestinal nutrient absorption and growth dynamics. An increase in luminal nutrient concentration results in increased absorption of nutrients, metabolic rate, and blood flow in the intestine (Krehbiel and Ferrell, 1999; Lappierre et al., 2000), as well as an increase in intestinal mass (Burrin et al., 1990; Wester et al., 1995). However, as the animal consumes more nutrients, intestinal absorption and transport of nutrients becomes less efficient (Huntington, 1990). In the current study, ME intake was not different between NP or PR heifers. Magness (1998) stated that increased blood flow to peripheral tissues in pregnant ewes was most likely a result of increased total cardiac output (NP = 5.2 L/min vs. PR = 7.0 L/min). However, increased cardiac output due to pregnancy is somewhat specific in its distribution throughout the body, with 40 to 55% of the increase going to the gravid uterus and mammary gland, which leaves 45 to 60% remaining to be circulated throughout the body (Rosenfeld, 1977).
Jejunal samples were analyzed morphometrically for nuclear area, crypt depth, and histological labelling of proliferating cells (Table 4). This analysis revealed that the nuclear area and crypt depth were unaffected by pregnancy. Analysis of jejunal samples for PCNA revealed an interaction (P < 0.03) between treatment and slaughter period. Pregnant heifers had less cellular proliferation in the crypt region at d 120 and 200 (P ≤ 0.02). The observed decrease of cells labelled for PCNA, in the absence of morphological or mass changes, suggests a decrease in cellular turnover due to pregnancy. This is substantiated by the greater cellularity estimate in Table 1, in which there was a greater DNA concentration in the jejunum of PR heifers relative to NP heifers (2.58 vs. 2.23 mg/g), indicating that more cells were present in the tissue and that villus length could be increasing. Thus, as the jejunum adapts to increase the absorptive surface due to the increase in nutrient demand associated with pregnancy, the villi likely have increased in length and enterocyte residence time.
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Estimates of total tissue hyperplasia and hypertrophy are shown in Table 5. Estimates of cell number, DNA, cell size, RNA, and protein for the duodenum were not affected by pregnancy (P > 0.1). These estimates were responsive to slaughter period, which arises from the increase in intestinal mass that has been shown previously (Scheaffer et al., 2001). A similar response, as slaughter period advanced, was observed for the protein concentration of the jejunum. The ileum and small intestine as a whole responded similarly to the duodenum when total cell number and size were analyzed (Table 5), with slaughter period having the most significant effect. However, the DNA content of the jejunum was greater due to pregnancy (NP = 3.52 vs. PR = 4.43 g; P = 0.05). The greater DNA content in the jejunum is indicative of greater villus length and greater cell concentration, which might be explained by a decline in cell turnover in the jejunum in light of the lack of response to pregnancy in jejunal mass. This would result in a greater number of epithelial cells within the jejunum for absorption. The response of decreasing cellular number shown by Sainz and Bently (1997) is attributed to the overall decrease in mass of the intestine due to decreased nutrient intake. The increase in DNA content of the jejunum due to pregnancy in our data is likely in response to the increased nutrient demand of pregnancy.
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Decreased cell proliferation in the jejunum (Table 4) is similar to that observed when animals were restricted on a total dietary basis (Goodlad and Wright, 1984) rather than nutrient dilution (Jin et al., 1994), which has been shown not to alter tissue mass. It appears that in total dietary restriction, the gastrointestinal tract transitions to a nutrient conservation state by decreasing its energy use and cellular proliferation. In the nutrient dilution state, the intestine seems to adapt in order to absorb more nutrients with greater cellular turnover (Jin et al., 1994), which results in greater energy use.
Total contents of protein, DNA, and RNA were primarily impacted by slaughter period in a majority of the tissues analyzed (Table 5). In the duodenum, an increase was observed as slaughter period advanced for the contents of protein, DNA, and RNA (P ≤ 0.03). The DNA content of the jejunum was increased in the PR heifers (P = 0.05). An interaction was observed for the RNA content of the jejunum; this was due to the NP heifers having greater RNA content in the d-40 slaughter period compared to the PR heifers (P = 0.03), with the remaining slaughter periods being similar. Total protein in the ileum was decreased by slaughter period (P = 0.03). The DNA content of the ileum was also affected by slaughter period, responding quadratically. The total protein in the liver tended (P = 0.11) to be reduced due to pregnancy (NP = 1,529 vs. PR = 1,027 g; Table 5) and increased linearly as slaughter period advanced. The tendency for an interaction between pregnancy and slaughter period was also observed (P = 0.10). The NP heifers responded with a linear increase, whereas the PR heifers were similar.
In conclusion, visceral cellularity and O2 consumption is attenuated in the maternal system due to pregnancy by decreased cellular proliferation in the jejunum, O2 consumption in the ileum, and decreased energy use in the ileum and total small intestine. Cell proliferation in the jejunum decreased due to pregnancy, similar to that observed in feed-restricted animals. Pregnancy did not collaterally decrease visceral mass. These data indicate that pregnancy reduces cell proliferation in visceral tissues. Energy consumption by visceral tissues was not increased due to pregnancy, contrary to what one might expect with the increased nutrient demand of pregnancy. Moreover, the viscera appear to use less energy in their contribution to whole-animal metabolism during pregnancy. Our data indicate that pregnancy does not substantially increase gut mass (Scheaffer et al., 2001) or metabolism, and that changes in visceral metabolism likely does not account for the majority of reported increases in basal metabolic rate resulting from pregnancy. Additional research is needed both in the area of energy use and in visceral metabolic responses during pregnancy.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Footnotes |
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Received for publication February 11, 2002. Accepted for publication March 17, 2003.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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-linked glucose on sodium-glucose cotransport activity along the small intestine in cattle. J. Anim. Sci. 79:1917–1924.This article has been cited by other articles:
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A. N. Scheaffer, J. S. Caton, D. A. Redmer, and L. P. Reynolds The effect of dietary restriction, pregnancy, and fetal type in different ewe types on fetal weight, maternal body weight, and visceral organ mass in ewes J Anim Sci, June 1, 2004; 82(6): 1826 - 1838. [Abstract] [Full Text] [PDF]
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