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Effect of dietary restriction, pregnancy, and fetal type on intestinal cellularity and vascularity in Columbia and Romanov ewes¹

Center for Nutrition and Pregnancy, Department of Animal and Range Sciences, North Dakota State University, Fargo 58105

	Abstract

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The objectives of this study were to evaluate intestinal cellularity and vascularity in mature ewes in response to dietary restriction and pregnancy status and to quantify the response of these variables to increased nutrient demand of fetal growth. In Exp. 1, 28 mature Dorset x crossbred white-faced ewes (61.6 ± 1.8 kg initial BW) were fed a pelleted, forage-based diet. Treatments were arranged in a 2 x 3 factorial, with dietary restriction (60% restriction vs. 100% maintenance for respective states of pregnancy) and pregnancy status (nonpregnant, NP; d 90 and 130) as main effects. Dietary treatments were initiated on d 50 of gestation and remained at 60 or 100% maintenance throughout the experiment. Nonpregnant ewes were fed dietary treatments for 40 d. In Exp. 2, four Romanov ewes were naturally serviced (Romanov fetus and Romanov dam; R/R); two Romanov embryos per recipient were transferred to four Columbia recipients (Romanov fetus and Columbia recipient; R/C), and three Columbia ewes were naturally serviced (Columbia fetus and Columbia dam; C/C). In Exp. 1, dietary restriction and pregnancy status interacted with regard to maternal jejunal DNA concentration (P < 0.01), with restricted ewes having a greater DNA concentration (mg/g; fresh basis) at d 130. Vascularity (percentage of total tissue area) in the jejunum was increased (P < 0.06) as a result of dietary restriction and pregnancy status. Total microvascular volume of jejunal tissue was not altered by dietary restriction and increased (P < 0.01) at d 130 of pregnancy. In Exp. 2, R/R ewes had less (P < 0.09) DNA (g) in the jejunum compared with R/C and C/C ewes. Jejunal vascularity (%) was increased (P < 0.05) in R/R ewes compared with R/C or C/C ewes, whereas total jejunal microvascular volume remained unchanged. These data demonstrate intestinal vascular density responds to changes in diet and physiological state. In addition, pregnancy increased total jejunal microvascular volume.

Key Words: Dietary Restriction • Growth • Pregnancy • Sheep • Small Intestine • Vascularity

	Introduction

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The gastrointestinal tract uses a disproportionate amount of energy considering its contribution to overall body mass (Reeds et al., 1999). Metabolic rate and portal-drained viscera (PDV) blood flow decreases when animals are fasted (Eisemann and Neinaber, 1990) or given restricted diets (Burrin et al., 1989). Milligan and McBride (1985) reported that Na⁺/K⁺-ATPase-dependent respiration increases with level of intake. The PDV also increases blood flow and absorption of total nutrients when a greater luminal nutrient concentration is supplied (Seal and Reynolds, 1993; Lappierre et al., 2000). Freetly and Ferrell (1997, 1998) have shown that, in ewes, PDV blood flow and metabolic rate are increased as gestation advances.

One hallmark of pregnancy is an increase in total cardiac output (Stock and Metcalfe, 1994; Magness, 1998). Pregnancy has been shown to increase maternal metabolic rate compared with preconception metabolism (Brody, 1938; Klieber, 1987). This increase is not completely explained by increased metabolic rate associated with conceptus development (Reynolds et al., 1986). It has previously been shown in our laboratory that crypt cell proliferation in the intestinal mucosa was decreased during midgestation in beef heifers fed similar diets, whereas intestinal mass was unchanged (Scheaffer et al., 2001, 2003). More recently, we have demonstrated in ewes that visceral organ mass (liver and small intestine) decreases during nutrient restriction, increases with advancing pregnancy, and is responsive to maternal genotype (Schaeffer et al., 2004). We hypothesize that previously observed small intestinal responses to diet and physiological state are likely explained by changes in cellularity, cell proliferation, and vascularity. Therefore, our objectives were to evaluate changes in cellularity, cell proliferation, and vascularity in the small intestine due to dietary restriction, pregnancy status, and increased nutrient demand of fetal growth.

	Materials and Methods

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Experiment 1
Animals
Twenty-eight mature Dorset x crossbred white-faced ewes (initial BW = 61.6 ± 1.8) were used to evaluate effects of dietary restriction and pregnancy on small intestinal cellularity and vascularity. Experimental protocols followed institutional animal care and use guidelines (Protocol No. A9907). All ewes were qualified as reproductively sound before exposure to rams by the exhibition of standing estrus during the previous cycle. Ewes were hand mated to one of three of similar phenotype rams and breeding dates were recorded. Ewes that were determined to be pregnant via ultrasonographic examination were randomly assigned to either the d 90 or 130 slaughter group, and those that were not pregnant were allotted to the NP treatment.

Animal care and management have been previously described (Scheaffer et al., 2004). Briefly, ewes were fed a pelleted forage-based diet (Table 1). Treatments were arranged in a 2 x 3 factorial, with dietary restriction (60% restriction [RES] vs. 100% maintenance [MAINT] for respective stages of pregnancy) and pregnancy status (nonpregnant, NP; d 90 and 130) as main effects. Dietary treatments were initiated at d 50 of gestation and remained at 60 or 100% maintenance (NRC 1985) throughout the experiment (Table 1). Non-pregnant ewes were fed dietary treatments for 40 d.

Intestinal tissues were located and the demarcation among duodenum, jejunum, ileum, cecum, and colon was made (Scheaffer et al., 2004). Duodenal tissue was determined to be the tissue from the pyloric valve to the point that was directly adjacent to the branch of the gastrosplenic vein and the anterior mesenteric vein. Jejunal tissue was determined to be the intestinal tissue associated with the proceeding 10 cm of the mesenteric vein distal to the first major branch of the mesenteric vein. Ileal tissue was the remaining small intestinal tissue up to the ileocecal junction. Once these tissues had been located, cellular and vascular samples were collected. In addition, the mesentery was dissected away, the digesta gently stripped, and tissue weighed. Maternal BW responses and organ mass data has been previously reported (Scheaffer et al., 2004).

Small Intestinal Cellularity
A cross section of the respective intestinal tissues was sampled for cellularity analysis. 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; Swanson et al., 1999). Tissue (fresh basis) was minced, and 0.5 g was homogenized in 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 (Burton, 1956; 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 (duodenal, jejunal, ileal) DNA, RNA, and protein content was calculated by multiplying DNA, RNA, and protein concentration by fresh tissue weight (Scheaffer et al., 2004).

Jejunal Cell Proliferation
Jejunal samples of 5 to 10 g were collected, immersed into 10% formalin, placed into paraffin blocks sectioned to 5 µm, and then mounted on glass slides. Tissue sections were treated with blocking buffer for 20 min consisting of PBS and 1.5% (vol/vol) normal horse serum (Vector Laboratories, Burlingame, CA). Sections of fixed tissue were incubated with mouse antiproliferating cell nuclear antigen (PCNA) monoclonal antibody (Clone PC-10; Roche Diagnostics Corp., Indianapolis, IN) at 1 µg/mL in blocking buffer (1.5% normal horse serum in PBS). Primary antibody was detected by using a biotinylated secondary antibody (horse anti-mouse immunoglobulin G, Vectastain, Vector Laboratories) and the Avidin-Biotin Complex system (Vetcatstain; Vector Laboratories). 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.

A computerized image analysis system (Roche Image Analysis Systems, Elon College, NC) was used to evaluate PCNA labeling (Scheaffer et al., 2003; Jin et al., 1994). The total area of PCNA-labeled crypt cell nuclei was determined within 10 random fields per tissue for each ewe (Jin et al., 1994; Swanson et al., 2000).

Small Intestinal Vascularity
Jejunal tissue was used to evaluate small intestinal vascularity (%; Reynolds and Redmer, 1992) or amount of blood vessels in proportion to the amount of tissue. The tissue was immediately transported to the laboratory and kept hydrated with 0.9% PBS. A mesenteric artery branch was located and dissected out of mesenteric tissue. A polyethylene (PE-60; o.d. = 1.22 mm; i.d. = 0.77 mm) beveled catheter was placed into the mesenteric artery branch and secured to surrounding tissue. The vasculature was defined by perfusing the tissue with Evan’s blue dye, and then flushing with PBS. Perfusate flow was regulated to a specific region of tissue by tying or clamping off vessels, which provided flow to tissue outside the studied margins. Flow was continuous through the localized tissue until effluent appeared at the mesenteric venous drainage of the localized tissue. Next, the localized vasculature was perfusion fixed with 10% formalin, and tissue was immersion fixed in 10% formalin. The vasculature was visualized immunohistochemically with antismooth muscle cell actin (mouse anti-SMCA monoclonal; Calbiochem, San Diego, CA; cat. No. CP47) in combination with a biotinylated secondary antibody (horse anti-mouse IgG; Vector Laboratories) and counterstained with nuclear fast red. Vascularity was calculated as described by Reynolds and Redmer (1992) and a total of 20 micrographs per ewe was analyzed.

Calculations and Statistics
Data were analyzed using analysis of variance with the GLM of SAS (SAS Inst., Inc., Cary, NC) for a 2 x 3 factorial arrangement treatments. The statistical model contained dietary treatment (restricted or maintenance), pregnancy status (NP vs. d 90 vs. d 130), and the interaction. Preplanned comparisons to determine the effect of pregnancy (NP vs. d 90 and d 130) and to evaluate the influence of advancing gestation (d 90 vs. d 130) were determined (P < 0.10). In the absence of interactions, main effect means are presented. When interactions were present (P < 0.10), both the main effect and the means of the respective treatments (interactive means) were included in the tables.

Experiment 2
Estrus was synchronized in Romanov (44.8 ± 2.6 kg BW) and Columbia (105.2 ± 17.0 kg BW) ewes using a Norgestomet implant (Sychromate-B; Merial Ltd., Duluth, GA) for 14 d. Estrus status (first estrus = d 0) was checked twice daily after implant removal using vasectomized rams. Recipients were identified and donor ewes were bred by rams of respective breeds (Columbia or Romanov). At d 2 of the estrous cycle, two-to four-cell Romanov embryos were collected by flushing the uterine horns with media during midventral laparotomy. Embryos were recovered from flushing media (ViGro Complete Flush Solution, Agtech, Inc., Manhattan, KS) and graded. Healthy embryos were transferred into the oviduct of recipients via catheters during midventral laparotomy, while ewes were under general anesthesia. Embryos were transferred into the oviduct of Columbia recipients, resulting in the Romanov embryo-Columbia ewe treatment (R/C). The Romanov embryo-Romanov ewe (R/R) treatment consisted of Romanov ewes naturally mated to Romanov rams. Similarly, for the Columbia embryo-Columbia ewe (C/C) treatment, Columbia ewes were naturally mated to Columbia rams.

After breeding and embryo transfer ewes were housed outdoors with free access to shelter. Ewes had ad libitum access to a cool-season pasture, fresh water, and trace mineral salt blocks (Na, 97.25%; Zn, 0.40%; Fe, 0.16%; Mn, 0.12%, Cu, 0.12%, and Co, 0.004%). Slaughter procedures and tissue collections, preparation, and analyses were as outlined for Exp. 1.

Calculations and Statistics
This experiment was a completely randomized design with three treatments. Differences between groups were analyzed using ANOVA with the GLM procedure of SAS. The LSD procedure was used to separate means in the presence of a significant (P < 0.10) F-test for treatment.

	Results

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Experiment 1
Initial BW was not different between the MAINT and RES ewes (62.6 vs. 60.5 ± 2.6 kg). Fetal number was not different between the MAINT and RES treatment groups (1.9 vs. 1.7 ± 0.21), and it did not differ between d 90 and 130 reproductive status (1.9 vs. 1.7 ± 0.20; Scheaffer et al., 2004).

In the duodenum, measures of hyperplasia (DNA, mg/g) and hypertrophy (RNA:DNA) were unresponsive to dietary restriction and pregnancy status. Conversely, duodenal protein:DNA was greater (P < 0.01) in NP compared with pregnant ewes. Duodenal RNA was unaffected by dietary restriction and pregnancy status. An interaction (treatment x pregnancy status) was observed (P < 0.06) for duodenal protein concentration. At d 90, RES ewes had lower (P < 0.03) duodenal protein concentration than MAINT ewes (Table 2).

Total duodenal protein content (g) was less (P < 0.01; Table 3) at d 90 compared with NP or d 130. Total jejunal protein content was decreased due to restriction (Table 3; P < 0.07). Jejunal and total small intestinal DNA and RNA contents were unaffected by dietary restriction or pregnancy status. Ileal DNA content was decreased (P < 0.01) in the RES ewes, and in the NP ewes and d 90 ewes (P < 0.02) compared with d 130 ewes. Ileal RNA content (g) was not altered by treatment, whereas total protein content tended to be decreased (P = 0.11) by dietary restriction.

View larger version (32K): [in this window] [in a new window]   Figure 1. Influence of dietary restriction (maintenance; MAINT vs. restricted; RES) and pregnancy status (NP = nonpregnant, d 90 = d 90 of pregnancy, and d 130 = d 130 of pregnancy) on vascularity (%) and total microvascular volume (mL) of jejunal mucosal tissue.

	Discussion

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The intestine is a luminal tissue that functions as the interface between internal tissues and dietary consumption, an absorptive surface for dietary nutrients, and a protective layer between the extracellular space of the animal and its environment. These functions result in mammalian intestinal epithelial cell turnover occurring every 24 to 96 h (GÖke and Podolsky, 1996). Cellular turnover, nutrient absorption, and secretion of enzymes and other proteins make the intestine a strong competitor for both metabolic energy and protein (Nieto and Lobley, 1999; Caton et al., 2001; Reynolds et al., 2001). When considering cellular turnover, overall tissue mass of the specific organ in question contributes substantially to overall net effect. It has been previously shown by our laboratory that in sheep, intestinal mass is responsive to dietary treatment, pregnancy status, and ewe type (Scheaffer et al., 2004). This is particularly true when expressed per unit of maternal BW. Intestinal tissue function and cellular turnover is a complex series of interactions between luminal nutrient content, demand for absorbed nutrients, and tissue specific metabolism (Ziegler et al., 1999).

In the present study, the hyperplastic estimate of duodenal cellularity (DNA, mg/g) was unresponsive to dietary treatment, pregnancy status, or ewe type (Tables 2 and 6). In a similar analysis with cattle (Scheaffer et al., 2003), duodenal cellularity was not responsive to pregnancy. Due to the functional organization of the small intestine, duodenal cellularity is perhaps less likely to be responsive to dietary treatments than either jejunal or ileal tissue. Decreases in the hypertrophic estimate (Protein:DNA) did not result in altered duodenal mass in response to pregnancy status. Protein:DNA ratio was not different as a result of ewe type; however, RNA:DNA was greater in the R/R than in either the R/C or C/C ewes. These responses are difficult to explain, but Swanson et al. (1999) noted that increased jejunal RNA could be an indication of increased cellular metabolic rate due to ewes being fed low-quality forage. Further investigation of this response of duodenal tissue is needed to understand more completely its metabolic significance.

Jejunal tissue has been shown be the most likely to respond to physiological demands of gestation (Scheaffer et al., 2003). The jejunum has also been shown to be responsive to diet quality (Jin et al., 1994) and quantity (Burrin et al., 1992; McLeod and Baldwin, 2000) when other sections of the small intestine have been unaffected. Jin et al. (1994) observed an increase of jejunal cell number in response to increasing dietary fiber. It seems that the jejunal mucosa is responsive to nutrient demand as well as to luminal nutrient supply. In the current experiments, the estimate of cell number in the jejunum (cellularity:DNA mg/g) interacted between dietary treatment and pregnancy status. This was due to a decrease in DNA in maintenance (4.25 to 3.10) and an increase (4.36 to 5.15) in DNA (mg/g; fresh basis) in restricted ewes as pregnancy status advanced from NP to d 130. The DNA concentration of jejunal tissue was not affected by dietary restriction.

Increased metabolic rate of the maternal system due to pregnancy has been the topic of several classical efforts in the area of energy metabolism (Brody, 1938; Klieber, 1987). There seems to be a required proportion of intestinal tissue mass to support the demands of the developing conceptus (Scheaffer et al., 2004). This functional intestinal tissue mass is maintained in the face of decreased nutrient availability supplying the needed nutrients for the physiological priority of pregnancy. As outlined by Scheaffer et al. (2004), small intestinal mass increases in proportion to maternal BW as gestation advances (16.3, 18.4, 25.3 and 15.3, 19.6, and 21.5 ± 2.3 g/kg of maternal BW for MAINT and RES ewes, respectively) Also, as fetal mass increases in relation to maternal BW, small intestinal mass also increases. These observations support the hierarchy of prioritization of metabolic substrates outlined by Pàlsson (1955) and illustrated that tissues with the greatest metabolic rate appeared to have a higher priority for substrates than tissues with less metabolic rate. Rapid intestinal mucosal cell turnover contributes to the high metabolic rate, which result in a high priority for metabolic substrates.

Reeds et al. (1999) has discussed the relationship of increased gastrointestinal tract metabolic rate and increased PDV mass proportional to whole animal BW in animals where growth was restricted by decreased dietary nutrient intake. They concluded that as level of diet was decreased the proportion of PDV mass to BW also decreased. This response could be attributed to a decrease in epithelial cell turnover, resulting in conservation of energy resources. Interpretations of their experiments are limited by the lack of cellular proliferation estimates. Our data indicate that intestinal cell number (DNA mg/g) and organ mass tend to decline due to dietary restriction in pregnant ewes; however, organ mass scaled to BW was increased as pregnancy status advanced in spite of dietary restriction (Scheaffer et al., 2004), indicating an increase in metabolic rate of the PDV due to pregnancy status. In addition, cellular proliferation was not altered by restriction, pregnancy status, or ewe type. This observation indicates that jejunal cell turnover is decreased due to the increased demand for nutrients by the maternal system during pregnancy and also indicates that the mechanism does not seem to be long-term changes in cellular proliferation per unit of tissue.

Areas of stained proliferating cells were generally unresponsive to dietary restriction, pregnancy, or ewe type. However, mass changes were evident (Scheaffer et al., 2004) with dietary restriction, with NP and R/R ewes having a lower total mass of jejunum when compared with nondiet-restricted, pregnant, and C/C ewes, respectively. Therefore, the total number of proliferating cells likely increased when total mass increased. This agrees with DNA data (Tables 3 and 6), which demonstrate that restriction decreases ileal DNA, whereas pregnancy increases it. Likewise, C/C ewes had greater total gut mass (Scheaffer et al., 2004) and higher total DNA contents compared with R/R ewes.

Changes in the percentage of vascularity and total microvascular volume (Tables 4 and 7) indicate that total absorptive capacity and potential nutrient delivery of the jejunum are modulated by diet and physiological state. The priority of substrate delivery to the developing fetus has been demonstrated (Arnold et al., 2001) by increased vascularity in the placenta of restricted and R/R ewes compared with MAINT, R/C, and C/C ewes, respectively. An increase in blood vessel density was observed in the caruncular tissue of the placenta indicating only a maternal response in vascularity (Arnold et al., 2001). In the present study, dietary restriction increased vascular density (%) and resulted in similar total microvascular volume in jejunal tissue of pregnant ewes compared with the maintenance treatment (Figure 1). These data indicate that gut tissue, during periods of dietary restriction, maintains absorptive capacity and nutrient delivery, even in the event of decreasing total tissue mass (Scheaffer et al., 2004). In addition, during pregnancy, the jejunum demonstrated an increased percentage of vascularity (Table 6; Figure 1A) and increased mass (Scheaffer et al., 2004), which resulted in nearly a doubling of total microvascular volume in d 130 pregnant vs. NP controls (Figure 1B). These data, in conjunction with previous data (Scheaffer et al., 2004), indicate that the gut responds to the increased nutrient demand of pregnancy by increasing mass, vascular density, and total microvascular volume.

Jejunal vascular density in R/R ewes was nearly twice that observed in R/C and C/C ewes. However, total microvascular volume remained unchanged, indicating that absorptive capacity and nutrient delivery potential was similar among R/R, R/C, and C/C ewes. Interestingly, fetal weights from these ewes were also similar (Scheaffer et al., 2004).

To our knowledge, this is the first report on vascular density and total microvascular volume of intestinal mucosal tissue in dietary restricted or pregnant ewes; however, blood flow through the intestine is an area of active investigation (Seal and Reynolds, 1993). MacRae et al. (1997) have shown an increase in blood flow of the mesenteric drained viscera (MDV) and the PDV due to increasing levels of dietary intake. In addition, they also demonstrated a greater MDV flux of essential AA from the lumen of the intestine into the venous drainage due to the increase in blood flow. In the current experiment, jejunal vascular density was increased by dietary restriction and advancing gestation. Additionally, evaluation of interactive means for vascular density indicated that, in NP ewes, restriction and maintenance were similar, whereas during pregnancy, restricted ewes had greater (P < 0.05) jejunal vascular density compared with those fed at maintenance (17.6 vs. 13.6% and 20.8 vs. 13.8% for RES and MAINT ewes at d 90 and 130, respectively). These data indicate that combined effects of dietary restriction and pregnancy results in the increased jejunal vascular density and total tissue microvascular volume (Table 4). Increased vascularity in the jejunal mucosa in response to dietary restriction, pregnancy, and ewe type may provide additional insight into increases in PDV blood flow due to pregnancy as observed by Freetly and Ferrell (1997).

An increase in maternal cardiac output is a wellestablished response to advancing gestation (Rosenfeld. 1977; Magness, 1998). The distribution of the increase in cardiac output has been primarily explained by the increase in blood flow to the uterine compartment, skin, and extremities (Magness, 1998). However, Freetly and Ferrell (1997) observed an increase in total blood flow through the PDV and splanchnic drained viscera in mature ewes. Freetly and Ferrell (1997) also demonstrated that oxygen consumption, a measure of metabolic rate, did not respond to advancing gestation or increasing fetal number associated with the increase in blood flow. These observations indicate that pregnancy is a somewhat unique physiological state regarding gastrointestinal physiology. Increased PDV blood flow due to increased cardiac output associated with pregnancy may result in increased nutrient absorption; however, increased blood flow and an increase in tissue metabolic rate (Gallavan et al., 1983) are simultaneous responses. Observed increases in nutrient absorption associated with increased levels of blood flow and nutrient flux across the PDV coincide with an increase in tissue metabolic rate by the measure of oxygen consumption (Huntington et al., 1988). Edelstone and Holzman (1981) observed that blood flow, nutrient absorption, and oxygen consumption were increased post-prandially in neonatal lambs, which indicates the cohesion of these three measures with increased nutrient absorption. A classical response of nutrients in the intestinal lumen is tissue-specific hyperemia or an increase in blood flow (Sheperd, 1979). Sheperd (1979) also observed that luminal glucose infusion increased blood flow, oxygen consumption, and glucose absorption.

Reeds et al. (1999) described the PDV to contribute 3 to 6 % of BW, while accounting for 20 to 35% of cardiac output, whole-body protein turnover, and energy expenditure. An increase in total cardiac output is a hallmark response of the maternal system to pregnancy (Magness, 1998; Stock and Metcalfe, 1994). The effect of increased cardiac output on the distribution of blood flow to the intestine is not completely clear. Although an increase in blood flow through the intestinal tract due to redistribution or merely increase in total cardiac output could result in an increased nutrient extraction from the lumen.

In an experiment where PDV blood flow was examined in fed or fasted sheep, Katz and Bergman (1969) found that total PDV blood flow decreased due to a 3-d fast in both pregnant and NP ewes. However, in sheep, they made the observation that this decrease due to fasting was not different when scaled to BW, indicating that pregnant ewes were more resistant to declining PDV blood flow than the NP ewes. Their results could be explained by our experiment, where we observed increased mucosal vascular density and total microvascular volume in the dietary restricted pregnant ewes.

	Implications

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Data from these experiments are interpreted to suggest that vascular density in gut mucosal tissue is more responsive to dietary restriction, pregnancy, and ewe type than are cellularity or cellular proliferation. During dietary restriction, the gut mucosa likely conserves nutrients by decreasing mass, while maintaining total absorptive capacity by keeping a constant total microvascular volume. During pregnancy, gut mucosa seems to increase potential absorptive capacity and nutrient delivery by increasing mass, vascular density, and total microvascular volume.

	Footnotes

 
¹ Gratitude is expressed to the NDSU Sheep Unit and the Anim. and Range Sci. Physiology and Nutrition Laboratories for their valuable assistance with the project.

² Current address: Colorado State Univ., Anim. Reprod. and Biotech. Lab., 3801 W. Rampart Rd., Fort Collins 80523.

⁴ Current address: Univ. of Montreal, CRRA Dept., 3200 Sicotte, St. Hyacinthe, QC J2S 7C6.

³ Correspondence—phone: 701-231-7653; fax: 701-231-7590; e-mail: joel.caton{at}ndsu.nodak.edu.

Received for publication January 9, 2004. Accepted for publication July 7, 2004.

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