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Effects of selenium supply and dietary restriction on maternal and fetal body weight, visceral organ mass and cellularity estimates, and jejunal vascularity in pregnant ewe lambs¹

J. J. Reed^*,2, M. A. Ward^*,2,3, K. A. Vonnahme^*, T. L. Neville^*, S. L. Julius^*, P. P. Borowicz^*, J. B. Taylor, D. A. Redmer^*, A. T. Grazul-Bilska^*, L. P. Reynolds^* and J. S. Caton^*,4

^* Center for Nutrition and Pregnancy, Animal and Range Sciences Department, North Dakota State University, Fargo; and USDA-ARS, US Sheep Experiment Station, Dubois, ID

	Abstract

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
To examine effects of nutrient restriction and dietary Se on maternal and fetal visceral tissues, 36 pregnant Targhee-cross ewe lambs were allotted randomly to 1 of 4 treatments in a 2 x 2 factorial arrangement. Treatments were plane of nutrition [control, 100% of requirements vs. restricted, 60% of controls] and dietary Se [adequate Se, ASe (6 µg/kg of BW) vs. high Se, HSe (80 µg/kg of BW)] from Se-enriched yeast. Selenium treatments were initiated 21 d before breeding and dietary restriction began on d 64 of gestation. Diets contained 16% CP and 2.12 Mcal/kg of ME (DM basis) and differing amounts were fed to control and restricted groups. On d 135 ± 5 (mean ± range) of gestation, ewes were slaughtered and visceral tissues were harvested. There was a nutrition x Se interaction (P = 0.02) for maternal jejunal RNA:DNA; no other interactions were detected for maternal measurements. Maternal BW, stomach complex, small intestine, large intestine, liver, and kidney mass were less (P 0.01) in restricted than control ewes. Lung mass (g/kg of empty BW) was greater (P = 0.09) in restricted than control ewes and for HSe compared with ASe ewes. Maternal jejunal protein content and protein:DNA were less (P 0.002) in restricted than control ewes. Maternal jejunal DNA and RNA concentrations and total proliferating jejunal cells were not affected (P 0.11) by treatment. Total jejunal and mucosal vascularity (mL) were less (P 0.01) in restricted than control ewes. Fetuses from restricted ewes had less BW (P = 0.06), empty carcass weight (P = 0.06), crown-rump length (P = 0.03), liver (P = 0.01), pancreas (P = 0.07), perirenal fat (P = 0.02), small intestine (P = 0.007), and spleen weights (P = 0.03) compared with controls. Fetuses from HSe ewes had heavier (P 0.09) BW, and empty carcass, heart, lung, spleen, total viscera, and large intestine weights compared with ASe ewes. Nutrient restriction resulted in less protein content (mg, P = 0.01) and protein:DNA (P = 0.06) in fetal jejunum. Fetal muscle DNA (nutrition by Se interaction, P = 0.04) concentration was greater (P < 0.05) in restricted ewes fed HSe compared with other treatments. Fetal muscle RNA concentration (P = 0.01) and heart RNA content (P = 0.04) were greater in HSe vs. ASe ewes. These data indicate that maternal dietary Se may alter fetal responses, as noted by greater fetal heart, lung, spleen, and BW.

Key Words: fetal • maternal • nutrient restriction • pregnancy • selenium

	INTRODUCTION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Selenium, in the form of high-Se wheat, fed to finishing beef steers at 9 times the daily requirement, increased jejunal mass and number of jejunal cells, but not percentage of proliferating jejunal cells (Soto-Navarro et al., 2004). Consequently, total number of proliferating jejunal cells increased 84% in steers fed high Se compared with adequate Se. Regardless of these effects in jejunal tissue, high dietary Se did not influence the other intestinal and organ masses (Soto-Navarro et al., 2004) or final BW (Lawler et al., 2004). Ewes fed at 60% of NRC (1985) requirement during mid to late gestation had less visceral mass (Scheaffer et al., 2004b) and jejunal DNA concentration (mg/g) at d 130 (Scheaffer et al., 2004a). Considering the effects of high dietary Se (Soto-Navarro et al., 2004), it is possible that Se may provide a sparing effect on visceral organs in nutrient-restricted gestating ewes. No published data are available evaluating the combined effects of nutrient restriction and high dietary Se on maternal and fetal visceral tissues. Therefore, the objectives of this study were to determine effects of nutrient restriction and supranutritional levels of organically bound Se on maternal and fetal visceral mass, growth, and estimates of cellularity and vascularity of nutrient-transferring tissues.

	MATERIALS AND METHODS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals and Treatments
The North Dakota State University Animal Care and Use Committee approved the care and use of the animals. Twenty-one days before breeding, 108 Targhee and Columbia crossed ewe lambs (8.5 ± 0.5 mo of age) were placed in group-fed pens at the US Sheep Experiment Station, Dubois, ID, were assigned randomly to Se treatments (adequate vs. high Se), and Se supplementation began and was continuous until the end of the study. Ewe lambs were fed (2.04 kg/ewe daily) a basal diet consisting of 47% alfalfa hay, 20% corn, 20% sugar beet pulp pellets, 8% malt barley straw, and 5% concentrated separator by-product (de-sugared molasses; DM basis). In addition to the basal diet, adequate-Se ewes received 100 g/ewe daily of a 96% corn and 4% molasses pellet and high-Se ewes received 100 g/ewe daily of a 88% corn, 4% molasses, and 8% Se-enriched yeast (Sel-Plex, Alltech Inc., Nicholasville, KY) pellet. At approximately d 50 of gestation, the ewes were pregnancy tested using ultrasound (Aloka, Tokyo, Japan) and 36 pregnant ewes were shipped to the Animal Nutrition and Physiology Center at North Dakota State University.

Ewes were individually housed in 0.91 x 1.2-m pens in a temperature controlled (12°C) and ventilated facility for the duration of the study. Lighting within the facility was automatically timed to mimic ambient daylight. On d 64 of gestation, the 36 pregnant ewe lambs (53.8 ± 1.3 kg) were assigned randomly to 1 of 4 treatments in a completely randomized design, with the treatments arranged as a 2 x 2 factorial. Main effects evaluated were dietary levels of Se (adequate vs. high Se), and plane of nutrition [100% (control) vs. 60% (restricted) of the NRC (1985) requirements for gestating ewe lambs]. The high-Se group received 80 µg/kg of BW (81 µg/kg of BW for control-high-Se and 78 µg/kg of BW for restricted-high-Se ewes) and the adequate group received 6 µg/kg of BW (7 µg/kg of BW for control- adequate-Se and 4 µg/kg of BW for restricted-adequate-Se ewes). Coupling ewe BW with Se intake values yielded total Se intakes that were 0.37, 4.41, 0.21, and 4.17 mg/d for control-adequate-Se, control-high-Se, restricted-adequate-Se, and restricted-high-Se ewes, respectively.

Diets were fed once daily, with free access to water and trace mineralized salt (containing no added Se; American Stockman, Overland Park, KS). Diets (DM basis) were similar in CP (16.0%) and ME (2.12 Mcal of ME/kg), were fed individually, and consisted of alfalfa hay (chopped, 3.8 cm in length), 0.42 ppm (mg/kg) of Se, whole corn (offered when additional energy was needed to meet ME requirements), and pelleted (0.48-cm diam.) supplements. The adequate-Se supplement (0.32 ppm of Se) contained 96% corn and 4% molasses, whereas the HSe supplement (43.2 ppm of Se) contained 88% corn, 4% molasses, and 8% Se-enriched yeast (DM basis; Sel-Plex; Table 1). Chopped alfalfa was top-dressed with supplement and corn. Diet samples were analyzed for DM, ash, N (methods 930.15, 942.05, and 990.02, respectively; AOAC, 1990), ADF, and NDF (Ankom, Fairport, NY), and Se by atomic absorption spectroscopy (Finley et al., 1996).

Slaughter Procedures and Tissue Harvesting
One ewe from each treatment was assigned randomly to each of 9 slaughter days [average gestation length was 135 ± 5 d (mean ± range)]. One hour before slaughter, ewes were weighed to obtain final BW, jugular venous blood was sampled for Se analyses, and the ewes were injected via jugular venipuncture with 5-bromo-2-deoxy-uridine (BrdU; 5 mg/kg of BW). Plasma (10 mL) was collected with sterile, EDTA vacuum tubes (BD, Franklin Lakes, NJ), centrifuged at 1,500 x g for 30 min, and stored frozen (–20°C) until analysis by ICP-MS at the Veterinary Diagnostic Laboratory, Logan, UT. Ewes were stunned with a captive-bolt gun (Supercash Mark 2, Acceles and Shelvoke Ltd, Birmingham, UK) and exsanguinated, and blood was obtained. After exsanguination, maternal and fetal tissues were harvested.

The gravid uterus was immediately removed, dissected from the vagina at the external os of the cervix, and weighed. The fetus was removed from the placenta, and fetal variables were measured (as described later). The ewe was eviscerated, and the whole viscera were weighed.

The liver, spleen, and pancreas were dissected from the visceral tissues and weighed. The stomach complex was divided from the esophagus from its entry at the dorsal sac of the rumen and from the intestine at the pyloric valve. Digesta and fat were removed, and the stomach complex was weighed. Intestinal tissues were located, and the demarcations of duodenum, jejunum, ileum, cecum, and colon were made as described by Scheaffer et al. (2004a). A 150-cm section of jejunum was immediately removed for vascular perfusion according to Soto-Navarro et al. (2004). After specific regions were identified, the mesentery was dissected from the tissue; digesta was gently stripped, and the segments were weighed. Digesta from the stomach complex and intestines were combined and weighed.

The heart, heart fat, lungs, kidneys, perirenal fat, and adrenals were dissected from the carcass and weighed. Mammary tissue (approximately 5 g) was harvested for DNA, RNA, and protein analysis. The weight of the carcass, including hide and head, was defined as the eviscerated BW.

Maternal jejunal tissue samples (15 cm) were collected for percent mucosa, RNA, DNA, protein, and cellular proliferation analysis. Samples were collected at a site 15 cm down the mesenteric vein distal to the mesenteric-ileocecal vein junction and then up the mesenteric arcade to the point of intestinal intersection. A subsample (5 cm) of the 15-cm jejunal tissue sample was gently washed in PBS buffer, weighed, placed on a polyethylene cutting board, and opened with the luminal side up. Mucosal tissue was separated (scraped) from the remaining tissues with a glass histological slide, and the remaining jejunal tissue was weighed. Mucosa was saved for analysis of DNA, RNA, and protein.

The fetus was immediately removed from the placenta by tying off and then severing the umbilical cord at the umbilicus and was then weighed to obtain a fetal BW. Heart girth dimension and crown-to-rump length were measured. The fetal carcass was processed similar to the maternal carcass. The full alimentary tract was weighed. The stomach complex (reticulum, rumen, omasum, abomasum) was removed at the entry of the esophagus and at the pyloric junction, drained of fluid, and weighed. Then, individual organs of the gastrointestinal tract were dissected, stripped of contents, and weighed. The small intestine was estimated to be from the pyloric junction and then caudally to the ileocecal valve. The remainder of the tract (including the cecum) was identified as large intestine. The kidney, adrenal glands, heart, lungs, and perirenal fat were removed from the fetal carcass and weighed.

Five-gram samples were collected from the fetal small intestine, heart, and muscle. Samples were collected at a site 3 mesenteric vein branches distal from the mesenteric-ileocecal vein junction and then up the mesenteric arcade to the point of intestinal intersection.

Maternal jejunum, mucosa, and mammary tissue, and fetal jejunum, heart, and muscle were preserved for RNA, DNA, and protein analysis. The samples were wrapped in foil, snap-frozen in supercooled isopentane (submerged in liquid nitrogen), and stored at (80°C (Reynolds et al., 1990; Reynolds and Redmer, 1992).

Cellularity Estimates
Freshly thawed tissue samples (0.5 g) were homogenized using a Polytron with a PT-10s probe (Brinkmann, Westbury, NY), in Trizma base (Sigma Chemical, T-6086, St. Louis, MO), sodium, and EDTA buffer (TNE buffer; 0.05 M Tris, 2.0 M NaCl, 2 mM EDTA, pH 7.4). Samples were analyzed for DNA and RNA using diphenylamine (Johnson et al., 1997) and orcinol procedures (Reynolds et al., 1990). Protein in tissue homogenates was determined with Coomassie brilliant blue G (Bradford, 1976) with bovine serum albumin (Fraction V; Sigma Chemical) as the standard (Johnson et al., 1997). The prepared samples were analyzed with a spectrophotometer (Beckman DU 640, Beckman Coulter Inc.) and were assessed against concentration curves of known standards (DNA; Calf Thymus D-1501 and RNA; Bakers yeast, S. cerevisiae RG750; Sigma Chemical). The concentration of DNA was used as an index of hyperplasia, with protein:DNA and RNA:DNA ratios used as indexes of hypertrophy (Swanson et al., 2000; Scheaffer et al., 2003; Soto-Navarro et al., 2004).

Jejunal Cell Proliferation
Five-bromo-2-deoxy-uridine was used for histological estimates of tissue cellular proliferation, as described previously (Jablonka-Shariff et al., 1993; Jin et al., 1994). Fresh jejunal tissue sections were immersed in a Carnoy’s fixative (60% ethanol, 30% chloroform, 10% glacial acetic acid VWR, West Chester, PA; J. T. Baker, Phillipsburg, NJ) for 3 h and transferred to a 70% ethanol solution. Tissues were embedded in paraffin (Reynolds and Redmer, 1992) and 4-µm tissue sections were made from the paraffin blocks, mounted on glass slides, and prepared for staining procedures (Fricke et al., 1997; Soto-Navarro et al., 2004). The prepared tissues were incubated with antiBrdU, formalin grade, mouse IgG, monoclonal antibody (Clone BMC, Roche Diagnostics, Indianapolis, IN) at 9 µL/1.8 mL of blocking buffer. Primary antibody was detected using 3, 3'-diaminobenzidine (Vector Laboratories, Burlingame, CA), and stained cells were undergoing proliferation in the S-stage of mitosis. Hematoxylin (EMD Chemicals Inc., Gibbstown, NJ) was used to counterstain the nondividing nuclei, and the periodic acid-Schiffs staining procedure (Luna, 1968) was utilized to highlight other structures present within the jejunal tissue cross-section. Cellular proliferation was quantified using Image-Pro Plus 5.0 software (MediaCybernetics Inc., Silver Spring, MD).

Small Intestine Vascularity
A portion of the freshly excised jejunum was perfusion-fixed according to Scheaffer et al. (2004a) and Soto-Navarro et al. (2004), with the exception that a different casting resin was used. For the current study, a latex resin (Microfil MV-132, 4 mL of latex compound combined with 5 mL of diluent, all from Flow Tech Inc., Carver, MA) was used as the casting resin. Cross-sections of perfused intestinal tissue were processed as described above. Four-micrometer tissue sections were stained using periodic acid-Schiff’s staining procedures (Luna, 1968) to contrast the vascular tissue. Mean capillary area, capillary number, and capillary circumference measurements were made in the intestinal villi using the Image-Pro Plus software (Media-Cybernetics Inc.).

Calculations
Empty BW was calculated as BW minus total digesta weight. Maternal BW was calculated as empty BW minus gravid uterine weight (Rattray et al., 1974; Robinson et al., 1978). To express organ mass on an empty BW basis or maternal BW basis, fresh organ mass (g) was divided by empty BW (kg) or maternal BW (kg). Percent jejunal mucosa was calculated by dividing the mucosal scrape mass by the sample mass before scraping. Total jejunal mucosa was calculated by multiplying the percent jejunal mucosa by total jejunal mass (g). Fetal organ mass data are expressed as grams of fresh tissue and grams per kilogram of empty carcass weight. Total digesta was not measured in the fetus; therefore, fresh organ mass was divided by empty carcass weight (including head, hide, and hoof mass).

Total DNA, RNA, and protein contents were calculated by multiplying DNA, RNA, and protein concentrations by fresh tissue weights (Swanson et al., 2000; Scheaffer et al., 2003, 2004a). Percent proliferating cells was estimated by dividing the number of diaminobenzidine (BrdU)-stained nuclei by the total number (BrdU-+ hematoxylin-stained) of nuclei present within the area of tissue analyzed. Number of proliferating cells was calculated by dividing total tissue DNA (mg) by 6.6 x 10^–12 g (the average amount of DNA per nucleus; Baserga, 1985) and then multiplying that value by the percentage of cell proliferation (Zheng et al., 1994).

Capillary area density was determined by dividing the total capillary area by the area of tissue analyzed (Scheaffer et al., 2004a; Soto-Navarro et al., 2004). Capillary number density was calculated by dividing the total number of vessels counted by tissue area. To estimate the capillary surface density (total capillary circumference per unit of tissue area), mean capillary perimeter (circumference) was divided by tissue area (Reynolds et al., 2005; Borowicz et al., 2007). Although capillary surface density actually represents the circumference of the capillary cross-sections, it is nevertheless proportional to their surface area. Finally, mean area per capillary was determined by dividing capillary area density by capillary number density. Total vascularity (mL) was calculated by multiplying the percentage of capillary area density by tissue mass.

Statistics
Data were analyzed as a completely randomized design with a 2 x 2 factorial arrangement of treatments using PROC GLM (SAS Inst. Inc., Cary, NC). Because ewe lambs carried both singles and twins, fetal number was included in the model. If the fetal number was significant (P < 0.10), it was retained in the model. If the fetal number was not significant, fetal number was dropped from the model. The model contained effects for nutrition (control and restricted), level of Se (adequate and high), and the nutrition x Se interaction. When interactions were present (P < 0.10), means were separated by the least significant difference test. Main effects were considered significant when P < 0.10.

	RESULTS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
No Se x nutrition interactions (P > 0.47) were detected for maternal BW, BW change, and reproductive tissue weights, thus main effects of nutrition and Se treatments are presented in Table 2. Initial ewe BW was not different (P > 0.45; Table 2) between treatments on d 64 of gestation before nutritional treatments commenced. At experiment termination, nutrient-restricted ewes were lighter (P = 0.001) compared with controls. Nutrient-restricted ewes gained 33 g/d compared with 163 ± 10 g/d for controls. Consequently, empty BW, maternal BW, and empty carcass weight were less (P = 0.001) in restricted than in control ewes. Ewe empty BW increased (P = 0.04) in high-Se compared with adequate-Se ewes. Total digesta content was less (P = 0.001) in restricted compared with control ewes. Gravid uterine mass (kg) was affected by nutrition and Se; restricted ewes had less (P = 0.01) gravid uterine mass (kg) than did control and high-Se ewes had greater (P = 0.02) gravid uterine mass than did adequate-Se ewes. However, there were no differences (P > 0.25) among treatments when gravid uterine mass was expressed on an empty BW or maternal BW basis.

Full viscera, stomach complex, liver, pancreas, spleen, heart, lung, kidney, perirenal fat, and mammary gland masses (g) were less (P 0.04) in restricted than in control ewes (Table 3). However, when expressed on an empty BW or maternal BW basis, stomach complex mass was less (P 0.002) in restricted compared with control ewes. In addition, lung mass was greater (P 0.09) in restricted ewes on both an empty BW and a maternal BW basis. However, restricted ewes had increased blood mass (P = 0.01) based on both an empty BW and a maternal BW basis compared with control ewes.

Duodenum and jejunum masses were less (P 0.002) in restricted ewes compared with control, but there were no differences (P > 0.14) when expressed on empty BW basis (Table 4). Restricted ewes had less (P = 0.001, 0.04, and 0.08 for g and g/kg of empty BW, and g/kg maternal BW basis, respectively) small intestine mass compared with control ewes. Large intestinal mass was less (P = 0.04; for g, g/kg empty BW, and g/kg maternal BW) in restricted compared with control ewes. Ewes receiving high Se had greater (P = 0.01) small intestinal mass (Table 4). However, individual components of the small intestine (duodenum, jejunum, jejunal mucosa, and ileum) and the large intestine were unaffected (P 0.20) by Se.

Neither plane of nutrition nor Se level had an effect (P 0.23) on percentage of proliferating nuclei, total jejunal cells, or total cells proliferating in the maternal jejunum (Table 6). Total mucosal cell number was less (P = 0.01) in restricted compared with control ewes. There were no differences observed in total proliferating mucosa cells in the maternal jejunum or jejunal mucosa.

A nutrition x Se interaction occurred in fetal muscle DNA concentrations (Table 10; Figure 1). Fetuses from restricted mothers fed high Se had greater (P < 0.05) muscle DNA concentrations compared with other treatments. Maternal nutrient restriction resulted in greater (P = 0.008) fetal muscle RNA concentration (mg/ g) and less (P = 0.02) protein concentration (Table 10). Consequently, fetal muscle protein:DNA was less (P = 0.009) in fetuses from restricted vs. control ewes.

View larger version (11K): [in this window] [in a new window]   Figure 1. Interaction means for DNA concentrations in fetal muscle tissue as influenced by level of nutrition and dietary Se in pregnant ewe lambs. Treatments were CASe = control nutrition-adequate Se, C-HSe = control nutrition-high Se, R-ASe = restricted nutrition-adequate Se, and R-HSe = restricted nutrition-high Se. Nutritional treatments were from d 64 of gestation until slaughter at 135 ± 5 (mean ± range) d of gestation. Selenium treatments (fed from 21 d before breeding until slaughter) were daily intake of organically bound Se; adequate Se (ASe; 6 µg/kg of BW) and high Se (HSe; 80 µg/kg of BW), respectively. a,bInteraction means having different superscripts differ (P 0.01).

	DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Nutrient restriction reduced most organ masses. Physiological responses to nutrient restriction have been documented previously (Brameld et al., 2000; Ferraris and Carey, 2000; Scheaffer et al., 2004b). Reductions in organ masses resulted from fewer nutrients available to maintain tissue size, but also likely reflected an adaptive response that allows the animal to survive on lower nutrient supply (Ferrell and Jenkins, 1985). Both nutrition and Se affected lung mass. On a gross (g) basis, lung mass was less in the restricted ewes; however, lung mass was greater when expressed on both an empty BW and a maternal BW basis. These data indicate an asymmetric change in lung mass. Scheaffer et al. (2004b) reported no differences in lung mass on a gross, empty BW, or maternal BW basis between nutrient-restricted- and control-fed ewes. Also, in the current study, high-Se ewes had greater lung mass on gross and empty BW basis compared with adequate-Se ewes. A possible explanation for this, is the excretory pathway of dimethylselenide (Se(CH₃)₂), a methylated form of Se, via respiration (Sunde, 1997). If the body is trying to excrete excess Se, increased respiration and thus lung capacity and mass may result as a response to elevated Se status in the body. Additionally, fetal lung mass as a proportion of BW was unaltered by maternal dietary Se.

Similar to the current study, Scheaffer et al. (2004a) reported reductions in full viscera, small intestine, stomach complex, and liver masses in pregnant ewes fed 60% of controls. In contrast with the current data set, Scheaffer et al. (2004a) reported no differences in the large intestine. Maternal large intestine in the current study was reduced on a gross, an empty BW, and a maternal BW basis in restricted ewes. A possible explanation for the different responses observed in the respective studies, were diet form and content. The diet utilized in the Scheaffer et al., (2004a) study was pelleted and consisted of both beet pulp and chopped alfalfa hay. As previously described, the diets offered in this study consisted of loose chopped alfalfa hay, corn, and pelleted supplement. Differences in rate of passage and extent of digestion could have been contributing factors in differences observed in the nutrient-restricted animals of the respective studies.

Nutrient restriction had no effect on DNA or RNA in maternal jejunal tissue. However, nutrient restriction resulted in decreased maternal jejunal protein content and protein:DNA ratio. Additionally, DNA and protein content were less in the maternal jejunal mucosa. These data indicate that there were fewer mucosal cells present in restricted compared with control ewes. Therefore, reductions in jejunal mass in restricted ewes may have been primarily due to reductions in hyperplasia rather than hypertrophy. This is supported by the fact that restricted ewes had decreased jejunal and total jejunal mucosa masses compared with control. These data agree with others (Sainz and Bently, 1997; Scheaffer et al., 2004b) who reported reduced DNA concentrations in jejunal tissues of nutrient-restricted ruminants. Conversely, Soto-Navarro et al. (2004) reported increased jejunal DNA concentration in steers fed 3 ppm Se from an organic source compared with steers that were fed no supplemental Se. However, their feedlot steers (Soto-Navarro et al., 2004) were on a high plane of nutrition, which may explain the more dramatic response in jejunal DNA concentrations in response to high Se.

The small intestine consumes approximately 20% of maintenance energy in ruminants (Ferrell and Jenkins, 1985; Bell, 1993; Caton et al., 2000). Therefore, it is one of the first organs affected by limited nutrition. ntestinal mucosa tissues are altered in structure and function when intake is diminished (Ferraris and Carey, 2000). Enterocyte proliferation and migration along the cryptvillis axis is less, resulting in a reduction in villus height in severely malnourished rats, mice, and hamsters (Ferraris and Carey, 2000). In the current study, total cells present in the maternal jejunal mucosal tissue were less in the restricted compared with control ewes; however, total jejunal cells were unaffected by plane of nutrition. Therefore, these data may indicate that jejunal mucosa is more sensitive to nutrient restriction than the organ as a whole.

Nutrient restriction resulted in less capillary area density, area per capillary, and total jejunal and mucosal vascularity. The combination of these data likely indicates reductions in blood flow in the jejunum. Burrin et al. (1989) reported reductions in O₂ consumption nd portal drained visceral blood flow in sheep consuming high concentrate diets fed at maintenance vs. ad libitum. Burrin et al. (1989) hypothesized that energy intake may be driving changes in vascularity. In contrast, Scheaffer et al. (2004a) reported increases in jejunal vascularity (%) in nutrient-restricted, mature, pregnant ewes.

Many of the fetal visceral tissue masses (including empty viscera, stomach complex, small intestine, liver, spleen, and pancreas) were less in restricted compared with control ewes. As a result, fetal BW and empty carcass were less. Numerous researchers (Osgerby et al., 2002; Vonnahme et al., 2003; Scheaffer et al., 2004a) have reported decreased fetal BW and/or organ mass in fetuses from nutrient-restricted dams. Potential implication of low fetal BW and birth weight include poor growth and lifelong performance (Barker, 1998; Greenwood et al., 1998).

Neither nutrient restriction nor dietary Se impacted fetal jejunal RNA or DNA concentrations or content; however, RNA:DNA ratio was greater in fetal jejunum from high-Se compared with adequate-Se ewes. These data suggest an increase in hypertrophy or synthetic capacity in the fetal jejunum in the high-Se vs. adequate-Se-fed ewes. Nutrient restriction resulted in less fetal jejunal protein content and decreased protein:DNA ratio, supporting the indication of smaller cell size in the fetal jejunum of restricted ewes.

Fetal heart mass (g) was not affected by nutrient restriction; however, cellularity estimates indicated that nutrient restriction increased RNA, decreased protein and protein:DNA ratios, and tended to increase DNA concentrations. These data indicate the nutrient restriction resulted in smaller more numerous cells in fetal hearts compared with controls.

There were nutrition x Se interactions in fetal heart, kidney, adrenals, perirenal fat, and stomach complex tissue masses when expressed as g/kg of ECW. Fetal heart mass (g/kg of ECW) of restricted was greater compared with control ewes. Increases in fetal heart mass in response to nutrient restriction have been previously reported (Han et al., 2004; Dong et al., 2005). Han et al. (2004) reported increased left ventricular hypertrophy in fetuses under nutrient stress. Based on current and previously reported data (Vonnahme et al., 2003; Han et al., 2004; Dong et al., 2005), it appears nutrient restriction results in asymmetric growth in the fetal heart. Fetal perirenal fat (g) was reduced by nutrient restriction. In addition a nutrition x Se interaction for fetal perirenal fat (g/kg of ECW) revealed that fetuses from restricted ewes fed high Se had less perirenal fat mass compared with all other treatments. A potential negative consequence of reduced perirenal fat in the high-Se fetuses, could be reduced ability to maintain internal heat production. Brown adipose tissue associated with the neonate, contains high levels of type 2 deiodinase selenoenzyme, and is responsible for increasing metabolic activity thus metabolic heat through the activation of triiodothyronine from thyroxine (Arthur et al., 1991). Therefore, reduced perirenal fat stores could impede the neonate’s ability to withstand environmental cold stress.

Of the cellularity estimates measured, fetal muscle was the most responsive to maternal dietary treatment. There was a nutrition x Se interaction in fetal muscle DNA concentration; where fetuses from restricted high Se ewes had muscle DNA concentrations approximately 1.6 times that of all other treatments. Increases in hyperplasia may allow for increases in tissue function (Baserga, 1985). Fetal muscle RNA concentrations were also greater in high-Se compared with adequate-Se groups. In terms of productive performance, Greenwood et al. (1998) reported slower growth rates and reduced feed efficiency in low birth weight lambs, which they attribute in part to increased catabolism of absorbed amino acids for energy. In comparison, growth rates were greater in lambs with greater birth weights and fed at similar energy levels. Therefore, it is also possible that fetuses from the restricted dams receiving added dietary Se may be more efficient in a production setting compared with fetuses from restricted ewes that received no additional dietary Se.

In summary, fetal BW increased in Se fed ewes and fetal muscle DNA concentrations increased in nutrient-restricted ewes fed high Se. Maternal and fetal visceral organ masses were generally reduced by nutrient restriction, while high dietary Se increased maternal and tended to increase fetal small intestinal mass. Maternal intestinal vascularity estimates were reduced by nutrient restriction and not altered by Se. Additional research investigating production and mechanistic responses seems warranted.

	Footnotes

 
¹ This project partially supported by National Research Initiative Competitive Grants No. 2003-35206-13621 and 2005-35206-15281 from the USDA Cooperative State Research, Education, and Extension Service, by NIH Grant HL 64141, and partially by USDA-IFAFS Grant No. 00-52102-9636. Gratitude is expressed to employees of the Animal Nutrition and Physiology Center and Ruminant Nutrition and Physiology Laboratories for their contributions to this project.

² Both authors contributed equally to this work.

³ Present address: Colby Community College, 1255 S Range, Colby, KS 67701.

⁴ Corresponding author: joel.caton{at}ndsu.edu

Received for publication November 30, 2006. Accepted for publication May 18, 2007.

	LITERATURE CITED

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 


AOAC. 1990. Official Methods of Analysis. Vol. I. 15th ed. Assoc. Off. Anal. Chem., Arlington, VA.

Arthur, J. R., F. Nicol, G. J. Beckett, and P. Trayhurn. 1991. Impairment of iodothryronine 5'-deiodinase activity in brown adipose tissue and its acute stimulation by cold in selenium deficiency. Can. J. Phys. Pharm. 69:782–785.[Medline]

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