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Journal of Animal Science - Animal Growth, Physiology, and Reproduction

Maternal nutritional plane and selenium supply during gestation impact visceral organ mass and intestinal growth and vascularity of neonatal lamb offspring1

 

This article in JAS

  1. Vol. 91 No. 6, p. 2628-2639
     
    Received: Oct 6, 2012
    Accepted: Mar 7, 2013
    Published: November 25, 2014


    4 Corresponding author(s): joel.caton@ndsu.edu
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doi:10.2527/jas.2012-5953
  1. A. M. Meyer*22,
  2. T. L. Neville*,
  3. J. J. Reed*33,
  4. J. B. Taylor,
  5. L. P. Reynolds*,
  6. D. A. Redmer*,
  7. C. J. Hammer*,
  8. K. A. Vonnahme* and
  9. J. S. Caton 4
  1. Center for Nutrition and Pregnancy, Department of Animal Sciences, North Dakota State University, Fargo 58108
    USDA-ARS, U.S. Sheep Experiment Station, Dubois, ID 83423

Abstract

To investigate effects of nutritional plane and Se supply during gestation on neonatal offspring visceral organ mass and intestinal growth and vascularity, 84 nulliparous Rambouillet ewes (age = 240 ± 17 d, BW = 52.1 ± 6.2 kg) were allocated to a 2 × 3 factorial design. Ewes were fed 1 of 2 Se diets [adequate Se (ASe, 11.5 µg/kg BW) or high Se (HSe, 77.0 µg/kg BW)], initiated at breeding, and 1 of 3 nutritional planes [60% (restricted; RES), 100% (control; CON), or 140% (high; HIH) of NRC requirements], initiated at d 40 of gestation. Ewes were fed individually and remained on treatments through parturition. All lambs were removed from their dams at birth and fed milk replacer. At 20.6 ± 0.9 d of age, lambs were necropsied, visceral organs dissected, and jejunal samples collected. Lambs born to ewes fed CON and HIH had greater (P < 0.05) BW, gastrointestinal tract, stomach complex, and liver masses at necropsy than RES. Large intestinal and pancreatic masses, as well as stomach complex, large intestinal, and liver proportional masses, demonstrated (P ≤ 0.08) a nutritional plane × Se supply interaction. Proportional pancreatic mass was greater (P = 0.03) for lambs born to RES ewes than HIH. Although small intestinal mass was not affected (P ≥ 0.18) by gestational treatments, lambs born to HIH-fed ewes had greater (P ≤ 0.09) jejunal DNA concentration than RES and CON, and greater (P = 0.01) total DNA than RES. Nutritional plane and Se supply interacted to affect (P ≤ 0.003) jejunal percent proliferation and total proliferating small intestinal cells, although jejunal crypt depth and villus length were not affected by gestational treatment (P ≥ 0.17). Jejunal glucagon-like peptide-2 mRNA expression was greater (P ≤ 0.07) in lambs born to ewes fed RES compared with CON and HIH. Jejunal capillary size was affected (P = 0.09) by the interaction of nutritional plane × Se supply. Lambs from CON ewes had greater (P ≤ 0.04) jejunal capillary surface density than RES. Nutritional plane and Se supply interacted to affect (P = 0.07) jejunal soluble guanylate cyclase mRNA expression in a manner opposite of capillary size. In conclusion, neonatal lamb visceral organ mass was affected by gestational nutrition, even when lambs had ad libitum intake and similar management postnatally. Despite similar small intestinal mass at 20 d of age, jejunal growth, vascularity, and gene expression were altered by maternal nutrition during gestation.



INTRODUCTION

Programming of livestock growth and development occurs in utero due to changes in nutrient availability, which may be caused by differences in maternal nutrient intake during gestation (Wu et al., 2006; Caton and Hess, 2010; Reynolds et al., 2010). Poor maternal nutrition during gestation has reduced preweaning growth, carcass quality, HCW, and reproductive performance in beef cattle and sheep (Caton and Hess, 2010; Du et al., 2010; Funston et al., 2010). Individual nutrients, such as Se (Reed et al., 2007; Meyer et al., 2010a), may be able to reverse these effects and can be found at high concentrations in forages and other feedstuffs (Rosenfeld and Beath, 1964).

Livestock gastrointestinal (GI) tissues are responsive to gestational nutrition, and we have demonstrated that maternal nutrient restriction in sheep alters offspring small intestinal mass (Reed et al., 2007), proliferation (Meyer et al., 2010b; Yunusova et al., 2013), vascularity (Meyer et al., 2010b; Neville et al., 2010b; Yunusova et al., 2013), and maltase activity (Yunusova et al., 2013). During the neonatal period, many structural and functional changes occur in the GI tract, but data are lacking to describe effects of maternal nutrition during pregnancy on neonatal GI tract development and function in ruminants. Because gestational nutrition influences colostrum and milk production (Swanson et al., 2008; Meyer et al., 2011), artificial rearing of offspring is necessary to clearly determine the effect of gestational nutrition on postnatal development (Meyer et al., 2010a; Neville et al., 2010a). The objective of this study was to determine effects of maternal nutritional plane and Se supply during gestation on offspring GI, and specifically small intestinal, growth and development. We hypothesized that even when lambs were raised independently of their dams, effects of maternal nutrition during gestation on the GI tract would persist to 20 d of age.


MATERIALS AND METHODS

Institutional Animal Care and Use Committees at North Dakota State University (NDSU), Fargo, and USDA-ARS, U.S. Sheep Experiment Station (USSES; Dubois, ID) approved animal care and use for this study.

Animal Management and Diets

Ewe and lamb management has been previously described in Meyer et al. (2010a; 2011; 2012b). Briefly, 84 nulliparous Rambouillet ewes (age = 240 ± 17 d; initial BW = 52.1 ± 6.2 kg) from USSES received either adequate dietary Se (ASe; 3.5 µg Se/kg BW daily, n = 42) or high dietary Se (HSe; 65 µg Se/kg BW daily, n = 42) from breeding (d 0) to d 36 of gestation. On d 36 of gestation, ewes were shipped from USSES to the Animal Nutrition and Physiology Center at NDSU for the remainder of the experiment.

At NDSU, ewes were housed individually (0.91- × 1.2-m pens) in a temperature-controlled (12 to 21°C) facility. Ewes continued their Se supply treatments (actual Se intakes: ASe, 11.5 µg Se/kg BW daily; HSe, 77.0 µg Se/kg BW daily) and on d 40 of gestation were assigned randomly to 1 of 3 nutritional plane treatments supplying 60% (restricted; RES), 100% (control; CON), or 140% (high; HIH) of ME and CP requirements (NRC, 1985). This resulted in a completely randomized design with a 2 × 3 factorial arrangement of Se supply × nutritional plane (ASe-RES, ASe-CON, ASe-HIH, HSe-RES, HSe-CON, HSe-HIH; n = 14 ewes/treatment).

Ewes had free access to water and a trace-mineralized salt block containing no additional Se. During gestation, diets were fed once daily in a complete pelleted ration and 3 pellet formulations (adequate Se pellet, high Se pellet, and concentrated Se pellet) were blended to meet ME and Se amounts necessary for each ewe, according to treatment (Meyer et al., 2010a). At USSES and NDSU, Se was provided to HSe ewes in the form of Se-enriched wheat millrun, resulting from processing of wheat grown in a seleniferous region near Pierre, SD. Purified selenomethionine was the Se source for the concentrated Se pellet fed at NDSU (fed to RES and CON ewes, as needed, to meet Se content of HSe treatment). Nutrient requirements were based on NRC (1985) recommendations for 60-kg BW, pregnant ewe lambs during mid to late gestation (weighted ADG of 140 g/d). Diets were adjusted for BW and BW gain for each 14-d interval of gestation. Orts were collected daily to calculate intake (feed offered – orts), but ewes generally ate the feed offered daily and rarely left refusals. Ewe DMI and nutrient intake, as well as BW, BCS, and ultrasonic body composition changes during gestation, have been previously reported by Meyer et al. (2010a; 2011).

Ewes were monitored closely during lambing and lambs were removed immediately after birth and before suckling. Lambs were fed artificial colostrum (45% CP, 15% crude fat, 0.15% crude fiber, 10% ash, 0.40% minimum Ca, 0.90% maximum Ca, 0.30% minimum NaCl, 0.50% maximum NaCl, 1.0% minimum Na, 1.5% maximum Na, 0.40% P, 0.75 mg/kg Se 110,000 IU/kg vitamin A, 22,000 IU/kg vitamin D, 440 IU/kg vitamin E; Acquire Colostrum Replacement; APC, Inc., Ankeny, IA) within 30 min of birth and at 2, 4, 8, 12, 16, and 20 h postpartum to achieve 10.64 g IgG/kg BW (19.1 mL of colostrum/kg BW for the first 2 feedings and 25.5 mL of colostrum/kg BW in subsequent feedings). This dosage of IgG was calculated to provide 50 g of IgG per 4.7-kg lamb, based on previous reports using a similar product (Hammer et al., 2011).

At 24 h of age, lambs were transitioned to milk replacer (24% CP, 30% crude fat, 0.10% crude fiber, 6.5% ash, 25% lactose, 0.50% minimum Ca, 1.0% maximum Ca, 0.65% P, 2 mg/kg minimum Cu, 6 mg/kg maximum Cu, 0.3 mg/kg Se, 66,000 IU/kg vitamin A, 22,000 IU/kg vitamin D3, 330 IU/kg vitamin E; Super Lamb Instant Milk Replacer; Merrick’s Inc., Middleton, WI), fed from a bottle, until a strong suckling response was observed. Once this occurred, lambs were moved to a pen with free access to water and creep feed (55% corn, 25% soybean meal, 12.5% oats, and 7.5% supplement; J&S Farmers Mill, Barnesville, MN), and adapted to a teat-bucket system for ad libitum access to milk replacer.

Visceral Organ Measurements and Tissue Collection

Within treatment groups, lambs were assigned randomly to necropsy between 19 and 22 d of age (average = 20.6 ± 0.9 d). At euthanasia, lambs were weighed, stunned by captive bolt (Supercash Mark 2; Acceles and Shelvoke Ltd., England), and exsanguinated before detailed dissections were performed.

Viscera were removed and weighed with digesta before dissection. The liver, pancreas, stomach complex, small intestine, and large intestine were dissected, gently stripped of fat and digesta, and weighed. A 100-cm section of the jejunum was removed and used for vascular perfusion, mucosal collection, and immersion fixation (Reed et al., 2007). The section began at a point adjacent to the mesenteric vein 3 vascular branches caudal from its junction with the ileocecal vein. The sample was taken by cutting around the main branches of the mesenteric arcade, including a section of the mesenteric vein. The tissue was placed in warm PBS, covered with cheese cloth, and returned to the lab for perfusion. A second 100-cm section was taken distal from the first for measurement of stripped weight and stripped length.

Intestinal Tissue Preparation and Analysis

A 15-cm segment was removed from the proximal end of the 100-cm jejunum sample and opened by cutting along the mesenteric side to expose the luminal surface. The lumen was rinsed with PBS and a mucosal sample was scraped with a glass microscope slide and flash-frozen by immersion in isopentane supercooled in liquid N, then stored at –80°C (Reynolds and Redmer, 1992) for cellularity and angiogenic factor mRNA analyses.

Next, a 10-cm segment was removed from the 100-cm jejunum sample and rinsed by immersion in PBS. Cross sections (<1 cm wide) were cut from the jejunal sample and immersion fixed in Carnoy’s solution (60% ethanol, 30% chloroform, 10% glacial acetic acid). After 6 h, fixed tissue samples were transferred to 70% ethanol for storage until they were embedded in paraffin.

The remainder of the 100-cm jejunal section was fixed via vascular perfusion using methods described by Meyer et al. (2012b). Briefly, within 10 min of sample collection, a main branch of the mesenteric vein was catheterized with polyethylene tubing, flushed with warm PBS, and pulse infused with Carnoy’s solution to fix the tissue. After allowing the tissue to fix, sections of jejunum that appeared to have the most perfusion with Carnoy’s solution were removed. Small cross sections (<1 cm wide) were immersed into Carnoy’s solution for 4 to 6 h before being placed into 70% ethanol.

Jejunal mucosal samples (1 g) were thawed, homogenized, and analyzed for DNA content using diphenylamine (Burton, 1956; Johnson et al., 1997), RNA content using orcinol (Kamali and Manhouri, 1969; Reynolds et al., 1990), and protein content using Coomassie brilliant blue G (Bradford, 1976) procedures. Concentration of DNA was used to estimate cell number and protein:DNA and RNA:DNA ratios were used to estimate cell size.

Jejunal tissues immersion fixed in Carnoy’s solution were embedded in paraffin (Reynolds and Redmer, 1992), mounted on glass slides, and processed for staining procedures (Fricke et al., 1997; Scheaffer et al., 2003). Specimens were blocked with PBS and 1.5% normal horse serum (Vector Laboratories, Burlingame, CA), and incubated with mouse antiproliferating cell nuclear antigen monoclonal antibody (1 µL/mL of blocking buffer; Clone PC-10; Roche Diagnostics Corp., Indianapolis, IN). Immunocomplexes were detected with horse antimouse IgG (Vectastain; Vector Laboratories) and Avidin-Biotin Complex system (Vectastain; Vector Laboratories). Tissues were counterstained with Nuclear Fast Red (Sigma-Aldrich, St. Louis, MO) to visualize all nuclei. Percentage of proliferating cells in the crypt region was then quantified (6 images per lamb) as recently described (Yunusova et al., 2013), using Image-ProPlus software (version 5.0, Media Cybernetics Inc., Silver Spring, MD). Crypt depth and villous length were also measured at this time (6 images per lamb; Jin et al., 1994).

Cross sections of perfusion-fixed jejunal tissue were embedded, sectioned, mounted, and processed, as described for immersion-fixed tissue, and tissue sections were stained using periodic acid-Schiff’s staining procedures to contrast the vascular tissue (Luna, 1968). Mean capillary area, number, and circumference measurements were made in the intestinal villi (5 images per lamb) using the Image-Pro Plus software and used to calculate capillary area density, capillary number density, capillary surface density, and area per capillary, as recently described (Yunusova et al., 2013).

Quantitative real-time RT-PCR was performed on frozen jejunal mucosal samples to determine mRNA expression of selected angiogenic factors and receptors {vascular endothelial growth factor [VEGF], VEGF receptor-1 [fms-related tyrosine kinase 1, FLT1], VEGF receptor-2 [kinase insert domain receptor, KDR], endothelial nitric oxide synthase 3 [NOS3; produces nitric oxide (NO)], and soluble guanylate cyclase [GUCY1B3; NO receptor]}, as well as glucagon-like peptide-2 (GLP-2) and its receptor (GLP-2R). Messenger RNA was extracted and quantified from 100 mg of tissue, using methods as described by Neville et al. (2010b), and previously published TaqMan probe and primer sequences (Redmer et al., 2005). TaqMan reagents and procedures (Applied Biosystems, Foster City, CA) were used to perform a multiplex reaction. Frozen jejunal mucosal samples had total cellular RNA (tcRNA) extracted using TriReagent (Molecular Research Center, Cincinnati, OH), which was then quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Wilmington, DE). Approximately 30 ng of tcRNA from each sample was reverse transcribed and then polymerization and amplification reactions were conducted in a 96-well PCR plate using the Applied Biosystems ABI Prism 7000 sequence detector (Foster City, CA). Forty cycles (50 cycles for NOS3 and GUCY1B3) were performed at 60°C for hybridization and polymerization. Expression of each angiogenic factor was normalized to expression of 18S in a multiplex reaction using the human 18S predeveloped assay reagent (Applied Biosystems). The predeveloped assay reagent solution, which is primer limited and contains a VIC®-labeled probe (a proprietary reporter dye; Applied Biosystems), was further adjusted by using one-fourth the normal amount so that it would not interfere with amplification of the FAM- (6-carboxy-fluorescein) labeled gene of interest. The multiplex reaction was also used to prepare standard curves for 18S and the gene of interest, based on dilutions of cDNA obtained from reverse transcription of RNA obtained from pooled, late-pregnancy sheep placentomal tissues.

Calculations

Digesta weight was calculated by difference (total full viscera weight – empty visceral tissue weight). Empty BW (EBW) was determined by subtracting digesta weight from the final BW obtained before slaughter. Total GI tract mass was calculated as the sum of the empty stomach complex, small intestine, and large intestine masses. Mucosal density (%) was determined by dividing the mucosal tissue weight from the tissue section used by the total tissue weight of the tissue section used.

Total intestinal cell number was calculated by dividing the estimated total DNA (jejunal DNA concentration × intestinal mass) by 6.6 × 10–12 g (Baserga, 1985). Total intestinal cell number was then multiplied by the percentage of proliferating nuclei to determine the total number of proliferating nuclei.

Capillary area density (%) was calculated as the total capillary area divided by the tissue area analyzed and multiplied by 100. Capillary number density (number/mm2) was determined by dividing the total number of vessels counted by the tissue area analyzed and multiplying this by 106. The capillary surface density [(µm/µm2) × 10], or total capillary circumference per unit of tissue area, was calculated as capillary circumference divided by the tissue area analyzed. Area per capillary (µm2) was determined by dividing total capillary area by capillary number. Total jejunal vascularity was calculated as the capillary area density multiplied by jejunal mass.

Statistical Analyses

Twins (n = 6) were removed from the data set. One ewe was not pregnant, and 5 lambs died between birth and necropsy. This resulted in the following lamb numbers for each treatment: ASe-RES (n = 13), ASe-CON (n = 14), ASe-HIH (n = 12), HSe-RES (n = 10), HSe-CON (n = 13), HSe-HIH (n = 13).

Data were analyzed as a 2 × 3 factorial design using a general linear model (SAS Inst. Inc., Cary, NC) with ewe Se supply (ASe vs. HSe), nutritional plane (RES vs. CON vs. HIH), and the corresponding interaction as fixed effects in the model. Lamb sex was included in the model when P ≤ 0.10. Least squares means were separated using least significant difference and considered significant when P ≤ 0.10. In the absence of interactions (P > 0.10), main effects of Se supply and nutritional plane are reported.


RESULTS

Visceral Organ Mass

Lamb BW and visceral organ masses at necropsy (d 20.6 ± 0.9 of age) are shown in Table 1. Lamb BW and EBW were affected (P ≤ 0.03) by maternal nutritional plane during gestation, but not Se supply (P > 0.38), where lambs born to ewes fed RES weighed less (P < 0.04) than CON or HIH. Empty GI tract, stomach complex, and liver masses followed BW (RES < CON, HIH) and were influenced by nutritional plane (P ≤ 0.06), independent of Se supply (P > 0.16). These masses were also less (P < 0.05) for lambs born to RES ewes compared with CON and HIH. Although small intestinal mass was unaffected (P > 0.17) by maternal gestational treatments, nutritional plane and Se supply interacted to affect (P ≤ 0.03) large intestinal and pancreatic masses. Within lambs born to ewes fed HSe, CON and HIH had greater (P < 0.006) large intestinal mass than RES; however, nutritional plane did not affect (P > 0.71) large intestinal mass among lambs born to ASe ewes. Additionally, large intestinal mass was greater (P = 0.09) for lambs born to ASe ewes than HSe within RES, whereas large intestinal mass was less (P = 0.05) for ASe than HSe within the HIH nutritional plane. Pancreatic mass was greater (P ≤ 0.07) for HIH than RES and CON among lambs born to ASe ewes, but less (P = 0.04) for HIH compared with CON among lambs born to HSe ewes. Within lambs whose dams were fed CON during gestation, HSe had greater (P = 0.05) pancreatic mass than ASe, whereas ASe were greater (P = 0.05) than HSe within lambs born to HIH ewes.


View Full Table | Close Full ViewTable 1.

Effects of maternal Se supply and nutritional plane during gestation on neonatal lamb BW and visceral organ mass1,2

 
Se supply3
Nutritional plane4
P-value5
Item ASe HSe SEM6 RES CON HIH SEM7 Se Nut Se by Nut
BW, kg 10.8 11.1 0.4 9.9a 11.2b 11.6b 0.4 0.57 0.02 0.51
Empty BW,8 kg 9.6 9.9 0.3 8.9a 10.1b 10.3b 0.4 0.39 0.03 0.46
Empty gastrointestinal tract, g 541 553 17 505a 562b 573b 21 0.60 0.05 0.75
Stomach complex, g 121 118 3 109a 126b 123b 4 0.50 0.005 0.31
Small intestine, g 337 345 13 318 345 360 17 0.67 0.18 0.68
Large intestine, g 0.53 0.03 0.03
    ASe 82.4y 84.2y 81.9xy 4.5
    HSe 71.2x 90.0yz 94.2z 5.0
Liver, g 301 304 11 278a 316b 315b 13 0.85 0.06 0.17
Pancreas, g 0.60 0.65 0.02
    ASe 12.7x 13.0x 14.7y 0.7
    HSe 13.8xy 14.8y 12.8x 0.8
a,bWithin an item, main effect means differ (P < 0.10).
x-zWithin an item, interactive means differ (P < 0.10).
1Lambs were raised independently of their dams.
2Portions of these data were previously published in Meyer et al. (2009) and Camacho et al. (2012).
3Lambs born to ewes fed 11.5 µg of Se/kg of BW (adequate Se, ASe) or 77.0 µg of Se/kg of BW (high Se, HSe) from breeding to parturition.
4Lambs born to ewes fed 60% (restricted, RES), 100% (control, CON), or 140% (high, HIH) of nutrient requirements from d 40 of gestation to parturition.
5P-values for Se supply (Se), nutritional plane (Nut), and their interaction.
6SEM for ASe (n = 39) and HSe (n = 36).
7SEM for RES (n = 23), CON (n = 27), and HIH (n = 25).
8Empty BW = BW – digesta weight.

Lamb visceral organ masses expressed proportionally to EBW are in Table 2. Proportional empty GI tract and small intestinal masses (g/kg EBW) were unaffected (P > 0.26) by maternal gestational treatments, whereas stomach complex, large intestinal, and liver proportional masses were affected (P ≤ 0.08) by the interaction of maternal nutritional plane and Se supply. Although there was no effect (P ≥ 0.13) of nutritional plane within lambs born to HSe ewes, CON had greater (P = 0.04) proportional stomach complex mass than RES among lambs born to ASe ewes. Additionally, within CON lambs, those whose dams were fed ASe during gestation had greater (P = 0.04) proportional stomach complex mass compared with HSe. There were no differences (P > 0.20) in proportional large intestinal mass due to nutritional plane within lambs born to HSe ewes, although RES was greater (P = 0.02) than HIH among ASe. Despite this, lambs born to HSe ewes had greater (P = 0.04) proportional large intestinal mass than ASe within the HIH nutritional plane. Proportional liver masses were unaffected (P ≥ 0.12) by nutritional plane among HSe lambs, but RES were greater (P ≤ 0.08) than CON and HIH within lambs born to ASe ewes. Lambs from ASe dams also had greater (P = 0.04) proportional liver mass compared with HSe within RES. Proportional pancreatic mass was affected (P = 0.10) by the nutritional plane, but not Se supply (P ≥ 0.14), during gestation, where lambs born to RES ewes were greater (P = 0.03) than HIH.


View Full Table | Close Full ViewTable 2.

Effects of maternal Se supply and nutritional plane during gestation on neonatal lamb visceral organ proportional mass (g/kg empty BW)1,2

 
Se supply3
Nutritional plane4
P-value5
Item ASe HSe SEM6 RES CON HIH SEM7 Se Nut Se by Nut
Empty gastrointestinal tract 56.6 56.4 1.0 57.5 56.5 55.5 1.3 0.86 0.53 0.27
Stomach complex 0.22 0.58 0.07
    ASe 12.0x 13.9y 12.8xy 0.7
    HSe 13.1xy 11.9x 11.6x 0.8
Small intestine 35.2 35.6 0.8 36.2 35.1 34.9 1.0 0.73 0.63 0.36
Large intestine 0.70 0.78 0.04
    ASe 9.27y 8.62xy 7.85x 0.45
    HSe 8.34xy 8.66xy 9.14y 0.47
Liver 0.52 0.26 0.08
    ASe 32.8y 30.4x 29.9x 1.0
    HSe 29.9x 31.9xy 29.9x 1.0
Pancreas 1.45 1.43 0.06 1.56b 1.42ab 1.33a 0.08 0.80 0.10 0.14
a,bWithin an item, main effect means differ (P < 0.10).
x,yWithin an item, interactive means differ (P < 0.10).
1Lambs were raised independently of their dams.
2Portions of these data were previously published in Camacho et al. (2012).
3Lambs born to ewes fed 11.5 µg of Se/kg of BW (adequate Se, ASe) or 77.0 µg of Se/kg of BW (high Se, HSe) from breeding to parturition.
4Lambs born to ewes fed 60% (restricted, RES), 100% (control, CON), or 140% (high, HIH) of nutrient requirements from d 40 of gestation to parturition.
5P-values for Se supply (Se), nutritional plane (Nut), and their interaction.
6SEM for ASe (n = 39) and HSe (n = 36).
7SEM for RES (n = 23), CON (n = 27), and HIH (n = 25).

Jejunal Growth and Glucagon-like Peptide-2 Expression

Although jejunal mucosal cellularity and density measures (Table 3) were not affected by gestational Se supply (P ≥ 0.11), both DNA concentration and total DNA were impacted by nutritional plane during gestation (P ≤ 0.07). Lambs born to ewes fed the HIH nutritional plane had greater (P ≤ 0.09) DNA concentration than RES and CON, and greater (P = 0.01) total DNA than RES. Protein, RNA, RNA:DNA, protein:DNA, and mucosal density were unaffected (P ≥ 0.11) by gestational treatments.


View Full Table | Close Full ViewTable 3.

Effects of maternal Se supply and nutritional plane during gestation on neonatal lamb jejunal mucosal cellularity and density1

 
Se supply2
Nutritional plane3
P-value4
Item ASe HSe SEM5 RES CON HIH SEM6 Se Nut Se by Nut
DNA, mg/g 5.40 5.38 0.15 5.17a 5.30a 5.71b 0.18 0.93 0.07 0.15
DNA, g7 1.53 1.57 0.08 1.38a 1.56ab 1.72b 0.09 0.67 0.04 0.26
RNA, mg/g 5.55 5.45 0.18 5.33 5.50 5.68 0.22 0.71 0.54 0.11
RNA, g7 1.59 1.59 0.09 1.43 1.61 1.72 0.12 0.97 0.21 0.14
Protein, mg/g 28.3 27.3 1.7 27.1 27.8 28.4 2.1 0.65 0.90 0.95
Protein, g7 8.35 8.16 0.71 7.52 8.42 8.83 0.90 0.85 0.56 0.83
RNA:DNA 1.03 1.03 0.02 1.04 1.06 1.00 0.03 0.90 0.29 0.12
Protein:DNA 5.37 5.15 0.32 5.31 5.40 5.06 0.40 0.62 0.80 0.80
Mucosal density, % 83.4 83.6 0.5 83.6 83.8 83.0 0.6 0.79 0.55 0.12
a,bWithin an item, main effect means differ (P < 0.10).
1Lambs were raised independently of their dams.
2Lambs born to ewes fed 11.5 µg of Se/kg of BW (adequate Se, ASe) or 77.0 µg of Se/kg of BW (high Se, HSe) from breeding to parturition.
3Lambs born to ewes fed 60% (restricted, RES), 100% (control, CON), or 140% (high, HIH) of nutrient requirements from d 40 of gestation to parturition.
4P-values for Se supply (Se), nutritional plane (Nut), and their interaction.
5SEM for ASe (n = 39) and HSe (n = 36).
6SEM for RES (n = 23), CON (n = 27), and HIH (n = 25).
7Total small intestinal mucosal DNA, RNA, and protein content (small intestinal mass × mucosal density × cellularity concentration).

Jejunal percent proliferation and total proliferating small intestinal cells (Table 4) were affected (P ≤ 0.003) by the interaction of nutritional plane and Se supply during gestation. In ewes fed ASe, HIH resulted in lambs with greater (P ≤ 0.08) percent jejunal proliferation than RES and CON. Conversely, in ewes fed HSe, HIH resulted in lambs with less (P = 0.06) percent proliferation than CON among lambs from HSe ewes. Within CON fed ewes, HSe had greater (P = 0.08) percent proliferation than ASe, whereas ASe was greater (P = 0.002) compared with HSe within lambs from HIH. Lambs born to ewes fed ASe-HIH had greater (P = 0.007) total proliferating small intestinal cells than lambs from all other treatments. Total small intestinal cells (Table 4) were affected (P = 0.03) by nutritional plane during gestation, where lambs born to RES ewes had less (P < 0.02) total cells than HIH. Despite these differences, jejunal crypt depth and villus length were unaffected by gestational treatment (P > 0.16).


View Full Table | Close Full ViewTable 4.

Effects of maternal Se supply and nutritional plane during gestation on neonatal lamb jejunal proliferation and villus morphology1

 
Se supply2
Nutritional plane3
P-Value4
Item ASe HSe SEM5 RES CON HIH SEM6 Se Nut Se by Nut
Proliferation, % 0.37 0.72 0.003
    ASe 5.26xy 3.78x 7.35z 0.86
    HSe 5.15xy 5.80yz 3.56x 0.94
Total small intestinal cells, × 1012 278 285 14 250a 280ab 314b 17 0.69 0.03 0.27
Total proliferating small intestinal cells, × 1012 0.34 0.16 <0.001
    ASe 11.8x 10.2x 23.3y 2.5
    HSe 14.4x 14.9x 10.4x 2.7
Crypt depth, µm 60.8 61.5 1.7 58.4 61.9 63.1 2.1 0.78 0.25 0.76
Villus length, µm 271 278 8 261 285 277 10 0.53 0.17 0.45
a,bWithin an item, main effect means differ (P < 0.10).
x−zWithin an item, interactive means differ (P < 0.10).
1Lambs were raised independently of their dams.
2Lambs born to ewes fed 11.5 µg of Se/kg of BW (adequate Se, ASe) or 77.0 µg of Se/kg of BW (high Se, HSe) from breeding to parturition.
3Lambs born to ewes fed 60% (restricted, RES), 100% (control, CON), or 140% (high, HIH) of nutrient requirements from d 40 of gestation to parturition.
4P-values for Se supply (Se), nutritional plane (Nut), and their interaction.
5SEM for ASe (n = 39) and HSe (n = 36).
6SEM for RES (n = 23), CON (n = 27), and HIH (n = 25).

Jejunal mucosal GLP-2 mRNA expression (Table 5) was impacted (P = 0.05) by nutritional plane but was unaffected (P > 0.18) by gestational Se. Lambs born to ewes fed the RES nutritional plane had greater (P ≤ 0.07) GLP-2 expression compared with CON and HIH. There were no effects (P > 0.12) of gestational treatments on GLP-2R expression in jejunal mucosa, however.


View Full Table | Close Full ViewTable 5.

Effects of maternal Se supply and nutritional plane during gestation on neonatal lamb jejunal mucosal angiogenic factor and glucagon like peptide-2 mRNA expression (gene of interest/18S)1,2

 
Se supply3
Nutritional plane4
P-values5
Item6 ASe HSe SEM7 RES CON HIH SEM8 Se Nut Se by Nut
VEGF 0.184 0.210 0.013 0.199 0.198 0.194 0.017 0.16 0.97 0.60
FLT1 0.030 0.030 0.002 0.031 0.030 0.031 0.002 0.97 0.97 0.83
KDR 0.098 0.105 0.010 0.104 0.097 0.103 0.012 0.59 0.91 0.90
NOS3 0.399 0.380 0.043 0.373 0.455 0.340 0.052 0.74 0.25 0.24
GUCY1B3 0.11 0.22 0.07
    ASe 0.138xy 0.228yz 0.300z 0.042
    HSe 0.199xyz 0.126x 0.172xy 0.047
GLP-2 0.393 0.472 0.043 0.537b 0.359a 0.401a 0.053 0.19 0.05 0.62
GLP-2R 0.771 0.898 0.050 0.799 0.794 0.880 0.063 0.13 0.53 0.92
a,bWithin an item, main effect means differ (P < 0.10).
x–zWithin an item, interactive means differ (P < 0.10).
1Lambs were raised independently of their dams.
2GLP-2 and GLP-2R data were previously published in Caton et al. (2009b).
3Lambs born to ewes fed 11.5 µg of Se/kg of BW (adequate Se, ASe) or 77.0 µg of Se/kg of BW (high Se, HSe) from breeding to parturition.
4Lambs born to ewes fed 60% (restricted, RES), 100% (control, CON), or 140% (high, HIH) of nutrient requirements from d 40 of gestation to parturition.
5P-values for Se supply (Se), nutritional plane (Nut), and their interaction.
6VEGF = vascular endothelial growth factor, FLT1 = VEGF receptor-1, KDR = VEGF receptor-2, NOS3 = endothelial nitric oxide synthase 3, GUCY1B3 = soluble guanylate cyclase, GLP-2 = glucagon-like peptide-2, GLP-2R = GLP-2 receptor.
7SEM for ASe (n = 39) and HSe (n = 36).
8SEM for RES (n = 23), CON (n = 27), and HIH (n = 25).

Jejunal Vascularity and Angiogenic Factor Expression

Jejunal area per capillary (Table 6) was affected (P = 0.09) by the interaction of gestational nutritional plane and Se supply. Although there were no differences (P > 0.21) due to nutritional plane within lambs born to HSe ewes, RES had greater (P ≤ 0.07) capillary size than CON and HIH among ASe. The main effect of nutritional plane affected (P = 0.05) capillary surface density, where lambs from CON ewes had greater (P = 0.01) density than RES. Gestational treatments did not affect (P ≥ 0.20) capillary area density or capillary number density, but nutritional plane tended to affect (P = 0.11) total small intestinal vascularity. Lambs born to ewes fed the CON nutritional plane had greater (P = 0.04) total vascularity compared with RES.


View Full Table | Close Full ViewTable 6.

Effects of maternal Se supply and nutritional plane during gestation on neonatal lamb jejunal vascularity1

 
Se supply2
Nutritional plane4
P-values5
Item ASe HSe SEM3 RES CON HIH SEM6 Se Nut Se by Nut
Capillary area density, % 20.6 21.0 0.6 20.5 21.7 20.1 0.7 0.61 0.20 0.59
Capillary no. density, per mm2 1,134 1,139 46 1,092 1,149 1,171 56 0.93 0.59 0.33
Capillary surface density, (µm/µm2) × 10 0.98 1.00 0.03 0.93a 1.05b 0.99ab 0.03 0.53 0.05 0.78
Area per capillary, µm2 0.60 0.37 0.09
    ASe 240z 188xy 179x 20
    HSe 197xyz 235yz 202xyz 22
Total small intestinal vascularity, mL 68.7 73.6 3.1 65.0 76.5 72.0 3.9 0.27 0.11 0.66
a,bWithin an item, main effect means differ (P < 0.10).
x–zWithin an item, interactive means differ (P < 0.10).
1Lambs were raised independently of their dams.
2Lambs born to ewes fed 11.5 µg of Se/kg of BW (adequate Se, ASe) or 77.0 µg of Se/kg of BW (high Se, HSe) from breeding to parturition.
3SEM for ASe (n = 39) and HSe (n = 36).
4Lambs born to ewes fed 60% (restricted, RES), 100% (control, CON), or 140% (high, HIH) of nutrient requirements from d 40 of gestation to parturition.
5P-values for Se supply (Se), nutritional plane (Nut), and their interaction.
6SEM for RES (n = 23), CON (n = 27), and HIH (n = 25).

Gestational treatments did not affect (P > 0.15) jejunal mucosal mRNA expression of VEGF, FLT1, KDR, and NOS3. Despite this, nutritional plane and Se supply interacted to affect (P = 0.07) GUCY1B3 mRNA expression in the jejunal mucosa. Within lambs born to ASe ewes, HIH had greater (P = 0.008) GUCY1B3 expression than RES, but there were no differences (P > 0.24) due to nutritional plane among HSe ewes. Additionally, lambs from ASe dams had greater (P ≤ 0.08) GUCY1B3 expression compared with HSe within CON and HIH nutritional planes.


DISCUSSION

To date, most research in ruminant livestock species relating gestational nutrition of the dam to offspring responses has focused on fetal, market weight, or mature time points. Periparturient GI tract function is essential for health and survival of neonatal ruminants during the transition from the intra- to extra-uterine environment and from parenteral to enteral nutrition. Results demonstrate that maternal nutrition during gestation seems to alter postnatal development and possibly function of the neonatal GI tract at 20 d of age, which may subsequently affect growth and performance. It has previously been reported that lamb birth weight, growth, health, and endocrine status were affected by maternal nutrition in this study (Meyer et al., 2010a; Hammer et al., 2011; Camacho et al., 2012), and GI data presented here aids in the understanding of these whole-animal observations.

Visceral Organ Mass

Previous work in our laboratory has demonstrated that visceral organ mass of late-term fetal lambs can be influenced by both the nutritional plane (Reed et al., 2007; Lemley et al., 2012) and Se supply (Reed et al., 2007) of the dam. In the current study, RES decreased actual (g) empty GI tract, stomach complex, and liver masses at 20 d of age, although HIH had no effect on these organs. Additionally, Se supply and nutritional plane during gestation interacted to affect large intestinal and pancreatic actual masses. Previously, high dietary Se throughout gestation has increased large intestinal mass in fetal lambs but had no effect on fetal pancreatic mass (Reed et al., 2007).

In previous studies using a similar nutrient restriction treatment during mid and late gestation, the masses of more organs were decreased due to maternal nutrient restriction (Reed et al., 2007; Lemley et al., 2012), but these measurements were taken in late-term fetuses. Thus, organ growth in lambs born to RES ewes may have increased during the final days of gestation and early postnatal life compared with their control counterparts, allowing masses at d 20 to be similar. Gestation length was affected by the nutritional plane × Se supply interaction in this study (Meyer et al., 2010a); therefore, because many visceral organs mature during late gestation, it is likely that necropsy of fetuses based strictly on fetal age may give a false impression of fetal organ size at term. Allowing parturition to take place naturally, followed by necropsy of lambs at a common postnatal age, should have eliminated some of this bias, however.

Growth of several organs to d 20 postnatally appeared to be disproportional or asymmetrical to EBW in this study, as effects of maternal dietary treatments on proportional (g/kg EBW) organ masses do not mirror those for actual organ masses. Proportional pancreatic mass was increased in lambs born to RES ewes, indicating that the pancreas grew at a greater rate than that of the overall body during prenatal, postnatal, or both periods. Stomach complex, large intestinal, liver, and kidney (Camacho et al., 2012) masses, as well as lamb weight and girth at birth (Meyer et al., 2010a), were also affected by this interaction, although d 20 lamb BW was not. This suggests that organ size at 20 d of age was influenced more by fetal growth, and thus gestational nutrition, than postnatal growth. In all organs but the stomach complex, HSe during gestation had no effect on lambs born to CON-fed ewes but altered organ mass at d 20 in lambs from ewes fed RES or HIH. Similarly, HSe only affected lamb birth weight in the divergent nutritional planes, where birth weight was increased to that of CON in lambs born to RES ewes and decreased numerically in ewes fed HIH (Meyer et al., 2010a). Although Se supply × nutritional plane did not interact to affect ewe BW gain or BCS change during gestation, ewes fed HSe-HIH also had increased omental and mesenteric fat and backfat at lambing (Meyer et al., 2010a). This suggests that high dietary Se during gestation caused both dams and fetuses to partition available nutrients differently.

Jejunal Growth and Glucagon-like Peptide-2 Expression

Gestational nutrition did not affect small intestinal mass in 20-d-old lambs. This is contrary to previous work where a similar nutrient restriction during mid and late gestation decreased fetal lamb (d 135) small intestinal mass (Reed et al., 2007), and where nutritional plane interacted with Se supply during gestation to affect both lamb small intestinal actual and proportional masses at 180 d of age (Yunusova et al., 2013). Additionally, when ewes were nutrient restricted (∼30% of ad libitum) from d 90 of gestation to parturition, their lambs had reduced small intestinal mass and jejunal length shortly after birth, but not at 28 wk of age (Gao et al., 2008). Reasons for the lack of differences in small intestinal mass in the current study are unclear but may be due to ad libitum postnatal intake of lambs or the timing of sampling.

Despite similar small intestinal mass at 20 d of age, gestational nutrition affected other indices of intestinal growth, including cellularity and proliferation. Lambs born to HIH-fed ewes had increased DNA (both concentration and total) and total intestinal cells, suggesting that these lambs either had more intestinal hyperplasia or less cell turnover than their counterparts to result in greater cell number. Previous studies in our laboratory have demonstrated that a similar nutrient restriction of ewes during mid and late gestation decreased fetal jejunal cell size (protein:DNA) and total protein (Reed et al., 2007). High dietary Se throughout gestation increased fetal jejunal cell size (RNA:DNA; Reed et al., 2007), although this was not observed in the current study.

Nutritional plane interacted with Se supply during gestation to affect small intestinal proliferation in 20-d-old lambs, where lambs born to ewes fed an ASe-HIH diet had increased proliferation compared with other treatments. Because there were no differences in villus length or crypt depth in these lambs, these lambs likely had an increased rate of cellular turnover in the small intestine, which would indicate an inefficient use of energy and nutrients to build more cells without increased intestinal surface area. In a previous study, there were no differences in jejunal proliferation due to maternal nutrition during gestation in late-term fetal lambs (Neville et al., 2010a), although form and quantity of high dietary Se fed during gestation have impacted fetal jejunal cell size (Neville et al., 2008). In another study, total proliferating jejunal cells were reduced in 180-d-old lambs born to nutrient-restricted or overnourished ewes (Yunusova et al., 2013), suggesting that alterations in proliferative rate may be sustained well into life.

As discussed for other visceral organ growth measures, differences noted between intestinal growth and development of fetal and 20-d-old lambs may be due to the timing of tissue sampling or postnatal ad libitum intake of lambs in the current study. During late gestation, cortisol and fetal swallowing of amniotic fluid play an important role in the maturation process of the small intestine, including rapid growth, increasing turnover rate, production of digestive enzymes (Sangild et al., 2000), and expression of the angiogenic factor VEGF (Holmes et al., 2008). Maternal cortisol followed nutritional plane (restricted < control < high; K. A. Vonnahme, unpublished data) in this study, and gestation length was affected by the Se supply × nutritional plane interaction (Meyer et al., 2010a). Additionally, lambs born to ewes fed HSe had decreased cortisol immediately after birth (Camacho et al., 2012). Although these are crude measures, they demonstrate that fetal maturation and signals for parturition were affected by maternal nutrition during gestation, which likely impacted maturation of fetal tissues. Additionally, the potential change in nutritional plane in the current study due to removal of lambs from their dams and subsequent ad libitum intake may have allowed for compensatory growth of the small intestine. It is likely, for example, that lambs born to RES ewes had an increase in their nutritional plane when allowed ad libitum access to milk replacer postnatally. Especially given the extremely plastic nature of the small intestine, this may be a mechanism by which catch-up growth (Hales and Ozanne, 2003) occurs in offspring that were growth restricted in utero.

Glucagon-like peptide-2 is a trophic peptide hormone that is known to impact not only intestinal growth but also absorption, blood flow, and barrier function, among other characteristics (Rowland and Brubaker, 2011). Although GLP-2 has been studied in several rodent and swine models, it has only recently been measured in sheep (Yunusova et al., 2013) and cattle (Taylor-Edwards et al., 2010). In the current study, 20-d-old lambs born to RES ewes had increased jejunal GLP-2 mRNA expression. Conversely, 180-d-old lambs born to nutrient-restricted ewes had decreased jejunal GLP-2 expression in another study (Yunusova et al., 2013). Lambs in the current study born to RES ewes may have been responding to an increase in nutritional plane postnatally, as increased DMI has been demonstrated to stimulate both circulating GLP-2 and ileal GLP-2R mRNA expression in cattle (Taylor-Edwards et al., 2010). While 20-d-old lambs could still be adjusting from decreased luminal nutrients from the swallowing of less nutrient-dense amniotic fluid of their nutrient-restricted dams (Kwon et al., 2004), 180-d-old lambs mentioned from the previous study would have been much past this point. The mechanisms by which GLP-2 increases tissue growth and function are not completely understood and it appears that GLP-2 acts mostly via indirect mediators, including IGF-I and II, nitric oxide, and vasoactive intestinal polypeptide (Rowland and Brubaker, 2011). In the current study, GLP-2 may have been involved in mechanisms that increased small intestinal mass of lambs born to nutrient-restricted ewes, allowing for possible catch-up growth described previously.

Jejunal Vascularity and Angiogenic Factor Expression

Vascularity and blood flow in the small intestine are imperative for nutrient transport and in fact, the small intestine receives 10 to 15% of resting cardiac output (Lundgren, 1984). Although intestinal vascularization has not been extensively studied in ruminants, previous research in our laboratory has demonstrated that vascularity and angiogenic factor expression in the small intestine can be affected by nutritional plane, Se supply, and physiological state in reproducing ewes and cows (Caton et al., 2009a; Neville et al., 2010b; Meyer et al., 2012b).

Both nutritional plane and its interaction with Se supply during gestation impacted 20-d-old lamb jejunal vascularity. Fetal lambs (d 135) from a similar nutrient restriction model had decreased total jejunal microvascular volume (Neville et al., 2010b) and lambs born to RES ewes in the current study also had decreased capillary surface density. Although it has been previously discussed that growth characteristics of the small intestine altered by maternal nutrition during gestation may dissipate in later life, these data suggest that changes in vascularization may persist well into life. In another study, nutrient restriction during mid and late gestation reduced jejunal capillary size (area per capillary) and high dietary Se throughout gestation increased jejunal capillary area density in 180-d-old lambs (Yunusova et al., 2013).

The relationship between small intestinal vascularization and small intestinal function, and how this extends to whole animal growth and metabolism is unknown. It is interesting to note, however, that jejunal capillary size in 20-d-old lambs responded in an opposite fashion to birth weight (Meyer et al., 2010a). Lambs born to ewes fed ASe-RES during gestation had the lowest birth weight but greatest jejunal capillary size at 20 d. It is possible that increased capillary area in the small intestine is a mechanism by which these growth-retarded lambs were attempting to increase their growth rate once in an external environment with ad libitum access to feed.

Angiogenesis, the formation of new blood vessels from present vasculature, is a highly regulated process controlled by many angiogenic growth factors (Aron and Anthony, 2004). Vascular endothelial growth factor is one of the most potent angiogenic growth factors and acts through its receptors, FLT1 and KDR, to promote vascular endothelial cell survival, proliferation, migration, and permeability (Ferrara, 2004). Also acting in this process is NO, which is produced by NOS3 from arginine and increases blood flow through vasodilation, increased vascular permeability, and stimulated VEGF production. These actions result in part from cyclic guanosine monophosphate, which is produced by GUCY1B3 after binding of NO (Martin et al., 2001; Roy et al., 2006).

In previous work, high dietary Se during gestation has decreased jejunal VEGF mRNA expression in fetal lambs (Neville et al., 2010b) and both gestational nutrient restriction or overnourishment have tended to decrease jejunal KDR expression in 180-d-old lambs (Yunusova et al., 2013). Despite this, maternal nutrition during gestation did not affect jejunal mRNA expression of the VEGF system or NOS3, although nutritional plane and Se supply interacted to affect GUCY1B3. This appears to be a gene that is sensitive to manipulation of gestational nutrition, as GUCY1B3 has previously been affected by gestational nutrient restriction in fetal lambs (Neville et al., 2010b) and gestational nutrient restriction and protein supplementation in market weight calves (Meyer et al., 2012a). Because of its role in producing the many effects of NO, GUCY1B3 expression may lead to greater vasodilation, angiogenesis, vascular permeability, and blood flow to the intestine, which could thereby lessen other negative impacts on intestinal development and/or growth. Interestingly, GUCY1B3 expression is opposite of jejunal capillary size (area per capillary) in this study. Because GUCY1B3 binds NO, it is possible that it was upregulated in the small intestines of lambs with small capillary size to increase vasodilation and blood flow to and from the tissue.


Conclusions

There is a growing body of evidence demonstrating that the early postnatal environment also has long-term effects on offspring, in addition to that in utero (e.g., Greenwood and Cafe, 2007). Based on the current data, it is likely that the GI tract, and specifically the small intestine, plays a role in postnatal programming. This study indicates that neonatal lamb visceral organ growth and small intestinal development to 20 d of age can be affected by maternal nutrition during gestation, even when offspring are raised independently of their dams. Further research is necessary to better understand the role of maternal nutrition during gestation to allow for development of improved management strategies both during gestation and the neonatal period.

 

References

Footnotes


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