Artificial rearing influences the morphology, permeability and redox state of the gastrointestinal tract of low and normal birth weight piglets
- Hans Vergauwen†1,
- Jeroen Degroote†2,
- Sara Prims1,
- Wei Wang2, 3,
- Erik Fransen4,
- Stefaan De Smet3,
- Christophe Casteleyn1,
- Steven Van Cruchten1,
- Joris Michiels2 and
- Chris Van Ginneken1Email author
© The Author(s). 2017
Received: 5 September 2016
Accepted: 16 March 2017
Published: 8 April 2017
In this study the physiological implications of artificial rearing were investigated. Low (LBW) and normal birth weight (NBW) piglets were compared as they might react differently to stressors caused by artificial rearing. In total, 42 pairs of LBW and NBW piglets from 16 litters suckled the sow until d19 of age or were artificially reared starting at d3 until d19 of age. Blood and tissue samples that were collected after euthanasia at 0, 3, 5, 8 and 19 d of age. Histology, ELISA, and Ussing chamber analysis were used to study proximal and distal small intestine histo-morphology, proliferation, apoptosis, tight junction protein expression, and permeability. Furthermore, small intestine, liver and systemic redox parameters (GSH, GSSG, GSH-Px and MDA) were investigated using HPLC.
LBW and NBW artificially reared piglets weighed respectively 40 and 33% more than LBW and NBW sow-reared piglets at d19 (P < 0.01). Transferring piglets to a nursery at d3 resulted in villus atrophy, increased intestinal FD-4 and HRP permeability and elevated GSSG/GSH ratio in the distal small intestine at d5 (P < 0.05). GSH concentrations in the proximal small intestine remained stable, while they decreased in the liver (P < 0.05). From d5 until d19, villus width and crypt depth increased, whereas PCNA, caspase-3, occludin and claudin-3 protein expressions were reduced. GSH, GSSG and permeability recovered in artificially reared piglets (P < 0.05).
The results suggest that artificial rearing altered the morphology, permeability and redox state without compromising piglet performance. The observed effects were not depending on birth weight.
KeywordsMilk replacer Oxidative stress Small intestine Suckling period Tight junction proteins
The neonatal period in the pig’s life is accompanied with high morbidity and mortality [1, 2]. In addition, increasing litter sizes in modern swine production have led to higher rates of piglets born with a low birth weight (LBW) . Both newborn LBW human [4–6] and LBW piglets [7–10] seem to have a lower capacity to mount an antioxidant response. Newborns transitioning from maternal mediated respiration to autonomous pulmonary respiration outside the uterus are suddenly exposed to O2-derived free radicals . This increased the production of reactive species in various organs . A redox imbalance affects cellular signaling, protein synthesis, and enhances proteolysis that can ultimately lead to a dysfunctional intestinal barrier and suboptimal regenerative potential as shown in vitro [12–15]. Consequently, the observed redox imbalance and the downstream effects could explain the abnormal absorption and metabolism of nutrients, reduced growth and impaired development of the small intestine, liver, and muscle observed in LBW piglets [10, 16–21]. This redox imbalance appears to persist beyond weaning . Wang et al. observed that mRNA expression of occludin, heme oxygenase 1, catalase, thioredoxin reductase genes and occludin protein expression continued to be lower in LBW pigs during the suckling period . This apparently conflicts with the observation that LBW piglets that survive the critical first days after birth show an intestinal morphology, digestive capacity, cytokine production, intestinal motility and permeability that is comparable with those seen in normal birth weight (NBW) littermates [22–24].
The increasing incidence of supernumerary and LBW piglets has raised an urgent need for innovative rearing strategies . Next to cross-fostering , supplementing piglets  and split nursing , piglets can be transferred to a nursery and artificially reared [28–30]. Similar to conventional weaning, the artificially reared piglet encounters psychological and physical stressors including maternal and littermate separation, abrupt changes in diet composition and environment, lower intake of bioactive substances, as well as unfamiliar drinking nipples, increased exposure to pathogens and antigens, comingling and establishment of social hierarchy with unfamiliar pigs from different litters. The physiological responses to the strategy of full artificial rearing are largely unknown. Conventional weaning is associated with the induction of intestinal oxidative stress [31–33] and LBW piglets have more difficulties to maintain a balanced redox state when exposed to weaning stressors . It is unknown at present if the response to artificial rearing, since it includes similar stressors as conventional weaning, is different in LBW piglets—which have a lower antioxidant capacity —compared to NBW piglets.
Therefore, we aimed to investigate the impact of artificial rearing on piglet performance, proximal and distal small intestinal (SI) morphology, mitosis, apoptosis, and tight junction protein expression, permeability, and SI, liver and systemic redox state development compared to conventional rearing. Given the similarities between conventional weaning stressors and artificial rearing stressors we hypothesized that artificial rearing results in a redox imbalance and negatively affects intestinal morphology and functionality. Secondly, given the differences observed between NBW and LBW piglets during the suckling period, we hypothesized that in view of their affected redox state, the morphology and functionality of the small intestine is suboptimal in LBW piglets and that artificial rearing has a greater negative impact in this birth weight category.
Pig model and tissue collection
Composition of the milk replacer used for piglets from 3 d of age until weaning at d 19
Ingredient composition, %
Coco fat filled whey 50/50
Skimmed milk powder
Soy protein concentrate Soycomil K
Cheddar whey powder
Whey protein concentrate80, DVN
Spray dried blood plasma P80
Dicalciumphosphate 18% P
Vitamin and mineral premixa
Calculated nutrient levels
dMET + CYS, g/kg
Pigs were killed by exsanguination by severing the carotid arteries and jugular veins following induction of terminal anesthesia by intramuscular injection of ketamine (15 mg/kg BW) combined with xylazine (2 mg/kg BW). All piglets were weighed prior to euthanasia.
Blood was collected in EDTA and heparinized tubes containing supplemental bathophenanthroline disulfonate sodium salt. Erythrocytes were isolated by centrifuging (3000 × g, 15 min) 0.5 mL of unclotted, heparinized blood. After removing the plasma, erythrocytes were lysed by adding 100 μL of a 70% metaphosphoric acid solution, 600 μL milli Q and intense vortexing. These extracts were then centrifuged (3000 × g, 15 min), and 0.5 mL of the remaining acid extract was transferred to a vial containing 50 μL of a γ-glu-glu internal standard solution. After opening the abdomen, the liver was isolated and samples of the left lateral lobe were dissected for acid and phosphate buffered aqueous extraction, as described for the small intestinal mucosa. Subsequently, the small intestine (SI), defined as the part of the gastrointestinal tract between the pylorus and the ileocecal valve, was dissected and its length was measured. A 10 cm segment of proximal and distal SI (5 and 75% of total SI length, respectively) was taken for Ussing chamber measurements. In addition, 20 cm segments at 5 and 75% of the total SI length were emptied and carefully flushed with saline. The tissue of these 20 cm segments was placed on an ice-cold surface and the mucosa was retrieved by gently scraping the mucosal surface with a glass slide. Aliquots of the mucosa were either used instantaneously for acid and phosphate buffered aqueous extracts or transferred to plastic 2 mL screw-capped tubes, snap-frozen in liquid nitrogen and stored at -80 °C pending redox state analysis. Furthermore, 5 cm segments at 5 or 75% of the total SI length were taken, flushed with saline, snap-frozen in liquid nitrogen and stored at -80 °C pending protein expression analysis. Finally, a 5 cm segment at 5 or 75% of the total SI length was flushed with saline, divided in smaller pieces of max 1.5 cm in length and fixated for 2 h in 4% freshly prepared paraformaldehyde (in 0.01 mol/L phosphate-buffered saline) (volume tissue/volume fixative: 1/5) and routinely processed for paraffin-embedding .
Small intestinal histo-morphological measurements
In brief, 4 μm sections of paraffin-embedded samples were mounted on slides and stained with hematoxylin-eosin. Villus height, mid-villus width, and crypt depth were measured at 10× magnification using an Olympus BX61 microscope and image analysis software (analySIS Pro, Olympus Belgium, Aartselaar, Belgium) in 1–3 well-oriented villi and associated crypts in at least 12–15 sections per tissue sample, to yield 30 measurements per small intestinal region.
Small intestinal protein expression profile analysis
The concentration of specific tight junction proteins and markers for apoptosis and mitosis of the proximal and distal intestinal tissue samples was investigated using commercially available enzyme-linked immunosorbent assays (ELISA) of occludin (SEC228Hu), claudin-3 (SEF293Hu), proliferating cell nuclear antigen (PCNA) (SEA591Hu) and caspase-3 (SEA626Hu) (Cloud-Clone Corporation®, Houston, TX, USA). All tissue samples were crushed, dissolved in phosphate-buffered saline solution (PBS, pH 7.4, 0.01 mol/L), sonicated 6 times for 5 s at 4 °C (Sonics Vibracell™, VCX130, Newtown CT, USA), and kept on ice for 30 min. Subsequently the samples were centrifuged for 2 min at 13,400 rpm at 4 °C (Heraeus X3R with TX-750, Thermo Scientific, Rockford, USA), after which the supernatant was isolated, total protein concentration was determined using a Pierce TM BCA Protein Assay Kit (Thermo Scientific, Rockford, USA) and finally the samples were diluted to a total protein concentration of 10 ng/μL. Then, samples were processed on a sandwich ELISA plate and the experiment was performed according to the manufacturer’s instructions. Absorbance was measured using an Infinite M200 Pro spectrophotometer with X-Fluor software at 450 nm at 25 °C (Tecan Group Ltd., Männedorf, Switzerland). Values of protein expression were determined per gram of total protein in a sample, measured using a PierceTM BCA Protein Assay Kit (ThermoFisher Scientific, Belgium), and expressed as fmol/mg.
Ex vivo measurement of small intestinal permeability
Whereby dc/dt is the change of serosal concentration in the 20- to 100-min period (cm/s); V is the volume of the chamber, c0 is the initial marker concentration in the mucosal reservoirs and A the area of the exposed intestine in the chambers (cm2).
Mucosal, liver and blood homogenate extracts and biochemical assays
An acid extract was prepared from 1 g of homogenized (Braun homogenizer at 900 rpm) intestinal mucosa or liver that was placed in 10 mL ice-cold perchloric acid (PCA) 10% solution and centrifuged at 15,000 × g for 15 min at 4 °C. The resulting acid extract (0.5 mL) was transferred to tubes containing 50 μL γ-glu-glu internal standard solution. Samples were snap frozen in liquid nitrogen and stored at -80 °C until analysis of GSH and glutathione disulfide (GSSG). The biuret reaction was applied to determine the total protein content. Mucosal GSH and GSSG were measured using a modified high performance liquid chromatography (HPLC) method [39, 40]. The derivation procedure included the reaction of 100 mmol/L iodoacetic acid solution with thiols to form S-carboxymethyl derivatives followed by chromophore derivation of primary amines with dinitrofluorobenzene (DNFB, 1% (v/v) in ethanol). GSH and GSSG were separated through EC250/4.6 Nucleosil 120-7 NH2 aminopropyl column (Machery-Nagel, Düren, Germany) protected by the same NH2 guard column (CC8/4). Chromatographic runs were performed at a flow-rate of 1.5 mL/min, starting at 80% solvent A/20% solvent B for 5 min followed by a 10 min linear gradient to 1% solvent A/99% solvent B and a 10 min isocratic period at 1% solvent A/99% solvent B (solvent A: water-methanol solution (1:4, v/v), solvent B: 0.5 mol/L sodium acetate–64% methanol). The column was re-equilibrated to the initial conditions for 15 min while maintaining the column temperature at 40 °C. The UV detector was set at 365 nm for absorption measurements. GSH and GSSG were identified by retention times of authentic standards. Concentrations were determined by using the internal and external standards and expressed as μmol/g protein. In addition, a phosphate buffered aqueous extract was made by mixing approximately 1 g of homogenized mucosa in 10 mL ice cold 1% Triton-X-100 phosphate buffer solution (pH = 7.0), by using an Ultra-Turrax dispensing machine (IKA-Werke GmbH & Co. KG, Staufen, Germany). The supernatant was transferred to 2 mL tubes, snap frozen and stored at -80 °C until analysis. Supernatants were used for the determination of GSH-Px activity and malondialdehyde (MDA; expressed as nmol/g protein) concentration. Assessment of GSH-Px activity (expressed as U/g protein) in EDTA plasma and mucosa was determined spectrophotometrically . The thiobarbituric acid reactive substances (TBARS) method was used to measure MDA concentration in EDTA plasma, liver and mucosa extracts .
Linear mixed models were fitted to assess the influence of birth weight category (NBW/LBW), feeding (artificially reared/sow-reared) and days postnatal (as a categorical variable) on the quantitative outcome variables. To model the dependence between observations within the same litter, random intercept terms for litter were added to the model. Depending on the research question, separate analyses were carried out in subgroups (e.g. only in sow-reared piglets) or time points were analyzed separately. In the subgroup analyses where no random effect terms was needed, a multiple linear regression model was fitted. Post hoc tests to compare mean values between the different time points (days postnatal) were carried out using Tukey’s honestly significant difference. Models were fitted using the Mixed Model procedure of the JMP Pro11 software (SAS Institute, Cary, NC, USA). Significance for the fixed effects was tested using an F-test with Kenward-Roger correction. Data are expressed as means and their standard errors (S.E.), and P < 0.05 was considered significant.
Body weight of piglets
Histo-morphological measurements in the proximal and distal small intestine
PCNA and caspase-3 protein expression in the proximal and distal small intestine
Caspase-3 protein expression significantly decreased in the proximal SI of sow-reared (P < 0.05) and artificially reared piglets (P < 0.001) piglets from d3 to d19 (Fig. 3b). In contrast, in the distal SI of sow- and artificially reared piglets, no age-related differences were observed. Caspase-3 protein expression was significantly lower in the proximal (P < 0.05) and distal (P < 0.05) SI of artificially reared piglets compared to sow-reared piglets at d19.
Occludin and claudin-3 protein expression in the proximal and distal small intestine
Claudin-3 expression in the proximal and in the distal SI of artificially reared piglets dropped significantly from d3 to d5 (P < 0.01; Fig. 4b). Claudin-3 expression increased from d5 to d19 and was significantly higher in the SI of artificially reared piglets compared to sow-reared piglets at d19 (P < 0.01). Claudin-3 expression in the distal SI of LBW sow-reared piglets was on average 33% higher compared to NBW sow-reared piglets at d3, d8 and d19 (P < 0.01).
Ex vivo permeability in the proximal and distal small intestine
Mucosal redox state represented by GSH concentration, GSSG concentration, GSSG/GSH ratio, GSH-Px activity and MDA concentration
In the proximal intestine of sow-reared pigs, the concentration of MDA abruptly dropped after birth (P < 0.001; Fig. 6e) and remained stable from d3 until d19, whereas in the distal intestine the decrease was more spread out in time (P < 0.01). Transferring piglets to a nursery decreased the concentration of GSH, whereas the GSSG concentration and GSSG/GSH ratio were increased in the SI of artificially reared piglets at d5. In the proximal SI of artificially reared piglets GSH concentration peaked, while GSSG concentration and GSSG/GSH ratio showed a minimum at d8 (P < 0.05). However by d19, these redox parameters returned to the values noted at birth (P < 0.05). GSH-Px activity showed a minimum at d5 (P < 0.05), but recovered from d8 to d19 (P < 0.01) in the SI of artificially reared piglets.
In the proximal SI, GSH concentration was significantly higher (P < 0.05), while GSSG concentration (P < 0.01) and consequently the GSSG/GSH ratio (P = 0.001) was significantly lower in artificially reared piglets compared to sow-reared piglets at d8. GSH-Px activity was significantly lower at d8 (P < 0.001) and significantly higher at d19 (P < 0.001) in both regions of the SI of artificially reared piglets compared to sow-reared piglets. GSH-Px activity in the SI of LBW sow-reared piglets was significantly higher than their NBW littermates (proximal: on average 1.38 U/g higher; distal: on average 0.64 U/g higher) (P < 0.001). A similar observation for both birth weight categories was made in the distal SI of artificially reared piglets (on average 1.24 higher, P < 0.01).
At d8, MDA concentration in the proximal SI of sow-reared piglets was significantly higher compared to artificially reared piglets (P < 0.01).
Liver redox state represented by GSH concentration, GSSG concentration, GSSG/GSH ratio, GSH-Px activity and MDA concentration
When piglets were introduced to a milk replacer, GSH (P < 0.05) and GSSG (P < 0.01) concentrations significantly decreased whereas GSSG/GSH ratio (P < 0.05) and MDA concentration (P < 0.001) significantly increased from d3 to d5. Meanwhile, GSH-Px activity remained unchanged. All redox parameters in the liver were stable from d5 to d8 in artificially reared piglets, except GSSG/GSH ratio that showed a significant decrease (P < 0.01). Towards d19, GSH concentration (P < 0.01) and GSH-Px activity (P < 0.05) significantly increased while MDA concentration (P < 0.01) significantly decreased in artificially reared piglets.
At d8, a lower GSH concentration and a higher MDA were observed in artificially reared piglets compared to sow-reared piglets (P < 0.001). At d19, higher GSH-Px activities were observed in artificially reared piglets compared to sow-reared piglets (P < 0.01).
Systemic redox state represented by GSH concentration, GSSG concentration, and GSSG/GSH ratio, GSH-Px activity and MDA concentration
Given the need for an alternative rearing strategy that lowers the challenges that LBW and supernumerary piglets face during the suckling period, we aimed to investigate the responses to full artificial rearing. Our data demonstrated that artificial rearing beneficially affected piglet performance, notwithstanding impairing effects on small intestinal architecture, permeability and redox state in both LBW and NBW piglets.
Artificial rearing influences piglet performance
This study documents the implications of full artificial rearing of LBW and supernumerary piglets. Under standard rearing conditions these pigs are at high risk of succumbing due to insufficient nutrient intake, increased disease susceptibility, and physiological deficits (e.g. lower energy reserves) . Our study demonstrated that transitioning piglets to a nursery with ad libitum access to a milk replacer led to significantly higher body weights of LBW and NBW piglets compared to sow-reared piglets at d19. The experiment was terminated on the same day as when the conventionally reared piglets were weaned on the farm with a 3-week batch system. For this specific farm, the average age at weaning is 19.6 d. Furthermore, milk production of the sow strongly decreases towards the end of the suckling phase. Around d 18–19, sow milk starts to be very limiting and becomes hard to compare to the ad libitum access for the artificially reared piglets . Using a weigh-suckle-weigh technique, De Vos et al.  showed that piglets with ad libitum access to milk replacer have a higher relative energy intake compared to sow-reared piglets . Our findings confirm this and other previous research where LBW piglets receiving an energy rich diet—comparable with our LBW piglets fed a milk replacer ad libitum—presented a comparable body weight gain as NBW piglets receiving a lower energy intake—comparable with our NBW piglets fed by the sow . In addition, the milk replacer used in our study contained spray-dried plasma which could have contributed further to the higher weight gain in the artificially reared group. Ermer et al.  showed that spray-dried porcine plasma increased feed intake. Thus next to ad libitum access to feed, diet composition cannot be neglected.
Artificial rearing influences small intestinal architecture
Small intestinal morphology is one of the major indicators reflecting gut health in pigs . However, caution should be taken when evaluating morphology alone as a measure of gut health. For example, Enterotoxigenic Escherichia coli, the major causal agent of neonatal diarrhea, may occur without histological changes in the intestine . Notwithstanding, stereological analysis of small intestinal morphology will provide the most accurate estimation of the intestinal absorptive surface area , a proxy of the surface can be calculated using villus height and villus width . In our study, the mucosal surface area of the proximal small intestine at d19 was markedly larger in artificially reared piglets than sow-reared piglets. In this regard, feeding a milk replacer shows promise as the increased mucosal surface suggests a higher ability to absorb nutrients. Moreover, previous research showed an increased activity of maltase and sucrase when piglets are fed a milk replacer [49–51]. Thus, artificial rearing seems to improve the digestive capacity at the level of the small intestine.
The deepening of the crypts could be a response to promote mucus secretion rather than lead to enterocyte maturation and proliferation and thus an increase in PCNA expression. Previous research showed that breast-fed infants showed a delay in the mucin degradation when compared to artificially reared infants . Phillips  showed that crypt goblet cells have the ability to restitute the mucus layer and showed a decrease in the percentage of villus epithelial volume occupied by mucin secretory granules.
Transferring piglets to a nursery at d3 exposed them to stressful effects caused by psychological, environmental or nutritional factors similar to those encountered during the conventional weaning process . However, it is difficult to unravel the separate contributions of these factors. Previous studies showed significant villus atrophy at d4 and deeper crypts at d7 in piglets that were separated from the sow and still fed sow’s milk compared to unweaned piglets [55, 56]. Similarly, our study showed transient villus atrophy in piglets transferred to a nursery at d3. This villus height reduction is analogue to the intestinal morphological changes as a result of inadequate food intake immediately after conventional weaning [56–59]. In contrast to conventional weaning, villus length is rapidly restored. This could be related to the inclusion of spray-dried plasma in the milk replacer since this is known to increase villus height [8, 60–63].
Artificial rearing affects small intestinal tight junction protein expression
We hypothesized that artificial rearing influences small intestinal physiology. The intestinal epithelium plays a critical role in the transport of nutrients and macromolecules. At the same time, it has to provide an effective barrier to harmful macromolecules and microorganisms . Epithelial cells constitute a dynamic barrier where large molecules can be transported by transcytosis and this can be measured by HRP . Tight junctions (TJs) are essential components of the physical intercellular barrier and their presence and functionality changes under different physiological and pathological conditions [66, 67]. Well-formed TJs are characterized by low solute permeability which can be determined by measuring FD-4 permeability . The family of junctional adhesion molecules, the claudin and occludin families, are structural transmembrane TJ components that have the potential to mediate cell–cell adhesion [66, 68]. Within TJs, claudins are the main determinants of the selective pore properties [68, 69], while the role of occludin in barrier functioning is more diverse [70, 71]. In mice, the expression of claudin-3 is promoted during the first 3 weeks of life concomitant with the establishment of the intestinal microbiota [72, 73]. Our study showed that claudin-3 expression rose particularly in artificially reared piglets. Possibly the different microbiota fingerprint  and the absence of milk born IgA’s  can be held responsible. Claudin-3 is known to be a “tightening” claudin . This could explain why permeability is seemingly unaffected by the observed increase in claudin-3. However, at the start of artificial rearing, claudin-3 as well as occludin protein levels transiently dropped. This drop is reflected in a concomitantly increased permeability for FD-4 and HRP. Previous studies showed lower abundances of occludin mRNA and protein, claudin-3 protein, and increased lactulose permeability after weaning [50, 76]. Thus artificial rearing induced a similar response as seen after conventional weaning.
Artificial rearing resulted in a redox imbalance
Previously, we investigated the link between oxidative stress, intestinal integrity, and permeability in intestinal epithelial cells in vitro  and in vivo during normal suckling . Vergauwen et al.  and others showed a redistribution of TJ proteins during times of imposing reactive species and could relate these responses to a compromised permeability [12, 77]. The current study demonstrated that transfer of piglets to a nursery resulted in oxidative stress.
Glutathione (GSH) is an important regulator of the redox status within intestinal epithelial cells . The liver is the major site of GSH biosynthesis and exports GSH via the bile to the proximal SI . Thus GSH originating from the liver supports mucosal GSH by decreasing lipid peroxidation and maintaining the GSSG/GSH redox homeostasis in the proximal intestine [11, 79, 80]. In our study, artificial rearing resulted in the liver in a decreased GSH content, an increased GSSG/GSH ratio and MDA concentration. After this first phase, the redox parameters returned to their initial values. GSH-Px activity in the liver seemed unaffected during the transition period, while GSH-Px activity transiently dropped in the proximal and distal SI when piglets were introduced to a nursery. Previously, conventional weaning caused a drop in GSH-Px activity but GSH-Px activity increased afterwards as part of a feedback mechanism . Our study shows that artificial rearing increases oxidative stress in the SI and as a result the GSH-Px activity increased and was higher than the activity seen in sow-reared piglets. Furthermore, LBW piglets showed a higher GSH-Px activity compared to their NBW littermates. It is clear that the antioxidant capacity of the liver helps protecting the proximal intestine by secreting GSH into the lumen of proximal SI [78, 79]. This could explain the increased mucosal GSH concentration of the proximal SI and the concomitant massive decrease of the liver GSH concentration. On the other hand, a remarkable drop of the GSH concentration in the distal SI was observed from d3 until d5. Consequently, this resulted in a high concentration of MDA and GSSG, resulting in a higher GSSG/GSH ratio in the distal SI. Furthermore, the GSSG/GSH ratio increased systemically upon transfer to a nursery. Degroote et al.  already showed that conventional weaning increased the GSSG/GSH ratio.
Our research presents a window of opportunity for antioxidant supplementation to protect piglets from redox imbalance due to artificial rearing. NBW artificially reared piglets were more susceptible to redox imbalance and loss of intestinal integrity upon transferal to a nursery (from d3 to d5). Taken together, these findings favor the transfer of both LBW and NBW piglets to a nursery as a solution for LBW and supernumerary piglets. Further research is necessary to elucidate how these artificially reared LBW and NBW piglets will respond to the introduction to solid food.
In conclusion, we demonstrated that artificial rearing influences morphological and functional parameters in the small intestine, liver and blood in a way similar to what is seen after conventional weaning. Nevertheless, growth performance of artificially reared piglets was positively influenced. In addition, artificially reared piglets rapidly recovered from redox imbalances and restored intestinal permeability within a couple of days. Further research is needed to explore the possibility to supplement LBW and NBW piglets with antioxidants prior to initiating artificial rearing. Thus, artificial rearing is a valuable alternative to raise LBW or supernumerary piglets.
Enzyme linked immunosorbent assay
Fluorescein isothiocyanate dextran 4 kDa
High performance liquid chromatography
Low birth weight
Normal birth weight
We thank K. Huybrechts, G. Vrolix, K. Jennes, M. De Reys, S. Coolsaet, A. Ovyn and T. Van der Eecken for their technical assistance.
This work was supported by a grant from the government agency for Innovation by Science and Technology (IWT-LO 100856).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
Author contributions: CVG, JM and SDM concept and design of research; HV, JDG, WW, CVG and JM performed experiments; HV and JDG analyzed the data; HV and EF design and implementation of the statistical model; HV, JDG, CVG and JM interpreted the results of the experiments; HV and CVG prepared the figures; HV and CVG drafted manuscript; HV, JDG, CVG, JM, SDS, WW, CC, SVC and SP edited and revised the manuscript; CVG and JM approved the final version of the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All husbandry and experimental procedures were approved by the Ethical Committee for Animal Testing (ECD) of the University of Antwerp (Belgium) (EC number: 2015-02) in accordance with the European Commission directive for the humane care and use of animals in research (2010/63/EU).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Lay Jr DC, Matteri RL, Carrol JA, Fangman TJ, Safranski TJ. Preweaning survival in swine. J Anim Sci. 2002;80(E. Suppl. 1):E74–86.Google Scholar
- Tuchscherer M, Puppe B, Tuchscherer A, Tiemann U. Early identification of neonates at risk: traits of newborn piglets with respect to survival. Theriogenology. 2000;54(3):371–88. doi:10.1016/s0093-691x(00)00355-1.View ArticlePubMedGoogle Scholar
- Milligan BN, Dewey CE, de Grau AF. Neonatal-piglet weight variation and its relation to pre-weaning mortality and weight gain on commercial farms. Prev Vet Med. 2002;56(2):119–27.View ArticlePubMedGoogle Scholar
- Friel JK, Diehl-Jones B, Cockell KA, Chiu A, Rabanni R, Davies SS, et al. Evidence of oxidative stress in relation to feeding type during early life in premature infants. Pediatr Res. 2011;69(2):160–4. doi:10.1203/PDR.0b013e3182042a07.View ArticlePubMedGoogle Scholar
- Ahola T, Levonen AL, Fellman V, Lapatto R. Thiol metabolism in preterm infants during the first week of life. Scand J Clin Lab Invest. 2004;64(7):649–58. doi:10.1080/00365510410002959.View ArticlePubMedGoogle Scholar
- Rook D, Te Braake FW, Schierbeek H, Longini M, Buonocore G, Van Goudoever JB. Glutathione synthesis rates in early postnatal life. Pediatr Res. 2010;67(4):407–11. doi:10.1203/PDR.0b013e3181d22cf6.View ArticlePubMedGoogle Scholar
- Yin J, Ren W, Liu G, Duan J, Yang G, Wu L, et al. Birth oxidative stress and the development of an antioxidant system in newborn piglets. Free Radic Res. 2013;47(12):1027–35. doi:10.3109/10715762.2013.848277.View ArticlePubMedGoogle Scholar
- Tran H, Bundy JW, Li YS, Carney-Hinkle EE, Miller PS, Burkey TE. Effects of spray-dried porcine plasma on growth performance, immune response, total antioxidant capacity, and gut morphology of nursery pigs. J Anim Sci. 2014;92(10):4494–504. doi:10.2527/jas.2014-7620.View ArticlePubMedGoogle Scholar
- Michiels J, De Vos M, Missotten J, Ovyn A, De Smet S, Van Ginneken C. Maturation of digestive function is retarded and plasma antioxidant capacity lowered in fully weaned low birth weight piglets. Br J Nutr. 2013;109(1):65–75. doi:10.1017/s0007114512000670.View ArticlePubMedGoogle Scholar
- Krueger R, Derno M, Goers S, Metzler-Zebeli BU, Nuernberg G, Martens K, et al. Higher body fatness in intrauterine growth retarded juvenile pigs is associated with lower fat and higher carbohydrate oxidation during ad libitum and restricted feeding. Eur J Nutr. 2014;53(2):583–97. doi:10.1007/s00394-013-0567-x.View ArticlePubMedGoogle Scholar
- Aw TY. Intestinal glutathione: determinant of mucosal peroxide transport, metabolism, and oxidative susceptibility. Toxicol Appl Pharmacol. 2005;204(3):320–8. doi:10.1016/j.taap.2004.11.016.View ArticlePubMedGoogle Scholar
- Vergauwen H, Tambuyzer B, Jennes K, Degroote J, Wang W, De Smet S, et al. Trolox and ascorbic acid reduce direct and indirect oxidative stress in the IPEC-J2 cells, an in vitro model for the porcine gastrointestinal tract. PLoS One. 2015;10(3):e0120485. doi:10.1371/journal.pone.0120485.View ArticlePubMedPubMed CentralGoogle Scholar
- Kelly FJ. Glutathione content of the small intestine: regulation and function. Br J Nutr. 1993;69(2):589–96.View ArticlePubMedGoogle Scholar
- Carrasco-Pozo C, Morales P, Gotteland M. Polyphenols Protect the Epithelial Barrier Function of Caco-2 Cells Exposed to Indomethacin through the Modulation of Occludin and Zonula Occludens-1 Expression. Journal of agricultural and food chemistry. 2013. doi:10.1021/jf400150p.
- Bhattacharyya A, Chattopadhyay R, Mitra S, Crowe SE. Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiol Rev. 2014;94(2):329–54. doi:10.1152/physrev.00040.2012.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang J, Chen L, Li D, Yin Y, Wang X, Li P, et al. Intrauterine growth restriction affects the proteomes of the small intestine, liver, and skeletal muscle in newborn pigs. J Nutr. 2008;138(1):60–6.PubMedGoogle Scholar
- Wang X, Wu W, Lin G, Li D, Wu G, Wang J. Temporal proteomic analysis reveals continuous impairment of intestinal development in neonatal piglets with intrauterine growth restriction. J Proteome Res. 2010;9(2):924–35. doi:10.1021/pr900747d.View ArticlePubMedGoogle Scholar
- Mickiewicz M, Zabielski R, Grenier B, Le Normand L, Savary G, Holst JJ, et al. Structural and functional development of small intestine in intrauterine growth retarded porcine offspring born to gilts fed diets with differing protein ratios throughout pregnancy. J Physiol Pharmacol. 2012;63(3):225–39.PubMedGoogle Scholar
- Ferenc K, Pietrzak P, Godlewski MM, Piwowarski J, Kilianczyk R, Guilloteau P, et al. Intrauterine growth retarded piglet as a model for humans--studies on the perinatal development of the gut structure and function. Reprod Biol. 2014;14(1):51–60. doi:10.1016/j.repbio.2014.01.005.View ArticlePubMedGoogle Scholar
- D’Inca R, Gras-Le Guen C, Che L, Sangild PT, Le Huerou-Luron I. Intrauterine growth restriction delays feeding-induced gut adaptation in term newborn pigs. Neonatology. 2011;99(3):208–16. doi:10.1159/000314919.View ArticlePubMedGoogle Scholar
- Wang W, Degroote J, Van Ginneken C, Van Poucke M, Vergauwen H, Dam TM et al. Intrauterine growth restriction in neonatal piglets affects small intestinal mucosal permeability and mRNA expression of redox-sensitive genes. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2015. doi:10.1096/fj.15-274779.
- Huygelen V, De Vos M, Willemen S, Tambuyzer B, Casteleyn C, Knapen D, et al. Increased intestinal barrier function in the small intestine of formula-fed neonatal piglets. J Anim Sci. 2012;90 Suppl 4:315–7. doi:10.2527/jas.53731.View ArticlePubMedGoogle Scholar
- Huygelen V, De Vos M, Willemen S, Fransen E, Casteleyn C, Van Cruchten S, et al. Age-related differences in mucosal barrier function and morphology of the small intestine in low and normal birth weight piglets. J Anim Sci. 2014;92(8):3398–406. doi:10.2527/jas.2014-7742.View ArticlePubMedGoogle Scholar
- Willemen S, Che L, De Vos M, Huygelen V, Tambuyzer B, Casteleyn C, et al. Perinatal growth restriction is not related to higher intestinal distribution and increased serum levels of 5-hydroxytryptamin in piglets. J Anim Sci. 2012;90 Suppl 4:305–7. doi:10.2527/jas.53730.View ArticlePubMedGoogle Scholar
- Ferrari CV, Sbardella PE, Bernardi ML, Coutinho ML, Vaz Jr IS, Wentz I, et al. Effect of birth weight and colostrum intake on mortality and performance of piglets after cross-fostering in sows of different parities. Prev Vet Med. 2014;114(3-4):259–66. doi:10.1016/j.prevetmed.2014.02.013.View ArticlePubMedGoogle Scholar
- De Vos M, Che L, Huygelen V, Willemen S, Michiels J, Van Cruchten S, et al. Nutritional interventions to prevent and rear low-birthweight piglets. J Anim Physiol Anim Nutr. 2014;98(4):609–19. doi:10.1111/jpn.12133.View ArticleGoogle Scholar
- Donovan TS, Dritz SS. Effect of split nursing on variation in pig growth from birth to weaning. J Am Vet Med Assoc. 2000;217(1):79–81.View ArticlePubMedGoogle Scholar
- De Vos M, Huygelen V, Willemen S, Fransen E, Casteleyn C, Van Cruchten S, et al. Artificial rearing of piglets: Effects on small intestinal morphology and digestion capacity. Livest Sci. 2014;159:165–73. doi:10.1016/j.livsci.2013.11.012.View ArticleGoogle Scholar
- Wedig J, Christian MS, Hoberman A, Diener RM, Thomas-Wedig R. A study to develop methodology for feeding 24-hour-old neonatal swine for 3 weeks. Int J Toxicol. 2002;21(5):361–70. doi:10.1080/10915810290096577.View ArticlePubMedGoogle Scholar
- Fiorotto ML, Reeds PJ, Cunningham JJ, Pond WG. A semiautomatic device for feeding liquid milk-replacer diets to infant pigs. J Anim Sci. 1993;71(1):78–85.PubMedGoogle Scholar
- Degroote J, Michiels J, Claeys E, Ovyn A, De Smet S. Changes in the pig small intestinal mucosal glutathione kinetics after weaning. J Anim Sci. 2012;90 Suppl 4:359–61. doi:10.2527/jas.53809.View ArticlePubMedGoogle Scholar
- Zhu LH, Zhao KL, Chen XL, Xu JX. Impact of weaning and an antioxidant blend on intestinal barrier function and antioxidant status in pigs. J Anim Sci. 2012;90(8):2581–9. doi:10.2527/jas.2012-4444.View ArticlePubMedGoogle Scholar
- Sauerwein H, Schmitz S, Hiss S. The acute phase protein haptoglobin and its relation to oxidative status in piglets undergoing weaning-induced stress. Redox Report. 2005;10(6):295–302. doi:10.1179/135100005x83725.View ArticlePubMedGoogle Scholar
- Klobasa F, Werhahn E, Butler JE. Composition of sow milk during lactation. J Anim Sci. 1987;64(5):1458–66.View ArticlePubMedGoogle Scholar
- Csapo J, Martin TG, Csapo-Kiss ZS, Hazas Z. Protein, fats, vitamin and mineral concentration in porcine colostrum and milk from parturition to 60 days. Int Dairy J. 1996;6(8-9):881–902.View ArticleGoogle Scholar
- Ontsouka CE, Bruckmaier RM, Blum JW. Fractionized milk composition during removal of colostrum and mature milk. J Dairy Sci. 2003;86(6):2005–11. doi:10.3168/jds.S0022-0302(03)73789-8.View ArticlePubMedGoogle Scholar
- Buesa RJ, Peshkov MV. How much formalin is enough to fix tissues? Ann Diagn Pathol. 2012;16(3):202–9. doi:10.1016/j.anndiagpath.2011.12.003.View ArticlePubMedGoogle Scholar
- McKie AT, Zammit PS, Naftalin RJ. Comparison of cattle and sheep colonic permeabilities to horseradish peroxidase and hamster scrapie prion protein in vitro. Gut. 1999;45(6):879–88.View ArticlePubMedPubMed CentralGoogle Scholar
- Yoshida T. Determination of reduced and oxidized glutathione in erythrocytes by high-performance liquid chromatography with ultraviolet absorbance detection. J Chromatogr B Biomed Appl. 1996;678(2):157–64.View ArticlePubMedGoogle Scholar
- Reed DJ, Babson JR, Beatty PW, Brodie AE, Ellis WW, Potter DW. High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal Biochem. 1980;106(1):55–62.View ArticlePubMedGoogle Scholar
- Hernandez P, Zomeno L, Arino B, Blasco A. Antioxidant, lipolytic and proteolytic enzyme activities in pork meat from different genotypes. Meat Sci. 2004;66(3):525–9. doi:10.1016/s0309-1740(03)00155-4.View ArticlePubMedGoogle Scholar
- Grotto D, Santa Maria LD, Boeira S, Valentini J, Charao MF, Moro AM, et al. Rapid quantification of malondialdehyde in plasma by high performance liquid chromatography-visible detection. J Pharm Biomed Anal. 2007;43(2):619–24. doi:10.1016/j.jpba.2006.07.030.View ArticlePubMedGoogle Scholar
- Aguinaga MA, Gomez-Carballar F, Nieto R, Aguilera JF. Production and composition of Iberian sow’s milk and use of milk nutrients by the suckling Iberian piglet. Animal. 2011;5(9):1390–7. doi:10.1017/s1751731111000474.View ArticlePubMedGoogle Scholar
- Han F, Hu L, Xuan Y, Ding X, Luo Y, Bai S, et al. Effects of high nutrient intake on the growth performance, intestinal morphology and immune function of neonatal intra-uterine growth-retarded pigs. Br J Nutr. 2013;110(10):1819–27. doi:10.1017/s0007114513001232.View ArticlePubMedGoogle Scholar
- Ermer PM, Miller PS, Lewis AJ. Diet preference and meal patterns of weanling pigs offered diets containing either spray-dried porcine plasma or dried skim milk. J Anim Sci. 1994;72(6):1548–54.PubMedGoogle Scholar
- Caspary WF. Physiology and pathophysiology of intestinal absorption. Am J Clin Nutr. 1992;55(1 Suppl):299s–308s.PubMedGoogle Scholar
- Egberts HJ, de Groot EC, van Dijk JE, Vellenga L, Mouwen JM. Tight junctional structure and permeability of porcine jejunum after enterotoxic Escherichia coli infection. Res Vet Sci. 1993;55(1):10–4.View ArticlePubMedGoogle Scholar
- Van Ginneken C, Van Meir F, Weyns A. Stereologic characteristics of pig small intestine during normal development. Dig Dis Sci. 2002;47(4):868–78.View ArticlePubMedGoogle Scholar
- Cera KR, Mahan DC, Reinhart GA. Effect of weaning, week postweaning and diet composition on pancreatic and small intestinal luminal lipase response in young swine. Journal of animal science. 1990;68(2). doi:10.2527/1990.682384x.
- Wang J, Zeng L, Tan B, Li G, Huang B, Xiong X, et al. Developmental changes in intercellular junctions and Kv channels in the intestine of piglets during the suckling and post-weaning periods. J Anim Sci Biotechnol. 2016;7:4. doi:10.1186/s40104-016-0063-2.View ArticlePubMedPubMed CentralGoogle Scholar
- Huygelen V, De Vos M, Prims S, Vergauwen H, Fransen E, Casteleyn C, et al. Birth weight has no influence on the morphology, digestive capacity and motility of the small intestine in suckling pigs. Livest Sci. 2015;182:129–36. http://dx.doi.org/doi:10.1016/J.LIVSCI.2015.11.003.View ArticleGoogle Scholar
- Midtvedt AC, Carlstedt-Duke B, Midtvedt T. Establishment of a mucin-degrading intestinal microflora during the first two years of human life. J Pediatr Gastroenterol Nutr. 1994;18(3):321–6.View ArticlePubMedGoogle Scholar
- Phillips TE. Both crypt and villus intestinal goblet cells secrete mucin in response to cholinergic stimulation. Am J Physiol. 1992;262(2 Pt 1):G327–31.PubMedGoogle Scholar
- Funderburke DW, Seerley RW. The effects of postweaning stressors on pig weight change, blood, liver and digestive tract characteristics. J Anim Sci. 1990;68(1):155–62.View ArticlePubMedGoogle Scholar
- van Beers-Schreurs HM, Nabuurs MJ, Vellenga L, Kalsbeek-van der Valk HJ, Wensing T, Breukink HJ. Weaning and the weanling diet influence the villous height and crypt depth in the small intestine of pigs and alter the concentrations of short-chain fatty acids in the large intestine and blood. J Nutr. 1998;128(6):947–53.PubMedGoogle Scholar
- Kelly D, Smyth JA, McCracken KJ. Digestive development of the early-weaned pig. 1. Effect of continuous nutrient supply on the development of the digestive tract and on changes in digestive enzyme activity during the first week post-weaning. Br J Nutr. 1991;65(2):169–80.View ArticlePubMedGoogle Scholar
- Kelly D, Smyth JA, McCracken KJ. Digestive development of the early-weaned pig. 2. Effect of level of food intake on digestive enzyme activity during the immediate post-weaning period. Br J Nutr. 1991;65(2):181–8.
- Spreeuwenberg MA, Verdonk JM, Gaskins HR, Verstegen MW. Small intestine epithelial barrier function is compromised in pigs with low feed intake at weaning. J Nutr. 2001;131(5):1520–7.PubMedGoogle Scholar
- Hedemann MS, Hojsgaard S, Jensen BB. Small intestinal morphology and activity of intestinal peptidases in piglets around weaning. J Anim Physiol Anim Nutr. 2003;87(1-2):32–41.View ArticleGoogle Scholar
- Pierce JL, Cromwell GL, Lindemann MD, Russell LE, Weaver EM. Effects of spray-dried animal plasma and immunoglobulins on performance of early weaned pigs. J Anim Sci. 2005;83(12):2876–85.View ArticlePubMedGoogle Scholar
- Touchette KJ, Allee GL, Matteri RL, Dyer CJ, Carroll JA. Effect of spray-dried plasma and Escherichia coli on intestinal morphology and the hypothalamic–pituitary–adrenal (HPA) axis of the weaned pig. J Anim Sci. 1999;77(Supplement 1):56.Google Scholar
- Jiang R, Chang X, Stoll B, Fan MZ, Arthington J, Weaver E, et al. Dietary plasma protein reduces small intestinal growth and lamina propria cell density in early weaned pigs. J Nutr. 2000;130(1):21–6.PubMedGoogle Scholar
- van Dijk AJ, Niewold TA, Margry RJ, van den Hoven SG, Nabuurs MJ, Stockhofe-Zurwieden N, et al. Small intestinal morphology in weaned piglets fed a diet containing spray-dried porcine plasma. Res Vet Sci. 2001;71(1):17–22. doi:10.1053/rvsc.2001.0478.View ArticlePubMedGoogle Scholar
- Ramanan D, Cadwell K. Intrinsic Defense Mechanisms of the Intestinal Epithelium. Cell host & microbe. 2016. doi:10.1016/j.chom.2016.03.003.
- Boudry G. The Ussing chamber technique to evaluate alternatives to in-feed antibiotics for young pigs. Animal Res. 2005;54:219–30.View ArticleGoogle Scholar
- Schneeberger EE, Lynch RD. The tight junction: a multifunctional complex. Am J Physiol Cell Physiol. 2004;286(6):C1213–28. doi:10.1152/ajpcell.00558.2003.View ArticlePubMedGoogle Scholar
- Lu Z, Ding L, Lu Q, Chen Y-H. Claudins in intestines: distribution and functional significance in health and diseases. Tissue Barriers. 2013;1(3):e24978. doi:10.4161/tisb.24978.View ArticlePubMedPubMed CentralGoogle Scholar
- Anderson JM, Van Itallie CM, Fanning AS. Setting up a selective barrier at the apical junction complex. Curr Opin Cell Biol. 2004;16(2):140–5. doi:10.1016/j.ceb.2004.01.005.View ArticlePubMedGoogle Scholar
- Furuse M, Tsukita S. Claudins in occluding junctions of humans and flies. Trends Cell Biol. 2006;16(4):181–8. doi:10.1016/j.tcb.2006.02.006.View ArticlePubMedGoogle Scholar
- Yu AS, McCarthy KM, Francis SA, McCormack JM, Lai J, Rogers RA, et al. Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells. Am J Physiol Cell Physiol. 2005;288(6):C1231–41. doi:10.1152/ajpcell.00581.2004.View ArticlePubMedGoogle Scholar
- Raleigh DR, Boe DM, Yu D, Weber CR, Marchiando AM, Bradford EM, et al. Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function. J Cell Biol. 2011;193(3):565–82. doi:10.1083/jcb.201010065.View ArticlePubMedPubMed CentralGoogle Scholar
- Patel RM, Myers LS, Kurundkar AR, Maheshwari A, Nusrat A, Lin PW. Probiotic bacteria induce maturation of intestinal claudin 3 expression and barrier function. Am J Pathol. 2012;180(2):626–35. doi:10.1016/j.ajpath.2011.10.025.View ArticlePubMedPubMed CentralGoogle Scholar
- Holmes JL, Van Itallie CM, Rasmussen JE, Anderson JM. Claudin profiling in the mouse during postnatal intestinal development and along the gastrointestinal tract reveals complex expression patterns. Gene Expression Patterns. 2006;6(6):581–8. doi:10.1016/j.modgep.2005.12.001.View ArticlePubMedGoogle Scholar
- Mackie RI, Sghir A, Gaskins HR. Developmental microbial ecology of the neonatal gastrointestinal tract. Am J Clin Nutr. 1999;69(5):1035s–45s.PubMedGoogle Scholar
- Milatz S, Krug SM, Rosenthal R, Gunzel D, Muller D, Schulzke JD, et al. Claudin-3 acts as a sealing component of the tight junction for ions of either charge and uncharged solutes. Biochim Biophys Acta. 2010;1798(11):2048–57. doi:10.1016/j.bbamem.2010.07.014.View ArticlePubMedGoogle Scholar
- Wang H, Zhang C, Wu G, Sun Y, Wang B, He B, et al. Glutamine enhances tight junction protein expression and modulates corticotropin-releasing factor signaling in the jejunum of weanling piglets. J Nutr. 2015;145(1):25–31. doi:10.3945/jn.114.202515.View ArticlePubMedGoogle Scholar
- Fukui A, Naito Y, Handa O, Kugai M, Tsuji T, Yoriki H et al. Acetyl salicylic acid induces damage to intestinal epithelial cells by oxidation-related modifications of ZO-1. American journal of physiology Gastrointestinal and liver physiology. 2012. doi:10.1152/ajpgi.00236.2012.
- Lee TK, Li L, Ballatori N. Hepatic glutathione and glutathione S-conjugate transport mechanisms. Yale J Biol Med. 1997;70(4):287–300.PubMedPubMed CentralGoogle Scholar
- Aw TY. Biliary glutathione promotes the mucosal metabolism of luminal peroxidized lipids by rat small intestine in vivo. J Clin Investig. 1994;94(3):1218–25.View ArticlePubMedPubMed CentralGoogle Scholar
- Ballatori N, Truong AT. Relation between biliary glutathione excretion and bile acid-independent bile flow. Am J Physiol. 1989;256(1 Pt 1):G22–30.PubMedGoogle Scholar