All diets were ground to pass a 5 mm sieve.In the Hemp Cake diet,the amount of wheat and rapeseed meal was reduced to ensure the same energy and protein level as in the other diets,and hemp seed cake was added prior to pelleting at a commercial feed factory.The Pellets and Hemp Hulls diets were based on the same ingredients,thus to distinguish between the different feeds before preparation of the experimental diets,micro grits were added to the Meal diet and the Hemp Cake diet.After pelleting,hemp seed hulls were added to the Hemp Hulls diet at Grønhøj Research Station via the feed facility Spotmix.No segregation of the hemp seed hulls from the pellets was observed.It was assumed that hemp seed hulls,added to the Hemp Hulls diet,do not contribute with energy or nutrients.The diets were optimised according to the nutritional recommendations for growing-finishing pigs from 30 to 105 kg BW and contained the same amount of energy per kg feed in all groups.The hemp seed products were delivered by Nørding Oil.A wet sieve analysis was performed to determine particle size distribution using an electronic sieve shaker ,and the diets were analysed for major nutritional composition at Eurofins Steins Laboratory A/S.Saliva samples were collected from clinically healthy pigs on the day before slaughter by allowing the pigs to chew on a gauze until it was soaked.The sampling started in the morning and finished after noon.No gustatory stimulation was used before saliva sampling.The gauze units with saliva were put into falcon tubes and immediately placed on ice.After completing the sampling,the saliva samples were transported to the laboratory at Aarhus University,Foulum,Denmark and processed immediately.Saliva was extracted from the gauze units by centrifugation at 10 000g for 10 min at 4 C.The saliva samples were stored at 20 C until sample preparation and analysis.Immediately after slaughter,drying racks stomachs were collected and subsequently transported to Laboratory for Swine Diseases,Kjellerup,Denmark for examination.
At the laboratory,the stomach content was collected to assess the consistency of the stomach content based on the solid particle sedimentation percentage.The stomachs were washed and scored using an ulcer index scale for changes from 0 to 8 and a total index scale from 0 to 10,which includes chronic changes,i.e.,scar tissue and oesophageal strictures.The saliva samples for metabolomics analysis were selected based on the ulcer index scores since we were interested in examining possible biomarkers for acute gastric ulcers and not for chronic changes,as these changes are not expected to result in biomarker responses at this stage.The groups were established as follows: healthy pigs: ulcer index score 0; pigs with hyperkeratosis : ulcer index score in the range of 1–3; pigs with active ulcers: ulcer index score in the range of 6–8.In the present study,the four experimental diets were formulated to create the following expected differences: the meal and pelleted diets were selected based on previous research revealing differences in prevalence of gastric ulcers ,and were formulated to explore potential salivary biomarkers for gastric ulceration.The hemp diets were formulated to investigate the potentially anti-inflammatory effect of hemp and the swelling capacity ,which are two mechanisms considered important to prevent gastric ulcers.The higher prevalence of gastric ulcers observed in pigs provided pelleted feed compared to pigs provided meal feed is in accordance to what other studies have reported.However,the odds of having severe gastric ulcers tended to be lower in pigs fed Hemp Hulls compared to pigs fed Pellets,suggesting that hemp seed hulls might have a positive effect in the prevention of severe gastric ulcers.The solid phase percentage of the gastric content was higher in pigs fed Meal compared to pigs fed Pellets,which is in agreement with other studies.In addition,the solid phase percentage of the gastric content in pigs fed Hemp Hulls was higher compared to that of pigs fed Pellets,indicating that the addition of hemp seed hulls to a pelleted diet results in a more solid stomach content,presumably due to the swelling capacity of hemp seed hulls.The feed conversion ratio was poorer in pigs provided meal feed compared to pigs provided pelleted feed,which is in line with the literature.
The main reason for this is considered to be the lower nutrient digestibility of coarsely ground diets due to impaired accessibility of endogenous enzymes to the substrates in the feed particles.Another contributing factor can be the higher feed waste when feeding meal feed as compared to pelleted feed.The main objective of this study was to examine a possible correlation between salivary metabolites and ulcer index score and to identify metabolites,which could be used as potential biomarkers for gastric ulceration in pigs.The argument for combining the healthy group and HK group is that the pigs in the group with HK are assessed as being in the very early stage of ulceration.In the stage of HK,no loss of epithelia has occurred,and thus no ulcer development has taken place yet.Instead,the stratified squamous epithelium around oesophagus has gradually changed structure observed as thickening and roughening of the epithelium.Thus,in the stage of HK,we do not expect a strong inflammatory response or effect on salivary metabolites.Several lipid metabolites including different oxylipins were detected in saliva as discriminating metabolites between the healthy/HK group and the group with ulcers.Lipid mediators play a role in the initiation,amplification and resolution of the inflammatory response as well as in signalling events.Oxylipins are oxidation products of polyunsaturated fatty acids,which are formed via one or more mono- or dioxygen-dependent reactions.In addition,oxylipins are important regulators and markers of a wide range of normal and pathological processes in the body.Eicosapentaenoic acid and arachidonic acid are two of the most important long-chain polyunsaturated fatty acids in terms of precursors to oxylipins with strong anti- and proinflammatory properties,respectively.The biosynthetic origin of oxylipins is still largely unknown; however,it is well established that oxylipins are found in all body tissues,urine,blood and saliva.The majority of the salivary oxylipins were tentatively identified to belong to the oxylipin group TriHOME,DHOME or DiHODE,and are all derivatives of linoleic acid.The linoleic acid metabolism,including the pathways of the identified salivary oxylipins that derive from linoleic acid,is shown in Fig.3 developed by KEGG.
Linoleic acid is an essential n-6 fatty acid and is a precursor of arachidonic acid.Arachidonic acid can via the cyclooxygenase pathway be converted into eicosanoids including prostaglandins.Prostaglandins,such as prostaglandin E2,are known to play a role in the induction of acute inflammation as well as in the protection and healing of gastric ulcers.Production of prostaglandins is generally low in uninflamed tissue but increases rapidly in acute inflammation before the recruitment of immune cells.In this study,a prostaglandin metabolite,13,14-dihydro-15-keto-tetranor prostaglandin F1a,was tentatively identified and found to be increased in saliva of pigs with gastric ulcers; however,the increase was not significant.The presence of a prostaglandin metabolite reflects the biosynthesis of prostaglandins via the cyclooxygenase pathway.Linoleic acid shares a common metabolic pathway with alpha-linolenic acid.Thus,linoleic acid competes with alphalinolenic acid in binding to the enzyme D6-desaturase to direct the metabolism towards more pro-inflammatory n-6 polyunsaturated fatty acid-derived compounds such as arachidonic acid and less anti-inflammatory eicosapentaenoic acid.In addition,arachidonic acid competes with eicosapentaenoic acid as substrates for lipoxygenase and cyclooxygenase to produce immunoregulatory eicosanoids,including prostaglandins.In contrast to arachidonic acid-derived eicosanoids which are mainly pro-inflammatory,eicosapentaenoic acid-derived eicosanoids have anti-inflammatory effects to rebuild immune homeostasis such as by reducing the expression of interleukins.Pigs with acute gastric ulcers might have greater synthesis of pro-inflammatory prostaglandins than healthy pigs to induce an inflammatory response,restore tissue structure and physiological function,and eventually heal the ulcer.Acute inflammation may favour the conversion of linoleic acid to arachidonic acid and eventually the generation of pro-inflammatory prostaglandins,and thereby the conversion of alpha-linolenic acid to eicosapentaenoic acid via enzyme D6- desaturase will be reduced.Instead,alpha-linolenic acid might be converted to other metabolites including oxylipins such as 2R-HpOTrE.This could explain the significantly higher concentration of the alpha-linolenic acid-derived oxylipin,2R-HpOTrE,observed in pigs with acute gastric ulcers compared to healthy pigs.In addition,an upregulation of the pathway of linoleic acid into arachidonic acid could explain the lower concentrations of linoleic acid-derived oxylipins,including 9,12,13-TriHOME,9,10-DHOME/12,13-DHOME and 9,10-DiHODE/15,16-DiHOD E/12,13-DiHODE,observed in pigs with gastric ulcers compared to healthy pigs.Furthermore,the requirement for production of pro-inflammatory prostaglandins is probably lower in healthy pigs than in pigs with acute gastric ulcers.Thus,linoleic acid might mainly be converted into oxylipins in healthy pigs.
In addition,linoleic acid-derived oxylipins might have antiinflammatory effects and may be beneficial in terms of gastric health.The pelleting process might affect the lipid composition in the diet due to the potential oxidation of fatty acids.Unfortunately,fatty acid composition was not analysed in the experimental diets,cannabis drying and thus it was not possible to demonstrate that the significant lower level of linoleic acid-derived oxylipins observed in pigs with acute gastric ulcers provided pelleted feed was not caused by any differences in fatty acid concentrations in the diets.Metabolite identification revealed metabolites deriving from the feed.Since the pigs were fed ad libitum,and collection of saliva samples was performed without cleaning the mouth of the pigs,the saliva samples were most likely contaminated with feed residues.The possible contaminants from feed residues were considered during the metabolomics analysis,and multiple cleaning steps were performed to extract these residues.Contamination with feed residues cannot be fully avoided; however,to minimise feed residues in saliva samples,deprivation of feed several hours before sampling could be an opportunity to minimise feed residuals in saliva samples as well as performing a cleaning procedure of the mouth prior to sampling.Deprivation of feed prior to saliva sampling,however,was not possible in this study,as it might affect the health status of the stomach.The metabolomics analysisrevealed the presence of a disaccharide,which might be sucrose,lactose or isomaltose,in the saliva of pigs,which probably derives from the feed as well as azelaic acid,which is a dicarboxylic acid found in cereals,such as wheat,barley and rye.Daidzein,a metabolite from soybean meal,was identified as a metabolite,which contributed to the separation of the groups,found at significantly higher levels in healthy/HK pigs compared to pigs with ulcers.However,since the majority of the pigs fed the Meal diet were assessed as healthy,the increased level of daidzein in healthy/HK pigs might be explained by the significantly greater feed intake observed in pigs provided meal feed compared to pigs provided pelleted feed.In addition,the structure of the Meal diet might leave more residues in the mouth.Furthermore,a partial least-squares discriminant analysis model for healthy pigs fed the Meal diet vs pigs with ulcers fed the Pellets diet was built and showed a good separation between groups.However,since diet and health status were confounded,the model was excluded.Another metabolite,which probably derived from the feed,floionolic acid,found at a significantly higher concentration in healthy/HK pigs compared to pigs with gastric ulcers,is a longchain fatty acid found in vegetable fats and oils.This difference might also be explained by the greater feed intake in pigs provided meal feed compared to pigs provided pelleted feed.A sulphated compound was found to be significantly increased in healthy/HK pigs compared to pigs with gastric ulcers.Sulphated compounds found in saliva are probably a result of the metabolism.Since salivary glands with high permeability are surrounded by capillaries,saliva contains a variety of locally synthesised compounds as well as systemic compounds.Thus,it could be speculated that sulphated compounds produced elsewhere in the body,including the liver and kidney,possibly enter the blood circulation and eventually end up in the salivary glands where they are secreted in saliva.However,the role of sulphated compounds in gastric ulceration remains to be determined.In the current study,the pigs used for saliva sampling were not clinically diseased and therefore we did not expect them to suffer from inflammation due to other diseases.Thus,we assume that the identified salivary oxylipins were a result of an inflammatory response caused by gastric ulcers.However,the possible role of oxylipins in inflammation associated to gastric ulceration needs further examination.The development of industrial methods for production of bio-based products has been gaining attention in recent years.For the production of such products,industrial hemp can be considered as one of the efficient and sustainable renewable resources in the world that has proven itself for several hundred years.Industrial hemp can be grown as a fiber,seed,or dual-purpose crop.Traditionally,it is grown for its long and strong fibers.From the fibers production process,large amount of short woody fibers ,called shives or hurds,are formed.These shives are considered as solid waste.Therefore,shives are mainly used as a high-performance bedding material for animals.