1. Introduction
The pig is a monogastric animal with endogenous enzymes playing a crucial role in its digestive process [
1]. It is therefore essential to provide pigs with high-quality feed that provides nutrients in a readily available form for the enzymes to digest. Apart from the composition of the feed, different feed structures and forms, such as particle size (PS), extrusion, pellet, flake and cooking, significantly affect the efficiency of feed and nutrient utilization. Therefore, they should be optimized for nutrient absorption [
2]. Ensuring the provision of adequate essential nutrients to meet the nutritional requirements of pigs presents a challenge due to diverse feed processing techniques and ingredient variations that can significantly impact nutrient utilization [
3]. In addition, these effects may vary depending on the growth stage of the pigs [
4]. However, finely ground materials can have a negative impact, particularly on gastric ulcers [
5].
Barley and wheat are quantitatively the most important components of diets for growing pigs in Denmark and most other European countries. Both cereals have a high concentration of carbohydrates, predominantly as polysaccharides, including starch and non-starch polysaccharides (NSP). The principal polysaccharides in cereal NSP are arabinoxylans (AX), cellulose and mixed linked (1- > 3; 1- > 4)-β-D-glucan (β-glucan), which, together with lignin, make up most of the cell wall and are referred to as dietary fiber (DF). The composition of the cell walls varies between the cellular tissues within the cereal grain and among similar tissues of different grains [
6]. Barley has a husk layer that may remain even after the threshing process, whereas wheat husk is lost during the threshing of wheat, thus the DF content is approximately 50% higher in barley than in wheat [
7]. These differences in DF content, which are counteracted by a higher starch concentration in wheat compared to barley [
7], are responsible for the higher apparent total tract digestibility (ATTD) of wheat diets compared to barley diets in pigs [
8].
Grinding is a physical process that reduces PS and increases the surface area, allowing better contact with digestive enzyme, which improves apparent ileal digestibility (AID) and ATTD [
9]. This enables optimal nutrient utilization and enhances animal performance. Furthermore, particle size has been associated with changes in the microbial population, and coarse particles stimulate microbial fermentation of DF, which contributes to improved intestinal health by reducing ulceration and
E. coli adhesion to the mucosa in the small intestine [
10]. The hypothesis of the present study was that finely ground feed improves the digestibility of nutrients and the concentration of short-chain fatty acids (SCFA) in the digesta.
The objective of this study was to investigate the influence of PS of barley and wheat in diets on AID, ATTD, recovery of nutrients and DF, mean transit time (MTT) in the small and large intestines, and SCFA concentration and composition in growing pigs.
2. Materials and Methods
The study complied with the guidelines of the Danish Ministry of Justice, Act no. 474 of 15 May 2014, concerning experiments with animals and the care of experimental animals, as stipulated in the executive order no. 12 of 7 January 2016.
2.1. Diets
Four experimental diets that differed in cereal type (barley or wheat) and PS (fine or coarse) were used (
Table 1). The grain components were ground for diets with a fine PS. To achieve approximately the same PS in the ground diets irrespective of grain type, it was found that barley should be ground using a 3 mm sieve and wheat should be ground using a 4.5 mm sieve in a hammer mill. The barley and wheat used in the coarse diets were rolled before inclusion. It was possible to produce rolled feed without the risk of whole grains in the feed. Chromic oxide (2 g/kg diet) was included in the diets as a marker for the determination of the AID and ATTD of nutrients and energy, and MTT.
2.2. Nimals and Experimental Designs
Two animal experiments were conducted to perform distinct analyses. The pigs (DanBred Genetics, Ballerup, Denmark) used in both experiments were from the pig herd of Aarhus University, Denmark.
2.3. Experiment 1
The experiment was conducted according to a 5 × 5 Latin square design, using five crossbred barrows [initial BW 35.9 ± 1.5 kg; (Danish Landrace × Yorkshire) × Duroc]. The pigs were fed five different diets, including the four experimental diets and a standard diet, over five periods, each with a duration of two weeks. However, the standard diet was not part of this study, and the results from the standard diet were not included in the statistical analyses. The pigs were fitted with a simple T-cannula at the ileum, approximately 15 cm anterior to the ileocecal junction, following previously outlined procedures [
11]. The pigs were fed the same amount of daily net energy, and the amount of feed was adjusted throughout the experiment to match the body weight of the pigs. The feed was provided in three meals of equal size at 07:00, 15:00 and 22:00 h, and the meal size was gradually increased following feeding units for growing pigs [
12]. Each experimental period consisted of 14 days: 8 days of adaptation to the experimental diets, followed by 3 days of feces collection and 3 days of ileal digesta collection. The pigs were placed in stainless steel metabolic crates on the last day of the adaptation period. Feces were collected from 07:00 to 15:00 on days 9 to 11. Digesta were collected from 07:00 to 15:00 on days 12 to 14. This approach has been shown to provide a representative sample of digesta that encompasses postprandial changes in nutrient flow [
13]. During the collection period, digesta were collected every hour, weighed and immediately frozen (−20 °C). After 6 day collection, period the pigs were returned to their pens for 8 days of adaptation to the next experimental diet.
2.4. Experiment 2
This study involved 32 castrated male pigs with an initial weight of 30.8 ± 1.3 kg [(Danish Landrace × Yorkshire) × Duroc]. The experimental design was a randomized block design with eight blocks, each consisting of four pigs fed one of the four diets. The animals were fed equal amounts of feed on an energy basis at approximately 10% below
ad libitum feed intake. The pigs were fed twice a day, and the meal size was gradually increased from 6.16 MJ to 7.70 MJ net energy per day as the animals grew [
14]. The pigs were individually housed on a concrete floor with no bedding material.
The pigs were fed one of the four experimental diets for a period of four weeks, after which they were euthanized, and samples were collected. The animals were stunned followed by exsanguinations. The digestive tract was rapidly removed and divided into the following sections: the stomach, the small intestine, the cecum and four equal sections of the colon (Colon1, Colon2, Colon3 and Colon4). The total digesta of the small intestine, the cecum and the four colonic sections (Colon1–4) were collected and weighed. The samples were frozen and stored at −20 °C until needed for further analysis.
2.5. Chemical Analyses
All analyses were made in duplicate. Cr
2O
3, nitrogen and starch determinations were performed on wet material, while all other analyses were carried out on freeze-dried materials. SCFA was performed on freeze-dried material in Experiment 1 and on wet material in Experiment 2. The dry matter content of feed, digesta and feces was determined by drying at 105 °C until a constant weight was achieved. Protein (N × 6.25) was determined using the Kjeldahl method with a Kjell-Foss 16,200 autoanalyzer. Gross energy was determined using bomb calorimetry with a LECO AC 300 automated calorimeter system 789–500 (LECO, St Joseph, MI, USA). Fat was extracted with diethyl ether after acid hydrolysis [
15]. Cr
2O
3 content was determined using the method described by Schurch et al. [
16]. Digesta samples were analyzed for SCFA by gas chromatography as described in detail by Jensen et al. [
17].
Sugars (glucose, fructose and sucrose) and fructans in feed, ileal digesta and fecal samples were analyzed using the enzymatic-colorimetric method of Larsson and Bengtsson [
18], and the sucrose present as part of fructans was corrected as described by Bach Knudsen and Hessov [
19]. Starch was analyzed using a modified enzymatic method as described by Bach Knudsen [
7]. In feed, starch determination was also conducted without further milling preceding the analysis. In digesta and feces, starch was determined in wet and freeze-dried ground samples. Total β-glucan was determined using an enzymatic-colorimetric method [
20]. Total non-starch polysaccharides (T-NSP) and their constituent sugars were determined as alditol acetates by gas-liquid chromatography for neutral sugars and by a colorimetric method for uronic acids, as described by Bach Knudsen [
7]. Soluble NSP (S-SNP) in the starch-free residue was extracted using a phosphate buffer at neutral pH (0.2 mol/L, 100 °C, pH 7.0) [
21], and the neutral and acidic sugars in insoluble NSP (I-NSP) were analyzed as previously described [
7]. The content of cellulose was calculated as follows:
non-cellulosic polysaccharides (NCP) as:
arabinoxylan (AX) as:
and S-NSP as:
Klason lignin was measured gravimetrically as the residue resistant to 12 mol/L H
2SO
4 [
22,
23].
2.6. Calculations and Statistical Analyses
The apparent digestibility of nutrients at the terminal ileum and total tract were calculated relative to the indigestible marker (Cr
2O
3) content:
where X is the nutrient in question. X
(diet) and X
(digesta) are concentrations of specific nutrients in the diet and digesta from the terminal ileum or feces.
The quantitative flow (recovery) of nutrient X was calculated as follows:
the mean transit time in the intestinal segments was calculated as follows:
where Cr
2O
3(GI) and Cr
2O
3day are the amounts of Cr
2O
3 in the specific GI segment and the daily intake of Cr
2O
3.Before the animal experiment, a power analysis was performed using SAS JMP based on previous experience with digestibility and SCFA concentration. Based on a power level of 80% and a significance level (α) of 0.05, the minimum required sample size for the study was determined to be 5 pigs. All data were analyzed as least squares means on the Fit Model platform of SAS JMP version 15. 0. 0 (SAS Inst. Inc., Cary, NC, USA). Statistical significance was determined at p < 0.05, and trends are considered for 0.05 ≤ p < 0.10. The least square means were calculated using a post-hoc Tukey test.
The data from Experiment 1 were analyzed as a Latin square design using two-way ANOVA:
where
Yijkl is the measured dependent variable,
μ is the overall mean,
pi is the random effect of period,
αj is the effect of animal,
ck is the main effect of cereal types (
k = barley and wheat),
pl is the main effect of PS (
l = fine or coarse),
cpkl is the interaction between cereal types and PS, and
εijkl is the residual component.
The data from Experiment 2 were analyzed as a randomized block design using two-way ANOVA:
where
Y is the measured variable,
is the overall mean,
bj is the random effect of block,
ck is the main effect of cereal types (
k = barley or wheat),
sl is the main effect of PS (
l = fine or coarse),
cpkl is the interaction between cereal types and PS, and
εjkl is the residual component.
4. Discussion
Grinding feed materials to reduce their PS is a conventional way to increase the surface area of the feed particles for improved nutrient digestibility and utilization. These aspects have been studied with different ingredients such as distillers’ dried grains with solubles, corn, soybean meal and soybean hulls. These studies have generally demonstrated improved AID and ATTD in growing pigs and improved feed efficiency without affecting gastric ulceration in growing pigs fed a corn-wheat-soybean meal-based diet [
24]. However, PS distribution at low and high DF levels may influence gut health in different ways [
25], and knowledge on how finely and coarsely ground European cereal feedstuffs such as wheat and barley with contrasting DF content influence AID and ATTD of nutrients and the degradation through the large intestine is lacking. In the current study, we found that pigs fed a coarse barley diet had lower AID and ATTD of nutrients compared with other diets. For the DF and its components, the ATTD of wheat and the fine PS diets were higher compared with coarse PS diets. The main reason for the difference in NSP and DF between barley and wheat is the presence of the husk layer in barley, which accounts for 10–15% of the whole grain [
26]. In barley, the husk is tightly attached to the pericarp layer, whereas wheat loses its husk layer during threshing and therefore only has the pericarp and testa layers left as part of the grain [
27,
28]. Total wet and solid materials and other nutrients in ileal and fecal materials of pigs fed a barley coarse diet were higher compared with other diets. Furthermore, the dry matter content of the ileal digesta after feeding the coarse barley diet was higher, indicating that it was primarily the undigested residues induced by the coarse structure that caused the higher ileal digesta flow rather than differences in the physicochemical properties. The weight of digesta in the colon, however, was only influenced by cereal type. Furthermore, the MTT in pigs fed a barley coarse diet was lower than that of pigs fed other diets, suggesting that the digesta of barley coarse diet did not have enough time to be fermented in the large intestine. This phenomenon has previously been seen when the diet contained high insoluble fiber such as cellulose and insoluble NCP [
29].
In the present study, the digestibility of NSP and its the main components—cellulose, AX and β-glucan—clearly increased during passage of the large intestine but at various rates according to the property of DF components and cereal type. The cellulose digestibility in ileum was not influenced by either cereal type or PS due to the insolubility of cellulose [
30]. However, the digestibility of total NSP, AX and β-glucan was influenced by PS at the ileal level. β-glucan was already extensively degraded at the terminal ileum, as found in other studies with barley and oats [
31], and almost completely degraded in the cecum, as also found with oats [
32]. The degradation of cellulose and AX occurred more slowly and with a significant influence of PS for cellulose and for both cereal type and PS for AX. The degradation of AX was consistently lower for barley than for wheat, which is most likely caused by the structure of the AX in the husk layer and ferulic acid cross-linkages. The ferulic acid content, 731 µg/g in whole grain barley compared to 689 µg/g in whole grain wheat is known to hinder fermentation and degradation of the cell wall in the large intestine [
33,
34]. Generally, ferulic acid cross-linkages profoundly affect the degradation and fermentation of cell walls, decreasing digestibility in the small and large intestines [
35].
The cereal type, PS, and level of DF did not influence the relative weight of the small intestine, cecum, and colon and digesta weight. The final body weight of the experimental pigs was approximately 52 ± 1.5 kg. Generally, smaller pigs have lower fermentation ability in the large intestine than larger pigs [
36], and although there was a larger inflow of potentially fermentable carbohydrates to the large intestine with the barley diets, the total degradation of carbohydrates in the large intestine was only higher for the barley coarse diet. However, this had no influence on the relative weight of the large intestine. In contrast, in pigs exceeding a body weight of 100 kg, it has been shown that the fermentation of DF could lead to an increase in the weight of the small and large intestines [
36,
37].
The NSP content in the barley diets was higher compared to the wheat diets, which was expected to induce more fermentation in the large intestine, thereby increasing SCFA concentration. However, the SCFA concentration in pigs fed the wheat diets was higher than in pigs fed barley diets. This is probably related to the generally higher digesta weight in the colon of pigs fed barley diets [
38]. The husk of barley has a rigid structure, and although the DF intake was higher for the barley diets compared to the wheat diets, it was only in the case of the barley coarse diet that the total degradation of carbohydrates in the large intestine was higher (245 g/d) compared to the other diets (151–158 g/d). In addition, the mean transit time, which was significantly lower for the barley coarse compared to the other diets, also seems to have limited importance for total SCFA in the large intestine. Unlike our results, Stewart and Slavin [
38] reported that a finely ground aleurone by-product of wheat and small particle size of wheat bran showed higher SCFA concentrations in vitro compared to large particle size or coarsely grounded by-products probably due to increased accessible surface area. This difference may be caused by different microbial fermentation between wheat and barley diets, as barley β-glucan can decrease the abundance of
Bacteroides,
Porphyromonas, and
Prevotella spp, which are related to DF fermentation [
39]. In the current study, the β-glucan level in the barley diet (2.1–2.4 g/kg) is 4–5 times higher than in the wheat diets (0.4–0.5 g/kg), and thus there is a potential for higher β-glucan content in barley-based diets to impede the fermentation process with specific microbiota, thereby the PS effect became blurred.