1. Introduction
The swine production system entails proper nutrition management based on less expensive raw materials, which contributes significantly to profitability. Due to fluctuating prices and a trend toward the partial or total substitution of classical feedstuffs, there is a growing interest in locating and utilizing available resources. Due to their availability and low cost, many types of alternative fibrous feedstuffs (e.g., byproducts) have been used in Romania and other parts of the world for a long time. The residual meals obtained after cold-pressing oil-rich seeds are rich in nutrients and bioactive compounds, making them suitable for animal feed. Diversifying the plant source matrix by using byproducts as production residues could result in lower feed costs, improved health, and a cleaner environment.
Fibers, positively influence animal gut health, although the animal’s growth performance falls short of its full potential [
1,
2].
The high cost of pig feed (65–70% of the total production cost) has led to increased interest in replacing or supplementing the classical soybean meal with more affordable and accessible ingredients such as sunflower meal and rapeseed meal. The issue is related to additional costs due to processing equipment, feed wastage, and increased risk to animal health, as well as reduced performance as a result of byproduct variability and suitability for pig feed. Although the proportion of lysine in sunflower meal is lower than that in soybean meal, it is increasingly being employed as a protein source in growing-finishing pig diets. Nevertheless, as the food sector has grown, a variety of byproducts have become available and can be used to substitute sunflower meal, particularly by farmers who cannot obtain them easily.
Two lesser-known byproducts as feed sources for pigs are mustard meal and grapeseed meal, resulting from the oil extraction industry. White mustard (
Sinapis alba) is a historical culture from the Hellenistic and Roman times commonly used in the medicinal treatment or as a spice or edible oil [
3]. Mustard seed has high oil content ranging between 38% and 44% [
4], along with a protein level in excess of 28% and a fiber level of ~14% [
3]. Previous research has focused on using mustard byproducts as a protein source [
3] as opposed to a source of bioactive substances. Grape (
Vitis vinifera) is a widely grown fruit culture. Previous studies have focused on the metabolic profile and the anti-inflammatory properties of grape pomace bio-compounds [
5,
6]. Antioxidant mechanisms resulting from the polyphenol concentration [
7] and their role in decreasing toxicity caused by aflatoxin B1 in the mesenteric lymph nodes [
8] have also been investigated. Research results indicates dietary fibrous ingredients impact the production of VFAs, also named short-chain fatty acids (SCFA). VFAs are important organic metabolites containing a maximum of five carbons, derived from the microbial anaerobic fermentation of dietary fiber, resistant starch, and amino acids. Their production depends on the degree of fermentation in the digestive tract [
9,
10,
11]. The most common VFAs are acetate (C
2H
4O
2), propionate (C
3H
6O
2), and butyrate (C
4H
8O
2). Additionally, branched SCFAs (BCFA) are end-products of aliphatic amino acid catabolism (leucine, isoleucine, and valine) produced in lower amounts. The predominant BCFAs, isobutyrate (C
4H
8O
2) and isovalerate (C
5H
10O
2), have attracted less attention.
In classical studies, it has been shown that considerable amounts of VFAs are produced in the cecum and colon of pigs, whereas, in the stomach and the small intestine, the VFA concentration is lower [
1,
9,
12], albeit slightly higher compared to rats and rabbits [
13]. Recently, Li et al. [
14] showed that the fermentative process of some fiber components could start in the small intestine, while Montoya et al. [
12] identified, in growing pigs, soluble kiwifruit fiber levels close to 80% fermenting in the distal section of the small intestine. Furthermore, in growing pigs used as human models, 25–30% of undigested material was fermented in the ileum upon employing a high-fiber human-type diet. Bugaut [
15] indicated that VFAs contribute to the basal metabolic requirement as follows: 15–28% for the total digestive tract 30% for the whole large intestine, and 1.9–2.7% in the cecum. Philippe and Nicks [
16] specified that the fermentative capacity of the hindgut and fiber level could change the production of enteric methane (E-CH
4).
A number of studies have highlighted the concentration of VFAs in different sections of the gastrointestinal tract in relation to the microflora community [
9,
17,
18,
19]. Moreover, E-CH
4 assessment has been the subject of numerous studies, especially in ruminants [
20,
21,
22,
23,
24]. Dämmgen et al. [
25] developed a model to quantify E-CH
4 emissions from pigs, while Philippe and Nicks [
16] described the processes involved in E-CH
4 emissions and reviewed the emission factors (including diets) and their effects. As far as we know, the influence of increased dietary fiber level on the concentration of SCFAs and BCFAs in the ileum and cecum, resulting from the use of a mixture of mustard meal and grapeseed meal as a replacement for sunflower meal, has not been studied.
Therefore, the aim of this study was to investigate the changes in VFA concentration in the ileum compared to the cecum, as well as their relationship with the microbial community and E-CH4 level, as a function of the effect of dietary fiber level in pigs fed different types of oil industry byproducts.
2. Materials and Methods
2.1. Animal and Housing
The biological protocol was endorsed by the INCDBNA Balotesti Ethical Committee (no. 7976/12/2020). All procedures and methods applied in the experiments were carried out at the Experimental Biobase of INCDBNA Balotesti according to Romanian Law no. 199/2018, in compliance with EU Directive 2010/63/EU for animal experiments.
The experiment was conducted over 38 days on 70 growing healthy Topigs hybrid pigs (female Large White × Hybrid (Large White × Pietrain) × male Talent (mainly Duroc), in an experimental modern indoor facility (21 °C, 60% rH, cage size 2.6 × 2.3 m). The pigs, 56 ± 3 days old, 20.96 ± 0.26 kg initial body weight (BW), were ear-tagged and distributed completely randomly into four mixed-sex pens per group, 35 pigs per group, with a similar sex ratio (18 castrated male and 17 female pigs in each group). The group size was determined according to Charan and Kantharia [
26].
2.2. Treatments
Two dietary treatments were formulated: (i) SM diet based on sunflower meal, with a 6.23% level of dietary fiber (Topigs guidance); (ii) MG diet based on MG-mixt, with a 7.28% level of dietary fiber. The sunflower meal was totally replaced by MG-mixt in the MG diet (
Table 1).
The mustard and grapeseed meal byproducts that resulted after oil extraction were delivered by 2E-Prod SRL, Alexandria, Romania. The price of acquisition was 24% less than that of sunflower meal. The final formula of the MG-mixt product (7:8, w/w) was obtained after several simulations of different proportions of the two byproducts. The MG-mixt was processed separately, dosed, and ground in a hammer mill. The resulting product was placed in a mixer for homogenization (4–6 min), and then compressed at 80 °C using a 6 mm diameter pellet press in the presence of a binder (a mixture between water + molasses in equal parts; PLT 100). Before being ground and mixed into the compound feed, the crumbly pellets were sampled for chemical analysis.
The weight of the two ingredients in the mixture was set in such a way as to cover the specific nutrient requirements as in the case of the SM diet. Whereas dietary ME and net energy (NE), as well as limiting crude amino acids were similar between groups, fiber, comprising crude fiber, neutral-detergent fiber (NDF), acid detergent fiber (ADF), and lignin (ADL), were higher in diets with MG-mixt added. Digestible amino acids were lower in the MG-mixt group. The combination MG-mixt led to a 22.1% higher crude fiber level compared to sunflower meal (g·kg DM
−1;
Table 2). The feed intake and leftovers were recorded daily.
The dry matter and fiber chemical composition of MG-mixt and the sunflower meal are shown in
Table 2. The levels of ADL and insoluble hemicellulose were calculated.
2.3. Measurements
The individual BW was determined using an electronic scale (26 and 64 days after weaning), covering a live weight range from 20 to 50 kg. The pigs fasted overnight before weighing. A total of 40 pigs randomly selected were slaughtered for biological samples (20 pigs per group, 20 female and 20 males; 37± 3.2. kg carcass) at the end of the trial. After stunning and exsanguination, the correctly labeled pigs were transported to the necropsy laboratory. The abdominal cavity was opened, and the intestinal mass was removed without perforation of the intestines. From each pig’s slaughtered, the entire cecum and approximately 15 cm of ileum distal part (~5 cm anterior to the ileocecal junction) were dissected for collecting digesta.
This study presents the value of the carcass dressing percentage. Immediately after euthanasia, the intestine content was rapidly removed and collected into sterile plastic bags, before being transported to the analytical laboratory on an ice bed in a special refrigerated container.
2.4. Chemical Analyses
2.4.1. Gross Chemical Composition
The gross chemical composition of ingredients and diets was determined in duplicate using standardized methods according to European Commission (EC) Regulation no. 152 (2009). Crude fiber (CF) extraction was performed by the intermediate filtration method, according to European Commission (EC) Regulation no. 152 (2009) and the standard SR EN ISO 6865:2002. According to Weende’s method, sugar and starch were extracted first by acid hydrolysis with H2SO4, then proteins, some hemicellulose, and lignin were removed by alkaline hydrolysis with NaOH. The residue was filtered, dried, calcined, and weighed. For neutral and acid detergent fiber (NDF, ADF) determination, Van Soest extractions were performed, according to SR EN ISO 16472:2006 and SR EN ISO 13906:2008. The analyses were carried out using the Raw Fiber Extractor FIWE 6 (Velp Scientifica, Usmate, Italy). The ADL was calculated by the difference between ADF, crude fiber, and ash.
2.4.2. Ileum and Cecum Microflora Analyses
Tenfold serial dilutions of 1 g sample content from the distal ileum and cecum digesta were homogenized with 7 mL of Brain Heart Infusion broth (Oxoid Basingstoke, Hampshire, UK) supplemented with 2 mL of glycerol and frozen at −20 °C until the analysis. After defrosting samples, decimal dilutions in phosphate-buffered saline (Dulbecco A; Oxoid Livingstone Ltd., London, England) were conducted. The samples were assessed for lactic acid bacteria (LAB),
Escherichia coli (
E. coli; biotype β-hemolytic),
Salmonella spp.,
Clostridium spp., coliform count,
Enterococcus spp., and Enterobacteriaceae. The LAB were cultured on de Man, Rogosa, and Sharpe agar (MRS, Oxoid CM0361) incubated in anaerobic conditions at 37 °C for 48 h (Thermo Scientific jar with Anaerogen 2.5 L, Oxoid Basingstoke, Hampshire, UK). Coliforms were cultured on MacConkey agar (Oxoid CM0007) incubated aerobically at 37 °C for 24 h.
E. coli (biotype β-hemolytic) was analyzed as previously described by [
27].
Clostridium spp. and
Enterococcus spp. were cultured anaerobically at 37 °C for 48 h on Reinforced Clostridia Agar (Oxoid CM0151) and Slanetz–Bartley agar (Oxoid CM0377), respectively. Enterobacteriaceae and
Salmonella spp. were enumerated on Oxoid selective medium (Eosin Methylene Blue Agar, Levine CM 0069, Basingstoke, UK) and
Salmonella–Shigella agar (CM0099) by incubation at 37 °C for 48 h in aerobic conditions. Each sample had three replicates. The microflora level was expressed as log
10 CFU· g
−1 content.
2.4.3. Volatile Fatty Acid Quantification and pH Analysis of Ileum and Cecum Content
The VFAs were quantified by gas chromatography in water extracts of distal ileum and cecum content (five replicates/sample). Briefly, the samples were mixed with distilled water in a proportion of 0.7:1 (w/w), and then centrifuged at 13,000 × g for 15 min. A sample volume of 1 μL from the centrifuged extract was injected under split mode into a gas chromatograph (Varian, 430-GC) equipped with a capillary column Elite-FFAP with a length of 30 m, an inner diameter of 320 μm, and a film thickness of 0.25 μm (Perkin Elmer, Seattle, Washington, USA). The carrier gas was hydrogen, with a flow rate of 1.5 mL/min. The injector temperature was set to 250 °C, and the split rate was 1:40. The flame ionization detector (FID) temperature was set to 200 °C, while the column oven temperature was set to 110 °C. The oven temperature was increased to 170 °C at 12 °C/min, where it was held for 9.5 min. The analysis time was 10 min. The sample concentration was calculated using a standard commercial mixture of VFA (CRM46975, Supelco, St. Louis, MO, USA). The final results were expressed as μmol/g.
To measure the pH, 1 g of fresh sample (ileum and cecum content of each pig) was collected and transferred into 9 mL of distilled water (1:10 dilution, w/v). The pH value was measured (mean of three readings) using a portable pH meter (pH 7 + DHS, XS Instruments, Carpi, MO, Italy). After each pH measurement, the electrode was carefully washed with water and calibrated between each animal.
2.5. E-CH4 Prediction
The E-CH
4 level equation developed by Philippe and Nicks [
16], expressed as carbon dioxide equivalent (g CO
2 eq·day
−1), is as follows:
where dRes (g/day) refers to digestible residues calculated according to INRA-AFZ (2004), as quoted by Philippe and Nick [
16]. The theoretical digestibility coefficient was obtained from the IBNA Balotesti database.
2.6. Calculations
Carcass weight was used to calculate the dressing percentage (carcass yield, %). The indicators Kleiber ratio (KR) and relative growth rate (RGR, %) were calculated using the following equations [
28]:
where ADG is the average daily gain and MBW
0.75 is the metabolic weight;
The cost of each diet, the feed intake, and the ADG were all taken into account when calculating economic efficiency.
For energy retention (ER) calculation (MJ·day
−1), the difference between
ME intake and heat production (HP) was used [
29].
The HP was calculated according to Aarnink’s equation [
30].
where
MEm (MJ) is the energy for maintenance (
MEm = 0.4398 × BW
0.75) [
29], and
kY is the efficiency of protein and lipid retention [
29].
The molar percentage for the main SCFAs (acetate, propionate, and butyrate) was calculated by dividing the total amount of VFAs (including valerate and BCFAs) by the concentration of each VFA.
The EvaPig tool, version 2.0.3.2 (2020), developed by the French National Institute for Agricultural Research, METEX NØØVISTAGO, and the French Association of Zootechnie, was used to calculate the energy, amino acid, and phosphorus values of pig feed.
2.7. Statistical Analyses
The descriptive statistics were calculated using IBM SPSS (2011). Data were analyzed using two-way ANOVA. In our model, the main effects were considered the diets (differing in fiber content) and the gut sections (ileum and cecum). The data distribution was verified by the Shapiro–Wilk test. Except for body weight and ADG, each pen was considered as an experimental unit irrespective of the analyses or measurements performed. The impact was considered as statistically significant at p ≤ 0.05 and as a trend at 0.10 > p > 0.05. The effect of replicates was omitted in the analysis due to their insignificance (p > 0.05).
The Pearson or nonparametric Spearman correlation was applied to evaluate the measure of bivariate association. The interpretation of correlation coefficients followed [
31]. Regression analyses were used to assess the strength of the relationship among total VFAs, predicted E-CH
4, bacteria, intake of fiber and associated fractions, and amino acid intake.
4. Discussion
Fiber can be considered an important component of the pig diets that attracted attention due to their health advantages. Fiber fermentation serves as the source of energy for bacterial communities [
37], influencing several physiological processes by filling the intestinal tract and producing gases and VFAs known as physiologically active compounds [
38]. Many studies have previously investigated VFAs levels in different segments of human and animal gastrointestinal tracts [
9,
39], but there is no information on the concentration of VFAs in the ileum and cecum of adult pigs fed mustard and grapeseed oil meals, which increase the level of dietary fiber.
Our study confirmed the theory of fiber-producing satiety through decreasing feed intake by delaying gastric emptying. As a result, despite the fact that pigs have increased their abilities to use dietary fiber across their life cycle, our data have shown a decrease in feed intake in groups fed fiber-rich diets, resulting in a slight decline in growth performance. In fact, a high fiber level has often adverse effects on the digestibility of other nutrients presumably linked to the rate of passage of digesta through the gastrointestinal tract [
14]. However, this lower performance in the MG group was accompanied by a decrease in the feeding prices and cost per kilogram of daily body gain. This result is consistent with previous studies [
14,
38] mentioning that fibrous byproducts are frequently used in many countries, although fiber-rich diets do not maximize and sometimes inhibit performance. Evidence of the nutritional potential of black and yellow mustard seed meal was also provided by Sarker et al., [
3].
Pigs’ growth performance and their intestinal health are linked to the intestinal microbial community. The intestine hosts a complex bacterial community (Firmicutes, Proteobacteria, etc.), releasing metabolites important for health. However, the small intestine hosts fewer bacteria than the large intestine; consequently, the fermentation process occurs at a lower rate in this gastrointestinal segment. In this study, a diverse community of microflora, within the reference limits [
32,
33,
34,
35,
36], was identified in the ileum and cecum. Wang et al. [
40] specified that Firmicutes are the most dominant bacteria, followed by Bacteroidetes across each age stage. Our results support this theory. Thus, as dominant phyla in both gut sections, we identified Firmicutes (
Lactobacillus spp.,
Enterococcus spp.,
Clostridium, and
Staphylococcus spp.), as well as Proteobacteria (
E. coli and coliforms related to Enterobacteriaceae) to a lower extent. We have observed that data pooled across all Firmicutes bacteria in each gut section indicated that their level was higher in the cecum than in the ileum. This is consistent with the explanation given by Jaworski and Stein [
37] who consider that the cecum plays a significant role in fiber fermentation. This should also explain the stimulating effect recorded for pigs fed the MG diet; however, in contrast, the data pooled across Proteobacteria showed a lower concentration in the cecum vs. the ileum. Hence, consumption of MG-mixt influenced the level of
E. coli bacteria. According to the previous study of Umu et al. [
41],
Lactobacillus bacteria almost disappear after the growing period, being negatively correlated with age. At the genus level, we found
Lactobacillus spp. as the major bacteria in both the ileum and the cecum digesta, followed by
Clostridium spp. in the growing pigs category considered in this work. Furthermore, we noticed that the fiber-rich diet in the MG group had a significantly positive effect on
Lactobacillus spp. Hence, the higher level in the ileum confirms the theory that the level of
Lactobacillus spp. is increased at a lower pH [
42]; however, this theory was found to be invalid in the cecum where both the pH and the
Lactobacillus level were higher. This may have been influenced by the type of fiber, their physicochemical characteristics, and the lignification grade, which all support diverse microflora. As mentioned previously by Gao et al. [
43], a high pH level increases intestinal osmotic pressure, whereas the digestion function decreases with changes in microflora composition. In contrast,
E. coli exhibited the lowest density, regardless of gut section or type of pig feed. However, the increase in ileal
Lactobacillus concentration in fiber-rich diets was accompanied by an increase in
E. coli levels. This could have been due to the low pH, but this correlation was not confirmed in the cecum. In contrast with the results obtained by Franklin et al. [
42], our data revealed a higher level of
E. coli. Many
E. coli species are harmless due to the presence of bacteriocins that prevent pathogenic bacteria from colonizing the gut. However, according to Schierack et al. [
44],
E. coli are among the most pathogenic bacteria dominating most samples from the gastrointestinal tract but not from feces [
45]. Enterobacteriaceae, including
E. coli, coliforms, and
Salmonella, are known as pathogenic bacteria [
46].
The higher level of the total bacteria identified in this study in pigs fed a fiber-rich diet, influenced the total VFAs. This is consistent with previous findings [
47], where alfalfa fiber was administered to 28–48-days-old pigs. The intensity of the fermentative process at the cecum converted dietary fiber into VFAs in a higher concentration than the ileum (more than double). Likewise, we found a total concentration of VFAs higher in the distal ileum than in the cecum of the fiber-rich fed group. The insignificant increase in total VFAs in the ileum of MG-fed pigs was especially due to the concentrations of acetic acid and propionic acid, as the concentration of both these VFAs decreased in the cecum. In contrast, the cecum’s elevated butyrate proportions were noted, maybe due to the cecum microflora’s metabolism and acidic fermentation.
Beneficial bacteria regulate the pH of the intestine. In the current investigation, a lower pH was linked to a higher generation of VFAs measured in the cecum. Acetic acid was the predominant VFA found both in the ileum and cecum. While the molar proportion of acetic acid decreased in the cecum, the concentration of propionate increased significantly. Results pointed toward a significant increase in propionic acid in pigs fed a fiber-rich diet probably due also to the pH level and associated bacteria. On the other hand, we observed a nonsignificant increase in butyrate concentration in the cecum as an effect of the fiber-rich diet. These data support the results obtained by Heinritz et al. [
17], despite a reduced dietary fat level in a group fed a fiber-rich diet. The concentration of total BCFAs was only significantly impacted in the ileum.
The lower pH in the cecum inhibited the concentration of E. coli but did not affect that of Clostridium bacteria, which are known to be acid-sensitive bacteria. In the cecum, the pH level was acidic, whereas it was near neutral in the ileum.
The Spearman correlations were strong between Clostridium spp. and total VFAs (rho = 0.65) and total BCFAs (rho = 0.71) due to their association with acetic acid and propionic acid (rho > 0.6). Although VFAs are known as possible inhibitors of certain pathogen bacteria, unexpected increases in total VFAs and E. coli were noticed in the MG-mixt group.
No support can be found in the literature regarding the low correlation between VFAs and
Lactobacillus. A possible explanation, given with caution, could be the lower intensity of the microbial fermentation process in the ileum and the difference in fermentability rate of the three fiber types. Many studies are contradictory; the amount and type of substrate, the source of fiber fractions, and their digestibility are essential factors. However, substantial fermentation of soluble dietary fiber was found in the pig small intestine by Jha and Leterme [
1].
Using regression analyses, we found: Clostridium bacteria, pH, and fiber intake to be potential good predictors for the concentration of total VFAs, with an R-square value of 0.65 (p < 0.0001), indicating that 65% of the variability of total VFAs can be explained by our predictors. Lysine intake was also identified as a good predictor (β coefficient = −15.12).
The production of E-CH
4 was altered but not significantly. To our knowledge, a higher efficacy in lowering the E-CH
4 level, using nutritional tools, has not been specified in the literature. In this study, by developing a model to predict E-CH
4 using our experimental data integrated into equations from the literature, we identified a significant decline in the MG-fed group. The values obtained were close to those mentioned by Guingand et al. [
48], higher than those obtained by Dong et al. [
49], and lower than those reported in other studies [
50,
51], as quoted in Philippe and Nicks [
16], which took into consideration the total CH
4 (enteric and from manure). Our values ranged between 0.26 and 1 kg CO
2 eq·LU
−1. DMI and ER significantly influenced the E-CH
4 level.
Upon applying Spearman correlation, we found a significant negative relationship of E-CH
4 with total VFAs (rho = −0.462,
p = 0.04), but a positive one with pH (rho = 0.227,
p = 0.03), in line with the theory of Yadah and Jha [
36] that the negative correlation between pH and VFAs can have a negative effect on E-CH
4 due to the decrease in methanogens and protozoa. The regression analysis revealed total VFAs and their major components, as well as ADF and lysine intake, as potential predictors for the E-CH
4 level.