3.1. Microbial Profiles of Pig Faeces
The number of bacterial reads in pig faeces before and after the experiment was quite similar: it varied between 35,000 and 40,000 reads among the groups. The number of species with a prevalence of at least 0.01% from the total amount of bacteria in different groups before the experiment was also similar: it varied from 400 in the RFnonV group to 473 in the SV group. The number of species with the same prevalence rate at the end of experiment varied from 340 to 387. Although the number of bacterial reads and bacterial composition among the groups was similar, we detected some distinct differences, particularly in the number of lactobacilli.
Before experiment, two bacterial genera—
Prevotella and
Lactobacillus, had the highest prevalence rates in all groups of pigs; they accounted for more than 40% of the total amount of bacterial composition (
Figure 2). The prevalence of
Prevotella varied from 21.8% to 38.2%, while
Lactobacillus prevalence ranged from 19.9% to 29.7%. The lowest
Lactobacillus prevalence was in the RF
V group, while the highest was in S
nonV group. In all groups, the most prevalent species among
Lactobacillus was
Lactobacillus amylovorus (
Tables S1–S4)). The other most prevalent genera included
Barnesiella,
Clostridium,
Blautia,
Faecalibacterium,
Roseburia and
Eubacterium, which ranged from 1.2% (
Eubacterium) up to 3.4% (
Barnesiella) in the faeces. Overall, the microbial profiles were similar among all the groups before the experiment (
Figure 2), however, it should be mentioned that
Bifidobacterium was detected only in two groups—RF
nonV and RF
V with the prevalence of 4.5% and 1.7% respectively.
At the end of experiment, the microbial profiles had changed depending on the pig group (
Figure 3). Overall, the main genera remained similar as before experiment, but there were obvious differences in the
Lactobacillus prevalence. The lowest prevalence occurred in faeces from the S
nonV group, while the highest was in the RF
V group (
p ≤ 0.05). In the latter group, lactobacilli were the most abundant bacteria: their prevalence reached 47.9% of the total bacteria, while in the rest of the groups the highest prevalence was
Prevotella. When comparing groups with fermented vs. non-fermented feed, pigs that received fermented feed had a notably higher
Lactobacillus prevalence compared to pigs fed non-fermented feed. The pigs from non-fermented feed groups had higher
Clostridium and
Terrisporobacter prevalence (
p ≤ 0.05), while the prevalence of other bacterial genera was low overall in all groups.
When comparing non-vaccinated with vaccinated groups, the main differences were also associated with the
Lactobacillus prevalence: it was higher in vaccinated compared to non-vaccinated groups (
p ≤ 0.05). At the same time, the amount of
Prevotella was higher in non-vaccinated groups, although the differences between RF
nonV and S
nonV groups were small (31.4% vs. 30.2%). The species prevalence and variety at the end of the experiment are presented in
Tables S5–S8).
The microbial communities in the pig gut perform a variety of beneficial functions [
31]. Previous studies of the gut microbial community have illustrated how populations of constituents are shaped by environmental exposure to microbes, diet, immunological pressures, host genetics and ecological forces within the ecosystem itself [
32,
33,
34]. Understanding which microbial populations are influenced by fermented feed provides insight into how dietary changes in pigs shape the gut microbiome. Although there have been studies about microbial communities within the gut of healthy pigs, the microbial composition can differ depending on breed, age, place, feed, hygiene conditions and other factors [
31,
35,
36]. This study demonstrated changes in microbial profiles using feed prepared by different technologies within similar groups of animals and the same farm. While the exact mechanism(s) by which vaccination can influence microbial changes within the gut of mammals remains unknown, this study suggests that vaccination has a certain influence, particularly on the number of
Lactobacillus spp.
Before the experiment, the microbial composition in all groups was very similar, with the highest prevalence for
Prevotella and
Lactobacillus, both of which accounted for 56% of the bacterial count. These genera include abundant microorganisms in young weaned healthy pigs [
37].
Prevotella spp. are usually dominant in pigs’ gut and gradually increase in number with age [
38,
39].
Prevotella spp. are key microbial members of the gastrointestinal tracts of adult animals; they are crucial for the degradation of starch and plant polysaccharides but also have a strong capacity for mucoprotein catabolism [
40,
41].
Lactobacillus spp. are common in both the proximal and distal regions of the porcine digestive tract; they colonise soon after birth [
42]. This genus influences intestinal physiology, regulates the immune system and balances the intestinal ecology of the host [
43]. In addition,
Lactobacillus spp. have been known to metabolise carbohydrates, including oligosaccharides and starch, which are fermented in the large intestine to short chain fatty acids by lactobacilli for subsequent utilisation by the pigs [
44]. Previous studies have indicated that stress greatly affects the gastrointestinal microbiota: it decreases total
Lactobacillus populations and thus provides an opportunity for pathogen overgrowth [
45]. Such stress usually occurs during the weaning period. According to the literature, when compared to diarrhoeic piglets, the gut microbiota of healthy piglets has a higher abundance of
Prevotellaceae,
Lachnospiraceae,
Ruminococcaceae and
Lactobacillaceae [
46]. These data suggest that the gut microbial composition may be used as a biomarker to predict the health status of piglets. The present study demonstrated the positive impact of fermented feed on the porcine microbial composition compared with a conventional feeding regimen in healthy pigs. At the end of the experiment (day 61 of the piglets’ life), the
Lactobacillus prevalence decreased on average 2.4 fold in the groups fed non-fermented feed, whereas the prevalence increased on average 1.8 fold in the groups fed fermented feed. Hence, providing fermented feed prevents the decrease in
Lactobacillus prevalence after weaning. This effect may help prevent digestive disorders associated with microbial changes because this period is one of the most critical regarding different infections [
46]. It is known that during this age,
Lactobacillus spp. in pigs normally decrease in number [
37]. The
Lactobacillus prevalence was also higher in vaccinated compared with non-vaccinated groups irrespective of the feed type. However, the data do not indicate whether vaccination against PCV2 leads to an increase in
Lactobacillus prevalence; more experiments are required in this area. Moreover, different types of vaccines and larger experiments should be performed to better understand the possible influence of vaccination on the microbial communities. According to this study, it may be assumed that at the very least vaccination against PCV2 does not negatively affect the microbial composition within the gastrointestinal tract of pigs.
3.2. LAB, TVC, TEC and M/Y Counts in Piglets’ Faeces
Microbiological parameters of the piglet faeces (from 25- and 61-day-old piglets) are shown in
Table 2 (
Table S9—Differences between microbiological parameters of the piglets’ faeces between all the tested groups). In most groups (except the S
V group), the LAB count was significantly lower at the end compared with the beginning of the experiment (20.1% lower in the S
nonV group; 37.9% lower in the RF
nonV group; and 25.3% lower in the RF
V). The LAB count at the end of experiment was significantly higher in both vaccinated compared with non-vaccinated groups (26.6% for S
V and 17.2% for RF
V). The LAB count was 26.3% higher in S
V compared to the RF
V group at the end of the experiment. In a previous study, the faeces of pigs receiving a diet with fermented feed contained significantly fewer total bacteria and fungi, as well as coliform bacteria (including Escherichia coli) and anaerobic
Clostridium perfringens counts. This phenomenon is due in part to the reduction in pH, an increase in the amount of lactic acid and other volatile fatty acids in the intestinal contents and a reduction in the number of
Enterobacteriaceae [
47,
48]. From birth until weaning and then during the post-weaning period, the gut microbiota is dynamic and undergoes major compositional changes that are driven by age, exposure to microbes, environmental conditions and diet [
49]. Many authors have described the great impact of early-life events in mammals, and particularly in pigs, on their future health. These experiences shape immune system development through changes in the pattern of microbial intestinal colonisation [
50,
51]. Colonisation is initiated at birth and is shaped by consumption of the sow’s milk, which provides nutritional advantages to the LAB population, building a milk-oriented microbiome that includes
Bacteroidaceae and
Lactobacillaceae. This composition rapidly changes after weaning when a (largely) plant-based diet is introduced [
35]. The rapidly varying microbiome of young piglets seems to increase in microbial diversity and richness in the suckling phase and gradually stabilises post-weaning [
35,
52,
53]. Our results are in agreement with Bian et al. [
54], namely that members of the
Lactobacillaceae became predominant on days 7, 14 and 28, but had a lower relative abundance again on day 49.
When comparing the TVC in piglets’ faeces, in all but the S
V group it was significantly lower at the end of experiment compared with the beginning (9.5% reduction in the S
nonV group; 23.6% reduction in the RF
nonV group; 3.4% reduction in the RF
V group). TVC was significantly higher in the vaccinated piglets compared to the non-vaccinated piglet faeces at the end of the experiment (22.2% for S
V and 13.8% for RF
V). Between the vaccinated groups, TVC was 10.0% higher in the S
V compared to the RF
V group. During weaning, there is gut microbiota dysbiosis, including a loss of microbial diversity. Studies have noted significant reductions in total bacterial number, including coliform bacteria and anaerobic
C. perfringens, as well as an increase in
Acetivibrio,
Dialister,
Oribacterium,
Prevotella and
Proteobacteriaceae, including
E. coli [
48,
49,
55,
56]. A decrease in
Lactobacillus spp. can lead to decrease in TVC. Zimmerman et al. [
57] demonstrated that
Lactobacillus,
Bifidobacterium and
Lactobacillus spp. have a positive immunomodulatory effect on vaccines in animals.
In all the groups, the TEC and Y/M counts were lower in piglets’ faeces at the end compared with the beginning of the experiment (11.9 and 5.5%, respectively, for the S
nonV group; 26.1 and 44.1%, respectively, for the S
V group; 7.2 and 7.9%, respectively, for the RF
nonV group; 6.1 and 5.6%, respectively, for the RF
V group). Both TEC and Y/M were significantly lower in the S
V compared to S
nonV group faeces at the end of experiment, but the opposite was true for the RF
V compared to the RF
nonV group. At the end of the experiment, the TEC and Y/M counts were higher in the RF
V compared to the S
V group (13.2 and 31.2%, respectively). Our results are in agreement with Dowarah et al. [
58], who showed that fermented feed improved the number of beneficial microbes (LAB and bifidobacteria) and reduced
E. coli and clostridia count, the pathogens responsible for diarrhoea, in faces. Arfken et al. [
59] investigated the mutualistic relationship between yeast in the piglet gastrointestinal (GI) tract and
Lactobacillus count. There was a significant reduction in the total number of fungi compared with the control group [
48].
The ANOVA results indicated that there were significant treatment duration, fermented feed and vaccination main effects, as well as significant treatment duration × fermented feed, treatment duration × vaccination, fermented feed × vaccination and treatment duration × fermented feed × vaccination interactions on microbiological parameters of the pig faeces. Indeed, only the fermented feed × vaccination interaction was not significant for TVC in pig faeces.
Finally, microbiological parameters of the piglets’ faeces were varied and, in most of the cases, influenced by the analysed factors and their interaction. Considering that genera and species as well as the interaction between strains can lead changes of piglets’ growth performance, correlation between the above-mentioned parameters were calculated.
3.3. Piglet Blood Parameters
The piglets’ blood parameters are shown in
Table 3 (
Table S10—Differences between blood parameters of the piglets’ between all the tested groups). At the end of experiment, there were significantly lower ALT, T3, T4, IP, Mg, K, Ca and vitamin B12 concentrations in the S
V compared with the S
nonV group. However, in the S
V group, Chol, HDL-C, TP and GLU concentrations were significantly higher compared with the SnonV group. At the end of experiment, there were significantly lower ALT, HDL-C, LDL-C, TP, ALB and Fe blood concentrations in the RF
V compared to RF
nonV group. However, in the RF
V group blood, TG and total bilirubin concentrations were significantly higher compared with the RF
nonV group.
When comparing blood parameters between the vaccinated groups (SV and RFV) at the end of experiment, ALT, TG, IgG, Mg, K, Fe and total bilirubin were significantly higher in the RFV group. By contrast, HDL-C, LDL-C, TP, ALB, T3, T4 and vitamin B12 were significantly lower in the RFV compared with the SV group.
In general, blood biochemical indices reflect comprehensive functions of piglet’s nutritional metabolism, health and welfare [
60]. The IgG level reflects immune status, plays important roles in the humoral immune response and controls bacterial infections in the body; it can also function to control diarrhoeal infections by binding multiple pathogenic antigens [
46]. Lu et al. [
59] reported that IgG and immunoglobulin M (IgM) concentrations were significantly greater and serum ALT and AST concentrations were decreased in pigs that received fermented feed (
p ≤ 0.05). The activities of the hepatic enzymes ALT and AST are key indices that reflect the acute injury of liver. The ALT blood concentration increases when the liver cell membrane is damaged. The AST content significantly increases when the liver mitochondrial membrane is impaired [
60]. The blood TP concentration reflects the relationship between protein absorption in vivo and humoral immunity [
60]. The hydrolysis products of glucosinolates are known to depress iodine metabolism in the thyroid gland and inhibit the synthesis of thyroid hormones T3 and T4. When these compounds, especially thiocyanates, interfere with iodine uptake, hypothyroidism and thyroid gland enlargement may ensue [
46]. However, in this study the utilised rapeseed meal contained very low concentrations of erucic acid and glucosinolates (7.5–11.2 μmol/g). Hence, the influence of the above-mentioned compounds on T3 and T4 should be minimal. The fermentation process can reduce dietary anti-nutrients (phytates, glucosinolates and trypsin inhibitors) and improve the absorption and use of nutrients, including amino acids and minerals (e.g., P, Ca, Zn, Cu and Fe) [
48,
61]. These changes can lead to haematological, biochemical and mineral profile variations in the blood [
61,
62]. However, it was published that the TP concentration was increased in the blood of piglets fed fermented feed [
63]. In opposition to above-mentioned findings, Min [
64] reported that the TP blood concentration was not affected by the fermented feed diets. In serum samples from piglets fed with fermented feed, there were higher ALP, TP, ALB and GLU concentrations [
62,
65,
66]. Liu et al. [
15] published that piglets fed fermented feed had lower levels of serum IgG, but there was no difference in the immunoglobulin IgA and IgM serum levels. Furthermore, the serum glucose level was decreased in piglets fed fermented feed; however, TP, Chol, TG, LDL-C, ALT, AST, Ca, P and Mg were not different among the treatments [
67]. Satessa et al. [
68] found that fermented rapeseed meal reduced the glucose, HDL-C and LDL-C concentrations, but TP, P, AST, ALT concentrations were increased, and significant changes in blood IgG were not established. According to Czech et al. [
61], the blood concentrations of Chol and TG AST in piglets fed with fermented rapeseed meal was reduced, and there was better availability of minerals.
The influence of the analysed factors and their interaction on piglets’ blood parameters is shown in
Table 4. Vaccination as a separate factor did not significantly influence piglets’ blood parameters. However, the treatment duration × fermented feed interaction was significant for T3 (
p = 0.038), T4 (
p = 0.041) and Fe (
p = 0.008) concentrations in piglets’ blood. There was a significant treatment duration × vaccination interaction on T4 (
p = 0.008) and K (
p = 0.007) concentrations in piglets’ blood. Finally, there was a significant fermented feed × vaccination interaction for T4 (
p = 0.033), vitamin B12 (
p = 0.003) and urea (
p = 0.014) concentrations in piglets’ blood. All three factors were significant with regard to the TP content in piglets’ blood (
p = 0.041).
3.4. Piglets’ Growth Performance
The piglets’ average FCR, ADG and the influence of analysed factors and their interaction on piglets’ growth performance parameters from day 25 to 61 are shown in
Table 5. For ADG, there were no significant differences among the groups. In addition, the analysed factors as well as their interactions did not exert significant effects on piglets’ ADG. However, there were significant differences in FCR between S
V and S
nonV (11.5% lower in the SV group), between RF
V and RF
nonV (10.2% lower in the RF
nonV group) and between S
V and RF
V (21.6% lower in the S
V group). There was a significant, very strong positive correlation between FCR and TEC in piglets’ faeces (R = 0.919,
p = 0.041). There were no significant correlations between the other analysed faecal microbiological parameters (LAB, TBC and Y/M count) and FCR or ADG.
Mortality and diarrhoea cases were similar in all groups throughout the experiment. The mortality of piglets in the non-vaccinated groups (SnonV and RFnonV) was 2%, while mortality in the vaccinated groups (SV and RFV) was 2% and 3%, respectively. There was more intense diarrhoea in the SnonV group at day 31 and 36. There were several diarrhoea cases in the SV, RFnonV and RFV groups from day 28 to 46.
The available published information about the influence of vaccination on piglets’ growth performance is varied. According to Oliver-Ferrando et al. [
69], the optimal time for PCV2 vaccination is at either 3 or 6 weeks of age. In our study, piglets were vaccinated on day 25 of life. Duivon et al. [
70] published that the added PCV2 valence in the vaccination protocol helps counter the negative impact of subclinical PCV2 infection on growth. Jeong et al. [
71] indicated that ADG showed improvement in vaccinated compared with non-vaccinated animals. Da Silva et al. [
72] compared the ADG between vaccinated and non-vaccinated animals from weaning to finishing for four commercial vaccine products. All products significantly increased ADG values. However, according to Woźniak et al. [
73] data from four farms where vaccination was used, the results were similar to those from non-vaccinated farms. The authors suggested revising the vaccination protocol. The above-mentioned findings can be explained by a major PCV2 genotype shift from the predominant PCV2 genotype 2b towards 2d [
74]. While the commercial vaccines that were first introduced to the US in 2006 have been highly effective in reducing clinical signs and improving production, recent studies have indicated a declining level of PCV2 prevalence and viraemia in the field. Hence, the efficiency of current vaccines against new and emerging strains, as well as new vaccine development, is crucial [
75]. Vaccination against PCV2 is an important co-working agent that confers unintended benefits in the protection against the other agents [
76]. Notably, the breed information is frequently omitted from experimental publications of vaccine studies, as well as environmental conditions (temperature, stocking density, etc.). These factors are very important for the further standardisation across studies before logical comparisons can be made among studies [
77].
While we found differences in the ADG between different groups, the influence of vaccination on different feed groups was the opposite. Specifically, the FCR was lower in the SV compared to the SnonV group. However, the effect was the opposite in the rapeseed meal groups: FCR was lower in the RnonV compared to the RFV group. The lowest FCR (1.38) occurred in the SV group. Notably, soya-based feed is much more expensive compared with the feed composed of local rapeseed meal. Overall, the data support the use of fermented local feedstock for piglet feeding because it provides similar growth parameters as imported soya.
3.5. Influence of Analysed Factors on Ammonia Emission
Table 6 presents the influence of the analysed factors on ammonia emission. At the end of experiment, the RF
V group had the lowest ammonia emission: 58.2, 23.8 and 47.33% lower than the S
nonV, S
V, and RF
nonV groups, respectively. There was lower ammonia emission in vaccinated groups at the end of the experiment: 45.2% lower in the S
V compared to S
nonV group and 47.33% lower in the RF
V compared to the RF
nonV group. ANOVA revealed a significant effect for all the analysed factors and their interaction on ammonia emission. Furthermore, there was a significant, very strong positive correlation between ammonia emission and Y/M count in piglets’ faeces at the end of experiment (R = 0.974;
p = 0.013).
There has been no information published about the influence of vaccination on ammonia emission; most of the published studies have focussed on dietary aspects. Yi et al. [
37] and Lee et al. [
78] reported that protected organic acids used for weanling pigs reduce faecal ammonia. Nguyen et al. [
79] concluded that diets with probiotics mixture decreased pigs’ ammonia emission. According to Wang et al. [
51], diets with fermented feed ingredients tended to decrease ammonia emissions, but this effect was not statistically significant. According to Bindas et al. [
80], ammonia in the faeces of experimental animals fed with fermented feed was significantly lower compared with the control animals. Reducing ammonia emissions by changing the dietary composition is considered economical and feasible because it can improve nitrogen utilisation and, consequently, reduce ammonia emissions [
53]. Lactobacilli-based feed fermentation decreases the emissions of total organic carbon and ammonium [
67]. Finally, many factors can contribute to ammonia emission on pig farms. Our data indicate that vaccination as a factor should be considered, as well as microbiological parameters of the piglets’ faeces. Indeed, we found a very strong correlation between ammonia emission and Y/M count in piglets’ faeces.