Growth performance and carcass characteristics
Growth performance results are shown in Table 3. At day 27 of the study, birds fed HTM had a significantly (P=0.0229) higher body weight than birds fed STM, regardless of mineral levels. This continued until day 34 as a strong trend (P=0.0542) towards an increased body weight in birds fed HTM compared to birds fed STM. Average daily gain (ADG) and average daily feed intake (ADFI) followed the same patterns as body weight throughout the study. In this case, between 10 and 27 days a tendency was observed towards an increased ADG (P=0.0738) and ADFI (P=0.0846) for birds fed HTM compared to birds fed STM. However, this tendency resulted in a significantly improved ADG (P=0.0130) and ADFI (P=0.0125) over the entire period for HTM fed birds compared to STM fed birds, regardless of mineral level. As a result, the feed conversion ratio (FCR) was not significantly different among treatments.
The effect of the treatments on carcass and breast meat yield were measured at the end of the study (Table 4). No significant differences were found in carcass yield. Breast meat yield was significantly higher in birds fed 80 ppm Zn compared to birds fed 20 ppm Zn (P=0.0171), while no significant differences were found between the sources or any source×level interaction.
Mineral content in ileal and cecal digesta
The mineral content in the digesta from ileum and cecum is shown in Table 5. Birds fed HTM had significantly higher Cu contents in the ileum compared to the birds fed STM (P=0.004). The Zn content in the ileum was proportional to the dietary Zn level, where birds fed a high dietary Zn presented significantly higher Zn than in birds fed low Zn (P<0.0001). In the cecum, the Cu level remained similar across the treatments, while Zn content depended significantly on the supplemented levels (P<0.0001). Surprisingly, there was a significant source×level interaction where birds fed 80 and 20 ppm Zn from HTM showed the highest and the lowest cecum values, respectively (P=0.0472).
In total, 42,854,714 sequences were obtained after removal of chloroplast and mitochondria reads (6046 sequences). Rarefactions curves of observed OTUs showed a plateau (Figure 1) and good coverage values close to 1 from 20,000 sequences (0.9999 ± 0.0001), showing that sampling depth for all the members of the microbial communities were sampled. Alpha and beta diversity showed a clear (and significant) separation between the ileum and cecum.
In the ileum, 23,998,507 sequences were recovered (521,707±174,002 sequences) representing 100 OTUs. Beta diversity ordination represented by redundancy analysis (Figure 1) showed significant distances on ANOSIM (P=0.020) and PERMANOVA (P=0.014) of unweighted unifrac and a trend in jaccard (P = 0.080), between microbial communities in the ileum of birds fed 80 ppm Zn from HTM to birds fed 20 ppm from HTM. The STM fed birds showed intermediate values not differing from any of the other treatments tested. The number of observed OTUs of alpha diversity was significantly higher in the group fed 80 ppm Zn from HTM (50±14.1) than 20 ppm Zn from HTM (39±8.3) (P=0.049), as well as the phylogenetic distances, whereas both STM groups were in-between (Table 6). In addition, Spearman correlations showed a tendency towards a positive correlation between the number of observed species in the ileum and the breast meat yield (P=0.093) (data not shown). The taxonomy at phyla level (data not shown) showed Firmicutes as main phyla (99.8%), followed by Actinobacteria (0.15%), Proteobacteria (0.08%) and Patescibacteria (0.01%). Actinobacteria phyla was significantly higher in group fed 80 ppm Zn from HTM (0.34%) in comparison with the group fed 20 ppm Zn from HTM (0.03%) (P=0.043). At genus level, the main organism present in the ileum was Lactobacillus (>88%) (Table 7).
Differences in the remaining genera in the ileum are shown in Table 7 and visualized in Figure 2. The different areas show that the diet containing 80 ppm Zn from HTM permitted some diversity strength in the ileum at day 34. The arrows in Figure 2 show significantly increased Enterococcus (P=0.012) and Streptococcus (P=0.042), in comparison to the diet containing 20 ppm Zn from STM, which in turn presented higher Lactobacillus than 80 ppm Zn from HTM (P=0.010). The group fed 80 ppm Zn from HTM also presented higher Bifidobacterium (P=0.018), Blautia (P=0.034), Streptococcus (P=0.043), Weissella (P=0.039), Eubacterium hallii group (P=0.033), Ruminococcus torques g (P=0.043) than the group fed 20 ppm Zn from HTM (Table 7). Dosage levels (20 versus 80 ppm) presented significant differences of abundance in Lactobacillus (20 ppm) and in Streptococcus and Enterococcus (80 ppm), mainly because of the high influence of the group fed 80 ppm Zn from HTM. No significant difference was found by the overall source effect, regardless of Zn level.
In the cecum, 17,250,171 sequences were recovered (359,379 ± 111,998 sequences) representing 644 OTUs. Beta diversity ordination represented by redundancy analysis (Figure 2) showed significant distances in ANOSIM per source of the unweighted unifrac and jaccard distances (P=0.019 and P=0.027, respectively), promoted by significant differences between both groups fed 20 ppm Zn (P=0.007 and P=0.031, respectively). Feeding 20 ppm Zn from HTM also presented marked differences in the ANOSIM and PERMANOVA of unweighted unifrac distances in comparison with feeding 80 ppm Zn from HTM (P=0.014 and P=0.011, respectively) or from STM (P=0.011 and P=0.022, respectively). The alpha diversity confirmed the differences found in the ordination of the bacterial communities (see Table 6). The Shannon index, an indication for population diversity, shows a tendency towards a higher diversity in the HTM fed groups compared to the STM groups (P=0.066), with the group fed 80 ppm Zn from STM having the lowest diversity in comparison with the group fed 20 ppm Zn from HTM (P=0.049). The observed species in the cecum tended to be higher in birds fed 20 ppm Zn from HTM compared to the birds fed 80 ppm Zn from STM (P=0.067). The evenness showed a trend towards an increased (P=0.099) richness in the HTM groups compared to the STM groups, regardless of Zn level. Taking the phylogenetic distances into account, the Faith_PD parameter did show a trend towards a different community in the groups fed 20 ppm Zn compared to the groups fed 80 ppm Zn (P=0.070), and specifically between 20 ppm Zn from HTM and 80 ppm Zn from STM (P=0.024) (Table 6). In addition, significant positives correlations were found between the carcass yield and Shannon (P=0.0322), evenness (P=0.046) or observed species (trend, P=0.062) in the cecum. Also the ADFI and ADG performance indexes tended to be positively correlated to the richness or evenness of the microbiota (P=0.053 and P=0.098, respectively) (data not shown).
The taxonomy in the cecum presented as the main phylum Firmicutes (87.3%), Actinobacteria (9.5%), Bacteroidetes (3.1%), and in less extend Proteobacteria (0.06%), Tenericutes (0.05%) and Cyanobacteria (0.01%). On family level, main species present in the cecum were the Lachnospiraceae, Ruminococcaceae, Lactobacillaceae and Bifidobacteriaceae.
At genus level, the main organisms present in the cecum were Lactobacillus, Faecalibacterium, unknown genus from Lachnospiraceae family, Bifidobacterium, several Ruminococcus groups, Butyricoccus, Blautia and Alistipes (Table 8). Being 80 ppm the most common commercial level used in broiler feed the most interesting comparison would be between groups fed 80 ppm Zn from either STM or HTM. In this case, significant differences were found in Streptococcus and two unidentified genus from the family vadin BB60 being higher in birds fed 80 ppm Zn from HTM than from STM. Only CHKCI002 from Eggerthellaceae family presented higher values in birds fed 80 ppm Zn from STM than from HTM. As in the ileum, the majority of the significant differences were in between HTM groups (Table 8). The group fed 20 ppm Zn from HTM contained higher relative abundances of Blautia, Anaerostipes, Christensenellaceae R-7 group, Ruminococcus 1, Ruminococcaceae UCG-004, Tyzzerella, Eubacterium nodatum group, Ruminococcaceae_uncultured, Family XIII (AD3011 and UCG-001 groups), Eubacterium brachy group, Pygmaiobacter, Peptococcaceae_other, whereas the group fed 80 ppm Zn from HTM showed significantly higher relative amounts of vadin BB60 group (Table 8). The group fed 20 ppm Zn from STM reached the highest Anaerotruncus level, Tyzzerella 3, Family XIII UCG-001, and Brachybacterium, and the group fed 80 ppm Zn from STM the highest CHKCI002 (Table 8). Dosage level 20 ppm presented higher significant abundance of Anaerostipes, Pygmaiobacter, unknown genus from Peptococcaceae family, Christensenellaceae R-7 group and family XIII in comparison with 80 ppm. This result is mainly cause by the high influence of the HTM20 group. Source (HTM vs STM) also changed the microbial composition of the cecum. In this case, HTM groups had an increased abundance of Streptococcus and Enteroccocaceae family, and STM groups of family vadin BB60 group and unknown genus from Christensenellaceae family.