Growth performance and feed efficiency
Growth performance is the most intuitive index for evaluating rapid growth in beef calves. This has economic implications and is thus associated with improving growth performance through breeding. Table 2 shows the growth performance parameters of the different groups at different times. The LAMR group had the best growth performance.
On days 1 and 90, there were no significant differences in the body weights of the calves in each group (P ≥ 0.050), whereas at the end of the experiment (day 122), the body weights of the animals in the LAMR group were greater than those in the other three groups (P = 0.016). There were no significant differences (P ≥ 0.050) in the ADG, DMI, or F/G values among the groups between days 1 and 89, which is consistent with the findings of Ding et al. [25] that the addition of AMR and its extracts to the diet did not significantly affect the growth performance of the experimental animals. However, after 90 days to 122 days, the ADG in the LAMR group was significantly greater than that in the other three groups (P = 0.030) and showed quadratic changes with increasing AMRP addition (P = 0.030). The DMI in the MAMR group was significantly lower than that in the other three groups (P < 0. 010) and showed linear decreases and quadratic changes as the AMRP dose increased (P = 0.005 and p < 0.010, respectively), while the F/G values showed quadratic changes with increasing AMRP addition (P = 0.035). A previous study revealed that the addition of olive cake (containing flavonoid and polyphenolic compounds) to a growing beef cattle diet markedly reduced the DMI while increasing feed efficiency[26], which may be related to earlier observations that the addition of olive cake alters the rumen microbiota and reduces the biohydrogenation of unsaturated fatty acids[27]. This study was conducted over 75 days, while the duration of the present study was 122 days. The self-regulation of the gastrointestinal microbiome after long-term AMRP feeding is not as good as the regulation of the gastrointestinal microbiome by AMRP, which leads to microbial changes and affects the growth performance of animals. Between days 1 and 122, the ADG values in the LAMR group were significantly greater than those in the other three groups (P = 0.002) and showed a quadratic change as the AMRP dose increased (P = 0. 019), which is consistent with the results obtained by Du et al. [28], in which the addition of AMR and its extracts to the diet increased the ADG of the animals, which may be related to the influence of AMR on the microbiome of the gastrointestinal tract, together with the anti-inflammatory effects of the flavonoid components of AMR. These flavonoid substances regulate the relative abundance of microbes in the gastrointestinal tract. It is known that there is a link between these microbes and the production of inflammatory factors; thus, AMR could enhance the body's immunity and play a positive role in the health and growth of animals [29]. A further study revealed that the addition of oregano essential oil, which contains flavonoids, to the diet significantly increased the ADG in Holstein calves, likely due to an alteration in rumen fermentation induced by the oil, together with increased concentrations of propionic acid, thus improving fermentation [30]. The DMI in the MAMR group was lower than that in the other three groups (P = 0.014), which may be related to the decrease in DMI on the 90th-122th days of life. Prolonged supplementation with AMRP impacted the microbes of the gastrointestinal tract, leading to reductions in DMI throughout the feeding period. The F/G values in all three experimental groups were significantly lower than those in the ZAMR group (P = 0.003); these values showed quadratic changes (P = 0.003) in response to the AMRP dose (P = 0.003), while the ADG values increased. The F/G values possibly correlated with the increased ADG and reduced DMI between 90 and 122 days. The LAMR group consistently maintained the highest ADG, resulting in the lowest F/G values and the highest feed efficiency.
These results indicated that a dietary dose of 10 g/d/calf AMRP was beneficial for increasing ADG and final body weight, decreasing F/G values, and increasing feed efficiency in Angus calves and that the active ingredients (flavonoids and polyphenolic compounds) in AMR possibly promoted growth and development and improved gastrointestinal flora, which is beneficial for healthy animal growth. The AMRP dose of 20 g/d/calf reduced the F/G values in the Angus calves, suggesting that too much AMR may reduce feed efficiency. These results suggest that AMR can improve animal production efficiency, although further investigation is needed to determine the precise relationship between the dose of AMR and growth performance.
Table 2
Effect of different doses of AMRP on the growth performance of Angus calves at different times.
Items | Diets | SEM | P value |
ZAMR | LAMR | MAMR | HAMR | L | Q | D |
Body weight(kg) |
Initial | 274.67 | 272.33 | 270.83 | 266.83 | 10.405 | - | - | - |
Day 1 | 288.50 | 293.33 | 286.33 | 280.33 | 4.129 | 0.423 | 0.536 | 0.760 |
Day 90 | 360.84 | 380.48 | 362.14 | 365.76 | 4.609 | 0.932 | 0.397 | 0.434 |
Day 122 | 376.50b | 410.17a | 381.67b | 381.50b | 5.06 | 0.743 | 0.077 | 0.016 |
Day 1 to 89 |
ADG (kg/d) | 0.81 | 0.98 | 0.85 | 0.96 | 0.026 | 0.149 | 0.540 | 0.054 |
ADFI (kg/d) | 8.40 | 8.33 | 8.19 | 8.34 | 0.068 | 0.645 | 0.454 | 0.774 |
F/G | 10.38 | 8.63 | 9.74 | 8.85 | 0.265 | 0.117 | 0.375 | 0.059 |
Day 90 to 122 |
ADG (kg/d) | 0.47b | 0.90a | 0.59b | 0.48b | 0.051 | 0.407 | 0.003 | 0.003 |
ADFI (kg/d) | 8.45a | 8.45a | 7.04b | 8.30a | 0.138 | 0.005 | <0.010 | <0.010 |
F/G | 19.48 | 9.55 | 13.69 | 25.09 | 2.514 | 0.331 | 0.035 | 0.136 |
Day 1 to 122 |
ADG (kg/d) | 0.72b | 0.96a | 0.78b | 0.83b | 0.025 | 0.382 | 0.019 | 0.002 |
ADFI (kg/d) | 8.41a | 8.35a | 7.91b | 8.33a | 0.734 | 0.269 | 0.085 | 0.014 |
F/G | 11.71a | 8.78c | 10.31b | 10.14bc | 0.307 | 0.146 | 0.008 | 0.003 |
L represents the linear model, Q represents the quadratic model, and D represents the impact of diet. ADG, average daily gain; ADFI, dry average daily feed intake; F/G, feed to gain values. Letters with different superscripts in the same row indicate significant differences (P < 0.050). The groups were as follows: ZAMR, 0 g/d/calf AMRP to the basic diet; LAMR: 10 g/d/calf AMRP was added to the basic diet; MAMR: 15 g/d/calf AMRP was added to the basic diet; HAMR: 20 g/d/calf AMRP was added to the basic diet.
Apparent nutrient digestibility
The apparent nutrient digestibility reflects an animal’s ability to digest and utilize dietary nutrients and is thus closely associated with growth performance. Table 3 shows the apparent nutrient digestibility measurements in each group at different sampling times, indicating that the LAMR group had the highest values of this parameter.
On day 1, there was no significant difference in apparent DM digestibility between the groups (P ≥ 0.050), while on day 90, significantly greater apparent DM digestibility was observed in the 3 treatment groups than in the ZAMR group (P < 0. 010), showing a linear increase and a quadratic change with the addition of AMRP (P < 0.010). On day 122, the apparent DM digestibility in the HAMR group was significantly greater than that in the ZAMR group (P = 0.015), while no significant differences were detected between the LAMR and ZAMR groups (P ≥ 0.050). This was not consistent with the best growth performance observed in the LAMR group and may be related to methane emissions. Zhao et al. [31] reported that the addition of high doses of AMRP extract increased the pH of the fermentation broth and the synthesis efficiency of 1-methylcyclopropene (MCP), which in turn promoted the proliferation of microbes and increased the apparent digestibility of DM in the diet; a higher pH of the fermentation broth also increased methane emission, resulting in feed waste. The apparent DM digestibility in the ZAMR group did not change significantly over time, in contrast to the apparent DM digestibility in the test groups, with the values in the LAMR and MAMR groups being significantly greater on day 90 than on day 122 (P = 0.009, p < 0.010, respectively), and that in the HAMR group being significantly greater on days 90 and 122 than on day 1 (P < 0.010). This suggests that AMRP supplementation may have affected both the intestinal flora and the activities of digestive enzymes, with different effects related to the different AMRP doses. On day 1, the apparent OM digestibility in the MAMR group was significantly greater than that in the other three groups (P < 0.010) and showed a quadratic change with AMRP addition (P = 0.032). On days 90 and 122, the apparent OM digestibility in the 3 treatment groups was significantly greater than that in the ZAMR group (P < 0.010 and p < 0.010, respectively), showing a linear increase and a quadratic change with increasing AMRP dose (P < 0.010 and p < 0.010, respectively). OM apparent digestibility was also observed to change significantly over time in all cases, and it is noteworthy that OM apparent digestibility in all the test groups increased on day 122 relative to that on day 1 (P < 0.010), whereas OM apparent digestibility in the ZAMR group decreased (P < 0.010). Zhao et al. [31] reported that the addition of AMR essential oil increased the apparent digestibility of OM, which was related to increased enzyme activity induced by the oil; this finding is consistent with the results of the present study and may be related to increased activities of digestive enzymes induced by AMRP. On day 1, there was no significant difference in the apparent digestibility of EE among the groups (P > 0.050), while on day 90, the apparent digestibility of EE in the MAMR group was significantly greater than that in the other three groups (P = 0.018). On day 122, the apparent digestibility of EE in the LAMR group was significantly greater than that in the other three groups (P = 0.004), showing a quadratic change with the addition of AMRP (P = 0.011). There was no significant change in EE apparent digestibility over time in the ZAMR group (P ≥ 0.050), in contrast to the significant changes in EE digestibility observed over time in the three experimental groups (P < 0.010, p = 0.008 and p = 0.048, respectively). This may be due to the promotion of fermentation by AMR, together with its ability to increase the diversity of the bacterial community, accompanied by increased lipase activity [31]. The gastrointestinal flora plays an important role in the digestion and absorption of nutrients, and the rumen microbiota is capable of degrading ingested cellulose. The digestibility of both neutral and acid detergent fibres may reflect the ability of ruminants to utilize fibrous materials [32]; however, the digestion and absorption of fibrous materials are influenced by the fermentation environment of the rumen, the composition of the diet, the residence time of the ingested material in the rumen, and the composition of the rumen microbiota [33]. An increase in the apparent digestibility of NDF reflects improved fibre digestion in sheep, which was found to have a synergistic effect on increased apparent Ca and P digestibility [34]. The present study revealed that on day 1, the apparent digestibility of NDF in the test groups was significantly lower than that in the ZAMR group (P = 0.003) and decreased linearly with the addition of AMR (P = 0.003), while there was no significant difference in the apparent digestibility of ADF between the groups (P ≥ 0.050). On day 90, both the apparent NDF and ADF digestibility in the MAMR and HAMR groups were significantly greater than that in the ZAMR group (P < 0.010), while on day 122, the apparent NDF digestibility did not differ significantly among the groups (P ≥ 0.050), and the apparent ADF digestibility in the HAMR group was significantly greater than that in the ZAMR group (P < 0.010), with the apparent ADF digestibility showing a linear increase and quadratic change with the addition of AMRP on days 90 and 122 (P < 0.010). Compared with that on day 1, the fibre digestibility in each group first increased and then decreased on days 90 and 122, respectively, while the digestibility in all the groups significantly decreased (P < 0.010) compared with that on day 1. This may be related to changes in fibre-degrading bacteria in the rumen; when the rumen pH was lower than 6.2, the growth and reproduction rates of fibre-degrading bacteria in the rumen decreased, ultimately leading to a reduction in fibre digestibility [33]. On day 1, the apparent Ca digestibility in the experimental groups was significantly greater than that in the ZAMR group (P = 0.004), showing quadratic variation with the addition of AMRP (P = 0.038), with no significant differences observed between the groups on days 90 and 122. On days 90 and 122, the apparent digestibility of P was significantly greater in the experimental groups than in the ZAMR group. On day 1, there were no significant differences in apparent P digestibility among the groups (P ≥ 0.050), while on days 90 and 122, the apparent P digestibility in the 3 treatment groups was significantly greater than that in the ZAMR group (P = 0.001 and p = 0.035, respectively), and the apparent P digestibility in the ZAMR and HAMR groups decreased significantly with time (P = 0.031 and p = 0.002, respectively). The results indicated that Ca apparent digestibility increased and P apparent digestibility decreased over time in all groups, with no synergistic effects shown by increased fibre digestibility on Ca and P apparent digestibility, possibly due to alterations in the microbial compositions induced by AMRP, leading to a decrease in the digestion and utilization of mineral elements by the host. On day 1, the apparent digestibility of CP in the test groups was not significantly different from that in the ZAMR group (P ≥ 0.050), while on day 90, the apparent digestibility of CP in the LAMR and HAMR groups was significantly lower than that in the ZAMR group (P < 0.010), with decreases of 4.09% and 10.32%, respectively, which may be related to decreased OTUs in the fecal microbiome. On day 122, the apparent digestibility of CP in the LAMR group was significantly greater than that in the ZAMR group (P < 0.010), with an increase of 6.82%. On days 90 and 122, the apparent digestibility of CP increased linearly and quadratically with increasing AMRP dose (P < 0.010, p < 0.010, p = 0.007 and p = 0.001, respectively). Xie et al. [35] reported that the addition of AMR extract to the diet significantly increased the apparent digestibility of CP, which may be related to the increase in esterase activity caused by the AMR extract. Zhao et al. [31] also reported that the addition of AMR extract to the diet increased the apparent digestibility of CP and increased the relative abundance of Prevotella at the genus level, which has been shown to be important for the degradation and absorption of protein and peptide fermentation in the rumen [36]. Zhao et al. [31] reported a decrease in the NH3-H content in the gastrointestinal tract with the addition of AMR extracts, suggesting increased synthesis of MCP and thus enhanced CP apparent digestibility. All the above studies showed that AMR and its extracts could increase the apparent digestibility of CP, possibly due to increases in the activities of digestive enzymes and the relative abundance of Prevotella, as well as enhanced MCP synthesis. The active constituents (flavonoids) of AMR lack long-term stability, often have low bioavailability, and need to be ingested at a specific dose to achieve the desired effect in animal studies, which is influenced by the number of additives and the feeding cycle [37]. The above results indicate that the addition of AMR to the diet could improve the digestion and absorption of nutrients in the diet by the animals, influenced by the amount of AMR added and the feeding cycle, with the doses of AMR at a dose of 10 g/day found to best promote digestion and nutrient absorption in Angus calves.
Table 3
Effect of different doses of AMRP on apparent nutrient digestibility in Angus calves at different sampling times.
Items | Diets | SEM | P value |
ZAMR | LAMR | MAMR | HAMR | L | Q | D |
DM |
1 d | 88.65 | 88.83B | 88.61C | 89.09B | 0.087 | 0.145 | 0.368 | 0.176 |
90 d | 88.29c | 90.68Ab | 91.92Aa | 90.03Ab | 0.302 | <0.010 | <0.010 | <0.010 |
122 d | 89.00b | 89.15Bb | 90.04Bab | 90.60Aa | 0.216 | 0.002 | 0.569 | 0.015 |
SEM | 0.183 | 0.288 | 0.343 | 0.179 | |
P | 0.303 | 0.009 | <0.010 | <0.010 |
OM |
1 d | 58.52Ab | 57.76Bbc | 59.68Ba | 57.67Bc | 0.209 | 0.604 | 0.032 | <0.010 |
90 d | 45.18Cd | 51.76Cc | 61.10Aa | 54.28Cb | 1.202 | <0.010 | <0.010 | <0.010 |
122 d | 53.38Bd | 63.58Aa | 59.97Bc | 62.19Ab | 0.834 | <0.010 | <0.010 | <0.010 |
SEM | 1.353 | 1.186 | 0.230 | 0.806 | |
P | <0.010 | <0.010 | <0.010 | <0.010 |
EE |
1 d | 42.23 | 31.56B | 36.85B | 36.32B | 2.961 | 0.846 | 0.502 | 0.802 |
90 d | 42.72b | 38.04Bb | 60.13Aa | 40.27ABb | 2.918 | 0.508 | 0.136 | 0.018 |
122 d | 49.99b | 68.95Aa | 52.57Ab | 51.59Ab | 2.294 | 0.471 | 0.011 | 0.004 |
SEM | 3.760 | 4.582 | 3.407 | 2.678 | |
P | 0.574 | <0.010 | 0.008 | 0.048 |
Ca |
1 d | 4.45Bc | 18.84Ba | 11.11Bbc | 14.54Bab | 1.588 | 0.054 | 0.038 | 0.004 |
90 d | 66.71A | 74.55A | 77.44A | 73.36A | 2.429 | 0.311 | 0.238 | 0.480 |
122 d | 72.05A | 81.78A | 82.66A | 77.38A | 2.301 | 0.419 | 0.116 | 0.359 |
SEM | 7.878 | 7.245 | 8.106 | 7.304 | |
P | <0.010 | <0.010 | <0.010 | <0.010 |
P |
1 d | 66.69A | 68.30 | 70.20 | 74.59A | 3.154 | 0.398 | 0.836 | 0.850 |
90 d | 35.89Bb | 62.28a | 68.24a | 63.77Aa | 3.477 | 0.001 | 0.005 | 0.001 |
122 d | 44.97ABb | 60.71a | 57.14a | 50.13Bab | 2.174 | 0.489 | 0.007 | 0.035 |
SEM | 5.144 | 2.559 | 2.594 | 3.256 | |
P | 0.031 | 0.468 | 0.077 | 0.002 |
NDF |
1 d | 65.31Aa | 56.74Ab | 59.07Ab | 56.36b | 1.058 | 0.003 | 0.084 | 0.003 |
90 d | 38.22Cc | 38.98Cc | 52.54Ba | 45.32b | 1.229 | <0.010 | <0.010 | <0.010 |
122 d | 49.53B | 49.55B | 49.09B | 49.32 | 1.205 | 0.926 | 0.968 | 0.999 |
SEM | 2.735 | 1.877 | 1.191 | 1.985 | |
P | <0.010 | <0.010 | <0.010 | 0.061 |
ADF |
1 d | 68.90A | 61.92A | 64.71A | 62.14A | 1.301 | 0.133 | 0.388 | 0.201 |
90 d | 33.72Cc | 34.40Cc | 49.21Ba | 42.83Cb | 1.386 | <0.010 | <0.010 | <0.010 |
122 d | 45.46Bb | 43.33Bb | 44.19Bb | 50.76Ba | 0.706 | <0.010 | <0.010 | <0.010 |
SEM | 3.623 | 2.974 | 2.346 | 1.990 | |
P | <0.010 | <0.010 | <0.010 | <0.010 |
CP |
1 d | 37.84Bab | 26.12Cb | 29.26Bb | 43.81Ba | 2.468 | 0.284 | 0.006 | 0.032 |
90 d | 55.05Aa | 50.96Bb | 55.67Aa | 44.73Bc | 0.983 | <0.010 | <0.010 | <0.010 |
122 d | 55.64Ab | 62.46Aa | 53.95Ab | 55.14Ab | 0.776 | 0.007 | 0.001 | <0.010 |
SEM | 2.771 | 4.064 | 3.074 | 1.281 | |
P | 0.004 | <0.010 | <0.010 | <0.010 |
L represents the linear model, Q represents the quadratic model, and D represents the impact of diet. DM, dry matter; OM, organic compound; EE, crude fat ether extract; Ca, calcium; P, phosphorus; NDF, neutral detergent fibre; ADF, acid detergent fibre; CP, crude protein. Lowercase letters with different superscript on the same row indicate significant differences (P < 0.050), while uppercase letters with different superscripts on the same columns indicate significant differences (P < 0.050). The same or no letters indicate no significant differences (P ≥ 0.050). The groups were as follows: ZAMR, 0 g/d/calf AMRP to the basic diet; LAMR: supplemented with 10 g/d/calf AMRP to the basic diet; MAMR: supplemented with 15 g/d/calf AMRP to the basic diet; HAMR: supplemented with 20 g/d/calf AMRP to the basic diet.
Analysis of the faecal microbiome composition using 16S rRNA gene sequencing
To further investigate temporal changes in fecal microbes in Angus calves, fecal samples collected on days 1, 90, and 122 were analysed by 16S rRNA gene sequencing. In total, we performed 16S rRNA sequencing on 72 samples (day 1, n = 24; day 90, n = 24; day 122, n = 24) and obtained a total of 6 937 919 raw reads (day 1; 2 972 043 sequences; day 90; 1 840 447 sequences; day 122; 2 125 429 sequences). After primer sequence removal and quality filtering, 5 72,438 valid 16S rRNA sequences were analysed further (day 1: 2 567 184 sequences; day 90: 1 450 496 sequences; day 122: 1 709 758 sequences). These sequences were found to cluster into 7 052 OTUs (day 1, 2 247; day 90, 2 667; day 122, 2 247). On day 1, there were a total of 1502 OTUs in the four groups, with the ZAMR, LAMR, MAMR, and HAMR groups containing 57, 56, 65, and 43 OTUs, respectively. On day 90, the total number of OTUs for the four groups was 1 599, with 162, 110, 134, and 93 OTUs in the ZAMR, LAMR, MAMR, and HAMR groups, respectively, while on day 122, the total was 1 171, with 85, 61, 82, and 144 OTUs in the ZAMR, LAMR, MAMR, and HAMR groups, respectively (Fig. 1a, b, and c). The sample dilution curves showed that the sequencing coverage of all samples in this assay was greater than 0.990 (Fig. 1d, e, f), indicating that almost all microbes were detected in the fecal. A study in mice showed that 93.3% of fecal OTUs were identical to those found in the hindgut [38]. This finding has been confirmed in other studies [39]. These studies suggest that fecal microbes are an effective reflection of gastrointestinal microbes and are easier to sample. Therefore, understanding the changes in fecal microbes in Angus calves after dietary supplementation with AMRP may provide insights for improving digestion and nutrient absorption.
Analysis of fecal microbiome alpha diversity
Alpha diversity reflects the richness and diversity of the microbial community. Of the measures used, the Chao1 and abundance-based coverage estimator (ACE) indices reflect the richness of the community structure, with higher values indicating greater richness, while the Shannon index and Simpson index indices are indicators of diversity, with higher values indicating greater diversity. The effects of different doses of AMRP on the alpha diversity of the microbiome communities of Angus calf fecal are shown in Table 5.
On day 1, the number of OTUs and the ACE and Shannon indices were significantly lower in the HAMR group than in the ZAMR group (P = 0.005, p = 0.023, and p = 0.014, respectively) and decreased linearly with the addition of AMRP (P = 0.010, p = 0.021, and p = 0.003, respectively), while the other indices differed significantly between the groups (P ≥ 0.050). On day 90, the number of OTUs in the LAMR group was significantly lower than that in the ZAMR group (P = 0.029), while there were no significant differences between the groups in terms of the other indicators (P ≥ 0.050). On day 122, both the number of OTUs and the Shannon index in the LAMR and MAMR groups were significantly lower than those in the ZAMR group (P = 0.023 and p = 0.042, respectively), with the Shannon index showing a quadratic change with increasing amounts of AMRP added (P = 0.043). The Simpson index in the MAMR group was significantly lower than that in the ZAMR group (P = 0.022), while the indices did not differ significantly between the groups (P ≥ 0.050). The number of OTUs, Chao1 index, and ACE index in all groups tended to initially increase and then decrease over time (P < 0.050), while the Shannon index and Simpson index in the LAMR and HAMR groups decreased over time (P < 0.050). Notably, the alpha diversity was significantly reduced (P < 0.050) in all groups on day 122. This may be related to the climate of Tianjin, where the weather was hot and rainy during the experimental period (Table S1), which could easily cause heat stress in beef calves. It has been shown that heat stress in beef calves can lead to changes in animal metabolism and can affect rumen microbiome disorders in beef calves [40]. Xie et al. [8] showed that AMR could significantly affect the gastrointestinal microbiome, with both the rumen and fecal microbiomes showing significant correlations, both positive and negative, with positive correlations being more strongly correlated than negative associations, suggesting that these positive associations could strengthen and negative interactions could weaken competition [41]. Therefore, it is hypothesized that the decrease in the alpha diversity of the rumen microbiome on day 122 might also be related to changes in the rumen microbiome. The above results showed that the dietary addition of AMRP decreased both the abundance and diversity of the fecal microbiome, with significant decreases in the number of OTUs and in the Shannon index, Simpson index, Chao1, and ACE indices in the LAMR and MAMR groups (P < 0.050) and significant decreases in the number of OTUs and in the Chao1 and ACE indices in the HAMR group (P < 0.050). The Shannon index and Simpson index did not change significantly (P ≥ 0.050). However, the findings on growth performance and nutrient digestibility described above suggested that the animals did not suffer any adverse effects.
Table 5
Effect of different doses of AMRP on the alpha diversity of the fecal microbiome in Angus calves at different time points.
Items | Diets | SEM | P -value |
ZAMR | LAMR | MAMR | HAMR | L | Q | D |
OTUs |
1 d | 1417.833Aa | 1410.667Aa | 1345.167Aab | 1274.333Ab | 17.807 | 0.010 | 0.270 | 0.005 |
90 d | 1428.333Aa | 1278.167Bb | 1380.833Aab | 1291.000Aab | 24.720 | 0.140 | 0.511 | 0.029 |
122 d | 1096.333Ba | 1053.167Cb | 1071.833Bb | 1076.667Bab | 18.890 | 0.824 | 0.555 | 0.023 |
SEM | 45.615 | 40.070 | 36.217 | 32.975 | |
P | <0.010 | <0.010 | <0.010 | 0.005 |
Shannon index |
1 d | 8.302a | 8.283Aa | 8.226Aab | 8.030b | 0.043 | 0.021 | 0.268 | 0.023 |
90 d | 8.260 | 8.037B | 8.135A | 8.169 | 0.046 | 0.674 | 0.177 | 0.411 |
122 d | 8.169a | 7.930Bb | 7.883Bb | 7.985ab | 0.041 | 0.050 | 0.043 | 0.042 |
SEM | 0.046 | 0.050 | 0.049 | 0.062 | |
P | 0.516 | 0.005 | 0.005 | 0.387 |
Simpson index |
1 d | 0.993 | 0.993A | 0.992A | 0.990 | 0.001 | 0.109 | 0.431 | 0.351 |
90 d | 0.991 | 0.989B | 0.989B | 0.991 | 0.000 | 0.967 | 0.063 | 0.276 |
122 d | 0.993a | 0.990Ba | 0.989Bb | 0.991ab | 0.000 | 0.078 | 0.013 | 0.022 |
SEM | 0.000 | 0.000 | 0.001 | 0.001 | |
P | 0.065 | 0.004 | 0.042 | 0.850 |
Chao1 |
1 d | 1487.549A | 1546.334A | 1407.034B | 1340.197A | 28.250 | 0.016 | 0.219 | 0.148 |
90 d | 1620.672A | 1424.926A | 1549.749A | 1443.090A | 34.767 | 0.179 | 0.504 | 0.148 |
122 d | 1147.283B | 1118.213B | 1134.305C | 1131.793B | 20.811 | 0.880 | 0.768 | 0.974 |
SEM | 58.443 | 54.274 | 46.389 | 43.343 | |
P | <0.010 | <0.010 | <0.010 | 0.004 |
ACE |
1 d | 1491.637Aa | 1511.082Aa | 1414.013Bab | 1347.830Ab | 21.277 | 0.003 | 0.238 | 0.014 |
90 d | 1614.731A | 1421.823A | 1550.119A | 1435.601A | 34.076 | 0.166 | 0.545 | 0.129 |
122 d | 1152.801B | 1121.211B | 1135.105C | 1138.931B | 20.537 | 0.889 | 0.690 | 0.966 |
SEM | 57.470 | 47.206 | 46.170 | 41.333 | |
P | <0.010 | <0.010 | <0.010 | 0.003 |
L represents the linear model, Q represents the quadratic model, and D represents the impact of diet. Lowercase letters with different superscripts in the same row Indicate significant differences (P < 0.050), while uppercase letters with different superscripts in the same columns indicate significant differences (P < 0.050). The same or no letters indicate no significant differences (P ≥ 0.050). The groups were as follows: ZAMR, 0 g/d/calf AMRP added to the basic diet; LAMR, 10 g/d/calf AMRP added to the basic diet; MAMR: 15 g/d/calf AMRP was added to the basic diet; HAMR: 20 g/d/calf AMRP was added to the basic diet.
Analysis of fecal microbiome beta diversity
Analysis of β diversity was used to identify differences in the composition of the microbial communities between the different samples. Principal component analysis (PCA) revealed that on day 1, the fecal microbiota of the test groups was markedly separated from that of the ZAMR group (Fig. 2A), while no significant separation was detected on day 90 (Fig. 2B), and on day 122, the groups overlapped (Fig. 2C).
Screening of core microbes predominant in the fecal microbiota of Angus calves
The core fecal microbes are predominant in the fecal microbiota, suggesting stable symbiosis between the host and core fecal microbes[42]. The core fecal microbes were detected at different times and in different subgroups and are essential for maintaining intestinal health. Thus, the core gut microbes may play vital regulatory roles in host physiology and growth. In this study, we screened the core microbes in Angus calves via 16S rRNA gene sequencing by considering the relative abundances of microbes and “persistent” core microbes measured across timescales. We analysed the bacterial taxonomic compositions (including phylum, class, order, family, and genus) at different times. The 10 most abundant bacterial species were observed in all the samples at days 1, 90 and 122 (Fig. 3). The top 10 most abundant bacteria were the same at days 1, 90 and 122. Thus, we determined that these 10 core fecal microbes (Firmicutes, Clostridia, Bacteroidota, Bacteroidia, Bcateroidales, Oscillospirales, Rikenellaceae, Oscillospiraceae, Ruminococcaceae_UCG-005, and Rikenellaceae_RC9_gut_group) were predominant in the fecal of the Angus calf.
Firmicutes are major cellulose-degrading bacteria that degrade cellulose into volatile fatty acids (VFAs) [43] and participate in the host immune response to prevent inflammation, which may indirectly promote digestion and metabolism in animals [44]. In this study, the relative abundances of Bacteroidia, Bactredales, and Bacteroidota were significantly greater on day 122 than on days 1 and 90 in the HAMR group, and the apparent ADF digestibility was also significantly greater on day 122 than on day 90 in the HAMR group, which may be related to the increased relative abundance of Firmicutes. The relative abundances of Bacteroidia, Bactredales, and Bacteroidota were not affected by the addition of different doses of AMRP on days 1 and 90, and the relative abundances of Bacteroidia, Bactredales, and Bacteroidota were significantly lower in the LAMR, MAMR, and HAMR groups than in the ZAMR group on day 122. The relative abundances of Bacteroidia, Bactredales, and Bacteroidota in the LAMR, MAMR, and HAMR groups were significantly lower than those in the ZAMR group (P = 0.016, P = 0.016, P = 0.025, respectively, Table S1); the relative abundances of Bacteroidia, Bactredales, and Bacteroidota in the ZAMR group increased gradually from day 1 to day 122; and those of Bacteroidia, Bactredales, and Bacteroidota in the LAMR, MAMR, and HAMR groups increased gradually from day 1 to day 90 and then decreased from day 90 to day 122, suggesting that prolonged AMRP feeding may lead to a significant decrease in the relative abundances of Bacteroidia, Bactredales, and Bacteroidota. Bacteroidia, Bactredales, and Bacteroidota play important roles in the digestion and absorption of food and feed components, and in humans, they are involved in the metabolism of polysaccharides and proteins[45]. Ding et al. [25] reported that the addition of AMR to the diet of meat goats significantly changed the relative abundance of Bacteroidota and improved the final weights of the goats. Clostridia are Bacteroidota, and their relative abundance did not change significantly. A study in mice showed that Clostridia plays a key role in obesity by preventing the absorption of fat from the intestinal tract and is also associated with the immune system, with the relative abundance of Clostridia being significantly lower in immunodeficient mice than in normal mice[46]. Oscillospirales and Oscillospiraceae belong to phylum Firmicutes. A study of calf diarrhea revealed that Oscillospirales dominated the gut microbes of healthy and recovered calves, while their relative abundance was low in infected calves[47]. Oscillospiraceae can produce butyrate[48], and butyrate can promote the development of the calf pancreas through antibacterial activity, enhancing the activity of digestive enzymes, etc., and can promote the absorption of nutrients[49]. Rikenellaceae, a member of Bacteroidota, can ferment glucose and produce propionate and succinic acid. Propionate can regulate the homeostasis of the intestinal microbiota, and succinic acid has certain antioxidant effects and can improve the body's immune ability[50]. Ruminococcaceae_UCG-005 is a fibre-degrading bacterium belonging to the Firmicutes, and Ruminococcaceae works synergistically with other microbes [51]. In this study, Ruminococcaceae_UCG-005 tended to increase and then decrease in all groups over time, which may be related to the presence of other Firmicutes; when cellulose and pentosan are broken down into organic acids, the pH decreases, and the relative abundance of Firmicutes decreases, while the relative abundance of Ruminococcaceae increases[52]. The Rikenellaceae_RC9_gut_group is associated with VFAs and may affect fat deposition in meat and is a member of the Bacteroidota [53]. In this study, the relative abundance of Rikenellaceae_RC9_gut_group in the ZAMR group gradually increased with time, while that in the experimental groups tended to increase and then decrease with time, indicating that the time of AMR supplementation had a greater effect than the AMR dose. In this study, the addition of different doses of AMRP did not change the status of Firmicutes or Bacteroidota as core microbes but affected their relative abundance, which may have affected their growth performance and apparent digestibility.
Screening for difference in microbes predominant in the fecal microbiota of Angus calves
Histograms of the LDA distribution (Fig. 4a, b, c) and evolutionary branching diagrams (Fig. 4d, e, f) of the different groups were obtained by LEfSe analysis. Histograms of the main differential microbes in the ZAMR group (Fig. 5a), LAMR group (Fig. 5b), MAMR group (Fig. 5c) and HAMR group (Fig. 5d) at different times were generated based on the results of LEfSe analysis.
The bacteria that showed differential abundance between the ZAMR group and the other groups were Bacteroidota, Bacteroidales, Rikenellaceae, UCG-010, and Alistipes. The relative abundance of UCG-010 decreased with time; UCG-010 plays an important role in fibre digestion, and a decrease in its abundance may lead to a decrease in the host's ability to digest fibre. In this study, the ZAMR group exhibited reduced NDF and ADF digestion over time. The relative abundance of other bacteria increased with time. Bacteroidota and Bacteroidales are gram-negative bacteria that are more capable of utilizing carbon sources and energy and can metabolize organic substances such as fatty acids, sugars, and aromatic compounds [45]. Alistipes belongs to the phylum Bacteroidota, which has been linked to obesity [54], and the increase in its relative abundance in the ZAMR group corresponded with reduced apparent digestibility and growth performance in this group.
The differentially abundant bacteria between the LAMR group and the other groups were Rikenellaceae, Rikenellaceae_RC9_gut_group, Alistipes, Bacilli, and Eubacterium_corprostanoligenes_group. Unlike in the ZAMR group, the relative abundance of Alistipes decreased in the LAMR group with time, suggesting that the addition of AMRP may have reduced the likelihood of host obesity and was thus more conducive to host health. The abundance of Bacilli, a potentially pathogenic bacterium in the gut, tended to decrease and then increase with time, and the change in abundance suggested that it was regulated by the dosage of AMRP; however, it may be that as the duration of AMRP treatment increased, the bacteria became unresponsive, thus increasing the chances of host disease. It has been found that the Rikenellaceae_RC9_gut_group is significantly and positively correlated with fat and volatile fatty acids such as butyric acid, which are found in meat, suggesting that it may affect fat accumulation in meat [53]. In the present study, the relative abundance of Rikenellaceae_RC9_gut_group increased, which may be associated with fat accumulation and increased volatile fatty acids. It has also been shown that the Eubacterium_corprostanoligenes_group may be associated with liver fibrosis and intestinal inflammation [55, 56]. Changes in the abundance of differentially abundant bacteria in the LAMR group favoured intestinal health and improved EE, P, NDF, and ADF apparent digestibility and ADG and feed efficiency.
The differentially abundant bacteria between the MAMR group and the other groups were Oscillospirales, Clostridia, Oscillospiraceae, Ruminococcaceae_UCG-005, Verrucomicrobiota, Akkermansia, Akkermansiaceae, and Verrucomicrobiae. The relative abundances of Oscillospirales, Clostridia, Oscillospiraceae, and Ruminococcaceae_UCG-005 did not change significantly over time and remained stable. The relative abundances of Verrucomicrobiota, Akkermansia, Akkermansiaceae, and Verrucomicrobiae significantly increased on day 122, and one study revealed that the deficiency or reduction of Akkermansia, a new genus of Verrucomicrobiota, is associated with a variety of diseases (e.g., obesity, diabetes, fatty liver, inflammation, and cancer), and this microbe has been described as representative of a new generation of beneficial bacteria [57]. Their relative abundance significantly increased in this study, which may have reduced the likelihood of host disease.
The bacteria that were differentially abundant between the HAMR group and the other groups were Spirochaetia, Peptostreptococcales-Tissierellales, Peptostreptococcacese, Muribaculaceae, Clostridiaceae, and Clostridium_sensu_stricto_1. The relative abundances of Spirochaetia and Peptostreptococcacese significantly decreased over time, while the relative abundances of Peptostreptococcales_Tissierellales and Muribaculacrae significantly increased. The relative abundances of Clostridiaceae and Clostridium_sensu_stricto_1 first decreased and then increased significantly. Peptostreptococcaceae can cause infections in various tissues and organs of the host, including abdominal, pelvic, and vaginal infections, which is detrimental to the health of the host [58]. Muribaculacrae are commensal intestinal bacteria that interact with the host's intestinal cells, degrade complex polysaccharides such as cellulose and pectin, and produce short-chain fatty acids, as well as inhibit the proliferation and infestation of some pathogenic bacteria, which is beneficial to the host's health. In the present study, their relative abundance was significantly greater, suggesting that they benefit the health of the host. These results suggest that AMRP, when added to the diet, can selectively regulate the intestinal flora by inhibiting bacteria associated with obesity and potential diseases (Alistipes, Bacilli, etc.) and stimulating health-promoting bacteria (Verrucomicrobiota, Akkermansia and Muribaculaceae). These different microbes are associated with a variety of diseases, and changes in their relative abundance are also relevant to host gut health. The addition of AMRP reduced the relative abundance of a variety of disease-associated microbes, which may be beneficial to the gut health of the host by promoting both digestion and nutrient absorption and improving growth performance.
Correlation analysis of differential fecal microbes with growth performance and apparent nutrient digestibility
Pearson's correlation analysis was used to investigate the relationships between different groups of differential bacterial flora and growth performance and apparent nutrient digestibility (Fig. 6). The abundances of Bacteroidota and Bacteroidales were significantly negatively correlated with ADG (r2 = -0.999 and r2 = -1.000, P = 0.011 and P = 0.007, respectively), and n increase in their relative abundance may have decreased ADG. Ruminococcaceae_UCG-010 was significantly negatively correlated with the F/G values, CP apparent digestibility, and Ca apparent digestibility (r2 = -0.997, r2 = -0.989, and r2 = -0.994, P = 0.023, P = 0.048, and P = 0.035, respectively), and its relative abundance decreased, which may have caused Ca and CP apparent digestibility to increase and feed efficiency to decrease. Rikenellaceae showed a significant negative correlation with EE apparent digestibility (r2 = -0.993, P = 0.037), and its relative abundance increased, potentially reducing EE apparent digestibility. Alistipe showed a significant positive correlation with apparent P digestibility (r2 = 0.997, P = 0.012) and a significant negative correlation with apparent CP digestibility (r2 = -0.999, P = 0.025), and an increase in its relative abundance may lead to an increase in apparent P digestibility and a decrease in apparent CP digestibility. Bacilli showed a significant positive correlation with the apparent digestibility of ADF (r2 = 0.994, P = 0.036) and a significant negative correlation with the apparent digestibility of OM (r2 = -0.999, P = 0.025), and an increase in its relative abundance may have resulted in an increase in the apparent digestibility of OM and a decrease in the apparent digestibility of ADF. Eubacterium_corprostanoligenes_group was positively correlated with apparent EE digestibility (r2 = 1.000, P = 0.001), and an increase in its relative abundance may lead to increased EE apparent digestibility. Oscillospirales was negatively associated with Ca, CP apparent digestibility, and DMI (r2 = -0.998, r2 = -0.999 and r2 = -0.988, P = 0.021, P = 0.011 and P = 0.049, respectively), and an increase in its relative abundance may lead to higher Ca, CP apparent digestibility, and DMI. Ruminococcaceae_UCG-005 was negatively associated with DM apparent digestibility (r2 = -0.999, P = 0.001), and a decrease in its relative abundance may lead to an increase in DM apparent digestibility. The abundance of Akkermansiaceae was negatively correlated with the apparent P digestibility (r2 = -0.990, P = 0.044), and an increase in its relative abundance may result in reduced apparent P digestibility. Spirochaetia showed a significant negative correlation with Ca and CP apparent digestibility (r2 = -0. 998 and r2 = -0.992, P = 0.020 and P = 0.040, respectively), and reduced relative abundance may lead to increased Ca and CP apparent digestibility. The abundance of Peptostreptococcaceae was significantly negatively correlated with the apparent digestibility of EE and CP (r2 = -0.991 and r2 = -0.999, P = 0.044 and P = 0.013, respectively), and a decrease in their relative abundance may thus result in increased apparent digestibility of EE and CP. Correlation analysis revealed significant associations between several differentially abundant microbes and growth performance and apparent nutrient digestibility among the groups, enhancing the understanding of the relationships among the gut microbiota, growth performance, and apparent nutrient digestibility, which can lay the foundation for subsequent studies. However, the specific mechanisms underlying the effects of these microbes on nutrient digestion and growth performance require further investigation.