Rumen fermentation
There were no significant differences in rumen pH for the same sampling intervals (0, 4, and 8 h; P > 0.05; Table 2) and all the collected mean among the HD, CON, and LD treatments (0, 4, and 8 h; P > 0.05). The variation of DCAD had no effect on ruminal BC and levels of acetic acid, propionic acid, butyric acid, total VFA (TVFA), and acetic acid/propionic acid (A/P) in the goats (P > 0.05).
Table 2 Effect of dietary cation–anion difference on the rumen pH of goats
Items
|
DCAD
|
SEM1
|
P-value
|
HD (+338)
|
CON +152)
|
LD (-181)
|
pH
|
|
|
|
|
|
Sampling time interval (h)
|
Sampling time
|
|
|
|
|
|
0
|
09:00
|
7.22 ± 0.19
|
7.21 ± 0.41
|
7.17 ± 0.36
|
0.14
|
0.97
|
|
13:00
|
7.10 ± 0.22
|
7.17 ± 0.57
|
6.99 ± 0.49
|
0.23
|
0.86
|
|
17:00
|
7.07 ± 0.25
|
7.20 ± 0.66
|
6.98 ± 0.54
|
0.26
|
0.83
|
4
|
13:00
|
7.05 ± 0.31
|
6.96 ± 0.36
|
7.04 ± 0.34
|
0.18
|
0.93
|
|
17:00
|
7.08 ± 0.38
|
6.96 ± 0.52
|
7.14 ± 0.47
|
0.24
|
0.87
|
8
|
17:00
|
7.15 ± 0.23
|
6.99 ± 0.38
|
7.08 ± 0.46
|
0.19
|
0.86
|
Mean
|
7.20 ± 0.22
|
7.18 ± 0.41
|
7.17 ± 0.34
|
0.14
|
0.98
|
BC2 (mL/L)
|
44.75 ± 0.88
|
43.35 ± 3.65
|
43.10 ± 3.81
|
1.38
|
0.67
|
Acetic acid (mmol/L)
|
42.22 ± 4.86
|
43.45 ± 5.71
|
44.81 ± 6.36
|
2.32
|
0.74
|
Propionic acid (mmol/L)
|
13.00 ± 1.96
|
13.07 ± 1.75
|
12.71 ± 2.37
|
0.84
|
0.95
|
Butyric acid (mmol/L)
|
8.00 ± 1.78
|
8.48 ± 2.28
|
8.11 ± 2.02
|
0.83
|
0.91
|
TVFA3 (mmol/L)
|
63.21 ± 8.08
|
65.01 ± 8.89
|
65.63 ± 8.10
|
3.41
|
0.87
|
A/P4
|
3.27 ± 0.34
|
3.34 ± 0.36
|
3.67 ± 1.13
|
0.29
|
0.60
|
1SEM=standard error of mean. The same as below.
2BC=Buffering capability.
3TVFA = acetic acid + propionic acid + butyric acid.
4A/P = acetic acid/propionic acid.
Rumen cellulolytic bacteria
The relative contents of F. succinogenes, R. flavefaciens, B. fibrisolvens, and R. albus were not significantly affected among goats fed HD, CON, and LD diets (P > 0.05; Table 3). The proportions of F. succinogenes and R. flavefaciens were markedly increased with lower DCAD compared to B. fibrisolvens and R. albus.
Table 3 Effect of dietary cation–anion difference on the rumen cellulolytic bacteria communities of goats
Items
|
DCAD
|
SEM
|
P-value
|
HD (+338)
|
CON (+152)
|
LD (-181)
|
Fibrobacter succinogenes (%)
|
0.443±0.003
(48.63 %)
|
0.362±0.003
(41.18 %)
|
0.324±0.001
(62.91%)
|
0.098
|
0.70
|
Ruminococcus flavefaciens (%)
|
0.343±0.21
(37.65 %)
|
0.414±0.86
(47.10 %)
|
0.119±0.09
(23.11 %)
|
0.210
|
0.51
|
Butyrivibrio fibrisolvens (%)
|
0.12±0.12
(13.17 %)
|
0.085±0.07
(9.67 %)
|
0.065±0.03
(12.62 %)
|
0.033
|
0.59
|
Ruminococcus albus (%)
|
0.005±0.01
(0.55 %)
|
0.018±0.03
(2.05 %)
|
0.007±0.01
(1.36 %)
|
0.008
|
0.50
|
Sequencing and diversity of ruminal microbiota
After Illumina Miseq high-throughput sequencing, a total of 698,626 valid reads were obtained with an average length of 410 bp. The VENN graph showed that there were 1075 of total 1261 OTU in 3 groups shared. There were 22, 25, 5 individual OTUs, accounting for 1.89%, 2.33%, and 0.43% for LD, HD, CON, respectively (Fig. 1). Rarefaction curves were established to quantify the OUT coverage of sampling and each rarefaction tended to be gentle with the increase of sequence number, and meanwhile, the OTU rank abundance in the 3 groups exhibited a gentler slope and wider distribution on the horizontal axis (Fig. 2).
Fig. 1 The VENN graph of rumen bacterial of goats fed diets with varying cation-anion difference levels, which displaying that the disposition of operational taxonomic units (OTUs) among the control group (CON, n=6), high dietary cation-anion difference (HD, n=6) and low dietary cation-anion difference (LD, n=6) in rumen fluid microbiota.
Fig. 2 Rarefaction curves (A) and the OTU rank abundance (B) of rumen bacterial of goats fed diets with varying cation-anion difference levels. The CON group: CON1, CON2, CON3, CON4, CON5, CON6. The HD group: HD1, HD2, HD3, HD4, HD5, HD6. The LD group: LD1, LD2, LD3, LD4, LD5, LD6.
Alpha diversity results showed that DCAD levels did not affect Chao, Ace, Simpson, and Shannon as listed in Table 4 (P > 0.05). The Chao, Ace, Shanno and Simpson chart of each group was also tended to be gentle corresponding to the increase of sequence number (Fig. 3).
Table 4 Effect of varying dietary cation-anion difference levels on rumen bacterial community richness and diversity of goats
Items
|
Dietary treatments1
|
SEM
|
P-value
|
HD (+338)
|
CON (+152)
|
LD (−181)
|
Bacterial Richness
|
Chao
|
906±89
|
843±129
|
811±116
|
4.28
|
0.333
|
Ace2
|
900±82
|
834±117
|
815±96
|
4.33
|
0.356
|
Bacterial Diversity
|
Simpson
|
0.027±0.02
|
0.033±0.01
|
0.028±0.01
|
0.04
|
0.760
|
Shannon
|
4.87±0.31
|
4.45±0.21
|
4.69±0.29
|
0.21
|
0.048
|
1Dietary treatments: HD: High dietary cation-anion difference; CON:Control group; LD: Low dietary cation-anion difference.
2Ace=abundance-based coverage estimator.
Fig. 3 Rarefaction curves (A) and the OTU rank abundance (B) of rumen bacterial of goats fed diets with varying cation-anion difference levels. The CON group: CON1, CON2, CON3, CON4, CON5, CON6. The HD group: HD1, HD2, HD3, HD4, HD5, HD6. The LD group: LD1, LD2, LD3, LD4, LD5, LD6.
According to Fig. 4, both weighted UniFrac (axis 1 + axis 2 = 66.7%, Fig. 4a) and unweighted UniFrac (axis 1 + axis 2 = 38.37%, Fig. 4b) were observed no difference in the composition of rumen microbiota for CON, HD and LD (P > 0.05).
Fig. 4 Principle coordinate analysis (PCoA) based on weighted UniFrac and unweighted UniFrac on rumen bacterial of goats fed diets with varying cation-anion difference levels. (a) PCoA based on weighted UniFrac of rumen bacterial of goats fed diets with varying cation-anion difference levels. (b) PCoA based on unweighted UniFrac of rumen bacterial of goats fed diets with varying dietary cation-anion difference levels. The percentage of variation explained by PC1 (first principal component) and PC2 (second principal component) was indicated in the axis. CON = the control group (n = 6); HD= high dietary cation-anion difference (n = 6); LD = low dietary cation-anion difference (n = 6).
Taxonomic classification summary indicated that 16 phyla were tested in all samples (Fig. 5A). At the phylum level, Bacteroidetes (61.60%) was the predominant phylum followed by Firmicutes, Synergistetes, Proteobacteria, Spirochaetae, Tenericutes with average relative abundances of 25.32%, 5.84%, 1.82%, 2.08%, 1.2%, respectively, but there was no difference (P > 0.05) among the groups on the above phylum levels except for Firmicutes, which was significantly higher in HD and LD compared to CON (P=0.008, Table 5).
At the genus level, taxon displayed that the relative abundance of 11 genera were not affected by DCAD among all samples (P > 0.05; Fig. 5B). At the same time, Prevotella, Paraprevotella, Selenomonas, Ruminococcus, Ruminococcus, Butyrivibrio, Quinella, Fretibacterium and Treponema showed no grouping difference of the genera across treatments (P > 0.05). Among the genera with relative abundance exceeded 0.1%, prevotella was the dominant genus in each group with the highest proportion (Table 5).
Fig. 5 Distributions of microbiota at phyla and genus level. (a) Relative abundances of phyla levels are depicted as mean values for the CON, the LD (Low dietary cation-anion difference) group and HD (High dietary cation-anion difference) group. (b) Distributions of genera in rumen fluid of the CON, the LD (Low dietary cation-anion difference) group and HD (High dietary cation-anion difference) group.
Table 5 Effect of varying dietary cation-cation difference on relative abundance (%) of bacteria taxa > 0.1% of average abundance in the rumen fluid of goats
Phylum
|
Genus
|
Dietary treatments1
|
SEM
|
P-value
|
HD
|
CON
|
LD
|
Bacteroidetes
|
|
60.40±6.24
|
65.08±4.41
|
59.33±9.13
|
2.81
|
0.328
|
|
Prevotella
|
18.92±3.66
|
24.16±2.72
|
16.09±2.68
|
3.05
|
0.223
|
|
Paraprevotella
|
3.58±1.23
|
2.76±0.89
|
3.51±1.52
|
1.24
|
0.877
|
Firmicutes
|
|
28.43±4.90a
|
18.71±5.30b
|
28.81±6.08a
|
2.23
|
0.008
|
|
Selenomonas
|
1.59±0.97
|
0.56±0.29
|
1.32±0.89
|
0.78
|
0.629
|
|
Ruminococcus
|
1.07±0.16
|
1.54±0.74
|
1.29±0.39
|
0.49
|
0.786
|
|
Succiniclasticum
|
1.34±0.36
|
0.69±0.09
|
0.68±0.24
|
0.25
|
0.143
|
|
Butyrivibrio
|
1.02±0.32
|
0.46±0.13
|
0.48±0.01
|
0.20
|
0.118
|
|
Quinella
|
2.29±0.37
|
0.71±0.30
|
3.73±2.15
|
1.27
|
0.277
|
Synergistetes
|
|
5.20±1.87
|
6.34±1.91
|
5.99±2.29
|
2.03
|
0.923
|
|
Fretibacterium
|
5.18±1.88
|
6.31±1.92
|
5.97±2.30
|
2.04
|
0.923
|
Spirochaetae
|
|
1.44±0.25
|
2.13±0.74
|
1.89±0.35
|
0.49
|
0.610
|
|
Treponema
|
0.72±0.16
|
0.98±0.52
|
1.39±0.35
|
0.37
|
0.458
|
Proteobacteria
|
|
1.10±0.42
|
4.49±2.38
|
0.67±0.07
|
1.40
|
0.140
|
Tenericutes
|
|
1.07±0.17
|
1.20±0.16
|
1.33±0.44
|
0.29
|
0.817
|
Growth performance
Levels of DMI were unaffected by DCAD variations (P > 0.05; Table 6). Lower DCAD had no effect (P > 0.05) on gwowth performance of final weight, ANG, ADG, and FCR and digestibility of crude protein, NDF, ADF, and OM for goats.
Table 6 Effect of dietary cation–anion difference on growth performance of goats
Items
|
DCAD
|
SEM
|
P-value
|
HD (+338)
|
CON (+152)
|
LD (-181)
|
Performance
|
|
|
|
|
|
Initial weight (kg)
|
30.35 ± 3.4
|
30.43 ± 2.6
|
29.44 ± 3.7
|
1.36
|
0.86
|
DMI (g/d)
|
899.0 ± 213.6
|
857.5 ± 120.9
|
864.0 ± 124.3
|
64.85
|
0.89
|
Final weight (kg)
|
31.33 ± 3.4
|
31.50 ± 3.8
|
30.41 ± 3.3
|
1.48
|
0.82
|
ANG (kg)
|
0.98 ± 0.3
|
1.07 ± 0.4
|
0.97 ± 0.3
|
0.21
|
0.89
|
ADG (g/d)
|
65.3 ± 12.2
|
71.3 ± 22.7
|
64.7 ± 18.8
|
8.28
|
0.63
|
FCR
|
13.76 ± 6.6
|
12.02 ± 3.5
|
13.36 ± 5.9
|
3.00
|
0.81
|
Digestibility (%)
|
|
|
|
|
|
CP (%)
|
56.37 ± 2.41
|
58.66 ± 5.91
|
57.68 ± 5.42
|
1.97
|
0.75
|
NDF (%)
|
50.26 ± 6.01
|
45.84 ± 8.39
|
45.58 ± 7.65
|
3.12
|
0.50
|
ADF (%)
|
51.19 ± 6.84
|
48.35 ± 7.74
|
47.59 ± 8.27
|
3.20
|
0.71
|
OM (%)
|
62.34 ± 3.59
|
57.73 ± 5.66
|
58.59 ± 3.92
|
2.11
|
0.36
|
Urine pH
There was no difference (P > 0.05) in urine pH for HD, CON, and LD during the observation period of 1–12 d, with pH values of 8.48, 8.43, and 8.46, respectively (Fig. 6a). Urine pH decreased slightly (8.45, 8.50, and 8.13 for HD, CON, and LD, respectively) during the dietary replacement period (13–18 d). Urine pH decreased significantly (8.43, 8.36, and 7.40 for HD, CON, and LD, respectively) with LD obviously lower than both HD and CON. During the trial period (31–45 d), compared with HD and CON, LD reduced urine pH over HD and CON (8.43, 8.42, and 6.75 for HD, CON, and LD, respectively; P < 0.05). Urine pH values were unaffected by DCAD variation between HD and CON (P > 0.05). Furthermore, urine pH was closely related to DCAD levels within the trial period (31–45 d; R2 = 0.9066, P < 0.05; Fig. 6b).
Fig. 6a Goat urine pH for dietary cation–anion difference levels throughout the experiment
Fig. 6b Association between urine pH and dietary cation-anion difference of goats in the trial period
Plasma Metabolites
Feeding of the LD diet resulted in the highest plasma Ca level (Table 7), which was significantly higher than both HD and CON (P < 0.05).There were no significant differences in plasma Glu, UN, ALT, AST, AKP, TP, Alb, GSH-Px, CAT, SOD, and MDA among the DCAD treatments (P > 0.05; Table 7).
Table 7 Effect of dietary cation–anion difference on the plasma metabolites of goats
Items
|
DCAD
|
SEM1
|
P-value
|
HD (+338)
|
CON (+152)
|
LD (−181)
|
Ca (mmol/L)
|
2.31 ± 0.21
|
2.38 ± 0.17
|
2.91 ± 0.17
|
0.08
|
<0.01
|
Glu (mmol/L)
|
4.57 ± 1.06
|
5.07 ± 0.91
|
4.60 ± 0.36
|
0.34
|
0.56
|
UN (mmol/L)
|
6.22 ± 0.66
|
5.63 ± 1.09
|
6.24 ± 1.16
|
0.41
|
0.50
|
ALT (IU/L)
|
8.61 ± 2.55
|
10.22 ± 3.21
|
10.76 ± 3.57
|
1.70
|
0.85
|
AST (IU/L)
|
11.25 ± 3.63
|
13.98 ± 4.77
|
11.91 ± 3.41
|
2.01
|
0.80
|
AKP
(King unit/100 mL)
|
20.85 ± 6.90
|
21.73 ± 12.57
|
17.81 ± 6.92
|
3.75
|
0.75
|
TP (g/L)
|
99.38 ± 14.10
|
112.89 ± 28.72
|
92.62 ± 21.62
|
11.47
|
0.37
|
Alb (g/L)
|
36.38 ± 4.34
|
37.11 ± 6.36
|
35.19 ± 6.31
|
2.35
|
0.85
|
GSH-Px (U/mL)
|
670.59 ± 127.49
|
755.29±296.72
|
709.41 ± 164.46
|
152.50
|
0.59
|
CAT (U/mL)
|
2.35 ± 0.79
|
1.95 ± 0.60
|
2.47 ± 1.03
|
0.66
|
0.84
|
SOD (U/mL)
|
68.53 ± 9.27
|
63.59 ± 8.59
|
67.98 ± 6.79
|
3.38
|
0.43
|
MDA (nmol/mL)
|
37.14 ± 5.57
|
34.61 ± 7.06
|
36.79 ± 5.94
|
2.54
|
0.75
|