The nutritional compositions of the raw alfalfa and acacia tree leaves are presented in Table 1. The empirical distribution of the observations for all observed traits was normal. The results of the MANOVA indicated that the groups were significantly (p < 0.001) different when investigated jointly in terms of all traits. The ANOVA results indicated that the main effects of the groups were significant for all observed traits, except for silage physical characteristics (smell, color, and structure: Table 3), crude ash (Table 2), and pH (Table 4). In the present study, we used three different levels of dried acacia leaves 3% (CT; 3.75 g/kg of silage), 6% (CT; 7.5 g/kg of silage), and 12% (CT; 15 g/kg of silage).
Table 1
Nutritional composition of raw alfalfa and acacia tree leaves (mean ± standard deviation).
Parameters | Feed |
| Alfalfa | Acacia leaves |
Dry Matter (DM) % | 94.57 ± 0.18 | 96.18 ± 1.27 |
Crude Protein (CP) % | 27.07 ± 0.72 | 30.43 ± 0.97 |
Neutral Detergent Fiber (NDF) % | 57.48 ± 1.50 | 42.91 ± 1.11 |
Acid Detergent Fiber (ADF) % | 36.86 ± 2.16 | 23.71 ± 2.78 |
Crude Fiber (CF) % | 33.75 ± 2.65 | 11.88 ± 0.32 |
Ether Extract (EE) % | 1.26 ± 0.49 | 1.83 ± 0.06 |
Crude Ash (CA) % | 7.49 ± 0.12 | 6.45 ± 0.49 |
Condensed Tannins (CT) % | | 12.5 ± 0.5 |
Dry Matter of Fresh Forage | 27.1 ± 1.21 | 32.2 ± 0.65 |
Table 2
Mean values and standard deviations (s.d.) as well as least significant differences (LSDs) and F statistics from one-way analysis of variance (ANOVA) for silage chemical compositions.
Trait | Group | C | M5 | A3 | A6 | A12 | M5A3 | M5A6 | M5A12 | LSD0.05 | F-ANOVA |
CP % | Mean | 26.22bc | 25.57cd | 25.46cd | 25.01d | 25.41cd | 26.91ab | 26.07bc | 27.59a | 1.042 | 5.83*** |
s.d. | 0.188 | 0.282 | 1.339 | 0.628 | 0.512 | 1.070 | 0.329 | 0.508 | | |
NDF % | Mean | 47.9c | 41.35e | 52.27b | 53.17b | 55.75a | 43.99d | 47.11c | 46.55c | 2.046 | 48.28*** |
s.d. | 0.371 | 1.172 | 0.532 | 1.339 | 0.561 | 1.484 | 2.768 | 1.398 | | |
ADF % | Mean | 37.34c | 30.14f | 40.52a | 38.66b | 39.44ab | 31.16ef | 34.23d | 32.35e | 1.22 | 92.41*** |
s.d. | 0.559 | 0.912 | 1.114 | 0.398 | 0.743 | 0.863 | 1.015 | 0.850 | | |
CF % | Mean | 35.85a | 28.03c | 36.66a | 31.7b | 31.96b | 27.57c | 28.93c | 25.14d | 1.598 | 55.12*** |
s.d. | 0.422 | 0.705 | 2.037 | 0.624 | 0.904 | 0.147 | 0.566 | 1.794 | | |
EE % | Mean | 2.082d | 2.72ab | 2.39c | 2.119d | 2.443c | 2.441c | 2.548bc | 2.769a | 0.21 | 11.98*** |
s.d. | 0.091 | 0.126 | 0.084 | 0.224 | 0.183 | 0.135 | 0.035 | 0.177 | | |
CA % | Mean | 8.545ab | 7.049c | 8.395ab | 7.837abc | 7.468bc | 8.656a | 8.728a | 8.263ab | 1.15 | 2.38 |
s.d. | 0.417 | 1.142 | 1.794 | 0.112 | 0.086 | 0.261 | 0.246 | 0.347 | | |
CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; CF, crude fiber; EE, ether extract; CA, crude ash. *** p < 0.001; In rows, mean values marked with the same letter are not statistically different.
Table 3
Alfalfa silage physical (smell, color and structure) and fermentation parameters
Trait Group
|
C
|
M5
|
A3
|
A6
|
A12
|
M5A3
|
M5A6
|
M5A12
|
LSD0.05
|
F-ANOVA
|
pH
|
Mean
|
5.793c
|
4.348e
|
5.97bc
|
6.133a
|
6.047ab
|
4.357e
|
4.675d
|
4.77cd
|
0.172
|
178.27***
|
s.d.
|
0.061
|
0.039
|
0.237
|
0.139
|
0.081
|
0.064
|
0.125
|
0.062
|
|
|
NH3-N (mg/dl)
|
Mean
|
7.112a
|
1.573c
|
6.89a
|
3.625b
|
3.458b
|
4.41b
|
3.493b
|
3.567b
|
1.287
|
17.93***
|
s.d.
|
1.423
|
0.317
|
0.245
|
1.048
|
1.160
|
0.677
|
0.864
|
0.625
|
|
|
DM %
|
Mean
|
23.29e
|
29.09d
|
24.89e
|
29.03d
|
32.99b
|
30.59cd
|
31.5bc
|
34.98a
|
1.943
|
34.63***
|
s.d.
|
1.248
|
1.695
|
1.561
|
0.532
|
1.192
|
1.938
|
0.618
|
1.211
|
|
|
LA %
|
Mean
|
90.61a
|
90.17a
|
88.4a
|
89.81a
|
89.69a
|
90.44a
|
89.61a
|
91.27a
|
1.825
|
20.36***
|
s.d.
|
0.574
|
0.682
|
2.372
|
0.878
|
1.218
|
1.826
|
0.435
|
0.553
|
|
|
AA %
|
Mean
|
3.491e
|
6.677c
|
5.032d
|
5.538d
|
6.724c
|
7.866b
|
7.218bc
|
7.409bc
|
0.783
|
38.15***
|
s.d.
|
0.299
|
0.221
|
1.080
|
0.593
|
0.534
|
0.503
|
0.318
|
0.074
|
|
|
PA %
|
Mean
|
1.69bc
|
0.654d
|
3.511a
|
1.828bc
|
1.786bc
|
1.291bcd
|
2.074b
|
1.125cd
|
0.9
|
7.55***
|
s.d.
|
0.255
|
0.374
|
0.750
|
0.367
|
0.531
|
1.183
|
0.550
|
0.392
|
|
|
BA %
|
Mean
|
4.211a
|
2.499b
|
5.053a
|
2.82b
|
1.799c
|
0.401d
|
1.099cd
|
0.201d
|
0.952
|
28.51***
|
s.d.
|
0.365
|
1.232
|
0.669
|
0.208
|
0.252
|
0.803
|
0.730
|
0.149
|
|
|
Smell
|
Mean
|
11ab
|
12.25a
|
8.67b
|
9.5ab
|
10.25ab
|
10.75ab
|
10.5ab
|
10ab
|
3.137
|
0.98
|
s.d.
|
1.155
|
2.062
|
1.886
|
3.416
|
1.708
|
2.217
|
2.380
|
1.633
|
|
|
Color
|
Mean
|
1.25a
|
1.75a
|
1.25a
|
1.25a
|
1a
|
1.5a
|
1.25a
|
1.25a
|
1.246
|
0.27
|
s.d.
|
0.957
|
0.500
|
0.957
|
0.957
|
0.817
|
1.000
|
0.957
|
0.500
|
|
|
Structure
|
Mean
|
3.25a
|
3a
|
2.667a
|
2.75a
|
3.25a
|
3.25a
|
2.5a
|
2.75a
|
1.616
|
0.29
|
s.d.
|
0.957
|
1.414
|
1.247
|
0.957
|
0.957
|
0.957
|
1.291
|
0.957
|
|
|
DM, dry matter; NH3 -N, ammonia nitrogen concentration; AA, acetic acid; PA, propionic acid; BA, butyric acid; LA, lactic acid. *** p < 0.001; In rows, mean values marked with the same letter are not statistically different.
Table 4
In vitro ruminal fermentation characteristics of alfalfa silage
Trait | Group | C | M5 | A3 | A6 | A12 | M5A3 | M5A6 | M5A12 | LSD0.05 | F-ANOVA |
pH | Mean | 6.957a | 6.95a | 6.967a | 6.987a | 6.97a | 6.967a | 6.96a | 6.97a | 0.038 | 0.71 |
s.d. | 0.029 | 0.014 | 0.025 | 0.038 | 0.014 | 0.017 | 0.008 | 0.042 | | |
TVFAs (mmol/L) | Mean | 71.75a | 71.47a | 63.92c | 63.3c | 60.7d | 67.8b | 64.67c | 64.22c | 2.307 | 25.35*** |
s.d. | 0.991 | 0.450 | 1.559 | 0.324 | 0.268 | 0.521 | 3.793 | 1.243 | | |
NH3-N (mg/dl) | Mean | 374a | 353ab | 316.3cd | 315.7cd | 299.3d | 327c | 351.7b | 309.7cd | 21.493 | 12.34*** |
s.d. | 8.160 | 5.350 | 3.400 | 4.030 | 16.440 | 3.740 | 36.310 | 3.090 | | |
Net gas (ml/200mg) | Mean | 25.05f | 40.31a | 22.68g | 27.27e | 25.34f | 36.9b | 33.35c | 31.86d | 0.943 | 376.59*** |
s.d. | 1.167 | 0.756 | 0.363 | 0.555 | 0.000 | 0.961 | 0.000 | 0.210 | | |
CH4 (ml/200mg) | Mean | 4.852ef | 7.505a | 4.195g | 4.993e | 4.773f | 6.755b | 6.238c | 5.879d | 0.211 | 245.71*** |
s.d. | 0.237 | 0.085 | 0.081 | 0.186 | 0.030 | 0.049 | 0.223 | 0.100 | | |
CH4 (%) | Mean | 19.37a | 18.62bc | 18.5bc | 18.3c | 18.83b | 18.32c | 18.71bc | 18.45bc | 0.509 | 4** |
s.d. | 0.092 | 0.142 | 0.251 | 0.313 | 0.119 | 0.443 | 0.670 | 0.355 | | |
TVFAs: total volatile fatty acids; NH3-N: ammonia nitrogen; CH4: methane. *** p < 0.001; In rows, mean values marked with the same letter are not statistically different.
Mean values of in vitro dry matter degradability after 48 h varied from 40.06% (for A3) to 59.63% (for M5). True neutral detergent fiber digestibility varied from 50.69% (for A12) to 66.92% (for M5). True organic matter digestibility 48 h varied from 93.91% (for A3) to 94.98% (for control). True dry matter digestibility varied from 230.6 mg (for A12) to 300.9 mg (for M5). Partioning factor varied from 3.233 mg/ml (for M5A3) to 4.557 mg/ml (for A3). Microbial protein varied from 85.6 mg/g (for M5A3) to 122.7 mg/g (for control). Microbial protein synthesis efficiency varied from 31.89% (for M5A3) to 51.62% (for A3). Truly degradable substrate varied from 50.37% (for A12) to 66.63% (for M5) (Table 5).
Table 5
In vitro dry matter degradability, true NDF degradability and true organic matter degradability parameters after 48 hours of daisy incubation
Trait | Group | C | M5 | A3 | A6 | A12 | M5A3 | M5A6 | M5A12 | LSD0.05 | F-ANOVA |
IVDMD % | Mean | 45.02d | 59.63a | 40.06e | 41.5e | 42.37e | 52.06b | 49.7c | 48.59c | 2.494 | 58.2*** |
s.d. | 2.781 | 1.398 | 1.791 | 0.837 | 0.133 | 1.588 | 1.246 | 2.382 | | |
TNDFD % | Mean | 57.11c | 66.92a | 52.66e | 53.34de | 50.69e | 60.55b | 58.52bc | 55.78cd | 2.786 | 29.3*** |
s.d. | 3.381 | 1.583 | 2.448 | 0.893 | 2.530 | 1.072 | 0.865 | 0.358 | | |
TOMD % | Mean | 94.98a | 94.81bc | 93.91f | 94.36e | 93.96f | 94.77c | 94.86b | 94.58d | 0.056 | 456.35*** |
s.d. | 0.017 | 0.036 | 0.031 | 0.030 | 0.063 | 0.060 | 0.028 | 0.010 | | |
TDMD (mg) | Mean | 246.6de | 300.9a | 232.4f | 236.5ef | 230.6f | 268.2b | 263.3bc | 254.9cd | 11.343 | 36.5*** |
s.d. | 13.104 | 6.843 | 10.861 | 2.710 | 10.130 | 4.578 | 3.962 | 0.446 | | |
PF (mg/ml) | Mean | 4.383a | 3.32d | 4.557a | 3.859b | 4.045b | 3.233d | 3.511cd | 3.557c | 0.21 | 46.29*** |
s.d. | 0.234 | 0.107 | 0.203 | 0.121 | 0.178 | 0.089 | 0.053 | 0.029 | | |
MP (mg/g) | Mean | 122.7a | 101.4b | 120.2a | 101.6b | 105.2b | 85.6c | 98.3b | 97.3b | 11.518 | 9.58*** |
s.d. | 11.579 | 8.525 | 10.453 | 5.411 | 10.130 | 5.707 | 3.962 | 1.459 | | |
MPSE % | Mean | 49.66a | 33.66d | 51.62a | 42.93b | 45.5b | 31.89d | 37.32c | 38.15c | 2.839 | 55.78*** |
s.d. | 2.615 | 2.149 | 2.226 | 1.813 | 2.458 | 1.840 | 0.943 | 0.508 | | |
TDS % | Mean | 56.63c | 66.63a | 52.22e | 52.97de | 50.37e | 60.29b | 58bc | 55.35cd | 2.688 | 31.82*** |
s.d. | 3.231 | 1.514 | 2.432 | 0.714 | 2.481 | 1.110 | 0.733 | 0.262 | | |
IVDMD, in vitro dry matter degradability after 48 h; TNDFD, true neutral detergent fiber degradability; TOMD, true organic matter degradability after 48 h; TDMD, true dry matter degradability; PF, partitioning factor; MP, microbial protein; MPSE, microbial protein synthesis efficiency; TDS, truly digestible substrate.
*** p < 0.001; In rows, mean values marked with the same letter are not statistically different.
Mean values of relative feed value varied from 105.6 (for A12) to 138.1 (for M5). Relative feed quality varied from 111.7 (for A12) to 147.3 (for M5A3). Fatty acids varied from 1.082 (for control) to 1.769 (for M5A12). Non-fibrous carbohydrates varied from 12.83 (for A12) to 26.21 (for M5). Crude protein in NDF varied from 2.895 (for M5) to 3.902 (for A12). Total digestible nutrients varied from 58.65 (for A12) to 69.22 (for M5A3). Dry matter intake varied from 2.342% (for A12) to 2.723% (for M5). Organic matter digestibility after 24 h varied from 38.69% (for control) to 51.45% (for M5). Metabolizable energy varied from 6.744 MJ/kg (for A3) to 9.149 MJ/kg (for M5). Net energy lactation varied from 3.692 MJ/kg (for A3) to 5.39 MJ/kg (for M5). Nitrogen-free NDF varied from 38.46 (for M5) to 51.85 (for A12) (Table 6).
Table 6
Relative feed value, relative feed quality, metabolizable energy and net energy lactation of alfalfa silage
Trait | Group | C | M5 | A3 | A6 | A12 | M5A3 | M5A6 | M5A12 | LSD0.05 | F-ANOVA |
RFV | Mean | 115.6d | 138.1a | 106.5e | 108.5e | 105.6e | 131.2b | 121.7c | 125.1c | 3.978 | 77.82*** |
s.d. | 1.054 | 2.659 | 1.921 | 1.583 | 1.098 | 3.729 | 5.024 | 2.170 | | |
RFQ | Mean | 128.5bc | 147.2a | 124.6c | 116.9d | 111.7e | 147.3a | 132.8b | 132.6b | 4.72 | 62.77*** |
s.d. | 3.331 | 3.482 | 1.567 | 3.096 | 1.552 | 3.982 | 4.901 | 2.479 | | |
FA | Mean | 1.082d | 1.72ab | 1.39c | 1.119d | 1.443c | 1.441c | 1.548bc | 1.769a | 0.21 | 11.98*** |
s.d. | 0.091 | 0.126 | 0.084 | 0.224 | 0.183 | 0.135 | 0.035 | 0.177 | | |
NCF | Mean | 18.6bc | 26.21a | 15.15de | 15.58d | 12.83e | 21.08b | 18.84bc | 18.09c | 2.501 | 22.92*** |
s.d. | 0.223 | 1.644 | 1.246 | 1.360 | 0.979 | 2.751 | 2.553 | 1.517 | | |
NDFCP | Mean | 3.353c | 2.895e | 3.659b | 3.722b | 3.902a | 3.079d | 3.298c | 3.259c | 0.143 | 48.28*** |
s.d. | 0.026 | 0.082 | 0.037 | 0.094 | 0.039 | 0.104 | 0.194 | 0.098 | | |
TDN | Mean | 63.43c | 66.48b | 63.98c | 60.35d | 58.65e | 69.22a | 64.74c | 64.4c | 1.416 | 46.32*** |
s.d. | 1.780 | 0.484 | 1.016 | 1.063 | 0.997 | 0.755 | 0.568 | 0.296 | | |
DMI % | Mean | 2.493a | 2.723a | 2.396a | 2.382a | 2.342a | 2.618a | 2.523a | 2.533a | 0.06 | 38.3*** |
s.d. | 0.010 | 0.052 | 0.010 | 0.023 | 0.007 | 0.056 | 0.073 | 0.043 | | |
OMD % | Mean | 38.69f | 51.45a | 36.63g | 40.45e | 38.82f | 48.76b | 45.71c | 44.51d | 0.834 | 344.97*** |
s.d. | 0.972 | 0.729 | 0.361 | 0.456 | 0.035 | 0.877 | 0.013 | 0.170 | | |
ME (MJ/kg) | Mean | 7.106e | 9.149a | 6.744f | 7.339d | 7.104e | 8.759b | 8.229c | 8.118c | 0.149 | 291.48*** |
s.d. | 0.170 | 0.115 | 0.045 | 0.065 | 0.031 | 0.184 | 0.018 | 0.004 | | |
NEL (MJ/kg) | Mean | 3.946e | 5.39a | 3.692f | 4.114d | 3.946e | 5.112b | 4.74c | 4.657c | 0.104 | 297.61*** |
s.d. | 0.120 | 0.081 | 0.029 | 0.046 | 0.021 | 0.128 | 0.012 | 0.002 | | |
NDFn | Mean | 44.55c | 38.46e | 48.61b | 49.45b | 51.85a | 40.91d | 43.82c | 43.29c | 1.903 | 48.28*** |
s.d. | 0.345 | 1.090 | 0.495 | 1.245 | 0.522 | 1.380 | 2.574 | 1.300 | | |
RFV, relative feed value; RFQ, relative feed quality; FA, fatty acids; NCF, non-fibrous carbohydrates; NDFCP, crude protein in NDF; TDN, total digestible nutrients; DMI, dry matter intake; OMD, organic matter degradability after 24 h; ME, metabolizable energy; NEL, net energy lactation; NDFn, nitrogen-free NDF. *** p < 0.001; In rows, mean values marked with the same letter are not statistically different.
In addition to between-group comparisons (Tables 2–6), we conducted a non-orthogonal contrast analysis to compare sets of groups (Tables 7–11). The control was significantly different from the mean of A3, A6 and A12 for all traits except the PA, smell, color and structure. The control was significantly different from the mean of M5A3, M5A6 and M5A12 for all traits except the LA, PA, smell, color and structure. M5 and A3 vs. M5A3 was significant for pH, DM, LA, PA and BA. Mean value of M5 and A6 was different from M5A6 for: pH, DM, AA, PA and BA. A comparison between M5 + A12 and M5A12 showed statistical significance for: pH, DM, AA and BA. Comparisons between A3, A6, A12 vs. M5, A3, A6, A12 vs. M5A3, M5A6, M5A12 as well as M5 vs. M5A3, M5A6, M5A12 showed significant differences for most of the observed traits (Table 7).
Table 7
Comparisons between particular levels of analyzed groups using the two-sample t-test for equal means of silage physical (smell, color and structure) parameters, silo acids, pH, dry matter, ammonia nitrogen concentration of alfalfa silage.
Trait | C vs. A3, A6, A12 | C vs. M5A3, M5A6, M5A12 | M5, A3 vs. M5A3 | M5, A6 vs. M5A6 | M5, A12 vs. M5A12 | A3, A6, A12 vs. M5 | A3, A6, A12 vs. M5A3, M5A6, M5A12 | M5 vs. M5A3, M5A6, M5A12 | |
pH | -0.24** | 1.19*** | 0.77*** | 0.57*** | 0.43*** | 1.68*** | 1.43*** | -0.25*** |
NH3-N (mg/dl) | 2.46*** | 3.29*** | -0.18 | -0.89 | -1.05 | 3.08*** | 0.83* | -2.25*** |
DM % | -5.68*** | -9.06*** | -3.6*** | -2.43** | -3.94*** | -0.12 | -3.38*** | -3.26*** | |
LA % | 3.31*** | 0.17 | -4.16*** | 0.38 | -1.33 | -2.87*** | -3.14*** | -0.27 |
AA % | -3.61*** | -4.01*** | -0.01 | -1.11** | -0.71* | 0.42 | -0.40 | -0.82* | |
PA % | -0.69 | 0.19 | 0.79* | -0.83* | 0.10 | 1.72*** | 0.88** | -0.84* | |
BA % | 0.99* | 3.64*** | 3.37*** | 1.56*** | 1.95*** | 0.72 | 2.66*** | 1.93*** | |
Smell | 1.50 | 0.60 | -0.30 | 0.40 | 1.20 | -2.8* | -0.94 | 1.80 |
Color | 0.08 | -0.08 | 0.00 | 0.25 | 0.12 | -0.58 | -0.17 | 0.42 |
Structure | 0.36 | 0.42 | -0.42 | 0.38 | 0.38 | -0.11 | 0.06 | 0.17 |
DM, dry matter; NH3 -N, ammonia nitrogen concentration; AA, acetic acid; PA, propionic acid; BA, butyric acid; LA, lactic acid. * p < 0.05; ** p < 0.01; *** p < 0.001.
The control was significantly different from the mean of A3, A6 and A12 for all traits except the crude ash. The control was significantly different from the mean of M5A3, M5A6 and M5A12 for all traits except crude protein and crude ash. M5 and A3 vs. M5A3 was significant for dry matter, crude protein, NDF, ADF and crude fibre. Mean value of M5 and A6 was different from M5A6 for dry matter and crude ash. A comparison between M5 + A12 and M5A12 showed statistical significance for all seven silage chemical compositions. Comparisons between A3, A6, A12 vs. M5, A3, A6, A12 vs. M5A3, M5A6, M5A12 as well as M5 vs. M5A3, M5A6, M5A12 showed significant differences for most of the observed traits (Table 8).
Table 8
Comparisons between particular levels of analyzed groups using the two-sample t-test for equal means for silage chemical compositions.
Trait | C vs. A3, A6, A12 | C vs. M5A3, M5A6, M5A12 | M5, A3 vs. M5A3 | M5, A6 vs. M5A6 | M5, A12 vs. M5A12 | A3, A6, A12 vs. M5 | A3, A6, A12 vs. M5A3, M5A6, M5A12 | M5 vs. M5A3, M5A6, M5A12 |
Dry matter % | -2.3*** | -3.59*** | -1.50* | -1.94** | -2.2*** | 1.18* | -1.29** | -2.47*** |
Crude protein % | 0.93* | -0.63 | -1.40** | -0.77 | -2.10*** | -0.28 | -1.56*** | -1.29** |
NDF % | -5.83*** | 2.02* | 2.82** | 0.15 | 2.00* | 12.38*** | 7.84*** | -4.53*** |
ADF % | -2.20*** | 4.76*** | 4.17*** | 0.17 | 2.44*** | 9.39*** | 6.95*** | -2.44*** |
Crude fiber % | 2.41*** | 8.64*** | 4.77*** | 0.94 | 4.86*** | 5.41*** | 6.23*** | 0.82 |
Ether extract % | -0.24** | -0.51*** | 0.11 | -0.13 | -0.19* | -0.40*** | -0.27*** | 0.13 |
Crude ash % | 0.65 | 0.00 | -0.93 | -1.29* | -1.00* | 0.85 | -0.65 | -1.50** |
* p < 0.05; ** p < 0.01; *** p < 0.001.
The control was significantly different from the mean of A3, A6 and A12 for: TVFA, NH3-N and methane. The control was significantly different from the mean of M5A3, M5A6 and M5A12 for: TVFA, NH3-N, net gas and methane. M5 and A3 vs. M5A3 was significant for net gas and methane in ml/200mg. Mean value of M5 and A6 was different from M5A6 for TVFA. A comparison between M5 + A12 and M5A12 showed statistical significance for net gas and methane in ml/200mg. Comparisons between A3, A6, A12 vs. M5, A3, A6, A12 vs. M5A3, M5A6, M5A12 as well as M5 vs. M5A3, M5A6, M5A12 showed significant differences for: TVFA, NH3-N, net gas and methane in ml/200mg (Table 9).
Table 9
Comparisons between particular levels of analyzed groups using the two-sample t-test for equal means for pH, total volatile fatty acids, ammonia nitrogen concentration, methane and gas production during in vitro experiment.
Trait | C vs. A3, A6, A12 | C vs. M5A3, M5A6, M5A12 | M5, A3 vs. M5A3 | M5, A6 vs. M5A6 | M5, A12 vs. M5A12 | A3, A6, A12 vs. M5 | A3, A6, A12 vs. M5A3, M5A6, M5A12 | M5 vs. M5A3, M5A6, M5A12 |
pH | -0.02 | -0.01 | -0.01 | 0.01 | -0.01 | 0.02 | 0.01 | -0.02 |
TVFA (mmol/L) | 9.11*** | 6.19*** | -0.11 | 2.72** | 1.87 | -8.83*** | -2.92*** | 5.91*** |
NH3-N (mg/dl) | 63.60*** | 44.60*** | 7.70 | -17.30 | 16.50 | -42.60*** | -19.00** | 23.60* |
Net gas (ml/200mg) | -0.05 | -8.99*** | -5.41*** | 0.44 | 0.96* | -15.22*** | -8.94*** | 6.27*** |
CH4 (ml/200mg) | 0.20* | -1.44*** | -0.91*** | 0.01 | 0.26** | -2.85*** | -1.64*** | 1.21*** |
CH4 (%) | 0.83*** | 0.88*** | 0.24 | -0.25 | 0.28 | -0.07 | 0.05 | 0.13 |
TVFAs: total volatile fatty acids; NH3-N: ammonia nitrogen; CH4: methane. * p < 0.05; ** p < 0.01; *** p < 0.001.
The control was significantly larger from the average of A3, A6 and A12 for: IVDMD, TNDFD, TOMD, TDMD, PF, MP, MPSE and TD. The control was significantly different from the average of M5A3, M5A6 and M5A12 for: IVDMD, TOMD, TDMD, PF, MP and MPSE. M5 and A3 vs. M5A3 was significant for IVDMD, TOMD, PF, MP and MPSE. Mean value of M5 and A6 was different smaller from M5A6 for TOMD. A comparison between M5 + A12 and M5A12 showed statistical significance for IVDMD, TNDFD, TOMD, TDMD and TD. Comparisons between A3, A6, A12 vs. M5, A3, A6, A12 vs. M5A3, M5A6, M5A12 as well as M5 vs. M5A3, M5A6, M5A12 showed significant differences for most of the observed traits (Table 10).
Table 10
Comparisons between particular levels of analyzed groups using the two-sample t-test for equal means for dry matter degradability, true NDF degradability and true organic matter degradability after 48 hours of daisy incubation, and in vitro degradability parameters from gas production after 24 hours.
Trait | C vs. A3, A6, A12 | C vs. M5A3, M5A6, M5A12 | M5, A3 vs. M5A3 | M5, A6 vs. M5A6 | M5, A12 vs. M5A12 | A3, A6, A12 vs. M5 | A3, A6, A12 vs. M5A3, M5A6, M5A12 | M5 vs. M5A3, M5A6, M5A12 |
IVDMD % | 3.71*** | -5.09*** | -2.20* | 0.90 | 2.40* | -18.31*** | -8.80*** | 9.51*** |
TNDFD % | 4.90*** | -1.20 | -0.80 | 1.60 | 3.00* | -14.70*** | -6.05*** | 8.60*** |
TOMD % | 0.90*** | 0.24*** | -0.42*** | -0.28*** | -0.20*** | -0.73*** | -0.66*** | 0.07** |
TDMD (mg) | 13.50** | -15.50** | -1.50 | 5.40 | 10.80* | -67.70*** | -29.00*** | 38.70*** |
PF (mg/ml) | 0.23* | 0.95*** | 0.71*** | 0.08 | 0.13 | 0.83*** | 0.72*** | -0.11 |
MP (mg) | 13.70** | 29.00*** | 25.20*** | 3.20 | 6.00 | 7.60 | 15.30*** | 7.70 |
MPSE % | 3.00* | 13.90*** | 10.70*** | 1.00 | 1.40 | 13.00*** | 10.90*** | -2.10 |
TD % | 4.80*** | -1.20 | -0.90 | 1.80 | 3.10** | -14.80*** | -6.03*** | 8.80*** |
IVDMD, in vitro dry matter degradability after 48 h; TNDFD, true neutral detergent fiber degradability; TOMD, true organic matter degradability after 48 h; TDMD, true dry matter degradability; PF, partitioning factor; MP, microbial protein; MPSE, microbial protein synthesis efficiency; TDS, truly degradable substrate. * p < 0.05; ** p < 0.01; *** p < 0.001.
The control was significantly different from the average of A3, A6 and A12 for: RFV, RFQ, FA, NCF, NDFCP, TDN, DMI and NDFn. The control was significantly different from the average of M5A3, M5A6 and M5A12 for: RFV, RFQ, FA, NDFCP, TDN, DMI, ME, NEL and NDFn. M5 and A3 vs. M5A3 was significant for: RFV, RFQ, NDFCP, TDN, DMI, ME, NEL and NDFn. Mean value of M5 and A6 was different smaller from M5A6 for TDN. A comparison between M5 + A12 and M5A12 showed statistical significance for: FA, NDFCP, TDN and NDFn. Comparisons between A3, A6, A12 vs. M5, A3, A6, A12 vs. M5A3, M5A6, M5A12 as well as M5 vs. M5A3, M5A6, M5A12 showed significant differences for most of the observed traits (Table 11).
Table 11
Comparisons between particular levels of analyzed groups using the two-sample t-test for equal means for relative feed value, relative feed quality, metabolizable energy and net energy lactation of alfalfa silage.
Trait | C vs. A3, A6, A12 | C vs. M5A3, M5A6, M5A12 | M5, A3 vs. M5A3 | M5, A6 vs. M5A6 | M5, A12 vs. M5A12 | A3, A6, A12 vs. M5 | A3, A6, A12 vs. M5A3, M5A6, M5A12 | M5 vs. M5A3, M5A6, M5A12 |
RFV | 8.70*** | -10.40*** | -8.90*** | 1.60 | -3.20 | -31.20*** | -19.10*** | 12.10*** |
RFQ | 10.80*** | -9.10*** | -11.40*** | -0.80 | -3.20 | -29.50*** | -19.90*** | 9.60*** |
FA | -0.24** | -0.51*** | 0.11 | -0.13 | -0.19* | -0.40*** | -0.27*** | 0.13 |
NCF | 4.08*** | -0.73 | -0.40 | 2.10 | 1.40 | -11.68*** | -4.81*** | 6.87*** |
NDFCP | -0.41*** | 0.14* | 0.20** | 0.01 | 0.14* | 0.87*** | 0.55*** | -0.32*** |
TDN | 2.43*** | -2.69*** | -3.99*** | -1.33* | -1.83** | -5.49*** | -5.13*** | 0.36 |
DMI % | 0.12*** | -0.07* | -0.06* | 0.03 | 0.00 | -0.35*** | -0.18*** | 0.17*** |
ME (MJ/kg DM) | 0.04 | -1.26*** | -0.81*** | 0.02 | 0.01 | -2.09*** | -1.31*** | 0.78*** |
NEL (MJ/kg DM) | 0.03 | -0.89*** | -0.57*** | 0.01 | 0.01 | -1.47*** | -0.92*** | 0.55*** |
NDFn | -5.42*** | 1.88* | 2.62** | 0.14 | 1.86* | 11.51*** | 7.29*** | -4.22*** |
RFV, relative feed value; RFQ, relative feed quality; FA, fatty acids; NCF, non-fibrous carbohydrates; NDFCP, crude protein in NDF; TDN, total digestible nutrients; DMI, dry matter intake; ME, metabolizable energy; NEL, net energy lactation; NDFn, nitrogen-free NDF. * p < 0.05; ** p < 0.01; *** p < 0.001.
Table 12
Mahalanobis distances between groups estimated on the basis of all traits.
Group | C | M5 | A3 | M5A3 | A6 | M5A6 | A12 | M5A12 |
C | 0 | | | | | | | |
M5 | 33.46 | 0 | | | | | | |
A3 | 36.95 | 32.91 | 0 | | | | | |
M5A3 | 58.30 | 33.60 | 31.34 | 0 | | | | |
A6 | 51.77 | 37.28 | 18.14 | 18.37 | 0 | | | |
M5A6 | 51.79 | 30.73 | 23.98 | 8.86 | 12.34 | 0 | | |
A12 | 62.20 | 46.41 | 27.87 | 19.95 | 10.97 | 17.08 | 0 | |
M5A12 | 66.44 | 44.53 | 38.08 | 13.87 | 22.08 | 16.47 | 17.69 | 0 |
The greatest variation in all the traits, based on the measured Mahalanobis distances, was found for the control and M5A12 (the Mahalanobis distance between them amounted to 66.44). The highest similarity was found between M5A3 and M5A6 (8.86). The Mahalanobis distances for all pairs of groups are listed in Table 12.
Multivariate comparisons
Figure 1 shows the variability of quantitative traits for the groups in terms of the first two canonical variables. In the graph, the coordinates of the points for particular groups are the values for the first and second canonical variables, respectively. The first two canonical variables accounted for 96.81% of the total multivariate variability among individual groups. Significant positive linear relationships with the first canonical variable were observed for butyric acid (0.73), TVFAs (0.78), and ruminal NH3 (0.80). The first canonical variable was negatively correlated with silage dry matter (–0.89) and fresh silage dry matter (–0.87). The second canonical variable was significantly positively correlated with smell (0.80), color (0.81), net gas (0.97), and methane (0.98); degradability parameters such as RFV (0.99), NCF (0.91), TDN (0.73), DMI (0.96), RFQ (0.88), OMD (0.97), ME (0.97), and NEL (0.97); and daisy incubator traits such as IVDMD (0.98), TNDFD (0.91), OMD (0.71), TDMD (0.96), and TDS (0.92). The second canonical variable was significantly negatively correlated with the chemical composition of silage: NDF (–0.93), ADF (–0.98), crude fiber (–0.78), silage quality parameter pH (–0.93), propionic acid (–0.80), two degradability parameters: NDFn (–0.93) and NDFCP (–0.93), and two daisy incubation traits: PF (− 0.87) and MPSE (− 0.89).