4.1. Induction of Acidosis and Recovery Rate
In all study sheep, acidosis was successfully induced and all acidosis induced sheep showed nervous depression and symptoms including watery, yellowish, and acidic smelling diarrhea, cessation of feed intake, and severe symptoms of foot pain and difficulty walking after the induction of acidosis. All the sheep were able to stand move but they move slowly with head hung down and dull eyes. The results of the present study revealed that most of the therapeutic regimens tested in the experiment were found to be effective in eliciting a favorable response in acidosis sheep. Except one sheep (20%) died from the treatment I (probiotic alone) there was no death due to induced acidosis in the treatment groups as compared to the death of two sheep(40%) in the control group.
4.2. Improvements of Physical Parameters
At the start of the experimental period (before treatment), the total mean heart rate of 139.84±9.290 in all groups of animals changed in to 127±0.20, 109±9.540 and 98.27±8.49 in the 1st,2nd and 3rd days, respectively after treatment so that difference being significant (p<0.05). Three treatment groups such as treatment group II, III and IV were statically significance difference comparing with control group at day three after treatment. The treatment group III, combination of probiotics and rumenotorics was most therapeutically effective for correction of heart rate on day three which was statically significance (p <0.05, p=0.001). The standard treatment had also high significant difference between control groups in the improvement of heart rate (p<0.05, p=0.008) next to probiotics and rumenotorics and the combination of probiotics and prebiotics (II) statically significance (p<0.05, p=0.01). Similarly rectal temperature was 37.9 ± 0.612°C in all groups, while the temperature was 38.36±0.97, 39.00± 0.617 and 40±0.0 with the 1st,2nd and 3rd days, respectively, after treatment showing improvement in body temperature of the sheep (Table4).
The standard treatment (IV) was improved rectal temperature from day one-day three compare with control groups and these improvements were statistically significant difference (P < 0.05) (Table4).The mean±SD respiration rate (breath/minute) was 36.96±8.085 before administered treatments in all groups of the animals at the induction phase but after treatment it was changed to 28±5.745, 26.00±0.18, 19.27±3.47 for three consecutive days, it was statically significant (p<0.05) (Table4).Therefore, the therapeutic regimens of probiotics with rumenotorics was highly statically significance (p<0.05, p=0.001 & p=0.000) to improve the heart rate and respiratory rate compared with untreated group.
Table4: Effect of various treatment regimens on physical parameters for induced acidosis
Parameter
|
Time of Rx
|
Control group
|
Treatment I
|
Treatment II
|
Treatment III
|
Treatment IV
|
Total mean
|
Heart rate
|
Before
|
134.40±9.21
|
142.40±10.88
|
140.80±14.45
|
142.80±8.67
|
138.8±7.16
|
139.84±9.29
|
(min)
|
After (RX)
|
|
|
|
|
|
|
|
Day1
|
138.00±8.72
|
127.20±7.16
|
123.20±8.67
|
123.20±126#
|
116.40±4.6
|
127±0.20
|
|
Day2
|
124.00±6.93
|
116.00±8.64
|
104.80±5.23#
|
108.8±7.64#
|
102.40±4.6#
|
109±9.540
|
|
Day3
|
112.67±3.05
|
107.00±8.87#
|
97.20±6.88#
|
91.20±7.69#
|
98.00±8.00#
|
98.27±8.49
|
Temperature
|
Before
|
38.40±8.764
|
38.00±.707
|
37.60±548
|
37.40±.548
|
38.20±.447
|
37.9± 0.612
|
(T0)
|
After (RX)
|
|
|
|
|
|
|
|
Day1
|
38.60±.89
|
38.20±.837
|
38.00±.707
|
38.80±.447#
|
39.20±1.05#
|
38.36±0.97
|
|
Day2
|
38.60±.000
|
39.00±.000
|
38.40±548
|
39.40±.548
|
39.20±.837#
|
39.00±0.67
|
|
Day3
|
38.80±000
|
39.00±39.00
|
39.00±.000
|
40.00±.000#
|
39.60±0.55#
|
40±0.00
|
Respiratory rate((min)
|
Before
|
40.40±8.764
|
35.2±3.347
|
33.60±8.295
|
44.00 ±.5.66
|
31.60±8.52
|
36.96±8.05
|
After(Rx)
|
|
|
|
|
|
|
Day1
|
33.60±7.266
|
30.40±4.561
|
25.60±4.561
|
26.00±4.472#
|
24.40±2.966#
|
28±5.745
|
Day2
|
28.67±1.16
|
24.00±3.266
|
23.20±3.347#
|
20.80±3.37#
|
20.40±2.96
|
26.00±0.18
|
|
Day3
|
22.67±5.03
|
22.00±2.309
|
19.60±2.966#
|
16.80±1.05#
|
17.20±2.63#
|
19.27 ±3.47
|
Values expressed as (#) superscript are significantly significant between control groups at p<0.05.
Values were expressed by means ±SD paired t-test before and after treatment by STATA version14
One way ANOVA followed by Bonferroni post hoc test was used to compare between group
4.3. Hematological Results
The hemoglobin concentration (mean ±SD) of experimental animals before treatment Group I, II, III, IV, and control group was 14.00±2.00, 15.80±.447, and 12.80 ±1.924, 14.40±1.517, 15.40 ±1.673 respectively and after treatment on day one was 13.20±2.168, 14.5±.707, 12.10 ± 2.19, 13.80±1.78 and 14.80±2.168 respectively, (p<0.05,). Similarly, all treatment groups were statically significance after treatment on day two and day three (p<0.05), obvious change (Table5).
Animals suffering from experimentally induced acidosis had increased packed cell volume (PCV) initially, however there was a decreased PCV level after treatment regimens were given significantly (p<0.05) between groups apparently treatment Group III (probiotics with rumenotorics) and control group, the mean±SD of PCV were statically significance (p<0.05, p=0.003, p=0.02 &0.006) from day one -day three, respectively. Standard treatment was the second level of treatment to improve the disordered of PCV value as well as the third level of treatment was treatment Group II (probiotics with prebiotics). The experimental trial on day two, treatment Group III (probiotics with rumenotorics) blood pH values was statistically significant (p<0.05), improved. Therefore, this result was shown that treatment Group III (probiotics with rumenotorics) had a significant effect on the value of PCV for treatment of acidotic sheep and increased blood pH on day two and day three after treatment,(p<0.05) whereas the blood pH was significant(p<0.05) in treatment Group IV at day one up to three .
Table5: Effect of various treatment regimens on hematological parameters on acidotic sheep (mean±SD)
Parameter
|
|
Time of Rx
|
Control group
|
Treatment I
|
Treatment II
|
Treatment III
|
Treatment IV
|
Blood pH
|
|
Before
|
6.20±0.447
|
6.60±.548
|
6.20±.447
|
6.60±.548
|
6.40±.48
|
|
|
After (RX)
|
|
|
|
|
|
|
|
Day1
|
6.40±0.548
|
6.59±.648
|
6.60±.548
|
6.80±.447
|
7.40±.548#
|
|
|
Day2
|
6.00±0.00
|
7.00±.00#
|
6.68±.547
|
7.100±.100#
|
7.480±.447#
|
|
|
Day3
|
6.67±.577
|
7.00±00
|
7.00±00#
|
7.50±00#
|
7.57±.577#
|
PCV
|
|
Before
|
44.00±4.183
|
42.00±5.7
|
41.80±3.768
|
38.00±6.819
|
44.20±2.387
|
(%)
|
|
After (RX)
|
|
|
|
|
|
|
|
Day1
|
43.20±3.70
|
39.00±3.742
|
37.40±2.88#
|
34.60±5.367#
|
42.00±2.345
|
|
|
Day2
|
39.00±1.00
|
37.25±4.272
|
35.60±1.881
|
29.80±6.261#
|
39.80±1.483#
|
|
|
Day3
|
35.55±7.12
|
33.00±3.559
|
29.40± 2.99#
|
24.80±10.134#
|
37.80±1.789#
|
hemoglobin
|
|
Before
|
15.40±1.67
|
14.00±2.000
|
15.80±.447
|
12.80 ±1.92
|
14.40±1.517
|
(gm/dl)
|
|
After (RX)
|
|
|
|
|
|
|
|
Day1
|
14.80±2.168
|
13.20±2.168
|
14.5±.707
|
12.10 ± 2.19
|
13.80±1.78
|
|
|
Day2
|
13.00±1.000
|
12.00±2.168
|
11.40±.548
|
11.40±1.817#
|
13.20±1.095
|
|
|
Day3
|
12.67±.577
|
11.50±1.732
|
11.10±0.89#
|
11.00±2.121#
|
13.00±1.000
|
Values expressed as (#) superscript are significantly significant between control groups at p<0.05.
Values were expressed by means ±SD paired t-test before and after treatment by STATA version14
One way ANOVA followed by Bonferroni post hoc test was used to compare between groups
4.4. Qualitative and Quantitative Ruminal Fluid Analysis Results
Chemical and physical examination of ruminal fluid was examined in the present experimental trials. During the induction phase of ruminal acidosis, the ruminal pH, protozoan count and motility were reduced. The cellulose digestion and glucose fermentation activity were completely stopped in almost all sheep as indicated in their respective tests in a laboratory in day zero or before treatment. The examination of ruminal fluid was watery and acidic smell.
Analysis of ruminal fluid pH was increased significantly (P<0.05) in acidotic sheep after treatment. From a comparison of each treatment groups with the control group, the combination of probiotics with rumenotorics treatment group(III), the mean±SD 4.96±.837, 5.92±.54, 6.30±.41 and 6.75±0.34 was statically significant (p= 0.024, 0.047, 0.002) respectively from day one - day three. The standard treatment Group IV (sodium bicarbonate) was also statically significant (p<0.05) improved ruminal pH from day two - day three where as treatment group II was significant difference at day three compare with control group ,it is more elaborated (Table6).
Sedimentation activity time and methylene blue reduction times were significantly differed (P<0.05) in all groups after employed the treatments for three successive days. From treatment groups, probiotics with rumenotorics were found to be significantly (P<0.05) decreased. The mean value of the methylene blue reduction time (min) before and after treatment was 9.40±2.302 and 4.60±3.507 respectively (Table6). Also, sedimentation activity time (min) was 1.60±.894 and 12.00±3.082 significantly increased after treatment (P<0.05). The analysis of qualitative ruminal fluid after treatment such as protozoan motility and concentration was statically significant (p <0.05).
Table6: Effect of various treatment regimens on of rumen liquor experimental induced acidosis in
Sheep
Parameter
|
Time of Rx
|
Control group
|
Treatment I
|
Treatment II
|
Treatment III
|
Treatment IV
|
|
Ruminal (pH)
|
Before
|
4.40±.54
|
4.80±.46188
|
5.00. ±447
|
4.96±.837
|
4.97±.637
|
|
|
After (RX)
|
|
|
|
|
|
|
Day1
|
4.96±.43
|
5.04±.27
|
5.18±.31
|
5.92±.54#
|
5.38±.540
|
|
|
Day2
|
5.31±.53
|
5.19±.33
|
5.380±.23
|
6.30±.41#
|
5.85±.540#
|
|
|
Day3
|
5.4333±.4934
|
5.50±.216
|
5.880±.19) #
|
6.75±0.34#
|
6.42±.570#
|
|
protozoan
|
Before
|
0.00*(0.-00)
|
0.00*(0-00)
|
0.00*(0-1.00)
|
0.00*(0-00)
|
0.0*(0 -00)
|
|
Motility test
|
After (RX)
|
|
|
|
|
|
|
|
Day1
|
0.00*(0 -1.00
|
0.00*(0 -2.00)
|
1.00*(0-2.00)
|
2.0*(0-2.00)
|
1.00*(0 -1.00)
|
|
|
Day2
|
1.00(1.0-1.00)
|
1.00*(0-1.00)
|
1.00*(1-2.00)
|
2.0*(2-3.00) #
|
1.00*(1-1.00)
|
|
|
Day3
|
1.00*(1 -2.00)
|
1.50*(1.00-3.00)
|
1.00*(1 -300)#
|
3.0*(2-3.0) #
|
2.00*(2-3.00) ) #
|
|
MBRT
|
Before
|
10.33±1.528
|
9.25±1.708
|
9.40±2.966
|
9.40±2.302
|
10.20±1.643
|
|
(min)
|
After (RX)
|
9.67±1.528
|
8.25±1.708
|
7.60±1.51
|
4.60±3.507#
|
6.40±1.673#
|
|
SAT
|
Before
|
1.33±.577
|
1.50±.577
|
1.40±548
|
1.60±.894)
|
1.20±.447
|
|
(Min)
|
After (RX)
|
4.00±1.000
|
3.25±.500
|
6.60±.219#
|
12.00±3.08#
|
4.00±1.000
|
|
Values expressed as (#) superscript are significantly significant between control groups at p<0.05.
Values are Mean ± S.D in Paired t-test, the values of protozoan motility was median (Q0 - Q3) and Kruskal-Wallis equality-of-populations rank test.
MBRT=Methylene blue reduction time; SAT= Sedimentation activity time of the ruminal fluid before and after treatment between groups
treatments
Figure2: The mean ruminal pH between treatment groups for three consecutive days.
pH0= ruminal pH on day zero; pH1=ruminal pH on day one; pH2=ruminal pH on day two; pH3=ruminal pH on day three. The x-axis is a list of treatments; treatment I –probiotics; Treatment II- probiotics with prebiotics; Treatment III- probiotics with rumenotorics; Treatment IV-standard treatment (sodium-bicarbonate).
4.5. Serum Analysis Results
The treatment regimens were revealed statically significant (p<0.05) between groups to improve the abnormal value of enzymatic and protein disorders. From all the treatment Groups II, III and IV were highly significant (p< 0.05) for the improvement of total protein, albumin, and AST. The mean values of total protein (g/dl), albumen (g/dl) and AST (u/L) before treatment (IV, III and II) were 5.5±.24, 2.08±.36, 254±59 then it was converted to, 7±.80, 3.3±.4, 109±3 respectively after treatment (Table7). The mean total protein (g/dl) and albumen concentration of experimental animals for treatment Group III were statistically significant (p<0.05), 5.2±.54, 2.08±.36 was before treatment then it was changed into 6.8±.40, 2.47±.27 after treatment respectively.
Table7: Various treatment regimens on serum proteins and liver enzyme profile in acidosis sheep
Parameter
|
|
Control group
|
Treatment
I
|
Treatment
II
|
Treatment
III
|
Treatment
IV
|
Total proteins
|
Before
|
5.32±.43
|
5.70±.46
|
5.75±.54
|
5.2±.54
|
5.5±.24
|
(g/dl)
|
After (RX)
|
5.7±.32
|
5.9±.35
|
6.00±.63*
|
6.8±.40
|
7±.80
|
Albumin
|
Before
|
2.17±.47
|
1.75±.86#
|
1.71±.36#
|
2.08±.36*
|
2.4±.38#*
|
(g/dl)
|
After (RX)
|
2.08±.18
|
2.52±.32
|
2.3±.10
|
2.47±.27#
|
3.3±.4*
|
AST
|
Before
|
219±39
|
241.6±44
|
202.4±.36
|
181±91*
|
254±59*
|
(U/l)
|
After (RX)
|
232±28
|
176±61.8
|
173.4±67
|
111±27
|
109±34
|
ALP
|
Before
|
285±86
|
404.3±46
|
366±12
|
415±31.
|
434±42
|
(U/l)
|
After (RX)
|
222±52
|
336. ±78
|
346.7±99
|
121.8±87
|
99±43
|
Values in each group are significantly different at p≤0.05 Values are Mean ± S.D in Paired t-test using and ANOVA-post hoc test between groups and control group.*-indicates significantly different from the control group; # -indicates significantly different between groups; *#- indicates significantly different between groups and control group