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Effect of Probiotic, Prebiotic and Synbiotic on Hematological Parameters of Crossbred Calves

Aashaq Hussain Dar S. K. Singh Jyoti Palod Kurat-ul-Ain Nitesh Kumar B .S. Khadda Faisal Farooq
Vol 7(4), 127-136
DOI- http://dx.doi.org/10.5455/ijlr.20170312053224

The present study was conducted to evaluate the impact of probiotic, prebiotic and synbiotic on haematological parameters of cross bred calves. Twenty four crossbred calves with average age of fifteen days were divided into 4 groups of 6 calves each. The calves were assigned to different dietary treatments viz. T0- basal diet (control), T1 (probiotic group) - basal diet + Lactobacillus acidophilus @ 1 g/calf/day (2 x 1010 cfu/ g), T2 (prebiotic group)- basal diet + Mannan oligosaccharide (MOS) @ 4 g per calf/day and T3 (synbiotic group) basal diet + Lactobacillus acidophilus @ 0.5 gm (2 x 1010 cfu/ g) + 2 g MOS per calf/day respectively for a period of 90 days. Significant weight gain was observed in probiotic and synbiotic group. The results showed that there was a significant increase in leucocyte count in T1, T2, and T3 on 90th day of experiment. Haemoglobin content was significantly higher from 30 days till completion of experimental study in T1, T2 and T3 groups than control group. T1, T2 and T3 supplemented groups showed significantly higher values of Packed Cell Volume on 60th and 90th days as compared to control group. Significantly higher values of Mean corpuscular haemoglobin concentration were observed in T1, T2 and T3 group at 30th day than control group. The treatment groups T2 and T3 showed significant increase in lymphocyte count on 90th day than control group (T0). Neutrophil count showed no significant difference except at 90th day where T1, T2 and T3 showed significant difference from control group. It may therefore be concluded that supplementation of probiotic, prebiotic and synbiotic is advantageous in improving leucocyte count, haemoglobin content, and packed cell volume in crossbred calves.


Keywords : Differential leucocyte count Haemoglobin Haematological indicies Prebiotic Probiotic Synbiotic

Introduction

The world wide criticism over the use of antibiotics as growth promoters due to their antibiotic resistance, probiotic, prebiotic and synbiotic came up as an alternative to antibiotics. Probiotics are the microorganism which contributes to the intestinal microbial balance. Literal meaning of probiotic is “for life” and has been listed in Generally Recognized as Safe (GRAS) ingredients by US Food and Drug Administration (USFDA). Lactic acid bacteria compete with the pathogenic bacteria by competitive attachment and production of lactic acid which results in lowering of pH of large intestine. Mannanoligosacchaides (prebiotic) are cell wall products of Saccharomyces cerevisiae and inhibit pathogenic bacterial adhesion to mucous epithelium by blocking their fimbrie (Kogan and Kocher, 2007). Synbiotics are the combination of probiotics and prebiotics which work in supplementary to each other. Probiotics have extensive range of favorable effects such improving body weight gain (BWG) and feed efficiency (Abdel-Raheem et al., 2012), immunomodulatory effects (Magalhaes et al., 2008), protection of young animals against gastrointestinal disorders like diarrhoea (Galvao et al., 2005) and decrease mortality (Magalhaes et al., 2008). Prebiotics enhance growth (Ghosh and Mehla, 2012), decrease faecal colliform count (Ghosh and Mehla, 2012), enhance immunity (Fleige et al., 2009) and are found to be most effective in times of stress or increased pathogen exposure throughout the calf’s lifetime (Heinrichs et al., 2009). Little work has been done on the haematological effects of probiotic, prebiotic and synbiotics. So the present study was undertaken to study the effect of probiotic, prebiotic and synbiotic on haematological parameters.

Materials and Methods

The present experiment was conducted on 24 crossbred calves (Bos indicus X Bos Taurus) in which 16 female calves and 8 male calves averaging 15 days old were used and experiment was carried out at the Instructional Dairy Farm (IDF) Nagla of College of Veterinary Sciences & Animal Sciences, Govind Ballabh Pant University of Agriculture & Technology, Pantnagar (Uttarakhand). The calves were randomly divided into 4 groups with 6 animals in each group (4 females and 2 males) and the experiment lasted for 90 days. The calves were fed as per the details given in Table 1.

Table 1: Feeding schedule of crossbred calves during experimental trial

Age of Calves (Weeks) Milk (part of BW) Calf Starter Mixed Green Fodder

(Berseem/ Oats/ Mustard)

2-4 1/10 ad lib ad lib
5-6 1/15 ad lib ad lib
7-8 1/20 ad lib ad lib
9-16 Quantity sufficient to mix the supplement ad lib ad lib

The basal diet fed to experimental calves consisted of calf starter and mixed greens (berseem+oats+mustard). The calf starter (commercial) given to the calves contained 89.75% dry matter (DM), 20% crude protein (CP) and 3% ether extract (EE). The mixed green fodder contained 25% dry matter, 14.5% crude protein and 4% ether extract. The diet containing feed and milk was given two times a day i.e. in the morning (6:00 am) and evening (6:00 pm). Water was offered adlibitum. The powder containing probiotic, prebiotic and synbiotic was mixed in milk up to end of 15 weeks age was fed early in the morning (6.00 am). Body weight was taken at the start and end of the experiment trial. Blood samples were taken at 30 days interval. The blood collection site was prepared aseptically by clipping the hairs and using sterile gauze piece and spirit. The blood sample (5 ml) was collected from jugular vein aseptically using a disposable syringe (Dispovan HSMD) with 18 gauge hypodermic needle early in morning at 8 am. 5 ml blood after the collection was transferred to two 4 ml EDTA coated tubes each and the blood samples were immediately transported to the laboratory in a box containing ice packs. For estimation of haemoglobin concentration, the method described by Drabkin and Austin (1932) was used. The haemoglobin concentration was expressed in gram percentage which was estimated by the optical density of Cyanomet – haemoglobin. PCV was done using Wintrobes method 1974. The total erythrocyte count was done as per method described by Jain (1986) using Hayem’s fluid and results were expressed in million per microliter (x106/µl).TLC was estimated with the help of haemocytometer (Jain, 1986) using Thomas diluting fluid and results were expressed in thousands per microliter (x103/µl). The DLC was studied by making thin blood smear over a clean grease free glass slide using a drop of blood. The smear was air-dried and stained with Giemsa stain. The different leukocytes were counted by Battlefield method and values were expressed in percentage using blood cell counter. The haematological indices were calculated using the formulae viz.

Mean Corpuscular Volume (MCV) = PCV X 10/TEC (expressed in femto litre)

Mean Corpuscular Hemoglobin (MCH) = HB X 10/TEC (expressed in picogram)

Mean Corpuscular Hemoglobin Concentration (MCHC) = HB X100/PCV (expressed in %)

The cost of production was calculated in terms of 1kg additional body weight in probiotic, prebiotic and synbiotic above the control group.

Statistical Methodology

Statistical analysis was done by using SPSS 16.0 software as a comparison of means. A p-value of 0.05 was considered statistically significant.

Result and Discussion

Body Weight Gain

The body weight gain in 90 days and weight gain per day is given in Table 2. It was observed that there was a significant weight gain in 90 days and weight gain per day in probiotic and synbiotic groups than control. Prebiotic group showed non significantly more weight gain than the control group. The better growth performance might be due to better intestinal microbial balance in case of probiotic and synbiotic group which might have lead to increased digestion and absorption of nutrients and minerals from the gastrointestinal tract.

Table 2: Average (Mean ±SE) body weight gain/ day (kg) of crossbred calves

Group T0 (Control) T(Probiotic) T(Prebiotic) T3 (Synbiotic)
Average initial body weight (kg) 32.00 ±0.96 32.08±0.74 32.08±1.08 32.08±0.83
Average final body weight (kg) 61.38±1.13a 70.11±1.27b 64.18±1.04a 72.18±1.21b
Average total body weight gain (kg) 29.38±0.88a 38.03±1.23b 32.10±1.36a 40.10±1.32b
Average body weight gain (g/day) 322.89±9.72a 417.91±13.54b 352.75±14.95a 440.66±14.53b

Means bearing different superscripts in a row differ significantly (P<0.05)

The present study was in agreement with the results of Abdel-Raheem et al. (2012) who reported better performance in probiotic and synbiotic group with improved final body weight, body weight gain than prebiotic and control group. Heinrichs et al. (2003) also reported no effect of mannanoligosaccharide supplementation on growth performance. However, Ghosh and Mehla, (2012) reported a significant increase in body weight gain in calves supplemented with prebiotics.

Total Erythrocyte Count

The mean total erythrocyte count at 0th , 30th, 60th and 90th day of experimental study of cross bred calves under various treatments have been presented in Table 3. There was no significant result found throughout the experimental study which was in conformity with the results of Kim et al. (2011) and Dimova et al. (2013) who reported that erythrocyte count remained unaffected by probiotic supplementation. Adams et al. (2008) also reported no significant effect of probiotic supplementation on erythrocyte count. Masanetz et al. (2011) also reported that there was no significant effect of prebiotic supplementation on total erythrocyte count. Contrary to our study, Gazanfar et al. (2015) reported a significant effect of probiotic supplementation on total erythrocyte count.

Total Leucocyte Count

The total leucocyte count values at different periods of study have been presented in Table 3. On the 60th day of the experimental trial, significant differences were observed in Tand T3 as compared to T0. Significant increase in leucocyte count was observed in T1, T2, and T3 on the 90th day of the experiment. There was an increase of 22.22, 14.93 and 19.24 percent in total leucocyte count of T1, T2, and Tthan T0. The increase in TLC in treatment groups may be due to better microenvironment in intestines which can cause pluripotent hemopoietic precursors to differentiate into clones of lymphocytes as reported by Glick (2000).

Table 3: Effect of probiotic, prebiotic and synbiotic on different haematological parameters of crossbred calves at different intervals

Day Parameter T0 (Control) T(Probiotic) T(Prebiotic) T3 (Synbiotic)
0 TEC(106/µl) 6.04±0.41 6.10±0.44 6.07±0.06 6.08±0.26
TLC(103/µl) 8.3±0.85 8.48±0.37 8.42±0.90 8.67±0.44
Hb (g/dl) 8.97±0.09 9.03±0.12 9.20±0.12 9.30±0.15
PCV (%) 28.97±0.55 28.67±0.88 30.33±0.88 29.00±0.58
30 TEC(106/µl) 6.8±0.36 7.11±0.35 6.88±0.04 6.85±0.055
TLC(103/µl) 8.51±0.45 8.91±0.89 8.65±0.67 8.72±0.29
Hb (g/dl) 8.67±0.15a 9.97±0.18b 10.27±0.19b 10.37±0.04b
PCV (%) 29.23±1.01 30.67±1.20 29.00±0.58 31.07±0.52
60 TEC(106/µl) 6.75±0.09 6.84±0.43 6.89±0.14 6.80±0.33
TLC(103/µl) 8.87±0.09a 9.90±0.06b 9.18±0.37ab 9.89±0.26b
Hb (g/dl) 10.22±0.17a 12.53±0.08b 12.63±0.18b 12.84±0.06b
PCV (%) 30.43±0.58a 36.13±0.98c 33.30±0.42b 39.22±0.62d
90 TEC(106/µl) 6.81±0.45 6.84±0.06 6.81±0.27 6.88±0.59
TLC(103/µl) 8.37±0.19a 10.23±0.21b 9.62±0.03b 9.98±0.28b
Hb (g/dl) 11.30±0.04a 13.70±0.06b 13.68±0.10b 13.98±0.02c
PCV (%) 32.90±0.96a 39.93±2.57b 37.13±0.39b 40.42±3.07b

Means bearing different superscripts in a row differ significantly (P<0.05)

Our results were in accordance with the results of Al-Siaidy et al. (2010) who reported significantly higher white blood cell count in calves fed with both commercial grade and culture of Lactobacillus acidophilus than control group. The same was also reported by Gazanfar et al.(2015) who reported significant increase in white blood cell count in probiotic supplemented group. Whereas Chen et al. (2005) and Agazzi et al. (2014) reported that there was no effect of supplementing probiotic on white blood cell count of growing pigs and calves respectively. The present results are in contrary with the results of Masanetz et al. (2011). Masanetz et al. (2011) reported that lactulose supplemented group showed significant decrease in peripheral leucocytes than control and inulin treated groups.

Total Hemoglobin

The hemoglobin values at different periods of experimental study are presented in Table 3. From 30th day till the completion of experiment, T1, T2 and Tgroups showed significantly higher haemoglobin content than control. The final day haemoglobin concentration showed 21.24%, 21.06%, 23.72% increase in T1, T2 and T3 group than Tgroup. The increase in haemoglobin concentration might be due to increased iron absorption due to thinning of intestines due to probiotic and prebiotic supplementation. The haemoglobin values in control group on day 30 decreased and then increased with time was in agreement with Mohri et al. (2007) who also observed a decrease of haemoglobin from birth to the age of 28 days and later the values increased with age. The results were in accordance with the Al-Saiady et al. (2010) and Gazanfar et al. (2015) who also reported a significant increase in haemoglobin level in probiotic administered groups. Abdel Fattah et al. (2009) reported that there was a significant increase in haemoglobin levels in broilers fed with prebiotic and synbiotic. Sayed (2003) also reported an increase in hemoglobin concentration in kids supplemented with probiotic whereas Kim et al.(2011) observed no significant effect on hemoglobin levels of Holstein calves supplemented with probiotics. Masanetz et al. (2011) also reported significant effect of feeding prebiotic on hemoglobin concentration at the time of slaughter in inulin treated calves compared to control and lactulose treated calves.

Packed Cell Volume (PCV)

Table 3 represents Packed Cell Volume (PCV) in percentage of different treatment groups. Packed Cell Volume at 60th and 90th days in T1, Tand Tsupplemented groups showed significantly higher values of Packed Cell Volume as compared to control group. There were 21.37, 12.86 and 22.86 percent increase in packed cell volume value of T1, Tand T3 groups than control group on 90th day. The increase in packed cell volume value in probiotic group were in accordance with the results of Al-Saidy et al. (2010) who also reported significant increase in PCV values in male calves supplemented with probiotic (Lactobacillus acidophilus 27c). Abdel-Fattah et al. (2009) reported that there was significant increase in Packed Cell Volume levels in broilers fed with prebiotic and synbiotic. Similar results of increased packed cell volume in probiotic supplemented group in kids were reported by Sayed (2003). However Kim et al. (2011) observed no significant difference between probiotic supplemented group and antibiotic treated group in Holstein calves. Similarly no significant effect was observed by Gazanfar et al. (2015) on packed cell volume values by probiotic supplementation. Masanetz et al. (2011) reported no significant difference between control group and prebiotic group on hematocrit value till 20 weeks. However their results showed significant increase in hematocrit value at slaughter time.

Mean Corpuscular Volume (MCV)

The mean corpuscular volumes of experimental crossbred calves in different groups at various intervals are presented in Table 4. No significant difference was observed between the groups throughout the experimental period. The present results were in conformity with the results of Adams et al. (2009) and Agazzi et al. (2014) who reported that there was no significant difference between probiotic supplemented group and control group on mean corpuscular volume. However Morill et al. (1995) reported significantly different values of mean corpuscular volume at day 10 in probiotic fed calves.

Mean Corpuscular Haemoglobin (MCH)

The mean corpuscular haemoglobin values at different periods of study under different treatments are presented in Table 4. The results for MCH indicated that there was no significant difference between the groups throughout the experiment. Our results are in conformation with the results of Adams et al. (2009) and Agazzi et al. (2014) who reported that there was no significant difference in mean corpuscular haemoglobin values between probiotic supplemented group and control group. Morill et al. (1995) also reported significantly different values of MCV at day 10 in probiotic fed calves.

Table 4: Effect of probiotic, prebiotic and synbiotic on haematological indices of crossbred calves at different time intervals

Day Parameter T0 (Control) T(Probiotic) T(Prebiotic) T3 (Synbiotic)
0 MCV (fl) 47.79±3.65 47.37±3.20 49.97±1.31 47.99±3.05
MCH (Pg) 14.63±0.88 14.94±1.03 15.16±0.13 15.38±0.90
MCHC (g/dl) 31.37±0.61 31.55±0.56 30.39±1.08 32.07±0.22
30 MCV (fl) 43.08±0.89 43.53±3.86 42.15±0.66 45.37±1.08
MCH (Pg) 12.81±0.60 14.11±0.90 14.93±0.35 15.14±0.07
MCHC (g/dl) 29.70±0.82a 32.56±0.85b 35.45±1.31b 33.41±0.67b
60 MCV (fl) 45.10±1.46 53.40±4.66 48.40±1.32 58.01±3.65
MCH (Pg) 15.14±0.33 18.46±1.24 18.36±0.64 18.97±0.99
MCHC (g/dl) 33.61±0.99 a 34.72±0.92 a 37.93±0.61b 32.75±0.38 a
90 MCV (fl) 48.83±3.90 58.43±4.31 54.72±2.47 60.39±9.67
MCH (Pg) 16.74±1.05 20.02±0.17 20.15±0.85 20.62±1.77
MCHC (g/dl) 34.40±0.98 34.59±2.19 36.83±0.13 34.98±2.60

Means bearing different superscripts in a row differ significantly (P<0.05)

Mean Corpuscular Haemoglobin Concentration (MCHC)

The mean corpuscular haemoglobin concentration values at different periods of experimental study under different treatments are presented in Table 4. Significantly higher values were observed in T1, T2 and Tgroup at 30th day of experimental trail than control group. T2 group showed significantly higher values of mean corpuscular haemoglobin concentration than T0, T1and Tat 60th day of experimental study. However, Agazzi et al. (2014) reported that there was no significant difference between probiotic supplemented group and control group on mean corpuscular haemoglobin concentration. Similarly, Adams et al. (2009) found no significant effect of feeding probiotic.

Differential Leucocyte Count (DLC)

The differential leucocyte count at different periods of study in experimental crossbred calves has been presented in Table 5. The treatment groups T2 and Tshowed significant increase in lymphocyte count on 90th day than control group (T0). Neutrophil count showed no significant difference except at 90th day where T1, T2 and T3 showed significant difference from control group. There was no significant difference observed between the different groups throughout the experimental study in monocyte, basophil and acidophil percentage. The increase in lymphocyte percentage in prebiotic and synbiotic group might be due to enhanced immunity which lead elevated lymphocyte level. Our results were in agreement with the results of Frizzo et al. (2008) and Agazzi et al. (2014) who reported no significant effect of feeding probiotic on any component of differential leucocyte count in calves. However Gazanfar et al. (2015) reported increase in eosinophil percentage by probiotic supplementation. Masanetz et al. (2011) reported no significant effect of feeding prebiotic on lymphocyte, basophil and percentage but showed significant difference for monocyte percentage.

Table 5: Average (Mean±SE) percentage of different components of differential luecocyte count of crossbred calves at different time intervals

Day DLC (%) T0 (Control) T(Probiotic) T(Prebiotic) T3 (Synbiotic)
0 Lymphocyte 53.33±6.49 55.00±5.69 54.00±5.29 56.00±5.20
Neutrophil 37.67±11.24 36±9.85 37±9.17 35±9
Monocyte 5.67±0.67 5.33±0.88 4.33±1.33 4.33±1.76
Basophil 0.33±0.33 0.33±0.33 0.33±0.33 0.33±0.33
Eosinophil 1.67±0.33 1.67±0.33 1.67±0.33 1.67±0.33
30 Lymphocyte 61.33±5.21 65.00±4.73 63.67±6.57 66.00±6.03
Neutrophil 32.67±9.03 29±8.16 30.33±11.37 28±10.44
Monocyte 3.00±0.58 3.67±0.33 3.67±0.33 2.67±0.67
Basophil 0.67±0.67 0.33±0.33 0.33±0.33 1.00±0.58
Eosinophil 2.00±0.00 1.67±0.33 1.67±0.33 2.33±0.33
60 Lymphocyte 71.00±1.16 72.67±1.76 73.33±1.76 73.67±1.67
Neutrophil 25.00±2.00 23.33±3.06 22.67±3.06 22.33±2.89
Monocyte 1.00±0.58 1.67±0.33 1.33±0.67 1.67±0.33
Basophil 0.33±0.33 0.33±0.33 0.33±0.33 0.33±0.33
Eosinophil 1.67±0.33 1.67±0.33 1.67±0.33 1.67±0.33
90 Lymphocyte 68.00±0.58a 71.67±0.88ab 73.33±0.33b 76.67±0.67c
Neutrophil 26±1.00c 22.33±1.53bc 20.67±0.58b 14.33±4.04b
Monocyte 3.67±0.33 3.33±0.67 3.00±0.58 6.67±2.03
Basophil 0.33±0.33 0.33±0.33 0.67±0.33 0.33±0.33
Eosinophil 1.67±0.33 1.67±0.33 2.00±0.58 1.67±0.33

Means bearing different superscripts in a row differ significantly (P<0.05)

Cost of Production

The cost of production of 1kg additional body weight over the control group in probiotic, prebiotic and synbiotic has been given in Table 6.

Table 6: Average expenditure (Rs) on per kg additional weight gain above control group in probiotic, prebiotic and synbiotic group

Probiotic Prebiotic Synbiotic
Weight Gain(kg) 8.65 2.72 10.72
Feed Additive Consumed(g) 90 360 45g probiotic+ 180g prebiotic
Cost 44.98 98.28 71.63
Cost/kg Weight 5.20 36.13 6.68

The probiotic group gained 8.65 kg more weight than control group. The cost for production of 1kg more weight gain than control group was Rs/-5.20. The prebiotic group gained 2.72 kg more weight than control group and the cost of production of 1kg more body weight was Rs/-36.13. The synbiotic group gained 10.72 kg more weight than control group and the cost of production of 1kg more body weight was Rs/-6.68. From the results it is obvious that probiotic and synbiotics are effective in increasing body weight gain and are less expensive than common feed supplements when compared in terms of cost per kg body weight gain.

Conclusion

The present study showed that probiotic, prebiotic and synbiotic are effective in improving some haematological parameters like leucocyte count, haemoglobin and packed cell volume. Also probiotic and synbiotic are effective in improving body weight gain and are affordable to farmers.

References

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