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Effect of Milk Fermented with Lactobacillus acidophilus NCDC15 on Nutrient Digestibility, Faecal Biomarkers and Immune Response in Murrah calves

Abstract

In neonates, rapid change in diet imbalances gut health allowing colonization of opportunistic pathogens that confer harmful effects on animal health causing reduced digestion and malabsorption of nutrients. In this milieu, probiotic feeding can be a promising approach in promoting animal health and stabilization of gastrointestinal microbiota. Hence, the present study was designed to investigate the effect of Lactobacillus acidophilus NCDC15 enriched fermented milk on nutrient digestibility, faecal biomarkers and immune response in Murrah buffalo calves. Twenty-four, neonatal calves (5-7 days) were randomly allocated into four groups for 90 days. The control group (CT) was provided a basal diet of calf starter and green fodder (maize and jowar), without any probiotic fermented milk (PFM) supplementation. Basal diet was supplemented with probiotic fermented milk at 100, 200 and 300 mL/calf/day, in PFM100, PFM200 and PFM300 groups, respectively. Nutrient digestibility remained similar among the groups. Faecal acetate was higher (P<0.05) in PFM200 and PFM300, while, faecal butyrate was increased (P<0.05) with all levels of probiotic supplementation than control. Faecal Lactobacillus and Bifidobacterium count were increased (P<0.05) with a concomitant reduction in coliform population (P<0.05) among all the treatments. Cell-mediated and humoral immune response were higher (P<0.001) in PFM200 and PFM300 than CT. Overall, it can be concluded that inclusion of Lactobacillus acidophilus NCDC15 in the form of fermented milk upto 300 mL/calf/day improved immunity and faecal biomarkers in Murrah buffalo calves without any adverse effect on nutrient utilization which may positively impact growth performance in Murrah buffalo calves.

Keywords:
Buffalo calves; Fermented milk; Faecal biomarkers; Immunity; Lactobacillus acidophilus NCDC15

HIGHLIGHTS

  • Inclusion of Lactobacillus acidophilus NCDC15 in the form of fermented milk at 100, 200 and 300 mL/calf/day in Murrah buffalo calves.

  • Fermented milk improved immunity and faecal biomarkers in Murrah buffalo calves without any adverse effect on nutrient utilization

  • Responses were more evident in 200 and 300 mL probiotic fermented milk-fed groups as compared to 100 mL.

HIGHLIGHTS

  • Inclusion of Lactobacillus acidophilus NCDC15 in the form of fermented milk at 100, 200 and 300 mL/calf/day in Murrah buffalo calves.

  • Fermented milk improved immunity and faecal biomarkers in Murrah buffalo calves without any adverse effect on nutrient utilization

  • Responses were more evident in 200 and 300 mL probiotic fermented milk-fed groups as compared to 100 mL.

INTRODUCTION

Gastrointestinal tract (GIT) of newborn calves is rapidly colonized by an array of microbiota during and after birth [11 Malmuthuge N, Griebel PJ, Guan LL. The gut microbiome and its potential role in the development and function of newborn calf gastrointestinal tract. Front Vet Sci. 2015;2:36.] and is a critical period for the gut formation. The immune system of neonates is immature in the initial weeks of life and often results in high morbidity and mortality, as results of inappropriate colostral supply or contact to pathogenic microbes [22 Ayrle H, Mevissen M, Kaske M, Nathues H, Gruetzner N, Melzig M, Walkenhorst M. Medicinal plants-prophylactic and therapeutic options for gastrointestinal and respiratory diseases in calves and piglets? A systematic review. BMC Vet Res. 2016;12(1):1-31.]. Furthermore, rapid change in diet, environment and other stresses [33 Krehbiel CR, Rust SR, Zhang G, Gilliland SE. Bacterial direct-fed microbials in ruminant diets: Performance response and mode of action. J Anim Sci. 2003; 81 (14_suppl_2): 120-32., 44 Ouwehand A, Isolauri E, Salminen S. The role of the intestinal microflora for the development of the immune system in early childhood. Eur J Nutr. 2002;41(1):32-7.] imbalances gut health and allows colonization of opportunistic pathogens that confer harmful effects on animal health [55 Maldonado NC, de Ruiz C.S, Otero MC, Sesma F, Nader-Macías ME. Lactic acid bacteria isolated from young calves-characterization and potential as probiotics. Res Vet Sci. 2012; 92(2):342-9., 66 Bauer E, Williams BA, Smidt H, Verstegen MW, Mosenthin R. Influence of the gastrointestinal microbiota on development of the immune system in young animals. Curr Issues Intest Microbiol. 2006; 7(2): 35-52.] particularly scours [77 Quezada-Mendoza VC, Heinrichs AJ, Jones CM. The effects of a prebiotic supplement (Prebio Support) on fecal and salivary IgA in neonatal dairy calves. Livest Sci. 2011; 142(1-3): 222-8.] accompanied by reduced digestion and malabsorption of nutrients [88 Signorini ML, Soto LP, Zbrun MV, Sequeira GJ, Rosmini MR, Frizzo LS. Impact of probiotic administration on the health and fecal microbiota of young calves: a meta-analysis of randomized controlled trials of lactic acid bacteria. Res Vet Sci. 2012; 93(1): 250-8.]. Hence, it becomes tough to diminish the occurrence of such gastrointestinal infections in young calves to gain optimum growth and succeeding productivity in later life.

Antibiotics have been used to treat and prevent intestinal illnesses since the 1940s, and these practices have resulted in the accumulation of antimicrobial residues in animal products as well as the emergence of microbial drug resistance [99 Samanta AK, Jayaram C, Jayapal N, Sondhi N, Kolte AP, Senani S, Sridhar M, Dhalin A. Assessment of fecal microflora changes in pigs supplemented with herbal residue and prebiotic. PloS one 10. 2015; 10(7): e0132961.]. Therefore, the European Union banned the application of antibiotics in food animals since 2006 [1010 Castanon JIR. History of the use of antibiotic as growth promoters in European poultry feeds. Poultry Sci. 2007; 86(11):2466-71.] and this necessitated a worldwide consciousness to discover possible alternatives to replace antibiotics while not negotiating animal safety and consumer health perspectives. In this milieu, probiotics can be a promising approach in promoting animal health and stabilization of gastrointestinal microbiota. Probiotics are live microorganisms when administrated in adequate amount confers beneficial health effects to host. Microorganism most extensively studied in these aspects are Lactobacilli species [1111 Sharma AN, Kumar S, Tyagi AK. Effects of mannan‐oligosaccharides and Lactobacillus acidophilus supplementation on growth performance, nutrient utilization and faecal characteristics in Murrah buffalo calves. J Anim Physiol Anim Nutr. 2018;102(3):679-89.]. It is well known that feeding probiotics to calves [1212 Corcionivoschi N, Drinceanu D, Stef L, Luca I, Julean C. Probiotics-identification and ways of action. Innov Romanian Food Biotechnol. 2010;6:1-11., 1313 Morrison SJ, Dawson S, Carson AF. The effects of mannan oligosaccharide and Streptococcus faecium addition to milk replacer on calf health and performance. Livest Sci. 2010;131(2-3):292-6., 1414 Riddell JB, Gallegos AJ, Harmon DL, McLeod KR. Addition of a Bacillus based probiotic to the diet of preruminant calves: Influence on growth, health, and blood parameters1, 2, 3. Int J Appl ResVet M. 2010;8:78-85.] enhanced gut health, digestive ability and growth performance [1515 Frizzo S, Soto LP, Zbrun MV, Bertozzi E, Sequeira G, Armesto RR, et al. Lactic acid bacteria to improve growth performance in young calves fed milk replacer and spray-dried whey powder. Anim Feed Sci Technol. 2010; 157(3-4):159-67., 1616 Kawakami SI, Yamada T, Nakanishi N, Cai Y. Feeding of lactic acid bacteria and yeast on growth and diarrhea of Holstein calves. J Anim Vet Adv. 2010;9(7):1112-4., 1717 Frizzo LS, Zbrun MV, Soto LP, Signorini ML. Effects of probiotics on growth performance in young calves: A meta-analysis of randomized controlled trials. Anim Feed Sci Technol. 2011;169(3-4):147-56.]. Probiotics can improve immunity by inducing serum immunoglobulin secretion in early-weaned calves [1818 Sun P, Wang JQ, Zhang HT. Effects of Bacillus subtilis natto on performance and immune function of preweaning calves. J Dairy Sci. 2010;93(12):5851-5.]. Humoral immunity also increased due to the combined effects of probiotic and prebiotic in calves [1919 Mohamadi P, Dabiri N. Effects of probiotic, prebiotic and synbiotic on performance and humoral immune response of female suckling calves. In proceeding of the 62nd annual meeting of the European Association for Animal Production. Stavanger, Norway. 29 August to 2 September, 2011; p. 204.]. Although these studies well proved the effectiveness of probiotic administration in cattle calves but till date studies on indigenous Murrah buffalo calves are limited. Furthermore, apart from host species the response of probiotic differs upon the type and efficacy of microorganisms administered in host.

In support of the earlier findings, it was assumed that the administration of fermented milk with probiotic to Murrah buffalo calves will improve the gut microbiota and may possibly enhance their performance along with advantageous repercussions on gut health and faecal characteristics of Murrah buffalo calves. This also believed that fortified milk supplemented with probiotics can have beneficial effects on the immune response in calves.

The primary concern of this experiment was to determine the effects of fermented milk with probiotic Lactobacillus acidophilus (L. acidophilus) NCDC15 on apparent nutrient digestibility, faecal biomarkers and immune responses in Murrah buffalo calves.

MATERIAL AND METHODS

The present study of 90 days was conducted in the Livestock Research Centre of ICAR-National Dairy Research Institute (NDRI), Karnal, Haryana, India. Experimental protocol involving handling and management of animals were carried out in compliance with applicable rules and guidelines laid down by the Institutional Animal Ethics Committee (IAEC) constituted under Committee for the Purpose of Control and Supervision of Experiments in Animals (CPCSEA), New Delhi, Government of India.

Preparation of probiotic enriched fermented milk for administration to calves

Pure, freeze-dried culture of L. acidophilus NCDC15 was procured from the National Collection for Dairy Culture (NCDC), Dairy Microbiology Department, ICAR-NDRI, Karnal and revived in MRS broth. L. acidophilus NCDC15 selected for milk fermentation had proven probiotic potential [2020 Kumar S, Pattanaik AK, Sharma S, Jadhav SE, Dutta N, Kumar A. Probiotic potential of a Lactobacillus bacterium of canine faecal-origin and its impact on select gut health indices and immune response of dogs. Probiotic Antimicro Prot. 2017;9(3):262-77.]. The 1 mL of inoculum was added to 9 mL phosphate-buffered saline (PBS) and vortexed thoroughly. Serial 10-fold dilutions were then made in PBS. A 100-μL volume of each dilution was inoculated onto MRS agar. The MRS plates were incubated anaerobically at 37°C for 48 h for isolation of lactic acid bacteria. Lactic acid bacteria were identified by colony morphology and colony counts were recorded. All testing was performed in triplicate. Based on previous studies we select the 108 cfu/ml and to make probiotic enriched fermented milk, a loop-full of inoculum was added to the milk and incubated for 24 hours at 37 °C. At every alternate day, colony-forming unit (CFU) per mL of fermented milk was counted to test the viability of bacterial cells and maintained at a level of 108 CFU/mL throughout experimental feeding.

Animal distribution, housing, treatments and feeding regime

Twenty-four neonatal Murrah buffalo calves aged 5-7 days and 31 ± 2.0 kg BW were randomly allocated into four groups of six animals in each. Buffalo calves were weaned from their dams and housed individually in well-ventilated calf pens having adequate access to sunlight. Before accommodating the experimental calves, all of pens were cleaned with detergent, disinfected with potassium permanganate solution and washed with diluted phenyl to ensure appropriate preventive measures against various contagious and infectious diseases. Individual pens were well equipped with detached feeder and waterer to allow free access to feed and water, respectively, during the entire experimental period. Calves were dewormed as per the standard deworming schedule.

The control (CT) group received a basal diet consisting of calf starter and green fodder as well as whole milk without any probiotic supplementation. In PFM100, PFM200 and PFM300 groups, the basal diet remained same except that the probiotic fermented milk was provided at 100, 200 and 300 mL/calf/day, respectively. Fermented milk was prepared from milk offered to the animals as per their schedule, to make sure that no additional intake by animals in treatment groups. The calf starter containing maize, bajra, groundnut cake (GNC), soybean meal (SBM), mustard oil cake (MOC), wheat bran, rice polish, mineral mixture was formulated (Table 1) using quality ingredients and offered from the second week onwards. The animals were given ad libitum freshly harvested green forage comprising maize and jowar. All the calves had access to clean water ad libitum 24h. Whole milk was fed to the calves at 1/10th of actual BW up to the 1st two weeks followed by 1/15th, 1/20th and 1/25th of actual BW in the 3rd to 4th week, 5th to 6th week and 7th to 8th week of respectively, after morning and evening diet distribution.

Table 1
Gross and chemical composition of the basal diet.

Structural growth measurements, digestion trial and proximate analysis of samples

Structural growth measurements were monitored by assessing body length, wither height, hip-height and heart girth using “tape measures". Following 60 days of experimental feeding, a digestion trial of seven days was conducted, in which daily dry matter intake (DMI) and total faeces voided were listed. For estimation of N, faecal samples (1/50th fraction of the total voiding) were pooled and conserved in 25% sulphuric acid for 7 days from each animal. Representative samples (feeds, orts and faeces) were oven-dried at 60°C for 48 h and grounded in a hammer mill of 1 mm sieve size and tested for proximate principles [2121 AOAC. Official Method of Analysis, 16th edn (Association of Official Analytical Chemists: Washington DC). 1995.] such as total ash (942.05), ether extract (920.39), Kjeldahl nitrogen (984.13), neutral detergent fibre (2002.04), acid detergent fibre (973.18).

Faecal collection, sampling and biomarkers estimation

Faeces of the calves were scored for faecal consistency using a 1-4 point scale (1 = Normal and firm faeces, 2 = Soft or loose faeces, 3=Runny or very loose faeces and 4 = Watery faeces) [2222 Larson LL, Owen FG, Albright JL, Appleman RD, Lamb RC, Muller LD. Guidelines toward more uniformity in measuring and reporting calf experimental data. J Dairy Sci. 1977;60(6):989-91.]. Calves of fecal consistency 3 or 4 have been graded as diarrhoeal. Hydrated intervention was given when the calve had pale and dry mucous membranes along with diarrhea: 8 g of NaCl, 8 g of NaHCO3, 2 g of KCl, 15 g of dextrose, and 2 L of warm water [2323 Salazar LF, Nero LA, Campos-Galvão ME, Cortinhas CS, Acedo TS, Tamassia LF, Marcondes MI. Effect of selected feed additives to improve growth and health of dairy calves. PloS one. 2019;14(5): e0216066.]. Faecal sample about 10-15 g was collected by a rectal massage from each animal following perianal cleansing with dilute betadine solution with sterile gloves at the monthly interval on d0, d30, d60, and d90 to evaluate faecal pH, ammonia (NH3), lactate, volatile fatty acids (VFA) and microbiota populations. The samples were collected in sterile 50 mL falcon tubes at approximately 07:00h and transferred to the laboratory for further analysis. The pH of the samples was determined directly with a digital pH meter before aliquoting of the faeces (pH Spear, Eutech Instruments, Klang Selangor, DE, Malaysia, pH Range: -1.00 to 14.00 pH, Resolution: 0.01 pH, Accuracy: ±0.01 pH). The pH meter was specially designed to test the semi-solid samples by direct pH measurement [2424 Kore KB, Pattanaik AK, Das A, Sharma K. Evaluation of alternative cereal sources in dog diets: effect on nutrient utilisation and hindgut fermentation characteristics. J Sci Food Agric. 2009;89(13):2174-80.]. Additional three faecal aliquots were prepared to evaluate fermentative end products i.e., ammonia, lactate, and VFA [2424 Kore KB, Pattanaik AK, Das A, Sharma K. Evaluation of alternative cereal sources in dog diets: effect on nutrient utilisation and hindgut fermentation characteristics. J Sci Food Agric. 2009;89(13):2174-80.]. Approximately 2.0g of freshly collected faeces was briefly mixed with 6 mL of 6.0 N HCl and processed at -20oC for subsequent ammonia analysis [2525 Chaney AL, Marbach EP. Modified reagents for determination of urea and ammonia. Clin. Chem. 1962;8(2):130-2.]. A 2g fresh faecal aliquot was blended with 4 mL of freshly prepared 25% (w/v) metaphosphoric acid and centrifuged (10,000 rpm) for 10 min. The resultant supernatant was used for the analysis of total VFA [2626 Cottyn BG, Boucque CV. Rapid method for the gas-chromatographic determination of volatile fatty acids in rumen fluid. J Agric Food Chem. 1968;16(1):105-7.]. The third aliquot of approximately 2g was diluted with 4 mL of distilled water and centrifuged for 10 min at 10,000 rpm, and the supernatant was processed for analysis of lactate [2727 Barker SB, Summerson WH. The colorimetric determination of lactic acid in biological material. J Biol Chem. 1941; 138: 535-54.].

Two sets of 10-fold (10-1 to 10-8) serial dilutions with a combined 10 mL volume consisting of 1 g of homogenized fresh faeces and 9 mL of NS (normal saline: 0.9% NaCl) were enumerated for bacterial populations and plated in duplicate onto selective media [2828 Kumar S, Pattanaik AK, Jose T, Sharma S, Jadhav SE. Temporal Changes in the Hindgut Health Markers of Labrador Dogs in Response to a Canine-origin Probiotic Lactobacillus johnsonii. Anim Nutr Feed Technol. 2016;16:251-70.]: for lactobacilli-MRS agar (Himedia), for coliforms-EMB Agar, Levine (Himedia), Clostridial agar (Himedia) for clostridia and Bifidobacteria agar (Himedia) for bifidobacterium. Specific agar plates were aerobically incubated for lactobacilli and coliforms for 24 and 48 hours at 37°C; respectively. The bifidobacteria and clostridial agar were anaerobically incubated at 37°C. After incubation the agar plates were appraised for bacterial growth. The bacterial colonies were counted as CFU/g faeces and converted into log10 CFU/g. CFU were described as distinct colonies with a diameter of at least 1 mm [2929 Swanson KS, Grieshop CM, Flickinger EA, Bauer LL, Healy HP, Dawson KA, Merchen NR, Fahey Jr GC. Supplemental fructooligosaccharides and mannanoligosaccharides influence immune function, ileal and total tract nutrient digestibilities, microbial populations and concentrations of protein catabolites in the large bowel of dogs. J Nutr. 2002;132(5):980-9.].

Cell-mediated and humoral immune response

After 75 days of experimental feeding, all the calves were assessed for cell-mediated immune (CMI) response by measurement of skin indurations using in vivo DTH (delayed-type hypersensitivity) test against phytohemagglutinin-phaseolus vulgaris (PHA-p; Sigma, St Louis, MO, USA) as a mitogen [3030 Masucci F, De Rosa G, Grasso F, Napolitano F, Esposito G, Di Francia A. Performance and immune response of buffalo calves supplemented with probiotic. Livest Sci. 2011;137(1-3):24-30.]. Until conducting the DTH test, the skin area to be examined (both sides of the neck region) was cleaned and shaved before 24 h. An area of about one square cm was encircled on both sides of the neck region, with a black marker pen. Skin thickness was measured using an electronic digital Vernier caliper (measuring range 0-150mm), which reflected a basal value (0 h). All the animals were injected with 100 μL PHA-p intradermally (50 µg/100 µL in phosphate-buffered saline, PBS) solution on one side and normal saline solution on the other side of neck area as a negative control. Skin thickness was measured post-inoculation at 6, 12, 18, 24, 36, 48 and 72 h and was expressed as percentage of skin thickness increase relative to the value at 0th h.

In case of humoral immune response (HIR), calves were injected intravenously (IV) with 10% suspension of 1 mL washed chicken red blood cell (CRBC) in 0.15M NaCl after 60 days of experimental feeding. Serum samples were collected before injection (0 days) and then on d7, d14, d21 and d28 and processed for antibody determination at -20°C. The sera samples were thawed, inactivated for 30 min at 56°C, and tested for antibody titre using the microtitre haemagglutination (HA) procedure [3131 Wegmann TG, Smithies O. A simple hemagglutination system requiring small amounts of red cells and antibodies. Transfusion. 1966;6(1):67-73.]. The HA titers were read after 3 h at room temperature and expressed as log2.

Statistical analysis

Data generated in the present experiment were analyzed by using the Statistical Package for Social Sciences (SPSS, version 20.0 Chicago, USA) and presented as mean ± standard error.

Data from digestibility trials (intake and digestibility) were analyzed as a randomized complete design using the General linear model of the SPSS based on the statistical model:

Yij=μ+Ti+eij, where,

Yij = dependent variable of the jth calf on the ith treatment

μ = overall mean

Ti = the fixed effect of ith treatment effect (i= 100, 200, 300 mL/calf/day of PFM)

eij = random residual (error) associated with the dependent variable from the jth calf on the ith treatment. Means were tested using Duncan’s multiple range tests.

Before statistical analysis, fecal microbial counts were transformed into log10. Continuous data collected over time (i.e., monthly structural growth measurements, forthnightly average fecal score, monthly faecal pH, faecal ammonia N, lactate, VFAs and humoral and cell mediated immunity) were analyzed using the linear model:

Yijk=μ+Ti+Pj+(TP)ij+eijk where

μ = general mean

Ti = effect of ith treatment (i= 100, 200, 300 mL/calf/day of PFM)

Pj = effect of jth 90 days period

(TP)ij = effect of interaction between treatment and 90 days period

eijk = random error. Treatment differences with (P < 0.05) were considered as a significant statistic.

RESULTS

Body structural Measurements and apparent nutrient digestibility coefficient

Data concerning to structural growth measurements (Table 2) implied that initial hip height (cm), heart girth (cm), wither height (cm) and body length (cm) were similar (P > 0.05) across the groups. Final hip height was improved (P < 0.05) by the inclusion of probiotics in all the supplemented groups. In a similar line, heart girth was increased in probiotics fed groups with the trend of PFM300~ PFM200> PFM100>CT. On the other hand, wither height and body length was higher (P < 0.05) in PFM200 and PFM300 compared to CT, whereas, PFM100 had a characteristic that was comparable to that of other groups. The values of apparent digestibility coefficient of various nutrients [Dry matter (DM), Organic matter (OM), Crude protein (CP), Neutral detergent fiber (NDF), and Acid detergent fiber (ADF)] are furnished in Table 3. There was no influence (P > 0.05) on supplementation of probiotic fermented milk on apparent digestibility coefficient among the groups.

Faecal biomarkers

Faecal score, examined per pen individually, remained unvaried (P > 0.05) in all groups but period-wise (Figure 1) significant (P < 0.05) impact was also observed. Fortnightly the average faecal consistency scores are given in Table 4. The average faecal pH (Table 4) was decreased (P < 0.05) in probiotic fermented milk administered groups as compared to control. The period-wise (Figure 2) comparison also revealed significant decreased (P < 0.05) effect on faecal pH.

Figure 1
Fortnightly faecal score in Murrah buffalo calves supplemented with probiotic enriched fermented milk

Figure 2
Faecal pH in Murrah buffalo calves supplemented with probiotic enriched fermented milk.

The data apropos of faecal microbiota (Table 5) indicated that Lactobacillus population (log10 cfu/g in fresh faeces) was improved (P < 0.05) in all probiotic supplemented groups as compared to control. In addition, there was also a treatment and time relationship for faecal Lactobacillus count (P = 0.048) and this indicated that the lactobacilli count was higher during the experimental period due to probiotic supplementation. The Bifidobacterium population in all supplemented groups responded positively (P < 0.001) to probiotic supplementation in comparison to control (CT). On the other hand, coliform count (log10 cfu/g in fresh faces) was significantly (P < 0.05) decreased in all the supplemented group compared to CT, however, no influence (P > 0.05) was found for clostridia count (log10 cfu/g in fresh faeces) in all the groups.

The data presented in Table 4 illustrates the effect of L. acidophilus supplementation on faecal ammonia and lactate. In all probiotic supplemented groups, the concentration of faecal ammonia (μmol/g fresh faeces) was decreased (P < 0.05) relative to the control (CT). While the opposite trend was observed for faecal lactate levels (µmol/g of fresh faeces) which was increased (P < 0.05) in all probiotics administered groups as compared to control. Moreover, there was significant effect of period-wise comparison on faecal ammonia (P < 0.001) and lactate (0.005) in Figure 3 and Figure 4 respectively, but period * treatment interaction remained unaffected. The total VFAs data illustrated in Table 6 showed that among the faecal VFAs, acetate level was increased (P < 0.05) in PFM200 and PFM300 as compared to the other two groups. However, propionate and butyrate were increased in all the probiotics fed groups as compared to control. On the other hand, there was no effect of period and treatment * period interaction on faecal VFA concentrations with supplementation of probiotics.

Figure 3
Faecal ammonia in Murrah buffalo calves supplemented with probiotic enriched fermented milk.

Figure 4
Faecal lactate in Murrah buffalo calves supplemented with probiotic enriched fermented milk.

Immune response

The DTH response results to PHA-p in the form of an absolute increase in skin induration (Figure 5). There was significant (P < 0.001) increases in absolute skin thickness (mm) in PFM200 and PFM300 when compared to CT. The HI response data (Figure 6) assessed as antibody responses to chicken-erythrocytes (CRBC) by HA test indicated that the antibody (HA) titre log2 was significantly higher (P < 0.05) in PFM200 and PFM300 groups as compared to CT and that of PFM100 was comparable to rest of groups. Additionally, the HI response data also showed that titre of antibody in all groups displayed a continuous increase up to 14-days post-inoculation followed by a 21-days decrease.

Figure 5
DTH response to intradermal PHA-P in in Murrah buffalo calves supplemented with probiotic enriched fermented milk.

Figure 6
Antibody titre against C-RBC in Murrah buffalo calves supplemented with probiotic enriched fermented milk.

Table 2
Body measurements in Murrah buffalo calves supplemented with probiotic enriched fermented milk.
Table 3
Nutrient utilization in Murrah buffalo calves supplemented with probiotic enriched fermented milk
Table 4
Faecal score, pH, ammonia and lactate in Murrah buffalo calves supplemented with probiotic enriched fermented milk.
Table 5
Faecal microbiota of Murrah buffalo calves supplemented with probiotic enriched fermented milk.
Table 6
Faecal VFAs of Murrah buffalo calves supplemented with probiotic enriched fermented milk

DISCUSSION

Body structural Measurements and apparent nutrient digestibility coefficient

Neonatal Murrah buffalo calves have high metabolizing and fast growth rates, but their growth performance can be limited by several factors [3232 Zhang R, Zhou M, Tu Y, Zhang NF, Deng KD, Ma T, Diao QY. Effect of oral administration of probiotics on growth performance, apparent nutrient digestibility and stress‐related indicators in Holstein calves. J Anim Physiol Anim Nutr. 2016;100(1): 33-8.]. The effect of probiotic can vary with the host’s dosages, regimens, bacterial strains, type, age, health and nutritional status [3333 Vlasova AN, Kandasamy S, Chattha KS, Rajashekara G, Saif LJ. Comparison of probiotic lactobacilli and bifidobacteria effects, immune responses and rotavirus vaccines and infection in different host species. Vet Immunol Immunopathol. 2016;172:72-84.]. In the present study, increases in structural growth measurements may indicate increased body capacity [3434 Lesmeister KE, Heinrichs AJ, Gabler MT. Effects of supplemental yeast (Saccharomyces cerevisiae) culture on rumen development, growth characteristics, and blood parameters in neonatal dairy calves. J. Dairy Sci. 2004; 87(6):1832-9.]. Improvement in structural body measurement can result from additional energy in calves receiving probiotic required for skeletal deposition due to increased initial DMI and growth rates in calves supplemented with probiotic [3535 Ojha L, Kumar S, Kewalramani N, Sarkar S, Tyagi AK. Growth and haematological parameters in murrah buffalo calves as affected by addition of Lactobacillus acidophilus in the diet. Indian J Anim Nutr. 2018;35(3):282-9.]. Consistent with our findings, partial replacement of probiotic yogurt for milk in calves significantly increased body length, wither height and hip depth can be attributed to higher DMI and weight gain [3636 Noori M, Alikhani M, Jahanian R. Effect of partial substitution of milk with probiotic yogurt of different pH on performance, body conformation and blood biochemical parameters of Holstein calves. J Appl Anim Res. 2016;44(1):221-9.]. Likewise, dietary supplementation of probiotics resulted in an increase in heart girth and wither height relative to control cross-bred calves [3737 Chandra R, Mehla RK, Sirohi SK, Rahman H. Effect of probiotic supplementation on growth of crossbred calves. Indian J Animal Sci. 2009;79(12):1254-7.]. Likewise, supplementation of species-specific probiotic in calves significantly improved heart girth measurements in treatment as compared to control [3838 Agazzi A, Tirloni E, Stella S, Maroccolo S, Ripamonti B, Bersani C, et al. Effects of species-specific probiotic addition to milk replacer on calf health and performance during the first month of life. Ann Anim Sci. 2014;14(1): 101-15.]. In a recent study carried it was also observed that feeding of probiotic, prebiotic and synbiotic in Murrah buffalo calves significantly improved final hip height and heart girth [1111 Sharma AN, Kumar S, Tyagi AK. Effects of mannan‐oligosaccharides and Lactobacillus acidophilus supplementation on growth performance, nutrient utilization and faecal characteristics in Murrah buffalo calves. J Anim Physiol Anim Nutr. 2018;102(3):679-89.]. However, in contrast to the current results, no significant difference in body measurement parameters with probiotic supplementation was reported [1414 Riddell JB, Gallegos AJ, Harmon DL, McLeod KR. Addition of a Bacillus based probiotic to the diet of preruminant calves: Influence on growth, health, and blood parameters1, 2, 3. Int J Appl ResVet M. 2010;8:78-85.].

Probiotic supplementation to cattle calves showed no significant difference on apparent digestibility of nutrients between the treatment and control groups which is similar to the observation of the present study [2222 Larson LL, Owen FG, Albright JL, Appleman RD, Lamb RC, Muller LD. Guidelines toward more uniformity in measuring and reporting calf experimental data. J Dairy Sci. 1977;60(6):989-91.]. However, contrary to present findings, digestibility coefficient of DM, CP, CF and NDF was significantly (P < 0.05) higher in the probiotic fed group [3939 Gupta P, Sharma K.S Porwal M, Joshi M. Biological performance of female calves fed diets supplemented with different strains of lactobacilli. Int J Environ Sci Technol. 2015;4:1181-7., 4040 Sadrsaniya DA, Raval AP, Bhagwat SR, Nageshwar A. Effects of Probiotics Supplementation on Growth and Nutrient Utilization in Female Mehsana Buffalo Calves. Indian Vet J. 2015;92(9):20-2.].

Faecal biomarkers

In the present study, all experimental animals were individually housed and maintained under proper feeding and hygiene program, and diarrhea was rarely recorded, indicating the calves were exposed to good conditions with few environmental stressors that could be the explanation for unchanged faecal score among the treatments. In a similar line, supplementation of live yeast product to young calves did not affect faecal scores [4141 Galvao KN, Santos JE, Coscioni A, Villaseñor M, Sischo WM, Berge ACB. Effect of feeding live yeast products to calves with failure of passive transfer on performance and patterns of antibiotic resistance in fecal Escherichia coli. Reprod Nutr Dev. 2005;45(4):427-40.]. Even administration of Bacillus sp. as a probiotic in neonatal calves found no variations in growth performance or risk of diarrhoea [4242 Bakhshi N, Ghorbani GR, Rahmani HR, Samie A. Effect of probiotic and milk feeding frequency on performance of dairy Holstein calves. Int J Dairy Sci. 2010;5(4):285-91., 1414 Riddell JB, Gallegos AJ, Harmon DL, McLeod KR. Addition of a Bacillus based probiotic to the diet of preruminant calves: Influence on growth, health, and blood parameters1, 2, 3. Int J Appl ResVet M. 2010;8:78-85.]. However, contrary to the present findings, supplementation of probiotic significantly reduced (P < 0.05) faecal scores and duration of diarrhoea in treatment groups as compared to control groups, possibly due to the the surrounding environment. [4343 Lee YE, Kang IJ, Yu EA, Kim S, Lee HJ. Effect of feeding the combination with Lactobacillus plantarum and Bacillus subtilis on fecal microflora and diarrhea incidence of Korean native calves. Korean J Vet Serv. 2012;35:343-6., 4444 Le OT, Dart PJ, Harper K, Zhang D, Schofield B, Callaghan MJ, et al. Effect of probiotic Bacillus amyloliquefaciens strain H57 on productivity and the incidence of diarrhoea in dairy calves. AnimProd Sci. 2017;57(5):912-9.]. Consequently, the benefit from probiotic administration for the health of neonatal calves can depend on the type used, the mode of administration and the environmental status.

Lowering of faecal pH in present study agrees to another study [4545 Bayatkouhsar J, Tahmasebi AM, Naserian AA, Mokarram RR, Valizadeh R. Effects of supplementation of lactic acid bacteria on growth performance, blood metabolites and fecal coliform and lactobacilli of young dairy calves. Anim Feed Sci Technol. 2013;186(1-2):1-11.] in which supplementation of probiotic caused a significant lowering of faecal pH in calves. It was due to the production of large concentrations of lactic acid during the carbohydrate fermentation by lactic acid bacteria.

The increased population of Lactobacillus and Bifidobacterium with a concomitant reduction of Coliforms in the current study might be due to increased lactic acid concentration in GIT of the probiotic supplemented group which in turn led to increase in beneficial gut microbes with a concomitant reduction in the growth of harmful microbes [1212 Corcionivoschi N, Drinceanu D, Stef L, Luca I, Julean C. Probiotics-identification and ways of action. Innov Romanian Food Biotechnol. 2010;6:1-11.]. Furthermore, the rumen of newborn calves were not functional and the microbial population is slowly controlled with age as the animals mature [4545 Bayatkouhsar J, Tahmasebi AM, Naserian AA, Mokarram RR, Valizadeh R. Effects of supplementation of lactic acid bacteria on growth performance, blood metabolites and fecal coliform and lactobacilli of young dairy calves. Anim Feed Sci Technol. 2013;186(1-2):1-11.,4646 Karney TL, Johnson MC, Ray B. Changes in the lactobacilli and coliform populations in the intestinal tract of calves from birth to weaning. J Anim Sci. 1986;63(1):446-7.]. Such causes may be the explanation for the significant treatment and period interaction for Lactobacillus count in this trial. Microbiota ferments amino acid to short-chain fatty acid (SCFA) and ammonia to obtain the energy. Probiotic bacteria increase SCFA formation by accelerating carbohydrate breakdown which is resistant to indigenous bacteria [4747 Sakata T, Kojima T, Fujieda M, Takahashi M, Michibata T. Influences of probiotic bacteria on organic acid production by pig caecal bacteria in vitro. Proc Nutr Soc. 2003;62(1):73-80.]. The SCFA acts as a host energy source, supplying 10-30% of the basal metabolic requirement along with energy for hepatic cells, colonocytes and peripheral tissue [4848 Cummings JH, Macfarlane GT. The control and consequences of bacterial fermentation in the human colon. J Appl Microbiol. 1991;70(6):443-59.]. Acceleration in net SCFA and lactic acid production by probiotic supplementation likely contributed to lower the net ammonia content. An improved faecal VFA level in probiotic supplemented group is an indicator of better adaptation of probiotic in the gut of calves. Probiotics also aid in the development of rumen in calves by elevating the concentration of VFA in rumen. Supplementation of probiotic as Bacillus amyloliquefaciens strain H57 increased the concentration of VFA such as valerate and butyrate in the rumen of dairy calves may have added more energy to the rumen epithelium and assisted in the rumen development [4444 Le OT, Dart PJ, Harper K, Zhang D, Schofield B, Callaghan MJ, et al. Effect of probiotic Bacillus amyloliquefaciens strain H57 on productivity and the incidence of diarrhoea in dairy calves. AnimProd Sci. 2017;57(5):912-9.]. Similarly, an increase in VFA concentration on supplementation of probiotic in calves was observed [4949 Pinos-Rodríguez JM, Robinson PH, Ortega ME, Berry SL, Mendoza G, Bárcena R. Performance and rumen fermentation of dairy calves supplemented with Saccharomyces cerevisiae1077 or Saccharomyces boulardii1079. Anim Feed Sci Technol. 2008;140(3-4):223-32.]. However, contrary to this, supplementation of bacteria based probiotic in Holstein calves did not affect ruminal VFA content [5050 Qadis AQ, Goya S, Ikuta K, Yatsu M, Kimura A, Nakanishi S, Sato S. Effects of a bacteria-based probiotic on ruminal pH, volatile fatty acids and bacterial flora of Holstein calves. J Vet Med Sci. 2014;76:877-85.].

Immune response

Proper production of microbiota in the gastrointestinal tract in early weeks of life is critical for developing a healthy immune system as calves are born with a naive immune system [5050 Qadis AQ, Goya S, Ikuta K, Yatsu M, Kimura A, Nakanishi S, Sato S. Effects of a bacteria-based probiotic on ruminal pH, volatile fatty acids and bacterial flora of Holstein calves. J Vet Med Sci. 2014;76:877-85.]. Lactobacillus strains augment the integrity of the intestinal barrier function that results in decreased translocation of bacteria across the intestinal mucosa and maintenance of immune tolerance [5151 Hemarajata P, Versalovic J. Effects of probiotics on gut microbiota: mechanisms of intestinal immunomodulation and neuromodulation. Therap. Adv. Gastroenterol. 2013;6(1):39-51.]. PHA-p, a lectin from Phaseolus vulgaris causes non-specific proliferation of T-cells which are responsible for DTH reactions and used mainly in vivo as an indicator of cell-mediated immune response [5252 Grasso F, Napolitano F, De Rosa G, Quarantelli T, Serpe L, Bordi A. Effect of pen size on behavioral, endocrine, and immune responses of water buffalo (Bubalus bubalis) calves. J Anim Sci. 1999;77(8):2039-46.]. The proliferative response potential of circulating T-lymphocytes to an injected mitogen such as PHA can be measured by skin thickness test [5353 Smits JE, Bortolotti GR, Tella JL. Simplifying the phytohaemagglutinin skin‐testing technique in studies of avian immunocompetence. Funct Ecol. 1999;13(4):567-72.]. The reaction depends on specific antigen-dependent T-cell recall response seen as an inflammatory reaction that reaches maximum intensity after antigenic challenge of 24 to 48 h [5454 Meydani SN, Ha WK. Immunologic effects of yogurt. Am J Clin Nut. 2000;71(4):861-72.] that justifies improved DTH response in PFM200 and PFM300 (Figure 3). The outcome of this study towards the positive impact of probiotics on CMI response aligns with the findings of other researchers who had shown that dietary probiotics enhanced specific immune functions in young dogs [5555 Benyacoub J, Czarnecki-Maulden GL, Cavadini C, Sauthier T, Anderson RE, Schiffrin EJ, Von der Weid T. Supplementation of food with Enterococcus faecium (SF68) stimulates immune functions in young dogs. J Nutr. 2003; 133(4): 1158-62.] and indicated enhanced DTH response on administration of E. faecalis in mice [5656 Shimada T, Cheng L, Ide M, Fukuda S, Enomoto T, Shirakawa T. Effect of lysed Enterococcus faecalis FK‐23 (LFK) on allergen-induced peritoneal accumulation of eosinophils in mice. Clin Exp Allergy. 2003;33(5):684-7.]. In another study, PHA-P intradermal injection increases the thickness of the skin fold in prebiotic and symbiotic treated calves, but the effect in probiotic treated calves is comparable from treatment and control groups [5757 Dar AH, Singh SK, Mondal BC, Palod J, Kumar A, Singh V, et al. Effect of probiotic, prebiotic and synbiotic on faecal microbial count and cellmediated immunity in crossbred calves. Indian J Anim Res. 2018;52(10):1452-6.]. On the contrary, DTH response to a percutaneous injection of PHA was not affected by the supplementation of probiotics [3030 Masucci F, De Rosa G, Grasso F, Napolitano F, Esposito G, Di Francia A. Performance and immune response of buffalo calves supplemented with probiotic. Livest Sci. 2011;137(1-3):24-30.]. The HIR is an immunity component that is mediated by secreted antibodies produced in the cells of the B lymphocyte lineage (B cell). Mohamadi [1919 Mohamadi P, Dabiri N. Effects of probiotic, prebiotic and synbiotic on performance and humoral immune response of female suckling calves. In proceeding of the 62nd annual meeting of the European Association for Animal Production. Stavanger, Norway. 29 August to 2 September, 2011; p. 204.] have observed increased humoral response after ovalbumin was injected into calves that received synbiotic supplementation against probiotic and prebiotic supplemented groups. Probiotics can prevent intestinal diseases through both humoral and cell-mediated immune modulation [2020 Kumar S, Pattanaik AK, Sharma S, Jadhav SE, Dutta N, Kumar A. Probiotic potential of a Lactobacillus bacterium of canine faecal-origin and its impact on select gut health indices and immune response of dogs. Probiotic Antimicro Prot. 2017;9(3):262-77.] as indicated in the present investigation.

CONCLUSIONS

Based on the above findings, it may be concluded that dietary supplementation of probiotic Lactobacillus acidophilus as fermented milk improved the body growth indices and neonatal health measured in terms of quality faecal attributes and immunity. Further in-depth analysis is indicated that the observed responses were more evident in 200 and 300 mL probiotic fermented milk-fed groups as compared to 100 mL. So 200 mL PFM is economical for raising Murrah calves. Overall, the findings of this study showed the efficacy of fermented milk enriched with probiotic is the potential feed additive to be used to promote health status and performance of Murrah calves.

Acknowledgments

The authors wish to acknowledge Director, ICAR-National Dairy Research Institute, Karnal, India for providing facilities required for successful completion of the research.

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  • Funding:

    No funding.

Edited by

Editor-in-Chief:

Alexandre Rasi Aoki

Associate Editor:

Renata Marino Romano

Publication Dates

  • Publication in this collection
    10 Jan 2022
  • Date of issue
    2021

History

  • Received
    28 Apr 2021
  • Accepted
    16 Aug 2021
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