In depth analysis of rumen microbial and carbohydrate-active enzymes profile in Indian crossbred cattle

Rumen houses a plethora of symbiotic microorganisms empowering the host to hydrolyze plant lignocellulose. In this study, NGS based metagenomic approach coupled with bioinformatic analysis was employed to gain an insight into the deconstruction of lignocellulose by carbohydrate-active enzymes (CAZymes) in Indian crossbred Holstein-Friesian cattle. Cattle rumen metagenomic DNA was sequenced using Illumina-MiSeq and 1.9 gigabases of data generated with an average read length of 871 bp. Analysis of the assembled sequences by Pfam-based Carbohydrate-active enzyme Analysis Toolkit identified 17,164 putative protein-encoding CAZymes belonging to different families of glycoside hydrolases (7574), glycosyltransferases (5185), carbohydrate-binding modules (2418), carbohydrate esterases (1516), auxiliary activities (434) and polysaccharide lyases (37). Phylogenetic analysis of putative CAZymes revealed that a significant proportion of CAZymes were contributed by bacteria belonging to the phylum Bacteroidetes (40%), Firmicutes (30%) and Proteobacteria (10%). The comparative analysis of HF cross rumen metagenome with other herbivore metagenomes indicated that Indian crossbred cattle rumen is endowed with a battery of CAZymes that may play a central role in lignocellulose deconstruction. The extensive catalog of enzymes reported in our study that hydrolyzes plant lignocellulose biomass, can be further explored for the better feed utilization in ruminants and also for different industrial applications.     

Presence of bifidobacteria in the rumen of calves fed different rations.

A study was made on the numbers and species of bifidobacteria present in the rumen of calves fed high-roughage and high-concentrate diets. With the roughage ration the bifidobacteria were not detectable in a 10(-3) dilution, whereas with the concentrate ration their number was high, usually in the order of 10(8) to 10(9)/ml of rumen fluid. The species most represented, identified by means of deoxyribonucleic acid-deoxyribonucleic acid hybridization tests, included Bifidobacterium ruminale, Bifidobacterium globosum, and an apparently new species.     

Susceptibility and resistance of anaerobic rumen fungi to antimicrobial feed additives

Listeria monocytogenes survived in meat, cheese and egg ravioli stored at 5°C for 14 d. Ravioli were considered edible for the first 9 d of storage. Initial L. monocytogenes populations of 3 × 10⁵ cfu/g of ravioli were reduced to non-detectable levels after heat treatment simulating that which would be used by the consumer.     

Relationship between rumen methanogens and methane production in dairy cows fed diets supplemented with a feed enzyme additive

Aims: To investigate the relationship between ruminal methanogen community and host enteric methane (CH₄) production in lactating dairy cows fed diets supplemented with an exogenous fibrolytic enzyme additive. Methods and Results: Ecology of ruminal methanogens from dairy cows fed with or without exogenous fibrolytic enzymes was examined using PCR–denaturing gradient gel electrophoresis (PCR–DGGE) analyses and quantitative real‐time PCR (qRT‐PCR). The density of methanogens was not affected by the enzyme additive or sampling times, and no relationship was observed between the total methanogen population and CH₄ yield (as g per head per day or g kg^?1 DMI). The PCR–DGGE profiles consisted of 26 distinctive bands, with two bands similar to Methanogenic archaeon CH1270 negatively correlated, and one band similar to Methanobrevibacter gottschalkii strain HO positively correlated, with CH₄ yield. Three bands similar to Methanogenic archaeon CH1270 or Methanobrevibacter smithii ATCC 35061 appeared after enzyme was added. Conclusions: Supplementing a dairy cow diet with an exogenous fibrolytic enzyme additive increased CH₄ yield and altered the composition of the rumen methanogen community, but not the overall density of methanogens. Significance and Impact of the Study: This is the first study to identify the correlation between methanogen ecology and host CH₄ yield from lactating dairy cows.     

Correlation between microbial enzyme activities in the rumen fluid of sheep under different treatments

Five total mixed rations prepared from finger millet (Eleusine Coracana) straw as a roughage (48%) and mixed concentrate (52%), supplemented with a 1% isoacid mixture (i-C4, i-C5, C5 and phenylacetic acid in equal proportions) or oil (groundnut oil, 5% more than the control) or urea (5% more nitrogen than the control), and protein (groundnut cake, 5% more nitrogen than the control) were given in a Latin square experiment to sheep. Enzymatic activities were estimated for urease, cellulase, protease, amylase, and lipase in various fractions of rumen fluid on the one hand and rumen microbial biomass on the other hand. Rumen samples were taken 3-4 hours after feeding and mixed rumen bacteria were separated as a strained rumen fluid without protozoa (SRFWP), cell free rumen fluid (CFRF) and enzymes associated with the bacteria cell (EABC). Samples of SRFWP and EABC contained higher enzyme activities than CFRF. Depending on the type of enzymes in each fraction, some significant coefficient of determination (r2) was seen. These values showed very close cooperative action between proteolytic and amylolytic enzymes under the experimental conditions, or perhaps the presence of some species of bacteria with both activities. Lipolytic bacteria are completely specialized for lipase production only (P < 0.05). The results showed oil, isoacid and crude protein enhanced microbial production (P < 0.05) and this can change the pattern of enzymes in the rumen of sheep.     

Effect of maize silage to grass silage ratio and feed particle size on protein synthesis and amino acid profile in different microbial fractions in a semi-continuous rumen simulation

The objective was to investigate the effect of variation in forage source and feed particle size of a diet, including interactions, on the amount and the composition of microbial crude protein (CP) in a semi-continuous culture system (Rusitec). Different microbial CP fractions were compared. Five diets with mean forage proportion of 0.88 and different maize silage to grass silage ratios (100 : 0, 79 : 21, 52 : 48, 24 : 76 and 0 : 100) were used. Diets were ground through sieves with a pore size of either 1 or 4 mm, matching the particle size of fine (F) and coarse (C), respectively. Diets were characterised by increasing concentrations of CP and fibre fractions, and decreasing concentrations of starch with ascending inclusion rates of grass silage. Microbial mass was isolated from feed residues after incubation from the liquid phase of the fermenter and from the liquid effluent. The amount of synthesised microbial CP was determined on the basis of 15N balance. It increased quite linearly by the stepwise replacement of maize silage by grass silage, and was higher in C treatments compared to F treatments. Efficiency of microbial CP synthesis (EMPS) was improved from 29 to 43 mg microbial N/g degraded organic matter (OM) by increasing the proportion of grass silage in the diet, but was unaffected by particle size. The N content as well as the profiles of amino acids of the three microbial fractions was affected by diet composition and particle size. The ratio of solid- to liquid-associated microbes was affected by diet composition and feed particle size. The amount and EMPS seemed to be improved by degradation of OM from grass silage and an increasing availability of N. Moreover, the results of this study indicated a shift in the composition of the microbial community caused by variation in forage composition and feed particle size.     

Effect of combined herbal feed additives on methane,total gas production and rumen fermentation

The present study was to evaluate effect of herbal feed additives on methane and total gas production during the rumen fermentation for environment and animal health concern. Different parts of the five medicinal plants were selected such as leaf and small stems of Ocimum sanctum (Tulsi), roots of Curcuma longa (Haldi), fruits of Emblica officinalis (Amla), leaves of Azadirachta indica (Neem) and leaves and small stem of Clerodendrum phlomidis (Arni) for our study. Addition of different herbal additive combinations did not influence IVDMD and total gas production however methane production (mg/g of substrate DM) was significantly (P<0.05) reduced in Amla: Neem and Neem: Arni combinations. Total nitrogen significantly (P<0.01) increased in the combinations of Tulsi: Haldi and Amla: Neem. TCA–ppt-N is significantly (P<0.01) increased in Tulsi: Haldi, Haldi: Amla, Amla: Neem and Neem: Arni however NH₃-N (mg/dl) significantly decreased in all treatments. We conclude that the screening of plant combinations, Amla: Neem and Neem: Arni have potential to decrease methane production and our herbal feed supplements have no side-effects on the ruminant in small amount.     

Studies on blood biochemical constituents and rumen fermentation in growing buffalo calves fed ammoniated straw-based rations supplemented with different protein sources

Studies were done on blood biochemical constituents and rumen fermentation pattern of growing buffalo calves fed ammoniated wheat straw (UAS)-based rations supplemented with three different protein supplements. Eighteen buffalo calves (140 kg) were divided into three groups of six calves each and were fed on UAS supplemented with a concentrate mixture (roughage:concentrate ratio 58:42) containing 8% (or 3.5% of total ration) untreated groundnut cake (GNC) (group I), 8% formaldehyde treated GNC (group II) and 8% fish meal (group III), respectively, for 19 weeks. During this growth study, blood biochemistry was studied. Rumen fermentation study was done on three fistulated growing buffalo calves using a 3×3 Latin square switch over design. Blood glucose and ammonia-N did not differ significantly among the treatments. However, blood urea-N of calves in group III was higher (P<0.05) than in groups I and II, but the latter two did not differ from each other in this respect. Total protein in group I was comparable to group II and group III which were significantly different from each other. The rumen pH, total volatile fatty acids, ammonia-N and total protozoal count were similar, however, total N and TCA insoluble N in group II and group III were higher (P<0.01) than in group I. Fish meal containing diet favoured holotrichs, whereas, protected GNC containing diet favoured spirotrichs as indicated by significantly higher count. It was concluded that the beneficial effects of supplementing formaldehyde protected GNC at 8% in the concentrate mixture to the UAS-based diets were greater than supplementation of unprotected GNC or low quality fish meal in ration of buffalo calves growing at 400 g because of improved rumen fermentation, absorption and metabolism of nutrients as observed in blood biochemistry.     

Diversity responses of rumen microbial communities to Fusarium-contaminated feed, evaluated with rumen simulating technology

The mycotoxin-producing fungus Fusarium culmorum causes major feed spoilages in agricultural livestock, but effects of F. culmorum -contaminated feed on the structural diversity of the rumen-inhabiting microbial community are not understood. Avoiding animal experiments, this study was conducted with the rumen simulating technique (Rusitec). Small subunit (SSU) rRNA gene copy numbers of bacteria and archaea, determined by quantitative polymerase chain reaction (PCR), indicated no differences between contaminated and non-contaminated digested feed, but fungal copy numbers, not attributable to F. culmorum itself, were elevated approximately fourfold in the contaminated feed, with 2.3 × 10⁹ g⁻¹ dry weight. Single-strand conformation polymorphism profiles of PCR-amplified partial SSU rRNA genes revealed a single but clear difference between contaminated and non-contaminated feed in profiles encompassing the phylogenetic clusters of Fibrobacteriales and Clostridiales. Minor quantitative differences were also seen in profiles of archaea and fungi. Positive correlations were found between fungal rRNA gene copy numbers and the degradability of different nutrients, but there was no correlation with degradation rates of the major mycotoxin contaminant deoxynivalenol. Thus, the diversity responses of the microbial community to F. culmorum -contaminated feed were caused by a fungus-induced, altered feed quality rather than by direct mycotoxicity.     

Effect of rare earth elements on in vitro rumen microbial fermentation and feed digestion

The objectives of this study were to investigate the effects of rare earth elements (REEs) on in vitro rumen fermentation, gas production, microbial protein synthesis and nutrient digestion using in vitro batch culture and continuous culture technique. A mixture of REE containing (g/kg) 380 g of LaCI₃·6H₂O, 521 g of CeCI₃·6H₂O, 30 g of PrCI₃·6H₂O and 69 g chlorides of other light REEs. The experimental diet consisted of 885 g/kg barley grain, 84 g/kg barley silage and 31 g/kg supplement (dry matter (DM) basis). Diet supplemented with different dosages of REE (control, no additional REE; low, 400 mg/kg REE; and high, 800 mg/kg REE, DM basis) were incubated for 4, 8, 14 and 24 h in diluted rumen fluid. At the end of 24 h of incubation, gas production and concentration of volatile fatty acid (VFA) linearly increased with increasing REE supplementation; whereas, influence of REE supplementation on VFA profile was marginal. Dry matter disappearance was not affected (P>0.10). Six dual-flow continuous culture fermenters were used in a replicated 3 × 3 Latin square with same treatments and same diet used in the batch culture. Mean ruminal pH (5.71) and total VFA (93.6 mM) concentration were not affected by supplementation of REE. The molar proportion (mol/100 mol) of acetate (39.1) and propionate (50.5) was similar among the treatments. However, the proportion (mol/100 mol) of butyrate was higher with the high REE (6.6) than with low REE (5.3) or the control (5.8). Ruminal true digestibilities of organic matter (OM) (0.785, 0.811 and 0.828), acid detergent fibre (0.360, 0.431 and 0.432) and crude protein (0.496, 0.590 and 0.589) for control, low and high REE, respectively, linearly increased with increasing REE supplementation, whereas, the increase in ruminal digestibility from low to high dosage of REE was minimal. Microbial nitrogen (N) production (g/day) and microbial efficiency (g N/kg of truly fermented OM) were not affected by treatments. Improvement of ruminal digestibility of OM due to REE supplementation was attributed to the increase in digestibility of fibre and degradability of protein. The results suggest that REE supplementation improved ruminal fibrolytic and proteolytic activities.     

Linkage of microbial ecology to phenotype: correlation of rumen microbial ecology to cattle's feed efficiency

Linkage of rumen microbial structure to host phenotypical traits may enhance the understanding of host-microbial interactions in livestock species. This study used culture-independent PCR-denaturing gradient gel electrophoresis (PCR-DGGE) to investigate the microbial profiles in the rumen of cattle differing in feed efficiency. The analysis of detectable bacterial PCR-DGGE profiles showed that the profiles generated from efficient steers clustered together and were clearly separated from those obtained from inefficient steers, indicating that specific bacterial groups may only inhabit in efficient steers. In addition, the bacterial profiles were more likely clustered within a certain breed, suggesting that host genetics may play an important role in rumen microbial structure. The correlations between the concentrations of volatile fatty acids and feed efficiency traits were also observed. Significantly higher concentrations of butyrate (P < 0.001) and valerate (P = 0.006) were detected in the efficient steers. Our results revealed potential associations between the detectable rumen microbiota and its fermentation parameters with the feed efficiency of cattle.     

Expression of rumen microbial fibrolytic enzyme genes in probiotic Lactobacillus reuteri

This study was aimed at evaluating the cloning and expression of three rumen microbial fibrolytic enzyme genes in a strain of Lactobacillus reuteri and investigating the probiotic characteristics of these genetically modified lactobacilli. The Neocallimastix patriciarum xylanase gene xynCDBFV, the Fibrobacter succinogenes beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene, and the Piromyces rhizinflata cellulase gene eglA were cloned in a strain of L. reuteri isolated from the gastrointestinal tract of broilers. The enzymes were expressed and secreted under the control of the Lactococcus lactis lacA promoter and its secretion signal. The L. reuteri transformed strains not only acquired the capacity to break down soluble carboxymethyl cellulose, beta-glucan, or xylan but also showed high adhesion efficiency to mucin and mucus and resistance to bile salt and acid.     

Effect of complete feed blocks or grazing and supplementation of lambs on performance, nutrient utilisation, rumen fermentation and rumen microbial enzymes

A study to compare two feeding systems, stall feeding (SF) and grazing plus supplementation (GR) was carried out, based on intake, performance and rumen fermentation characteristics of lambs. While SF animals received ad libitum complete feed blocks (CFB), GR animals were allowed grazing for 8 h on a pasture and supplemented with concentrate mixture at 250 g per head per day. Intake in grazing animals was determined using chromium III oxide as internal marker. Intake of dry matter (DM), crude protein (CP) and organic matter (OM) were higher ( P < 0.01) in SF than in GR animals. Similarly, digestibility of OM, CP and energy were higher ( P < 0.01) in SF animals. Average daily gain in SF animals (101 g) was significantly ( P < 0.01) higher than in GR animals (78 g) but total wool yield was similar for the two groups (856 g, SF; 782 g, GR). The pH of the rumen content, concentration of total volatile fatty acids and total activities of carboxymethyl cellulase, xylanase and esterase in the rumen liquor were similar. The concentrations (mg/dl) of total nitrogen (125, SF; 63, GR) and NH₃-nitrogen (42, SF; 31, GR) were higher in SF animals than that of GR animals. A significantly higher activity ( P < 0.05) of microcrystalline cellulase (24.5 v. 7.7 units) and lower activity ( P < 0.05) of protease (309 v. 525 units), was observed in the rumen of SF animals than in GR animals. SF animals could therefore harness more energy through degradation of plant cell walls thus reducing breakdown of plant proteins as gluconeogenic source. The SF system of feeding where CFB was offered to sheep appeared superior to GR in terms of intake, nutrient utilisation and animal performance. Therefore the SF feeding system where CFB are offered to animals can be advocated as an alternative to grazing and supplementation feeding strategy for sheep production, especially where the pastures are highly eroded and need resting for regeneration or curing. The CFB feeding can also be adopted under adverse conditions like drought and famine, a common phenomenon in arid and semiarid conditions.     

Effects of different sources of physically effective fiber on rumen microbial populations

Physically effective fiber is needed by dairy cattle to prevent ruminal acidosis. This study aimed to examine the effects of different sources of physically effective fiber on the populations of fibrolytic bacteria and methanogens. Five ruminally cannulated Holstein cows were each fed five diets differing in physically effective fiber sources over 15 weeks (21 days/period) in a Latin Square design: (1) 44.1% corn silage, (2) 34.0% corn silage plus 11.5% alfalfa hay, (3) 34.0% corn silage plus 5.1% wheat straw, (4) 36.1% corn silage plus 10.1% wheat straw, and (5) 34.0% corn silage plus 5.5% corn stover. The impact of the physically effective fiber sources on total bacteria and archaea were examined using denaturing gradient gel electrophoresis. Specific real-time PCR assays were used to quantify total bacteria, total archaea, the genus Butyrivibrio, Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens and three uncultured rumen bacteria that were identified from adhering ruminal fractions in a previous study. No significant differences were observed among the different sources of physical effective fiber with respect to the microbial populations quantified. Any of the physically effective fiber sources may be fed to dairy cattle without negative impact on the ruminal microbial community.     

Behaviour and welfare of veal calves fed different amounts of solid feed supplemented to a milk replacer ration adjusted for similar growth

Veal calves in Europe are typically fed large quantities of milk replacer and small amounts of solid feed, a diet known to lead to the development of abnormal oral behaviours in these animals. These abnormal oral behaviours are thought to be an indication of frustration, chronic stress, and hence poor welfare. The present study investigated how different feeding strategies, differing in solid feed and milk replacer provision, affected the behaviour and welfare of veal calves across time. Four treatment groups (A–D) comprising of 12 Holstein–Friesian bull calves each (7.6 ± 0.1 weeks old and 54.7 ± 0.3 kg at arrival), penned in groups of three, were fed one of four amounts of a solid feed mixture, i.e. 50% concentrates, 25% fresh maize silage, and 25% wheat straw (on dry matter [DM] basis): A = 0, B = 9, C = 18, and D = 27 g DM/kg^0.75/d. Provision of milk replacer was adjusted to achieve similar average daily gain across treatments. Behaviour was recorded around feeding (10 min continuous focal observations of individual calves) and throughout the day (7 sessions of 30 min scan sampling at 5 min interval every 2 h from 06:30 h) every week for four months. In an attempt to find an easy practical method to measure behavioural response to feeding strategy, two 3-min behavioural tests were carried out: (1) in months 1 and 3, calves were presented with a ball and latency to make oral contact with it was recorded; and (2) in month 1, calves were presented with an overall and time spent orally manipulating (i.e. chewing or licking) it was recorded using scan sampling every 10 s. Calves in treatment D displayed less abnormal oral behaviours around feeding, less tongue playing throughout the day, and more chewing in the first two months, compared to treatment A. Treatment B only led to lower tongue playing levels compared to A and treatment C had no benefit in terms of reducing abnormal oral behaviours. Although a solid feed dose–response was expected on the display of abnormal behaviours in veal calves, treatment C did not fit within this expectation. These findings point to a more complex relationship between solid feed and abnormal oral behaviour frequency in veal calves. The two behavioural tests distinguished the different treatments as expected, and thus showed a solid feed dose–response. Because of an increase in chewing and ruminating efficiency over time, amounts of solid feed should be increased with age to maintain high levels of chewing and ruminating. Moreover, high levels of chewing and ruminating may have to be maintained long enough at the beginning of the fattening period to lead to a reduction in abnormal oral behaviours.     

Influence of ciliate protozoa on biochemical changes and hydrolytic enzyme profile in the rumen ecosystem

AIMS: To assess the effect of presence or absence of rumen protozoa on fermentation characteristics and enzyme profile in growing lambs. METHODS AND RESULTS: Weaner lambs (G1, G2, G3, G4, G5 and G6 groups) were defaunated by oral administration of sodium laurel sulphate (at 8 g 100 kg(-1) body weight). The lambs of G4, G5 and G6 groups were refaunated. The roughage and concentrate ratio in the diet of G1 and G4, G2 and G5, and G3 and G6 were 50:50 (R1), 65:35 (R2) and 80:20 (R3), respectively. Daily dry matter intake was similar in defaunated and faunated lambs. However, digestibility of organic matter (OM), cellulose and gross energy were lower in defaunated lambs while crude protein (CP) digestibility was similar in both defaunated and faunated lambs. The rumen pH and NH3-N were lower (P < 0.01) while TVFA, total-N and TCA-ppt-N were higher (P < 0.01), in defaunated lambs. Ruminal activity of carboxymethyl cellulase was lower (P < 0.01) in defaunated lambs and amylase, xylanase, protease and urease were similar in faunated and defaunated lambs. Nutrient utilization, rumen metabolites and ciliate protozoal count were higher, whereas digestibility of fibre fractions was lower in high rather than low concentrate fed lambs. The rumen protozoa present before defaunation were B-type and the protozoa which re-established on refaunation were also B-type. CONCLUSIONS: Absence of ciliate protozoa decreased nutrient digestibility and increased ruminal TVFA and total-N with lower NH3-N concentration, indicating better energy and protein utilization in defaunated lambs. SIGNIFICANCE AND IMPACT OF THE STUDY: Defaunation improved energy and protein utilization in lambs.     

Metagenomic analysis exploring taxonomic diversity of rumen microbial communities in Vechur and crossbred cattle of Kerala state,India

Rumen, one of the most productive diverse microbial habitats plays a vital role in the breakdown of feed to produce energy for maintenance and milk production in cattle. Culture-based procedures...     

Effects of nickel supplementation on nutrient utilization,mineral balance,haematology and antioxidant status of crossbred dairy calves

BackgroundNickel has been identified as an important newer trace mineral playing essential role in animals however, its precise mechanism of action in the body is yet to be determined. Interaction of Ni with other essential minerals is suggested in reports limited to lab animals and needs to be explored further in large animals.AimThis study was conducted to study the influence of Ni supplementation at different levels, on minerals and health status of crossbred dairy calves.MethodTwenty-four Karan Fries crossbred (Tharparkar × Holstein Friesian) male dairy calves were selected based on body weight (137.09 ± 5.68) and age (10.78 ± 0.61) and were divided into 4 treatment groups (n = 6) given basal diet supplemented with 0 (Ni0), 5 (Ni5), 7.5 (Ni7.5) and 10 (Ni10) ppm Ni/kg of DM. Nickel was supplemented in the form of nickel sulphate hexahydrate (NiSO₄.6 H₂O) solution. To ensure the intake of the required amount of nickel by each animal, the calculated quantity of solution was mixed with 250 g concentrate mixture and offered individually to the calves. The calves were fed total mixed ration (TMR) consisting of green fodder, wheat straw and concentrate mixture in the ratio of 40:20:40 and the nutritional requirements were met according to NRC (2001) guidelines. Growth performance was recorded at fortnightly interval whereas, plasma minerals, haematology, antioxidant and immunity parameters were studied at monthly interval during the 150-day experimental period. Nutrient utilization and mineral balances were estimated with the help of a metabolism trial conducted at the end of feeding trial.ResultsSupplementation of Ni exhibited no influence on dry matter intake (DMI), body weight, average daily gain (ADG) and nutrient digestibility of dairy calves. However, the absorption and balance of minerals such as Ni, Fe, Cu, Zn and their respective plasma concentration increased (P < 0.05) with Ni supplementation and highest values were observed in calves fed 10 mg Ni/kg DM. The red blood cell (RBC) count, haemoglobin (Hb) concentration, haematocrit (HCT) and activity of superoxide dismutase (SOD) and catalase antioxidant enzymes showed highest increase (P < 0.05) in calves supplemented with Ni at level of 10 mg/kg DM as compared to other treatment groups. However, white blood cell (WBC) count, glutathione peroxidase (GPx), total antioxidant status (TAS), total immunoglobulins and IgG plasma concentration remained unaltered with addition of different levels of Ni in the diet of calves.ConclusionsThe supplementation of Ni at level of 10 mg/kg DM shows a positive effect on status of trace minerals such as Fe, Cu, Zn and improves the physiological conditions and health status of crossbred dairy calves indicated by improved haematology and antioxidant parameters.