Differential expression of miRNAs and related mRNAs during heat stress in buffalo heifers.

The present experiment was aimed to study differential expression of miRNAs and related mRNAs during heat stress (HS) in buffalo heifers. Twelve Murrah buffalo heifers aged between 1.5 and 2.0 years, weighting between 250 and 300 Kg were randomly assigned into two equal groups. The animals were kept in the psychrometric chamber under Thermo-neutral (TN; THI = 72) and HS (THI = 87-90) conditions for 6 h every day between 1000 and 1600 h for 21 days. The blood sampling was done at 1500 h on 15th day of the experiment and physiological parameters viz. pulse rate (PR), respiratory rate (RR) and rectal temperature (RT) were recorded at 1500 h on day -5, -3, -1, 0, +1, +3, +5 with respect to blood sampling. PBMCs were used for extraction of miRNAs and total RNA; and first strand cDNA was synthesized. qPCR was performed for relative gene expression studies. Physiological, hematological (erythrocytic indices), biochemical (triglycerides, urea, ALT, AST, LDH), redox (SOD, ROS) and endocrine parameters (T4) altered significantly (P < 0.05) during HS as compared to TN. Out of eight targeted miRNAs only four were expressed in buffalo heifers. The relative expression of bta-mir-142, bta-mir-1248 and bta-mir-2332 was significantly (P < 0.05) up-regulated whereas expression of bta-mir-2478 was significantly (P < 0.05) down-regulated during HS as compared to TN. The relative expression of the predicted target genes i.e. HSF1, HSP60, HSP70, HSPA8 and HSP90 were significantly (P < 0.05) up-regulated whereas HSF4 expression was significantly (P < 0.05) down-regulated during HS as compared to TN. It can be concluded that a THI of 87-90 could lead to a moderate HS in buffalo heifers. Differential expression studies of miRNAs and related mRNAs in present study deciphers the role of miRNAs in the heat tolerance in buffalo heifers.

Biochemical and blood gas alterations in buffalo (Bubalus bubalis) whole blood stored in CPDA-1 and CPD/SAG-M bags.

OBJECTIVE: The objective of this study was to evaluate the biochemical and blood gas alterations of whole blood of buffaloes that was stored in citrate-phosphate-dextrose with adenine (CPDA-1) and CPD/SAG-M blood bags for 42 days. DESIGN: Prospective study. INTERVENTIONS: Ten male buffaloes were used in this study. A total volume of 900 mL of blood was collected from each buffalo so that 450 mL was stored in CPDA-1 and 450 mL was stored in CPD/SAG-M bags at 2-6°C for 42 days. The stored blood was evaluated at 7 time points (D): D0 (immediately after blood collection) and 7 (D7), 14 (D14), 21 (D21), 28 (D28), 35 (D35), and 42 (D42) days after collection. Blood gas, biochemical, and microbiological parameters were monitored. MEASUREMENTS AND MAIN RESULTS: The overall blood pH decreased from 6.997 ± 0.05 at D0 to 6.784 ± 0.09 at D42, differing from baseline from D14 onward (P < 0.05). There were increases in partial pressure of oxygen (pO2 ), partial pressure of carbon dioxide (pCO2 ), lactate, and potassium (K) and decreases in the concentrations of sodium, bicarbonate, glucose, and pH (P < 0.05) during storage in both bags but no alterations in total protein concentration. Most of the variables were consistently similar between the 2 types of blood bags (P > 0.05) evaluated, with the exception of pCO2 , HCO3, cholesterol, and total protein, which had higher values in the CPDA-1 bag (P < 0.05). The K, pO2 , and lactate had the highest alterations during storage, with increases from baseline to D42 of 563%, 317%, and 169%, respectively. CONCLUSION: In general, no significant changes of clinical importance were observed after storage of whole blood samples from buffaloes for 42 days in the 2 types of blood bags that are indicated for use with this species.

Comparative biochemical profiles, utero-ovarian function, and fertility of the postpartum buffalo with and without subclinical endometritis.

In postpartum buffaloes, the process of uterine involution and changes in blood metabolic profile has not been studied in relation to development of subclinical endometritis (SCE). In this study, buffaloes (n = 100) approaching calving were identified. Weekly blood samples were collected on the day of calving up to 6 weeks post-calving. The diameter of uterine horns and onset of ovarian cyclicity (corpus luteum) were recorded through ultrasonography. On the basis of polymorphonuclear cell (PMN) cell count in endometrial cytology at days 45-50 postpartum, buffaloes were divided into two groups, viz., with SCE (> 5% PMN; n = 38) and without SCE (≤ 5% PMN; n = 62). Buffaloes with SCE took longer (P < 0.05) time to complete uterine involution and had larger (P < 0.05) uterine horn diameter between 3rd and 6th weeks postpartum and lower prostaglandin F2α metabolite (PGFM) concentration on the day of calving (P < 0.05) and 1 week (P < 0.001) post-calving than without SCE group. Buffaloes with SCE had lower (P < 0.001) concentration of glucose at weeks 2 and 3, higher (P < 0.001) ß-hydroxybutyric acid (BHBA) at week 3, and lower serum albumin concentration throughout the sampling period (P < 0.05 to 0.001) except at 1 week post-calving as compared to without SCE group. The urea concentration was significantly lower (P < 0.05 to 0.001) in buffaloes with SCE from 4 weeks post-calving onwards than without SCE group. The calcium concentration was lower in buffaloes with SCE at weeks 5 (P < 0.001) and 6 (P < 0.05) postpartum, whereas the concentration of magnesium and phosphorus was uniform between the two groups. No significant (P > 0.05) difference in onset of ovarian cyclicity between the 2 groups was observed, whereas buffaloes with SCE had longer (P = 0.001) median days open (141 days) than their counterpart (117 days). The first service conception rate, cumulative pregnancy rate, and pregnancy rate at 150 days postpartum were lower (P < 0.05) in buffaloes with SCE than without SCE group. In summary, higher BHBA and lower serum concentrations of glucose, albumin, urea, and calcium control onset of subclinical endometritis which in turn has negative impact on fertility of buffaloes.

Mineral availability in soil and fodders affecting blood profiles in Nili-Ravi dairy buffaloes.

A mineral assessment study for dairy buffaloes, Nili-Ravi breed, was carried out seasonally at silvopasture farm at Sargodha, Pakistan. Sampling for soil, forage, and buffalo serum was done seasonally for a period of 1 year for mineral (P, Mg, Na, K, Ca) evaluation. In the study, experiments on forages showed significant differences in mean concentrations of Mg and P with changing seasons, whereas the values for Ca, Mg, Na, and K did not vary significantly. Three groups of buffaloes were checked for mineral concentration in blood serum, viz., calves, lactating and non-lactating. Ca mean concentration varied significantly for non-lactating buffaloes and calves between two seasons. Na mean level showed a significant difference for two sampling seasons among calves and lactating buffaloes. Mean level for Mg only differed significantly in serum of non-lactating buffaloes between the two seasons. Mean levels for Ca and P in soil and forage remained higher than the optimal level, whereas soil Na was below the critical level. K concentrations in soil and blood serum were above the critical level, while Mg levels remained below the optimal range in soil, forage, and blood serum. K concentration in forage remained below the optimal level. All buffalo categories displayed lower levels of Ca and P in serum. Na concentration in forage and serum exceeded the critical level. Furthermore, Ca yielded significant and positive correlation between soil and serum level, whereas K had significant but negative correlation for soil-forage and forage-serum. Overall, it was determined that P deficiency existed among buffaloes, which could be improved via supplementation. Similarly, K deficiency and Mg deficiency were exhibited for forage and soil respectively which could be countered through addition of fertilizers rich for these minerals. In brief, the mineral utilization of buffaloes is affected by reduced availability of essential nutrients and may result in lower productivity. It is suggested to add mineral supplementation in addition to natural diet of buffaloes to enhance the productivity of these animals.

Uterine, vaginal and placental blood flows increase with dynamic changes in serum metabolic parameters and oxidative stress across gestation in buffaloes.

The aims of the present study were to determine uterine, vaginal and placental blood flows by Doppler ultrasound cross-buffalo gestation and to evaluate the relationships among reproductive Doppler parameters and serum metabolic parameters as well as oxidative stress. Uterine (UA) and vaginal (VA) arteries were scanned every month, and placentome was scanned from month 4 till 8 in gestation. Time-averaged maximum velocity (TAMV), pulsatility index (PI), resistance index (RI), systolic/diastolic ratio (SD) and arterial diameter (AD) were used for accessing UA and VA hemodynamics. Time-averaged maximum velocity positively correlated with and AD, and both negatively correlated with their PI, RI and SD in UA and VA. TAMV and AD increased constantly in pregnancy, with maximum increase in months 4 and 9. Pulsatility index, RI and AD of UA decreased between months 4 and 9, while PI, RI and AD of VA decreased between months 5 and 9 and then increased in month 10 in pregnancy. Time-averaged maximum velocity of placentome blood flow increased exponentially from months 4 to 8, but decreased at the last two months in pregnancy. Serum lipids were significantly higher in the first month compared to all other months, while glucose was significantly lower in months 9 and 10. Malondialdehyde increased from month 3 till term, but peaked in month 5 and 10. Glutathione and catalase were highest in the first month and remained after. Time-averaged maximum velocity and AD for both UA and VA negatively correlated with serum lipids, glucose, catalase and glutathione, while positively correlated with malondialdehyde and total protein. Thus, increases in uterine blood flow (UtBF), vaginal blood flow (VaBF) and placental blood flow (PaBF) are associated with increased metabolism and oxidative stress in buffalo pregnancy.

Use of the MILLIPLEX® bovine cytokine/chemokine multiplex assay to identify Mycobacterium bovis-infection biomarkers in African buffaloes (Syncerus caffer).

As a recognized Mycobacterium bovis maintenance host, the African buffalo (Syncerus caffer) poses transmission risks to livestock, humans and other wildlife. Early detection of M. bovis infection is critical for limiting its spread. Currently, tests detecting cell-mediated immune responses are used for diagnosis in buffaloes. However, these may have suboptimal sensitivity or specificity, depending on the blood stimulation method. Recent evidence suggests that assays using combinations of host cytokine biomarkers may increase diagnostic performance. Therefore, this study aimed to investigate the application of a MILLIPLEX® bovine cytokine/chemokine multiplex assay to identify candidate biomarkers of M. bovis infection in buffaloes. Whole blood from twelve culture-confirmed M. bovis-infected buffaloes, stimulated with the QuantiFERON® TB Gold Plus in-tube system, was tested using the MILLIPLEX® platform. Results indicated binding of bovine antibodies to fifteen buffalo cytokine/chemokine targets. Moreover, there was a significant difference in concentrations between unstimulated and TB antigen-stimulated buffalo samples for seven cytokines/chemokines included in the kit. Although these preliminary results require further investigation in larger sample sets and a comparison between M. bovis-infected and uninfected cohorts, the utility of the MILLIPLEX® platform in a novel species was demonstrated, in addition to identifying potential African buffalo cytokines for future research.

Acute phase proteins and markers of oxidative status in water buffalos during the transition from late pregnancy to early lactation.

The transition period, from pregnancy to lactation, implies comprehensive metabolic and endocrine changes including a systemic inflammatory reaction and oxidative stress around calving in dairy cows. The aim of the present study was a longitudinal characterization of the serum concentration of acute phase proteins (APP), i.e., haptoglobin (Hp), serum amyloid A (SAA) and acidic glycoprotein (AGP), as well as of markers for oxidative stress in another large dairy animal, i.e. water buffalo, during the transition from late pregnancy to early lactation. As indicators of oxidative status, derivatives of reactive oxygen metabolites (dROM), ferric reducing ability (FRAP), thiobarbituric acid reactive substances (TBARS), and advanced oxidation protein products (AOPP) were determined in serum. Indicators for metabolic stress included nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB) and adiponectin. Bovine specific ELISA methods for Hp and adiponectin were adapted and validated for their application to water buffalo samples. Blood samples were collected weekly from 11 pluriparous water buffalo cows (lactation number 4.6 ± 1.6; daily milk yield 9.0 ± 1.9 kg; means ± SD) from 6 weeks (wk) ante partum (ap) until 8 wk post partum (pp). The maximum concentrations of Hp were observed in wk 1 pp, followed by a decrease towards values lower than before calving starting from wk 3 pp. The concentrations of SAA also peaked in wk 1 pp and then returned to basal values. The AGP serum concentrations increased suddenly from the first to the second wk pp and remained elevated for all the observation period. Indicators of oxidative status which changed in concentration during the transition period were dROM, AOPP and the oxidative stress index (OSi) (dROM/FRAP ratio). Briefly, dROM and AOPP values were lower pp as compared to ap, and OSi was largely following the pattern of dROM due to the constant FRAP values. The TBARS values did not change during the observation period. From the metabolic indicators, adiponectin was not changing with time, whereas greater NEFA and BHB values were observed ap than pp. The time course of NEFA and of some indicators for oxidative status (dROM, OSi and AOPP) point to greater metabolic load in late pregnancy as compared with the first wk of lactation - contrary to the common situation in dairy cows. Both BHB and NEFA values remained below the thresholds applied for dairy cows to define subclinical or clinical ketosis, thus indicating that the buffaloes included in this study were not under metabolic stress. The increase in concentration of the APP around calving supports the concept that an inflammatory reaction is a physiological epiphenomenon of the onset of lactation in water buffalos that is independent of metabolic stress.

Serum kisspeptin: New possible biomarker for sexual behaviour and sperm concentration in buffalo bulls.

The study was designed to decipher the inter-relationship between peripheral hormones (kisspeptin and testosterone), sexual behaviour and seminal variables of Murrah buffalo bulls (n = 134). In this study, we recorded that 13%, 37%, 40%, 6% and 4% Murrah buffalo bulls had reaction time of <30, 31-60, 61-180, 181-300 and >300 s, respectively. Further, it was observed that 4%, 85% and 10% buffalo bulls were sexually aggressive, active and dull, respectively, during semen collection. The courtship behaviour was not found to be desirable for the bulls used for the semen collection. Mean of ejaculate volume, sperm concentration and mass motility (0-5 scale) were 3.57 ml, 977.11 million/ml, 2.7, respectively. Correlation studies revealed that the reaction time was positively correlated with courtship behaviour and body weight, and negatively correlated with sexual aggressiveness and sperm concentration. Serum kisspeptin in buffalo bulls, measured for the first time, was found to 3.8 ± 0.7 ng/ml. Serum kisspeptin and testosterone level are negatively correlated to each other and kisspeptin level influenced the sexual behaviour (reaction time, sexual aggressiveness and penile erection) of study bulls. Serum kisspeptin was higher in the buffalo bulls with higher sperm concentration indicating its role in spermatogenesis. In conclusion, for the first time basic information related to sexual behaviour of Murrah buffalo bulls in large population along with its inter-relationship with peripheral hormones (kisspeptin and testosterone) has been documented.

Serum proteomics analysis reveals the thermal fitness of crossbred dairy buffalo to chronic heat stress.

Chronic heat stress (CHS) reduces the production efficiency of the buffalo dairy industry. Relatively low-abundance proteins with particular functions in biological processes are changed by CHS. The present study aimed to quantify the differences in low-abundance proteins of crossbred dairy buffaloes under CHS and thermal-neutral (TN) conditions. With label-free quantification, 344 low-abundance proteins were identified in serum. Of these, 17 differentially expressed low-abundance proteins with known functions were detected, and six of the differentially expressed proteins related to heat stress were validated with parallel reaction monitoring. Lipase (LPL), glutathione peroxidase 3 (GPX3), cathelicidin-2 (CATHL2), ceruloplasmin (CP), and hemoglobin subunit alpha 1 (HBA1) cooperatively played roles in the thermal fitness of dairy buffalo by decreasing heat production and increasing blood oxygen delivery. Also, dairy buffaloes may adapt to CHS and hypoxia with high levels of RBCs, HBA1 and CP to increase blood oxygen delivery capacity.

Effect of plastic foreign body impaction on rumen function and heavy metal concentrations in various body fluids and tissues of buffaloes.

Rumen impaction by plastic waste (PW) is a common condition in the developing countries where ruminants are reared under extensive grazing. As heavy metals (HM) are used in the manufacture of plastic, we hypothesized that buffaloes with PW impaction would have suboptimal rumen function and higher content of HM in the rumen fluid, sera and body tissues. Buffaloes at an abattoir were categorized into impacted with PW or not (Group C and B, respectively; n = 30/group). From the animals of group C and B, rumen fluid and sera were collected ante-mortem, while muscle, liver and kidney were collected after exsanguination. Blood and rumen fluid of stall-fed buffaloes (Group A; n = 15) served as known negative control. Rumen function was assessed by protozoal density, motility, pH, methylene blue reduction time (MBRT) and sedimentation activity time (SAT). Concentration of HM such as mercury (Hg), lead (Pb), cadmium (Cd), chromium (Cr) and copper (Cu) in the body fluids, tissues and impacted PW was estimated by atomic absorption spectrometry (AAS). A significant decrease in the rumen protozoal density, motility and increase (P < 0.05) in the rumen fluid pH, MBRT and SAT were observed in group C as compared to group A and B. Concentration of Pb, Hg and Cd showed a moderate to strong significant negative correlation with rumen function indicators. Quantitatively, Cu content (ppm) was highest in PW with 0.9 ± 0.04. The concentration of HM in the body fluids and tissues was significantly (P < 0.05) elevated in group C, but was below the toxic levels. In stall-fed buffaloes, Hg, Pb and Cd were undetectable in body fluids. Collectively, the results indicate that long term exposure of buffaloes to HM from PW affect the health, productivity and increase the risk of entering the food chain.

Epidemiological survey of Anaplasma marginale in cattle and buffalo in Sri Lanka.

Bovine anaplasmosis caused by Anaplasma marginale represents a serious threat to cattle farming worldwide, especially in the tropics and subtropics. In the present study, archived DNA samples from the blood of cattle (n=437) in the Nuwara Eliya, Galle, Ampara, Polonnaruwa, and Jaffna districts and buffalo (n=327) in the Galle, Polonnaruwa, Mannar, and Mullaitivu districts in Sri Lanka, were screened for A. marginale using a major surface protein 5 (msp5) gene-based PCR assay. The findings showed that 32.7 and 57.5% of cattle and buffalo, respectively, were A. marginale-positive. The rate of positivity differed significantly among geographical regions. In conclusion, the high rates of A. marginale infection in cattle and buffalo highlight the importance of effective control measures in Sri Lanka.

Profiling circulating microRNAs in serum of Fasciola gigantica-infected buffalo.

Circulating miRNAs are stably existed in serum and plasma and can serve as a novel class of biomarkers for the diagnosis of helminthic infection. Fasciola gigantica, the causative agents of fascioliasis, live in the liver of in humans and ruminants, especially cattle, goat and sheep. In this study, a total of 121 host circulating miRNAs were differentially expressed (2 ≥ fold change, p < 0.05), of which 44 miRNAs were up-regulated and 77 miRNAs were significantly down-regulated. Consistent with the sequencing data, qRT-PCR results showed that the expression levels of bta-miR-21-5p and bta-miR-23a were elevated gradually and bta-miR-125a was decreased gradually at the F. gigantica infection time points. Four F. gigantica-specific miRNAs, including three known miRNAs (fgi-miR-87, fgi-miR-71, and fgi-miR-124), and one novel miRNA (novel miR-1) were identified in the sera of F. gigantica-infected buffaloes. Further analyses demonstrated that two parasite-derived miRNAs (fgi-miR-87 and fgi-miR-71) were specifically detected in sera of F. gigantica-infected buffaloes. These findings will be helpful to understand the roles of circulating miRNAs in host-parasite interaction and to potentiate serum miRNAs as diagnostic targets for F. gigantica.

Parallel measurement of IFN-γ and IP-10 in QuantiFERON®-TB Gold (QFT) plasma improves the detection of Mycobacterium bovis infection in African buffaloes (Syncerus caffer).

The QuantiFERON®-TB Gold (QFT) stimulation platform for cytokine release is a novel approach for diagnosis of bovine tuberculosis in wildlife species. Plasma interferon gamma (IFN-γ) is routinely measured to detect immune sensitization to Mycobacterium bovis. However, the cytokine interferon gamma-inducible protein 10 (IP-10) has been proposed as an alternative, more sensitive, diagnostic biomarker. In this study, we investigated the use of the QFT system with measurement of IFN-γ and IP-10 in parallel to identify M. bovis-infected African buffaloes. The test results of either biomarker in a cohort of M. bovis-unexposed buffaloes (n = 70) led to calculation of 100% test specificity. Furthermore, in cohorts of M. bovis culture-positive (n = 51) and M. bovis-suspect (n = 22) buffaloes, the IP-10 test results were positive in a greater number of animals than the number based on the IFN-γ test results. Most notably, when the biomarkers were measured in parallel, the tests identified all M. bovis culture-positive buffaloes, a result neither the single comparative intradermal tuberculin test (SCITT) nor Bovigam® IFN-γ release assay (IGRA) achieved, individually or in parallel. These findings demonstrate the diagnostic potential of this blood-based assay to identify M. bovis-infected African buffaloes and a strategy to maximise the detection of infected animals while maintaining diagnostic specificity and simplifying test procedures.

Serum haptoglobin and protein electrophoretic fraction modifications in buffaloes (Bubalus bubalis) around calving and during early lactation.

Serum protein distribution and concentration can be affected by different physiological and pathological conditions. The aim of this study was to evaluate the changes in the concentration of serum protein fractions and haptoglobin in clinically healthy dairy buffaloes during late pregnancy and early lactation. Blood and milk samples were collected from 30 buffaloes at around 7 d before expected calving (blood only) and 7, 30 and 50 d after calving. In serum samples, the total protein, haptoglobin, albumin, α1-, α2-, ß1-, ß2-, γ-globulins, and albumin/globulin ratio (A/G) values were evaluated. In milk, fat%, protein%, lactose%, somatic cell score (SCS) were assessed, along with milk yield (MY) and daily milk production (DMP). The peripartum period significantly influenced (P < 0.005) total protein, albumin, haptoglobin, α2-, ß2- and γ-globulins (P < 0.005). Milk yield, DMP and fat% changed significantly throughout the monitoring period (P < 0.005). Milk yield and DMP were positively correlated with total protein, albumin, ß2-globulins and A/G ratio, and negatively correlated with haptoglobin and α2-globulins. These results provide new knowledge about the serum protein electrophoretic pattern in Italian Mediterranean Buffaloes during the last phase of pregnancy and early stages of lactation.

Blood lymphocyte subpopulations in healthy water buffaloes (Bubalus bubalis, Mediterranean lineage): Reference intervals and influence of age and reproductive history.

There is an increasing interest toward infectious diseases and mechanisms of immune response of water buffaloes, mainly because of the growing economic impact of this species and of its high-quality milk. However, little is known about the immune system of these animals in physiological conditions. Recently, a wide number of antibodies cross reacting with buffalo antigens has been validated for use in flow cytometry (FC), allowing detailed characterization of the lymphocytic population in this species. The aim of the present study was to describe the lymphocyte subpopulations in a large number of healthy water buffaloes, providing reference intervals (RIs), and to assess whether the composition of blood lymphocyte population significantly varied with age and reproductive history. Our final aim was to lay the ground for future studies evaluating the role of host immune response in water buffaloes. One-hundred-twelve healthy buffaloes from four different herds in the South of Italy were included in the study. All animals had been vaccinated for Infectious Bovine Rhinotracheitis (IBR), Salmonellosis, Colibacillosis and Clostridiosis, and all herds were certified Brucellosis- and Tuberculosis-free. Venous blood collected into EDTA tubes was processed for FC, and the percentage of cells staining positive for the following antibodies was recorded: CD3, CD4, CD8, CD21, TCR-δ-N24, WC1-N2, WC1-N3 and WC1-N4. Absolute concentration of each lymphoid subclass was then calculated, based on automated White Blood Cell (WBC) Count. Reference Intervals were calculated according to official guidelines and are listed in the manuscript. The composition of the lymphocyte population varied with age and reproductive history, with animals <2-years-old and heifers having higher concentration of most of the subclasses. The present study provides RIs for the main lymphocytic subclasses in healthy water buffaloes, highlighting gross differences between young and old animals. Establishment of age-specific RIs is recommended in water buffaloes. The data we present may be useful as a basis for further studies concerning mechanisms of immune response toward infectious agents in water buffaloes.

Toxicological potential of cobalt in forage for ruminants grown in polluted soil: a health risk assessment from trace metal pollution for livestock.

The trace metal pollution in the environment is a highly concerned issue in these days. One of the important causes of trace metal pollution is the exhaust gases released from the vehicles on the roads. These dangerous gases pose life-threatening effects on the forage plants grown along the roadside as these plants are at direct risk to these trace metals. The aims of the present study were to determine the cobalt (Co) concentrations in soil, forages, and blood plasma of the buffaloes and to evaluate the Co deficiencies and toxicities in these samples. All samples were collected from six sites (Faisalabad roadside, Bhalwal roadside, Shaheenabad roadside, Mateela roadside, 50 Chak roadside, and Dera Saudi-control) of Sargodha city. The Co concentrations in these samples were determined by atomic absorption spectrophotometer (AA-6300 Shimadzu Japan). In soil samples, Co level ranged from 1.958 to 3.457 mg/kg in the six sampling sites. The highest Co level was observed at site 6 and the lowest at site 2. In forage samples, Co level ranged from 0.770 to 2.309 mg/kg in the six sampling sites. The highest Co level was observed at site 3 and the lowest at site 2. In blood plasma samples, Co level ranged from 2.644 to 4.927 mg/kg in the six sampling sites. The highest Co level was observed at site 1 and the lowest at site 3. The results showed higher Co values in the samples collected from the site IV while the bioconcentration factor for forage-soil was found highest in the samples collected from Site III. On the other hand, a correlation was found positively significant when soil and forage were correlated, and it was found negatively significant when blood and forage were correlated.

Comparative proteomic analysis of high- and low-fertile buffalo bull spermatozoa for identification of fertility-associated proteins.

The present study identified few potential proteins in the spermatozoa of buffalo bulls that can be used as an aid in fertility determination through comparative proteomics. The sperm proteome of high-fertile buffalo bulls was compared with that of low-fertile buffalo bulls using two-dimensional difference gel electrophoresis (2D-DIGE), and the differentially expressed proteins were identified through mass spectrometric method. The protein interaction network and the functional bioinformatics analysis of differentially expressed proteins were also carried out. In the spermatozoa of high-fertile bulls, 10 proteins were found overexpressed and 15 proteins were underexpressed at the level of twofold or more (p ≤ 0.05). The proteins overexpressed in high-fertile spermatozoa were PDZD8, GTF2F2, ZNF397, KIZ, LOH12CR1, ACRBP, PRSS37, CYP11B2, F13A1 and SPO11, whereas those overexpressed in low-fertile spermatozoa were MT1A, ATP5F1, CS, TCRB, PRODH2, HARS, IDH3A, SRPK3, Uncharacterized protein C9orf9 homolog isoform X4, TUBB2B, GPR4, PMP2, CTSL1, TPPP2 and EGFL6. The differential expression ranged from 2.0- to 6.1-fold between the two groups, where CYP11B2 was high abundant in high-fertile spermatozoa and MT1A was highly abundant in low-fertile spermatozoa. Most of the proteins overexpressed in low-fertile spermatozoa were related to energy metabolism and capacitation factors, pointing out the possible role of pre-mature capacitation and cryo-damages in reducing the fertility of cryopreserved buffalo spermatozoa.

Responses of pro-inflammatory cytokines, acute phase proteins and cytological analysis in serum and cerebrospinal fluid during haemorrhagic septicaemia infection in buffaloes.

Sudden death is usually the main finding in field animals during haemorrhagic septicaemia outbreaks caused by Pasteurella multocida type B:2 that causes acute, fatal and septicaemic disease in cattle and buffaloes. This situation may be due to failure in early detection of the disease where early treatment of antibiotics may improve the prognosis of the animal and other surviving animals. Thus, there is a grey area on the knowledge on the potential usage of pro-inflammatory cytokines and acute phase proteins as early biomarkers in the diagnosis of haemorrhagic septicaemia. In addition, exploration of the cerebrospinal fluid during infection has never been studied before. Therefore, this study was designed to fill up the grey areas in haemorrhagic septicaemia research. Twenty-one buffalo calves were divided into seven treatment groups where group 1 was inoculated orally with 10 mL of sterile phosphate-buffered saline pH 7 which act as a negative control group. Groups 2 and 3 were inoculated orally and subcutaneously with 10 mL of 1012 colony-forming unit of P. multocida type B:2. Group 4 and 5 buffaloes were inoculated orally and intravenously with 10 mL of lipopolysaccharide broth. Groups 6 and 7 were administered orally and subcutaneously with 10 mL of outer membrane protein broth. During the post-infection period of 21 days, blood and cerebrospinal fluid were sampled for the analyses of pro-inflammatory cytokines, acute phase proteins and cytological examination. Buffalo calves infected with P. multocida and its immunogens via different routes of inoculation showed significant changes (p < 0.05) of pro-inflammatory cytokines, acute phase proteins and cytological changes in both the serum and cerebrospinal fluid. Buffalo calves from groups 3 and 7 showed the highest pro-inflammatory cytokines, whereas group 6 had the highest acute phase protein concentration and group 5 revealed the highest value for cytology changes. In summary, results obtained in this study could be used as a profiling study to add novel knowledge to the haemorrhagic septicaemia research as well as the development of biomarkers.

Chemical capture of wild swamp buffalo (Bubalus bubalis) in tropical northern Australia using thiafentanil, etorphine and azaperone combinations.

BACKGROUND: Studying wild animals in situ is fundamental to collecting baseline information, but generally they need to be immobilised for examination, sampling, marking and/or equipping with tracking apparatus. Capturing wild animals is inherently risky and there is a need for immobilisation methods that are safe for both the animals and researchers. METHODS: A total of 16 free-ranging swamp buffalo (Bubalus bubalis) were chemically captured by dart for the application of satellite tracking collars in tropical northern Australia; 7 animals were anesthetised with a thiafentanil-etorphine-azaperone (TEA) combination and 9 animals with a thiafentanil-azaperone (TA) combination. Anaesthesia was reversed with intravenous naltrexone. Mean dosages of etorphine and thiafentanil for animals in the TEA group were 0.01 mg/kg of each drug and mean dosage of thiafentanil for animals in the TA group was 0.02 mg/kg. Total dose per animal of azaperone and naltrexone was 80 mg and 150 mg, respectively. Anaesthetic monitoring was by physical observation of physiological variables, pulse oximetry and capnography. Blood laboratory parameters including creatine kinase (CK), aspartate transaminase (AST), serum bicarbonate and anion gap were measured. RESULTS: All subject animals recovered well from anaesthesia despite the occurrence of subclinical acidosis in some patients. There was no significant difference between the treatment groups. Conversely, chase time had an adverse effect on body temperature, irrespective of the anaesthetic combination used. CONCLUSIONS: Thiafentanil and azaperone, with or without etorphine, delivered rapid safe, effective, reversible field anaesthesia in healthy swamp buffalo.

Immune status and haemato-biochemical profile of buffalo calves supplemented with phytogenic feed additives rich in tannins, saponins and essential oils.

The present study was performed to ascertain the synergistic effects of phytogenic feed additives (PFA-7) supplementation on immune status and haemato-biochemical profile of buffalo calves. The PFA-7 is a mixture of neem seed cake (Azadirachta indica), mahua seed cake (Madhuca longifolia), fennel seed (Foeniculum vulgare), harad (Terminalia chebula), fruit pulp of bahera (Terminalia bellirica), fruit pulp of amla (Phyllanthus emblica) and ajwain seed (Trachyspermum ammi) mixed in 2:2:2:1:1:1:1 proportion. Male buffalo calves (n = 21) having similar age and body weight were allotted to three groups in a completely randomised design. The dietary treatments were viz. T1: control (without PFA-7) and T2 and T3: provided with PFA-7 at 2 and 4% of dry matter intake (DMI), respectively, plus sodium sulphate at 0.06% of DMI. The feeding trial was carried out for 4 months, and serum isolation was done on days 0, 60 and 120 post-feeding. The concentrations of total protein, albumin, globulin, aspartate transaminase and alanine transaminase increased, whereas cortisol and glucose decreased in the supplemented groups as compared to the control. The levels of triglycerides, urea, albumin/globulin ratio, calcium, phosphorus and alkaline phosphatase were not affected by the supplementation of PFA-7. Both cell-mediated and humoral immune response increased in the supplemented groups. The results revealed that PFA-7 positively impacted haemato-biochemical profile and both cellular and humoral immunity of the growing calves. The PFA-7 can be used as an alternative for chemical feed additives in the diet of growing calves.