Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

Effect of incorporation of additives in tris-based egg yolk extender on buffalo (Bubalus bubalis) sperm tyrosine phosphorylation during cryopreservation

Phosphorylation of tyrosine residues on sperm protein is a known indicator of capacitation and a major intracellular signalling event. There is evidence that sperm cryopreservation promotes tyrosine phosphorylation and is associated with reduced fertility of spermatozoa. Under this study, cryoprotective role of different additives namely taurine, trehalose, catalase and 4-bromophenacyl bromide on buffalo sperm quality was evaluated. Buffalo semen was cryopreserved in tris-based egg yolk extender supplemented with additives like taurine (50 mm) or trehalose (100 mm) or 4-bromophenacyl bromide (200 μm) or catalase (100 U/ml) and used for assessment of levels of tyrosine phosphorylation in frozen-thawed spermatozoa. The results obtained were compared with the level of protein tyrosine phosphorylation of semen cryopreserved in tris-based egg yolk extender without additives. Proteins were extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random and analysed for tyrosine phospho-proteins using SDS-PAGE followed by immunoblotting. Monoclonal anti-phosphotyrosine antibody (Clone pT-154) was used as primary antibody followed by treatment with HRP-conjugated secondary antibody. Signals were detected on X-ray film using chemiluminescence. Nine proteins (p20, p30, p32, p38, p49, p56, p59, p72 and p86) were found to be tyrosine phosphorylated in cryopreserved spermatozoa. Supplementation of additives significantly (p<0.05) reduced the level of protein tyrosine phosphorylation in spermatozoa. Moreover, this study showed improved (p<0.05) post-thaw motility, viability and membrane integrity of spermatozoa on addition of these additives. The results obtained clearly indicate reduced level of capacitation like changes on supplementation of additives in terms of protein tyrosine phosphorylation.     

Genetic relationship and diversity analysis of Indian water buffalo (Bubalus bubalis)

The water buffalo (Bubalus bubalis) is an important dairy animal on the Indian subcontinent and in Southeast Asian countries. The diversity and differentiation among 12 populations or breeds of buffalo were studied. Data were generated and analyzed from 527 animals belonging to 10 recognized breeds and 2 additional populations of Indian buffalo by using 22 microsatellite loci. Relationships among buffalo breeds and populations were estimated based on genetic distances. The Bayesian analysis grouped 12 populations into 8 distinctive clusters. Geographically close breeds clustered together, except for the Jaffarabadi and Murrah, which were not in geographic contiguity. The Mantel test revealed nonsignificant correlations between genetic and geographic distances. This supports the hypothesis that buffaloes have been domesticated at different places for specific purposes. The phylogenetic relationship based on microsatellite loci supported the breed classification based on body size. The Toda breed, which is considered to be endangered, had genotypes similar to those of the surrounding buffalo populations.     

Assessment of buffalo (Bubalus bubalis) sperm DNA fragmentation using a sperm chromatin dispersion test

Chromosomal fragmentations or damage in sperm DNA has considerable value in determination of semen quality. However, rapid and/or simple method to assess sperm DNA integrity in buffalo has apparently not been reported. In the present study, SCD was used for the first time in buffalo bulls for assessment of sperm DNA fragmentation. A modified SCD protocol, under bright field microscope was developed and validated by comparison with other routine tests which can be used for processing of samples. The DNA fragmentation index (DFI) from SCD was correlated with semen quality parameters viz. viability (r=-0.68, p<0.05), membrane integrity (r=-0.74, p<0.05) and capacitation status (r=-0.69, p<0.05). The amount of DNA fragmentation assessed by SCD was highly correlated (R=0.874, p<0.05) with results of acridine orange test (AOT), a traditional method of assessing DNA damage. There were no significant differences between two observers with regards to scoring dispersion patterns. Therefore, the SCD test can be routinely used for detection of DNA fragmentation in buffalo sperm, with potential for replacing conventional time consuming tests.     

Detomidine-diazepam-ketamine anaesthesia in buffalo (Bubalus bubalis) calves

Eight buffalo calves (8-12 months, 70-100 kg) were randomly assigned to two groups of four animals each. Animals of group I were given detomidine (100 micrograms/kg), whereas animals of group II received a mixture of detomidine (100 micrograms/kg), diazepam (100 micrograms/kg) and ketamine (3 mg/kg) (DDK) intravenously. Various clinical parameters, such as weak time, down time, pedal and pinprick reflexes, muscle relaxation and extent of sedation, as well as heart and respiratory rates and electrocardiograms were measured before (time 0) and 15, 30, 45, 60, 75 and 90 min after treatment. In all the animals of group II (DDK), the pedal reflex was completely abolished (score: 3.00 +/- 0.00) within 5 min, the pinprick response was either very weak or it was completely abolished at this interval. Muscle relaxation and sedation were excellent within 5 min of DDK administration. The depth of sedation and analgesia was maximum from 5 to 15 min postinjection. Detomidine alone, however, failed to produce appropriate depression of the pedal and pinprick reflexes, sedation was mild and muscle relaxation was inadequate. Heart rate showed a significant (P < 0.05) decrease in group I, but the decrease was non-significant in group II. A more pronounced increase in respiratory rate was observed in group I as compared to group II. Animals of both groups recovered within 90 min without any complication. Minimal changes in the cardiovascular system in the group given the DDK combination were an advantage over the group given detomidine. The results indicated that DDK combination is safe and suitable for 15 min of anaesthesia with excellent muscle relaxation and has only limited cardiorespiratory effects in buffaloes.     

Prenatal development of retina in buffalo (Bubalus bubalis)

The development of retina in Indian buffalo (Bubalus bubalis) has not been reported previously. The aim of the present study was therefore to report the major landmarks and the time course in the development of retina. Serial histological sections of Indian buffalo embryos and foetuses were used as group1 (<20.0 cm CVRL), group2 (>20.0 but <40.0 cm CVRL) and group3 (>40.0 cm CVRL). Age estimation was made on the basis of crown vertebral‐rump length (CVRL), which ranged between 36 and 286 days (1.6–94.0 cm). The retina in Indian buffalo was developed in a similar manner to that of the other mammals with the principal differences in the time of occurrence of various layers of this nervous tunic. In 36 days (1.6 cm stage), the foetal retina was composed of pigmented layer and the layer of neuroblasts. Differentiation of layers was first observed in 47 days (4.0 cm CVRL) which became prominent in 52 days (5.1 cm stage). At 120 days (20.5 cm stage), the differentiation of inner plexiform layer and inner nuclear layer was evident. At 143 days (31.0 cm) foetal age, the faint line in neuroblastic layer was the first evidence of the future outer plexiform layer. In foetuses of group III, the retina was comprised of all 10 layers (eight cell layers and two membranes) viz. pigmented epithelium, layer of rods and cones, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, layer of nerve fibres and the inner limiting membrane.     

Neosporosis in water buffalo (Bubalus bubalis) in Southern Italy

A study was carried on 1377 water buffalo serum samples from 50 farms in southern Italy to test the presence of Neospora caninum antibodies by indirect fluorescence antibody test (IFAT). Rabbit anti-buffalo immunoglobulins conjugated to fluorescein were used in the test. Fluorescence in sera dilutions above 1:200 was considered as indicative of the presence of N. caninum antibodies. The overall prevalence of infection in the animals was 34.6%. The prevalence increased in relation to the age of subjects and most of the herds examined (82%) were found infected. In two farms abortions and neurological signs were reported. No suppurative inflammatory lesions were seen, but few protozoan-like cysts were observed on foetal tissues by histology.     

Effect of caffeine on metabolic activity of ejaculated and epididymal spermatozoa of buffalo (Bubalus bubalis)

Split fractions of 25 ejaculated semen samples and spermatozoa from 5 caudae epididymides were used to study the effect of different levels of caffeine on the motility and fructolytic activity. During the first hour of incubation at 37°C, addition of caffeine to unwashed ejaculated buffalo sperm significantly increased the percentage of motility and amount of fructose utilized. In presence of 2–8 mM caffeine, sperm maintained their initial motility at least 2 hours at 37°C. Maximal stimulation of fructolytic activity was obtained with 2 mM whereas the minimal stimulation was found with higher concentration of 8–10 mM caffeine. Compared to ejaculated spermatozoa, epididymal sperm appeared to be more influenced by caffeine. Fructolytic activity was stimulated at least 1.5 times in the presence of 2 mM caffeine. Nearly during all incubation periods, the amounts of fructose utilized by caffeine-treated sperm were greater than in control samples. Data on epididymal sperm motility demonstrated that caffeine (2–10 mM) significantly stimulated and maintained the initial motility at least 3 hours at 37°C .     

A study on renal function in the Indian buffalo (Bubalus bubalis)

Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) in buffalo species ( Bubalus bubalis ) were estimated using a single injection technique. The total body clearances of inulin and para-aminohippuric acid (PAH) served as estimates of GFR and ERPF, respectively. Inulin and PAH were administered to animals as a single i.v. bolus. The time—concentration curves were determined for each compound. Three mathematical models were applied to the data. The two compartment model gave the best fit to the data. The single compartment model gave slightly higher values, but could be used in clinical and certain research situations to estimate renal functions when it is not practical to take large number of samples.     

Toxocara vitulorum in the milk of buffalo (Bubalus bubalis) cows

The duration of excretion of Toxocara vitulorum larvae in the milk of buffalo cows determines the optimum time for treating calves. Studies on 10 cows showed that a few larvae occur in the colostrum of some cows before the calf has suckled, but most are present from the day after calving and for a further five days. From day 9 onwards, very few larvae were found in the milk. The total number of larvae found was comparable with the number of adult parasites collected from the calves of cows with similar histories. The larvae were 1254 +/- 60 microns long and 36 +/- 6.7 microns in diameter at the ventriculus, figures which are substantially different from some published results.     

PCR-SSCP analysis of leptin gene and its association with milk production traits in river buffalo (Bubalus bubalis)

Leptin gene has been found to be associated with various economic traits including milk production and fat quality in dairy animals. In the present study, we investigated genetic variations in intron 1 region of leptin gene in riverine buffaloes (Bubalus bubalis) using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing methods and associated them with milk traits. The study revealed three SSCP variants A, B and C among a total of 301 buffaloes from nine breeds. The frequency of variant C was found invariably high among all the breeds except in Marathwada buffalo. Variant A was found to be absent in Chilika, Nili-Ravi, Nagpuri and Pandharpuri breeds and also had the lowest frequencies in Mehsana, Jaffarabadi, Murrah and Toda breeds. Sequencing of SSCP variants revealed a total of five polymorphic sites, with three haplotypes. Statistical analysis revealed significantly high fat percentage at 150?days in SSCP variant B in Mehsana buffaloes. However, the associations of SSCP variants of leptin gene with total milk yield, 305?days milk yield and total fat yield were found to be non-significant. The present study is the first report on association analysis of leptin gene polymorphisms with milk production and milk quality traits in river buffalo.     

The egg production of Toxocara vitulorum in Asian buffalo (Bubalus bubalis)

The egg production of Toxocara vitulorum in Asian buffalo has been studied. Eggs were first present in the faeces of calves when they were 22.3 +/- 1.6 days old. In calves treated with pyrantel when 3 days old, the age at first patency was extended by 3.5 days indicating that there was no pre-natal transmission. Calves on only half the milk of the cow had a significantly longer prepatent period of 27.7 +/- 2.2 days. The peak egg output occurred in calves 35.7 +/- 2.6 days old and had a duration of 5.5 +/- 2.5 days with 98,000 +/- 63,700 eggs g-1 of faeces. The duration of the patent period was 35 +/- 12 days. The average lengths of populations of mature female parasites from different hosts at the time of peak egg output or older, ranged from 15.0 to 31.0 cm and was correlated with those of the males in the same populations (10.6-20.4 cm). The size of females was not affected by intraspecific competition. The proportion of males in the populations was 0.39 +/- 0.11. The egg output per female per day at the peak was 110,000 +/- 58,000 and was correlated with the size of the females at autopsy, but the egg output per female per day at the time of autopsy was lower and was not correlated, so it was concluded that the drop in egg counts was the result of reduced fecundity. The fertility of the eggs from faeces was greater than 92% throughout.(ABSTRACT TRUNCATED AT 250 WORDS)