Effect of Bradykinin on Murrah buffalo (Bubalus bubalis) semen cryopreservation

Keeping in view the poor freezability of bubaline semen in conventionally used extenders, this study was conducted on three Murrah bulls to improve semen cryopreservation with the incorporation of Bradykinin (0.5, 1.0 and 2.0 ng ml(-1)) in routinely used egg yolk tris-glycerol (EYTG) extender. Bradykinin (2.0 ng ml(-1)) had significant (P<0.05) beneficial effect on live sperm % (81.6+/-1.8) and hypo osmotic swelling (HOS) % (63.0+/-1.3) as compared to their respective control values of 73.4+/-2.1 and 56.3+/-2.0 at 0 h post freezing. The post-thaw progressive sperm motility in semen samples diluted with EYTG containing 2.0 ng ml(-1) Bradykinin (65.5+/-1.4) was also significantly (P<0.01) higher than control (60.3+/-1.9) at 0 h post freezing. Thus incorporation of 2 ng ml(-1) Bradykinin in buffalo semen diluted in EYTG extender may be useful in improving the quality of cryopreserved bubaline semen.     

Chromosome analysis of Murrah buffalo (Bubalus bubalis ) spermatozoa using zona-free hamster oocytes

An interspectific in vitro fertilization system was adopted to analyse sperm chromosomes of Murrah buffalo (Bubalus bubalis ). Superovulation was induced in mature female golden hamsters (Mesocricetus auratus ) to obtain a large number of oocytes. The zona pellucidae were digested by trypsin treatment. Zona free hamster oocytes were penetrated by buffalo spermatozoa capacitated with calcium ionophore A23187. Fertilized ova were cultured in TC 199 medium supplemented with 10% fetal calf serum (FCS). Podophyllotoxin and vinblastine were used to interrupt karyogamy and tubulin polymerization, respectively. Oocytes were fixed by modified gradual fixation air drying method. Slides were stained with 2% Giemsa for 45 min. Analyzable metaphase chromosome spreads were obtained from 22.4+/-3.8% of the penetrated oocytes. Of the 70 sexed spermatozoa, 38 were X-bearing and 32 were Y-bearing spematozoa.     

Alterations in hypophysial responsiveness to synthetic GnRH at different postpartum intervals in Murrah buffalo (Bubalus bubalis )

The objective of this study was to investigate the hypophysial responsiveness to GnRH at different intervals post partum in Murrah buffalo. Plasma LH and FSH levels were measured at 1 h before and upto 6 h subsequent to the administration of GnRH (1 ug/kg body weight) or saline on Days 2, 20 and 35 post partum in 2 groups of buffalo (n=4 each). Plasma progesterone levels were measured in samples collected once daily from Day 3 to Day 46 post partum. Pretreatment basal LH levels exhibited a progressive increase from Day 2 through Day 35 post partum, while the basal FSH levels increased only until Day 20 post partum. Following a highly subdued LH response to GnRH on Day 2 post partum, a 408% increase (P < 0.01) was observed in the total LH released in response to GnRH on Day 20 post partum, followed by a 20% reduction (non-significant) over Days 20 to 35 post partum. The interval from parturition was highly correlated with total LH released (r = 0.711, P < 0.01). Unlike LH, a substantial amount of FSH was released following GnRH treatment on Day 2 post partum, which was not significantly different from the FSH response on Days 20 and 35 post partum. The LH and FSH response to GnRH was not significantly different between animals in which luteal activity resumed and in those which showed no luteal activity post partum. While pointing to a dramatic enhancement in the hypophysial responsiveness to GnRH between Days 2 and 20 post partum, these results suggest that pituitary responsiveness to GnRH does not appear to be the limiting factor for resumption of estrous cycles by Day 35 post partum in Murrah buffalo.     

Effect of Spermine-NONOate on acrosome reaction and associated protein tyrosine phosphorylation in Murrah buffalo (Bubalus bubalis) spermatozoa

The present study was conducted to know the role of Nitric Oxide (NO) on the acrosome reaction (AR) in Murrah buffalo (Bubalus bubalis) spermatozoa. Ejaculated buffalo spermatozoa were washed, suspended in sp-TALP media containing 6 mg BSA/mL and cell concentration was adjusted to 50×10(6) cells/mL. The cells were incubated for 6h in the absence or presence of heparin (10 μg/mL) to induce capacitation. Fully capacitated spermatozoa were incubated in presence of 100 μg/mL Lysophosphatidyl choline (LPC, T1) or 100 μM Spermine-NONOate (T2) or 100 mM L-NAME (T3) or 100 μM Spermine-NONOate+100 mM L-NAME (T4) or 1 mM db-cAMP + 0.1 mM IBMX (T5) or 100μM H-89 (T6) or 100 μM Spermine-NONOate+100 μM H-89 (T7) in combination to induce acrosome reaction. The extent of AR was assessed by dual-staining of spermatozoa with trypan blue/Giemsa stain. AR-associated tyrosine-phosphorylated proteins were detected by SDS-PAGE followed by immunoblotting using monoclonal anti-phosphotyrosine antibody. Significant (P<0.05) number of spermatozoa were acrosome reacted in Spermine-NONOate (T2) treated cells but it was significantly (P<0.05) lower than LPC (T1) induced AR. Addition of Spermine-NONOate + L-NAME (T4) resulted in non significant (P>0.05) decrease in acrosome reaction. On addition of H-89 + Spermine-NONOate (T7) to sperm culture medium, resulted in significant (P<0.05) decrease in the percent acrosome reaction. Conversely, addition of db-cAMP+IBMX (T5, cAMP analogue) resulted in the significantly (P<0.05) higher number of acrosome reacted spermatozoa. Pattern of sperm protein tyrosine phosphorylation was also different in NO induced acrosome reaction compared to that of LPC. The present study concluded that nitric oxide is involved in acrosome reaction of buffalo spermatozoa by causing the tyrosine phosphorylation of proteins mainly p17 and p20 and through activation of cAMP/PKA pathway.     

Vitrification of buffalo (Bubalus bubalis) oocytes

The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then placed in 15-microL of VS and immediately loaded into 0.25-mL French straws, each containing 150 microL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at least 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P<0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P<0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-microL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 microg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5+/-10.7 and 31.5+/-1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of buffalo oocytes.     

Storage of buffalo (Bubalus bubalis) semen

Characteristics of buffalo semen, diluents used for liquid storage, aspects involved in freezing and thawing of semen are reviewed, and fertility results after artificial insemination (AI) with frozen-thawed semen are given.     

Embryo transfer in water buffalo (Bubalus bubalis)

A normal, live 35-kg water buffalo bull calf was born 300 days after it was nonsurgically collected as a 7-day blastocyst from a water buffalo donor and transferred nonsurgically to an unrelated water buffalo recipient. The development of estrus synchronization, superovulation and estrus detection methods in water buffalo are described.     

Nutrient utilisation,growth performance and blood metabolites in Murrah buffalo calves (Bubalus bubalis) divergently selected for residual feed intake

The aim of this study was to evaluate differences in efficiency of feed utilisation between buffalo calves with low and high residual feed intake (RFI) by comparing feed intake, nutrient digestibility, growth traits and blood metabolites. Eighteen male Murrah buffalo calves (aged 4–6 months; 70 ± 1.0 kg body weight) were fed ad libitum with a total mixed ration for 120 d. Based on linear regression models involving dry matter intake (DMI), average daily gain (ADG) and mid-test metabolic body size, calves were assigned into low and high RFI groups. The RFI varied from ?0.33 to +0.28 kg DM/d with an average RFI of ?0.14 and 0.14 kg DM/d in low and high RFI calves, respectively. Calves had a mean DMI of 1.9 and 2.4 kg/d and an ADG of 0.5 and 0.6 kg/d in low and high RFI groups, respectively. Low RFI calves ate 19.0% less DM each day and required significantly less metabolisable energy for maintenance compared with high RFI calves (12.5 vs. 16.7 MJ/d). Nutrient digestibility and nitrogen balance did not differ among low and high RFI calves. In more efficient animals (low RFI calves) higher (p < 0.05) plasma level of growth hormone, insulin-like growth factor-1 (IGF-1), triiodothyronine (T₃) and lower concentration of thyroxin hormone were detected. No significant differences in levels of insulin, hydroxyproline, plasma and urine creatinine, total protein and albumin between high and low RFI groups were found. Blood metabolites showed significant (p < 0.05) differences at initial and final stages of study in both groups. At final stage of study, RFI showed negative correlations with growth hormone, IGF-1, T₃, urine creatinine and albumin. Low RFI buffalo calves are more efficient in feed utilisation and the differences in blood metabolites are probably due to differences in feed intake and body metabolism.     

Theileria lawrencei: infection in the Indian water buffalo, Bubalus bubalis

Two Indian water buffalo (Bubalus bubalis), experimentally infected with Theileria lawrencei (Serengeti) by inoculation of stabilate material, died from acute theileriosis. Macroschizonts were first detected in both buffalo in the peripheral lymph node regional to the site of inoculation 8 days later. No microschizonts were seen in either animal and intraerythrocytic theilerial piroplasms were only detected in one in very low numbers. Both buffalo had marked febrile responses and died on Days 15 and 16 after exposure, when only low numbers of lymphoid cells were parasitized with macroschizonts, the vast majority of which contained few nuclei. Both buffalo showed evidence of terminal leucopenia and thrombocytopenia. On post mortem both animals showed generalised hyperplasia and edema of all lymphoid tissues, gross edema of the lungs, and hemorrhages in the heart and kidneys. T. lawrencei (Serengeti) infections of water buffalo and of cattle are compared and shown to be very similar.     

Experimental Sarcocystis hominis infection in a water buffalo (Bubalus bubalis)

A water buffalo (Bubalus bubalis) was fed 5.0 x 10(5) Sarcocystis hominis sporocysts from a human volunteer who had ingested S. hominis cysts from naturally infected cattle. A necropsy was performed on the buffalo 119 days after inoculation, and a large number of microscopic sarcocysts (approximately 5,000/g) were found in skeletal muscles. Ultrastructurally, the sarcocyst wall from buffalo muscles has upright villar protrusions measuring about 5.6 x 0.8 microm with numerous microtubules that run from the base to the apex. Sarcocysts from this buffalo were infective to 2 human volunteers, confirming their identity as S. hominis. Therefore, we believe that buffaloes can act experimentally as the intermediate host for S. hominis.     

Monosomy X and gonadal dysgenesis in a buffalo heifer (Bubalus bubalis)

Investigations on a 3-yr-old river buffalo heifer presenting anestrus revealed a chromosome make-up of 2n=49 in the lymphocyte cultures, compared with the 2n=50 characteristic of riverine buffalo. The missing chromosome was identified as one of the Xs by karyotypic analysis, and monosomy X was confirmed by C and G-banding techniques. Both ovaries of the heifer were underdeveloped, although the other components of the internal genitalia were normal. The phenotypic and karyotypic features confirmed this to be a case of ovarian dysgenesis with 49,XO karyotype similar to that of the Turner's syndrome in man and other mammals.     

Ciliate protozoa in the rumen of Brazilian water buffalo, Bubalus bubalis Linnaeus.

Total numbers and distribution of genera, subgenera and species were determined for the ciliate protozoa in rumen contents of 4 Brazilian water buffalo Bubalus bubalis Linnaeus. The fauna of one animal, housed in close proximity to European and zebu-type cattle, differed considerably from that of the remaining animals, which were somewhat isolated on a large ranch. Several of the protozoan species observed in the semi-isolated animals were first described in rumen contents from humped Indian cattle, and their subsequent occurrence in other hosts and geographic locations has been limited or absent. In all, 49 different species of protozoa were found, 8 of which have not been previously described. Three of the new species belong to the genus Entodinium: E. ciculum sp. n., E. spinonucleatum sp. n. and E. triangulum sp. n.; 4 to Diplodinium (Ostracodinium): D. (O.) brazili sp. n., D. (O.) esalqum sp. n., D. (O.) nucleolobum sp. n., and D. (O.) tiete sp. n.; and one to Diplodinium (Eudiplodinium): D. (E.) bubalus sp. n.     

Effect of naloxone on GnRH-induced LH and FSH release in buffalo, Bubalus bubalis

The effect of naloxone on GnRH-induced LH and FSH release was measured in buffaloes in luteal phase of estrous cycle. Animals were administered intravenously, naloxone/saline (50 mg/injection) every 15 min for 3 hr followed by GnRH (100 micrograms). Peripheral plasma LH and FSH concentrations were measured in blood samples collected at 15 min intervals from 1 hr prior to beginning of naloxone/saline treatment up to 3 hr post GnRH administration and every 30 min for the subsequent 3.5 hr. Between the animals of Group I administered naloxone and those of Group II given saline, GnRH-induced peak LH and FSH concentrations, the total LH and FSH released in response to GnRH, and the time to peak LH and FSH concentrations were not significantly different. The results of the present study suggest the absence of a direct effect of naloxone on pituitary responsiveness to GnRH.     

Early embryonic development in Thai swamp buffalo (Bubalus bubalis )

A total of 33 nonsurgical embryo collections was carried out to investigate early embryo development in Thai swamp buffalo. Collections were performed on Days 5.5, 6.0, 6.5, 7.0 and 7.5. The different stages of embryo development on these days were the 16-cell stage, compact morula, blastocyst, hatched blastocyst and hatched expanding blastocyst, respectively. In addition, some degenerating embryos and unfertilized ova were also recovered. A higher recovery rate was obtained with single embryo collection after natural estrus than after induced estrus or superovulation, 78% (7 9 ) vs 46% (6 13 ) vs 54.5% (6 11 ), respectively. A higher percentage of normal embryos was also obtained with single embryo collection after either natural or induced estrus than after superovulation, 71% (5 7 ), 83% (5 6 ) and 38% (6 16 ), respectively.     

Total lipids and phospholipids in buffalo semen (Bubalus bubalis)

Spermatozoal and seminal plasma concentrations of total lipids from 50 ejaculates and phospholipids and their fractions from 30 ejaculates were quantified in the semen of five Murrah buffalo bulls. Sperm lipid content ranged from 0.93 to 1.72 mg/10(9) cells with an overall average 1.32 +/- 0.03 mg/10(9) cells. Its concentration in seminal plasma varied from 1.39 to 2.22 mg/ml with overall average of 1.75 +/- 0.03 mg/ml. Spermatozoal total phospholipid content ranged from 0.44 to 0.94 mg/10(9) cells with overall mean being 0.64 +/- 0.02 mg/10(9) cells. The corresponding values for seminal plasma were 0.53 and 0.88 mg/ml with an overall mean of 0.69 +/- 0.02 mg/ml. Phosphatidyl choline constituted the major fraction both in the spermatozoa and and seminal plasma.     

The serum lipoprotein pattern of water buffalo (Bubalus bubalis)

1. The serum lipoprotein pattern of water buffalo was studied by means of electrophoresis and the lipoproteins were isolated by ultracentrifugation on the basis of their hydrated density. 2. High density lipoproteins (HDL) showed a higher level of cholesterol than did the other lipoproteins. Moreover, the level of phospholipids was higher in HDL than in very low density lipoproteins (VLDL). 3. The buffalo B100 apoprotein was similar to that of man and rat. Three apoproteins similar to human apo E, apo AI and AII were found in buffalo HDL, buffalo VLDL contained essentially apo B protein.     

The postnatal histology of the epididymis in buffalo (Bubalus bubalis).

The epididymis of buffalo is differentiated into 6 regions. The head represents the first 3 regions, the body the 4th region and the tail the 5th and 6th region. At 3-30 weeks, the epithelium is simple high columnar with few basal cells in region I, simple low columnar with few basal cells in region II, pseudostratified low columnar in regions III, IV, V, and pseudostratified low columar in region VI. As age advances, the epithelium increases in height and shows a tendency toward advanced pseudostratification. The basal cells are greater in number in regions III, IV and V than in the other regions of the epididymis. The epithelial cells contain stereocilia in region I at 3 weeks and regions III, IV and V at 30 weeks, whereas they are absent in regions II and VI. The tubules in region I are the smallest in diameter while the tubules in region VI have the largest diameter.     

Dominance of Methanomicrobium phylotype in methanogen population present in Murrah buffaloes (Bubalus bubalis)

Aims:  To study the diversity of rumen methanogens in Murrah buffaloes ( Bubalus bubalis ) from North India by using 16S rRNA gene libraries obtained from the pooled rumen content from four animals and using suitable software analysis.
Methods and Results:  Genomic DNA was isolated and PCR was set up by using specific primers. Amplified product was cloned into a suitable vector and the positive clones were selected on the basis of blue–white screening and sequenced. The resulting nucleotide sequences were arranged in the phylogenetic tree. A total of 108 clones were examined, revealing 17 different 16S rRNA gene sequences or phylotypes. Of the 17 phylotypes, 15 (102 of 108 clones) belonged to the genus Methanomicrobium , indicating that the genus Methanomicrobium is the most dominant component of methanogen populations in Murrah buffaloes ( Bubalus bubalis ) from North India. The largest group of clones (102 clones) was more than 98% similar to Methanomicrobium mobile . BLAST analysis of the rumen contents from individual animals also revealed 17 different phylotypes with a range of 3–10 phylotypes per animal.
Conclusion:  Methanomicrobium phylotype is the most dominant phylotype of methanogens present in Murrah buffaloes ( Bubalus bubalis ).
Significance and Impact of the Study:  Effective strategies can be made to inhibit the growth of Methanomicrobium phylotype to reduce the methane emission from rumen contents and thus help in preventing global warming.