Buffalo Bulls Research Articles

Assessment of surface temperatures of buffalo bulls (Bubalus bubalis) raised under tropical conditions using infrared thermography

This paper aimed to evaluate the surface temperatures of buffalo bulls using infrared thermography, considering four distinct anatomical parts over time, and to correlate surface temperatures and thermal comfort indexes. The humid tropical climate (Köppen's Afi) was predominant in the research station where the experiment was performed and the trial lasted from April to August. Ten bulls (n=10) were evaluated every 25 days (morning: 6:00-9:00; afternoon: 12:00-15:00) and the parameters assessed were respiratory rate (RR), rectal temperature (RT), and the thermograms of surface temperature for orbital area (ORB), right flank (RF), left flank (LF) and scrotum (SCR). Climatological data was continuously monitored and the Temperature and Humidity Index (THI) and the Index of Comfort of Benezra (ICB) were calculated. The average values of THI were ≥78, and significant differences between shifts were observed (P<0.05). The ICB ranged from 1.96 to 2.25 and significant differences were observed for shifts and throughout the months (P<0.05). The averages of surface temperatures were RT=38.2±0.5°C, ORB=36.1±0.8°C, LF=33.5±2.5°C, RF=35.4±1.7ºC and SCR=33.3±1.1°C, which exhibited significant differences for shifts and throughout the months (P<0.05). Positive correlations were obtained between THI and ORB (0.72), RF (0.77), LF (0.75) and SCR (0.41) (P<0.0001). The maximum temperature of ORB showed the highest correlation with RT (0.58, P<0.0001). Therefore, the surface temperatures are subject to climatic variations and increase throughout the day, due to the variation in thermal comfort indexes, and the maximum ORB temperature was the parameter most related to rectal temperature. Lastly, the results indicate that IRT may be a useful non-invasive and accurate tool to detect the variations in ORB, LF, RF and SCR temperature in buffalo bulls.

L-CARNITINE ENHANCING ROLES ON BUFFALO SEMEN FREEZABILITY, ULTRA STRUCTURE AND FERTILIZING POTENTIALS

Recently, male impact has been recognized as a momentous cause of infertility. Male infertility isn’t an entity but it evokes a variety of mechanisms and explanations to understand it. In between, a biochemical semen additive that improves or overcome certain semen situations so can enhance or treat semen fertility and infertility, respectively. L-carnitine is one of these biochemical semen additives. Till now, the exact effects of L-carnitine on buffalo semen processing outcomes haven’t been discovered. The current study aimed to clarify L-carnitine roles during buffalo semen cryopreservation. Semen samples were obtained from six fertile buffalo bulls (aged 3 to 5 years). Weekly, two consecutive ejaculates were collected from each bull for successive six weeks using artificial vagina. The ejaculates were pooled to eliminate samples variability. Semen samples were extended with Tris-based extender supplemented with different concentrations of L-carnitine (0.01, 0.05 and 0.1 mg/ml) Vs. Tris-based extender only (control). Then they were processed to cryopreservation and thawing to assess different semen characteristics. It had been found that L-carnitine (0.05mg/ml) significantly (p < 0.05) improved post thawing motility, viability index, acrosomal integrity, in vitro fertilization, blastocyst and conception rates of treated buffalo semen (57.00±2.55%, 126.5±8.33, 14.40 ± 1.43%, 56.45%, 15.79% and 64.71%, respectively), compared with control (42.00±2.56%, 87.00±10.43, 23.80±1.86%, 36.53%, 2.13% and 42.86%, respectively). Conclusion: L-carnitine supplementation to buffalo semen extender significantly enhanced its characteristics and protecting its plasma membrane and mitochondrial functional integrity. Moreover, the preceding results focusing more light on the potential roles of L-carnitine in regulating male fertility and infertility.

Characterization of tissue inhibitor metalloproteinases in semen and their relationship with vital sperm function tests vis-à-vis fertility of breeding buffalo bulls

The present study was undertaken to separate and compare the tissue inhibitor metalloproteinases (TIMP) of seminal plasma and frozen-thawed sperm extracts from 30 buffalo bulls by immunoblotting and determine a relationship between various TIMP with post-thaw sperm function tests vis-à-vis bull fertility. Seven immunoreactive bands in seminal plasma (65, 55, 48, 33, 31, 24 and 11 kDa) and 5 in frozen-thawed spermatozoa (75, 65, 55, 24 and 16 kDa) were detected in Western blots following incubation (TIMP–140) and subsequent washing in vitro, indicating that TIMP is bound to sperm membranes. The frozen-thawed semen was evaluated for first service conception rate (FSCR), per cent HOST, acrosome reaction, viability, DNA integrity and total motility and linked to TIMP. In seminal plasma, the bulls positive for 48, 33 and 24 kDa TIMP had significantly higher FSCR (57.0 ± 2.6 vs 27.0 ± 2.4%, 55.7 ± 3.0 vs 31.3 ± 3.2% and 45.0 ± 3.8 vs 32.8 ± 4.7%, respectively) as compared to their negative counterparts. Except per cent viability, almost all seminal parameters (acrosome reaction, per cent HOST, DNA integrity and total motility) were significantly higher in bulls positive for TIMP of 48, 33, 31 and 24 kDa than in their negative contemporary mates. In frozen-thawed sperm extracts, the bulls positive for TIMP–24 had significantly higher FSCR (51.7 ± 3.7 vs 27.2 ± 3.0%), higher percentage of acrosome-reacted (55.9 ± 2.8 vs 48.9 ± 2.2%) and HOS-positive (69.2 ± 1.5 vs 65.3 ± 1.9%) spermatozoa in comparison to their negative herd mates. These results suggested that TIMP influences semen quality and subsequent fertility of buffalo bulls through inhibition of metalloprotease activity in semen.

Identification of Heparin Binding Proteins in Frozen-thawed Capacitated/Acrosome-reacted Spermatozoa and Their Relationship with Fertility of Buffalo Bull Semen

The present was conducted to separate heparin binding proteins (HBP) of capacitated sperm extracts and determine their relationship with acrosome reaction and semen fertility of bulls (n=30). Fourteen immunoreactive bands in capacitated spermatozoa of bulls (135, 100, 75, 70, 65, 55, 48, 45, 33, 31, 26, 24, 20 and 18 kDa) were detected in western blots following incubation with anti-HBP antibody. The frozen-thawed semen was evaluated for first service conception rate (FSCR) and percent acrosome reaction and linked to HBP. In capacitated spermatozoa, bulls positive for 70, 31, 20 and 18 kDa had significantly higher (P 0.05) in bulls positive for 135 and 100 kDa HBP than in their negative contemporary mates and exhibited an increase of 7.4% for the two proteins. A significantly higher (P < 0.05) rate of acrosome reaction was recorded in bulls with HBP of 135, 100, 70 and 18 kDa in comparison to their contemporary mates with a difference of 7.4, 7.4, 8.7 and 9.4%, respectively. Alternatively, FSCR and acrosome reaction of bulls with 75, 55 and 48 kDa were lower (P < 0.05) than their corresponding counterparts. In conclusion, HBP of 135, 100, 70 and 18 kDa did seem to activate in vitro sperm acrosome reaction and subsequent fertility of bulls.

Isolation of bacteria in semen and evaluation of antibiotics in extender for cryopreservation of buffalo (Bubalus bubalis) bull spermatozoa.

This study was designed to investigate the occurrence of bacterial species in water buffalo semen at the time of collection/processing and to assess the efficacy of some selected antibiotics (GTLS; gentamycin, tylosin and linco-spectin or SP; streptomycin and penicillin) in cryodiluent on bacterial control and quality including invivo fertility of buffalo spermatozoa. For this purpose, four experiments were conducted. In experiment 1, a total of 11 bacterial species were isolated from buffalo ejaculates. In experiment 2, total aerobic bacterial counts at post dilution and thawing were lower (P<0.05) in GTLS than in SP or control. The majority of the bacterial isolates from ejaculates were more susceptible to GTLS than SP. In experiment 3, sperm acrosome integrity was higher (P<0.05) in GTLS and SP compared to control. In experiment 4, the invivo fertility results for GTLS were higher (P<0.05) than that for SP. In conclusion, a number of bacterial species were isolated from the bubaline semen, which requires an efficient control before its use in artificial insemination program. The GTLS combination of antibiotics may be incorporated into a freezing extender/protocol without compromising the post-thaw quality and invivo fertility of buffalo bull spermatozoa.

Royal jelly supplementation in semen extender enhances post-thaw quality and fertility of Nili-Ravi buffalo bull sperm

Two experiments were conducted to evaluate the effect of royal jelly (RJ) on post-thaw sperm quality, in vitro and in vivo fertility rate of cryopreserved buffalo bull sperm. The semen was collected from three mature regular donor buffalo bulls, ejaculates were pooled and semen evaluated initially. In Experiment 1, the ejaculates were extended in tris-citric acid diluter supplemented with different RJ concentrations (0, 0.05, 0.1, 0.2, 0.3 or 0.4%). The diluted semen was cooled to 4°C, packaged into 0.5mL straws and frozen using standard procedure. The straws were thawed and assessed for sperm progressive motility, viability, plasma membrane, acrosome, and chromatin integrity. The results indicated that sperm progressive motility was significantly greater (P<0.05) in 0.05, 0.1, 0.2 and 0.3% RJ than 0.4% RJ supplemented and control groups. The sperm viability, plasma membrane and acrosome integrity were significantly improved (P<0.05) in 0.1% RJ supplemented group the compared to other treatment groups.In Experiment 2, cryopreserved sperm with 0.1% RJ supplementation and control (without RJ supplementation) were used to observe the in vitro fertilizing potential and in vivo fertility. In vitro fertilization method was applied to assess the cleavage rate; whereas, AI was performed in buffalo during in vivo fertility trial. The buffaloes were inseminated 12h after standing estrus and pregnancy diagnosis was performed through ultrasonography. The results revealed that the cleavage rate was higher (P<0.05) in 0.1% RJ as compared to control group. However, the pregnancy rate was similar (P>0.05) between 0.1% RJ supplemented and control groups. It is concluded that supplementation of RJ in freezing extender can improve the cryosurvival rate and in vitro fertilizing capacity of buffalo bull sperm.

Effect of incubation on freezability of cholesterol-loaded cyclodextrin treated buffalo (Bubalus bubalis) spermatozoa.

Aim:The aim of this study was to investigate the effect of incubation on freezability of cholesterol loaded cyclodextrin (CLC) treated buffalo spermatozoa.Materials and Methods:Semen samples with mass motility of 3+ and greater, collected from Murrah buffalo bulls were utilized. Immediately after collection, four equal groups of semen sample were made. Group I was kept as control and diluted with Tris upto concentration of 60×106 sperm/ml, where as Groups II, III, and IV were treated with CLC at 3 mg/120× 106 spermatozoa, incubated at 37°C for action of CLC for 10, 15 and 20 min, respectively, and diluted with tris upto concentration of 60×106 sperm/ml. All groups were subjected to equilibration and freezing. The evaluation of semen samples from all groups was carried out at fresh, pre-freeze and post-thaw stage for progressive motility, viability and hypo-osmotic swelling response (HOS response).Results:At the pre-freeze stage, significantly (p<0.05) higher percentage of progressive motility and viability was observed in treatment groups as compared to control with no significant difference among treatment groups. HOS response was significantly (p<0.05) higher in treatment groups as compared to control at pre-freeze stage. At post-thaw stage, significantly (p<0.05) higher percentage of progressive motility, viability and HOS response was recorded in Group II as compared to control and other treatment groups (III and IV). Group II retained significant post-thaw motility and viability at various post-thaw incubation periods.Conclusion:Incubation period of 10 min for CLC treated buffalo spermatozoa yielded significantly higher results in terms of freezability as compared to incubation for 15 and 20 min.

L-Cysteine improves antioxidant enzyme activity, post-thaw quality and fertility of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa.

The effects of l-cysteine in extender on antioxidant enzymes profile during cryopreservation, post-thaw quality parameters and invivo fertility of Nili-Ravi buffalo bull spermatozoa were studied. Semen samples from 4 buffalo bulls were diluted in Tris-citric acid-based extender having different concentrations of l-cysteine (0.0, 0.5, 1.0, 2.0 and 3.0mm) and frozen in 0.5-ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase and reductase)] were significantly higher (P<0.05) at pre-freezing and post-thawing in extender containing 2.0mm l-cysteine as compared to other groups. Post-thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μms-1 ), straight line velocity (μms-1 ), curvilinear velocity (μms-1 ), beat cross frequency (Hz), viable spermatozoa with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with the addition of 2.0mm l-cysteine as compared to other groups (P<0.05). The fertility rates (59 versus 43%) were higher (P<0.05) in buffaloes inseminated with doses containing 2.0mm of l-cysteine than in the control. In conclusion, the addition of 2.0mm l-cysteine in extender improved the antioxidant enzymes profile, post-thaw quality and invivo fertility of Nili-Ravi buffalo bull spermatozoa.

Quantification of leptin in seminal plasma of buffalo bulls and its correlation with antioxidant status, conventional and computer-assisted sperm analysis (CASA) semen variables

The present study is the first to quantify leptin in seminal plasma of buffalo and investigate its relationship with seminal attributes. Ten ejaculates each from 10 Murrah buffalo bulls were collected. Semen quality variables such as semen volume, sperm concentration, sperm abnormalities, membrane integrity, antioxidant enzyme activities (superoxide dismutase, catalase and total antioxidant capacity), malondialdehyde (MDA) concentration, as well as sperm kinetics and motility variables were evaluated. The leptin concentration in serum and seminal plasma were estimated by the ELISA method. Bulls were classified in two groups on the basis of sperm concentration with Group I having >800 million sperm/mL and Group II <500 million sperm/mL. Greater (P<0.05) mean sperm abnormalities, seminal leptin concentrations and MDA concentrations were recorded in Group II than Group I. The seminal leptin was positively correlated with sperm abnormalities and MDA concentration while being negatively correlated with sperm concentration, but there was no correlation with sperm kinetic and motility variables, sperm membrane integrity and seminal plasma antioxidant enzyme activity. Thus, the data suggest that seminal leptin has a role in spermatogenesis and can be used as a marker for spermatogenesis to predict the capacity of buffalo bulls for semen production.

Determination of Quality Characteristics of Kundhi Buffalo Bull Semen

The objective of present study was to investigate the quality characteristics of semen collected from kundhi bulls. Semen was collected by an artificial vagina and transported into laboratory immediately for evaluation. Total of 96 (Twenty four from each bull) semen the samples were evaluated and found creamy white. The mean (±SEM) were mass activity (+++), volume (2.25 ± 0.01 ml), pH (6.10 ± 0.007), progressive motility (69 ± 0.34%,) sperm concentration (1542 ± 9.20x106/ml), morphology (79 ± 1.37%) and sperm membrane (55.56 ± 1.37%) respectively. Non-significant (P&gt;0.05) difference between the bulls for the characteristics except percentages morphology and sperm membrane where a significant (P

Effect of Exogenous Diethylstilbestrol on Semen Quality of Kundhi Buffalo Bull

The study was conducted to figure out the impact of exogenous Diethylstilbestrol on semen quality of Khundi buffalo bulls. Six Kundhi buffaloes of aged 5-6 years were divided into two groups, three bulls were placed in group-1 treated as experimental group and three bulls were kept as an untreated external control. 20mg of Diethylstilbestrol was injected intramuscularly to each experimental bull, parameters of semen and serum estrogen level was determined through ELISA. The comparative average semen volume, concentration, pH and motility in control and experimental animals were 4.7 vs. 5.39 ml, 1.43 vs. 1.27 x109, 6.76 vs. 6.66 and 73 vs. 71% respectively; the color of semen was creamy in control animals than experimental animals, except than the concentration of bull semen of control and experimental animals the all studied seminal parameters of bulls were statistically non-significant. The serum estrogen level was significantly declined after Diethylstilbestrol treatment. It was concluded that the administration of exogenous estrogens in buffalo bull has no or little impact in the improvement of semen quality and resulted in decline of serum estrogen level bulls. Further studies at molecular level like relative gene expression of estrogen receptors at mRNA level and immunohistochemistry are required to confirm the specific cellular localization estrogen receptor in the testes of buffalo bulls.

Serological study for detection of foot-and-mouth disease virus activity in breeding bulls of an elite herd of North India

A serological study using 3AB nonstructural protein ELISA and liquid phase blocking ELISA to assess foot-and-mouth disease virus (FMDV) non-structural and structural protein antibodies (NSP-and SP-Abs) was performed through sampling from 103 cattle and 37 buffalo bulls of an elite herd of north India. A total of 53 (51.46%) cattle bulls and 14 (37.84%) buffalo bulls were found positive for NSP-Ab indicating an exposure to FMDV. The proportion of animals showing >1.8 log10titre against all three serotypes in the vaccine varied from 58.25% for cattle bulls and 48.64% for buffalo bulls indicating poor vaccinal herd immunity, which assumably might have made the animals susceptible to virus exposure in the farm.

Dimensional characteristics of spermatozoa of Nili-Ravi buffalo bulls: seasonaland climatic influences

This study was conducted in 2 different climatic zones of Punjab Province of Pakistan, the Sahiwal and Bahawalpur divisions, simultaneously for the period of 1 year divided into seasons. Seven adult Nili-Ravi buffalo breeding bulls from each division were used. Dimensional characteristics of spermatozoa were determined. Season had a nonsignificant effect on the sperm head length in both divisions and a nonsignificant difference was also observed between bulls of the Sahiwal and Bahawalpur divisions. The buffalo bulls of both divisions exhibited significantly increased sperm head breadth in autumn, whereas a nonsignificant difference was observed between divisions. Sperm head shape values were significantly different among the seasons in both the divisions whereas they were nonsignificantly different between the two divisions. Midpiece length value was significantly higher in winter than spring in both divisions and also significantly different between the divisions. Sperm tail length was significantly increased in autumn in Sahiwal and in autumn and spring in Bahawalpur. It was significantly longer in Sahiwal than Bahawalpur. The pattern of seasonal and climatic influence on total sperm length was similar to that of sperm tail length. In conclusion, autumn and spring seasons and the climate of the Sahiwal division have additive effects on sperm dimensions, except midpiece length.

Fermentation Pattern in Murrah Buffalo Bulls Fed Crop Residue Based Complete Rationsvis-a-visConventional Feeding System

The experiment compared the effect of feeding complete rations with conventional feeding system on rumen fermentation pattern of Murrah buffalo bulls. Four adult rumen fistulated buffalo bulls (5 y; 331±9.36 kg BW) were divided into four groups in a 4 x 4 Latin square switch over design and offered three isonitrogenous complete rations comprising of locally available crop residues viz. maize stover (T1), red gram straw (T2) and black gram straw (T3), and concentrate mixture in 60: 40 ratio, respectively, and a conventional ration (C) for a period of 28 days. The rumen-N fractions (NH3-N, TCA-insoluble nitrogen, Residual nitrogen), TVFA and pH were higher (P<0.01) in the SRL of animals fed rations T1 to T3 when compared to those animals fed C. The total N, food and protozoal N was similar among the treatment groups. It is therefore, concluded that feeding of complete rations comprising of locally available crop residues as roughage source provided conducive rumen environment for better nitrogen utilization and higher TVFA production than conventional ration.

Association between Genotypes of Growth Hormone Gene and Semen Quality Traits in Cow Bulls and Buffalo Bulls

Association between Genotypes of Growth Hormone Gene and Semen Quality Traits in Cow Bulls and Buffalo Bulls

Activity of Enzymatic Antioxidants and Total Antioxidant Capacity in Seminal Plasma of Murrah Bulls during Cryopreservation

Semen samples with mass motility of 4+ or more and an initial progressive motility ≥ 80% collected from murrah buffalo bulls were utilized for study. Semen samples were evaluated for various seminal attributes, enzymatic antioxidants (superoxide dismutase, catalase and glutathione peroxidase) and total antioxidant capacity at fresh, pre-freeze and post-thaw stage. Seminal attributes (progressive motility, viability, acrosomal integrity and hypo-osmotic swelling response) were significantly (p<0.05) higher in bull II as compared to bulls (I & III) at pre-freeze and post-thaw stage. Superoxide dismutase was significantly higher in bulls (II & III) than bull I at fresh stage (p<0.05) and significantly (p<0.01) higher in bull II as compared to bulls (I & III) at pre-freeze and post-thaw stage. Catalase was significantly higher in bull II than bulls (I & III) at fresh (p<0.05) and pre-freeze (p<0.01) stage. Significantly, higher total antioxidant capacity was noticed at pre-freeze (p<0.05) and post-thaw (p<0.01) stage in bull II and bulls (II & III), than bulls (I & III) and bull I, respectively. On the basis of our findings, it could be concluded that seminal enzymatic antioxidants (superoxide dismutase & catalase) and total antioxidant capacity varied among bulls at various stages of cryopreservation. There was progressive reduction in activity of enzymatic antioxidants and total antioxidant capacity from fresh to post-thaw stage.

Endocrine Status of Serum Testosterone, Estrogen and Thyroid Hormones in High Fertility Breeding Buffalo Bulls and their Male Calves

Endocrine Status of Serum Testosterone, Estrogen and Thyroid Hormones in High Fertility Breeding Buffalo Bulls and their Male Calves

Effect of supplementation of glutamic acid and aspartic acid in egg yolk tris and egg yolk-citrate-glucose diluents on cryopreservation of Murrah buffalo bull spermatozoa

Present study was designed to assess the effect of supplementation of glutamic acid and aspartic acid in egg yolk tris and egg yolk-citrate-glucose diluents on spermatozoa quality of Murrah buffalo bull after equilibration and thawing. Semen was collected from four Murrah buffalo bulls biweekly from each bull with the help of artificial vagina (AV) on dummy animal between 08:00 to 09:00 hours using standard protocol. A total of thirty two ejaculates were taken from four bulls. In fresh ejaculates were pooled and divided into eight equal parts. The two basic diluents used for extending semen were Egg yolk Tris (EYT) and Egg yolk-citrate glucose (EYCG). Glutamic acid and aspartic acid were supplemented in different combinations. Diluted semen was equilibrated and cryopreserved. At two stages viz. after equilibration and after 12 hours of cryopreservation (thawed samples) progressive motility, percent live and percent acrosomal damage of spermatozoa was assessed. Post thaw acrosomal damage was significantly higher ( P <0.05) in EYCG than EYT. Supplementation of aspartic acid to EYT and EYCG significantly ( P <0.05) decreased the post thaw acrosomal damage as compared to control. Supplementation of glutamic acid to EYT and EYCG significantly ( P <0.05) improved all the studied spermatozoa qualities. The addition of both aspartic and glutamic acid together did not produce any additive effect on spermatozoa quality. It can be concluded that tris buffer was a better substitute to citrate buffer and glutamic acid alone may be supplemented to semen extender to improve the spermatozoa quality after equilibration and thawing.

B mode ultrasonography for diagnosis of urethral calculi in buffalo bulls

Study was performed to evaluate B mode ultrasonography for diagnosis of site of urethral obstruction in male buffalo bulls presented with complaint of anuria or dysuria. Ten animals were included in the study. Pre and post scrotal urethra were scanned. Ultrasonography was able to detect pre-scrotal obstruction in 6 animals. Hyperechoicdensity within urethra, with anechoic acoustic shadow and dilatation of urethra proximal to the site of obstruction indicated urethral obstruction by calculi. In other 4 animals, no calculi were detected ultrasonographically. In these animals post-scrotal urethrotomy was performed and calculi were retrieved. Superficial placement and a relatively fixed anatomical location of pre-scrotal urethra were found helpful in localizing the pre-scrotal urethral calculi. Ultrasonographic examination was found helpful in detecting the obstruction by prescrotal urethral calculi and may improve the chances of successful surgical treatment of urethral calculi in buffalo bulls.

24 EFFECT OF DONOR CELL TYPE ON IN VITRO AND IN VIVO DEVELOPMENTAL COMPETENCE OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

Selection of the donor cell type for somatic cell NT is very important based on its capability to be reprogrammed by the oocyte cytoplasm. A very wide variety of donor cells of different origin have been used for somatic cell NT, having differences in the overall efficiency. The aim of this study was to compare the cloning efficiency of donor cells derived from the ventral side of origin of tail skin and seminal plasma of a buffalo bull (age: 3 years old). Somatic cells from skin and seminal plasma were isolated and cultured as described by Selokar et al. (2014 PLOS ONE 9(3), e90755). Cultured seminal plasma cells had classic epithelial morphology, grew in clusters, were hexagonal in outline shape, and were positive for immunocytochemical detection of keratin marker, indicating that they were of epithelial origin, whereas tail-derived cells were spindle in shape and found positive for vimentin expression, indicating the fibroblast origin. To determine their reprogramming potential, these cells between passages 5 to 8 were used for the production of buffalo cloned embryos by handmade cloning as per the method described by Selokar et al. (2012 Theriogenology 78, 930–936). In brief, oocytes were isolated from slaughter-house ovaries and matured in vitro. After 21 h of maturation, cumulus cell mass and zona pellucida were removed by enzymatic treatment, hyaluronidase and pronase, respectively. Zona-free buffalo oocytes were enucleated on the basis of protrusion cone. A single somatic cell was attached to an enucleated oocyte with addition of phytohemagglutinin, followed by sandwich type of electrofusion between the somatic cell-bearing oocyte and enucleated oocyte using BTX electrofusion machine. Fused oocytes were activated by 4 μM calcium ionophore for 5 min and incubated in 2 mM 6-DMAP for 4 h and were cultured in K-RVCL-50® medium for 7 days on a flat surface in a 4-well dish in an incubator (5% CO2 and 38.5°C temperature). The total numbers of embryos reconstructed from tail-derived cells and semen-derived cells were 132 and 158, respectively. Cleavage and blastocyst rate were calculated from total embryos cultured, and data were analysed by Student’s t-test. We found no significant effect on both cleavage (89.30 ± 2.1 v. 94.1 ± 0.6) and blastocyst rate (40.7 ± 4.0 v. 43.1 ± 9.6) for the embryos produced from cells derived from tail and seminal plasma. To study the in vivo developmental competence of embryos derived from the 2 donor cell types, one embryo of each cell type was transferred into 6 recipient animals. Pregnancies were confirmed by ultrasonography at 30 to 35 days after transfer and monitored regularly at 15-day intervals up to 90 days. Three pregnancies were found for tail-derived cells, whereas no pregnancy was obtained for semen-derived cells. Out of 3 pregnancies obtained, 1 embryonic death was observed before 45 days, and 2 are continuing at advance stage. In conclusion, tail-derived cells are the better donor cell choice for buffalo somatic cell NT research. Currently, our focus is on epigenetic reprogramming behaviour of these 2 different cell types to elucidate the possible reprogramming mechanism.