We have established trophoblast cell lines, from parthenogenesis-derived buffalo blastocysts. The buffalo trophoblast cells were cultured continuously over 200 days and 21 passages. These cells were observed by phase-contrast microscopy for their morphology and characterized by reverse transcriptase polymerase chain reaction and immunofluorescence against trophoblast-specific markers and cytoskeletal proteins. Trophoblast cells showed positive staining for CDX2, a marker of these cells at both blastocyst and cell line levels. Epithelial morphology of these cells was revealed by positive staining against cytokeratins and tubulin but not against vimentin and dolichos biflorus agglutinin. Gene expression profiles of many important placenta-specific genes were studied in the primary trophectoderm outgrowths, which were collected on days 0, 5, 9, 12 and 15 of culture and trophoblast cell line at passages 12–15. Therefore, the trophoblast cell line derived can potentially be used for in vitro studies on buffalo embryonic development.

This study investigates a novel early pregnancy marker in water buffaloes, focusing on RSAD2 mRNA expression, known to be upregulated by interferon-tau (IFNT) during pregnancy. While RSAD2 is primarily recognized for its antiviral effect, we hypothesized its role as a conceptus-induced component in regulating pregnancy in buffaloes. Given its differential expression compared to other IFNT-induced genes in cows, RSAD2 may serve as a biomarker for early pregnancy detection in buffaloes. RNA, cDNA, and plasma samples were obtained from archived samples collected before insemination (d0) and at d20, d25 and d40 after insemination. Twelve RNA samples, having optimal optical density and concentration, from six pregnant and six non-pregnant buffaloes were selected. The cDNA was analyzed to measure the abundance of RSAD2 mRNA using real-time quantitative PCR (RT-qPCR) and plasma for protein expression analysis using Western blot. The RT-qPCR analysis showed a transcript of RSAD2 increased significantly by 7-fold and 6-fold on d20 and d25, compared to both d0 and d40 in the pregnant group only. At d20, the sensitivity of RSAD2 was 100% and the specificity was 83.3%, and at d25-d both the sensitivity and specificity was 100%, indicating low incidences of misdiagnosing early pregnancy in buffaloes. In the non-pregnant group, RSAD2 expression remained low and did not change after insemination. Western blot analysis revealed an immunoreactive RSAD2 protein band. Densitometry analysis of the RSAD2-specific protein band, based on gray mean value, showed significantly increased expression of RSAD2 at d25 compared to d0 in the pregnant group. In conclusion, these results indicated that RSAD2 expressions at both the mRNA and protein levels show promising potential for detecting pregnancy at d25 post-insemination.

The aim of this study was to examine the suitability of different growth functions (linear, sinusoidal, Gompertz, Schumacher and Richards) to fit cumulative milk production data from buffalo cows. Cumulative milk production at each day in milk was calculated from two published datasets reporting (i) fortnightly test-day milk yield records of the first lactation of Murrah buffalo that had calved during 1977–2012 and (ii) the first lactation records of Jaffarabadi buffalo collected from history-cum-pedigree registers for each quinquennium between 1991 and 2010. Each function was fitted to the lactation curves using nonlinear regression procedures. The Richards and sinusoidal equations provided the smallest root mean square error values, Akaike's and Bayesian information criteria and, therefore, the best fit for the cumulative lactation curves for milk yield. The Richards equation appeared to provide the most accurate estimate of the cumulative milk production at peak milk yield. Sinusoidal and flexible classical growth functions are appropriate to describe cumulative milk production curves and estimate lactation traits in buffalo.

An adult Indian buffalo (Bubalus bubalis) presented with corneal opacity, irritation, and excessive lacrimation from the left eye in the Referral Veterinary Polyclinic-Teaching Veterinary Clinical Complex (RVC-TVCC), Indian Veterinary Research Institute, Izatnagar. Clinical examination revealed a whitish thread-like worm in the left eye’s anterior chamber. The worm was surgically removed from the eye with supportive nerve blocks. Light microscopy was used for parasite morphological identification, which provided insight into the worm as female Setaria sp. Genomic DNA was isolated, and polymerase chain reaction amplification of 12S rRNA was conducted for molecular confirmation of the parasite. The amplicon was sequenced and analysed by bioinformatics software. Sequence data showed an amplicon size of 243 bp. Phylogenetic analysis with reference data from the NCBI Genbank database revealed the worm was S. digitata, with a similarity of 99.17%. The common predilection site of S. digitata is in the peritoneal cavity of natural hosts like cattle and buffalo and is mostly non-pathogenic. The aberrant migration of the parasite larva to the brain and eye commonly occurs in goats, sheep, and horses, causing clinical conditions like cerebrospinal nematodiasis (lumbar paralysis) and ocular setariasis, respectively. Nevertheless, until now, there have been no reports of ocular setariasis in buffalo. This report is the first unusual occurrence of ocular setariasis in buffalo and its molecular confirmation and phylogenetic analysis using 12S rRNA.

This study is a comparative analysis of the biochemical, hormonal, and mineral compositions of follicular fluid in preovulatory and cystic follicles of water buffalo (Bubalus bubalis). In total, reproductive tracts from 215 buffalo along with intact ovaries were collected randomly from an abattoir. The incidence of cystic conditions found in this study was 3.72% (8/215), involving the right ovary in 62.5% of instances and the left ovary in 37.5% of instances during the non-breeding season. Follicular fluid was aspirated from preovulatory follicles (12–15 mm diameter, oestrogen-active, follicular phase or stage IV corpus luteum on one of the two ovaries, n = 10) and cystic follicles (at least 20 mm diameter, no corpus luteum on any one of the two ovaries, n = 8). The follicular fluid samples were assayed for biochemical components (uric acid, creatinine, blood urea nitrogen, cholesterol, total protein, glucose, ascorbic acid, and alkaline phosphatase), hormones (progesterone, estradiol, and insulin), and minerals (calcium, magnesium, phosphorus, copper, zinc, and cobalt). Cystic follicles had greater (P < 0.05) concentrations of creatinine, blood urea nitrogen, cholesterol, progesterone, copper, zinc, and cobalt, and lesser (P < 0.05) concentrations of uric acid, glucose, ascorbic acid, estradiol, insulin, calcium, magnesium, and phosphorus compared with preovulatory follicles. These results indicated the marked differences in follicular fluid composition between preovulatory and cystic follicles in buffalo. Some of the changes were indicative of oxidative stress and disturbed steroidogenesis, two important mechanisms shown to be associated with cystic ovarian disease in various species. Further studies are warranted to investigate whether these differences are directly or indirectly involved in the formation of cystic follicles or are mere manifestations of the condition.

The starting objective of this research communication was to determine the prevalence of subclinical mastitis in buffalo in Turkey. We also seeked to isolate and identify staphylococci, determine their antimicrobial susceptibilities and biofilm-forming abilities as well as investigating the presence of biofilm-related genes and microbial surface components recognizing adhesive matrix molecules. A total of 107 (66.9%) staphylococci (28 S. aureus and 79 coagulase-negative staphylococci, CoNS) were isolated from 160 mastitic milk samples collected from 200 lactating water buffalos. The staphylococci were especially resistant to beta-lactams except for cefoxitin but were less resistant to the other antimicrobials that were tested. Based on the Congo red agar method, 92.9% of the S. aureus and 70.9% of the CoNS isolates were positive for biofilm-forming ability, while all S. aureus and 97.5% of CoNS isolates were positive by a microtiter plate analysis. The presence of icaA and icaD genes was not always correlated with biofilm synthesis, and even in the absence of these genes, the isolates were able to synthesize biofilm.

The aim of this study was to evaluate the intra-observer repeatability of some animal-related variables which could be used in welfare-assessment protocols at farm level. Recordings were performed on seven dairy farms (four for cattle and three for buffaloes). The animals were observed on three occasions at three-week intervals. The variables collected for each animal were behaviour during milking (stepping and kicking), avoidance distance, lameness, and cleanliness. For each farm and each variable, intra-observer repeatability was computed using the Kendall coefficient of concordance (W). A ratio between the variance of the animal and the sum of the latter with the error variance was also calculated using a model of analysis of variance with one factor (animal), to give a further measure of repeatability. These two methods yielded similar results. In particular, in dairy cattle, a high repeatability for avoidance distance, stepping, lameness and cleanliness was observed, whereas for buffaloes avoidance distance and stepping were the repeatable variables.

On-farm welfare-assessment protocols should be based on valid, reliable and feasible indicators which reflect the animal's state in the context of the housing and management system. This paper focuses on the selection of parameters for cattle and buffalo from welfare research, from assessment protocols used in different European countries and from the literature. Three groups of parameters are described: (1) parameters which can readily be included, such as lameness, injuries, body condition score, cleanliness, getting up/lying down behaviour, agonistic social behaviour, oral abnormal behaviours, human behaviour toward the animals and measures of the animal-human relationship; (2) parameters which require more information on reliability, such as indicators of good welfare and housing factors; and (3) parameters which are regarded as important but so far lack reliability in most countries, such as the incidence of clinical diseases and mortality.

Meat from low-volume farm animals such as farmed deer and wild boar is currently widely available to the consumer within the UK. Despite a rapid growth in production of these species there is a paucity of research that focuses on their welfare. This paper reviews the current literature and legislation relating to the transport and slaughter of farmed deer, wild boar, ostriches, buffalo and bison in order to identify priorities for future research. Research on low-volume farm animals has focused on red deer and their welfare is safeguarded with comprehensive legislation and guidelines. Studies have indicated the importance of appropriately designed facilities and sympathetic handling of red deer to ensure humane slaughter with minimal stress. However, the impact of transport and slaughter on the welfare of other deer species has not been recorded and this should be recognised. Much research has been conducted on the welfare of ostriches during transport and at slaughter. However, many of the results are discordant and research is required to clarify, in particular, posture during transport and stunning methods. Research on the welfare of wild boar, buffalo and bison is scarce. Best practice for both transport and slaughter of these species needs further research and clarification within legislation. For the low-volume species discussed in this review, priorities for further research include (1) appropriate methods of handling to minimise stress during transport and slaughter; (2) identification/clarification of appropriate slaughter methods and (3) training of transport and abattoir personnel in handling and slaughter methods.

Adult male and female Murrah buffalo fibroblast cells were used as donors for the production of embryos using handmade cloning. Both donor cells and reconstructed embryos were treated with 50 nM trichostatin-A (TSA) and 7.5 nM 5-aza-2′-deoxycytidine (5-aza-dC). The blastocyst rate of both treated male (40.1% ± 2.05) and female (37.0% ± 0.83) embryos was significantly lower than in untreated control males (49.7% ± 3.80) and females (47.2% ± 2.44) but their apoptotic index was lower (male, control: 5.90 ± 0.48; treated: 4.96 ± 0.31): (female, control: 8.11 ± 0.67; treated: 6.65 ± 0.43) and epigenetic status in terms of global acetylation and methylation of histone was significantly improved. The expression level of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was higher (P < 0.05) and that of PGK, G6PD, OCT 4, IFN-tau and CASPASE3 was significantly lower (P < 0.05) in treated male blastocyst than control and the expression levels of DNMT1, IGF1R and BCL-XL were not significantly different between the two groups. In the female embryos, the relative mRNA abundance of OCT4 was significantly higher (P < 0.05), and that of XIST and CASPASE3 was significantly lower (P < 0.05) in the epigenetic modifier-treated group compared with that of the control group, whereas the expression levels of HPRT, PGK, G6PD, DNMT1, IFN-tau, IGF1R and BCL-XL were not significantly different between the two groups. In both embryos, a similar effect of treatment was observed on genes related to growth and development, but the effect on the expression of X-linked genes varied. These results indicate that not all X-linked genes respond to TSA and 5-aza-dC treatment in the same manner.

The effects of presumed long distance transport on blood parameters were examined in cattle and water buffalo exported from India to Bangladesh. The aim was to assess the welfare of the animals in terms of the frequency of dehydration, metabolic depletion and muscle injury or activation, when they reached their destination. The physiological indicators were total plasma protein, serum sodium, plasma glucose, serum non-esterified fatty acids (NEFA) and serum creatine kinase (CK). Ninety-eight cattle and 57 water buffalo were randomly selected from 24 trucks as they arrived at the market between 0700 and 1000h each day during the hot season (April). In the majority of animals, all five blood variables were higher than published normal values. In all the cattle, total plasma protein concentration, serum NEFA concentration and serum CK activity were higher than the normal range and these values were higher than normal in over 84% of the buffalo. Seventy-two percent of all the animals were hypernatraemic. About 90% of the buffalo and 86% of the cattle were hyperglycaemic. There were no differences in total plasma protein and serum sodium between cattle and water buffalo. By contrast, cattle had significantly higher NEFA and CK and lower glucose than water buffalo. It was evident that this long distance export trade was associated with dehydration, lipolysis and muscle injury or activation. It is recommended that both cattle and water buffalo are given adequate feed and water whenever they are off-loaded from vehicles during the course of journeys.

We investigated the effect of different types of mother-young contacts (fenceline, restricted and no contact) on the health, growth performance, behaviour and physiological state of Murrah buffalo calves. A total of 24 calves were allocated to three groups of 8: NCM (no calf–mother contact), RCM (restricted calf–mother contact) and FCM (fenceline calf–mother contact). At three months of age, the FCM calves had a higher average body weight (58.6 ± 1.5 kg) than the RCM (52.8 ± 1.3 kg) and NCM (53.6 ± 1.3 kg) calves (P < 0.05). The average daily gain (ADG) at three months of age was greater in FCM (0.6 ± 0.1 kg/d) than RCM (0.5 ± 0.1 kg/d) and NCM (0.5 ± 0.1 kg/d) calves (P < 0.05). The mean immunoglobulin G (IgG) concentrations were significantly greater on d 7, 28, 42 and d 56 of sampling (P < 0.05 for all comparisons) in FCM than RCM and NCM calves. The mean cortisol levels were highest in the NCM calves followed by RCM and the lowest values were observed in FCM calves on d 0, d 7, d 28, d 42 and d 56, respectively (P < 0.05, for all recorded days). The diarrhoea score and eggs per gram of faeces (EPG) were significantly greater in the NCM group compared to the RCM and FCM groups. The time spent in cross sucking, licking inanimate objects and self-licking was greatest (P < 0.05) in NCM calves, followed by RCM calves, and was almost non-existent in the FCM group on all the recorded weeks. Time spent in backward ear position was greater (P < 0.05) in FCM than RCM and NCM calves. The average time spent in forward ear position was greatest (P < 0.05) in the RCM calves followed by NCM with the lowest time observed in FCM calves. The mean duration of time spent by calves in the asymmetrical and axial ear position differed significantly (P < 0.05) among NCM, RCM and FCM calves, P < 0.05. The full and fenceline buffalo calf–mother contact system showed higher growth rates, lower levels of stress responses, oral stereotypies and eggs per gram of faeces. In addition, ear postures may be used as a dependable, and reliable measure of positive, low arousal emotional states.

Since embryonic stem cells (ESCs) were first identified, significant progress has been achieved. However, the establishment of buffalo ESCs (bESCs) is still unclear. This study was undertaken to explore the effect of the blastocyst stage on the isolation of bESCs. Firstly, our results indicated that the pluripotent genes were mainly expressed at the early stages of blastocyst, and the attachment and colony formation rates of bESCs derived from expanded blastocyst and hatched blastocyst were significantly higher than early blastocyst and blastocyst. In the meantime, bESCs showed positive alkaline phosphatase activity and expressed genes like OCT4, NANOG, SOX2, c-MYC, CDH1, KLF4, and TBX3. Immunofluorescence also confirmed the expression of OCT4, SOX2. Embryoid bodies expressing three marker genes were generated from the differentiation experiment, and fibroblast, epithelial, and neuron-like cells were induced. Moreover, naive-related genes KLF4, TBX3, primed-related genes FGF5, ACTA2 were expressed in the cells, but not REX-1. Immunofluorescence and western blot confirmed the FGF5 expression. Furthermore, bESCs could maintain pluripotency with the signal of LIF and bFGF. In summary, our results indicated that expanded blastocyst and hatched blastocyst are more suitable for bESCs isolation.

Buffalo has many excellent economic traits and it is one of the greatest potential livestock. Compared with cattle, buffalo has poorer reproductivity, it is of great significance to improve the development potential of oocytes. Buffalo oocyte in vitro maturation (IVM) has been widely used in production, but the poor development ability of bovine oocytes IVM limits the development of buffalo reproductivity. Milrinone as a phosphodiesterase inhibitor could affect the maturation of oocytes in goat and mice, but there have been few reported studies in water buffalo. To optimize buffalo oocyte in vitro maturation systems, the effects of phosphodiesterase inhibitor (milrinone) on pre-maturation culture of buffalo oocytes were investigated in this study. Buffalo cumulus–oocyte complexes (COCs) were cultured in medium with different concentrations (0, 12, 25, 50 and 100 mol/l) of milrinone for different times (0, 4, 8, 12, 16, 22 and 24 h). The results showed that the buffalo COCs nuclear maturation process could be inhibited by milrinone (25–100 mol/l) in a dose-dependent manner. The inhibitory effect of milrinone on in vitro maturation of buffalo oocytes did not decrease with the extension of time. This indicated that milrinone can be used as a nuclear maturation inhibitor during the maturation process in buffalo oocytes. In addition, milrinone can inhibit the effect of follicle stimulating hormone (FSH)-induced IVM of buffalo oocytes, but with time FSH partially eliminated the inhibition. Therefore, inhibition of milrinone on the nuclear maturation of buffalo oocytes was reversible, and buffalo oocytes can mature normally after the inhibition is lessened.

The study was undertaken to examine the relative abundance (RA) of the major developmental important candidate genes in different grades of immature oocytes (A-grade, B-grade, C-grade and D-grade) and various stages of in vitro-produced embryos (2-cell, 4-cell, 8–16-cell, morula, and blastocyst) of buffalo using RT-qPCR. Results showed that the RA of GLUT1, CX43, HSP70.1 and GDF9 was significantly higher (P < 0.05) in the A-grade of oocytes than the C-grade and D-grade but did not differ significantly from the B-grade of oocytes. Similarly, RA of BMP15 and Survivin were significantly higher (P < 0.05) in A-grade than the other grades of oocytes, however, poly(A) polymerase expression was not significantly different (P > 0.05) among the immature oocytes. The expression of GLUT1 was significantly higher (P < 0.05) in the blastocysts, but the expression of CX43 (P < 0.05; P > 0.05), HSP70.1 (P < 0.05; P > 0.05) and GDF9 (P > 0.05) was higher at the 2-cell stage than the other stages of embryos. Interestingly, the expression levels of poly(A) polymerase (P < 0.05), BMP15 (P < 0.05; P > 0.05) and Survivin (P > 0.05) were higher at the 8–16-cell stage than the other stages of embryos. It is concluded that A-grade of immature oocytes has shown more mRNA abundance for the major developmental important genes; therefore A-grade oocytes may be considered as the most developmentally competent and suitable for handmade cloning research in buffalo.

Insulin-induced gene 2 (INSIG2) is a recently identified gene that is implicated in the regulation of cholesterol metabolism and lipogenesis in mammals. Although the data in goats emphasizes a role for INSIG2 in milk fat synthesis, the regulatory mechanism in buffalo is not clear. In this study, we analyzed the protein abundance of INSIG2 at peak lactation and dry-off period in buffalo mammary tissue. The results indicated that, relative to the peak lactation, the protein abundance of INSIG2 in the dry-off period was higher. To determine the function of INSIG2 in milk fat synthesis, INSIG2 was overexpressed and knocked down by lentiviral transfection in buffalo mammary epithelial cells (BuMECs). The response to overexpressing INSIG2 included down-regulation of SREBP, PPARG, FASN, ELOVL6, SCD, APGAT6 and TIP47 coupled with a decrease in content of triacylglycerol (TAG). However, in response to knockdown of INSIG2, the significant increase in content of TAG along with marked up-regulation of SREBP, PPARG, FASN, ELOVL6, SCD, APGAT6 and TIP47 suggests that INSIG2 negatively affects milk fat synthesis in BuMECs. No significant difference in mRNA abundance of GPAM and DGAT2 in response to overexpression or interference of INSIG2 indicates that they might also be influenced by other regulatory factors. Taken together, our results provide strong support for the negative effect of INSIG2 on milk fat synthesis in BuMECs.

The present study was undertaken to analyze the relative abundance (RA) of pluripotency-associated genes (NANOG, OCT4, SOX2, c-MYC, and FOXD3) in different grades of immature oocytes and various stages of in vitro-produced buffalo embryos using RT-qPCR. Results showed that the RA of NANOG, OCT4, and FOXD3 transcripts was significantly higher (P < 0.05) in A grade oocytes compared with the other grades of oocytes. The RA of the c-MYC transcript was significantly higher (P < 0.05) in A grade compared with the C and D grades of oocytes, but the values did not differ significantly from the B grade of oocytes. The RA of the SOX2 transcript was almost similar in all grades of the oocytes. The expression levels of NANOG (P > 0.05), OCT4 (P > 0.05), c-MYC (P > 0.05) and SOX2 (P < 0.05) were higher in the blastocysts compared with the other stages of the embryos. Markedly, FOXD3 expression was significantly higher (P < 0.05) in 8–16-cell embryos compared with the 2-cell and 4-cell embryos and blastocyst, but did not differ significantly from the morula stage of the embryos. In the study, the majority of pluripotency-associated genes showed higher expression in A grade immature oocytes. Therefore, it is concluded that the A grade oocytes appeared to be more developmental competent and are suitable candidates for nuclear cloning research in buffalo. In buffalo, NANOG, OCT4, SOX2, and c-MYC are highly expressed in blastocysts compared with the other stages of embryos.

This study examined the effects of zinc chloride (ZnCl2) and sodium selenite (Na2SeO3) supplementation in maturation medium on in vitro maturation (IVM) rate, oxidative biomarkers and gene expression in buffalo oocytes. Ovaries from a slaughterhouse were aspirated and good quality cumulus–oocyte complexes (COCs) with at least four layers of compact cumulus cells and evenly granulated dark ooplasm were selected. COCs were randomly allocated during IVM (22 h) to one of four treatment groups: (1) control maturation medium (basic medium), or basic medium supplemented with (2) ZnCl2 (1.5 µg/ml), (3) Na2SeO3 (5 µg/l), or (4) ZnCl2 + Na2SeO3 (1.5 µg/ml + 5 µg/l, respectively). Oocytes were denuded after 22 h of IVM in the first four replicates. Specimens were fixed and stained to evaluate the stage of nuclear maturation. The spent medium was collected for biochemical assays of total antioxidant capacity (TAC), malondialdehyde (MDA) and hydrogen peroxide concentrations. A second four replicates were used for COCs for RNA extraction. The expression levels of antioxidant (SOD1, GPX4, CAT and PRDX1), antiapoptotic (BCL2 and BCL-XL) and proapoptotic (BAX and BID) genes were measured. Supplementation with ZnCl2 and Na2SeO3 during IVM increased the ratio of oocytes reaching metaphase II at 22 h, increased TAC and decreased MDA and H2O2 concentrations in the maturation medium (P < 0.05). Moreover, beneficial effects were associated with complementary changes in expression patterns of antioxidative, antiapoptotic and proapoptotic genes, suggesting lower oxidative stress and apoptosis. Supplementation medium with zinc chloride and sodium selenite improves the maturation rate, reduces oxidative stress and increases expression levels of antioxidative and antiapoptotic genes.

Somatic cell nuclear transfer (SCNT) holds vast potential in agriculture. However, its applications are still limited by its low efficiency. Histone 3 lysine 9 trimethylation (H3K9me3) was identified as an epigenetic barrier for this. Histone demethylase KDM4D could regulate the level of H3K9me3. However, its effects on buffalo SCNT embryos are still unclear. Thus, we performed this study to explore the effects and underlying mechanism of KDM4D on buffalo SCNT embryos. The results revealed that compared with the IVF embryos, the expression level of KDM4D in SCNT embryos was significantly lower at 8- and 16-cell stage, while the level of H3K9me3 in SCNT embryos was significantly higher at 2-cell, 8-cell, and blastocyst stage. Microinjection of KDM4D mRNA could promote the developmental ability of buffalo SCNT embryos. Furthermore, the expression level of ZGA-related genes such as ZSCAN5B, SNAI1, eIF-3a, and TRC at the 8-cell stage was significantly increased. Meanwhile, the pluripotency-related genes like POU5F1, SOX2, and NANOG were also significantly promoted at the blastocyst stage. The results were reversed after KDM4D was inhibited. Altogether, these results revealed that KDM4D could correct the H3K9me3 level, increase the expression level of ZGA and pluripotency-related genes, and finally, promote the developmental competence of buffalo SCNT embryos.

This study was carried out to compare the efficacy of different methods to activate buffalo A + B and C + D quality oocytes parthenogenetically and to study the in vitro developmental competence of oocytes and expression of some important genes at the different developmental stages of parthenotes. The percentage of A + B oocytes (62.16 ± 5.06%, range 53.8–71.3%) was significantly higher (P < 0.001) compared with that of C + D oocytes (37.8 ± 5.00%, range 28.6–46.1%) retrieved from slaughterhouse buffalo ovaries. Among all combinations, ethanol activation followed by culture in research vitro cleave medium gave the highest cleavage and blastocyst yields for both A + B and C + D grade oocytes. Total cell numbers, inner cell mass/trophectoderm ratio and apoptotic index of A + B group blastocysts were significantly different (P < 0.05) from their C + D counterpart. To determine the status of expression patterns of developmentally regulated genes, the expression of cumulus–oocyte complexes, fertilization, developmental competence and apoptotic-related genes were also studied in parthenogenetically produced buffalo embryos at different stages, and indicated that the differential expression patterns of the above genes had a role in early embryonic development.