Culture conditions for blastogenic responses of bovine mammary mononuclear cells

Several experimental parameters were examined to determine optimal conditions for proliferative responses of mammary mononuclear cells (MMC) obtained from six nonlactating dairy cows. These parameters were: pre-incubation of cells in medium prior to assay, mitogen concentration, assay incubation time, and type of culture medium. Response variables included viability of cells and the rate of proliferation as assessed by tritiated thymidine incorporation. Pre-incubation of cells in medium had no effect on the proliferative response of MMC. Whereas Concanavalin A (ConA; 3.3 or 6.6 micrograms/ml) and phytohemagglutinin (PHA; 1, 5, 10 micrograms/ml) did stimulate proliferation of MMC, the higher doses did not stimulate greater proliferation than the lower doses of mitogens. The greatest mitogenic response was obtained on days 2 and 3 of incubation. Proliferative responses were significantly higher at all mitogen levels tested in a 50-50 mixture of Rosewell Park Memorial Institute medium 1640 and Liebovitz-15 medium (RPMI/L-15) than in RPMI alone. Viability of MMC was also significantly higher in the RPMI/L-15 medium. To test whether the significant effect of media on blastogenesis was specific for mononuclear cells from the bovine mammary gland, peripheral blood lymphocytes (PBL) from four dairy cows were cultured with ConA and PHA in a mitogen assay in both RPMI and RPMI/L-15. Viability was measured on day of collection and on all culture days. PBL were stimulated equally in both media. PBL viability decreased significantly on day 1 in both RPMI and RPMI/L-15. These results suggest that the optimal culture conditions for blastogenic responses of mammary mononuclear cells and peripheral blood lymphocytes may differ.     

Induction of blastogenesis in bovine peripheral blood lymphocytes by oxidation with sodium periodate

Suitable treatment and culture conditions are defined for the induction of blast transformation in bovine peripheral blood lymphocytes by oxidation with sodium metaperiodate (NaIO4). Stimulation with NaIO4 required slight modification of techniques used routinely for activation of lymphocytes in vitro with lectins and antigens. Gradient-separated mononuclear leukocytes responded with maximal [3H]TdR incorporation after oxidation with 0.50 to 1.0 mM NaIO4 for 30 minutes at 25 C. Oxidized cells cultured at 1 to 2 X 10(6)/ml responded better than cells cultured at any other concentration, when compared with untreated cells. Blastogenesis in response to oxidation reached its maximum rate within 48 hours of treatment, after which it declined rapidly. Partial removal of glass wool-adherent cells reduced periodate-triggered blastogenesis by 95%, but did not significantly affect activation with phytohemagglutinin, concanavalin A, pokeweed mitogen, or purified protein derivative. Reintroduction of macrophages restored responses to their precolumn level. Oxidation with NaIO4 provided a simple, rapid means of inducing blastogenesis in bovine lymphocytes. Manipulation of the well-defined triggering conditions may help to explain the mechanisms involved in lymphocyte activation.     

Culture requirements of Ascaris suum larvae using a stationary multi-well system: Increased survival,development and growth with cholesterol

Ascaris suum third-stage larvae (L₃) were converted from infected rabbits and cultured in a stationary multi-well plate system. Several different culture media including Mediuim 199, NCTC-135, Minimum Essential Medium-Eagle, Dulbecco's Modified Eagle's Medium, RPMI 1640, McCoy's 5A and Neuman-Tytell mdium were tested to determine which was best for overall larval survival, development and growth. Larvae developed from L₃ to fourth-stage (L₄) in all media tested, but larval survival and the yield and growth of L. were superior in RPMI 1640. Pyruvate is an important medium component because its addition to RPMI 1640 enhanced the yield and growth of L₄ while its removal from DMEM reduced larval survival and the yield and growth of L₄. Cholesterol markedly enhanced larval survival and the yield and growth of L₄ when added to RPMI 1640 as either a soluble supplement in Tween 80 or to a lesser degree, as a component of liposomes. The multi-well culture plate system is a convenient method for determining the effects of different media and changes in media composition on larvae in vitro.     

Assessment of lymphocyte function during vitamin A deficiency

Two variables of immune status were measured in vitamin A-deficient (A-), vitamin A-sufficient pair-fed (A + PF), and healthy control (A+) rats to examine the mechanisms of immune regulation by vitamin A. Parallel samples were run in bovine fetal serum (BFS)-containing medium and serum-free medium to control BFS-origin vitamin A in the cultures. Splenocytes derived from A- rats showed significantly depressed blastogenic response to all lectins tested in both mediums when compared with responses of A + PF and A+ rats. The splenocyte blastogenic response of A + PF rats was significantly lower than that of A+ (control) rats only when cultured in BFS medium and stimulated by lentil lectin, lipopolysaccharide, or concanavalin A mitogens. Thymic lymphocyte blastogenic transformation assays were equivocal. Splenic immunosuppression could not be linked to significant reductions in surface glycoprotein receptor availability, since splenocytes of A- rats, as well as thymocytes, were capable of binding fluorescein isothiocyanate-conjugated lectins in the same capacity as splenocytes of A+ and A + PF rats. The combination of depressed cellular function and adequate lectin binding to viable cells implies that the regulation of immunohomeostasis by vitamin A was achieved through intracellular mechanisms, possibly selective genomic expression.     

In vitro stimulation of bovine milk lymphocytes standardization of the assay for bovine brucellosis

Lymphocytes of bovine milk origin were investigated by immunostimulation in vitro to standardize the assay for measuring the immune responses of the cells which might be useful in further understanding the immunopathology and diagnosis of bovine brucellosis. The lymphocytes were separated from whole freshly collected milk by centrifugation. The pellet of lymphocytes was washed in RPMI-1640 medium, cultured at different concentrations for different days and with Brucella abortus soluble antigen strain 1119-3 and Concanavalin A. Each culture was labelled with 1.0 μCi of methyl-[³H]thymidine 16–18 hours prior to termination of incubation at 37 C. Termination was done by cooling to 4 C. The cells were harvested for liquid scintillation counting spectrometry. In the groups of calfhood vaccinated cows and nonexposed milkers, a milk lymphocyte concentration of 2.0 × 10⁶/ml of medium yielded a statistically significant blastogenesis. The Brucella abortus soluble antigen concentration of 4.4 μg of protein/well was found optimal to induce significant immunostimulation. A period of 4 days of incubation of the milk lymphocyte in the test was found optimal in inducing statistically significant blastogenesis in this system.     

Specific in vitro lymphocyte response of chickens injected with killed Mycobacterium tuberculosis.

An in vitro microassay for lymphocyte transformation, using 3H-thymidine incorporated into avian peripheral blood leukocytes, is described. The transformation responses of 1 X 10(6) leukocytes stimulated by the nonspecific mitogen, phytohemagglutinin-M (PHA-M), were equivalent with 68- and 92-hour incubation durations. The PHA-M in concentration of 50 micronl/ml produced greater stimulation than did that of 25 or 100 micronl/ml. The transformation response to PHA-M was significantly affected by the lot of bovine fetal serum used to supplement the RPMI 1640 culture medium. The specific antigen, purified protein derivative (PPD) of tuberculin, stimulated significant transformation in cultures of 1 X 10(6) leukocytes from Mycobacterium tuberculosis-sensitized fowl. The PPD at concentration of 50 microng/ml was superior to 100 or 150 microng/ml, and 68-hour incubation was significantly better than 92-hour incubation. The magnitude of the in vitro transformation response to PPD was greater at 6 weeks after sensitization than at 2 or 4 weeks, and it was not directly related to the magnitude of the in vivo wattle test response in sensitized chickens.     

Addition of D-penicillamine,hypotaurine, and epinephrine (PHE) mixture to IVF medium maintains motility and longevity of bovine sperm and enhances stable production of blastocysts in vitro

The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF) that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 10⁶ cells/ml) on fertilization and blastocyst developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 were obtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 10⁶ cells/ml using sperm from 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at 12 h after IVF with a sperm concentration of 1 × 10⁶ cells/ml. Moreover, high linearity, high flagellar beat cross frequency, and low amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combination of the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes. Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase of cAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF medium at a sperm concentration of 2 × 10⁶ cells/ml, we can achieve stable normal fertilization and blastocyst development in any bull.     

Effect of serum on lymphocyte blastogenesis I. Basic characteristics of action by diseased dog serum

The immunoregulatory effect of serum on phytomitogen-induced lymphocyte blastogenesis was studied in 4 sera from diseased dogs and 1 serum from a clinically healthy dog. The results indicated that: (1) Each of the diseased animals responded to the given infection with a specific pattern of blastogenesis inhibition. (2) The blastogenesis suppression $\text{in vitro}$ was proportional to the content of the suppressive serum in the medium. (3) A simultaneous presence of the mitogen and the suppressing “serum's lymphocyte immunoregulatory factors” (SLIF) was necessary for inducing blastogenesis suppression. (4) The suppressive sera most probably acted directly on the cells. (5) The final effect of the sera on lymphocyte blastogenesis was a result of an orchestrated action of blastogenesis-supporting, augmenting, and suppressing SLIF cooperating with the mitogen. (6) The suppressive pattern varied with the individual peripheral blood lymphocytes populations used in the test. (7) The blastogenesis-suppressing SLIF was heat-stable, noncytotoxic, and was not or only partially removable by absorption with peripheral blood lymphocytes. (8) The testing of SLIF activities required the use of various animal lymphocytes and a relatively complex setup of mitogens and control serum combinations for correct interpretations.     

Effect of Growth Factors on Bovine Blastocyst Development in a Serum-Free Medium

Shamsuddin, M.: Effect of growth factors on bovine blastocyst development in a serum-free medium. Acta vet. scand. 1994, 35, 141-147. - To investigate the effect of growth factors on pre-implantation development, bovine zygotes, produced by in vitro fertilization (IVF) of in vitro-matured (IVM) oocytes, were cultured in a serum-free medium to which the following growth factors were added one at a time: epidermal growth factor (EGF), acidic fibroblast growth factor (a-FGF), insulin-like growth factor-II (IGF-II), platelet-derived growth factor from human platelets (PDGF), and platelet-derived growth factor-AB, human, recombinant (PDGF-AB). All growth factors were added at a dose of either 10 or 50 ng/ml, except PDGF which was added at a dose of either 5 or 15 ng/ml. The control medium was TCM 199 supplemented with sodium pyruvate (0.25 mmol/1), BSA (10 mg/ml), insulin (5 μg/ml), transferrin (5 μg/ml), and sodium selenite (5 ng/ml). Embryos were cultured for 8 days (day of insemination = Day 0). The mean percentages of first cleavage on Day 2 varied from 67% to 86% and the differences between the 2 doses, or between the control and growth factor- treated groups were not significant (p≥0.13). The effects of the two doses on subsequent development up to the blastocyst stage did not differ either (p≥0.12). There was no stimulatory effect of any of the used exogenous growth factors on embryo development up to the morula or blastocyst stage on Day 7, or blastocyst stage on Day 8. Moreover, medium supplemented with PDGF had fewer blastocysts than the control (p≤0.03). The results indicate that growth factor supplementation may not necessarily increase the yield of blastocysts from bovine IVM-IVF oocytes in a serum-free medium.     

The influence of different doses of α-tocopherol and ascorbic acid on selected oxidative stress parameters in in vitro culture of leukocytes isolated from transported calves

The objective of the study was to assess in vitro the influence of various doses of two different antioxidants, α-tocopherol and ascorbic acid, on protective mechanisms against ROS in white blood cells obtained from calves exposed to transport, and to compare these results with those obtained from non-transported animals.The concentrations of lipid peroxidation products in leukocytes and in the retained medium were assessed by determining the level of ThioBarbituric Acid Reactive Substances (TBARS). Total antioxidant status in the leukocytes and the medium were estimated using a ferric-reducing ability of plasma (FRAP) assay. Leukocyte viability was determined using the trypan blue reduction test.The study demonstrated that after bovine leukocytes (WBC) were incubated in vitro with α-tocopherol and ascorbic acid, peroxidation intensity decreased and total antioxidant capacity increased. The results of the study reveal that these antioxidants in concentrations over 0.1 mg/ml have a major impact on free radical activity on bovine white blood cells and on cell viability during transport of animals.Based on this study, we suggest that incubation of the leukocytes with antioxidants decreases the oxidative stress development, which can be helpful in protection of the immunological cells during bovine respiratory disease.     

Growth and metabolism of murine and bovine embryos in bovine uterine flushing-supplemented culture media.

The aim of this study was to compare the development and metabolic activity of cultured murine and bovine embryos in 2 standard media (HAM F-10 and RPMI) in the presence or absence of bovine uterine flushings. Murine morulae (n = 653) and day 7 bovine embryos (n = 273) were cultured for 18 h or 36 h in either HAM F-10 or RPMI in the presence or absence of bovine uterine flushings. After culture, the development, quality, and metabolic activity (glucose utilization or methionine uptake and incorporation) of embryos was assessed. It was found that HAM F-10 (without uterine flushings) was a more suitable medium than RPMI for optimal development and metabolism of murine and bovine embryos. Poor quality and development, as well as decreased metabolism, were evident after culture of murine embryos in RPMI; in contrast, this medium had no adverse effects on bovine embryos in culture. Supplementation of HAM F-10 with bovine uterine flushings improved the growth of murine embryos and the protein synthesis (as measured by an increased methionine incorporation) for both murine and bovine embryos. However, supplementation with bovine uterine flushings could not overcome deficiencies of an inappropriate medium (RPMI) for murine embryos. Supplementation of a well-defined culture medium with uterine flushings increased metabolism of embryos in culture, and thus might help to increase pregnancy rates after transfer of such embryos to recipient cows.     

Growth of Foot-and-Mouth Disease Virus in Dispersed Tissue Cells: I. Methods of Production

Methods are described for rapid and economical production of large quantities of foot-and-mouth disease virus in stationary cultures of trypsin-dispersed bovine kidney cells in a simple medium. Yields of between 10⁷ and 10⁸ plaque-forming units per milliliter were obtained from serum-free cultures containing approximately a million and a half viable trypsin-dispersed cells per milliliter.

Some of the advantages and disadvantages of these methods of virus production are discussed.

     

Inhibition of in vitro immunocyte function by sera from cattle with bovine leukosis

Most sera from leukaemic cattle inhibited phagocytic activity of normal bovine peripheral polymorphonuclear leukocytes, growth of interleukin 2-dependent bovine T cells and mitogen-induced (phytohaemagglutinin, concanavalin A, pokeweed mitogen, lipopolysaccharide and protein A) blastogenesis of normal bovine lymphocytes. By contrast, antibody-dependent, and spontaneous cell-mediated cytotoxicity were suppressed by only a few sera. The antibody titer against bovine leukaemia virus in these sera correlated with the percent inhibition of lymphocyte blastogenesis. These leukotic sera had no direct cellular cytotoxicity and the inhibitory activity was not lost by dialysis or heat inactivation at 62°C for 30 min. However, the activity was reduced by heating at 80°C for 30 min. Neither the concanavalin A sepharose 4B effluent fraction nor 3.5% polyethyleneglycol-treated serum was found to contain significant lymphocyte-inhibitory activity. Blastogenic transformation of lymphocytes prepared from leukaemic cattle was hardly detectable; however, the mitogen responsiveness of these lymphocytes was improved by a 37°C 1-h preincubation followed by washing.     

The role of insulin, glucagon, dexamethasone, and leptin in the regulation of ketogenesis and glycogen storage in primary cultures of porcine hepatocytes prepared from 60 kg pigs

A study was conducted to elucidate hormonal control of ketogenesis and glycogen deposition in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (54-68 kg) by collagenase perfusion and seeded into collagen-coated T-25 flasks. Monolayers were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the culture period. Hepatocytes were maintained in DMEM/M199 containing 1% DMSO, dexamethasone (10(-6) or 10(-7) M), linoleic acid (3.4 x 10(-5) M), and carnitine (10(-3) M) for 3 days. On the first day of serum-free culture, insulin was added at 1 or 100 ng/ml and glucagon was added at 0, 1, or 100 ng/ml. Recombinant human leptin (200 ng/ml) was added during the final 24 h; medium and all cells were harvested on the third day. Concentrations of acetoacetate and beta-hydroxybutyrate (ketone bodies) in media and glycogen deposition in the cellular compartment were determined. Ketogenesis was highly stimulated by glucagon (1 and 100 ng/ml) and inhibited by insulin. In contrast, glycogen deposition was stimulated by insulin and attenuated by glucagon; high insulin was also associated with a reduction in the ketone body ratio (acetoacetate:beta-hydroxybutyrate). High levels of dexamethasone stimulated ketogenesis, but inhibited glycogen deposition at low insulin. Culture of cells with leptin for 24 h, over the range of insulin, glucagon, and dexamethasone concentrations had no effect on either glycogen deposition or ketogenesis. These data suggest that while adult porcine hepatocytes are indeed sensitive to hormonal manipulation, leptin has no direct influence on hepatic energy metabolism in swine.     

The In Vitro Secretion of Acetylcholinesterase by Adult Stages of Heligmosomoides polygyrus: the Effects of Broad‐Spectrum Anthelmintics

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 ± 0.07 µmol min^?1 l^?1 mg^?1) than for female (0.56 ± 0.04 µmol min^?1 mg^?1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug‐free medium (RPMI medium supplemented with 0.5 % BSA) for 24 h or continuously exposed to the drugs in supplement‐free medium (24 h), the concentration‐ and time‐dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50 % (IC₅₀) relative to drug‐free controls were estimated. The IC₅₀ values ranged from 0.012 µM (IVM) to 2.96 µM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad‐spectrum anti‐nematodal activity is discussed.     

Non-immune erythrocyte rosette formation of bovine peripheral blood and thymus lymphocytes

An E-rosetting reaction is described which gave 92.1%±2.4 (mean±S.D.) E-rosettes with bovine fetal thymocytes and 48.2%±8.4 with with bovine peripheral blood leukocyte (PBL) preparations. Both culture conditions and culture medium were critical factors in obtaining maximal and reproducible E-rosette numbers. Optimum rosette formation occurred when bovine PBL and neuraminidase treated sheep erythrocytes (nSRBC) were reacted in L-15 culture medium supplemented with 10% fetal calf serum (FCS). Other media including 100% FCS, MEM with 10% FCS, and RPMI-1640 with 10% FCS were less satisfactory. Cultural conditions found to be optimal for enumeration of bovine E-rosettes are similar to those reported as optimal for detection of human T cells. The specificity of rosette formation by bovine thymus derived (T) lymphocytes was shown by demonstration of (1) rosettes and surface membrane immunoglobulins (mIg) on different cells in PBL, (2) rosette formation by the majority of fetal thymocytes, and (3) no inhibition of rosette formation by anti-immunoglobulin serum. Using the E-rosette and mIg assays for presumptive bovine T and B lymphocytes, respectively, it was possible to differentiate from 57.5 to 90% (75.2%±9.3) of cells in bovine PBL preparations, and from 90.2 to 97.5% (94.2%±2.1) of cells in bovine fetal thymocyte preparations into T and B cells.     

Ochratoxin A as a suppressor of mitogen-induced blastogenesis of porcine blood lymphocytes

The in vitro effect of ochratoxin A on pig lymphocytes stimulated by the mitogen Concanavalin A was studied by measuring the rates of ³H-thymidine incorporation in DNA.Ochratoxin A inhibited the mitogenic response to Concanavalin A in a dose dependent way. An almost total inhibition was obtained with ≥ 1 mg ochratoxin A/1, approximately 60 % inhibition was produced by 0.5 mg ochratoxin A/1 and approximately 10 % inhibition by 0.06 mg ochratoxin A/1.The immunosuppressive effect of ochratoxin A was not altered much by different contents of bovine serum albumin, 0.1 or 0.3 %, in the cell culture medium.     

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

Effect of insulin,transferrin and selenium and epidermal growth factor on development of buffalo oocytes to the blastocyst stage in vitro in serum-free,semidefined media

The in vitro development of buffalo oocytes up to the blastocyst stage was studied in serum-free, semidefined media containing bovine serum albumin, follicle-stimulating hormone (FSH), insulin, transferrin and selenium (ITS) and epidermal growth factor (EGF). In experiment 1, oocytes aspirated from abattoir-derived ovaries were cultured in eight serum-free, semidefined culture media containing different combinations of these four factors. In experiment 2, the maturation of buffalo oocytes and the development of the embryos were compared in a complex co-culture system and in the serum-free, semidefined media. Supplementation with FSH and EGF significantly (P < 0.05) increased the maturation rates of buffalo oocytes, and the yield of blastocysts was higher (P < 0.05) in media containing EGF and ITS. The yield of blastocysts was lower in the serum-free semidefined media (P < 0.05) than in the complex co-culture system.