Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: Cloning efficiency,growth in suspension,and selection of drug-resistant mutant phenotypes

Summary Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured in reduced serum with or without additives (1 μg/ml insulin, 3×10⁻⁷ M linoleic acid, 1×10⁻⁸ M H₂SeO₃) or bovine serum albumin (BSA, 1% wt/vol). With the additives and ≤0.5% fetal bovine serum (FBS), Ham’s F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle’s minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology. In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5% FBS; in Ham’s F12, 1% FBS+deoxycytidine+BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at theaprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethyl-nitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels. This work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under Contract W-7405-ENG-48 and supported by the Environmental Protection Agency under Interagency Agreements IAG-D5-E681-AN and AO.     

Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos.

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved basal medium for Y-1 mouse adrenal cortex tumor cells in culture. I. Dependence of growth and steroid response on calcium ion concentration.

An improved basal medium is presented that required only minimal supplementation with dialyzed fetal bovine serum or bovine serum albumin and fetuin to be comparable to Ham's F-10, which requires 15% horse serum (HS) and 2.5% fetal bovine serum (FBS) for the growth and function of Y-1, mouse adrenal cortex tumor, cells. Cell monolayers maintained for up to 2 weeks without any protein supplementation have retained their steroid response to ACTH. The medium differs from Ham's F-10 in its buffer composition and higher calcium-ion concentration. This medium should be a useful adjunct to studies pertaining to steroid and lipid intermediary metabolism, the retention of a specialized physiological function in a chemically defined medium, and the mechanism of hormonal response.     

In vitro maturation and fertilization of goat oocytes

Goat oocytes were isolated from 3-5 mm diam. follicles. The oocytes with compact cumulus mass were matured and fertilized in vitro. Three different media, viz. modified Krebs-Ringer bicarbonate, Dulbecco's and Ham's F-12 with three different additives (bovine serum albumin, BSA; follicle stimulating hormone, FSH and fetal calf serum, FCS) were tested. The three basal media gave almost similar results with Ham's F-12 being slightly better. Addition of BSA (10 mg/ml) increased the rates of maturation and penetration. FSH + BSA (2.5 micrograms/ml + 10 mg/ml) further enhanced the rates while FCS (10%) proved to be even more effective. In modified Krebs-Ringer bicarbonate and Dulbecco media with additives FCS + BSA, around 60% oocytes matured to metaphase II of which 53% were penetrated by capacitated goat spermatozoa while in F-12 medium 70% reached metaphase II and 63% were penetrated. Ham's F-12 medium with additives FCS + BSA was slightly better for maturation and penetration of goat oocytes in comparison to two other media tested.     

Improved basal medium for Y-1 mouse adrenal cortex tumor cells in culture

Summary An improved basal medium is presented that requires only minimal supplementation with dialyzed fetal bovine serum or bovine serum albumin and fetuin to be comparable to Ham's F-10, which requires 15% horse serum (HS) and 2.5% fetal bovine serum (FBS) for the growth and function of Y-1, mouse adrenal cortex tumor, cells. Cell monolayers maintained for up to 2 weeks without any protein supplementation have retained their steroid response to ACTH. The medium differs from Ham's F-10 in its buffer composition and higher calcium-ion concentration. This medium should be a useful adjunct to studies pertaining to steroid and lipid intermediary metabolism, the retention of a specialized physiological function in a chemically defined medium, and the mechanism of hormonal response. Supported by the Medical Research Service of the Veterans Administration.     

Calcitonin-responsive clonal cell line from porcine kidney (PK(15)) in serum-free medium

PK(15), a homogeneous epithelial cell line from porcine kidney which was originally established through single cell cloning from PK-2a, was found to respond to [Asu1,7]eel calcitonin with an increase in adenosine 3':5'-cyclic monophosphate (cAMP) content, but do not respond to parathyroid hormone or arginine vasopressin. These cells were able to grow in a synthetic medium (a 1:1 mixture of Dulbecco's modified Eagle's MEM and Ham's F12 medium) without any supplementary factor. The medium supplemented with selenous acid, transferrin, and insulin permitted a growth rate equivalent to those in serum containing medium. When grown in the serum-free defined medium, these cells showed an increase in cAMP content in response to [Asu1,7]eel calcitonin to approximately the same degree as in the serum containing medium (10% fetal calf serum). Our present study first indicates that PK(15) cells are capable of growing in the serum-free defined medium retaining the calcitonin responsiveness of the original cells.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Selective growth and serial passage of mouse melanocytes from neonatal epidermis in a medium supplemented with bovine pituitary extract

Suspensions of disaggregated epidermal cells from skins of newborn C57BL/10JHir mice were plated in a growth medium that consisted of Ham's F-10 plus bovine pituitary extract (BPE), insulin, and transferrin. Fetal bovine serum (FBS) was added to the culture medium at a concentration of 4% at the time of plating. On the second day of culture, a small number of melanocytes was randomly distributed among large sheets of keratinocytes. From the third day onward, FBS was excluded from the culture medium to prevent the proliferation of keratinocytes and fibroblasts. The melanocytes began to grow preferentially, and after 12 days pure and enriched populations of melanocytes could be harvested. In the absence of the proliferation of keratinocytes and fibroblasts, melanocytes could be serially passaged in the growth medium supplemented with a conditioned medium (CM) prepared from keratinocyte-enriched cultures, namely, those at the early stages of the primary culture. FBS was added at a concentration of 1% for the first day. These results suggest that both BPE and keratinocyte CM contain growth factors required for proliferation of melanocytes.     

Modification of MCDB 110 medium to support prolonged growth and consistent high cloning efficiency of diploid human fibroblasts

In preparation for studies on the growth factor requirements of normal and transformed human fibroblasts, we have developed a serum-free medium that supports vigorous long-term serial subculture of diploid human fibroblasts and allows them to form large-sized colonies with high efficiency (40 to 60%) when plated at cloning density (2 to 5 cells/cm2). This medium, which is a modification of Ham's MCDB 110 base medium with its serum replacement supplements, is relatively easy to prepare and the cost of the serum replacements is approximately the same as that of fetal bovine serum supplied at 10%. The ingredients of "Supplement B" of MCDB 110 medium were added in an ethanol solution, rather than in the form of liposomes, and were combined with bovine serum albumin (0.5%), a lipid carrier. Gelatin and fetuin were included as attachment factors instead of polylysine. Bioassays indicated that none of the ingredients in the medium were contaminated with either epidermal growth factor or platelet-derived growth factor. In this modified serum-free medium, which we have designated McM+SR1, diploid human fibroblasts grew for 21 days at the same rate as in the base medium, McM, supplemented with 10% FBS (i.e., 21 population doublings). During the next 20 days, they underwent 15 population doublings which was 75% of the rate of cells growing in the medium containing serum.     

Effects of oxygen tension and supplements to the culture medium on activation and development of bovine follicles in vitro

Our laboratory developed a method for culturing small pieces of bovine and baboon ovarian cortex, rich in primordial follicles, that supports the initiation of follicle growth and development to the primary stage. However, only a few follicles progressed to the secondary stage. The purpose of the current experiments was to determine if changes in culture conditions, specifically oxygen concentration and supplements to the culture medium, would facilitate the primary to secondary follicle transition. In Experiment 1, ovarian cortical pieces from late-gestation bovine fetuses were cultured with 2, 5, 20, or 60% oxygen in Waymouth's medium plus ITS+ (insulin, transferrin, selenium plus linoleic acid and BSA). Although the three lower concentrations of oxygen were generally equivalent in promoting follicle activation and growth, the highest concentration (60%) had deleterious effects on follicle survival after 7 days in culture, reducing the number of healthy follicles to about 35% of the number observed with 20% oxygen (P<0.05). In Experiment 2, bovine ovarian cortical pieces were cultured in the standard gas mixture (5% CO(2) in air) with graded doses of fetal bovine serum (FBS, 2.5, 5, or 10%) in the presence or absence of 0.5 or 1x ITS+. All concentrations of FBS alone were much less effective at maintaining follicular health and supporting the initiation and progression of follicular growth than was ITS+. However, 5 and 10% FBS alone increased the percentage of healthy primordial and primary follicles by about twofold (P<0.05) in the absence of ITS+ and in the presence of 0.5x ITS+, they enhanced the primary to secondary follicle transition by 10- and 9-fold, respectively. Thus, of the culture conditions evaluated, 20% oxygen and medium containing 0.5x ITS+ plus 5% or 10% FBS were the most effective for promoting follicular health and development.     

Contribution of exogenous progesterone to human adrenal cortisol synthesis in vitro: a comparison of early gestation fetal and adult tissues

The ability of human adult adrenal to utilize progesterone (P4) for cortisol (F) synthesis in vitro has been compared with that of fetal adrenal tissue. Explant cultures were studied for 6 days using Ham's F10 medium supplemented with 10% fetal bovine serum (FBS), with or without added P4 as substrate. Short-term (4h) incubations of fresh tissue minces were carried out in Ham's F10, with or without P4, in the absence of FBS. In contrast with the fetal gland, F production by cultured adult tissue was unaffected by addition of P4. During short-term incubations, P4 increased F production 150-fold in the fetal tissue as compared to 2- to 4-fold in the adult. ACTH had no acute effect on the P4 to F conversion in either tissue. These results demonstrate that the fetal adrenal exhibits a greater ability to utilize P4 for F production than the adult adrenal.     

Improved in vitro development of porcine embryos with different energy substrates and serum

The effect of replacing 5.5 mM glucose in North Carolina State University (NCSU)-23 medium with 0.5 mM pyruvate/5.0 mM lactate on porcine IVF embryo development was investigated in Experiment 1. Culturing embryos with pyruvate/lactate for 7 days or with pyruvate/lactate from Days 0 to 2, and then glucose from Days 2 to 7 improved cleavage rates. In Experiment 2, embryos were cultured for 7 days in pyruvate/lactate containing NCSU-23 medium supplemented with 0.05% PVA, 0.4% BSA or 10% fetal bovine serum (FBS). The BSA supplement increased the rates of cleavage, blastocyst formation, and the number of total cells in blastocysts. In Experiment 3, embryos were cultured in pyruvate/lactate containing NCSU-23 medium supplemented with 0.4% BSA for 7 days (BSA-PL), 0.4% BSA from Days 0 to 4 and then 10% FBS from Days 4 to 7 (BSA-PL-->F ) or 0.4% BSA from Days 0 to 7 with addition of 10% FBS (BSA-PL + F ) at Day 4. More blastocysts in BSA-PL--> F and hatching or hatched blastocysts in BSA-PL-->F and BSA-PL+F were obtained. Total cell number in blastocysts derived from BSA-PL-->F and BSA-PL+F were increased. Our results demonstrated that supplementing pyruvate/lactate containing NCSU-23 medium with 0.4% BSA for 4 days and replacing it with 10% FBS for another 3 days improved porcine IVF embryo development.     

Induction of chromosomal aberrations in cultured Chinese hamster cells by short-term treatment with cadmium chloride

Inducibility of chromosomal aberrations and cytotoxicity in cultured Chinese hamster cells by cadmium chloride (CdCl2) was investigated under 3 different treatment conditions: (i) 2-h treatment in MEM medium supplemented with 10% fetal bovine serum (MEM + 10% FBS) or (ii) in HEPES-buffered Hanks' solution (HEPES-Hanks), and (iii) continuous treatment for 24 h in MEM + 10% FBS. Two-h treatment with CdCl2 in HEPES-Hanks or continuous treatment for 24 h in MEM + 10% FBS was respectively 2 or 3 times more cytotoxic than 2-h treatment with the metal in MEM + 10% FBS. Continuous treatment for 24 h with a CdCl2 concentration in excess of 5 X 10(-6) M was too toxic to the cells to allow chromosomal analysis, and moreover, only a slight increase in incidence of chromosomal aberrations was observed at a concentration of 5 X 10(-6) M CdCl2. In contrast, a marked and concentration-dependent increase in incidence of chromosomal aberrations was observed after post-treatment culture for 22 h follows 2-h treatment with 1 X 10(-6) M to 5 X 10(-5) M of CdCl2 in both MEM + 10% FBS and HEPES-Hanks. Two-h treatment with cadmium in HEPES-Hanks was approximately 3 times more potent for the induction of chromosomal aberrations than that in MEM + 10% FBS. Types of aberrations induced by CdCl2 mainly consisted of chromatid gaps and breaks, although a few exchanges, dicentrics and fragmentations were observed at high concentrations of cadmium. Increase in incidence of tetraploidy was also observed with a concentration dependency after 2-h treatment with CdCl2. Potency of CdCl2 to induce chromosomal aberrations after 2-h exposure was comparable to that of benzo[a]pyrene activated with S9 at equitoxic concentrations. Two-h treatment with cadmium markedly inhibited incorporation of [3H]thymidine, even at concentrations at which incorporation of [3H]uridine or [3H]leucine was less inhibited. However, the inhibition of [3H]thymidine incorporation by cadmium was reversible and the incorporation restored to the control level during 2-6 h of post-treatment incubation. These findings suggest that restoration of DNA synthesis after cadmium exposure is required for the efficient detection of chromosomal aberrations induced by the metal.     

Mitogenic activity of fetal bovine serum, fish fry extract, insulin-like growth factor-I, and fibroblast growth factor on brown bullhead catfish cells--BB line

Bioassays were performed to assess the effects of different levels of growth medium supplementation with fetal bovine serum (FBS), fish fry extract (FE), combinations of FBS and FE, and addition of insulin-like growth factor I (IGF-I) and fibroblast growth factor (FGF) on the proliferation of brown bullhead catfish cells (BB line). Treatments (n = 4) were: 2.5, 5, 10, and 15.0% FBS or FE and 5/2.5, 5/5, 10/2.5, and 10/5 of a FBS/FE combination as supplement to the growth medium, or the addition of 0.1, 1, 2.5, 10, 25, and 75 ng/ml of either IGF-I or FGF to the growth media. Initial cell density was 1.1 x 10(6) cells per well on uncoated 24-well plates. Incubation temperature was 29.5 +/- 0.7 degrees C. Six hours after plating, initial culture medium was removed, plates rinsed with Dulbecco's phosphate buffered saline, treatment media added, and cells allowed to proliferate for 24 hours. Another bioassay was performed with rat myoblast omega cells (RMo) using the same levels of growth medium supplemented with FBS, FE and FBS/FE. Base growth medium was Dulbecco's MEM. The initial cell density was 7.2 x 10(6) cells per well, and the bioassay was carried out at 36.0 +/- 0.5 degrees C, on a 95% air, 5% CO2 incubator. Increasing levels of FBS had a positive effect (P < 0.05) on the proliferation of both BB and RMo cells. Increasing levels of FE had a negative effect (P < 0.05) on the proliferation of BB cells and totally inhibited the proliferation of RMo cells at any level of supplementation. Higher levels of FE on the FBS/FE combinations presented a negative effect on the proliferation of both BB and RMo cells (P < 0.05). Insulin-like growth factor I had a positive quadratic effect (P < 0.05) on the proliferation of BB cells. Apparently, mammalian growth factors slightly stimulated mitogenic activity in fish cells, while FE contained factors which inhibited the mitogenic activity of RMo and BB cell lines.     

Successful pregnancy after transfer of rabbit blastocysts grown in vitro from single-cell zygotes

To establish successful pregnancy in rabbits after the transfer of blastocysts cultured in vitro for 72 h, pregnancy rates were compared according to synchronization methods of recipient and embryo transfer sites. Also, the effect of RDH (1:1:1 mixture of RPMI, DMEM and Ham's F10) medium with additives such as BSA and taurine was evaluated for developmental capacity and cell number. Developmental capacity and cell number were considered important for implantation. When we evaluated the relative survival of rabbit one-cell embryos after culture in Ham's F10, in RD or in RDH for 72 h, embryos cultured in RDH and RD developed much better than in Ham's F10. When the effects of BSA and taurine in RDH medium were tested for rabbit embryo development, BSA or taurine promoted transition to the blastocyst stage and increased cell numbers of cultured embryos in RDH medium. The BSA and taurine together in RDH medium had a synergistic effect on embryo development. By transferring cultured blastocysts to the oviduct of the recipient doe synchronized one day behind the donor, live-born pups were obtained successfully. These results demonstrated that rabbit blastocysts can develop to normal pups after in vitro culture and embryo transfer.     

Partial differentiation of rat egg cylinders in serum-free and protein-free medium

Modified organ cultures of rat egg cylinders were grown for 2 weeks in Eagle's MEM without serum or with serum added at different times. Explant survival was decreased only in cultures grown for the entire 2 weeks in serum-free medium, whereas the explant growth was impeded in all but the cultures grown for 2 weeks in 50% MEM plus 50% serum. Differentiation of epidermis and cartilage in the cultures deprived of serum for the entire 2-week period was comparable to that in fully serum-supplemented medium, whereas other differentiated tissues were rare or absent. In explants cultivated without serum for only the first week, neuroblasts were scarce.     

Serum and alpha 2-macroglobulin induce transient hyperpolarizations in the membrane potential of an osteoblastlike clone

Using microelectrode techniques, we have observed that the application of serum or alpha 2-macroglobulin (alpha 2M) induces transient hyperpolarizations in the membrane potential of a rat osteosarcoma clone (ROS 17/2). Hyperpolarizations arose from activation of Ca2+-dependent K+ channels by transient increases in the concentration of intracellular free Ca2+. Hyperpolarizing spikes were observed for several h following the addition of fetal bovine serum (FBS) to cell cultures. Application of small volumes of FBS or alpha 2M rapidly induced synchronized bursts of hyperpolarizing spikes. No response was elicited by serum-free medium, latex beads, or bovine serum albumin (BSA). Immunofluorescence labeling patterns were consistent with the receptor-mediated endocytosis of alpha 2M but not BSA. The ligand specificity and kinetics of these hyperpolarizations suggest that they are associated with a receptor-mediated event, possibly an early stage of receptor-mediated endocytosis.     

Cryopreservation of chum salmon blastomeres by the straw method

In order to preserve genetic resources of chum salmon, Oncorhynchus keta, optimum conditions for cryopreservation of isolated blastomeres were investigated. Survival rates under various conditions were compared: the nature and the concentration of cryoprotectants before and after freezing, the seeding temperature, and the developmental stages of donor embryos. Isolated blastomeres immersed for 30 min in Eagle's MEM containing both a cryoprotectant and 10% fetal bovine serum (FBS) at 10 degrees C were transferred into a straw and frozen at 1 degrees C/min to -30 degrees C by a programmable freezer before being plunged into liquid nitrogen. Ice seeding was carried out at -5 to -15 degrees C. Frozen blastomeres were thawed in water at 15 degrees C. Blastomeres cryopreserved with MEM containing 10% dimethyl sulfoxide (Me(2)SO) and 10% FBS (10% Me(2)SO/MEM10) showed higher survival rates than those cryopreserved with MEM containing 10% FBS and 10% glycerol, ethyleneglycol, 1, 2-propanediol, or sucrose. Blastomeres treated with 10% Me(2)SO/MEM10 showed higher survival rates than those treated with MEM containing only 10% Me(2)SO. Blastomeres seeded above -10 degrees C showed higher survival rates than non-seeded ones. Frozen blastomeres at advanced stages demonstrated high survival rates. Blastomeres cryopreserved under optimum conditions showed survival rates of 59.3+/-2.8%. These results indicate that 10% Me(2)SO/MEM10 is a suitable cryoprotectant medium to cryopreserve chum salmon blastomeres, that seeding should be carried out above -10 degrees C on pre-freezing, and that blastomeres at the blastula stage should be used as material.     

Primary culture and maintenance of steroid-secreting human placenatal monolayers.

A simple method is described for primary culture and for maintenance of hormone-producing cells from normal human placenta. A consistent yield of cells was obtained and an average survival of 3 to 4 months in culture using 1 mm3 explants from the most vascular area of the placentas. These explants were placed in a variety of culture media in 30 ml flasks and incubated at 37 degrees C in an atmosphere of 5% CO2 and 95% air. The best yields in terms of cell growth were observed with Eagle's MEM (minimum essential medium) with supplements of horse serum and fetal calf serum or human cord serum. (Ham's F-10 with supplement of horse serum and fetal calf serum supports growth for the longest period and media containing human cord serum had the best yield of steroids.     

Replacement of serum and hormone additives with follicular fluid in the IVM medium: effects on maturation, fertilization and subsequent development of buffalo oocytes in vitro

Buffalo follicular fluid was used in the IVM medium in place of serum and hormone additives for stimulating nuclear and cytoplasmic maturation of buffalo oocytes in vitro. Follicular fluid (buFF) was aspirated from visible surface follicles from buffalo ovaries. Cumulus oocyte complexes (COCs) were matured for 24 to 26 h at 38.5 degrees C, 5% CO(2) in air in the maturation medium (TCM-199). When used, the concentration of fetal bovine serum (FBS) was 10% and that of FSH-P was 5 mug/ml. In Experiment 1 TCM-199 was supplemented with 1) FBS, 2) FBS + FSH-P, 3) 20% buFF and 4) 40% buFF. The matured oocytes were denuded and stained with Giemsa stain to study nuclear maturation. The proportion of oocytes which completed nuclear maturation was similar in medium containing FSH (74%) and 20 or 40% buFF (67%), which was higher (P < 0.05) than in medium with FBS but without FSH or buFF (47%). In Experiment 2, which was aimed at examining the effects of buFF on cumulus expansion and rates of fertilization and subsequent development to the blastocyst stage after IVF, the maturation medium was supplemented with 1) FBS + FSH-P, 2) 20% buFF and 3) 40% buFF. The COCs matured in medium containing 20 or 40% buFF had significantly higher (P < 0.01) cumulus expansion than those matured in medium with FBS + FSH-P. Of the COCs matured in medium with FBS + FSH-P and 20 or 40% buFF, the fertilization rates indicated by the incidence of cleavage (56, 51 and 52%, respectively) and the proportion of cleaved COCs developing to morula (58, 54 and 57%, respectively) and blastocyst stage (30, 31 and 35%, respectively) were not significantly different. In Experiment 3, supplementation of the maturation medium with 1) FBS + FSH-P and 2) FBS + FSH-P + 20% buFF resulted in similar rates of morulae (41 and 38%, respectively) and blastocysts (31 and 25%, respectively), indicating that simultaneous presence of FBS, FSH-P and buFF did not have an additive effect on embryo yield. The results show that the gonadotropin and serum source in the IVM medium can be replaced by buFF at the 20% level to achieve comparable morula and blastocyst yields.