Effect of Vancomycin on the Growth of Psittacosis-Trachoma Agents Cultivated in Eggs and Cell Culture

The antibiotic vancomycin was effective in preventing bacterial contamination during studies with psittacosis and trachoma (PT) agents. This antibiotic was not toxic to chick embryos at 80 mg per egg, or to HeLa 229 cells cultivated in a medium containing 3,200 μg/ml of vancomycin; however, it was toxic to LLC-MK2 cells at a concentration of 1,600 μg/ml. Vancomycin did not affect the growth of a variety of PT agents at a concentration of 2 mg per egg or 800 μg per ml of cell culture medium, but it did inhibit the growth of common gram-positive bacterial air contaminants.     

Transport of L-lysine in the fission yeast Schizosaccharomyces pombe

Systems of L-lysine transport in Schizosaccharomyces pombe are not constitutive, as at no phase of growth in a rich medium is lysine taken up. Transport activity appears only after preincubation of harvested cells with glucose or another suitable source of energy. If cycloheximide is added during this preincubation no transport systems are synthesized. After removal of glucose, the activity of the transport system decays with a half-time of 13 min. The transport of L-lysine into S. pombe cells from the stationary phase of growth preincubated for 60 min with 1% D-glucose is mediated by at least two systems, the high-affinity one with a Kt of 26 mumol/l and Jmax of 4.95 nmol/min per mg dry wt., the low-affinity one with a KT of 1.1 mmol/l and Jmax of 11.8 nmol/min per mg dry wt. The transport of lysine mediated by these two systems proceeds uphill. The high-affinity system has a pH optimum at 4.0-4.2, the accumulation ratio is highest at a cell density 2-5 mg dry wt. per ml and decreases with increasing lysine concentrations. Lysine accumulated by this system does not exit from cells. The only potent competitive inhibitors are L-arginine, L-histidine and D-lysine. The other amino acids tested do not behave as competitive inhibitors. Of the various metabolic inhibitors tested, the most potent were proton conductors and antimycin A.     

The Ability of 2-Deoxyglucose to Promote the Lysis of Streptococcus bovis JB1 via a Mechanism Involving Cell Wall Stability

The non-metabolizable glucose analog, 2-deoxyglucose (2-DG), decreased the growth rate and optical density of Streptococcus bovis JB1 20%, but it had an even greater effect on stationary phase cultures. Control cultures receiving only glucose (2 mg/ml) lysed very slowly (<5% decline in optical density in 48 h), but cultures that had been grown with glucose and 2-DG (2 mg/ml each) lysed much faster (>85% decline in optical density in 48 h). Cultures that were treated with inhibitors that decreased intracellular ATP (sodium fluoride, nigericin, and valinomycin or tetrachlorosalicylanilide) or membrane potential (sodium fluoride, nigericin, and valinomycin, tetrachlorosalicylanilide, or phenylmethylsulfonyl fluoride) did not promote lysis. 2-DG had its greatest effect when it was added at inoculation. If 2-DG was added at later times, less lysis was observed, and cells that were given 2-DG just prior to stationary phase were unaffected. Cells that were grown with glucose and 2-DG were more susceptible to cell wall-degrading enzymes (lysozyme and mutanolysin) than cells that had been grown only with glucose, but sublethal doses of penicillin during growth did not promote lysis after the cells had reached stationary phase. The idea that 2-DG might be affecting autolytic activity was supported by the observation that cultures washed and resuspended in fresh medium with or without 2-DG lysed at a slower rate than cultures that were not centrifuged or were resuspended in the culture superntant. Received: 11 April 1997 / Accepted: 10 June 1997     

Requirement of heme for growth of Bacteroides fragilis.

Heme or protoporphyrin IX was required for growth of Bacteroides fragilis in a defined medium. The amount of heme necessary for half-maximal growth was 2 to 10 ng/ml (3.8 to 15 pmol/ml) among the Bacteroides species and strains tested. The growth rate, metabolic products from glucose fermentation, and cell yields were affected by the concentration of heme in the medium and by the length of time the culture was incubated. When heme was growth limiting (4 ng/ml), growth rates decreased by 50%, cultures started producing lactic and fumaric acids, and the cell yields declined. The cell yield for B. fragilis (ATCC 25285) at 24 h in medium containing 6.5 microgram of heme per ml was 69 g (dry weight) of cells per mol of glucose compared to 16 g (dry weight) of cells per mol of glucose with 4 ng of heme per ml. B. fragilis was unable to grow in defined medium when a porphyrin precursor, delta-aminolevulenic acid or porphobilinogen, was added in place of heme.     

Effect of initial cell density on hybridoma growth, metabolism, and monoclonal antibody production.

A murine hybridoma cell line (167.4G5.3) was cultivated in batch mode with varying inoculum cell densities using IMDM media of varying fetal bovine serum concentrations. It was observed that maximum cell concentrations as well as the amount of monoclonal antibody attainable in batch mode were dependent on the inoculum size. Specifically, cultures with lower inoculum size resulted in lower cell yield and lower antibody concentrations. However, in the range of 10(2) to 10(5) cells per ml, the initial cell density affected the initial growth rate by a factor of only 20%. Furthermore, specific monoclonal antibody production rates were independent of initial cell density and the serum concentration. Glutamine was the limiting nutrient for all the cultures, determining the extent of growth and the amount of antibody produced. Serum was essential for cell growth and cultures with initial cell concentrations up to 10(6) cells per ml could not grow without serum. However, when adapted, the cells could grow in a custom-made serum-free medium containing insulin, transferrin, ethanolamine, and selenium (ITES) supplements. The cells adapted to the ITES medium could grow with an initial growth rate slightly higher than in 1.25% serum and the growth rate showed an initial density dependency-inocula at 10(3) cells per ml grew 30% slower than those at 10(4) or 10(5). This difference in growth rate was decreased to 10% with the addition of conditioned ITES medium. The addition of conditioned media, however, did not improve the cell growth for serum-containing batches.     

Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Effect of 2-deoxyglucose on Schizosaccharomyces pombe

Megnet, Roland (Institut für Allgemeine Mikrobiologie der Universit?t, Bern, Switzerland). Effect of 2-deoxyglucose on Schizosaccharomyces pombe. J. Bacteriol. 90:1032-1035. 1965.-Cultivation of Schizosaccaromyces pombe in a medium containing 2-deoxyglucose (100 mug/ml) results in the death of the cells after an initial period of apparently normal growth. At higher deoxyglucose concentration (400 mug/ml), the cells die immediately after inoculation. Only growing cells are killed, and microscopic inspection of the cultures reveals cell-wall fragments of lysed cells. A mutant resistant to 2-deoxyglucose, which cannot use glucose as a carbon source, was found to be partially deficient in hexokinase. The data constitute evidence for the inhibition of some reaction(s) in the synthesis of cell-wall polysaccharides by metabolites of 2-deoxyglucose in this organism.     

Growth of Physarum flavicomum and Physarum rigidum in Chemically Defined Minimal Media

Physarum flavicomum, P. polycephalum, and P. rigidum grew at pH 4.2 in a medium composed of mineral salts, glucose, biotin, thiamine, hematin, and four amino acids. Important differences in pH tolerance were noted among the species. The minimal medium of P. flavicomum and P. polycephalum contained the amino acids methionine, glycine, and arginine, but valine was also required by P. rigidum. Starting with an inoculum of about 0.3 mg of protein per 25 ml of minimal medium, P. flavicomum and P. polycephalum grew to 23 mg and P. rigidum to 12 mg of protein per 25 ml in 3, 2, and 4 weeks, respectively. P. flavicomum and P. polycephalum grew with valine or leucine replacing arginine in the minimal medium but the growth yields and growth rates were decreased. All three species utilized homocysteine thiolactone in the minimal media in place of methionine. Serine adequately replaced glycine for P. rigidum but was inhibitory in the minimal medium of P. flavicomum or P. polycephalum unless homocysteine thiolactone also replaced methionine. Growth rates of all three organisms were increased in the presence of seven amino acids (original four plus leucine, lysine, and isoleucine).     

Inhibition of intracellular proteolysis in muscle cultures by multiplication-stimulating activity. Comparison of effects of multiplication-stimulating activity and insulin on proteolysis, protein synthesis, amino acid uptake, and sugar transport

The effects of the insulin-like growth factor, multiplication-stimulating activity (MSA), on chick myotube cultures were investigated. In serum-free media, MSA at levels reported to be present in fetal serum (5 ng/ml) significantly inhibited overall rates of protein degradation and stimulated protein synthesis and amino acid uptake. Half-maximal effects on protein degradation (-30%), synthesis (+25%), and amino acid uptake (+50%) occurred at approximately 0.05 micrograms/ml. In contrast, 10(2)-10(3)-fold higher concentrations (5 micrograms/ml) were required to stimulate transport of the glucose analog 2-deoxyglucose. The results indicate that MSA is an effective anabolic agent regulating protein metabolism and amino acid uptake, but not sugar transport in these cells. Parallel studies conducted with insulin demonstrated similar size effects on protein metabolism and amino acid uptake in serum-free media. However, unlike MSA, insulin levels (10(-2) units/ml) well in excess of its normal physiological range were required to produce significant effects. In addition, the relative sensitivity of sugar transport with respect to protein metabolic effects differed for insulin and MSA. Thus, 2-deoxyglucose transport was approximately 10 times more sensitive to insulin than protein synthesis, proteolysis, or amino acid uptake in contrast to MSA where the reverse was true. However, despite the relatively higher sensitivity of sugar transport to insulin, supraphysiological levels (10(-3) units/ml) of this hormone were still required for significant stimulation. These results suggest a generally low insulin sensitivity in cultured chick myotubes relative to adult tissues. In contrast, the effects of MSA are consistent with a possible role of this or similar factors in regulating growth and development of embryonic muscle.     

Effect of Type of Carbohydrate on Growth and Protein Synthesis by Tetrahymena pyriformis

SYNOPSIS. In chemically defined media at carbohydrate concentrations ≧ 0.5% (w/v) Tetrahymena pyriformis W multiplied more rapidly, developed larger cells, and achieved greater growth as measured by optical density when carbohydrate was provided as dextrin rather than glucose. In media containing 0.3 mg/ml of amino acid nitrogen, growth increased with glucose concentration from 0.1 to 1%, did not change significantly to 3%, and was sharply inhibited at higher glucose levels. With dextrin, maximum growth paralleled carbohydrate concentration from 0.1 to 3%. At higher N levels the inhibitory concentration of glucose was lowered, but growth in dextrin media was not affected except at N concentrations that were inhibitory independent of carbohydrate source. At 1% carbohydrate levels, total cell protein per ml of culture was 60% greater, protein per cell approximately 50% greater, and cells were 1.5 to 2 times larger in media with dextrin than with glucose. Comparable differences in protein synthesis were observed at 2% carbohydrate levels and efficiency of conversion of substrate-N to protein-N was greater in the medium with dextrin than glucose.
Growth as measured by optical density in media with 0.3 mg/ml of N and 1 or 2% (w/v) of dextrin was not significantly reduced by the simultaneous presence of 1 or 2% glucose. This observation appeared to negate osmotic pressure as an explanation of reduced growth in the presence of glucose. At higher osmolar concentrations osmotic pressure appeared to be a major determinant of overall growth but not of cell size.     

Growth Factors of Bacterial Origin for the Culture of the Rumen Oligotrich Protozoon, Entodinium caudatum

Attempts were made to develop an artificial medium suitable for axenic culture of Entodinium caudatum. Agnotobiotic cultures of the protozoon were established as stock cultures for testing the suitability of various growth media. A cell-free extract of mixed bacteria isolated from the rumen was shown to contain one or more growth factors for the protozoon when supplied with activated charcoal as a carrier. The medium (CYSE medium), which supported the growth of the protozoon in the presence of 50 μg/ml each of penicillin and chloramphenicol, consisted of activated charcoal (20 mg), heat-treated yeast (Y) (80 mg), 13%β-sitosterol-coated rice starch (S) (120 mg), and cell-free extract of rumen bacteria (1 ml) in 40 ml buffer solution. When culturing the protozoon, the CYSE medium was supplemented daily with 20 mg each of Y and S and half of the medium was replaced with fresh medium once every 5 d. The possible use of this method to establish an axenic culture of E. caudatum is discussed.     

Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free culture medium

In Exp. 1, Medium 199 and Medium RD (RPMI-1640 and Dulbecco's MEM, 1:1 v/v) were compared in a 2 x 2 factorial design by supplementing each with 15 mg bovine serum albumin (BSA)/ml of 1 mg polyvinyl alcohol (PVA)/ml. All media contained 5 micrograms insulin/ml, 5 micrograms transferrin/ml, 5 ng selenium/ml (ITS), and 10 ng epidermal growth factor (EGF)/ml. One-cell embryos were cultured at 39 degrees C with 5% CO2 in air for 65 h and then stained with Hoechst 33342 to determine blastomere number. Embryos in Medium 199 developed poorly (P less than 0.001) when PVA was used instead of BSA (30 vs 76 cells/embryo), but developed rapidly in Medium RD with PVA or BSA (118 and 121 cells). Similar results were obtained in Exp. 2 in BSA- and PVA-free medium. In Exp. 3, the development of 1-cell embryos after 65 h in unsupplemented (protein-free) Medium RD (68% blastocysts, 117 cells) did not differ (P greater than 0.37) from that obtained using Medium RD with insulin, ITS or EGF alone. Culture in protein-free Medium RD for 96 h resulted in 82% of the 1-cell embryos forming blastocysts and 40% hatching through the zona pellucida. In a preliminary test of viability, 1-cell embryos cultured in this medium for 48 or 65 h and transferred to synchronous recipients resulted in 5/18 (28%) and 3/24 (12%) Day-15 viable fetuses. Cell counts of approximately 120 per blastocyst after culturing 1-cell embryos for 65 h in Medium RD indicated that cell division was more rapid than that obtained with all other media tested previously in this laboratory. This is the first report of rabbit embryo development from the 1-cell to the hatching blastocyst stage in a defined protein-free culture medium.     

Growth ofChlamydia psittaci strain menigopneumonitis in mouse L cells cultivated in a defined medium in spinner cultures

Summary L cells were grown in spinner cultures in a defined medium consisting of Waymouth medium MB752/1 (19) supplemented with 2 mg of fatty acid-free bovine serum albumin (BSA) per ml and 5 μg of oleate per ml (WO₅ medium). Growth in WO₅ medium was comparable to spinner L cell growth in two serum-containing media. The optimal concentration of oleate in the WO medium was 5 to 10 μg per ml. The use of 20 to 80 μg of oleate per ml of medium resulted in lower peak populations and earlier declines in viable cell counts. Cell death occurred rapidly in WO₁₆₀ medium. Cell growth in WO medium containing 5 to 80 μg of oleate per ml was well above the level of growth observed when no oleate was present in the medium. Since the total lipid and fatty acid compositions of the BSA used in this study have been characterized by the authors, the WO medium may be considered a defined medium. L cells have been continuously maintained in spinner cultures in WO₅ medium for over 50 passages with no major variation in the growth pattern. A 1000-fold increase inChlamydia psittaci strain meningopneumonitis, with a peak titer of 9.3×10⁷ plaque-forming units per ml, was observed when the chlamydial agents were grown in spinner L cells in WO₅ medium. This investigation was supported by Public Health Service Research Grant HE 08214 from the Program Projects Branch, Extramural Programs, National Heart and Lung Institute; The World Health Organization; and The Hormel Foundation.     

Isoniazid Uptake in Relation to Growth Inhibition of Mycobacterium tuberculosis

(14)C-isoniazid (INH) was used to study the relationship between drug uptake or binding by Mycobacterium tuberculosis and growth inhibition of the organism, which is dependent upon the concentration of drug and the duration of exposure. When strain H37R(a), grown in modified Sauton's liquid medium, was treated with 0.1 mug of INH per ml for 2 to 6 hr, followed by 10 mug of nicotinic hydrazide (NH) per ml to block further INH uptake, growth was retarded but not completely inhibited upon continued incubation. NH itself did not retard growth. However, cells treated in a similar manner with INH alone grew normally when diluted 1:100 in fresh, drug-free media. Uptake data showed that bacilli exposed to 0.1 mug of INH per ml accumulated 5.5, 9.7, and 12 mmug/mg of dry cells at 2, 4, and 6 hr, respectively. Other experiments suggested that once isoniazid is bound, it is not rapidly lost when NH is added or when the cells are diluted in fresh media. In the presence of 1.0 mug of INH per ml, tubercle bacilli took up 10 to 37 mmug/mg of dry cells in 20 to 90 min. These cells were not markedly inhibited when diluted 1:40 in fresh NH-containing media and incubated for 6 days. Growth inhibition of tubercle bacilli by INH depends on the uptake of sufficient drug, but the evidence obtained in this study suggests that the absolute concentration of bound INH is not as important in the action of the drug as is the maintenance of a critical cellular concentration for a requisite period of time.     

The effect of tube closures and media on the enrichment of Campylobacter jejuni

Enrichment of C. jejuni in metal, Morton-style capped tubes gave no growth. Cotton wool or sponge rubber plugged tubes yielded enrichment of C. jejuni to 1.6 × 10³ cfu per ml from an inoculation of 0.18 cfu per ml in 10 ml medium.Enrichment of C. jejuni from egg melange in cotton plugged tubes and bottles showed that the ratio of egg melange to broth should not exceed 4:1 in bottles and 2:3 in tubes. When enriching from incubating fertile eggs infected by C. jejuni a decreasing quantitative and qualitative recovery was experienced with increasing time of egg incubation. Five enrichment broths and two selective plating media were compared in this experiment. The medium of Doyle and Roman (Appl. Environ. Microbiol. 43, 1343–1353 (1982)) and a routine enrichment broth plus rifampin (brucella broth containing per litre: 50 ml lysed horse blood, 10 mg rifampin, 5000 IU polymyxin B, 10 mg vancomycin, 5 mg trimethoprim lactate and the reductants of George et al. (J. Clin. Microbiol. 8, 36–41 (1978)) were superior to BNP broth. All media showed decreased enrichment of C. jejuni with increasing time of egg incubation, when growth was only of the order of 2 × 10² cfu per ml at day 15 of egg incubation. Rifampin was required to suppress contamination by Proteus species and Gram-positive cocci.     

Development of insulin-sensitivity at weaning in the rat. Role of the nutritional transition.

This study was undertaken to determine the factors involved in the development of insulin-sensitivity at weaning. Glucose kinetics were studied in suckling rats and in rats weaned on to a high-carbohydrate (HC) or a high-fat (HF) diet, in the basal state and during euglycaemic-hyperinsulinaemic-clamp studies. These studies were coupled with the 2-deoxyglucose technique, allowing a measure of glucose utilization by individual tissues. In the basal state, the glycaemia was higher in HF-weaned rats (124 +/- 4 mg/dl) than in suckling (109 +/- 1 mg/dl) and HC-weaned rats (101 +/- 3 mg/dl). Glucose turnover rates were similar in the three groups of animals (14 mg/min per kg). Nevertheless, basal metabolic glucose clearance rate was 20% lower in HF-weaned rats than in the other groups. During the euglycaemic-hyperinsulinaemic experiments, hepatic glucose production was suppressed by 90% in HC-weaned rats, whereas it remained at 40% of basal value in suckling and HF-weaned rats, indicating an insulin resistance of liver of these animals. Glucose clearance rate during the clamp was 18.3 +/- 0.9 ml/min per kg in suckling rats, whereas it was 35.3 +/- 1.2 ml/min per kg in HC-weaned rats and 27.8 +/- 1.1 ml/min per kg in HF-weaned rats, indicating an insulin resistance of glucose utilization in suckling, and to a lower extent, in HF-weaned rats. The deoxyglucose technique showed that peripheral insulin resistance was localized in muscles and white adipose tissue of suckling and HF-weaned rats. These results indicate that the switch from milk to a HC diet is an important determinant of the development of insulin-sensitivity at weaning in the rat.     

Adenylate energy charge in Acholeplasma laidlawii.

Adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate were produced by Acholeplasma laidlawii B-PG9 growing in modified Edward medium. The adenylate energy charge was calculated to be 0.84 +/- 0.07 and ranged from 0.91 to 0.78 during exponential growth (12 to 24 h). During exponential growth, A. laidlawii contained, at 17.5 h, 2.3 X 10(-17) mol of adenosine 5'-triphosphate per colony-forming unit and, at 16 h, 27.3 nmol of adenosine 5'-triphosphate per mg (dry weight). The medium supported a doubling time of 0.95 h. The molar growth yields (Yglucose = grams [dry weight] per mole of glucose used) were 40.2 +/- 3.4 (16 h) and 57.1 +/- 9.7 (20 h) during midexponential growth. A maximum yield of 8.3 X 10(9) colony-forming units was reached at 24 h, when 56% of the initial concentration of glucose had been used. At 40 h, during the stationary phase, 14.95 +/- 3.75 mumol of glucose per ml of medium had been used. At this time, the culture fluids contained 21.86 +/0 mumol of lactate per ml and 3.14 +/- 0.13 mumol of pyruvate per ml.     

Induction of Prodigiosin Biosynthesis after Shift-Down in Temperature of Nonproliferating Cells of Serratia marcescens

Nonpigmented bacteria obtained by growth of Serratia marcescens at 38 C synthesized prodigiosin at 25 C if certain individual amino acids were added to cultures of nonproliferating cells. In order of effectiveness, the amino acids were: DL-histidine, L-proline, L-hydroxyproline, DL-alanine, L-alanine, DL-aspartic acid, D-alanine, DL-proline, L-serine, L-ornithine, L-glutamic acid, and D-proline. DL-Histidine at its optimal concentration (20 mg/ml) induced formation of prodigiosin (198 mug of prodigiosin per mg of bacterial protein) after incubation of cultures for 54 hr. Lower concentrations (10 mg/ml) of the other amino acids usually were optimum but less prodigiosin was synthesized, and the maximal amount of pigment occurred between 36 and 48 hr. DL-Methionine was not effective alone but at a low concentration (40 mug/ml) enhanced and accelerated biosynthesis of prodigiosin in the presence of other suitable amino acids. Addition of 2 mg of L-proline per ml at 0 hr induced formation of only 30 mug of prodigiosin after incubation for 42 hr, but addition at 36 hr of 5 mg more of L-proline per ml increased synthesis to 120 mug at 42 hr. Again, DL-methionine markedly augmented prodigiosin biosynthesis in these cultures. Synthesis of prodigiosin ceased if cultures were shifted from 25 to 38 C. Prodigiosin biosynthesis by the nonproliferating cells was maximum when cultures were aerated, the amount of bacterial protein was about 2.0 mg/ml, and amino acids were added at 0 hr. Bacteria synthesized prodigiosin most efficiently when they were harvested from aerated cultures grown at 38 C for 24 hr in a complete medium in a fermentor.     

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

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.     

In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium

The effect of BSA, caffeine and calcium was studied on the penetration of pig oocytes by frozen-thawed spermatozoa in a modified Tris-buffered medium (mTBM) without added bicarbonate. Pig cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22 h. The COC were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with spermatozoa for 6 h in mTBM containing caffeine (5 mM) and 0.1 or 0.4% BSA (Experiment 1). In Experiment 2, oocytes were inseminated in mTBM containing 0.1% BSA and various concentrations of caffeine (0 to 5 mM). In Experiment 3, insemination was carried out in mTBM containing 0.1% BSA, 1 mM caffeine and various concentrations of Ca(2+) (0.5 to 10 mM). Supplementation of mTBM with either 0.1 or 0.4% BSA resulted a high penetration rate with a high polyspermy rate. However, the mean number of spermatozoa per oocyte was significantly higher at 0.4% than at 0.1% BSA. The penetration rate, polyspermy rate and mean number of spermatozoa per oocyte were all significantly higher when 1 to 5 mM caffeine were added to the medium than in caffeine-free medium. No penetration was observed in the presence of 0.5 mM Ca(2+). The penetration rate was significantly increased from 12 to 92% at 2.5 to 10 mM Ca(2+). The mean number of spermatozoa per oocyte did not differ between 2.5 and 5 mM Ca(2+) but increased significantly at 7.5 and 10 mM. These results show the successful in vitro penetration of pig oocytes in a chemically semidefined medium without added bicarbonate. Although BSA and caffeine can modulate the rate of sperm penetration, calcium seems to be an important regulatory ion.