Pharmacokinetics in plasma and alveolar regions of a healthy calf intramuscularly administered a single dose of orbifloxacin

This study analyzed the pharmacokinetics of orbifloxacin (OBFX) in plasma, and its migration and retention in epithelial lining fluid (ELF) and alveolar cells within the bronchoalveolar lavage fluid (BALF). Four healthy calves received a single dose of OBFX (5.0 mg/kg) intramuscularly. Post-administration OBFX dynamics were in accordance with a non-compartment model, including the absorption phase. The maximum concentration (C_max) of plasma OBFX was 2.2 ± 0.1 μg/ml at 2.3 ± 0.5 hr post administration and gradually decreased to 0.3 ± 0.2 μg/ml at 24 hr following administration. The C_max of ELF OBFX was 9.3 ± 0.4 μg/ml at 3.0 ± 2.0 hr post administration and gradually decreased to 1.2 ± 0.1 μg/ml at 24 hr following administration. The C_max of alveolar cells OBFX was 9.3 ± 2.9 μg/ml at 4.0 hr post administration and gradually decreased to 1.1 ± 0.2 μg/ml at 24 hr following administration. The half-life of OBFX in plasma, ELF, and alveolar cells were 6.9 ± 2.2, 7.0 ± 0.6, and 7.8 ± 1.6 hr, respectively. The C_max and the area under the concentration-time curve for 0–24 hr with OBFX were significantly higher in ELF and alveolar cells than in plasma (P<0.05). These results suggest that OBFX is distributed and retained at high concentrations in ELF and alveolar cells at 24 hr following administration. Hence, a single intramuscular dose of OBFX (5.0 mg/kg) may be an effective therapeutic agent against pneumonia.     

Evaluation of route and frequency of administration of three antimicrobial drugs in cattle

This study in six cows compared serum concentrations of trimethoprim and sulphadoxine (16 mg/kg body weight (BW)) after once daily and twice daily administration, and of procaine penicillin G (20,000 IU/kg BW) after subcutaneous (SQ) and intramuscular (IM) administration, and evaluated postmortem tissue concentrations of penicillin following SQ treatment. Trimethoprim and penicillin were measured microbiologically, and sulphadoxine colorimetrically. Using minimum inhibitory concentrations (MIC), trimethoprim reached serum concentrations above 0.5 μg/mL from 15 minutes to 120 minutes, and sulphadoxine exceeded 9.5 μg/mL from 10 minutes to 12 hours, after administration. At 24 hours after treatment, both had declined to below the MIC of most organisms. A second treatment at 12 hours maintained concentrations of sulphadoxine above 9.5 μg/mL for a further 24 hours. For penicillin administered IM and SQ, concentrations that peaked at 0.88 μg/mL would inhibit most common grampositive bacteria for the entire 24 hour period and fastidious gram-negative organisms from 90 minutes to 12 hours after SQ treatment, but for virtually the entire period after IM administration. Mean ± SD concentrations (μg/mL) of penicillin at euthanasia, five days after the last SQ administration, were 1.15 ± 1.27 (injection site), 1.00 ± 0.80 (liver), 0.90 ± 0.58 (renal cortex), 0,58 ± 0.17 (renal medulla), 0.13 ± 0.11 (diaphragm), 0.10 ± 0.08 (gluteal muscle), and 0.06 ± 0.04 (fat). Therefore, except for the most sensitive organisms, twice daily injection of trimethoprim/sulphadoxine (16 mg/kg BW) may be required. Penicillin G administered SQ at 20,000 IU/kg BW should provide effective serum levels for as long as IM administration against gram-positive organisms, but for only about half as long against gram-negative bacteria. The label withdrawal time of five days cannot be used when penicillin is given SQ at 20,000 IU/kg BW for three days.     

The Effect of pH and Potassium Phosphate Buffer on the Toxicity of Cadmium for Bacteria

An increase in the pH of Plate Count Agar led to increased toxicity of cadmium (Cd) for Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC 12600, Clostridium perfringens ATCC 8798, Escherichia coli ATCC 11775 and Pseudomonas aeruginosa ATCC 10145. The effect of the pH on toxicity was not clearly observed with Streptococcus bovis ATCC 9809 and was absent with Bacillus subtilis ATCC 8473.When potassium phosphate buffer was added to the growth medium, the Cd toxicity for M. luteus and B. subtilis was enhanced. The toxicity of Cd for S. bovis decreased when potassium phosphate buffer was added to the medium. When the pH increased at differing phosphate concentrations, the sensitivity of M. luteus to Cd decreased.In the authors’ opinion, the effect of pH on Cd toxicity for bacteria is due to a continuously increasing negative charge towards an alkaline value by most bacteria which increases the affinity of cations towards the cell wall.     

Antimicrobial susceptibility of porcine Brachyspira hyodysenteriae and Brachyspira pilosicoli isolated in Sweden between 1990 and 2010

Background

The anaerobic spirochetes Brachyspira hyodysenteriae and Brachyspira pilosicoli cause diarrheal diseases in pigs. Their fastidious nature has hampered standardization of methods for antimicrobial susceptibility testing. For monitoring of antimicrobial susceptibility wild type cutoff values are needed to define where the wild type distribution of MICs ends and no approved cutoffs are available for Brachyspira spp. In this study antimicrobial susceptibility data for both species (in total 906 isolates) were compiled and analyzed and wild type cut off values for B. hyodysenteriae proposed.

Methods

The MICs of tiamulin, valnemulin, tylosin, tylvalosin, doxycycline and lincomycin were determined by broth dilution in brain heart infusion broth supplemented with 10% fetal calf serum.

Results

The compiled MICs from the broth dilution tests of the B. hyodysenteriae type strain, B78^T (ATCC® 27164^T), showed that the method yields reproducible results. In an international perspective the frequencies of isolates with decreased antimicrobial susceptibility were low among both B. hyodysenteriae and B. pilosicoli. However, in B. pilosicoli a constant level of 10-15% isolates with tiamulin MICs >4 μg/ml was detected between 2002 and 2010 and in B. hyodysenteriae a gradual increase in tiamulin MICs was seen between 1990 and 2003 although this increase has ceased during the last years. The wild type cutoff values proposed for B. hyodysenteriae are: tiamulin >0.25 μg/ml, valnemulin >0.125 μg/ml, tylosin >16 μg/ml, tylvalosin >1 μg/ml, lincomycin >1 μg/ml and doxycycline >0.5 μg/ml.

Conclusions

The broth dilution method used in this study has over the years generated tightly grouped MIC populations for the field isolates and reproducible results for the control strain B78^T and is therefore a suitable antimicrobial susceptibility test method for monitoring of Brachyspira spp. Here we propose wild type cutoff values for six antimicrobial agents for B. hyodysenteriae tested by broth dilution based on MIC distributions and the current knowledge on mechanisms of resistance in this species. There are few studies on antimicrobial resistance mechanisms and MIC distributions in B. pilosicoli but to some extent the cutoff values proposed for B. hyodysenteriae may be applicable also for monitoring of antimicrobial susceptibility in B. pilosicoli.     

The Concentrations of Molybdenum and Zinc in Liver in Relation to Copper Accumulation in Normal and Copper Poisoned Sheep

The concentrations of copper, molybdenum and zinc were measured in the liver of normal grazing sheep and lambs from Eastern Norway, and in sheep dead of chronic copper poisoning. The following mean values were found: Normal sheep: 173 ± 130 μg Gu/g wet weight, 1.0 ±0.3 μg Mo/g, and 49 ± 10 μg Zn/g; lambs: 129 ± 59 μg Gu/g, 0.9 ± 0.3 μg Mo/g, and 46 ±9 μg Zn/g; sheep dead of copper poisoning: 429 ± 249 μg Gu/g, 0.4 ± 0.1 μg Mo/g, and 43 ± 2d μg Zn/g. Sheep with low liver copper (Gu < 10 μg/g) were also analyzed for molybdenum and zinc, with the following results: 1.0 ± 0.2 μg Mo/g, and 45 ± 8 μg Zn/g wet weight. The differences in liver copper between all the groups, and the differences in molybdenum concentrations between the normal sheep and the lambs and between the normal sheep and the poisoned sheep were significant (P < 0.001). No significant correlations between liver copper/liver molybdenum or liver copper/liver zinc were detected.     

Comparison of Different Agar Diffusion Methods for the Detection of Antimicrobial Residues in Slaughter Animals

The different agar diffusion methods were compared using antibiotic and sulphonamide-impregnated filter-paper discs and the kidneys of healthy and emergency-slaughtered pigs and cows after slaughter.No method used seemed to be sensitive to all antimicrobial drugs preimpregnated onto discs. Tetracycline yielded a greater zone of inhibition at pH 6 than at pH 8 and aminoglycosides, erythromycin, polymyxin B and lin cornycin at pH 8 than at pH 6. It therefore seems necessary to use different pHs (6 and 8). The addition of trimethoprim to the medium is necessary for the detection of sulphonamides. Bacillus subtilis BGA used as the test organism was more sensitive to sulphonamides on the “Test agar for the inhibitor test” containing trimethoprim than on the “Iso-Sensitest agar” also containing trimethoprim. The addition of trimethoprim to “Test agar for the inhibitor test” is recommended at pH 8 but not at pH 6 because false-positive cases (with inhibition zones > 2 mm) were observed at pH 6 with trimethoprim on the kidneys of healthy pigs.     

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.     

Distribution of neomycin in bull calves after intramuscular injection

Neomycin sulfate was injected intramuscularly in calves. Blood and tissue samples were taken at zero, one, two, four, six, eight and 24 hours after administration. The tissues with high levels (greater than 10 μg/g) of drug at the one hour period were kidney cortex and medulla, urine, blood serum and the injection site. By 24 hours after administration only the kidney cortex and urine had high levels of neomycin. The drug could not be detected in any brain tissues and very small amounts (less than 1 μg/g) were present in the bile, thymus and vitreous humor. Levels greater than 5 μg/g were present in lung tissues for less than four hours but were greater than 2 μg/g for more than 24 hours.

The mean level in the injection site was greater than 700 μg/g at one hour but only trace amounts were found at 24 hours.

On the basis of the tissue drug concentration intramuscularly administered neomycin was suggested as therapeutically useful for respiratory and urinary tract infections caused by susceptible bacteria.

     

Pharmacokinetics and plasma concentrations of acetylsalicylic acid after intravenous, rectal, and intragastric administration to horses

Six healthy adult horses (5 mares and 1 stallion) were given a single dose of acetylsalicylic acid (ASA), 20 mg/kg of body weight, by intravenous (IV), rectal, and intragastric (IG) routes. Serial blood samples were collected via jugular venipuncture over a 36-h period, and plasma ASA and salicylic acid (SA) concentrations were determined by high-performance liquid chromatography. After IV administration, the mean elimination rate constant of ASA (± the standard error of the mean) was 1.32 ± 0.09 h⁻l, the mean elimination half-life was 0.53 ± 0.04 h, the area under the plasma concentration-versus-time curve (AUC) was 2555 ± 98 μg · min/mL, the plasma clearance was 472 ± 18.9 mL/h/kg, and the volume of distribution at steady state was 0.22 ± 0.01 L/kg. After rectal administration, the plasma concentration of ASA peaked at 5.05 ± 0.80 μg/mL at 0.33 h, then decreased to undetectable levels by 4 h; the plasma concentration of SA peaked at 17.39 ± 5.46 μg/mL at 2 h, then decreased to 1.92 ± 0.25 μg/mL by 36 h. After rectal administration, the AUC for ASA was 439.4 ± 94.55 μg · min/mL and the bioavailability was 0.17 ± 0.037. After IG administration, the plasma concentration of ASA peaked at 1.26 ± 0.10 μg/mL at 0.67 h, then declined to 0.37 ± 0.37 μg/mL by 36 h; the plasma concentration of SA peaked at 23.90 ± 4.94 μg/mL at 4 h and decreased to 0.85 ± 0.31 μg/mL by 36 h. After IG administration, the AUC for ASA was 146.70 ± 24.90 μg · min/mL and the bioavailability was 0.059 ± 0.013. Administration of a single rectal dose of ASA of 20 mg/kg to horses results in higher peak plasma ASA concentrations and greater bioavailability than the same dose given IG. Plasma ASA concentrations after rectal administration should be sufficient to inhibit platelet thromboxane production, and doses lower than those suggested for IG administration may be adequate.     

Drug residues in milk after intrauterine injection of oxytetracycline, lincomycin-spectinomycin, and povidone-iodine in cows with metritis

A study was conducted to document the maximum retention times of antimicrobial residues in milk after their use in intrauterine treatment of metritis in lactating cows and to evaluate several risk factors hypothesized to influence the retention time of these drugs. Oxytetracycline (3 g), lincomycin-spectinomycin (2 g of one-third lincomycin and two-thirds spectinomycin), or povidone-iodine (6 g) were given to cows with metritis by intrauterine route. The Bacillus stearothermophilus var calidolactis disk assay was performed on each milk sample. Of the 61 cows treated with oxytetracycline, 30 had residues in their postinjection milk for variable periods (range, 12.5 to 44.0 hours; mean, 26.6 +/- 10.3). Of the 47 cows treated with lincomycin-spectinomycin, 17 had residues in their postinjection milk for various periods (range, 14.5 to 24 hours; mean, 19.5 +/- 8.9). Povidone-iodine was not detected in milk. Because a high number of cows (n = 61) were treated with oxytetracycline, only data from these cows were used in testing the influence of 3 factors (severity of metritis, time after parturition when cows with metritis were treated, and parity) on maximum retention of the drug in milk. Severity of metritis did not have a significant influence (P greater than or equal to 0.1) on the maximum retention time of the drug. The retention time decreased linearly with the increase of time after parturition when the cow with metritis was treated. First lactation cows had a significantly (P less than or equal to 0.01) shorter retention time than did older cows.     

A modified method for the detection of antibiotic residues in slaughter animals.

Biological methods in current use for the detection of antibiotic residues in slaughter animals are reviewed. A modified method is suggested in which the conditions for the control have been standardized. By the use of a semi-defined medium, the batch-to-batch variations are minimized. In order to facilitate the detection of sulfonamides the medium is supplemented with trimethoprim. The standardized conditions included the use of a sporulating organism, Bacillus subtilis, an inoculum size of 0.5 × 10⁵ spores per ml medium, and 5 ml medium of pH 6.0 per plate. A preincubation-diffusion time of 1 h in room temperature is recommended before incubation.The modified method was compared with the currently prescribed Swedish method. The new method was easier to perform and showed a more uniform sensitivity to most of the antibiotics used.     

禽蛋中泰乐菌素残留量的微生物学检测方法

试验建立了泰乐菌素在禽蛋残留中微生物学检测方法,将试样均质1min(5000r/min)后,称取20g置于离心管内,加入5ml水,充分搅匀,放置60min后,离心20min(8000r/min),取上清液作为样液。再用藤黄微球菌作为检定菌进行含量测定。结果显示,泰乐菌素在0.1~0.8μg/ml的浓度范围内具有良好线性关系(r=0.9982),禽蛋中3种添加浓度2.00、4.00、8.00μg/ml的平均回收率为86.6%(n=9),RSD分别为5.1%、4.3%、3.9%(n=3)。     

Characterization of Alpha-fetoprotein Levels in Three Dolphin Species: Development of Sensitive Immunoassays for Analysis of the Pregnancy-associated Variations

A single radial immunodiffusion (SRID) assay and a chemiluminescent immunoassay (CLIA) were initially developed for alpha-fetoprotein (AFP) of the striped dolphin. Utilizing these developed assays, we investigated pregnancy-associated changes in the levels of AFP in the sera of fetuses and pregnant females of three dolphin species; samples were either collected from captive individuals or obtained as fishery by-products. The concentrations of AFP in the fetal serum ranged from 419.0 to 2026.3 μg/ml in the striped dolphin, 12.6 to 1218.7 μg/ml (for an AFP equivalent; eqAFP) in the common bottlenose dolphin and 770.6 to 3129.1 μg eqAFP/ml in the Risso''s dolphin. AFP levels decreased with increased fetal size in fetuses over 20 cm in length. The concentrations of AFP in sera of pregnant females ranged from 7.18 to 8068.7 ng/ml in the striped dolphin, 6.6 to 1241.1 ng eqAFP/ml in the common bottlenose dolphin and 3.4 to 2868.7 ng eqAFP/ml in the Risso''s dolphin. The levels in most pregnant females were equal to or lower than those found in males and nonpregnant individuals, although a few pregnant females exhibited extremely high levels (in the range of hundreds to thousands of nanograms per milliliter). Such high levels of AFP were not observed during pseudopregnancy. To our knowledge, this is the first report on basal profiles for serum AFP levels in small odontocetes. The profiles indicated that AFP may play a significant role during embryonic development, although maternal levels do not appear to be a diagnostic biomarker for monitoring pregnancy.     

Iodine Content in Colostrum and Milk of Cows and Sows

The concentration of total iodine in colostrum and normal milk of cows and sows has been determined using a Technicon Autoanalyzer. In cows as well as in sows a lowering of the level of iodine in milk was observed during the first few days after parturition. At the first sampling within 8 hrs. after parturition the concentration of iodine in colostrum of cows from 2 herds was on average 3.4 and 2.4 μg/100 ml, respectively. Corresponding value for colostrum of the sows was 67 μg/100 ml. Based on informations about the composition of the food and daily food consumption it could be estimated that 0.5–1 and 20–45 % of the daily intake of iodine were secreted per 1 milk or colostrum of cows and sows, respectively. It can be concluded that the mammary gland of the sow has a considerably higher ability to concentrate iodine than that of the cow. Furthermore the concentration mechanism is more efficient immediately after parturition than at later stages of lactation.     

Maximum volume of nasal administration using a mucosal atomization device without aspiration in Japanese White rabbits

Recently, a mucosal atomization device (MAD) has been applied in veterinary medicine. In the present study, the maximum volume of nasal atomization without aspiration using MAD was examined in eight healthy female Japanese White (JW) rabbits. Each rabbit had their head and neck examined by computed tomography before and after nasal atomization with four different doses (0.15, 0.3, 0.45, and 0.6 ml per nostril) of diluted contrast medium (1:2 mixture of iohexol and saline). This was done under general anesthesia by an intramuscular administration of alfaxalone 2.5 mg/kg, medetomidine 40 μg/kg, and butorphanol 0.4 mg/kg, with a 7-day washout period between each treatment. The diluted contrast medium was distributed in the nasal cavity, external nares, and/or oral cavity in all rabbits receiving each treatment. The intranasal distribution volumes of the contrast medium were 287 (250–333) mm³ [median (interquartile range)] for 0.15 ml, 433 (243–555) mm³ for 0.3 ml, 552 (356–797) mm³ for 0.45 ml, and 529 (356–722) mm³ for 0.6 ml of treatment. The intranasal distribution volume for 0.15 ml treatment tended to be lower than that for 0.6 ml treatment (P=0.083). The contrast medium was deposited in the trachea in one rabbit (12.5%) and four rabbits (50%) receiving treatments of 0.45 and 0.6 ml per nostril, respectively. The maximum volume of nasal atomization without aspiration into the trachea was 0.3 ml per nostril for the JW rabbits.     

A Modified Screening Method for Estimating Erythrocyte Glucose Phosphate Isomerase

A fluorimetric method to estimate erythrocyte glucose phosphate isomerase is described. Five μl of blood is added to 100 μl of water. Ten μl of the resulting hemolysate is incubated with a reaction mixture (200 μl) containing Tris-HCl buffer pH 8.0, 20 μmoles, MgCl₂ 2 μmoles, nicotinamide adenine dinucleotide phosphate 0.08 μmoles, glucose-6-phosphate dehydrogenase 0.2 EU and fructose-6-phosphate 0.12 μmoles. After ten minutes of 25°C 20 μl of the mixture is added to 2 ml of 0.01 M phosphate buffer pH 7.4 and the nicotinamide adenine dinucleotide phosphate fluorescence determined. Two ml of water was added to the remaining reaction mixture and the hemoglobin concentration determined at 410 nm. The technique is primarily designed for use with small amounts of blood, of widely varying activity, from various animal species.     

Prevalences of resistance to seven antimicrobials among fecal Escherichia coli of swine on thirty-four farrow-to-finish farms in Ontario, Canada

Fecal specimens were composited and a hydrophobic-grid membrane-filter method was used to measure antimicrobial resistance to ampicillin 16 μg/ml, carbadox 30 μg/ml, gentamicin 4 μ/ml, nitrofurantoin 32 μg/ml, spectinomycin 16 μg/ml, sulfisoxazole 32 μg/ml and tetracycline 8 μg/ml among 8119 Escherichia coli isolates from 68 fecal samples collected on 34 farrow-to-finish swine farms marketing over 500 hogs/yr. The overall prevalences of resistance to antimicrobials among these isolates were: ampicillin 29%, carbadox 3.5%, gentamicin 0.6%, nitrofurantoin 27%, spectinomycin 28%, sulfasoxizole 38% and tetracycline 71%. Thirty to seventy-six per cent of the variations in prevalences were explained by between-farm differences.     

Comparison of Four Enrichment Media in the Recovery of Campyloracter Jejuni

The effectiveness of 4 enrichment media for the recovery of low levels of inoculated cells of Campylobacter jejuni was evaluated. The media contained antibiotics or antibiotics and bile acids as selective compounds. Three of the media recovered most of the inoculated low numbers of 6 C. jejuni strains. In the 3 media the growth rate of 3 strains, indicated by the increase in the log number of cells during 24 h or 48 h incubation at 42 ° C, was about the same as in the control medium without selective compounds. The same 3 media also recovered a low number of Campylobacter cells from artificially contaminated raw milk or ground meat samples. The enrichment medium B containing 40 I.U. Colistin, 5 μg novobiocin, 2 mg Na-cholic acid and 50 mg cycloheximide per ml was inhibitory for most Campylobacter strains studied.     

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.