Effects of egg yolk and salt on Micrococcaceae heat resistance.

The heat resistance and growth possibilities of various members of the Micrococcaceae in egg yolk and egg yolk with added salt were determined. Egg yolk alone protected members of the Micrococcaceae considerably against heat. Whereas in water Staphylococcus aureus S6 had a decimal reduction time (D) value of 66 s at 55 degrees C, its D value in egg yolk at the same temperature was 246 s. In salted egg yolk (water activity, 0.95), S. aureus S6 had a D value of 180 s at 66 degrees C and was largely inactivated during the pasteurization processes currently applied. Micrococcus saprophyticus and S. epidermidis (D value of each under the same conditions, 390 s) could survive such treatments to a certain extent and can thus spoil commercial egg yolk.     

Heat Resistance of Salmonella in Various Egg Products

The heat-resistance characteristics of Salmonella typhimurium Tm-1, a reference strain in the stationary phase of growth, were determined at several temperatures in the major types of products produced by the egg industry. The time required to kill 90% of the population (D value) at a given temperature in specific egg products was as follows: at 60 C (140 F), D = 0.27 min for whole egg; D = 0.60 min for whole egg plus 10% sucrose; D = 1.0 min for fortified whole egg; D = 0.20 min for egg white (pH 7.3), stabilized with aluminum; D = 0.40 min for egg yolk; D = 4.0 min for egg yolk plus 10% sucrose; D = 5.1 min for egg yolk plus 10% NaCl; D = 1.0 min for scrambled egg mix; at 55 C (131 F), D = 0.55 min for egg white (pH 9.2); D = 1.2 min for egg white (pH 9.2) plus 10% sucrose. The average Z value (number of degrees, either centigrade or fahrenheit, for a thermal destruction time curve to traverse one logarithmic cycle) was 4.6 C (8.3 F) with a range from 4.2 to 5.3 C. Supplementation with 10% sucrose appeared to have a severalfold greater effect on the heat stabilization of egg white proteins than on S. typhimurium Tm-1. This information should be of value in the formulation of heat treatments to insure that all egg products be free of viable salmonellae.     

Heat inactivation of staphylococcus epidermidis at various water activities.

Members of the family Micrococcaceae play an important role in food spoilage and even in food poisoning. In contrast to members of the family Enterobacteriaceae, these bacteria can grow in media with low water activities. Therefore, the heat resistance of Staphylococcus epidermidis, a rather resistant member of the family Micrococcaceae, was studied at water activities between 0.87 and 1.00. The heat inactivation curves were clearly biphasic at all temperatures and water activities tested. Especially at low water activities, the D-values of the tail phase were extremely high (at 0.87 water activity, a D-value at 70 degrees C of 500 s was recorded).     

Effect of turbulent-flow pasteurization on survival of Mycobacterium avium subsp. paratuberculosis added to raw milk

A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity of Mycobacterium avium subsp. paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72 degrees C in duplicate trials. No strains survived at 72 degrees C for 15 s; and only one strain survived at 69 degrees C. Means of pooled D values (decimal reduction times) at 63 and 66 degrees C were 15.0 +/- 2.8 s (95% confidence interval) and 5.9 +/- 0.7 s (95% confidence interval), respectively. The mean extrapolated D72 degrees C was <2.03 s. This was equivalent to a >7 log10 kill at 72 degrees C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6 degrees C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72 degrees C for 15 s, the minimum M. avium subsp. paratuberculosis kill was >4 log10. Properly maintained and operated equipment should ensure the absence of viable M. avium subsp. paratuberculosis in retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2 degrees above 72 degrees C.     

Adaptational changes in Staphylococcus aureus MF31 grown above its maximum temperature when protected by NaCl: physiological studies

Staphylococcus aureus MF31 can grow at 46 degrees C, 2 degrees C above its normal maximum temperature of growth if 1 M NaCl is added to the medium. In the present work we show that monosodium glutamate, proline, threonine, aspartic acid, and betaine (in order of decreasing effectiveness) also enabled cells to grow at 46 degrees C. Cells grown at 46 degrees C in he presence of salt (protected or P cells) accumulated glutamate more rapidly than cells grown at 37 degrees C without salt (normal or N cells) and contained an increased amino acid pool. The principal constituents of this pool were dicarboxylic amino acids and proline. Turbidimetric evidence suggests that NaCl caused plasmolysis in S. aureus. The P cells, although grown in 1 M NaCl, had about the same Cl- and K+ content as the N cells grown without added NaCl. P cells had increased heat resistance but high concentrations of CaCl2 in the heating menstruum reduced their D55 value from a maximum of 214 min to less than 30 s. We suggest that growth at 46 degrees C in 1 M NaCl can be explained, in part at least, by the increased amino acid pool internal to the cell and the external osmotic support given by Cl- anions excluded by the cell.     

Effects of thermoradiation on bacteria.

A 60Co source was used to determine the effects of thermoradiation on Achromobacter aquamarinus, Staphylococcus aureus, and vegetative and spore cells of Bacillus subtilis var. globigii. The rate of inactivation of these cultures, except vegetative-cell populations of B. subtilis, was exponential and in direct proportion to temperature. The D10 (dose that inactivates 90% of the microbial population) value for A. aquamarinus was 8.0 Krad at 25 degrees C and 4.9 Krad at 35 degrees C. For S. aureus, D10 was 9.8 and 5.3 Krad at 35 and 45 degrees C, respectively. Vegetative cells of B. subtilis demonstrated a rapid initial inactivation followed by a steady but decreased exponential rate. The D10 at 25 degrees C was 10.3 Krad, but at 35 and 45 degrees C this value was 6.2 and 3.8 Krad, respectively. Between 0 and 95 Krad, survival curves for B. subtilis spores at 75 degrees C showed slight inactivation, increasing in rat at and above 85 degrees C. The D10 values for spores at 85 and 90 degrees C were 129 and 92 Krad, respectively. Significant synergism between heat and irradiation was noted at 35 degrees C for A. aquamarinus and 45 degrees C for S. aureus. The presence of 0.1 mM cysteine in suspending media afforded protection to both cultures at these critical temperatures. On the other hand, cysteine sensitized B. subtilis spores at radiation doses greater than 100 Krad. The combined effect of heat and irradiation was more destructive to bacteria than either method alone.     

Studies on liquefaction and storage of ejaculated dromedary camel (Camelus dromedarius) semen

The purpose of this study was to evaluate seminal liquefaction and quality of ejaculated camel semen during storage in different extenders at room (23 degrees C) and refrigeration (4 degrees C) temperature. Semen was collected using an artificial vagina and diluted immediately (1:1), using a split-sample technique, in five extenders [(1) Tris-tes egg yolk, (2) Tris-lactose egg yolk, (3) citrate egg yolk, (4) sucrose egg yolk and (5) Tris-fructose egg yolk], while one fraction was kept without an extender to act as control. The semen was transported to the lab at 37 degrees C, in a portable incubator within half an hour, and thereafter liquefaction of semen was monitored every 15 min. After complete liquefaction of the semen it was evaluated for sperm concentration and morphology and then was extended to a final ratio of 1:3. Aliquots of each semen sample were then stored at refrigeration and room temperature. The average volume of an ejaculate was 4.3+/-0.4 mL and it had a very viscous consistency. The average concentration of spermatozoa was 230.4+/-10.7 x 10(6)mL(-1) and the proportion of spermatozoa with protoplasmic droplets averaged 1.02+/-0.2, while 2.7+/-0.6 and 9.7+/-2.9% had mid-piece and tail abnormalities, respectively. All extended semen samples liquefied within 1.5h at 37 degrees C, however, there was slow liquefaction in the sample without an added extender (control). Best liquefaction was observed in Tris-lactose extender followed by Tris-fructose and citrate egg yolk diluents whereas in the other two extenders there was head-to-head agglutination of the spermatozoa. There was no difference in the initial motility of the spermatozoa in extenders 1-5 after its liquefaction, however, after 24 and 48 h of storage a higher proportion of spermatozoa were motile in extenders 1, 2 and 4 (P<0.05) at both the temperatures. There was a gradual decline in viability of the spermatozoa in all extenders at both the temperatures, although, a high portion of the spermatozoa had intact acrosomes throughout the storage period. It may be concluded that dromedary semen, when added to an extender (1:1) immediately after collection, liquefies within 60-90 min at 37 degrees C. It maintains a high proportion of motile and viable spermatozoa that can survive storage up to 48 h in Tris-lactose egg yolk, Tris-tes egg yolk and sucrose egg yolk diluents. However, best liquefaction and progressive sperm motility is achieved in Tris-lactose egg yolk extender.     

Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system

The lactoperoxidase system (LPS) enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus. After LPS activation, biphasic survival curves were observed for L. monocytogenes at 57.8 degrees C and for S. aureus at 55.2 degrees C. The data were consistent with a model that assumed two bacterial populations differing in heat sensitivity. The more heat-sensitive fractions (93% of the L. monocytogenes, 92% of the S. aureus) were killed almost instantly. For these biphasic survival curves, D values were based on the much smaller, less-heat-sensitive fractions. For L. monocytogenes, the D52.2 degrees C values were 30.2 min (untreated milk) and 10.7 min (LPS activated); corresponding D55.2 degrees C values were 8.2 and 1.6 min; corresponding D57.8 degrees C values were 2.3 and 0.5 min. For S. aureus, the D52.2 degrees C values were 33.3 min (untreated milk) and 2.2 min (LPS activated), and the corresponding D55.2 degrees C values were 7.6 and 1.1 min, respectively. The most rapid killing of L. monocytogenes occurred when samples were heated soon after activation of the LPS. Activation of the LPS followed by heating can increase the margin of safety with respect to milkborne pathogens.     

Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system.

The lactoperoxidase system (LPS) enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus. After LPS activation, biphasic survival curves were observed for L. monocytogenes at 57.8 degrees C and for S. aureus at 55.2 degrees C. The data were consistent with a model that assumed two bacterial populations differing in heat sensitivity. The more heat-sensitive fractions (93% of the L. monocytogenes, 92% of the S. aureus) were killed almost instantly. For these biphasic survival curves, D values were based on the much smaller, less-heat-sensitive fractions. For L. monocytogenes, the D52.2 degrees C values were 30.2 min (untreated milk) and 10.7 min (LPS activated); corresponding D55.2 degrees C values were 8.2 and 1.6 min; corresponding D57.8 degrees C values were 2.3 and 0.5 min. For S. aureus, the D52.2 degrees C values were 33.3 min (untreated milk) and 2.2 min (LPS activated), and the corresponding D55.2 degrees C values were 7.6 and 1.1 min, respectively. The most rapid killing of L. monocytogenes occurred when samples were heated soon after activation of the LPS. Activation of the LPS followed by heating can increase the margin of safety with respect to milkborne pathogens.     

Occurrence of Bacillus sporothermodurans and other aerobic spore-forming species in feed concentrate for dairy cattle

AIMS: To determine the aerobic spore composition and presence of Bacillus sporothermodurans spores in feed concentrate for dairy cattle. METHODS AND RESULTS: Six feed concentrate samples from five different farms were analysed. High levels of spores (up to 10(6) spores g(-1)) were found. Identification of 100 selected isolates was obtained by a combination of fatty acid methyl esters analysis, amplified ribosomal DNA restriction analysis and 16S rDNA sequencing. Ninety-seven isolates could be identified to the species level or assigned to a phylogenetic species group. Most of the isolates obtained after a heat treatment of 10 min at 80 degrees C were identified as members of the B. subtilis group (32 isolates), B. pumilus (25 isolates), B. clausii (eight isolates) and B. licheniformis (eight isolates). The isolates with very heat-resistant spores, obtained after a heat treatment of 30 min at 100 degrees C, were identified as members of the B. subtilis group (five isolates), B. sporothermodurans (three isolates), B. amyloliquefaciens (one isolate), B. oleronius (one isolate) and B. pallidus (one isolate). Bacillus cereus was present in each feed concentrate sample and was isolated using a selective mannitol egg yolk polymyxin agar medium. CONCLUSIONS: Feed concentrate for dairy cattle contains known as well as as yet unknown species of Bacillus and related genera with properties relevant to the dairy sector. SIGNIFICANCE AND IMPACT OF THE STUDY: The results formulate the hypothesis that feed concentrate can be a contamination source of spores, including those of B. sporothermodurans, for raw milk at the farm level.     

A modified pork plasma agar for the enumeration of Staphylococcus aureus in foods

The coagulase reaction of Staphylococcus aureus on the PPSA (pork plasma for S. aureus) agar of Devoyod et al. was found to be fibrinogen-deficient. By including bovine fibrinogen (BFG) in the medium, the fibrin halos around S. aureus colonies became more distinct, preparations of pork plasma previously unacceptable for inclusion in the original PPSA agar were performing well, and the amount of pork plasma required in PPSA agar could be reduced by nearly 90%. In the modified medium, designated PPF (pork plasma fibrinogen) agar, the agar base (Baird-Parker agar without egg yolk) was unchanged. After surface plating, the base was covered with 8 mL of a modified overpour agar: 2.5% pork plasma, 0.38% BFG, and 0.0015% soy trypsin inhibitor in 0.7% Bacto agar. Most S. aureus strains could be enumerated after 24 h of incubation at 35 degrees C; the others required 44 h. Without soy trypsin inhibitor, a number of strains showed considerable fibrinolysis between 24 and 44 h of growth; this activity was neutralized by the inhibitor. The S. aureus counts of 27 food samples on PPF agar were essentially the same as the confirmed S. aureus counts obtained by the Baird-Parker method.     

Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa

We investigated the use of duck egg yolk (DEY), Guinea fowl egg yolk (GFEY) and Indian indigenous hen (Desi) egg yolk (IDEY) in extender for improving the post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa, and compared it with commercial hen egg yolk (CHEY; control). For this purpose, two consecutive ejaculates of semen from each of two Nili-Ravi buffalo bulls were collected on 1 day each week for 5 weeks (replicates; n=5) with artificial vagina (42 degrees C). Split pooled ejaculates, were diluted in tris-citric acid glycerol extender containing either DEY or GFEY or IDEY or CHEY at 37 degrees C. Extended semen was cooled to 4 degrees C in 2 h and equilibrated for 4 h at 4 degrees C. Cooled semen was then filled in 0.5 ml straws at 4 degrees C and frozen in programmable cell freezer. Thawing of semen was performed at 37 degrees C for 30 s. Sperm motility, plasma membrane integrity and sperm morphology (acrosome integrity, head, mid-piece and tail abnormalities) of each semen sample were assessed at 0, 3 and 6 h after thawing and incubation at 37 degrees C. Visual motility (%) and percentage of intact plasma membranes assessed at 6h post-thaw of buffalo bull spermatozoa were highest (P<0.05) due to DEY as compared to GFEY, IDEY and control. The percentage of spermatozoa with normal acrosomes at 0, 3 and 6 h post-thaw was highest (P<0.05) in DEY extender than GFEY, IDEY and CHEY. Sperm tail abnormalities (%) observed at 0, 3 and 6 h post-thaw in samples cryopreserved with freezing extender having DEY were lower (P<0.05) as compared to extender containing GFEY, IDEY and CHEY. In conclusion, DEY compared to other avian yolks in extender improves the frozen-thawed quality of buffalo bull spermatozoa.     

Comparison of two dilution rates on canine semen quality after cryopreservation in a coconut water extender

The objective of the present study was to evaluate the effect of sperm dilution (one part semen:one part extender or at 200 x 10(6) spermatozoa/mL) using a coconut water extender on the post-thaw sperm quality. Twelve ejaculates were collected from six dogs. Semen was divided into two aliquots, one for dilution one part semen:one part extender (group 1) and another for a concentration of 200 x 10(6) spermatozoa/mL (group 2). Semen was initially extended at 37 degrees C at a proportion of one part semen:half part extender (1:1/2) for group 1 (A-fraction). For group 2, the volume for a concentration of 200 x 10(6) spermatozoa/mL was calculated and a half of this volume was used for the initial dilution (A-fraction, 37 degrees C). Coconut water extender containing 20% egg yolk was used for this initial dilution in both groups. After dilution, the semen was cooled for 40 min in a thermal box (15 degrees C) and for 30 min in a refrigerator. The other half of the extender (B-fraction) containing egg yolk and glycerol (12%) was added to semen in both groups. Subsequently, the final concentration of glycerol in the extender was 6%. Ejaculates were frozen in 0.25 mL straws 5 cm above the surface of liquid nitrogen and stored at -196 degrees C. After 1 week, straws were thawed at 37 degrees C for 1 min and the microscopic criteria were evaluated. The dilution method had no influence on sperm motility, vigor and normal spermatozoa (71.4 compared with 67.7%). There was no effect of dog, ejaculate within male on post-thaw semen quality. Moreover, there was not a male x treatment interaction. Both treatments were efficient in preserving sperm quality.     

The enumeration of sublethally injured populations of Staphylococcus aureus in foods

Substantiating earlier investigations, pure cultures of Staphylococcus aureus were found to be equally well recovered on Baird-Parker agar at 37°C as at 42°C, whereas Micrococcus spp. are suppressed at the latter temperature to an extent exceeding 5 log₁₀ cycles. It was also established that egg yolk dissimilation by Staph. aureus is intensified at 42°C. Heat treated (60°C) populations of Staph aureus were quantitatively recovered on Baird-Parker agar at 42°C, though acid-injured populations were not. Acid-injury (2% lactic acid at 37°C) could be completely restored by solid medium repaiar during at least 6 h at 23°C on tryptone soya peptone yeast extract egg yolk pyruvate agar. Pure culture studies were confirmed in surveys on trade samples of foods.     

Inactivation of Mycobacterium paratuberculosis in cows' milk at pasteurization temperatures.

The thermal inactivation of 11 strains of Mycobacterium paratuberculosis at pasteurization temperatures was investigated. Cows' milk inoculated with M. paratuberculosis at two levels (10(7) and 10(4) CFU/ml) was pasteurized in the laboratory by (i) a standard holder method (63.5 degrees C for 30 min) and (ii) a high-temperature, short-time (HTST) method (71.7 degrees C for 15 s). Additional heating times of 5, 10, 15, 20, and 40 min at 63.5 degrees C were included to enable the construction of a thermal death curve for the organism. Viability after pasteurization was assessed by culture on Herrold's egg yolk medium containing mycobactin J (HEYM) and in BACTEC Middlebrook 12B radiometric medium supplemented with mycobactin J and sterile egg yolk emulsion. Confirmation of acid-fast survivors of pasteurization as viable M. paratuberculosis cells was achieved by subculture on HEYM to indicate viability coupled with PCR using M. paratuberculosis-specific 1S900 primers. When milk was initially inoculated with 10(6) to 10(7) CFU of M. paratuberculosis per ml, M. paratuberculosis cells were isolated from 27 of 28 (96%) and 29 of 34 (85%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. Correspondingly, when 10(3) to 10(4) CFU of M. paratuberculosis per ml of milk were present before heat treatment, M. paratuberculosis cells were isolated from 14 of 28 (50%) and 19 of 33 (58%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. The thermal death curve for M. paratuberculosis was concave in shape, exhibiting a rapid initial death rate followed by significant "tailing." Results indicate that when large numbers of M. paratuberculosis cells are present in milk, the organism may not be completely inactivated by heat treatments simulating holder and HTST pasteurization under laboratory conditions.     

Use of two freezing extenders to cool stallion spermatozoa to 5 degrees C with and without seminal plasma

Motion characteristics of cooled stallion spermatozoa in 2 freezing extenders were studied. Ejaculates from 8 stallions were split into treatments and cooled in thermoelectric cooling units at each of 2 rates. Cooling started at 37 degrees C for Experiments 1 and 3 and at 23 degrees C for Experiments 2 and 4, at a rate of -0.7 degrees C/min to 20 degrees C and from 20 to 5 degrees C, at either -0.05 degrees C/min (Rate I) or -0.5 degrees C/min (Rate II). Percentages of motile (MOT) and progressively motile spermatozoa (PMOT) were determined at 6, 24 and 48 h. Treatments in Experiment 1 were modified skim milk extender (SM); SM + 4% egg yolk (EY); SM + 4% glycerol (GL); and SM + 4% egg yolk + 4% glycerol (EY + GL). At 24 and 48 h, MOT and PMOT were lowest (P < 0.05) for spermatozoa extended in SM + EY; spermatozoa in SM + GL had the highest MOT and PMOT. Thus, glycerol partially protected spermatozoa against the effects of cooling after long-term storage. Treatments in Experiment 2 were SM, semen centrifuged and pellet resuspended in SM (SMc), SM + EY, and semen centrifuged and pellet resuspended in SM + EY (EYc). Spermatozoa in SM + EYc had the highest (P < 0.05) PMOT at 24 h and MOT and PMOT at 48 hours. Spermatozoa in SM + EY (not centrifuged) had the lowest MOT and PMOT at 24 and 48 h, respectively. There was a detrimental interaction between egg yolk and seminal plasma. Extenders in Experiment 3 were Colorado extender (CO3), CO3 + 4% egg yolk (EY), CO3 + 4% glycerol (GL), and CO3 + 4% egg yolk + 4% glycerol (EY + GL). Spermatozoa in CO3 + EY had the lowest (P < 0.05) PMOT at 24 and 48 h. CO3 did not protect spermatozoa cooled in the presence of seminal plasma. Therefore, in Experiment 4 we tested CO3 with seminal plasma present (control) and semen centrifuged and pellet resuspended in CO3 (CO3c), CO3 + EY (EYc), CO3 + GL (GLc) and CO3 + EY + GL (EY + GLc). Spermatozoa in CO3 had the lowest (P < 0.05) MOT and PMOT at all time periods, which suggested a detrimental interaction of this extender with seminal plasma.     

The protective effect of some food ingredients on Staphylococcus aureus MF31

The upper limiting temperature of growth of Staphylococcus aureus MF31 in heart infusion broth (HI) was about 44 degrees C but addition of monosodium glutamate (MSG) and soy sauce permitted the organism to grow above this temperature. This effect is similar to that of NaCl. Tomato ketchup, Worcestershire and HP sauces added to HI did not allow growth at the non-permissive temperature of 46 degrees C but death was delayed. Staphylococcus aureus died in unsupplemented chicken meat slurry at 46 degrees C but grew at 48 degrees C in slurry supplemented with 5.8% NaCl and survived incubation for 18 h at 50 degrees C in slurry supplemented with 5.8% NaCl and 5% MSG. Cultures grown at 37 degrees C had a D60 value of 2 min in 50 mmol/l Tris (pH 7.2) buffer. Cultures grown at 46 degrees C in HI containing 5.8% NaCl had a D60 value of 8 min in Tris buffer. Addition of 5.8% NaCl plus 5% MSG to the buffer increased the D60 by a factor of about 7 for both cultures. In storage experiments at room temperature, the culture grown at 37 degrees C and at 46 degrees C plus 5.8% NaCl died at about the same rate in salami. In milk powder, however, the count of 37 degrees C culture decreased from 10% g to 10(6)/g in 5 weeks while the count of 46 degrees C culture remained unchanged. In cottage cheese, freeze-dried rice and macaroni, the 37 degrees C cultures also died more rapidly. It is suggested that cultures grown at 46 degrees C plus 5.8% NaCl may be suitable for experiments with artificially contaminated foods.     

Fluorometric evaluation of cryopreserved bovine spermatozoa extended in egg yolk and milk

A comparison of fluorogenically quantifiable parameters of cryopreserved, bovine spermatozoa that had been processed in homogenized milk and egg yolk citrate-based extenders was made using flow cytometry. Semen from four bulls was processed in egg yolk-citrate or homogenized milk extenders, packaged in straws and frozen at -196 degrees C. Samples were thawed at 37 degrees C, subdivided into three portions and stained after 0, 1.5 and 3 h of incubation at 37 degrees C. Spermatozoa were stained using a combination of carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) and analyzed by dual parameter flow cytometry. The sperm cells were quantified fluorometrically at each time interval for both green and red fluorescence. The proportion of spermatozoa retaining the fluorescent CFDA derivative was larger at each time interval for samples in egg yolk citrate than those in milk. Differences in the retention of spermatozoal viability were detected between identical samples of bovine spermatozoa extended in milk or egg yolk based media.     

Long-term preservation of chilled canine semen: effect of commercial and laboratory prepared extenders

The present study was conducted to evaluate chilled semen conservation over time in 3 commercial and 4 laboratory prepared extenders, including a new Tris-glucose extender. The beneficial effect of adding egg yolk to these media was also analyzed. The effects of these extenders on motility and acrosome reaction were characterized objectively using a computer-aided semen analyzer and the chlortetracycline staining, respectively. No significant differences were observed when comparing the different commercial extenders without egg yolk, but addition of egg yolk improved all motility parameters significantly (preservation of 50% of motility was observed at 3.2+/-1, 2.9+/-0.5, 2.3+/-0.5, 8.5+/-0.2, 5.4+/-1.1, 5.2+/-0.4 d, for Biladyl, green extender and fresh-phos extenders without and with egg yolk, respectively). Motility parameters were best preserved in egg yolk supplemented Biladyl extender with a mean percentage of 86.3+/-10.5 motile spermatozoa after 7 d at 4 degrees C. Efficacy of egg yolk-supplemented commercial extenders on sperm motility at 4 degrees C was (in decreasing order) as follows: Biladyl > green extender > fresh-phos. However, high quality motility and the percentage of motile spermatozoa were highest with some of the laboratory prepared extenders: a 50% conservation rate of motile spermatozoa was observed following the use of supplemented egg yolk extenders. These are classified in decreasing order as follows: Tris-glucose (13+/-1 d) > Tris-fructose (9.7+/-0.6) > EDTA (4.+/-0.6 d) > Tris-bes (3.6+/-1.1 d). A low concentration of motile spermatozoa was still observed in the Tris-glucose egg yolk extender 16 d after collection, clearly demonstrating the importance of the medium and the beneficial effect of egg yolk on sperm motility of 4 degrees C chilled semen. Similar effects of extender were observed for acrosome reactions. Egg yolk clearly had a protective effect reducing acrosome reactions significantly in all media tested as follows: the highest acrosome losses were observed in the fresh-phos and EDTA extenders without egg yolk; the lowest rate was observed with Tris-glucose supplemented egg yolk extender. In conclusion, at 4 degrees C, egg yolk extender best-protected sperm motility parameters. Differences in osmolarity between the extenders in terms of substrate related to sperm metabolic activity may explain the optimal results obtained using egg yolk-supplemented Tris-glucose extender, which preserved motility and acrosome integrity in chilled dog semen. These results indicated that good quality dog spermatozoa could be preserved for up to 10 d.     

The unique pentagonal structure of an archaeal Rubisco is essential for its high thermostability

We have previously determined the crystal structure of a novel pentagonal ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. Here we have carried out biochemical studies to identify the necessities and/or advantages of this intriguing pentagonal structure. The structure indicated the presence of three neighboring residues (Glu-63, Arg-66, and Asp-69), participating in ionic interactions within unique dimer-dimer interfaces. We constructed three single mutant proteins (E63S, R66S, and D69S) and one triple mutant protein (E63S/R66S/D69S) by replacing the charged residues with serine. The wild type (WT) and all mutant proteins were purified and subjected to gel permeation chromatography at various temperatures. WT and D69S proteins were decameric at all temperatures examined between 30 and 90 degrees C. The majority of E63S and R66S were decamers at 30 degrees C but were found to gradually disassemble with the elevation in temperature. E63S/R66S/D69S was found in a dimeric form even at 30 degrees C. An interesting correlation was found between the subunit assembly and thermostability of the proteins. Circular dichroism and differential scanning calorimetry analyses indicated that the denaturation temperatures of dimeric enzymes (E63S, R66S, and E63S/R66S/D69S) were approximately 95 degrees C, whereas those of the enzymes retaining a decameric structure (WT and D69S) were approximately 110 degrees C. Disassembly into tetramer or dimer units did not alter the slopes of the Arrhenius plots, indicating that the decameric structure had no effect on catalytic performance per se. The results indicate that the decameric assembly of Tk-Rubisco contributes to enhance the thermostability of the enzyme. Taking into account the growth temperature of strain KOD1 (65-100 degrees C), the decameric structure of Tk-Rubisco can be considered essential for the stable presence of the enzyme in the host cells. This study provides an interesting example in which the thermostability of a protein can be enhanced by formation of a unique quaternary structure not found in mesophilic enzymes.