The reaction of acclimated isolated gill epithelium of mollusc to a short-term superoptimal heating

Thermoresistance (TR) of isolated gill epithelium of molluscs Anodonta anatina L. (n = 20) acclimated for 72 h at 24 degrees C was studied. In 1, 2, 12, 24, 36, 48, 60, and 72 h, the gills were submitted to a provoking action: a 10-minutes heating at 36 degrees C. As a criterion of the tissue TR level served a logarithmic index of the time of survival at 40 degrees C. During 24 h, negative correlation (about 0.50) was revealed between the acclimated epithelium TR level and the value and direction (sign) of its change under the provoking action. In 36 h, this coefficient fell to 0.13. As a result, the range of interorganism variability of the tissue TR level enlarged. Meanwhile, a decrease in the acclimated tissue mean TR level occurred as late as in 60 h of the experiment, being equal to 21%. In 72 h, it reached 30%, which traditionally indicates a deterioration of the functional state of tissue cells and a subsequent death. However, we believe that the deterioration of this state actually had happened earlier, in 36 h, when the range of the interorganism variability of the tissue TR level became enlarged. It was at this moment that the acclimated tissue lost its ability to regulate the shift in stability with the TR level at this period of acclimation.     

A rapid spectrophotometric method of determining heat-resistant lysosomes in animal tissues

Rapid spectrophotometric method for the determination of thermoresistance in tissue animal lysosomes is described. The of analysis is decreased by 5-6 h, in comparison with enzymatic technique. The determination regimen was chosen in such a way that the process of lysosomal lysis was linear. The dependence of the incubation mixture temperature on the degree of lysosomal lysis was complex. The rate of lysosomal lysis rapidly increased at greater than 37 degrees C. Lysosome incubation at 0-4 degrees C for 24 h decreased its hypothermal (t = 10-30 degrees C), but not hyperthermal (t greater than 37 degrees C) sensitivity. Isolated lysosome thermoresistance may be used as an index of its stability and labialization in vivo and in vitro by various physico-chemical factors. The percentage of initial absorption (A520) and the initial rate of lysosomal lysis (delta A520/min), as well as melting temperature (Tmel) and biological half-life (t1/2) may be the measurements of such effect.     

Development of a dynamic continuous-discrete-continuous model describing the lag phase of individual bacterial cells

AIMS: A previous model for adaptation and growth of individual bacterial cells was not dynamic in the lag phase, and could not be used to perform simulations of growth under non-isothermal conditions. The aim of the present study was to advance this model by adding a continuous adaptation step, prior to the discrete step, to form a continuous-discrete-continuous (CDC) model. METHODS AND RESULTS: The revised model uses four parameters: N(0), initial population; N(max), maximum population; p0, mean initial individual cell physiological state; SD(p0), standard deviation of the distribution of individual physiological states. A truncated normal distribution was used to generate tables of distributions to allow fitting of the CDC model to viable count data for Listeria monocytogenes grown at 5 degrees C to 35 degrees C. The p0 values increased with increasing SD(p0) and were, on average, greater than the corresponding population physiological states (h0); p0 and h0 were equivalent for individual cells. CONCLUSION: The CDC model has improved the ability to simulate the behaviour of individual bacterial cells by using a physiological state parameter and a distribution function to handle inter-cell variability. The stages of development of this model indicate the importance of physiological state parameters over the population lag concept, and provide a potential approach for making growth models more mechanistic by incorporating actual physiological events. SIGNIFICANCE AND IMPACT OF THE STUDY: Individual cell behaviour is important in modelling bacterial growth in foods. The CDC model provides a means of improving existing growth models, and increases the value of mathematical modelling to the food industry.     

Reproducibility of the cold-induced vasodilation response in the human finger.

Cold-induced vasodilation (CIVD) is a cyclic oscillation in blood flow that occurs in the extremities on cold exposure and that is likely associated with reduced risk of cold injury (e.g., frostbite) as well as improved manual dexterity and less pain while working in the cold. The CIVD response varies between individuals, but the within-subject reproducibility has not been adequately described. The purpose of this study was to quantify the within-subject variability in the CIVD response under standardized conditions. Twenty-one volunteers resting in a controlled environment (27 degrees C) immersed the middle finger in warm water (42 degrees C) for 15 min to standardize initial finger temperature and then in cold water (4 degrees C; CWI) for 30 min, on five separate occasions. Skin temperature (Tf) and blood flow (laser-Doppler; expressed as percent change from warm-water peak) responses that describe CIVD were identified, including initial nadir reached during CWI, onset time of CIVD, initial apex during CIVD, time of that apex, and overall mean during CWI. Within-subject coefficient of variation for Tf across the five tests for the nail bed and pad, respectively, were as follows: nadir, 9 and 21%; onset, 18 and 19%; apex, 12 and 17%; apex time, 23 and 24%; mean 10 and 15%. For blood flow, these values were as follows: nadir 52 and 64%; onset, 6 and 5%; apex, 33 and 31%; apex time 9 and 8%; and mean 43 and 34%. Greater variability was found in the temperature response of the finger pad than the nail bed, but for blood flow the variability was similar between locations. Variability in onset and apex time between sites was similar for both temperature and blood flow responses. The reproducibility of the time course of CIVD suggests this methodology may be of value for further studies examining the mechanism of the response.     

Effects of sodium butyrate and dibutyryl cyclic AMP on thermosensitivity of HeLa cells and their production of heat shock proteins

When HeLa cells were incubated at 42 degrees C for 6 h with 1 mM sodium butyrate or when cells treated with 1 mM dibutyryl cyclic AMP for 24 h were incubated at 42 degrees C for 6 h, they were more thermoresistant than heated control cells without such drugs. The production of heat shock proteins was not enhanced by the drug treatment. These results suggest that there is a factor (or factors) other than heat shock proteins that accounts for the thermoresistance of HeLa cells.     

Effects of summer frost exposures on the cold tolerance strategy of a sub-Antarctic beetle

The sub-Antarctic beetle Hydromedion sparsutum (Coleoptera, Perimylopidae) is common locally on the island of South Georgia where sub-zero temperatures can be experienced in any month of the year. Larvae were known to be weakly freeze tolerant in summer with a mean supercooling point (SCP) around -4 degrees C and a lower lethal temperature of -10 degrees C (15min exposure). This study investigated the effects of successive freezing exposures on the SCP and subsequent survival of summer acclimatised larvae. The mean SCP of field fresh larvae was -4.2+/-0.2 degrees C with a range from -1.0 to -6.1 degrees C. When larvae were cooled to -6.5 degrees C on 10 occasions at intervals of 30min and one and four days, survival was 44, 70 and 68%, respectively. The 'end of experiment' SCP of larvae surviving 10 exposures at -6.5 degrees C showed distinct changes and patterns from the original field population depending on the interval between exposure. In the 30min interval group, most larvae froze between -6 and -8 degrees C, a depression of up to 6 degrees C from the original sample; all larvae were dead when cooling was continued below the SCP to -12 degrees C. In the one and four day interval groups, most larvae froze above -6 degrees C, showing no change as a result of the 10 exposures at -6.5 degrees C. As with the 30min interval group, some larvae froze below -6 degrees C, but with a wider range, and again, all were dead when cooled to -12 degrees C. However, in the one and four day interval groups, some larvae remained unfrozen when cooled to -12 degrees C, a depression of their individual SCP of at least 6 degrees C, and were alive 24h after cooling. In a further experiment, larvae were cooled to their individual SCP temperature at daily intervals on 10 occasions to ensure that every larva froze every day. Most larvae which showed a depression of their SCP of 2-4 degrees C from their day one value became moribund or died after six or seven freezing events. Survival was highest in larvae with SCPs of -2 to -3 degrees C on day one and which froze at this level on all 10 occasions. The results indicate that in larvae in which the SCP is lowered following sub-zero exposure, the depression of the SCP is greatest in individuals that do not actually freeze. Further, the data suggest that after successive frost exposures in early winter the larval population may become segregated into two sub-populations with different overwintering strategies. One group consists of larvae that freeze consistently in the temperature range from -1 to -3 degrees C and can survive multiple freeze-thaw cycles. A second group with lower initial SCPs (around -6 degrees C), or which fall to this level or lower (down to -12 degrees C) after freezing on one or more occasions, are less likely to freeze through extended supercooling, but more likely to die if freezing occurs.     

A dramatic accumulation of glycogen in the brown adipose tissue of rats following recovery from cold exposure

In a morphological study of brown adipose tissue (BAT) of rats returned after exposure to cold (+5 degrees C) to neutral temperature (+25 degrees C), striking periodic acid Schiff staining was observed, indicating substantial glycogen accumulation. Enzymatic analysis revealed that the glycogen content increased from the 4.05 +/- 0.51 (micromol glucose unit per gram of tissue, mean +/- SE) control value to 57.3 +/- 9.66 when the animals were returned to neutral temperature for 24 h after a 1-week cold period. Glycogen repletion was also observed in liver and skeletal muscle; however, the glycogen levels in these tissues never exceeded the control values. The accumulation of glycogen in the BAT started by the 3rd hour of replacement and peaked by the 24th hour. This glycogen was readily utilized during the next short cold exposure of the animals. The plasma leptin concentration was reduced at the cold temperature. The hexokinase II activity in the BAT increased to 29.3 +/- 1.46 vs the 11.8 +/- 1.06 control (mU/mg protein +/- SE) after a 1-week cold exposure and this level was maintained during the return to neutral temperature. The total glycogen synthetase (GStot) and the glycogen synthetase a activity also increased after a 1-week cold exposure and increased further during the replacement. The level of GStot reached 26.9 +/- 1.39 vs 9.54 +/- 1.43 control by the 24th hour of replacement. At the same time, the glycogen phosphorylase a activity declined during the replacement. The concentration of glucose 6-phosphate (an activator of GS) decreased in the cold but returned to normal during the replacement. These changes in the BAT are in favor of glycogen synthesis.     

Effect of freezing temperature,at which straws were plunged into liquid nitrogen,on the post-thaw motility and acrosomal status of ram spermatozoa

The present study was conducted to observe the effect of initial freezing temperature on subsequent survival and acrosomal integrity of Malpura and Bharat Merino ram spermatozoa during post-thawing incubation. Semen samples were diluted in TEST-yolk-glycerol extender, loaded in 0.25 ml straws and cooled down to -25, -75 or -125 degrees C freezing temperature using a programmable cell freezer. Computer assisted sperm analysis and acrosomal integrity of thawed samples were assessed after thawing and at hourly intervals during incubation at 37 degrees C for 4 h. The percentage of motile cells in samples frozen at -125 degrees C were 80.3 and 63.7 after post-thawing and -thawing incubation, compared to 75.9 and 39.7 at -25 degrees C or 73.9 and 51.8 at -75 degrees C temperatures, respectively. The spermatozoa with normal acrosome were also significantly, respectively, higher in samples frozen at -125 degrees C, compared to -25 and -75 degrees C temperatures. There were no significant breed variations on percentage of motile, percentage of rapidly motile cells, percentage of normal acrosomes, curvilinear velocity and lateral head displacement except straight line velocity and average path velocity of spermatozoa. The results indicated that -125 degrees C initial freezing temperature conferred the best cryopreserving ability to ram spermatozoa for post-thawing thermoresistance test compared to -25 or -75 degrees C freezing temperature.     

Immobilization of a soluble chemically thermostabilized enzyme

Cellobiase was coupled to a dialdehyde dextran by reductive alkylation in the presence of sodium cyanoborohydride. The resulting conjugate, obtained without loss of enzymic activity, presents properties of thermoresistance largely superior to those of native enzyme: the rate of inactivation is reduced compared to that of native enzyme and its optimal temperature of activity is 70-75 degrees C instead of 65 degrees C. Finally the conjugate presents increased longevity when subjected to experiments of operational stability; its hydrolytic activity is maintained at 60 degrees C in a 10% (w/v) cellobiose solution for more than 100 h whereas the native enzyme is inactivated after 45 h. The cellobiase-dextran conjugate was immobilized by covalent coupling on aminated silica by reductive alkylation in the presence of NaBH(3)CN. The characteristics of thermoresistance of this stabilized and immobilized conjugate were studied and compared to those of a preparation of native cellobiase immobilized on a silica support activated with glutaraldehyde. Analysis of the thermoresistance of these two cellobiase preparations clearly shows that immobilization has maintained and even enhanced their properties. In particular, the operational stability, measured at 68 degrees C on 10% (w/v) cellobiose shows an increased longevity of the stabilized and immobilized enzyme for 120 h compared to 60 h for the native immobilized enzyme. Two successive incubations of these cellobiase derivatives show that it is possible to obtain 2.5 times more glucose with the stabilized-immobilized enzyme than with the immobilized preparation. The procedure described above enables us to prepare a thermostabilized immobilized cellobiase.     

Changes in primary heat sensitivity of rhabdomyosarcoma PA-2 cells in rats during selection for heat resistance

Using the technique of multistep selection, a thermoresistant substrain (TR RA-2T45) was obtained from a rhabdomyosarcoma RA-2 strain. The cells of this substrain retain the ability to form lung metastasis after a 20 minute treatment at 45 degrees C in vitro. The selection of cells for their ability to retain their reproduction activity is accompanied by the increase in their primary thermoresistance. The increased reproductive thermoresistance and primary thermoresistance of the selected cells is hereditary stabilized. Cells of both original strain and its substrain are capable of thermal tolerance, which makes perspective the selection for further increase in thermoresistance in RA-2T4t cell population.     

General and primary thermoresistance in genetically differing variants of murine neuroblastoma

General and primary thermoresistance of mouse neuroblastoma clonal cell lines derived from N18A subline was studied: the N18A1 clonal cell line was not treated by heat, the NTR1 was obtained by one-step selection for resistance to the long action of the temperature 40 degrees C, the NHSR1 was obtained by multistep selection for resistance to short-time treatment at 44 degrees C. The NHSR1 clonal line was shown to have higher general and primary thermoresistance by comparison with that of N18A1 cells. The NTR1 cell line, capable of unlimited proliferation at 40 degrees C, did not differ in general resistance but displayed a slower primary resistance compared to that in the N18A1 cells. Cells of all the three clones were found to be capable of temporary increasing in primary thermoresistance, i.e. hardening. A possible contribution of the primary resistance into the general one in cells of all the selected clones has been discussed.     

Influence of environmental stress on distributions of times to first division in Escherichia coli populations, as determined by digital-image analysis of individual cells

The distributions of times to first cell division were determined for populations of Escherichia coli stationary-phase cells inoculated onto agar media. This was accomplished by using automated analysis of digital images of individual cells growing on agar and calculation of the "box area ratio." Using approximately 300 cells per experiment, the mean time to first division and standard deviation for cells grown in liquid medium at 37 degrees C and inoculated on agar and incubated at 20 degrees C were determined as 3.0 h and 0.7 h, respectively. Distributions were observed to tail toward the higher values, but no definitive model distribution was identified. Both preinoculation stress by heating cultures at 50 degrees C and postinoculation stress by growth in the presence of higher concentrations of NaCl increased mean times to first division. Both stresses also resulted in an increase in the spread of the distributions that was proportional to the mean division time, the coefficient of variation being constant at approximately 0.2 in all cases. The "relative division time," which is the time to first division for individual cells expressed in terms of the cell size doubling time, was used as measure of the "work to be done" to prepare for cell division. Relative division times were greater for heat-stressed cells than for those growing under osmotic stress.     

Heat shock-induced acquisition of thermotolerance at the levels of cell survival and translation in Xenopus A6 kidney epithelial cells.

In this study we have investigated the acquisition of thermotolerance in a Xenopus laevis kidney A6 epithelial cell line at both the level of cell survival and translation. In cell survival studies, A6 cells were incubated at temperatures ranging from 22 to 35 degrees degrees C for 2 h followed by a thermal challenge at 39 degrees degrees C for 2 h and a recovery period at 22 degrees C for 24 h. Optimal acquisition of thermotolerance occurred at 33 degrees degrees C. For example, exposure of A6 cells to 39 degrees degrees C for 2 h resulted in only 3.4% survival of the cells whereas prior exposure to 33 degrees C for 2 h enhanced the survival rate to 69%. This state of thermotolerance in A6 cells was detectable after 1 h at 33 degrees C and was maintained even after 18 h of incubation. Cycloheximide inhibited the acquisition of thermotolerance at 33 degrees C suggesting the requirement for ongoing protein synthesis. The optimal temperature for the acquisition of translational thermotolerance also occurred at 33 degrees C. Treatment of A6 cells at 39 degrees C for 2 h resulted in an inhibition of labeled amino acid incorporation into protein which recovered to approximately 14% of control after 19 h at 22 degrees C whereas cells treated at 33 degrees C for 2 h prior to the thermal challenge recovered to 58% of control levels. These translationally thermotolerant cells displayed relatively high levels of the heat shock proteins hsp30, hsp70, and hsp90 compared to pretreatment at 22, 28, 30, or 35 degrees C. These studies demonstrate that Xenopus A6 cells can acquire a state of thermotolerance and that it is correlated with the synthesis of heat shock proteins.     

Freeze-induced membrane damage in ram spermatozoa is manifested after thawing: observations with experimental cryomicroscopy.

Cryoinjury in ram sperm was investigated by direct observation, using cryomicroscopy, to validate model hypotheses of freezing injury in such a specialized cell. Fluorescein diacetate was used to determine when during the freeze-thaw cycle the sperm membrane became permeable. In noncryoprotected sperm plasma membrane, integrity was maintained throughout the cooling and freezing process, but fluorescein leakage occurred during rewarming. The temperature of post-thaw permeabilization varied in relation to the minimum temperature reached during freezing; cells cooled to -10 degrees C retained fluorescence into the post-thaw temperature range of 9-24 degrees C (mean +/- SEM; 13.25 +/- 0.91 degrees C), whereas cells cooled to -20 degrees C lost fluorescence shortly after thawing (mean +/- SEM; 2.62 +/- 0.91 degrees C). Sperm cooled to 5 degrees C, but not frozen, retained fluorescence during rewarming up to 20-30 degrees C. The inclusion of glycerol and egg yolk in the freezing medium significantly and independently increased the post-thaw permeabilization temperature. Maintenance of fluorescence was also correlated with ability to resume motility after thawing. Sperm reactivation experiments were undertaken to examine deleterious effects of freezing upon the flagellar microtubular assembly. No direct evidence for such effects was obtained. Instead, a highly significant correlation between minimum freezing temperature and post-thaw temperature of initial reactivation was detected.     

High temperature stress and spikelet fertility in rice (Oryza sativa L.)

In future climates, greater heat tolerance at anthesis will be required in rice. The effect of high temperature at anthesis on spikelet fertility was studied on IR64 (lowland indica) and Azucena (upland japonica) at 29.6 degrees C (control), 33.7 degrees C, and 36.2 degrees C tissue temperatures. The objectives of the study were to: (i) determine the effect of temperature on flowering pattern; (ii) examine the effect of time of day of spikelet anthesis relative to a high temperature episode on spikelet fertility; and (iii) study the interactions between duration of exposure and temperature on spikelet fertility. Plants were grown at 30/24 degrees C day/night temperature in a greenhouse and transferred to growth cabinets for the temperature treatments. Individual spikelets were marked with paint to relate fertility to the time of exposure to different temperatures and durations. In both genotypes the pattern of flowering was similar, and peak anthesis occurred between 10.30 h and 11.30 h at 29.2 degrees C, and about 45 min earlier at 36.2 degrees C. In IR64, high temperature increased the number of spikelets reaching anthesis, whereas in Azucena numbers were reduced. In both genotypesor=33.7 degrees C at anthesis caused sterility. In IR64, there was no interaction between temperature and duration of exposure, and spikelet fertility was reduced by about 7% per degrees C>29.6 degrees C. In Azucena there was a significant interaction and spikelet fertility was reduced by 2.4% degrees Cd-1 above a threshold of 33 degrees C. Marking individual spikelets is an effective method to phenotype genotypes and lines for heat tolerance that removes any apparent tolerance due to temporal escape.     

Thermal inactivation and injury of Moraxella-Acinetobacter cells in ground beef.

The thermal inactivation and injury (sensitivity to 0.8% NaCl) of a radiation-resistant culture of Moraxella-Acinetobacter mixed in minced beef were determined. Survival curves for Moraxella-Acinetobacter cells in beef had an initial shoulder preceding a logarithmic decline when the cells were heated at 65, 70, and 75 degrees C, but not at 80 degrees C. In all cases, the experimental points not included in the shoulder were linearized by means of a least-squares straight line, and the latter was used to determine D values. Shoulder values of 12.2, 4.1, and 0.6 min at temperatures of 65, 70, and 75 degrees C were added to the respective D values of 35.4, 6.6, and 1.4 min to determine the time required to destroy one log cycle. The Z value was 7.3 degrees C. Moraxella-Acinetobacter cells in meat were more rapidly injured than inactivated, on initial exposure to heat. The number of cells injured by this initial exposure increased as the temperature was increased. At 65 degrees C the percentage of injured cells increased more rapidly with exposure time than did the inactivated cells. As the temperature was increased, the rates of inactivation and injury became more and more similar.     

Alterations of the composition and size of the free fatty acid pool of Tetrahymena responding to low-temperature stress

Cells of Tetrahymena mimbres (formerly T. pyriformis NT-1) in midlogarithmic growth under isothermal conditions (at 39 degrees C) contained a very small, compositionally discrete pool of free fatty acids, principally (60.6% of the total free fatty acid mass) palmitic and stearic acids. The composition, degree of unsaturation, and size of this free fatty acid pool were rapidly (15 min or less) altered in response to chilling. During the acclimation period following chilling to 15 degrees C, the size of the free fatty acid pool increased from a mean value of 15.5 nmol free fatty acid/mumol lipid phosphorus in 39 degrees C cells to 24.2 nmol free fatty acid/mumol lipid phosphorus. The degree of free fatty acid saturation rapidly increased over the initial hour following the onset of hypothermal conditions, but by 24 h the unsaturated free fatty acid/saturated free fatty acid ratio was 0.35 (equivalent to a 2.7-fold increase in unsaturation relative to 39 degrees C controls (unsaturated/saturated ratio = 0.13) and 4.4-fold greater than cells acclimated for 1 h (unsaturated/saturated ratio = 0.08)). By 24 h the percentage of palmitic and stearic acids had decreased to 45.6%. Similar, and in some instances more pronounced, changes were observed to occur in triacylglycerol-bound fatty acids. Modulation of steady-state free fatty acid composition could also be achieved by the addition of exogenous fatty acids to the growth medium. The ability to manipulate the level of intracellular free fatty acids should prove to be a valuable experimental tool in determining how specific fatty acids regulate various lipid-modifying enzymes.     

Effects of cooling rate and storage temperature on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storage at 4 degrees C, and 5) fast cooling rate to and storage at 4 degrees C. Total spermatozoal motility (TSM), progressive spermatozoal motility (PSM), and spermatozoal velocity (SV) were estimated at 6, 12, 24, 48, 72, 96 and 120 h postejaculation. The longevity of spermatozoal motility was greatly reduced when spermatozoa were stored at 37 degrees C as compared to lower spermatozoal storage temperatures. At 6 h postejaculation, TSM values (mean % +/- SEM) of semen stored at 37 degrees C, slowly cooled to and stored at 25 degrees C or slowly cooled to and stored at 4 degrees C were 5.4 +/- 1.1, 79.8 +/- 1.6, and 82.1 +/- 1.6, respectively. Mean TSM for semen that was cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a moderate rate for four of seven time periods (6, 24, 72 and 120 h), and it was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a fast rate for five of seven time periods (6, 12, 24, 72 and 120 h). Mean TSM of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 25 degrees C for five of seven time periods (24 to 120 h). A similar pattern was found for PSM. Mean SV of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean SV of semen cooled to 25 degrees C for all time periods. A slow cooling rate (initial cooling rate of -0.3 degrees /min) and a storage temperature of 4 degrees C appear to optimize liquid preservation of equine spermatozoal motility in vitro.     

Conversion of steroids in bovine blood in vitro

Blood samples collected from eight Braunvieh cows between the sixth and eighth month of gestation were allowed to stand with and without anticoagulant at 20 degrees C and 0 degrees C for different time periods. In these samples the degree of in vitro conversion of gestagens, androgens and estrogens was investigated. The concentrations were measured by radioimmunoassay. After 24 h at 20 degrees C, the levels of pregnenolone, progesterone, 17alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone and estrone decreased to 62, 29, 25, 10, 34 and 44%, respectively, of the initial value and those of 17alpha, 20beta-dihydroxyprogesterone, epitestosterone and estradiol-17alpha increased to 385, 800 and 852%, respectively. The conversion was slower in clotted blood. The concentrations of testosterone and estradiol-17beta were consistent over the 24 h period. There was no marked decrease of the steroid concentration after 24 h of incubation of whole blood at 0 degrees C and of plasma at 20 degrees C. After the addition of (3)H-steroids, conversion could be demonstrated by thin-layer chromatography and autoradiography. These results demonstrate that all investigated hormones except testosterone and estradiol-17beta were metabolized by bovine blood cells.     

Progesterone metabolism during storage of blood samples from Gyr cattle: effects of anticoagulant, time and temperature of incubation

Ten Gyr cows with a functional corpus luteum were used to evaluate the effects of time and temperature of incubation of blood samples on progesterone (P4) concentrations detected in plasma or serum. From each cow, a blood sample was collected into a flask containing no anticoagulant, another into an heparinized flask and a third into a flask containing sodium fluoride. The blood from each flask was divided into 46 aliquots. One of them was centrifuged within 5 min of collection. The remaining 45 aliquots were divided into three groups and kept at three different temperatures: 4 degrees C, 17 degrees C, or 37 degrees C. For each anticoagulant, aliquots from every cow and incubation temperature were centrifuged every 30 min for 6 h, and then at 8, 12 and 24 h. Plasma or serum were separated immediately after centrifugation and were kept frozen at -20 degrees C until assayed for progesterone. The mean initial concentration of P4 in serum (8.3 ng/ml) significantly diminished (P<0.05) to 6.7 ng/ml after 5 h of incubation at 4 degrees C, 3 h at 17 degrees C, or 2 h at 37 degrees C. In plasma from heparinized blood the initial concentration (7.8 ng/ml) declined significantly after 6 h of incubation at 4 degrees C, 2 h at 17 degrees C, or 1 h at 37 degrees C. Sodium fluoride used as anticoagulant prevented the degradation of P4 since the initial concentration of P4 (6.7 ng/ml) never declined during incubation at either 4 degrees C or 37 degrees C; the only significant reduction occurred after 24 h of incubation at 17 degrees C.