Comparative Studies of Fresh and Postmortem Isolated Rat Cortical Synaptosomes: Morphology, K+ Transport, and Respiration

Abstract: Synaptosomal fractions were isolated from rat cerebral cortex immediately and at 1, 2, 3, and 4 h postmortem. The capability to undergo function-dependent configurational changes in relation to ouabain- and protoveratrine-sensitive K⁺ uptake, and the rate of O₂ consumption were compared between fresh and postmortem synaptosomal fractions. Our results suggest that a 2 h postmortem storage at room temperature does not decrease substantially the functional performance of synaptosomes, while longer storage periods interfere with their capacity to maintain intracellular cation levels. The findings appear to be relevant to the use of human autopsy material for the preparation of synaptosomes     

Comparison of assessment of fowl sperm viability by eosin-nigrosin and dual fluorescence (SYBR-14/PI)

The kinetics of fowl sperm viability/mortality following short-term and long-term in vitro storage were studied using 2 different staining methods: eosin/nigrosin (observed under light microscopy) and SYBR-14/PI (dual fluorescence). Based on data obtained at 0, 30 min and at 2, 4 and 24 h (T0, T30, T2, T4, and T24) after in vitro storage (4 degrees C, agitated) of fresh or frozen-thawed semen, the dual association SYBR-14/PI was more effective than eosin/nigrosin (P < 0.05) staining for the detection of sperm viability/mortality at early stages (30 min) in nonfrozen ejaculates stored above 0 degree C. In cryopreserved preparations, the 2 techniques were comparable for assessing viable spermatozoa immediately after thawing, but higher percentages (P < 0.05) of nonviable spermatozoa were detected by the SYBR-14/PI procedure for up to 4 h of in vitro storage post thawing (4 degrees C, agitated). Finally, comparable results were observed between the 2 techniques 24 h after beginning in vitro storage post thawing. It is concluded that the dual association SYBR-14/PI procedure is more effective (or, at least, more rapid) than eosin/nigrosin staining for the assessment of sperm viability/mortality in both fresh and cryopreserved samples of fowl semen. However, in the latter case, the thawing stage needs to be followed by a period of in vitro storage lasting at least 4 h to allow for easier discrimination between viable and nonviable populations of spermatozoa.     

POSTMORTEM AUTOLYTIC RESPONSE IN RAT BRAIN LYSOSOMES

Rat cerebral lysosomes were investigated during postmortem autolysis to determine the effect of the length and temperature of storage. A lysosome enriched fraction was isolated by differential ultracentrifugation and there was found to be a progressive increase in fragility as the length of storage increased. However, those lysosomes which survived the homogenisation procedure exhibited similar properties for at least 6 h after death if storage was at 21°C. Changes apparent after further storage for 18 h at 4°C were also detected in a shorter period if the temperature of storage was increased. The results suggest that, coupled with an all-or-none rupture of the lysosomes, there is a small increase in the permeability of the lysosomes shortly after death. In many respects the characteristics of the intact lysosomes up to 6 h after death resemble those obtained immediately.     

A cell-free approach to the study of membrane trafficking in frozen rat brains potentially applicable to frozen autopsy material

Summary A cell-free transfer system was used to measure capacity of brain membranes to support membrane renewal. To study transfer in brain, radiolabeled donor microsome fractions were prepared using brain slices from rats or frozen human brain autopsy specimens. Acceptor fractions, prepared from fresh or frozen rat brain or frozen human brain autopsy specimens, were immobilized on nitrocellulose. The complete reconstituted transfer system contained ATP plus ATP-regenerating system (or NADH) as a source of energy and brain cytosol. Slices of frozen brain incorporated acetate into membrane lipids with approximately the same efficiency as fresh brains. This efficiency declined with storage at 4 °C but only slowly. Donor fractions labeled with acetate from frozen slices exhibited specific transfer (37 °C minus 4 °C) of labeled membrane lipids with efficiencies comparable to fresh. The acceptor fraction could be prepared either from fresh or frozen material. Transfer was on the average two-fold stimulated by ATP at 37 °C compared to no ATP. Transfer also was stimulated by NADH. Analysis of linear transfer rates between 0 and 30 min revealed no significant effect of delay time or of time of prolonged storage on transfer efficiency beyond an initial decline of ca. 25% observed within the first two weeks after freezing. A decline of transfer was obtained with brains as the animals aged.     

Subcellular fractionation of postmortem brain

Abstract— Procedures used to separate subcellular organelles from fresh brain were applied to brains which had been removed from guinea pigs (1) immediately after death; (2) after the dead animal was maintained at room temperature for 3 h, followed by 16–17 h at 4°C; or (3) after the dead animal was maintained for 19–20 h at room temperature. Subcellular fractionation of the brains in 0.32 M sucrose was followed by discontinuous density gradient centrifugation of the crude mitochondrial fraction. After overnight storage of brains at room temperature, there was a moderate shift in succinate dehydrogenase activity from sub-fraction C (mitochondria) to subfraction B (synaptosomes). There was little change in the distribution of galactolipid among particulate subfractions. There was little change in distributions of monoamine oxidase or acetylcholinesterase activities. Under the less extreme postmortem conditions, there were no shifts in the subcellular distributions of brain enzymes. Ultrastructural changes were much more profound and consisted of losses of identifiable mitochondria and synaptosomes and a progressive increase in very dense bodies. Our results suggest that in spite of the marked morphological changes, meaningful separation of subcellular organelles can be achieved with postmortem tissue.     

Spring cabbage peroxidases--potential tool in biocatalysis and bioelectrocatalysis

Two fractions of peroxidase activity, cationic Px-cat and anionic Px-ani, were isolated and partially purified (143.5- and 5.49-fold, respectively) from homogenate of spring cabbage heads. Optimum pH for both fractions is 6.0; however, Px-cat is almost equally active at neutral pH (7.0) while Px-ani reveals high activity in more acidic pHs (with 60% of maximum activity at pH 3.0). Optimal temperature for both fractions was 40 degrees C. Px-ani possessed much higher thermal stability at 40-50 degrees C (60% of remaining activity after 144h of incubation) than Px-cat. The peroxidases remained fully active during 4 weeks of storage at 4 degrees C. Kinetic studies revealed that Px-cat and Px-ani had lower apparent Km values for ABTS (0.0377 and 0.0625mM) and o-dianisidine (0.357 and 0.286mM) than for guaiacol (6.41 and 13.89mM). The best substrate for Px-cat was pyrogallol and for Px-ani-o-dianisidine. Px-cat immobilized on polyanionic PyBA-modified carbon electrode was found to produce linear repetitive signals upon consecutive additions of hydrogen peroxide during at least 1-week period and to work effectively under buffered and non-buffered conditions. These properties were comparable with those of commercially available horseradish peroxidase. Stability of the hybrid bioelectrocatalytic film and low costs of extraction and partial purification of Px-cat make it a highly promising enzyme for practical applications, including construction of bioelectrodes.     

Effect of culture conditions and media on the frequency of chromosomal abnormalities in human sperm chromosome complements

Human sperm chromosomes can be visualized after fusion with hamster eggs. Most laboratories use one of two methods of sperm treatment for capacitation: incubation in a modified Krebs-Ringer medium (BWW) for 5-7 h at 37 degrees C or storage in a TES-Tris yolk buffer (TYB) for 24-72 h at 4 degrees C. To determine whether data from the two methods were comparable, we performed a series of controlled experiments on one normal donor in which ejaculates were split and one aliquot of sperm was capacitated in BWW for 5-7 h at 37 degrees C (fresh) and the second aliquot was capacitated in TYB for 48 h at 4 degrees C (TYB). After capacitation, the technique used to obtain human sperm chromosome complements was identical for both aliquots. Both fresh and TYB sperm were further subdivided into two groups, which were subjected to either a short (1 h) or a long (3 h) gamete coincubation in BWW. This experiment was performed to determine if the longer incubation in BWW might induce chromosomal fragile sites and breaks because of nutritional depletion of the medium. A total of 458 human sperm chromosome complements was analysed. There was no significant difference in the frequency of sperm chromosomal abnormalities or in the sex ratio in the sperm coincubated with eggs for a short (1 h) or long (3 h) time in BWW. When sperm pretreatments were compared, there was a significant increase in the frequency of total sperm chromosomal abnormalities after TYB storage compared to fresh treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of sperm subpopulations in canine ejaculates: effects of cold storage and egg yolk concentration

The aim of this study was to evaluate the effects of cold storage and egg yolk concentration on the distribution of spermatozoa within the different subpopulations. Twenty ejaculates from 4 dogs were collected, diluted in either TRIS buffer containing 20% (TEY20) or 10% centrifuged egg yolk (TEY10) and cooled following a conventional protocol. The kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates and after 24 and 72 h of preservation at 5°C. A multivariate clustering procedure separated 54,261 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (19.80%), Subpopulation 2 consisting of slow and low-linear spermatozoa (25.21%), Subpopulation 3 consisting of high speed and progressive spermatozoa (23.88%), and Subpopulation 4 consisting of highly active but non-progressive spermatozoa (31.11%). Although, cold storage had a significant (P<0.05) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after cold storage. Subpopulations 1 and 2 significantly (P<0.001) decreased during cold storage (Subpopulation 1: 26.6, 16.9 and 18.4%; and Subpopulation 2: 33.6, 21.3 and 24.0%, respectively, for fresh, 24 and 72 h post-cooled), whereas Subpopulations 3 and 4 significantly (P<0.05) increased (Subpopulation 3: 16.7, 27.6 and 24.3%, and Subpopulation 4: 23.1, 34.1 and 33.4%, respectively, for fresh, 24 and 72 h post-cooled). Regarding the relative percentage of spermatozoa within each extender, Subpopulation 3 was more frequently observed in TEY20 after both 24 and 72 h of cold storage. Significant correlations (P<0.05) were found between the proportions of spermatozoa assigned to Subpopulation 3 in the fresh ejaculates and those in stored samples after 24 h (r=0.48498). In conclusion, cold storage significantly modified both the specific parameters and the distribution of spermatozoa within subpopulations. These changes did not affect the general motile sperm structure present in dog, which is conserved during cold storage. The analysis of the changes observed in structures of subpopulations also suggests that the TEY20 provide more effective preservation of dog semen during cold storage.     

Heavy metal composition of polysomal fractions following cadmium challenge

Endogeneous levels of zinc and copper were found to be 1.2±0.1×10⁻² and 0.3±0.1×10⁻² μg/A260 unit, respectively, in polysomal fractions from control animals; cadmium, however, was undetectable. In experimental animals (injected with cadmium) zinc, copper, and cadmium were found in polysomal fractions isolated by two different methods. One hour after a cadmium injection there was a rise in both the zinc and copper content of the polysomal fractions, which then declined steadily to below control levels by 16 h. Neither zinc nor cadmium were dialyzable from these fractions by a TRIS buffer; however, addition of 0.01M EDTA to the buffer resulted in removal of 75% of the zinc and all of the detectable cadmium. The addition of cadmium (CdCl₂) to control supernatants (adjusted to the cadmium concentration present in supernatants 6 h after in vivo exposure) resulted in metal binding to polysomal fractions in levels comparable to those observed after in vivo exposures to the metal. When cadmium was added in the form of cadmium thionein, a smaller fraction of the metal was isolated with the polysomal fraction. Cadmium bound to polysomal fractions in vivo (24 h after exposure) was sensitive to release by protease digestion, but insensitive to release by ribonuclease digestion.     

Effect of Postmortem Factors on Muscarinic Receptor Subtypes in Rat Brain

Although prior studies have supported the validity of measuring total muscarinic receptor binding in postmortem brain, there has not been a study of postmortem effects on muscarinic receptor subtypes, M1 and M2, defined by high and low affinity for pirenzepine, respectively. We have examined in rat brain the effect of postmortem delay at room temperature, storage at 4 degrees C and -20 degrees C, and multiple freeze/thaw cycles on total muscarinic binding, measured with [3H]quinuclidinylbenzilate ([3H]QNB) and on M1 muscarinic binding, measured with [3H]pirenzepine ([3H]Pir). We found that delay at room temperature up to 4 h, or storage at 4 degrees C for 24 h or at -20 degrees C for 4 weeks, or 3 freeze/thaw cycles had no effect on [3H]QNB or [3H]Pir binding. Exposure of brain to room temperature for 15 h, however, led to an increase in [3H]QNB binding, without change in [3H]Pir. Scatchard analysis showed an increase in binding sites without a change in affinity. We conclude that [3H]QNB and [3H]Pir are valid measures of total and M1 muscarinic binding, respectively, under these circumstances, but that caution must be used in the interpretation of indirect measures of M2 binding.     

Microsomal membrane proteins and vanadate-sensitive ATPase from Vicia faba root tips after clinostat treatment.

Microsomal and soluble protein fractions from Vicia faba root tips were used for SDS-PAGE and Western-immunoblot analysis with anti-ubiquitin antibodies after 9 h clinostat treatment of the plants. In contrast to soluble proteins omnilateral gravistimulation (9 h) resulted in an enhanced proteolytic capacity for microsomal proteins. The increase of vanadate-sensitive ATPase activity was 83% after 9 h clinostat treatment, when the enzyme activity was measured directly after membrane preparation. Enhanced ATPase activity was correlated with the appearance of a polypeptide of about 100 kDa and its fragments (93 and 80 kDa). ATPases are not the only membrane bound proteins, which are changed during clinostat treatment, as several ubiquitinated polypeptides were also affected. A 1 h storage of microsomal fractions led to a shift of band intensities on ubiquitin-specific Western-blots. The demonstrated effect could not be observed, when fractions were isolated in the presence of protease inhibitors. In accordance with the polypeptide analysis omnilateral gravistimulation resulted in an enhanced capacity to degrade specific microsomal ubiquitin-conjugates, whereas the soluble ubiquitin-pool was not visibly affected.     

Comparison between Ageing of Slices and Ethylene or Rindite Treatments of Whole Potato Tubers: Studies on Microsomal and Mitochondria1 Enzymatic Activities

When potato tubers (Solanum tuberosum L.) are treated with Rindite(ethylene chlorohydrin, ethylene dichloride, carbon tetrachloride,7/3/1, v/v/v) for 2 d, and then stored at 20°C, importantchanges in enzymatic activities are observed in mitochondrialas well as in microsomal fractions isolated from these tubers.In mitochondria isolated from Rindite-treated tubers, the oxidationofsuccinate was more active and more resistant to cyanide thanin mitochondria from untreated tubers. The functioning of acyanide-resistant, SHAM-sensitive oxidative activity was similarto that observed after an ageing treatment of tuber slices oran ethylene treatment of whole tubers. However, in the lattercase, changes in oxidative properties or cyanide-resistancewere less important. Moreover, all the observed changes disappearedafter 4 d of storage. The NADH-femcyanide reductase and NADHcytochromec reductase activities of microsomal fractions isolated fromRindite-treated tubers increased after 1 d or 4 d of storage,relative to the activities of untreated tubers. Moreover, theoleatedesaturase activity, which could not be detccted in microsomesprepared from untreated tubers, was high in microsomes fromtreated tubers stored for 1 d and 4 d, and then the activitydecreased. The induction of an oleatedesaturase activity byRindite treatment can be compared to the effect of the ageingtreatment on tuber slices. The enhancement of oleatedesaturaseactivity was linked to an increase in cytochrome b5content ofmicrosomes of treated tubers. However, all these changes werenot observed when whole tubers were treated with ethylene alone. Key words: Rindite, Mitochondria, Microsomes, Solanum tuberosum     

Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. I. Distribution of the enzyme activity in microsomes, mitochondria, and golgi complex

The subcellular distribution of NADH-cytochrome b5 reductase in rat liver cells was reinvestigated. In fresh heavy and light Golgi fractions (GF3 and GF1 + 2) and in mitochondria, the specific activity of rotenone-insensitive NADH-cytochrome c reductase was approximately 100, 60, and 30%, respectively, of the value found in microsomes. However, the Golgi enzyme was unstable inasmuch as pelleting and resuspending the fresh fractions resulted in a considerable inactivation (40--60%), which was further increased with subsequent storage at 4 degrees C. A similar inactivation was observed using cytochrome b5 but not ferricyanide as electron acceptor. The inactivation of Golgi NADH-cytochrome c reductase activity was independent of the protein concentration of the fractions during storage, was unaffected by the addition of the antioxidant butylated hydroxytoluene, but was partly prevented by buffering the fractions at neutral pH and by storage at--20 degrees C. A total Golgi fraction was analyzed by density equilibration on continuous sucrose gradients after exposure to digitonin. As expected, the distribution of both protein and galactosyl transferase were shifted to higher densities by this treatment. However, not all galactosyl transferase-bearing elements were shifted to the same extent by exposure to the detergent, suggesting a biochemical heterogeneity of the Golgi complex. In contrast to their behavior in microsomes, the distribution of NADH- cytochrome c reductase and cytochrome b5 of Golgi fractions was shifted by digitonin, although to a lesser extent than that of galactosyl transferase. These results indicate that NADH-cytochrome b5 reductase is an authentic component of Golgi membranes, as well as of microsomes and of mitochondria. The conflicting results reported in the past on the Golgi localization of the enzyme could be due, on the one hand, to the differential lability of the activity in its various subcellular locations and, on the other, to the heterogeneity of the Golgi complex in terms of both cholesterol and enzyme distribution.     

Effect of different fractions of seminal plasma on the fertilizing ability of fowl spermatozoa stored in vitro.

This paper describes the effects of whole seminal plasma and of dialysed seminal plasma on the fertilizing ability of fowl spermatozoa stored for 24 h at 4 degrees C. The fertilizing ability of fowl semen diluted 1:1 with Beltsville Poultry Semen Extender and stored for 24 h at 4 degrees C was enhanced after replacement of the homologous seminal plasma by the diluent (89 versus 77% fertilization rate). Better results were obtained with seminal plasma dialysed against water before sperm storage to discard the less than 1 kDa or the less than 50 kDa fractions. It was concluded that low molecular weight seminal plasma fractions could damage the fertilizing ability of spermatozoa during storage at 4 degrees C, whereas high molecular weight fractions appeared to enhance fertilizing ability.     

Stored xylem sap from wheat and barley in drying soil contains a transpiration inhibitor with a large molecular size

Xylem sap was collected from wheat and barley growing in a drying soil, and the effect of the sap on transpiration was detected by a bioassay with detached wheat leaves. The inhibitory activity of fresh sap was small, and could be largely accounted for by the abscisic acid content (about 2×10^-5mol m^-3). When fresh sap was stored at -20°C for several days, the activity increased. Maximum activity developed after a week. This increase in activity was due to a compound that increased in size with storage at -20°C. When fresh sap was fractionated with filters of different molecular size exclusion characteristics, and the separated fractions stored at -20°C for a week, activity developed only in the fraction containing compounds smaller than 0·3 kDa. However, when sap already stored at -20°C was fractionated, activity was only in fractions containing compounds larger than 0·3 kDa. The increase in activity and in size did not occur with storage in liquid nitrogen (-196°C) or at -80°C. These results suggest that storage at -20°C causes the aggregation or polymerization of a small compound with low activity to form a large compound with high activity. This change is not catalysed by an enzyme because it can occur in a fraction from which molecules larger than 0·3 kDa are removed. It is probably promoted by high solute concentrations when ice crystals form. Sap collected from plants in soils of high water potential had little or no activity after storage at -20°C.     

Effect of semen collection practices on sperm characteristics before and after storage and on fertility of stallions

This study analyzed effects of different methods and intervals of semen collection on the quantity and quality of fresh, cool-stored, and frozen-thawed sperm and fertility of AI stallions. In Experiment 1, ejaculates were obtained from six stallions (72 ejaculates per stallion) using fractionated versus non-fractionated semen collection techniques. Initial sperm quality of the first three jets of the ejaculate was not different from that of total ejaculates. Centrifugation of sperm-rich fractions before freezing improved post-thaw motility and sperm membrane integrity when compared to non-centrifuged sperm-rich fractions or non-fractionated centrifuged ejaculates (P<0.05). In Experiment 2, semen from four stallions (60-70 ejaculates per stallion) was collected either once daily or two times 1h apart every 48 h. The first ejaculates of double collections had significantly higher sperm concentrations, percentages of progressively motile sperm (PMS) after storage for 24h at 5 degrees C and lower percentages of midpiece alterations than single daily ejaculates. Semen collected once daily showed significantly lower values of live sperm after freezing and thawing than the first ejaculate of two ejaculates collected 1h apart every 48 h. In Experiment 3, semen was collected from 36 stallions (> or =12 ejaculates per stallion) during the non-breeding season and the time to ejaculation and the number of mounts was recorded. When time to ejaculation and the number of mounts increased, volume and total sperm count (TSC) also increased (P<0.05), whereas a decrease was observed in sperm concentration, percentage of PMS after storage for 24 h at 5 degrees C, percentage of membrane-intact sperm in fresh semen (P<0.05) as well as motility and percentage of membrane-intact sperm of frozen-thawed sperm (P<0.05). In Experiment 4, AI data of 71 stallions were retrospectively analyzed for the effect of number of mounts per ejaculation and frequency, time interval of semen collections on pregnancy, and foaling rates (FRs) of mares. Semen volume increased, but sperm concentration and percentage of PMS after 24-h cool-storage decreased with increasing number of mounts on the phantom (P<0.05). A statistically significant inter-relationship was demonstrated between frequency and interval of semen collection and FR. Mares inseminated with stallions from which semen was collected frequently (> or =1 on an average per day) showed significantly higher FRs than mares inseminated with semen from stallions with a daily collection frequency of 0.5-1 or <0.5. FR of mares inseminated with stallions having 0.5-1 days between semen collections was significantly better than FR of mares that were inseminated with stallions having semen collection intervals of 1-1.5 days or >2.5 days.     

Viability of nuclei of two-cell mouse embryos stored at 4 degrees C and fused with blastomeres of fresh two-cell embryos.

This study compares the resistance of the nuclei and the cytoplasm of two-cell mouse embryos to short-term storage at low temperature above 0 degrees C. Two-cell embryos were stored at 4 degrees C for 24-96 h in PB1 containing 0.25, 0.5, 0.75, and 1.0 M sucrose. The development to blastocysts in culture was highest in the presence of 0.5 M sucrose. However, only 3% of the embryos developed into blastocysts after 96 h of storage. On the other hand, the viability of the nuclei of two-cell embryos stored at 4 degrees C was significantly prolonged when they were transplanted into a blastomere of enucleated fresh F1 (C57BL/6JXCBA) two-cell embryos. The proportions of chimeric embryos that developed to blastocysts were 88, 67, 76, 71, 64, 45, 32, and 20% following storage for 0, 48, 72, 96, 120, 144, 168, and 192 h, respectively. In addition, there was no difference in the coat color of the young derived from nuclei stored at 4 degrees C or fresh nuclei, although the proportions of chimeric embryos that developed into live young after transfer tended to decrease with increased storage time. Moreover, the viability of nuclei stored at 4 degrees C for 192 h was confirmed in the germ cell population of chimeric mice mated with albino mice. These results demonstrated that the nuclei in the two-cell mouse embryos were more resistant to storage at low temperature than the cytoplasm.     

Recovery of motile,membrane-intact spermatozoa from canine epididymides stored for 8 days at 4 degrees C

The purpose of this study was to determine how long canine spermatozoa remain motile and with intact membranes when maintained within epididymides stored at 4 degrees C, and to determine whether such stored spermatozoa are able to bind to canine zonae pellucidae. Testes with attached epididymides, obtained from 32 dogs (26 purebred; six mixed breeds) at orchiectomy, were refrigerated at 4 degrees C, and spermatozoa were collected from caudae epididymides at nine time intervals ranging from 5 to 192 h. The effects on spermatozoa that had been refrigerated within epididymides for various times were determined by assaying sperm motility, integrity of plasma membranes and of acrosomes, and measuring binding of membrane-intact spermatozoa to canine zonae pellucidae. Membrane integrity was assessed using a double fluorescent dye, and acrosome integrity by staining with Pisum sativum agglutinin. For the zona-binding assay at various refrigeration time points, duplicate sets of six oocytes each, isolated from ovaries retrieved at elective ovariohysterectomy, were placed into 100 microl droplets of sperm capacitation medium containing 5 x 10(6) spermatozoa/ml. One minute later, oocytes were rinsed vigorously by pipetting, and then incubated for 1 h at 38.5 degrees C in a humidified atmosphere of 5% CO2 in air; the number of membrane-intact spermatozoa bound to zonae were counted. There was no significant decrease in membrane integrity and acrosome integrity of spermatozoa recovered from epididymides stored at 4 degrees C within the first 48 h of refrigeration. In contrast, sperm motility decreased significantly within the first 5 h of refrigeration (P < 0.05), but then declined more gradually thereafter. Some spermatozoa recovered from epididymides that had been refrigerated for 192 h retained their capability to bind to zonae pellucidae, although the mean number of refrigerated spermatozoa (0.4) bound to zonae was less than that of fresh samples (9.0). Membrane integrity of spermatozoa recovered from epididymides refrigerated for various times was highly correlated (r = 0.88) with sperm motility. Even after storage for 192 h (8 days) at 4 degrees C, motile spermatozoa could be recovered from the epididymides, and such refrigerated spermatozoa were capable of binding to zonae. We interpreted these data to indicate that it might be possible to recover functional spermatozoa from postmortem specimens of domestic and nondomestic canids.     

Effects of storage and incubation conditions on human granulocyte phagocytic, bactericidal, and chemotactic functions

This study was undertaken to determine the effects of different incubation conditions on human granulocyte (PMN) bactericidal, phagocytic, and chemotactic functions. Specifically, (1) how long may a patient's blood be held before assay and maintain original PMN function, and (2) how long may isolated PMNs be incubated for the purpose of exposure to various agents and still maintain original function? PMNs isolated following storage of whole heparinized blood at 4 °C for 24 and 48 hr phagocytized as well as fresh cells and their bactericidal activity was 96 and 85% of control values after 24 and 48 hr, respectively. Chemotaxis decreased to 62% of control after 24 hr. The bactericidal capacity of isolated PMNs stored at 4 °C for 24, 48, and 72 hr decreased to 85, 81, and 78% of controls, respectively. Phagocytosis after 24 hr storage was equal to controls. Chemotaxis was decreased to 59 and 34% of controls after 24 and 48 hr, respectively. Isolated PMNs incubated at 37 °C demonstrated impairment in phagocytic capacity after only 4 hr.