Effects of cooling and warming rate to and from -70 degrees C, and effect of further cooling from -70 to -196 degrees C on the motility of mouse spermatozoa

We have previously reported high survival in mouse sperm frozen at 21 degrees C/min to -70 degrees C in a solution containing 18% raffinose in 0.25 x PBS (400 mOsm) and then warmed rapidly at approximately 2000 degrees C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to -70 degrees C at rates ranging from 0.3 to 530 degrees C/min. The survival curve was an inverted "U" shape, with the highest motility occurring between 27 and 130 degrees C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130 degrees C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was -70 degrees C. When sperm were frozen to -70 degrees C at optimum rates, lowering the temperature to -196 degrees C had no adverse effect.     

Computer-assisted sperm analysis of fresh epididymal cat spermatozoa and the impact of cool storage (4 degrees C) on sperm quality

Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll^® centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR^®14-PI) and DNA fragmentation (TUNEL). After addition of a Tris–glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4 °C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P < 0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation.     

The effects of cooling to 5 degrees C and freezing and thawing on the ultrastructure of bull spermatozoa.

Semen from 6 bulls was examined under the transmission electron microscope immediately after collection, after dilution and cooling to 5 degrees C and after freezing and thawing. Conception rates were determined following artificial insemination of the frozen and thawed semen. Dilution and cooling to 5 degrees C caused acrosomal swelling in about 50% of the spermatozoa. Subsequent freezing and thawing caused considerable ultrastructural changes to the acrosomes (disruption of the plasma and outer acrosomal membranes and dispersion of the acrosomal contents) and middle pieces (breakage of the plasma membrane and a reduction in the electron density of the mitochondrial matrix) of a high proportion of spermatozoa. The average non-return rate following insemination of semen from 5 of the bulls was 61.6% and higher (P greater than 0.001) than for the sixth bull (15%). Although this difference in semen viability was also demonstrated in the structural studies (acrosome, P greater than 0.05: middle piece, P greater than 0.001), more work is required to assess the relationship between structure and function of spermatozoa.     

Acetate Degradation at 70 degrees C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70 degrees C

Anaerobic acetate degradation at 70 degrees C and at 55 degrees C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70 degrees C and less than 15 days was needed at 55 degrees C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55 degrees C up to 90% of the COD was removed. Batch assays showed that sludges from two 70 degrees C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70 degrees C. Both 70 degrees C sludges, as well as the 55 degrees C sludge, produced methane at temperatures of 37 to 73 degrees C. The 55 degrees C sludge exhibited shorter lag phases than the 70 degrees C sludges and higher specific methane production rates between 37 and 65 degrees C.     

Effects of equex STM paste on viability of frozen-thawed dog spermatozoa during in vitro incubation at 38 degrees C

In the canine, artificial insemination with cryopreserved semen generally yields lower pregnancy rates with vaginal deposition than with uterine deposition, one of the reasons being the shortened life span of frozen-thawed spermatozoa. The incubation of spermatozoa at body temperature partially mimics the situation in vivo, and evaluation of the kinetics of viability loss under these conditions can be used to measure the damage caused by freezing and thawing procedures. In this study, 2 aliquots were separated from split ejaculates collected from 7 dogs and were frozen by lowering the straws, in 3 steps, into an LN(2) tank after dilution with egg yolk Tris-citrate-glucose extender with or without the addition of 0.5% Equex STM paste. Motility and plasma membrane integrity (evaluated with the combined fluorescent probes 6-carboxyfluorescein diacetate and propidium iodide) were assessed immediately after thawing and over the next 3 h at 38 degrees C. The addition of Equex STM paste significantly increased the proportion of spermatozoa having an intact plasmalemma immediately after thawing compared with the control. It also increased the longevity of the thawed spermatozoa, prolonging the maintenance of both motility and plasma membrane integrity.     

The post-thaw quality of ram sperm held for 0 to 48 h at 5 degrees C prior to cryopreservation

The effects of holding diluted ram semen at 5 degrees C for up to 48 h prior to cryopreservation were investigated. Semen from six rams was collected by electro-ejaculation in the autumn and again from six different rams in the spring. The sperm concentration and motility were determined using spectrophotometry and computerized automated semen analysis, respectively. Samples were diluted at 23 degrees C to 400 x 10(6)cells/ml in a one-step Tris-egg yolk-glycerol (5%, v/v) media, cooled to 5 degrees C over 2h and maintained at 5 degrees C for the duration of the experiments. Aliquots were loaded into 0.5 ml French straws at 0, 24 or 48 h after cooling, frozen in liquid nitrogen vapor for 12-13 min, 4.5 cm above the liquid nitrogen, and plunged into liquid nitrogen for storage. After thawing, autumn samples frozen after 0, 24, or 48 h of storage exhibited similar percentages of motility (29, 31, 36%, respectively), progressively motility (16, 15, 17%, respectively), plasma membrane integrity (28, 35, 29%, respectively) and live acrosome-reacted cells (0.4, 0.6, 0.8%, respectively; P>0.05). In addition, the quantity of sperm that bound to hen's egg perivitelline membranes after being held at 5 degrees C for 0, 24, or 48 h was not significantly different when the values were expressed as means of the quantity of sperm (155, 177, 106 sperm, respectively) or as the proportion of sperm inseminated (0.39, 0.49, 0.34, respectively; P>0.05). Likewise, ram sperm collected in the spring and frozen at 0, 24 and 48 h after cooling had similar (P>0.05) total motility (21, 25, 20%, respectively), progressive motility (14, 15, 11%, respectively), plasma membrane integrity (26, 33, 31%, respectively) and live acrosome-reacted cells (3.7, 3.5, 3.2%, respectively; P>0.05). The 0 h holding time had significantly less sperm bound to a hen's egg perivitelline membrane compared to the 48 h holding time (250 and 470 sperm, respectively) although the 24h holding time was not different from the 0 or 48 h holding time (281 sperm; P<0.05) but analysis of the proportion of the total sperm inseminated resulted in no significant differences observed (P>0.05). These results indicate that ram sperm can be held at 5 degrees C for up to 48 h prior to freezing with no injurious effects on motility, membrane integrity, or fertilizing potential as indicated by membrane binding ability.     

Epididymal and ejaculated cat spermatozoa are resistant to cold shock but egg yolk promotes sperm longevity during cold storage at 4 degrees C

The aims were to evaluate the susceptibility of feline ejaculated and epididymal spermatozoa to cold shock and to evaluate the effect of egg yolk in the preservation extender. Ejaculated and epididymal spermatozoa from eight males were subjected to a slow (0.5 degrees C/min) or a fast (3 degrees C/min) cooling rate with controls kept in room temperature. Ejaculated and epididymal spermatozoa from another eight males were cooled in a plain Tris buffer (Tris) or in Tris with 20% egg yolk (EYT) and evaluated for 96 h. Subjective motility (MOT), plasma membrane integrity (PMI), and acrosome integrity (ACRI) were evaluated. Cooling did not induce sperm damage regarding PMI (P=0.6) or ACRI (P=0.19) and chilled spermatozoa had better overall MOT (P=0.046) than controls. EYT was better for MOT (P>0.05) from 48 h of cold storage than Tris. EYT was also better for overall ACRI (P<0.0001) while Tris was better for overall PMI (P=0.0004). There were no interactions between time and treatment (P>0.05) for PMI or ACRI. Ejaculated spermatozoa had better overall MOT (P<0.05) and PMI (P<0.05) than epididymal spermatozoa, and higher ACRI in experiment 1 (P=0.0003) but not in experiment 2 (P=0.117). Source of spermatozoa did not affect the susceptibility to cooling or the effect of egg yolk as there were no interactions (P>0.05) between source of spermatozoa and treatment (cooling or control) or between time, source and extender (P>0.05). In conclusion cat spermatozoa were tolerant to cold shock and egg yolk was beneficial for preservation of MOT and ACRI but not PMI.     

Thawing and processing of cryopreserved bovine spermatozoa at various temperatures and their effects on sperm viability, osmotic shock and sperm membrane functional integrity

The objective of this study was to evaluate the effects of thawing and processing temperatures on post-thaw sperm viability, occurrence of osmotic shock and sperm membrane functional status. The occurrence of osmotic shock, characterized by increased spermatozoa with coiled tails, eventually results in reduced sperm viability and sperm membrane integrity. The effects of different thawing temperatures were assessed by thawing frozen specimens at 37, 21 or 5 degrees C for 1 to 2-min, followed by processing at these temperatures. A subset of frozen specimens were thawed at 37 degrees C for 10 to 15-sec and transferred to a water bath at 21 or 5 degrees C for 1 to 2-min to complete thawing, followed by processing at these temperatures. Sperm processing (washing) consisted of dilution, centrifugation and resuspension to remove glycerol from the medium and to gradually return the spermatozoa to isotonic conditions. Post-thawed specimens (0.5 mL) were slowly diluted 1:1 (v/v) at a rate of 0.1 mL/min, centrifuged, and resuspended to 0.5 mL (37 degrees C). Diluted specimens were equilibrated for 1 to 2-min after dilution and for 5-min after resuspension. The specimens were then incubated for 2-h (37 degrees C) and assessed at 60-min intervals for the percentage of motility, for progressive motility (Grades 0 to 4), for the percentage of spermatozoa with coiled tails, and for the percentage of swollen spermatozoa. The percentage of swollen spermatozoa (measurement of sperm membrane integrity) was assessed by exposing spermatozoa to a modified hypoosmotic swelling (HOS) test. The results obtained seem to indicate that physiological thawing and processing temperatures (37 degrees C) are required to maintain sperm motility. However, thawing and processing at lower temperatures (< 37 degrees C) seems to prevent the occurrence of osmotic shock and to maintain sperm membrane functional integrity. In this study, thawing at 37 degrees C (10 to 15-sec) and transfer to a water bath at 21 degrees C (1-min) to complete thawing, followed by processing at 21 degrees C, yielded better results in terms of increased sperm viability, reduced occurrence of osmotic shock and higher reactivity to the HOS test.     

Lipase activity in stallion seminal plasma and the effect of lipase on stallion spermatozoa during storage at 5 degrees C

Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during cooled semen storage. In the first experiment, seminal plasma (1.0 ml) was assayed for lipase activity based upon hydrolysis of triglycerides (olive oil substrate) into free fatty acids and subsequent titration of pH change (SigmaDiagnostic Lipase Kit). Lipase activity in stallion seminal plasma was 0.36 +/- 0.02 Sigma units/ml, (mean + S.E.M.; n = 16 ejaculates from six stallions). In the second experiment, equine semen (three ejaculates from each of four stallions) was divided into five treatment aliquots. In Treatment 1, semen was extended 1:3 with nonfat dried skim milk extender (NFDSM). In treatment groups 2 through 5, spermatozoa were washed by centrifugation (300 x g for 15 min) and resuspended in NFDSM to a final concentration of 25 x 10(6) spermatozoa/ml. Porcine pancreatic lipase (pPL) was added to Treatment 3 (10 pPL units/ml), Treatment 4 (100 pPL units/ml) and Treatment 5 (100 pPL units/ml, heat inactivated at 100 degrees C for 5 min) while Treatment 2 had no pancreatic lipase added and served as the control. Samples were cooled slowly to 5 degrees C, and stored at 5 degrees C until evaluation. Sperm motility was evaluated at time 0, 24, 48 and 72 h by computerized semen analysis, and data were analyzed via repeated measures ANOVA. The addition of 100 units/ml but not 10 units/ml of pPL decreased (P < 0.01) total and progressive motility of stored sperm. Heat-inactivated pPL (Treatment 5) did not significantly decrease motility of spermatozoa during storage. Because the lipase activity assayed (Sigma units) and the lipase activity added to cooled semen (pPL units) were not equivalent, pPL was assayed in the Sigma Diagnostic Lipase assay. The relationship between Sigma Units (Y) and pPL units (X) appeared to be a log-linear relationship with log(Y) = -0.912 + 0.007X; R2 = 0.90. Mean lipase activity assayed in stallion seminal plasma was equivalent to approximately 64 pPL units/ml. These data suggest that endogenous lipase activity in stallion seminal plasma may be a factor in the adverse effects of seminal plasma on cooled spermatozoa in some stallions.     

Morphological change of the acrosome on motile bovine spermatozoa due to storage at 4 degrees C

Swelling of the apical ridge and anterior acrosome of motile bovine spermatozoa was observed during in-vitro storage using differential interference-contrast optics. This morphological alteration is different from that described as the false acrosome reaction on immotile spermatozoa, apparent in ageing semen samples and which has been associated with cell death. In this study, transmission electron microscopy revealed that the apical ridge acrosomal matrix was extended into complex folds and/or projections. Acrosomal and plasma membrane integrity was retained. Storing spermatozoa (1500 X 10(6)/ml) in seminal plasma at 4 degrees C for 1 day was most conducive to the swelling of the apical ridge. Replacing seminal plasma with egg yolk-citrate inhibited swelling. However, incubating semen at 37 degrees C in egg yolk-Tris-fructose extender (25 X 10(6) spermatozoa/ml) after storage in egg yolk-citrate at 4 degrees C for greater than or equal to 3 days restored the swelling characteristic.     

Effect of breed and sperm concentration on the changes in structural, functional and motility parameters of ram-lamb spermatozoa during storage at 4 degrees C

The objectives of this study were (1) to determine the changes in structural, functional and motility parameters of ram-lamb semen stored at two different concentrations at 4 degrees C for 8 days in egg-yolk based extender and (2) to determine the effect of breed of ram-lambs on the changes in structural, functional and motility parameters of ram-lamb semen from different breeds stored at two different concentrations at 4 degrees C for 8 days in egg-yolk based extender. Two different concentrations suitable for laparoscopic and cervical insemination were employed in this experiment. A total of 14 ram-lambs (Polled Dorset-5, Suffolk-5, Katahdin-4) with satisfactory breeding potential were selected. Semen samples were collected by electro-ejaculation. Semen samples were extended to 50 and 200 million sperm per ml with a commercial egg yolk based extender (Triladyl, Minitube of America, Verona, WI, USA) at room temperature and were stored at 4 degrees C. The sperm DNA fragmentation index (DFI), percentages of high mitochondrial membrane potential (hMMP) and plasma membrane integrity (PMI) were assessed using flow cytometry as part of structural and functional parameters on Days 0, 1, 4, 6, and 8. A computer assisted sperm analyser (HTM-IVOS, Version 10.8, Hamilton Thorne Research, Beverly, MA, USA) was used to assess the sperm motility parameters on Days 0, 1, 4, 6, and 8. PROC MIXED procedure was used to determine the effect of days of storage, concentration and breed. The concentration and days of storage significantly affected the sperm structural, functional and motility parameters (P<0.0001). Significant concentration x days of storage interaction was found for all structural and functional parameters. There was a significant concentration x days of storage interaction for average path velocity, curvilinear velocity, straightness and linearity. Overall changes in the sperm structural, functional and sperm motility parameters over the storage period were less dramatic in the 200 x 10(6) ml(-1) concentration when compared to 50 x 10(6) ml(-1) concentration. The hMMP and total progressive motility were influenced by breed. In conclusion, the quality of structural, functional and motility parameters declined as days of storage were increased and the magnitude of changes in the parameters was less dramatic at the higher concentration.     

The use of comet assay to assess DNA integrity of boar spermatozoa following liquid preservation at 5 degrees C and 16 degrees C

The comet assay, under neutral conditions, allows the assessment of DNA integrity influenced by sperm ageing, which is manifested in DNA double-strand breaks. Here, we attempted to use a modified neutral comet assay test (single-cell gel electrophoresis), to our knowledge for the first time, to assess DNA integrity of boar spermatozoa during liquid storage for 96 h at 5 degrees C and 16 degrees C. In this comet assay protocol we used 2% beta-mercaptoethanol prior to the lysis procedure, to aid in removing nuclear proteins. Ejaculates from 3 boars (designated A, C and G) were diluted with a standard semen extender, Kortowo-3 (K-3), which was supplemented with lipoprotein fractions extracted from hen egg yolk (LPFh) or ostrich egg yolk (LPFo). Irrespective of the extender type, the percentage of comet-detected spermatozoa with damaged DNA increased gradually during prolonged storage at 5 degrees C and 16 degrees C. Spermatozoa stored in K-3 extender exhibited elevated levels of DNA damage at both storage temperatures. Significant differences in DNA damage among the boars were more pronounced during storage in LPF-based extenders at 5 degrees C: spermatozoa of boars A and G were less susceptible to DNA damage. The percent of tail DNA in comets was lower in LPF-based extenders, and there were individual variations among the boars. We observed that changes in DNA integrity were dependent on the extender type and storage temperature. A higher level of DNA instability was observed in K-3 extended semen compared with K-3/LPFh or K-3/LPFo extended semen during storage at 5 degrees C. No significant difference in the level of DNA damage between K-3/LPFh and K-3/LPFo was observed. It seems that a long-term storage can affect genomic integrity of boar spermatozoa. The modified neutral comet assay can be used to detect low levels of DNA damage in boar spermatozoa during liquid preservation. Therefore, screening for sperm DNA damage may be used as an additional test of sperm function that can have diagnostic value in practice.     

Changes in lipid content of fowl spermatozoa after liquid storage at 2 to 5 degrees C

Quantitative and qualitative changes may occur in the lipids of spermatozoa during in vitro storage of gametes and may indicate possible degradations occurring within the cells under these conditions. The aim of the present study was to investigate such changes. The motility, viability, morphological integrity and lipid content were measured in fowl semen stored for 48 h at 2 to 5 degrees C and diluted 1:1 in Beltsville Poultry Semen Extender (BPSE) under aerobic agitation. The total lipid content of spermatozoa decreased (P < 0.05) from 820 to 620 micrograms/10(9) cells over 48 h. There was no significant evolution in the total lipid content of seminal plasma (1000 to 850 micrograms/10(9) cells). The proportion of phospholipids in spermatozoa decreased from 75 to 60% of the total lipids. Among the phospholipids, the proportions of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin decreased (P < 0.05) from 58, 13 and 10% at 0 h to 42, 10 and 9% at 48 h, respectively. In contrast, lysophosphatidylcholine, which was marginally represented at 0 h (2%), increased considerably (24%) at 48 h. During the same period, the proportion of motile spermatozoa decreased from 87.5 to 46% and the proportion of viable and morphologically normal cells decreased from 84 to 48%. These results indicate the occurrence of lipid lysis, peroxidation and/or endogenous metabolism able to modify the structure of the spermatozoal membranes and to alter their metabolism and fusion abilities.     

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4 degrees C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22 degrees C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n = 8) using 51Cr labeled autologous platelets. The mean +/- SD recovery 15 min after reinfusion of the BC-PC was 30.5% +/- 13.3% and for PRP-PC 41.4% +/- 7.9% (p less than 0.0001). The survival in vivo for BC-PC was 2.4 days +/- 0.4 days and for PRP-PC 7.0 days +/- 1.4 days (p less than 0.0001). Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4 degrees C, we advocate storage of platelets at 22 degrees C.     

The effect of buffer osmolality on the survival of cat (Felis catus ) spermatozoa at 5 degrees C

Cat semen was diluted at 37 degrees C in Tes-Tris buffer (TesT), pH 7.5, at osmolalities ranging from 195 to 390 m0sm/kg, cooled to 5 degrees C over 90 minutes and stored for 24 hours at that temperature. Motility and percentage of spermatozoa staining with a supravital stain were estimated before cooling, after cooling and after storage for 24 hours. The osmolality of undiluted pooled ejaculates from five animals was measured, and also that of different diluents (citrate with phosphate buffer, lactose and TesT-egg yolk) used for cat semen. The osmolality measurements of cat semen suggested an osmolality of less than 320 m0sm/kg at ejaculation, increasing with time after ejaculation. Varying the egg yolk concentrations (2% to 20%) did not affect the osmolality of TesT diluent. Diluent osmolalities of less than 292 m0sm/kg were found to reduce sperm motility significantly (P <0.001 ) although there was no significant increase in the percentage of cells staining with a supravital stain, while those greater than 325 m0sm/kg increased the variation of response among animals. Cooling and storage significantly reduced motility (P <0.01 to P <0.001 ) and increased the number of stained cells (P <0.001 ). There were significant differences between ejaculates (P <0.01 ) and significant interactions between osmolality and cooling/storage (P <0.05 to P <0.001 ). The best overall results were seen with a TesT diluent of 292 to 325 m0sm/kg which supported good motility for at least 24 hours.     

Relative contributions of biological and chemical reactions to the overall rate of pyrite oxidation at temperatures between 30 degrees C and 70 degrees C

The stoichiometry and kinetics of the spontaneous, chemical reaction between pyrite and ferric iron was studied at 30, 45, and 70 degrees C in shake flasks at pH 1.5 by monitoring the ferrous iron, total iron, elemental sulfur, and sulfate concentration profiles in time. It was found that the sulfur moiety of pyrite was oxidized completely to sulfate. Elemental sulfur was not produced in detectable amounts. The iron moiety of pyrite was released as ferrous iron. All observed initial reaction rates could be fitted into an empirical equation. This equation includes the concentrations of ferric iron and pyrite, and a constant which is dependent on the temperature and the nature of the main anion present. It was observed that ferrous iron formed during the reaction slowed down the oxidation of pyrite by ferric iron. The extent of this effect decreased with increasing temperature. With the aid of the empirical equation, the contribution of the chemical oxidation of pyrite by ferric iron to the overall oxidation in a hypothetical plug-flow reactor, in which biologically mediated oxdidation of pyrite and ferrous iron by oxygen also takes place, can be assessed. At 30, 45, and 70 degrees C, respectively, 2, 8-17, and 43% of the pyrite was oxidized chemically by ferric iron. Therefore, it is expected that only in reactors operating at high temperatures with extremely thermophilic bacteria, will chemical oxidation cause a significant deviation from the apparent first order overall kinetics of biological pyrite oxidation.