Numerical simulation of cooling rates in vitrification systems used for oocyte cryopreservation

Oocyte cryopreservation is of key importance in the preservation and propagation of germplasm. Interest in oocyte cryopreservation has increased in recent years due to the application of assisted reproductive technologies in farm animals such as in vitro fertilization, nuclear transfer and the need for the establishment of ova/gene banks worldwide. However, the cryopreservation of the female gamete has been met with limited success mainly due to its small surface-area:volume ratio.In the past decade, several vitrification devices such as open pulled straws (OPS), fine and ultra fine pipette tips, nylon loops and polyethylene films have been introduced in order to manipulate minimal volumes and achieve high cooling rates. However, experimental comparison of cooling rates presents difficulties mainly because of the reduced size of these systems. To circumvent this limitation, a numerical simulation of cooling rates of various vitrification systems immersed in liquid nitrogen was conducted solving the non-stationary heat transfer partial differential equation using finite element method.Results indicate the nylon loop (Cryoloop®) is the most efficient heat transfer system analyzed, with a predicted cooling rate of 180,000 °C/min for an external heat transfer coefficient h = 1000 W/m² K when cooling from 20 to −130 °C; in contrast, the open pulled straw method (OPS) showed the lowest performance with a cooling rate of 5521 °C/min considering the same value of external heat transfer coefficient. Predicted cooling rates of Miniflex® and Cryotop® (polyethylene film system) were 6164 and 37,500 °C/min, respectively, for the same heat transfer coefficient.     

Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time

Specific protocols for the cryopreservation of endangered Cantabrian brown bear spermatozoa are critical to create a genetic resource bank. The aim of this study was to assess the effect of cooling rates and equilibration time before freezing on post-thawed brown bear spermatozoa quality. Electroejaculates from 11 mature bears were extended to 100 × 10⁶ spermatozoa/mL in a TES–Tris–Fructose–based extender, cryopreserved following performance of the respective cooling/equilibration protocol each sample was assigned to, and stored at −196 °C for further assessment. Before freezing, after thawing, and after 1 hour's incubation post-thawing at 37 °C (thermal stress test), the quality of the samples was assessed for motility by computer-assisted semen analysis, and for viability (SYBR-14/propidium iodide), acrosomal status (peanut agglutinin–fluorescein isothiocyanate /propidium iodide), and sperm chromatin stability (SCSA) by flow cytometry. In experiment 1, three cooling rates (0.25 °C/min, 1 °C/min, and 4 °C/min) to 5 °C were assessed. After thawing, total motility (%TM) was higher and percentage of damaged acrosomes (%dACR) was lower (P < 0.05) for 0.25 °C/min than for 4 °C/min. The thermal stress test data indicated equally poor quality (P < 0.05) for the 4 °C/min cooled samples in viability (%VIAB), %dACR, %TM, and progressive motility (%PM). In experiment 2, the effect of a pre-freezing equilibration period at 5 °C for 1 hour (cooling at 0.25 °C/min) was evaluated. Samples kept at 5 °C for 1 hour showed higher (P < 0.05) values than the nonequilibrated ones for both thawing (%dACR) and thermal stress test (%VIAB, %TM, and %PM). In experiment 3, samples stored without cooling and equilibration (direct freezing) were compared with the samples cooled at 0.25 °C/min and equilibrated for 1 hour (control freezing). Using thermal stress test, we observed that direct freezing causes damage in viability, acrosomal status, and motility of spermatozoa compared with the control group (P < 0.05). In conclusion, our results suggest that slow cooling rates to 5 °C and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm.     

Effects of temperature and thermal history on neuromuscular properties of two crustacean species

Summary The effect of temperature on the ability of neuromuscular junctions and muscle fibers to contract, release neurotransmitter, and maintein postsynaptic membrane properties, was measured in vivo and in vitro in claw closer muscles in stone crabsMenippe mercenaria (Say) and blues crabsCallinectes sapidus (Rathbun).In vivo muscle stress (defined as force generated per unit of muscle cross-sectional area) was measured in crabs of both species collected from northern populations (annual temperature range 2–30°C) and southern populations (annual temperature range 15–30°C). Muscle stress was compared between (1) crabs of both species maintained in the laboratory at 30°C (laboratory warmed); (2) crabs given a brief acclimation period (4 weeks for blue crabs; 7 weeks for stone crabs) at 8°C in the laboratory (laboratory colled), and (3) stone crabs that had been naturally acclimated from summer (30°C) to winter (8°C) temperatures over a 6 month period in the field (naturally cooled). No differences were found in the abilities of the northern and southern populations of either species to generate muscle stress when tested at summer temperatures (30°C) common to both populations. Northern and southern blue crabs produced similar levels of muscle stress whether laboratory warmed (30°C) or laboratory cooled (8°C). Conversely, northern and southern stone crabs showed significantly reduced muscle stress in laboratory cooled crabs compared with laboratory warmed crabs. Stone crabs from both populations generated the same amount of muscle stress after having been naturally cooled to 8°C as they had generated the previous summer (30°C).In vitro neuromuscular properties (i.e. (1) muscle stress as a measure of contractile ability; (2) excitatory junction potential (EJP) amplitude as a measure of neurotransmitter release; (3) specific membrane resistance (R_m) as a measure of postsynaptic membrane properties) were compared at 8, 20, and 30°C between northern cold acclimated (naturally cooled stone crabs and laboratory cooled blue crabs) and non-cold acclimated (laboratory cooled stone crabs. Muscle fibers in claws of stone crabs and blue crabs showing cold acclimation had higher R_m at 8°C than non-cold acclimated crabs. This higher R_m resulted in a broadening of EJP's which enhanced EJP summation, muscle fiber depolarization, and muscle stress.Abbreviations EJP excitatory junction potential - E _r resting membrane potential - F _e lacilitation - R _m specific membrane resistance     

Habituation of the metabolic and ventilatory responses to cold-water immersion in humans

An experiment was undertaken to answer long-standing questions concerning the nature of metabolic habituation in repeatedly cooled humans. It was hypothesised that repeated skin and deep-body cooling would produce such a habituation that would be specific to the magnitude of the cooling experienced, and that skin cooling alone would dampen the cold-shock but not the metabolic response to cold-water immersion. Twenty-one male participants were divided into three groups, each of which completed two experimental immersions in 12 °C water, lasting until either rectal temperature fell to 35 °C or 90 min had elapsed. Between these two immersions, the control group avoided cold exposures, whilst two experimental groups completed five additional immersions (12 °C). One experimental group repeatedly immersed for 45 min in average, resulting in deep-body (1.18 °C) and skin temperature reductions. The immersions in the second experimental group were designed to result only in skin temperature reductions, and lasted only 5 min. Only the deep-body cooling group displayed a significantly blunted metabolic response during the second experimental immersion until rectal temperature decreased by 1.18 °C, but no habituation was observed when they were cooled further. The skin cooling group showed a significant habituation in the ventilatory response during the initial 5 min of the second experimental immersion, but no alteration in the metabolic response. It is concluded that repeated falls of skin and deep-body temperature can habituate the metabolic response, which shows tissue temperature specificity. However, skin temperature cooling only will lower the cold-shock response, but appears not to elicit an alteration in the metabolic response.     

Physiological and productive responses of multiparous lactating Holstein cows exposed to short-term cooling during severe summer conditions in an arid region of Mexico

Heat stress generates a significant economic impact for the dairy industry in arid and semi-arid regions of the world, so that heat abatement is an important issue for dairy producers. The objective of this study was to evaluate effects of two short-term cooling periods on physiological and productive status of lactating Holstein cows during hot ambient temperatures. Thirty-nine multiparous cows were blocked by milk yield and assigned to one of three treatments including: control group (C), cows cooled before milking time (0500 and 1700 h daily, 1 h cooling); AM group, cows cooled at 1000 h and before milking (2 h cooling); and AM?+?PM group, cows cooled at 1100, 1500 and 2200 h, as well as before milking (4 h cooling). The cooling system was placed in the holding pen which the cows were moved through for cooling. Respiratory rate, and temperatures of thurl and right flank, were lower (P?P?P?P?相似文献   

Thermoregulatory responses of Holstein and Brown Swiss Heat-Stressed dairy cows to two different cooling systems

Thirty-seven Holstein and 26 Brown Swiss dairy cows were used to evaluate the effect of two different cooling systems on physiological and hormonal responses during the summer. A control group of cows had access only to shade (C). A second group was cooled with spray and fans (S/F) and the third group was under an evaporative cooling system called Korral Kool (KK). The maximum temperature humidity index during the trial was from 73 to 85. Rectal temperatures and respiration rates of the C group were higher (P < 0.05) than those of the S/F and KK groups in both Holstein and Brown Swiss cows. Triiodothyronine levels in milk were higher (P < 0.05) in the KK group than in the S/F and C groups, while cortisol levels were lower (P < 0.05) in the C group than in S/F and KK. There was no significant difference in the hormonal response of the two breeds. These results demonstrate that both cooling systems may be used increase the comfort of Holstein and Brown Swiss cows during summer in hot, dry climates.     

Intracellular ice formation in yeast cells vs. cooling rate: Predictions from modeling vs. experimental observations by differential scanning calorimetry

To survive freezing, cells must not undergo internal ice formation during cooling. One vital factor is the cooling rate. The faster cells are cooled, the more their contents supercool, and at some subzero temperature that supercooled cytoplasm will freeze. The question is at what temperature? The relation between cooling rate and cell supercooling can be computed. Two important parameters are the water permeability (Lp) and its temperature dependence. To avoid intracellular ice formation (IIF), the supercooling must be eliminated by dehydration before the cell cools to its ice nucleation temperature. With an observed nucleation temperature of −25 °C, the modeling predicts that IIF should not occur in yeast cooled at <20 °C/min and it should occur with near certainty in cells cooled at ?30 °C/min. Experiments with differential scanning calorimetry (DSC) confirmed these predictions closely. The premise with the DSC is that if there is no IIF, one should see only a single exotherm representing the freezing of the external water. If IIF occurs, one should see a second, lower temperature exotherm. A further test of whether this second exotherm is IIF is whether it disappears on repeated freezing. IIF disrupts the plasma membrane; consequently, in a subsequent freeze cycle, the cell can no longer supercool and will not exhibit a second exotherm. This proved to be the case at cooling rates >20 °C/min.     

Effect of freezing and thawing on cell membranes of Lentinus edodes,the shiitake mushroom

When cell membranes of Lentinus edodes mycelium were rapidly frozen at either 50 or 160°C/min, viability was lost and this correlated with rupture of the plasmalemma and residual membrane material and with alterations in the organelles. Although with slow cooling (1°C/min) 80% of the samples recovered viability, some cells still showed similar changes to those cooled rapidly, indicating that individual cells of the mycelium do not respond in the same way.     

Survival kinetics of Salmonella enterica serotype senftenberg (S. senftenberg) after heat and acid stress

The survival kinetics of two clinical isolates of Salmonella senftenberg were studied after heat and acid stress. The strains survived better at 53 and 55 °C after heat shock of 30 min at 50 °C or overnight heat adaptation at 45 °C. An increase in the decimal reduction time, D, of heat-shocked [10.2 min (53 °C) and 9.37 min (55 °C)] and heat-adapted [8.12 min (53 °C) and 7.8 min (55 °C)] cells was observed compared with the non-stressed cells [6.87 min (53 °C), 6.56 min 55 °C)]. A significant difference was also observed in the survival of acid-adapted to acid non-adapted S. senftenberg bacteria.     

Survival of Pacific oyster, Crassostrea gigas, oocytes in relation to intracellular ice formation

The effect of IIF in Pacific oyster oocytes was studied using cryo and transmission electron microscopy (TEM). The viability of oocytes at each step of a published cryopreservation protocol was assessed in an initial experiment. Two major viability losses were identified; one when oocytes were cooled to −35 °C and the other when oocytes were plunged in liquid nitrogen. Although the cryomicroscope showed no evidence of IIF in oocytes cooled with this protocol, TEM revealed that these oocytes contained ice crystals and were at two developmental stages when frozen, prophase and metaphase I. To reduce IIF, the effect of seven cooling programmes involving cooling to −35 or −60 °C at 0.1 or 0.3 °C min⁻¹ and holding for 0 or 30 min at −35 or −60 °C was evaluated on post-thaw fertilization rate of oocytes. Regardless of the cooling rate or holding time, the fertilization rate of oocytes cooled to −60 °C was significantly lower than that of oocytes cooled to −35 °C. The overall results indicated that observations of IIF obtained from cryomicroscopy are limited to detection of larger amounts of ice within the cells. Although the amount of cellular ice may have been reduced by one of the programmes, fertilization was reduced significantly; suggesting that there is no correlation between the presence of intracellular ice and post-thaw fertilization rate. Therefore, oyster oocytes may be more susceptible to the effect of high solute concentrations and cell shrinkage than intracellular ice under the studied conditions.     

Vorläufige Ergebnisse von Untersuchungen an eingefrorenen und bei tiefen Temperaturen bestrahlten Lymphozyten

Zusammenfassung Lymphozytenkulturen werden nach Zugabe von Glyzerin als Gefrierschutzsubstanz zunächst gemeinsam mit ungefähr 1 °C/min Abkühlgeschwindigkeit bis zum vollständigen Erstarren eingefroren und dann mit verschiedener Geschwindigkeit ( 200 °C pro min und 3 bis 5 °C/min) auf verschieden tiefe Temperaturen (–22, –80 und –196 °C) gebracht. Alle eingefrorenen Proben weisen nach 24 h Kulturdauer eine kleinere, nach 48 h Kulturdauer eine wesentlich höhere Mitoserate als die Kontrollgruppe (unbehandelte, nicht eingefrorene Plasmaproben) auf. In den schnell auf –196 °C abgekühlten Proben wurden in beiden Fällen (24 h und 48 h Kulturdauer) keine Metaphaseplatten gefunden. Die Zellkonzentrationen waren nur bei den schnell auf –196 °C abgekühlten Kulturen stark verringert. Die Chromosomenaberrationsrate der eingefrorenen Kulturen ist nicht signifikant erhöht.

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Preliminary results of investigations of lymphocytes, frozen and irradiated at low temperaturesI. The effects of cooling rate, low temperature, and incubation time on frozen lymphocytes

Summary Peripheral human lymphocytes were cooled to the temperature of solidification at a rate of less than 1 °C/min using glycerol as a protective agent against the effects of freezing. After solidification at temperatures of –10 to –15 °C and cooling to about –22 °C one group of the samples was thawn and the others were cooled rapidly to –80 or –196 °C by immersion into solid carbon dioxide or liquid nitrogen. Other samples of the frozen cell suspension were cooled down to the same temperatures much slower at a cooling rate of 3 to 5 °C/min. After rapid thawing in a 25 °C water bath the cell suspensions were removed from glycerol and cultured for 24 or 48 h before stopping mitoses by adding colchicine. The samples frozen at –22 °C, the samples cooled both rapidly and slowly to –79 °C, and the ones cooled slowly to –196 °C showed a lower rate of mitosis when colchicine was added after 24 h and a significantly higher rate of mitosis after 48 h of incubation before adding colchicine as compared to the controls (untreated, unfrozen plasma). In the culture frozen rapidly to –196 °C no metaphases could be found. The cell concentrations before and after freezing showed no significant differences except those of the culture frozen rapidly to –196 °C. The chromosome aberration rate is not significantly increased.

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Herrn Prof. Dr. H. A. Künkel zum 60. Geburtstag gewidmet.     

The proteomic response of sea squirts (genus Ciona) to acute heat stress: a global perspective on the thermal stability of proteins

Congeners belonging to the genus Ciona have disparate distributions limited by temperature. Ciona intestinalis is more widespread with a cosmopolitan distribution ranging from tropical to sub-arctic zones, while Ciona savignyi is limited to temperate-latitudes of the northern Pacific Ocean. To compare the heat stress response between congeners, we quantified changes in protein expression using proteomics. Animals were exposed to 22 °C, 25 °C, and 28 °C for 6 h, then recovered at a control temperature (13 °C) for 16 h (high heat stress experiment). In a second experiment we exposed animals to lower levels of heat stress at 18 °C, 20 °C, and 23 °C, with a 16 °C control. A quantitative analysis, using 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry (with a 69% and 93% identification rate for Ciona intestinalis and Ciona savignyi, respectively), showed changes in a number of protein functional groups, including molecular chaperones, extracellular matrix proteins, calcium-binding proteins, cytoskeletal proteins and proteins involved in energy metabolism. Our results indicate that C. intestinalis maintains higher constitutive levels of molecular chaperones than C. savignyi, suggesting that it is prepared to respond faster to thermal stress. Systematic discrepancies between estimated versus predicted molecular masses of identified proteins differed between protein families and were more pronounced under high heat conditions, suggesting that thermal sensitivities are lower for cytoskeletal proteins and ATP-synthase than for any other protein group represented on 2D gels.     

The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm

Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10 to 12 weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode’s lactate (TL-HEPES), modified Kreb’s Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon’s Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1 M raffinose or 0.1 M sucrose. The sperm samples in each extender were cooled to 4 °C and held for 45 min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of −150 °C by using various cooling rates (10, 40, 70, and 100 °C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37 °C slide warmer and held for 1 min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P < 0.05). Sperm motility increased as cooling rate was increased for both strains (P < 0.05). Highest cryosurvival was achieved when 100 °C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1 M sucrose or raffinose (P < 0.05). This study showed that TES extender containing 0.1 M raffinose or sucrose with 70 °C/min and 100 °C/min cooling rate improved post-thaw motility of rat sperm.     

Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification

There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to −196 °C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95 to 69,250 °C/min to −196 °C and for each cooling rate, subjecting them to five warming rates back above 0 °C at rates ranging from 610 to 118,000 °C/min. In samples warmed at the highest rate (118,000 °C/min), survivals were 70% to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610 °C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95 to 69,000 °C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming.     

Thermal adaptability of large white pigs in the tropical environment

Twenty-four Large White weaners-twelve males and twelve females, were randomly divided into three groups of eight (four males and four females per two separate pens) and were assigned to three groups of two pens each for the males and the females weaners.One group of two pens was without wallow facility (control), one other group was provided with wallows (wet) and the third group was in air-conditioned room (cold). Twice a day, the respiratory rate and the rectal temperatures were measured early in the mornings at 8.00–9.00 hrs (A.M.) and late in the early evenings at 16.00–18.00 hrs.The mean respiratory frequency (A.M) ranged from 7 to 9; 6–12 and 8–13 breaths per minute for the cold, wet and control respectively while the mean respiratory frequency (P.M.) ranged from 6 to 9, 10 to 17 and 13 to 19 breaths per minute for the cold, wet and control respectively.The mean rectal temperatures (A.M.) ranged very slightly from 38.54° to 39.12°C; 38.50° to 39.05°C and 38.61°C to 39°C for the cold, wet as control respectively while the mean rectal temperatures (P.M.) ranged from 39.00° to 39.22°C; 38.97° to 39.29°C and 39.28° to 39.55°C for the cold, wet and control respectively.The animals were maintained for another seven to ten weeks and were slaughtered. The slaughter characteristics did not indicate an appreciable thermal stress except for the reproductive organs which showed weight increase indicating reduced efficiency of the thermally stressed animals as is the case in the tropical environment.     

The effects of elevated temperature and humidity on rectal temperature and respiration rate in the New Zealand white rabbit

In 2 replicated factorial experiments, 7-h climate chamber exposures were used to study the responses of adult NZW rabbits to a range of elevated temperatures and humidities. At 18 mm Hg water vapour pressure, 23.8° C was well tolerated, rectal temperature (RT) and respiration rate (RR) averaging 38.6±0.3° C and 82.9±15.5 breaths/min, respectively. Both parameters were elevated (P<0.001) at 32.2°, 37.8° and 43.3° C. RT and RR reached plateau levels of 39.5–40.1° C and 410–460/min at 32.2° C, which was tolerated for the full 7-h test period. Test temperatures of 37.8° and 43.3° C, on the other hand, could be tolerated for only 80 and 40 min respectively, before RT reached the safe upper limit of 41.7° C. Final RR values at 37.8° and 43.3° C were 701.6±42.7 and 812±55.1/min, respectively. In a 34.5° C atmosphere a humidity of 21 mm Hg water vapour pressure was classified as dry, and was tolerated for 323±123 min. RT and RR increased by 0.6° C and 316/min during the first 20 min of exposure (P<0.05). Thereafter both parameters increased progressively, but with no significant differences between successive recording periods, until RR reached 550.3±88.8/min at 41.7° C RT. Humidities of 25, 29 and 33 mm Hg water vapour pressure were, on the other hand, classified as wet and were tolerated for only 92±22, 81±16 and 119±50 min, respectively. RR at the times that RT reached 41.7° C at these 3 humidities was 732±26, 789±30 and 764±23/min, respectively. The results point to the likelihood that thermal stress will adversely affect the productivity and welfare of NZW rabbits in the tropics unless adequate housing environments are provided. Significant between-individual phenotypic differences in heat tolerance suggest the need for genetic studies of the possibility of selecting for improved heat tolerance.     

Seasonality of phytoplankton in relation to silicon cycling and interstitial water circulation in large,shallow lakes of central Canada

The main basins of Lake Winnipeg (52°N 97°E) and Southern Indian Lake (57°N 99°W) had similar phytoplankton cycles during their open water seasons. A brief spring algal maximum was followed by an early summer minimum and, subsequently, an extended autumnal increase when highest biomasses were observed. The maxima were dominated by Melosira spp. The seasonal cycle of Melosira followed closely the seasonal cycle of dissolved Si. These basins exhibited a typical phytoplankton cycle for dimictic lakes even though they did not form a significant thermocline (1°C per meter).The lakes were well-mixed because they were shallow and had large wind fetches. Although thermal stability of the water column was always low, it was positive until maximum heat content was achieved at which time it became nil or negative. These lakes heated and cooled rapidly, and sediment heat storage was a substantial fraction of the total heat budget. Because heating and cooling of water and of sediments were out of phase, heat exchange at the sediment surface could control vertical circulation of interstitial water, nutrient exchange across the sediment-water interface and the seasonality of phytoplankton. Thermal gradients in the sediments during the heating season would be quite pronounced (4°C per meter).It is proposed that positive stability in interstitial waters during the heating season would impose molecular diffusive transport on the sediment column. When the lakes begin to cool, the upper interstitial water column would become thermally unstable and circulation would occur within the sediments. This would result in the observed net flux of dissolved Si, and other nutrients, out of the sediments into the overlying waters. As a consequence, in Lake Winnipeg and Southern Indian Lake the highest phytoplankton biomasses and productivity occurred in the late summer and autumn.     

Suprazero cooling rate,rather than freezing rate,determines post thaw quality of rhesus macaque sperm

Sperm become most sensitive to cold shock when cooled from 37 °C to 5 °C at rates that are too fast or too slow; cold shock increases the susceptibility to oxidative damage owing to its influence on reactive oxygen species (ROS) production, which are significant stress factors generated during cooling and low temperature storage. In addition, ROS may be a main cause of decreased motility and fertility upon warming. They have been shown to change cellular function through the disruption of the sperm plasma membrane and through damage to proteins and DNA. The objective of this study was to determine which cryopreservation rates result in the lowest degree of oxidative damage and greatest sperm quality. In the rhesus model, it has not been determined whether suprazero cooling or subzero freezing rates causes a significant amount of ROS damage to sperm. Semen samples were collected from male rhesus macaques, washed, and resuspended in TEST-yolk cryopreservation buffer to 100 × 10⁶ sperm/mL. Sperm were frozen in 0.5-mL straws at four different combinations of suprazero and subzero rates. Three different suprazero rates were used between 22 °C and 0 °C: 0.5 °C/min (slow), 45 °C/min (medium), and 93 °C/min (fast). These suprazero rates were used in combination with two different subzero rates for temperatures 0 °C to −110 °C: 42 °C/min (medium) and 87 °C/min (fast). The different freezing groups were as follows: slow-med (SM), slow-fast (SF), med-med (MM), and fast-fast (FF). Flow cytometry was used to detect lipid peroxidation (LPO), a result of ROS generation. Motility was evaluated using a computer assisted sperm motion analyzer. The MM and FF treated sperm had less viable (P < 0.0001) and motile sperm (P < 0.001) than the SM, SF, or fresh sperm. Sperm exposed to MM and FF treatments demonstrated significantly higher oxidative damage than SM, SF, or fresh sperm (P < 0.05). The SM- and SF-treated sperm showed decreased motility, membrane integrity, and LPO compared with fresh semen (P < 0.001). Slow cooling from room temperature promotes higher membrane integrity and motility post thaw, compared with medium or fast cooling rates. Cells exposed to similar cooling rates with differing freezing rates were not different in motility and membrane integrity, whereas comparison of cells exposed to differing cooling rates with similar freezing rates indicated significant differences in motility, membrane integrity, and LPO. These data suggest that sperm quality seems to be more sensitive to the cooling, rather than freezing rate and highlight the role of the suprazero cooling rate in post thaw sperm quality.     

Cytochemical characterization of deoxyribonucleoprotein by UV-microspectrophotometry on heat denatured cell nuclei

Denaturation of double-stranded DNA into a single-stranded state can be studied by heating fixed cells attached to quartz slides and then determining the increase in nuclear UV-absorption at 265 nm by microspectrophotometry at room temperature. In order to prevent renaturation as the slides are cooled, formaldehyde is added to the solution in which heat denaturation is performed. The influence of formaldehyde concentration, duration of heating and ionic strength on the stability of DNA to heat denaturation has been examined. The standard method involves heating of ethanol/acetone fixed cells to temperatures between 22°C and 100°C in 0.15 M NaCl, 0.015 M sodium citrate containing 4% formaldehyde for 20 min followed by cooling to room temperature and mounting in glycerol.     

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.