Effect of holding time and temperature of bovine whole blood on concentration of progesterone, estradiol-17beta and estrone in plasma and serum samples

Bovine jugular venous blood was collected, with and without heparin, and aliquoted into 140 12-ml tubes. Four subsamples (two heparinized and two coagulated) were centrifuged immediately (time zero) and plasma or serum was aspirated and stored at -20 degrees C. One-half of the remaining subsamples were stored at 4 degrees C and the other one-half at 25 degrees C (room temperature). At 1-h intervals (0 to 24 h), 6-h intervals (24 to 72 h) and at 96 and 120 h, four subsamples (heparinized and coagulated at both 4 degrees C and 25 degrees C) were centrifuged, plasma or serum was aspirated and stored at -20 degrees C. Whole blood incubation for 1 h at 25 degrees C reduced mean plasma and serum progesterone (P(4)) concentration (P<0.05). Similarly, whole blood incubation at 4 degrees C for 2 and 3 h, respectively, reduced mean plasma and serum P(4) concentration (P<0.05). No difference was found in mean P(4) concentration between plasma and serum samples harvested from whole blood incubated at 4 degrees C or 25 degrees C. Concentration of estradiol-17beta (E(2)) and estrone (E(1)) fluctuated over time, irrespective of holding temperature. There was a blood type, heparinized or coagulated, by time interaction (P<0.01) for both E(2) and E(1) concentrations It was concluded that incubation time and temperature between collection and centrifugation of bovine blood samples influenced the assayable P(4) concentration in both plasma and serum. In contrast, incubation temperature had no effect on assayable E(2) and E(1) concentrations, but assayable E(2) and E(1) over time were differentially affected, depending on whether plasma or serum was assayed.     

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

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

Effect of time and temperature of incubation of heparinized caprine blood on concentrations of progesterone detected in plasma

Blood samples from 12 pregnant does were collected in flasks containing heparin. Each sample was divided into 31 aliquots, the first of which was immediately centrifuged for plasma separation. Fifteen of the remaining aliquots from each goat were kept at 17 degrees C, while the remaining is were refrigerated at 4 degrees C. Centrifugation of these aliquots was carried out at half-hour intervals until completion of 6 h, and then at 8, 12 and 24 h post collection. The mean concentration of progesterone in the aliquots centrifuged initially was 4.0 +/- 0.3 ng/ml, and it was not significantly altered during incubation of the blood at 17 degrees C. In contrast, the concentrations of progesterone increased significantly to 5.0 +/- 0.3 (P < 0.05) during the first hour of incubation at 4 degrees C, remaining elevated most of the time. The stability of progesterone during incubation of heparinized caprine blood at 17 degrees C indicates that immediate centrifugation is not necessary. Refrigeration during the storage of blood samples is not recommended since progesterone levels are altered, probably due to separation of progesterone from the membranes of red blood cells during the hemolysis that is caused by incubation at low temperatures.     

Effect of time and temperature on bovine serum and plasma progesterone concentration

Whole blood, with and without anticoagulant, from 5 pregnant cows was incubated at 40°C for 0 (30 minutes after collection), 6 and 24 hours (hr) before the blood was centrifuged and the plasma or serum was frozen for later progesterone assay. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 1.7 ng/ml at 6 hr (P < 0.01) and to 2.8 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 3.9 ng/ml at 6 hr (P < 0.01) and to 4.4 ng/ml at 24 hr (P < 0.01). Whole blood samples with and without EDTA were also incubated at 4°C for 24 hr. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 4.2 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 4.7 ng/ml at 24 hr (P < 0.01). The incubation time and temperature of whole blood, from collection of blood to the separation of serum or plasma, significantly affects assayable concentration of progesterone.     

The effects of storage time and temperature and anticoagulant on laboratory measurements of canine blood progesterone concentrations

The effects of anticoagulant, storage time, storage temperature, and assay method, on laboratory measurements of blood progesterone concentrations of dogs is unclear; these factors have had a dramatic effect on blood progesterone concentrations in other species (particularly cows). In six experiments, we determined the effects of assay technique (chemiluminescence versus radioimmunoassay (RIA)), storage time, and temperature, as well as the use of heparinized plasma versus serum (coagulated blood) on measured progesterone concentrations of bitches. The studies showed that: (a) RIA measured significantly higher serum progesterone concentration (SPC) than chemiluminescence; (b) refrigeration of whole blood during the first 2 h after sample collection significantly decreased measured SPC; (c) progesterone concentration in heparinized plasma was not affected by storage temperature of whole blood for at least 5 h; (d) refrigeration of whole, clotted blood did not affect SPC, provided that samples were held at room temperature for the first 2 h after collection. These findings are of particular importance when blood samples are collected for determination of the initial rise in SPC that is associated with the LH surge in estrous bitches.     

Time-dependent loss of radioimmunoassayable levels of progesterone following ambient temperature incubation of heparinized bovine blood

Plasma progesterone levels in heparinized blood collected at 10 min intervals for 8 continuous hours from four nulliparous Holstein cows on day 3 (early luteal), day 10 or 11 (mid-luteal) and day 18 or 19 of the estrous cycle were found to decline over time when blood was incubated at ambient temperature. The loss was more obvious during the mid-luteal collection period than either the day 3 or day 18 or 19 periods in all cows. This appeared to be associated more with high progesterone levels on day 10 or 11 rather than with differences in the period of the estrous cycle. There was an average decrease in progesterone levels of 3.4, 1.0 and 1.5 ng/ml between samples having the shortest and longest incubation periods on day 10 or 11, day 3 and day 18 or 19, respectively. This apparent decrease in levels of progesterone from bovine blood indicates need in the future for careful consideration concerning the handling of bovine blood collected for subsequent radioimmunoassay (RIA) of progesterone. Further work to elucidate the mechanism which is responsible for the apparent loss is also warranted.     

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 degrees 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 degrees 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 degrees C demonstrated impairment in phagocytic capacity after only 4 hr.     

Ethylene glycol and glycerol loading and unloading in porcine meniscal tissue

The development of a long-term storage method for meniscus, a complex tissue of the knee prone to injury, would improve the procedure and outcomes of meniscus transplantation. Cryopreservation uses cryoprotective agents (CPAs) including ethylene glycol (EG) and glycerol to preserve a variety of live tissues, and understanding of the CPA permeation kinetics will be critical in designing a vitrification protocol for meniscus.The purpose of this preliminary study was to understand the loading and unloading behaviours of EG and glycerol in meniscus by observing their efflux. For the main experiment, lateral and medial porcine menisci were incubated with CPA for 24 h at three temperatures (i.e., 4, 22, and 37 °C). Then, the menisci were immersed in 25 ml of X-VIVO™10 and CPA efflux was recorded by monitoring the molality of two consecutive washout solutions at different time points. In a subsequent experiment, menisci were incubated in the CPA solutions for 48 h at 22 °C, and the results were compared to those obtained at 22 °C in the main experiment.Results showed a rapid efflux of CPA from meniscus at the beginning of each wash. With increasing temperature, the amount of CPA efflux (and hence loading) increased. Using 24 h incubation, EG loaded the menisci more completely than glycerol. But after 48 h of incubation, both EG and glycerol achieved approximately the same degree of meniscus loading.This study provides preliminary data that will facilitate future design of experiments aimed at development of meniscus permeation studies.     

Temperature Effects on the Onset of Sporulation by Phytophthora ramorum on Rhododendron ‘Cunningham's White’

The effect of temperature and moist period on the onset of sporangia production by Phytophthora ramorum on Rhododendron ‘Cunningham's White’ was examined with misted detached leaves held in humid chambers. Following wound inoculation with sporangia, leaves were pre‐incubated at 20°C for either 24 or 72 h prior to placement at six different temperatures (4, 10, 15, 20, 25 and 30°C). The overall mean moist period required for first occurrence of sporulation over all six temperatures was 3.24 days with the 24‐h pre‐incubation time, compared with 1.49 days for the 72‐h pre‐incubation time. Following 24 h pre‐incubation at 20°C and at an incubation temperature of 15°C, sporangia were first collected from leaves following a 24 h incubation. At 10 and 20°C, sporangia were first collected after 48 h, whereas at 4, 25 and 30°C, sporangia were first collected after 3 days. Following 72 h pre‐incubation at 20°C, sporulation generally occurred within 1 day, even at temperatures such at 4 and 30°C that are suboptimal for sporulation. The highest levels of P. ramorum sporulation were observed at 20°C. P. ramorum formed sporangia on host tissue under moist conditions within the same time frame reported for P. phaseoli, P. palmivora and P. nicotianae, but substantially more slowly than certain other species such as P. infestans. Quantifying moisture and temperature conditions for initiation of sporangia production provides knowledge which leads to a greater understanding of the epidemic potential of P. ramorum.     

Stability of cytokines,chemokines and soluble activation markers in unprocessed blood stored under different conditions

BackgroundBiomarkers such as cytokines, chemokines, and soluble activation markers can be unstable when processing of blood is delayed. The stability of various biomarkers in serum and plasma was investigated when unprocessed blood samples were stored for up to 24 h at room and refrigerator temperature.MethodsBlood was collected from 16 healthy volunteers. Unprocessed serum, EDTA and heparinized blood was stored at room (20–25 °C) and refrigerator temperature (4–8 °C) for 0.5, 2, 4, 6, 8, and 24 h after collection before centrifugation and separation of serum and plasma. Samples were batch tested for various biomarkers using commercially available immunoassays. Statistically significant changes were determined using the generalized estimating equation.ResultsIFN-γ, sIL-2Rα, sTNF-RII and β₂-microglobulin were stable in unprocessed serum, EDTA and heparinized blood samples stored at either room or refrigerator temperature for up to 24 h. IL-6, TNF-α, MIP-1β and RANTES were unstable in heparinized blood at room temperature; TNF-α, and MIP-1β were unstable in unprocessed serum at room temperature; IL-12 was unstable in unprocessed serum at refrigerator temperature; and neopterin was unstable in unprocessed EDTA blood at room temperature. IL-1ra was stable only in unprocessed serum at room temperature.ConclusionAll the biomarkers studied, with the exception of IL-1ra, were stable in unprocessed EDTA blood stored at refrigerator temperature for 24 h. This indicates that blood for these biomarkers should be collected in EDTA and if delays in processing are anticipated the unseparated blood should be stored at refrigerator temperature until processing.     

Effect of storage conditions of blood on radiation-induced chromosomal aberrations and apoptosis in human lymphocytes

To evaluate the effect of storage conditions of blood on the direct relationship between radiation-induced chromosome aberrations and apoptosis in human peripheral blood lymphocytes, whole blood was irradiated with 3 Gy X-rays. Directly after irradiation, a sample of blood was analyzed for chromosome damage and proliferation index, after phytohaemagglutinin stimulation and incubation at 37 °C for 56 h. Blood samples were stored for 48 h at 4 and 20 °C with or without phytohaemagglutinin and analyzed for cell viability and apoptosis at 0, 24 and 48 h storage time. After 48 h of storage, unstimulated cultures were stimulated to proliferate. These samples and cultures stimulated immediately before storage were incubated at 37 °C for 56 h and analyzed for chromosome damage and proliferation index. Metaphases were examined for the presence of dicentrics, excess acentrics, and rings. Storage at 20 °C without phytohaemagglutinin for 48 h increases significantly the yield of apoptosis and decreases significantly the yield of dicentrics. During 48 h of storage time the presence of phytohaemagglutinin and the temperature of 4 °C protected the irradiated lymphocytes from apoptosis allowing accurate estimation of the real yield of radiation-induced chromosome damage. Therefore these blood-storage conditions enable analysis in metaphase and may offer some advantages for biodosimetry of absorbed radiation dose.     

The effects of different types of syringes on equine spermatozoa

Control extender was incubated at 4 degrees C for 24 hours. Rubber or plastic syringe plungers were separately incubated in semen extender for 24 hours at 4 degrees C. Following incubation, the extender was stored at -20 degrees C until the time of semen collection. The treatments consisted of the following: Group A = equine semen plus control extender; Group B=equine semen plus extender incubated with rubber plungers and Group C=equine semen plus extender incubated in plastic plungers; Group D=equine semen plus control extended in rubber plunger syringes and Group E=equine semen plus control extender in plastic plunger syringer. Each group contained a 5-ml volume of semen and extender at a concentration of 1.0 x 10(8) sperm/ml. The number of live spermatozoa, percentage of progressively motile spermatozoa and rate of progressive motility were taken following collection and every 15 minutes for 1 hour following application of treatments. In experiment 2, treatments were allowed to incubate with semen for 45 minutes, then the extender was removed and was replaced with fresh extender. The rate of progressive motility and the percentage of progressively motile spermatozoa were taken immediately, at 45 minutes, and then every 15 minutes for 1 hour. In experiment 1, the number of live spermatozoa was not affected among the 5 groups. However, there was a decrease (P<0.01) in the rate of progressive motility and in the percentage of progressively motile spermatozoa in Group B compared with the remaining 4 treatment groups at 30, 45 and 60 minutes, with no differences noted when semen was held in syringes with a rubber or a plastic plunger. In experiment 2, the percentage of progressively motile spermatozoa increased after the addition of the control extender.     

Influence of storage time and temperature on in vitro digestion of neutral detergent fibre at 48 h, and comparison to 48 h in sacco neutral detergent fibre digestion

The effect of within-day delays of 0.5, 2.5, 4.5 and 6.5 h between collection of rumen fluid from a cow and initiation of in vitro fermentation, as well as storage of rumen fluid for 48 h at either −24°C or 6, 22 and 39°C, on in vitro digestion of neutral detergent fibre (NDF) at 48 h was determined. In addition, the 48 h in vitro digestion of NDF, determined with a minimum time delay (i.e., 0.5 h) between collection from the cow and initiation of incubation, was compared to NDF digestion determined in sacco at 48 h. Rumen inoculum from a single cow was utilized in a thrice replicated incubation with whole crop alfalfa, corn, cereal and sudangrass forages of a lower and higher quality. The same cow was used as the host for the in sacco bags. The in vitro procedure used a bulk procedure with 5.0 cm × 5.5 cm multi-weave polyethylene polyester polymer bags that retained particles of 25 mm and larger. The in sacco procedure used the same bags retained in a large mesh bag. A within-day time delay of up to 6.5 h between collection of rumen fluid from the cow and initiation of in vitro fermentation had no impact on measured 48 h in vitro digestion of NDF. In contrast, no temperature dependent storage procedure maintained 48 h in vitro digestion of NDF at levels determined with no 48 h storage, although high quality alfalfa was least affected by any storage procedure. The 48 h in vitro digestion of NDF, determined using the minimum time delay between collection from the cow and initiation of the incubation, was higher than values obtained in sacco. Results show that this bulk in vitro procedure resulted in higher 48 h digestion of NDF than those determined with a similar in sacco procedure, thereby suggesting that laboratories located some distance from the donor animal can utilize in vitro procedures to accurately estimate 48 h digestion of NDF. However, storage of rumen fluid for 48 h, by any temperature dependent procedure examined, in order to facilitate fewer trips to the donor animal, or trips of substantially longer duration, will underestimate 48 h digestion of NDF to an extent that depends upon the forage incubated.     

Behavior of Vibrio cholerae in hot foods.

Four food types held hot at 45 to 60 degrees C were deliberately contaminated with O1 and non-O1 Vibrio cholerae strains. These organisms were assayed for survival and recovery from the foods within 1 h of the time the food was kept hot. The results showed no growth of V. cholerae non-O1 on thiosulfate-citrate bile-sucrose agar plates after 24 h of incubation at 37 degrees C for food held hot at 50 to 60 degrees C. Growth was low for V. cholerae O1 and was not achieved in some instances in which foods were held at either 55 or 60 degrees C after 40 or 60 min of from the time the food was kept hot. Both organisms, however, were recovered equally from all food types held at all temperatures after 48 h of incubation. When incubated for an additional 24 h, the organisms grew to unusually small-sized colonies, measuring 0.1 to 0.3 mm in diameter, on the same agar plates that were negative for growth after an initial 24 h of incubation. It was concluded that V. cholerae survived the period of time held at hot temperatures. Although the organisms were not recovered from some foods when held at some of the temperatures and times after 24 h of incubation, they remained viable. An incubation period of 48 h at 37 degrees C was found to be appropriate for the recovery of V. cholerae from hot foods.     

Behavior of Vibrio cholerae in hot foods

Four food types held hot at 45 to 60 degrees C were deliberately contaminated with O1 and non-O1 Vibrio cholerae strains. These organisms were assayed for survival and recovery from the foods within 1 h of the time the food was kept hot. The results showed no growth of V. cholerae non-O1 on thiosulfate-citrate bile-sucrose agar plates after 24 h of incubation at 37 degrees C for food held hot at 50 to 60 degrees C. Growth was low for V. cholerae O1 and was not achieved in some instances in which foods were held at either 55 or 60 degrees C after 40 or 60 min of from the time the food was kept hot. Both organisms, however, were recovered equally from all food types held at all temperatures after 48 h of incubation. When incubated for an additional 24 h, the organisms grew to unusually small-sized colonies, measuring 0.1 to 0.3 mm in diameter, on the same agar plates that were negative for growth after an initial 24 h of incubation. It was concluded that V. cholerae survived the period of time held at hot temperatures. Although the organisms were not recovered from some foods when held at some of the temperatures and times after 24 h of incubation, they remained viable. An incubation period of 48 h at 37 degrees C was found to be appropriate for the recovery of V. cholerae from hot foods.     

Isolation,identification and some cultural conditionsof a protease-producing thermophilic Streptomyces straingrown on chicken feather as a substrate

Six strains of thermophilic actinomycetes were isolated from soil using an enrichmenttechnique with feathers as the sole carbon and nitrogen source. They showed clear proteolyticactivity on casein agar medium. The most active strain was tentatively identified as Streptomycesthermonitrificans. This isolate was grown in a basal medium with feathers and:or other carbon andnitrogen sources. Supernatant from centrifuged cultures was examined for protease activity andtemperature and pH optima were determined for enzyme activity. Optimum proteolytic activity onbasal liquid medium containing 1% chicken feather pieces was obtained at 50°C, in a mediumadjusted at pH8 and incubated for 72 h at 150 rpm. Proteolytic activity was further increased by1.5% feather pieces and the time required for maximal activity was 96 h. The keratinolytic activityof S. thermonitrificans was examined by incubation with native chicken feather pieces and it wasfound that it is significantly active. The degradation of whole intact feathers by S.thermonitrificans was obtained after 48 h of incubation at 50°C. The pH and temperature optimafor proteolytic activity were 9.0 and 50°C, respectively. The proteolytic activity was stable at40°C for 1 h. The proteolytic activity was inhibited by DFP but not by EDTA or pCMB. Theseresults inidicated that the enzyme(s) can be classified as an alkaline protease. 1999 ElsevierScience Ltd. All rights reserved.     

A rapid radioimmunoassay for determining plasma concentrations of LH in dogs

A heterologous dog LH radioimmunoassay was modified to provide accurate results for LH concentrations in blood plasma of dogs within 3-4 h. This assay utilizes radioiodinated ovine LH (LER-1056-C2), antiserum against ovine LH (GDN-15) at a final dilution of 1:48,000 and dog LH (LER-1685-1) as standard. A 60-min incubation, including a 30-min delay in the addition of tracer, was carried out at 37 degrees C. The free and antibody-bound hormone were separated by addition of a Micro Sepharose bead suspension containing anti-gamma-globulin, followed by incubation at room temperature for 30 min. The minimum detectable concentration in this assay, calculated from the precision profile, was 1.5 micrograms/l. The amount of dog LH needed to cause 50% reduction of the initial binding was 1.57 +/- 0.13 ng/tube (15.7 micrograms/l for 100-microliters samples). Daily blood samples were collected in heparinized tubes from the cephalic vein of 5 pointer and 7 beagle bitches from the onset of pro-oestrus until 3-4 days after either the last mating or artificial insemination with frozen semen or until metoestrus. Samples were assayed for LH content by the short and normal incubations as well as for progesterone and oestradiol-17 beta content. In all bitches plasma concentrations of progesterone increased rapidly within 1 week after the LH peak which indicates that they had ovulated. Comparison of the short (1.5 h) with the normal (24 h) incubation system resulted in a regression equation: y = 1.0 + 0.7 x (r = 0.95, n = 153 samples).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in selected aspects of immune function in the leopard frog, Rana pipiens, associated with exposure to cold

The effect of exposure to low temperatures (5 °C) on lymphocyte proliferation, leukocyte populations, and serum complement levels was examined in the northern leopard frog, Rana pipiens. Proliferation of T lymphocytes in response to phytohemagglutinin stimulation was significantly decreased in frogs kept for 2, 3, and 5 months at 5 °C compared to that of animals kept at 22 °C. A significant increase in the average percentage of neutrophils and a decrease in the mean percentage of eosinophils was observed in the blood of frogs held for 5 months in the cold compared to animals held at 22 °C for the same length of time. Mean serum complement activity after 1 month at 5 °C was significantly reduced in comparison to animals held at 22 °C and was not detectable after 5 months in the cold. Recovery of complement levels at room temperature (22 °C) was also examined after cold exposure. Complement levels were significantly higher than controls (at 22 °C) in frogs returned to 22 °C for 7 and 14 days after 5 months in the cold. After frogs were held at 5 °C for 1 month, serum complement levels increased significantly within 2 days after returning to 22 °C and continued to rise 5 and 9 days after warming. Injections with Aeromonas hydrophila following a 5-week exposure to 5 °C failed to cause death or observable symptoms of disease in frogs that were returned to 22 °C. Accepted: 20 November 1996     

Effects of incubation temperatures on sexual differentiation in the turtle,Chelydra serpentina

Eggs of the common snapping turtle, Chelydra serpentina, were incubated at constant temperatures ranging from 20°C to 30°C, At hatching, the oviducts were absent or incomplete in males; the testes were differentiated. In females at hatching, the oviduct was intact hut in some cases the gonad retained bisexual characteristics. Three months after hatching, the ovary was differentiated and contained follicles. Eggs incubated at 20°C and at 30°C developed into females in 100% of the cases. At 26°C, 99% of the individuals were males; at 24°C, 100% were males. More males than females developed at incubation temperatures of 22°C and 28°C.     

Refrigeration of blood samples prior to separation is essential for the accurate determination of plasma or serum zinc concentrations

An evaluation of refrigeration (7°C) to prevent falsely high plasma or serum zinc concentrations owing to elapsed time between blood collection and centrifugation was performed. At room temperature (23°C), both plasma and serum zinc concentrations increased significantly, if blood samples were stored uncentrifuged. Plasma zinc concentrations increased 6.3% at 1 h and 40.7% at 24 h, whereas serum zinc concentrations increased only 0.9% at 1 h and 12.5% at 24 h at room temperature. When blood samples were stored uncentrifuged in the refrigerator for up to 24 h, there were no significant increases in zinc concentrations in either plasma or serum. These findings suggest that plasma or serum separation should be performed immediately after blood drawing to obtain accurate zinc concentrations, and if this is not feasible, the samples should be immediately refrigerated and separation performed within eight hours.