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.     

Effects of delayed phlebotomy on plasma steroid hormone concentrations in two elasmobranch species

Measuring circulating concentrations of steroid hormones can be used as a method for determining reproductive maturity and cycles in elasmobranchs. However, it is unknown how long steroid hormones remain stable in elasmobranch blood following capture, and thus how quickly these samples should be collected for the results of subsequent steroid hormone analyses to be accurate. The objectives of this study were to determine if the sex steroid hormones progesterone, testosterone and estradiol would remain at stable concentrations in the blood of the Spiny Dogfish (Squalus acanthias Linnaeus, 1758) and the Atlantic Sharpnose Sharks (Rhizoprionodon terraenovae Richardson, 1836) that were captured, left on deck and un‐refrigerated for 24 hr. Blood samples were serially drawn from five initially live sharks over a period of 24 hr. While concentrations of all three hormones did significantly fluctuate over the sampling period in both species, the resulting hormone concentrations from each sampling period still fell within the range of previously reported values for each species in their respective reproductive stage. Additionally, no significant changes in hematocrit were detected in either species over the 24‐hr period. This research represents an extreme situation in which sharks were left on deck and un‐refrigerated, and suggests that even when subjected to these conditions steroid hormone concentrations may fluctuate, but the resulting values may still be useful for assessing reproductive stage.     

Effect of low intensity laser beam on steroid dehydrogenase activity and steroid hormone production in cultured porcine granulosa cells

Effect of the beam emitted by a helium-neon (He-Ne) laser of a low power (2.8 mW) on the activity of delta 5, 3 beta-hydroxysteroid dehydrogenase (delta 5, 3 beta-HSD), as well as on the estradiol and progesterone production was studied in cultured granulosa cells obtained from porcine ovary. It has been found that the laser beam stimulates the activity of steroid dehydrogenase, increases estrogen levels and exerts a variable influence on the progesterone level. In control cultures the highest level of both hormones occurred simultaneously, while in cultures exposed to the laser beam progesterone level rose at a slower rate and reached maximum when the estrogen level dropped. Hyperplasia and epithelisation were observed only in the experimental cultures. The most intense cytochemical reaction for delta 5, 3 beta-HSD, as well as the most pronounced increase in estrogen and progesterone level occurred in cells exposed to a pulsating beam for 60 sec.     

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 chronic metoclopramide therapy on serum pituitary hormone concentrations

The effect of chronic (3--9 months) therapy with metoclopramide on serum levels of pituitary and thyroid hormones was studied in 4 males and 1 female. The mean serum prolactin concentration during metoclopramide therapy was significantly higher than after discontinuation of metoclopramide. Serum prolactin concentrations increased acutely after each dose of metoclopramide. Serum prolactin concentrations increased acutely after each dose of metoclopramide, and gradually returned to normal by 6--12 h. There was no sifgnificant differences in the serum TSH, T3, T4, GH, and gonadotrophin levels during and after metoclopramide administration. In the male subjects the mean serum testosterone was normal, but significantly lower during metoclopramide therapy.     

Effect of thyroid hormone on concentrations of plasma calcitonin in broiler chicks

The purpose of these studies was to determine the effect of thyroidectomy (Tx), and thyroid hormone (T₃/T₄) treatment on concentrations of plasma CT in chicks. In addition, the turnover of CT in Tx- and T₃/T₄-treated chicks was estimated using a novel nonradioactive salmon CT preparation. One-week-old broiler chicks (Gallus domesticus) (n=75) were divided into three groups. Group I was sham-injected daily (i.m. saline), Group II was injected with 50 μg/day of T₃/T₄ while Group III was injected with the goitrogen, methimazole, (150 mg/kg BW per day) for 8 weeks. Chicks (8–9 weeks old) were implanted with catheters in the brachial wing vein and administered ruthenium-labeled salmon CT. Blood samples were collected at 30 s, 1, 2, 4, 8, 20 min, and 3 h after injection. Results showed that concentrations of plasma CT were decreased in T₃/T₄-injected birds. There was no significant effect of methimazole on circulating concentrations of plasma CT. The half-life of CT was significantly increased (P<0.05) in both T₃/T₄-injected (n=6; 1.34±0.16 min) and goitrogen-treated birds (n=2; 5.81±2.83 min) compared to controls (n=7; 54±3 s) The results demonstrate that changes in concentrations of plasma thyroid hormones can significantly affect concentrations of plasma CT.     

Effect of storage time and temperature on stallion sperm DNA and fertility

We used the sperm chromatin structure assay (SCSA) to study the change in stallion sperm DNA susceptibility to denaturation after exposure of extended semen to three different storage temperatures (5, 20, or 37 degrees C) at 7, 20, 31, and 46 h. In addition, we compared the rates of sperm DNA denaturation in fertile and subfertile stallions. Among fertile stallions, spermatozoa stored at 20 and 37 degrees C showed a significant (P < 0.05) rise in the SCSA measures (Mean(alpha1), S.D.(alpha(t)), and percent cells outside the main population-COMP(alpha(t))) overtime, with the degree of rise being more dramatic at 37 degrees C. Over all stallions, samples stored at 5 degrees C showed no significant (P > 0.05) changes in the SCSA values measured over time, indicating maintenance of chromatin quality for up to 46 h. The COMP(alpha(t)) from stallions classified as subfertile showed an increased susceptibility to denaturation or decline in chromatin quality between 20 and 31 h when stored at 5 degrees C; however, spermatozoa from fertile stallions did not change during the time intervals analyzed. These data suggest that sperm DNA from some subfertile stallions may decline at a greater rate than spermatozoa from fertile stallions when exposed to similar storage conditions.     

Effect of time-of-day-specific strength training on serum hormone concentrations and isometric strength in men

A time-of-day influence on the neuromuscular response to strength training has been previously reported. However, no scientific study has examined the influence of the time of day when strength training is performed on hormonal adaptations. Therefore, the primary purpose of this study was to examine the effects of time-of-day-specific strength training on resting serum concentrations and diurnal patterns of testosterone (T) and cortisol (CORT) as well as maximum isometric strength of knee extensors. Thirty eight diurnally active healthy, previously untrained men (age 20-45 yrs) underwent a ten-week preparatory strength training period when sessions were conducted between 17:00-19:00 h. Thereafter, these subjects were randomized into either a morning (n=20, training times 07:00-09:00 h) or afternoon (n=18, 7:00-19:00 h) training group for another ten-week period of time-of-day-specific training (TST). Isometric unilateral knee extension peak torque (MVC) was measured at 07:00, 12:00, 17:00, and 20:30 h over two consecutive days (Day 1 & Day 2) before and after TST. Blood samples were obtained before each clock-time measurement to assess resting serum T and CORT concentrations. A matched control group (n=11) did not train but participated in the tests. Serum T and CORT concentrations significantly declined from 07:00 to 20:30 h on all test days (Time effect, p<.001). Serum CORT at 07:00 h was significantly higher on Day 1 than Day 2 in the control and afternoon group, both in Pre and Post conditions (Day x Time interaction, p<.01). In the morning group, a similar day-to-day difference was present in the Pre but not Post conditions (Time x Group interaction, p<.05). MVC significantly increased after TST in both the morning and afternoon groups (Pre to Post effect, p<.001). In both groups, a typical diurnal variation in MVC (Time effect, p<.001) was found, especially on Day 2 in the Pre condition, and this feature persisted from Pre to Post in the afternoon group. In the morning group, however, diurnal variation was reduced after TST on both Day 1 and Day 2 (Pre to PostxDay x TimexGroup interaction, p<.05). In conclusion, 10 weeks of morning time-of-day-specific strength training resulted in reduced morning resting CORT concentrations, presumably as a result of decreased masking effects of anticipatory psychological stress prior to the morning testing. The typical diurnal pattern of maximum isometric strength was blunted by the TST period in the morning but not the afternoon group. However, the TST period had no significant effect on the resting total T concentration and its diurnal pattern and on the absolute increase in maximum strength.     

Glucocorticoid influence on porcine granulosa cell IGF-I and steroid hormone production in vitro

The effect of cortisol on granulosa cell (GC) insulin-like growth factor I (IGF-I) synthesis, and IGF-mediated steroid production was examined at various stages of follicle maturation. Granulosa cells were recovered from gilts on Days 14, 18, and 20 of the estrous cycle, while luteinizing GC were recovered on Day 21, just prior to ovulation. The cells were cultured in serum-free medium with increasing concentrations of cortisol (0, 1, 10, and 100 microg/mL) for 5 d with or without IGF-I stimulation (10 ng/mL). During culture all cells were supplemented with FSH and androstenedione (A4). Cellular IGF-I, progesterone (P4) and estradiol-17beta (E2) production was determined by specific radioimmunoassays (RIA), and cell proliferation was assessed. Granulosa cell IGF-I and steroid hormone synthesis increased (P<0.05) with follicle maturation. Direct exposure to high cortisol concentrations, however, altered both IGF-I synthesis and action. Cortisol treatment lowered (P<0.05) IGF-I production by GC recovered on Days 18, 20, and 21. Furthermore, it reduced (P<0.05) IGF-stimulated P4 synthesis at all stages and decreased (P<0.05) IGF-stimulated E2 synthesis by cells recovered on Day 14. In contrast, cortisol enhanced (P<0.05) FSH-stimulated P4 production by GC collected on Days 14 and 18. The opposing effects on FSH and IGF-I action indicate that cortisol did not promote an overall suppressive effect on cell function, nor did it impair cell proliferation. Hence, these results demonstrate that elevated cortisol concentrations can disrupt both IGF-I synthesis and IGF-mediated actions by porcine GC under in vitro conditions, and that specific disruptions are dependent on the stage of follicle maturation.     

Effects of variations in thyroid hormone serum concentrations on serum ACE-activity

Serum angiotensin converting enzyme activities were significantly increased in 26 untreated hyperthyroid patients (20.3 +/- 5.4 U/ml; P less than 0.001) compared with healthy control subjects (13.1 +/- 2.3 U/ml). In 12 patients a significant fall in enzyme activities was observed after treatment compared with pretreatment serum ACE levels (P less than 0.001). Eight patients with hypothyroidism (15.7 +/- 5.1 U/ml) and 11 athyreotic patients, totally thyroidectomized for well-differentiated thyroid cancer, showed no significant differences in serum ACE activities (14.3 +/- 2.2 U/ml) compared with control subjects. After thyroid hormone supplementation a significant increase in serum ACE activity (P less than 0.05) was found in the athyreotic patients. Addition of increasing amounts of L-thyroxine to a serum sample of an athyreotic patient showed no significant effect on ACE activity in vitro. We suggest that the elevated serum ACE activity in hyperthyroidism is not from the thyroid gland, but represents a direct effect of thyroid hormone on ACE synthesis and/or release from endothelial cells.     

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.     

不同固定液及保存温度、时间对小鼠组织DNA的影响

比较10％甲醛、95％乙醇、Saccomanno3种固定液及不同保存温度、保存时间对小鼠肝、肺组织DNA的影响。取材后将标本分为无固定液组、甲醛组、乙醇组和Saccomanno组,不同温度保存至一定时间后提取DNA进行比较。结果无固定液时,保存3d后,室温且与低温组差别不大。甲醛固定时,对不同保存温度、时间、不同组织的DNA影响不同。乙醇、Saccomanno法室温放置1个月后DNA仍保存良好。短时间室温保存对DNA影响不大。10％甲醛使DNA降解,95％乙醇、Saccomanno法固定则可以保护DNA的完整性。     

Effect of heat stress on plasma concentrations of prolactin and luteinizing hormone in ewes.

Basal concentrations of prolactin but not luteinizing hormone were elevated in ewes by 8--10 h of heat stress given daily during the first 11 days of their oestrous cycle. However, the prolactin and luteinizing hormone responses to thyrotrophin releasing hormone and gonadotrophin releasing hormone were unaffected.     

Effect of calf removal on serum luteinizing hormone and cortisol concentrations in postpartum beef cows

Serum luteinizing hormone (LH) and cortisol concentrations were measured in ten fall calving, Angus cows averaging 38 +/- 8 days postpartum. Calves from five cows were weaned at the beginning of the study. Blood samples were collected at 20 min. intervals for 48 h after weaning and for 8 h on day 4 and day 6 postweaning. Mean serum LH concentrations increased (P<0.01) in weaned cows (W) from 0.55 +/- 0.01 ng/ml at time of calf removal to 1.3 +/- 0.04 ng/ml 48 h afterwards. Comparable LH concentrations for suckled cows (S) were 0.65 +/- 0.08 ng/ml and 0.62 +/- 0.03 ng/ml respectively. Average serum LH concentrations at 48 h after weaning were greater (P<0.01) for W cows than S cows and a treatment by time interaction occurred (P<0.01) with serum LH concentrations increasing (P<0.01) from time of calf removal to 48 h after calf removal in W cows. Frequency of LH peaks increased (P<0.01) in W cows and by 48 h after weaning was greater (P<0.01) in W cows than in S cows. Magnitude of LH peaks did not differ between the two groups. Serum cortisol concentrations were not different between W and S cows except for a transient elevation (P<0.01) in W cows from 7.6 +/- 0.9 ng/ml to 11.9 +/- 1.0 ng/ml 9 to 12 h after calf removal. Since serum LH concentrations were increased in W cows but not in S cows at 48 h and serum cortisol concentrations increased transiently in W cows we suggest that circulating cortisol levels may not be a physiological inhibitor of LH secretion in the suckled postpartum beef cow.     

Effect of adjuvant-induced arthritis on serum luteinizing hormone and testosterone concentrations in the male rat

Male Lewis rats were made arthritic by injecting 1 mg Mycobacterium butyricum suspended in Freund's incomplete adjuvant into their right hind footpad. Arthritic and non-arthritic animals were sacrificed on days 18, 21, 24 or 27 after the injection of the adjuvant. Body weight, left and right hind paw volume, thymus weight, and serum luteinizing hormone (LH) and testosterone concentrations were determined on each day. Adjuvant injection resulted in a significant enlargement in the left and right hind paws on days 18 through 27. In contrast, body and thymus weights were reduced significantly in the arthritic rats compared to the non-arthritic animals. Serum concentrations of testosterone were also reduced significantly in arthritic rats on days 18, 21 and 24 after the injection of the adjuvant. However, by day 27 serum testosterone concentrations recovered to near control values. Serum concentrations of LH in the arthritic animals were elevated on days 18 through 27. These results demonstrate that serum testosterone concentrations were reduced in rats with adjuvant-induced arthritis. The reduction in serum testosterone is probably not the result of an impaired hypothalamic-pituitary axis.     

Combined effects of spray‐drying conditions and postdrying storage time and temperature on Salmonella choleraesuis and Salmonella typhimurium survival when inoculated in liquid porcine plasma

The objective of this study was to determine the effectiveness of the spray‐drying process on the inactivation of Salmonella choleraesuis and Salmonella typhimurium spiked in liquid porcine plasma and to test the additive effect of immediate postdrying storage. Commercial spray‐dried porcine plasma was sterilized by irradiation and then reconstituted (1:9) with sterile water. Aliquots of reconstituted plasma were inoculated with either S. choleraesuis or S. typhimurium, subjected to spray‐drying at an inlet temperature of 200°C and an outlet temperature of either 71 or 80°C, and each spray‐drying temperature combinations were subjected to either 0, 30 or 60 s of residence time (RT) as a simulation of residence time typical of commercial dryers. Spray‐dried samples were stored at either 4·0 ± 3·0°C or 23·0 ± 0·3°C for 15 days. Bacterial counts of each Salmonella spp., were completed for all samples. For both Salmonella spp., spray‐drying at both outlet temperatures reduced bacterial counts about 3 logs at RT 0 s, while there was about a 5·5 log reduction at RT 60 s. Storage of all dried samples at either 4·0 ± 3·0°C or 23·0 ± 0·3°C for 15 days eliminate all detectable bacterial counts of both Salmonella spp.

Significance and Impact of the Study

Safety of raw materials from animal origin like spray‐dried porcine plasma (SDPP) may be a concern for the swine industry. Spray‐drying process and postdrying storage are good inactivation steps to reduce the bacterial load of Salmonella choleraesuis and Salmonella typhimurium. For both Salmonella spp., spray‐drying at 71°C or 80°C outlet temperatures reduced bacterial counts about 3 log at residence time (RT) 0 s, while there was about a 5.5 log reduction at RT 60 s. Storage of all dried samples at either 4.0 ± 3.0°C or 23.0 ± 0.3°C for 15 days was effective for eliminating detectable bacterial counts of both Salmonella spp.     

Effect of low-calorie diets on plasma retinol-binding protein concentrations in overweight women

The concentrations of total protein, albumin and retinol-binding protein, a major transport protein for vitamin A, are significantly decreased by protein-calorie malnutrition. Weight-loss diets, sometimes involving severe energy deficits over prolonged periods of time, are common in the United States. The effect, if any, of prolonged low calorie weight-loss diets with normal intakes of protein on albumin, total protein and retinol-binding protein concentrations (and potentially on vitamin A metabolism) had not been extensively studied. We measured total protein, albumin, apo + holo retinol-binding protein and holo-free- and holo-transthyretin-bound retinol-binding protein concentrations during the course of a nutritionally adequate weight-loss diet (50% calorie restriction). We found that this type of dieting did not affect total protein, albumin or apo + holo, holo-free or holo-transthretin-bound retinol-binding protein concentrations significantly. This suggests that protein intake is more critical than caloric intake for retinol-binding protein status.     

The effect of storage time and molybdate on steroid receptors in gynaecological tissues

Human female reproductive tract tissues were analysed for estrogen and progestogen receptor content in the presence or absence of sodium molybdate immediately after removal at surgery. Other fractions of the tissue were stored in liquid nitrogen and similarly analysed after 2, 4, 6 and 8 weeks storage. The results showed that at all times the apparent receptor content for both steroids was significantly higher (P<0.001) and Kd values were significantly lower (P<0.02) in assays carried out with 10mM molybdate added to the buffer systems.Furthermore, as soon as either whole tissue or tissue cytosol was frozen for storage, receptors were “lost” with values decreasing by approximately 30% for both steroid receptors. However, once frozen in liquid nitrogen tissue receptor content remained stable over the eight weeks of study. It is recommended that laboratories standardize techniques to allow valid comparisons of results.