Biomedical subjects

F Ellendorff

Publications and source records attributed to F Ellendorff.

At least 19 recordsLinked to original sources

PubMed2005

Detection of testosterone, nandrolone and precursors in horse hair.

Growing interest among several horse-breeder associations has initiated the development of a screening procedure to test for anabolic agents in hair, which has the advantage over blood and urine specimens of allowing long-term detection. An analytical method was established to monitor in tails or manes several anabolic substances available as veterinary medicines or as so-called nutritional supplements (clenbuterol, different esters or prohormones of nandrolone and testosterone). The analytical procedure to detect steroids in hair samples consists of the following steps: decontamination of the hair strand or segment with methanol/water (1:1), milling, extraction of the hair material in an ultrasonic bath using methanol, purification by liquid-liquid extraction (n-pentane/methanol, 25:1) and HPLC cleanup, derivatisation of the relevant LC fractions with MSTFA, and measurement using GC-MS/MS technique. The first objective of our study was the detection of exogenous nandrolone (nortestosterone, NT) in the horse hair; therefore nandrolone-associated compounds [nandrolone dodecanoate administered intramuscularly (i.m.) and a mixture of 4-estrenediol and 4-estrenedione, transdermal] were administered to four geldings. The highest concentrations of NT following i.m. treatment were measured after 10 days in a 2-cm hair segment (up to 18 pg/mg); NT was detectable for up to 120 days and in some cases up to 330 days in tail hair (limit of detection 0.3 pg/mg). Following transdermal application, nandrolone as well as the administered prohormones were identified in tail and mane until the latest sampling at 3 months. Furthermore, untreated stallions (128) were investigated to estimate the range of endogenous levels of NT and testosterone (T) in hair. Maximum values of 3 pg/mg (NT) and 1 pg/mg (T) were quantified originating from endogenous formation in the male horse. Additionally, a possible relationship between steroid concentrations in hair specimens and the age of stallions was appraised. NT and T were not detected in hair samples of control geldings. Following nandrolone treatment of geldings, highest values in hair exceeded the endogenous amount detected in untreated stallions. Therefore comparison of concentrations measured in control samples with the estimated endogenous levels could give a clue to exogenous application in cases of abnormally high amounts of NT or T. The possibility of the evaluation of threshold values is discussed as a means to verify an exogenous administration of NT and T in hair samples. Furthermore, the detection of a synthetic substance in hair, e. g. the parent steroid ester by itself, would be unequivocal proof of an exogenous origin of NT or T and the previous medication of the stallion.

Administration, Cutaneous↗

PubMed2005

A new tool to monitor training and performance of sport horses using global positioning system (GPS) with integrated GSM capabilities.

Global Positioning Systems (GPS) are considered suitable to monitor the position and velocity of horses during cross-country competition or in training. Furthermore, simultaneous recording of life data such as heart rate could be useful to assess the horse's condition during exercise. To test the suitability and reliability of a commercially available GPS system with integrated heart rate recording system and with built in GSM for data transmission, the Fidelak Equipilot Type EP-2003-15/G-2.11 (EP-15/G) was evaluated first for reliability of pulse recording from a pulse generator within the physiological range of horses; furthermore distance, velocity and heart rate recordings were carried out on a standard 1000 m field track with five repetitions. Agreement (% deviation from actually measured distance and from stopwatch-distance based velocity calculations) and variability (Coefficient of Variation for distance, velocity, heart rate) were calculated. From the results it was safe to assume that the heart rate sensor recorded horse heart rates at a high degree of accuracy. Overall distances and velocities are in high agreement with actually measured values. However, overall variability expressed in terms of relative variability (C.V.) is smaller for distance recording (C.V. 0.68%) when compared to velocity (C.V. 1.01%). The system tested is suitable and reliable for simultaneously recording of distance, velocity and heart rates for horses during cross country exercise. GPS-based monitoring of movement along with simultaneous recording of physiological data and the possibility to call upon data will not only be of benefit for training horses or for surveillance during competition, it may also be suitable for distant patient monitoring and in behavioural studies as well as in veterinary medicine in general.

Animals↗

PubMed2004

The beta-agonist clenbuterol in mane and tail hair of horses.

REASONS FOR PERFORMING STUDY: The beta2-agonist clenbuterol is commonly administered for therapeutic purposes in the horse, but its use an an anabolic agent is illegal. Clenbuterol can be detected in blood and urine for a relatively short period after administration and detection in hair could enhance the analytical range and be used to determine the history of clenbuterol application. HYPOTHESIS: That detection in mane or tail hair is possible over an extended period. METHODS: Four horses received 0.8 microg clenbuterol hydrochloride/kg bwt b.i.d. for 10 days. Four other horses were used as untreated controls. Blood, urine, mane and tail hair samples were taken on Day 0 (before) and 5, 10, 30, 35, 40, 60, 90, 120, 150 and 360 days after start of treatment. Gas chromotography/high resolution mass spectrometry (GC/HRMS) was developed for clenbuterol analysis: limit of detection was 0.2 pg/mg; intra-assay repeatability limit r = 0.06 (confidence level 95%); interassay repeatability limit r = 0.03 (confidence level 95%). Prior to treatment, clenbuterol was absent from all samples analysed. RESULTS: Clenbuterol was detectable as early as Day 5 in tail and mane hair of Segment 1 (0-20 mm from the roots) and was maximal on Day 90. However, as time progressed, shift into lower 20 mm segments was observed. On Day 360, the maximum concentration (up to 21 pg/mg) was located in Segment 13, i.e. 26-28 cm from roots of hair. Clenbuterol was not detectable in blood or urine after Day 30. Mane and tail hair results were very similar. CONCLUSIONS: The study showed that the beta-agonist clenbuterol can be found in mane and tail hair of horses after extended periods. POTENTIAL RELEVANCE: It will be possible to detect clenbuterol in breeding and show horses where anabolic drugs have been used illegally to improve conformation. This method may also be helpful to monitor therapeutic clenbuterol treatment.

Administration, Oral↗

PubMed2002

Localization of the fertilized germinal disc in the chicken egg before incubation.

At the time of oviposition, the embryo in the fertilized chicken egg has developed the germinal disc on top of the yolk and contains thousands of blastodermal cells. The germinal disc escapes exact localization because it is surrounded by albumen and shell membranes under a calcified shell. This study investigated whether or not ultrasonography or nuclear magnetic resonance imaging (MRI) are suited to localize the germinal disc and, if so, to estimate the accuracy of localization. Hatchability of treated eggs was also recorded. The ultrasound waves were reflected by the shell. The germinal disc could be seen only after removal of the eggshell and the outer shell membrane. In all, 39 intact eggs, MRI localized the germinal disc within 2 mm thick image slices. The mean position of the germinal disc deviated 3.0+/-2.1 mm from the maximum vertical plane of the egg with a mean distance to the inner surface of the shell of 2.7+/-1.1 mm. Incubation resulted in 84.2 and 77.5% hatched chicks for imaged eggs and controls, respectively. The localization results of MRI were verified manually in 38 open shell cultures. Correlation coefficients for the position of the germinal disc were r = 0.86 to 0.99 for the x-axis, r = 0.75 to 0.89 for the y-axis, and r = 0.63 to 0.76 for the z-axis. The study thus shows that MRI is a reliable tool to localize the germinal disc within the intact freshly laid egg.

Animals↗

PubMed2001

Neuroendocrine regulation of growth hormone and luteinizing hormone in fetal and neonatal pig.

Ontogeny of the production and the regulation of growth hormone and Luteinizing hormone is studied in a series of experiments utilizing male and female pigs at different fetal and neonatal stages. Growth hormone mRNA is detectable in both sexes as early as d. 50 p.c. The mRNA levels increase to reach the maximum levels at d. 95-110 p.c. Plasma levels of GH follow the developmental patterns of GH mRNA. A sex difference is evident around d. 80-90 p.c. with males having higher GH levels than females. The stimulatory but not the inhibitory mechanisms of GH secretion are fully functioning in the pig fetus. LHbeta mRNA is detectable earlier in females (d. 50 p.c.) than in males (d. 65 p.c.). Plasma concentrations of LH increase with fetal age in female fetuses, but in male fetuses there is no distinct developmental pattern evident. Basal LH secretion achieves maximum levels in both sexes after birth. Opioids do modulate fetal LH secretion, however, the mode of their action is age-dependent.

Animals↗

PubMed2001

[ACTH stimulation test for the determination of salivary cortisol and of cortisol responses as markers of the training status/fitness of warm-blooded sports horses].

Previous work (Marc et al., 2000) suggested that plasma cortisol responses to treadmill exercise or ACTH injection are a reliable marker for performance evaluation in warmblood horses. For practical purposes blood sample collections and treadmill exercise tests are somewhat troublesome and time consuming. The goal of this study was thus to evaluate the use of saliva for cortisol determination (by direct EIA) as a marker for performance and to investigate the reliability and repeatability of plasma cortisol responses to a single i.v. injection of ACTH (50 micrograms or 250 micrograms). Furthermore, the effect of training horses for 8 weeks 3 times per week covering the same distance (increasing from 3.5 km during the first week to 8 km during the last week) either by trotting (approximately 240 m/min) or by cantering (375 m/min) was investigated. For this purpose initially ten four-year-old Hannovarian geldings, all reared in the same State stud, were used. Mean overall correlation between salivary cortisol and plasma cortisol concentrations was 0.64 when samples of various points of time were used. However, in spite of attempts to standardize saliva sample collection, correlation between salivary cortisol levels and plasma cortisol levels at distinct points of time in different tests were low and significant (r = 0.85, p < 0.02) only in one test. Thus, salivary cortisol measurements for diagnostic purposes are not reliable or useful. The repeatability of plasma cortisol responses to ACTH for untrained and trained horses were r = 0.86 and r = 0.8 respectively (p < or = 0.01 and p < or = 0.05 respectively). Training horses either by trotting or cantering did not affect the cortisol response either to treadmill exercise or to stimulation by ACTH. It is concluded that the relationship between salivary cortisol levels and plasma cortisol levels is not close enough to allow the use of salivary cortisol determination as marker of the training status/fitness of horses. The repeatability of the cortisol response to ACTH is similar to the cortisol response to treadmill exercise. Based on plasma cortisol responses to ACTH or treadmill exercise training horses by cantering at low speed is not superior to training by trotting for the fitness of horses.

Adrenocorticotropic Hormone↗

PubMed2000

Localization of Xho1 repetitive sequences on autosomes in addition to the W chromosome in chickens and its relevance for sex diagnosis.

Repetitive sequences of the Xho1 family comprise about 70% of the W chromosome in chickens, are considered to be restricted to this sex chromosome and are used in sex diagnosis. This study investigated the reliability of sex diagnosis on small cell numbers. Fluorescent in situ hybridizations (FISH) with the Xho1 repeat sequence were performed on metaphases and signals were detected on the W chromosome and on some autosomes. Differences in signal intensity suggested that the number of repeats ranged from several thousand on the W chromosome to only a few on the autosomes. These results confirm the report from Clinton (1994) of minor male products in sex specific PCR of the Xho1 repeat. We adopted his PCR protocol to analyze small numbers of cells. FISH on blastomeres and on blood cells from E3 to E11 embryos were compared to PCR results. The FISH procedure as well as the PCR protocol are suitable for sexing. For single cells FISH was slightly more consistent.

Animals↗

PubMed2000

Plasma cortisol and ACTH concentrations in the warmblood horse in response to a standardized treadmill exercise test as physiological markers for evaluation of training status.

Reliable physiological markers for performance evaluation in sport horses are missing. To determine the diagnostic value of plasma ACTH and cortisol measurements in the warmblood horse, 10 initially 3-yr-old geldings of the Hannovarian breed were either exposed to a training schedule or served as controls. During experimental Phase 1, horses were group-housed, and half of the horses were trained for 20 wk on a high-speed treadmill. During Phase 2, groups were switched and one group was trained for 10 wk as during Phase 1, whereas the control group was confined to boxes. During Phase 3 horses were initially schooled for riding. Thereafter, all horses were regularly schooled for dressage and jumping, and half of the horses received an additional endurance training for 24 wk. During all phases horses were exposed at regular intervals to various standardized treadmill exercise tests. During and after the tests frequent blood samples were taken from an indwelling jugular catheter for determination of ACTH and cortisol. Treadmill exercise increased both hormones. Maximum ACTH concentrations were recorded at the end of exercise, and maximum cortisol levels were recorded 20 to 30 min later. Except for one test there were no differences in ACTH levels between trained horses and controls. There was no significant effect of training on the cortisol response (net increase) to treadmill exercise in any of the tests during Phase 1. During Phase 2 higher cortisol responses were recorded in controls than in trained horses (P < .05) after 10 wk of training (controls confined to boxes). During Phase 3 plasma cortisol responses were also higher in controls than in trained horses (P < .05 after 6, 18, and 24, P < or = .07 after 12 wk of training) when the inclination of the treadmill was 5%, but not at 3%. There was no overlap in net cortisol responses at 30 min between trained and untrained horses. An ACTH application after 24 wk of training resulted in higher cortisol responses in controls than in trained horses (P < or = .05), without any overlap between the groups at 30 min after ACTH. Plasma cortisol responses to either treadmill exercise or ACTH injection may be a reliable physiological marker for performance evaluation. Prerequisites are sufficient differences in training status and sufficient intensity of exercise test conditions.

Adrenocorticotropic Hormone↗

PubMed1999

Concentrations of free steroids in the jugular and hepatic portal veins of pigs after ingestion of testosterone, estrogen, or progesterone or transplantation of ovaries to the intestine.

Estrogen, progesterone or testosterone were administered orally to ovariectomized gilts fitted with indwelling catheters. Blood samples were taken from the jugular and hepatic portal veins at intervals varying from a few minutes to daily over periods that varied from 1 d to several months. Concentrations of free steroids rose dramatically in the hepatic portal vein within a few min of feeding steroids and remained high for 3-8 hr before declining to base levels. Concentrations in the jugular vein sometimes rose very slightly for the same period. At 48 hr after administration the concentrations remained at baseline in the hepatic portal vein, but rose several-fold in the jugular and remained elevated for several days. Autotransplantation of the ovaries to the intestine resulted in very large ovaries consisting of many large, heavily luteinized cystic follicles. Concentrations of progesterone and estrogen in the hepatic portal vein were very high, but were low in the jugular vein. The gut wall allows for passage of some orally administered free steroids to the liver via the hepatic portal vein. The free steroids are essentially metabolized before they reach the jugular vein, but can be recirculated via the enterohepatic route.

Administration, Oral↗

PubMed1998

Analysis of chicken embryonic development after removal of blastodermal cells for sexing.

1. The present study evaluated developmental characteristics in the chick embryo throughout incubation following cell removal from the freshly laid egg. 2. Between 10 and a few hundred cells of the blastoderm were removed for sex diagnosis. Incubation of the treated embryos was then continued in an open egg shell culture system. 3. Cell recovery from different regions within the blastoderm was performed. 4. The experiments presented here demonstrate the persistence of the developmental potential of chicken embryos in an in ovo culture system after removal of different numbers of cells from the germinal disc prior to incubation. 5. No deviations in developmental characteristics were recorded when compared to untreated control cultures. 6. No detrimental effects of double cell biopsies could be observed. 7. A similar number of chicks developed to hatch regardless of the location of manipulation within the blastoderm.

Animals↗

PubMed1997

Ontogeny of growth hormone and LH beta-, FSH beta- and alpha-subunit mRNA levels in the porcine fetal and neonatal anterior pituitary.

The mechanism underlying the ontogenetic increase in plasma growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentration during fetal life in mammalian species and the prenatal sex difference in these hormones in some species is not fully understood. To this end anterior pituitaries were collected from German Landrace fetuses and piglets at day (d) 50, 65, 80, 95, 110 pc and d 6 pp and pituitary GH, LH beta, FSH beta and alpha-subunit mRNA levels were determined by measuring Northern blot hybridization signals. GH mRNA was detected in both sexes as early as d 50 pc. The mRNA level markedly increased with age in both sexes (males > females, P < or = 0.05) reaching its maximum at d 95/110 pc. LH beta mRNA signals were first detected at d 50 pc in females and at d 65 pc in males increasing thereafter to a maximum at d 6 pp in both sexes (P < or = 0.05). In males the augmentation in LH beta mRNA was delayed compared to females (P < or = 0.01). Before d 80 pc no FSH beta mRNA hybridization signals were apparent. Thereafter the mRNA level continuously increased with age (P < or = 0.01) in both sexes reaching its maximum at d 6 pp. The FSH beta mRNA level in females was always higher than in males (P < or = 0.01). As early as d 50 pc the alpha-subunit mRNA level was high in both sexes and further increased without sex difference to d 6 pp (P < or = 0.05). In conclusion, the mRNA levels of GH, LH beta and FSH beta are age and sex dependent during fetal development. We suggest that the fetal increase in plasma GH concentration can be accounted for by changes in GH mRNA levels, while the dramatic perinatal decrease in plasma GH concentration seems to be primarily controlled at the posttranslational and/or secretion level. The fetal sex difference and the increase in plasma LH and FSH concentrations seems to be primarily dependent on the cellular concentration of the gonadotropin beta-subunit mRNAs and/or number of gonadotrophs.

Age Factors↗

PubMed1996

Expression of GH, TSH beta, LH beta and FSH beta genes during fetal pituitary development in the pig.

The development of the anterior pituitary gland involves the proliferation and differentiation of ectodermal cells in Rathke's pouch to generate distinct cell types, each of which produces its corresponding trophic hormone. Studying pituitary development will therefore reveal novel aspects of organogenesis. In the present study, we examined by in situ hybridization the expression of genes for anterior pituitary hormones during development of the fetal pig pituitary. We found that the beta-subunit gene of thyroid-stimulating hormone (TSH beta) was first expressed at E40, (E = day of embryonal/fetal life), growth hormone (GH) mRNA appeared between E40 and E50, and the gonadotrophin genes (LH beta and FSH beta) were expressed at E50. The transcripts for TSH beta, LH beta and FSH beta were abundantly expressed at about E80, while GH mRNA continued to be richly expressed until after birth. The GH gene was first expressed in the mantle layer of the anterior lobe, while the TSH beta and gonadotrophin (LH beta and FSH beta) mRNAs were found in the central and the basal regions of the anterior lobe, respectively. All of these mRNAs (GH, TSH beta, LH beta, and FSH beta) remained concentrated until the end of gestation in the area where they first appeared. The distinctive pattern of developmental expression of these hormone genes in the fetal pig anterior pituitary makes this tissue an excellent system in which to study tissue-specific gene activation and regulation.

Animals↗

PubMed1994

Proopiomelanocortin gene expression during pig pituitary and brain development.

The expression of proopiomelanocortin (POMC) mRNA, which plays an important role in neural, endocrine, neuroendocrine and immune systems, was studied by in situ hybridization during the development of pituitary in the domestic pig. The POMC gene was activated as early as fetal day 30 (E30). The signal for POMC mRNA in the anterior lobe progressively increased from E30 to E80 and then remained at relatively constant level. In contrast, POMC transcripts in the intermediate lobe first appeared at E40 and steadily increased during development. POMC transcripts in the brain were first found at E40 and were scattered in the arcuate nucleus (AN) and nucleus medialis thalami (NMT). At E50 the extra-pituitary POMC mRNA was located not only in the AN and NMT but also in the fasciculus tegmenti and entorhinal cortex. In the posterior lobe, no signal was detected. The specific pattern of expression of the pig POMC gene in the pituitary and in specific regions of the central nervous system suggests important roles for POMC in fetal development. These results also suggest that POMC is excellent for studying the expression and regulation of pituitary hormone genes because of its tissue-specific regulation and developmental pattern.

Animals↗

PubMed1994

Tissue distribution and residues of clenbuterol, salbutamol, and terbutaline in tissues of treated broiler chickens.

To examine the tissue distribution and residues after withdrawal of various beta-agonists (i.e., clenbuterol, salbutamol, and terbutaline) 160 1-d-old broiler chickens were assigned to four groups. During treatment (16 to 35 d), the birds were fed a control diet or a diet containing 1 ppm of clenbuterol, 10 ppm of salbutamol, or 10 ppm of terbutaline. After d 35 all groups received the control diet. Five birds of each group were then slaughtered and tissues were collected on d 0, 1, 2, 3, 7, 14, and 43 following withdrawal of beta-agonists from the feed. Extraction of beta-agonists from the tissues was carried out by a new method using hetero-bifunctional solid phase extraction. The amount of beta-agonists in the extracts was measured by an enzyme immunoassay (EIA). The highest concentrations of beta-agonists were found in feathers: 224 ng of clenbuterol/g, 1,140 ng of salbutamol/g, and 1,159 ng of terbutaline/g. Clenbuterol accumulated above plasma levels in all tissues that were investigated (liver, kidney, stomach, muscle, fat, feather, eye). Salbutamol was most concentrated in feather, eye, liver, and kidney; terbutaline accumulated only in feather, liver, and kidney. Overall, clenbuterol showed the highest accumulation in the tissues analyzed. A withdrawal period of greater than 2 wk was required for residues in edible tissues to decline below detectable levels.

Adipose Tissue↗

PubMed1993

Arginine vasotocin gene expression and hormone synthesis during ontogeny of the chicken embryo and the newborn chick.

Chicken embryos at different developmental stages (embryonal day (E) 6 to 21) and chicks at posthatch day 1 (D1) were monitored for the development of their hypothalamo-neurohypophysial system as indicated by the kinetics of arginine vasotocin (AVT) gene expression via mRNA concentration and brain AVT content. Our data concerning the onset of gene expression support previous results from our laboratory and others about an early activation of the AVT gene transcriptional and translational activity around E6. We could detect measurable amounts of AVT in chicken embryo brains at E6 and an exponential increase during further development until D1. Dot blots of hypothalamic RNA extracts indicated that AVT gene transcript concentrations rose between E12 and E17 and slightly dropped thereafter. Northern hybridization showed that this drop was caused by a decrease of full length message and an increase of smaller transcripts during late embryonal and D1 stages, probably an AVT mRNA specific degradation phenomenon. The dissociation between the increase of AVT concentration and AVT mRNA concentration visible at the D1 stage might be due to accumulation and storage of AVT in the magnocellular neurons, preferentially in their axon terminals in the neurohypophysis. Blood samples taken from E14 onwards revealed a constant increase in plasma osmolality and plasma AVT concentration. Our data suggest that, in the chicken, AVT seems to be required early during embryonal development, either for osmoregulatory or further unknown functions.

Animals↗

PubMed1993

[Plasmid profiles of different Salmonella serovars from poultry flocks in Germany].

151 salmonella isolates belonging to 6 serotypes and originating from diverse material collected in broiler chicken farms, hatcheries and poultry slaughteries in Northern Germany during 1984 and 1990 were investigated for their plasmid DNA content. This is the first report describing plasmids of S. saint-paul, isolated from poultry lines. The different plasmids of 22 Md, 3.1 Md, 2.4 Md and 1.1 Md molecular weight can be used for further epidemiological studies of salmonellosis in poultry. The 62 Md plasmid of S. typhimurium and the 36 Md plasmid of S. enteritidis have been found to be serovar specific. The smaller plasmids (m. w. less than 5.0 Md), found in S. virchow and S. blockley can be used for further epidemiological studies.

Animals↗

PubMed1992

Functional maturation of arginine vasotocin secretory responses to osmotic stimulation in the chick embryo and the newborn chicken.

The time-course of appearance of plasma vasotocin and the ontogeny of the vasotocin response to osmotic stimulation during embryonic development was examined in the chicken. In the first experiment, blood was obtained by heart puncture of chicken embryos on embryonic days (E) 15-20, and by decapitation of chickens on the day of hatching (D1 = E21). Plasma was separated for measurement of osmolality and vasotocin. Basal plasma osmolality remained constant until E20 (285-291 mosmol/kg) and was significantly increased at D1 (304 +/- 2 mosmol/kg). Plasma vasotocin was first detectable at E16 (5.5 +/- 0.8 (S.E.M.) pmol/l) and increased thereafter with a peak at E18 (21.8 +/- 2.3 pmol/l). The concentration decreased again at D1 (9.0 +/- 1.7 pmol/l). Osmotic stimulation with hypertonic NaCl 60 min before the animals were killed in the second experiment increased plasma osmolality, at all time points, by 9 +/- 0.2 mosmol/kg in all animals, but increased vasotocin in an age-dependent manner. The vasotocin response was greatest in D1 chickens, and the increase in vasotocin concentration relative to the control group value was 61 pmol/l, whereas the corresponding value in young (E15-E18) and older (E19-E20) embryos was only approximately 9 and 18 pmol/l respectively. In a third experiment the time-course of vasotocin secretion after osmotic stimuli in catheterized chickens (E18 and D1) was established. The basal plasma osmolality was significantly increased after 15 min in both groups. Vasotocin levels were significantly raised after 15 min in E18 and after 30 min in D1 chickens and further increased thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia↗

PubMed1991

Comparative studies on in vitro reactivity of fresh and cryopreserved pig lymphocytes.

The effect of cryopreservation on in vitro reactivity of pig lymphocytes was studied. Peripheral blood mononuclear cells (PBMC) were frozen by controlled-rate freezing and stored in liquid nitrogen (LN2) between 4 and 36 days. Following thawing 74.7 +/- 2.6% of cells were recovered of which 94.5 +/- 0.9% were viable as determined by trypan blue exclusion. Functional parameters measured included the concentration of free intracellular Ca2+ ([Ca2+]i) in resting and mitogen-stimulated PBMC, mitogen and alloantigen-induced blastogenesis, as well as cell-mediated cytotoxicity. Irrespective of storage time and cell donor, [Ca2+]i in frozen-thawed PBMC (67.7 +/- 4.3 nM) was significantly lower (P less than 0.001) when compared to fresh cells (96.2 +/- 4.5 nM). In addition, cryopreserved PBMC only weakly responded with an increase of [Ca2+]i after stimulation by various concentrations of phytohemagglutinin (PHA). Following activation by PHA (2 micrograms/ml) for 4 days fresh lymphocytes (84,047 +/- 5475 cpm) incorporated significantly more (P less than 0.005) [3H]thymidine than frozen PBMC (66,001 +/- 4117 cpm). A similar difference in proliferation rates (P less than 0.05) between fresh (10,046 +/- 1915 cpm) and frozen-thawed PBMC (5852 +/- 1304 cpm) was observed in one-way mixed lymphocyte cultures (MLC), while the spontaneous incorporation of radiolabel was unchanged in frozen stored cells. By using MLC-derived cytotoxic effector cells (E) and [3H]thymidine-labeled concanavalin A blasts as targets (T), cryopreserved PBMC displayed a severe deficiency of cytotoxic effector functions at all tested E:T ratios. These results indicate that pig PBMC are very sensitive to LN2 storage although some immunological functions are more affected by cryopreservation than others.

Animals↗