RIA Procedure Research Articles

Evidence of NSAID partitioning into splanchnic lymphatics: Possible route to avoid first‐pass hepatic metabolism

Aspirin and related NSAIDs rapidly undergo hydrolysis in blood and first‐pass hepatic metabolism. To better understand aspirin bioavailability in rodents we investigated whether a significant fraction of the drug may directly partition into the splanchnic lymphatics to promote NSAID circulatory half‐life and activity.MethodsFasted male Sprague Dawley rats, some of which were orally pretreated 0.5, 1 and 5 hrs earlier with aspirin (pulverized Immediate‐release Walgreens OTC aspirin in water at doses of 40 and 100 mg/kg), were placed under isoflurane anesthesia and after laparotomy the splachnic lymphatic was cannulated and lymph collected for the subsequent 60–120 min. At euthanasia, blood was also collected. Samples of lymph and blood were analyzed for the presence of salicylic acid (SA) and platelet thromboxane (TxB2) conc, using established colorimetiric and RIA procedures.ResultsWe determined that lymph SA conc increased in a time‐ and dose‐dependent fashion after oral administration of aspirin and represented ~60% of circulating SA levels (using the 1 hr time point). Furthermore, aspirin dosing inhibited both lymph and plasma TxB2 conc by >;80%, 5hrs post‐treatment.ConclusionA significant fraction of intragastrically administered aspirin partitions into the lymphatic circulation in rats, which may be a critical factor in assuring the drug's bioavailability and therapeutic activity.

Spectrophotometric assay for serum glutathione transferase: A re-examination

The aim of the present paper is a careful re-examination of an automated spectrophotometric procedure for glutathione transferase (GST) activity in human serum described previously and used in many laboratories. GST activity in human serum has been assayed spectrophotometrically under various experimental conditions. Recombinant human GSTs and specific inhibitors were also used to check the possible occurrence of artifacts. Basal level of the enzyme calculated using this method turns out to be much higher than that found using RIA and ELISA procedures. Relevant pH-dependent artifacts deeply affect this spectrophotometric assay. Notably, spectral changes previously interpreted as a measure of basal activity, are mainly due to an increase of the spontaneous reaction between the two substrates. GST activity in normal serum cannot be correctly determined with the spectrophotometric assay described previously because of the very low enzyme concentration and the pH-dependent artifacts.

Changes in plasma levels of BDNF and NGF reveal a gender-selective vulnerability to early adversity in rhesus macaques

Early stressful events can increase vulnerability for psychopathology, although knowledge on the effectors is still limited. Here we tested the hypothesis that peripheral levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which are involved in the response to stress and in the pathophysiology of anxiety and depression, might be affected in a non-human primate model of adverse rearing. Males and females rhesus macaques reared with their mothers (MR) or in peer-only groups (PR) were used as experimental subjects. BDNF, NGF, adrenocorticotropic hormone (ACTH), cortisol and growth hormone (GH) were determined at baseline on postnatal days (PND) 14, 30 and 60 by means of specific ELISA and RIA procedures. In addition, behavior was assessed on PND 7, 14, 21, 30 (Brazelton test) and 60 (home cage observation). Data indicate gender differences in basal levels of BDNF throughout development. Peer-rearing increased significantly BDNF levels only in females. In addition, while all peer-reared subjects showed high levels of stereotypies and self-directed behaviors, behavioral passivity was selectively increased in females. By contrast, NGF levels were increased in response to peer-rearing only in males, and correlated positively with other "classic" endocrine responses to stress, such as cortisol and GH. Our data identify BDNF and NGF as neuroendocrine markers underlying differential responses to maternal deprivation in males and females rhesus macaques. The selective changes in BDNF levels in females could help explain the greater vulnerability to mood disorders of this gender reported in humans.

Reference levels for 17-hydroxyprogesterone, 11-desoxycortisol, cortisol, testosterone, dehydroepiandrosterone sulfate and androstenedione in infants from birth to six months of age

Reference plasma adrenal steroid levels during early infancy are frequently used to verify hormone measurements when any adrenal abnormality is suspected. We aim to obtain longitudinal reference plasma levels for 17-hydroxyprogesterone (17OHP), 11-desoxycortisol (11DOC), cortisol, dehydroepiandrosterone sulfate (DHEAS), testosterone, and androstenedione in healthy infants from birth to 6 months of age. In 138 term infants, 80 males and 58 females, plasma steroid levels were measured using specific RIA procedures at birth and on the 3rd, 15th, 30th, 60th, 90th, 120th, 150th, and 180th days of life. Smoothed percentiles for each variable were calculated according to the LMS method (LMS program version 1.16, Institute of Child Health, London). Except for cortisol, plasma levels of adrenal steroids decreased progressively from birth to 6 months of age. Plasma concentrations of 17OHP, 11DOC, and cortisol did not show gender differences, but testosterone and androstenedione were significantly higher in boys, and DHEAS levels were higher in girls. Longitudinal reference plasma levels for 17OHP, 11DOC, cortisol, DHEAS, testosterone, and androstenedione have been described in an adequate sample of healthy infants from birth to 6 months of age. These standards, displayed as smoothed percentiles, may be used as reference values in the management of congenital endocrine (adrenal or gonadal) abnormalities that appear in the first weeks of life.

Development of a Biotin-streptavidin Amplified Enzyme Immunoassay for Oxytocin and its Application During Milk Ejection and the Reproductive Cycle in the Mithun (Bos frontalis)

Oxytocin is a key hormone involved in milk ejection. It plays a key role in regulation of reproductive cyclicity in female mammals by taking part in the process of luteolysis. Determination of oxytocin is, therefore, important for studying the control of its secretion and its role in reproduction of the mithun. A simple and sufficiently sensitive enzyme immunoassay (EIA) for oxytocin determination in mithun plasma using the biotin-streptavidin amplification system and second antibody coating technique was therefore developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in a competitive assay. The EIA was conducted directly in 200 microl of unknown mithun plasma. Standards prepared in hormone-free plasma were used. The lowest detection limit was 0.5 pg/ml plasma. Plasma volumes for the EIA (50, 100, and 200 microl) did not influence the shape of standard curve, even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare endogenous mithun oxytocin with a bovine oxytocin standard. The former showed good parallelism with the bovine standard curve. For biological validation of the assay, plasma oxytocin was measured in the blood samples collected before, during, and after milking in three mithun cows and in six non-lactating cyclic mithuns during the entire estrous cycle. A sharp release of oxytocin shortly after udder stimulation was observed. A high level of oxytocin was maintained during milking, falling sharply thereafter. The mean plasma oxytocin concentration was different on different days of the estrous cycle (P < 0.001). Two peaks of oxytocin were recorded, one at day 6 and another at day 18 of the estrous cycle. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine plasma oxytocin levels in mithuns. Apart from being non-radioactive, the EIA procedure described here also utilizes a highly stable biotinalyted hormone which has a shelf life of several years, unlike the short shelf life of iodinated tracer used in RIA procedures.

Is the yak ( Poephagus grunniens L.) really a seasonal breeder?

Yaks are considered to be seasonally polyestrous and breeding occurs from July to November. Here we show that some yaks in peak non-breeding season do exhibit cyclic luteal activity without exhibiting any behavioral signs around expected estrus. A total of eight non-lactating yaks were selected from the Yak Farm belonging to National Research Centre on Yak for various sets of experiments. The animals were maintained as per semi range system of management. They were allowed to graze during daytime and fed concentrate mixture @2 kg/animal/day as per standard farm practices of the center. Blood samples were collected on alternate days for 30 days by jugular venipuncture from the yaks during peak breeding season (July to November) and from the same yaks in non-breeding season (February to March). The plasma samples were analysed for progesterone and estradiol-17β by RIA and EIA procedures, respectively. During breeding season, the mean plasma progesterone at estrus was basal (≤0.2 ng/ml) and there after started to increase and reached a peak at days 15–16 of the cycle and then declined rapidly to basal levels at estrus. Mean plasma estradiol-17β concentrations were high at estrus and then declined to basal level at days 2–3 of the cycle. The mean plasma estradiol-17β concentrations stayed low for the remaining part of the estrous cycle except for a small elevation, which was exhibited between days 6 and 12 of the cycle. During non-breeding season, in 50% of the animal studied, plasma progesterone and estradiol-17β levels were at basal level. However, in the remaining animals distinct luteal activity was recorded in terms of plasma progesterone profile. High concentrations of progesterone were maintained in one animal, which suggested existence of cystic corpus luteum. The trend of progesterone and estradiol-17β profiles in other three animals indicated that they could be cycling although they did not exhibit estrus symptoms. It was concluded from the present study that with improved nutritional support some yaks exhibit estrous cyclicity even during the non-breeding period of the year.

Reduced plasma ghrelin concentration in celiac disease after gluten-free diet treatment

ObjectiveCeliac disease (CD) is characterized by malabsorption, weight loss and increased energy expenditure. The aim of this study was to investigate the relationship between circulating ghrelin and leptin, which are produced at gastrointestinal level and are involved in energy balance regulation, and changes in body composition and energy metabolism in CD patients before and after gluten-free diet (GFD)-induced restoration of the intestinal mucosa. Material and methodsBody composition (by dual-energy X-ray absorptiometry), resting metabolic rate and substrate oxidation rates (by indirect calorimetry) were assessed in 18 adult women with the classic form of CD (age 31.4±7.8 years, body mass index (BMI) 20.6±2.1 kg/m2) before and at least 2 years after GFD treatment and in 22 age-matched healthy women (age 33.1±7.2 years, BMI 22.9±2.1 kg/m2). Plasma leptin and ghrelin concentrations were assessed by the ELISA and RIA procedures, respectively. ResultsFat-free mass was reduced before and after GFD compared to control subjects (p<0.01), while fat mass increased after treatment (p<0.01). Plasma leptin concentration was similar between groups and correlated only with BMI (r=0.84; p<0.0001) and percentage body fat (r=0.86; p<0.0001). Circulating ghrelin levels (pg/ml) were similar between untreated patients and control subjects, but decreased after GFD treatment (untreated CD: 282.6±55.5 versus treated 109.2±49.9; p<0.0001 and versus control subjects 262.2±30.0; p<0.0001) and were negatively correlated with BMI in CD patients (r=−0.32; p<0.01). ConclusionsThe low plasma ghrelin concentration found in CD patients after GFD treatment could only be partially explained by the slight increase in body-weight and fat mass. Further studies are needed to better ascertain the role played by an incomplete functional or quantitative recovery of ghrelin-producing cells in CD.

Serum 25-Hydroxyvitamin D Is Unaffected by Multiple Freeze-Thaw Cycles

Vitamin D deficiency is a common disorder believed to affect as many as 57% of hospitalized patients (1) and 9–50% of outpatients, depending on the characteristics of the study population (2)(3)(4)(5)(6). Vitamin D deficiency can cause secondary hyperparathyroidism and, if severe, osteomalacia. Both of these conditions are associated with loss of bone density, and both are reversible by repletion of vitamin D (7)(8)(9)(10). Recently, the association of vitamin D deficiency with an increased risk for a broader range of diseases, such as prostate cancer (11)(12) and colon cancer(13), has come to attention. Dietary vitamin D supplementation has even been implicated in decreasing the risk of developing type 1 diabetes mellitus (14), and ultraviolet irradiation has been shown to decrease systolic blood pressure in patients with mild untreated hypertension (15). Determining whether vitamin D deficiency is present usually involves assessing the stores of vitamin D by measuring serum 25-hydroxyvitamin D (25OHD) concentrations, in combination with an assessment of parathyroid hormone concentrations. Currently, the preferred clinical 25OHD assay is a two-step procedure that involves a rapid extraction step of 25OHD from serum or plasma, followed by an equilibrium RIA procedure. The …

209 NORGESTOMET IMPLANTS REDUCE LH RELEASE PATTERN IN ZEBU COWS UNDERGOING REPEATED OOCYTE PICK-UP

Ultrasound-guided follicular puncture (OPU) has become the most used technique to recover cumulus-oocyte complexes (COCs) from valuable donors for in vitro embryo production, because of the low risk and the possibility of collecting COCs at intervals as short as twice-a-week. However, repeated aspiration of ovarian follicles may induce endocrine abnormalities due to partial luteinization of punctured follicles and interference with follicular development. The use of exogenous progestagens is an alternative used to control these side effects, and is under evaluation. The aim of this study was to evaluate whether the effect of norgestomet treatment on intra-follicular and systemic steroid concentrations and on ovarian follicular dynamics is related to changes in LH releasing pattern. Pluriparous non-lactating Gir breed (Bos indicus) cows (n = 10) were randomly distributed between treatment (norgestomet ear implants, replaced weekly) and control (no hormone used) groups, and had their ovarian follicles larger than 3 mm in diameter aspirated twice a week, during the next two consecutive weeks. Follicular dynamics were evaluated every 12 h between OPU sessions, and the largest follicles present were used to recover samples of follicular fluid. Blood samples were collected daily for progesterone evaluation in all cows, and 3 times within a 4-h window interval, at 24, 48, 72, and 96 h after follicle puncture for LH evaluation, in 6 cows (3 from treated and 3 from control groups). LH was measured by a standardized RIA procedure. Data was analyzed by ANOVA, and means were compared by Tukey's test. Results are presented as means ± SEM. Treatment with norgestomet reduced mean progesterone plasma concentration during the evaluated period (36.3 ± 14.0 vs. 250.3 ± 49.3 pg/mL; P &lt; 0.0001), the incidence of follicles growing above 9 mm (30% vs. 65%; P &lt; 0.05) in the intervals between OPU sessions, and intrafollicular estradiol and progesterone concentrations in the largest follicles (n = 27) present (265.5 ± 47.4 and 34.9 ± 5.4 ng/mL vs. 765.2 ± 169.1 and 173.3 ± 43.4 ng/mL, respectively; P &lt; 0.05). Plasma LH concentrations were consistently lower during the 3 session intervals in cows treated with norgestomet (0.16 ± 0.04, 0.22 ± 0.03, 0.22 ± 0.09 and 0.17 ± 0.01 vs. 0.44 ± 0.15, 0.53 ± 0.04, 0.42 ± 0.05 and 0.39 ± 0.11 for 24, 48, 72, and 96 h after OPU, respectively; P &lt; 0.05). These results confirm the theory that norgestomet treatment is associated with a reduction in the LH-release pattern, as expected due to the reduction in both luteinization of punctured follicles and in the steroidogenic activity of growing follicles observed during the experiment. The use of norgestomet ear implants can be an alternative in the management of donor cows undergoing oocyte pickup. This work was sponsored by FAPEMIG and CNPq. The authors thank Dr. Robert Douglas for aiding with LH RIA.

Analysis of plasma IGF-I hormone level and its correlation with live weight and age in Holstein-Friesian heifers

Abstract. The aim of this study was to evaluate the changes and relation of live weight and IGF-I plasma concentration during the rearing period in HF heifers. Number of animals was 85 kept in similar housing and feeding conditions. Blood samples were collected monthly at the same hour of the day and weight of the calves were scaled simultaneously up to the 14th month of age. Plasma IGF-I concentration was measured by RIA procedure. Data were analysed by STATISTICA, SPSS 10.0 version and POST HOC TEST programs. Results of this work allow to conclude that change of plasma IGF-I c.c. and live weight show similar trend. Rearing phase of calves shows three peaks: 1st at birth; 2nd closed to sexual maturity; 3rd closed to first service. IGF-I-profile can be taken as similar for all individual and animals can be categorised into groups with low or high hormone concentrations. There is only negative correlation (r = −0.24) between IGF-I levels at birth and at 4 months of age. Bulls have special effect on IGF-I c.c. at birth, at 5th, 13th and 14th months of age (P &lt; 0.1). On contrary to previous findings IGF-I level and weight of calves correlated negatively (r = −0.31, P &lt; 0.05) in the whole population, while within progeny groups positive and negative correlation may occur, too. Negative correlation prove the compensation ability of animals in growth rate. In comparison with the overall mean of IGF-I c.c., the subpopulation below average has lower live weight, too and with high IGF-I level a higher live weight is bound.

The monoclonal antibody MAC252 does not react with the (-) enantiomer of abscisic acid.

An RIA procedure has been developed for ABA quantification using MAC62, a monoclonal antibody raised against (+)-cis, trans -ABA. This widely used method now relies on MAC252, a recloned version of the exhausted MAC62. Recently, it has been suggested that MAC252 was not able to discriminate between the (+) and (-) enantiomers of ABA. As this can be misleading when interpreting RIA results, it has been carefully examined here whether MAC252 reacts with (-)-ABA. MAC252 exhibited negligible cross-reactivity with (-)-ABA, which was confirmed with commercial mixtures of ABA isomers. It is concluded that the RIA protocol can continue to be used with MAC252 as it was with MAC62.

Hypothermia During or After Severe Perinatal Asphyxia Prevents Increase in Cyclic Gmp Levels - in the Newborn Rat Striatum 1882

In the rat striatum, the enzyme nitric oxide synthase (NOS) is present in a dense fiber network and in few medium sized non-spinal interneurons. This enzyme is activated by glutamate throughout the influx of Ca2+ to the cell. A sustained activation of NOS caused by excessive release of glutamate, results in strongly increased NO that is a potent activator of the soluble guanylyl cyclase resulting in the synthesis of intracellular cGMP. Previous work showed that NO-containing striatal neurons suffer short-and long-term changes after perinatal asphyctic insult and that changes were prevented by hypothermic exposure. In order to know if this morphological changes were due to an excess of the NOS activity we designed the meassurements of striatal cGMP levels, with the aim to evaluate NOS activity in asphyctic and hypothermic treated pups using a model of severe perinatal asphyxia (SPA). The levels of striatal cGMP of normal pups (PN10) using a RIA procedure were compared to those subjected to SPA and SPA treated with hypothermia during or after insult. cGMP levels in striatal tissue from control pups (PN10) were 25.8 pmol/mg protein and in the SPA group 38.1 pmol/mg which is an increase of approx 70%(p<0.01). Hypothermia during as well as after insult was capable to prevent this increase of cGMP. L-NAME at 0.1 mM decreased cGMP levels in control, SPA and hypothermic treated pups whithout differences between them. Nitroprusside stimulated cGMP showed no differences between groups. These findings indicate that high cGMP levels in the striatum of rats subjected to SPA are under control of an hyperactive NOS leading to excessive NO production which could be harmful to the cerebral tissue. Hypothermia may prove a useful therapeutic agent against cerebral oxidative stress as it reduces NOS activity.

Radioimmunoassay of bound and free melatonin in plasma

We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin in human plasma. All procedures showed a diurnal rhythm of melatonin in human subjects, with interindividual differences greater than interprocedure differences. However, further investigations demonstrated considerable variability of recovery in the nonextraction procedure, suggesting a variability of binding proteins between samples. Combining recovery and dialysis experiments in the extraction procedures, we demonstrated that chloroform was unable to extract albumin-bound melatonin from a human serum albumin solution but, paradoxically, was able to extract bound and free melatonin from a plasma sample. The methanol extraction procedure extracted free and bound melatonin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays should be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.

Improved techniques for the rapid radioimmunoassay of ecdysteroids and other metabolites

Summary An RIA procedure for assaying a large number of samples in a relatively short time has been developed. It utilizes multi-channel repeater pipettors for the addition of reagents, specially designed racks which accommodate 48 6 x 50-mm tubes and which fit directly into the centrifuge, multitube vortexers, and a specially designed aspiration device. The racks allow the tubes to remain in the same support throughout most of the assay. The aspirator permits the rapid aspiration of eight tubes at a time, 192 in 2 min. One hundred ninety-two samples can be accurately assayed in as little as 2.5 h.

A research note: an analysis of RIA and GC/MS split hair samples from the New Orleans pretrial diversion program

This paper compares the outcomes of hair analysis for cocaine conducted by two different, independently developed analytical methods, one using radioimmunoassay and the other GC/MS chemical ionization. In this study two hair samples were collected from each subject and a sample was sent simultaneously to each laboratory. The assays were done independently with each laboratory being unaware of the results of the other laboratory. The laboratories each developed their own cutoff criteria, and did not utilize the same cutoff criteria. Analysis of the comparative performance shows a strong correlation between the two procedures, although the GC/MS method, which used a lower cutoff value, identified more positive specimens than did the RIA method. Additionally, there were no cases in which the RIA procedure identified a positive sample which was negative according to GC/MS, although a number of GC/MS positive assays were reported as negative by the RIA laboratory.

The concentration of GnRH in hypothalamus, LH and FSH in pituitary, LH, PRL and sex steroids in peripheral and ovarian venous plasma of hypo- and hyperthyroid, cysts-bearing gilts.

The aim of this work was to investigate the hormonal pattern in hypo- and hyperthyroid gilts with experimentally induced cystic ovarian disease (COD). A total of 70 adult, nulliparous gilts divided into six groups were used for the experiment. Group I was euthyroid and control. Group II was made hypothyroid by oral administration of methylthiouracyl for 24 days. Group III represented euthyroid gilts injected with gonadotropins (PMSG and hCG). Group IV consisted of hypothyroid gilts injected with gonadotropins. Group V was treated with L-thyroxine for 24 days and Group VI with thyroxine and with gonadotropins for the last 10 days of the test. The treatment of all groups was terminated on the 4th-5th day of the next estrous cycle. The peripheral blood of the gilts was collected on Day 0, and on Day 24. On the 25th day the gilts were laparotomized and cannulas were inserted into utero-ovarian veins of each ovary for blood collection. Simultaneously, peripheral blood samples were collected during 1 to 3 consecutive days. The animals were then slaughtered and the hypothalamus, pituitary and ovaries were frozen and preserved for further analyses. In hypothalamic tissue the content of GnRH: in the pituitary the concentration of LH and FSH; and in peripheral and ovarian blood plasma the level of LH, PRL, E1, P4, A4, T and cortisol (Cl) were estimated by RIA procedure. The level of GnRH in the hypothalamus, and LH and FSH in the pituitary showed a tendency to parallel with thyroid function which may indicate a role of this gland in their production or secretion. In hypothyroid animals an increase of LH and PRL and a slight decrease of secretory function of the ovaries were noted. Injections of gonadotropins in euthyroid or hypo- and hyperthyroid gilts intensified the function of the ovaries, which was manifested by numerous follicular cysts and corpora lutea. The hormonal milieu of gilts from these groups showed a low level of LH, PRL and an increased content of sex steroids in peripheral and ovarian blood. The ovarian steroidogenesis of cyst-bearing gilts was disturbed, which was indicated by an increased level of E1, P4, A4, T, and Cl, but a low level of E2. These disturbances in steroidogenesis in cystic gilts may be caused by a deficiency in LH secretion as the consequence of the pituitary gonadotropin suppression by the used gonadotropins. The steroid hormone pattern of cyst-bearing gilts strongly resembles the endocrine profile noted in polycystic ovarian disease in women.

Monoclonal antibodies specific for the binding site on the LH receptor alter progesterone production in cultured granulosa cells.

Monoclonal antibodies (mAbs) specific for the LH receptor (LHR) were generated through a modified auto-anti-idiotypic approach in which human chorionic gonadotropin (hCG) was used as the immunogen followed by cyclophosphamide to induce anti-idiotypic antibodies. The purpose of this study was to investigate the effectiveness of these antibodies to alter progesterone production in porcine granulosa cells in vitro. Anti-LHR mAbs were incubated with granulosa cells in the presence or absence of a stimulatory dose of hCG. Progesterone output by treated cells was measured using a RIA procedure. Most of the mAb could inhibit stimulated progesterone production by cultured granulosa cells. It was speculated that two possible mechanisms may cause the inhibition effect observed. Several of the antibodies appeared to block hCG binding thus removing the stimulatory effects of hCG. However, the most potent inhibiting mAbs for progesterone production had little or no effect on hCG binding, suggesting that some other mechanism was responsible for the observed inhibition. In addition, several of the antibodies were found to have a stimulatory effect on progesterone production by granulosa cells even in the absence of a stimulating dose of hCG. It is proposed that these antibodies were able to mimic hCG.

Clinical observations on changes in concentrations of hormones in plasma of two stallions with thermally-induced testicular degeneration

Summary To investigate effects of thermally-induced testicular degeneration on hormonal and seminal parameters in stallions, the scrotum was insulated for 36 hours in two mature (5-year-old mixed breed and 11-year-old Throughbred) stallions. Semen was collected daily for 10 days (DSO) prior to, and at intervals after, scrotal insulation. When DSO determinations were not being made, semen was collected 3 times weekly. Jugular blood samples were collected at 15-minute intervals for 6 hours from each stallion prior to, and at intervals after, scrotal insulation. A mouse interstitial cell testosterone assay was modified to quantify biologic activity of equine luteinizing hormone (BLH) in plasma samples. Immunoactive luteinizing hormone (ILH) and testosterone (T) concentrations were determined in plasma samples by routine RIA procedures. Percentages of progressively motile and morphologically normal spermatozoa began to decrease by 1 to 2 weeks postinsulation, reached nadir values at 3 to 3-1/2 weeks postinsulation, and returned to preinsulation values by 7 weeks postinsulation. Total number of spermatozoa and total number of progressively motile, morphologically normal spermatozoa in ejaculates at DSO returned to normal by 8 weeks postinsulation in stallion 2 and 12 weeks postinsulation in stallion 1. Concentrations of BLH and ILH increased, and while T concentrations decreased, immediately postinsulation. The increase in ILH concentrations was greater than the increase in BLH concentrations, resulting in a decrease in the BLH:ILH (B:I) ratio. Following the peak in LH secretion immediately postinsulation, LH concentrations gradually decreased while T concentrations increased. The B:I ratio was elevated from 1 to 13 weeks postinsulation compared to immediately postinsulation. In addition to changes in spermatozoal quality in ejaculates, stallion response to scrotal insulation included increased secretion of luteinizing hormone and impaired Leydig cell function (as determined by reduced testosterone concentration in circulating plasma). The proportion of biologically active LH secreted in response to thermal testicular injury increased during the recovery phase.

Superior analytic characteristics of an immunometric assay for gonadotropins also provides useful clinical prediction of in vitro fertilization outcomes

To determine whether an analytically superior gonadotropin assay, the monoclonal, two-site immunometric assay, also provided superior prediction of clinical outcomes when compared with conventional RIA methodology. Methods comparison study. Tertiary academic center. One hundred fifty-seven consecutive IVF patients. Comparisons of FSH and LH levels on cycle day 3 were made using paired RIA and immunometric assay procedures. The ability of day 3 LH, FSH, and their ratio in predicting IVF performance was determined using regression analyses. The predictive ability of FSH as assayed by immunometric assay at least equaled that obtained by RIA for both peak E2 levels and the number of mature oocytes retrieved. Results from this study indicate that gonadotropin levels as measured by immunometric assay represent an effective clinical tool in predicting IVF outcomes that may prove superior to RIA.

Exogenous PGF 2α enhanced GnRH-induced LH release in postpartum cows

This study evaluated the effect of exogenous PGF 2α on circulating LH concentrations in postpartum multiparous (n = 32) and primiparous (n = 46) Brahman cows. The cows were randomly allotted within parity and calving date to receive 0, 1, 2 or 3 mg im PGF 2α (alfaprostol)/100 kg body weight (BW), with or without GnRH on Day 30 after calving. Blood samples were collected at weekly intervals from calving through treatment. Serum progesterone concentrations were determined using RIA procedures to assure that only anestrous cows were treated. Sterile marker bulls were maintained with cows on Coastal bermudagrass pastures until the first estrus was detected. Multiparous cows had a shorter (P < 0.05) interval from calving to estrus than did primiparous cows. Serum LH was affected by time (P < 0.0001), PGF 2α dose (P < 0.0002), GnRH (P < 0.0001), parity by PGF 2α dose (P < 0.0003), PGF 2α dose by GnRH (P < 0.0009), parity by GnRH (P < 0.0008), and by parity by PGF 2α dose by GnRH (P < 0.0005). Multiparous cows not receiving GnRH had higher mean serum LH (P < 0.02), LH peak pulse height (P < 0.03), and area under the LH release curve (P < 0.03) compared with primiparous cows. The number of LH pulses/6 h was greater (P < 0.06) in multiparous than primiparous cows, and was greater (P < 0.02) in multiparous cows receiving 3 mg/100 kg BW than in cows receiving 2 mg/100 kg BW, but not in the controls or in cows receiving 1 mg/100 kg BW. Exogenous GnRH resulted in increased (P < 0.0001) serum LH concentrations in all cows, and LH was enhanced (P < 0.0009) by simultaneous treatment with PGF 2α. Primiparous cows had a greater response (P < 0.0005) to PGF 2α and GnRH compared with multiparous cows. Pituitary release of LH in response to GnRH was enhanced by simultaneous exposure to PGF 2α in Day 30 postpartum cows.