The melatonin system is expressed in the ovine uterus: effect of the day of the oestrous cycle and undernutrition.

CONTEXT: Melatonin influences female reproduction, but expression of the melatonin system has not been characterised in the ovine uterus. AIMS: We aimed to determine whether synthesising enzymes (arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin-O-methyltransferase (ASMT)), melatonin receptors 1 and 2 (MT1 and MT2), and catabolising enzymes (myeloperoxidase (MPO) and indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and 2)), are expressed in the ovine uterus, and if they are influenced by the oestrous cycle (Experiment 1) or by undernutrition (Experiment 2). METHODS: In Experiment 1, gene and protein expression was determined in sheep endometrium samples collected on days 0 (oestrus), 5, 10 and 14 of the oestrous cycle. In Experiment 2, we studied uterine samples from ewes fed either 1.5 or 0.5times their maintenance requirements. KEY RESULTS: We have demonstrated the expression of AANAT and ASMT in the endometrium of sheep. AANAT and ASMT transcripts, and AANAT protein were more elevated at day 10, then decreased to day 14. A similar pattern was observed for MT2 , IDO1 , and MPO mRNA, which suggests that the endometrial melatonin system might be influenced by ovarian steroid hormones. Undernutrition increased AANAT mRNA expression, but seemed to decrease its protein expression, and increased MT2 and IDO2 transcripts, whereas ASMT expression was unaffected. CONCLUSIONS: The melatonin system is expressed in the ovine uterus and is affected by oestrous cycle and undernutrition. IMPLICATIONS: The results help explain the adverse effects of undernutrition on reproduction in sheep, and the success of exogenous melatonin treatments in improving reproductive outcomes.

Effects of extending the length of pro-oestrus in an oestradiol- and progesterone-based oestrus synchronisation program on ovarian function, uterine environment and pregnancy establishment in beef heifers.

The aim of the present study was to investigate the effects of a strategy for extending pro-oestrus (the interval between luteolysis and ovulation) in an oestrus synchronisation protocol (named J-Synch) in beef heifers on follicular growth, sexual steroid concentrations, the oestrogen receptor ERα and progesterone receptors (PR) in the uterus, insulin-like growth factor (IGF) 1 and pregnancy rates. In Experiment 1, heifers treated with the new J-Synch protocol had a longer pro-oestrus period than those treated with the conventional protocol (mean (±s.e.m.) 93.7±12.9 vs 65.0±13.7h respectively; P<0.05). The rate of dominant follicle growth from the time of progesterone device removal to ovulation was greater in heifers in the J-Synch than conventional group (P<0.05). Luteal area and serum progesterone concentrations were greater in the J-Synch Group (P<0.05) for the 12 days after ovulation. Progesterone receptor (PGR) staining on Day 6 after ovulation in the uterine stroma was lower in the J-Synch than conventional group (P<0.05), and the expression of PR gene (PGR) and IGF1 gene tended to be lower in J-Synch-treated heifers (P<0.1). In Experiment 2 (n=2349), the pregnancy rate 30-35 days after fixed-time AI (FTAI) was greater for heifers in the J-Synch than conventional group (56.1% vs 50.7% respectively). In conclusion, our strategy for extending pro-oestrus (i.e. the J-Synch protocol) significantly improves pregnancy establishment in beef heifers. This improvement was related to an increased rate of growth of the dominant ovulatory follicle, greater progesterone concentrations during the ensuing luteal phase and different uterine patterns of PGR and IGF1, which may have favoured embryo development and pregnancy establishment.

Long-term effect of early nutrition on endocrine parameters and liver and endometrial gene expression of the members of the somatotrophic axis in Hereford heifers.

This study compared the effect of different management systems on endocrine parameters, and gene expression of members of the somatotrophic axis in the liver and endometrium of beef heifers. Twenty-two 709-days-old heifers submitted to Early Weaning (EW, n = 8), Traditional Weaning (TW, n = 7) and TW plus creep feeding (TW+CF, n = 7) were used. Animals were synchronized with two prostaglandin (PG) injections at 11-day interval (Oestrus = Day 0). Blood samples were collected daily for progesterone (P4) determination, and endometrial and liver biopsies on Days 7 and 16 for transcript determination of members of the somatotrophic axis. Progesterone concentrations were greater on Days 15 and 16 (p < .02) of the cycle in TW+CF than TW and EW heifers. On Day 7, TW+CF heifers expressed greater liver total growth hormone receptor transcripts than TW heifers (p = .05) and greater insulin-like growth factor (IGF)-binding protein 3 mRNA than both EW and TW groups (p < .05). On Days 7 and 16, TW+CF expressed more endometrial IGF1 mRNA than the other groups (p < .01). We conclude that increasing the plane of nutrition of nursing calves may have a long-term effect on the functioning of the somatotrophic axis both in the liver and in the endometrium.

Milk fatty acid profile from cows fed with mixed rations and different access time to pastureland during early lactation.

Milk fatty acid (FA) profiles were determined in Holstein cows (n = 27) fed total mixed rations (TMR) ad libitum (G0) or diet composed by TMR (50% dry matter [DM] offered) plus grazing of pasture with 6 hr of access time to paddock in one session (G1) or 9 hr in two sessions (G2) at 45 days in milk (DIM). Moreover, milk FA was determined at 65 DIM when G0 cows turned out to G1 diet without adaptation period (Post-G0), G1 remained as controls. Milk FA was quantified using gas chromatography and mass spectrometry. Preformed FA at 45 DIM was greater (+27%) for G2 than G0 cows (p < .05). Stearic acid (C18:0) was 30% greater for G2 cows (p < .05). De novo FA was lowest for G2 cows (p < .05). Conjugated linoleic acid (CLA) did not differ (p < .12), while vaccenic acid (C18:1trans) was twofold greater for grazing treatments (p < .01). Linolenic acid [C18:3(n-3)] was greatest for G2 and lowest for G0 cows (p < .01). Omega 6 FA was greater for G0 than grazing cows, mainly due to linoleic acid [18:2cis(n-6); p < .05]. These results determined that n-6/n-3 ratio was almost threefold greater for G0 than grazing cows (p < .001). When diet of G0 cows changed to include pasture (Post-G0), preformed FA increased (p < .05), explained mainly by the increase (p < .05) of stearic (C18:0) and C18:1trans, while de novo FA tended to decrease (p < .1). Moreover, the amount of CLA and C18:3(n-3) tended to increase (p < .1) in Post-G0 cows. Offering 50% of dietary DM from pasture modified milk FA profile in early lactation potentially beneficial for human health. When TMR-fed cows were turned out to 50% pasture, milk FA profile reflected dietary change without need of an adaptation period.

Endometrial gene expression in primiparous dairy cows at the end of the voluntary waiting period is affected by nutrition: Total mixed ration vs increasing levels of herbage allowance.

The study postulated that differential nutritional management during the early lactation period would be reflected in endometrial expression of genes related to embryo growth at the end of the voluntary waiting period. Thus, the effect of the combined use of total mixed ration (TMR) and grazing under different herbage allowances during the first 75 days post-partum (DPP) on endometrial gene expression was evaluated in primiparous dairy cows. Cows were blocked by body weight, age and body condition score and randomly assigned to three grazing treatments: high (HA, 30 kg DM per cow per day), medium (MA, 15 kg DM per cow per day) and low (LA, 7.5 kg DM per cow per day) herbage allowance (mixed pasture, 2,600 kg DM per ha) plus 8 kg DM of supplement or TMR (55% forage, 45% concentrate) fed ad libitum (TMR) from calving to 75 DPP. At 57 DPP, cows were synchronized for oestrus (day 0, 68 DPP) and at day 7, endometrial biopsies were obtained. The nutritional treatment did not affect insulin, IGF-1 and leptin concentrations on days 0, 4 or 7. Expression of IGF1, IGFBP3, IGFBP4, ADIPOR1 and ADIPOR2 mRNA was significantly affected by the nutritional treatment. Endometrial IGF1 and IGFBP4 mRNA were twofold greater in TMR and HA than MA and LA cows. Expression of IGFBP3 and ADIPOR1 mRNAs was greater in TMR and HA than MA cows, but did not differ from LA cows. All groups had greater expression of ADIPOR2 mRNA than MA cows. This study provided solid evidence of the importance of nutritional management during early lactation on uterine environment at the end of the voluntary waiting period. The greater expression of genes related to embryo growth and uterine function (IGF system, progesterone and adiponectin receptors) in cows fed diets maximizing energy intake suggests a favourable environment for embryonic growth, which may explain the improved reproductive performance of cows in good energy balance.

LC-MS/MS Measurement of Parathyroid Hormone-Related Peptide.

INTRODUCTION: Parathyroid hormone-related peptide (PTHrP) is involved in activating pathways, allowing tumor cells to form bone metastases. Measurement of PTHrP is used for the diagnosis and clinical management of patients suspected of hypercalcemia of malignancy. We developed an LC-MS/MS method for measuring PTHrP, established sex-specific reference intervals, and assessed the method's performance. METHODS: PTHrP was enriched from plasma samples with rabbit polyclonal anti-PTHrP antibody conjugated to magnetic beads. Enriched PTHrP was digested with trypsin, and PTHrP-specific tryptic peptide was analyzed with 2-dimensional LC-MS/MS in multiple reaction monitoring mode. RESULTS: The lower limit of quantification was 0.6 pmol/L, and the upper limit of linearity was 600 pmol/L. Total imprecision was <10%. Very poor agreement was observed with the RIA (n = 207; Deming regression RIA = 0.059 × LC-MS/MS - 1.8, r = 0.483; Sy|x = 3.9). Evaluation of the clinical performance of the assay using samples from patients with and without hypercalcemia (n = 199) resulted in an area under the ROC curve of 0.874. In sets of consecutively analyzed routine samples of patients assessed for hypercalcemia, the PTHrP positivity rate by RIA (n = 1376) was 1.9%, and 26.6% by LC-MS/MS (n = 1705). Concentrations were below the lower limit of quantification in 95.6% of the samples by RIA and 2.0% by LC-MS/MS. CONCLUSIONS: PTHrP is a normal constituent in circulating blood and its concentrations are substantially underestimated by commercial RIAs, causing false-negative results in samples from patients suspected of hypercalcemia. Our observations suggest a link between increased concentrations of PTHrP in postmenopausal women with low body mass index and increased incidence of osteoporosis.

Undernutrition affects embryo quality of superovulated ewes.

To determine the effect of undernutrition on embryo production and quality in superovulated sheep, 45 ewes were allocated into two groups to be fed diets that provided 1.5 (control, C; n = 20) or 0.5 (low nutrition, L; n = 25) times daily requirements for maintenance, from oestrous synchronization with intravaginal sponges to embryo collection. Embryos were collected 7 days after the onset of oestrus (day 0). Low nutrition resulted in lower live weight and body condition at embryo collection (P < 0.05). Diet (P < 0.01) and day of sampling (P < 0.001) significantly affected plasma non-esterified fatty acid (NEFA) and insulin concentrations. Plasma leptin concentrations decreased on day 7 only in L ewes. A significant effect of dietary treatment (P < 0.05) and day (P < 0.0001) was observed on plasma insulin-like growth factor (IGF)-I concentrations. The number of recovered oocytes and embryos did not differ between the groups (L: 15.4 ± 0.4; C: 12.4 ± 0.4). Recovery rate was lower (P < 0.05) in the L (60%) than in the C group (73%). The total number of embryos and number of viable-transferable embryos (5.0 ± 0.3 and 3.4 ± 0.3 embryos, respectively) of the L group were lower (P < 0.1) when compared with controls (8.4 ± 0.4 and 6.2 ± 0.4 embryos, respectively). Undernutrition during the period of superovulation and early embryonic development reduced total and viable number of embryos. These effects might be mediated by disruption of endocrine homeostasis, oviduct environment and/or oocyte quality.

Oestrogen and Progesterone Receptors and COX-2 Expression in Endometrial Biopsy Samples During Maternal Recognition of Pregnancy in Llamas (Lama glama).

Endometrial expression of oestrogen receptor-α (ERα), progesterone receptor (PR) and cyclooxigenase-2 (COX-2) was evaluated in non-pregnant and pregnant llamas during the period when luteolysis/maternal recognition of pregnancy is expected to occur. Females (n = 28) were divided into two groups: non-pregnant llamas were induced to ovulate with a Buserelin injection, and endometrial biopsies were obtained on day 8 (n = 5) or 12 (n = 5) post-induction of ovulation. Animals of the pregnant group (n = 18) were mated with a fertile male. Pregnancy was confirmed by the visualization of the embryo collected by transcervical flushing in 5 of 9 animals on day 8 post-mating and by progesterone profile on day 12 post-mating in 4 of 9 animals, when endometrial biopsies were obtained. An immunohistochemical technique was used to evaluate receptors population and COX-2 expression. Pregnant llamas showed a higher percentage of positive cells and stronger intensity for ERα than for non-pregnant llamas in stroma on day 8 and in the luminal epithelium on day 12 post-induction of ovulation, while a deep decrease in endometrial PR population was reported in pregnant llamas on that day in luminal and glandular epithelia and stroma. In the luminal epithelium, COX-2 expression was lower in pregnant than in non-pregnant animals. Briefly, the increase of ERα in pregnant llamas gives further support to the hypothesis that oestrogens are involved in the mechanism of maternal recognition of pregnancy. Endometrial PR decrease in pregnant llamas might be a necessary event to allow the expression of proteins involved in conceptus attachment, a mechanism widely accepted in other species. Moreover, embryo seems to attenuate maternal PGF(2α) secretion during early pregnancy by decreasing the endometrial expression of COX-2 in the luminal epithelium of pregnant llamas.

Periconceptional undernutrition modifies endocrine profiles and hepatic gene expression in sheep.

This study investigated whether a 22-day period of undernutrition (half maintenance) could affect maternal endocrine responses and liver gene expression during early pregnancy (day 7). Thirty-five ewes were fed 1.5 (n = 15) or 0.5 (n = 20) their maintenance requirements and slaughtered on day 7 of the oestrus cycle or pregnancy (oestrus = day 0). Insulin, IGF, leptin and non-esterified fatty acids (NEFA) were determined on days -14, 0 and 7. Transcripts of the IGF family and adipokines receptors were determined in the liver by real-time RT-PCR. Underfed animals presented lower body weight and body condition, greater plasma concentration of NEFA, and lower plasma concentrations of leptin, insulin and IGF1 compared to adequately fed animals. Underfed ewes presented greater hepatic expression of IGFBP2 than well-fed ewes, but tended to have lesser expression of IGFBP5. While no effect of undernutrition on IGFBP4 and ADIPOR2 mRNA expressions was observed, they were increased by pregnancy in underfed animals. This study shows that undernutrition modifies endocrine profiles and hepatic gene expression of IGFBP2 and 5. The pregnancy status increased hepatic gene expression of IGFBP4 and ADIPOR2 mRNA in undernourished ewes.

Periconceptional undernutrition increases quantity and quality of oocyte population, but not cognitive or emotional response of 60-day-old lambs.

Maternal periconceptional undernutrition is associated with altered development and increased risks of adverse outcomes in the offspring. The aim of this work was to determine the effect of periconceptional undernutrition on behavioural and reproductive aspects of the offspring. Fifty ewes were synchronized in oestrus (day 0) and allocated to two groups (n = 25) to be fed diets that provided 1.5 (C) or 0.5 (L) times the requirements for maintenance until day 15. Ewes were mated and fed the control diet from day 16 until lambing. Two months after lambing, 26 lambs were exposed to tests to determine their cognitive/emotional responses. Six ewe lambs were euthanized and in vitro oocyte maturation and fertilization procedures performed. The experimental diets produced no changes of mean live weight (LW) of C ewes, L ewes presenting a reduction in their initial LW with significant differences at day 15, in comparison with C ewes (p < 0.05). L ewes experienced a significant reduction in their body condition (BC) in comparison with C ewes (p < 0.05). Fourteen days after the onset of the experimental diets, mean LW and BC of L ewes was significantly lower than those of C ewes (p < 0.05). Undernourished ewes presented a trend to a reduction of prolificacy and fecundity (p < 0.10) in comparison with C ewes. Emotional and cognitive test revealed a similar response between groups. Ewe lambs from the undernourished ewes presented a population of oocytes 1.7 times higher than ovaries from control ewe lambs (66.0 ± 0.73 vs. 113.7 ± 15.6 oocytes; p < 0.05) and had more oocytes in the 'good' (p < 0.05) and 'healthy' (p < 0.05) categories. In conclusion, a low plane of nutrition around conception significantly increases quantity and quality of the oocyte population of 60-day-old female descendants. Modifications of the cognitive and emotional responses of the progeny have not been evidenced.

Accessibility of blood affects the attractiveness of cattle to horn flies.

The burden of infestation of the horn fly, Haematobia irritans (Linnaeus) (Diptera: Muscidae), differs among bovines within the same herd. We hypothesized that these differences might be related to the epidermal thickness of the cattle and the blood intake capacity of the fly. Results showed that dark animals carried more flies and had a thinner epidermis than light-coloured animals, which was consistent with the greater haemoglobin content found in flies caught on darker cattle. Similarly, epidermal thickness increased with body weight, whereas haemoglobin content decreased. Overall, we suggest that accessibility of blood is a factor that partially explains cattle attractiveness to flies.

Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase.

Pregnenolone (PREG) can be converted to PREG esters (PE) by the plasma enzyme lecithin: cholesterol acyltransferase (LCAT), and by other enzyme(s) with unknown identity. Acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) convert various sterols to steryl esters; their activities are activated by cholesterol. PREG is a sterol-like molecule, with 3-ß-hydroxy moiety at steroid ring A, but with much shorter side chain at steroid ring D. Here we show that without cholesterol, PREG is a poor ACAT substrate; with cholesterol, the V(max) for PREG esterification increases by 100-fold. The binding affinity of ACAT1 for PREG is 30-50-fold stronger than that for cholesterol; however, PREG is only a substrate but not an activator, while cholesterol is both a substrate and an activator. These results indicate that the sterol substrate site in ACAT1 does not involve significant sterol-phospholipid interaction, while the sterol activator site does. Studies utilizing small molecule ACAT inhibitors show that ACAT plays a key role in PREG esterification in various cell types examined. Mice lacking ACAT1 or ACAT2 do not have decreased PREG ester contents in adrenals, nor do they have altered levels of the three major secreted adrenal steroids in serum. Mice lacking LCAT have decreased levels of PREG esters in the adrenals. These results suggest LCAT along with ACAT1/ACAT2 contribute to control pregnenolone ester content in different cell types and tissues.

Measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies.

BACKGROUND: Measurement of serum thyroglobulin (Tg) is used to monitor patients after treatment for differentiated thyroid carcinoma (TC). Difficulty in using Tg as a biomarker of the recurrence of TC in many patients stems from the presence of endogenous anti-Tg autoantibodies (Tg-AAbs), which can interfere with immunoassays (IAs) and cause false-negative results. METHODS: We enriched Tg from serum samples using rabbit polyclonal anti-Tg antiserum and protein precipitation. Unrelated proteins were partially depleted in the process. Enriched proteins were then denatured, reduced, and digested with trypsin after the addition of a winged internal standard peptide. A Tg-specific tryptic peptide was purified by immunoaffinity extraction and analyzed by 2-dimensional LC-MS/MS. Instrument cycle time was 6.5 min per sample. RESULTS: The lower limit of quantification was 0.5 ng/mL (0.76 fmol/mL dimer). Total imprecision of triplicate measurements in serum samples over 5 days was <10%. Comparison with a commercial IA using serum samples free of Tg-AAb (n = 73) showed Deming regression, IA = 1.00 * LC-MS/MS - 2.35, r = 0.982, standard error of the estimate (S(y|x)) = 9.52. In a set of Tg-AAb-positive samples that tested negative for Tg using IA (n = 71), concentrations determined by LC-MS/MS were ≥0.5 ng/mL in 23% of samples (median 1.2, range 0.7-11 ng/mL). CONCLUSIONS: The introduced method has acceptable performance characteristics for use in clinical diagnostic applications. The most substantial disagreement between methods was observed in Tg-AAb-positive samples with concentrations <2 ng/mL (determined with LC-MS/MS). The affinity-assisted enrichment strategy used for Tg in this method should be applicable to other biomarkers that have endogenous autoantibodies.

Oestrogen, progesterone and oxytocin receptors and COX-2 expression in endometrial biopsy samples from the induction of ovulation to luteolysis in llamas (Lama glama).

Endometrial expression of oestrogen (ERα), progesterone (PR) and oxytocin receptor (OR) and cyclooxygenase-2 (COX-2) was evaluated from the induction of ovulation to luteolysis in llamas. Ovarian activity was daily assessed by ultrasonography in five females. Ovulation was induced immediately after the detection of an ovulatory follicle by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left and right horns on day 0 and days 4, 8, 10 and 12 post-GnRH. Blood samples were collected daily for progesterone and estradiol-17ß determinations by RIA. An immunohistochemical technique was used to study receptors population and COX-2 expression which were then evaluated by two independent observers. The expression of ERα and PR was highest on day 0 in the luminal epithelium and stroma in association with high plasma estradiol-17ß concentrations. Thereafter, a decrease in ERα population was registered on day 4 and a new increase of its expression was observed between days 8 and 12 in those cell types. Conversely, PR population was gradually down-regulated until its lowest expression was reached on day 10 post-GnRH in the luminal epithelium. Content of OR was similar throughout the study in all cell types. The expression of COX-2 was highest from day 8 to 12 post-GnRH in the luminal epithelium, in relation to the time of maximal PGF2α release. Both steroid receptors populations and COX-2 expression were similar between horns. Meanwhile, OR expression was higher in the right than in the left uterine horn. In summary, this study showed that the loss of endometrium sensitivity to progesterone by days 8-10 post-induction of ovulation and the concomitant increase of COX-2 expression could play a key role in the mechanism of luteolysis and somehow be related to the short corpus luteum lifespan of llamas.

Milk casein and fatty acid fractions in early lactation are affected by nutritional regulation of body condition score at the beginning of the transition period in primiparous and multiparous cows under grazing conditions.

The objective was to evaluate the effect of body condition score (BCS) at 30 days before calving (-30 days) induced by a differential nutritional management, parity and week of lactation (WOL) on milk yield and composition, and milk casein and fatty acid composition. Primiparous and multiparous Holstein cows with high BCS (PH, n = 13; MH, n = 9) and low BCS (PL, n = 9; ML = 8) under grazing conditions were sampled at WOL 2 and 8 (before and after peak of lactation). Milk yield was greater in multiparous than in primiparous cows and tended to decrease from WOL 2 to 8 only in ML cows. Milk protein, fat and casein yields were greater in multiparous than in primiparous cows and decreased from WOL 2 to 8. Milk casein concentration in milk protein was greater in MH cows than in ML, PH and PL cows at WOL 2. Milk κ-casein was greater, and ß-casein was less in multiparous than in primiparous cows. As lactation progressed, proportion of casein fractions were not altered. Only κ-casein fraction was affected by BCS at -30 days as PL showed a higher concentration than PH. The de novo (4:0-15:1) and mixed-origin fatty acids (16:0-16:1) in milk fat increased, whereas preformed fatty acids (≥17:0) decreased from WOL 2 to 8. Saturated (SAT) fatty acids tended to be greater and monounsaturated fatty acids (MUFA) were less in multiparous than in primiparous cows. High-BCS cows had greater concentrations of polyunsaturated (PUFA), conjugated linoleic acid (CLA) as well as n-6 and n-3 fatty acids in milk fat than low-BCS cows. The results indicate that casein and fatty acid fractions in milk were affected by parity and may be modified by a differential nutritional management during the pre-calving period (BCS at -30 days) in cows under grazing conditions.

Metabolic memory determines oviductal gene expression of underfed ewes during early gestation.

Our aim was to investigate the oviduct environment by studying oviduct gene expression after undernutrition in day-5 pregnant ewes with different initial (i) BCS, and its association with the number of embryos recovered. Thirty-six ewes were divided into 2 groups with different iBCS: iBCS ≥2.75 (n = 19; high, H) and iBCS ≤2.25 (n = 17; low, L), and were randomly assigned to two nutritional treatments for 20 days: 1.5 (control, C) or 0.5 (underfed, U) times the daily maintenance requirements. Thus, the final four groups were: high-iBCS control (HC, n = 9), high-iBCS underfed (HU, n = 10), low-iBCS control (LC, n = 9) and low-iBCS underfed (LU, n = 8). Samples of oviduct were collected and the expression of target genes was quantified using real-time PCR. While high-iBCS control ewes presented more ADIPOR1 mRNA than the high-iBCS underfed group (P < 0.05) and low-iBCS control ewes (P = 0.01), high-iBCS underfed group presented higher ADIPOR2 gene expression than low-iBCS underfed ewes (P < 0.01) evidencing a differential oviductal gene expression for these receptors. In high-iBCS ewes, control animals presented higher IGFBP2 gene expression than underfed ewes (P < 0.05), associated these results with a poor oviductal environment. High-iBCS underfed ewes presented higher IGFBP4 gene expression than high-iBCS control ewes (P < 0.05). Stepwise regression models, using various combinations of data on metabolic and reproductive hormones, and oviduct gene expression as independent variables, identified a set of variables that accounted for 75% of the variation in the number of embryos recovered. In conclusion, the oviductal gene expression depends on body reserves and nutritional treatment, and the effect is gene-specific.

Measurement of thyroid stimulating immunoglobulins using a novel thyroid stimulating hormone receptor-guanine nucleotide-binding protein, (GNAS) fusion bioassay.

Hyperthyroidism, defined by overproduction of thyroid hormones, has a 2-3% prevalence in the population. The most common form of hyperthyroidism is Graves' disease. A diagnostic biomarker for Graves' disease is the presence of immunoglobulins which bind to, and stimulate, the thyroid stimulating hormone receptor (TSHR), a G-protein coupled receptor (GPCR). We hypothesized that the ectopically expressed TSHR gene in a thyroid stimulating immunoglobulin (TSI) assay could be engineered to increase the accumulation of the GPCR pathway second messenger, cyclic AMP (cAMP), the molecule measured in the assay as a marker for pathway activation. An ectopically expressing TSHR-mutant guanine nucleotide-binding protein, (GNAS) Chinese hamster ovary (CHO) cell clone was constructed using standard molecular biology techniques. After incubation of the new clone with sera containing various levels of TSI, GPCR pathway activation was then quantified by measuring cAMP accumulation in the clone. The clone, together with a NaCl-free cell assay buffer containing 5% polyethylene glycol (PEG)6000, was tested against 56 Graves' patients, 27 toxic thyroid nodule patients and 119 normal patients. Using receiver operating characteristic analysis, when comparing normal with Graves' sera, the assay yielded a sensitivity of 93%, a specificity of 99% and an efficiency of 98%. Total complex precision (within-run, across runs and across days), presented as a percentage coefficient of variation, was found to be 7·8, 8·7 and 7·6% for low, medium and high TSI responding serum, respectively. We conclude that the performance of the new TSI assay provides sensitive detection of TSI, allowing for accurate, early detection of Graves' disease.

Legionella pneumophila serogroup 3 infection: importance of serology.

We present a case of Legionella pneumophila serogroup 3 (LP3) infection in a patient with severe community-acquired pneumonia (CAP). The diagnosis was complicated by an initial equivocal L. pneumophila urinary antigen test, followed by two negative samples. LP3 was cultured from a sputum sample and the diagnosis was confirmed by serology 15 days into the admission. This case highlights the importance of considering non-LP1 serogroups as causes of CAP and the role of serological testing in diagnosis.

Estradiol cypionate administered at the end of a progesterone-based protocol for FTAI induces ovulation and improves postovulatory luteal function and uterine environment in anestrous beef cows.

The objective of this study was to evaluate the effect of the administration of estradiol cypionate (ECP) at the end of an estradiol and progesterone-based protocol for fixed time artificial insemination (FTAI) on ovarian response and uterine function in postpartum anestrous beef cows. Multiparous suckled cows were randomly assigned to receive ECP at doses of 0 (control, n = 15), 0.5 (n = 15) or 1.0 mg (n = 15) im at the time of progesterone intravaginal insert removal. Serum 17ß-estradiol concentrations at 24 h after insert removal were greater (P < 0.05) in both ECP treatments than in controls. No differences in estradiol were found between 0.5 mg and control cows (P > 0.1) from 48 h after insert removal until ovulation, although greater (P < 0.05) concentrations were maintained until ovulation in 1.0 mg ECP treated cows. Maximum 17ß-estradiol concentration attained in each female was greater as ECP dose was greater (10.4 ± 0.4, 11.8 ± 0.5 and 13.5 ± 0.7, for control, 0.5 and 1.0 mg ECP treated cows, respectively; P < 005). Proportion of cows that ovulated tended to be greater (P = 0.06) in ECP treated than in control cows. Ovulation occurred earlier and the size of the ovulatory follicle was smaller (P < 0.05) for 1.0 mg but not for 0.5 mg (P > 0.1) when compared with control cows. After ovulation (Day 13 and 14), serum progesterone concentrations were greater (P < 0.05) in 0.5 and 1.0 mg ECP than control cows. Uterine environment on Day 6 after ovulation was affected by treatment; transcript expression of three of nine evaluated genes (i.e., estrogen, IGF-1 and insulin receptors genes) were upregulated (P < 0.05) after ECP treatment. In conclusion, ECP administration at progesterone insert removal in anestrous cows i) induces greater serum estradiol concentrations and tended to induce greater ovulation rate, ii) acts in a dose-dependent manner, as ECP dose increases ovulation occurs earlier and the size of the ovulatory follicle is smaller, iii) improves postovulatory luteal function and affects uterine gene expression. Altogether, this information contributes with the understanding of the effect of preovulatory estradiol exposure on ovulation and postovulatory ovarian and uterine function in anestrous beef cows.

Liquid chromatography-tandem mass spectrometry assay for androstenedione, dehydroepiandrosterone, and testosterone with pediatric and adult reference intervals.

BACKGROUND: Measurement of serum androgens is important in adult, geriatric, pediatric endocrinology, and oncology patients. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of androstenedione, dehydroepiandrosterone (DHEA), and testosterone in these patients. METHODS: We spiked 200 muL of serum or plasma with isotope-labeled internal standards and performed extraction with methyl t-butyl ether. We then derivatized the extracts with hydroxylamine and analyzed them by LC-MS/MS using a 2-dimensional chromatographic separation with a 3.5-min analysis time. RESULTS: Total imprecision for each analyte was <11.2%. Limits of quantification were 10, 50, and 10 ng/L for androstenedione, DHEA, and testosterone, respectively. Reference intervals were established for children (age 6 months to 17 years), men, and women. Androstenedione and DHEA concentrations were lowest in 2- to 3-year-old children. Adult concentrations were achieved in girls at Tanner stage 3 and in boys at Tanner stage 4-5. In premenopausal and (postmenopausal) women the median concentrations of androstenedione, DHEA, and testosterone were 810 (360), 3000 (1670), 270 (180) ng/L, respectively. In postmenopausal women, concentrations of testosterone were age independent, whereas androstenedione and DHEA concentrations decreased with age. In men the median concentrations of androstenedione, DHEA, and testosterone were 440, 2000, and 3700 ng/L, respectively. In men older than 40 years, median concentrations decreased at rates of 5%, 10%, and 20% per decade for androstenedione, DHEA, and testosterone, respectively. CONCLUSIONS: This LC-MS/MS method has the required lower limit of quantification and specificity for analysis of endogenous concentrations of androgens in all groups studied. Reference intervals were established for healthy children and adults.