Reimagining the Framework Supporting the Static Analysis of Transporter Drug Interaction Risk; Integrated Use of Biomarkers to Generate Pan-Transporter Inhibition Signatures.

Solute carrier (SLC) transporters present as the loci of important drug-drug interactions (DDIs). Therefore, sponsors generate in vitro half-maximal inhibitory concentration (IC50 ) data and apply regulatory agency-guided "static" methods to assess DDI risk and the need for a formal clinical DDI study. Because such methods are conservative and high false-positive rates are likely (e.g., DDI study triggered when liver SLC R value ≥ 1.04 and renal SLC maximal unbound plasma (Cmax,u )/IC50 ratio ≥ 0.02), investigators have attempted to deploy plasma- and urine-based SLC biomarkers in phase I studies to de-risk DDI and obviate the need for drug probe-based studies. In this regard, it was possible to generate in-house in vitro SLC IC50 data for various clinically (biomarker)-qualified perpetrator drugs, under standard assay conditions, and then estimate "% inhibition" for each SLC and relate it empirically to published clinical biomarker data (area under the plasma concentration vs. time curve (AUC) ratio (AUCR, AUCinhibitor /AUCreference ) and % decrease in renal clearance (ΔCLrenal )). After such a "calibration" exercise, it was determined that only compounds with high R values (> 1.5) and Cmax,u /IC50 ratios (> 0.5) are likely to significantly modulate liver (AUCR > 1.25) and renal (ΔCLrenal > 25%) biomarkers and evoke DDI risk. The % inhibition approach supports integration of liver and renal SLC data and allows one to generate pan-SLC inhibition signatures for different test perpetrators (e.g., SLC % inhibition ranking). In turn, such signatures can guide the selection of the most appropriate individual (or combinations of) biomarkers for testing in phase I studies.

First-principles calculations and Raman scattering evidence for local symmetry lowering in rhombohedral ilmenite: temperature- and pressure-dependent studies.

ATiO3-type materials may exist in two different crystalline forms: the perovskite and ilmenite. While many papers have devoted their attention to evaluating the structural properties of the perovskite phase, the structural stability of the ilmenite one still remains unsolved. Here, we present our results based on the lattice dynamics and first-principles calculations (density functional theory) of the CdTiO3 ilmenite phase, which are confronted with experimental data obtained through micro Raman spectroscopy that is a very good tool to probe the local crystal structure. Additional Raman bands, which are not foreseen from group-theory for the ilmenite rhombohedral structure, appeared in both low temperature (under vacuum condition) and high-pressure (at room temperature) spectra. The behavior can be explained by considering the local loss of inversion symmetry operation which reduces the overall space group from [Formula: see text] ([Formula: see text]) to [Formula: see text] ([Formula: see text]). Our results can also be extended to other ilmenite-type compositions.

Impacts of estrus expression and intensity during a timed-AI protocol on variables associated with fertility and pregnancy success in Bos indicus-influenced beef cows.

This experiment evaluated the impacts of estrus expression and intensity, estimated by physical activity during a timed-AI protocol, on reproductive performance of Bos indicus-influenced beef cows. A total of 290 lactating, primiparous, and multiparous nonpregnant Nelore × Angus cows received a 2 mg injection of estradiol benzoate and an intravaginal progesterone (P4) releasing device (CIDR) on d -11, a 12.5 mg injection of PGF2α on d -4, CIDR removal in addition to 0.6 mg injection of estradiol cypionate and 300 IU injection of eCG on d -2, and timed-AI on d 0. Cows were fitted with a pedometer behind their left shoulder on d -4. An estrus detection patch was attached to the tail-head of each cow on d -2. Pedometer results were recorded on d -2 and 0. Estrus expression was defined as removal of >50% of the rub-off coating from the patch on d 0. Net physical activity during estrus was calculated by subtracting total steps from d -4 to -2 (nonestrus basal activity) from total steps from d -2 to 0 (proestrus + estrus period) of each cow. Cows that did not express estrus were classified as NOESTR. Cows that expressed estrus were ranked by net physical activity; those above the median were classified as HIESTR and the remaining cows as LWESTR. Ovarian ultrasonography was performed on d 0 and 7. Blood was collected on d 0, 7, 20, and 30. Pregnancy status was verified by ultrasonography on d 30. Only data from cows responsive to the estrus synchronization protocol were utilized (NOESTR, n = 59; LWESTR, n = 100; HIESTR, n = 98). Diameter of dominant follicle on d 0, corpus luteum volume on d 7, and plasma P4 concentrations on d 7 were greater (P ≤ 0.05) in HIESTR vs. LWESTR and NOESTR and also greater (P ≤ 0.05) for LWESTR vs. NOESTR. Plasma P4 concentrations on d 0 were greater (P < 0.01) in NOESTR vs. HIESTR and LWESTR and similar (P = 0.93) between HIESTR and LWESTR. Whole blood mRNA expression of myxovirus resistance 2 on d 20 was greater (P ≤ 0.05) in HIESTR vs. LWESTR and NOESTR, and similar (P = 0.72) between LWESTR and NOESTR. Pregnancy rates were less (P ≤ 0.04) in NOESTR vs. HIESTR and LWESTR (52.4%, 68.9%, and 73.5%, SEM = 7.2), and similar (P = 0.57) between HIESTR and LWESTR. Hence, expression of estrus during a timed-AI protocol improved ovarian dynamics and pregnancy success, whereas estrus intensity modulated key biological markers associated with fertility but not pregnancy rates in B. indicus-influenced cows beef cows.

Impact of a timed-release FSH treatment from 2 to 6 months of age in bulls II: Endocrinology, puberty attainment, and mature sperm production in Holstein bulls.

The use of genomic testing in the cattle industries has renewed an interest in hastening bull puberty. In prepubertal males, FSH facilitates Sertoli cell proliferation and testis maturation. The aim of this study was to determine the effect of prepubertal administration of a timed-release FSH (delivered in a hyaluronan solution) on hormone secretion, puberty attainment, and mature sperm production in Holstein bulls in an AI center. Bulls (n = 29) were randomly assigned to one of two treatment groups based on birth date and pedigree. Beginning at 62 days of age (Day 62), bulls were injected im every 3.5 days with either 30 mg FSH (Folltropin-V; NIH-FSH-P1 units) in a 2% hyaluronan solution (FSH-HA, n = 17) or saline (control, n = 12) until Day 170.5. Blood samples to assess FSH, activin A, and testosterone were collected prior to each treatment. Scrotal circumference (SC) and BW were measured monthly. Puberty assessment (ability to ejaculate 5 × 107 sperm, 10% motile) was initiated at Day 244. Average mature daily sperm production (3× wk collection, combined 2 ejaculates) was assessed from Day 571-627. In blood collected every 3.5 days, FSH concentrations within FSH-HA bulls were increased (P < 0.05) over initial Day 62 concentration from Day 93.5-170.5. Concentrations of FSH did not differ between treatments from Day 62-93.5, but were greater (P < 0.05) in FSH-HA than control bulls from Day 97-170.5. Concentrations of activin A assessed for Day 62, 86.5, 107.5, 139, and 170.5 were greater (P < 0.05) in FSH-HA than control bulls on Day 86.5 and 107.5. Treatments did not differ (P > 0.1) in testosterone, BW, or SC. FSH-HA bulls attained puberty at a younger age than control bulls (278 ± 7.7 vs. 303 ± 9.1 days of age, P < 0.05), but mature daily sperm production was not different when measured from Day 571-627 (average 5.84 ± 0.11 billion cells/day, P = 0.5). In summary, FSH administration every 3.5 days from Day 62-170.5 resulted in an increase in FSH concentration beginning at 97 days of age and a hastened age of puberty. We propose this exogenous FSH delivered in hyaluronan initiates a positive feedback loop that includes an increase in activin A production observed on Day 86.5 and 107.5. However, differences in mature sperm production were not realized in this experiment.

Impact of a timed-release FSH treatment from 2 to 6 months of age in bulls I: Endocrine and testicular development of beef bulls.

In prepubertal males, FSH facilitates Sertoli cell proliferation and testis maturation. The study aimed to determine the effect of an exogenous FSH treatment on hormone secretion and testis development in Angus bulls. Bulls (n = 22) weaned at 53 ± 3.8 days of age were randomized into two treatment groups based on age and pedigree. Beginning at Day 59, bulls were injected im every 3.5 days with either 30 mg FSH (Folltropin-V; NIH-FSH-P1 units) in a 2% hyaluronan solution (FSH-HA, n = 11) or saline (control, n = 11) until Day 167.5. Blood samples to assess FSH, activin A, and testosterone were collected prior to each treatment. To determine how FSH profiles surrounding treatment were affected, three intensive blood sampling periods, each encompassing two treatment administrations, began at Day 66, 108, and 157, and blood was collected at 0, 6, 12, 18, 24, 36, 60, and 84 h respective to time of treatment. Scrotal circumference (SC) and BW were measured monthly. Bulls were castrated at Day 170 to measure testis size, seminiferous tubule diameter, and the number of Sertoli and germ cells per tubule cross-section. During intensive FSH sampling, FSH-HA bulls experienced an increase (P < 0.05) in FSH over control bulls for at least 18 h post-injection in all instances. In blood collected every 3.5 days, FSH concentrations in FSH-HA bulls were increased (P < 0.05) over initial Day 59 concentration from Day 97.5-167.5. FSH concentrations did not differ between treatments from Day 59-90.5, but were greater (P < 0.05) in FSH-HA from Day 94-167.5. Concentrations of activin A assessed for Day 59, 83.5, 94, 129, and 167.5 were greater (P < 0.05) in FSH-HA than control bulls on Day 83.5 and 94. The treatments did not differ (P > 0.1) in testosterone, BW, SC, testis size, tubule diameter, or number of germ cells per tubule. However, the number of Sertoli cells per tubule was greater in FSH-HA than control bulls (45.2 ± 1.4 vs. 41.6 ± 0.9 cells, P < 0.05). In summary, FSH-HA treatment every 3.5 days from Day 59-167.5 maintained elevated FSH for a minimum of 18 h post-injection, likely attributable to the addition of HA. We propose the exogenous FSH-HA treatment initiates a positive feedback loop that includes an increased density of Sertoli cells per tubule cross-section, which is related to increased activin A concentrations on Day 83.5 and 94. Furthermore, this activin A increase preceded an increase in endogenous FSH from Day 94-167.5 in FSH-HA bulls.

Endogenous Probes for Drug Transporters: Balancing Vision With Reality.

Various endogenous probes have been identified for a number of hepatic and renal drug transporters and available clinical data indicate that they could be leveraged in phase I trials to facilitate subject phenotyping and drug-drug interaction (DDI) assessment. Despite the progress, however, it is recognized that the menu of probes needs expanding, that existing probes need further characterization and validation, and that compound files need to be built in support of probe absorption-metabolism-distribution-excretion-DDI modeling exercises.

Transporter-Mediated Hepatic Uptake Plays an Important Role in the Pharmacokinetics and Drug-Drug Interactions of Montelukast.

Montelukast, a leukotriene receptor antagonist commonly prescribed for treatment of asthma, is primarily metabolized by cytochrome P450 (CYP)2C8, and has been suggested as a probe substrate for investigating CYP2C8 activity in vivo. We evaluated the quantitative role of hepatic uptake transport in its pharmacokinetics and drug-drug interactions (DDIs). Montelukast was characterized with significant active uptake in human hepatocytes, and showed affinity towards organic anion transporting polypeptides (OATPs) in transfected cell systems. Single-dose rifampicin, an OATP inhibitor, decreased montelukast clearance in rats and monkeys. Clinical DDIs of montelukast were evaluated using physiologically based pharmacokinetic modeling; and simulation of the interactions with gemfibrozil-CYP2C8 and OATP1B1/1B3 inhibitor, clarithromycin-CYP3A and OATP1B1/1B3 inhibitor, and itraconazole-CYP3A inhibitor, implicated OATPs-CYP2C8-CYP2C8 interplay as the primary determinant of montelukast pharmacokinetics. In conclusion, hepatic uptake plays a key role in the pharmacokinetics of montelukast, which should be taken into account when interpreting clinical interactions.

Post-artificial insemination supplementation with calcium salts of soybean oil influences pregnancy establishment factors in beef cows.

The objective of this experiment was to compare hormonal, uterine, and conceptus factors associated with pregnancy establishment in beef cows supplemented or not with Ca salts of soybean oil (CSSO) for 21 d beginning after timed AI. One hundred lactating multiparous Nelore cows were allocated to 20 groups of 5 cows/group and timed inseminated on d 0 of the experiment. After AI, groups were randomly assigned to receive (as-fed basis) 100 g of protein-mineral mix + 100 g of ground corn per cow per day, in addition to 1) 100 g/cow daily of CSSO ( = 10) or 2) 100 g/cow daily of kaolin (CON; rumen-inert indigestible substance; = 10). Groups were maintained in 4 pastures (5 groups from the same treatment within each pasture) with ad libitum access to forage. Groups were segregated daily and individually offered treatments from d 0 to 21. Blood samples were collected and transrectal ultrasonography was performed to verify ovulation and corpus luteum (CL) volume immediately before AI (d 0) and on d 7 and 15. After ultrasonography on d 15, 60 cows (30 cows/treatment and 3 cows/group) diagnosed without the presence of a CL on d 0 but with a CL greater than 0.38 cm3 in volume on d 7 and 15 were assigned to conceptus collection via transcervical flushing with PBS followed by endometrial biopsy in the uterine horn ipsilateral to the CL. Additional blood samples were collected for whole-blood RNA extraction (d 20), and pregnancy status was verified by transrectal ultrasonography (d 30) in cows not assigned to conceptus collection. Cows receiving CSSO had greater ( ≤ 0.04) mean plasma linoleic acid concentration, plasma linoleic:linolenic acid ratio, plasma progesterone (P4) concentration, and CL volume during the experiment compared with CON cows. Moreover, CSSO supplementation increased ( ≤ 0.04) length and mRNA expression of and by the conceptus as well as blood mRNA expression of interferon-stimulated genes on d 20 in gestating cows. No treatment differences were detected ( ≥ 0.30) for endometrial mRNA expression of and . In summary, post-AI CSSO supplementation to B. indicus beef cows increased plasma concentration of linoleic acid and enhanced pregnancy establishment factors, which included CL development and plasma P4 concentration, conceptus growth, and mRNA expression of as well as blood mRNA expression of interferon-stimulated genes.

Optical and transport properties correlation driven by amorphous/crystalline disorder in InP nanowires.

Indium phosphide nanowires with a single crystalline zinc-blend core and polycrystalline/amorphous shell were grown from a reliable route without the use of hazardous precursors. The nanowires are composed by a crystalline core covered by a polycrystalline shell, presenting typical lengths larger than 10 µm and diameters of 80-90 nm. Raman spectra taken from as-grown nanowires exhibited asymmetric line shapes with broadening towards higher wave numbers which can be attributed to phonon localization effects. It was found that optical phonons in the nanowires are localized in regions with average size of 3 nm, which seems to have the same order of magnitude of grain sizes in the polycrystalline shell. Regardless of the fact that the nanowires exhibit a crystalline core, any considerable degree of disorder can lead to a localized behaviour of carriers. In consequence, the variable range hopping was observed as the main transport instead of the usual thermal excitation mechanisms. Furthermore the hopping length was ten times smaller than nanowire cross-sections, confirming that the nanostructures do behave as a 3D system. Accordingly, the V-shape observed in PL spectra clearly demonstrates a very strong influence of the potential fluctuations on the exciton optical recombination. Such fluctuations can still be observed at low temperature regime, confirming that the amorphous/polycrystalline shell of the nanowires affects the exciton recombination in every laser power regime tested.

Effects of different supplemental soya bean oil levels on the performance of prepubertal Saanen goats: Oestrogen and progesterone release.

The aim of this study was to investigate the effects of different levels of soya bean oil in the total diet on the growth rate, metabolic changes, and oestrogen and progesterone release in Saanen goats. After dietary adaptation, 21 prepubertal goats (weight of 29.12 ± 0.91 kg, 230 days old) were randomly distributed among three diets of D2: inclusion of 2% soya bean oil in the total diet; D3: basal diet - inclusion of 3% soya bean oil in the total diet; and D4: inclusion of 4% soya bean oil in the total diet. The basal diet (D3) was formulated to promote a daily gain of 0.140 kg. The goats were weighed, and their blood samples were collected weekly. Glucose, cholesterol, triglycerides, total protein, urea, non-esterified fatty acids, beta-hydroxybutyrate, oestrogen and progesterone in the plasma were measured. Prepubertal goats that were fed D4 exhibited a significantly lower dry matter intake, urea and cholesterol levels compared with the goats that were fed D2 and D3. Indeed, goats that were fed D4 displayed a significantly lower final weight than goats that were fed D2 and D3. In contrast, the inclusion of soya bean oil in the diet increased the progesterone and oestrogen concentrations, and goats that were fed D4 released a significantly higher concentration of progesterone than those that were fed D2 and D3. Furthermore, the percentage of goats with a progesterone level greater than 1 ng/ml (functional Corpus luteum) was significantly higher among the goats that were fed D3 and D4 than among those that were fed D2. In this study, although the inclusion of 4% soya bean oil in the diet decreased dry matter intake and growth rate, it increased progesterone concentration and the percentage of goats with a functional Corpus luteum, suggesting that the inclusion of soya bean oil accelerated puberty in prepubertal goats.

Organic anion transporting polypeptide-mediated transport of, and inhibition by, asunaprevir, an inhibitor of hepatitis C virus NS3 protease.

Asunaprevir (ASV), an investigational, highly protein-bound inhibitor of hepatitis C virus NS3 protease, shows considerable hepatic compartmentalization in animal models. Preclinical data showed ASV inhibition of human OATP1B1 (IC50 = 0.3 µM), OATP2B1 (0.27 µM), and, to a lesser extent OATP1B3 (3.0 µM), confirmed by modest (<2-fold) clinical elevations in rosuvastatin exposure with concomitant ASV. Although no significant OATP transport of ASV was observed in vitro at standard micromolar assay concentrations, clinical coadministration of ASV with a single dose of the OATP inhibitor rifampin gave large, variable increases in ASV plasma Cmax (21-fold mean) and AUCinf (15-fold mean), consistent with reduced hepatic uptake. In vitro reevaluation at therapeutically relevant low-nanomolar concentrations of unbound ASV showed active, saturable human hepatocyte uptake (Km = 0.685 µM) and rifampin-reversible transport by OATP1B1 and OATP2B1, but not OATP1B3. At therapeutically relevant concentrations, ASV is therefore a sensitive substrate for, and weak inhibitor of, human OATP1B1, 1B3 and 2B1.

Effect of interval from induction of puberty to initiation of a timed AI protocol on pregnancy rate in Nellore heifers.

Prepubertal Bos indicus heifers (n = 774) were submitted to an E2/P4-based timed artificial insemination (TAI) protocol at three different intervals after induction of their pubertal ovulation by insertion of an intravaginal progesterone (P4) device for 12 days. Heifers were randomly assigned to start the TAI protocol at 10 (group 10; n = 253), 12 (group 12; n = 265), or 14 (group 14; n = 256) days after the P4 device was removed. The TAI protocol consisted of the following: insertion of intravaginal device containing P4 (Controlled internal drug release [CIDR]; previously used twice for 9 days each) + estradiol benzoate (2 mg) on Day 0, CIDR withdrawal + estradiol cypionate (0.5 mg) and PGF2α (12.5 mg) on Day 9, and TAI on Day 11. A subgroup of heifers (n = 472) was evaluated by ultrasound on Days 9 and 11 to evaluate the ovaries and to determine P4 concentrations on Day 9. On Day 9, more (P < 0.05) CLs were present, and follicular diameter was smaller (P < 0.05) for group 10 than for groups 12 and 14 (38.4%, 29.3%, and 23.3% with CL and 9.4 ± 0.1, 9.9 ± 0.1, and 9.8 ± 0.1 mm diameter, respectively), but P4 concentrations did not differ (P > 0.1) between treatments (2.4 ± 0.06 ng/mL). Follicular diameter at TAI (11.08 ± 0.09 mm) and ovulation rate (88.4%) did not differ between treatments (P > 0.1). However, conception and pregnancy rates for all heifers were greater (P < 0.05) in group 12 (50.4% and 45.5%, respectively) than in group 10 (38.2% and 33.7%, respectively), with group 14 intermediate to other treatments (45.6% and 40.6%, respectively). The final pregnancy rate did not differ between treatments (80.9%). In conclusion, a 12-day interval from the end of the puberty induction protocol to the start of the TAI protocol resulted in greater conception and pregnancy rates in prepubertal Nellore heifers.

Increasing length of an estradiol and progesterone timed artificial insemination protocol decreases pregnancy losses in lactating dairy cows.

Our hypothesis was that increasing the length of an estradiol and progesterone (P4) timed artificial insemination (TAI) protocol would improve pregnancy per artificial insemination (P/AI). Lactating Holstein cows (n=759) yielding 31 ± 0.30 kg of milk/d with a detectable corpus luteum (CL) at d -11 were randomly assigned to receive TAI (d 0) following 1 of 2 treatments: (8d) d -10 = controlled internal drug release (CIDR) and 2.0mg of estradiol benzoate, d -3 = PGF2α(25mg of dinoprost tromethamine), d -2 = CIDR removal and 1.0mg of estradiol cypionate, d 0 = TAI; or (9 d) d -11 = CIDR and estradiol benzoate, d -4 = PGF2α, d -2 CIDR removal and estradiol cypionate, d 0 TAI. Cows were considered to have their estrous cycle synchronized in response to the protocol by the absence of a CL at artificial insemination (d 0) and presence of a CL on d 7. Pregnancy diagnoses were performed on d 32 and 60. The ovulatory follicle diameter at TAI (d 0) did not differ between treatments (14.7 ± 0.39 vs. 15.0 ± 0.40 mm for 8 and 9 d, respectively). The 9 d cows tended to have greater P4 concentrations on d 7 in synchronized cows (3.14 ± 0.18 ng/mL) than the 8d cows (3.05 ± 0.18 ng/mL). Although the P/AI at d 32 [45 (175/385) vs. 43.9% (166/374) for 8d and 9 d, respectively] and 60 [38.1 (150/385) vs. 40.4% (154/374) for 8d and 9 d, respectively] was not different, the 9 d cows had lower pregnancy losses [7.6% (12/166)] than 8d cows [14.7% (25/175)]. The cows in the 9 d program were more likely to be detected in estrus [72.0% (269/374)] compared with 8d cows [62% (240/385)]. Expression of estrus improved synchronization [97.4 (489/501) vs. 81% (202/248)], P4 concentrations at d 7 (3.22 ± 0.16 vs. 2.77 ± 0.17 ng/mL), P/AI at d 32 [51.2 (252/489) vs. 39.4% (81/202)], P/AI at d 60 [46.3 (230/489) vs. 31.1% (66/202)], and decreased pregnancy loss [9.3 (22/252) vs. 19.8% (15/81)] compared with cows that did not show estrus, respectively. Cows not detected in estrus with small (<11 mm) or large follicles (>17 mm) had greater pregnancy loss; however, in cows detected in estrus, no effect of follicle diameter on pregnancy loss was observed. In conclusion, increasing the length of the protocol for TAI increased the percentage of cows detected in estrus and decreased pregnancy loss.

Effect of progesterone concentrations, follicle diameter, timing of artificial insemination, and ovulatory stimulus on pregnancy rate to synchronized artificial insemination in postpubertal Nellore heifers.

Two experiments were designed to evaluate the effects of treatments with low versus high serum progesterone (P4) concentrations on factors associated with pregnancy success in postpubertal Nellore heifers submitted to either conventional or fixed timed artificial insemination (FTAI). Heifers were synchronized with a new controlled internal drug release device (CIDR; 1.9 g of P4 [CIDR1]) or a CIDR previously used for 18 days (CIDR3) plus 2 mg of estradiol (E2) benzoate on Day 0 and 12.5 mg of prostaglandin F2α on Day 7. In experiment 1 (n = 723), CIDR were removed on Day 7 or 9 and heifers were inseminated after estrus detection. In experiment 2 (n = 1083), CIDR were all removed on Day 9 and FTAI was performed either 48 hours later in heifers that received E2 cypionate (ECP) on Day 9 (0.5 mg; E48) or 54 or 72 hours later in conjunction with administration of GnRH (100 µg; G54 or G72). Synchronization with CIDR1 resulted in greater serum P4 concentrations and smaller follicle diameters on Days 7 and 9 in both experiments. In experiment 1, treatment with CIDR for 9 days decreased the interval from CIDR removal to estrus (Day 7, 3.76 ± 0.08 days vs. Day 9, 2.90 ± 0.07; P < 0.01) and improved conception (Day 7, 57.1% vs. Day 9, 65.8%; P = 0.05) and pregnancy rates (Day 7, 37.6% vs. Day 9, 45.3%; P = 0.04). In experiment 2, treatment with ECP improved (P < 0.01) the proportion of heifers in estrus (E48, 40.9%(a); G54, 17.1%(c); and G72, 32.0%(b)), but the pregnancy rate was not affected (P = 0.64) by treatments (E48, 38.8%; G54, 35.5%; G72, 37.5%). Synchronization with CIDR3 increased follicle diameter at FTAI (CIDR1, 11.07 ± 0.10 vs. CIDR3, 11.61 ± 0.10 mm; P < 0.01), ovulation rate (CIDR1, 82.8% vs. CIDR3, 88.0%; P < 0.01) and did not affect conception (CIDR1, 42.2 vs. CIDR3, 45.1%; P = 0.38) or pregnancy rates (CIDR1, 34.7 vs. CIDR3, 39.4%; P = 0.11). In conclusion, length of treatment with P4 affected the fertility of heifers bred based on estrus detection. When the heifers were submitted to FTAI protocol, follicle diameter at FTAI (≤10.7 mm, 23.6%; 10.8-15.7 mm, 51.5%; ≥15.8 mm, 30.0%; P < 0.01) was the main factor that affected conception and pregnancy rates.

Timed artificial insemination programs during the summer in lactating dairy cows: comparison of the 5-d Cosynch protocol with an estrogen/progesterone-based protocol.

The objective of this study was to compare a GnRH-based to an estrogen/progesterone (E2/P4)-based protocol for estrous cycle synchronization and fixed timed artificial insemination (TAI), both designed for synchronization of ovulation and to reduce the period from follicular emergence until ovulation in cows with a synchronized follicular wave. A total of 1,190 lactating Holstein cows (primiparous: n=685 and multiparous: n=505) yielding 26.5 ± 0.30 kg of milk/d at 177 ± 5.02 d in milk were randomly assigned to receive one of the following programs: 5-d Cosynch protocol [d -8: controlled internal drug release (CIDR) + GnRH; d -3: CIDR removal + PGF2α; d -2: PGF2α; d 0: TAI + GnRH] or E2/P4 protocol (d -10: CIDR + estradiol benzoate; d -3: PGF2α; d -2: CIDR removal + estradiol cypionate; d 0: TAI). Rectal temperature and circulating progesterone (P4) were measured on d -3, -2, 0 (TAI), and 7. The estrous cycle was considered to be synchronized when P4 was ≥ 1.0 ng/mL on d 7 in cows that had luteolysis (P4 ≤ 0.4 ng/mL on d 0). To evaluate the effects of heat stress, cows were classified by number of heat stress events: 0, 1, and 2-or-more measurements of elevated body temperature (≥ 39.1 °C). Pregnancy success (pregnancy per artificial insemination, P/AI) was determined at d 32 and 60 after TAI. The cows in the 5-d Cosynch protocol had increased circulating P4 at the time of PGF2α injection (2.66 ± 0.13 vs. 1.66 ± 0.13 ng/mL). The cows in the E2/P4 protocol were more likely to be detected in estrus (62.8 vs. 43.4%) compared with the cows in the 5-d Cosynch protocol, and expression of estrus improved P/AI in both treatments. The cows in the 5-d Cosynch protocol had greater percentage of synchronized estrous cycle (78.2%), compared with cows in the E2/P4 protocol (70.7%). On d 60, the E2/P4 protocol tended to improve P/AI (20.7 vs. 16.7%) and reduced pregnancy loss from 32 to 60 d (11.0 vs. 19.6%), compared with the 5-d Cosynch protocol. In cows withtheir estrous cycle synchronized, the E2/P4 protocol had greater P/AI (25.6 vs. 17.7%) on d 60 and lower pregnancy loss from 32 to 60 d (6.7 vs. 21.7%) compared with cows in the 5-d Cosynch protocol. Follicle diameter affected pregnancy loss from 32 to 60d only in the cows in the 5-d Cosynch protocol, with smaller follicles resulting in greater pregnancy loss. Pregnancy per AI at d 60 was different between protocols in the cows with 2 or more measurements of heat stress (5-d Cosynch=12.2% vs. E2/P4=22.8%), but not in the cows without or with 1 heat stress measurement. In conclusion, the 5-d Cosynch protocol apparently produced better estrous cycle synchronization than the E2/P4 protocol but did not improve P/AI. The potential explanation for these results is that increased E2 concentrations during the periovulatory period can improve pregnancy success and pregnancy maintenance, and this effect appears to be greatest in heat-stressed cows when circulating E2 may be reduced.

Timing of prostaglandin F2α treatment in an estrogen-based protocol for timed artificial insemination or timed embryo transfer in lactating dairy cows.

Objectives were to investigate progesterone concentrations and fertility comparing 2 different intervals from PGF(2α) treatment and induced ovulation in an estrogen-based ovulation synchronization protocol for timed artificial insemination (TAI) or timed embryo transfer (TET) in lactating dairy cows. A total of 1,058 lactating Holstein cows [primiparous (n=371) and multiparous (n=687)], yielding 34.1 ± 0.33 kg of milk/d at various days in milk were randomly assigned to receive treatment with PGF(2α) on either d 7 or 8 of the following protocol: d 0: 2mg of estradiol benzoate + controlled internal drug release device; d 8: controlled internal drug release device removal + 1.0mg of estradiol cypionate; d 10: TAI or d 17: TET. Only cows with a corpus luteum at d 17 received an embryo and all cows received GnRH at TET. Pregnancy diagnoses were performed by detection (transrectal ultrasonography) of an embryo on d 28 or a fetus on d 60. Fertility [pregnancy per artificial insemination (P/AI) or pregnancy per embryo transfer (P/ET)] was affected by breeding technique (AI vs. ET) and time of PGF(2α) treatment (d 7 vs. 8) at the 28-d pregnancy diagnosis for TAI [32.9% (238) vs. 20.6% (168)] and TET cows [47% (243) vs. 40.7% (244)] and at the 60-d pregnancy diagnosis for TAI [30% (238) vs. 19.2% (168)] and TET cows [37.9% (243) vs. 33.5% (244)]. The progesterone (P4) concentration at d 10 altered fertility in TAI cows, with higher P/AI in cows with P4 concentration <0.1 ng/mL compared with cows with P4 concentration ≥ 0.1 ng/mL, and in ET cows, with higher P/ET in cows with P4 concentration <0.22 ng/mL compared with cows with P4 concentration ≥ 0.22 ng/mL. Prostaglandin F(2α) treatment at d 7 increased the percentage of cows with P4 <0.1 ng/mL on d 10 [39.4 (85) vs. 23.2 (54)]. Reducing the period between PGF(2α) and TAI from 72 to 48 h in dairy cows resulted in a clear reduction in fertility in cows bred by TAI and a subtle negative effect in cows that received TET. The earlier PGF(2α) treatment benefits are most likely mediated through gamete transport, fertilization, or early embryo development and a more subtle effect of earlier PGF(2α) treatment that may be mediated through changes in the uterine or hormonal environment that manifests itself after ET on d 7.

Progesterone-based strategies to induce ovulation in prepubertal Nellore heifers.

Four experiments were conducted to evaluate hormonal strategies to induce ovulation in Nellore heifers. In experiment 1, heifers (N = 1039) received a controlled internal drug release (CIDR) of fourth use (CIDR-4) on Day -12 or no CIDR (CIDR-0). The CIDR was removed on Day 0 in the CIDR-4 treatment, and estrus detection and AI were performed from Days 1 to 7. On Day 8, heifers not detected in estrus were evaluated for CL presence and received the same treatment again, followed by estrus detection and AI from Days 21 to 27. All heifers in experiments 2 (N = 896), 3 (N = 839), and 4 (N = 948) received the CIDR-4 treatment on Day -12. In experiment 2, heifers were randomly assigned to a control group (no additional treatment) or to receive equine chorionic gonadotropin (eCG; 200 IU eCG im) on Day 0. In experiment 3, heifers received the same treatments as in experiment 2, or a treatment that included eCG and estradiol cypionate (ECP) (eCG+ECP; 200 IU im eCG plus 0.5 mg ECP im) on Day 0. In experiment 4, heifers received the treatments described in experiment 3 or only ECP (0.5 mg) on Day 0. In experiments 2 and 3, estrus detection and AI was performed from Days 1 to 7 and on Day 8, heifers not detected in estrus were evaluated for CL presence. In experiment 4, heifers were evaluated for presence of a CL between Days 10 and 14. In experiment 1 heifers treated with CIDR-4 had greater estrus detection, ovulation induction, and pregnancy rates than in the CIDR-0 group. In experiment 2, heifers treated with eCG had greater estrus detection, ovulation induction, and pregnancy rates in 7 days than heifers in the control group. In experiment 3, heifers treated with eCG+ECP had greater estrus detection, ovulation induction, and pregnancy rates than the control and eCG treatments. In experiment 4, ovulation induction was greater for heifers treated with eCG and eCG+ECP relative to control, but did not differ from the ECP treatment. In conclusion, the use of a CIDR of fourth use for 12 days and the addition of eCG and/or ECP at CIDR removal efficiently induced ovulation and increased pregnancy rates in prepubertal Nellore heifers.

Factors associated with metabolic syndrome in climacteric women of southern Brazil.

AIM: To investigate the association between socioeconomic, demographic, behavioral, and reproductive factors and the metabolic syndrome (MS) in climacteric women. METHOD: This cross-sectional study was carried out in a sample of 527 women aged 40-65 years seen at an outpatient menopause and gynecologic surgery clinic in Southern Brazil. MS was defined according to NCEP-ATP III diagnostic criteria. Poisson regression was used to calculate crude and adjusted prevalence ratios and their respective 95% confidence intervals (CI). RESULTS: The prevalence of MS was 54.8% (95% CI 50.6-59.1%), varying with menopausal status (45.7% before menopause, 56.3% in perimenopause, and 57.5% in postmenopausal women). Among the components of MS, hypertension and abdominal obesity were the most prevalent (84.8% and 66.8%, respectively). The prevalence of MS rose with advancing age and increasing parity. Women with low education (years of schooling) showed a higher prevalence of MS compared to those with a high education level (64% vs. 36.8%). Women with early menarche (≤11 years of age) showed an increase of 32% in MS prevalence (95% CI 1.08-1.62) compared to those with a late menarche (≥14 years of age). CONCLUSION: These findings are relevant to public health, particularly as they show the significance of exposure to long-term, hard-to-reverse effects, such as early menarche and low educational achievement, in the development of metabolic syndrome.

Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-ß-glucuronide of gemfibrozil.

In previous studies, gemfibrozil acyl-ß-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-ß-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-ß-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-ß-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-ß-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.