Urinary free metanephrines measurement in dogs with adrenal gland diseases using a new simple liquid chromatography tandem mass spectrometry method

Measurement of urinary metanephrines in spot samples is used for the diagnosis of canine pheochromocytoma (PC). We describe a simple analytical method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for measuring free metanephrine (MN) and normetanephrine (NMN) in spot urine samples. Using the developed method, we evaluated the stability of urinary free-MN and free-NMN at various storing conditions. In addition, we assessed the feasibility of urinary free-MN and -NMN measurement for diagnosing PC. Urine samples were mixed with stable isotope internal standards and thereafter purified by ultrafiltration. The purified samples were analyzed by LC-MS/MS in the multiple reaction monitoring mode after separation on a multimode octa decyl silyl column. The coefficient of variation of free-MN and -NMN measurement was 7.6% and 5.5%, respectively. The linearity range was 0.5–10 µg/l for both analytes. Degradation was less than 10% for both analytes under any of the storage conditions. The median free-NMN ratio to creatinine of 9 PC dogs (595, range 144–47,961) was significantly higher (P<0.05) than that of 13 dogs with hypercortisolism (125, range 52–224) or 15 healthy dogs (85, range 50–117). The developed method is simple and may not require acidification of spot urine. The results of this preliminary retrospective study suggest that the measurement of urinary free metanephrines is a promising tool for diagnosing canine PC.     

Characteristics of cytochrome P450-dependent metabolism in the liver of the wild raccoon,Procyon lotor

Wildlife is exposed to a wide range of xenobiotics in the natural environment. In order to appropriately assess xenobiotic-induced toxicity in wildlife, it is necessary to understand metabolic capacities. Carnivores, in general, have low metabolic abilities, making them vulnerable to a variety of chemicals. Raccoons (Procyon lotor) in the wild have been found to have high levels of xenobiotics. However, little is known about the metabolic capacity of the cytochrome P450 (CYP) enzymes in this species. Thus, this study used liver samples to investigate the characteristics of CYP enzymes in wild raccoons. In 22 wild raccoons, CYP concentrations in hepatic microsomes were examined. To better understand the properties of CYP-dependent metabolism, in vitro metabolic activity studies were performed using ethoxyresorufin, pentoxyresorufin and testosterone as substrates. In addition, three raccoons were fed commercial dog food in the laboratory for one week, and the effects on CYP-dependent metabolism were investigated. In comparison to other mammalian species, raccoons had very low concentrations of CYP in their livers. In an in vitro enzymatic analysis, raccoons’ ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD) metabolic capacities were less than one-fifth and one-tenth of rats’, respectively. These results indicate the possible high risk in raccoons if exposed to high levels of environmental xenobiotics because of their poor CYP activity. In this study, the features of CYP-dependent metabolism in wild raccoons are described for the first time.     

Antral Follicle Populations and Embryo Production – In Vitro and In Vivo – of Bos indicus–taurus Donors from Weaning to Yearling Ages

Interest in indicus–taurus cattle has been increasing, as these animals are likely to present the best characteristics of Zebu and European bovine breeds. The aim of this study was to compare the embryo production of indicus–taurus donors with high vs low antral follicle counts obtained by ovum pickup/in vitro production (OPU/IVP) and superovulation (SOV)/embryo collection. Braford females at weaning age (3/8 Nelore × 5/8 Hereford, n = 137, 9 ± 1 month old) were subjected to six serial ovarian ultrasonographs and were assigned to two groups according to the number of antral follicles ≥3 mm as follows: G‐High antral follicular count (AFC, n = 20, mean ≥40 follicles) and G‐Low AFC (n = 20, mean ≤10 follicles). When the females (n = 40) reached 24 months of age, they were subjected to both OPU/IVP and SOV/embryo collection. The average number of follicles remained highly stable throughout all of the ultrasound evaluations (range 0.90–0.92). The mean number of COCs recovered (36.90 ± 13.68 vs 5.80 ± 3.40) was higher (p < 0.05) for females with high AFC, resulting in higher (p < 0.05) numbers of total embryos among females with high vs low AFC (6.10 ± 4.51 vs 0.55 ± 0.83). The mean number of embryos per collection was also higher (p < 0.05) for G‐High vs G‐Low (6.95 ± 5.34 vs 1.9 ± 2.13). We conclude that a single ultrasound performed at pre‐pubertal ages to count antral follicles can be used as a predictor of embryo production following IVP and SOV/embryo collection in indicus–taurus females.     

Effect of Insemination–Ovulation Interval and Addition of Seminal Plasma on Sow Fertility to Insemination of Cryopreserved Sperm

In swine, the use of frozen-thawed (FT) sperm for artificial insemination (AI) is limited because of poor sow fertility, possibly associated with a post-thaw capacitation-like status resulting in fewer fully viable sperm. Sow fertility to AI with FT sperm may improve with deeper deposition of sperm within the female tract, insemination very close to ovulation, or reversal of cryocapacitation by seminal plasma (SP). We performed two experiments to examine these suggestions. In experiment 1, 122 multiparous Yorkshire sows received 600 IU equine chorionic gonadotrophin at weaning and 5 mg pLH 80 h later to control time of ovulation. The predicted time of ovulation (PTO) was 38 h after pLH injection. Thereafter, sows were assigned on the basis of parity to a single AI of FT sperm at 2 h before PTO, or at 12 h before PTO, or FT sperm supplemented with 10% SP at 12 h before PTO. Control sows received fresh semen at 12 h before PTO. All semen doses were adjusted to 3 x 10(9) live cells and deposited into the cervix. Experiment 2 employed 99 multiparous crossbred sows and repeated the treatments of experiment 1 except that all FT inseminations were intrauterine. In both experiments, farrowing rates were lower (p < 0.01) following FT inseminations with no effect of time of insemination or of supplemental SP. In experiment 1, litter size was smaller following FT insemination (p < 0.05), but no effect on litter size was evident in experiment 2. Supplemental SP had no effect on litter size in either experiment. The lack of effect of either SP or timing of FT insemination on sow fertility suggests that the non-lethal sperm cryoinjury affecting fertility involves more than just cryocapacitation.     

Uterine Responses to Exogenous Oxytocin Before and After Pre-partum Luteolysis in the Cow

The aim of this study was to test the functional status of uterine oxytocin receptors in cows in vivo around parturition. The animals received consecutive, intra‐arterial injections of 800, 1600 and 3200 mU of oxytocin at three different stages: during late gestation (days 260–274), at 12 h and at 24 h after intramuscular injection of a prostaglandin F_2α analogue at day 275 to induce parturition. Cows (n=6) had been provided with myometrial electrodes and a catheter had been installed in the aorta and in a branch of the uterine vein (UV). Regular blood samples were obtained from the UV from 5 min before until 45 min after each oxytocin injection to measure plasma levels of prostaglandin F_2α (PGF_2α) and oxytocin. Uterine electromyographic (EMG) activity was registered continuously during each experiment. The increase of oxytocin levels in UV plasma after intra‐arterial injections was dose dependent (p < 0.02). Pre‐ and post treatment oxytocin levels at 24 h after induction of parturition were significantly increased (p=0.0313). Both during late pregnancy and at 12 h after induction of parturition, oxytocin caused a significant increase in EMG activity (p=0.022). After the 3200 mU dose the increase was significantly higher than with the other 2 doses (p=0.004). After each dose, EMG activity returned to baseline levels within some 15 min. At 24 h after induction of parturition, the pre‐treatment level of EMG activity had increased. Doses of 800 mU and 1600 mU of oxytocin produced a significant (p=0.022) increment of EMG activity, which was of the same magnitude as during the preceding stages; after 3200 mU of oxytocin the response was significantly higher than before (p=0.008). No significant increases of PGF_2α levels in UV plasma could be measured after oxytocin injections at any of the three stages. It is concluded that the myometrium of the pregnant cow responds in vivo to physiological doses of oxytocin. At 24 h after induction of parturition, when luteolysis has occurred and a parturient pattern of parturient myometrial activity has already started to develop, the response is enhanced. Physiological doses of oxytocin did not evoke a spurt release of PGF_2α in uterine venous blood during the peripartal period.     

Effect of stale seedbed preparations and subsequent weed control in lettuce (cv. Iceboll) on weed densities

The effects of stale seedbed preparations and several weed control methods on the emergence of weeds in lettuce were studied. The specific goal was to evaluate the use of a stale seedbed in combination with chemical or mechanical weed control methods in the field. Depending on location and year, stale seedbed preparations followed by weed control prior to planting reduced the amount of weeds during crop growth by 43–83%. Control of the emerged seedlings after a stale seedbed preparation was more effective with glyphosate than with a rotary harrow. Covering the rotary harrow during control to prevent light reaching the soil improved its effect on the weed density during crop growth in two of 3 years. Radiation with far red light (FR) did not reduce the number of emerging weeds in this study. Mechanical control by finger weeder, torsion weeder and hoe was applied without stale seedbed preparations. These measures reduced the weed densities by 88–99%, compared with the untreated control and were more effective than chemical weed control with carbetamide and chlorpropham. The results show that the stale seedbed technique in combination with mechanical control of emerging weeds can reduce the weed population during crop growth as effectively as chemical control. The technique may therefore help reduce the use of herbicides in lettuce crops in the future.     

Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p < .05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2‐deoxy‐d ‐glucose, DOG). Furthermore, treatment with DOG also led to a dose‐dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance.