The effect of thymol on acetylcholine-induced contractions of the rat ileum and uterus under ex vivo conditions.

Thyme (Thymus vulgaris L.) is a well-known medicinal plant, the aerial parts of which have long been used internally or externally as a traditional remedy for various diseases. Thyme essential oils have important pharmaceutical applications and are regularly used in the pharmaceutical, food and cosmetic industries. In folk medicine, thyme preparations are used to treat respiratory, digestive, cardiovascular and nervous disorders, as well as to relieve dysmenorrhea. Thymol, a major constituent of Thymus vulgaris essential oil, has been shown to affect skeletal and smooth muscle excitation and contraction. Therefore, the main objective of this study was to evaluate its effect on acetylcholine (ACh)-induced rat intestinal and uterine preparations. Isolated ileum and uterine horn preparations were placed in a 20-ml organ bath containing Tyrode or De Jalon solution and exposed to graded concentrations of ACh (0.036, 0.109, 0.36, 1.09, 3,6, 10.9, and 36 µM) and either 0.02 or 0.2 mM thymol. Additionally, the dose-response relationship of thymol impact on intestinal and uterine contraction was evaluated. Contraction changes were monitored using an isometric transducer. Thymol at the higher dose (0.2 mM) significantly reduced ACh-induced intestinal and uterine contractions. Thus, this study provides new important data on competitive actions between thymol and ACh. In the dose-response study, the IC50 values were calculated as 5.26 mM for the ileum and 5.35 mM for the uterus. Our results demonstrated the efficacy of thymol in reducing ileal and uterine smooth muscle contractions, thus supporting the use of thyme in traditional medicine in the treatment of digestive disorders and painful menstrual cramps.

Insect Protein-Based Diet as Potential Risk of Allergy in Dogs.

Before insects can be used widely as an alternative source of dietary protein, their allerginicity should be investigated. Therefore, the aim of our study was to assess the potential adverse reactions of the immune system of dogs against Tenebrio molitor proteins. Dogs sensitised to storage mites T. putrescentiae and A. siro were included. Clinically healthy and clinically allergic dogs were compared. Proteins were extracted from mealworm larvae and their digestibility determined by in vitro incubation with digestive proteases. Mealworm protein extracts and digests were analysed by SDS-PAGE. Canine sera tested for the presence of mite-specific IgEs were used for subsequent Western blotting. LC-MS/MS analysis was used to identify mealworm proteins and their allergenic potential was predicted with the AllermatchTM tool. The binding of canine sera IgEs to mealworm proteins was confirmed; however, the differences between the two groups of dogs were not significant. Moreover, no clear correlation was found between sensitisation to storage mites and clinical status of the dogs. Altogether, 17 different proteins were identified, including tropomyosin, α-amylase, and Tm-E1a cuticular protein that are known cross-reacting IgE-binding allergens. Our results suggest that dogs allergic to mites may clinically express also the cross-reactivity with mealworm proteins.

Proteomic Analysis of Fresh and Liquid-Stored Boar Spermatozoa.

In this study comparative proteomics was used to define changes in the expression of the spermatozoa proteins during liquid storage. Semen from eight boars was analyzed on the day of collection and after liquid preservation at 15-17 °C for three days. Sperm parameters (concentration, motility, morphology, vitality) and percentage of non-capacitated and acrosomal-reacted spermatozoa were determined. Sperm proteins were extracted and separated by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteomic profiles were computationally compared to highlight differentially expressed protein spots that were, in turn, identified by mass spectrometry. The intensities of four spots were significantly different between fresh and liquid stored sperm. Namely: ATP citrate lyase, chaperonin containing T-complex polypeptide 1 (TCP1) subunit ε and probable phospholipid-transporting ATP-ase were over-expressed in liquid stored sperm, whereas cytosolic non-specific dipeptidase was over-expressed in fresh sperm. These differentially expressed proteins could be used as plausible biomarkers for the evaluation of boar semen quality and spermatozoa survival after liquid storage and could help to address problems associated with sperm preservation.

Dialysis of the goat semen and its effect on the quality of frozen/thawed spermatozoa processed in the presence of egg yolk.

The aim of the present study was to analyze the effect of dialysis on the quality of frozen/thawed buck semen. Ejaculates (n = 15) from three Saanen bucks were divided into three experimental groups. Semen in Group Ce (centrifugation) was processed by standard method and washed two times at 1.085 × g for 20 min. During this time, the diluted control semen (Co) was stored at room temperature. Semen in Group D was dialyzed using 300 kDa cut-off semi-permeable cellulose tubing. The semen from all groups was diluted with extender containing 20% egg yolk and frozen in liquid nitrogen vapor. After thawing, semen samples were evaluated by microscopic and biochemical analyses. Phospholipase A2 was in amounts that was 72.0 ± 11.7% less after dialysis and 21.3 ± 10.0% less after washing with centrifugation compared to the control semen (P < 0.05). Spermatozoa from Group Co had a lesser motility and viability and greater percentage of morphological abnormal spermatozoa in comparison to Groups D and Ce at 3 h after thawing and incubation on 37 °C. At the same time motility and percentage of HOST positive spermatozoa were greater in Group Ce compared with D (P < 0.05). There, however, was no difference in morphology and viability (CFDA/PI analysis) of spermatozoa between Ce and D group. Results from the present study suggest the dialysis is the promising alternative method for reducing phospholipase A2 in the buck semen before cryopreservation.

Retrospective study of bull semen quality - possible correlation with pesticide use?

Decline in semen quality in humans and increased incidence of male reproductive problems could be caused by different factors, including pesticides that could mimic or block the action of endogenous hormones. If the decline in semen quality is real, and environmental chemicals are at least partially responsible for this decline, similar changes should be observed in animals that live in close connection with humans and are exposed to similar levels of pollutants. In the present study, the semen quality of bulls in the last 30 years was examined with respect to the year of birth. Furthermore, semen quality results were correlated to the total pesticide use in a limited geographical area. The results indicate a notable decrease in both ejaculate volume and total number of spermatozoa in ejaculates of bulls born in the late 1970s, while after that (until 2006) there was no obvious downward or upward trend either in ejaculate volume or in the total number of sperm cells. The amount of pesticides released into the environment increased about twofold in the given period, and linear regression analysis revealed a strong and statistically significant correlation between the amount of pesticides used and the total number of spermatozoa in the ejaculate.

Dermatophyte Trichophyton mentagrophytes Produces Cysteine Protease Inhibitor.

The protein inhibitor of cysteine proteases was isolated from an important zoophilic dermatophyte species Trichophyton mentagrophytes (T. mentagrophytes) and partially characterized. The isolation process involved affinity chromatography, followed by ion-exchange chromatography and reverse phase high performance liquid chromatography. The fungal inhibitor appears to exist in a high (24 kDa) and low (12 kDa) molecular mass form. It inhibits proteolytic activity of papain, cathepsins B and L but not of cathepsin H or trypsin. Results of immunoblotting procedures indicate that sera of T. mentagrophytes infected rabbits contain antibodies against higher molecular mass forms of the inhibitor. Since no sequence homology has been found between partial protein sequences of T. mentagrophytes inhibitor and other known cysteine protease inhibitors so far, we can speculate that this inhibitor has some structurally unique characteristics. The T. mentagrophytes inhibitor shares some biochemical similarities (molecular mass, high and low molecular mass forms, inhibitory profiles) with clitocypin from Clitocybe nebularis and macrocypins from Macrolepiota procera.