Michał Dzięcioł

Expression of microRNAs and their target genes and pathways associated with ovarian follicle development in cattle

Development of ovarian follicles is controlled at the molecular level by several gene products whose precise expression leads to regression or ovulation of follicles. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through sequence-specific base pairing with target messenger RNAs (mRNAs) causing translation repression or mRNA degradation. The aim of this study was to identify miRNAs expressed in theca and/or granulosa layers and their putative target genes/pathways that are involved in bovine ovarian follicle development. By using miRCURY microarray (Exiqon) we identified 14 and 49 differentially expressed miRNAs (P < 0.01) between dominant and subordinate follicles in theca and granulosa cells, respectively. The expression levels of four selected miRNAs were confirmed by qRT-PCR. To identify target prediction and pathways of differentially expressed miRNAs we used Union of Genes option in DIANA miRPath v.2.0 software. The predicted targets for these miRNAs were enriched for pathways involving oocyte meiosis, Wnt, TGF-beta, ErbB, insulin, P13K-Akt, and MAPK signaling pathways. This study identified differentially expressed miRNAs in the theca and granulosa cells of dominant and subordinate follicles and implicates them in having important roles in regulating known molecular pathways that determine the fate of ovarian follicle development.

Effects of cycle stage and sampling procedure on interpretation of endometrial cytology in mares

The aim of this study was to ascertain if (1) the stage of reproductive cycle influences cytological results, (2) cytology obtained from an endometrial biopsy is more accurate than cytology derived using the cytobrush, and (3) different methods used for evaluation of cytological samples produce similar diagnostic results. Material was collected from 46 mares in estrus, 48 in diestrus and from 33 mares in anestrus. Smears were evaluated using two criteria. In criterion I, a total of 300cells were counted and the percentage of polymorphonuclear cells was recorded. In criterion II, the number of polymorphonuclear cells/high power field was determined. Endometrial biopsy samples were also examined histologically, and comparisons were made between cytological and histological results. Regardless of the method used for sample collection, endometritis was diagnosed significantly more frequently if smears were evaluated according to criterion I. The agreement between the number of polymorphonuclear cells infiltrated into the endometrial luminal epithelium and stratum compactum and the cytology results for smears evaluated according to criterion I was fair for anestrous mares, poor for diestrous mares, and moderate for mares in estrus. When cytological smears were evaluated according to criterion II the agreement between the histological and cytological and results was always poor. In summary, cytological evaluation of the mare endometrium should be based on counting the numbers of polymorphonuclear cells in relation to epithelial cells rather than counting the number of polymorphonuclear cells in relation to high power field.

Dynamic tissue perfusion measurement in the reproductive organs of the female and male dogs

Abstract The aim of this study was to investigate the usefulness of new software Pixel Flux (PXFX) for clinical evaluation of tissue perfusion in the field of reproduction in dogs. The experiment was performed on six adult Beagle dogs. Different organs and tissues of the animals were examined with the MyLab25 Gold ultrasound system. Blood flow in the ovary, testicle, prostate, the ramification of the penile artery, and the network of blood vessels of the pampiniform plexus were examined with the use of colour coded Doppler technique, and obtained data was evaluated with the PXFX software. The more objective digital evaluation of data obtained with colour Doppler sonography through the application of dynamic tissue perfusion measurements provides new opportunities for diagnosis, as well as continuous monitoring of the function of the examined tissues and organs. The use of PXFX software is strongly indicated as a tool in small animal practice as an additional method for evaluation of tissue perfusion, especially in the cases when other methods like pulsed wave Doppler techniques are difficult to be performed

The influence of Sildenafil citrate on uterine tissue perfusion and the cardiovascular system during the luteal phase of the ovarian cycle in cows

The aim of the study was to evaluate the influence of the Sildenafil citrate on the blood flow in the uterus of cows during dioestrus. Uterine blood flow was examined in five, healthy, adult cows. Between day 6-8 of the ovarian cycle, each cow received 200mg of sildenafil diluted in 10ml of warm saline into the body of the uterus. Analysis of the blood pressure, ECG and the maximum velocity in m/s (V max) in the aorta was performed and selected parameters of the blood flow (PI, pulsatile index; RI, resistance index; SPV, systolic peak velocity; EDV, end diastolic velocity; FVI, flow velocity integral; SV/DV, systolic peak velocity: end-diastolic velocity ratio) were measured in the uterine artery (Arteria uterine) before and after sildenafil infusion. In addition, Color Doppler examination of the uterine wall perfusion was analyzed. A significant decrease of values of PI and SV/DV ratio as well as an increase of end diastolic velocity and time averaged maximum velocity was noted. With the use of color coded sonography, the increased intensity of the blood flow in the uterine wall was observed. It was concluded that intrauterine administration of sildenafil during dioestrus can increase uterine tissue perfusion.

Identification of putative volatile sex pheromones in female domestic dogs (Canis familiaris)

More than hundred urine samples, vaginal secretions, vulval and anal imprints as well as anal sac secretions, collected during various phases of the ovarian cycle, were evaluated with the HS-SPME/GC-MS technique. The results indicate there were differences among samples of urine collected during particular phases of the ovarian cycle. Unequivocally, the amount of carbonyl aromatic compounds, such as acetophenone (hypnone) and benzaldehyde, increased during the period of proestrus and estrus of the estrous cycle. This was accompanied by increases in methylketones, e.g., 2-octanone, 2-pentanone and 3-hexanone. Simultaneously, amounts of sulfide compounds (1-methylotiopropane; 1-methylotiobutane, 1-methylotipentane and dimethyl trisulfide) decreased during the period of estrus and abruptly increased in diestrus. These observations suggest a possible dual mechanism of interaction between males and females during and subsequent to the mating period, including the existence of both attracting and repelling signals. No significant changes were detected in samples other than urine. Further studies including a proteomic approach as well as behavioral assessments are suggested to identify the complete range of estrual semiochemical signals and to verify the semiochemical activities of identified substances.

Semiochemical Communication in Dogs in the Context of Reproductive Behavior

The attempts of identification of sex pheromones playing crucial role in creation and modulation of the sexual behavior have been taken in many various domestic and wild species. In the present study, we evaluated the presence of methyl paraben, considered as a main sex pheromone in dogs, in secretions of bitches (GC/MS analysis) and, via behavioral tests, the ability of this substance to stimulate sexual arousal in stud dogs. We verified that some dogs can detect the most fertile period during estrus and we examined various objective methods for the evaluation of sex pheromone activity. Results obtained demonstrated that methyl paraben is not present in the urine of the bitch during estrus. Also, the effectiveness of methyl paraben (Eau D’Estrus, Synbiotics, USA) in causing sexual stimulation in male dogs has been questioned. The ability of male dogs to identify the most fertile period in bitches was confirmed, what suggest more complex characteristics of the semiochemical signal emitted by the bitch during estrus. We also concluded that bacteria present in the bitch’s vagina during estrus could significantly influence the production of semiochemicals. Moreover we found the evaluation of the blood flow (color Doppler) in ramifications of the penile artery in response to specific semiochemical signals, as a useful and specific method for detection of pheromonal stimulation in males. Taking into account the postulated complex character of sex pheromones in dogs, further studies including also detection of specific proteins and evaluation of reactivity of specific brain structures (fMRI) toward pheromones should be considered.

Intravaginal administration of sildenafil citrate increases blood flow in the bovine uterus

Abstract The aim of the study was to evaluate the influence of sildenafil citrate administrated intravaginaly on the blood flow in the bovine uterus during dioestrus. Uterine blood flow was examined in six healthy adult cows. Sildenafil was administrated intravaginaly to each co w between the 6th and 8th d of the ovarian cycle, in the form of vaginal suppositories containing 100 mg of active substance at a dose of 100, 200, or 300 mg per animal. Uterine perfusion was estimated by the colour Doppler examination, and obtained results were analysed with the Pixel Flux Software (Chameleon, Germany). Moreover, cardiovascular parameters were also evaluated. Animals were examined before and five times after drug application (two times at 15 min intervals, and three times at 2 h intervals). A placebo suppository was also given to the cows. The analysis of the intensity and velocity of blood flow in the uterus proved that sildenafil administrated intravaginaly significantly increased blood flow in the uterus and the effect of increased perfusion was observed for 4 h and 30 min after administration. The effect of increased uterine perfusion was observed after low as well as high doses of sildenafil. Significant changes in the cardio-vascular parameters were not detected. There were no changes in the uterine perfusion as well as in cardiovascular parameters after placebo administration.

The Influence of Experimentally Reduced Vaginal Flora in Oestrus Females on the Mating Behaviour of Male Domestic Dogs (Canis familiaris)

This study assessed the effect of reduced vaginal flora during oestrus on the mating behaviour of four male dogs. The experiment included a total of 32 bitches in oestrus that were allocated to three groups. Group A included untreated bitches (n = 15), group B, bitches treated by an intravaginal infusion of an antibiotic (n = 15) and group C, bitches treated by an intramuscular injection of antibiotic (n = 15). Bitches in oestrus (i.e. with progesterone concentrations of 10–15 ng/mL) were presented to the male dogs. Our results demonstrate that antibiotic-treated bitches with reduced vaginal bacterial flora (females in groups B and C) were less attractive to males than untreated females. There was no significant difference between the two methods of antibiotic administration. These results indicate that vaginal flora produces semiochemical substances (pheromones) during oestrus that induce sexual arousal in male dogs.