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Kolesarova A.,Slovak University of Agriculture | Roychoudhury S.,Slovak University of Agriculture | Slivkova J.,Slovak University of Agriculture | Sirotkin A.,Institute of Farm Animal Genetics and Reproduction | And 2 more authors.
Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering | Year: 2010

The heavy metals lead (Pb) and mercury (Hg) pose potential risks to sustainability of environment and thus to our future generations. General objective of this in vitro study was to examine the secretory activity of porcine ovarian granulosa cells after Pb and Hg administration and to outline the potential intracellular mediators of its effects. For this purpose, release of insulin-like growth factor I (IGF-I) and steroid hormone progesterone (P 4), expression of proliferation- related (cyclin B1) and apoptosis-related (caspase-3) peptides was examined in porcine ovarian granulosa cells after heavy metals administration. Obtained data indicate Pb-induced inhibition of IGF-I release at lower doses (0.063 mg/mL and 0.046 mg/mL) by ovarian granulosa cells. However, P4 release was not influenced by Pb addition, while the expression of cyclin B1 and caspase-3 was induced by Pb addition. These results indicate that Pb can affect the pathway of proliferation and apoptosis of porcine ovarian granulosa cells through intracellular substances such as cyclin B1 and caspase-3. On the other hand, the P4 release by ovarian granulosa cells of pregnant gilts was stimulated by experimental Pb administration at doses of 0.25 mg/mL and 0.063 mg/mL and experimental Hg administration at doses 0.25 mg/mL and 0.083 mg/mL. P4 release by ovarian cells of pregnant gilts was not influenced by a combinatory dose of FSH (1.0 ng/mL) + Pb (0.083 mg/mL) + Hg (0.083 mg/mL) but it was inhibited by experimental administration of FSH (10 ng/mL) + Pb (0.25 ng/mL) + Hg (0.25 ng/mL). Possible involvement of heavy metals - Pb and Hg and pituitary hormone FSH, in the regulation of P4 release by porcine ovarian granulosa cells of pregnant gilts was noted. Data obtained from in vitro studies suggest the dose dependent association of heavy metals administration with the hormonal release by porcine ovarian granulosa cells. This association also depended on pregnancy of the gilts. Source


Vasicek J.,Institute of Farm Animal Genetics and Reproduction | Vasicek J.,Slovak University of Agriculture | Pivko J.,Institute of Farm Animal Genetics and Reproduction | Chrenek P.,Institute of Farm Animal Genetics and Reproduction | Chrenek P.,Slovak University of Agriculture
Folia Biologica (Poland) | Year: 2014

The objective of this study was to assess the utility of the magnetic-activated cell sorting (MACS) technique used for improving characteristics and quality of insemination doses by the elimination of apoptotic rabbit spermatozoa from a heterospermic pool (Experiment 1) as well as from the ejaculates of individual bucks (Experiment 2). Superparamagnetic microbeads conjugated with annexin V eliminated spermatozoa with externalized phosphatidylserine via MACS. The control (untreated) and magnetically separated spermatozoa (in both E1 and E2) were used for artificial insemination of hormonally treated rabbit does. MACS separation of spermatozoa yielded two fractions: annexin V-negative (AnV-) and annexin V-positive (AnV+). The CASA analysis after MACS sperm sorting revealed that the proportion of apoptotic spermatozoa in the semen of New Zealand White bucks varied from 7 to 20%. Transmission electron microscopy revealed that MACS treatment might eliminate spermatozoa with membrane damages and released acrosomal matter. However, the MACS separation (in both E1 and E2) did not affect the reproductive parameters of rabbit does. © Institute of Systematics and Evolution of Animals, PAS, Kraków, 2014. Source


Vasicek J.,Institute of Farm Animal Genetics and Reproduction | Kuzelova L.,Slovak University of Agriculture | Kulikova B.,Constantine the Philosopher University | Chrenek P.,Institute of Farm Animal Genetics and Reproduction | Chrenek P.,Slovak University of Agriculture
Avian Biology Research | Year: 2015

The objective of this study was to assess the effect of different diluent type and storage time on the quality of rooster insemination doses in terms of the spermatozoa motility and apoptosis (in vitro) and spermatozoa fertility potential (in vivo). The motility parameters were significantly (P<0.001) higher in the spermatozoa diluted in saline solution than those diluted in avian diluent at each storage time (0.5, 1 and 2 hours). The proportion of apoptotic spermatozoa was significantly (P < 0.05) higher in the semen samples diluted in the avian diluent in comparison to the semen samples diluted in the saline solution after 1 hour of storage. The proportion of dead spermatozoa was significantly (P < 0.05) higher in avian diluent in comparison to the semen samples diluted in the saline solution after 2 hours of storage. The fertility of spermatozoa diluted in the avian diluent significantly (P < 0.01) decreased 1 hour after the ejaculate collection. In conclusion, saline solution maintained better quality of rooster spermatozoa in terms of their motility values and the proportion of apoptotic and dead cells (in vitro) than the commercial avian diluent when stored at 4 °C for 2 hours. According to in vivo results, the saline solution seems to be suitable for semen dilution at low temperature, since similar fertility results were obtained over the whole storage time. Thus the commercial avian diluent could be replaced by the saline solution under certain conditions. Furthermore, the saline solution is cheaper and more accessible for practical use than the commercial avian diluent. Source


Vasicek J.,Institute of Farm Animal Genetics and Reproduction | Vasicek J.,Slovak University of Agriculture | Makarevich A.V.,Institute of Farm Animal Genetics and Reproduction | Chrenek P.,Institute of Farm Animal Genetics and Reproduction | Chrenek P.,Slovak University of Agriculture
Central European Journal of Biology | Year: 2011

Magnetic-activated cell sorting (MACS) separates apoptotic spermatozoa by the use of annexin V-conjugated nanoparticles which bind to phosphatidylserine that is externalized on the outer leaflet of the sperm plasma membrane. This technique yields two fractions: annexin V-negative (AnV -) and annexin V-positive (AnV +). The aim of the study was to evaluate the effect of MACS application on the motility parameters of rabbit spermatozoa. Rabbit semen samples collected separately from 4 bucks (I, II, III, and IV) were filtered and separated in a MACS system. The semen samples from a control (untreated) group, AnV - and AnV + fraction were evaluated using CASA system. The experiment was replicated 4 times for each buck. The AnV + sperm had significantly lower concentration than the AnV - fractions and the control samples (P<0. 05 for bucks I, II, III, but not IV). We observed that the proportion of apoptotic spermatozoa in the semen of NZW bucks is about 20%. There was no significant difference in the percentage of motile and progressively motile spermatozoa between the AnVfractions and control samples. In conclusion, the MACS technique has no harmful effect on the rabbit sperm concentration and motility. © 2011 Versita Warsaw and Springer-Verlag Wien. Source


Kuzelova L.,Slovak University of Agriculture | Vasicek J.,Institute of Farm Animal Genetics and Reproduction | Chrenek P.,Slovak University of Agriculture | Chrenek P.,Institute of Farm Animal Genetics and Reproduction
Reproduction in Domestic Animals | Year: 2015

Contents: The goal of this study was to evaluate the occurrence of macrophages in rooster semen and to investigate their impact on the spermatozoa quality. Ross 308 breeder males (n = 30) with no evidence of genital tract infections were used to determine the concentration of macrophages using fluorescently conjugated acetylated low-density lipoprotein (AcLDL). Subsequently, the roosters were divided into two groups on the basis of semen macrophage concentration, and semen quality was compared in two heterospermic samples. We applied computer-assisted semen analysis (CASA) system to determine motility parameters. Fluorescence microscopy and flow cytometry were used to evaluate occurrence of apoptotic and dead spermatozoa. Spermatozoa fertility potential was examined after intravaginal artificial insemination of hens. Eighteen roosters (control group) contained 0.2-3% of macrophages within spermatozoa population and ten roosters (macrophage group) had 10-15% of macrophages. Males from macrophage group had lower (p < 0.05) motility parameters (total and progressive movement, velocity curved line) and increased concentration of dead spermatozoa detected by flow cytometry and fluorescence microscopy (p < 0.001 and p < 0.05, respectively). Differences (p < 0.05) between fluorescent microscopy and flow cytometry in results on spermatozoa apoptosis and viability were observed. No significant difference was found between groups in fertility of spermatozoa. In conclusion, the higher presence of macrophages in rooster semen may have a negative effect on some parameters of rooster spermatozoa evaluated in vitro. Furthermore, our study suggests that flow cytometry allows more precise examination of spermatozoa viability and apoptosis in a very short time compared with the fluorescent microscopy. © 2015 Blackwell Verlag GmbH. Source

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