Aim: The aim of this study was to analyze the effect of bovine follicular fluid on the survival, morphology and kinetic parameters of bovine thawed spermatozoa under laboratory conditions. Materials and methods: The semen from 5 bulls of proven fertility was incubated in follicular and physiological fluid for 8 hours. During this time assessment using the CASA system was performed. At the beginning and the end of incubation process evaluation by flow cytometry was conducted. Results: The results of the sperm motility assessment showed a significant decrease in the analyzed parameters both in the follicular and physiological fluid. A significant reduction in all parameters characterizing movement properties in the semen incubated in the follicular fluid was found. In the physiological fluid, a similar trend was demonstrated only for the following proper- ties: VAP, VSL, VCL, ALH, BCF. A significant difference was found for both fluids in: VCL (p=0.026), ALH (p=0.038) and LIN (p<0.001) at the beginning of incubation. The results of the plasma membrane integrity assessment showed a statistically significant increase in the percent- age of dying sperm at the 8th hour of the incubation in the follicular fluid. In the case of semen incubation in physiological fluid, a statistically significant decrease in the percentage of live non-damaged cells was found with a simultaneous increase in the subpopulation of undamaged dead cells. Conclusions: Follicular fluid rapidly accelerates the capacitation process. The results of flow cytometry support the hypothesis concerning the ability of follicular fluid to prolong sperm sur- vival.
We have developed an effective protocol for in vitro micropropagation in order to obtain large numbers of identical plants and another protocol for in vitro polyploidization of Ajuga reptans, based on the use of oryzalin. Two donor plants of A. reptans (AR 4, AR 7) were treated with 0, 1, 5, 10 μM oryzalin for 2 weeks. The analysis of the ploidy level of these plants was verified by flow cytometric analysis using the internal standardization method. The effects of polyploidization on growth as well as morphological and stomatal size were also measured. After in vitro polyploidization, some plants became tetraploids or octoploids. The most efficient conditions for inducing tetraploidy were the treatments with 10 μM oryzalin.
We used germination tests to assess the frequency of polyembryony in 9 asparagus cultivars with a high propensity to produce double embryos with different ploidy levels: Alpha, Andreas, Boonlim, Cipres, Eposs, Helios, Limbras, Ravel and Sartaguda. Twin embryos inside a single seed were found in 3 cultivars: Eposs 2n, Ravel 2n and Sartaguda 2n, at 0.60% frequency (15 seeds with twin embryos out of 2500 seeds). Of 30 obtained seedlings, 14 were separated diploid-diploid twins, 6 were conjoined diploid pairs, 8 were separated diploid-haploid and 2 were diploid-haploid pairs conjoined in the hypocotyl region. Some embryos showed unilateral dominance of one embryo (size and shape). The haploid status of the smallest embryo was confirmed by chromosome number (n=x=10) and flow cytometry (nuclear C DNA amount 1.95 pg). The haploid obtained in this manner possessed enough vegetative vigor to undergo chromosome doubling.