Can linoleic acid improve the quality of frozen thawed bull sperm?

BACKGROUND: Cryopreservation is known to have a detrimental effect on the motility, viability and membrane integrity of sperm cells. OBJECTIVE: The aim of this study was to investigate the effect of various amount of linoleic acid supplementation to the Tris extender, on bull sperm parameters, DNA integrity and oxidative stress after freeze-thawing. METHODS: Ejaculates were split into five aliquots and extended to a final concentration of 18x10(6) spermatozoa per ml with the base extender containing different doses of linoleic acid 0.125 ml, (L125); 0.250 ml (L250); 0.5 ml (L500), 1 ml (L1000) and no additive (control; L0). The extended samples were equilibrated slowly to 4 degree C for 4 h and then froze using a digital freezing machine. Frozen straws were thawed individually in water bath at 37 degree C for 30 s to analyse progressive motility and sperm motion characteristics as well as membrane integrity. Biochemical assays were performed in a spectrophotometer using commercial kits. DNA damage was evaluated by Comet Assay. RESULT: The addition of various linoleic acid did not improve the sperm subjective, CASA and progressive motilities, sperm motility characteristics and DNA integrity (P>0.05). L500 exhibited the greatest values for membrane integrity than that of the other groups (P<0.001). All supplementation groups led to lower percentages of tail abnormalities in comparison to the control (P<0.001). L500 and L1000 significantly decreased total abnormalities. In conclusion, our findings showed that L500 linoleic acid supplementation in semen extender was of great beneficial effect on frozen-thawed bull semen in terms of morphology and plasma membrane integrity.

Effects of various cryoprotectants on bull sperm quality, DNA integrity and oxidative stress parameters.

The objectives of this study was to compare the effects of type and concentration of cryoprotectants glycerol (G), ethylene glycol (EG) and dimethyl sulfoxide (DMSO) on the plasma membrane and DNA integrity as well as antioxidant activity of cryopreserved Eastern Anatolian red bull sperm. Ejaculates were collected from the three bulls using an artificial vagina twice a week. The ejaculates were pooled to increase the semen volume for replication and to eliminate variability among the evaluated samples. The pooled ejaculates were also split into seven equal experimental groups and diluted with the modified base extender to a final spermatozoa concentration of 15×10(6)/ml. The extended samples were cooled slowly to 4°C and equilibrated for 4h. They were then loaded into 0.25ml French straws and frozen using a digital freezing machine at 3 programmed rates: -3°C/min from +4°C to -10°C, -40°C/min from -10°C to -100°C, and -20°C/min from -100°C to -140°C. Thereafter, the straws were plunged into liquid nitrogen at -196°C. Frozen straws were thawed individually at 37°C for 30s in a water bath to analyse progressive motility and sperm motion characteristics as well as membrane integrity using hypo-osmotic swelling test. Biochemical assays were performed in a spectrophotometer using commercial kits. DNA damage was evaluated by Comet Assay using Image Analysis System. 6% G exhibited the greatest percentages of CASA (43.7±2.92%) and progressive (26.4±2.64%) motilities when compared to the other groups (P<0.001). 6% G and 6% EG showed the greatest values of preserved membrane integrity (P<0.001). 6% DMSO and 3% EG + 3% DMSO resulted in greater chromatin damage than the other groups (P<0.001). The antioxidant activities of GPx, GSH, and CAT as well as the total antioxidant activity were affected by the type of cryoprotectant; notably, 2% G+2% EG+2% DMSO yielded the lowest activities when compared to the other groups (P<0.001). In conclusion, no advantages were found in using EG or DMSO to replace G in bull sperm cryopreservation. Freezing with cryoprotectant 6% G yielded the best post-thaw sperm characteristics for Eastern Anatolian Red bull spermatozoa.

[Comparison of two different methods for the investigation of in vitro susceptibilities of planktonic and biofilm forming Candida species to antifungal agents]. / Candida türlerinin biyofilm olusturan ve planktonik formlarinin antifungal ajanlara karsi in vitro duyarliliklarinin arastirilmasinda iki farkli yöntemin karsilastirilmasi

Microdilution method that determines the minimum inhibitory concentrations (MIC) of antifungal agents against Candida spp. is still the only method used in laboratories for both biofilm and planktonic forms. However, it was determined in several studies that there were susceptibility differences between the biofilm and planktonic forms of the same microorganism. The aims of this study were the determination of in vitro susceptibilities of planktonic and biofilm forms of Candida strains against antifungal agents, the comparison of the data obtained from planktonic and biofilm forms and the evaluation of two different methods used for the detection of susceptibilities of biofilm forms. Candida albicans ATCC 10231, Candida parapsilosis ATCC 90028 and Candida krusei ATCC 6258 were used as reference strains together with clinical isolates of one of each C.albicans, C.parapsilosis and Candida tropicalis. Microdilution method was used to determine the susceptibilities of planktonic forms of the strains according to CLSI M27-A3 standards, and MIC values of fluconazole, itraconazole, flucytosine, amphotericin B and nystatin were determined. For the detection of antifungal susceptibilities of Candida spp. biofilm forms, Calgary biofilm method (CBM) and BioTimer assay (BTA) were used, and minimum biofilm eradication concentration (MBEC) and minimum biofilm inhibition concentration (MBIC) values of the same antifungals were determined. The difference between MIC and CBM-MBEC, CBM-MBEC and BTA-MBEC, CBM-MBEC and BTAMBIC values were found statistically significant (p < 0.05). In general CBM-MBEC values were found to be higher than MIC values. However, MBEC values were not always very reliable since the exact number of the microorganisms in biofilm can not be determined. BTA-MBIC values were also generally lower than the MBEC values and higher than the MIC values. Statistically significant difference between two methods was determined only for the MBEC values of flucytosine (p= 0.002) and itraconazole (p = 0.025). For flucytosine (p = 0.001) and itraconazole (p = 0.001), there was also a significant difference between CBM-MBEC and BTA-MBIC values, however, the difference was not significant (p > 0.05) for the other antifungal agents. These findings supported that antifungal susceptibilities of biofilm forming Candida strains should also be investigated. However, MBEC and MBIC of the antifungal agents should not always be expected to be higher than the MIC values since the mechanism of action of the specific antifungal agents and the first inoculum concentration of the microorganisms might differ.