Determination of antibiotic resistance profiles and biofilm production of Staphylococcus spp. isolated from Anatolian water buffalo milk with subclinical mastitis.

Mastitis is one of the most crucial diseases of dairy animals. Especially subclinical mastitis (SCM) has negative impacts on of dairy economy in term of reducing milk quality and quantity also premature culling and cost of therapy. Staphylococci are important etiological agents in SCM. The aim of the study was to investigate the biofilm production and antibiotic resistance profiles of Staphylococcus spp. other than S. aureus isolated from milks of Anatolian water buffalo with subclinical mastitis. Twenty-two coagulase negative staphylococci (CNS) identified phenotypically were also identified with PCR as Staphylococcus spp. other than S. aureus. Biofilm productions were investigated both by Congo Red Agar Method and PCR. The antibiotic resistance profiles of the isolates were determined by Disc Diffusion Method and they were antibiotyped. Only three (13.6%) isolates were biofilm positive both phenotypically and genotypically. All isolates except for two were resistant against at least two antibiotics. Multidrug-resistance among the isolates was low (13.6%). Antibiotyping results showed that the similarities among the strains were between 30-100%. Genotyping of the strains revealed that a genetic heterogeneity was found among CNS isolates and their similarities were between 43% and 93%. In conclusion, CNS isolates identified as subclinical mastitis agents in buffaloes showed a high antibiotic resistance profile especially against oxacillin and vancomycin. Further studies should be conducted to investigate new mechanisms and/or genes responsible for antibiotic resistance in buffaloes.

Suitability of the hemi-zona assay for the evaluation of the effect of the length of the equilibration period before cryopreservation.

The aim of this study was to test the suitability of the interspecific hemizona assay (HZA) to predict the fertilizing capacity of bovine sperm after modifying the length of the equilibration period before freezing and thawing. Ejaculates from 10 proven fertile bulls were split after dilution, equilibrated at 4 °C for either 24 h (control sperm = CS) or 6, 48, 72 or 96 h (test sperm = TS) and cryopreserved. Hemizona (HZ) pairs from in vitro matured pig oocytes were used for the heterologous HZA: After thawing and swim-up (1 h) CS and TS were co-incubated with matching HZ (125,000 S/HZ in 25 µL Fert-TALP) for 4 h. Spermatological analyses (progressive motile sperm (PMS), plasma membrane- and acrosome-intact sperm (PMAI), sperm showing a high degree of DNA fragmentation (%DFI)) were performed after 0 and 3 h of incubation after thawing. After an equilibration time of 48 h and 72 h values for PMAI0h were higher (P < 0.05) compared to PMAI0h values of sperm equilibrated for 6 h, and %DFI3h values were higher after 96 h (P < 0.05) compared to 6 h equilibration. Between 12 and 90 TS and 13-97 CS were tightly bound to each HZ, respectively. The mean Hemizona Index (HZI) after a sperm equilibration for 48 h (HZI = 92.3 ± 12.7) or 72 h (HZI = 98.9 ± 16.23) was higher (P < 0.01) than after an equilibration for 6 h (HZI = 73.3 ± 13.93) or 96 h (HZI = 81.3 ± 11.41). The HZI for 96 h equilibration was moderately negatively related to PMS0h and PMS3h (r < -0.35, P < 0.05). Furthermore the HZI for 6 h equilibration was highly negatively correlated with DFI0h (r = -0,46, P < 0.01). On the basis of these results it can be concluded that the hemi-zona assay is a suitable test to detect alterations in the fertilizing capacity of bovine sperm after modifying the equilibration period.

Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.

The objective was to examine if there are relationships between alterations in sperm viability, reactive oxygen species (ROS) synthesis, and DNA integrity induced by cryopreservation of bovine sperm. Four ejaculates were collected from each of six bulls. Each ejaculate was diluted and divided into two aliquots; one was incubated for 24 hours at 37 °C, and the other frozen, thawed, and incubated for 24 hours at 37 °C. Analyses of quality of sperm were performed after 0, 3, 6, 12, and 24 hours of incubation. Progressive motile sperm was determined with computer assisted sperm analysis. Percentages of plasma membrane- and acrosome-intact sperm, sperm with a high mitochondrial membrane potential, sperm showing a high degree of DNA fragmentation (%DFI), and their reactive oxygen species content were assessed with dichlorofluorescein-diacetate, dihydrorhodamine, diaminofluorescein diacetate, and mitochondrial superoxide indicator using flow cytometry. Although all other sperm parameters showed alterations (P < 0.05) during the 24-hour incubation time, %DFI stayed constant (P > 0.05, 0.91 ± 0.23) in nonfrozen sperm. Cryopreservation induced changes of all sperm parameters (P < 0.05). In contrast to all other sperm parameters, dichlorofluorescein-diacetate-fluoroescence indicating the synthesis of H2O2 showed a similar exponential rise (P < 0.05) like the %DFI values in frozen sperm. In conclusion, changes of DNA integrity in frozen sperm seem to be related to synthesis of H2O2 but not to sperm viability and synthesis of other reactive oxygen species.