Assessment of different functional parameters of frozen-thawed buffalo spermatozoa by using cytofluorimetric determinations

Flow cytometry is a useful tool that provides an accurate, objective and rapid evaluation of semen quality. The use of this technique could significantly improve the quality of buffalo semen samples used in artificial insemination. This study was carried out to evaluate, by flow cytometry, frozen-thawed buffalo spermatozoa quality parameters such as: sperm viability by SYBR-14/propidium iodide staining; mitochondrial function by JC-1 potentiometric probe; sperm chromatin stability (SCSA) by acridine orange and acrosome reaction by FITC-PNA staining. Semen samples from 5 Italian Mediterranean buffalo bulls were used. Sperm viability was not different between bulls and ranged from 33.4 to 43.6%. A consistent rate (55.1±10.8%) of sperm cells showed high mitochondrial membrane potential (Δψhigh), with no significant differences between subjects. SCSA differed significantly between the 5 buffalo bulls, moreover data showed high stability within each buffalo. DNA fragmentation indexes (DFI), such as %-DFI, -DFI, SD-DFI, were 11.2±8.6, 153.3±24.6 and 81.6±21.2, respectively. Regarding acrosome reaction (AR), the percentage of acrosome-reacted live (ARL) and acrosome-reacted dead (ARD) spermatozoa was 0.3±0.2 and 15.3±5.5 respectively. This functional parameter differed significantly between buffalo bulls and showed high stability. Following to Ca2+ ionophore A23187 for 3 hrs, AR significantly differed between subjects and was characterized by an increase of both ARL (10.8%) and ARD population (22.0%). The present study indicates that flow cytometry could be a useful tool for a quick multiparametric evaluation of sperm quality in buffalo. In particular, SCSA and AR resulted sperm functional parameters sensitive enough for the diagnosis of frozen-thawed semen fertilizing potential.