Sperm DNA integrity in frozen-thawed semen from Italian Mediterranean Buffalo bulls and its relationship to in vivo fertility

• Comparison of three sperm DNA assays in Italian Mediterranean Buffalo (IMB) bulls.
• Relationship among sperm quality features and in vivo fertility by ROC curve.
• Cut-off values of significant sperm features to predict the likelihood of pregnancy.

The relationship among sperm attributes of DNA integrity, sperm motility, morphology, viability, acrosome integrity and in vivo fertility of frozen-thawed Italian Mediterranean Buffalo (IMB) sperm has not been reported. Straws of frozen-thawed semen samples from three bulls were examined. Sperm DNA assays (i.e., neutral Comet assay, Sperm Bos Halomax-SBH and Sperm Chromatin Structure Assay-SCSA) were not correlated to each other (P > 0.05). Many neutral Comet assay measures were correlated to total sperm motility-TMOT (% head-H-DNA, r = 0.74; Olive moment, r = −0.76; P < 0.05) and coiled tails (r-values ranged from% H-DNA, r = −0.80 to tail length, r = −0.71; P < 0.05). The COMP-αt was negatively correlated to viable acrosome intact (VAI) sperm, and distal droplets (r = −0.60 and −0.61; P < 0.05), whereas Mean-αt and Mode-αt were positively correlated to bent midpieces (r = 0.63 and 0.61; P < 0.05). The SBH assay was positively correlated to non-viable acrosome damaged (NVAD) sperm (r = 0.60; P < 0.05) and negatively correlated to viable acrosome damaged (VAD) sperm (r = −0.63; P < 0.05). The overall pregnancy rate (PR-at 30 and 45 d post artificial insemination-AI) and the calving rate were 57%, 55% and 45%, respectively. Among sperm features analyzed the area under the Receiver Operating Characteristic (ROC) Curve was significant (P < 0.05) for TMOT, NVAD, Standard Deviation-αt (SD-αt) and neutral comet measures (Olive tail moment and tail moment, % H- DNA and tail area) in estimating pregnancy.