Flow cytometry and Laser Tweezers Raman spectroscopy have been used to investigate Nili-Ravi buffalo (Bubalus bubalis) sperm from different samples (fresh, stained, sorted and frozen-thawed) of the flow-sorting process to optimize sperm sex sorting procedures. During the sorting and freezing-thawing processes, the two detection methods both indicated there were differences in mitochondrial activity and membrane integrity. Moreover, a dispersive-type NIR (Near Infrared Reflection) use of the Raman system resulted in the ability to detect a variety of sperm components, including relative DNA, lipid, carbohydrates and protein contents. The use of the Raman system allowed for PCA (principal components analysis) and DFA (discriminant function analysis) of fresh, stained, sorted and frozen thawed sperm. The methodology, therefore, allows for distinguishing sperm from different samples (fresh, stained, sorted and frozen-thawed), and demonstrated the great discriminative power of ANN (artificial neural network) classification models for the differentiating sperm from different phases of the flow-sorting process. In conclusion, the damage induced by sperm sorting and freezing-thawing procedures can be quantified, and in the present research it is demonstrated that Raman spectroscopy is a valuable technology for assessing sperm quality. (C) 2016 Elsevier B.V. All rights reserved.
