Semen cryopreservation is one of the reproductive biotechnologies that have revolutionized the dairy sector through dissemination of the germplasm of elite bulls. Adoption of artificial insemination technique using frozen-thawed semen on the world wide scale has resulted in marked genetic improvement in the dairy animals [1,2]. However, the process of semen cryopreservation affects the functional resilience of spermatozoa that might result in loss of about 50% viable spermatozoa at post-thaw stage [[3], [4], [5]]. The reduced cryosurvival and vigor of frozen-thawed spermatozoa may be attributed to functional and ultra-structural insults to the spermatozoa in the form of mitochondrial and plasma membrane damage [6,7] and oxidative damage by uncontrolled reactive oxygen species (ROS) production [[8], [9], [10], [11], [12]]. The uncontrolled ROS production during various stages of semen preservation may result in lipid peroxidation of plasma membrane [8], reduction in mitochondrial membrane potential [13], development of capacitation [14] and apoptotic like changes and DNA damage [15]. All these features have been described as induction of early apoptosis features in the spermatozoa following cryopreservation [15]. The ultimate consequence is poor viability and motility of the spermatozoa at post-thaw stage and compromised fertility with frozen-thawed semen [[16], [17], [18]]. The buffalo spermatozoa are inherently more susceptible to cryoinjuries than cattle spermatozoa due to differences in the biochemical composition and lipid ratio of plasma membrane [19,20]. The amelioration of cryoinjuries has been performed in the past using various techniques and antioxidants [1] with varying degree of success. A molecule which possesses multiple modes of action and provides wholesome cyto and cryoprotection to the spermatozoa is the need of hour to improve the freezability and fertility of buffalo semen.

Humanin (HN) is a mitochondrial derived peptide (MDP) which possesses very strong antioxidant and cytoprotective properties. It is supposed to be the first discovered peptide of MDPs. The credit of discovery goes to Hashimoto et al. [21] who discovered it from the patient's brain suffering from Alzheimer disease. The other cells and tissue where HN is expressed include liver, kidney and colon [22], skeletal muscles [23], cardiomyocytes [24], retinal pigment epithelium cells [25], tumors [26], and ovaries [27,28]. The HN levels also have been detected in body fluids like seminal plasma [29] and follicular fluid [27]. However, it is still not clear that which kind of cells or tissues contribute to the circulating HN pool [30]. In the reproductive system, Humanin has been reported to be expressed in the testicular tissue of mice [22] and testicular tissue and Leydig cells of rats [31]. Moretti et al. [32] first discovered the presence of HN in human spermatozoa and testicular tissue by immunocytochemistry and immunoelectron microscopy. Recently, Rao et al. [29] documented the estimation of HN levels in human seminal plasma for the very first time. Their observations revealed that HN levels were significantly higher in patients with normal sperm quality in comparison to oligopsermic and asthenospermic patients.

Humanin is supposed to encode in the mitochondrial genome by MT-RNR2, which is a 16S ribosomal gene. It prevents apoptosis either by interfering with Bax which is a pro-apoptotic factor [33] or by interacting with insulin like growth factor-binding protein 3 (IGFBP-3) [34]. It also acts as an antioxidant by neutralizing the hydrogen peroxide (H2O2) and by mitochondrial up regulation of Glutathione (GSH). The HN is known to provide cytoprotection to various kinds of cells under stress conditions. Humanin analogue (HNG) was also used to validate its cryoprotective role in the human semen [35].

The perusal of literature says that no report is available documenting the presence of HN like MDP in the spermatozoa and seminal plasma of domestic animals in general and buffalo in particular. Only one pilot study was conducted in our laboratory to standardize the dose of Humanin for supplementation in extender, in relation to physico-morphological and oxidative stress status of buffalo spermatozoa [11]. Also, no report could be traced out validating the role of HN in improving the kinematics, fertility and overall freezability of frozen-thawed buffalo spermatozoa. Establishing the presence of Humanin-like peptide may pave a way to develop a new fertility marker in the buffalo bulls. Therefore, the present study was designed with an objective to establish the presence of Humanin-like MDP in the spermatozoa, testicular tissue, seminal vesicles and seminal plasma of buffalo bulls and effect of HN supplementation on the freezability and fertility of buffalo spermatozoa.