Online First Article Fertility of Cryopreserved Buffalo Semen Can be Improved by Supplementation of Arachidic Acid in Extender Rabea Ejaz 1 *, Saima Qadeer 2 , Muhammad Sajjad Ansari 3 , Bushra Allah Rakha 4 , Shazia Shamas 5 , Iram Maqsood 1 , Asma Ul Husna 6 , Asima Azam 1 and Shamim Akhter 6 1 Department of Zoology, Shaheed Benazir Bhutto Women University, Peshawar - 25000, Pakistan, 2 University of Education, Jauharabad Campus, Jauharabad-41200, Pakistan 3 Division of Science and Technology, University of Education, Lahore-54000, Pakistan 4 Department of Wildlife Management, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi-46300, Pakistan, 5 Department of Zoology, University of Gujrat, Gujrat-50700, Pakistan 6 Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi-46300, Pakistan Article Information Received 12 July 2019 Revised 11 September 2019 Accepted 20 September 2019 Available online 02 June 2020 Authors’ Contribution RE, SA, BAR and MSA participated in development of study design, statistical analysis of data and writing the manuscript. AA, SQ, SS and AUH participated in the practical work and writing of the manuscript. Key words Arachidic acid, Buffalo, Cryopreservation, Semen, Saturated fatty acid The study aimed to evaluate in vivo fertility of cryopreserved buffalo spermatozoa by addition of arachidic acid in semen extender. For this purpose, semen was collected from 3 adult buffalo bulls (Bubalus bubalis) of same age by artifcial vagina keeping temperature at 42°C for a period of 5 weeks (replicates; n=30). Ejaculates were mixed in tris citric acid extender having 0.0 (control), 20.0, 25.0 and 30.0 ng/mL of arachidic acid at a temperature of 37°C (>1 mL volume, >0.5 billion per mL conc., >60% motility) and cryopreserved using standard procedures. Percent sperm motility, liveability, plasmalemma integrity and viability were increased in extender (P<0.05) having 20.0 ng/mL of arachidic acid compared to 25.0 ng/ mL, 30.0 ng/mL and control. However, sperm chromatin integrity was equally improved in experimental extenders having arachidic acid compared to control. Sperm abnormalities were reduced in experimental extender with 20 ng/mL of arachidic acid compared to other experimental extenders containing arachidic acid and control. In experiment 2, a total of 533 inseminations were carried out by the extender containing best level of arachidic acid (20 ng/mL of extender). In vivo fertility was signifcantly improved in buffaloes inseminated with semen containing 20.0 ng/mL of arachidic acid (58.64%) compared to control (46.06%). In conclusion, addition of arachidic acid (20.0 ng/mL) in extender signifcantly enhanced quality and in vivo fertility of post thaw buffalo semen. INTRODUCTION D uring cryopreservation, the freeze thaw damages are unavoidable that result in reduced semen quality (Yoshida, 2000). Cryopreservation without protective agents causes loss of lipids (Pickett and Komarek, 1964) and reorientation of phospholipids from sperm membrane that disrupts plasma membrane functions (Lessard et al., 2000). In buffalo spermatozoa, the cold shock and freezing causes 6.49% and 19.1% loss of phospholipids respectively (Sarmah et al., 1984). Lipids are lost from sperm membranes due to lipid peroxidation reactions * Corresponding author: rabeaejaz29@gmail.com 0030-9923/2020/0001-0001 $ 9.00/0 Copyright 2020 Zoological Society of Pakistan (Aitken, 1995) and generation of acetyl CoA (Cook et al., 1982) by β-oxidation. The loss of lipids from sperm membrane can be mitigated by enriching the semen extender with fatty acids (Ejaz et al., 2014, 2017). High ratio of polyunsaturated fatty acids (PUFAs) in sperm plasmalemma causes high fuidity that in turn resulted in low resistance to thermal shock (Giraud et al., 2000). However, saturated fatty acids (SFAs) are not as much susceptible to peroxidation (Rael et al., 2004) compared to unsaturated fatty acids. Arachidic acid (AA) is naturally found in buffalo semen and is involved in membrane functions (Jain and Anand, 1976). Previously, when AA was evaluated at 0, 5.0, 10.0 and 20.0 ng/mL in extender, improvement in buffalo sperm quality was recorded at the highest level i.e., 20 ng/mL of tris citric acid extender (Ejaz et al., 2014). We speculated that ABSTRACT Pakistan J. Zool., pp 1-8, 2020. DOI: https://dx.doi.org/10.17582/journal.pjz/20190712110704