Egypt. J. Vet. Sci. Vol. 53, No. 2, pp. 241-248 (2022) A MINO acids are an important component of culture media and their benefcial role is reported in different species. It was hypothesized that supplementation of amino acids to in vitro maturation (IVM) media may affect IVM and fertilization of buffalo oocytes. The objective was to study the effect of supplementing IVM media with essential amino acids (EAA), non- essential amino acids (NEAA) or both (EAA+NEAA) on in vitro maturation (Experiment I) and fertilization (Experiment II) of buffalo oocytes. In experiment I, cumulus-oocyte complexes aspirated from follicles (2-8 mm in diameter) were matured in IVM media: (1) Maturation medium (MM; TCM-199 enriched with BSA 6mg/mL, 1 µg/mL estradiol-17β, 10 IU/mL LH, 0.5 µg/mL FSH and 50 ug/mL gentamicin) alone as control; (2) MM supplemented with 2% EAA solution; (3) MM enriched with 1% NEAA solution; and (4) MM added with 2% EAA solution + 1% NEAA solution at 38.5°C in an atmosphere with 5% CO 2 and 95% humidity under mineral oil.. The degree of cumulus expansion and degree of nuclear maturation (Aceto orcein staining) was measured after 24 h of incubation. In experiment II, following maturation as in experiment I, the oocytes were co incubated with sperms at 39 °C in 5% CO 2 in air with maximum humidity. After 18 h, oocytes were stained as described earlier and sperm penetration rate was recorded. Cumulus expansion was higher (P<0.05) in medium supplemented with EAA, NEAA and EAA+NEAA compared to control. Nuclear maturation rate and Sperm penetration rate remained similar in media supplemented with EAA, NEAA, EAA+NEAA and control. Results indicated that supplementation of maturation media with amino acids have benefcial effect on oocyte maturation in terms of cumulus expansion. However, non-signifcant effect on sperm penetration rate suggested further studies to evaluate the effect of amino acids presence of in vitro maturation media on subsequent culturing of embryos. Keywords: Amino acids, Buffalo, Oocytes, in vitro maturation, Serum free media. 27 Supplementation of Amino Acids in Serum Free IVM-Media and its Effect on In Vitro Maturation and Fertilization of Buffalo Oocytes Surrya Khanam *1 , Asima Azam 2 , Rabea Ejaz 2 , Saima Qadeer 3 Asma-ul-Husna 4 , Nemat Ullah 5 and Shamim Akhter 6 1 Department of Zoology, Women University Swabi 2 Department of Zoology, Shaheed Benazir Bhutto Women University, Peshawar 3 Department of Zoology, Division of Science & Technology, University of Education Lahore, 54000, Pakistan 4 Department of Zoology, The University of Haripur, 22620, Pakistan 5 Riphah International University, Lahore 6 Department of Zoology, PMAS-Arid Agriculture University, Rawalpindi Egyptian Journal of Veterinary Sciences https://ejvs.journals.ekb.eg/ Corresponding author: Surrya Khanam, E-mail: surryiamalik@gmail.com (Received 11/10/2021; accepted 20/02/2022) DOI. 10.21608/ejvs.2022.92571.1272 ©2022 National Information and Documentation Centre (NIDOC) Introduction Buffalos are important part of livestock industry in Pakistan and are considered as black gold of Pakistan [1]. However, different factors such as low number of follicles, poor ovulatory response and atretic follicles leads to low reproductive potential in buffalos. To deal with these problems different types of assisted reproductive techniques such as superovulation, artifcial insemination and