The Indian red jungle fowl (IRJF; Gallus gallus murghi) native to South-Asia is facing threats in its natural habitat and needs immediate conservation employing ex-situ and in-situ approaches. For ex-situ, in vitro conservation of IRJF, semen banking is one of the potential techniques that require an extender with appropriate cryoprotectants having adequate retrieval capacity for functional spermatozoa. Therefore, study was designed to evaluate semen production capacity (semen characteristics, timing, frequency of collection and seasonal changes), identification of efficient extender, cryo-damage estimation, permeable (glycerol, DMA, DMSO and DMF) for short and long term storage of semen and the use of non-permeable cryoprotectants (PVP and egg yolk) and fertility outcomes for ex situ in vitro conservation of IRJF germplasm. Semen was collected from eight male birds of IRJF (housed at main campus PMAS-AAUR) in a graduated plastic tube through abdominal massage and transferred to the laboratory for assessment of semen volume, initial motility and concentration. The ejaculates having at least 60% motility were processed for further experimentation. The qualifying ejaculates were studied for motility, volume, concentration, plasma membrane integrity, viability and acrosomal integrity of spermatozoa. Semen production in IRJF was quite low compared to domestic fowl but could be collected safely on daily basis preferentially in the evening. A number of diluents (Beltsville poultry, turkey, Lake, EK, Tselutin poultry and chicken semen extender) were tested against their efficacy to preserve IJRF spermatozoa in liquid (5ºC) and frozen state (LN2; -196ºC). The turkey semen extender was found superior compared to all xxvii 4 4 experimental extenders for storage in liquid and frozen state showing remarkably higher fertility rate. Cryopreservation with 11% glycerol caused maximum loss to motility (50%) followed by plasma membrane integrity (45%), viability (25%) and minimal to acrosomal integrity (20%). Glycerol optimization (11, 15 and 20%) study demonstrated the superiority (P < 0.05) of 20% glycerol in freezing for cryopreservability and fertility after artificial insemination. Various levels of DMA, DMSO, DMF and PVP (4, 6, 8, and 10%) were evaluated; 6% DMA, 8% DMSO, 8% DMF and 6% PVP maintained higher (P < 0.05) semen quality and fertility after artificial insemination compared to control treatment where spermatozoa were exposed to 20% glycerol. Among four levels of egg yolk (10, 15, 20 and 25%) evaluated; 15% egg yolk was superior (P < 0.05) for semen quality and fertility compared to the control. It is concluded that IJRF semen with maximum efficiency can be collected once in a day either in the morning or evening. The germplasm of IJRF can be conserved in liquid (for two days) and in frozen state (for indefinite period) using cryopreservation protocol based on DMA or DMF or DMSO or PVP and egg yolk can be used efficiently in artificial breeding program for conservation.