Evidence of venom variability associated with venom extraction interval in the monocled cobra (Najakaouthia)

Snake venom variability is a critical factor in antivenom production. Although several influencing factors have been studied, the impact of extraction interval on venom properties remains poorly understood. This study evaluated the effects of various extraction intervals on venom yield, the percentage of dry weight (%Dw), and composition in the monocled cobra (Naja kaouthia). Twenty-five captive snakes were randomly allocated to five groups, each undergoing a control extraction followed by a repeated extraction at 1, 4, 7, 10, or 14 days. Venom profiles were characterized using reversed-phase high-performance liquid chromatography (RP-HPLC) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Results indicated that while fresh venom yield remained stable across all intervals, %Dw significantly decreased in extractions performed between 1 and 10 days, returning to control levels only by day 14. RP-HPLC analysis revealed that early repeated extraction significantly reduced the relative abundance of long-chain α-neurotoxins-rich fraction, which contains the principal neurotoxic components of the venom. Conversely, fractions containing cytotoxins and phospholipase A₂ (PLA₂) showed a higher relative abundance in the 1-day repeated extraction. In conclusion, N. kaouthia requires a minimum recovery period of 14 days for most measured properties but not all of them approached the control level. These findings suggest that standardized venom extraction protocols may be important for maintaining a consistent dry venom production and representative toxin profile, which could potentially contribute to the quality and effectiveness of antivenom production.