ABSTRACT: Sperm capacitation is essential for fertilization and is characterized by a cascade of biochemical signaling and membrane remodeling events. This process is highly dependent on membrane composition. Profiling lipid alterations provides a critical window into the molecular underpinnings of capacitation and the regulatory influence of zinc ions (Zn2+). Metabolomic studies in boar sperm have shown that capacitation coincides with broad shifts in metabolite abundance and that extracellular zinc attenuates or redirects many of these changes, highlighting its role as a key modulator. To extend this framework to the lipidome, we profiled boar sperm under three conditions: non-capacitated (0 h), capacitated in vitro (4 h), and capacitated with extracellular zinc (4 h + Zn), using liquid chromatography-mass spectrometry and image-based flow cytometry to validate capacitation status. Relative to 0 h, capacitation was associated with altered abundances of 30 lipids (P < 0.05) spanning several lipid categories: fatty acyls (n = 8), sterol lipids (n = 7), sphingolipids (n = 1), glycerolipids (n = 3), glycerophospholipids (n = 4), and unannotated lipids (n = 9). When exogenous Zn2+ was supplemented during in vitro capacitation, 12 of these shifts were maintained at 0 h-like levels (P < 0.05), suggesting an inhibiting or stabilizing role. In 2 of 16 hits, exogenously supplemented Zn2+ enhanced the capacitation-associated change (P < 0.05), whereas in the remaining 14 it exerted no measurable effect (P > 0.05). When exogenous Zn2+ was supplemented during in vitro capacitation, distinct lipid shifts were identified and organized using Tukey's lipid-pattern classification based on significance (P < 0.05) and directionality, organizing them into four categories: Type-1 lipids (capacitation-associated), Type-2 lipids (zinc-inhibited), Type-3 lipids (zinc-specific response), and Type-4 lipids (zinc-enhanced). These categories describe distinct modes of lipid regulation, where some species remained unaffected by zinc (Type-1, n = 16), others were stabilized or inhibited from progressing toward capacitation-associated levels (Type-2, n = 12), a subset responded exclusively to zinc independent of capacitation (Type-3, n = 4), and a small group exhibited amplified capacitation-linked shifts under zinc supplementation (Type-4, n = 2). Together, these data reveal a class-specific, zinc-dependent architecture of lipid remodeling that integrates metabolic and membrane regulation within the broader capacitation cascade.
