189 | Fluorescent analysis by confocal microscopy of lipid droplets as a marker of oocyte quality in Sarda sheep under different seasonal conditions

Silvia Zaccardi¹, Domenica Cocciolone¹, Guido Macchiarelli¹, Sara Succu², Giovanni Giuseppe Leoni³, Maria Grazia Palmerini¹ | ¹Department of Life, Health and Environmental Sciences, University of L’Aquila, Italy; ²Department of Veterinary Medicine, University of Sassari, Italy; ³Department of Biomedical Sciences, University of Sassari, Italy.

Seasonal reproduction is a widespread adaptive strategy in mammals, ensuring that offspring are born during periods favorable for survival and growth. Sheep exhibit marked annual cycles of reproductive activity: females alternate between phases of ovarian activity and ovulation (the breeding season) and phases of ovarian quiescence and anovulation (anestrus) (Karsch et al., 1980). In Sarda sheep, the breeding season typically begins in early summer (May–June) and ends in the following spring (March–April). This seasonality is regulated by photoperiod, with long days suppressing and short days stimulating reproductive activity (Mara et al., 2013). Mammalian oocytes contain species specific stores of lipids, which play essential roles during maturation and early embryonic development. Ovine oocytes, similar to bovine and porcine ones, contain lipid droplets (LDs) that serve as energy reserves. In mature oocytes, LDs tend to be more uniformly distributed, reflecting metabolic activity and developmental competence. Lipid content and metabolism—rather than total lipid quantity alone—are key indicators of oocyte quality and are associated with successful maturation and embryo development (Reader et al., 2017). This study investigated seasonal effects on oocyte morphology and LDs organization in Sarda sheep using phase-contrast and confocal microscopy, focusing on LDs as markers of oocyte quality. Oocytes were collected from ovaries of adult ewes at slaughter during winter (January–March; W) and spring/summer (May–July). Summer samples were further classified as oocytes from non pregnant (SNG) and pregnant (SG) ewes. Morphology was first assessed by phase contrast microscopy. LDs were then visualized by confocal laser scanning microscopy using BODIPY 493/503, with DAPI nuclear counterstaining. Quantitative and morphometric analyses of LDs—including numerical density and fluorescence intensity—were performed using ImageJ. Phase contrast microscopy revealed well rounded oocytes in the W and SNG groups, whereas SG oocytes displayed irregular shapes with spikes in the perivitelline space. Confocal imaging showed distinct patterns of lipid distribution: W oocytes contained numerous small, homogeneous, highly fluorescent LDs; SNG oocytes exhibited larger, more dispersed, and less fluorescent LDs; SG oocytes displayed even larger LDs, some appearing partially depleted. The lighter cytoplasm observed in W oocytes suggests differences in lipid composition compared with the darker cytoplasm of SNG and SG oocytes. In conclusion, both oocyte morphology and lipid organization vary seasonally in Sarda sheep. The presence of cytoplasmic spikes and altered LDs patterns in SG oocytes may indicate delayed cytoplasmic maturation and metabolic imbalance. Confocal lipid imaging thus represents a valuable tool for assessing oocyte quality. Future ultrastructural studies may further elucidate how environmental factors, including climate change, influence oocyte competence.