New molecular findings in llama spermatozoa plasma membrane

Abstract

The lipid composition of the spermatozoa plasma membrane influences physiological processes required for fertilization, including motility, capacitation, acrosomal exocytosis, and fusion with the oocyte membrane. Furthermore, it is a relevant factor for cryopreservation methodologies. As its evaluation has not yet been conducted in camelids, the objective of this study was to determine the lipid composition of the plasma membrane of raw llama semen spermatozoa. Fifty-two ejaculates from six Lama glama males were obtained through electroejaculation under general anaesthesia. Volume, sperm motility, concentration, plasma membrane functionality, viability, and acrosomal status were evaluated in all samples. Then, each ejaculate was diluted 1:1 with PBS-2.5 mM EDTA and centrifuged. The pellets were diluted with PBS-EDTA and preserved in chloroform-methanol (1:2). Subsequently, lipid partitioning was performed according to the Blight and Dyer method. Cholesterol (Chol) was quantified using an enzymatic-colorimetric method (Colestat enzymatic AA kit), and total phospholipid (PL) phosphorus was determined using the method described by Rouser et al. (Lipids, 5 494-496, 1970). Gas chromatography was employed for fatty acid (FA) analysis. Descriptive statistics were used for lipid characterization, and correlations with routine seminal characteristics were ana- lysed using Pearson or Spearman tests. Values for Chol and PL were 97.68 ± 28.46 and 127.16 ± 9.94 nmol/100 million spermatozoa, respectively (mean ± SD), with a Chol/PL ratio of 0.78 ± 0.25. Of the total FA comprising membrane PL, 31.10 ± 6.97% were saturated fatty acids (SFA), 10.21 ± 2.63% were monounsaturated (MFA), 2.87 ± 0.93% were dienoic (DFA), and 55.82 ± 10.32% were polyunsaturated (PUFA), with docosahex- aenoic acid (22:6n-3) being predominant at 43.30 ± 7.41% of total composition. Additionally, Chol and Chol/PL were positively correlated with live spermatozoa with intact acrosomes and negatively correlated with dead spermatozoa with reacted acro- somes. SFA and MFA were positively correlated with oscillatory sperm motility, whereas PUFA, mainly 22:6n-3, correlated nega- tively with this variable. This study allowed us to determine the lipid composition of the plasma membrane of raw llama semen spermatozoa, providing novel data for the species and enhancing understanding of its reproductive physiology and behaviour in cryopreservation processes.