ASSESSMENT OF MOLECULAR BIOMARKERS IN FROZEN-THAWED STALLION SPERMATOZOA BY FLOW CYTOMETRY

EBEL BARRERA F; A C. LIAUDAT; VERÓN GL; FERRANTE A; MIRAGAYA M; LOSINNO L; BOSCH P; VAZQUEZ-LEVIN MH

Congreso; XX Jornadas Anuales de la Sociedad Argentina de Biología (SAB)-XVII Jornadas de la Sociedad Uruguaya de Biociencias (SUB), Segundas Jornadas Rioplatenses de Biología; 2018

ASSESSMENT OF MOLECULAR BIOMARKERS IN FROZEN-THAWED STALLION SPERMATOZOA BY FLOW CITOMETRYEbel-Barrera F1,2, Liaudat A2, Verón GL1, Ferrante A3, Miragaya M3, Losinno L4, Bosch P2, Vazquez-Levin MH1. 1Instituto de Biología y Medicina Experimental (IBYME-CONICET), 2Departamento de Biología Molecular (FCEFQyN-UNRC, 3Cátedra de Teriogenología (INITRA-FAV-UBA) y 4Laboratorio de Producción Equina (FAyV-UNRC). Email: francisca.ebelb@gmail.com. Cryopreservation damages spermatozoa and negatively affects their function. With the increasing international trade of animal semen, many stallion breeds use frozen-thawed samples. However, its use causes considerable frustration and economic losses due to unexpected poor pregnancy rates in numerous cases. Routine semen evaluation techniques are limited, since they only assess presence, amount and certain properties of sperm cells, which may not completely reflect the negative impact of cryopreservation. The use of molecular biomarkers of sperm structure and function will contribute to the assessment of cryopreservation-related sperm membrane damage, which is mainly reflected in sperm acrosomal loss. Flow cytometry (FC) has become the prefer method to objectively assess molecular biomarkers in large cell populations in an objective and reproducible fashion. The aim of the present study was to define standard conditions to evaluate frozen-thawed stallion spermatozoa using molecular biomarkers in FC protocols. Sperm nuclear staining was done using Propidium Iodide (PI) and acrosomal staining with Pisum Sativum Agglutinin (PSA) coupled to Fluororescein Isothiocyanate (FITC-PSA). Staining patterns for both biomarkers were first validated using fluorescence microscopy in fixed spermatozoa mounted in glass slides. Sperm cells incubated for 15 min with IP (5 µg/mL) displayed a specific nuclear signal in 100% of the cases. In spermatozoa treated for 15 min with FITC-PSA (50 µg/mL), three specific patterns were observed in the acrosomal cap (mean±standard deviation of the mean; SDM): P1= strong (30.3±9.1%), P2= patchy (54.1±11.7%) and P3= absent signal (15.6±4.1%). FC protocols runs were done with 100000 events. First, IP was tested at 0.1, 1 and 5µg/mL. A subpopulation was gated according to the forward and side scatter properties of sperm cells, obtaining on average 41.7±6.8% cells. Of this subpopulation, three subpopulations was identified, two IP positive (IP1= 60.2±21.1%; IP2= 1.0±0.2%) and one IP negative (IP3=36.1±19.4%), identified as cell debris or egg yolk and discarded. Then, FITC-PSA was co-incubated at 2.5 and 10 µg/mL with IP. This evaluation revealed the presence of three cell population: Q1= cells IP positive and FITC-PSA negative (10.4±5.7%); Q2= cells IP and FITC-PSA positive (26.6±4.1%) and Q3-Q4= cells IP negative (63.1±6.6%). Sperm cells were present in Q1 and Q2. Similar results were obtained with sperm samples from three stallions. These studies show the use of FC in the evaluation of molecular biomarkers in frozen-thawed equine spermatozoa. Studies with functional molecular biomarkers are underway.