Fluorometric assessments of acrosomal integrity and viability in cryopreserved bovine spermatozoa

• Published in: Biology of reproduction Vol. 56; no. 4; pp. 991 - 998
• Main Authors: Thomas, C.A, Garner, D.L, DeJarnette, J.M, Marshall, C.E
• Format: Journal Article
• Language: English
• Published: Madison, WI Society for the Study of Reproduction 01.04.1997
• Subjects: Acrosin - analysis; ACROSOMA; ACROSOME; Acrosome - ultrastructure; Animals; Biological and medical sciences; BIOLOGICAL PRESERVATION; Birth control; BULLS; Cattle; CELL COUNTING; Cell Survival; CONGELACION; CONGELATION; CONSERVACION BIOLOGICA; CONSERVATION BIOLOGIQUE; CONTEO DE CELULAS; CRYOPRESERVATION; Cryopreservation - methods; ESPERMATOZOO; FLOW CYTOMETRY; Flow Cytometry - methods; Fluorescein-5-isothiocyanate; FLUORESCENCE MICROSCOPY; Fluorescent Antibody Technique; Fluorescent Dyes; FLUORESCENT STAINING; FLUORIMETRIA; FLUORIMETRIE; FLUORIMETRY; FREEZING; Fundamental and applied biological sciences. Psychology; Gynecology. Andrology. Obstetrics; Immunoglobulins; Lectins; Male; Mammalian male genital system; Medical sciences; METHODE; METHODS; METODOS; MICROSCOPIA; MICROSCOPIE; MICROSCOPY; Microscopy, Fluorescence - methods; Microscopy, Interference; Morphology. Physiology; MOTILITY; MOUVEMENT; MOVEMENT; MOVIMIENTO; NUMERATION CELLULAIRE; Peanut Agglutinin; Rabbits; Semen Preservation - methods; Serine Proteinase Inhibitors; SPERM MOTILITY; SPERM VIABILITY; SPERMATOZOA; Spermatozoa - cytology; Spermatozoa - physiology; Spermatozoa - ultrastructure; SPERMATOZOIDE; STAINING; Sterility. Assisted procreation; TAUREAU; TORO; Vertebrates: reproduction; VIABILIDAD; VIABILITE; VIABILITY; Flow cytometry; Spermatozoa; Bovine; Sperm conservation; Assisted procreation; Exploration; Male; Germinal cell; Fluorescence spectrometry; Vertebrata; Mammalia; Cryopreservation; Quality; Artiodactyla; Acrosome; Viability; Ungulata
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod56.4.991

The combination of specific fluorometric staining and flow cytometry provides a rapid and precise means of assessing the functional status of cells. We sought to utilize this approach to quantify two important seminal characteristics, acrosomal integrity and sperm viability, and to compare these with classical microscopic measurements of acrosomal integrity and sperm motility. Samples of thawed, cryopreserved sperm packaged in 0.5-ml French straws were obtained from 12 Holstein bulls. Classical acrosomal assessments and sperm motility estimates were made using differential interference contrast microscopy. Fluorescent acrosomal probes included LysoTracker Green DND-26 (LYSO-G), fluorescein-labeled peanut agglutinin, the biotinylated isocoumarin serine protease inhibitor Bi-Aca-Aca-OMe-IC that was secondarily labeled with fluorescein-avidin, and rabbit antibodies to bovine acrosin that were secondarily labeled with fluoresceinated anti-rabbit immunoglobulin. The fluorescent probes for sperm viability were a combination of SYBR-14 and propidium iodide (PI) or of SYTO-17 and PI. Significant differences were found among methods and among bulls, but not among straws (n = 3). All four fluorescent measures of acrosomal integrity showed highly significant correlations with both classical measurements. These data indicated that the quality of cryopreserved bovine sperm samples could be readily quantified using a variety of organelle-specific fluorescent staining techniques