ARTIFICIAL INSEMINATION IN FARM ANIMALS - Phenix-Vet

60Artificial Insemination in Farm AnimalsTests Aims Procedure and Analysis ReferencesHypo-osmoticswelling test.Spz membraneintegrity(indirectmethod)Computer Objectiveassisted sperm evaluation ofanalysis sperm cell[CASA] motilityZonapellucidabinding assayFluorescentprobes andflowcytometryAssessment ofspermfertilizingpotentialMembraneintegrity andevaluation oflive and deadcellsCapacitationstatusSperm incubation with hypo-osmoticsolutions for 30 minutes at 37ºCSpz with intact plasmalemma becomeswolled and show coiled tailsDetermination of motility parametersfor individual spzCharacterization of Spz movementaccording to the average velocity, thetrajectory, the amplitude of movementand beat cross frequency. It allowsidentification of Spz subpopulationsEngland &Plummer, 1993;Kumi-Diaka, 1993Verstegen et al.,2001;Rijsselaere et al.,2003;Niżański et al.,2009- ZP-binding assay ( ZBA) using intact Hermansson ethomologous oocytesal., 2006;- hemizona binding assay (HZA) using Kawakami et al.,bisected hemizonae1998;The number of spermatozoa bound to Rijsselaere et al.,ZP is counted with contrast-phase 2005;microscopy. The number of bound Spz Ström-Holst et al.,reflects its fertilizing potential 2001Combined use of several fluorescentdyes (i.e, propidium iodide PI andcarboxyfluorescein diacetate, SYBR-14/PI) allow the identification of livecellsLive cells activate fluorescence(deacylation) which is maintainedintracellularly in intact membranecells. Dead Spz are stained red due tothe influx of PI through damagedplasma membrane.Fluorescent antibioticchlorotetracycline (CTC), when boundto free calcium ions, is fluorescent.Combined with Hoechst 33258 allowsalso assessment of percentage of livecells and capacitation statusThree classes of sperm cells may beassessed: uncapacitated and acrosomeintact (F-pattern), capacitated andacrosome intact (B-pattern) andcapacitated and acrosome reacted (ARpattern)Hewitt &England, 1998;Peña et al., 1998;Rijsselaere et al,2005;P.F. Silva &Gadella, 2006Guérin et al.,1999;Hewitt &England, 1998;Petrunkina et al.,2004;Rota et al. 1999b