Cryoprotective Effect Of Three Penetrating Cryoprotectants Combined With Three Basic Diluents On Rabbit Semen

Glycerol, dimethyl suffixed, acetamide were adopted to verify thecryoprotective ability of pentrating cryoprotectant with three kinds ofextenders.Sperm mobility, intact-acrosome ratio,plasma membrane int-egrity(hypoosmotic swelling test,HOST) were assessed in fresh, cooled,frozen-thawed sample and ultrastructural observation was carried outto analyze the effect of frozed-thawed. Three extenders are as followed:(1 )3.52%tris, 1.96%citric acid, 1.59%glucose;(2)3.03%tris, 1.68% citricacid, 1.25%glucose; (3), 5%sucrose, 5%lactose, and three cryoprotect-ants: glycerol and DMSO end concentration (2%,3%,4%) and acetamideconcentration (2%,4%, 5%). 1: ldiluention, equilibration at 4℃ for 3hours,freezing at -90─95℃ for 10 minutes and thawing at 42℃ for 20seconds .The results were as followed:1:The influence on frozen-thawed sperm mobility is different whendifferente extender is combined with different cryoprotectant.The perc-entage of motile sperm is higher in 4% glycerol+A and 5%acetamide+C(P<0.05);The mobility is lower in 4% glycerol +C and 5% acetamide+ B(P<0.05).The influence of DMSO on frozen-thawed sperm mobilityhas no relationship with diluents in this experiment(P>0.05).2:The influence on intact-acrosome is different when different exte-nder is combined with different cryoprotectant and acetamide had bett-er protective effect than the others in designed concentrations(P<0.05).With A,B and Cextender,different concentration of acetamide, DMSOand glycerol had different effect on intact-acrosome ratio respectively(P<0.05): The intact-acrosome ratio is higher in 5%acetamide + B. Theintact-acrosome ratio is lower in 2% glycerol+C (P<0.05).3:The percentage of sperm tail swelling showed a significantpositive correlation with sperm motility (r=0.7704). With A extender,three cryoprotectants had no significant different effect on frozen-thawed sperm membrane functional integrity (P>0.05). With B diluent,3%glycerol had better cryoprotective ability on membrane functionalintegrity than 4%acetamide (P<0.05). With C diluent, 3% glycerol hadbetter cryoprotective effect on membrane functional integrity than3%DMSO(P<0.05).HOS test can be used as a method to examine rabbitsperm membrane functional integrity.4:After freezing-thawing of semen, morphological changes of spermwere that the plasma membrane became swelled, thined, folded,disrupted and structural changes were that the acrosome swelled, theouter or inner acrosomal membrane vesiculated and even lost. Themidpiece membrane of some sperm was disrupted, mitochondrion wasbared,cracked, and electric concentration is lower. The membrane ofprinciple and endpiece was disrupted and axoneme was cracked andd is p ers ed.5:Sperm mobility, intact-acrosome ratio, HOST positive ratio hasc o n s istency.Master Candidate: Li Xiaoj uanSpecialty: Animal reproduction physiologyand animal reproduction techniqueSupervisor: Professor Zhang Guixue...