Experiments were conducted to study the effect of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa after cryopreservation in straws. Semen (split ejaculate) in maxi-straws (6 mm o.d.) was frozen using a programmable freezing chamber. Three methods for in vitro sperm evaluation were used: motility (MOT), acrosome integrity (NAR) and flow cytometric analysis of sperm treated with carboxyfluorescein diacetate and propidium iodide to assess sperm plasma membrane integrity (PMI). No interactions were found among the three variables evaluated. Length of prefreeze exposure to glycerol, ranging from .5 min to 75 min, had no effect on post-thaw sperm viability. Exposure of sperm to a glycerol-containing extender medium at 5 degrees C gave improved post-thaw viability over that exposed at 0 degree C (P less than .05). Glycerol at a concentration of 3 or 4% resulted in maximum post-thaw MOT. Acrosome integrity values were greatest for 2 and 3% glycerol, whereas PMI was greatest when glycerol concentration was 4 to 6%. The primary cryoprotective effect of glycerol on boar semen may be extracellular. It is concluded that 3 or 4% glycerol gives maximum viability of frozen-thawed spermatozoa when the present methods are employed.

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Contents:Thirteen ejaculates f r o m each of t w o boars that weregreatly different in postthaw sperm quality were f r o z e n in either 2 or 4 % glycerol (final concentrationlsplitsample). Frozen-thawed sperm were evaluated in vitro, f o r motility, acrosomal integrit y ( N A R ) , plasma membrane integrity (PMI), and f o r sperm penetration ( S P A ) o f zona-jree hamster eggs. T o evaluate in vivo fertilizing capacity, 5 2 sows were inseminated once, 30-34 hours after onset o f estrus. Ova were recovered f r o m the reproductive tract 2-4 days following insemination and evaluated for cleauage and sperm binding. T h e t w o boars differed significantly f o r all in vivo and i n vitro parameters. Motilit y and PMI were higher (P < 0.05) f o r sperm f r o z e n with 4 % than 2% glycerol whereas N A R was higher with 2% glycerol. Higher numbers o f sperm were bound t o eggs i n vivo f o r sperm f r o z e n with 4% than with 2% glycerol (P < 0.05). T h e fertilization rate f o r sperm f r o z e n with 4 % glycerol was n o t significantly higher than t h e fertilization rate f o r sperm f r o z e n with 2% glycerol (P = 0.12). On the basis of these data, 4 % rather than 2% glycerol may be close t o o p t i m u m when freezing boar semen in straws. However, more extensive in vivo fertility testing is required t o confirm this trend. L o w correlations were observed between in vitro (and in vivo) evaluation of sperm quality and fertility when the effect o f boar and glycerol concentrations was k e p t constant. Inhalt: Die Brauchbarkeit von in vitro-Methoden fur die Voraussage der in vivo-Befruchtungskapazitat von Eberspermatozoen nach Tiefgefrierung mit 2% oder 4% GlycerinVon 2 Ebern, die sich in der Qualitat ihres Auftauspermas deutlich unterschieden, wurden j e 13 Ejakulate mit 2% oder 4 % Glycerinanted (Endkonzentrationsplit sample) tiefgefroren. Die aufgetauten Samenproben wurden in vitro nach Motilitat, akrosomaler Integritat ( N A R ) , Plasmamembran-Integritat (PMI) und im Spermapenetrationstest ( S P A ) a m zonafreien Hamsterei uberpruft. Zur Beurteilung der Befruchtungskapazitat in vivo wurden 5 2 Sauen 30-34 h nach Brunstbeginn einmalig besamt. 2-4 Tage nach der Besamung wurden die Eier aus d e m Genitaltrakt gewonnen und auf Teilung und auf A n h e f t u n g von Samenzellen an der Zona pellucida beurteilt. Die beiden Eber waren in allen In vitro-und In vivo-Parametern signifikant verschieden. Motilitat und PMI waren besser ( p < 0,05) fur Spermien, die in 4% Glycerin konserviert waren, N A R besser in 2%igem Glycerin. Nach Konservierung in 4 % Glycerin waren mehr Spermien a n der Eihiille angeheftet als nach 2% Glycerin ( p < 0,05). A b e r die Befruchtungsraten waren nach 4% und 2% Glycerinkonservierung praktisch nicht verschieden ( p = 0,l.Z). 9 Auf der Grundlage dieser Ergebnisse ist zu folgern, daj3 die Tiefgefrierkonservierung von Pelletsperma mit 4% Glycerin giinstiger ist als mit 2% Glycerin; dieser Trend mu& jedoch in umfangreicheren In vivo-Fertilitatstests noch bestatigt werden. Wenn die Effekte...