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Effect of progesterone on unwashed and nonpreincubated boar spermatozoa in homologous in vitro fertilization
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Theriogenology EFFECT OF PROGESTERONE ON UNWASHED AND NONPREINCUBATED BOAR SPERMATOZOA IN HOMOLOGOUS IN VITRO FERTILIZATION J.M. Vaquez; E.A. Martinez, C. Matas axi J. Rota Department of Animal Phatology, Mmcia University, Mu&t, 30.071, Spain
There is a growing bcdy of evidence ixlicating that progesteroon: has several effects on capacitated boar spermatozoa. However, the effect of progesterone on unwashed and nonprekubated boar spermatozoa has not been determkd. Recently, we have shown that boar spermatozoa undergoes capacitation and true acrosome reaction during coincubation with oocytes even when not washed or prekubated (Martkz et al., Biol. Reprcd., 55:134. 1996). This study was performed to determine if the addition of progesterone in the culture nxxlium would enhance acrosome reaction, penetration rates or affect viability of unwashed and nonprek&ati boar spem~toy In experkmt 1, spemutozoa fnrm sperm-rich tixtions of two boars were directly kubated at 5x10 ceIIs/mI in a 42-mm plastic dish contain@ 2 ml control medium (M; TCM199 modified as decribed by Mattioli et al., Gamete Res., 20:177. 1988), M+DMSO (0.16%; solvent for progesterone) (MD) or MD+ progesterone (15 pM) (MP) at 39’ C under 5% CR, in air. The physiological acrosome reaction and the viability of spermatozoa were assessed using PNA-F’ITC in comb&ion with propidium iodide and two-&our flow cytometry at 0, 15, 45, 90, 180 and 240 minutes of incubation. The data were analyzed by ANOVA and comparisons between means were made by Tukey-test. Results are summarized below.
Mins of incubation
Mins of incubation
Figure 1.- Mean tiidence of viability (a) and true acrosome reaction (b) in boar spermatozoa for threedifferentmedia.+:M;,O:MD;m:MP.( *, ** p< 0.01). Data were pooled Tom 4 replicates. In experiment2, groups of 15 pig oocytes matured in vitro as described by Yoshida et al. (Mol. Reprod. Dev., 31: 68. 1992) were co&Wed with 5~10~ cells/ml kom two boars in a 42-mm plastic dish contain@ 2 ml of M, MD or MP at 39’ C u&r 5% CR in air. After 1618 hours, the oocytes were fixed, skined and assessed for penetration. Results are summarized in the table below. Table 1. Effect of progesterone on in vitm penetration indicate that These results rates (rneankSEM). Data were pooled progesterone in the cuhure medium from 4 replicates may contribute to induce the acrosome Media Matured Sperm Sperm/ooW reaction and, at the same time, may Oocytes (n) Pextration (W) (n) preserve sperm viability but does not M 91.66k2.3 8.11kO.4 160 increase the mtion rates of MD 90.53&l .8 7.91zk0.6 151 matured pig ocqtes. 95.03k2.5 MP 172 8.34k0.3 Tbis research was supported by CICYT (AGF 95/1009), CDTI (94/0059) and IFRM (94/085).
Theriogenology EFFECT OF PROGESTERONE ON UNWASHED AND NONPREINCUBATED BOAR SPERMATOZOA IN HOMOLOGOUS IN VITRO FERTILIZATION J.M. Vaquez; E.A. Martinez, C. Matas axi J. Rota Department of Animal Phatology, Mmcia University, Mu&t, 30.071, Spain
There is a growing bcdy of evidence ixlicating that progesteroon: has several effects on capacitated boar spermatozoa. However, the effect of progesterone on unwashed and nonprekubated boar spermatozoa has not been determkd. Recently, we have shown that boar spermatozoa undergoes capacitation and true acrosome reaction during coincubation with oocytes even when not washed or prekubated (Martkz et al., Biol. Reprcd., 55:134. 1996). This study was performed to determine if the addition of progesterone in the culture nxxlium would enhance acrosome reaction, penetration rates or affect viability of unwashed and nonprek&ati boar spem~toy In experkmt 1, spemutozoa fnrm sperm-rich tixtions of two boars were directly kubated at 5x10 ceIIs/mI in a 42-mm plastic dish contain@ 2 ml control medium (M; TCM199 modified as decribed by Mattioli et al., Gamete Res., 20:177. 1988), M+DMSO (0.16%; solvent for progesterone) (MD) or MD+ progesterone (15 pM) (MP) at 39’ C under 5% CR, in air. The physiological acrosome reaction and the viability of spermatozoa were assessed using PNA-F’ITC in comb&ion with propidium iodide and two-&our flow cytometry at 0, 15, 45, 90, 180 and 240 minutes of incubation. The data were analyzed by ANOVA and comparisons between means were made by Tukey-test. Results are summarized below.
Mins of incubation
Mins of incubation
Figure 1.- Mean tiidence of viability (a) and true acrosome reaction (b) in boar spermatozoa for threedifferentmedia.+:M;,O:MD;m:MP.( *, ** p< 0.01). Data were pooled Tom 4 replicates. In experiment2, groups of 15 pig oocytes matured in vitro as described by Yoshida et al. (Mol. Reprod. Dev., 31: 68. 1992) were co&Wed with 5~10~ cells/ml kom two boars in a 42-mm plastic dish contain@ 2 ml of M, MD or MP at 39’ C u&r 5% CR in air. After 1618 hours, the oocytes were fixed, skined and assessed for penetration. Results are summarized in the table below. Table 1. Effect of progesterone on in vitm penetration indicate that These results rates (rneankSEM). Data were pooled progesterone in the cuhure medium from 4 replicates may contribute to induce the acrosome Media Matured Sperm Sperm/ooW reaction and, at the same time, may Oocytes (n) Pextration (W) (n) preserve sperm viability but does not M 91.66k2.3 8.11kO.4 160 increase the mtion rates of MD 90.53&l .8 7.91zk0.6 151 matured pig ocqtes. 95.03k2.5 MP 172 8.34k0.3 Tbis research was supported by CICYT (AGF 95/1009), CDTI (94/0059) and IFRM (94/085).