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Removal of seminal plasma by centrifugation, before cooled storage, enhances membrane stability of stallion spermatozoa
Animal Reproduction Science 121S (2010) S188–S190
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Abstract
Removal of seminal plasma by centrifugation, before cooled storage, enhances membrane stability of stallion spermatozoa夽 I. Barrier-Battut a,∗ , C. Bonnet a , A. Giraudo a , C. Dubois a , M. Caillaud a , M. Vidament b a b
French National Studs, la Jumenterie du Pin, 61310 Exmes, France UMR INRA-CNRS-Université de Tours, 37380 Nouzilly, France
1. Introduction
2. Materials and methods
Several genetically valuable stallions are known as “poor coolers”, because fertility of their semen after cooled storage is considerably reduced, compared to fresh semen. Toxicity of seminal plasma was suggested as an explanation, and the removal of seminal plasma by centrifugation was tested on a few stallions. However the results remain controversial: some experiments demonstrated a beneficial effect (Jasko et al., 1991; Pagl et al., 2006; Webb et al., 2009), whereas other studies did not (Bedford et al., 1995; Dawson et al., 2000; Jasko et al., 1992; Kareskoski et al., 2006). Most of the time, semen quality after storage was evaluated using only motility. Furthermore, variability between stallions was high, and the beneficial effect of centrifugation was observed only for “poor coolers” (Brinsko et al., 2000). However the low number of ejaculates for each stallion (3 ejaculates) made statistical analysis of the individual variability questionable. The purpose of the present study was to test the effect of centrifugation, followed or not by removal of seminal plasma, on several parameters indicating semen quality after 48 h at 4 ◦ C: motility, plasma membrane integrity, acrosome integrity, and response to a pharmacological induction of acrosome reaction. A sufficient number of ejaculates was selected for each stallion, in order to analyse the individual variability.
2.1. Animals and semen preparation
夽 This paper is part of the supplement entitled “Proceedings of the Tenth International Symposium on Equine Reproduction”, Guest Edited by Margaret J. Evans. ∗ Corresponding author. Tel.: +33 233121204. E-mail address: [email protected] (I. Barrier-Battut). 0378-4320/$ – see front matter doi:10.1016/j.anireprosci.2010.04.017
Fourteen stallions used for artificial insemination with cooled transported semen were included in the study. During the breeding season, 5 ejaculates were analysed for each stallion, except stallion G (only 3 ejaculates) and stallions E and H (4 ejaculates). For each ejaculate, three standard insemination doses were prepared with 20 × 106 spermatozoa/mL in INRA96® (IMV, France) (Batellier et al., 2001): one control dose and two doses for centrifugation. After 10 min centrifugation at 600 × g, the supernatant was aspirated and replaced by INRA96® in the dose “centri SP−”, whereas in the dose “centri SP+” sperm were re-suspended without removal of seminal plasma. All doses were then stored at 4 ◦ C for 48 h.
2.2. Semen analysis After 48 h storage, each dose was homogenized, then warmed for 10 min at 36 ◦ C. Samples were subjected to computer-assisted motility analysis (HTM-IVOS, Hamilton Thorne Research, USA). Plasma membrane integrity was assessed using the hypo-osmotic swelling test: after 15 min at 36 ◦ C in a 50 mOsm saline solution, the percentage of sperm showing swollen flagella (HOS positive) was observed microscopically. Acrosomal labelling was performed using fluoresceinated Pisum sativum agglutinin after permeabilisation with ethanol, with or without induction of acrosome reaction at 36 ◦ C for 1 h with 6 M ionophore A23187. Fluorescence of the sperm heads was observed microscopically.
I. Barrier-Battut et al. / Animal Reproduction Science 121S (2010) S188–S190
S189
Fig. 1. Percentages of (A) progressively motile spermatozoa, (B) intact plasma membrane, (C) intact acrosome and (D) response to induction of the acrosome reaction (difference between the percentages of acrosome reacted sperm with and without ionophore), after 48 h storage at 4 ◦ C. Letters indicate significant differences between treatments, within stallions (A–N) (P < 0.05, Kruskal–Wallis test), and for the total of 66 ejaculates (P < 0.0001, ANOVA).
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I. Barrier-Battut et al. / Animal Reproduction Science 121S (2010) S188–S190
3. Results In our conditions, centrifugation without removal of seminal plasma did not affect sperm parameters compared to control (Fig. 1). However, removal of seminal plasma significantly enhanced plasma membrane integrity after storage, and reduced responsiveness to an induction of the acrosome reaction. Motility and acrosome integrity without ionophore challenge were not affected. A significant effect of the stallion was observed for all variables. When data were analysed individually within stallions, motility was significantly higher in the “centri SP−” dose compared to control for one stallion (J) (P < 0.05, Kruskal–Wallis test). Motility was significantly lower in the “centri SP−” dose than in control for one stallion (F). Response to HOS test was significantly higher in the “centri SP−” dose for 6 of the 14 stallions, and response to induction of acrosome reaction was significantly lower in the “centri SP−” dose for stallion A. The final percentage of seminal plasma in semen doses depended on the initial sperm concentration, and was between 4.5% and 33% in “control” and “centri SP+” doses, and between 0.5% and 3.3% in “centri SP−” doses. However none of the variables studied was significantly correlated with the percentage of seminal plasma. The effect of seminal plasma is therefore not dose-dependent, at least when that percentage is less than 33%. 4. Discussion Our results demonstrated that removal of seminal plasma before cooled storage contributes to the stabilisation of sperm membranes: it enhanced the resistance of sperm to an hypo-osmotic shock, and reduced the responsiveness to an induction of the acrosome reaction. The effect of seminal plasma was not dose-dependent, which agrees with data from Brinsko et al. (2000). However in our conditions, removal of seminal plasma did not affect sperm motility. In bovine species, major seminal plasma proteins associate with sperm membrane choline-phospholipids, and stimulate cholesterol and phospholipid efflux, with a timeand concentration-dependent effect, thereby potentiating sperm capacitation (Thérien et al., 1998). Proteins belonging to the same family were identified in stallion seminal plasma, but no support of lipid efflux was demonstrated (Töpfer-Petersen et al., 2005). Those proteins could be responsible for the destabilising effect of seminal plasma on sperm plasma membrane observed during cooled storage. Apart from elimination of seminal plasma, centrifugation could be deleterious by itself, either by causing mechanical damage (Kareskoski et al., 2006), or by inducing generation of reactive oxygen species and peroxidation of the sperm plasma membrane, as suggested in human (Parinaud et al., 1997). In our conditions, centrifugation without further removal of seminal plasma did not adversely affect sperm quality. This agrees with previous
data (Jasko et al., 1991) using a similar force (500 × g), but a longer centrifugation time (18 min). We observed significant variations between stallions, but independently of the ability of the semen to withstand cold storage. This contradicts previous results (Brinsko et al., 2000). Individual variability between stallions is most likely responsible for the heterogeneity observed between results of different experiments. Our results demonstrated that removal of seminal plasma contributes to the stabilisation of sperm membranes during cooled storage: it enhances the resistance of sperm to hypo-osmotic conditions, and reduces the response to a pharmacological induction of the acrosome reaction, without a dose-dependent effect in our conditions. The influence of such a stabilisation in relation to the fertilising ability needs to be investigated. Acknowledgement We thank the French National Studs for financial support. Conflict of interest None. References Batellier, F., Vidament, M., Fauquant, J., Duchamp, G., Aranaud, G., Yvon, J.M., Magistrini, M., 2001. Advances in cooled semen technology. Anim. Reprod. Sci. 68, 181–190. Bedford, S.J., Graham, J.K., Amann, R.P., Squires, E.L., Pickett, B.W., 1995. Use of two freezing extenders to cool stallion spermatozoa to 5 ◦ C with and without seminal plasma. Theriogenology 43, 939–953. Brinsko, S.P., Crockett, E.C., Squires, E.L., 2000. Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. Theriogenology 54, 129–136. Dawson, G.R., Webb, G.W., Pruitt, J.A., Loughlin, T.M., Arns, M.J., 2000. Effect of different processing techniques on motility and acrosomal integrity of cold-stored stallion spermatozoa. J. Equine Vet. Sci. 20, 191–194. Jasko, D.J., Moran, D.M., Farlin, M.E., Squires, E.L., 1991. Effect of seminal plasma dilution or removal on spermatozoal motion characteristics of cooled stallion semen. Theriogenology 35, 1059–1067. Jasko, D.J., Hathaway, J.A., Schaltenbrand, V.L., Simper, W.D., Squires, E.L., 1992. Effect of seminal plasma and egg-yolk on motion characteristics of cooled stallion spermatozoa. Theriogenology 37, 1241–1252. Kareskoski, A.M., Reilas, T., Andersson, M., Katila, T., 2006. Motility and plasma membrane integrity of spermatozoa in fractionated stallion ejaculates after storage. Reprod. Domest. Anim. 41, 3–38. Pagl, R., Aurich, J.E., Müller-Schlösser, F., Kankofer, M., Aurich, C., 2006. Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 degrees C. Theriogenology 66, 1115–1122. Parinaud, J., LeLannou, D., Vieitenz, G., Griveau, J.F., Milhet, P., Richoilley, G., 1997. Enhancement of motility by treating spermatozoa with an antioxidant solution (Sperm-Fit) following ejaculation. Hum. Reprod. 12, 2434–2436. Thérien, I., Moreau, R., Manjunath, P., 1998. Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm. Biol. Reprod. 59, 768–776. Töpfer-Petersen, E., Ekhlasi-Hundrieser, M., Kirchhoff, C., Leeb, T., Sieme, H., 2005. The role of stallion seminal proteins in fertilisation. Anim. Reprod. Sci. 89, 159–170. Webb, G.W., Dean, M.M., Humes, R.A., Heywood, J.S., 2009. A comparison of the ability of three commercially available diluents to maintain the motility of cold stored stallion semen. J. Equine Vet. Sci. 29, 229–232.
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Abstract
Removal of seminal plasma by centrifugation, before cooled storage, enhances membrane stability of stallion spermatozoa夽 I. Barrier-Battut a,∗ , C. Bonnet a , A. Giraudo a , C. Dubois a , M. Caillaud a , M. Vidament b a b
French National Studs, la Jumenterie du Pin, 61310 Exmes, France UMR INRA-CNRS-Université de Tours, 37380 Nouzilly, France
1. Introduction
2. Materials and methods
Several genetically valuable stallions are known as “poor coolers”, because fertility of their semen after cooled storage is considerably reduced, compared to fresh semen. Toxicity of seminal plasma was suggested as an explanation, and the removal of seminal plasma by centrifugation was tested on a few stallions. However the results remain controversial: some experiments demonstrated a beneficial effect (Jasko et al., 1991; Pagl et al., 2006; Webb et al., 2009), whereas other studies did not (Bedford et al., 1995; Dawson et al., 2000; Jasko et al., 1992; Kareskoski et al., 2006). Most of the time, semen quality after storage was evaluated using only motility. Furthermore, variability between stallions was high, and the beneficial effect of centrifugation was observed only for “poor coolers” (Brinsko et al., 2000). However the low number of ejaculates for each stallion (3 ejaculates) made statistical analysis of the individual variability questionable. The purpose of the present study was to test the effect of centrifugation, followed or not by removal of seminal plasma, on several parameters indicating semen quality after 48 h at 4 ◦ C: motility, plasma membrane integrity, acrosome integrity, and response to a pharmacological induction of acrosome reaction. A sufficient number of ejaculates was selected for each stallion, in order to analyse the individual variability.
2.1. Animals and semen preparation
夽 This paper is part of the supplement entitled “Proceedings of the Tenth International Symposium on Equine Reproduction”, Guest Edited by Margaret J. Evans. ∗ Corresponding author. Tel.: +33 233121204. E-mail address: [email protected] (I. Barrier-Battut). 0378-4320/$ – see front matter doi:10.1016/j.anireprosci.2010.04.017
Fourteen stallions used for artificial insemination with cooled transported semen were included in the study. During the breeding season, 5 ejaculates were analysed for each stallion, except stallion G (only 3 ejaculates) and stallions E and H (4 ejaculates). For each ejaculate, three standard insemination doses were prepared with 20 × 106 spermatozoa/mL in INRA96® (IMV, France) (Batellier et al., 2001): one control dose and two doses for centrifugation. After 10 min centrifugation at 600 × g, the supernatant was aspirated and replaced by INRA96® in the dose “centri SP−”, whereas in the dose “centri SP+” sperm were re-suspended without removal of seminal plasma. All doses were then stored at 4 ◦ C for 48 h.
2.2. Semen analysis After 48 h storage, each dose was homogenized, then warmed for 10 min at 36 ◦ C. Samples were subjected to computer-assisted motility analysis (HTM-IVOS, Hamilton Thorne Research, USA). Plasma membrane integrity was assessed using the hypo-osmotic swelling test: after 15 min at 36 ◦ C in a 50 mOsm saline solution, the percentage of sperm showing swollen flagella (HOS positive) was observed microscopically. Acrosomal labelling was performed using fluoresceinated Pisum sativum agglutinin after permeabilisation with ethanol, with or without induction of acrosome reaction at 36 ◦ C for 1 h with 6 M ionophore A23187. Fluorescence of the sperm heads was observed microscopically.
I. Barrier-Battut et al. / Animal Reproduction Science 121S (2010) S188–S190
S189
Fig. 1. Percentages of (A) progressively motile spermatozoa, (B) intact plasma membrane, (C) intact acrosome and (D) response to induction of the acrosome reaction (difference between the percentages of acrosome reacted sperm with and without ionophore), after 48 h storage at 4 ◦ C. Letters indicate significant differences between treatments, within stallions (A–N) (P < 0.05, Kruskal–Wallis test), and for the total of 66 ejaculates (P < 0.0001, ANOVA).
S190
I. Barrier-Battut et al. / Animal Reproduction Science 121S (2010) S188–S190
3. Results In our conditions, centrifugation without removal of seminal plasma did not affect sperm parameters compared to control (Fig. 1). However, removal of seminal plasma significantly enhanced plasma membrane integrity after storage, and reduced responsiveness to an induction of the acrosome reaction. Motility and acrosome integrity without ionophore challenge were not affected. A significant effect of the stallion was observed for all variables. When data were analysed individually within stallions, motility was significantly higher in the “centri SP−” dose compared to control for one stallion (J) (P < 0.05, Kruskal–Wallis test). Motility was significantly lower in the “centri SP−” dose than in control for one stallion (F). Response to HOS test was significantly higher in the “centri SP−” dose for 6 of the 14 stallions, and response to induction of acrosome reaction was significantly lower in the “centri SP−” dose for stallion A. The final percentage of seminal plasma in semen doses depended on the initial sperm concentration, and was between 4.5% and 33% in “control” and “centri SP+” doses, and between 0.5% and 3.3% in “centri SP−” doses. However none of the variables studied was significantly correlated with the percentage of seminal plasma. The effect of seminal plasma is therefore not dose-dependent, at least when that percentage is less than 33%. 4. Discussion Our results demonstrated that removal of seminal plasma before cooled storage contributes to the stabilisation of sperm membranes: it enhanced the resistance of sperm to an hypo-osmotic shock, and reduced the responsiveness to an induction of the acrosome reaction. The effect of seminal plasma was not dose-dependent, which agrees with data from Brinsko et al. (2000). However in our conditions, removal of seminal plasma did not affect sperm motility. In bovine species, major seminal plasma proteins associate with sperm membrane choline-phospholipids, and stimulate cholesterol and phospholipid efflux, with a timeand concentration-dependent effect, thereby potentiating sperm capacitation (Thérien et al., 1998). Proteins belonging to the same family were identified in stallion seminal plasma, but no support of lipid efflux was demonstrated (Töpfer-Petersen et al., 2005). Those proteins could be responsible for the destabilising effect of seminal plasma on sperm plasma membrane observed during cooled storage. Apart from elimination of seminal plasma, centrifugation could be deleterious by itself, either by causing mechanical damage (Kareskoski et al., 2006), or by inducing generation of reactive oxygen species and peroxidation of the sperm plasma membrane, as suggested in human (Parinaud et al., 1997). In our conditions, centrifugation without further removal of seminal plasma did not adversely affect sperm quality. This agrees with previous
data (Jasko et al., 1991) using a similar force (500 × g), but a longer centrifugation time (18 min). We observed significant variations between stallions, but independently of the ability of the semen to withstand cold storage. This contradicts previous results (Brinsko et al., 2000). Individual variability between stallions is most likely responsible for the heterogeneity observed between results of different experiments. Our results demonstrated that removal of seminal plasma contributes to the stabilisation of sperm membranes during cooled storage: it enhances the resistance of sperm to hypo-osmotic conditions, and reduces the response to a pharmacological induction of the acrosome reaction, without a dose-dependent effect in our conditions. The influence of such a stabilisation in relation to the fertilising ability needs to be investigated. Acknowledgement We thank the French National Studs for financial support. Conflict of interest None. References Batellier, F., Vidament, M., Fauquant, J., Duchamp, G., Aranaud, G., Yvon, J.M., Magistrini, M., 2001. Advances in cooled semen technology. Anim. Reprod. Sci. 68, 181–190. Bedford, S.J., Graham, J.K., Amann, R.P., Squires, E.L., Pickett, B.W., 1995. Use of two freezing extenders to cool stallion spermatozoa to 5 ◦ C with and without seminal plasma. Theriogenology 43, 939–953. Brinsko, S.P., Crockett, E.C., Squires, E.L., 2000. Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. Theriogenology 54, 129–136. Dawson, G.R., Webb, G.W., Pruitt, J.A., Loughlin, T.M., Arns, M.J., 2000. Effect of different processing techniques on motility and acrosomal integrity of cold-stored stallion spermatozoa. J. Equine Vet. Sci. 20, 191–194. Jasko, D.J., Moran, D.M., Farlin, M.E., Squires, E.L., 1991. Effect of seminal plasma dilution or removal on spermatozoal motion characteristics of cooled stallion semen. Theriogenology 35, 1059–1067. Jasko, D.J., Hathaway, J.A., Schaltenbrand, V.L., Simper, W.D., Squires, E.L., 1992. Effect of seminal plasma and egg-yolk on motion characteristics of cooled stallion spermatozoa. Theriogenology 37, 1241–1252. Kareskoski, A.M., Reilas, T., Andersson, M., Katila, T., 2006. Motility and plasma membrane integrity of spermatozoa in fractionated stallion ejaculates after storage. Reprod. Domest. Anim. 41, 3–38. Pagl, R., Aurich, J.E., Müller-Schlösser, F., Kankofer, M., Aurich, C., 2006. Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 degrees C. Theriogenology 66, 1115–1122. Parinaud, J., LeLannou, D., Vieitenz, G., Griveau, J.F., Milhet, P., Richoilley, G., 1997. Enhancement of motility by treating spermatozoa with an antioxidant solution (Sperm-Fit) following ejaculation. Hum. Reprod. 12, 2434–2436. Thérien, I., Moreau, R., Manjunath, P., 1998. Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm. Biol. Reprod. 59, 768–776. Töpfer-Petersen, E., Ekhlasi-Hundrieser, M., Kirchhoff, C., Leeb, T., Sieme, H., 2005. The role of stallion seminal proteins in fertilisation. Anim. Reprod. Sci. 89, 159–170. Webb, G.W., Dean, M.M., Humes, R.A., Heywood, J.S., 2009. A comparison of the ability of three commercially available diluents to maintain the motility of cold stored stallion semen. J. Equine Vet. Sci. 29, 229–232.