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Evidence for the presence of ATP-binding cassette transporter (ABC transporter) A1 in stallion spermatozoa
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
36.0 10.06b) and membrane integrity (32.5 9.98; 42.0 10.61 and 36.0 7.44) were assessed for BS, Fert-talp and Sperm-Talp, respectively. Incubating epididymal spermatozoa with Sperm and Fert-Talp increased sperm parameters, as observed in previous studies [1]. As these media did not have a negative influence on fertility, these procedures can be safely used to freeze equine epididymal sperm. The results of the present experiment reinforce that the incubation of equine epididymal semen with either Sperm-Talp or Fert-Talp medium improves sperm parameters without reducing fertility. Thus, these media can be used with either ejaculated or epididymal sperm as an alternative for stallions with history of poor semen freezability.
497
epifluorescence microscope. Immunocytochemistry resulted in a distinct expression pattern in stallion spermatozoa. Strong immunofluorescence was detected in the acrosomal area, i.e. the cranial two thirds of the sperm heads, with enhanced staining in the equatorial zone. In contrast, no immunoreactivity could be observed in the caudal third of the sperm heads. Staining was also detectable in the capitulum. In the flagellum, the principle piece also revealed strong immunostaining, but only weak to no signal was observed in the middle piece (Fig. 1). No difference in the expression pattern of ABCA1 was observed between individual animals or between different ejaculates of one stallion. The present results provide clear evidence for the presence of ABCA1 in different domains of the stallion sperm plasma membrane.
Reference [1] Papa FP, et al. Anim Reprod Sci 2008;107:293-301.
Reference [1] Quazi F, Molday RS. Lipid transport by mammalian ABC proteins. Essays Biochem 2011;50:265-90.
Evidence for the presence of ATP-binding cassette transporter (ABC transporter) A1 in stallion spermatozoa M. Merkl 1, S. Schäfer-Somi 1, I. Walter 2, M. Helmreich 2, M. Glösmann 3, and C. Aurich 1 1 Centre for Artificial Insemination and Embryo Transfer, Vienna, Austria, 2 Institute for Anatomy, Histology and Embryology, Vienna, Austria, 3 VetCore - Facility for Research, University of Veterinary Science, Vienna, Austria Little is known on the regulation of cholesterol efflux from the sperm plasma membrane in stallion spermatozoa. The ATP-binding cassette transporter A1 (ABCA1) represents an interesting candidate since it supports the release of cholesterol in mouse spermatozoa and other cell types (QUAZI & MOLDAY, 2011). In the present study, stallion spermatozoa were assessed for i) the expression of ABCA1 transporter and ii) differences in its expression pattern between individual stallions or ejaculates. Semen was collected from six pony stallions aged between 4 and 19 years. In order to provide largely standardized ejaculates, all stallions underwent a preparation procedure prior to sampling, which included daily semen collection for five days. Thereafter, semen was collected from each stallion on three consecutive days. Ejaculates were evaluated immediately after collection. For immunocytochemistry, samples were taken from every ejaculate and washed with 0.9% NaCl-solution. After washing, spermatozoa were smeared on microscope slides and fixed in methanol. For immunostaining, blocking buffer containing bovine serum albumin, and goat serum were used to prevent non-specific binding reactions. Slides were incubated at 4 C with polyclonal anti-ABCA1 antibodies (ABCA1, NB400-105, rabbit, 1:100, Novus Biologicals). For negative controls, primary antibodies were incubated with ABCA1 peptide prior to this step. Samples were subsequently labelled with secondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG, 1:200, Invitrogen), counterstained with 40 ,6-diamidino-2phenylindol (DAPI, 5ml/50mlPBS), and mounted in Mowiol. Immunofluorescence was evaluated using an
Efficiency of ground semen collection in the stallion G. Meroni 1, 2, H. Sieme 3, and D. Burger 2 1 School of Agricultural, Forest and Food Sciences, Zollikofen, Switzerland, 2 Swiss Institute of Equine Medicine, ALP-Haras and University of Berne, Avenches, Switzerland, 3 Clinic for Horses, Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Germany The collection of semen on the ground from the standing stallion is an alternative method to dummy mount collection of semen and this method is of increasing importance. Ground collection is particularly popular for stallions that suffer from health problems, or in studs that do not have a dummy or a suitable mare at their disposal. General opinion appears to be that stallions place less stress on their hindquarters if the procedure is performed while standing rather than when mounted. This has made standing semen collection popular for stallions that are actively engaged in equestrian sports. The aim of this study was to collect data on libido and semen parameters associated with ground semen collection. The experiment was carried out on twelve Franches-Montagnes stallions with covering experience between 3 and 15 years of age. After an acceptance and subsequent training period of both semen collection techniques, they were tested in a cross over experimental protocol evaluating semen parameters collected with an artificial vagina during 2 periods of 5 successive days on a dummy and while standing, respectively. Collection of semen was always carried out in the same room, in the presence of an oestrogen-primed ovarectomized mare. Ground vs. dummy mount semen collection was accompanied by a longer time to ejaculation (P < 0.05), higher seminal volume (P < 0.05) and lower total sperm count (P < 0.05). Our results indicate that ground semen collection is a viable option for stallions that cannot mount a phantom or a mare. However, stallion owners should consider that ground semen collection is associated
36.0 10.06b) and membrane integrity (32.5 9.98; 42.0 10.61 and 36.0 7.44) were assessed for BS, Fert-talp and Sperm-Talp, respectively. Incubating epididymal spermatozoa with Sperm and Fert-Talp increased sperm parameters, as observed in previous studies [1]. As these media did not have a negative influence on fertility, these procedures can be safely used to freeze equine epididymal sperm. The results of the present experiment reinforce that the incubation of equine epididymal semen with either Sperm-Talp or Fert-Talp medium improves sperm parameters without reducing fertility. Thus, these media can be used with either ejaculated or epididymal sperm as an alternative for stallions with history of poor semen freezability.
497
epifluorescence microscope. Immunocytochemistry resulted in a distinct expression pattern in stallion spermatozoa. Strong immunofluorescence was detected in the acrosomal area, i.e. the cranial two thirds of the sperm heads, with enhanced staining in the equatorial zone. In contrast, no immunoreactivity could be observed in the caudal third of the sperm heads. Staining was also detectable in the capitulum. In the flagellum, the principle piece also revealed strong immunostaining, but only weak to no signal was observed in the middle piece (Fig. 1). No difference in the expression pattern of ABCA1 was observed between individual animals or between different ejaculates of one stallion. The present results provide clear evidence for the presence of ABCA1 in different domains of the stallion sperm plasma membrane.
Reference [1] Papa FP, et al. Anim Reprod Sci 2008;107:293-301.
Reference [1] Quazi F, Molday RS. Lipid transport by mammalian ABC proteins. Essays Biochem 2011;50:265-90.
Evidence for the presence of ATP-binding cassette transporter (ABC transporter) A1 in stallion spermatozoa M. Merkl 1, S. Schäfer-Somi 1, I. Walter 2, M. Helmreich 2, M. Glösmann 3, and C. Aurich 1 1 Centre for Artificial Insemination and Embryo Transfer, Vienna, Austria, 2 Institute for Anatomy, Histology and Embryology, Vienna, Austria, 3 VetCore - Facility for Research, University of Veterinary Science, Vienna, Austria Little is known on the regulation of cholesterol efflux from the sperm plasma membrane in stallion spermatozoa. The ATP-binding cassette transporter A1 (ABCA1) represents an interesting candidate since it supports the release of cholesterol in mouse spermatozoa and other cell types (QUAZI & MOLDAY, 2011). In the present study, stallion spermatozoa were assessed for i) the expression of ABCA1 transporter and ii) differences in its expression pattern between individual stallions or ejaculates. Semen was collected from six pony stallions aged between 4 and 19 years. In order to provide largely standardized ejaculates, all stallions underwent a preparation procedure prior to sampling, which included daily semen collection for five days. Thereafter, semen was collected from each stallion on three consecutive days. Ejaculates were evaluated immediately after collection. For immunocytochemistry, samples were taken from every ejaculate and washed with 0.9% NaCl-solution. After washing, spermatozoa were smeared on microscope slides and fixed in methanol. For immunostaining, blocking buffer containing bovine serum albumin, and goat serum were used to prevent non-specific binding reactions. Slides were incubated at 4 C with polyclonal anti-ABCA1 antibodies (ABCA1, NB400-105, rabbit, 1:100, Novus Biologicals). For negative controls, primary antibodies were incubated with ABCA1 peptide prior to this step. Samples were subsequently labelled with secondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG, 1:200, Invitrogen), counterstained with 40 ,6-diamidino-2phenylindol (DAPI, 5ml/50mlPBS), and mounted in Mowiol. Immunofluorescence was evaluated using an
Efficiency of ground semen collection in the stallion G. Meroni 1, 2, H. Sieme 3, and D. Burger 2 1 School of Agricultural, Forest and Food Sciences, Zollikofen, Switzerland, 2 Swiss Institute of Equine Medicine, ALP-Haras and University of Berne, Avenches, Switzerland, 3 Clinic for Horses, Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Germany The collection of semen on the ground from the standing stallion is an alternative method to dummy mount collection of semen and this method is of increasing importance. Ground collection is particularly popular for stallions that suffer from health problems, or in studs that do not have a dummy or a suitable mare at their disposal. General opinion appears to be that stallions place less stress on their hindquarters if the procedure is performed while standing rather than when mounted. This has made standing semen collection popular for stallions that are actively engaged in equestrian sports. The aim of this study was to collect data on libido and semen parameters associated with ground semen collection. The experiment was carried out on twelve Franches-Montagnes stallions with covering experience between 3 and 15 years of age. After an acceptance and subsequent training period of both semen collection techniques, they were tested in a cross over experimental protocol evaluating semen parameters collected with an artificial vagina during 2 periods of 5 successive days on a dummy and while standing, respectively. Collection of semen was always carried out in the same room, in the presence of an oestrogen-primed ovarectomized mare. Ground vs. dummy mount semen collection was accompanied by a longer time to ejaculation (P < 0.05), higher seminal volume (P < 0.05) and lower total sperm count (P < 0.05). Our results indicate that ground semen collection is a viable option for stallions that cannot mount a phantom or a mare. However, stallion owners should consider that ground semen collection is associated