ORIGINAL ARTICLE Ultrastructure of sperm ‘tail stump’ defect in wild boar M. L. Fischman1, A. Bolondi2 & H. Cisale1 1 Laboratorio de Calidad Seminal y Criopreservación de Gametas, Área Fı́sica Biológica, INITRA, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Argentina; 2 Unidad de Microscopı́a Electrónica y Bioquı́mica Aplicada, Instituto Nacional de Tecnologı́a Agropecuaria (INTA-Castelar), Argentina Keywords Semen—sperm tail abnormality—tail stump sperm—wild boar Correspondence Maria Laura Fischman, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Area Fı́sica Biológica, Chorroarı́n 280, C1427CWO Ciudad de Buenos Aires, Argentina. Tel.: +54 11 4524 8432; Fax: +54 11 4524 8480; E-mail: fischman@fvet.uba.ar Accepted: August 28, 2008 Summary Sperm ‘tail stump’ defect was found in ejaculates of a wild boar maintained in captivity. It was in good physical condition, the testes and genital tract were found to be of normal size and consistency. There was no evidence of macroscopic abnormalities at the clinical analysis and at necropsy. The volume and concentration of the semen samples obtained by electroejaculation were lower than normal. The slides examined contained a high level of abnormal spermatozoa (52.7%). The most frequent morphological finding was a droplet-like form attached to the base of the head or a very short stump. The non-stumped spermatozoa had no normal tail but a shortened one. Analysing the histological structure with light microscopy, no ring of spermatozoa was observed lining the lumen of the seminiferous tubules and the characteristically cellular structure was not conserved. The ultrastructural examination evidenced a disorganisation of the normal tubular structure of the flagellum, with lost of regular pattern of the axial bundle of fibrils and the mitochondrial helix. The origin of this abnormality is unknown. Introduction Sperm abnormalities have been long associated with male infertility and sterility in most species. These abnormalities vary from morphological defects that are evident upon routine semen analysis, to those which are more difficult to detect. The causes of defective sperm structure may be environmental, genetic or a combination of both. Between them, it is possible to find different tail disorders such as coiled tails, tail stump defect or primary ciliary dyskinesia (Chenoweth, 2005). By definition, stump tail spermatozoa have very short flagella, are completely or nearly completely immotile, and have severe aberrations of the fibrous sheath. It has been suggested that this defect has a genetic origin, probably involving a centriolar alteration (Baccetti et al., 1993). The tail stump defect was first reported in bulls in 1925 (Williams & Savage, 1925). It has also been encountered in the mouse, rabbit, dog, stallion and man (Barth & Oko, 1989). Coubrough & Barker (1964) described three sterile Canadian bulls (Ayrshire, Shorthorn and Holstein) that showed a small stump or stub replacing the mid-piece and tail in the majority of ejaculated ª 2009 The Authors Journal Compilation ª 2009 Blackwell Publishing Ltd Æ Andrologia 41, 35–38 sperm. In addition, sperm concentration was very low and sperm motility was virtually absent. Monitoring of sperm morphology in these bulls indicated that the percentage of sperm with the defect increased with their age. It has been suggested that the prevalence of more than 25% of tail stumped spermatozoa in the ejaculate is indicative of a genetic fertility problem (Barth & Oko, 1989). Other reports had linked this defect with sterility in bulls (Coubrough & Barker, 1964; Blom, 1976; Williams, 1987). In some cases, a genetic cause had been demonstrated (Coubrough & Barker, 1964; Blom, 1976; Vierula et al., 1987; Foote et al., 1992). Even so, there were other reports in which the origin remained unknown (Arriola et al., 1985; Williams, 1987; Revell et al., 2003). In boars, several sperm tail defects have been described in ejaculates obtained from sterile individuals. There were coiled spermatozoa (Holt, 1982) or abaxial implantation of the middle piece in spermatozoa and spermatids (Thilander et al., 1985). In all cases, over 95% of the spermatozoa were affected. A hereditary short-tail sperm defect was described in Finnish Yorkshire boars (Andersson et al., 2000; Sironen et al., 2001). To our knowledge, 35 Wild boar sperm tail defect there are no reports of tail stump defect in boars and wild boars. In this study, we report a tail stump defect in a young wild boar. The morphology of the spermatozoa and the histology of the testicles are described. Materials and methods Semen characterisation and sperm morphology The wild boar was 2 years old when examined for the first time, it was in good physical condition and had no history of illnesses and offspring during its previous use in field. Semen sample were obtained by electroejaculation as previously described (Fischman et al., 2003). Semen was collected once a week, during 8 weeks and standard semen evaluation was performed each time. Subjective sperm motility was estimated in fresh semen samples using a phase-contrast light microscope (400·). The volume of the ejaculate was determined directly on a graduated vase and the sperm concentration was measured by the haematocytometric method, with a Neubauer chamber (Hafez, 1989). In order to evaluate viability, semen smears were stained with eosin-nigrosin and 200 spermatozoa were evaluated with phase-contrast light microscope (1000·) (Pintado et al., 2000). To visualise morphology, rose bengal staining was performed on semen smears and at least 200 spermatozoa were evaluated (1000·). Testicular histology Wild boar necropsy was performed. The testicular tissue samples were fixed in Bouin’s solution, transferred to 10% formaldehyde buffer, dehydrated in alcohol and embedded in paraffin. Sections (5 lm) were cut, stained with haematoxylin-eosin and examined with light microscopy (1000·). M. L. Fischman et al. at necropsy. The volume (mean ± SEM) of semen samples was 7 ± 1.5 ml and the mean concentration was 250 ± 150 million sperm ml)1. No motile spermatozoa were observed in any ejaculates. The slides examined contained a high level of abnormal spermatozoa. The most frequent morphological finding was a droplet-like form attached to the base of the head or a very short stump (Fig. 1). Non stumped spermatozoa had no normal tail but a shortened one, and represented 47.3% of the whole samples. Spermatozoa with dropletlike morphology were more prevalent (32.4%) than shortstump cells (20.3%). The heads were generally of normal size and shape. At necropsy, epididymal spermatozoa were analysed. Values obtained did not differ from ejaculated spermatozoa (data not presented). Analysing the histological structure with light microscopy, we found a little quantity of visible sperm tails in the lumen of each seminiferous tubule. No ring of spermatozoa was observed lining the lumen of the tubules and the characteristically cellular structure was not conserved (Fig. 2). Ultrastructural examination evidenced a disorganisation of the normal tubular structure of the flagellum, with loss of regular pattern of the axial bundle of fibrils and the mitochondrial helix. The acrosomes showed a completed structure, and heads showed dense and homogeneous chromatin (Fig. 3). Discussion The male studied had not only abnormal sperm morphology, but also the volume and concentration of (a) (b) Sperm transmission electron microscopy (TEM) Semen samples were fixed in glutaraldehyde 2% in Millonig buffer (pH 7.2) and post-fixed in osmium tetroxide 2%. The samples were dehydrated in increasing concentrations of ethanol solutions and embedded in Epon. The ultra thin sections were stained with the uranyl-Reynold’s double stain technique and examined using a TEM (Jeol 1200EX II; Joel Ltd, Tokyo, Japan). Results Upon palpation, the testes and genital tract were found to be of normal size and consistency. There was no evidence of macroscopic abnormalities at the clinical analysis and Fig. 1 Wild boar sperm showing (a) short-stump tail and (b) a droplet-like appendage instead of a tail. Eosin-nigrosin staining. Original magnification 1000·. ª 2009 The Authors 36 Journal Compilation ª 2009 Blackwell Publishing Ltd Æ Andrologia 41, 35–38 M. L. Fischman et al. Wild boar sperm tail defect Fig. 2 Testicular histological slides showing the absence of sperm tails in the seminiferous tubules. Haematoxylin-eosin staining. Original magnification 200·. images of the testicular histological slides, which showed the absence of sperm tails in the seminiferous tubules, suggest a low rate of spermatogenesis. This finding is consistent with the low sperm concentration observed. In relation to the sperm ultrastructure, the centriolar microtubules seemed to be not functioning like the organisation points for the assemblage of the double microtubules. These are the starting point of the axonema in a normal spermatozoon. Ultrastructural changes showed a severe alteration in the cellular surface around the postnuclear region, where the microtubular protein should be polymerised. In spite of that, in some transversal sections it was possible to detect the typical structure 9 + 2, in others the flagellum lost its structure and showed a globular-like pattern immediately after the annulus, where a dense ring indicated a fusion between different structures. We do not know the origin of this case reported. Comparison of the DNA of this wild boar with that obtained from Finnish Yorkshire boars reported by Andersson et al. (2000) showed that the pathology is not the same (M. Andersson, personal communication). Ejaculates from a direct brother were analysed. Samples were of nonsuperlative quality but droplet-like tails or tail stump defect appeared only sporadically. Nowadays, artificial insemination of domestic sows with wild boar semen is frequent practice by breeders looking for wild flavoured meat but obtaining more prolificacy. The finding of this kind of pathologies shows the importance of a sperm control before the incorporation of these types of animals to a farm. Acknowledgements This paper was supported by funds from UBACyT, Universidad de Buenos Aires, Argentina, grant no. V028. The authors thank the Pathology Laboratory staff of the Facultad de Ciencias Veterinarias for performing the necropsy and testicles slides. References Fig. 3 Transmission electron microscopy of wild boar sperm. Longitudinal section of droplet-stump appendage. Original magnification 10 000·. the ejaculates obtained were lower (volume: 7 ± 1.5 ml, concentration: 250 ± 150 million sperm ml)1) than in normal wild boars (volume: 15.5 ± 8.8 ml, concentration: 830 ± 420 million sperm)1) (Fischman et al., 2003). 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