Effects of Filtration of Semen Doses from Subfertile Boars through Neuter Sephadex Columns E Bussalleu 1 , E Pinart 1 , MM Rivera 2 , X Arias 2 , M Briz 1 , S Sancho 1 , N Garcı´a-Gil 1 , J Bassols 1 , A Pruneda 1 , M Yeste 1 , I Casas 1 , T Rigau 2 , JE Rodriguez-Gil 2 and S Bonet 1 1 Biotecnologia de la Reproduccio ´ Porcina, Departament de Biologia, Facultat de Cie `ncies, Universitat de Girona, Girona; 2 Dept. Medicina i Cirurgia Animals, Unitat de Reproduccio ´ Animal, Facultat de Veterina `ria, Universitat Auto `noma de Barcelona, Bellaterra, Spain Contents This study was designed to develop a method of improving the quality of sperm obtained from subfertile Pie´train boars. Seminal doses were filtered through neuter Sephadex TM columns (G-25 Medium, G-50 Fine, G-50 Medium and G- 75, length 10 ± 0.5 cm, flow rate 1 ml/20 s). Doses were prepared by pooling 10 ml semen samples collected from 58 asthenoteratospermic boars and diluted the sperm-cell rich fraction 1 : 6 in Betsville thawing solution extender. Sperm quality was determined before and after the filtering process. Sperm morphology and motility were assessed using the computer program SCAÒ2002 production, and sperm vitality was evaluated by fluorescence multistaining. ORT and HRT tests were used to determine the osmotic resistance of spermatozoa, and metabolic performance was assessed by measuring L-lactate production. Results indicate that the filtration process rendered increased proportions of mature spermatozoa and of viable spermatozoa with an intact acrosome, nucleus and mitochondrial sheath. Sperm filtra- tion led to decreased percentages of spermatozoa with proximal and distal droplets and of agglutinated spermato- zoa, along with slightly diminished ORT values. HRT scores and L-lactate production were unaffected. Our findings indicate that filtering through a Sephadex column improves the sperm morphology and vitality of seminal doses obtained from subfertile boars, but produces no functional changes in the spermatozoa. All four column types yielded similar results. Introduction Seminal doses obtained from boars of reproductive and genetic interest do not always show the optimal quality required for their use in artificial insemination (AI) programmes. Throughout their reproductive life, male pigs usually experience transient decreases in fertility, which are usually related to husbandry or stress factors. Furthermore, at advanced ages, the sperm quality of reproductive males progressively and irreversibly de- clines, despite their continued genetic interest. In effect, sperm anomalies in the ejaculates of reproductive males account for considerable economic losses at farms and AI centres. Further, the presence of dead or aberrant spermatozoa in ejaculates can have toxic (Shannon and Curson 1972) and lytic (Lindemann et al. 1982) effects on adjacent spermatozoa. The use of techniques designed to improve the sperm quality of these males can help resolve these problems. In several species of productive interest, different meth- ods of filtration have been used to improve the sperm quality of seminal doses obtained from reproductive males, including columns comprised of glass wool or glass beads, and Leucoscorb TM or Sephadex TM col- umns. Filtration serves to increase the number of viable and motile spermatozoa, although effects vary consid- erably depending on the nature of the matrix and the species (Johnson et al. 1996; Adiga and Kumar 2001). The principle of filtration is based on the fact that non- viable spermatozoa tend more to adhere to a matrix than motile and seemingly functional spermatozoa (Jeyendran 2002). In addition, viable spermatozoa are able to cross the filtration barrier without modifying their functional characteristics (Adiga and Kumar 2001). In fertile and subfertile bulls (Anzar and Graham 1995; Januskauskas et al. 2005), buffalos (Ahmad et al. 2003), horses (Samper et al. 1995; Alghamdi et al. 2002) and dogs (Mogas et al. 1998), semen filtration through glass wool and Sephadex columns is current practice. The objective of this study was to determine the effect of filtration through columns of neuter Sephadex on the sperm quality of seminal doses obtained from subfertile Pie´ train boars. Material and Methods Semen collection Semen samples were obtained from 58 post-pubertal Pie´train boars with asthenoteratonecrospermia by the gloved-hand method with males mounted on a dummy sow. The sperm-rich fraction was diluted 1:6 in Betsville thawing solution (BTS) (Cidosa, Tecnovit, Spain) after removing the gelatinous fraction by filtering through gauze. This diluted cell-rich fraction was aliquoted into 80 ml seminal doses, which were refri- gerated at 15°C until Sephadex filtration. Sephadex columns and packaging Prepared seminal doses were filtered through four different columns of neuter Sephadex: G-25 Medium, G-50 Fine, G-50 Medium and G-75 (Amersham Biosciences, Uppsala, Sweden). These four types of Sephadex differ according to the diameter of beads comprising the gel once hydrated. According to data provided by the manufacturer, bead diameters are: 85–260 lm for Sephadex G-25, 40–160 lm for Sephadex G-50 Fine, 100–300 lm for Sephadex G-50 Medium and 90–280 lm for Sephadex G-75. Optimal pHs are 2–13 for Sephadex G-25 Medium and, 2–10 for Sephadex G-50 Fine, G-50 Medium and G-75. Reprod Dom Anim 43, 48–52 (2008); doi: 10.1111/j.1439-0531.2007.00853.x ISSN 0936-6768 Ó 2007 The Authors. Journal compilation Ó 2007 Blackwell Verlag