Pérez Llano B, García Casado P 1, Sala Echave R, Enciso M 2, Gosálvez J 2
Gestión Veterinaria Porcina S.L.Pol.Ind.P-29 C/Calibre 121 28400 C.Villalba, Madrid, Spain
1 Dpto. Reproducción Animal, INIA, Ctra.Coruña km.5.9, 28040 Madrid, Spain
2 Unidad de Genética, Facultad de Biología, Universidad Autónoma de Madrid, 28049 Madrid, Spain
INTRODUCTION
The evaluation of boar semen quality for breeding purposes includes several tests that cover sperm characteristics as motility, vitality, morphology, acrosomal status, functionality of the sperm plasma membrane. The sperm DNA integrity is a very important factor that must be taking into account in semen evaluation because it is related to the pregnancy outcome and the normal embryo development (1, 3, 4), but until now, this feature has not been included in the routine semen evaluation. Several techniques have been used to check sperm DNA integrity. Recently, a new technique, Sperm Chromatin dispersion (SCD) Test, has been developed for human sperm and adapted to boar sperm (SPERM-Sus-HALOMAX®).
This method gives rise to sperm nucleoids with a central core and a peripheral halo of dispersed DNA loops. DNA fragmentation in sperm nuclei can be accurately determined by assessing halo size (nuclei with low DNA fragmentation produce very small or absence of halos in the head of the spermatozoon, whereas those sperm nuclei with high levels of DNA fragmentation release their DNA loops forming large halos)(2). The aim of this work is to study several sperm quality parameters as, motility, acrosomal status, plasma membrane integrity (sHOST) and DNA fragmentation level in boar raw semen compared to the same samples diluted in ACROMAX ® (GVP, Madrid, Spain), a long term extender,using the new SCD Test SPERM-Sus-HALOMAX® (ChromaCell SL, Madrid, Spain)
MATERIALS AND METHODS
Semen was collected once from seven adult boars aged 18-24 months.Semen quality was assessed for percentage of sperm motility, percentage of normal acrosomes (Pursel and Johnson 1974), percentage of abnormal sperm, percentage of cells positive to sHOS test (Pérez-Llano et al.1998) and percentage of DNA fragmentation. All these tests were performed in undiluted and in diluted semen samples the same day of semen collection (day 1).
From every ejaculate two samples were taken: one sample of 20 ml was kept in a plastic tube without extender and other sample of 2 ml was extended in 18 ml of ACROMAX® (GVP, SL, Madrid, Spain). After an equilibration period of 3 hours at room temperature, both samples of each ejaculate were preserved at 15ºC during 21 days. Along the preservation period, samples were taken at days 2, 4, 5, 6, 8, 9, 11, 16 and 22 to check the same parameters of semen quality stated above.
To determine DFI in boar sperm cells, the Sperm-Sus-Halomax® kit (ChromaCell SL, Madrid, Spain) were used (Figure 1a,b). The method is fundamentally based on inclusion of sperm cells in an agarose microgel spreaded on the slide, lysis and staining for light microscopy. The percentage of sperm cells that develops a big halo of chromatin dispersion around the head is calculated by counting 200 cells in an optic microscope (X400).To assure that under all experimental circumstances the production of a big halo of chromatin dispersion was correlated with presence of fragmented DNA, in situ nick translation (ISNT) was performed at day 4, 11 and 22.
Figure 1a. Boar sperm cells processed with the Sperm Sus Halomax® kit and stained for bright field microscopy (a) or in situ nick translated (b). In both cases, sperm cells with large halos of chromatin dispersion corresponds with sperm heads containing fragmented DNA while those showing absence of halos or minimal chromatin dispersion contains unfragmented DNA.
Figure 1b. Detail of boar selected sperm heads containing fragmented and unfragmented DNA.