Epsilon Archive for Student Projects

Abstract

Shahiduzzaman, A.K.M, 2007. Induced immotility during long-term storage at +5°C does not prolong survival of dog spermatozoa. Master’s thesis. ISSN 1403-
2201. Report number 64 Preservation of canine semen by extension and chilling has provided the AI
veterinarians with an opportunity to overcome limitations of both time and space. Several parameters are assessed to predict the fertility of canine spermatozoa for a
given sample. Motility is one of them. Spermatozoa stay immotile while preserved in CLONE (Cryogenetic Laboratories of New England, Inc, USA) Chilled Semen Extender whereas they maintain motility over the whole 23 days period of study while preserved in a Tris egg yolk glucose extender at 5°C. The aim of this study was to investigate whether the immotility induced by the CLONE chilled semen extender prolongs the lifespan of dog spermatozoa stored at
5°C, compared with a Tris-egg yolk-glucose (TG) extender, which maintains motility. Semen from eleven dogs was pooled, split in four aliquots, centrifuged, and the supernatants removed. The four sperm pellets were mixed with a TG extender; with the CLONE chilled semen (CL) extender; with TG extender mixed with TG but without egg yolk and with less glucose (TG+ATG); or with the CLONE
extender mixed with the CLONE activator (CL+ACL) to a final sperm concentration of 200 x 106 spermatozoa / mL. The samples were stored at 5ºC for 23 days and examined twelve times for sperm motility by computer-assisted sperm
analysis (CASA), as well as for plasma membrane and acrosome integrity, glucose consumption, and DNA fragmentation index (DFI). The experiment was performed
in triplicate. The third replicate was examined for presence of bacteria. For the statistical calculations, the 23 experimental days were divided into three periods,
the first period comprising days 1–8, the second period days 9–14, and the third period days 15–23. Total motility (TM%) did not differ between extenders in the first period, but was higher in TG and TG+ATG compared with CL+ACL in the
second and third periods, and with CL in the third period (P<0.05). Progressive motility (PM%) was higher in TG (P<0.0001), TG+ATG (P=0.0007) and CL+ACL
(P=0.0176) than in CL in the first period. Both TG and TG+ATG maintained higher PM% than did CL and CL+ACL in the second and third period. Tris-egg yolkglucose
and TG+ATG maintained higher straight line velocity (VSL) and average path velocity (VAP) compared with CL and CL+ACL during the whole experimental period. Curvilinear velocity (VCL) was higher in TG, CL, and TG+ATG than in CL+ACL samples (P<0.05) in the first and third periods. No
differences in lateral head displacement (LHD) were observed between extenders or over time. Acrosome and plasma membrane integrity decreased over time, but
there were no differences among the extenders in the first and second periods (P>0.05). In the third period, however, TG, TG+ATG, and CL maintained acrosome and plasma membrane integrity better than CL+ACL. Glucose consumption was not
significantly different between extenders until the third period, when it was higher in CL and CL+ACL than in TG (P=0.0055) and TG+ATG (P=0.0010). No breakdown of DNA chromatin (P>0.05) occurred until day 14. A lower DFI was
observed in TG+ATG than in TG and CL in the third period. Microorganisms found were mainly a sparse growth of Escherichia coli (days 1, 8, and 14) and a mixed
flora (days 1, 8, 14, and 23), but also included yeast (days 14 and 23) and Pseudomonas fluorescens (day 23). In conclusion, spermatozoa preserved in TG or TG+ATG showed better values for all the different parameters throughout the experiment compared with sperm subjected to CL or CL+ACL. Consequently, the immotility induced by the CLONE chilled semen extender during long-term cold
storage at 5°C did not prolong the lifespan of spermatozoa compared with the lifespan following storage in Tris-egg yolk-glucose. In addition, our results indicate that good quality dog semen may possibly be stored for up to 14 days in TG extender at 5°C, with retained fertilizing capacity. In vivo studies should,however, be performed to further support this conclusion.