Abstract
The aim of this study was to evaluate the use of low concentrations of natural and lyophilized low density lipoprotein (LDL) from hen's egg yolk for cryopreservation of canine semen. Different ammonium sulphate concentrations were tested to extract LDL from egg yolk. The yolk was centrifuged, and LDL was isolated using 10, 20, 40, 45, or 50% ammonium sulphate solution (ASS). The LDL-rich floating fraction was collected for chemical characterization. Dry matter content was lowest (P<0.05) in the LDL extracted with the 50% ASS. The purification of LDL increased in association with increasing ammonium sulphate concentrations. SDS-PAGE showed that the 50% ASS solution yielded a purer fraction of LDL from egg yolk. For semen cryopreservation, TRIS extender was used replacing 20% egg yolk (control) by natural or lyophilized LDL using 1, 2, and 3% (w/v). Semen was centrifuged (755Xg for 7 min), diluted with one of the extenders, packed into 0.5mL straws (100x106 sperm/mL), and placed in a programmable cryopreservation machine. Thawed semen (37°C/ 30s) was analyzed for sperm motility, morphology, and by the hypoosmotic and epifluorescence tests (CFDA/ PI). Natural LDL extracted with 50% ASS was as effective as whole egg yolk to preserve canine frozen sperm when using low concentrations. The lyophilized LDL, mainly in the two higher concentrations tested (2 and 3%), was unsuitable to maintain the effectiveness of the LDL cryoprotective effect on dog sperm.
Highlights
The cryoprotective effect of egg yolk on cooled and frozen sperm from domestic animals has been primarily attributed to low density lipoproteins (LDL; Pace and Graham, 1974)
Dry matter content was lowest (P
LDL isolated with the 10% saturated solution had the lowest value for fresh lipid content and highest value for protein content (P
Summary
The cryoprotective effect of egg yolk on cooled and frozen sperm from domestic animals has been primarily attributed to low density lipoproteins (LDL; Pace and Graham, 1974). Attempts have been made to use natural LDL in lieu of egg yolk in semen extenders (Foulkes, 1977), with promising results (Moussa et al, 2002; Hu et al, 2010). The extraction of LDL from egg yolks facilitates the use of specific amounts of defined substances, thereby excluding undesirable components, eg.: high density lipoprotein (Demianowicz and Strzezek, 1996), which are deleterious to semen cryopreservation. Moussa et al (2002) proposed an easy, quick, and efficient method to extract LDL, based on the use of 40% of ammonium sulphate. Some factors might alter LDL purity and yield, such as temperature, quantity of ammonium sulphate used and the way it is added (Harris, 1992)
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