Cryoprotection effectiveness of low concentrations of natural and lyophilized LDL (low density lipoproteins) on canine spermatozoa

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 (100x10 6 sperm/mL), and placed in a programmable cryopreservation machine. Thawed semen (37ºC/ 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.

Antioxidant effects of supplementation of 3,4‐dihydroxyphenyl glycol on sperm parameters and oxidative markers following cryopreservation in canine semen

“…In agreement with the present study, other studies showed that adding antioxidants could improve viability percentage of dog semen during the freezing process. Neves et al., (2014) demonstrated that low concentrations of low‐density lipoprotein (LDL) could be used to support the canine sperm viability after the freezing‐thawing process. Strzeżek et al., (2009) reported that the Glutathione reductase (GSH) concentration in canine semen was closer to 5 mM, which was recommended for supplementation of semen extender/freezing solution.…”

A comparison of liquid and lyophilized egg yolk plasma to low density lipoproteins for freezing of canine spermatozoa

“…To our knowledge, our study is the first time that EYP was lyophilized, and there are no previous reports about the use of lyophilized EYP for the cryopreservation of canine sperm. Interestingly, native LDL have been lyophilized but failed to preserve post‐thaw motility and integrity (plasma membrane and acrosome) of ram (Moustacas et al, ) and dog (Neves et al, ) spermatozoa. In view of our results, the lyophilization of EYP does not seem to interfere with cryoprotective properties of plasma LDL.…”

“…The simplest and fastest procedure was described by Moussa et al () and achieved 97% purity and gave hope for industrial applications. However, all previously reported analytical procedures failed to be adapted to an industrial scale, and LDL could be neither successfully sterilized (unpublished data) nor lyophilized (Moustacas et al, ; Neves, Heneine, & Henry, ).…”

Assisted reproductive techniques for canines: preservation of genetic material in domestic dogs

“…Consequently, studies on alternative extenders that can replace egg yolk are of great importance. As shown in Table 1 , several materials have been examined as candidates for cryoprotectants that can replace egg yolk [ 11 , 12 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]. Among them, only low-density lipoprotein (LDL) [ 25 , 27 ] and skim milk [ 12 , 35 , 36 ] have been assessed as alternative components of extenders, for their benefits in improving fertility by means of artificial insemination.…”