Natural, but not lyophilized, low density lypoproteins were an acceptable alternative to egg yolk for cryopreservation of ram semen

Abstract

The objective was to evaluate the suitability of using natural or lyophilized low density lipoproteins (LDL), in lieu of whole egg yolk, in extenders for cryopreserving ram semen. Once extragonadal sperm reserves were depleted in 10 fertile Santa Inês cross rams, two ejaculates per ram were collected for cryopreservation. Nine extenders were used: Tris-16% egg yolk extender with 5% glycerol as a control (T1), and substitution of whole egg yolk with 8, 12, 16 or 20% natural LDL (T2-T5, respectively), or with 8, 12, 16, or 20% lyophilized LDL (T6-T9). Semen was diluted to 100 × 10(6) sperm/mL, packaged into 0.25 mL straws, cooled, held at 5 °C for 3 h, and then frozen in liquid nitrogen vapor. Immediately after thawing (37 °C for 30 s), sperm total and progressive motility, and kinetic parameters were analyzed with computer assisted semen analysis (CASA). Percentage of sperm with plasma membrane functional integrity was assessed by the hypoosmotic swelling test (HOST), sperm membrane physical integrity with propidium iodide (PI), and acrosome integrity with FITC-PSA using an epifluorescent microscope. For all sperm end points, there was no difference between the control and natural LDL treatments (P > 0.05): total motility (T1: 20.9 ± 11.9 and average of T2-T5: 25.9 ± 13.6%; mean ± SD), progressive motility (T1: 6.6 ± 4.2 and average of T2-T5: 11.7 ± 7.5%), HOST(+) (T1: 23.7 ± 6.9 and average of T2-T5: 23.2 ± 8.7 %) and PI(-)/PSA(-) (T1: 13.8 ± 7.8 and average of T2-T5: 18.1 ± 7.8%). However, lyophilization was apparently unable to preserve the protective function of LDL; every sperm end point was significantly worse than in the control and natural LDL groups. We concluded that natural LDL was appropriate for cryopreserving ram semen, as it yielded results similar to those obtained with whole egg yolk.

Low density lipoproteins added to an extender frozen or lyophilized are evenly efficient in cryoprotecting ovine sperm cells than when 16% whole egg yolk was added

“…In the present study, we demonstrated that total egg yolk used in freezing extenders can be replaced by LDL at very low concentrations, such as 2% (v/v), during ovine spermatozoa cryopreservation. This concentration is smaller than those reported in previous studies (ALVAREZ et al, 2008;TONIETO, 2008;HU et al, 2010;MOUSTACAS et al, 2011). Furthermore, Silva et al (2014) reported that LDL concentrations below 4% are not ideal for preserving ovine sperm during freezing.…”

“…However, extenders containing LDL in both aqueous and lyophilized form at concentrations of 2% to 8% provided a similar or superior cryoprotective capacity as that by extenders containing 16% egg yolk. Moustacas et al (2011) demonstrated that LDL at concentrations of 8-20% had the same cryoprotective capacity as the whole egg yolk from ovine sperm, indicating that the use of concentrations above 8% is not required. In the present study, we demonstrated that total egg yolk used in freezing extenders can be replaced by LDL at very low concentrations, such as 2% (v/v), during ovine spermatozoa cryopreservation.…”

“…Therefore, many researchers have aimed to improve sperm cryopreservation. Recently, several studies have shown that replacing the egg yolk in extenders with low-density lipoproteins (LDL) enhanced motility, membrane integrity, and the fertilization ability of frozen sperm from various species compared to the use of commercial extenders containing total egg yolk (MOUSSA et al, 2002;AMIRAT et al, 2004;JIANG et al, 2007;BENCHARIF et al, 2008;VARELA JÚNIOR et al, 2009;MOUSTACAS et al, 2011;VERA-MUNOZ et al, 2009;HU et al, 2010;SILVA et al, 2014;HENEINE;.…”

“…The addition of LDL to ovine sperm freezing extenders at concentrations ranging from 5 to 20% has been tested, and a concentration of 8% had the same cryoprotective capacity as higher concentrations (ALVAREZ et al, 2008, TONIETO, 2008MOUSTACAS et al, 2011). Additionally, it has been shown that when LDL were lyophilized and, then, resuspended to compose the diluting extender, this resulted on a substantial reduction in their cryoprotective capacity (MOUSTACAS et al, 2011).…”

“…Additionally, it has been shown that when LDL were lyophilized and, then, resuspended to compose the diluting extender, this resulted on a substantial reduction in their cryoprotective capacity (MOUSTACAS et al, 2011).…”