Effect of storage conditions on the LDL effectiveness in ovine sperm cryopreservation

Abstract

The present study evaluated the effect of storage conditions on the LDL efficacy for cryopreserving ovine sperm. In this way, we compared egg yolk extender with three different forms for LDL storing, LDL diluted in Tris-glucose extender and stored in frozen (i) and freeze-dried (ii) states and LDL stored pure and added into the extender prior to use (iii). We also tested the effect of two storage temperatures (-20 and -80 °C) and three storage times (30, 60, 120 d). Frozen and freeze-dried extenders containing LDL, as well as LDL stored pure, improved post-thaw sperm quality. Storage temperatures did not influence negatively the cryoprotectiveness of LDL extenders. Furthermore, lyophilised LDL extenders stored at -20 °C were more effective in preserving sperm longevity than the other extenders stored at -20 °C. Finally, LDL extenders stored for 30 and 120 d were more efficient than 60 d in preserving ram sperm freezability.

Can we use LDL instead of egg yolk in BotuCrio® extender to cryopreserve sperm from the Mangalarga Marchador stallion?

“…The cryoprotective capacity of Merk-egg yolk extender containing acetamide, methyl cellulose and trehalose on the viability of the equine sperms had been described previously (Henry et al, 2002;Snoeck et al, 2007;. However, this cryoprotectant association had not yet been studied in INRA 82 extender.…”

“…The BotuCrio® extender containing methylformamide and glycerol was superior to preserve equine semen during cryopreservation than the extenders Merk-egg yolk and INRA 82 modified with the addition of acetamide, methyl cellulose and trehalose in substitution of glycerol. Other researchers have also reported this superiority of BotuCrio® on equine semen frozen with extenders containing glycerol as its main cryoprotectant agent (Terraciano et al, 2008;Candeias et al, 2012;Costa et al, 2014;Ramires Neto et al, 2014), especially to freeze semen from "bad freezer" stallions (Alvarenga et al, 2003;Squires et al, 2004;Ramires Neto et al, 2014) as were considered the majority of Mangalarga Marchador stallions breed (Alvarenga et al, 2003).…”

“…Despite the results obtained previously, it can be noticed that the use of the acetamide, methyl cellulose and trehalose in Merk-egg yolk extender and INRA 82 did not reach the cryoprotective capacity desired when compared to the BotuCrio®. It can be inferred that the composition of the BotuCrio® extender is superior to the composition of the Merk and INRA 82, given the combination of intra and extracellular cryoprotectant substances that can play an important role in sperms preservation at cooling and freezing process (Snoeck et al, 2007;Hoffman et al, 2011;Ramires Neto et al, 2014). Or even that the association between methylformamide and glycerol offers a better cryoprotection to the equine sperm as it has already been described by Rossi et al (2003) and Melo et al (2007).…”

“…The LDL was extracted from egg yolk according to the protocol described by Moussa et al (2002) and modified by Neves et al (2014). The LDL was added to the extender after its extraction and a short period of frozen maintenance according to the protocol described by Snoeck et al (2017).…”

“…Identifying alternative cryoprotectants to the glycerol to cryopreserve equine sperm has become an area of extensive investigation. Extenders based on egg yolk as the Lactose-EDTA (Merk-egg yolk) (Martin et al, 1979;Henry et al, 2002) and from milk and egg yolk as the INRA 82 (Magistrini et al, 1992;Vidament et al, 2002;Pillet et al, 2008;Candeias et al, 2012;Álvarez et al, 2014) containing glycerol or after replacing this by other cryoprotectors based on amides were tested to preserve sperm viability and/or the fertility during cryopreservation, in an attempt to prevent the cytotoxic effect of glycerol (Alvarenga et al, 2000;Henry et al, 2002;Gomes et al, 2002;Medeiros et al, 2002;Papa et al, 2002;Squires et al, 2004;Snoeck et al, 2007;Gibb et al, 2013). The amides have lower viscosity and low molecular weight which favors a greater permeability of this compound through the plasmatic membrane, causing less osmotic damages (Alvarenga et al, 2005;Melo et al, 2007) and were effective to preserve the sperms from stallions considered bad freezers Ramires Neto et al, 2014).…”