The effects of EDTA and propylene glycol on sperm quality and levels of extender elements during cryopreservation

The fluid medium of semen that spermatozoa are suspended within it is called seminal plasma. There was the very complex and variable biochemical composition of seminal plasma among species that is made up of energy substrates (fructose, sorbitol, glycerylphosphocholine), organic compounds (citric acid, amino acids, peptides, low and high-molecular-weight proteins, lipid, hormones, cytokines), and also ions that the sperm function is highly dependent on the ionic environment (Juyena et al. 2012; Hamamah and Gatti 1998). Cations such as calcium and magnesium that belong to the same family in the periodic table, are used in osmotic equilibrium (Liang et al. 2016; Cevic et al. 2007). They have similar homeostatic regulatory systems and can potentially antagonize each other in many physiological activities and are components of many important enzymes (Liang et al. 2016; Cevic et al. 2007).
Magnesium is the second most prevalent intracellular cation and is involved in the metabolic activity of the cell as the main cofactor for kinase enzymes (Eghbali et al. 2010; Hashemi et al. 2017). Magnesium acts as an intracellular calcium antagonist so that increased magnesium levels in human seminal plasma compared with calcium improve erection and ejaculation processes (Hashemi et al. 2017). Within the cell, most of the magnesium is bound to proteins and negatively-charged molecules, 80% of cytosolic magnesium is bound to ATP, which is the substrate for numerous enzymes (Eghbali et al. 2010). Depletion of intracellular magnesium affects activities dependent on this ion, such as glycolysis, protein synthesis, respiration and reproduction (Wong et al. 2001).There is an evidence that alteration in magnesium and calcium levels of seminal plasma can affect sperm quality with decreasing fertility potential (Bassey et al. 2013).
Calcium may have a role in steroidogenesis by influencing delivery, utilization of cholesterol by mitochondria. Calcium also stimulates the conversion of pregnenolone to progesterone (Hurley and Doane 1989). Calcium plays an important role in sperm physiology including motility, capacitation and signaling pathways in the acrosomal reaction (Liang et al. 2016; Aisen et al. 1999). Since calcium influx into sperm cells is involved in the acrosome reaction, it seems that high calcium concentrations in sperm freezing media could increase the risk of premature reactions (Braud et al. 2016).
Sperm storage is a useful way to store genetic resources for some endangered species (Hurley and Doane 1989). Freezing is a branch of cryobiology that includes long-term protection and preservation of cells and tissues under very low conditions (Mortimer 1994). However, investigations have shown that freezing and thawing processes not only generate oxygen free radicals that impair post-thaw motility, viability, intracellular enzymatic activity, fertility, and sperm function (Silva et al. 2012; Ozkavukcu et al. 2008; Peris et al. 2007) but also changes the membrane permeability to some ions including calcium. Semen cryopreservation has increased levels of intracellular calcium that leads to dysfunction and cell death (Bittencourt et al. 2014; Nateq et al 2021). Binding to the zona pellucida or progesterone leads to an increase in the intracellular calcium concentration of sperm due to the opening of the calcium channel and also the releasing of calcium from intracellular stores (Keshtgar et al. 2016). This process increased the production of free radicals in the cell and leads to de-polymerization and membrane fusion, which the final result is acrosome reaction (Keshtgar et al. 2016). Ethylene diamine tetra acetate (EDTA) is the chelator of divalent metal ions such as calcium, magnesium, copper, zinc, etc. (Bourinbaiar and Lee 1996). The EDTA main function is to chelate the extracellular calcium, reducing its influx to the intracellular environment, which minimizes the deleterious effect of calcium on the sperm (Bittencourt et al. 2014).Cryoprotectants is another factor that influences sperm survival during cryopreservation. Low molecular weight cryoprotectants, such as Ethylene glycol (EG), glycerol, and, 1,2 propylene glycol (PG), may cause less damage to spermatozoa because its low molecular weight allows them to cross the plasma membrane more easily (Li et al. 2005; Büyükleblebici et al. 2014).
Previous studies indicated that EDTA and PG improved sperm quality. However, there is no report about the interaction of them on calcium and magnesium of semen in rams. The aim of this study was to investigate the effects of calcium and magnesium of semen seminal plasma, as well as the addition of EDTA and PG to the diluent during the cryopreservation on calcium and magnesium of seminal plasma, and the sperm parameters after freezing.
The present study was designed to evaluate the effect of adding ethylene diamine tetra acetate (EDTA) and propylene glycol (PG) on semen calcium and magnesium and quality traits of frozen-thawed Ghezel ram sperm. Semen samples were collected from five rams (3-4 years) during the non-reproductive season once a week with 15 replicates. Samples were diluted with Tris-based extender without additive (control) and supplemented with 1.75 mM EDTA, 2% PG, and 7% PG (instead of glycerol). Sperm quantitative characteristics were studied on 0, 20, 40, and 60 days of the storage after freeze-thawing. Results indicated a negative correlation between the amount of calcium in seminal plasma and sperm parameters (P<0.01). Also, there was a negative correlation between magnesium and sperm membrane integrity (P<0.01). Furthermore, 1.75 mM EDTA and 2% PG significantly decreased calcium and magnesium levels in freeze-thawed samples compared to the control group (P<0.05). Adding of 1.75 mM EDTA and 2% PG also significantly improved sperm quality parameters compared to the control group (P<0.05). It may be concluded that the addition of 1.75 mM EDTA and 2% PG to diluent significantly decreased the amount of calcium and magnesium of seminal plasma and improved the sperm parameters after the freeze-thawing process.