Effect of cholesterol‐loaded cyclodextrin treatment of bovine sperm on capacitation timing

Abstract Pre‐loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not been evaluated. The objective of this study was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC‐PNA), intracellular calcium levels (Propidium Iodide/FLUO 3‐AM) and membrane fluidity (Merocyanine 540/YO‐PRO‐1). Cholesterol‐loaded cyclodextrin (CLC) (2 mg/mL) improved post‐thaw viability to 61% from 45% in control sperm (p < .05). The addition of ionomycin (0.05 mM) induced capacitation in sperm by 1 h, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 h. Treatment with CLC significantly decreased membrane fluidity in sperm (p < .05). In conclusion, CLC‐treated sperm required 1 h more to capacitate when compared with non‐treated sperm based on percentage of live cells with high membrane disorder (p < .05). Increased cryosurvival and viability over time was observed, but longer time to capacitate may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization.

important determining factor of membrane stability and fluidity at low temperature. Cholesterol maintains phospholipids in a random, lamellar arrangement as temperature decreases (Amann & Pickett, 1987) and modulates fluidity of membranes by interacting with the fatty acyl chains of phospholipids (Watson, 1981). In model membranes, increasing the cholesterol: phospholipid ratio broadens the phase transition and reduces membrane leakage and membrane phase separations (Drobnis et al., 1993). Therefore, treatment of sperm with cholesterol prior to cryopreservation could reduce sensitivity of sperm membranes to cooling damage, thus reducing or eliminating the lateral phase separation of lipids (Watson, 1981).
Cyclodextrins can be used to insert or remove cholesterol from cell membranes. Cyclodextrins are cyclic oligosaccharides obtained by the enzymatic degradation of starch, and possess an external hydrophilic face and an internal hydrophobic core (Dodziuk, 2006) that can encapsulate hydrophobic compounds, such as cholesterol (Mocé et al., 2010). These molecules have a high affinity for sterols in vitro, are very efficient in stimulating efflux of cholesterol from the membrane of sperm (e.g. Companyó et al., 2007;Visconti et al., 1999) and, if pre-loaded with cholesterol, can insert cholesterol into cell membranes (Navratil et al., 2003). The addition of methyl residues to cyclodextrins enhance their solubility in water and ability to dissolve hydrophobic compounds (Yancey et al., 1996). Therefore, methylβcyclodextrin (MβCD) is most commonly used to pre-load sperm with cholesterol, creating a CLC (Mocé et al., 2010). Treating sperm with CLC prior to cryopreservation increases sperm cryosurvival rates (Mocé & Graham, 2006;Purdy & Graham, 2004a), although even with better survival no evidence has emerged that indicates this result is associated with increased fertility rates in vitro. This lack of evidence may suggest compromised sperm fertilization ability and/ or the need to alter the timing of in vitro fertilization (IVF) with CLCtreated sperm.
The objective of this study was to investigate the role of CLC on in vitro timing of capacitation in frozen-thawed bull sperm.

| Preparation of cyclodextrin-loaded cholesterol
In this study, cyclodextrins were used to modify sperm membranes.
The procedures of Purdy and Graham (2004a) were followed. Briefly, 1 g MβCD was dissolved in 2 mL methanol, and 200 mg cholesterol dissolved in 1 mL chloroform. The solution was mixed until clear and the solvents evaporated. The resulting powder was stored at 23°C until use, and hereafter referred to as CLC. A working solution of CLC was prepared by adding 50 mg of cholesterol-loaded cyclodextrin to 1 mL of Tyrode's albumin lactate pyruvate (TALP) (Graham et al., 1986) following the procedures of Purdy and Graham (2004a), and mixed thoroughly to create a CLC concentration of 50 mg/mL (345 mOsm).

| Semen collection and cryopreservation
One ejaculate from 10 bulls was collected by artificial vagina method (Alexander, 2014). All ejaculates had a minimum concentration of 1 x 10 9 /mL, >70% normal morphology, and greater than 60% motile sperm. Ejaculates were transported to the lab immediately and then, prior to cryopreservation, each ejaculate was split into two aliquots.
CLC treatment and cryopreservation of sperm were conducted as described by Mocé and Graham (2006). One aliquot served as the control, and the other was treated with 2 mg CLC/120x10 6 sperm.
Both aliquots were incubated for 15 min at 22°C, then diluted with 20% egg yolk-Tris diluent to a concentration of 100 x 10 6 sperm/ mL and cooled to 5°C over 2 h. Once cooled, aliquots were diluted 1:1 (vol:vol) with 14% glycerolated egg yolk-Tris diluent (final glycerol concentration 7%), loaded into 0.5 mL straws, frozen in nitrogen vapor 4 cm above the surface of liquid nitrogen for 15 min, and plunged into liquid nitrogen for storage at −196°C until thawing.

| Flow cytometric evaluation
Cryopreserved semen from each of the 10 ejaculates was used to evaluate the effect of cyclodextrin concentration and the effect of ionomycin treatment on capacitation timing between control and CLC-treated sperm over 180 min. For both groups (control and CLCtreated sperm), straws were thawed in a 37°C water bath for 30 s, and motile sperm were isolated by washing thawed semen through a 45/90 Percoll® gradient (Parrish et al., 1995) at 800 g for 20 min.
Supernatant was removed and sperm washed again in 2 mL TALP by centrifugation for 5 min at 300g. Washed sperm samples were pooled and diluted with TALP medium to 15 mL total, then separated into 2.5 mL borosilicate glass tubes suitable for analysis by flow cytometry with 2-5 x 10 6 sperm/mL.
Within one sub-sample group, 0.5 mL of washed, diluted sample was treated with MβCD at concentrations of 0, 1 or 2 mg/mL to test the effect of MβCD on capacitation timing by cholesterol efflux.
Within a second sub-sample group, 2 μl of 0.05 mM ionomycin was added to 0.5 mL of washed, diluted sample to act as a capacitating agent by increasing intracellular calcium levels.

| Statistical analysis
All statistical analyses were performed with SAS software (version 9.4; SAS Institute Inc., Cary, NC, USA). Separate analyses were performed for the percent live sperm, live acrosome-reacted sperm, live sperm with high intracellular calcium levels, and live sperm with high membrane fluidity. Values for these parameters are presented as the mean ± SEM (Figures 1-4). Percentage data were transformed (arcsin) prior to analysis. Initially, MβCD levels (0, 1and 2 mg/mL) were analysed by ANOVA, at each time point within each treatment (control, control+ionomycin, CLC and CLC + ionomycin) to determine if MβCD administration affected sperm parameters. No differences (p > .05) were determined in any sperm parameter for the different MβCD levels within the four sperm treatments at any specific time point. Therefore, data for the three MβCD treatments were pooled in the final analyses. A Repeated Measurements Analysis of Variance was used to detect differences temporally within each treatment.
Within each time point, treatment differences were determined using ANOVA and means were separated using Student-Newman-Keuls (SNK).

| RE SULTS
From the 10 ejaculates used in this split ejaculate study, there are four treatment groups (control, control + ionomycin, CLC and CLC + ionomycin) which were evaluated for acrosomal integrity, intracellular calcium level and membrane disorder by flow cytometry over time up to 180 min. The treatment of CLC prior to cryopreservation was the main effect, with ionomycin as a secondary treatment being tested. Varied MβCD concentration levels of 0, 1 and 2 mg/mL revealed no difference at any time points (Table 1) Ionomycin-treated control and CLC sperm were both different at TA B L E 1 Average percentage of live cells observed between time 0 and 180 min across treatments concentrations in mg/mL. No significant differences were found (p > .05)

| DISCUSS ION
Cryopreservation of bovine sperm negatively effects viability, induces synthesis of reactive oxygen species, and causes DNA damage (Gürler et al., 2016). In this and previous studies, treatment of bovine sperm with CLC prior to freezing supports increased post-thaw viability both initially after thaw and over time (Mocé & Graham, 2006;Purdy & Graham, 2004a). Our results displayed significantly higher percentage of live post-thaw sperm cells treated with CLC prior to freezing compared with control sperm at time zero (p < .01). These improved cryosurvival rates suggest that CLC-treated sperm can more effectively withstand the damaging conditions of cryopreservation, and yield otherwise unlikely survivability. Recent studies have indicated increased post thaw viability when CLC is added prior to cryopreservation of bovine sperm packaged for low dose insemination, which is a potential application for the use of CLC to optimize the preservation of valuable genetic donors (Lone et al., 2021).
In previous studies, MβCD improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)-binding ability (Águila et al., 2015), and increased rates of activation and fertilization (Kato et al., 2011) in bovine sperm in vitro. However, our results indicated no significant difference (p > .05) at 0, 1 and 2 mg/mL at all time points. This difference may indicate that the concentration of MβCD used in our present study was not high enough to induce cholesterol efflux in CLC-treated sperm, as in previous studies there was a decrease observed in a dose-dependent manner compared to the control sperm (Águila et al., 2015). High concentrations of MβCD were not used due to indication of DNA integrity being compromised at 5 mM (Águila et al., 2015), however, treatment at these levels may be necessary to see an effect on CLC-treated sperm. CLC-treated sperm are estimated to have an increased cholesterol: phospholipid ratio from 0.45 to 0.9 or higher (Purdy & Graham, 2004a), therefore, to see an effect of MβCD on these sperm, treatment with up to twice the concentration used in the current study may be required. Further studies assessing MβCD effect at concentrations greater than 2 mg/ mL would be required to evaluate cholesterol efflux in CLC-treated sperm, while also assessing potential damage to DNA integrity.
When assessing percent of live acrosome-reacted sperm, the group treated with CLC was associated with the lowest percent of live cells reacted at all time points, supporting the hypothesis that CLC-treated sperm require more time to react acrosomally, and therefore capacitate more slowly compared with control sperm.
Previous studies evaluated this reaction over 30 min (Purdy & Graham, 2004b), however evaluation over a longer 3 h timeframe allowed for a more in depth assessment. This hypothesis is also supported when assessing percent of live cells exhibiting membrane disorder. Our results reinforce the inference that adding cholesterol to frozen-thawed sperm cells cultured in the above conditions induces membranes to become more rigid (Purdy et al., 2005), an observation based on the finding of increased membrane stability of CLC-treated sperm. This was true for sperm cells both treated with ionomycin and non-treated CLC sperm, with significant difference seen between most time points (p < .05).
Ionomycin, a potent and selective calcium ionophore, has been used as a research tool to stimulate hyperactivation in bovine sperm.
A similar calcium ionophore A23187 has been used to induce capacitation of bovine sperm (Purdy & Graham, 2004b), although A23187 acts by incorporating into the sperm plasma membrane and transporting calcium stores across the plasma membrane (Talbot et al., 1976), whereas Ionomycin acts by inducing the release of cytosolic calcium stores (Morgan & Jacob, 1994). Our results support the work of Purdy and Graham (2004b) with CLC-treated sperm and control sperm exhibiting similar percentages of cells with high intracellular calcium. Figure 3

| CON CLUS ION
In conclusion, treatment of bovine sperm pre-loaded with cholesterol prior to freezing via CLC (2 mg/mL) improved cryosurvival and post-thaw viability, initially and over time by significantly decreasing membrane fluidity in sperm. However, CLC-treated sperm exhibit delayed capacitation by 1 h based on assessment of viability and acrosomal integrity, intracellular calcium levels, and membrane disorder. MβCD treatment (1 mg/mL and 2 mg/mL) did not impact capacitation, though ionomycin (0.05 mM) induced capacitation by 1 h, resulting in increased intracellular calcium and acrosome reaction.
Further investigation of retained cholesterol post-thaw is required, as longer time to capacitate may hinder fertilization capacity and/or require adjustments to IVF timing.

AUTH O R CO NTR I B UTI O N S
GL and BC conceived and designed the study. GL performed the flow cytometric assay, statistical analyses and drafted the manuscript. All authors edited, revised and accepted the article.

ACK N OWLED G EM ENTS
The authors thank Laura Starbuck, Austin LaVelle, Maria Alexandra Marquez Lema and staff of the Barfield laboratory at Colorado State University for their assistance in material collection and processing.
We also thank Rocky Mountain Sire Services, Bennett, CO for assistance with bull handling and semen collection, Select Sires, Inc., Plain City, OH for providing the semen and Dr. Ann Hess for her assistance with statistical analyses. This work was supported by CryoGam Colorado, LLC.

FU N D I N G I N FO R M ATI O N
This work was privately funded by CryoGam Colorado, LLC.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y
The data that support the findings of this study are available from the corresponding author upon reasonable request.