Cholesterol-loaded Cyclodextrin Research Articles

Cholesterol-loaded cyclodextrin improves motility and survival of cryopreserved zebrafish (Danio rerio) sperm

We studied the impact of modulating cholesterol levels in zebrafish sperm plasma membranes using cholesterol-loaded methyl-β-cyclodextrin (CLC) and unloaded methyl-β-cyclodextrin (MβC). Zebrafish sperm were treated with these substances before cryopreservation, and post-thaw sperm motility and in vitro fertilization (IVF) rates were compared between treated and untreated samples. Our findings indicate that adding cholesterol to sperm membranes increases post-thaw motility, motile cell count, and motile cell survival within a 0.5–4.0 mg per 1.2 × 108 cell concentration range. Conversely, depleting cholesterol using MβC at 1.0 and 2.0 mg per 1.2 × 108 cells reduced these parameters. On average, all CLC-treated sperm samples produced a 15 % higher IVF rate compared to untreated sperm. Including CLC in the extender before cryopreservation is beneficial for post-thaw sperm quantity and quality in zebrafish.

Effect of cholesterol-loaded cyclodextrin treatment on boar sperm cryopreservation.

This study investigated the efficacy of different concentrations of cholesterolloaded cyclodextrin (CLC) on cryopreservation in boar sperm quality. In this study, we treated boar sperm with different concentrations of CLC before freezing and analyzed the sperm cholesterol concentration, plasma membrane, acrosome integrity rate and total motility rate before and after freeze-thawing. We also investigated the levels of reactive oxygen species (ROS), malondialdehyde (MDA), adenosine triphosphate (ATP), and structural- and oxidative-damage related proteins in all groups after thawing. The results revealed that the cholesterol concentration of the CLC-treated groups was higher than that of the control group, both before freezing and after thawing (p<0.05). The plasma membrane integrity rate, acrosome integrity rate, and total motility rate of sperm were also enhanced after thawing in the CLC-treated group (all p<0.05). Moreover, ROS and MDA production and ATP loss were reduced in CLC-treated sperm during freezing and thawing (p<0.05). Finally, CLC pretreatment partially prevented the consumption of various proteins involved in metabolism including capping actin protein of muscle Z-line subunit beta (CAPZB), heat shock protein 90 alpha family class A member 1 (HSP90AA1) and phosphoglycerate mutase 2 (PGAM2) (p<0.05). The CLC treatment increased cholesterol concentration and decreased structural injury and oxidative damage during boar sperm freezing and thawing, improving the efficacy of sperm cryopreservation in boar.

Sperm characteristics of cryopreserved Prochilodus lineatus semen after adding cholesterol-loaded cyclodextrin

The experiment evaluated the effect of adding cholesterol-loaded cyclodextrin (CLC) to Prochilodus lineatus fish (Curimata) semen on post-thaw sperm quality. Twelve adult fish were used for sperm collection after induced spermiation with carp pituitary gland. The semen was diluted and treated with CLC in concentrations of 0 (control), 0.5, 1.0, 2.0, 3.0, and 4.0 mg for 120 × 106 spermatozoa/ml, loaded in 0.5 ml straws, packaged and placed in dry vapor vessel cylinders for 24 h before being submerged in liquid nitrogen for storage. The samples were thawed in a water bath at 60 °C for 8 s, and the sperm parameters evaluated were motility, activation duration, longevity, plasma membrane integrity, and morphology. Data were tested for normal distribution and ANOVA, followed by Friedman test (P < 0.05). Spermatozoa treated with CLC displayed higher motility than the control (P < 0.05). The duration of sperm activation was longer in sperm treated with 0.5, 1.0, and 2.0 mg of CLC than in control (P < 0.05). The membrane integrity was higher in sperm treated with 0.5, 1.0, 2.0, and 3.0 mg of CLC than in control and four mg-treated samples (P < 0.05). The sperm longevity and morphology alterations did not differ between treatments (P > 0.05). Adding 0.5, 1.0, or 2.0 mg of CLC in Prochilodus lineatus semen before cryopreservation improves sperm motility and membrane integrity.

Addition of low concentration of cholesterol-loaded cyclodextrin (CLC) has a positive effect on cryopreserved canine spermatozoa evaluated by andrological and biophysical methods

BackgroundThis study was conducted to find the best concentration of cholesterol-loaded cyclodextrin (CLC) which has a positive impact on canine post thaw semen quality. Three different concentrations of CLC (0.83 mg/ml; 1.66 mg/ml; 3.32 mg/ml) and 2-hydroxylpropyl-beta-cyclodextrin (HBCD) (1.66 mg/ml) were used in addition to cryopreservation extender and compared with the control after thawing. Samples were assessed using computer-assisted semen analyzer (CASA), flow cytometry, fluorimeter by measuring the fluorescence anisotropy (ANISO) and determining the generalized membrane polarization (GP).ResultsAn addition of 0.83 mg/ml CLC significantly increased the percentage of progressive motile (PROG) and rapid spermatozoa (RAP) (P < 0.05). 1.66 mg/ml HBCD decreased progressive motility of spermatozoa and population with rapid movement relative to the control (P < 0.05). Furthermore, the groups with an addition of 1.66 mg/ml and 3.32 mg/ml of CLC, as well as the group with only cyclodextrin, increased percentage of dead spermatozoa without lipid peroxidation and decreased percentage of viable spermatozoa without LPO which was lower in these groups than in the control (P < 0.05). Other sperm parameters assessed on flow cytometer were not significantly different. The addition of CLC at 0.83 mg/ml and 3.32 mg/ml concentrations and 1.66 mg/ml of HBCD caused an increase in ANISO measured at 23 ºC (P < 0.05).ConclusionsIn conclusion, the results suggest that increasing cholesterol in the plasma membrane of canine spermatozoa can improve their freezability. However, only low concentrations of CLC may improve semen quality after thawing without adversely affecting other parameters.

Protective Efficacy of Cholesterol-Loaded Cyclodextrin (CLC) Against Sperm Morphological Abnormalities in Marwari Stallions

The present study aimed to examine the protective potential of cholesterol loaded cyclodextrin (CLC) at varying concentrations against sperm morphological abnormalities of cooled and frozen thawed semen of Marwari stallions. A total of forty eight ejaculates (six stallions and eight ejaculates per stallion) were promptly processed following semen collection and subjected to gross assessment of semen quality attributes. The assessment of sperm morphologies in fresh equine semen was conducted using Eosin and Nigrosine staining technique. The semen samples from individual Marwari stallions were evenly divided into five fractions and incubated in a water bath at 37°C for 15 min after dilution with primary extender to obtain the spermatozoa concentration at 120 × 106 sperm/ml. During incubation, the fractions of every semen sample were exposed to different concentrations of CLC as 0, 1.0, 1.5, 2.0 and 3.0 mg/ml of extender (C, T1, T2,T3, and T4, respectively). All the 5 aliquots were cooled and subsequently subjected to cryopreservation. Each cooled and frozen- thawed (at 37°C for 30 sec) semen samples were analyzed to assess the morphologies of the spermatozoa. Based on the findings, it was concluded that none of the CLC treatment could render any significant protection against the sperm morphological abnormalities at either pre-freeze and/or post-thaw stage of semen samples from Marwari stallions.

Soybean lecithin and cholesterol-loaded cyclodextrin in combination to enhances the cryosurvival of dairy goat semen

The objective of the study was to examine the effect of soy lecithin (SL) and cholesterol loaded cryclodestrin (CLC) on cryo-survival of sperm cryopreserved in the presence or absence of seminal plasma in Saanen dairy goats. Tris-based dilutions containing various concentrations of SL (0, 0.5%, 1.0% or 2.0%) and CLC (0, 2.0 g/L, 4.0 g/L or 6.0 g/L CLC) were used to cryopreserve Saanen dairy goat sperm. The quality of frozen-thawed sperm, including progressive motility, viability, acrosome and plasma membrane integrity, as well as fertility were detected. Results found that the optimal combination of the two cryoprotectants was 1.0% SL+4.0 g/L CLC, which significantly increased progressive motility, viability, acrosome and plasma membrane integrity of frozen thawed sperm. The impact of the two cryoprotectants in combination was not affected by the presence of seminal plasma. The conception rates obtained after artificial insemination using sperm cryopreserved with and without seminal plasma were 88.89% and 91.67% (P > 0.05), respectively. The respective values for average number of litter sizes were 1.55 ± 0.17 and 1.56 ± 0.21 (P > 0.05). Therefore, this study improved the cryopreservation efficiency of goat semen, enhanced the sperm cryosurvival, and layed a foundation for the wide application of frozen goat semen.

Post-thaw addition of pentoxifylline to equine semen previously cryopreserved with different concentrations of cholesterol-loaded cyclodextrin

Cholesterol-loaded cyclodextrin (CLC) grants resistance to sperm during cryopreservation but may compromise sperm capacitation and fertility (Zahn et al. Theriogenology, 2002;58(2):237:240). Stimulants of sperm kinetics like pentoxifyllin (PTX) may increase sperm energy production and stimulate metabolic pathways responsible for sperm capacitation (Guasti et al. Anim.Reprod.Sci. 2017;179:27-34). The present study aimed to stimulate semen motility by adding PTX after thawing of semen previously cryopreserved with different concentrations of CLC. Mangalarga Marchador stallions (n=12; 4-14 years) were assigned to four groups: (G1) untreated control, (G2) inclusion of cholesterol-loaded cyclodextrin (CLC) at concentrations of 1mg, (G3) 1.5mg and (G4) 2mg. Two straws of each stallion per group were thawed and divided into two aliquots, pentoxifylline (2 mg/mL, 7.18 mM) homogenized in BotuCrio®, Botupharma, Brazil (PTX: G1, G2, G3 e G4) was added to one and not to other aliquot (No-PTX: G1, G2, G3 and G4). Sperm kinetics was analyzed by CASA (Sperm Class Analyzer, SCA® v.4.0 Microptic®) immediately after thawing (T0) or at 40 minutes (T40), 80 minutes (T80) and 120 minutes (T120) after thawing after incubation at 37°C. For statistical analysis, ANOVA F-test was performed.Intracellular hydrogen peroxide production was estimated by flow cytometry with dichlorodihydrofluorescein diacetate. Fertility was tested by deep horn insemination 0 to 6h after ovulation: No-PTX G1, No-PTX G3 and PTX G3. PTX reduced sperm velocity parameters at all times (p<0.05) compared to the groups without addition of pentoxifylline (No-PTX G1, G2, G3 and G4). Egg yolk based semen extenders may interfere with PTX cell penetration as they reduce permeability of the cell membrane. Regarding the integrity of the sperm plasma membrane and production of hydrogen peroxide, the use of pentoxifylline conferred an increase in sperm possibly under oxidative stress in the control group, with injured plasma membrane and with intracellular hydrogen peroxide (G1 PTX). When PTX was added to the group with highest concentration of cholesterol (G4 PTX), the intactness of the plasma membrane was improved, but with intracellular hydrogen peroxide. Mitochondrial potential and plasma membrane destabilization were also evaluated (n.s.). Pregnancy rates were 50% (2/4) for No-PTX G1, 67% (2/3) for No-PTX G3 and 67% (2/3) for PTX G3. In conclusion, addition of PTX did not interfere with mitochondrial potential, membrane destabilization and conception rate but was detrimental to cell longevity and to sperm kinetics.

Effect of cholesterol inclusion on kinetics, mitochondrial potential and membrane stability of equine spermatozoa after thawing

Cholesterol loss occurs in equine semen during cryopreservation and induces early capacitation and reduced viability in the female reproductive tract (Blanch et al., Reproduction in Domestic Animals. 2017;47(6):959-64). In order to increase cell resistance and longevity, the addition of cholesterol has shown satisfactory results. However, attention to the amount of cholesterol added is important as high concentrations may inhibit sperm capacitation (Contreras et al, Theriogenology. 2022;189:1-10). The object of the present study was to test the effect of cholesterol inclusion on equine semen characteristics, thus, addition of cholesterol-loaded cyclodextrin (CLC) was performed as described by Purdy and Graham (Cryobiology. 2004;48:36-45). Twelve healthy Mangalarga Marchador stallions, aged 4 to 14 years were assigned into four groups for the same ejaculate in a cross-over experimental design: without cholesterol (G1; control) or inclusion of CLC at concentrations of 1mg/120 × 106 sperm per mL (G2), 1.5mg/120 × 106 sperm per mL (G3) and 2mg/120 × 106 sperm per mL (G4).. After centrifugation, the pellet was resuspended in a freezing egg yolk-based extender (Botu-CrioTM, Botupharma, Brazil) at a final concentration of 100 × 106 sperm/mL. Semen samples were packed into 0.5 mL straws. Sperm kinetics were performed using CASA (Sperm Class Analyzer, SCA® v.4.0 Microptic®) and longevity wasevaluated by thermoresistance test at 37°C. For statistical analysis, the ANOVA F-test was performed. Evaluations were carried out at four times: T0 (immediately after thawing); T40 (after 40 minutes); T80 (after 80 minutes); and T120 (after 120 minutes). At T0, mitochondrial potential (probe JC-1, Sigma-Aldrich®) and plasma membrane destabilization (probe M540, Sigma-Aldrich®) were evaluated by flow cytometry (BD FACS Verse TM®) and Tukey test was performed for statistical analysis. At T0, higher total motility (TM) and progressive motility (PM) parameters were observed in G3 than in G1 (p<0.05). Progressive linear velocity (VSL) was higher in G3 when compared to G2 and in G4 (p<0.05). However, no difference was observed between G1 and G3. Mitochondrial potential and plasma membrane stability were similar in all experimental groups at T0 (p>0.05). After incubation at 37°C, at T40, T80 and T120, MT, MP and RAP parameters did not differ among groups. However, parameters related to cellular velocity (VCL, VSL and VAP) were higher in G3 than in G2 at T40 (VCL), T80 (VCL, VSL and VAP) and T120 (VCL and VAP). G4 showed superior VCL and VSL values at T80 than G2 (p<0.05). Therefore, 1.5mg and 2.0mg of CLC inclusion resulted in higher sperm kinetics, in addition it did not interfere with mitochondrial potential and the stability of plasma membrane and may therefore be a good option for cryopreservation of equine semen.

Effect of FertTalp addition to stallion semen on kinetic parameters and production of intracellular hydrogen peroxide after thawing

Motility stimulants increase sperm energy production and their addition after semen thawing will increase motility (Stephens et al., J. Equine Vet. Sci. 2013;33:615-621), and stimulate metabolic pathways responsible for sperm capacitation (Guasti et al., Anim. Reprod. Sci. 2017;179:27-34). Semen of 12 Mangalarga Marchador stallions was cryopreserved with addition of different concentrations of cholesterol-loaded cyclodextrin (CLC): G1 (control – without cholesterol), G2 (1mg of cholesterol), G3 (1.5mg of cholesterol) and G4 (2mg of cholesterol). To run the experiment, two straws from each stallion per experimental group were thawed and divided into two aliquots of 400µL. This was supplemented with 100µL of Fert Talp, a Tyrode's albumin lactate pyruvate medium with (FT) or without (No-FT) heparin, a motility stimulant. This subdivided the four groups into eight. Sperm kinetics were performed analyzed by CASA (Sperm Class Analyzer, SCA® v.4.0 Microptic®). Sperm longevity was analyzed by using a thermoresistance test at 37°C (TTR). Statistical analysis was done by ANOVA F-test. Complementary to sperm kinetics, the production of intracellular hydrogen peroxide was estimated by flow cytometry using Dichlorodihydrofluorescein diacetate (DCFDA®). Results were classified as: Q1 [Plasma membrane (PM) injured without intracellular hydrogen peroxide]; Q2 (PM injured with intracellularhydrogen peroxide); Q3 (intact MP with intracellular hydrogen peroxide); Q4 (intact PM without intracellular hydrogen peroxide) and Tukey test was performed for statistical analysis. Immediately after thawing, there was no difference between FT and No-FT (p>0.05). After incubation at 37°C, the addition of FT showed higher values for RAP (percentage of rapid sperm, %), VAP (average path velocity, μm/s) and LIN (linearity of the curvilinear trajectory, %) after 80 minutes of incubation and also superior rates of RAP, VSL (straight line velocity, μm/s), VAP and LIN after 120 minutes of incubation (p<0.05), demonstrating satisfactory results both with regard to sperm kinetics and longevity. It can be concluded that the use of FT was beneficial for sperm velocity. These findings suggest that the addition of this motility stimulant after thawing may improve fertility because sperm velocity – VCL (curvilinear velocity, μm/s), VSL and VAP are the most reliable parameters to assess sperm quality (Verstegen et al. Theriogenology, 2002;57:149-179). Significant differences were also observed for Q4 (intact PM, without hydrogen peroxide), where the G2 FT group demonstrated superiority when compared to all groups not supplemented with FT (p>0.05), providing satisfactory plasmatic membrane results against oxidative stress.

Does Better Post-Thaw Motility of Dog Sperm Frozen with CLC Mean Better Zona Pellucida Binding Ability?

Even though the search for methods improving cryopreservation of canine spermatozoa led to an improvement of post-thaw quality, fertilizing results after insemination with frozen-thawed semen are still not satisfying. In this study, we focused on modification of spermatozoa membrane fluidity and investigated whether kinematic parameters as assessed by computer-assisted semen analyzer (CASA) can be improved. The primary aim of our study was to investigate whether the use of cholesterol-loaded cyclodextrins (CLC; 0.5 mg, 1 mg, 2 mg) and 2-Hydroxypropyl-ß-cyclodextrin (HBCD; 1 mg) positively influence capacitation status as examined by tyrosinphosphorylation, cholesterol efflux and zona binding assay (ZBA) of spermatozoa. The use of 0.5 mg of CLC increased the percentage of motile, progressive and rapid spermatozoa compared to the control. Addition of HBCD decreased motility and progressive motility of spermatozoa and the population with rapid movement in comparison to the control. The percentage of live spermatozoa without efflux of cholesterol compared to the control was increased when extender with 0.5 mg of CLC was used. There was no change in capacitation status. The zona binding ability of spermatozoa was significantly lower in the group with 0.5 mg of CLC than in the control. In conclusion, these results suggest that improvement of kinematic parameters does not necessarily coincide with better zona pellucida binding ability of spermatozoa.

Optimizing Bull Semen Cryopreservation Media Using Multivariate Statistics Approaches

Cryo-injury reduces post-thaw semen quality. Extender components play a protective role, but existing experimental approaches do not elucidate interactions among extender components, semen samples, and post-thaw quality. To identify optimal concentrations for 12 extender ingredients, we ran 122 experiments with an I-optimal completely random design using a large dataset from our previous study. We obtained a maximum predicted total motility of 70.56% from an I-optimal design and 73.75% from a Monte Carlo simulation. Individual bull variations were significant and interacted with extenders independently. 67% of bulls reliably preferred extender formulations to reach maximum motility. Multifactor analysis suggests that some antioxidants may offer superior protection over others. Partial least squares path modeling (PLS-PM) found the highest positive loadings for glutathione in the antioxidant class, glycerol in the CPA class, and fructose in the basic compounds class. The optimal ranges for milk, water, and ethylene glycol were extremely narrow. Egg yolk, cholesterol-loaded cyclodextrin, and nerve growth factor had medium-loading impacts. PLS-PM showed that CPA, osmoregulators, and basic components were the most efficient contributors to motility, while the antioxidant and extracellular protectant classes had less efficiency. Thus, ingredients, concentrations, and interactions of extender compounds are critical to extender formulation, especially when using multiple compounds with the same function.

Effect of cholesterol-loaded cyclodextrin treatment of bovine sperm on capacitation timing.

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) (2mg/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.

Effect of cholestanol and cholesterol-loaded cyclodextrin on stallion sperm function and capacitation post-cryopreservation

Cryopreservation of stallion semen is less efficient than other species such as bovine. This is mainly because of the greater susceptibility of stallion sperm to the freezing damage that generates oxidative stress and plasma membrane injury, resulting in DNA fragmentation and cell death.These data suggest the need to develop new strategies of sperm cryopreservation that can improve the efficiency of this technique in stallions by reducing or preventing membrane damage and cell death. The present study aimed to evaluate the effect of adding membrane stabilizers to the freezing medium and assess the quality and in vitro capacitation of stallion sperm after thawing. Semen samples from three stallions frozen with membrane stabilizers (cholesterol-loaded cyclodextrin and cholestanol-loaded cyclodextrin) were evaluated in two experiments: i) sperm quality and functional analysis after thawing, and ii) sperm quality and functional analysis after 4 h of post-thaw incubation in capacitating conditions. Plasma membrane integrity, mitochondrial membrane potential, membrane lipid disorder, intracellular Ca2+, tyrosine phosphorylation, acrosome reaction, DNA damage, sperm motility, and binding to the zona pellucida were assessed. The results showed that cholesterol-loaded cyclodextrin was the stabilizer that most efficiently reduced the membrane disruption and post-thaw cell damage. In addition, this stabilizer made it possible to obtain in vitro capacitated sperm showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, binding to the zona pellucida and better response to in vitro capacitating conditions.

Effect of cholesterol-loaded cyclodextrin enriched extenders on the quality of prefrozen and frozen buffalo semen

Objective: To investigate the effects of non-permeable cryoprotectant, cholesterol-loaded cyclodextrin, when added at different concentrations into cooled and frozen-thawed semen extended with Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders. Methods: A total of 40 ejaculates from four buffalo bulls were collected using artificial vagina. Ejaculates were extended with one of Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders which contained different concentrations [0 (control), 0.75, 1.50, 2.25 and 3.00 mg/mL] of cholesterol-loaded cyclodextrin. The extended semen samples were cooled to 5 °C and then frozen slowly to -196 °C in 0.25 mL ministraws before being stored in liquid nitrogen pending its evaluation. Sperm motility, live sperm, normal sperm morphology, sperm membrane integrity and acrosome morphology were measured. Results: Supplementation of cholesterol-loaded cyclodextrin improved progressive motility, viability, morphology and acrosome as well as plasma membrane integrities at 1.50-2.25 mg/mL depending upon types of used extenders and stages of pre- and post-freezing process (P&lt;0.01). The best concentration was 1.50 mg/mL at pre-freeze stage and 2.25 mg/mL at post- freezing. However, greater concentration (3.00 mg/mL) of cholesterol-loaded cyclodextrin had a detrimental effect compared to the control group with the two evaluated extenders (P&lt;0.01). Conclusions: Cholesterol-loaded cyclodextrin supplementation at 1.50-2.25 mg/mL concentration could improve pre-frozen and post-thawed buffalo sperm quality. The most suitable concentration is 1.50 mg/mL at pre-freeze stage and 2.25 mg/mL at post-freezing.

Effects of partially replacing glycerol with cholesterol-loaded cyclodextrin on protamine deficiency, in vitro capacitation and fertilization ability of frozen–thawed Yanbian Yellow cattle sperm

Glycerol is widely used as a cryoprotectant to protect the sperm from freezing damage during cryopreservation. However, glycerol at a high concentration has toxic effects on the sperm. Therefore, we explored the effects of partially replacing glycerol with cholesterol-loaded cyclodextrin (CLC) in a cryoprotectant on protamine deficiency, in vitro capacitation, and fertilization ability of freeze–thawed Yanbian Yellow cattle sperm. We used fresh semen, control (6% glycerol), and four treatment—I, II, III, and IV (3% glycerol + 0, 0.75, 1.5, and 3 mg/mL CLC, respectively)—groups. Computer-assisted semen analysis; JC-1, CMA3, and FluoZin-3-AM staining; flow cytometry; and IVF were conducted. Replacing a portion of glycerol with 1.5 mg/mL CLC significantly improved sperm motility, viability, plasma membrane integrity, acrosome integrity, and membrane lipid disorders, mitochondrial membrane potential (MMP), capacitation, and fertilization ability (P < 0.05) compared with the control. Additionally, in group I and III, the protamine deficiency were significantly lower (P < 0.05) than in the control group. It was found that 6% glycerol has a higher degree of damage to sperm DNA integrity than 3% glycerol. Overall, this study revealed that partial replacement of glycerol with CLC can be used as a novel cryoprotection method to reduce the toxicity of glycerol and improve the quality of thawed Yanbian Yellow cattle sperm.

Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

The bacterial mechanosensitive channel of small conductance (MscS) has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel's sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the "force-from-lipids" principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.

Cholesterol-loaded cyclodextrin attenuates dilution effect and improves quality of bovine low sperm insemination doses during cryopreservation.

In the present study, the effect of cholesterol-loaded cyclodextrin (CLC) on the quality of low sperm doses at post-thaw was evaluated. Twenty four ejaculates (6 from each bull) were collected and split into eight aliquots. First four aliquots were diluted up to 20-, 15-, 10- and 5-million sperm/0.25ml, and remaining four were treated with CLC at the rate of 1mg/120 million spermatozoa, followed by dilution up to 20-, 15-, 10- and 5-million sperm/0.25ml. The diluted semen was equilibrated, cryopreserved and evaluated post-thaw. The averages of total motility, progressive motility, average path velocity, straight linear velocity, membrane intact spermatozoa and noncapacitated spermatozoa were higher (p<.05) in CLC-treated sperm doses compared to control ones. However, the moribund spermatozoa, capacitated spermatozoa and acrosome-reacted spermatozoa were reduced (p<.05) in CLC-treated spermatozoa compared to control. The curvilinear velocity and linearity did not differ (p>.05) between control and CLC-treated sperm doses. In conclusion, treatment of spermatozoa with CLC at the rate of 1mg/120 million spermatozoon attenuates the dilution effect and improves the quality of bovine low sperm insemination doses during cryopreservation; hence it could be a favourable cryoprotectant for preserving bovine semen at higher dilutions.

Cholesterol-Loaded Cyclodextrin Addition to Skim Milk-Based Extender Enhances Donkey Semen Cooling and Fertility in Horse Mares

The present study aimed to compare semen parameters and fertility of cooled donkey semen extended in a commercially available skim milk (SKM) based extender and the same extender with cholesterol-loaded cyclodextrin (SKM-CLC). In Experiment 1, thirty-five ejaculates from seven jacks were split in SKM and SKM-CLC, extended at 50 million sperm/mL and stored at 5°C for 48 hours. Total motility (TM), progressive motility (PM), percentage of sperm with rapid motility (RAP) were assessed with CASA. Plasma membrane stability (PMS), and high mitochondrial membrane potential (HMP) were assessed with the combination of Yo-Pro and MitoStatusRed with flow cytometry. Semen was assessed before (0), 24 and 48h after cooling. In Experiment 2, two estrous cycles of 15 mares were used for fertility assessment. Mares were examined every other day by transrectal ultrasonography and had ovulation induced with 250 µg of histrelin acetate when a ≥35 mm follicle was first detected. Mares were randomly inseminated with semen obtained from one jack. Semen was extended in either SKM or SKM-CLC and cooled-stored for 24 hours. Pregnancy diagnosis was carried out 15-day post-ovulation. Data were analyzed with a mix model and Tukey's as posthoc and logistic regression model. Significance was set at P ≤ .05. There were no differences in TM, PM, RAP, PMS, and HMP for semen extended in either extender immediately before cooling (P > .05). There was a reduction in TM, PM, RAP, PMS, and HMP overtime across groups (P < .05); however, semen extended with SKM-CLC had superior TM, PM, RAP, PMS, and HMP than semen extended in SKM at 24- and 48-hours post-cooling (P < .05). Mares bred with semen extended in SKM had a lower conception rate (13%, 2/15 cycles) than cycles bred with SKM-CLC (47%, 7/15 cycles; P < .05). In conclusion, incorporating CLC into SKM extender improved cooling ability and fertility of donkey semen in horse mares. It remains to be determined if similar results can be obtained in clinical practice with mares and jennies.

Implications of cryopreservation on structural and functional attributes of bovine spermatozoa: An overview.

Sperm cryopreservation is an important adjunct to assisted reproduction techniques (ART) for improving the reproductive efficiency of dairy cattle and buffaloes. Improved understanding of mechanisms and challenges of bovine semen cryopreservation is vital for artificial insemination on a commercial basis. Although cryopreservation of bovine spermatozoa is widely practiced and advanced beyond that of other species, there are still major gaps in the knowledge and technology. Upon cryopreservation, disruption of spermatozoal plasma membrane configuration due to alterations in metabolic pathways, enzymes and antioxidants activity add to lower efficiency with loss of sperm longevity and fertilising ability. Therefore, the effective amalgamation of cryo-variables like ambient temperature, cooling and thawing rates, nucleation temperature, type and concentration of the cryoprotectant, seminal plasma composition, free radicals and antioxidant status are required to optimise cryopreservation. Novel strategies like supplementation of cholesterol-loaded cyclodextrins (CLC), nanovesicles, osteopontin, antioxidants, etc., in an extender and recent techniques like nano-purification and modified packaging have to be optimised to ameliorate the cryodamage. This article is intended to describe the basic facts about the sperm cryopreservation process in bovine and the associated biochemical, biophysical, ultra-structural, molecular and functional alterations.

The Impact of Simvastatin on Lipidomic Markers of Cardiovascular Risk in Human Liver Cells Is Secondary to the Modulation of Intracellular Cholesterol.

Statins are the first-line lipid-lowering therapy for reducing cardiovascular disease (CVD) risk. A plasma lipid ratio of two phospholipids, PI(36:2) and PC(18:0_20:4), was previously identified to explain 58% of the relative CVD risk reduction associated with pravastatin, independent of a change in low-density lipoprotein-cholesterol. This ratio may be a potential biomarker for the treatment effect of statins; however, the underlying mechanisms linking this ratio to CVD risk remain unclear. In this study, we investigated the effect of altered cholesterol conditions on the lipidome of cultured human liver cells (Hep3B). Hep3B cells were treated with simvastatin (5 μM), cyclodextrin (20 mg/mL) or cholesterol-loaded cyclodextrin (20 mg/mL) for 48 h and their lipidomes were examined. Induction of a low-cholesterol environment via simvastatin or cyclodextrin was associated with elevated levels of lipids containing arachidonic acid and decreases in phosphatidylinositol species and the PI(36:2)/PC(18:0_20:4) ratio. Conversely, increasing cholesterol levels via cholesterol-loaded cyclodextrin resulted in reciprocal regulation of these lipid parameters. Expression of genes involved in cholesterol and fatty acid synthesis supported the lipidomics data. These findings demonstrate that the PI(36:2)/PC(18:0_20:4) ratio responds to changes in intracellular cholesterol abundance per se, likely through a flux of the n-6 fatty acid pathway and altered phosphatidylinositol synthesis. These findings support this ratio as a potential marker for CVD risk reduction and may be useful in monitoring treatment response.