Effect Of Cholesterol-loaded Cyclodextrin Research Articles

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.

Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation

Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 °C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility.

Effect of cholesterol-loaded cyclodextrin on cryosurvival and fertility of cryopreserved carp (Cyprinus carpio) sperm

Addition of cholesterol-loaded cyclodextrin (CLC) to the diluents of mammalian semen increased stability and rigidity of phospholipid hydrocarbon chains of plasma membrane during sperm cryopreservation process. CLC has been tested successfully as cryoprotectant in various livestock sperm cryopreservation protocols but its efficacy for cryopreserving of fish sperm has not previously been tested. In the present study, different cholesterol loaded cyclodextrin concentrations were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with Ovopel. The extenders were prepared by using 300mM glucose and 10% DMSO supplemented with different concentrations of CLC (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0mg per 120×106 spermatozoa) and without CLC (control). The pooled semen was diluted separately at a ratio of 1:3 (v/v) by using CLC extenders. Diluted semen placed into 0.25ml straws were equilibrated at 4°C for 15min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1×105 spermatozoa/egg.Fresh sperm with no treatment showed the greatest sperm motility, duration of motility, viability, and fertilization results compared to the other tested cryopreserved and control groups (p<0.05). Supplementation of 1.5mg CLC to the extender showed the best cryoprotective effect for sperm motility, duration of motility, and viability against freezing damage in comparison to extenders containing 2.5mg, 3.0mg CLC, and control group (p<0.05). Cryopreserved sperm containing 1.5mg CLC provided greater result in term of fertilization success when compared to other extenders containing 0.5, 2.5, and 3.0mg CLC or control (p<0.05). The amount of CLC effected post-thaw sperm quality and fertility as a dose-dependent manner.It is concluded that treatment of cholesterol-loaded cyclodextrin for carp sperm cryopreservation significantly improves cell cryosurvival and fertilization.

66 EFFECT OF ADDITION OF CHOLESTEROL-LOADED CYCLODEXTRIN BEFORE FREEZING ON QUALITY AND FERTILITY OF STALLION FROZEN SEMEN

The aim of the present study was to evaluate the effect of cholesterol-loaded cyclodextrin on the quality and fertility of stallion frozen semen. Three ejaculates from each of 4 stallions were used. The semen was diluted (1 : 1) with a skim milk-based extender (Botu-SemenTM, Botupharma, Brazil). The samples were divided into 2 groups: control group (CG), composed of semen diluted only with extender, and treated group (TG), composed of semen diluted with extender plus 750 mg mL–1 of cholesterol-loaded cyclodextrin. Both groups were incubated for 15 min at 20°C. The semen was frozen with Botu-CryoTM (Botupharma, Brazil) extender according to the manufacturer's protocol in 0.5-mL straws containing 100 × 106 of total sperm. The sperm kinetic parameters were analysed by computer-assisted semen analysis, and plasma membrane integrity by flow cytometer (propidium iodide and fluorescein isothiocyanate -PSA) on post-thaw. The fertility trial was carried out inseminating 2 cycles of 20 mares (total of 40 cycles) immediately post-ovulation using 4 straws of CG or TG (400 × 106 total sperm), one from each stallion in a randomised design. Comparison of sperm parameters was performed by t-test and fertility by Fisher's exact test. No difference (P &gt; 0.05) was observed in total motility (%, CG = 57.9 ± 6.5 v. TG = 60.2 ± 6.7), progressive motility (%, CG = 26.9 ± 4.8 v. TG = 28.5 ± 4.8), percentage of rapid sperm (%, CG = 43.5 ± 8.8 v. TG = 45.7 ± 7.6), membrane integrity (%, CG = 20.1 ± 5.1 v. TG = 20.3 ± 6.3), and fertility (CG = 60% v. TG = 70%) between the groups. The results of this study showed that the use of cholesterol-loaded cyclodextrin did not affect sperm kinetic parameters and fertility in stallion with good quality in post-thaw semen. Further studies must be performed with stallions sensitive to freeze-thawing process.

Effect of cholesterol-loaded cyclodextrins on bull and goat sperm processed with fast or slow cryopreservation protocols

Cholesterol-loaded cyclodextrins (CLC) added to the sperm before cryopreservation enhance sperm quality after freeze-thawing in several cold shock-sensitive species, including cattle and goats. However, all studies conducted to date have used conventional protocols, in which sperm are cooled slowly to 5°C before freezing. As cholesterol plays a significant role in sperm cold shock resistance, it is possible that CLC-treated sperm can withstand cooling damage when the sperm are not cooled slowly to 5°C before freezing. In this study, we determined whether CLC-treated goat (1 mg CLC/120×106 sperm) and bull (2 mg CLC/120×106 sperm) sperm quality, after thawing, was different for sperm frozen using conventional protocols (including a slow cooling phase to 5ºC) and protocols in which the sperm were frozen from room temperature, without cooling the sperm slowly to 5°C before freezing. CLC-treated sperm exhibited higher percentages of plasma membrane-intact sperm than control sperm when cryopreserved using conventional protocols. In addition, CLC treatment enhanced both sperm motility and plasma membrane integrity when sperm were frozen directly from room temperature. However, this treatment did not fully prevent the damage of the sperm after cooling rapidly and subsequent freezing, as the sperm quality was lower than that presented by the samples frozen using the conventional protocol. The results are promising, but studies to optimize the protocols for freezing sperm directly from room temperature need to be conducted, as well as studies to determine how cryopreserving sperm in this manner affects other sperm functions.

Cholesterol-loaded cyclodextrin pretreatment of ram spermatozoa protects structural integrity of plasma membrane during osmotic challenge and reduces their ability to undergo acrosome reaction in vitro

The effect of cholesterol-loaded cyclodextrin (CLC) pretreatment on the structural integrity of ram sperm membranes during osmotic challenges and the ability of those spermatozoa, in the presence of cholesterol, to undergo acrosome reaction in vitro were examined in two successive experiments. In experiment 1, ejaculates (n=9) were extended with Tris–citric acid–glucose (TCG) solution to a concentration of 400×106spermatozoa/mL and then divided into two equal aliquots. The first aliquot was treated with CLC (3mg/120×106 spermatozoa), and the second aliquot remained as control. The sperm samples were then incubated at 35°C for 15min in one of the five fructose solutions, adjusted to 20, 80, 290, 500, or 1500mOsm/L. After osmotic challenges, the percent live sperm and the sperm plasma membrane integrity were estimated in both groups by a modified hypo-osmotic swelling test associated with supravital eosin staining. CLC pretreatment significantly increased the percentage of live, intact, and live-intact spermatozoa (P<0.05) following exposure to different osmotic challenges. In experiment 2, ejaculates (n=8) were first centrifuged twice to remove seminal plasma and extended with TCG to concentration of 200×106spermatozoa/mL. After CLC supplementation, the ability of spermatozoa to undergo acrosome reaction was evaluated in the presence of an acrosome reaction inducing substance, lysophosphatidylcholine. The percentage of sperm that underwent acrosome reaction in CLC-supplemented group was lower (P<0.05) compared to control. In conclusion, CLC pretreatment enhanced the structural integrity of ram sperm membranes in wide range of osmotic pressures, between 20 and 1500mOsm/L. Furthermore, CLC pretreatment reduced the ability of ram spermatozoa to undergo acrosome reaction in vitro.

Effects of cholesterol-loaded cyclodextrin during freezing step of cryopreservation with TCGY extender containing bovine serum albumin on quality of goat spermatozoa

The susceptibility of mammalian spermatozoa to cold shock and freezing damage is due to changes in membrane lipid composition, particularly cholesterol depletion in plasma membrane during cryopreservation. The aim of this study was to investigate the effects of different concentrations of cholesterol-loaded cyclodextrin (CLC) and bovine serum albumin (BSA) on the cryopreservation of goat spermatozoa in tris-citrate egg yolk extender. Semen was collected from four mature goats and divided into seven aliquots prior to cryopreservation. The first aliquot remained untreated and was mixed with TCG, the second aliquot was mixed with TCG and egg yolk (TCGY), third aliquot was mixed with TCGY and 2.5% BSA (TCGYB) and other aliquots were mixed with TCGYB containing 0.75, 1.5, 2.5 and 3 mg/ml CLC. All samples were cryopreserved in straws over liquid nitrogen vapor and sperm motion Kinetics were measured by computer-assisted semen analysis (CASA) (percent motility (MOT), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and amplitude of lateral head displacement (ALH)). Acrosome status and vitality was observed by the triple-stain technique. CLC addition to extender resulted in significant ( p < 0.05) enhancement of MOT, STR, and VCL of post-thawing sperm. Post-thawed motility, progressive motility and recovery rate were significantly ( p < 0.05) higher in 1.5 mg/ml CLC with 2.5% BSA in TCGY extender compared to other groups. The 1.5 CLC sperm yielded a significant increase in percentage of spermatozoa with intact acrosome ( P > 0.05). These results indicate that treating goat sperm with CLC and BSA in TCGY extender improved motility and vitality after freezing and thawing.

Cholesterol-loaded cyclodextrin enhances osmotic tolerance and inhibits the acrosome reaction in rabbit spermatozoa

The effects of cholesterol-loaded cyclodextrin (CLC) treatment on the osmotic tolerance and ability to undergo the acrosome reaction of rabbit spermatozoa, with an unusually high cholesterol/phospholipid ratio in plasma membranes, were examined in two successive experiments. In the first experiment, CLC-pretreated and untreated sperm cells were exposed for 15 min to one of five fructose solutions, adjusted to 20, 80, 290, 500 or 1500 mOsm/L. After the anisoosmotic challenge, the integrity of sperm membranes in the CLC-supplemented (at a dose level of 3 mg/120 × 10 6 spermatozoa) and control groups was estimated by a modified hypoosmotic swelling test (HOST) associated with a supravital eosin staining test (HE-test). In the second part of the study, the influence of cholesterol supplementation on the acrosome reaction of sperm cells stimulated by either calcium ionophore A23187 (CI) or lysophosphatidylcholine (LPC) was evaluated. CLC pretreatment increased viable and live-HOST-responsive sperm rates ( P < 0.01) after incubation in anisoosmotic solutions varying from 80 to 1500 mOsm/L. However, CLC supplementation did not influence the percentage of HOST-responsive sperm cells ( P > 0.05). A significant interaction was determined between CLC pretreatment and the level of osmotic pressure in maintaining the functional and physical integrities of sperm membranes undergoing osmotic challenges. Both CI and LPC successfully induced the acrosome reaction in rabbit spermatozoa ( P < 0.001). Compared with CI, LPC was more effective ( P < 0.0001). CLC pretreatment resulted in a significant reduction ( P < 0.01) in the percentage of acrosome reacted sperm cells irrespective of the inducing agent, either CI or LPC. In conclusion, CLC treatment enhanced the anisoosmotic tolerance of rabbit spermatozoa and reduced their ability to undergo the acrosome reaction after stimulation by CI or LPC.

80 CHOLESTEROL-LOADED METHYL-BETA-CYCLODEXTRIN IMPROVES BOAR SPERMATOZOA CRYOPROTECTANT ADDITION AND REMOVAL TOLERANCE

Difficulties associated with the cryopreservation of boar sperm include their sensitivities to osmotic stresses and chilling sensitivity. We investigated the effects of cholesterol-loaded cyclodextrin (CLC) on boar sperm motility and membrane integrity following exposure to various osmolalities. Samples were collected using the gloved hand method from crossbred boars, and ejaculates having greater than 75% motility were extended 1:3 with Androhep (Minitube Inc., Verona, WI) for this study. Samples were centrifuged at 700g for 5 min, and the resulting pellets were resuspended to 1.2 × 108 cells mL–1 in Androhep. Samples were then treated with 0, 1.5, or 3.0 mg of CLC/1.2 × 108 cells mL–1 for 10 min at room temperature. In experiment 1, samples were aliquoted into 1.5-mL centrifuge tubes, centrifuged at 700g for 5 min and the sperm exposed to Dulbecco’s PBS at different osmolalities for 5 min before being returned to 300 mOsm by adding Dulbecco’s PBS solutions at differing osmolalities. After returning the sperm to isosmotic conditions, sperm motility was analyzed. In experiment 2, samples were treated as in experiment 1 and following exposure to the various osmolalities, sperm were stained with Alexa 488-PNA and propidium iodide to determine sperm membrane integrity. Ten thousand sperm per treatment were analyzed by flow cytometry. Data were analyzed by standard ANOVA. The CLC-treated sperm (normalized means ± SEM; 33 ± 16, 80 ± 8, 86 ± 5, 100, 64 ± 4, 7 ± 3, 0 ± 0, respectively) exhibited greater percentages of motile cells following hypo-isosmotic exposure than control sperm (4 ± 1.6, 33 ± 9.6, 84 ± 7.1, 100, 37 ± 5.5, 3 ± 1.6, 0 ± 0, respectively), and there was a tendency for CLC-treated sperm (P = 0.0225) to maintain motility following hyper-isosmotic exposure. In addition, CLC-treated sperm (87 ± 4, 93 ± 1, 95 ± 1, 93 ± 2, 88 ± 4, 83 ± 3, 41 ± 9, respectively; P &lt; 0.05) maintained greater percentages of membrane integrity following treatment with anisosmotic solutions compared with controls (29 ± 8, 63 ± 10, 81 ± 7, 92 ± 3, 73 ± 8, 44 ± 5, 21 ± 9, respectively). Using a combination of these osmotic tolerance data with previously published boar sperm membrane permeability characteristics, we mathematically modeled the number of steps needed for the addition or removal of cryoprotectants. Computer simulations indicate that an abrupt addition of 1 m glycerol will cause boar sperm to exceed their osmotic tolerance limits unless they are treated with 3 mg of CLC. Moreover, the addition of 1 m EG causes boar sperm to exceed all osmotic tolerance limits and therefore, the addition and removal of EG requires multiple-step protocols. However, the addition and removal of 1 m DMSO maintains volume excursions well within the osmotic tolerance limits with the addition of cholesterol (1.5 and 3 mg). Empirical data for addition of CPA have shown similar results as seen with the computer simulation. These data support the hypothesis that adding cholesterol to porcine sperm broadens their osmotic tolerance limits and potentially provide a mechanism to increase post-thaw survival of porcine sperm.

Effect of cholesterol-loaded cyclodextrin on motility and plasma membrane integrity of cooled stallion sperm

Effect of cholesterol-loaded cyclodextrin on motility and plasma membrane integrity of cooled stallion sperm

Effect of cholesterol-loaded cyclodextrin on the cryosurvival of bull sperm

Bull sperm were treated with several levels of cholesterol-loaded cyclodextrin (CLC) and frozen in egg yolk diluents containing either Tris or sodium citrate, to determine the CLC concentration that best benefits bull sperm cryosurvival. After thawing, higher percentages of motile (60%) and viable (55%) sperm were obtained when 1.5 mg/ml CLC was added to sperm prior to freezing, than for sperm frozen in egg yolk Tris alone (42 and 46%, respectively; P<0.05). Increasing concentration of CLCs, maintained higher percentages of viable sperm up to addition of 6.0 mg/ml CLC when the percentages of viable sperm began to decline (50%; P<0.05). Addition of 1.5 mg/ml CLC to sperm frozen in sodium citrate diluent resulted in 53% motile sperm compared to 37% for control, although these were not different ( P>0.05). The beneficial effects of CLC addition were observed regardless of whether sperm incubated with CLC at 22 or 37 °C ( P>0.05) and maximum effects were observed when sperm incubated with CLC for 15 min. Longer incubation times, up to 60 min, resulted in similar results ( P>0.05). The amount of cholesterol that incorporated into sperm, increased with increasing CLC concentration, in a linear fashion, and each sperm incorporates a similar amount of cholesterol (coefficient of variation=12.9 ± 0.7%). In addition, the cholesterol incorporates into all sperm membranes. Increasing membrane cholesterol levels, by adding CLCs to cells, prior to freezing, is a simple technology that increases the cryosurvival of bull sperm, and may benefit the cryosurvival of many cell types.