Use of Cholesterol in Sperm Cryopreservation: Present Moment and Perspectives to Future

a Carmela por los

This study evaluated the effect of adding different concentration of cholesterol-loaded-cyclodextrin (CLC) on sperm quality after thawing. Thirty ejaculates were diluted, centrifuged and resuspended to 120 million cells/mL in a Tris diluent. Semen was treated with 0 (Control), 0.75, 1.5, 3.0, 4.5, 6.0 or 7.5 mg of CLC. Then, the samples were cooled at 4 °C for 2 h, diluted with Tris-egg yolk and 2% glycerol, packaged into 0.5 mL straws, frozen in liquid nitrogen (N2 ) vapor for 20 min before being plunged into N2 . Straws were thawed at 37 °C for 30 sec, and evaluated for thermal resistance test (TRT); progressive motility using CASA; hypoosmotic test and for binding capacity of sperm to perivitelline membrane (PM). The variables were determined using ANOVA at 5% probability. Higher percentage of motile sperm were maintained after thawing and TTR when 0.75 mg CLC was added, evaluated at 0, 60 and 120 min of incubation (50.4, 33.8 and 22.5%, respectively) compared to other treatments (P<0.05). The percentage of coiling was higher in sperm treated with 6.0 and 7.0 mg of CLC than other treatments (P<0.05). Addition of 0.75 mg CLCs also resulted in more sperm binding to the PM after cryopreservation than control sperm (166 vs 65; P<0.05). However, when the spermatozoa binding potential was determined on a motile sperm basis by dividing the average number of spermatozoa bound to PM for each bucks by the percentage of motile spermatozoa, CLC treatment provided higher binding efficiency (1.52) than control (1.00; P<0.05). In conclusion, CLCs improved the percentage of post-thaw of motility in caprine sperm as well as increased the number of sperm that bind to PM. Addition of 0.75 mg of CLC to caprine sperm prior to cryopreservation improved the quality sperm motility for up to 2 h.

Commercial use of sex-sorted stallion sperm will depend upon the ability to cryopreserve the sperm. In many cases the desired sperm for sorting is already cryopreserved; therefore, this will necessitate the need to thaw, sex-sort, and subsequently refreeze the sperm for future use in intracytoplasmic sperm injection (ICSI). Experiments were conducted to determine if previously frozen stallion spermcould be thawed, sex-sorted, and cryopreserved. Ejaculates from 6 stallions were frozen in FR5 cryopreservation medium at 400 million sperm mL–1 (0.5-mL straws). Samples were thawed for 30 s in 37�C water and diluted (100 million sperm mL–1) in Kenny's modified Tyrode's medium (KMT). Samples (2 mL) were stained with 36.53 µm Hoechst 33342 in 34�C water for 45 min and then diluted to 75 million sperm mL–1 with KMT containing 0.002% food coloring dye (FD&amp;C #40). Sperm were sorted using an SX MoFlo™ (Dako, Fort Collins, CO, USA), and the sorted samples centrifuged at 850g for 10 min. The sperm were suspended to 4.6 million sperm mL–1 in FR5 + 0.54 m glycerol (control) or the following treatments: FR5 + 0.52 m dimethylformamide (DMF); FR5 + 0.54 m glycerol + 1.5 mg cholesterol-loaded cyclodextrins (CLC)/120 million cells; FR5 + 0.52 m DMF + 1.5 mg CLC/120 million cells. Non-sorted control sperm (NSC) were removed from original frozen/thawed samples and suspended to 4.6 million sperm mL–1 in FR5 + 0.52 m DMF. Sorted and non-sorted samples were cooled from 22�C to 4�C over 2 h, loaded into 0.25-mL straws, and frozen in liquid nitrogen vapor. Straws were thawed in 37�C water for 30 s and the percentages of motile and viable sperm were determined using computer-assisted sperm analysis and flow cytometry (5 million cells were stained with 10 µL propidium iodide and 20 µL SYBR-14), respectively. Data were analyzed by analysis of variance and treatment means were separated using the Student-Newman-Keuls mean separation test. The percentages of total motile spermatozoa were higher for frozen/thawed sexed refrozen (FTR) sperm refrozen in DMF + CLC (10%) than for the glycerol control (2%; SEM � 1; P &lt; 0.05), but this motility was similar to that of the DMF control (5%), the glycerol + CLC treatment (4%), and the NSC (7%; SEM � 1; P &gt; 0.05). The percentages of viable spermatozoawere higher for FTR (glycerol and DMF) sperm treated with CLC prior to freezing (68% and 71%) than for control sperm (58% and 56%; SEM � 3; P &lt; 0.05), respectively. Frozen/thawed sexed refrozen samples resulted in significantly higher percentages of viable spermatozoa compared to NSC (42%; SEM � 3; P &lt; 0.05). These findings indicate that cryopreserved stallion sperm can be thawed, sex-sorted, and refrozen and still maintain a high percentage of viable spermatozoa; treating sperm with CLC prior to refreezing improved cryosurvival for both cryoprotectants. This may provide adequate numbers of viable sperm for use in intracytoplasmic sperm injection procedures. This work was funded by XY, Inc.

Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival

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.

Propagation of genetically diverse felid populations would benefit from more effective assisted reproduction strategies, including enhanced methods for sperm cryopreservation. In felids, sperm cryopreservation has been improved by substituting soy-lecithin for egg yolk in cryomedium (Vick et al. 2012 Theriogenology 78, 2120–2128). In other species, such as elephants (Kiso et al. 2012 Reprod., Fert. Dev. 24, 1134–1142) and cattle (Purdy et al. 2004 Cryobiology 48, 36–45), the addition of cholesterol-loaded cyclodextrins (CLC) to sperm before freezing has been shown to produce superior cryopreservation results. In this study, our objectives were to (1) assess cholesterol content of cat sperm membranes and capacitation status following incubation with CLC; (2) evaluate post-thaw sperm motility, acrosome status, and fertility in vitro following CLC treatment and freezing in a soy-based cryomedium; and (3) conduct a preliminary assessment of cholesterol content in nondomestic cat sperm. Freshly collected domestic cat sperm (n = 2 males, 3–4 ejaculates/male) were incubated with CLC (0, 1.5, or 3.0 mg mL–1), and cholesterol levels were measured using an Amplex Red Cholesterol Assay. Sperm aliquots from each CLC concentration were treated with calcium ionophore (2 µM, 30 min) during in vitro incubation and stained with fluorescein isothiocyanate/PNA to evaluate induced acrosomal loss. To assess post-thaw parameters, cat sperm treated with CLC were frozen in straws using soy-lecithin cryomedium, thawed, and cultured in vitro over time. To evaluate fertility, oocytes were collected laparoscopically from gonadotropin-treated domestic cats (n = 7 females, 147 oocytes total) and inseminated with low numbers of thawed-frozen sperm pretreated with 0 or 1.5 mg mL–1 CLC. Data were analysed using ANOVA and mean differences assessed with Fisher l.s.d. or chi-squared analysis. Sperm cholesterol levels were increased (P &amp;lt; 0.05) after exposure to both 1.5 and 3.0 mg mL–1 CLC. Prefreeze motility was decreased (P &amp;lt; 0.05) and capacitation was delayed at 3.0 mg mL–1 CLC relative to treatment with 0 or 1.5 mg mL–1 CLC. Both post-thaw motility and percentage of acrosome intact sperm were reduced (P &amp;lt; 0.05) with the highest CLC concentration, but results were similar (P &amp;gt; 0.05) for 0 and 1.5 mg mL–1 CLC. Fertilization percentages did not differ (P &amp;gt; 0.05) between treatment groups (0 CLC, 33.3%, 25/75; 1.5 mg mL–1 CLC, 26.4%, 19/72). Preliminary results from a single cheetah (Acinonyx jubatus) and single fishing cat (Prionailurus viverrinus) suggest that sperm membrane cholesterol may be lower compared to the domestic cat. Cholesterol content appeared to increase in both species after exposure to 1.5 mg mL–1 CLC. In summary, our findings suggest CLC treatment increased cholesterol content of felid sperm membranes. The higher CLC concentration was detrimental to sperm motility, capacitation, and post-thaw sperm traits. The lower CLC concentration did not improve post-thaw sperm function in domestic cats. Research supported by the Procter &amp; Gamble Wildlife Conservation Scholarship Program.

Altering the lipid composition of plasma membranes not only affects the ability of sperm to capacitate and acrosome react, but also affects the way sperm respond to cryopreservation. When cyclodextrins are preloaded with cholesterol to form cholesterol-loaded-cyclodextrin (CLC) and then incubated with bull sperm before cryopreservation, higher percentages of motile and viable cells are recovered after freezing and thawing compared with control sperm. The amount of cholesterol in a membrane is important for maintaining its integrity during cryopreservation, and CLC alters the lipid composition of sperm, affecting their cryosurvival. This study evaluated the effect of adding cholesterol to boar sperm on cryosurvival rates and the ability of cryopreserved sperm to bind to the zona pellucida. Methyl-β-cyclodextrin was loaded with cholesterol as follows: 0.45 mL of cholesterol (200 mg mL–1 in chloroform) was added to 1 g of methyl-β-cyclodextrin dissolved in 2 mL of methanol, and the solution was stirred until clear. The mixture was poured into a glass dish and the solvents removed using a stream of nitrogen gas. The resulting crystals were allowed to dry for an additional 24 h, at which time they were removed from the dish and stored in a glass container at 22°C. A working solution of the cholesterol-loaded cyclodextrin was prepared by adding 50 mg of CLC to 1 mL TALP at 37°C and mixing the solution briefly using a vortex mixer. Ejaculates from each of 8 boars were collected, diluted 1:1 in BTS® (Minitub, Brazil), and maintained for 2 h at room temperature. The ejaculates were then cooled to 15°C over 60 min. The ejaculates were then centrifuged at 400 × g for 10 min (at 15°C), the supernatant was discarded, and the sperm were suspended to 120 × 106 cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk). The sperm were divided into 2 treatments (T): T1 = control and T2 = 1.5 mg of CLC mL–1. The samples incubated for 15 min at 15°C, after which they were cooled to 5°C over 90 min and diluted 1:1 (v:v) with Freeze diluent (2.5 mL of lactose solution 11%, 6 mL of glycerol, and 1.5 mL of Orvus-es-Paste). The sperm were then packaged into 0.5-mL French straws and frozen in static liquid nitrogen vapor (4.5 cm above the liquid nitrogen) for 20 min before being plunged into liquid nitrogen for storage. Straws were thawed and the efficiency of the sperm to bind to both the chicken egg perivitelline membrane (EPM) and porcine zona pellucida (PZP) were determined using epifluorescence microscopy. The post-thaw motility and binding efficiency of sperm to salt-stored EPM and PZP were analysed by analysis of variance. Boar sperm treated with CLC maintained higher post-thaw motility than control sperm (47 and 34%, respectively; P &lt; 0.05) and had higher numbers of sperm binding to the PZP and EPM (101 sperm/EPM and 166 sperm/PZP) than control samples (77 sperm/EPM and 65 sperm/PZP; P &lt; 0.05). In addition, sperm were easier to visualise on the EPM than the porcine zona pellucida. Adding CLC to boar sperm before cryopreservation increased the number of sperm surviving cryopreservation. Fapemig, CNPq, and CAPES from Brazil.

Sperm cryosurvival is affected by altering the lipid composition of sperm plasma membranes and causes damage to spermatozoa during the cryopreservation process as loss of motile cells and functionality, compared with fresh sperm. Our objective was to compare the effect of adding cholesterol-loaded cyclodextrin (CLC) on sperm quality after freezing boar sperm. The CLC was prepared as described: 200 mg of cholesterol was dissolved in 1 mL of chloroform, and 1 g of methyl-β-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass dish and the solvents removed using a hot plate for 24 h. The crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL of BTS at 37°C. Thirty-five ejaculates from 5 boars were collected, diluted 1:1 in Beltsville thawing solution, and kept to 2 h at 22°C. The ejaculates were held at 15°C for 60 min and centrifuged at 15°C for 400g for 10 min; the pellet was suspended to 120 million cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk) and divided in 2 treatments: control and 1.5 mg of CLC/mL. The samples were incubated for 15 min at 15°C, cooled to 5°C over a 90-min period, and diluted 1:1 with freeze diluent (72.5 mL of lactose solution 11%, 6 mL of glycerol, 1.5 mL of Equex). Sperm were packaged into 0.5-mL straws, frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen. Straws were thawed in a water bath at 37°C for 30 sec, extended in Beltsville thawing solution, and analyzed by optic microscopy. Sperm were stained with 35 μg mL-1 of Hoechst 33342 and incubated for 15 min at 37°C, centrifuged at 400g for 5 min, and suspended in BTALP to a final concentration of 2 million spermatozoa/mL. A total of 10 000 spermatozoa (5 μL) from each sample were added to droplets containing 10 porcine oocytes. Porcine cumulus oocyte complexes were aspirated and placed in BTALP. The cumulus cells of the oocytes were removed by vortexing for 2 min at maximum speed. Denuded oocytes were washed 4 times in BTALP and incubated for 1 h at 38.5°C in an atmosphere of 5% CO2 in air, following which 10 oocytes per treatment were randomly placed into 45 μL droplets of BTALP, using a small bore fire polished glass pipette to remove loosely bound spermatozoa. Five oocytes were placed onto glass slides and covered with a cover slip supported by a mix of paraffin wax and petroleum jelly. Oocytes were viewed using an epifluorescence microscope, and the total number of spermatozoa bound to each zona pellucida (ZP) was determined at 400× magnification. Treatment differences for sperm motility and zona binding were determined using ANOVA. The addition of CLC to boar sperm before cryopreservation resulted in higher percentages of motile sperm and higher numbers bound to the ZP (35% and 67 sperm/ZP) compared with control cells (26% and 36 sperm/ZP; P &lt; 0.01). In summary, adding CLC to boar sperm before cryopreservation improved cells. FAPEMIG, Piglandia, CNPq, FACEPE.

Altering the lipid composition of sperm plasma membranes affects sperm cryosurvival. Cryopreservation induces many stresses on the spermatozoa, including destabilization of the plasma membrane, which results in the loss of sperm motility and function. Treating bull spermatozoa with cholesterolloaded cyclodextrin (CLC) prior to cryopreservation increases sperm cryosurvival rates. This study compared the effect of adding other sterols, which should incorporate into the membrane and increase membrane fluidity at low temperatures, thereby increasing cryosurvival. Ejaculates from four bulls were divided into two experiments (E). In E1, ejaculates were extended with Tris, and then subdivided into four treatments: No additive (control), 1.5 mg CLC/120 million sperm (positive control), and 1.5 mg/120 million sperm in cyclodextrin pre-loaded with either cholestanol or desmosterol. Spermatozoa were incubated for 15 min at 22�C after which both the ability of fresh spermatozoa to bind to the zona pellucida (ZP) and chicken egg perivitelline membrane (EPM) and their osmotic tolerance were evaluated. In E2, sperm were diluted to 120 million cells mL–1 in a Tris diluent and treated as described for E1. Then, samples were diluted 1:1 (v:v) in Tris with 20% Egg Yolk (EY) and cooled to 5�C. After dilution 1:1 (v:v) with Tris containing 10% EY and 16% glycerol, samples were allowed to equilibrate for 15 min, and then were packaged into 0.5-mL straws, frozen in static liquid nitrogen vapor for 20 min, and plunged into liquid nitrogen for storage. Straws were thawed and the motility and zona-binding ability were determined using a Hamilton Thorne Motility Analyzer (Hamilton Thorne Biosciences, Beverly, MA, USA) and epifluorescence microscopy, respectively. Treatment differences for sperm motility, osmotic tolerance, and zona binding were determined using analysis of variance. Treating spermatozoa with CLC resulted in more fresh bull spermatozoa binding to the EPM and ZP compared to cholestanolor desmosterol-loaded cyclodextrin-treated spermatozoa or control cells (P &lt; 0.05). No differences were observed between EPM and ZP binding (P &gt; 0.05). The percentages of total and progressively motile spermatozoa were higher for fresh samples treated with cholesterol-, cholestanol-, or desmosterol-loaded cyclodextrin than for control cells (P &lt; 0.05) when spermatozoa were exposed to anismotic conditions, and then returned to isosmolality. After cryopreservation, the percentages of motile spermatozoa and number of spermatozoa binding to ZP were similar for spermatozoa treated with CLC (56% and 115 sperm/ZP) and cholestanol (53% and 108 sperm/ZP) compared to spermatozoa treated with desmosterol (42% and 86 sperm/ZP; P &lt; 0.05). All treatments provided higher motility and binding efficiency than control spermatozoa (32% and 62 sperm/ZP; P &lt; 0.05). Therefore, adding cholesterol or cholestanol to bull sperm membranes improved cell cryosurvival. Studies to determine if cholestanol affects sperm capacitation need to be conducted.

Damage occurring to spermatozoa during cryopreservation results in a loss of motile cells and cells that are functionally normal, compared with fresh sperm samples. Treating bull sperm with cholesterol-loaded cyclodextrins (CLC) before cryopreservation results in increased sperm cryosurvival. However, in previous studies, CLC were always added to sperm samples that had been highly diluted. The aim of this study was to develop a procedure for adding CLC to whole bull ejaculates that would optimize sperm cryosurvival. Adding 2 or 4 mg of CLC/120 x 10(6) sperm to sperm samples ranging in concentration from 120 to 2,000 x 10(6) sperm/mL resulted in greater (17 to 28 percentage points; P < 05) numbers of live cells compared with control samples (no CLC treatment), regardless of the sperm concentration, except for samples at 120 x 10(6) sperm/mL treated with 4 mg of CLC. Incubating sperm with CLC at 23 or 37 degrees C before cryopreservation resulted in similar sperm cryosurvival. The cooling rate used to cryopreserve CLC-treated cells did not affect sperm cryosurvival. Finally, adding CLC to undiluted ejaculates (2 mg of CLC/120 x 10(6) sperm) resulted in greater percentages of live sperm compared with the control samples (62 vs. 45%; P < 0.05), although the percentages of motile sperm were similar for both CLC-treated and control samples (58%). In conclusion, bull sperm cryo-survival can be improved if spermatozoa are treated with CLC before freezing. In addition, CLC can be added to fresh ejaculates at either 23 or 37 degrees C. This technique is simple, practical, and can be easily integrated into current cryopreservation protocols.

When cholesterol is added to sperm membranes before cryopreservation, higher percentages of motile and viable cells are recovered after thawing. However, because one of the first steps in sperm capacitation is cholesterol efflux from the sperm plasma membrane, adding cholesterol to enhance cryosurvival may retard sperm capacitation. These studies evaluated the ability of sperm treated with cholesterol-loaded cyclodextrins (CLC) to capacitate, acrosome react, and fertilize oocytes. Control (non-CLC-treated) and CLC-treated sperm were treated with heparin, dilauroylphosphatidylcholine (PC12), or calcium ionophore A23187 (A23187) to capacitate and induce the acrosome reaction. Sperm capacitation, assessed by an increase in intracellular calcium level, and acrosome-reacted sperm were measured using flow cytometry. Fresh CLC-treated sperm cells underwent capacitation and/or the acrosome reaction at rates different from control samples, and the differences detected were dependent on the method used to induce sperm capacitation and the acrosome reaction. After cryopreservation, however, CLC-treated and control sperm underwent capacitation and the acrosome reaction at similar rates regardless of the method used to induce capacitation and the acrosome reaction. The primary concern for CLC-treated sperm, however, is whether this treatment would affect in vitro or in vivo fertility. Adding either control or CLC-treated cryopreserved sperm to bovine oocytes in vitro resulted in similar oocyte cleavage rates and blastocyst formation rates. In addition, when inseminated into heifers, pregnancy rates for control and CLC-treated sperm were also similar. Therefore, treating bull sperm with CLC permits greater numbers of sperm to survive cryopreservation while preserving the fertilizing potential of each individual sperm.

Treating boar sperm with cholesterol-loaded cyclodextrins widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability

Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol

C-1012: Cholesterol-loaded cyclodextrin improves goat semen cryosurvival by enhancing sperm cholesterol content and osmotic tolerance

Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival