Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival

Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sperm were maintained after thawing when 1.5 mg CLC was added to sperm from stallions whose sperm do not survive freezing well, compared to control sperm from those same stallions (67% vs. 50%; P<0.05). Addition of CLCs increased the percentages of membrane intact sperm surviving cryopreservation compared to untreated sperm for all stallions (P<0.05). The amount of cholesterol that incorporated into the membranes of the sperm cells increased in a polynomial fashion (R2=0.9978) and incorporated into all sperm membranes. In addition, there was a significant loss of cholesterol from sperm membranes after cryopreservation; however, addition of CLCs to sperm prior to cryopreservation maintained higher cholesterol levels in the sperm after freezing and thawing than untreated sperm (P<0.05). Addition of CLCs also resulted in more sperm binding to the zona pellucida of bovine oocytes after cryopreservation than control sperm (48 vs. 15; P<0.05). In conclusion, CLCs improved the percentage of post-thaw viability in equine sperm as well as increased the number of sperm that bind to zona pellucida. Addition of CLCs to stallion sperm prior to cryopreservation is a simple procedure that increases the cryosurvival of cells.     

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.     

Treatment with cholesterol-loaded methyl-β-cyclodextrin increased the cholesterol in rabbit oocytes,but did not improve developmental competence of cryopreserved oocytes

Membrane cholesterol:phospholipids ratio is an important determinant of cell chilling sensitivity. At low temperatures, major membrane destabilisation occurs when the membrane undergoes a phase transition. To increase membrane fluidity and stability during cooling and thus increase oocyte cryoresistance, cholesterol has been added to the plasma membrane. This study was conducted to determine if cholesterol could be incorporated into rabbit oocytes by incubation with cholesterol-loaded methyl-β-cyclodextrin (CLC) and if added cholesterol could improve the developmental ability of cryopreserved oocytes after parthenogenetic activation or intracytoplasmic sperm injection. Fresh, frozen and vitrified oocytes incubated with CLC containing 20% NBD-labelled cholesterol (NBD-CLC) were evaluated using confocal microscopy. Fluorescence intensity was higher in fresh oocytes than in cryopreserved ones. Pre-treating rabbit oocytes with 1 mg of NBD-CLC/mL did not improve cleavage and developmental rates after cryopreservation. Results showed that treatment with CLC increased the cytoplasmic cholesterol content, but did not improve cleavage rate and developmental competence of cryopreserved oocytes.     

Cholesterol-loaded cyclodextrin is efficient in preserving sperm quality of cryopreserved ram semen with low freezability

Semen freezability is positive correlated with the cholesterol content in the sperm cell. Freeze-thawing mainly cause temperature chock and change on media osmolarity, which can modify plasma membrane lipids content and sperm conformation, resulting in decreased fertility. Therefore, the aim of this study is to investigate the effect of adding cholesterol-loaded cyclodextrin (CLC) to the cryopreservation process of ram semen with low freezability. For that, two experiments were performed using 5 ejaculates of 6 rams, totalizing 30 samples. For experiment 1 the following treatments were tested: in natura (IN), Tris solution (CON), CLC + Tris solution (CLC), and pure methyl-β-cyclodextrin + Tris solution (MCD). For experiment 2 treatments CON and CLC were tested in samples subdivided into three freezability classes: high (n = 10), intermediate (n = 10) and low (n = 10). Freezability classes were based on the variation of sperm motility between IN and CON groups from the first experiment. Sample analyzes included sperm motility, sperm morphology, plasma and acrosome membrane integrity, mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and fluidity of plasma membrane. Results showed that CLC treatment was more efficient in maintaining sperm motility, integrity of plasma membrane, integrity of acrosome, and mitochondria membrane potential. In addition, CLC treatment in the groups with low and intermediate freezability showed improvement on progressive motility and percentage of rapid cells. In contrast, no difference was noted between CLC and CON treatments in the high freezability group. Therefore, the addition of CLC to semen extender improved sperm cryopreservation, especially in rams with low freezability.     

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 1.5mg/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 6.0mg/ml CLC when the percentages of viable sperm began to decline (50%; P < 0.05). Addition of 1.5mg/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 degrees C (P > 0.05) and maximum effects were observed when sperm incubated with CLC for 15min. Longer incubation times, up to 60min, 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.     

Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process

Experiments were conducted to investigate the effect of seminal plasma on sperm motility during the cryopreservation process. Ejaculated and epididymal spermatozoa from the ram and the bull were washed by centrifugation and resuspended in either seminal plasma or a modified Tyrode's medium (TALP) prior to dilution in medium suitable for cryopreservation. Resuspension of washed ejaculated ram spermatozoa in seminal plasma resulted in higher percentages of motile spermatozoa than resuspension in TALP after the spermatozoa were cooled to 5 degrees C (52 vs 35%), and after thawing (14 vs 9%), respectively. Resuspension of epididymal ram spermatozoa in seminal plasma had no beneficial effect in maintaining sperm motility after cooling (78 vs 73%); however, seminal plasma was beneficial to epididymal ram spermatozoa after thawing (34 vs 3%), respectively. Resuspension of washed ejaculated bull spermatozoa in either seminal plasma or TALP had no effect on the percentage of motile spermatozoa after cooling to 5 degrees C (73 vs 75%) or after thawing (60 vs 60%), respectively. In addition, seminal plasma had no beneficial effect on the percentage of motile epididymal bull spermatozoa when compared with that of TALP-treated spermatozoa after cooling (75 vs 72%) or after thawing (66 vs 63%), respectively. Seminal plasma from different sires (ram and bull) affected epididymal sperm motility. The ability of sperm cells to withstand damage during cryopreservation, however, appears to reside in the sperm cells themselves, probably due to sperm cell composition.     

Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm

Different cholesterol conjugates-loaded-cyclodextrin was added to bull sperm to improve sperm quality after freezing. Ejaculates from four bulls were diluted to 120 million cells/ml in Tris (T) diluent and then sub-divided into 10 treatments as follow: no additive (control); 1.5 mg CLC (positive control); 0.75 mg or 1.5 mg of cyclodextrin pre-loaded with cholesterol conjugated to heptanoate, palmitate, pelargonate or stearate, and incubated for 15 min at 22 °C. The samples were then diluted 1:1 with 20% egg yolk (EY) in T diluent and cooled to 5 °C over a 2 h. Upon reaching 5 °C, the samples were diluted 1:1 with T diluent containing 10% EY + 16% glycerol, and allowed to equilibrate for 15 min, and packaged into 0.5 ml straws and frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen for storage. Straws were thawed and the sperm motility, viability and number sperm binding to perivitelline membrane were determined. The ability of bull sperm to bind to the oocyte membranes was conducted using the chicken egg perivitelline membrane (CEPM) as described by Barbato et al. [G.F. Barbato, P.G. Cramer, R.H. Hammerstedt, A practical in vitro sperm-egg binding assay that detects subfertile males. Biol. Reprod. 58 (1998) 686-699] and modified by Amorim et al. [E. Amorim, J.K. Graham, B. Spizziri, M. Meyers, L. Amorim, C. Torres, The effect of adding cholesteryl-heptanoate, -palmitate, -pelargonate, or -stearate loaded cyclodextrin on bull sperm cryosurvival, in: Proceeding 40th Annual Meeting of the Society for the Study of Reproduction (SSR), July, San Antonio, TX, EUA, 2007], where these authors did not observe difference between bovine zona pellucide and CEPM. Higher percentages of motile and viable sperm were maintained after thawing from bull sperm treated with CLC and pelargonate compared to all other treatments (P < 0.05). Control samples and sperm treated with heptanoate, palmitate, or stearate loaded cyclodextrin exhibited similar motility percentages and viable sperm after freezing (P > 0.05). The percentage of motile sperm and number sperm binding to chicken egg perivitelline membrane was higher for CLC and pelargonate compared to control (P < 0.05). Therefore, adding either cholesterol or pelargonate to bull sperm membranes improved cell cryosurvival, whereas treatments with cyclodextrins pre-loaded with other cholesterol-like molecules did not.     

Membrane changes during different stages of a freeze-thaw protocol for equine semen cryopreservation

Many theories have been postulated concerning the possible effects of cryopreservation on spermatozoa, including suggestions the freeze-thawing process produces membranes that have greater fluidity and are more fusogenic, thus inducing changes similar to those of capacitation. The main objectives of this study were to determine at what stage of the freeze-thaw process membrane changes occur and whether evaluation with chlortetracycline (CTC) stain could predict the freezability of stallion sperm. Sperm viability and state of capacitation were simultaneously evaluated using CTC and Hoechst 33258 (H258) techniques. Membrane function was evaluated using the hypo-osmotic swelling test (HOS) and progressive motility (PM) was evaluated under light microscopy at each stage of a freeze-thaw protocol. Evaluated were raw semen; after dilution and centrifugation; after redilution and equilibration at room temperature; after cooling to 5 degrees C; after super cooling to -15 degrees C; and after thawing. The most pronounced functional damage to membranes and the greatest decrease in PM occurred in samples of all stallions after thawing (P<0.05). Cryopreservation, as evaluated by CTC/H258 staining, significantly (P<0.05) affected sperm membrane integrity after centrifugation, after redilution and equilibration at room temperature and after cooling to 5 degrees C. The HOS and H258 tests gave similar results (R values of approximately 0.75) and correlated inversely with the number of live noncapacitated sperm cells (R values of approximately -0.75). Remarkably, the subpopulation of capacitated live cells was unaffected in all freeze-thawing steps and the number of live acrosome reacted cells increased by a factor of 4. However, it was not possible to determine whether the changing CTC patterns reflect a true capacitation phenomenon or an intermediate destabilized state of the sperm cell membrane. This increase may indicate that the subpopulation of functional sperm cells capable of binding to the zona pellucida increases after freeze-thawing despite the deteriorative effect of this procedure for the entire live sperm population.     

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

Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5° and 22 °C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 °C than 22 °C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC’s to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.     

Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction, and fertility

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.     

Membrane status of boar spermatozoa after cooling or cryopreservation

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.     

Vitrification of bovine oocytes after treatment with cholesterol-loaded methyl-beta-cyclodextrin

A major site of cryoinjury during cryopreservation of mammalian oocytes is the plasma membrane. Chilling can irreversibly damage plasma membrane integrity during the lipid phase transition that occurs upon cooling. Membranes containing higher cholesterol concentrations are more fluid at lower temperatures and therefore less sensitive to cooling. The purpose of this study was to determine if cryosurvival of vitrified oocytes could be improved by incubation with cholesterol-loaded methyl-beta-cyclodextrin (CLC) prior to vitrification in the presence or absence of fetal calf serum (FCS), and if cholesterol could enter oocytes through cumulus cells and the zona pellucida. Cumulus-enclosed oocytes incubated with various concentrations (0, 0.75 or 1.5 mg/mL) of CLC in the presence of FCS for 25-45 min prior to vitrification did not result in different rates of development after warming of vitrified oocytes, followed by in vitro fertilization. However, there was an increase (P<0.05) in cleavage and number of eight-cell embryos from oocytes preincubated for 1h with 2mg/mL CLC in a chemically defined system and then handled and vitrified in chemically defined media, in comparison to those not exposed to CLC prior to vitrification or to those handled and vitrified in the presence of FCS (55, 41 and 38% eight-cell embryos, respectively). Fluorescence was seen in cumulus-oocyte complexes (COCs) previously exposed to CLC containing cholesterol labeled with a fluorescent dye; fluorescence was also seen in oocytes after removal of the cumulus cells. Oocytes not exposed to the labeled cholesterol did not fluoresce. Cholesterol from CLC readily entered cumulus cells and oocytes and improved survival in chemically defined vitrification systems.     

The effect of 45 degrees C hyperthermia on the membrane fluidity of cells of several lines.

The membrane fluidity of cells of human (AG1522 human foreskin fibroblasts), rodent [Chinese hamster ovary (CHO) and radiation-induced mouse fibrosarcoma], and feline (Crandall feline kidney) cell lines after heating at 45 degrees C was measured by flow cytometry. In addition, a heat-resistant variant of radiation-induced mouse fibrosarcoma cells and two heat-sensitive CHO strains were studied. Fluorescence polarization of the plasma membrane probe trimethylammonium-diphenylhexatriene was used as a measure of membrane fluidity. The sensitivity of all cell lines to 45 degrees C hyperthermia was compared. The baseline membrane fluidity varied among the cell lines, but did not correlate with sensitivity to hyperthermia. However, CHO cells, especially the heat-sensitive mutants, had the largest increase in membrane fluidity after heating at 45 degrees C, while the heat-resistant mouse fibrosarcoma variants and Crandall feline kidney cells resisted changes in fluidity. In general, the more resistant the cell line was to killing by heat, the more resistant it was to changes in membrane fluidity.     

Use of cholesterol-loaded cyclodextrin: An alternative for bad cooler stallions

During the cooling process, sperm may suffer irreversible damage that compromises the fertility rate. Incorporating cholesterol-loaded cyclodextrin (CLC) represents a strategy to increase sperm resistance at low temperatures; however, high levels of cholesterol in the cell membrane can interfere with sperm capacitation. The goals of this study were to determine the CLC concentration and cooling temperature that produce optimal kinetic parameters and viability of sperm from stallions identified as bad coolers (BCs) and good coolers (GCs), as well as the effect of adding CLC on the occurrence of the acrosome reaction (ACR) and on the fertility rate of cooled sperm. In experiment 1, each ejaculate was divided into four groups: Control and treated with 1 (CLC-1), 1.5 (CLC-1.5), or 2 mg (CLC-2) of CLC/120 × 10⁶ sperm and cooled for 48 hours at 5 °C. In experiment 2, each ejaculate was divided into four groups: Control and CLC-1.5 cooled at 15 °C or 5 °C for 24 hours. For experiment 3, GC and BC stallions were used, and the ejaculates were divided into control and CLC-1.5 cooled at 5 °C for 48 hours. According to experiment, the sperm kinetics (SK) and plasma membrane integrity (PMI) were analyzed before and after 24 and 48 hours of cooling. In experiment 4, the ejaculates were divided into four groups: Control and CLC-1.5 maintained at room temperature or cooled at 5 °C for 24 hours. Each group was incubated with ionophore calcium at 37 °C for 3 hours. The incidence of ACR was analyzed before and after 1, 2, and 3 hours of incubation. For experiment 5, two cycles of 10 mares for a GC stallion and two cycles of 25 for a BC stallion were used. The inseminations were performed with control and CLC-1.5 groups cooled at 5 °C for 24 hours. According to results, all groups treated with CLC exhibited higher PMI compared with controls, and CLC-1.5 and CLC-2 exhibited the best SK results. The cooling temperature of 5 °C was superior to 15 °C when the sperm was treated with CLC. The GC and BC stallions benefited from the CLC-1.5 treatment, but the BCs were more evident, which presented greatly increased PMI and SK. There was a delay in capacitation of at least 3 hours for the fresh sperm and at least 1 hour for cooled sperm supplemented with CLC-1.5. After adding CLC-1.5, the fertility of BC stallion significantly increased, but that of the GC was not altered. Thus, incorporating CLC is an effective technique to cool equine semen, although it is indicated mainly for BC stallions.     

Heat-induced changes in the membrane fluidity of Chinese hamster ovary cells measured by flow cytometry.

Flow cytometry was used to measure the fluorescence polarization of the lipid probe trimethylammonium-diphenylhexatriene as an indicator of plasma membrane fluidity of Chinese hamster ovary (CHO) cells heated under various conditions. Fluorescence polarization was measured at room temperature about 25 min after heating. When cells were heated for 45 min at temperatures above 42 degrees C, fluorescence polarization decreased progressively, signifying an increase in plasma membrane fluidity. The fluorescence polarization of cells heated at 42 degrees C for up to 55 h was nearly the same as for unheated control populations, despite a reduction in survival. The fluorescence polarization of cells heated at 45 degrees C decreased progressively with heating time, which indicated a progressive increase in membrane fluidity. The fluorescence polarization distributions broadened and skewed toward lower polarization values for long heating times at 45 degrees C. Thermotolerant cells resisted changes in plasma membrane fluidity when challenged with subsequent 45 degrees C exposures. Heated cells were sorted on the basis of their position in the fluorescence polarization distribution and plated to determine survival. The survival of cells which were subjected to various heat treatments and then sorted from high or low tails of the fluorescence polarization histograms was not significantly different. These results show that hyperthermia causes persistent changes in the membrane fluidity of CHO cells but that membrane fluidity is not directly correlated with cell survival.     

Effects of cholesterol-loaded cyclodextrins on the quality of frozen-thawed equine epididymal sperm

Equine epididymal sperm are known to be severely sensitive to cryopreservation, in terms of sperm quality and pregnancy rate. The objective of this study was to examine the effects of cholesterol loaded cyclodextrins (CLCs) on the quality of stallion epididymal sperm during cryopreservation.In experiment I, sperm were treated with different concentrations of CLCs: (1) 0 mg (control), (2) 1.5 mg, (3) 3 mg, and (4) 6 mg per 120 × 10⁶ sperm. The sperm viability and amount of cholesterol were determined at 15, 30 and 45 min after CLC treatment using viability markers (Ethidium homodimer-1 and Calcein AM) and gas chromatography, respectively. In experiment II, CLC treated sperm (1.5 mg CLC per 120 × 10⁶ sperm) were fixed and stained with filipin to examine the cholesterol distribution. In experiment III, sperm were treated with CLCs at concentrations of 1.5, 3.0, 6.0 mg per 120 × 10⁶ sperm for 15 min, then equilibrated with freezing extender at 4 °C for 1 h prior to cryopreservation. Epididymal sperm without CLC loading (0 mg) were used as the control group. The sperm quality was examined at post-equilibration and 10 min, 2 h and 4 h after freezing and thawing.The cholesterol was successfully loaded into the plasma membrane of stallion epididymal sperm. The amount of cholesterol was increased in a manner of dose and time dependence, and the filipin–sterol complexes were increasingly labeled over the sperm head. CLCs at 1.5 mg/120 × 10⁶ sperm significantly improved sperm quality during sperm equilibration and cryopreservation compared to other doses of CLCs and non-CLC control. An increasing concentration and incubation time of CLCs was detrimental to sperm quality.It is concluded that cholesterol loading to the sperm plasma membrane via CLCs decreases chilling sensitivity and also improves epididymal sperm cryopreservability.     

Effects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved buffalo spermatozoa.

The principal objective of this study was to derive an improved procedure for cryopreservation of swamp buffalo (Bubalus bubalis) spermatozoa. Experiments were conducted to determine effects of cooling rate, intermediate plunge temperature and warming rate on motility and acrosome integrity of spermatozoa. Spermatozoa were obtained from three bulls (three ejaculates/bull) and were subjected to nine cooling conditions before being frozen in liquid nitrogen: cooling at 10, 20, or 30 degrees C/min each to -40, -80, or -120 degrees C before being plunged into liquid nitrogen. The spermatozoa frozen under a given condition were then thawed either at 1000 or 200 degrees C/min. Cooling rate, intermediate temperature and warming rate significantly affected survival of spermatozoa obtained from the three bulls. Cooling spermatozoa from 4 to -120 degrees C either at 20 or 30 degrees C/min yielded better progressive motility compared to other cooling conditions (50 versus 30%). Rapid warming was superior to slow warming. In an additional study, motility and fertility of spermatozoa frozen after being cooled to -120 degrees C at 20 degrees C and 30 degrees C/min and those frozen by a standard protocol used routinely for semen processing were assessed. Progressive motility of cryopreserved spermatozoa cooled at 20 degrees C and 30 degrees C/min was 40%, while that of spermatozoa cryopreserved using a standard protocol was 25%. A total of 178 buffalo cows were inseminated with cryopreserved spermatozoa obtained from one bull, and their pregnancy status was assessed 60 days later by rectal palpation. Out of the 60, 26 (43%) and 23 of 58 (40%) cows inseminated with sperm cooled at 20 and 30 degrees C/min, respectively, became pregnant, whereas 17 of 60 (28%) cows inseminated with sperm frozen by a standard protocol became pregnant. This study demonstrates that an effective cryopreservation procedure for buffalo spermatozoa can be derived by systematic examination of various cryobiological factors.     

Effect of various cooling rates (from 30 degrees C to 5 degrees C) and thawing temperatures on the deep-freezing of Bos Taurus and Bos Bubalis semen

A total of 36 semen ejaculates, six from each of three Holstein-Friesian bulls and three Murrah buffalo bulls, were frozen in tris citric acid-fructose-egg-yolk-glycerol diluent after 1 hour of equilibration to study the effect of various cooling rates (15, 30, 60 and 120 minutes from 10 degrees to 5 degrees C vs a control sample cooled for 120 minutes from 28 degrees to 5 degrees C) and thawing temperatures (40 degrees C 60 seconds , 60 degrees C 15 seconds and 80 degrees C 5 seconds ) on prefreeze and post-thaw sperm motility. Sperm motility differed significantly (P < 0.01) between various cooling rates in both the Holstein-Friesian bull semen and the Murrah buffalo semen at prefreezing, immediately post-thawing, and after 1 hour of post-thaw incubation at 38 degrees C. Post-thaw sperm motility and survival at 38 degrees C were significantly (P<0.01) higher in Holstein-Friesian bulls at 60 degrees C and 80 degrees C than at 40 degrees C (39.79+/-2.46% and 38.15+/-2.18% Vs 35.16+/-2.19%, and 20.22+/-2.14% and 19.05+/-2.05% vs 14.83+/-1.64%, respectively). In Murrah buffalo bulls the recovery percentage and survival rate increased significantly (P<0.01) with the increase in temperature from 40 degrees C to 80 degrees C (41.72+/-2.45%, 47.45+/-2.09% and 51.61+/-2.06%; and 9.22+/-1.47%, 11.79+/-1.63% and 12.27+/-1.53%, respectively). Prefreeze motility did not differ between cattle and buffalo bulls (64.97+/-1.08% Vs 67.11+/-0.89%, respectively) but post-thaw motility was significantly (P<0.01) higher in the buffalo (46.93+/- 1.39% Vs 37.70+/-1.32%). While incubation survival was higher in the cattle (18.04+/-1.16% Vs 10.96+/-0.89%). A fast cooling rate was found to be detrimental for cattle spermatozoa, whereas the post-thaw buffalo sperm motility deteriorated very quickly at 38 degrees C. The influence of species-by-cooling rate interaction was significant (P<0.01) for post-thaw motility and survival rate, but the species-by-thawing or cooling-by-thawing interactions were not significant. These results suggest that a cooling rate of 2 hour either at 10 degrees C or 28 degrees C is essential for cattle semen. However, buffalo semen can be frozen successfully after 30 minutes of cooling at 10 degrees C. A thawing temperature of 60 degrees C yielded a higher sperm motility rate than 40 degrees C. Thus, our findings can be applied under tropical conditions for the successful freezing-thawing of bovine semen provided conception rates are not affected adversely.     

Characterization of sperm plasma membrane properties after cholesterol modification: consequences for cryopreservation of rainbow trout spermatozoa

During cryopreservation, the cell plasma membrane faces severe perils, including lipid phase separation, solute effects, and osmotic stresses associated with ice crystallization. How the initial biophysical properties of the plasma membrane can be modulated before cryopreservation in order to influence cellular resistance to the freeze-thaw stress is addressed in this study. Rainbow trout (Oncorhynchus mykiss) spermatozoa were chosen because the lack of an acrosome in this species suppresses potential interactions of cryopreservation with capacitation. Methyl-beta cyclodextrin-induced modulation of membrane cholesterol revealed the presence of a significant cholesterol exchangeable pool in the trout sperm plasma membrane, as membrane cholesterol content could be halved or doubled with respect to the basic composition of the cell without impairing fresh sperm motility and fertilizing ability. Biophysical properties of the sperm plasma membrane were affected by cholesterol changes: membrane resistance to a hypo-osmotic stress increased linearly with membrane cholesterol whereas membrane fluidity, assessed with DPH (1,6-diphenyl-1,3,5-hexatriene) and with several spin-labeled analogues of membrane lipids, decreased. Phosphatidyl serine translocation between the bilayers was slowed at high cholesterol content. The increased cohesion of fresh trout sperm plasma membrane as cholesterol increased did not improve the fertilizing ability of frozen-thawed sperm whereas the lowest cholesterol contents impaired this parameter of sperm quality. Our study demonstrated that cholesterol induced a stabilization of the plasma membrane in rainbow trout spermatozoa, but this stabilization before cryopreservation brought no improvement to the poor freezability of this cell.     

Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports

The aim of this study was to improve the freezing protocol of bull sperm, by investigating the influence on sperm viability after freeze/thawing of different freezing medium components, as well as the effect of cooling rates in the different stages of the cooling protocol, in single factor experiments. The experimental variables were: (1) salt-based versus a sugar-based medium (Tris versus sucrose); (2) glycerol concentration; (3) detergent (Equex) concentration; (4) presence of bicarbonate; (5) rate of cooling from 22 degrees C to holding temperature (CR1); (6) holding temperature (HT); (7) rate of cooling from holding temperature to -6 degrees C (CR2); (8) rate of cooling from -10 to -100 degrees C (CR3). All experiments were performed using five bulls per experiment (three ejaculates per bull). Sperm motility after freezing and thawing was assessed by CASA system, and sperm membrane integrity was assessed by flow cytometry. Sucrose-based medium did not offer a clear significant benefit compared to Tris medium. The concentration of Equex that gave the best results in Tris-based media group and sucrose-based media group was in a range between 2-7 and 4-7 g/l, respectively. In both media groups, a glycerol concentration of 800 mM was the best in any post-thaw viability parameters. In the Tris media group, the presence of bicarbonate had a negative effect on sperm viability. CR1 and CR2 had no significant effect on any of the post-thaw sperm viability parameters, but a CR1=0.2 degrees C/min and CR2=4 degrees C/min appeared to give better results in both media. The holding temperature (HT) that gave the best results was found to be in the range of 5-9 degrees C. There was a significant disadvantage of using a low CR3 of 10 degrees C/min, while 150 degrees C/min appeared to be the best cooling rate for either medium.