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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Department of Biomedical Sciences, Colorado State University, Fort Collins 80523
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Abstract |
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Key Words: bull sperm • cholesterol • cryopreservation • cyclodextrin • freezing rate
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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; Medeiros et al., 2002) that reduce cell survival and alter surviving sperm function (Curry, 2000), in turn, reducing cell longevity and fertility compared with fresh sperm. Previous research has improved sperm survival, but still only about one-half of the sperm survive freezing (Vishwanath and Shannon, 2000). One dairy industry goal is to reduce the number of sperm in each breeding unit so that more breeding units can be made from each ejaculate (Vishwanath and Shannon, 2000), but this can only be achieved by improving cell cryosurvival.
Sperm sensitivity to "cold shock" damage is determined by the membrane phospholipid composition and the membrane cholesterol to phospholipid ratio (Holt, 2000). Sperm possessing high cholesterol:phospholipid ratios (e.g., rabbit and human) are more resistant to cold shock damage than sperm having low cholesterol:phospholipid ratios (e.g., stallion, ram, and bull; Watson, 1981; Parks and Lynch, 1992; White, 1993).
Cyclodextrins can be used to alter the cholesterol content of cell membranes (Christian et al., 1997; Visconti et al., 1999), and if cyclodextrins are preloaded with cholesterol they insert cholesterol into membranes (Navratil et al., 2003). If stallion (Combes et al., 2000; Moore et al., 2005a), bull (Purdy and Graham, 2004), or ram (Morrier et al., 2004) sperm are treated with cholesterol-loaded cyclodextrins (CLC) before freezing, they exhibit greater cryosurvival rates than untreated sperm. However, CLC were added to sperm at sperm concentrations (~120 x 106 sperm/mL) that are impractical for industry settings. If CLC could be added to fresh ejaculates and still benefit sperm cryosurvival, this technology could have practical application.
These studies were conducted to determine if the sperm concentration alters the beneficial effects of CLC on sperm cryosurvival, and to develop a procedure for adding CLC to fresh bull ejaculates to improve sperm cryosurvival.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Animals
Semen was collected using an artificial vagina from 10 mature Holstein bulls housed at the Animal Reproduction Laboratory at Colorado State University (Fort Collins). The bulls were fed a diet that provided 100% of their nutritional needs (NRC, 2000) and provided water ad libitum. All animal care and procedures used to collect semen were approved by the Animal Care and Use Committee of Colorado State University.
Materials
All chemicals were reagent grade and purchased from Sigma (St. Louis, MO), except for SYBR-14 and propidium iodide (PI), which were purchased from Molecular Probes (Eugene, OR).
Cyclodextrin Preparation
Methyl-ß-cyclodextrin was preloaded with cholesterol as described by Purdy and Graham (2004). Briefly, 200 mg of cholesterol was dissolved in 1 mL of chloroform. In a separate test tube, 1 mg of cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of cholesterol was added to the cyclodextrin, and the mixture was stirred until the combined solution was clear. The solution was then poured into a glass Petri dish, and the solvents were removed and allowed to dry for 24 h, after which the resulting crystals were removed from the dish and stored in a glass container at room temperature. A working solution of CLC was prepared by adding 50 mg of CLC to 1 mL of Tris diluent (250 mM tris[hydroxymethyl]aminomethane, 83 mM citric acid monohydrate, 69 mM D-(+)-glucose, pH = 7.0, 300 mOsm) at 37°C and mixing the solution briefly using a vortex mixer (Purdy and Graham, 2004).
Analysis of Spermatozoa
The percentages of motile and progressively motile spermatozoa in each sample were determined using a computer-assisted sperm analysis system (IVOS Version 10.7s; Hamilton Thorne Research, Bedford, MA) with settings of 30 frames acquired to avoid sperm track overlapping, minimum contrast = 50, minimum average path velocity = 25 µm/s, straightness = 80%, nonmotile head size = 5, and nonmotile head intensity = 90. For each sample, 6-µL subsamples were placed on slides and a minimum of 200 cells per subsample were analyzed. A sperm was defined as being progressively motile if the average path velocity was >60 µm/s, and straightness was >80%. Sperm straightness is the ratio of the straight-line velocity divided by the average path velocity, as defined by Budworth et al. (1988).
The percentages of viable spermatozoa in the samples were determined using flow cytometry, as described by Purdy and Graham (2004). Briefly, spermatozoa were stained for flow cytometric analysis by transferring a 0.1-mL aliquot from each sample into a tube containing 0.45 mL of Tris-BSA (6 mg/mL) diluent, 8.5 µL of SYBR-14 (10 µM solution in dimethyl sulfoxide), and 5 µL of PI (1 mg/mL solution in distilled water). The samples were incubated for 10 min at 22°C and filtered through a 40-µm nylon mesh before being analyzed using an Epics V flow cytometer (Coulter Electronics, Miami, FL) equipped with an argon ion laser tuned to 488 nm at 100 mW of power. Fluorescence from 50,000 cells was measured using a 515-nm long-pass filter and a 525-nm band-pass filter to detect SYBR-14, and a 590-nm dichroic mirror and a 630-nm long-pass filter to detect PI. Using this protocol, all cells stained with SYBR-14, which permitted the cells to be distinguished from egg yolk particles, but only nonviable cells stained with PI.
Experiment 1: Effect of Sperm and CLC Concentrations on Cell Cryosurvival
Immediately after collection, the concentration of spermatozoa in each of 10 ejaculates was determined photometrically, and the spermatozoa were diluted to 5 sperm concentrations (from 120 to 2,000 x 106 cells/mL) with the Tris diluent. Only 3 ejaculates had a sperm concentration of at least 2,000 x 106 cells/mL; therefore, this treatment had only 3 replicates. Each of the 5 sperm concentrations was then split into 3 CLC-treatment aliquots (0.5 mL each for the samples at 120 and 360 x 106 cells/mL, 0.4 mL for the samples at 720 x 106 cells/mL, and 0.2 mL for the samples at 1,200 and 2,000 x 106 cells/mL). One of the aliquots was used as a control (non-CLC treated), 1 was treated with 2 mg of CLC/120 x 106 sperm, and the other was treated with 4 mg of CLC/120 x 106 sperm. Samples were incubated for 15 min at 22°C, after which each sample was diluted to 60 x 106 cells/mL with egg yolk-Tris diluent (Tris diluent with 20% egg yolk).
Spermatozoa were then cooled slowly to 5°C over 2 h, and then diluted 1:1 (vol:vol) with glycerolated (17.5% glycerol) egg yolk-Tris diluent (resulting in a final glycerol concentration of 8.75%), and equilibrated for 15 min. Sperm were then packaged into 0.5-mL straws (IMV Technologies, L’Aigle, France) and frozen in liquid nitrogen vapor, with the straws being suspended horizontally at 4.5 cm above the liquid nitrogen for 10 min before being plunged into the liquid nitrogen for storage.
Two straws from each treatment were thawed in a water bath at 37°C for 30 s before analysis. The contents of one straw were used to determine the percentage of motile sperm in each treatment, and that of the second straw used to determine the percentage of viable sperm for each treatment.
Analysis of variance (2005 version, SAS Inst. Inc., Cary, NC) was used to determine the main effects of sperm concentration (5 levels), CLC level (3 levels), and the interaction of these effects. Individual treatment means were separated using Student-Newman-Keuls multiple range test (SAS Inst. Inc.). However, we also were interested in knowing if the sperm concentration affected cell survival; therefore, ANOVA was used to determine if different sperm concentrations within a CLC level resulted in different cryosurvival rates; these data are presented in the tables. In this and all experiments, the effects were considered significant if P < 0.05.
Experiment 2: Effect of Sperm Concentration, CLC Level, and Temperature of Incubation on Sperm Cryosurvival
Nine ejaculates were collected as described above, the concentration of sperm in each ejaculate was determined photometrically, and each ejaculate was split into 4 fractions. Three of the fractions were then diluted to 120, 1,200, or 
1,500 x 106 cells/mL (this highest sperm concentration utilized the fresh ejaculates, which ranged from 1,500 to 2,000 x 106 cells/mL) with the Tris diluent. Each of these was then split into 4 treatment aliquots (for 120 x 106 sperm/mL, 0.5 mL aliquots were used, and for the samples at 1,200 and 1,500 x 106 cells/mL, 0.2 mL aliquots were used). One set of 2 aliquots was treated with a total of 2 and 4 mg CLC/120 x 106 cells, and was incubated for 15 min at 22°C. The other set of 2 aliquots was treated similarly, but was incubated at 37°C. The fourth fraction was not diluted, but was maintained as the control ejaculate (non-CLC treated); it was split and 1 aliquot was maintained at 22°C, whereas the other was maintained at 37°C for 15 min. After incubation, each sample was diluted to 60 x 106 cells/mL in egg yolk-Tris diluent and cooled to 5°C as described above. Upon reaching 5°C, each aliquot was diluted 1:1 with glycerolated egg yolk-Tris diluent, frozen, thawed, and evaluated as described above.
As in Exp. 1, ANOVA (SAS Inst. Inc.) was used to determine the main effects of sperm concentration (3 levels), CLC level (3 levels), and temperature of incubation (2 levels), and all of their interactions. Individual treatments within each main effect were separated using Student-Newman-Keuls multiple range test (SAS Inst. Inc.). In this study, we were interested in the sperm concentration x CLC level treatment differences within each incubation temperature. Therefore, the data are presented as such in the tables.
Experiment 3: Effect of Freezing Rate
Immediately after collection, the concentration of spermatozoa in each of 9 ejaculates was determined and each ejaculate was split into 2 aliquots. One aliquot was used as a control (no CLC treatment) and the other was treated with 2 mg of CLC/120 x 106 sperm, and incubated for 15 min at 22°C. After incubation, 60 x 106 cells were added to 1 mL of the egg yolk-Tris diluent and then cooled to 5°C, diluted 1:1 with glycerolated egg yolk-Tris diluent, and packaged into 0.5-mL plastic straws.
Sperm were frozen either in the liquid nitrogen vapor, or in a programmable freezer (Planer Kryo 10 Series III, TS Scientific, Perkasie, PA). Straws were frozen in the programmable freezer at 8 freezing rates (–5, –8, –10, –12, –15, –25, –35, or –50°C/min) from 5°C until they reached –80°C, and then were stored in liquid nitrogen. For the samples frozen in liquid nitrogen vapor, straws were suspended horizontally for 10 min at 8 heights (1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, or 8.5 cm) above the liquid nitrogen before being plunged into the liquid nitrogen for storage. Straws were thawed and evaluated as described above.
The cooling rates used for the straws frozen in liquid nitrogen vapor were determined by measuring the temperature inside a straw at each level using a thermocouple, and calculating the initial cooling rate.
For this experiment, ANOVA (SAS Inst. Inc.) was used to evaluate the main effects of CLC treatment and cooling rates, and the interaction between them. The Student-Newman-Keuls multiple range test (SAS Inst. Inc.) was used to separate the cooling rate means.
Experiment 4: Addition of CLC to Undiluted Ejaculates
Immediately after collection, the concentration of spermatozoa in each of 61 ejaculates was determined, and each ejaculate was split into 2 aliquots. One aliquot was used as a control (no CLC treatment) and the other was treated with 2 mg of CLC/120 x 106 sperm, and incubated for 15 min at 22°C. After incubation, 60 x 106 cells were added to 1 mL of the egg yolk-Tris diluent, cooled to 5°C, diluted 1:1 with glycerolated egg yolk-Tris diluent, frozen, thawed, and evaluated as described above.
In this experiment, ANOVA (SAS Inst. Inc.) was used to determine if the cryosurvival rate of CLC-treated sperm was different from control (nontreated) sperm.
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RESULTS |
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The initial concentration to which sperm were diluted before CLC treatment and subsequent cryopreservation did not affect the percentage of motile sperm after thawing. However, sperm concentration at the time of CLC addition did affect the percentages of progressively motile sperm and live sperm after thawing. Progressive motility was less for CLC-treated sperm diluted to 120 x 106 sperm/mL (14%) than for sperm treated with CLC at 2,000 x 106 sperm/mL (28%; P < 0.05; data not shown). Similarly, the percentage of live sperm was less for samples diluted to 120 x 106 sperm/mL before CLC treatment (37%) than for samples treated with CLC at greater sperm concentrations (49 to 56%; P < 0.05; data not shown). Analysis for the main effect of CLC revealed that samples treated with 2 or 4 mg of CLC/120 x 106 sperm exhibited greater percentages of live cells (61 and 48%, respectively) than control (38%) sperm (P < 0.05; data not shown). In addition, a significant interaction between sperm concentration and CLC level was observed in the percentages of live cells. This interaction was specifically due to a detrimental effect of CLC treatment to samples diluted to 120 x 106 sperm/mL before CLC addition, whereas this same CLC treatment benefited sperm at all other sperm concentration treatments.
The concentration of sperm to which cells were initially diluted before adding CLC did not affect postthaw percentages of total motile and live cells, except for sperm treated with 4 mg of CLC/120 x 106 sperm. At this CLC concentration, sperm diluted to 120 x 106/mL before CLC addition exhibited lower percentages of live cells, total motile cells, and progressively motile cells than samples at greater initial sperm concentrations (P < 0.05; Table 1).
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Similar to Exp. 1, sperm concentration affected the percentages of total motile and live sperm, after cryopreservation (P < 0.05). Again, the percentages of motile sperm and live sperm were less for samples diluted to 120 x 106 sperm/mL (47 and 40%, respectively; data not shown) than for samples at greater sperm concentrations (56 to 63% and 51 to 52%, respectively; P < 0.05). In addition, adding CLC to the samples improved the percentages of total motile (52 to 58%), and live cells (44 to 51%) compared with control samples (44 and 21%, respectively; P < 0.05; data not shown). No interactions between the main effects of sperm concentration, CLC concentration, and temperature were found for any sperm variable.
Temperature of incubation did not affect the percentages of total motile or live sperm except for sperm diluted to 120 x 106/mL and treated with 2 mg of CLC (Table 2). Cells diluted to 2,000 x 106 sperm/mL and treated with CLC exhibited greater percentages of both motile and live sperm after thawing than control sperm (P < 0.05; Table 2). However, sperm at lower concentrations generally showed greater percentages of live cells when treated with CLC, although percentages of motile cells were often not different from controls (Table 2).
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Similar to previous experiments, samples treated with CLC before freezing exhibited greater percentages of total motile, progressively motile, and live sperm (72, 14, and 33%, respectively) than control sperm (55, 10, and 21%, respectively; P < 0.05; data not shown).
The cooling rates for sperm frozen at different distances above the liquid nitrogen level ranged from –51 to –114°C/min (Table 3). However, samples frozen at different levels above the liquid nitrogen level exhibited similar percentages of total and progressively motile sperm at all cooling rates and CLC levels (0 or 2 mg/ 120 x 106). Differences in the percentages of live sperm were observed among cooling rates when CLC-treated sperm were frozen 1.5 cm above the liquid nitrogen level. This rapid cooling rate resulted in lower percentages of live cells (21%) than samples frozen at 6.5 cm (38 to 41%; P < 0.05; Table 3). Control and CLC-treated sperm exhibited a difference in percentage of total motile sperm only when cells were frozen 3.5 cm above the liquid nitrogen (53 vs. 77%, respectively; P < 0.05; Table 3). For the other cooling rates, similar percentages of motile control and CLC-treated sperm were observed, although the percentages of total motile sperm were between 10 and 24% greater for the CLC-treated samples than for the control samples (Table 3). Sperm treated with CLC exhibited greater percentages of progressively motile sperm than control samples when sperm were frozen 1.5 and 4.5 cm above the liquid nitrogen level (P < 0.05; Table 3). Percentages of live cells were significantly greater (P < 0.05) for CLC-treated sperm than for control samples (between 14 and 20%; Table 3) when sperm were frozen 5.5, 6.5, 7.5, and 8.5 cm above the liquid nitrogen level.
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When undiluted ejaculates were treated with 2 mg of CLC/120 x 106 sperm before cryopreservation, greater percentages of live cells were observed (62%) than for control sperm (45%; P < 0.05; Table 5), although the percentages of total motile sperm and progressively motile sperm were similar for the control and the CLC-treated sperm (Table 5).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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; Purdy and Graham, 2004; Moore et al., 2005a). Regardless of sperm concentration, 2 mg of CLC/120 x 106 sperm increased both the percentages of motile sperm and live sperm at all sperm concentrations, including when CLC were added to fresh ejaculates. However, when the sperm concentration was adjusted to 120 x 106 sperm/mL before CLC treatment, treatment with 4 mg of CLC/120 x 106 cells reduced sperm cryosurvival. This could be due to a reduced egg yolk concentration in the final sample when the sperm concentration was adjusted to 120 x 106 cells/mL (15% egg yolk) with respect to the other treatments (between 18.3 and 19.75% egg yolk), or because this CLC concentration is detrimental for sperm survival when added to low sperm concentrations. However, neither possibility explains this result completely, because Graham and Hammerstedt (1992) did not observe differences in bull sperm cryosurvival when sperm were frozen in diluents containing either 1% or 20% egg yolk, and Purdy and Graham (2004) did not observe a decrease in sperm cryosurvival when sperm were treated with CLC until CLC concentration reached >6 mg/120 x 106 cells. Even then, cryosurvival of CLC-treated sperm remained greater than that of control samples. Therefore, the reason for decreased sperm cryosurvival for samples containing this low sperm concentration when treated with 4 mg CLC in this particular experiment is not known.
The temperature at which sperm were incubated with CLC had no effect on subsequent sperm cryosurvival. Only when samples were adjusted to 120 x 106 sperm/mL before treatment with 2 mg of CLC, did incubation temperature affect cryosurvival. In this situation, sperm incubated at room temperature exhibited greater percentages of total and progressively motile sperm than did the sperm incubated at 37°C. These results are similar to those reported previously by Purdy and Graham (2004) for bull sperm. Although we did not observe differences between incubation temperatures for the control samples, the trend was the same (sperm incubated at 22°C exhibited 5 percentage points greater motile and live sperm than the sperm incubated at 37°C). Because sperm can be incubated with CLC at either of the temperatures tested, this protocol is sufficiently flexible with respect to the incubation temperature to make its use feasible for industry application.
When sperm are frozen at suboptimal freezing rates, sperm cryosurvival decreases. A cooling rate that is too slow exposes spermatozoa to the "solution effects" of increasing salt concentration (osmolality increases) and changing pH. Moreover, a cooling rate that is too fast does not allow intracellular water sufficient time to leave the cell, which leads to intracellular ice nucleation (Vishwanath and Shannon, 2000). This experiment was conducted to determine if CLC-treated sperm require a different cooling rate than that for control sperm. However, sperm cryosurvival was similar for all freezing rates tested for sperm frozen in a programmable freezer. For sperm frozen at different heights above the liquid nitrogen level, differences were only observed in the percentages of live cells for CLC-treated sperm, which exhibited greater percentages of live cells when samples were frozen at 6.5, 7.5, and 8.5 cm above the liquid nitrogen level (estimated cooling rates between –51 and –65°C/min). Previous studies report contradictory results with respect to the optimal rate for freezing bull sperm. Thus, optimal freezing rates for bull sperm have been reported between –15 to –52°C/min (Robbins et al., 1976; Chen et al., 1993; Kumar et al., 2003) and –40 to –140°C/min (Woelders et al., 1997). Differences in the percentages of live cells between CLC-treated and control sperm were only observed when sperm were frozen at a rate of –8, –12, or –50°C/min, for sperm frozen in a programmable freezer, whereas differences in the percentages of live cells between CLC-treated and control sperm were only observed when sperm were frozen at 5.5, 6.5, 7.5, and 8.5 cm above the liquid nitrogen level. However, the percentages of motile and live cells were greater at all the freezing rates for CLC-treated sperm, compared with control sperm.
Addition of CLC to fresh ejaculates resulted in greater percentages of live cells after thawing, compared with the non-CLC treated samples, although the percentages of motile sperm were similar between CLC-treated and control sperm. As discussed above, the increase in the percentages of live cells is similar to that reported for sperm from bulls and stallions treated with CLC (Combes et al., 2000; Purdy and Graham, 2004; Moore et al., 2005a). However, previous studies generally report an increase in the percentages of motile sperm after cryopreservation (Combes et al., 2000; Purdy and Graham, 2004; Moore et al., 2005a) as well. The reason we did not observe a similar increase in motile sperm is not known. However, the motility analyses for stallion sperm were performed after cryopreserved sperm were further diluted 2- to 4-fold after thawing (Combes et al., 2000; Moore et al., 2005a), similar to the dilution performed for the viability analysis in this study (dilution 1:4.5). Perhaps differences in motility between CLC-treated and nontreated sperm are more easily separated for sperm subjected to osmotic stress. This might be expected because CLC treatment increases the osmotic tolerance limits of stallion sperm compared with the control samples (Moore et al., 2005b). However, Purdy and Graham (2004) reported an increase in the percentage of motile sperm after thawing for bull sperm that had been treated with CLC before cryopreservation, when motility analyses were performed on samples that were not diluted further after thawing. The fact that treating sperm with CLC before cryopreservation resulted in increased percentages of live cells is very important, because these cells have experienced all the processes that sperm have to undergo osmotically when inseminated into a female.
The exact mechanism by which added cholesterol protects sperm membranes is not known. As stated earlier, species with high cholesterol to phospholipid ratios (rabbit and human) are resistant to cold shock. At least part of the sperm damage induced from cold shock is due to the lipid phase transition that the membrane experiences during the cooling process. Human sperm are resistant to cold shock, and an abrupt lipid phase transition has not been detected when they are cooled (Drobnis et al., 1993). High cholesterol levels stabilize membranes during cooling. The cholesterol content in the membranes of bull and stallion sperm increased 2-to 3-fold compared with control sperm after treatment with CLC (Purdy and Graham, 2004; Moore et al., 2005a), and it remains greater than in control sperm after cryopreservation (Moore et al., 2005a). This increased cholesterol content in bull and stallion sperm raises the cholesterol to phospholipid ratio in these sperm to values similar (0.82 to 0.9; Purdy and Graham, 2004; Moore et al., 2005a) to those of sperm that are cold-shock resistant (0.88 and 0.99 for rabbit and human sperm, respectively; Watson, 1981). It is likely that the lipid phase transition is eliminated or the temperature at which it occurs is lower for CLC-treated bull and stallion sperm than for control sperm. Supporting this idea, Purdy et al. (2005) demonstrated increased membrane fluidity at lower temperatures for bull sperm treated with CLC than for untreated sperm. On the other hand, cholesterol could be increasing sperm membrane permeability to cryoprotectants and lessening osmotic cell damage, because CLC treatment increases the osmotic tolerance of stallion sperm (Moore et al., 2005b). This is very important because sperm experience large volume changes when cryoprotectants are added or removed, and their membranes can suffer damage during these processes (Graham, 1996). Further studies need to be conducted to determine the exact mechanism by which cholesterol affects sperm membranes during cooling.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Corresponding author: jkgraham{at}colostate.edu
Received for publication August 8, 2005. Accepted for publication November 9, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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