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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. Author contributions
  11. References

This study was conducted to determine the optimum level of glycerol and cholesterol-loaded cyclodextrin (CLC) in a Tris-based diluent for cryopreservation of ram spermatozoa. Ram semen was treated with 0, 1.5, 3 or 4.5 mg CLC/120 × 106 cells in Tris-based diluents containing 3, 5 or 7% glycerol in a factorial arrangement 3 × 4 and frozen in liquid nitrogen vapour. Sperm motility, viability (eosin–nigrosin staining) and functional membrane integrity (hypo-osmotic swelling test) were assessed immediately after thawing (0 h) and subsequently after 3 and 6 h at 37°C. There was an interaction between CLC and glycerol on the functional membrane integrity (p < 0.05). In the presence of 3% glycerol, the highest functional membrane integrity (32.2%) was found in the spermatozoa treated with 1.5 mg CLC/120 × 106 sperm. Post-thaw sperm motility was highest in 1.5 mg CLC immediately after thawing (40.5%) and after 3-h (30.6%) incubation at 37°C (p < 0.05). Viability of spermatozoa was higher in all CLC treatments than in the untreated samples, and it was highest (33.9%) in the spermatozoa treated with 1.5 mg CLC (p < 0.05). These data indicate that the addition of cholesterol to sperm membranes by 1.5 mg CLC/120 × 106 cells may allow the use of a lower concentration of glycerol (3%), which is sufficient to mitigate the detrimental effects of freezing and thawing.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. Author contributions
  11. References

Freezing is a destructive procedure for cell viability. During cryopreservation, spermatozoa encounter a stressful status that includes osmotic stresses produced by adding and removing cryoprotectant (Gilmore et al. 1995; Guthrie et al. 2002), alterations in membrane lipids induced by shifting liquid crystalline to gel state (Hammerstedt et al. 1990; Medeiros et al. 2002), and intracellular and extracellular ice formation (Mazur 1984).

Glycerol is commonly utilized as plasma membrane-permeant cryoprotectant to inhibit intracellular ice formation (Storey et al. 1998; Salamon and Maxwell 2000). It was reported that addition and removal of cryoprotectant, such as glycerol, induce osmotic damages (Hammerstedt et al. 1990; Gilmore et al. 1995; Guthrie et al. 2002). Shrinking and swelling are sperm responses to osmotic changes that can result in a significant loss of functional integrity (Guthrie et al. 2002) and consequently cell death.

Plasma membrane lipids go through a transition from liquid crystalline phase to a gel phase (lipid-phase transition) at low temperatures (Horvath and Seidel Jr. 2006). Specific phase transition temperatures are reported for the different phospholipids. In this situation, cooling results in lateral migrating and aggregating lipids in microdomains, which induce membrane gaps between the gel and remaining fluid membrane domains (Amann 1999). This cryodamage causes the membrane to become transiently leaky, thus compromising membrane integrity (Drobnis et al. 1993; Medeiros et al. 2002).

The cholesterol content of the sperm membrane and the ratio of cholesterol to phospholipids are species specific. This may be the reason for the differences in the sperm tolerance to cold shock (Darin-Bennett and White 1977). In comparison with bull and human spermatozoa, ram spermatozoa have lower molar rate of cholesterol/phospholipids (0.38 vs 0.45 and 0.99; Darin-Bennett and White 1977) and are more sensitive to cold shock (Muiño-Blanco et al. 2008). The detrimental effect of the osmotic stress and cold shock can be diminished by increasing cholesterol content of the membrane (Glazar et al. 2009; Mocé et al. 2010). Cyclodextrins, cyclic oligosaccharides, can be used to alter the cholesterol content of cell membranes (Visconti et al. 1999) and to insert cholesterol into membranes if they are pre-loaded with cholesterol (cholesterol-loaded cyclodextrin; Navratil et al. 2003). Recent studies have demonstrated that treatment with cyclodextrin pre-loaded with cholesterol (CLC) improves cryosurvival rates of pig (Zeng and Terada 2001), bull (Purdy and Graham 2004a), stallion (Moore et al. 2005) and ram (Mocé et al. 2010) spermatozoa. Loading cholesterol into sperm membrane increases membrane thickness by packing of the lipids, increases membrane condensation and modulates the membrane permeability to water (Müller et al. 2008) and cryoprotectants, such as glycerol (Li et al. 2006; Glazar et al. 2009). Glycerol is commonly used for freezing ram semen, but, its chemical toxicity was reported (Hammerstedt et al. 1990). There were no published reports on the concurrent effect of different concentrations of CLC and glycerol on ram semen freezing until now.

This study was conducted to determine the optimum level of glycerol and CLC in a Tris-based diluent for ram sperm cryopreservation in viability, motility and membrane functionality just after thawing and in vitro incubation.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. Author contributions
  11. References

Chemical reagents

The following chemicals were used: methyl-β-cyclodextrin (Sigma-Aldrich, St. Louis, MO, USA), tris[hydroxymethyl]aminomethane, citric acid monohydrate, glucose, fructose and sodium citrate dihydrate (AppliChem GmbH, Darmstadt, Germany), nigrosin, chloroform, methanol and glycerol (Merck, Darmstadt, Germany) and eosin (Panreac, EU, Spain).

Cholesterol-loaded cyclodextrin preparation

Cyclodextrins (methyl-β-cyclodextrin) were pre-loaded with cholesterol as described by Purdy and Graham (2004a). Briefly, 1 g of methyl-β-cyclodextrin was dissolved into 2 ml of methanol in a glass test tube. A 0.45 ml aliquot of cholesterol, dissolved in chloroform (200 mg/ml), was added to the methyl-β-cyclodextrin and stirred until the combined solution was clear, after which the mixture was poured into a glass Petri dish and the solvents were removed using a stream of nitrogen gas. The resulting crystals were allowed to dry for 24 h and were stored in a glass vial at 22°C until use. A working solution of CLC was prepared by adding 50 mg of CLC to 1 ml of a Tris diluent at 37°C and mixing the solution using a vortex mixer for 30 s.

Animals

Four mature Taleshi rams, aged between 3 and 5 years, were used. They were healthy and fertile. The animals were housed at University of Guilan, Faculty of Agricultural Sciences, Education Research and Practice Farm, South of Rasht (it is located at 37° 12′ North latitude and 49° 39′ longitude). Rams were fed daily with a diet of 1300 g alfalfa hay, 100 g rice straw, 200 g barely and 500 g concentrates (2.35 Mcal/kg ME, 14% CP, 0.9% Ca and 0.8% P). Animals had free access to salt lick and fresh water.

Semen collection

Semen was collected by artificial vagina four times with 2-day intervals between sessions, over two consecutive weeks in the breeding season from autumn to early winter. In each collection session, one ejaculate per ram was collected (a total of sixteen ejaculates). After collection, semen was diluted separately 1 : 1 (v:v) with Tris diluent (300 mm tris[hydroxymethyl]aminomethane, 95 mm citric acid monohydrate, 28 mm glucose, pH 7.0) and transported to the laboratory in an insulated Styrofoam box (30–33°C) within 45 min of collection.

Semen dilution, freezing and thawing

Immediately upon reaching the laboratory, the percentage of sperm motility and the concentration of spermatozoa in each ejaculate were determined (see 'Sperm assessment'). The initial dilution was considered, and all diluted ejaculates were tested to possess acceptable volume (>0.5 ml), motility (>70%) and concentration (>2.5 × 109 sperm/ml), and then, they were pooled.

Pooled semen was split into four fractions, and a total of 0 (control), 1.5, 3 and 4.5 mg CLC were added per 120 × 106 cells, and after that the samples were diluted to 1200 × 106 sperm/ml by Tris diluent without egg yolk and glycerol. The samples were incubated for 15 min at 22°C. Each fraction was split into three equal volumes and diluted 1 : 1 (v:v) with Tris diluent containing 6%, 10% or 14% glycerol and 40% egg yolk (resulting in final concentrations of glycerol = 3%, 5% and 7%, egg yolk = 20% and sperm = 600 × 106 cells/ml).

Diluted samples were packaged into 0.25-ml French straws, sealed with polyvinyl alcohol powder and cooled to 5°C over 2 h. The straws were frozen in liquid nitrogen vapour, with the straws horizontally suspended 4.5 cm above the liquid nitrogen for 13 min, before being plunged into liquid nitrogen for storage.

After 2 weeks, three straws from each treatment and replicate were thawed in a water bath at 37°C for 30 s, prior to analysis. Thawed semen was incubated at 37°C for 6 h. Motility, viability and functional membrane integrity were assessed immediately after thawing (0 h) and subsequently after 3- and 6-h incubation post-thawing.

Sperm assessment

The concentration of spermatozoa was determined by means of a Neubauer haemocytometer.

The percentage of sperm motility was assessed by phase-contrast microscopy (400× magnification) on a warm stage at 37°C. Samples were diluted with Tris–glucose 1 : 8, and then, a wet mount was made using a 5 μl drop of semen placed directly on a microscope slide and covered by a cover slip. Sperm motility was estimated in 3-7 different microscopic fields for each semen sample. The subjective estimations were approximated to the nearest 5% by single technician. The mean of the successive estimations was recorded as the final motility score (Evans and Maxwell 1987).

The viability was assessed by means of a one-step eosin–nigrosin staining (Björndahl et al. 2003). Briefly, equal volumes of semen and stain solution (0.67 g eosin Y, 0.9 g sodium chloride and 10 g nigrosin in 100 ml distilled water) were incubated for 30 s at room temperature (22°C). One drop of mixture was put on a slide, instantly smeared and air dried. A total of 200 sperm were evaluated under light microscope (1000× magnification, oil immersion). Sperm showing partial or complete pink or red colour was considered dead, and sperm showing strict exclusion of the stain was considered to be alive.

The hypo-osmotic swelling test (HOST) was used to evaluate the functional integrity of the sperm membrane. The procedure was described by Jeyendran et al. (1992) and adapted for ram semen by García-Artiga (1994). HOST was performed by incubating 5 μl of semen with 500 μl of a 100 mOsm hypo-osmotic solution (7.35 g sodium citrate dihydrate and 13.51 g fructose in 1 L distilled water) at 37°C for 30 min. One drop of the mixture was placed on a pre-warmed slide, covered with a cover slip and examined under a phase-contrast microscope (400× magnification). The sperm with swollen tails were considered intact. To assess the percentages of intact sperm, a total of 200 sperm were evaluated in at least five different microscopic fields.

Statistical analysis

All data on motility, viability and functional membrane integrity of sperm were recorded at 0-, 3- and 6-h incubation after thawing and analysed by the MIXED procedure of SAS (SAS Institute Inc 2002). The statistical design was a 3 × 4 factorial arrangement of the twelve treatment combinations as fixed effects and the three incubation times as a repeated measure. Pooled semen was considered as subjects in this experiment. The CLC levels, glycerol percentage, incubation time and their interactions were defined as class variables. Results are reported as least-squares means (LSM) ± SE. Differences were considered to be statistically significant at p < 0.05.

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. Author contributions
  11. References

Motility, viability and functional membrane integrity of spermatozoa were higher in all CLC treatments than untreated samples, and they were highest in the spermatozoa treated with 1.5 mg CLC, regardless of glycerol concentration (p < 0.05; Table 1). The main effect of glycerol showed that sperm motility was highest in 3% glycerol (p < 0.05), and there was no difference between 5 and 7% glycerol (p > 0.05).

Table 1. Main effect of cholesterol-loaded cyclodextrin (CLC) and glycerol on motility, viability (eosin–nigrosin staining) and functional membrane integrity (HOST) of ram spermatozoa after freezing and thawing
Main effect Motility (%)Viability (%)HOST (%)
  1. a-dDifferent superscripts within main effect denote significant differences (p < 0.05).

  2. Presented data are LSM ± SE.

CLC

(mg/120 × 106 sperm)

019.0 ± 0.7c23.7 ± 0.9c25.2 ± 0.6d
1.530.6 ± 0.7a33.9 ± 0.9a31.5 ± 0.6a
326.5 ± 0.7b31.4 ± 0.9b29.3 ± 0.6b
4.525.3 ± 0.7b30.7 ± 0.9b26.9 ± 0.6c
Glycerol (%)327.2 ± 0.6a29.5 ± 0.827.7 ± 0.5
525.2 ± 0.6b30.3 ± 0.828.4 ± 0.5
723.6 ± 0.6b30.0 ± 0.828.6 ± 0.5

There was no interaction between incubation time and glycerol or CLC in response to HOST (p > 0.05). On the contrary, an interaction was observed between CLC and glycerol in response to HOST (p < 0.05, Table 2). In the presence of 3% glycerol, 1.5 mg CLC/120 × 106 sperm resulted in the highest response to HOST, and the highest difference was recorded between 0 and 1.5 mg CLC (p < 0.0001). The response to HOST was higher in the spermatozoa treated with 3 mg CLC/120 × 106 sperm than those treated with 0 and 4.5 mg CLC/120 × 106 sperm in the presence of 5% glycerol (p < 0.05). The highest response to HOST was found in the spermatozoa treated with 1.5 and 3 mg CLC/120 × 106 sperm in the presence of 7% glycerol (p < 0.05). In the spermatozoa treated with 1.5 mg CLC, there was no difference in the functional membrane integrity among the levels of glycerol (p > 0.05). In the spermatozoa treated with 3 mg CLC, the functional membrane integrity was lower in the treatment containing 3% glycerol than those containing 5 and 7% glycerol (p < 0.05); in addition, there was no difference between 5 and 7% glycerol (p > 0.05).

Table 2. Percentage of functional membrane integrity (HOST) of thawed ram spermatozoa treated with 0, 1.5, 3 or 4.5 mg CLC/120 × 106 sperm in the presence of different glycerol levels
Glycerol (%)CLC (mg/120 × 106 sperm)HOST (%)
  1. a–cDifferent superscripts within the column denote significant differences (p < 0.05).

  2. Data are LSM ± SE.

3025.0 ± 1.6c
1.532.2 ± 1.6a
326.4 ± 1.6c
4.527.3 ± 1.6bc
5025.5 ± 1.6c
1.529.8 ± 1.6ab
330.9 ± 1.6a
4.527.2 ± 1.6bc
7025.1 ± 1.6c
1.532.7 ± 1.6a
330.4 ± 1.6a
4.526.2 ± 1.6c

There was an interaction between concentration of glycerol and incubation time on sperm motility (p < 0.05). Post-thaw sperm motility was highest in 3% glycerol at 3- and 6-h incubation (p < 0.05; Fig. 1a). Due to significant effect between CLC and incubation time on the sperm motility, corresponding interaction is presented in Fig. 2 (p < 0.05). Post-thaw sperm motility was highest in 1.5 mg CLC immediately after thawing (0 h) and 3-h incubation at 37°C (p < 0.05).

image

Figure 1. (a) Percentage of motility and (b) viability of thawed ram spermatozoa preserved with either 3 (▲), 5 (●) or 7% (-□-) glycerol during incubation at 37°C.a-f Different superscripts indicate significant differences among treatments (p < 0.05)

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image

Figure 2. Percentage of motility of thawed ram spermatozoa treated with either 0 (×), 1.5 (●), 3 (-□-) or 4.5 (♦) mg CLC/120 × 106 sperm during incubation at 37°C.a-h Different superscripts indicate significant differences among treatments (p < 0.05)

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There was an interaction between concentration of glycerol and incubation time on sperm viability (p < 0.05). At 0, 3 or 6 h, there was no difference among the levels of glycerol on sperm viability (p > 0.05; Fig. 1b). There was no interaction between CLC and incubation time on the viability (p > 0.05).

Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. Author contributions
  11. References

Ram spermatozoa are especially sensitive to cold shock due to their lower molar rate of cholesterol/phospholipids in comparison with other species. For this reason, it is important to reduce the detrimental effect of cold shock, for example increasing the cholesterol content on cell membrane.

We observed that post-thaw sperm motility and sperm viability were higher in all spermatozoa treated with CLC than untreated spermatozoa; there was, although, no difference in the functional membrane integrity between the spermatozoa treated with 0 and 4.5 mg CLC at all levels of glycerol. Therefore, our data demonstrate that the application of up to 3 mg CLC prior to freezing incorporates cholesterol into the plasma membrane, thus improving post-thaw sperm longevity. This was in agreement with previous studies (Zeng and Terada 2001; Purdy and Graham 2004a; Moore et al. 2005). Mocé et al. (2010) suggested that maintaining higher cholesterol concentrations in sperm increases longevity after thawing, possibly via decreasing the capacitation-like changes due to freezing. In contrast, the 6 mg/ml (1.8 mg/120 × 10sperm) CLC did not provide a protective effect on sex-sorted spermatozoa during freezing and thawing; this fact is likely due to the detrimental effect of the high membrane fluidity prior to flow cytometric sorting (de Graaf et al. 2007). In bovine, control and CLC-treated sperm (1.5 mg CLC/120 × 10cell) undergo capacitation and the acrosome reaction at similar rates after freezing–thawing, and pregnancy rates are also similar for control and CLC-treated sperm (Purdy and Graham 2004b). Moreover, fertility and prolificacy rates were not affected by treating 2 mg CLC/120 × 106 sperm and storing for 24 h at 5°C prior to cryopreservation comparing with fresh semen in ovine (Purdy et al. 2010). Therefore, under routine methods of semen dilution and freezing, it seems that increase in sperm membrane cholesterol may provide a protective effect for cryosurvival.

In this experiment, there was no interaction between glycerol and CLC on sperm motility and viability. The lack of an interaction implies that these two treatments act in additive manner (Zar 1999). The sperm motility was highest in the 3% glycerol, and the highest viability was found in the 1.5 mg CLC. Moreover, the functional membrane integrity was higher in 1.5 mg CLC-3% glycerol than 0 mg CLC-5% glycerol. Consequently, we suggest that 3% glycerol and 1.5 mg CLC per 120 × 106 cells may be the best to enhance sperm longevity. Several observations demonstrated that, comparing with the control group, treating spermatozoa with CLC before cryopreservation increases the percentages of motile and viable cells after freezing and thawing; moreover, the addition of ≤2 mg CLC/120 × 106 cells results in the highest quality (Purdy and Graham 2004a; Mocé and Graham 2006; Mocé et al. 2010; Awad 2011). It was demonstrated that high level of cholesterol increases the membrane permeability to cryoprotectants such as glycerol (Li et al. 2006; Glazar et al. 2009). Treating sperm with CLC increases membrane resistance to a hypo-osmotic stress through altering membrane permeability (Müller et al. 2008; Glazar et al. 2009). Therefore, under treating with 1.5 mg CLC/120 × 106 sperm, the minimum level of glycerol (3%) may be helpful for enhancing the cryosurvival of ram spermatozoa.

The novelty of this study, which is the application of low CLC, is the possibility to reduce the amount of glycerol needed for freezing. The best results have been obtained with 4–6% glycerol (Byrne et al. 2000; Anel et al. 2003). The addition of a lower amount of glycerol would possibly reduce osmotic stress and cell volume changes; therefore, more longevity occurs when the cryoprotectants are added or removed. For this reason, there is a tendency to use a low concentration of glycerol. Our results support the hypothesis that treating spermatozoa with 1.5 mg CLC will decrease the amount of glycerol needed for freezing ram spermatozoa.

Our results showed that sperm motility and sperm viability are higher in spermatozoa treated with 4.5 mg CLC than in those untreated. Nevertheless, these parameters were lower in spermatozoa treated with 4.5 mg CLC than in those treated with 1.5 mg CLC. Moreover, there was no difference in the functional membrane integrity between spermatozoa treated with 4.5 and 0 mg CLC. Consequently, our results demonstrate that 4.5 mg CLC/120 × 106 sperm cannot improve sperm survival under freezing procedure when compared to spermatozoa treated with 1.5 mg CLC. An increase in membrane cholesterol content affects membrane condensation and membrane permeability to small polar molecules (Bloom et al. 1991). It is expected to reduce passive molecules' movement across the condensed plasma membrane. Although the membrane fluidity is considered to be closely related to the membrane condensation, which decreases membrane fluidity (Bloom et al. 1991), it seems that membrane fluidity and permeability diverge at high cholesterol membrane content; in contrast to the continuous increase in membrane resistance to osmotic stress, fluidity no longer decreases (Müller et al. 2008). Sperm cell undergoes a reduction in cell volume about a half of the isotonic volume when glycerol is added or water is lost during freezing (Parks and Graham 1992). It expands to over 2-fold the isotonic volume when suspended in isotonic solution after thawing (Parks and Graham 1992). Membrane thickness may affect the ability of cell volume changes without lysis. Therefore, the high protection of the low level of CLC in comparison with the higher level of CLC may be due to suitable cholesterol content and thickness of membrane, which may help to tolerate changes in cell volume in the presence of cooling and freezing stressors.

Conclusion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. Author contributions
  11. References

Adding a little cholesterol to sperm membranes by CLC technology (1.5 mg/120 × 106 cells) may enable a reduction in the concentration of glycerol (3%) sufficient to protect against detrimental effects of freezing–thawing.

Acknowledgements

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. Author contributions
  11. References

The authors wish to thank Dr. M. Zavareh and Dr. N. Ghavi Hossein-Zadeh (Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran) for their valuable help with the statistical analysis and Prof. C. Tamanini (Dipartimento di Morfofisiologia Veterinaria e Produzioni Animali, Universita, di Bologna, Bologna, Italy) for English revision.

Author contributions

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. Author contributions
  11. References

R Motamedi-Mojdehi performed the assays, M Roostaei-Ali Mehr designed the experiment and R Rajabi-Toustani performed technical assistance.

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  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. Author contributions
  11. References
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