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Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm
Animal Reproduction Science 118 (2010) 194–200
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm C.H. Oliveira a , A.B. Vasconcelos b , F.A. Souza a , O.A. Martins-Filho c , M.X. Silva a , F.C. Varago a , M.A. Lagares a,∗ a
Department of Veterinary Clinics and Surgery, Veterinary School of the Federal University of Minas Gerais, Av. Antonio Carlos 6627, CEP: 31270-901, Belo Horizonte, MG, Brazil Instituto de Estudos Avanc¸ados em Veterinária José Caetano Borges/Universidade de Uberaba/FAZU/ABCZ, Av. Tutunas 720, CEP: 38061-500, Uberaba, MG, Brazil c Laboratório de Biomarcadores de Diagnóstico e Monitorac¸ão, Centro de Pesquisas René Rachou - Fiocruz, Av. Augusto de Lima 1715, CEP: 30190-002, Belo Horizonte, MG, Brazil b
a r t i c l e
i n f o
Article history: Received 29 April 2009 Received in revised form 24 July 2009 Accepted 18 August 2009 Available online 25 August 2009 Keywords: Cholesterol Membrane Semen Stallion Cryopreservation Methyl--cyclodextrin
a b s t r a c t Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen–thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 × 106 spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 ◦ C, and cryopreserved in liquid nitrogen; (T2) T1 with the addition of cholesterol before cooling (the cholesterol was incorporated to the sperm membranes with the methyl--cyclodextrin-cholesterol complex); (T3) T2 with post-thaw removal of the cholesterol with 0.052 mg methyl--cyclodextrin/50 × 106 sperm; and (T4) T3 with 0.156 mg methyl--cyclodextrin/50 × 106 sperm. Sperm progressive motility and functional integrity of sperm plasma membranes were evaluated microscopically and by the hyposmotic swelling test, respectively. Using flow cytometry, physical integrity of sperm plasma membranes was assessed with propidium iodide, acrosomal integrity with fluoresceinated lectin peanut agglutinin, and rate of sperm acrosome reaction induced with of the calcium ionophore A23187. Cholesterol inclusion (T2) increased the proportion of frozen–thawed sperm with intact plasma membrane. Nevertheless, sperm from T2 (9.3 ± 5.9%) had a lower rate of acrosome reaction after induction, compared to the control group (16.5 ± 11.0%). After cholesterol removal, there was no increase in the induced acrosome reaction rate (T3: 11.3 ± 7.1% and T4: 11.8 ± 9.9%). Perhaps the cyclodextrin concentrations used were too low to remove sufficient cholesterol from sperm membranes to restore the ability of cryopreserved sperm to undergo an acrosome reaction. Regardless, the addition of cholesterol to improve post-thaw sperm integrity, and its subsequent removal, still has potential for cryopreservation of stallion sperm. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author. Tel.: +55 313134092245; fax: +55 313134092230. E-mail address: [email protected] (M.A. Lagares). 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.08.011
One of the characteristics of the sperm plasma membrane which confers a greater sensitivity to temperature reduction is the cholesterol phospholipids ratio. In general, the greater the cholesterol concentration, the less flexible
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or less fluid the plasma membrane (Amann and Pickett, 1987; Amann and Graham, 1992). The cholesterol phospholipids ratio in the plasma membrane of equine sperm is 0.36, an intermediate value relative to boar and bull sperm (Amann and Graham, 1992; Parks and Lynch, 1992). The inclusion of cholesterol in the extender has been reported as an alternative to increase sperm plasma membrane stability in various species during cooling, thereby, improving post-thaw semen quality (Combes et al., 2000; Purdy and Graham, 2004a,b; Moore et al., 2005; Álvarez et al., 2006). Nevertheless, mares inseminated with cholesterolincorporated semen were less fertile than controls (Zahn et al., 2002); this fact was attributed to the anti-fertilizing action of cholesterol, because it inhibits the acrosome reaction (Davis, 1980). However, the post-thaw removal of cholesterol from the plasma membrane of stallion sperm to improve fertility has not yet been reported. Cyclodextrins are cyclic oligosaccharides of glucose that contain a hydrophobic center capable of incorporating lipids (Klein et al., 1995). “In vitro” the methyl-cyclodextrin (MCD) has high affinity to steroids, mainly to cholesterol (Yancey et al., 1996). Water molecules, which have greater energy and lesser entropy, are localized into the cyclodextrin cavity. After cyclodextrins are in contact to a compound that has lesser polarity than water, the formation of supra molecular complexes occurs (Saenger, 1980), so that when a plasma membrane is incubated with a cyclodextrin, it removes the cholesterol from the membrane, transferring it to its cavity, with subsequent efflux of water molecules (Atger et al., 1997). The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen–thawed sperm with the MCD.
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artificial vagina. Immediately after collection, semen was filtered to remove the gel fraction, and progressive motility, sperm concentration and morphology, were evaluated as described. Progressive motility was evaluated using bright-field microscopy (100×). Sperm concentration was calculated using a Neubauer hemocytometer. An aliquot of semen was put into 12.5% formalin solution and the morphology of 200 sperm was evaluated under phase contrast microscopy (1000×). Ejaculates with ≥50% progressive motility and ≥70% morphologically normal sperm were frozen. 2.3. Sperm cryopreservation
2. Materials and methods
The ejaculates were divided into two aliquots according to the treatments. One quarter of the semen was conventionally frozen (Control). It was diluted 1:1 with Kenney extender (Kenney et al., 1975), the aliquots were centrifuged (500 × g for 10 min) and the supernatant was removed leaving approximately 10% of the initial semen volume. The sediment was re-suspended by shaking and diluted to 100 × 106 sperm/mL in INRA 82 freezing extender (Vidament et al., 1997). The semen was packaged into 0.5 mL straws and cooled to 5 ◦ C (0.53 ◦ C/min) in 50 min. The straws were then placed 3 cm above liquid nitrogen for 20 min, followed by immersion. For the remainder of the semen, modified Tyrode’s medium (TALP) (Bavister and Yanagimashi, 1977, modified by Christensen et al., 1996) was added to obtain a concentration of 120 × 106 sperm/mL. For each 120 × 106 spermatozoa, 1.5 mg of methyl--cyclodextrin-cholesterol (MCD-chol) was added to incorporate the cholesterol into the sperm membranes. The aliquot was incubated for 15 min at room temperature, then diluted in Kenney extender (1:1), centrifuged (500 × g for 10 min), and frozen as described for the control group.
2.1. Cyclodextrin-cholesterol complex preparation
2.4. Post-thawing sperm evaluation
The MCD-cholesterol complex was prepared as described by Purdy and Graham (2004b). In a glass test tube, 1 g of MCD (C-4555, Sigma Chemical, St. Louis, MO, USA) was dissolved in 2 mL of methanol. In a second glass test tube, 200 mg of cholesterol (C-8503, Sigma Chemical, St. Louis, MO, USA) was dissolved in 1 mL of chloroform and an aliquot of 450 L of this solution was added to the MCD solution. The combined solution of MCD/cholesterol was thoroughly mixed until a clear solution was obtained. The solvents were removed by “speed-vac” using Univapo 100H equipment (Uniequip, Matinsried, BA, GE) and the resulting crystals were stored at room temperature until use. To incorporate cholesterol into the sperm, a working solution of cholesterol-loaded cyclodextrin (CLC) was made by adding 50 mg of MCD-chol to 1 mL of a modified Tyrode’s medium (TALP), and incubated in a 37 ◦ C water bath until use.
Semen was thawed at 75 ◦ C, for 7 s (Cochran et al., 1984) and kept at 37 ◦ C for 30 s. Total and progressive sperm motility, physical and functional integrity of the sperm plasma membrane, percentage of reactive spermatozoa before and after induced an acrosome reaction, were evaluated. Total and progressive motility and sperm morphology were evaluated as described for fresh semen. The percentage of spermatozoa with functional membrane was assessed using the hyposmotic test with distilled water (Lomeo and Giambersio, 1991, modified by Lagares et al., 1998) at a ratio of 1:2 (semen:distilled water). This was conducted by adding 100 L from semen to 200 L of distilled water to obtain a hypo-osmotic solution with an osmolarity of approximately 100 mOsmol/L. Semen samples were diluted with distilled water for 5 min at 37 ◦ C and 200 spermatozoa were evaluated (400×) for sperm swelling (tail curling) and subtracted from the number of sperm with tail swelling before the osmotic stress. Plasma membrane physical integrity and sperm acrosome integrity were evaluated using propidium iodide (PI) and FITC-PNA dyes, respectively (García-Macías et al.,
2.2. Semen collection and evaluation Twelve stallions, ranging from 4 to 12 years of age, were used. Semen was collected using a Hannover model
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Fig. 1. Evaluation of stallion sperm with flow cytometry. The quantity of fluorescence emitted by a FITC-PNA probe, detected by photodetector FL-1, is indicated on the X-axis, whereas the quantity of fluorescence emitted by a vital PI dye and detected by FL-3 is indicated on the Y-axis. (a) PI+ /PNA− : non-intact plasma membrane/intact acrosome; (b) PI− /PNA− : intact plasma membrane and acrosome; (c) PI+ /PNA+ : non-intact plasma membrane and acrosome; and (d) PI− /PNA+ : intact plasma membrane/non-intact acrosome.
2007). Frozen–thawed semen samples were diluted to 1:20 L in PBS (250 L of semen:5 mL of PBS). An aliquot of 200 L of this suspension was diluted once more by adding 1 mL of PBS, to minimize the contamination of extender particles during the flow cytometry acquisition. After dilution, samples were stained with 15 L of a stock solution of FITC-PNA (1.125 g/mL, lectin agglutinin of Arachis Hypogaea, L-7381, Sigma Chemical, St. Louis, MO, USA) and incubated for 10 min in a water bath at 37 ◦ C. Next, the samples were stained with 30 L of a stock solution of PI (1.5 mM; PI 4170, Sigma Chemical, St. Louis, MO, USA) and incubated for another 10 min, at room temperature. Immediately thereafter, samples were submitted
to flow cytometry (FACScalibur; Becton Dickinson® , San Jose, CA, USA). Ten thousand cells (500 cell/s) were analyzed for each sample. The red fluorescence of PI (FL3 photodetector) and green fluorescence of FITC-PNA (FL1 photodetector) of the cells were detected. Sperm showing red fluorescence were classified as plasma membrane non-intact, whereas those with green fluorescence were classified as not having an intact acrosome membrane and were, therefore, considered acrosome reacted. Four classes of sperm were considered: (a) PI+ /PNA: non-intact plasma membrane/intact acrosome, (b) PI− /PNA− : intact plasma membrane and acrosome, (c) PI+ /PNA+ : non-intact plasma membrane and acrosome, and (d) PI− /PNA+ : intact plasma
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Fig. 2. Evaluation of frozen–thawed stallion sperm. Mean ± S.D. percentage of (A) progressively motile sperm; (B) sperm with an intact membrane (based on PI); (C) sperm with an intact membrane and acrosome; and (D) swollen sperm (HOST).
membrane/non-intact acrosome. For all groups, the proportion of acrosome reacted sperm was determined before and after induction of an acrosome reaction. 2.5. Induction of acrosome reaction To evaluate the “in vitro” fertilizing ability of equine sperm post-thawing, an acrosome reaction was induced (IAR) with calcium ionophore A23187 (Sigma Chemical, St. Louis, MO, USA) in all treatments. After diluting the calcium ionophore A23187 in DMSO (0.5 mg/mL), 5 L of the solution was added to an aliquot of 0.5 mL of thawed semen (50 × 106 spermatozoa), with a final concentration of 9.5 M. After incubating for 30 min, the samples were re-assessed to acrosome integrity using flow cytometry, as described previously. Fluorescence emitted by the sperm cells stained with FITC-PNA and PI dyes in post-thaw sperm and after induction of the acrosome reaction are shown (Fig. 1). 2.6. Methods for cholesterol removal To determine if removal of cholesterol from the sperm membranes increased ability of frozen–thawed sperm to undergo an induced acrosome reaction, two concentrations of MCD (Treatments 3 and 4, respectively) were tested for cholesterol removal. In Treatment 3, concentration 1 [MCD 1×] was the same used to incorporate cholesterol to the membranes (0.052 mg/50 × 106 spermatozoa). To prevent the binding of MCD to lipids in the freezing extender, the concentration used in Treatment 4 was three times higher [MCD 3×] than that used in
Treatment 3 (0.156 mg/50 × 106 spermatozoa). Aliquots of 10.4 L and 31.2 L for the concentrations [MCD 1×] and [MCD 3×], respectively, were removed from a work solution containing 50 mg of MCD in 1 mL of TALP to dilute the lipid components of the extender and, then, added to the frozen–thawed semen (0.5 mL) containing 50 × 106 sperm/straw. The samples were incubated for 15 min at room temperature and, after incubation, acrosome rates were evaluated before and after an induced acrosome reaction. 2.7. Statistical analysis The experiment was designed in randomized blocks, with each stallion as a block. Analysis of variance was used to determine the effect of group on the proportion of frozen–thawed sperm with sperm progressive motility, physical and functional sperm membrane and acrosome integrity and acrosome reaction induction. If there was an effect of group, differences were estimated with a Student–Newman–Keuls test (SNK). Data were analyzed using the SAS program (Statistical Analysis System). A probability of P < 0.05 was considered significant. 3. Results Progressive motility and plasma membrane functionality tested by the HOST on post-thawed semen were not significantly different among treatments (Fig. 2). However, the addition of cholesterol to fresh semen (T2) increased the percentage of spermatozoa with plasma membrane integrity post-thawing, as compared to control (T1). When
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Fig. 3. Mean ± S.D. percentage of frozen–thawed stallion sperm that were acrosome reacted. Percentage of sperm with an intact plasma membrane with a spontaneous and induced acrosome reaction (A and B, respectively).
an acceptor of cholesterol, as MCD at different concentration, was used (T3 and T4), a reduction of the percentage of sperm with plasma membrane integrity, as compared to T2, was observed. When the acrosome reaction rate after induction was calculated based on the percentage of sperm with plasma membrane integrity, the reaction rate was less (P < 0.05) in sperm with added cholesterol (T2: 9.3 ± 5.9%) compared to the control group (16.5 ± 11.0%; Fig. 3). However, the percentage of acrosome reacted sperm in the two groups with MCD (T3: 11.3 ± 7.1% and T4: 11.8 ± 9.9%) were not significantly different from either of the other two groups (T1 and T2). 4. Discussion The effect of cholesterol on sperm motility is still controversial. In the present study, the inclusion of cholesterol to the extender did not significantly increase the percentage of progressively motile frozen–thawed sperm. These results were similar to those reported in studies on frozen–thawed semen in horses (Zahn et al., 2002; Moore et al., 2005) and cattle (Mocé and Graham, 2006). However, in other studies on frozen–thawed equine (Combes et al., 2000; Álvarez et al., 2006) and bovine (Purdy and Graham, 2004a,b) semen, sperm motility increased after thawing when cholesterol was incorporated with MCD. In the present study, cholesterol incorporation was conducted as described for bulls (Purdy and Graham, 2004a,b) and equine (Moore et al., 2005) semen. Differences between the current study and those in cattle (Purdy and Graham, 2004a,b), may be due to species differences in size, shape, lipid composition (Parks et al., 1981; Parks and Lynch, 1992) and permeability of the sperm membranes (Guthrie et al., 2002).
In the present study, an increase of the osmotic sperm resistance was not observed in T2. Nevertheless, it was reported that cholesterol inclusion increased the osmotic tolerance of stallion (Moore et al., 2005) and donkey cryopreserved semen (Álvarez et al., 2006), and cooled semen of stallions with a greater concentration of cholesterol (2.5 mg/120 × 106 sperm; Torres et al., 2006). For unknown reasons, in the present study the cholesterol inclusion did not increase of the osmotic sperm tolerance. In the present study, incorporating cholesterol into the membranes protected the spermatozoa against the deleterious effects of freezing on plasma membrane physical integrity. A similar increase, when cholesterol was added to the diluent, was detected with frozen–thawed bull (Purdy and Graham, 2004a,b; Mocé and Graham, 2006), stallion (Combes et al., 2000; Zahn et al., 2002; Moore et al., 2005) and cooled boar semen (Galantino-Homer et al., 2006). In the present study, there was a reduction in the induced acrosome reaction of the cholesterol-incorporated spermatozoa (T2). This may have been due to changes in membrane permeability and fusion ability (Holt and North, 1986) and increased plasma membrane stability, which impaired the entrance of calcium (Yeagle, 1985), thereby not stimulating adenylcyclase activity, preventing capacitation and subsequent acrosome reaction. This may suggest an inhibiting action of cholesterol on stallion sperm acrosome reaction. The hypothesis that cholesterol affect sperm capacitation and fusion ability could be supported by the evidence of a reduction in fertility (pregnancy rates of 25% compared with 75% in mares inseminated with sperm with and without the addition of cholesterol, respectively; Zahn et al., 2002). In contrast with evidence in the stallion, the addition of cholesterol to bull sperm did not significantly affect in vivo or in vitro fertility (Purdy and Graham, 2004a).
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An important aspect when using CLC in the incorporation of cholesterol to the membrane is its incubation with sperm diluted in a lipid free medium (Purdy and Graham, 2004a,b). Because the semen freezing medium is rich in lipids, the same concentration of MCD used to incorporate cholesterol may not be sufficient to remove it. Therefore, a concentration three times greater than the one used to incorporate cholesterol was tested. However, reduction of the physical integrity of the sperm membrane, as compared to T2, was observed with both concentrations, suggesting a deleterious effect of MCD. This effect was reported in mammal sperm (Visconti et al., 1999) and in fibroblasts (Kilsdonk et al., 1995). After the cholesterol incorporated before freezing was removed, sperm capacitation and acrosome reaction could be induced, thus, restore the fertilizing ability of the sperm. The percentage of acrosome reacted sperm with intact membrane after induction, could be considered as sperm with true acrosome reaction. Conversely, when the acrosome reaction rate was based on all sperm, include the false and true acrosome reacted sperm (Didion et al., 1989). Therefore, based on the percentage of sperm with an induced acrosome reaction with intact membrane, we inferred that the MCD partially removed the added cholesterol. Perhaps some of the MCD added to the semen after thawing (T3 and T4) complexed with the egg yolk lipids of the extender. Consequently, cholesterol was not sufficiently removed from the membranes to reach physiological concentrations. Therefore, greater MCD concentrations or an increase of its incubation time with semen after thawing may result in a greater rate of cholesterol removal from the membranes as well as an increase of the acrosome reacted sperm rate after induction. However, it may also increase the deleterious effect of MCD on the membrane (Visconti et al., 1999) and reduce sperm longevity (Watson, 2000). Although in the present study, the incorporation of cholesterol to stallion semen before freezing increased the rate of sperm membrane integrity, studies on cholesterol markers may determine the adequate MCD concentration to remove cholesterol from the membrane to physiological concentrations. To remove cholesterol from the sperm membrane, a greater rate of MCD-cholesterol binding could have been obtained post-thawing by removing the sperm from the extender containing lipids, with subsequent addition of MCD. Other possibility is the use of other cholesterol-reducing compounds, such as albumin, which is a physiological cholesterol reducer; to remove the incorporated cholesterol from sperm membranes. This is the first report on cholesterol removal from the stallion sperm membrane following incorporation by means of MCD binding. After determining the adequate MCD concentration for the removal of cholesterol from the sperm membrane to physiological concentrations, the fertilization ability of cryopreserved stallion semen could increase. 5. Conclusions The incorporation of cholesterol to equine semen before freezing increased the rate of sperm membrane integrity, and the MCD partially removed the added cholesterol,
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm C.H. Oliveira a , A.B. Vasconcelos b , F.A. Souza a , O.A. Martins-Filho c , M.X. Silva a , F.C. Varago a , M.A. Lagares a,∗ a
Department of Veterinary Clinics and Surgery, Veterinary School of the Federal University of Minas Gerais, Av. Antonio Carlos 6627, CEP: 31270-901, Belo Horizonte, MG, Brazil Instituto de Estudos Avanc¸ados em Veterinária José Caetano Borges/Universidade de Uberaba/FAZU/ABCZ, Av. Tutunas 720, CEP: 38061-500, Uberaba, MG, Brazil c Laboratório de Biomarcadores de Diagnóstico e Monitorac¸ão, Centro de Pesquisas René Rachou - Fiocruz, Av. Augusto de Lima 1715, CEP: 30190-002, Belo Horizonte, MG, Brazil b
a r t i c l e
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Article history: Received 29 April 2009 Received in revised form 24 July 2009 Accepted 18 August 2009 Available online 25 August 2009 Keywords: Cholesterol Membrane Semen Stallion Cryopreservation Methyl--cyclodextrin
a b s t r a c t Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen–thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 × 106 spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 ◦ C, and cryopreserved in liquid nitrogen; (T2) T1 with the addition of cholesterol before cooling (the cholesterol was incorporated to the sperm membranes with the methyl--cyclodextrin-cholesterol complex); (T3) T2 with post-thaw removal of the cholesterol with 0.052 mg methyl--cyclodextrin/50 × 106 sperm; and (T4) T3 with 0.156 mg methyl--cyclodextrin/50 × 106 sperm. Sperm progressive motility and functional integrity of sperm plasma membranes were evaluated microscopically and by the hyposmotic swelling test, respectively. Using flow cytometry, physical integrity of sperm plasma membranes was assessed with propidium iodide, acrosomal integrity with fluoresceinated lectin peanut agglutinin, and rate of sperm acrosome reaction induced with of the calcium ionophore A23187. Cholesterol inclusion (T2) increased the proportion of frozen–thawed sperm with intact plasma membrane. Nevertheless, sperm from T2 (9.3 ± 5.9%) had a lower rate of acrosome reaction after induction, compared to the control group (16.5 ± 11.0%). After cholesterol removal, there was no increase in the induced acrosome reaction rate (T3: 11.3 ± 7.1% and T4: 11.8 ± 9.9%). Perhaps the cyclodextrin concentrations used were too low to remove sufficient cholesterol from sperm membranes to restore the ability of cryopreserved sperm to undergo an acrosome reaction. Regardless, the addition of cholesterol to improve post-thaw sperm integrity, and its subsequent removal, still has potential for cryopreservation of stallion sperm. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author. Tel.: +55 313134092245; fax: +55 313134092230. E-mail address: [email protected] (M.A. Lagares). 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.08.011
One of the characteristics of the sperm plasma membrane which confers a greater sensitivity to temperature reduction is the cholesterol phospholipids ratio. In general, the greater the cholesterol concentration, the less flexible
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or less fluid the plasma membrane (Amann and Pickett, 1987; Amann and Graham, 1992). The cholesterol phospholipids ratio in the plasma membrane of equine sperm is 0.36, an intermediate value relative to boar and bull sperm (Amann and Graham, 1992; Parks and Lynch, 1992). The inclusion of cholesterol in the extender has been reported as an alternative to increase sperm plasma membrane stability in various species during cooling, thereby, improving post-thaw semen quality (Combes et al., 2000; Purdy and Graham, 2004a,b; Moore et al., 2005; Álvarez et al., 2006). Nevertheless, mares inseminated with cholesterolincorporated semen were less fertile than controls (Zahn et al., 2002); this fact was attributed to the anti-fertilizing action of cholesterol, because it inhibits the acrosome reaction (Davis, 1980). However, the post-thaw removal of cholesterol from the plasma membrane of stallion sperm to improve fertility has not yet been reported. Cyclodextrins are cyclic oligosaccharides of glucose that contain a hydrophobic center capable of incorporating lipids (Klein et al., 1995). “In vitro” the methyl-cyclodextrin (MCD) has high affinity to steroids, mainly to cholesterol (Yancey et al., 1996). Water molecules, which have greater energy and lesser entropy, are localized into the cyclodextrin cavity. After cyclodextrins are in contact to a compound that has lesser polarity than water, the formation of supra molecular complexes occurs (Saenger, 1980), so that when a plasma membrane is incubated with a cyclodextrin, it removes the cholesterol from the membrane, transferring it to its cavity, with subsequent efflux of water molecules (Atger et al., 1997). The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen–thawed sperm with the MCD.
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artificial vagina. Immediately after collection, semen was filtered to remove the gel fraction, and progressive motility, sperm concentration and morphology, were evaluated as described. Progressive motility was evaluated using bright-field microscopy (100×). Sperm concentration was calculated using a Neubauer hemocytometer. An aliquot of semen was put into 12.5% formalin solution and the morphology of 200 sperm was evaluated under phase contrast microscopy (1000×). Ejaculates with ≥50% progressive motility and ≥70% morphologically normal sperm were frozen. 2.3. Sperm cryopreservation
2. Materials and methods
The ejaculates were divided into two aliquots according to the treatments. One quarter of the semen was conventionally frozen (Control). It was diluted 1:1 with Kenney extender (Kenney et al., 1975), the aliquots were centrifuged (500 × g for 10 min) and the supernatant was removed leaving approximately 10% of the initial semen volume. The sediment was re-suspended by shaking and diluted to 100 × 106 sperm/mL in INRA 82 freezing extender (Vidament et al., 1997). The semen was packaged into 0.5 mL straws and cooled to 5 ◦ C (0.53 ◦ C/min) in 50 min. The straws were then placed 3 cm above liquid nitrogen for 20 min, followed by immersion. For the remainder of the semen, modified Tyrode’s medium (TALP) (Bavister and Yanagimashi, 1977, modified by Christensen et al., 1996) was added to obtain a concentration of 120 × 106 sperm/mL. For each 120 × 106 spermatozoa, 1.5 mg of methyl--cyclodextrin-cholesterol (MCD-chol) was added to incorporate the cholesterol into the sperm membranes. The aliquot was incubated for 15 min at room temperature, then diluted in Kenney extender (1:1), centrifuged (500 × g for 10 min), and frozen as described for the control group.
2.1. Cyclodextrin-cholesterol complex preparation
2.4. Post-thawing sperm evaluation
The MCD-cholesterol complex was prepared as described by Purdy and Graham (2004b). In a glass test tube, 1 g of MCD (C-4555, Sigma Chemical, St. Louis, MO, USA) was dissolved in 2 mL of methanol. In a second glass test tube, 200 mg of cholesterol (C-8503, Sigma Chemical, St. Louis, MO, USA) was dissolved in 1 mL of chloroform and an aliquot of 450 L of this solution was added to the MCD solution. The combined solution of MCD/cholesterol was thoroughly mixed until a clear solution was obtained. The solvents were removed by “speed-vac” using Univapo 100H equipment (Uniequip, Matinsried, BA, GE) and the resulting crystals were stored at room temperature until use. To incorporate cholesterol into the sperm, a working solution of cholesterol-loaded cyclodextrin (CLC) was made by adding 50 mg of MCD-chol to 1 mL of a modified Tyrode’s medium (TALP), and incubated in a 37 ◦ C water bath until use.
Semen was thawed at 75 ◦ C, for 7 s (Cochran et al., 1984) and kept at 37 ◦ C for 30 s. Total and progressive sperm motility, physical and functional integrity of the sperm plasma membrane, percentage of reactive spermatozoa before and after induced an acrosome reaction, were evaluated. Total and progressive motility and sperm morphology were evaluated as described for fresh semen. The percentage of spermatozoa with functional membrane was assessed using the hyposmotic test with distilled water (Lomeo and Giambersio, 1991, modified by Lagares et al., 1998) at a ratio of 1:2 (semen:distilled water). This was conducted by adding 100 L from semen to 200 L of distilled water to obtain a hypo-osmotic solution with an osmolarity of approximately 100 mOsmol/L. Semen samples were diluted with distilled water for 5 min at 37 ◦ C and 200 spermatozoa were evaluated (400×) for sperm swelling (tail curling) and subtracted from the number of sperm with tail swelling before the osmotic stress. Plasma membrane physical integrity and sperm acrosome integrity were evaluated using propidium iodide (PI) and FITC-PNA dyes, respectively (García-Macías et al.,
2.2. Semen collection and evaluation Twelve stallions, ranging from 4 to 12 years of age, were used. Semen was collected using a Hannover model
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Fig. 1. Evaluation of stallion sperm with flow cytometry. The quantity of fluorescence emitted by a FITC-PNA probe, detected by photodetector FL-1, is indicated on the X-axis, whereas the quantity of fluorescence emitted by a vital PI dye and detected by FL-3 is indicated on the Y-axis. (a) PI+ /PNA− : non-intact plasma membrane/intact acrosome; (b) PI− /PNA− : intact plasma membrane and acrosome; (c) PI+ /PNA+ : non-intact plasma membrane and acrosome; and (d) PI− /PNA+ : intact plasma membrane/non-intact acrosome.
2007). Frozen–thawed semen samples were diluted to 1:20 L in PBS (250 L of semen:5 mL of PBS). An aliquot of 200 L of this suspension was diluted once more by adding 1 mL of PBS, to minimize the contamination of extender particles during the flow cytometry acquisition. After dilution, samples were stained with 15 L of a stock solution of FITC-PNA (1.125 g/mL, lectin agglutinin of Arachis Hypogaea, L-7381, Sigma Chemical, St. Louis, MO, USA) and incubated for 10 min in a water bath at 37 ◦ C. Next, the samples were stained with 30 L of a stock solution of PI (1.5 mM; PI 4170, Sigma Chemical, St. Louis, MO, USA) and incubated for another 10 min, at room temperature. Immediately thereafter, samples were submitted
to flow cytometry (FACScalibur; Becton Dickinson® , San Jose, CA, USA). Ten thousand cells (500 cell/s) were analyzed for each sample. The red fluorescence of PI (FL3 photodetector) and green fluorescence of FITC-PNA (FL1 photodetector) of the cells were detected. Sperm showing red fluorescence were classified as plasma membrane non-intact, whereas those with green fluorescence were classified as not having an intact acrosome membrane and were, therefore, considered acrosome reacted. Four classes of sperm were considered: (a) PI+ /PNA: non-intact plasma membrane/intact acrosome, (b) PI− /PNA− : intact plasma membrane and acrosome, (c) PI+ /PNA+ : non-intact plasma membrane and acrosome, and (d) PI− /PNA+ : intact plasma
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Fig. 2. Evaluation of frozen–thawed stallion sperm. Mean ± S.D. percentage of (A) progressively motile sperm; (B) sperm with an intact membrane (based on PI); (C) sperm with an intact membrane and acrosome; and (D) swollen sperm (HOST).
membrane/non-intact acrosome. For all groups, the proportion of acrosome reacted sperm was determined before and after induction of an acrosome reaction. 2.5. Induction of acrosome reaction To evaluate the “in vitro” fertilizing ability of equine sperm post-thawing, an acrosome reaction was induced (IAR) with calcium ionophore A23187 (Sigma Chemical, St. Louis, MO, USA) in all treatments. After diluting the calcium ionophore A23187 in DMSO (0.5 mg/mL), 5 L of the solution was added to an aliquot of 0.5 mL of thawed semen (50 × 106 spermatozoa), with a final concentration of 9.5 M. After incubating for 30 min, the samples were re-assessed to acrosome integrity using flow cytometry, as described previously. Fluorescence emitted by the sperm cells stained with FITC-PNA and PI dyes in post-thaw sperm and after induction of the acrosome reaction are shown (Fig. 1). 2.6. Methods for cholesterol removal To determine if removal of cholesterol from the sperm membranes increased ability of frozen–thawed sperm to undergo an induced acrosome reaction, two concentrations of MCD (Treatments 3 and 4, respectively) were tested for cholesterol removal. In Treatment 3, concentration 1 [MCD 1×] was the same used to incorporate cholesterol to the membranes (0.052 mg/50 × 106 spermatozoa). To prevent the binding of MCD to lipids in the freezing extender, the concentration used in Treatment 4 was three times higher [MCD 3×] than that used in
Treatment 3 (0.156 mg/50 × 106 spermatozoa). Aliquots of 10.4 L and 31.2 L for the concentrations [MCD 1×] and [MCD 3×], respectively, were removed from a work solution containing 50 mg of MCD in 1 mL of TALP to dilute the lipid components of the extender and, then, added to the frozen–thawed semen (0.5 mL) containing 50 × 106 sperm/straw. The samples were incubated for 15 min at room temperature and, after incubation, acrosome rates were evaluated before and after an induced acrosome reaction. 2.7. Statistical analysis The experiment was designed in randomized blocks, with each stallion as a block. Analysis of variance was used to determine the effect of group on the proportion of frozen–thawed sperm with sperm progressive motility, physical and functional sperm membrane and acrosome integrity and acrosome reaction induction. If there was an effect of group, differences were estimated with a Student–Newman–Keuls test (SNK). Data were analyzed using the SAS program (Statistical Analysis System). A probability of P < 0.05 was considered significant. 3. Results Progressive motility and plasma membrane functionality tested by the HOST on post-thawed semen were not significantly different among treatments (Fig. 2). However, the addition of cholesterol to fresh semen (T2) increased the percentage of spermatozoa with plasma membrane integrity post-thawing, as compared to control (T1). When
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Fig. 3. Mean ± S.D. percentage of frozen–thawed stallion sperm that were acrosome reacted. Percentage of sperm with an intact plasma membrane with a spontaneous and induced acrosome reaction (A and B, respectively).
an acceptor of cholesterol, as MCD at different concentration, was used (T3 and T4), a reduction of the percentage of sperm with plasma membrane integrity, as compared to T2, was observed. When the acrosome reaction rate after induction was calculated based on the percentage of sperm with plasma membrane integrity, the reaction rate was less (P < 0.05) in sperm with added cholesterol (T2: 9.3 ± 5.9%) compared to the control group (16.5 ± 11.0%; Fig. 3). However, the percentage of acrosome reacted sperm in the two groups with MCD (T3: 11.3 ± 7.1% and T4: 11.8 ± 9.9%) were not significantly different from either of the other two groups (T1 and T2). 4. Discussion The effect of cholesterol on sperm motility is still controversial. In the present study, the inclusion of cholesterol to the extender did not significantly increase the percentage of progressively motile frozen–thawed sperm. These results were similar to those reported in studies on frozen–thawed semen in horses (Zahn et al., 2002; Moore et al., 2005) and cattle (Mocé and Graham, 2006). However, in other studies on frozen–thawed equine (Combes et al., 2000; Álvarez et al., 2006) and bovine (Purdy and Graham, 2004a,b) semen, sperm motility increased after thawing when cholesterol was incorporated with MCD. In the present study, cholesterol incorporation was conducted as described for bulls (Purdy and Graham, 2004a,b) and equine (Moore et al., 2005) semen. Differences between the current study and those in cattle (Purdy and Graham, 2004a,b), may be due to species differences in size, shape, lipid composition (Parks et al., 1981; Parks and Lynch, 1992) and permeability of the sperm membranes (Guthrie et al., 2002).
In the present study, an increase of the osmotic sperm resistance was not observed in T2. Nevertheless, it was reported that cholesterol inclusion increased the osmotic tolerance of stallion (Moore et al., 2005) and donkey cryopreserved semen (Álvarez et al., 2006), and cooled semen of stallions with a greater concentration of cholesterol (2.5 mg/120 × 106 sperm; Torres et al., 2006). For unknown reasons, in the present study the cholesterol inclusion did not increase of the osmotic sperm tolerance. In the present study, incorporating cholesterol into the membranes protected the spermatozoa against the deleterious effects of freezing on plasma membrane physical integrity. A similar increase, when cholesterol was added to the diluent, was detected with frozen–thawed bull (Purdy and Graham, 2004a,b; Mocé and Graham, 2006), stallion (Combes et al., 2000; Zahn et al., 2002; Moore et al., 2005) and cooled boar semen (Galantino-Homer et al., 2006). In the present study, there was a reduction in the induced acrosome reaction of the cholesterol-incorporated spermatozoa (T2). This may have been due to changes in membrane permeability and fusion ability (Holt and North, 1986) and increased plasma membrane stability, which impaired the entrance of calcium (Yeagle, 1985), thereby not stimulating adenylcyclase activity, preventing capacitation and subsequent acrosome reaction. This may suggest an inhibiting action of cholesterol on stallion sperm acrosome reaction. The hypothesis that cholesterol affect sperm capacitation and fusion ability could be supported by the evidence of a reduction in fertility (pregnancy rates of 25% compared with 75% in mares inseminated with sperm with and without the addition of cholesterol, respectively; Zahn et al., 2002). In contrast with evidence in the stallion, the addition of cholesterol to bull sperm did not significantly affect in vivo or in vitro fertility (Purdy and Graham, 2004a).
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An important aspect when using CLC in the incorporation of cholesterol to the membrane is its incubation with sperm diluted in a lipid free medium (Purdy and Graham, 2004a,b). Because the semen freezing medium is rich in lipids, the same concentration of MCD used to incorporate cholesterol may not be sufficient to remove it. Therefore, a concentration three times greater than the one used to incorporate cholesterol was tested. However, reduction of the physical integrity of the sperm membrane, as compared to T2, was observed with both concentrations, suggesting a deleterious effect of MCD. This effect was reported in mammal sperm (Visconti et al., 1999) and in fibroblasts (Kilsdonk et al., 1995). After the cholesterol incorporated before freezing was removed, sperm capacitation and acrosome reaction could be induced, thus, restore the fertilizing ability of the sperm. The percentage of acrosome reacted sperm with intact membrane after induction, could be considered as sperm with true acrosome reaction. Conversely, when the acrosome reaction rate was based on all sperm, include the false and true acrosome reacted sperm (Didion et al., 1989). Therefore, based on the percentage of sperm with an induced acrosome reaction with intact membrane, we inferred that the MCD partially removed the added cholesterol. Perhaps some of the MCD added to the semen after thawing (T3 and T4) complexed with the egg yolk lipids of the extender. Consequently, cholesterol was not sufficiently removed from the membranes to reach physiological concentrations. Therefore, greater MCD concentrations or an increase of its incubation time with semen after thawing may result in a greater rate of cholesterol removal from the membranes as well as an increase of the acrosome reacted sperm rate after induction. However, it may also increase the deleterious effect of MCD on the membrane (Visconti et al., 1999) and reduce sperm longevity (Watson, 2000). Although in the present study, the incorporation of cholesterol to stallion semen before freezing increased the rate of sperm membrane integrity, studies on cholesterol markers may determine the adequate MCD concentration to remove cholesterol from the membrane to physiological concentrations. To remove cholesterol from the sperm membrane, a greater rate of MCD-cholesterol binding could have been obtained post-thawing by removing the sperm from the extender containing lipids, with subsequent addition of MCD. Other possibility is the use of other cholesterol-reducing compounds, such as albumin, which is a physiological cholesterol reducer; to remove the incorporated cholesterol from sperm membranes. This is the first report on cholesterol removal from the stallion sperm membrane following incorporation by means of MCD binding. After determining the adequate MCD concentration for the removal of cholesterol from the sperm membrane to physiological concentrations, the fertilization ability of cryopreserved stallion semen could increase. 5. Conclusions The incorporation of cholesterol to equine semen before freezing increased the rate of sperm membrane integrity, and the MCD partially removed the added cholesterol,
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