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Iodixanol density gradient centrifugation for selecting stallion sperm for cold storage and cryopreservation
Animal Reproduction Science 133 (2012) 184–190
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Iodixanol density gradient centrifugation for selecting stallion sperm for cold storage and cryopreservation Gesa Stuhtmann a,b , Harriëtte Oldenhof a , Pamela Peters a,b , Jutta Klewitz a , Gunilla Martinsson b , Harald Sieme a,∗ a b
Clinic for Horses – Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Germany National Stud Lower Saxony, Celle, Germany
a r t i c l e
i n f o
Article history: Received 5 December 2011 Received in revised form 19 June 2012 Accepted 21 June 2012 Available online 27 June 2012 Keywords: Stallion spermatozoa Sperm cleanup Density gradient centrifugation Iodixanol
a b s t r a c t Density gradient centrifugation can be used for selection of sperm of superior quality and removal of seminal plasma for use in artificial insemination. In this study, the use of two-layer iodixanol density gradient centrifugation was evaluated for processing of stallion semen. The protocol includes centrifugation through a 16% iodixanol top layer of 1.090 g mL−1 and collection of motile and intact sperm on a 30% iodixanol bottom layer of 1.165 g mL−1 . Sperm recovery and effects on sperm quality were determined during cold storage as well as after cryopreservation and compared with ordinary dilution and centrifugation. Two-layer iodixanol density gradient centrifugation allows for selection of greater percentages of morphologically normal and progressively motile sperm compared to ordinary centrifugation. This likely results from collecting sperm on the bottom layer that functions as cushion fluid, which prevents mechanical forces as occur when sperm are packed in a pellet. In addition, percentages of membrane and chromatin integrity are increased when cells are selected based on their density via centrifugation through the top and bottom layers. Removal of seminal plasma and increased initial percentages of motile and membrane intact sperm after iodixanol density gradient centrifugation also result in greater percentages of progressively motile and membrane intact sperm during cold storage as well as after freezing and thawing. In conclusion, the two-layer iodixanol density gradient centrifugation protocol described in this manuscript allows for selection of stallion sperm with greater survival rates for cold storage and cryopreservation. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The increased use of artificial insemination in horse breeding has demanded the development of semen preparation methods that provide spermatozoa of good quality for insemination, with minimal variation amongst
∗ Corresponding author at: Clinic for Horses – Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Bünteweg 15, 30559 Hannover, Germany. Tel.: +49 0511 953 8530; fax: +49 0511 953 82 8530. E-mail address: [email protected] (H. Sieme). 0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2012.06.017
ejaculates and individuals, and maintenance of quality during cold storage as well as cryopreservation (Loomis, 2001; Aurich, 2008). Sperm separation techniques are used in assisted reproduction to select spermatozoa of superior quality and remove non-viable, morphologically abnormal or immature spermatozoa and seminal plasma. Sperm quality is considered good when a sample contains large numbers of progressively motile sperm, with greater percentages of membrane and chromatin intactness. Separation techniques are based on differences in sperm viability, motility, morphology, size and density. They include: (i) dilution and washing via ordinary centrifugation,
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(ii) sperm migration/sedimentation methods (swim-up), (iii) filtration/adherence methods (glass wool or beads, sephadex, leucosorb), and (iv) density gradient centrifugation (polyvinylpyrrolidone or silane-coated silica particles, iohexol, ficoll) (Sieme et al., 2003; Mortimer, 2000; Morrell, 2006). After Percoll was withdrawn for clinical use, alternative solutions for density gradient centrifugation were tested including iodixanol solutions. Density gradient centrifugation protocols using iodixanol exist for selection of human and bull sperm (Harrison, 1997; Smith et al., 1997), but its use for processing of stallion semen has not been evaluated. Iodixanol, however, has been used for cushion centrifugation of stallion sperm (Ecot et al., 2005; Sieme et al., 2006). Using this method, spermatozoa are collected as a layer on top of the iodixanol layer, which has a greater density than sperm. This is in contrast with ordinary centrifugation or density gradient protocols that use Percoll (Sieme et al., 2003), EquiPure (Macpherson et al., 2002) or Androcoll (Morrell et al., 2009b), in which sperm are collected in a pellet at the bottom of the tube. Collecting sperm in a pellet is thought to be deleterious, because sperm will be tightly packed together with cell debris and reactive oxygen species (Pickett et al., 1975; Smith et al., 1997). Two-layer density gradient centrifugation using a top and bottom layer of different densities can be used for selecting spermatozoa on the basis of their density properties through the top and bottom layers, where the bottom layer also functions as cushion fluid thereby preventing pelleting of the cells. The aim of the present study was to test the use of two-layer iodixanol density gradient centrifugation for selection of stallion sperm, and compare the efficiency with ordinary centrifugation methods. Sperm recovery rates of these methods were determined, and effects on sperm morphology and motility as well as membrane and chromatin integrity. Sperm quality parameters were assessed during storage at 5 ◦ C. In addition, application of iodixanol density gradient centrifugation for cryopreservation of sperm from stallions with so-called ‘good’ and ‘poor’ freezing characteristics was evaluated. 2. Materials and methods 2.1. Semen collection, dilution, and ordinary centrifugation For the experiments described in this study, three ejaculates were collected from each of six stallions (ages 5–11 years) of the Hanoverian warmblood breed, that participated in the artificial insemination program of the National Stud of Lower Saxony in Celle, Germany. Semen was collected at the beginning of the breeding season. Prior to collecting semen for experiments, semen collections were performed for 1 week to stabilize the extra-gonadal sperm reserves. Stallions were divided into those with sperm with ‘good’ and ‘poor’ freezing qualities (three in each group), based on previous experiments. This was done according to the criteria described by Vidament et al. (1997): stallions with a post-thaw progressive sperm motility less than or equal to 35% in three or more ejaculates out of ten were
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considered to be with ‘poor’ freezing qualities, whereas stallions with sperm above these criteria were designated as those with ‘good’ freezing qualities. Semen was collected every other day, using an artificial vagina (Hanoverian model; Minitüb, Landshut, Germany). A ‘phantom’ device for stallions was used with a ‘teaser’ mare fixed in front. The collection device was equipped with a sterile gauze filter to remove the gel portion. Gel-free semen was evaluated for volume, and the sperm concentration was estimated via measurement of the optical density (Spermacue; Minitübe, Tiefenbach, Germany). A 1 mL aliquot of raw semen was frozen in liquid nitrogen for later analysis of chromatin integrity, and the remainder was diluted with pre-warmed EquiPro extender (Minitübe, Tiefenbach, Germany) to 100 × 106 cells mL−1 . Diluted semen was divided into three parts. One part was diluted with an equal volume of EquiPro to 50 × 106 cells mL−1 , and is further referred to as diluted semen. The other two parts were processed by ordinary centrifugation or iodixanol density gradient centrifugation as subsequently described. Ordinary centrifugation was performed by centrifuging 40 mL diluted semen in a 50 mL tube at 600 × g for 10 min. The supernatant was removed using a syringe with a needle. The re-suspended sperm pellet was transferred to a clean tube, after which the cell concentration was determined using a haemocytometer (Thoma Neu, Hecht, Sontheim, Germany). The total number of re-suspended sperm was determined, and the recovery rate was calculated by comparing with the total number of sperm before centrifugation. 2.2. Iodixanol density gradient centrifugation of diluted semen A 60% iodixanol solution in water with a density of 1.320 g mL−1 at room temperature is commercially available as OptiPrep (Axis-shield, Oslo, Norway). Before density gradient centrifugation, isotonic solutions of the desired densities were prepared by dilution with Hanks buffered salt solution (HBSS; 5.33 mM KCl, 0.441 mM KH2 PO4 , 4.17 mM NaHCO3 , 137.92 mM NaCl, 0.338 mM NaH2 PO4 , 5.56 mM glucose, pH 7.4). Dilution of iodixanol with HBSS did not change the pH or osmolality; these variables remained 7.4 and 300 mOsm kg−1 , respectively. A 16% iodixanol solution of 1.090 g mL−1 was used as top layer, and a 30% solution of 1.165 g mL−1 as bottom layer. Densities were measured using a bench-top refractometer (Zeiss, Jena, Germany). Density gradients were prepared in a 50 mL conical plastic tube: 10 mL of the top layer was added first, after which 10 mL bottom layer was added underneath. Twenty mL of diluted semen was carefully layered on top of the iodixanol solutions. After centrifugation at 1000 × g for 20 min, spermatozoa show up as a band between the upper and bottom layer. The supernatant was aspirated carefully, when the band that contained the sperm was reached this was transferred into a clean tube using a 25 mL syringe with attached a 500 L straw. The cell concentration and recovery rate were determined as described above.
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2.3. Cold storage and cryopreservation of diluted and centrifuged sperm The sperm as harvested after centrifugation was divided into two parts: one part was used for storage at 5 ◦ C, whereas the other part was used for cryopreservation. For cold storage, samples were diluted with EquiPro to a final concentration of 50 × 106 cells mL−1 , and remained at room temperature for 1 h, after which the temperature was reduced to 5 ◦ C. For cryopreservation, the centrifuged samples were diluted with EquiPro to a concentration of 100 × 106 cells mL−1 . Then the samples were diluted with an equal volume of EquiPro freezing extender (containing 5% glycerol) resulting in 50 × 106 cells mL−1 and 2.5% glycerol, and cooled to 5 ◦ C with 0.1 ◦ C min−1 . This cooling rate was reached via equilibration of diluted semen in a beaker with water of room temperature in a cold handling cabinet of 5 ◦ C. After reaching 5 ◦ C (after about 2.5 h) diluted semen was packaged in 0.5 mL straws, cooled from 5 ◦ C to −15 ◦ C at 10 ◦ C min−1 , and from −15 ◦ C to −140 ◦ C at 25 ◦ C min−1 using a controlled rate freezer (IMV-Technologies, L’Aigle, France). Straws were transfered into liquid nitrogen, in which they were stored until analysis. Thawing was done in a water bath at 37 ◦ C for 30 s. 2.4. Assessment of sperm motility, morphology, and chromatin and membrane integrity Sperm morphology, motility parameters, membrane integrity, acrosomal status and chromatin integrity were assessed immediately after dilution or centrifugation, after 24, 48 and 72 h of storage at 5 ◦ C, as well as after cryopreservation. Sperm motility parameters were assessed via computer assisted sperm analysis (SpermVision; Minitübe, Tiefenbach, Germany) using 20 m analyzing chambers (Leja, Nieuw-Vennep, Netherlands). Motility parameters that were determined included: percentage of progressively motile sperm (PMS), curvilinear velocity (VCL in m s−1 ), average path velocity (VAP in m s−1 ), and the amplitude of lateral head movement (ALH in m). Evaluation of sperm morphology was done for 200 cells per sample according to Brito (2007). Therefore, 100 L semen was stained with 300 L nigrosin–eosin solution (10% nigrosin, 0.7% eosin, 3.75 mM Na2 HPO4 , 1.88 mM KH2 PO4 , 5.78 mM NaK tartrate, 3.75 mM glucose). Plasma and acrosomal membrane integrity of spermatozoa were evaluated using a flow cytometer (Cell Lab Quanta SC MPL; Beckman-Coulter, Krefeld, Germany). This flow cytometer is equipped with a 488 nm argon ion laser of 22 mW for excitation, and band pass 525/30, 590/30 and long pass 670 nm filters for green, orange and red fluorescence, respectively. HBS of 300 mOsm kg−1 (20 mM HEPES pH 7.4, 137 mM NaCl, 10 mM glucose, 2.5 mM KOH) was used as sheath fluid. A double staining with propidium iodide (PI; Sigma–Aldrich, St. Louis, MO, USA) and fluorescein labeled peanut agglutinin (FITC-PNA; Vector Laboratories, Burlingame, CA, USA) was performed, according to Rathi et al. (2001) with minor modifications. Ten
L 50 × 106 cells mL−1 in extender was diluted in 480 L HBS, and 2.5 L 500 g mL−1 propidium iodide (PI) and 7.5 L 600 g mL−1 fluorescein labeled peanut agglutinin (FITC-PNA) were added. This results in 1 × 106 cells mL−1 , 2.5 g mL−1 PI and 9 g mL−1 FITC-PNA. Samples were incubated for 15 min at 37 ◦ C in darkness, and 10,000 cells were measured. Sperm were selected based on their sideward scatter and electronic volume properties. Cells that have a damaged plasma membrane show red fluorescence of PI bound to DNA, cells that have a damaged acrosome will show green fluorescence of FITC-PNA bound to glycoproteins. Sperm that were both PI- and FITC-PNA-negative were considered viable with intact plasma and acrosomal membranes. The sperm chromatin structure assay (SCSA) was used to evaluate chromatin integrity. In this assay, sperm chromatin is treated with acid after which the extent of DNA fragmentation is determined (Evenson et al., 2002). In short, thawed samples were diluted in TNE (0.15 M NaCl, 0.01 M Tris–HCl, 1 mM disodium EDTA, pH 7.4), at approximately 2 × 106 cells mL−1 . From this 200 L was taken, diluted with 400 L acid solution (0.08 N HCl, 0.15 M NaCl, 0.1% Triton X-100, pH 1.2), and mixed for 30 s. Then, 1.2 mL acridine orange (Polysciences, Warrington, PA, USA) staining solution (0.15 M NaCl, 0.037 M citric acid, 0.126 M Na2 HPO4 , 0.0011 M disodium EDTA, pH 6.0, containing 6 g mL−1 acridine orange) was added. Samples were incubated on ice for 3 min, after which 10,000 cells were analyzed using a FACScan flow cytometer (BectonDickinson, Heidelberg, Germany). This flow cytometer is equipped with a 488 nm argon ion laser of 15 mW for excitation, and band pass 530/30, 582/42 and long pass 650 nm filters for green, orange and red fluorescence, respectively. Acridine orange stains normal double stranded DNA green, and denatured single-stranded DNA red; the acid treatment potentially denatures damaged DNA. The DNA fragmentation index (DFI) was calculated from the fractions of cells with single and double stranded DNA.
2.5. Statistical analysis Statistical analysis was done using ‘SAS’ software (SAS Institute Inc., Cary, NC, USA), using the generalized linear model procedure. To confirm normal distribution of residuals the ‘univariate’ procedure was used, and the mean and standard deviation were calculated. Different sperm processing methods were tested on the same ejaculate, for statistical analysis we therefore analyzed using paired analysis. Fixed effects were the three different semen preparation methods, whereas random effects were stallion and ejaculate. In addition, for cryopreserved samples, the fixed effects were the ‘poor’ and ‘good’ freezing groups. Correlations were analyzed using Pearson correlation coefficient analysis, while differences between semen preparation methods, and ‘poor’ and ‘good’ freezing groups were analyzed using Tukey multiple pair wise comparisons. Differences were taken to be statistically significant when the probability was P < 0.05.
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Fig. 1. Percentages of morphologically abnormal sperm (A), progressively motile sperm (B), membrane intact sperm (C), and the DNA fragmentation index as a measure for chromatin integrity (D); for diluted semen (black bars) and semen that was processed via ordinary centrifugation (light gray bars) as well as iodixanol density gradient centrifugation (dark gray bars). Averages ± standard deviations were determined from three ejaculates from six stallions. Differences (P < 0.05) between processing methods are indicated with different letters.
3. Results 3.1. Sperm characteristics In Fig. 1 and Table 1, sperm characteristics including morphology and motility as well as membrane and chromatin integrity are shown, for semen that was diluted and processed using ordinary centrifugation or two-layer iodixanol density gradient centrifugation. Whereas 74% of the sperm are recovered after ordinary centrifugation, only 33% are recovered after iodixanol density gradient centrifugation. Ordinary centrifugation results in an increase in the percentage of morphologically abnormal sperm. In addition, centrifugation and collection of sperm in a pellet results in a decrease in progressively motile and membrane intact cells. For semen processed using iodixanol density gradient centrifugation, the percentages of morphologically abnormal and progressively motile sperm are not different from diluted semen. In addition, iodixanol density gradient centrifugation results in greater percentages of membrane intact sperm and greater chromatin integrity, as compared to both diluted semen and semen processed via ordinary centrifugation.
The percentage of membrane intact cells decreases about 8% for sperm that was diluted and stored at 5 ◦ C for up to 72 h. In contrast, membrane integrity remains constant during this period for sperm obtained after ordinary centrifugation as well as iodixanol density gradient centrifugation (Fig. 2). Percentages of progressively motile cells decrease when sperm is stored for 72 h at 5 ◦ C: 38% for diluted sperm and 20% for sperm selected using ordinary centrifugation or iodixanol density gradient centrifugation. Because the initial percentage of progressively motile sperm for diluted and iodixanol processed sperm is 46–48% and that of ordinary centrifuged sperm only 38%, the percentage of progressively motile sperm after 72 h storage at 5 ◦ C is greatest for semen processed using iodixanol density gradient centrifugation. 3.2. Cryopreservation Whether stallion sperm selected via iodixanol density gradient centrifugation could be used for cryopreservation, was tested as well as if differences exist in cryosurvival after different centrifugation protocols for sperm from stallions with sperm with ‘good’ and ‘poor’ freezing qualities
Table 1 Sperm characteristics for semen that was diluted, and processed using ordinary centrifugation as well as iodixanol density gradient centrifugation. Evaluation of sperm morphology (MAS: morphologically abnormal sperm) was done by microscopic observations, chromatin integrity (DFI: DNA fragmentation index) and plasma and acrosomal membrane integrity (PMI: plasma membrane intact, PAS: positive acrosomal status) were determined using flow cytometry. Motility parameters (PMS: progressively motile sperm, VCL: curvilinear velocity, VAP: average path velocity, ALH: amplitude of lateral head displacement) were determined using computer assisted sperm analysis. Averages ± standard deviations were determined from three ejaculates from six stallions. Differences (P < 0.05) between processing methods are indicated with different letters. Diluted Sperm recovery (%) PMS (%) VCL (m s−1 ) VAP (m s−1 ) ALH (m1 ) PMI (%) PAS (%) DFI (%) MAS (total %) Abnormal acrosomes (%) Abnormal heads (%) Abnormal neck region (%) Mid-piece abnormalities (%) Abnormal principal/end piece (%)
Centrifugation
– 46.6a 136.4 95.7 2.8 68.9a 6.8a,b 15.1a 37.7 10.5a 6.1 4.8 8.8 7.4
± ± ± ± ± ± ± ± ± ± ± ± ±
18.8 24.5 20.5 0.3 11.2 4.1 9.0 9.8 4.0 2.6 3.7 5.2 4.9
74.4b 38.0b 131.8 93.2 2.9 60.1a 8.4a 15.8a 45.7 12.9a 9.6 6.0 5.7 11.2
± ± ± ± ± ± ± ± ± ± ± ± ± ±
15.6 17.3 23.1 20.3 0.4 11.7 4.8 8.9 13.5 6.3 7.4 3.0 4.2 5.1
Iodixanol density gradient centrifugation 33.1a 48.2a. 134.8 89.5 2.7 79.0b 3.5b 4.6b 37.4 5.7b 8.0 4.3 7.7 11.6
± ± ± ± ± ± ± ± ± ± ± ± ± ±
10.9 21.7 26.0 19.9 0.4 10.9 2.1 3.3 15.2 3.2 5.2 3.7 4.6 9.1
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Fig. 2. Percentages of progressively motile sperm (A; solid lines) as well as membrane intact sperm (B; dashed lines) during storage at 5 ◦ C for up to 72 h; for diluted semen (black squares) and semen that was processed via ordinary centrifugation (light gray circles) as well as iodixanol density gradient centrifugation (dark gray triangles). Averages ± standard deviations were determined from three ejaculates from six stallions. Differences (P < 0.05) between processing methods at a particular time point are indicated with different letters.
Fig. 3. Percentages of progressively motile sperm (A, C) as well as membrane intact sperm (B, D) before cryopreservation (A, B) and after freezing and thawing (C, D); for semen that was processed via ordinary centrifugation as well as iodixanol density gradient centrifugation. Averages ± standard deviations were determined from three ejaculates from three stallions that were classified as semen from stallions with ‘good’ freezing qualities (light gray bars) and three that were classified as those with semen with ‘poor’ freezing qualities (dark gray bars), as well as all six stallions together (black bars). Differences (P < 0.05) between processing methods and between stallions with semen with ‘good’ and ‘poor’ freezing qualities are indicated with different letters and asterisks, respectively.
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(Fig. 3). Post-freeze survival is greater for sperm selected using iodixanol density gradient centrifugation prior to cryopreservation compared to ordinary centrifugation. The percentage of membrane intact cells is about 30% for sperm cryopreserved after ordinary centrifugation, and this is increased up to about 50% after iodixanol density gradient centrifugation, both for stallions with sperm with ‘good’ and ‘poor’ freezing qualities. Differences between sperm from stallions with sperm with ‘good’ and ‘poor’ freezing qualities become evident as differences in progressive motility. For stallions with sperm with ‘good’ freezing qualities, iodixanol density gradient centrifugation prior to cryopreservation, results in 39% progressively motile cells compared to 28% after ordinary centrifugation. For stallions with sperm with ‘poor’ freezing qualities, both preparation methods result in less then 10% progressively motile sperm with slightly greater percentages for sperm prepared using iodixanol density gradient centrifugation. Stallions with sperm with ‘good’ and ‘poor’ freezing qualities already exhibit differences in pre-freeze motility percentages, that likely underlie differences after freezing and thawing. Prefreeze motility and membrane integrity are greater after iodixanol density gradient centrifugation as compared to ordinary centrifugation, which may explain differences in post-thaw survival between these two sperm preparation methods. 4. Discussion In the present study, a two-layer iodixanol density gradient centrifugation protocol for processing of stallion semen was evaluated. Using a 16% iodixanol solution top layer of 1.090 g mL−1 and a 30% solution of 1.165 g mL−1 as bottom layer allows for selection of higher percentages of morphological normal sperm as compared to ordinary centrifugation. Moreover, percentages of progressively motile sperm and membrane and chromatin integrity are increased when cells are selected via density gradient centrifugation. Collecting sperm on a so-called cushion fluid allows for using greater centrifugation speeds while limiting mechanical forces on cells. Ordinary cushion fluid centrifugation using 60% iodixanol of 1.320 g mL−1 collects both sperm and cell debris (Sieme et al., 2003; Mousset-Simeon et al., 2004). Collection of sperm as a layer on a dense solution reduces contact with the cell debris collected in the pellet. With cell debris there is likely accumulated reactive oxygen species which may cause damaging reactions. In contrast with ordinary cushion fluid centrifugation, selection of cells through a density gradient solution results in selection of sperm with less DNA fragmentation (Morrell et al., 2009a). Cells with decreased chromatin integrity most likely have a different density from normal sperm. Use of sperm with decreased DNA fragmentation for artificial insemination, is described to correlate with increased fertility rates and pregnancies (Evenson et al., 2002). Iodixanol density gradient centrifugation and ordinary dilution yield similar percentages of progressively motile sperm directly after processing, whereas motility is decreased for sperm exposed to ordinary centrifugation. As for cold storage, however, a centrifugation protocol
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is needed to remove seminal plasma and minimize the decrease in motility during the first 24 h of storage (Jasko et al., 1992; Brisko et al., 2000). Also for cryopreservation, seminal plasma should be removed (Jasko et al., 1992; Love and Kenney, 1998). Similar as for cold storage, increased percentages of motile and membrane intact sperm after iodixanol density gradient centrifugation also result in greater yields of survival after freezing and thawing. Sperm recovery and quality after density gradient centrifugation are affected by the density solutions that are used, the centrifugation protocol applied, as well as the method for retrieval of the spermatozoa (Smith et al., 1997). Recovery rates after sperm selection procedures including density gradient centrifugation are generally poor (Sieme et al., 2006). It is anticipated that this loss can be compensated by using smaller doses for insemination, while sperm of superior quality is used (Morrell et al., 2009a; Macias Garcia et al., 2009). Sperm selection procedures, including two-layer iodixanol density gradient centrifugation, allow for improvement of sperm quality in terms of increased motility and membrane and chromatin integrity. It remains to be determined, however, how functional properties are affected. Acknowledgments The authors would like to thank the National Stud of Lower Saxony for taking care of the stallions and help with semen collections. Jane Morrell is acknowledged for critical reading of the manuscript and helpful advice. References Aurich, C., 2008. Recent advances in cooled-semen technology. Anim. Reprod. Sci. 107, 268–275. Brisko, J.W., Crockett, E.C., Squires, E.L., 2000. Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. Theriogenology 54, 129–136. Brito, L.F.C., 2007. Evaluation of stallion sperm morphology. Clin. Tech. Equine Pract. 5, 249–264. Ecot, P.G., Decuadro-Hansen, G., Delhomme, G., Vidament, M., 2005. Evaluation of cushioned centrifugation technique for processing equine semen for freezing. Anim. Reprod. Sci. 89, 245–248. Evenson, D.P., Larson, L.K., Jost, K.L., 2002. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparison with other techniques. J. Androl. 23, 25–43. Harrison, K., 1997. Iodixanol as a density gradient medium for the isolation of motile spermatozoa. J. Assist. Reprod. Genet. 14, 385–387. Jasko, D.J., Hathaway, J.A., Scaltenbrand, V.L., Simper, W.D., Squires, E.L., 1992. Effect of seminal plasma and egg yolk on motion characteristics of cooled stallion spermatozoa. Theriogenology 37, 1241–1252. Loomis, P.R., 2001. The equine frozen semen industry. Anim. Reprod. Sci. 68, 191–200. Love, C.C., Kenney, R.M., 1998. The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion. Theriogenology 50, 955–972. Macpherson, M.L., Blanchard, T.L., Love, C.C., Brinsko, S.P., Varner, D.D., 2002. Use of siliane-coated silica particle solution to enhance the quality of ejaculated semen in stallions. Theriogeniology 58, 317–320. Macias Garcia, B., Morrell, J.M., Ortega-Ferusola, C., Gonzalez-Fernandez, L., Tapia, A., Rodriguez-Martinez, H., Pena, J., 2009. Centrifugation on a single layer of colloid selects improved quality spermatozoa from frozen–thawed stallion semen. Anim. Reprod. Sci. 114, 193–202. Morrell, J.M., 2006. Update on semen technologies for animal breeding. Reprod. Domest. Anim. 41, 63–67. Morrell, J.M., Johannisson, M.A., Dalin, A.M., Rodriguez-Martinez, H., 2009a. Morphology and chromatin integrity of stallion spermatozoa prepared by density gradient and single layer centrifugation through silica colloids. Reprod. Domest. Anim. 44, 512–517.
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Morrell, J.M., Johannisson, M.A., Strutz, H., Dalin, A.M., RodriguezMartinez, H., 2009b. Collodial centrifugation of stallion semen: changes in sperm motility, velocity and chromatin integrity during storage. J. Equine Vet. Sci. 29, 24–32. Mortimer, D., 2000. Sperm preparation methods. J. Androl. 21, 357–366. Mousset-Simeon, N., Rives, N., Masse, L., Chevallier, F., Mace, B., 2004. Comparison of six gradient media for selection of cryopreserved donor spermatozoa. J. Androl. 25, 881–884. Pickett, B.W., Sullivan, J.J., Beyers, W.W., Pace, M.M., Remmenga, E.E., 1975. Effect of centrifugation and seminal plasma on motility and fertility of stallion and bull spermatozoa. Fertil. Steril. 26, 167–174. Rathi, R., Colenbrander, B., Bevers, M.M., Gadella, B.M., 2001. Evaluation of in vitro capacitation of stallion spermatozoa. Biol. Reprod. 65, 462–470.
Sieme, H., Martinsson, G., Rautenberg, H., Walter, K., Aurich, C., Petzoldt, R., Klug, E., 2003. Application of techniques for sperm selection in fresh and frozen–thawed stallion semen. Reprod. Domest. Anim. 38, 134–140. Sieme, H., Knop, K., Rath, D., 2006. Effects of cushioned centrifugation on sperm quality in stallion semen stored cooled at 5 ◦ C for 24 h and stored cooled for 2 or 24 h and then frozen. Anim. Reprod. Sci. 94, 99–103. Smith, T.T., Byers, M., Kaftani, D., Whitford, W., 1997. The use of iodixanol as a density gradient material for separating human sperm from semen. Arch. Androl. 38, 223–230. Vidament, M., Dupere, A.M., Julienne, P., Evain, A., Noue, P., Palmer, E., 1997. Equine frozen semen: freezability and fertility field results. Theriogenology 48, 907–917.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Iodixanol density gradient centrifugation for selecting stallion sperm for cold storage and cryopreservation Gesa Stuhtmann a,b , Harriëtte Oldenhof a , Pamela Peters a,b , Jutta Klewitz a , Gunilla Martinsson b , Harald Sieme a,∗ a b
Clinic for Horses – Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Germany National Stud Lower Saxony, Celle, Germany
a r t i c l e
i n f o
Article history: Received 5 December 2011 Received in revised form 19 June 2012 Accepted 21 June 2012 Available online 27 June 2012 Keywords: Stallion spermatozoa Sperm cleanup Density gradient centrifugation Iodixanol
a b s t r a c t Density gradient centrifugation can be used for selection of sperm of superior quality and removal of seminal plasma for use in artificial insemination. In this study, the use of two-layer iodixanol density gradient centrifugation was evaluated for processing of stallion semen. The protocol includes centrifugation through a 16% iodixanol top layer of 1.090 g mL−1 and collection of motile and intact sperm on a 30% iodixanol bottom layer of 1.165 g mL−1 . Sperm recovery and effects on sperm quality were determined during cold storage as well as after cryopreservation and compared with ordinary dilution and centrifugation. Two-layer iodixanol density gradient centrifugation allows for selection of greater percentages of morphologically normal and progressively motile sperm compared to ordinary centrifugation. This likely results from collecting sperm on the bottom layer that functions as cushion fluid, which prevents mechanical forces as occur when sperm are packed in a pellet. In addition, percentages of membrane and chromatin integrity are increased when cells are selected based on their density via centrifugation through the top and bottom layers. Removal of seminal plasma and increased initial percentages of motile and membrane intact sperm after iodixanol density gradient centrifugation also result in greater percentages of progressively motile and membrane intact sperm during cold storage as well as after freezing and thawing. In conclusion, the two-layer iodixanol density gradient centrifugation protocol described in this manuscript allows for selection of stallion sperm with greater survival rates for cold storage and cryopreservation. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The increased use of artificial insemination in horse breeding has demanded the development of semen preparation methods that provide spermatozoa of good quality for insemination, with minimal variation amongst
∗ Corresponding author at: Clinic for Horses – Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Bünteweg 15, 30559 Hannover, Germany. Tel.: +49 0511 953 8530; fax: +49 0511 953 82 8530. E-mail address: [email protected] (H. Sieme). 0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2012.06.017
ejaculates and individuals, and maintenance of quality during cold storage as well as cryopreservation (Loomis, 2001; Aurich, 2008). Sperm separation techniques are used in assisted reproduction to select spermatozoa of superior quality and remove non-viable, morphologically abnormal or immature spermatozoa and seminal plasma. Sperm quality is considered good when a sample contains large numbers of progressively motile sperm, with greater percentages of membrane and chromatin intactness. Separation techniques are based on differences in sperm viability, motility, morphology, size and density. They include: (i) dilution and washing via ordinary centrifugation,
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(ii) sperm migration/sedimentation methods (swim-up), (iii) filtration/adherence methods (glass wool or beads, sephadex, leucosorb), and (iv) density gradient centrifugation (polyvinylpyrrolidone or silane-coated silica particles, iohexol, ficoll) (Sieme et al., 2003; Mortimer, 2000; Morrell, 2006). After Percoll was withdrawn for clinical use, alternative solutions for density gradient centrifugation were tested including iodixanol solutions. Density gradient centrifugation protocols using iodixanol exist for selection of human and bull sperm (Harrison, 1997; Smith et al., 1997), but its use for processing of stallion semen has not been evaluated. Iodixanol, however, has been used for cushion centrifugation of stallion sperm (Ecot et al., 2005; Sieme et al., 2006). Using this method, spermatozoa are collected as a layer on top of the iodixanol layer, which has a greater density than sperm. This is in contrast with ordinary centrifugation or density gradient protocols that use Percoll (Sieme et al., 2003), EquiPure (Macpherson et al., 2002) or Androcoll (Morrell et al., 2009b), in which sperm are collected in a pellet at the bottom of the tube. Collecting sperm in a pellet is thought to be deleterious, because sperm will be tightly packed together with cell debris and reactive oxygen species (Pickett et al., 1975; Smith et al., 1997). Two-layer density gradient centrifugation using a top and bottom layer of different densities can be used for selecting spermatozoa on the basis of their density properties through the top and bottom layers, where the bottom layer also functions as cushion fluid thereby preventing pelleting of the cells. The aim of the present study was to test the use of two-layer iodixanol density gradient centrifugation for selection of stallion sperm, and compare the efficiency with ordinary centrifugation methods. Sperm recovery rates of these methods were determined, and effects on sperm morphology and motility as well as membrane and chromatin integrity. Sperm quality parameters were assessed during storage at 5 ◦ C. In addition, application of iodixanol density gradient centrifugation for cryopreservation of sperm from stallions with so-called ‘good’ and ‘poor’ freezing characteristics was evaluated. 2. Materials and methods 2.1. Semen collection, dilution, and ordinary centrifugation For the experiments described in this study, three ejaculates were collected from each of six stallions (ages 5–11 years) of the Hanoverian warmblood breed, that participated in the artificial insemination program of the National Stud of Lower Saxony in Celle, Germany. Semen was collected at the beginning of the breeding season. Prior to collecting semen for experiments, semen collections were performed for 1 week to stabilize the extra-gonadal sperm reserves. Stallions were divided into those with sperm with ‘good’ and ‘poor’ freezing qualities (three in each group), based on previous experiments. This was done according to the criteria described by Vidament et al. (1997): stallions with a post-thaw progressive sperm motility less than or equal to 35% in three or more ejaculates out of ten were
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considered to be with ‘poor’ freezing qualities, whereas stallions with sperm above these criteria were designated as those with ‘good’ freezing qualities. Semen was collected every other day, using an artificial vagina (Hanoverian model; Minitüb, Landshut, Germany). A ‘phantom’ device for stallions was used with a ‘teaser’ mare fixed in front. The collection device was equipped with a sterile gauze filter to remove the gel portion. Gel-free semen was evaluated for volume, and the sperm concentration was estimated via measurement of the optical density (Spermacue; Minitübe, Tiefenbach, Germany). A 1 mL aliquot of raw semen was frozen in liquid nitrogen for later analysis of chromatin integrity, and the remainder was diluted with pre-warmed EquiPro extender (Minitübe, Tiefenbach, Germany) to 100 × 106 cells mL−1 . Diluted semen was divided into three parts. One part was diluted with an equal volume of EquiPro to 50 × 106 cells mL−1 , and is further referred to as diluted semen. The other two parts were processed by ordinary centrifugation or iodixanol density gradient centrifugation as subsequently described. Ordinary centrifugation was performed by centrifuging 40 mL diluted semen in a 50 mL tube at 600 × g for 10 min. The supernatant was removed using a syringe with a needle. The re-suspended sperm pellet was transferred to a clean tube, after which the cell concentration was determined using a haemocytometer (Thoma Neu, Hecht, Sontheim, Germany). The total number of re-suspended sperm was determined, and the recovery rate was calculated by comparing with the total number of sperm before centrifugation. 2.2. Iodixanol density gradient centrifugation of diluted semen A 60% iodixanol solution in water with a density of 1.320 g mL−1 at room temperature is commercially available as OptiPrep (Axis-shield, Oslo, Norway). Before density gradient centrifugation, isotonic solutions of the desired densities were prepared by dilution with Hanks buffered salt solution (HBSS; 5.33 mM KCl, 0.441 mM KH2 PO4 , 4.17 mM NaHCO3 , 137.92 mM NaCl, 0.338 mM NaH2 PO4 , 5.56 mM glucose, pH 7.4). Dilution of iodixanol with HBSS did not change the pH or osmolality; these variables remained 7.4 and 300 mOsm kg−1 , respectively. A 16% iodixanol solution of 1.090 g mL−1 was used as top layer, and a 30% solution of 1.165 g mL−1 as bottom layer. Densities were measured using a bench-top refractometer (Zeiss, Jena, Germany). Density gradients were prepared in a 50 mL conical plastic tube: 10 mL of the top layer was added first, after which 10 mL bottom layer was added underneath. Twenty mL of diluted semen was carefully layered on top of the iodixanol solutions. After centrifugation at 1000 × g for 20 min, spermatozoa show up as a band between the upper and bottom layer. The supernatant was aspirated carefully, when the band that contained the sperm was reached this was transferred into a clean tube using a 25 mL syringe with attached a 500 L straw. The cell concentration and recovery rate were determined as described above.
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2.3. Cold storage and cryopreservation of diluted and centrifuged sperm The sperm as harvested after centrifugation was divided into two parts: one part was used for storage at 5 ◦ C, whereas the other part was used for cryopreservation. For cold storage, samples were diluted with EquiPro to a final concentration of 50 × 106 cells mL−1 , and remained at room temperature for 1 h, after which the temperature was reduced to 5 ◦ C. For cryopreservation, the centrifuged samples were diluted with EquiPro to a concentration of 100 × 106 cells mL−1 . Then the samples were diluted with an equal volume of EquiPro freezing extender (containing 5% glycerol) resulting in 50 × 106 cells mL−1 and 2.5% glycerol, and cooled to 5 ◦ C with 0.1 ◦ C min−1 . This cooling rate was reached via equilibration of diluted semen in a beaker with water of room temperature in a cold handling cabinet of 5 ◦ C. After reaching 5 ◦ C (after about 2.5 h) diluted semen was packaged in 0.5 mL straws, cooled from 5 ◦ C to −15 ◦ C at 10 ◦ C min−1 , and from −15 ◦ C to −140 ◦ C at 25 ◦ C min−1 using a controlled rate freezer (IMV-Technologies, L’Aigle, France). Straws were transfered into liquid nitrogen, in which they were stored until analysis. Thawing was done in a water bath at 37 ◦ C for 30 s. 2.4. Assessment of sperm motility, morphology, and chromatin and membrane integrity Sperm morphology, motility parameters, membrane integrity, acrosomal status and chromatin integrity were assessed immediately after dilution or centrifugation, after 24, 48 and 72 h of storage at 5 ◦ C, as well as after cryopreservation. Sperm motility parameters were assessed via computer assisted sperm analysis (SpermVision; Minitübe, Tiefenbach, Germany) using 20 m analyzing chambers (Leja, Nieuw-Vennep, Netherlands). Motility parameters that were determined included: percentage of progressively motile sperm (PMS), curvilinear velocity (VCL in m s−1 ), average path velocity (VAP in m s−1 ), and the amplitude of lateral head movement (ALH in m). Evaluation of sperm morphology was done for 200 cells per sample according to Brito (2007). Therefore, 100 L semen was stained with 300 L nigrosin–eosin solution (10% nigrosin, 0.7% eosin, 3.75 mM Na2 HPO4 , 1.88 mM KH2 PO4 , 5.78 mM NaK tartrate, 3.75 mM glucose). Plasma and acrosomal membrane integrity of spermatozoa were evaluated using a flow cytometer (Cell Lab Quanta SC MPL; Beckman-Coulter, Krefeld, Germany). This flow cytometer is equipped with a 488 nm argon ion laser of 22 mW for excitation, and band pass 525/30, 590/30 and long pass 670 nm filters for green, orange and red fluorescence, respectively. HBS of 300 mOsm kg−1 (20 mM HEPES pH 7.4, 137 mM NaCl, 10 mM glucose, 2.5 mM KOH) was used as sheath fluid. A double staining with propidium iodide (PI; Sigma–Aldrich, St. Louis, MO, USA) and fluorescein labeled peanut agglutinin (FITC-PNA; Vector Laboratories, Burlingame, CA, USA) was performed, according to Rathi et al. (2001) with minor modifications. Ten
L 50 × 106 cells mL−1 in extender was diluted in 480 L HBS, and 2.5 L 500 g mL−1 propidium iodide (PI) and 7.5 L 600 g mL−1 fluorescein labeled peanut agglutinin (FITC-PNA) were added. This results in 1 × 106 cells mL−1 , 2.5 g mL−1 PI and 9 g mL−1 FITC-PNA. Samples were incubated for 15 min at 37 ◦ C in darkness, and 10,000 cells were measured. Sperm were selected based on their sideward scatter and electronic volume properties. Cells that have a damaged plasma membrane show red fluorescence of PI bound to DNA, cells that have a damaged acrosome will show green fluorescence of FITC-PNA bound to glycoproteins. Sperm that were both PI- and FITC-PNA-negative were considered viable with intact plasma and acrosomal membranes. The sperm chromatin structure assay (SCSA) was used to evaluate chromatin integrity. In this assay, sperm chromatin is treated with acid after which the extent of DNA fragmentation is determined (Evenson et al., 2002). In short, thawed samples were diluted in TNE (0.15 M NaCl, 0.01 M Tris–HCl, 1 mM disodium EDTA, pH 7.4), at approximately 2 × 106 cells mL−1 . From this 200 L was taken, diluted with 400 L acid solution (0.08 N HCl, 0.15 M NaCl, 0.1% Triton X-100, pH 1.2), and mixed for 30 s. Then, 1.2 mL acridine orange (Polysciences, Warrington, PA, USA) staining solution (0.15 M NaCl, 0.037 M citric acid, 0.126 M Na2 HPO4 , 0.0011 M disodium EDTA, pH 6.0, containing 6 g mL−1 acridine orange) was added. Samples were incubated on ice for 3 min, after which 10,000 cells were analyzed using a FACScan flow cytometer (BectonDickinson, Heidelberg, Germany). This flow cytometer is equipped with a 488 nm argon ion laser of 15 mW for excitation, and band pass 530/30, 582/42 and long pass 650 nm filters for green, orange and red fluorescence, respectively. Acridine orange stains normal double stranded DNA green, and denatured single-stranded DNA red; the acid treatment potentially denatures damaged DNA. The DNA fragmentation index (DFI) was calculated from the fractions of cells with single and double stranded DNA.
2.5. Statistical analysis Statistical analysis was done using ‘SAS’ software (SAS Institute Inc., Cary, NC, USA), using the generalized linear model procedure. To confirm normal distribution of residuals the ‘univariate’ procedure was used, and the mean and standard deviation were calculated. Different sperm processing methods were tested on the same ejaculate, for statistical analysis we therefore analyzed using paired analysis. Fixed effects were the three different semen preparation methods, whereas random effects were stallion and ejaculate. In addition, for cryopreserved samples, the fixed effects were the ‘poor’ and ‘good’ freezing groups. Correlations were analyzed using Pearson correlation coefficient analysis, while differences between semen preparation methods, and ‘poor’ and ‘good’ freezing groups were analyzed using Tukey multiple pair wise comparisons. Differences were taken to be statistically significant when the probability was P < 0.05.
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Fig. 1. Percentages of morphologically abnormal sperm (A), progressively motile sperm (B), membrane intact sperm (C), and the DNA fragmentation index as a measure for chromatin integrity (D); for diluted semen (black bars) and semen that was processed via ordinary centrifugation (light gray bars) as well as iodixanol density gradient centrifugation (dark gray bars). Averages ± standard deviations were determined from three ejaculates from six stallions. Differences (P < 0.05) between processing methods are indicated with different letters.
3. Results 3.1. Sperm characteristics In Fig. 1 and Table 1, sperm characteristics including morphology and motility as well as membrane and chromatin integrity are shown, for semen that was diluted and processed using ordinary centrifugation or two-layer iodixanol density gradient centrifugation. Whereas 74% of the sperm are recovered after ordinary centrifugation, only 33% are recovered after iodixanol density gradient centrifugation. Ordinary centrifugation results in an increase in the percentage of morphologically abnormal sperm. In addition, centrifugation and collection of sperm in a pellet results in a decrease in progressively motile and membrane intact cells. For semen processed using iodixanol density gradient centrifugation, the percentages of morphologically abnormal and progressively motile sperm are not different from diluted semen. In addition, iodixanol density gradient centrifugation results in greater percentages of membrane intact sperm and greater chromatin integrity, as compared to both diluted semen and semen processed via ordinary centrifugation.
The percentage of membrane intact cells decreases about 8% for sperm that was diluted and stored at 5 ◦ C for up to 72 h. In contrast, membrane integrity remains constant during this period for sperm obtained after ordinary centrifugation as well as iodixanol density gradient centrifugation (Fig. 2). Percentages of progressively motile cells decrease when sperm is stored for 72 h at 5 ◦ C: 38% for diluted sperm and 20% for sperm selected using ordinary centrifugation or iodixanol density gradient centrifugation. Because the initial percentage of progressively motile sperm for diluted and iodixanol processed sperm is 46–48% and that of ordinary centrifuged sperm only 38%, the percentage of progressively motile sperm after 72 h storage at 5 ◦ C is greatest for semen processed using iodixanol density gradient centrifugation. 3.2. Cryopreservation Whether stallion sperm selected via iodixanol density gradient centrifugation could be used for cryopreservation, was tested as well as if differences exist in cryosurvival after different centrifugation protocols for sperm from stallions with sperm with ‘good’ and ‘poor’ freezing qualities
Table 1 Sperm characteristics for semen that was diluted, and processed using ordinary centrifugation as well as iodixanol density gradient centrifugation. Evaluation of sperm morphology (MAS: morphologically abnormal sperm) was done by microscopic observations, chromatin integrity (DFI: DNA fragmentation index) and plasma and acrosomal membrane integrity (PMI: plasma membrane intact, PAS: positive acrosomal status) were determined using flow cytometry. Motility parameters (PMS: progressively motile sperm, VCL: curvilinear velocity, VAP: average path velocity, ALH: amplitude of lateral head displacement) were determined using computer assisted sperm analysis. Averages ± standard deviations were determined from three ejaculates from six stallions. Differences (P < 0.05) between processing methods are indicated with different letters. Diluted Sperm recovery (%) PMS (%) VCL (m s−1 ) VAP (m s−1 ) ALH (m1 ) PMI (%) PAS (%) DFI (%) MAS (total %) Abnormal acrosomes (%) Abnormal heads (%) Abnormal neck region (%) Mid-piece abnormalities (%) Abnormal principal/end piece (%)
Centrifugation
– 46.6a 136.4 95.7 2.8 68.9a 6.8a,b 15.1a 37.7 10.5a 6.1 4.8 8.8 7.4
± ± ± ± ± ± ± ± ± ± ± ± ±
18.8 24.5 20.5 0.3 11.2 4.1 9.0 9.8 4.0 2.6 3.7 5.2 4.9
74.4b 38.0b 131.8 93.2 2.9 60.1a 8.4a 15.8a 45.7 12.9a 9.6 6.0 5.7 11.2
± ± ± ± ± ± ± ± ± ± ± ± ± ±
15.6 17.3 23.1 20.3 0.4 11.7 4.8 8.9 13.5 6.3 7.4 3.0 4.2 5.1
Iodixanol density gradient centrifugation 33.1a 48.2a. 134.8 89.5 2.7 79.0b 3.5b 4.6b 37.4 5.7b 8.0 4.3 7.7 11.6
± ± ± ± ± ± ± ± ± ± ± ± ± ±
10.9 21.7 26.0 19.9 0.4 10.9 2.1 3.3 15.2 3.2 5.2 3.7 4.6 9.1
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Fig. 2. Percentages of progressively motile sperm (A; solid lines) as well as membrane intact sperm (B; dashed lines) during storage at 5 ◦ C for up to 72 h; for diluted semen (black squares) and semen that was processed via ordinary centrifugation (light gray circles) as well as iodixanol density gradient centrifugation (dark gray triangles). Averages ± standard deviations were determined from three ejaculates from six stallions. Differences (P < 0.05) between processing methods at a particular time point are indicated with different letters.
Fig. 3. Percentages of progressively motile sperm (A, C) as well as membrane intact sperm (B, D) before cryopreservation (A, B) and after freezing and thawing (C, D); for semen that was processed via ordinary centrifugation as well as iodixanol density gradient centrifugation. Averages ± standard deviations were determined from three ejaculates from three stallions that were classified as semen from stallions with ‘good’ freezing qualities (light gray bars) and three that were classified as those with semen with ‘poor’ freezing qualities (dark gray bars), as well as all six stallions together (black bars). Differences (P < 0.05) between processing methods and between stallions with semen with ‘good’ and ‘poor’ freezing qualities are indicated with different letters and asterisks, respectively.
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(Fig. 3). Post-freeze survival is greater for sperm selected using iodixanol density gradient centrifugation prior to cryopreservation compared to ordinary centrifugation. The percentage of membrane intact cells is about 30% for sperm cryopreserved after ordinary centrifugation, and this is increased up to about 50% after iodixanol density gradient centrifugation, both for stallions with sperm with ‘good’ and ‘poor’ freezing qualities. Differences between sperm from stallions with sperm with ‘good’ and ‘poor’ freezing qualities become evident as differences in progressive motility. For stallions with sperm with ‘good’ freezing qualities, iodixanol density gradient centrifugation prior to cryopreservation, results in 39% progressively motile cells compared to 28% after ordinary centrifugation. For stallions with sperm with ‘poor’ freezing qualities, both preparation methods result in less then 10% progressively motile sperm with slightly greater percentages for sperm prepared using iodixanol density gradient centrifugation. Stallions with sperm with ‘good’ and ‘poor’ freezing qualities already exhibit differences in pre-freeze motility percentages, that likely underlie differences after freezing and thawing. Prefreeze motility and membrane integrity are greater after iodixanol density gradient centrifugation as compared to ordinary centrifugation, which may explain differences in post-thaw survival between these two sperm preparation methods. 4. Discussion In the present study, a two-layer iodixanol density gradient centrifugation protocol for processing of stallion semen was evaluated. Using a 16% iodixanol solution top layer of 1.090 g mL−1 and a 30% solution of 1.165 g mL−1 as bottom layer allows for selection of higher percentages of morphological normal sperm as compared to ordinary centrifugation. Moreover, percentages of progressively motile sperm and membrane and chromatin integrity are increased when cells are selected via density gradient centrifugation. Collecting sperm on a so-called cushion fluid allows for using greater centrifugation speeds while limiting mechanical forces on cells. Ordinary cushion fluid centrifugation using 60% iodixanol of 1.320 g mL−1 collects both sperm and cell debris (Sieme et al., 2003; Mousset-Simeon et al., 2004). Collection of sperm as a layer on a dense solution reduces contact with the cell debris collected in the pellet. With cell debris there is likely accumulated reactive oxygen species which may cause damaging reactions. In contrast with ordinary cushion fluid centrifugation, selection of cells through a density gradient solution results in selection of sperm with less DNA fragmentation (Morrell et al., 2009a). Cells with decreased chromatin integrity most likely have a different density from normal sperm. Use of sperm with decreased DNA fragmentation for artificial insemination, is described to correlate with increased fertility rates and pregnancies (Evenson et al., 2002). Iodixanol density gradient centrifugation and ordinary dilution yield similar percentages of progressively motile sperm directly after processing, whereas motility is decreased for sperm exposed to ordinary centrifugation. As for cold storage, however, a centrifugation protocol
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is needed to remove seminal plasma and minimize the decrease in motility during the first 24 h of storage (Jasko et al., 1992; Brisko et al., 2000). Also for cryopreservation, seminal plasma should be removed (Jasko et al., 1992; Love and Kenney, 1998). Similar as for cold storage, increased percentages of motile and membrane intact sperm after iodixanol density gradient centrifugation also result in greater yields of survival after freezing and thawing. Sperm recovery and quality after density gradient centrifugation are affected by the density solutions that are used, the centrifugation protocol applied, as well as the method for retrieval of the spermatozoa (Smith et al., 1997). Recovery rates after sperm selection procedures including density gradient centrifugation are generally poor (Sieme et al., 2006). It is anticipated that this loss can be compensated by using smaller doses for insemination, while sperm of superior quality is used (Morrell et al., 2009a; Macias Garcia et al., 2009). Sperm selection procedures, including two-layer iodixanol density gradient centrifugation, allow for improvement of sperm quality in terms of increased motility and membrane and chromatin integrity. It remains to be determined, however, how functional properties are affected. Acknowledgments The authors would like to thank the National Stud of Lower Saxony for taking care of the stallions and help with semen collections. Jane Morrell is acknowledged for critical reading of the manuscript and helpful advice. References Aurich, C., 2008. Recent advances in cooled-semen technology. Anim. Reprod. Sci. 107, 268–275. Brisko, J.W., Crockett, E.C., Squires, E.L., 2000. Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. Theriogenology 54, 129–136. Brito, L.F.C., 2007. Evaluation of stallion sperm morphology. Clin. Tech. Equine Pract. 5, 249–264. Ecot, P.G., Decuadro-Hansen, G., Delhomme, G., Vidament, M., 2005. Evaluation of cushioned centrifugation technique for processing equine semen for freezing. Anim. Reprod. Sci. 89, 245–248. Evenson, D.P., Larson, L.K., Jost, K.L., 2002. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparison with other techniques. J. Androl. 23, 25–43. Harrison, K., 1997. Iodixanol as a density gradient medium for the isolation of motile spermatozoa. J. Assist. Reprod. Genet. 14, 385–387. Jasko, D.J., Hathaway, J.A., Scaltenbrand, V.L., Simper, W.D., Squires, E.L., 1992. Effect of seminal plasma and egg yolk on motion characteristics of cooled stallion spermatozoa. Theriogenology 37, 1241–1252. Loomis, P.R., 2001. The equine frozen semen industry. Anim. Reprod. Sci. 68, 191–200. Love, C.C., Kenney, R.M., 1998. The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion. Theriogenology 50, 955–972. Macpherson, M.L., Blanchard, T.L., Love, C.C., Brinsko, S.P., Varner, D.D., 2002. Use of siliane-coated silica particle solution to enhance the quality of ejaculated semen in stallions. Theriogeniology 58, 317–320. Macias Garcia, B., Morrell, J.M., Ortega-Ferusola, C., Gonzalez-Fernandez, L., Tapia, A., Rodriguez-Martinez, H., Pena, J., 2009. Centrifugation on a single layer of colloid selects improved quality spermatozoa from frozen–thawed stallion semen. Anim. Reprod. Sci. 114, 193–202. Morrell, J.M., 2006. Update on semen technologies for animal breeding. Reprod. Domest. Anim. 41, 63–67. Morrell, J.M., Johannisson, M.A., Dalin, A.M., Rodriguez-Martinez, H., 2009a. Morphology and chromatin integrity of stallion spermatozoa prepared by density gradient and single layer centrifugation through silica colloids. Reprod. Domest. Anim. 44, 512–517.
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Sieme, H., Martinsson, G., Rautenberg, H., Walter, K., Aurich, C., Petzoldt, R., Klug, E., 2003. Application of techniques for sperm selection in fresh and frozen–thawed stallion semen. Reprod. Domest. Anim. 38, 134–140. Sieme, H., Knop, K., Rath, D., 2006. Effects of cushioned centrifugation on sperm quality in stallion semen stored cooled at 5 ◦ C for 24 h and stored cooled for 2 or 24 h and then frozen. Anim. Reprod. Sci. 94, 99–103. Smith, T.T., Byers, M., Kaftani, D., Whitford, W., 1997. The use of iodixanol as a density gradient material for separating human sperm from semen. Arch. Androl. 38, 223–230. Vidament, M., Dupere, A.M., Julienne, P., Evain, A., Noue, P., Palmer, E., 1997. Equine frozen semen: freezability and fertility field results. Theriogenology 48, 907–917.