Characterizing the glycocalyx of poultry spermatozoa: III. Semen cryopreservation methods alter the carbohydrate component of rooster sperm membrane glycoconjugates1

Characterizing the glycocalyx of poultry spermatozoa: III. Semen cryopreservation methods alter the carbohydrate component of rooster sperm membrane glycoconjugates1 J. Peláez, D. C. Bongalhardo, and J. A. Long2 USDA, Agricultural Research Service, Animal and Natural Resources Institute, Animal Biosciences and Biotechnology Laboratory, Beltsville, MD 20705 ABSTRACT The carbohydrate-rich zone on the sperm surface is essential for inmunoprotection in the female tract and early gamete interactions. We recently have shown the glycocalyx of chicken sperm to be extensively sialylated and to contain residues of mannose, glucose, galactose, fucose, N-acetyl-galactosamine, Nacetyl-glucosamine, and N-acetyl-lactosamine. Our objective here was to evaluate the effects of 3 different cryopreservation methods on the sperm glycocalyx. Semen from roosters was pooled, diluted, cooled to 5°C, and aliquoted for cryopreservation using 6% dimethylacetamide (DMA), 11% dimethylsulfoxide (DMSO), or 11% glycerol (GOH). For the DMA method, semen was equilibrated for 1 min with cryoprotectant and rapidly frozen by dropping 25-μL aliquots into liquid nitrogen. For the other methods, semen was equilibrated for either 1 min (DMSO) or 20 min (GOH), loaded into straws, and frozen with a programmable freezer. Thawing rates mimicked the freezing rates (e.g., rapid for DMA; moderate for DMSO and GOH). Aliquots of thawed and fresh, unfrozen semen were incubated with 1 of 12 fluorescein isothiocyanate-conjugated lectins

and counterstained with propidium iodide, and mean fluorescence intensity (MFI) was assessed by flow cytometry. For each lectin, the MFI of propidium iodidenegative (viable sperm) was compared among the fresh and frozen-thawed treatments (n = 5). For sperm frozen with GOH and DMA, the MFI of most lectins was similar (P > 0.05) to that of fresh sperm, whereas only 5 of 12 lectins were similar between fresh and DMSOfrozen sperm. Sperm from all 3 methods had higher (P < 0.05) MFI for lectins specific for N-acetyl-glucosamine and β-galactose than did fresh sperm. Fewer sperm were damaged (P < 0.001) with GOH than with DMA or DMSO, and membrane integrity was correlated with MFI for 9 of 12 lectins (P < 0.05). These data indicate that surface carbohydrates are altered during cryopreservation, and that cryoprotectant type and freezing-thawing rates affect the degree of modification. Although the glycoconjugates have not yet been identified, it is likely that these cryopreservationinduced changes contribute to the reduced fertility of frozen-thawed chicken semen.

Key words: chicken, sperm glycocalyx, semen cryopreservation, flow cytometry, lectin 2011 Poultry Science 90:435–443 doi:10.3382/ps.2010-00998

INTRODUCTION

Despite the fact that this scientific breakthrough was accomplished with rooster semen (Polge, 1951), the overall fertility rates with frozen-thawed (F-T) poultry semen remain highly variable and not reliable enough for use in commercial production or preservation of genetic stocks. Most of the scientific efforts to improve the cryosurvival of poultry sperm have focused on empirical approaches, such as cryoprotectant type and freezing rate (reviews: Lake, 1986; Hammerstedt, 1995; Lake, 1995; Etches, 1996; Donoghue and Wishart, 2000); however, limited attention has been directed toward understanding how and why poultry sperm lose functional competence after cryopreservation (Long, 2006). The sperm glycocalyx is a dense carbohydrate layer extending 20 to 60 nm from the cell surface (Bearer

More than 50 yr ago, the discovery of the cryoprotective properties of glycerol (GOH) pioneered the success of modern cryobiology and led to the development of semen cryopreservation for a wide range of species. ©2011 Poultry Science Association Inc. Received July 8, 2010. Accepted October 26, 2010. 1 Supported in part by the USDA-Agricultural Research Service project “Analysis of Sperm Storage Mechanisms in Poultry” (Project no. 1265-31000-83-00D) and the Ministerio de Educación y Ciencia (Madrid, Spain). Mention of a trade name, proprietary product, or vendor does not constitute a guarantee or warranty of the product by USDA or imply its approval to the exclusion of other suitable products or vendors. 2 Corresponding author: [email protected]

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and Friend, 1990) that emanates from either plasma membrane proteins (glycoproteins) or lipids (glycolipids). The sperm glycocalyx is modified extensively during sperm transport and maturation, and represents the primary interface between the male gamete and its environment. Glycoconjugates known to be critical for poultry gamete interaction include sialic acid, which has been implicated for both sperm passage through the vagina (Steele and Wishart, 1996) and sperm sequestration in the sperm storage tubules of the hen (Froman and Thursam, 1994), and N-acetyl-d-glucosamine, which is necessary for sperm-egg interaction (Robertson et al., 2000). We recently have shown that the glycocalyx of chicken sperm is extensively sialylated and contains residues of α-mannose, α-glucose, α- and β-galactose, α-fucose, α- and β-N-acetyl-galactosamine, and N-acetyl-lactosamine, as well as monomers and dimers of N-acetyl-glucosamine in variable amounts (Peláez and Long, 2007). Monomers of N-acetyl-glucosamine and glycoconjugates containing mannose residues recognized by GNA lectin (from Galanthus nivalis) appear to be clearly restricted to the acrosomal region in both turkey and chicken spermatozoa. Further, our work has shown that the glycocalyx of turkey sperm is highly sensitive to storage at 4°C (Peláez and Long, 2008). Impaired fertility has been associated with alterations in the carbohydrate content of poultry spermatozoa (Froman and Engel, 1989), most likely from a reduction in the number of sperm in the sperm storage tubules of the hen, because the mechanism governing this storage is thought to involve head-to-head agglutination mediated by sperm surface carbohydrate residues (Bakst, 1987), and sperm release during the fertile period would be effected by changes in these interactions (van Krey et al., 1981; Bakst, 1987). It therefore seems reasonable to hypothesize that alterations in the glycocalyx caused by cryopreservation might be largely responsible for the reduced fertility observed after insemination with this type of semen. It is not known, however, whether the glycocalyx is actually damaged by cryopreservation. Therefore, the objectives of this study were to identify potential alterations occurring in the glycocalyx of chicken spermatozoa after cryopreservation and to elucidate whether such changes are associated with other potential alterations important to sperm selection, such as morphology and plasma membrane integrity. Finally, because different freezing methods and cryoprotectants are used for chicken sperm, we examined the effect of the 3 most commonly used cryopreservation protocols on the glycocalyx composition.

MATERIALS AND METHODS Birds Male roosters (aged 32 wk; Hy-Line W-36 strain, Hy-Line International, Elizabethtown, PA) used in the study were maintained in the Beltsville Agricultural Research Center Poultry Facility under standard man-

agement practices, which included a 16L:8D light cycle for semen production. Management complied with the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (Allen, 2000). The care, treatment, housing, and use of roosters for this project were approved by the Beltsville Area Animal Care and Use Committee. Males were prescreened for sperm mobility as described by Froman and McLean (1996), with minor modifications (Long and Kulkarni, 2004). Six birds of average mobility were randomly chosen for the study.

Semen Collection and Handling Semen from the 6 males was collected manually (Burrows and Quinn, 1937) twice per week, with a minimum of a 48-h interval allowed between the 2 collections. Shortly after collection, ejaculates were pooled and diluted 1:1 with Lake’s prefreeze diluent [magnesium acetate 4.92 mM; sodium glutamate 113.53 mM; potassium acetate 50.95 mM; fructose 44.40 mM; N,N-bis(2hydroxyethyl)-2-aminoethanesulfonic acid 4.69 mM; polyvinylpyrrolidone 8.33 µM; l-glutathione 1.63 mM; pH 7.1; 340 mOsm/kg; Sigma-Aldrich, St. Louis, MO]. An aliquot of raw semen was removed before dilution for analysis of surface membrane sugar residues, plasma membrane integrity, and sperm morphology in fresh semen. Diluted semen was split into 3 aliquots and cooled to 5°C (20 min) before cryopreservation.

Freeze-Thaw Protocols GOH Protocol. Lake’s prefreeze diluent + 33% GOH (Sigma-Aldrich) was added drop-wise to diluted semen to yield a final concentration of 11% GOH. The mixture was allowed to equilibrate for 20 min at 5°C, and 0.2 mL was loaded into 0.25-mL plastic straws. A computer-controlled semen freezing system (IceCube 14 M-B, Minitube of America, Verona, WI) was used to cool straws first at a rate of 7°C/min from +5 to −35°C, followed by a rate of 20°C/min from −35 to −140°C. Straws were then plunged into liquid nitrogen and stored at −196°C until use. Straws were thawed by immersion in a 5°C water bath. The contents of multiple straws were split in 2 aliquots after thawing to evaluate the effect of GOH on the sperm glycocalyx. The first aliquot was further analyzed without removing GOH, whereas GOH was removed from the second aliquot by using a sperm-washing technique based on centrifugation through an Accudenz density gradient as described by Long and Kulkarni (2004). After centrifugation, the extender and GOH remained above the 12% layer, whereas sperm cells were present at the interface between the 12 and 30% layers. Spermatozoa were recovered and used for further assessments. Dimethylsulfoxide Protocol. Crypreservation was performed similarly to the previous protocol, except that dimethylsulfoxide (DMSO; Sigma-Aldrich) instead of GOH was used as cryoprotectant, and the

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preloading equilibration time was only 1 min. Frozen straws were thawed by immersion in a 5°C water bath. Dimethylsulfoxide was not removed from thawed semen before analysis. DMA Protocol. Pure dimethylacetamide (DMA; Sigma-Aldrich) was added in bulk to diluted semen, using a volume necessary to attain 6% concentration in the final solution. After 1 min of equilibration at 5°C, the mixture was frozen directly by dropping 25-μL aliquots into liquid nitrogen. The formed pellets were placed into plastic cryovials and stored at −196°C until use. At the time of analysis, 2 to 3 pellets were taken out from the cryovials and allowed to thaw in a glass beaker, the bottom of which remained in contact with water at 60°C until complete melting of the sample. Dimethylacetamide was not removed from the thawed semen before analysis.

Analysis of Surface Membrane Sugar Residues Twelve lectins conjugated with fluorescein isothiocyanate (FITC; EY Laboratories Inc., San Mateo, CA) were used to detect residues of 9 carbohydrate groups: lectin from Limax flavus (LFA; Reference No. F-5101) for sialic acid; lectins from Canavalia ensiformis (ConA; Reference No. F-1104) and Galanthus nivalis (GNA; Reference No. F-7401) for α-mannose and α-glucose; lectin I from Griffonia simplicifolia (GS-I; Reference No. F-2401) for α-galactose; lectin I from Ricinus communis (RCA-I; Reference No. F-2001) and lectin from Arachis hypogaea (PNA; Reference No. F-2301) for β-galactose; lectin from Lotus tetragonolobus (Lotus; Reference No. F-1601) for α-fucose; lectins from Glycine max (SBA; Reference No. F-1301), and Wisteria floribunda (WFA; Reference No. F-3101) for α- and β-N-acetyl-galactosamine; lectin from Erythrina cristagalli (ECA; Reference No. F-5901) for N-acetyllactosamine; lectin II from G. simplicifolia (GS-II; Reference No. F-2402) for monomers of N-acetyl-glucosamine; and lectin from Triticum vulgare (succinylated form; s-WGA; Reference No. F-2102) for dimers of N-acetyl-glucosamine. The specificity of these lectins for the respective carbohydrate group was demonstrated previously (Peláez and Long, 2007). For all experiments, lectins were used at a concentration of 100 μg/ mL in Tris buffer (TBS: 0.05 M Tris, 0.15 M NaCl; pH 7.6; Sigma-Aldrich). Lectins GS-I, GS-II, and ConA were prepared in TBS containing 1 mM CaCl2 and 1 mM MgCl2 (Sigma-Aldrich). Washed (400 × g, 5 min) fresh and F-T sperm were resuspended in TBS to a concentration of 2.5 × 109 sperm/mL. A 2.5-μL aliquot of each sperm suspension was added to a 62.5-μL volume of lectin solution to yield a final concentration of 100 × 106 sperm/mL. Sperm-lectin mixtures were incubated for 30 min at room temperature and protected from light. After incubation, they were washed twice by centrifugation (700

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× g, 5 min) and the pellets were resuspended in the appropriate buffer (e.g., TBS with or without cations). Ten microliters of the final resuspension of each sample was diluted in 0.5 mL of the appropriate buffer and then counterstained with 12 μM propidium iodide (PI; Reference No. P3566; Molecular Probes, Eugene, OR) for a minimum of 5 min at room temperature. A Coulter EPICS XL-MCL Flow Cytometer (Coulter Corporation, Miami, FL) equipped with a single 488nm excitation source was used for all analyses. Forward-scatter gating and side-scatter gating were used to select single sperm from clumps and debris. The fluorescence from FITC-stained and PI-stained spermatozoa was collected in FL1 (a 525-nm band pass filter) and FL3 (a 620-nm band pass filter) fluorescence detectors, respectively. Because cells with intact plasma membranes preclude lectins from binding to internal structures, only FITC-fluorescence signals generated by PI-negative spermatozoa were considered in the analysis. The mean FITC fluorescence intensity per cell (MFI) of the viable sperm population was recorded from the FL1 detector output as an indicator of lectin binding. The experiment was repeated 5 times using the same semen donors.

Analysis of Plasma Membrane Integrity Data for this analysis were generated from the same flow cytometry assessment described previously. Fluorescence signals collected in the FL3 detector from both PI-negative and PI-positive spermatozoa were considered in the analysis. Sperm excluding PI (PI-negative spermatozoa) represented the population of cells with intact plasma membrane (i.e., live sperm), the percentage of which was calculated by the cytometer.

Analysis of Sperm Morphology Ten microliters of sperm suspension was diluted in 500 μL of a 1% wt/vol paraformaldehyde (Sigma-Aldrich) solution prepared in Lake’s diluent. After fixation for a minimum of 30 min, an aliquot of 5 μL was placed on a glass slide, coverslip-mounted, and observed by phase-contrast microscopy. The viewed sperm were classified into normal or abnormal morphological cells. Normal spermatozoa were filiform-shaped (snake-like), featuring a slightly curved head followed by undisrupted midpiece and tail regions. Tails could also be observed as slightly curved at their distal tiers. A minimum of 200 total spermatozoa were counted and then the percentage of each class was calculated. Abnormal morphologies included isolated heads (cells with no apparent tail), bent sperm (cells with an acute bend at the midpiece or tail), coiled sperm (cells with an evident swelling at the end of the tail), short-tailed sperm (cells showing a tail broken at its proximal, medium, or distal part), knotted (i.e., constricted) sperm heads, and others (decondensing nucleus, swollen mitochondria, etc.).

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Statistical Analysis All analyses were conducted using Statistica software for Windows (version 7, 2004; StatSoft, Inc., Tulsa, OK). A P-value of <0.05 was considered significant. Differences among samples (1 prefreeze data set and 3 postthaw data sets) were tested using the KruskalWallis test, followed by Tukey’s honestly significant difference analysis for post hoc comparisons. Variables assessed in this way included 1) fluorescence intensity (MFI values) within lectins, 2) percentage of spermatozoa with an intact plasma membrane, and 3) percentages of sperm with either normal or abnormal morphologies. The only exception was the study of GOH-removal effects, wherein, for each lectin, differences in MFI between samples (pre- and postremoval) were evaluated using the Mann-Whitney U test. Pearson’s coefficient of correlation was obtained for assessing eventual associations that existed between changes (when passing from fresh to F-T status) in sperm surface sugar residues and changes in either plasma membrane integrity or normal morphology, as well as between changes in the latter 2 sperm traits (Carrasco, 1995).

RESULTS Membrane Surface Carbohydrates Overall, the MFI for most lectins was similar (P > 0.05) between fresh sperm and sperm cryopreserved using the GOH protocol (10 of 12) or the DMA protocol (9 of 12), whereas fewer than half of the lectins (5 of 12) were similar between sperm frozen with the DMSO protocol compared with fresh, unfrozen sperm (Table 1). For all 3 freezing treatments, the lectins s-WGA and

RCA-I (corresponding to the carbohydrates N-acetylglucosamine and β-galactose) had higher MFI than did fresh sperm. For the DMSO protocol, the lectins ConA (mannose/glucose), ECA (N-acetyl-lactosamine), PNA (galactose), SBA (N-acetyl-galactosamine), and WFA (N-acetyl-galactosamine) also had higher MFI than did fresh sperm (P < 0.01). For LFA (the lectin used to detect terminal sialic acid residues), the MFI was lower (P < 0.05) for DMSO-cryopreserved sperm than for fresh sperm. Removal of the cryoprotectant GOH from thawed sperm by means of Accudenz centrifugation decreased the fluorescence intensity of the lectins s-WGA and RCA-I (P = 0.014 and P = 0.021, respectively; Figure 1); however, the MFI for all other lectins were not different (P > 0.32) after GOH removal (Figure 1).

Plasma Membrane Integrity All 3 cryopreservation protocols reduced the percentage of sperm cells with an intact plasma membrane compared with fresh, unfrozen spermatozoa (P < 0.03; Figure 2). Among the 3 freezing methods, fewer spermatozoa were damaged (P < 0.001) when using the GOH protocol (27% reduction of live sperm from fresh sample) than when using the DMA or DMSO protocol (74 and 72% reduction of live sperm from fresh sample, respectively).

Sperm Morphology Different types of abnormal morphologies were observed in fresh and F-T semen, including isolated heads, bent sperm, coiled tails, short-tailed sperm, knotted sperm heads, and others, such as decondensed nuclei

Table 1. Fluorescence intensity values (mean ± SE; n = 5; df = 3) of fresh and frozen-thawed sperm, cryopreserved using glycerol (GOH), dimethylacetamide (DMA), or dimethylsulfoxide (DMSO) incubated in the absence (control) or presence of 12 different fluorescein isothiocyanate-labeled lectins Fluorescence intensity Lectin1 (carbohydrate group2) Control (NA) ConA (α-Man; α-Glc) ECA (LacNAc) GNA (α-Man) GS-I (α-Gal) GS-II (GlcNAc) LFA (NANA) Lotus (α-Fuc) PNA (β-Gal) SBA (GalNAc) WFA (α- or β-GalNAc) s-WGA [GlcNAc(2)] RCA-I (β-Gal) a–cValues

Fresh 0.15 0.16 0.21 0.24 0.24 0.17 0.97 0.23 0.24 0.21 0.33 16.29 61.48

± ± ± ± ± ± ± ± ± ± ± ± ±

0.01a 0.00a 0.01a 0.01a 0.03a 0.00a 0.07a 0.01a 0.01a 0.02a 0.03a 3.22a 7.33a

GOH 0.15 0.20 0.35 0.27 0.36 0.22 0.76 0.23 0.35 0.37 0.53 79.37 156.93

± ± ± ± ± ± ± ± ± ± ± ± ±

0.00a 0.01ab 0.03b 0.03a 0.05a 0.02a 0.05ab 0.01a 0.03a 0.03a 0.09ab 3.10b 12.50b

DMA 0.15 0.21 0.49 0.44 0.74 0.26 0.62 0.32 0.46 0.93 0.77 152.13 214.73

± ± ± ± ± ± ± ± ± ± ± ± ±

0.01a 0.02ab 0.06ab 0.12a 0.27a 0.03a 0.07b 0.04a 0.03ab 0.21ab 0.17ab 26.66c 27.43b

DMSO 0.16 0.28 0.70 0.58 1.00 0.56 0.81 0.42 0.66 1.10 0.85 133.82 168.32

± ± ± ± ± ± ± ± ± ± ± ± ±

0.01a 0.03b 0.12b 0.16a 0.29a 0.20a 0.11ab 0.08a 0.12b 0.37b 0.13b 24.36bc 19.77b

within a row lacking a common superscript differ significantly (P < 0.05). = lectin from Canavalia ensiformis; ECA = lectin from Erythrina cristagalli; GNA = lectin from Galanthus nivalis; GS-I = lectin I from Griffonia simplicifolia; GS-II = lectin II from G. simplicifolia; LFA = lectin from Limax flavus; Lotus = lectin from Lotus tetragonolobus; PNA = lectin from Arachis hypogaea; SBA = lectin from Glycine max; WFA = lectin from Wisteria floribunda; s-WGA = lectin from Triticum vulgare; RCA-I = lectin I from Ricinus communis. 2Man = mannose; Glc = glucose; LacNAc = N-acetyl-lactosamine; Gal = galactose; GlcNAc = N-acetyl-glucosamine; NANA = N-acetyl-neuraminic acid; Fuc = fucose; GalNAc = N-acetyl-galactosamine; GlcNAc(2) = dimers of N-acetyl-glucosamine. 1ConA

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Figure 1. Fluorescence intensity values (mean + SE; n = 5; df = 1) of sperm frozen and thawed using the glycerol protocol before (white columns) and after (dark columns) removal of cryoprotectant. Each pair of columns represents values for sperm samples incubated with either none (control) or 1 of 12 different fluorescein isothiocyanate-labeled lectins [lectin from Canavalia ensiformis (ConA), lectin from Erythrina cristagalli (ECA), lectin from Galanthus nivalis (GNA), lectin I from Griffonia simplicifolia (GS-I), lectin II from G. simplicifolia (GS-II), lectin from Limax flavus (LFA), lectin from Lotus tetragonolobus (Lotus), lectin from Arachis hypogaea (PNA), lectin from Glycine max (SBA), lectin from Wisteria floribunda (WFA), lectin from Triticum vulgare (s-WGA), lectin I from Ricinus communis (RCA-I)]. Different letters (a, b) indicate differences (P < 0.05) within lectins.

Figure 2. Percentages of sperm with an intact plasma membrane (mean + SE; n = 5; df = 3) before (fresh) and after cryopreservation using 3 different methodologies (GOH: method using glycerol as cryoprotectant; DMA: method using dimethylacetamide as cryoprotectant; DMSO: method using dimethylsulfoxide as cryoprotectant). Different letters (a–c) indicate differences (P < 0.05) among sperm categories.

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and swollen mitochondria. The main abnormality observed in fresh semen was short-tailed sperm (27.7 ± 8.8%), whereas bent sperm (range, 12.4 to 40.4%) and short-tailed sperm (range, 19.4 to 32.5%) were the primary abnormalities in F-T sperm. Cells classified as isolated heads, coiled sperm, knotted sperm heads, and others, however, were relatively infrequent in fresh semen (0.61 ± 0.31%, 0.00 ± 0.00%, 1.1 ± 0.12%, and 0.24 ± 0.24%, respectively). Cryopreservation-induced changes in sperm morphology varied among protocols (Figure 3). The percentage of morphologically normal sperm thawed from the DMA protocol was similar to that observed in fresh semen (P = 0.999), whereas the percentages of morphologically normal sperm in both the DMSO and GOH protocols were lower than that in fresh semen (P = 0.033 and P < 0.001, respectively). The highest percentage of bent sperm was observed in the GOH treatment (P < 0.001), followed by the DMSO (P < 0.01) and DMA (P = 0.163) treatments. Percentages of other morphological classes did not change after cryopreservation, regardless of the methodology used [range, isolated heads: 1.82 to 3.07% (P = 0.053); coiled: 0.12 to 0.39% (P = 0.40); knotted heads: 0.86 to 1.21% (P = 0.81); others: 0.20 to 0.25% (P = 0.95)].

Association Between Sperm Characteristics Plasma membrane integrity was found to be correlated with fluorescence intensity in sperm incubated with ECA (r = −0.69), GNA (r = −0.57), GS-I (r = −0.58),

Lotus (r = −0.56), PNA (r = −0.64), SBA (r = −0.67), WFA (r = −0.71), s-WGA (r = −0.90), and RCA-I (r = −0.81; P < 0.05 for all). On the contrary, such a relationship was not found between percentage of normal spermatozoa and fluorescence intensity in any lectin (r = from −0.23 in RCA-I to 0.37 in LFA; P > 0.05 for all). A significant correlation was not found between sperm with normal morphology or sperm with an intact plasma membrane (P > 0.05).

DISCUSSION We report the first comprehensive assessment of the effects of cryopreservation on the glycocalyx of sperm. To our knowledge, similar research findings have not been published for other species. Earlier work from our laboratory demonstrating that the turkey sperm glycocalyx was altered by storage at 4°C provided the basis for further exploration of the effects of low-temperature storage on poultry sperm carbohydrate content. These alterations could cause malfunctioning of the sperm glycocalyx, thereby compromising reproductive events mediated by this structure. If such a situation has functional significance for fertility, then preventing these changes would be a valid strategy for increasing the efficiency of cryopreserved semen. As in our previous work, we used flow cytometry to quantify the intensity of fluorescence emitted by sperm incubated with FITC-labeled lectins. Because lectins specifically recognize and bind to carbohydrate structures (Leathem and Brooks, 1997), fluorescence inten-

Figure 3. Percentages (mean + SE; n = 5; df = 3) for normal and 2 main abnormal sperm morphology categories before (fresh) and after cryopreservation using 3 different protocols (DMA: method using dimethylacetamide as cryoprotectant; DMSO: method using dimethylsulfoxide as cryoprotectant; GOH: method using glycerol as cryoprotectant). Values within a morphology class lacking a common letter (a–c) differ significantly (P < 0.05).

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sity is considered proportional to the amount of sugar that is available for binding, such that increased fluorescence intensity indicates that more sugar residues are available for lectin binding. Sperm cryopreserved with all 3 protocols exhibited alterations of this type; however, the magnitude of changes in individual carbohydrates varied among and within protocols. Glycocalyx composition was most affected by the DMSO protocol, moderately affected with the DMA protocol, and comparatively less altered with the GOH protocol. Within a protocol, β-galactose and N-acetyl-glucosamine residues recognized by lectins RCA-I and s-WGA consistently underwent the most remarkable changes of all moieties investigated, suggesting that these 2 residues, their glycoconjugates, or both are more sensitive to cryopreservation than others. These differences suggest that 1) freeze-thaw methods vary in their capacity to induce changes on the sperm plasma membrane surface, and 2) variability also exists in the susceptibility of glycoconjugates to withstand cryopreservation. The sperm plasma membrane is known to be altered by cryopreservation, with several changes occuring at the molecular and structural levels (Parks and Graham, 1992). Modifications in proteins or lipids are frequently reported (Hinkovska-Galcheva et al., 1989; Cerolini et al., 2001; Blesbois et al., 2005; Cheng et al., 2005). The current study adds to this knowledge by clearly demonstrating that surface carbohydrates are also affected. The major question that then arises is how these changes are produced and what effect these changes have on sperm function. Although our work does not provide experimental evidence to elucidate the mechanism(s) involved, a few hypotheses can be proposed. One possibility is that the increased lectin binding results from a shedding phenomenon of terminal sialic acid moieties, which would unmask subterminal sugars, thereby increasing their exposure on the surface of the plasma membrane. This hypothesis is supported by the fact that the fluorescence intensity obtained with the LFA lectin decreased after cryopreservation, suggesting that lower numbers of sialic acid residues exist on the surface of thawed sperm. Sialic acid is a common constituent of cell glycocalyx, where it acts as a masking agent on surface recognition sites (Schauer, 1985). We have previously shown that the glycocalyx of chicken spermatozoa is extensively sialylated, with all its other terminal carbohydrates being coated by neuraminic acid residues to a greater or lesser extent (Peláez and Long, 2007). Therefore, loss of sialic acid caused by cryopreservation (as appeared to occur in our experiment) may have been responsible, at least in part, for the quantitative changes in glycocalyx composition that we observed. An interaction between the cryoprotectant agents and the sperm membrane is another possible source of alteration. Glycerol, for instance, is thought to interact with membrane glycoproteins (Parks and Graham, 1992) and has been reported to cause alterations of the plasma membrane (Hammerstedt and Graham, 1992).

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Our study is in agreement with these facts because it revealed that postthaw removal of GOH significantly reduced the magnitude of alteration for some carbohydrates, suggesting that an interaction between this cryoprotectant and glycoconjugates may exist. Similar interactions between the cryoprotectants DMA and DMSO, however, cannot be inferred from this study. Considering the known roles of sperm surface carbohydrates for a normal development of events leading to successful reproduction, it seems that the glycocalyx alterations described in this paper could have functional significance for fertility. The modifications we observed could affect sperm transport and storage within the reproductive tract of the hen based on the known sperm selection mechanism in the vagina, consisting of immunological recognition of surface antigenicity (Steele and Wishart, 1992). It has been reported that removal of sialic acid from the sperm surface renders these cells less capable of reaching the storage tubules (Froman and Thursam, 1994). Therefore, proper masking of sugars by terminal sialic acid residues appears to be essential for ensuring the transvaginal migration of spermatozoa, as specifically demonstrated by Steele and Wishart (1996). The increased exposure of certain carbohydrates after cryopreservation is likely to modify the antigenicity of sperm and could interfere with sperm being selected in the vagina. Likewise, the ability of sperm to be stored in or released from the tubules, or both, could be also compromised because these phenomena are thought to be mediated by specific surface carbohydrate interactions (van Krey et al., 1981; Bakst, 1987). An altered sperm surface could be detrimental to the fertilization process itself. The basis for this suggestion is that saccharide moieties on the sperm surface appear to be involved in the regulation of early gamete interaction (Diekman, 2003). In addition, specific sperm surface-associated glycoproteins have been shown to contribute in different ways to the fertilization process of various species and taxa (Kopecný and Flechon, 1987; Veselský et al., 1992; Bérubé and Sullivan, 1994; Lassalle and Testart, 1996; Evans, 1999; Yu et al., 2002; Srivastav et al., 2004). In poultry, N-acetyl-glucosamine is necessary for the sperm-egg interaction (Robertson et al., 2000). It is not known if premature exposure of the glycoprotein (before reaching the ovum) would adversely affect the ability of a sperm cell to bind to the perivitelline layer. Certainly the possibility exists for modification of the carbohydrate component of glycocalyx to impair the process of fertilization in poultry. An interesting outcome was the fact that alterations in N-acetyl-glucosamine and β-galactose (recognized by lectins s-WGA and RCA-I) were of significantly lower magnitude after removal of GOH from the sperm suspension. This finding revealed that changes in the status of those residues caused by the GOH protocol were partially reversible. We specifically included this treatment in the study because GOH has a contraceptive effect on intravaginally inseminated sperm, which

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can be overcome by reducing the GOH concentration before insemination (Lake, 1986; Hammerstedt, 1995; Etches, 1996; Donoghue and Wishart, 2000; Long and Kulkarni, 2004). The mechanisms for the contraceptive effect of GOH are not understood, although they are thought to be related to interactions of a different nature being established between GOH, sperm, and even the vaginal tissue (Westfall and Howarth, 1977; Delee et al., 1991). It has been suggested that such interactions could consist of alterations of the surface proteins (Donoghue and Wishart, 2000), and our study supports this assertion. Further, our work suggests that the contraceptive mechanism could be mediated in some fashion by premature exposure of N-acetyl-glucosamine or β-galactose, or both. The fact that cryopreservation significantly reduced the percentage of sperm cells with an intact plasma membrane was an expected consequence of the process. Differences noted among the 3 protocols in the percentage of live sperm, primarily higher survival rates in the GOH protocol, have been reported previously (Chalah et al., 1999). The significant correlation between sperm viability and lectin fluorescence intensity for 9 of the 12 of sugar residues supports the concept that differences in plasma membrane integrity and glycocalyx alterations among the GOH, DMA, and DMSO protocols were related to the cryoprotectant, the cryopreservation method, or both. With regard to sperm morphology, however, the GOH protocol had a high number of bent sperm after thawing compared with the DMA and DMSO protocols. Morphological abnormalities such as this have been attributed to adverse osmotic conditions surrounding spermatozoa during the cooling process (Bakst and Sexton, 1979); however, DMA is generally thought to cause a greater degree of osmotic damage in the form of abnormal morphology than GOH (Tselutin et al., 1999; Chełmońska et al., 2006) These discrepancies may be due to the differing methodologies for evaluating sperm morphology and viability. In the earlier studies, the nigrosin-eosin technique was used to assess viability and morphology simultaneously, whereas, in our experiment, sperm morphology was assessed independently of viability to allow for direct comparison of potential relationships between these 2 characteristics. One drawback of the current study is that sperm morphology was not assessed in semen samples after removal of GOH. It is possible that removal of GOH after thawing could have reduced or reversed the number of abnormal sperm, or both, because this treatment positively affected the viability and glycocalyx alterations. In conclusion, the carbohydrate component of the chicken sperm glycocalyx was modified during the cryogenic cycle by an increased exposure of certain sugar residues from a loss of terminal sialic acid residues or rearrangement of the actual glycoprotein or glycolipid within the membrane bilayer, or both. The different cryopreservation protocols varied in their capacity to cause glycocalyx alterations, and some glycoconjugates were more vulnerable than others regardless of the freez-

ing-thawing methodology. The GOH-based cryodiluent induced a lower magnitude of glycocalyx alteration and plasma membrane disruption than did cryodiluents using DMA or DMSO, suggesting that GOH is a superior cryoprotectant; however, more morphological damage occurred with GOH than with DMA or DMSO. The glycoconjugates affected by cryopreservation and their function are yet unknown and merit further study. Of particular interest is the role(s) played by glycoconjugates recognized by lectins s-WGA and RCA-I, which were highly vulnerable to freezing-thawing and partially recovered their original status after removing GOH from the sperm suspension. This practice is known to result in the recovery of fertility of sperm cryopreserved using GOH. Research is thus needed to clarify how the changes described in this paper affect the function and fate of sperm after artificial insemination, to increase our current knowledge of the effects of cryopreservation on fertility.

ACKNOWLEDGMENTS We thank W. Smoot [Beltsville Agricultural Research Center (BARC), Beltsville, MD] for assistance in semen collection, as well as T. Conn (BARC) and G. Welch (BARC) for valuable expertise in laboratory management and flow cytometry analysis. The sponsorship provided by the Office of International Relations of the Smithsonian Institution (Washington, DC) for the postdoctoral stay of J. Peláez is greatly appreciated.

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