|Pelaez, Jesus - FORMER ARS EMPLOYEE|
Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 15, 2009
Publication Date: February 1, 2011
Citation: Pelaez, J., Bongalhardo, D.C., Long, J.A. 2011. Characterizing the glycocalyx of poultry spermatozoa; semen cryopreservation methods alter the carbohydrate component of rooster sperm membrane glycoconjugates. Poultry Science. 90(2):435-443. Interpretive Summary: Egg-type chicken producers would benefit from the capability of using frozen/thawed semen in commercial practice. Current methodology, however, does not provide the fertility rates necessary for profitability. Our research is focused on learning why and how chicken sperm lose functional competence during in vitro storage. We have recently identified the carbohydrates present on the surface of the chicken sperm membrane. In this study, our objective was to determine if these carbohydrates are altered during semen cryopreservation, and if different freezing methods have any effect on the modulation of sperm membrane glycoconjugates. We found several differences that have implications for the reduced fertility of frozen/thawed chicken semen. In particular, the impact of cryopreservation on membrane carbohydrates was influenced by the cryoprotectant used.
Technical 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 contain residues of mannose, glucose, galactose, fucose, N-acetyl-galactosamine, N-acetyl-glucosamine and N-acetyl-lactosamine. Our objective here was to evaluate the effects of cryopreservation on the sperm glycocalyx. Semen was pooled from 6 roosters, diluted 1:1 (Lake’s pre-freeze diluent), cooled to 5°C and aliquoted for cryopreservation using 6% DMA, 11% DMSO or 11% glycerol. For the DMA method, semen was equilibrated for 1 min with DMA and rapidly frozen by dropping 25 µl aliquots into liquid nitrogen. For the DMSO and glycerol methods, semen was equilibrated for either 1 min (DMSO) or 20 min (glycerol), loaded into 0.25 ml straws and frozen (5 to -35°C, 7°C/min; -35 to -140°C, 20°C/min; nitrogen plunge). Thawed (rapid, DMA; moderate, DMSO, glycerol) semen was stained with 1 of 12 FITC-conjugated lectins (100 µg/mL; 30 min; 25°C; 100x106 cells/mL). Samples counterstained with PI were assessed by flow cytometry. On the day of cryopreservation, aliquots of fresh semen were stained with the panel of lectins and PI. For each lectin, the Mean Fluorescence Intensity (MnFI) of live sperm was compared among fresh and frozen/thawed treatments (n=5 replicates). For the majority of lectins (10/12), the MnFI was higher (p<0.05) for frozen/thawed than fresh sperm. Exceptions included lectins specific for sialic acid and a-fucose, where DMSO and glycerol treatments, respectively, had MnFI similar (p>0.05) to fresh sperm. Among the frozen/thawed treatments, the MnFI of sperm cryopreserved with DMSO was higher (p<0.05) for 4/10 lectins, including those specific for N-acetyl-lactosamine and N-acetyl-glucosamine. These data indicate that surface carbohydrates are altered during cryopreservation, and that cryoprotectant type and freeze/thaw rates affect the degree of modification. While the specific functions of these glycoconjugates are not known, it is likely that the observed differences in frozen/thawed sperm contribute to the reduced fertility of cryopreserved chicken semen. Other possible functional implications are discussed.