Current perspectives on ruminant sperm freezability: Harnessing molecular changes related to semen quality through omics technologies

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Marvin Bryan Salinas
Phongsakorn Chuammitri
Korawan Sringarm
Sukolrat Boonyayatra
Anucha Sathanawongs


The recent advances in sperm cryopreservation transcend cryobanking and other assisted reproductive technologies. Since its discovery, cryopreservation has contributed positive impacts on animal breeding as well as in genetic exchange, improvement, and conservation efforts. However, cryoinjury and variabilities in cryopreservation outcomes remain as key challenges to sperm cryobiology. The present work explored the molecular bases for such freezability differences and freezing-thawing injuries in the ruminant sperm. Relevant biomarkers identified in the seminal plasma and the spermatozoa were highlighted, including lipids, proteins, metabolites, transcripts, and genes. Specific molecular mechanisms concerning sperm structures and functions were also examined relative to their association to cryotolerance, and spermiogram or seminogram modifications following cryopreservation procedures. Current conflicts and gaps in the knowledge base on ruminant spermatozoa were also emphasized. Further investigation of these areas using the available breakthrough molecular tools such as omics technologies is therefore proposed to improve, optimize, or even predict the overall quality of frozen-thawed ruminant semen towards reproductive efficiency.


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Bryan Salinas, M. ., Chuammitri, P. ., Sringarm, K. ., Boonyayatra, S. ., & Sathanawongs, A. . (2021). Current perspectives on ruminant sperm freezability: Harnessing molecular changes related to semen quality through omics technologies: Veterinary Integrative Sciences, 19(3), 487–511. Retrieved from
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Author Biography

Marvin Bryan Salinas, Department of Veterinary Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand  Department of Morphophysiology and Pharmacology, College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz 3120, Nueva Ecija, Philippines


Ahmad, M., Ahmad, N., Riaz, A., Anzar, M., 2015. Sperm survival kinetics in different types of bull semen: progressive motility, plasma membrane integrity, acrosomal status and reactive oxygen species generation. Reprod Fertil Dev, 27(5), 784-793. doi:10.1071/rd13400

Ahmed, S., Khan, M. I.-u.-R., Ahmad, M., Iqbal, S., 2018. Effect of age on lipid peroxidation of fresh and frozen-thawed semen of Nili-Ravi buffalo bulls. Ital J Anim Sci, 17(3), 730-735. doi:10.1080/1828051X.2018.1424569

Aitken, R. J., 1995. Free radicals, lipid peroxidation and sperm function. Reprod Fertil Dev, 7(4), 659-668. doi:10.1071/rd9950659

Amorim, E. A. M., Graham, J. K., Spizziri, B., Meyers, M., Torres, C. A. A., 2009. Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm. Cryobiology, 58(2), 210-214. doi:

Andrabi, S. M. H., 2009. Factors Affecting the Quality of Cryopreserved Buffalo (Bubalus bubalis) Bull Spermatozoa. Reprod Domest Anim, 44(3), 552-569. doi:

Anghel, A., Stela, Z., 2010. Role of antioxidant additives in the protection of the cryopreserved semen against free radicals. Rom Biotechnol Lett, 15, 33-41.

Anzar, M., He, L., Buhr, M. M., Kroetsch, T. G., Pauls, K. P., 2002. Sperm apoptosis in fresh and cryopreserved bull semen detected by flow cytometry and its relationship with fertility. Biol Reprod, 66(2), 354-360. doi:10.1095/biolreprod66.2.354

Arando, A., Gonzalez, A., Delgado, J. V., Arrebola, F. A., Perez-Marín, C. C., 2017. Storage temperature and sucrose concentrations affect ram sperm quality after vitrification. Anim Reprod Sci, 181, 175-185. doi:10.1016/j.anireprosci.2017.04.008

Aurich, C., Schreiner, B., Ille, N., Alvarenga, M., Scarlet, D., 2016. Cytosine methylation of sperm DNA in horse semen after cryopreservation. Theriogenology, 86(5), 1347-1352. doi:

Bailey, J., Buhr, M. J., 1994. Cryopreservation alters the Ca2+ flux of bovine spermatozoa. Can J Anim Sci, 74, 45-51.

Bailey, J. L., Bilodeau, J. F., Cormier, N., 2000. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J Androl, 21(1), 1-7.

Barbagallo, F., La Vignera, S., Cannarella, R., Aversa, A., Calogero, A. E., Condorelli, R. A., 2020. Evaluation of Sperm Mitochondrial Function: A Key Organelle for Sperm Motility. J Clin Med, 9(2), 363. doi:10.3390/jcm9020363

Batissaco, L., Arruda, R. P. d., Alves, M. B. R., Torres, M. A., Lemes, K. M., Prado-Filho, R. R., . . . Celeghini, E. C. C., 2020. Cholesterol-loaded cyclodextrin is efficient in preserving sperm quality of cryopreserved ram semen with low freezability. Reprod Biol, 20(1), 14-24. doi:

Bilodeau, J. F., Chatterjee, S., Sirard, M. A., Gagnon, C., 2000. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol Reprod Dev, 55(3), 282-288. doi:10.1002/(sici)1098-2795(200003)55:3<282::Aid-mrd6>3.0.Co;2-7

Bissonnette, N., Lévesque-Sergerie, J. P., Thibault, C., Boissonneault, G., 2009. Spermatozoal transcriptome profiling for bull sperm motility: a potential tool to evaluate semen quality. Reproduction, 138(1), 65-80. doi:10.1530/rep-08-0503

Boe-Hansen, G. B., Fortes, M. R. S., Satake, N., 2018. Morphological defects, sperm DNA integrity, and protamination of bovine spermatozoa. Andrology, 6(4), 627-633. doi:10.1111/andr.12486

Bollwein, H., Bittner, L., 2018. Impacts of oxidative stress on bovine sperm function and subsequent in vitro embryo development. Anim Reprod, 15, 703-710. doi:10.21451/1984-3143-AR2018-0041

Capra, E., Lazzari, B., Turri, F., Cremonesi, P., Portela, A. M. R., Ajmone-Marsan, P., . . . Pizzi, F., 2019. Epigenetic analysis of high and low motile sperm populations reveals methylation variation in satellite regions within the pericentromeric position and in genes functionally related to sperm DNA organization and maintenance in Bos taurus. BMC Genomics, 20(1), 940. doi:10.1186/s12864-019-6317-6

Capra, E., Turri, F., Lazzari, B., Cremonesi, P., Gliozzi, T. M., Fojadelli, I., . . . Pizzi, F., 2017. Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between high- and low-motile sperm populations. BMC Genomics, 18(1), 14. doi:10.1186/s12864-016-3394-7

Card, C. J., Anderson, E. J., Zamberlan, S., Krieger, K. E., Kaproth, M., Sartini, B. L., 2013. Cryopreserved bovine spermatozoal transcript profile as revealed by high-throughput ribonucleic acid sequencing. Biol Reprod, 88(2), 49. doi:10.1095/biolreprod.112.103788

Card, C. J., Krieger, K. E., Kaproth, M., Sartini, B. L., 2017. Oligo-dT selected spermatozoal transcript profiles differ among higher and lower fertility dairy sires. Anim Reprod Sci, 177, 105-123. doi:

Castro, L. S., Hamilton, T. R., Mendes, C. M., Nichi, M., Barnabe, V. H., Visintin, J. A., Assumpção, M. E., 2016. Sperm cryodamage occurs after rapid freezing phase: flow cytometry approach and antioxidant enzymes activity at different stages of cryopreservation. J Anim Sci Biotechnol, 7, 17. doi:10.1186/s40104-016-0076-x

Champroux, A., Cocquet, J., Henry-Berger, J., Drevet, J. R., Kocer, A., 2018. A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance. Front Cell Dev Biol, 6, 50. doi:10.3389/fcell.2018.00050

Chatterjee, S., de Lamirande, E., Gagnon, C., 2001a. Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione. Mol Reprod Dev, 60(4), 498-506. doi:10.1002/mrd.1115

Chatterjee, S., Gagnon, C., 2001b. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Mol Reprod Dev, 59(4), 451-458. doi:10.1002/mrd.1052

Chen, X., Wang, Y., Zhu, H., Hao, H., Zhao, X., Qin, T., Wang, D., 2015. Comparative transcript profiling of gene expression of fresh and frozen-thawed bull sperm. Theriogenology, 83(4), 504-511. doi:10.1016/j.theriogenology.2014.10.015

Codognoto, V. M., Yamada, P. H., Schmith, R. A., de Ruediger, F. R., Scott, C., de Faria Lainetti, P., … Oba, E., 2018. Functional insights into the role of seminal plasma proteins on sperm motility of buffalo. Anim Reprod Sci, 195, 251–258. doi:

Cormier, N., Bailey, J. L., 2003. A differential mechanism is involved during heparin- and cryopreservation-induced capacitation of bovine spermatozoa. Biol Reprod, 69(1), 177-185. doi:10.1095/biolreprod.102.011056

Dai, L., Zhao, Z., Zhao, R., Xiao, S., Jiang, H., Yue, X., . . . Zhang, J., 2009. Effects of novel single nucleotide polymorphisms of the FSH beta-subunit gene on semen quality and fertility in bulls. Anim Reprod Sci, 114(1), 14-22. doi:

Dalal, J., Kumar, A., Honparkhe, M., Deka, D., Singh, N., 2016. Minimization of apoptosis-like changes in cryopreserved buffalo bull sperm by supplementing extender with Bcl-2 protein. Vet World, 9, 432-436. doi:10.14202/vetworld.2016.432-436

De Leeuw, F. E., Chen, H. C., Colenbrander, B., Verkleij, A. J., 1990. Cold-induced ultrastructural changes in bull and boar sperm plasma membranes. Cryobiology, 27(2), 171-183. doi:10.1016/0011-2240(90)90009-s

Del Valle, I., Mendoza, N., Casao, A., Cebrián-Pérez, J. A., Pérez-Pé, R., Muiño-Blanco, T., 2010. Significance of Non-conventional Parameters in the Evaluation of Cooling-induced Damage to Ram Spermatozoa Diluted in Three Different Media. Reprod Domest Anim, 45(6), e260-e268. doi:

Dhanju, C. K., Cheema, R. S., Kaur, S. P., 2001. Effect of Freezing on Proteins and Protein Profiles of Sperm Membrane Extracts and Seminal Plasma of Buffalo Bulls. Asian-Australas J Anim Sci, 14(12), 1678-1682. doi:10.5713/ajas.2001.1678

Dogan, S., Mason, M. C., Govindaraju, A., Belser, L., Kaya, A., Stokes, J., . . . Memili, E., 2013. Interrelationships between apoptosis and fertility in bull sperm. J Reprod Dev, 59(1), 18-26. doi:10.1262/jrd.2012-068

Dogan, S., Vargovic, P., Oliveira, R., Belser, L. E., Kaya, A., Moura, A., . . . Memili, E., 2015. Sperm protamine-status correlates to the fertility of breeding bulls. Biol Reprod, 92(4), 92. doi:10.1095/biolreprod.114.124255

Dorado, J., Muñoz-Serrano, A., Hidalgo, M., 2010. The effect of cryopreservation on goat semen characteristics related to sperm freezability. Anim Reprod Sci, 121(1), 115-123. doi:

Dutta, S., Majzoub, A., Agarwal, A., 2019. Oxidative stress and sperm function: A systematic review on evaluation and management. Arab J Urol, 17(2), 87-97. doi:10.1080/2090598x.2019.1599624

Erickson, L., Kroetsch, T., Anzar, M., 2015. Relationship between sperm apoptosis and bull fertility: in vivo and in vitro studies. Reprod Fertil Dev. doi:10.1071/rd14417

Esteso, M. C., Soler, A. J., Fernández-Santos, M. R., Quintero-Moreno, A. A., Garde, J. J., 2006. Functional significance of the sperm head morphometric size and shape for determining freezability in iberian red deer (Cervus elaphus hispanicus) epididymal sperm samples. J Androl, 27(5), 662-670. doi:10.2164/jandrol.106.000489

Evans, H. C., Dinh, T. T. N., Ugur, M. R., Hitit, M., Sajeev, D., Kaya, A., . . . Memili, E., 2020. Lipidomic markers of sperm cryotolerance in cattle. Sci Rep, 10(1), 20192. doi:10.1038/s41598-020-77089-9

Fang, Y., Zhao, C., Xiang, H., Jia, G., Zhong, R., 2020. Melatonin improves cryopreservation of ram sperm by inhibiting mitochondrial permeability transition pore opening. Reprod Domest Anim, 55(9), 1240-1249. doi:

Felipe-Pérez, Y. E., Valencia, J., Juárez-Mosqueda, M. D. L., Pescador, N., Roa-Espitia, A. L., Hernández-González, E. O., 2012. Cytoskeletal proteins F-actin and β-dystrobrevin are altered by the cryopreservation process in bull sperm. Cryobiology, 64(2), 103-109. doi:

Fortes, M. R., Satake, N., Corbet, D. H., Corbet, N. J., Burns, B. M., Moore, S. S., Boe-Hansen, G. B., 2014. Sperm protamine deficiency correlates with sperm DNA damage in Bos indicus bulls. Andrology, 2(3), 370-378. doi:10.1111/j.2047-2927.2014.00196.x

Ganguly, I., Gaur, G. K., Kumar, S., Mandal, D. K., Kumar, M., Singh, U., . . . Sharma, A., 2013. Differential expression of protamine 1 and 2 genes in mature spermatozoa of normal and motility impaired semen producing crossbred Frieswal (HF×Sahiwal) bulls. Res Vet Sci, 94(2), 256-262. doi:10.1016/j.rvsc.2012.09.001

Gomes, F. P., Park, R., Viana, A. G., Fernandez-Costa, C., Topper, E., Kaya, A., . . . Moura, A. A., 2020. Protein signatures of seminal plasma from bulls with contrasting frozen-thawed sperm viability. Sci Rep, 10(1), 14661. doi:10.1038/s41598-020-71015-9

Gonçalves, F. S., Barretto, L. S., Arruda, R. P., Perri, S. H., Mingoti, G. Z., 2010. Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development. Reprod Domest Anim, 45(1), 129-135. doi:10.1111/j.1439-0531.2008.01272.x

Gosálvez, J., López-Fernández, C., Fernández, J. L., Gouraud, A., Holt, W. V., 2011. Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species. Mol Reprod Dev, 78(12), 951-961. doi:10.1002/mrd.21394

Govindaraju, A., Uzun, A., Robertson, L., Atli, M. O., Kaya, A., Topper, E., . . . Memili, E., 2012. Dynamics of microRNAs in bull spermatozoa. Reprod Biol Endocrinol, 10, 82. doi:10.1186/1477-7827-10-82

Grötter, L. G., Cattaneo, L., Marini, P. E., Kjelland, M. E., Ferré, L. B., 2019. Recent advances in bovine sperm cryopreservation techniques with a focus on sperm post-thaw quality optimization. Reprod Domest Anim, 54(4), 655-665. doi:10.1111/rda.13409

Gürler, H., Malama, E., Heppelmann, M., Calisici, O., Leiding, C., Kastelic, J. P., Bollwein, H., 2016. Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm. Theriogenology, 86(2), 562-571. doi:10.1016/j.theriogenology.2016.02.007

Harayama, H., Nishijima, K., Murase, T., Sakase, M., Fukushima, M., 2010. Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP-dependent hyperactivation in the spermatozoa of Japanese Black bulls. Mol Reprod Dev, 77(10), 910-921. doi:

He, Y., Wang, K., Zhao, X., Zhang, Y., Ma, Y., Hu, J., 2016. Differential proteome association study of freeze-thaw damage in ram sperm. Cryobiology, 72(1), 60-68. doi:10.1016/j.cryobiol.2015.11.003

Hering, D. M., Lecewicz, M., Kordan, W., Majewska, A., Kaminski, S., 2015. Missense mutation in glutathione-S-transferase M1 gene is associated with sperm motility and ATP content in frozen-thawed semen of Holstein-Friesian bulls. Anim Reprod Sci, 159, 94-97. doi:10.1016/j.anireprosci.2015.06.001

Hitit, M., Ugur, M. R., Dinh, T. T. N., Sajeev, D., Kaya, A., Topper, E., . . . Memili, E., 2020. Cellular and Functional Physiopathology of Bull Sperm With Altered Sperm Freezability. Front Vet Sci, 7, 581137-581137. doi:10.3389/fvets.2020.581137

Holt, W. V., North, R. D., 1991. Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa. J Reprod Fertil, 91(2), 451-461. doi:10.1530/jrf.0.0910451

Hozyen, H., Elshamy, A., Farghali, A., 2020. Supplementation of Nano Selenium Minimizes Freeze-Thaw Induced Damage to Ram Spermatozoa. Int J Vet Sci, 8, 249-254.

Januskauskas, A., Johannisson, A., Rodriguez-Martinez, H., 2001. Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen-thawed semen from Swedish AI bulls. Theriogenology, 55(4), 947-961. doi:10.1016/s0093-691x(01)00456-3

Januskauskas, A., Johannisson, A., Rodriguez-Martinez, H., 2003. Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility. Theriogenology, 60(4), 743-758. doi:10.1016/s0093-691x(03)00050-5

Jenkins, T. G., Carrell, D. T., 2012. The sperm epigenome and potential implications for the developing embryo. Reproduction, 143(6), 727-734. doi:10.1530/rep-11-0450

Jobim, M. I., Oberst, E. R., Salbego, C. G., Souza, D. O., Wald, V. B., Tramontina, F., Mattos, R. C., 2004. Two-dimensional polyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semen freezability. Theriogenology, 61(2-3), 255-266. doi:10.1016/s0093-691x(03)00230-9

Kadirvel, G., Kathiravan, P., Kumar, S., 2011. Protein tyrosine phosphorylation and zona binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa. Theriogenology, 75(9), 1630-1639. doi:

Kadirvel, G., Kumar, S., Kumaresan, A., 2009a. Lipid peroxidation, mitochondrial membrane potential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen. Anim Reprod Sci, 114(1-3), 125-134. doi:10.1016/j.anireprosci.2008.10.002

Kadirvel, G., Kumar, S., Kumaresan, A., Kathiravan, P., 2009b. Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium. Anim Reprod Sci, 116(3-4), 244-253. doi:10.1016/j.anireprosci.2009.02.003

Khalifa, T., Lymberopoulos, A., 2013. Changeability of sperm chromatin structure during liquid storage of ovine semen in milk-egg yolk- and soybean lecithin-based extenders and their relationships to field-fertility. Cell Tissue Bank, 14(4), 687-698. doi:10.1007/s10561-012-9357-6

Khalil, W. A., El-Harairy, M. A., Zeidan, A. E. B., Hassan, M. A. E., 2019. Impact of selenium nano-particles in semen extender on bull sperm quality after cryopreservation. Theriogenology, 126, 121-127. doi:

Khalil, W. A., El-Harairy, M. A., Zeidan, A. E. B., Hassan, M. A. E., Mohey-Elsaeed, O., 2018. Evaluation of bull spermatozoa during and after cryopreservation: Structural and ultrastructural insights. International Journal of Veterinary Science and Medicine, 6, S49-S56. doi:

Khan, D. R., Ahmad, N., Anzar, M., Channa, A. A., 2009. Apoptosis in fresh and cryopreserved buffalo sperm. Theriogenology, 71(5), 872-876. doi:

Khan, I. M., Cao, Z., Liu, H., Khan, A., Rahman, S. U., Khan, M. Z., Sathanawongs, A., & Zhang, Y., 2021. Impact of Cryopreservation on Spermatozoa Freeze-Thawed Traits and Relevance OMICS to Assess Sperm Cryo-Tolerance in Farm Animals. Front Vet Sci, 8, 609180. doi:

Khosravizadeh, Z., Hassanzadeh, G., Tavakkoly Bazzaz, J., Alizadeh, F., Totonchi, M., Salehi, E., . . . Abolhassani, F., 2020. The effect of cryopreservation on DNA methylation patterns of the chromosome 15q11–q13 region in human spermatozoa. Cell Tissue Bank, 21(3), 433-445. doi:10.1007/s10561-020-09828-1

Kropp, J., Carrillo, J. A., Namous, H., Daniels, A., Salih, S. M., Song, J., Khatib, H., 2017. Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos. BMC Genomics, 18(1), 280. doi:10.1186/s12864-017-3673-y

Kumar, A., Kroetsch, T., Blondin, P., Anzar, M., 2015. Fertility-associated metabolites in bull seminal plasma and blood serum: 1H nuclear magnetic resonance analysis. Mol Reprod Dev, 82(2), 123-131. doi:10.1002/mrd.22450

Kumar, A., Prasad, J. K., Srivastava, N., Ghosh, S. K., 2019. Strategies to Minimize Various Stress-Related Freeze-Thaw Damages During Conventional Cryopreservation of Mammalian Spermatozoa. Biopreserv Biobank, 17(6), 603-612. doi:10.1089/bio.2019.0037

Kumar, R., Atreja, S. K., 2012. Effect of incorporation of additives in tris-based egg yolk extender on buffalo (Bubalus bubalis) sperm tyrosine phosphorylation during cryopreservation. Reprod Domest Anim, 47(3), 485-490. doi:10.1111/j.1439-0531.2011.01908.x

Kumar, R., Jagan Mohanarao, G., Arvind, Atreja, S. K., 2011. Freeze–thaw induced genotoxicity in buffalo (Bubalus bubalis) spermatozoa in relation to total antioxidant status. Mol Biol Rep, 38(3), 1499-1506. doi:10.1007/s11033-010-0257-1

Kumar, R., Singh, V. K., Atreja, S. K., 2014. Glutathione-S-transferase: Role in buffalo (Bubalus bubalis) sperm capacitation and cryopreservation. Theriogenology, 81(4), 587-598. doi:

Kumaresan, A., Johannisson, A., Al-Essawe, E. M., Morrell, J. M., 2017. Sperm viability, reactive oxygen species, and DNA fragmentation index combined can discriminate between above- and below-average fertility bulls. J Dairy Sci, 100(7), 5824-5836. doi:10.3168/jds.2016-12484

Lemma, A., 2011. Effect of Cryopreservation on Sperm Quality and Fertility. Artif Insemin Farm Anim,191–216.

Lone, S. A., Prasad, J. K., Ghosh, S. K., Das, G. K., Balamurugan, B., Verma, M. R., 2018. Study on correlation of sperm quality parameters with antioxidant and oxidant status of buffalo bull semen during various stages of cryopreservation. Andrologia. doi:10.1111/and.12970

Longobardi, V., Albero, G., De Canditiis, C., Salzano, A., Natale, A., Balestrieri, A., . . . Gasparrini, B., 2017. Cholesterol-loaded cyclodextrins prevent cryocapacitation damages in buffalo (Bubalus bubalis) cryopreserved sperm. Theriogenology, 89, 359-364. doi:10.1016/j.theriogenology.2016.09.048

Longobardi, V., Kosior, M. A., Pagano, N., Fatone, G., Staropoli, A., Vassetti, A., . . . Gasparrini, B., 2020. Changes in Bull Semen Metabolome in Relation to Cryopreservation and Fertility. Animals, 10(6), 1065. doi:10.3390/ani10061065

López Armengol, M. F., Jurado, S. B., Pelufo, V., Aisen, E. G., 2012. A quantitative ultramorphological approach for systematic assessment of sperm head regions: An example in rams. Cryobiology, 64(3), 223-234. doi:

Losano, J., Angrimani, D., Dalmazzo, A., Rui, B. R., Brito, M. M., Mendes, C. M., . . . Nichi, M., 2017a. Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality. Reprod Domest Anim, 52(2), 289-297. doi:10.1111/rda.12901

Losano, J. D. A., Padín, J. F., Méndez-López, I., Angrimani, D. S. R., García, A. G., Barnabe, V. H., Nichi, M., 2017b. The Stimulated Glycolytic Pathway Is Able to Maintain ATP Levels and Kinetic Patterns of Bovine Epididymal Sperm Subjected to Mitochondrial Uncoupling. Oxid Med Cell Longev, 2017, 1682393. doi:10.1155/2017/1682393

Lv, Y. Q., Ji, S., Chen, X., Xu, D., Luo, X. T., Cheng, M. M., . . . Jin, Y., 2020. Effects of crocin on frozen-thawed sperm apoptosis, protamine expression and membrane lipid oxidation in Yanbian yellow cattle. Reprod Domest Anim, 55(8), 1011-1020. doi:10.1111/rda.13744

Magalhães, M. J., Martins, L. F., Senra, R. L., Santos, T. F. d., Okano, D. S., Pereira, P. R. G., . . . Baracat-Pereira, M. C., 2016. Differential abundances of four forms of Binder of SPerm 1 in the seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability. Theriogenology, 86(3), 766-777.e762. doi:

Mahmoud, K. G., Sakr, A. M., Ibrahim, S. R., Sosa, A. S., Hasanain, M. H., Nawito, M. F., 2021. GnRHR gene polymorphism and its correlation with semen quality in Buffalo bulls (Bubalus bubalis). Iraqi J Vet Sci, 35(2), 381-386. doi:10.33899/ijvs.2020.126886.1407

Mahmoud, K. G. M., El-Sokary, A. A. E., Abdel-Ghaffar, A. E., Abou El-Roos, M. E. A., Ahmed, Y. F., 2015. Analysis of chromatin integrity and DNA damage of buffalo spermatozoa. Iran J Vet Res, 16(2), 161-166.

Maia, M. d. S., Bicudo, S. D., Sicherle, C. C., Rodello, L., Gallego, I. C. S., 2010. Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen cryopreserved in extenders with antioxidants. Anim Reprod Sci, 122(1), 118-123. doi:

Mandal, R., Badyakar, D., Chakrabarty, J., 2014. Role of Membrane Lipid Fatty Acids in Sperm Cryopreservation. Adv Androl, 2014, 190542. doi:10.1155/2014/190542

Martí, E., Marti, J. I., Muiño-Blanco, T., Cebrián-Pérez, J. A., 2008a. Effect of the cryopreservation process on the activity and immunolocalization of antioxidant enzymes in ram spermatozoa. J Androl, 29(4), 459-467. doi:10.2164/jandrol.107.003459

Martí, E., Pérez-Pé, R., Colás, C., Muiño-Blanco, T., Cebrián-Pérez, J. A., 2008b. Study of apoptosis-related markers in ram spermatozoa. Anim Reprod Sci, 106(1), 113-132. doi:

Martí, J. I., Martí, E., Cebrián-Pérez, J. A., Muiño-Blanco, T., 2003. Survival rate and antioxidant enzyme activity of ram spermatozoa after dilution with different extenders or selection by a dextran swim-up procedure. Theriogenology, 60(6), 1025-1037. doi:10.1016/s0093-691x(03)00105-5

Martin, G., Cagnon, N., Sabido, O., Sion, B., Grizard, G., Durand, P., Levy, R., 2007. Kinetics of occurrence of some features of apoptosis during the cryopreservation process of bovine spermatozoa. Hum Reprod, 22(2), 380-388. doi:10.1093/humrep/del399

Martin, G., Sabido, O., Durand, P., Levy, R., 2004. Cryopreservation induces an apoptosis-like mechanism in bull sperm. Biol Reprod, 71(1), 28-37. doi:10.1095/biolreprod.103.024281

Martínez-Fresneda, L., Castaño, C., Bóveda, P., Tesfaye, D., Schellander, K., Santiago-Moreno, J., García-Vázquez, F. A., 2019. Epididymal and ejaculated sperm differ on their response to the cryopreservation and capacitation processes in mouflon (Ovis musimon). Sci Rep, 9(1), 15659. doi:10.1038/s41598-019-52057-0

Martínez-Fresneda, L., E, O. B., Velázquez, R., Toledano-Díaz, A., Martínez-Cáceres, C. M., Tesfaye, D., . . . Santiago-Moreno, J., 2019. Seasonal variation in sperm freezability associated with changes in testicular germinal epithelium in domestic (Ovis aries) and wild (Ovis musimon) sheep. Reprod Fertil Dev, 31(10), 1545-1557. doi:10.1071/rd18511

Martínez-Fresneda, L., Sylvester, M., Shakeri, F., Bunes, A., Del Pozo, J. C., García-Vázquez, F. A., . . . Santiago-Moreno, J., 2021. Differential proteome between ejaculate and epididymal sperm represents a key factor for sperm freezability in wild small ruminants. Cryobiology, 99, 64-77. doi:10.1016/j.cryobiol.2021.01.012

Mendoza, N., Casao, A., Pérez-Pé, R., Cebrián-Pérez, J. A., Muiño-Blanco, T., 2013. New insights into the mechanisms of ram sperm protection by seminal plasma proteins. Biol Reprod, 88(6), 149. doi:10.1095/biolreprod.112.105650

Menezes, E. B., Velho, A. L. C., Santos, F., Dinh, T., Kaya, A., Topper, E., . . . Memili, E., 2019. Uncovering sperm metabolome to discover biomarkers for bull fertility. BMC Genomics, 20(1), 714. doi:10.1186/s12864-019-6074-6

Minervini, F., Guastamacchia, R., Pizzi, F., Dell'Aquila, M. E., Barile, V. L., 2013. Assessment of different functional parameters of frozen-thawed buffalo spermatozoa by using cytofluorimetric determinations. Reprod Domest Anim, 48(2), 317-324. doi:10.1111/j.1439-0531.2012.02152.x

Mocé, E., Blanch, E., Tomás, C., Graham, J. K., 2010. Use of cholesterol in sperm cryopreservation: present moment and perspectives to future. Reprod Domest Anim, 45 Suppl 2, 57-66. doi:10.1111/j.1439-0531.2010.01635.x

Moraes, C. R., Meyers, S., 2018. The sperm mitochondrion: Organelle of many functions. Anim Reprod Sci, 194, 71-80. doi:10.1016/j.anireprosci.2018.03.024

Morató, R., Prieto-Martínez, N., Muiño, R., Hidalgo, C. O., Rodríguez-Gil, J. E., Bonet, S., Yeste, M., 2018. Aquaporin 11 is related to cryotolerance and fertilising ability of frozen-thawed bull spermatozoa. Reprod Fertil Dev, 30(8), 1099-1108. doi:10.1071/rd17340

Mostek, A., Dietrich, M. A., Słowińska, M., Ciereszko, A., 2017. Cryopreservation of bull semen is associated with carbonylation of sperm proteins. Theriogenology, 92, 95-102. doi:10.1016/j.theriogenology.2017.01.011

Müller, K., Pomorski, T., Müller, P., Zachowski, A., Herrmann, A., 1994. Protein-dependent translocation of aminophospholipids and asymmetric transbilayer distribution of phospholipids in the plasma membrane of ram sperm cells. Biochemistry, 33(33), 9968-9974. doi:10.1021/bi00199a020

Nagdas, S. K., Buchanan, T., McCaskill, S., Mackey, J., Alvarez, G. E., Raychoudhury, S., 2013. Isolation of a calcium-binding protein of the acrosomal membrane of bovine spermatozoa. Int J Biochem Cell Biol, 45(4), 876-884. doi:10.1016/j.biocel.2013.01.016

Nakidkina, A. N., Kuzmina, T., 2019. Apoptosis in Spermatozoa and Its Role in Deteriorating Semen Quality. Russ J Dev Biol, 50, 165 - 172.

Naresh, S., 2016. Effect of cooling (4°C) and cryopreservation on cytoskeleton actin and protein tyrosine phosphorylation in buffalo spermatozoa. Cryobiology, 72(1), 7-13. doi:10.1016/j.cryobiol.2015.12.004

Naresh, S., Atreja, S. K., 2015. The protein tyrosine phosphorylation during in vitro capacitation and cryopreservation of mammalian spermatozoa. Cryobiology, 70(3), 211-216. doi:10.1016/j.cryobiol.2015.03.008

Narud, B., Klinkenberg, G., Khezri, A., Zeremichael, T. T., Stenseth, E.-B., Nordborg, A., . . . Kommisrud, E., 2020. Differences in sperm functionality and intracellular metabolites in Norwegian Red bulls of contrasting fertility. Theriogenology, 157, 24-32. doi:

Nazari, H., Ahmadi, E., Hosseini Fahraji, H., Afzali, A., Davoodian, N., 2020. Cryopreservation and its effects on motility and gene expression patterns and fertilizing potential of bovine epididymal sperm. Veterinary Medicine and Science, 7(1), 127-135. doi:10.1002/vms3.355

Nikbin, S., Panandam, J. M., Yaakub, H., Murugaiyah, M., Sazili, A. Q., 2014. Novel SNPs in heat shock protein 70 gene and their association with sperm quality traits of Boer goats and Boer crosses. Anim Reprod Sci, 146(3-4), 176-181. doi:10.1016/j.anireprosci.2014.03.001

Pereira, R. M., Mesquita, P., Pires, V. M. R., Baptista, M. C., Barbas, J. P., Pimenta, J., . . . Marques, C. C., 2018. Prion protein testis specific (PRNT) gene polymorphisms and transcript level in ovine spermatozoa: Implications in freezability, fertilization and embryo production. Theriogenology, 115, 124-132. doi:

Peris-Frau, P., Soler, A. J., Iniesta-Cuerda, M., Martín-Maestro, A., Sánchez-Ajofrín, I., Medina-Chávez, D. A., . . . Garde, J. J. (2020). Sperm Cryodamage in Ruminants: Understanding the Molecular Changes Induced by the Cryopreservation Process to Optimize Sperm Quality. Int. J. Mol. Sci, 21(8). doi:10.3390/ijms21082781

Peris, S. I., Bilodeau, J. F., Dufour, M., Bailey, J. L., 2007. Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm. Mol Reprod Dev, 74(7), 878-892. doi:10.1002/mrd.20686

Perumal, P., Srivastava, S., Ghosh, S., Baruah, K. K., 2014. Computer-Assisted Sperm Analysis of Freezable and Nonfreezable Mithun (Bos frontalis) Semen. J Anim, 2014, 1-6. doi:10.1155/2014/675031

Pini, T., Leahy, T., Soleilhavoup, C., Tsikis, G., Labas, V., Combes-Soia, L., . . . de Graaf, S. P., 2016. Proteomic Investigation of Ram Spermatozoa and the Proteins Conferred by Seminal Plasma. J Proteome Res, 15(10), 3700-3711. doi:10.1021/acs.jproteome.6b00530

Pool, K. R., Rickard, J. P., de Graaf, S. P., 2020. Global Methylation and Protamine Deficiency in Ram Spermatozoa Correlate with Sperm Production and Quality but Are Not Influenced by Melatonin or Season. Animals, 10(12), 2302. doi:10.3390/ani10122302

Pradieé, J., Sánchez-Calabuig, M. J., Castaño, C., O'Brien, E., Esteso, M. C., Beltrán-Breña, P., . . . Rizos, D., 2018. Fertilizing capacity of vitrified epididymal sperm from Iberian ibex (Capra pyrenaica). Theriogenology, 108, 314-320. doi:10.1016/j.theriogenology.2017.11.021

Pukazhenthi, B. S., 2016. Saving wild ungulate diversity through enhanced management and sperm cryopreservation. Reprod Fertil Dev, 28(8), 1133-1144. Retrieved from

Rajoriya, D. J., Prasad, J., Ghosh, S., Ramteke, S., Barik, N. C., Das, G., Pande, M., 2014. Cholesterol loaded cyclodextrin increases freezability of buffalo bull (Bubalus bubalis) spermatozoa by increasing cholesterol to phospholipid ratio. Vet World, 7, 702-706. doi:10.14202/vetworld.2014.702-706

Rajoriya, D. J., Prasad, J., Ramteke, S., Perumal, P., De, A., Ghosh, S., . . . Kumaresan, A., 2020. Exogenous cholesterol prevents cryocapacitation like changes, membrane fluidity and enhances in‐vitro fertility in bubaline spermatozoa. Reprod Domest Anim, 55. doi:10.1111/rda.13674

Rajoriya, J. S., Prasad, J. K., Ramteke, S. S., Perumal, P., Ghosh, S. K., Singh, M., . . . Srivastava, N., 2016. Enriching membrane cholesterol improves stability and cryosurvival of buffalo spermatozoa. Anim Reprod Sci, 164, 72-81. doi:

Ramón, M., Pérez-Guzmán, M. D., Jiménez-Rabadán, P., Esteso, M. C., García-Álvarez, O., Maroto-Morales, A., . . . Garde, J. J., 2013. Sperm Cell Population Dynamics in Ram Semen during the Cryopreservation Process. PLOS ONE, 8(3), e59189. doi:10.1371/journal.pone.0059189

Rasul, Z., Ahmad, N., Anzar, M., 2001. Changes in motion characteristics, plasma membrane integrity, and acrosome morphology during cryopreservation of buffalo spermatozoa. J Androl, 22(2), 278-283.

Rego, J. P., Martins, J. M., Wolf, C. A., van Tilburg, M., Moreno, F., Monteiro-Moreira, A. C., . . . Moura, A. A., 2016. Proteomic analysis of seminal plasma and sperm cells and their associations with semen freezability in Guzerat bulls. J Anim Sci, 94(12), 5308-5320. doi:10.2527/jas.2016-0811

Rickard, J. P., Leahy, T., Soleilhavoup, C., Tsikis, G., Labas, V., Harichaux, G., . . . de Graaf, S. P., 2015. The identification of proteomic markers of sperm freezing resilience in ram seminal plasma. J Proteomics, 126, 303-311. doi:10.1016/j.jprot.2015.05.017

Riesco, M. F., Alvarez, M., Anel-Lopez, L., Neila-Montero, M., Palacin-Martinez, C., Montes-Garrido, R., . . . Anel, L., 2021. Multiparametric Study of Antioxidant Effect on Ram Sperm Cryopreservation-From Field Trials to Research Bench. Animals, 11(2). doi:10.3390/ani11020283

Ros-Santaella, J. L., Domínguez-Rebolledo, A. E., Garde, J. J., 2014. Sperm flagellum volume determines freezability in red deer spermatozoa. PLOS ONE, 9(11), e112382. doi:10.1371/journal.pone.0112382

Ryu, D. Y., Song, W.-H., Pang, W., Yoon, S.-J., Rahman, M. S., Pang, M., 2019. Freezability biomarkers in bull epididymal spermatozoa. Sci Rep, 9, 12797. doi:10.1038/s41598-019-49378-5

Sang, L., Du, Q.-Z., Yang, W.-C., Tang, K.-Q., Yu, J.-N., Hua, G.-h., . . . Yang, L.-G., 2011. Polymorphisms in follicle stimulation hormone receptor, inhibin alpha, inhibin beta A, and prolactin genes, and their association with sperm quality in Chinese Holstein bulls. Anim Reprod Sci, 126(3), 151-156. doi:

Santiani, A., Evangelista, S., Sepúlveda, N., Risopatrón, J., Villegas, J., Sánchez, R., 2014. Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa. Theriogenology, 82(6), 884-889. doi:

Sellappan, S., Sivashanmugam, P., Lakshminarayana, S., Kolte, A., B Krishnan, B., Arangasamy, A., Ravindra, J., 2017. Occurrence and functional significance of the transcriptome in bovine (Bos taurus) spermatozoa. Sci Rep, 7, 42392. doi:10.1038/srep42392

Shah, N., Singh, V., Yadav, H. P., Verma, M., Chauhan, D. S., Saxena, A., . . . Swain, D. K., 2017. Effect of reduced glutathione supplementation in semen extender on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa. Anim Reprod Sci, 182, 111-122. doi:10.1016/j.anireprosci.2017.05.006

Shangguan, A., Zhou, H., Sun, W., Ding, R., Li, X., Liu, J., . . . Zhang, S., 2020. Cryopreservation Induces Alterations of miRNA and mRNA Fragment Profiles of Bull Sperm. Front Genet, 11, 419. doi:10.3389/fgene.2020.00419

Shi, L., Ren, Y., Zhou, H., Hou, G., Xun, W., Yue, W., . . . Yang, R., 2014. Effect of rapid freezing–thawing techniques on the sperm parameters and ultrastructure of Chinese Taihang black goat spermatozoa. Micron, 57, 6-12. doi:

Singh, M., Ghosh, S. K., Prasad, J. K., Kumar, A., Tripathi, R. P., Bhure, S. K., Srivastava, N., 2014. Seminal PDC-109 protein vis-à-vis cholesterol content and freezability of buffalo Spermatozoa. Anim Reprod Sci, 144(1), 22-29. doi:

Sivakumar, A., Kumar, S., Yathish, H. M., Mishra, C., Modi, R. P., Chaudhary, R., . . . Sarkar, M., 2018. Expression Profiling and Identification of Novel SNPs in CatSper2 Gene and Their Influence on Sperm Motility Parameters in Bovines. Anim Biotechnol, 29(1), 34-40. doi:10.1080/10495398.2017.1294597

Soleilhavoup, C., Tsikis, G., Labas, V., Harichaux, G., Kohnke, P. L., Dacheux, J. L., . . . Druart, X., 2014. Ram seminal plasma proteome and its impact on liquid preservation of spermatozoa. J Proteomics, 109, 245-260. doi:10.1016/j.jprot.2014.07.007

Song, W. H., Ryu, D. Y., Pang, W. K., Yoon, S. J., Rahman, M. S., Pang, M. G., 2020. NT5C1B and FH are closely associated with cryoprotectant tolerance in spermatozoa. Andrology, 8(1), 221-230. doi:10.1111/andr.12653

Srivastava, N., Srivastava, S.K., Ghosh, S.K., Kumar, A., Perumal, P., Jerome, A., 2013. Acrosome membrane integrity and cryocapacitation are related to cholesterol content of bull spermatozoa. Asian Pac J Reprod, 2(2): 126-131.

Sukardi, S., Elliott, R. M., Withers, J. O., Fontaine, U., Millar, J. D., Curry, M. R., Watson, P. F., 2001. Calcium-binding proteins from the outer acrosomal membrane of ram spermatozoa: potential candidates for involvement in the acrosome reaction. Reproduction, 122(6), 939-946.

Talukdar, D., Ahmed, K., Deka, B. C., Sinha, S., Deori, S., Das, G., 2016. Cryo-capacitation changes during cryopreservation of swamp buffalo spermatozoa. Indian J Anim Sci, 86, 397-400.

Tiwari, A., Singh, D., Kumar, O. S., Sharma, M. K., 2008. Expression of cytochrome P450 aromatase transcripts in buffalo (Bubalus bubalis)-ejaculated spermatozoa and its relationship with sperm motility. Domest Anim Endocrinol, 34(3), 238-249. doi:

Treulen, F., Arias, M. E., Aguila, L., Uribe, P., Felmer, R., 2018. Cryopreservation induces mitochondrial permeability transition in a bovine sperm model. Cryobiology, 83, 65-74. doi:10.1016/j.cryobiol.2018.06.001

Ugur, M. R., Saber Abdelrahman, A., Evans, H. C., Gilmore, A. A., Hitit, M., Arifiantini, R. I., . . . Memili, E., 2019. Advances in Cryopreservation of Bull Sperm. Front Vet Sci, 6, 268. doi:

Varela, E., Rojas, M., Restrepo, G., 2020. Membrane stability and mitochondrial activity of bovine sperm frozen with low-density lipoproteins and trehalose. Reprod Domest Anim, 55(2), 146-153. doi:10.1111/rda.13599

Velho, A., Bezerra de Menezes, E., Dinh, T., Kaya, A., Topper, E., Moura, A., Memili, E., 2018. Metabolomic markers of fertility in bull seminal plasma. PLOS ONE, 13, e0195279. doi:10.1371/journal.pone.0195279

Verma, A., Rajput, S., De, S., Kumar, R., Chakravarty, A. K., Datta, T. K., 2014. Genome-wide profiling of sperm DNA methylation in relation to buffalo (Bubalus bubalis) bull fertility. Theriogenology, 82(5), 750-759.e751. doi:10.1016/j.theriogenology.2014.06.012

Wang, P., Wang, Y. F., Wang, H., Wang, C. W., Zan, L. S., Hu, J. H., . . . Ma, G. J., 2014. HSP90 expression correlation with the freezing resistance of bull sperm. Zygote, 22(2), 239-245. doi:10.1017/s096719941300004x

Waterhouse, K. E., Haugan, T., Kommisrud, E., Tverdal, A., Flatberg, G., Farstad, W., . . . De Angelis, P. M., 2006. Sperm DNA damage is related to field fertility of semen from young Norwegian Red bulls. Reprod Fertil Dev, 18(7), 781-788. doi:10.1071/rd06029

Watson, P. F., 2000. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci, 60-61, 481-492. doi:10.1016/s0378-4320(00)00099-3

Westfalewicz, B., Dietrich, M., Słowińska, M., Judycka, S., Ciereszko, A., 2019. Seasonal changes in the proteome of cryopreserved bull semen supernatant. Theriogenology, 126, 295-302. doi:10.1016/j.theriogenology.2018.12.015

Wojtusik, J., Wang, Y., Pukazhenthi, B. S., 2018. Pretreatment with cholesterol-loaded cyclodextrins prevents loss of motility associated proteins during cryopreservation of addra gazelle (Nanger dama ruficollis) spermatozoa. Cryobiology, 81, 74-80. doi:10.1016/j.cryobiol.2018.02.007

Yadav, H. P., Kumar, A., Shah, N., Chauhan, D. S., Saxena, A., Yadav, S., Swain, D. K., 2017. Effect of cholesterol loaded cyclodextrin supplementation on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa. Theriogenology, 96, 164-171. doi:10.1016/j.theriogenology.2017.04.016

Yang, W.-C., Tang, K.-Q., Yu, J.-N., Zhang, C., Zhang, X.-X., Yang, L.-G., 2010. Effects of MboII and BspMI polymorphisms in the gonadotropin releasing hormone receptor (GNRHR) gene and sperm quality in Holstein bulls. Mol Biol Rep, 38, 3411-3415. doi:10.1007/s11033-010-0450-2

Yathish, H. M., Kumar, S., Chaudhary, R., Mishra, C., A, S., Kumar, A., . . . Mitra, A., 2018. Nucleotide variability of protamine genes influencing bull sperm motility variables. Anim Reprod Sci, 193, 126-139. doi:

Yoon, S. J., Rahman, M. S., Kwon, W. S., Park, Y. J., Pang, M. G., 2016a. Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome. PLOS ONE, 11(3), e0152690. doi:10.1371/journal.pone.0152690

Yoon, S. J., Rahman, M. S., Kwon, W. S., Ryu, D. Y., Park, Y. J., Pang, M. G., 2016b. Proteomic identification of cryostress in epididymal spermatozoa. J Anim Sci Biotechnol, 7(1), 67. doi:10.1186/s40104-016-0128-2

Zhang, X. G., Hu, S., Han, C., Zhu, Q. C., Yan, G. J., Hu, J. H., 2015. Association of heat shock protein 90 with motility of post-thawed sperm in bulls. Cryobiology, 70(2), 164–169.

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