Optimization of conditions for long-term prefreezing storage of brown bear sperm before cryopreservation
Brown bear ejaculates are usually collected in field conditions and may need to be shipped to a laboratory for the application of reproductive biotechnologies before cryopreservation. The aim of this study was to extend the prefreezing step to 48 hours (1 hour vs. long-term storage [LS] to 24 and 48...
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| Veröffentlicht in: | Theriogenology 2015-10, Vol.84 (7), p.1161-1171 |
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| creator | López-Urueña, E. Alvarez, M. Gomes-Alves, S. Anel-López, L. Martínez-Rodríguez, C. Manrique, P. Borragan, S. Anel, L. de Paz, P. |
| description | Brown bear ejaculates are usually collected in field conditions and may need to be shipped to a laboratory for the application of reproductive biotechnologies before cryopreservation. The aim of this study was to extend the prefreezing step to 48 hours (1 hour vs. long-term storage [LS] to 24 and 48 hours) to enable the sample to be transported. The effects of storage temperature (experiment 1), glycerol concentration (experiment 2), and dilution rate (experiment 3) on sperm were evaluated. Electroejaculates from brown bears were stored under different experimental conditions and cryopreserved. The sperm motility and viability, apoptotic status, and acrosomal status of sperm were assessed before freezing (prefreezing), after thawing, and after 2-hour incubation at 37 °C (thermal stress test). In all experiments, one control sample was frozen using a standard protocol (control). In experiment 1, three temperatures during LS with 6% glycerol were tested: 5 °C (T5), 15 °C (T15), and room temperature (RT). The LS-T5 sample yielded the highest postthawing results for viability (42.4%), progressive motility (15.6%), and intact acrosome (83.1%) after 24 hours in comparison with the other temperatures (P |
| doi_str_mv | 10.1016/j.theriogenology.2015.06.017 |
| format | Article |
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The aim of this study was to extend the prefreezing step to 48 hours (1 hour vs. long-term storage [LS] to 24 and 48 hours) to enable the sample to be transported. The effects of storage temperature (experiment 1), glycerol concentration (experiment 2), and dilution rate (experiment 3) on sperm were evaluated. Electroejaculates from brown bears were stored under different experimental conditions and cryopreserved. The sperm motility and viability, apoptotic status, and acrosomal status of sperm were assessed before freezing (prefreezing), after thawing, and after 2-hour incubation at 37 °C (thermal stress test). In all experiments, one control sample was frozen using a standard protocol (control). In experiment 1, three temperatures during LS with 6% glycerol were tested: 5 °C (T5), 15 °C (T15), and room temperature (RT). The LS-T5 sample yielded the highest postthawing results for viability (42.4%), progressive motility (15.6%), and intact acrosome (83.1%) after 24 hours in comparison with the other temperatures (P < 0.05); for 48 hours, the LS-T5 sample reached higher total and progressive motility (25.9% and 9%, respectively) and nonapoptotic values (36.5%). Recovery rates revealed susceptibility to freezing at LS-15 or LS-RT samples at 24 hours (viability) or 48 hours (viability and motility). In experiment 2, samples were stored at 5 °C up to 48 hours and three glycerol concentrations were evaluated: 0% (0Gly), 3% (3Gly), and 6% (6Gly). Postthawing viability and motility increased progressively with the percentage of glycerol for 24 hours at 5 °C; 6% glycerol during 48-hour storage had beneficial effects on sperm cryopreservation. Besides, 6% glycerol had a clearly superior freezability for viability (42.7% and 40.8% for 24 hours and 48 hours, respectively) and motility (24 hours: total, 44.1%; progressive, 17.1%; 48 hours: total, 38.4%; progressive, 16%). In experiment 3, samples were stored up to 48 hours at 5 °C with 6% of glycerol and two dilution methods were evaluated: dilution 1:1 (average: 1782 × 106 sperm/mL; low) or final dilution (100 × 106 sperm/mL; high). Both dilution rates showed similar postthawing and postincubation results within 24 hours of long-term storage. After 48 hours, high dilution supported better postthawing quality. Both dilutions showed similar resistance to cryopreservation, except after 48 hours, when the high dilution reached a higher percent recovery rate of viability (38.8% vs. 21.6%, P < 0.05). In conclusion, our results suggested that the best conditions for long-term prefreezing storage (up to 48 hours) of brown bear electroejaculates are at 5 °C, at a concentration of 100 × 106 sperm/mL, and with 6% glycerol.</description><identifier>ISSN: 0093-691X</identifier><identifier>EISSN: 1879-3231</identifier><identifier>DOI: 10.1016/j.theriogenology.2015.06.017</identifier><identifier>PMID: 26234462</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Acrosome - physiology ; Animals ; Apoptosis ; Brown bear ; Cell Survival ; Cryopreservation - methods ; Cryopreservation - veterinary ; Cryoprotective Agents ; Dilution rate ; Glycerol - analysis ; Glycerol concentration ; Long-term storage ; Male ; Semen Preservation - methods ; Semen Preservation - veterinary ; Solutions - chemistry ; Sperm Motility ; Spermatozoa - physiology ; Spermatozoa cryopreservation ; Temperature ; Ursidae</subject><ispartof>Theriogenology, 2015-10, Vol.84 (7), p.1161-1171</ispartof><rights>2015 Elsevier Inc.</rights><rights>Copyright © 2015 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c386t-3091a8f0ef5a725ad06f2ba11b8c3dbd76a5b32fcf30f3a89da7cc966a9bd6083</citedby><cites>FETCH-LOGICAL-c386t-3091a8f0ef5a725ad06f2ba11b8c3dbd76a5b32fcf30f3a89da7cc966a9bd6083</cites><orcidid>0000-0003-2531-595X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.theriogenology.2015.06.017$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,777,781,3537,27905,27906,45976</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26234462$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>López-Urueña, E.</creatorcontrib><creatorcontrib>Alvarez, M.</creatorcontrib><creatorcontrib>Gomes-Alves, S.</creatorcontrib><creatorcontrib>Anel-López, L.</creatorcontrib><creatorcontrib>Martínez-Rodríguez, C.</creatorcontrib><creatorcontrib>Manrique, P.</creatorcontrib><creatorcontrib>Borragan, S.</creatorcontrib><creatorcontrib>Anel, L.</creatorcontrib><creatorcontrib>de Paz, P.</creatorcontrib><title>Optimization of conditions for long-term prefreezing storage of brown bear sperm before cryopreservation</title><title>Theriogenology</title><addtitle>Theriogenology</addtitle><description>Brown bear ejaculates are usually collected in field conditions and may need to be shipped to a laboratory for the application of reproductive biotechnologies before cryopreservation. The aim of this study was to extend the prefreezing step to 48 hours (1 hour vs. long-term storage [LS] to 24 and 48 hours) to enable the sample to be transported. The effects of storage temperature (experiment 1), glycerol concentration (experiment 2), and dilution rate (experiment 3) on sperm were evaluated. Electroejaculates from brown bears were stored under different experimental conditions and cryopreserved. The sperm motility and viability, apoptotic status, and acrosomal status of sperm were assessed before freezing (prefreezing), after thawing, and after 2-hour incubation at 37 °C (thermal stress test). In all experiments, one control sample was frozen using a standard protocol (control). In experiment 1, three temperatures during LS with 6% glycerol were tested: 5 °C (T5), 15 °C (T15), and room temperature (RT). The LS-T5 sample yielded the highest postthawing results for viability (42.4%), progressive motility (15.6%), and intact acrosome (83.1%) after 24 hours in comparison with the other temperatures (P < 0.05); for 48 hours, the LS-T5 sample reached higher total and progressive motility (25.9% and 9%, respectively) and nonapoptotic values (36.5%). Recovery rates revealed susceptibility to freezing at LS-15 or LS-RT samples at 24 hours (viability) or 48 hours (viability and motility). In experiment 2, samples were stored at 5 °C up to 48 hours and three glycerol concentrations were evaluated: 0% (0Gly), 3% (3Gly), and 6% (6Gly). Postthawing viability and motility increased progressively with the percentage of glycerol for 24 hours at 5 °C; 6% glycerol during 48-hour storage had beneficial effects on sperm cryopreservation. Besides, 6% glycerol had a clearly superior freezability for viability (42.7% and 40.8% for 24 hours and 48 hours, respectively) and motility (24 hours: total, 44.1%; progressive, 17.1%; 48 hours: total, 38.4%; progressive, 16%). In experiment 3, samples were stored up to 48 hours at 5 °C with 6% of glycerol and two dilution methods were evaluated: dilution 1:1 (average: 1782 × 106 sperm/mL; low) or final dilution (100 × 106 sperm/mL; high). Both dilution rates showed similar postthawing and postincubation results within 24 hours of long-term storage. After 48 hours, high dilution supported better postthawing quality. Both dilutions showed similar resistance to cryopreservation, except after 48 hours, when the high dilution reached a higher percent recovery rate of viability (38.8% vs. 21.6%, P < 0.05). In conclusion, our results suggested that the best conditions for long-term prefreezing storage (up to 48 hours) of brown bear electroejaculates are at 5 °C, at a concentration of 100 × 106 sperm/mL, and with 6% glycerol.</description><subject>Acrosome - physiology</subject><subject>Animals</subject><subject>Apoptosis</subject><subject>Brown bear</subject><subject>Cell Survival</subject><subject>Cryopreservation - methods</subject><subject>Cryopreservation - veterinary</subject><subject>Cryoprotective Agents</subject><subject>Dilution rate</subject><subject>Glycerol - analysis</subject><subject>Glycerol concentration</subject><subject>Long-term storage</subject><subject>Male</subject><subject>Semen Preservation - methods</subject><subject>Semen Preservation - veterinary</subject><subject>Solutions - chemistry</subject><subject>Sperm Motility</subject><subject>Spermatozoa - physiology</subject><subject>Spermatozoa cryopreservation</subject><subject>Temperature</subject><subject>Ursidae</subject><issn>0093-691X</issn><issn>1879-3231</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkE1r3DAQhkVoSTZp_kLQIYde7I6stWxDLiE0HxDIpYXehD5GjhbbciRvyubXx84mgd56Ggae9x3mIeScQc6AiR-bfHrE6EOLQ-hCu8sLYGUOIgdWHZAVq6sm4wVnX8gKoOGZaNifI3Kc0gYAuBDskBwVouDrtShW5PFhnHzvX9Tkw0CDoyYM1i9Loi5E2oWhzSaMPR0juoj44oeWpilE1eLC6xj-DlSjijSNC6dxziE1cRfmSML4_Nb9jXx1qkt4-j5PyO_rn7-ubrP7h5u7q8v7zPBaTBmHhqnaAbpSVUWpLAhXaMWYrg232lZClZoXzjgOjqu6saoyphFCNdoKqPkJ-b7vHWN42mKaZO-Twa5TA4ZtkqxirFxDWS_oxR41MaQ0vyfH6HsVd5KBXFzLjfzXtVxcSxBydj3Hz94vbXWP9jP8IXcGrvcAzv8-e4wyGY-DQesjmkna4P_v0ivRpJzZ</recordid><startdate>20151015</startdate><enddate>20151015</enddate><creator>López-Urueña, E.</creator><creator>Alvarez, M.</creator><creator>Gomes-Alves, S.</creator><creator>Anel-López, L.</creator><creator>Martínez-Rodríguez, C.</creator><creator>Manrique, P.</creator><creator>Borragan, S.</creator><creator>Anel, L.</creator><creator>de Paz, P.</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0003-2531-595X</orcidid></search><sort><creationdate>20151015</creationdate><title>Optimization of conditions for long-term prefreezing storage of brown bear sperm before cryopreservation</title><author>López-Urueña, E. ; Alvarez, M. ; Gomes-Alves, S. ; Anel-López, L. ; Martínez-Rodríguez, C. ; Manrique, P. ; Borragan, S. ; Anel, L. ; de Paz, P.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-3091a8f0ef5a725ad06f2ba11b8c3dbd76a5b32fcf30f3a89da7cc966a9bd6083</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Acrosome - physiology</topic><topic>Animals</topic><topic>Apoptosis</topic><topic>Brown bear</topic><topic>Cell Survival</topic><topic>Cryopreservation - methods</topic><topic>Cryopreservation - veterinary</topic><topic>Cryoprotective Agents</topic><topic>Dilution rate</topic><topic>Glycerol - analysis</topic><topic>Glycerol concentration</topic><topic>Long-term storage</topic><topic>Male</topic><topic>Semen Preservation - methods</topic><topic>Semen Preservation - veterinary</topic><topic>Solutions - chemistry</topic><topic>Sperm Motility</topic><topic>Spermatozoa - physiology</topic><topic>Spermatozoa cryopreservation</topic><topic>Temperature</topic><topic>Ursidae</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>López-Urueña, E.</creatorcontrib><creatorcontrib>Alvarez, M.</creatorcontrib><creatorcontrib>Gomes-Alves, S.</creatorcontrib><creatorcontrib>Anel-López, L.</creatorcontrib><creatorcontrib>Martínez-Rodríguez, C.</creatorcontrib><creatorcontrib>Manrique, P.</creatorcontrib><creatorcontrib>Borragan, S.</creatorcontrib><creatorcontrib>Anel, L.</creatorcontrib><creatorcontrib>de Paz, P.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Theriogenology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>López-Urueña, E.</au><au>Alvarez, M.</au><au>Gomes-Alves, S.</au><au>Anel-López, L.</au><au>Martínez-Rodríguez, C.</au><au>Manrique, P.</au><au>Borragan, S.</au><au>Anel, L.</au><au>de Paz, P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Optimization of conditions for long-term prefreezing storage of brown bear sperm before cryopreservation</atitle><jtitle>Theriogenology</jtitle><addtitle>Theriogenology</addtitle><date>2015-10-15</date><risdate>2015</risdate><volume>84</volume><issue>7</issue><spage>1161</spage><epage>1171</epage><pages>1161-1171</pages><issn>0093-691X</issn><eissn>1879-3231</eissn><abstract>Brown bear ejaculates are usually collected in field conditions and may need to be shipped to a laboratory for the application of reproductive biotechnologies before cryopreservation. The aim of this study was to extend the prefreezing step to 48 hours (1 hour vs. long-term storage [LS] to 24 and 48 hours) to enable the sample to be transported. The effects of storage temperature (experiment 1), glycerol concentration (experiment 2), and dilution rate (experiment 3) on sperm were evaluated. Electroejaculates from brown bears were stored under different experimental conditions and cryopreserved. The sperm motility and viability, apoptotic status, and acrosomal status of sperm were assessed before freezing (prefreezing), after thawing, and after 2-hour incubation at 37 °C (thermal stress test). In all experiments, one control sample was frozen using a standard protocol (control). In experiment 1, three temperatures during LS with 6% glycerol were tested: 5 °C (T5), 15 °C (T15), and room temperature (RT). The LS-T5 sample yielded the highest postthawing results for viability (42.4%), progressive motility (15.6%), and intact acrosome (83.1%) after 24 hours in comparison with the other temperatures (P < 0.05); for 48 hours, the LS-T5 sample reached higher total and progressive motility (25.9% and 9%, respectively) and nonapoptotic values (36.5%). Recovery rates revealed susceptibility to freezing at LS-15 or LS-RT samples at 24 hours (viability) or 48 hours (viability and motility). In experiment 2, samples were stored at 5 °C up to 48 hours and three glycerol concentrations were evaluated: 0% (0Gly), 3% (3Gly), and 6% (6Gly). Postthawing viability and motility increased progressively with the percentage of glycerol for 24 hours at 5 °C; 6% glycerol during 48-hour storage had beneficial effects on sperm cryopreservation. Besides, 6% glycerol had a clearly superior freezability for viability (42.7% and 40.8% for 24 hours and 48 hours, respectively) and motility (24 hours: total, 44.1%; progressive, 17.1%; 48 hours: total, 38.4%; progressive, 16%). In experiment 3, samples were stored up to 48 hours at 5 °C with 6% of glycerol and two dilution methods were evaluated: dilution 1:1 (average: 1782 × 106 sperm/mL; low) or final dilution (100 × 106 sperm/mL; high). Both dilution rates showed similar postthawing and postincubation results within 24 hours of long-term storage. After 48 hours, high dilution supported better postthawing quality. Both dilutions showed similar resistance to cryopreservation, except after 48 hours, when the high dilution reached a higher percent recovery rate of viability (38.8% vs. 21.6%, P < 0.05). In conclusion, our results suggested that the best conditions for long-term prefreezing storage (up to 48 hours) of brown bear electroejaculates are at 5 °C, at a concentration of 100 × 106 sperm/mL, and with 6% glycerol.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>26234462</pmid><doi>10.1016/j.theriogenology.2015.06.017</doi><tpages>11</tpages><orcidid>https://orcid.org/0000-0003-2531-595X</orcidid></addata></record> |
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| subjects | Acrosome - physiology Animals Apoptosis Brown bear Cell Survival Cryopreservation - methods Cryopreservation - veterinary Cryoprotective Agents Dilution rate Glycerol - analysis Glycerol concentration Long-term storage Male Semen Preservation - methods Semen Preservation - veterinary Solutions - chemistry Sperm Motility Spermatozoa - physiology Spermatozoa cryopreservation Temperature Ursidae |
| title | Optimization of conditions for long-term prefreezing storage of brown bear sperm before cryopreservation |
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