{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,15]],"date-time":"2025-10-15T10:39:05Z","timestamp":1760524745493,"version":"build-2065373602"},"reference-count":35,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2025,8,9]],"date-time":"2025-08-09T00:00:00Z","timestamp":1754697600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004281","name":"Polish National Science Centre grant","doi-asserted-by":"publisher","award":["Opus Lap 2021\/43\/I\/NZ9\/01077","N1-0281","P1\u20130386","P4\u20130107"],"award-info":[{"award-number":["Opus Lap 2021\/43\/I\/NZ9\/01077","N1-0281","P1\u20130386","P4\u20130107"]}],"id":[{"id":"10.13039\/501100004281","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Slovenian Research and Innovation Agency","award":["Opus Lap 2021\/43\/I\/NZ9\/01077","N1-0281","P1\u20130386","P4\u20130107"],"award-info":[{"award-number":["Opus Lap 2021\/43\/I\/NZ9\/01077","N1-0281","P1\u20130386","P4\u20130107"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>The aim was to evaluate the effectiveness of semen cryopreservation and oocyte vitrification in roe deer as a potential method of gamete preservation for endangered deer species. Sperm were isolated from the cauda epididymis of fourteen bucks (n = 14). The motility measure (CASA) and morphology of fresh semen (FS) and frozen\u2013thawed semen (TS) were compared. A hyaluronic binding assay was used to distinguish between mature FS spermatozoa expressing hyaluronan receptors and immature FS lacking these receptors, and the mitochondrial membrane potential (MMP) in TS was determined (flow cytometry). A Sperm\u2013Hyaluronan Binding Assay (HBA) showed a viability rate of 61.9% in FS and 78.2% in TS. Oocytes received from eight does (n = 8) underwent a viability test and vitrification, and fresh oocytes from the other eight does (n = 8) were fertilized with TS and embryos were cultured until the blastocyst stage. The number of isolated oocytes, cumulus\u2013oocyte complexes (COCs), cleaved embryos, and expanded blastocysts was evaluated. Higher percentages of morphological factors (acrosome, head, midpiece, and tail shape) were observed in FS compared to TS, whereas the motility and progressive movement were greater in TS (p \u2264 0.001). The viability was 50.5% and MMP was 40.6% in TS. A total of 311 oocytes were collected and from 150 COCs and 125 blastocysts developed. The viability of thawed oocytes after vitrification was 81%. The viability of vitrified oocytes and cryopreserved sperm confirmed the effectiveness of freezing protocols and highlights the potential for their implementation in other deer species.<\/jats:p>","DOI":"10.3390\/ani15162335","type":"journal-article","created":{"date-parts":[[2025,8,11]],"date-time":"2025-08-11T08:10:32Z","timestamp":1754899832000},"page":"2335","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Effective Cryopreservation of Post Mortem-Collected Roe Deer Gametes by Evaluation of Post-Thaw Oocyte and Sperm Characteristics and In Vitro Fertilization"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1044-9288","authenticated-orcid":false,"given":"Anna Justyna","family":"Korzekwa","sequence":"first","affiliation":[{"name":"Reasearch Team of Biodiversity Protection, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences (IAR&FR PAS), Trylinskiego 18 Str., 10-683 Olsztyn, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0714-5301","authenticated-orcid":false,"given":"Elena","family":"Buzan","sequence":"additional","affiliation":[{"name":"Faculty of Mathematics, Natural Sciences and Information Technologies, University of Primorska, Glagolja\u0161ka 8, 6000 Koper, Slovenia"},{"name":"Faculty of Environmental Protection, College of Environmental Protection, Trg Mladosti 7, 3320 Velenje, Slovenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2981-5516","authenticated-orcid":false,"given":"Bostjan","family":"Pokorny","sequence":"additional","affiliation":[{"name":"Faculty of Environmental Protection, College of Environmental Protection, Trg Mladosti 7, 3320 Velenje, Slovenia"},{"name":"Slovenian Forestry Institute, Ve\u010dna Pot 2, 1000 Ljubljana, Slovenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9277-6310","authenticated-orcid":false,"given":"Gulsum Ummu","family":"Boztepe","sequence":"additional","affiliation":[{"name":"Reasearch Team of Biodiversity Protection, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences (IAR&FR PAS), Trylinskiego 18 Str., 10-683 Olsztyn, Poland"},{"name":"University Centre of Veterinary Medicine JU-UA, University of Agriculture in Krak\u00f3w, 31-120 Krakow, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marek","family":"Lecewicz","sequence":"additional","affiliation":[{"name":"Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-957 Olsztyn, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"W\u0142adys\u0142aw","family":"Kordan","sequence":"additional","affiliation":[{"name":"Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-957 Olsztyn, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Wilson, D.E., and Reede, D.M. (2005). Mammal Species of the World, Johns Hopkins University Press.","DOI":"10.56021\/9780801882210"},{"key":"ref_2","unstructured":"Andersen, R., Duncan, P., and Linnell, J.D.C. (1998). The European Roe Deer: The Biology of Success, Scandinavian University Press."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"3873","DOI":"10.1111\/mec.13292","article-title":"Immunogenetic heterogeneity in a widespread ungulate: The European roe deer (Capreolus capreolus)","volume":"24","author":"Galan","year":"2015","journal-title":"Mol. Ecol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1285","DOI":"10.1046\/j.1365-294X.2002.01534.x","article-title":"The genetic structure of natural and reintroduced roe deer (Capreolus capreolus) populations in the Alps and central Italy, with reference to the mitochondrial DNA phylogeography of Europe","volume":"11","author":"Vernesi","year":"2002","journal-title":"Mol. Ecol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3071","DOI":"10.1111\/j.1365-294X.2004.02279.x","article-title":"Phylogeography of roe deer (Capreolus capreolus) populations: The effects of historical genetic subdivisions and recent nonequilibrium dynamics","volume":"13","author":"Randi","year":"2004","journal-title":"Mol. Ecol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1016\/j.cvfa.2019.02.009","article-title":"Multigenerational Effects","volume":"35","author":"Roberts","year":"2019","journal-title":"Vet. Clin. N. Am. Food Anim. Pract."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1007\/s10344-019-1274-6","article-title":"Assessing the complex relationship between landscape, gene flow, and range expansion of a Mediterranean carnivore","volume":"65","author":"Barros","year":"2019","journal-title":"Eur. J. Wildl. Res."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Buzan, E., Potu\u0161ek, S., Duni\u0161, L., and Pokorny, B. (2022). Neutral and selective processes shape MHC diversity in roe deer in Slovenia. Animals, 12.","DOI":"10.3390\/ani12060723"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1111\/j.1469-7998.1997.tb02795.x","article-title":"Conservation of deer: Contributions from molecular biology, evolutionary ecology, and reproductive physiology","volume":"243","author":"Jabbour","year":"1997","journal-title":"J. Zool."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"965","DOI":"10.1023\/A:1008813532763","article-title":"Sex and wildlife: The role of reproductive science in conservation","volume":"8","author":"Wildt","year":"1999","journal-title":"Biodivers. Conserv."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Korzekwa, A.J., and Kotlarczyk, A.M. (2021). Artificial reproductive technology (ART) applied to female cervids adapted from domestic ruminants. Animals, 11.","DOI":"10.3390\/ani11102933"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1530\/jrf.0.0620049","article-title":"Relationship between antler development and plasma androgen concentrations in adult roe deer (Capreolus capreolus)","volume":"62","author":"Boissin","year":"1981","journal-title":"Reproduction"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Apollonio, M., Merli, E., Chirichella, R., Pokorny, B., Alagi\u0107, A., Flaj\u0161man, K., and Stephens, P.A. (2020). Capital-income breeding in male ungulates: Causes and consequences of strategy differences among species. Front. Ecol. Evol., 8.","DOI":"10.3389\/fevo.2020.521767"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3199","DOI":"10.1242\/dev.148213","article-title":"The enigma of embryonic diapause","volume":"144","author":"Renfree","year":"2017","journal-title":"Development"},{"key":"ref_15","first-page":"136","article-title":"Assisted reproductive technologies in deer (Artiodactyla, Cervidae): A review","volume":"45","author":"Pintus","year":"2014","journal-title":"Sci. Agric. Bohem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"695","DOI":"10.1016\/j.theriogenology.2016.02.023","article-title":"Cryopreservation of captive roe deer (Capreolus capreolus) semen","volume":"86","author":"Hildebrandt","year":"2016","journal-title":"Theriogenology"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.anireprosci.2018.03.010","article-title":"Slow and ultra-rapid freezing protocols for cryopreserving mouflon (Ovis musimon) and fallow deer (Dama dama) epididymal sperm","volume":"192","author":"Esteso","year":"2018","journal-title":"Anim. Reprod. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1007\/s10561-008-9081-4","article-title":"Cryopreservation of domestic animal sperm cells","volume":"10","author":"Barbas","year":"2009","journal-title":"Cell Tissue Bank."},{"key":"ref_19","first-page":"1","article-title":"Slow and ultra-rapid freezing protocols for cryopreserving roe deer (Capreolus capreolus) epididymal sperm collected at different times of year","volume":"67","author":"Esteso","year":"2021","journal-title":"Eur. J. Wildl. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"100741","DOI":"10.1016\/j.animal.2023.100741","article-title":"Review: Sperm cryopreservation in wild small ruminants: Morphometric, endocrine and molecular basis of cryoresistance","volume":"17","author":"Esteso","year":"2023","journal-title":"Animal"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/S0940-9602(03)80071-7","article-title":"Variations in lectin-binding on the zona pellucida during oocyte growth in some wild ungulates","volume":"185","author":"Parillo","year":"2003","journal-title":"Ann. Anat.-Anat. Anz."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Korzekwa, A.J., Kotlarczyk, A.M., Szczepa\u0144ska, A.A., Grzyb, M., Siergiej, A., and Woc\u0142awek-Potocka, I. (2020). Antioxidative potential of red deer embryos depends on reproductive stage of hind as a oocyte donor. Animals, 10.","DOI":"10.3390\/ani10071190"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"856","DOI":"10.1071\/RD09197","article-title":"Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments","volume":"22","author":"Bisbal","year":"2010","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1002\/jemt.10311","article-title":"Biology of spermatozoa maturation: An overview with an introduction to this issue","volume":"61","author":"Toshimori","year":"2003","journal-title":"Microsc. Res. Tech."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1016\/j.theriogenology.2004.03.003","article-title":"Decay of sperm obtained from epididymes of wild ruminants depending on postmortem time","volume":"63","author":"Guerra","year":"2005","journal-title":"Theriogenology"},{"key":"ref_26","first-page":"113","article-title":"Effect of different cryoprotectant concentrations for ultrarapid freezing of immature goat follicular oocytes on their subsequent maturation and fertilization in vitro","volume":"4","author":"Garg","year":"2007","journal-title":"Anim. Reprod."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1005","DOI":"10.1016\/j.theriogenology.2011.12.018","article-title":"Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 \u00b0C in presence of several antioxidants","volume":"78","author":"Garde","year":"2012","journal-title":"Theriogenology"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.anireprosci.2018.05.004","article-title":"Cinnamtannin B-1, a novel antioxidant for sperm in red deer","volume":"195","author":"Soler","year":"2018","journal-title":"Anim. Reprod. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"620","DOI":"10.1016\/j.rbmo.2012.08.003","article-title":"Correlation study between sperm concentration, hyaluronic acid-binding capacity and sperm aneuploidy in Hungarian patients","volume":"25","author":"Ujfalusi","year":"2012","journal-title":"Reprod. Biomed. Online"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"642","DOI":"10.1111\/bju.12523","article-title":"Varicocele decreases epididymal neutral \u03b1-glucosidase and is associated with alteration of nuclear DNA and plasma membrane in spermatozoa","volume":"113","author":"Camejo","year":"2014","journal-title":"BJU Int."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"590","DOI":"10.1016\/j.theriogenology.2012.11.011","article-title":"Supplementation of maturation medium with L-carnitine improves cryo-tolerance of bovine in vitro matured oocytes","volume":"79","author":"Chankitisakul","year":"2013","journal-title":"Theriogenology"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"107361","DOI":"10.1016\/j.anireprosci.2023.107361","article-title":"Effects of different cryopreservation methods on canine isolated preantral follicles","volume":"258","author":"Uno","year":"2023","journal-title":"Anim. Reprod. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.cryobiol.2020.02.011","article-title":"Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection","volume":"93","author":"Piras","year":"2020","journal-title":"Cryobiology"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1262\/jrd.19150","article-title":"Effect of centrifugation treatment before vitrification on the viability of porcine mature oocytes and zygotes produced in vitro","volume":"54","author":"Somfai","year":"2008","journal-title":"J. Reprod. Dev."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e13596","DOI":"10.1111\/asj.13596","article-title":"Effect of vitrification at different meiotic stages on epigenetic characteristics of bovine oocytes and subsequently developing embryos","volume":"92","author":"Yodrug","year":"2021","journal-title":"Anim. Sci. J."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/15\/16\/2335\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:27:04Z","timestamp":1760034424000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/15\/16\/2335"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,9]]},"references-count":35,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2025,8]]}},"alternative-id":["ani15162335"],"URL":"https:\/\/doi.org\/10.3390\/ani15162335","relation":{},"ISSN":["2076-2615"],"issn-type":[{"type":"electronic","value":"2076-2615"}],"subject":[],"published":{"date-parts":[[2025,8,9]]}}}