{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T23:23:31Z","timestamp":1785194611148,"version":"3.55.0"},"reference-count":54,"publisher":"Oxford University Press (OUP)","issue":"5","license":[{"start":{"date-parts":[[2009,5,1]],"date-time":"2009-05-01T00:00:00Z","timestamp":1241136000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/pages\/standard-publication-reuse-rights"}],"content-domain":{"domain":["rep.bioscientifica.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2009,5,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Zona pellucida (ZP) hardening (resistance to proteolysis) has been classically identified as a post-fertilization event that contributes to the block to polyspermy. Di-(N-succinimidyl)-3,3\u2032-dithiodipropionate (DSP), a permeable amine-reactive cross-linker, was recently shown to induce pre-fertilization ZP hardening and to improve porcine IVF productivity. The objectives of this study were to investigate i) how DSP affects pre-fertilization ZP hardening and IVF in cattle, ii) if a non-permeable amine-reactive cross-linker such as bis(sulfosuccinimidyl) suberate (BS3) affects ZP hardening and IVF in cattle and pigs, and iii) whether DSP or BS3, if improvement in IVF productivity was demonstrated in either species, affects in vitro embryo development. Bovine and porcine in vitro matured oocytes were incubated with the cross-linkers (0.06, 0.3, and 0.6\u200amg\/ml) for 30\u200amin. Then they were subjected to ZP digestion or IVF. In cattle, both DSP and BS3 induced ZP hardening and decreased the penetration rate, although monospermy, penetration, or male pronuclear formation was not affected. In pigs, BS3 treatment induced ZP hardening, decreased penetration and male pronuclear formation, and increased monospermy. IVF productivity only improved when porcine oocytes were exposed to DSP. When porcine zygotes derived from this treatment were further cultured in vitro, the cleavage and blastocyst formation rates increased. These results support the idea that mechanisms involved in the prevention of polyspermic fertilization in cattle and pigs have different efficiencies, and ZP hardening induced by DSP cross-linker may be useful for improving porcine embryo production.<\/jats:p>","DOI":"10.1530\/rep-08-0340","type":"journal-article","created":{"date-parts":[[2009,3,4]],"date-time":"2009-03-04T21:38:10Z","timestamp":1236202690000},"page":"803-812","update-policy":"https:\/\/doi.org\/10.1530\/crossmarkpolicy-1","source":"Crossref","is-referenced-by-count":14,"title":["Pre-fertilization zona pellucida hardening by different cross-linkers affects IVF in pigs and cattle and improves embryo production in pigs"],"prefix":"10.1093","volume":"137","author":[{"given":"Sebastian","family":"Canovas","sequence":"first","affiliation":[{"name":"Department of Physiology Faculty of Veterinary Science 2Department of Cell Biology and Histology School of Medicine, University of Murcia, Murcia 30071, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raquel","family":"Romar","sequence":"additional","affiliation":[{"name":"Department of Physiology Faculty of Veterinary Science 2Department of Cell Biology and Histology School of Medicine, University of Murcia, Murcia 30071, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luis Alberto","family":"Grullon","sequence":"additional","affiliation":[{"name":"Department of Physiology Faculty of Veterinary Science 2Department of Cell Biology and Histology School of Medicine, University of Murcia, Murcia 30071, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manuel","family":"Aviles","sequence":"additional","affiliation":[{"name":"Department of Physiology Faculty of Veterinary Science 2Department of Cell Biology and Histology School of Medicine, University of Murcia, Murcia 30071, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pilar","family":"Coy","sequence":"additional","affiliation":[{"name":"Department of Physiology Faculty of Veterinary Science 2Department of Cell Biology and Histology School of Medicine, University of Murcia, Murcia 30071, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2009,5,1]]},"reference":[{"key":"2026010712485974400_bib1","first-page":"109","article-title":"Maturation of redox regulatory mechanisms in the epididymis","volume":"53","author":"Aitken","year":"1998","journal-title":"Journal of Reproduction and Fertility"},{"key":"2026010712485974400_bib2","doi-asserted-by":"crossref","first-page":"551","DOI":"10.1042\/bj20030466","article-title":"Effect of a null mutation of the oviduct-specific glycoprotein gene on mouse fertilization","volume":"374","author":"Araki","year":"2003","journal-title":"Biochemical Journal"},{"key":"2026010712485974400_bib3","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1038\/172082b0","article-title":"Polyspermy in mammals","volume":"172","author":"Austin","year":"1953","journal-title":"Nature"},{"key":"2026010712485974400_bib4","first-page":"686","article-title":"La maturation de l'\u00f3euf, la f\u00e9condation et les premi\u00e9res phases du d\u00e9veloppement embryonnaire des mamm\u00edferes d'apr\u00e9s des recherches faites chez le lapin","volume":"40","author":"van Beneden","year":"1875","journal-title":"Bulletin de l'Acad\u00e9mie royale de m\u00e9decine de Belgique"},{"key":"2026010712485974400_bib5","first-page":"48","article-title":"Adherence of bovine viral diarrhea virus to bovine oocytes and embryos with a hardened zona pellucida cultured in vitro","volume":"67","author":"Bielanski","year":"2003","journal-title":"Canadian Journal of Veterinary Research = Revue Canadienne de Recherche V\u00e9t\u00e9rinaire"},{"key":"2026010712485974400_bib6","doi-asserted-by":"crossref","first-page":"S156","DOI":"10.1016\/j.theriogenology.2007.05.048","article-title":"Calcium and other ion dynamics during gamete maturation and fertilization","volume":"68","author":"Boni","year":"2007","journal-title":"Theriogenology"},{"key":"2026010712485974400_bib7","doi-asserted-by":"crossref","first-page":"1324","DOI":"10.2527\/jas1989.6751324x","article-title":"Effects of the oviduct and wheat germ agglutinin on enzymatic digestion of porcine zona pellucidae","volume":"67","author":"Broermann","year":"1989","journal-title":"Journal of Animal Science"},{"key":"2026010712485974400_bib8","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1071\/RD01102","article-title":"In vitro production of pig embryos: a point of view","volume":"14","author":"Coy","year":"2002","journal-title":"Reproduction, Fertility and Development"},{"key":"2026010712485974400_bib9","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1530\/rep.0.1240279","article-title":"Effect of in vitro fertilization medium on the acrosome reaction, cortical reaction, zona pellucida hardening and in vitro development in pigs","volume":"124","author":"Coy","year":"2002","journal-title":"Reproduction"},{"key":"2026010712485974400_bib10","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1530\/rep.1.00299","article-title":"Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro","volume":"129","author":"Coy","year":"2005","journal-title":"Reproduction"},{"key":"2026010712485974400_bib11","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1530\/REP-07-0280","article-title":"Hardening of the zona pellucida of unfertilized eggs can reduce polyspermic fertilization in the pig and cow","volume":"135","author":"Coy","year":"2008","journal-title":"Reproduction"},{"key":"2026010712485974400_bib12","doi-asserted-by":"crossref","first-page":"15809","DOI":"10.1073\/pnas.0804422105","article-title":"Oviduct-specific glycoprotein and heparin modulate sperm\u2013zona pellucida interaction during fertilization and contribute to the control of polyspermy","volume":"105","author":"Coy","year":"2008","journal-title":"PNAS"},{"key":"2026010712485974400_bib13","first-page":"359","article-title":"The enigma of sperm\u2013egg recognition in mice","volume":"63","author":"Dean","year":"2007","journal-title":"Society of Reproduction and Fertility Supplement"},{"key":"2026010712485974400_bib14","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/0012-1606(90)90006-5","article-title":"Precocious loss of cortical granules during mouse oocyte meiotic maturation and correlation with an egg-induced modification of the zona pellucida","volume":"137","author":"Ducibella","year":"1990","journal-title":"Developmental Biology"},{"key":"2026010712485974400_bib15","doi-asserted-by":"crossref","first-page":"1251","DOI":"10.1095\/biolreprod48.6.1251","article-title":"Regulation of the polyspermy block in the mouse egg: maturation-dependent differences in cortical granule exocytosis and zona pellucida modifications induced by inositol 1,4,5-trisphosphate and an activator of protein kinase C","volume":"48","author":"Ducibella","year":"1993","journal-title":"Biology of Reproduction"},{"key":"2026010712485974400_bib16","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/B978-012515400-0\/50007-5","article-title":"Fertilization in mammals","volume-title":"Knobil and Neill's Physiology of Reproduction","author":"Florman","year":"2006","edition":"3 edn"},{"key":"2026010712485974400_bib17","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1530\/jrf.0.0640495","article-title":"In vitro fertilization of zona-free bovine oocytes matured in culture","volume":"64","author":"Fulka","year":"1982","journal-title":"Journal of Reproduction and Fertility"},{"key":"2026010712485974400_bib18","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1530\/rep.1.00295","article-title":"Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine","volume":"128","author":"Funahashi","year":"2004","journal-title":"Reproduction"},{"key":"2026010712485974400_bib19","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1095\/biolreprod57.1.49","article-title":"Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization","volume":"57","author":"Funahashi","year":"1997","journal-title":"Biology of Reproduction"},{"key":"2026010712485974400_bib20","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1002\/mrd.1120110107","article-title":"The effects of short-term incubation (aging) of mouse oocytes on in vitro fertilization, zona solubility, and embryonic development","volume":"11","author":"Gianfortoni","year":"1985","journal-title":"Gamete Research"},{"key":"2026010712485974400_bib21","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1530\/ror.0.0020147","article-title":"Three-dimensional structure of the zona pellucida","volume":"2","author":"Green","year":"1997","journal-title":"Reviews of Reproduction"},{"key":"2026010712485974400_bib22","first-page":"103","article-title":"Polyspermy of pig ova","volume":"1","author":"Hancock","year":"1959","journal-title":"Animal Production"},{"key":"2026010712485974400_bib23","doi-asserted-by":"crossref","first-page":"726","DOI":"10.1095\/biolreprod.106.052589","article-title":"Osteopontin reduces polyspermy during in vitro fertilization of porcine oocytes","volume":"75","author":"Hao","year":"2006","journal-title":"Biology of Reproduction"},{"key":"2026010712485974400_bib24","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1002\/mrd.20794","article-title":"Osteopontin improves in vitro development of porcine embryos and decreases apoptosis","volume":"75","author":"Hao","year":"2008","journal-title":"Molecular Reproduction and Development"},{"key":"2026010712485974400_bib25","first-page":"409","article-title":"A comparative analysis of molecular mechanisms for blocking polyspermy: identification of a lectin-ligand binding reaction in mammalian eggs","volume":"63","author":"Hedrick","year":"2007","journal-title":"Society of Reproduction and Fertility"},{"key":"2026010712485974400_bib26","first-page":"211","article-title":"Fertilization of pig eggs in vivo and in vitro","volume":"40","author":"Hunter","year":"1990","journal-title":"Journal of Reproduction and Fertility"},{"key":"2026010712485974400_bib27","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1530\/jrf.0.1170395","article-title":"Disulfide formation in bovine zona pellucida glycoproteins during fertilization: evidence for the involvement of cystine cross-linkages in hardening of the zona pellucida","volume":"117","author":"Iwamoto","year":"1999","journal-title":"Journal of Reproduction and Fertility"},{"key":"2026010712485974400_bib28","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1016\/j.anireprosci.2007.03.006","article-title":"Effects of modification of in vitro fertilization techniques on the sex ratio of the resultant bovine embryos","volume":"105","author":"Iwata","year":"2008","journal-title":"Animal Reproduction Science"},{"key":"2026010712485974400_bib29","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1016\/j.anireprosci.2007.03.010","article-title":"Changes to the meiotic spindle and zona pellucida of mature mouse oocytes following different cryopreservation methods","volume":"105","author":"Ko","year":"2008","journal-title":"Animal Reproduction Science"},{"key":"2026010712485974400_bib30","doi-asserted-by":"crossref","first-page":"1695","DOI":"10.1016\/j.theriogenology.2004.07.023","article-title":"Differences in proteinase digestibility of the zona pellucida of in vivo and in vitro derived porcine oocytes and embryos","volume":"63","author":"Kolbe","year":"2005","journal-title":"Theriogenology"},{"key":"2026010712485974400_bib31","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1095\/biolreprod63.1.242","article-title":"Effects of the porcine oviduct-specific glycoprotein on fertilization, polyspermy, and embryonic development in vitro","volume":"63","author":"Kouba","year":"2000","journal-title":"Biology of Reproduction"},{"key":"2026010712485974400_bib32","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.theriogenology.2007.09.007","article-title":"In vitro-produced bovine embryos: dealing with the warts","volume":"69","author":"Lonergan","year":"2008","journal-title":"Theriogenology"},{"key":"2026010712485974400_bib33","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1095\/biolreprod59.2.451","article-title":"Development of early porcine embryos in vitro and in vivo","volume":"59","author":"Machaty","year":"1998","journal-title":"Biology of Reproduction"},{"key":"2026010712485974400_bib34","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1530\/jrf.0.1130323","article-title":"Enhancement of bovine oocyte fertilization in vitro with a bovine oviductal specific glycoprotein","volume":"113","author":"Martus","year":"1998","journal-title":"Journal of Reproduction and Fertility"},{"key":"2026010712485974400_bib35","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1210\/rp.57.1.37","article-title":"Molecular basis of mammalian gamete binding","volume":"57","author":"Miller","year":"2002","journal-title":"Recent Progress in Hormone Research"},{"key":"2026010712485974400_bib36","doi-asserted-by":"crossref","first-page":"2565","DOI":"10.2741\/1991","article-title":"Up date of in vitro production of porcine embryos","volume":"11","author":"Nagai","year":"2006","journal-title":"Frontiers in Bioscience: a Journal and Virtual Library"},{"key":"2026010712485974400_bib37","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1177\/153537020623100206","article-title":"Fourier transform infrared spectroscopic analysis of the intact zona pellucida of the mammalian egg: changes in the secondary structure of bovine zona pellucida proteins during fertilization","volume":"231","author":"Nara","year":"2006","journal-title":"Experimental Biology and Medicine"},{"key":"2026010712485974400_bib38","doi-asserted-by":"crossref","first-page":"1805","DOI":"10.1007\/s00018-007-6552-x","article-title":"New insights into the molecular mechanisms of sperm\u2013egg interaction","volume":"64","author":"Nixon","year":"2007","journal-title":"Cellular and Molecular Life Sciences"},{"key":"2026010712485974400_bib39","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1530\/rep.1.00460","article-title":"Fluctuations in bovine ovarian follicular fluid composition throughout the oestrous cycle","volume":"129","author":"Orsi","year":"2005","journal-title":"Reproduction"},{"key":"2026010712485974400_bib40","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1016\/0093-691X(86)90143-3","article-title":"Bovine in vitro fertilization with frozen\u2013thawed semen","volume":"25","author":"Parrish","year":"1986","journal-title":"Theriogenology"},{"key":"2026010712485974400_bib41","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1002\/mrd.20805","article-title":"A novel approach to identify bovine sperm membrane proteins that interact with receptors on the vitelline membrane of bovine oocytes","volume":"75","author":"Pate","year":"2008","journal-title":"Molecular Reproduction and Development"},{"key":"2026010712485974400_bib42","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1016\/j.theriogenology.2007.03.022","article-title":"Risk assessment of transmission of bovine viral diarrhea virus (BVDV) in abattoir-derived in vitro produced embryos","volume":"68","author":"Perry","year":"2007","journal-title":"Theriogenology"},{"key":"2026010712485974400_bib43","first-page":"61","article-title":"Culture of pig embryos","volume":"48","author":"Petters","year":"1993","journal-title":"Journal of Reproduction and Fertility"},{"key":"2026010712485974400_bib44","doi-asserted-by":"crossref","first-page":"99","DOI":"10.2527\/jas1975.40199x","article-title":"Freezing of boar spermatozoa: fertilizing capacity with concentrated semen and a new thawing procedure","volume":"40","author":"Pursel","year":"1975","journal-title":"Journal of Animal Science"},{"key":"2026010712485974400_bib45","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/S1534-5807(03)00195-3","article-title":"Fertility and taxon-specific sperm binding persist after replacement of mouse sperm receptors with human homologs","volume":"5","author":"Rankin","year":"2003","journal-title":"Developmental Cell"},{"key":"2026010712485974400_bib46","doi-asserted-by":"crossref","first-page":"3346","DOI":"10.2527\/1999.77123346x","article-title":"In vitro production of sexed embryos for gender preselection: high-speed sorting of X-chromosome-bearing sperm to produce pigs after embryo transfer","volume":"77","author":"Rath","year":"1999","journal-title":"Journal of Animal Science"},{"key":"2026010712485974400_bib47","first-page":"20","article-title":"Molecular mechanisms of the interaction between sperm and the zona pellucida in mammals: studies on the pig","volume":"2","author":"Topfer-Petersen","year":"1995","journal-title":"International Journal of Andrology"},{"key":"2026010712485974400_bib48","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1530\/jrf.0.0890253","article-title":"The hardening effect of dimethylsulphoxide on the mouse zona pellucida requires the presence of an oocyte and is associated with a reduction in the number of cortical granules present","volume":"89","author":"Vincent","year":"1990","journal-title":"Journal of Reproduction and Fertility"},{"key":"2026010712485974400_bib49","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1002\/jemt.10346","article-title":"How does polyspermy happen in mammalian oocytes?","volume":"61","author":"Wang","year":"2003","journal-title":"Microscopy Research and Technique"},{"key":"2026010712485974400_bib50","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.anireprosci.2006.06.014","article-title":"Improvement in bovine embryo production in vitro by treatment with green tea polyphenols during in vitro maturation of oocytes","volume":"100","author":"Wang","year":"2007","journal-title":"Animal Reproduction Science"},{"key":"2026010712485974400_bib51","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.mce.2004.08.017","article-title":"Recent aspects of mammalian fertilization research","volume":"234","author":"Wassarman","year":"2005","journal-title":"Molecular and Cellular Endocrinology"},{"key":"2026010712485974400_bib52","first-page":"1","article-title":"Defending the zygote: search for the ancestral animal block to polyspermy","volume":"72","author":"Wong","year":"2006","journal-title":"Current Topics in Developmental Biology"},{"key":"2026010712485974400_bib53","first-page":"189","article-title":"Mammalian fertilization","volume-title":"The Physiology of Reproduction","author":"Yanagimachi","year":"1994","edition":"2 edn"},{"key":"2026010712485974400_bib54","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1017\/S0967199400002720","article-title":"Comparison of the acrosome reaction-inducing ability of the outer and inner surfaces of the zona pellucida and oolemma: a study using the golden hamster","volume":"3","author":"Yazawa","year":"1995","journal-title":"Zygote"}],"container-title":["Reproduction"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/rep.bioscientifica.com\/view\/journals\/rep\/137\/5\/803.xml","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/academic.oup.com\/reproduction\/article-pdf\/137\/5\/803\/64529154\/803.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/reproduction\/article-pdf\/137\/5\/803\/64529154\/803.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,7]],"date-time":"2026-01-07T17:50:47Z","timestamp":1767808247000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/reproduction\/article\/137\/5\/803\/8242973"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2009,5,1]]},"references-count":54,"journal-issue":{"issue":"5","published-print":{"date-parts":[[2009,5,1]]}},"URL":"https:\/\/doi.org\/10.1530\/rep-08-0340","relation":{},"ISSN":["1470-1626","1741-7899"],"issn-type":[{"value":"1470-1626","type":"print"},{"value":"1741-7899","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2009,5,1]]},"published":{"date-parts":[[2009,5,1]]}}}