Sanitary requirements for bovine gametes and embryos in ... - CBRA

Ponsart and Pozzi. Sanitary requirements for gametes and embryos.Technical staff and animal managementAs mentioned in the OIE Animal Health Code,the laboratory personnel should be technically competentand observe high standards of personal hygiene topreclude the introduction of pathogenic organisms duringsemen evaluation, processing, and storage. In CSS(2011b; AI center animal management guidelines),complete recommendations for animal management, sireand hygiene procedures, feeding and housing conditionsand veterinary and professional care are described.Specific sanitary requirements for bovine semenIn general terms, microorganisms can be presentin the semen of an infected male or can gain entry to semenduring collection, processing, or storage. In order tomaintain a controlled status of semen batches, tests must beapplied on semen and genital tract, semen production, andsanitary control of the bulls present in the center.For given pathogenic agents, semen can becertified as free if the donor bull originated from a freeherd, the dam of the bull is free, the donor bull is free,the donor bull is introduced to a center where all otherbulls are free, and all the bulls present in the center aresubmitted to regular tests regarding this agent. For eachagent, specific OIE requirements aim to maintain thehealth of animals on an artificial insemination center ata level which permits the international distribution ofsemen with a negligible risk of infecting other animalsor humans with pathogens transmissible by semen, asdescribed in the Table 4.Sanitary requirements for embryos used ininternational tradeAlthough transfer of bovine embryos is muchless likely to result in disease transmission than transport oflive animals (Thibier and Wrathall, 2012), the sanitary riskassociated with bovine embryo transfer remains the subjectof scientific investigations (Van Soom et al., 2008) andadaptations of national and international legislations (OIE,2012; Chapters 4.7 and 4.8).Physiological backgroundInteraction of oocytes and embryos withpathogenic agents has been extensively reviewed byBielanski (2006). Oocytes and early embryo stages up toapproximately day 8 after fertilization, are surrounded byan acellular glycoprotein shell with a sponge-like surface,the zona pellucida (ZP). Visualized by scanning electronmicroscopy, the ZP is composed of a fibrous networkwith numerous pores. The pores are larger at the outersurface (outer diameter of embryos range from 155 to223 µm for different embryonic developmental stages)but decrease in size centripetally in both animals andhuman embryos (Bielanski, 2006). Such anatomicalstructures of the ZP allows for adhesion of pathogens, butprevents them from fully penetrating the ZP.Since the ZP is acellular in character, viruses arenot able to replicate there and they must cross the ZP andthe cell plasma membrane to infect an embryo. In general,ova or embryos can become contaminated at differentstages: before the ZP is formed (under physiologicalconditions, the ZP is formed in the ovarian preantralsecondary follicles), later by agents present in thefollicular fluid, by pathogen-contaminated semen duringfertilization or during passage through the oviduct and theuterus, even if integrity of the ZP prevents contaminationof embryonic cells for most pathogens (Bielanski, 2006;Van Soom et al., 2008). With the application of in vitrofertilization techniques, immature oocytes surrounded bya multilayer of compacted granulosa cells (cumulusoocytecomplexes) are collected from ovarian folliclesusing Ovum Pick Up (OPU) or from slaughterhousederived ovaries and placed in the maturation medium.During this period, the granulosa cells become expandedand their connections with the ZP loosen. Later, thegranulosa cells are mechanically removed, deleting theconnection between these cells and the ZP. Specific risksare linked to artificial culture conditions, rather than theutero-tubal environment which occurs with in vivofertilization (Bielanski, 2006).Requirements applied to in vivo derived embryosAssessment of disease transmission via in vivoderived embryosExperience and experimental evidence hasindicated a low potential for transmission of infectiouspathogens via in vivo-derived (IVD) embryos (Givens etal., 2007; Thibier 2011). Pathogens may be shed into thegenital tract and contaminate the surface of embryos, ifthose pathogens are present at the time of collection orbetween fertilization and collection. Scientific reflectionson the sanitary risks associated with ET have focused onthe probability that embryos can be contaminated either viathe oocyte, the semen, or adhesion to the zona pellucida.There have been many investigations to evaluateinteractions between pathogens and embryos, usingdifferent in vivo or in vitro infection approaches (Table 5).In vivo approaches are the most suitable toevaluate the likelihood of transmission through embryotransfer, however such experiments require expensiveprotocols in order to infect donor animals and performsubsequent transfer to recipients. As an example, facilitieswith a controlled environment and that are insect-proof arerequired to investigate vector transmitted diseases. Fordiseases with a long incubation period (e.g., prions),experiments may last several years. Complementary invitro approaches can be used to gain knowledge withmore accessible costs (Table 5). Indeed, in vitroexperiments are ultimately more conservative and arenot as likely to be influenced by factors like minimuminfective dose, innate immunity etc. Realistically, if an invitro experiment does not reveal the presence of infectiousagents, the likelihood of an in vivo experiment showing itspresence is very unlikely.Anim. Reprod., v.10, n.3, p.283-296, Jul./Sept. 2013 287