Evaluation of risks of viral transmission to recipients of bovine embryos arising from fertilisation with virus-infected semen

Theriogenology 65 (2006) 247–274 www.journals.elsevierhealth.com/periodicals/the

Review

Evaluation of risks of viral transmission to recipients of bovine embryos arising from fertilisation with virus-infected semen A.E. Wrathall a,*, H.A. Simmons a, A. Van Soom b a

Animal Services Unit, Veterinary Laboratories Agency, Woodham Lane, New Haw, Weybridge, Surrey KT15 3NB, UK b Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, 9820 Ghent, Belgium Received 16 March 2005; received in revised form 21 May 2005; accepted 24 May 2005

Abstract This scientific review was prompted by recent legislation to curtail the use of semen from potentially virus-infected bulls to produce embryos for import into the European Union. From studies in laboratory animals, humans and horses, it is apparent that viruses may sometimes attach to, or be integrated into, spermatozoa, although in domestic livestock, including cattle, this seems to be a rare phenomenon, and carriage of virus through the zona pellucida into the oocyte by fertilising sperm has never been described in these species. Four specific viruses; enzootic bovine leukosis (EBLV), bovine herpesvirus-1 (BoHV-1), bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV), all of which tend to cause subclinical infections in cattle, but which can occur in bovine semen, are examined with regard to the risks that use of infected semen might lead to production of infected embryos. With regard to in vivo-derived embryos, when internationally approved embryo processing protocols are used, the risks from EBLV- and BTV-infected semen are negligible, and the same is almost certainly true for semen infected with BoHV-1 if the embryos are also treated with trypsin. For BVDV, there is insufficient data on how the virus is carried in semen and how different BVDV strains can interact with sperm, oocytes and embryos. There is a potential, at least, that in vivo-derived embryos resulting from infected semen might carry BVDV, although field studies so far suggest that this is very unlikely. With regard to in vitro-produced embryos, use of semen infected with any of the four viruses, with the probable exception of EBLV, will often lead to contaminated embryos, and * Corresponding author at: Veterinary Laboratories Agency, Animal Services Unit, 25 Serpentine Road, Kendal, Cumbria LA9 4PF, UK. Tel.: +44 1539 741428; fax: +44 1932 347046. E-mail address: [email protected] (A.E. Wrathall). 0093-691X/$ – see front matter. Crown Copyright # 2005 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.05.043

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virus removal from these embryos is difficult even when the internationally approved embryo processing protocols are used. However, it has never been demonstrated that such embryos have resulted in transmission of infection to recipients or offspring. Crown Copyright # 2005 Published by Elsevier Inc. All rights reserved. Keywords: Viruses; Bovine; Semen; Embryos; Import–export

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The basis for asserting that embryo transfer in livestock species is safe . . . . . . . . Evidence that viruses can attach to or be integrated into spermatozoa and carried into the oocyte at fertilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The situation in domestic livestock, including cattle. . . . . . . . . . . . . . . . . . . . . . Enzootic bovine leukosis virus (EBLV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bovine herpesvirus-1 (BoHV-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bovine viral diarrhoea virus (BVDV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bluetongue virus (BTV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Member States of the European Union (EU) have traditionally authorised importation of bovine embryos from non-EU countries (‘third countries’) if the embryos conform to guarantees laid down in EU Council Directive 89/556/EEC [1] and EU Commission Decision 92/471/EEC [2] as further amended by EU Commission Decision 94/280/EC [3]. Both in vivo-derived and in vitro-produced embryos are covered by this Directive which does not appear to differentiate between the level of risk associated with the two types. Although initially the type of semen used for fertilisation of the embryos was not a factor limiting their importation into the EU, it became apparent in 2004 that the animal health conditions laid down in the above Directive for intra-Community trade in bovine embryos were stricter than the conditions which apply to importation of such embryos from third countries. Thus, on 26 November 2004, an attempt was made (Decision 2004/786/EC) [4] to insist that embryos imported from third countries must be conceived as a result of artificial insemination (AI) or in vitro fertilisation (IVF) with semen complying with EU Council Directive 88/407/EEC—the Bovine Semen Directive [5]. The semen would also have to come from approved semen collection centres/storage centres in accordance with Directive 88/407/EEC. A further Commission Decision (2004/639/EC) came into force on 1 January 2005 [6] which means that, from that date, semen from sires seropositive for, or vaccinated against, infectious bovine rhinotracheaitis (IBR) will not be able to be used. The proposed new Decisions (2004/786/EC and 2004/639/EC) led to complaints from several countries, both within and outside the EU, essentially claiming that further

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restrictions on embryo importations based on potential risks of disease transmission via the semen are scientifically unjustified. As a consequence, on 9 March 2005 the Commission published a new Decision (2005/217/EC) [7] which will permit, until 31 December 2006, continued importation of bovine embryos (both in vivo-derived and in vitro-produced) that were collected or produced under the old conditions up to 31 December 2005. Meanwhile the Commission will seek opinions and evidence from interested parties in the EU and elsewhere. The purpose of this paper is to review the scientific evidence on whether viral pathogens that occur in bovine semen can be transmitted into the oocyte at fertilisation and, if so, whether the resulting embryos are likely to transmit disease.

2. The basis for asserting that embryo transfer in livestock species is safe There is a wealth of experimental and field evidence to show that, if approved sanitary protocols are followed, the risks of transmitting infectious diseases via embryo transfer are extremely small. Sanitary protocols developed by the International Embryo Transfer Society (IETS) and published in the IETS Manual [8], and also published as Appendices by the Office International des Epizooties in its Terrestrial Animal Health Code [9], have been widely accepted and applied in international trade. In contrast to the well known and real risks of disease transmission through use of contaminated semen for AI, there is little if any evidence that use of contaminated semen to produce embryos for transfer (especially in vivo-derived embryos) poses a risk to the health status of those embryos, provided that they are properly processed using the approved sanitary protocols. Many hundreds of thousands of bovine embryos have been collected and transferred both within and between countries with no confirmed reports of disease transmission by this route. Embryo processing, as recommended in Chapter 6 of the IETS Manual [8], entails rotating the embryos under a stereomicroscope, using at least 50 magnification, to examine them for defects, for example cracks in the zona pellucida, and for cellular and other debris adhering to the zona. Such debris can often be removed, for example by repeated pipetting, but if not, those embryos, and those with a defective zona (including any that have hatched from the zona) must be discarded because they cannot be washed effectively. Acceptable embryos, i.e. those with an intact and visibly clean zona pellucida are then washed by passing them, in groups of not more than 10, sequentially through a series of at least 10 wells of sterile, buffered saline medium, usually containing 0.4% bovine serum albumin (BSA), and with broad spectrum antibiotics. Each wash must involve at least a 100-fold dilution of the previous wash and a fresh, sterile pipette is used to move the embryos between each well, and to gently stir them in each well. After they have been taken through the 10 washes, embryos must be re-checked microscopically to ensure the zona pellucida is still intact and free of adherent material. Where the additional trypsin treatment protocol is used, the embryos are transferred through the first five buffered saline washes, as described above, then through two aliquots of sterile 0.25% trypsin in a buffered saline solution without Ca2+ or Mg2+, but with antibiotics, for a total exposure of 60–90 s. The trypsin (1:250) should have an activity such that 1 g will hydrolyse 250 g of casein at 25 8C, pH 7.6, in 10 min. After the trypsin treatment the embryos are transferred through the remaining five washes in buffered saline, as with the first five but with 2% serum or

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0.4% BSA to serve as a substrate for removal of the remaining enzymatic activity of the trypsin. A fundamental concept enshrined in the sanitary protocols set out in the Terrestrial Animal Health Code [9] and the IETS Manual [8] is accountability of the ‘Officially Approved Embryo Collection Team’ (in the case of in vivo-derived embryos), or ‘Embryo Production Team’ (in the case of in vitro-produced embryos). Teams in the exporting countries are held responsible for adhering to these sanitary protocols for handling embryos, including their collection, processing, identification and certification before export. The technical objective of embryo processing, including evaluation of the integrity of and absence of cellular and other debris from the zona pellucida, washing, trypsin treatment and use of antibiotics in the media, is to enable ‘disinfection’, i.e. the removal or inactivation of any potential pathogens that may be present, but without affecting embryo viability. When these joint OIE/IETS protocols are followed, the costly multiple testing regimens that were formerly (prior to about 1985) stipulated for donors (sires as well as dams) and herds of origin in the exporting country, and the quarantines sometimes used for recipients and embryo transfer offspring in the importing country, can be avoided. The principles of the OIE/IETS protocols, as above, were incorporated into the EU Council Directive 89/556/EEC [1], and EU Commission Decision 92/471/EEC [2], which regulate intra-Community trade in bovine embryos, and their importation from Third Countries. It is emphasised that the evidence for low risks associated with embryo movement applies primarily to in vivo-derived embryos, but, as explained by Bielanski in Chapter 3 of the IETS Manual [8], the evidence for low risk is less secure for IVF embryos. However, the EU Directive and EU Decision, referred to above, do not appear to differentiate between the risks associated with the two types of embryo. With regard to semen that is used to produce embryos for international trade, in the majority of cases this is selected from batches of frozen semen from bulls located in accredited AI Centres. Such bulls are normally certified negative for acute, epidemic diseases such as foot-and-mouth disease, and also for chronic diseases such as brucellosis, tuberculosis, leptospirosis, campylobacteriosis, trichomoniasis. An increasing number of AI Centres also ensure their bulls are certified negative for enzootic bovine leukosis, infectious bovine rhinotracheitis, and sometimes also for bovine viral diarrhoea.

3. Evidence that viruses can attach to or be integrated into spermatozoa and carried into the oocyte at fertilisation Brackett et al. [10] found that when rabbit spermatozoa were exposed in vitro to DNA of the polyomavirus; simian virus 40 (SV40), not only was the viral DNA adsorbed onto the sperm but also, when the sperm were used for in vitro fertilisation, it was transmitted into the rabbit oocyte. In later work, Nussbaum et al. [11] exposed bull spermatozoa to three different viruses: Sendai virus, influenza virus and Semliki Forest virus, all of which are typical enveloped viruses, but are not natural bovine pathogens. In contrast to that of Brackett’s study, these were whole, biologically active viruses, and, when appropriate pH adjustments were made, all three were shown (by fluorescent labelling) to have fused with the sperm cells. Fusion did not occur, however, when the viral envelope was removed by

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neuraminidase treatment, which led Nussbaum et al. to conclude that sperm may readily serve as carriers of viral genes, especially those of enveloped viruses such as herpesviruses and exogenous retroviruses whose fusion activity is expressed at pH 7.4. The conclusion of Nussbaum et al. [11] seemed to have been fulfilled when Baccetti et al. [12] demonstrated that the retrovirus; human immunodeficiency virus type 1 (HIV-1; an enveloped RNA virus), could sometimes bind to and enter human spermatozoa both in vitro and in vivo, the latter being the case in sperm of patients with acquired immunodeficiency syndrome—AIDs. By application of electron microscopy and in situ hybridisation (DNA probe) techniques, Baccetti et al. also claimed to have demonstrated virus particles in zygotes and blastocysts that were morphologically similar to those seen in the spermatozoa. They concluded that the virus had been carried by infected sperm into oocytes at fertilisation. It should be noted, however, that these findings by Baccetti et al. [12] are disputed by other workers [13–15] who believe that HIV-1 occurs in seminal lymphocytes and macrophages rather than within the spermatozoa. Another human virus of concern by reason of its carriage by spermatozoa is hepatitis B virus (HBV), a DNA virus, which in naturally occurring infections can be carried within the sperm chromatin [16,17]. Ali et al. [18] have also reported that when motile human spermatozoa carrying HBV DNA were cultured in vitro with zona pellucida-free hamster oocytes the latter underwent fertilisation and early embryonic development. They then used fluorescence in situ hybridisation (FISH) to show that the HBV had integrated into the pronucleus, nucleus and chromosomes of these early embryos. Dot hybridisation, polymerase chain reaction (PCR) tests and other techniques revealed that the viral DNA was also being expressed at the one and two cell embryo stages. These results suggest that HBV-infected spermatozoa can carry the virus into the oocyte and might thereby lead to vertical transmission of the virus to the next generation. Yet another virus which appears to have the capacity to enter into spermatozoa is the human papilloma virus (HPV). Chan [19], for example, reported not only that exogenous DNA derived from HPV was actively taken up by mouse spermatozoa but the DNA-carrying sperm were also able to migrate into and transfect mouse blastocysts. The UK Human Fertilisation and Embryology Authority requires all sperm donors to be screened for HIV, and for hepatitis B and C viruses, and their semen to be quarantined for 6 months before use for AI [20]. This screening enables measures to be taken to reduce the risk of virus transmission by AI to the partner, fetus and newborn baby. Safety measures are also needed especially when the semen is intended for in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). Processing semen by centrifugation through Percoll differential density gradients, or through Sephadex columns has been used to separate the motile spermatozoa from other semen components, and has thereby successfully reduced or eliminated the viral load in semen from HIV-positive men for use in IVF and ICSI. Other methods shown to be effective for virus removal from semen that has been experimentally ‘spiked’ with viruses include the use of trypsin washes [21], and passage through density gradients incorporating trypsin [22,23]. Semen that has been ‘cleaned-up’ by such methods has been used in human assisted reproduction techniques without leading to HIV seroconversion of either mother or child [24,25]. From the above it would appear that the semen ‘clean-up’ techniques being applied in humans could provide valuable risk-reduction measures in domestic livestock also, but

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they may not be effective in all circumstances. For example, although they might remove viruses from semen samples that have been experimentally ‘spiked’, they might not work with naturally occurring infections. In the latter cases the viral DNA or RNA may be attached to the surface of the sperm or be integrated into the sperm chromatin, but this is less likely to occur in artificially ‘spiked’ semen. Unpublished work by G.R. Holyoak (personal communication) suggests that equine arteritis virus (EAV), a highly contagious, enveloped RNA virus that can be transmitted venereally in horses, becomes tightly bound to the sperm membrane in naturally infected stallions. Thus, whereas Holyoak and his colleagues were able to remove EAV from experimentally ‘spiked’ semen samples by Percoll differential gradient centrifugation, Sephadex column separation, and even by simple washing, these methods did not remove the virus from semen collected from a naturally infected stallion.

4. The situation in domestic livestock, including cattle Many pathogens have been reported to occur in the semen of cattle and other livestock [26–28] but in most cases they have been found to be free in the seminal plasma, or in the cellular constituents of semen other than spermatozoa. Bovine immunodeficiency virus (BIV), for example, a retrovirus closely related to HIV (see above), was reported by Nash et al. [29] to occur in commercially processed bovine semen but in this case the virus was demonstrated in seminal leukocytes only, and not within the spermatozoa themselves. In other studies, Gradil et al. [30] and Burger et al. [31] were unable to demonstrate BIV in either spermatozoa or in seminal leukocytes from seropositive bulls in the USA. Moreover, when Bielanski et al. [32] exposed zona pellucida-intact oocytes to BIV during maturation, and also during IVF with semen that had been artificially ‘spiked’ with BIV, the resulting embryos, when tested with a nested-PCR assay, were found to be negative for BIV. Catastrophic pathogens such as foot-and-mouth disease (FMD) virus that can occur in bull semen pose a major threat if the semen is used [33]. However, in most such cases, the short incubation period, clear clinical expression of disease, and the mandatory postcollection cryostorage of semen whilst bulls are tested and/or observed for clinical signs, effectively overcomes the risks of international transmission of these kinds of pathogen via semen, so they are not considered here. Other important pathogens which can sometimes occur in bull semen include four viruses that are of special significance for international trade: enzootic bovine leukosis virus (EBLV); bovine herpesvirus-1 (BoHV-1) (also called infectious bovine rhinotracheitis/infectious pustular vulvovaginitis virus); bovine viral diarrhoea virus (BVDV), and bluetongue virus (BTV). These pathogens often cause subclinical infections, so it is sometimes possible for high value bulls to become infected yet still to be used as semen donors for AI or for natural service. When it is wished to export the semen, or to export embryos that have been fertilised with such semen, problems can arise, as exemplified by the new legislation from the EU Commission. In view of the fact that some countries in the EU have eradication programmes, or measures to exclude these four diseases, such legislation is not surprising. The four diseases will now be considered in more detail.

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5. Enzootic bovine leukosis virus (EBLV) Enzootic bovine leucosis (EBLV) (syn. bovine leukaemia) is a Deltaretrovirus, of family Retroviridae, which primarily infects B-lymphocytes. Although seldom causing clinical manifestations, infection does produce a persistent lymphocytosis which may eventually result in lymphatic tumours in adult cattle. Cattle that are seropositive to BLV can generally be said to be virus-positive also. The infection is widely distributed in cattle populations across the world, although some countries (e.g. Denmark) have eradicated it, and other countries have programmes to achieve this. Transmission of EBLV between animals is primarily by the transfer of blood lymphocytes which can occur by a variety of routes. For example, it can occur transplacentally during pregnancy, by feeding contaminated colostrum or milk, by injections where single needles are shared between multiple animals, and by surgical procedures where instruments are not sterilised after use. The virus has been found in semen only rarely [34] and when it does occur it is probably associated with leakage of EBLV-infected lymphocytes into the genital tract, and it is not incorporated within the spermatozoa [26]. While there is substantial evidence that BLV is rarely if ever transmitted by AI, transmission as a result of inoculating infected lymphocytes into the uterus of cows has been reported by Van Der Maaten and Miller [35]. Much work has been done to ascertain whether the EBLV is liable to be transmitted by embryo transfer, but all the evidence indicates otherwise. As documented in Appendix B of the IETS Manual [8] several hundreds of in vivo-derived, zona pellucida-intact embryos collected from EBLV-seropositive (i.e. infected) dams, which in some cases were inseminated with semen from EBLV-seropositive bulls, have been transferred into seronegative recipients without resulting in transmission of the virus. None of the recipients seroconverted and all the calves remained seronegative. In most experiments the embryos were washed and processed using the IETS Manual protocols although in one instance involving 1306 fresh or frozen-thawed, in vivo-derived embryos, approximately 20% of which had damaged zonae pellucidae, the embryos were not specifically washed before being transferred from donors in North America to EBLV-seronegative recipients in France [36]. While many of the donor cows, and possibly some of the bulls, were thought to have been BLV-seropositive, the exact level of infection was unknown because importation protocols did not require prior testing for the disease. All of the recipients in France remained seronegative. Based on all these negative transmission data, EBLV is listed by the IETS in Category 1, i.e. it is a ‘‘. . . disease agent for which there is sufficient evidence to show that the risk of transmission is negligible provided the embryos are properly handled between collection and transfer’’—see Appendix 3.3.5 of the Terrestrial Animal Health Code [9]. Other studies have involved EBLV exposure during in vitro production of embryos. When 170 matured oocytes were artificially exposed to semen to which a cell-free suspension of EBLV had been added, Bielanski et al. [37] reported that none of the resulting embryos tested virus-positive after washing. The risk of such embryos being infected with EBLV, even if they are obtained from oocytes fertilised in vitro by semen from infected bulls, is therefore likely to be very small.

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6. Bovine herpesvirus-1 (BoHV-1) BoHV-1 is classed as an a-herpesvirus. In cattle it causes a variety of economically important respiratory and reproductive problems, the latter including vulvovaginitis, endometritis and abortion in females, and balanoposthitis in males. In the bull the virus often replicates initially in the mucosae of the prepuce, penis and urethra, and semen is probably contaminated by virus shedding from the infected mucosae during ejaculation. Subsequent to primary infection by BoHV-1, including infection by attenuated live vaccine strains, the virus moves to the cranial or spinal ganglia of the host where it remains latent for life, so clinically normal animals can be virus carriers. Factors such as stress can lead to recrudescence of infection from the ganglia, and thus the virus may once again be excreted, including in the semen. In the latter case excretion of virus can be with or without a serum antibody response [38]. Although infected bulls must be regarded as potentially lifelong excretors of virus in their semen, studies in North America have suggested that semen from BoHV-1 seropositive bulls can be free of virus for long periods of time if the bulls are well managed in a low-stress environment [27]. Conversely, Kupferschmeid et al. [39] reported seroconversion of cattle in Switzerland after AI with imported semen from a donor bull that was seronegative at the time of semen collection. Semen samples from this bull initially tested negative for the virus in cell culture but they were positive in a ‘modified Cornell semen test’, so the authors concluded that some laboratory methods are insufficiently reliable to exclude presence of BoHV-1. The fact that Switzerland was undergoing BoHV-1 eradication when the contaminated semen was imported highlights the need to ensure that semen is free from the virus. In addition to Switzerland, several other European countries, including Austria, Denmark, Finland, Norway and Sweden, have eradicated the disease, and some others have schemes in progress to achieve freedom [40]. Measures to satisfy importing countries that semen is free from BoHV-1 are given in Chapter 2.3.5 of the OIE Terrestrial Animal Health Code [9]. For frozen semen they include certification that the donor bull was kept in a BoHV-1-free herd or AI Centre at the time of collection, or that the bull was held in isolation during the period of collection and 30 days following collection, and was blood tested for antibody to BoHV-1 with negative results at least 21 days after the last collection of semen for the export consignment. Where the serological status of the donor bull is positive, or unknown, an aliquot of semen from each collection should be subjected to virus isolation testing, with negative results. Whether BoHV-1 infection becomes established after a cow is inseminated with contaminated semen depends on the properties of the virus strain and the amount of virus in the semen straw. The same principle applies with embryo transfer. Some BoHV-1 strains appear to have more affinity for the reproductive tract than others [41]. Studies in Australia showed that when cows were inseminated with doses higher than 105.3 TCID50 they invariably became infected [42,43] but only 6 of 25 cows seroconverted with doses below 200 TCID50 [44]. Since semen contamination is thought to arise from virus shedding during ejaculation it is generally believed the virus is in the seminal plasma rather than the spermatozoa, and Van Engelenburg et al. [45] used a PCR-based assay to confirm this. Nevertheless, in an earlier study, Elazhary et al. [46] claimed to have detected virus antigen in or on the area of

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the post-nuclear cap of the spermatozoa when they used fluorescent antibody techniques to examine semen from a naturally infected bull. The latter finding prompted Hare [47] to comment: ‘‘. . . in which case, theoretically, BoHV-1 could be carried into the ovum at fertilisation’’. However, as Van Oirschot [38] pointed out, this matter remains controversial because while Elazhary et al. demonstrated virus antigen in association with spermatozoa they did not show conclusively whether it was within them or attached to their surfaces. Several studies have been carried out in which in vivo-derived, zona pellucida-intact embryos have been collected from BoHV-1 infected donor cows approximately 7 days after fertilisation. Some of the cows had been experimentally infected by intrauterine or intranasal inoculation at or close to the time of fertilisation while others were naturally infected or seropositive embryo donors. The collected embryos were either tested for the virus in vitro or transferred into seronegative recipients. In other instances in vivo-derived embryos were exposed to BoHV-1 in culture, washed with or without trypsin washes (as per IETS Manual protocols), then assayed for presence of the virus. In all these studies, provided that trypsin washes were included in the protocol, the results, so far as presence of virus on the embryos, or transmission to recipients and their offspring, were concerned, were negative—see Appendix B of the IETS Manual [8]. In most cases embryos were washed in the manner recommended in the IETS Manual, but in a large trial reported by Thibier and Nibart [36], 1306 fresh or frozen-thawed, in vivo-derived embryos, approximately 20% of which had damaged zonae pellucidae, were transferred without washing or trypsin treatment from donors in North America to recipients in France that were BoHV-1 seronegative. In their report the authors claimed that approximately 95% of the donor cows were seropositive to BoHV-1 at the time of embryo collection but, despite this, none of the recipients in France seroconverted. The percentage, if any, of semen donors (i.e. bulls) that were seropositive was not stated. In consequence of the many negative transmission studies, BoHV-1 has been listed in IETS Category 1, i.e. it is a ‘‘. . . disease agent for which there is sufficient evidence to show that the risk of transmission is negligible provided that the embryos are properly handled between collection and transfer’’—see Appendix 3.3.5 of the OIE Terrestrial Animal Health Code [9]. In the case of BoHV-1, proper handling of the embryos must include trypsin treatment (see above). At this juncture it should be noted that in only one study [48] of the many that have been done were the embryos collected specifically from cows that had been inseminated with semen containing BoHV-1. Two donor cows were involved, each having been inseminated with ‘spiked’ (not naturally infected) semen. Since one of the two cows yielded no embryos, and the other had just two undeveloped (but virus-free) embryos, the results of this study are of limited relevance. Other experiments to evaluate the risks of BoHV-1 transmission have been done with in vitro fertilised (IVF) embryos and have included the use of infected semen and/or exposure of oocytes to the virus at the time of fertilisation. Guerin et al. [49] studied the effect of BoHV-1 on groups of oocytes that were exposed to the virus during maturation and fertilisation, then washed 10 times before being tested for presence of virus. The virus appeared to have no effect on oocyte maturation but significantly (P < 0.01) reduced the IVF rate to 65%, compared to 85% in controls. It also led to an increased level of sperm decondensation abnormalities (49% as compared to 4% in controls: P < 0.001). The

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authors concluded that BoHV-1 was not only absorbed onto the gametes but it also impaired their ability to undergo IVF, possibly due to an effect on sperm penetration or an interaction with the intracellular fusion mechanisms. When Bielanski and Dubuc [50] added 106 TCID50/mL BoHV-1 to the embryo production system at fertilisation they saw only a small trend towards retarded embryonic development but were able to isolate the virus from all the embryos despite the fact that their post-culture processing included more than the IETS Manual recommended number of washes. In a later experiment Bielanski and Dubuc [51] used oocytes from both experimentally and naturally infected donor cows and found again that the IVF systems became infected, as also did the embryos. The rate of embryo development and the proportion of morphologically normal (transferable blastocysts) produced in the infected IVF system were significantly reduced in this instance. Moreover, despite washing and trypsin treatment to IETS-recommended standards, the blastocysts produced were shown to be infected and probably had the potential for disease transmission. After further studies Bielanski et al. [52] concluded that, compared with in vivo-derived embryos, in vitro-produced embryos have a greater propensity to carry BoHV-1 after experimental exposure to the virus, and are more difficult to disinfect by means of the trypsin treatment protocol recommended in the IETS Manual [8]. In another study, D’Angelo et al. [53] exposed ‘‘8 or 9 day’’ in vitro-produced embryos to BoHV-1, then assayed them by virus isolation, and they too found that neither washing, nor washing in conjunction with trypsin, were effective for the complete removal of virus from the embryos. Likewise Edens et al. [54] found that an infective dose of BoHV-1 remained associated with individual day-7 embryos that had been exposed to the virus in vitro, then washed and transferred individually onto cultures of susceptible oviductal cells. However, when individual embryos were not only washed with culture medium but also passed through two trypsin washes prior to their being co-cultured with oviductal cells there was no transmission of virus. These authors [54] concluded that washing plus trypsin treatment of the embryos would probably prevent BoHV-1 transmission if they were to be transferred individually into the uterus of susceptible recipients. Studies to elucidate how BoHV-1 interacts with spermatozoa, and mechanisms whereby it interferes with fertilisation of oocytes, have been made by Vanroose and colleagues in Belgium. They applied immunofluorescence techniques to locate the viral antigen in zona pellucida-intact and zona pellucida-free embryos exposed to BoHV-1 at different stages of development and showed viral replication in a subset of cells in the zona pellucida-free, morula-stage embryos [55]. The zona pellucida-intact embryos were protected from infection but hatched blastocysts exposed to BoHV-1 expressed viral antigen in approximately 13% of their cells and these blastocysts were seen to become degenerate [56]. In another study, Vanroose et al. [57] found that rates of cleavage and blastocyst formation were significantly reduced by exposure to the virus during the IVF stage of in vitro embryo production. To explore this further the same authors studied the effect on cumulus-free, zona pellucida-intact oocytes of the presence of different titres of BoHV-1 during IVF with different numbers of spermatozoa. To ascertain if fertilisation was completed, the presumed zygotes were fixed and stained 24 h post-IVF, and the numbers of sperm bound to the zona of each zygote were counted. The results showed that presence of

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increasing amounts of the virus during IVF led to significant and proportional reductions in the numbers of sperm attached to the zona. Fertilisation rates were found to increase in proportion to the number of sperm cells used, but as the virus titres were increased so fertilisation rates were also reduced. In further studies Vanroose et al. [58] used spermatozoa for the IVF process that had previously been incubated with BoHV-1, and dilutions of four different monoclonal antibodies known to neutralise the virus were added in different experimental groups. As before, the numbers of sperm attached to each presumed zygote were assessed by fixation and staining 24 h after IVF. In this experiment they found that with sperm previously exposed to BoHV-1, but without addition of monoclonal antibody, there was a 60% reduction in numbers of sperm bound to the zona as compared to the numbers in a control (sperm without virus exposure) group. In addition they found that two of the four monoclonal antibodies (anti-gC and anti-gD) interfered with the inhibition of sperm-zona binding that was caused by BoHV-1. The authors suggested that molecules on the surface of the sperm cell plasma membrane may act as receptors for the BoHV-1 glycoproteins gC and gD, and, when sperm have viruses attached to their surface in this way, their ability to attach to the zona pellucida is impaired. The same group of workers followed this up by showing that monoclonal antibody directed against gC completely stopped the inhibition of sperm–oocyte binding by BoHV-1 whereas the monoclonal directed against gD only partially prevented the inhibitory effect [59]. Thus it would appear that glycoproteins gC and gD are important for the binding of sperm cells to oocytes. Although it is evident from these experiments that BoHV-1, if present in semen, has a tendency to bind onto the plasma membrane of the spermatozoa and to inhibit their ability to fertilise oocytes, there is no suggestion that infected sperm might penetrate through the zona pellucida and thereby lead to infected embryos. A more plausible risk scenario is that some of the normal sperm in a batch of BoHV-1 infected semen might fertilise the oocytes whilst other, virus-infected, sperm might be passively carried onto or into the surface of the zonae. In this case it should be possible to treat such embryos with trypsin, or some other effective anti-viral substance, to render the zona pellucida free from infective virus. This is the basis for the trypsin washing protocol that is advocated in the IETS Manual [8] for processing embryos prior to their transfer (see above). Alternatively it might be possible inactivate the virus when semen is known or suspected to contain BoHV-1 by treating the semen before it is used for AI in the case of in vivo-derived embryos, or prior to its use for IVF in the case of in vitro-produced embryos. It has been shown by Schultz et al. [60] that treatment of BoHV-1-infected semen with gamma globulins from hyperimmune serum can neutralise the virus and reduce the risk of viral transmission via AI without affecting fertility. In the future, specific monoclonal antibody (such as anti-gC; see above) might also be tried to achieve this. Another method to ‘clean-up’ BoHV-1-infected semen was advocated by Bielanski et al. [61] who found that virus titres of 103 and 104 TCID50 could be removed by using a 0.3% trypsin solution. The fertilising capacity of such treated semen was reported by Bielanski [62] to be unaffected, but results of a study by Silva et al. [21] disagreed. The latter indicated that while trypsin concentrations at or above 0.3% were effective against the virus, they did reduce semen fertility, decreasing motility of the spermatozoa, and damaging their plasma membranes. Consequently Silva et al. [21] recommended a maximum concentration of 0.25% trypsin

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be used, and this only if the concentration and motility of sperm in the ejaculate was high before freezing. If it is wished to treat very small amounts of semen, for example prior to its use for IVF, then more specialised techniques, such as the trypsin density gradients advocated for virus removal by Loskutoff et al. [22,23], may be appropriate. However, in the long term, the goal should be to establish AI Centres in which all bulls are BoHV-1 negative [63]. Methods to achieve disease-free bull studs in AI Centres are described by Howard and Pace [64] and in Appendix 3.2.1 of the OIE Terrestrial Animal Health Code [9].

7. Bovine viral diarrhoea virus (BVDV) A detailed review of the risks of BVDV transmission by in vivo-derived and in vitroproduced bovine embryos has been published recently by Stringfellow et al. [65], so only those aspects of particularly relevance to risks of infected semen being involved in the transmission of BVDV to embryos will be covered here. The genus Pestivirus comprises three single stranded, small, enveloped, RNA viruses: BVDV, classical swine fever virus (CSFV) and border disease virus (BDV) of sheep. Two genetically distinct genotypes (I and II) and two biotypes (cytopathic and non-cytopathic) of BVDV are observed [66]. The virus is endemic in most countries of the world, although some (e.g. in Scandinavia) have eradication programmes for it [67]. Exposure of cattle to BVDV may result in either an acute, transient (often subclinical) infection, or a persistent infection, the latter being initiated by transplacental infection of the developing fetus in early (prior to about 125 days) pregnancy. In the latter case the calf may survive through birth and develop normally to adulthood, but is immunotolerant to the strain of BVDV involved, and can be persistently or intermittently viraemic without seroconversion. Such individuals are usually identified by virological testing of two blood samples collected 4 weeks apart [27]. The samples should be taken when such cattle are at least 9 months old, after their colostral antibody to BVDV has waned. With regard to the presence of virus in semen, acute transitory BVDV infection in adult bulls produces a brief viraemia followed by seroconversion, and virus can often be demonstrated in their semen for up to about 14 days by virus isolation testing [68]. Congenitally infected (viraemic and immunotolerant) bulls, on the other hand, may excrete the virus in semen permanently [69]. A third, rare phenomenon occurs where bulls, as was the case with one named ‘Cumulus’ in New Zealand [70], may shed infectious virus in semen for prolonged periods without demonstrating viraemia, but with a constant very high serum antibody level. The latter suggests that such bulls undergo repeated viral challenge. Post mortem examination of the bull ‘Cumulus’ revealed testicular BVDV infection which, it was postulated, may have originated at about the time of puberty. The implication is that, once established in the testis, the virus would be protected from serum antibody by the blood–testis barrier, but leakage of viral antigen back across the barrier would lead to hyperimmunisation of the bull. There have been several studies of the presence of BVDV infection in the semen of both acutely infected and persistently infected bulls. Paton et al. [68], in addition to reporting presence of the virus in semen samples from acutely infected bulls, also reported a marked

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deterioration in their semen quality, with reduced sperm density and motility, and an increase in sperm abnormalities. Kirkland et al. [71] found virus in the semen of three of five acutely infected bulls but, in contrast to Paton’s findings, there was no obvious effect on semen quality. Revell et al. [72] studied the semen from two persistently infected bulls and found quality was poor in both cases, and there were gross abnormalities, especially collapsed sperm heads, in the semen of one of the bulls. Howard et al. [73], who studied 12 persistently infected bulls, found that the presence of virus in their semen was not always accompanied by diminished semen quality, and Afshar and Eaglesome [26] believed that the abnormalities seen by Revell et al. were unlikely to have been linked to the BVDV infection but, instead, may have been a result of individual bull effects. Givens et al. recently carried out additional studies on BVDV excretion in semen following acute infection of post-pubertal bulls [74,75]. Serum and semen samples were collected at intervals over a 7-month period from three seronegative bulls after they had been inoculated with BVDV when 2 years old. The samples were tested by both virus isolation and reverse transcriptase-nested polymerase chain reaction (RT-nPCR) tests. Testicular biopsies were taken at the end of the 7 months and these were tested by the same two methods and also by immunohistochemistry. Virus isolation and RT-nPCR tests on serum showed a transient viraemia lasting up to 17 days post-inoculation in all three bulls, and excretion of infectious BVDV in semen was shown by virus isolation tests to have occurred over a similar period in two of the bulls. Using the more sensitive RT-nPCR method, however, presence of virus was demonstrated in the semen of these two bulls for up to 7 months. Givens et al. [74] acknowledged that PCR tests will detect non-infectious as well as infectious virus, so confirmation of such long-term excretion of infectious virus in semen would require positive virus isolation tests. As mentioned, the latest time the semen was found positive by virus isolation in their study was 17 days post-inoculation. However, when raw semen that had been collected 5 months post-inoculation from one of the two bulls was injected intravenously into a 6-month-old seronegative calf, this calf did seroconvert to BVDV, and the same virus strain was later isolated from it. This indicates that infectious virus was present in the semen of this bull 5 months after inoculation. In addition to evidence from the calf, testing of the testicular biopsies taken at 7 months postinoculation gave positive results with RT-nPCR and immunohistochemistry in the two bulls with virus-positive semen samples, and in one of them infectious virus was also isolated from the testis, showing that persistent testicular infection with BVDV had become established in these bulls. Viral antigen was detected within the seminiferous tubules of the testes, adjacent to the basement membrane. Chronic inflammatory lesions were also present [74]. In contrast to the New Zealand bull, ‘Cumulus’, which had very high serum antibody levels and prolonged presence of infectious BVDV in semen [70], antibody titres in the bulls of Givens et al. [74] were not particularly high, perhaps indicating that severity of testicular infection in the former was greater than in the latter. The prevalence of bulls such as these which shed BVDV in their semen for long periods following acute infection is unknown but data reviewed by Givens and Waldrop [75] and additional data provided by Gaede et al. [76] indicate that it is very low. Experimental studies by Meyling and Jensen [69] and field studies by McGowan et al. [77] have shown that heifers exposed to BVDV at or around the time of AI may have

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significantly reduced conception rates and increased abortion rates. However, when the virus is present in semen of acutely or persistently infected bulls, it does not appear to have been determined whether it is within the spermatozoa, free within the seminal plasma or within the non-sperm cellular components. Radostits and Littlejohns [78] and Brownlie [79] have suggested that the virus occurs mainly within lymphocytes but little seems to have been done to confirm this. In persistently infected cattle it is debatable whether BVDV can occur within the gametes as a result of a gonadal infection in prenatal life. While the situation in cattle is unclear, when Choi and Chae [80] infected year-old boars experimentally with CSFV, a virus closely related to BVDV, post mortem examination of testicular tissue by in situ hybridisation showed CSFV nucleic acid localised in spermatogonia, spermatocytes and spermatids. This suggests, at least in that disease, that the spermatozoa themselves might sometimes carry the virus. Using similar techniques the same authors [81] demonstrated CSFV in the ovaries of experimentally infected sows, but in this case the viral nucleic acid was located almost exclusively in the cytoplasm of macrophages rather than in the oocytes. Some years ago, Gardiner [82] reported the presence of BDV in ‘gonocytes and oocytes’ of fetuses and lambs from sheep that had been exposed to the infection during pregnancy; however, this could not be confirmed in similar studies by Waldvogel et al. [83]. More recently, Brownlie et al. [84], and Fray et al. [85] examined ovarian follicles from persistently infected cows for the presence of BVDV. Brownlie et al. used both immunohistochemistry and an in situ hybridisation technique and with these they detected viral antigen in over 6% of oocytes and viral nucleic acid in 18% of the follicles. Fray et al., on the other hand, used an indirect immunofluorescence assay with monoclonal antibodies to study cryostat sections of ovaries, and found 18.7% of 1939 oocytes examined contained BVDV antigen. The developmental competence of these infected oocytes could not be determined, but the authors of these two papers suggested that, if they were viable at ovulation, and if fertilised and subsequently transferred, irrespective of whether or not they were washed as advocated by the IETS, they might be capable of transmitting BVDV to recipient cows and their offspring. Other evidence relevant to whether BVDV can occur within oocytes has come from attempts to collect and transfer embryos from persistently infected heifers inseminated with BVDV-free semen. These attempts have had only limited success, mainly because such heifers respond poorly to superovulation, but in four cases where embryos were recovered, washed and transferred into recipients, a total of five normal, uninfected calves have been born [86–89]. Moreover, when seronegative recipients were used, these did not seroconvert to BVDV after embryo transfer, which indicates that washed embryos from persistently infected animals do not necessarily contain an infective dose of BVDV within their cells or on the zona pellucida. Studies were done in the early 1980s by Singh et al. [90] to ascertain whether, after exposing in vivo-derived, zona pellucida-intact bovine embryos in vitro to cytopathic strains of BVDV, then washing them 10 times, had detectable, infectious virus associated with them. The results of these studies were negative and consequently BVDV was listed in IETS Category 3 (Appendix 3.3.5 of the OIE Terrestrial Animal Health Code [9]), i.e. a ‘‘. . . disease agent for which preliminary evidence indicates that the risk of transmission is negligible provided that the embryos are properly handled between collection and transfer,

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but for which additional in vitro and in vivo experimental data are required to substantiate the preliminary findings’’. Little further information about transmission of BVDV via in vivo-derived embryos was obtained until Waldrop et al. [91] evaluated the IETS washing procedure for removal of two non-cytopathic strains of BVDV. In essence they exposed 93 zona pellucida-intact, in vivo-derived embryos from seronegative cows to BVDV strain SD-1 (genotype 1), and 92 similar embryos to BVDV strain CD-87 (genotype 2). Following in vitro exposure to these viruses the embryos were washed (without trypsin), sonicated then tested by virus isolation and by the reverse transcriptase-nested PCR. In contrast to the results obtained by Singh et al. [90] with their cytopathic BVDV strains, Waldrop et al. [91] found that a quarter to a third of the embryos exposed to strain SD-1 still had virus attached after washing, while none of those exposed to CD-87 had attached virus. Thus it seems that the IETS washing procedures are more effective for removing some BVDV strains than others. Further research by Waldrop et al. [92] has shown that even when trypsin treatment was applied in addition to the 10 washes, embryos exposed to high affinity BVDV strains retained a low level of viral contamination. The same group of workers [93] exposed some further in vivoderived embryos to BVDV, washed and in some cases trypsin treated them, sonicated them, then injected the sonicated material from individual embryos intravenously into seronegative calves. Seroconversion occurred in 38 and 13%, respectively, of the calves injected with washed sonicates and washed plus trypsin-treated sonicates. Thus, while trypsin treatment was beneficial, it was not completely effective for removing the BVDV from the embryos. Schlafer et al. [48] inseminated two superovulated cows during oestrus with semen which had been ‘seeded’ with a cytopathic strain of BVDV, but viable embryos were not recovered from either cow for meaningful studies. Otherwise it seems that no experiments have been performed in which in vivo-derived embryos have been collected from seronegative heifers or cows inseminated with semen that was naturally or experimentally infected with BVDV. This is a significant gap in the context of the present paper since information is needed as to whether such semen would transmit the virus to the embryos, and, if so, whether the virus would be removed by the IETS washing protocols. With regard to in vitro-produced embryos, the effect of BVDV infection at the time of IVF has been the focus of several experimental studies. In France, Guerin et al. [94] found that fertilisation and embryo cleavage rates were significantly reduced when semen from a persistently infected bull was used to inseminate oocytes, and development to the blastocyst stage was only 2.1% compared to 19.6% with uninfected, control bull semen. In a subsequent study [95] there were three groups of in vitro-matured oocytes: the first group was exposed to semen from a persistently infected bull (positive control); the second was exposed to the same semen after it had been treated during the ‘swim-up’ phase with a specific anti-BVDV immunoglobulin, and the third group was exposed to semen from a BVDV-free bull (negative control). Proportions of oocytes developing to the blastocyst stage in the three groups were 4.0, 8.2 and 13.9%, respectively. The authors suggested that the low rate of embryo development in the first group was a specific effect of the virus, but they also claimed that the marginally reduced rate in the second group in which the virus was neutralised, may have been due to poor semen quality in the persistently infected bull. In contrast to these results from Guerin’s group [94,95], a study by Bielanski and Loewen

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[96] in Canada found that when a non-cytopathic BVDV strain (105 TCID50/mL) was used to ‘spike’ the semen from an uninfected bull prior to IVF, 22% of embryos developed to blastocysts. Moreover, when they used samples of semen from three persistently infected bulls to produce embryos by IVF, the proportions developing to blastocysts were 13, 17 and 20%. It is unclear why the results from the French and the Canadian workers differed, but one possibility is that the different rates of embryo development were due to individual bull fertility effects and were unrelated to persistent BVDV infection. Other studies [97–100] have shown that the presence of non-cytopathic BVDV in culture systems for most if not all stages of in vitro production of bovine embryos has little if any effect on their development. There is now a considerable amount of evidence to show that removal of virus from in vitro-produced embryos exposed to BVDV, even when the zona pellucida is intact, can be more difficult than it is with in vivo-derived embryos. For example Trachte et al. [101] found that, after artificial exposure to either cytopathic or non-cytopathic strains of BVDV, neither the standard IETS washing protocol nor washing followed by trypsin treatment effectively removed the virus. Similar findings were reported by Bielanski and Jordan [102]. Attempts were made by Bielanski et al. [103] to remove BVDV from semen samples from persistently infected bulls. The samples, which contained 105 to 106 TCID50 virus/ mL semen, were subjected to a variety of the treatments commonly used prior to IVF, i.e. washing, swim-up, Percoll gradients, glass wool filtration, and glass bead filtration. The final pellets of sperm were then tested by the immunoperoxidase technique. Although in some cases there were up to two log reductions in virus titres, these physical methods did not remove the BVDV completely so a possibility remains that some of the virus might have been attached to (or within) the sperm. Other techniques such as exposing infected semen to specific immunoglobulins [95], or to antiviral compounds active against a range of RNA viruses [104], or processing it through trypsin density gradients as advocated by Loskutoff et al. [22,23] might be more effective for removing BVDV, but the long-term goal should be to establish AI Centres in which all the animals are tested and maintained free from BVDV infection. Since in many cattle populations the virus is still almost ubiquitous, the safest policy in these circumstances would be to test and exclude persistently infected animals and to ensure that all others have experienced active infection prior to onset of sexual maturity and are consistently seropositive to BVDV before entry to the AI Centre. To exclude the possibility of virus-containing semen from transiently infected bulls being used for AI, the screening of semen samples from suspect bulls by the reverse transcription PCR appears to be an appropriately sensitive method, although positive results should be confirmed by virus isolation [75,76]. Research is needed to determine whether BVDV vaccines could be used and whether vaccinated bulls are ever liable to excrete virus in their semen afterwards.

8. Bluetongue virus (BTV) BTV is a the type species of the Orbivirus genus, family Reoviridae, and at least 24 different serotypes of BTV have been identified. A considerable degree of cross reaction

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exists between the BTV serogroup and the epizootic haemorrhagic disease (of deer) serogroup. The virus is naturally transmitted by just a small number of the many species of midges (Culicoides spp.), and cattle are probably the major host, although infected cattle rarely if ever show clinical signs [105]. Other hosts include sheep and deer, which in endemic areas (very approximately between latitudes 408N and 358S) tend to have a natural resistance, but elsewhere they can experience devastating disease epidemics. Infection of a susceptible animal is followed by a significant viraemia which (assuming the animal survives) lasts for about 10 days, although low levels of BTV associated with red blood cells can occasionally be detected for up to 100 days [106]. Very little virus is found in the secretions and excretions of infected animals and the disease is not normally regarded as contagious by the oral route, or via aerosols. Although BTV can be demonstrated sporadically in the semen of viraemic bulls [107,108] its presence there is probably associated with traces of virus-infected blood that have leaked from the genital tract, and once the viraemia ceases the virus is no longer found in semen. Ensuring that semen does not contain BTV, especially when donor bulls reside in areas where virus vectors are seasonally active, or active all year round, is difficult. If semen is to be exported, especially to countries where BTV does not occur, it is usual for those countries to expect it to come from bulls kept in a BTV-free country or zone for at least 100 days prior to, and during, collection of the semen, or from bulls that are serologically negative when tested between 28 and 60 days after the last collection. Where vectors are present all year round it is recommended that donor bulls be protected from Culicoides (which is not easy) for at least 100 days before commencement of, and during, semen collection. Alternatively, if such bulls are serologically positive, they should have virus isolation tests, or PCR tests done on blood samples taken at the start of, and conclusion of, and at least every 7 days (for virus isolation) or at least every 28 days (for PCR testing) during semen collections to confirm the absence of virus. In zones where BTV occurs seasonally, semen may be approved for export if it is collected at least 100 days before commencement of the season, or blood tests for serum antibodies, or for viraemia, may be used to confirm the donor bull was not infected during and up to 28–60 days after collecting for the export consignment. Young bulls intended for use as semen donors should ideally be moved out of endemic areas before they reach 6 months of age, which is when they are expected to lose their passive immunity. The regular banking of serum samples from bulls in AI Centres facilitates the retrospective testing of semen long after its collection, or after death of the donors. More details of procedures whereby semen may be guaranteed free from BTV for export are given in Chapter 2.2.13 of the OIE Terrestrial Animal Health Code [9]. The risks of BTV transmission plus the potential of clinically normal cattle to serve as reservoirs for spread of the virus by the insect vectors have led to major constraints on international trade in ruminants and their semen and embryos, particularly trade to countries (such as some in Europe) with large sheep populations and where competent vectors might be present. There is, however, no confirmed evidence that persistently BTVinfected carrier animals can occur such as those which are so important a feature in the epidemiology of BVDV (see above). To understand the fears about BTV which led to the constraints on international trade it is necessary to mention some studies done in the USA in the 1970s. In these Luedke et al.

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[109] claimed to have shown not only that transplacental BTVoccurs in cattle but also that, as a consequence, some offspring became immunotolerant and were persistently viraemic with BTV. Later, one of these offspring, a Hereford bull, number B28A, was reported to have excreted BTV in his semen over a prolonged period [110] and to have produced spermatozoa containing virus-like particles and ultrastructural abnormalities [111]. Bull B28A was also reported to have sired 12 persistently BTV-viraemic calves [112]. Not surprisingly, these findings led to severe restrictions on international trade in semen, and later to restrictions on trade in embryos, from BTV-endemic regions. However, they also led to a number of attempts to confirm Luedke’s findings experimentally (e.g. [113,114]). Despite the fact that insect-derived BTV (serogroup 11), or BTV derived from bull B28A, was used in the latter experiments, the results of none of them supported Luedke’s findings. Consequently Parsonson [115] strongly refuted the existence of persistent BTV infection with immunotolerance in cattle, and, in the decade since his report, his view has become accepted widely in the veterinary profession. However, it is also accepted that BTV can and does occur sporadically in the semen of viraemic bulls. With regard to the possibility that BTV might be transmitted via in vivo-derived embryos, there have been several experiments which convincingly show, at least for embryos washed according to the IETS protocols, that the virus is not so transmitted. In the one experiment in which BTV-infected semen was used, Thomas et al. [116] collected 20 zona pellucida-intact embryos from three BTV-seronegative donor cows 6 days after AI. The semen used for AI was from a bull which had been inoculated with BTV (serotype 17) and contained approximately 103 chick embryo lethal doses per millilitre. Two of the three donors became viraemic, and 18 of the 20 embryos recovered were washed 10 times using buffered saline containing BTV-antibody-negative fetal calf serum (but without trypsin treatment) and transferred into 16 recipients, with nine pregnancies. BTV was not isolated from any of the uterine flush fluids from the donors. Six of the nine pregnant recipients delivered live, healthy calves, but one had to be euthanased due to dystocia. One set of twins and one single calf died during calving. None of the recipients or live calves developed antibodies to BTV following embryo transfer. In another, larger, experiment by Acree et al. [117] in the USA in 1987–1988, heifers were deliberately infected with BTV (serotype 11) by exposure to bites from Culicoides midges and by inoculation with insect-origin virus. During the acute, viraemic stage 12–16 days after infection, 169 embryos were collected from 59 of the heifers and subjected to the IETS-recommended protocols (10 washings but not trypsin treatment). Fifty-seven embryos from these ‘acute stage’ heifers were tested for BTV in vitro by inoculating them onto cell cultures or into embryonated chicken eggs, and all were negative. The remaining 110 embryos were transferred into susceptible recipients and 36 calves were subsequently born. Again, all the recipients and their offspring remained BTV negative. As a result of these and several other experiments, references to which can be found in Appendix B of the IETS Manual [8], BTV has been listed in IETS Category 1, i.e. it is a ‘‘. . . disease agent for which there is sufficient evidence to show that the risk of transmission is negligible provided that the embryos are properly handled between collection and transfer’’—see Appendix 3.3.5 of the OIE Terrestrial Animal Health Code [9]. Additionally, in a simulation study to assess the risks when in vivo-derived, washed bovine embryos were moved from an area of South America where BTV is endemic, Sutmoller and Wrathall

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[118] were able to show that there would be approximately a one in 30,000 risk of transmission when embryos are collected in the vector season, and a one in a million risk in the season with low vector activity. Two experiments to determine the effects of BTV on in vitro-produced embryos have been reported but in neither was BTV-infected semen used. Tsuboi and Imada [119] exposed bovine oocytes (still surrounded by their cumulus cells) to BTV whilst the oocytes were being matured in vitro for 24 h. The oocytes were then fertilised and allowed to develop to the blastocyst stage, which they did without the virus having any obvious effect on development. In the other experiment, Langston et al. [120] exposed a total of 77 zona pellucida-intact embryos (morulae and blastocysts that had been fertilised in vitro) to between 8  105 and 2  108 plaque-forming units per millilitre of BTV (serotype 17) for 2 h. The embryos were then washed according to the IETS protocols except that 12 rather than 10 washes were given. Trypsin treatment was not used. The washed embryos, in groups of 5 to 10, were then sonicated and tested for the presence of the virus by inoculation onto bovine embryonic cell monolayers. The tenth, eleventh and twelfth washing fluids were likewise tested. BTV was recovered from all the groups of washed embryos and from almost all the washing fluids. Consequently the authors concluded that a protocol slightly more rigorous than that proposed by the IETS, shown to be effective for removal of BTV from in vivo-derived embryos, was ineffective for removal of the virus from in vitroproduced embryos. The difficulty of removing of BTV from zona pellucida-intact, in vitro-produced bovine embryos when using the IETS washing protocols, as compared to the ease with which similar washings will remove the virus from in vivo-derived embryos, is a reflection of wider a phenomenon which occurs with other pathogenic agents also. Appendix 3.3.2 of the OIE Terrestrial Animal Health Code [9], which recommends health standards for international movement of in vitro-produced bovine embryos, stipulates the following (see Article 3.3.2.5, para 1): ‘‘After the culture period is finished but prior to freezing, storage and transport, the embryos should be subjected to washing and other treatments similar to those specified for in vivo-derived embryos, in accordance with the IETS Manual’’. Although such washings are undoubtedly beneficial, sanitary controls for IVF embryos must also rely heavily on other risk-reduction measures. In Chapter 3 of the IETS Manual [8] Bielanski comes to the following conclusion ‘‘for the present, international trade in bovine IVF embryos is likely to present a lower risk of disease transmission when the embryos are produced from donors of known sanitary status, compared to those produced by fertilisation of oocytes from commercially slaughtered animals’’. Perry [121], in a recent paper, describes a Monte Carlo simulation model he devised to evaluate the risks associated with in vitro-produced bovine embryos originating from ovaries collected from cattle slaughtered in commercial abattoirs.

9. Conclusions New legislation from the EU prompted this re-examination of the risks that transfer of bovine embryos fertilised by semen from bulls infected with certain viruses might transmit those viruses to recipients and offspring. The new EU legislation is not surprising in view of

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the fact that some prospective importing countries within the EU have schemes in progress, or completed, for eradicating certain bovine virus diseases. Current recommendations and protocols for the international movement of embryos that are set out in the IETS Manual [8] and the OIE Terrestrial Animal Health Code [9] are based on earlier assessments of the risks, so it seemed appropriate to review the evidence again to enable valid comment on the new legislation. In general it should be noted that the relevant EU legislation covering livestock embryos does incorporate recommendations made by the IETS and the OIE. However, there is an important omission: the EU does not seem to differentiate between risks associated with in vivo-derived embryos, which for most disease agents are negligible if processed by IETS/ OIE recommended protocols, and the risks with in vitro-produced embryos, which, compared with the in vivo type, can be greater. The review looks initially at evidence from studies in laboratory animals, humans and horses in which some viruses have been found to integrate very closely with, or into, the spermatozoa, and where carriage by the infected sperm into oocytes at fertilisation is a distinct possibility. Some of the evidence from laboratory animals is from in vitro, i.e. artificial, rather than natural situations, and evidence for similar phenomena in cattle or other domestic livestock is still lacking or at least tenuous. In humans, semen processing protocols are currently used to ‘clean-up’ the semen from donor males, thereby reducing the risk of transmitting infectious agents such as HIV via AI, IVF and ICSI. The protocols include centrifugation of semen through Percoll differential density gradients to separate the motile spermatozoa from constituents such as lymphocytes and other potentially virusinfected cells. Semen ‘clean-up’ techniques could provide valuable risk-reductions in cattle and other domestic livestock also, but they would probably be ineffective if viral RNA or DNA is attached to or integrated into the spermatozoa. Examples of viruses in the latter category in other species include HBV in humans and EAV in horses where possibilities of infected sperm adhering to or penetrating through the intact zona pellucida into the oocyte cannot be ruled out. More research is needed on interactions between viruses and spermatozoa in cattle, particularly where the spermatogonia, and/or other sperm precursor cells might have a potential to be infected. Four bovine viruses which are, or might be, of particular concern to veterinary authorities in the EU are reviewed in detail here, namely enzootic bovine leukosis virus (EBLV), bovine herpesvirus-1 (BoHV-1), bovine viral diarrhoea virus (BVDV), and bluetongue virus (BTV). Unlike more acute, epidemic diseases such as foot-and-mouth disease (FMD), these four disease agents tend to cause subtle, often chronic manifestations, and are frequently subclinical. In each case the pathogenesis, epidemiology and possibilities of virus transmission by semen and by embryos are examined, and the risks of transmission by in vivo-derived and in vitro-produced embryos are assessed and compared. Controls used to reduce or prevent transmission, and legitimate requirements of importing countries are also described. There is good evidence in all four cases that semen from infected bulls can contain the virus in question. In the case of EBLV, however, semen infection is rare, and when it does occur the virus tends to be in seminal lymphocytes which should be removed when in vivoderived embryos are examined and washed under the microscope. In the same way, presence of BTV in semen is usually associated with red blood cells, and these too would be

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removed from in vivo-derived embryos by proper washing. Thus, at least for in vivoderived embryos, the risk of transmitting the disease when semen infected with EBLV or BTV is used for AI or natural service of the embryo donors, is negligible. With regard to in vitro-produced embryos there is evidence from one experiment that when semen artificially infected with EBLV was used for IVF there was no residual virus on these embryos after washing, but this was not the case for BTV. In fact, with BTV, even the use of a more rigorous embryo washing protocol than is usually recommended was ineffective for removal of the virus. The situation for the other two viruses, BoHV-1 and BVDV, is more complex in that their presence in semen might in some circumstances involve viral adherence to or integration into the spermatozoa. In the case of BoHV-1 it is also possible, at least in theory, that such infected sperm might actually adhere to or even penetrate into the zona pellucida of the oocyte at fertilisation. However, from the work of Vanroose and colleagues, it is much more likely that when BoHV-1 is present on the sperm, attachment of the latter to the zona pellucida will be inhibited, so fertilisation would tend to be by an uninfected spermatozoon rather than by an infected one. The IETS-recommended embryo washing protocol, including the trypsin treatment that is recommended for BoHV-1, should be completely effective for removal of any virus that remains on zona pellucida-intact embryos after fertilisation. However it must be noted that the experimental evidence on this point includes only one small experiment in which semen infected with BoHV-1 was used to inseminate two donors, and the semen used was ‘spiked’ with virus rather than being collected from a naturally infected bull. Additionally it must be emphasised that the evidence for effectiveness of washing applies to in vivoderived embryos only, and not in vitro-produced ones. In fact, whatever the route whereby virus exposure of in vitro-produced embryos takes place, even the washing-plus-trypsin treatment protocol is ineffective for removing BoHV-1. With BVDV the situation as regards infection in semen is particularly complex. Some workers have suggested that the virus occurs in the lymphocytes or other cellular constituents, but from a study of pigs infected with CSFV, which is closely related to BVDV, there is evidence that virus can integrate into spermatogonia or other precursor cells, and it might, therefore, be present inside the mature sperm. Results of experiments to investigate whether the IETS washing protocols, or washing plus trypsin, are effective for the removal of BVDV from zona pellucida-intact, in vivoderived embryos have differed depending on which virus strains are involved. Early work in the 1980s was done mainly with cytopathic BVDV, and washing alone (without trypsin) was effective for its removal. In more recent studies, however, where embryos were exposed to various non-cytopathic BVDV strains, and then washed with or without trypsin, the amounts of virus remaining on the embryos differed according to the BVDV strain, but amounts were significant nevertheless. Unfortunately there have been no experiments in which in vivo-derived embryos have been collected and tested for virus, or collected and transferred to seronegative recipients, following insemination of embryo donors with BVDV infected semen. So we do not know if either ‘spiked’ or naturally infected semen could transmit the virus onto or into the embryos, and thence to recipients. Whether any BVDV carried by semen to in vivo-derived embryos can be removed by washing is also unknown.

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There is a considerable amount of information about the interaction of BVDV with in vitro-produced embryos but little to indicate whether semen used for IVF can lead to carriage of this virus by spermatozoa into the oocyte at fertilisation. The data that are available suggest it is unlikely, although some workers found that infected semen used for IVF had detrimental effects on the number of embryos that developed to blastocysts. Other workers found no such detrimental effects. As is the case with several other viruses, it is very difficult to completely remove BVDV (whatever the strain) from in vitro-produced embryos by application of the IETS washing protocols. That stud bulls should ideally be free from the above four viruses is unquestioned, and protocols for AI Centres and production of bull semen published in Appendix 3.2.1 of the OIE Terrestrial Animal Health Code [9] describe how this can be achieved. However, in some countries bulls may not be virus-free, even in AI Centres, or their freedom might be questioned. The demand for their semen continues nevertheless, and the risks of transmitting their particular virus(es) are real and have to be assessed and tackled, particularly for export. Nevertheless the risks that using semen from such bulls for the fertilisation of embryos for export are believed to be much lower than they are for the semen per se. This is the case particularly with in vivo-derived embryos which have, apart possibly from those exposed to BVDV, an extremely low health risk if the internationally (OIE/IETS) recognised protocols for embryo collection and processing are followed. Many hundreds of thousands of in vivo-derived bovine embryos have been collected and transferred both within and between countries with no confirmed reports of disease transmission by this route. The potential disease risks associated with in vitro-produced bovine embryos are less predictable, although this is due not so much to possible presence of virus in semen used for IVF than to the fact that infection may occur at any stage in the production process. In view of the very small amounts of semen required for IVF, it should in any case be possible to develop semen ‘clean-up’ techniques to minimise the risks, especially for bovine viruses that are not intimately associated with the sperm.

Acknowledgements The authors are grateful to colleagues in the Health and Safety Advisory Committee of the IETS for helpful discussions and comments on this paper. Financial support from the Chief Scientist’s Group of the Department of Environment, Food and Rural Affairs, UK, is acknowledged.

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