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APPENDIX CAnimal Genetic Resources: Sperm fan Parsonson The importance of using semen in the dissemination of the ge- netic diversity of domesticated animal species is well recog- nized. Increases in highly desirable genetic characteristics and in the productivity of the various animal species have established artificial breeding (AB) as an efficient and reliable method in animal husbandry. Technical progress in the cryopreservation of semen and in mod- ern handling, storing, and transport systems have made international trade in animal semen an important segment of the commerce of many countries. These technical achievements have been a critical factor in the improvement of livestock worldwide. The portability and keeping qualities of cryopreserved semen have provided access to gene pools around the world. As techniques are developed for the collection, handling, and storage of semen from additional animal species not widely used at present, further expansion of world ge- netic resources will occur (Seidel, 1986~. Authorities responsible for disease control within a country usu- ally require specific health standards for the donors standing at AB centers, as well as general standards for the operation of the centers and for the handling, storage, and quality of the gametes produced from the centers. International trade requires standards and tests for Ian Parsonson was assistant chief of the Australian Animal Health Laboratory at the Com- monwealth Scientific and Industrial Research Organization. He is a member of the Australian Commonwealth Government Genetic Manipulation Advisory Committee. 215
216 / Appendix C semen, which are set out in protocols by the importing countries. These protocols and regulations define the standards for the donor animals, the AB centers, and the bacteriological control of the prod- uct. Generally, the quality control requirements set high standards for most AB establishments. The standards include disease control conditions for entry of animals into centers, for health standards for animals once in the centers, and for sale of semen from donors at the AB center. Standards for the importation of semen from countries in Europe, North America, and Australasia are comparable; they include tests for disease agents known to be exotic to the importing country. The advantage of cryopreserved semen is that it can be held pending verifi- cation of the accredited health status of the donors before being released for sale. An additional advantage of holding cryopreserved semen is that progeny testing can be conducted to evaluate genetic gain or to detect genetic defects before general release of the semen samples. Transmission of disease agents in semen or in spermatozoa has been seen as potentially the most likely method of spread of infec- tious disease both domestically and internationally. The realization that cryopreservation, antibiotics, and semen extenders such as milk and egg yolk citrate also assisted in the preservation of many patho- gens led to the development of health standards for donors and codes of practice for AB centers. Adherence to protocols for the health status of animals in AB centers and use of strict aseptic collection, handling, and preservation methods can lead to a high-quality, mini- mally infected product. Artificial breeding also provides a method of disease control through the use of specific pathogen free semen. Artificial breeding centers are the basis of national disease con- trol programs in many countries and are usually a requirement for an export trade in semen. The Office International des Epizooties (OIE) has developed recommendations for certification of AB centers on the basis of sanitary codes for control of disease and standards for processing semen (Office International des Epizooties, 1986~. Three disease-free categories regional freedom, herd freedom, and indi- vidual donor and its gametes freedom from specific diseases-form the basis for international trade in animal gametes and in particular semen. MAJOR SPECIES OF DOMESTIC LIVESTOCK INVOLVED IN INTERNATIONAL GERMPLASM TRADE The major species of domestic livestock involved in international trade in germplasm are cattle, sheep, pigs, goats, horses, and poultry.
Appendix C / 217 Species involved at a much reduced level include buffalo, deer, dogs, and various types of zoological animals. In addition, there is some trade in insect germplasm (bees), in fish germplasm, and in certain lines of laboratory animals, usually as frozen embryos. The major species used in AB programs throughout, however, is cattle (Bos taurus and Bos indicus), and it is through the production of semen from this species that the advantages and problems of artificial breeding have been identified. When bovine semen was first used for artificial breeding, the distribution of the semen was restricted to local regions because of the limited viability of the product and the lack of mobility of the . . Inseminators. ^. ~ There was a relationship between the fertility of par- ticular bulls and the pregnancy rate of cows inseminated with their semen, and soon, bulls whose nonreturn rates were lower than ex- pected standards were detected. It also soon became obvious that certain venereal diseases were being transmitted with the semen. Some of the diseases, such as brucellosis (Brucella abortus) and trichomoniasis (Trichomonas foetus), had been identified as causes of reproductive failure, and attempts were made to diagnose infected bulls. As bo- vine semen became an article of commerce, quality control standards became mandatory in many countries. Several other infectious agents affecting fertility were added to the growing list of pathogens that might be present in semen, including Campylobacteriosis (Campylobacter fetus var. venerealis and C. intermedius). The cryopreservation of extended semen added to the problem in that correctly frozen semen also preserved the microorganisms that were present as contaminants. Antibiotics were added to semen ex- tenders not so much to eradicate the microorganisms present, but rather to reduce their potential to cause harm. Along with these steps, research into improved aseptic collection and handling of fresh semen showed the importance of careful attention to temperature and temperature gradients for cryopreservation. ARTIFICIAL BREEDING CENTERS AND DONOR ANIMAL REQUIREMENTS The Australian Standing Committee on Agriculture has required that AB centers comply with Australian standards to be eligible for registration with the Australian Quarantine Inspection Service for the export of semen. Bovine semen production centers are licensed un- der state legislation to produce semen for sale from bulls kept within the center and judged to be disease free according to minimum stan- dards set out in the Minimum Health Standards for Stock Standing at
218 / Appendix C Licensed or Approved Artificial Breeding Centers in Australia (Australian Quarantine and Inspection Service, 1988~. A center is a quarantine area and is under veterinary supervision. The center must have facilities within the fully health-tested area for accommodation of stock and for collection of semen from the stock. It must have a processing laboratory and storage facilities for semen derived from the stock. Other requirements include providing for treatment of sick animals, introducing new stock without endanger- ing those already present, and maintaining the center as a strict quar- antine area. Prior to entering the center, cattle are examined for freedom from infectious disease and, as possible, tested for hereditary defects (for example, mannosidase tests for Angus cattle and crosses of Angus extraction) to detect carriers. The health tests require freedom from brucellosis (Brucella abortus), tuberculosis (Mycobacterium bovis), and leptospirosis (L. Pomona and L. hardjo). Treatment with dihydrostrepto- mycin is also required but only after tests have been made for free- dom from campylobacteriosis and trichomoniasis. Cattle cannot be derived from a property on which Johnes disease (Mycobacterium paratuberculosis) has been diagnosed within the previous 5 years. In addition, they must have negative tests for paratuberculosis. Cattle must also be free of antibodies to enzootic bovine leukosis, and the property must have a history of freedom from the disease. All of these diseases are listed as "B" or "Others" on the OIE disease lists (discussed below). Once the animals are in the fully health-tested area of the licensed center, they must undergo annual testing for freedom from the above-mentioned diseases. Semen production centers in Australia must comply with the Aus- tralian and OIE standards to be eligible to export semen. By defini- tion, a licensed bovine semen production center is licensed under state legislation to produce semen for sale from bulls kept within the center and judged to be disease free according to the minimum stan- dards described by the Australian Quarantine Inspection Service (1988~. Similar requirements are laid out in regulations governing AB centers in the United States, Canada, Great Britain, and New Zealand, and they form part of the OIE standards for artificial breeding. Ad- ditional disease control tests are required of countries in which the diseases on the OIE's List A (discussed below) are present. Even for many of these important disease agents, however, information on their transmission in semen is minimal. Often, the evidence is ob- tained during the viremic phase of the disease, when the agent is at its highest titer in the blood. Evidence of the persistent excretion of viruses in semen is available only for a few diseases, and it may be
Appendix C / 219 an insignificant problem, particularly when health standards and tests are applied to donors prior to and following semen collection. Of the diseases listed by the OIE (Hare, 1985), lists A, B. and Others include a number of agents that have been isolated in semen from bulls, rams, bucks, and boars. The evidence, however, is often minimal or restricted in scope, and in some cases confusing reports on individual agents may be given. For many infectious agents there is an obvious need for further studies to identify their potential to transmit disease through the genital route. Current techniques for artificial breeding provide access to the cervix and uterus, which is not presented normally in natural mat- ing. It is the necessity to deliver semen through this route in many species, or through laparoscopy in some species, that enables the agents to bypass the immunological and physiological defenses of the female. CONTROL OF COLLECTION, HANDLING, PRESERVATION, AND STORAGE OF SEMEN Standards have been developed in many countries for donor ani- mals and the collection and storage of semen. With the advent of liquid nitrogen cryopreservation and the development of extender recipes and antibiotic regimes, the quality of preserved semen has improved. Even with all previous research and continuing improve- ment in techniques for collection, handling, and storage, semen re- mains a product contaminated with microorganisms. Further attempts to reduce the level of contamination have concentrated on the health of the donor animals, their freedom from specific diseases, and as far as possible, asepsis during collection and subsequent handling of se- men. The latter includes ensuring the sterility of extender liquids and the addition of a suitable antibiotic cover. Because of the need to remove or control mycoplasmas, the antibiotic cover has now been changed in many countries and will soon become a standard compo- nent of semen for international use. In the United States, the use of penicillin, dihydrostreptomycin, and polymyxin B sulphate has been replaced in the minimum requirements of the Certified Semen Ser- vices (CSS) with the new combination of tylosin, gentamicin, linco- mycin, and spectinomycin (soak, 1986~. The change to the use of this antibiotic combination has shown that processing procedures, extender composition, and antibiotic combinations may affect the ef- ficacy of microbial control or fertility. The CSS has listed the following requirements (soak, 1986~:
220 / Appendix C Extender must be of a type approved by the CSS. Contact between antibiotics and raw semen must be for less tin 3 minutes. · Cooling of semen and nonglycerol fraction of less than 2 hours to 5°C. Glycerol cannot be added as an extender component until after cooling to 5°C. The new antibiotics and procedures have been accepted in the United States for intra- and interstate movement of semen for artifi- cial breeding. International movement of semen will require export- ing countries to comply with the changes. Many countries outside the United States are moving to adopt similar changes (Shin, 1986), and such changes may be adopted as part of the quarantine protocols for international movement of germplasm. MAJOR DISEASES OF CONCERN IN ARTIFICIAL BREEDING List A Agents The major diseases of concern in the international transfer of ga- metes for artificial breeding are mostly those caused by viruses. The JOE's List A Agents are communicable diseases that (1) have the po- tential for very serious and rapid spread irrespective of borders, (2) are of serious socioeconomic or public health consequences, and (3) are of major importance in the international trade of livestock and livestock products (Gibbs, 1981; Hare, 1985; Odend'hal, 1983; Parez, 1985~. Some of these diseases are described below. Foot-and-Mouth Disease Virus The most important virus disease on the OIE's List A is that caused by the foot-and-mouth disease virus (FMDV) because of its presence on all continents, except Australia and North America, and because of the number of animal species affected by FMDV. Cottral et al. (1968) identified the presence of FMDV in semen of bulls and its subsequent transmission by artificial insemination. This finding was confirmed by Sellers et al. (1968), who also showed that bulls ex- posed by indirect-contact infection had high titers of FMDV in the semen before the lesions of clinical disease appeared. These findings precluded the use of semen from FMDV-infected countries for a number of years. As new testing systems were developed, protocols were also developed by some countries to allow international movement
Appendix C /221 of semen. Acceptance of these protocols has enabled considerable headway to be made in relation to other disease agents on List A, such as bluetongue virus (BTV). Bluetongue Virus BTV can infect a wide range of ruminant species. During the past 10 years, research into the excretion of BTNI in bovine and ram semen has shown that, in the vast majority of cases, BTV is only shed during the period of viremia, and then at a titer that is considerably lower than in the blood (Bowen et al., 1983; Breckon et al., 1980; Parsonson et al., 1981b, 1987a,b). When semen contaminated with BTV is instilled into the cervix and uterus of susceptible cattle, it usually causes infection, generally without clinical signs, and it ap- pears to have little or no effect on subsequent pregnancy (Bowen and Howard, 1984; Parsonson et al., 1987a,b). However, when cattle were experimentally infected with insect-passaged (Culicoides variipennis) BTV serotypes 11 and 17, congenitally deformed calves were pro- duced (Luedke et al., 1977~. Similar experiments have not produced infected calves, nor was serological or virological evidence of trans- placental transfer of BTV found in those studies (Parsonson, 1990; Parsonson et al., 1987b). In a study in which bovine fetuses were inoculated in utero with BTV at 125 days gestation, MacLachlan et al. (1984) showed that the virus did not persist in the fetus at birth. Thus, fetuses infected in utero are unlikely to be reservoirs for the virus. Additional studies on the fetal transmission of BTV seem unnec- essary in light of the mounting evidence against transmission by this route. There is need, however, for further studies of the initial repro- ductive stages in sheep and cattle to ensure that BTlI is not a cause of reproductive losses in endemic areas. Despite the experimental evi- dence, Osburn (1986) suggested a need for further studies in dairy cattle in California. Rinderpest Virus Although rinderpest virus can infect a wide range of ruminant species, there is very little evidence for its spread through artificial breeding. Scott (1964), however, reported isolation of the virus from all secretions and excretions of infected animals, and Plowright (1968) cited Curasson (1921) and lacotot (1931) as finding the virus in vagi- nal secretions for 5 to 12 weeks after abortion had occurred. Because rinderpest and peste des petite in ruminants cause obvious disease
222 / Appendix C on a herd basis, it is unlikely that semen or ova for artificial breeding would be collected from animals in an area where an outbreak has occurred. In an extensive series of tables, Scott (1964) has detailed animal species known to be infected with rinderpest virus. The lists are important when considering species that are endangered or at risk. Lumpy Skin Disease (Neethling Virus) In experimentally infected animals, Weiss (1968) demonstrated the presence of lumpy skin disease virus (LSDV) in bull semen for 22 days following fever and generalized skin lesions. Weiss, however, also demonstrated the presence of virus in semen at days 7 and 12 in bulls undergoing inapparent infections. Almost certainly, virus would be present in secretions of cows from which embryos were collected during infection. The similarity between LSDV and capripox has been noted in several reports, and LSDV has been classified in the genus Capripoxvirus by Penner (1976~. Sheep and Goat Pox Virus These viruses were originally thought to be species specific. However, Kitching and Taylor (1985), however, have shown a close serological antigenic relationship, and cross-protection studies have supported that firlding. As a result, Kitching and Taylor have suggested that the term capripox be used in the future, irrespective of the species involved. Because of the similarities between these viruses and LSDV, ex- cretion in both semen and vaginal discharges are extremely likely during infection of donors. Because collection of semen or ova would be unlikely during the occurrence of clinical disease, there should be little chance of venereal transmission. This group of viruses is ex- tremely resistant to environmental factors, however, and all precau- tions to avoid venereal transmission must be taken. A suitable at- tenuated virus vaccine for capripox, the 0240LT/1 BHK/2 LT/4 strain of Kenyan sheep and goat pox virus, has been shown to be stable and safe and to provide substantial protection for at least 1 year (Kitching et al., 1987~. Semen and embryos could therefore be collected from donor animals vaccinated against Capripoxvirus. Rift Valley Fever Virus Rift Valley fever virus (RVFV) is an arthropod-borne virus that can also be spread by contamination of the environment and by aero
Appendix C / 223 sots. It is a particularly difficult agent to control. Vaccination is carried out in some areas of the African continent where epizootics have occurred and in endemic areas where sporadic outbreaks cause problems to livestock. Although R~FV causes abortions in pregnant animals of many species, including humans, and although it can be fatal to neonatal and young animals, there is a paucity of information on its effect on reproduction. This lack of information may be related to the severity of the clinical condition and the insignificant role that reproductive disorders would play in an outbreak of the disease. However, there is a need to know how to avoid transfer of this agent in gametes originating in endemic areas of Africa and destined for international trade. Several wild animal species from Africa are also susceptible, and any efforts to store gametes to preserve animal ge- netic resources would have to take cognizance of this fact (Easterday, 1965; Meegan et al., 1979~. Vesicular Stomatitis Virus Vesicular stomatitis viruses (Indiana and New Jersey serotypes) are confined to the Americas. Although some understanding of the epidemiological behavior of the virus group is available, there are great gaps in essential knowledge. Virtually no studies have been carried out on transmission of the virus through the reproductive tract. Domestic species of animals affected by these viruses include cattle, horses, and pigs; humans are also affected. Some wild animal spe- cies, including deer, are affected. The virus propagates readily in laboratory animals, such as rabbits, guinea pigs, and mice. A carrier state for these viruses has not been demonstrated and hence, the serological tests used by U.S. authorities to introduce zebu cattle from Brazil (House et al., 1983) were standardized to reject recently in- fected animals. Perhaps similar tests should be set for semen donors. This agent is one of the remaining enigmas of veterinary science and pathogenesis, and epidemiological studies are urgently needed be- cause animals and gametes from the Americas constitute a large seg- ment of international trade in livestock germplasm. Contagious Bovine Pleuropneumonia Contagious bovine pleuropneumonia (Mycoplasma mycoides subsp. mycoides) is a disease of cattle present in Africa, Portugal, Spain, oc- casionally other parts of Europe, Asia minor, some east European countries, and China.
224 / Appendix C Very little is known of the reproductive disorders caused by this microorganism. In studies carried out in Australia before the disease was eradicated in 1968, there was no indication of semen contamina- tion, nor of intrauterine infection of the fetus (Seddon, 1965~. Swine Vesicular Disease Virus Swine vesicular disease virus (SVDV) and (FMDV) have both been isolated from boar semen. Sows inseminated with SVDV seroconverted (McVicar and Eisner, 1977), thus demonstrating the potential to transmit SVDV infection through this route. Although difficulty with mount- ing was seen in boars affected with FMDV, it may not prevent collec- tion of semen contaminated with FMDV or SVDV before manifesta- tion of clinical disease (McVicar, 1984; Thacker et al., 1984~. African Swine Fever Virus African swine fever virus (ASFV) has been described as causing a highly contagious, fatal disease in domestic pigs, but it also can be manifested as a mild to moderately severe disease, almost indistin- guishable clinically from hog cholera. The virus is present in Africa, Italy, Portugal, Sardinia, and Spain. At present, ASFV appears to have been eradicated from parts of South America and the Carib- bean. A report that ASPS, present in cryopreserved semen collected during viremia from an experimentally infected boar, was transmit- ted to a recipient female and produced infection (Schlafer, 1983) indi- cates the need to treat semen as a possible source of infection with this agent. Hog Cholera Virus Successful eradication programs in several countries have reduced the incidence of hog cholera virus (HCV) in herds of pigs. Because the most serious chronic manifestations of HCV are on reproductive conditions, including fertility, abortions, teratogenic defects, and neonatal deaths, control of this disease has widespread benefits (Cheville and Mengeling, 1969~. Evidence that a country is free of HCV is required for interna- tional movement of swine semen. Serological tests and disease con- trol practices are becoming required conditions for members of the European Economic Community. Outbreaks of hog cholera are still occurring in Europe, however, as evidenced by an outbreak in the United Kingdom (Anonymous, 1986~.
Appendix C / 225 Summary The diseases of greatest concern in the international movement of semen are those discussed above. Generally, the high pathogenicity and economic risks resulting from an infection by these agents en- sure that production of gametes for international trade will be from countries free of these diseases. Increased risks may be involved and may be unavoidable, however, to procure access to highly desirable genetic material. Countries that largely depend on livestock indus- tries have had to develop protocols to enable collection of semen and embryos from areas where diseases on the OIE's List A occur. Be- cause of advances in testing methods for the agents listed and better disease controls within the countries where the agents are present, semen and gametes from high-risk areas of Africa and South America can now be transferred through offshore quarantine facilities to the United States, Australia, and New Zealand. Such transfers of ga- metes have increased prospects for international movement of animal germplasm and also have improved prospects for cryopreservation and use of rare, potentially desirable genetic materials. List B Agents The diseases on the OIE's List B are generally diseases for which adequate test systems are available in most countries. They are con- sidered to be of socioeconomic or public health importance within countries and are significant in the international trade of livestock and livestock products. Some of these agents are described below. Bacterial and Protozoal Agents As stated by Wierzbowski (1984), the methods of testing bulls in artificial breeding centers for brucellosis, tuberculosis, paratuberculosis, leptospirosis, and trichomoniasis are well established and effective. Bartlett (1981) defined the need for uniformity of diagnostic methods for the diseases listed above as a basis for semen derived from spe- cific pathogen-free donors, and Wierzbowski (1984) listed the princi- pal diseases of concern for artificial breeding. With modern diagnos- tic tests, all the agents listed can be detected readily. For several of the diseases, either vaccines or reasonably adequate antibiotic treat- ment methods are available. For campylobacteriosis, diagnostic tests that can identify carrier bulls in AB centers are available (Clark, 1985a). A vaccine developed in Australia can ensure eradication of campylobacteriosis from AB
226 / Appendix C centers (Clark et al., 1974, 1976, 1979~. Bovine trichomoniasis can be readily diagnosed (Clark, 1985b), and under some circumstances con- trolled or eradicated from AB centers (Clark et al., 1983~. For both of the above venereal infections, diagnostic systems have been standardized in Australia. In addition, the vaccination of bovine donors and teas- ers in AB centers in Australia will help ensure freedom from Campylobacter fetus and its variants. There are no apparent reasons why these tech- niques cannot be used in other countries, with the same beneficial results of keeping donor bulls free of C. fetus and without the doubt- ful reliance on antibiotics in semen or in the treatment of carrier bulls. Investigations on campylobacteriosis in donor cattle in Austria were reported by Flatscher and Holzman (1985) at the Fifty-Third General Session of the OIE. Perhaps as a result of these and similar studies, campylobacteriosis will cease to be a disease of concern in international trade and for long-term storage of germplasm. Mycoplasmas Examinations of bovine semen carried out in a series of surveys over recent years have noted the presence of a number of myco- plasma and ureaplasma contaminants. Mycoplasma bovigenitalium is usually the predominant organism along with ureaplasma. Other isolates, however, such as M. bovis, M. canadense, M. californicum, M. alkalescens, and Mycoplasma species group 7 have been recognized as pathogens in the bovine (Ball et al., 1987~. Fresh semen (332 samples), processed semen (137 samples), and preputial washes yielded myco- plasmas or ureaplasmas from 46 percent, 31 percent, and 80 percent, respectively, and stored, processed semen straws taken as long ago as 1975 contained M. canadense and M. bovigenitalium (Ball et al., 1987~. Addition of lincomycin and spectinomycin to the semen extender eliminated the capability to detect mycoplasmas and reduced the iso- lation of ureaplasmas. The role of M. bovis infections of the bovine genital tract has not been clearly defined (Hartman et al., 1964; Hirth and Nielsen, 1966), although the infection is found in the reproductive tract at a lower incidence than M. bovigenitalium, M. canadense, or ureaplasmas (Friis and Blom, 1983~. However, M. bovigenitalium has been found as a genital tract infection in bulls (Al-Aubaidi et al., 1972), and the dis- ease has been reproduced experimentally (Parsonson et al., 1974~. This has allowed identification of the acute and chronic inflamma- tory response to this organism, an agent often present among the microbiological contaminants of the preputial cavity. There have been
Appendix C / 227 several reports of the effect of mycoplasmas in artificial breeding and, in particular, their effect on fertility and freezability of bovine semen (Bartlett et al., 1976; Fejes, 1986; Kissi et al., 1985; Nielsen et al., 1987~. In a report to the Committee on Infectious Diseases of Cattle of the U.S. Animal Health Association (Osburn, 1986), it was recommended that the antibiotic additives to control bacterial con- tamination in processed bull semen be replaced with gentamicin, lincomycin, spectinomycin and tylosin, specifically for mycoplasma. Certified Semen Services has adopted similar recommendations, and in order to comply with the CSS minimum requirements, the new antibiotic formula was to be phased in by January 1, 1988 (soak, 1986~. Although these requirements apply to artificial breeding in the United States, in order to supply germplasm to the U.S. market, countries around the world will have to adopt these standards, and so international trade will follow the dictates of the market (soak, 1986). The changes in antibiotic cover for bovine semen should be ex- tended to buck and ram semen, for which the mycoplasma-induced diseases are of much greater importance. Jones (1983) reviewed the mycoplasmas of sheep and goats and identified those of proven patho- genicity. Because the urogenital tract is frequently colonized, it is important to minimize the effect of these organisms in artificial breeding. Mycoplasmas isolated from boar semen have been reviewed by Thacker et al. (1984), who concluded that the effect of these mycoplasma spe cies in swine semen is poorly understood. Because of the difficult growth requirements for bacterial isola- tion of mycoplasmas, isolation and identification from boar semen are difficult. However, the addition of the antibiotics lincomycin and spectinomycin would have an effect on controlling mycoplasmas in boar semen. Infectious Bovine Rhinotracheitis Because infectious bovine rhinotracheitis (IBR) viruses are so readily transmitted in semen and already found in cattle populations world- wide, importing countries usually require evidence of donor freedom from infection. Some strains of IBR, particularly those in North America, appear to be abortogenic, whereas strains in Australia and New Zealand normally do not cause abortions. Bovine herpesvirus 1 (BHV 1) causes mild to severe syndromes and often varies in clinical manifestations. Because the BHV 1 group is virtually impossible to eradicate from the general cattle population, steps have been taken in Australia and Britain to maintain IBR-free bull herds in AB centers. Extensive test
228 / Appendix C ing of bovine semen to detect IBR virus, using a semen culture test (Sheffey and Krinsky, 1973) or a system devised by Kahrs and Littell (1980), has shown the difficulty of detecting minimal amounts of BHV 1. Inoculation of semen contaminated with IBR virus into the uterus of cattle at estrus is followed by infection within days and seroconversion to the virus (Kendrick and McEntee, 1967; Parsonson and Snowdon, 1975~. If semen batches are suspected of being contaminated with IBR virus and require testing, this may be the most reliable method at present. Loewen and Darcel (1985) compared two methods of isolat- ing IBR virus from either milk or egg-yolk citrate semen extenders: centrifugation and dilution of samples with phosphate-buffered sa- line (pH 7.4~. Both systems improved isolation of the virus. Animal inoculation, either parentally or through the uterus at estrus, appears to be the most reliable method of detecting IBR (bo- vine herpesvirus 1) virus in semen (Parsonson and Snowdon, 1975; Schultz et al., 1982~. The use of seronegative donors would reduce the likelihood of transmission and would be preferable to all other options. Bovine Leukosis Virus Testing cattle herds for bovine leukosis virus (BLV) antibodies has identified carrier herds in many countries. Using the reliable gly- coprotein antigen in the agar gel immunodiffusion test, carrier bulls can be identified and not used as donors for international transfer of semen. There is a strong body of opinion, however, that leukocyte- free bovine semen will not transfer virus. Monke (1986) examined the use of bull semen originating from seven AB centers in the United States in a closed Jersey breed herd of 200 cows that had remained serologically negative for BE virus antibodies for 5 years. During this time, 24 of the 66 donor bulls were consistently serologically positive and a further 2, which provided 1,019 units (48.3 percent) of the semen used, became seropositive for BLV during the study. Bovine Viral Diarrhea Virus During evaluations of a procedure to test large numbers of semen samples for viral contamination, using an in vivo system to test pooled semen samples, called the Cornell semen test, Schultz et al. (1982) found that the only consistent viral contaminant present at infectious levels was bovine viral diarrhea virus (BVDV). The authors sug- gested that it would not be necessary for bulls to have overt clinical infections; subclinical infections are common.
Appendix C / 229 Over a 4-year period, 40,000 samples from ejaculates were tested. No IBRV, bovine herpes mammillitis virus, BLV, or BTV were de- tected at levels sufficient to cause infection. The significance of the finding of BVDNI in semen had not then been established (McClurkin et al., 1979; Whitmore et al., 1978~; however, studies by Littlejohns (1982), McClurkin et al. (1984), and Roeder and Harkness (1986) have since established BSIDV as a major viral disease of reproduction. Bulls, rams, or bucks persistently infected with BVDV can shed virus in semen. Semen contaminated with the virus could then introduce BVDV infection into closed cattle, sheep, or goat herds and cause subsequent losses in production through reproductive failure (Littlejohns, 1982; Lucas, 1986; Roeder and Harkness, 1986~. The production of cattle immunotolerant to BVDV (McClurkin et al., 1984) highlights the ease with which this disease could spread in a noninfected herd. Control strategies for avoiding fetal infection in the early stages of gestation were included by Roeder and Harkness (1986) in a discus- sion of prospects for control of BVD viral infections. In a series of letters to the editor of the Veterinary Record (Brownlie et al., 1984; Harkness et al., 1984), control systems were discussed and evaluated. Control and eradication of the major viral diseases in bulls in artifi- cial breeding centers, as in Britain, offer the most pragmatic solution (Lucas, 1986~. Ephemeral Fever Virus Ephemeral fever of bovines and buffaloes is known to occur in many countries of southeast Asia, Africa, and Australia. In epizoot- ics that have occurred in Australia, the morbidity in cows and heifers was approximately 90 percent, and in bulls 67 percent (Spradbrow and Francis, 1969), and temporary infertility was reported in bulls with clinical infection (Chenoweth and Burgess, 1972~. In experimen- tally infected bulls, spermatozoa were found to have detached heads and tails, or bent and coiled tails, and ephemeral fever virus (EFV) was isolated from the semen of one bull. Ten cows inseminated at estrus with semen contaminated with EFV did not become infected, but remained susceptible to a subsequent challenge with EFV 30 days after insemination (Parsonson and Snowdorr, 1974~. Akabane Virus Parental inoculation of Akabane virus in eight bulls was followed by clinical, virological, and serological studies. Semen were collected regularly and examined for the presence of Akabane virus by inocu
230 / Appendix C ration of cell cultures and subcutaneous inoculation of susceptible cattle. Akabane virus was not detected in the semen by either isola- tion method. From the results of this study, it was concluded that Akabane viral infection of the bull would not affect reproduction (Parsonson et al., 1981a). Parainfluenza 3 Virus An ubiquitous virus in cattle and sheep populations, parainflu- enza 3 (PI-3) virus, has been isolated from bull semen on occasion. Abraham and Alexander (1986) reported isolation of PI-3 in a group of 15 bulls one month after vaccination with a live PI-3 vaccine, but they could not detect any relationship between this virus and the fertility of the bulls. Porcine Parvovirus The reproductive losses associated with parvovirus infections in susceptible pregnant sows and gilts (young females) have been shown to cause major problems in piggeries Coo and Johnson, 1976; Mengeling, 1979~. Moreover, the infection is usually attributed to viral contami- nation of the environment, either by direct contact through the oral- nasal route or through the recently introduced pigs or from contami- nated instruments. Bonte et al. (1984) experimentally introduced porcine parvovirus through the oral-nasal route into boars and found evi- dence of the virus in the genital tract before immunity was induced. They concluded that it would be possible to introduce infection through semen. To avoid transmission of infectious agents through boar semen, constraints and disease testing of boars in special centers developed for the purpose of artificial breeding would be required, much as they are in bovine AB centers. Control measures ensuring freedom from specific porcine pathogens have been called for in pig-breeding centers in the United States and in the United Kingdom (Thacker et al., 1984~. The same high standards of freedom from specific diseases and maintenance of such freedom by a disease-testing program, quaran- tine and testing of new introductions, and strict attention to hygiene in the swine AB centers would be essential. By using the same ap- proach with swine AB centers as that adopted for controlling bovine semen, similarly high-quality standards for semen could be attained. Trying to assess the microorganism contamination of semen is very unrewarding because of the difficulty of culturing semen from ani- mals. Inoculation into animals, as in the Cornell semen test, offers a
Appendix C / 231 possible means for monitoring contamination, but it has a very low benefit-cost relationship in comparison with achieving disease free- dom from important pathogens through vaccination and the hygienic handling, preservation, and storage of boar semen. Diseases of Sheep and Goats The major disease of sheep and goats that is currently inhibiting the international movement of semen from these species is scrapie. The countries of the world that are free of scrapie do not want to introduce the disease. Australia, New Zealand, and South Africa are among the only major sheep-producing countries of the world that are free of both scrapie and maedi-visna. Recently, efforts have been made in Australia and New Zealand to introduce new sheep and goat genotypes using systems based on artificial breeding and long-term quarantine (up to 5 years). Early results of experimental trials in the United States with scrapie-in- fected breeding stock and noninfected recipients have been reported by Foote et al. (1986~. The experiments consist of groups in which embryo transfer in sheep with artificially induced scrapie embryo transfer in goats with artificially induced scrapie and embryo trans- fer in sheep with naturally occurring scrapie are being conducted. After 5 years of the trial, only the positive control group (both donors and recipients were scrapie exposed), have expressed the disease. An additional project is the artificial insemination of scrapie-free ewes with semen from scrapie-infected sires. After 3 years, there has been no evidence of scrapie in the progeny. If these results continue, artificial breeding may provide the means to control this disease and to ensure the safe collection of genetic material from sheep and goats in countries where scrapie is a problem. For the long-term storage of germplasm from sheep and goats, such assurances as freedom from scrapie, maedi-visna, and caprine arthritis and encephalitis would be mandatory. Scrapie has become the most difficult of all animal diseases to diagnose and control be- cause of the inability to diagnose it except on clinical signs, which then have to be followed by histological examination and reproduc- tion of the disease in homologous and heterologous species based on intracerebral inoculation of brain and other tissues. In those countries in which scrapie does not exist, the opportuni- ties for a structured AB program are present. Development of such programs has taken place in Australia and New Zealand, often at existing cattle AB centers. The standard disease controls are similar to those established for cattle. Requirements for quality control, han
232 / Appendix C cling, storage, and transport of semen are also similar to those for cattle. Now, with the movement to storage of ram and buck semen in ministraws instead of pellets, there is less problem with contamina- tion in storage. Because none of the diseases of sheep and goats caused by the retroviruses of maedi-visna and pulmonary adenomatosis in sheep or by caprine arthritis and encephalitis in goats, has been shown to be transmitted by artificial breeding, use has been made of AB to control these diseases in countries where they are present. Diseases of Horses Although there are some severe technical problems with artificial breeding of horses, there are requirements for superior genetic mate- rial, notwithstanding the restrictions on AB by some equine breed societies. Without the inhibition of breed-governing groups that op- pose AB, the techniques for artificial breeding and preservation of gametes will evolve. Most AB in horses is carried out within stan- dard-bred horse societies and by some companion and pleasure horse groups, but AB is not generally used for commercial purposes and is still not widely accepted. A major bacterial venereal disease of horses, contagious equine metritis (CEM), caused by Haemophilus equigenitalis (Taylor et al., 1978), appeared in Thoroughbred horses in several countries of Europe (Timoney et al., 1977), in North America (Swerczek, 1978), and in Australia and New Zealand in 1977 (Hughes et al., 1978~. The spread of the agent was traced to the movement of several stallions used for breeding, and it highlights how rapidly such agents can be dispersed with the aid of modern transportation and commercialization. Because of the value of the Thoroughbred industry, many millions of dollars can be lost when mares are infertile for a breeding season. The stallion with CEM is an asymptomatic carrier and can transmit infection for ex- tended periods of time. Although CEM has been eradicated from the horse populations in Australia, New Zealand, North America, Brit- ain, and Ireland, there is a continuing need for quarantine vigilance against reintroduction of this disease agent. A number of viral diseases of horses are arthropod borne, for example, African horse sickness, equine encephalosis, Venezuelan equine encephalitis and Eastern and Western equine encephalitis, vesicular stomatitis, and equine infectious anaemia. There is little or no evi- dence for the involvement of the equine encephalitides in equine reproduction, other than the disease state of the animals during breeding. Several equine viral diseases have a marked effect on reproduc- tion, including the equine herpes viruses, EHV 1, EHV 2, and EHV 3.
Appendix C / 233 . Of these, EHV 1 is a major pathogen of horses; it causes respiratory disease (subtype 2) and abortion (subtype 1~. Equine arteritis has been shown to infect some stallions persis- tently, and the route of spread can be by venereal transmission from semen (Timoney et al., 1986~. There are several viral diseases of horses for which there is insuf- ficient information to identify clearly the role the causal agents play in reproduction. The agents include the equine influenzas, A equine 1 (H7N7) and A equine 2 (H3N8), Borna disease virus, Japanese en- cephalitis virus, and Getah virus. An increasing demand for cryopreserved equine semen has led to more effort being devoted to developing better semen extender sys- tems (Francl et al., 1987) and to improving the methods of freezing semen to ensure preservation of fertility in equine semen (Volkmann, 1987). Poultry Diseases The intensification and integration of the poultry industry has concentrated large flocks of birds, both broilers and layers, within relatively small areas. These concentrations of birds require a very high level of disease control. Most preventive methods are based on quarantine of important birds, close disease monitoring, and the de- velopment of specific pathogen-free breeder units, especially elite and grandparent flocks. Even in such flocks, great care must be taken in regard to the use of preventive vaccines, food and water supplies, and general hygiene, as well as isolation from environmental factors (Allen, 1981~. The major pathogens of concern are those that are known to be transmitted by the vertical route. Diseases of concern include Salmo- nella pullorum, Mycoplasma gallisepticum, M. synoviae, and M. meleagridis, and the viral diseases of infectious laryngotracheitis, adenovirus 127, and avian leukosis complex. In the majority of elite and grandparent flocks, these diseases have been eradicated and are regularly moni- tored. Eradication of all diseases from the commercial areas is too difficult, and in these flocks, vaccines play a large role in disease control (Allen, 1981~. For artificial breeding of poultry, disease control in the donor flocks is essential. For long-term preservation of germplasm, it will be imperative to use only specific pathogen free flocks. For preserva- tion of endangered or rare bird species, a high standard of disease control will be necessary, but because the genetic material will be rare, some accommodation may have to be made.
234 / Appendix C I The cryopreservation of chicken semen from inbred and special- .zed strains will be a vital resource for specific pathogen free elite and grandparent flocks. Storage of semen from these sources will provide a safeguard in the event of a change in disease status. More important, however, it will provide a valuable genetic library for the future. Studies by Sexton (1979), Lake et al. (1981), Lake (1986), and Bacon et al. (1986) have established the possibilities for long-term cryopreservation of chicken semen. Established methods for artifi- cial breeding of poultry will enable storage of inbred and elite lines of chickens and other birds, including endangered wild species. ARTIFICIAL BREEDING OF ENDANGERED AND RARE SPECIES The numbers of rare and endangered species of animals and birds are rapidly increasing as humans move into the breeding ranges of these species. The importance of utilizing all knowledge available from artificial breeding to attempt the continuation of endangered species and, where possible, the long-term preservation of their ga- metes for posterity, is a vital issue for this generation (Durrant et al., 1986; Mason, 1974~. The development of animal model systems for embryo technolo- gies for rare and endangered species of wildlife has generated great interest among several concerned groups (Wilds et al., 1986~. The Convention on International Trade in Endangered Species of Wild Flora and Fauna (U.S. Department of the Interior, 1984) has recog- nized more than 200 mammalian species as being threatened by ex- tinction. The accelerated rate of habitat destruction is leaving conser- vationists very little choice other than managing most wildlife in captive situations, such as in zoological gardens or parks and wild- life reservations. Many of the wildlife species that are threatened require very careful, expert handling and treatment to ensure that the techniques adopted for their preservation are suitable and not harm- ful or stressful to their well-being. Because of the lack of knowledge of the genetic background of many endangered species, with respect to the size of the genetic base, any information that is pertinent is urgently required. This also ap- plies to reproductive technologies, including gamete collection, han- dling, storage, and treatment. Until the information is available and organized into developing strategies for propagation of species by artificial means, there is little chance of using AB as a viable alterna
Appendix C / 235 five. Yet, it offers the only major hope for the preservation of many species. RECOMMENDATIONS FOR FURTHER RESEARCH The OIE has set standards for the hygienic collection and han- dling of fresh and preserved bovine semen in artificial breeding cen- ters, appendix 5.2.1.1 (1986~. The OIE states that observation of the standards will result in bovine semen almost free of common bacteria. In the OIE code for bovine semen (1986) from AB centers accred- ited for export, the purpose of official sanitary control in semen pro- duction is to maintain the health of animals at an AB center at a standard that permits the international distribution of semen free of specific pathogenic organisms that can be carried in semen and cause infection in recipient female cattle. In discussing the cryopreservation of genetic material and the disease risks and controls, Wierzbowski (1984) pointed out the danger of contaminated single ejaculates being spread over a long time period. Hare (1985) emphasized the advan- tages of semen as a disease-control method over animal-to-animal contact. Collection of semen can be made from donors known to be free of specific pathogens, and a statistically defined sample of any individual collection can be tested for freedom from specific microor- ganisms before insemination. An additional benefit is that the do- nors can be tested for evidence of previous infection for periods of time, extending into months if necessary, before their semen is used in artificial breeding. Hare (1985) elaborates on the historical development of AB cen- ters and the development of legislative controls based on country of origin, herds, and finally individual donors and their semen. Such controls have become recognized internationally. Proposals for freedom from specific pathogenic microorganisms for bovine semen were made originally by Bartlett et al. (1976~. However, some countries, such as Australia, already had a strict code for the production of semen free of specific pathogens. Disease-free require- ments are now being recommended internationally, and the disease lists generally adopted are based on the OIE's A, B. and Other lists. It is expected that in a member country, donor animals producing semen will be free of diseases on List A, the AB herd should also be free of diseases on List B. and the individual donors should be free of diseases on the Other list (Office International des Epizooties, 1986~. Although the OIE has been cited as having an influence on AB, Wierzbowski (1984) has correctly suggested that the national or state veterinary authorities should be responsible for the health status of
236 / Appendix C AB centers producing germplasm for international trade. In addi- tion, the Food and Agriculture Organization of the United Nations should produce lists of suitable AB centers, with annual updates to establish standards of quality internationally. Irrespective of the offi- cial body that undertakes the task, continuous monitoring of the dis- eases to be excluded and notification of newly recognized pathogenic microorganisms would be essential to the effectiveness of such stan- dards. The international trade in germplasm has functioned with exceptional efficiency, based on the requirements of the importing countries and the standards for AB centers of the exporting countries. Only very rarely is transmission of disease in germplasm recognized as occurring following international transfer (Kupferschmied et al., 1986~. When such cases do occur in a country in which the disease has been controlled or eradicated, the consequences for the disease control authorities of the recipient country can be extremely difficult, and the country may incur considerable financial costs in reinstitut- ing freedom from the disease. Diagnostic tests for many diseases likely to be transmitted by semen are being improved, and new tests are being developed in many laboratories engaged in this research around the world. The problems of searching for agents in semen are manifold, however, and have been the subject of extensive research. Such methods are far too costly and often are grossly inefficient when compared with testing donor animals before and after collection of semen and prior to release of semen (Kahrs and Littell, 1980; Parez, 1984; Richmond et al., 1977; Schultz et al., 1982~. For the majority of viral diseases in domestic animals, there is a concurrent viremia whenever the virus is excreted in semen, and therefore, it is preferable to test for antibodies or virus in the blood. The recent development of technologies, such as the polymerase chain reaction (Erlich, 1989), will enable screening of semen and embryo samples for evidence of specific pathogens and should enhance the confidence in international movement of germplasm. For microorganisms present in the preputial cavity of animals that are commonly transferred into the semen collection, other than viral or protozoa! contaminants, the dilution of semen in extenders tends to lower the probability of infection. In addition, the use of antibiotics provides a control on the proliferation of bacterial and fungal contaminants. For the diseases for which vaccination can be used as a prophylactic measure, such as campylobacteriosis, excre- tion of immunoglobulins into the preputial cavity enables control of the infection. Similarly, if vaccines are used to protect against or control general infections, secreted immunoglobulins will reduce the possibility of semen contamination. In some tropical environments,
Appendix C / 237 the climate and the normal behavior of animals can make it difficult to control microorganisms, for example, water buffalo and bucks in India (Gangadhar et al., 1986; Sharma and Deka, 1986~. In AB centers, there will always be populations of microorgan- isms, many of which will reside in the preputial sheath and on the surface of the penis. The aims of the OIE recommendations and the majority of AB centers are to confine and restrict pathogenic microor- ganisms. Many of the major bacterial diseases of concern in the first AB centers, including brucellosis, tuberculosis, leptospirosis, campylo- bacteriosis, and trichomoniasis, have now been eradicated or are con- trolled, and are no longer considered problem diseases. New disease agents have emerged, however, such as mycoplasmas, ureaplasmas, And Haemophilus spp. In addition, some of the viral diseases not considered previously are now recognized as potentially spread through semen. The most important viral disease in this category is BVDV, and some attempts are now being made to eliminate bulls carrying this agent from AB centers in Britain (Lucas, 1986~. Pestiviruses also cause a disease of great importance in sheep flocks (Border disease), and thus, ram semen should be free of this group of viruses. Bulls that have been infected in utero with BVDV and appear clinically normal and test seronegative will frequently excrete the virus (Littlejohns, 1982~. Although BVDV has been found in semen (McClurkin et al., 1979), it is very difficult to culture from semen and could be present as a contaminant. Thus, BVD seronegative bulls in AB centers could best be checked by attempting to isolate the virus from blood and by carrying out serological testing to ensure freedom from BVDV. Infectious bovine rhinotracheitis virus is known to be a contami- nant of semen when there is a recrudescence of the disease on the penis and prepuce of infected bulls (Snowdon, 1964; Studdert et al., 1964~. Once bulls have exhibited penile infection, the disease can intermittently recur with shedding of virus. Bulls that are seronegati~re for IBR virus are the only animals which can reliably be used for the production of semen for export (Kupferschmied et al., 1986~. Following intranasal IBR virus vaccination of bulls and testing large numbers of semen samples in the Cornell semen test (Schultz et al., 1982), IBRV was not detected in the semen of IBR seropositive bulls. The authors suggested that there may be a variation in the excretion sites between vaccinated (intranasal) and naturally infected bulls. Because of the detection of bluetongue virus in the semen of bulls (Bowen et al., 1983; Breckon et al., 1980; Luedke et al., 1975; Parsonson et al., 1981b), and the possibility of a bull remaining persistently
238 / Appendix C infected and excreting virus intermittently in his semen (Luedke et al., 1975), many countries have required a seronegative status for BTV from all donor bulls. At present, this requirement is essential to ensure semen free of BTV. There are indications, however, that this requirement may in fact be far too rigid and restrict access to desir- able germplasm because experimental and field experience has not verified the "carrier state" for BTN1 (Monke et al., 1986; Parsonson et al., 1987b). Bovine leukosis (BL) virus has not been found to be shed in se- men (Schultz et al., 1982~. Monke (1986) found that seropositive bulls infected with BLV did not transmit the virus in their semen. Ideally, bulls at AB centers should be specific pathogen free, par- ticularly where storing semen for long periods of time is being con- sidered (Bartlett, 1981~. However, because not all viral diseases pro- duce a "carrier state" in bulls, serologic evidence to show that there has not been a rise in antibody titer, but rather that there has been a fall in antibody titer, may be sufficient to enable collection of semen from seropositive bulls. With BVD virus, seropositive bulls, or seronegative bulls from whom virus could not be isolated from the blood, could be used for the long-term storage of semen. For IBR virus, seronegative animals are preferable, but, as Schultz et al. (1982) showed, under some circumstances even these animals could be used. Obviously, a specific virus free status for the important viral agents, as is required in many AB centers in Britain (Lucas, 1986) and Austra- lia, is the ideal status to achieve. Within the United States, the Certified Semen Services provides a voluntary code, Minimum Requirements for Health of Bulls Producing Semen for A.I., for the member organizations of the National Associa- tion of Animal Breeders to establish standards for herd health and standards of hygiene for AB establishments (Howard, 1986~. Although these standards are proposed for bull AB centers, the principles would be applicable for other species of domestic animals used in artificial breeding. REFERENCES Abraham, A., and R. Alexander. 1986. Isolation of parainfluenza-3 virus from bull's semen. Vet. Rec. 119:502. Al-Aubaidi, J. M., K. McEntee, D. H. Lein, and S. J. Roberts. 1972. Bovine seminal vesiculitis and epididymitis caused by Mycoplasma bovigenitalium. Cornell Vet. 62:581-596. Allan, W. H. 1981. Virus diseases of poultry. Pp. 205-216 in Virus Diseases of Food Animals, Vol. 1, E. P. J. Gibbs, ed. London: Academic Press. Anonymous. 1986. Classical swine fever in Shropshire. Vet. Rec. 118:438.
Appendix C / 239 Australian Quarantine and Inspection Service. 1988. Minimum Health Standards for Stock Standing at Licensed or Approved Artificial Breeding Centers in Australia, 3d ed. Canberra: Australian Government Publishing Service. Bacon, L. D., D. W. Salter, J. V. Motta, L. B. Crittenden, and F. S. Ogasawara. 1986. Cryopreservation of chicken semen of inbred or specialized strains. Poult. Sci. 65:1965-1971. Ball, H. J., E. F. Logan, and W. Orr. 1987. Isolation of mycoplasmas from bovine semen in Northern Ireland. Vet. Rec. 121:322-324. Bartlett, D. E., L. L. Larson, W. G. Parker, and T. H. Howard. 1976. Specific pathogen free (SPF) frozen bovine semen: A goal? Proc. Tech. Conf. A. I. Reprod. 6:11-22. Bartlett, D. E. 1981. Bull semen: Specific micro-organisms. Pp. 29~8 in Disease Control in Semen and Embryos. FAO Animal Production and Health Paper 23. Rome, Italy: Food and Agriculture Organization of the United Nations. Bonte, P., M. Vandeplassche, and P. Biront. 1984. Porcine parvovirus in boars. II. Influence on fertility. Zentralbl. Veterinaermed. B. 31:391-395. Bowen, R. A., and T. H. Howard. 1984. Transmission of bluetongue virus by intrau- terine inoculation or insemination of virus-containing bovine semen. Am. J. Vet. Res. 45:1386-1388. Bowen, R. A., T. H. Howard, K. W. Entwistle, and B. W. Pickett. 1983. Seminal shedding of bluetongue virus in experimentally infected mature bulls. Am. J. Vet. Res. 44:2268-2270. Breckon, R. K., A. J. Luedke, and T. E. Walton. 1980. Bluetongue virus in bovine semen: Viral isolation. Am. J. Vet. Res. 41:439~42. Brownlie, J., M. C. Clarke, and C. J. Howard. 1984. Mucosal disease in cattle. Vet. Rec. 115:158. Cheville, N. F., and W. L. Mengeling. 1969. The pathogenesis of chronic hog cholera (swine fever). Histologic, immunofluorescent and electron microscopic studies. Lab. Invest. 20:261-274. Chenoweth, P. J., and G. W. Bulr'~. 17~. 1vilu-plece aDnormallnes 1n Dovme semen following ephemeral fever. Aust. Vet. J. 48:37-38. Clark, B. L. 1985a. Bovine campylobacteriosis. Pp. 1-5 in Australian Standard Diagnos- tic Techniques for Animal Diseases, No. 22. Melbourne, Australia: Common- wealth Scientific and Industrial Research Organization. Clark, B. L. 1985b. Bovine trichomoniasis. Pp. 1 - in Australian Standard Diagnostic Techniques for Animal Diseases, No. 23. Melbourne, Australia: Commonwealth Scientific and Industrial Research Organization. Clark, B. L., J. H. Dufty, M. J. Monsborough, and I. M. Parsonson. 1974. Immuniza- tion against bovine vibriosis.4. Vaccination of bulls against infection with Cam~lahar~t~r fefus subsp. venerealis. Aust. Vet. J. 50:407~09. . ~A ~ ~, . . .. . - -r .7 ~ Clark, B. L., J. H. Dufty, M. J. Monsborough, and I. M. Parsonson. 1976. Immuniza- tion against bovine vibriosis due to Campylobacterfetus subsp.fetus biotype intermedius. Aust. Vet. J. 52:362-365. Clark, B. L., J. H. Dufty, M. J. Monsborough, and I. M. Parsonson. 1979. A dual vaccine for immunization of bulls against vibriosis. Aust. Vet. J. 55:43. Clark, B. L., J. H. Dufty, and I. M. Parsonson. 1983. Immunization of bulls against trichomoniasis. Aust. Vet. J. 60:178-179. Cottral, G. E., P. Gailuinas, and B. F. Cox. 1968. Foot-and-mouth disease virus in semen of bulls and its transmission by artificial insemination. Arch. Gesamte Virusforsch. 23:362-377. Doak, G. A. 1986. Certified Semen Services implementation of new antibiotic combi- nation. Pp. 42-43 in Proceedings of the 11th Technical Conference on Artificial Insemination and Reproduction, Milwaukee, Wisconsin, April 25-26, 1986.
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Appendix C / 241 Kitching, R. P., J. M. Hammond, and W. P. Taylor. 1987. A single vaccine for the control of capripox infection in sheep and goats. Res. Vet. Sci. 40:53-60. Kupferschmied, H. U., U. Kihm, P. Bachmann, K. H. Muller, and M. Ackermann. 1986. Transmission of IBR/IPV virus in bovine semen: A case report. Theriogenology 25:439~43. Lake, P. E. 1986. The history and future of the cryopreservation of ovine germ plasm. Poult. Sci. 65:1-15. Lake, P. E., O. Ravie, and J. McAdam. 1981. Preservation of fowl semen in liquid nitrogen: Application to breeding programmer. Br. Poult. Sci. 22:71-77. Littlejohns, I. R. 1982. Bovine mucosal disease and its familial virus. Pp. 665-667 in Viral Diseases in South-East Asia and the Western Pacific, J. S. McKenzie, ed. Sydney, Australia: Academic Press. Loewen, K. G., and Darcel, C. le Q. 1985. A comparison of two methods for the isolation of bovine herpesvirus 1 (BHV-1) from extended bovine Reman Th~r'n~Pn~lno~' 23:935-943. %, . . ~ ~ A, _ , ~. ~vie ~ ~ ~4 I. ~ ~ &~$ UVULA LVIV5~ Lucas, M. t1. 1986. Control of virus diseases in bulls in artificial insemination centres in Britain. Vet. Rec. 119:15-16. Luedke, A. J., T. E. Walton, and R. H. Jones. 1975. Detection of bluetongue virus in bovine semen. Proc. 20th World Vet. Cong. 38:2039-2042. Luedke, A. J., M. M. Jochim, and R. H. Jones. 1977. Bluetongue in cattle: Effects of Culicoides varzipennis transmitted bluetongue virus on pregnant heifers and their calves. Am. J. Vet. Res. 38:1687-1695. MacLachlan, N. J., C. E. Schore, and B. I. Osburn. 1984. Antiviral responses of blue- tongue virus-inoculated bovine fetuses and their dams. Am. J. Vet. Res. 45:1469- 1473. Mason, I. L. 1974. Conservation of animal genetic resources. Pp. 13-94 in First World Congress on Genetics Annli~1 tO l.i`7=ct^~k Pr^~ll~ti^^ 11~1 ~ MA C:. Graficas Orbe. ~, ~ ~. . . .. _ _ _r, ~vat v~^v$~ ~ VA. i. \~1~11~ ~alll. McClurkin, A. W., M. F. Coria, and R. C. Cutlip. 1979. Reproductive performance of apparently healthy cattle persistently infected with bovine viral diarrhea virus. J. Am. Vet. Med. Assoc. 174:1116-1119. McClurkin, A. W., E. T. Littledike, R. C. Cutlip, G. H. Frank, M. F. Coria, and S. R. Bolin. 1984. Production of cattle immunotolerant to bovine viral diarrhea virus. Can. J. Comp. Med.~48:156-161. McVicar, J. W. 1984. Boar semen. Pp. 70-79 in Proceedings International Symposium on Microbiological Tests for the International Exchange of Animal Genetic Mate- rial, O. H. V. Stalheim, ed. Columbia, Mo.: American Association of Veterinary Laboratory Diagnosticians. McVicar, J. W., and R. J. Eisner. 1977. Foot-and-mouth disease and swine vesicular disease viruses in boar semen. Proc. Annul Meet. U. S. Anim. Health Assoc. 81:221-230. Meegan, J. M., H. Hoogstraal, and M. I. Moussa. 1979. An epizootic of Rift Valley fever in Egypt in 1977. Vet. Rec. 105:124-125. Mengeling, W. L. 1979. Prenatal infection following maternal exposure to porcine parvovirus on either the seventh of fourteenth day of gestation. Can. J. Comp. Med. 43:106-109. Monke, D. R. 1986. Noninfectivity of semen from bulls infected with bovine leukosis virus. J. Am. Vet. Med. Assoc. 188:823-826. Monke, D. R., W. D. Hueston, and J. W. Call. 1986. Veterinary management of an artificial insemination center containing bluetongue seropositive bulls. Proc. Annul Meet. U. S. Anim. Health Assoc. 90:139-143. . .
242 / Appendix C Nielsen, K. H., R. B. Stewart, M. M. Garcia, and M. D. Eaglesome. 1987. Enzyme immunoassay for detection of Mycoplasma bovis antigens in bull semen and prepu- tial washings. Vet. Rec. 120:596-598. Odend'hal, S., ed. 1983. The Geographical Distribution of Animal Viral Diseases. New York: Academic Press. Office International des Epizooties. 1986. International Zoo-Sanitary Code, 5th ed. Paris: Office International des Epizooties. Osburn, B. I. 1986. Report of the Committee on Bluetongue and Bovine Leukosis. Proc. Annul Meet. U. S. Anim. Health Assoc. 90:154-159. Parez, M. 1984. The most important genital diseases of cattle control, treatment and hygiene of semen collection. Pp. 175-203 in Proceedings of the 11th Conference of the Office International des Epizooties, Regional Commission for Europe, Vienna, September 25-28, 1984. Paris: Office International des Epizooties. Parsonson, I. M. 1990. Pathology and pathogenesis of bluetongue infections. Pp. 119- 141 in Current Topics in Microbiology and Immunology, Vol. 162, P. Ross and B. M. Gorman, eds. Berlin: Springer-Verlag. Parsonson, I. M., and W. A. Snowdon. 1974. Ephemeral fever virus: Excretion in the semen of infected bulls and attempts to infect female cattle by the intrauterine inoculation of virus. Aust. Vet. J. 50:329-334. Parsonson, I. M., and W. A. Snowdon. 1975. The effect of natural and artificial breeding using bulls infected with, or semen contaminated with, infectious bo- vine rhinotracheitis virus. Aust. Vet. J. 51:365-369. Parsonson, I. M., J. 'M. Al-Aubaidi, and K. McEntee. 1974. Mycoplasma bovigenitalium: Experimental induction of genital disease in bulls. Cornell Vet. 64:240-264. Parsonson, I. M., A. J. Della-Porta, W. A. Snowdon, and M. L. O'Halloran. 1981a. Experimental infection of bulls with Akabane virus. Res. Vet. Sci. 31:157-160. Parsonson, I. M., A. J. Della-Porta, D. A. McPhee, D. H. Cybinski, K. R. E. Squire, H. A. Standfast, and M. F. Uren. 1981b. Isolation of bluetongue virus serotype 20 from the semen of an experimentally infected bull. Aust. Vet. J. 57:252-253. Parsonson, I. M., A. J. Della-Porta, D. A. McPhee, D. H. Cybinski, K. R. E. Squire, and M. F. Uren. 1987a. Bluetongue virus serotype 20: Experimental infection of pregnant heifers. Aust. Vet. J. 64:14-17. Parsonson, I. M., A. J. Della-Porta, D. A. McPhee, D. H. Cybinski, K. R. E. Squire, and M. F. Uren. 1987b. Experimental infection of bulls and cows with bluetongue virus serotype 20. Aust. Vet. J. 64:10-13. 'Plowright, W. 1968. Rinderpest virus. Pp. 27-110 in Virology Monographs 3, S. Gard, C. Hallauer, and K. F. Meyer, eds. New York: Springer-Verlag. Richmond, J. V., J. W. McVicar, R. J. Eisner, L. A. J., Johnson, and V. G. Pursel. 1977. Diagnostic problems for virus detection in boar semen. Proc. Annul Meet. U. S. Anim. Health Assoc. 81:231-243. Roeder, P. L., and J. W. Harkness. 1986. BVD virus infection: Prospects for control. Vet. Rec. 118:143-147. Schlafer, D. H. 1983. Boar semen. Pp. 65-69 in Proceedings of an International Symposium on Microbiological Tests for the International Exchange of Animal Genetic Material, O. H. V. Stalheim, ed. Columbia, Mo.: American Association of Veterinary Laboratory Diagnosticians. Schultz, R. D., L. S. Adams, G. Letchworth, B. E. Sheffey, T. Manning and B. Bean. 1982. A method to test large numbers of bovine semen samples for viral contami- nation and results of a study using this method. Theriogenology 17:115-123. Scott, G. R. 1964. Rinderpest. Pp. 113-223 in Advances in Veterinary Science, Vol. 9, C. grandly, and E. L. Jungherr, eds. New York: Academic Press.
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