Introduction

The control of semen-borne pathogens in the bovine is an essential task in the cattle artificial insemination (AI) industry to prevent the dissemination of disease to multiple locations. Each year, millions of units of frozen bovine semen are produced and distributed both domestically in the United States and also internationally throughout the world. To reduce the risk of disease spread, standards for the production of semen from donor bulls have been established and donor bulls must complete a rigorous testing program in order to qualify for entry into an AI center (AIC) and be continually monitored while being collected for semen production. As new tests are developed and new research is conducted, testing programs to monitor donor bulls in AICs are continually assessed and improved to reduce the probability of spread of diseases through frozen bovine semen.

In the United States, Certified Semen Services, Inc. (CSS) establishes minimum testing requirements for donor bulls entering and residing in AICs for domestic distribution of semen. Additional testing requirements may have to be met if the frozen semen from the donor bull is intended for international export and these requirements are negotiated by the United States Department of Agriculture (USDA) and the importing country. Many countries establish their own requirements for testing and these are often based on the guidelines published by the World Organisation for Animal Health (OIE) for the collection and processing of bovine semen. One of the intended purposes of these OIE guidelines is to maintain the health of animals in an AIC at a level that allows the international distribution of semen with negligible risk of infecting other animals or humans with pathogens transmissible by semen.¹

In general, donor bulls and animals intended for the use as mount animals undergo testing in three phases. Selected animals are first tested prior to entering quarantine facilities of the AIC. The second phase consists of assembling animals for a quarantine period to included testing throughout and at the end of the quarantine period. And finally, once the animal has entered the resident population in the AIC, it is tested on an annual or semi-annual basis to monitor the disease status of the AIC. Additional testing may be required depending on the requirements of different export markets.

This chapter covers the major diseases of concern that are at risk for transmission through frozen bovine semen. The pathogens involved and the testing strategies to minimize the risk of transmission to other cattle are reviewed.

Bovine viral diarrhea

Bovine viral diarrhea virus (BVDV) is a single-stranded RNA Pestivirus belonging to the Flaviviridae family. It occurs worldwide and is a significant disease with regard to economic consequences in infected herds. Clinical consequences vary and include respiratory, gastrointestinal, and reproductive effects, with the major source of infection coming from persistently infected (PI) animals present in a herd. PI animals will transmit BVDV to susceptible animals in the herd causing an acute transient infection that may lead to reduced pregnancy rates in heifers and cows. Several studies have shown BVDV can be present in semen from bulls that are persistently and acutely infected and are able to transmit BVDV to susceptible recipient cattle.^2–5 In addition, there are reports of bulls that have testicular infections that are able to transmit BVDV in the semen without being viremic.^6,7

PI animals are a result of exposure to the virus through transplacental infection of the developing fetus prior to 125 days of gestation. Bulls that are PI can develop normally but are immunotolerant to the strain of BVDV the animal was exposed to.⁸ The PI bull is capable shedding BVDV for long periods of time and in large amounts, despite producing semen with normal concentration, motility, and morphology.² The effect of using semen from PI bulls and exposing susceptible females is significant. One study showed that 12 of 12 seronegative and virus-negative heifers artificially inseminated with semen from a PI bull seroconverted within 2 weeks after exposure.³ Another study showed that 161 of 162 cows that were inseminated with semen from a PI bull were seropositive compared with 95 of 143 cows that were inseminated with semen from other bulls, and concluded that semen from PI bulls has the potential to transmit BVD infection in susceptible herds.² This highlights the importance of properly screening bulls for PI status prior to entry into an AIC.

Testing requirements for BVD PI status of bulls prior to entry to an AIC includes two tests for the BVDV antigen in serum or whole blood at least 10 days apart. CSS requires that serum or whole blood is tested using a virus isolation (VI), polymerase chain reaction (PCR), or antigen capture enzyme-linked immunosorbent assay (ELISA) test with the first sample collected on the farm of origin within 30 days prior to entry into the AIC isolation facility with a negative result. The bull is sampled for a second BVDV antigen test on serum or whole blood during the isolation period at least 10 days following entry into the AIC isolation facility with a negative result. Because of the interference of maternal antibodies on circulating BVDV in the serum, bulls that are less than 6 months of age must be tested using whole blood while in isolation.⁹

Susceptible bulls exposed to BVDV can become acutely or transiently infected and are viremic for a period of 6–21 days following exposure. The period of viremia also coincides with a significant increase in antibody production to the infecting strain of BVDV, indicating exposure to the virus.¹⁰ BVDV can be detected in semen from acutely infected bulls throughout the duration of viremia, although the levels of virus in the semen are significantly less than those found in bulls that are persistently infected.^2,7,11 As a result, the seroconversion rates of exposed animals inseminated using semen from acutely infected bulls that have virus present are lower than those of animals inseminated using semen from PI bulls (1.8–15.4%).^4,5 Despite the lower seroconversion rates, testing for acutely infected bulls in an AIC is important to prevent the spread of BVDV to susceptible herds.

Testing requirements for BVDV acute infections is similar to testing of PI bulls. CSS requires that the bull must be tested on-farm using VI, PCR, or antigen capture ELISA on serum or whole blood within 30 days of entry into the AIC isolation facility with a negative result. The bull is tested again at least 10 days from the beginning of the period in the AIC isolation facility with a negative result. Similar to testing for PI status, bulls less than 6 months of age require testing to be completed on whole blood. If the result is positive, the bull is isolated from the remaining animals in the isolation facility and tested again at least 21 days later, in addition to the remaining animals in the isolation group. If the second result is positive, this indicates the bull is a PI animal and is not allowed entry into the resident herd. If the second result is negative, the bulls must be tested a third time at least 10 days from the second sample with a negative result prior to entering the production herd.⁹ In the event that BVDV is isolated from a bull as a result of an acute infection, all semen batches collected 30 days prior and 30 days after the positive result are to be tested with VI or PCR before they can be distributed.

Optional testing for BVDV acute infections recommended by the OIE includes a test for the detection of BVDV antibodies. Serum samples are collected prior to or at the beginning of the isolation period and at least 21 days after entry into the isolation facility. Seroconversion indicates exposure during the isolation period and the bull must be retested prior to entering the resident herd. The OIE also recommends that bulls are sampled on an annual basis and tested for BVDV antibodies to monitor for seroconversion while they are residents of the production herd of the AIC.¹

There have been two reports of bulls with persistent testicular infection that were able to shed virus particles in the semen despite absence of a viremic state and the presence of circulating antibodies for BVDV.^6,12 This is believed possible because an antibody response was generated from the initial infection, but virus in the testicles was protected from circulating antibodies due to the blood–testes barrier. Both reports demonstrated that the bulls consistently produced semen containing virus over an extended period of time, although the viral load in the insemination dose was less than that of semen produced by PI bulls but more than that present in semen from acutely affected bulls.⁷ One study reported that use of frozen semen from one of the reported bulls in three heifers resulted in seroconversion in one of the three heifers that were inseminated.¹³

There are reports of bulls with prolonged testicular infection (PTI) that had detectable BVDV virus antigen in the semen for 2.75 years following experimental acute infections. However, BVDV could only be detected in semen using VI testing during the viremic period of infection, although infectious virus could be detected in testicular tissue 12.5 months after exposure.^7,14 The positive result was due to detection of neutralized virus with PCR but subsequent investigation was unable to detect infectious virus 17 days after infection using VI of the semen. Despite this, one experiment in one of the studies demonstrated that raw semen collected from one of the positive PTI bulls at 5 months after infection resulted in the seroconversion of a 6-month-old calf when the semen was injected intravenously.¹⁴ In the other study, a repeated trial resulted in no seroconversions of calves inoculated intravenously with raw semen, and breeding trials using frozen semen from a PTI bull to inseminate heifers also resulted in no seroconversions. It was concluded that the risk of viral transmission from prolonged testicular infections with BVDV may be negligible, but additional information is required to substantiate this conclusion.⁷

CSS requires that bulls housed in an AIC must be tested in one of two ways for PTI prior to semen release for distribution. The first method is to test all bulls in isolation with a BVDV serum neutralization test. Bulls that test positive are required to have one batch code of frozen processed semen tested using VI or PCR. The second method is to require all bulls in isolation, regardless of their antibody status, to have one batch code of frozen processed semen tested using VI or PCR. Any bull found to have persistent testicular infection for BVDV is not eligible for semen collection and is not permitted to remain in the resident production herd.⁹

There are currently no recommendations regarding testing of bulls to determine their PTI status. As more information is made available, further assessment may be required to include additional testing.