Mel DeJarnette, Dr. Kristina McDonald, Dr. David Brown, and Dr. Tony Good
Select Sires, USA
We empathize with NZ dairy producers and the industry as they find their way forward and work through herd depopulations in attempts to eradicate Mycoplasma bovis. In a number of media posts there are implications that germplasm may be involved in the transmission. There are many more theories than facts being shared and with social media, those do not stop at the NZ shoreline. Many of you may have questions, or may be asked questions about Mycoplasma bovis. What follows is a brief white paper, so to speak, highlighting Select Sires’ experiences and research related to mycoplasma in bovine semen.
What are mycoplasma?
Mycoplasma are fastidious organisms that lack a cell wall. They attach to host cells in a parasitic nature. This characteristic makes them very difficult to detect using conventional culture techniques and extremely resistant to many in vivo antibiotic treatments. Not all mycoplasma are created equal. Indeed there are literally hundreds of strains and variants of mycoplasma, yet only a few have been deemed of significant pathologic concern. Of relevance to the current conversation is Mycoplasma bovis which has been shown to be a causative agent for pneumonia and joint issues in calves and mastitis in cows.
Control of mycoplasma in frozen bovine semen processed in US AI centers.
Aside from the discovery that glycerol allows sperm to survive the freezing process, the addition of antibiotics to semen extenders, even before we started to freeze them, was one of the greatest advances in male reproduction in our lifetime. Although tremendous efforts go into health testing to ensure bulls are free of relevant potentially contagious diseases, bulls do not live in a sterile environment and thus collecting semen is not a sterile process. Any organism in the environment can also be in the semen and the addition of antibiotics to semen and extenders was attributed to significant improvements in field fertility of non-frozen semen in the 1950’s.
In the 1980’s, Certified Semen Services (CSS; the regulatory authority of US AI centers) sanctioned an extensive antibiotic trial that served as the basis for the present protocol that includes addition of a gentamycin, tylosin, lincomycin, and spectinomycin (GTLS) cocktail into semen and for frozen semen extenders (Shin et al., 1988). The minutely defined quantities of the GTLS cocktail was determined to provide effective “control” of growth of relevant microorganisms, including Mycoplasma bovis, in “frozen” semen without having significant negative impact on the sperm cells themselves. The key words are control and frozen. Antibiotics do not kill all organisms, including mycoplasma, but serve to control their replication and growth while in solution. Similarly, the freezing process itself at liquid nitrogen temperatures serves to kill a few cells but totally prevents replication of all cells. Finally, the dilution process itself, necessary to capitalize on efficient distribution of superior genetics, also serves to reduce the infectious dose of any organisms that may happen to survive the entire process.
It is important to note that the studies above were conducted in recognition of a separate study (Hirth et., 1967) that concluded it is nearly impossible to intentionally infect cows with Mycoplasma bovis using semen as a vector. The combination of these studies led CSS to conclude the transmission of Mycoplasma bovis, even if present in frozen bovine semen, is of negligible risk and thereby concluded testing sires/semen for mycoplasma was unnecessary, unwarranted, and offers no return on investment for the dairy industry.
It is important for readers to understand that the biosecurity of CSS approved and processed semen is NOT the result of a single factor but rather the combination of many factors, including but not limited to:
Health testing of the sire
Hygiene of the sire and the collection process
Antibiotic addition to semen and extenders
Freezing control of replication
Dilution ratio of organisms below minimally infectious dosages
It is also important to note that since approval of the GTLS cocktail, there have been in excess of 1.085 billion doses of frozen bovine semen produced and sold under the CSS logo without a single documented case of transmission of mycoplasma bovis (NAAB statistics).
What about non-frozen semen?
The use of non-frozen semen was not addressed in the 1980’s CSS study of antibiotic efficacy for controlling growth of microorganisms in frozen semen. However, it was immediately recognized that the absence of freezing removes one of several very important control mechanisms. Thereby, CSS has offered no officially approved procedures for a non-frozen semen program. Instead, CSS provided guidelines for fresh semen programs to be evaluated on a trial/research basis provided each sire tests negative for “mycoplasma” on three consecutive weekly tests.
There are several very important qualifiers to understand this recommendation/guideline for non-frozen semen. First, CSS made no distinction between Mycoplasma bovis and other non-pathogenic strains of mycoplasma. Second, the guideline was proposed prior to polymerase chain reaction (PCR) when culture was the “gold standard” in diagnostic procedures. Finally, the guidelines were NOT imposed to conclude that fresh semen represents an increased risk relative to frozen semen but simply an “unknown” risk relative to frozen semen. In the absence of freezing, it was simply logical to question the level of control.
What is Select Sires’ interest in this field of study?
In the late 1990’s to early 2000’s, Select Sires was very interested in replicating the fresh semen programs used commercially in New Zealand. We had no illusion of improving fertility relative to frozen semen. Instead we were simply interested in capitalizing on the efficiencies gained by maintaining equal fertility at lower cell number dosages and thereby more efficient utilization of limited supply high-value genetics.
Following CSS research guidelines and state of the art culture technologies of the time, our endeavors were abruptly halted by reality. Using a split ejaculate technique, we promptly found that laboratories varied tremendously in their ability to detect mycoplasma even when present. Some labs were very efficient while other labs could not replicate the results. It did not take long to deduce that if you want a negative result, send the sample to lab A, but if you want to know the truth send it to lab B. Additionally, we concluded that even in the best of labs, presence was not always detected using culture even when known to be present. We had bulls that tested negative for 10 or more consecutive tests, then went positive and then back to negative.
It is also important to note that in these initial studies we were generically looking for mycoplasma. The CSS guideline did not specify that their recommendations targeted Mycoplasma bovis over any other strain and thus neither did we. The hurdle to the introduction of a CSS approved non-frozen semen program in the US seemed insurmountable and our interest waned.
Enter Dr. Kristina McDonald
Shortly after the futility of the endeavor described above, Select Sires was fortunate to have Kristina McDonald respond to an ad for a laboratory semen processing technician. Kristina came to us with a microbiology degree and an interest to continue and build upon our efforts of the past. Select Sires sponsored Kristina’s MS and PhD at The Ohio State University. Her master’s degree targeted the exploitation of emerging PCR based technologies that would be much more accurate and specific diagnostics than culture alone. This PCR technique was then used in her PhD studies that surveyed the bull population in our facilities for the presence of various mycoplasma species. As a tangent to the official research efforts toward her degree, Kristina also continued to lead Select Sires’ proprietary efforts toward the development of semen processing protocols that could be deemed effective by CSS for control of Mycoplasma bovis in non-frozen semen. Though much of Dr. McDonald’s research is yet to be published, below are several important and relevant findings.
Using semen samples spiked with known quantities of Mycoplasma bovis, Dr. McDonald confirmed the efficacy of CSS antibiotic cocktail to control growth of Mycoplasma bovis, Mycoplasma bovigenitalium, ureaplasma, Haemophilus somni and Campylobacter fetus in semen and frozen semen extenders.
With over 250 total sires tested by qPCR, mycoplasma bovis has not been detected in the semen of any sire on our premises.
What about milk extender?
Select Sires uses a proprietary formulated milk-based extender. Through years of research and commercial application we have documented that this extender provides a significant fertility advantage to our product and to our customers. Nonetheless, milk comes from cows and cows can be exposed to numerous pathogens. The DNA fragments from these on-farm nuisance organisms that were destroyed during pasteurization may occasionally be found in extender using today’s highly sensitive PCR based technologies. The source of our milk is commercially processed USDA approved, pasteurized, homogenized whole milk. Thereby, these are NOT live organisms and the product is perfectly safe for human consumption or use in semen extenders.
Back to New Zealand
New Zealand discovered Mycoplasma bovis in a herd on the South Island in July 2017 and further investigations have raised the total count to 38 reported properties infected as of June 2018. These discoveries have created quite a stir in the New Zealand dairy industry as it has been previously declared that the country was “free” of Mycoplasma bovis. However, this declaration was made years ago when the efficacy and specificity of testing procedures were much less precise than those of today. It was indeed puzzling to many in our industry back then how such confidence could be claimed. How do you prove a negative using tests that often cannot confirm a positive? How do you become proficient at detection of a disease that purportedly does not exist in the environment? Thus, many in our industry wonder if this is really a “new” infection or simply a reflection of the ability of modern more sophisticated PCR based technologies to detect what has likely been present in New Zealand all along. It would be interesting to know what we’d find if we subject some 20 to 30-year old NZ cheese or powdered milk to these new procedures. If we found something would it help to relieve concerns and clarify steps forward?
Otherwise, there is broad speculation as to how the disease found its way into the country. Cattle movements, vaccines, and frozen semen and embryos have all been implicated but with no clear conclusion to date. There is some speculation there could be calls for increased testing for mycoplasma for imported semen. There is no question this would be easy enough to do, however the science simply does not support that this is necessary or offers any return on investment for our customers. Yielding for mycoplasma places the industry on the slippery slope to test for the next and the next and the next disease of perceived concern until the cost of testing becomes burdensome to the end user who ultimately has to pay the real cost.
While the rest of the world has learned to manage and deal with Mycoplasma bovis, this is new for New Zealand. The Ministry of Primary Industries (MPI, equivalent to our USDA) is operating based on confidence in the prior decree of a Mycoplasma bovis-free island and wants to return to that status via depopulation of herds and eradication of the disease. Producers are rightly concerned and confused about their industry and their future. It is easy to second guess these situations but that generally only creates more frustrations and confusion while solving no one’s real issues.
The Finnish publication
A recent publication out of Finland is casting even greater confusion and consternation on this subject. It is very important to distinguish that the Finnish publication was NOT a controlled research trial but rather a summary of the efforts and conclusions from an anecdotal, after the fact search for the source for a known Mycoplasma bovis infection in a herd in Finland (Haapala et al., 2018). Their observation that antibiotics are bacteriostatic and not bactericidal was not novel and it is well-documented that one can occasionally culture microbes once removed from the antibiotics. However, there was no direct evidence in a control situation that the semen in question could be used to infect females with Mycoplasma bovis. The observations and conclusions were based totally on circumstantial evidence and are not irrefutable.
An important take home message for Select Sires’ customers is that the clear majority of published research and Select Sires’ on-going research efforts reconfirm that semen processed according to CSS protocol presents negligible risk for transmission of any disease including Mycoplasma bovis. Select Sires and our customer-owners empathize with our New Zealand counterparts and wish them wisdom, patience, and good fortune in their endeavors for a speedy resolution to this troubling industry issue.
Who is Select Sires?
Select Sires is one of the largest dairy farmer owned cooperatives in the world. In effect it is a federation of nine farmer owned and controlled cooperatives. The cooperative sells more than 19m straws of semen annually to 90 countries around the world and, over several decades, has developed semen production and processing procedures which are amongst the most rigorous in the world; procedures verified and endorsed by the US AI industry standards organization NAAB (National Association of Animal Breeders). Select Sires is represented in New Zealand by World Wide Sires NZ.
Haapala, V., T. Pohjanvirta, N Vähänikkilä, J. Halkilahti, H. Simonen, S. Pelkonen, T. Soveri, H. Simojoki and T. Autio. 2018. Semen as a source of Mycoplasma bovis mastitis in dairy herds. Veterinary Microbiology 216:60-66.
Hirth, R. S., W. N. Plastridge, & M. E. Tourtellotte. 1967. Survival of mycoplasma in frozen bovine
semen. American Journal of Veterinary Research, 28:97-99.
Shin, S. J., D. H. Lein, V. H. Patten, and H. L. Ruhnke. 1988. A new antibiotic combination for frozen bovine semen: 1. Control of mycoplasmas, ureaplasmas, Campylobacter fetus subsp. Venerealis and Haemophilius somnus. Theriogenology 29:577-591.