Brian Melville and Professor Beverly Roeder, Department of Biology
Dairy cows supply an important food source for humans, and any disease that hurts production threatens this source. Once a cow has a PRMD (production related metabolic disease) there are many signs and symptoms that are readily observed and tested for, but it would be useful to use diagnostic techniques to discover if an animal is at risk of a future disease when the cow is clinically normal. These diagnostic tools could be used with clinically normal healthy appearing cows, empowering producers to make more informed decisions about animal care to improve milk production. Other exciting possibilities exist, such as breeding cattle to have the disease resistant traits. We can use the information from this research to improve and protect this significant food source.
Dr. Roeder has conducted research in this area of study (Roeder et al 2011) to predict resistance or susceptibility to PRMDs using fecal and plasma isotopic signatures. Clinically normal, pregnant Holstein cows and heifers were randomly chosen and their feces, hair, plasma, and red blood cells were analyzed isotopically. Samples were taken 21 days prepartum (before giving birth), at parturition (around the time of giving birth), and 21 days postpartum (after giving birth). Stable isotope ratios of carbon (13C/12C; δ13C) and nitrogen (15N/14N; δ15N) were determined for these samples using an elemental analyzer-isotope ratio mass spectrometer (EA-IRMS). Statistical analysis was done with the program SAS. Health records were used to determine which cows developed PRMDs such as ketosis, mastitis, milk fever, etc. Weekly milk production was also recorded. Using prepartum and parturient isotopic signatures in feces, it was determined that the change in δ13C values could accurately predict approximately twothirds of the cows that would subsequently develop a PRMD in the first five months of lactation.
Our analysis targets fatty acids. Changes in fatty acid metabolism or elevation of fatty acids play a role in many metabolic diseases. Dairy cow metabolic diseases are often associated with high levels of blood serum fatty acids. This combined with Dr. Roeder’s demonstration of predictive carbon isotopic values lead us to believe there might be types of fatty acids, or abnormal quantities of certain fatty acids, that may be predictive for susceptibility or resistance to metabolic diseases. Our lipid analysis has uncovered clues about which fatty acids are present in PRMD resistant or susceptible cows.
Kyle Houston, another student, and I worked with individuals in the Chemistry department to find a good method to extract the lipids from our samples and to analyze them. Chi et al (2005) analyzed pig manure using a capillary electrophoresis method, and we originally planned to use this method. This method would have given us clues to the types of lipids in the samples, but would not have the specificity of more sensitive lipid analyses. Gas chromatography using the methyl-ester derivatives of lipid samples is sometimes used for analysis of lipids, and we explored the idea of using gas chromatography for quite some time. Ultimately we found a lab in the Chemistry department that was already had a successful method for analyzing lipid samples. Dr. John T Prince and his students, including biochemistry major Brendan Coutu, have been doing impressive research in lipidomics using orbitrap mass spectrometry. Their lab had been doing exactly what were wanted to do. They had a great method, and we had a great problem.
Of course we needed to adapt our samples to their protocols. It was still necessary to develop an extraction method for our specific samples (cow feces). Working with Dr. Prince’s student Brendan Coutu, a liquid-liquid extraction method was worked out with some trial and error. The lipid contents of the samples were extracted, and some preliminary analysis with the orbitrap mass spectrometer was done. Because of the quality of the data the extractions were all redone. The samples were analyzed to produce our results.
The orbitrap mass spectrometer analyses of our feces samples gave us some interesting clues into what fatty acids might be involved in PRMDs in dairy cows. Three negatively charged glycerophospholipids, one negatively charged prenol lipid, one positively charged sterol lipid, and one positively charged polyketide lipid were detected that are significantly different between PRMD resistant and susceptible dairy cows. Dr. Roeder, Dr. Prince, and their students continue to work on the project. They obtained more samples from 220 heifers and 220 cows from November 2012 to March 2013 and are currently doing orbitrap mass spectrometer analysis of these samples. We hope results from age and lactation, and parity matched dairy cows will confirm our results.
Looking forward, finding diagnostic techniques and underlying physiological causes will be helpful in the cultivation of dairy cows. Mass spectrometry is not the most efficient way to find health information about a dairy cow. But, If we can confirm a certain lipid, or group of lipids, is in a different concentration a biochemical test might be developed to determine if a cow is more or less susceptible to PMRDs. The feces samples are usually easier to obtain than other types of samples such as blood or milk, which are difficult to obtain or not always available. Dairy cow breeding could take into account the resistance to PRMDs. Lipid differences in PRMD susceptible dairy cows might suggest underlying physiological reasons for their predisposition to PRMDs. Perhaps, a preventative treatment could be used to protect a cow from a future disease, and protecting an expensive investment and important food source.
References
- Chi F, Lin PH, Leu M. 2005. Quick determination of malodor-causing fatty acids in manure by capillary electrophoresis. Chemosphere 60:1262–1269.
- Roeder BL, Yamada N, Ford JCA, Maharjan S, Nguyen JD, Eggett DL, Hatch KA. 2011. Resistance or susceptibility to production-related metabolic diseases (PRMDs) can be predicted with fecal and erythrocyte isotopic signatures. 44th Annual Conf Am Assoc Bovine Practitioners, St. Louis, MO, p 26, poster #9.