No abstract

Simple SummaryMethane is a greenhouse gas with a global warming potential 28 times that of CO2. Enteric methane accounts for 17% of global methane emissions and 3.3% of total global greenhouse gas emissions from human activities. There is, therefore, significant research interest in finding ways to reduce enteric methane emissions by ruminants. Partners in Expert Working Group 2 (WG2) of the European Cooperation in Science and Technology (COST) Action METHAGENE have used several methods for measuring methane output by individual dairy cattle under various environmental conditions. Methods included respiration chambers, the sulphur hexafluoride (SF6) tracer technique, breath sampling during milking or feeding, the GreenFeed system, and the laser methane detector. Respiration chambers are considered the ‘gold standard’, but are unsuitable for large-scale measurements of methane emissions, which are needed for genetic evaluations. In this study, the suitability of methods for large-scale studies was reviewed and compared. All methods showed high correlations with respiration chambers, but comparisons among alternative methods generally had lower correlations. Results confirm, however, that there is sufficient correlation between methods for measurements from all methods to be combined, with appropriate weightings, for use in international genetic studies. This will pave the way for breeding cattle with lower methane emissions.AbstractPartners in Expert Working Group WG2 of the COST Action METHAGENE have used several methods for measuring methane output by individual dairy cattle under various environmental conditions. Methods included respiration chambers, the sulphur hexafluoride (SF6) tracer technique, breath sampling during milking or feeding, the GreenFeed system, and the laser methane detector. The aim of the current study was to review and compare the suitability of methods for large-scale measurements of methane output by individual animals, which may be combined with other databases for genetic evaluations. Accuracy, precision and correlation between methods were assessed. Accuracy and precision are important, but data from different sources can be weighted or adjusted when combined if they are suitably correlated with the ‘true’ value. All methods showed high correlations with respiration chambers. Comparisons among alternative methods generally had lower correlations than comparisons with respiration chambers, despite higher numbers of animals and in most cases simultaneous repeated measures per cow per method. Lower correlations could be due to increased variability and imprecision of alternative methods, or maybe different aspects of methane emission are captured using different methods. Results confirm that there is sufficient correlation between methods for measurements from all methods to be combined for international genetic studies and provide a much-needed framework for comparing genetic correlations between methods should these become available.

Primary bovine mammary epithelial cells (pbMEC) are often used in cell culture to study metabolic and inflammatory processes in the udder of dairy cows. The most common source is udder tissue from biopsy or after slaughter. However, it is also possible to culture them from milk, which is non-invasive, repeatable and yields less contamination with fibroblasts. Generally, not much is known about the influence of cell origin and cell culture techniques such as cryopreservation on pbMEC functionality. Cells were extracted from milk and udder tissue to evaluate if milk-derived pbMEC are a suitable alternative to tissue-derived pbMEC and to test what influence cryopreservation has. The cells were cultivated for three passages and stored in liquid nitrogen. The relative gene expression of the five target genes kappa-casein, lingual antimicrobial peptide (LAP), lactoferrin, lysozyme (LYZ1) and the prolactin receptor normalised with keratin 8 showed a tendency to decrease in the tissue cultures, but not in the milk-derived cultures, suggesting a greater influence of the cultivation process on tissue-derived cells, freezing lowered expression levels in both cultures. Overall expression of LAP and LYZ1 tended to be higher in milk cells. Cholesterol efflux was measured to compare passages one to seven in milk-derived cells. Passage number did not alter the efflux rate (p ≤ 0.05). We showed for the first time that the extraction of pbMEC from milk can be a suitable alternative to tissue extraction.

Bovine mastitis, the inflammation of the udder, is a major problem for the dairy industry and for the welfare of the animals. To better understand this disease, and to implement two special techniques for studying mammary gland immunity in vitro, we measured the innate immune response of primary bovine mammary epithelial cells (pbMEC) from six Brown Swiss cows after stimulation with the heat-inactivated mastitis pathogens, Escherichia coli 1303 and Staphylococcus aureus 1027. The cells were extracted and cultivated from milk instead of udder tissue, which is usually done. The advantages of this technique are non-invasiveness and less contamination by fibroblasts. For the first time, pbMEC gene expression (GE) was measured with a microfluidic high-throughput real-time reverse transcription-quantitative PCR platform, the BioMark HD TM system from Fluidigm. In addition to the physiological analysis, the precision and suitability of this method was evaluated in a large data set. The mean coefficient of variance (6 s.e.) between repeated chips was 4.3 6 0.4% for highly expressed and 3.3 6 0.4% for lowly expressed genes. Quantitative PCR (qPCR) replicate deviations were smaller than the cell culture replicate deviations, indicating that biological and cell culture differences could be distinguished from the background noise. Twenty-two genes (complement system, chemokines, inflammatory cytokines, antimicrobial peptides, acute phase response and toll-like receptor signalling) were differentially expressed (P , 0.05) with E. coli. The most upregulated gene was the acute phase protein serum amyloid A3 with 618-time fold. S. aureus slightly induced CCL5, IL10, TLR4 and S100A12 expression and failed to elicit a distinct overall innate immune response. We showed that, with this milk-derived pbMEC culture and the high-throughput qPCR technique, it is possible to obtain similar results in pbMEC expression as with conventional PCR and with satisfactory precision so that it can be applied in future GE studies in pbMEC.Keywords: bovine mastitis, gene expression profiling, microfluidic qPCR, primary bovine mammary epithelial cells, innate immune response ImplicationsWe show that a time-and cost-efficient high-throughput quantitative PCR (qPCR) system, applied on primary bovine mammary epithelial cells (pbMEC) cultured from milk, is a convenient alternative to the two major standard procedures in measuring gene expression. We obtained similar results as studies with pbMEC from udder tissue and measurements on DNA microarrays or conventional qPCR. We suggest that the milk-derived pbMEC culture and the microfluidic high-throughput qPCR system could be applied in future experiments with pbMEC.

No abstract

The handheld, portable laser methane detector (LMD) was developed to detect gas leaks in industry from a safe distance. Since 2009, it has also been used to measure the methane (CH4) concentration in the breath of cattle, sheep, and goats to quantify their CH4 emissions. As there is no consensus on a uniform measurement and data-analysis protocol with the LMD, this article discusses important aspects of the measurement, the data analysis, and the applications of the LMD based on the literature. These aspects, such as the distance to the animal or the activity of the animals, should be fixed for all measurements of an experiment, and if this is not possible, they should at least be documented and considered as fixed effects in the statistical analysis. Important steps in data processing are thorough quality control and reduction in records to a single point measurement or “phenotype” for later analysis. The LMD can be used to rank animals according to their CH4 breath concentration and to compare average CH4 production at the group level. This makes it suitable for genetic and nutritional studies and for characterising different breeds and husbandry systems. The limitations are the lower accuracy compared to other methods, as only CH4 concentration and not flux can be measured, and the high amount of work required for the measurement. However, due to its flexibility and non-invasiveness, the LMD can be an alternative in environments where other methods are not suitable or a complement to other methods. It would improve the applicability of the LMD method if there were a common protocol for measurement and data analysis developed jointly by a group of researchers.

Mastitis is the most cost intensive production disease in dairy industry. Medical treatment, reduced fertility, extra labour, and reduced milk yield cause a considerable financial burden. Calculations of annual losses due to mastitis revealed an amount of 10% of total value of farm milk sales, two thirds being a result of reduced milk yield caused by subclinical udder inflammation (Schroeder, 2010). During early lactation, high energy requirements for milk production cannot be adjusted by increasing feed intake and result in negative energy balance (NEB) often followed by metabolic imbalance. Energy deficit leads to extensive mobilization of body fat reserves and may result in increased blood nonesterified fatty acid (NEFA) and β-hydroxybutyrate (BHB) concentrations. Elevated NEFA and BHB levels are considered to have inhibiting effects on immune cells (Suriyasathaporn et al., 2000) and to assist the state of impaired immune system (Loor et al., 2007;Roche et al., 2009 ABSTRACT: Negative energy balance (NEB), if followed by metabolic imbalance, is a common problem in high-yielding dairy cows frequently associated with inflammation of the mammary gland. After entering the teat canal, mammary epithelium is the first line of defense against a pathogen invasion. To investigate the effect of NEB on the innate host defense of the mammary epithelium, primary bovine mammary epithelial cell (pbMEC) cultures were generated by cell extraction of milk derived from energy restricted and control feeding cows. pbMEC were obtained from 8 high-yielding dairy cows affected by induced NEB in mid-lactation due to a reduction to 51 ± 2% of total energy requirement (restriction group) and from 7 control cows (control group). They were exposed to heat-inactivated Escherichia coli and Staphylococcus aureus for 24 and 72 h to investigate the influence of NEB on gene expression profiles of cytokines, chemokines, genes associated with apoptosis and antimicrobial peptides plus their receptors (AMPR) of the innate immune response. The immune challenge of pbMEC demonstrated an effect of immune capacity and NEB in 15 differential expressed genes. NEB induced a substantial up-regulation in restriction compared to control cells by trend in E. coli and a down-regulation in S. aureus exposed cells. Our investigations showed that the dietary-induced NEB in vivo influenced the immune response of pbMEC in vitro and altered the expression of immunological relevant genes due to a difference in energy supply. These results demonstrate that pbMEC are a suitable model for mastitis research, in which even effects of feeding regimes can be displayed.

Characteristics of low methane emitting cows as categorized by midinfrared spectra and respiration chamber measurements. Denninger et al. Mid-infrared spectra (MIR) were used to identify low and high methane emitting dairy cows within the Swiss, Brown Swiss population. Thirty individuals were selected for methane measurements using respiration chambers and laser methane detectors. The MIR predictions were fairly persistent across different environments and differently developed equations. However, correlations with methane measurements were too weak to use MIR as a tool to select low emitting cows. Cows categorized as low emitters by respiration chamber data expressed distinct characteristics in digestion and efficiency.