Assessment of Stress Levels in Lactating Cattle: Analyzing Cortisol Residues in Commercial Milk Products in Relation to the Temperature-Humidity Index.

Chronic stress in the dairy cattle industry has negative impacts on animal health, productivity, and welfare. It has been confirmed that cortisol transfers to milk and resists the high temperature during milk processing. This study evaluated the relationship between the milk cortisol concentration (MCC) in commercial milk products and the temperature-humidity index (THI) at the time of milk production. Eleven commercially produced pasteurized and sterilized milk products, purchased in Chuncheon, Korea, with production dates ranging from July to October 2021 were analyzed. The MCC was extracted using diethyl ether and analyzed using an enzyme immunoassay. The average THI values based on microclimate data provided by the Korea Meteorological Administration were 77 ± 0.8, 75 ± 1.4, 69 ± 1.4, and 58 ± 1.8, in July, August, September, and October, respectively. The average MCC levels were 211.9 ± 95.1, 173.5 ± 63.8, 109.6 ± 53.2, and 106.7 ± 33.7 pg/mL in July, August, September, and October, respectively. The MCC in July was higher than in August, September, and October (p < 0.05), while it was lower in September and October than in August (p < 0.05). Significant variations in the MCC were observed in commercial milk products across the four production months (p < 0.05), except for two milk products. Overall, monitoring the cortisol residue in commercial dairy milk products can be an alternative indicator of stress in dairy cattle of farms.

Measurement of greenhouse gas emissions from a dairy cattle barn in Korea.

Korea is currently developing country-specific emission factors to support the 2050 zero-carbon campaign. Dairy cattle represent one of the largest livestock industries in Korea, and the industry is estimated to continue increasing because of an increase in milk demand. However, country-specific emission factors for dairy cattle are currently only available for calculating methane (CH4) emissions from enteric fermentation. Two experiments were conducted to evaluate CH4 and nitrous oxide (N2O) fluxes from sawdust-bedded barn in dairy cow and steer, as well as dairy cattle manure composting lots. The greenhouse gas (GHG) fluxes were quantified using the open-chamber method and gas chromatography. CH4 fluxes from steer, dairy cow, and manure compost were 27.88 ± 5.84, 36.12 ± 10.85, and 259.44 ± 61.78 µg/head/s, respectively. N2O fluxes from steer, dairy cow, and manure compost were 14.04 ± 1.27, 4.11 ± 1.57, and 3.97 ± 1.08 µg/head/s, respectively. The result of this study can be used to construct country-specific data for GHG emissions from manure management. Thus, the application of mitigation strategies can be prioritized based on the GHG profile and targeted source.

Stress Concepts and Applications in Various Matrices with a Focus on Hair Cortisol and Analytical Methods.

When studying stress in animals, it is important to understand the types of stress and their classification, and how to assess the stress levels in different animal species using different matrices accurately and precisely. The classification of stress types helps to distinguish between good stress (eustress) and bad stress (distress). Hence, first, it is crucial to assess the animal's level of stress in a non-intrusive manner and second to identify the type of stress that is best suited to its environment. Third, it is also important to analyze the obtained samples using a suitable method to increase the validity of stress hormone measurements. Therefore, in this review, we aim to: (1) explain the classification of stress, (2) discuss the wide range of body matrices (e.g., saliva, milk, hair, urine, feces, sweat, fins, etc.) that can be used as samples to evaluate stress levels, as well as their comparisons and limitations, and present the reliable matrices for measuring stress hormones with special emphasis on hair, (3) compare the analytical methods for measuring stress hormones after sample preparation. Despite some literature that does not include hair as a reliable matrix for evaluating stress levels, hair is one of the matrices for measuring long-term stress hormone accumulations. This review discusses some factors that influence the level of stress hormones in the hair. By understanding these issues, the scientific community will not only be able to improve the understanding of stress and biomarker evaluation but also suggest how to deal with the consequences of stress in future research.

Estimating milk production losses by heat stress and its impacts on greenhouse gas emissions in Korean dairy farms.

Meteorological disasters caused by climate change like heat, cold waves, and unusually long rainy seasons affect the milk productivity of cows. Studies have been conducted on how milk productivity and milk compositions change due to heat stress (HS). However, the estimation of losses in milk production due to HS and hereby environmental impacts of greenhouse gas (GHG) emissions are yet to be evaluated in Korean dairy farms. Dairy milk production and milk compositions data from March to October 2018, provided by the Korea Dairy Committee (KDC), were used to compare regional milk production with the temperature-humidity index (THI). Raw data for the daily temperature and relative humidity in 2018 were obtained from the Korea Meteorological Administration (KMA). This data was used to calculate the THI and the difference between the maximum and minimum temperature changing rate, as the average daily temperature range, to show the extent to which the temperature gap can affect milk productivity. The amount of milk was calculated based on the price of 926 won/kg from KDC. The results showed that the average milk production rate was the highest within the THI range 60-73 in three regions in May: Chulwon (northern region), Hwasung (central region), and Gunwi (southern region). The average milk production decreased by 4.96 ± 1.48% in northern region, 7.12 ± 2.36% in central region, and 7.94 ± 2.57% in southern region from June to August, which had a THI range of 73 or more, when compared to May. Based on the results, the level of THI should be maintained like May. If so, the farmers can earn a profit of 9,128,730 won/farm in northern region, 9,967,880 won/farm in central region, and 12,245,300 won/farm in southern region. Additionally, the average number of cows raised can be reduced by 2.41 ± 0.35 heads/farm, thereby reducing GHG emissions by 29.61 ± 4.36 kg CO2eq/day on average. Overall, the conclusion suggests that maintaining environmental conditions in the summer that are similar to those in May is necessary. This knowledge can be used for basic research to persuade farmers to change farm facilities to increase the economic benefits and improve animal welfare.

Selection of appropriate biomatrices for studies of chronic stress in animals: a review.

Cortisol and corticosterone, hormones traditionally considered biomarkers of stress, can be measured in fluid biomatrices (e.g., blood, saliva) from live animals to evaluate conditions at sampling time, or in solid biomatrices (e.g., hair, feather) from live or dead animals to obtain information regarding long-term changes. Using these biomarkers to evaluate physiological stress responses in domestic animals may be challenging due to the diverse characteristics of biomatrices for potential measurement. Ideally, a single measurement from the biomatrix should be sufficient for evaluating chronic stress. The availability of appropriate and cost-effective immunoassay methods for detecting the biomarkers should also be considered. This review discusses the strengths and limitations of different biomatrices with regard to ensuring the highest possible reliability for chronic stress evaluation. Overall, solid biomatrices require less frequent sampling than other biomatrices, resulting in greater time- and cost-effectiveness, greater ease of use, and fewer errors. The multiplex immunoassay can be used to analyze interactions and correlations between cortisol and other stress biomarkers in the same biomatrix. In light of the lack of information regarding appropriate biomatrices for measuring chronic stress, this review may help investigators set experimental conditions or design biological research.

Effects of feather processing methods on quantity of extracted corticosterone in broiler chickens.

Corticosterone is known as a biological stress index in many species including birds. Feather corticosterone concentration (FCC) has increasingly been used as a measure for chronic stress status in broiler chickens. As sample preparation is the first step of analytical process, different techniques of feather matrix disruption need to be validated for obtaining better result in analysing corticosterone extraction. The current study was a validation of pulverizing the feather by bead beater (BB) and surgical scissors (SS) processing prior to corticosterone extraction in feather of broiler chickens. The type of feather processing prior to the hormone extraction may alter the final output. Thereby, finding a standard method according to laboratory facilities is pivotal. This study carried out to determine the effects of feather pulverization methods on the extraction amount of corticosterone in broiler chickens. Feathers were sampled from four weeks old Ross 308 broiler chickens (n = 12 birds). All broiler chickens were kept under the same environmental condition and had access to feed and water. Feather samples were assigned to one of the following processing methods 1) using a BB for pulverizing and 2) using a SS for chopping into tiny pieces. Each sample was duplicated into two wells during enzyme immunoassay (EIA) analysis to improve the accuracy of the obtained data. The results showed lower standard errors and constant output of FCC by using the BB method compared with the SS method. Overall comparison of FCC showed a significantly higher (p < 0.001) amount of the FCC in the BB compared with the SS. Overall, using the BB method is recommended over the SS method for feather processing due to the ability to homogenize a large number of samples simultaneously, ease of use and greater extraction of feather corticosterone.

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices.

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 ± 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80 rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 °C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 ± 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 °C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

Methodological validation of measuring Hanwoo hair cortisol concentration using bead beater and surgical scissors.

Different methodologies in hair cortisol extraction may alter the final output. Thus finding the standard methodology according to a laboratory facilities is pivotal. This study was carried out to validate the feasibility of two methods of grinding hair for cortisol extraction in Korean native (Hanwoo) cattle. Hair from nine cattle including mature cows, heifers, and calves were assigned to one of the following methods for grinding hair; 1) using bead beater (BB) and 2) using surgical scissors (SS). Hair samples (> 1 g) were harvested from forehead of each individual twice (first and second measurement) to validate the results. To improve the accuracy of the obtained data, each sample was duplicated into two wells during enzyme immunoassay (EIA) analysis. Overall comparison of hair cortisol concentration (HCC) showed that the data within the range (out of the range) of standards provided by the EIA kit were 88.9% (11.1%) and 66.7% (33.3%) for BB compared with SS, respectively. In the first measurement, application of BB was tended to show higher (p = 0.056) amount of HCC compared with SS. In the second measurement application of BB showed higher (p = 0.0028) amount of HCC compared with SS. Among the cattle, calves showed higher HCC using BB compared with SS (p < 0.05). Application of BB in hair grinding methodology for Hanwoo cattle may improve cortisol extraction in comparison to application of SS method, with more consistency. Thus, it would be the preferable method to use.