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Methane Emission and Metabolic Status in Peak Lactating Dairy Cows and Their Assessment Via Methane Concentration Profile

Methane Emission and Metabolic Status in Peak Lactating Dairy Cows and Their Assessment Via... Acta Veterinaria-Beograd 2023, 73 (1), 71-86 UDK: 636.2.09:616-008.9:547.211 DOI: 10.2478/acve-2023-0006 Research article METHANE EMISSION AND METABOLIC STATUS IN PEAK LACTATING DAIRY COWS AND THEIR ASSESSMENT VIA METHANE CONCENTRATION PROFILE 1 1 2 Dušan BOŠNJAKOVIĆ , Danijela KIROVSKI , Radiša PRODANOVIĆ , 2 2 1 1 Ivan VUJANAC , Sveta ARSIĆ , Milica STOJKOVIĆ , Slavica DRAŽIĆ , 2 1 Sreten NEDIĆ *, Ljubomir JOVANOVIĆ University of Belgrade, Faculty of Veterinary Medicine, Department of Physiology and Biochemistry, Bul. Oslobodjenja 18, Belgrade, Serbia; University of Belgrade, Faculty of Veterinary Medicine, Department of Ruminants and Swine Diseases, Bul. Oslobodjenja 18, Belgrade, Serbia (Received 22 October, Accepted 21 December 2022) Ruminant husbandry contributes to global methane (CH ) emissions and beside its negative impact on the environment, enteric CH emissions cause a loss of gross energy intake in cows. The study is aimed to estimate CH emission and metabolic status in dairy cows via the methane concentration profi le as a tool for analyzing the CH production pattern. The study included eighteen cows whose enteric CH emission was measured during three consecutive days in three periods: 2 hours before (P1), 2–4 hours (P2) and 6–8 hours (P3) after the morning feeding. Based on CH enteric emissions, cows were divided into two groups (n=6, respectively): HM (average CH concentration: 5430.08 ± 365.92 ppm) and LM (average CH concentration: 1351.85 ± 205.20 ppm). Following CH measurement, on day 3, venous blood was sampled to determine the indicators of the metabolic status. HM cows had signifi cantly higher average CH concentrations, maximum and average CH peak amplitude than LM cows 4 4 in all measuring periods (P1-P3), while the number of CH peaks tended to be higher in HM than in LM cows in P2. There were no differences in the maximum and average CH peak width and average distance among two CH peaks between examined groups 4 4 of cows. HM cows had signifi cantly higher total protein concentrations and signifi cantly lower total bilirubin and NEFA concentrations than LM cows. In conclusion, HM cows have a greater number of eructations and release more CH per eructation than LM cows, hence the differences in metabolic status are most likely related to the differences in their liver function. Keywords: dairy cows, methane emission, metabolic status *Corresponding author: e-mail: sreten.nedic@vet.bg.ac.rs 71 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 INTRODUCTION Methane (CH ) is one of the most potent greenhouse gases (GHG), with a warming potential of 25 to 28 times greater compared to carbon dioxide (CO ) [1]. Ruminant husbandry releases approximately 80 million tons of CH into the Earth’s atmosphere annually, representing around 28% of the total anthropogenic CH emissions [2,3]. Enteric CH is predominantly generated in the rumen (87-90%) and, to a lesser extent, in the large intestine (10-13%) by the metabolic activity of methanogenic microorganisms such as archaea [1,4]. The rumen represents an anaerobic environment made up of microorganisms that participate in the fermentation processes by degrading the feed organic matter such as plant structural carbohydrates, proteins and other organic polymers to volatile fatty acids (VFAs) and gases, including CO and molecular hydrogen (H ) [5]. Released VFAs such as acetate, propionate, and butyrate are absorbed by the rumen and are utilized as an energy source for milk production in dairy cows. On the other hand, methanogenic archaea use H to reduce CO and thereby produce CH , as one of the fi nal products of ruminal fermentation, 2 4 in a process known as methanogenesis [6,7]. Enteric CH produced in the rumen is almost completely eliminated by eructation (95-99%) providing a possibility that methanogenesis could also be seen as a loss of gross energy intake of 2% to 12% [8]. Considering the environmental effects of enteric CH emissions, as well as the loss of gross energy intake, it is important to examine all the factors that infl uence methanogenesis. The large proportion of the variation in CH emissions can be explained by diet composition and feed intake but there is additional variation among animal phenotypes. Although methanogenesis and energy metabolism are closely related, to the best of our knowledge no research on dairy cows has been performed to examine the differences in the blood metabolic parameters in cows with different levels of enteric CH emissions. There is only one research conducted by Kim et al. [9] on Japanese Black steers which showed that these cattle have different metabolic statuses based on the enteric CH emissions. Since lactating cows are exposed to metabolic challenges [10], it is reasonable to expect some differences in their metabolic status in relation to the enteric CH emissions level. Despite the numerous studies related to CH emissions, the methodology of determining CH emissions from individual animals is very diverse. Methods could be classifi ed into long-term, intended for measuring CH emissions during a 24-hours period, and short-term methods, based on measurements of enteric CH emissions in exhaled or eructated gas in short (minute) intervals over a few consecutive days [3,11- 15]. The respiration chamber as a long-term method of measuring CH emissions is used as the „gold standard“. However, this method is accompanied by disruption of the animal’s natural behavior and needs great technical and personnel requirements, limiting its widespread use for practical and research purposes [12,13]. Consequently, numerous short-term methods for measuring enteric CH emissions have been established, such as GreenFeed and methane hood system, sniffer method and laser 72 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le methane detection, which are applicable to a larger number of animals at lower costs and enable the accurate measurement of enteric CH emissions in the natural environment of animals [11-18]. Bearing in mind that a large number of factors infl uence methane emission, the average CH emission values are not suffi cient to defi ne the phenotype of cows according to CH production. Therefore, it is necessary to examine additional parameters such as variables of CH concentration profi le, including the average CH 4 4 concentrations and CH peak-related variables (number of CH peaks, maximum CH 4 4 4 peak amplitude, average CH peak amplitude, maximum CH peak width, average 4 4 CH peak width and distance among two CH peaks). These variables would clearly 4 4 indicate how increased CH production interacts with physiological processes such as gas elimination through eructation, but also CH production interactions with ration composition and management. Since some countries, like New Zealand, provide fi nancial support for farmers who implement protocols for the mitigation of CH emissions on dairy farms [19], the use of methods that provide enteric CH concentration profi les may contribute to understand the cause of high CH emissions from dairy cows and thus enable the development of protocols with CH emissions mitigation as the outcome. This study aimed to examine differences in CH concentration profi les (examined through average CH concentration, number of CH peaks, maximum CH peak 4 4 4 amplitude, average CH peak amplitude, maximum CH peak width, average CH 4 4 4 peak width and distance among two CH peaks) and metabolic status in peak lactating dairy cows regarding the level of enteric CH emissions estimated by the short-term method using a gas analyser that operates on the principle of infrared spectroscopy. MATERIAL AND METHODS Ethics statement The experiment was performed during June 2022 on a commercial dairy farm (Lepušnica, AlDahra Corporation) in the vicinity of Belgrade, Serbia (44°56’08.6” N, 20°28’44.5” E). The experimental protocol was evaluated and approved by the Veterinary Directorate, Ministry of Agriculture, Forestry and Water Economy of the Republic of Serbia (approval number 323-07-11720/2020-05/4) in accordance with the National Regulation on Animal Welfare. Animals and experimental design Eighteen clinically healthy, multiparous and peak lactating Holstein-Friesian dairy cows were involved in the study. At the beginning of the study the cows were averaging (mean ± standard error of the mean – SEM) 600 ± 13.35 kg body weight (BW), 2.72 ± 0.06 body condition score (BCS), 2.77 ± 0.26 parity, 49.33 ± 2.58 days in milk (DIM), 37.88 ± 2.96 L/day milk production and 21.67 ± 0.44 kg dry matter intake (DMI). The experimental cows were housed under equal husbandry conditions (tie- 73 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 stall barn) and fed with the same total mixed ration (TMR; Table 1) to meet or exceed National Research Council (NRC, 2001) requirements [20]. The daily amount of TMR was divided into three meals and offered to the animals three times a day at 7:00 a.m. (35%), 12:00 a.m. (20%) and 7:00 p.m. (45%), respectively. Water was freely available via automated water bowls. The experimental cows were milked individually three times a day at 6.00 a.m., 12.00 a.m. and 6.00 p.m. Cow health status was monitored on a daily basis and the cows did not show any symptoms of illness during the entire study. Table 1. Composition and nutritional value of the TMRs for lactating cows. Ingredients, kg DM/day HM and LM groups Corn silage 6.8 Alfalfa haylage 1.9 Brewers grain (wet) 1.9 Molasses 1.5 Cottonseed meal 2.3 Corn grain 5.1 Barley 0.3 Rye grain 0.2 Wheat grain 0.3 Sunfl ower meal (34%CP) 4.1 Sodium bicarbonate 0.1 Calcium carbonate 0.2 NaCl 0.1 Monocalcium phosphate 0.1 Vitamin/Mineral Mix 0.1 Nutritional value Dry matter (%) 50.13 Ash % of DM 6.04 Fat % of DM 4.90 Cellulose % of DM 20.47 Starch % of DM 12.84 Sugar % of DM 2.69 Protein CP % of DM 17.85 RDP (%) 11.84 RUP (%) 5.96 MP (g/kg) 110.75 NDF (%) 35.07 ADF (%) 20.32 Ca (%) 0.68 P (%) 0.48 Energy value – Metabolic energy (MJ/kg DM) 15.0 pH 4.63 RDP – rumen degradable protein; RUP – rumen undegradable protein; MP – metabolizable protein; NDF – neutral detergent fi ber; ADF – acid detergent fi ber. 74 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le Measurement of enteric CH emissions Enteric CH emissions were measured for each cow in three periods of three consecutive days: 2 hours before the morning feeding (P1), 2 – 4 hours (P2) and 6 – 8 hours (P3) after the morning feeding resulting in a total of 9 measurements per cow. After the initial measurement of enteric CH emissions, twelve of the initial eighteen Holstein-Friesian cows were selected and divided into two numerically equal groups (6 cows per group) in a way that was described by Kim et al. [9]. The fi rst group included six Holstein-Friesian cows with a high enteric CH emission (HM group; average CH concentration: 5430.08 ± 365.92 ppm), and the second group included six Holstein-Friesian cows with a low enteric CH emission (LM group; average CH 4 4 concentration: 1351.85 ± 205.20 ppm). A portable gas analyser (BIOGAS 5000 ATEX, IECEx, Geotech, UK) equipped with an integrated gas sensor, pump (sampling rate of 550 mL/min), sampling tube with fi lter and exhaust tube was used to measure enteric CH emissions in the eructated gas of experimental cows. Namely, a standard (orogastric) fl exible stainless steel stomach probe with polished bulb end having open end (180 cm length; 1.5 cm internal diameter) was placed in the caudal third of the esophagus before each measurement. Immediately, the sampling tube of the gas analyser was placed into the stomach probe lumen to collect the eructated gas, enabling the gas analyser to measure CH concentration (ppm) using the dual beam infrared absorption spectroscopy method. Afterward, the probe was checked for clogging and fl ushed with water. The gas analyser was set to draw gas continuously for 3 minutes and read the CH concentration at 5-second intervals resulting in a total of 36 readings per measurement. The obtained values were Figure 1. The CH concentration profi le described by CH concentrations (blue line), CH 4 4 4 peaks (solid blue triangle), CH peak amplitude (orange line), CH peak width (yellow line) 4 4 75 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 downloaded to the computer using Gas Analyser Manager software (Geotech, UK) and used for the estimation of enteric CH emissions and CH concentration profi les 4 4 based on CH eructation peaks analysis. These analyses were performed in the MatLab Signal Process ing Toolbox (version R2022b; The MathWorks, Inc., Natick, USA) [21] as recommended by Bell et al. [22]. Accordingly, for each experimental cow the following variables of CH concentration were calculated: average CH concentration, 4 4 number of CH peaks, maximum CH peak amplitude, average CH peak amplitude, 4 4 4 maximum CH peak width, average CH peak width and distance among two CH 4 4 4 peaks (Figure 1). Finally, in the presented study, a profi le was defi ned as a recording of the CH concentration in the eructated gas of the cows examined for over 3 minutes. These profi les were used to calculate and compare different phenotypes for the CH emissions following the suggestions presented by Sorg et al. [23]. Blood sampling and biochemical analyses Blood sampling was performed one day after enteric CH emission measurement was completed. Blood samples were collected by jugular venipuncture from each cow approximately one hour before the morning feeding using a sterile 18-gauge needle (BD Vacutainer, Plymouth, Devon, UK) into 10.0-mL vacutainer tubes (BD Vacutainer, Plymouth, Devon, UK), containing a clot activator. The samples were placed in an icebox immediately and transferred to a laboratory within 1 hour, where they were kept on ice for 2 hours to clot spontaneously. After clotting, samples were centrifuged at 1500 x g for 10 minutes to harvest the serum which was transferred into 1.5-mL polypropylene tubes (Eppendorf AG, Hamburg, Germany) and then stored at −20 °C until biochemical analyses. Each blood sample was analysed for total protein (g/L), albumin (g/L), blood urea nitrogen (BUN; mmol/L), total bilirubin (µmol/L), aspartate aminotransferase (AST; U/L), triacylglycerols (TAG; mmol/L), total cholesterol (mmol/L), HDL-cholesterol (HDL-C; mmol/L), glucose (mmol/L), beta-hydroxybutyrate (BHB; mmol/L), and non-esterifi ed fatty acids (NEFA; mmol/L). Biochemical metabolites were analyzed using the respective methods/kits: total protein (biuret reaction); albumin (bromcresol green method), BUN (urease/ glutamate dehydrogenase method); total bilirubin (diazotized sulphanilic acid method); AST (IFCC method); TAG (glycerol phosphate oxidase/peroxidase); total cholesterol (cholesterol oxidase/peroxidase method), HDL-C (direct detergent method) and BHB (enzymatic method) by BioSystems S.A. (Barcelona, Spain); and NEFA (colorimetric method) by Randox Laboratories Ltd. (Crumlin, UK). Analyses were performed automatically with spectrophotometer (A15; BioSystems S.A., Barcelona, Spain). Glucose was measured immediately after blood collection in a drop of whole blood from the tip of a vacutainer needle using commercial test strips (Abbott Diabetes Care Ltd., Oxon, UK) based on enzymatic method (glucose dehydrogenase, GDH-NAD method). 76 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le Data analysis The data were statistically processed using the software STATISTICA, v. 8.0 (StatSoft, Inc., Tulsa, OK, USA). The results are presented as means ± SEM. The normality of data distribution was tested using Shapiro-Wilk test. All data were within the normal distribution (p>0.05). The signifi cance of differences in the observed parameters between the different experimental groups was determined using the independent Student’s t-test. In addition, the signifi cance of differences in the observed parameters between measurement periods within the same experimental group was estimated using dependent Student’s t-test. Correlations between enteric CH emissions and indicators of metabolic status of observed cows were calculated using Pearson’s test. Signifi cance was declared at p<0.05 and p<0.01, and a tendency was acknowledged at 0.05≤ p<0.10. RESULTS Variables of CH concentration profi le Average values (±SEM) of CH concentration profi le variables in peak lactating dairy cows with different levels of enteric CH emissions are shown in Table 2. It is noticeable that average CH concentrations were statistically signifi cantly higher in the HM group compared to the LM group in all three measurement periods (p<0.05 for P1, p<0.01 for both P2 and P3). Additionally, the average CH concentrations within the HM group were statistically signifi cantly higher in P3 than in P1 and P2 (p=0.01 and p<0.05, respectively). On the contrary, no statistically signifi cant differences (p>0.05) were found in average CH concentrations between the three measurement periods in LM cows; however, average CH concentrations in P3 tended to be higher compared to P1 (p=0.06) in these cows. By analysing the numerical values, it can be seen that the average CH concentration in both groups of cows increased from P1 to P3. Although the number of CH peaks was numerically higher in HM cows than in LM cows in all three measurement periods, it was determined that this variable tended to be higher (p=0.09) only in P2 in HM cows compared to LM cows. It was also found that the number of CH peaks tended to be higher in P3 than in P1 (p=0.08) within HM cows, while it was signifi cantly higher in P3 than in P1 (p=0.01) and tended to be higher in P3 than in P2 (p=0.07) within LM cows. The maximum CH peak amplitude was signifi cantly higher in HM cows than in LM cows in all three measurement periods (p<0.05 for P1, p<0.01 for both P2 and P3). Furthermore, within HM cows, the maximum CH peak amplitude was signifi cantly higher in P2 than in P1 (p<0.05) and tended to be higher in P3 than in P1 (p=0.09). In contrast, no statistically signifi cant differences were recorded in the values of this variable between the three periods of measurement within the LM cows. The average CH peak amplitude was statistically signifi cantly higher in HM than in LM cows in all three measurement periods (p<0.05 for P1, p<0.01 for both P2 and P3). Additionally, the average CH peak amplitude 77 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 in HM cows tended to be higher in P3 than in P2 (p=0.08), while in LM cows, no signifi cant differences between three measurement periods in the average CH peak amplitude were determined. Finally, no statistically signifi cant differences were noticed in maximum CH peak width, average CH peak width and average distance among 4 4 two peaks, neither between HM and LM cows, nor between three measurement periods within respective groups of cows. Table 2. Variables of CH concentration profi le (mean ± SEM) in peak lactating dairy cows with different level of enteric CH emissions. Periods of measurement Variables Units Groups P1 P2 P3 Aa Aa Ab HM 4170.5 ± 1038.8 4520.8 ± 849.6 9032.4 ± 1932.3 Average CH ppm Ba Ba Ba concentrations LM 908.2 ± 330.4 1433.6 ± 429.2 1641.9 ± 458.9 Aa Aa Aa HM 2.4 ± 0.2 2.7 ± 0.4 3.1 ± 0.4 Number of CH Aa Aab Ab peaks LM 1.8 ± 0.4 1.9 ± 0.2 2.4 ± 0.4 Aa Ab Aab HM 33305.6 ± 9043.9 48583.3 ± 5186.8 91777.8 ± 21644.7 Maximum CH peak ppm Ba Ba Ba amplitude LM 10250.0 ± 4612.9 11555.0 ± 5426.1 19277.8 ± 6753.1 Aa Aa Aa HM 19079.0 ± 5028.3 23981.0 ± 2990.3 53848.0 ± 13749.4 Average CH ppm Ba Ba Ba peak amplitude LM 4166.7 ± 1730.1 5309.3 ± 2560.0 5951.4 ± 1311.3 Aa Aa Aa HM 13.0 ± 1.1 12.3 ± 1.2 11.7 ± 0.6 Maximum CH peak Aa Aa Aa width LM 12.1 ± 1.8 10.5 ± 0.9 12.5 ± 1.2 Aa Aa Aa HM 11.1 ± 1.6 9.0 ± 0.8 9.8 ± 0.5 Average CH Aa Aa Aa peak width LM 6.5 ± 2.01 9.7 ± 1.1 8.9 ± 1.2 Aa Aa Aa HM 46.4 ± 7.8 44.1 ± 8.5 50.4 ± 3.1 Average distance Aa Aa Aa between two peaks LM 68.8 ± 20.4 45.3 ± 7.1 44.3 ± 7.6 HM – high enteric CH emissions group; LM – low enteric CH emissions group. 4 4 P1 – period of 2 hours before the morning feeding; P2 – period of 2 – 4 hours after the morning feeding; P3 – period of 6 – 8 hours after the morning feeding. A,B – different uppercase letters in superscript indicate statistically signifi cant differences (p<0.05) a,b,c between HM and LM groups of cows in the same period; – different lowercase letters in superscript indicate statistically signifi cant differences (p<0.05) between measurement periods in the same group of cows. Indicators of metabolic status Results obtained for blood metabolic indicators are summarized in Table 3. The results show that HM cows had a statistically signifi cantly higher concentrations of total protein (p<0.05), and a statistically signifi cant lower concentrations of total bilirubin (p<0.05) and NEFA (p<0.01). On the other hand, no statistically signifi cant differences (p>0.05) were found in the concentrations of albumin, BUN, AST, TAG, total cholesterol, HDL-C, glucose and BHB between HM and LM cows. Interestingly, the results of the Pearson’s correlation test indicate that there is a signifi cant moderately negative correlation between enteric CH emission and total bilirubin concentration 78 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le (r=-0.61; p<0.05) and a signifi cantly strong negative correlation between enteric CH emission and NEFA concentrations (r=-0.82; p<0.01). No correlation was found between the enteric CH emission and other indicators of metabolic status of the observed cows. Table 3. Comparison of metabolic status indicators (mean ± SEM) between cows with different level of methane emission. Indicator (unit) HM LM A B Total protein (g/L) 81.83 ± 2.0 71.58 ± 4.1 A A Albumin (g/L) 38.22 ± 1.8 37.23 ± 1.7 A A BUN (mmol/L) 4.70 ± 0.41 5.00 ± 0.3 A B Total bilirubin (µmol/L) 2.04 ± 0.1 3.33 ± 0.5 A A AST (U/L) 108.63 ± 17.1 92.50 ± 10.2 A A TAG (mmol/L) 0.17 ± 0.01 0.17 ± 0.04 A A Total cholesterol (mmol/L) 6.04 ± 0.3 5.04 ± 0.6 A A HDL-C (mmol/L) 159.80 ± 7.4 141.64 ± 15.1 A A Glucose (mmol/L) 2.77 ± 0.1 2.72 ± 0.1 A A BHB (mmol/L) 0.46 ± 0.04 0.45 ± 0.1 A B NEFA (mmol/L) 0.27 ± 0.02 0.42 ± 0.03 A,B HM – high enteric CH emissions group; LM - low enteric CH emissions group; – different 4 4 uppercase letters in superscript indicate statistically signifi cant differences (p<0.05) between HM and LM groups of cows. DISCUSSION The results of the presented study reveal signifi cant differences in the variables of CH concentration profi les and some indicators of metabolic status in peak lactating dairy cows with different levels of enteric CH production. In this study, a novel approach of signal processing was used to determine the variables of the CH concentration profi le of peak lactating dairy cows. It is known that this approach is widely applied in medical science, but it is also considered appropriate for processing potentially noisy and dense data such as those obtained by measuring the concentration of CH in the exhaled or eructated gas of animals [22,24,25]. Using this approach, the obtained results for the average CH concentrations indicate that HM cows had signifi cantly higher values of this variable compared to LM cows in all measurement periods. These results suggest that HM and LM cows truly belong to different phenotypes for CH production (high and low), which is needed for comparisons of the observed variables between HM and LM cows to be justifi ed and relevant. At the same time, the average CH concentrations showed an increasing trend from P1 to P3 in both groups of cows, which agrees with the results of Crompton et al. [26], who examined fl uctuations in enteric CH emissions associated with feeding patterns in Holstein-Friesian dairy cows in mid-lactation. Additionally, 79 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 similar results were obtained by Jonker et al. [27] and Rooke et al. [28], who observed enteric CH emission in relation to time after feeding and feed allowance in beef cattle. In the mentioned studies [26-28], enteric CH emission was determined in open-circuit respiration chambers and it was shown that CH emission was the lowest before the morning feeding. Furthermore, these studies have also documented that CH emissions rise gradually and reach a peak between 40 (for beef cattle) and 140 (for dairy cows) minutes after each feeding, and then gradually decline until the next feeding. Although, based on these fi ndings, a decrease in enteric CH emissions in P3 could be expected in our study because this measurement was performed 6 to 8 hours after the morning feeding, this was not the case. This can be explained by the fact that the cows in our study had one more meal between P2 and P3 measurements, which constitutes approximately 20% of the total daily amount of TMR offered, and could have contributed to CH concentrations in P3 being higher than in P2. Finally, our results for the average CH concentrations suggest that, despite the different levels of CH emissions, HM and LM cows have a comparable daily pattern of CH 4 4 emissions. The lowest average CH concentrations could describe this pattern before the morning feeding (P1) and those rises 2-4 hours after the morning feeding (P2) and further rise 6-8 hours after the morning feeding (P3). The number of CH peaks in the presented study did not statistically differ between HM and LM cows in different measurement periods. However, the CH number of peaks tended to be higher in P2 (p=0.09) and was numerically higher in the remaining two measurement periods in HM cows compared to LM cows. Moreover, similar to the average CH concentration, the obtained values for the CH number of peaks 4 4 indicates an increasing trend from P1 to P3 in both groups of cows. Given that the methodology for measuring CH emissions used in this study is based on determining its concentration in the eructated gas, it is important to explain that the appearance of each CH peak could be attributed to an eructation event in the examined cow. A similar interpretation of the appearance of CH peaks was given by Bell et al. [22] and Hardan et al. [25], who used infrared gas analysers to measure enteric CH emissions in the exhaled air of dairy cows and applied the same mathematical approach for the CH concentration profi le analysis. Unfortunately, there is no similar study in the available literature that examined the CH number of peaks or eructation rate in cows with different levels of CH emissions, with which our results could be compared. Nevertheless, the higher number of eructations in HM cows than in LM cows and its increasing trend from P1 to P3 in both groups of cows could be associated with the levels of enteric CH production. It is obvious that a higher number of eructation events followed higher average CH concentrations in this study. Therefore, it can be hypothesized that there is a stronger stimulation of the eructation refl ex through the activation of the vagal afferents of the dorsal rumen by produced gas [29] in cows with high CH production, as well as in the periods after feeding that were coupled with increased CH production in both groups of cows. This hypothesis is additionally supported by our results obtained for the maximum CH peak amplitude, average CH 4 4 80 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le peak amplitude, maximum CH peak width and average CH peak width. Namely, HM 4 4 cows had a signifi cantly higher maximum CH peak amplitude and average CH peak 4 4 amplitude than LM cows in all three measurement periods, whereby in both groups of cows, there was an increasing trend in both parameters from P1 to P3. Also, there were no signifi cant differences in maximum CH peak width and average CH peak 4 4 width between HM and LM groups in different measurement periods or between measurement periods within the respective groups of cows. Accordingly, the described results suggest that higher production of enteric CH more powerfully stimulates the eructation refl ex and leads to a greater number of eructations (CH peaks) and a greater release of CH per eructation event (CH amplitude variables) of approximately equal 4 4 duration (CH width variables). This is clearly noticeable both in HM cows compared to LM cows, as well as in later measurement periods after the morning feeding compared to the previous ones (P3 vs. P2) or those before the morning feeding (P2 vs. P1 and P3 vs. P1). However, additional studies are needed in the physiology of the eructation refl ex and its connection with the level of enteric CH production to confi rm the presented hypothesis. Finally, it should be noted that the average number (±SEM) of eructation events in both groups of cows in all measurement periods was in the general range of 0.7 to 1.5 eructations per minute [30,31]. Furthermore, our results for the average distance among two CH peaks (eructations) did not differ signifi cantly between HM and LM cows or between different measurement periods within the same group of cows. They were also in the general range that predicts the appearance of one eructation event every 40 to 90 seconds in cattle [32]. To the best of our knowledge, this is the fi rst study on the metabolic status of dairy cows with different levels of enteric CH emissions. Nevertheless, the results obtained for the indicators of the metabolic status in our study could be compared with the results reported by Kim et al. [9], who observed differences in metabolic status in various fattening phases of Japanese Black steers with high (HME) and low (LME) methane emissions. In the mentioned study, it was found that HME steers have only signifi cantly higher concentrations of BHB compared to LM steers in all fattening phases, while differences between these groups of steers in the concentration of total protein, total cholesterol, NEFA and glucose and AST activity were not recorded. These fi ndings are not in agreement with ours as we observed signifi cantly higher total protein concentrations and signifi cantly lower total bilirubin and NEFA concentrations in HM cows compared to LM cows. The possible explanation for the described disagreements can be addressed to the cattle breed and production category used in the compared studies. Kim et al. [9] included Black Japanese steers in their study, while Holstein-Friesian dairy cows in peak lactation were used in our study. As these two categories of cattle have different production purposes and different metabolomics and proteomics signatures [33], it is reasonable to expect differences in the metabolic response between these cattle in relation to enteric CH production. Additionally, the mentioned authors performed the study in three phases of fattening, which differ in their metabolic challenge for the steers, while our study included cows 81 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 averaging (±SEM) 49.33 ± 2.58 DIM, avoiding the most pronounced negative energy balance and its infl uence on the monitored indicators of the metabolic status [34]. The values obtained for NEFA concentrations in our study indicate that HM cows have a more favorable energy balance compared to LM cows. Namely, NEFA concentrations refl ect the magnitude of peripheral fat breakdown and energy balance of cows, while BHB concentrations refl ect the ability of the liver to manipulate infl owing NEFA [35]. Although there are no signifi cant differences in BHB concentration between HM and LM cows, attention should be paid to other parameters of liver function observed in this study, such as concentrations of total protein and total bilirubin. In this regard, signifi cantly lower total protein concentrations and signifi cantly higher total bilirubin concentrations in LM cows compared to HM cows indicate that the energy balance of LM cows affects their functional liver capacity [36,37]. Мoreover, the signifi cant negative correlation between enteric CH emissions and total bilirubin and NEFA concentrations, respectively, found in the presented study additionally supports these considerations. On the other hand, the absence of differences in the concentration of BHB between HM and LM cows can be explained by the fact that blood BHB concentration may be affected by the production of butyrate in the rumen [38]. In other words, higher production of butyrate in the rumen of HM cows could have caused an increase in BHB blood concentrations in these cows and masked any differences in the values of this parameter between HM and LM cows. Moreover, the higher production of butyrate in HM cows would not be surprising because there are indications that butyrate synthesis is a metabolic pathway in the rumen that may favor higher CH production because it is associated with the release of H [7,9]. However, 4 2 additional studies are needed in the ruminal microbiome and metabolome and liver metabolome and transcriptome fi elds that will elucidate the nature of the differences found in the metabolic status between HM and LM cows in the present study. Finally, it should be highlighted that all observed indicators of the metabolic status of peak lactating dairy cows in this study were within the reference ranges proposed by Cozzi et al. [39] and Moretti et al. [40]. CONCLUSION In conclusion, this study showed that peak lactating dairy cows with different phenotypes for CH emissions (high and low) have similar daily pattern of CH 4 4 emissions, since the lowest CH emission, detected before the morning feeding, gradually increased after each feeding. This fi nding is in accordance with other studies in which different methods of CH emissions measurement were used. However, our study revealed that dairy cows with a high CH emission phenotype have more eructations and release more CH per eructation. Additionally, the metabolic status of peak lactating dairy cows is affected by the level of CH emission. This is most noticeable in the parameters that depict different aspects of liver function since in cows with high CH production, higher concentrations of total proteins and lower 82 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le concentrations of total bilirubin and NEFA were found. Finally, further research in the fi eld of liver transcriptome and metabolome is needed in order to determine the origin and signifi cance of these differences. Acknowledgments The research was conducted with the support of the Science Fund of the Republic of Serbia, project number 7750295, “Mitigation of methane production from dairy cattle farm by nutritive modulation of cow`s metabolism-MitiMetCattle”. Authors’ contributions DB, DK, SN and LJ contributed to the design and conception of the study. RP, IV, SA and SN conducted the fi eld experiment. RP and SA performed biochemical analyses of blood serum samples. DB, MS and SD participated in data analyses. DB, DK, and LJ were involved in draft manuscript preparation. All authors read and approved the fi nal manuscript. Declaration of confl icting interests The author(s) declared no potential confl icts of interest with respect to the research, authorship, and/or publication of this article. REFERENCES 1. Hook SE, Wright ADG, McBride BW: Methanogens: methane producers of the rumen and mitigation strategies. Archaea 2010, 1-11. 2. Lassen J, Løvendahl P: Heritability estimates for enteric methane emissions from Holstein cattle measured using noninvasive methods. J Dairy Sci 2016, 99:1959-1967. 3. Zhao Y, Nan X, Yang L, Zheng S, Jiang L, Xiong B: A review of enteric methane emission measurement techniques in ruminants. Animals 2020, 10:1004. 4. Broucek J: Production of methane emissions from ruminant husbandry: a review. Journal of Environmental Protection 2014, 5: 1482. 5. 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Res Vet Sci 2017, 114:322-331. 85 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 EMISIJA METANA I METABOLIČKI STATUS MLEČNIH KRAVA U PIKU LAKTACIJE I NJIHOVA PROCENA PUTEM PROFILA KONCENTRACIJE METANA Dušan BOŠNJAKOVIĆ, Danijela KIROVSKI, Radiša PRODANOVIĆ, Ivan VUJANAC, Sveta ARSIĆ, Milica STOJKOVIĆ, Slavica DRAŽIĆ, Sreten NEDIĆ, Ljubomir JOVANOVIĆ Uzgoj preživara doprinosi globalnoj emisiji metana (CH ) i pored negativnog uticaja na životnu sredinu, enterični CH kod krava izaziva gubitak energije unete hranom. Cilj ove studije je bio da se izvrši procena emisije CH i metaboličkog statusa krava određi- vanjem profi la koncentracije metana kao alata kojim se analizira način produkcije CH . Istraživanjem je obuhvać eno osamnaest krava čija je enterička emisija CH merena tokom tri uzastopna dana u tri perioda: 2 sata pre (P1), 2–4 sata (P2) i 6–8 sati (P3) po- sle jutarnjeg hranjenja. Na osnovu emisije CH , krave su podeljene u dve grupe (n=6, pojedinačno): HM (prosečna CH koncentracija: 5430,08 ± 365,92 ppm) i LM (pro- sečna CH koncentracija: 1351,85 ± 205,20 ppm). Nakon merenja CH , treć eg dana, 4 4 uzorkovana je venska krv radi utvrđivanja indikatora metaboličkog statusa. HM krave su imale statistički značajno već e prosečne koncentracije CH , maksimalnu i prosečnu amplitudu CH pika u odnosu na LM krave u svim periodima merenja (P1-P3), dok je broj CH pikova imao tendenciju da bude već i kod HM nego kod LM krava u P2. Nije bilo razlika u maksimalnoj i prosečnoj širini pika CH i prosečnoj udaljenosti između dva pika CH između ispitivanih grupa krava. HM krave su imale značajno već e kon- centracije ukupnih proteina i značajno niže koncentracije ukupnog bilirubina i NEFA od LM krava. Zaključuje se da su HM krave imale već i broj ruktusa i time oslobađale više CH nego LM krave, a razlike u metaboličkom statusu između HM i LM krava su najverovatnije bile povezane sa razlikama u funkciji jetre. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta veterinaria de Gruyter

Methane Emission and Metabolic Status in Peak Lactating Dairy Cows and Their Assessment Via Methane Concentration Profile

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Acta Veterinaria-Beograd 2023, 73 (1), 71-86 UDK: 636.2.09:616-008.9:547.211 DOI: 10.2478/acve-2023-0006 Research article METHANE EMISSION AND METABOLIC STATUS IN PEAK LACTATING DAIRY COWS AND THEIR ASSESSMENT VIA METHANE CONCENTRATION PROFILE 1 1 2 Dušan BOŠNJAKOVIĆ , Danijela KIROVSKI , Radiša PRODANOVIĆ , 2 2 1 1 Ivan VUJANAC , Sveta ARSIĆ , Milica STOJKOVIĆ , Slavica DRAŽIĆ , 2 1 Sreten NEDIĆ *, Ljubomir JOVANOVIĆ University of Belgrade, Faculty of Veterinary Medicine, Department of Physiology and Biochemistry, Bul. Oslobodjenja 18, Belgrade, Serbia; University of Belgrade, Faculty of Veterinary Medicine, Department of Ruminants and Swine Diseases, Bul. Oslobodjenja 18, Belgrade, Serbia (Received 22 October, Accepted 21 December 2022) Ruminant husbandry contributes to global methane (CH ) emissions and beside its negative impact on the environment, enteric CH emissions cause a loss of gross energy intake in cows. The study is aimed to estimate CH emission and metabolic status in dairy cows via the methane concentration profi le as a tool for analyzing the CH production pattern. The study included eighteen cows whose enteric CH emission was measured during three consecutive days in three periods: 2 hours before (P1), 2–4 hours (P2) and 6–8 hours (P3) after the morning feeding. Based on CH enteric emissions, cows were divided into two groups (n=6, respectively): HM (average CH concentration: 5430.08 ± 365.92 ppm) and LM (average CH concentration: 1351.85 ± 205.20 ppm). Following CH measurement, on day 3, venous blood was sampled to determine the indicators of the metabolic status. HM cows had signifi cantly higher average CH concentrations, maximum and average CH peak amplitude than LM cows 4 4 in all measuring periods (P1-P3), while the number of CH peaks tended to be higher in HM than in LM cows in P2. There were no differences in the maximum and average CH peak width and average distance among two CH peaks between examined groups 4 4 of cows. HM cows had signifi cantly higher total protein concentrations and signifi cantly lower total bilirubin and NEFA concentrations than LM cows. In conclusion, HM cows have a greater number of eructations and release more CH per eructation than LM cows, hence the differences in metabolic status are most likely related to the differences in their liver function. Keywords: dairy cows, methane emission, metabolic status *Corresponding author: e-mail: sreten.nedic@vet.bg.ac.rs 71 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 INTRODUCTION Methane (CH ) is one of the most potent greenhouse gases (GHG), with a warming potential of 25 to 28 times greater compared to carbon dioxide (CO ) [1]. Ruminant husbandry releases approximately 80 million tons of CH into the Earth’s atmosphere annually, representing around 28% of the total anthropogenic CH emissions [2,3]. Enteric CH is predominantly generated in the rumen (87-90%) and, to a lesser extent, in the large intestine (10-13%) by the metabolic activity of methanogenic microorganisms such as archaea [1,4]. The rumen represents an anaerobic environment made up of microorganisms that participate in the fermentation processes by degrading the feed organic matter such as plant structural carbohydrates, proteins and other organic polymers to volatile fatty acids (VFAs) and gases, including CO and molecular hydrogen (H ) [5]. Released VFAs such as acetate, propionate, and butyrate are absorbed by the rumen and are utilized as an energy source for milk production in dairy cows. On the other hand, methanogenic archaea use H to reduce CO and thereby produce CH , as one of the fi nal products of ruminal fermentation, 2 4 in a process known as methanogenesis [6,7]. Enteric CH produced in the rumen is almost completely eliminated by eructation (95-99%) providing a possibility that methanogenesis could also be seen as a loss of gross energy intake of 2% to 12% [8]. Considering the environmental effects of enteric CH emissions, as well as the loss of gross energy intake, it is important to examine all the factors that infl uence methanogenesis. The large proportion of the variation in CH emissions can be explained by diet composition and feed intake but there is additional variation among animal phenotypes. Although methanogenesis and energy metabolism are closely related, to the best of our knowledge no research on dairy cows has been performed to examine the differences in the blood metabolic parameters in cows with different levels of enteric CH emissions. There is only one research conducted by Kim et al. [9] on Japanese Black steers which showed that these cattle have different metabolic statuses based on the enteric CH emissions. Since lactating cows are exposed to metabolic challenges [10], it is reasonable to expect some differences in their metabolic status in relation to the enteric CH emissions level. Despite the numerous studies related to CH emissions, the methodology of determining CH emissions from individual animals is very diverse. Methods could be classifi ed into long-term, intended for measuring CH emissions during a 24-hours period, and short-term methods, based on measurements of enteric CH emissions in exhaled or eructated gas in short (minute) intervals over a few consecutive days [3,11- 15]. The respiration chamber as a long-term method of measuring CH emissions is used as the „gold standard“. However, this method is accompanied by disruption of the animal’s natural behavior and needs great technical and personnel requirements, limiting its widespread use for practical and research purposes [12,13]. Consequently, numerous short-term methods for measuring enteric CH emissions have been established, such as GreenFeed and methane hood system, sniffer method and laser 72 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le methane detection, which are applicable to a larger number of animals at lower costs and enable the accurate measurement of enteric CH emissions in the natural environment of animals [11-18]. Bearing in mind that a large number of factors infl uence methane emission, the average CH emission values are not suffi cient to defi ne the phenotype of cows according to CH production. Therefore, it is necessary to examine additional parameters such as variables of CH concentration profi le, including the average CH 4 4 concentrations and CH peak-related variables (number of CH peaks, maximum CH 4 4 4 peak amplitude, average CH peak amplitude, maximum CH peak width, average 4 4 CH peak width and distance among two CH peaks). These variables would clearly 4 4 indicate how increased CH production interacts with physiological processes such as gas elimination through eructation, but also CH production interactions with ration composition and management. Since some countries, like New Zealand, provide fi nancial support for farmers who implement protocols for the mitigation of CH emissions on dairy farms [19], the use of methods that provide enteric CH concentration profi les may contribute to understand the cause of high CH emissions from dairy cows and thus enable the development of protocols with CH emissions mitigation as the outcome. This study aimed to examine differences in CH concentration profi les (examined through average CH concentration, number of CH peaks, maximum CH peak 4 4 4 amplitude, average CH peak amplitude, maximum CH peak width, average CH 4 4 4 peak width and distance among two CH peaks) and metabolic status in peak lactating dairy cows regarding the level of enteric CH emissions estimated by the short-term method using a gas analyser that operates on the principle of infrared spectroscopy. MATERIAL AND METHODS Ethics statement The experiment was performed during June 2022 on a commercial dairy farm (Lepušnica, AlDahra Corporation) in the vicinity of Belgrade, Serbia (44°56’08.6” N, 20°28’44.5” E). The experimental protocol was evaluated and approved by the Veterinary Directorate, Ministry of Agriculture, Forestry and Water Economy of the Republic of Serbia (approval number 323-07-11720/2020-05/4) in accordance with the National Regulation on Animal Welfare. Animals and experimental design Eighteen clinically healthy, multiparous and peak lactating Holstein-Friesian dairy cows were involved in the study. At the beginning of the study the cows were averaging (mean ± standard error of the mean – SEM) 600 ± 13.35 kg body weight (BW), 2.72 ± 0.06 body condition score (BCS), 2.77 ± 0.26 parity, 49.33 ± 2.58 days in milk (DIM), 37.88 ± 2.96 L/day milk production and 21.67 ± 0.44 kg dry matter intake (DMI). The experimental cows were housed under equal husbandry conditions (tie- 73 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 stall barn) and fed with the same total mixed ration (TMR; Table 1) to meet or exceed National Research Council (NRC, 2001) requirements [20]. The daily amount of TMR was divided into three meals and offered to the animals three times a day at 7:00 a.m. (35%), 12:00 a.m. (20%) and 7:00 p.m. (45%), respectively. Water was freely available via automated water bowls. The experimental cows were milked individually three times a day at 6.00 a.m., 12.00 a.m. and 6.00 p.m. Cow health status was monitored on a daily basis and the cows did not show any symptoms of illness during the entire study. Table 1. Composition and nutritional value of the TMRs for lactating cows. Ingredients, kg DM/day HM and LM groups Corn silage 6.8 Alfalfa haylage 1.9 Brewers grain (wet) 1.9 Molasses 1.5 Cottonseed meal 2.3 Corn grain 5.1 Barley 0.3 Rye grain 0.2 Wheat grain 0.3 Sunfl ower meal (34%CP) 4.1 Sodium bicarbonate 0.1 Calcium carbonate 0.2 NaCl 0.1 Monocalcium phosphate 0.1 Vitamin/Mineral Mix 0.1 Nutritional value Dry matter (%) 50.13 Ash % of DM 6.04 Fat % of DM 4.90 Cellulose % of DM 20.47 Starch % of DM 12.84 Sugar % of DM 2.69 Protein CP % of DM 17.85 RDP (%) 11.84 RUP (%) 5.96 MP (g/kg) 110.75 NDF (%) 35.07 ADF (%) 20.32 Ca (%) 0.68 P (%) 0.48 Energy value – Metabolic energy (MJ/kg DM) 15.0 pH 4.63 RDP – rumen degradable protein; RUP – rumen undegradable protein; MP – metabolizable protein; NDF – neutral detergent fi ber; ADF – acid detergent fi ber. 74 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le Measurement of enteric CH emissions Enteric CH emissions were measured for each cow in three periods of three consecutive days: 2 hours before the morning feeding (P1), 2 – 4 hours (P2) and 6 – 8 hours (P3) after the morning feeding resulting in a total of 9 measurements per cow. After the initial measurement of enteric CH emissions, twelve of the initial eighteen Holstein-Friesian cows were selected and divided into two numerically equal groups (6 cows per group) in a way that was described by Kim et al. [9]. The fi rst group included six Holstein-Friesian cows with a high enteric CH emission (HM group; average CH concentration: 5430.08 ± 365.92 ppm), and the second group included six Holstein-Friesian cows with a low enteric CH emission (LM group; average CH 4 4 concentration: 1351.85 ± 205.20 ppm). A portable gas analyser (BIOGAS 5000 ATEX, IECEx, Geotech, UK) equipped with an integrated gas sensor, pump (sampling rate of 550 mL/min), sampling tube with fi lter and exhaust tube was used to measure enteric CH emissions in the eructated gas of experimental cows. Namely, a standard (orogastric) fl exible stainless steel stomach probe with polished bulb end having open end (180 cm length; 1.5 cm internal diameter) was placed in the caudal third of the esophagus before each measurement. Immediately, the sampling tube of the gas analyser was placed into the stomach probe lumen to collect the eructated gas, enabling the gas analyser to measure CH concentration (ppm) using the dual beam infrared absorption spectroscopy method. Afterward, the probe was checked for clogging and fl ushed with water. The gas analyser was set to draw gas continuously for 3 minutes and read the CH concentration at 5-second intervals resulting in a total of 36 readings per measurement. The obtained values were Figure 1. The CH concentration profi le described by CH concentrations (blue line), CH 4 4 4 peaks (solid blue triangle), CH peak amplitude (orange line), CH peak width (yellow line) 4 4 75 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 downloaded to the computer using Gas Analyser Manager software (Geotech, UK) and used for the estimation of enteric CH emissions and CH concentration profi les 4 4 based on CH eructation peaks analysis. These analyses were performed in the MatLab Signal Process ing Toolbox (version R2022b; The MathWorks, Inc., Natick, USA) [21] as recommended by Bell et al. [22]. Accordingly, for each experimental cow the following variables of CH concentration were calculated: average CH concentration, 4 4 number of CH peaks, maximum CH peak amplitude, average CH peak amplitude, 4 4 4 maximum CH peak width, average CH peak width and distance among two CH 4 4 4 peaks (Figure 1). Finally, in the presented study, a profi le was defi ned as a recording of the CH concentration in the eructated gas of the cows examined for over 3 minutes. These profi les were used to calculate and compare different phenotypes for the CH emissions following the suggestions presented by Sorg et al. [23]. Blood sampling and biochemical analyses Blood sampling was performed one day after enteric CH emission measurement was completed. Blood samples were collected by jugular venipuncture from each cow approximately one hour before the morning feeding using a sterile 18-gauge needle (BD Vacutainer, Plymouth, Devon, UK) into 10.0-mL vacutainer tubes (BD Vacutainer, Plymouth, Devon, UK), containing a clot activator. The samples were placed in an icebox immediately and transferred to a laboratory within 1 hour, where they were kept on ice for 2 hours to clot spontaneously. After clotting, samples were centrifuged at 1500 x g for 10 minutes to harvest the serum which was transferred into 1.5-mL polypropylene tubes (Eppendorf AG, Hamburg, Germany) and then stored at −20 °C until biochemical analyses. Each blood sample was analysed for total protein (g/L), albumin (g/L), blood urea nitrogen (BUN; mmol/L), total bilirubin (µmol/L), aspartate aminotransferase (AST; U/L), triacylglycerols (TAG; mmol/L), total cholesterol (mmol/L), HDL-cholesterol (HDL-C; mmol/L), glucose (mmol/L), beta-hydroxybutyrate (BHB; mmol/L), and non-esterifi ed fatty acids (NEFA; mmol/L). Biochemical metabolites were analyzed using the respective methods/kits: total protein (biuret reaction); albumin (bromcresol green method), BUN (urease/ glutamate dehydrogenase method); total bilirubin (diazotized sulphanilic acid method); AST (IFCC method); TAG (glycerol phosphate oxidase/peroxidase); total cholesterol (cholesterol oxidase/peroxidase method), HDL-C (direct detergent method) and BHB (enzymatic method) by BioSystems S.A. (Barcelona, Spain); and NEFA (colorimetric method) by Randox Laboratories Ltd. (Crumlin, UK). Analyses were performed automatically with spectrophotometer (A15; BioSystems S.A., Barcelona, Spain). Glucose was measured immediately after blood collection in a drop of whole blood from the tip of a vacutainer needle using commercial test strips (Abbott Diabetes Care Ltd., Oxon, UK) based on enzymatic method (glucose dehydrogenase, GDH-NAD method). 76 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le Data analysis The data were statistically processed using the software STATISTICA, v. 8.0 (StatSoft, Inc., Tulsa, OK, USA). The results are presented as means ± SEM. The normality of data distribution was tested using Shapiro-Wilk test. All data were within the normal distribution (p>0.05). The signifi cance of differences in the observed parameters between the different experimental groups was determined using the independent Student’s t-test. In addition, the signifi cance of differences in the observed parameters between measurement periods within the same experimental group was estimated using dependent Student’s t-test. Correlations between enteric CH emissions and indicators of metabolic status of observed cows were calculated using Pearson’s test. Signifi cance was declared at p<0.05 and p<0.01, and a tendency was acknowledged at 0.05≤ p<0.10. RESULTS Variables of CH concentration profi le Average values (±SEM) of CH concentration profi le variables in peak lactating dairy cows with different levels of enteric CH emissions are shown in Table 2. It is noticeable that average CH concentrations were statistically signifi cantly higher in the HM group compared to the LM group in all three measurement periods (p<0.05 for P1, p<0.01 for both P2 and P3). Additionally, the average CH concentrations within the HM group were statistically signifi cantly higher in P3 than in P1 and P2 (p=0.01 and p<0.05, respectively). On the contrary, no statistically signifi cant differences (p>0.05) were found in average CH concentrations between the three measurement periods in LM cows; however, average CH concentrations in P3 tended to be higher compared to P1 (p=0.06) in these cows. By analysing the numerical values, it can be seen that the average CH concentration in both groups of cows increased from P1 to P3. Although the number of CH peaks was numerically higher in HM cows than in LM cows in all three measurement periods, it was determined that this variable tended to be higher (p=0.09) only in P2 in HM cows compared to LM cows. It was also found that the number of CH peaks tended to be higher in P3 than in P1 (p=0.08) within HM cows, while it was signifi cantly higher in P3 than in P1 (p=0.01) and tended to be higher in P3 than in P2 (p=0.07) within LM cows. The maximum CH peak amplitude was signifi cantly higher in HM cows than in LM cows in all three measurement periods (p<0.05 for P1, p<0.01 for both P2 and P3). Furthermore, within HM cows, the maximum CH peak amplitude was signifi cantly higher in P2 than in P1 (p<0.05) and tended to be higher in P3 than in P1 (p=0.09). In contrast, no statistically signifi cant differences were recorded in the values of this variable between the three periods of measurement within the LM cows. The average CH peak amplitude was statistically signifi cantly higher in HM than in LM cows in all three measurement periods (p<0.05 for P1, p<0.01 for both P2 and P3). Additionally, the average CH peak amplitude 77 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 in HM cows tended to be higher in P3 than in P2 (p=0.08), while in LM cows, no signifi cant differences between three measurement periods in the average CH peak amplitude were determined. Finally, no statistically signifi cant differences were noticed in maximum CH peak width, average CH peak width and average distance among 4 4 two peaks, neither between HM and LM cows, nor between three measurement periods within respective groups of cows. Table 2. Variables of CH concentration profi le (mean ± SEM) in peak lactating dairy cows with different level of enteric CH emissions. Periods of measurement Variables Units Groups P1 P2 P3 Aa Aa Ab HM 4170.5 ± 1038.8 4520.8 ± 849.6 9032.4 ± 1932.3 Average CH ppm Ba Ba Ba concentrations LM 908.2 ± 330.4 1433.6 ± 429.2 1641.9 ± 458.9 Aa Aa Aa HM 2.4 ± 0.2 2.7 ± 0.4 3.1 ± 0.4 Number of CH Aa Aab Ab peaks LM 1.8 ± 0.4 1.9 ± 0.2 2.4 ± 0.4 Aa Ab Aab HM 33305.6 ± 9043.9 48583.3 ± 5186.8 91777.8 ± 21644.7 Maximum CH peak ppm Ba Ba Ba amplitude LM 10250.0 ± 4612.9 11555.0 ± 5426.1 19277.8 ± 6753.1 Aa Aa Aa HM 19079.0 ± 5028.3 23981.0 ± 2990.3 53848.0 ± 13749.4 Average CH ppm Ba Ba Ba peak amplitude LM 4166.7 ± 1730.1 5309.3 ± 2560.0 5951.4 ± 1311.3 Aa Aa Aa HM 13.0 ± 1.1 12.3 ± 1.2 11.7 ± 0.6 Maximum CH peak Aa Aa Aa width LM 12.1 ± 1.8 10.5 ± 0.9 12.5 ± 1.2 Aa Aa Aa HM 11.1 ± 1.6 9.0 ± 0.8 9.8 ± 0.5 Average CH Aa Aa Aa peak width LM 6.5 ± 2.01 9.7 ± 1.1 8.9 ± 1.2 Aa Aa Aa HM 46.4 ± 7.8 44.1 ± 8.5 50.4 ± 3.1 Average distance Aa Aa Aa between two peaks LM 68.8 ± 20.4 45.3 ± 7.1 44.3 ± 7.6 HM – high enteric CH emissions group; LM – low enteric CH emissions group. 4 4 P1 – period of 2 hours before the morning feeding; P2 – period of 2 – 4 hours after the morning feeding; P3 – period of 6 – 8 hours after the morning feeding. A,B – different uppercase letters in superscript indicate statistically signifi cant differences (p<0.05) a,b,c between HM and LM groups of cows in the same period; – different lowercase letters in superscript indicate statistically signifi cant differences (p<0.05) between measurement periods in the same group of cows. Indicators of metabolic status Results obtained for blood metabolic indicators are summarized in Table 3. The results show that HM cows had a statistically signifi cantly higher concentrations of total protein (p<0.05), and a statistically signifi cant lower concentrations of total bilirubin (p<0.05) and NEFA (p<0.01). On the other hand, no statistically signifi cant differences (p>0.05) were found in the concentrations of albumin, BUN, AST, TAG, total cholesterol, HDL-C, glucose and BHB between HM and LM cows. Interestingly, the results of the Pearson’s correlation test indicate that there is a signifi cant moderately negative correlation between enteric CH emission and total bilirubin concentration 78 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le (r=-0.61; p<0.05) and a signifi cantly strong negative correlation between enteric CH emission and NEFA concentrations (r=-0.82; p<0.01). No correlation was found between the enteric CH emission and other indicators of metabolic status of the observed cows. Table 3. Comparison of metabolic status indicators (mean ± SEM) between cows with different level of methane emission. Indicator (unit) HM LM A B Total protein (g/L) 81.83 ± 2.0 71.58 ± 4.1 A A Albumin (g/L) 38.22 ± 1.8 37.23 ± 1.7 A A BUN (mmol/L) 4.70 ± 0.41 5.00 ± 0.3 A B Total bilirubin (µmol/L) 2.04 ± 0.1 3.33 ± 0.5 A A AST (U/L) 108.63 ± 17.1 92.50 ± 10.2 A A TAG (mmol/L) 0.17 ± 0.01 0.17 ± 0.04 A A Total cholesterol (mmol/L) 6.04 ± 0.3 5.04 ± 0.6 A A HDL-C (mmol/L) 159.80 ± 7.4 141.64 ± 15.1 A A Glucose (mmol/L) 2.77 ± 0.1 2.72 ± 0.1 A A BHB (mmol/L) 0.46 ± 0.04 0.45 ± 0.1 A B NEFA (mmol/L) 0.27 ± 0.02 0.42 ± 0.03 A,B HM – high enteric CH emissions group; LM - low enteric CH emissions group; – different 4 4 uppercase letters in superscript indicate statistically signifi cant differences (p<0.05) between HM and LM groups of cows. DISCUSSION The results of the presented study reveal signifi cant differences in the variables of CH concentration profi les and some indicators of metabolic status in peak lactating dairy cows with different levels of enteric CH production. In this study, a novel approach of signal processing was used to determine the variables of the CH concentration profi le of peak lactating dairy cows. It is known that this approach is widely applied in medical science, but it is also considered appropriate for processing potentially noisy and dense data such as those obtained by measuring the concentration of CH in the exhaled or eructated gas of animals [22,24,25]. Using this approach, the obtained results for the average CH concentrations indicate that HM cows had signifi cantly higher values of this variable compared to LM cows in all measurement periods. These results suggest that HM and LM cows truly belong to different phenotypes for CH production (high and low), which is needed for comparisons of the observed variables between HM and LM cows to be justifi ed and relevant. At the same time, the average CH concentrations showed an increasing trend from P1 to P3 in both groups of cows, which agrees with the results of Crompton et al. [26], who examined fl uctuations in enteric CH emissions associated with feeding patterns in Holstein-Friesian dairy cows in mid-lactation. Additionally, 79 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 similar results were obtained by Jonker et al. [27] and Rooke et al. [28], who observed enteric CH emission in relation to time after feeding and feed allowance in beef cattle. In the mentioned studies [26-28], enteric CH emission was determined in open-circuit respiration chambers and it was shown that CH emission was the lowest before the morning feeding. Furthermore, these studies have also documented that CH emissions rise gradually and reach a peak between 40 (for beef cattle) and 140 (for dairy cows) minutes after each feeding, and then gradually decline until the next feeding. Although, based on these fi ndings, a decrease in enteric CH emissions in P3 could be expected in our study because this measurement was performed 6 to 8 hours after the morning feeding, this was not the case. This can be explained by the fact that the cows in our study had one more meal between P2 and P3 measurements, which constitutes approximately 20% of the total daily amount of TMR offered, and could have contributed to CH concentrations in P3 being higher than in P2. Finally, our results for the average CH concentrations suggest that, despite the different levels of CH emissions, HM and LM cows have a comparable daily pattern of CH 4 4 emissions. The lowest average CH concentrations could describe this pattern before the morning feeding (P1) and those rises 2-4 hours after the morning feeding (P2) and further rise 6-8 hours after the morning feeding (P3). The number of CH peaks in the presented study did not statistically differ between HM and LM cows in different measurement periods. However, the CH number of peaks tended to be higher in P2 (p=0.09) and was numerically higher in the remaining two measurement periods in HM cows compared to LM cows. Moreover, similar to the average CH concentration, the obtained values for the CH number of peaks 4 4 indicates an increasing trend from P1 to P3 in both groups of cows. Given that the methodology for measuring CH emissions used in this study is based on determining its concentration in the eructated gas, it is important to explain that the appearance of each CH peak could be attributed to an eructation event in the examined cow. A similar interpretation of the appearance of CH peaks was given by Bell et al. [22] and Hardan et al. [25], who used infrared gas analysers to measure enteric CH emissions in the exhaled air of dairy cows and applied the same mathematical approach for the CH concentration profi le analysis. Unfortunately, there is no similar study in the available literature that examined the CH number of peaks or eructation rate in cows with different levels of CH emissions, with which our results could be compared. Nevertheless, the higher number of eructations in HM cows than in LM cows and its increasing trend from P1 to P3 in both groups of cows could be associated with the levels of enteric CH production. It is obvious that a higher number of eructation events followed higher average CH concentrations in this study. Therefore, it can be hypothesized that there is a stronger stimulation of the eructation refl ex through the activation of the vagal afferents of the dorsal rumen by produced gas [29] in cows with high CH production, as well as in the periods after feeding that were coupled with increased CH production in both groups of cows. This hypothesis is additionally supported by our results obtained for the maximum CH peak amplitude, average CH 4 4 80 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le peak amplitude, maximum CH peak width and average CH peak width. Namely, HM 4 4 cows had a signifi cantly higher maximum CH peak amplitude and average CH peak 4 4 amplitude than LM cows in all three measurement periods, whereby in both groups of cows, there was an increasing trend in both parameters from P1 to P3. Also, there were no signifi cant differences in maximum CH peak width and average CH peak 4 4 width between HM and LM groups in different measurement periods or between measurement periods within the respective groups of cows. Accordingly, the described results suggest that higher production of enteric CH more powerfully stimulates the eructation refl ex and leads to a greater number of eructations (CH peaks) and a greater release of CH per eructation event (CH amplitude variables) of approximately equal 4 4 duration (CH width variables). This is clearly noticeable both in HM cows compared to LM cows, as well as in later measurement periods after the morning feeding compared to the previous ones (P3 vs. P2) or those before the morning feeding (P2 vs. P1 and P3 vs. P1). However, additional studies are needed in the physiology of the eructation refl ex and its connection with the level of enteric CH production to confi rm the presented hypothesis. Finally, it should be noted that the average number (±SEM) of eructation events in both groups of cows in all measurement periods was in the general range of 0.7 to 1.5 eructations per minute [30,31]. Furthermore, our results for the average distance among two CH peaks (eructations) did not differ signifi cantly between HM and LM cows or between different measurement periods within the same group of cows. They were also in the general range that predicts the appearance of one eructation event every 40 to 90 seconds in cattle [32]. To the best of our knowledge, this is the fi rst study on the metabolic status of dairy cows with different levels of enteric CH emissions. Nevertheless, the results obtained for the indicators of the metabolic status in our study could be compared with the results reported by Kim et al. [9], who observed differences in metabolic status in various fattening phases of Japanese Black steers with high (HME) and low (LME) methane emissions. In the mentioned study, it was found that HME steers have only signifi cantly higher concentrations of BHB compared to LM steers in all fattening phases, while differences between these groups of steers in the concentration of total protein, total cholesterol, NEFA and glucose and AST activity were not recorded. These fi ndings are not in agreement with ours as we observed signifi cantly higher total protein concentrations and signifi cantly lower total bilirubin and NEFA concentrations in HM cows compared to LM cows. The possible explanation for the described disagreements can be addressed to the cattle breed and production category used in the compared studies. Kim et al. [9] included Black Japanese steers in their study, while Holstein-Friesian dairy cows in peak lactation were used in our study. As these two categories of cattle have different production purposes and different metabolomics and proteomics signatures [33], it is reasonable to expect differences in the metabolic response between these cattle in relation to enteric CH production. Additionally, the mentioned authors performed the study in three phases of fattening, which differ in their metabolic challenge for the steers, while our study included cows 81 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 averaging (±SEM) 49.33 ± 2.58 DIM, avoiding the most pronounced negative energy balance and its infl uence on the monitored indicators of the metabolic status [34]. The values obtained for NEFA concentrations in our study indicate that HM cows have a more favorable energy balance compared to LM cows. Namely, NEFA concentrations refl ect the magnitude of peripheral fat breakdown and energy balance of cows, while BHB concentrations refl ect the ability of the liver to manipulate infl owing NEFA [35]. Although there are no signifi cant differences in BHB concentration between HM and LM cows, attention should be paid to other parameters of liver function observed in this study, such as concentrations of total protein and total bilirubin. In this regard, signifi cantly lower total protein concentrations and signifi cantly higher total bilirubin concentrations in LM cows compared to HM cows indicate that the energy balance of LM cows affects their functional liver capacity [36,37]. Мoreover, the signifi cant negative correlation between enteric CH emissions and total bilirubin and NEFA concentrations, respectively, found in the presented study additionally supports these considerations. On the other hand, the absence of differences in the concentration of BHB between HM and LM cows can be explained by the fact that blood BHB concentration may be affected by the production of butyrate in the rumen [38]. In other words, higher production of butyrate in the rumen of HM cows could have caused an increase in BHB blood concentrations in these cows and masked any differences in the values of this parameter between HM and LM cows. Moreover, the higher production of butyrate in HM cows would not be surprising because there are indications that butyrate synthesis is a metabolic pathway in the rumen that may favor higher CH production because it is associated with the release of H [7,9]. However, 4 2 additional studies are needed in the ruminal microbiome and metabolome and liver metabolome and transcriptome fi elds that will elucidate the nature of the differences found in the metabolic status between HM and LM cows in the present study. Finally, it should be highlighted that all observed indicators of the metabolic status of peak lactating dairy cows in this study were within the reference ranges proposed by Cozzi et al. [39] and Moretti et al. [40]. CONCLUSION In conclusion, this study showed that peak lactating dairy cows with different phenotypes for CH emissions (high and low) have similar daily pattern of CH 4 4 emissions, since the lowest CH emission, detected before the morning feeding, gradually increased after each feeding. This fi nding is in accordance with other studies in which different methods of CH emissions measurement were used. However, our study revealed that dairy cows with a high CH emission phenotype have more eructations and release more CH per eructation. Additionally, the metabolic status of peak lactating dairy cows is affected by the level of CH emission. This is most noticeable in the parameters that depict different aspects of liver function since in cows with high CH production, higher concentrations of total proteins and lower 82 Bošnjaković et al.: Methane emission and metabolic status in peak lactating dairy cows and their assessment via methane concentration profi le concentrations of total bilirubin and NEFA were found. Finally, further research in the fi eld of liver transcriptome and metabolome is needed in order to determine the origin and signifi cance of these differences. Acknowledgments The research was conducted with the support of the Science Fund of the Republic of Serbia, project number 7750295, “Mitigation of methane production from dairy cattle farm by nutritive modulation of cow`s metabolism-MitiMetCattle”. Authors’ contributions DB, DK, SN and LJ contributed to the design and conception of the study. RP, IV, SA and SN conducted the fi eld experiment. 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Res Vet Sci 2017, 114:322-331. 85 Acta Veterinaria-Beograd 2023, 73 (1), 71-86 EMISIJA METANA I METABOLIČKI STATUS MLEČNIH KRAVA U PIKU LAKTACIJE I NJIHOVA PROCENA PUTEM PROFILA KONCENTRACIJE METANA Dušan BOŠNJAKOVIĆ, Danijela KIROVSKI, Radiša PRODANOVIĆ, Ivan VUJANAC, Sveta ARSIĆ, Milica STOJKOVIĆ, Slavica DRAŽIĆ, Sreten NEDIĆ, Ljubomir JOVANOVIĆ Uzgoj preživara doprinosi globalnoj emisiji metana (CH ) i pored negativnog uticaja na životnu sredinu, enterični CH kod krava izaziva gubitak energije unete hranom. Cilj ove studije je bio da se izvrši procena emisije CH i metaboličkog statusa krava određi- vanjem profi la koncentracije metana kao alata kojim se analizira način produkcije CH . Istraživanjem je obuhvać eno osamnaest krava čija je enterička emisija CH merena tokom tri uzastopna dana u tri perioda: 2 sata pre (P1), 2–4 sata (P2) i 6–8 sati (P3) po- sle jutarnjeg hranjenja. Na osnovu emisije CH , krave su podeljene u dve grupe (n=6, pojedinačno): HM (prosečna CH koncentracija: 5430,08 ± 365,92 ppm) i LM (pro- sečna CH koncentracija: 1351,85 ± 205,20 ppm). Nakon merenja CH , treć eg dana, 4 4 uzorkovana je venska krv radi utvrđivanja indikatora metaboličkog statusa. HM krave su imale statistički značajno već e prosečne koncentracije CH , maksimalnu i prosečnu amplitudu CH pika u odnosu na LM krave u svim periodima merenja (P1-P3), dok je broj CH pikova imao tendenciju da bude već i kod HM nego kod LM krava u P2. Nije bilo razlika u maksimalnoj i prosečnoj širini pika CH i prosečnoj udaljenosti između dva pika CH između ispitivanih grupa krava. HM krave su imale značajno već e kon- centracije ukupnih proteina i značajno niže koncentracije ukupnog bilirubina i NEFA od LM krava. Zaključuje se da su HM krave imale već i broj ruktusa i time oslobađale više CH nego LM krave, a razlike u metaboličkom statusu između HM i LM krava su najverovatnije bile povezane sa razlikama u funkciji jetre.

Journal

Acta veterinaria – de Gruyter

Published: Mar 1, 2023

Keywords: dairy cows; methane emission; metabolic status

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