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Peripheral blood mononuclear cell mitochondrial enzyme activity is associated with parity and lactation performance in early lactation Holstein dairy cows

Open AccessPublished:July 01, 2022DOI:https://doi.org/10.3168/jds.2021-21599

      ABSTRACT

      Mitochondria are central to metabolism and are the primary energy producers for all biosynthesis, including lactation. The objectives of this study were to determine if high- and low-producing dairy cows exhibit differences in peripheral blood mononuclear cell mitochondrial enzyme activities of citrate synthase, complex I, complex IV, and complex V during early lactation and, thus, to determine whether those differences were related to differences in lactation performance in the dairy cow. Fifty-six Holstein cows were assigned to 1 of 4 groups: (1) primiparous high, (2) primiparous low, (3) multiparous high, or (4) multiparous low. Primiparous and multiparous cows were analyzed separately. Then, cows were divided into high or low production groups for each production parameter [peak milk, average milk, energy-corrected milk (ECM), fat-corrected milk (FCM), milk lactose, milk fat, milk protein, total solids (TS), solids-not-fat, feed efficiency, and somatic cell count (SCC)]. For all data analysis, production parameters are expressed as yields (kg/d) and SCC (103 cells/mL). High and low production groups were defined by their respective mean production parameters for the 56 cows, with below average cows defined as low and above average cows defined as high. Whole blood samples were collected at one time point, approximately 70 d in milk at 0800 h, and processed for crude mitochondrial extracts from peripheral blood mononuclear cells to determine the activity rates of mitochondrial enzymes. Milk samples were collected 9 times (3 d, 3 times per d) during the week of blood collection and analyzed for major components (fat, protein, lactose, TS, and SCC). Multiparous cows had lower citrate synthase activity than primiparous cows across all production parameters. High-producing cows had greater complex I activity for peak milk, milk yield, ECM, FCM, milk fat, TS, and feed efficiency, and greater complex V activity for ECM, FCM, milk lactose, milk fat, and TS across parities. These findings imply that the most influential respiratory chain enzymes on the level of milk production are those responsible for electron transport chain initialization and ATP production.

      Key words

      INTRODUCTION

      Mitochondria are central to metabolism and are the primary energy producers for all biosynthesis, including lactation. Cows with metabolically efficient mitochondria may have superior ATP output to support the energy demands of biosynthesis of milk, ultimately leading to greater lactational performance. It is estimated that approximately 50% of the chemical energy available to an organism is converted to ATP within a cell, with the remainder being lost to the system as heat via proton leak (
      • Rolfe D.F.
      • Brand M.D.
      Contribution of mitochondrial proton leak to skeletal muscle respiration and to standard metabolic rate.
      ;
      • Rolfe D.F.
      • Newman J.M.
      • Buckingham J.A.
      • Clark M.G.
      • Brand M.D.
      Contribution of mitochondrial proton leak to respiration rate in working skeletal muscle and liver and to SMR.
      ;
      • Stuart J.A.
      • Brindle K.M.
      • Harper J.A.
      • Brand M.D.
      Mitochondrial proton leak and the uncoupling proteins.
      ). Cows with increased mitochondrial enzyme activity may be able to apply the proton gradient to oxidize substrate and meet energetic demands with reduced loss of energy as heat, compared with low enzymatic activity cows. Because there are upper limits on how efficiently energy can be converted to ATP, differences in mitochondrial throughput may explain differences in milk production.
      Despite the active role of mitochondria in metabolism, little research has been done on their relationship to milk production in dairy cattle. Bovine literature exploring mitochondrial function has primarily focused on the relationship between beef cattle mitochondrial oxygen consumption and residual feed intake, with the results being inconclusive across studies (
      • Lancaster P.A.
      • Carstens G.E.
      • Michal J.J.
      • Brennan K.M.
      • Johnson K.A.
      • Davis M.E.
      Relationships between residual feed intake and hepatic mitochondrial function in growing beef cattle.
      ;
      • Acetoze G.
      • Weber K.L.
      • Ramsey J.J.
      • Rossow H.A.
      Relationship between liver mitochondrial respiration and proton leak in low and high RFI steers from two lineages of RFI Angus bulls.
      ;
      • Duarte D.A.S.
      • Newbold C.J.
      • Detmann E.
      • Silva F.F.
      • Freitas P.H.F.
      • Veroneze R.
      • Duarte M.S.
      Genome-wide association studies pathway-based meta-analysis for residual feed intake in beef cattle.
      ).
      • Brown D.R.
      • DeNise S.K.
      • McDaniel R.G.
      Mitochondrial respiratory metabolism and performance of cattle.
      examined mitochondrial oxygen consumption in dairy and beef breeds and found that respiration was associated with milk production in dairy cattle, but it did not reflect beef growth traits. Previous studies faced logistical challenges for large scale mitochondrial analysis, due to the sensitivity of the method. Measuring mitochondrial enzyme activity offers an alternative high throughput method that could be used to evaluate mitochondrial function in commercial dairy settings.
      Mitochondrial enzymatic assays have been used to identify impairment of the respiratory chain (RC) complexes and the citric acid cycle enzymes in humans (
      • Rustin P.
      • Chretien D.
      • Bourgeron T.
      • Gerard B.
      • Rötig A.
      • Saudubray J.M.
      • Munnich A.
      Biochemical and molecular investigations in respiratory chain deficiencies.
      ). These enzymatic pathways are important to the production of ATP and related to an animal's ability to maintain energy balance and energy availability for biosynthetic pathways, which could influence animal production. Therefore, novel methods of assessing mitochondrial function of the dairy cow, such as the use of mitochondrial RC and citric acid cycle enzyme activity, may allow for the identification and selection of cows with greater production potential.
      Early studies by
      • Bell B.R.
      • McDaniel B.T.
      • Robison O.W.
      Effects of cytoplasmic inheritance on production traits of dairy cattle.
      and
      • Brown D.R.
      • DeNise S.K.
      • McDaniel R.G.
      Mitochondrial respiratory metabolism and performance of cattle.
      suggested that milk production could be increased by considering mitochondrial cytoplasmic inheritance, as mitochondria are solely acquired via maternal lines. Because the mitochondrial genome codes for proteins within the energy producing RC, maternal mitochondrial influence on lactation performance is not included in the genomic assessment of dairy cattle. Additionally, the importance of mitochondrial function in dairy reproduction has been strengthened by the findings that dairy cow infertility and oocyte quality have been associated with mitochondrial DNA (mtDNA) number, ATP content, and reduced mitochondrial resilience in aged cows (
      • Iwata H.
      • Goto H.
      • Tanaka H.
      • Sakaguchi Y.
      • Kimura K.
      • Kuwayama T.
      • Monji Y.
      Effect of maternal age on mitochondrial DNA copy number, ATP content and IVF outcome of bovine oocytes.
      ;
      • Ferreira R.M.
      • Chiaratti M.R.
      • Macabelli C.H.
      • Rodrigues C.A.
      • Ferraz M.L.
      • Watanabe Y.F.
      • Smith L.C.
      • Meirelles F.V.
      • Baruselli P.S.
      The infertility of repeat-breeder cows during summer is associated with decreased mitochondrial DNA and increased expression of mitochondrial and apoptotic genes in oocytes.
      ;
      • Kansaku K.
      • Takeo S.
      • Itami N.
      • Kin A.
      • Shirasuna K.
      • Kuwayama T.
      • Iwata H.
      Maternal aging affects oocyte resilience to carbonyl cyanide-m-chlorophenylhydrazone-induced mitochondrial dysfunction in cows.
      ).
      This study examines the use of peripheral blood mononuclear cells (PBMC) as a model tissue for detecting mitochondrial impairment and its effects on dairy cow milk production. Although little research has explored the use of PBMC mitochondrial function in dairy cattle, new research suggests that enzymatic activity of adipose tissue reflects stage of lactation (early vs. late), and peripheral blood mtDNA content correlates to liver mtDNA content in early lactation dairy cows (
      • Laubenthal L.
      • Hoelker M.
      • Frahm J.
      • Dänicke S.
      • Gerlach K.
      • Südekum K.H.
      • Sauerwein H.
      • Häussler S.
      Mitochondrial DNA copy number and biogenesis in different tissues of early-and late-lactating dairy cows.
      ). In human research, PBMC is commonly used to evaluate mitochondrial function (
      • Karabatsiakis A.
      • Böck C.
      • Salinas-Manrique J.
      • Kolassa S.
      • Calzia E.
      • Dietrich D.E.
      • Kolassa I.T.
      Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive subsymptoms and severity of major depression.
      ;
      • Delbarba A.
      • Abate G.
      • Prandelli C.
      • Marziano M.
      • Buizza L.
      • Arce Varas N.
      • Novelli A.
      • Cuetos F.
      • Martínez C.
      • Lanni C.
      • Memo M.
      • Uberti D.
      Mitochondrial alterations in peripheral mononuclear blood cells from Alzheimer's disease and mild cognitive impairment patients.
      ;
      • Hsiao C.P.
      • Hoppel C.
      Analyzing mitochondrial function in human peripheral blood mononuclear cells.
      ). Additionally,
      • Rustin P.
      • Chretien D.
      • Bourgeron T.
      • Gerard B.
      • Rötig A.
      • Saudubray J.M.
      • Munnich A.
      Biochemical and molecular investigations in respiratory chain deficiencies.
      showed that mitochondrial enzymatic activity of PBMC was able to detect RC impairment with minimal invasiveness, when compared with mitochondrial respiration assays. This indicates that PBMC could serve as a minimally invasive model tissue for identifying mitochondrial impairment. The objectives of this study were to determine if high- and low-producing dairy cows, and primi- and multiparous cows, exhibit differences in PBMC mitochondrial enzyme activities of citrate synthase (CS), complex I, complex IV, and complex V during early lactation, and to determine if those differences are associated with improved lactation performance or change with parity.

      MATERIALS AND METHODS

      Animal Management and Housing

      This study was approved by the University of California, Davis Animal Care and Use Committee and by the Purina Animal Nutrition Center Institutional Animal Care and Use Committee. Fifty-six Holstein cows (21 primiparous, 35 multiparous) from the Purina Animal Nutrition Center research herd in Gray Summit, Missouri, were enrolled between April 2017 and June 2017. The sample size of cows per treatment (high vs. low) was estimated to be 11, based on a 2-tailed test, with a difference of 30% between electron transport chain enzyme complex activities, with a power of 0.90 and an α of 0.05. Estimates of sample size were based on the cows available for the project and past studies, involving mitochondrial measurements (
      • Acetoze G.
      • Kurzbard R.
      • Klasing K.C.
      • Ramsey J.J.
      • Rossow H.A.
      Liver mitochondrial oxygen consumption and proton leak kinetics in broilers supplemented with dietary copper or zinc following coccidiosis challenge.
      ;
      • Niesen A.M.
      • Rossow H.A.
      The effects of relative gain and age on peripheral blood mononuclear cell mitochondrial enzyme activity in preweaned Holstein and Jersey calves.
      ), and the minimum sample size was 10 cows per treatment. To be included in the study, cows needed to be at a similar stage of lactation (70 ± 11 DIM) and were fed the same diet using Calan Broadbent Feeding System (American Calan Inc.). The time point of 70 ± 11 DIM was chosen, as cows would be near peak milk production and unlikely to be experiencing transitional stress. Cows were excluded from the study if they did not have 4 functional quarters or had a previous incidence of mastitis due to Staphylococcus aureus infection, as this form of mastitis can cause chronic subclinical infections that may affect the results of PBMC. Because this was a research herd, cows with known health conditions were housed separately and, therefore, were already excluded. Cows were housed in a fully enclosed freestall pen holding mattresses bedded with straw and sawdust. Each cow was assigned to a Calan feeder 1 wk before sample collection and fed a proprietary diet designed to meet or exceed lactation requirements, ad libitum, throughout the study. Training to the Calan feeder system occurred before the study, and intake data were collected −7 to 4 d relative to blood sample collection. Farm staff monitored cows daily, using radio-frequency-based electronic identification tags by dairy staff, to ensure all sensors were functioning properly and that cows were using their assigned Calan feeder. Refusals were collected and weighed daily to estimate daily DMI.

      Milk Sampling and BW Measurements

      Individual cow milk yield was recorded 3 times a day in a double-5 auto-flow parlor equipped with radio-frequency-based electronic identification, data recording, and a Taxatron 5000 (GEA) walk-over scale at the parlor exit. Milk samples were collected for 1 wk at all 3 milkings on Mondays, Wednesdays, and Fridays and samples from each milking were analyzed for fat, true protein, TS, SNF, and MUN, using a MilkoScan FT2 by Foss to determine average milk production at the time of mitochondrial isolation. Somatic cell count was analyzed on the day of blood collection (Wednesdays) using a DeLaval cell counter (DCC, DeLaval International AB).

      Blood Collection and Genetic Scoring

      Two whole blood samples (20 and 4 mL) were obtained via coccygeal venipuncture, into tubes containing K2 EDTA as an anticoagulant (BD Biosciences), at one time point during early lactation, within the 70 ± 11 DIM window, and processed within 1 h of sample collection. The single time point was chosen to evaluate mitochondrial function of PBMC during the same week milk samples were collected as the true production data for the cows would be known. Samples were taken as quickly as possible to ensure minimal stress to the animals. Well-mixed blood (4 mL) from a K2 EDTA tube was used to determine white blood cell count (109/L, %), red blood cell count (1012/L, %), hemoglobin (g/dL), hematocrit (%), mean corpuscular volume (MCV, fL), mean corpuscular hemoglobin (pg), mean corpuscular hemoglobin concentration (%), red cell distribution width (%), platelet count (109/L, %), platelet hematocrit (%), mean platelet volume (fL), platelet distribution width (%), and neutrophil (109/L, %), lymphocyte (109/L, %), monocyte (109/L, %), eosinophil (109/L, %), and basophil (109/L, %) yield using a VetScan HM5 Hematology Analyzer (Abaxis). Before evaluating samples, quality control samples (Multi-trol, Drew Scientific) were tested to ensure equipment functioned within specification.
      Genomic data were collected before this study. At this research facility, calves are sampled for genomic analyses at 3 d of age. Briefly, whole blood from calves was collected into a tube containing K2 EDTA (BD Biosciences) and transferred to a blood card for genetic analysis (Clarifide, Zoetis).

      PBMC Isolation

      Well-mixed blood from the remaining K2 EDTA tubes was transferred to sodium citrate cell preparation tubes (BD Biosciences). Cell preparation tubes were centrifuged at 1,800 × g for 30 min at 20°C. After centrifugation, platelet-rich plasma was discarded without disrupting the density-gradient separating PBMC. The PBMC were collected, washed, and pelleted at 300 × g for 10 min at 20°C in autoMACS Rinsing Solution (PBS, pH 7.2, and 2 mM EDTA; Miltenyi Biotec). During a second wash, red cell contaminants were lysed via osmotic shock using distilled water, vortexed, and immediately diluted with autoMACS Rinsing Solution, followed by centrifugation at 300 × g for 10 min at 20°C. During the third and final wash, PBMC were then pelleted at 300 × g for 10 min at 20°C and the supernatant was discarded. Before the study, cell preparation tubes were validated for isolation of PBMC in bovine blood via university veterinary pathologist and differential staining. Cell counting and viability of the isolated PBMC were determined using trypan blue and a hemocytometer. The viability of PBMC collected from multiparous and primiparous cows was not different, and the mean viability was 90% live cells.

      Mitochondrial Isolation and Protein Quantification

      Mitochondria were extracted from PBMC using the Mitochondria Isolation Kit for Cultured Cells (ab110170, Abcam). Lysate protein concentration from PBMC was measured by the Pierce BCA Protein Assay Kit (Thermo Scientific) and PBMC were frozen at −80°C for 10 min to weaken cellular membranes, followed by the addition of 0.2 μL of universal nuclease (PI88700, Fisher Scientific Co.). Samples were resuspended to a final protein concentration of 5 mg/mL with reagent A, followed by homogenization and centrifugation at 1,000 × g for 10 min at 4°C. The resulting supernatant was retained and the pellet was resuspended in reagent B in the same volume used for reagent A. Homogenization and centrifugation steps were repeated. The supernatant from reagent B was combined with that kept from reagent A and centrifuged at 12,000 × g for 15 min at 4°C. This final supernatant was discarded, and the resulting crude mitochondrial pellet was dissolved in 500 μL of reagent C supplemented protease inhibitor (ab201111, Abcam), and aliquoted into 5 micro-centrifuge tubes and stored at −80°C for enzymatic analyses. The crude mitochondrial protein concentration of one aliquot per cow was measured by BCA (ab102536, Abcam). Before downstream enzymatic analysis, the protein concentration of each sample was adjusted to meet the specifications of each assay protocol.

      Measurement of Mitochondrial Complex I, Complex IV, Complex V, and CS Enzyme Activities

      All mitochondrial enzyme activities were measured using the prepared crude mitochondrial extracts. Microplates for each kit were incubated for 3 h, to allow enzyme adherence to the antibodies in the plate before the collection of absorbance data, using a FLUOstar Omega microplate reader (BMG Labtech) in kinetic mode. Enzymatic assays were performed after all samples had been collected and processed for crude mitochondrial extracts. All assay kits were from the same manufacturer lot, were bovine species reactive, and the intra-assay coefficient of variation for all kits was <3%. Assay sensitivity data, where appropriate, can be found in the manufacturer's protocol. For each kit, spontaneous product conversion (background) was determined by measuring the slope of blank wells containing only the reaction solution for each kit. This activity was determined for each plate and subtracted from the activity of each sample run per plate. Each enzymatic activity was determined with the following assay kits.
      Complex I (EC 1.6.5.3) Enzyme Activity Microplate Assay Kit (ab109721, Abcam) was used to determine the activity of complex I via immunocapture and spectrophotometric analysis. Activity was determined by an increase in absorbance at 450 nm, following the oxidation of NADH to NAD+ and the simultaneous reduction of dye. Kinetic readings were measured at room temperature, 450 nm, and 20-s intervals for 30 min with shaking between readings. Interassay coefficient of variation of this kit was <15%.
      Complex IV (EC 1.9.3.1) activity was measured using the Complex IV Human Enzyme Activity Microplate Assay Kit (ab109909, Abcam). Complex IV was immunocaptured and activity was determined by decreased absorbance at 550 nm, resulting from the oxidation of reduced cytochrome c. Kinetic readings were measured at 30°C for 1-min intervals for 60 min without shaking between readings. Interassay coefficient of variation of this kit was <7%.
      Complex V (EC 3.6.3.14) enzyme activity was determined using the ATP Synthase Enzyme Activity Microplate Assay Kit (ab109714, Abcam). ATP hydrolyzed to ADP, facilitated by immunocaptured ATP Synthase, was coupled with the oxidation of NADH to NAD+, resulting in reduced absorbance at 340 nm. Kinetic readings were measured at 30°C for 1-min intervals for 60 min without shaking between readings. Interassay coefficient of variation of this kit was <13%.
      The activity of CS (EC 4.1.3.7) was measured spectrophotometrically by increased absorbance at 412 nm, via the development of 1,3,5-trinitrobenzene (TNB) from 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), using the Citrate Synthase Activity Assay Kit (ab119692, Abcam). Kinetic readings were measured at room temperature for 20-s intervals for 30 min with shaking between readings. Interassay coefficient of variation of this kit was <8%.

      Statistical Analysis

      Cows were used as the experimental unit of interest, and enzyme activity was defined as the linear rate of change of the absorbance per minute per microgram of crude mitochondrial protein loaded into the well. Only pre-steady state kinetics were evaluated. The slope for each sample was determined using the GLM procedure of SAS (SAS v. 9.4, SAS Institute Inc.) to regress optical density on time, with outlier removal set at 2 standard deviations, and final activities corrected by crude mitochondrial protein. The model was YOD = β0 + β1Time + εOD, in which YOD = optical density, β0 = y intercept, β1 = regression coefficient of time, and εOD = the error.
      Cows were divided into high or low production groups for each production parameter (peak milk, average milk, ECM, FCM, milk lactose, milk fat, milk protein, TS, SNF, feed efficiency, and SCC). For all data analysis, production parameters are expressed as yields (kg/d), SCC as 103 cells/mL, and feed efficiency is unitless [(milk yield kg/d)/(DMI kg/d)]. High and low production groups were defined by their respective mean production parameters for the 56 cows, with below average cows defined as low and above average cows defined as high.
      Analysis of variance was conducted, using the GLM procedure of SAS, to determine if the cow grouping by production group (high or low) resulted in significant differences between high and low production group yields. The model was Ylpdk = μ + Al + Bp + Cd + Dk + εlpdk, where Ylpdk = production parameter (peak milk, average milk, ECM, FCM, milk lactose, milk fat, protein, TS, SNF, feed efficiency, and SCC), μ = overall mean, Al = production group (high or low), Bp = parity (primiparous or multiparous), Cd = DIM at sample collection, Dk = The interaction between production group and parity, and elpdk = the error. Production parameters peak milk, average milk, ECM, FCM, milk lactose, milk fat, milk protein, TS, SNF, and SCC were different by group (high vs. low) and parity indicating that the method used to group cows was successful. Feed efficiency was the only parameter that did not differ by parity (Table 1).
      Table 1Least squares means of production parameters from high- and low-producing Holstein dairy cows by parity
      ItemMultiparousPrimiparousSEMP-value
      Low
      Below average for production parameter.
      High
      Above average for production parameter.
      LowHighProduction group
      Comparison of production group between high and low Holstein dairy cows.
      Parity
      Comparison of production parameters between multiparous and primiparous Holstein dairy cows.
      Milk production
       Peak milk (kg/d)52.568.735.945.11.82<0.001<0.001
       Average milk (kg/d)45.458.232.942.91.80<0.001<0.001
       ECM (kg/d)44.356.833.141.81.40<0.001<0.001
       FCM (kg/d)42.656.532.941.01.40<0.001<0.001
       Lactose (kg/d)2.272.951.652.140.09<0.001<0.001
       Milk fat (kg/d)1.392.001.131.480.05<0.001<0.001
       Milk protein (kg/d)1.371.710.9701.270.05<0.001<0.001
       TS (kg/d)5.366.933.925.060.18<0.001<0.001
       SNF (kg/d)3.955.062.823.730.14<0.001<0.001
       Feed efficiency
      Units of feed efficiency are (milk yield, kg/d)/(DMI, kg/d).
      1.582.041.532.300.07<0.0010.11
       SCC (103 cells/mL)26.11,57416.975.1206<0.001<0.001
      1 Below average for production parameter.
      2 Above average for production parameter.
      3 Comparison of production group between high and low Holstein dairy cows.
      4 Comparison of production parameters between multiparous and primiparous Holstein dairy cows.
      5 Units of feed efficiency are (milk yield, kg/d)/(DMI, kg/d).
      Multivariate regression analyses were conducted to determine if mitochondrial enzymatic activity was associated with production parameters using backward selection. Production parameters were regressed on mitochondrial enzyme activities, peak DIM (PDIM), BW, and hematological parameters using the GLM procedure of SAS. The model was YProd = β0 + β1Enz1 + β2PDIM + β3Hem + ε, in which YProd = production parameter (peak milk, milk yield, fat yield), β0 = y intercept, β1 = regression coefficient of enzyme activity for complex I (Enz1), β2 = regression coefficient of PDIM, β3 = regression coefficient of hematological parameter (Hem), and εProd = the error. Tests for goodness of fit, coefficient of determination (R2), mean bias (%), error due to bias of prediction (%), error due to slope ≠ 0 (%), and error due to random variation (%) were evaluated according to
      • Bibby J.
      • Toutenburg H.
      Prediction and Improved Estimation in Linear Models.
      .
      To determine if genetic markers of milk and component yields were correlated with mitochondrial enzyme activities, mitochondrial enzyme activity was regressed on genetic markers of lactation performance using the GLM procedure of SAS. The model used was YEnz = β0 + β1Gen + εENZ, in which YEnz = enzymatic activity of CS, complex I, complex IV, and complex V, β0 = y intercept, β1 = regression coefficient of genetic score (fluid merit, milk yield, fat yield, and protein yield), and εENZ = the error. A P-value of 0.05 was used for the determination of statistical significance.

      RESULTS AND DISCUSSION

      This study explored the relationship between mitochondrial enzymatic activities of CS, complex I, complex IV, and complex V and milk production in primiparous and multiparous dairy cows. Additional variables that influenced lactation performance of the cow were determined via regression analysis and error partitioning.

      Mitochondrial Enzyme Activity and Milk Production

      To evaluate the effect of PBMC mitochondrial enzyme activities on parity and milk production parameters, cow lactation performance was compared by production group (high or low) within parity and between parity for peak milk, average milk, ECM, FCM, milk lactose, milk fat, milk protein, TS, SNF, feed efficiency, and SCC (Table 2). Citrate synthase, the first enzyme of the citric acid cycle, initiates a series of reactions that are responsible for the generation of high energy electron carriers critical to the downstream production of ATP. Across all production parameters, CS activity was less for multiparous cows, compared with primiparous cows (Table 2). This difference in activity may indicate that multiparous cows have reduced rates of citric acid cycle initiation, compared with primiparous cows, or could represent differences in metabolic states and mitochondrial number between parity. Because primiparous cows are still growing, increased CS activity may be required to meet both their developmental and lactation needs.
      Table 2Least squares means of mitochondrial enzyme activities of peripheral blood mononuclear cells from high- and low-producing Holstein dairy cows by parity
      ItemMultiparousPrimiparousSEMP-value
      Low
      Below average for production parameter.
      High
      Above average for production parameter.
      LowHighGroup
      Comparison of production group between high and low Holstein dairy cows.
      Parity
      Comparison of mitochondrial enzyme activities between multiparous and primiparous Holstein dairy cows.
      Peak milk yield (kg/d)Mean = 60.7Mean = 41.2
       Citrate synthase
      Units of enzyme activity are milli optical density/min per μg of mitochondrial protein.
      0.8630.8881.511.310.140.50.0002
       Complex I0.01360.02450.02700.03360.0050.050.02
       Complex IV0.18860.12550.29030.25210.040.20.003
       Complex V0.021710.021650.015850.026610.00330.070.9
      Average milk yield (kg/d)Mean = 52.1Mean = 37.5
       Citrate synthase0.9650.8871.311.340.150.90.006
       Complex I0.016140.024300.025850.032800.00520.090.04
       Complex IV0.20910.13460.23440.25850.0400.50.06
       Complex V0.023300.020140.016880.026550.00310.30.9
      ECM (kg/d)Mean = 51.5Mean = 37.1
       Citrate synthase0.9200.9311.371.270.160.70.006
       Complex I0.012680.025500.025290.033480.00500.020.02
       Complex IV0.18950.15680.22670.26700.0460.90.07
       Complex V0.020780.022350.015950.027580.00320.020.9
      FCM (kg/d)Mean = 51.0Mean = 36.7
       Citrate synthase0.88480.95321.35201.29810.160.960.005
       Complex I0.010780.026150.024100.034730.00430.0020.01
       Complex IV0.18550.16110.21030.28490.0450.50.07
       Complex V0.018990.023540.015500.027910.00310.0030.9
      Milk lactose yield (kg/d)Mean = 2.6Mean = 1.9
       Citrate synthase0.90050.94751.37341.28290.160.90.005
       Complex I0.014180.025640.026880.031000.0050.070.04
       Complex IV0.17020.17200.24790.24290.0450.90.07
       Complex V0.018710.024180.016110.026370.00320.0070.9
      Milk fat yield (kg/d)Mean = 1.8Mean = 1.3
       Citrate synthase0.73231.05611.26981.41500.170.090.002
       Complex I0.010350.026390.022340.038780.0050.00010.003
       Complex IV0.15880.17900.20040.31930.0490.080.02
       Complex V0.019310.023300.016710.029120.00350.0050.6
      Milk protein yield (kg/d)Mean = 1.5Mean = 1.1
       Citrate synthase0.95220.89531.27331.38920.160.80.005
       Complex I0.016550.024280.026320.032370.00530.120.05
       Complex IV0.17950.16140.21990.27460.0440.60.06
       Complex V0.020090.023360.018030.025290.00520.070.9
      TS yield (kg/d)Mean = 6.3Mean = 4.6
       Citrate synthase0.90180.94411.37891.28660.170.90.005
       Complex I0.012050.025910.025240.032090.00490.020.02
       Complex IV0.18970.15700.24460.24620.0480.70.08
       Complex V0.020670.022410.015480.026010.00340.040.8
      SNF (kg/d)Mean = 4.5Mean = 3.3
       Citrate synthase0.94410.90651.37301.28400.180.70.005
       Complex I0.015300.025720.026810.031210.00490.090.05
       Complex IV0.20310.13720.24780.24330.0450.40.06
       Complex V0.022430.020830.016060.026480.00310.20.06
      Feed efficiency
      Units of feed efficiency are (milk yield, kg/d)/(DMI, kg/d).
      Mean = 1.81Mean = 1.87
       Citrate synthase0.88650.96011.24591.43930.170.30.003
       Complex I0.017530.022680.023130.038250.00540.020.02
       Complex IV0.16230.17870.21140.29210.0480.20.04
       Complex V0.021550.021780.017040.027440.00290.070.8
      SCC (103 cells/mL)Mean = 293.3Mean = 36.0
       Citrate synthase0.90751.00431.20851.52540.200.20.01
       Complex I0.018350.028480.020710.041740.00570.0010.09
       Complex IV0.17440.15160.19590.32850.0560.20.03
       Complex V0.021480.022690.018510.026280.00430.20.9
      1 Below average for production parameter.
      2 Above average for production parameter.
      3 Comparison of production group between high and low Holstein dairy cows.
      4 Comparison of mitochondrial enzyme activities between multiparous and primiparous Holstein dairy cows.
      5 Units of enzyme activity are milli optical density/min per μg of mitochondrial protein.
      6 Units of feed efficiency are (milk yield, kg/d)/(DMI, kg/d).
      Within the electron transport chain, complexes I and IV are 2 of the 3 enzymes that form the electrochemical gradient that drives ATP production through complex V. High-producing cows had greater complex I activity for peak milk, milk yield, ECM, FCM, milk fat, TS, and feed efficiency, compared with low producers across parities (Table 2).
      • Niesen A.M.
      • Rossow H.A.
      The effects of relative gain and age on peripheral blood mononuclear cell mitochondrial enzyme activity in preweaned Holstein and Jersey calves.
      found that preweaning Jersey calves with increased complex I activity had increased BW gain, compared with those with lesser BW gain. High gain Holstein calves tended to have increased complex I activity. Because complex I is the initial electron acceptor in the electron transport chain, the subsequent complexes receive electrons from it to produce ATP. This implies that the enzymatic activity of complex I would be critical in meeting the energy demands of an organism. This may explain why reduced activity of complex I was associated with decreased milk yield in this study and reduced calf weight gain in the previous study. In contrast to complex I, complex IV activity did not differ by production group, but was greater in primiparous cows, compared with multiparous cows for peak milk, milk fat, and feed efficiency (Table 2). These results are in contrast with a previous study by
      • Niesen A.M.
      • Rossow H.A.
      The effects of relative gain and age on peripheral blood mononuclear cell mitochondrial enzyme activity in preweaned Holstein and Jersey calves.
      , which showed no age-related changes in complex IV activity in preweaning Holstein dairy calves. The lack of change in complex IV enzyme activity in calves may be attributed to the support role of complex IV within the electron transport chain or its link to cellular apoptosis pathways. Because decreased complex IV activity is known to induce cell death, it is important that the enzyme is relatively stable across cell types in healthy mitochondria. Multiparous cows with decreased complex IV activity could be showing signs of greater mitochondrial dysfunction and aging stress than primiparous cows. Complex V is the enzyme responsible for ATP synthesis and relies on the proton gradient created by the electron transport chain. High-producing cows had greater complex V activity for ECM, FCM, milk lactose, milk fat, and TS across parities (Table 2). These findings are similar to those observed for complex I, and they suggest that the enzymes responsible for electron transport chain initialization and ATP production are the most influential on the level of milk production. Because complex V differed by production group, but did not reflect parity-related changes, it may be a biomarker of lactation performance.

      Additional Variables That Influence Milk Production

      Because milk production can be influenced by a variety of factors, multivariate regression models were developed to identify variables that correlate with peak milk, milk yield, and milk fat (Table 3). Insignificant variables were energy balance state (positive or negative, determined visually from graphs of daily BW vs. time), sire, number of health events, date of sampling, genetic trait scores, and hematological data, with the exception of MCV. For multiparous cows, complex I activity and peak DIM were most correlated with peak milk and milk yield (R2 = 0.34 and R2 = 0.33, respectively). Additionally, complex I activity in multiparous cows explained 34% of the variation observed for fat yield. For primiparous cows, peak milk yield was most influenced by MCV, a measurement of red blood cell capacity, which accounted for 30% of the variation. For primiparous milk yield, complex I and MCV were most correlated (R2 = 0.28), and primiparous fat yield was explained by complex I activity (R2 = 0.43). The association of complex I activity and milk production traits across parities was likely the result of its role as an initial electron acceptor within the electron transport chain. The reoccurrence of peak DIM in multiparous cow models is explained by its positive association with peak milk and milk yield (data not shown). With primiparous cows, the repeated influence of MCV could represent the relationship between red blood cell oxygen carrying capacity and the need for oxygen as the final electron acceptor.
      Table 3Multivariate regression variables that contribute to production in multiparous and primiparous cows
      ItemModel variable SSE
      The model was YProd = β0 + β1Enz1 + β2PDIM + β3Hem + ε, in which YProd = production parameter (peak milk, milk yield, fat yield), β0 = y intercept, β1 = regression coefficient of enzyme activity for complex I (Enz1), β2 = regression coefficient of peak DIM, β3 = regression coefficient of hematological parameter (Hem), and εProd = the error with the criteria for inclusion being P ≤ 0.1. SSE = sum of squares error.
      Complex I
      Complex I enzyme activity units are milli optical density/min per μg of mitochondrial protein.
      Peak DIMMCV
      Mean corpuscular volume units are fL.
      R2
      Multiparous
       Peak milk yield (kg/d)445.70679.450.34
       Milk yield (kg/d)262.27550.280.33
       Milk fat yield (kg/d)1.270.34
      Primiparous
       Peak milk yield (kg/d)246.730.30
       Milk yield (kg/d)90.5397.730.28
       Milk fat yield (kg/d)0.430.43
      1 The model was YProd = β0 + β1Enz1 + β2PDIM + β3Hem + ε, in which YProd = production parameter (peak milk, milk yield, fat yield), β0 = y intercept, β1 = regression coefficient of enzyme activity for complex I (Enz1), β2 = regression coefficient of peak DIM, β3 = regression coefficient of hematological parameter (Hem), and εProd = the error with the criteria for inclusion being P ≤ 0.1. SSE = sum of squares error.
      2 Complex I enzyme activity units are milli optical density/min per μg of mitochondrial protein.
      3 Mean corpuscular volume units are fL.
      Model fit statistics for variables that contribute to production in multiparous and primiparous cows are shown in Table 4. Multiparous cow models for peak milk and milk yield had the highest correlation (R2 = 0.34 and R2 = 0.33, respectively), compared with primiparous cows (R2 = 0.30 and R2 = 0.23, respectively). For milk fat, primiparous cows had the highest correlation compared with multiparous cows (R2 = 0.58 and R2 = 0.35, respectively). When evaluating the sources of error within these models, the error due to random variation was greatest (67–85%) across models and parities. This suggests that most of the prediction error was due to variation in the data set. The error due to the slope ≠ 0 was the second greatest error source (15–32%) and accounts for model error. Across all models, the error due to bias of prediction was lowest (0–1%) indicating that the models predicting fat, milk, and peak yield have the best fit to observed data. These findings suggest that predicting lactation performance can be improved by considering peak DIM, MCV, and complex I activity.
      Table 4Model fit statistics of regression variables that contribute to production in multiparous and primiparous cows
      ItemMultiparous (n = 35)Primiparous (n = 21)
      Peak milk yield (kg/d)
       Observed mean (kg/d)60.6541.15
       Predicted mean (kg/d)60.5941.15
       Observed SD (kg/d)10.716.44
       Predicted SD (kg/d)6.303.51
       R20.340.30
       Mean bias (%)0.090.00
       Error due to bias of prediction (%)0.000.00
       Error due to slope ≠ 0 (%)26.0029.00
       Error due to random variation (%)74.0071.00
      Milk yield (kg/d)
       Observed mean (kg/d)52.1037.49
       Predicted mean (kg/d)51.9537.03
       Observed SD (kg/d)9.216.09
       Predicted SD (kg/d)5.333.07
       R20.330.23
       Mean bias (%)0.291.24
       Error due to bias of prediction (%)0.001.00
       Error due to slope ≠ 0 (%)26.0032.00
       Error due to random variation (%)74.0067.00
      Milk fat yield (kg/d)
       Observed mean (kg/d)1.761.26
       Predicted mean (kg/d)1.751.25
       Observed SD (kg/d)0.330.22
       Predicted SD (kg/d)0.200.16
       R20.350.58
       Mean bias (%)0.110.40
       Error due to bias of prediction (%)0.000.00
       Error due to slope ≠ 0 (%)25.0015.00
       Error due to random variation (%)75.0085.00

      Mitochondrial Enzyme Activity and Genomics

      The genetic selection of high-producing dairy cattle has moved the industry forward in regard to milk production, but these gains have come at a cost in some farm systems, most notably declining dairy cow fertility rates (
      • Cai Z.
      • Guldbrandtsen B.
      • Lund M.S.
      • Sahana G.
      Prioritizing candidate genes for fertility in dairy cows using gene-based analysis, functional annotation and differential gene expression.
      ;
      • Ma L.
      • Cole J.B.
      • Da Y.
      • VanRaden P.M.
      Symposium review: Genetics, genome-wide association study, and genetic improvement of dairy fertility traits.
      ). Selecting a cow with high genetic merit for production may take away from energy available for reproduction, due to the finite pool of energy available to meet biological needs. Because mitochondria are the primary site of cellular energy production, identifying cows with more efficient mitochondria would provide more energy available to the cow. The role of the influence of mitochondria on milk production and reproduction has likely been undervalued. The maternal inheritance of mitochondria, and their possession of DNA independent of the nuclear genome, means that traditional paternal genetic selection does not directly encompass mitochondrial traits.
      To evaluate the contribution of mitochondrial enzymes and genetic markers to lactation performance, correlation models comparing the two were analyzed (Table 5). No relationship was found, with one exception, between mitochondrial enzyme activities and genetic scores for fluid merit, milk, fat, or protein yield (Table 5). The exception was the genetic score for primiparous milk fat yield and complex I activity, which were correlated (R2 = 0.24). To further evaluate this relationship, fat yield at the time of sampling for primiparous and multiparous cows was plotted against the genetic score for fat (Figure 1A) and complex I activity (Figure 1B). The genetic score for fat production explained 40% of the variation in fat yield for primiparous cows, but this decreased to 5% for multiparous cows (Figure 1A). If mitochondrial enzyme activity alone was used to predict yields, complex I activity explained 43% of the variation of fat yield in primiparous cows, decreasing to 35% for multiparous cows. The genetic score for fat yield had the weakest relationship to actual fat yield for multiparous cows. Milk and protein yield genetic scores also showed limited ability to predict actual yields for multiparous cows (Figure 1C, Figure 1D). Early lactation health events (ketosis, milk fever, displaced abomasum, and metritis) were similar in primiparous and multiparous cows (0.6 and 0.5 events/cow, respectively), but they were not different between high- and low-producing cows in either parity (data not shown). The decreased genetic predictability of milk and fat yield observed in multiparous cows was not the result of increased health events. Additionally, genetic scoring did not accurately predict production beyond the first lactation in this sample of cows (n = 56). However, our study evaluated a small number of cows and more research is needed to see if these results are consistent across dairy systems and with a greater number of cows. Because complex I activity better accounted for the variability in fat production across parities (Figure 1B), it may be a useful tool in assessing cow performance. Evaluating the activities of complexes I, IV, V, and CS could provide novel insight into traits that influence dairy cow performance. Future research should explore differences in mitochondrial enzyme activity across tissues, determine if mitochondrial enzymatic activity changes with the stage of lactation, evaluate the effect of mitochondrial enzyme activity on reproduction, disease states, and aging, and assess if interventions could be implemented to improve mitochondrial function of the dairy cow.
      Table 5Coefficients of determination between mitochondrial enzyme activity and genomic predictors of lactation performance
      ItemMultiparousPrimiparous
      R2P-valueR2P-value
      Genetic score fluid merit ($)
       Citrate synthase0.000.60.010.6
       Complex I0.000.80.040.4
       Complex IV0.000.70.010.7
       Complex V0.001.00.000.8
      Genetic score milk yield (kg)
       Citrate synthase0.010.60.020.6
       Complex I0.000.80.070.3
       Complex IV0.010.50.000.9
       Complex V0.020.40.000.8
      Genetic score fat yield (kg)
       Citrate synthase0.050.20.001.0
       Complex I0.070.10.240.03
       Complex IV0.000.90.010.7
       Complex V0.100.070.110.1
      Genetic score protein yield (kg)
       Citrate synthase0.000.70.001.0
       Complex I0.010.50.150.08
       Complex IV0.010.60.000.9
       Complex V0.000.70.050.3
      Figure thumbnail gr1
      Figure 1Regression of actual fat, milk, and protein yield versus predictors of production: genetic fat score versus actual fat yield (A); complex I activity versus actual fat yield (B); genetic milk score versus actual milk yield (C); and genetic protein score versus actual protein yield (D) for primiparous (♦) and multiparous (▪) cows. mOD = milli optical density.

      CONCLUSIONS

      Mitochondrial enzyme activities in PBMC were examined at 1 time point within early lactation (70 ± 11 DIM) in primiparous and multiparous Holstein cows. Parity-related differences were observed in mitochondrial enzymes CS, complex I, and complex IV. Complexes I and V showed differences for the level of production, but only complex V had no parity-related changes. These findings suggest that mitochondrial enzyme activity of mononuclear cells may be used as a marker of milk production in dairy cattle.

      Acknowledgments

      This study was funded by Purina Animal Nutrition and California Dairy Research Foundation and performed at Purina Animal Nutrition Center (Gray Summit, MO). Therefore, both laboratories (UC Davis Veterinary Medicine Teaching and Research Center Nutrition Lab and Purina Animal Nutrition Center) had input into experimental design and interpretation. This study was performed in collaboration with and at the Purina Animal Nutrition Center as a graduate student internship. Therefore, both laboratories had input into experimental design and interpretation. Data available upon request. The authors have not stated any conflicts of interest.

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