Synthesis, characterization, and cytotoxic activity of N-metallated rhenium(I) pincer complexes with (thio)phosphoryl pendant arms

J. Dairy Sci. 100:1502–1506 https://doi.org/10.3168/jds.2016-11277 © American Dairy Science Association®, 2017. Short communication: Analytical method and amount of preservative added to milk samples may alter milk urea nitrogen measurements Holley L. Weeks and Alexander N. Hristov1 Department of Animal Science, The Pennsylvania State University, University Park 16802 ABSTRACT Thus, it is important to maintain consistency in milk sample preservation and analysis to ensure precision of Milk urea N (MUN) is used by dairy nutritionists MUN results. and producers to monitor dietary protein intake and is Key words: milk urea nitrogen, bronopol, dairy cow indicative of N utilization in lactating dairy cows. Two experiments were conducted to explore discrepancies in Short Communication MUN results provided by 3 milk processing laborato- ries using different methods. An additional experiment Indicators for monitoring dietary N adequacy in was conducted to evaluate the effect of 2-bromo-2-ni- dairy cattle include BUN, urinary urea N, and MUN. tropropane-1, 3-diol (bronopol) on MUN analysis. In Of these, MUN is the most practical analysis because experiment 1, 10 replicates of bulk tank milk samples, individual cow or bulk tank milk samples are routinely collected from the Pennsylvania State University’s collected on dairy farms (Roy et al., 2011). Milk urea Dairy Center over 5 consecutive days, were sent to 3 N is used to monitor dietary CP and N utilization in milk processing laboratories in Pennsylvania. Aver- lactating cows and is linearly related to urinary N ex- age MUN differed between laboratory A (14.9 ± 0.40 cretion (Broderick and Clayton, 1997; Hof et al., 1997). mg/dL; analyzed on MilkoScan 4000; Foss, Hillerød, High MUN levels can indicate excess CP or RDP in the Denmark), laboratory B (6.5 ± 0.17 mg/dL; MilkoScan diet, which could increase N excretion and ammonia FT + 6000), and laboratory C (7.4 ± 0.36 mg/dL; emission from manure, may have negative effects on re- MilkoScan 6000). In experiment 2, milk samples were production, and also increase feed costs to the producer spiked with urea at 0 (7.3 to 15.0 mg/dL, depending (Nousiainen et al., 2004; Burgos et al., 2007; Lean et on the laboratory analyzing the samples), 17.2, 34.2, al., 2012). It should be pointed out, however, that some and 51.5 mg/dL of milk. Two 35-mL samples from analyses have shown no relationship between individual each urea level were sent to the 3 laboratories used in cow milk urea concentration and reproduction in dairy experiment 1. Average analyzed MUN was greater than cows on pasture (Trevaskis and Fulkerson, 1999), or predicted (calculated for each laboratory based on the bulk tank MUN and ammonia emissions from manure control; 0 mg of added urea): for laboratory A (23.2 vs. (Weeks et al., 2015). Jonker et al. (2002b) concluded 21.0 mg/dL), laboratory B (18.0 vs. 13.3 mg/dL), and that farms provided with their bulk tank MUN data on laboratory C (20.6 vs. 15.2 mg/dL). In experiment 3, a monthly basis for 6 mo had 0.52 mg/dL lower MUN replicated milk samples were preserved with 0 to 1.35 than farms who did not received their bulk tank MUN mg of bronopol/mL of milk and submitted to one milk data over the course of their study. This decrease in processing laboratory that analyzed MUN using 2 dif- MUN resulted from an 11 g/d per cow decrease in N ferent methods. Milk samples with increasing amounts intake. Milk analysis laboratories use different analyti- of bronopol ranged in MUN concentration from 7.7 to cal equipment and methods for MUN analysis, which 11.9 mg/dL and from 9.0 to 9.3 mg/dL when analyzed can result in variability in MUN data (Arunvipas et on MilkoScan 4000 or CL 10 (EuroChem, Moscow, al., 2003; Kohn et al., 2004). This can, in turn, cause Russia), respectively. In conclusion, measured MUN confusion on how MUN for a particular farm ranks concentrations varied due to analytical procedure used relative to optimal MUN values suggested in the litera- by milk processing laboratories and were affected by ture (for example, Jonker et al., 2002a; Powell et al., the amount of bronopol used to preserve milk sample, 2014). Therefore, the objectives of this study were to when milk was analyzed using a mid-infrared analyzer. (1) investigate consistency among laboratories’ MUN analytical methods, and (2) investigate differences in MUN due to milk preservative added. In experiment 1, bulk tank milk samples were col- lected from the Pennsylvania State University’s Dairy 1502 Received April 5, 2016. Accepted October 23, 2016. 1 Corresponding author: anh13@psu.edu SHORT COMMUNICATION: MILK UREA NITROGEN 1503 Research and Teaching Center herd. After 5 min of 5 min of continuous agitation in the cooling tank. The continuous agitation, six 35-mL milk samples were 2 L of milk were divided into four 500-mL samples. collected at 1400 h daily over 5 d. Two samples from Samples were spiked with urea at 0, 17.2, 34.2, and each day were shipped overnight to 3 milk processing 51.5 mg urea/dL milk (i.e., added urea levels control, 1, laboratories (laboratories A, B, and C) to be analyzed 2, and 3, respectively). Two 35-mL samples from each for MUN, milk fat, and milk true protein (see Table 1). urea level were preserved and sent to laboratories A, Milk samples were analyzed by mid-infrared spectros- B, and C for MUN analysis according to the methods copy at laboratory A using MilkoScan 4000 (MS; Foss, listed for experiment 1 (Table 1). Based on the amount Hillerød, Denmark), at laboratory B using MilkoScan of added urea and the analyzed MUN concentration FT+ 6000 (Foss), and at laboratory C using MilkoScan of the control milk samples (0 mg of added urea/dL) 6000 (Foss). Samples from 4 of the 5 d submitted to from each laboratory, expected MUN concentrations laboratory A were also analyzed for MUN using a CL were calculated for each laboratory and each urea level 10 analyzer (CL; EuroChem, Moscow, Russia), accord- (i.e., concentration of MUN in control milk sample plus ing to procedures described by Luzzana and Giardino amount of added urea for levels 1, 2, and 3). Data in (1999). The CL 10 method is considered the standard experiment 2 were analyzed using the MIXED proce- to which other methods are compared due to its accu- dure of SAS. The model contained laboratory, urea racy (Dairy One Cooperative Inc., Ithaca, NY, personal level, and laboratory × urea level interaction. Similar communication). Milk samples sent to laboratory A and to experiment 1, when the main effect of laboratory laboratory B were preserved with 2-bromo-2-nitropro- was significant (P ≤ 0.05), means were separated by pane-1, 3-diol (bronopol, Janssen Pharmacauticalaan, pairwise t-test (pdiff option of PROC MIXED). Beerse, Belgium). Milk samples sent to laboratory C In experiment 3, 48 milk vials were prepared with 16 were shipped refrigerated and without preservative due levels (0 to 1.35 mg/mL) of bronopol (Janssen Phar- to the inability of laboratory C’s equipment to analyze macauticalaan). Bronopol was added as 15% aqueous milk preserved with bronopol. Milk MUN data were solution. Each bronopol level was replicated 3 times. analyzed using the MEANS and MIXED procedures A 2-L bulk tank milk sample was collected from The of SAS (version 9.4; SAS Institute Inc., 2003). The Pennsylvania State University’s Dairy Research and MIXED model contained milk sampling day, labora- Teaching Center following the procedure outlined for tory, and sampling day × laboratory interaction, and experiments 1 and 2. Thirty-five mL of milk was added data were analyzed as repeated measures assuming to each vial, resulting in final bronopol concentrations an AR(1) covariance structure. When the main effect of 0, 0.10, 0.19, 0.29, 0.38, 0.47, 0.54, 0.65, 0.72, 0.80, of laboratory was significant (P ≤ 0.05), means were 0.91, 0.98, 1.07, 1.16, 1.24, and 1.35 mg/mL. All milk separated by pairwise t-test (pdiff option of PROC vials were sent to laboratory A for MUN analysis us- MIXED). ing mid-infrared spectroscopy on a MilkoScan 4000 In experiment 2, a 2-L bulk tank milk sample was col- (Foss) and a CL 10 analyzer. Data in experiment 3 lected from The Pennsylvania State University’s Dairy were analyzed using the MIXED procedure of SAS with Research and Teaching Center on a single day following bronopol concentration in the model and contrasts to Table 1. Experimental procedures and sample preparation used in the study1 Laboratory Laboratory Laboratory Laboratory Item A—MS A—CL B C Experiment 1 Equipment used MilkoScan 4000 CL 10 MilkoScan FT + 6000 MilkoScan 6000 Preservative used (bronopol) Yes Yes Yes No Refrigerated Yes Yes No Yes Analytical procedure Infrared analyzer Wet chemistry Infrared analyzer Infrared analyzer Experiment 22 Added urea (mg/dL of milk) Control: 0 Not analyzed Control: 0 Control: 0 Sample 1: 17.5 Sample 1: 17.5 Sample 1: 17.5 Sample 2: 34.2 Sample 2: 34.2 Sample 2: 34.2 Sample 3: 51.5 Sample 3: 51.5 Sample 3: 51.5 Experiment 32 Bronopol added at 0 to 1.35 mg/mL of milk Analyzed Analyzed Not analyzed Not analyzed 1MS = MilkoScan (Foss, Hillerød, Denmark); CL = CL 10 (EuroChem, Moscow, Russia); bronopol (Janssen Pharmacauticalaan, Beerse, Belgium). 2Method of analysis, equipment, and sample preparation as in experiment 1. Journal of Dairy Science Vol. 100 No. 2, 2017 1504 WEEKS AND HRISTOV Table 2. Average MUN, milk fat, and milk true protein concentrations in milk samples analyzed by laboratories A, B, and C (experiment 11) Laboratory Laboratory Laboratory Laboratory Item A—MS2 A—CL2 B C SEM P-value3 Mean MUN, mg/dL 14.9a 14.5a 6.5c 7.4b 0.32 <0.001 SD 1.56 0.26 0.43 0.63 Minimum 12.7 14.1 6.0 6.6 Maximum 17.3 14.9 7.2 8.4 CV 10.5 1.8 6.7 8.5 Mean milk fat, % 3.69a — 3.55c 3.58b 0.003 <0.001 SD 0.082 — 0.082 0.071 Minimum 3.57 — 3.45 3.49 Maximum 3.79 — 3.65 3.67 CV 3.6 2.3 2.0 Mean true protein, % 3.17b — 3.18a 3.16b 0.004 0.01 SD 0.029 — 0.046 0.038 Minimum 3.11 — 3.10 3.10 Maximum 3.20 — 3.25 3.20 CV 3.1 — 1.4 1.2 a–cMeans within a row having different letter superscripts differ at P ≤ 0.05. 1Mean values in table are LSM; n = 38 (MUN) and 30 (milk fat and protein), n represents the number of observations used in the statistical analysis. 2Laboratory A analyzed MUN using 2 methods: MilkoScan 4000 (MS; Foss, Hillerød, Denmark) and CL 10 (CL, EuroChem, Moscow, Russia); see Table 1 for details. Laboratory B used MilkoScan FT + 6000 and laboratory C used MilkoScan 6000. 3Main effect of laboratory. MUN data: effect of day of milk sampling, P = 0.76; laboratory × day of milk sampling interaction, P = 0.22; milk fat data: effect of day of milk sampling, P < 0.001; laboratory × day of milk sampling interaction, P = 0.03; milk true protein data: effect of day of milk sampling, P < 0.001; laboratory × day of milk sampling interaction, P = 0.10. evaluate linear and quadratic effects of bronopol con- the lowest variation with the CL 10. In the present centration. study, average MUN concentrations differed between The objective of experiment 1 was to set baseline laboratories A (MilkoScan 4000), B (MilkoScan FT+ MUN concentrations and determine differences in MUN 6000), and C (MilkoScan 6000), likely due to variation analysis among laboratories. Average MUN concentra- in analytical procedure and equipment (Arunvipas et tion analyzed at laboratory A was greater (P < 0.001) al., 2003; Kohn et al., 2004). If a theoretical benchmark than laboratories B and C, as shown in Table 2. The for MUN is assumed at 10 to 12 mg/dL (Jonker et al., 2 methods used by laboratory A, MilkoScan 4000 and 2002a; Powell et al., 2014), the present study indicated CL 10, returned similar MUN values. Average MUN that MUN results from laboratory A are above the concentration for laboratory C was also greater (P < benchmark and results from laboratories B and C are 0.001) than laboratory B. There was no effect of day below the benchmark for identical milk samples. Thus, of milk sampling (P = 0.76) and there was no day of data from experiment 1 indicate that laboratory and sampling × laboratory interaction (P = 0.22) for MUN. analytical method may cause significant variability in Variability in MUN data (SD and CV) was larger for bulk tank MUN data. laboratory A (MS method) than B and C. The low- The objective of experiment 2 was to quantify the est variability was associated with the CL method accuracy of each laboratory’s MUN analysis. Average (laboratory A). As shown in Table 2, the 3 laboratories analyzed MUN and the difference between analyzed and returned different results for milk fat and true protein predicted MUN are presented in Table 3. The differ- (P ≤ 0.01) concentrations, although differences were ence between analyzed and expected (calculated based subtle, particularly for milk protein. Kohn et al. (2004) on MUN concentration in the control milk samples for concluded that 34% of the variation in bulk tank MUN each laboratory and the amount of added urea) MUN for Foss (MilkoScan) 4000 was attributed to labora- concentration was largest (P < 0.001) for laboratory tory. In their study, milk samples analyzed on Foss 4000 C followed by laboratory B, and was least for labo- resulted in the largest SD (±2.51 mg/dL) when com- ratory A. The difference was clearly increasing (P < pared with CL 10. Conversely, the Bentley (±0.45 mg/ 0.001) for all laboratories with increasing the amount dL), Foss 6000 (±0.62 mg/dL), and Skalar (±0.55 mg/ of added urea, but the ranking among laboratories re- dL) instruments resulted in smaller SD when compared mained the same. The relationship between amount of with CL 10 (Kohn et al., 2004). Similarly, Peterson et added urea and the difference between analyzed and al. (2004) concluded that the highest variation among predicted MUN was linear for all laboratories (R2 = methods occurred with the Foss 4000 analyzer and 0.82 to 0.99; P ≤ 0.001). Peterson et al. (2004) com- Journal of Dairy Science Vol. 100 No. 2, 2017 SHORT COMMUNICATION: MILK UREA NITROGEN 1505 pared recovery of urea N among 5 analytical methods: 0.05 mg/dL and tended to increase linearly (P = 0.06) Bentley, CL 10, Foss 4000, Foss 6000, and Skalar us- with increasing bronopol concentration. MilkoScan ing milk samples from 100 individual cows. Recovery 4000 uses mid-infrared spectroscopy, in which a beam by CL 10 (85.0 ± 2.76%) was lower than for Bentley, of light at specified wavelength for the component be- Foss 6000, and Skalar, but greater compared with Foss ing measured is passed through milk and the amount 4000 (47.1 ± 9.88%). Furthermore, no differences were of light absorbed is measured (Arunvipas et al., 2003). detected for Bentley and CL 10 among laboratories; Data for MilkoScan 4000 from experiment 3 suggest however, results for Foss 4000, Foss 6000, and Skalar that the absence of bronopol in experiment 1, labora- varied among laboratories. In the present study, all 3 tory C milk samples may have resulted in lower MUN laboratories overestimated MUN compared with the values than if those samples contained bronopol. In the expected concentrations, but the least overestimation CL 10 method, the amount of ammonia formed from was for laboratory A, which used MilkoScan 4000. An urea after hydrolysis with urease is used to calculate important point in any analysis, including MUN, is MUN (Arunvipas et al., 2003; Kohn et al., 2004). Previ- calibration of the analytical equipment. All laboratories ous studies indicate greater variability with MilkoScan have established calibration procedures but as pointed 4000 compared with CL 10 for multiple laboratories out by Kohn et al. (2004), better calibration methods (Kohn et al., 2004; Peterson et al., 2004). Conversely, across laboratories may improve the consistency of Arunvipas et al. (2003) reported high reliability and MUN results. In experiment 2 of the current study, the repeatability for both MilkoScan 4000 and CL 10 range of calibration standards used by the laboratories when analyzed at a single laboratory. In the present may have not been adequate to cover the range of MUN study, bronopol concentration of 0.54 mg/mL analyzed in the milk samples with added urea. on MilkoScan 4000 resulted in about 12% greater (P The objective of experiment 3 was to determine the < 0.05) MUN than the control (9.6 vs. 8.5 mg/dL, effect of increasing concentrations of bronopol on MUN respectively). According to one milk processing labora- as analyzed by laboratory A using 2 MUN methods tory, recommended bronopol concentration is 0.085%, (MilkoScan 4000 and CL 10). Data from this experi- which is 0.85 mg/mL of milk (Dairy One Cooperative ment are shown in Figure 1. Concentration of MUN Inc., personal communication). At this concentration, analyzed on MilkoScan 4000 ranged from 7.7 to 11.9 and based on data from experiment 3 with MilkoScan ± 0.27 mg/dL and linearly increased (P < 0.001) with 4000 (Figure 1), MUN concentration would likely be increasing bronopol concentration. Milk urea N concen- overestimated by about 30%. Some have suggested that trations analyzed on CL 10 ranged from 9.0 to 9.3 ± bronopol should be used to preserve milk samples at Table 3. MUN concentrations in milk samples with added urea (experiment 21) Laboratory Laboratory Laboratory Item A—MS B C SEM P-value2 Average analyzed MUN,3 mg/dL 23.2a 18.0c 20.6b 0.20 <0.001 Overall difference,4 mg/dL 2.2c 4.6b 5.4a 0.20 <0.001 Added urea level 1, mg/dL Analyzed 21.0 14.4 16.5 Predicted 19.0 11.3 13.2 Difference 2.2 3.1 3.3 0.36 0.21 Added urea level 2, mg/dL Analyzed 25.4 21.5 24.4 Predicted 23.0 15.3 17.2 Difference 2.4b 6.2a 7.2a 0.55 0.02 Added urea level 3, mg/dL Analyzed 31.2 28.6 32.3 Predicted 27.0 19.3 21.2 Difference 4.2c 9.2b 11.2a 0.41 0.003 a–cMeans within a row having different letter superscripts differ at P ≤ 0.05. 1Difference data are LSM; n = 24 (analyzed MUN) and 6 (added urea levels 1–3), n represents the number of observations used in the statisti- cal analysis. 2Main effect of laboratory. 3Average MUN across added urea levels. Effect of added urea level, P < 0.001; laboratory × added urea level interaction, P < 0.001. 4Difference between analyzed and expected MUN concentrations (analyzed – expected MUN) across added urea levels. Expected MUN concen- trations were calculated from amount of urea added and MUN of the control samples from each laboratory. Added urea levels: control = 0, level 1 = 17.2, level 2 = 34.2, and level 3 = 51.5 mg of urea/dL of milk. Differences between analyzed and expected MUN concentrations were close to zero (10−32 to 9−16 mg/dL) for the control (i.e., 0 mg of added urea) samples. Journal of Dairy Science Vol. 100 No. 2, 2017 1506 WEEKS AND HRISTOV lyzer, MUN concentration may also be affected by the concentration of bronopol used. Consistently using the same laboratory and sampling procedure is advisable, if MUN concentration (bulk tank or individual cows) is used to monitor protein status of the herd. Thus, es- tablishing a MUN benchmark relative to the laboratory and analytical method used may be helpful for on-farm management purposes. REFERENCES Arunvipas, P., J. A. VanLeeuwen, I. R. Dohoo, and G. P. Keefe. 2003. Evaluation of the reliability and repeatability of automated milk urea nitrogen testing. Can. J. Vet. Res. 67:60–63. Barbano, D. M., K. L. Wojciechowski, and J. M. Lynch. 2010. Effect of preservatives on the accuracy of mid-infrared milk component testing. J. Dairy Sci. 93:6000–6011. Broderick, G. A., and M. K. Clayton. 1997. A statistical evaluation of animal and nutritional factors influencing concentrations of milk Figure 1. Average MUN concentration (±SE) in milk preserved with urea nitrogen. J. Dairy Sci. 80:2964–2971. increasing concentrations of bronopol (Janssen Pharmacauticalaan, Burgos, S. A., J. G. Fadel, and E. J. DePeters. 2007. Prediction of Beerse, Belgium) analyzed at laboratory A—MS (MilkoScan 4000, ammonia emission from dairy cattle manure based on milk urea Foss, Hillerød, Denmark) and laboratory A—CL (CL 10, EuroChem, nitrogen: Relation of milk urea nitrogen to urine urea nitrogen Moscow, Russia; experiment 3). Effect of bronopol concentration: lin- excretion. J. Dairy Sci. 90:5499–5508. ear P < 0.001 (MilkoScan 4000) and P = 0.06 (CL 10). *Indicates Hof, G., M. D. Vervoorn, P. J. Lenaers, and S. Tamminga. 1997. Milk significant difference (P < 0.05) between control (0 mg of bronopol) urea nitrogen as a tool to monitor the protein nutrition of dairy and specified concentration of bronopol when analyzed on MilkoScan cows. J. Dairy Sci. 80:3333–3340. 4000. Data are arithmetic means. Jonker, J. S., R. A. Kohn, and J. High. 2002a. Dairy herd management practices that impact nitrogen utilization efficiency. J. Dairy Sci. 85:1218–1226. concentrations of 0.01 to 0.02% (Barbano et al., 2010), Jonker, J. S., R. A. Kohn, and J. High. 2002b. Use of milk urea nitro- gen to improve dairy cow diets. J. Dairy Sci. 85:939–946. or 0.1 to 0.2 mg/mL. At these low concentrations, MUN Kohn, R. A., K. R. French, and E. Russek-Cohen. 2004. A comparison analysis is likely not going to be affected by bronopol of instruments and laboratory used to measure milk urea nitrogen (Figure 1). It is noted that MUN analysis by CL 10 in bulk-tank milk samples. J. Dairy Sci. 87:1848–1853. Lean, I. J., P. Celi, H. Raadsma, J. McNamara, and A. R. Rabiee. was more consistent and was less affected by bronopol 2012. Effects of dietary crude protein on fertility: Meta-analysis concentration. The CL 10 is widely accepted as the and meta-regression. Anim. Feed Sci. Technol. 171:31–42. most accurate measurement of MUN; however, due to Luzzana, M., and R. Giardino. 1999. Urea determination in milk by a differential pH technique. Le Lait, INRA Editions 79:261–267. cost and additional labor needed to analyze samples, Nousiainen, J., K. J. Shingfield, and P. Huhtanen. 2004. Evaluation it is not practical for commercial use (Arunvipas et of milk urea nitrogen as a diagnostic of protein feeding. J. Dairy al., 2003). We are not aware of published data on Sci. 87:386–398. Peterson, A. B., K. R. French, E. Russek-Cohen, and R. A. Kohn. bronopol interference with MUN analysis. Other milk 2004. Comparison of analytical methods and the influence of milk components, such as fat, protein, and SCC, do interfere components on milk urea nitrogen recovery. J. Dairy Sci. 87:1747– with the mid-infrared analysis of MUN (Arunvipas et 1750. Powell, J. M., C. A. Rotz, and M. A. Wattiaux. 2014. Potential use of al., 2003), but the calibration process and computer milk urea nitrogen to abate atmospheric nitrogen emissions from algorithm correct for these interferences. Barbano et al. Wisconsin dairy farms. J. Environ. Qual. 43:1169. (2010) studied various milk preservatives and reported Roy, B., B. Brahma, S. Ghosh, P. K. Pankaj, and G. Mandal. 2011. Evaluation of milk urea concentration as useful indicator for dairy that bronopol-based preservatives interfered with mid- herd management: A review. Asian J. Anim. Vet. Adv. 6:1–19. infrared analyses, particularly for milk protein, com- SAS Institute Inc.. 2003. SAS/STAT User’s Guide: Statistics, Version pared with K Cr O -preserved milk. 8 Edition. SAS Inst. Inc., Cary, NC. 2 2 7 Trevaskis, L. M., and W. J. Fulkerson. 1999. The relationship between Milk urea N is widely used on farm as an indicator various animal and management factors and milk urea, and its of dietary CP and RDP concentrations, intake, and N association with reproductive performance of dairy cows grazing utilization in dairy cows. Data from the current ex- pasture. Livest. Prod. Sci. 57:255–265. Weeks, H. L., T. W. Frederick, L. M. Hagan, K. Heyler, J. Oh, and A. periments indicate that reported MUN concentrations N. Hristov. 2015. Case Study: Farm-level evaluation of implement- vary between laboratories depending on method and ing nitrogen and phosphorus feeding best management practices equipment used. When analyzed by a mid-infrared ana- on Pennsylvania dairy farms. Prof. Anim. Sci. 31:473–483. Journal of Dairy Science Vol. 100 No. 2, 2017