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Item Supplementary figures for Reproductive physiological outcomes of dairy cows with different genomic merit for fertility: biomarkers, uterine health, endocrine status, estrus features, and response to ovarian synchronizationSitko, Emily; Laplacette, Ana; Duhatschek, Douglas; Rial, Clara; Perez, Martin M.; Tompkins, Sheridan; Kerwin, Allison L.; Giordano, Julio O. (Journal Dairy Science, 2024-06-07)Our overarching objective was to characterize associations between genomic merit for fertility and the reproductive function of lactating dairy cows in a prospective cohort study. In this manuscript, we present results of the association between genomic merit for fertility and indicators of metabolic status and inflammation, uterine health, endocrine status, response to synchronization, and estrous behavior in dairy cows. Lactating Holstein cows entering their first (n = 82) or second (n = 37) lactation were enrolled at parturition and fitted with an ear-attached sensor for automated detection of estrus. Ear-notch tissue samples were collected from all cows and submitted for genotyping using a commercial genomic test. Based on genomic predicted transmitting ability values for daughter pregnancy rate (gDPR) cows were classified into a high (Hi-Fert; gDPR >0.6; n = 36), medium (Med-Fert; gDPR -1.3 to 0.6; n = 45), and low (Lo-Fert; gDPR <-1.3; n = 38) group. At 33 to 39 d in milk (DIM), cohorts of cows were enrolled in the Presynch-Ovsynch protocol for synchronization of estrus and ovulation. Body weights, body condition scores (BCS), and uterine health measurements (i.e., vaginal discharge, uterine cytology) were collected from parturition to 60 DIM and milk yield was collected through 90 DIM. Blood samples were collected weekly through 3 wk of lactation for analysis of β-hydroxybutyrate, non-esterified fatty acids, and haptoglobin plasma concentrations. Body weight, BCS, NEFA, BHB, and Haptoglobin were not associated with fertility groups from 1 to 9 wk after parturition. The proportion of cows classified as having endometritis at 33 to 36 DIM tended to be greater for the Lo-Fert than the Hi-Fert group. The proportion of cows that resumed cyclicity did not differ at any timepoint evaluated and there were no significant associations between probability or duration and intensity of estrus with fertility group. Cows of superior genetic merit for fertility were more likely to ovulate, have a functional CL, have greater circulating P4, and have larger ovulatory size than cows of inferior fertility potential at key time points during synchronization of estrus and ovulation. Despite observing numerical differences with potential performance consequences for the proportion of cows that responded to synchronization of ovulation and were both cyclic and responded to the Ovsynch portion of the synchronization protocol, we did not observe significant differences between fertility groups. Although not consistent and modest in magnitude, the collective physiological and endocrine differences observed suggested that cows of superior genetic fertility potential might have improved reproductive performance, at least in part, because of modestly improved endocrine status, uterine health, and ability to ovulateItem The ovarian function and endocrine phenotypes of lactating dairy cows during the estrous cycle were associated with genomic-enhanced predictions of fertility potentialSitko, Emily; Laplacette, Ana; Rial, Clara; Duhatschek, Douglas; Giordano, Julio O.; Perez, Martin M.; Tompkins, Sheridan; Kerwin, Allison L.; Wiltbank, Milo C.; Domingues, Rafael R. (Journal of Dairy Science, 2024-04-29)The objectives of this prospective cohort study were to characterize associations among genomic merit for fertility with ovarian and endocrine function and the estrous behavior of dairy cows during an entire, non-hormonally manipulated estrous cycle. Lactating Holstein cows entering their first (n = 82) or second (n = 37) lactation had ear-notch tissue samples collected for genotyping using a commercial genomic test. Based on genomic predicted transmitting ability values for daughter pregnancy rate (gDPR) cows were classified into a high (Hi-Fert; gDPR >0.6 n = 36), medium (Med-Fert; gDPR -1.3 to 0.6 n = 45), and low fertility (Lo-Fert; gDPR <-1.3 n = 38) group. At 33 to 39 DIM, cohorts of cows were enrolled in the Presynch-Ovsynch protocol for synchronization of ovulation and initiation of a new estrous cycle. Thereafter, the ovarian function and endocrine dynamics were monitored daily until the next ovulation by transrectal ultrasonography and concentrations of progesterone (P4), estradiol, and FSH. Estrous behavior was monitored with an ear-attached automated estrus detection system that recorded physical activity and rumination time. Overall, we observed an association between fertility group and the ovarian and hormonal phenotype of dairy cows during the estrous cycle. Cows in the Hi-Fert group had greater circulating concentrations of P4 than cows in the Lo-Fert group from day 4 to 13 after induction of ovulation and from day -3 to -1 before the onset of luteolysis. The frequency of atypical estrous cycles was 3-fold greater for cows in the Lo-Fert than the Hi-Fert group. We also observed other modest associations between genomic merit for fertility with the follicular dynamics and estrous behavior. There were several associations between milk yield and parity with ovarian, endocrine, and estrous behavior phenotypes as cows with greater milk yield and in the second lactation were more likely to have unfavorable phenotypes. These results demonstrate that differences in reproductive performance between cows of different genomic merit for fertility classified based on gDPR may be partially associated with circulating concentrations of P4, the incidence of atypical phenotypes during the estrous cycles, and to a lesser extent the follicular wave dynamics. The observed physiological and endocrine phenotypes might help explain part of the differences in reproductive performance between cows of superior and inferior genomic merit for fertility.Item Combining reproductive outcomes predictors and automated estrus alerts recorded during the voluntary waiting period identified subgroups of cows with different reproductive performance potentialRial, Clara (Elsevier, 2024-03-11)The objective was to compare differences in reproductive performance for dairy cows grouped based on the combination of data for predictors available during the prepartum period and before the end of the VWP, automated estrus alerts (AEA) during the VWP, and the combination of both factors. In a cohort study, data for AEA and potential predictors of the percentage of cows inseminated in estrus (AIE) and pregnancies per AI (P/AI) for first service, and the percentage of cows pregnant by 150 DIM (P150) were collected from -21 to 49 DIM for lactating Holstein cows (n=886). The association between each reproductive outcome with calving season (cool, warm), calving-related events (yes, no), genomic daughter pregnancy rate (gDPR; high, medium, low), days in the close-up pen (ideal, not ideal), health disorder events (yes, no), rumination time (high or low CV prepartum and high or low increase rate postpartum), and milk yield (MY) by 49 DIM (high, medium, low) were evaluated in univariable and multivariable logistic regression models. Individual predictors (health disorders, gDPR, and MY) associated with the three reproductive outcomes in all models were used to group cows based on risk factors (RF; yes, n=535 or no, n=351) for poor reproductive performance. Specifically, cows were included in the RF group if any of the following conditions were met: the cow was in the high MY group, had low gDPR, or had at least one health disorder recorded. Cows were grouped into estrus groups during the VWP based on records of AEA (E-VWP, n=476 or NE-VWP, n=410). Finally, based on the combination of levels of AEA and RF cows were grouped into an estrus and no RF (E-NoRF, n=217), no estrus and RF (NE-RF, n=276), no estrus and no RF (NE-NoRF, n=134), and estrus and RF (E-RF, n=259) groups. Cows received AIE up to 31 d after the end of the VWP, and if not AIE, received timed AI after an Ovsynch plus progesterone protocol. Logistic and Cox proportional hazard regression compared differences in reproductive outcomes for different grouping strategies. The NoRF (AIE:76.9%; P/AIE:53.1%; P150:84.5%) and E-VWP (AIE:86.8%; P/AIE:44.8%; P150:82.3%) groups had more cows AIE, P/AI, and P150 than the RF (AIE:64.5%; P/AIE:34.9%; P150:72.9%) and NE-VWP (AIE:50.0%; P/AIE:38.9%; P150:72.1%) groups, respectively. When both factors were combined, the largest and most consistent differences were between the E-NoRF (AIE:91.3%; P/AIE:58.7%; P150:88.5%) and NE-RF groups (AIE:47.3%; P/AIE:35.8%; P150:69.5%). Compared with the whole population of cows or cows grouped based on a single factor, the E-NoRF and NE-RF groups had the largest and most consistent differences with the whole cow cohort. The E-NoRF and NE-RF group also had statistically significant differences of a large magnitude when compared with the remaining cow cohort after removal of the respective group. We conclude that combining data for AEA during the VWP with other predictors of reproductive performance could be used to identify groups of cows with larger differences in expected reproductive performance than if AEA and the predictors are used alone.Item Effects of targeted clinical examination based on alerts from automated health monitoring systems on herd health and performance of lactating dairy cowsM. M. Perez, E. M. Cabrera, and J. O. Giordano (Journal of Dairy Science, 2023-07-05)Our objectives were to compare the proportion of lactating dairy cows diagnosed with health disorders (HD) and herd performance when using a health monitoring program designed to rely primarily but not exclusively on alerts from automated health monitoring (AHM) systems or a health monitoring program based primarily on systematic clinical examinations, milk yield monitoring, and visual observation of cows. In a clinical trial, at ~30 d before expected parturition, nulliparous and parous Holstein cows, stratified by parity and days in gestation, were randomly assigned to the high intensity clinical monitoring (HIC-M; n = 625) or automated monitoring (AUT-M; n = 624) treatment. Cows were fitted with a neck-attached rumination and physical activity monitoring tag and individual daily milk yield data was collected from parlor milk meters. For cows in HIC-M, clinical examination was conducted daily until 10 days in milk (DIM) and then in response to milk yield reduction alerts or visual observation of clinical signs of HD in the course of 21 DIM. For cows in AUT-M, clinical examination until 21 DIM was because of health index (HI) score alerts generated with a combination of rumination time and physical activity and reduced milk yield alerts. Visual observation of clinical signs of HD was used for identifying cows potentially missed by automated alerts. Binomial and quantitative data were analyzed by logistic regression and ANOVA with repeated measures, respectively. The percentage of cows diagnosed with at least one HD during the experimental treatments risk period tended to be greater and the incidence rate ratio of HD diagnosed was greater for the HIC-M than AUT-M treatment. There was no difference between treatments for cows that exited the herd to 60 or 150 DIM, but there tended to be more cows that exited the herd from 61 to 150 DIM for the HIC-M than the AUT-M treatment. There were no differences between treatments for daily or total milk yield to 21 DIM or for weekly mean milk yield and total milk yield to 150 DIM. More cows were inseminated in estrus for first service if in the HIC-M treatment and had no HD diagnosed than if in the HIC-M treatment but with HD diagnosed, or in the AUT-M treatment and had no HD diagnosed. Cows in the AUT-M treatment with HD diagnosed did not differ from other groups. No differences between treatments were observed for pregnancies per AI or pregnancy loss for first service. Despite a reduction in the risk of diagnosis of HD, there was no evidence that a health monitoring program that relied on AHM systems alerts to select cows for clinical examination reduced herd performance when compared with a more intensive program that included systematic clinical examinations of all cows for the first 10 DIM, reduced milk yield alerts, and visual observation. However, to obtain the same herd performance than with the HIC-M treatment, the AUT-M treatment required use of VO. In conclusion, a health monitoring program designed to rely primarily on targeted clinical examination based on alerts from automated health monitoring systems might be a feasible alternative to programs that rely more on clinical examination provided that visual observation is used to identify cows not detected by automated alerts.Item Characteristics of the Beef x Dairy Industry in New York State: A summary of survey data collected from New York State farmers in 2020-2021Quaassdorff, Margaret; Hicks, Betsy (2023-07)Data presented in this white paper are from a Qualtrics survey conducted online between the months of October 2020 and June 2021. Farmers were surveyed to assess how they utilize beef sires in their dairy herds, their criteria in selecting dairy animals to breed to beef sires, and sire selection criteria. Farmers were also surveyed on their management practices of producing, raising, marketing and selling BxD cattle, as well as information needed. The survey was open to all farmers in New York State who had an interest in, or were currently producing or growing, BxD animals.Item Metabolic-digestive clinical disorders of lactating dairy cows were associated with alterations of rumination, physical activity, and lying behavior monitored by an ear-attached sensorRial, CLara (Journal of Dairy Science, 2023-06-28)The objective of this observational cohort study was to characterize the pattern of rumination time (RT), physical activity (PA), and lying time (LT) monitored by an automated health monitoring system, based on an ear-attached sensor, immediately before, during, and after clinical diagnosis (CD) of metabolic-digestive disorders. Sensor data were collected from 820 lactating Holstein cows monitored daily from calving up to 21 DIM for detection of health disorders (HD). Cows were grouped retrospectively in the no-clinical health disorder group (NCHD; n = 616) if no HD were diagnosed, or the metabolic-digestive group (METB-DIG; n = 58) if diagnosed with clinical ketosis or indigestion only. Cows with another clinical health disorder within -7 to +7 d of CD of displaced abomasum, clinical ketosis, or indigestion were included in the metabolic-digestive plus one group (METB-DIG+1; n = 25). Daily RT, PA, and LT, and absolute and relative changes within -7 to +7 d of CD were analyzed with linear mixed models with or without repeated measures. Rumination time and PA were smaller, and LT was greater for the METB-DIG and METB-DIG+1 group than for cows in the NCHD group for most days from -7 to +7 d of CD of HD. In general, daily RT, PA, and LT differences were larger between the METB-DIG+1 and NCHD groups than between the METB-DIG and NCHD groups. In most cases, RT and PA decreased to a nadir and LT increased to a peak immediately before or after CD of HD, with a return to levels similar to the NCHD group within 7 d of CD. Absolute values and relative changes from 5 d before CD to the day of the nadir for RT and PA or peak for LT were different for cows in the METB-DIG and METB-DIG+1 group than for the NCHD group. For PA, the METB-DIG+1 group had greater changes than the METB-DIG group. For cows affected by metabolic-digestive disorders, RT, PA, and LT on the day of CD and resolution of clinical signs were different than for cows in the NCHD group, but an increase in RT and PA or a decrease in LT was observed from the day of CD to the day of resolution of clinical signs. We conclude that dairy cows diagnosed with metabolic-digestive disorders including displaced abomasum, clinical ketosis, and indigestion presented substantial alterations in the pattern of RT, PA, and LT captured by an ear-attached sensor. Thus, automated health monitoring systems based on ear-attached sensors might be used as an aid for identifying cows with metabolic-digestive disorders. Moreover, RT, PA, and LT changes after CD might be positive indicators of recovery from metabolic-digestive disorders.Item Metritis and clinical mastitis events in lactating dairy cows were associated with altered patterns of rumination, physical activity, and lying behavior monitored by an ear-attached sensorC. Rial, A. Laplacette, L. Caixeta, C. Florentino, F. Peña-Mosca, and J. O. Giordano (Journal of Dairy Science, 2023-06-18)Understanding changes of behavioral parameters recorded by automated health monitoring systems based on ear-attached sensors on the days immediately before and after diagnosis of metritis and clinical mastitis can help develop dairy cow health monitoring and management strategies. Therefore, the objective of this observational cohort study was to characterize rumination time, physical activity, and lying time monitored by an ear-attached sensor immediately before, during, and after clinical diagnosis (CD) of metritis and clinical mastitis. Lactating Holstein cows monitored daily for up to 21 DIM for detection of health disorders were retrospectively included in the no-clinical health disorder group (NCHD; n = 616) if no clinical health disorders were diagnosed. Cows were included in the metritis (MET; n = 69) or clinical mastitis (MAST; n = 36) group if diagnosed only with non-severe metritis (i.e., no systemic signs of disease) or clinical mastitis (i.e., no pyrexia and no systemic signs of disease), respectively. Cows diagnosed with severe metritis (i.e., systemic signs of disease) or clinical mastitis (i.e., pyrexia and systemic signs of disease), or cows diagnosed with non-severe metritis or clinical mastitis plus another clinical disorder within -7 to +7 d of CD of metritis or clinical mastitis diagnosis were included in the metritis plus (MET+; n = 25) or the clinical mastitis plus (MAST+; n = 15) group, respectively. All cows were fitted with an ear-attached accelerometer to measure rumination time, physical activity, and lying time. Data were summarized as mean daily values, mean value absolute change, and relative change for the mean daily value from 3 or 5 d before CD to the nadir for cows with metritis and clinical mastitis, respectively. Data were analyzed with linear mixed models with or without repeated measures as appropriate. Rumination time and physical activity were lesser, and lying time was greater for the MET and MET+ groups than for the NCHD group for most days from -4 to +7 d of CD of metritis. Generally, daily rumination time, physical activity, and lying time differences were larger and more prolonged between the MET+ and NCHD groups than between the MET and NCHD groups. Similarly, cows in the MAST and MAST+ groups had lesser rumination time and physical activity than cows in the NCHD group for several days before diagnosis of clinical mastitis. Lying time was greater for the MAST+ than the NCHD group on day -1 and 0 relative to CD, but there were no differences with the MAST group. Absolute values and relative changes from 3 d before CD to the day of the nadir for rumination time and physical activity, or peak for lying time were different for cows in the MET and MET+ groups than for the NCHD group. Similar results were observed for the MAST and MAST+ groups compared with the NCHD group. For cows affected by metritis, either an increase in rumination time and physical activity or a decrease in lying time was observed from the day of CD to resolution of clinical signs but no changes were observed for the NCHD. Similarly, cows with clinical mastitis and the NCHD group had different rumination time, physical activity, and lying time on the day of CD and resolution of clinical signs, but cows with clinical mastitis had no significant changes from the day of CD to resolution of clinical signs. We conclude that dairy cows affected by metritis and clinical mastitis presented substantial alterations of the pattern of rumination time, physical activity, and lying time captured by an ear-attached sensor. Thus, automated health monitoring systems based on ear-attached sensors might be used as an aid for identifying cows with metritis and clinical mastitis. Moreover, rumination time, physical activity, and lying time changes after CD might be good indicators of resolution of clinical signs of metritis but not mastitis.Item Supplementary tables for Effect of reproductive management programs that prioritized AI at detected estrus or timed AI on the economic performance of primiparous Holstein cows of different genetic merit for fertilitySitko, Emily; Giordano, Julio O.; Di Croce, Fernando A.; McNeel, Anthony K.; Weigel, Dan J. (2023-06-23)The objective of this randomized controlled experiment was to evaluate the effect of reproductive management programs that prioritized or AI at detected estrus (AIE) or timed AI (TAI) during the first lactation on the economic performance of dairy cows of different genomically-enhanced predicted transmitting ability for fertility. Lactating primiparous Holstein cows from six commercial farms were stratified into high, medium, and low fertility groups based on a Reproduction Index (RI) value calculated from multiple genomically enhanced predicted transmitting abilities to predict the number of days to achieve pregnancy. Within herd and fertility group, cows were randomly assigned either to a program that prioritized AIE (P-AIE; n = 1,416) and used TAI for cows not AIE for all AI services or another that prioritized TAI and had an extended VWP for first service and prioritized TAI for second and greater AI services (P-TAI; n = 1,338). Cash flow (CF) per cow accumulated for the experimental (first) and second calving interval and cash flow per slot per 28 mo after calving in the experimental lactation were calculated. Market and rearing heifer cost values were used for estimating CF. For cows in the Hi-Fert group, a positive effect of delayed pregnancy on milk income during the first lactation was observed but was insufficient to generate significant differences in CF between treatments mainly because of milk income compensation in the second lactation and minor reductions in reproductive cost and gains in calf value for the P-AIE treatment. In this regard, CF for two CI was greater for the P-TAI treatment. Similarly, CF per slot was favorable to the P-TAI treatment. For cows in the Lo-Fert group, CF was numerically in favor of the P-AIE treatment due to a pregnancy and herd exit dynamics that resulted in gains in milk IOFC during the first and second lactation. Differences in CF between cows of different genetic merit for fertility were consistent across treatment and estimation method. Of note, cows in the Lo-Fert group had greater cash flow than cows in the Hi-Fert group in all comparisons. For the Lo-Fert group, greater milk production contributed directly (milk IOFC) and indirectly (reduced culling) to increased cash flow. We concluded that genetic merit for fertility and cash flow are associated because cows of inferior genetic potential for fertility had greater cash flow than cows of superior genetic for fertility despite some increased costs and reduced revenues. Also, the magnitude of the cash flow differences observed for cows of different genetic merit for fertility managed with the P-AIE or P-TAI program may be valuable to commercial dairy farms but did not allow to conclusively support the choice of a type of reproductive management strategy for cows of different genetic merit for fertility.Item Supplementary tables for Effect of reproductive management programs that prioritized AI at detected estrus or timed AI on the reproductive performance of primiparous Holstein cows of different genetic merit for fertilitySitko, Emily M.; Giordano, Julio O.; Perez, Martin M.; Granados, German E.; Masello, Magdalena; Sosa Hernandez, Froylan; Cabrera, Elisa M.; Schilkowsky, Ellen M.; Di Croce, Fernando A.; McNeel, Anthony K.; Weigel, Dan J. (2023-06-23)Our objective was to compare reproductive outcomes of primiparous lactating Holstein cows of different genetic merit for fertility submitted for insemination with management programs that prioritized AI at detected estrus (AIE) or timed AI (TAI). Moreover, we aimed to determine if subgroups of cows with different fertility potential would present a distinct response to the reproductive management strategies compared. Lactating primiparous Holstein cows (n = 6 commercial farms) were stratified into high (Hi-Fert), medium (Med-Fert), and low (Lo-Fert) genetic fertility groups (FG) based on a Reproduction Index (RI) value calculated from multiple genomically enhanced predicted transmitting abilities (gPTA). Within herd and fertility group, cows were randomly assigned either to a program that prioritized TAI and had an extended VWP (P-TAI; n = 1,338) or another that prioritized AIE (P-AIE; n = 1,416) and used TAI for cows not AIE. Cows in P-TAI received first service by TAI at 84 ± 3 DIM after a Double-Ovsynch protocol, were AIE if detected in estrus after a previous AI and received TAI after an Ovsynch-56 protocol at 35 ± 3 d after a previous AI if a corpus luteum (CL) was visualized at nonpregnancy diagnosis (NPD) 32 ± 3 d after AI. Cows with no CL visualized at NPD received TAI at 42 ± 3 d after AI after an Ovsynch-56 protocol with progesterone supplementation (P4-Ovsynch). Cows in P-AIE were eligible for AIE after a PGF2α treatment at 53 ± 3 DIM and after a previous AI. Cows not AIE by 74 ± 3 DIM or by NPD 32 ± 3 d after AI received P4-Ovsynch for TAI at 74 ± 3 DIM or 42 ± 3 d after AI. Binary data were analyzed with logistic regression, count data with Poisson regression, continuous data by ANOVA, and time to event data by Cox’s proportional hazard regression. Pregnancy per AI (P/AI) to first service was greater for cows in the Hi- (59.8%) than the Med- (53.6%) and Lo-Fert (47.7%) groups, and for the P-TAI (58.7%) than the P-AIE (48.7%) treatment. Overall, P/AI for all second and subsequent AI combined did not differ by treatment (P-TAI = 45.2%; P-AIE = 44.5%) or FG (Hi-Fert = 46.1%; Med-Fert = 46.0%; Lo-Fert = 42.4%). The hazard of pregnancy after calving was greater for the P-AIE than the P-TAI treatment [hazard ratio (HR) = 1.27, 95% CI: 1.17 to 1.37)], and for the Hi-Fert than the Med-Fert (HR = 1.16, 95% CI: 1.05 to 1.28) and Lo-Fert (HR = 1.34, 95% CI: 1.20 to 1.49) groups. More cows in the Hi- (91.2%) than the Med- (88.4%) and Lo-Fert (85.8%) groups were pregnant at 200 DIM. Within FG, the hazard of pregnancy was greater for the P-AIE than the P-TAI treatment for the Hi-Fert (HR = 1.41, 95% CI: 1.22 to 1.64) and Med-Fert (HR = 1.28, 95% CI: 1.12 to 1.46) groups but not for the Lo-Fert group (HR = 1.13, 95% CI: 0.98 to 1.31). We conclude that primiparous Holstein cows of superior genetic merit for fertility had better reproductive performance than cows of inferior genetic merit for fertility regardless of the type of reproductive management used. In addition, the effect of programs that prioritized AIE or TAI on reproductive performance for cows of superior or inferior genetic merit for fertility depended on the outcomes evaluated. Thus, programs that prioritize AIE or TAI could be used to affect certain outcomes of reproductive performance or management.Item Supplemental tables for Balancing dairy cattle diets for rumen nitrogen and methionine or all essentail amino acids relative to metabolizable energyLaPierre, P. Andrew; Van Amburgh, Michael; Higgs, Ryan; Chase, Larry; Schwab, Charles; Sloan, Brian; Lucini, Daniel (2023-01)Improving the ability of diet formulation models to more accurately predict AA supply while appropriately describing requirements for lactating dairy cattle pro- vides an opportunity to improve animal productivity, reduce feed costs, and reduce N intake. The goal of this study was to evaluate the sensitivity of a new version of the Cornell Net Carbohydrate and Protein System (CNCPS) to formulate diets for rumen N, Met, and all essential AA (EAA). Sixty-four high-producing dairy cattle were randomly assigned to 1 of the 4 following diets in a 14-wk longitudinal study: (1) limited metabo- lizable protein (MP), Met, and rumen N (Base), (2) adequate Met but limited MP and rumen N (Base + M), (3) adequate Met and rumen N, but limited MP (Base + MU), and (4) adequate MP, rumen N, and bal- anced for all EAA (Positive). All diets were balanced to exceed requirements for ME relative to maintenance and production, assuming a nonpregnant, 650-kg ani- mal producing 40 kg of milk at 3.05% true protein and 4.0% fat. Dietary MP was 97.2, 97.5, 102.3, and 114.1 g/kg of dry matter intake for the Base, Base + M, Base + MU, and Positive diets, respectively. Differences were observed for dry matter intake and milk yield (24.1 to 24.7 and 39.4 to 41.1 kg/d, among treatments). Energy corrected milk, fat, and true protein yield were greater (2.9, 0.13, and 0.08 kg/d, respectively) in cows fed the Positive compared with the Base diet. Using the updat- ed CNCPS, cattle fed the Base, Base + M, and Base + MU diets were predicted to have a negative MP balance (−231, −310, and −142 g/d, respectively), whereas cattle fed the Positive diet consumed 33 g of MP/d excess to ME supply. Bacterial growth was predicted to be depressed by 16 and 17% relative to adequate N supply for the Base and Base + M diets, respectively, which corresponded with the measured lower apparent total-tract NDF degradation. The study demonstrates that improvements in lactation performances can be achieved when rumen N and Met are properly supplied and further improved when EAA supply are balanced relative to requirements. Formulation using the revised CNCPS provided predictions for these diets, which were sensitive to changes in rumen N, Met, all EAA, and by extension MP supply.