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In a previously established animal model, 38 multiparous Holstein cows were assigned to 2 groups fed different diets to achieve either a normal (NBCS) or high (HBCS) body condition score (BCS) and backfat thickness (BFT) until dry-off at -49 d before calving (NBCS: BCS <3.5 [3.02 ± 0.24) and BFT <1.2 cm [0.92 ± 0.21]; HBCS: BCS >3.75 [3.82 ± 0.33] and BFT >1.4 cm [2.36 ± 0.35], mean ± SD). The groups were also stratified for comparable milk yields (NBCS: 10,361 ± 302 kg; HBCS: 10,315 ± 437 kg; mean ± SD). The cows were then fed the same diet during the dry period and subsequent lactation, maintaining the differences in BFT and BCS throughout the study. Using the serum metabolomics data, we created a classification model that identified different metabotypes. Machine learning classifiers revealed a distinct cluster labeled HBCS-PN (HBCS predicted normal BCS) among over-conditioned cows. These cows showed higher feed intake and better energy balance than the HBCS-PH (high BCS predicted high BCS) group, while milk yield was similar. The aim of this study was to investigate the changes in the hepatic transcriptome of cows differing in serum-metabotype postpartum. We performed hepatic transcriptome analysis in cows from 3 metabolic clusters: HBCS-PH (n = 8), HBCS-PN (n = 6), and normal BCS predicted normal BCS (NBCS-PN, n = 8) on d 21 (±2) postpartum. Liver tissue from cows expressed a total of 13,118 genes aligned with the bovine genome. A total of 48 differentially expressed genes (DEG; false discovery rate ≤0.1 and fold-change >1.5) were found between NBCS-PN and HBCS-PH cows, whereas 24 DEG (14 downregulated and 10 upregulated) were found between HBCS-PN and HBCS-PH cows. The downregulated DEG (n = 31) in NBCS-PN cows compared with HBCS-PH cows are involved in biosynthetic processes such as lipid, lipoprotein, and cholesterol synthesis (e.g., APOA1, MKX, RPL3L, CANT1, CHPF, FUT1, ZNF696), cell organization, biogenesis, and localization (e.g., SLC12A8, APOA1, BRME1, RPL3L, STAG3, FBXW5, TMEM120A, SLC16A5, FGF21), catabolic processes (e.g., BREH1, MIOX, APOBEC2, FBXW5, NUDT16), and response to external stimuli (e.g., APOA1, FGF21, TMEM120A, FNDC4), whereas upregulated DEG (n = 17) are related to signal transduction and cell motility (e.g., RASSF2, ASPN, SGK1, KIF7, ZEB2, MAOA, ACKR4, TCAF1), suggesting altered metabolic adaptations during lactation. Our results showed 24 DEG between HBCS-PN and HBCS-PH in the liver. The expression of SLC12A8, SLC16A5, FBXW5, OSGIN1, LAMA3, KDELR3, OR4X17, and INHBE, which are responsible for regulating cellular processes was downregulated in HBCS-PN cows compared with HBCS-PH cows. In particular, the downregulation of SLC12A8 and SLC16A5 expression in HBCS-PN cows indicates lower metabolic load and reduced need for NAD+ biosynthesis to support mitochondrial respiratory processes. The upregulation of MAOA, ACKR4, KIF27, SFRP1, and CAV2 in the liver of HBCS-PN cows may indicate adaptive mechanisms to maintain normal liver function in response to increased metabolic demands from over-conditioning. These molecular differences underscore the existence of distinct metabolic types in cows and provide evidence for the role of the liver in shaping different metabolic patterns.
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Período Pós-Parto , Transcriptoma , Feminino , Bovinos , Animais , Período Pós-Parto/metabolismo , Lactação/fisiologia , Leite/química , Fígado/metabolismoRESUMO
Vegetable fat blends are commonly used as fat sources in milk replacers (MR) for calves, but their composition differs considerably from that of bovine milk fat. The aim of this study was to investigate the serum lipid profile of pre-weaned calves fed twice-daily MR containing 30% fat (% DM). Upon arrival, 30 male Holstein-Friesian calves (BW = 45.6 ± 4.0 kg, age = 2.29 ± 0.8 d) were randomly assigned to 2 experimental diets (n = 15 per treatment): one MR was derived from either vegetable fats (VG; 80% rapeseed and 20% coconut fats) or animal fats (AN; 65% Packer's lard and 35% dairy cream). The 2 MR formulas contained 30% fat, 24% CP, and 36% lactose. Calves were housed indoors in individual pens with ad libitum access to chopped straw and water. Daily milk allowances were 6.0 L from d 1 to 5, 7.0 L from d 6 to 9, and 8.0 L from d 10 to 35, divided into 2 equal meals and prepared at 13.5% solids. An untargeted liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) method was employed to analyze the lipid profiles in the serum of calves sampled from the jugular vein at 35 d of age. In total, 594 lipids were characterized, comprising 25 different lipid classes. Principal component analysis (PCA) showed significant separation between VG and AN, indicating different lipid profiles in the serum. An orthogonal partial least squares discriminant analysis (OPLS-DA) classification model was used to further validate the distinction between the 2 treatment groups. The model exhibited a robust class separation and high predictive accuracy. Using a Volcano plot (fold change threshold ≥1.5 and false discovery rate ≤0.05), it was observed that calves fed AN had higher levels of 39 lipid species in serum than calves fed VG, whereas 171 lipid species were lower in the AN group. Lipid classes, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), triglycerides (TG), lysophosphatidylcholine (LPC), and lysophosphatidylethanolamine (LPE), were different. In particular, PC and PE were observed at lower levels in calves fed AN, possibly indicating shifts in cell membrane characteristics, intracellular signaling, and liver functions. In addition, a decrease in certain triglyceride (TG) species was observed in calves fed AN, including a decrease in TG species such as TG 36:0 and TG 38:0, possibly related to variations in the content of certain fatty acids (FA) within the AN MR, such as C10:0, C12:0, C14:0, and C18:0 compared with the VG MR. Calves fed AN had lower levels of LPC and LPE, and lyso-phosphatidylinositol (LPI), SM, and phosphatidylinositol (PI) species than calves fed VG, suggesting shifts in lipoprotein and lipid metabolic pathways. In conclusion, these results deepen the understanding of how lipid sources in MR can modulate the serum lipidome profiles of dairy calves.
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To investigate the effects of supplemental monensin administration on the metabolic responses of dairy cows, a systematic review and dose-response meta-analysis were conducted. Initially, 604 studies were identified through comprehensive database searches, including Google Scholar, Scopus, Science Direct, and PubMed, using key words related to dairy cows, monensin, and metabolic outcomes. After a 2-stage screening process, 51 articles with a total of 60 experiments were selected for meta-analysis based on criteria such as study implementation date between 2001 and 2022, presence of a control group that did not receive monensin supplementation, reporting of at least 1 outcome variable, and presentation of means and corresponding errors. The meta-analysis used the 1-stage random-effects method, and sensitivity analyses were performed to assess the robustness of the results. The results showed that the administration of monensin at a dosage of 19 to 26 mg/kg was inversely related to methane emissions and that the administration of monensin at a dosage of 18 to 50 mg/kg resulted in a significant decrease in dry matter intake. Administration of monensin at doses of 13 to 28 and 15 to 24 mg/kg also resulted in a significant decrease in ruminal acetate proportion and an increase in propionate proportion, respectively, with no effects on ruminal butyrate, NH3, or pH levels. We found no effects on blood parameters or nitrogen retention, but a significant negative correlation was observed between monensin supplementation and fecal nitrogen excretion. Based on the analysis of all variables evaluated, the optimal dose range of monensin was estimated to be 19 to 24 mg/kg.
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Leite , Monensin , Feminino , Bovinos , Animais , Leite/química , Fermentação , Metano/metabolismo , Rúmen/metabolismo , Nitrogênio/metabolismo , Suplementos Nutricionais/análise , Dieta/veterinária , Lactação/fisiologiaRESUMO
Quantifying the water and mineral losses in feces is essential to determine the optimal composition of oral rehydration solutions (ORS) for diarrheic animals. In a randomized complete block design, this study evaluated water, mineral, and blood acid-base balance of calves with naturally occurring diarrhea receiving ORS or a placebo. On d 0, 45 calves (age: 18 ± 3.2 d; mean ± SD) were selected based on the presence of visual signs of diarrhea, such as dirty tail or wet feces, along with clinical symptoms evaluated by measuring the skin turgor and the degree of enophthalmos. On d 1, calves were divided into blocks of 3 animals based on blood base excess (BE) measured at 0900 h, and within each block, calves were randomly assigned to 1 of 3 treatments (15 calves per treatment) including (1) a hypertonic ORS (HYPER; Na+ = 110 mmol/L; 370 mOsm/kg; strong ion difference [SID] = 60 mEq/L), (2) a hypotonic ORS with low Na+ (HYPO; Na+ = 77 mmol/L; 278 mOsm/kg; SID = 71 mEq/L), and (3) a placebo consisting of lukewarm water with 5 g/L of whey powder (CON). Milk replacer (MR) was fed through teat buckets twice daily at 0630 h and 1700 h in 2 equally sized meals of 2.5 L from d 1 to 3 and of 3.0 L on d 4 and 5. Treatments consisting of 2.0 L lukewarm solutions were administered between milk meals from d 1 to 3 at 1200 h and 2030 h through teat buckets. Refusals of MR and treatments were recorded daily, and blood samples were collected from the jugular vein once daily at arrival in the afternoon of d 0 and at 0900 h from d 1 to 5 after arrival. Urine and feces were collected quantitatively over a 48-h period from 1200 h on d 1 to 1200 h on d 3, and a representative sample of each 24-h period was stored. In addition, the volume of extracellular fluid was evaluated on d 2 by postprandial sampling over a 4-h period relative to the injection of sodium thiosulfate at 1300 h. Total daily fluid intake (MR, treatment, and water) from d 1 to 3 was greater in HYPER (LSM ± SEM; 8.9 ± 0.36 L/d) and HYPO (7.8 ± 0.34 L/d) than in CON (6.6 ± 0.34 L/d). This resulted in a greater water balance (water intake - fluid output in urine and feces) in calves receiving ORS (59.6 ± 6.28 g/kg BW per 24 h vs. 39.6 ± 6.08 g/kg BW per 24 h). Fecal Na+ losses were greater in HYPER than in the other treatments (81 ± 12.0 mg/kg BW per 24 h vs. 24 ± 11.8 mg/kg BW per 24 h). Blood pH was higher in HYPO (7.41 ± 0.016) than CON (7.35 ± 0.016) over the 5 monitoring days, whereas HYPER (7.37 ± 0.017) did not differ with other treatments. In this experimental model, diarrheic calves were likely unable to absorb the high Na+ load from HYPER, resulting in greater Na+ losses in feces, which might have impaired the alkalinizing capacity of HYPER. In contrast, HYPO significantly sustained blood acid-base balance compared with CON, whereas HYPER did not. This suggests that low tonicity ORS with a high SID are more suitable for diarrheic calves.
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Equilíbrio Ácido-Base , Águas Minerais , Animais , Bovinos , Soluções para Reidratação/uso terapêutico , Diarreia/veterinária , Diarreia/tratamento farmacológico , Sódio , Leite , Minerais , Águas Minerais/uso terapêutico , Ração Animal , Dieta/veterinária , Peso Corporal , DesmameRESUMO
The objective of this study was to characterize changes in the serum metabolome and various indicators of oxidative balance in dairy cows starting 2 wk before dry-off and continuing until wk 16 of lactation. Twelve Holstein dairy cows (body weight 745 ± 71 kg, body condition score 3.43 ± 0.66; mean ± SD) were housed in a tiestall barn from 10 wk before to 16 wk after parturition. Cows were dried off 6 wk before the expected calving date (mean dry period length = 42 d). From 8 wk before calving to 16 wk after calving, blood samples were taken weekly to study redox metabolism by determining antioxidant capacity, measured as the ferric-reducing ability of plasma, reactive oxidative metabolites, oxidative stress index, oxidative damage of lipids, measured as thiobarbituric acid reactive substances, and glutathione peroxidase activity. According to these results, dairy cows had the lowest serum antioxidant capacity and greater levels of oxidative stress during the dry-off period and the early postpartum period. For metabolomics, a subset of serum samples including wk -7 (before dry-off), -5 (after dry-off), -1, 1, 5, 10, and 15 relative to calving were used. A targeted metabolomics approach was performed using liquid chromatography and flow injection with electrospray ionization triple quadrupole mass spectrometry using the MxP Quant 500 kit (Biocrates Life Sciences AG). A total of 240 metabolites in serum were used in the final data analysis. Principal component analysis revealed a clear separation by days of sampling, indicating a remarkable shift in metabolic phenotype between the dry period and late and early lactation. Changes in many non-lipid metabolites associated with one-carbon metabolism, the tricarboxylic acid cycle, the urea cycle, and AA catabolism were observed in the study, with changes in AA serum concentrations likely related to factors such as energy and nitrogen balance, digestive efficiency, and changing diets. The study confirmed an extensive remodeling of the serum lipidome in peripartum dairy cows, highlighting the importance of changes in acylcarnitine (acylCN), phosphatidylcholines (PC), and triacylglycerols (TG), as they play a crucial role in lipid metabolism. Results showed that short-chain acylCN increased after dry-off and decreased thereafter, whereas lipid-derived acylCN increased around parturition, suggesting that more fatty acids could enter mitochondria. Phospholipids and sphingolipids in serum showed changes during lactation. In particular, concentrations of sphingomyelins, PC, and lysoPC decreased around calving but increased in mid- and late lactation. In contrast, concentrations of TG remained consistently low after parturition. The serum concentrations of bile acids fluctuated during the dry period and lactation, with glycocholic acid, cholic acid, glycodeoxycholic acid, and taurocholic acid showing the greatest concentrations. These changes are likely due to the interplay of diet, liver function, and the ability of the gut microbiota to convert primary to secondary bile acids. Overall, these descriptive results may aid in hypothesis generation and in the design and interpretation of future metabolite-based studies in dairy cows. Furthermore, they contribute to our understanding of the physiological ranges in serum metabolites relative to the lactation cycle of the dairy cow.
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Antioxidantes , Leite , Feminino , Bovinos , Animais , Leite/química , Antioxidantes/metabolismo , Soro , Lactação/fisiologia , Período Pós-Parto/metabolismo , Dieta/veterinária , Metaboloma , Metabolismo Energético , Ácidos e Sais BiliaresRESUMO
Corn grain with a high phosphorus (P) content (mainly in the form of phytate-P) may need to be processed to improve the digestibility of nutrients for young calves. Processing corn grains can improve the accessibility of phytate-P to the rumen enzymes and increase the bioavailability of P, which benefits the growth and development of calves. The objective of this study was to investigate the effects of feeding starter diets with steam-flaked corn (SFC) compared with ground corn (GC) with 2 P contents of 0.4% and 0.7% DM basis on intake, growth performance, nutrient digestibility, blood metabolites and urinary purine derivatives in dairy calves. A total of 48 female Holstein dairy calves (3 d old; average initial weight 39.7 ± 3.9 kg) were randomly assigned to a 2 × 2 factorial arrangement of treatments (12 calves/treatment) in a randomized complete block design. The treatment groups were: 1) a starter diet of GC with 0.4% P (GC-0.4P); 2) a starter diet of GC with 0.7% P (GC-0.7P); 3) a starter diet of SFC with 0.4% P (SFC-0.4P); 4) a starter diet of SFC with 0.7% P (SFC-0.7P). Calves received 6 L/d of transition milk on d 2-3 and 5 L/d of whole milk on d 4-30, which was increased to 7 L/d on d 31-45, then decreased to 5 L/d on d 46-60 and reduced to a single feeding of 2 L on d 61-62. All calves had free access to starter feed and water. All calves were weaned on d 63 and remained in the study until d 83. Rumen fluid samples were collected on d 38 (pre-weaning) and d 76 (post-weaning). Blood samples were collected on d 40 and 80 and urine samples were collected on 4 consecutive days from d 79 to 82 to analyze urinary excretion of PD. The phytate-P content ranged from 0.23 to 0.17 for GC and SFC, respectively. In particular, the interaction between corn processing method and P content showed that the SFC-0.7P diets had a greater intake of starter feed during the pre- and post-weaning periods compared with the other experimental groups. In addition, calves fed the SFC-0.7P diet had greater average daily gain, body weight, withers height at weaning, better organic matter digestibility, higher blood ß-hydroxybutyrate levels and higher microbial protein synthesis compared with all other groups. Feeding the SFC diet also resulted in improved feed efficiency, improved P digestibility and a tendency toward a lower rumen pH, albeit with a tendency toward an increase in blood glucose concentration during the pre-weaning period. In addition, the inclusion of 0.7% P to the starter diet resulted in increased fiber digestibility and a slight improvement in growth performance, which was particularly evident in hip height. Overall, the inclusion of SFC in the calf starter diet, especially in combination with a 0.7% DM basis P supplement, improved growth performance and nutrient utilization in dairy calves compared with GC.
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Milk replacers (MR) for calves contain alternative fat sources as substitute for milk fat. This substitution leads to differences in fat properties, such as the fatty acid profile and the triglyceride structure. This study evaluated how fat composition in MR affects gastrointestinal health, blood redox parameters, and postprandial metabolism in calves fed twice daily. Forty-five individually housed male Holstein-Friesian calves (2.3 ± 0.85 d of age) were assigned to 1 of 15 blocks based on the age and the day of arrival. Within each block, calves were randomly assigned to 1 of 3 experimental diets and received their respective diet from arrival until 35 d after arrival. The 3 experimental diets (n = 15 per treatment group) consisted of an MR with a blend of vegetable fats containing rapeseed and coconut (VG), an MR with only animal fats from lard and dairy cream (AN), and an MR containing a mixture of animal and vegetable fats including lard and coconut (MX). The fatty acid profile of each MR was formulated to resemble that of bovine milk fat while using only 2 fat sources. All MR were isoenergetic, with 30% fat (% DM), 24% crude protein, and 36% lactose. Chopped straw and water were available ad libitum from arrival onward but no starter feed was provided. Daily milk allowances were 6.0 L from d 1 to 5, 7.0 L from d 6 to 9, and 8.0 L from d 10 to 35, divided into 2 equal meals and prepared at 135 g/L (13.5% solids). Fecal appearance was scored daily; calves were weighed and blood was drawn on arrival and weekly thereafter. Urine and feces were collected over a 24-h period at wk 3 and 5 to determine apparent total-tract digestibility and assess gastrointestinal permeability using indigestible markers. Postprandial metabolism was evaluated at wk 4 by sequential blood sampling over 7.5 h, and the abomasal emptying rate was determined by acetaminophen appearance in blood. Fat composition in MR did not affect growth, MR intake, gastrointestinal permeability, nor nutrient digestibility. The percentage of calves with abnormal fecal scores was lower at wk 2 after arrival in calves fed VG than MX, whereas AN did not differ from the other treatments. Calves fed AN and MX had higher thiobarbituric acid reactive substances measured in serum than VG, whereas plasma ferric-reducing ability was greater in calves fed MX than VG. Postprandial acetaminophen concentrations did not differ across treatment groups, but the area under the curve was smaller in calves fed VG than in the other 2 treatments, which is indicative of a slower abomasal emptying. Postprandial serum triglyceride concentration was greater in calves fed AN than VG, whereas MX did not differ from the other treatments. Based on these outcomes, all 3 fat blends can be considered suitable for inclusion in MR for calves.
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Fat composition in milk replacers (MR) for calves differs from bovine milk fat in multiple ways. The aim of the study was to investigate the impact of different approaches of formulating fat in MR on growth, ad libitum intakes of MR and solid feeds, as well as blood metabolites in dairy calves. Upon 24 to 96 h after birth, 63 calves were acquired from dairy farms and incorporated into the study. Calves were blocked based on arrival day and randomly assigned within each block to one of 3 treatments differing in MR fat composition (n = 21 per group): VG was based on vegetable fats including 80% rapeseed and 20% coconut fats; AN was formulated with animal fats including 65% lard and 35% dairy cream; and MX with a mixture of 80% lard and 20% coconut fats. All 3 MR contained 30% fat, 24% crude protein, and 36% lactose and were formulated to have a fatty acid profile resembling that of milk fat. From arrival onward (3.1 ± 0.84 d of age; means ± standard deviation), calves were group housed and were offered an ad libitum supply of MR at 135 g/L (13.5% solids). Weaning was gradual and induced between wk 7 and 10, after which calves were fed only solid feeds. Starter feed, chopped straw, and water were offered ad libitum throughout the study. Calves were weighed, and blood was collected weekly until d 84 after arrival. Preweaning average daily gain was greater in calves fed AN (915 g/d) than other treatments (783 g/d), whereas no differences were detected in the weaning and postweaning phases. Preweaning MR intake was greater in calves fed AN than MX from wk 2 to 6 and was also higher in calves fed AN than VG in wk 5 and 6. Consistently, the number of rewarded visits during the ad libitum phase was greater in calves fed AN than MX, whereas VG showed no differences. This led to a higher preweaning total metabolizable energy intake in calves fed AN than in calves fed VG and MX. Serum cholesterol was higher, and serum albumin was lower in calves fed VG than other treatments. The proportion of high-density lipoprotein cholesterol in total plasma cholesterol was lower and that of low-density lipoprotein (LDL) cholesterol was higher in calves fed VG compared with other treatments. Overall, the fatty acid profile of plasma largely mirrored the MR fat composition during the preweaning period. Feeding AN enhanced MR intake and improved preweaning growth compared with other treatments. Feeding VG resulted in a marked increase in plasma cholesterol, particularly in the form of LDL cholesterol, which could be linked to an excessive intake of polyunsaturated fatty acids. These findings underscore the importance of formulating the fat content of MR to be similar to bovine milk fat.
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Significant differences exist in the composition of current milk replacers (MR) and bovine whole milk. This study investigated how the macronutrient profile of 3 different MR formulations containing varying amounts of fat, lactose, and protein, and a whole milk powder (WP), affect postprandial metabolism and gut permeability in male Holstein calves. Sixty-four calves (45.4 ± 4.19 kg [mean ± SD] and 1.8 ± 0.62 d of age) were blocked in order of arrival to the facility and within each block, calves were randomly assigned to 1 of 4 treatments. Treatments included a high-fat MR (HF: 25.0% dry matter [DM] fat, 22.5% protein, 38.6% lactose; n = 14), a high-lactose MR (HL: 44.6% lactose, 22.5% protein, 18.0% fat; n = 17), a high-protein MR (HP: 26.0% protein, 18.0% fat, 41.5% lactose; n = 17), and WP (26.0% fat, 24.5% protein, 38.0% lactose; n = 16). Calves were fed 3.0 L (135 g/L) 3 times daily at 0600, 1200, and 1800 h with a teat bucket. Milk intake was recorded daily for the first 28 d after arrival, and blood sampling and body weight measurements were performed at arrival and on d 7, 14, 21, and 27. Gut permeability was estimated from fractional urinary excretion of indigestible markers (Cr-EDTA, lactulose, and d-mannitol) administered as a single dose on d 21 instead of the morning milk meal. Digestibility was determined simultaneously from a total collection of feces over 24 h. Postprandial dynamics were measured on d 28 by sequential blood sampling over 7.5 h. Dry matter intake of MR over 28 d was slightly greater in calves fed HL and HP than in WP. Recovery of Cr-EDTA and d-mannitol over a 24-h urine collection was greater in calves fed WP and HP than HL calves. Apparent total-tract digestibility of crude ash, protein, and fat did not differ among treatments; however, DM digestibility was lower in calves fed WP than in other treatment groups. In addition, abomasal emptying, as indicated by the area under the curve (AUC) for acetaminophen, was slower in calves fed WP than in calves fed HF and HL. The AUC for postprandial plasma glucose was lower in calves fed HL than WP and HF and lower in calves fed HP than WP. The AUC for postprandial serum insulin was greater in calves fed HP than WP and HF, whereas calves fed HL did not differ from the other treatments. Postprandial triglycerides were greater in calves fed WP, and postprandial adiponectin was higher in calves fed HL than other treatments. The high content of lactose and protein in MR had a major effect on postprandial metabolism. This raises the possibility of optimizing MR formulations to maintain metabolic homeostasis and influence development.
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Substitutos do Leite , Leite , Animais , Bovinos , Masculino , Leite/metabolismo , Pós , Dieta/veterinária , Lactose/metabolismo , Ácido Edético , Nutrientes , Permeabilidade , Ração Animal/análise , Manitol , Peso Corporal , DesmameRESUMO
This study aimed to investigate the metabolic changes in the livers of dairy cows from 1 wk before dry off to 1 wk after calving. Twelve high-yielding Holstein cows were included in a longitudinal study and housed in a tiestall barn. The cows were dried off at 6 wk before the expected calving date (dry period length = 42 d). During the entire lactation, the cows were milked twice daily at 0600 and 1700 h. Liver biopsies were taken from each cow at 4 different times: wk -7 (before drying off), -5 (after drying off), -1 and +1 relative to calving. A targeted metabolomics approach was performed by liquid chromatography and flow injection with electrospray ionization triple quadrupole mass spectrometry using the MxP Quant 500 kit (Biocrates Life Sciences AG). A total of 185 metabolites in the liver were used for the final data analysis. Principal component analysis revealed a clear separation by days of sampling, indicating a notable shift in metabolic phenotype from late lactation to the dry period and further changes after calving. Changes were observed in several classes of compounds, including AA and biogenic amines. In particular, the changes in acylcarnitines (AcylCN), phosphatidylcholines (PC), sphingomyelins (SM), and bile acids (BA) indicated extensive remodeling of the hepatic lipidome. The changes in AcylCN concentrations in early lactation suggest incomplete fatty acid oxidation in the liver, possibly indicating mitochondrial dysfunction or enzymatic imbalance. In addition, the changes in PC and SM species in early lactation indicate altered cell membrane composition, which may affect cell signaling and functionality. In addition, changes in BA concentrations and profiles indicate dynamic adaptations in BA synthesis, as well as lipid digestion and absorption during the observation period. In particular, principal component analysis showed an overlapping distribution of liver metabolites in primiparous and multiparous cows, indicating no significant difference between these groups. In addition, Volcano plots showed similar liver metabolism between primiparous and multiparous cows, with no significant fold changes (>1.5) in any metabolite at significant P-values (false discovery rate <0.05). These results provide valuable insight into the physiological ranges of liver metabolites during dry period and calving in healthy dairy cows and should contribute to the design and interpretation of future metabolite-based studies of the transition dairy cow.
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Lactação , Fígado , Metaboloma , Animais , Bovinos , Feminino , Fígado/metabolismo , Estudos LongitudinaisRESUMO
Mobilization of body reserves including fat, protein, and glycogen is necessary to overcome phases of negative nutrient balance typical for high-yielding dairy cows during the periparturient period. Skeletal muscle, the largest internal organ in mammals, plays a crucial role in maintaining metabolic homeostasis. However, unlike in liver and adipose tissue, the metabolic and regulatory role of skeletal muscle in the adaptation of dairy cows to the physiological needs of pregnancy and lactation has not been studied extensively. The functional integrity and quality of skeletal muscle are maintained through a constant turnover of protein, resulting from both protein breakdown and protein synthesis. Thus, muscle protein breakdown (MPB) and synthesis are intimately connected and tightly controlled to ensure proper protein homeostasis. Understanding the regulation of MPB, the catabolic component of muscle turnover, and its assessment are therefore important considerations to provide information about the timing and extent of tissue mobilization in periparturient dairy cows. Based on animal models and human studies, it is now evident that MPB occurs via the integration of 3 main systems: autophagy-lysosomal, calpain Ca2+-dependent cysteine proteases, and the ubiquitin-proteasome system. These 3 main systems are interconnected and do not work separately, and the regulation is complex. The ubiquitin-proteasomal system is the most well-known cellular proteolytic system and plays a fundamental role in muscle physiology. Complete degradation of a protein often requires a combination of the systems, depending on the physiological situation. Determination of MPB in dairy cows is technically challenging, resulting in a relative dearth of information. The methods for assessing MPB can be divided into either direct or indirect measurements, both having their strengths and limitations. Available information on the direct measures of MPB primarily comes from stable isotopic tracer methods and those of indirect measurements from assessing expression and activity measures of the components of the 3 MPB systems in muscle biopsy samples. Other indirect approaches (i.e., potential indicators of MPB), including ultrasound imaging and measuring metabolites from muscle degradation (i.e., 3-methylhistidine and creatinine), seem to be applicable methods and can provide useful information about the extent and timing of MPB. This review presents our current understanding, including methodological considerations, of the process of MPB in periparturient dairy cows.
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Lactação , Proteínas Musculares , Músculo Esquelético , Período Periparto , Prenhez , Proteólise , Animais , Bovinos , Feminino , Gravidez , Tecido Adiposo/metabolismo , Dieta/veterinária , Lactação/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Período Periparto/metabolismo , Prenhez/metabolismoRESUMO
The purpose of this article is to review body condition scoring and the role of body fat reserves in relation to insulin sensitivity and metabolic phenotyping. This article summarizes body condition scoring assessment methods and the differences between subcutaneous and visceral fat depots in dairy cows. The mass of subcutaneous and visceral adipose tissue (AT) changes significantly during the transition period; however, metabolism and intensity of lipolysis differ between subcutaneous and visceral AT depots of dairy cows. The majority of studies on AT have focused on subcutaneous AT, and few have explored visceral AT using noninvasive methods. In this systematic review, we summarize the relationship between body fat reserves and insulin sensitivity and integrate omics research (e.g., metabolomics, proteomics, lipidomics) for metabolic phenotyping of cows, particularly overconditioned cows. Several studies have shown that AT insulin resistance develops during the prepartum period, especially in overconditioned cows. We discuss the role of AT lipolysis, fatty acid oxidation, mitochondrial function, acylcarnitines, and lipid insulin antagonists, including ceramide and glycerophospholipids, in cows with different body condition scoring. Nonoptimal body conditions (under- or overconditioned cows) exhibit marked abnormalities in metabolic and endocrine function. Overall, reducing the number of cows with nonoptimal body conditions in herds seems to be the most practical solution to improve profitability, and dairy farmers should adjust their management practices accordingly.
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Doenças dos Bovinos , Resistência à Insulina , Feminino , Bovinos , Animais , Insulina/metabolismo , Lactação , Tecido Adiposo/metabolismo , Lipólise , Dieta/veterinária , Doenças dos Bovinos/metabolismoRESUMO
The composition of milk replacer (MR) for calves greatly differs from that of bovine whole milk, which may affect gastrointestinal development of young calves. In this light, the objective of the current study was to compare gastrointestinal tract structure and function in response to feeding liquid diets having a same macronutrient profile (e.g., fat, lactose, protein) in calves in the first month of life. Eighteen male Holstein calves (46.6 ± 5.12 kg; 1.4 ± 0.50 d of age at arrival; mean ± standard deviation) were housed individually. Upon arrival, calves were blocked based on age and arrival day, and, within a block, calves were randomly assigned to either a whole milk powder (WP; 26% fat, DM basis, n = 9) or a MR high in fat (25% fat, n = 9) fed 3.0 L 3 times daily (9 L total per day) at 135 g/L through teat buckets. On d 21, gut permeability was assessed with indigestible permeability markers [chromium (Cr)-EDTA, lactulose, and d-mannitol]. On d 32 after arrival, calves were slaughtered. The weight of the total forestomach without contents was greater in WP-fed calves. Furthermore, duodenum and ileum weights were similar between treatment groups, but jejunum and total small intestine weights were greater in WP-fed calves. The surface area of the duodenum and ileum did not differ between treatment groups, but the surface area of the proximal jejunum was greater in calves fed WP. Urinary lactulose and Cr-EDTA recoveries were greater in calves fed WP in the first 6 h post marker administration. Tight junction protein gene expression in the proximal jejunum or ileum did not differ between treatments. The free fatty acid and phospholipid fatty acid profiles in the proximal jejunum and ileum differed between treatments and generally reflected the fatty acid profile of each liquid diet. Feeding WP or MR altered gut permeability and fatty acid composition of the gastrointestinal tract and further investigation are needed to understand the biological relevance of the observed differences.
Assuntos
Substitutos do Leite , Leite , Animais , Bovinos , Masculino , Leite/metabolismo , Pós , Dieta/veterinária , Ácido Edético/metabolismo , Lactulose/metabolismo , Trato Gastrointestinal/metabolismo , Ácidos Graxos/metabolismo , Ração Animal/análise , Desmame , Substitutos do Leite/metabolismo , Peso CorporalRESUMO
The current study was conducted to examine the effect of l-carnitine (LC) supplementation on telomere length and mitochondrial DNA copy number (mtDNAcn) per cell in mid-lactation cows challenged by lipopolysaccharide (LPS) in blood and liver. The mRNA abundance of 31 genes related to inflammation, oxidative stress, and the corresponding stress response mechanisms, the mitochondrial quality control and the protein import system, as well as the phosphatidylinositol 3-kinase/protein kinase B pathway, were assessed using microfluidics integrated fluidic circuit chips (96.96 dynamic arrays). In addition to comparing the responses in cows with or without LC, our objectives were to characterize the oxidative and inflammatory status by assessing the circulating concentration of lactoferrin (Lf), haptoglobin (Hp), fibrinogen, derivates of reactive oxygen metabolites (dROM), and arylesterase activity (AEA), and to extend the measurement of Lf and Hp to milk. Pluriparous Holstein cows were assigned to either a control group (CON, n = 26) or an LC-supplemented group (CAR; 25 g LC/cow per day; d 42 ante partum to d 126 postpartum (PP), n = 27). On d 111 PP, each cow was injected intravenously with LPS (Escherichia coli O111:B4, 0.5 µg/kg). The mRNA abundance was examined in liver biopsies of d -11 and +1 relative to LPS administration. Plasma and milk samples were frequently collected before and after the challenge. After LPS administration, circulating plasma fibrinogen and serum dROM concentrations increased, whereas AEA decreased. Moreover, serum P4 initially increased by 3 h after LPS administration and declined thereafter irrespective of grouping. The Lf concentrations increased in both groups after LPS administration, with the CAR group showing greater concentrations in serum and milk than the CON group. After LPS administration, telomere length in blood increased, whereas mtDNAcn per cell decreased; however, both remained unaffected in liver. For mitochondrial protein import genes, the hepatic mRNA abundance of the translocase of the mitochondrial inner membrane (TIM)-17B was increased in CAR cows. Moreover, TIM23 increased in both groups after LPS administration. Regarding the mRNA abundance of genes related to stress response mechanisms, 7 out of 14 genes showed group × time interactions, indicating a (local) protective effect due to the dietary LC supplementation against oxidative stress in mid-lactating dairy cows. For mtDNAcn and telomere length, the effects of the LPS-induced inflammation were more pronounced than the dietary supplementation of LC. Dietary LC supplementation affected the response to LPS primarily by altering mitochondrial dynamics. Regarding mRNA abundance of genes related to the mitochondrial protein import system, the inner mitochondrial membrane translocase (TIM complex) seemed to be more sensitive to dietary LC than the outer mitochondrial membrane translocase (TOM complex).
Assuntos
Doenças dos Bovinos , Lactação , Feminino , Bovinos , Animais , Lactação/fisiologia , Lipopolissacarídeos/efeitos adversos , Carnitina/metabolismo , DNA Mitocondrial , Variações do Número de Cópias de DNA , Dinâmica Mitocondrial , Inflamação/veterinária , Suplementos Nutricionais , Fígado/metabolismo , Leite/metabolismo , Dieta/veterinária , Expressão Gênica , Fibrinogênio/efeitos adversos , Fibrinogênio/metabolismo , RNA Mensageiro/metabolismo , Proteínas Mitocondriais/metabolismo , Telômero , Doenças dos Bovinos/metabolismoRESUMO
The ratio of n-6 to n-3 fatty acid (FA) is between 2 and 10 times higher in milk replacer (MR) than in whole milk, which may promote inflammation and compromise the integrity of the intestinal epithelium. To evaluate how decreasing the n-6:n-3 FA ratio of MR affects gastrointestinal (GIT) permeability and inflammatory status, 30 dairy calves (2.8 ± 1.06 d of age; mean ± standard deviation) were randomly assigned to be fed an MR with an n-6:n-3 FA ratio of 40:1 (CON; 29.3% crude fat of DM; n = 15) or 6.5:1 (n-3; 29.1% crude fat of DM; n = 15). Calves were fed 7.0 L/d in 2 meals. Calves were weighed and fecal consistency was analyzed weekly. On d 22, calves were administered Cr-EDTA, lactulose, and d-mannitol to assess GIT permeability. Blood and total urine were sequentially collected for 6 and 24 h, respectively, and analyzed for marker content. Whole blood collected 4 h after the meal was subjected to an ex vivo lipopolysaccharide (LPS) challenge to evaluate cytokine secretion from blood cells. Calves were euthanized on d 25 for collection of intestinal tissue samples. Tissue samples were processed to assess FA composition by gas chromatography, histomorphology by bright-field microscopy, and gene expression of tight junction proteins, lipid metabolism enzymes, and immune molecules by real-time quantitative PCR. Data were analyzed using PROC GLIMMIX in SAS (version 9.4, SAS Institute Inc.). Growth performance and fecal consistency were unaffected. Calves fed MR with a lower ratio of n-6 to n-3 FA had 2-fold higher n-3 FA contents and 2-fold lower ratios of n-6 to n-3 FA in proximal jejunum and ileum tissues. Total urinary recovery (0-24 h relative to marker administration) and plasma concentrations of the markers were unaffected. Expression of TJP1 tended to be higher in proximal jejunum tissue and lower in ileum tissue of n-3 calves. The expression of TLR4 and TNFA tended to be higher and CD14 was higher in ileum tissue of n-3 calves. Plasma concentrations of interleukin-4 were decreased in response to the ex vivo LPS challenge in n-3 calves. Histomorphology and GIT permeability were largely unaffected by treatment. Furthermore, the inclusion of linseed and algae oil may promote inflammation, as suggested by greater concentrations of the acute-phase proteins haptoglobin and serum amyloid A postprandially, demonstrating that fat sources should be evaluated for their suitability for MR formulations. Understanding how MR composition affects dairy calf health may improve nutritional strategies on farm.
Assuntos
Ácidos Graxos Ômega-3 , Substitutos do Leite , Animais , Bovinos , Leite , Dieta/veterinária , Lipopolissacarídeos , Permeabilidade , Ração Animal/análise , Desmame , Peso CorporalRESUMO
The present study aimed to evaluate the effects of different supplemental fat sources (soyabean oil (SBO) as a source of n-6 fatty acid (FA) and fish oil (FO) as a source of n-3 FA) in the starter feed of milk-fed dairy calves during the hot season. Forty Holstein calves (3 d of age; 39·67 kg of body weight; ten calves per group) were randomly assigned to the experimental treatments as follows: (1) starter feed supplemented with no fat source (CON), (2) starter feed supplemented with 3 % SBO (DM basis), (3) starter feed supplemented with 3 % FO (DM basis) and (4) starter feed supplemented with an equal mixture of SBO and FO (1·5 % each, DM basis). The milk feeding schedule was constant for treatments and all calves were weaned on day 65 of age. Results show that calves had greater starter intake, average daily gain and body length when fed SBO compared with the other treatments. However, feed efficiency was increased and inflammatory indicators (TNF-α, serum amyloid A and haptoglobin) concentrations were reduced in the calves fed FO compared with the other treatments. In summary, it was revealed that SBO rich in n-6 FA improved starter intake and growth performance, while FO rich in n-3 FA could improve the immune function of calves. Due to the current experimental condition, an equal mixture of SBO and FO (1·5 % each, DM basis) can be recommended to have an optimum growth performance and immune function while the calves are reared under the heat conditions.
Assuntos
Ração Animal , Leite , Ração Animal/análise , Animais , Peso Corporal , Bovinos , Dieta/veterinária , Ácidos Graxos/farmacologia , Resposta ao Choque Térmico , Imunidade , DesmameRESUMO
We aimed to evaluate the effects of dietary supplementation with magnesium oxide and calcium-magnesium dolomite on milk fat synthesis and milk fatty acid profile or persistency in milk fat synthesis after their cessation in dairy cows under milk fat depression conditions. Twenty-four multiparous dairy cows in early lactation (mean ± standard deviation; 112 ± 14 d in milk) were used in a randomized complete block design. Milk fat depression was induced in all cows for 10 d by feeding a diet containing 35.2% starch, 28.7% neutral detergent fiber, and 4.8% total fatty acid (dry matter). The experiment was conducted in 2 periods. During the Mg-supplementation period (d 1-20), cows were randomly assigned to (1) the milk fat depression diet used during the induction phase (control; n = 8), (2) the control diet plus 0.4% magnesium oxide (MG; n = 8), or (3) the control diet plus 0.8% calcium-magnesium dolomite (CMC; n = 8). Compared with the control group, feeding the magnesium-supplemented diets increased milk fat concentration and yield by 12% within 4 d. During the 20-d Mg-supplementation period, both the MG and CMC diets increased milk fat concentration and yield, as well as 3.5% fat-corrected milk and energy-corrected milk yield, without affecting dry matter intake, milk yield, and milk protein and lactose concentrations. In the Mg-cessation period (d 21-30), all cows received the control diet, which resulted in a greater milk fat concentration and yield in the cows that had already received the MG and CMC diets in the Mg-supplementation period. Whereas, milk fat concentration and yield remained high after discontinuation of the magnesium-containing alkalizer until d 27. The difference in milk fat synthesis was associated with lower trans-10 C18:1 (-22%) and higher trans-11 C18:1 (+12.5%) concentrations in milk during the Mg-supplementation period. Furthermore, it was evident that within 2 d of supplementation, the trans-10:trans-11 ratio was lower in MG and CMC cows compared with cows receiving the control. This suggested that the effect of magnesium-based alkalizers on milk fat synthesis was mediated via a shift in ruminal biohydrogenation of cis-9,cis-12 C18:2 in the rumen. In conclusion, abrupt addition of magnesium oxide and calcium-magnesium dolomite increased milk fat synthesis, which persisted for 7 d after cessation of magnesium-based alkalizers. A similar ability to recover milk fat synthesis and normal fatty acid biohydrogenation pathways was observed for magnesium oxide and calcium-magnesium dolomite.
Assuntos
Ração Animal , Magnésio , Ração Animal/análise , Animais , Bovinos , Depressão , Dieta/veterinária , Suplementos Nutricionais , Ácidos Graxos/metabolismo , Feminino , Lactação , Magnésio/metabolismo , Rúmen/metabolismoRESUMO
Telomeres cap the ends of eukaryotic chromosomes, and the telomere length (TL) is related to cellular age. The mitochondrial DNA copy number (mtDNAcn) reflects the abundance of mitochondria in a cell. In addition to generating energy, mitochondria are also the main producers of reactive oxygen species, which in turn can accelerate TL attrition and impair mitochondrial function. Nutrition in early life could influence mtDNAcn and TL in later life. In the present study, we investigated the effects of feeding different levels of milk replacer (MR) on TL shortening and energetic status by examining mtDNAcn of heifers during their first year of life. In this study, whole blood samples were obtained from German Holstein heifer calves 36 to 48 h after birth (wk 1) and at wk 12 and wk 16 of life (n = 37), as well as from 31 calves when reaching 1 yr of age. Calves were fed either a high level of MR (14% solids) at 10 L/d (1.4 kg of MR/d; n = 18) or a restrictive low level at 5.7 L/d (0.8 kg of MR/d; n = 19) until linear weaning in wk 13 to 14 of life. Additional whole blood samples were taken from their respective dams 36 to 48 h after calving. Relative TL (qT) and mtDNAcn in cells from whole blood were measured by multiplex quantitative PCR. The greatest qT values were observed in neonates (36-48 h after birth), with decreasing qT values thereafter. Delta qT values were calculated as ΔqT = qT (first year of life) - initial qT (36-48 h after birth). We found no effect of the feeding regimen on qT values, but qT decreased with age. The mtDNAcn was lowest in neonates, increased until wk 12 of life, and then remained at a constant level until after weaning (wk 16). After the first year of life, mtDNAcn was decreased and returned to levels comparable to those of the neonatal stage. No differences in mtDNAcn were detectable between feeding groups within each time point. When comparing the values of qT and mtDNAcn between the calves and their dams after calving (36-48 h after birth and after calving), greater values were observed in calves than in dams. Delta qT values were negative in all but 2 calves (on the restricted diet), indicating that the change in TL with age was not uniform among individual animals, whereas no difference in mean ΔqT values occurred between the feeding groups. Additional analyses of the correlation between qT, mtDNAcn, and various indicators of oxidative status from birth until wk 16 of life did not indicate major interactions between oxidative status, qT and mtDNAcn. The results of this study support an age-dependent decrease of TL in calves independent of the MR feeding level and show the dynamic changes of mtDNAcn in early life.
Assuntos
Ração Animal , Dieta , Ração Animal/análise , Animais , Bovinos , Variações do Número de Cópias de DNA , DNA Mitocondrial , Dieta/veterinária , Feminino , Leite/metabolismo , Mitocôndrias , Espécies Reativas de Oxigênio/metabolismo , Telômero , DesmameRESUMO
Forty-eight newborn Holstein dairy calves [40 ± 3.4 (SD) of kg of body weight (BW); 24 females and 24 males] were used in a completely randomized design to investigate the effects of a mixture of phytobiotic-rich herbal extract (Immunofin, IMPE) incorporated into milk on performance, ruminal fermentation, bacterial population, and serum biochemical metabolites during the preweaning period. Calves had free access to calf starter and clean water from d 6 until weaning. The treatments were the control (CON; without additive) and IMPE at 4, 8, and 12 mL/d. The treatments had no significant effect on total dry matter intake, weight gain, and BW at weaning. The incidence of diarrhea was lower in calves fed 8 mL of IMPE/d compared with CON. At weaning, body measurements (except for front leg circumference) were not affected by IMPE treatment. Relative to the CON group, front leg circumference was significantly decreased by IMPE supplementation. Serum IgG concentration was not significantly increased by IMPE supplementation compared with the CON group. Triglyceride concentration decreased in calves receiving 4, 8, and 12 mL/d of IMPE compared with the CON groups. In contrast to the CON group, serum albumin and total serum protein concentrations increased with IMPE supplementation. Calves receiving 4 mL/d of IMPE had a greater abundance of total bacteria, Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes compared with the other treatments. Molar proportions of acetate increased in calves fed IMPE (at 12 mL/d) compared with calves fed CON. Ruminal N-NH3 concentrations decreased linearly with the increase in IMPE supplementation. The results of the present study suggest that the addition of IMPE to milk may improve some health and immunity conditions, blood metabolite concentrations, and increase the abundance of some cellulolytic bacteria in the rumen of Holstein dairy calves. The use of IMPE may be an alternative to feeding antibiotics at subtherapeutic concentrations to improve calf health and immunity status.
Assuntos
Ração Animal , Rúmen , Ração Animal/análise , Animais , Peso Corporal , Bovinos , Dieta/veterinária , Feminino , Fermentação , Masculino , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Rúmen/metabolismo , DesmameRESUMO
The objective of this study was to investigate the effects of milk allowances equal to 526 g/d as moderate (MOD) versus 790 g/d of milk dry matter as high (HI), and starter diets containing 18% or 23% crude protein (CP), on growth performance, blood metabolites, and purine derivative (PD) excretion in the urine of dairy calves. A total of 52 female Holstein dairy calves (40.8 kg of body weight) were randomly assigned to the experimental diets. The treatments were (1) moderate milk and 18% CP starter diet (MOD-18CP); (2) MOD and 23% CP starter diet (MOD-23CP); (3) high milk and 18% CP starter diet (HI-18CP); and (4) HI and 23% CP starter diet (HI-23CP). Calves had free access to a starter feed and water and were weaned on d 53 but remained in the study until d 73. Urine samples were collected during the preweaning period (for 6 consecutive days between d 35 and 40) and postweaning period (for 6 consecutive days between d 65 and 70) to investigate urinary excretion of PD. Starter feed intake, ß-hydroxybutyrate (BHB), and blood urea concentrations were reduced; however, average daily gain (ADG) and blood glucose levels increased in calves fed HI before weaning compared with MOD. During the preweaning period, high milk feeding increased total urinary PD excretion but decreased it after weaning. The 23CP diet resulted in higher feed intake and ADG before weaning and higher excretion of allantoin and total excretion of PD compared with the 18CP diet. The HI-23CP treatment resulted in the greatest withers and hip heights at weaning and final measurement, as well as the highest preweaning blood insulin concentrations. In terms of rumen development, MOD-23CP showed the greatest benefits based on starter intake, blood BHB concentration, and urinary excretion of PD. Based on the higher urinary excretion of PD found in HI-fed calves before weaning, it is possible that milk feeding overestimates estimated microbial yield. The results suggest that feeding starters with a higher proportion of CP may help maintain a more balanced ratio of CP to ME during high milk feeding, to avoid protein deficiency due to low starter intake. When calves are fed a high milk allowance, urine excretion of PD may be misinterpreted as a measure of estimated microbial growth and rumen development; this should be considered during calculations of estimated microbial yield in milk-fed calves.