Heat stress-associated changes in the intestinal barrier, inflammatory signals, and microbiome communities in dairy calves.

Recent studies indicate that heat stress pathophysiology is associated with intestinal barrier dysfunction, local and systemic inflammation, and gut dysbiosis. However, inconclusive results and a poor description of tissue-specific changes must be addressed to identify potential intervention targets against heat stress illness in growing calves. Therefore, the objective of this study was to evaluate components of the intestinal barrier, pro- and anti-inflammatory signals, and microbiota community composition in Holstein bull calves exposed to heat stress. Animals (mean age = 12 wk old; mean body weight = 122 kg) penned individually in temperature-controlled rooms were assigned to (1) thermoneutral conditions (constant room temperature at 19.5°C) and restricted offer of feed (TNR, n = 8), or (2) heat stress conditions (cycles of room temperatures ranging from 20 to 37.8°C) along with ad libitum offer of feed (HS, n = 8) for 7 d. Upon treatment completion, sections of the jejunum, ileum, and colon were collected and snap-frozen immediately to evaluate gene and protein expression, cytokine concentrations, and myeloperoxidase activity. Digesta aliquots of the ileum, colon, and rectum were collected to assess bacterial communities. Plasma was harvested on d 2, 5, and 7 to determine cytokine concentrations. Overall, results showed a section-specific effect of HS on intestinal integrity. Jejunal mRNA expression of TJP1 was decreased by 70.9% in HS relative to TNR calves. In agreement, jejunal expression of heat shock transcription factor-1 protein, a known tight junction protein expression regulator, decreased by 48% in HS calves. Jejunal analyses showed that HS decreased concentrations of IL-1α by 36.6% and tended to decrease the concentration of IL-17A. Conversely, HS elicited a 3.5-fold increase in jejunal concentration of anti-inflammatory IL-36 receptor antagonist. Plasma analysis of pro-inflammatory cytokines showed that IL-6 decreased by 51% in HS relative to TNR calves. Heat stress alteration of the large intestine bacterial communities was characterized by increased genus Butyrivibrio_3, a known butyrate-producing organism, and changes in bacteria metabolism of energy and AA. A strong positive correlation between the rectal temperature and pro-inflammatory Eggerthii spp. was detected in HS calves. In conclusion, this work indicates that HS impairs the intestinal barrier function of jejunum. The pro- and anti-inflammatory signal changes may be part of a broader response to restore intestinal homeostasis in jejunum. The changes in large intestine bacterial communities favoring butyrate-producing organisms (e.g., Butyrivibrio spp.) may be part of a successful response to maintain the integrity of the colonic mucosa of HS calves. The alteration of intestinal homeostasis should be the target for heat stress therapies to restore biological functions, and, thus highlights the relevance of this work.

Physiological responses of Holstein calves to heat stress and dietary supplementation with a postbiotic from Aspergillus oryzae.

Increased ambient temperature causes heat stress in mammals, which affects physiological and molecular functions. We have recently reported that the dietary administration of a postbiotic from Aspergillus oryzae (AO) improves tolerance to heat stress in fruit flies and cattle. Furthermore, heat-induced gut dysfunction and systemic inflammation have been ameliorated in part by nutritional interventions. The objective of this study was to characterize the phenotypic response of growing calves to heat stress compared to thermoneutral ad libitum fed and thermoneutral feed-restricted counterparts and examining the physiologic alterations associated with the administration of the AO postbiotic to heat-stressed calves with emphasis on intestinal permeability. In this report, we expand previous work by first demonstrating that heat stress reduced partial energetic efficiency of growth in control (45%) but not in AO-fed calves (62%) compared to thermoneutral animals (66%). While heat stress increased 20% the permeability of the intestine, AO postbiotic and thermoneutral treatments did not affect this variable. In addition, AO postbiotic reduced fecal water content relative to thermoneutral and heat stress treatments. Heat stress increased plasma concentrations of serum amyloid A, haptoglobin and lipocalin-2, and administration of AO postbiotic did not ameliorate this effect. In summary, our findings indicated that heat stress led to reduced nutrient-use efficiency and increased systemic inflammation. Results suggest that the AO postbiotic improved energy-use efficiency, water absorption, and the intestinal permeability in heat stress-mediated increase in gut permeability but did not reduce heat stress-mediated rise in markers of systemic inflammation.

A postbiotic from Aspergillus oryzae attenuates the impact of heat stress in ectothermic and endothermic organisms.

Heat stress is detrimental to food-producing animals and animal productivity remains suboptimal despite the use of heat abatement strategies during summer. Global warming and the increase of frequency and intensity of heatwaves are likely to continue and, thus, exacerbate the problem of heat stress. Heat stress leads to the impairment of physiological and cellular functions of ectothermic and endothermic animals. Therefore, it is critical to conceive ways of protecting animals against the pathological effects of heat stress. In experiments with endothermic animals highly sensitive to heat (Bos taurus), we have previously reported that heat-induced systemic inflammation can be ameliorated in part by nutritional interventions. The experiments conducted in this report described molecular and physiological adaptations to heat stress using Drosophila melanogaster and dairy cow models. In this report, we expand previous work by first demonstrating that the addition of a postbiotic from Aspergillus oryzae (AO) into the culture medium of ectothermic animals (Drosophila melanogaster) improved survival to heat stress from 30 to 58%. This response was associated with downregulation of genes involved in the modulation of oxidative stress and immunity, most notably metallothionein B, C, and D. In line with these results, we subsequently showed that the supplementation with the AO postbiotic to lactating dairy cows experiencing heat stress decreased plasma concentrations of serum amyloid A and lipopolysaccharide-binding protein, and the expression of interleukin-6 in white blood cells. These alterations were paralleled by increased synthesis of energy-corrected milk and milk components, suggesting enhanced nutrient partitioning to lactogenesis and increased metabolic efficiency. In summary, this work provides evidence that a postbiotic from AO enhances thermal tolerance likely through a mechanism that entails reduced inflammation.

A redefinition of the modeled responses of mammary glands to once-daily milking.

Milking cows once daily is a management tool that has been implemented to improve physical and financial results of seasonal pasture-based dairy farms. The Molly cow model integrates physiology and metabolism of dairy cattle; however, milk production during short-term changes in milking frequency (e.g., 1× milking) is not well represented. The model includes a representation of variable rates of cell quiescence and death. However, the rate constants governing cell death and the return of quiescent to active cells are not affected by milking frequency. An empirical assessment of the problem was conducted, and it was hypothesized that changing the current representation of the rate of cell death in response to short-term 1× milking would more accurately represent active and quiescent cells and improve predictions of milk production. An extra senescent cell flux was added to account for cell loss during periods of 1× milking. Additional changes included a gradual decline in the rate of 1× stimulated senescence during 1× milking, and a structural change in cell cycling between active and quiescent cells during and after short-term 1× milking. Data used for parameter estimation were obtained from 5 studies where 1× milking or different feeding strategies were tested. Parameter estimates of cell loss indicated that 1× milking would affect a small proportion of quiescent cells to cause extra cell death. This added cell senescence was influenced by the length of 1× milking such that cell senescence peaked on d 1 of 1× milking and decayed from that point. The new structure in the model includes a variable rate of cell death in response to 1× milking and a gradual rate of return of quiescent cells back to the active pool in response to switching to 2× milking after short-term 1× milking. Root mean square errors, mean bias, and slope bias declined by at least 50% for predictions of energy-corrected milk yield and fat percent. The model showed quantitative agreement with production data from short-term 1× milking. The accuracy of predictions was improved and the error was reduced by implementing modifications in the model in response to changes in milking frequency.

Short communication: High incubation temperature in bovine mammary epithelial cells reduced the activity of the mTOR signaling pathway.

Hyperthermia alters utilization of AA in protein synthesis and cell-signaling activity in bovine mammary cells. Essential AA and insulin regulate translation of proteins by controlling the activity of mammalian target of rapamycin (mTOR) signaling pathway. The objectives of this study were to evaluate (1) the effects of incubation temperature on the mTOR signaling pathway and transcription of AA transporters in a bovine mammary alveolar cell line (MAC-T) and (2) the combined effects of incubation temperature and insulin on the mTOR signaling pathway in this cell line. Cells were cultured in medium with 10% fetal bovine serum at 37°C and 5% CO2. In experiment 1, cells were subjected to 37°C (control) or 41.5°C (high incubation temperature; HT) for 12 h. In experiment 2, cells were assigned to 1 of 4 treatments as a 2 × 2 factorial arrangement, including 2 cell culture temperatures (control and HT) and absence or presence of 1.0 µg/mL of insulin. Proteins were harvested and separated by gel electrophoresis. In experiment 1, gene expression of AA transporters (SLC1A1, SLC1A5, SLC3A2, SLC7A1, SLC7A5, and SLC36A1) were evaluated, and changes of ≥2 fold were deemed significantly different. In experiments 1 and 2, immunoblotting was used to identify total and site-specific phosphorylated forms of protein kinase B (Akt1; Ser473), p70 S6 kinase (S6K1; Thr389), ribosomal protein S6 (rpS6; Ser235/236), and eukaryotic elongation factor 2 (eEF2; Thr56). Phosphorylated and total forms of Akt1, S6K1, rpS6, and eEF2 were quantified and expressed as the ratio of phosphorylated to total protein. In experiment 1, HT resulted in a ≥2-fold increase expression of SLC1A1 and SLC3A2. High incubation temperature reduced the phosphorylated to total ratio of Akt1 and rpS6 and increased the phosphorylated to total ratio of eEF2. In experiment 2, we found no temperature by insulin interactions on phosphorylation state of the protein factors of interest. High incubation temperature reduced the phosphorylated to total ratio of Akt1. The addition of insulin increased the phosphorylated to total ratio of Akt1, S6K1, and rpS6. In summary, HT reduced the activity of the mTOR signaling pathway and increased the expression of AA transporters. High incubation temperature possibly reduced protein translation by reducing the mTOR signaling pathway activity in an effort to adapt to thermal stress. These results may help explain the direct effect of elevated temperature on AA metabolism and protein translation in heat-stressed animals.

Short communication: Relationships among temperature-humidity index with rectal, udder surface, and vaginal temperatures in lactating dairy cows experiencing heat stress.

The objective of this study was to evaluate relationships between measurements of temperature-humidity index (THI) and rectal, vaginal, and udder surface temperatures in lactating cows exposed to heat stress (HS). In experiment 1, 12 multiparous and 8 primiparous Holstein cows experienced a THI ranging from 69 to 76 at 2000 to 1000 h and THI from 74 to 82 at 1000 to 2000 h (peaked at 82 from 1400 to 1800 h). Cows were exposed to HS 10 h daily for 21 d. Measurements of rectal temperature (RT) and udder surface temperature were collected at 1000 and 1500 h (±30 min). Vaginal temperature was monitored every 10 min using digital loggers, averaged over 1 h, and paired with corresponding rectal and udder surface temperature data. In experiment 2, 12 multiparous Holstein cows experienced a THI ranging from 60 to 76 at 2000 to 1000 h and THI from 69 to 83 at 1000 to 2000 h (peaked at 83 from 1600 and 1900 h), eliciting 10 h/d of HS for 7 d. Rectal and udder surface temperatures were analyzed at 0700 and 1500 h (±30 min). Vaginal temperature was recorded and analyzed as indicated in experiment 1. Afternoon THI showed weak correlations with surface temperature (r = 0.19, n = 420 in experiment 1; r = 0.23, n = 84 in experiment 2), weak to moderate correlations with RT (r = 0.34, n = 366 in experiment 1; r = 0.26, n = 84 in experiment 2), and moderate correlations with vaginal temperature (r = 0.34, n = 175 in experiment 1; r = 0.35, n = 40 in experiment 2). Moreover, vaginal temperature increased 0.10 and 0.22°C per unit of THI (R2 = 0.15 in experiment 1; R2 = 0.40 in experiment 2). Afternoon vaginal temperature strongly correlated with RT (r = 0.69, n = 131 in experiment 1; r = 0.63, n = 37 in experiment 2) and explained 57 (experiment 1) and 68% (experiment 2) of variation in RT. Surface temperature showed moderate to strong correlations with RT (r = 0.57, n = 84) and vaginal temperature (r = 0.74, n = 37) in experiment 2. In conclusion, THI showed a weak to moderate relationship with core body temperatures and explained the increase in rectal and vaginal temperatures experienced by HS cows. Compared with rectal temperature, vaginal temperature showed stronger relationships with THI and can be used to determine thermal load. Udder surface temperature showed a moderate to strong relationship with core body temperature, and this relationship may support the use of surface temperature data to manage thermal load in HS cows.

Lowering rumen-degradable and rumen-undegradable protein improved amino acid metabolism and energy utilization in lactating dairy cows exposed to heat stress.

The objective of this study was to evaluate the effects of reducing dietary rumen-degradable protein (RDP) and rumen-undegradable protein (RUP) on protein and energy metabolism in heat-stressed dairy cows. Eighteen primiparous and 30 multiparous mid-lactation Holstein cows were used in a completely randomized design arranged in a 2 × 2 factorial (n = 12/treatment). Cows were randomly assigned to 1 of 4 dietary treatments that included 2 levels of RDP (10 and 8%; D) and 2 levels of RUP (8 and 6%; U) of dry matter for 21 d as (1) 10D:8U, (2) 8D:8U, (3) 10D:6U, and (4) 8D:6U. Diets were isoenergetic and contained 50% forage and 50% concentrate (dry matter basis). Cows were housed in a freestall barn. Three weeks before start of treatments, all animals were fed the 10D:8U diet and received supplemental cooling to prevent heat stress. During the treatment period, cows experienced a daily increment in temperature-humidity index from 74 to 82 for 1000 to 2000 h. Blood samples were collected on d -1 and 21 of the treatment period to determine plasma concentrations of AA, glucose, insulin, fatty acids, and ß-hydroxybutyrate. For primiparous cows, reducing from 10 to 8% RDP decreased insulin concentrations. For multiparous cows, we found significant RDP by RUP interactions for insulin, ß-hydroxybutyrate, fatty acids, total essential AA, and 3-methylhistidine concentrations. Reducing from 10 to 8% RDP decreased insulin concentrations at 6% RUP, but concentrations did not change when reducing RDP at 8% RUP. Reducing from 10 to 8% RDP decreased ß-hydroxybutyrate concentrations at 8% RUP, but concentrations did not change when reducing RDP at 6% RUP. Reducing from 10 to 8% RDP increased nonesterified fatty acid and total essential AA concentrations at 8% RUP, but concentrations did not change when reducing RDP at 6% RUP. Reducing from 8 to 6% RUP decreased 3-methylhistidine concentration at 8% RDP, but not at 10% RDP. Reducing from 8 to 6% RUP increased milk protein yield efficiency in primiparous and multiparous cows. These results indicate that reducing RDP and RUP lowers circulating insulin, which was associated with mobilization and utilization of fatty acids. Reduced RDP and RUP increases the use of AA to maintain milk protein synthesis and limit AA catabolism in cows exposed to warm climates.

Short communication: Circulating fatty acylcarnitines are elevated in overweight periparturient dairy cows in association with sphingolipid biomarkers of insulin resistance.

Defects in mitochondrial fatty acid processing are associated with the development of fatty liver disease, inflammation, and insulin resistance in overweight nonruminants. Surplus fatty acids (FA) and defects in FA oxidation favor the accumulation of fatty acylcarnitines (FAC) and the sphingolipid ceramide. Moreover, elevated circulating FAC and ceramide concentrations are inversely related to insulin action. Because we have previously demonstrated that plasma ceramide levels increase during the transition from gestation to lactation, our aim was to determine whether changes in plasma medium- and long-chain FAC levels are related to circulating FA and sphingolipids in peripartal dairy cows. We hypothesized that plasma FAC levels would be higher in overweight cows experiencing increased lipolysis, and that FAC levels would be positively associated with elevations in plasma ceramides. Twenty-one multiparous Holstein cows were grouped according to body condition score (BCS) at d -30 prepartum as lean (BCS <3.0; n = 10) or overweight (BCS >4.0; n = 11). Blood was collected at d -30, -15, -7, and 4, relative to parturition. Circulating FAC and ceramide levels were determined using liquid chromatography and tandem mass spectrometry. To investigate the potential contributions of sphingomyelin (SM) hydrolysis to ceramide accrual, we also determined plasma SM levels during the peripartum period. Data were analyzed under a mixed model with the fixed effects of adiposity and time, and the random effect of cow. Relative to lean cows, overweight cows had elevated FAC during the transition from gestation to lactation. Circulating FAC levels were positively associated with FA, ceramide, and dihydro-SM levels. Although circulating FAC levels increased in all cows during the peripartum, enhanced prepartum adiposity contributed to a greater rise in plasma FA and FAC. Our results support on-going efforts to determine whether altered mitochondrial FA processing promotes the accumulation of the insulin resistance biomarker ceramide in blood and liver.

Lowering rumen-degradable protein maintained energy-corrected milk yield and improved nitrogen-use efficiency in multiparous lactating dairy cows exposed to heat stress.

The objective of this study was to examine the effect of reducing rumen-degradable protein (RDP) and rumen-undegradable protein (RUP) proportions on feed intake, milk production, and N-use efficiency in primiparous and multiparous cows exposed to warm climates. Eighteen primiparous and 30 multiparous mid-lactation Holstein cows were used in a completely randomized design with a 2 × 2 factorial arrangement of treatments. Cows were randomly assigned to 1 of 4 dietary treatments formulated to contain 2 proportions of RDP (10 and 8%) and 2 proportions RUP (8 and 6%) of dry matter (DM) indicated as follows: (1) 10% RDP, 8% RUP; (2) 8% RDP, 8% RUP; (3) 10% RDP, 6% RUP; and (4) 8% RDP, 6% RUP. Protein sources were manipulated to obtain desired RDP and RUP proportions. Diets were isoenergetic and contained 50% forage and 50% concentrate (DM basis). Cows were individually fed the 10% RDP, 8% RUP diet 3 wk before treatment allocation. Cows were exposed to the prevailing Tennessee July and August temperature and humidity in a freestall barn with no supplemental cooling. Main effects and their interaction were tested using the Mixed procedure of SAS (least squares means ± standard error of the mean; SAS Institute Inc., Cary, NC). Observed values of nutrient intake and milk production were used to obtain NRC (2001) model predictions. Cows showed signs of heat stress throughout the study. Reducing from 10 to 8% RDP decreased dry matter intake (DMI; 0.9 kg/d) at 8% RUP, but increased DMI (2.6 kg/d) at 6% RUP in primiparous cows. Reducing from 10 to 8% RDP decreased milk yield (10%) at 8% RUP, but increased yield (14%) at 6% RUP. Treatments did not affect yield of energy-corrected milk. For multiparous cows, treatments did not affect DMI. Reducing from 10 to 8% RDP decreased yield of energy-corrected milk (3.4%) at 8% RUP, but increased yield (8.8%) at 6% RUP. Reducing from 10 to 8% RDP and 8 to 6% RUP both increased N-use efficiency for primiparous and multiparous cows. The NRC model underestimated metabolizable protein and RUP supply, and overestimated RUP requirements, resulting in predictive losses of milk yield 1.4 to 5.8 times greater than observed values. In summary, the reduction of RDP and RUP proportions did not affect DMI, whereas the RUP reduction at 10% RDP had a small negative effect on energy-corrected milk yield. However, reduction of RDP and RUP consistently improved N-use efficiency of heat-stressed multiparous cows. The reduction of RDP and RUP proportions reduced DMI and milk yield but did not affect energy-corrected milk yield in primiparous cows, indicating a limited supply of nutrients.

Jugular-infused methionine, lysine and branched-chain amino acids does not improve milk production in Holstein cows experiencing heat stress.

Poor utilization of amino acids contributes to losses of milk protein yield in dairy cows exposed to heat stress (HS). Our objective was to test the effect of essential amino acids on milk production in lactating dairy cows exposed to short-term HS conditions. To achieve this objective, 12 multiparous, lactating Holstein cows were assigned to two environments (thermoneutral (THN) or HS) from days 1 to 14 in a split-plot type cross-over design. All cows received 0 g/day of essential amino acids from days 1 to 7 (negative control (NC)) followed by an intravenous infusion of l-methionine (12 g/day), l-lysine (21 g/day), l-leucine (35 g/day), l-isoleucine (15 g/day) and l-valine (15 g/day, methionine, lysine and branched-chain amino acids (ML+BCAA)) from days 8 to 14. The basal diet was composed of ryegrass silage and hay, and a concentrate mix. This diet supplied 44 g of methionine, 125 g of lysine, 167 g of leucine, 98 g of isoleucine and 109 g of valine per day to the small intestine of THN cows. Temperature-humidity index was maintained below 66 for the THN environment, whereas the index was maintained above 68, peaking at 76, for 14 continuous h/day for the HS environment. Heat stress conditioning increased the udder temperature from 37.0°C to 39.6°C. Cows that received the ML+BCAA treatment had greater p.m. rectal and vaginal temperatures (0.50°C and 0.40°C, respectively), and respiration rate (8 breaths/min) compared with those on the NC treatment and exposed to a HS environment. However, neither NC nor ML+BCAA affected rectal or vaginal temperatures and respiration rates in the THN environment. Compared with THN, the HS environment reduced dry matter intake (1.48 kg/day), milk yield (2.82 kg/day) and milk protein yield (0.11 kg/day). However, compared with NC, the ML+BCAA treatment increased milk protein percent by 0.07 points. For the THN environment, the ML+BCAA treatment increased concentrations of milk urea nitrogen. For the HS environment, the ML+BCAA treatment decreased plasma concentrations of arginine, ornithine and citrulline; however, differences were not observed for the THN environment. In summary, HS elicited expected changes in production; however, infusions of ML+BCAA failed to increase milk protein yield. Lower dry matter intake and greater heat load in response to ML+BCAA contributed to the lack of response in milk production in HS cows. The ML+BCAA treatment may have reduced the breakdown of muscle protein in heat-stressed cows.

FORAGES AND PASTURES SYMPOSIUM: Improving efficiency of production in pasture- and range-based beef and dairy systems.

Despite overall increased production in the last century, it is critical that grazing production systems focus on improving beef and dairy efficiency to meet current and future global food demands. For livestock producers, production efficiency is essential to maintain long-term profitability and sustainability. This continued viability of production systems using pasture- and range-based grazing systems requires more rapid adoption of innovative management practices and selection tools that increase profitability by optimizing grazing management and increasing reproductive performance. Understanding the genetic variation in cow herds will provide the ability to select cows that require less energy for maintenance, which can potentially reduce total energy utilization or energy required for production, consequently improving production efficiency and profitability. In the United States, pasture- and range-based grazing systems vary tremendously across various unique environments that differ in climate, topography, and forage production. This variation in environmental conditions contributes to the challenges of developing or targeting specific genetic components and grazing systems that lead to increased production efficiency. However, across these various environments and grazing management systems, grazable forage remains the least expensive nutrient source to maintain productivity of the cow herd. Beef and dairy cattle can capitalize on their ability to utilize these feed resources that are not usable for other production industries. Therefore, lower-cost alternatives to feeding harvested and stored feedstuffs have the opportunity to provide to livestock producers a sustainable and efficient forage production system. However, increasing production efficiency within a given production environment would vary according to genetic potential (i.e., growth and milk potential), how that genetic potential fits the respective production environment, and how the grazing management fits within those genetic parameters. Therefore, matching cow type or genetic potential to the production environment is and will be more important as cost of production increases.

Temporary alterations to postpartum milking frequency affect whole-lactation milk production and the energy status of pasture-grazed dairy cows.

This study investigated the immediate and long-term effects of temporary alterations to postpartum milking frequency (MF) on milk production, body condition score (BCS), and indicators of energy status in pasture-grazed cows supplemented with concentrates. Multiparous Holstein-Friesian cows (n = 150) were randomly assigned to 1 of 5 groups at calving: milked twice daily (2 ×) throughout lactation (control), or milked either once daily (1 ×) or 3 times daily (3 ×) for 3 or 6 wk immediately postpartum, and then 2 × for the remainder of lactation. During wk 1 to 3 postpartum, cows milked 1 × produced 15% less milk and 17% less energy-corrected milk (ECM) than cows milked 2 ×. This immediate production loss increased to 20% less milk and 22% less ECM during wk 4 to 6 postpartum for cows that remained on 1 × milking; these animals also produced less than 1 × cows switched to 2 × milking after 3 wk. During wk 8 to 32, when all cows were milked 2 ×, those previously milked 1 × had sustained reductions in milk (-6%) and ECM (-8%) yields, which were not affected by the duration of reduced postpartum MF. In contrast, cows milked 3 × postpartum had 7% greater milk yields during wk 1 to 6 compared with 2 × controls, irrespective of the duration of increased MF. Milk yields also remained numerically greater (+5%) during wk 8 to 32 in cows previously milked 3 ×. Nevertheless, yields of ECM were not increased by 3 × milking, because of lower milk fat and protein contents that persisted for the rest of lactation. In addition, indicators of cow energy status reflected an increasing state of negative energy balance with increasing MF. Cows milked 1 × postpartum had greater plasma glucose and lower plasma nonesterified fatty acid concentrations during the reduced MF, and plasma glucose remained lower for 2 wk after cows had switched to 2 × milking. Moreover, BCS was improved relative to 2 × controls from wk 5 to 6. In contrast, cows milked 3 × had lower plasma glucose concentrations, greater plasma nonesterified fatty acid concentrations, and greater BCS loss during wk 1 to 3; however, greater body fat mobilization was not sustained, indicating that additional energy supplements may be required to achieve better milk production responses. In conclusion, temporary 1 × milking had lactation-long negative effects on milk and milk component yields but improved cow energy status and BCS, whereas temporary 3 × milking immediately increased milk yield but did not improve milk fat and protein yields in pasture-grazed cows.

Gene expression in liver and adipose tissue is altered during and after temporary changes to postpartum milking frequency.

Short-term changes to milking frequency can alter the metabolic status of dairy cows depending on the duration, magnitude, and stage of lactation at which the milking frequency changes occur. Additionally, effects of altered milking frequency that are subsequent to cows returning to a normal twice-daily (2×) milking regimen are not well established. This study tested the hypothesis that plasma concentrations of key hormones and metabolites and transcription of genes involved in the somatotropic axis and lipid metabolism would be altered in liver and subcutaneous adipose tissue from cows milked with different frequencies. Multiparous Holstein-Friesian dairy cows were allocated to 2× milking for the whole lactation, or once-(1×) or 3 times-(3×) daily milking for 3 or 6 wk, immediately postpartum, and then 2× milking for the remainder of the lactation. Liver and subcutaneous fat were biopsied at wk 1 (liver only), 3, 6, and 9 postpartum, and transcription of genes involved in the somatotropic axis and lipid metabolism were measured. At wk 3, cows milked 3× had lower hepatic expression of growth hormone receptor (GHR1A) compared with cows milked 2× or 1×, and lower IGF1 expression compared with cows milked 1×, indicating greater uncoupling of the somatotropic axis. At wk 6, reduced transcription of total GHR and GHR1B occurred in the adipose tissue of cows milked 3×. Cows milked 1× had greater transcription in adipose tissue of lipogenesis genes at wk 3 and 6, and lipolysis genes at wk 6, compared with cows milked 2×, indicating a period of increased fatty acid storage, followed by increased fatty acid reesterification. At wk 9, cows previously milked 3× for 6 wk maintained lower transcription of genes involved in lipogenesis, lipolysis, and ketolysis in adipose tissue compared with cows milked 2×, indicating that the effects of 3× milking persist for at least 3 wk after switching to 2× milking. Results indicate that alterations to milking frequency affect the transcription of genes involved in lipid mobilization and storage, enabling the animal to manage the energy demands associated with the change in milk production. Some of these gene transcription changes were maintained in cows previously milked 3×, indicating that the adipose tissue gene expression changes were still required even after 3 wk of the less-demanding 2× milking regimen.

The expression of genes involved in hepatic metabolism is altered by temporary changes to milking frequency.

Changes to milking frequency (MF) affect the metabolic and energetic status of dairy cows. However, the duration of altered MF necessary to modify hepatic transcription during early lactation is less clear. Additionally, long-term responses to short-term alterations in MF have not been established. Holstein-Friesian dairy cows (n = 120) were allocated to 3 or 6 wk of either once-daily (1 ×) or thrice-daily (3 ×) milking, immediately postpartum. Following treatment, cows were switched to twice-daily (2 ×) milking. These 4 treatment groups were compared with cows milked 2 × (n = 30) for the whole lactation. Liver tissue was collected by biopsy at 1, 3, 6, and 9 wk postpartum from 12 cows per treatment, RNA was extracted, and transcript abundance of genes involved in hepatic metabolism was quantified. Milking frequency altered the expression of most of the genes measured; however, we observed no effects caused by the length of time on the alternative milking frequency and no interactions between MF and length. During the MF treatment, mRNA expression of some, but not all, genes involved in gluconeogenesis (G6PC, PCK1), fatty acid ß-oxidation (CPT1A, CPT2), ketogenesis (HMGCS2), lipid transport (APOA1), and lipolysis (PNPLA2) were lower for cows milked 1 × and plasma glucose and insulin concentrations were greater. Cows milked 3 × had reduced mRNA expression for some of the genes involved in fatty acid synthesis (ACACA) and lipid transport (APOB) and had greater plasma NEFA concentrations at wk 1. At 9 wk postpartum, expression data indicated that cows previously milked 3 × had a greater capacity for gluconeogenesis (PCK1), ketogenesis (HMGCS2), and urea cycling (ASL, CPS1) and lower glucose concentrations than cows previously milked 1 ×, because some of the genes involved in these processes were still altered. Milking cows 1 × relative to 2 ×, however, did not result in significant carryover effects on the expression of the genes measured in this study, indicating that metabolic changes are not sustained beyond the period of reduced MF. Changes to MF altered the hepatic response during early lactation; however, this was not dependent on the duration of MF change. Although we observed only minimal carryover effects on hepatic metabolism from short periods of reduced MF postpartum, there may be long-term effects on urea cycling (ASL, CPS1) and ketogenesis (HMGCS2) when 3 × milking occurs immediately postpartum.

Reducing milking frequency during nutrient restriction has no effect on the hepatic transcriptome of lactating dairy cattle.

The objective of this study was to investigate if a reduced milking frequency altered the effect of dietary energy restriction on the hepatic transcriptome of grazing dairy cows during early lactation. Multiparous Holstein-Friesian and Holstein-Friesian × Jersey cows (n = 120) were milked twice daily (2×) from calving until 34 ± 6 days in milk (mean ± SD). Cows were then allocated to one of four treatments in a 2 × 2 factorial arrangement. Treatments consisted of two milking frequencies [2× or once daily (1×)] and two feeding levels for 3 wk: adequately fed (AF) or underfed (UF, 60% of AF). Liver tissue was biopsied from 12 cows per treatment after 3 wk of treatment, and the hepatic transcriptome was profiled with an Agilent 4 × 44k bovine microarray. Over 2,900 genes were differentially expressed in response to the energy restriction; however, no effects resulted from changes to milking frequency. This may indicate that after 3 wk of 1× milking, any changes to the liver transcriptome that may have occurred earlier have returned to normal. After 3 wk of energy restriction, gene expression patterns indicate that glucose-sparing pathways were activated, and gluconeogenesis was increased in UF cows. Genes involved in hepatic stress were upregulated in response to the energy restriction indicative of the pressure energy restriction places on liver function. Other pathways upregulated included "cytoskeletal remodeling," indicating that a 3 wk energy restriction resulted in molecular changes to assist tissue remodeling. Overall, 1× milking does not modify the hepatic transcriptome changes that occur in response to an energy restriction.

Expression of key lipid metabolism genes in adipose tissue is not altered by once-daily milking during a feed restriction of grazing dairy cows.

The objective of this study was to investigate the effect of reduced milking frequency, at 2 feeding levels, on gene expression in adipose tissue of grazing dairy cows during early lactation. Multiparous Holstein-Friesian and Holstein-Friesian × Jersey cows (n=120) were grazed on pasture and milked twice daily (2×) from calving to 34±6d in milk (mean ± standard deviation). Cows were then allocated to 1 of 4 treatments in a 2×2 factorial arrangement. Treatments consisted of 2 milking frequencies (2× or once daily; 1×) and 2 feeding levels for 3 wk: adequately fed (AF), consuming 14.3 kg of dry matter/cow per day, or underfed (UF), consuming 8.3 kg of dry matter/cow per day. After the treatment period, all cows were fed to target grazing residuals ≥1,600 kg of DM/cow per day and milked 2× for 20 wk. Adipose tissue was collected from 12 cows per treatment by subcutaneous biopsy at -1, 3, and 5 wk relative to treatment start, RNA was extracted, and transcript abundance of genes involved in lipid metabolism was quantified using a linear mixed model. At the end of the 3-wk treatment period, transcript abundance of genes involved in fatty acid (FA) uptake into adipose tissue (LPL), FA synthesis [FA synthase (FASN) and stearoyl-coenzyme A desaturase (SCD)], FA oxidation [acyl-coenzyme A synthetase long-chain family member 1 (ACSL1) and carnitine palmitoyltransferase 2 (CPT2)], glyceroneogenesis [glycerol-3-phosphate dehydrogenase 1 (GPD1) and pyruvate carboxylase (PC)], and triacylglyceride synthesis [diacylglycerol O-acyltransferase 2 (DGAT2)] were greater in AF1× cows compared with all other treatments. However, when cows were underfed, no effects of milking frequency were observed on transcript abundance of genes involved in adipose lipid metabolism. Despite increases in plasma NEFA concentrations in UF cows, no effects of underfeeding were observed on the transcription of lipolytic genes. At 5 wk, after cows were returned to 2× milking and standard feed allowance, transcript abundances of genes involved in FA synthesis [acetyl-coenzyme A carboxylase α (ACACA) and SCD)] were increased in cows previously UF. Expression of ACSL1 was decreased in UF1× cows relative to UF2× cows and CPT2 expression was greater in AF1× cows compared with AF2× cows. In conclusion, after 3 wk of reduced milking frequency during a feed restriction, transcription of genes involved in lipid metabolism in adipose tissue were not altered, possibly due to the reduced milk production in these animals. However, 3 wk of 1× milking in AF cows increased transcription of genes involved in FA synthesis, oxidation, and triacylglyceride synthesis.

Technical note: Nitrogen isotopic fractionation can be used to predict nitrogen-use efficiency in dairy cows fed temperate pasture.

The objective of this study was to investigate the relationship between nitrogen isotopic fractionation (δ(15)N) and nitrogen-use efficiency (milk nitrogen/nitrogen intake; NUE) in pasture-fed dairy cows supplemented with increasing levels of urea to mimic high rumen degradable protein pastures in spring. Fifteen cows were randomly assigned to freshly cut pasture and either supplemented with 0, 250, or 336 g urea/d. Feed, milk, and plasma were analyzed for δ(15)N, milk and plasma for urea nitrogen concentration, and plasma for ammonia concentration. Treatment effects were tested using ANOVA and relationships between variables were established by linear regression. Lower dry matter intake (P = 0.002) and milk yield (P = 0.002) occurred with the highest urea supplementation (336 g urea/d) compared with the other two treatments. There was a strong linear relationship between milk δ(15)N - feed δ(15)N and NUE: [NUE (%) = 58.9 - 10.17 × milk δ(15)N - feed δ(15)N (‰) (r(2) = 0.83, P < 0.001, SE = 1.67)] and between plasma δ(15)N - feed δ(15)N and NUE: [NUE (%) = 52.4 - 8.61 × plasma δ(15)N - feed δ(15)N (‰) (r(2) = 0.85, P < 0.001, SE = 1.56)] . This study confirmed the potential use of δ(15)N to predict NUE in cows consuming different levels of rumen degradable protein.

Once-daily milking during a feed deficit decreases milk production but improves energy status in early lactating grazing dairy cows.

The objective of this study was to investigate the effect of milking frequency (MF) at 2 feeding levels (FL) on milk production, body condition score, and metabolic indicators of energy status in grazing dairy cows during early lactation. Multiparous Holstein-Friesian and Holstein-Friesian × Jersey cows (n=120) grazed pasture and were milked twice daily (2×) from calving until 34 ± 6 d in milk (mean ± standard deviation). Cows were then allocated to 1 of 4 treatments in a 2 × 2 factorial arrangement. Treatments consisted of 2 FL: adequately fed [AF; 14.3 kg dry matter intake (DMI)/cow per d] or underfed (UF; 8.3 kg of DMI/cow per d) and 2 MF: 2× or once daily (1×). Treatments were imposed for 3 wk. After the treatment period, all cows were offered a generous pasture allowance (grazing residuals >1,600 kg of dry matter/ha) and milked 2×. During the 3-wk treatment period, we observed an interaction between FL and MF for energy-corrected milk (ECM), such that the decrease due to 1× milking was greater in AF than in UF cows (20 and 14% decrease, respectively). No interactions were found posttreatment. Cows previously UF produced 7% less ECM than AF cows during wk 4 to 12; however, no subsequent effect was observed of the previous underfeeding. Cows previously milked 1× produced 5% less ECM during wk 4 to 12, and differences remained during wk 13 to 23. During the 3-wk treatment period, UF cows lost 0.2 body condition score units (1-10 scale) and this was not affected by 1× milking. During the treatment period, UF cows had lower plasma glucose, insulin, and insulin-like growth factor I, and greater nonesterified fatty acids and ß-hydroxybutyrate concentrations than AF cows. Cows milked 1× had greater plasma glucose, insulin, and insulin-like growth factor I, and lower nonesterified fatty acids and ß-hydroxybutyrate concentrations compared with cows milked 2×. In conclusion, energy status was improved by 1× milking; however, when UF cows were milked 1×, milk production was reduced by more than underfeeding alone. The immediate and residual responses to 1× milking need to be considered when using this management strategy during a feed deficit.

Short communication: immediate and deferred milk production responses to concentrate supplements in cows grazing fresh pasture.

The objective of this study was to determine the increase in milk production from supplementation that occurred after supplementation ceased. This portion of the total response (i.e., the deferred response), although accepted, is generally not accounted for in short-term component research projects, but it is important in determining the economic impact of supplementary feeding. Fifty-nine multiparous Holstein-Friesian dairy cows were offered a generous allowance of spring pasture [>45 kg of dry matter (DM)/cow per day) and were supplemented with 0, 3, or 6 kg (DM)/d of pelleted concentrate (half of the allowance at each milking event) in a complete randomized design. Treatments were imposed for the first 12 wk of lactation. Treatments were balanced for cow age (5.4 ± 1.68 yr), calving date (July 27 ± 26.0 d), and genetic merit for milk component yield. During the period of supplementation, milk yield and the yield of milk components increased (1.19 kg of milk, 0.032 kg of fat, 0.048 kg of protein, and 0.058 kg of lactose/kg of concentrate DM consumed), but neither body condition score nor body weight was affected. After concentrate supplementation ceased and cows returned to a common diet of fresh pasture, milk and milk component yields remained greater for 3 wk in the cows previously supplemented. During this 3-wk period, cows that previously received 3 and 6 kg of concentrate DM per day produced an additional 2.3 and 4.5 kg of milk/d, 0.10 and 0.14 kg of fat/d, 0.10 and 0.14 kg of protein/d, and 0.10 and 0.19 kg of lactose/d, respectively, relative to unsupplemented cows. This is equivalent to an additional 0.19 kg of milk, 0.006 kg of fat, 0.006 kg of protein, and 0.008 kg of lactose per 1 kg of concentrate DM previously consumed, which would not be accounted for in the immediate response. As a result of this deferred response to supplements, the total milk production benefit to concentrate supplements is between 7% (lactose yield) and 32% (fat yield) greater than the marginal response measured during the component experiment. Recommendations to dairy producers based on component feeding studies must be revised to include this deferred response.

Nitrogen metabolism and rumen microbial enumeration in lactating cows with divergent residual feed intake fed high-digestibility pasture.

Dairy cattle selected for negative residual feed intake (n-RFI; efficient) should maintain production while reducing dry matter intake over a lactation because of improvements in feed digestion and efficient use of nutrients. The objective of this study was to measure nitrogen (N) digestibility and rumen microbial community composition over a short period during early lactation in lactating Holstein-Friesian cows selected previously for divergent RFI. It was proposed that n-RFI cows would have greater apparent digestibility of N than the positive RFI (p-RFI; inefficient) animals, to compensate for the lower dry matter intake determined during selection for divergence. Sixteen 3-yr-old rumen-cannulated, lactating cows (56 ± 10d in milk) selected for n-RFI (n = 8) and p-RFI (n = 8) were housed in metabolism stalls and fed fresh vegetative ryegrass (Lolium perenne L.) pasture ad libitum as a sole diet during an 8-d digestibility study. Intake of nutrients and outputs of milk, feces, and urine were determined. Rumen parameters were determined by removing, weighing, and sampling digesta, and by cobalt-EDTA dilution. Intakes of N, dry matter, organic matter, or its components did not differ with RFI. Compared with p-RFI cows, n-RFI cows had a greater apparent N digestibility (77.2 vs. 75.5%), and a tendency toward greater dry matter and organic matter digestibilities. The n-RFI cows had a lower fecal N output (126 vs. 138 g/d) and a lower partition of feed N to fecal N (23.1 vs. 24.7%) compared with p-RFI animals. We found no differences between phenotypes in the partition of N to urinary N or milk crude protein but did observe a trend for n-RFI cows to partition less N to milk casein (16.8 vs. 17.9%). Rumen digesta mass was similar for both groups, despite differences in calculated fractional liquid outflow rates, and most bacterial, archaeal, protozoal, and fungal communities were similar for both phenotype groups. In conclusion, dry matter intake and rumen function were similar for both phenotypes when the animals were fed highly digestible fresh ryegrass, but apparent digestibility of dietary N was higher in the efficient (n-RFI) cows. Future research should measure digestion parameters in cows with divergent RFI when fed diets differing in chemical composition (e.g., divergent crude protein contents).