Abstract Essential amino acids (EAA) may alter bovine mammary epithelial cell (BMEC) activities through regulation of gene transcription. For this study, we used pathway analysis of RNA-sequencing data to identify upstream transcription factors (TF) that potentially mediate the effects of single EAA deficiency on BMEC. Differentiated BMEC were cultured in 1 of 4 treatment media representing normal physiological concentrations of all amino acids (CTL), or ¼ the normal concentration of histidine (LH), lysine (LK), or methionine (LM). After 48 h, rates of DNA synthesis were reduced 30 to 40% by each of the deficiencies. Protein synthesis rate was 30 to 50% lower in LK and LM but was unaffected by LH. Up-regulated upstream TF included those related to nutrient stress, interferon signaling, and suppression of cell population growth. Down-regulated TF were primarily related to cell cycle progression. An array of classic ATF4 targets increased in response to EAA deficiencies including amino acid transporters, branched-chain aminotransferase, aminoacyl-tRNA synthetases, sestrin2 (a leucine sensor), and activators of apoptosis. Interferon signaling emerged as a novel pathway activated by EAA deficiencies, possibly contributing to reduced cell proliferation and protein synthesis. In contrast, the MAPK signaling pathway was unaffected. Expression of hypoxia-inducible factor 1α (HIF1A) increased, consistent with an anti-proliferative role. Downregulation of Forkhead box protein M1 (FOXM1) likely contributed to slower cell proliferation. These findings reveal a complex network of transcriptional regulators and signaling pathways influenced by EAA availability and shed light on the nutritional regulation of milk protein production by dairy cows.

The aim of this study was to describe the utility of Doppler ultrasonography of the milk vein (MV) to assess mammary plasma flow (MPF) and determine whether a relationship exists between MPF assessed via Doppler ultrasound of the MV (MPFMV) and the Fick principle (MPFFick) on the day of calving and at 7 DIM. A total of 11 parity 2 (P2) and 10 parity 3 (P3) Holstein cows were enrolled in the study immediately after calving. The MPFFick was estimated using the mammary arteriovenous difference of Phe and Tyr as internal markers at 2, 4, 6 and 8 h after colostrum collection and the morning milking at 7 ± 1 DIM. Milk was collected at milking 2 and the evening milking at 7 ± 1 DIM for determination of milk protein output. The MPFMV was also estimated by Doppler ultrasound of the left and right MV at 2 and 8 h after colostrum collection and the morning milking at 7 ± 1 DIM. Both MPFMV and MPFFick increased by 15.8 and 19.5% from calving to 7 DIM, respectively. No differences were detected between parity in MPFFick; however, P3 cows had greater MPFMV than P2 cows, as well as greater MV diameter. No relationship existed between MPFMV and MPFFick during the calving and fresh periods, which may have been due to the violation of multiple assumptions of the Fick principle as a result of the fluctuating rate of milk secretion, tissue metabolism, and blood flow associated with the onset of lactation. Although Doppler ultrasound of the MV may be a time-efficient, non-invasive technique to estimate fresh period MPF, further work is needed to validate its accuracy with techniques estimating MPF directly in the pudendal artery, such as the dye dilution technique. Such knowledge would allow for the widespread utilization of this non-invasive technique to uncover knowledge gaps pertaining to the relationship between fresh period mammary metabolism and milk production.

The aim of this study was to characterize dry and fresh period mammary metabolism as estimated via mammary plasma flow of the milk vein (MV; MPFMV) and mammary acetate and glucose (glc) metabolism and to determine potential cow- and mammary-level factors that influence colostrum production. A total of 21 Holstein cows (n = 11 parity 2 [P2] and 10 parity 3 [P3] cows) were dried off at d-58 ± 3.4 relative to calving and followed until 7 ± 1 d in milk (DIM). All cows were provided the same dry and lactating diets supplying 46.4 and 54.4 Mcal ME/d, respectively. Starting at d-49 relative to expected calving, estimation of MPFMV via Doppler ultrasonography, and mammary acetate and glc arteriovenous difference, uptake and clearance, were determined every other wk until wk-1 before expected calving, then every 4 d until actual calving, at calving, and at 7 ± 1 DIM by collection of frequent pairs of coccygeal vessel and MV blood samples. Colostrum was collected within 1.4 ± 0.76 h of calving, components were determined via mid-infrared spectroscopy, and IgG was determined by radial immunodiffusion. Mammary metabolism as estimated via MPFMV and glc and acetate uptake was lowest from wk-6 to wk-4 relative to calving. Total MPFMV began to steadily increase starting at wk-2, while mammary acetate and glc uptake only began to increase at wk-1 and d-4, respectively. The earlier increase in mammary acetate uptake may suggest its role in providing energy for mammogenesis and lactogenesis I, while mammary glc uptake may coincide with lactogenesis II and copious milk production. Assuming the complete oxidation of mammary acetate and glc uptakes, the average daily rate of energy expenditure of the dry mammary gland was 0.93 Mcal/d and equated to a total energy expenditure of 56.1 Mcal throughout the entire dry period. Correlation and regression analyses highlighted the importance of achieving adequate dry-off during the far-off period of high-producing cows in promoting colostrum production, as greater previous lactation milk yield and far-off mammary acetate uptake were negatively associated with colostrum yield. In summary, the mammary gland appears to achieve a non-secretory, low metabolic state between wk-6 to wk-4 relative to calving, and this period appears to be of greater importance in driving colostrum production than the close-up period. Future work should aim to uncover the relationships between dry period mammary metabolism, especially the non-secretory state during the far-off period, and prepartum mammogenesis and colostrogenesis.

There is limited research investigating the impact of closeup nutrition on colostrum and transition milk (TM) production and composition in both primiparous (PP) and multiparous (MP) cattle, despite the potential of alterations in prepartum metabolism to modify the availability of substrates for colostrum and milk synthesis. The objective of this study was to determine the effect of altering closeup dietary NDF content on colostrum, transition and mature milk composition and to characterize potential associations between cow and metabolic factors and colostrum yield and composition. Primiparous heifers were blocked by expected calving date and genetic parent average for milk yield and MP cows were blocked by parity, expected calving date, completed 305-d average milk yield and body condition score (BCS) at dry-off. From d -57 ± 5.8 before expected calving, PP (n = 20) and MP (n = 28) cattle were fed a high NDF diet (HI; 55.7% DM basis; 2.37 Mcal ME/kg DM). From d -19 ± 4.0, cattle were randomly assigned within block to a close-up diet (CUD) to either remain on HI or move to a low NDF diet (LO; 40.6% DM basis; 2.48 Mcal ME/kg DM). Blood samples were collected at wk -4, -3, -2, -1, at calving (within 12 h), and at d +4, +7, +14 and +21 relative to calving. Colostrum (milking [M] 1) was collected within 8 h of calving, followed by TM (M2 to M7) and mature milk (d 16 ± 2.0, wk +2), and total yield (kg) was measured at each milking. Component (fat, protein, lactose, TS, MUN, SCC, BHB), insulin, and immunoglobulin G (IgG) concentrations were analyzed by Fourier-Transform Infrared spectroscopy, ELISA, and radial immunodiffusion, respectively, and multiplied by milk yield at each respective milking to determine component (g), insulin (μg) and IgG (g) yields. Feeding LO had no effect on colostrum yield, component concentrations and yields, and IgG content and yield. However, cows fed LO had greater colostrum insulin concentration and yield, albeit this difference only existed between LO-MP and HI-MP cows and not between LO-PP and HI-PP heifers. The LO cows tended to have greater colostrum SCC than HI cows; yet, no differences were observed between parity with CUD. Interestingly, correlation analyses demonstrated that colostrum production was differentially associated with serum metabolites and hormones in PP heifers and MP cows. In terms of milk production, LO-MP cows produced greater transition and mature milk yields and component yields than HI-MP cows. In contrast, no differences existed between LO-PP and HI-PP heifers. In summary, the results suggest that colostral insulin levels and SCC are responsive to alterations in closeup NDF content. Further, providing low compared with high NDF diets during closeup improved transition and mature milk production in MP, but not PP, cattle, emphasizing the need for future research investigating the underlying differential metabolic and production responses to closeup dietary NDF content between parity groups.

As dog owners continue to seek to feed their dogs similarly to themselves, there is demand for high protein, low carbohydrate (HPLC) diets. The consumption of HPLC diets can improve glycemic control, similarly to high fiber diets. However, the effects of HPLC and high fiber diets on cardiac function have yet to be evaluated in healthy dogs. The objective of the present study was to investigate the glucose, insulin, glucagon and amino acid (AA) postprandial response and echocardiographic measurements in laboratory-housed, adult large breed dogs fed a commercially available HPLC, a moderate protein, moderate carbohydrate (MPMC), or a commercially available MPMC, high fiber, "metabolic" diet for 42 d. This study was conducted as a 3 x 3 Latin square where dogs received: 1) a commercial HPLC diet (48% of metabolizable energy (ME) from protein, 10% of ME from nitrogen-free extract; NFE), 2) a MPMC diet (28% of ME from protein, 39% of ME from NFE) formulated with the same ingredients as HPLC or 3) a MPMC, high fiber, "metabolic" (MET) diet (30% of ME from protein, 37% of ME from NFE) as a commercial control. An echocardiogram and a 12-h glucose, insulin and glucagon response and 6-h AA meal response were performed on day 42 of feeding. Data were analyzed using proc glimmix in SAS (version 9.4). All echocardiographic parameters remained within a healthy reference range for dogs of this size. Dogs fed HPLC had a larger net area under the curve (NetAUC) for plasma glucagon (P < 0.001) compared to dogs fed MPMC and MET, a smaller NetAUC for glucose: insulin (P = 0.039) compared to dogs fed MPMC but MET was similar to both. Glucose NetAUC tended to be different among treatments (P = 0.057), where dogs fed MPMC had a greater netAUC than dogs fed HPLC and dogs fed MET tended to have a greater netAUC than HPLC. Dogs fed HPLC had greater concentrations of Ile, Leu, Lys, Thr, Tyr and Val over time compared to dogs fed MPMC and MET, and dogs fed MET had greater concentrations of Gln and Met over time compared to dogs fed HPLC and MPMC (P < 0.05). Dogs fed a HPLC diet may have improved glucose uptake compared to dogs fed a MPMC diet. This research provides the first insight into the cardiometabolic health of dogs consuming three diets differing in their protein, carbohydrate and fiber content.

The primary objective of the study was to characterize concentrations and yields of lactoferrin (LF), insulin, and IGF-I in colostrum, transition milk (TM), and whole milk (WM) of multiparous (MP) and primiparous (PP) cows. A secondary objective was to determine associations between colostrum and TM components (fat, protein, lactose), IgG and bioactive compounds (oligosaccharides, LF, insulin, IGF-I; defined as compounds present in micro quantities that stimulate physiological responses systemically or locally within the neonate). Holstein cows (10 MP and 10 PP) were assigned to the study at calving and colostrum was collected 5.3 ± 0.7 h after calving, followed by twice daily sampling of milkings 2–5 (TM) and milking 12 (WM). Colostrum, TM, and WM samples were analyzed by commercial ELISA to determine concentrations of LF, insulin, and IGF-I. Concentrations of insulin, IGF-I and LF were greatest in colostrum compared with all other milkings. Similarly, IGF-I and LF yields were greatest in colostrum, while insulin yield was greatest in colostrum and milking 2 compared with milking 4, 5 and 12. Specifically, insulin, IGF-I and LF yields were 36.3, 14.7 and 2.3 × greater in colostrum compared with WM, respectively. Primiparous cows had greater insulin and lower LF concentrations in colostrum compared with MP cows. Additionally, lower LF concentrations in PP cows compared with MP cows persisted through milkings 2 and 3. The majority of associations between components and bioactive compounds in colostrum were positive; however, correlations were different between PP and MP cows. Specifically, there were a greater number of strong (ρ > 0.80) correlations between components and IgG in PP cows. In contrast, MP cows demonstrated a greater number of strong correlations among bioactive compounds, as well as between components and total oligosaccharides. To the author's knowledge, this study is the first to characterize the yields of IGF-I, LF, and insulin during the colostral (collection within 6 h) and TM period in both MP and PP cows. The study findings indicate that colostrum contains higher levels of bioactive peptides compared with WM and that parity influences the levels of LF and insulin, but not IGF-I. Our study also uniquely demonstrates associations between early lactation components and bioactive compounds, which are not consistent among parities.

Abstract Recent optimization methods have demonstrated the potential to maximize marginal profits in beef (Marques et al., 2020) and dairy (Campos et al., 2023) by optimizing diet formulation. These methods present an opportunity to explore how diets could be optimized for individual dairy cows, such as in a closed-loop precision feeding system. Therefore, our objectives were to 1) compare individual income over feed costs (IOFC), calculated with observed dry matter intake (DMI), milk yield (MY), milk protein and milk fat, with a predicted IOFC for the same diet and cows using the NASEM (2021) model, and 2) optimize the inclusion rate of ingredients in the diet to maximize predicted IOFC. Previously, 20 multiparous (lactation 2 through 4) and 9 primiparous cows, ranging from 22 to 472 d in milk (DIM), were housed in a free-stall pen at the Ontario Dairy Research Centre (Canada) equipped with sensors to record DMI, body weight (BW; walk-over-weight) and body condition score (BCS; DeLavel 3D camera), in addition to daily MY, weekly milk component tests, and 4 X/d respiratory gas exchange measurements (GreenFeed, C-Lock Inc, USA) for estimation of daily energy flows (Kedzierski, 2020). Weekly means for each cow were used to evaluate IOFC in this study (n = 116). Each IOFC was calculated as milk revenue minus total feed cost. Milk revenue was calculated from milk fat, protein and other solids, consistent with pricing on Canadian dairy farms. The observed IOFC was calculated using the observed MY, milk components and DMI, and the same diet formulation and costs for all cows, whereas the predicted IOFC estimated these values using the NASEM (2021) model with all available observed animal inputs (e.g., BW, BCS, DIM). However, the observed DMI and MY had to be provided initially for the model, as the DMI prediction equation requires a target MY, but the prediction of MY requires DMI. Final predicted DMI and MY values were used in the calculation of predicted IOFC. The differential evolution algorithm from the ‘SciPy’ package (v 1.11.4) in python (v 3.12.0) was used to maximize IOFC by adjusting the inclusion of wheat straw, alfalfa silage, corn silage and high moisture corn grain, while fixing minerals to their original proportions. Overall, the concordance correlation coefficient (CCC) indicated moderate agreement (0.62) between the predicted IOFC and observed IOFC and good agreement (0.99) between the predicted IOFC and the optimized IOFC, suggesting the optimization method was able to select diets to meet a similar objective. Results indicate that a strategy is needed to minimize the residual error between predicted and observed values for individual cows, such as algebraic reparameterization of the NASEM model, before using an optimization method to automatically formulate diets.

While there is generally no consensus about how nutrients determine milk synthesis in the mammary gland, it is likely that the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) plays a role as a key integrator of nutritional and mitogenic signals that can influence a multitude of catabolic and anabolic pathways. The objectives of this study were to evaluate acute changes (<24 h) in translational signaling, in addition to chronic changes (14 d) in mammary gland structure and composition, in response to a severe feed restriction. Fourteen lactating Holstein dairy cows were assigned to either ad libitum feeding (n = 7) or a restricted feeding program (n = 7). Feed-restricted cows had feed removed after the evening milking on d 0. Mammary biopsies and blood samples were collected 16 h after feed removal, after which cows in the restricted group were fed 60% of their previously observed ad libitum intake for the remainder of the study. On d 14, animals were slaughtered and their mammary glands dissected. In response to feed removal, an acute increase in plasma nonesterified fatty acid concentration was observed, concurrent to a decrease in milk yield. In mammary tissue, we observed downregulation of the mTORC1-S6K1 signaling cascade, in addition to reductions in mRNA expression of markers of protein synthesis, endoplasmic reticulum biogenesis, and cell turnover (i.e., transcripts associated with apoptosis or cell proliferation). During the 14 d of restricted feeding, animals underwent homeorhetic adaptation to 40% lower nutrient intake, achieving a new setpoint of 14% reduced milk yield with 18% and 29% smaller mammary secretory tissue DM and CP masses, respectively. On d 14, no treatment differences were observed in markers of protein synthesis or mammary cell turnover evaluated using gene transcripts and immunohisto chemical staining. These findings implicate mTORC1-S6K1 in the early phase of the adaptation of the mammary gland's capacity for milk synthesis in response to changes in nutrient supply. Additionally, changes in rates of mammary cell turnover may be transient in nature, returning to basal levels following brief alterations that have sustained effects.

It has been established that essential amino acids (EAA) regulate protein synthesis in mammary epithelial cells by rapidly altering the phosphorylation state of translation factors. However, the long-term transcriptional response to EAA supply has been investigated much less. Eight transcription factors were selected as candidate mediators of EAA effects on mammary cell function via the amino acid response ( ATF4 , ATF6 ), mitogen-activated protein kinase ( JUN , FOS , EGR1 ), and mechanistic target of rapamycin complex 1 ( MYC , HIF1A , SREBF1 ). The objective was to determine if and when expression of these candidate genes was affected in primary cultures of bovine mammary epithelial cells more than 24 h after imposing an EAA deficiency, and to evaluate effects of EAA deficiency on protein synthesis, endoplasmic reticulum size, cell proliferation, and lipogenesis. Differentiated cells were cultured in 1 of 3 treatment media representing normal physiological concentrations of all amino acids (CTL), low lysine (LK), or low methionine (LM) for 24, 40, 48, or 60 h. Both LK and LM suppressed protein synthesis and activated ATF4 expression, indicating the classic amino acid response pathway had been triggered. However, there was no effect of LK or LM on endoplasmic reticulum size, possibly related to elevated ATF6 expression on LM. Expression of early response genes JUN , FOS , EGR1 and MYC was not elevated by EAA deficiency but LM decreased EGR1 expression. LM also increased expression of HIF1A . The EGR1 and HIF1A expression results are consistent with the decrease in cell proliferation rate observed. Variable responses in SREBF1 expression to LK and LM at different timepoints may have contributed to a lack of effect on lipogenesis rates. These findings indicate that EAA deficiency may inhibit mammary protein synthesis and cell proliferation through transcription factors.

Although postruminal glucose infusion into dairy cows has increased milk protein yield in some past experiments, the same trend has not been observed in others. A meta -regression of 64 sets of observations from 29 previously published glucose and propionate infusion studies in dairy cattle, treating study and experiment (study) as random effects, was performed to establish the general effects of glucose equivalent (GlcE) infusion rate on milk true protein (MTP) yield and content, if any, and to identify independent, fixedeffect variables that accounted for the changes in MTP yield and content that were observed. Candidate explanatory variables included rate and site of infusion, diet composition and intake, body weight and lactation stage of the cows, and the change in nutrient intake between GlcE and control treatments. Across all studies, according to a model containing only the random effects of study and experiment, GlcE infusion at an average of 954 g/d increased MTP yield by 26 g/d, on average, whereas mean MTP content was not affected. Backward stepwise elimination of potential explanatory variables from a full mixed model produced a final, reduced model for MTP yield that retained a positive, second -order quadratic effect of infusion rate of GlcE and a positive, linear effect of the change in crude protein intake (CPI) between GlcE treatment and control. This change in CPI due to GlcE infusion ranged from -0.546 to 0.173 kg/d in the dataset. The model fit indicated that when CPI was allowed to drop during GlcE infusion, the effect of GlcE on MTP yield was smaller than when CPI was maintained or increased, in a manifestation of the classic protein: energy interaction. The final reduced model for MTP content contained the same explanatory variables as for MTP yield, plus a negative effect of intravenous compared with gastrointestinal infusion. Overall, the meta -analysis revealed that both MTP yield, and content were positively related to GlcE infusion rate and to the change in CPI between glucose treatment and control.

Abstract Essential (E) amino acids (AA) may alter bovine mammary epithelial cell functions by impacting mRNA expression of genes through their upstream transcription factors. The objective of this research was to study the impact of EAA deficiencies on gene expression and to identify upstream transcriptional factors mediating their effects in bovine mammary epithelial cells. Epithelial cells were collected from the mammary glands of lactating dairy cows and cultured in combined media of DMEM/F12 and Medium 170, differentiated for 5 d, and subsequently treated in triplicate for 60 h with a control medium containing all EAA at normal plasma concentrations for cows (CTL), or a medium with 20 μM Lys (LY), 5 μM Met (LM), or 10 μM His (LH), which are each 25% of normal concentration. RNA was isolated from the cells and sequenced on an Illumina NovaSeq 6000 producing 150 base pair paired-end reads with a minimum sequencing depth of 150 million reads. Reads were cleaned with ‘fastp’ (Chen et al., 2018), aligned to the bovine reference genome (ARS-UCD1.2) using ‘STAR’ aligner (Dobin et al., 2013), and genes counted using ‘featureCounts’ (Liao et al., 2014). Differential expression of genes between treatment pairs was determined using the ‘DESeq2’ package (Love et al., 2014) in R. Genes with an absolute log2 fold change ≥ 1 and a false discovery rate of < 0.05 were considered differentially expressed. The number of differentially expressed genes relative to CTL was 780 for LY, 383 for LH, and 536 for LM. QIAGEN Ingenuity Pathway Analysis (IPA) identified four pathways consistently affected across LY, LM, and LH: kinetochore metaphase signaling, mitotic roles of polo-like kinase, cell cycle: G2/M DNA damage checkpoint regulation, and interferon signaling (Figure 1-3). Notably, the kinetochore metaphase signaling pathway was identified as the most significant across all treatments, suggesting a strong link to cell proliferation and apoptosis during EAA deficiency. The interferon signaling might be related to the Janus kinase 2/signal transducer and activator of transcription 5 (Jak2/STAT5) pathway which can promote the expression of genes encoding milk proteins such as β-casein and β-lactoglobulin. Furthermore, the IPA identified upstream transcription factors related to cell proliferation, apoptosis, autophagy, protein translation, endoplasmic reticulum biogenesis, and amino acid metabolism including NUPR1, FOXM1, MYC, CCND1, TP53, DDIT3, and ATF4 across all treatments. The findings suggest that AA deprivation regulates bovine mammary epithelial cell function by instigating amino acid response pathways and altering secretory cell number.

https://doi.org/10.1093/af/vfad031 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © Hare, Fischer-Tlustos, Wood, Cant, Steele Feature Article Prepartum nutrient intake and colostrum yield and composition in ruminants

A 305-d lactation followed by a 60-d dry period has traditionally been considered economically optimal, yet dairy cows in modern intensive dairy systems are frequently dried off while still producing significant quantities of milk. Managing cows for an extended lactation has reported production, welfare and economic benefits, but not all cows are suitable for an extended lactation. Implementation of an extended lactation strategy on-farm could benefit from use of a decision support system, based on a mathematical lactation model, that can identify suitable cows during early lactation that have a high likelihood of producing above a target milk yield (MY) at 305 d in milk (DIM). Therefore, our objectives were 1) to compare the suitability of 3 commonly used lactation models for modeling extended lactations (Dijkstra, Wood, and Wilmink) in primiparous and multiparous cows under a variety of lactation lengths, and 2) to determine the amount of early lactation daily MY data needed to accurately forecast MY at d 305 by using the most suitable model and determine if this is sufficient for identifying cows suitable for an extended lactation before the end of a typical voluntary waiting period (50 to 90 d). Daily MY data from 467 individual Holstein-Friesian lactations (DIM >305 d; 379 ± 65 d lactation length [mean ± SD]) were fitted by the 3 lactation models using a nonlinear regression procedure. The parameter estimates of these models, lactation characteristics (peak yield, time to peak yield, and persistency), and goodness-of-fit were compared between parity and different lactation lengths. The models had similar performance and differences between parity groups were consistent with previous literature. Then, data from only the first i DIM for each individual lactation, where i was incremented by 30 d from 30 to 150 DIM and by 50 d from 150 to 300 DIM, were fitted by each model to forecast MY at d 305. The Dijkstra model was selected for further analysis as it had superior goodness-of-fit statistics for i = 30 and 60. The data set was fit twice by the Dijkstra model, with parameter bounds either unconstrained or constrained. The quality of predictions of MY at d 305 improved with increasing data availability for both models and assisting the model fitting procedure with more biologically relevant constraints on parameters improved the predictions, but neither was reliable enough for practical use on-farm due to the high uncertainty of forecasted predictions. Using 90 d of data, the constrained model correctly classified 66% of lactations as being above or below a target MY at d 305 of 25 kg/d, with a probability threshold of 0.95. The proportion of correct classifications became smaller at lower targets of MY at d 305 and became greater when using more lactation days. Overall, further work is required to develop a model that can forecast late lactation MY with sufficient accuracy for practical use. We envisage that a hybridized machine learning and mechanistic model that incorporates additional historical and genetic information with early lactation MY could produce meaningful lactation curve forecasts.

During heat stress, dairy cows spend less time lying down to dissipate heat. Heat stress abatement strategies generally target cows outside of their resting areas. However, cooling cows while in their stalls could help alleviate heat stress without compromising lying behavior. The objective of this study was to assess the effects of an inspired-air supplemental cooling system (SCS) on respiration rate, rectal temperatures, lying behavior, rumination time, and milk production (energy-corrected milk, ECM) of lactating dairy cows. A free-stall pen was retrofitted with custom stall partitions to deliver cooled air and mist. The pen, including the stall platform, was divided into two separate sides. Twenty-eight lactating Holstein cows were randomly sorted into two groups, each housed on one side of the experimental pen. Cows experienced four treatments (control, CTRL; cooled air, AIR; mist, MIST; cooled air and mist, AIR + MIST) in a four-treatment, four-period, two-sequence crossover design, with each period lasting seven days. Cooled air was provided continuously, and mist was cycled 3 min on, 12 min off from 0900 to 2100 h. Respiration rates were observed hourly between 0900 and 1500 h, and only measurements recorded while cows were lying down were used in the analysis. Rectal temperatures of 16 focal cows were recorded at 1545 h once per day. Lying behavior and rumination were recorded continuously, and milk yields recorded twice daily were used to calculate ECM. Throughout the experimental period, the average temperature-humidity index was 66.4 & PLUSMN; 6.07. During the MIST treatment, the respiration rate was lower than the CTRL (45.7 vs 49.0 & PLUSMN; 1.92 breaths/min) and AIR (45.7 vs 48.7 & PLUSMN; 1.92 breaths/min). CTRL and AIR did not differ (48.7 vs 49.0 & PLUSMN; 1.92 breaths/min), and MIST and AIR + MIST (45.7 vs 47.1 & PLUSMN; 1.92 breaths/min) did not differ. All other variables were not significantly different between treatments. In conclusion, the SCS appeared to be tolerated by cows and shows the potential to assist in alleviating heat stress. The cooling capacity needs to be evaluated under more extreme environmental conditions causing heat stress than those experienced during this study. Further testing is required to determine the cooled air temperature specifications and mist delivery frequency. & COPY; 2023 The Author(s). Published by Elsevier B.V. on behalf of The Animal Consortium. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

This is the first time the indicator amino acid oxidation (IAAO) technique has been applied in cats. Our results suggest that this methodology has potential to be used in this species; however, adaptations to the isotope protocol are required to successfully apply the IAAO method to determine amino acid (AA) requirements and metabolic availability of AA in cats. The aim of this study was to evaluate whether the indicator amino acid oxidation (IAAO) method could be applied in the domestic cat. Six adult male cats were used in a replicated 3 x 3 Latin square design. Three semi-synthetic diets were developed: a methionine (Met) and total sulfur AA (TSAA) deficient diet (T-BASAL; 0.24% Met+Cys - DM basis) and two Met and TSAA-sufficient diets in which either dl-Met (T-DLM) or 2-hydroxy-4-(methylthio)-butanoic acid (T-MHA) were supplemented, respectively, on an equimolar basis to meet the TSAA requirement (0.34%). After a 2-d diet adaptation, IAAO studies were performed. Cats were offered 13 small meals. The sixth meal contained a priming dose (4.8 mg/kg-BW) of l-[1-C-13]-Phe and the remaining meals a constant dose (1.04 mg/kg-BW). Breath samples were collected every 25 min to measure (CO2)-C-13 enrichment. The following morning, fasted blood samples were collected. Cats returned to the T-BASAL top dressed with a dl-Met solution for 4 d prior to being fed a new dietary treatment. Isotopic steady state was evaluated through visual inspection. Data were analyzed using PROC GLIMMIX procedure in SAS 9.4. While (CO2)-C-13 enrichment was successfully captured in breath samples, cats failed to reach (CO2)-C-13 steady state. Thus, a non-steady state isotope model was developed and coded in ACSLX (V3.1.4.2) individually for each cat on each study day to predict (CO2)-C-13 enrichment, and then, calculate oxidation of l-[1-C-13]-Phe ((FCO2)-C-13). A higher predicted (FCO2)-C-13 was observed for cats fed T-BASAL compared to the others (P < 0.05), while no differences were observed between T-DLM and T-MHA (P > 0.05). Cats fed T-DLM tended to have higher plasma Met concentrations compared to those fed T-BASAL with cats fed T-MHA intermediate (P = 0.0867). Plasma homocysteine concentrations were higher in cats fed T-BASAL compared to the others (P < 0.05), while threonine concentrations tended to be higher in cats fed T-BASAL compared to those fed T-MHA (P = 0.0750). In conclusion, short-term provision of a semi-synthetic diet deficient in Met may elicit a metabolic response aiming to conserve Met. The successful quantification of (CO2)-C-13 enrichment in breath and the higher predicted (FCO2)-C-13 in cats fed a Met deficient diet suggest that the IAAO technique may be used in cats. Adaptations in the isotope protocol should be made to achieve (CO2)-C-13 steady state in breath and avoid mathematical modeling to predict (FCO2)-C-13. Lay Summary It is necessary to apply more sensitive techniques to improve our limited understanding of amino acid (AA) requirements of adult cats. The non-invasive indicator amino acid oxidation (IAAO) technique is highly sensitive in mature animals. However, while it has been widely applied in different species, this technique has yet to be used in cats. We used six healthy adult cats to evaluate whether the IAAO method could be successfully applied in this species. A similar continuous small meals regimen as reported in IAAO studies in dogs was used. An oral primed-constant isotope infusion protocol was applied where l-C-13-Phenylalanine (l-[1-C-13]-Phe) was used as the tracer and the oxidation of l-[1-C-13]-Phe as the response of interest. Breath samples were collected to determine enrichment of (CO2)-C-13 in breath and calculate oxidation of l-[1-C-13]-Phe. While we were able to collect breath samples using calorimetry chambers and capture enrichment of (CO2)-C-13 in breath, cats did not achieve steady state, which is necessary to calculate oxidation of l-[1-C-13]-Phe. Modifications in the isotope protocol should be made to achieve steady state of (CO2)-C-13 in breath, and thus, to successfully apply the IAAO technique to determine requirement of AA in adult cats.

The longitudinal slope of a stall is important for positioning cows in the stall and maintaining cleanliness of the stall surface. The objectives of this study were to determine the short-term effect of increasing free-stall slope from 4.5% to 9.3% on dairy cow lying behavior, rumination, milk production, cleanliness, and preference. In experiment 1, 60 multiparous Holstein cows were divided randomly into two groups and exposed to a 4.5% stall slope (standard in the research facility) and a 9.3% stall slope in a replicated crossover design with four 7-day periods. Each group of cows were housed in a pen with 30 free-stalls and switched between treatments weekly. Lying behavior and rumination were recorded continuously, milk yields were recorded twice per day, and cleanliness scores were collected on the last day of each week. Experiment 2 consisted of an 8-day preference test conducted with 14 cows from experiment 1 in an experimental pen with 32 stalls (16 stalls with a 4.5% slope and 16 stalls with a 9.3% slope). Continuous video monitoring was used to record time spent lying, standing, and perching in each treatment during the last 5 days, and lying time was used to assess preference. In experiment 1, cows spent 12 min/day less time lying down (12.8 vs 12.6 ± 0.28 h/d) and had 0.6 more bouts/day (9.5 vs 10.1 ± 0.38 no./d) with a shorter duration by 6 min/day (1.4 vs 1.3 ± 0.03 h/bout) in stalls with a 9.3% slope. Rumination was 5.5 min less per day in stalls with a 9.3% slope (578.4 vs 572.9 ± 16.56 min/d) and milk yield did not differ between treatments (33.6 vs 33.4 ± 0.78 kg/d). Stall slope did not affect cow cleanliness (1.6 vs 1.6 ± 0.05 points on a 1–5 scale). In experiment 2, cows showed no clear preference for lying, standing, or perching in either stall slope (4.5% and 9.3%) when given a choice. These results indicate that increasing stall slope from 4.5% to 9.3% marginally altered lying behavior, but did not interfere with stall use or influence cow preferences.

Free-stalls for dairy cows promote cleanliness, provide cows with a defined space to lie down, and decrease labor and bedding required for maintenance. However, current stall features can restrict behavior and reduce stall use. The objective of this study was to assess the short-term effect of a novel free-stall design (flexible single-bar partition, no neck rail, increased slope) on stall cleanliness and the lying behavior, rumination, milk production, and preferences of dairy cows in comparison to standard free-stalls (metal loop partitions, neck rail). In the first experiment, 60 Holstein cows were randomly divided into two groups and switched between standard and novel stalls in a replicated crossover design with four 7-day periods. Lying behavior and rumination were recorded continuously. Milk yields were recorded 2x/day, and stall cleanliness scores were collected on the last four days of weeks 3 and 4. The second experiment was a 6-day preference test where 14 cows from experiment 1 were given free access to 16 standard and 16 novel stalls. Lying behavior was recorded continuously. On day 6, standing, perching, and intention, lying, and rising movements were recorded. In experiment 1, cows spent 12 min less time lying down (14.1 vs 14.3 ± 0.48 h/d), had one fewer lying bouts (8.8 vs 9.8 ± 0.23 bouts/d), and had longer lying bouts by 6 min (1.4 vs 1.5 ± 0.05 h/bout) in novel stalls. Rumination time (547.3 vs 548.9 ± 4.66 min/d) and milk yield (35.0 vs 35.2 ± 0.51 kg/d) did not differ between stall types. However, novel stalls were more than twice as soiled as standard stalls (32.8 vs 14.2 ± 1.38% of rear half soiled). In experiment 2, the cows most recently housed in the novel stalls showed a clear preference for lying down in those stalls, whereas cows most recently housed in standard stalls showed no preference. Intention and lying down movements were longer in standard stalls when other cows were present in diagonally opposite stalls, but were similar between stall types when not occupied. All cows preferred standing in novel stalls. Novel stalls may have improved stall use compared to standard free-stalls, and the design requires further development to address cleanliness concerns. Future work is required to provide comfortable stalls without sacrificing cleanliness.

Light from the environment is important for vision and regulating various biological processes. Providing supplemental lighting in the stall area could allow for individually targeted or group-level control of light. This study aimed to determine whether dairy cattle had preferences for short-term exposure to white (full-spectrum) light-emitting diode (LED) light or no LED light, yellow-green or white LED light, and blue or white LED light in the stall area. In total, 14 lactating cows were housed in a free-stall pen with unrestricted access to 28 stalls. LED light was controlled separately for each side of the stall platform. Two combinations of light were tested per week, and each week consisted of three adaptation days and four treatment days. Lying behaviour and video data were recorded continuously using leg-mounted pedometers and cameras, respectively. Preference was assessed by the amount of time spent lying and the number of bouts under each light treatment. No differences occurred between treatments within each week for daily lying time and number of bouts. Similarly, no differences occurred between treatments within each time period. Further controlled studies of long-term exposure to different LED wavelengths and intensities are required to determine potential benefits on metabolic processes.