Time profiles of energy balance in dairy cows in association with metabolic status, inflammatory status, and disease.

The early lactation period in dairy cows is characterized by complex interactions among energy balance (EB), disease, and alterations in metabolic and inflammatory status. The objective of this study was to cluster cows based on EB time profiles in early lactation and investigate the association between EB clusters and inflammatory status, metabolic status, oxidative stress, and disease. Holstein-Friesian dairy cows (n = 153) were selected and monitored for disease treatments during wk 1 to 6 in lactation. Weekly EB was calculated based on energy intake and energy requirements for maintenance and milk yield in wk 1 to 6 in lactation. Weekly plasma samples were analyzed for metabolic variables in wk 1 to 6, and inflammatory and oxidative stress variables in wk 1, 2, and 4 in lactation. Liver activity index (LAI) was computed from plasma albumin, cholesterol, and retino-binding protein concentration. First, cows were clustered based on time profiles of EB, resulting in 4 clusters (SP: stable positive; MN: mild negative; IN: intermediate negative; SN: severe negative). Cows in the SN cluster had higher plasma nonesterified fatty acids and ß-hydroxybutyrate concentrations, compared with cows in the SP cluster, with the MN and IN cluster being intermediate. Cows in the SN cluster had a higher milk yield, lower dry matter intake in wk 1, lower insulin concentration compared with cows in the SP cluster, and lower glucose and IGF-1 concentration compared with cows in the SP and MN clusters. Energy balance clusters were not related with plasma haptoglobin, cholesterol, albumin, paraoxonase, and liver activity index (LAI). Second, cows were grouped based on health status [IHP: cows with treatment for inflammatory health problem (endometritis, fever, clinical mastitis, vaginal discharge or retained placenta); OHP: cows with no IHP but treatment for other health problem (milk fever, cystic ovaries, claw, and leg problems, rumen and intestine problems or other diseases); NHP: cows with no treatments, in the first 6 weeks after calving]. Energy balance was not different among health status groups. The IHP cows had lower nonesterified fatty acids and greater insulin concentration in plasma compared with OHP. The IHP cows had lower plasma albumin concentration, lower LAI and higher haptoglobin concentration compared with OHP and NHP. Overall, EB time profiles were associated with the metabolic status of dairy cows in early lactation, but were only limitedly related with markers of inflammation and oxidative stress status. Inflammatory and metabolic status were related to disease events in early lactation and caused prolonged effects on liver metabolism.

Impact of nutrient restriction at dry-off on performance and metabolism.

Thanks to improvements in genetics, nutrition, and management, modern dairy cows can still produce large amounts of milk at the end of lactation, with possible negative effects on health and welfare, particularly when milking is stopped abruptly. To limit yield at dry-off, strategies involving different types of dietary restriction have been used worldwide. Thus, we aimed to investigate the effects of a reduced nutrient density at dry-off on milk production, metabolism, the pattern of rumen fermentation, and milk fatty acid profile around dry-off and in the ensuing periparturient period. During the last week before dry-off, 26 Holstein cows were enrolled in pairs according to the expected calving date and either fed ad libitum ryegrass hay (nutrient restricted, NR; 13 cows) or continued to receive lactation diet (control group, CTR; 13 cows). After dry-off, both groups received only grass hay for 7 d, and free access to water was always provided. Blood, milk, and rumen fluid samples were collected from 7 d before dry-off to 28 DIM. Milk production, DMI (during the periparturient period), and rumination times were recorded daily. At dry-off, NR cows had decreased milk yield (-62%) and milk lactose compared with CTR but had higher fat and protein contents. In the subsequent lactation, no significant differences were observed in milk yield and composition. The BCS did not differ between groups during the transition period, but it decreased in NR after dry-off. Before dry-off, NR had decreased glucose, urea, and insulin, but higher creatinine, BHB, and nonesterified fatty acids (NEFA). The day after dry-off, NEFA were lower in NR, but they were higher 7 d after calving. At dry-off, NR had higher rumen pH, lower lactate, urea, and total volatile fatty acid concentrations. Considering volatile fatty acid molar proportions, NR had increased acetate but decreased propionate and butyrate at dry-off. Rumination time dropped 6 d before dry-off in NR and after dry-off in CTR, but no differences were observed in the periparturient period. Milk fatty acid profile revealed a remarkably lower proportion of short-chain fatty acids in NR at dry-off and a higher proportion of medium- and long-chain ones. These results confirmed that decreasing nutrient density reduce milk yield before dry-off. However, metabolism around dry-off was significantly affected, as suggested by plasma, rumen fluid, and milk analyses. Further research is required to investigate the impact of the metabolic effects on the inflammatory response, liver function, and immune system, particularly concerning the mammary gland.

Graduate Student Literature Review: The challenge of drying-off high-yielding dairy cows.

The cessation of lactation (i.e., dry-off) in dairy cattle is an area of research that has received much focus in recent years. The dry period is necessary to optimize tissue remodeling of the mammary gland, but represents a stressful event, incorporating several changes in daily routine, diet, and metabolism. Moreover, the high milk yields achieved by modern cows in late gestation exacerbate the need for relevant manipulations in the days around dry-off, as excessive accumulation of milk might jeopardize the success of the dry period, with potential negative effects on future lactation. Production levels over 15 kg/d are an additional risk factor for udder health, delay mammary involution, and worsen metabolic stress and inflammatory responses. Furthermore, the pressure to reduce antibiotic usage in farm animals has resulted in increased attention on the dry period, given that historically most dairy cattle were provided prophylactic intramammary antibiotic treatment at dry-off as a means to reduce the risk of intramammary infections in the subsequent lactation. Several strategies have been proposed over the years to cope with these challenges, aiming to gradually reduce milk yield before dry-off, promoting at the same time the start of mammary involution. Among them, the most common are based on feed or nutrient restriction, a decrease in milking frequency, or administration of prolactin inhibitors. These practices have different capacities to reduce milk yield through different mechanisms and entail several implications for udder health, animal welfare, behavior, endocrine status, metabolism, and inflammatory conditions. The present review aims to provide a comprehensive overview of the dry-off phase in high-yielding cows and of the impact of high milk production at dry-off, and to describe possible strategies that might be implemented by farmers and veterinarians to optimize this critical phase in an integrated way.

Effect of supplementing live Saccharomyces cerevisiae yeast on performance, rumen function, and metabolism during the transition period in Holstein dairy cows.

Dairy cows have to face several nutritional challenges during the transition period, and live yeast supplementation appears to be beneficial in modulating rumen activity. In this study, we evaluated the effects of live yeast supplementation on rumen function, milk production, and metabolic and inflammatory conditions. Ten Holstein multiparous cows received either live Saccharomyces cerevisiae (strain Sc47; SCY) supplementation from -21 to 21 d from calving (DFC) or a control diet without yeast supplementation. Feed intake, milk yield, and rumination time were monitored until 35 DFC, and rumen fluid, feces, milk, and blood samples were collected at different time points. Compared with the control diet, SCY had increased dry matter intake (16.7 vs. 19.1 ± 0.8 kg/d in wk 2 and 3) and rumination time postpartum (449 vs. 504 ± 19.9 min/d in wk 5). Milk yield tended to be greater in SCY (40.1 vs. 45.2 ± 1.7 kg/d in wk 5), protein content tended to be higher, and somatic cell count was lower. In rumen fluid, acetate molar proportion was higher and that of propionate lower at 21 DFC, resulting in increased acetate:propionate and (acetate + butyrate):propionate ratios. Cows in the SCY group had lower fecal dry matter but higher acetate and lower propionate proportions on total volatile fatty acids at 3 DFC. Plasma analysis revealed a lower degree of inflammation after calving in SCY (i.e., lower haptoglobin concentration at 1 and 3 DFC) and a likely better liver function, as suggested by the lower γ-glutamyl transferase, even though paraoxonase was lower at 28 DFC. Plasma IL-1ß concentration tended to be higher in SCY, as well as Mg and P. Overall, SCY supplementation improved rumen and hindgut fermentation profiles, also resulting in higher dry matter intake and rumination time postpartum. Moreover, the postcalving inflammatory response was milder and liver function appeared to be better. Altogether, these effects also led to greater milk yield and reduced the risk of metabolic diseases.

Metabolic and physiological adaptations to first and second lactation in Holstein dairy cows: Postprandial patterns.

Dairy cows during their first and second lactation have different milk yield, body development, feed intake, and metabolic and endocrine statuses. However, large diurnal variations can also exist in terms of biomarkers and hormones related to feeding behavior and energy metabolism. Thus, we investigated the diurnal patterns of the main metabolic plasma analytes and hormones in the same cows during their first and second lactations in different stages of the lactation cycle. Eight Holstein dairy cows were monitored during their first and second lactation, during which they were reared under the same conditions. Blood samples were collected before the morning feeding (0 h) and after 1, 2, 3, 4.5, 6, 9, and 12 h on scheduled days between -21 d relative to calving (DRC) and 120 DRC for the assessment of some metabolic biomarkers and hormones. Data were analyzed using the GLIMMIX procedure of SAS (SAS Institute Inc.). Regardless of parity and stage of lactation, glucose, urea, ß-hydroxybutyrate, and insulin peaked a few hours after the morning feeding, whereas nonesterified fatty acids decreased. The insulin peak was attenuated during the first month of lactation, whereas postpartum growth hormone spiked on average 1 h after the first meal in cows during their first lactation. This peak occurred earlier than during the second lactation. Most of the differences in diurnal trends between lactations were observed in the postpartum period (and in some cases even in early lactation). Glucose and insulin were higher during the first lactation throughout the day, and the differences increased 9 h after feeding. Conversely, nonesterified fatty acids and ß-hydroxybutyrate showed the opposite trend, and their plasma concentrations at 9 and 12 h after feeding differed between lactations. These results confirmed the differences observed between the first 2 lactations in prefeeding metabolic marker concentrations. Furthermore, plasma concentrations of investigated analytes showed high variability during the day, and thus we advise caution when interpreting metabolic biomarker data in dairy cows, especially during the periods close to calving.

Metabolic and physiological adaptations to first and second lactation in Holstein dairy cows.

Huge differences exist between cow yields and body sizes during their first and second lactations. The transition period is the most critical and investigated phase of the lactation cycle. We compared metabolic and endocrine responses between cows at different parities during the transition period and early lactation. Eight Holstein dairy cows were monitored at their first and second calving during which they were reared under the same conditions. Milk yield, dry matter intake (DMI), and body weight (BW) were regularly measured, and energy balance, efficiency, and lactation curves were calculated. Blood samples were collected on scheduled days from -21 d relative to calving (DRC) to 120 DRC for the assessment of metabolic and hormonal profiles (biomarkers of metabolism, mineral status, inflammation, and liver function). Large variations in the period in question for almost all variables investigated were observed. Compared with their first lactation, cows during their second lactation had higher DMI (+15%) and BW (+13%), their milk yield was greater (+26%), lactation peak was higher and earlier (36.6 kg/d at 48.8 DRC vs. 45.0 kg/d at 62.9 DRC), but persistency was reduced. Milk fat, protein, and lactose contents were higher during the first lactation and coagulation properties were better (higher titratable acidity, faster and firmer curd formation). Postpartum negative energy balance was more severe the during the second lactation (1.4-fold at 7 DRC) and plasma glucose was lower. Circulating insulin and insulin-like growth factor-1 were lower in second-calving cows during the transition period. At the same time, markers of body reserve mobilization (ß-hydroxybutyrate and urea) increased. Moreover, albumin, cholesterol, and γ-glutamyl transferase were higher during second lactation, whereas bilirubin and alkaline phosphatase were lower. The inflammatory response after calving was not different, as suggested by the similar haptoglobin concentrations and only transient differences in ceruloplasmin. Blood growth hormone did not differ during the transition period but was lower during the second lactation at 90 DRC, whereas circulating glucagon was higher. These results agree with the differences in milk yield and confirmed the hypothesis of a different metabolic and hormonal status between the first and second lactation partly related to different degrees of maturity.

Impact of dry-off and lyophilized Aloe arborescens supplementation on plasma metabolome of dairy cows.

Positive effects have been observed as a result of Aloe arborescens supplementation in the dry-off phase in dairy cows. Metabolomic approaches can provide additional information about animal physiology. Thus, we characterized plasma metabolome around dry-off in 12 cows supplemented (AL) or not (CTR) with 10 g/d of lyophilized A. arborescens with an untargeted metabolomic approach. Overall, 1658 mass features were annotated. Regardless of treatment, multivariate statistics discriminated samples taken before and after dry-off. Overall, 490 metabolites were different between late lactation and early dry period, of which 237 were shared between AL and CTR. The most discriminant compounds (pentosidine and luteolin 7-O-glucoside) were related to the more fibrous diet. Pathway analysis indicated that pyrimidine and glycerophospholipid metabolisms were down-accumulated, suggesting reduced rumen microbial activity and liver load. Samples from AL were discriminated from CTR either the day of dry-off or 7 days after. At dry-off, aloin and emodin were the most discriminant metabolites, indicating that Aloe's bioactive compounds were absorbed. Seven days later, 534 compounds were different between groups, and emodin was among the most impacted. Pathway analysis highlighted that glycerophospholipid, pyrimidine, and folate metabolisms were affected. These results might indicate that Aloe has positive effects on liver function and a modulatory effect on rumen fermentation.

Effect of stage of lactation and dietary starch content on endocrine-metabolic status, blood amino acid concentrations, milk yield, and composition in Holstein dairy cows.

Milk yield and composition are modified by level and chemical characteristics of dietary energy and protein. Those factors determine nutrient availability from a given diet, and once absorbed, they interact with the endocrine system and together determine availability of metabolites to the mammary gland. Four multiparous dairy cows in early lactation and subsequently in late lactation were fed 2 diets for 28 d in a changeover design that provided, within the same stage of lactation, similar amounts of rumen fermentable feed with either high (HS) or low starch (LS). All diets had similar dietary crude protein (15.5% dry matter) and rumen-undegradable protein (∼40% of crude protein) content. Profiles of AA were calculated to be similar to that of casein. On d 28, [1-13C] Leu was infused into one jugular vein with blood samples taken at 0, 2, 4, 6, and 8 h, and cows milked at 0, 2, 4, 5, 6, 7, and 8 h from start of infusion. Isotopic enrichments of plasma Leu, keto-isocaproic acid, and milk casein were determined for calculation of Leu kinetics. Data were subjected to ANOVA using the MIXED procedure of SAS (SAS Institute Inc.), with time as repeated factor and cow as the random effect. Dry matter intake within each stage of lactation was similar between groups. Feeding LS resulted in lower blood glucose and greater ratio of bovine somatotropin to insulin. This response was associated with greater blood concentrations of nonesterified fatty acids and ß-hydroxybutyrate, which might have contributed to greater milk fat content in LS-fed cows. Except for His, average concentrations of all AA in blood were higher in late than early lactation. Diet did not alter average plasma concentrations of AA. However, for most of the essential AA (particularly branched-chain), the HS diet led to a marked decrease in concentrations after the forage meal, resulting in significant differences between dietary groups in early lactation. In early-lactating cows fed HS, a greater reduction in plasma concentrations at 8 h relative to pre-feeding values (time zero) was observed for Met, Lys, and His, resulting in decreases of 27.9%, 33.6%, and 38.5%, respectively. A higher bovine somatotropin/insulin ratio in early lactation and in cows fed LS could possibly have led to greater breakdown and, consequently, higher AA flux from peripheral tissues. In LS-fed cows, higher mobilization of body fat and protein was confirmed by the greater body weight loss in both stages of lactation. Higher irreversible loss of [1-13C] Leu in early lactation suggested lower protein retention in peripheral tissues during early compared with late lactation. Milk yield, protein output, and composition were similar between groups at both stages of lactation, whereas milk coagulation was faster (lower curd firming rate) and with higher curd firmness in response to feeding HS in late lactation. Overall, data indicated that rate of carbohydrate fermentability in the rumen can modify the availability of metabolites to the mammary gland and consequently modify milk protein coagulation.

Effects of supplementing Saccharomyces cerevisiae fermentation products to dairy cows from the day of dry-off through early lactation.

The scope of this experiment was to study the effects of Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V) on milk yield, milk composition, somatic cell count, rumination activity, and immunometabolic profile (inflammation) of dairy cows during the peripartum period. Postpartum inflammation severity was evaluated as the liver functionality index (LFI). The LFI is based on profiles of specific blood inflammatory markers in the first month of lactation. We hypothesized that SCFP could increase the rumination time in dairy cows. Treatments were control (CTR; no supplement, n = 17) or SCFP (19 g/d of NutriTek, n = 17) included into a pellet delivered at robotic milking unit. Treatments were fed from d -60 to 42 relative to parturition. Cows were fed the same basal rations formulated to pre- or postpartum requirements. Cows were voluntarily milked with robotic milking unit. Blood samples were collected at d -60, -28, -7, 7, and 28 relative to parturition. To study the effect of the treatment and severity of inflammation during periparturient period on subsequent cow performance, cows were retrospectively divided into 2 groups based on their LFI score: low (LLFI) and high (HLFI). Thus, LFI grouping and supplementation treatment groups were as follows: LLFI-CTR, LLFI-SCFP, HLFI-CTR, HLFI-SCFP. Data were analyzed with ANOVA using a mixed model for repeated measures; the model included the effect of the diet, LFI group, time relative to parturition, and their interaction. The nonesterified fatty acids concentrations were greatest at d 7 of lactation for LLFI-CTR compared with other groups. No other differences in plasma metabolites were observed. The LLFI-CTR cows had a greater reduction of body condition score from d -7 until 28 relative to parturition compared with other groups. Somatic cell counts were not different among groups, with averages of 175, 169, 384, and 126 × 1,000 cells/mL for the HLFI-CTR, HLFI-SCFP, LLFI-CTR, and LLFI-SCFP group, respectively, regardless of day. However, the LLFI-CTR had greater somatic cell count on d 42 compared with other groups. During the week before parturition, the LLFI-CTR group had reduced rumination time of 46 min compared with the other 3 groups. However, the minutes of rumination per day was only different between LLFI-CTR and the LLFI-SCFP groups. Milk production of cows was different for LFI scores as follows: 50.2 versus 46.7 kg/d for HLFI and LLFI, respectively. Interestingly, there were no differences of milk production due to supplementation treatment of the HLFI cows. However, the LLFI-SCFP group produced 49.1 kg/d compared with 44.3 kg/d of the LLFI-CTR group during the first month of lactation. Milk composition did not differ throughout the experimental period for the 4 groups of cows. In conclusion, SCFP supplementation assisted cows experiencing low LFI to maintain milk production, somatic cell count, and plasma nonesterified fatty acid concentrations similar to cows with high LFI.

Plasma albumin-to-globulin ratio before dry-off as a possible index of inflammatory status and performance in the subsequent lactation in dairy cows.

The dry-off of dairy cows represents an important phase of the lactation cycle, influencing the outcome of the next lactation. Among the physiological changes, the severity of the inflammatory response can vary after the dry-off, and this response might have consequences on cow adaptation in the transition period. The plasma protein profile is a diagnostic tool widely used in humans and animals to assess the inflammatory status and predict the outcome of severe diseases. The albumin-to-globulin ratio (AG) can represent a simple and useful proxy for the inflammatory condition. In this study, we investigated the relationship between AG before dry-off and inflammation, metabolic profile, and performance of 75 Holstein dairy cows. Blood samples were collected from -62 (7 d before dry-off) to 28 d relative to calving (DFC) to measure metabolic profile biomarkers, inflammatory variables, and liver function. Daily milk yield in the first month of lactation was recorded. Milk composition, body condition score, fertility, and health status were also assessed. The AG calculated 1 wk before dry-off (-62 DFC) was used to retrospectively group cows into tertiles (1.06 ± 0.09 for HI, 0.88 ± 0.04 for IN, and 0.72 ± 0.08 for LO). Data were subjected to ANOVA using the PROC MIXED program in SAS software. Differences among groups observed at -62 DFC were almost maintained throughout the period of interest, but AG peaked before calving. According to the level of acute-phase proteins (haptoglobin, ceruloplasmin, albumin, cholesterol, retinol-binding protein), bilirubin, and paraoxonase, a generally overall lower inflammatory condition was found in HI and IN than in the LO group immediately after the dry-off but also after calving. The HI cows had greater milk yield than LO cows, but no differences were observed in milk composition. The somatic cell count reflected the AG ratio trend, with higher values in LO than IN and HI either before dry-off or after calving. Fertility was better in HI cows, with fewer days open and services per pregnancy than IN and LO cows. Overall, cows with high AG before dry-off showed an improved adaptation to the new lactation, as demonstrated by a reduced systemic inflammatory response and increased milk yield than cows with low AG. In conclusion, the AG ratio before dry-off might represent a rapid and useful proxy to evaluate the innate immune status and likely the ability to adapt while switching from the late lactation to the nonlactating phase and during the transition period with emphasis on early lactation.

A mycotoxin-deactivating feed additive counteracts the adverse effects of regular levels of Fusarium mycotoxins in dairy cows.

Little is known about the effects of commonly found levels of Fusarium mycotoxins on the performance, metabolism, and immunity of dairy cattle. We investigated the effects of regular contamination levels, meaning contamination levels that can be commonly detected in dairy feeds, of deoxynivalenol (DON) and fumonisins (FB) in total mixed ration (TMR) on the performance, diet digestibility, milk quality, and plasma liver enzymes in dairy cows. This trial examined 12 lactating Holstein dairy cows using a 3-period × 3-treatment Latin square design. The experimental period was 21 d of mycotoxin exposure followed by 14 d of washout. During treatment periods, cows received one of 3 diets: (1) CTR (control) diet of TMR contaminated with 340.5 µg of DON/kg of dry matter (DM) and 127.9 µg FB/kg of DM; (2) MTX diet of TMR contaminated with Fusarium mycotoxins at levels higher than CTR but below US and European Union guidelines (i.e., 733.0 µg of DON/kg of DM and 994.4 µg of FB/kg of DM); or (3) MDP diet, which was MTX diet supplemented with a mycotoxin deactivator product (i.e., 897.3 µg of DON/kg of DM and 1,247.1 µg of FB/kg of DM; Mycofix, 35 g/animal per day). During washout, all animals were fed the same CTR diet. Body weight, body condition score, DM intake, dietary nutrient digestibility, milk production, milk composition and rennet coagulation properties, somatic cell count, blood serum chemistry, hematology, serum immunoglobulin concentrations, and expression of multiple genes in circulating leucocytes were measured. Milk production was significantly greater in the CTR group (37.73 kg/d) than in the MTX (36.39 kg/d) and the MDP (36.55 kg/d) groups. Curd firmness and curd firming time were negatively affected by the MTX diet compared with the other 2 diets. Furthermore, DM and neutral detergent fiber digestibility were lower after the MTX diet than after the CTR diet (67.3 vs. 71.0% and 42.8 vs. 52.3%). The MDP diet had the highest digestibility coefficients for DM (72.4%) and neutral detergent fiber (53.6%) compared with the other 2 diets. The activities of plasma liver transaminases were higher after the MTX diet than after the CTR and MDP diets. Compared with the CTR diet, the MTX diet led to slightly lower expression of genes related to immune and inflammatory functions, indicating that Fusarium mycotoxins had an immunosuppressive effect. Our results indicated that feed contaminated with regular levels of Fusarium mycotoxins adversely affected the performance, milk quality, diet digestibility, metabolic variables, and immunity of dairy cows, and that supplementation with mycotoxin deactivator product counteracted most of these negative effects.

Association of postpartum uterine diseases with lying time and metabolic profiles of multiparous Holstein dairy cows in the transition period.

The objective of this study was to assess how uterine disorders alter the lying behaviour and plasma biomarkers in dairy cows. 34 multiparous cows were retrospectively classified into three groups according to the first uterine disorder that cows were diagnosed with: retained placenta (RP), metritis (MET), or healthy (H; cows without any clinical disease). Lying time (LT) and duration of lying bouts (LB) were monitored between 6 weeks prior to and 8 weeks after calving via the AfiAct II pedometer. Blood samples were collected routinely between 14 days before and 28 days after calving. Data was analysed using Proc MIXED of SAS ver. 9.4. Regardless of grouping, both LT and LB were longer (P < 0.01) in the prepartum period (774 ± 16.6 min/day and 89.9 ± 2.1 min/bout) than in the first 28 days after calving (DFC; 653 ± 16.7 min/day and 63.7 ± 2.1 min/bout). Cows with RP had longer LT than healthy cows during the last 3 weeks before calving (837 ± 30.9 vs. 735 ± 27.1 min/day; P < 0.05). LT in cows with MET and healthy cows were not significantly different. The LB was similar among groups, averaging 76.1 ± 3.4 min/bout in healthy cows, 73.2 ± 3.8 min/bout in cows with RP, and 75.2 ± 3.7 min/bout in cows with MET (P > 0.05). Compared with healthy cows, cows with RP laid down longer and stood up for shorter times (P < 0.05), particularly before calving. In addition, cows with RP had increased mobilization of body stores and more pronounced inflammatory status, as demonstrated by plasma haptoglobin (P = 0.04) and albumin (P < 0.01) concentrations. Our data suggest that automatic monitoring of lying behaviour could help identify cows at increased risk of developing certain disorders, such as RP.

Methyl donor supply to heat stress-challenged polymorphonuclear leukocytes from lactating Holstein cows enhances 1-carbon metabolism, immune response, and cytoprotective gene network abundance.

Mechanisms controlling immune function of dairy cows are dysregulated during heat stress (HS). Methyl donor supply-methionine (Met) and choline (Chol)-positively modulates innate immune function, particularly antioxidant systems of polymorphonuclear leukocytes (PMN). The objective of this study was to investigate the effect of Met and Chol supply in vitro on mRNA abundance of genes related to 1-carbon metabolism, inflammation, and immune function in short-term cultures of PMN isolated from mid-lactating Holstein cows in response to heat challenge. Blood PMN were isolated from 5 Holstein cows (153 ± 5 d postpartum, 34.63 ± 2.73 kg/d of milk production; mean ± SD). The PMN were incubated for 2 h at thermal-neutral (37°C; TN) or heat stress (42°C; HS) temperatures with 3 levels of Chol (0, 400, or 800 µg/mL) or 3 ratios of Lys:Met (Met; 3.6:1, 2.9:1, or 2.4:1). Supernatant concentrations of IL-1ß, IL-6, and tumor necrosis factor-α were measured via bovine-specific ELISA. Fold-changes in mRNA abundance were calculated separately for Chol and Met treatments to obtain the fold-change response at 42°C (HS) relative to 37°C (TN). Data were subjected to ANOVA using PROC MIXED in SAS (SAS Institute Inc., Cary, NC). Orthogonal contrasts were used to determine the linear or quadratic effect of Met and Chol for mRNA fold-change and supernatant cytokine concentrations. Compared with PMN receiving 0 µg of Chol/mL, heat-stressed PMN supplemented with Chol at 400 or 800 µg/mL had greater fold-change in abundance of CBS, CSAD, GSS, GSR, and GPX1. Among genes associated with inflammation and immune function, fold-change in abundance of TLR2, TLR4, IRAK1, IL1B, and IL10 increased with 400 and 800 µg of Chol/mL compared with PMN receiving 0 µg of Chol/mL. Fold-change in abundance of SAHH decreased linearly at increasing levels of Met supply. A linear effect was detected for MPO, NFKB1, and SOD1 due to greater fold-change in abundance when Met was increased to reach Lys:Met ratios of 2.9:1 and 2.4:1. Although increasing Chol supply upregulated BAX, BCL2, and HSP70, increased Met supply only upregulated BAX. Under HS conditions, enhancing PMN supply of Chol to 400 µg/mL effectively increased fold-change in abundance of genes involved in antioxidant production (conferring cellular processes protection from free radicals and reactive oxygen species), inflammatory signaling, and innate immunity. Although similar outcomes were obtained with Met supply at Lys:Met ratios of 2.9:1 and 2.4:1, the response was less pronounced. Both Chol and Met supply enhanced the cytoprotective characteristics of PMN through upregulation of heat shock proteins. Overall, the modulatory effects detected in the present experiment highlight an opportunity to use Met and particularly Chol supplementation during thermal stress.

The effect of parity number on the metabolism, inflammation, and oxidative status of dairy sheep during the transition period.

The objective of this study was to evaluate whether dairy sheep during the transition period are affected by their parity numbers with regard to (1) body weight (BW), body condition score (BCS), and production performance (milk yield and composition) and (2) metabolic, inflammation, and stress biomarkers. For this purpose, 30 Sarda dairy ewes [15 primiparous (PRP) and 15 multiparous (MUP) ewes] were recruited on d 90 of gestation. Each group was homogeneous according to age, BW, and BCS. Sampling was carried out at -60, -30, -7, 0, +30, and +60 d from lambing. The MUP ewes showed a higher BW (46.32 vs. 38.71 kg) and larger litter size (1.45 vs. 1.06 kg) but a lower BCS (2.47 vs. 2.70) than the PRP ewes. Furthermore, the MUP ewes had lower concentrations of glucose (3.49 vs. 4.27 mol/L), cholesterol (1.63 vs. 1.81 mmol/L), free fatty acids (0.47 vs. 0.62 mmol/L), and triglycerides (0.22 vs. 0.25 mmol/L) compared with PRP ewes. With regard to inflammation and oxidative stress parameters, the PRP group had higher haptoglobin (0.48 vs. 0.18 g/L) and paraoxonase (187.90 vs. 152.11 U/L) activity than the MUP group. Overall, the MUP ewes were characterized by greater milk production performance and greater feed intake, resulting in a better energy balance, than the PRP ewes. Interestingly, these findings highlighted a different metabolic and inflammatory response over the transition period between PRP and MUP ewes, with the latter displaying lower concentrations of inflammatory-related biomarkers.

Effect of litter size on prepartum metabolic and amino acidic profile in rabbit does.

The use of modern prolific lines of rabbit does in intensive production systems leads to an increase in productivity but also causes a rise in several problems related to the does' health status. Hence, the aim of this study was to investigate the effect of the litter size on the metabolic, inflammatory and plasma amino acid profile in rabbit does. The blood of 30 pregnant does was sampled on the 27th day of pregnancy. The does were retrospectively grouped according to the number of offspring into a high litter size group (HI, does with ≥ 12 kits; n = 16) and a low litter size group (LO, does with ≤ 11 kits; n = 14). Data were subjected to Pearson's correlation analysis. Further, data were analysed in agreement to a completely randomized design in which the main tested effect was litter size. The linear or quadratic trends of litter size on parameters of interests were post hoc compared by using orthogonal contrasts. In addition, compared with the LO group, the HI group had lower levels of glucose (-5%; P < 0.01), zinc (-19%; P < 0.05), albumin (-6%; P < 0.05) and total cholesterol (-13%; P < 0.07), but the total bilirubin level was higher in the HI group (+14%; P < 0.05). Regarding the plasma amino acids, the HI group had lower concentrations of threonine (-15%), glycine (-16%), lysine (-16%) and tryptophan (-26%) and a higher level of glutamic acid (+43%; P < 0.05) compared with the LO group. The exclusively ketogenic amount of amino acids was lower (P < 0.06) in the HI (55.8 mg/100 ml) does compared with the LO does (56.8 mg/100 ml). These results show that a few days before delivery, rabbit does that gave birth to a higher number of offspring had a metabolic profile and an inflammatory status that was less favourable with respect to does who gave birth to a lower number of offspring. Moreover, the plasma amino acid profile points out that there was an enhanced catabolic condition in the rabbit does with a high number of gestated foetuses; it was likely related to the greater energy demand needed to support the pregnancy and an early inflammatory response.

Technical note: Capillary electrophoresis as a rapid test for the quantification of immunoglobulin G in serum of newborn lambs.

Finding a rapid and simple method of serum IgG determination in lambs is essential for monitoring failure of passive transfer of immunity. The aim of this study was to assess the ability of capillary electrophoresis (CE), an instrument mainly used in blood serum protein analysis, to estimate IgG content in serum of newborn lambs through determination of only total Ig percentage by comparing the results with those obtained with radial immunodiffusion (RID), the reference method for serum IgG quantification. Serum samples were collected at 24 h after birth from 40 Sarda lambs. The IgG concentration measured by RID and serum total Ig concentration measured by CE were (mean ± standard deviation) 29.8 ± 16.1 g/L and 37.8 ± 15.0%, respectively. Data provided by RID and CE analysis showed a polynomial trend (RID = 0.02CE2 - 0.04CE + 4.13; coefficient of determination, R2 = 0.96), displaying a strong relationship between these 2 methods. Applying the polynomial equation, the IgG values were predicted. Predicted IgG values were highly correlated (r = 0.98) and related (R2 = 0.96) to IgG values obtained by RID assay. These data were subjected to Bland-Altman analysis, revealing a high level of agreement between CE and RID methods with a bias that was not different from 0 (-0.04 g/L) and agreement limits of -6.38 g/L (low) and +6.30 g/L (high). In addition, the linear regression analysis between differences (dependent variable) and average of IgG concentration by CE and RID (independent variable) did not show proportional bias (R2 = 0.01). In conclusion, CE is a reliable instrument for a lamb health monitoring program, where Bland-Altman analysis also confirmed that the CE method can be a suitable alternative to RID.

Short communication: Inflammation, migration, and cell-cell interaction-related gene network expression in leukocytes is enhanced in Simmental compared with Holstein dairy cows after calving.

The aim of this study was to investigate changes in the abundance of genes involved in leukocyte function between cows highly specialized for milk production (Holstein, n = 12) and cows selected for meat and milk (Simmental, n = 13). Blood was collected on d 3 after calving in PAXgene tubes (Preanalytix, Hombrechtikon, Switzerland) to measure mRNA abundance of 33 genes. Normalized gene abundance data were subjected to MIXED model ANOVA using SAS (SAS Institute Inc. Cary, NC). Simmental cows had greater transcript abundance of proinflammatory cytokines and receptor genes (IL1B, TNF, IL1R, TNFRSF1A), cell migration- and adhesion-related genes (CX3CR1, ITGB2, CD44, LGALS8), and the antimicrobial IDO1 gene. In contrast, compared with Holstein cows, Simmental cows had lower abundance of the toll-like receptor (TLR) recognition-related gene TLR2, the antimicrobial-related gene LTF, and S100A8, which is involved in cell maturation, regulation of inflammatory processes, and immune response. These results revealed that breed plays an important role in the modulation of genes involved in immune adaptation and inflammatory response, and the immune system of Simmental cows could potentially have a more acute response in early lactation. In turn, this might be beneficial for mounting a more efficient response after calving and allow for a smoother homeorhetic adaptation to lactation.

The role of altered immune function during the dry period in promoting the development of subclinical ketosis in early lactation.

Subclinical ketosis (SCK) may impair white blood cell (WBC) function and thus contribute to the risk of disease postpartum. This preliminary study investigated changes occurring in the immune system before disease onset to elucidate their role in the occurrence of SCK. A group of 13 Holstein dairy cows were housed in tie-stalls and retrospectively divided into 2 groups based on their levels of ß-hydroxybutyrate (BHB) measured in plasma between calving day and 35 d from calving (DFC). Levels of BHB <1.4 mmol/L were found in 7 cows (control cows, CTR group) and levels >1.4 mmol/L were found in 6 cows at ≥1 of 6 time points considered (cows with SCK, KET group). From -48 to 35 DFC, body condition score, body weight, dry matter intake, rumination time, and milk yield were measured, and blood samples were collected regularly to assess the hematochemical profile and test the WBC function by ex vivo challenge assays. Data were submitted for ANOVA testing using a mixed model for repeated measurements that included health status and time and their interactions as fixed effects. Compared with CTR cows, KET cows had more pronounced activation of the immune system (higher plasma concentrations of proinflammatory cytokines, myeloperoxidase, and oxidant species, and greater IFN-γ responses to Mycobacterium avium), higher blood concentrations of γ-glutamyl transferase, and lower plasma concentrations of minerals before calving. Higher levels of nonesterified fatty acids, BHB, and glucose were detected in KET cows than in CTR cows during the dry period. The effect observed during the dry period was associated with a reduced dry matter intake, reduced plasma glucose, and increased fat mobilization (further increases in nonesterified fatty acids and BHB) during early lactation. A reduced milk yield was also detected in KET cows compared with CTR. The KET cows had an accentuated acute-phase response after calving (with greater concentrations of positive acute-phase proteins and lower concentrations of retinol than CTR cows) and impaired liver function (higher blood concentrations of glutamate-oxaloacetate transaminase and bilirubin). The WBC of the KET cows, compared with CTR cows, had a reduced response to an ex vivo stimulation assay, with lower production of proinflammatory cytokines and greater production of lactate. These alterations in the WBC could have been driven by the combined actions of metabolites related to the mobilization of lipids and the occurrence of a transient unresponsive state against stimulation aimed at preventing excessive inflammation. The associations identified here in a small number of cows in one herd should be investigated in larger studies.

Immunometabolic status and productive performance differences between periparturient Simmental and Holstein dairy cows in response to pegbovigrastim.

In the present study, we aimed to investigate the side effects of pegbovigrastim, injected approximately 7 d before parturition and on the day of calving, on a panel of plasma biomarkers to evaluate energy, inflammatory, oxidative, and liver function status. We also addressed treatment responses in different breeds during the transition period. Holstein and Simmental cows were randomly assigned into 2 groups based on expected calving date and according to parity: the treated group (PEG; 14 Holstein and 12 Simmental cows) received pegylated recombinant bovine granulocyte colony stimulating factor (pegbovigrastim, Imrestor; Elanco Animal Health, Greenfield, IN), and the control group (CTR; 14 Holstein and 14 Simmental cows) received saline solution. The PEG or CTR treatments were administered via subcutaneous injection in the scapular region at approximately 7 d (mean 7.80 ± 5.50 d) before expected parturition and within 24 h after calving. Blood samples were collected at -21, -7 (before injection), 1, 3, and 28 d relative to calving. Milk production was recorded at 7, 15, 21, 30, and 42 d. A mixed model with repeated measures was fitted to the normalized data using Proc MIXED of SAS (SAS Institute Inc., Cary, NC). Simmental PEG cows showed higher plasma protein concentrations at 1 and 3 d after calving compared with Simmental CTR and Holstein PEG cows, whereas no differences were detected between Holstein PEG and CTR cows. Albumin was greater at 1 d in Simmental PEG compared with Simmental CTR cows. In contrast, γ-glutamyl transferase was higher overall (across breed) in PEG than in CTR. The PEG group had higher values throughout the postcalving period compared with CTR. Cows treated with pegbovigrastim had also higher alkaline phosphatase (ALP) activity at 1 and 3 d after calving. The Holstein PEG group had higher ALP activity at 3 d compared with the Holstein CTR and Simmental PEG groups, and higher ALP at 1 d compared with the Simmental CTR group. The PEG group had higher levels of IL-6 at 3 and 28 d but higher IL-1ß only at 28 d after calving compared with the CTR group. Overall, Holstein cows were characterized by a greater response in the production of inflammation biomarkers (cytokines, haptoglobin, and ceruloplasmin). In addition, PEG cows had higher values of zinc at 1 and 3 d after calving compared with CTR cows. The response observed in plasma biomarkers for energy metabolism and liver functionality after pegbovigrastim treatment in Simmental and Holstein cows was not different from that in control cows. However, our data shed light on the different metabolic adaptations during the transition period between Simmental and Holstein cows, characterized by different energy, inflammatory, and oxidative pattern responses. For the first time, we have highlighted the effect of pegbovigrastim in maintaining stable cytokine levels during the first month after parturition, reflecting greater regulation of neutrophil recruitment, trafficking, and maturation during the inflammatory response. These results provide evidence of the immunomodulatory action of pegbovigrastim around parturition, when dairy cows are highly immunosuppressed. At the same time, these data demonstrate that increasing release of cytokines after parturition is not linked to exacerbation of a systemic inflammation evaluated based on haptoglobin and ceruloplasmin levels.