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 d in milk. Milk production, DMI (during the periparturient period), and rumination times were recorded daily. At dry-off, compared with CTR, NR decreased milk yield (- 62%) and milk lactose 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, ß-hydroxybutyrate, 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 impacted, 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.

Observational study on the associations between milk yield, composition, and coagulation properties with blood biomarkers of health in Holstein cows.

The considerable increase in the production capacity of individual cows owing to both selective breeding and innovations in the dairy sector has posed challenges to management practices in terms of maintaining the nutritional and metabolic health status of dairy cows. In this observational study, we investigated the associations between milk yield, composition, and technological traits and a set of 21 blood biomarkers related to energy metabolism, liver function or hepatic damage, oxidative stress, and inflammation or innate immunity in a population of 1,369 high-yielding Holstein-Friesian dairy cows. The milk traits investigated in this study included 4 production traits (milk yield, fat yield, protein yield, daily milk energy output), 5 traits related to milk composition (fat, protein, casein, and lactose percentages and urea), 11 milk technological traits (5 milk coagulation properties and 6 curd-firming traits). All milk traits (i.e., production, composition, and technological traits) were analyzed according to a linear mixed model that included the days in milk, the parity order, and the blood metabolites (tested one at a time) as fixed effects and the herd and date of sampling as random effects. Our findings revealed that milk yield and daily milk energy output were positively and linearly associated with total cholesterol, nonesterified fatty acids, urea, aspartate aminotransferase, γ-glutamyl transferase, total bilirubin, albumin, and ferric-reducing antioxidant power, whereas they were negatively associated with glucose, creatinine, alkaline phosphatase, total reactive oxygen metabolites, and proinflammatory proteins (ceruloplasmin, haptoglobin, and myeloperoxidase). Regarding composition traits, the protein percentage was negatively associated with nonesterified fatty acids and ß-hydroxybutyrate (BHB), while the fat percentage was positively associated with BHB, and negatively associated with paraoxonase. Moreover, we found that the lactose percentage increased with increasing cholesterol and albumin and decreased with increasing ceruloplasmin, haptoglobin, and myeloperoxidase. Milk urea increased with an increase in cholesterol, blood urea, nonesterified fatty acids, and BHB, and decreased with an increase in proinflammatory proteins. Finally, no association was found between the blood metabolites and milk coagulation properties and curd-firming traits. In conclusion, this study showed that variations in blood metabolites had strong associations with milk productivity traits, the lactose percentage, and milk urea, but no relationships with technological traits of milk. Specifically, increasing levels of proinflammatory and oxidative stress metabolites, such as ceruloplasmin, haptoglobin, myeloperoxidase, and total reactive oxygen metabolites, were shown to be associated with reductions in milk yield, daily milk energy output, lactose percentage, and milk urea. These results highlight the close connection between the metabolic and innate immunity status and production performance. This connection is not limited to specific clinical diseases or to the transition phase but manifests throughout the entire lactation. These outcomes emphasize the importance of identifying cows with subacute inflammatory and oxidative stress as a means of reducing metabolic impairments and avoiding milk fluctuations.

Blood biochemical changes upon subclinical intramammary infection and inflammation in Holstein cattle.

The aim of this study was to investigate the associations between subclinical intramammary infection (IMI) from different pathogens combined with inflammation status and a set of blood biochemical traits including energy-related metabolites, indicators of liver function or hepatic damage, oxidative stress, inflammation, innate immunity, and mineral status in 349 lactating Holstein cows. Data were analyzed with a linear model including the following fixed class effects: days in milk, parity, herd, somatic cell count (SCC), bacteriological status (positive and negative), and the SCC × bacteriological status interaction. Several metabolites had significant associations with subclinical IMI or SCC. Increased SCC was associated with a linear decrease in cholesterol concentrations which ranged from -2% for the class ≥50,000 and <200,000 cells/mL to -11% for the SCC class ≥400,000 cells/mL compared with the SCC class <50,000 cells/mL. A positive bacteriological result was associated with an increase in bilirubin (+24%), paraoxonase (+11%), the ratio paraoxonase/cholesterol (+9%), and advanced oxidation protein product concentration (+23%). Increased SCC were associated with a linear decrease in ferric reducing antioxidant power concentrations ranging from -3% for the class ≥50,000 and <200,000 cells/mL to -9% for the SCC class ≥400,000 cells/mL (respect to the SCC class <50,000 cells/mL). A positive bacteriological result was associated with an increase in haptoglobin concentrations (+19%). Increased SCC were also associated with a linear increase in haptoglobin concentrations, which ranged from +24% for the class ≥50,000 and <200,000 cells/mL (0.31 g/L) to +82% for the SCC class ≥400,000 cells/mL (0.45 g/L), with respect to the SCC class <50,000 cells/mL (0.25 g/L). Increased SCC were associated with a linear increase in ceruloplasmin concentrations (+15% for SCC ≥50,000 cells/mL). The observed changes in blood biochemical markers, mainly acute phase proteins and oxidative stress markers, suggest that cows with subclinical IMI may experience a systemic involvement.

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.

Associations between ultrasound hepatic measurements, body measures, and milk production traits in Holstein cows.

Ultrasound (US) imaging has been proposed as a noninvasive tool for monitoring liver dysfunction in dairy cows. This study, carried out on 306 clinically healthy Holstein cows in the first 120 d of lactation kept in 2 herds in northern Italy, aimed at investigating the association between US imaging-derived traits, namely predicted liver triacylglycerol content (pTAG, mg/g), liver depth (LD, mm), portal vein depth (PVD, mm) and area (PVA, mm2), and body size measurements, body condition score (BCS), and milk productivity indicators. Transcutaneous US examination, milk sampling, body size measurements (withers height and heart girth), and BCS were collected once from all cows in 10 sampling batches. The body weights (BW) of a subsample of 73 cows were recorded and used together with an existing data set of BW and measures of Holstein Friesian cows (n = 399) to develop a regression equation to predict BW, which was then used to compute productivity indicators by scaling the milk production traits to predicted BW. Body size measures, BCS, milk traits, and productivity indicators were classified (low, medium, and high) in 0.75 units of standard deviation of the residuals generated from a linear model that included the effects of parity, days in milk, and sampling batch. Liver pTAG, PVA, PVD, and LD were analyzed with a sequence of linear mixed models that included the fixed effects of days in milk and parity and the random effect of sampling batch as common terms, whereas the classes of body and milk traits and the productivity indicators were included one by one. The US-related traits were found to be associated with body size measurements and BCS. Specifically, pTAG was inversely related to BCS, whereas PVD and LD increased with increasing heart girth, BCS, and predicted BW. Generally, no relevant associations were observed between the US parameters and milk production traits, including when expressed in terms of productivity. In conclusion, this study suggests that US measures of liver dimensions of clinically healthy cows are related to their size, whereas pTAG concentrations reflect body condition status, with no particular implications for milk production and productivity. Moreover, healthy cows seemed able to counteract the metabolic stress of the first 120 d of the lactation period without straining liver functionality. Finally, US imaging proved to be a promising technique to assess liver metabolic conditions. However, further studies are needed to confirm its potential as a noninvasive tool for monitoring liver conditions in healthy cows.

Quarter-level analyses of the associations among subclinical intramammary infection and milk quality, udder health, and cheesemaking traits in Holstein cows.

In this study, we investigated associations among subclinical intra-mammary infection (IMI) and quarter-level milk composition, udder health indicators, and cheesemaking traits. The dataset included records from 450 Holstein cows belonging to three dairy herds. After an initial screening (T0) to identify animals infected by Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, and Prototheca spp., 613 quarter milk samples for 2 different sampling times (T1 and T2, 1 mo after T1) were used for analysis. Milk traits were analyzed using a hierarchical linear mixed model including the effects of days in milk, parity and herd, and bacteriological and inflammatory category [culture negative with somatic cell count (SCC) <200,000 cells/mL; culture negative with SCC ≥200,000 cells/mL; or culture positive]. All udder health indicators were associated with increased SCC and IMI at both sampling times. The largest effects were detected at T2 for milk lactose (-7% and -5%) and milk conductivity (+9% and +8%). In contrast, the increase in differential SCC (DSCC) in samples with elevated SCC was larger at T1 (+17%). Culture-negative samples with SCC ≥200,000 cells/mL had the highest SCC and greatest numbers of polymorphonuclear-neutrophils-lymphocytes and macrophages at both T1 and T2. Regarding milk cheesemaking ability, samples with elevated SCC showed the worst pattern of curd firmness at T1 and T2. At T2, increased SCC and IMI induced large decreases in recoveries of nutrients into the curd, in particular recovered protein (-14% and -16%) and recovered fat (-12% and -14%). Different behaviors were observed between Strep. agalactiae and Prototheca spp., especially at T2. In particular, samples that were positive for Strep. agalactiae had higher proportions of DSCC (+19%) compared with negative samples with low SCC, whereas samples that were positive for Prototheca spp. had lower DSCC (-11%). Intramammary infection with Prototheca spp. increased milk pH compared with culture-negative samples (+3%) and negative samples that had increased SCC (+2%). The greatest impairment in curd firmness at 30 min from rennet addition was observed for samples that were positive for Prototheca spp. (-99% compared with negative samples, and -98% compared with negative samples with high SCC). These results suggest that IMI caused by Prototheca spp. have detrimental effects on milk technological traits that deserve further investigation of the mechanisms underlying animals' responses to infection.

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.

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.

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.

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.

Circulating amino acids in blood plasma during the peripartal period in dairy cows with different liver functionality index.

The liver functionality index (LFI) measures the changes of albumin, cholesterol, and bilirubin concentrations between 3 and 28 d postpartum. This composite index, based on variables with direct relevance to liver-specific plasma protein synthesis (albumin), hepatic/intestinal lipoprotein synthesis (cholesterol), and clearance of breakdown products of heme catabolism (bilirubin), provides a tool for evaluating manifestations of hepatic disease. Both energy and protein metabolism are likely to be affected by various physiological challenges in this period but have not been tested systematically. The present study was conducted to profile AA in cows with high or low LFI during the peripartal period and relate this to production outcomes. Eighteen multiparous cows were used from -21 through 28 d around parturition and divided retrospectively into the high or low LFI group. Blood samples were obtained on -21, -14, -7, 1, 3, 7, 10, 14, 17, 21, and 28 d relative to calving, and biomarkers and AA in plasma were measured. Grouping based on LFI resulted in 8 cows with high LFI (HLFI) and 10 cows with low LFI (LLFI). Although the temporal response in dry matter intake (DMI, 16.3 kg/d) and body condition score (2.56) did not differ, cows with high compared with low LFI had greater overall milk production (37.9 vs. 32.9 kg/d) although energy-corrected milk yield did not differ (42.6 vs. 38.7 kg/d). As expected, cows grouped as LLFI had lower cholesterol and albumin but greater bilirubin after calving compared with HLFI animals. Despite similar temporal responses in DMI between groups, concentrations of total AA were greater in HLFI, particularly after calving. Although concentrations of total essential AA (EAA) and branched-chain AA did not differ with LFI status, cows in HLFI had greater concentrations of Thr and Ile postpartum. Nearly all plasma AA concentrations followed the general trend of a nadir at 1d after calving followed by a gradual increase to prepartal levels before 28 d. Glycine was the only AA exhibiting a gradual increase in concentration through the transition, with a maximum at 7d postpartum followed by a gradual decrease. We detected no effect of LFI status on plasma Lys, which decreased markedly from -21d to calving, followed by an increase to prepartal values by d7. In contrast, concentrations of Met and His decreased markedly between -21 and 10d and did not reach prepartal values by 28 d. The marked decrease in Gln concentration after calving regardless of LFI might compromise immune function during this period. Overall, the results indicate the existence of an association among inflammation, liver function postpartum, and AA plasma concentrations, irrespective of temporal differences in DMI. Cows with better indices of liver function produced more milk and maintained greater concentrations of total AA and some EAA such as Thr and Ile. Whether these AA played a direct role in the greater milk production remains to be determined.

Effect of summer season on milk protein fractions in Holstein cows.

Milk characteristics are affected by heat stress, but very little information is available on changes of milk protein fractions and their relationship with cheesemaking properties of milk. The main objective of the study was to evaluate the effect of hot season on milk protein fractions and cheesemaking properties of milk for Grana Padano cheese production. The study was carried out in a dairy farm with a cheese factory for transforming the milk to Grana Padano cheese. The study was carried out from June 2012 to May 2013. Temperature and relative humidity of the inside barn were recorded daily during the study period using 8 electronic data loggers programmed to record every 30 min. Constant managerial conditions were maintained during the experimental periods. During the experimental period, feed and diet characteristics, milk yield, and milk characteristics were recorded in summer (from June 29 to July 27, 2012), winter (from January 25 to March 8, 2013), and spring (from May 17 to May 31, 2013). Milk yield was recorded and individual milk samples were taken from 25 cows selected in each season during the p.m. milking. Content of fat, proteins, caseins (CN), lactose and somatic cell count (SCC), titratable acidity, and milk rennet coagulation properties were determined on fresh samples. Milk protein fraction concentrations were determined by the sodium dodecyl sulfate-PAGE. Data were tested for nonnormality by the Shapiro-Wilk test. In case of nonnormality, parameters were normalized by log or exponential transformation. The data were analyzed with repeated measures ANOVA using a mixed model procedure. For all the main milk components (fat, protein, total solids, and solids-not-fat), the lowest values were observed in the summer and the greatest values were observed in the winter. Casein fractions, with the exception of γ-CN, showed the lowest values in the summer and the greatest values in the winter. The content of IgG and serum albumin was greater in summer than in the winter and spring. A mild effect of season was observed for milk SCC, with greater values in summer than in the winter and spring. A worsening of milk coagulation properties was observed in summer season. The alteration of cheesemaking properties during hot season seems strictly linked with changes of milk protein fractions mainly with the decrease of αS-CN and ß-CN and the increase of undefined proteins.

Metabolic and biochemical changes in plasma of the periparturient rabbit does with different litter size.

The aim of this study was to investigate the metabolic and biochemical changes in plasma that occur in the reproductive rabbit doe close to the parturition, as well as if the number of offspring affects the metabolism and the health status of the doe. At -3, 4 and 12 days from parturition (-D3, D4, and D12, respectively) nine rabbit does at their third parity from a commercial hybrid line (HYPLUS PS 19) selected for high prolificacy were weighted and blood was collected for a wide inflammometabolic profile. According to the number of offspring the does were retrospectively divided in two groups: high litter size group (HI; n=5) and low litter size group (LO; n=4). BW was higher (P<0.01) at -D3 and had the lowest values at D4. At D12, the BW was lower (P<0.05) in LO compared with HI. Several metabolites significantly changed from dry to lactation period. Glucose and cholesterol had the lowest levels at -D3; non-esterified fatty acid (NEFA) and aspartate aminotransferase had the highest values before parturition (P<0.05); creatinine and ß-hydroxybutyrate (BHBA) were higher at -D3 with respect to D4 (P<0.05). The lowest value of paraoxonase was observed in does at -D3 (P<0.05), whereas at this time ceruloplasmin and total bilirubin had the highest concentration (P<0.05). The differences for blood profile parameters between does grouped according to litter size were mainly evident before parturition (-D3). In particular, BHBA, NEFA and total bilirubin had higher concentrations (P<0.05) in HI v. LO group, whereas albumin and PON were lower in HI group (P<0.01). After parturition there were no significant differences for the metabolic parameters between the two groups. The results show that for reproductive rabbit doe the last days of gestation are very stressful from a metabolic and inflammatory point of view. The genetic selection of does for higher litter size has increased their ability to mobilize body reserves in order to guarantee the nutrients to a high number of kits. This exposes them to a more severe metabolic and inflammatory challenge during the transition period. Consequently, feeding and managerial strategies for high prolificacy periparturient rabbit does should be revised.

Effect of dietary starch level and high rumen-undegradable protein on endocrine-metabolic status, milk yield, and milk composition in dairy cows during early and late lactation.

Diet composition defines the amount and type of nutrients absorbed by dairy cows. Endocrine-metabolic interactions can influence these parameters, and so nutrient availability for the mammary gland can significantly vary and affect milk yield and its composition. Six dairy cows in early and then late lactation received, for 28 d in a changeover design, 2 diets designed to provide, within the same stage of lactation, similar amounts of rumen fermentable material but either high starch plus sugar (HS) content or low starch plus sugar content (LS). All diets had similar dietary crude protein and calculated supply of essential amino acids. Dry matter intake within each stage of lactation was similar between groups. Milk yield was similar between groups in early lactation, whereas a higher milk yield was observed in late lactation when feeding HS. At the metabolic level, the main difference observed between the diets in both stages of lactation was lower blood glucose in cows fed LS. The lower glucose availability during consumption of LS caused substantial modifications in the circulating and postprandial pattern of metabolic hormones. Feeding LS versus HS resulted in an increase in the ratio of bovine somatotropin to insulin. This increased mobilization of lipid reserves resulted in higher blood concentrations of nonesterified fatty acids and ß-hydroxybutyrate, which contributed to the higher milk fat content in both stages of lactation in the LS group. This greater recourse to body fat stores was confirmed by the greater loss of body weight during early lactation and the slower recovery of body weight in late lactation in cows fed LS. The lower insulin to glucagon ratio observed in cows fed LS in early and late lactation likely caused an increase in hepatic uptake and catabolism of amino acids, as confirmed by the higher blood urea concentrations. Despite the higher catabolism of amino acids in LS in early lactation, similar milk protein output was observed for both diets, suggesting similar availability of amino acids for peripheral tissue and mammary gland. The latter could be the result of sparing of amino acids at the gut level due to starch that escaped from the rumen, and to the balanced amino acid profile of digestible protein. This last aspect appears worthy of further research, with the aim to enhance the efficiency of protein metabolism of dairy cows, reducing environmental nitrogen pollution without affecting milk yield potential.

Experimental acute rumen acidosis in sheep: consequences on clinical, rumen, and gastrointestinal permeability conditions and blood chemistry.

Acute acidosis was induced in sheep, and gastrointestinal permeability was assessed by using lactulose as a permeability marker. Metabolism was evaluated by monitoring blood metabolites. Four rams (72.5 ± 4.6 kg BW) were used in a 2 × 2 changeover design experiment. The experimental period lasted 96 h from -24 to 72 h. After 24 h of fasting (from -24 to 0 h) for both controls and acidosis-induced rams (ACID), 0.5 kg of wheat flour was orally dosed at 0 and 12 h of the experimental period to ACID, while the basal diet (grass hay, ad libitum) was restored to control. At 24 h, a lactulose solution (30 g of lactulose in 200 mL of water) was orally administered. Blood samples were collected at -24, 0, 24, 48, and 72 h of the experimental periods for the analysis of metabolic profiles and during the 10 h after lactulose dosage to monitor lactulose changes in blood. In addition, rumen and fecal samples were collected at 24 h of the experimental period. The acidotic challenge markedly reduced (P < 0.01) rumen pH and VFA but increased rumen d- and l-lactic acid (P < 0.01). Concurrently, a decrease of fecal pH and VFA occurred in ACID (P < 0.01), together with an abrupt increase (P < 0.01) of lactate and fecal alkaline phosphatase. Blood lactulose was significantly increased in ACID peaking 2 h after lactulose dosage. Blood glucose, ß-hydroxybutyrate, Ca, K, Mg, and alkaline phosphatase showed a significant reduction (P < 0.05) at 24 h, whereas urea and NEFA declined (P < 0.05) from 48 to 72 h. A strong inflammatory acute phase response with oxidative stress in ACID group was observed from 24 to 72 h; higher values of haptoglobin (P < 0.01) were measured from 24 to 72 h and of ceruloplasmin from 48 (P < 0.05) to 72 h (P < 0.01). Among the negative acute phase reactants, plasma albumin, cholesterol, paraoxonase, and Zn concentration also decreased (P < 0.05) in ACID at different time points between 24 and 72 h after acidotic challenge start. A rise (P < 0.05) of reactive oxygen metabolites and a drop of vitamin E (P < 0.01) between 24 and 72 h were indicative of oxidative stress in ACID. The perturbation of these blood metabolites suggests that acute acidosis was effectively induced by our model. The increase of lactulose in blood in ACID indicates that gastrointestinal permeability for the marker increased and the large increment after 2 h from dosage suggests that most of the passage occurred through the rumen or abomasal walls.

Assessment of gastrointestinal permeability by lactulose test in sheep after repeated indomethacin treatment.

The aim of the study was to assess the small intestine permeability by using lactulose as a sugar probe and blood metabolites in sheep after a challenge with repeated indomethacin injections. According to a changeover design, 7 adult sheep (4 males and 3 females) were subjected to 4 intramuscular injections (every 12 h) of saline [control (CRT); 7 animals] or indomethacin (INDO; 7 animals). Two hours after the last injection, 30 g of lactulose were administered orally to both CTR and INDO. Blood samples were collected daily for the analysis of the metabolic profile and 5 samples were collected at 2-h intervals following lactulose ingestion to monitor changes in blood levels of lactulose as an index of intestinal permeability. The INDO challenge induced clinical symptoms such as lack of appetite, dullness, weakness, depression, and diarrhea with traces of blood in the feces. In INDO group, haptoglobin and ceruloplasmin increased (P < 0.01) after INDO challenge whereas a decrease (P < 0.05) of negative acute phase reactants (e.g., cholesterol, albumin, and paraoxonase) was observed. Reactive oxygen metabolites increased (P < 0.01) from 60 to 204 h after the INDO challenge start, with a decrease of vitamin E concentration from 12 (P < 0.01) to 132 h (P < 0.05). Blood lactulose concentrations were increased (P < 0.05) in INDO animals and the highest mean values (17.67 µg/mL in INDO vs. 0.17 µg/mL in CRT; P < 0.01) were observed 6 h after oral dosage of lactulose. These changes indicate that the INDO challenge led to severe inflammatory responses with oxidative stress by enhancing small intestinal permeability in sheep that allowed lactulose to enter in blood. The results of this experiment demonstrate that lactulose can be used as a probe to assess gastrointestinal permeability in adult ruminants to test the consequences of stressing conditions on animal welfare. For this purpose, the most suitable time for blood sampling is between 2 and 8 h after the oral dosage of lactulose.