Search

Precalving feeding level and body condition score (BCS) alter postcalving energy balance and oxidant status of dairy cows. We hypothesized that the reported benefits of a controlled restriction precalving depend on precalving BCS. The objective was to identify alterations in activity and intermediates of the hepatic one-carbon metabolism, transsulfuration, and tricarboxylic acid pathways. Twenty-eight pregnant and nonlactating grazing dairy cows of mixed age and breed (Friesian, Friesian × Jersey) were randomly allocated to 1 of 4 treatment groups in a 2 × 2 factorial design: 2 prepartum BCS categories [4.0 (thin, BCS4) and 5.0 (optimal, BCS5); 10-point scale], by managing cows in late lactation to achieve the 2 groups at dry-off, and 2 levels of energy intake during the 3 wk preceding calving (75 or 125% of estimated requirements), obtained via allowance (m2/cow) of fresh pasture composed of mostly perennial ryegrass and white cover. Average (± standard deviation) age was 6 ± 2, 6 ± 3, 5 ± 1, and 7 ± 3 yr for BCS4 fed 75 and 125%, and BCS5 fed 75 and 125%, respectively. Breed distribution (average ± standard deviation) for the 4 groups was 79 ± 21, 92 ± 11, 87 ± 31, and 74 ± 23% Friesian, and 17 ± 20, 8 ± 11, 13 ± 31, and 25 ± 23% Jersey. Liver tissue was collected by biopsy at -7, 7, and 28 d relative to calving. Tissue was used for 14C radio-labeling assays to measure betaine-homocysteine S-methyltransferase, 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and cystathionine-β-synthase (CBS) activity. Liver metabolomics was undertaken using a targeted liquid chromatography with tandem mass spectrometry-based profiling approach. After initial liquid chromatography separation, mass spectra were acquired under both positive and negative ionization, whereas multiple reaction monitoring was used to measure target compound signal response (peak area count). Enzyme activity and metabolite peak area count were normalized with the homogenate protein concentration. Repeated measures analysis of variance via PROC MIXED in SAS (SAS Institute Inc., Cary, NC), with BCS, feeding, and time as fixed effects, and cow as random effect was used. All enzyme activities were affected by time, with betaine-homocysteine S-methyltransferase activity peaking at 7 d, whereas CBS and MTR activity decreased postpartum. Overall, thin cows had greater MTR activity, whereas cows fed 125% requirements had greater CBS activity. An interaction was detected between BCS and feeding for CBS activity, as thin cows fed 125% of requirements had greater overall activity. Compared with liver from BCS4 cows, BCS5 cows had overall greater betaine, glycine, butyrobetaine/acetylcholine, serine, and taurine concentrations. The same metabolites, plus choline and N-N-dimethylglycine, were overall greater in liver of cows fed 75% compared with those fed 125% of requirements. An interaction of BCS and feeding level was detected for the aforementioned metabolites plus methionine, cystathionine, cysteinesulfinate, and hypotaurine, due to greater overall concentrations in BCS5 cows fed 75% of requirements compared with other groups. Overall, differences in hepatic enzyme activity and intermediate metabolites suggest that both BCS and feeding level can alter the internal antioxidant system (e.g., glutathione and taurine) throughout the periparturient period. Further studies are needed to better understand potential mechanisms involved.

Reverse-transcription quantitative-PCR (RT-qPCR) is commonly used for assessing the cellular response to changes in physiologic and pathologic conditions. The selection of stable endogenous control genes is an important step of any RT-qPCR study, as expression can vary depending on the experimental environment. Our objective was to identify endogenous control genes in circulating neutrophils isolated from cows during the peripartum period. To do this, we used microfluidics gene expression arrays (Fluidigm, San Francisco, CA) for RT-qPCR analysis. Selection of the endogenous control genes was based on previous research investigating gene expression in neutrophils. The selected genes included ACTB, B2M, G6PD, GAPDH, GCH1, GOLGA5, OSBPL2, PGK1, RPL13A, RPL19, RPS9, SDHA, SMUG1, SNRPA, TBP, UXT, and YWHAZ. Four genes (GAPDH, GOLGA5, PGK1, and UXT) did not provide satisfactory quantification results using the selected method and were therefore excluded from the analyses. The suitability of the remaining 13 genes for use as endogenous control genes was assessed using geNorm and Normfinder. The gene pair with the greatest stability using geNorm was RPL13A and RPL19, whereas Normfinder ranked RPL19 and YWHAZ as the most stable pair. The 2 genes deemed most suitable for the experimental design were RPL19 and YWHAZ, which were selected for subsequent gene expression analysis. This study highlights that genes used as endogenous controls for relative quantification should be assessed on an experimental basis, even if the genes have been used in previous research.

The common practice of increasing dietary energy density during the close-up dry period (last ∼3 wk prepartum) has been recently associated with a higher incidence of metabolic disorders after calving. Despite these reports, over-feeding of metabolizable energy (ME) during the far-off, nonlactating period is a common management policy aimed at achieving optimum calving body condition score (BCS) in pasture-based systems, as cows are generally thinner than total mixed ration cows at the end of lactation. Our hypothesis was that both far-off and close-up overfeeding influence the peripartum adipose tissue changes associated with energy balance and inflammatory state. Sixty mid-lactation, grazing dairy cows of mixed age and breed were randomly allocated to 1 of 2 groups that were managed through late lactation to achieve a low and high BCS (approximately 4.25 and 5.0 on a 10-point scale) at dry-off. The low BCS cows were then overfed ME to ensure that they achieved the same BCS as the higher BCS group by calving. Within each rate of BCS gain treatment, cows were offered 65, 90, or 120% of their pre-calving ME requirements for 3 wk pre-calving in a 2 × 3 factorial arrangement of treatments (i.e., 10 cows/treatment). Subcutaneous adipose tissue was collected via biopsy at -1, 1, and 4 wk relative to parturition. Quantitative PCR was used to measure mRNA and microRNA expression of targets related to adipogenesis and inflammation. Cows overfed in the far-off period had increased expression of miR-143 and miR-378 prepartum (-1 wk) indicating greater adipogenesis, consistent with their rapid gain in BCS following dry-off. Furthermore, the lower postpartum expression of IL6, TNF, TLR4, TLR9, and miR-145, and a higher abundance of miR-99a indicated lower body fat mobilization in early lactation in the same group. In the close-up period, feeding either 65 or 120% of ME requirements caused changes in FASN, IL1B, IL6R, TLR9, and the microRNA miR-143, miR-155, and miR-378. Their respective expression patterns indicate a tentative negative-feedback mechanism in metabolically compromised, feed-restricted cows, and a possible immune-related stimulation of lipolysis in apparently static adipocytes in overfed cows. Data from cows fed 90% of ME requirements indicate the existence of a balance between lipolytic (inflammatory-related) and anti-lipolytic signals, to prime the mobilization machinery in light of imminent lactation. Overall, results indicate that far-off dry cow nutrition influences peripartum adipose tissue metabolism, with neither strategy negatively affecting the physiological adaptation to lactation. Furthermore, to ensure a favorable transition, cows should be subjected to a small feed restriction in the close-up period, irrespective of far-off nutritional management.

Extensive metabolic, physiological, and immunological changes are associated with calving and the onset of lactation. As a result, cows transitioning between pregnancy and lactation are at a greater risk of metabolic and infectious diseases. The ability of neutrophils to mount an effective immune response to an infection is critical for its resolution, and increasing evidence indicates that precalving nutrition affects postpartum neutrophil function. The objectives of the current study were to investigate the effect of 2 precalving body condition scores (BCS; 4 vs. 5 on a 10-point scale) and 2 levels of feeding (75 vs. 125% of estimated maintenance requirements) on gene expression in circulating neutrophils. We isolated RNA from the neutrophils of cows (n = 45) at 5 time points over the transition period: precalving (-1 wk), day of calving (d 0), and postcalving at wk 1, 2, and 4. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 dynamic array), were used to quantify the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using repeated measures ANOVA with Tukey-Kramer multiple-testing correction to determine treatment effects among weeks. Precalving BCS altered the inflammatory state of neutrophils, with significant increases in overall gene expression of antimicrobial peptides (BNBD4 and DEFB10) and the anti-inflammatory cytokine IL10, and significantly decreased expression of proinflammatory cytokine IL23A in thinner cows (BCS 4) compared with cows calving at BCS 5. Feeding level had a time-dependent effect on gene expression; for example, increased expression of genes involved in leukotriene synthesis (PLA2G4A and ALOX5AP) occurred only at 1 wk postcalving in cows overfed (125% of requirements) precalving compared with those offered 75% of maintenance requirements. Results indicate that precalving body condition and changes in prepartum energy lead to altered gene expression of circulating neutrophils, highlighting the importance of transition cow nutrition for peripartum health.

Biomarkers that identify prepathological disease could enhance preventive management, improve animal health and productivity, and reduce costs. Circulating extracellular vesicles, particularly exosomes, are considered to be long-distance, intercellular communication systems in human medicine. Exosomes provide tissue-specific messages of functional state and can alter the cellular activity of recipient tissues through their protein and microRNA content. We hypothesized that exosomes circulating in the blood of cows during early lactation would contain proteins representative of the metabolic state of important tissues, such as liver, which play integral roles in regulating the physiology of cows postpartum. From a total of 150 cows of known metabolic phenotype, 10 cows were selected with high (n = 5; high risk) and low (n = 5; low risk) concentrations of nonesterified fatty acids, β-hydroxybutyrate, and liver triacylglycerol during wk 1 and 2 after calving. Exosomes were extracted from blood on the day of calving (d 0) and postcalving at wk 1 and wk 4, and their protein composition was determined by mass spectroscopy. Extracellular vesicle protein concentration and the number of exosome vesicles were not affected by risk category; however, the exosome protein cargo differed between the groups, with proteins at each time point identified as being unique to the high- and low-risk groups. The proteins α-2 macroglobulin, fibrinogen, and oncoprotein-induced transcript 3 were unique to the high-risk cows on d 0 and have been associated with metabolic syndrome and liver function in humans. Their presence may indicate a more severe inflammatory state and a greater degree of liver dysfunction in the high-risk cows than in the low-risk cows, consistent with the high-risk cows’ greater plasma β-hydroxybutyrate and liver triacylglycerol concentrations. The commonly shared proteins and those unique to the low-risk category indicate a role for exosomes in immune function. The data provide preliminary evidence of a potential role for exosomes in the immune function in transition dairy cows and exosomal protein cargo as biomarkers of metabolic state.

Extensive metabolic and physiologic changes occur during the peripartum, concurrent with a high incidence of infectious disease. Immune dysfunction is a likely contributor to the increased risk of disease at this time. Studies using high-yielding, total mixed ration-fed cows have indicated that neutrophil function is perturbed over the transition period; however, this reported dysfunction has yet to be investigated in moderate-yielding, grazing dairy cows. Therefore, we investigated changes in the expression of genes involved in neutrophil function. Blood was collected from cows at 5 time points over the transition period: precalving (−1 wk; n = 46), day of calving (d 0; n = 46), and postcalving at wk 1 (n = 46), wk 2 (n = 45), and wk 4 (n = 43). Neutrophils were isolated by differential centrifugation and gene expression was investigated. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 Dynamic Array, San Francisco, CA) were used to investigate the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using a repeated measures ANOVA. Genes that were differentially expressed over the transition period included those involved in neutrophil adhesion (SELL, ITGB2, and ITGBX), mediation of the immune response (TLR4, HLA-DRA, and CXCR2), maturation, cell cycle progression, apoptosis (MCL1, BCL2, FASLG, and RIPK1), and control of gene expression (PPARG, PPARD, and STAT3). We noted reduced gene expression of proinflammatory cytokines (IFNG, TNF, IL12, and CCL2) on the day of calving, whereas anti-inflammatory cytokine gene expression (IL10) was upregulated. Increased gene expression of antimicrobial peptides (BNBD4, DEFB10, and DEFB1) occurred on the day of calving. Collectively, transcription profiles are indicative of functional changes in neutrophils of grazing dairy cows over the transition period and align with studies in cows of conventional total mixed ration systems. This altered function may predispose cows to disease over the transition period and is likely to be a natural change in function due to parturition.

Precalving feeding level affects dry matter intake, postcalving energy balance, the risk of hepatic lipidosis and metabolic disease, and gene expression in liver and adipose tissue. These coincide with a higher risk of disease postpartum and, very likely, a failure to reach optimum production as well as reproductive targets. Current interpretation of the available evidence suggest that metabolic stressors affect the immune system of transition dairy cows and lead to reduced immunocompetence. The objective of the current study was to investigate the effect of precalving body condition score (BCS) and level of feeding on immunocompetence during the peripartum period. Twenty-three weeks before calving, 78 cows were allocated randomly to 1 of 6 treatment groups (n = 13) in a 2 × 3 factorial arrangement: 2 precalving BCS categories (4.0 and 5.0, based on a 10-point scale) and 3 levels of energy intake during the 3 wk preceding calving (75, 100, and 125% of estimated requirements). Blood was sampled precalving and at 1, 2 and 4 wk after calving. Cells were analyzed by flow cytometry and quantitative real-time PCR. The numbers of T helper lymphocytes (CD4+), cytotoxic T lymphocytes (CD8+), natural killer cells (CD335+), and γδ T lymphocytes (WC1+) as well as their activation status [IL-2 receptor (CD25)+ cells] were highly variable between animals, but there was no evident effect of BCS, feeding level, or time. All groups presented with an increase in expression of cytokines in unstimulated blood cells in the week after calving, although this was significant only for IFNG in the BCS 4.0 group. Analysis of in vitro-stimulated cells allowed 2 general observations: (1) cows with high energy intake precalving (125%) had increased cytokine expression precalving, and (2) all cows had increased cytokine expression in the week after calving. The present study provides evidence that prepartum feed management can affect immunocompetence during the transition period. Considering the current results, optimally conditioned animals might benefit from a restricted precalving diet, whereas underconditioned cows can be fed to requirements.

Recent studies demonstrating a higher incidence of metabolic disorders after calving have challenged the management practice of increasing dietary energy density during the last ~3 wk prepartum. Despite our knowledge at the whole-animal level, the tissue-level mechanisms that are altered in response to feeding management prepartum remain unclear. Our hypothesis was that prepartum body condition score (BCS), in combination with feeding management, plays a central role in the peripartum changes associated with energy balance and inflammatory state. Twenty-eight mid-lactation grazing dairy cows of mixed age and breed were randomly allocated to 1 of 4 treatment groups in a 2 × 2 factorial arrangement: 2 prepartum BCS categories (4.0 and 5.0, based on a 10-point scale; BCS4, BCS5) obtained via differential feeding management during late-lactation, and 2 levels of energy intake during the 3 wk preceding calving (75 and 125% of estimated requirements). Subcutaneous adipose tissue was harvested via biopsy at -1, 1, and 4 wk relative to parturition. Quantitative polymerase chain reaction was used to measure mRNA and microRNA (miRNA) expression of targets related to fatty acid metabolism (lipogenesis, lipolysis), adipokine synthesis, and inflammation. Both prepartum BCS and feeding management had a significant effect on mRNA and miRNA expression throughout the peripartum period. Overfed BCS5 cows had the greatest prepartum expression of fatty acid synthase (FASN) and an overall greater expression of leptin (LEP); BCS5 was also associated with greater overall adiponectin (ADIPOQ) and peroxisome proliferator-activated receptor gamma (PPARG), whereas overfeeding upregulated expression of proadipogenic miRNA. Higher postpartum expression of chemokine ligand 5 (CCL5) and the cytokines interleukin 6 (IL6) and tumor necrosis factor (TNF) was detected in overfed BCS5 cows. Feed-restricted BCS4 cows had the highest overall interleukin 1 (IL1B) expression. Prepartum feed restriction resulted in greater chemokine ligand 2 (CCL2) expression. Overall, changes in mRNA expression were consistent with the expression pattern of inflammation-related miRNA. These data shed light on molecular mechanisms underlying the effect of prepartum BCS and feeding management on metabolic and inflammatory status of adipose tissue during the peripartum period. Data support the use of a controlled feed restriction prepartum in optimally conditioned cows, as well as the use of a higher level of dietary energy in under-conditioned cows.

Precalving feeding level alters postcalving energy balance, dry matter intake, the liver and adipose tissue transcriptome, hepatic lipidosis, and the risk of metabolic diseases in both high-production cows consuming total mixed rations and moderate-production cows grazing pasture. We hypothesized that the reported benefits of a controlled restriction before calving are dependent on precalving body condition score (BCS): low BCS animals would not benefit from reduced feeding levels precalving, but high BCS cows would have metabolic and immunomodulatory profiles indicative of an improved health status. One hundred sixty-one days before calving, 150 cows were allocated randomly to 1 of 6 treatment groups (n = 25) in a 2 × 3 factorial arrangement: 2 precalving BCS categories (4.0 and 5.0; based on a 10-point scale: BCS4 and BCS5, respectively) and 3 levels of energy intake during the 3 wk preceding calving (75, 100, and 125% of estimated requirements). Cows in the BCS4 and BCS5 groups were managed through late lactation to ensure that target calving BCS was achieved at dry off. Cows were then fed to maintain this BCS target until 3 wk before expected calving date, at which point they were managed within their allotted precalving energy intake treatments by offering different allowances of fresh pasture/cow per day. Milk production, body weight, and BCS were measured weekly; blood was sampled weekly before and after calving and on d 0, 1, 2, 3, and 4 relative to calving. Aspirated plasma was assayed for nonesterified fatty acids, β-hydroxybutyrate, total protein, albumin, cholesterol, haptoglobin, IL-1β, IL-6, total antioxidant capacity, and reactive oxygen species. Liver was sampled wk 1, 2, and 4 postcalving for triacylglycerol analysis. Results confirm that precalving BCS and precalving feeding level have both independent and interdependent effects on production and health characteristics of transition dairy cows. Irrespective of precalving BCS, a controlled restriction precalving reduced the net release of nonesterified fatty acids from adipose tissue postpartum and increased plasma calcium concentrations, reducing the risk of milk fever. Fatter cows produced more milk but lost more BCS postcalving and had greater blood β-hydroxybutyrate concentrations and increased hepatic lipidosis. In comparison, after calving, indicators of reduced immune competence were accentuated in BCS4 cows subjected to a feed restriction before calving, probably increasing the risk of infectious diseases. It would appear from these results that optimally conditioned cows will benefit from a short-term (2-3 wk) controlled feed restriction (75-90% of requirements), whereas cows in less than optimal condition should be fed to requirements before calving.

Peripartum, and especially during the transition period, dairy cows undergo dramatic physiological changes. These coincide with an increased risk of disease during the first 2 wk after calving and have been linked to dairy cows failing to achieve production as well as reproductive targets. Previous evidence suggests that these physiological changes affect the immune system and that transition dairy cows experience some form of reduced immunocompetence. However, almost all of these studies were undertaken in high-production, housed dairy cows. Grazing cows have much lower levels of production and this study aimed to provide clarity whether or not the dysfunctional attributes of the peripartum immune system reported in high production housed cows are evident in these animals. Therefore, cell culture techniques, flow cytometry, and quantitative PCR were applied to analyze the cellular composition of peripheral blood mononuclear cells from transition dairy cows as well as the performance of these cells in an in vitro assay. First, a combination of in vitro stimulation and quantitative PCR for cytokines was validated as a quantifiable immunocompetence assay in 29 cattle and a correlation of quantitative PCR and ELISA demonstrated. Second, the relative number of T helper cells, cytotoxic T cells, B cells, γδ T cells, natural killer cells, and monocytes in peripheral blood was measured, of which B cells and natural killer cells increased in number postcalving (n = 29) compared with precalving. Third, following in vitro stimulation cytokine profiles indicated decreased expression of IFNγ, tumor necrosis factor, and IL-17 and increased expression of IL-10 wk 1 after calving, which later all returned to precalving values (n = 39). Additionally, treatment of transition cows with a nonsteroidal anti-inflammatory drug (i.e., carprofen) administered on d 1, 3, and 5 postcalving (n = 19; untreated control n = 20) did not affect the cytokine expression at any time point. In conclusion, an immunocompetence assay has been developed that highlights a characteristic expression pattern for IFNγ, tumor necrosis factor, IL-17, and IL-10 that reflects a state of reduced immunocompetence in moderate-yielding pasture-based transition cows after calving, which is similar to that described for higher-yielding housed cows.

Four cohorts of nonlactating, pregnant dairy cows (n=50, 47, 45, and 42) were individually fed indoors to determine the amount of feed required for body weight (BW) gain from autumn pasture and commonly used supplementary feeds. These results were used to estimate the apparent efficiency with which metabolizable energy (ME) is used for BW gain (app_k g ). Control cows were offered autumn pasture to estimated maintenance requirements (~0.55MJ of ME/kg of BW 0.75 ), with an additional 20 MJ of ME/d allocated for pregnancy and activity. All other cows received the same allowance of autumn pasture and an additional allowance (2.5 or 5.0kg of dry matter/d) of autumn pasture (Past), spring pasture silage (Psil), maize silage (Msil), cracked maize grain (Mgr), or palm kernel expeller (PKE), resulting in a total of 11 treatments. Individual cow dry matter intake was determined daily; BW was recorded once per week for cohorts 1 and 2, and 3 times per week for cohorts 3 and 4. The ME contents of feeds were estimated from feed quality assays. Regression analyses were used on each feed to determine the ME requirement for 1kg of BW gain. The app_k g of Past and Msil was 0.34 and 0.47, respectively; these estimates are in line with published literature. The app_k g of Psil (0.50) was consistent with the published k g for spring pasture, from which the silage was made. Palm kernel expeller had the greatest app_k g (0.61). The reasons for this cannot be deduced from the current study but may reflect the relatively high fat content of the feed and the high k g of fat. The app_k g for Mgr was low (0.38) in comparison with the other supplementary feeds and, in particular, relative to its feed ME and published k g estimates. Although the reason for the low app_k g cannot be deduced from the current data, the most plausible reason is the preferential use of propionate-derived glucose for conceptus metabolism rather than BW gain, a factor not accounted for in previous experimental models that did not use late-gestation cows. In summary, the app_k g for autumn pasture was low but consistent with historical growth rate trials in other ruminant species. In comparison, Msil, Psil, and PKE were used with a greater apparent efficiency (app_k g =0.47 to 0.61), but Mgr resulted in a relatively low rate of gain per MJ of ME (app_k g =0.38). These differences have implications for accurate feed budgeting on farm.

In Table 3 (page 6478), the means and standard errors of the difference (SED) for 9 of the parameters were tabulated as log values, rather than in the original units. All P-values and Tukey comparisons are correct. The corrected table is shown below, with corrected parameters and values in bold. The authors regret the errors.