Higher Nonesterified Fatty Acid Concentrations Research Articles

Metabolic and productive parameters of lactating dairy cows under heat stress conditions supplemented with plant polyphenol extract.

The impact of heat stress on dairy cattle leads to significant economic losses and a negative impact on the welfare of the animals. The objective of this research was to evaluate the effect of the nutritional additive (Thermoplus®) in dairy cows under postpartum heat stress conditions, and its effects on the metabolic profile, production and quality of milk. Eighteen lactating Holstein cows (8 multiparous and ten primiparous), in a free-stall system, with a mean body condition score (BCS) of 3.14 ± 0.05, live weight of 624.55 ± 18, 61kg, with initial mean days in milk (DIM) of 90 ± 10.11, were selected. The animals were grouped into a control (CG, n = 9) and a treatment (TG, n = 9). Both groups underwent 14days of diet adaptation, the TG received the basal diet supplemented with 50g of the additive, once a day, individually, while the control group received only the total diet. Data collection of metabolic and productive parameters were evaluated on days -14 (before adaptation), 1 (after the diet adaptation period), 16, 30, and 44. Milk, blood, and body condition score (BCS) were collected once a day, and heart rate, respiratory rate, and rectal temperature were collected twice a day. Serum concentrations of albumin, calcium, magnesium, glucose, gamma-glutamyl transferase (GGT), beta-hydroxybutyrate (BHBA), non-esterified fatty acids (NEFAs), and paraoxonase-1 (PON-1) were evaluated. In the milk, the percentage of fat, protein, lactose, and total solids were determined in each sampling. Milk yield was measured daily. Humidity and ambient temperature values were collected on the days of the collection every 30min, from 5:30 am to 5:00pm, to calculate the temperature-humidity index (THI). Statistical analyzes were performed using the SAS software (version 9.3, SAS Institute Inc., Cary, NC, USA). The THI ranged from 62.22 to 79.47. Our findings showed that when the THI was greater than 72, the animals in the TG were able to maintain milk yield (Odds ratio (OD) = -0.0577,), and the animals in the CG had a greater chance of reducing it (OD = -0.2301). Multiparous cows in the TG had higher milk yield than CG (32.57 ± 0.34 vs 30.50 ± 0.36kg per day; P = 0.0078) and lower SCC (34.110 ± 6,940 vs 665.50 ± 214.41 cells per ml; P = 0.03), with the same percentages of total solids (P > 0.05). In multiparous metabolic markers, TG when compared CG had higher albumin concentrations (2.50 ± 0.07 vs 2.12 ± 0.07g/dl; < 0.001), equal PON-1 (P > 0.05), and higher BHBA levels (0.49 ± 0.03 vs 0.39 ± 0.04mmol/l). Primiparous from the CG had higher concentrations of NEFA (0.18 ± 0.02mmol/l) than multiparous from the same group (0.09 ± 0.02mmol/l) P = 0.0265. The use of the plant polyphenol extract in postpartum Holstein cows challenged by heat stress had beneficial effects on the production and health of the mammary gland in multiparous cows without decreasing milk solids. The non-reduction of the activities of the acute phase proteins indicates an immunomodulatory and inflammatory-reducing effect of the product used.

Effect of high NEFA concentration on lipid metabolism disorders in hepatocytes based on lipidomics.

Introduction: High concentrations of nonesterified fatty acids (NEFA) is the key of characteristic of fatty liver in dairy cows. Therefore, the aim of this study was to investigate the effect of high concentration of NEFA on lipid metabolism in hepatocytes through the lipidomic approach and molecular biology techniques. Methods: Stimulate AML-12 cells with different concentrations of NEFA, observe the cellular lipid accumulation, and select 0.6mM NEFA stimulation concentration for subsequent experiments. Collect cells for lipidomics analysis. Results: High concentration of NEFA (0.6-2.4mM) significantly reduced the cell viability in a concentration-dependent manner, indicating that high concentrations of NEFA have lipotoxicity on hepatocytes. In addition, NEFA promoted triglycerides (TAG) accumulation, increased the mRNA expression of the lipogenic molecules SREBP1c and FASN, and decreased the mRNA expression of lipolytic molecules CPT1A and HSL in hepatocytes. Mechanistically, high concentration of NEFA induced lipid metabolism disorders in hepatocytes by regulating metabolic pathways such as glycerol phospholipid metabolism, glycosyl phosphatidylinositol anchored biosynthesis, triglyceride metabolism, sphingolipid metabolism, and inositol phosphate metabolism. Discussion: High concentration of NEFA is lipotoxic to cells, promoting lipid accumulation. LPE (18:2), LPE (18:3), LPE (18:1) via glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, glycerolipid metabolism, sphingolipid metabolism, and inositol phosphate metabolism, indicating their potential regulation role in the pathogenesis of fatty liver.

Effect of two recombinant bovine somatotropin formulations on feeding behavior and metabolism of Holstein cows

This study evaluated the effect of two commercial forms of recombinant bovine somatotropin (rbST; Later® and Fast®; 500 mg) on feed variability, feeding behavior, zootechnical parameters, and milk production of Holstein cows after peak production. Eighteen cows were randomly divided into two groups and treated with injectable of rbST every 14 days for 70 days. Blood samples were collected on day 1, 4, and 7 of each cycle, to assess circulating levels of non-esterified fatty acids (NEFA). Weight and body condition score (BCS) assessments were performed. Milk yields were measured daily using the DelPro™ software (DeLaval®). Feed consumption and feeding behavior was obtained daily using automatic individual feeders (Intergado®). No difference was observed in dry matter intake (DMI) between groups (P=0.07), and no impact was noted on dietary variability (P=0.64). In addition, animals treated with rbST-Fast exhibited more frequent visits to feeders, longer time in consumption, and higher relative DMI (per percentage of live weight) (P&lt;0.01). Animals in the rbST-Fast group produced more milk than those in the rbST-Later group (P=0.03). Furthermore, the weight of the cows was significantly different (P=0.05) between groups. The weight of the rbST-Fast animals was smaller and varied more than thot of the rbST-Later animals (P &lt;0.01). Nonetheless, no difference was noted in BCS (P=0.30) between the groups. The animals in the rbST-Fast group had higher concentrations of NEFA than those in the rbST-Later group (P&lt;0.01). In conclusion, behavioral and metabolic shifts that sustained greater milk production in the cows receiving rbST-Fast. Nonetheless, these shifts are likely linked to lipomobilization and not dietary variability or consumption, as evidenced by the increase in NEFA and greater weight loss in that group.

Comparison of Uterine Involution and the Resumption of Ovarian Cyclicity between Lame and Sound Holstein Cows

The aim of the study was to examine the effect of lameness and energy status on the involution of the uterus and the resumption of ovarian cyclicity in dairy cows. Lame (lameness score of four and the presence of hoof lesions, n = 22) and sound (normal gait and the absence of hoof lesions, n = 25) multiparous cows with healthy puerperium were enrolled simultaneously in the study and were monitored from day 10 antepartum (ap) to day 50 post-partum (pp). Ultrasonography of the cervix, the formerly gravid uterine horn and the ovarian structures was performed on d 8, 11, 14, 23, 30, and 42 pp. Blood sampling for progesterone, β-hydroxybutyrate (BHBA), and non-esterified fatty acids (NEFAs) was used to assess cyclicity and energy status. Lame compared to sound cows had higher NEFA concentrations on day 14 pp (0.54 ± 0.05 vs. 0.37 ± 0.05, respectively, p = 0.005), delayed involution of the cervix and the formerly pregnant uterine horn (p = 0.0003 and p = 0.02, respectively), lower ovulation rates within the experimental period (63.6% vs. 88%, respectively, p = 0.05), and higher rates of atresia or cyst formation on day 50 pp (36.4% vs. 12%, respectively, p = 0.05). Independently of lameness status, cows with high NEFA concentrations had lower ovulation rates within the experimental period (65.5% vs. 94.4%, p = 0.02), lower normal ovarian activity on day 50 pp (58.6% vs. 88.9%, p = 0.03), and higher rates of atresia or cyst formation on day 50 pp (34.5% vs. 5.6%, p = 0.02) compared to cows with optimal NEFA concentrations. Furthermore, an interaction between lameness and increased NEFA concentrations was observed regarding the ovulation rate within the experimental period and the percentage of atresia or cyst formation on day 50 pp. Sound cows with low NEFA levels had the lowest mean cervical diameter compared to cows with lameness (both with elevated and optimal NEFA concentrations, p = 0.009 and p = 0.002, respectively). Conclusively, lameness during puerperium negatively affected ovarian function and uterine involution. These effects were exacerbated (through interaction or cumulation) in relation to elevated NEFA concentrations.

Inositol-requiring enzyme 1α and c-Jun N-terminal kinase axis activation contributes to intracellular lipid accumulation in calf hepatocytes

During the perinatal period, dairy cows undergo negative energy balance, resulting in elevated circulating levels of nonesterified fatty acids (NEFA). Although increased blood NEFA concentrations are a physiological adaptation of early lactation, excessive NEFA in dairy cows is a major cause of fatty liver. Aberrant lipid metabolism leads to hepatic lipid accumulation and subsequently the development of fatty liver. Both inositol-requiring enzyme 1α (IRE1α) and c-Jun N-terminal kinase (JNK) have been validated for their association with hepatic lipid accumulation, including their regulatory functions in calf hepatocyte insulin resistance, oxidative stress, and apoptosis. Meanwhile, both IRE1α and JNK are involved in lipid metabolism in nonruminants. Therefore, the aim of this study was to investigate how IRE1α and JNK regulate lipid metabolism in bovine hepatocytes. An experiment was conducted on randomly selected 10 healthy cows (hepatic triglyceride [TG] content <1%) and 10 cows with fatty liver (hepatic TG content >5%). Liver tissue and blood samples were collected from experimental cows. Serum concentrations of NEFA and β-hydroxybutyrate (BHB) were greater, whereas serum concentrations of glucose and milk production were lower in cows with fatty liver. The western blot results revealed that dairy cows with fatty liver had higher phosphorylation levels of JNK, c-Jun, and IRE1α in the liver tissue. Three in vitro experiments were conducted using primary calf hepatocytes isolated from 5 healthy calves (body weight: 30-40 kg; 1 d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h, which showed that the phosphorylated levels of JNK, c-Jun, and IRE1α increased in both linear and quadratic effects. In the second experiment, hepatocytes were treated with high concentrations of NEFA (1.2 mM) for 12 h with or without SP600125, a canonical inhibitor of JNK. Western blot results showed that SP600125 treatment could decrease the expression of lipogenesis-associated proteins (PPARγ and SREBP-1c) and increase the expression of fatty acid oxidation (FAO)-associated proteins (CPT1A and PPARα) in NEFA-treated hepatocytes. The perturbed expression of lipogenesis-associated genes (FASN, ACACA, and CD36) and FAO-associated gene ACOX1 were also recovered by JNK inhibition, indicating that JNK reduced excessive NEFA-induced lipogenesis and FAO dysregulation in calf hepatocytes. Third, short hairpin RNA targeting IRE1α (sh-IRE1α) was transfected into calf hepatocytes to silence IRE1α, and KIRA6 was used to inhibit the kinase activity of IRE1α. The blockage of IRE1α could at least partially suppressed NEFA-induced JNK activation. Moreover, the blockage of IRE1α downregulated the expression of lipogenesis genes and upregulated the expression of FAO genes in NEFA-treated hepatocytes. In conclusion, these findings indicate that targeting the IRE1α-JNK axis can reduce NEFA-induced lipid accumulation in bovine hepatocytes by modulating lipogenesis and FAO. This may offer a prospective therapeutic target for fatty liver in dairy cows.

Inhibition of cluster antigen 36 protects against fatty acid-induced lipid accumulation, oxidative stress, and inflammation in bovine hepatocytes

When ketosis occurs, supraphysiological concentrations of nonesterified fatty acids (NEFA) display lipotoxicity and are closely related to the occurrence of hepatic lipid accumulation, oxidative stress, and inflammation, resulting in hepatic damage and exacerbating the progression of ketosis. However, the mechanism of these lipotoxic effects caused by high concentrations of NEFA in ketosis is still unclear. Cluster antigen 36 (CD36), a fatty acid transporter, plays a vital role in the development of hepatic pathological injury in nonruminants. Thus, the aim of this study was to investigate whether CD36 plays a role in NEFA-induced hepatic lipotoxicity in dairy cows with clinical ketosis. Liver tissue and blood samples were collected from healthy (n = 10) and clinically ketotic (n = 10) cows at 3 to 15 d in milk. In addition, hepatocytes isolated from healthy calves were treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h; or infected with CD36 expressing adenovirus or CD36 silencing small interfering RNA for 48 h and then treated with 1.2 mM NEFA for 12 h. Compared with healthy cows, clinically ketotic cows had greater concentrations of serum NEFA and β-hydroxybutyrate and activities of aspartate aminotransferase and alanine aminotransferase but lower serum glucose. In addition, dairy cows with clinical ketosis displayed excessive hepatic lipid accumulation. More importantly, these alterations were accompanied by an increased abundance of hepatic CD36. In the cell culture model, exogenous NEFA (0, 0.6, 1.2, or 2.4 mM) treatment could dose-dependently increase the abundance of CD36. Meanwhile, NEFA (1.2 mM) increased the content of triacylglycerol, reactive oxygen species and malondialdehyde, and decreased the activities of glutathione peroxidase and superoxide dismutase. Moreover, NEFA upregulated phosphorylation levels of nuclear factor κB (NF-κB) and the inhibitor of NF-κB (IκB) α, along with the upregulation of protein abundance of NLR family pyrin domain containing 3 (NLRP3) and caspase-1, and mRNA abundance of IL1B, IL6, and tumor necrosis factor α (TNFA). These alterations induced by NEFA in bovine hepatocytes were associated with increased lipid accumulation, oxidative stress and inflammation, which could be further aggravated by CD36 overexpression. Conversely, silencing CD36 attenuated these NEFA-induced detriments. Overall, these data suggest that CD36 may be a potential therapeutic target for NEFA-induced hepatic lipid accumulation, oxidative stress, and inflammation in dairy cows.

Body condition score and serum metabolites and minerals concentrations as indicators of ovarian activity and pregnancy success in goats on rangeland

Aim of the study: To investigate potential differences in ovarian structures relative to serum metabolite and mineral concentrations at mating. Also, body condition score (BCS), serum metabolites, and mineral profiling at mating were compared between pregnant and non-pregnant goats. Area of study: Hot zone of northern Mexico (26 °N). Material and methods: Mixed-breed goats (n= 89) on arid rangeland were exposed to bucks during the non-breeding season. Ovarian structures were recorded at mating and ten days after breeding using ultrasonography. Pregnancy was detected at 30 and 120 days post-mating. BCS, blood metabolites, and minerals were determined at mating. Main results: Pregnant goats had higher BCS at mating than non-pregnant goats. The mean serum glucose concentration was higher (p&lt;0.05) for pregnant goats than that for non-pregnant ones (87.3 ± 12.1 vs. 74.4 ± 11.6 mg/dL). Significantly lower (p&lt;0.01) serum urea nitrogen levels at mating were recorded in non-pregnant (10.7 ± 3.5 mg/dL) than in pregnant goats (12.4 ± 3.7 mg/dL). Lower serum glucose (72.2 ± 6.9 vs. 89.4 ± 11.2) and higher non-esterified fatty acids concentrations (NEFA; 0.43 ± 0.23 vs. 0.18 ± 0.12) were significantly associated (p&lt;0.05) with pregnancy loss. Higher serum total protein concentrations were associated with a greater number and larger ovulatory follicles. High serum phosphorus was significantly associated with larger ovulatory follicles. Goats with ovulatory follicles ≥7.6 mm were more likely (p&lt;0.05) to get pregnant than goats with smaller ovulatory follicles. Research highlights: Monitoring BCS, serum glucose, blood urea nitrogen, and NEFA could be used to identify goats at risk for infertility.

P-035 Exposure to high concentrations of non-esterified fatty acids impairs bovine sperm quality

Abstract Study question Do high concentrations of non-esterified fatty acids (NEFA) affect sperm quality? Summary answer In vitro exposure to high NEFA concentrations induced detrimental effects on acrosome integrity, mitochondrial membrane potential, plasma membrane integrity, and DNA damage in bovine spermatozoa. What is known already High NEFA levels present in obese individuals and in cows experiencing negative energy balance has been linked with impaired reproductive success. In vitro bovine models have showed that a high NEFA microenvironment (i.e. steric acid [SA], palmitic acid [PA], oleic acid [OA]) during oocyte maturation (Reproduction 2013, 145:33-44), fertilisation (ESHRE meeting 2021, P-176), and embryo culture (BMC Genomics 2016, 17:1004) is detrimental for blastocyst formation. However, the effect of high NEFA levels on sperm quality has been less explored. Study design, size, duration Bovine sperm from two bulls was prepared to a maximum concentration of 20x106/mL then incubated in in vitro fertilisation (IVF) medium for 4 h under different NEFA levels representing physiological (Control-1[C1], 28 μM SA, 23 μM PA, 21 μM OA) and pathophysiological (High-NEFA, 280μM SA, 230μM PA, 210μM OA), serum levels found in cows experiencing negative energy balance) concentrations. A second control (C2) group contained solvent. Participants/materials, setting, methods Fluorescence assays (Toxicity 2018, 393:52-50) were used to evaluate simultaneously acrosome integrity (FITC-PSA), mitochondrial membrane potential (JC-1) and plasma membrane integrity (DRAQ7, Hoechst 33342). Spermatozoa (n = 800 per group, four replicates) were assessed with epifluorescence microscopy and Ziess ZEN software. Sperm DNA damage was also assessed with the Halosperm G2® kit (C1; n = 812, C2; n = 879, High-NEFA; n = 930, six replicates). Data were arcsine transformed before analysis (ANOVA, followed by LSD post hoc test). Main results and the role of chance There was no significant difference between the control groups or between bulls in any of the variables analysed. The percentage (mean ± SEM) of spermatozoa with intact acrosome was higher in the control groups compared with high-NEFA (C1=64.50 ± 6.34%, C2=69.13 ± 4.08%, High-NEFA=49.75 ± 6.14%), but it reached statistical significance only when compared to C2 (C2 vs High-NEFA P = 0.037, C1 vs High-NEFA P = 0.096). Similarly, the percentage of spermatozoa with low mitochondrial membrane potential was increased following exposure to high concentrations of NEFA (C1=50.88 ± 5.69%, C2=45.00 ± 4.03%, High-NEFA=61.62 ± 3.38%), but a significant difference was only observed when compared to C2 (C2 vs High-NEFA P = 0.027, C1 vs High-NEFA P = 0.124). The high-NEFA group showed a higher plasma membrane damage (59.50 ± 1.06%) compared to controls (C1=47.50 ± 4.07% P = 0.01, C2=46.75 ± 1.79% P = 0.008). DNA damage was also increased in the High-NEFA group (20.00 ± 0.64%) compared to controls (C1=8.90 ± 0.71% P &amp;lt; 0.001, C2=6.82 ± 0.78% P &amp;lt; 0.001). Limitations, reasons for caution In vitro animal models do not reflect accurately in vivo conditions and cannot be directly extrapolated to humans, but they are useful for the development of conceptual models that could eventually be tested in the target species (e.g. obese individuals). Wider implications of the findings Our results indicate that high NEFA concentrations can impair sperm quality, and given that sperm with DNA damage can achieve fertilization (Human Reproduction 2010, 25:1594-1608), our data partially explain the decreased blastocyst formation previously observed in this model of high NEFA exposure (ESHRE meeting 2021, P-176). Trial registration number 000

The Impact of Overstocking and Negative Energy Balance on Quantitative Measurement of Non-typhoidal Salmonella in Periparturient Dairy Cattle.

Stressful conditions in animal production facilities may exacerbate the fecal shedding and foodborne transmission of non-typhoidal Salmonella enterica subsp. enterica. Dairy cows are reservoirs of this zoonotic microorganism, and its prevalence has roughly doubled in the past decade on dairy farms in United States. Dairy cows are commonly overstocked at the feed bunk, and stressors placed on the animal prior to parturition may exacerbate Salmonella shedding. However, no studies have evaluated the impact of overstocking and metabolic stress on fecal concentrations of the pathogen. Therefore, we conducted a field trial with 120 multiparous dairy cows randomized into one of four treatment groups with different stocking densities at the feed bunk during the periparturient period as follows: US, understocked from −60 to −1 DRC; OS, overstocked from −60 to −1 DRC; USOS, understocked from −60 to −26 DRC/overstocked from −25 to −1 DRC; and OSUS, overstocked from −60 to −26 DRC/ understocked from −25 to −1 DRC. Fecal and blood samples were collected at four time points relative to calving. qPCR assays were used to quantify Salmonella invA gene and total bacterial community from fecal samples, and a subset of isolates recovered from fecal bacterial culture were characterized using pulsed field gel electrophoresis and serotyping. Serum non-esterified fatty acids (NEFA) were measured as a metabolic stress indicator using an immunoassay. Multivariable analyses were performed to test if changes in Salmonella concentrations were associated with stocking density, energy balance, or days relative to calving. From fecal isolates, three Salmonella serovars were identified, S. Cerro; Kentucky; Meleagridis. Concentrations of Salmonella increased as cows approached calving. Higher stocking densities at the feed bunk did not impact total bacterial community or NEFA; however, cows in the overstocked groups had higher Salmonella fecal concentrations. Further, cows with higher NEFA concentrations after calving had a higher likelihood of detection of Salmonella. Future farm interventions should aim to reduce environmental and metabolic stress during the periparturient period to decrease the dissemination of Salmonella to cattle, the environment, and humans.

Sirtuin 3 inhibits nuclear factor-κB signaling activated by a fatty acid challenge in bovine mammary epithelial cells

Susceptibility to mastitis is highest during the peripartal (transition) period and is often concomitant with other comorbidities such as ketosis. Although infection with pathogenic microorganisms and immune-dysfunction around calving clearly play key roles in mastitis development, other metabolic factors also contribute. Sirtuin 3 (SIRT3), a mitochondrial deacetylase regulating energy and redox homeostasis, antagonizes the lipotoxic effects of nonesterified fatty acids (NEFA). Thus, we hypothesized that increases in circulating NEFA concentrations, as observed in the transition period, provokes inflammatory responses that can be reversed via activation of SIRT3. Here we aimed to study (1) proinflammatory NF-κB signaling and SIRT3 abundance in mammary tissue of ketotic cows and healthy controls, and (2) the effect of SIRT3 on NF-κB activation in bovine mammary epithelial cells (BMEC) treated with high levels of NEFA. The mammary gland biopsy samples were from a previous study, which included 15 healthy cows and 15 ketotic cows. Primary BMEC were isolated from 3 healthy Holstein cows with collagenase III digestion. Purified BMEC were incubated with or without SIRT3 overexpression adenovirus for 48 h, then treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 24 h. Mammary tissue of ketotic cows was associated with lower protein abundance of SIRT3 along with greater NF-κB P65 phosphorylation levels (p-NF-κB P65), p-NF-κB P65:NF-κB P65 ratio, and mRNA abundance of IL1B and IL6. In BMEC, exogenous NEFA dose-dependently reduced protein abundance of SIRT3, but increased p-NF-κB P65, p-NF-κB P65:NF-κB P65 ratio, and mRNA abundance of IL1B and IL6. Compared with green fluorescent protein adenovirus vector + NEFA, overexpression of SIRT3 in NEFA-treated BMEC downregulated p-NF-κB P65 and mRNA abundance of IL1B and IL6. Immunofluorescence indicated that overexpression of SIRT3 inhibited nuclear translocation of NF-κB P65. Overall, our data demonstrated that ketosis is associated with a reduction in SIRT3 abundance and activation of NF-κB signaling in the mammary gland. In vitro data provided evidence that high NEFA concentrations inhibit SIRT3, which contributes to enhanced NF-κB signaling including nuclear translocation and a pro-inflammatory response. The data suggest a promising role of SIRT3 as a target for helping alleviate localized inflammation of the mammary gland resulting from exposure to high concentrations of NEFA.

Effect of diet and nonesterified fatty acid levels on global transcriptomic profiles in circulating peripheral blood mononuclear cells in early lactation dairy cows

After calving, lipid mobilization caused by increased nutrient demands for lactation leads to elevated circulating concentrations of nonesterified fatty acids (NEFA). Excessive NEFA levels have previously been identified as a major risk factor for postpartum immunosuppression. The aim of this study was to investigate changes in global transcriptomic gene expression of peripheral blood mononuclear cells (PBMC) in dairy cows offered different early lactation diets (high concentrate, n = 7; medium, n = 8; or low, n = 9) and with differing circulating levels of NEFA. Cows were classified as having NEFA concentrations of either <500 µM (low, n = 6), 500 to 750 µM (medium, n = 8) or >750 µM (high, n = 10) at 14 d in milk. Plasma urea concentrations were greater for cows on the high concentrate diet but β-hydroxybutyrate and glucose concentrations did not differ significantly between either dietary treatments or NEFA groups. Cows with high NEFA weighed more at drying off and suffered greater body condition score loss after calving. The PBMC were isolated at 14 d in milk, and RNA was extracted for RNA sequencing. Differential gene expression was analyzed with DESeq2 with q-value for false discovery rate control followed by Gene Ontology Enrichment. Although there were no differentially expressed genes associated with lactation diet, 304 differentially expressed genes were identified between cows with high and low circulating NEFA, with 118 upregulated and 186 downregulated. Gene Ontology enrichment analysis demonstrated that biological adhesion and immune system process were foremost among various PBMC functions which were altered relating to body defenses and immunity. High NEFA concentrations were associated with inhibited cellular adhesion function by downregulating 20 out of 26 genes (by up to 17-fold) related to this process. Medium NEFA concentrations altered a similar set of functions as high NEFA, but with smaller enrichment scores. Localization and immune system process were most significant, with biological adhesion ranking only eleventh. Our results demonstrated that increased circulating NEFA concentrations, but not diet, were associated with immune system processes in PBMC in early lactation cows. Leukocyte cell-to-cell adhesion was inhibited when the NEFA concentration exceeded 750 µM, which would reduce the efficiency of diapedesis and so contribute to decreased body defense mechanisms and predispose animals to infection.

Prediction of Reproductive Success in Multiparous First Service Dairy Cows by Parameters from In-Line Sensors

The aim of the current study was to evaluate the relationship of different parameters from an automatic milking system (AMS) with the pregnancy status of multiparous cows at first service and to assess the accuracy of such a follow-up with regard to blood parameters. Before the insemination of cows, blood samples for measuring biochemical indices were taken from the coccygeal vessels and the concentrations of blood serum albumin (ALB), cortisol, non-esterified fatty acids (NEFA) and the activities of aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) were determined. From oestrus day to seven days after oestrus, the following parameters were registered: milk yield (MY), electric milk conductivity, lactate dehydrogenase (LDH) and β-hydroxybutyric acid (BHB). The pregnancy status was evaluated using ultrasound “Easy scan” 30–35 days after insemination. Cows were grouped by reproductive status: PG− (non-pregnant; n = 48) and PG+ (pregnant; n = 44). The BHB level in PG− cows was 1.2 times higher (p &lt; 0.005). The electrical conductivity of milk was statistically significantly higher in all quarters of PG− cows (1.07 times) than of PG+ cows (p &lt; 0.05). The arithmetic mean of blood GGT was 1.61 times higher in PG− cows and the NEFA value 1.23 times higher (p &lt; 0.05) compared with the PG+ group. The liver function was affected, the average ALB of PG− cows was 1.19 times lower (p &lt; 0.05) and the AST activity was 1.16 times lower (p &lt; 0.05) compared with PG+ cows. The non-pregnant group had a negative energy balance demonstrated by high in-line milk BHB and high blood NEFA concentrations. We found a greater number of cows with cortisol &gt;0.0.75 mg/dL in the non-pregnant group. A higher milk electrical conductivity in the non-pregnant cows pointed towards a greater risk of mastitis while higher GGT activities together with lower albumin concentrations indicated that the cows were more affected by oxidative stress.

Patterns of blood biochemical parameters of peripartum dairy cows raised in either smallholder or semi-commercial dairy farms in Thailand.

Background and Aim:Data about dynamics of serum biochemical parameters and negative energy balance (NEB) related conditions differ among geographical areas or farm types. It should be cautious about applying those data to justify control and prevention strategies of NEB problems. Therefore, dynamics of blood biochemical parameters related to NEB condition of peripartum dairy cows raised in either smallholder (SH) or semi-commercial (SC) farms were studied.Materials and Methods:Thirty-two healthy pregnant dry cows were selected from five dairy farms in Western part of Thailand, including 15 and 17 cows from three SH and two SC farms, respectively. Blood samples were collected at 2 weeks before the expected calving date, and 1, 2, 4, and 8 weeks postpartum to determine concentrations of glucose, non-esterified fatty acid (NEFA), and b-hydroxybutyrate (BHBA). Body condition scores (BCSs) and milk yields were also recorded.Results:Dairy cows in both farm types suffered from NEB by elevation of serum NEFA and BHBA, and loss of BCS postpartum. Degrees of elevation in serum NEFA and BHBA were different between farm types. The SC cows showed more BCS loss postpartum, but lower serum NEFA. In the SH cows, even with less BCS loss, the cows showed high serum NEFA concentrations.Conclusion:Dairy cows in our study entered NEB condition even with low milk yields. Moreover, elevation of serum NEFA and BHBA postpartum was higher in our studied cows as compared with other studies in high producing cows of commercial dairy farms in temperate areas.

Cardiac, Energy and Hormonal Blood Markers, and Lactatemia in Cows with Displaced Abomasum

Background: Displaced abomasum (DA) is a common and economically important disorder that affects dairy cattle. Nutritional factors and adaptive responses that occur in the peripartum play a central role in the pathogenesis. The measurement of blood metabolites represents a useful tool for monitoring and prognostic determination in affected animals. Therefore, the objective was to evaluate cardiac, energy and hormonal blood markers, lactatemia, and insulin sensitivity in cows diagnosed with right displaced abomasum (RDA) and left displaced abomasum (LDA), comparing them with each other.Materials, Methods &amp; Results: Nineteen cases of abomasum displacement in cows were studied, including 9 cases of RDA and 10 cases of LDA. The diagnosis was established by means of physical examination and measurement of the concentration of chlorides in the ruminal fluid (&gt; 30mEq/L). After diagnosis, clinical-surgical therapeutic management was instituted. At the time of diagnosis (M1) and at the resolution of the case (M2), blood samples were collected to assess the variables: non-esterified fatty acids (NEFA), beta hydroxybutyrate (βHB), L-lactate, creatine kinase (CK), creatine kinase MB (CK-MB), cardiac troponin I (cTnI), lactate dehydrogenase (LDH), glucose, insulin, and cortisol. In addition, insulin sensitivity was estimated using the Revised Quantitative Insulin Sensitivity Check Index (RQUICKI) and RQUICKI-βHB. The means of the variables were compared, separating the effects of groups (RDA and LDA) and moments (M1 and M2), at the level of 5% probability. The concentrations of NEFA, CK-MB, L-lactate, glucose, insulin, and cortisol were higher at M1 and the RQUICKI and RQUICKI-βHB indices were lower at this moment. L-lactate, CK, and CK-MB were higher in the RDA group, while cTnI, βHB, and LDH did not present a group or moment effect. Cardiac markers correlated with the energy profile metabolites, L-lactate, and cortisol.Discussion: The high concentrations of NEFA at M1 reflected the condition of negative energy balance. βHB concentrations were stable, that may be related to the number of days postpartum in which the animals were diagnosed. The hyperglycemic condition and the increase in serum cortisol concentrations found at M1 can be induced by the condition of metabolic stress resulting from the disease. Hyperinsulinemia were recorded in the present study could be an important factor related to the pathogenesis of DA, since there seems to be a correlation between hyperinsulinemia and decreased abomasal emptying rate. The RQUICKI and RQUICKI-βHB indices was significantly lower at M1, which may indicate lower sensitivity of peripheral tissues to insulin at this time. Changes in serum activity of LDH and CK may result from tissue damage due to organ displacement, in addition to damage associated with surgery and the administration of injectable drugs, mainly intramuscularly. The elevation in plasma L-lactate at M1 and in the RDA group may be associated with abomasal hypoperfusion. The high positive correlations found between L-lactate and the variables glucose, insulin, and cortisol reinforcing the association between the concentration of L-lactate and the moment of greatest stress. The increase in cardiac biomarkers may be related to the occurrence of ischemia/reperfusion injury in the abomasum, which involves oxidative stress and the production of inflammatory mediators. The hyperglycemic condition and the higher concentrations of NEFA can also contribute to the occurrence of myocardial injury. The correlations found between cardiac biomarkers and plasma L-lactate, strengthen the idea that there is a relationship between L-lactate and myocardial injury. In this sense, the measurement of blood concentrations of cTnI, CK-MB, and L-lactate could contribute as severity markers and prognosis indicators in cattle with DA.

Altered embryotrophic capacities of the bovine oviduct under elevated free fatty acid conditions: an in vitro embryo--oviduct co-culture model.

Maternal metabolic stress conditions are of growing importance in both human and dairy cattle settings as they can have significant repercussions on fertility. Upregulated lipolysis is a common trait associated with metabolic disorders and results in systemically elevated concentrations of non-esterified fatty acids (NEFAs). The effects of high NEFA concentrations on the follicular environment, oocyte and embryo development is well documented. However, knowledge on the effects of NEFAs within the oviduct, representing the initial embryonic growth environment, is currently lacking. Therefore, the experiments outlined here were designed to obtain fundamental insights into both the direct and indirect interactions between NEFAs, bovine oviductal cells and developing zygotes. Hence, zygotes were co-cultured with NEFA-pre-exposed bovine oviductal cells or subjected to simultaneous NEFA exposure during the co-culture period. The outcome parameters assessed were embryo development with cleavage (48h post insemination (pi)), morula (120-126h pi) and blastocyst (192h pi) rates, as well as morula intracellular lipid content and blastocyst quality using Bodipy and differential staining respectively. Our data suggest a direct embryotoxicity of NEFAs as well as impaired embryo development through a reduced oviductal ability to support and protect early embryo development.

The effects of non-esterified fatty acids and β-hydroxybutyrate on the hepatic CYP2E1 in cows with clinical ketosis.

Dairy cows with ketosis display severe oxidative stress as well as high blood concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHB). Cytochrome P4502E1 (CYP2E1) plays an important role in the induction of oxidative stress. The aim of this study was to investigate CYP2E1 expression and activity in the liver of clinically ketotic cows (in vivo) and the effects of NEFA and BHB on CYP2E1 expression and activity in hepatocytes (in vitro). Dairy cows with clinical ketosis exhibited a low blood concentration of glucose but high concentrations of NEFA and BHB. Hepatic mRNA, protein expression, and activity of CYP2E1 were significantly higher in cows with clinical ketosis than in control cows. In vitro, both NEFA and BHB treatment markedly up-regulated the mRNA and protein expressions as well as activity of CYP2E1 in cow hepatocytes. Taken together, these results indicate that high levels of NEFA and BHB significantly up-regulate the expression and activity of hepatic CYP2E1, and may be influential in the induction of oxidative stress in cows with clinical ketosis.

Milk production in Saanen goats treated with repeated low doses of intermediate-release insulin during early lactation

ABSTRACT: The effect of insulin administration on the productive responses of Saanen goats during early lactation was investigated. Ten of 20 adult females were subjected to subcutaneous administration of intermediate-acting insulin (0.14UI/kg body weight) at 2, 9, and 14 days postpartum. Milk yield was measured twice daily for 13 weeks and milk samples were collected to measure protein and fat contents. Plasma levels of progesterone, insulin, non-esterifies fatty acids, glucose and other metabolites were measured. Results showed a significantly increased effect of insulin treatment on the content of milk fat and protein; moreover, milk production in the first and second postpartum weeks were higher than control group. The peak of lactation in the insulin group was achieved one week earlier in comparison to the control group. In addition, the milk production rate showed lower persistency (milk yield 13 week/milk yield at peak) in the same group. During the first four weeks of postpartum, treated animals showed greater weight loss and higher non-esterified fatty acid concentration, whereas no effect was observed on the concentration of progesterone and other metabolites. The above results indicated that repeated administration of insulin in dairy goats during early lactation increase yield and qualitative components of milk, but has substantial consequences on animal productive rate and metabolic response.

Metabolic status of crossbreed F1 Holstein × Gyr dairy cows during the transition period in two different seasons in Brazil

Crossbreed F1 Holstein x Gyr cows are proving to be a good alternative for milk production under tropical conditions. In order to contribute to that affirmative, our work focused on clarifying the metabolic patter of F1 Holstein x Gyr dairy cows during the most critical time in the lactation cycle, the transition period, and to compare their performance in two seasons. Blood sampling was performed on 15 cows during summer (January to April) and on 13 cows during winter (May to August), beginning three weeks before the estimated calving date, until 30 days relative to calving. The season had a great influence on the metabolic status. Almost all evaluated metabolites, with exception for aminotransferase (AST), non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA), that have shown no variation among seasons, demonstrated a different patter between summer and winter. Liver functions were enhanced during the postpartum, with increased liver enzymes activity and increased concentration of cholesterol and BHB. Animals went through negative energy balance (NEB) and an alarming number of animals had experienced subclinical ketosis and high NEFA concentrations.

Non esterified fatty acid levels in indian cross bred jersey cows under small holder farming systems

Data on Production diseases assessment in Indian dairy cattle is less. The objective of this study was to assess the Non-esterified fatty acids level in three different small holder farms of Cauvery Delta region of Tamilnadu. Sixty cross bred dairy cattle were selected at different levels of lactation in the three farms. Serum samples were analyzed for non esterified fatty acids (NEFA). Mean ± standard deviations of the values were arrived and the data were statistically analyzed. NEFA values of early, mid, late lactation, Far off dry and close up dry cows were 0.39±0.09, 0.24±0.03, 0.19±0.03, 0.18±0.03 and 0.23±0.03mmol/L, respectively. No significant variations were recorded in between the farms. Cows at early lactation had higher NEFA concentrations where as lowest concentrations was observed in Far off Dry cows.

Effects of non‐esterified fatty acids on the expression and secretion of angiopoietin‐like protein 4 and fibroblast growth factor 21 in calf hepatocytes cultured in vitro

IntroductionNegative energy balance (NEB) is considered as the pathological basis of energy metabolic disorders, such as fatty liver and ketosis, in periparturient dairy cows. An elevated serum non‐esterified fatty acids (NEFA) concentration is one of the most important indicators of NEB. Angiopoietin‐like protein 4 (ANGPTL4) and fibroblast growth factor 21 (FGF21) have been identified as hepatokines, which involved in regulation of metabolic adaptations. In dairy cows, most circulating ANGPTL4 and FGF21 originate from the liver. However, the direct effects of NEFA on the expression and secretion of ANGPTL4 and FGF21 in bovine hepatocytes are not entirely clear. The purpose of the present study was to investigate the effects of different NEFA concentrations on the expression and secretion of these two hepatokines in calf hepatocytes cultured in vitro.MethodsBovine hepatocytes were cultured using the collagenase perfusion method. After culture for 72 h, the hepatocytes were treated with NEFA at final concentrations of 0 (control), 0.6, 1.2, 1.8 and 2.4 mmol/L. After 24 h of continuous culture, the mRNA and protein expression levels of ANGPTL4 and FGF21 in the hepatocytes were determined by real‐time PCR and Western blot, respectively. ANGPTL4 and FGF21 secretions in the supernatant were determined by enzyme‐linked immunosorbent assay (ELISA).ResultsThe results showed similar levels of ANGPTL4 expression and secretion following treatment with 0.6 and 1.2 mmol/L NEFA, which were higher than those observed for the controls (P&lt;0.05). ANGPTL4 expression and secretion were also similar at 1.8 and 2.4 mmol/L NEFA and significantly higher than those observed for controls (P&lt;0.01). The expression and secretion of FGF21 at 0.6 mmol/L NEFA‐treated hepatocytes was higher than that of the control group (P&lt;0.05). The FGF21 expression and secretion were similar at 1.2, 1.8 and 2.4 mmol/L NEFA‐treated hepatocytes and significantly higher than those observed for controls (P&lt;0.01).ConclusionThe results of this study indicate that high concentrations of NEFA significantly increase the expression and secretion of ANGPTL4 and FGF21 in bovine hepatocytes cultured in vitro in a dose‐dependent manner. Specifically, NEFA promotes the expression and secretion of ANGPTL4 and FGF21, which may worsen fat mobilization under NEB conditions and thereby lead to the pathological processes of energy metabolism disorders in dairy cows in the peripartum period. However, further studies are needed to elucidate the underlying mechanisms.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.