Higher Plasma Nonesterified Fatty Acids Research Articles

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

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

Effects of calving interval of dairy cows on development, metabolism, and milk performance of their offspring

Extending the voluntary waiting period (VWP) for insemination in dairy cows is of interest as a strategy to reduce the frequency of calving events and inseminate at a moment with fewer fertility problems. Little is known about the calves born from dams with a different VWP followed by a different calving interval (CInt). The objective of the current study was to identify the effect of dam's CInt on body condition, metabolic status, and milk production of their offspring from birth until 100 DIM of the offspring's first lactation. Holstein-Friesian dairy cows (n = 154, 41 primiparous, 113 multiparous) were blocked according to parity, milk yield, and SCC and randomly assigned to a VWP of 50, 125, or 200 d. Female calves (n = 62) from cows with different CInt were monitored from birth until their first calving event as heifer. Certain dams were not successfully inseminated soon after the planned VWP, resulting in differences between the intended VWP and the actual CInt. Calves were regrouped according to their dam's actual CInt (CInt_1: 324-408 d; CInt_2: 409-468 d; CInt_3: 469-586 d). The dam's CInt did not affect calf birth weight. From birth to weaning, the calves born to dams in CInt_1 had a higher plasma nonesterified fatty acids (NEFA) concentration (0.34 mmol/L; CI: 0.30, 0.37) than CInt_2 (0.28 mmol/L; CI: 0.26, 0.31) and CInt_3 (0.26 mmol/L; CI: 0.24, 0.29) calves. Calves born to dams with a shorter CInt (CInt_1) had greater IgG and IgM against keyhole limpet hemocyanin (KLH) than CInt_3 (IgG: 6.05 ± 0.30 vs. 4.64 ± 0.30; IgM: 6.45 ± 0.17 vs. 5.89 ± 0.16, respectively; mean ± SE) before weaning. After weaning till calving, CInt_1 calves tended to have greater plasma NEFA concentration than CInt_3-calves. During the first 100 DIM, a longer CInt of the dams resulted in lower plasma IGF_1 (CInt_2), lower milk lactose (CInt_3), and lower fat- and protein-corrected milk (FPCM; CInt_2) in offspring, compared with shorter CInt of the dams (CInt_1). Collectively, a longer CInt in dams did not affect birth weight of their calves or BW during the weaning or rearing phase. From birth till weaning, a longer CInt in dams resulted in less anti-KLH IgG and lower plasma NEFA concentration in plasma of the calves. During the first lactation of their offspring, a longer CInt in dams can result in a lower plasma IGF_1 and FPCM during the first 100 DIM, although effects were not present in all CInt categories.

Phenotyping metabolic status of dairy cows using clustering of time profiles of energy balance peripartum

Due to a combination of a relatively low energy intake and a high demand of energy required for milk production, dairy cows experience a negative energy balance (EB) at the start of lactation. This energy deficit causes body weight reduction and an increased risk for metabolic diseases. Severity and length of negative EB can differ among cows. Peripartum time profiles of EB for dairy cows are not described yet in the literature. Creating EB-derived time profiles with corresponding metabolic status and disease treatments could improve understanding the relationship between EB and metabolic status, as well as enhance identification of cows at risk for compromised metabolic status. In this research we propose a novel method to cluster EB time series and examine associated metabolic status and disease treatments of dairy cows in the peripartum period. In this study, data of 3 earlier experiments were merged and examined. Four dairy cow clusters for time profiles of EB from wk -3 until +7 relative to calving were generated by the global alignment kernel algorithm. For each cluster, mean of body weight prepartum was distinguishable, indicating this might be a possible on-farm biomarker for the peripartum EB profile. Moreover, cows with severe EB drop postpartum were more treated for milk fever and had high plasma nonesterified fatty acids and β-hydroxybutyrate concentration, and low IGF-1, insulin, and glucose concentration in the first 7 wk of lactation. Overall, this study demonstrated that cows can be clustered based on EB time profiles and that characteristics such as prepartum body weight, and postpartum nonesterified fatty acids and glucose concentration are promising biomarkers to identify the time profile of EB and potentially the risk for metabolic diseases.

NEFA Promotes Autophagosome Formation through Modulating PERK Signaling Pathway in Bovine Hepatocytes.

Simple SummaryNegative energy balance can lead to the mobilization of large amounts of body fat. A large amount of non-esterified fatty acids (NEFA) produced by lipolysis not only can be used for energy generation by β-oxidation in the liver but can also act as a potential regulator of lipid metabolism. The present study provides evidence that NEFA can activate hepatocyte autophagy through the protein kinase R-like endoplasmic reticulum kinase (PERK) signaling pathway. Autophagy has been reported to contribute to lipid metabolism through promoting the breakdown of intracellular lipids. These findings enable a better understanding of the redistribution and utilization of energy during the perinatal period of dairy cows.During the perinatal period, the abnormally high plasma non-esterified fatty acids (NEFA) concentration caused by the negative energy balance (NEB) can impose a significant metabolic stress on the liver of dairy cows. Endoplasmic reticulum (ER) stress is an important adaptive response that can serve to maintain cell homeostasis in the event of stress. The protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is the most rapidly activated cascade when ER stress occurs in cells and has an important impact on the regulation of hepatic lipid metabolism and autophagy modulation. However, it is unknown whether NEFA can affect autophagy through modulating the PERK pathway, under NEB conditions. In this study, we provide evidence that NEFA treatment markedly increased lipid accumulation, the phosphorylation level of PERK and eukaryotic initiation factor 2α (eIF2α), and the expression of glucose-regulated protein 78 (Grp78), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). More importantly, NEFA treatment can cause a substantial increase in the protein levels of autophagy-related gene 7 (ATG7), Beclin-1 (BECN1), sequestosome-1 (p62), and microtubule-associated protein 1 light chain 3 (LC3)-II, and in the number of autophagosomes in primary bovine hepatocytes. The addition of GSK2656157 (PERK phosphorylation inhibitor) can significantly inhibit the effect of NEFA on autophagy and can further increase lipid accumulation. Overall, our results indicate that NEFA could promote autophagy via the PERK pathway in bovine hepatocytes. These findings provide novel evidence about the potential role of the PERK signaling pathway in maintaining bovine hepatocyte homeostasis.

Reducing hepatic endoplasmic reticulum stress ameliorates the impairment in insulin signaling induced by high levels of β-hydroxybutyrate in bovine hepatocytes

Ketotic dairy cows exhibit a state of negative energy balance (NEB) characterized by elevated circulating levels of β-hydroxybutyrate (BHB) and fatty acids. Impaired hepatic insulin signaling in dairy cows occurs frequently during the transition into lactation, but its role on liver function during this period is not well known. In nonruminants, endoplasmic reticulum (ER) stress is a causal factor contributing to impaired insulin signaling in the liver. Thus, the aim of this study was to investigate the status of hepatic insulin and ER stress signaling and whether ER stress contributes to impaired insulin signaling in dairy cows with ketosis. Healthy (control cows, n = 10, BHB ≤0.6 mM) and ketotic (ketotic cows, n = 10, BHB ≥1.2 mM) cows at 3 to 10 d in milk were selected for liver biopsy and blood sampling before feeding. In vitro experiments were conducted with isolated hepatocytes from 5 healthy calves (1 d old, fasted female, 30-40 kg of body weight). Treatments included BHB (0, 0.9, 1.8, 3.6 mM), tauroursodeoxycholic acid (TUDCA, a canonical inhibitor of ER stress), and different incubation times (0.5, 1, 2, 3, 5, 7, 9, or 12 h). Ketotic cows had lower daily milk yield (median: 29.50 vs. 23.00 kg), higher plasma nonesterified fatty acid (NEFA) (median: 0.33 vs. 1.17 mM), BHB (median: 0.43 vs. 3.22 mM), aspartate aminotransferase (median: 70.58 vs. 155.70 U/L), alanine aminotransferase (median: 18.31 vs. 37.90 U/L), lower plasma glucose (median: 4.32 vs. 2.37 mg/dL), and revised quantitative insulin sensitivity check index (median: 0.39 vs. 0.37) compared with healthy cows. Increased abundance of phosphorylated insulin receptor substrate-1 (IRS1) and decreased abundance of phosphorylated protein kinase B (AKT) and glycogen synthase kinase-3β (GSK3β) in ketotic cows indicated a state of insulin resistance. In addition, abundance of phosphorylated protein kinase RNA-like ER kinase (PERK) and inositol requiring protein-1α (IRE1α), and cleavage of activating transcription factor-6 (ATF6) were greater in the liver of ketotic cows. In vitro, at the early stages of incubation, treatment with BHB upregulated abundance of phosphorylated of IRE1α, PERK, and the cleavage of ATF6, as well as several unfolded protein response genes [X-box-binding protein-1 (XBP1), 78 kDa glucose-regulated protein (GRP78), and C/EBP homologous protein (CHOP)]. Furthermore, in response to increasing doses of BHB, the phosphorylation level of PERK, IRE1α, and the cleavage of ATF6, and the abundance of XBP1, GRP78, and CHOP increased. In addition, BHB treatment increased phosphorylation of IRS1 and decreased phosphorylation of AKT and GSK3β, and upregulated abundance of gluconeogenic genes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase). Importantly, these changes were reversed by inhibiting ER stress with TUDCA treatment. Overall, the present study indicated that reversing ER stress during ketosis might help alleviate hepatic insulin resistance. Targeting ER stress may represent a potential therapeutic target for controlling the negative aspects of ketosis on liver function.

Dietary Isomalto/Malto-Polysaccharides Increase Fecal Bulk and Microbial Fermentation in Mice.

The prevalence of metabolic-syndrome-related disease has strongly increased. Nutritional intervention strategies appear attractive, particularly with novel prebiotics. Isomalto/malto-polysaccharides (IMMPs) represent promising novel prebiotics that promote proliferation of beneficial bacteria in vitro. The present study investigates for the first time the in vivo effects of IMMP in mice. C57BL/6 wild-type mice received control or IMMP-containing (10%, w/w) diets for 3 weeks. IMMP leads to significantly more fecal bulk (+26%, p<0.05), higher plasma non-esterified fatty acids (colorimetric assay, +10%, p<0.05), and lower fecal dihydrocholesterol excretion (mass spectrometry, -50%, p<0.05). Plasma and hepatic lipid levels (colorimetric assays following lipid extraction) are not influenced by dietary IMMP, as are other parameters of sterol metabolism, including bile acids (gas chromatography/mass spectrometry). IMMP is mainly fermented in the cecum and large intestine (high-performance anion exchange chromatography). Next-generation sequencing demonstrates higher relative abundance of Bacteroides and butyrate producers (Lachnospiraceae, Roseburia Odoribacter) in the IMMP group. The combined results demonstrate that IMMP administration to mice increases fecal bulk and induces potentially beneficial changes in the intestinal microbiota. Further studies are required in disease models to substantiate potential health benefits.

Physical exercise prepartum to support metabolic adaptation in the transition period of dairy cattle: A proof of concept.

In dairy cattle, the hormonal changes around calving induce large metabolic changes to support milk production. Mobilization of adipose reserves is one of the changes involved, imposing a metabolic load on the liver. We hypothesized that the risk for excessive lipolysis and hepatic lipidosis postpartum can be reduced by starting fat mobilization and processing during the prepartum period by physical exercise, especially in cows with a high body condition score (BCS). As a proof of concept, 32 pregnant Holstein‐Friesian dairy cows were selected for a 2 × 2 experimental design. Sixteen cows had a BCS < 3.25 (group LOW) and 16 cows a BCS ≥ 3.25 (group HIGH). Cows within each group were randomly allocated to one of two treatments: group STEP was walked twice daily for 45 min during the dry period while group CON remained indoors. Treatment was stopped at calving and cows were monitored until 6 weeks after calving. Liver biopsies were taken in a subset of 16 cows to determine liver triglyceride (TG) concentration. We found that calculated energy balance was more negative for group STEP prepartum, resulting in higher plasma non‐esterified fatty acids and β‐hydroxybutyrate concentrations. During the first 6 weeks postpartum, neither dry matter intake nor milk yield was affected by exercise. As expected, the cows in group HIGH had increased liver TG concentrations postpartum relative to group LOW with increased plasma non‐esterified fatty acids directly after calving. Exercise during the dry period mitigated postpartal liver TG accumulation, but this did not seem to be related to increased plasma lipoprotein transport. We conclude that substantial physical activity prepartum can induce lipolysis and lipid utilization, thereby starting an early adaptation to lactation. This may be instrumental to reduce the risk for excessive liver TG accumulation postpartum, especially in cows with a high BCS at dry‐off.

Characterization of the endocannabinoid system in subcutaneous adipose tissue in periparturient dairy cows and its association to metabolic profiles.

Adipose tissue (AT) plays a major role in metabolic adaptations in postpartum (PP) dairy cows. The endocannabinoid (eCB) system is a key regulator of metabolism and energy homeostasis; however, information about this system in ruminants is scarce. Therefore, this work aimed to assess the eCB system in subcutaneous AT, and to determine its relation to the metabolic profile in peripartum cows. Biopsies of AT were performed at 14 d prepartum, and 4 and 30 d PP from 18 multiparous peripartum cows. Cows were categorized retrospectively according to those with high body weight (BW) loss (HWL, 8.5 ± 1.7% BW loss) or low body weight loss (LWL, 2.9 ± 2.5% BW loss) during the first month PP. The HWL had higher plasma non-esterified fatty acids and a lower insulin/glucagon ratio PP than did LWL. Two-fold elevated AT levels of the main eCBs, N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), were found 4 d PP compared with prepartum in HWL, but not in LWL cows. AT levels of the eCB-like molecules oleoylethanolamide, palmitoylethanolamide, and of arachidonic acid were elevated PP compared with prepartum in all cows. The abundance of monoglyceride lipase (MGLL), the 2-AG degrading enzyme, was lower in HWL vs. LWL AT PP. The relative gene expression of the cannabinoid receptors CNR1 and CNR2 in AT tended to be higher in HWL vs. LWL PP. Proteomic analysis of AT showed an enrichment of the inflammatory pathways’ acute phase signaling and complement system in HWL vs. LWL cows PP. In summary, eCB levels in AT were elevated at the onset of lactation as part of the metabolic adaptations in PP dairy cows. Furthermore, activating the eCB system in AT is most likely associated with a metabolic response of greater BW loss, lipolysis, and AT inflammation in PP dairy cows.

NEFA-Sensitive Orai1 Expression in Regulation of De Novo Lipogenesis

Background/Aims: Non-esterified fatty acids (NEFAs) are important inducers of inflammatory responses and hepatic lipid accumulation, which lead to non-alcoholic fatty liver disease (NAFLD). High plasma NEFA is found in NAFLD patients, and associated with metabolic syndrome and type-2 diabetes. NFκB is known to upregulate Orai1, the Ca²⁺ channel responsible for store-operated Ca²⁺ entry. The present study explored the role of NEFA-sensitive NFκB-dependent Orai1 expression in the regulation of lipid synthesis. Methods: BRL-3A rat liver hepatocyte lines were studied in the absence and presence of NEFA. Transcript and protein expression levels of factors involved in lipid synthesis were quantified by quantitative polymerase chain reaction (qPCR) and western blot analyses. Fatty acids were measured by immunofluorescence. Results: NEFA significantly increased, as indicated by the expression of sterol regulatory element-binding protein 1 (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA carboxylase α (ACC1), Orai1, and NFκB p65 by qPCR and western blot analyses. These effects were reversed by the Orai1 inhibitor, 2-aminoethoxydiphenyl borate, and the NFκB inhibitor, wogonin. Furthermore, SREBP-1c, FAS, ACC1, and Orai1 were significantly decreased by Orai1 silencing. Conclusions: Taken together, these results demonstrated that NEFA-sensitive NFκB-dependent Orai1 expression regulates de novo lipogenesis.

Metabolic and Ruminal Fluid Markers of Dairy Cows Supplemented with a Combination of Yeast Culture and Hydrolyzed Yeast

Background: In order to reduce the effects of a negative energy balance, some measures have been taken into account in nutritional management during the transition period. The use of yeast, has been a good alternative used to improve the rumen metabolism and helping the adjustment of the microbiotato the new diet. The aim of the study was to evaluate the effects of supplementing a combination of yeast culture and hydrolyzed yeast on the metabolism of dairy cows during the transition period.Materials, Methods &amp; Results: The experiment was conducted in a semi-extensive system, using 20 Holstein cows, divided equally into a control group (CG) and a supplemented group (SG). The SG received 28 g/animal/day of a combination of yeast culture and hydrolyzed yeast from 20 ± 2 days pre-calving until early lactation (18 ± 3 days). Serum concentrations of non-esterified fatty acids (NEFA), albumin and urea were determined at calving, and for three time points during the early postpartum period and three time points during the early lactation period. Regarding energy metabolism, prepartum concentrations of NEFA were higher than the physiological standard in both groups. However, NEFA, albumin and urea decreased during the early postpartum period in the supplemented animals and could be attributed to the yeast in enhancing ruminal microorganisms’ cellulolytic capacity, increasing fibre digestibility and starch utilization.Discussion: The increased concentration of non-esterified fatty acids (NEFA) due to the mobilization of fat deposits that happens in the transition period, especially in the postpartum period reflects the cow’s adaptation to the negative energy balance (NEB). The lower concentrations of NEFA observed in the present study could be attributed to the effect of the yeast in enhancing the ruminal microorganisms’ cellulolytic capacity. The control cows had a BCS within the recommended range while the supplemented group had it close to the minimal limit proposed for this period. Thus, supplemented cows lost less BCS during the early postpartum period, had a lower BCS loss during the experimental period and had lower NEFA concentration that the CG. It was possible to observe a difference in serum albumin and urea between treatments only in the postpartum period. Besides showing no significant effect in BCS on prepartum period, control cows had a BCS within the recommended range while the supplemented group had it close to the minimal limit proposed for this period. Cows with high BCS prepartum had higher plasma NEFA before and after calving. It can be observed in the present study in both groups. However, a positive effect in prevent subclinical disorders might be attributed to YC, since the SG showed low NEFA plasma levels compared to the CG. Thus, supplemented cows lost less BCS during the early postpartum period, had a lower BCS loss during the experimental period and had lower NEFA concentration that the CG. There is a negative correlation between BCS and NEFA in the early postpartum period and this information explains the results observed in the present study where BCS declines in the SG are followed by a NEFA increase. This is not so marked in the CG, indicating that SG supplementation can act by improving digestibility. Yeast supplementation promotes higher output energy, enhancing postpartum performance in dairy cows. Yeast supplementation showed benefits in early lactation compared to the prepartum and early postpartum periods, suggesting that supplementation has to have an adaptation period to be effective in protein synthesis. In conclusion, supplementation with a combination of yeast culture and hydrolyzed yeast to cows during the transition period can positively influence the energy and protein metabolism, reducing the collateral effects of negative energy balance.

The metabolic response in fish to mildly elevated water temperature relates to species-dependent muscular concentrations of imidazole compounds and free amino acids

Fish species show distinct differences in their muscular concentrations of imidazoles and free amino acids (FAA). This study was conducted to investigate whether metabolic response to mildly elevated water temperature (MEWT) relates to species-dependent muscular concentrations of imidazoles and FAA. Thirteen carp and 17 Nile tilapia, housed one per aquarium, were randomly assigned to either acclimation (25°C) or MEWT (30°C) for 14 days. Main muscular concentrations were histidine (HIS; P<0.001) in carp versus N-α-acetylhistidine (NAH; P<0.001) and taurine (TAU; P=0.001) in tilapia. Although the sum of imidazole (HIS+NAH) and TAU in muscle remained constant over species and temperatures (P>0.05), (NAH+HIS)/TAU ratio was markedly higher in carp versus tilapia, and decreased with MEWT only in carp (P<0.05). Many of the muscular FAA concentrations were higher in carp than in tilapia (P<0.05). Plasma acylcarnitine profile suggested a higher use of AA and fatty acids in carp metabolism (P<0.05). On the contrary, the concentration of 3-hydroxyisovalerylcarnitine, a sink of leucine catabolism, (P=0.009) pointed to avoidance of leucine use in tilapia metabolism. Despite a further increase of plasma longer-chain acylcarnitines in tilapia at MEWT (P=0.009), their corresponding beta-oxidation products (3-hydroxy-longer-chain acylcarnitines) remained constant. Together with higher plasma non-esterified fatty acids (NEFA) in carp (P=0.001), the latter shows that carp, being a fatter fish, more readily mobilises fat than tilapia at MEWT, which coincides with more intensive muscular mobilization of imidazoles. This study demonstrates that fish species differ in their metabolic response to MEWT, which is associated with species-dependent changes in muscle imidazole to taurine ratio.

Fetuin-A dynamics in transition cows' plasma

Around parturition and during early lactation, cows enter a state of negative energy balance (NEB) driven by the onset of lactation and a reduced dry matter intake. The major metabolic adaptation to NEB is adipose tissue lipolysis that releases nonesterified fatty acids (NEFA) into systemic circulation. High plasma NEFA predisposes cows to metabolic and inflammatory based diseases that negatively impact animal welfare and increase economic losses to dairy producers. However, not all cows with increased plasma NEFA concentrations develop a disease, thus underscoring the need for novel lipid mobilization-related biomarkers that could improve accuracy of disease prediction. In humans, high fetuin-A concentrations have been identified as an independent marker of insulin resistance and a predictor of type 2 diabetes and non-alcoholic fatty liver. Furthermore, elevated human fetuin-A concentrations are strongly associated with dyslipidemias. Fetuin-A is an abundant carrier of NEFA in plasma and is mainly expressed in hepatocytes, monocytes/macrophages, and adipocytes. Fetuin-A inhibits insulin receptor signaling and therefore increases lipolysis and NEFA efflux from adipose tissues. Despite its strong link with lipolytic processes, fetuin-A expression in plasma and its relationship with metabolic markers in transition cows remains poorly understood. The objective of this study was to evaluate the dynamics of serum fetuin-A and its association with NEFA during the transition period.

89 EFFECT OF NONESTERIFIED FATTY ACIDS ON IN VIVO OR VITRO EMBRYO PRODUCTION IN KOREAN NATIVE CATTLE (HANWOO)

Recent reports suggest that high concentrations of nonesterified fatty acids (NEFA) negatively affect oestrous cycle, fertility, in vitro oocyte maturation, embryo quality, and viability. This study was performed to determine the relationship between plasma concentration of NEFA and embryo quality in Hanwoo cattle. In experiment 1, embryo recovery rate from superovulated donor cows 7 days after AI was evaluated. Donors, at random stages of the oestrous cycle, received a CIDR (Day 0). On Day 7, 200 mg of FSH was administered followed by 40, 30, 20, and 10 mg of FSH in declining doses twice daily by intramuscular injection for 4 days. On the third day of FSH administration, 25 mg of prostaglandin F2a was given and the CIDR was withdrawn. After FSH injections were complete, donors were artificially inseminated twice on Day 11 and 12 at 12-h intervals. At first AI, 250 μg of gonadotropin-releasing hormone (GnRH) was administered. During embryo collection, plasma samples were obtained from jugular veins to measure NEFA concentrations by chemistry analyzer. Next, the effect of NEFA on embryo development in vitro was examined (experiment 2). After in vitro maturation and fertilization of abattoir oocytes using standard procedures, zygotes were cultured in mSOFaa supplemented with 5% (vol/vol) oestrous cow serum (ECS) collected from 8 random cows representing 8 different NEFA concentrations (72.6, 126, 175.5, 244.6, 311, 393, 527.3, and 979 g dL–1) and compared with mSOFaa without serum (no-serum control). Statistical analysis was performed by ANOVA (SAS 9.1, SAS Institute Inc., Cary, NC, USA) and Duncan's multiple range tests where appropriate. Recovery of total and transferable morulae and blastocysts was related to NEFA levels of donor cows. The donor group with the lowest plasma NEFA levels yielded the most embryos, of which most were high quality (n = 3, 173 ± 11 g dL–1 NEFA; 14 ± 3 total recovered embryos of which 85 ± 7% were transferable). Higher NEFA plasma levels reduced both the absolute number of embryos recovered and the fraction of transferable embryos (n = 8, 301 ± 20 g dL–1 NEFA with more than the 10 recovered of which 56 ± 5% were transferable; n = 6, 301.5 ± 37 g dL–1 NEFA with 8 ± 2 total embryos recovered of which 19 ± 8% were transferable; n = 4, 288.5 ± 58 g dL–1 NEFA with &lt;7 embryos recovered of which 45 ± 4% were transferable). In experiment 2, cleavage and blastocyst rates were not significantly different (P &gt; 0.05) between the groups. However, embryos exposed to the 2 lowest concentrations of NEFA showed a higher hatching rate compared with control and embryos exposed to the 3 highest NEFA levels (126 and 175.5 g dL–1 NEFA: 75.33, 77.67% hatching respectively v. control, 979, 527.3, 393 g dL–1 NEFA: 48.33, 32.78, 45.33, 44.52% hatching; P &lt; 0.05). However, embryo developmental rate was highly variable. Our data suggest that high plasma NEFA concentrations can have negative effects on in vivo and in vitro embryo production, whereas low levels may be beneficial.This research was supported by Bio-industry Technology Development Program (No.112130-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Seasonal fertility differences in synchronised dairy cows: Ultrasonic, metabolic and endocrine findings

The aim of this study was to identify relationships among seasonal differences of fertility, metabolic parameters and appearance of irregular luteal forms in high-yielding dairy cows. Holstein-Friesian cows were put on the Provsynch regimen in winter (n = 10) and in summer (n = 10). Blood sampling (starting 35 days post partum) and rectal ultrasound examinations (starting post insemination) were carried out once a week in each examination period. Metabolic [plasma nonesterified fatty acid (NEFA) and beta-hydroxybutyrate (BHB) levels, ferric reducing ability of plasma (FRAP) and serum beta-carotene] and endocrine parameters [plasma thyroxine (T4), triiodothyronine (T3), insulin-like growth factor (IGF-I) and insulin levels] were measured. In summer, two cows were excluded from the study because of metritis and none of the remaining animals became pregnant, but 6 of the 8 cows had irregular luteal forms (ILF) on their ovaries. In winter, one cow was excluded because of metritis and 6 of the 9 cows became pregnant, while 2 of the 3 open cows had irregular luteal forms. In summer the mean plasma NEFA and BHB concentrations were significantly higher, while serum carotene and plasma IGF-I concentrations were significantly lower than in winter. The high plasma NEFA concentration found in summer seemed to be in association with the lower body condition score (BCS) caused by depressed appetite. In conclusion, statistical analysis supports the hypothesis that increased plasma NEFA and BHB and decreased plasma IGF-I concentrations may result in reduced fertility in summer. These changes may be associated with the more frequent appearance of ILFs and probably have a negative effect on ovarian function and/or oocyte quality.

Effects of plane of nutrition and 2,4-thiazolidinedione on insulin responses and adipose tissue gene expression in dairy cattle during late gestation

Specific mechanisms by which dry period dietary energy affects transition cow metabolism have been intensively investigated but those of thiazolidinedione (TZD) administration have not. We hypothesized that effects of both are mediated via changes in insulin, glucose, or fatty acid metabolism. The objective of this experiment was to determine the effects of the insulin-sensitizing agent TZD and dietary energy level on glucose and fatty acid metabolism during late gestation in dairy cows. Multiparous Holstein cows (n=32) approximately 50 d before expected calving date were dried-off and assigned to 1 of 2 dietary energy levels for 3 wk (high: 1.52Mcal/kg of NEL, or low: 1.34Mcal/kg of NEL) and treated daily during the final 14 d with 4.0mg of TZD/kg of body weight (BW) or saline in a completely randomized design. Cows fed the low energy diet had lower dry matter intake (12.8 vs. 16.1kg/d) and higher plasma nonesterified fatty acid (NEFA) concentrations (103.3 vs. 82.4μEq/L) compared with cows fed the high energy diet. Cows administered TZD had higher plasma glucose concentrations (62.5 vs. 59.6mg/dL) than saline controls and cows fed the high energy diet had higher plasma insulin concentrations (35.1 vs. 25.3μU/mL) compared with those fed the low energy diet. After 2 wk of TZD treatment, all cows were subjected to an intravenous glucose tolerance test (GTT; 0.25g of dextrose/kg of BW) followed 110min later by an insulin challenge (IC; 1.0μg of insulin/kg of BW). Differences in plasma glucose response to GTT were minimal based on diet; however, cows fed the low energy diet had more negative NEFA areas under the curve (AUC; −4,838 vs. −2,137μEq/L × min over 90min) and greater rates of NEFA decrease (1.35 vs. 0.63%/min) during GTT, suggesting differential responses of tissue glucose and fatty acid metabolism in response to dietary energy level. During IC, the TZD-treated cows tended to have more negative glucose AUC (−45.0 vs. −12.1mg/dL × min over 15min) than controls, suggesting that TZD-treated cows had greater responses to insulin. Limited interactions were observed between dietary and TZD treatments in all response variables measured. Adipose tissue biopsies performed on the final day of treatment suggested higher expression of peroxisome proliferator-activated receptor-γ (0.71 vs. 0.50 relative expression) and lipoprotein lipase (0.71 vs. 0.40 relative expression) in cows fed the high energy diet as measured by quantitative real-time PCR. These results indicate that energy level and insulin-sensitizing agents affect glucose and lipid metabolism during the dry period.

Coordinated gene expression in adipose tissue and liver differs between cows with high or low NEFA concentrations in early lactation

Dairy cows with high and low plasma non-esterified fatty acid (NEFA) concentrations in early lactation were compared for plasma parameters and mRNA expression of genes in liver and subcutaneous adipose tissue. The study involved 16 multiparous dairy cows with a plasma NEFA concentration of >500 μmol/l [n = 8, high NEFA (HNEFA)] and <140 μmol/l [n = 8, low NEFA (LNEFA)] in the first week post-partum (pp). Blood samples, adipose and liver tissues were collected on day 1 (+1d) and at week 3 pp (+3wk). Blood plasma was assayed for concentrations of metabolites and hormones. Subcutaneous adipose and liver tissues were analysed for mRNA abundance by real-time qRT-PCR encoding parameters related to lipid metabolism. Results showed that mean daily milk yield and milk fat quantity were higher in HNEFA than in LNEFA cows (p < 0.01), and the NEB was more negative in HNEFA than in LNEFA in +3wk too (p < 0.05). HNEFA cows had slightly lower (p < 0.1) insulin concentrations than LNEFA cows across the study period, and the body condition score decreased more from +1d to +3wk in HNEFA than in LNEFA (p = 0.09). The mRNA abundance of genes in the liver related to fatty acid oxidation (carnitine palmitoyltransferase 2 and very long chain acyl-coenzyme A dehydrogenase) and ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2) were lower in HNEFA than in LNEFA cows. No differences between the two groups were observed for mRNA expression of genes in adipose tissue. The number of calculated significant correlation coefficients (moderately strong) between parameters in the liver and in adipose tissue was nearly similar on +1d, and higher for HNEFA compared with LNEFA cows in +3wk. In conclusion, dairy cows with high compared with low plasma NEFA concentrations in early lactation show differentially synchronized mRNA expression of genes in adipose tissue and liver in +3wk that suggests a different orchestrated homeorhetic regulation of lipid metabolism.

Changes of glycogen content in liver, skeletal muscle, and heart from fasted rats

Glycogen content of white and red skeletal muscles, cardiac muscle, and liver was investigated in conditions where changes in plasma levels of non-esterified fatty acids (NEFA) occur. The experiments were performed in fed and 12 and 48 h-fasted rats. The animals were also submitted to swimming for 10 and 30 min. Glycogen content was also investigated in both pharmacologically induced low plasma NEFA levels fasted rats and pharmacologically induced high plasma NEFA levels fed rats. The participation of Akt and glycogen synthase kinase-3 (GSK-3) in the changes observed was investigated. Plasma levels of NEFA, glucose, and insulin were determined in all conditions. Fasting increased plasma NEFA levels and reduced glycogen content in the liver and skeletal muscles. However, an increase of glycogen content was observed in the heart under this condition. Akt and GSK-3 phosphorylation was reduced during fasting in the liver and skeletal muscles but it remained unchanged in the heart. Our results suggest that in conditions of increased plasma NEFA levels, changes in insulin-stimulated phosphorylation of Akt and GSK-3 and glycogen content vary differently in liver, skeletal muscles, and heart. Akt and GSK-3 phosphorylation and glycogen content are decreased in liver and skeletal muscles, but in the heart it remain unchanged (Akt and GSK-3 phosphorylation) or increased (glycogen content) due to consistent increase of plasma NEFA levels.

Dyslipidemia in obese cats

Obesity is an important endocrine disorder in cats and is a risk factor for diabetes similar to humans. The goal of this study was to examine the effect of long-term obesity and different diets (high protein, and high carbohydrate supplemented with saturated fatty acids or n-3 polyunsaturated fatty acids) on plasma lipids in the fasted and fed states in 12 lean (LEAN) and 12 obese (OBESE) cats with ultracentrifugation, and nuclear magnetic resonance spectroscopy. OBESE had higher plasma non-esterified fatty acids and triglycerides, as well as very-low-density-lipoproteins (VLDL) consisting primarily of medium-sized particles. The concentration of low-density-lipoproteins (LDL) was comparable between the groups, although OBESE had mostly very small, whereas LEAN had mostly large particles. The concentration of high-density-lipoproteins (HDL) was lower in OBESE and consisted primarily of small particles. Plasma triglycerides, and triglycerides and cholesterol in all lipoproteins increased postprandially. Different diets had little effect on lipids. Our results show that long-term obese cats develop similar lipoprotein changes to humans, yet, hypertension and atherosclerosis have not been described in obese cats. This suggests that dyslipidemia alone is not sufficient to induce hypertension and atherosclerosis. Other anti-atherogenic factors may be present in the obese, dyslipidemic cat.

Effect of nutrition from mid to late pregnancy on the performance of twin- and triplet-bearing ewes and their lambs

This experiment was conducted to determine the effect of two different feeding levels on ewes during mid and late pregnancy on lamb birthweight and growth to weaning. Twin- and triplet-bearing Romney ewes (n = 80 and 56, respectively) were allocated to one of two feeding regimes and provided pastures with an average sward height of less than 2 cm (~700 kg dry matter/ha) or greater than 4 cm (~1300 kg dry matter/ha). Ewes were allocated to these feeding regimes during mid pregnancy (day 70–107 of pregnancy; P70–P107) and were reallocated in late pregnancy (P108–P147). This resulted in four sward height treatments: 2–2, 2–4, 4–2 and 4–4. During mid pregnancy, ewes provided with &gt;4-cm-high swards had liveweight gains of 262–290 g/day compared with 12–31 g/day for ewes provided with &lt;2-cm-high swards. In late pregnancy, ewe liveweight gains were influenced by the ewes’ previous nutritional treatment. Ewes in the 2–4 treatment had higher daily gains (538 g/day) than 4–4 ewes (343 g/day). In addition, 4–2 ewes gained 90 g/day compared with 247 g/day for 2–2 ewes. Throughout pregnancy, triplet-bearing ewes were heavier (P &lt; 0.05) than twin-bearing ewes, but during lactation ewe weights were similar. On P130, ewes provided with &lt;2-cm-high swards (4–2 and 2–2) had greater β-hydroxybutyrate (P &lt; 0.05) and lower glucose (P &lt; 0.05) plasma concentrations than ewes provided with &gt;4-cm-high swards (2–4 and 4–4). On P139, however, ewes provided with 4–4 swards had higher plasma non-esterified fatty acid and β-hydroxybutyrate concentrations than ewes in all other treatments. Lambs born to ewes in the 4–4 treatment had the greatest birthweights (P &lt; 0.05), whereas lambs born to 2–2 ewes had the lowest birthweights. The birthweight of lambs born to 2–4 ewes was similar to that of lambs born to ewes provided with 4–2 and 4–4 swards. At weaning, ewe weights were similar between ewe nutritional treatments and ewes bearing twin or triplet fetuses. These findings suggest that when pasture growth is inadequate to provide pasture sward heights of 4 cm throughout pregnancy, restricted intake in mid-pregnancy may be partially compensated by providing additional pasture in late pregnancy.

Skeletal muscle subsarcolemmal mitochondrial dysfunction in high-fat fed rats exhibiting impaired glucose homeostasis

To investigate whether changes in body energy balance induced by long-term high-fat feeding in adult rats could be associated with modifications in energetic behaviour and oxidative stress of skeletal muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial populations. Adult rats were fed low-fat or high-fat diet for 7 weeks. Body energy balance and composition analysis together with plasma insulin and glucose level determination in the whole animal. Oxidative capacity, basal and induced proton leaks as well as aconitase and superoxide dismutase activities in SS and IMF mitochondria from skeletal muscle. High-fat fed rats exhibit increased body lipid content, as well as hyperinsulinemia, hyperglycaemia and higher plasma non-esterified fatty acids. In addition, SS mitochondria display lower respiratory capacity and a different behaviour of SS and IMF mitochondria is found in the prevention from oxidative damage. A deleterious consequence of decreased oxidative capacity in SS mitochondria from rats fed high-fat diet would be a reduced utilization of energy substrates, especially fatty acids, which may lead to intracellular triglyceride accumulation, lipotoxicity and insulin resistance development. Our results thus reveal a possible role for SS mitochondria in the impairment of glucose homeostasis induced by high-fat diet.