Comparative assessment of emulsifiers for in vitro ruminal gas production and fermentation measurements: Tween 80 is a suitable emulsifier.

Emulsifiers are essential for achieving a homogenous distribution of lipophilic supplements in in vitro rumen fluid incubations. Since emulsifiers can alter rumen fermentation, it is crucial to select one that minimally impacts fermentation parameters to reduce potential biases. This study aimed to evaluate seven emulsifiers' impact on in vitro ruminal fermentation using the Hohenheim Gas Test in order to identify the most inert emulsifier. Rumen fluids were collected from three non-lactating Original Brown-Swiss cannulated cows before morning feeding and incubated for 24 h with a basal diet in triplicates. The emulsifiers tested were ethanol, ethyl acetate, propylene glycol, glycerol, ethylene glycol, soy lecithin, and Tween® 80, each in two dosages (0.5% or 1% v/v). The untreated basal diet served as control. Compared to control, in vitro organic matter digestibility was enhanced by ethyl acetate (by 36.9 and 48.2%), ethylene glycol (by 20.6 and 20.1%), glycerol (by 46.9 and 56.8%) and soy lecithin (by 19.7 and 26.8%) at 0.5 and 1% dosage, respectively. Additionally, the 24-h methane production increased for ethanol (by 41.9 and 46.2%), ethylene glycol (by 50.5 and 51.5%), and glycerol (by 63.1 and 65.4%) for the 0.5 and 1% dosage, respectively, and 0.5% dosage for ethyl acetate (by 31.6%). The acetate molar proportion was 17.2%pt higher for ethyl acetate, and 25.5%pt lower for glycerol at 1% dosage, compared to the control. The propionate concentration was 22.1%pt higher 1% glycerol, and 15.2%pt and 15.1%pt higher for 0.5 and 1% propylene glycol, respectively, compared to the control. In summary, Tween® 80 did not significantly affect in vitro rumen fermentation parameters, making it the most suitable choice for in vitro incubations involving lipophilic substances in rumen fluid. Ethanol may be considered as an alternative emulsifier if methane production is not the variable of interest.

Bioactive Peptide Discovery from Edible Insects for Potential Applications in Human Health and Agriculture.

In the past decade, there has been fast-growing interest among researchers to discover bioactive peptides from edible insects and to evaluate their potential applications in the management of human, livestock, and plant health. This review summarizes current knowledge of insect-derived peptides and their potential role in tackling human health issues and solving agriculture problems by protecting crops and livestock against their pathogens. Numerous bioactive peptides have been identified from edible insect species, including peptides that were enzymatically liberated from insect proteins and endogenous peptides that occur naturally in insects. The peptides exhibited diverse bioactivities, encompassing antioxidant, anti-angiotensin-converting enzyme, anti-dipeptidyl peptidase-IV, anti-glucosidase, anti-lipase, anti-lipoxygenase, anti-cyclooxygenase, anti-obesity, and hepatoprotective activities. Such findings point to their potential contribution to solving human health problems related to inflammation, free radical damage, diabetes, hypertension, and liver damage, among others. Although most of the experiments were performed in vitro, evidence for the in vivo efficacy of some peptides is emerging. Evidence of the protective effects of insect-derived endogenous antimicrobial peptides in combating farm animal and plant pathogens is available. The ability of insect-derived endogenous neuropeptides to protect plants against herbivorous insects has been demonstrated as well. Nevertheless, the potency of peptides identified from insect protein hydrolysates in modulating livestock and plant health remains a knowledge gap to be filled.

In vitro ruminal fermentation, methane production and nutrient degradability as affected by fruit and vegetable pomaces in differing concentrations.

Pomaces are food industry by-products and may serve as animal feed to increase sustainability of meat and milk production. The aim of the present study was to evaluate fermentation characteristics of dried fruit and vegetable pomaces in a short-term in vitro experiment using the Hohenheim Gas Test. A selection of six fruit (apple, aronia, orange, pomegranate, red, white grape) and three vegetable (beetroot, carrot, tomato) pomaces was tested in three concentrations (150, 300, 500 g kg-1 of dry matter (DM)) as supplement to the basal diet (hay, used as control). Three runs were performed, using rumen fluid from one of three different rumen-cannulated cows in each run. Per run, each compound was tested in duplicate. After 24 h incubation, total gas production, methane and CO2 concentration, short-chain fatty acids, in vitro organic matter digestibility as well as microbial counts were determined. In addition, the pomaces' polyphenol content including the fractions non-tannin phenols, condensed tannins and hydrolysable tannins were analysed. Most pomaces did not significantly affect rumen fermentation characteristics in any of the tested dosages and may thus be applied in ruminant nutrition without adverse effects. Aronia significantly decreased (-14.5%) the organic matter digestibility in the highest concentration whereas apple (+12%), carrot (+10%) and beetroot (+8%) increased gas formation related to digestible organic matter. The 500 g kg-1 dosage of pomegranate significantly decreased methane formation by about 28% without impairing digestibility. Pomegranate was the only pomace of those high in total tannins that contained exceptionally high amounts of hydrolysable (90% of total tannins) and proportionally low amounts of condensed tannins (10% of total tannins), indicating that the hydrolysable tannins most likely reduced the methane production. Therefore, pomegranate pomace may be an interesting option for a methane mitigating feed supplement in ruminants and should be considered for following in vivo testing.

Antioxidant and Inflammatory Gene Expression Profiles of Bovine Peripheral Blood Mononuclear Cells in Response to Arthrospira platensis before and after LPS Challenge.

Oxidative stress and inflammatory diseases are closely related processes that need to be controlled to ensure the desirable high performance of livestock. The microalga spirulina has shown antioxidant and anti-inflammatory properties in monogastric species. To investigate potential beneficial effects in ruminants, we replaced soybean meal (SOY) in the diets of dairy cows and fattening bulls by spirulina (SPI) and analyzed plasma concentrations of antioxidants (ß-carotene, α-tocopherol, polyphenols) and serum total antioxidant capacity. Following in vitro stimulation with lipopolysaccharide (LPS), peripheral blood mononuclear cells (PBMCs) were isolated for expression analysis of inflammation- and antioxidant-defense-related genes. Plasma ß-carotene concentration was higher in SPI, compared to SOY cows, but did not differ in bulls. Plasma total phenol concentration was significantly higher in SPI, compared to SOY bulls, but not in cows. Stimulation of bovine PBMCs with LPS increased the expression of most cytokines and some antioxidant enzymes. Gene expression of PBMCs derived from SPI animals, compared to SOY animals, hardly differed. Our results indicate that in ruminants, spirulina might not have potent antioxidant and anti-inflammatory properties. Future studies should evaluate the microbial degradation of spirulina and its bioactive compounds in the rumen to provide further data on potential beneficial health effects in ruminants.

Partitioning of fatty acids into tissues and fluids from reproductive organs of ewes as affected by dietary phenolic extracts.

Evaluation of dietary interventions with regard to fertility problems often observed in ruminant livestock is of global interest. Though the effects of polyphenol supplementation in ruminants on digestion and food quality are well described, the impact on reproductive tissues and fluids remains scarcely investigated. These compounds protect dietary unsaturated fatty acids (FA) from oxidation and biohydrogenation and thus saturation. In addition, modification of the expression of genes associated with FA metabolism may occur. Therefore, we characterized for the first time the FA profiles of reproductive tissues and fluids and investigated their potential modification by dietary polyphenols in 22 cyclic ewes. The animals were randomly divided into four groups and fed a basal diet of meadow hay and one of four concentrate types either non-supplemented (control) or supplemented with grape seed extract, Acacia mearnsii bark extract (13 g/kg dry matter (DM) each) or a combination of both (26 g/kg DM). After 10 weeks of feeding, the animals were slaughtered. Samples of reproductive (oviduct, uterus) and metabolically differently active tissues (liver, muscle, adipose) as well as of plasma and fluids from oviduct and uterus were analysed for their FA composition. In addition, the expression of lipid metabolic and antioxidant genes was analysed in all tissues except the adipose tissue. Fatty acid profiles in tissues and fluids as well as gene expression in tissues significantly differed between the different fluids and tissues. In contrast, only a few diet and matrix (fluid or tissue) × diet interactions were observed. Still, the FA profile of the uterus was the only one not at all affected by the diet. The mRNA expression was not affected by the diet for most of the genes investigated, which might in part be explained by the similar plasma polyphenol concentrations found at slaughter. Overall, our findings contribute to an improved understanding of the characteristic FA composition of reproductive tissues and fluids in sheep. In addition, the effect of polyphenols on different tissues, fluids and tissue gene expression has been confirmed as described in other animal species.

Vascular Endothelial Growth Factor A and VEGFR-1 Change during Preimplantation in Heifers.

Vascular endothelial growth factor A (VEGFA) plays a critical angiogenic role in the endometrium of placentalia during preimplantation. The role of VEGFA and its receptors is not fully characterised in bovine reproduction. We analysed the mRNA expression of VEGFA isoforms 121, 165 and 189, and VEGF receptors 1 and 2 in three experimental settings (A, B and C). We compared intercaruncular endometrium of cyclic to pregnant heifers at Days 12, 15 and 18 post insemination (Day 0), and between Day 15 and Day 18 conceptuses (A). We further compared caruncular versus intercaruncular endometrium at Day 15 (B), and endometrium of heifers carrying embryos originating from somatic cell nuclear transfer (SCNT) versus in vitro fertilisation (IVF) at Day 18 (C). Endometrial VEGFA protein was localised and quantified. Pregnant heifers displayed lower intercaruncular endometrial mRNA expression of VEGFA-121 (p = 0.045) and VEGFA-189 (p = 0.009) as well as lower VEGFA protein abundance (p < 0.001) at Day 15. The VEGFA protein was localised in intercaruncular luminal, glandular epithelium and in tunica muscularis of blood vessels. At Day 15, caruncular endometrium displayed higher VEGFA mRNA expression than intercaruncular endometrium (p < 0.05). Intercaruncular endometrial VEGFA protein at Day 18 was higher in abundance in SCNT than in IVF (p = 0.038). Therefore, during preimplantation in cattle, there may be a need for timely physiological reduction in intercaruncular endometrial VEGFA expression in favour of the caruncular area to facilitate a gradient towards the implantation sites. A higher expression of VEGFA in SCNT may predispose for later placentation abnormalities frequently observed following SCNT.

Alpine and lowland grazing differentially alter the reproductive tract redox milieu and amino acid composition in cattle.

An alpine environment is unique due to pasture biodiversity, with an abundant content of natural antioxidant polyphenols. The present study investigated the effects of lowland and alpine grazing on the oviduct and uterine tissue redox status and amino acid concentrations in plasma and reproductive fluids. In the first experiment, heifers grazed on lowland (H-LOW: n = 13) and on alpine (H-ALP: n = 15) pastures. In the second experiment, heifers grazed on the same lowland (HS-LOW: n = 6) and on a different alpine (HS-ALP: n = 6) pasture. The abundance of mRNA transcripts for antioxidant enzymes in the oviduct (glutathione S-transferase alpha 2, glutathione synthetase (GSS)) and the endometrium (catalase, glutathione-disulfide reductase, GSS) was less (P <  0.05), and for glutathione peroxidase 4 in the endometrium greater (P =  0.006) in the H-LOW than in the H-ALP group. The abundance of mRNA transcript for catalase was less in the endometrium in the H-LOW than in the H-ALP (P =  0.001) group. Catalase and NAD(P)H quinone dehydrogenase 1 concentrations in the oviduct were greater in the HS-LOW than in the HS-ALP group (P <  0.05). Of 32 amino acids analysed, there were differences in concentrations in the H-LOW and H-ALP group of 13, seven and 15 in plasma, oviduct and uterine fluids, respectively (P <  0.05). Comparing the HS-LOW to the HS-ALP groups, there were 13, one and three amino acids in the plasma, oviduct and uterine fluids, respectively, that were differentially abundant (P <  0.05). The grazing systems had some effect on the redox status and amino acid patterns in reproductive tissues.

Effect of supplementation of pelleted hazel (Corylus avellana) leaves on blood antioxidant activity, cellular immune response, and heart beat parameters in sheep1.

Hazel leaves (Corylus avellana) fed to sheep resulted in decreased methane emissions without negatively affecting feed intake and were found to have antioxidant properties in vitro. The objective of this study was to evaluate effects of hazel leaves, rich in tannins, on blood antioxidant activity, cellular immune response, and heart beat parameters in sheep. Four experimental pellets were produced by mixing alfalfa and hazel leaves in different proportions, including alfalfa alone as a control, 30% and 60% of hazel leaves, the latter also with 3.8% polyethylene glycol (PEG). Six adult, nonpregnant, nonlactating female sheep (71 ± 5.7 kg of body weight) were allocated to 4 treatments in a 6 × 4 crossover design with four 18-d periods. The diet consisted of experimental pellets and ryegrass-dominated hay (ratio 80% to 20% in dry matter), resulting in hazel leaf proportions of approximately 0%, 25%, and 50% in the total diet. Blood samples were collected at the end of each period to determine plasma total phenol concentration and markers of oxidative status as well as peripheral blood mononuclear cells (PBMC) activation and proliferation response in vitro. Heart rate (HR) and HR variability parameters were measured for 2 consecutive days in each period, during different activities (i.e., eating pellets or hay, or lying). Treatments were compared with multiple comparisons and contrast analysis was used to test for linear and quadratic relations. Compared with control, feeding a high dosage of hazel leaves enhanced (P = 0.006) the plasma total antioxidant capacity, which linearly (P = 0.016) increased with increasing level of hazel leaves in the diet. The total phenol concentration and activities of the antioxidant enzymes superoxide dismutase, catalase, and glutathione reductase in the plasma were not different (P ≥ 0.23) among the treatments; however, the latter slightly increased linearly (P = 0.047) with increasing hazel leaves proportion. No differences were observed in the activation and proliferation of PBMC among treatments. The HR decreased linearly (P ≤ 0.009) during pellet eating and lying and the root mean square of successive differences of interbeat intervals (RMSSD) increased linearly (P = 0.037) when lying with increasing level of hazel leaves in the diet. In conclusion, our findings indicate that hazel leaves are a promising supplement to improve oxidative status with no effect on cellular immune response and cardiac stress level of sheep.

Bovine embryo elongation is altered due to maternal fatty acid supplementation.

The pre-implantation period is prone to embryonic losses in bovine. Embryo-maternal communication is crucial to support embryo development. Thereby, factors of the uterine fluid (UF) are of specific importance. The maternal diet can affect the UF composition. Since omega 3 fatty acids (omega 3 FA) are considered to be beneficial for reproduction, we investigated if dietary omega 3 FA affected factors in the UF related to embryo elongation. Angus heifers (n = 37) were supplemented with either 450 g of rumen-protected fish oil (omega 3 FA) or sunflower oil (omega 6 FA) for a period of 8 weeks. Following cycle synchronization and artificial insemination, the uteri were flushed post mortem to recover the embryos on day 15 of pregnancy. The UF and tissue samples of endometrium and corpus luteum (CL) were collected. Strikingly, the embryo elongation in the omega 3 group was enhanced compared to the omega 6 group. No differences were observed in uterine prostaglandins, even though the endometrial concentration of their precursor arachidonic acid was reduced in omega 3 compared to omega 6 heifers. The dietary FA neither led to differential expression of target genes in endometrium nor CL nor to a differential abundance of low-density lipoprotein cholesterol, cortisol or amino acids in the UF. Interestingly, the omega 3 group displayed a higher plasma progesterone concentration during luteal growth than the omega 6 group, possibly promoting embryo elongation. Further research should include an ovarian perspective to understand the functional link between dietary omega 3 FA and reproductive outcome.

Nutrition and healthy ageing: calorie restriction or polyphenol-rich "MediterrAsian" diet?

Diet plays an important role in mammalian health and the prevention of chronic diseases such as cardiovascular disease (CVD). Incidence of CVD is low in many parts of Asia (e.g., Japan) and the Mediterranean area (e.g., Italy, Spain, Greece, and Turkey). The Asian and the Mediterranean diets are rich in fruit and vegetables, thereby providing high amounts of plant bioactives including polyphenols, glucosinolates, and antioxidant vitamins. Furthermore, oily fish which is rich in omega-3 fatty acids is an important part of the Asian (e.g., Japanese) and also of the Mediterranean diets. There are specific plant bioactives which predominantly occur in the Mediterranean (e.g., resveratrol from red wine, hydroxytyrosol, and oleuropein from olive oil) and in the Asian diets (e.g., isoflavones from soybean and epigallocatechin gallate from green tea). Interestingly, when compared to calorie restriction which has been repeatedly shown to increase healthspan, these polyphenols activate similar molecular targets such as Sirt1. We suggest that a so-called "MediterrAsian" diet combining sirtuin-activating foods (= sirtfoods) of the Asian as well as Mediterranean diet may be a promising dietary strategy in preventing chronic diseases, thereby ensuring health and healthy ageing. Future (human) studies are needed which take the concept suggested here of the MediterrAsian diet into account.

Effects of blackcurrant-based juice on atherosclerosis-related biomarkers in cultured macrophages and in human subjects after consumption of a high-energy meal.

Regular consumption of fruit and vegetables may be associated with decreased CVD risk. In the present study, we investigated the effects of blackcurrant (BC) juice, rich in polyphenols and ascorbic acid, on oxidative and inflammatory biomarkers in cultured macrophages in vitro and in human subjects with an atherosclerosis-prone phenotype (after consumption of a high-energy meal). In cultured macrophages (RAW264.7), BC treatment significantly inhibited lipopolysaccharide-induced inflammation as indicated by lower mRNA levels of TNF-α, IL-1ß and inducible NO synthase (iNOS) and lower nuclear p65 levels indicating decreased NF-κB activity. iNOS protein levels were lower and haem oxygenase 1 levels higher in BC-treated cells when compared with untreated controls. Subjects given a high-energy meal had elevated serum glucose and insulin levels with no significant difference between the BC-based juice and placebo treatment groups. TAG following meal ingestion tended to be attenuated after the BC treatment. Plasma ascorbic acid and radical-scavenging capacity were decreased following placebo meal consumption; however, BC significantly elevated both parameters compared with baseline and placebo ingestion. Plasma oxidised LDL, α-tocopherol and paraoxonase activity were unchanged in both treatment groups. Furthermore, production of TNF-α and IL-1ß was not significantly changed by BC meal consumption. The present results suggest potential antioxidative and anti-inflammatory properties of BC in vitro in cultured macrophages. Although the observations were not directly transferable to a postprandial in vivo situation, the present results show that BC juice consumption may improve postprandial antioxidant status as indicated by higher ascorbic acid levels and free radical-scavenging capacity in plasma.

CLA does not impair endothelial function and decreases body weight as compared with safflower oil in overweight and obese male subjects.

OBJECTIVE: Conjugated linoleic acid (CLA) showed a wide range of beneficial biological effects with relevance for cardiovascular health in animal models and humans. Most human studies used olive oil as a reference. This study assessed the effect of CLA as compared with safflower oil on endothelial function and markers of cardiovascular risk in overweight and obese men. Heated safflower oil and olive oil were given for additional descriptive control. METHODS: Eighty-five overweight men (aged 45-68 years, body mass index 25-35 kg/m(2)) were randomized to receive 4.5 g/d of the CLA isomeric mixture, safflower oil, heated safflower oil, or olive oil in a 4-week double-blind study. Endothelial function was assessed by peripheral arterial tonometry (PAT) index determination in the fasting and postprandial state (i.e., 4 hours after consumption of a fat- and sucrose-rich meal). RESULTS: CLA as compared with safflower oil consumption did not impair fasting or postprandial PAT index but decreased body weight. CLA as compared with safflower oil did not change total, low-density lipoprotein (LDL), or high-density lipoprotein (HDL) cholesterol; triglycerides; insulin sensitivity indices; C-reactive protein; soluble adhesion molecules; oxidized LDL; lipoprotein a (Lp[a]); paraoxonase; or platelet-activating factor acetylhydrolase (PAF-AH) activity, but significantly reduced arylesterase activity and increased concentrations of the F(2)-isoprostane 8-iso-prostaglandin F (PGF)(2α). CONCLUSION: CLA did not impair endothelial function. Other parameters associated with metabolic syndrome and oxidative stress were not changed or were slightly improved. Results suggest that CLA does not increase cardiovascular risk. Increased F(2)-isoprostane concentrations in this context may not indicate increased oxidative stress.