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Daily and seasonal temperature fluctuations are inevitable due to climate change, which highlights the importance of studying the detrimental effects of temperature fluctuations on the health, productivity, and product quality of farm animals. Muscle membrane composition and the molecular signals are vital for muscle cell differentiation and muscle growth, but their response to temperature stress is not well characterized. Temperature changes can lead to modification of membrane components of the cell, which may affect its surroundings and intracellular signaling pathways. Using C2C12 myoblast cells as a model of skeletal muscle development, this study was designed to investigate the effects of high temperature (39 °C and 41 °C) and low temperature (35 °C) on molecular pathways in the cells as well as the cell membrane fatty acid composition. Our results show that several genes were differentially expressed in C2C12 cells cultured under heat or cold stress, and these genes were enriched important KEGG pathways including PI3K-Akt signaling pathway, lysosome and HIF- signaling pathway, Wnt signaling pathway and AMPK signaling pathway. Our analysis further reveals that several membrane transporters and genes involved in lipid metabolism and fatty acid elongation were also differentially expressed in C2C12 cells cultured under high or low temperature. Additionally, temperature stress shifts the fatty acid composition in the cell membranes, including the proportion of saturated, monounsaturated and polyunsaturated fatty acids. This study revealed an interference between fatty acid composition in the membranes and changing molecular pathways including lipid metabolism and fatty acids elongation mediated under thermal stress. These findings will reinforce a better understanding of the adaptive mechanisms in skeletal muscle under temperature stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Besides classical semen parameters, semen stress tests (SSTs) are helpful tools to assess the fertilizing capacity of sperm. However, valid studies on SSTs in relation to fertility are rare because several corrections of common fertility parameters for female and male effects are required. Therefore, over a one-year period, we analyzed semen parameters of 260 ejaculates obtained from 130 Pietrain boars aged between 8 and 9 months in one AI center as well as 1521 corresponding insemination records for these ejaculates. Two consecutive ejaculates (4th and 5th) were collected from each boar and extended in DiluPorc™ BTS. In addition to routine semen evaluation, sperm motility was assessed after heat-resistance test (300 min incubation at 38 °C after seven days storage at 16 °C, HRT) and cold-resistance test (10 min incubation at 38 °C after three days storage at 6 °C, CRT). Generalized linear models (GLMs) were applied to analyze effects for the following predictors of farrowing rate (FR), number of total born (NTB) and live born (NLB) piglets: farm (P = 0.013 [FR], P = 0.001 [NTB], P = 0.023 [NLB]), parity (P = 0.679, P = 0.01, P < 0.001), weekday (P = 0.012, P = 0.08, P = 0.009) and year × season (P < 0.001, P = 0.688, P = 0.574). On boar level, GLMs revealed significant effects on FR, NTB and NLB for the predictors sow (all P < 0.001), total sperm number per dose (P = 0.007, P = 0.002, P < 0.001), total sperm motility (P = 0.002, P = 0.2, P = 0.003) and mitochondrial activity (P = 0.004, P < 0.001, P = 0.002). Moreover, FR and NTB were influenced by membrane integrity (both P < 0.001), FR and NLB by cold-resistance (P < 0.001, P = 0.043), and NTB and NLB by sperm morphology (P = 0.001, P < 0.001) and boar (both P < 0.001). NLB was additionally influenced by heat-resistance (P = 0.004) and farm (P = 0.018) and solely NTB was influenced by sperm output (P = 0.03). Boar and semen related factors explained 9% of the total variation in NTB and 7% of the total variation in NLB. Only 14.2% (n = 37) of the samples were both cold- and heat-resistant (≥65% of motile sperm). Cold- and heat-resistance were dependent factors (Chi-square, P = 0.001) and sperm motility after CRT and HRT showed a moderate positive correlation (r s  = 0.40, P < 0.001, Spearman's rho). Finally, ROC curves demonstrated that neither SST can be used as a sole test for predicting the fertilizing capacity of boar ejaculates., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In smallholder agriculture, the fast-growing and perennial accumulator plant comfrey (Symphytum spp.) was used to supply pigs with protein and minerals. Comfrey leaves show similar values in dry matter as soybean or blue lupine in crude protein content, but much higher levels of calcium and phosphorus. However, in terms of increased efficiency in animal husbandry, comfrey has been displaced by mainly soybean and cereals. Due to its profile of macro- and micronutrients the use of comfrey could have the potential to re-establish local resource cycles and help remediate over-fertilized soils. The aim of the study was to evaluate whether a modern pig breed accepts a continuous feed supplement of dried comfrey leaves. After an initial adaptation period post-weaning, German Landrace piglets were subjected to either a standard control diet or a diet supplemented with 15% dried comfrey leaves for 4 weeks. Body weight was reduced in comfrey-supplemented piglets compared to controls, which might be attributed to reduced palatability in the experimental setting. Nevertheless, comfrey-supplemented piglets exhibited adequate bone mineralization and intestinal integrity. The microbiome profile in feces and digesta revealed higher diversity in comfrey-supplemented piglets compared to controls, with pronounced effects on the abundances of Treponema and Prevotella. This may be due to described bio-positive components of the comfrey plant, as data suggest that the use of comfrey leaves may promote intestinal health. Digestive tract phosphorus levels were reduced in piglets receiving comfrey supplementation, which may ultimately affect phosphorus levels in manure. Results indicate that comfrey leaves could serve as a feed component in integrated agricultural systems to establish regional nutrient cycles. The trial provides a basis for further work on comfrey as a regionally grown protein source and effective replacement for rock mineral supplements., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The measurement of feather corticosterone concentrations (fCORT) is a comparatively new method for the evaluation of stress in wild and captive birds and may be a useful indicator in animal welfare research. The aim of this study was to assess the suitability of fCORT as an indicator of stress, and for this purpose a prolonged stress period was experimentally simulated by oral intake of corticosterone via drinking water and corticosterone concentrations were analyzed in feathers grown during this period. Layer pullets of both a control group (n = 20) and a CORT group (n = 20) were offered drinking water ad libitum throughout the entire experimental phase. The drinking water of the CORT group was supplemented with corticosterone at a concentration of 20 mg/l from the 64th to the 114th day of life. The vaned parts of the primaries 5 (P5s) were clipped on d 114 and fCORT was analyzed by ELISA after extraction. Body weights increased from day 64 until d 114 in both groups, however, at the end of the experiment, mean body mass in the CORT group was significantly lower than in the control group (P < 0.001). Pullets of the CORT group also showed shorter and lighter P5s as well as a retarded molt of the primaries. The supplementation of drinking water with corticosterone increased the average fCORT in the P5s of the CORT pullets compared with the control group (median: 110.3 pg/mm [interquartile range (IQR): 47.2] vs. 10.0 pg/mm [IQR: 2.5], P < 0.001). The results show that experimentally increased systemic corticosterone concentrations over a period of seven weeks in layer pullets are reflected in corticosterone concentrations of feathers grown during that time. This indicates that the measurement of fCORT may be a useful and minimally invasive tool for the evaluation of long-term stress in chicken and provides the basis for further investigations on its use in animal welfare research., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In tropical environments, dairy cattle production is constrained by several factors, including climate. The seasonal loss of milk due to heat stress is a recurring challenge for many dairy producers. The objective of this study was to detect heat stress thresholds, milk yield loss and individual animal variations using random regression models for dairy cattle from test-day milk records. Data were obtained from the Kenya Livestock Breeders Organization for the years 2000-2017 and merged with weather data. The weather parameters were grid-interpolated solar and meteorological data obtained from the National Aeronautics and Space Administration/Prediction Of Worldwide Energy Resources (NASA/POWER). After editing, the records comprised 49 993, 45 251 and 36 136 test-day records for first, second, and third lactations, respectively, for the four main dairy breeds: Friesian (68.0%), Ayrshire (21.1%), Jersey (7.6%) and Guernsey (3.3%). Variance components were estimated using Restricted Maximum Likelihood in ASReml software. Random regression models with third-order Legendre polynomials were fitted to the average and individual lactation curves and the reaction norms. An extended factor analytic variance structure for the random cow effects was used to estimate (co)variances between days in milk and thermal load. The daily average temperature (TA) and temperature humidity index (THI) were identified as the most suitable thermal load indicators for assessing milk yield losses. Considering a one day lag, the estimated heat stress thresholds were about 22 °C and 69 index units for TA and THI, respectively. Almost no differences were observed for estimated residual variances between the thermal load indicators, indicating there was no better model fit by TA or THI. The heat stress thresholds and milk loss patterns are important for management of dairy production systems in the tropics with climatic conditions similar to this study. Data recording should be improved as a tool to monitor the expected impacts of climate change and mitigation measures., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Telmisartan prevents diet-induced obesity (DIO) in rodents. Given that the precise underlying mechanism is not known, we examined whether a gut-related mechanism might be involved. Sprague-Dawley rats received cafeteria diet (CD) for 3 months to develop DIO and were administered either telmisartan (8 mg/kg bw ) or vehicle. In addition, pair-fed (PF) rats received CD adjusted to TEL and control rats (CON) only received chow. Stool samples were analysed by 16 S rRNA gene amplicon sequencing. CD-fed rats became obese while TEL, PF and CON rats remained lean. Alpha diversity analyses indicated that bacterial diversity was similar before the study but changed over time. Beta diversity revealed a time-, CD- and telmisartan-dependent effect. The Firmicutes/Bacteroidetes ratio and the abundance of Blautia, Allobaculum and Parasutterella were higher in DIO and PF than in CON, but not in TEL. Enterotype (ET)-like clustering analyses, Kleinberg's hub network scoring and random forest analyses also indicated that telmisartan induced a specific signature of gut microbiota. In response to stool transfer from telmisartan-pre-treated donor to high-fat fed acceptor mice, body weight gain was slightly attenuated. We attribute the anti-obesity action of telmisartan treatment to diet-independent alterations in gut microbiota as the microbiota from telmisartan-treated, CD-fed rats clearly differed from those of DIO and PF rats. ET-like clustering network, random forest classification and the higher stability in bacterial co-occurrence network analyses indicate that there is more than one indicator species for telmisartan's specific signature, which is further strengthened by the fact that we cannot identify a single indicator species., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Analysing hair cortisol concentrations (HCCs) is a minimally invasive way to retrospectively assess long-term stress, and its application in studies of animal welfare and stress has attracted considerable interest. However, not only stress-related effects but also hair-specific characteristics and external influences can affect HCCs and interfere with the interpretation of results. Thus, it was the aim of this study to investigate the impact of daylight and UV irradiation on cortisol concentrations in the hairs of pigs and cattle. We also examined whether a potential irradiation effect on HCCs depended on the colour of the hair. For this purpose, black and white hair samples from 18 Saddleback pigs and 18 Holstein Friesian cattle were exposed to artificial light (both visible and UV) and compared with control hair samples from the same animals kept in the dark. Exposure to artificial light significantly decreased HCCs in both pigs (P < 0.05) and cattle (P < 0.001), and hair colour had an influence on HCCs, with black hair showing higher cortisol levels than white hair (cattle: P < 0.001, pigs: P = 0.07). The interaction between light exposure and hair colour was significant in both pigs (P < 0.01) and cattle (P < 0.001), so light exposure reduced HCCs in porcine white hair but not black hair. In cattle, light-exposed white hair exhibited lower hair cortisol levels than control white hair or black hair. These results demonstrate that artificial light irradiation degrades hair cortisol or favours its elimination by structural changes of the hair matrix. However, this effect was only detectable in white hair, indicating that the melanin pigments in black hair absorbed radiation, thereby reducing the effects of photodegradation. Compared with other known influencing factors on HCCs, such as age and body region, the influence of light irradiation was relatively low in this in vitro experiment. However, further studies should investigate this influence under real-life animal conditions, such as outdoor and indoor housing., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

This paper reviews existing on-farm GHG accounting models for dairy cattle systems and their ability to capture the effect of dietary strategies in GHG abatement. The focus is on methane (CH 4 ) emissions from enteric and manure (animal excreta) sources and nitrous oxide (N 2 O) emissions from animal excreta. We identified three generic modelling approaches, based on the degree to which models capture diet-related characteristics: from 'none' (Type 1) to 'some' by combining key diet parameters with emission factors (EF) (Type 2) to 'many' by using process-based modelling (Type 3). Most of the selected on-farm GHG models have adopted a Type 2 approach, but a few hybrid Type 2 / Type 3 approaches have been developed recently that combine empirical modelling (through the use of CH 4 and/or N 2 O emission factors; EF) and process-based modelling (mostly through rumen and whole tract fermentation and digestion). Empirical models comprising key dietary inputs (i.e., dry matter intake and organic matter digestibility) can predict CH 4 and N 2 O emissions with reasonable accuracy. However, the impact of GHG mitigation strategies often needs to be assessed in a more integrated way, and Type 1 and Type 2 models frequently lack the biological foundation to do this. Only Type 3 models represent underlying mechanisms such as ruminal and total-tract digestive processes and excreta composition that can capture dietary effects on GHG emissions in a more biological manner. Overall, the better a model can simulate rumen function, the greater the opportunity to include diet characteristics in addition to commonly used variables, and thus the greater the opportunity to capture dietary mitigation strategies. The value of capturing the effect of additional animal feed characteristics on the prediction of on-farm GHG emissions needs to be carefully balanced against gains in accuracy, the need for additional input and activity data, and the variability encountered on-farm., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Urine and fecal excretions from cattle contribute to global nitrogen (N) emissions. The milk urea nitrogen (MUN) concentration in dairy cows is positively correlated with urinary urea N (UUN) emissions, and both decline with the reduction in crude protein intake. However, MUN concentration may differ between individual cows despite feeding the same ration. Thus, we hypothesized that due to differences in endogenous N utilization cows with high MUN concentration excrete more UUN than cows with a low MUN concentration. The objective of the present study was to elucidate N partitioning and urea metabolism in dairy cows with divergent MUN concentrations fed two planes of crude protein. Twenty Holstein dairy cows with high (HMU; n = 10) and low (LMU; n = 10) milk urea concentrations were fed two isocaloric diets with a low (LP) and normal (NP) crude protein level. Methane and ammonia emissions were recorded in respiration chambers. Feed intake, feces and urine excretions and milk yield were recorded for four days and subsamples were analyzed for total N and N-metabolites. A carbon-13 labeled urea bolus was administered intravenously followed by a series of plasma samplings. Total N and UUN excretions and ammonia emissions from excreta were lower on the LP diet, however, methane emissions, urinary N excretions and ammonia emissions were comparable between groups. Although plasma and salivary urea concentrations, urea pool size and urea turnover were higher, HMU cows had lower renal urea clearance rates. Additionally, HMU cows had lower renal clearance rates for creatinine, uric acid and creatine and excreted less uric acid (on the LP diet only) and creatine with urine. In conclusion, contrary to our hypothesis, HMU cows did not excrete more UUN than LMU cows. The lower urinary creatine excretion of HMU cows suggests that these animals have a lower environmental nitrogen footprint., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The objective of this study was to compare 2 laying hen strains in 5 production periods regarding phytase activity, phytate (InsP 6 ) degradation, and myo-inositol (MI) release in the digestive tract and phosphorus (P) and calcium (Ca) utilization. One offspring of 10 nonrelated roosters per strain (Lohmann Brown-classic (LB) or Lohmann LSL-classic (LSL)) was placed in one of 20 metabolic units in a completely randomized block design in week 8, 14, 22, 28, and 58 of life. All hens were fed the same corn and soybean meal-based diet at one time, but the diet composition was adjusted to the requirements in the respective period. For 4 consecutive days, excreta were collected quantitatively at 24-hour intervals. In week 10, 16, 24, 30, and 60, the blood plasma, digesta of crop, gizzard, jejunum, ileum, and ceca, and mucosa of the jejunum was collected. The concentration of inorganic P in the blood plasma was higher in LB than in LSL hens (P = 0.026). Plasma Ca concentrations increased with each period (P < 0.001) in both strains. In jejunum digesta, the MI concentration did not differ between strains, but InsP 6 concentration was higher in LB than in LSL hens (P = 0.002) and the highest in week 30 and 60. Total phosphatase and phytase activities were higher in LB than in LSL hens (P ≤ 0.009). Period effects were also significant for these enzymes. Concentrations of some constituents of the cecal content were different between the strains. The MI concentration in the egg albumen and yolk was higher in LB than in LSL hens. Differences in InsP 6 - and MI-related metabolism of the 2 hen strains existed. These differences were partly dependent of the period. Especially, week 24 was a period of remarkable change of metabolism. Great differences also existed among individuals, making it worth to have a closer look at the metabolism of individuals in addition to evaluating treatment means. Further studies on metabolic, genetic, and microbiome level may help explain these differences., (Copyright © 2020. Published by Elsevier Inc.)

Local cultivars of comfrey (Symphytum spp.) have been used to cover protein and mineral requirements of farm animals in low-input systems. Due to its known health-promoting (e.g. allantoin), but also anti-nutritive ingredients (e.g. pyrrolizidine alkaloids), multidisciplinary approaches are essential in order to quantify the nutritional value and the potential of its use in poultry and farm animals in terms of meeting animal needs, using local resources as well as remediating over-fertilized soils. Focusing on animal effects, here one-day old sexed Cobb500 broiler chickens were subjected to either a standard control diet or a standard diet supplemented with 4% dried comfrey leaves for 32 days. Performance traits indicate good acceptance of supplementation with comfrey leaves. Parameters for liver function, mineral homeostasis, bone mineral density as well as intestinal microanatomy revealed no signs of impairment. Quantified pyrrolizidine alkaloids were below the detection limit in liver and breast muscle (<5 μg/kg tissue). Comfrey supplemented male broiler chickens showed higher ash content in breast muscle and revealed altered gene expression profiles for metabolic pathways in blood cells. In healthy broiler chickens, the transcriptome analyses revealed no aberrations in the immune-related pathways due to comfrey supplementation. The results imply that the use of comfrey leaves in a high-performance broiler line seems feasible and offers the potential for closed nutrient cycles in site-adapted local agricultural systems. Further analyses need to focus on possible growth-promoting and health-improving components of comfrey and the safe use of chicken products for human consumption., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Despite the crucial role of glucocorticoid receptor (GR) in proper immune responses, the effect of GR hypersensitivity on inflammation is rarely reported. To fill this knowledge gap, we exploited the natural gain-of-function substitution in the porcine glucocorticoid receptor (GR Ala610Val ) and challenged pigs carrying normal or hypersensitive GR using 50 µg/kg lipopolysaccharide (LPS) following pretreatment with either saline or single bolus of 60 µg/kg dexamethasone (DEX). The GR Ala610Val substitution reduced baseline cortisol, adrenocorticotropic hormone (ACTH), and triglyceride concentration and granulocyte proportion whereas baseline platelet counts were elevated. Val-carriers, i.e. AlaVal as well as ValVal pigs, showed less LPS-induced cortisol rise but the cortisol fold change was similar in all genotypes. Differently, ACTH response to LPS was most significant in GR Ala610Val heterozygotes (AlaVal). LPS-induced disorders, including sickness behaviors, anorexia, thrombocytopenia, cytokine production, and metabolic alterations were more intense in Val-carriers. On the other hand, Val-carriers were more sensitive to DEX effect than wild types (AlaAla) during endotoxemia, but not under unchallenged conditions. This is the first report revealing aggravated responses to endotoxemia by GR gain-of-function. Together, these results imply that GR hypersensitivity is difficult to diagnose but may represent a risk factor for endotoxemia and sepsis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Faecal egg counting techniques (ECTs) are useful tools for assessing anthelmintic efficacy and selecting hosts resistant to parasite infection. McMaster (MM) is one of the most commonly used ECTs, but it suffers from low sensitivity and precision. Mini-FLOTAC (MF) has been proposed to replace MM, but so far has not been evaluated for gastro-intestinal nematode infections in chickens. This study compared sensitivity, precision, and accuracy of MM and MF with two trials using egg-spiked faecal samples ranging from 50-1250 eggs per gram of faeces (EPG). In addition, effects of two flotation fluids with different specific gravities (SG), namely salt (SG = 1.20) and sucrose solutions (SG = 1.32), on accuracy and time-spent for both ECTs were evaluated. Overall sensitivity based on the composite reads across all EPG-levels was 97.1 % for MM and 100 % for MF. MF was, however, more sensitive (P = 0.003) or tended to (P = 0.087) be more sensitive than MM at only the lowest EPG-level (i.e. 50 EPG) using one of the duplicate reads, whereas there was no significant difference at any EPG-level using composite reads. Overall average precision of MF (79.5 %) was higher (P < 0.001) than that of MM (63.4 %) across all EPG-levels. Precision of MM increased from 22 to 87 % with increasing EPG-levels from 50-1250 EPG. Corresponding precision estimates for MF ranged from 76 to 91 %. Overall recovery rate of MM (74.6 %) was significantly higher (P < 0.001) than that of MF (60.1 %). There was no significant difference in recovery rate of spiked-eggs among different EPG-levels (P = 0.833). Recovery rate of MM ranged from 64 % to 79 % across different EPG-levels, while it ranged from 54 % to 64 % with MF without an interaction between ECT and EPG-level (P = 0.701). It took more time (P < 0.001) to process (prepare and read) samples with MF than with MM using the same flotation fluid. The sugar solution tended to (P = 0.100) increase egg-recovery with both ECTs, while increasing (P < 0.001) time-spent for processing the samples. Our data collectively suggest that MM is less sensitive than MF only at around minimum detection level of MM when using unrepeated reads. We conclude that McMaster is faster, relatively more accurate but less precise than Mini-FLOTAC. The sugar solution with higher SG increases accuracy of both techniques at the expense of increased labour time., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The endometrium undergoes cyclical changes during the estrous cycle and pregnancy. These alterations are controlled by various factors, including cytokines. The present study aimed to screen the effect of several chemokines (CCL2, CCL4, CCL5, CCL8, CXCL2, CXCL8, CXCL9, CXCL10, and CXCL12) on endometrial stromal and endothelial cells. Real-time PCR analysis revealed mRNA expression of all examined chemokines and their receptors in primary stromal cells and undetectable levels of CXCL9, CXCL10, and CXCR3 in endothelial cells. Immunocytochemical staining showed variable distribution of chemokine receptors in stromal and endothelial cells. All examined chemokines enhanced stromal cell proliferation, and CCL2 and CXCL12 also increased the migratory potential of these cells. The evaluation of a possible indirect effect of chemokines on angiogenesis and lymphangiogenesis demonstrated that CXCL12 may potentially negatively affect lymphatic vessel creation. Downregulation of VEGFC mRNA and protein expression was noticed after CXCL12 stimulation. Among all examined chemokines, CCL4 and CCL8 positively affected the proliferation and migration of endothelial cells. The number of capillary-like structures was significantly reduced after CXCL8, CXCL10, and CXCL12 stimulation. In conclusion, among all examined chemokines, CCL2 is thought to act as the modulator of stromal cell functions, whereas CCL4 and CCL8 are suggested to be potent factors directly stimulating blood vessel creation., (Copyright © 2020. Published by Elsevier Inc.)

Both blood- and milk-based biomarkers have been analysed for decades in research settings, although often only in one herd, and without focus on the variation in the biomarkers that are specifically related to herd or diet. Biomarkers can be used to detect physiological imbalance and disease risk and may have a role in precision livestock farming (PLF). For use in PLF, it is important to quantify normal variation in specific biomarkers and the source of this variation. The objective of this study was to estimate the between- and within-herd variation in a number of blood metabolites (β-hydroxybutyrate (BHB), non-esterified fatty acids, glucose and serum IGF-1), milk metabolites (free glucose, glucose-6-phosphate, urea, isocitrate, BHB and uric acid), milk enzymes (lactate dehydrogenase and N-acetyl-β-D-glucosaminidase (NAGase)) and composite indicators for metabolic imbalances (Physiological Imbalance-index and energy balance), to help facilitate their adoption within PLF. Blood and milk were sampled from 234 Holstein dairy cows from 6 experimental herds, each in a different European country, and offered a total of 10 different diets. Blood was sampled on 2 occasions at approximately 14 days-in-milk (DIM) and 35 DIM. Milk samples were collected twice weekly (in total 2750 samples) from DIM 1 to 50. Multilevel random regression models were used to estimate the variance components and to calculate the intraclass correlations (ICCs). The ICCs for the milk metabolites, when adjusted for parity and DIM at sampling, demonstrated that between 12% (glucose-6-phosphate) and 46% (urea) of the variation in the metabolites' levels could be associated with the herd-diet combination. Intraclass Correlations related to the herd-diet combination were generally higher for blood metabolites, from 17% (cholesterol) to approximately 46% (BHB and urea). The high ICCs for urea suggest that this biomarker can be used for monitoring on herd level. The low variance within cow for NAGase indicates that few samples would be needed to describe the status and potentially a general reference value could be used. The low ICC for most of the biomarkers and larger within cow variation emphasises that multiple samples would be needed - most likely on the individual cows - for making the biomarkers useful for monitoring. The majority of biomarkers were influenced by parity and DIM which indicate that these should be accounted for if the biomarker should be used for monitoring.

Background: Cys is limiting for reduced glutathione (GSH) synthesis and can be synthesized from Met. We hypothesized that the dietary Met hydroxyl analogue dl-2-hydroxy-4-methylthiobutyric acid (dl-HMTBA) affects Cys and GSH metabolism and oxidative stress defense differently than Met., Objective: The objective was to elucidate whether dl-HMTBA supplementation of a Met-deficient diet affects Cys flux, GSH fractional synthetic rate (FSR), and the basal oxidative stress level relative to Met supplementation in pigs., Methods: Twenty-nine male German Landrace piglets aged 28 d were allocated to 3 dietary groups: a basal diet limiting in Met (69% of Met plus Cys requirement) supplemented with either 0.15% l-Met (LMET; n = 9), 0.15% dl-Met (DLMET; n = 11), or 0.17% dl-HMTBA (DLHMTBA; n = 9) on an equimolar basis. At age 54 d the pigs received a continuous infusion of [1-13C]-Cys to calculate Cys flux and Cys oxidation. After 3 d, GSH FSR was determined by [2,2-2H2]-glycine infusion, and RBC GSH and oxidized GSH concentrations were measured. At age 62 d the animals were killed to determine hepatic mRNA abundances of enzymes involved in GSH metabolism, GSH concentrations, and plasma oxidative stress defense markers., Results: The Cys oxidation was 21-39% and Cys flux 5-15% higher in the fed relative to the feed-deprived state (P < 0.001). On average, GSH FSR was 49% lower (P < 0.01), and RBC GSH and total GSH concentrations were 12% and 9% lower, respectively, in DLHMTBA and DLMET relative to LMET pigs (P < 0.05). In the feed-deprived state, Gly flux, the GSH:oxidized glutathione (GSSG) ratio, RBC GSSG concentrations, plasma oxidative stress markers, and the hepatic GSH content did not differ between groups., Conclusions: Although GSH FSR was higher in LMET compared with DLMET or DLHMTBA feed-deprived pigs, these differences were not reflected by lower oxidative stress markers and antioxidant defense enzymes in LMET pigs., (Copyright © The Author(s) 2019.)

Feeding management of the postnatal and preweaning calf has an important impact on calf growth and development during this critical period and affects the health and well-being of the calves. After birth, an immediate and sufficient colostrum supply is a prerequisite for successful calf rearing. Colostrum provides high amounts of nutrient as well as non-nutrient factors that promote the immune system and intestinal maturation of the calf. The maturation and function of the neonatal intestine enable the calf to digest and absorb the nutrients provided by colostrum and milk. Therefore, colostrum intake supports the start of anabolic processes in several tissues, stimulating postnatal body growth and organ development. After the colostrum feeding period, an intensive milk feeding protocol, that is, at least 20% of BW milk intake/day, is required to realise the calf potential for growth and organ development during the preweaning period. Insufficient milk intake delays postnatal growth and may have detrimental effects on organ development, for example, the intestine and the mammary gland. The somatotropic axis as the main postnatal endocrine regulatory system for body growth is stimulated by the intake of high amounts of colostrum and milk and indicates the promotion of anabolic metabolism in calves. The development of the forestomach is an important issue during the preweaning period in calves, and forestomach maturation is best achieved by solid feed intake. Unfortunately, intensive milk-feeding programmes compromise solid feed intake during the first weeks of life. In the more natural situation for beef calves, when milk and solid feed intake occurs at the same time, calves benefit from the high milk intake as evidenced by enhanced body growth and organ maturation without impaired forestomach development during weaning. To realise an intensive milk-feeding programme, it is recommended that the weaning process should not start too early and that solid feed intake should be at a high extent despite intensive milk feeding. A feeding concept based on intensive milk feeding prevents hunger and abnormal behaviour of the calves and fits the principles of animal welfare during preweaning calf rearing. Studies on milk performance in dairy cows indicate that feeding management during early calf rearing influences lifetime performance. Therefore, an intensive milk-feeding programme affects immediate as well as long-term performance, probably by programming metabolic pathways during the preweaning period.

Oestrus detection remains a problem in the dairy cattle industry. Therefore, automatic detection systems have been developed to detect specific behavioural changes at oestrus. Vocal behaviour has not been considered in such automatic oestrus detection systems in cattle, though the vocalisation rate is known to increase during oestrus. The main challenge in using vocalisation to detect oestrus is correctly identifying the calling individual when animals are moving freely in large groups, as oestrus needs to be detected at an individual level. Therefore, we aimed to automate vocalisation recording and caller identification in group-housed dairy cows. This paper first presents the details of such a system and then presents the results of a pilot study validating its functionality, in which the automatic detection of calls from individual heifers was compared to video-based assessment of these calls by a trained human observer, a technique that has, until now, been considered the 'gold standard'. We developed a collar-based cattle call monitor (CCM) with structure-borne and airborne sound microphones and a recording unit and developed a postprocessing algorithm to identify the caller by matching the information from both microphones. Five group-housed heifers, each in the perioestrus or oestrus period, were equipped with a CCM prototype for 5 days. The recorded audio data were subsequently analysed and compared with audiovisual recordings. Overall, 1404 vocalisations from the focus heifers and 721 vocalisations from group mates were obtained. Vocalisations during collar changes or malfunctions of the CCM were omitted from the evaluation. The results showed that the CCM had a sensitivity of 87% and a specificity of 94%. The negative and positive predictive values were 80% and 96%, respectively. These results show that the detection of individual vocalisations and the correct identification of callers are possible, even in freely moving group-housed cattle. The results are promising for the future use of vocalisation in automatic oestrus detection systems.

Modern chickens have been genetically developed to perform high under optimal conditions. We hypothesized that high-performance is associated with a higher sensitivity to environmental challenges in laying hens. By using nematode infections as an environmental stressor, we assessed performance-level associated host responses in a high (i.e. Lohmann Brown Plus, LB) and in a lower performing, a so-called dual-purpose chicken genotype (i.e. Lohmann Dual, LD). The hens were infected with 1000 eggs of Ascaridia galli and Heterakis gallinarum at 24 weeks of age. Hen performance parameters, humoral immune responses in plasma and egg yolks and worm burdens were assessed at several occasions over a period of 18 weeks post infection (wpi). While infections had no significant effect on feed intake (P = 0.130) and body weight in both genotypes (P = 0.392), feed conversion efficiency was negatively affected by infections (P = 0.017). Infections reduced both laying rate and egg weight and thereby per capita egg mass in both genotypes (P < 0.05). While laying rate in infected LB hens decreased significantly (P < 0.05) in the early infection period (i.e. by 3 wpi), the decrease in LD hens appeared much later (i.e. by 14 wpi). Worm burdens resulting from the experimental infection were not different between the genotypes for both worm species (P > 0.05), whereas LB hens were more susceptible (P < 0.05) to re-infections than LD hens. Changes in humoral immune responses (i.e. ascarid-specific IgY antibodies in plasma and egg yolks) of the two genotypes over time reflected closely the corresponding changes in larval counts of the hens, descending from both experimental and subsequent natural infections in both genotypes. Infections caused a shift in egg size classes, leading to smaller frequency of larger eggs in both genotypes. Infections reduced egg weight (P = 0.018) and led to a reduced fat content in the egg yolks (P = 0.045). The proportion of poly-unsaturated fatty acids (PUFA), especially n-6-PUFA, was also lower in egg yolks of the infected hens (P = 0.032). We conclude that tolerance to nematode infections in laying hens is dependent on host-performance level. The impairment in host tolerance was both genotype and time dependent, likely due to differences in genetic programming for production peak and persistency of the two genotypes. The two genotypes exhibited similar levels of resistance after a fully controlled experimental infection, but the high performing hens were more susceptible to subsequent natural infections. Infections negatively affected economically important egg-quality traits, including egg weight, fat content and fatty acid profiles in egg yolks., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In order to evaluate the influence of thermal stress on physiological parameters, and the oocyte quality of Girolando (n = 12) and adapted Pantaneira (n = 12) cattle, twelve sessions of ultrasound guided follicular aspiration (OPU) were performed, between January and November 2014 (during dry (May-September) and rainy season (October-April) in Brazil). The recovered cumulus-oocyte complexes (COCs) were selected and classified, according to quality, immediately after OPU. The oocytes were then stored in 3% paraformaldehyd before conducting immunofluorescence analysis under confocal microscopy to identify HSP70 and 90 proteins. Before each OPU session, the rectal temperature (RT) and respiratory frequency (RF) of each animal were measured. The black globe humidity index (BGHI) was calculated on the day of the OPUs and 90 days before each OPU session, and related to the thermal stress of the animals. The quality of oocytes from Girolando cattle, but not Pantaneira, showed a negative relationship with BGHI of 90 days before OPU. RT of both breeds did not exceed normal values for cattle below BGHI 95. BGHI variation on the day of OPU did not affect RF of the adapted Pantaneira breed (p = 0.3221). On the other hand, Girolando cattle showed a positive relationship between RF and BGHI (p = 0.0103). With increasing BGHI, the amount of HSP70 increased in Girolando oocytes, however, decreased in the Pantaneira breed. We have not observed a relationship between HSP 90 and BGHI, however Girolando cattle produced a greater amount of this protein in relation to the Pantaneira breed. In conclusion, higher BGHIs, 90 days before OPU session, negatively affect oocyte quality of Girolando cattle and positively affect oocyte quality of the Pantaneira breed. Higher BGHIs on the day of the OPU session negatively affected the respiratory frequency of the Girolando breed, and lead to a higher recruitment of HSP70 to protect oocyte maturation. The opposite pattern was observed for Pantaneira. In addition, Pantaneira cattle produced twice as much as HSP70 as Girolando cattle, suggesting that a natural higher production of this protein could be involved in the mechanisms of adaptation to heat conditions., (Copyright © 2019. Published by Elsevier Inc.)

The present study was conducted to assess the effects of the probiotic Enterococcus faecium AL41 (EF) and of the enteric pathogen Salmonella Enteritidis PT4 (SE) on the development of posthatch pectoralis major muscle (PM) of broiler chicks. The four experimental groups were control (CON), EF, SE, and EF+SE (EFSE). EF and SE were given per os from days 1 to 7 and at day 4 posthatch, respectively. Muscle samples from 6 chicks per group were taken at day 8 (D8) and day 11 (D11) to evaluate PM myofiber growth, capillarization, DNA, RNA, and protein content, as well as enzyme activities (isocitrate dehydrogenase, lactate dehydrogenase, creatine kinase). PM growth rate was 7.45 ± 2.7 g/d in non-SE groups (CON, EF) and 5.10 ± 1.82 g/d in SE-infected groups (P < 0.02). Compared with group CON, application of bacteria (groups EF and SE) reduced the fiber cross-sectional area (246 and 262 vs. 347 ± 19 μm2) and the number of myonuclei per fiber (0.66 and 0.64 vs. 0.79 ± 0.03). At D11, hypertrophic myofiber growth normalized in the EF group, but negative effects persisted in SE and EFSE birds contributing to lower daily PM gain. In addition, SE infection strongly disturbed PM capillarization. Negative effects on capillary cross-sectional area and on the area (%) covered by capillaries persisted until D11 in the SE group, whereas pre-feeding of EF restored capillarization in the EFSE group to control levels. We conclude that supplementation of the probiotic bacteria EF AL41 had positive effects on PM capillarization and, thus, on delivery of O2, supply of nutrients, and removal of metabolites. Supplementation of probiotic bacteria might therefore reduce energetic stress and improve muscle health and meat quality during SE infection., (© The Author(s) 2018. Published by Oxford University Press on behalf of Poultry Science Association.)

Mitochondria are key intracellular targets of hypoxia-reoxygenation (H/R) stress due to their central role in generation of ATP and reactive oxygen species (ROS). Intertidal oysters Crassostrea gigas are adapted to frequent H/R cycles and maintain aerobic function despite frequent oxygen fluctuations. To gain insight into the molecular mechanisms of H/R tolerance, we assessed changes in mitochondrial respiration and (phospho)proteome of C. gigas during hypoxia and recovery. Oyster mitochondria maintained OXPHOS capacity despite a decline in cytochrome c oxidase activity during H/R stress. Rearrangements of the mitochondrial proteome during H/R stress involved upregulation of mitochondrial electron transport system and iron-binding proteins, and suppression of the pathways that channel electrons to ubiquinone, possibly as a mechanism to limit ROS production. H/R stress led to upregulation of a mitophagic activator PGAM5 and dephosphorylation of metalloendopeptidase OMA1, indicating stimulation of mitochondrial quality control mechanisms. Changes in abundance and phosphorylation levels of key proteins involved in mitochondrial protein homeostasis indicate suppression of protein synthesis during hypoxia, likely as an energy-saving mechanism, and its subsequent reactivation during reoxygenation. Thus, shifts in the mitochondrial (phospho-)proteome might play an important role in H/R stress resistance of oysters ensuring mitochondrial integrity and function during oxygen fluctuations. SIGNIFICANCE: Hypoxia-reoxygenation (H/R) stress elicits shifts in proteome and phosphoproteome of mitochondria in a hypoxia-tolerant model bivalve, oyster Crassostrea gigas, upregulating electron transport system, limiting electron flow to ubiquinone and activating mitochondrial quality control and protein homeostasis mechanisms. These findings provide insights into the potential role of proteomic shifts in adaptive response to H/R stress and serve as an important benchmark to understand the mechanisms of mitochondrial sensitivity to hypoxia and reoxygenation., (Copyright © 2018. Published by Elsevier B.V.)

Background: DL-2-hydroxy-4-methylthiobutyric acid (DL-HMTBA), an L-methionine (L-Met) hydroxyl analogue, has been suggested to be a dietary L-Met source. How dietary DL-HMTBA compared with L-Met affects whole-body L-Met kinetics in growing individuals is unknown., Objectives: We determined to what extent DL-HMTBA supplementation of an L-Met-deficient diet affects whole-body L-Met and L-cysteine (L-Cys) kinetics, protein synthesis (PS), and the L-Met incorporation rate in liver protein (L-MetInc) compared with L-Met and DL-Met supplementation in a piglet model., Methods: Forty-five, 28-d-old weaned piglets (male, German Landrace) were allocated to 4 dietary groups: L-Met-deficient diet [Control: 69% of recommended L-Met plus L-Cys supply; 0.22% standardized ileal digestible (SID) L-Met; 0.27% SID L-Cys; n = 12] and Control diet supplemented equimolarly to 100% of recommended intake with either L-Met (n = 12; LMET), DL-Met (n = 11; DLMET), or DL-HMTBA (n = 10; DLHMTBA). At 47 d of age, the piglets were infused with L-[1-13C; methyl-2H3]-Met and [3,3-2H2]-Cys to determine the kinetics and PS rates. Plasma amino acid (AA) concentrations, hepatic mRNA abundances of L-Met cycle and transsulfuration (TS) enzymes, and L-MetInc were measured., Results: During feed deprivation, L-Met kinetics did not differ between groups, and were ≤3 times higher in the fed state (P < 0.01). Remethylation (RM) was 31% and 45% higher in DLHMTBA than in DLMET and Control pigs, respectively, and the RM:transmethylation (TM) ratio was 50% higher in DLHMTBA than in LMET (P < 0.05). Furthermore, TS and the TS:TM ratio were 32% lower in DLHMTBA than in LMET (P < 0.05). L-MetInc was 42% lower in DLMET and DLHMTBA than in L-Met-deficient Control pigs, whereas plasma AA and hepatic mRNA abundances were similar among DL-HMTBA-, L-Met-, and DL-Met-supplemented pigs., Conclusions: In piglets, DL-HMTBA compared with L-Met and DL-Met supplementation increases RM and reduces the TS rate to conserve L-Met, but all 3 Met isomers support growth at a comparable rate., (© 2019 American Society for Nutrition.)

The influence of variously processed feedstuffs on the gastrointestinal tract, various blood parameters, and T cell subsets of different localizations was investigated in 36 broilers. Birds were fed a similarly composed feed mixture differing in particle size (coarse vs. fine). Feedstuffs with different particle size distribution were additionally hydrothermally treated (HTT) (without-non-compacted, pelleted, expanded, and pelleted). The HTT affected BW at slaughtering day and feed intake (P < 0.001). Birds fed non-compacted feed consumed less feed and were lighter than animals of other feeding groups irrespective of grinding fineness. Proventricular size was influenced by HTT (P < 0.001) but not the weight. Generally, feeding of pelleted feed increased proventricular size. Gizzard weight and length was affected by particle size and HTT (P < 0.01). Non-compacted feed resulted in heavier but shorter gizzards compared to other feeds except for finely ground expanded and pelleted feed. Organ traits of gizzard and proventriculus were strongly related to the feed intake. The jejunum was heavier in birds fed expanded and pelleted feed compared to animals fed solely pelleted feed (P = 0.030). The intestinal segment weight to length ratio (WL ratio) of duodenum, jejunum, and total small intestine was affected by HTT (P < 0.01). Generally, non-compacted feed resulted in lowest WL ratios regardless of the particle size distribution. Feeding variously processed feedstuffs also affected white blood cells, blood sodium, and chloride concentrations (P < 0.05) and tended to influence blood carbon dioxide partial pressure and blood total carbon dioxide levels (P < 0.1) in venous blood. Subsets of CD4/CD8 double negative T cells of caecal tonsils were influenced by HTT (P < 0.043). Non-compacted feed tended to increase these subsets in this localization compared to other feeds. In conclusion, the present study shows that an increased feed intake provoked by feed compaction evoke proventricular and ventricular alterations. Furthermore, feeding of variously processed feedstuffs influences the acid base homeostasis and the local immune system of caecal tonsils.

The domestic fowl (Gallus gallus f. dom.) is highly motivated to roost on elevated structures. Previous studies indicated that broiler chickens hardly use elevated perches but frequently use elevated platforms. However, it is unclear which height and type of elevated structures broilers prefer at various daytimes.We investigated the use of elevated perches and grids varying in height (10, 30, and 50 cm above the floor) by chickens of 3 strains differing in growth performance. In 2 successive trials, male chickens of Ross 308 (Ross, fast growing, n = 200), Lohmann Dual (Dual, medium growing, n = 200), and Lohmann Brown Plus (LB, slow growing, n = 200) were tested in 12 experimental compartments (each strain in 4 compartments), respectively. Usage of structures was recorded on video and analyzed with time-sampling observation for each week of age during dawn, the light period, dusk, and the dark period. In addition, behavioral activity was measured with an antenna-transponder system.The results showed that Ross and Dual chickens preferred grids to perches throughout the entire observation period. With increasing age, chickens of all strains increasingly used the elevated structures during all daytimes. In contrast to Dual and LB, Ross chickens reduced the use of structures from the 3rd to 4th week of age during the light but not the dark period. Additionally, during the dark period, chickens of all 3 strains at the end of fattening period used elevated structures at 50 cm height most often. The behavioral activity decreased significantly in all 3 strains but was much lower in Ross chickens compared with Dual and LB.The results indicated that chickens are motivated to roost at nighttime on high-elevated and suitable structures such as grids. Further studies are needed to assess the space requirements for elevated structures that should be offered to chickens.

Horse serum is commonly used as an additive to support the maintenance of hematopoietic progenitor cells in culture. However, the wide variability in the performance of different lots calls for parallel testing of multiple batches over extended periods of culture. Identification of the serum components that determine hematopoietic support would therefore save considerable time and effort and would help to standardize culture procedures. We report here that the ability of horse serum to support the self-renewal of multipotent murine hematopoietic progenitor FDCP-Mix cells is correlated to the concentration of specific fatty acid products of phospholipase A2 and more closely to the spectrum of eicosanoids generated by their further processing through cyclooxygenase and lipoxygenase pathways. Supportive sera have low levels of lysophosphatidylcholine and inflammatory eicosanoids. This links known markers of inflammation, infection and platelet activation to the ability of serum to maintain progenitor cells in an undifferentiated state, providing a means for prospective identification of suitable sera as well as quality control of the production process., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Pigment epithelium-derived factor (PEDF) is evolving as metabolic regulatory protein. Albeit mostly considered in only pathological conditions related to excess energy intake resulting in obesity and insulin resistance, PEDF is likely to be involved in other physiological processes such as the homeorhetic adaptation of metabolism to lactation. We aimed to characterize the expression of PEDF and its association to the concomitant mobilization of body reserves during lactation in nonobese subjects. This mobilization is particularly distinct in dairy cows, and we therefore assessed the mRNA expression of PEDF and its putative receptors in different tissues in 2 trials with dairy cows fed with or without conjugated linoleic acids (CLAs). Conjugated linoleic acids depress milk fat synthesis and may thus reduce the drain of energy via milk. In pluriparous cows, the serum PEDF concentrations and the mRNA abundance in subcutaneous adipose tissue (scAT), as well as the hepatic and scAT mRNA abundance of the putative receptors, adipose triglyceride lipase, and laminin receptor 1, changed over time of sampling (day -21 until day 252 relative to calving). Conjugated linoleic acid treatment was associated with reduced PEDF concentrations in serum and lower PEDF mRNA abundance in scAT on day 21 postpartum. Comparing different tissues from primiparous cows, PEDF mRNA was highest in the liver, followed by scAT, visceral adipose tissue (AT), and mammary gland, and lowest in the muscle. Significant changes in PEDF expression with time of sampling were limited to AT in primiparous and pluriparous cows. Our data support a regulatory role for PEDF. The similarities between the time course of the serum concentrations of PEDF and its mRNA abundance in scAT may point to a regulatory role for AT rather than the liver for PEDF in dairy cows., (Copyright © 2018 Elsevier Inc. All rights reserved.)

High performing dairy cows experience distinct metabolic stress during periods of negative energy balance. Subclinical disorders of the cow's energy metabolism facilitate failure of adaptational responses resulting in health problems and reduced performance. The autonomic nervous system (ANS) with its sympathetic and parasympathetic branches plays a predominant role in adaption to inadequate energy and/or fuel availability and mediation of the stress response. Therefore, we hypothesize that indices of heart rate variability (HRV) that reflect ANS activity and sympatho-vagal balance could be early markers of metabolic stress, and possibly useful to predict cows with compromised regulatory capacity. In this study we analysed the autonomic regulation and stress level of 10 pregnant dried-off German Holstein cows before, during and after a 10-h fasting period by using a wide range of HRV parameters. In addition heat production (HP), energy balance, feed intake, rumen fermentative activity, physical activity, non-esterified fatty acids, β-hydroxybutyric acid, cortisol and total ghrelin plasma concentrations, and body temperature (BT) were measured. In all cows fasting induced immediate regulatory adjustments including increased lipolysis (84%) and total ghrelin levels (179%), reduction of HP (-16%), standing time (-38%) and heart rate (-15%). However, by analysing frequency domain parameters of HRV (high-frequency (HF) and low-frequency (LF) components, ratio LF/HF) cows could be retrospectively assigned to groups reacting to food removal with increased or decreased activity of the parasympathetic branch of the ANS. Regression analysis reveals that under control conditions (feeding ad libitum) group differences were best predicted by the nonlinear domain HRV component Maxline (L MAX, R 2=0.76, threshold; TS=258). Compared with cows having L MAX values above TS (>L MAX: 348±17), those with L MAX values below TS (L MAX cows (18.5±0.4 and 47.3 kg/day). From the present study, it seems conceivable that L MAX can be used as a predictive marker to discover alterations in central autonomic regulation that might precede metabolic disturbances.

The insulin-like growth factors IGF-1 and IGF-2 play important roles in the growth, development, and metabolism of teleost fish. We isolated cDNA sequences of igf1, and igf2 genes from maraena whitefish. We quantified the mRNA and protein expressions of IGFs in different tissues of marketable juvenile maraena whitefish. Moreover, we analyzed the gene expression profiles during maraena whitefish development from unfertilized egg to fingerling and examined the effect of incubation temperature on igf1, and igf2 gene expression during embryonic and early larval development. Transcripts encoding IGF-1 or IGF-2 were detected in all tested tissues, with the greatest abundance in the liver. We measured higher igf2 than igf1 copy numbers in all tissues and at all developmental stages examined, even at advanced juvenile stages. Using the Western blot technique, we demonstrated that several isoforms of IGF-1 are expressed in the liver and gills but not in muscle tissue, indicating tissue-specific protein expression of IGF-1. We observed an accelerated embryonic development with increasing temperature, resulting in shortened hatching periods. Out of the three tested temperatures, we observed the highest hatching rate, larval hatching size, and larval growth at 6 °C. At 9 °C, hatching rate, larval hatching size and larval growth were reduced compared to the values we observed at 4 °C and 6 °C, since incubation temperature might have exceeded the optimum. To our knowledge, our data show for the first time that both igf1 and igf2 expression were upregulated due to elevated incubation temperature within embryonic development of fish. Further, we found significantly higher igf expression for the best-developing larvae (6 °C group) at specific life stages of maraena whitefish., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Broiler eggs were either incubated at 37.8°C during the whole incubation period (control), or at higher (38.8°C, group H) and lower temperatures (36.8°C, group L) from embryonic day (ED) 7 up to ED 10 (ED 7 to 10) or from ED 10 up to ED 13 (ED 10 to 13). Before and after this temperature treatment the eggs were incubated at 37.8°C. The day-old chicks were weighted, sexed and fed up to day 35. On days 1 and 35 samples were taken from the breast and leg muscles for analyzing of the mitochondrial respiratory activity (MRA) and from the breast muscles for analysis of the cross-sectional areas (CSA) and the glycogen phosphorylase (GP), phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS) and cytochrome oxidase (COX) activities. Statistical analysis showed that treatment (control, group H, group L), sex and their interaction, but not the treatment period (ED 7 to 10; ED 10 to 13), significantly influenced the results. Group H chicks had lower (P⩽0.05) body and heart weights but higher (P⩽0.05) liver weights, CSA values, leg MRA as well as PFK, LDH, CS, GP and COX activities compared with the group L chicks. The results of the control chicks differ (P⩽0.05) from those of the group H (body, heart weight, COX), the group L chicks (liver weight, PFK, LDH, CS, GP) or the birds of both other groups (CSA). The group H broiler had higher (P⩽0.05) body and leg weights as well as LDH, CS, COX and GP activities than the group L broilers. The BWs and the LDH and GP results of the control broiler differ (P⩽0.05) from those of both other groups or from the results of the group H (CS) and group L broiler (COX). Female broilers had lower (P⩽0.05) body, breast and leg weights, but higher (P⩽0.05) CSA, LDH, CS and GP activities than the male animals. Analysis of treatment×sex interaction showed that group H hens had higher (P⩽0.05) body and breast weights, LDH and GP activities compared with the group L hens, whereas in the male broiler no effect of the interaction could be found, except for the lower (P⩽0.05) CSA values in the group H than group L cocks. The treatment effects are probably due to altered embryonic activity and related molecular mechanisms. The sex-related differences in the broiler indicate that these alterations already occur in the embryos and chicks, but become significant with the sexual dimorphism after hatch.

Factors affecting the development of Ascaridia galli-specific humoral responses and their protective roles are largely unknown. We investigated the effects of time and infection dose on A. galli-specific IgY antibody levels following experimental infection. The acquisition and development of new infections and reinfections were also monitored by using tracer birds. Relationships between the retrospective IgY and the final worm burden of the birds were investigated to determine whether humoral immune responses generated during infection provide protection to the host animal. Young chickens were infected (+) with either 100 or 1000 embryonated eggs of A. galli (100+: n = 45; 1000+: n = 45) or kept as uninfected controls (CON: n = 10). Uninfected birds were also added to each infection group as tracer (T) birds (T100+; n = 5 and T1000+; n = 5). Faecal egg counts and IgY antibody concentrations in plasma and egg yolk were determined at selected intervals. Final worm burdens were quantified at 28 weeks post infection (wpi). The plasma antibody (PAB) and egg yolk antibody (EAB) levels of experimentally infected birds were compared to those of control and tracer birds throughout the study period, and PAB levels were found to depend initially on the infection dose but thereafter mainly on reinfections. Starting at wpi 2, 1000+ had consistently higher PAB levels than CON did (P < 0.05). With exceptions at wpi 0, 2 and 12, PAB levels were also higher (P < 0.05) or tended to be higher (P < 0.10) in 100+ than in CON. An earlier and higher increase was observed in the PAB levels of T1000+ than in those of T100+, implying that (re-)infection occurrence depended on the infection dose. Although 1000+ showed higher (P < 0.05) EAB levels than CON did at both wpi 14 and 18, EAB levels were higher in 100+ than in CON only at wpi 28 (P < 0.05). The total worm burdens in the initial experimentally infected birds were similar (P = 0.257); they were also highly comparable between experimentally and naturally infected birds, indicating that final worm burden was mainly determined by the naturally occurring infections resulting from continuous exposure. When all available information on the retrospective plasma and egg yolk IgY levels was collectively evaluated to estimate the larval or total worm burdens of the experimentally infected birds, both PAB and EAB levels at particular wpi were significantly associated with worm burden, especially with larval count. In conclusion, our data support the hypothesis that the number of larvae, rather than the number of mature worms, affects the antibody levels in both plasma and egg yolk. Despite the increased levels of A. galli-specific antibodies in plasma and egg yolk throughout the study period, only a weak indication was found that antibodies might be directly associated with protection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Residual feed intake (RFI), the difference between actual feed intake and predicted feed requirements, is suggested to impact various aspects of meat quality. The objective of this study was to investigate the molecular mechanisms underpinning the relationship between RFI and meat quality. Technological, sensory and nutritional analysis as well as transcriptome profiling were carried out in Longissimus thoracis et lumborum muscle of pigs divergent in RFI (n=20). Significant differences in sensory profile and texture suggest a minor impairment of meat quality in more efficient pigs. Low RFI animals had leaner carcasses, greater muscle content and altered fatty acid profiles compared to high RFI animals. Accordingly, differentially expressed genes were enriched in muscle growth and lipid & connective tissue metabolism. Differences in protein synthesis and degradation suggest a greater turnover of low RFI muscle, while divergence in connective tissue adhesion may impact tenderness. Fatty acid oxidation tending towards decrease could possibly contribute to reduced mitochondrial activity in low RFI muscle., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Exposure to psychosocial stress can have a profound impact on immune reactivity and health mediated by hypothalamic-pituitaryadrenal (HPA) axis activation. However, current knowledge regarding the mechanisms involved in cross-sensitization between stress and the immune system is limited. Here, we investigated the effects of a single social isolation followed by repeated oral Escherichia coli (E. coli) applications on cortisol, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), haptoglobin and C-reactive protein (CRP) concentrations in the blood; on clinical signs of disease; and on mRNA expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11β-hydroxysteroid dehydrogenase 1 and 2 (11β-HSD1 and 11β-HSD2), TNF-α and IL-6 in the hypothalamus, prefrontal cortex (PFC) and spleen of 7-, 21- and 35-day-old piglets. Additionally, the protein levels of splenic TNF-α and IL-6 were analyzed. Non-isolated, E. coli-challenged piglets served as a control. Social isolation for 4 h induced a rise in the plasma cortisol concentrations immediately after social treatment and after repeated E. coli applications in isolated compared to non-isolated piglets. The circulating TNF-α concentration was not affected by social treatment. Furthermore, previously isolated piglets showed a higher frequency of signs of disease in response to E. coli challenge than non-isolated piglets, while the haptoglobin and CRP concentrations did not significantly differ between social treatments. In the brain, 11β-HSD1, 11β-HSD2 and IL-6 mRNA expression in the hypothalamus and GR, and 11β-HSD1 and 11β-HSD2 mRNA expression in the PFC were higher in isolated, E. coli-challenged piglets than in the corresponding controls. Moreover, isolated piglets also displayed higher MR, 11β-HSD1 and IL-6 mRNA expression levels and TNF-α concentrations in the spleen. The stress-induced alterations in the hypothalamus and spleen were particularly pronounced in younger piglets. The present findings may contribute to a better understanding of the complex interplay between early psychological stress and an increased risk of disease and might also have implications on aspects of the health and welfare of farm animals and humans., (Copyright © 2017. Published by Elsevier Inc.)

Cytokines are required for normal growth and development of the mammary gland and TGF-β prominently represents an established effector of apoptosis, e.g., during involution of the mammary gland. By the control of intracellular signaling pathways, including JAK/STAT, MAPK, PI-3K, and NF-κB, cytokines efficiently regulate cell proliferation and inflammation in the breast. Therefore, cytokines are discussed also in a context of malignant mammary growth. As a group of tissue hormones produced by somatic cells or by cells from the immune system, cytokines are defined by their immunomodulatory potential. Over the past 40 years, multiple cytokines were identified in colostrum and milk. Importantly, cytokines derived from mammary secretions after birth are required for maturation of the immune system in the developing gastrointestinal tract from the suckling. Moreover, recent studies have further assessed the particular interactions between probiotic bacterial strains and cytokines. In light of the increasing prevalence of inflammatory diseases of the gastrointestinal system, the effects of probiotic microorganisms during milk fermentation may have immunotherapeutic potential in the future., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Salmon lice (Lepeophtheirus salmonis) are parasitic copepods, living mainly on Atlantic salmon and leading to large economical losses in aquaculture every year. Due to the emergence of resistances to several drugs, alternative treatments are developed, including treatment with hydrogen peroxide, freshwater or thermal treatment. The present study gives a first overview of the thermotolerance and stress response of salmon lice. Sea lice nauplii acclimated to 10 °C can survive heat shocks up to 30 °C and are capable of hardening by a sublethal heat shock. We searched in the genome for heat shock protein (HSP) encoding genes and tested their inducibility after heat shock, changes in salinity and treatment with hydrogen peroxide, employing microfluidic qPCRs. We assessed 38 candidate genes, belonging to the small HSP, HSP40, HSP70 and HSP90 families. Nine of these genes showed strong induction after a non-lethal heat shock. In contrast, only three and two of these genes were induced after changes in salinity and incubation in hydrogen peroxide, respectively. This work provides the basis for further work on the stress response on the economically important parasite L. salmonis.

Evidence in the current literature suggests that expression and activity of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a key regulatory enzyme in glucocorticoid metabolism, is elevated in the liver and reduced in visceral adipose tissue and skeletal muscle following caloric restriction (CR). In order to investigate the influence of CR on 11β-HSD1 in more detail, we assessed expression and activity of 11β-HSD1 in several tissues in two independent CR and re-feeding animal models. Levels and activity of 11β-HSD1 after CR and re-feeding were measured [mouse liver and pig liver, pig visceral adipose tissue and pig skeletal muscle] using semi-quantitative RT-PCR, Western Blot analysis, and HPLC. After CR, no significant difference on mRNA levels was detected in mouse liver. But 11β-HSD1 mRNA expression was upregulated after subsequent re-feeding. In contrast, 11β-HSD1 protein expression after CR was significantly up-regulated, while no difference was detected after re-feeding. Interestingly, upregulation of protein after CR (1.4-fold) was lower than the increase in enzymatic activity (2.6-fold). Furthermore, while no difference was observed in protein levels after two weeks re-feeding, 11β-HSD1 activity increased 2.5-fold. In pig tissues neither 11β-HSD1 mRNA levels, protein expression or enzyme activity were influenced after CR and re-feeding. Overall, the results demonstrate species-dependent differences in regulation of 11β-HSD1 following CR and suggest the presence of an additional regulation step for 11β-HSD1 activity in mouse liver., (Copyright © 2017 Elsevier B.V. All rights reserved.)

To improve production efficiency, the sheep meat industry has increased flock prolificacy. However, multiple-born lambs have lower birth weights, increased mortality and reduced growth rate compared with single-born lambs. Lamb mortality is a major issue for livestock farming globally and solutions are required to increase survival to realise the value of increased flock fecundity. Nutrition during gestation can influence maternal-foetal placental nutrient transfer and thus foetal growth and organ/tissue development, as well as improve postnatal productivity. This review covers the challenges and opportunities associated with increased prolificacy, highlights gaps in our knowledge and identifies some opportunities for how targeted intervention with specific nutrients during mid-to-late pregnancy may influence lamb survival and productivity with a specific focus on pasture-based systems. This time frame was selected as intervention strategies in short-time windows post-pregnancy scanning and before lambing to improve lamb survival in high-risk groups (e.g. triplets) are likely to be the most practical and economically feasible options for pasture-based extensive farming systems.

IGFs and IGF-binding proteins (IGFBPs) are abundantly present in milk and in dairy products. Compared to the IGFs, the IGFBP have received less attention in milk, although truncated IGFBPs and IGFBP-glycosylation have been described in milk. Thereby, complex control of local IGF-effects can be assumed on the levels of IGFBPs, proteases, and protease inhibitors. The present review collects the current knowledge both on presence and regulation of IGFs and IGFBPs in milk particularly from dairy animal species. As a rule higher levels of IGF-I, IGF-II, and IGFBPs are measured around parturition if compared to later time-points of lactation. In all farm animal species included in this review, it is found that the relative abundancies of IGFBPs in milk and serum are similar, with IGFBP-3 and -2 characterized by higher concentrations if compared to IGFBP-4 or -5. The concentrations of IGFs and IGFBPs in milk or dairy products can be altered by hormones, dairy processing, or fermentation. Because milk can be used for non-invasive biomarker research, quality management, and health monitoring, we discuss novel directions of IGF-analysis and potential on-site biomarker research in milk., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The development of milk during evolution is considered a more recent step to provide the neonate with adequate amounts of energy, nutrients, and specific hormonal signals thereby, granting a fast and efficient rate of postnatal growth and development. Since the insulin- or the insulin-like growth factor (IGF) systems were evolved much earlier, it can be assumed that the functionality of the IGF-system has been integrated into the novel matrix milk containing casein and whey proteins from the beginnings. In fact, IGFs and IGF-binding proteins (IGFBPs) are abundantly present in milk, which is particularly true for fore-milk or colostrum and the potential effects of milk-borne IGF-compounds on the consuming organisms have in fact been addressed by several studies. Those studies examined, if orally administered IGFs can be absorbed by the consumer's gastro-intestinal tract and thus contribute e.g. to the somatic growth of infants. A second line of studies assessed local effects of milk-borne IGFs on growth and development of the gastro-intestinal tract itself. Finally, distinct functions of isolated IGF-compounds for growth and involution of the mammary gland have also been provided in the past. While the consumption of milk seems not to represent a major source of endogenous IGFs, accumulating evidence indicates secondary effects of milk on the endogenous IGF-system, which may be mediated by micronutrients such as branched amino acids and metabolic programming. By contrast, direct effects on growth and development of oesophageal and intestinal cells have been observed if IGFs were administered orally., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

There is considerable interest in enhancing beneficial fatty acids, particularly 18:3n-3, conjugated linoleic acid and long chain n-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in beef to provide benefits to human health. Here, the potential to enhance these fatty acids by feeding a n-3 polyunsaturated fatty acid (PUFA)-rich plant extract (PX) from lucerne is presented. Cattle (n=8/diet) were assigned to the following finishing diets: straw and concentrate (S-CC); straw and concentrate containing 25% PX and 2000IUvitaminE/kg PX-concentrate (S-PXC); grass silage (GS); GS plus 75gPX/kg dry matter intake (DMI) (GS-LPX) or GS plus 150g PX/kg DMI (GS-HPX). Addition of PX to concentrates or GS increased the n-3 fatty acid content of loin muscle and had favourable effects on the n-6:n-3 PUFA ratio (P<0.05), without detrimental effects on loin muscle sensory characteristics., (Copyright © 2017. Published by Elsevier Ltd.)

Data on nutrient sensing by free fatty acid receptors (FFAR1, FFAR2, FFAR3, FFAR4) and hydroxycarboxylic acid receptors (HCAR1, HCAR2) are increasing for human or rodent models. Both receptor families link intestinal fermentation by the microbiota and energy metabolism with cellular responses. Therefore, this finding provides a link that is independent of the only function of the fermentation products as energy substrates. For example, these reactions are associated with insulin secretion, regulation of lipolysis, adipose tissue differentiation and innate immune responses. In farm animals, the available data on both receptor families from the intestine and other tissues increase. However, currently, the data are primarily linked with the distribution of receptor messenger RNAs (mRNAs) and more rarely with proteins. Functional data on the importance of these receptors in farm animal species is not abundant and is often associated with the immune system. In certain farm animal species, the receptors were cloned and ligand binding was characterised. In chicken, only one FFAR2 was recently identified using genome analysis, which is contradictory to a study using an FFAR1 small interfering RNA. The chicken FFAR2 is composed of more than 20 paralogs. No data on HCAR1 or HCAR2 exist in this species. Currently, in pigs, most available data are on the mRNA distribution within intestine. However, no FFAR1 expression has been shown in this organ to date. In addition to FFAR2, an orthologue (FFAR2-like) with the highest abundance in intestine has been reported. The data on HCAR1 and HCAR2 in pigs is scarce. In ruminants, most of the currently available information on receptor distribution is linked to mRNA data and shows the expression, for example, in mammary gland and adipose tissue. However, some protein data on FFAR2 and FFAR1 protein has been reported and functional data availability is slowly increasing. The receptor mRNAs of HCAR1 and HCAR2 are expressed in bovine. The HCAR2 protein has been demonstrated in certain tissues, such as liver and fat. Because of the physiological importance of both receptor families in human life science, more studies that analyse the physiological significance of both receptor families in animal science may be performed within the next several years.

Objective: This study is a first time assessment of safety and tolerability, pharmacokinetics, and pharmacodynamics of RO5046013 in human, in comparison with unmodified rhIGF-I., Design: The study was conducted as a single-center, randomized, double-blinded, placebo-controlled, single ascending dose, parallel group study in a clinical research unit in France. A total of 62 healthy volunteers participated in this clinical trial. RO5046013 was given as single subcutaneous injection, or as intravenous infusion over 48h, at ascending dose levels. The active comparator rhIGF-I was administered at 50μg/kg subcutaneously twice daily for 4days. Safety and tolerability, pharmacokinetics, and pharmacodynamics of RO5046013 were evaluated., Results: PEGylation resulted in long exposure to RO5046013 with a half-life of 140-200h. Exposure to RO5046013 increased approximately dose proportionally. RO5046013 was safe and well tolerated at all doses, injection site erythema after SC administration was the most frequent observed AE. No hypoglycemia occurred. Growth hormone (GH) secretion was almost completely suppressed with rhIGF-I administration, whereas RO5046013 caused only a modest decrease in GH at the highest dose given IV., Conclusions: PEGylation of IGF-I strongly enhances half-life, reduces the negative GH feedback and hypoglycemia potential, and therefore offers a valuable alternative to rhIGF-I in treatment of relevant diseases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Brain growth and function are regulated by insulin-like growth factors I and II (IGF-I and IGF-II) but also by IGF-binding proteins (IGFBPs), including IGFBP-2. In addition to modulating IGF activities, IGFBP-2 interacts with a number of components of the extracellular matrix and cell membrane via a Cardin-Weintraub sequence or heparin binding domain (HBD1). The nature and the signalling elicited by these interactions are not fully understood. Here, we examined transgenic mice (H1d-hBP2) overexpressing a mutant human IGFBP-2 that lacks a specific heparin binding domain (HBD1) known as the Cardin-Weintraub sequence. H1d-hBP2 transgenic mice have the genetic background of FVB mice and are characterized by severe deficits in brain growth throughout their lifetime (p<0.05). In tissue lysates from brain hemispheres of 12-21day old male mice, protein levels of the GTPase dynamin-I were significantly reduced (p<0.01). Weight reductions were also found in distinct brain regions in two different age groups (12 and 80weeks). In the younger group, impaired weights were observed in the hippocampus (-34%; p<0.001), cerebellum (-25%; p<0.0001), olfactory bulb (-31%; p<0.05) and prefrontal cortex (-29%; p<0.05). At an age of 12weeks expression of myelin basic protein was reduced (p<0.01) in H1d-BP-2 mice in the cerebellum but not in the hippocampus. At 80weeks of age, weight reductions were similarly present in the cerebellum (-28%; p<0.001) and hippocampus (-31; p<0.05). When mice were challenged in the elevated plus maze, aged but not younger H1d-hBP2 mice displayed significantly less anxiety-like behaviour, which was also observed in a second transgenic mouse model overexpressing mouse IGFBP-2 lacking HBD1 (H1d-mBP2). These in vivo studies provide, for the first time, evidence for a specific role of IGFBP-2 in brain functions associated with anxiety and risk behaviour. These activities of IGFBP-2 could be mediated by the Cardin-Weintraub/HBD1 sequence and are altered in mice expressing IGFBP-2 lacking the HBD1., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Maternally derived antibodies can provide partial protection against certain bacterial and viral infections. We investigated whether chicks descending from nematode-infected hens are more resistant against Ascaridia galli, a prevalent gastrointestinal nematode, than chicks from nematode-free mothers. One-day-old chicks (N=153) from infected (mab+; maternal antibody+) or uninfected control dams (mab-) were experimentally infected with A. galli at two different levels (100 or 1000 eggs/chick). The worm burdens of the chicks were determined at 6 weeks post infection. There was a high correlation (r=0.89) between A. galli-specific antibody concentrations in dam plasma and egg yolk. There was no difference between worm burdens of chicks descending from infected or uninfected dams (P=0.892), indicating no maternally derived protection against A. galli. Chicks receiving the higher infection dose had higher worm burdens (P<0.05). Although there was no difference (P>0.05) between worm counts of female and male chicks infected with 100 eggs, females chicks infected with 100 eggs harboured longer and heavier female worms. We conclude that there is no protective maternal immunity against A. galli infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of our study was to characterize the immediate phenotypic and adaptive regulatory responses of fetuses to different in utero conditions reflecting inadequate maternal protein supply during gestation. The gilts fed high- (250% above control) or low- (50% under control) protein diets isoenergetically adjusted at the expense of carbohydrates from the day of insemination until the fetuses were collected at day 64 or 94 of gestation. We analyzed body composition, histomorphology, biochemistry, and messenger RNA (mRNA) expression of fetal skeletal muscle. Both diets had only marginal effects on body composition and muscular cellularity of fetuses including an unchanged total number of myofibers. However, mRNA expression of myogenic regulatory factors (MYOG, MRF4, P ≤ 0.1), IGF system (IGF1, IGF1R, P ≤ 0.05) and myostatin antagonist FST (P = 0.6, in males only) was reduced in the fetal muscle exposed to a maternal low-protein diet. As a result of excess protein, MYOD, MYOG, IGF1R, and IGFBP5 mRNA expression (P ≤ 0.05) was upregulated in fetal muscle. Differences in muscular mRNA expression indicate in utero regulatory adaptive responses to maternal diet. Modulation of gene expression immediately contributes to the maintenance of an appropriate fetal phenotype that would be similar to that observed in the control fetuses. Moreover, we suggest that the modified gene expression in fetal skeletal muscle can be viewed as the origin of developmental muscular plasticity involved in the concept of fetal programming., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The production rates of viable embryos using sexed semen through the conventional methodologies of multiple ovulation and embryo transfer are generally not satisfactory. However, the cryopreservation of these embryos is considered efficient. Knowledge of epigenetics can provide new tools or allow for adapting new protocols that could enhance the efficiency of reproductive biotechnologies. The aim of this study was to characterize the pattern of trimethylation of histone 3 at lysine 4 (H3K4me3) in bovine embryos produced in vivo with sexed semen that were submitted to cryopreservation. Bos taurus × Bos indicus cows (n = 5) were superovulated and inseminated with sexed (two sessions) or conventional (two sessions) semen. A portion of the embryos collected on Day 7 was immediately stored in paraformaldehyde (3%) and another portion was stored in paraformaldehyde after cryopreservation/thawing. All embryos from the four groups (fresh, conventional semen; fresh, sexed semen; cryopreserved, conventional semen; and cryopreserved, sexed semen; 15 embryos per group) were evaluated by immunofluorescence under confocal microscopy to identify and quantify the H3K4me3 status. In total, 190 embryos were recovered, 100 of which were produced with conventional semen and 90 with sexed semen. The use of conventional semen after superovulation yielded 72% (72 of 100) viable embryos, which were mostly (81%; 59 of 72) in advanced stages of development (blastocysts and expanded blastocysts). Embryos produced with sexed semen had a lower viability rate (36.7%; 33 of 90), and most of them were collected at earlier stages of development (morulae and early blastocysts; P < 0.05). The H3K4me3 signal was similar among groups; however, there was a difference between morulae and blastocysts. A high intensity of H3K4me3 was observed in bovine embryos produced in vivo, and this pattern did not vary using sexed semen and the slow cryopreservation process. The lower viability of bovine embryos produced with sexed semen could be not explained by differences in H3K4me. Cryopreservation did not alter the pattern of H3K4me3; in this sense, we suggest that it is a process that exerts minimal damage to the embryos., (Copyright © 2016 Elsevier Inc. All rights reserved.)