The interest in biodiesel production from oil-bearing seeds rather than soybean necessitates the scientific validation of other good quality protein sources that could substitute soybean meal in animal diets, particularly, broiler chickens where soybean meal constitutes a large portion of their diet. Therefore, the present study was conducted to investigate the effect of sun-dried Azolla leaf meal (ALM) as an unconventional dietary protein source in broiler chicken diet on growth performance, meat quality, skeletal muscle cell growth and protein synthesis through regulation of ribosomal protein S6 kinase (p70S6 kinase α). A total of 120 male Ross 308 broiler chicks were randomly allocated to three dietary treatments. Each treatment had four cages (i.e. replicates) with 10 birds/cage. The control group was fed with a corn–soy-based diet, the AZ5 group was supplemented with 5% ALM and the AZ10 group was supplemented with 10% ALM for 37 days. A 5-day trial was also conducted to measure the apparent nutrient digestibility. Growth performance parameters were measured weekly. At the end of the experiment, 12 birds from each group (3/cage) were euthanized and used for samplings. Inclusion of ALM tended to improve BW gain (P = 0.06) and increased feed intake (P < 0.01). Additionally, ALM decreased the percentage of breast meat cooking loss linearly (P < 0.01). In addition, ALM at a dose of 5% increased the production of propionate in the cecum (P = 0.01). Activation of breast muscle p70S6 kinase was higher when ALM was included in a dose-dependent manner (P < 0.01). The inclusion of ALM increased breast meat redness (P < 0.01); however, the lightness was within the normal range in all groups. Findings from our study suggest that ALM could be included in a broiler chicken diet up to 5% without any major negative effect on meat quality or performance, and it regulates muscle protein synthesis through activation of mammalian target of rapamycin/6S kinase signaling.

Nuclear factor erythroid 2-related factor 2 (NRF2) plays a key role in the response to oxidative stress. Diets containing known NRF2 modulators could be used to minimize oxidative stress in dairy cows. Currently, studies evaluating the activity of NRF2 in bovine have used the classical in vitro approach using synthetic media, which is very different than in vivo conditions. Furthermore, studies carried out in vivo cannot capture the short-term and dynamic response of NRF2. Thus, there is a need to develop new approaches to study NRF2 modulation. The aim of the present study was to establish an in vitro–in vivo hybrid system to investigate activation of NRF2 in bovine cells that can serve as an intermediate model with results closer to what is expected in vivo. To accomplish the aim, we used a combination of a gene reporter assay in immortalized bovine mammary cells, synthetic NRF2 modulators, and blood serum from periparturient cows. Synthetic agonist tert-butylhydroquinone and sulforaphane confirmed to be effective activators of bovine NRF2 with acute and large effect at 30 and 5 μM, respectively, with null response after the above doses due to cytotoxicity. When the agonists were added to blood serum the response was more linear with maximum activation of NRF2 at 100 and 30 μM, respectively, and the cytotoxicity was prevented. High concentration of albumin in blood serum plays an important role in such an effect. Brusatol (100 nM) was observed to be an effective NRF2 inhibitor while also displaying general protein synthesis inhibition and cytotoxicity when added to synthetic media. A consistent inhibition of NRF2 was observed when brusatol was added to the blood serum but the cytotoxicity was reduced. The synthetic inhibitor ML385 had no effect on modulation of bovine NRF2. Hydrogen peroxide activates NRF2 in bovine mammary cells starting from 100 μM; however, strong cytotoxicity was detected starting at 250 μM when cells were cultivated in the synthetic media, while blood serum prevented cytotoxicity. Overall, our data indicated that the use of synthetic media can be misleading in the study of NRF2 in bovine and the use of blood serum appears necessary.

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Metabolic challenges experienced by dairy cows during the transition between pregnancy and lactation (also known as peripartum), are of considerable interest from a nutrigenomic perspective. The mobilization of large amounts of non-esterified fatty acids (NEFA) leads to an increase in NEFA uptake in the liver, the excess of which can cause hepatic accumulation of lipids and ultimately fatty liver. Interestingly, peripartum NEFA activate the Peroxisome Proliferator-activated Receptor (PPAR), a transcriptional regulator with known nutrigenomic properties. The study of PPAR activation in the liver of periparturient dairy cows is thus crucial; however, current in vitro models of the bovine liver are inadequate, and the isolation of primary hepatocytes is time consuming, resource intensive, and prone to errors, with the resulting cells losing characteristic phenotypical traits within hours. The objective of the current study was to evaluate the use of precision-cut liver slices (PCLS) from liver biopsies as a model for PPAR activation in periparturient dairy cows. Three primiparous Jersey cows were enrolled in the experiment, and PCLS from each were prepared prepartum (−8.0 ± 3.6 DIM) and postpartum (+7.7± 1.2 DIM) and treated independently with a variety of PPAR agonists and antagonists: the PPARα agonist WY-14643 and antagonist GW-6471; the PPARδ agonist GW-50156 and antagonist GSK-3787; and the PPARγ agonist rosiglitazone and antagonist GW-9662. Gene expression was assayed through RT-qPCR and RNAseq, and intracellular triacylglycerol (TAG) concentration was measured. PCLS obtained from postpartum cows and treated with a PPARγ agonist displayed upregulation of ACADVL and LIPC while those treated with PPARδ agonist had increased expression of LIPC, PPARD, and PDK4. In PCLS from prepartum cows, transcription of LIPC was increased by all PPAR agonists and NEFA. TAG concentration tended to be larger in tissue slices treated with PPARδ agonist compared to CTR. Use of PPAR isotype-specific antagonists in PCLS cultivated in autologous blood serum failed to decrease expression of PPAR targets, except for PDK4, which was confirmed to be a PPARδ target. Transcriptome sequencing revealed considerable differences in response to PPAR agonists at a false discovery rate-adjusted p-value of 0.2, with the most notable effects exerted by the PPARδ and PPARγ agonists. Differentially expressed genes were mainly related to pathways involved with lipid metabolism and the immune response. Among differentially expressed genes, a subset of 91 genes were identified as novel putative PPAR targets in the bovine liver, by cross-referencing our results with a publicly available dataset of predicted PPAR target genes, and supplementing our findings with prior literature. Our results provide important insights on the use of PCLS as a model for assaying PPAR activation in the periparturient dairy cow.

Fishmeal (FM) is the main protein source in fish feed. However, it is quite expensive due to its limited resources. Therefore, finding a dietary alternative to the FM to sustain fish production is crucial, and the current study was performed to assess the impact of poultry offal silage (POS) with or without betaine supplementation; as an effective and cheaper alternative to FM; on feed efficiency, growth performance, spleen morphology and intestinal morphometry of Nile tilapia (Oreochromis niloticus) fingerlings. Four dietary treatments were formulated: (1) FM based diet, (2) FM‐B; FM diet +0.7% betaine, (3) POS diet and (4) POS‐B; POS diet +0.7% betaine. Each dietary treatment consisted of three replicates (n = 10/replicate), and the experiment was continued for 16 weeks. By the end of the experiment, spleen and intestine specimens were collected from 15 fish (n = 5/replicate) for histopathological assessment. The results were statistically analysed using GLM procedures of SAS 9.4. Feed efficiency increased in both POS‐B and FM‐B groups (p = 0.01), while body weight and body weight gain showed only weak tendencies towards an increase (p = 0.10 and 0.12, respectively). The villi length was the highest in POS‐B fed group (p < 0.01). In addition, melanomacrophage centres of the spleen increased in both betaine‐supplemented groups (p < 0.01). From our findings, we conclude that betaine supplementation with poultry offal silage improved production performance and immune status of Nile tilapia fish.