Propolis alleviates the negative effects of heat stress on egg production, egg quality, physiological and immunological aspects of laying Japanese quail.

The present work was carried out to investigate the effects of dietary propolis supplementation to laying Japanese quail (Coturnix coturnix japonica) on egg production, egg quality, physiological and immunological aspects under heat stress conditions. A total of 200, 21-day-old, Japanese quail females were distributed equally into standard wired cages in two identical environmentally-controlled rooms (10 cages per room, 10 birds per cage). From 29-70 d of age, the quail birds in the first room remained at a normal temperature of 24°C (C group), whereas the quail birds in the second room were kept under heat stress at 35°C (HS group). Each group was further assigned to 2 propolis subgroups (5 cages per subgroup); one of them received a basal diet without propolis supplementation (-PR subgroup), while, the other received 1 g propolis/ kg basal diet (+PR subgroup). In the present study, performance and egg production of laying quail were significantly (P<0.001) impaired by HS treatment and improved by the PR treatment. Similarly, the negative and positive effects of HS and PR, respectively, were appeared on the egg shell thickness and yolk index. Stress indicators in laying quail were significantly (P<0.001) increased by HS, while, PR significantly (P<0.05) moderated these levels in the HS+PR group when compared to the HS-PR quail group. In addition to the positive impact of PR on the plasma levels of calcium, phosphorus, and albumin, it also normalized the plasma levels of alanine aminotransferase and cholesterol in the heat-stressed quail birds. Moreover, the quail birds in the HS groups expressed lower immunological aspects than those in the C group, while, the addition of propolis to the diets enhanced the immune status of laying quail birds under HS conditions. These results strongly suggest that dietary propolis supplementation could be a successful attempt to maintain the performance and egg production of laying Japanese quail at convenient levels under heat stress conditions.

Antioxidant and developmental capacity of retinol on the in vitro culture of rabbit embryos.

SummaryOxidative stress is a major cause of defective embryo development during in vitro culture. Retinoids are recognized as non-enzymatic antioxidants and may have an important role in the regulation of cell differentiation and vertebrate development. However, there are not enough reports discussing the antioxidant and developmental capacity of retinoids, including retinol (RT), on the in vitro development of embryos recovered from livestock animals, particularly in rabbit species. Therefore, morula embryos obtained from nulliparous Red Baladi rabbit does were cultured for 48 h in TCM199 medium in the absence of RT (control group) or in the presence of RT at concentrations of 10, 100 and 1000 nM. The developmental capacity to the hatched blastocyst stage, the antioxidant biomarker assay and the expression of several selected genes were analyzed in each RT group. The data show that RT significantly (P<0.001) promoted the embryo hatchability rate at the concentration of 1000 nM to 69.44% versus 29.71% for the control. The activity of malondialdehyde (MDA) level was significantly (P<0.05) lower in the RT groups than in the control group, while the total antioxidant capacity (TAC), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were significantly (P<0.05) higher following treatment with RT. Furthermore, RT treatment considerably upregulated the relative expression of gap junction protein alpha 1 (GJA1), POU class 5 homeobox 1 (POU5F1) and superoxide dismutase 1 (SOD1) genes compared with the control group. The current study highlights the potential effects of RT as antioxidant in the culture medium on the in vitro development of rabbit embryos.

The importance of propolis in alleviating the negative physiological effects of heat stress in quail chicks.

Heat stress is one of the most detrimental confrontations in tropical and subtropical regions of the world, causing considerable economic losses in poultry production. Propolis, a resinous product of worker honeybees, possesses several biological activities that could be used to alleviate the deleterious effects of high environmental temperature on poultry production. The current study was aimed at evaluating the effects of propolis supplementation to Japanese quail (Coturnix coturnix japonica) diets on the production performance, intestinal histomorphology, relative physiological and immunological parameters, and selected gene expression under heat stress conditions. Three hundred one-day-old Japanese quail chicks were randomly distributed into 20 wired-cages. At 28 d of age, the birds were divided into 2 temperature treatment groups; a normal at 24°C (C group) and a heat stress at 35°C (HS group). The birds in each group were further assigned to 2 subgroups; one of them was fed on a basal diet without propolis supplementation (-Pr subgroup) while the other was supplemented with propolis (+Pr subgroup). Production performance including body weight gain, feed intake and feed conversion ratio were measured. The intestinal histomorphological measurements were also performed for all treatment groups. Relative physiological parameters including body temperature, corticosterone hormone level, malondialdehyde (MDA) and free triiodothyronine hormone (fT3), as well as the relative immunological parameters including the total white blood cells count (TWBC's), heterophil/lymphocyte (H/L) ratio and lymphocyte proliferation index, were also measured. Furthermore, the mRNA expression for toll like receptor 5 (TLR5), cysteine-aspartic protease-6 (CASP6) and heat shock proteins 70 and 90 (Hsp70 and Hsp90) genes was quantified in this study. The quail production performance was significantly (P<0.05) impaired by HS treatment, while Pr treatment significantly improved the quail production performance. The villus width and area were significantly (P<0.05) lower in the HS compared to the C group, while Pr treatment significantly increased crypts depth of quail. A negative impact of HS treatment was observed on the physiological status of quail; however, propolis significantly alleviated this negative effect. Moreover, quail of the HS group expressed lower immunological parameters than C group, while propolis enhanced the immune status of the quail. The relative mRNA expression of TLR5 gene was down-regulated by HS treatment while it was up-regulated by the Pr treatment. Furthermore, the positive effects of propolis in HS-quail were evidenced by normalizing the high expressions of CASP6 and Hsp70 genes when compared to the C group. Based on these results, the addition of propolis to quail diets as a potential nutritional strategy in order to improve their performance, especially under heat stress conditions, is recommended.

Propolis supplementation attenuates the negative effects of oxidative stress induced by paraquat injection on productive performance and immune function in turkey poults.

Paraquat (PQ) is used as a herbicide in agriculture and causes oxidative and inflammatory damage to animal tissues. The current study was conducted to investigate the positive effects of dietary propolis (PR), as a potent naturally produced antioxidant, on growth performance and immune function of turkey poults exposed to oxidative stress induced by PQ injection. Native male turkey poults (n = 120, 49-d-old) were randomly assigned into 4 groups: poults received a basal diet with a daily subcutaneous PQ injection of 5 mg/kg BW for 7 consecutive days (PQ group), an experimental diet containing 1 g/kg PR with a daily subcutaneous PQ injection for 7 days (PR+PQ group), or received the experimental PR diet with a daily subcutaneous injection of 0.5 mL sterile saline for 7 days (PR group); while the control poults received a basal diet with a daily subcutaneous saline injection for 7 consecutive days (C group). The productive performance in the PQ group showed a significant (P < 0.05) reduction in the weight gain (WG) and feed intake (FI), and impaired feed conversion ratio (FCR). Propolis supplementation in the PR+PQ group significantly ameliorated the PQ effects on WG and FCR. Turkey poults of the PQ and PR+PQ groups had a significant augmentation in the blood malondialdehyde (MDA), tumor necrosis factor-α (TNFα), and corticosterone levels, and in contrast, a significant reduction in the triiodothyronine (T3), when compared to the C group. While propolis significantly reduced the MDA and corticosterone, and increased the T3 levels in the PR+PQ group compared to the PQ group. Furthermore, the dietary PR supplementation significantly limited the PQ-suppressive effects on cell- and humoral-mediated immunity and lymphocyte proliferation of turkey poults. In addition, propolis supplementation in the PR and PR+PQ groups markedly reversed the PQ-induced DNA fragmentation and heat shock protein 70 (Hsp70) over-expression in blood cells. It can be concluded that PR could improve turkey immunity and performance, particularly under inflammation and oxidative stress induced by PQ exposure.

Depression of leukocyte protein synthesis, immune function and growth performance induced by high environmental temperature in broiler chickens.

In tropical and semitropical regions, raising broiler chickens out of their thermal comfort zone can cause an added economic loss in the poultry industry. The cause for the deleterious effects on immunity and growth performance of broilers under high environmental temperatures is still poorly understood. Therefore, the aim of the current investigation was to evaluate the effect of heat stress on leukocytes protein synthesis and immune function as a possible direct cause of low performance in broiler chickens under such condition. In this study, 300 one-day-old male broiler chicks (Cobb500™) were randomly assigned into 2 groups with 5 replicates of 30 chicks each. From 21 to 42 days of age, one group was exposed to non-stressed condition at 24 °C and 50% relative humidity (control group), while the other group was exposed to heat stress at 35 °C and 50% relative humidity (HS group). At 42 days of age, blood samples were collected from each group to evaluate stress indicators, immune function, and leukocytes protein synthesis. Production performance was also recorded. Noteworthy, protein synthesis in leukocytes was significantly (P < 0.05) inhibited in HS group by 38% compared to control group. In contrast, the phosphorylation level on threonine 56 site (Thr56) of eukaryotic elongation factor (eEF2), which indicates the suppression of protein translation process through altering the protein elongation phase, was significantly threefold higher in HS group than in control (P < 0.05). In addition, an increase in stress indicators was markedly (P < 0.05) presented in the HS birds by twofold increase in heterophil/lymphocyte (H/L) ratio and threefold increase in plasma corticosterone level compared to control. Furthermore, the immune function was significantly (P < 0.05) suppressed in HS birds than control (0.99 vs. 1.88 mg/mL plasma IgG, 89.2 vs. 148.0 µg/mL plasma IgM, 4.80 vs. 7.20 antibody titer against SRBC, and 1.38 vs. 3.39 stimulation index of lymphocyte proliferation in HS vs. control group, respectively). Moreover, results on the broiler performance indicate that HS birds had a significant (P < 0.05) lower body weight gain by 58%, lower feed consumption by 39%, higher conversion ratio by 27%, and higher mortality by more than three times, compared to control birds. In conclusion, our results demonstrate that the inhibition of leukocyte protein synthesis through increasing the level of eEF2 Thr56 phosphorylation may play a key role in the observed decrease in immune function and growth performance with the high mortality rate encountered in broiler chickens under heat stress environment.

Comprehensive growth performance, immune function, plasma biochemistry, gene expressions and cell death morphology responses to a daily corticosterone injection course in broiler chickens.

The massive meat production of broiler chickens make them continuously exposed to potential stressors that stimulate releasing of stress-related hormones like corticosterone (CORT) which is responsible for specific pathways in biological mechanisms and physiological activities. Therefore, this research was conducted to evaluate a wide range of responses related to broiler performance, immune function, plasma biochemistry, related gene expressions and cell death morphology during and after a 7-day course of CORT injection. A total number of 200 one-day-old commercial Cobb broiler chicks were used in this study. From 21 to 28 d of age, broilers were randomly assigned to one of 2 groups with 5 replicates of 20 birds each; the first group received a daily intramuscular injection of 5 mg/kg BW corticosterone dissolved in 0.5 ml ethanol:saline solution (CORT group), while the second group received a daily intramuscular injection of 0.5 ml ethanol:saline only (CONT group). Growth performance, including body weight (BW), daily weight gain (DG), feed intake (FI) and feed conversion ratio (FC), were calculated at 0, 3 and 7 d after the start of the CORT injections. At the same times, blood samples were collected in each group for hematological (TWBC's and H/L ratio), T- and B-lymphocytes proliferation and plasma biochemical assays (total protein, TP; free triiodothyronine hormone, fT3; aspartate amino transaminase, AST; and alanine amino transaminase, ALT). The liver, thymus, bursa of Fabricius and spleen were dissected and weighed, and the mRNA expression of insulin-like growth factor 1 gene (IGF-1) in liver and cell-death-program gene (caspase-9) in bursa were analyzed for each group and time; while the apoptotic/necrotic cells were morphologically detected in the spleen. From 28 to 35 d of age, broilers were kept for recovery period without CORT injection and the same sampling and parameters were repeated at the end (at 14 d after initiation of the CORT injection). In general, all parameters of broiler performance were negatively affected by the CORT injection. In addition, CORT treatment decreased the plasma concentration of fT3 and the mRNA expression of hepatic IGF-1. A significant increase in liver weight accompanied by an increase in plasma TP, AST and ALT was observed with CORT treatment, indicating an incidence of liver malfunction by CORT. Moreover, the relative weights of thymus, bursa and spleen decreased by the CORT treatment with low counts of TWBC's and low stimulation of T & B cells while the H/L ratio increased; indicating immunosuppressive effect for CORT treatment. Furthermore, high expression of caspase-9 gene occurred in the bursa of CORT-treated chickens, however, it was associated with a high necrotic vs. low apoptotic cell death pathway in the spleen. Seven days after termination of the CORT treatment in broilers, most of these aspects remained negatively affected by CORT and did not recover to its normal status. The current study provides a comprehensive view of different physiological modulations in broiler chickens by CORT treatment and may set the potential means to mount a successful defense against stress in broilers and other animals as well.

Expression of Inflammatory and Cell Death Program Genes and Comet DNA Damage Assay Induced by Escherichia coli in Layer Hens.

Modern methods of industrial poultry and egg production systems involve stressful practices that stimulate Escherichia coli (E. coli) activity causing endotoxic shock. This investigation was conducted to evaluate the expression of pro-inflammatory cytokines and cell death program genes and DNA damage induced by E. coli in the brain and liver tissues of laying hens. A total of two hundred and ten H&N brown layer hens with 20 week age, were used in this research. First, preliminary experiments were designed (60 hens in total) to establish the optimal exposure dose of E. coli and to determine the nearest time of notable response to be used in the remainder studies of this research. At 35-wk of age, 150 hens were randomly assigned into 2 groups with 3 replicates of 25 birds each; the first group was injected in the brachial wing vein with 107 E. coli colony/hen, while the second group was injected with saline and served as a control. The body temperature and plasma corticosterone concentration were measured 3 hr after injection. Specimens of liver and brain were obtained from each group and the gene expression of p38 mitogen-activated protein kinase, interlukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), Bax, and caspase-3 genes were measured by quantitative real-time PCR. DNA damage in the brain and liver tissues were also measured by comet assay. Hens treated with E. coli showed significant (P<0.05) increase of body temperature and plasma corticosterone (42.6°C and 14.5 ng/ml, respectively) compared to the control group (41.1°C and 5.5 ng/ml, respectively). Additional remarkable over-inflammation gene expression of p38, IL-1ß and TNF-α.genes were also detected in the brain (2.2-fold, 2.0-fold and 3.3-fold, respectively) and the liver (2.1-fold, 1.9-fold and 3.0-fold, respectively) tissues of the infected chickens. It is also important to note that hens injected with E. coli showed an increase in DNA damage in the brain and liver cells (P<0.05). These results were synchronized with activating cell death program since our data showed significant high expression of Bax gene by 2.8- and 2.7-fold and caspase-3 gene by 2.5- and 2.7-fold in the brain and liver tissues of infected chickens, respectively (P<0.05). In conclusion, the current study indicates that E. coli injection induces inflammatory physiological response and triggers cell death program in the brain and liver. Our results provide more understanding to endotoxic shock by E. coli in chickens at cellular level. Further studies are required to confirm if such responses are destructive or protective to set the means through which a chicken mounts a successful defense against avian pathogenic E. coli.

Antioxidant Capacity of Melatonin on Preimplantation Development of Fresh and Vitrified Rabbit Embryos: Morphological and Molecular Aspects.

Embryo cryopreservation remains an important technique to enhance the reconstitution and distribution of animal populations with high genetic merit. One of the major detrimental factors to this technique is the damage caused by oxidative stress. Melatonin is widely known as an antioxidant with multi-faceted ways to counteract the oxidative stress. In this paper, we investigated the role of melatonin in protecting rabbit embryos during preimplantation development from the potential harmful effects of oxidative stress induced by in vitro culture or vitrification. Rabbit embryos at morula stages were cultured for 2 hr with 0 or 10-3 M melatonin (C or M groups). Embryos of each group were either transferred to fresh culture media (CF and MF groups) or vitrified/devitrified (CV and MV groups), then cultured in vitro for 48 hr until the blastocyst stage. The culture media were used to measure the activity of antioxidant enzymes: glutathione-s-transferase (GST) and superoxide dismutase (SOD), as well as the levels of two oxidative substrates: lipid peroxidation (LPO) and nitric oxide (NO). The blastocysts from each group were used to measure the expression of developmental-related genes (GJA1, POU5F1 and Nanog) and oxidative-stress-response-related genes (NFE2L2, SOD1 and GPX1). The data showed that melatonin promoted significantly (P<0.05) the blastocyst rate by 17% and 12% in MF and MV groups compared to their controls (CF and CV groups). The GST and SOD activity significantly increased by the treatment of melatonin in fresh or vitrified embryos, while the levels of LPO and NO decreased (P<0.05). Additionally, melatonin considerably stimulated the relative expression of GJA1, NFE2L2 and SOD1 genes in MF and MV embryos compared to CF group. Furthermore, melatonin significantly ameliorated the reduction of POU5F1 and GPX1 expression induced by vitrification. The results obtained from the current investigation provide new and clear molecular aspects regarding the mechanisms by which melatonin promotes development of both fresh and vitrified rabbit embryos.