Gamal Mehaisen, PhD

Poultry genetics resources, including commercial selected lines, indigenous breeds, and experimental lines, are now being irreversibly lost at an alarming rate due to multiple reasons, which further threats the future livelihood and academic purpose. Collections of germplasm may reduce the risk of catastrophic loss of genetic diversity by guaranteeing that a pool of genetic variability is available to ensure the reintroduction and replenishment of the genetic stocks. The setting up of biobanks for poultry is challenging because the high sensitiveness of spermatozoa to freezing–thawing process, inability to cryopreserve the egg or embryo, coupled with the females being heterogametic sex. The progress in cryobiology and biotechnologies have made possible the extension of the range of germplasm for poultry species available in cryobanks, including semen, primordial germ cells, somatic cells and gonads. In this review, we introduce the state-of-the-art technologies for avian genetic resource conservation and breed reconstruction, and discuss the potential challenges for future study and further extending of these technologies to ongoing and future conservation efforts.

Spirulina platensisis (SP) is a blue-green microalgae with a high value for animal and poultry nutrition. The study employed 250 40-week-old, HY-Line W-36 commercial laying hens. The layers received one of five experimental diet substitutes in five groups for 10 consecutive weeks (five replicates of 10 hens each group); a soybean-corn basal diet formulation without SP (Control group) or the soybean partially substituted with 3% SP, 6% SP, 9% SP, and 12% SP (for the remaining four groups). The results showed that dietary SP treatment significantly (p < 0.05) improved the productive performance, egg quality, blood metabolites, and hematological parameters of laying hens. In addition, there were linear and quadratic effects for increasing the levels of SP inclusion into the layer diets; however, the highest values of most parameters were observed when using 9% SP (90 g/kg of the layer diets). Furthermore, the results showed that 4.7% of the soybean meal ingredient in the layer diet could be replaced by 1% of SP. In conclusion, the partial replacement of soybean meal by SP into layer diets could be used as a promising nutritional approach to optimize the performance of laying hens.

Sperm cryopreservation is of great importance for the poultry industry but still needs to be optimized. The high susceptibility of poultry sperm to cryodamage leads to low fertility rates after cryopreservation. Therefore, the present study aimed at evaluating the effect of including a cryoprotectant, dimethylacetamide (DMA), in the chicken semen freezing extenders at a final concentration of 3%, 6%, or 9% on the post-thawed sperm motility, quality, antioxidant biomarkers, anti-freeze gene expression, and fertilizing ability. Results showed that the total motile sperm, progressivity, and viability were quadratically increased (p < 0.05) in the 6% DMA group. The antioxidant enzyme activity and lipid peroxidation were negatively (p < 0.05) affected by the increase in DMA concentration. Furthermore, some anti-freeze-associated genes such as heat shock protein 70 (HSP70) and ras homolog family member A (RHOA) were linearly and quadratically down-regulated (p < 0.05) with the high concentration of DMA. Finally, the fertility and hatchability rates did not indicate statistical differences between DMA groups. It can be concluded that using the low concentration of 3–6% DMA in the freezing semen extender is preferable to obtain acceptable results in the post-thawed sperm quality and fertility.

The long-term semen cryopreservation is increasingly crucial for conservation of endangered livestock and poultry species. Glycerol is the most widely used cryoprotectant for freezing chicken semen. Continuous improvement in details with glycerol may help increase the fertility of post-thawed semen. Two experiments were performed in the present study to investigate the effects of glycerol concentration, removal method, and straw type on the quality of post-thawed sperm. In experiment 1, glycerol concentration (3%, 5%, 7%, 9%, 11%, and 13%) and glycerol removal method (final dilution ratio 1:1, 1:2, 1:4, 1:8, 1:16, and 1:20) combination groups were investigated for post-thawed sperm quality, residual glycerol concentration, and fertility to find the best combinations. Experiment 2 was performed to evaluate the effects of straw type (0.25 and 0.5 mL) and glycerol concentration (3%, 5%, 7%, 9%, 11%, and 13%) on the post-thawed sperm quality. Results showed that post-thawed sperm motility of 6 glycerol concentration groups were different (P < 0.01). Sperm motility of 5%, 7%, 9%, 11% and 13% was higher than that of 3% (P < 0.01). There was no difference among different concentrations of glycerol in VSL, VCL, VAP, ALH, WOB, BCF, LIN, or STR (P > 0.05). As for the glycerol removal method, sperm motility of 1:8 dilution was the highest, followed by 1:1 and 1:2, while the difference among groups was not statistically significant (P = 0.11). Glycerol concentration and removal method had no interaction effect on sperm motion parameters (P > 0.05). The highest fertility (48.70%) was found for the 5% and 1:2 combination. There was no difference for sperm motility between 0.25 and 0.5 mL straws (P > 0.05). Glycerol concentration and straw type had no interaction effect on the sperm motion parameters (P > 0.05). It can be concluded from these observations that the combination of 5% glycerol and 1:2 dilution rendered higher fertility should be suggested in practice, and that both 0.25 and 0.50 mL straws fit the present procedure.

Heat stress (HS) induces deleterious effects on the performance of laying hens and causes economic losses for poultry industry. This study was carried out to investigate the organic effect of selenium-enriched yeast (SY) on relieving the performance, immunity and physiological deterioration induced by heat stress in laying hens. A total of 324, 28-week-old, Hy-Line Brown commercial chicken layers were randomly distributed into 4 treatments according to a 2 × 2 factorial design, with 9 hens × 9 replicates per treatment (n = 81). From 30 to 34 weeks of age, layers were exposed to 2 temperature treatments (the HS treatment groups): a thermoneutral temperature at 24°C and a heat stress at 35°C. Layers were further assigned into the 2 subgroups according to dietary supplementation with organic selenium-enriched yeast (the SY treatment groups) at either 0 or 0.4 mg/kg diet. Results indicated that all the aspects of the layer performance during the experimental period were impaired by exposure to HS, while SY supplementation improved the layer performance in both the HS and non-HS layers. Intestinal villi disruptions and liver necrotic hepatocytes were observed in the layers exposed to HS, while villi integrity and hepatocytic normality were enhanced by SY treatment. A significant (P < 0.05) decrease in the total leukocyte count, sheep red blood cell (SRBC) antibody titer, and T- and B-lymphocyte proliferation along with an increase in the heterophils/lymphocytes (H/L) ratio were observed in the HS layers compared to non-HS layers. On the contrary, SY treatment significantly (P < 0.05) improved the immune function traits in both the HS layers and non-HS layers. Furthermore, the SY treatment plays an important role in mitigating the oxidative stress and inflammation induced by HS, displaying lower levels of plasma corticosterone, lipid peroxidation, interleukin-1β, and tumor necrosis factor-α in HS layers supplemented with SY compared to HS layers without SY supplementation. These results conclude that addition of SY to the diet of laying hens could be applied as a potential nutritional approach to relieve the deterioration effects of heat stress on the immunity, physiological status, and productive performance of laying hens.

Vitrification is an economically effective method for embryo cryopreservation in human and livestock animals; however, it carries the risk of damage by the exposure to severe oxidative stress. The present study was conducted to evaluate the effect of leptin at different levels on the in vitro development of fresh and vitrified preimplantation embryos in a rabbit model. Normal embryos at morulae stage were randomly cultured for 2 h with 0, 10, 20 or 100 ng/mL of leptin, then were cultured for further 48 h as freshly or after vitrification. Thereafter, developed blastocysts form the best leptin level in fresh and vitrified embryos along with their controls were allocated to analyze the pro-oxidant (malondialdehyde, MDA; nitric oxide, NO), antioxidant (total antioxidant capacity, TAC; superoxide dismutase, SOD; glutathione peroxidase, GPx), apoptotic (Bcl-2 associated X protein, BAX; heat shock 60kD protein member 1, HSP60; tumor necrosis factor alpha, TNFα) and developmental (sex determining region Y box protein 2, SOX2; Nanog homeobox protein, NANOG; Octamer-binding protein 4, OCT4) biomarkers. Results indicate that expanding and hatching rates of embryos were significantly higher at 20 ng/mL leptin than the other levels, while vitrification had an independent suppression effect on the in vitro development rates. The MDA and NO were significantly higher, while TAC, SOD and GPx were significantly lower in the vitrified than fresh embryos. In contrast, leptin treatment significantly decreased the pro-oxidant biomarkers and increased the antioxidant biomarkers in both fresh and vitrified embryos. Vitrification significantly increased the antiapoptotic biomarkers, and decreased the developmental biomarkers in embryos. In contrast, leptin decreased the BAX and TNFα, increased the HSP60, and moreover, ameliorated the reduction of developmental biomarkers in the vitrified embryos. These results conclude that leptin could be used as antiapoptotic and antioxidant promotor to support the in vitro embryonic development, particularly under oxidative stress emerged from cryopreservation programs.

There is an extensive search for natural products that can be introduced to broiler rations to improve performance, especially during the unfavorable breeding conditions. Under heat-stress conditions, the immune response seriously deteriorates, which consequently impairs broiler production performance. Thus, the present study aimed to investigate the potentials of Citrullus colocynthis seeds (CCs) supplementation to modulate the immune response of broilers subjected to chronic heat stress. A total of 300 Cobb-500 male broiler chickens aged 21 days were randomly divided into two equal groups and reared under either thermo-neutral condition (24 ± 1 °C) or subjected to cyclic heat stress (34 ± 1 °C for 8 h). Each group was further divided into two groups (5 replicate × 15 chicks) and was fed either the basal diet or the basal diet with 0.1% CCs supplementation. The results showed that heat stress impaired the production performance by lowering the final body weight and feed intake as well as impairing feed conversion. The levels of stress markers (i.e., malondialdehyde, tumor necrosis factor-α and corticosterone) increased (p < 0.05), whereas the activity of antioxidant enzymes decreased in broilers exposed to heat stress. Further, heat stress caused direct suppression of broiler humoral and cell-mediated immune responses. The stimulating index of T and B lymphocytes proliferation, as well as the antibody titer against sheep red blood cells, were significantly (p < 0.05) reduced by heat-stress exposure. However, CCs supplementation to broilers subjected to heat stress improved (p < 0.05) the final body weight, feed intake, and feed conversion ratio (FCR), compared to the non-supplemented stressed group. The cellular and cell-mediated immune response indicators significantly enhanced (p < 0.05) with CCs supplementation. Supplementation of CCs to broilers reared under similar environmental conditions elevated the total white blood cells (TWBCs) count and the broiler stimulating index of T and B lymphocytes. It can be concluded that CC seeds can be effectively used to stimulate the immune response and improve the production performance of broilers reared under heat-stress condition.

Citrullus colocynthis (CC) has been known as a natural medicinal plant with wide biological activities, including antioxidant, anti-inflammatory, and antilipidemic effects. The aim of this study was to investigate the effect of inclusion of the ethanolic extract of CC seeds (ECCs) into layer diets on the lipid profile, stress indicators, and physiological and productive performance of laying hens. A total of 216 forty-week-old commercial Hy-Line brown laying hens were randomly assigned into four equal groups (3 birds × 18 replicates per group) that received a basal diet supplemented with 0, 0.5, 1.0, and 2.0 g/kg of ECCs for 12 consecutive weeks. The first group served as a control. The results showed that ECCs at 1.0 and 2.0 g/kg significantly (p < 0.05) improved the productive and physiological performance compared to the other groups. In addition, stress indicators examined in the laying hens, including lipid peroxidation (malondialdehyde (MDA)), corticosterone hormone (CORT), tumor necrosis factor alpha (TNFα), and heat shock protein 70 (HSP70), were significantly alleviated after inclusion of ECCs into layer diets at the three levels compared to the control group. Furthermore, all ECC levels induced a significant reduction in plasma triglyceride (TG) and cholesterol (CH) levels in the plasma, liver, and egg yolk, whereas the highest levels were obtained with 2.0 g/kg of ECCs. Particularly important, a high linear correlation (R2 = 0.60–0.79) was observed between increasing doses of ECCs and MDA, liver CH, and egg yolk CH concentrations and egg weight, feed intake, and feed conversion ratio; moreover, the correlation was extremely high (R2 = 0.80–0.100) with the level of TG, CH, low-density lipoprotein CH, high-density lipoprotein CH, and CORT. These results indicated that dietary supplementation with 2.0 g/kg of ECCs could be considered a successful nutritional approach to producing healthier, lower-cholesterol eggs for consumers, in addition to enhancing the physiological and productive performance of laying hens by alleviating the stress of intensive commercial production.

Conjugated linoleic acid (CLA) is known for its multiple benefits including improvement of growth, increasing lean mass, and anti-carcinogenic effects. However, when used in long-term supplementations CLA does not improve semen parameters in boar and bull and reduces fertility in Japanese quails. The content of unsaturated fatty acids in dietary lipids plays a significant role in spermatogenesis owning the high proportion of unsaturated fatty acids in plasma membrane of sperms. Whether CLA plays a role in testicular tissue and epididymal fat is still unknown. Therefore, in this study we hypothesize that long-term supplementation of equal proportion of CLA isomer mix (c9,t11-CLA and t10,c12- CLA) in rabbit bucks might alter male reproductive potentials. Twelve V-Line weaned male rabbits were used in 26 weeks trial, rabbits were individually raised and randomly allocated into three dietary groups. Control group (CON) received a basal diet, a group received 0.5% CLA (CLA 0.5%), and a group received 1% CLA (CLA 1%). Rabbits were euthanized at the end of the trial and several parameters were evaluated related to growth, semen quality, and testicular and epididymal tissue histopathology and transcriptome. The long-term supplementation of CLA increased feed intake by 5% and body weight by 2–3%. CLA 1% decreased sperm progressive motility. In testicular tissue L-carnitine and α-tocopherol were decreased by CLA supplementation. In epididymal fat, CLA tended to decrease concentration of polyunsaturated fatty acids, the expression of SCD5 gene was upregulated by CLA 1% and CASP3 gene was upregulated by CLA 0.5%. Transcription of PPARG was downregulated by CLA. Feeding 1% CLA also decreased testicular epithelial thickness. Long-term supplementation of CLA modestly enhanced male rabbit growth, but negatively impacted male reproduction, especially at high dose of CLA.

Reactive oxygen species (ROS) and free radicals are one of the major detrimental factors that can negatively affect the quality of sperm during cryopreservation. Melatonin is an effective antioxidant and free radical scavenger in various cells. In this study, therefore, the aim was to evaluate the post-thawed quality of spermatozoa after cryopreservation of rooster semen in freezing extender supplemented with melatonin. Semen samples from seven Green-legged Partridge roosters were pooled and diluted with EK extender supplemented with 10−3, 10−6, or 10−9 M melatonin (control sample was prepared without supplementation with melatonin), and the pooled sample was subjected to cryopreservation. Post-thawed sperm motility was determined using the IVOS system, whereas plasma membrane status, acrosome integrity, mitochondrial activity, lipid peroxidation, chromatin status, and apoptotic-like changes were determined using fluorochromes and flow cytometry. Results, indicate post-thaw motile sperm cell count was greater (P < 0.05) in the frozen samples supplemented with melatonin (10−3 and 10−6 M) than the control sample. Although no significant differences were observed in post-thawed acrosomal integrity, plasma membrane integrity and mitochondrial activity were greater (P < 0.05) in samples frozen with melatonin (10−3 and 10−6 M) than that of the control sample. In addition, with supplementation of melatonin there was a decrease (P < 0.05) in the amount of lipid peroxidation, DNA fragmentation, and apoptotic-like changes after thawing. These results indicate there is a positive effect of melatonin supplementation in rooster semen freezing extenders on post-thaw sperm quality.

This study examines the effect of dietary supplementation with Lactobacillus acidophilus (LA) on the cholesterol levels, immune response, and productive performance of laying hens. A total of 216, 40-week-old, commercial Hy-Line brown chicken layers were randomly assigned into four treatment groups (18 birds × three replicates per group) and fed diet supplemented with 0 (control), 1 × 109, 21 × 109, and 31 × 109 colony forming units (CFUs) of Lactobacillus acidophilus (LA) per kg of feed for six consecutive weeks. Results show that plasma triglycerides, low-density lipoprotein (LDL) and total cholesterols became lesser, while high-density lipoprotein (HDL) cholesterol became higher in LA-supplemented groups compared to the control. In addition, a significant reduction occurred in the liver and egg yolk cholesterol by LA supplementation. Moreover, the immunological parameters including antibody titer against sheep red blood cells (SRBCs), phytohemagglutinin (PHA)-wattle swelling test, and T- & B-lymphocyte proliferation were enhanced in laying hens supplemented with LA compared to the control hens. While the heterophil to lymphocyte (H/L) ratio decreased with LA supplementation, indicating low stress conditions in the treated hens. These positive effects for LA were further reflected on the productive performance of laying hens and improved egg production, egg weight, egg mass, and feed efficiency. Our findings indicate that LA probiotic could be recommended in laying hens’ diets for lowering egg yolk cholesterol with positive impacts on health and performance.

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.

Oxidative 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.

Safeguarding biodiversity is an important goal for animal production in developed countries. This study investigated genetic diversity among native Middle-Egypt rabbit (NMER) populations in North Upper-Egypt province by using microsatellite polymorphism. Nineteen microsatellite loci were used in the study and an area of 231 km was surveyed, as native rabbits covered 14 points belonging to four Northern Upper Egypt governorates (South Giza, Fayoum, Beni Suef and Minya). Standard statistical parameters of genetic variability within and between populations confirmed that the highest genetic diversity was found towards the south. Among NMER populations, the mean number of alleles per locus was lowest in South Giza (5.32), while it was highest in Minya (6.00). This study found that NMER featured a high number of private alleles ranging between 7 and 11 (mean value was 10.5). Results also showed a high genetic diversity in NMER populations and that heterozygosity ranged between 0.384 and 0.445, strongly indicating extensive genetic variation in the NMER populations. The mean values of observed and expected heterozygosity were 0.405 and 0.612, respectively. Factorial correspondence analysis and neighbour joining trees (NJ) showed 2 main NMER rabbit groups: the Northern group (South Giza and Fayoum) and the Southern group (Beni Suef and Minya). All populations showed a high percentage of assignment in this study (0.913 to 0.946). The structure analysis showed that each population existed in separate clusters. This research provides an overview of genetic diversity of NMER populations in the Northern Upper Egypt province for the first time. In conclusion, results of this study could be used to designate priorities for conservation of NMER populations.

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.

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.

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.

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.

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-1β (IL-1β), 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-1β 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.

This study aimed to investigate the effect of melatonin supplementation at different levels in culture medium on embryo development in rabbits. Embryos of 2-4 cells, 8-16 cells and morula stages were recovered from nulliparous Red Baladi rabbit does by laparotomy technique 24, 48 and 72 h post-insemination, respectively. Normal embryos from each stage were cultured to hatched blastocyst stages in either control culture medium (TCM-199 + 20% fetal bovine serum) or control supplemented with melatonin at 10(-3) M, 10(-6) M or 10(-9) M. No effect of melatonin was found on development of embryos recovered at 24 h post-insemination. The high level of melatonin at 10(-3) M adversely affected the in vitro development rates of embryos recovered at 48 h post-insemination (52 versus 86, 87 and 80% blastocyst rate; 28 versus 66, 78 and 59% hatchability rate for 10(-3) M versus 10(-9) M, 10(-6) M and control, respectively, P< 0.05). At the morula stage, melatonin at 10-3 M significantly increased the in vitro development of embryos (92% for 10(-3) M versus 76% for control, P < 0.05), while the hatchability rate of these embryos was not improved by melatonin (16-30% versus 52% for melatonin groups versus control, P < 0.05). Results show that a moderate level of melatonin (10(-6) M) may improve the development and hatchability rates of preimplantation rabbit embryos. The addition of melatonin at a 10-3 M concentration enhances the development of rabbit morulae but may negatively affect the development of earlier embryos. More studies are needed to optimize the use of melatonin in in vitro embryo culture in rabbits.

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.

The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against
cryopreservation injuries on rabbit sperms of 2 lines selected for hyper-prolificacy (H) and longevity (L).
Ejaculates were collected and pooled from ten sexually mature rabbit bucks per each line. A total number
of 196 pooled semen ejaculates per line were used to evaluate post-thawing semen by 252 artificial
inseminations using White New Zealand rabbit does. Semen was equally divided into 3 volumes and
diluted (1:1) with different TRIS-citric acid based extenders (A, B and C). In extender A, 3 M dimethyl
sulfoxide and 0.1 M sucrose were added as cryoprotectants. For extender B, the sucrose in extender A was
replaced by 20% egg-yolk, and for extender C, the TRIS-based extender was supplemented with 2 M
acetamide and 20% egg-yolk. No interaction effect was detected between rabbit line and semen extender
on studied traits. Moreover, no significant differences were detected between H- and L-lines for all traits
except for prolificacy that was higher in L- line (6.04 vs. 4.37 young born at birth, respectively). However,
freezing semen with extender A and B showed better post-thawing semen quality characteristics, fertility
and prolificacy than frozen semen with extender C. It could be concluded that extenders A and B are
preferable for freezing semen of H- and L-rabbit lines than extender C to obtain higher fertility and
prolificacy. A moderate and interesting relationship was found between acrosomal integrity of frozen
semen and fertility rate. (r=0.17; P=0.04).

Seventeen microsatellite loci were used to identify the phylogenetic relationship among four Egyptian breeds and one Spanish line of rabbits. A total of 114 bucks rabbits belonging to four Egyptian breeds -Black Baladi (EBB), Gabali (EG), Red Baladi (ERB) and White Giza (EWG)- and Spanish White New Zealand line from Universidad Politécnica de Valencia (NZW) were studied. All microsatellite loci typed were polymorphic. The average number of alleles per locus was 5.41, ranging from 2 to 12. A total of 16 private alleles were found in 7 out of 17 microsatellite loci used. Mean observed heterozygosity was 0.527, ranging from 0.477 in the NZW breed to 0.581 in the EWG. Lower values for Ho were found for all populations. The inbreeding coefficient of individuals relative to the total population (FIT) was 0.279. The overall within-population heterozygote deficit (FIS) was 0.165, ranging from 0.045 in NZW breed to 0.266 in EBB breed. The overall variation between population (FST) was 0.137, where the NZW breed showed the most differentiated population (FST = 0.194). The Neighbour-Joining tree of the Reynolds genetic distances (DR) among populations shows a clear separation of the Spanish population (NZW) from the Egyptians breeds and there is a population mixture in the Egyptian populations. Only the ERB may to cluster in one independent population.

This study aimed to investigate the effect of linseed oil as a natural source rich in omega-3 fatty acids on egg yolk cholesterol and per- formance of laying hens. A total of 180 commercial Hy-Line brown laying hens were randomly divided into 4 groups and were fed for 28 d on control diet and diets containing 2, 4 and 6% linseed oil. Egg production performance and feed consumption were recorded during 42 d of the study. Blood and egg samples were collected from laying hens at 14, 28 and 42 d of the experiment to measure the total pro- tein, albumin, globulin, calcium and phosphorus in plasma as well as yolk cholesterol concentration in eggs. Results revealed that egg pro- duction performance was significantly improved by supplementation of linseed oil in the diets (egg number was 39.07 vs. 37.78 eggs/hen and egg mass was 2432.29 vs. 2358.13 g/hen for linseed groups vs. control group, respectively, P < 0.05). Feed consumption was signifi- cantly (P < 0.05) lower in linseed groups than in control group (110.78 vs. 121.05 g/hen/day), and consequently, the feed conversion ratio decreased (2.05 vs. 2.31). Plasma protein, albumin, globulin, calcium and phosphorus were not influenced by the linseed oil levels in the diet. Egg yolk cholesterol significantly decreased by linseed supple- mentation (11.57, 11.18 and 10.98 mg/g cholesterol in 2%, 4% and 6% linseed oil groups vs. 12.80 mg/g cholesterol in control group, P < 0.05). Therefore, the dietary supplementation of linseed oil in chicken diets as natural source of omega-3 fatty acids is healthier for egg con- sumers.

Two experiments were conducted with male broiler chickens to study the effect of light program
and melatonin injection on enhancing the immune response and reducing the inflammatory response
induced by lipopolysaccharide (LPS) injection. In the first experiment, one-day-old broiler chicks were divided
into two groups: The first group was exposed to continuous light (CL) (23L: 1D), whereas the other group
was exposed to intermittent light (IL) (1L: 3D). At 6 wks of age, within each light program, chickens were
injected intravenously (iv.) with either 3 mg/kg BW LPS, or with sterile saline. Blood samples were collected
at 0, 3, 12 and 24 h post-injection. Total white blood cells (WBC), at 3, 12 and 24 h, were significantly higher
in the IL-LPS group compared to the CL-LPS group. Body temperature and plasma corticosterone
concentration at 3 and 12 h, were significantly lower in the IL-LPS group, compared to the CL-LPS group.
Interleukin-1-like activity and IL-6 concentration were significantly lower in the IL-LPS group compared to the
CL-LPS group at 3, 12 and 24 h. In the second experiment, 6 wk-old broiler chicks raised under CL, were
divided into three groups. The first group was injected i.v. with 40 mg/kg BW melatonin, followed by 3 mg/Kg
BW LPS 1 hour later. The second group was injected with saline followed by LPS 1 hour later. The third group
received two saline injections 1 hour apart. Blood samples were collected at 0, 3, 12 and 24 h after the
second injection. T lymphocyte proliferation were significantly higher in the melatonin-LPS group compared
to the saline-LPS group. Plasma corticosterone concentration and body temperature were significantly lower
in the melatonin-LPS group compared to the saline-LPS group at 3 h. IL-1-like activity and IL-6 concentration
were significantly higher in the saline-LPS group compared to the other two groups at 3 and 12 h post
injection. Total WBC was significantly higher in the melatonin-LPS group compared to the saline-LPS group
at 12 and 24 hr. Our results indicate that intermittent light and melatonin injection can enhance the immune
response and reduce the inflammation induced by LPS injection.

This study aimed to evaluate the in vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatments in rabbit does. Ninety-six nulliparous does were randomly subjected to consecutive superovulation treatments with eCG (20 IU/kg body weight intramuscularly (i.m.), eCG group), FSH (3 x 0.6 mg/doe at 24 h intervals i.m., FSH group), or without superovulation treatment (control group). Does were artificially inseminated 3 days later and ovulation was induced immediately by hCG (75 IU/doe intravenous). Seven experimental groups were differentiated: first FSH and eCG treatment, second FSH and eCG treatment, eCG-interchanged group (does with previous FSH treatment), FSH-interchanged group (does with previous eCG treatments) and control group. Embryos were collected in vivo by laparoscopy 76-80 h post-insemination in the first and second recovery cycles and post mortem in the third recovery cycles. The ovulation rate was significantly higher in does treated with the first-FSH than in those treated with eCG or in control does (25.2+/-2.0 versus 19.2+/-1.4 to 11.0+/-1.5, and 12.2+/-1.2, first-FSH, first-eCG to second-eCG and control groups, respectively, P < 0.05). Significant differences were observed in the total recovery influenced by ovulation rate in each group (20.3+/-2.2 to 9.4+/-1.2, first-FSH to control groups). Embryo donor rate (donor with at least one normal embryo) was similar among groups with an overall of 75.1%. The number of normal embryos recovered per doe with at least one normal embryo increased significantly in relation to ovulation rate (17.7+/-2.2 to 8.41+/-3, first-FSH and control groups). The vitrification of embryos negatively affected their in vitro development to hatched blastocyst in all groups (88.1% versus 48%, P > 0.05). However, after embryo transfer, this negative effect was only observed in superovulated vitrified embryos (16.8 and 12.8% versus 39.4% total born rate from eCG, FSH and control vitrified groups, P < 0.05). Results indicated that the primary treatments with eCG or FSH increased the number of normal embryos recovered per donor doe, but these embryos are more sensitive to vitrification protocols.

The aim of this work was to evaluate the effect of different doses of eCG administered subcu- taneously (0, 50 and 200 IU) and the hormonal induction of ovulation (GnRH or hCG) on embryo recovery and in vitro development of embryos post-vitrification in two selected lines of rabbit does. The two selected lines were line V (selected for the litter size at weaning) and line R (selected for growth rate). Administration of 200 IU of eCG significantly increased ovulation rate (19.2±1.2 ver- sus 15.5±1.1 and 12.2±1.3, and the number of haemorrhagic follicles (13.8±1.6 versus 3.8±1.4 and 3.8±1.7), but significantly decreased recovery rate (28.8±6.3 versus 47.7±5.7 and 48.7±6.7, 200 IU versus 50 IU and 0 IU eCG, respectively), the number of normal embryos recovered per doe with at least one embryo (5.8±0.9 versus 8.2±0.9, 200 IU versus 50 IU eCG doses) and the in vitro development of embryos post-vitrification (51.9% versus 66.1%, 200 IU versus 50 IU eCG doses, re- spectively). Inducing ovulation with hCG significantly increased ovulation rate when compared with GnRH (17.3±0.8 versus 13.8±1.4), but no significant differences in embryo recovery and embryo development post-vitrification were observed between the two treatments. No significant differences were observed between the two selected lines in ovulation and recovery rates, the number of haem- orrhagic follicles and the number of recovered embryos per doe. However, the post-vitrification in vitro rate of development was 59.7% for line R and 51.9% for line V (p<0.05). It was concluded that the use of 50 IU of eCG subcutaneous with hCG or GnRH prior to embryo cryopreservation programmes in rabbits achieves the best results for embryo recovery, with the best development of recovered embryos post-vitrification.

Rabbit does from R line selected for growth rate present a low reproductive performance and this study aimed to evaluate both the recovery efficacy and viability of recovered embryos after vitrification and the reproductive performance of donor does subjected to in vivo recovery. Does were divided into three groups: 28 does without in vivo recovery (control), 25 does in which in vivo recovery was started in the nulliparous state (group 1) and 30 does with at least one litter before in vivo recovery (group 2). Does were superovulated with a single subcutaneous injection of 50 IU of equine chorionic gonado- tropin (eCG) per female, and were then artificially inseminated 60 h later and immediately administered an intravenous dose of 75 IU of human chorionic gonadotropin (hCG) per female. Does from group 1 and 2 were recovered in vivo 76–80 h post- insemination by repeated laparoscopies at one to four times and permitted one or two parturitions between recoveries [in vivo (IV) recovery]. At the end of the experiment, about 16 does of all groups were recovered post-mortem (PM recovery). All normal embryos were vitrified, devitrified and then cultiva- ted in vitro to evaluate the viability after thawing. A significant increase in the ovulation rate was found in does recovered PM than in those recovered IV in the nulliparous state. However, no significant differences were observed in the recovery rate, the donor rate, the number of normal embryos recovered with at least one normal embryo per doe and the viability after thawing between the PM and IV groups. A significant decrease in the fertility rate, total born, live born and weaned kids was found for does from group 1 in comparison with does from group 2. Results support the use of repeated laparoscopy to increase the number of recovered embryos per donor doe especially in suchRline does, if they are permitted to produce at least one litter before the beginning of in vivo recovery.