Dietary guanidinoacetate reduces spaghetti meat myopathy risk in the breast muscle of broiler chickens.

The global demand for white chicken meat along with the increase in the occurrence of growth-related breast muscle myopathies (BMMs) [namely white striping (WS), wooden breast (WB), and spaghetti meat (SM)] highlights the need for solutions that will improve meat quality while maintaining the high productivity of modern broilers. Guanidinoacetate (GAA), a precursor of creatine, is used as a feed additive and has previously shown the potential to affect the quality of breast meat. This study investigated growth performance, meat quality and the risk ratio for the development of BMMs in broilers assigned to two dietary treatments: control (CON) group, fed a commercial basal diet, and supplemented GAA (sGAA) group, receiving the control diet supplemented on top with 0.06% GAA. Growth performance indicators such as BW, daily weight gain, daily feed intake, feed conversion ratio and cumulative feed conversion ratio were recorded on a pen basis. As a trait affecting animal welfare, the occurrence of foot pad dermatitis was also evaluated. At day 43, birds were processed, and breasts were scored for the incidence and severity of BMMs (n = 166 and 165 in CON and sGAA groups, respectively). Quality traits (ultimate pH, colour) and technological properties (i.e., drip and cooking losses, marinade uptake, shear force, and oxidation levels of the lipid and the protein fractions) of breast meat were assessed in both treatments on samples not showing any macroscopic sign of BMMs (n = 20 breast fillets per group). Data of myopathy risk ratio were analysed as the risk for each group to develop WS, WB, and SM myopathies. Our results show that while sGAA and control groups did not differ significantly in growth performance, a remarkably beneficial effect of GAA was observed on the incidence of BMMs with significantly reduced risk of sGAA group to develop SM myopathy. The risk of sGAA group to develop SM was 30% lower compared to CON (P = 0.028). Finally, a significantly lower drip loss was observed in sGAA in comparison with CON (1.78 vs 2.48%, P = 0.020). Together, our results show that the inclusion of 0.06% GAA in feed can improve the water-holding capacity of meat and reduce the risk to develop SM myopathy without compromising the performance of broilers.

Effect of chronic heat stress on gastrointestinal histology and expression of feed intake-regulatory hormones in broiler chickens.

Heat stress (HS) dramatically impairs the growth performance of broiler chickens, mainly as a consequence of reduced feed intake due to the loss of appetite. This study was aimed at evaluating the alterations induced by chronic HS conditions on the morphological and morphometric features of the gastrointestinal (GI) tract and on the expression of some enteroendocrine cells (EECs) involved in the regulation of feed intake in chickens. Three hundred male chickens (Ross 308) were divided into two experimental groups and raised either in thermoneutral environment for the whole fattening period (0-41 days) (TNT group) or subjected to chronic HS conditions (30 °C for 24 h/day) from 35 to 41 days (HS group). Samples of proventriculus, duodenum, jejunum and cecum were collected from 24 broilers (12/group). Haematoxylin-eosin was used for the morphometric evaluations, while immunohistochemistry was applied for the evaluation of EECs expressing ghrelin (GHR), cholecystokinin (CCK), neuropeptide Y (NPY), glucagon-like peptide-1 (GLP-1), and serotonin (5-HT). In the proventriculus, HS reduced total wall thickness and mucous layer height (P ≤ 0.01) as well as mean diameter, circumference, and area of the compound tubular glands (P ≤ 0.001) with respect to TNT. The small intestine of HS birds was characterised by decreased villous height and total thickness (duodenum, P ≤ 0.01; jejunum, P ≤ 0.001), whereas crypt depth and width were reduced only in the jejunum (P ≤ 0.01). HS had negligible effects on the morphological aspects of the cecum. In the proventriculus, an increase in GHR and NPY EECs was observed in response to HS (P ≤ 0.001). Similarly, the small intestine villi of the HS group showed greater GLP-1 (P ≤ 0.05), 5-HT (P ≤ 0.001) and CCK (P ≤ 0.01) EECs. Moreover, the expression of 5-HT EECs was higher in the duodenal (P ≤ 0.01) and jejunal (P ≤ 0.01) crypts of HS birds, whereas GLP-1 and CCK EECs increased only in jejunal crypts (P ≤ 0.05). Finally, 5-HT EEC expression was increased in the cecum of HS group (P ≤ 0.01). In conclusion, these outcomes demonstrate that chronic HS induces morphometric alterations not only in the small intestine but also in a key organ such as the proventriculus. Furthermore, HS conditions affect the presence and distribution of EECs, suggesting that some GI peptides and biogenic amine may be implicated in the regulation of appetite and voluntary feed intake in heat-stressed broiler chickens.

Effects of genotype and age on eggshell cuticle coverage and color profile in modern laying hen strains.

The aim of this research was to investigate the effects of laying hen genotype and age on eggshell cuticle deposition. A total of 4,320 brown eggs were obtained from 3 modern hen strains (A, B, and C), currently used worldwide for commercial egg production, at different intervals of age (20-30, 40-50, and 60-70 wk). Four samplings of 120 randomly collected eggs were carried out for each genotype/interval of age. Eggs were individually weighed and cuticle blue staining was used to assess quality and degree of cuticle coverage. On each egg, the eggshell color profile was assessed before and after staining using the CIE L*a*b* system and these values were used to calculate ΔE*ab. A 4-point scale visual score (VS) system was also applied to estimate the degree of cuticle coverage after staining (0 = no coverage, 1 = partial coverage, 2 = total coverage - low degree, 3 = total coverage - high degree). The effects of genotype and age and their interaction on eggshell color attributes were assessed by means of factorial ANOVA, while omnibus Chi-Square and Chi-squared Automatic Interaction Detector algorithm were applied for the analysis of VS data. Overall, both genotype and age affected the eggshell color profile as well as the degree of cuticle coverage. Hen strain A showed better cuticle deposition in comparison with B and particularly C one, being ΔE*ab values significantly higher. The VS evaluation revealed that eggs with impaired cuticle coverage degree increased with the hen age (23, 34, and 37%, respectively for 20-30, 40-50, and 60-70 wk; P < 0.05). However, a significant interaction between genotype and age was observed: transition from early to late hen age resulted in a significantly different pattern of ΔE*ab changes in each genotype. The classification tree analysis confirmed that the hen genotype has a greater effect than the age on cuticle deposition. In conclusion, considering the importance of the cuticle in table egg production, these results highlight the crucial role exerted by the genotype on eggshell cuticle coverage.

Breast muscle and plasma metabolomics profile of broiler chickens exposed to chronic heat stress conditions.

Understanding the variations of muscle and plasma metabolites in response to high environmental temperature can provide important information on the molecular mechanisms related to body energy homeostasis in heat-stressed broiler chickens. In this study, we investigated the effect of chronic heat stress conditions on the breast muscle (Pectoralis major) and plasma metabolomics profile of broiler chickens by means of an innovative, high-throughput analytical approach such as the proton nuclear magnetic resonance (1H NMR) spectrometry. A total of 300 Ross 308 male chicks were split into two experimental groups and raised in either thermoneutral conditions for the entire rearing cycle (0-41 days) (TNT group; six replicates of 25 birds/each) or exposed to chronic heat stress conditions (30 °C for 24 h/day) from 35 to 41 days (CHS group; six replicates of 25 birds/each). At processing (41 days), plasma and breast muscle samples were obtained from 12 birds/experimental group and then subjected to 1H NMR analysis. The reduction of BW and feed intake as well as the increase in rectal temperature and heterophil: lymphocyte ratio confirmed that our experimental model was able to stimulate a thermal stress response without significantly affecting mortality. The 1H NMR analysis revealed that a total of 26 and 19 molecules, mostly related to energy and protein metabolism as well as antioxidant response, showed significantly different concentrations respectively in the breast muscle and plasma in response to the thermal challenge. In conclusion, the results obtained in this study indicated that chronic heat stress significantly modulates the breast muscle and plasma metabolome in fast-growing broiler chickens, allowing to delineate potential metabolic changes that can have important implications in terms of body energy homeostasis, growth performance and product quality.

Orexin system is expressed in avian liver and regulates hepatic lipogenesis via ERK1/2 activation.

Orexins are originally characterized as orexigenic hypothalamic neuropeptides in mammals. Subsequent studies found orexin to be expressed and perform pleiotropic functions in multiple tissues in mammals. In avian (non-mammalian) species, however, orexin seemed to not affect feeding behavior and its physiological roles are poorly understood. Here, we provide evidence that orexin and its related receptors are expressed in chicken hepatocytes. Double immunofluorescence staining showed that orexin is localized in the ER, Golgi, and in the lysosomes in LMH cells. Brefeldin A treatment reduced orexin levels in the culture media, but increased it in the cell lysates. Administration of recombinant orexins upregulated the expression of orexin system in the liver of 9-day old chicks, but did not affect feed intake. Recombinant orexins increased fatty acid synthase (FASN) protein levels in chicken liver, activated acetyl-CoA carboxylase (ACCα), and increased FASN, ATP citrate lyase(ACLY), and malic enzyme (ME) protein expression in LMH cells. Blockade ERK1/2 activation by PD98059 attenuated these stimulating effects of orexin on lipogenic factors. Overexpression of ERK1/2 increased the expression of lipogenic genes, and orexin treatment induced the phosphorylated levels of ERK1/2Thr202/Tyr204, but not that of p38 Thr180/Tyr182 or JNK1/2 Thr183/Tyr185 in chicken liver and LMH cells. Taken together, this is the first report evidencing that orexin is expressed and secreted from chicken hepatocytes, and that orexin induced hepatic lipogenesis via activation of ERK1/2 signaling pathway.

Impact of galactooligosaccharides delivered in ovo on mitigating negative effects of heat stress on performance and welfare of broilers.

Galactooligosaccharides (GOS) delivered in ovo improve intestinal health of broiler chickens. This study aimed to demonstrate the impact of in ovo stimulation with GOS prebiotic on day 12 of egg incubation on performance and welfare traits in broiler chickens. The incubating eggs were divided into 3 groups, based on the substance injected in ovo: 3.5 mg of GOS dissolved in 0.2 mL physiological saline (GOS), 0.2 mL physiological saline (S), or uninjected controls (C). Constant heat stress (HS) was induced on days 32 to 42 post-hatch by increasing environmental temperature to 30°C. Thermoneutral (TN) animals were kept at 25°C. The performance (body weight [BW], daily feed intake [DFI], daily weight gain [DWG], and feed conversion rate [FCR]) were measured and mortality was scored for starter (days 0 to 13), grower (days 14 to 27), and finisher (days 28 to 42) feeding phases. Rectal temperature was scored on days 32 to 42. Food-pad dermatitis (FPD) was scored post-mortem (day 42). GOS increased (P < 0.01) BW on day 42 (2.892 kg in GOS vs. 2.758 kg in C). Heat stress significantly reduced (P < 0.01) final BW (2.516 kg in TN vs. 3.110 kg in HS). During finisher phase, DFI was significantly higher in GOS vs. C (173.2 g vs. 165.7 g; P < 0.05). FCR calculated for the entire rearing period (days 0 to 42) ranged from 1.701 in C to 1.653 in GOS (P < 0.05). GOS improved FCR in HS animals during finisher phase (P < 0.05). Rectal temperature of GOS chickens under HS reached 42.5°C 1 day earlier than C and S (P < 0.05), which suggests that those birds recovered earlier from the high environmental temperature. Heat stress increased (P < 0.05) mortality about 5 times compared to TN during finisher phase (from 1.59% in TN to 7.69% in HS). GOS decreased FPD in TN conditions by 20% (no lesions in 81% in GOS vs. 60% in C). GOS delivered in ovo mitigated negative effects of HS on performance and welfare in broiler chickens.

Effect of galactooligosaccharides delivered in ovo on meat quality traits of broiler chickens exposed to heat stress.

A study was carried out to evaluate meat quality traits in fast-growing chickens stimulated in ovo with trans-galactoolighosaccarides (GOS) and exposed to heat stress. On day 12 of egg incubation, 3,000 fertilized eggs (Ross 308) were divided into prebiotic group (GOS) injected with 3.5 mg GOS/egg, saline group (S) injected with physiological saline, and control group (C) uninjected. After hatching, 900 male chicks (300 chicks/treatment) were reared in floor pens in either thermoneutral (TN; 6 pens/group, 25 birds/pen) or heat stress conditions (HS, 30°C from 32 to 42 D; 6 pens/group, 25 birds/pen). At 42 D of age, 15 randomly chosen birds/treatment/temperature were slaughtered and the pectoral muscle (PM) was removed for analyses. Data were analyzed by GLM in a 3 × 2 factorial design. In ovo treatment had no effect on PM weight, pH, water-holding capacity, and shear force. GOS and S birds had lighter (L*, P < 0.01) PM than C group, whereas the latter showed a higher (P < 0.05) yellowness index (b*) compared to S group. Proximate composition, cholesterol, and intramuscular collagen properties were not affected by treatment. As for fatty acid composition, only total polyunsaturated fatty acids (PUFA) content and n-6 PUFA were slightly lower in GOS group compared to S. Heat stress had a detrimental effect on PM weight (P < 0.01) and increased meat pH (P < 0.01). PM from HS chickens was darker with a higher b* index (P < 0.05) and had a higher (P < 0.01) lipid content and a lower (P < 0.05) total collagen amount. Total saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and PUFA were similar among groups. Significant interactions between factors were found for fatty acid composition: GOS decreased (P < 0.01) SFA and increased (P < 0.05) MUFA contents in HS birds. In conclusion, in ovo injection of GOS could mitigate the detrimental effect of heat stress on some meat quality traits.

Black soldier fly defatted meal as a dietary protein source for broiler chickens: effects on carcass traits, breast meat quality and safety.

Finding insect meals as alternative sources of poultry feedstuffs is a recent research topic; therefore, the present study aimed to evaluate the effects of defatted black soldier fly (Hermetia illucens L., HI) larvae meal in broiler chicken diets on the carcass characteristics and meat quality parameters, proximate composition, fatty acid profile and the heavy metal content of the breast meat. Four dietary treatments were designed: a control diet (HI0) and three experimental diets (HI5, HI10 and HI15), corresponding to 50, 100 and 150 g/kg HI inclusion levels, respectively. The inclusion of 50, 100 and 150 g HI meal per kg feed supply 16.56%, 33.01% and 49.63% of required crude protein. The broilers were slaughtered at day 35, the carcasses were weighed and the breast muscles were excised from 16 birds per each feeding group (two birds per replicate pens) and used for meat quality evaluation. Linear and quadratic responses were observed, for increasing HI meal levels, in the live and carcass weights (maximum for HI10). As far as the colour of the breast meat is concerned, redness (a*) showed a linear response, while yellowness (b*) linearly decreased with increasing HI meal levels (minimum for HI15). As the HI larvae meal increased in the diets, the moisture content linearly decreased and the protein content increased. The total saturated fatty acid and total monounsaturated fatty acid proportions rose to the detriment of the polyunsaturated fatty acid fraction. The HI larvae meal, used in the current study, represents a valuable protein source for broiler chickens when included by up to 100 g/kg in their diets, as an improved slaughtering performance was observed without any detrimental effects on meat quality parameters or heavy metal residues in the meat.

Comparison of 2 commercial turkey hybrids: productivity, occurrence of breast myopathies, and meat quality properties.

This study was undertaken to compare productive performance, occurrence of breast myopathies, chemical composition, and technological properties of the meat in 2 dominant commercial turkey hybrids. A total of 972 1-day-old male turkey poults (equally divided in hybrid A and B) were randomly distributed in 18 floor pens. Overall, productive performance resulted similar between the genotypes, although they showed different growth profile (turkeys from group B grew up faster up to 84 d). Regarding the occurrence of myopathies, the percentage of breasts affected by white striping was markedly higher in both genotypes (46 vs. 60% of severe lesions, respectively for A and B; P < 0.05), while the occurrence of spaghetti meat-like condition was negligible. The histological features of the different categories of meat abnormalities resulted similar to those previously described for chicken hybrids. The technological traits such as ultimate pH, lightness, redness, marinade uptake, cooking losses, and shear force were not significantly affected by the genotype. However, turkeys from group B exhibited lower yellowness (b*, 0.50 vs. 1.04; P < 0.05) and higher drip losses (1.34 vs. 1.26%; P < 0.05). The shelf-life test on thigh meat showed no significant changes in meat color over the storage time in both hybrids, whereas thigh meat from group A showed absolute lower values of lightness (L*) and yellowness (b*) (P < 0.05). Lipid oxidation of thigh meat significantly increased during storage, although no significant difference was found between the hybrids. Proximate composition and intramuscular collagen properties resulted similar between genetic lines with the exception of total fat content (1.55 vs. 1.21%, respectively for A and B; P < 0.05). The genotype had a moderate effect on fatty acid families of breast meat as only monounsaturated fatty acid content was significantly affected (31.7 vs. 29.8%, respectively for A and B). In conclusion, the overall productive traits of commercial turkeys, including the occurrence of muscle myopathies, as well as quality attributes of fresh and refrigerated meat were only slightly affected by the genotype.

Effect of different arginine-to-lysine ratios in broiler chicken diets on the occurrence of breast myopathies and meat quality attributes.

This study was carried out to evaluate the effect of different digestible arginine-to-lysine (dig Arg:Lys) ratio in broiler diets on the occurrence of breast myopathies, productivity, and meat quality traits. A total of 1,755 1-day-old Ross 308 male chicks was randomly divided in 3 experimental groups (9 replications/group): CON, fed a 4 feeding-phases commercial diet (dig Arg:Lys ratio = 1.05, 1.05, 1.06, 1.07, respectively in starter, grower I, grower II and finisher phase), and ARG2 and ARG3 groups fed CON diet supplemented respectively with 20 and 30% higher levels of crystalline L-arginine (dig Arg:Lys ratio = 1.25, 1.25, 1.26, 1.27 and 1.35, 1.35, 1.36, 1.37, respectively). Productive performance was recorded throughout the rearing cycle. At processing (43 d), breasts (n = 150/group) were randomly collected to evaluate the incidence and severity of white striping (WS), wooden breast (WB), and spaghetti meat (SM) defects (3-points scale). Meat quality traits, such as color, pH, drip and cook losses, marinade performances, and proximate composition were determined on 12 fillets/group. Although productive performance and meat quality traits resulted not significantly affected, the occurrence of some breast myopathies was modified by the dietary treatments. ARG2 and ARG3 exhibited a significantly higher percentage of breast without WS compared to CON (28 and 41 vs. 17%, respectively; P < 0.001). ARG3 reported the lowest percentage of breasts with severe WS (11 vs. 27 and 31%, respectively for ARG3, ARG2, and CON; P < 0.001) and the highest of those showing no SM abnormality (81 vs. 69 and 65%, respectively for ARG3, ARG2, and CON; P < 0.01). Furthermore, a numerical reduction of breasts with severe WB was observed in ARG3 (12 vs. 25 and 19%, respectively for ARG3, ARG2, and CON; P = 0.12). In conclusion, the dietary supplementation of arginine to increase the dig Arg:Lys ratio by about 30% in respect to the current recommendations for broiler chickens has positive implications on the occurrence of some breast meat abnormalities.

Avian model to mitigate gut-derived immune response and oxidative stress during heat.

The tissue injury at the early stages of the heat stress response triggers release of inflammatory and oxidative agents from intestinal content into the milieu of the body. Intestinal homeostasis (i.e., eubiosis) improves the barrier function and mitigates the gut-derived influx of endotoxins. In this study we have analyzed the mitigating role of embryonic stimulation of the gut homeostasis in chickens on immune and oxidative responses to heat. The animal trial was conducted on broiler chickens. The treatment included a single in ovo injection of the galactooligosaccharides (GOS) prebiotic into the air cell of the egg on day 12 of incubation. Control eggs were in ovo injected with the same volume of sterile physiological saline. After hatching, birds were raised in group pens (6 pens/group, 25 birds/pen). Short-term, mild heat stress was induced on day 32 post-hatching by increase in the ambient temperature above the thermal comfort (30 °C for 8.5 h). The spleen was harvested from randomly selected individuals. The relative gene expression study was conducted with RT-qPCR. The two gene panels were analyzed: (1) immune response genes (IL-1ß, IL-2, IL-4, IL-6, IL-12p40 and IL-17) and (2) stress response genes (HSP25, HSP70, HSP90, BAG3, CAT and SOD). Data were evaluated by the analysis of variance in a 2 × 2 factorial design that included in ovo treatment and ambient temperature as factors. We have found that the immune-related and stress-related gene expression signatures were triggered in animals subjected to heat but with unbalanced intestinal flora (i.e., dysbiotic, without in ovo stimulation with GOS). These animals had increased expression of the genes involved in the immune responses (IL-4 and IL17) and stress responses (HSP25, HSP70, HSP90, CAT and SOD) to short-term heat stress that indicated presence of inflammatory and oxidative mediators (P < 0.05). The individuals that were in ovo stimulated with GOS did not mount the anti-inflammatory or antioxidative responses. Heat shock proteins (HSP25 and HSP70) were increased in both groups challenged with heat, which indicated their role in adaptation to heat.

Variability and interaction of some egg physical and eggshell quality attributes during the entire laying hen cycle.

The aim of this study was to investigate the variability and relationships between some egg physical (egg weight, width, length, shape index, and surface area) and eggshell parameters (weight and percentage, thickness, breaking strength, and L*, a*, and b* values) during the entire laying hen cycle. A total of 8,000 eggs was collected every 5 wk, from 30 to 81 wk of hen age (10 samplings of 400 eggs/house), in 2 identical poultry houses equipped with enriched cages. For the statistical analysis, ANOVA, Bivariate Correlation, Principal Component Analysis (PCA), and Hierarchical Cluster Analysis were used. An increase of egg weight, length, and eggshell lightness (L*) associated with a reduction of eggshell percentage, breaking strength, and redness (a*) was observed as the hen aged (P < 0.05). Overall, the coefficients of variation resulted in <5% in width, length, shape index, and egg surface area; from 5 to 10% of egg weight, shell weight, shell percentage, shell thickness, L*, and b*; and >10% of eggshell breaking strength and a*. According to the PCA, the highest changes during the laying cycle are related to egg physical parameters (32%) and to eggshell breaking strength, percentage, and thickness (26%). The egg physical parameters appeared to be strongly correlated to each other, whereas a slight correlation between eggshell breaking strength and color attributes were evidenced (-0.231 and 0.289, respectively, for L* and a*; P < 0.01). Hierarchical cluster analysis, based on principal components of the overall egg attributes, is hereby considered, and evidenced dissimilarities for eggs laid from peak production up for 39 wk of hen age from the eggs laid afterwards. The latter group could also be divided into 2 subgroups, one comprising eggs laid from 44 and 53 wk of hen age and the other from 58 wk to the end. In conclusion, the large dataset created in this study allowed to extrapolate some robust information regarding the variability and correlations of the egg physical and eggshell quality attributes throughout the entire laying hen cycle.

Effect of EU electrical stunning conditions on breast meat quality of broiler chickens.

Electrical stunning is still the main stunning method used worldwide in commercial poultry plants. The stunning procedures in water bath stunners affect both bird welfare and meat quality attributes. The European Union (EU) Council Regulation 1099/2009 on the protection of the animal at the time of killing established the minimum current flow through an individual bird at a specified frequency to assure an effective stun that must last until the bird's death. The aim of this study was to compare the effect of the application of different stunning current flows on the prevalence of hemorrhages (classified as 1 = no lesion, 2 = moderate, and 3 = severe lesion) and some quality traits (pHu, color, drip and cooking losses, and shear force) of chicken breast meat. A total of 12 flocks of broiler chickens, each equally divided into light, medium, and heavy sizes, was submitted either to the stunning condition usually adopted before the entry into force of the current EU regulation (90 mA/bird, 400 Hz) (OLD) or to that enforced by it (150 mA/bird, 400 Hz) (NEW). Overall, the prevalence of severe hemorrhages dramatically increased in the NEW group in comparison with the OLD one (55 vs. 27%; P < 0.001) and particularly in heavy-sized birds (72 vs. 25%; P < 0.001). In general, meat quality attributes were not affected by the stunning conditions with the exception of drip loss that resulted lower in NEW than OLD birds (1.01 vs. 1.27; P < 0.001). In conclusion, the adoption of a higher current flow, as suggested by the EU regulation to protect animals at the time of killing, increases the prevalence of breast hemorrhages while maintaining meat quality traits with a possible beneficial effect on water holding capacity of fresh meat.