Effect of precursor amino acids for carnosine synthesis on breast fiber microstructures and myofiber differentiation-related gene expression in slow-growing chicken.

Objective: : The effects of carnosine synthesis on the structural and microstructural determinants of meat quality have not been studied to date. Therefore, this study aimed to investigate the effect of supplementation with carnosine synthesis precursors on the characteristics and microstructure of breast muscle fibers in slow-growing Korat chickens (KR). Methods: : Slow-growing KR were fed a non-supplemented commercial diet (control group) or a commercial diet supplemented with 1.0% ß-alanine, 0.5% L-histidine, or a combination of both 1.0% ß-alanine and 0.5% L-histidine. At 10 weeks, KR were slaughtered, and the breast muscle was collected. Samples were fixed and extracted to study the microstructure, fat level, and porosity of the meat using X-ray and scanning electron microscopy, and real-time polymerase chain reaction was performed to analyze the expression of genes related to myofiber differentiation. Results: : L-histidine supplementation significantly altered myofiber diameter and muscle fiber density and compactness by regulating muscle fiber-type differentiation via carnosine synthase (CARNS1) and myocyte enhancer factor 2C (MEF2C) expression, as well as myogenic differentiation antigen (MyoD) and myogenic regulatory factor 5 (Myf5) expression. While excess L-histidine potentially stimulated CARNS1 to modify muscle fiber arrangement and tenderness in breast meat, dietary ß-alanine supplementation alone or in combination with L-histidine supplementation induced a relatively less remarkable but not significant (p<0.05) effect on the breast meat characteristics studied. Conclusion: : Interestingly, the combination of ß-alanine and L-histidine supplementation had no effect on meat microstructure, meat porosity, and fat content in comparison with the control group. Thus, this combination had the best selectivity for improving meat quality. However, further studies are required to clarify the effects of carnosine levels on meat processing.

Omega-3 meat enrichment and L-FABP, PPARA, and LPL genes expression are modified by the level and period of tuna oil supplementation in slow-growing chickens.

This study proposes a strategy to manipulate the fatty acid (FA) content in slow-growing Korat chicken (KRC) meat using tuna oil (TO). To determine the optimal level and feeding period of TO supplementation, we conducted a study investigating the effects of dietary TO levels and feeding periods on meat quality, omega-3 polyunsaturated fatty acid (n-3 PUFA) composition, and gene expression related to FA metabolism in KRC breast meat. At 3 wk of age, 700 mixed-sex KRC were assigned to seven augmented factorial treatments with a completely randomized design, each consisting of four replicate pens containing 25 chickens per pen. The control group received a corn-soybean-based diet with 4.5% rice bran oil (RBO), while varying amounts of TO (1.5%, 3.0%, or 4.5%) replaced a portion of the RBO content in the experimental diets. The chickens were fed these diets for 3 and 6 wk, respectively, before being slaughtered at 9 wk. Our results indicated no significant interactions between TO levels and feeding periods on the growth performance or meat quality of KRC (P > 0.05). However, the liver fatty acid-binding protein gene (L-FABP, also known as FABP1), responsible for FA transport and accumulation, showed significantly higher expression in the chickens supplemented with 4.5% TO (P < 0.05). The chickens supplemented with 4.5% TO for a longer period (3 to 9 wk of age) exhibited the lowest levels of n-6 PUFA and n-6 to n-3 ratio, along with the highest levels of eicosapentaenoic acid, docosahexaenoic acid, and n-3 PUFA in the breast meat (P < 0.05). However, even a short period of supplementation with 4.5% TO (6 to 9 wk of age) was adequate to enrich slow-growing chicken meat with high levels of n-3 PUFA, as recommended previously. Our findings indicated that even a short period of tuna oil supplementation could lead to desirable levels of omega-3 enrichment in slow-growing chicken meat. This finding has practical implications for the poultry industry, providing insights into optimal supplementation strategies for achieving desired FA profiles without adversely affecting growth performance or meat quality.

This study investigated the effect of different levels and feeding periods of tuna oil (TO), a source of omega-3 polyunsaturated fatty acids (n-3 PUFA), was used to modify the fatty acid (FA) profile in slow-growing Korat chicken (KRC) meat. The interaction between TO supplementation levels and feeding periods did not influence growth performance or meat quality in KRC. However, higher level of TO supplementation led to increased expression of the liver fatty acid-binding protein gene, which is involved in FA transport and accumulation. The highest levels of eicosapentaenoic acid, docosahexaenoic acid, and n-3 PUFA were detected in the chickens that were fed 4.5% TO supplementation for a long period (3 to 9 wk of age). These chickens also had the lowest amounts of omega-6 polyunsaturated fatty acids (n-6 PUFA) and n-6 to n-3 ratio. Interestingly, even a short period of 4.5% TO supplementation (6 to 9 wk of age) in slow-growing chickens was sufficient to enrich the KRC meat with n-3 PUFA. These findings highlight the potential for improving the nutritional profile of chicken meat by regulating TO supplementation in the diet.

Effects of ß-alanine and L-histidine supplementation on carnosine contents in and quality and secondary structure of proteins in slow-growing Korat chicken meat.

Carnosine enrichment of slow-growing Korat chicken (KRC) meat helps differentiate KRC from mainstream chicken. We aimed to investigate the effects of ß-alanine and L-histidine supplementation on the carnosine synthesis in and quality and secondary structure of proteins in slow-growing KRC meat. Four hundred 21-day-old female KRC were used, and a completely randomized design was applied. The chickens were divided into 4 experimental groups: basal diet (A), basal diet supplemented with 1.0% ß-alanine (B), 0.5% L-histidine (C), and 1.0% ß-alanine combined with 0.5% L-histidine (D). Each group consisted of 5 replicates (20 chickens per replicate). On d 70, 2 chickens per replicate were slaughtered, and the levels of carnosine, anserine, and thiobarbituric acid reactive substances were analyzed. Biochemical changes were monitored using synchrotron radiation-based Fourier transform infrared microspectroscopy; 5 chickens per replicate were slaughtered, and the meat quality was analyzed. Statistical analysis was performed using ANOVA and principal component analysis (PCA). Group D chickens exhibited the highest carnosine meat content, followed by those in groups B and C. However, amino acid supplementation did not affect anserine content and growth performance. Higher carnosine levels correlated with increasing pH45 min and decreasing drip loss, cooking loss, shear force, and lipid oxidation. PCA revealed that supplementation with only ß-alanine or L-histidine was related to increased content of ß-sheets, ß-turns, and aliphatic bending groups and decreased content of α-helix groups. This study is the first to report such findings in slow-growing chicken. Our findings suggest that KRC can synthesize the highest carnosine levels after both ß-alanine and L-histidine supplementation. Higher carnosine contents do not adversely affect meat quality, improve meat texture, and alter the secondary structures of proteins. The molecular mechanism underlying carnosine synthesis in chickens needs further study to better understand and reveal markers that facilitate the development of nutrient selection programs.

Jejunal Transcriptomic Profiling for Differences in Feed Conversion Ratio in Slow-Growing Chickens.

Improving feed efficiency is an important breeding target for the poultry industry; to achieve this, it is necessary to understand the molecular basis of feed efficiency. We compared the jejunal transcriptomes of low- and high-feed conversion ratio (FCR) slow-growing Korat chickens (KRs). Using an original sample of 75 isolated 10-week-old KR males, we took jejunal samples from six individuals in two groups: those with extremely low FCR (n = 3; FCR = 1.93 ± 0.05) and those with extremely high FCR (n = 3; FCR = 3.29 ± 0.06). Jejunal transcriptome profiling via RNA sequencing revealed 56 genes that were differentially expressed (p < 0.01, FC > 2): 31 were upregulated, and 25 were downregulated, in the low-FCR group relative to the high-FCR group. Functional annotation revealed that these differentially expressed genes were enriched in biological processes related to immune response, glutathione metabolism, vitamin transport and metabolism, lipid metabolism, and neuronal and cardiac maturation, development, and growth, suggesting that these are important mechanisms governing jejunal feed conversion. These findings provide an important molecular basis for future breeding strategies to improve slow-growing chicken feed efficiency.

Avian sperm increase in vitro the release of exosomes from SST-enriched organoids.

In birds, oviductal cells play a crucial role in the storage of sperm via cell-to-cell communication including extracellular vesicles (EV). We developed a culture of oviductal organoids enriched in sperm storage tubules (SSTorg) to demonstrate the release of EV. SSTorg were cultured for 24 h and added to live (LV), frozen (FZ) and lysed (LY) avian sperm, seminal plasma (SP), avian sperm conditioned medium (CM), or bovine sperm (BV). Western blot demonstrated that SSTorg contained EV protein markers, valosin-containing protein (VCP), heat shock proteins (HSP90AA1, HSPA8), and annexins (ANXA2, A4, A5). Co-culture with LV significantly decreased the intracellular level of all these proteins except HSPA8. Immunohistochemistry confirmed this result for VCP and ANXA4. LY, CM, SP and BV had no effect on the intracellular level of these proteins, whereas FZ induced a decrease in ANXA2, A4 and A5. In culture media, VCP and HSP90AA1 signals were detected in the presence of LV, FZ, BV, LY, CM and SP, but no ANXA4 signal was observed in the presence of FZ and SP. ANXA2 and A5 were only detected in the presence of LV. The most abundant EV were less than 150 nm in diameter. ANXA4 and A5 were more abundant in EV isolated from the SSTorg culture medium. This study provides a useful culture system for studying interactions between SST cells and sperm. We demonstrated the release of EV by SSTorg in vitro, and its regulation by sperm. This may be of crucial importance for sperm during storage in hens.

Avian uterine fluid proteome: Exosomes and biological processes potentially involved in sperm survival.

Uterine fluid is an aqueous milieu to which sperm are exposed during their storage and ascent. In this study, a bottom-up proteomic strategy and bioinformatic analysis of hen uterine fluid was performed to improve the understanding of this fluid and its potential role in sperm survival mechanisms. The proteomic data were submitted to ProteomeXchange. Among the 913 proteins identified, 160 are known to be secreted and 640 are referenced in exosomes databases. We isolated exosomes from the avian uterine fluid, analyzed them using electron microscopy, and targeted several exosomes markers (ANXA1/2/4/5, VCP, HSP90A, HSPA8, PARK7, and MDH1) using immunoblotting. Electron microscopy and immunohistochemistry were also used to analyze uterovaginal junctions for the exosomal proteins ANXA4, VCP, and PARK7. Exosomes were observed both at the surface epithelium and inside sperm storage tubules. Our data were compared with two previously published studies on proteomic of hen uterine fluid, and with one study describing the proteomic content of rooster seminal plasma and sperm. In conclusion, we demonstrated for the first time that avian uterine fluid contains exosomes. These may play a key role in preserving sperm functions within the female genital tract. Their presence in the sperm storage tubules may represent an important mechanism regarding interaction between the female genital tract and sperm.

Proteomic analysis of uterine fluid of fertile and subfertile hens before and after insemination.

Avian uterine fluid (UF) and uterovaginal sperm storage tubules (SST) are key components in accepting sperm in SST, maintaining sperm function for several weeks, releasing sperm from SST and their ascent through the uterus. To improve the understanding of sperm storage processes requires investigating UF and SST. This study aimed to identify proteins modulated by sperm in the hen's genital tract and to highlight their role during sperm storage. Two genetic lines of hens exhibiting long (F+) or short (F-) sperm storage ability were used. GeLC MS/MS analysis was used to establish a quantitative inventory of proteins regulated after insemination in both lines. The proteomic data are available via ProteomeXchange with identifier PXD013514. Immunohistochemistry was used to identify high (ANXA4/ANXA5/OCX32) and low (HSPA8/PIGR) fertility markers in the uterovaginal junction. Our results demonstrated that sperm induced a significant and rapid change in the UF proteomic content and also in the SST epithelium. In F+ hens, mobilization of the ANXA4 protein in the apical part of SST cells after insemination was associated with increased levels of some proteoglycans and binding proteins, and also antimicrobial eggshell matrix protein (OCX32) in the UF. We also observed increased levels of lipid transporters involved in egg formation (VTG1-2, APOA1-4-H). In F- hens, insemination induced increased levels of PIGR in both UF and SST, of ANXA5 in SST, of UF enzymes exhibiting metallopeptidase activity and mucins. In conclusion, sperm induced significant changes in the UF proteomic content. This study also provides evidence that the SST immune system plays a major role in regulating sperm storage.

Eggshell matrix proteins OC-116, OC-17 and OCX36 in hen's sperm storage tubules.

While uterine epithelium secretes eggshell matrix proteins to regulate eggshell structural organization, uterovaginal junction (UVJ) epithelium supports sperm storage in tubules (SST). Here, we examined the presence of OCX36, OC-116 and OC-17 eggshell matrix proteins in SSTs. Two experimental lines of hens displaying either a long (F+ line) or a short (F- line) potential to store sperm were used, before and 24h after insemination. Using immunohistochemistry and western blot, we analyzed the presence of OC-116, OC-17 and OCX36 proteins in the SSTs. Using lectin and calcium staining, we examined the presence in SSTs of Gal/GalNAc (Galactose/N-acetylgalactosamine) and Glc/GlcNAc (Glucose/N-acetylglucosamine) glycans, as well as calcium ions. Our results indicate that in both F+ and F- hens, the eggshell matrix proteins OC-116 and OCX36 were identified in SST cells and lumen, in contact with spermatozoa. The OC-17 protein was found associated with calcium in F+ and F- hens, only in the SST lumen 24h after insemination. Glycans Gal/GalNAc and Glc/GlcNAc were found to be more abundant in the apical cytoplasmic area of the SST cells of F+ hens than in that of F- hens after insemination. This is the first report demonstrating the presence in SSTs of the OC-116, OC-17 and OCX36 eggshell matrix proteins, and their concomitant presence with Gal/GalNAc and Glc/GlcNAc glycans, as well as with calcium. Our results suggest that the OC-116, OC-17 and OCX36 eggshell matrix proteins may be involved in sperm storage.

Proteomes of the Female Genital Tract During the Oestrous Cycle.

The female genital tract includes several anatomical regions whose luminal fluids successively interact with gametes and embryos and are involved in the fertilisation and development processes. The luminal fluids from the inner cervix, the uterus and the oviduct were collected along the oestrous cycle at oestrus (Day 0 of the cycle) and during the luteal phase (Day 10) from adult cyclic ewes. The proteomes were assessed by GeLC-MS/MS and quantified by spectral counting. A set of 940 proteins were identified including 291 proteins differentially present along the cycle in one or several regions. The global analysis of the fluid proteomes revealed a general pattern of endocrine regulation of the tract, with the cervix and the oviduct showing an increased differential proteins abundance mainly at oestrus while the uterus showed an increased abundance mainly during the luteal phase. The proteins more abundant at oestrus included several families such as the heat shock proteins (HSP), the mucins, the complement cascade proteins and several redox enzymes. Other proteins known for their interaction with gametes such as oviductin (OVGP), osteopontin, HSPA8, and the spermadhesin AWN were also overexpressed at oestrus. The proteins more abundant during the luteal phase were associated with the immune system such as ceruloplasmin, lactoferrin, DMBT1, or PIGR, and also with tissue remodeling such as galectin 3 binding protein, alkaline phosphatase, CD9, or fibulin. Several proteins differentially abundant between estrus and the luteal phase, such as myosin 9 and fibronectin, were also validated by immunohistochemistry. The potential roles in sperm transit and uterine receptivity of the proteins differentially regulated along the cycle in the female genital tract are discussed.