In-ovo feeding with creatine monohydrate: implications for chicken energy reserves and breast muscle development during the pre-post hatching period.

The most dynamic period throughout the lifespan of broiler chickens is the pre-post-hatching period, entailing profound effects on their energy status, survival rate, body weight, and muscle growth. Given the significance of this pivotal period, we evaluated the effect of in-ovo feeding (IOF) with creatine monohydrate on late-term embryos' and hatchlings' energy reserves and post-hatch breast muscle development. The results demonstrate that IOF with creatine elevates the levels of high-energy-value molecules (creatine and glycogen) in the liver, breast muscle and yolk sac tissues 48 h post IOF, on embryonic day 19 (p < 0.03). Despite this evidence, using a novel automated image analysis tool on day 14 post-hatch, we found a significantly higher number of myofibers with lower diameter and area in the IOF creatine group compared to the control and IOF NaCl groups (p < 0.004). Gene expression analysis, at hatch, revealed that IOF creatine group had significantly higher expression levels of myogenin (MYOG) and insulin-like growth factor 1 (IGF1), related to differentiation of myogenic cells (p < 0.01), and lower expression of myogenic differentiation protein 1 (MyoD), related to their proliferation (p < 0.04). These results imply a possible effect of IOF with creatine on breast muscle development through differential expression of genes involved in myogenic proliferation and differentiation. The findings provide valuable insights into the potential of pre-hatch enrichment with creatine in modulating post-hatch muscle growth and development.

A deep learning-based automated image analysis for histological evaluation of broiler pectoral muscle.

Global market demand for chicken breast muscle with high yield and quality, together with the high incidence rate of breast muscle abnormalities in recent years highlights the need for tools that can provide a rapid and precise evaluation of breast muscle development and morphology. In this study, we used a novel deep learning-based automated image analysis workflow combining Fiji (ImageJ) with Cellpose and MorphoLibJ plugins to generate an automated diameter and cross-sectional area quantification for broiler breast muscle. We compared data of myofiber diameter from 14-day-old broiler chicks, generated either by manual analysis or by automated analysis. Comparison between manual and automated analysis methods exhibited a striking accuracy rate of up to 99.91%. Moreover, the automated analysis method was much faster. When the automated analysis method was implemented on 84 breast muscle cross-section images it characterized 59,128 myofibers within 4.2 h, while manual analysis of 27 breast muscle cross-section images enabled analysis of 17,333 myofibers in 54 h. The automated image analysis method was also more productive, producing data sets of both diameter and cross-sectional area at an 80-fold higher rate than the manual analysis (26,279 vs. 321 data sets per hour, respectively). In order to demonstrate the ability of this automated image analysis tool to detect differences in breast muscle histomorphology, we applied it on cross sections from chicks of control and in ovo feeding group, injected with a methionine source [2-hydroxy-4-(methylthio) butanoic calcium salt (HMTBa)], known to effect skeletal muscle histomorphology. Analysis was performed on 19,807 myofibers from the control group and 21,755 myofibers from the HMTBa group and was completed in less than 1 h. The clear advantages of this automated image analysis workflow characterized by high precision, high speed, and high productiveness demonstrate its potential to be implemented as a reproducible and readily adaptable research or diagnostic tool for chicken breast muscle development and morphology.

Supply and demand of creatine and glycogen in broiler chicken embryos.

Optimal embryonic development and growth of meat-type chickens (broilers) rely on incubation conditions (oxygen, heat, and humidity), on nutrients and on energy resources within the egg. Throughout incubation and according to the embryo's energy balance, the main energy storage molecules (creatine and glycogen) are continuously utilized and synthesized, mainly in the embryonic liver, breast muscle, and the extraembryonic yolk sac (YS) tissue. During the last phase of incubation, as the embryo nears hatching, dynamic changes in energy metabolism occur. These changes may affect embryonic survival, hatchlings' uniformity, quality and post hatch performance of broilers, hence, being of great importance to poultry production. Here, we followed the dynamics of creatine and glycogen from embryonic day (E) 11 until hatch and up to chick placement at the farm. We showed that creatine is stored mainly in the breast muscle while glycogen is stored mainly in the YS tissue. Analysis of creatine synthesis genes revealed their expression in the liver, kidney, YS tissue and in the breast muscle, suggesting a full synthesis capacity in these tissues. Expression analysis of genes involved in gluconeogenesis, glycogenesis, and glycogenolysis, revealed that glycogen metabolism is most active in the liver. Nevertheless, due to the relatively large size of the breast muscle and YS tissue, their contribution to glycogen metabolism in embryos is valuable. Towards hatch, post E19, creatine levels in all tissues increased while glycogen levels dramatically decreased and reached low levels at hatch and at chick placement. This proves the utmost importance of creatine in energy supply to late-term embryos and hatchlings.

Intra-amniotic administration of l-glutamine promotes intestinal maturation and enteroendocrine stimulation in chick embryos.

Initial nutritional stimulation is a key driving force for small intestinal maturation. In chick embryos, administration of l-glutamine (Gln) into the amniotic fluid stimulates early development of the small intestinal epithelium by promoting enterocyte differentiation. In this study, we evaluated the effects of intra-amniotic administration of Gln on enterocyte morphology and function, and elucidated a potential enteroendocrine pathway through which Gln stimulates small intestinal maturation. Our results show that Gln stimulation at embryonic day 17 significantly increased enterocyte and microvilli dimensions by 10 and 20%, respectively, within 48 h. Post-hatch, enterocytes and microvilli were 20% longer in Gln-treated chicks. Correspondingly, Gln stimulation significantly upregulated mRNA expression of brush border nutrient transporters PepT-1 and SGLT-1 and tight junction proteins TJP-1 and TJP-2, before and after hatch (P < 0.05). Since GLP-2 signaling from intestinal L-cells is associated with enterocyte growth, functionality and integrity, we examined the effects of Gln stimulation on mRNA expression of key hormones and receptors within this enteroendocrine pathway and found significant increases in GLP-2R, IGF-1 and IGF-1R expression before and after hatch (P < 0.05). In conclusion, our findings link primary nutrient stimulation in the developing small intestine with enterocyte morphological and functional maturation and enteroendocrine signaling.

Nutritional stimulation by in-ovo feeding modulates cellular proliferation and differentiation in the small intestinal epithelium of chicks.

Nutritional stimulation of the developing small intestine of chick embryos can be conducted by in-ovo feeding (IOF). We hypothesized that IOF of glutamine and leucine can enhance small intestinal development by promoting proliferation and differentiation of multipotent small intestinal epithelial cells. Broiler embryos (n = 128) were subject to IOF of glutamine (IOF-Gln), leucine (IOF-Leu), NaCl (IOF-NaCl) or no injection (control) at embryonic d 17 (E 17). Multipotent, progenitor and differentiated cells were located and quantified in the small intestinal epithelium between E 17 and d 7 after hatch (D 7) in all treatment groups by immunofluorescence of SRY-box transcription factor 9 (Sox9) and proliferating cell nuclear antigen (PCNA), in-situ hybridization of leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) and peptide transporter 1 (PepT1) and histochemical goblet cell staining. The effects of IOF treatments at E 19 (48 h post-IOF), in comparison to control embryos, were as follows: total cell counts increased by 40%, 33% and 19%, and multipotent cell counts increased by 52%, 50% and 38%, in IOF-Gln, IOF-Leu and IOF-NaCl embryos, respectively. Only IOF-Gln embryos exhibited a significance, 36% increase in progenitor cell counts. All IOF treatments shifted Lgr5+ stem cell localizations to villus bottoms. The differentiated, PepT1+ region of the villi was 1.9 and 1.3-fold longer in IOF-Gln and IOF-Leu embryos, respectively, while goblet cell densities decreased by 20% in IOF-Gln embryos. Post-hatch, crypt and villi epithelial cell counts were significantly higher IOF-Gln chicks, compared to control chicks (P < 0.05). We conclude IOF of glutamine stimulates small intestinal maturation and functionality during the peri-hatch period by promoting multipotent cell proliferation and differentiation, resulting in enhanced compartmentalization of multipotent and differentiated cell niches and expansions of the absorptive surface area.

Incubation temperature affects yolk utilization through changes in expression of yolk sac tissue functional genes.

The yolk sac tissue (YST) is a multifunctional metabolic organ supporting chicken embryonic development. This study examined whether incubation temperatures (ITs) affect YST functions. For this purpose, 300 eggs were assigned to 3 groups and incubated at control IT of 37.8°C, at 1.5°C below, 36.3°C (cold IT), and at 1.5°C above, 39.3°C (hot IT). For each group, 6 embryos' whole body mass and residual yolk (RSY) weights were recorded during incubation, and YST was sampled for both histology and gene expression analysis. YST functionality during incubation was examined by regression analysis, comparing changes in expression patterns of genes involved in lipid uptake and metabolism (LRP2, ApoA1), oligopeptides uptake (PepT1), gluconeogenesis (FBP1), glycogenesis (GYS2), and thyroid hormones regulation (TTR, DIO1, DIO2). Results show that hot and cold ITs affected YST gene expression and yolk utilization. PepT1 expression decreased towards hatch, in both hot and cold ITs, while in the Control IT, it reached a plateau. ApoA1 and DIO2 expression showed a moderate linear fit compared to polynomial fit in the control. GYS2 expression had no change along incubation, while in the control IT, it showed a polynomial fit. Expression of LRP2, FBP1, and DIO1 genes was affected by either cold or hot IT's. TTR expression patterns were similar in all IT groups. The variations in gene expression patterns observed in the 3 ITs can explain the changes in yolk utilization, an important parameter for hatchling quality. While the control IT showed optimal utilization, with an RSY value of 11.12% at the day of hatch, the cold and hot IT groups exhibited lower utilization with an RSY value of 18.18 and 29.99%, respectively. These findings are the first to show that ITs change the expression of key YST genes, leading to variations in yolk utilization by the embryo.

It's All About Timing: Early Feeding Promotes Intestinal Maturation by Shifting the Ratios of Specialized Epithelial Cells in Chicks.

The small intestine (SI) of chicks (Gallus gallus) matures rapidly during the initial post-hatch period and acquires digestive, absorptive, and secretive capabilities. The effects of the timing of first feeding on the quantities and distribution of specialized epithelial cells, which generate and maintain SI morphology and functionality, have not yet been examined. In this study, we identified specialized SI epithelial cell sub-types, including stem, progenitor, proliferating, and differentiated cells within crypts and villi of chicks during the first 10 days post-hatch, by in situ hybridization (ISH), immunofluorescence (IF), and histochemical staining. We then examined their quantities and ratios between day of hatch and d10 in chicks that were fed upon hatch [early feeding (EF)], compared to chicks that were fed 24 h post-hatch [delayed feeding (DF)]. Results showed that EF increased total cell quantities in the crypts and villi at days 1, 3, 7, and 10, compared to DF (p < 0.0001). At d3, EF, in comparison to DF, decreased crypt stem cell proportions (p < 0.0001), increased crypt proliferating (p < 0.01) and differentiated (p < 0.05) cell proportions, and increased villus enterocyte proportions (p < 0.01). By d10, EF increased both the quantities and proportions of villus enterocytes and goblet cells, compared to DF. We conclude that feeding upon hatch, compared to 24 h-delayed feeding, enhanced SI maturation and functionality by increasing the quantities and proportions of proliferating and differentiated cells, thus expanding the digestive, absorptive, and secretive cell populations throughout the initial post-hatch period.

Dysregulation of Dicer1 in beta cells impairs islet architecture and glucose metabolism.

microRNAs (miRNAs) play important roles in pancreas development and in regulation of insulin expression in the adult. Here we show that loss of miRNAs activity in beta-cells during embryonic development results in lower beta-cell mass and in impaired glucose tolerance. Dicer1-null cells initially constitute a significant portion of the total beta-cell population. However, during postnatal development, Dicer1-null cells are depleted. Furthermore, wild-type beta cells are repopulating the islets in complex compensatory dynamics. Because loss of Dicer1 is also associated with changes in the distribution of membranous E-cadherin, we hypothesized that E-cadherin activity may play a role in beta cell survival or islet architecture. However, genetic loss of E-cadherin function does not impair islet architecture, suggesting that miRNAs likely function through other or redundant effectors in the endocrine pancreas.

miRNAs control insulin content in pancreatic ß-cells via downregulation of transcriptional repressors.

MicroRNAs (miRNAs) were shown to be important for pancreas development, yet their roles in differentiated ß-cells remain unclear. Here, we show that miRNA inactivation in ß-cells of adult mice results in a striking diabetic phenotype. While islet architecture is intact and differentiation markers are maintained, Dicer1-deficient ß-cells show a dramatic decrease in insulin content and insulin mRNA. As a consequence of the change in insulin content, the animals become diabetic. We provide evidence for involvement of a set of miRNAs in regulating insulin synthesis. The specific knockdown of miR-24, miR-26, miR-182 or miR-148 in cultured ß-cells or in isolated primary islets downregulates insulin promoter activity and insulin mRNA levels. Further, miRNA-dependent regulation of insulin expression is associated with upregulation of transcriptional repressors, including Bhlhe22 and Sox6. Thus, miRNAs in the adult pancreas act in a new network that reinforces insulin expression by reducing the expression of insulin transcriptional repressors.