More than 15 papers were published between 1996 and 2003 summarizing the findings of M. Sc. and PhD students from my lab (Uni et al., 1996; Uni et al. 1998a; Uni et al. 1998b; Uni et al. 1999; Uni et al. 2000; Uni et al., 2023a). Publications characterized localization of enterocyte cells, their turnover at early age, ontogeny of intestinal enzymes, transporters, goblet cells, villi and microvilli structure during the transition from yolk nutrition to exogenous feeding. Also the critical time windows for intestinal maturation and for nutrient absorption was defined as well as the adaptation of gut tissues to nutritional and environmental signals (Geyra et al., 2001a; Geyra et al., 2001b; Gal-Garber et al., 2003; Uni et al., 2000; Uni et al. 2001; Uni et al. 2023b).

The impact of this new body of knowledge provided the conceptual and practical foundation for nutritional management of newly hatched chicks in the poultry industry and led to the idea of early and in ovo feeding (Uni and Ferket, 2004).

The research identifies key factors limiting the development and survival of pre-hatch broiler embryos and hatchlings, including nutrient availability in the egg, digestive capacity, and reliance on yolk reserves pre and post-hatch. These constraints contribute to poor chick quality and early mortality. Approaches such as early feeding (providing feed immediately post-hatch) and in ovo feeding (nutrient administration before hatch) can mitigate these issues. Combining both methods hold significant potential to improve early growth, feed efficiency, and overall bird performance, especially given to the rapid growth rates of modern broilers.

Introducing and promoting the concept of in ovo feeding, a terminology which refer to nutrient supplementation directly to the embryo amniotic fluid before hatching, was done hand by hand with a great colleague and partner - Prof. Peter Ferket from North Carolina State University. Together we patented the idea (Uni and Ferket, 2003; US Patent No. 6,592,878) and developed few in ovo feeding solutions which are suitable for enriching the amniotic fluid of the embryo 3 days before hatch, during transfer time at the hatchery (Uni et al., 2005; Foye et al., 2007). The pioneering experiments were done with Embrex Inovoject machine who developed the technology for mass injection and for targeting the amniotic fluid of the broiler embryo at E18. Experiments with specific nutrients/minerals and vitamins were preformed successfully. For example, in ovo feeding with zinc-methionine lead to changes in chicken intestinal zinc exporter mRNA expression and to small intestinal functionality (Tako et al., 2005). A formula that contained also carbohydrates improves energy status of late-term chicken embryos and promote hatchability (Uni, et al., 2005; Foye et al., 2007). Exploring in ovo delivery of carbohydrates, amino acids, minerals, and bioactive compounds (see reviews by Kadam et al., 2013; Peebles., 2018; Das et al., 2021) linked early feeding to improved and accelerated intestinal and muscular development (Kornasio et al., 2011), growth, feed efficiency, immune function, and even bone mineralization. For example,: Eggs injected in ovo at E17 with a solution containing minerals, vitamins (including vitamin D3), and carbohydrates produced chicks with improved bone mechanical properties, cortical and trabecular structure, and mineralization at various stages compared to controls. Notably, enhancements were seen pre-hatch and during early post-hatch days, with lasting improvements in bone architecture and mineralization at later stages (d 28 and 54). The results indicate that embryonic nutrition can positively influence both early and long-term skeletal development in broilers (Yair et al., 2013; Yair et al., 2015).

Research from the last 5 years focused on the dynamic changes in the subpopulations of intestinal cells. Using RNAscope methodology we were able to show that nutritional stimulation by in-ovo feeding modulates cellular proliferation, differentiation and maturation in the small intestinal epithelium of pre-post hatch chick by shifting the ratios of specialized epithelial cells (Reicher et al., 2020; Reicher et al., 2022a; Reicher et al., 2022b).

In ovo feeding is now widely used in poultry science. Our publications transformed the understanding of how prenatal nutrition can shape lifetime performance in poultry, influencing both academic research and industrial feeding strategies (Noy and Uni, 2010). An excellent review publication by Oliveira G. et al. (2023) provides a bibliographical mapping of research related to in ovo injection practice and shows the global research from various laboratories using more than 100 different substances and ingredients delivered in ovo. I was glad to see the names of the three most frequently cited authors: Uni (573 citations), Ferket (376 citations) and Peebles (260 citations).

In ovo feeding has taken a great step forward due to the attention of the poultry industry as a practice that can strengthen and leverage poultry production systems.

The exploration of the chicken embryo nutrition lead to investigation of the yolk - The major source of nutrients for the embryo during the 21 days of incubation. The yolk is composed of nutrient-rich content and surrounded by a tissue (yolk sac tissue = YST) derived from the embryo’s midgut. Our findings pointed towards limitations in yolk mineral (P, Fe, Zn, Cu, and Mn) and fat availability and in their utilization by embryo during incubation (Uni et al., 2012). Content and uptake of minerals in the yolk of broiler embryos during incubation and effect of nutrient enrichment - by in ovo feeding - were studied (Yair and Uni, 2011; Yair et al., 2015) and exhibit positive, long term effects of elevating mineral reserves in the yolk on bone structure, composition, and mechanical properties.

YST functionality was examined by gene expression from E 13 to day of hatch, revealing its dynamic roles in embryonic development (Yadgary et al., 2014). Over 3,500 genes changed expression during this period, reflecting shifts in YST function. Early on, the YST showed high erythropoietic (blood-forming) activity, while later it upregulated genes for lipid digestion, transport, and metabolism. The YST also produced plasma proteins typically made by the liver. Toward hatch, epithelial cell degradation was observed. When we asked the question if incubation temperature has an effect on YST development and functionality, we found that even a variation of less than 2 Celsius (from the optimal incubation temperature) led to impaired functionality. Both “cold” (36.3 °C), and “hot” (39.3 °C) incubation temperatures altered YST gene expression and reduced yolk utilization, impairing yolk utilization and potentially hatchling quality (Dayan et al., 2020). These results demonstrate that non-optimal incubation temperatures disrupt YST metabolic gene regulation, Overall, the YST functions as a multifunctional organ, which function temporarily substituting for the intestine, liver, and bone marrow to support the embryo until hatching (Wang and Uni 2023).

Since curiosity is one of the main characters needed for a researcher, we continued asking questions about the chicken intestine. The question about taste perception in broiler chicken is an important one as it may have an effect on appetite and on feeding behavior. In one of our studies taste receptor genes were identified in the gastrointestinal tract (GIT) of embryonic and post-hatch chickens. Bitter (ggTas2r1, ggTas2r2, ggTas2r7) and umami (ggTas1r1, ggTas1r3) receptors and their signalling proteins were expressed in the gut, suggesting that chickens use taste pathways in the gastrointestinal tract to sense nutrients and regulate digestion. We used a two-choice test to determine taste detection thresholds in chickens and found that chickens were as sensitive to bitterness as mammals but less sensitive to sweet and umami tastes (Cheled-Shoval et al., 2015; Cheled-Shoval et al., 2002).

Other current research activities in my lab are: efforts for elucidated growth, energy dynamics, metabolic disorders in fast-growing broilers, energy partitioning in the pre-post hatch period and subsequently, in ovo feeding of guanidinoacetate (GAA) supplementation, a precursor for creatine production (an organic compound that facilitates recycling of ATP, primarily in muscle and brain tissue). We also study muscle myopathies (e.g., spaghetti meat, woody breast) and their nutritional determinants, including energy dynamics in developing muscle, and the introducing of AI-assisted histological analysis for early detection of breast muscle tissue abnormalities. (Dayan et al., 2023a; Dayan et al., 2023b; Dayan et al., 2023c).

The impact of this research is via bridging molecular physiology through precision livestock management, offering innovative approaches to meat quality and food loss reduction.