# In-ovo supplementation with bovine milk osteopontin improves hatchability, chick quality, growth performance, and enhances intraepithelial lymphocyte populations in specific-pathogen-free layer chicks

## TL;DR

Adding bovine milk osteopontin to chicken eggs before hatching improves hatching success, chick quality, growth, and immune cell development.

## Contribution

This study is the first to demonstrate that in-ovo supplementation with bovine milk osteopontin enhances hatchability and immune development in layer chicks.

## Key findings

- In-ovo supplementation with 1 mg and 25 mg of bmOPN increased hatchability by approximately 8% compared to the control.
- bmOPN increased intestinal T-cell and non-T intraepithelial lymphocyte populations in chicks.
- Chicks supplemented with 1 mg and 10 mg of bmOPN showed higher body weights and growth performance by day 14.

## Abstract

To evaluate the effect of in-ovo supplementation of bovine milk-derived Osteopontin (bmOPN) in chicks, fertilized specific-pathogen-free eggs were randomly allocated into six treatment groups (n = 26 eggs/group) on embryonic day 18. The hatchability was collected from six independent experiments. Eggs in each treatment received a 200 μL in-ovo injection of PBS containing 0 mg, 0.1 mg, 1 mg, 10 mg, 25 mg, or 50 mg of bmOPN. On day of hatch (DOH), hatchability and chick quality parameters were assessed. Body weights were recorded on DOH, 7, and 14 days of age. On day 7 of age, intestinal histomorphometric parameters, including villus height (VH), crypt depth (CD), and VH:CD ratio, were measured in the 0, 1, and 25 mg groups. Peripheral blood mononuclear cells (PBMCs) and intraepithelial lymphocytes (IELs) were isolated from 0 and 1 mg groups for flow cytometry analysis. Data were analyzed using Generalized Linear Mixed Model, one-way ANOVA, Kruskal-Wallis or Mann-Whitney U test followed by Tukey HSD/Dunn test. The hatchability increased by approximately 8% in the 1 mg and 25 mg groups compared to the control, reaching 93.6% and 93.9%, respectively. We observed dose-dependent decreases in chick length and residual yolk percentage, along with an increase in navel score up to 25 mg bmOPN. Hatched chick body weights increased slightly (1–2 g) in the 1, 10, and 25 mg groups, and by day 14, chicks in the 1 mg and 10 mg groups maintained higher body weights and body weight gains. On day 7, bmOPN administration increased the number of intestinal T-cell IELs (TCRαβ+ subsets and TCRγδ+), as well as non-T IELs (including iCD8α+ cells), while no changes were observed in peripheral blood mononuclear cells. These findings suggest that in-ovo supplementation of bmOPN enhances hatchability, chick quality, growth performance, and mucosal immune development. However, further studies are warranted to investigate the effects of bmOPN administered through different routes on intestinal function and immune responses during inflammation in both broiler and layer chickens.