Feather follicles are dynamic and heterogeneous mini-organs containing epithelial, mesenchymal, pigmentary, and progenitor cell populations. However, cryopreservation studies have focused mainly on fibroblasts, and other follicular populations remain poorly explored. This study aimed to establish favorable cryopreservation conditions for cells isolated from growing feathers of Gallus gallus domesticus using chicken serum. Primary cultures were assessed by morphology, differentiation-associated staining, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Cells were cryopreserved for 60 days in media containing 10% or 80% chicken serum combined with 5%, 7.5%, or 10% dimethyl sulfoxide (Me₂SO). Post-thaw cell status was evaluated by Annexin V/propidium iodide flow cytometry immediately after thawing and after 72 h of recovery, and metabolic activity was assessed using a colorimetric tetrazolium assay. Cultures progressively became dominated by small, rounded cells arranged in compact colonies. Morphological and molecular findings were consistent with preferential maintenance of a population displaying epithelial-associated characteristics after culture and cryopreservation. Media containing 80% chicken serum produced more favorable post-thaw outcomes than media containing 10%, with lower late apoptosis immediately after thawing, greater metabolic recovery, and higher retention of adherent cells. Serum concentration accounted for 77.87% of the variation in metabolic activity and had a stronger effect than Me₂SO concentration. Overall, the 80% chicken serum formulations containing 5% or 7.5% Me₂SO showed the most consistent recovery across the evaluated endpoints. These findings support the use of chicken serum-based media for cryopreservation of feather follicle-derived cells and broaden the range of cellular resources available for avian biobanking and conservation-oriented biotechnologies.

This study evaluated the effects of increasing dietary inclusion levels of branched-chain volatile fatty acids, also called isoacids (ISO), on the growth performance, carcass traits, intestinal health, and breast muscle metabolomic profile of broiler chickens. A total of 700 one-day-old male Cobb 500 chicks were assigned to five corn–soybean meal-based diets with 10 replicate pens (14 birds/pen) in a completely randomized design: A Positive Control (PC) diet formulated to meet nutritional requirements was supplemented with 0.0, 0.10, 0.20, 0.40, or 0.60% of ISO (supplied as a blend of isobutyrate and 2-methylbutyrate). Data were analyzed using General Linear Models with orthogonal polynomial contrasts, PERMANOVA for microbiome beta diversity and ANOVA with FDR correction for metabolomics. Growth performance was monitored across feeding phases. At day 42, carcass traits, breast meat quality, intestinal permeability, cecal short-chain fatty acids (SCFAs), and cecal and ileal microbiota were assessed, alongside liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) metabolomics of the Pectoralis major muscle. Dietary ISO significantly improved average daily weight gain (p < 0.0001) and feed conversion ratio (p < 0.0001) during the first 21 days (early-life), with an optimal inclusion level of 0.40%. While overall carcass and breast yields were unaffected, ISO supplementation significantly and quadratically reduced abdominal fat deposition (p < 0.0001), with an optimal inclusion range between 0.10% and 0.40%. ISO did not compromise intestinal permeability or alter microbial alpha diversity, but it significantly restructured the cecal microbiota (beta diversity; Bray–Curtis p = 0.0213) and linearly increased cecal isobutyric acid accumulation in the cecal content (p = 0.0443). Breast muscle metabolomics revealed a dose-dependent, nonlinear response; the 0.40% inclusion level maximized the relative abundance of galactolipids, whereas the highest dose (0.60% ISO) upregulated sphingomyelins, lysophospholipids, and acylcarnitines. In conclusion, a specific dietary ISO supplementation dose of 0.4% serves as an effective nutritional strategy to boost early feed efficiency and reduce abdominal adiposity. Doses exceeding this optimal level, such as 0.60%, offer no further productive benefits and may trigger cellular stress.