Abstract
Background:
Weaning is a critical and stressful phase in the early life of lambs, often causing reduced feed intake, impaired gut integrity, and increased susceptibility to gastrointestinal diseases. Therefore, maintaining intestinal barrier function and digestive capacity is essential for supporting healthy growth and reducing post-weaning challenges.

Aim:
This study examined how the intestinal barrier integrity and digestive enzyme activity of newly weaned lambs were affected by nano-encapsulated black seed (Nigella sativa) and thyme (Thymus vulgaris) oils.

Methods:
Four treatment groups (n = 16) were randomly assigned to 64 Awassi lambs (45 days old, 12.5 ± 1.2 kg body weight): Control (C), Nano-encapsulated black seed oil (NBS), Nano-encapsulated thyme oil (NTO), and combination (NBS + NTO). For 56 days after weaning, 200 mg/kg of nano-encapsulated oils were added to the feed. Western blotting and immunofluorescence were used to examine the presence of tight junction proteins [claudin-1, occludin, and zonula occludens-1 (ZO-1] in intestinal tissue samples. Serum and duodenal contents were used to test the activity of pancreatic enzymes (amylase, lipase, and protease).

Results:
After being weaned, 64 Awassi lambs were divided into four groups (n = 16): control, nano-encapsulated black seed oil, nano-encapsulated thyme oil, and their combination. ZO-1 (3.1-fold), occludin (2.5-fold), and claudin-1 (2.8-fold) were all significantly more expressed in the combination group than in the control group (p < 0.001). There was also a significant increase in lipase (178 ± 19 vs. 98 ± 11 U/l), protease (892 ± 78 vs. 512 ± 56 U/mg protein), and amylase (485 ± 42 vs. 289 ± 31 U/l). The average daily gain increased by 24% (285 ± 28 vs. 230 ± 24 g/day; p < 0.01), and intestinal permeability decreased by 58% (lactulose/mannitol ratio: 0.18 ± 0.02 vs. 0.43 ± 0.05; p < 0.001).

Conclusion:
The combination treatment increased tight junction proteins (2.5–3.1-fold), enhanced enzyme activities (68%–82%), reduced intestinal permeability by 58%, and improved feed efficiency and growth performance. Nanoencapsulated black seed and thyme oils synergistically enhance intestinal integrity, digestive enzyme activity, and growth performance in weaned lambs.

Key words: Digestive enzymes; Nano-encapsulation; Nigella sativa; Thymus vulgaris; Weaning stress.