Use of a Non-Ionic Surfactant to Facilitate Carvacrol Diffusion Through Embryonic Structures After ‘In Ovo’ Injection

Enteric diseases can severely affect the health and welfare of broiler chicks, often requiring antibiotic treatment. A key focus of livestock research has been the development of preventive measures to avoid the need for antibiotic products. Within this context, the use of dietary essential oils (EO) has been widely studied, showing consistent positive effects on gut health (Brenes & Roura, 2010). However, little is known regarding in ovo applications of EOs, and if they improve intestinal development in broiler chicks. Transdermal absorption of carvacrol, the main component of oregano EO (OEO), increased when emulsified using a non-ionic surfactant (polysorbate 80) (Laothaweerungsawat et al., 2020). Further understanding of EO diffusion patterns into egg compartments and dispersion into embryonic structures following in ovo injection is required. The main objective of the current experiment was to determine the effects of a non-ionic surfactant on the diffusion of carvacrol through, or into different embryonic structures after in ovo injection at day 17.5 of incubation (E17.5). It was hypothesised that in ovo injection of OEO emulsified with a surfactant would increase the diffusion of carvacrol to embryonic structures, such as amniotic fluid, blood, and yolk.

Three treatments were injected in two sites (air cell or amnion); 1) 0.9% saline control, 2) OEO (1.75% v/v) in 0.9% saline with (1:1) v/v polysorbate 80 (Tween-80, Sigma-Aldrich, St. Louis, USA) or 3) OEO (1.75% v/v) in 0.9% saline without (0:1) polysorbate 80. Eggs were incubated, and at E17.5 the blunt side of the egg was injected with 0.5mL of each solution with a 23G 1¼” needle. For injection in the air cell a guard was used. Amniotic fluid, blood and yolk were collected at 3, 6 and 9h after injection. Samples were analysed (n ≥ 3 per treatment) for carvacrol concentration using gas-chromatography mass-spectrometry (GC-MS). Differences in carvacrol concentration and mortality were analysed using PROC MIXED in SAS 9.4.

Polysorbate 80 administered with OEO (1:1) through amnion or air cell resulted in a significant increase in carvacrol diffusion into amniotic fluid (P = 0.004), blood (P = 0.049), and yolk (P < 0.0001). In contrast, no carvacrol was detected in amniotic fluid or blood without the surfactant, and only trace amounts were detected in yolk. In addition, the OEO treatment with polysorbate 80 in air cell was associated with 100% embryonic mortality (P < 0.0001), indicating a potential toxic effect of the surfactant when injected in air cell. Detection of carvacrol in the yolk, which is not directly on the transmission route of OEO injection, could be due to the lipophilic nature of carvacrol emulsified with the surfactant, favouring diffusion into lipophilic environments such as yolk. Yolk is absorbed into the embryonic GIT during late incubation. This indicates that carvacrol, when administered with a non-ionic surfactant, may reach the GIT through this route in sufficient amounts to improve gut health and development.

In conclusion, the use of polysorbate 80 facilitates carvacrol diffusion to embryonic structures after injection of OEO but has a potential toxic effect when injected in the air cell.

ACKNOWLEDGEMENTS: This work was supported by AgriFutures and Delacon Biotechnik.