The Effect of intra egg injection of different levels of Thyme essential oil nanoemulsion on reproductive and blood parameters in Japanese quail

In recent years, injection of stimulant solutions into fertilized eggs has been considered to improve reproductive and functional parameters in birds. So far, much research has been conducted to improve embryonic development in birds by manipulating egg nutrients. In one study, injection of dietary supplements into the eggs of birds improved hatching rate, reduced mortality rate, shortened breeding time duration, increased growth rate, and increased weight gain at the end of the rearing period (Johnston et al., 1997). Egg injection technology is not only a way to vaccinate birds against certain diseases, but it is also a practical way of injecting some of the nutrients needed by the developing embryo (Foye et al., 2005 and Uni et al., 2005). Shirazi thyme (Zataria multiflora) is one of the most well-known medicinal plants. Essential oils (EOs) of shirazi thyme, that is a yellow or dark reddish brown liquid with a strong smell and a spicy and long-lasting, cooling flavour, extracted from the distillation of leaves and flowering shoots of this herb (Momeni and Shahrokhi, 1992). Essential oils comprise a complex mixture of volatile and non-volatile compounds produced by aromatic plants as secondary metabolites (Bakkali et al 2008). The main constituents of thyme EOs are flavonoide, glycosids, tannins, saponins, steroids (such as cytosterol), triterpenoids (such as oleanolic acid and ursolic acid), and volatile oils rich in monoterpenes (Martínez-Vázquez et al., 1996).Using nanoemulsions can be a great choice for the use of essential oils in foods (Jo et al., 2005). Changing the particle size to nanoparticles (size less than 100 nm) increases the surface-to-volume ratio and increases the reactivity, making it possible to use smaller amounts of these materials than materials with larger particles (Hernández-Sierra et al., 2008). The very small size of the synthesized nanoparticles can be effective in enhancing its antimicrobial effect (Krishna et al., 2011).

This study was conducted to investigate the effect of in-ova injection of different levels of nanoemulsion of thyme essential on reproductive (fertility, total hatchability, hatch of fertile eggs and embryonic mortality rates) and blood parameters (estrogen, testosterone, glucose, triglyceride, cholesterol, LDL, HDL, total protein) in Japanese quail.

The experiment was done by using 600 Japanese quail eggs in a completely randomized design with 5 treatments, 4 replications and 30 eggs per replicate. Treatments were including: negative control (without injection), positive control (with water and emulsifier injection) as well as intra ova injection of nanoemulsified thyme essential oil at levels of 50, 100 and 150 ppm. The experimental solutions were injected into the air sac of the eggs before incubation.The daily collected eggs were kept in a room at a temperature of about 17-18 °C. The male to female quail ratio was 1:3 in the flock and the birds had free access to water and feed at all stages of the experiment. Lighting length was set at the production stage, 16 hours brightness and 8 hours dark. Experimental solutions were injected into the egg sac immediately before incubation. At the end of incubation, fertility or embryonic development, hatchability, embryonic mortality rates and age of embryonic mortality were determined. To investigate the percentage of embryonic mortality during the incubation period, the eggs that had not been hatched were broken and the eggs with embryonic losses were selected. To determine the stage of embryonic mortality, the incubation period was divided into three stages: 1 to 9 days old (black eye and feather absent), 10 to 17 days old (embryo with feather and yellow sac visible) and 17 to 18 days old (grown embryos without yolk visible externally) (Aygun and Sert, 2012). The hatched chicks from each treatment and replicate were weighed and then transferred to their own pan. Hatched chicks were reared for 8 weeks to evaluate blood parameters and genital characteristics. The ration used for all groups was similar throughout the trial period according to the recommendations of the National Research Association (NRC, 1994).

Results showed that not only the experimental treatments did not improve fertility, total hatchability, hatch of fertile eggs and embryonic mortality rates, but the highest concentration of thyme nanoemulsion (150 ppm) had a detrimental effect on these parameters compared with the control groups (p < 0.05). At the end of the breeding period, uterine weight and length, ovarian weight, weight and diameter of F1 to F3 follicles in female birds as well as testosterone concentration in male birds were not affected by levels of thyme essential oil (P>0.05). Sera estrogen concentration of female birds due to intra ova injection of higher dose of nanoemulsified thyme essential oil was higher compared to the positive control group (p < 0.05). Blood glucose in experimental treatments was not significantly different from untreated control group (P>0.05).Total serum triglyceride was lower in the treatment group receiving 100 ppm nanoemulsion essential oil than the control groups (p < 0.05). The highest cholesterol and LDL levels were in the water plus emulsifier group and the lowest levels of these parameters numerically were belonged to the 150 nm emulsion treatment. There was no significant difference in sera HDL and total protein levels between experimental treatments (P>0.05). Therefore, Blood parameters, except triglyceride, which were lower at 100 ppm concentration, had no significant changes compared to controls.

Injection of different concentrations of thyme essential oil nanoemulsions into the air sac of Japanese quail egg, not only did not improved reproductive parameters in, but also its high concentration (150 ppm) had a negative effect on these parameters. Changes in the morphometric characteristics of the reproductive system and blood parameters were also not significant. However, the concentration of triglyceride at 100 ppm level of thyme essential oil decreased compared to the control group. In general, in-ova injection of thyme essential oil nanoemulsion is not recommended to improve reproductive performance in Japanese quail.