Effect of in ovo injection of Lactobacillus isolates from the gastrointestinal tract of Iranian native poultry on hatchability, immune response, and growth performance in broilers

Introduction: Modern industrial poultry production removes the natural relationship between day-old chicks and hens. Consequently, the establishment of gut microbiota largely depends on the bacteria present in the hatchery and barn environment. On the other hand, chicks may be exposed to pathogenic agents while still at the hatchery, during hatching, sex determination, vaccination, and transportation, even before consuming their first feed. Since the initial contact with microbes might involve pathogenic bacteria, introducing beneficial bacteria into the gastrointestinal tract of chicks can help reduce the risk of pathogen settlement. The primary colonization of beneficial microbes is not only crucial for competing against pathogenic bacteria but also plays a significant role in stimulating factors associated with the growth and maturation of the immune system. Additionally, in the agricultural industries, the use of antibiotic growth promoters (AGPs) in animal diets has been reduced or even eliminated due to concerns regarding bacterial antibiotic resistance and antibiotic residues in animal products. As a result, several alternatives to AGPs have appeared in the poultry market. One of the many alternatives that are currently being investigated is the use of probiotics. Probiotics are defined as a “live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance”. Among the probiotics used in poultry, Lactobacillus isolates have been particularly valued for their positive effects on gut health. Furthermore, Lactobacillus naturally dominates the gastrointestinal tract of poultry. The beneficial effects of these bacteria occur through various mechanisms, including competitive exclusion, maintenance of protective intestinal barriers, and immune system enhancement. Probiotics can upregulate the expression of immune-related genes such as cytokines, interferons, and interleukins, which directly or indirectly increase the serum levels of immunoglobulins in response to antigens. Ideally, the best way to avoid the need for drugs is to prevent pathogen proliferation as early as possible. It has been commonly assumed that microbial colonization occurs after hatching in poultry; however, scientific evidence suggests that live bacteria may be present in the intestine in small quantities even before hatching. One favorable method of probiotic administration is the in ovo injection of probiotics into the amniotic sac at the late stage of embryonic development. This approach can facilitate the early establishment of beneficial bacteria in the gastrointestinal tract to create an environment that prevents pathogen colonization. Therefore, the objective of this study was to evaluate in ovo administration as a method for delivering some Lactobacillus isolates derived from the gut microbiota of Iranian native poultry to the intestines of broiler chicks before hatching and assess their effects on hatchability, immune system development, serum metabolites, and growth performance.
Materials and methods: To investigate the effect of in ovo injection of Lactobacillus isolates of Iranian native poultry in broilers, on day 18^th of incubation, 672 embryonated eggs (Ross 308 strain) were assigned into 24 experimental units, in a completely randomized design with six treatments and four replicates (28 eggs per replicate). The hatched chicks, based on the same division as the in ovo injection stage, were raised for six weeks. Experimental treatments included: 1. In ovo injection of physiological saline (saline 9%; negative control), 2. In ovo injection of physiological saline (saline 9%) and antibiotic growth promoter (avilamycin) in the diet (positive control), 3. In ovo injection of Lactobacillus salivarius isolate Pls2 (MH595986; 10⁵ CFU/egg), 4. In ovo injection of Lactobacillus reuteri isolate Plr6 (MF686483; 10⁵ CFU/egg), 5. In ovo injection of Lactobacillus reuteri isolate Plr4 (MF686463­; 10⁵ CFU/egg), and 6. In ovo injection of Lactobacillus reuteri isolate Plr2 (MG547731; 10⁵ CFU/egg). Percent of hatchability was calculated as the percent of healthy hatched chicks from fertile eggs. To evaluate the immune response of the broilers, hemagglutination assay and hemagglutination inhibition methods were separately used to measure the antibody titers against sheep red blood cells (SRBC) and Newcastle disease virus (NDV), respectively, and were expressed as the log₂. To measure glucose, triglyceride, and total cholesterol from serum, a spectrophotometric method via liquiform kits was used. Important growth performance metrics, such as body weight gain, feed intake, feed conversion ratio, and European Production Efficiency Factor (EPEF), were evaluated throughout the entire rearing period. Results are reported as means, and differences among treatments were compared using the Tukey test, and significance was determined at P<0.05.
Results and discussion: In ovo injection of Lactobacillus, isolates had no negative effect on hatchability (P>0.05). In ovo injection of Lactobacillus isolates and positive control groups showed lower feed conversion ratio compared to the negative control group (P<0.05). The EPEF in the positive control group and in ovo administered Lactobacillus isolates was higher than the negative control group (P<0.05). Among the lactobacilli used, in ovo injection of Plr2 showed similar EPEF to the positive control group (P>0.05). Broiler dietary supplementation of antibiotic growth promoter and in ovo injection of Plr4 and Plr2 increased total and secondary antibody titer against SRBC compared to the negative control group (P<0.05). The positive control group and in ovo injection of Plr2 showed higher antibody titers against NDV than the negative control group (P<0.05).  In ovo injection of Pls2, Plr6, and Plr4 increased serum glucose compared to control groups (P<0.05). In ovo injection of Plr4 decreased the triglyceride concentration of serum compared to the control groups at 42 days of age (P<0.05). In ovo injection of the Lactobacillus isolates reduced the cholesterol concentration of serum in broilers (P<0.05).
Conclusions: In general, the injection of Lactobacillus isolates used in this study, particularly Plr4 and Plr2, at the late embryonic stage as appropriate microbial compounds, may improve broiler growth performance without adversely affecting hatchability and by positively influencing the immune response and serum metabolites.