Effect of in-ovo feeding of beta-hydroxy beta-methylbutyrate, dextrin and first access to feed on humoral and cellular immune responses of broilers

The time between the first and the last chick hatching is called the hatch window and the time required for all eggs to hatch is between 24 and 48 hours (Decuypere et al. 2001). In addition, processes such as sex determination, vaccination, and on-farm transfers require more time, resulting in hatched chicks not being able to access water and feed for 72 hours. (Willemsen et al. 2010). Post-hatch fasting leads to high chicken mortality, low growth in the first week and a reduction in disease resistance, due to insufficient storage of yolk sac and incomplete bird's immune system in this period. One suggested option for overcoming these challenges is in ovo feeding (IOF), (Zhai et al. 2008). The in ovo injection of nutrients around days of 17 to18 called in ovo feeding (Foye et al 2006), supplies the embryo with additional nutrients via amniotic fluid prior hatching and those nutrients continue to be utilized by the chick post-hatch during this fasting period. Yolk protein is a source of maternal antibodies. The results suggest that IOF increases immunoglobulins, weight of bursa of fabricius, and lymphocyte proliferation on 28 days of age (Dibnere et al 1998). Results from another study indicate that, drinking water contains glucose leads to decrease in gluconeogenic enzymes, which means reduction in proteins breakdown and beneficial antibodies (Donaldson 1995). In most studies, egg is inoculated with a single nutrient, while other factors assumed to be constant. In such studies, the interrelationships between factors are ignored. Therefore, designing experiments using modern statistical mathematical methods in order to optimize test conditions and obtain more accurate results will be useful (Ahmadi and Golian 2010). Response surface methodology (RSM) is a proper model, because it is a useful set of statistical and mathematical procedures to survey responses from the combinations of the factors for searching optimum results, optimizing the interactions between factors and their levels, and to reduce investigation costs. In this method, in addition to examine the effect of each factor, all coefficients of the quadratic regression model and the interaction of factors can be estimated. Box-Behnken design (BBD) is a multivariate experimental design, where multiple factors are studied simultaneously. The BBD is considered to be more efficient than full factorial designs, because it requires a significantly smaller number of treatments and allows researchers to estimate and optimize processes (Box and Hunter 1957). This study was performed to determine the immune response of broilers to in ovo feeding of beta-hydroxy beta-methylbutyrate (HMB) and dextrin and the first time of access to feed, using Box-Behnken design.

A total of 1800 fertile eggs of 65 wk-old broiler breeders was obtained from a commercial broiler breeder flock (Ross 308) and stored under commercial conditions for two days before incubation begins. Eggs were weighed, and those within a similar weight distribution (66.4 ± 1.3 g) were randomly distributed into 60 groups of 30 eggs. Eggs were then incubated in a commercial hatchery at 37.5°C and 56%relative humidity. On the 15th day of incubation all eggs were candled and cracked and unfertilized or contained dead embryos eggs were discard (Kornasio et al. 2011). On the 18th day, 1500 eggs were randomly assigned into 15 treatments based on a Box-Behnken design (Box et al. 1987) with four replicates and three levels of in ovo administration of beta-hydroxy beta-methylbutyrate (0, 0.5 and 1.0 %) and dextrin (0, 20 and 40 %) in a 0.9 % saline as well as timing of the first feeding (6, 21 and 48 h). The injection was performed manually through a hole made in the wide part of the egg, using needle number 23 and injection of 0.5 ml of sterile solution, which was inserted to a depth of 17 mm of amniotic fluid (Uni et al. 2005). From the hatched chicks of each replication, 14 chicks were randomly selected, weighed and transferred to the farm, and according to the box-Benken design in 15 experimental treatments (each treatment has four replications and totally 60 experimental units were reared). Access to water and food was free during the 42-day trial period. Chickens were fed three diets of starter, grower, and finisher based on the Ross 308 strain nutrients requirement (Ross Broiler Guide 2014). The feed form and composition, were in crumble form with 2850 kcal/kg metabolizable energy and 21.92% crude protein) during starter period (1 to 10 days), in pellet form (3 mm) with 2880 kcal/kg ME and 19.97% CP during grower period (11 to 24 days) and in pellet form (6 mm) with 2950 kcal/kg metabolic energy and 17.98%crude protein during finisher (25 to 42 days) period (Ghanaatparast et al. 2018 a,b). Experimental data (60 data lines) gained by BBD were fitted to the second-order polynomial equation by SAS through the RSREG procedure. A ridge analysis (Draper, 1963, SAS Institute 2011) was used to compute the optimal response for TPB and PPB maximization (RIDGE MAX procedure in SAS). Using ANOVA and corresponding absolute t-value of the model parameters, a process of the sensitivity analysis was performed on developed RSM models to find which model term is considered more important throughout the modeling process (Box et al. 1987).

Increasing the time of the first access to feed and post hatch fasting reduced the humoral and cellular immunity (P < 0.05). In ovo feeding of HMB and dextrin improved primary humoral immune response (P < 0.05). In Ovo feeding of HMB and dextrin resulted in improving primary humoral immune response (P <0.05), and in Ovo injection of HMB enhanced primary and secondary humoral immune response simultaneously with fasting. By increasing fasting time, in ovo feeding of HMB and dextrin increased the cutaneous basophilic hypersensitivity response in both times of PHA-P injection (24 and 48 hours) and the second injection of PHA-P (48 hours), respectively. Limited access to the post-hatch feed causes stress and corticosteroid secretion and prevents the proper growth of immune cells. Therefore, increased immunity in chickens depends on the first access to feed, during which the acquired immunity is strengthened and the bird is protected against pathogens through the respiratory tract, which is known to be a source of infection (Yi et al. 2005). The interaction between inoculated nutrients and their concentrations in egg, as well as post hatch starvation affected broiler’s performance and such interactions can affect the optimal levels determined in an experiment (Tako et al.2004; Kornasio et al. 2011). In general, injections of carbohydrates such as fructose or ribose improve the humoral and cellular immune response in broilers, and of course the extent of the effect on the poultry immune system depends on the type of carbohydrate (Bhanja et al. 2014).

The first access of day- old chick to feed is considered as effective factor to prevent declined bird's immune system function. If access to water and feed is delayed during post-hatch (for reasons such as vaccination, sex determination, and transport to farm), in-ovo feeding of HMB and dextrin is effective to improv the humoral and cellular immune response. The Box-Benken design for minimally invasive experiments is a good method to explain the relationship between in-ovo feeding of nutrients and the first access to feed on the immune response of broilers.