Early (in-ovo) administration of probiotics to promote growth in broiler chicken

Objective 1: To investigate the efficacy of in-ovo supplementation of probiotics on growth and performance in broiler chicken.

Probiotic culture conditions and preparation of probiotic spray: Probiotic strains (L. paracasei DUP-13076, and L. rhamnosus NRRL-B-442) were obtained from the USDA NRRL culture collection. These probiotic isolates were selected based on our preliminary studies that revealed their ability to inhibit Salmonella, adhere to chicken cecal epithelium and absence of adverse effects on embryo survival and development. Each strain was cultured separately in 10 ml of de Mann, Rogosa, Sharpe broth (MRS) under at 37°C for 24 h. The cells were then sedimented by centrifugation (3600 X g for 15 min), washed twice with sterile phosphate buffered saline (PBS, pH 7.0), and resuspended in 10 ml PBS. The approximate bacterial count in each culture was determined spectrophotometrically. Equal portions from each of the three strains were combined to make a three-strain probiotic cocktail.  The bacterial population in the three-strain mixture was determined by plating 0.1-ml portions of appropriate dilutions on MRS agar, followed by anerobic incubation at 37°C for 24 h. Appropriate dilutions of the three-strain mixture in PBS were used to obtain the desired level of inoculum (9 log CFU).

Experimental design: Freshly laid fertile eggs from single-comb White Leghorn chickens were collected from the University of Connecticut poultry farm and randomly assigned to the following 4 groups. A total of 280 eggs were used in each of the two trials with 70 eggs per group. The 4 treatment groups include:

Egg treatment, incubation and sampling: Freshly laid eggs were weighed. Eggs in group 2 and 4 were sprayed with 200 μl of the three strain probiotic cocktail (~9 log CFU/egg) while eggs in group 1and 3 were sprayed with PBS (solvent control; 200 μl of PBS/egg) using an atomizer. The sprayed eggs were then incubated in a thermostat incubator (2362N hova-bator, GQF Manufacturing Company Inc., GA) with automatic egg turner (1611 egg turner with 6 universal racks, GQF Manufacturing Company Inc.), temperature and humidity control for 18 days at 37.8°C and 55% relative humidity. During this period, seven eggs per treatment were sampled on days 1, 7, 14 and 18. These eggs were weighed, candled and dipped in 50 ml PBS, rubbed for one minute, and the surviving probiotic population was enumerated by plating the PBS solution on MRS agar as previously described.

Morphometry of embryos: In addition to probiotic enumeration, growth performance parameters including organ and embryo weight, crown-rump length, tarsal length, tibial length and the length of the third digit were measured (6, 12).

Hatchability and post-hatch performance parameters: On day 18, the remaining eggs were sprayed with probiotics or PBS and transferred to the hatcher and held at 37.8°C and 65% relative humidity for 3 days or until hatch. A repeat inoculation was performed on day 18, since our preliminary studies revealed that the probiotic population on the egg surface reduced from 7 log on day 1 to 3 log CFU on day 18 of incubation.  On day of hatch (day 21), percent hatchability was recorded and hatchlings were weighed prior to placement on floor pens. Seven hatchlings from each treatment group were sacrificed and morphometric measurements were performed as described above. Blood was collected in non-heparinized vials to obtain serum for further analysis (10).  Entire gizzard and liver were collected, weighed and expressed relative to body weight (2).

Broiler chicken, diet and management: Day-old broiler chicks were transferred to floor pens and feed/water will administered ad libitum for the entire 5 week experimental period. All birds were grouped in separate pens depending on the treatment type. Birds of each group received feed supplementation similar to the treatment they obtained while they were embryos. Briefly, Group 1 (control) and 2 (in-ovo only) were fed with feed that is not supplemented with probiotics for the entire duration. Groups 3 (in-feed only) and 4 (in-ovo + in-feed) were fed with feed supplemented with probiotic cocktail for 5 weeks, starting on day 0. For the in-feed supplementation, probiotic cocktail was prepared as described previously. Appropriate volume of the cocktail culture was added to the feed and mixed thoroughly to obtain the desired concentration in the feed. Protocols and procedures recommended by Institutional Animal Care and Use Committee (IACUC) were followed throughout the experiment.

Body weight and Feed conversion ratio: Prior to feeding, individual body weights were obtained on week 1, 2, 3, 4 and 5. Feed consumed was recorded daily on per pen basis, the uneaten feed was collected once daily before morning feeding and feed conversion ratio will be calculated (10).

Organ weights, abdominal fat deposition and carcass yield percentages:  On weeks 1, 2, 3, 4 and 5, seven birds from each treatment group were sacrificed. Internal organs, gizzard, heart, liver,  cecum, and colon were collected. Both absolute and relative percent of organ weight to the body weight was calculated (10). Head and feet of the bird were removed, followed by defeathering, and evisceration so that the carcass was in a ready to cook (RTC) state. RTC carcass weight was measured and expressed as a percentage of live body weight.

Serum lipid analysis: Blood samples collected at necropsy were centrifuged at 2000g for 10 min and the serum was transferred to vials and stored at -20° until lipid analysis. Serum samples were analyzed for total cholesterol, LDL and HDL cholesterol and triglycerides by colorimetric assay (10).

Statistical analysis: A completely randomized design with factorial treatment structure was followed. For the first part of the experiment involving the eggs, the experimental unit was a hovabator that receives different treated-diets (70 eggs per incubator), and the sampling unit was the egg. For the morphometric measurements, the factors included 4 treatments (control, in-ovo only, in-feed only, in-ovo+in-feed), 6 parameters (organ and embryo weight, crown-rump length, tarsal length, tibial length and the length of the third digit), and 4 time points (day 1, 7, 14 and 18). For the second part of the study involving hatchling/chicken, the experimental unit was the pen of chickens that receives different diets (42 birds/pen), and the sampling unit was the bird.  In the chicken performance data, the factors included the 4 treatments, 4 parameters (body weight, carcass weight, feed intake, feed conversion rate), and 5 time points (week 1, 2, 3, 4 and 5). In organ weight data, the factors included 4 treatments, 3 samples (gizzard, heart and liver), and 5 time points (week 1, 2, 3, 4 and 5). For serum lipid analysis, the factors included 4 treatments, 4 parameters (total cholesterol, LDL and HDL cholesterol and triglycerides) and 5 time points (week 1, 2, 3, 4 and 5). Proc-mixed procedure of the statistical analysis software (version 9.3, SAS Institute Inc. Cary, NC) was used. Differences among the means were detected at P ≤ 0.05 using Fisher’s Least Significance Difference (LSD) test with appropriate correction for multiple comparisons.