Final report for GNE16-128

Project Type: Graduate Student
Funds awarded in 2016: $14,999.00
Projected End Date: 08/31/2018
Grant Recipient: University of Connecticut
Region: Northeast
State: Connecticut
Faculty Advisor:
Mary Anne Amalaradjou
University of Connecticut
Faculty Advisor:
Michael Darre
University of Connecticut
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Project Information


Increasing concerns over antibiotic use in food animals and the emergence of antibiotic resistant pathogens resulted in the U.S Food and Drug Administration directive curbing the use of antibiotic growth promoters (AGPs) in poultry production. This has led to an urgent need for safe and natural alternatives to AGPs in promoting poultry health and performance. In this regard, several researchers have demonstrated the efficacy of probiotic supplementation to day-old chicks in improving performance in market birds. However, the period of embryonic growth and immediate post-hatch development account for almost 50% of the productive life of modern broilers. Furthermore, this developmental period is critical to attaining quality broiler performance at marketing.  Therefore, in-ovo probiotic administration would provide for an effective means to influence embryogenesis, post-hatch growth, performance and health in chicken. Through this research will provide an economical, safe and practical alternative to AGPs that can promote embryonic growth. Further, it is anticipated that the embryonic growth will translate into increased growth in broiler chickens, better disease prevention and improved economic opportunities for the poultry industry.

Project Objectives:

The specific objective includes:

  1. To investigate the efficacy of in-ovo supplementation of probiotics on growth and performance in broiler chicken. We will evaluate the probiotic effect on (a) broiler hatchability b) growth performance (embryonic and post-hatch), (b) organ weights, abdominal fat, serum lipids and (c) intestinal histomorphology.

Over the last two decades, increased consumer awareness and demand for a healthy diet has highlighted the significant role of poultry meat as a source of high quality protein for human consumption. In order to meet this ever-growing demand, the poultry industry adopted intensive rearing practices which brought about new challenges in the form of various diseases. This led to the use of antibiotics for prophylaxis, therapeutics and growth promotion purposes.  However, administration of antibiotics in food animals has led to the emergence of antibiotic-resistant foodborne pathogens, development of cross resistance to antibiotics used in human medicine and accumulation of antibiotic residues in various poultry and meat products.   Furthermore, in intensive poultry rearing, newly hatched chicks rarely have contact with their mothers and therefore experience a delay in the establishment of the gut microflora. This early period when the gut microflora is not established is when the chicks are highly susceptible to infections. This susceptibility is further aggravated by the administration of antibiotic growth promoters (AGP) which can have damaging effects on the poultry gut microflora and overall performance. To contain and eventually eliminate risks associated with antibiotic use, the U.S. Food and Drug Administration issued a directive to control antimicrobial use in food animals by restricting drug use only to assure the health status of the animals, and not as a feed additive.The restrictions on AGP use in poultry feeds has led to the need for suitable alternatives that can not only maintain production performance of birds but also ensure the microbiological safety of animal products.

Probiotics or direct fed microbials (DFM) are one of the options that have been evaluated as alternatives to AGPs and shown to be effective in promoting growth and reducing infections. Probiotics or DFM are “Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”. These microorganisms promote growth and performance through multiple mechanisms including i) maintenance of healthy gut microflora, ii) enhanced feed intake and digestion, iii) preventing pathogen survival and colonization iv) production of antimicrobial metabolites, v) preservation of  the structure and function of the intestinal epithelium and vi) modulating the immune response. In this regard, several studies have demonstrated the use of probiotics and competitive exclusion cultures as an effective alternative to antibiotics in the animal feed industry. In addition, in feed supplementation of probiotics has been shown to increase the growth and performance in chicken. Although, these studies have demonstrated the growth promoting effects of probiotics, their application is limited to in-feed or in-water supplementation of probiotics to day-old chicks.  However, the period of embryonic and neonatal development accounts for almost 50% of the productive life of modern broilers. Furthermore, the embryonic and immediate post-hatch developmental period represents a significant phase that is critical to attaining quality broiler performance at marketing.  Therefore, early administration of probiotics, i.e., in-ovo probiotic supplementation could be a potential and viable alternative to promote sustained growth and development in broilers.

Our preliminary studies pertaining to this proposal demonstrate that in-ovo application of probiotics (billion bacterial cells consisting of Lactobacillus delbreuckii sub sp. bulgaricus NRRL-B-548, L. paracasei DUP-13076, and L. rhamnosus NRRL-B-442) influenced the embryogenesis by promoting the growth and development of the embryo. Briefly, freshly laid eggs were sprayed with the probiotic cocktail using an atomizer. The eggs were then incubated by placing in a thermostat incubator with an automatic egg turner for 18 days at 37.8°C and 55% relative humidity. The eggs were periodically sampled to monitor probiotic survival on the egg surface. Additionally, all embryos were sacrificed on day 18 of incubation and growth parameters including crown-rump length, embryo weight, third-digit lenth, tibial length and tarsal length were measured.  Results from this study demonstrated that in-ovo probiotic supplementation significantly increased (P≤0.05) all tested parameters when compared with the control. Further, we did not observe any gross morphological changes in the internal organs and organ weights. Based on these preliminary results, we expect that in ovo probiotic supplementation will not only promote embryonic growth but also translate into increased growth and performance in broiler chicken.


Click linked name(s) to expand
  • Mary Anne Amalaradjou
  • Michael Darre


Materials and methods:

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:



1.    EC: Egg control

No treatment was applied

2.     IO: Probiotic cocktail treatment on eggs (in-ovo only)

Eggs were sprayed with 200 µl (~9 log cfu/egg) probiotic cocktail on day 1 and day 18 of incubation.

3.     IF: In-feed PCT supplementation on hatch (in-feed only)

No egg treatment, only in-feed supplementation (~ 9 log CFU/kg of feed) following hatch until sacrifice

4.     IOIF: In-ovo + in-feed PCT supplementation

Eggs were sprayed with 200 µl (~9 log cfu/egg) probiotic, followed by in-feed supplementation (~ 9 log CFU/kg of feed) following hatch until sacrifice

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.

Research results and discussion:

Results and Discussion:

Table 1: Treatment scheme

Treatment groups (Pre-hath)

Treatment groups (Post-hatch)




No treatment applied


Probiotic cocktail applied in feed only



Probiotic cocktail applied in ovo only


Probiotic cocktail applied both in feed and in ovo

Embryo and hatchling

On 7th, 14th and 18th day of incubation, relative embryo weights were significantly higher (p<0.05) in the probiotic group (16.05%, 18.55%, and 32.5%) compared to the control group (13.32%, 17.49%, and 28.11%; Fig. 1). With the other parameters including crown-rump length, radioulnar length and length of the third digit (Fig. 2), significant difference was observed between the control and probiotic group (p<0.05).  Further, we did not observe any gross morphological changes in the internal organs.  Following 18 days of incubation, the eggs were transferred to the hatcher and monitored for hatch.

Spray application of probiotics on to layer embryos led to a significant increase in hatchability (p<0.05). Hatchability of the probiotic-treated group was 78.5% compared to the control which was 75.55% (Table 2). We have screened all the three probiotics individually to test their efficacy on hatchability and found that Lactobacillus delbreuckii sub sp. bulgaricus NRRL-B-548 (LD) had significantly lower hatchability than the control eggs and therefore LD was eliminated from further experiments. Lactobacillus. paracasei DUP-13076 (LP) and L. rhamnosus NRRL-B-442 (LR) were used to prepare the cocktail mixture.

Table 2: Percent hatchability

Treatment Group




Control (no in-ovo probiotic supplementation)


L. rhamnosus NRRL-B-442


Lactobacillus. paracasei DUP-13076


Probiotic cocktail of Lactobacillus. paracasei DUP-13076 and L. rhamnosus NRRL-B-442



The hatchability significantly increased with probiotic treatment compared to the control group. Hatchability of Lactobacillus delbreuckii sub sp. bulgaricus NRRL-B-548 was <50% and was omitted from the study.

On the day of the hatch, the live weight of the hatchling did not differ (p>0.05) between the control group (39.775 ± 0.54 g) and the probiotic-treated group (40.2 ± 0.53 g). Further, absolute breast, leg, and liver weights were significantly higher in the probiotic group compared to the control group. The absolute breast, leg, and liver weights increased by 14%, 7%, and 11% respectively, as shown in Figure 3. In accordance with the absolute weights, relative weights of breast, leg, and liver were also higher (p<0.05) in the probiotic group. Also, in ovo probiotic application increased crown-rump length by 7%, radio-ulna length by 6% and third digit by 3% when compared to the control (Figure 3). Overall, chicks hatched from probiotic sprayed eggs were longer and heavier than the control.

Post-hatch (pullets)

During the post-hatch period, supplementation with probiotics was associated with higher live weights when compared to the control. For instance, the mean live weight of the chicks by 5th week was higher (p<0.05) in the IOIF (511.84 ± 17.22 g) and IF (509.43 ± 18.79 g) groups when compared with IO (452.21 ± 21.9 g) and EC (414.47 ± 20.1 g; Fig. 4). Similarly, breast weight increased in all the treatment groups compared to EC, IOIF increased breast weight by 9.2%, while with IF and IO, it was 4.4% (Fig. 5). The leg weight did not differ between the EC and IO. However, it was greater in IOIF and IF by 3% and 1.7% respectively compared to the control.

All the treatments resulted in an increase in the absolute mean liver weight by 0.01 – 0.05%, however, the treatments were not significantly different from EC. Crown-rump, radio-ulna and tibiotarsal lengths were greater (p<0.05) in IOIF compared to the EC (Fig. 6-7). All the length measurements were significantly lower in IF compared to the EC. IO did not vary with EC in crown-rump lengths, however, tibiotarsal and radio-ulnar lengths were higher (p<0.05) in IO compared to the EC. Furthermore, the tibial bone weight was significantly higher (p<0.05) in IOIF (5.016 ± 0.19 g) compared to the IF (4.902± 0.2 g), IO (4.515± 0.23 g) and EC (4.14 ± 0.28 g; Fig. 5). In this study, chicks with dietary probiotic supplementation (IF and IOIF) had higher body weight, RTC weight, breast weight and leg weight.

Serum cholesterol (Figure 8a) and triglyceride (Figure 8b) levels were estimated from week 5 samples to study the effect of probiotics. Total cholesterol level (95 µg/dL) was significantly (p<0.05) more in IOIF and no significant difference was observed in HDL levels among different groups. LDL lipid level in IOIF (50 µg/dL) was significantly (p<0.05) higher compared to IF (25 µg/dL). Serum triglyceride level was 1 mmol/L in IO and 0.6 mmol/L in IOIF and were significantly different (p<0.05) from each other and from EC and IF (1.6 – 1.8 mmol/L).

In the present study supplementation of probiotics was found to improve FCR (Table 3). It can be seen that FCR was significantly lower when probiotic treatment was applied either on egg surface in feed compared to the control group. The average FCR (week 5) was lowest in IF (4.3) and IOIF (4.4) followed by IO (4.8) and was highest in the EC (5.3).

Table 3: Feed conversion ratio of different treatment groups









































Research conclusions:

In conclusion, early in ovo probiotic administration enhanced the growth and development of the chicken embryo and hatchling and subsequently improved post-hatch growth in pullets. Specifically, the improvement in embryo and hatchling growth was associated with an improvement in post-hatch growth following in ovo probiotic supplementation (IO). This clearly demonstrates that improving embryonic growth can promote subsequent development in post-hatch birds.

Participation Summary

Education & Outreach Activities and Participation Summary

2 Consultations
2 Webinars / talks / presentations

Participation Summary

20 Farmers
2 Number of agricultural educator or service providers reached through education and outreach activities
Education/outreach description:
  1. The work was presented as “three-minute thesis” at “Our Farms, Our Future – 2018 National SARE Conference”. 3MT-presentation-SARE-03072018 ; (7:15′ – 11:00′)
  2. Poster presented at “Our Farms, Our Future – 2018 National SARE Conference”.Muyyarikkandy-Northeast_SARE_poster_Final
  3. Aviagen and Primalac companies were interested in the research and expressed willingness of collaboration.
  4. Contacted by farmers at “Our Farms, Our Future – 2018 National SARE Conference” and “2018 Annual Poultry Science Association Meeting” to learn about the scope of this research.

Project Outcomes

2 Farmers reporting change in knowledge, attitudes, skills and/or awareness
2 Grants applied for that built upon this project
Project outcomes:


The administration of sub-therapeutic doses of antibiotics as growth promoters in food animals including chickens can lead to antibiotic resistance in humans and animals. Further, the presence of antibiotic residues in poultry and meat products can also be deleterious to humans. This increased concern over antibiotic resistance led to the FDA directive that curbed the use of antibiotics in food animals. Therefore, the poultry industry is facing a great challenge to maintain production performance of birds due to increased feed costs and restricted antimicrobial use in feed. Traditionally probiotics have been administered in feed or water to day-old chicks to improve performance and market weight in broiler chicken. However, in modern broilers, embryonic and immediate post-hatch development period represents almost 50% of their productive life. Therefore, early in-ovo administration of probiotics would provide for an effective means to not only influence embryonic growth but also the post-hatch growth, performance, and health of chicken.

Knowledge Gained:

It was noticed that early in ovo probitoic supplementation along with dietary supplementation has an added advantage over either in ovo or in-feed supplementation alone. We also noticed that there was a significant improvement in bone growth of chicken with in ovo probiotic supplementation in addition to the muscle growth. 

Assessment of Project Approach and Areas of Further Study:
  1. The effect of early probiotic supplementation on poultry egg production should be conducted.
  2. In addition, it will be worth studying whether the beneficial effects of probiotic application is translating to their progenies.
Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.