An Approach to Building a Sustainable Small Flock Poultry Operation Through Improvement in Nutrition, Food safety, and Marketing

1. Establish specific dietary energy level that is optimal to calcium absorption for an egg type  breed in a free-range pastured operation

Treatment diets and husbandry practices

A commonly reared egg- type backyard breed, Isa-Brown Red, was utilized in the study. Two types of production setting was studied- one cage based study and another one was in pastured free-range setting. 

The study was conducted at the Poultry Research Unit, College of Agriculture, Tennessee State University, Tennessee. All experimental procedures for the study were performed following the guidelines set by the Local Experimental Animal Care Committee and received approval from the ethics committee of the College of Agriculture, TSU, Tennessee.

This study consisted of a total of thirty-six ISA brown laying hen strain at the late phase of production (~65 weeks) randomly allocated in 36 cages to one of three dietary treatments with low (T1), medium (T2), and high levels (T3) of energy (11.50, 12.75, and 13 MJ/kg, respectively). Treatment diets were formulated to be isonitrogenous and with the same inclusion level of calcium. Feed was made in the feed mill of the poultry research unit, TSU. Each treatment consisted of 6 replicates, considering two consecutive cages as a replicate (1 bird/cage). The experiment was conducted over a 6-week period.  The experiment design was a completely randomized design (CRD). All birds were each given 110 g feed per day. Water was provided ad libitum by a nipple drinker.

Birds were also reared in free-range in pasture with the same treatment diets (six replicate pen units- 5’ X 6’ pen size). The detail on the methods and results on this study component is underway, and will be presented in the next progress report. There were three replicate plots per treatment, with 9 birds per replicate at production phase (~ 30 sq. foot run/replicate). Pasture were rotated every 7 days. Forage coverage % were accounted for each rotation made. The experiment was evaluated for two weeks for each dietary energy level. 

Experimental diets (corn-soy based) consisted of three different energy levels (~135 kcal/lb difference). This experiment will also test organic soy-free diet (TBD). The dietary energy level of soy-free diet will be selected based on field practice. The major ingredient for soy-free diet will be corn and other minor ingredient will include wheat. Calcium levels and other nutrient profile of test diets will be kept constant (Table 1). All the ingredients used in test diets: corn, soy, and wheat, are the major crops grown in TN.

Table 1. An illustration of nutrient profile of experimental diets.

*The energy level chosen for treatment 1 is the recommended energy levels for a typical commercial egg-type breed. TBD: To Be Determined. TRT= Treatment. TRT 1-3: conventional corn-soy diet; TRT 4: Organic soy-free diet.

Other details on the methods for the variables measured for the cage-type setting is presented below:

Performance and egg quality materials

Birds were weighed at the beginning of the experiment (d0), and on d14, d28 and d42 of the experimental period. Feed intake was measured for each bird’s weight period. Similarly egg weight, egg production percentage were measured in daily basis. Feed intake and egg weight data were utilized to calculate the FCR for d0-d14, d15-d28, d29-d42, and d0-d42 for the experimental period.

On the last day of the experiment, eggs from each cage were collected and stored at 4ºC for subsequent measurements and later eggshell thickness was measured to the nearest 0.001 mm at the blunt and sharp ends and the equatorial region using a micrometer.

Blood Sample

Five millilitres of blood (with anticoagulant) from the wing vein of one randomly sampled bird per replication were obtained after oviposition, after 8 hours and again after 24 hours on day 42 of experiment. Plasma was separated by centrifugation of the blood at 1500 rpm for 15 min at 4C and stored at 56ºC until use. Calcium level in blood and the circadian dynamics of Ca- metabolism related hormones in blood (dihydroxy cholecalciferol (1,25 (OH)₂D₃), 17β-Estradiol-2, Parathyroid Hormone (PTH), calcitonin, and Parathyroid Hormone-Related Peptide (PTHrP)) will be measured.

Amino acid digestibility

A digestibility assay to understand apparent dietary ileal digestibility of amino acids was conducted on d42 of the experiment. The ileal digesta was collected by carefully excising the small intestine from Meckel’s diverticulum to 2 cm anterior to the ileo-cecal junction. The contents were gently flushed out with ice-cold saline, and the digesta from the one randomly selected bird per replication were pooled. Ileal digesta samples were frozen in liquid nitrogen and stored in a freezer at − 64◦C and later subjected to freeze drying before amino acid analysis. TiO2 was used as an indigestible marker at a rate of 0.5 % in the diets. TiO2 was thoroughly mixed into the diets using a standard feed mixer to ensure uniform distribution throughout the feed. The diet was provided in mash form to ensure a well-balanced intake, as mash prevents birds from separating out ingredients.

Tissue collection

Uterus, kidney and duodenum tissue samples were collected with RNA later from one bird per replication and stored in a freezer at − 64◦C until further analysis.

ELISA Analysis

The concentration of ovalbumin (OVA), ovotransferrin (OVO) were measured through commercial ELISA kit from the uterus, kidney and duodenum samples. The ovalbumin was measured using General Ovalbumin (OVA) ELISA kit (Catalog No.- MBS2000240, MyBioSource INC, San Diego, CA 92195) and the ovotranferrin were measured by Chicken Ovotransferrin (OT) ELISA kit (Catalog No.- MBS944289, MyBioSource INC, San Diego, CA 92195).

Quantitative PCR Analysis

Tissue samples were stored with RNA later for real-time PCR analysis, and total RNA was extracted from the uterine tissues, duodenum, and kidney using RNeasy Mini Kit (QIAGEN, Hilden, Germany) and then RNA concentrations were measured using a NanoDrop 2000c (Thermo Scientific, Waltham). The cDNA was synthesized using QuantiNova Reverse Trancription Kit (QIAGEN, Hilden, Germany).

Finally eggshell calcification related genes (calbindin d28k and carbonic anhydrase II (CA2)) for differential expression in the duodenum, kidney, and uterine tissue were measured. The β -actin gene was used as a housekeeping gene. The quantitative PCR (qPCR) was performed in triplicate with SYBR Green supermix (Bio-Rad, Hercules, CA, USA) in CFX Duet Real-Time PCR machine (Bio-Rad, Hercules, CA, USA). The qPCR conditions were as follows: 95ºC for 2 min and then 40 cycles of 95ºC for 5s, 60ºC for 30s, and 72ºC for 5s per step as melt curve. Each group had 3 replicates, and 3 samples were examined for each uterine, kidney and duodenal tissue.

Statistical Analysis:

All results were expressed as the mean standard error of the means (SEM). Data were analyzed by one-way analysis of variance (ANOVA) with the post hoc Duncan’s test using SAS software (SAS Institute, Inc., Cary, NC, USA). The level was considered up to statistically significant when P < 0.05.

Educational material output:

• Bird responses to dietary energy levels on performance
• Specific dietary energy level that is optimal to calcium absorption

2. Evaluate performance and egg quality differences for conventional and organic soy-free diets between farms

 The on-farm rearing of the birds will be performed by the three co-operating farmers (Susan Kyle, Ron Bridges, and Cynthia Capers) in their respective farms, with either conventional or organic based diets (Table 3). At least 50 birds will be utilized at each farm, and study will be conducted for the 12-week period at the same time as TSU research farm utilizing the same batch of birds. Treated and untreated drinking water supplies will be alternated weekly for these farms with co-operating farmers and at TSU research farm for Treatment 4 diet. Environmental friendly UV-based water treatment technology will be applied to  sanitize farm water supplies. Feed conversion values, daily egg production, egg weight, eggshell quality, and livability will calculated for conventional and organic based dietary approaches for each farm (of co-operating farmers) and will be compared with performance values obtained at TSU research farm. The fatty acids profile of eggs will also be measured between these two feeding approaches. The co-operating farmers have been informed  about their roles (research and education components) in the project. Furthermore, they have actively participated in planning of project and the farm trial. We have had opportunity to meet in-person through on-site farm visits (Rustic Farm and Heniscity Farm) and remotely via (multiple) zoom meetings.

Table 3: Dietary table for on-farm studies by co-operating farmers

Educational Output:

• Possible farm to farm variation in performance outputs for same dietary approaches
• Impacts of poultry water supplies treatment on performance (within farm comparison).

3. Assessing prevalence of Salmonella species, E. coli O157:H7 and Listeria monocytogenes under treated and untreated water supplies

Three farms under co-operating farmers and the TSU research farm (Treatment 4 replicates) will be subjected for this evaluation. During the 12-week experimental period, poultry drinking water samples, egg rinse samples and cloacal swab samples will be taken biweekly and assessed for the prevalence of foodborne pathogens (Salmonella spp., E. coli and Listeria). There will be total of six sampling occasions- three occasions will be utilized on the 7^th day during when water treatment is in place, whereas other three occasions will be during when water supplies are not treated.   Eggs (n= 15), and cloacal swabs (n=10 birds) and water supply source will be aseptically collected from each farm on each occasion. All samples will be transported in an insulated foam box with ice and processed in the laboratory within 24 h of collection or stored overnight at 4°C until processed the next day.

For this reporting cycle: 

Samples collected:

Egg Swabs

1. Layer Hens in Cages: 3 egg swabs per cage (18 cages) = 54 samples
2. Layer Hens in pasture: 6 egg swabs per cage (6 cages) = 36 samples

Procedure for microbial analysis

Each egg will be placed separately in a sterile bag containing 300 mL of buffered peptone water (BPW) and manually rinsed for 2 min, ensuring that all external parts have contact with the rinse. Sterile cotton swabs in 0.1% BPW will be used to take cloacal samples and thereafter will be immersed in 10 ml of 0.1% BPW and vortexed for 30 s. Farm drinking water (10 ml) and 90 mL of BPW will be added and pummeled in a stomacher 400 circulator at 230 rpm for 2 min. All homogenates will be evaluated for Salmonella spp, E. coli O157:H7, Listeria monocytogenes by using both traditional and biochemical methods.

Detection of Salmonella spp, E. coli O157:H7 and Listeria monocytogenes characterized according to (Liu and Kilonzo-Nthenge, 2017). Briefly, 0.1 ml of each pre-enriched sample will be added to 10 mL of Rappaport-Vassiliadis (RV), enterohemorrhagic E. coli enrichment broth, and Listeria enrichment broth base, respectively, and then incubated at 37°C for 24 h. Similarly, for each water sample, 1 ml will be added directly to 10 ml of each respective broth and incubated at 37°C for 24 h. Consequently, a loop (10 μl) of cultured broths will be streaked onto respective plates: Xylose-Lysine-Desoxycholate (XLD) selective agar (Oxoid, Basingstoke, Hants, England) for Salmonella spp, Sorbitol MacConkey agar (CM0813; Oxoid) supplemented with cefixime (50 ng/ ml) and potassium tellurite (25 mg/ml) supplement (SR0172E; Oxoid) for Escherichia coli O157:H7, and Listeria selective agar for Listeria monocytogenes. Plates will be incubated at 37°C for 24 to 48 h. Presumptive Salmonella spp colonies of red, yellow with black centers, colorless E. coli O157:H7, and brownish-black colonies Listeria monocytogenes presumptive colonies will be preserved in sterilized 80% glycerol at -80 ºC for confirmation with PCR.

Educational outputs:

• Understand the relationship of food pathogen prevalence that exist between environment (treated versus untreated water supplies), bird (cloaca) and farm produce (eggs)

4. Assess cost associated with production inputs and performance outputs in each conventional and organic based dietary approaches by preparing budget sheet and evaluate profitability (Dr. Lawani and co-operating farmers)

During the experimental phase, data will be collected to estimate production and marketing costs in each conventional and organic based dietary approaches for small flock production. The data will be used to generate sample budgets for each approach and analyzed to compare their profitability.

The budget will consider three time-horizons: raising of rearing phase (before egg production), production phase, and the marketing phase. In addition to the cost commonly associated with rearing chickens, some specific costs will vary and will be related to each time horizon. During the rearing phase, we will collect variable costs associated with the pullet diet. In the production phase, the cost of the experimental diets with the associated three different energy levels will also be evaluated in addition to the cost related to feed consumption and other operating costs. During the marketing phase, we will examine different scenarios. We assume that most of the production will be sold directly to consumers through farmers markets, roadside stands, or directly on the farm. We will also examine indirect marketing options where producers can sell for example as a group to retailers. The rationale behind separating the time horizon into three phases is that i) it allows us to identify the source of difference of performance between the conventional and organic based dietary approaches for small flock production, ii) it offers farmers the possibility to change/adapt their strategy to the market conditions (e.g., if after the day-old chicks raising farmers realize that the market price will not lead to a profitable enterprise, they can change and use the conventional approach instead of the organic based dietary one).

We will also conduct research on consumers’ willingness to pay for organic based dietary eggs. This research will help us estimate how much more consumers are willing to pay for eggs produced using this approach and their market share. We will use a choice experiment for this research. A choice experiment is a tool to elicit consumer utility and is widely applied in consumer studies. Choice experiment allows researchers to uncover how consumer value attributes of a product by asking them to state their choice over different alternatives. The stated choices are econometrically estimated and transformed into utility.  This research will be conducted using a representative sample of U.S. consumers through Qualtrics.

The results will be published in extension outlets. Sample budgets will also be developed in Excel and made accessible to farmers. Farmers will also be trained on using the budgets and the marketing strategies for profitable small flock poultry operation.

Education outputs:

• Hands-on on budget sheets, and product pricing, and marketing options 

PS: The flow diagram for bird trial and activities involved are outlined herewith. Farm trial_ outline

LOS_ Research Dean_SSARE Research and Education

5. Educate small flock producers, county agents, and poultry stakeholders on basics of feeding, food safety practices, and farm economics for sustainable poultry operation 

Detailed out in the Outreach Plan.