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Supplemental effect of Pediococcus acidilactici CNCM I-4622 probiotic on the laying characteristics and calcium and phosphorus metabolism in laying hens – Scientific Reports

Animal care

The animal care and experimental procedures described in this experiment were conducted according to the Animal Welfare Committee guidelines and received the approval of ethics committee of Animal Resource and Science College and Dankook University (DK-1-2040, Cheonan, South Korea). The experiments were performed in accordance with ARRIVE guidelines (

Source of additive

The probiotic “Pediococcus acidilactici CNCM I-4622” (Bactocell®; PA) was produced and commercialized by Lallemand SAS (Blagnac, France) and guaranteed to contain a minimum of 1 × 1010 cfu viable bacterial cells/g.

Birds and husbandry management

480 Hy-Line brown laying hens (50 weeks old) of approximately similar body weight (BW) were assigned to two dietary treatment groups in a 14-week trial. The hens were fostered in large double-sided four-tier battery cages with 20 replicates for each treatment (1 bird/cage (45 × 35 × 37 cm)), with 3 adjacent cages representing a replicate. During the experimental period, birds received feed and water ad libitum, with 16 h of daily lighting. The temperature and relative humidity of the air inside the fostering room was maintained at minimum and maximum of 26 °C and 30 °C respectively, and 60% of humidity was maintained until the end of the trial. Hens were allotted into replicates of 3 hens, i.e., 80 replicates/group and 240 birds per group. Feed intake was individually measured, while egg record datasheet reported the laying performance for each replicate of 3 hens. A wire egg collection tray was fixed in front of all cages to avoid eggs from being mixed between replicates.

Experimental design and animals

Laying hens were randomly allocated into 1 of 2 dietary treatment groups CON—Control (Corn-soybean meal based basal diet) and PA-basal diet supplemented with PA probiotic (2 × 1012 CFU Pediococcus acidilactici CNCM I-4622/ton of feed during the first 3 weeks of the trial and 1 × 1012 CFU PA/ton of feed for weeks 4–14 inclusively). The trial lasted 15 weeks, with one week of adaptation (week 0) and 14 weeks of supplementation. The ingredients and the chemical composition of the experimental diets are shown in Table 1. Basal diets (mash form) were formulated according to NRC11 recommendation.

Table 1 Composition of laying hen diets (as fed-basis).

Sampling and analysis

Laying performance

The percentage of downgraded eggs (dirty, broken, soft, small eggs) (%) and the total and marketable laying rate (%) were recorded per replicate of 3 hens at each day of the trial, then averaged per week. The marketable laying rate was calculated as follows: % total laying rate—% downgraded eggs and the number of marketable eggs as total number of eggs—number of downgraded eggs. The cumulated weight of marketable eggs and downgraded eggs was recorded per replicate of 3 hens at each day of the trial, then averaged per week. The average egg weight was determined by dividing the weight of the collected eggs by the number of eggs laid per replicate. The exported egg mass (all eggs and marketable eggs) was calculated as follows: laying rate (%) × average egg weight. The feed intake was recorded individually weekly and calculated as follows: total of distributed feed each day—refusals at the end of each week, and then averaged per replicate of 3 hens. The feed conversion ratio (FCR) was calculated at every week (1 to 14) for all eggs and for marketable eggs, based on feed intake and egg production data with the following calculation: feed intake/(nb of eggs × average egg weight).

Egg quality traits

All the eggs produced on the last day of each week were individually weighed and graded (Ministry of Agriculture, Food and Rural Affairs). The 4 categories recorded for egg size were extra-large (> 68 g), large (61 to 67 g), medium (53 to 60 g), and small (44–52 g) previously described by Mikulski et al.10. Then, eggs having medium weight were randomly collected to analyze egg quality parameters. The egg quality traits such as eggshell color, yolk color, eggshell strength, eggshell thickness, the relative weight of egg compartments (yolk, albumen, eggshell), albumen height and Haugh units (HU) were evaluated at the end of weeks 1, 3, 5, 7, 9, 11, and 13 (2-week intervals) on 30 eggs per treatment for each time point. The eggs were randomly collected (30 eggs/treatment) from each group at 5:00 pm, weighed individually using digital precision scale (0.01 g) and brought to laboratory for egg quality analysis. In brief, first, the eggshell color was checked using eggshell color fan (Daeho Co. Ltd). Second, the egg weight, eggshell strength, albumen height, yolk color, Haugh Unit (HU) were measured using fully automatic DET 6000 egg tester (Kyoto, Japan) and noted for statistical analysis. The yolk was then separated from the albumen using a Teflon spoon and placed on the blotting paper towel to remove adhering albumen. Then, the yolk and albumen were stored in clean paper cup. The yolk, albumen and eggshell weights were measured separately using digital Sartorius BCA2201i—1S weigh scale (Gangnam, Korea) and expressed as a percentage of the weight of whole egg (%). To determine eggshell weight, the eggshells were cleaned, and the inner membrane was removed. Then the eggshells were allowed to dry at room temperature. Later, the eggshell thickness was measured at 3 points (rounded end (top), pointed end (bottom), and the middle) using digital micrometer gauge (Baxlo Instrument, Spain) and the mean value was taken as thickness. Above stated internal and external egg quality analysis were done within 24 h of egg collection.

Intestinal morphology

10 hens per group and per time point were humanely killed by cervical dislocation at the beginning and end of the experiment for small intestinal morphology analysis in 3 intestinal sections (duodenum, jejunum, ileum), after staining for microscopic observation (LEICA ICC50 E, LEICA, Germany) and imaging. The following parameters were recorded: villus height (VH), crypt depth (CD) and the ratio VH/CD.

Nutrient digestibility

Chromic oxide (0.3%) as an indigestible marker was added to the diet of layers at the end of week 13 and provided for about one week until the end of the experiment to measure the nutrient digestibility. Representative feed samples were collected using sterilized plastic bags from each treatment group right after mixing the marker. At the end of week 14, approximately 50 g fresh excreta samples were collected from 20 birds/treatment (1bird/cage) using stainless steel collection tray. Then excreta samples were brought to the laboratory within 30 min and stored at -20℃ to examine the apparent total tract nutrient digestibility (ATTD) of dry matter (DM), nitrogen (N), energy (GE), fat, calcium, and phosphorus. Prior to analysis, all feed and feces samples were placed in a hot air-drying oven for 24 h at 105 °C. Later the samples were grounded to pass 1 mm screen sieve mesh. DM, N, E and fat digestibility was carried out according to the procedure of AOAC12. However, ATTD of Ca and P retention digestibility were analyzed as described by Luh Huang and Schulte13. The chromium absorption was identified using UV-1201 spectrophotometry. GE was analyzed using Parr 6400 oxygen bomb calorimeter (Parr Instrument Co., Moline, IL, USA) and N was analyzed using TecatorTM Kjeltec8400 analyzer (Hoeganaes, Sweden). The content of calcium and phosphorus in feed sample was determined by the optical emission spectrometry with excitation in the inductively coupled argon plasma in the Optima 2,000 DV camera. The apparent total tract digestibility was calculated using: ATTD (%) = 100 − [(NF/ND) × (CrD/CrF)] × 100], where NF, ND, CrD, and CrF were referred as nutrient concentration in the excreta sample (feces, F), nutrient concentration in the diet (D), chromium concentration in the diet (D) and chromium concentration in the excreta sample (feces, F), respectively.

Blood biomarkers

At the beginning and at the end of the experiment, 20 birds were randomly selected from each treatment and blood samples were collected from the brachial vein with sterile K3EDTA vacuum tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and stored at 4 °C. For serum analysis, approximately 3 mL blood samples were centrifuged at 4000×g for 15 min at 4 °C, and serum calcitriol was assessed using enzyme-linked immunosorbent assay kits (Calcitriol ELISA Kit (OKEH02542), Aviva Systems Biology Corporation, UAS) following the manufacturer’s protocol. Serum concentrations of osteocalcin (OC), parathyroid hormone (PTH), and alkaline phosphatase (ALP) were determined using commercial kits (Immutopics, Inc., San Clemente, CA), the immunoradiometric assay method, and a gamma counter (BioSource International, Camarillo, CA), respectively. The blood minerals (Ca and P) were determined using the commercial kits (MyBioSource, San Diego, CA; BioAssay Systems, Hayward, CA).

Bone parameters

At the beginning and at the end of the trial, 20 birds from each treatment were randomly selected to analyze the bone parameters such as hardness, cohesiveness, and bone calcium and phosphorous contents. After thawing the tibias for at least 24 h at 15 °C, they were broken with the Three-Point Bending Test of Animal Bone following the ASABE Standards 2007 [ANSI/ASAE S459 MAR1992 (R2007)] using a Zwick and Roell universal testing machine with a 2.5 kN load cell. The fulcrum was adjusted at 55 mm to get the requested length to bone diameter ratio (greater than 10). The smallest diameter of the tibias was measured. The bones were laid in the test apparatus with the flattest side down, and the force was applied to the midshaft with a crosshead speed of the loading bar of 10 mm/min. The force–deformation curve was read from the texture analyzer. From this curve, the ultimate force required to break a bone was recorded in Newton. To take bone size into account, the value of the ultimate force was divided by the cross-section of the bone (A), which was calculated as the product of π and the square of the radius of the thinnest part of the bone (using the small diameter, see ANSI/ASAE 8.2). This (F/2 × A) was taken as an approximation of the ultimate shear strength. Bones were too thin to measure the outer and inner diameters to get a more exact measurement of ultimate shear strength. Bending strength (force applied when the bone fractures) was automatically derived from the slope of the load/ displacement graph13, and the slope of the load-deformation curve, which is an estimate for the bone stiffness, was derived by the regression between 0.3 and 0.5 mm.

Gene expression analysis in the small intestine, ovarian tissue, bone and blood

At the end of the experiment, 10 birds/treatment (randomly selected) were slaughtered (by cutting carotid artery) and samples of bones, small intestine and ovarian tissue were isolated from individual hens. The tissue samples were stored in a − 80 °C deep freezer immediately after collection. A portion of each bone, small intestine, ovarian tissue and blood was homogenized in 500 µl of TRIzol reagent (Invitrogen, Carlsbad, CA) using a taco™Prep homogenizer (GeneReach Biotechnology Corp., Taiwan). Total RNA was isolated from the tissue homogenates of each sample according to the manufacturer’s instructions. Immediately after RNA extraction, RNA quality and quantity were determined spectrophotometrically using an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE) and were assessed using an Automated Electrophoresis, TapeStation system (Agilent, Santa Clara, CA). The purity of the RNA was determined from the ratio of the absorbance at 260 nm to that at 280 nm (A260/A280). 10 hens were sampled per group, but some samples did not have a high concentration of RNA. So, qRT-PCR was performed with only RNA samples having a concentration that could be used for the experiment, the number of samples actually used was then smaller than the number of samples collected: n = 5 for bone gene expression and n = 3 for blood gene expression (vs n = 10 for small intestine and ovarian gene expression). Each sample was analyzed in triplicate. Small intestine samples were collected from duodenum, jejunum or ileum, but as the RNA concentration was not high enough in some samples, the small intestine samples from each intestinal section were combined and qRT-PCR was performed with only RNA samples at the concentration usable for the analysis. The following procedure was applied for mRNA expression analysis:

cDNA synthesis and RT-qPCR validation

cDNA synthesis on 500 ng of RNAs was performed to validate treatment-dependent gene expression changes using the ReverTra Ace qPCR RT Master Mix (Toyobo; Osaka, Japan). The RT master mix contains reverse transcriptase, RNase inhibitor, oligo dT primer, random primer, MgCl2, and dNTPs. The cDNA synthesis reactions were carried out for 15 min incubation at 37 °C followed by incubation at 50 °C for 5 min, and 98 °C for 5 min using a thermocycler (Bio-Rad Laboratories; Berkeley CA, USA) at the Center for Biomedical Engineering Core Facility (Dankook University, South Korea). As a housekeeping gene, a primer pair of glycerol aldehyde-3-phosphate dehydrogenase (GAPDH) was designed in the Gallus gallus GAPDH sequences of Ensembl (ENSGALG00000014442) and the synthesized cDNA was verified. A positive RT-qPCR reaction was detected by fluorescent signal accumulation, with cycle threshold (CT: the number of cycles required for the fluorescent signal to cross a fixed threshold) being inversely proportional to the amount of the target nucleic acid in a sample, using CFX96 Real-Time PCR System (Bio-Rad Laboratories; Berkeley CA, USA). The GAPDH as an endogenous control (housekeeping gene) was used to normalize the expression level of the gene of interest. Validation of gene expression changes using the comparative 2-ddCT method for mRNA quantification showed CT values for each sample. For each cDNA sample, RT-qPCR validation was performed in triplicates. For statistical analyses, max Ct values were fixed at 35. Delta Ct was calculated by the difference between the Ct of the gene of interest (for a given sample/replicate) and the Ct of GAPDH gene (for a given sample/replicate). Delta Ct was then averaged per sample (n = 3 replicates). For each sample, delta delta Ct were calculated by the difference between delta Ct of the 2 groups, and 2-DDCT (fold change) was then calculated. Gene specific primer sequences are presented in Table 2. Eight genes (mRNA expression analysis) were analyzed in the intestinal tissue: CALB1 (Calbindin 1 Protein Coding gene), ATP2B1 (ATPase Plasma Membrane Ca2+ Transporting 1 Protein Coding gene), SLC34A2 (Solute Carrier Family 34 Member 2 Protein Coding gene) and occludin, claudin-1, claudin-5, ZO-1, ZO-2 (genes involved in tight junction proteins). Three genes (mRNA expression analysis) were analyzed in the ovarian tissue: CALB1 (Calbindin 1 Protein Coding gene), ATP2B1 (ATPase Plasma Membrane Ca2+ Transporting 1 Protein Coding gene) and SPP1 (Secreted Phosphoprotein 1 Protein Coding gene). Five genes (mRNA expression analysis) were analyzed in the bone tissue: GUSB1 (Glucuronidase Beta Protein coding gene), SLC34A2 (Solute Carrier Family 34 Member 2 Protein Coding gene), CYP24A1 (Cytochrome P450 Family 24 Subfamily A Member 1 Protein Coding gene), VDR (Vitamin D Receptor Protein Coding gene) and ACTB (Actin Beta Protein Coding gene). Two genes (mRNA expression analysis) were analyzed in the blood tissue: CALB1 (Calbindin 1 Protein Coding gene) and FGF23 (Fibroblast Growth Factor 23 Protein Coding gene). CALB1 was therefore analyzed in the small intestine, the blood and the ovarian tissue. ATP2B1 was analyzed in the small intestine and the ovarian tissue. SLC34A2 was analyzed in the bone tissue and the small intestine. CALB, CYP24A1 and VDR are Ca homeostasis-related genes modulated by vitamin D metabolism. ATP2B21 (plasma membrane Ca2+-transporting ATPase I) and SPP1 (osteopontin) are genes involved in ossification and in osteocytes/osteoblasts differentiation. SLC34A2 (solute carrier family 34 member 2), GUSB1 (glucuronidase beta 1), ACTB (actin B) and FGF23 (fibrobast growth factor) are genes involved in hormone and growth metabolism. Occludin, claudin-1, claudin-5, ZO-1 and ZO-2 are genes involved in tight junction proteins.

Table 2 Gene specific primer sequences.

Statistical analysis

Laying performance (total laying rate, marketable laying rate, % downgraded eggs/total eggs, total egg weight, weight of marketable eggs, exported egg mass, feed intake and FCR) and egg quality parameters were analyzed by a mixed model with repeated measures where the group, the week and their interaction were set as fixed effects and the laying hen or the replicate, depending on the parameters, as random factor. Nutrient digestibility, parameters of intestinal morphology, bone parameters and blood biomarkers were analyzed by a mixed model applied to the final time with the group as fixed effect and the laying hen or the replicate, depending on the parameters, as random factor. Owing to the low number of replicates for gene expression data, they were analyzed by the non-parametric Mann–Whitney test at each time point (initial/final). Significance level was set at 5% (P ≤ 0.05) and statistical trends were reported at 10% (P ≤ 0.1). Graphics present means and SD per group for each time point. Tables present the estimated marginal means and SEM provided by the mixed model. Statistical analyses were performed using IBM SPSS 26.0 and figures were prepared with Excel.