An ethics committee “Comité d’Ethique en Expérimentation Animale Val de Loire » (CEEA VdL N°19) protocol registered approved all experimental studies, which were in accordance with the French National Guidelines for the care and use of animals for research purposes (certificate of authorisation to experiment on living animals APAFIS number 10237-201706151202940v3).
Three hundred and twenty-four broiler breeder females chicks from Hendrix Genetics (Saint Laurent de la Plaine, France) were studied from day 1 to 40 weeks of age. Animals were divided in homogeneous groups of 10 to11 birds in 32 pens, each pen with an area of 3 m2. The animals were reared at “Pôle Expérimental Avicole de Tours” (INRA, Nouzilly, France) according to the conventional conditions of breeding: 24 h of light on arrival, day length being reduced to approximately 8 h at two days of age, then kept constant until the age of photostimulation (21st week). From 21 weeks of age, there was a gradual increase in exposure to light up to 15 h per day at 25 weeks. Animals were maintained under this light regime until the end of the experiment and then they were killed by electrical tunning and bled out as recommended by the ethical committee.
From one to the fourth week of age, female breeder chicks received an ad libitum diet (free access to food), called a starting diet. We used two types of diets: control and supplemented with 1% of GSE. At week 4, all animals received a restricted diet according to Hendrix Genetics recommendation. Animals from the control group were segregated in 3 groups: control, supplemented with 0.5% and supplemented with 1% of GSE. The resulting four groups were: group A (control, n = 92), group B (supplemented with 0.5% of GSE since week 4, n = 80), group C (supplemented with 1% of GSE since week 4, n = 80) and group D (supplemented with 1% of GSE since the hatch, n = 72). From 4 week to 40 week, animals received three different diets: growing (from 4 to 18 week), before laying (from 18 week to 21 week) and laying (from 21 week to 40 week). Remaining feed was weighed. The composition of GSE used for this study was analysed by HPLC (High Performance Liquid Chromatography). The most important component was the procyanidins (> 90%). The supplement was manually mixed with the diet at the proper concentration of the total diet. The composition of the diets are shown in the additional file 1 (Table S1) and a schema of the experimental design is showed in additional file 2 (Figure S1)
Measurement of egg production and quality
From the 24th week, the eggs from each pen were collected twice a day, counted and weighted using a balance (Ohauss, Pionner) The numbers of normal and double (eggs with tw egg yolks) eggs were collected and counted. The weight of the albumen, the egg yolk and the dehydrated shell were determined. At the beginning of the laying period (26th week), the width and the length of the eggs laid for each group of animals were measured using a digital calliper (Mitutoyo, CD-20DCX). Using an Instron instrument (Instron, UK527), we analysed the static stiffness (Sd, N/mm), the tensile strength (F), the elastic modulus (Eshell, N/mm2), the toughness (Kc, N/mm3) of the eggs. The weight and the thickness of the shell were also measured. These parameters were assessed for 30 eggs of each group of animals.
Determination of weight and number of follicles
At 40 weeks old, 10 hens per group were selected and the preovulatory follicles (from 1 to 6) were collected, hierarchically defined and weighed. Granulosa cells and theca cells were collected from the F1.
For artificial insemination, we used the semen of 48 cocks (Cobb500), collected and pooled to form a single sample. The hens were inseminated with 2 × 108 spermatozoa, at 28th and 33rd week. Eggs were collected and counted daily for 3 weeks following the artificial insemination and incubated every seven days. We assessed the number of unfertilised eggs, early (EEM) and late (LEM) embryonic mortality by breaking eggs and candling on the 7th (EEM) and 14th (LEM) day of incubation. The different percentages (EEM, LEM, hatchability, hatchability of fertile eggs and fertility) were calculated using the following formulae:
% EEM = number of EEM / (number of incubated eggs – unfertilised eggs) * 100
% LEM = number of LEM / (number of incubated eggs – unfertilised eggs + number of EEM) * 100
% Hatchability set = (number of hatched chicks / number of incubated eggs) * 100
% Hatchability of fertile eggs = (number of hatched chicks / number of fertile eggs after 14 days of incubation) * 100
Measurement of progesterone, androstenedione, testosterone and oestradiol deposition in egg yolk
We assessed steroids concentration from 30 egg yolks per group. Steroids were extracted with diethyl ether after intense agitation and centrifugation. The steroid-containing diethyl ether phase was decanted after freezing the tubes in nitrogen for 10 s. The organic solvents were then evaporated and the extracts taken up in phosphate buffer. Steroid hormones (progesterone, oestradiol, testosterone and androstenedione) were then measured in the extracts using ELISA assays. For progesterone, the ELISA assay was performed as described by (31). The sensitivity of the assay was 0.4 ng/mL. Oestradiol and testosterone concentrations were determined using commercial ELISA assays from Cayman Chemicals and the sensitivity of these assays was 0.01 ng/mL. Androstenedione levels were analysed using an ELISA assay from Abcam and the sensitivity of the assay was 0.01 ng/mL. The intra-assay and inter-assay coefficients of variation (CV) for each assay averaged < 10%.
Measurements of the expression of steroidogenic genes in granulosa cells
Total RNA from granulosa cells of preovulatory follicle 1 of ten animals per group was extracted by homogeneisation in the TRIzol tissue reagent using an ULTRATURAX instrument, according to the manufacturer’s recommendations (Invitrogen, by Life Technologies, Villebon sur Yvette, France). The cDNA was generated by reverse transcription of total RNA (2 µg) in a mixture comprising 0.5 mM of each deoxyribonucleotide triphosphate (dATP, dTTP, dGTP, dCTP), 2 M of RT Buffer, 15 µg/µL of oligodT, 0.125 U of ribonuclease inhibitor, and 0.05 U of Moloney murine leukemia virus reverse transcriptase (MMLV) for one hour at 37 °C. Real-time PCR was performed using the MyiQ Cycle Device (Bio-Rad, Marnes-la-Coquette, France), in a mixture with SYBR Green Supermix 1X Reagent (Bio-Rad, Marnes-la-Coquette, France), 250 nM specific primers (Invitrogen by Life Technologies, Villebon-sur-Yvette, France) and 3 µL of cDNA diluted 1:5 in water) for a total volume of 11 µL. The samples were set up in duplicate on the same plate according to the following procedure: after an incubation of 2 min at 50 °C and a denaturation step of 10 min at 95 °C, samples were subjected to 40 PCR cycles (30 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C). The primers used are shown in the additional file 3 (Table S2). For each gene, the relative abundance of transcription was determined by calculating e− ct. Then, the relative expression of the gene of interest was related to the relative expression of the geometric mean of the three reference genes (GAPDH, ACTB and EIF3).
In vitro culture of hen granulosa cells
Granulosa cells from preovulatory follicles 1 (F1) of hens fed with GSE dietary supplementation or with a control diet were collected from 7 animals for each group. Then, cells were dispersed in 0.3% collagenase type A (Roche Diagnostic, Meylan, France) in F12 medium containing 5% foetal bovine serum (FBS), at 37 °C. Cells were pelleted by centrifugation, washed twice with fresh medium and counted in a haemocytometer. The viability of granulosa cells was estimated by Trypan Blue exclusion. Cells were cultured in a medium composed of DMEM supplemented with 100 U/mL penicillin, 100 mg/L streptomycin, 3 mmol/L L-glutamine and 5% FBS. The cells were initially cultured for 24 h with no treatment. After overnight serum deprivation, cells collected from hens Rhode Island were stimulated with GSE (0.01, 0.1, 1, 50 and 100 µg/mL) or left untreated for 48 h. Cells collected from hens fed with GSE supplementation were stimulated with IGF1 (10− 8M), LH (10− 8M), IGF1 + LH (10− 8M) or left untreated. All cultures were maintained under a water-saturated atmosphere of 95% air/ 5% CO2 at 37 °C.
In vitro measurement of progesterone secretion by granulosa cells
The concentration of progesterone secreted into the medium by granulosa cells under various conditions was determined according to an ELISA protocol described by (31). The sensitivity of the kit was 0.4 ng/mL. The intra- and inter-assay coefficients of variation were < 10% and < 4.3% respectively. This experiment was carried out using four replicates of three hens for each group.
Plasma concentration of three adipokines were determined using ELISA assays. Chicken-specific kits, MBS269004 (sensitivity 5 pg/mL), MBS016609 (sensitivity 0.1 µg/mL) and MBS738819 (sensitivity 0.1 ng/mL), were used for NAMPT, ADIPOQ and RARRES2 respectively (My BioSource). The experiment was performed following the manufacturer’s protocol with an intra-assay coefficient of variation ≤ 8%, < 15% and < 5.6 respectively. The absorbance was measured at 450 nm and then compared with reference values.
Measurement of ROS (hydrogen peroxide (H₂O₂) in yolk
The content of ROS, hydrogen peroxide (H₂O₂) from 10 egg yolks per group were measured by Ros-Glo H2O2 assay (Promega Corporation, Charbonnières-les-Bains, France). In each analysis, one gram of each egg yolk for each group was precisely analysed following the manufacturer’s protocol.
Data are represented as mean ± s.e.m., with a level of significance less than 0.05 (*P < 0.05). An analysis of variance using repeated measurements (Proc.Mix procedure) was used to compare the average numbers of normal eggs among the different hen groups over time. An analysis of variance (Proc.GLM procedure) was used to compare the number of double-yolk eggs, the weight of follicles, the content of ROS, the average concentrations of secreted steroids and androgen and levels of expression of adipokines and their receptors among the different groups. Two factors were analysed: the time of GSE supplementation (from birth (one day of age, D group) compared from 28 days (week 4, B and C groups)) and a diet effect. If the time of GSE supplementation was significant, we analysed separately the diets A and D, and A,B and C and if not we compared the whole A,B,C and D groups. A chi-square test was used for analysis of percentage fertility between the different parameters. A Pearson test was used to analyse correlations between ROS content and steroid in yolk egg and plasma adipokine concentration. The correlation was noted ‘r’ and the P value was considered significant if P < 0.05. SAS software (version 9.3, Cary, USA) was used for all analyses. Different letters indicate significant differences (P < 0.05).