Effects of the in ovo injection of vitamin D₃ and 25-hydroxyvitamin D₃ in Ross 708 broilers subsequently fed commercial or calcium and phosphorus-restricted diets. II. Immunity and small intestine morphology^1,2,3

Abstract

Effects of the in ovo injection of vitamin D₃ (D₃) and 25-hydroxyvitamin D₃ (25OHD₃) on the immunity and small intestine morphology of broilers fed calcium and phosphorus-restricted diets were investigated. At 18 d of incubation (doi), live embryonated Ross 708 broiler hatching eggs were in ovo-injected with a 50 μL solution of one of the following treatments using an Inovoject multiegg injector: 1) diluent (control); diluent containing either 2) 2.4 μg D₃; 3) 2.4 μg 25OHD₃; or 4) 2.4 μg D₃ + 2.4 μg 25OHD₃. At hatch, 18 randomly selected male broilers belonging to one of the 4 in ovo injection treatments were placed in each of 12 floor pens and were fed either a commercial diet or a diet restricted by 20% in calcium and available phosphorus (ReCaP) content for the starter, grower and finisher dietary phases. Concentrations of plasma IgG and IgM at 14 d of age (doa) and α-1-acid glycoprotein at 40 doa were determined. Bursa, liver, spleen, duodenum, jejunum, and ileum weights were recorded at 7, 14, and 40 doa and small intestine histology was evaluated at 14 and 40 doa. Blood and organ samples were randomly collected from 1 bird in each of the 6 replicate pens within each of the 8 (4 in ovo x 2 dietary) treatment groups. Plasma IgG levels were higher in 25OHD₃ than in diluent or D₃ in ovo-injected birds. At 14 doa, a higher jejunal villus length (VL) to crypt depth (CD) ratio (RVC) was observed in birds that were in ovo-injected with 25OHD₃ alone as compared to all other in ovo injection treatments. At 40 doa, ileal VL increased and jejunal CD decreased in commercial diet-fed birds compared to ReCaP diet-fed birds. In conclusion, the in ovo injection of 25OHD₃ alone increased the immune response and improved the small intestine morphology and subsequent nutrient uptake of Ross 708 broilers. However, a ReCaP diet was observed to be detrimental to their small intestine morphology.

INTRODUCTION

The in ovo injection of broiler hatching eggs is widely used in the poultry industry. In comparison to the traditional method of broiler vaccination, in ovo injection has been shown to be less stressful, and is faster and provides uniform delivery of vaccines or nutrients to broiler embryos (Williams, 2007). “The poultry industry commercially uses in ovo vaccination against various diseases including Marek`s disease (Williams, 2007), while the in ovo injection of nutrients is not yet applied commercially. Rapid development of the small intestine takes place at 15 d of incubation (doi) and includes changes in relative intestinal weight and villi morphology. Furthermore, at that time, the expression and activity of brush-border enzymes and transporters prepare the embryo for exogenous feed ingestion (Uni et al., 2003). It is well documented that an earlier improvement in small intestine development has a positive long-term effect on broiler performance (Uni et al., 1999). The in ovo injection of mannan oligosaccharides has been shown to increase villus length (VL) and villus area (Cheled-Shoval et al., 2011). Improvements in small intestine morphology are associated with improvements in growth performance characteristics such as BW and feed conversion ratio (FCR) under commercial conditions (Yang et al., 2007a,b), as well as during a coccidiosis challenge (Dalloul et al., 2005). In addition, in ovo injection of different nutrients such as CpG oligodeoxynucleotides, vitamins C and E, and plant extracts have the potential to enhance immunity (Gore and Qureshi, 1997; Dalloul et al., 2005; Saki and Salary, 2015; El-Senousey et al. 2018).

Vitamin D₃ (D₃) is a fat soluble vitamin and its absorption in the upper region of the small intestine is facilitated by bile salts. After intestinal absorption, D₃ is transported to the liver where it is converted by 25-hydroxylase to the second metabolite of D₃, 25-hydroxyvitamin D₃ (25OHD₃). Subsequently, in renal cells, 25OHD₃ is converted by 1α-hydroxylase to the most active form of D₃, 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂-D₃; Henry, 1980). Vitamin D is crucial for proper embryo development (Narbaitz, 1987), and is involved in calcium (Ca) and phosphorous absorption (Bar et al, 1980), immunity (Adams and Hewison, 2008), and intestinal development (Chou et al., 2009) in chickens. When included at the same dietary level, 25OHD₃ is approximately twice as active as D₃ in promoting Ca absorption (Myrtle and Norman, 1971), and has a higher retention in chicks compared to D₃ (93% and 80% respectively; Bar et al., 1980). Inclusion of 25OHD₃ in broiler breeder diets has been shown to increase VL in the duodenum of broiler embryos at 19 doi and during the first 2 d of posthatch age (doa) (Ding et al, 2011). Additionally, supplemental dietary 25OHD₃ at 2,760 IU/ Kg has been shown to increase VL and decrease crypt depth (CD) in broiler chickens (Chou et al., 2009). An increase in VL is linked to increased nutrient absorption (Onderci et al., 2006) and decreased CD is associated with less frequent epithelial cell turnover, leading to a lower energy requirement in the gut (Yang et al., 2008). Maternal 25OHD₃ has also been shown to increase the innate immunity of chicks relative to D₃ (Saunders-Blades and Korver, 2015). Additionally, dietary supplementation of 25OHD₃ has been shown to increase humoral immunity in early posthatch broiler chickens (Chou et al., 2009). Rodriguez-Lecompte et al. (2016) reported that the immunomodulatory function of vitamin D is associated with the levels of Ca and phosphorous in the diet.

Effects of the in ovo injection of D₃ and 25OHD₃ on the small intestine morphology and immunity of broilers have not been previously investigated. It was hypothesized that the vitamin D₃ sources utilized in this study would positively influence the immune competency and small intestine morphology of the broilers, especially in those fed Ca and available phosphorous (aP)-restricted diets. Therefore, the objectives of this study were to determine the effects of the in ovo injection of D₃ and 25OHD₃ on small intestine morphology, humoral immunity, and the inflammatory response of broilers fed either commercial diets that were and were not restricted in Ca and aP content by 20%.

MATERIALS AND METHODS

Experimental Design and Treatments

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Mississippi State University (Protocol #IACUC-17-406). Fertile broiler hatching eggs laid by 35 wk-old Ross 708 breeder hens were obtained from a commercial source and stored for 24 h. Prior to set, eggs were selected as described by Sokale et al. (2017) and a total of 2,880 eggs (average weight = 59.8 ± 0.81) were subsequently set and incubated in a Jamesway model PS 500 setter unit (Jamesway Incubator Company Inc., Cambridge, Ontario, Canada) under standard conditions (37.5° C and 29.4° C dry and wet bulb temperatures, respectively) as is described by Fatemi et al (2021b). Thirty eggs was assigned to each of 4 preassigned treatment groups on each of 24 incubator tray levels (blocks). Treatment groups on each tray were randomly placed to avoid positional effects, and monitoring of incubational conditions were as described by Fatemi et al (2020a, b). In ovo injection treatments were prepared according to the procedures of Fatemi et al. (2020a, b), and injections were administrated at 18 doi using a Zoetis Inovoject m in ovo injection machine (Zoetis Animal Health, Research Triangle Park, NC). At injection day, 8 eggs on each of the 12 tray levels were injected with Coomassie Brilliant Blue G-250 (colloidal) dye for determination of site of injection as described by Sokale et al. (2017). The sites of injections in this study were confirmed to be 97.92 and 2.7% in the amnion, and embryo body respectively. Williams (2007, 2011) has reported that amniotic and body proper injections are efficacious. In ovo injection treatments were: 1) diluent (control; 50 μL of commercial diluent [commercial Marek's Disease vaccine diluent; Merial Co., Duluth, GA]); 2) D₃ (50 μL of commercial diluent containing 2.4 μg D₃), 3) 25OHD₃ (50 μL of commercial diluent containing 2.4 μg 25OHD₃), and D₃ + 25OHD₃ (50 μL of commercial diluent containing 2.4 μg D₃ and 2.4 μg 25OHD₃). For the posthatch period, 18 randomly selected male chicks belonging to 1 of the 4 in ovo injection treatments were placed in each of 12 floor pens and were subjected to 1 of 2 dietary treatments. Therefore, 8 treatment groups (4 in ovo injection x 2 dietary treatments) were randomly represented in each of 6 replicate blocks of pens (48 total pens) in the grow-out facility. Bird housing and husbandry during the posthatch period were as described by Fatemi et al. (2021a). Commercial diets were formulated according to Ross 708 guidelines (Aviagen, 2015). The 2 dietary treatments were: 1) commercial diet; 2) the commercial diet restricted in Ca and aP content by 20% (ReCaP diet) for the starter (Ca = 0.768%, aP = 0.384%), grower (Ca = 0.696%, aP = 0.348%) and finisher (Ca = 0.624%, aP = 0.312%) dietary phases (Table 1).

Table 1.

Feed composition of the experimental diets from 0 to 41 d of age (doa).

Starter (0-14 doa)
Item	Commercial diet	Calcium and available phosphorus restricted (ReCap) diet
Ingredient	(%)
Yellow corn	53.23	53.23
Soybean meal	38.23	38.23
Animal fat	2.60	2.60
Dicalcium phosphate	2.23	1.71
Limestone	1.27	1.01
Salt	0.34	0.34
Choline chloride 60%	1.00	1.00
Lysine	0.28	0.28
DL-Methionine	0.37	0.37
L-threonine	0.15	0.15
Premix1	0.25	0.25
Coccidiostat2	0.05	0.05
Cellulose	0	0.78
Total	100	100
Calculated nutrients
Crude protein	23	23
Calcium	0.96	0.768
Available phosphorus	0.48	0.384
Apparent metabolizable energy (AME; Kcal/kg)	3,000	3,000
Digestible Methionine	0.51	0.51
Digestible Lysine	1.28	1.28
Digestible Threonine	0.86	0.86
Digestible total sulfur amino acids (TSAA)	0.95	0.95
Sodium	0.16	0.16
Choline	0.16	0.16
Grower (15−28 doa)
Item	Commercial diet	Calcium and available phosphorus restricted (ReCap) diet
Ingredient	(%)
Yellow corn	57.13	57.13
Soybean meal	34.80	34.80
Animal fat	3.50	3.50
Dicalcium phosphate	2.00	1.52
Limestone	1.17	0.94
Salt	0.34	0.34
Choline chloride 60%	0.10	0.10
Lysine	0.21	0.21
DL-Methionine	0.32	0.32
L-threonine	0.16	0.16
Premix	0.25	0.25
Coccidiostat	0.05	0.05
Cellulose	0	0.71
Total	100	100
Calculated nutrients
Crude protein	21.5	21.5
Calcium	0.87	0.696
Available phosphorus	0.435	0.348
AME (Kcal/kg)	3,100	3,100
Digestible Methionine	0.47	0.47
Digestible Lysine	1.15	1.15
Digestible Threonine	0.77	0.77
Digestible TSAA	0.87	0.87
Sodium	0.16	0.16
Choline	0.16	0.16
Finisher (29−45 doa)
Item	Commercial diet	Calcium and available phosphorus restricted (ReCap) diet
Ingredient	(%)
Yellow corn	54.23	54.23
Soybean meal	38.23	38.23
Animal fat	2.50	2.50
Dicalcium phosphate	2.23	1.71
Limestone	1.27	1.01
Salt	0.34	0.34
Choline chloride 60%	0.10	0.10
Lysine	0.28	0.28
DL-Methionine	0.37	0.37
L-threonine	0.15	0.15
Premix	0.25	0.25
Coccidiostat	0.05	0.05
Cellulose	0	0.78
Total	100	100
Calculated nutrients
Crude protein	19.5	19.5
Calcium	0.78	0.624
Available phosphorus	0.39	0.312
AME (Kcal/kg)	3,200	3,200
Digestible methionine	0.43	0.43
Digestible lysine	1.02	1.02
Digestible threonine	0.68	0.68
Digestible TSAA	0.8	0.8
Sodium	0.16	0.16
Choline	0.16	0.16

¹The broiler premix provided per kilogram of diet: vitamin A (retinyl acetate), 10,000 IU; cholecalciferol, 4,000 IU; vitamin E (DL-α-tocopheryl acetate), 50 IU; vitamin K, 4.0 mg; thiamine mononitrate (B₁), 4.0 mg; riboflavin (B₂), 10 mg; pyridoxine HCL (B₆), 5.0 mg; vitamin B₁₂ (cobalamin), 0.02 mg; D-pantothenic acid, 15 mg; folic acid, 0.2 mg; niacin, 65 mg;biotin, 1.65 mg; iodine (ethylene diamine dihydroiodide), 1.65 mg; Mn (MnSO₄H₂O), 120 mg;Cu, 20 mg; Zn, 100 mg, Se, 0.3 mg; Fe (FeSO₄.7H₂O), 800 mg.

²Decocx (Zoetis, Parsippany, NJ).

Blood Sampling and Immunological Assessment

One bird from each of the 48 floor pens was randomly selected, individually weighed, and euthanized for organ sampling at 0, 7, 14, and 40 doa. At 0 doa, weights of the liver, spleen, bursa and small intestine (SI, between terminus of the gizzard and origin of the cecal ducts) were obtained. At 7, 14, and 40 doa, individual weights of the liver, spleen, bursa, duodenum, jejunum, and ileum were obtained. All weights were subsequently expressed as percentages of BW. From the same birds that were selected for organs sampling at the aforementioned time periods, blood samples were also collected from the chorioallantoic vasculature. Plasma was subsequently extracted for immunological assay. Plasma IgG concentration was determined for samples collected at 7 and 14 doa according to the manufacturer's protocol (MyBioSource, San Diego, CA). Duplicate 100 μL volumes of standard and experimental samples were loaded into plate wells precoated with capture antibody, and were then incubated at 37°C for 90 min. Plates were washed 2 times with wash solution (50 mM Tris-buffered saline, 0.14 M NaCl, 0.05% Tween 20, pH 8.0) and 100 μL of biotinylated chicken IgG antibody was added to each well, and plates were incubated at 37°C for 60 min. Plates were washed 3 times with wash solution and 100 μL of enzyme-conjugate liquid was added to each well in order to initiate a TMB substrate color reaction. Reactant was thoroughly washed out by PBS or TBS. Plates were incubated at 37°C for 30 min and were washed 5 times with wash solution. A 100 μL volume of color reagent was added to each well in order to produce a blue color in each sample. Samples were then incubated in a dark incubator at 37°C for 30 min. Finally, the color of each sample was changed from blue to yellow under the action of a 100 μL volume of stop solution. Plasma IgM concentration was determined at 14 doa according to the procedure of Perez-Carbajal et al. (2010) and plasma α-1-acid glycoprotien (AGP) was determined at 40 doa according to the procedure of Kaab et al. (2018). Optical densities (OD) at 450 nm (OD450) for IgG, IgM and AGP were measured with a SpectraMax M5 Microplate Reader (Molecular Devices, San Jose, CA).

Small Intestine Morphology

From each intestine sample collected from the 1 bird in each of the 48 floor pens at 14 and 40 doa, a 2 cm sample from the middle region of the duodenum, jejunum, and ileum was excised and fixed in 10% formalin. Intestinal samples were then gradually dehydrated and embedded in paraffin. The slides used for histomorphological analysis were prepared according to the procedure of Wang et al. (2015). Villus and crypt measurements from 3 different and randomly selected locations within each of the 3 intestinal regions for a particular bird were included on each slide. Slides were examined using a light microscope at 40X magnification (Micromaster, Fisher Scientific, Pittsburgh, PA) according to the method described by Fasina et al. (2010). Microscopic images were measured using ImageJ software (Wayne Rasband, NIMH, Bethesda, MD) to measure VL, villus width (VW) and CD. Villus length was considered as the distance from the tip to the base of the lamina propria and VW was measured between the top third and bottom third of the villus. Crypt depth was the length from the base of the villus to the mucosa layer. The ratio of VL to CD (RVC) was calculated by dividing VL by CD. Villus surface area was calculated for all sections using the following formula presented below by Nain et al. (2012). Villus surface area (VSA) = 2π × (average VW/2) × VL; where average VW is the average of 3 measurements per bird (2 VW measurements from the 3 sample sections examined on each slide). Mean values of the observation variables within each of the 3 intestinal regions for each bird were subsequently subjected to statistical analysis.

Statistical Analysis

The experimental unit was floor pen, and the experimental design was a randomized complete block. A group of pens was the blocking factor with all 8 treatment combinations randomly represented in each of 6 blocks. All data were analyzed at each time period separately using 2-way analysis of variance in a 2 dietary treatment x 4 in ovo injection treatment factorial design. The general linear mixed models procedure (PROC GLIMMIX) of SAS 9.4 (SAS Institute, 2013) was employed. Differences were considered significant at P ≤ 0.05. The following model was used for analysis of the data:

$${\mathrm{Y}}_{\text{ijk}}=\mu +{\mathrm{B}}_{\mathrm{i}}+{\mathrm{D}}_{\mathrm{j}}+{\mathrm{I}}_{\mathrm{k}}+{\left(\text{DI}\right)}_{\text{jk}}+{\mathrm{E}}_{\text{ijk}},$$

Where μ was the population mean; B_i was the block factor (i = 1 to 2); D_i was the effect of each dietary treatment (j = 1 to 2); Ik was the effect of in ovo injection treatment (k = 4); (DI)ij was the interaction of each dietary treatment with in ovo injection treatment; and E_ij was the residual error.

RESULTS

Body and Organ Weights

No significant effects due to in ovo injection treatment were observed for BW and all relative organ data at 0 doa (Table 2), and no significant interactions were observed between diet and in ovo injection treatment for BW and relative organ weights at 7, 14, and 40 doa. Furthermore, at 7 and 14 doa, there were no significant main effects due to in ovo or dietary treatment for any of the variables examined. There were also no significant main effects due to in ovo treatment for any of the variables examined at 40 doa. However, BW was lower and relative duodenum weight was greater in birds fed ReCaP diets in comparison to those fed commercial diets (Table 2).

Table 2.

Broiler mean BW, relative weight of the whole small intestine (SI), liver, spleen, bursa, duodenum, jejunum, and ileum at 0, 7, 14, and 40 d of age (doa) in diluent in ovo-injected (50 μL) control eggs, and eggs in ovo-injected with diluent containing 2.4 μg of vitamin D₃ (D₃) or 25-hydroxycholecalciferol (25OHD₃) alone, or a combination of 2.4 μg of D₃ and 2.4 μg of 25OHD₃ (D₃ + 25OHD₃), and fed a commercial diet or a diet in which calcium and available phosphorus were restricted by 20%.

Treatment	BW (g)	SI1 (%)	Liver (%)	Spleen (%)	Bursa (%)
			0 doa
In ovo injection
Diluent2	41.3	2.88	1.02	0.017	0.087
D33	41.0	3.38	0.99	0.015	0.143
25OHD34	41.8	2.82	0.95	0.023	0.041
D3+25OHD35	40.6	2.99	0.98	0.016	0.093
Pooled SEM	0.78	0.191	0.036	0.0029	0.0528
P value	0.759	0.177	0.588	0.158	0.599

Treatment	BW (g)	Duodenum (%)	Jejunum (%)	Ileum (%)	Liver (%)	Spleen (%)	Bursa (%)
			7 doa
In ovo injection
Diluent	158.8	1.63	2.83	2.46	3.12	0.076	0.36
D3	156.8	1.77	2.54	2.62	3.18	0.077	0.17
25OHD3	147.8	2.16	2.81	2.62	3.15	0.084	0.26
D3+25OHD3	157.4	1.52	2.75	2.29	3.08	0.087	0.17
Diet
Commercial	153.5	1.88	2.74	2.62	3.08	0.077	0.16
ReCaP6	156.9	1.67	2.73	2.45	3.18	0.085	0.32
Pooled SEM	4.55	0.123	0.300	0.193	0.155	0.007	0.112
P value
In ovo	0.413	0.072	0.845	0.539	0.952	0.634	0.566
Diet	0.506	0.230	0.966	0.510	0.437	0.226	0.142
In ovo x Diet	0.177	0.132	0.742	0.881	0.451	0.887	0.530

Treatment	BW (g)	Duodenum (%)	Jejunum (%)	Ileum (%)	Liver (%)	Spleen (%)	Bursa (%)
			14 doa
In ovo injection
Diluent	452	1.85	1.87	1.58	2.68	0.106	0.493
D3	449	1.15	1.76	1.53	2.93	0.169	0.178
25OHD3	465	1.10	1.62	1.42	2.53	0.139	0.210
D3+25OHD3	445	1.27	1.61	1.63	2.75	0.069	0.153
Diet
Commercial	454	1.46	1.63	1.53	2.73	0.149	0.188
ReCaP	450	1.23	1.80	1.55	2.72	0.092	0.161
Pooled SEM	9.8	0.255	0.084	0.110	0.070	0.0477	0.021
P value
In ovo	0.563	0.439	0.241	0.514	0.055	0.634	0.493
Diet	0.731	0.524	0.087	0.891	0.875	0.318	0.370
In ovo x Diet	0.285	0.280	0.399	0.463	0.061	0.327	0.481

Treatment	BW (g)	Duodenum (%)	Jejunum (%)	Ileum (%)	Liver (%)	Spleen (%)	Bursa (%)
			40 doa
In ovo injection
Diluent	2,408	0.497	1.025	0.738	2.26	0.100	0.118
D3	2,261	0.564	1.038	0.818	2.14	0.091	0.145
25OHD3	2,402	0.532	0.963	0.755	2.14	0.090	0.138
D3+25OHD3	2,254	0.510	1.021	0.657	2.50	0.112	0.119
Diet
Commercial	2,573a	0.476b	0.969	0.718	2.30	0.096	0.125
ReCaP	2,089b	0.575a	1.055	0.766	2.22	0.101	0.136
Pooled SEM	69.3	0.0250	0.044	0.0549	0.088	0.0055	0.0120
P value
In ovo	0.228	0.575	0.838	0.350	0.260	0.290	0.342
Diet	0.001	0.013	0.204	0.415	0.566	0.537	0.422
In ovo x Diet	0.114	0.819	0.635	0.389	0.518	0.475	0.379

Treatment means within the same variable column within type of treatment with no common superscript differ significantly (P < 0.05).

¹The weight of and gastrointestinal tract from the bottom of proventriculus to the end of the ceca.

²Eggs injected with 50 μL commercial diluent at d 18 of incubation.

³Eggs injected with 50 μL commercial diluent containing vitamin D₃ at 2.4 μg at d 18 of incubation.

⁴Eggs injected with 50 μL commercial diluent containing 25OHD₃ at 2.4 μg at d 18 of incubation.

⁵Eggs injected with 50 μL commercial diluent containing D₃ at 2.4 and 25OHD₃ at 2.4 μg at d 18 of incubation.

⁶A diet restricted Ca and available P by 20% throughout the rearing period.

Immunity

No significant interactions were observed between diet and in ovo treatment for any of the immunological measurements determined at their specified time periods (Table 3). There were also no significant main effects due to diet for plasma IgG concentrations at 7 and 14 doa, IgM at 14 doa, and AGP at 14 doa, or due to in ovo injection treatment for plasma IgG concentrations at 7 doa, and AGP at 40 doa. However, at 14 doa, birds that received 25OHD₃ alone had higher plasma IgG concentrations compared to those that were injected with diluent or D₃ alone (Table 3). An injection of D₃ + 25OHD₃ also resulted in plasma IgG concentrations that were higher than those in birds injected with D₃ alone. Furthermore, at 14 doa, birds that received the combination of 25OHD₃ and D₃ had higher plasma IgM levels in comparison to those that received diluent or D₃ alone, and those that received 25OHD₃ alone had higher IgM concentrations than those that were in ovo-injected with only diluent (Table 3).

Table 3.

Broiler mean plasma IgG at 7 and 14 d of age (doa), IgM at 14 doa, and alpha-1-acid glycoprotein (AGP) at 40 doa in diluent in ovo-injected (50 μL) control eggs, and eggs in ovo-injected with diluent containing 2.4 μg of vitamin D₃ (D₃) or 25-hydroxycholecalciferol (25OHD₃) alone, or a combination of 2.4 μg of D₃ and 2.4 μg of 25OHD₃ (D₃ + 25OHD₃), and fed a commercial diet or a diet in which calcium and available phosphorus were restricted by 20%.

Treatment	IgG-7doa1	IgG-14doa2	IgM-14doa3	AGP-40doa4
μM
In ovo injection
Diluent5	2.39	6.52bc	2.37c	2.92
D36	2.26	6.43c	2.42bc	2.93
25OHD37	2.34	6.63a	2.48ab	2.72
D3+25OHD38	2.34	6.58ab	2.57a	2.86
Diet9
Commercial	2.32	6.58	2.42	2.86
ReCaP	2.35	6.50	2.50	2.82
Pooled SEM	0.049	0.053	0.045	0.009
P value
In ovo	0.302	0.013	0.005	0.079
Diet	0.570	0.090	0.080	0.580
In ovo x Diet	0.302	0.984	0.974	0.129

^a,bTreatment means within the same variable column within type of treatment with no common superscript differ significantly (P < 0.05).

¹Plasma IgG concentrations at 7 doa.

²Plasma IgG concentrations at 14 doa.

³Plasma IgM concentrations at 14 doa.

⁴Plasma AGP concentrations at 40 doa.

⁵Eggs injected with 50 μL commercial diluent at d 18 of incubation.

⁶Eggs injected with 50 μL commercial diluent containing vitamin D₃ at 2.4 μg at d 18 of incubation.

⁷Eggs injected with 50 μL commercial diluent containing 25OHD₃ at 2.4 μg/ at d 18 of incubation.

⁸Eggs injected with 50 μL commercial diluent containing D₃ at 2.4 and 25OHD₃ at 2.4 μg at d 18 of incubation.

⁹A diet restricted Ca and available P by 20% through the rearing period.

Small Intestine Morphology

No significant interactions were observed between diet and in ovo injection treatments at any time period for any of the histomorphological measurements (Tables 4 and 5). Due to the large number of variables examined, only those variables exhibiting significant main effects due to in ovo injection and dietary treatment are discussed. At 14 doa, VL was higher in the ileum in birds that were injected with 25OHD₃ alone compared to those that received only diluent, and duodenal CD was shallower in birds injected with 25OHD₃ alone in comparison to all other treatments. Additionally, a lower jejunal CD was observed in birds that received 25OHD₃ alone in comparison to those that received diluent or D₃ alone, and was lower in the D₃ + 25OHD₃ group in comparison to the D₃ alone group (Table 4). Greater duodenal, jejunal, and ileal RVC were observed in birds that received 25OHD₃ alone in comparison to all other treatments. In comparison to the diluent-injected group, ileal VSA was decreased in response to the in ovo injection of 25OHD₃ alone, and VW in the jejunum was lower in birds that received D₃ and 25OHD₃ together than if they were received separately. At 14 doa, VL in the duodenum was decreased in birds fed ReCaP diets in comparison to those fed commercial diets. Conversely, jejunal CD was higher in birds fed ReCaP diets in comparison to those fed commercial diets (Table 4).

Table 4.

Broiler mean small intestine morphology measurements at 14 d of age (doa) in diluent in ovo-injected (50 μL) control eggs, and eggs in ovo-injected with diluent containing 2.4 μg of vitamin D₃ (D₃) or 25-hydroxycholecalciferol (25OHD₃) alone, or a combination of 2.4 μg of D₃ and 2.4 μg of 25OHD₃ (D₃ + 25OHD₃), and fed a commercial diet or a diet in which calcium and available phosphorus were restricted by 20%.

	Duodenum					Jejunum					Ileum
Treatment	villus length	Villus width	Crypt depth	RVC1	VSA2	villus length	Villus width	Crypt depth	RVC	VSA	villus length	Villus width	Crypt depth	RVC	VSA
	µm			length/depth	mm2	µm			length/depth	mm2	µm			length/depth	mm2
In ovo injection
Diluent3	994	89.4	136a	7.7b	0.28	848	89.3ab	116b	7.4b	0.34	336b	83.1	90	4.0b	0.40a
D34	999	93.8	137a	7.4b	0.29	922	98.9a	137a	7.0b	0.34	391ab	87.1	107	3.8b	0.35ab
25OHD35	1088	82.8	89b	12.2a	0.24	902	93.0a	88c	10.0a	0.33	480a	81.2	77	6.4a	0.27b
D3+25OHD36	983	83.2	122a	8.2b	0.27	780	73.3b	107cb	7.5b	0.30	428ab	75.4	90	4.8b	0.30ab
Diet7
Commercial	1065a	93.3	124	9.5	0.28	849	85.7	104b	8.6	0.32	415	85.8	88	5.0	0.34
ReCaP	967b	81.4	114	9.0	0.27	877	91.5	120a	7.7	0.33	403	77.5	94	4.6	0.31
Pooled SEM	50.4	9.62	10.0	0.69	0.028	45.8	6.92	6.6	0.58	0.025	39.4	10.35	8.9	0.52	0.039
P value
In ovo	0.092	0.549	0.001	0.001	0.195	0.168	0.050	0.001	0.001	0.543	0.020	0.864	0.067	0.001	0.039
Diet	0.005	0.062	0.129	0.311	0.644	0.555	0.380	0.021	0.150	0.778	0.746	0.362	0.455	0.376	0.409
In ovo x Diet	0.606	0.192	0.598	0.942	0.190	0.176	0.351	0.068	0.845	0.595	0.390	0.326	0.570	0.178	0.531

^a,bTreatment means within the same variable column within type of treatment with no common superscript differ significantly (P < 0.05).

¹Ratio of villus length to crypt depth.

²Villus surface area (VSA) calculated with average villus length and width = 2π × (width/2) × length.

³Eggs injected with 50 μL commercial diluent at d 18 of incubation.

⁴Eggs injected with 50 μL commercial diluent containing vitamin D₃ at 2.4 μg at d 18 of incubation.

⁵Eggs injected with 50 μL commercial diluent containing 25OHD₃ at 2.4 μg at d 18 of incubation.

¹Eggs injected with 50 μL commercial diluent containing D₃ at 2.4 and 25OHD₃ at 2.4 μg at d 18 of incubation.

⁷A diet restricted Ca and available P by 20% throughout the rearing period.

Table 5.

Broiler mean small intestine morphology measurements at 40 d of age (doa) in diluent in ovo-injected (50 μL) control eggs, and eggs in ovo-injected with diluent containing 2.4 μg of vitamin D₃ (D₃) or 25-hydroxycholecalciferol (25OHD₃) alone, or with a combination of 2.4 μg of D₃ and 2.4 μg of 25OHD₃ (D₃ + 25OHD₃), and fed a commercial diet or a diet in which calcium and available phosphorus were restricted by 20%.

	Duodenum					Jejunum					Ileum
Treatment	Villus length	Villus width	Crypt depth	RVC1	VSA2	Villus length	Villus width	Crypt depth	RVC	VSA	Villus length	Villus width	Crypt depth	RVC	VSA
	µm			Length/Depth	mm2	µm			Length/Depth	mm2	µm			Length/Depth	mm2
In ovo injection
Diluent3	1182b	101	124a	9.9b	0.27	975	111	123a	8.2b	0.36	565ab	80.9	97a	5.9bc	0.43b
D34	1200b	113	126a	9.7b	0.30	903	107	119a	7.7b	0.38	458c	86.1	95a	4.9c	0.57a
25OHD35	1388a	106	96b	15.1a	0.24	1005	110	91b	11.3a	0.35	658a	88.2	72b	9.6a	0.43b
D3+25OHD36	1253ab	98	112ab	11.5b	0.25	918	106	104ab	9.5ab	0.37	538bc	80.1	76b	7.5b	0.47ab
Diet7
Commercial	1290	106	109	12.4a	0.26	957	111	100b	10.0	0.37	599a	91.4a	89	7.1	0.47
ReCaP	1222	103	120	10.7b	0.27	944	106	118a	8.4	0.36	501b	76.3b	80	6.9	0.48
Pooled SEM	50.4	45.9	7.7	5.8	0.72	0.025	71.4	10.37	8.5	0.98	0.031	22.9	7.61	4.1	0.60
P value
In ovo	0.023	0.474	0.006	0.001	0.276	0.590	0.983	0.011	0.020	0.833	0.003	0.834	0.007	0.001	0.031
Diet	0.166	0.681	0.063	0.032	0.734	0.834	0.560	0.019	0.074	0.490	0.017	0.049	0.115	0.827	0.701
In ovo x Diet	0.606	0.773	0.608	0.672	0.361	0.939	0.541	0.983	0.103	0.117	0.698	0.949	0.820	0.585	0.830

^a,bTreatment means within the same variable column within type of treatment with no common superscript differ significantly (P < 0.05).

¹Ratio of villus length to crypt depth.

²Villus surface area (VSA) calculated with average villus length and width = 2π × (width/2) × length.

³Eggs injected with 50 μL commercial diluent at d 18 of incubation.

⁴Eggs injected with 50 μL commercial diluent containing vitamin D₃ at 2.4 μg at d 18 of incubation.

⁵Eggs injected with 50 μL commercial diluent containing 25OHD₃ at 2.4 μg at d 18 of incubation.

⁶Eggs injected with 50 μL commercial diluent containing D₃ at 2.4 and 25OHD₃ at 2.4 μg at d 18 of incubation.

⁷A diet restricted Ca and available P by 20% throughout the rearing period.

At 40 doa, VL in the duodenum was greater in birds injected with 25OHD₃ alone in comparison to those that were injected with diluent or D₃ alone. However, ileal VL increased in response to the injection of 25OHD₃ alone when compared to the D₃ alone or 25OHD₃ + D₃ treatments. Furthermore, VL in the D₃ treatment was lower than that in the diluent control group. Crypt depth in the duodenum was shallower in birds that received 25OHD₃ alone in comparison to those in the D₃ or diluent alone treatment groups, but ileum CD in birds having been in ovo- injected with 25OHD₃ alone or in combination with D₃ was shallower in comparison to those in the D₃ or diluent alone treatments (Table 5). In the duodenum and jejunum, CD was only lower in the 25OHD₃ treatment in comparison to the D₃ alone and diluent treatment groups, and in the jejunum, a higher RVC was observed in birds injected with 25OHD₃ alone in comparison to those injected with diluent or D₃ alone. Also, 25OHD₃ alone resulted in a higher RVC in the duodenum in comparison to all other treatments. In the ileum, a higher RVC was observed in the 25OHD₃ alone treatment in comparison to all other treatments, and the combination of 25OHD₃ and D₃ resulted in a higher RVC compared to D₃ alone. In the ileum, VSA was higher in the D₃ alone treatment in comparison to the 25OHD₃ or diluent alone treatments. At 40 doa, birds fed ReCaP diets had a lower duodenal RVC, and lower ileal VL and VW, but conversely had a deeper jejunal CD in comparison to those fed commercial diets (Table 5).

DISCUSSION

Development of the small intestine in response to vitamin D₃ occurs mainly through changes in its morphology. Inclusion of 25OHD₃ in breeder diets has resulted in an increased VL in broiler embryos at 19 doi as well as in broilers at 2 doa (Ding et al., 2011). In addition to its early posthatch effects, dietary supplementation of 25OHD₃ at 2,670 IU/kg has been shown to increase duodenal and jejunal VL and decrease jejunal CD in broilers at 28 and 35 doa (Chou et al., 2009). Various effects of the in ovo injection of vitamin D₃ sources including D₃, 25OHD₃, 1- α hydroxyvitamin D₃, and 1α,25-(OH)₂-D₃ have been reported ( Bello et al., 2014; Abbasi et al., 2017; Mansour et al., 2017). However, their effects on the histomorphology of the small intestine of the embryo or posthatch broiler have not been previously reported. Nevertheless, findings in the current study reveal that the in ovo injection of 2.4 μg of 25OHD₃ improved the small intestine morphology of broilers in comparison to diluent or D₃ alone. In mammals, 1-α hydroxylase, which converts 25OHD₃ to 1α,25-(OH)₂-D_3, has been identified in dendritic cells, macrophages (Overbergh et al., 2000; Veldman et al., 2000), B-cells (Chen et al., 2007), T-cells (Veldman et al., 2000), and duodenal sections of the small intestine (Gawlik et al., 2015). Additionally, in contrast to D₃, 25OHD₃ has been shown to increase the activity of 1-α hydroxylase in the chicken (Morris et al., 2015). This suggests that cells of the immune system and those in the intestine may display a greater response to this secondary metabolite of vitamin D₃. In comparison to D₃, the half-life of 25OHD₃ is longer (Smith and Goodman, 1971; Hollis and Wagner, 2013) and it is absorbed at a higher rate (Bar et al., 1980). The improvement in small intestine morphology observed in response to the in ovo injection of 25OHD₃ alone, may be due to the longer half-life and higher rate of absorption of 25OHD₃. Previous findings in our laboratory revealed that breast meat yield increased (Fatemi et al., 2021 a,b) and FCR decreased (Fatemi et al., 2020a) in response to the in ovo injection of 2.4 μg of 25OHD₃. An improvement in small intestine morphology and humoral immunity is associated with increased breast meat yield and an improvement in the growth performance of broilers (Chou et al., 2009; Wang et al., 2019). Therefore, an enhancement of breast meat yield and growth performance observed in response to the in ovo injection of 25OHD₃ could be due to an enhancement of small intestine morphology and immunity in birds.

It is well documented that at least 2 wk are required for lymphocytes to be fully developed in posthatch chickens (Bar-Shira and Friedman, 2006). Additionally, various vitamin D₃ sources affect humoral immunity. In the current study, it was shown that in comparison to the injection of diluent or D₃ alone, the in ovo injection of 25OHD₃ resulted in increased IgG and IgM levels at 14 doa when compared to that those injected with diluent or D₃ alone. Abbasi et al. (2017) also showed that an increase in antibody titers against Newcastle Disease was observed in hatchlings in response to the in ovo injection of a 0.5 ml of solution containing 0.4 μg of 25OHD₃ and 6 μg of vitamin K. Thus, increased Ig levels of early-hatch broilers within the first 2 wk of life can be beneficial for the enhancement of their immunity. These results indicate that the in ovo administration of 2.4 μg of 25OHD₃ might be a suitable candidate for boosting the humoral immunity of early posthatch broilers. In addition to humoral immunity, the inflammatory response of the broilers was monitored by measuring AGP at 40 doa. It has been proposed in several reports that AGP levels in chickens can be used to monitor an increase in inflammation (Lee et al., 2010; Asasi et al., 2013; Fatemi, 2016). Furthermore, the AGP concentrations of modern broiler lines have been observed to be higher than those of broiler lines used in the 1990's (O’Reilly et al., 2018). However, the AGP concentrations of the birds in the current study were not significantly affected by in ovo injection or dietary treatment, indicating that a general systemic inflammatory response in the birds was not significantly affected by either treatment.

In this study, a 20% reduction in dietary Ca and aP resulted in a decline in duodenal VL at 14 doa, and other negative effects included a lower duodenal RVC and a higher jejunal CD at 40 doa. A reduction in dietary aP has been shown to exert a greater effect on broiler gut morphology than a reduction in dietary Ca. A diet containing 0.6 % of Ca and 0.3 % of aP has been shown to have no negative effects on VCR in comparison to recommended levels of Ca and aP (0.90 % of Ca and 0.45 % of aP) in broilers. However, a 30% reduction in aP in combination with a 0.90% level of Ca resulted in a lower VCR in the duodenum and ileum (Paiva et al., 2014). The inclusion of supplemental dietary phytase at 1,000 FTU/kg was sufficient to overcome the negative effects of a reduction in aP on small intestine morphology and broiler performance. In addition to this, an increase in dietary Ca levels from 0.90 to 1.05% has been shown to have no effect on small intestine morphology in broilers at different ages ( Xing et al., 2020). An increase in intestine weight relative to BW is associated with a decline in small intestine morphology (Chou et al., 2009; Wu et al., 2013, 2016), and performance (Wu et al., 2016). In the current study, relative duodenal and jejunal weights increased at 41 doa, and relative jejunal weight tended to increase at 41 doa, in birds fed ReCaP rather than commercial diets. Therefore, the decline in small intestine morphology in response to a 20% reduction in Ca and aP may have been partially linked to an increase in intestine weight.

In conclusion, the influence of the in ovo injection of various vitamin D₃ sources on small intestine morphology and immunity of Ross 708 broilers fed commercial and ReCaP diets were investigated. These results showed that the ReCaP diet adversely affected broiler intestinal morphology and growth, but that it did not influence effects of the injection of these vitamin D₃ sources on their immunity and small intestine morphology. Regardless of dietary treatment, the small intestine morphology and humoral immunity of the birds were improved in response to the in ovo injection of 25OHD₃ alone, but these effects were not observed when D₃ was injected alone. This improvement in response to the in ovo injection of 2.4 μg of 25OHD₃ could be due to its longer half-life or greater rate of absorption. Further research is required to determine the effects of the in ovo injection of the vitamin D₃ sources on the molecular mechanisms of intestinal development and immunity in broilers.