Dietary supplementation and in ovo injection of 1α-OHD3 in a low-calcium and low-phosphorous diets for broilers

Abstract

Three hundred and twenty day-old chicks were assigned to four experimental treatments of positive control (standard diets with recommended levels of calcium and phosphorus), negative control (diets containing calcium and phosphorus at the 50% of recommended levels), Negative control plus in ovo injection of 1α-OHD3 (50 ng/egg 1α-OHD3 on day 18 of incubation), and negative control plus feeding of 1α-OHD3 (5 µg/kg of diet). Four replications assigned for each treatment with 20 birds per each. Diets consisted of a starter phase from 0 to 14 days and a grower phase from 15 to 28 days. At 28 days, feed intake (FI), body weight gain (BWG) and feed conversion ratio (FCR) were determined. Two birds per pen were killed to evaluate the carcass and tibia traits. No differences in FI, FCR, carcass traits, tibia weight and length, ash, Ca, P and Seedor index were observed at 28 days of age. Supplementation of dietary 1α-OHD3 significantly increased BWG in compare to negative control. Between treatments, negative control had lowest breaking strength. The current study indicated that supplementation of dietary 5 µg/kg of 1α-OHD3 may improve performance and tibia strength of broilers fed low-calcium and low-phosphorus diets.

Keywords:

• 1α-OHD3
• broiler
• in ovo injection
• tibia characteristics

Introduction

Broiler chicken production has undergone drastic changes and developments over the last few decades. Constant improvements in nutrition and genetic selection over the last two decades have led to a fast growth rate in modern broiler strains, to the extent that the average time required to grow a broiler chicken to 2 kg has reduced nearly by half (from 63 days to 37 days). Unfortunately, early fast growth rate in broiler chickens is accompanied by a number of problems – namely, increased body fat deposition, a high incidence of metabolic diseases, high mortality and a high incidence of skeletal diseases, e.g. tibial dyschondroplasia (Zubair & Leeson 1999). To combat such broiler feeding planners have also come up with new innovations to boost broiler performance efficiency further. Uni and Ferket (2004) emphasized that the few days pre- and post-hatch are critical for the development and survival of commercial chickens. Early feeding programme such as in ovo feeding is an innovation that has attracted many researchers. A favourable influence of injection of vitamin C into broiler breeder eggs was reported by Ipek et al. (2004). However, no information is available on the effect of vitamin D3 as in ovo feeding that has direct effect on bone development.

Vitamin D3 is required for normal growth and bone development of chicks. Several derivatives, such as 1α-hydroxycholecalciferol (1α-OHD3), and 25-OHD3, have been reported to have higher biological potency compared to cholecalciferol (Edwards et al. 2002). It has been demonstrated that feeding of 1α-OHD3 may improve the performance and phosphorus utilization of broilers (Biehl et al. 1995; Biehl & Baker 1997; Snow et al. 2004). 1α-OHD3 facilitates Ca absorption by removing a portion of Ca from the digesta, the phytin molecule becomes more soluble and accessible to the hydrolytic actions of intestinal phytase. Therefore, the efficiency of intestinal phytase on phytate phosphorus hydrolysis may increase when 1α-OHD3 is supplemented. Broilers until 2 weeks of age can not produce enough cholecalciferol hydroxylase enzyme in the endoplasmic reticulum of liver hepatocytes [cholecalciferol (vitamin D3) hydroxylated to 25-OHD3 by 25-hydroxylase]. Therefore, they are sensitive to lameness. Then, inclusion of 1α-OHD3 in broilers diets can help them to provide active physiological form of vitamin D (1,25(OH)₂D3). A number of studies have clearly demonstrated the importance and requirement of vitamin D in chick embryonic development. As Chorioallantoic membrane has vitamin D3 receptor, calcium transfer from egg shell to embryo is affected by vitamin D3 (Tuan et al. 1991). It could be hypothesized that in ovo injection of this vitamin or other analogues (such as 1α-OHD3) may affect hatchling performance and leg health.

Thus, This study was conducted to determine the effects of dietary feeding and in ovo injection of 1α-OHD3 in a low-calcium and low-phosphorus diets for broilers on performance, tibia traits and serumic levels of calcium and phosphorous.

Materials and methods

In this experiment 400 fertilized eggs were collected from a commercial breeder flock (Ross 308). A hundred fertilized eggs were injected with 50 ng/egg 1α-OHD3 on day 18. 1α-OHD3 was purchased from Vitamin Derivatives, Inc. (Georgia, USA). Sixteen floor pens containing 320 day-old chicks were assigned to four experimental treatments. The four treatments were: positive control (standard diets with recommended levels of calcium and phosphorus), negative control (diets containing calcium and phosphorus at the 50% of recommended levels), negative control plus in ovo injection of 1α-OHD3 (50 ng/egg 1α-OHD3 on day 18 of incubation) and negative control plus feeding of 1α-OHD3 (5 µg/kg of diet). Diets consisted of a starter phase from 0 to 14 days and a grower phase from 15 to 28 days. All diets (Table 1) were fed as a mash.

Table 1. Composition of experimental diets.

	Starter (0–14 days)		Grower (15–28 days)
Ingredients	Control +	Control –	Control +	Control –
Corn grain	55.13	55.13	59.33	59.33
Soybean meal	35.98	35.98	31.48	31.48
Corn gluten meal	2.96	2.96	3.37	3.37
Dicalcium phosphate	2.16	0.67	1.88	0.54
Soy oil	1.4	1.4	1.9	1.9
CaCO3	0.67	0.17	0.50	0.11
L-lysine HCl	0.32	0.32	0.26	0.26
DL-methionine	0.31	0.31	0.25	0.25
L-threonine	0.06	0.06	0.03	0.03
Common salt	0.22	0.22	0.22	0.22
Sodium bicarbonate	0.19	0.19	0.18	0.18
Toxin binder	0.1	0.1	0.1	0.1
Vitamin & mineral premix^a	0.5	0.5	0.5	0.5
Celite (as diet filler)	0.00	1.99	0.00	1.73
Calculated composition
Metabolisable energy (Kcal/kg)	2900	2900	3000	3000
Crude protein	21.5	21.5	20	20
Calcium	1.05	0.525	0.9	0.45
Available phosphorous	0.5	0.25	0.45	0.225
Methionine	0.64	0.64	0.57	0.57
Methionine + cysteine	1.00	1.00	0.91	0.91
Lysine	1.34	1.34	1.18	1.18
Threonine	0.88	0.88	0.79	0.79
Arginine	1.39	1.39	1.26	1.26

^aThe vitamin and mineral premix provided the following (per kg of diet): 11,000 IU vitamin A; 4000 IU vitamin D₃; 50 IU vitamin E; 3 mg menadione; 3 mg thiamine; 8 mg riboflavin; 60 mg niacin; 4 mg pyridoxine; 0.016 mg vitamin B12; 15 mg pantothenic acid; 2 mg folic acid; 0.15 mg biotin; 40 mg iron; 100 mg zinc; 16 mg copper; 120 mg manganese; 1 mg iodine; 0.1 mg cobalt and 0.3 mg selenium.

Feed intake (FI), body weight gain (BWG) and feed conversion ratio (FCR) were recorded at the end of experiment. On day 28 for measuring carcass traits and collection of blood and tibias, two chicks from each replicate were randomly selected and weighed. Blood samples of 5 ml were collected into a 5-ml anti-coagulated syringe by cardiac puncture, immediately centrifuged for 10 min at 3000 × g at 20°C, and frozen at −20°C until further analysis. Birds were killed by cervical dislocation. The left and right tibias from individual birds were excised, sealed in plastic bags, and stored at −20°C for further analysis. According to the method reported by Hall et al. (2003), the left tibias were boiled for 5 min to loosen muscle tissue. Then, the meat, connective tissue and the fibula bone removed completely using scissors and forceps. After the tibias were cleaned, they were put into a container of ethanol for 48 h (removing water and polar lipids). Bones were then further extracted in anhydrous ether for 24 h (removing non-polar lipids). Tibias were dried at 105°C for 24 h and then weighed. Tibia weight was recorded. Tibia ash content was determined by ashing the bone in a muffle furnace for 18 h at 600°C. The left tibia was used for determine the amount of ash, Ca and P. The right tibia was used to analyze the breaking-strength. Tibia breaking-strength was determined using an all-digital electronic universal testing machine (Instron Universal Testing Machine Model 4502, Canton, MA). Tibias were cradled on two support points measuring 4 cm apart. Using a 50 kg load cell and a crosshead speed of 10 mm/min, the force was applied to the midpoint of the same facial of each tibia (Jendral et al. 2008). The Seedor index is the value obtained when the bone weight is divided by its length, as proposed by Seedor et al. (1991). It is used as a bone density indicator, the higher the value the denser the bone.

A completely randomized design was used in this experiment. Results were analyzed by Analysis of variance (ANOVA) using the GLM procedures of SAS software (SAS 2001). Differences between treatments were compared by the Duncan test following ANOVA, and values were considered statistically different at P < 0.05.

Results and discussion

Compared with those of birds fed low-calcium and low-phosphorus diets, dietary supplementing 5 μg/kg 1α-OHD3 increased BWG (P < 0.05; Table 2), in fact the feeding of deficient diet resulted in lowest BWG, and apply 1α-OHD3 (injection or feeding) increased BWG even more than the birds fed with basal diet. Feed intake, FCR and carcass traits were unaffected by treatments. Vitamin D and its metabolites may affect growth performance (Yarger et al. 1995). As vitamin D undergoes hydroxylation, it is likely that the molecule tends to become more polar and more easily absorbed in the small intestines (Applegate & Angel 2005). Furthermore, birds in the starter period do not have a complete enzymatic system to perform hydroxylation in the liver, which favours the administration of active metabolites (Świątkiewicz et al. 2006), thus explaining the increased use of already hydroxylated metabolites such as 1α-OHD3.

Table 2. Effect of experimental treatments on performance and carcass traits.

Treatments	FI (g/bird)	BWG (g/bird)	FCR (g:g)	Carcass (%)	Breast (%)	Thigh (%)	Liver (%)
Control+	1775.79	1228.46^ab	1.445	70.262	34.553	27.283	3.138
Control–	1779.67	1208.43^b	1.473	71.310	36.062	27.996	3.399
Control– & in ovo injection	1769.42	1234.60^ab	1.432	72.073	36.300	27.814	3.002
Control– & 1α-OHD3 feeding	1800.51	1262.81^a	1.425	70.819	37.192	27.502	3.304
SEM	8.740	7.703	0.008	0.490	0.415	0.435	0.076

FI, feed intake; BWG, body weight gain; FCR, feed conversion ratio; SEM, standard error mean.

^ab Values in the same row without a common superscript letter are significantly different (P < 0.05).

Feeding the 1α-OHD3 increases BWG, FI, FCR and tibia ash (Biehl et al. 1995; Biehl & Baker 1997; Edwards et al. 2002; Snow et al. 2004; Han et al. 2012). Weight gain was higher in chicks fed the phosphorus-deficient diet containing 1a-OHD3 in compared to those fed the phosphorus-adequate positive-control diet (Biehl & Baker 1997). This result about BWG was in agreement with those reported by Fritts and Waldroup (2003), they reported that 25-OHD3 improved both weight gain and feed efficiency in growing broilers. Biehl and Baker (1997) reported that 1α-OHD3 increased the growth of 8 to 21-day-old broilers fed a corn-soybean meal diet lowering in phosphorus and adequate in vitamin D3, but in purified diets with adequate vitamin D3, it had no effect on performance, which suggests that 1α-OHD3 might improve the growth of broilers by increasing phytate phosphorus utilization.

Decreasing Ca and P decreased breaking strength in compare with positive control (P < 0.05), but feeding and in ovo injection of 1α-OHD3 increased this parameters (Table 3). Between all groups, the birds fed low-calcium and low-phosphorus diet with supplementation of 1α-OHD3 had highest breaking strength, that it’s difference with other groups was significant (P < 0.05). In fact application of 1α-OHD3 in low-calcium and low-phosphorus diets (in ovo injection or dietary supplementation) compensated the reduction of tibia strength as a result of feeding with deficient diets. As shown in this table, the birds fed with low-calcium and low-phosphorus diets had lowest content of ash, calcium and phosphorus of tibia, that this difference in case of tibia phosphorus was significant (P < 0.05). In this experiment, tibia breaking strength and tibia phosphorus content were improved when 1α-OHD3 was supplemented in diet, which agrees with the findings of Han et al. (2012). Similar results were also found in the feeding of vitamin D3 (Qian et al. 1997) and 25-OHD3 (Ledwaba & Roberson 2003). Edelstein et al. (1978) reported that 1α-OHD3 metabolizes to 1,25-(OH)2D3 in intestinal and bone tissue, and the latter facilitates the absorption and retention of calcium and phosphate (Tanaka et al. 1971, 1972; Tanaka & DeLuca 1974), thereby improving tibia breaking strength. Vitamin D and its derivatives may also increase phosphorus absorption simply by increasing calcium absorption. Less calcium increases the proportion of soluble phosphorus in contact with the gut mucosa as calcium forms the Ca2PO4 salt at the normal intestinal pH.

Table 3. Effect of experimental treatments on tibia traits.

Treatments	Weight (g)	Length (mm)	Seedor index	Breaking strength (N)	Ash (%)	Calcium (%)	Phosphorous (%)
Control+	7.241	77.55	93.257	162.275^b	30.852	10.952	5.952^a
Control–	6.675	77.56	86.025	134.019^c	28.852	10.340	5.305^b
Control– & in ovo injection	6.768	77.94	86.765	164.800^b	29.150	11.136	5.503^b
Control– & 1α-OHD3 Feeding	6.968	77.49	89.908	186.403^a	29.753	10.980	5.620^ab
SEM	0.151	0.488	1.698	5.487	0.321	0.191	0.080

^abcValues in the same row without a common superscript letter are significantly different (P < 0.05).

Determinations of blood components are summarized in Table 4. Birds fed with standard diet (positive control group) show highest calcium and phosphorus levels in compare to other groups which fed dietary insufficient calcium and phosphorus levels. Decreasing in the level of dietary calcium and phosphorus resulted in lower serum calcium and phosphorus, because uptake of these two elements by birds fed deficient diets were decreased. Plasma phosphorus content had a tendency to increase by addition of 1α-OHD3. 1α-OHD3 facilitates absorption of Ca and Pi. There might be two ways of 1α-OHD3 action on phosphorus: (1) increasing non-phytate phosphorus utilization by facilitating phosphorus absorption; (2) improving phatate utilization by facilitating calcium absorption and decreasing the restriction of calcium on endogenous phytase. In addition, 1α-OHD3 stimulates small intestinal NaPi-IIb co-transporter gene expression and facilitates phosphorus transport (Han et al. 2009). It was suggested that in broilers raised in stressful conditions (high density, heat stress, some diseases or immune disorders) the intestinal absorption or liver hydroxylation of vitamin D3 may be impaired (Yarger et al. 1995). In conclusion, application of 1α-OHD3 in the diet and/or in ovo injection may partly improve broiler performance and bone quality in broilers fed diets lowering in calcium and phosphorus content.

Table 4. Effect of experimental treatments on serum parameters.

Treatments	Calcium (mg/dl)	Phosphorous (mg/dl)
Control+	8.443	4.692^a
Control–	7.181	4.052^b
Control– & in ovo injection	7.109	3.874^b
Control– & 1α-OHD3 feeding	6.763	4.165^ab
SEM	0.254	0.116

^abValues in the same row without a common superscript letter are significantly different (P < 0.05).