Influence of Supplemental Vitamin D 3 on Production Performance of Aged White Leghorn Layer Breeders and Their Progeny

An experiment was conducted to elucidate the effect of graded levels of vitamin D3 in White Leghorn (WL) layer breeders on egg production, shell quality, hatchability of eggs and juvenile performance of offspring during their late laying period (7288 wk). White Leghorn breeder females were randomly divided into 5 groups of 50 each and were housed in individual California cages in an open-side housing system. Considering birds in five cages as a replicate, 10 such replicates were randomly allotted to each treatment. A basal diet was formulated containing all the nutrients as recommended for WL layers except vitamin D3, which served as control. Another, four diets were formulated by supplementing graded levels of feed grade crystalline cholecalciferol to the basal diet that contained 300, 600, 1,200 and 2,400 ICU of vitamin D3 per kg. Each diet was offered ad libitum to one of the above five treatment groups. The egg production, egg weight, daily feed consumption and the feed intake per dozen eggs or kg egg mass of the birds fed diet without any supplemental vitamin D3 was comparable with those of supplemental groups. Similarly, the level of vitamin in the diet did not have any effect on any of the above parameters. However, the specific gravity of eggs laid by the birds fed the diet without supplemental vitamin D3 was comparable with either 600 or 2,400 ICU supplemental groups but significantly higher when compared to the 300 and 1,200 ICU groups. The egg -shell breaking strength was significantly lowered in the 600 ICU supplemental groups as compared to the strength of other dietary groups. The Haugh unit, egg shell weight, shell thickness, tibia breaking strength, bone ash and calcium content were not influenced by vitamin D3 concentration in the diet. Serum Ca concentration was influenced by vitamin D3 level in the diet. The serum Ca concentration of birds fed either control or the vitamin supplemented diet up to 1200 ICU/kg diet was comparable. However, increasing the concentration of vitamin D3 to 2,400 ICU/kg diet significantly enhanced the concentration of Ca in the serum, which was significantly higher compared to other dietary groups. The serum concentration of P and protein, however, was not influenced by level of vitamin D3 in the diet. Neither fertility nor hatchability was influenced by vitamin D3 concentration in the diet. Feeding a vitamin D3 deficient diet or supplementation of vitamin to hens did not have any influence on their progeny chicks. It can be concluded that dietary supplementation of vitamin D3 may not be essential for optimum production, shell quality, hatchability, and juvenile performance of WL breeders during 72 to 88 weeks of age. (


INTRODUCTION
Vitamin D 3 (cholecalciferol) is one of the most important dietary factor responsible for normal growth, egg production, shell quality and reproduction in fowls (Ameenuddin et al., 1982).It is also a required component of the endocrine system of birds and regulates calcium (Ca) and phosphorus (P) homeostasis and bone mineralization.The 1, 25 (OH) 2 D 3 stimulates Ca resorption from bone and re-absorption from glomerular filtrate, induces intestinal epithelium to synthesize calcium binding proteins (CaBP) and increase Ca absorption from the guts (Bar et al., 1972).This CaBP has also been identified in the uterus of laying hens (Fuller et al., 1976) and responsible for Ca deposition of egg shell at the on set of egg production (Bar and Hurwitz, 1973).
Resorption of bone Ca for egg shell formation in high producing hens is likely the cause of osteoporosis in layers during post peak production (Abe et al., 1982).It has also been suggested that the efficiency of the hydroxylation reactions necessary to convert cholecalciferol into its metabolically active form i.e. 1, 25 (OH) 2 D 3 may be reduced in aged hens (Frost et al., 1990;Elaroussi et al., 1994) ).Adequate level of 1, 25 (OH) 2 D 3 is required for the regulation of Ca absorption and excretion and initiate the mobilization of Ca from the bone to provide adequate Ca required for egg shell formation (Abe et al., 1982).
The elucidation of metabolism of vitamin D 3 and the role of this vitamin in Ca and P metabolism and egg shell quality has been one of the phenomena of nutritional research over the last three decades.Investigations into the ability of the older hens to metabolize or respond to vitamin D 3 have shown that shell quality and bone strength deteriorates more rapidly (Bar and Hurwitz, 1987).However, supplementation of vitamin D 3 to the deficient diets alleviated the decline in productivity and shell quality (Newman and Leeson, 1997).
Birds can synthesize cholecalciferol from cholesterol when they receive adequate sunlight.Vitamin D 3 insufficiency may be a common problem when birds reared in environmentally controlled house.However, no informations in literature are available whether there is a need to supply vitamin D 3 in the diet of layer in tropical country like India, where the birds are reared in cages with open side housing system.Therefore, the present study was conducted to elucidate the effect of graded level of vitamin D 3 in aged White Leghorn (WL) breeders on egg production, shell quality, hatchability of eggs and juvenile performance of offspring during their late laying period.

Birds and management
The White Leghorn breeders of 72 weeks of age were randomly divided into 5 groups of 50 each and were housed in individual California cages in open side housing system.Considering birds in five cages as a replicate, 10 such replicates were randomly allotted to each treatment.A continuous 16-h light per day was provided using incandescent bulbs.All the birds were maintained under uniform managemental conditions throughout the experimental period.The temperature of the house varied between 36-38°C during the entire experimental period.

Experimental diets
A basal diet was formulated containing all the nutrients as recommended for WL layers except vitamin D 3, which served as control (Table 1).Another, four diets were formulated by supplementing graded levels of feed grade crystalline cholecalciferol (vitamin D 3 , Dulphar Interfran, Mumbai, India) to the basal diet that contained 300, 600, 1,200 and 2,400 ICU of vitamin D 3 per kg.To ensure proper mixing of cholecalciferol in diets, the vitamin was dissolved in 100 ml of propylene glycol-ethanol solution (95 ml propylene glycol and 5 ml ethanol).A separate premix of cholecalciferol was prepared by mixing the above solution with finely ground maize and finally the vitamin premix was mixed with respective experimental feed.Each diet was provided ad libtum to one of the above five groups, from 72 to 88 weeks of age.

Response criterion
Body weight, egg production and egg weight : Individual body weight of the bird was recorded at the beginning and end of the experiment.Egg production on individual basis was recorded daily and percent hen day egg production (HDEP) was calculated.All the eggs laid during the last three consecutive days of every 28 day period, were collected to measure the egg weight.
Egg shell quality : Fifteen eggs were randomly chosen from each treatment from the eggs laid during the last three consecutive days of each 28-day period to determine the specific gravity (Densitometer, Mettler-Toledo, ISO-14001, Switzerland), shell weight, shell thickness and shell breaking strength (Universal Testing Machine, EZ test, 120891-04, Japan).The cleaned egg-shells, dried for twenty-four hours, were weighed and expressed as % of whole egg.The shell thickness was measured at three different locations (middle, broad and narrow end) using a micrometer gauge (Mitutoyo Code, 7027, Japan) and mean value was calculated.
Serum bio-chemical studies : At the end of experimental period, 5 ml of blood was collected from brachial vein from 10 birds in each dietary treatment.Subsequently serum was separated and the levels of Ca (AOAC, 1990), inorganic P (Fiske and Subba row, 1925) and protein (Doumas et al., 1971) in serum were analysed.

Hatchability and performance of progeny
Hatchability of eggs laid by WL layers fed diet with or without vitamin D 3 supplementation was evaluated at 86 weeks of age.All the hens in each treatment were inseminated with pooled semen from males of the same age.Eggs were collected through the 3 rd to 8 th day following insemination and incubated to determine the fertility and hatchability.The chicks hatched were individually weighed to record day old body weight.The chicks were wing banded and reared to 14 days of age in stainless steel battery brooders under uniform feeding and managemental condition to determine the survivability and body weight gain.

Bone mineralization
Six birds from each treatment were selected at random and sacrificed by cervical dislocation at the end of experiment.Both the tibiae were freed from soft tissue and diaphysis, defatted by soaking in petroleum ether for 48 h and dried at 100°C for 12 h.The right and left tibiae were used for determination of bone ash and bone strength, respectively.Dried bone samples were ashed at 600±30°C for 12 h for estimation of bone ash and Ca (AOAC, 1990).Breaking strength on the left tibia was determined by universal testing machine (EZ test, 120891-04, Shimadzu-Japan).

Statistical analysis
Data were subjected to statistical analysis under completely randomized design employing one-way analysis of variance (Snedecor and Cochran, 1989).The means of different treatments were compared with Duncan multiple range tests (Duncan, 1955).Significance was considered at p<0.05 level.

RESULTS
The egg production and egg weight of the birds fed diet without any supplemental vitamin D 3 was comparable with those of supplemental groups (Table 2).Similarly, the levels of the vitamin in the diet did not have any effect either on egg production or egg weight.The average daily feed intake and the feed consumed per dozen eggs or kg egg mass was comparable among all the dietary groups.However, the specific gravity of eggs laid by the birds fed diet without supplemental vitamin D 3 was comparable with either 600 or 2,400 ICU supplemental groups but significantly higher as compared to 300 and 1,200 ICU groups.The egg -shell breaking strength was significantly lowered in the 600 ICU supplemental groups as compared to the comparable strength of other dietary groups.The Haugh unit, egg shell weight, shell thickness, tibia breaking strength, bone ash and calcium content were not influenced due to vitamin D 3 concentration in the diet.
Serum Ca concentration was influenced by vitamin D 3 levels in the diet.The serum Ca concentration of birds fed either control or the vitamin supplemented diet up to 1200 ICU/kg diet was comparable.However, increasing the concentration of vitamin D 3 to 2,400 ICU/kg diet significantly enhanced the concentration of Ca in the serum, which was significantly higher as compared to other dietary groups.The serum concentration of P and protein, however, was not influenced by increasing levels of vitamin D 3 in the diet.
Neither fertility nor hatchability (TES and FES) was influenced by vitamin D 3 concentration in the diet (Table 3).The fertility and hatchability of eggs from vitamin D 3 deficient diet were comparable with those fed diets supplemented with vitamin D 3 upto 2,400 ICU/kg.Supplementing vitamin D 3 at 300 ICU/kg diet to meet the NRC (1994) requirement or linearly increasing to 2,400 ICU/kg diet did not reveal any additional advantage in either fertility or hatchability.Feeding vitamin D 3 deficient diet or supplementation of vitamin to dams did not have any influence on their progeny chicks.The day old and 14 th day body weight of chicks obtained from dams fed either deficient or supplemental vitamin D 3 was comparable.

DISCUSSION
Majority of the parameters including egg production and egg shell quality were not affected by vitamin D 3 concentrations in the breeder diet.Even without supplemental vitamin D 3 , the birds continued to laid eggs without any influence on egg production.The egg weight and shell quality of the eggs of birds fed without vitamin D 3 was also comparable with those of vitamin D 3 supplemental groups.Supplementing vitamin D 3 at 300 ICU/kg diet to meet the NRC (1994) requirement or linearly increasing the concentration to 2,400 ICU/kg did not have any additional advantage on any of the production parameters.Though, egg specific gravity and breaking strength varied due to levels of vitamin D 3 in the diet, no specific trend could be observed.Serum Ca varied significantly due to vitamin D 3 supplementation.Though, serum Ca concentration was highest in the birds fed diet supplemented with 2400ICU/kg, no influenced could be reflected on any of the production parameters.Fertility, hatchability and post-hatch performance of the progeny (14 th day post hatch) was also not influenced due to vitamin D 3 levels in the diet.
Contrary to the findings of the present study, Tsang and Grunder (1990) reported poor egg shell quality in WL layers fed diet without supplemental vitamin D 3 during 59-80 wks of age, which were on a diet containing 3.1% Ca and 1,100 ICU/kg supplemental D 3 prior to the experiment.Withdrawing of supplemental D 3 also reduced the egg production drastically (59 wk-73%: 80 wk-3.8%).However, no effect was observed on average daily feed intake.Hatchability was significantly lowered in the birds fed vitamin D 3 deficient diet.In a 13 weeks experiment with 21 weeks old single comb WL pullets, Abdulrahim et al. (1979) observed significantly lower egg production of the pullets fed the vitamin D 3 deficient basal diets as compared to those fed diets supplemented with 120 to 720 ICU/kg vitamin D 3 in the diet.The egg shell quality was also the poorest in the vitamin D 3 deficient diet.No difference in egg production or shell quality was observed due to variation in the vitamin D 3 (120-720 ICU/kg) content in the diet.Feed consumption, however, was not influenced due to vitamin D 3 levels in the diet.Though egg production, egg weight, shell weight and specific gravity were not influenced due to variation in vitamin D 3 concentration (250, 500 or 2,000 ICU/kg) in the diet, the highest percentage of cracked eggs were observed for hen (54 wk) fed 250 ICU vitamin D 3 per kg diet (Keshavarz, 1996).During a 48week period study with 20 wk old laying hen, Matilla et al. (2004) reported no variation in production performances by supplementing enhanced levels of vitamin D 3 (6,000 or 15,000 ICU/kg) to a control diet (2,500 ICU/kg).Similarly Park et al. (2005) did not find any difference in egg production in laying hens (87 wk) by supplementing higher levels of vitamin D 3 (4,000 or 8,000 ICU/kg) to diet containing 2,000 ICU/kg of D 3.
Most of the studies conducted on requirement and optimization vitamin D 3 in layers was in either temperate countries or environmentally controlled house where vitamin D 3 insufficiency is a common problem because of restricted ultraviolet light exposure (Scharla, 1998).Probably, this may be the reason why in most of the studies vitamin D 3 deficient diet had adverse effect on egg production and shell quality.Enhancing the vitamin D 3 concentration in the diet beyond the requirement (NRC, 1994) did not found to be beneficial in further increasing the production performances (Keshavarz, 1996;Matilla et al., 2004).However, in tropical countries like India where sunlight is more potent and most of the poultry farming is operated in open sided housing system, vitamin D 3 insufficiency may not be a problem that becomes nutritionally important.The birds might have synthesized enough cholecalciferol through adequate sunlight thereby optimizing calcium metabolism (Shen et al., 1981).This could be one of the probable cause that no influence of dietary supplementation of the vitamin on production performance of WL breeders could be observed during the experimental period.
In addition to sun-light many other factors influence the vitamin D 3 requirement such as amount and ratio of dietary Ca and P, their availability, species and physiological factors (MCDowell, 1989).In the present study, the birds from the beginning of the laying period (18 wks) were on a standard diet containing 3.8% Ca and 0.71% P till 72 weeks of age.The same diet was continued during the experimental period of 16 weeks.Probably, these concentration and proportion of Ca and P in the diet were optimum to meet Ca and P requirement of the breeder diet devoid of vitamin D 3 .
Besides the persistence of vitamin D 3 in animals during the period of vitamin D 3 deficiency may be explained by slow turnover rate of vitamin D 3 in certain tissues such as skin and adipose tissue.During the time of deprivation, vitamin D 3 in these tissues is released slowly, thus meeting vitamin D 3 needs of the animal over a long period of time (Miller and Norman, 1984).The birds in the present study had fed diet supplemented with 2400 ICU vitamin D3 /kg diet prior to the experimental study (20-72 wk).The excess vitamin D 3 fed prior to the experiment might have stored in the body and consequently met the requirement during deficient period, thereby maintaining the optimum performance.
Sign of vitamin D 3 deficiency begin to occur in laying hens in confinement about 1 to 2 months after they are deprived of vitamin D 3 and the first sign of deficiency is thinning of the egg shell and subsequent decline in egg production (McDowell, 1989).However, in the present experiment no symptoms of vitamin D 3 deficiency could be noticed even after 4 months of experimental period in birds fed vitamin D 3 deficient diet.Thus, it can be concluded that dietary supplementation of vitamin D 3 may not be essential for optimum production, shell quality, hatchability, and juvenile performance of WL breeders during 72 to 88 weeks of age.

Table 2 .
Influence of vitamin D 3 supplementation on production performance of White Leghorn layer breeders a, b Means with different superscripts in a row differ significantly (p<0.05).* FCR: Feed conversion ratio.

Table 3 .
Influence of Vitamin D3 supplementation on hatchability and performance of progeny chicks Levels of vitamin D 3 (ICU per kg diet) * TES: Total egg set.** Fertile egg set.