Experimental birds and collection of eggs
The study was carried out during the period of February 2019 to June 2020, in Department of Veterinary Physiology and Biochemistry and Poultry Farm situated in College of Veterinary and Animal Sciences, CSKHPKV, Palampur, Himachal Pradesh. Total 48 healthy layer birds with 24 from DND and 24 from DR of 16-18 weeks of age were randomly selected and reared upto 70 weeks in different pens separately on deep litter system with nest boxes. Birds were offered standard layer feed (Table 1) and
ad libitum drinking water. Total 90 egg samples were randomly collected with 45 from DND and 45 from DR. Freshly laid eggs were randomly collected in morning, labelled and dry-cleaned with sandpaper to remove feaces and dirt from outer egg shell for further investigations.
External physical egg characteristics
Egg weight was measured using digital balance; egg length and breadth was measured with help of digital vernier caliper to the nearest. Egg shell thickness was measured at broad, narrow and middle portion of egg using digital vernier caliper. Egg shell weight was measured after washing egg shell under running tap water to remove sticking albumen and chalazae residue and further air dried overnight and weighed on a digital balance.
Total egg surface as S = 4.68*P
2/3
Where
S is total egg surface and P is egg weight in grams.
(Sirri et al., 2018) were also determined.
Internal physical egg characteristics
Egg was broken on flat surface and allowed to stand for five minutes. Egg yolk and albumen heights were measured with help of spherometer. Albumen height was recorded twice and average value was calculated. Yolk and albumen were carefully separated. Yolk was placed on Whatmann filter paper no. 1 and rolled to remove albumen residues and chalazae. After that outer membrane of yolk was punctured with rat tooth forceps and pure yolk was poured into pre-weighed graduated glass beaker. Thus weight and volume of yolk and albumen was determined with electronic balance and by observing graduated scale on beaker respectively. Haugh Unit (HU) was estimated by using the following formula-
HU = 100 * log (h - 1.7W0.37 + 7.6)
Where,
h is height of albumen in mm and W is weight of egg in grams.
Biochemical parameters of egg
Total proteins estimation
Total proteins (grams/egg) were estimated by Biuret method. Standard protein solution of different concentrations
viz. 1, 2, 3, 4, 5 mg/ml was prepared by dissolving different concentrations of serum bovine albumin in distilled water. For blank 6 ml of distilled water was taken. For protein standard 1 ml of protein standard solution, 4 ml of 0.75 Normal (N) sodium hydroxide and 1 ml of biuret reagent were added and mixed. Let it stand for 20 minutes at room temperature. Absorbance was measured at 545 nm in spectrophotometer. Calibration curve was prepared (Fig 1).
1 ml of albumen and 0.5 ml of yolk samples were diluted with 1ml of distilled water in different test tubes. For blank 5 ml of 0.75 N sodium hydroxide and 1 ml of biuret agent were added. For test 100 µl of diluted sample, 4.9 ml of 0.75 N sodium hydroxide and 1 ml of biuret reagent were added and mixed. Let it stand for 20 minutes at room temperature. Absorbance measured at 545 nm in spectrophotometer. Protein concentration was measured by using calibration curve after multiplying with dilution factor. Protein concentrations of albumen and yolk fractions were added to determine total protein in mg/ml.
Total cholesterol estimation
Total cholesterol in yolk (mg/egg yolk) was estimated by cholesterol oxidase peroxidase method
(Pasin et al., 1998).
Total glucose estimation
Total glucose (mg/egg) was estimated by glucose oxidase and peroxidase method (
Lott and Turner, 1975).
Total lipids estimation
Lipids (grams/egg) were estimated by modified Folch method
(Folch et al., 1957, Washburn and Nix, 1974 and
Washburn, 1989).
1 gram of yolk and 1 gram of albumen samples was weighed in different test tubes with screw cap. Add 15 ml of 2:1 chloroform: methanol to each sample. Test tubes were shaken vigorously with hand. Add 5 ml of distilled water and again shake with hand. Centrifuge test tubes for 10 minutes at 2500 rpm. There was formation of biphasic layer. Top water-methanol layer was discarded. Bottom layer of chloroform was filtered through Whatmann filter paper no. 1 into pre-weighed test tubes. These test tubes were placed in boiling water bath with agitation until chloroform was evaporated. Test tubes were kept in dessicator containing copper sulphate overnight. After that test tubes were weighed on electronic balance. Grams of total lipids per egg were obtained by adding lipids in yolk and albumen samples.
Incubation, candling and hatching
200 eggs were taken with 100 from DND and 100 from DR at 30-32 weeks of age. Immediately after collection eggs were weighed. Fumigation of eggs was done for 20 minutes in fumigation chamber by adding 1 part of potassium permagnate and 2 parts of formalin. Egg setter trays were stored at 65°F in cold storage room with 75% relative humidity (R.H.). Egg setter trays were loaded in setter on batch basis for first 18 days at 99.5°F to 99.75°F temperature and 55-60% R.H. Autorotation of eggs was done after every one hour at 45° angle. Candling of eggs was done on 18
th day using mass candler. Entire tray of hatching eggs was placed over mass candler in dark room and eggs through which light passed were infertile and were discarded. Eggs were transferred to hatcher. Eggs were kept in horizontal position in hatcher for last 4 days at 98.5°F temperature and 65-70% R.H.
Upon hatching vaccination of day-old chicks was done against Marek’s disease in nape of neck by subcutaneous route. Weight of day old chicks was also recorded. The chicks were reared in brooder with 2" heighted litter material, 1.5’ heighted brooder guard for one week at 95°F temperature, 60% R.H. and proper ventilation to determine chick survivability. Chicks were fed crumbled feed which was further broken into fine particles and
ad libitum drinking water.
Percentage of fertility and hatchability of eggs were determined by following formulas-
Hatchability (%) was determined on fertile egg set (FES) basis and on total egg set (TES) basis:
Health of progeny of each breed was determined by estimating chick survivability (%)-
Statistical analysis
All the experimental data were analyzed using Graph Pad INSTAT by using unpaired-‘t’ test at 5% level of significance.
