Impact of Manipulating Quail Eggs with Betaine During Early Embryogenesis on Chick Development, Hatchability, Hematological Variables, and Some Physiological Estimates

This study evaluated the effect of spraying incubated quail eggs with betaine on chick development, hatchability and some physiological estimates of Japanese quails during early embryogenesis. A total of 750 eggs were equally divided into two groups (2 groups × 5 treatments × 5 replicates × 15 eggs). Eggs in the 1 st group were incubated at normal incubation temperature (37.5°C/NIT), while those in the 2 nd group were incubated at chronic incubation temperature (39.5 °C/CIT) for 3 hours daily from the 4 th up to and 6 th day of incubation. Eggs in NIT and CIT groups were subjected to ve treatments, T1 (negative control), T2 sprayed distilled water (positive control), while T3, T4, T5 groups sprayed distilled water supplemented with 500, 1000 and 2000mg betaine/L respectively. The chick weight at hatch and slaughter weight and rst egg weight was signicantly impaired by CIT treatment. CIT group revealed a signicant increase in the H/L ratio and a signicant decrease in T3 hormone and blood protein levels than the NIT group. Regarding betaine effects, the embryonic mortality rates, hatchability, hatched chick weight, and slaughter weight were signicantly improved compared with the control. Also, betaine signicantly increased blood protein and T3 hormone levels and signicantly decrease the liver enzymes levels and total feed consumption compared with untreated group. The RV/TV ratio of quails in CIT group was signicantly increased, while betaine treatment signicantly decreased this ratio. Considering these results, it’s strongly suggested that spraying of betaine on eggs at 1000 or 2000 mg//L optimizes Japanese quails performance.


Introduction
Japanese quail (Coturnix coturnix japonica) farming offers potential alternatives for meat and eggs . The injection hatching eggs technique during incubation phase is considered a stressful procedure for embryos growing; in addition to, it requires experience and skill for application. Therefore, this study was planned to investigate the effect of spraying hatching eggs exposed to high incubation temperatures with betaine solutions during early incubation phase on chick development, hatchability, hematological variables and physiological status of Japanese quails.

Materials And Methods
The experiment was conducted at the Experimental Poultry Farm, Poultry Production Department, Faculty of Agriculture, Assiut University, Assiut, Egypt.

Preparation of betaine solution
Betaine solutions were freshly prepared by dissolving betaine powder in distilled water. Both betaine mixture solutions and distilled water were set in an incubator at 37.5°C for 2 hours before spraying.

Egg incubation
A total of 750 freshly fertilized Japanese quail eggs were collected from the ock reared under Assiut governorate atmosphere, at 20 weeks of age. All eggs were classi ed into two equal experimental groups (3 group × 5 treatments × 5 replicates × 15 eggs). In the rst group (control), eggs were incubated at 37.5 o C and 55-60% RH (normal incubation temperature/NIT) from day 0 up to 14 days of incubation. In the second group (chronic incubation temperature/CIT), eggs were daily exposed to 39.0 o C and 55-60% for 3 successive days (E4-E6) during the early embryogenesis for 3 hours (14:00-17:00). Eggs in each group were classi ed into ve spraying betaine treatments; in the 1 st one, eggs were not treated (negative control), while those in the 2 nd treatment were sprayed with distilled water only (Positive control). Eggs in the 3 rd , 4 th and 5 th treatments were sprayed with 500, 1000 and 2000 mg betaine per liter distilled water, respectively.

Eggs treatments
Egg surfaces were cleaned by using ethanol 100% before incubation. Eggs in the second group were transferred to the separate incubator and exposed to 39.0°C and 55%-60% relative humidity for three hours daily on days 4, 5 and 6 of incubation. All eggs were horizontally placed, and the solution was sprayed on the egg surface opposite to the air chamber. The eggshell was sprayed with 50 ml betaine per 1000 eggs after heat exposure. All eggs were daily turned automatically 12 times i.e. every 2 hours (Aygun and Sert 2013).

Studied variables
Egg weight loss (amount of moisture loss) The difference in the egg weight before setting in the incubator and on the 6 th day of incubation was used to determine the water loss amount and represented as the initial egg weight percentage.
Embryonic mortality, hatchability traits of incubation and chick weight at hatch At the end of incubation period, the un-hatched eggs were broken to determine the embryonic mortality rate by using the equation: (number of dead embryos/whole viable egg numbers) × 100, the chick cull rate was calculated as abnormal chick numbers/whole viable chick numbers at hatch) × 100. At hatch, the hatchability rate was calculated as (hatch chick numbers/whole egg numbers) × 100 (Molenaar et al., 2011). The incubation time (TEPH/h) was calculated as the hour numbers from setting eggs in the incubator till hatch. All hatched chicks were weighed at hatch by using a digital balance, while relative chick weight (RCW/%) were determined as (hatch chick weight / initial egg weight) × 100. Ninety newlyhatched quails (2 groups × 5 treatments × 3 replicates × 3 birds) were used to cloaca temperatures (°C) temperatures by using a digital thermometer throughout inserting it one cm deep into the hatched-chick cloaca.

Newly-hatched chick quality traits
Approximately after 12 hours from the hatch, 100 dry chicks (2 groups × 5 treatments × 5 replicates × 2 chicks) were randomly taken to determine chick weight and then killed by cervical dislocation to determine the residual yolk sac weight (YSW/g). After drying excessive quail embryonic uids using absorbent papers, yolk sacs, heart, intestine, gizzard, liver and empty chick weight (ECW/%) were weighed and represented as the chick weight at hatch percentage.
Productive traits 300 chicks (2 groups × 5 treatments × 5 replicates × 6 chicks) from hatched chicks were individually weighed and placed in oor wooden pens from the rst day of the growing period up to 15 days and then kept in the battery cages from 15 up to 42 days of age. From one day old till 14days, all chick quails were fed a ration containing 22.52% CP and ME 12.78 MJ kg−1, from days 15 to 28d they were fed a diet containing 22.12% CP and ME 14.14MJ kg−1 of diet, while from 28 to 42 day the chick was fed on a diet 21.0% CP and ME 13.56 MJ kg−1 of diet. The feed and fresh tap water was offered ad-libitum all over the experimental period. The chick quails were daily exposed to 23 lighting hours (30-40 lux m 2 ) during the experiment. All chicks were weekly weighed from hatch up to 42 days of age, while total weight gain (TWG) was measured according to the equation (The nal body weight -initial body weight)/ number of days) during the experiment. Total feed consumption (TFC/g) was measured during the experiment, while feed conversion ratio (FCR) was calculated by dividing total feed consumption (TFC) by total weight gain (g) of chicks. Age at laying rst egg (AFE/day) was calculated and the weight of rst egg (WFE/g) was weighted and recorded.
Hematological parameters, blood proteins, liver enzymes, cholesterol, T3 hormone At 42 days of age, 100 blood samples were collected from females (2 groups × 5 treatments × 5 replicates × 2 birds) into heparinized tubes for hematological analysis. The plasma samples were taken after centrifugation the blood samples at 3000 rpm for 15 minutes. Total protein (mg/dl), albumin (mg/dl) and globulin (mg/dl) were measured by using commercial kits according to (Trinder, 1969) and triiodothyronine (T3) hormone concentration determined by commercial kits. Hemoglobin (Hb) concentration was spectrophotometrically measured; the numbers of red blood cell (RBCs/10 6 ) and white blood cells (WBC/10 3 ) were counted using a hemocytometer as described by (Schalm et al., 1975), while heterophils to lymphocyte (H/L) ratio were estimated as described by (Gross and Siegel 1983). Serum triglyceride, cholesterol, aspartate amino transferase (AST) and alanine amino transferase (ALT) were measured by using their accompanying commercial kits.

Statistical analysis
The data were subjected to statistically analysis by using to general linear model (GLM) of SAS (SAS Institute 2004) according to the model: Y ijk =μ + I i + B j + IB ij + e ijk , Where Y ijk is an observation, μ is the overall mean, I i is the effect of incubation temperature (i=1, 2), B j is the effect of the betaine dose (j=1, 2, 3, 4, 5), IB ij is the effect of interaction between incubation temperature and betaine dose, and e ij is the random error. The differences between least squares means (LSM) were tested at P <0.05 level by using Duncan's multiple range test (Duncan, 1955).

Embryonic mortalities
No signi cant differences were found among treatments for chick cull rate, egg weight, relative loss weight, and embryo quality traits (Table 1). No impact of the embryonic thermal manipulation was observed on the mortality rate. Regarding the effect of betaine treatments, the results revealed a signi cant decrease (P=0.02 & 0.03) in early and late embryonic mortalities. The interactions of incubation temperature and betaine treatments had no signi cant effect on these parameters.
Hatchability traits, chick weight at hatch and cloaca temperature The data presented in Table 2 reveal no signi cant effects of incubation temperatures on hatchability percentages and relative chick weight. The high incubation temperature signi cantly (p=0.023) decreased the incubation period, the TEPH as well as chick weight at hatch than the control group. Furthermore, the cloacal temperature in the NIT (39.61°C) group was signi cantly (p=0.002) lower than those reported in the CIT (39.97°C) group.
Regarding betaine treatments, the results revealed a signi cant increase (p=0.01) in the hatchability percentage and chick weight at hatch. No effect of the interaction between the betaine treatments and incubation temperature on these parameters.

Carcass traits of newly-hatching chicks
After hatch, the results revealed that chick weight before (7.29g) and after the slaughter (7.88g) in NIT group was signi cantly higher (p=0.01) than (6.88 and 7.34g) in CIT group (Tables 3). Also, the heart percentage (1.05%) was signi cantly lower (p=0.01) in the CIT group than (0. 86%) in the NIT group. The liver percentage in the CIT group (3.73%) was signi cantly higher (p=0.002) than (2.92%) in NIT group. The weights of chick before and after slaughtering were signi cantly higher (p=0.04) in betaine-treated eggs (T4 and T5) than the control group. No effect of betaine treatment or interaction between incubation temperature and betaine treatment on the percentages of the intestine, liver and gizzard.

Productive performance
At 42 days after hatch, the results revealed that chick weight at 42 days of age (212.2g) and the weight of the rst egg (7.59g) in CIT group was signi cantly higher (p=0.02) than (217.5 and 8.60g) in the NIT group (Tables 4). No effect of incubation temperatures on the total weight gain, total feed consumption, feed conversion ratio and age at laying rst egg.
The betaine treatments signi cantly (p=0.04 & 0.002) increase the chick weight at 42d, total weight gain and the weight of rst egg. Also, the total feed consumption and feed conversion ratio were signi cantly (p=0.0001) decreased compared with control group. No impact of the interaction between incubation temperature and betaine treatment on these parameters except for total feed consumption and feed conversion rate were signi cantly decreased.

Carcass traits of female quails
At 42 days of age, the results presented in Tables 5 revealed that RV and RV/TV ratio in the CIT group were (0.493 and 0.308g) signi cantly higher (p≤0.05) than (0.418 and 0.205g) in the NIT group. While, spleen percentage (0.049%) signi cantly (p=0.02) decreased than (0.065%) in the NIT group. The RV/TV ratio in quails treated with betaine signi cantly (p=0.001) decreased than those of the control group. No signi cant effect due to interaction between incubation temperature and betaine treatment on these parameters.

Effects of incubation temperature and betaine treatment on female reproductive traits
Data presented in Table 6 showed no signi cant effect of the incubation temperatures on the percentages of ovary and oviduct as well as follicle weights. Regarding betaine treatments (T4, T5) signi cantly (p=0.013) increased oviduct percentage compared with other groups. No effect of the interaction between incubation temperature and betaine treatment on female reproductive traits.
Hematological parameters and blood proteins of female quails From data in Table 7, the RBCs (×10 6 ) and H/L ratio in the CIT group were signi cantly (p=0.01 & p=0.0001) increased, while WBC (×10 3 ), total protein, albumin and globulin were signi cantly (p=0.001) decreased than in the NIT group. Hb (g dL−1) was not affected. Regarding the effect of betaine treatments the WBC (×10 3 ), total protein, albumin and globulin were signi cantly (p=0.001) increased than those of the control groups. No effect due to interaction between incubation temperature and betaine treatment on all parameters.
Liver enzyme triglyceride, cholesterol and T3 hormone of female quails From data in Table 8, the ALT, AST, triglyceride and cholesterol in the CIT group were signi cantly (p=0.001) increased, while T3 hormone (μg L−1) signi cantly (p=0.001) decreased than in the NIT group. Regarding the effect of betaine, the ALT, AST, triglyceride and cholesterol in the treated groups were signi cantly (p≤0.05) decreased, while T3 hormone signi cantly (p=0.027) increased than those of the control group.
No effect of the interaction between incubation temperature and betaine treatment on the liver enzymes, triglyceride, cholesterol and T3 hormone of female quails.

Discussion
In the present study, Incubation temperature manipulation during embryogenesis had negative long-lasting effects on quail development and physiology, including weight and production performance traits. The results revealed incubation period and TEPH were signi cantly decreased by thermal stress similar to Previous studies in broilers revealed heat stress impact on weights of newly hatched chicks as well as weights before and after slaughtering (Abuoghaba, 2017). Similarly in quails, the results showed a negative impact of thermal stress on newly hatched quail weights. The heart weight was signi cantly lower in CIT group than in the control group. Also, in broilers the heart weight was decreased in the heat-stressed group may be related to the increased susceptibility of cardiovascular disorders such as ascites (Leksrisompong et al., 2007, Abuoghaba, 2017. The liver weight was signi cantly higher (p≤0.05) in CIT group than in the control group. These ndings agree with (Sgavioli et al., 2015) who found that incubation heat stress led to an increase in liver weight as well as a decrease in heart weight in broilers. At slaughter age (D42) in female quails, the quail's weight and weight of the rst egg were signi cantly decreased by incubation thermal manipulation this may be due to decrees in the initial body weight of the chick that affected the nal body weight. These results agreed with the ndings of (Hulet et al., 2007).
The slaughter weights of TV (g), and spleen (%) were signi cantly decreased, while RV and RV/TV ratio The betaine spraying in the eggs would improve the feed intake, feed conversion, and body weight gain in post-hatch life this was con rmed by starting egg-laying at earlier ages and improve the weight of the rst egg (Table 4). In this study, when spraying betaine in heat-stressed eggs, the carcass traits were subsequently improved.
Interaction between betaine and incubation heat stress signi cantly increases the EC weight and In conclusion, the spraying betaine in eggs during incubation phase would likely reduce the mortality rate, feed intake and feed conversion as well as improve hatchability rate, chick weight at hatch, chick weight at slaughter age, so that quail birds start the egg-laying at earlier ages. This could be re ected positively on the egg weight and egg quality traits. Moreover, betaine positively improves the WBC, blood proteins, liver enzymes, blood lipids, and thyroid activity. Under heat stress conditions the results showed increase cloacal temperature, RV/TV ratio, H/L ratio, ALT, AST, cholesterol, triglyceride levels in quail. While, the hatching time, chick weight at hatch, chick weight at slaughter age, the weight of the rst egg, TWBC's and blood proteins were signi cantly decreased compared to the control group.
The present data showed that spraying of betaine could reduce the adverse effects of heat stress on RWL, TFC, FCR, carcass weight and RV/TV ratio indicating that betaine has an anti-heat stress role. The bene cial impacts of betaine on quail's performance may be due to its osmoregulatory action, methyl group donors and anti-oxidative effect. Funding This work was not nancially supported by the Assiut University, but was self-nanced at our own expense.
Data availability Data will be made available on reasonable request.
Code availability Data were analyzed using SAS (9.1).
Ethics approval All the procedures used in this study have been approved by the Institutional Animal Ethics Committee (IAEC) of Assiut University, Egypt.

Con ict of interest
The authors declare that no con ict of interest.

Tables
Table1 Impact of incubation temperature, betaine manipulations and their interaction on relative water loss of incubated eggs at 6 days of age and embryonic mortality rates    TWG (g) = Total weight gain (g), TFC (g) = Total feed consumption, FCR (g feed/g meat) = Feed conversion ratio, AFE (day) = Age at laying rst egg, WFE (g) = Weight rst egg (g), NIT= Normal incubation temperature, CIT= Chronic incubation temperature.