Embryo Development and Post-Hatch Performances of Kampung Chicken by in Ovo Feeding of L-Arginine

The research was conducted to evaluate embryo development, post-hatch performances, and growth rate of kampung chicken treated in-ovo feeding of L-Arginine. A total of 135 kampung chicken fertile eggs (weight 42-43 g) were used and divided into 5 treatment groups of three replications. They were placed in the semi-automatic incubator. The first group was without in-ovo feeding (negative control); the second group was in-ovo feeding of saline 0.9% (positive control); the 3, 4, and 5 groups were in-ovo feeding of 0.5, 1.0, and 1.5% L-Arginine, respectively. In-ovo feeding of L-Arginine were injected into albumen on day 10 of incubation period using automatic syringe in the narrow end side of egg by inserting needle through a small hole at 10 mm depth. After hatching, all day old chicks were placed in floor pens (1 x 0.5 x 0.5 m) accordance with the previous egg groups. The results showed that in-ovo feeding of L-Arginine increased weight and circumference of the embryo, but did not affect the length of embryo. In-ovo feeding of L-Arginine resulted in a higher body weight gain and a lower feed conversion even though feed intake was not significantly different compared to the control groups. The growth rate performance up to 6 weeks rearing increased significantly by increasing L-Arginine administration to 1.0%. It can be concluded that embryo development and post-hatch performances of kampung chicken were markedly increased by in-ovo feeding of L-arginine.


INTRODUCTION
Kampung chicken is one of native chicken breeds in Indonesia. It is mostly found in rural areas reared in conventional, semi intensive and full intensive systems. Compared with commercial breed, kampung chicken in their original habitat showed a better adaptability and disease resistance (Sulistyoningsih et al., 2013;Azahan et al., 2014;Tamzil et al., 2015), but lower performance as an animal-protein producer (meat and egg).
Muscle mass is an important indicator in determining body weight. Increased muscle mass has a very close correlation with the higher final body weight (Fernandes et al., 2009;Fouad et al., 2012). On the optimum environmental conditions such as nutrition, management, and climate, the number of muscle cells will be the determinant of muscle mass. Grodzik et al. (2013) reported that more muscle number is formed in the embryonic phase, and then more muscle cells will hypertrophy. These conditions will have an impact on the increasing of growth rate, so higher body weight can be achieved in a short time.
Change in the number of muscle cells in poultry is only occurred during the incubation period (embryonic development) (Guo-song et al., 2012;Grodzik et al., 2013). The number of muscle cells in this period will not be changed and only experienced magnification (hypertrophy) after passing through the embryonic phase. The level of growth hormone in the embryonic phase determines the activity of the cell proliferation (Fernandes et al., 2009;De Carvalho et al., 2012). Manipulation of growth hormone levels can be done by increasing the availability of nutrients for embryos, especially protein (Coskun et al., 2014). However, the chicken embryo development occurs outside the body and nutritional needs of embryos have been determined in the process of egg formation, so we need the right method to add exogenous nutrients into the egg.
In-ovo feeding is a technique of adding exogenous nutrition into eggs in the incubation period. In-ovo feeding was reported to increase the performance of chickens after hatching (Daneshyar et al., 2010;Kornasio et al., 2011;Chen et al., 2013;Shafey et al., 2014;Al-Shamery & Al-Suhaib, 2015). This condition is believed to occur because of the optimal development of the embryo during embryogenesis.
L-Arginine is an amino acid that is classified as a semi-essential amino acid . Foye (2005) reported that the administration of L-Arginine in the embryonic phase could increase the growth rate of turkey after hatching, while Murakami et al. (2012) suggested that L-Arginine was an important stimulator of growth hormone release. Another study report that L-Arginine can increase muscle mass (Fernandes et al., 2009). In addition, L-Arginine is also a basic amino acid which has a major role as a stimulator of other amino acids like proline, ornithine, glutamine, and creatine (AlDaraji et al., 2012); as well as compounds that play important roles in the physiological activities of chicken such as nitric oxide, polyamines, and dimethylargininesa (Fouad et al., 2012).
Administration of L-Arginine at a dose of 0.7% in turkeys (Foye et al., 2006) and 1.0% in quail  by in-ovo feeding could increase body weight and post-hatch performance. The present study was conducted to evaluate the effect of in-ovo feeding of L-Arginine on embryo development and post-hatch performance of kampung chicken.

MATERIALS AND METHODS
This research used 135 fertile eggs with an average weight range of 42-43 g which were obtained from 50 weeks of age hens kept in an intensive rearing system. All eggs were placed into a semi-automatic incubator with the temperature of 37.5-38.5 °C and relative humidity of ±65%. The experimental eggs were arranged in a completely randomized design (CRD) with 5 treatments and 3 replications. Each replication consisted of nine eggs. From day 3 until 17 of the incubation period, the eggs were turned 3 times a day (at 07:00, 15:00, and 22:00). On day 7 of the incubation period, the eggs were checked for fertility. The infertile eggs were then replaced with fresh prepared fertile eggs.
In-ovo feeding was applied according to the design of 5 treatment groups. The first group was without injection (negative control), the 2 nd group was injected with saline solution of 0.9% (positive control), the 3 rd group was injected with 0.5% L-arginine (0.5 g per 100 mL saline m/v), the 4 th group was injected with 1.0% L-arginine (1.0 g per 100 mL saline m/v), and the 5 th group was injected with 1.5% L-arginine (1.5 g per 100 mL saline m/v). The material was injected into albumen (0.5 mL/egg) on day 10 of the incubation period. Before being injected, the broad end of eggs was on the top position. The injection was carried out using an automatic syringe into the narrow end of the egg with the depth of 10 mm of the needle length. The injected area was cleaned with alcohol and covered using paraffin and nail paint. Then all eggs were returned back into the incubator.
After hatching, all day old chick (DOC) were placed in arranged floor pens (length x width x height/1 x 0.5 x 0.5 m) in accordance with the treatment arrangement.
Each pen consists of 3 unsexed chickens. Each pen was equipped with 60 watt light bulb, a feeder, and a drinker. Feed (Table 1)   ages of 1, 2, 3, 7, 8, and 9 d, while antibiotics were given at the age of 4, 5, 6, 15, 16, and 17 d. At the age of 4 th day, the vaccination (ND live) was administered via eye drops. The observation of embryo development begins with the breaking of eggs on day 18 of the incubation period (3 eggs per treatment in each replication).
Variables measured were body weight, embryo length, and pectoral circumference of the embryo that were measured after the embryo was separated from the other components such as albumen, allantoin, amnion, and yolk sac. Post-hatch performance was evaluated by measuring variables of feed intake, body weight gain, and feed conversion efficiency. The growth rate was obtained by measuring the body weight of chicken at the ages of 1, 2, 3, 4, 5, and 6 weeks. Data were analyzed following the analysis of variance. If the treatment showed significant effect it was then continued to be tested with Duncan's multiple range test (Gaspersz, 1991).

RESULTS
In-ovo feeding of L-Arginine into kampung chicken eggs on day 10 th of incubation period significantly af-fected (P<0.05) embryo weight, pectoral circumference, body weight gain, feed conversion, and weekly body weight, but did not affect embryo length, and feed intake (Tables 2, 3, and 4). The results showed that inovo feeding of L-Arginine increased embryo weight, pectoral circumference, body weight gain, and feed conversion, and weekly body weight compared to control group. Negative and positive control treatments showed no significant effect on the variables measured.

DISCUSSION
In-ovo feeding of L-Arginine resulted in a higher embryo weight. The increasing of embryo weight may be due to the increase in muscle mass as it is affected by administration of in-ovo feeding. L-Arginine was reported as one of the amino acids that could stimulate muscle development (Zhao et al., 2011;Fu et al., 2016). Based on this report, we assumed that in-ovo feeding of L-Arginine increased L-Arginine content in the muscle and resulting the increased muscle mass and heavy embryo.
L-Arginine has been reported to increase growth hormone (Sahin et al., 2006;McKnight et al., Note: Means in the same rows with different superscripts differ significantly (P<0.05).

Variables
In-ovo feeding Negative control Positive control L-Arginine 0.5% L-Arginine 1.0% L-Arginine 1.5%  Table 2. Embryo development of in-ovo feeding of L-Arginine kampung chickens on day 18 of incubation period Note: Means in the same rows with different superscripts differ significantly (P<0.05).

Variables
In-ovo feeding Negative control Positive control L-Arginine 0.5% L-Arginine 1.0% L-Arginine 1.5%  Table 3. Post-hatch performance of in-ovo feeding of L-Arginine kampung chickens at the age of 42 days Note: Means in the same rows with different superscripts differ significantly (P<0.05). Week
Averages feed intakes of all treatments were not significantly different. This may occur due to the type and form of feed used in all treatments were similar, therefore the feed palatability was not different. This level of palatability proved that the feed intake did not differ.
The value of feed conversion showed a decrease with higher concentrations of L-arginine. Injection of L-arginine about 1.0% and 1.5% resulted in lower feed conversion ratio than the other treatments. Group of chicken with a lower feed conversion ratio showed an improvement of digestive tract performance as might be influenced by the L-Arginine administration. Therefore, the absorption of nutrients was more effective and impact on the derivation of feed conversion. Similar results were also reported by Foye et al. (2009)  As reported by Fouad et al. (2012), L-Arginine stimulated cell proliferation of small intestine. Increasing L-arginine levels in the embryonic phase would lead to an increase in the number of small intestine cells (Lin et al., 2011). Tako et al. (2004) and Cheled-Shoval et al. (2011) explained that an increase in the size of the villi and crypts were the result of the increase in the number of cells because proliferation activities of the small intestine were high at the beginning of growth. Increasing the size of the villi and crypts will lead to an increase in the surface area of the small intestine (Foye, 2005).
This increase caused an increase in nutrient absorption. While Smirnov et al. (2006) and Ospina-Rojas et al. (2013) explained that the increasing of the surface area of small intestine was the result of the increased mucin gene expression by goblet cells.
Although the injection of L-Arginine resulted in a higher body weight, at the early week of rearing period, different concentrations of L-Arginine that were injected did not affect body weight. However, at the late weeks of the rearing period, chicken injected with the higher dose of L-Arginine had the higher body weight. This condition indicated that the higher concentration of L-Arginine injected resulted the higher number of muscle cells. Changes in the number of muscle cells is actually not directly influenced by L-Arginine. L-Arginine is a stimulator of IGFs which will work as a stimulator of proliferation activity of muscle cells in the embryonic phase. Therefore, L-Arginine stimulates the production of IGFs (IGF-1 and IGF-2) through the mechanism of GH/IGFs axis (Fernandes et al., 2009;Hazim et al., 2012). The increasing levels of IGFs is caused by the increased sensitivity of hormone receptors in the mechanism of GH/IGFs axis by L-Arginine (Foye et al., 2009;De Carvalho et al., 2012). While Fouad et al. (2012) reported that Arginine was an essential raw material in the chemical structure of IGFs. Increasing levels of IGFs on embryonic phase would inhibit the activity of myostatin and activate the MAP kinase pathway (Kollias & McDermott, 2008). MAP kinase pathway activity causes cell proliferation process for a long time.

CONCLUSION
In-ovo feeding of L-Arginine increased embryo development of kampung chicken. Injection of 1.0% concentration of L-Arginine in-ovo increased post-hatch performances and growth rate.