The Effectiveness of Using a Combination of Saponin and Vaccine Given In Ovo on Hatchability and Early Growth Performance of Local Chickens

. A study has been carried out to determine the correct dosage for using a combination of saponin and vaccine in an in ovo formula on hatchability and early growth performance. There were four levels of saponin use (0.0; 1.5; 3.0; 4.5 microgram) arranged using a factorial design with two factors, namely saponin level as the first factor, while whether or not vaccine was used as the second factor, so there were eight formula combinations. Each formula combination was dissolved in phosphate buffered saline, then injected into the amnion of fertile eggs of Sentul chickens during incubation for 18 days (0.5 ml/egg) using an automatic injector machine with a 20G needle. The results showed that increasing saponin levels combined with vaccines as an ingredient in the in ovo feeding formula results in decreased hatchability. A very significant decrease occurred at the 4.5 µg level. The average hatchability of vaccine treatment was significantly better than those treated without vaccine. There is no interaction effect of the two materials on hatchability. However, the interaction effect was seen in all parameters of early growth performance of Sentul chicken during the 4 weeks of observation. The vaccine treatment will be more effective if it is used separately or not mixed with saponin (0.0 µg), while the use of saponin without vaccine will produce the best weight gain and feed efficiency when used at the highest dose level (4.5 µg).


Introduction
Local chicken farms play a pivotal role in meeting the demand for both meat and eggs, thus making a significant contribution to the industry.Consequently, there is a pressing need to elevate the productivity of these chicken breeds by offering them enhanced care and attention.The introduction of superior local chicken breeds represents a commendable step forward, especially when accompanied by refined feed management practices.Indeed, the performance of these superior breeds can be further optimized through the implementation of effective feed management strategies [1].The feed management approach is to apply in ovo feeding (IOF) technology.
In ovo feeding (IOF) is a technique that entails injecting liquid nutrients directly into the amnion, prompting the embryo to orally consume these nutrients before hatching [2,3].Providing supplements in the form of nutrients during the critical period of embryo growth using in ovo feeding technology is known to improve the nutritional quality of embryos, so that after hatching it is known to provide many benefits, namely: increasing the efficiency of feed use, eliminating aberrant bone growth, increasing muscle growth (especially chest muscles), and increasing the immune response to digestive antigens and reducing posthatching mortality and morbidity [4,5].
Providing nutrients as early as possible (in ovo feeding) is a big opportunity to produce DOC (day old chick) for local chickens that are enriched with important nutrients before hatching.Chicks (DOC) treated with IOF usually have good growth with a low mortality rate.Therefore, the availability of IOF formula ingredients is very necessary.Broadly, the users (stakeholders) of this formula product are the breeder industry that produces DOC.This is the reason why it is important to develop an in ovo feeding formula that has a specific target for increasing local chicken productivity.The use of in ovo feeding techniques in poultry began with the success of preventing Marek's disease through ovo vaccination techniques in the early 80s, so it became known as in ovo vaccine [6].Uni and Ferket [2] advanced this technique by administering precise volumes of nutrition (0.4 -1.2 ml) directly into the amniotic fluid of chicken and turkey eggs.Since then, research into IOF ingredient formulations has continued to develop.The ingredients that can become IOF nutrients are quite diverse, namely: carbohydrates [7 -9], amino acids [10 -13], minerals [7], vitamins [14], fatty acids and other modulators.
It is interesting to combine vaccines with important nutrient components or other compounds as ingredients for in ovo feeding formulas, for example with flavonoid compounds such as saponins.Flavonoids are known to protect cell structure, increase the effectiveness of vitamin C, are anti-inflammatory, and act as antibiotics.In addition, saponins are often used as adjuvants for vaccines.Therefore, a study was carried out aimed at finding out the effectiveness of using a combination of vaccine and saponin as an in ovo feeding formula in local chickens.
The research used 320 fertile eggs from Sentul chickens so that 40 eggs could be distributed per treatment combination.All in ovo feeding (IOF) materials were dissolved in PBS; the volume was 0.5 ml/injection/egg.The procedure of IOF injection in this study refers to the method of Sogunle et al [15] which was modified by Krisnan et al [16] into another method.This modification was necessitated by the utilization of local chicken eggs in this study, which inherently differ in size from eggs laid by purebred chickens.The injection process was carried out to the egg amnion using a 20G needle (0.9 mm diameter and 8 mm long).The injection was done manually when the incubated eggs on day 18 were transferred from setter to hatcher.
Parameters observed included hatchability and early growth performance for 4 weeks (body weight, feed consumption and feed conversion).All data were analyzed using Analysis of Variance (ANOVA) which was processed using SAS 9.

Hatchability
Hatchability is an indicator that shows the level of an egg's ability to hatch.This value will describe the eggs' percentage that hatch from fertile or germinated eggs.Providing treatment during the incubation period (embryo phase) will certainly have an influence on hatchability.Data on the effect of the IOF formula containing a combination of saponin and vaccine on the hatchability of Sentul chickens is presented in Table 1.The results of the study did not show an interaction effect (P>0.05), however, the presence of the vaccine in the formula actually gave significantly different average hatchability results (P<0.05).Differences in treatment effects also occurred due to differences in levels of saponin use.The 4.5 µg level produced a significantly different hatchability percentage (P<0.05)from other levels.
The data in Table 1 shows that the hatchability of Sentul chickens ranges from 66.50 -92.50%.The combination of treatment containing 0.0 µg saponin and vaccination showed the highest hatchability value (92.50%), while the lowest was shown by the treatment 0.0 µg and 4.5 µg saponin and without vaccine.There was a tendency that increasing the use of saponin to 3.0 µg in the unvaccinated treatment showed increasing hatchability, but this would decrease when the level of saponin use was at 4.5 µg.This trend was different from the vaccinated treatment which showed decreasing hatchability as the level of saponin use increased.

Early Growth Performance of Sentul Chicken
Generally, chicken performance can be presented through body weight and growth, feed consumption, and feed conversion value.All data measuring these parameters were presented in Table 2 and Figure 1.Based on the data in Table 2, it can be seen that there was an interaction effect (P<0.05) caused by the combination of treatments on body weight gain.The use of 0.0 µg saponin (without saponin) combined with the vaccine showed better body weight gain compared to without the vaccine.However, when the level of saponin use increased to 1.5 µg, the weight gain looked the same between treatments combined with and without vaccine.Vaccine use showed a significant difference (P<0.05) when the level of saponin use increased to 3.0 µg.The resulting weight gain value was better than the combination without vaccine.The same thing also happened when the level of saponin use increased to 4.0 µg, but the combination with the vaccine resulted in a lower weight gain compared to the combination treatment without the vaccine.These results indicate that the combination of vaccine with saponin as an ingredient in the in ovo feeding formula will be effective at certain doses of saponin use.The data in Table 2 and Figure 1 provide the conclusion that the combination of vaccines with 0.0 µg saponin (without saponin) produces the highest weight gain.In other words, the vaccine will be more effective when used alone or not mixed with other ingredients.Beside that, the use of saponin (without vaccine) will be effective if used at the highest dose (4.5 µg).The combination of vaccines with 0.0-3.0 µg saponin tends to increase feed consumption, but the increase will stop when saponin use reaches the level of 4.0 µg.Similar to the use of saponin without vaccine, feed consumption increased at saponin use levels of 1.5 µg and 4.5 µg, while at the 3.0 µg level it showed the same level of feed consumption as the 1.5 µg level.An interaction effect occurred when the use of saponin increased from 3.0 µg to 4.0 µg.The combination treatment with vaccine showed almost the same feed consumption, while the combination treatment without vaccine was seen to result in increased feed consumption along with increasing levels of 1.0 µg saponin use.
Data on weight gain and feed consumption will influence the resulting feed conversion value.Feed conversion is obtained from the comparison between feed consumption and body weight gain.The smaller the value, the more efficient feed consumption.Based on the data in Table 1 and Figure 2, it can be seen that conversion feed has the same trend as body weight gain.There was an interaction effect (P<0.05) caused by the combination of treatments on the feed conversion value.The combination of vaccines with 0.0 µg saponin (without saponin) produces the best feed conversion.As previously discussed, there is a tendency that vaccines will be more effective when used separately or not mixed with other ingredients.These results are in line with research by Sharma and Burmester (1982) who discovered the in ovo vaccine technique in treating Marek's disease which also underlies the development of the in ovo feeding technique.Besides, the use of the highest dose of saponin (4.5 µg) and without vaccine will produce a better feed efficiency.

Conclusion
Increasing saponin levels combined with vaccines as an ingredient in the in ovo feeding formula results in decreased hatchability.A very significant decrease occurred at the 4.5 µg level.The average hatchability of vaccine treatment was significantly better than those treated without vaccine.There is no interaction effect of the two materials on hatchability.However, the interaction effect was seen in all parameters of early growth performance of Sentul chicken during the 4 weeks of observation.The vaccine treatment will be more effective if it is used separately or not mixed with saponin (0.0 µg), while the use of saponin without vaccine will produce the best weight gain and feed efficiency when used at the highest dose level (4.5 µg).

Fig. 1 .
Fig. 1.Effect of treatment on body weight gain, consumption and feed conversion on Sentul chickens during 4 weeks of observation.

Table 1 .
Effect of IOF Formula (containing a combination of saponin and vaccine) on average hatchability of Sentul chickens (%)

Table 2 .
Effect of treatment on body weight gain, consumption and feed conversion on Sentul chickens during 4 weeks of observation