Volume 11, Issue 3: 103-108; May 29, 2021  
CHICKENS (Gallus gallus domesticus)  
Lilik KRISMIYANTO, Hanny Indrat WAHYUNI and dan Nyoman SUTHAMA  
Department of Animal Science, Faculty of Animal and Agricultural Science Universitas Diponegoro, Semarang City 50275, Indonesia  
Email: lilikkrismiyanto@lecturer.undip.ac.id;  
Supporting Information  
ABSTRACT: Pelung chicken (Gallus gallus domesticus) is a typical Indonesian local breed originating from  
West Java. Pelung chicken breeding, especially male bird, was initially used as a hobby livestock because  
they have tunable voice. This study aimed to evaluate the effect of feeding lime (Citrus aurantifolia) juice as a  
source of vitamin C on blood parameter and performance of male pelung chickens. The experimental birds  
were 64 male pelung chicken aged 12 weeks, divided into 4 weight groups namely group 1: 740 910 g,  
group 2: 910 1,080 g, group 3: 1,080 1,250 g, and group 4: 1,250 1,420. The treatments applied were  
T0: formulated diet; FD, T1: FD+ lime juice 1%, T2: FD + lime juice 2%, and T3: FD + lime juice 3%. The  
present experiment was assigned in body weight-based randomized block design. Measured parameters were  
heterophile, lymphocyte, heterophile-lymphocyte ratio (H/L), total plasma protein, the relative weight of  
lymphoid organs (spleen and bursa of fabricius) and performance (feed intake, daily body weight gain and  
feed conversion ratio) of birds. The results showed that the feeding diet added with 1-3% lime juice significant  
on heterophile, H/L ratio, the relative weight of spleen, total plasma protein, daily body weight gain and feed  
conversion ratio, but not significant on lymphocyte, the relative weight of bursa of fabricius and feed intake.  
Male pelung chicken fed diet added with 3% lime juice (T3) indicates better blood parameter stability (H/L  
ratio and lymphoid organs), total plasma protein and performance.  
Keywords: Blood parameter, Lime, Pelung chicken, Performance, Vitamin C.  
The are many kinds of local chickens in Indonesia throughout the country, such as Kedu, Sentul, Nunukan, Pelung,  
Sumatra, Bekisar chickens and others. The local chicken population in the last 10 years has increased significantly  
between 249,963,499 to 310,959,951 birds (Directorate General of Livestock and Animal Health, 2020). Pelung chicken  
are local Indonesian poultry originated from Cianjur, West Java, which has existed since 1850 (Asmara et al., 2019). This  
type of chicken is well known as a fancy bird and the body weight of the ready-to-cut at 12 weeks old can achieve  
between 700 800 g (Hidayat and Asmarasari, 2015). Indonesian local poultry, including pelung chickens, are generally  
known to be adaptive well to the tropical environment (Sumantri et al., 2020). However, efforts to maintain body  
resistance in order to be more adaptive to the fluctuating tropical environment need providing natural additives are  
important. Body resistance is generally related to the ability of natural additives work as a source of antioxidants (Zhou et  
The provision feeding diet with additives containing antioxidant and anti-stress substances are very relevant with the  
aim to increase immune-related blood parameter (Surai et al., 2019; Marimuthu et al., 2020). A suitable natural additive  
that is locally available as an antioxidant and anti-stress is lime juice which is rich in vitamin C (Elwan et al., 2019; Ali,  
2020). Shrestha et al. (2012) reported that the content of vitamin C in lime juice is equal to 118.2 140.8 mg per 100 g.  
high temperatures (Sahin et al., 2003) and very efficient in regulating the activity of amylase enzyme and uric acid  
metabolisms in broilers (Imik et al., 2013). Amylase activity and uric acid metabolisms are both in related to the ability of  
glucose and blood protein regulations due to heat stress (Belhadj Slimen et al., 2016). Ferreira et al. (2015) showed that  
vitamin C can prevent the effect of heat on broilers reared at 39 °C which is characterized by the difference in body  
weight gain with the area of meat fiber specifically increase as compared to control.  
Although chickens capable in producing their own endogenous vitamin C, but is not enough to overcome stress  
when reared in the tropical region (Abidin and Khatoon, 2013). Therefore, they require exogenous vitamin C  
supplementation. The use of lime juice as a source of vitamin C is very relevant for raising chickens in the tropical  
environments that possible for the adaptation process. Dietary addition of lime juice is expected to increase blood  
parameter of Pelung chickens, since vitamin C functions as an antioxidant. Although, free radical was not observed in the  
Citation: Krismiyanto L, Wahyuni HI and Suthama dN (2021). Effect of lime (Citrus aurantifolia) juice as source of vitamin c on performance and some blood parameters  
in Pelung chickens (Gallus gallus domesticus). Online J. Anim. Feed Res., 11(3): 103-108. DOI: https://dx.doi.org/10.51227/ojafr.2021.17  
present study, vitamin C is known to be able. Vitamin C is able to donate electrons and hydrogen to the free radicals;  
consequently free radicals become relatively stable. Criteria for blood parameter in present study included ratio of  
heterophile-lymphocytes (H/L) and blood plasma protein. H/L ratio is the main stress indicator in poultry, because the  
higher the ratio number, the severe stress level is experienced, and vice versa (Kusnadi, 2009). Plasma protein is a  
secondary indicator of blood parameter besides the H/L ratio, because it is an indication of an adequacy supply of amino  
acids for antibody formation. The general phenomenon is that the higher plasma protein, the better blood parameter of  
chickens that support productivity.  
Experimental animal and diet  
The experiment used 12-week old of 64 male birds obtained from in situ maintenance (native habitat) in Cianjur,  
West Java, divided into 4 groups based on body weight (group 1: 740910 g, group 2: 9101,080 g, group 3: 1,080–  
1,250 g, and group 4: 1,2501,420 g) and reared for 6 weeks. Basal diet composed of rice bran, yellow corn, white bran,  
soybean meal, meat bone meal, and mix mineral which are prepared with a crude protein content of 14.57% and  
metabolizable energy (ME) of 2,892.03 kcal/kg (Table 1). Lime juice according to treatment level at 1, 2 and 3% were  
mixed with approximately 20 g of diet and given in the morning, to ensure all lime juice can be complete consumed, and  
followed by diet without lime juice to fulfill daily requirement. Experimental diets were provided from 13 week to 18 week  
old after condition period for one week (12-13 week).  
Table 1 - Compositions and nutrient content for formulated diet.  
Rice bran  
Yellow corn  
Wheat bran  
Soybean meal  
Meat bone meal  
Mineral and vitamin mix3  
Metabolizable energy (kcal/kg)2  
Crude Protein1  
Crude Fiber1  
Compositions (%)  
Ether extract1  
Total phosphor  
Sources: Based on chemical analysis at the Laboratory of Feed and Nutrition Science, Diponegoro University; Based on Bolton (1967) as  
follow: 40.81 {0.87 [crude protein + 2.25 crude fat + nitrogen free extract] + 2.5}; Provided minerals per kg of the feed: calcium 32.5%,  
phosphor 1%, iron 6 g, mangan 4 g, iodine 0,075 g, copper 0,3, zinc 3,75 g, vitamin B12 0,5 mg, vitamin D3 50,000 IU.  
Experimental Parameters  
Observed parameters were heterophile, lymphocytes, heterophile-lymphocytes ratio, plasma protein and lymphoid  
organs (spleen and bursa of fabricius) measured at fifth the last week of experimental rearing period. Blood samples were  
taken through the brachial vein with 3 mL syringe. Blood was inserted into tube containing anticoagulant as was used  
sample for heterophile-lymphocyte test and total plasma protein based on Enzyme-Linked Immunosorbent Assay (ELISA)  
method. Chickens were further decapitated and dissected to obtain lymphoid organs (spleen and bursa of fabricius) to  
measure relative weight by dividing with final body weight and multiplied by 100%.  
Experimental design and statistical analysis  
The study was arranged in a randomized block design (RBD) based on body weight and as follows:  
K1 = body weight range 740 910 g with average 835.51 ± 49.48 g  
K2 = body weight range > 910 1,080 g with average 985.69 ± 42.04 g  
K3 = body weight range >1,080 1,250 g with average 1,160.63 ± 55.69 g  
K4 = body weight range >1,250 1,420 g with average 1,369.06 ± 65.97 g  
The study was designed in 4 treatments and 4 groups as replications (4 birds each). The treatment applied:  
T0 = formulated diet  
T1 = formulated diet + lime juice 1%  
T2 = formulated diet + lime juice 2%  
T3 = formulated diet + lime juice 3%  
Data subjected to analysis of variance to determine the effect of treatment. If the treatment indicated significant  
effect, it then followed by Duncan's test at 5% probability (Gomez and Gomez, 1984).  
Citation: Krismiyanto L, Wahyuni HI and Suthama dN (2021). Effect of lime (Citrus aurantifolia) juice as source of vitamin c on performance and some blood parameters  
in Pelung chickens (Gallus gallus domesticus). Online J. Anim. Feed Res., 11(3): 103-108. DOI: https://dx.doi.org/10.51227/ojafr.2021.17  
Ethical Approval  
The in vivo study was supervised by the Animal Ethics Committee of the Faculty of Animal and Agricultural Sciences,  
Universitas Diponegoro and managed in accordance with the recommendations in the basic animal husbandry and  
health protocols referred to in Legislation of the Republic of Indonesia No. 18, 2009.  
Blood parameter and immune organ  
Dietary addition of lime juice indicated significant effect on heterophile, heterophile-lymphocyte (H/L) ratio, total  
plasma protein, and spleen relative weight (p<0.05), but it did not affect lymphocytes and the weight of the bursa of  
fabricius (Table 2). Heterophile and H/L ratio in the treatment with the addition of 3% lime juice (T3) was the same as  
does the feeding of 2% lime juice (T2), but lower than the control/without lime juice (T0) and 1% lime juice treatment  
(T1). The total plasma protein due to with the feeding effect of 2% (T2) and 3% (T3) lime juice was higher than that in T0  
and T1.  
Growth performance  
The dietary inclusion with lime juice had a significant effect (p <0.05) on daily body weight gain (DBWG) and feed  
conversion ratio (FCR), but it did not affect feed intake of diet had the same value. The addition of lime juice at 1% (T1),  
2% (T2), and 3% (T3) significantly (p<0.05) increased on DBWG and higher compared to formulated diet (T0). Feed  
conversion ratio with lime juice addition at 2% (T2) and 3% (T3) were higher than that of T0, but between that of T2 and  
T3 treatments were not different from T1.  
Table 2 - Feeding lime juice on blood parameters and immune organs in male pelung chicken.  
Heterophile (%)  
Lymphocyte (%)  
H/L ratio  
0.080 ns  
Total plasma protein (g/dl)  
Relative weight of spleen (%)  
Relative weight of bursa of fabricius (%)  
abmeans values without common letter are different at p<0.05; T0: formulated diet/FD, T1: FD + lime juice 1%, T2: FD + lime juice 2%, T3: FD  
+ lime juice 3%; * = P<0.05; ns= non significant.  
Table 3 - Feeding lime juice on growth performance of male pelung chicken.  
Feed Intake (g/bird/day)  
DBWG (g/bird/day)  
abmeans values without common letter are different at p<0.05. T0: formulated diet/FD, T1: FD + lime juice 1%, T2: FD + lime juice 2%, T3: FD  
+ lime juice 3%. FCR: feed conversion ratio; DBWG: daily body weight gain; * = P<0.05; ns= non significant.  
Blood parameter and immune organ  
The decreased heterophile and H/L ratio in T3 (Table 2) gave an indication that the vitamin C of lime juice functions  
effectively and the chickens could cope with stress, due to the changes environmental, temperature, from in situ to ex  
situ conditions. The changes in environmental condition are greatly possible for the chickens suffer from the complexity of  
stressor. Vitamin C, or called as the chemical ascorbic acid, is a 6-carbon lactone that can be synthesized from glucose by  
poultry and some other animal species. Even though chicken can synthesis ascorbic acid (vitamin C), it is not enough to  
be able to overcome heat stress which is reported by Ahmadu et al. (2016). Therefore, chicken absolutely needs  
additional vitamin C from exogenous sources which in this study derived from lime juice. The H/L ratio in present study  
was consistent with the report by Kusnadi et al. (2005) that dietary addition of vitamin C at 500 ppm produced lower H/L  
ratio with the average 0.61 than control (0.93). The decreased H/L ratio was supported by lower heterophile even though  
lymphocytes did not change much (Table 2). This condition means that more vitamin C supplementation (from lime juice)  
increases body resistance. The increased some blood parameters impacted to the chicken’s life more comfortable  
because it connected with the function of vitamin C that could reduce heat production due to low body protein catabolism  
In contrast, endogenous vitamin C synthesis in the group of chickens given diet without lime juice supplementation  
(T0) and also those fed diet with low vitamin C supplementation of lime juice (T1) were not able or not enough to cope  
Citation: Krismiyanto L, Wahyuni HI and Suthama dN (2021). Effect of lime (Citrus aurantifolia) juice as source of vitamin c on performance and some blood parameters  
in Pelung chickens (Gallus gallus domesticus). Online J. Anim. Feed Res., 11(3): 103-108. DOI: https://dx.doi.org/10.51227/ojafr.2021.17  
with environmental influences. When environmental condition change, especially in the tropic region with open house  
rearing system, show that it is great possible the adaptation mechanisms is occurred in relation to the decrease in body  
resistance (Dantzer and Mormède, 1983). Vitamin C in the condition of T0 treatment is absolutely needed to be function  
as an antidote or stress control for chickens. In the tropical country, heat stress could suppress blood parameter when the  
intake of micronutrients as antioxidants, especially vitamins C and E was low (Ahmadu et al., 2016) and negatively affect  
that greatly disrupted metabolic physiology, growth, and detrimental to the production performance in poultry (Attia et al.,  
The low effectiveness of vitamin C results in an increase in the H/L ratio that occurs in T0 and T1 treatments is in  
line with the increase in spleen weight (Table 2). The large size of the spleen indicates more and more antigens are  
accommodated so that free lymphocytes in the blood decreases because the spleen is tasked with taking antigens from  
the blood that have been bound to lymphocytes, ultimately increasing the H/L ratio (Jamillah et al 2013). As discussed  
earlier, the H/L ratio is an indicator of the comfort level of poultry life, in which the higher the ratio is, and then the higher  
the discomfort is experienced by poultry (Kusnadi, 2009). Therefore, an unfavorable condition can cause an increase in  
the number of heterophiles and the total white blood cell according to Table 2.  
The results (Table 2) indicated the phenomenon as evidence that vitamin C is indeed as an antioxidant that can  
reduce the negative effects of environmental temperature changes. Gursu et al. (2003) reported that vitamin C can  
reduce the negative effects of new environments through the work of antioxidant systems that important to over-come.  
Vitamin C has 2 hydroxyl groups which are easily oxidized, therefore they can easily release electrons and hydrogen to be  
donated to free radicals so that free radicals were not reactive or became stable (Kusnadi, 2006). Vitamin C is powerful  
antioxidant with 2 mechanisms activity namely conversion to L-dehydroascorbic acid and the formation of ascorbic  
radicals (Ajakaiye et al., 2011). First, conversion to L-dehydroascorbic acid is a back and forth reaction, and molecular  
interchange can form a redox system physiologically back to its original vitamin C activity. Second, through the formation  
of ascorbate radicals that can destroy free radicals that formed by oxygen, such as hydroxyl (OH*), mono-oxygen (O*), and  
superoxide (O2*). The mechanism mentioned above is closely related to other advantages of vitamin C which is able to  
restore the tocopherol radicals to a stable alpha tocopherol (Tamzil, 2014). These mechanisms may be proved by the  
decrease in H/L ratio and relative weight spleen supported by increase total plasma protein (Table 2).  
Growth performance  
The effectiveness of vitamin C in overcoming stress and increasing the ability to adapt to the ex situ condition  
(Semarang) with higher ambient temperatures can also be seen from the increase in plasma protein which further affect  
the productivity (Table 3). Total blood plasma protein in the treatment in T2 (2% lime juice) and T3 (3% lime juice) was  
higher as compared to T0 (without lime juice) and T1 (1% lime juice) (Table 2). The addition of lime juice 2-3% can  
increase total plasma protein. Examples of the normal range of total plasma proteins in broilers was between 4.14-4.53  
g/dL (Fattah et al., 2008). The increase in total plasma protein can be associated with a decrease in uric acid which is an  
indicator of the effect of giving vitamin C, and could increase the efficiency of protein use (Kusnadi, 2006), and decrease  
blood uric acid in laying hens (Saki et al., 2010). The use of protein was related to the effectiveness of controlling stressed  
by vitamin C which could further suppress the body's protein catabolism; thereby, reducing plasma protein 2.6 to 2.9  
mg/dL in blood broilers (Gursu et al., 2003), and decrease plasma uric acid from 6.25 mg/dL (control) become 3.50  
mg/dL (Saki et al., 2010). This phenomenon was supported by the data vitamin C as much as 250 mg/kg can increase  
plasma protein. The result of this study indicated that blood with increased supplementation level of lime juice. Heat  
stress, as indicated that Attia et al. (2017), that were significant decreased protein total (14.1%), albumin (29.2%),  
glucosa (6.8%), and increased H/L ratio (+17.7). Vitamin C supplementation is very helpful in reducing the negative  
effects of heat stress on metabolic parameters by increasing blood parameter and productivity in broiler.  
Vitamin C levels derived from lime juice did not directly affect the feed intake of however; vitamin C had more  
beneficial impact on body resistance (Table 2). Antioxidant compounds in lime juice could increase daily body weight gain,  
especially in T2 and T3 treatments. Antioxidant functions of vitamin C exert of its effect as free radical breaker and  
enzyme cofactor. The present result was supported by Bikrisima et al. (2014) that vitamin C derived from guava juice has  
been known function antioxidant functions as a free radical breaker and enzyme cofactor. Similarly, PokuJnr et al. (2018)  
reported that an addition of vitamin C at 10-30 mg/bird/day could increase feed intake, body weight gain, and FCR in  
Guinea fowl.  
Supplemented vitamin C is the level of 3% lime juice (T3) was indicated blood stability, to increase growth performance  
(daily body weight gain) and feed conversion ratio. The use of 3% lime juice resulted in the best outcomes for male pelung  
chicken growth. Lime juice needs to be extracted to get the real vitamin C.  
Corresponding Author  
E-mail: lilikkrismiyanto@lecturer.undip.ac.id; ORCID: 0000-0001-5634-6965  
Citation: Krismiyanto L, Wahyuni HI and Suthama dN (2021). Effect of lime (Citrus aurantifolia) juice as source of vitamin c on performance and some blood parameters  
in Pelung chickens (Gallus gallus domesticus). Online J. Anim. Feed Res., 11(3): 103-108. DOI: https://dx.doi.org/10.51227/ojafr.2021.17  
Authors’ Contribution  
All authors contributed in research and writing, equally.  
Conflict of interests  
The authors declare that they have no competing interests.  
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Citation: Krismiyanto L, Wahyuni HI and Suthama dN (2021). Effect of lime (Citrus aurantifolia) juice as source of vitamin c on performance and some blood parameters  
in Pelung chickens (Gallus gallus domesticus). Online J. Anim. Feed Res., 11(3): 103-108. DOI: https://dx.doi.org/10.51227/ojafr.2021.17