DESCRIPTION OF PROBLEM
Nowadays, the growth and production of broilers is a crucial source of meat. Many years of genetic selection resulted in the rapid growth of modern breeds of broiler chickens in a short period. Commercial breeding programs, balanced nutrition, and the good health status of birds result in the high effectiveness of poultry production (Sobolewska et al., 2017). Hatchery objectives are to obtain high hatchability with day-old chickens of optimal quality to maximize profitability while commercial farms prefer high-quality chicks as key determinants of a successful and profitable production cycle (Narinç, 2022). Improving hatchability, chick quality, and performance will mean additional profit for hatchery and farm managers (Kuka et al., 2023).
In ovo feeding of substances such as antioxidants during incubation may improve the antioxidant status of the chicken embryo and posthatch growth phases (Yigit et al., 2014; Elsaadany, 2019). Moreover, in ovo feeding of chickens with extracts from numerous plant products has enhanced their defenses against the contagious bursal virus, avian influenza virus, and fowl pox virus (Sood et al., 2013; Nyandoro et al., 2014) The antioxidant level of the chicken embryo may be enhanced by in ovo injection of antioxidants because they have an effective defense against free radicals (Salary et al., 2014). Recently, attention has been shifted to the use of herbal additives as growth promoters and antioxidant components from herbs, spices, and their products (Oke et al., 2016; Oke et al., 2017; Oke, 2018; Voemesse et al., 2019; Tokofai et al., 2020; Kpomasse et al., 2021; Adjei-Mensah et al., 2022; Kpomasse et al., 2023).
Though the results are promising, there exist some contradictions mainly due to the varying nutrient compositions of the substances, extraction techniques employed, the dose and the moment of injection (N’nanle et al., 2017; Oke et al., 2021; Sogunle et al., 2022; Adjei-Mensah et al., 2022), which necessitate the exploration of more materials, especially plant materials to validate the in ovo injection approach. Plants are a rich source of essential nutrients (Samtiya et al., 2021), chemicals and other active components (Radha et al., 2021).
The seeds in citrus fruits are classified as part of the citrus waste. According to Nobakht (2013), the seeds of citrus fruits contain active antioxidants, including a mixture of flavonoids, isoflavones, flavones, anthocyanins, coumarins, lignans, catechins and epicatechins. Lime (Citrus aurantiifolia) seed contains approximately 8.78% crude protein, 37.95% carbohydrate and 5.01% fiber (Boubekri, 2014). Hence, it is hypothesized that injecting lime seed extracts into broiler eggs during prehatch will enhance the hatching profile and growth performance as well as improve the health and intestinal morphology of birds. Therefore, the current study was designed to evaluate the effects of in ovo feeding of Citrus aurantiifolia seeds extract on hatchability, chick quality, blood and thyroid parameters, intestinal morphology and posthatching growth performance of broiler chickens.
MATERIALS AND METHODS
Ethics
This study was approved by the ethics and scientific committee of the Regional Center of Excellence in Poultry Science, University of Lome (CERSA/UL) and was carried out at the Centre’s experimental unit and laboratory (CERSA/UL).
Experimental Design
A total of 750 Cobb 500 breeder eggs with an average weight of 66.9±1.2 g from a 48-wk-old flock from the research farm of CERSA, University of Lome were used for this study. The eggs were incubated at standard incubation conditions including a temperature of 37.6°C, relative humidity of 50 to 60% and turning once an hour until d 18 of incubation in a Royal Pas Reform (SmartPro) combi incubator, Netherlands. At d 18 of incubation, the incubated eggs were candled and the eggs with evidence of a living embryo were divided into 4 groups of 126 eggs each. The groups were divided into 3 replicates of 42 eggs and each replicate was in a hatch basket. The baskets were randomly put in the hatcher. These groups were (1) Control: Eggs without injection, (2) Ext0.5µg: eggs injected with 0.5 μg/ml of Citrus aurantiifolia seed extract (CASE), (3) Ext0.75µg: eggs injected with 0.75 μg/ml of CASE and (4) Ext1µg: eggs injected with 1.0 μg/ml of CASE. The injection volume for all groups was 0.2 ml/egg.
Citrus Aurantiifolia Seeds Extraction
Citrus aurantiifolia seeds used in this study were collected from fresh matured and unaffected fruits and air-dried at 16°C for 3 wk. The dried seeds were pulverized into powder using a laboratory crusher (Culatti, France) equipped with a 10 µm mesh sieve and weighed. The extraction was done with water and ethanol. A mass of 400 g of crushed Citrus aurantiifolia seeds was dissolved in 4 l of an ethanol-water mixture (70/30, v/v). The mixture was brought to room temperature and macerated with stirring for 72 h then the recovered extract was filtered. The filtrate obtained was evaporated in a rotary evaporator (G1 Heidolph, Germany) at 45°C. The extract was then placed in a Petri dish in an oven at 40°C until drying (Boubekri, 2014) to obtain 112 g of hydro-ethanolic extract.
Citrus Aurantiifolia Seeds Phytochemical Elements and Antioxidant Capacity
The hydroalcoholic extract stock solution was tested for chemical groups (alkaloids, flavonoids, polyphenols, saponins, limonins) by qualitative chemical analysis (coloring tests) according to methods described by Harborne and Harborne (1973) and Chhabra and Uiso (1990). The antioxidant activity of the hydroalcoholic extracts of the seeds was examined by the diphenylpicrylhydrazine (DPPH) test and the ferric reducing antioxidant power (FRAP).
In the DPPH radical test, the purple-colored DPPH was reduced to a yellow compound, diphenylpicrylhydrazine, whose color intensity is inversely proportional to the reducing capacity of the antioxidants present in the medium (Sanchez-Moreno, 2002). The antioxidant activity was estimated according to the following equation (Fatima et al., 2016):
With AA: Antioxidant Activity and Abs: Absorbance at 515 nm. The effective concentration (EC50) was determined according to line The ratio is given as follows:
with, a = the directing coefficient of line f(C) = % inhibition; b = the ordinate at the origin (Patricia et al., 2014).
The ferric-reducing antioxidant power, in the ethanolic extracts, was determined according to the method described by Bougandoura and Bendimerad (2013). It is based on the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) by antioxidants which give a blue color according to the following reaction:
with RH: Antioxidant active substance. R*: Oxidized active substance. Ascorbic acid was used for the positive control, whose absorbance was measured under the same conditions as the samples. An increase in absorbance thus corresponds to an increase in antioxidant activity (Ghaisas et al., 2008). The reducing power of iron is expressed by the RP50, which corresponds to the concentration of the sample giving an absorbance of 0.5.
Citrus Aurantiifolia Seeds Extract Injection
Approximately, 20 ml of saline solution (0.9% of NaCl) was added to 10 mg of CASE and homogenized with a vortex to obtain a 500 ug/ml solution. After successive dilutions, a series of injectable solutions containing respectively 0.5 μg/ml, 0.75 μg/ml and 1 μg/ml were obtained. Injection of CASE was made in the air chamber of the living embryo grouped in treatment during d 18 of incubation. A needle with a gauge size of 18 G was utilized to create 2 holes in the shell, specifically above the air chamber. This was done to reduce the pressure inside the chamber and aid in the retention of the injected solution. The injection volume for all groups was 0.2 ml per egg, and it was administered within the air cell using an automatic syringe equipped with a 13 mm needle. After injection into 1 of the holes, both holes were sealed with adhesive tape and the egg was placed in the hatching baskets.
Hatching Event and Chick Quality Assessment
Beginning from 462 until 506 h of incubation, eggs were observed every 2 h individually for hatching events such as internal piping, external piping, and chick emergence. The following parameters were evaluated for each treatment according to Zhong et al. (2018).
The incubators were stopped at 510 h and unhatched eggs were cracked, classified as “infertile eggs” (candling error) and eggs with dead embryos and hatchability was expressed as the percentage of fertile eggs:
At hatch, day-old chicks were recorded according to treatment, weighed and subjected to chick quality assessment using the Tona scoring system (Tona et al., 2003). The quality score for a chick was defined as the sum of the scores assigned to each quality parameter (activity, down and appearance, eyes, conformation of legs, navel area, yolk sac, and remaining membranes and yolk were scored).
Sample and Data Collection
At hatch, blood samples were collected, from 6 birds per treatment (2 birds per replicate). The blood samples collected were stored in 2 Ethylenediaminetetraacetic acid (EDTA) tubes and 1 dry tube. The samples in 1 EDTA tube and the 1 in a dry tube were then centrifuged at 3,000 rpm for 15 minutes to obtain either plasma or serum. The serum and plasma samples obtained after centrifugation were stored at -20°C until used for subsequent analysis. On the serum, colorimetric method by means of commercial kits with Mindray Biochemical analyzer was employed for the determination of serum total protein, glucose, creatinine, total cholesterol and serum thyroid hormones: T4 (thyroxine) and T3 (triiodothyronine) was also measured using immunofluorescence technique on FineCare kit (FS-113) with an automatic system (Biomerieux Mini Vidas). The measurements of antioxidant capacities in plasma by superoxide dismutase (SOD) and Malondialdehyde (MAD) were done. Superoxide dismutase (SOD) was determined spectrophotometrically as described by (Nishikimi et al., 1972) and malondialdehyde (MDA) was measured using the colorimetric method with a spectrophotometer at a wavelength of 532 nm (Placer et al., 1966). The samples in the second EDTA tube were analyzed for the following parameters: Hemoglobin, white blood cell (WBC), red blood cell (RBC), Hematocrit, lymphocyte, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) using an automatic analyzer (DYMIND H36®).
After blood collection, birds were humanely slaughtered. The weights of some organs: liver, gizzard, heart, spleen, bursa of Fabricius and hatching muscle were measured. The relative weight of each organ was then determined by the ratio of the weight of the organ to the live weight of the bird expressed as a percentage.
Intestinal Morphology of Day-Old Chick
To study the intestinal morphology, the jejunum and ileum sections of the small intestine of the 6 birds per treatment slaughtered, were separated and their length was measured. Then, 2 centimeters were removed from the middle part of each intestinal segment (jejunum and ileum). The contents of the intestine were emptied from the isolated segments, and tissue blocks were prepared after fixation, dehydration, clearing, and being placed in paraffin. After staining (Alcian blue), a light microscope (Thermo Fisher Scientific, US, Massachusetts) was used to examine and capture images of the slides, and the villus height and crypt depth were measured and then their ratio was determined (Poorghasemi et al., 2018). The pH in the intestinal compartments was measured with Hanna® pH meter.
Posthatch Performance
According to each treatment, the hatched chicks were divided into 6 replicates of 15 birds and were assigned to a corresponding floor pen. Chickens were reared for 6 wk. In this period, breeding conditions were the same for all chicks in terms of temperature changes and vaccination time and free access to feed and water was provided. The diets (Table 1) used in this experiment were the same for all experimental groups. Weekly, body weight and feed intake of each replication were recorded to determine the feed conversion ratio and weight gain of each treatment correct for mortality.
Table 1. Ingredients and chemical composition of the standard broiler diet.1
| Items | 1–21 d | 22–42 d |
|---|---|---|
| Ingredients (kg/100kg) | ||
| Yellow maize | 57 | 60 |
| Wheat bran | 4 | 7 |
| Soybean cake | 18 | 15 |
| Soybean meal | 15.6 | 12.6 |
| Oyster shell | 2 | 2 |
| Lysine | 0.2 | 0.15 |
| Methionine | 0.2 | 0.2 |
| Dicalcium Phosphate | 1.1 | 1.35 |
| NaCl | 0.4 | 0.2 |
| Concentrate2 | 1.5 | 1.5 |
| Nutrient composition | ||
| Metabolizable energy (Kcal/kg) | 2,902.70 | 3,059.45 |
| Crude protein (%) | 22.33 | 20.59 |
| Crude fiber (%) | 4.18 | 3.88 |
| Crude ash (%) | 2.77 | 2.86 |
| Crude fat (%) | 5.6 | 5.00 |
| Calcium (%) | 1.10 | 1.07 |
| Available phosphorus (%) | 0.67 | 0.63 |
| Lysine (%) | 1.16 | 1.34 |
| Methionine (%) | 0.50 | 0.61 |
| Methionine + cystine (%) | 0.85 | 0.81 |
- 1
-
Diets were formulated based on total amino acids basis and not digestible amino acids.
- 2
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Concentrate: Soybean meal, rapeseed meal, sunflower seed meal, corn gluten feed, vinasse, soybean oil, palm fatty acids, sodium chloride. Vit. A, 12000 IU, vit. E, dl-α-tocopheryl acetate) 20 mg, menadione 2.3 mg, Vit. D3, 2200 ICU, riboflavin 5.5 mg, calcium pantothenate 12 mg, nicotinic acid 50 mg, Choline 250 mg, vit. B12 10 μg, vit. B6 3 mg, thiamine 3 mg, folic acid 1 mg, d-biotin 0.05 mg. Trace mineral, mg/kg of diet): Mn 80 Zn 60, Fe 35, Cu 8, Selenium 0.1 mg.
Statistical Analysis
The analysis of the differences between the groups was carried out using 1-way analysis of variance (ANOVA) with the post hoc Tukey multiple comparison test using Minitab statistical software (Minitab 21) following the model: yij = μ + τj + εij, where μ = general mean, τ = treatment effect and ε= random error. A Chi-square test was used for the mortality and hatchability percentage analyses. The CASE injection concentrations served as the experimental units grouped in a completely randomized design. The data were adjusted to satisfy the assumptions of ANOVA, and all of the assumptions were checked using the Shapiro-Wilk test for normality (Levene’s test for homogeneity of variance). The results are expressed as mean ± SEM and considered significant at P < 0.05.
RESULTS AND DISCUSSION
Phytogenic feed additives have been used in animal feed for many years, and their popularity has recently increased since consumers demand natural products (Alagawany et al., 2021). With the growing interest in the use of in ovo phytobiotics, this study evaluated the effect of in ovo feeding of Citrus aurantiifolia seeds extract on the hatching profile and overall performance of broiler chickens posthatch.
Citrus Aurantiifolia Seeds Phytochemical Elements and Antioxidant Capacity
The phytochemical screening carried out on CASE revealed that polyphenols and flavonoids were strongly present in Citrus aurantiifolia seed while the presence of alkaloids, saponins and limonin were moderate (Table 2). The antioxidant capacity of the CASE evaluated in this study is presented in Table 2. Based on the calibration curve, which used ascorbic acid as a reference for the antioxidant DPPH test, the hydroethanolic extract of the studied seed has an effective concentration (EC50) of 49.52 ± 0.06 µg/ml. The reducing activity, determined from the calibration curve using ascorbic acid as a reference antioxidant for the FRAP test, showed an average RP50 of 13.21 ± 0.12 µg/ml. The growing interest in the beneficial effects of antioxidants on health has led to the development of a large number of tests to determine the antioxidant capacities of natural plant extracts (Mohammed et al., 2022). Two methods were used in the present study. This present finding showed a good capacity for reducing the DPPH radical of the hydroethanolic extract of the Citrus aurantiifolia seed. The ferric-reducing antioxidant power revealed for the Citrus aurantiifolia seed studied could be explained by its content of phenolic compounds which are also influenced by the method of extraction and analysis. High quantities of phenolic compounds are present in extracts of citrus seed which exhibit antioxidant activity, total flavonoids, free radical activity and reducing power. These constituents help the seed extracts to be effective as natural antioxidants (Al-Anbari and Hasan, 2015). The findings of this present study are similar to those observed by Mohanapriya et al. (2013) and a report by Rahman et al. (2019) on lemon seed extract which showed a strong presence of flavonoids and polyphenols. Previous studies have shown the correlation between the presence of phenolic compounds in an extract and its antioxidant activity (Diouf et al., 2009). This implies that introducing Citrus aurantiifolia seeds into the diet of animals may help combat oxidative stress.
Table 2. Results of phytochemical screening and contents of total polyphenols and flavonoids of Citrus aurantiifolia seed extract.
| Phytonutrients | Hydro-ethanolic extract from the seed of C. aurantiifolia |
|---|---|
| Flavonoids | +++1 |
| Polyphenols | +++1 |
| Alkaloids | ++2 |
| Saponin | ++2 |
| Limonins | ++2 |
| Tannins | +3 |
| Lectins | +3 |
| DPPH test (Effective concentration of extract which inhibits 50% of the DPPH radical) | 49.52 ± 0.06 |
| FRAP test (Concentration of the sample giving an absorbance of 0.5) | 13.21 ± 0.12 |
- 1
-
+++: Highly present.
- 2
-
++: Moderate present.
- 3
-
+: Present.
In ovo Feeding of CASE on Hatching Event, Hatching Parameters and the Quality of Hatched Chicks
Hatching events (Table 3) were significantly advanced by in ovo feeding of CASE (P < 0.05). The hatch window was significantly decreased (P < 0.001) in the incubated eggs injected at 0.75 and 1.0 µg/ml compared to the control and in ovo injection at 0.5 µg/ml. However, the average hatch time significantly decreased (P < 0.001) in the experimental CASE-injected treatments relative to the control. Hatch duration was significantly shortened (P = 0.043) in the incubated eggs injected with 1.0 µg/ml concentration compared to the control. Internal pipping duration was not affected by in ovo injection of CASE (P > 0.05) but in ovo injection of CASE at 0.5 µg/ml markedly shortened (P = 0.037) external pipping compared to the control. Table 4 shows the results on the effect of in ovo feeding of CASE on hatching parameters and the quality of the newly hatched chicks. The hatchability and mortality of incubated eggs that received 1.0 μg/ml had significantly lower and higher percentages, respectively compared to control eggs and CASE injected at 0.5 μg/ml (P = 0.001). The body weight at hatch was significantly heavier (P = 0.001) in the group that had CASE injected at 0.5 μg/ml compared to the other groups. The chick quality score did not differ across the treatments except for the eye score (P > 0.05). The eye score obtained in the control was significantly lower (P < 0.001) compared to other treatments.
Table 3. Effect of in ovo feeding of Citrus aurantiifolia seeds extract on the hatching event of chicken eggs.
| Empty Cell | Experimental groups | Empty Cell | Empty Cell | |||
|---|---|---|---|---|---|---|
| Parameters | Control | Ext0.5µg | Ext0.75µg | Ext1µg | SEM1 | P-value2 |
| Average hatch time (h) | 493.05a | 492.41b | 492.42b | 492.26c | 0.053 | <0.001 |
| Internal piping duration (h) | 12.70 | 11.95 | 11.83 | 11.87 | 0.541 | 0.323 |
| External piping duration (h) | 11.10a | 9.87b | 10.75ab | 10.66ab | 0.438 | 0.037 |
| Hatch duration (h) | 23.68a | 22.60ab | 22.54ab | 21.81b | 0.715 | 0.043 |
| Hatch window (h) | 24.00a | 24.00a | 22.00b | 22.00b | 0.516 | <0.001 |
Abbreviations: h (hours); Control (eggs without injection); Ext0.5µg (eggs injected with 0.5 μg/ml of Citrus aurantiifolia seeds extract, CASE); Ext0.75µg (eggs injected with 0.75 μg/ml of CASE); Ext1µg (eggs injected with 1.0 μg/ml of CASE).
- a,b,c
-
Different letters indicate significant differences between means within rows (P < 0.05).
- 1
-
SEM: pooled standard error of means.
- 2
-
P-value: Probability.
Table 4. Effect of in ovo feeding of Citrus aurantiifolia seeds extract on hatching parameters and the quality of hatched chicks.1
| Empty Cell | Experimental groups | Empty Cell | Empty Cell | |||
|---|---|---|---|---|---|---|
| Parameters | Control | Ext0.5µg | Ext0.75µg | Ext1µg | SEM2 | P-value3 |
| Hatchability of fertile (%) | 90.83a | 90.44a | 89.16ab | 87.50b | 0.711 | 0.001 |
| Mortality (%) | 9.17b | 9.56b | 10.83ab | 12.50a | 0.714 | 0.001 |
| Egg weight at setting (g) | 66.94 | 66.94 | 66.93 | 66.94 | 0.005 | 0.895 |
| BW of hatched chicks (g) | 46.57b | 47.87a | 46.69b | 46.51b | 0.317 | 0.001 |
| BW at hatch/Egg Weight (%) | 69.57b | 71.51a | 69.75b | 69.48b | 0.474 | 0.001 |
| Overall chick quality | 96.86 | 97.33 | 97.20 | 97.13 | 1.17 | 0.982 |
| Activity | 5.8 | 5.8 | 5.6 | 5.6 | 0.34 | 0.877 |
| Down and appearance | 9.93 | 10.00 | 10.00 | 9.86 | 0.08 | 0.295 |
| Retracted yolk | 12.00 | 12.00 | 12.00 | 11.00 | 0.39 | 0.108 |
| Eyes | 14.53b | 16.00a | 16.00a | 16.00a | 0.25 | <0.001 |
| Legs | 15.20 | 16.00 | 16.00 | 16.00 | 0.31 | 0.059 |
| Navel | 11.40 | 11.20 | 11.20 | 10.86 | 0.57 | 0.829 |
| Remaining membrane | 11.73 | 11.73 | 11.60 | 11.66 | 0.279 | 0.957 |
| Remaining yolk | 15.73 | 15.60 | 15.06 | 15.86 | 0.344 | 0.108 |
Abbreviations: Control (eggs without injection); Ext0.5µg (eggs injected with 0.5 μg/ml of Citrus aurantiifolia seeds extract, CASE); Ext0.75µg (eggs injected with 0.75 μg/ml of CASE); Ext1µg (eggs injected with 1.0 μg/ml of CASE).
- a,b,c,d
-
Different letters indicate significant differences between means within rows (P < 0.05).
- 1
-
n= 3 replicates of 10 chicks per replicate for chick quality.
- 2
-
SEM: pooled standard error of means.
- 3
-
P-value: Probability.
After hatch, the chicks may undergo a further holding time of up to 72 h. This period can be stressful for the chicks, as they are deprived of feed and water, leading to a period of starvation (Akosile et al., 2023). It was found in this present study that the hatch window, hatch duration and average hatch time decreased with the increasing level of CASE injection. This result might be explained by a higher metabolic rate in these embryos. Probably, the extract injected increased the swallowing of the amniotic fluid by stimulating the production of corticosteroids which can have led to the activation of thyroid hormones which play an important role in the hatching process (Decuypere and Bruggeman, 2007). Similarly, Ngueda et al. (2021) found that injection of Manihot esculenta extract solution on d 18 of incubation reduced the total incubation duration. In contrast, Tona et al. (2021) observed a longer hatching time when incubated eggs were injected with selenium. This variation might be attributed to the pure mineral nature of the selenium while this present study used a citrus seed which could have several mineral compounds.
By employing various assessment methods and supplementing hatchery eggs with the appropriate amount of phytogenic feed additives, we can enhance embryo growth. Consequently, this results in improved chick weight and quality parameters, ultimately optimizing the overall quality of chicks and fostering the healthy growth of broiler chickens (Akosile et al., 2023). In this experiment, the injection of CASE at 1.0 µg/ml into the incubated eggs, decreased the hatchability rate which is inconsistent with the results of Hajati et al. (2014) who reported that in ovo injection of grape seed extract increased hatchability. Interestingly, similar hatchability was recorded for the control group and CASE group at 0.5 µg/ml. This may be due to the potential role of bioactive substances such as flavonoids, and polyphenols with free-radical-scavenging ability that could reduce oxidative stress (Fasuyi, 2006). However, the high mortality rate in the Ext 1µg/ml group might be due to the high concentration of CASE in the injected solution. Citrus aurantiifolia seeds contain antinutritional compounds (saponins and tannins) which are toxic at high concentrations. According to Akosile et al. (2023), the effects of in ovo administration of phytogenic feed additive extracts on hatchability can vary depending on the specific extract and dosage used. The improvement in the weight of chicks at hatch in the Citrus aurantiifolia seed extract treatment could be linked to the presence of glucide, a protein in citrus seed (Williams et al., 2020). According to Uni et al. (2005), embryonic nutrition of carbohydrates increases the glycogen stores of the liver and can ultimately increase the weight of the hatched chicks. As can be seen in the results of this study, from the eggs that were injected with CASE, chicks with higher physical quality similar to the control, which is consistent with the results of Khaligh et al. (2018). The reason might be due to the high quantity of carbohydrates content in the citrus seed (Williams et al., 2020) which can be a good solution for the embryo to easily use the energy source, because carbohydrates can reduce the consumption of muscle protein, which is used as an energy source, by providing the necessary energy for the embryo, and thus produce chickens with better quality (Retes et al., 2018). Additionally in this study, it was found that the citrus seed had a good antioxidant capacity while Zhang et al. (2018) stated that high levels of antioxidants in the embryonic egg and tissues can act as a mechanism consistent with protecting tissues during normal oxidative stress at hatching and produce high-quality chicks.
In ovo Feeding of Citrus Aurantiifolia Seeds Extract on Thyroid Hormones, Immune Organs and Some Giblet Relative Weight at Hatch
The experimental treatments had a significant influence (P < 0.05) on thyroid hormones, the bursa of Fabricius and gizzard relative weight (Table 5). T3 and T4 increased when the CASE was augmented, with significantly higher (P = 0.007; P < 0.001) values obtained in the Ext1µg group. There were significantly lower (P = 0.011; P < 0.001) values of the relative weight of the bursa of Fabricius and gizzard for CASE injected at 0.75 and 1.0 µg /ml than the control.
Table 5. Effect of in ovo feeding of Citrus aurantiifolia seeds on day-old chick thyroid hormones and immune organs relative weight.1
| Empty Cell | Experimental groups | Empty Cell | Empty Cell | |||
|---|---|---|---|---|---|---|
| Parameters | Control | Ext0.5µg | Ext0.75µg | Ext1µg | SEM2 | P-value3 |
| Thyroids hormones | ||||||
| T3 | 1.03b | 1.12ab | 1.24ab | 1.36a | 0.08 | 0.007 |
| T4 | 1.49c | 1.75b | 2.19a | 2.38a | 0.07 | <0.001 |
| Immune organs (%) | ||||||
| Spleen | 0.27 | 0.29 | 0.27 | 0.28 | 0.01 | 0.582 |
| Bursa of Fabricius | 0.48a | 0.43ab | 0.28b | 0.30b | 0.06 | 0.011 |
| Relative organ weight (%) | ||||||
| Hatching muscle | 0.98 | 0.99 | 1.01 | 1.05 | 0.091 | 0.880 |
| Heart | 0.80 | 0.86 | 0.87 | 1.14 | 0.132 | 0.080 |
| Liver | 1.40 | 1.59 | 1.47 | 2.22 | 0.431 | 0.247 |
| Gizzard | 2.17a | 2.08ab | 1.80bc | 1.68c | 0.105 | <0.001 |
Abbreviations: T3 (triiodothyronine); T4 (thyroxine); Control (eggs without injection); Ext0.5µg (eggs injected with 0.5 μg/ml of Citrus aurantiifolia seeds extract, CASE); Ext0.75µg (eggs injected with 0.75 μg/ml of CASE); Ext1µg (eggs injected with 1.0 μg/ml of CASE).
- a,b,c
-
Different letters indicate significant differences between means within rows (P < 0.05).
- 1
-
n= 3 replicates of 2 chicks per replicate.
- 2
-
SEM: pooled standard error of means.
- 3
-
P-value: Probability.
From the present study, the eggs that were injected with CASE showed a higher concentration of thyroxine hormone while the gizzard and bursa of Fabricius relative weight were lower. Hyperthyroidism in the CASE groups could be associated with a fast metabolism rate which may potentially influence body composition, including the weight of organs like the gizzard and the bursa of Fabricius (Koutsos and Klasing, 2014). The findings of this current study are contrary to the finding of Madej et al. (2015) which stated that the in ovo injection of prebiotics does not influence the weight bursa of Fabricius. This may be due to the botanic source.
Blood Numerous Counts, Biochemical and Oxidative parameters
Table 6 presents the effect of in ovo feeding of CASE on day-old chick blood profile. From the results, the values of red blood cells, hemoglobin, neutrophils and lymphocytes were significantly higher (P < 0.05) in the Ext1µg group of birds compared to the other groups. Overall, the concentration of blood hematological increased with increasing CASE concentration. The results of the effect of the in ovo feeding of CASE on the blood biochemical and oxidative parameters of the newly hatched chicks are presented in Table 7. There were significantly lower (P < 0.05) concentrations of total protein, total cholesterol and MDA in the 1.0 µg/ml CASE injected group. Superoxide dismutase concentration in the hatched chicks from in ovo injection of Citrus aurantiifolia seeds extract treatments was significantly increased compared to the control (P < 0.001).
Table 6. Effect of in ovo feeding of citrus aurantiifolia seeds extract on day-old chick hematological parameters.1
| Empty Cell | Experimental groups | Empty Cell | Empty Cell | |||
|---|---|---|---|---|---|---|
| Parameters | Control | Ext0.5µg | Ext0.75µg | Ext1µg | SEM2 | P-value3 |
| White blood cell (103/ul) | 168.97 | 168.62 | 167.98 | 167.89 | 0.522 | 0.152 |
| Red blood cell (106/ul) | 1.99b | 2.08b | 2.55b | 3.93a | 0.493 | 0.003 |
| Hemoglobin (g/dl) | 11.63b | 12.86b | 13.13b | 16.41a | 1.13 | 0.003 |
| Hematocrit (%) | 35.36 | 35.83 | 37.53 | 38.18 | 2.06 | 0.485 |
| Neutrophil (Neut) (%) | 10.98b | 12.16ab | 12.88ab | 14.99a | 1.23 | 0.028 |
| Lymphocyte (Lym) (%) | 41.91b | 43.29b | 44.02ab | 46.95a | 1.11 | 0.002 |
| Neut/Lym | 0.26 | 0.28 | 0.29 | 0.31 | 0.023 | 0.152 |
Abbreviations: Control (eggs without injection); Ext0.5µg (eggs injected with 0.5 μg/ml of Citrus aurantiifolia seeds extract, CASE); Ext0.75µg (eggs injected with 0.75 μg/ml of CASE); Ext1µg (eggs injected with 1.0 μg/ml of CASE).
- a,b
-
Different letters indicate significant differences between means within rows (P < 0.05).
- 1
-
n = 3 replicates of 2 chicks per replicate.
- 2
-
SEM: pooled standard error of means.
- 3
-
P-value: Probability.
Table 7. Effect of in ovo feeding of Citrus aurantiifolia seeds extract on the blood biochemical and oxidative parameters of the newly hatched chicks.1
| Empty Cell | Experimental groups | Empty Cell | Empty Cell | |||
|---|---|---|---|---|---|---|
| Parameters | Control | Ext0.5µg | Ext0.75µg | Ext1µg | SEM2 | P-value3 |
| Total protein (g/L) | 3.30a | 3.39a | 3.47a | 3.84b | 0.154 | 0.003 |
| Glucose (g/L) | 2.18 | 2.36 | 2.07 | 2.06 | 0.220 | 0.518 |
| Creatinine (mg/dl) | 0.61 | 0.58 | 0.57 | 0.63 | 0.031 | 0.262 |
| Total cholesterol (g/L) | 1.69a | 1.36ab | 1.30ab | 1.06b | 0.196 | 0.034 |
| MDA (nmol /ml) | 0.31a | 0.30a | 0.31a | 0.19b | 0.012 | <0.001 |
| SOD (nmol /ml) | 0.09c | 0.13b | 0.15ab | 0.17a | 0.011 | <0.001 |
Abbreviations: MDA (Malondialdehyde); SOD (Superoxide dismutase; Control (eggs without injection); Ext0.5µg (eggs injected with 0.5 μg/ml of Citrus aurantiifolia seeds extract, CASE); Ext0.75µg (eggs injected with 0.75 μg/ml of CASE); Ext1µg (eggs injected with 1.0 μg/ml of CASE).
- a,b
-
Different letters indicate significant differences between means within rows (P < 0.05).
- 1
-
n = 3 replicates of 2 chicks per replicate.
- 2
-
SEM: pooled standard error of means.
- 3
-
P-value: Probability.
In this study, the values of blood hematological parameters increased with increasing CASE concentration. According to the results, the counts of red blood cells and the hemoglobin of the chicks hatched from the eggs that were injected with CASE had a significant increase compared to the control. Consistent with the results of the present study, Panda and Cherian, (2014) stated in their experimental results that the increase in the hemoglobin concentration of the newly hatched broilers is directly related to the embryo’s nutrition during the incubation period. According to a study by Naeem et al. (2022), dehydration and nutrient deficiency can adversely affect the hematopoietic process in broiler embryos. Therefore, the decrease in blood cells in the control group can be attributed to the loss of humidity and insufficiency of nutrients in the stored eggs (Campbell et al., 1994). The significantly lower concentrations of cholesterol related to the injected eggs in this study may indicate a hypocholesterolemia effect of the citrus waste (Ebrahimi et al., 2016). Serum antioxidants are considered the key parameters for evaluating oxidative status in the enzymatic system. Meanwhile, immune function was strongly associated with antioxidant function which could be a crucial index of immune function (Eisa et al., 2022). In this study, CASE administration affected the oxidative status of the broilers. The plasma SOD activity had been improved while the MDA levels were lowered. Similarly, Gouda et al. (2020) demonstrated an improved SOD activity of broiler chickens when they were fed a diet supplemented with vitamin C (200 mg/kg). Chen et al. (2017) discovered that supplementing diets with more threonine (3 g/kg) lowered MDA levels in the serum in broilers.
In ovo Feeding of Citrus Aurantiifolia Seeds Extract on Intestinal Morphology of Day-Old Chicks
The results associated with the effect of the in ovo feeding of CASE on the intestinal morphology of day-old chicks are presented in Table 8. The histological presentation of the jejunal and ileal tissues of broiler chicks is shown in Figure 1. The jejunum length, as well as the ratio of jejunum length to the total length of the small intestine of the chicks at hatch in the CASE treatments, had a significant increase compared to the control (P < 0.05). The ileum pH was significantly higher (P = 0.017) in the control group than in the 0.75 and 1.0 µg/ml CASE injection groups. The villi height in the jejunum was significantly longer (P = 0.044) in the 1.0 µg/ml CASE injection group but was markedly shorter (P < 0.001) in the ileum compared to the control group. The crypt depth in the jejunum in the Ext0.5µg treatment increased significantly (P < 0.004) compared to the control and Ext1µg groups. The ratio of villus height to crypt depth of jejunum was significantly superior (P < 0.023) in the 1.0 µg/ml CASE injection group compared to the control.
Table 8. Effect of in ovo feeding of Citrus aurantiifolia seeds on intestinal morphology of day-old chick.1
| Empty Cell | Experimental groups | Empty Cell | Empty Cell | |||
|---|---|---|---|---|---|---|
| Parameters | Control | Ext0.5µg | Ext0.75µg | Ext1µg | SEM2 | P-value3 |
| Ileum | ||||||
| length (cm) | 9.66 | 8.78 | 8.79 | 8.81 | 0.690 | 0.511 |
| length/length of small intestine (%) | 24.74 | 20.86 | 22.06 | 22.28 | 1.38 | 0.066 |
| Ph | 6.55a | 5.75ab | 5.65b | 5.72b | 0.289 | 0.017 |
| Villi height (VH) (µm) | 299.37a | 294.53a | 292.87a | 258.53b | 3.25 | <0.001 |
| Crypt depth (CD) (µm) | 71.86 | 70.87 | 74.03 | 71.86 | 2.22 | 0.539 |
| VH/CD | 4.16a | 4.17a | 3.95a | 3.61b | 0.093 | <0.001 |
| Jejunum | ||||||
| length (cm) | 15.66b | 18.58a | 17.75a | 17.36ab | 0.661 | 0.002 |
| length/length of small intestine (%) | 40.45b | 44.16a | 44.63a | 43.82ab | 1.24 | 0.012 |
| pH | 6.18 | 6.10 | 5.89 | 5.86 | 0.166 | 0.184 |
| Villi height (VH) (µm) | 316.86b | 373.37ab | 347.37ab | 381.0a | 22.8 | 0.044 |
| Crypt depth (CD) (µm) | 65.86b | 71.53a | 70.03ab | 65.20b | 1.76 | 0.004 |
| VH/CD | 4.81b | 5.22ab | 4.97ab | 5.83a | 0.318 | 0.023 |
Abbreviations: Control (eggs without injection); Ext0.5µg (eggs injected with 0.5 μg/ml of Citrus aurantiifolia seeds extract, CASE); Ext0.75µg (eggs injected with 0.75 μg/ml of CASE); Ext1µg (eggs injected with 1.0 μg/ml of CASE).
- a,b
-
Different letters indicate significant differences between means within rows (P < 0.05).
- 1
-
n= 3 replicates of 2 chicks per replicate.
- 2
-
SEM: pooled standard error of means.
- 3
-
P-value: Probability.
Figure 1. Photomicrograph showing jejunum and ileum segments of experimental broilers. (A) (Control), (B) (Ext0.5µg), C (Ext0.75µg), and D (Ext1µg).
Phytogenic feed additives, such as herbal plants and their extracts, have been reported to have positive effects on the gastrointestinal tract, including spasmolytic and laxative effects (Windisch and Kroismayr, 2006). It has been demonstrated that in ovo feeding with bioactive compounds that boost the activity of digestive enzymes enhances digestion in hatchling chicks (Siwek et al., 2018). In this present study, CASE injection caused a significant increase in the length, villi height and the ratio of villi height to crypt depth of jejunum, probably due to the increased proliferation of intestinal cells (Ostaszewski and Nissen, 1988) in this part of the small intestinal. According to Tako et al. (2004), embryonic nutrition of carbohydrates can also increase the growth and development of the digestive system by increasing the proliferation and differentiation of enterocytes and/or reducing the breakdown of protein. Consistent with the results of the present experiment, Murakami et al. (2007) stated that in ovo injection of vitamin E protects enterocytes, thereby increasing cell proliferation and increasing villus growth. Many studies showed that organic acids have effects on the pH of the gastrointestinal tract ((Salahi et al., 2015). The lower values of pH in the small intestine of the chick from injected eggs in this experiment may be due to the acid character of the citrus seed. This result is in line with Salahi et al. (2015) who showed that injection of butyric acid affected the pH of the jejunum and ileum on the hatch day with a decrease in the pH of the jejunum. Similarly, Adil et al. (2011) reported that the inclusion of organic acids in the diets of broiler chickens diminished pH of the gastrointestinal tract.
In ovo feeding of Citrus Aurantiifolia Seeds Extract on the Growth Performance of Broilers
The results on the effect of in ovo feeding of CASE on the growth performance of broilers are presented in Table 9. During the grower period (22–42 d) and the entire rearing period, the increase in the body weight gain and feed conversion ratio of broiler chickens in the Ext0.5µg group was significantly improved compared to the other treatment groups (P < 0.001). Figure 2 shows body weight increased with increasing age of broiler chickens. From d 21 onward, chickens from the Ext1µg group had the lowest body weight (P < 0.05) until d 42 while chickens from eggs subjected to Ext0.5µg administration of CASE had higher weights during the entire rearing period.
Table 9. Effect of in ovo feeding of Citrus aurantiifolia seeds extract on the growth performance of broiler chickens.1
| Empty Cell | Experimental groups | Empty Cell | Empty Cell | |||
|---|---|---|---|---|---|---|
| Parameters | Control | Ext0.5µg | Ext0.75µg | Ext1µg | SEM2 | P-value3 |
| d 1–d 21 | ||||||
| BWG (g) | 816.8 | 841.91 | 841.6 | 800.7 | 23.1 | 0.238 |
| FI (g) | 1,112.16 | 1,112.52 | 1,114.87 | 1,112.80 | 0.972 | 0.058 |
| FCR (g:g) | 1.36 | 1.32 | 1.32 | 1.39 | 0.037 | 0.248 |
| D 21–d 42 | ||||||
| BWG (g) | 891.3b | 991.4a | 898.2b | 835.4b | 26.4 | <0.001 |
| FI (g) | 1,370.6 | 1,381.4 | 1,372.2 | 1,392.5 | 13.3 | 0.353 |
| FCR (g:g) | 1.54ab | 1.39c | 1.52bc | 1.67a | 0.050 | <0.001 |
| d1-d42 | ||||||
| BWG (g) | 1,708.0bc | 1,833.3a | 1,739.8b | 1,636.1c | 33.2 | <0.001 |
| FI (g) | 2,482.7 | 2,493.9 | 2,487.1 | 2,505.34 | 13.6 | 0.395 |
| FCR (g:g) | 1.45ab | 1.36c | 1.42bc | 1.53a | 0.030 | <0.001 |
Abbreviations: BWG (body weight gain); FI (feed intake); FCR (feed conversion ratio); Control (eggs without injection); Ext0.5µg (eggs injected with 0.5 μg/ml of Citrus aurantiifolia seeds extract, CASE); Ext0.75µg (eggs injected with 0.75 μg/ml of CASE); Ext1µg (eggs injected with 1.0 μg/ml of CASE).
- a,b,c
-
Different letters indicate significant differences between means within rows (P < 0.05).
- 1
-
n = 6 replicates of 15 birds per pen.
- 2
-
SEM: pooled standard error of means.
- 3
-
P-value: Probability.
Figure 2. Growth curve of broiler chickens in days.
It could be noticed in this present study that there is no effect of CASE on the starter period. The effect of in ovo feeding on growth performance was not evident in the first few weeks of life and could be noticed during the grower phase which probably may be due to the bioactive components in the extract used which improves digestive enzymes. This result is in line with the finding of N’nanle et al. (2017) who stated that Moringa leaves extract injected on d 18 of incubation significantly affected the body weight at 6 and 7 wk of age of layer chick. This supports the results of Bhanja et al. (2012) and Shafey et al. (2014) who observed body weight improvement in broiler chickens fed in ovo with vitamins and amino acids. The best values of body weight gain and feed conversion ratio were recorded for birds that received 0.5 µg/ml of CASE. There is thus a dose effect. Citrus aurantiifolia seed extract at a higher dose could slow down intestinal development, which would likely decrease intestinal absorption functions which may be the reason for the slower growth than normal shown in the group injected with 1 µg/ml. This finding suggests that embryos stored components of the CASE in their body tissues likely what Makkar and Becker (1996) found with the extract of Moringa oleifera leaves. During the first days of rearing, chickens mobilize progressively the nutrients (carbohydrates stored as glycogen or triglycerides; proteins used as energy source or stored as glycogen or triglycerides) as an additional substance to the starter diet given that feed intake is not affected (N’nanle et al., 2017).
CONCLUSION AND APPLICATIONS
- 1.
Citrus aurantiifolia seeds extract in broiler eggs had a significant improvement in the hatching characteristics, physical quality, blood indices, development of small intestine and growing performance of chickens in a dose-dependent manner.
- 2.
In ovo administration of 0.2 ml/egg of Citrus aurantiifolia seeds extract solution of 0.5 μg/ml at d 18 of incubation improves day-old chick weight and jejunum morphology.
- 3.
Hatchery operators can integrate Citrus aurantiifolia seed extract into chick production during incubation as a method to reduce delays in early feeding.
Source: Science Direct
