Abstract
This study aimed to evaluate the effect of in ovo injection and post-hatch supplementation of Citrus aurantiifolia seed on hatchability, growth performance, antioxidant capacity, immune organs weight, and intestinal morphology of Cobb 500 broilers chickens. On day 18 of incubation, 450 fertile eggs were randomly allocated into three sets: non-injected group, 0.5 µg/ml Citrus aurantiifolia seed extract (CASE) group and 0.75 µg/ml CASE group. After hatch, each set was allocated into two groups (5 replicates of 10 birds/replicate each) and was fed on a standard diet with or without Citrus aurantiifolia seed powder (CASP) supplementation (5 g/kg). The groups were divided as follows: IoCA0-C, IoCA0-CA, IoCA5-C, IoCA5-CA, IoCA75-C and IoCA75-CA where IoCA0-C was: non-injected group fed standard diet (negative control group), IoCA0-CA: non-injected and fed CASP supplemented diet (positive control group), IoCA5-C: 0.5 µg/ml of CASE-injected and fed standard diet, IoCA75-C: 0.75 µg/ml of CASE-injected and fed standard diet, IoCA5-CA: 0.5 µg/ml of CASE-injected and fed CASP supplemented diet and IoCA75-CA: 0.75 µg/ml of CASE-injected and fed CASP supplemented diet. The results revealed that the interaction between CASE in ovo injection at 0.5 µg/ml and dietary CASP supplementation at 5 g/kg in the diet increased body weight and weight gain (p < 0.001). The interaction between CASE in ovo injection and dietary CASP supplementation decreased the serum malondialdehyde concentration, the relative weight of the thymus and improved duodenum and ileum villus height (p < 0.05). In conclusion, in ovo injection and dietary supplementation of Citrus aurantiifolia seed powder can improve growth performance and the intestinal morphology of broiler chickens.
HIGHLIGHTS
In ovo feeding of Citrus aurantiifolia seed extract improves chick weight at hatch.
In ovo feeding and post-hatch supplementation of Citrus aurantiifolia seed improves feed utilisation.
The supplementation of Citrus aurantiifolia seed in the diets of broilers enhances intestinal health.
Introduction
It is anticipated that the growing human population, rising income levels, growing nutritional and health consciousness, and urbanisation will all contribute to the rising demand for chicken (Miller et al. Citation2022). Disease control, effective production, product quality, and affordable production costs have been challenges faced by the chicken business over the years (Hafez and Attia Citation2020). Chicks’ small intestinal development depends critically on the nutrition they receive right after hatching (Eisa et al. Citation2022). It was discovered that whereas prolonged access to external feed resulted in delayed development of the small intestine’s mucosal layer, feeding immediately post-hatch accelerated intestinal morphological development (Uni et al. Citation1998; Uni and Ferket Citation2003). Increased mortality is the most severe effect of delayed feeding (Willemsen et al. Citation2010). Various early feeding strategies have been proposed and developed to mitigate or perhaps reverse the detrimental consequences of delayed nutrition. These initiatives encompass everything from in ovo feeding to specially designed post-hatch diets (Uni and Ferket Citation2003, Citation2004; Leeson Citation2008). For poultry production to be optimised, intestinal development must be functional. Digestion and nutrient absorption, which are directly impacted by the small intestine’s morphological and functional development, are essential for this optimisation (Tako et al. Citation2004; Cheled-Shoval et al. Citation2011). The last phases of digestion and nutrient absorption are significantly influenced by the small intestine, namely the crypts and villi of the epithelium (Wang and Peng Citation2008). Furthermore, a key component of determining a bird performance (growth, meat, and egg quality) is the gastrointestinal tract’s (GIT) effective functionality and health.
In ovo feeding is a well-known early nutrition method that may present additional opportunities to affect a chick’s development inside the egg and get around the late incubation phase’s nutrient limitations (Eisa et al. Citation2022). This approach gives the chance to give the embryo the nutrients it needs to grow and develop, as well as to improve its status through injection. It has been demonstrated that this technique can give the late-term embryo enough nutrition to shield it from the harmful effects of starvation during the longer hatching window (Uni et al. Citation2005). It is anticipated that early nutrition will stimulate the immune system by either giving substrates for immunomodulator activity that results in the generation of several immunoglobulins or by providing nutrients for cell proliferation and differentiation (Jha et al. Citation2019). Citrus fruit seeds, according to Nobakht (Citation2013), are rich in flavonoids, isoflavones, flavones, anthocyanins, coumarins, lignans, catechins, and epicatechins, among other active antioxidants. In the present study, 5 g/kg of citrus aurantiifolia seed was selected as the highest level for the extra Citrus aurantiifolia seed powder supplementation and assumed that extra Citrus aurantiifolia seed powder supplementation would exert a beneficial effect on broilers at the early age. Therefore, this study was performed to compare the effects of in ovo injection on hatchability and post hatch supplementation of Citrus aurantiifolia seed powder on the growth performance of broilers, antioxidant capacity, lymphoid organs relative weight, and intestinal morphometry of broilers.
Materials and methods
Study site and ethics
The present study was conducted at the hatchery and experimental units of the Regional Centre of Excellence in Poultry Science (CERSA), in Badja, 41 km from Lomé, Togo, located in a hot (28.85 °C ± 0.62) and humid (71.62% ± 1.75) climate following the guidelines of the Canadian Council on Animal Care (2009), under the approval (008/2021/BC-BPA/FDS-UL) of the ethics and scientific committee of the Regional Centre of Excellence in Poultry Science, University of Lome (CERSA/UL).
Preparation of the seed extract
A mass of 400 g of crushed Citrus aurantiifolia seeds dried from fresh matured and unaffected fruits 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 is evaporated in a steam rota (rotary evaporator) at 45 °C. The extract is then placed in a Petri dish in an oven at 40 °C until drying (Boubekri Citation2014). Ten (10) mg of the extract were reconstituted with 10 mL of saline solution (0.9%) to obtain the stock solution (1000 µg). To produce a 10 µg working solution, 1 mm of the stock solution was once more reconstituted with 100 mL of saline solution. Finally, 5.0 mL and 7.5 ml of the working solution were reconstituted with 10 mL of saline solution, respectively, to obtain 0.5 µg and 0.75 µg concentrations used for the injection.
Incubation and in ovo supplementation
For the experiment, a completely randomised design was adopted. Six hundred (600) hatchable eggs of Cobb 500 chicken were obtained based on the hatching performance records from 58-weeks-old breeders flock at the research farm of CERSA, University of Lome. Each egg was weighed before being incubated in a Royal Pas Reform (SmartProTM) combi incubator from the Netherlands, which was set at 37.7 °C and 60% relative humidity. The eggs were automatically turned every hour. On the 18th day of incubation, eggs were candled to remove infertile ones and eggs with evidence of living embryos were randomly assigned to three groups of 150 eggs each. Groups one and two were injected with the following concentrations of Citrus aurantiifolia extract 0.5 µg and 0.75 µg; the eggs were returned to the incubator and waited 6 h before being moved to the baskets for hatching. In the hatcher, the temperature was maintained but relative humidity increased to 70% and the eggs were observed for hatching according to their groups. To facilitate the in ovo delivery of the extract and prevent stress in the eggs during incubation, two holes were made on the broad side of the eggs using 18 G needles. An automatic syringe was then used to inject the extract, and the holes were then sealed with adhesive tape. No group of eggs was injected with saline solution because previous studies (Atan and Kop-Bozbay Citation2021; Karamik and Kop-Bozbay Citation2021), reported no significant difference between the saline-injected and non-injected eggs. All the injected eggs received 0.2 ml of the extract in the air chamber.
Management and diets
Hatchlings (n = 300) were weighed individually and distributed according to a completely randomised design with six treatments of five replicates with 10 birds per replicate and were fed on a standard diet supplemented with 0 and 0.5% Citrus aurantiifolia seed powder (CASP) as presented in Table . The dietary treatments comprised: non-injected group fed standard diet (negative control group), IoCA0-CA: non-injected and fed CASP supplemented diet (positive control group), IoCA5-C: 0.5 µg/ml of CASE-injected and fed standard diet, IoCA75-C: 0.75 µg/ml of CASE-injected and fed standard diet, IoCA5-CA: 0.5 µg/ml of CASE-injected and fed CASP supplemented diet and IoCA75-CA: 0.75 µg/ml of CASE-injected and fed CASP supplemented diet. This experiment follows up on a previous study (unpublished) on the use of Citrus aurantiifolia seeds in broiler chicken diets, revealing that a 0.5% (5 g/kg) supplementation of the seeds powder enhanced bird performance, while in ovo feeding with Citrus aurantiifolia seed extract at dosages of 0.5 μg/ml and 0.75 μg/ml proved effective. The ingredient composition of the standard diet (starter diet from d 1 to 21, and grower diet from d 22 to 42) and its nutrient levels are presented in Table . Birds were reared in-house under a light schedule of 23 h of light and 1 h of darkness and were provided with mash feed and water ad libitum.
Table 1. Experimental design of groups.
Table 2. Ingredients and nutrient composition of the standard diet.
Growth performance and immune organs
During the rearing period, the chick weight, feed intake and feed conversion ratio (FCR) were recorded weekly. At 42 d of age, birds were weighed individually after a 12-h feed withdrawal, and feed intake was recorded to calculate FCR by dividing feed intake by weight gain. For slaughter, two birds were chosen at random from each replicate. After slaughter, lymphoid immune organs weights (spleen, thymus, and bursa of Fabricius) were recorded.
Antioxidant capacity
Five (5) mL of whole blood samples were collected to separate serum and stored at −20 °C until analysis. Lipid peroxidation (MDA marker) was assayed according to Ohkawa et al. (Citation1979) using a lipid peroxide (MDA) colorimetric kit (Biodiagnostic, Egypt). The method is based on the reaction between Thiobarbituric acid (TBA) and malondialdehyde (MDA) in an acidic medium at a temperature of 95 °C for 30 min to form thiobarbituric acid reactive product. The absorbance of the resultant pink product can be measured at 534 nm. SOD was assayed according to Nishikimi et al. (Citation1972) using SOD colorimetric kit (Biodiagnostic, Egypt). This assay relies on the ability of the enzyme to inhibit the phenazine methosulphate-mediated reduction of nitroblue tetrazolium dye. The absorbance was measured at 560 nm.
Intestinal morphometry and histo-morphological analysis
The duodenum, jejunum, and ileum lengths, as well as the empty weight of each segment, were measured and expressed in relative weight (g/kg live weight), respectively (Liu et al. Citation2010; Shang et al. Citation2020) and relative length (cm/m length of the small intestine). Approximately, 2 cm of each segment of the small intestine (duodenum, jejunum and ileum) from sampled chickens was collected and fixed in a 10% buffered formaldehyde solution. These were the definitions of these segments: The intestines were divided into three sections: 1) the gizzard to the pancreatic and bile ducts (duodenum); 2) the jejunum to Meckel’s diverticulum and the bile duct entrance site; and 3) Meckel’s diverticulum to the ileo-cecal junction (ileum) (Onderci et al. Citation2006). The samples were rinsed with phosphate buffer saline solution to remove the digesta. The samples were then fixed in 10% neutral-buffered formalin for histology. In the laboratory, the samples were dehydrated, cleared, and embedded in paraffin and a 5-μm section of each sample was prepared on a glass slide, and then stained with hematoxylin-eosin following the procedure of Al-Sabaawy et al. (Citation2021). A light microscope (Thermo Fisher Scientific, US, Massachusetts) was used to examine and capture images of the slides. Villus height (VH), from villi tip to crypt junction and crypt depth (CD), from villi base to sub mucosa were measured with ImageJ software (http://rsb.info.nih.gov/ij/, Maryland, USA).
Statistical analysis
One-way analysis of variance (ANOVA) with the post-hoc Tukey multiple comparison tests using Minitab statistical software (Minitab 21) was used to analyse hatch parameters data. A power analysis was performed to determine the sample size required during incubation to get the intended power, guaranteeing that the research had adequate sensitivity to identify significant effects based on a preliminary study. For post-hatch parameters, the data were analysed in a 3 × 2 factorial design, in which we had three levels of in ovo injection of CASE and two post-hatch diets supplemented (no supplementation or supplementation). Initially, the data were submitted to the Shapiro–Wilk test for normality (Levene’s test for homogeneity of variance), and the data were transformed in order to meet ANOVA assumptions and then to the analysis of variance using a two-way analysis of variance with the statistical program Minitab® 21. The means of the factors were compared with the post hoc Tukey multiple comparison. The results are expressed as mean ± SEM and considered significant at p < 0.05. P1 was the p-value for in ovo levels, P2 was the p-value for post-hatch supplementation levels and Int was the p-value for P1 and P2 interaction.
Results and discussion
Hatchability
The effects of non-injected and two doses of CASE in ovo injection on the hatchability of broiler chicks are presented in Table . No significant effect was observed on the hatchability across all the treatment groups (p > 0.05). The study’s outcome can be linked to the high metabolic activity during the later stages of incubation, which may cause oxidative stress in the embryo and lower the chick’s performance and survival rate after extract injection. Embryos grow and develop quickly during the later stages of incubation, which increases metabolic activity and produces reactive oxygen species (ROS) (Querin et al. Citation2001). This may lead to oxidative stress, which might disturb the normal balance between the production of ROS and antioxidant defence systems (Querin et al. Citation2001; Covarrubias et al. Citation2008). This can lower hatchability and post-hatch survival rates by damaging cellular components, compromising membrane integrity, and impairing the supply of nutrients and oxygen. According to studies by Naeem Asa et al. (Citation2022) and Das et al. (Citation2021), it has been demonstrated that the potential effects can be reversed by in ovo injection of nutrients, antioxidants, and compounds comprising either or both of them. In this present study, the in ovo injection of CASE improved the chick weight at hatch (p = 0.006). This increase may be due to the antioxidant status of the extract which alleviates hatch-related oxidative stress and protects skeletal muscle stem cells from oxidative damage (El-Kholy et al. Citation2021). While N'nanle et al. (2017) reported increased hatchability and chick weight at lower concentrations of in ovo feeding of moringa leaf extract but reduced hatchability at higher concentrations, Bozbay and Göneci (Citation2023) reported that groups receiving cinnamon and ginger had higher hatchability and chick quality. Comparably, Akosile et al. (Citation2023) observed higher chick weight after injecting clove and cinnamon extract into broiler chicken eggs, whereas Ngueda et al. (Citation2021) observed comparable hatchability and mortality rate after injecting 0.5 µg of cassava leaf extract into broiler chicken eggs. Additionally, the finding of this current study is consistent with Kuka et al. (Citation2023) who administered in ovo Soursop leaf extract and reported increased chick weight and similar hatchability rate.
Table 3. Hatchability and chick weight of fertile.
Growth performance
The effect of egg injection and early nutrition with diets supplemented with or without Citrus aurantiifolia seed powder (CASP) on growth performance is shown in Table . The interaction between in ovo injection and diet supplemented was significant for final body weight and body weight gain with higher value in 5 g/kg of CASP groups. The group of chicks that were injected with 0.5 µg/ml of CASE and chicks fed on a diet supplemented with 5 g/kg of CASP showed a significant (P1 and P2 < 0.001) increase in final body weight and body weight gain and recorded significantly (P1 = 0.005 and P2 < 0.001) improved FCR compared to the other groups.
Table 4. Feed consumption, body weight gain, FCR and final body weight in experimental groups.
The elevated final body weight may be a result of the administration of CASE which potentially contains amino acids and vitamins (Bendich et al. Citation1986), throughout the embryonic phase, encouraging higher protein synthesis. According to Xiao et al. (Citation2020), dietary amino acids can improve protein synthesis, and growth and modulate enterocyte metabolism by supplying cell energy as amino acids have the ability to enhance mitochondrial respiration, and increase the intracellular content of pyruvic acid and lactic acid. This implies that the combinatory effect of citrus seed through in ovo feeding and post-hatch supplementation is superior to the individual effect. Thus, injecting 0.5 µg/ml of CASE and feeding chicks on a diet supplemented with 5 g/kg of CASP yield better performance. The present results are close to those of Readh et al. (Citation2023), who supplemented the by-products of the mixture of peel and dried seeds resulting from the extraction of the juice of Citrus sinensis in the feed of broiler chickens. In order to affect gut functions and feed efficiency, aromatic herbs and their extracts can favourably promote endogenous digestive secretions and create intestinal epithelial structures (Jang et al. Citation2007; Yang et al. Citation2019). This present finding agrees with Tahmasebi and Toghyani (Citation2016) who indicated that in ovo threonine administration improved the body weight of broilers throughout the entire rearing period which most likely was mediated by its impact on GIT development. Similarly, Bhanja and Mandal (Citation2005) found that injecting various amino acid combinations boosted the body weight of chicks on days 7 and 21 of age. This high total feed intake may be explained as a low digestive ability of the birds and compensate for it by increasing feed intake. De Verdal et al. (Citation2010) noted that broilers exhibited higher feed intake in response to their low digestive ability. The group of birds from in ovo injected eggs with CASE and fed diets supplemented with CASP recorded significantly better FCR compared with the other groups. A low feed conversion ratio indicates that the birds have gained muscle mass that is closely correlated with the quantity of feed they consumed. The farmer benefits when the flock’s feed conversion ratio is the lowest because feeding incurs the largest production costs. This is supported by the observation made by Bhanja and Mandal (Citation2005) that the chicks who received amino acid injections had a higher FCR than the untreated controls. Meanwhile, Citrus aurantiifolia seed extract may possess a component that facilitates the functions of mucin and gastric enzymes (Oulebsir et al. Citation2022). Better FCR in our trial compare to the negative control may be attributed to the extra level of dietary Citrus aurantiifolia seed.
Serum antioxidants
The results of serum antioxidant capacity are presented in Table . The interaction between in ovo injection and diet supplemented was significant for the reduction of MDA concentration with a lower value in 5 g/kg of CASP groups. The group of chicks that were injected with CASE and chicks fed on a diet supplemented with CASP showed significantly higher SOD activity (P1 = 0.004; P2 < 0.001) while serum MDA concentration reduced significantly with an injection of CASE (P1 = 0.001) and a diet supplemented with CASP (P2 < 0.001).
Table 5. Serum antioxidants (SOD and MDA) of broilers in experimental groups.
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. Citation2022). Reactive oxygen species (ROS) are produced during normal cell metabolism, however, quantities of ROS that surpass the antioxidant protection levels of cells can cause widespread damage to DNA, proteins, and endogenous lipids (Yu Citation1994). In this study, CASE administration affected the oxidative status of the broilers. The interactive effect of in ovo injection of CASE and supplementation of CASP at 5 g/kg reduced MDA but the increased SOD activity was observed in only the main effects. Similarly to our findings, Gouda et al. (Citation2020) demonstrated an improved SOD activity of broiler chickens when they were fed a diet supplemented with vitamin C (200 mg/kg). The efficacy of vitamin C and its absorption in broilers can be improved by citrus extracts, especially those that include bioflavonoids (Elwan et al. Citation2019). The antioxidant qualities and vitamin C-synergistic effects of citrus bioflavonoids, such as hesperidin, naringin, and quercetin, are well-established (Elwan et al. Citation2019). Vitamin C is more stable and bioavailable as a result of this combination, which aids absorption in the gut. Because of the relationship between immune function, oxidative stress, and serum antioxidant levels, nutritional interventions aimed at improving antioxidant status are crucial for the general health of the animal. Immune system function, oxidative stress reduction, and broiler chickens’ health and performance may all be enhanced by increasing antioxidant defences through diet supplementation. However, the precise effects of dietary antioxidants on blood antioxidant levels and general health may vary depending on factors including inclusion levels, duration of supplementation, and combinations with other dietary components. More research is needed to fully comprehend the mechanisms underlying these effects and provide dietary solutions that promote the health and welfare of the birds.
Immune organs
Table summarises the effect of Citrus aurantiifolia seed in ovo injection and dietary supplementation on the relative weight of immune organs. There was no significant difference in the relative weight of the spleen between the groups (p > 0.05). The thymus of chicks hatched from injected eggs and chicks fed extra Citrus aurantiifolia seed diet was significantly (P1 and P2 < 0.001) lower than those of noninjected eggs and fed standard diet. Moreover, there was a significant (p = 0.005) interaction between in ovo injection and diet supplemented on thymus relative weight of chicks. The significantly lower (P1 and P2 < 0.001) values of the relative weight of the bursa of Fabricius were related to groups of chicks injected with 0.75 µg/ml of CASE and chicks fed on diets supplemented with CASP.
Table 6. Relative immune organ weight expressed to body weight of experimental birds.
The spleen is the organ responsible for antibody synthesis and immune cell proliferation (Pozo et al. Citation2009; Tarantino et al. Citation2013). The immune system is the hub for T-cell proliferation and maturation in the thymus gland (Gordon and Manley, Citation2011). For this present finding, it can be deduced that CASP supplemented in the diets of birds could aid metabolism which may potentially influence body composition, including the weight of organs like the thymus and the bursa of Fabricius. In contrast with Corzo et al. (Citation2007) and Ren et al. (Citation2014), dietary CASP supplementation decreased the relative weight of the thymus in the present study, implying that a higher level of citrus waste in broilers diet could affect the development of immune organs at a young age. One of the main organs of adaptive immunity is the thymus, which is necessary for T-cell growth. Poor T-cell development or function can thus be indicated by a decrease in thymic weight, which could impair the birds’ immunological responses (Fairbrother et al. Citation2004; Ratcliffe and Härtle Citation2022). Likewise, a noteworthy disparity in the Fabricius bursa between birds given CASE injections and those fed diets supplemented with CASP underscores potential alterations in bursal development. These variations may be a sign of abnormality in B-cell development or function, which might affect the humoral immunity of broilers. Fabricius bursa is responsible for B-cell maturation and antibody production (Ratcliffe and Härtle Citation2022). In future studies, it might be interesting to look at the T and B cell populations and activity.
Small intestinal morphometry
The effect of C. aurantiifolia seed on the morphological development of the small intestine segments is shown in Table . There was no significant difference in the relative length of the duodenum and ileum across the treatment groups (p > 0.05). The jejunum relative length of chicks hatched from injected eggs was significantly higher (p = 0.009) than those of the other groups. Dietary supplementation markedly reduced the relative weight of the jejunum (P2 = 0.010) and ileum (P2 < 0.001). A significant (p < 0.001) interaction was found between in ovo injection of CASE and supplementation of diet with CASP on the relative weight of ileum. Citrus aurantiifolia seed did not affect duodenum and ileum pH (p > 0.05) while the pH in the Jejunum of chicks injected with CASE and chicks fed on diets supplemented with CASP was significantly lower (P1 and P2 < 0.001) than those of the other groups. Table shows the results of the intestinal morphometry of the birds. The group of birds that were fed on the diet supplemented with CASP and birds that were injected with CASE showed a significant increase (p < 0.05) in villi height of all the segments of the small intestine. Moreover, CASP supplementation increased the ratio of villi height to crypt depth in the ileum (P2 < 0.001) while an inverse trend was observed in the duodenum (P2 = 0.036). There were no significant differences (p > 0.05) in the crypt depth of all segments of the small intestine and the ratio of villi high and crypt depth in the jejunum. In addition, the interaction between in ovo injection of CASE and diet supplemented with CASP was significant (p < 0.05) for the villi height of duodenum and ileum.
Table 7. Morphological development and pH of the small intestine of experimental birds.
Table 8. Intestinal morphometry of the small intestine of experimental birds.
It has been shown that in ovo feeding hatchling chicks bioactive chemicals that increase the activity of digestive enzymes improve digestion (Siwek et al. Citation2018). Morphometric results of the duodenum, jejunum, and ileum in broilers in this study on day 42 of the experiment revealed that in ovo and dietary supplementation of CASP in the diet of the birds improved the intestinal health of the birds by modifying the height of the villi. Nutrient absorption is enhanced by longer villi and deeper crypts with a larger surface area, activating intestinal cell maturation and digestive enzyme activity (Yang et al. Citation2009; Sarica et al. Citation2009). In this present study, CASP supplementation did not change the crypt depth of the segments of the small intestine of the birds compared to the control group. However, the villi height of all small intestinal segments increased significantly with the supplementation of CASP. This suggests that supplementing broilers with CASE in ovo and CASP in the diet improves nutrient absorption and boosts overall growth performance (Alyileili et al. Citation2020) showing that C. aurantifolia seeds have a good effect in the intestinal development of broilers. Thus, birds will benefit from in ovo injection of CASE and post-hatch supplementation with CASP by increasing nutrient absorption in the gut. According to Noleto-Mendonça et al. (Citation2021), phenolic substances, like flavonoids, have antioxidant action that may support intestinal mucosa development and health by shielding intestinal cells from oxidative stress-induced inflammation brought on by reactive species. The action of CASP on villi height could be attributed to the phenolic compounds contained in these seeds (Russo et al. Citation2015). Furthermore, it has been reported that a high intestinal villus is associated with a well-differentiated intestinal mucosa with high digestive and absorptive capabilities (Jeurissen et al. Citation2002).
Conclusion
In ovo injection and post-hatch dietary supplementation of Citrus aurantiifolia seed powder have been shown to enhance the growth performance of broiler chickens and improve their intestinal morphology. This is evidenced by an increase in villus height and a higher villus height to crypt depth ratio. Additional research is needed to investigate the impact of supplementing with Citrus aurantifolia seeds on immune organ development and gene expression profiles. Furthermore, it is important to assess the interaction between in ovo injection and dietary supplementation in different environmental conditions in order to effectively integrate C. aurantiifolia seed as a feed additive in poultry nutrition.
Author contributions
Conceptualisation, RF Kpossou, N Everaert, K Tona. Data Collection and Analysis, R F Kpossou, B Adjei-Mensah. Writing original draft preparation, RF Kpossou. Writing review, B Adjei-Mensah. Supervision, review and editing, N Everaert, K Tona.
Acknowledgements
We express our gratitude to the World Bank Group for funding the project through the Regional Center of Excellence in Poultry Science (CERSA) and to the staff University of Lome, Togo, for their enormous support.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
The data that support the findings of this study are available from the corresponding author, [RF Kpossou] on request.
Additional information
Funding
References
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