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Impact of Phytobiotic Growth Promoters Supplementation on the Morphology and Biometry of the Vital Organs in Broilers

ABSTRACT

Supplementation of Cv (clove) and Ts (tulsi) augments the growth rate and gut health of broilers. However, studies on their impact on the vital organs and male reproductive system remain scarce. Therefore, the current study aimed to investigate the impact of Cv and Ts supplementation on hematological profiles, as well as the heart, lungs, liver, kidney, and testes of broilers, both morphologically and biometrically. Sixty one-day-old broiler chicks, divided into four homogenous groups (control-T0 and three supplement groups-T1, T2, and T3), were fed 0.5% Cv and 2% Ts (T1), 1% Cv and 3% Ts (T2), and 1.5% Cv and 4% Ts (T3) with drinking water for 21 d. Organ samples were collected on d 14 and d 28 for macroscopic and microscopic (hematoxylin and eosin stain) investigation. Cv and Ts didn’t affect the hematological profile, organ weight gain, and their general histoarchitectures. However, the histomorphometric investigation showed an increased (p<0.05) parabronchus surface area of the lungs, size of the renal glomeruli, the diameter of the proximal and distal convoluted tubules of the kidney in the T1 and T2 groups, and size of the seminiferous tubules of the testes in group T1. These findings suggest that unlike antibiotic or steroid growth promoters (GPs), Cv and Ts have beneficial impacts on the vital organs as well as on the male gonad of broilers from the morphologic and biometric perspectives.

Keywords:
Broiler; Clove; Tulsi; Vital organs; Testes

INTRODUCTION

Broiler farming is a major sector across the globe, with broiler meat being among the most popularly consumed protein sources. The stakeholders of the poultry industries in Bangladesh are currently moving towards safe organic eggs and meat, as well as better health and living conditions. By 2030, 373 million tons of meat will be produced worldwide, an increase of nearly 44 million tons, with 84% of the growth taking place in emerging countries (OECD/FAO, 2021). This rise has been especially apparent in nations like China, the USA, and Brazil, which are among the biggest broiler producers in the world (OECD/FAO, 2021). Despite its expansion, broiler production has encountered difficulties, including worries about animal welfare, environmental impacts, and food safety. These challenges have prompted the desire to develop alternative, environmentally friendly production techniques, such as the production of organic and free-range broilers.

The use of antibiotic growth promoters (GPs) in poultry has been linked to the development of antibiotic resistance, while steroid GPs cause severe immune suppression, let alone their residual effects on human health (Vidovic & Vidovic, 2020Vidovic N, Vidovic S. Antimicrobial resistance and food animals: Influence of livestock environment on the emergence and dissemination of antimicrobial resistance. Antibiotics 2020;9(2):52. https://doi.org/10.3390/antibiotics9020052
https://doi.org/10.3390/antibiotics90200...
; Islam et al., 2022cIslam R, Sultana N, Ayman U, et al. Role of steroid growth promoter on growth performance and meat quality traits in broiler. Poultry Science 2022c;101(7):101904. https://doi.org/10.1016/j.psj.2022.101904
https://doi.org/10.1016/j.psj.2022.10190...
, Islam et al., 2023b). To replace synthetic GPs like antibiotics or steroids for the production of organic meat, stakeholders and researchers are working to build a unique, phytobiotic-based feeding approach for broilers (Islam et al., 2022c; Islam et al., 2023a). Organic GPs are additives to broiler feed that boost growth and increase feed efficiency (Dhama et al., 2014Dhama K, Ruchi T, Khan RU, et al. Growth promoters and novel feed additives improving poultry production and health, bioactive principles and beneficial applications: the trends and advances-a review. International Journal of Pharmacology 2014;10:129-59. https://doi.org/10.3923/ijp.2014.129.159
https://doi.org/10.3923/ijp.2014.129.159...
; Islam et al., 2023a). The use of antibiotics to promote growth performance has been a common practice for many years (David et al., 2012David LS, Vidanarachchi JK, Samarasinghe K, et al. Effects of moringa based feed additives on the growth performance and carcass quality of broiler chicken. Tropical Agricultural Research 2012;24:12-20. http://dx.doi.org/10.4038/tar.v24i1.7985
http://dx.doi.org/10.4038/tar.v24i1.7985...
). However, there has been an increasing interest in developing alternative solutions due to increasing concerns regarding the development of antibiotic resistance and associated human health risks (Wierup, 2001Wierup M. The Swedish experience of the 1986 year ban of antimicrobial growth promoters, with special reference to animal health, disease prevention, productivity, and usage of antimicrobials. Microbial Drug Resistance 2001;7:183-90. https://doi.org/10.1089/10766290152045066
https://doi.org/10.1089/1076629015204506...
; Islam et al., 2023a).

Organic growth boosters may be found in the form of yeast derivatives, essential oils, pre- and probiotics, and herbal extracts (Agostini et al., 2012Agostini PS, Sola-Oriol D, Nofrarías M, et al. Role of in-feed clove supplementation on growth performance, intestinal microbiology, and morphology in broiler chicken. Livestock Science 2012;147(1-3):113-8. https://doi.org/10.1016/j.livsci.2012.04.010
https://doi.org/10.1016/j.livsci.2012.04...
; Laudadio et al., 2012Laudadio V, Passantino L, Perillo A, et al. Productive performance and histological features of intestinal mucosa of broiler chickens fed different dietary protein levels. Poultry Science 2012;91(1):265-70. https://doi.org/10.3382/ps.2011-01675
https://doi.org/10.3382/ps.2011-01675...
; Dhama et al., 2014Dhama K, Ruchi T, Khan RU, et al. Growth promoters and novel feed additives improving poultry production and health, bioactive principles and beneficial applications: the trends and advances-a review. International Journal of Pharmacology 2014;10:129-59. https://doi.org/10.3923/ijp.2014.129.159
https://doi.org/10.3923/ijp.2014.129.159...
; Chowdhury et al., 2018Chowdhury S, Mandal GP, Patra AK, et al. Different essential oils in diets of broiler chickens: 2. Gut microbes and morphology, immune response, and some blood profile and antioxidant enzymes. Animal Feed Science and Technology 2018;236:39-47. https://doi.org/10.1016/j.anifeedsci.2017.12.003
https://doi.org/10.1016/j.anifeedsci.201...
; Thuekeaw et al., 2022Thuekeaw S, Angkanaporn K, Nuengjamnong C. Microencapsulated basil oil (Ocimum basilicum Linn.) enhances growth performance, intestinal morphology, and antioxidant capacity of broiler chickens in the tropics. Animal Bioscience 2022;35(5):752-62. https://doi.org/10.5713/ab.21.0299
https://doi.org/10.5713/ab.21.0299...
). These ingredients are thought to promote gastrointestinal health and functionality, improve nutrient absorption, and strengthen broilers’ immune systems, all of which contribute to enhancing growth and feed efficiency (Agbola et al., 2015; Alagawany et al., 2021Alagawany M, El-Saadony MT, Elnesr SS, et al. Use of lemongrass essential oil as a feed additive in quail's nutrition: its effect on growth, carcass, blood biochemistry, antioxidant and immunological indices, digestive enzymes and intestinal microbiota. Poultry Science 2021;100(6):101172. https://doi.org/10.1016/j.psj.2021.101172
https://doi.org/10.1016/j.psj.2021.10117...
; Islam et al., 2023aIslam R, Sultana N, Bhakta S, et al. Modulation of growth performance, gut morphometry, and cecal microbiota in broilers by clove (Syzygium aromaticum) and tulsi (Ocimum sanctum) supplementation. Poultry Science 2023a;102(1):102266. https://doi.org/10.1016/j.psj.2022.102266
https://doi.org/10.1016/j.psj.2022.10226...
).

Due to the many health advantages of Cv, it has been utilized in traditional medicine for millennia (Batiha et al., 2020Batiha GE, Alkazmi LM, Wasef LG, et al. Syzygium aromaticum L. (Myrtaceae): traditional uses, bioactive chemical constituents, pharmacological and toxicological activities. Biomolecules 2020;10(2):202. https://doi.org/10.3390/biom10020202
https://doi.org/10.3390/biom10020202...
; Arif et al., 2022Arif M, ur Rehman A, Naseer K, et al. Effect of Aloe vera and clove powder supplementation on growth performance, carcass and blood chemistry of Japanese quails. Poultry Science 2022;101(4):101702. https://doi.org/10.1016/j.psj.2022.101702
https://doi.org/10.1016/j.psj.2022.10170...
). It has been found to have analgesic, anti-inflammatory, antibacterial, and antioxidant effects (El-Maati et al., 2016El-Maati MF, Mahgoub SA, Labib SM, et al. Phenolic extracts of clove (Syzygium aromaticum) with novel antioxidant and antibacterial activities. European Journal of Integrative Medicine 2016;8(4):494-504. https://doi.org/10.1016/j.eujim.2016.02.006
https://doi.org/10.1016/j.eujim.2016.02....
; Batiha et al., 2020). Eevuri & Putturu (2013Eevuri TR, Putturu R. Use of certain herbal preparations in broiler feeds-A review. Veterinary World 2013;6:172-9. https://doi.org/10.5455/vetworld.2013.172-179
https://doi.org/10.5455/vetworld.2013.17...
) reported that traditional Indian medicine has been exploited. Ts, also known as holy basil, has been valued as a sacred plant in Hinduism for hundreds of years. Chaudhary et al. (2018Chaudhary SK, Rokade JJ, Aderao GN, et al. Saponin in poultry and monogastric animals: A review. International Journal of Current Microbiology and Applied Sciences 2018;7:3218-25. https://doi.org/10.20546/ijcmas.2018.707.375
https://doi.org/10.20546/ijcmas.2018.707...
) explored its therapeutic characteristics and validated its potential health benefits. It has been traditionally used for its medicinal properties in humans, but its potential benefits for livestock, particularly broilers, have also been explored in recent studies (Baliga et al., 2012Baliga MS, Shivashankara AR, Azmidah A, et al. Gastrointestinal and hepatoprotective effects of Ocimum sanctum L. Syn (holy basil or tulsi): validation of the ethnomedicinal observation. Bioactive Food as Dietary Interventions for Liver and Gastrointestinal Disease 2012;325-35. https://doi.org/10.1016/B978-0-12-397154-8.00039-7
https://doi.org/10.1016/B978-0-12-397154...
; Eevuri & Putturu, 2013; Islam et al., 2023aIslam R, Sultana N, Bhakta S, et al. Modulation of growth performance, gut morphometry, and cecal microbiota in broilers by clove (Syzygium aromaticum) and tulsi (Ocimum sanctum) supplementation. Poultry Science 2023a;102(1):102266. https://doi.org/10.1016/j.psj.2022.102266
https://doi.org/10.1016/j.psj.2022.10226...
). These researches have shown the benefits of Ts supplementation on broiler development, immunological function, and general health status.

Ts supplementation in broiler diets improves feed efficiency, weight gain, and carcass traits, while also reducing oxidative stress (Agostini et al., 2012Agostini PS, Sola-Oriol D, Nofrarías M, et al. Role of in-feed clove supplementation on growth performance, intestinal microbiology, and morphology in broiler chicken. Livestock Science 2012;147(1-3):113-8. https://doi.org/10.1016/j.livsci.2012.04.010
https://doi.org/10.1016/j.livsci.2012.04...
; Wati et al., 2015Wati T, Ghosh TK, Syed B, et al. Comparative efficacy of a phytogenic feed additive and an antibiotic growth promoter on production performance, caecal microbial population and humoral immune response of broiler chickens inoculated with enteric pathogens. Animal Nutrition 2015;1(3):213-9. https://doi.org/10.1016/j.aninu.2015.08.003
https://doi.org/10.1016/j.aninu.2015.08....
; Chowdhury et al., 2018Chowdhury S, Mandal GP, Patra AK, et al. Different essential oils in diets of broiler chickens: 2. Gut microbes and morphology, immune response, and some blood profile and antioxidant enzymes. Animal Feed Science and Technology 2018;236:39-47. https://doi.org/10.1016/j.anifeedsci.2017.12.003
https://doi.org/10.1016/j.anifeedsci.201...
; Chakma et al., 2020Chakma J, Samanta A, Dutta T, et al. Effect of supplementing Moringa oleifera leaf extract and clove bud oil to the diet on microflora population and intestinal morphology of broiler birds. Indian Journal of Animal Health 2020;59:222-7. https://doi.org/10.36062/ijah.59.2SPL.2020.222-227
https://doi.org/10.36062/ijah.59.2SPL.20...
; Islam et al., 2023aIslam R, Sultana N, Bhakta S, et al. Modulation of growth performance, gut morphometry, and cecal microbiota in broilers by clove (Syzygium aromaticum) and tulsi (Ocimum sanctum) supplementation. Poultry Science 2023a;102(1):102266. https://doi.org/10.1016/j.psj.2022.102266
https://doi.org/10.1016/j.psj.2022.10226...
). Many scientific researches have shown that eugenol, a major active ingredient of both Cv and Ts, has antibacterial effects against various pathogens, such as Escherichia coli and Staphylococcus aureus (El-Maatiet al., 2016El-Maati MF, Mahgoub SA, Labib SM, et al. Phenolic extracts of clove (Syzygium aromaticum) with novel antioxidant and antibacterial activities. European Journal of Integrative Medicine 2016;8(4):494-504. https://doi.org/10.1016/j.eujim.2016.02.006
https://doi.org/10.1016/j.eujim.2016.02....
; Milind & Deepa, 2011Milind P, Deepa K. Clove: a champion spice. International Journal of Research Ayurveda and Pharmacy 2011;2:47-54.; Islam et al., 2023a). The earlier study findings showed that feeding 2-5% Ts to broilers promotes antioxidant and anti-stress activities, as well as improving broiler growth performance (Bhosale et al., 2015Bhosale DS, Bhagwat SR, Pawar MM, et al. Comparative efficacy of dietary addition of tulsi (Ocimum sanctum) leaf powder and vitamin E on broiler performance. Indian Journal of Animal Nutrition 2015;32(3):348-50.; Hasan et al., 2016Hasan MN, Mostofa M, Sorwar MG, et al. Effects of tulsi leaf extract on body weight gain in broiler production. Bangladesh Journal of Veterinary Medicine 2016;14(1):21-5. https://doi.org/10.3329/bjvm.v14i1.28818
https://doi.org/10.3329/bjvm.v14i1.28818...
; Khalil et al., 2017Khalil AA, ur Rahman U, Khan MR, et al. Essential oil eugenol: Sources, extraction techniques and nutraceutical perspectives. RSC Advances 2017;7(52):32669-81. https://doi.org/10.1039/C7RA04803C
https://doi.org/10.1039/C7RA04803C...
). In another study, Al-Mufarrej et al. (2019Al-Mufarrej SI, Al-Baadani HH, Fazea EH. Effect of level of inclusion of clove (Syzygium aromaticum) powder in the diet on growth and histological changes in the intestines and livers of broiler chickens. South African Journal of Animal Science 2019;49(1):166-75. https://doi.org/10.4314/sajas.v49i1.19
https://doi.org/10.4314/sajas.v49i1.19...
) fed 1-6% Cv to broilers and found that feeding clove at a concentration of 2% or more negatively affects broiler growth performance. Islam et al. (2023a) used the same combinations of Cv (0.5-1.5%) and Ts (2-4%) in broiler to investigate their effects on growth performance and gut health. Herbal supplements, like other alternative medicinal therapies, should be given prime focus so that farmers can gain maximum production without harmful effects (Saxena, 2008Saxena MJ. Herbs a safe and scientific approach. International Poultry Production 2008;16(2):11-3.). However, no study has been conducted to elucidate the role of Cv and Ts on the heart, lungs, liver, kidney, and testes of broilers and therefore, it remains ambiguous. Gross morphometric and biometric attributes can be an effective tool to study the impacts of Cv and Ts supplementation on these organs in broilers. In this context, the current study was conducted to investigate how the supplementation of Cv and Ts affects the aforementioned organs, both morphologically and biometrically.

MATERIALS AND METHODS

Statement of the experiment

The study was conducted on sixty (60) healthy male broiler chicks (one-day-old, Cobb-500 strain) to investigate changes in vital organs (heart, lungs, liver, kidney, and testis), both at the macroscopic and microscopic levels, if any. The experimental trial and laboratory works were conducted at the Department of Anatomy and Histology, Bangladesh Agricultural University (BAU), Bangladesh, following the proper guidelines set by the institutional “Animal Welfare and Experimentation Ethics Committee”, BAU [AWEEC/BAU/2021(05)].

Experimental shed preparation

The broiler chicks were housed in an acclimatized shed, with the provision of sufficient artificial light and ventilation and standard bio-security measures. The shed was fumigated using 17.5 g of KMnO4 and 35 ml of 37% formaldehyde. The broiler cages were disinfected with a 3% potassium permanganate spray and the feeding trays and drinking pots were cleaned regularly with 1% bleached water.

Preparation of Clove bud powder and Tulsi extract

Cv (Syzygium aromaticum) buds were ground into a very fine powder using an electric grinder and stored in an airtight container. An aqueous extract of Ts (Ocimum sanctum) was prepared from fresh leaves by blending them in water. Finally, the required amount of Cv powder and water were added to prepare different combinations, i.e., 0.5% + 2%, 1% + 3%, and 1.5% + 4% of Cv and Ts, respectively.

Broiler management

Standard temperature (brooding temperature: 35°C for the first three days followed by a gradual decrease of 2-3°C daily until reaching 21°C, rearing temperature: 21°C) and relative humidity (50-60%) were maintained throughout the study period. Following the seven days of the acclimatization period, broiler chicks (N=60) were randomly distributed to four experimental groups (15 broiler chicks in each group). Dimension of the pen: 5×4 square meters. Broilers were fed a commercial broiler starter diet (pellet form) up to d 14, and then shifted to a grower diet (feed composition is given in supplementary table 1). 24 hours of light were provided on the first three days, followed by a 30-minutes reduction of the light period each day up to d 28, with the provision of adequate ventilation. The experimental feeding trial was conducted from d 8 to d 28.

Table 1
Effects of Clove and Tulsi on the hematological profile of 28-days-old broilers.

The control group (T0) received the commercial diet and normal fresh drinking water without any additional supplementation of Cv or Ts. The treatment groups received 0.5% Cv and 2% Ts (T1), 1% Cv and 3% Ts (T2), and 1.5% Cv and 4% Ts (T3) with drinking water for 21 d (from d 8 to d 28).

Sample collection

Blood samples were collected from the wing vein to analyze the hematological profile of broilers. Heart, lungs, liver, kidney, and testis samples were collected from five broilers (sacrificed by the cervical subluxation method) from each group on d 14 and d 28 of the experiment. As part of the macroscopic study, the weight of each organ was then recorded using a high-precision electric balance (AND FGH Series Digital Balance, Korea). The final live weights of broilers of different experimental groups are given in supplementary table 2. The relative weights (%) of the organs were calculated using the following formula:

Table 2
Effects of Clove and Tulsi on the histomorphometric attributes of the heart (heart wall thickness) and lungs (surface area of lung parabronchus) in broilers.

Microscopic investigation

The collected tissue samples were fixed with 10% neutral buffered formalin for 72 hours. The samples were processed for paraffin section preparation and stained with Hematoxylin and Eosin stain, following the standard procedure described by Sultana et al. (2020Sultana N, Amin T, Afrose M, et al. Effects of Dexamethasone on the Morphometry and Biometry of Immune Organs of Broiler Chicken. International Journal of Morphology 2020;38(4):1032-8. http://dx.doi.org/10.4067/S0717-95022020000401032.
http://dx.doi.org/10.4067/S0717-95022020...
). The histological features were examined under a photomicroscope (Model: IS.1153.EPL, Euromex, Netherlands). Morphometric measurements, i.e., heart wall thickness (µm), lung parabronchus surface area (SAP, µm2), glomeruli size and the diameter of the proximal (PCT) and distal convoluted tubules (DCT) of the kidney (µm), and the size of the seminiferous tubules (SST) of the testis (µm) were measured using Image Focus Alpha software. A total of five microscopic fields from each tissue section (five tissue sections for each organ) were evaluated for each organ from each group.

Statistical analysis

All the data sets obtained in the current study were statistically analyzed using the IBM Statistical Package for the Social Sciences-V22, following a completely randomized design. At first, the normality of the data sets was tested by Levene’s test. Comparisons between the experimental groups were made using the one-way ANOVA test, followed by post-hoc Duncan’s Multiple Range Test (DMRT) and Least Significant Difference (LSD) Test. Differences were considered significant when p-value was less than 0.05. On the other hand, p-values greater than 0.05 but less than 0.1 (0.05< p<0.1) were considered as a tendency to increase or decrease.

RESULTS

Effects of Clove and Tulsi on the hematological profile

The hematological profile of 28-days-old broilers supplemented with Cv and Ts are presented in Table 1. The experimental groups had no significant difference (p>0.05) in RBC (mil/mm3), WBC (×103/µl), Hb (g/dl), PCV (%), ESR (mm in the first hour), neutrophil (%), eosinophil (%), lymphocyte (%), and monocyte (%) levels. The RBC, WBC, Hb, PCV, ESR, neutrophil, eosinophil, lymphocyte, and monocyte levels were found to be between 2.6-2.75 million/mm3, 16.67-17.10 × 103/µl, 6.40-7.33 g/dl, 24.33-25.67 %, 2-2.67 mm in the first hour, 65.67-67.70 %, 1.60-2.67 %, 27.67-29.33 %, and 2-2.67 %, respectively.

Effects of Clove and Tulsi on the relative weights of different organs

The relative weights of the heart, lungs, liver, kidney, and testes of different experimental groups are shown in Figure 1. No significant difference (p>0.05) in heart weight was observed among the experimental groups on d 14 or d 28. However, relative heart weights tended to be higher in the broilers of groups T0 and T3 on d 28.

Figure 1
The effects of clove and tulsi on the relative weights (%) of the vital organs (heart, lungs, liver, kidney, and testis) of broilers. Data are presented as the mean ± SEM.

On d 14, the lungs of the different experimental groups had no substantial difference (p>0.05) in their relative weights. However, they differed markedly (p<0.01) on d 28, where the broilers of group T0 had the highest relative lung weight and the T2 had the lowest.

The livers, kidneys, and testes of different groups had no significant difference (p>0.05) in their relative weights on d 14 or d 28.

Effects of Clove and Tulsi on the heart

Macroscopic attributes

The hearts revealed a normal macroscopic appearance without any visible alteration in both the control and the phytobiotic-supplemented groups. They appeared triangular in shape, bright red in color with a slight greyish tinge, having yellowish-white coronary fat at their base.

Microscopic attributes (heart wall thickness)

The microscopic details of the heart are shown in Figure 2. Microscopically, the hearts were composed of three distinct layers - endocardium, myocardium, and epicardium. No histoarchitectural alteration was observed in any of the experimental groups. Histomorphometric investigation of the heart wall thickness also showed no noticeable variation among the groups on d 14 and d 28 (Table 2).

Effects of Clove and Tulsi on the lungs

Macroscopic attributes

The lungs of the experimental groups appeared wedge-shaped and reddish-pink in color. No visible gross abnormality was observed, either in shape or color.

Figure 2
Histoarchitecture of the heart wall showing the effects of clove and tulsi (hematoxylin-eosin stain). Ep - Epicardium, M - Myocardium, and En - Endocardium. Magnification - 40X, Scale bar - 200 µm.

Microscopic attributes (surface area of lung parabronchus)

The microscopic details of the lungs are shown in Figure 3. Microscopically, the lung parenchyma was composed of tubular parabronchi separated by thin fibrous septa. The parabronchi were somewhat pentagonal in shape. Blood vessels were observed in between the parabronchi. However, no noticeable histoarchitectural alteration was found in the lungs of the experimental groups. The histomorphometric investigation of the SAP showed significant variation (p<0.05) among the experimental groups on both d 14 and d 28. The T2 group had the highest SAP, followed by T1, T0, and T3 on d 28 (Table 2).

Figure 3
Histoarchitecture of the lungs showing the effects of clove and tulsi (hematoxylin-eosin stain). PW - Parabronchial wall, PL - Parabronchial lumen, and PS - Parabronchial septum. Magnification - 100X, Scale bar - 100 µm.

Effects of Clove and Tulsi on the liver

Macroscopic attributes

The livers of the experimental groups had normal size and shape, without any noticeable alteration.

Microscopic attributes

The histoarchitecture of the liver is shown in Figure 4. The liver of the experimental groups exhibited normal histological architecture. The hepatic parenchyma was characterized by hexagonal hepatic lobules containing polyhedral hepatocytes arranged in hepatic cords and central veins.

Figure 4
Histoarchitecture of the liver showing the effects of clove and tulsi (hematoxylin-eosin stain). CV - Central vein, H - Hepatocyte, and S - Sinusoid. Magnification - 400X, Scale bar - 20 µm.

Effects of Clove and Tulsi on the kidney

Macroscopic attributes

The kidneys of the experimental groups appeared quadrangular in shape and dark brown in color. No visible gross abnormality, either in shape or color, was observed.

Microscopic attributes (glomerular size and tubular diameter)

The microscopic details of the kidney are shown in Figure 5. Microscopically, the kidney exhibited normal histoarchitectures with regularly distributed glomeruli throughout the cortex. The glomeruli were surrounded by Bowman’s capsule, with Bowman’s space in between them. The PCTs were lined by simple cuboidal epithelia with apical brush borders. The DCTs showed smooth apical surfaces with comparatively wider lumens. No noticeable histoarchitectural alteration was found in the kidneys of the experimental groups. The histomorphometric investigation of the size of the glomerulus as well as the diameter of the PCTs and DCTs showed no mentionable variation (p<0.05) among the experimental groups on d 14. However, substantial differences (p<0.05) in the glomerulus size as well as the diameter of PCTs and DCTs were observed on d 28, with the T2 group having the largest diameter, followed by T1, T3, and T0 (Table 3).

Table 3
Effects of Clove and Tulsi on the histomorphometric attributes of the kidney (diameter of the proximal convoluted tubules and distal convoluted tubules) in broilers.

Figure 5
Histoarchitecture of the kidney showing the effects of clove and tulsi (hematoxylin-eosin stain). G - Glomerulus, PCT - Proximal Convoluted Tubule, and DCT - Distal Convoluted Tubule. Magnification - 100X, Scale bar - 100 µm.

Effects of Clove and Tulsi on the testis

Macroscopic attributes

No gross visual macroscopic changes were observed in the testes of the experimental groups. Uniform size, shape, and color were found in both the control and treated groups of broilers.

Microscopic attributes (size of the seminiferous tubules)

The microscopic details of the testes are presented in Figure 6. Microscopically, the testes were enclosed with a fibrous capsule (tunica albuginea). Numerous seminiferous tubules containing spermatogenic cells were distributed throughout the testicular parenchyma. Single or cluster Leydig cells were observed in between the seminiferous tubules. The broilers of different experimental groups showed no noticeable histoarchitectural alteration. In the histomorphometric study, no mentionable variation (p>0.05) in the SSTs was found among the experimental groups. However, SSTs differed substantially (p<0.05) on d 28. The SST size was highest in group T2, followed by T1, T0, and T3 (Table 4).

Table 4
Effects of Clove and Tulsi on the histomorphometric attributes of the testes (size of the seminiferous tubules) in broilers.

Figure 6
Histoarchitecture of the testes showing the effects of clove and tulsi (hematoxylin-eosin stain). ST - Seminiferous tubules, SC - Spermatogenic cell, and LC - Leydig cell. Magnification - 400X, Scale bar - 20 µm.

DISCUSSION

Cv and Ts have been proven to be effective phytobiotic feed additives that improve growth performance and gut health in poultry (Agostini et al., 2012Agostini PS, Sola-Oriol D, Nofrarías M, et al. Role of in-feed clove supplementation on growth performance, intestinal microbiology, and morphology in broiler chicken. Livestock Science 2012;147(1-3):113-8. https://doi.org/10.1016/j.livsci.2012.04.010
https://doi.org/10.1016/j.livsci.2012.04...
; Al-Mufarrej et al., 2019Al-Mufarrej SI, Al-Baadani HH, Fazea EH. Effect of level of inclusion of clove (Syzygium aromaticum) powder in the diet on growth and histological changes in the intestines and livers of broiler chickens. South African Journal of Animal Science 2019;49(1):166-75. https://doi.org/10.4314/sajas.v49i1.19
https://doi.org/10.4314/sajas.v49i1.19...
; Islam et al., 2023aIslam R, Sultana N, Bhakta S, et al. Modulation of growth performance, gut morphometry, and cecal microbiota in broilers by clove (Syzygium aromaticum) and tulsi (Ocimum sanctum) supplementation. Poultry Science 2023a;102(1):102266. https://doi.org/10.1016/j.psj.2022.102266
https://doi.org/10.1016/j.psj.2022.10226...
). However, their effects on the vital organs of broilers need to be explored to get a clear insight into their efficacy. Morphologic and biometric evaluation of the vital organs can be an important tool in this context (Islam et al.,2021; Islam et al., 2022a, b; Sultana et al., 2021Sultana N, Islam R, Akter A, et al. Biochemical and morphological attributes of broiler kidney in response to dietary glucocorticoid, dexamethasone. Saudi Journal of Biological Sciences 2021;28(12):6721-9. https://doi.org/10.1016/j.sjbs.2021.07.047
https://doi.org/10.1016/j.sjbs.2021.07.0...
; Sultana et al., 2022). Therefore, the current study investigates the impact of Cv and Ts on the morphologic and biometric attributes of the vital organs (heart, lungs, liver, kidney, and testes) of broilers for the first time.

Effects of Clove and Tulsi on the hematological profile

The maintenance of optimal blood cellular profile is crucial to broiler. The hematological profile analysis in broilers fed Cv and Ts showed no mentionable effect on blood cellular constituents. Erythrocyte is one of the major cellular constituents that transport nutrition and oxygen (Hb) around different body parts (Odunitan-Wayas et al., 2018Odunitan-Wayas F, Kolanisi U, Chimonyo M. Haematological and serum biochemical responses of Ovambo chickens fed provitamin A biofortified maize. Brazilian Journal of Poultry Science 2018;20:425-34. https://doi.org/10.1590/1806-9061-2016-0444
https://doi.org/10.1590/1806-9061-2016-0...
). Nath et al. (2012Nath DD, Rahman MM, Akter F, et al. Effects of tulsi, black pepper and cloves extract as a growth promoter in broiler. Bangladesh Journal of Veterinary Medicine 2012;10:33-9. https://doi.org/10.3329/bjvm.v10i1-2.15644
https://doi.org/10.3329/bjvm.v10i1-2.156...
) reported that supplementation of tulsi, clove, and black pepper does not affect broiler hematological indices (TEC, PCV, Hb, and ESR). Hasan et al. (2016Hasan MN, Mostofa M, Sorwar MG, et al. Effects of tulsi leaf extract on body weight gain in broiler production. Bangladesh Journal of Veterinary Medicine 2016;14(1):21-5. https://doi.org/10.3329/bjvm.v14i1.28818
https://doi.org/10.3329/bjvm.v14i1.28818...
) reported higher PCV % while ESR % was comparatively lower than the previous report. Neutrophil, eosinophil, lymphocyte, and monocyte counts were also lower than the earlier report by Baudouin et al., 2021Baudouin KA, Soualio KA, Mathieu BN, et al. Hematological profile of broilers and local chickensin Korhogo, Cote d'Ivoire. International Journal of Agriculture, Environment and Bioresearch 2021;6(2):14-23. https://doi.org/10.35410/IJAEB.2021.5618
https://doi.org/10.35410/IJAEB.2021.5618...
. WBCs play a critical role in the body’s defense and are indicators of stress response (Glenn & Armstrong, 2019Glenn A, Armstrong CE. Physiology of red and white blood cells. Anaesthesia & Intensive Care Medicine 2019;20(3):170-4. https://doi.org/10.1016/j.mpaic.2019.01.001
https://doi.org/10.1016/j.mpaic.2019.01....
). The decrease in these parameters might be linked to the composition of the supplied feed and environmental factors (Baudouin et al., 2021). However, the current study results strongly suggests that supplementation of Cv and Ts has no harmful or toxic effect on the hematological profile of broilers.

Effects of Clove and Tulsi on the relative weights of different organs

The morphologic and biometric features of the vital organs of broilers have been widely studied due to the potential negative physiological effects associated with genetic selection for growth (Chen et al., 2017Chen CY, Huang YF, Ko YJ, et al. Obesity-associated cardiac pathogenesis in broiler breeder hens: Development of metabolic cardiomyopathy. Poultry Science 2017;96(7):2438-46. https://doi.org/10.3382/ps/pex016
https://doi.org/10.3382/ps/pex016...
; Harash et al., 2019Harash G, Richardson KC, Alshamy Z, et al. Heart ventricular histology and microvasculature together with aortic histology and elastic lamellar structure: A comparison of a novel dual-purpose to a broiler chicken line. PloS ONE 2019;14(3):e0214158. https://doi.org/10.1371/journal.pone.0214158
https://doi.org/10.1371/journal.pone.021...
). In avian species, the liver is larger than in mammals compared to body size. The size and color of the liver depend on the broiler’s age and body weight (Akers & Denbow, 2013Akers RM, Denbow DM. Digestive system. In: Hoboken, NJ. Anatomy and physiology of domestic animals. 2nd ed. London: John Wiley & Sons; 2013. p.483-527. ISBN-13 978-0813803296; Denbow, 2015). The morphologic and biometric investigation of the testes was done as it is the primary organ for male reproduction, being responsible for producing sperm and male hormones. Fragaso et al. (2013) observed that male fertility is directly linked to testicular weight. The present study findings highlight that Cv and Ts supplementation had no negative impact on broiler heart, lung, liver, kidney, and testis. The relative weights of the heart, right lung, liver, and right kidney found in the current study ranged between 0.45-0.69%, 0.25-0.39%, 2.5-3.02%, and 0.30-0.35%, respectively. The relative weights of the heart, lung, liver, kidney, and testis have been found to be 0.52±0.07% (Gaya et al., 2007Gaya LG, Costa AM, Ferraz JB, et al. Genetic trends of absolute and relative heart weight in a male broiler line. Genetics and Molecular Research 2007;6(4):1091-6.), 0.36±0.02% (Azizian & Saki, 2021Azizian M, Saki AA. Effect of mash, pellet, and extrude diet form on ascetic gene expression (HIF-1? mRNA) and heart index in broiler chicken. Journal of Agricultural Science and Technology 2021;23(1):17-25. https://doi.org/20.1001.1.16807073.2021.23.1.4.7
https://doi.org/20.1001.1.16807073.2021....
), 2.6% (Nath et al., 2012Nath DD, Rahman MM, Akter F, et al. Effects of tulsi, black pepper and cloves extract as a growth promoter in broiler. Bangladesh Journal of Veterinary Medicine 2012;10:33-9. https://doi.org/10.3329/bjvm.v10i1-2.15644
https://doi.org/10.3329/bjvm.v10i1-2.156...
), 0.609±0.02% (Xiao et al., 2011Xiao J, Cui HM, Yang F, et al. Effect of dietary high molybdenum on the cell cycle and apoptosis of kidney in broilers. Biological Trace Element Research 2011;142:523-31. https://doi.org/10.1007/s12011-010-8772-4
https://doi.org/10.1007/s12011-010-8772-...
), and 0.81±0.14 (Mahmood et al., 2011Mahmood F, Khan MZ, Khan A, et al. Lysine induced modulation of toxico-pathological effects of cottonseed meal in broiler breeder males. Pakistan Journal of Zoology 2011;43(2):357-65.), respectively, which is in line with the findings of our current study.

Effects of Clove and Tulsi on the macroscopic and microscopic characteristics of the heart

The histoarchitectural integrity of the heart is crucial for maintaining its normal function. The myocardium, which is the muscular layer of the heart, is the most important component of the heart. According to the study reports conducted by Harash et al. (2019Harash G, Richardson KC, Alshamy Z, et al. Heart ventricular histology and microvasculature together with aortic histology and elastic lamellar structure: A comparison of a novel dual-purpose to a broiler chicken line. PloS ONE 2019;14(3):e0214158. https://doi.org/10.1371/journal.pone.0214158
https://doi.org/10.1371/journal.pone.021...
), broilers have increased myocardial cell size compared to the non-broiler chicken, which is accompanied by increased myofibril size, number, and fibrosis of the heart wall. The implications of these histological changes in broiler hearts are significant. Chen et al. (2017Chen CY, Huang YF, Ko YJ, et al. Obesity-associated cardiac pathogenesis in broiler breeder hens: Development of metabolic cardiomyopathy. Poultry Science 2017;96(7):2438-46. https://doi.org/10.3382/ps/pex016
https://doi.org/10.3382/ps/pex016...
) reported that the increase in myocardial cell size and fibrosis can lead to reduced cardiac function and increased susceptibility to heart failure. The use of steroid GPs in broilers causes severe morphologic and biometric alterations like suppression of cardiomyocyte development (Rudolph, 2000Rudolph AM. Myocardial growth before and after birth: clinical implications. Acta Paediatrica 2000;89:129-33. https://doi.org/10.1111/j.1651-2227.2000.tb01201.x
https://doi.org/10.1111/j.1651-2227.2000...
; Islam et al., 2022aIslam R, Sultana N, Khan MAHNA. Sero-biochemical and cardiac morphological alterations in broiler chicken triggered by dietary dexamethasone. Advances in Animal and Veterinary Sciences 2022a;10:829-37. http://dx.doi.org/10.17582/journal.aavs/2022/10.4.829.837
http://dx.doi.org/10.17582/journal.aavs/...
). In the current study, the hearts of the different experimental groups supplemented with Cv and Ts showed normal histoarchitectures (Eurell & Frappier, 2006Eurell JA, Frappier BL. Dellmann's textbook of veterinary histology. 6th ed. Ames: Blackwell Publishing; 2006., Islam et al., 2022a), without any visible alterations. Therefore, phytobiotic-based (Cv and Ts) dietary inclusion can be a safer option in this context.

Effects of Clove and Tulsi on the macroscopic and microscopic characteristics of the lungs

The histological examination of broiler lungs from both the morphologic and morphometric aspects can provide important information about the presence and distribution of pathological changes, such as inflammation, fibrosis, and cellular proliferation (Islam et al., 2022bIslam R, Sultana N, Alam R, et al. Dose and time-dependent effects of glucocorticoid: A morphologic and morphometric study in the broiler lung. Turkish Journal of Veterinary & Animal Sciences 2022b;46(4):617-28. https://doi.org/10.55730/1300-0128.4233
https://doi.org/10.55730/1300-0128.4233...
). The parabronchi of broiler chickens are composed of air capillaries and blood capillaries. Air capillaries are lined with simple squamous epithelium, while blood capillaries are lined with endothelial cells. Eurell & Frappier (2006Eurell JA, Frappier BL. Dellmann's textbook of veterinary histology. 6th ed. Ames: Blackwell Publishing; 2006.) elucidated that the close proximity of the air and blood capillaries facilitates efficient gas exchange. The histological investigation of the lungs showed normal histoarchitectures in the broiler (Eurell & Frappier, 2006). However, the findings of the current study showed that the surface area of the lung parabronchus notably increased in the broilers supplemented with 1% Cv and 3% Ts. This might be beneficial in reducing or eliminating hypoxic stress in broilers through the augmentation of oxygen absorption in the lungs and increased availability of oxygen at the cellular level (Olkowski et al., 2005Olkowski AA, Duke T, Wojnarowicz C. The aetiology of hypoxaemia in chickens selected for rapid growth. Comparative biochemistry and physiology. Molecular & Integrative Physiology - Part A 2005;141:122-31. https://doi.org/10.1016/j.cbpb.2005.04.011
https://doi.org/10.1016/j.cbpb.2005.04.0...
; Özyiğit et al., 2015).

Effects of Clove and Tulsi on the macroscopic and microscopic characteristics of the liver

Avian liver is histologically different from that of mammals, having less connective tissue and lacking true hepatic lobulation (Eurell & Frappier, 2006Eurell JA, Frappier BL. Dellmann's textbook of veterinary histology. 6th ed. Ames: Blackwell Publishing; 2006.; Mushawwir et al., 2021Mushawwir A, Permana R, Darwis D, et al. Enhancement of the liver histologic of broiler induced by irradiated chitosan (IC). Proceedings of International Conference on Nuclear Science, Technology, and Application; 2020. Jakarta (ID): AIP Publishing LLC; 2021.). The liver is a vital organ due to its detoxifying activities within the body, as reported by Zhao et al. (2019Zhao M, Zhao H, Deng J, et al. Role of the CLOCK protein in liver detoxification. British Journal of Pharmacology 2019;176:4639-52. https://doi.org/10.1111/bph.14828
https://doi.org/10.1111/bph.14828...
). Denbow (2015Denbow DM. Gastrointestinal anatomy and physiology. In: Whittow GC, editor. Sturkie's avian physiology. 6th ed. New York: Academic Press; 2015. p.299-325. ISBN-13 978-012407160) reported that the synthesis, storage, and distribution of lipids, carbohydrates, and proteins are regulated by the liver, which thus plays critical roles in digestion and metabolism. Hence, a healthy liver is a must for maintaining the health of an organism. Duma et al. (2006Duma D, Jewell CM, Cidlowski JA. Multiple glucocorticoid receptor isoforms and mechanisms of post-translational modification. The Journal of Steroid Biochemistry and Molecular Biology 2006;102(1-5):11-21. https://doi.org/10.1016/j.jsbmb.2006.09.009.
https://doi.org/10.1016/j.jsbmb.2006.09....
) noted that drugs are commonly responsible for hepatic damage depending on their dose and duration of administration. Residues of antibiotics and steroids reportedly accumulate in the liver, which is accompanied by severe liver damage, according to earlier study reports (Amine et al., 2020Amine B, Tarek K, Riad B, et al. Evaluation of adverse effects of antibiotics on broiler chickens. Journal of World's Poultry Research 2020;10:145-50. https://dx.doi.org/10.36380/jwpr.2020.19
https://dx.doi.org/10.36380/jwpr.2020.19...
; Sultana et al., 2022Sultana N, Islam R, Das RR, et al. Steroid growth promoter modified glucose profile and liver morphology in broiler by altering the localization and expression pattern of hepatic glucocorticoid receptors. Research in Veterinary Science 2022;152:277-88. https://doi.org/10.1016/j.rvsc.2022.08.024
https://doi.org/10.1016/j.rvsc.2022.08.0...
; Ulomi et al., 2022Ulomi WJ, Mgaya FX, Kimera Z, et al. Determination of sulphonamides and tetracycline residues in liver tissues of broiler chicken sold in Kinondoni and Ilala municipalities, dar es Salaam, Tanzania. Antibiotics 2022;11(9):1222. https://doi.org/10.3390/antibiotics11091222
https://doi.org/10.3390/antibiotics11091...
). Histological investigation of the liver in different experimental groups also showed no alteration in their histoarchitecture. Therefore, Cv and Ts can be safer alternatives to antibiotic or steroid growth promoters.

Effects of Clove and Tulsi on the macroscopic and microscopic characteristics of the kidney

Histologically, broiler kidneys are similar to those of other avian species, but they have some unique features that reflect their rapid growth and high metabolic rate, as noticed by Nabipour et al. (2009Nabipour A, Alishahi E, Asadian M. Some histological and physiological features of avian kidney. Journal of Applied Animal Research 2009;36(2):195-8. https://doi.org/10.1080/09712119.2009.9707058
https://doi.org/10.1080/09712119.2009.97...
). The avian kidney consists of two distinct regions: the cortex and the medulla. The cortex contains glomeruli and proximal and distal convoluted tubules, while the medulla contains the loops of Henle and collecting ducts. The glomeruli are responsible for filtering blood, and the tubules and ducts are responsible for reabsorbing useful substances and excreting waste products (Eurell & Frappier, 2006Eurell JA, Frappier BL. Dellmann's textbook of veterinary histology. 6th ed. Ames: Blackwell Publishing; 2006.; Nabipour et al., 2009). Therefore, maintaining structural integrity is indispensable to preserve proper renal functionality. Synthetic growth promoters like antibiotics or steroids reportedly damage broiler kidneys, hence phytobiotic-based feeding practices can be an effective solution to this problem (Amine et al., 2020Amine B, Tarek K, Riad B, et al. Evaluation of adverse effects of antibiotics on broiler chickens. Journal of World's Poultry Research 2020;10:145-50. https://dx.doi.org/10.36380/jwpr.2020.19
https://dx.doi.org/10.36380/jwpr.2020.19...
; Sultana et al., 2021Sultana N, Islam R, Akter A, et al. Biochemical and morphological attributes of broiler kidney in response to dietary glucocorticoid, dexamethasone. Saudi Journal of Biological Sciences 2021;28(12):6721-9. https://doi.org/10.1016/j.sjbs.2021.07.047
https://doi.org/10.1016/j.sjbs.2021.07.0...
). The present study showed no visible alteration in the histoarchitectures of the kidneys of the experimental groups. However, the size of the glomerulus, and the diameter of both PCT and DCT substantially increased on d 28 in the broilers supplemented with 1% Cv and 3% Ts, indicating a higher capacity of filtration and resorption of water and electrolytes (Nabipour et al., 2009).

Effects of Clove and Tulsi on the macroscopic and microscopic characteristics of the testes

The testes of broiler chickens are composed of seminiferous tubules, which are responsible for the production of sperm, and interstitial tissue, which produces testosterone (Metayer et al., 2019Metayer JP, Travel A, Mika A, et al. Lack of toxic interaction between fusariotoxins in broiler chickens fed throughout their life at the highest level tolerated in the European Union. Toxins 2019;11(8):455. https://doi.org/10.3390/toxins11080455
https://doi.org/10.3390/toxins11080455...
). The seminiferous tubules are lined with germ cells that undergo a complex process of spermatogenesis to produce mature sperm. The interstitial tissue contains Leydig cells, which produce testosterone, a male sex hormone that plays a crucial role in the growth and development of broiler chickens, as reported by Mfoundou et al. (2022Mfoundou JD, Guo Y, Yan Z, et al. Morpho-Histology and Morphometry of Chicken Testes and Seminiferous Tubules among Yellow-Feathered Broilers of Different Ages. Veterinary Sciences 2022;9(9):485. https://doi.org/10.3390/vetsci9090485
https://doi.org/10.3390/vetsci9090485...
). Histological investigation of the testes also showed no noticeable change in their histoarchitectures. However, SST substantially increased in broilers fed 1% Cv and 3% Ts. Research has shown that the size of the seminiferous tubules is positively correlated with sperm count and sperm quality, both of which are important factors in male fertility. Larger seminiferous tubules have been associated with higher sperm counts, as well as a greater proportion of morphologically normal and motile sperm (Fragaso et al., 2013; Metayer et al., 2019; Mfoundou et al., 2022).

Research on phytobiotic-based dietary inclusion is getting immense popularity in recent years due to the severe health risks associated with supplementing antibiotic or steroid GPs to broilers. Following these studies, Cv and Ts have been proven to be effective phytobiotic supplements in promoting growth performance, gut health, and meat quality in poultry species. This study reported the impact of Cv and Ts on the vital organs and male gonads of broilers for the very first time. The results suggest that Cv and Ts have no toxic or negative impact, but rather exert some beneficial impacts like increased parabronchial surface area, size of the glomerulus, diameter of the kidney tubules, and size of the seminiferous tubules. However, further study is recommended to investigate the serum biochemical indices linked to these organs, in order to elucidate the overall health and functionality of these organs.

ACKNOWLEDGMENTS

We sincerely thank the Ministry of Science and Technology, Bangladesh for funding the research (BS/56). We would like to acknowledge the laboratory facilities provided by the Department of Anatomy and Histology, Bangladesh Agricultural University, Bangladesh. We are also thankful to the Nourish Poultry and Hatchery Ltd, Bangladesh for providing the day-old chicks.

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Publication Dates

  • Publication in this collection
    13 Oct 2023
  • Date of issue
    2023

History

  • Received
    19 Apr 2023
  • Accepted
    26 July 2023
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