Plant derived additives have been identified to contain bioactive compounds with effects similar to antibiotic growth promoters in three main areas, i.e., gut micro-flora, antioxidant properties, and liver function without compromising intestinal health and/or the bird’s genetic potential (Hernandez, et al., 2004; Singh et al., 2021). The addition of plants and their extracts into diets is aimed at improving the productivity of livestock through amelioration of undesired feed properties, promotion of the animal’s production performance and improving the quality of food derived from those animals (Kolodziej-Skalska et al., 2011; Agubosi et al., 2022; Shittu et al., 2021). Herbs such as herbs, spices, and various plant extracts have received increased attention as possible antibiotic growth promoters (Kim et al., 2009; Alagbe and Ushie, 2022; Oluwafemi et al., 2021).
Phytochemicals come from natural sources and are generally recognised as safe (GRAS) which make them good candidates to be used as feed additives in livestock production in comparison with antibiotics (Windisch et al., 2008; Alagbe, 2017). The biological mechanism of action of phytochemicals depends on their chemical structure (Kim et al., 2010; Alagbe et al., 2022). Phytochemicals used as poultry feed additives can improve animal’s health and performance because of their anti-microbial, anti-bacterial, anti-helminthic and anti-stress (Peric et al., 2009; Shittu et al., 2022) and anti-oxidant properties (Applegate et al, 2010; Alagbe et al., 2021), and their ability to modulate gut microbiota (Donoghue, 2009; Alagbe et al., 2021) and enhance immune responses (Kim et al., 2008; Adewale et al., 2021). Environment factors such as nutrition, temperature, humidity, seasonal changes and animal management (health and housing) have a measurable effect on semen quality (Cross, 1998). Sperm morphology, motility, sperm concentration and volume per ejaculate are common criteria for evaluating semen quality (Ademola, 2003).
Rubia cordifolia also known as Indian madder belongs to the family Rubiaceae is a perennial flowering plant widely distributed in Asia (Bhatt and Kushwah, 2013). The stem, root and leaves of Rubia cordifolia is traditionally used for the treatment of gastro-intestinal disorder, skin disease, malaria, typhoid, diabetes, liver diseases, menstrual and urinary diseases, cancer, inflammations, pneumonia, cough, chronic bronchitis, hemoptysis and other viral infections (Bhat et al., 2018; Prajapati and Parmar, 2011; Musa et al., 2020; Alagbe, 2017).
Several studies have demonstrated the anti-bacterial, antimicrobial, hepato-protective, antiviral, anti-rheumatic, immunomodulatory, cytotoxic, anti-ulcer, anti-fibrotic, antioxidant, anti-proliferative, antifungal and anti-helmithic properties in Rubia cordifolia extract (Boldizs et al., 2006; Kalyoncu et al., 2006; Gao et al., 2003; Alagbe, 2020). Aqueous Rubia cordifolia root, leaf and stem bark extract have proven to suppress the activities of some pathogenic bacteria such as: Salmonella spp, Staphyllococcus pyogenes, Staphyllococcus aureus, Bacteriodes spp and E. coli in animals (Ino et al., 1995; Kamuhabwa et al., 2000; Wei et al., 2011).
In view of these abundant potentials in Rubia cordifolia, this experiment was designed examine the growth performance, semen quality characteristics and hormonal profile of male rabbit bucks fed Rubia cordifolia root extracts.
Materials and methods
Site of the experiment
The experiment was carried out at Division of Animal Nutrition, Sumitra Research Institute, Gujarat, India with a coastline of 1,600 Km, 23o 13’N 72o41’E.
Collection, preparation and gas chromatography mass spectrometry (GC-MS) of Rubia cordifolia root extract (RCE)
Rubia cordifolia root was collected from Sumitra Research Institute in the month of April 2021. It was identified and authenticated by a certified taxonomist Dr. Xing Liu at the Department of Biological Sciences. Samples of Rubia cordifolia root were thoroughly washed with running tap water and air dried for 2 weeks to obtain a constant weight. Dried Rubia cordifolia root was pounded into powder with a mortar and pestle it was thereafter stored in a well labeled container.
Two hundred grams of dried Rubia cordifolia root powder was soaked in 1 liter for 72 hours, kept in the refrigerator at 4oC and stirred at 3 times daily using a spatula. The sample was filtered via a Whatman’s No.1 filter paper (10 cm). Thereafter, the filterate was stored in a laboratory labeled container and sent to the laboratory for further analysis.
Gas chromatography mass spectrometry (GC-MS) analysis of Rubia cordifolia root extracts (RCE) was carried out with Varian 450 GC system (Model 1100 series, China) with temperature and pressure range of 50oC to 450 oC isothermal 1079 PTV injector and 0 to 100 psi, consisting of splitless injector with total flow of 500 mL/minutes at 10 psi. The introduced sample (RCE) was passed through a rapid column at a cool down rate of 40 oC to 50 oC at 4.5 minutes with an electron range of 150eV. Bioactive compounds were identified with standard compounds in National Institute of Standard and Technology (NIST).
Animal management, diet formulation and experimental set-up
32 – 7 weeks weaned male rabbits (Newzealand × Chinchilla) weighing 611.3 ± 10 g were purchased from a reputable commercial breeding farms in Gujarat india and housed individually in an all wired galvanized cage measuring 50 cm × 50 cm × 30 cm: length × width × height suspended 120 cm above the ground, equipped with automatic nipple drinker and a metallic manual clay feeder was kept in each cage. Before the arrival of the animals, cages and pens were properly disinfected with Cid 2000 at10 mL per 20 liters of water. On arrival, rabbits were given anti-stress and randomly distributed into 4 groups of 8 rabbits per treatment with one animal per replicate in a completely randomized design. Rabbits were acclimatized before the commencement of the experiment during which they were given prophylactic treatment against parasites (endo and ecto-parasites) with ivermectin injection and bacterial infections (Oxytrox® ) strictly adhering to the manufacturers recommendation on drug administration.
Basal diet was formulated to meet the nutrient requirements of growing rabbits according to the recommendation of National Research Council (NRC, 1977) as presented in Table A. Rabbits in treatment 1 (T1) was fed basal diet with 0 mL Rubia cordifolia root extracts (RCE) while T2, T3 and T4 were fed basal diet with 20 mL, 40 mL and 60 mL per litre of water/day. The experiment lasted for 12 weeks during which strict biosecurity measures were observed. Feed and water were also given ad libitum.
Daily feed intake (g)
Daily feed intake was calculated by subtracting feed served from left over. It can be expressed as:
Daily feed intake (g) = Feed served (g) – Feed left over (g)
Body weight gain (g)
Body weight gain was calculated by subtracting final body weight from initial body weight
Body weight gain (g) = Final body weight (g) – Initial weight gain (g)
Feed conversion ratio (g)
Feed conversion ratio was calculated by dividing feed consumed by body weight gain as expressed below:
Feed conversion ratio (g) = Feed consumed (g)
Body weight gain (g)
Mortality was recorded as it occurs
Blood samples were collected from the marginal ear veins into vacutainer bottles without ethylene diamine tetra acetic acid from 4 randomly selected rabbits per treatment for hormonal assay using commercial diagnostic kit (AIA-360 Automated Immunoassay analyzer, USA) with dimension 1016 mm × 665 mm × 762 mm (width × depth × height).
Semen collection and evaluation
A 2-week period was used to train the bucks for semen collection. Semen was finally collected from the buck using the artificial vagina (AV) described by Herbert and Adejumo (1995). Prior to semen collection, the AV was warmed for a few minutes in warm water at a temperature slightly above body temperature and thereafter drained. Semen collection was done between 7.00 and 9.00 am to ensure that optimum quality semen were obtained.
The semen was promptly assessed for semen quality parameters such as semen colour, semen volume, mass activity, sperm motility, sperm concentration and percentage live sperm using Computer Assisted Semen Analyzer Bonraybio (Taichung City, Taiwan).
All data were subjected to one -way analysis of variance (ANOVA) using SPSS (23.0) and significant means were separated using Duncan’s test of the same statistical package.
The model: Dij = µ + αi + βij was used in this experiment:
Where Dij = any of the response variables; i = the overall mean; αi = effect of the xth treatment and βij = random error due to experimentation
Results and discussion
Bioactive compounds in Rubia cordifolia root extracts using GC-MS analysis
Medicinal plants contain several of bioactive compounds, such as phenolics, flavonoids, terpenoids, carotenoids, saponins and alkaloids in their stems, leaves, roots, seeds, flowers and twigs (Adewale et al., 2021). These compounds are widely used in the food, cosmetic and pharmaceutical industries because they posses; antioxidant (Shittu et al., 2020), antimicrobial (Singh et al., 2021), anti-inflammatory (Agubosi et al., 2022), hepato-protective (Olafadehan et al., 2021), antifungal and antiviral (Oluwafemi et al., 2020), immune-modulatory, cytotoxic, hypolipidemic, antibacterial, anti-tumor, antipyretic, antiplasmodial, antifibrotic (Alagbe et al., 2022) and antiproliferative properties (Agubosi et al., 2022). Gas chromatography mass spectrometry of Rubia cordifolia root extracts reveals the presence of 21 bioactive compounds which accounts for 92.46 %. 3-deoxy-d-mannoic acid contains (0.44 %), 4-Methoxy-2-nitroformanilide (0.02 %), γ-terpinene (1.10 %), β-fenchol (0.40 %), 3-Allyl-6-methoxyphenol (1.67 %), Glycidol stearate (0.10 %), 2-Methyl -4-vinylphenol (2.05 %), α-cubebene (2.09 %), Carbonic acid (0.77 %), 9,12-Octadecanoic acid (25.06 %), α-longipinene (0.75 %), Terpinen-4-ol (1.04 %), 1,3 propanediol, 2-ethyl 2-hydroxymethyl (14.71 %), γ-terpinene (0.94 %), γ-eudesmol (1.13 %), 9-Octadecenoic acid (29.16 %), Torreyol-α-cadinol (0.07 %), 1,2-Cyclopentanedione (0.30 %), Ethylene diacrylate (0.50 %) and 4-methyl-2,3-hexadien -1-ol (11.20 %). 9-Octadecenoic acid had the highest concentration while 4-Methoxy-2-nitroformanilide had the lowest concentration. The result obtained in this study agrees with the findings of Mohammad et al. (2018); Kaur et al. (2008); Singh et al. (2021). A synergistic combination of these bioactive compounds allows Rubia cordifolia root extracts to be used in the treatment of various ailments due to their therapeutic properties (Bsau et al., 2005; Gupta et al., 2011); Raghad and Abdul (2017).
Growth performance of rabbits fed different levels of Rubia cordifolia root extracts (RCE)
Growth performance of rabbits fed different levels of Rubia cordifolia root extracts (RCE) is presented in Table 3. Initial body weight (IBW), final body weight (FBW), weight gain (WG), average daily weight gain (ADWG), total feed intake (TFI), average daily feed intake (ADFI) and feed conversion ratio (FCR) ranged from 611.3 – 619.5 g, 1900.8 – 2300.4 g, 1282.1 – 1680.9 g, 8652.1 – 9103.1 g, 103.0 – 108.4 g, 4.00 – 4.84, 0.50 – 2.51 % respectively. ADWG value was highest in T4, intermediate in T2 and T3 and lowest in T1 (P˂0.05). Conversely, FCR value was maximum in T1, intermediate in T2 and T3 and lowest in T4 (P˂0.05). ADFI were not significantly (P˃0.05) affected among the treatments. Highest mortality rate was recorded in T1 (2.51 %) followed by T2 (0.50 %) while none were recorded in the other treatments (P˂0.05). Higher ADWG recorded among rabbits fed different levels of Rubia cordifolia root extract (RCE) indicates that the test ingredients enhanced nutrient utilization by stimulating the activities of enzymes and preventing dysbiosis due to the presence of several bioactive compounds (Table 2) thus enhancing performance (Sandip et al., 2022; Muritala et al., 2022). The result obtained in this study agrees with the findings of Ogbuewu et al. (2010); Oluwfemi and Alagbe (2019). The enhanced nutrient digestibility consequently enhances feed intake and health status of rabbits (Shittu et al., 2021). RCE also posses antioxidant and immune-modulatory properties due to the presence of α-cubebene, 3-deoxy-d-mannoic acid, Torreyol-α-cadinol, α-longipinene, 2-Methyl -4-vinylphenol and β-fenchol (Topala et al., 2014; Kokila et al., 2016).
Hormonal profile of rabbits fed different levels of Rubia cordifolia root extracts (RCE)
Table 4 shows the hormonal profile of rabbits fed different levels of Rubia cordifolia root extracts (RCE). The hormones determined includes: testosterone (TES), follicle stimulating hormone (FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH) which ranged from 2.06 – 4.00 (I.U/L), 6.50 – 6.98 (I.U/L), 8.80 – 12.30(I.U/L) and 0.93 – 2.03 (I.U/L) respectively. TES, LH and TSH values were significantly (P˂0.05) influenced among the treatments. The values follow similar pattern and were highest in T4, intermediate in T2 and T3 and lowest in T1. This is a clear indication that the bioactive compounds in Rubia cordifolia root extract (RCE) is capable of activating the activities gonadotropin releasing hormone which stimulates the secretion of LH, which in turns stimulates gonadal secretion of testosterone, estrogen and progesterone. Conversely, FSH values were not significantly (P˃0.05) different among the groups. The result obtained in this study agrees with the findings of Olatundun and Ogunlade (2020). According to Amao et al. (2013); Brucker et al. (1998), FSH and LH are secreted from the anterior pituitary cells of animals (gonadotrophs) with the aim of stimulating the gonads - in males, the testes and in females, the ovaries. Diminished secretion of LH or FSH can result in failure of gonadal function (hypogonadism), thus leading to poor sperm cell production (Ahemen et al., 2013). FSH plays for sperm production. It supports the function of Sertoli cells, which in turn support many aspects of sperm cell maturation (Ghassabian and Trasande, 2018; Kay, 2014). Kilgour et al. (1984); Jiang (2007) reported that FSH is necessary for the establishment of the normal population of Sertoli cell and the stimulation of the production of androgen-binding protein from the Sertoli cells. Androgen-binding protein binds with the testosterone making it available for its function in spermatogenesis (Bearden and Fuquay, 1997; Lovecamp and Devis, 2003). Testosterone is responsible in maintaining optimum conditions for spermiogenesis, spermatozoa transport and semen deposition near the site of fertilisation in the female (Kay, 2014; Castellini, 2003).
Semen parameters of rabbit buck fed different levels of Rubia cordifolia root extracts (RCE)
Semen parameters of rabbit buck fed different levels of Rubia cordifolia root extracts (RCE) is presented in Table 5. The semen colour was milky across the treatment while sperm volume, semen pH, sperm concentration, live sperm percentage, abnormal sperm percentage and motility percentage ranged from 0.51 – 0.66 mL, 7.00 – 7.18, 21.60 – 32.34 (×106/mL), 75.12 – 84.12 %, 10.04 – 14.21 % and 54.18 – 70.40 % respectively. Semen pH and colour were not significantly (P˃0.05) different among the treatments. Conversely, sperm volume, sperm concentration, live sperm percentage, abnormal sperm percentage and motility percentage were significantly (P˂0.05) influenced by the treatment. According to Abd-Azim and El-kamash (2015) variation in semen colour, semen pH, semen density and motility could be attributed differences in breed of rabbit bucks. Daader and Saleem (2005); El-Sheikh and Saleem (2010) reported semen volume to increase with age and body weight. Sperm concentration and live sperm concentration in bucks fed T2, T3 and T4 were better (P˂0.05) than those fed T1. This variation in values could be attributed to the antioxidant properties in RCE due to the presence of some secondary metabolites. Hoogenboezem and Swanepoel (2000) reported that semen quality and scrotal circumference are affected by factors related to underdevelopment of the testes and testicular degeneration. The frequency of abnormal sperm cells has been found to increase with factors such as extreme in temperature, malnutrition, toxins or anti-nutrients as well as activities of free radicals (Jimoh and Ewuola, 2018; Marai et al., 2001). This has been observed to result in lower ejaculate volume and sperm motility, increase in the percentage of abnormal sperm and a decrease in the total live sperm especially among rabbit bucks in T1 (Rathore, 1970). Underdevelopment of the testis has been reported as one of the factors that can affect the quality of the semen (Ganaie et al., 2013). However, the result observed in this experiment is in agreement with the findings of Ajuogu et al. (2018); Andrej et al. (2013) on the effect of herbal additive (Yucca) on rabbit spermatozoa characteristics.
It was concluded that Rubia cordifolia root extracts (RCE) is loaded with several secondary metabolites which allows it to perform multiple biological activities such as: anti-inflammatory, antioxidant, antifungal, antivirus, antioxidant, cytotoxic, hypolipidemic, immunomodulatory etc. RCE is relative cheap, available, effective, environmentally friendly and could be tolerated by rabbit bucks up to 60 mL per litre without causing any negative effect on the general health and performance of animals.