Growth performance and physiological response of weaned pigs fed diet supplemented with novel a phytogenics

The aim of this experiment was to investigate the growth performance and physiological response of weaned pigs fed diets supplemented with a novel phytogenics (FCTNC). A total of 100 cross bred male pigs (Landrace × Duroc) with an initial weight of 6.31 ± 0.25 kg and weaned at 21 days were individually housed in a semi-open sided pens. Pigs were distributed into five treatments groups with five replicates, each replicates comprises of 20 pigs. Experimental diets were adequate in all nutrients recommended by NRC (2012). Treatment one (T1): basal diet without antibiotics; T2 (basal diet with neomycin at 1.5 g/kg -1 ); T3, T4 and T5 were fed basal diet with 5 g/kg -1 , 10 g/kg -1 and 15 g/kg -1 respectively. Feed and water were offered unrestricted throughout the 56 days trial. Result revealed that there was effect of treatments ( P ˂ 0.05) in average body weight gain (ADG), daily feed intake (ADFI), mortality and feed conversion ratio (FCR). Pigs in T4 (24.54 kg) and T5 (24.22 kg) had the highest weight gain, intermediate in T2 (21.55 kg) and T3 (22.51 kg) and lowest in T1 (12.83 kg) ( P ˂ 0.05). There was a remarkable improvement in all the immune parameters examined among pigs fed FCTNC ( P ˂ 0.05). Microbial population of Escherichia coli and Salmonella spp decreased as the level of FCTNC increases ( P ˂ 0.05). Conversely, Lactobacillus spp count were highest in T4 and T5 relative to the other treatments. Haematological parameters were significantly influenced ( P ˂ 0.05) by the treatments. However, all values were within the normal range for a clinically healthy pigs. The study established that FCTNC could be fed to weaned pigs up to 15 g/kg -1 without having any negative effect on the health of animals.


Introduction
The utilization of alternative feed additives is on the rise in the swine industry recently to support most effective production, especially since the use of antibiotic growth promoters was banned due to antimicrobial resistance and the presence of toxic residue in animal products, which is the primary cause of several human illnesses (Andreas, 2021).Phytogenic feed additives are a class of organic compounds made from extracts of herbs, spices, and related plants.These naturally occurring bioactive chemicals are made up of a variety of distinct constituent types, including tannins, essential oils, saponins, flavonoids, and alkaloids (Singh et al., 2021;Vera, 2020).By serving as defense mechanisms against diseases, predators, and environmental stress, these substances give plants a competitive edge (Liliana;Melina, 2019).
Owing to the presence of phytochemicals, phytogenic feed additives are known to have the ability to enhance the growth performance, nutrient digestibility, and gut health of livestock species, including pigs (Musa et al., 2021;Adewale et al., 2021).Additionally, plant-derived compounds have a variety of qualities, including flavoring, antibacterial, antifungal, antiviral, hepatoprotective, immunological modulating, and physiological effects, all of which are critical to their ability to improve performance in animals (Ines, 2020).
Tests of active substances derived from plants are currently the more promising branch of preparations that can be efficient and used on a large scale while maintaining relatively reasonable economic relations.This is due to the increasing bacterial resistance and more frequent notation of multi-drug resistance strains along with the lack of work on the development of new antimicrobial agents.Numerous research have demonstrated the immunostimulatory properties of phytogenic feed additives and their ability to reduce the activity of pathogenic bacteria such Escherichia coli, Staphylococcus spp., Salmonella spp., and others (Ahmed;Tobias, 2020).Due to the presence of phytochemicals, they also possess special and potent instruments for reducing oxidative stress (Roberto, 2018), which has been shown to improve animal health and performance (Ahmed; Tobias, 2020).Despite this, there is lack of information on the phytochemical properties of FCTNC as a novel feed additive.Therefore, this study was carried out to evaluate the quantitative phytochemical properties of the test ingredient on the growth performance, haematological parameters, intestinal microbiota and immune response of weaned pigs this will further promote food safety and add to our knowledge of its ethno-medicinal use of FCTNC.

Experimental ground, ethical guidelines, collection and processing of phytogenic additive
At the Sumitra Research Institute in Gujarat, India, that is located at (23 o 13'N and 72 o 41'E) and boasts a 1600 km beachfront, the investigation was carried out in the Division of Animal Nutrition and Biochemistry.It was done in accordance with the AD/08A/2023 ethical standards for managing livestock.
Fresh Fenugreek, clove, thyme, nutmeg and cumin leaves were harvested from Sumitra botanical gardens, Gujarat, India in the month of January 2023.It was identified by a qualified taxonomist and assigned a voucher number DF/003A, DF/004B, DF/005C, DF/006D and DF/006E respectively, washed with water to remove sand and placed separately in a plastic sieve to allow excess water to drain before spreading in a flat aluminum tray to air dry for 11 days.Leaves were grinded into powder using a mortar and pestle and stored separately in a clean transparent labeled container.Thousand (1000) grams of each sample was measured from the stored grinded sample and mixed together form a novel phytogenic feed additive (FCTNC) as specified below; FCTNC = 1000 g (DF/003A) + 1000 g (DF/004B) + 1000 g (DF/005C) + 1000 g (DF/006D) + 1000 g (DF/006E)

Animal rearing/management and experimental layout
In India, 100 cross bred male pigs (Landrace × Duroc) with an initial weight of 6.31 ± 0.25 kilograms weaned at 21 days were used for this experiment.Pigs were quarantined for 14 days on arrival to Sumitra Research Farm, Gujarat.They were fed basal diet and given prophylactic treatments of Ivermectin® (against parasites, according to manufacturer's prescription) at (0.2 mL per kilogram).Weights of the animals were balanced before they were allotted into 5 pens (20 pigs/pen) (semi open sided pens each measuring 25 m × 16 m: length × breath) during a 56 -day trial.Completely randomized experimental design was adopted and pigs were distributed into five treatments groups with five replicates, each replicates comprises of 20 pigs.Experimental diets were adequate in all nutrients recommended by NRC (2012).Dietary treatments were; • Control diet: corn-soya meal based diets (basal diet: without therapeutic levels of antibiotics or phytogenic feed additives)treatment one (T1) • Treatment two (T2) -The basal diet with neomycin (1.5 grams/kilogram) (positive control) • Treatment three (T3) -The basal diet supplemented with 5 grams/kilogram FCTNC (negative control) • Treatment four (T4) -The basal diet supplemented with 10 grams/kilogram FCTNC (negative control) • Treatment five (T5) -The basal diet supplemented with 15 grams/kilogram FCTNC (negative control)

Traits associated with performance
The total quantity of feed taken was calculated by deducting the quantity of feed discarded from the quantity of feed provided.The proportion of average feed intake to average body weight growth was used to determine the feed conversion ratio, or the amount of feed required to create one unit of gain.The difference between the subject's initial and final body weights was used to compute the weight increase.By splitting the weight acquired for each course of treatment by the overall number of trial days, the average daily body weight was calculated.

Proximate investigation of experimental diet
Investigation on experimental diet was carried out using Automated Phoenix 5000 NIR feed analyzer, USA to determine for fat, moisture, protein, crude fibre and metabolizable energy.The machine has the following technical features; wave length range (1100-2500 nm), wavelength selection (scanning diffraction grating monochromator with nominal bandwidth of 10 nm, configurable data presented in 1 nm or 2 nm increments), temperature and humidity (35-105 oF ˂ 85 percent), lamp life (10,000 hours) and power requirements (100-240 V).

Haematological investigation
Before the commencement of the study and end (56th day), 2 mL of blood was collected very early in the morning from the culinary vein of 10 randomly selected pigs for haemtological studies.Blood samples were collected into an ethylene diaamine tetraacetic acid using OM -2206 Auto Haematology Analyzer.The machine has the following technical specifications; operating environment (temperature 15-35 ºC; humidity 10-90%), principles (electrical resistance for counting WBC, RBC, and PLT), sample volume (pre-diluted 20 µL), throughput (up to 60 samples per hour), input/output (RS-232, USB, LAN, keyboard and mouse interface).

Caecal microbial investigation
Microbial investigation in the caecum was examined using MALDI Biotyper ® System, USA.The procedure goes through the following process; • Adding target plate to a MALDI Biotyper project list, • Selecting the isolated colony, • Transferring sample onto the target plate and add matrix, • MALDI -TOF spectrum automatically generated by the software, • Spectrum instantly matched against the reference library to give identification, • Final review and result validation.

Analysis of immune parameters
Blood samples from animals collected from haematological test was used to determine the immune response of analysis via Microlisa plus versatile elisa plate reader (India) with 96-well reader control and data analysis for raw data ABS OD, Cutoff and multi standard up to 10 calibrators.Absorbance (dynamic range: 0 to 3.5 OD and resolution: 0.001 OD), light source (tungsten halogen), filters (405, 450, 492 and 630 nm), temperature (10 to 40 ºC) and memory (100 user programmable tests).

Identifying the alkaloid
Alkaloid measurement was carried out using the procedure outlined by Harborne (1973).Gravimetric analysis was used to figure out the alkaloid concentration.A total of five grams of the sample were weighed and mixed in a 1:10 (10%, v/v) ratio with 10 percent acetic acid solution in ethanol.At 28 °C, the blend was let to stand for 4 h.Subsequently, it underwent filtering using Whatman No. 42 filter paper.By using evaporation to reduce the filtrate to a quarter of its initial volume, concentrated aqueous NH4OH was then added drop by drop until the alkaloid crystallized.The alkaloid residue was collected in weighted filter paper, cleaned with a solution of 1percent ammonia, and dried at 80 °C in the oven.

Measurement of saponins
According to Brunner (1984), the spectrophotometric approach was utilized for saponin analysis.A 250 mL beaker was filled with one teaspoon of the test substance and 100 mL of isobutyl alcohol.To ensure even mixing, the liquid was shaken on a UDY shaker (UDY Corporation, Fort Collins, CO) for 5 h.A Whatman No. 1 filter paper was used to filter the resulting mixture onto a 100 mL beaker, and 20 milliliters of a forty percent saturated magnesium carbonate solution was then added.To create a transparent, neutral solution, the resultant mixture was then passed using Whatman No. 1 filter paper.
A 50 mL volumetric jar containing a single milliliter of the solution that was colorless were standardized, to which two milliliters of 5% FeCl3 solution were added.The mixture was then brought to the proper volume with purified water, and let sit for 30 minutes so that the solution could turn scarlet red.Using a saponin baseline solution, standardized saponin solutions (0-10 ppm) were produced and then diluted in 2 milliliters of five percent FeCl solution, just like the samples being studied were.Upon the creation of color, the sample's absorbance and that of reference saponin solutions were measured using a Spectronic 2D spectrophotometer (Milton Roy, Houston, TX) at a wavelength of 380 nm.

Measurement of tannins
The amount of tannin in the samples was measured employing the techniques outlined by Swain (1979).The sample (two grams) was put into a 50 mL container, to which twenty milliliters of 50 percent methanol was applied.The mixture was subsequently coated with a homogenizer and heated in a water bath at 77-80 °C for one hour while being agitated with a glass rod to avoid lumping.Using a double-layered Whatman No. 1 filter paper and 50 percent methanol as a rinse, the blend was passed through into a volumetric flask measuring 100 milliliters.
With distilled water, this was precisely prepared and well-combined.A milliliter of the sample gather was combined with 20 mL of distilled water, 2.5 mL of the Folin-Denis reagent, and a total of 10 mL of 17 per cent Na2CO3 in a volumetric flask with a capacity of 50 m after being homogenized.Distilled water was added to the proper measurements, the mixture was well-mixed, and it was let to stand for 20 minutes until a bluish-green hue appeared.The one milliliters sample mentioned was treated the same way as standard tannic acid solutions in the 0-10 ppm range.After the creation of colors, collections and standard solutions of tannic acid's absorbances were examined using a Spectronic 21D spectrophotometer at 760 nm.Tannin concentration was determined.

Measurement of total phenols
One hundred grams of the materials were extracted by stirring in 250 mL of methanol for three hours.After filtering the extracted materials using Whatman No. 1 filter paper and cleaning the remainder with one hundred milliliters of methanol, the extracts were chilled.Using a rotary evaporator and vacuum, the extracts were evaporated to dry.
To measure the entire amount of phenolic chemicals, the leftovers were dissolved in 10 milliliters of methanol.This measurement was made using the Folin-Ciocalteau phenol reagent and expressed as mg/100 g -1 of gallic acid analogues.In the specified sequence, from 0.2 milliliters of the concentrated methanol extracts, 0.8 milliliters of the Folin-Ciocalteau phenol reagent, and 2.0 milliliters of sodium carbonate (7.5%) were administered.After being thoroughly vortex-mixed, the blends had been diluted with 7 milliliters of deionized water.The process was permitted to finish for two hours at room temperature and in the dark before being spun up for five minutes at a force of 125 g.A spectrophotometer was used to gauge the supernatant at 756 nm.By using methanol in place of the sample, a control was applied.The outcomes were computed as gallic acid equivalents (mg/100 g -1 ) of the sample using gallic acid as the standard.The reaction was performed three times, and the outcomes were averaged.

Measurement of total flavonoid
The above was likewise established using the procedure described by Harborne (1973).A total of five grams of the sample were cooked for 30 minutes over reflux in fifty milliliters of a 2 mol/L -1 HCl solution.After allowing the mixture to cool, Whatman No. 42 filter paper was used to remove the impurities.Beginning with a drop, an equal volume of ethyl acetate was added to the determined volume of the extract.Using weighted filter paper, the precipitated flavonoid was retrieved.The total weight of the flavonoid in the sample was determined by the ensuing weight discrepancy.

Analyzing the oxalate content
Oxalate was measured using AOAC's (2005) methodology.A 100 mL conical flask weighed one gram of the sample.75 mL of 3 mol/L -1 H2SO4 was added, and the mixture was sporadically agitated with a magnetic stirring device for around one hour before it was passed using Whatman No. 1 filter paper.A twenty-five milliliter sample of the sample filtrate (extract) was taken, and it was titrated against a hot (80-90 °C) 0.1 N KMnO4 solution until a light pink color developed and remained for at least 30 seconds.The formula one milliliter of 0.1 permanganate = 0.006303 g oxalate yielded the amount of oxalate present in each sample.

Statistical evaluation
Applying the Statistical Analysis System Software (SAS), all collected data underwent a one-way analysis of variance.The SAS Turkey test was used to separate the means, and significant differences were identified at P ˂ 0.05.

Diets' gross composition
Table 1 describes the results of the composition of the diet offered to the pigs during the experiment.

Growth performance of weaned pigs fed novel phytogenic feed additive (FCTNC)
Table 3 revealed growth performance of weaned pigs fed novel phytogenic feed additive (FCTNC).Final body weight and weight gain values varies from 12.83 to 24.54 kg and 12.83 to 24.54 kg/pig -1 .Average daily weight gain (0.23 to 0.44 kg), total feed intake (38.04 to 48.25 kg), average daily feed intake (0.70 to 0.86 kg), feed conversion ratio (2.00 to 2.96) and mortality (1.00 to 2.7%).Average daily weight gain of pigs fed FCTNC significantly (P ˂ 0.05) against a positive control group.Likewise, average daily feed intake increased numerically.As a result, feed conversion ratio was better for FCTNC supplemented animals relative to the other groups (P ˂ 0.05).
Table 3.Growth performance of weaned pigs fed novel phytogenic feed additive FCTNC.
Note: Values in cells with various characters vary markedly (P ˂ 0.05); SEM: standard error of mean.Source: Authors, 2023.

Discussion
The practice of caring for, treating, and managing animals using local or indigenous knowledge and techniques is known as ethno veterinary medicine.Ethno veterinary medicine encompasses social behaviors and methods for managing animals in farming systems in addition to the use of plants as medicines (Chris and Abel, 2019).Animal defense mechanisms are aided by the high amounts of flavonoids, which function as pharmacological inhibitors of illnesses (Oluwafemi et al., 2021).
Additionally, they are known to have a variety of pharmacological and biological effects that lower the risk of cardiovascular illnesses, such as anti-inflammatory, immune-modulatory, and anti-allergic qualities (Musa et al., 2021).Phenols lower the incidence of cardiovascular infections (Lan, 2009) and enhance blood circulation by strengthening capillaries (Mbabie et al., 2012).Alkaloids function as substances that have anti-plasmodic and analgesic characteristics (Koncić et al., 2012;Edeoga et al., 2005).Saponins may increase the permeability of the gut wall, resulting in better vitamin and mineral absorption (Manu, 2019).Oxalates present in high concentrations in the diet can prevent the body from absorbing calcium (Alagbe, 2023).
The increase in weight gain recorded among pigs in T4 (10 g/kg -1 FCTNC) and T5 (15 g/kg -1 FCTNC) along with increased feed consumption reveals better feed utilization by the animals.The presence of some bioactive compounds in FCTNC, fenugreek (trigonelline), clove (eugenol), thyme (thymol), nutmeg (sabinene) and cumin (cuminaldehyde) could improve palatability (organoleptic stimulation), reduce retention time of feed and nutrient competition by reducing microbial pressure in the gut which would translate to better weight gain (Alagbe et al., 2022;Chen et al., 2018).
These bioactive compounds are also capable of rejuvenating intestinal epithelial cells and helps to increase the surface area of the intestinal villi to promote absorption (Franz et al., 2010) as well as perfect liver protection and other organs to reduce mortality in animals (Gabler et al., 2019).Bioactive substances have antioxidant, anti-inflammatory, antibacterial, amongst others (Gogoi et al., 2018;Agubosi et al., 2021).The outcome of this investigation agrees with the findings of Gois et al. (2016); Yang et al. (2019) when essential oil was supplemented in the diet of weaned pigs.
For disease prognosis, treatment monitoring, and feed stress monitoring, haematological tests have been found to be helpful (Togun;Oseni, 2005).According to Khan and Zafar (2005), hematological indices are reliable markers of an animal's physiological state.It was observed from this experiment that there the blood values before the commencement of the experiment were not significantly (P ˃ 0.05) affected.However, all values were within the reference values for healthy weaned pig recorded by Research Animal Resource (2009).
Result after the dietary supplementation of FCTNC showed that there was a remarkable increase in pack cell volume, haemoglobin, mean platelet volume, red blood cell, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration among birds in T2 to T5 relative to the other treatment groups (P ˂ 0.05).Similarly, white blood cells, neutrophils, monocytes and lymphocytes values recorded a numerical increase in T2 to T5.The pack cell volume (PCV), haemoglobin and red blood cell range recorded among pigs fed FCTNC; 28.10-34.81%,90.10-115.9g/L and 7.71-9.67(×10 12 /L) were within the values reported by Friedrichs et al. (2012).
Etiology of morphologic anaemia is assessed based on the PCV values (Alagbe et al., 2023).PCV is elevated in dehydration and polycythemia (Oluwafemi et al., 2021).Red blood cells helps to carry oxygen through the body and filter carbon dioxide (Singh et al., 2021).Platelets or thrombocytes are component of the blood whose function is to stop bleeding by clumping and clotting blood vessels (Shittu et al., 2021).In iron deficiency, mean corpuscular volume, mean corpuscular heamoglobin and mean corpuscular haemoglobin concentration are low (Adewale et al., 2021;Egeli et al., 1998).Monocytes helps to kill microorganisms, ingest foreign material and remove dead cells (Singh et al., 2021;Alagbe et al., 2019).Basophils are triggered during allergic reactions, excrete heparin for blood clotting and histamine to induce inflammation (Olafadehan et al., 2021).Neutrophils are produced in the bone marrow and are responsible in fighting microorganisms by releasing enzymes to kill pathogens (Olafadehan et al., 2021).Basophils release cytokines and also functions in signaling tissues (Adewale et al., 2021).However, all values were within the normal physiological range for a healthy weaned pig reported by Ventrella et al. (2017) and Klem et al. (2010).
The digestion and absorption of food, host metabolism, energy production, a balanced gut microbiota, mucus layer, barrier function, and mucosal immunity are all physiological and functional aspects of a healthy gut (Anna, 2021).The results of this study demonstrated that adding FCTNC to the diet considerably (P 0.05) decreased the number of harmful microorganisms, including Escherichia coli and Salmonella spp.Through the upregulation of antioxidant enzymes and the direct scavenging of reactive oxygen species, this demonstrated that the test material may both prevent the adherence of pathogens and provide significant antioxidant benefits (Anna, 2021;Alagbe, 2017).
It takes more than just the presence of disease-causing microbes in the stomach to bring about a disease.Once the gut defense is compromised by microorganisms, disease develops (Anna, 2021).Despite the incorporation of conventional antibiotics in diet of pigs in T2, the population of pathogenic bacteria were higher compared to other treatments fed FCTNC.Beneficial bacteria's (Lactobacillus spp) significantly (P ˂ 0.05) increased from T1 through T5.A protective barrier between the host and the bacteria is created by commensal microbiota, which also stimulates the immune system's growth, including the mucus layer (Cremonesi et al., 2022;Li et al., 2021;Li et al., 2022).Since the gastro intestinal tract is the immune system's largest organ and serves as a vital physiological function as a barrier against viruses and antigens, maintaining its integrity is essential for immune system performance (Hu et al., 2019).The findings of Zhe et al. (2023), who fed weaned pigs Litsea cubeba essential oil, are consistent with the results of this investigation.
Immunity is a collection of responses to various aggressors that the body has identified as antigens (Marie, 2020).Pigs fed FCTNC (T3 to T5) had significantly (P ˂ 0.05) increased immunological parameters, indicating that FCTNC may be able to alter immune responses through diverse pathways through interactions with the immune system.The control of cytokine expression is one of these ways, and it is essential for both the development of the innate immune system and the adaptive immune system (Andreas, 2021).
Due to the inclusion of phytochemicals or other bioactive substances in the test materials, they may also control the expression of pro-inflammatory mediators and enzymes implicated in the NF-kB and mitogen activated protein kinase pathways (Marie, 2020).Animals bred without the use of antibiotics can be more productive and have healthier immune systems with the help of dietary immunomodulation (Alagbe, 2023).
Immunoglobulin A (IgA) is the first line of defense in the resistance against infection through inhibiting bacteria adhesion to epithelial cells and by neutralization of bacteria toxins both intracellular and extracellular (Neeray, 2021).Immunoglobulin M (IgM) and IgG aids in the excretion of pathogens and neutralization of toxins respectively (Adreas, 2021).This study's findings is in agreement with the reports of Wang et al. (2021) when Macleaya cordata extract was supplemented in the diet of weaned pigs.Chen et al. (2019) also recorded a numerical increase (P ˂ 0.05) in immune parameters of pigs fed phytogenic feed additive.

Conclusions
Phytogenic compounds are a promising class of additions in the antibiotic alternatives.This viewpoint is supported by the existence of an unlimited number of plants that contain extremely potent compounds.Numerous antibacterial components and maybe new modes of action can be found in phytogens.Weaned pigs' growth performance, gut microbiota, immunological response, and blood profile were all improved by adding up to 15 g/kg -1 of FCTNC to their diet without having a negative impact on the animals' overall health.

Table 4 :
Table4represents the haematological indices of weaned pigs before the commencement of the experiment.Haematological indices of weaned pigs fed FCTNC before the commencement of the experiment.
Note: Values in cells with various characters vary markedly (P ˂ 0.05); SEM: standard error of mean.Source:

Table 7 .
Immune response of weaned pigs fed FCTNC.