In vivo study: The effects of Carica pubescens seed extract on the anthelmintic activity, feed digestibility, performance, and clinical parameters of thin-tailed sheep

This research serves to follow up on the results of an in vivo study of the administration of aqueous seed extract (ASE) of Carica pubescens waste on the performance and health parameters of thin-tailed sheep. This research was conducted at Al Baihaqi farm in Kediri, East Java. Thirty-two thin-tailed sheep aged 12 months were randomly given four treatments with P0 = negative control, P1 = 2.5% ASE, P2 = 5% ASE, and P4 = positive control (Albendazole). This study used sheep that were naturally exposed to Haemonchus contortus (from the farmer’s field) with the epg qualification being above 1000. The in vivo study was carried out for 9 weeks with the administration of Carica seed extract on the initial 7 days in a row, and the positive control was given Albendazole once on the seventh day. The number of ASE grants was based on the average fluid in the abomasum per kg of body weight. The feed design used in this study was wheat bran and Pennisetum purpureum cv. Mott according to the livestock’s needs. The data taken in this study were the feed consumption, digestibility, average daily gain, percent fecal egg count reduction, body condition score, and clinical parameters. The results showed that the FECR, ADG, and clinical parameters (AST and ALT) (P>0.05) underwent significant changes to support good performance while the BUN, creatinine, feed consumption, and digestibility were normal. This study indicates that ASE of up to 5% can be used as an anthelmintic alternative and support the recovery of sheep performance.


Introduction
Small ruminants are one of the meat-producing commodities [1].The Livestock and Animal Health Statistics [2] stated that the population of sheep in Indonesia in 2022 reached 15,615,000, dominantly in West Java.The development of sheep livestock in Indonesia has experienced several obstacles, and one of which is an infectious disease caused by gastrointestinal worms.Baihaqi et al. [3] added that IOP Publishing doi:10.1088/1755-1315/1341/1/012119 2 small ruminant livestock in Indonesia have been confirmed to experience gastrointestinal nematode infections in all seasons, and the most dominant type of nematode is Haemonchus contortus.Fernandes et al. [4] stated that increased emissions of methane result from gastrointestinal nematode infections.Baihaqi et al. [5] added that methane has a negative effect on the environment.
Meanwhile, resistance cases are a major problem in the development of ruminants due to the use of chemical anthelmintic drugs.One effort to deal with anthelmintic resistance is the use of natural anthelmintics [6].The bark of Paraserianthes falcataria has been confirmed to have active compounds that succeed in killing H. contortus in vitro, and when observed using SEM, it shows that there is damage to the cuticle structure [7]; the same results have also been found in the fruit peel of Carica pubescens.
Recent in vitro research by Baihaqi et al. [8] added that in vitro studies indicated that the seeds of C. pubescens inhibit H. contortus, leading to a damaged cuticle structure.The seeds of C. pubescens do not interfere with rumen fermentation characteristics but have a significant effect on methane reduction.
Evaluation of the status of local sheep infected by intestinal worms includes analyzing the hematological and blood chemistry parameters [9].Kotze and Hunt [10] stated that, for the sheep industry, internal parasites make up the list of top veterinary cost components at around AUD 665M.Ramsay et al. [11] stated that many alternatives to anthelmintic drugs from active plant compounds are currently being studied.This research was conducted to observe the administration of ASE up to a concentration of 5% as an anthelmintic alternative and its effect on the performance and health parameters of sheep 2.

Ethical approval
The study was approved by the Institutional Ethical Committee of the Faculty of Veterinary Medicine at the Universitas Gadjah Mada, Yogyakarta, Indonesia.Number: 0013/EC-FKH/Int./2019.

Plant material and extraction
Carica pubescens (herbarium voucher number: PPN-cc-003) seeds were collected from small-and medium-sized enterprises (CV.Gemilang Kencana,Wonosobo, Central Java, Indonesia).For the aqueous extracts, 150 g of powdered seed material was macerated with 750 mL of distilled water and incubated for 24 h.Macerated extracts were then filtered on cotton and concentrated under reduced pressure in a rotary evaporator at 40°C-50°C before being stored at 4°C.The dry extracts were subjected to phytochemical screening to identify the major phytochemical groups [12].

Determination of plant phytochemicals
Qualitative detection was performed on C. pubescens seeds to detect active compounds such as tannins, alkaloids, flavonoids, and steroids using the procedure described by Trease and Evans [13].The total phenolic content in the plant extracts was measured using the Folin-Ciocalteu method, and the results were expressed as mg gallic acid equivalents [14].The total flavonoid content of plant waste extracts was determined using a colorimetric method, and the results were expressed as milligram rutin, as previously reported by Nabavi et al. [15].

Experiment
This study used thirty-two female Thin-tailed sheep aged 12 months were randomly assigned to 4 treatments.Namely; P0 as negative control, P1= 2,5% ASE, P2= 5% ASE and P4= Positive control (Albendazole).Sheep used for the in vivo study were sheep naturally infected with Haemonchus contortus (from the farmer's area) with epg > 1000.In vivo treatment period was 6 weeks with treatment given ASE at doses of 2.5 and 5% for 7 consecutive days in the first week of treatment, while the positive control was given once on the 7th day with orally.Sheeps are placed in individual cages and given feed in the form of concentrate (soybean meal and polar) and forage (Napier grass) according to their needs and drinking water ad libitum.After a period of adaptation to the cage and feed for one week the study animals were divided into 4 groups, the negative group was not given any treatment, the ASE Carica pubescens 2.5% group, the ASE Carica pubescens 5% group for 7 consecutive days, and the positive control group with gave albendazole at a dose of 3.8 mg/kg BW once.
2.4.1.Parasitologic examination.Parasitological examination was carried out using faeces taken by grab sampling method from the rectum of the sheep in the pre-study period, days 0, 7, 14, 21, 28.Feces samples were counted Eggs per Gram Feces (TTGF) using the McMaster method.
Examination was carried out using the McMaster method.A total of 2 grams of stool sample was put into a beaker.58 ml of saturated sugar solution is added so that the total volume becomes 60 ml, then stirred and filtered using a tea strainer.The faecal suspension was taken using a pipette and then immediately put into the McMaster counting chamber.The samples were allowed to stand for 2-3 minutes and then examined under a microscope with a magnification of 40 times.Haemonchus contortus worm eggs found were counted to determine the amount of TTGF. 2. 4 weighed.The observation period was carried out for 7 days at the end of the treatment of mountain papaya fruit peel extract.Stool samples were taken 10% of the total and then dried in the sun followed by oven drying at 60°C.Sampling was carried out for 7 days, dry feces were ground with a Wiley Mill grinding machine with a 1 mm sieve size.The fecal samples in the composite were then analyzed for dry matter, organic matter and crude protein.

Examination of the degree of anemia (FAMACHA). The use of Famacha is by closing the
eyes by opening the upper and lower eyelids.The upper or lower eyelids can be seen for changes in the mucosa.The color of the eyelids is compared to the color on Famacha©.The color that is considered in accordance with the image is determined by a score to determine the degree of anemia.Comparing the degree of anemia with the results of PCV examination in thin-tailed sheep.Famacha© is used to help determine the degree of anemia due to H. contortus infection.

Examination of Body Condition Score (BCS).
Body condition score assessment is based on observation and palpation of eight parts of the animal's body.These body parts include: the spinous processes, the transverse processes, the flanks, the tuber coxae, and the ischiadicus tubers.The BCS assessment diagram uses a scale of 1-4.BCS (1=very thin; 2=skinny; 3=moderate; 4=fat).2.4.8.Examination Dag score.Examination of traces of runny feces/feces in conditions of diarrhea stuck to the sheep's buttocks.Scores range from 1 to 5. Score 1 is the lowest dag score, there are no traces of feces stuck to the buttocks.As the score increases, the stool that sticks to the buttocks is getting bigger and wider.

Data analysis
Data from in vivo experiment parameters were analyzed using a one-way analysis of variance.Statistical differences between the means were determined using Duncan's multiple-range test (DMRT).Results were considered statistically significant at p < 0.05.

Qualitative and quatitative phytochemical analyses
The result of the qualitative and quantitative phytochemical analyses are presented on Table 1 and 2 Observations on C. pubescens seeds proved qualitatively positive, while the total phenol was 9.5 (mg GAE/g dw), flavonoids content 3,1 (mg RE/g dw) and 5.4 % total tannins.The use of active plant compounds from agoindustrial waste can be used as an alternative to worm infection control, methane reduction and protein protection winth in vitro study as done by Baihaqi on plant waste Paraserianthes falcatria bark [7], fruit peel of Carica pubescens [17] and seed of Carica pubescens [8].

EPG-FECR parameters
The results of the Egg per gram (EPG) -Faecal Egg Count Reduction (FECR) in sheep in the negative control group, 2.5% ASE of Carica pubescens, 5% ASE treatment and positive control are presented in Table 3.The effect of ASE administration on gastrointestinal nematode eggs was studied with the EPG-FECR parameters, and the EPG at the end of the study period showed a decrease in all study groups.The negative control group also showed a decrease in the number of worm eggs because it was possible to obtain qualitative and quantitative nutritional improvement compared to when they were reared in a farm.Meanwhile, the positive control group (Albendazole) had 100% EPG reduction.Giving 2.5% and 5% ASE was able to suppress gastrointestinal nematode eggs (Haemonchus contortus) in thin-tailed sheep.Molla et al. [18] stated that M. Koenigii plant extract containing tannins, saponins, flavonoids, and phenols significantly reduced FECR.Morais-Costa et al. [19] also added that administration of the water extract of P. viridiflora plant increased FECR by almost 50% in the first week of treatment.
González-Cortazar et al. [20] found that L. acapulcensis with its flavonoids succeeded in suppressing the EPG value of gastrointestinal nematodes.

Nutrient intakes
The results of the examination of the Dry Matter Intake (DMI), Organic Matter Intake (OMI), and Crude Protein Intake (CPI) are presented in Table 4.The statistical analysis showed that the average consumption of DM, OM, and CP by the groups of ASE treatment at both concentrations (up to 5%), positive control, and negative control showed no significant difference between the pre-treatment and post-treatment (P>0.05).Maxselley et al. [21] stated that the supplementation of 100-300 g/day of coffee cherry pulp containing phenolic compounds had no effect on the feed intake.Ardeshiri et al. [22] added that the addition of Albizia lebbeck containing tannins had no effect on the consumption of DM, OM, and CP in Arabi sheep.

Nutrient digestibility
The statistical results of the digestibility parameters of DM, OM, and CP of the sheep in the negative control group, 2.5% ASE treatment group, 5% ASE treatment group, and positive control group are presented in Table 5.Based on the results of the study, the digestibility of DM, OM, and CP in the sheep given up to 5% ASE, albendazole (positive control), and negative control was observed to have no significant changes (P>0.05) between the pre-treatment and post-treatment.This indicates that the provision of 5% ASE may affect the feed digestibility in the digestive tract.In an in vivo study, Ibidhi and Salim [23] demonstrated that the administration of Trigonella foenum graecum L. seeds, which have high flavonoids, saponins, and phenolics, at concentrations of up to 30 g in the concentrate, did not affect the nutrient digestibility of the food in Barbarine sheep.However, every plant has different active compounds such as in the case study by Maxselley et al. [21], which showed that the supplementation of CoCP at 300 g/day could have a negative impact on the digestion of feed possibly due to the presence of high phenolic compounds that affect the feed digestibility.

Effect of ASE on Hematology parameters
The results of hematology and blood chemistry examination in the thin-tailed sheep at the beginning and end of the study are presented in Table 6.The hematological analysis showed that there were significant differences in the hemoglobin (Hb), red blood cells (RBC), and hematocrits (HCT) values in the 2.5% ASE, 5% ASE, and positive control groups compared to the negative control group between before the treatment and after the treatment (P<0.05).In the sheep infected with gastrointestinal nematodes and untreated (negative control), these hematological parameters did not show any increases.The effect of ASE has been shown to suppress EPG from the nematode Haemonchus contortus.Meenakshisundaram et al. [24] added that the ethanol extract of I. tinctoria containing active compounds is successful in increasing the Hb value and PCV in livestock positively-infected with GIN possibly because helminthic infections are eliminated by the active compounds of this plant.The nature of Haemonchus contortus is sucking blood and causing inflammation of the digestive tract as well as causing anemia and diarrhea.Sambodo et al. [25] stated that the RBC and leukocyte parameters when sheep are infected with GIN are observed to be low because it is possible to lose blood during infection, thus leading to anemia.The total leukocyte count and eosinophil value in the 2.5% ASE, 5% ASE, and positive control groups were observed to decrease significantly after treatment (P<0.05),whereas in group I (no treatment or negative control), the number of leukocytes did not show any significant changes (P>0.05).

Effect of ASE Carica pubescens on chemical blood parameters
The blood chemistry values in the thin-tailed sheep during the pre-treatment and post-treatment of the study are presented in Table 7 The results of the blood chemistry analysis showed that the activity of AST and ALT in the blood of the negative control sheep remained unchanged until the end of the study, whereas in the sheep in the 2.5% ASE, 5% ASE, and positive control groups these parameters showed a marked decrease (P<0.05).This is possibly related to the status of damage to the digestive tract and liver as well as muscle degeneration due to gastrointestinal worm infestation.Radotits et al. [26] stated that a decrease in protein in the blood is possibly caused by increased plasma leakage or decreased absorption through the intestine that is injured/damaged by parasites.Tanwar and Misra [27] added that the low albumin value is possible due to increased albumin catabolism and protein malabsorption through the damaged intestinal mucosa.The decrease in AST and ALT values in the sheep in the 2.5% ASE, 5% ASE, and positive control groups in this study could indicate that the administration of the extract indicated a liver tissue repair and did not result in organ/tissue damage.Sakna et al. [28] stated that several studies reported that a group of triterpenoid compounds, both free triterpenoids and triterpenoids bound to one or more glucose and saponin molecules, were shown to have hepatoprotective activity.The BUN and creatinine parameters in the treated sheep were at the levels comparable to normal values for sheep, reaching 10-35 mg/dL and 1.2-1.9mg/dL.The rams fed of Kyasuwa grass had a BUN value of 14 mg/dL.The reference interval for BUN in desert Bighorn sheep was 5 -28 mg/dL and the creatinine was in the range of 1.6 to 2.6 mg/dL.The results of this study indicated that the administration of 5 mg/mL Carica fruit seed extract and albendazole in this study did not cause changes in the kidney function.Therefore, administering Carica seed extract at such concentration does not lead to interference/damage to the kidneys.Meenakshisundaram et al. [24] found the blood urea nitrogen and serum creatinine values being in a normal state, indicating that the administration of plant extract does not have adverse effects on the kidneys.

Effect of ASE on FAMACHA Parameters
The results of the FAMACHA parameters in the treatment and control groups are presented in table 8.The statistical results showed that the positive control and 5% ASE treatment groups on the FAMACHA parameter were observed to be significant (P<0.05) while the negative control and 2.5% ASE groups were not significantly different.Sambodo et al. [29] stated that in general the level of anemia can be evaluated by looking at the color of the conjunctiva of the eye because the blood capillaries are on the surface, so the color of the tissue reflects the color of the blood.The increase in FAMACHA value is in line with the changes in hematology after ASE administration.

Effect of ASE on body weight
The in vivo test results related to body weight are presented in table 9.The results showed that the average body weight of the thin-tailed sheep increased significantly (P<0.05)in the groups that received 2.5% ASE, 5% ASE, and positive control, while the sheep that did not receivetreatment (negative control) experienced a significant decrease in the body weight.This is in line with the findings of Morais-Costa et al. [19] in which the administration of P. viridiflora water extract significantly (P<0.05)gave an average increase in the body weight.The sheep that were given the extract treatment did not show changes in the behavior, submandibular edema, weakness, or feed consumption during the experiment.The body weight of the sheep with 2.5% ASE, 5% ASE, and positive control tends to increase possibly due to the loss of Haemonchus contortus worms and no disturbance of feed consumption and digestibility.The clinical conditions, hematology, and blood chemistry also showed no disturbance in the liver and kidneys, indicating that these parameters support the increase in the body weight of sheep.

Effect of ASE on BCS parameters
The results of this in vivo test showed that the average BCS increased insignificantly (P> 0.05) in the treatment groups (2.5% ASE and 5% ASE) and positive control group, while in the negative control group, the sheep were observed to experience a decrease in BCS ( Disappearance or decrease in gastrointestinal helminthic parasitic infections as indicated by a decrease in the number of nematode worm eggs in the feces and followed by an improvement in the hematologyand blood chemistry levels to normal values for sheep indicate a physiological condition of the digestive system and perfect body metabolism.Maximum feed intake will increase digestive performance and livestock production (BCS value).Mavrot et al. [30] also suggested that gastrointestinal nematode wormparasites greatly affect the production performance of sheep which is characterized by a decrease in body weight gain associated with an increase in the number of nematode worm eggs in the feces.Soto-Barrientos et al. [31] also stated that low BCS in sheep has a strong association with heavy gastrointestinal nematode worm infestation rates.Furthermore, a study in sheep showed that treatment for gastrointestinal nematode worms resulted in an improvement in BCS performance.

Conclusion
The use of seeds from Carica pubescens at concentrations of 2.5% and 5% can become an anthelmintic alternative and does not interfere with the productivity and health status of livestock.

Hematologic and blood chemistry examination. Blood
[17]Body weight.Examination of body weight was carried out by weighing the livestock in the morning before the morning feeding, carried out every weekend during the study period.2.4.3.samples from sheep were taken through the jugular vein with 3 mL venoject.Blood sampling was carried out on day 0 and on day 28.Blood tests include Hb levels, erythrocyte count, PCV, leukocyte count and leukocyte differential, and blood chemistry with Frye et al.[17].2.4.4.Feed consumption.Feed consumption is calculated by reducing the amount of feed given with leftover feed or uneaten feed.The feed to be given is weighed first, while the remaining feed is weighed for the calculation of the feed given the next day.Concentrate and forage animal feed samples were analyzed for dry matter, organic matter and crude protein.2.4.5.Feed digestibility.Stool is collected for 24 hours during the observation period and then .

Table 3 .
Effect of ASE on EPG-FECR parameters a,b Different superscripts on the same line show a real difference ASE: Aquesous seed extract of Carica pubescens

Table 4 .
Effect of giving ASE to DMI, OMI dan CPI (g/ day) a,b Different superscripts on the same line show a real difference (P<0,05) ASE: Aquesous seed extract of Carica pubescens

Table 6 .
Hematology parameters a,b Different superscripts on the same line show a real difference (P<0,05) ASE: Aquesous seed extract of Carica pubescens .

Table 9 .
Body weight parameters

Table 10
a,b Different superscripts on the same line show a real difference (P<0,05) ASE: Aquesous seed extract of Carica pubescens