Effect of Consortium Probiotic Addition on the Health of Pre-Weaned Buffalo at BPTU Siborong-borong

Low immune systems and bacterial infections are the main cause of the high mortality of buffalo calves. The study aimed to investigate the effect of probiotics addition (Saccharomyces cerevisiae and Lactobacillus acidophilus) on the performance of pre-weaned buffalo. The study used 20 pre-weaned buffalo (4-6 months old and an average body weight of 163.53 kg). They were grouped into the Treatment group fed fresh chopped elephant grass and concentrate plus the probiotics; and Control group without the probiotics. The probiotics were offered daily orally using a drenching gun. The parameters measured were body weight, feed consumption, and hematological parameters. The results showed that probiotics increase the average daily gain of calves (P<0.05). The two probiotics have no significant effect on dry matter, protein and energy intake, and feed digestibility. There was an increase in the white blood cell content on calves supplemented with the probiotics, indicating the improvement of the immune system. Conversely, decreasing the white blood cell content in calves who did not receive the probiotics showed a decline in the immune system of the calves. It is concluded that the probiotic improved animal health and body weight gain in pre-weaned buffalo.


Introduction
In the last 8 years, the buffalo population in Indonesia significantly declines from 3.2 million to 1.3 million, or 59.38%, with an average of 4.7% per year [1].The main cause is the maintenance system, which is generally traditional-extensive depending on the supply of natural feed availability.The quality of natural feed fluctuates greatly, the calves' mortality rate is quite high the female reproduction is low [2].The mortality rate in buffaloes mostly occurs at the age of < 3 months (pre-weaning), causing breastfeeding disorders due to rejection by cows; lack of nutrient intake, low body weight, low immune system, and bacterial infections.The calves' buffalo mortality rate is still relatively high at around 11% for Murrah buffalo in East Kalimantan [3] with the highest mortality rate at < 2 months of age or in the pre-weaning period.
Nutritional experts, microbiologists, and biochemists have placed a high priority on the creation of methods to enhance animal productivity, reduce production costs, and lessen the detrimental effects of IOP Publishing doi:10.1088/1755-1315/1341/1/012079 2 livestock production on the environment.To accomplish the aforementioned objectives, animal feed supplementation with probiotics and/or antibiotics has been used [4].Toxic effects on animals, the emergence of bacterial resistance, adverse effects on human health, and a negative impact on the safety and quality of products derived from ruminants and poultry, such as meat, dairy, and eggs, have led to a decrease in social acceptance of the use of antibiotics in animal production.Usually to overcome this, antibiotics and synthetic antimicrobial agents are often used to reduce stress and increase growth and feed efficiency.
Antibiotic use in livestock is currently banned and may not be used as feed additives to hasten the growth or weight gain of the livestock to increase feed efficiency [5].As a result, probiotics like yeast Saccharomyces cerevisiae have been investigated as prospective feed supplements to optimize feed consumption, increase feed digestibility, decrease the number of infections, and improve animal performance and health [6] [7].Supplementing the diet with live yeast cells S. cerevisiae did not produce consistent results, according to the current scientific literature.It has been shown that the presence of S. cerevisiae has both positive benefits on animal health and performance [8] as well as no effects [9].The use of different types and doses of live S. cerevisiae cells as well as variations in feed composition, animal species, physiological status, and age of the animals could all be factors in the explanations for the discrepancies in the results.
Bacteria Lactobacillus acidophilus has been used as probiotics supplemented with buffalo and dairy calves.This probiotic improved feed efficiency, final body weight (BW) gain, dry matter (DM) intake, and average daily gain (ADG).The probiotic also has a positive effect on the overall health index, including fecal consistency, calve morbidity, and mortality.Therefore, the present study was conducted to determine the supplementation effect of S. cerevisiae, which was combined with L. acidophilus in buffalo calves' health and performance.

Material and Method
The research was conducted under animal ethical approval with a registration number of Balitbangtan/Balitnak/Rm/01/2021.

Probiotic production.
The probiotics strain used in this study consisted of two microbes, L. acidophilus, and S.cerevisiae.The two probiotics are isolated, collected, and developed at Indonesian Research Institute for Animal Production (IRIAP).Both probiotics were cultured in separate broth agar plates (1 L) at 2% (v/v) and incubated at 37 °C for 48 h.The bacteria and yeast cells were harvested after centrifugation (10000g for 10 min at 4 °C).The standard plate count method was used to enumerate the bacterial numbers (colony forming unit (CFU)/g following the method of [10].The populations of each L. acidophilus and S. cerevisiae microbes per unit weight were 2.4 x 10 4 CFU/g.The product of L. acidophilus and S. cerevisiae were used for the feeding trial experiment

Animals and feeding management
Twenty healthy swamp buffalo calves (4-6 months old; 163.53 ± 43 kg average BW) were randomly allocated to two groups based on BW as follows; 1) Group I (n=10) fed basal diet consisted of fresh chopped elephant grass (70%) dan concentrate (30%) (control); 2) Group II (n=10) fed basal diet supplemented with two probiotics S. cerevisiae with the dose rate of 15 g/day/head (36 × 10 4 CFU) and L. acidophilus with the dose rate of 100 g/day/head (2.4 x 10 6 CFU) (Treatment).The calves were housed in individual pens on concrete floors equipped with individual feed and drinking water bins.The pens, feed, and drinking water bins were cleaned every day.
Each animal was examined for any indications of illness, dehydration, or injury before the trial experiment, and those that were initially considered unhealthy were excluded from the study.All the calves have free access to the basal diet and drinking water.The nutrient content of the grass and concentrate used in the experiment is given in Table 1.The probiotics product containing at least 36 × IOP Publishing doi:10.1088/1755-1315/1341/1/0120793 10 4 CFU of S. cerevisiae and 2.4 x 10 6 CFU of L. acidophilus of each dose was diluted into 10 ml of sterilized distilled water immediately before being given to the calves.The probiotics were fed once a day in the morning to the treatment group by using a drenching gun.

Parameters measurement
The BW of calves was measured once a week before morning feeding using an automated electronic weighing scale.Daily measurements of the feed offered feed residue, and feces excreted were conducted to determine DM intake (DMI) and feed digestibility.The ADG and feed digestibility values were calculated later.Proximate analysis of feed offered and residue were analyzed based on standard procedures of the Association of Official Analytical Chemists [11].
Blood samples were collected twice, the firstly was before the calves received the probiotics and the second was two months after the calves received the probiotics.The blood samples were taken from the jugular vein of the calves using sterile vacutainer tubes containing EDTA (1mg/ml).The tubes containing blood samples were brought immediately to the lab for hematological parameter examination.Hematological Analyzer (Convergys X3 Convergent) was used to analyze the content of red blood cells (RBC), white blood cells (WBC), hematocrit, and hemoglobin in the blood of calves.All data collected and recorded were tabulated using Excel and then statistically analyzed using one-way ANOVA using IBM SPSS statistics software version 28.0.0.0 (190).

Growth performance and feed consumption
The data on body weight, feed intake, and digestibility of the buffalo calves fed basal diets compared to the calves supplemented with the probiotics are presented in Table 2.There was no effect of probiotics supplementation on the daily feed intake and digestibility of calves fed elephant grass and concentrate (P>0.05).The calves in the Control group consuming similar amounts of DM, protein, and energy compared to the calves that received the probiotics.However, the calves that received the probiotics during the 57 days of observation had higher weight gain than those of the calves in the Control group (P<0.05).The finding of the current study supported the result of [12], which indicated that the yeast S. cerevisiae as a probiotic supplied to ruminant feed improved the growth performance and feed efficiency in fattening calves.
The results of observations on the body weight of the calves during the 57-day measurement period showed that there were differences in the average body weight gain (ADG) of the calves in the treatment group compared to the control group (P<0.05).The calves in the treatment group had a higher weight gain of around 24.64% compared to the calves in the control group.These results are in line with previous studies that probiotics containing S. cerevisiae and L. acidophilus can promote higher growth and feed conversion efficiency [13] [14], which also reported higher growth rates.Moreover, a metanalyses study concluded that S. cerevisiae supplementation improved growth performance, rumen development, and immunocompetence in buffalo calves, particularly in preweaning calves [15].The addition of bacteria L. acidophilus in the current study does not affect feed consumption and digestibility.The results were coherent with the study by [16], which identified no effect on nutrient utilization, or average daily gain when the calves received L. acidophilus up to 10 8 CFU on the diet.However, our findings support the results that indicated that particular probiotics for the buffalo calves' health may improve their growth [17].
The current results also support the earlier studies [18] [19], which showed the importance of particular yeast and bacteria in preserving the intestinal health of cattle.It is widely known that L. acidophilus uses simple fermentable carbohydrates to make lactic acid, and their proliferation rate controls the local pH in the intestine [20].[21] evaluated the state of the neonatal buffalo calves' guts using fecal pH as an indication.Additionally, ammonia is produced as a result of dangerous microorganisms fermenting proteins in the colon.According to [22] the altered levels of fecal metabolites (lactate and ammonia) may be related to a potential change in the fermentation pattern (from proteolysis to saccharolytic activity) and decreased urease activity of gut bacteria.Another reason for this observation could be that taking probiotics supplements led to a rise in the number of healthy bacteria and a decrease in pathobionts in buffalo.

Blood hematological
The data on blood hematological parameters of buffalo calves fed basal diets compared with calves fed basal diets supplemented with the probiotics are presented in Figures 1a; 1b; 2a; and 2c.The hemoglobin in calves' blood increased by 12.18% from 11.82 to 13.26 (g/dl) in the Control group and only increased by 5.72% from 12.55 to 13.26 g/dl in the Treatment group (Figure 1a).Conversely, the percentage of red blood cells (HCT) of calves receiving the probiotics increased by 12.53% from 26.57 to 29.90 %, while the HCT of calves in the Control group increased by only 8.49% from 28.47 to 30.89% (Figure 1b).
There was no effect of the probiotic's inclusion in the diet on the WBC and RBC content of the calves.The WBC content of calves was increased by 1.6% in the Treatment group and decreased by 13.18% in the Control group (Figure 2a).While the RBC of calves increased by 0.88% and by 22.28% in Treatment and Control groups, respectively (Figure 2b).Finding on the current study have similarity with other studies.Although the effects of S. cerevisiae supplementation on the blood characteristics of ruminant animals have been found to vary [23] [24] [25].The studies indicated that S. cerevisiae had no impact on the RBC, packed cell volume (PCV), or hemoglobin (Hb) concentration in growing goats.However, addition of S. cerevisiae on the diet enhanced WBC [26] [27] [28] [29] indicating S. cerevisiae increased animal immunity.This conclusion agrees with other studies conducted on lambs [23] and young goats [30].It's yet unclear exactly how S. cerevisiae strengthens goats' immune systems, although one or a combination of mechanisms may be happened.
The WBC content of calves receiving S. cerevisiae and L. acidophilus slightly increased (1.6%), while the WBC content of calves did not receive S. cerevisiae and L. acidophilus declined up to 13.18%, indicating improvement of calves' immunity when they received the probiotics.The probiotics are used in feed through pharmacokinetic and pharmacodynamic processes [31].Through a number of methods, such as the dissociation of pathogens from microbial antagonists, the activation of the animal's immune system, the attachment and dissociation of pathogens, and an increase in the activity of bacteria that make enzymes, the probiotics can be employed as a probiotic and prebiotic.The previous study demonstrated that S. cerevisiae positively impacted broiler growth, demonstrating probiotic's capacity to control the development of gut bacteria and facilitate the synthesis of volatile fatty acids to enhance nutrient absorption [32].
In term of L. acidophilus supplementation, the current study's finding was coherent with the study by [16].The study using dairy calves showed that addition of this bacteria up to 10 8 CFU increased faecal counts of lactobacilli and bifidobacteria (log10 (CFU)/g of fresh feces), but the counts of coliforms and clostridia were lower (P <0.001).In the calves received probiotic, the cell-mediated immunity was enhanced (P 0<0.05).Although addition of L. acidophilus has no effect on daily weight gain and intake, the bacteria was superior in improving the gut health in terms of its microbiota and metabolites and cell-mediated immunity response in calves.

Conclusion
In conclusion, the addition of the probiotics S. cerevisiae and L. acidophilus increases the average daily gain in calves fed elephant grass and concentrate.The two probiotics have no significant effect on dry matter, protein and energy intake, and feed digestibility.There was an increase in the white blood cell content on calves supplemented with the probiotics, indicating the improvement of the immune system.Conversely, decreasing the white blood cell content of calves who did not receive the probiotics showed a decline in the immune system of the calves.

Figure 1a .
Figure 1a.The hemoglobin of the blood of buffalo calves in the control group and the treatment group receiving a combination of two probiotics Figure 1b.The percentage of red blood cells (HCT) of buffalo calves in the control group and treatment group receiving the combination of two probiotics

Figure 2a .
Figure 2a.The white blood cell (WBC) content of buffalo calves in the control group and treatment group receiving a combination of two probiotics Figure 2b.The red blood cell (RBC) content of buffalo calves in the control group and treatment group receiving a combination of two probiotics

Table 1 .
The nutrients content of elephant grass and concentrate was used as a basal diet during the experiment

Table 2 .
The body weight, feed intake, and digestibility of the buffalo calves fed basal diets compared to the calves supplemented with the probiotics.