Jurnal Bioteknologi dan Biosains Indonesia

MOLECULAR IDENTIFICATION OF PHOSPHATE SOLUBILIZING BACTERIA FROM PGPR PRODUCT DERIVED FROM TEMANGGUNG REGENCY

Muhammad Falih Akbar — 2023-12-07

T Phosphate solubilizing bacteria are soil bacteria that can dissolve phosphates from insoluble phosphate bonds so that they can be absorbed by plants. Phosphates in the soil are naturally present in organic and inorganic forms. Both forms are insoluble or slightly soluble forms of phosphate, so their availability for soil biota is very limited. This study aimed to identify the phosphate solubilizing bacteria from locally PGPR product from Temanggung, Indonesia, namely BPF 1. The method in this study was carried out by molecular identification using the 16S rRNA encoding gene which includes bacterial DNA isolation, DNA amplification using PCR, bacterial DNA sequencing and the creation of phylogenetic trees. The results of the study were obtained isolate bacteria solubilizing phosphate BPF 1 from PGPR products of local farmers of Temanggung Regency identified as the genus Bacillus, having the closest kinship with B. subtilis strain SBMP4 (NR_118383.1) with a similarity of 98.42%.

IN-SILICO ANALYSIS OF SYMBIONT BACTERIA DIVERSITY IN THE MIDGUT OF Aedes aegypti USING 16S rDNA MOLECULAR MARKERS DATABASE

Syubbanul Wathon — 2023-12-07

Dengue Hemorrhagic Fever (DHF) is caused by the dengue virus, which is transmitted through Aedes aegypti mosquitoes when they feed on human blood. To effectively control the DHF vector, it is crucial to accurately characterize the symbiont bacteria associated with Ae. aegypti through an in-silico approach to identify potential targets. This study utilized in- silico analysis based on the 16S rDNA molecular marker to explore the diversity of symbiont bacteria obtained from bioinformatics databases. The analysis and visualization of bacterial diversity were conducted using the Pathosystem Resource Integration Center (PATRIC). The analysis results revealed that bacterial diversity in the midgut of Ae. aegypti, categorized as culturable and non-culturable bacteria, exhibited similar abundance patterns at the family level, albeit with varying detection rates. The most dominant taxa included the phylum Proteobacteria, class Gammaproteobacteria, order Enterobacterales, and family Enterobacteriaceae. Within the culturable bacteria category, the dominant taxa were the genus Salmonella and species Salmonella enterica, whereas the non-culturable bacteria category indicated the prevalence of the genus Escherichia and species Escherichia coli.

EVALUATION AND INHIBITORY MECHANISM ANALYSIS OF NATURAL COMPOUNDS AGAINTS DIHYDROOROTATE DEHYDROGENASE AS ANTI-CANCER AGENTS

Aji Wibowo — 2023-12-07

Cancer remains one of the deadliest diseases worldwide, and currently cancer treatment is facing several problems related to adverse effects and drug resistance. To address these problems, new prospective anticancer medications are required. Natural compounds, which have been extensively used in the drug research, including for the treatment of cancer, are emerging as viable candidates. This study aimed to evaluate 33 in-house natural compounds against dihydroorotate dehydrogenase (DHODH) enzyme, a viable target to develop anticancer agent, and to analyze the hit inhibitory mechanism against protein target. In the activity assay, atovaquone was the sole substance to have activity against DHODH, with an inhibition rate of 47.44% at 10 µM. However, discrepancies were shown in the molecular docking result, where atovaquone were identified as hits. Molecular dynamic analysis revealed that atovaquone initially bound to the active site before being forced to the outside due to cleavage of hydrogen bond between the ligand and responsible residue. This study clearly demonstrated the importance of molecular dynamic analysis to study inhibitory mechanism of compound against target protein that may be useful for further development.

ANALYSIS OF INTERLEUKIN-10 LEVELS IN MESENCHYMAL STEM CELL SECRETOME CREAM WITH ELISA METHOD

Vika Amelia — 2023-12-20

Mesenchymal Stem Cells (MSC) are cells that are able to differentiate into other cells and have immunomodulatory properties that are used to treat inflammatory diseases. MSCs have the ability to repair damaged tissue by regenerating damaged tissue. MSCs produce a secretome, where the MSC secretome has various cytokines, chemokines, growth factors, anti-inflammatory factors and proteins that are produced in molecular form. One of the molecules secreted by the secretome is Interleukin-10.IL-10 is an anti-inflammatory cytokine which functions to inhibit the production of several other types of cytokines such as TNF, IL-1, chemokine and IL-12). Apart from that, IL-10 is able to inhibit the function of macrophages in assisting T cell activity. One of the products from Secretome MSC is a cream preparation. To find out whether the MSC cream contains IL-10 protein, Interleukin-10 levels were analyzed in the MSC secretome cream with 3 different MSC cream concentration formulations (2%, 3% and 5%). Analysis of IL-10 levels was carried out using ELISA to detect IL-10 antigen. The results of the examination of 9 samples showed that all samples were positive for IL-10 with a concentration of 2% dilution 1:2 showing the best results with an Optical Density (OD) of 0.069 and followed by a concentration of 5% dilution 1:2 showing an OD value of 0.065.

EVALUATING THE LONG-TERM STORAGE TIME VIABILITY AND SIZE DYNAMICS OF BACILLUS SP. BIOENCAPSULATION IN SODIUM ALGINATE MATRIX

Mochammad Mirza Saputra — 2023-12-20

The use of biological agents such as Bacillus sp. bacteria has begun to be widely used by farmers as a new form of control. Bacillus sp. needs to require special methods in its application because it has certain living conditions, and Bacillus sp. is vulnerable to environmental pressures. Bioencapsulated formulation in the form of granules is considered effective as a form of bacterial application in soil because it is able to protect Bacillus sp. and maintain survival. This study aims to determine the best concentration of sodium alginate in maintaining the viability of Bacillus sp. at the in vitro stage and to see changes in the size of the beads. The beads were made using extrusion method by combining Bacillus sp. suspension and sodium alginate suspension at 1%, 1.5%, and 2% concentration. The concentration of sodium alginate was not a major factor in changes in bead size and viability test results experienced significant changes in each observation. 1.5% and 2% concentrations can reach the highest viability value of 3x10⁶ CFU/mL and decreased during 1 month of observation. Changes in bead size and viability were caused by the alginase enzyme produced by Bacillus sp.

DEVELOPMENT OF PLASMID-BASED FOR EXTERNAL CONTROL MATERIALS OF CYP2D6*10 (rs1065852) GENE PCR-BASED DETECTION

Jekmal Malau — 2023-12-29

Reliable clinical diagnosis of Single Nucleotide Polymorphisms (SNPs) is necessary for personalizing tamoxifen medication according to CYP2D6*10 genetic variations. Our research aimed to create a recombinant plasmid for external control material with a molecular size of 3812 bp. The recombinant plasmid was achieved by cloning an 838 bp gene insert of CYP2D6*10 rs1065852 into a 2974 bp pJET1.2 plasmid into Escherichia coli DH10B and selection on ampicillin agar medium. Isolated E. coli recombinants provided the plasmid molecules for analysis. Bi-directional sequencing and Real-Time PCR confirmed the presence of wild-type and mutant rs1065852 DNA fragments in the plasmid, namely homozygote CC and TT. The conclusion is that we have successfully introduced a novel recombinant plasmid developed by cloning the SNP rs1065852, which carries the 100C>T mutation, using pJET 1.2/blunt system, which could significantly enhance the accuracy of clinical SNP diagnostics for personalized medicine in breast cancer treatment.

MICROBIAL L-ASPARAGINASES AND STRATEGIES TO IMPROVE THEM

Dini Achnafani — 2023-12-29

L-asparaginase is a type of hydrolase enzyme that has been used in anticancer treatment, mainly Acute Lymphoblastic Leukemia (ALL). L-asparaginase reduces the blood supply of L-asparagine needed by cancer cells to survive. The commercially approved L-asparaginase by the FDA originated from E. coli and E. chrysantemi. However, reports of immunogenic effects in more than 50% of cases due to the use of these enzymes have become the driving force for the need to explore other sources of L-asparaginase. In this review, various alternative sources of L-asparaginase other than these two microbes will be explained. Microbes from the group of Gram-positive bacteria, actinomycetes, and fungi produce L-asparaginase with a higher affinity for L-asparagine than L-glutamine. Protein engineering is an alternative strategy to produce L-asparaginase that is not recognized by antibodies to reduce the immune reaction. Besides, the fermentation process also needs to be considered to determine the appropriate substrate and bioprocess system to obtain the enzyme.

BIOTECHNOLOGY OF PRODIGIOSIN: RECENT DEVELOPMENTS AND TECHNOLOGICAL CHALLENGES

Wani Devita Gunardi — 2023-12-29

Background: Prodigiosin is produced by Serratia marcescens. It has several pharmacological benefits, such as anticancer, antimicrobial, and antidaibetic. However, prodigiosin production still faces problems because it cannot be produced effectively, efficiently, and cheaply. Objective: This study aimed to conduct a review that can explain the upstream and the downstream process in prodigiosin production. Methods: Articles were searched from PubMed and ScienceDirect with the keywords prodigiosin and Serratia marcescens from Juny until September 2023 including review and original article. Relevant data and information were then extracted. Results: Prodigiosin has spectrometrical characteristics, which are crucial for evaluating its production, extraction, and purification identification. Submerged or solid-state fermentation is applicable for prodigiosin production, but solid-state fermentation is better. The kind of growing substrates and the cultural condition influence it. The use of oil-based carbon sources is recommended for the high productivity of prodigiosin. In order to have a cheap, effective, and efficient production process, different experiments have been conducted. Standard extraction and purification methods can carry out the downstream process. Conclusion: Prodigiosin can be produced via submerged or solid-state fermentation. Using cheap and readily available substrate are the key to success for the upstream and downstream process. The standard extraction and purification methods are available.This findings can be used as a basis for further research regarding large-scale production of prodigiosin with the cheap, effective, and efficient methode.

ANALYSIS OF KIDNEY HISTOPATHOLOGY IN SEPSIS MODEL OF MICE (MUS MUSCULUS) WITH PREVENTIVE TREATMENT OF LEAF EXTRACT FROM 'DAUN KENTUT' PLANT (PAEDERIA FOETIDA L.) AGAINST ESCHERICHIA COLI INFECTION

Lisa Savitri — 2023-12-29

Sepsis is a critical health concern characterized by organ dysfunction due to uncontrolled host reactions to infections. The prevalence of microorganisms causing sepsis varies, with Gram-positive bacteria and fungal sepsis gaining significance. Sepsis often leads to acute kidney injury (AKI), and its association with sepsis is known as sepsis-associated AKI (SA-AKI). The "Daun Kentut" plant, known for its diverse bioactive compounds, has shown potential in combating inflammation and oxidative stress. In this study, histopathological changes in mouse kidneys induced by Escherichia coli were investigated. Hemorrhage was the primary change observed. Dehydration caused by E. coli infection potentially led to kidney injury through hormonal responses. The plant extract's anti-inflammatory potential, attributed to compounds like saponins and flavonoids, was also explored. The study emphasizes the need for understanding sepsis-related kidney damage and suggests possible preventive strategies using natural compounds.

ANALYSIS OF BIOMETHANE PRODUCTION POTENTIAL AND MITIGATION OF BIOMETHANE EMISSIONS FROM PIG FARMS IN NORTH SULAWESI PROVINCE

Efrando Manullang — 2023-12-29

The potential for biomethane production from livestock manure as renewable energy has good prospects, considering that the number of pigs in North Sulawesi Province increases yearly. Therefore, it is necessary to analyze the potential for biomethane production, replacing the use of LPG, and mitigate biomethane emissions from pig farms. The results showed that from 2015-2021, the most significant biomethane production was in 2021, amounting to 7,887 tons of CH₄/year in the Minahasa Regency area, 2,496 tons of CH₄/year (31.65%) in Tomohon City, 805 tons of CH₄/year (10.20%) in South Minahasa Regency, 779 tons of CH₄/year (9.88%), and in other areas 3,807 (48.27%). Biomethane can replace 8.59 million kg of Liquefied Petroleum Gas (LPG). The results of mitigating biomethane emissions in 2021 reached 34.22 Gg CO_2eq, and using biomethane to replace LPG reached 11.59 Gg CO_2eq. Total greenhouse gas emissions can be reduced to 45.81 Gg CO_2eq. Biomethane from pig manure has promising potential as a new renewable energy and requires further investigation.

BACTERIAL CONTAMINATION TEST IN POWDER-FORMULATED Helicoverpa armige-ra NUCLEAR POLYHEDROSIS VIRUS (HaNPV1) SUBCULTURE

Mia Miranti — 2023-12-29

The Helicoverpa armigera Nuclear Polyhedrosis Virus (HaNPV1) is a subculture derived from the original HaNPV, and it has been cultivated in Spodoptera litura larvae as an alternative host. HaNPV1 was subsequently formulated using gypsum and talcum as carrier media. Following this formulation, a bacterial contamination test was conducted to assess the quality of the viral formulation. The experiment was arranged in the randomized factorial block design (RFBD) with 2 replications. The viral formulations was stored for 16 weeks and the samples were taken every two weeks for contamination analysis. The data was then analyzed with the analysis of variance (ANOVA) and a post-hoc using Duncan’s Multiple Range test. The variable observed was the number of the bacterial colonies cultivated on the specific media i.e., Nutrient Agar (NA), Salmonella Shigella Agar (SSA) and Eosin Methilen Blue Agar (EMB). The results showed that the bacterial contaminants was detected from 0 to 12 weeks of storage time. However, the highest contamination was found in viral formulation after 8 weeks of storage time and the highest bacterial contaminations were recorded from all viral formulation tested in NA. The results indicated that the bacterial contamination were found around 1.45 × 10⁹ cfu/gram and 1.97 × 10⁹ cfu/gram in gypsum and talcum formulations, respectively. On SSA and EMB media, the bacteria contaminants from all formulation found in 8 weeks of storage time, but Salmonella, Shigella, or Escherichia coli (aspathogenic bacteria) were not found. After 12 weeks storage time, there was no indication of contamination found in all media. Furthermore, Bacillus species was found as a most dominant contaminant in all samples. In conclusion, although the viral formulations using gypsum and talc were not contaminated by pathogenic bacteria such Salmonella, Shigella, or E. coli. Nevetherless, the viral formulation was still easily contaminated by other non-pathogenic bacterial species. Thus, a more standardized and stricted strategy needs to be developed for a better viral formulation product.

POTENTIAL OF HAIR TONIC SARANG BANUA (Clerodendrum fragrans) LEAF EXTRACTS AS ANTI ALOPECIA IN VIVO

Zhafira Dwika Ananda — 2023-12-29

Alopecia merupakan suatu keadaan hilang atau tidak tumbuhnya rambut kepala yang dapat terjadi akibat dari penuaan, kekurangan gizi, ketidakseimbangan hormon. Hair tonic merupakan sediaan cair yang digunakan sebagai penumbuh dan penyubur rambut. Sediaan hair tonic sebagai penumbuh rambut membutuhkan antioksidan yang tinggi. Sarang banua (Clerodendrum fragrans) memiliki antioksidan yang tinggi. Riset ini bertujuan untuk mengetahui potensi hair tonic ekstrak daun sarang banua sebagai anti alopecia secara in vivo. Metode riset dilakukan secara in vivo pada tikus putih wistar (Rattus norvegicus). Penelitian ini merupakan penelitian eksperimental Rancangan Acak Lengkap (RAL) dengan 5 kelompok, K– (tanpa ekstrak), K+ (minoxidil 2%), F1 (5% ekstrak), F2 (7,5% ekstrak), dan F3 (10% ekstrak) dilakukan 3 perlakuan. Perlakuan dilakukan setiap hari dioleskan ke tikus yang mengalami stress dan kebotakan alami. Hasil uji ANOVA menunjukkan bahwa F1 (ekstrak 5%) tidak ada perbedaan signifikan dengan K+ (minoxidil 2%).

Alopecia is a condition where head hair is missing or not growing which can occur as a result of aging, malnutrition, hormonal imbalance. Hair tonic is a liquid preparation that is used to grow and fertilize hair. Hair tonic preparations as hair growth require high levels of antioxidants. Sarang Banua (Clerodendrum fragrans) contains has high antioxidant. This research aims to determine the potential of hair tonic sarang banua leaf extract as an anti-alopecia in vivo. The research method is in vivo with white wistar rats (Rattus norvegicus). This research was an experimental LSD with 5 groups, K– (no extract), K+ (minoxidil 2%), F1 (5% extract), F2 (7.5% extract), and F3 (10% extract) carried out 3 treatments. The treatment is carried out every day and applied to mice that experience stress and natural baldness. The ANOVA test results showed that F1 (5% extract) had no significant difference from the K+ (2% minoxidil).

EVALUATION OF ANTIBACTERIAL ACTIVITY OF PHYCOCYANIN PEPTIDES FROM SPIRULINA PLATENSIS THROUGH MULTIPLE MOLECULAR DOCKING STUDY

Ni Putu Yunika Arindita — 2023-12-29

Peptides derived from the phycocyanin pigment of Spirulina plantesis were screened to identify potential natural antibacterial candidates. In silico protein hydrolysis was performed to obtain active peptides, and a multiple molecular docking (MLD) approach was used to to predict the synergistic mechanisms of various peptide combinations with their respective receptors. Nine phycocyanin peptides showed interaction values ranging from -5.1 to -6.0 kcal/mol, compared to the native ligand (BB-78485) with a value of -9.7 kcal/mol. The results demonstrated a synergistic effect, as the binding affinity of peptide combinations exceeded that of individual peptides and the native ligand. Hydrophobic bonds and van der Waals forces played a crucial role in binding to the active site of 2VES proteins. However, ADME studies revealed that two peptides, YCL and ASYF, had poor pharmacokinetic properties. Overall, this study highlights the potential of phycocyanin peptides as antibacterial agents through the application of MLD.

ENVIRONMENTAL ENHANCEMENT INCREASED SPATIAL MEMORY BUT NO SINGNIFICANT EFFECT ON ANXIETY IN RAT WITH PROPYLTHIOURACIL INDUCTION

Zulkhah Noor — 2023-12-30

This study aims to determine the effect of environmental enrichment on spatial memory and anxiety in Propylthiouracil (PTU)-induced thyroid disorder rats. The design of this study was an experimental post test and controlled group design to analyze differences in spatial memory in rats at the end of the study. The subjects in this study were normal rat pups and Rat with PTU induction. Subjects were divided into 4 groups namely, the control group, the normal group with environmental enrichment, the rat group with PTU induction, and the rat group with PTU induction with environmental enrichment. Each group consisted of 8 rats. At the time the rats were born, the rats were given 0.025% PTU which was mixed in their drink and given to the mother until the rats were born until they were 4 weeks old. Environmental enrichment was carried out for 6 weeks. The spatial memory test in Rat was carried out when the pups were 60 days old. Data analysis used One Way Annova, Kruskal Wallis, Post Hoc Test and Mann-Whitney Test. Enrichment of the environment increased the learning latency time of normal rats by 27.8 ± 1.509 seconds to 9.9 ± 4.434 seconds with memory retention increasing by 6.85% (p <0.05). Environmental enrichment in PTU-induced rats accelerated from 84.5 ± 8.70 to 77 ± 1.383 with memory retention increasing by 7.06% (p <0.05). Environmental enrichment did not significantly affect the behavior and anxiety of normal rats or PTU induced (p>0.05). Environmental enrichment to normal rats and impaired thyroid function can increase spatial memory but has no effect on reducing anxiety.

THE PATHOPHYSIOLOGY OF CUTANEOUS MANIFESTATIONS RELATED TO COVID-19

Prasetyadi Mawardi — 2023-12-30

Covid-19 is a respiratory infection caused by the newly emerging coronavirus. Covid-19 can also cause skin abnormalities, although the pathophysiology of manifestations of skin disorders associated with Covid-19 has not been understood with certainty. This study aims to discuss the pathophysiology of cutaneous manifestation related to Covid-19. The study was conducted based on literature and data mining related to Covid-19 using PubMed and Google Scholar. Eligible papers provided pathophysiology of cutaneous manifestations related to Covid-19. Papers not available in English were excluded. As a result of the presence of edema, vasodilation, and cellular infiltration, skin lesions such as rash, urticarial, or extensive purpura, or even thrombosis and haemorrhage, arise if damage to the vessels became severe. The pathophysiology of Covid-19-associated skin manifestations is estimated through some different mechanisms, such as lesions found in various Covid-19 patients, namely, the presence of varicella such as eruption, non-characteristic rash, and urticarial. Further studies are required to establish a Covid-19 diagnosis based on skin manifestations.

DETERMINATION OF OPTIMAL ANNEALING TEMPERATURE Vibrio alginolyticus PRIMERS USING POLYMERASE CHAIN REACTION METHOD

Gladys Indira Putri — 2023-12-30

Food poisoning is a global issue of grave concern. If food is not properly cooked, it can be a medium for the spread of pathogenic bacteria. Vibrio alginolyticus is one of the pathogenic bacteria that can cause food poisoning. real-time Polymerase Chain Reaction (rt-PCR) can detect pathogenic bacteria in food, so it is necessary to determine the optimal annealing temperature. This research aims to obtain the optimal annealing temperature of the Va_Chr1_FR primer using Gradient PCR. The DNA concentration used was 174.5 with an A₂₆₀/A₂₈₀ purity of 1.94. The temperature range tested, 53°C-62°C, corresponds to the melting temperature of the Va_Chr1_FR primers. The primers designed were F5'-TTCTTCTGTTGTAGGTTCCG-F3' and R5'-CCAGCCCTCACATCTAATAC-R3'. Based on these results, a temperature of 60°C is deemed as the most optimal annealing temperature because it produces one of the brightest bands on electrophoresis with an amplicon length of 146 bp. The findings of this study will be beneficial to the development of Va_Chr1_FR Vibrio alginolyticus primers testing on food samples using the real-time PCR method.

EFFICIENT REGENERATION LOCAL BANANA TISSUE CULTURE USING FLORAL APICES BY CYTOKININ COMBINATION

Isnina Tasya Makhsunah — 2023-12-30

Banana (Musa sp.) is an important crop that is widely cultivated and consumed in Indonesia. Banana plants production can be increased through tissue culture with the use of explants and the right combination of hormones. This study was conducted to determine the use of flower apices explants on local banana varieties using a combination of cytokinin hormones containing BAP (Benzylaminopurine) and several levels of TDZ (Thidiazuron). The result of this research was analyzed using a one-way analysis of variance. The hormone combination in this study consists of MS + BAP 2 mg/L + three concentrations of TDZ (2 mg/L, 2.5 mg/L, and 3 mg/L) as a regeneration medium. Three local banana varieties (Kepok Putih, Kepok Kuning, and Pisang Susu) was cultured on the three combination medium. The results showed that the use of flower apices explants contained in banana flowers grown on MS media with a combination of cytokinin hormones could increase the efficiency of explant regeneration. Media MS + BAP 2 mg/L + TDZ 3 mg/L gave the best regeneration rate for Kepok Putih (62.50%), Kepok Kuning (62.50%) and Banana Susu (56.25%) compared to TDZ 2. mg/L and TDZ 3 mg/L. The novelty of this study can provide information in the propagation of local banana plants using flower apices explants.

THE EFFECT OF INCUBATION PERIOD ON ETHANOL CONTENT OF JASMINE AND GREEN TEA KOMBUCHA

Nisa Ihsani — 2023-12-30

Kombucha is a fermented tea that provides many benefits for health. The alcohol (ethanol) content of kombucha has withdrawn a lot of attention because of human health risks and halal concerns. Alcohol and Tax and Trade Bureau (TTB), BC Center for Disease, and MUI fatwa have defined that kombucha is safe to be consumed as long as the alcohol content is less than 0.5%. This study was conducted to compare the alcohol (ethanol) concentration of jasmine and green tea kombucha during the fermentative stage (5 day at 25^oC) and post-fermentative stage (7 and 21 day, both at 4^oC). The result showed that ethanol concentrations for jasmine and green tea kombucha rose from the fifth d of incubation to the seventh d of the post-fermentative stage, followed by a decline at 21th d of the post-fermentative stage (p>0.05). The level of organic acids, Acetic Acid Bacteria (AAB), and pH confirmed that there were no significant differences at all (p>0.05). In conclusion, jasmine and green tea kombucha at fermentative stage (5 d at 25^oC) and post-fermentative stage (7 and 21 day, both at 4^oC) did not produce ethanol more than 0.5%, without any significant differences in organic acids and AAB levels, respectively.

CONCEPTION RATE OF ARTIFICIAL INSEMINATION OF BALI CATTLE ON DIFFERENT SEMEN DEPOSITIONS

Sri Firmiaty — 2023-12-30

This study aims to analyze the pregnancy rate resulting from artificial insemination of Bali cattle with different semen depositions. The research method used was a field experiment with three treatments: semen deposition at positions 3, 4, and 4+ (right side/dexter side). Pregnancy rate data were evaluated through Non-Return Rate (NRR1 and NRR2), Service per Conception (S/C), and Conception Rate (CR). The study was conducted on 120 Bali cattle in Bulukumba District, South Sulawesi. The results showed an increase in NRR in semen deposition 3, 4, and 4+ during the observation period of NRR 31 – 60. Although some breeders reported arousal symptoms late, the increase in NRR indicates a good understanding of arousal detection. Semen deposition at position 3 produces the lowest NRR, which can be caused by reproductive function abnormalities, silent heat, endometritis, or late reporting by farmers. The results of the S/C evaluation showed the lowest value and the highest of CR at semen deposition 4, which means that the semen deposition was closer to the fertilization site. Semen deposition at positions 4+ on dexter side althrough closer to the fertilization site was lower results of CR than posisition 4, because sperma only toward in right tubafolopii. The effectiveness of AI is significantly impacted by accurately detecting and timing the insemination process based on the ovulation period. This study provides important information regarding AI techniques in Bali cattle that can be used to increase the population and genetic quality of beef cattle in Indonesia.