The purpose of this study was to identify seminal plasma proteins in Bali bull and their potential as biomarkers of fertility. Semen was collected from 10 bulls aged 5–10 years using an artificial vagina. Fresh semen was then centrifuged (3000× g for 30 min). The supernatant was put into straws and stored in liquid nitrogen. The semen plasma protein concentration was determined using the Bradford method, and the protein was characterized using 1D-SDS-PAGE. Coomassie Brilliant Blue (CBB) was used to color the gel, and the molecular weight of the protein was determined using PM2700. A total of 94 proteins were identified in the seminal plasma of Bali bulls analyzed using LC-MS/MS (liquid chromatography–mass spectrometry). Proteins spermadhesin 1 (SPADH1), C-type natriuretic peptide (NPPC), clusterin (CLU), apoliprotein A-II (APOA2), inositol-3-phosphate synthase 1 (ISYNA1), and sulfhydryl oxidase 1 (QSOX1) were identified as important for fertility in Bos javanicus. These proteins may prove to be important biomarkers of fertility in Bali bulls. These proteins are important for reproductive function, which includes spermatozoa motility, capacitation, and acrosome reactions. This study provides new information about the protein content in seminal plasma in Bali bulls. The LC-MS/MS-based proteome approach that we applied in this study obtained 94 proteins. The identification of these seminal plasma proteins of Bali bulls and their potential as fertility biomarkers may have an impact on the success of future artificial insemination (AI).

Background and Aim: Protamine (PRM) is the major protein in the sperm nucleus and plays an essential role in its normal function. Moreover, PRM has great potential as a protein marker of semen production and quality. This study aimed to assess the potential of sperm bovine PRM as a protein marker of semen production and quality in bulls at the National Artificial Insemination (AI) Center of Indonesia. Materials and Methods: The semen production capacity of each bull was collected from frozen semen production data at the Singosari AI Center for 6 months, and was then divided into two groups (high and low). A total of 440 frozen semen straws from six Limousin (LIM), six Friesian Holstein (FH), six Peranakan Ongole (PO), and four Aceh bulls aged 4-5 years were used in the study. The frozen semen was used to measure the concentration of PRM1, PRM2, and PRM3 using the enzyme immunoassay method. The frozen semen was also used to assess the quality of the semen, including progressive motility (PM) through computer-assisted semen analysis, sperm viability through eosin–nigrosin analysis, and the DNA fragmentation index through Acridine Orange staining. Results: PRM1 was significantly higher in all bull breeds included in the study (p<0.00), followed by PRM2 (p<0.00) and PRM3 (p<0.00). PRM1 significantly affected semen production in LIM, FH, PO, and Aceh bulls (p<0.05). Moreover, PRM2 significantly affected semen production only in FH and Aceh bulls (p<0.05), whereas PRM3 affected this parameter in PO and Aceh bulls exclusively (p<0.05). Consistently and significantly, PRM1 was positively correlated with the PM and viability of sperm and negatively associated with its DNA fragmentation in LIM, FH, PO, and Aceh bulls (p<0.05; p<0.01). The correlation analysis between PRM2 and PRM3 and semen quality parameters varied across all bull breeds; some were positively and negatively correlated (p<0.05; p<0.01), and some were not correlated at all. Conclusion: PRM1 has excellent potential as a protein marker of semen production and quality in bulls at the National AI Center of Indonesia.

Background and Aim: Indonesia has two National Artificial Insemination centers and 17 Regional Artificial Insemination Centers. The frozen semen production techniques differed between the centers, including the type of diluent and semen dilution technique. The aim of the research was to compare the quality of frozen Limousin bull semen diluted using different techniques. Materials and Methods: Semen was collected from three sexually mature Limousin bulls using an artificial vagina. Immediately after collection, the semen was evaluated macroscopically and microscopically. Semen that had >70% motile sperm and 0.05). However, sperm viability and membrane intactness of one-step dilutions were higher than those of three-step dilutions. The concentrations of MDA and AST enzymes of sperm in one-step dilutions were lower than those of three-step dilutions (p<0.05). Conclusion: It was concluded that the one-step-dilution technique was better than three-step dilution for cryopreservation of Limousin bull semen.

Background and Aim: Fertility is crucial for enhancing the efficiency of livestock production, as it directly impacts the reproductive rates. A comprehensive understanding of the relationship between sperm quality and embryo development is key to optimizing reproductive outcomes and improving the quality of livestock. This study analyzed the developmental competence of in vitro embryos recovered from Bali cattle with normal or poor sperm motility. Materials and Methods: Nine bulls with normal fresh semen (NFS) or poor fresh semen (PFS) motility were ejaculated for semen. Semen ejaculates, including volume, motility, and sperm concentration, were evaluated immediately after collection to measure the quality of the fresh semen. Frozen semen was evaluated using computer-assisted semen analysis (CASA) for motility, progressive sperm motility, distance curve path, distance curve linear, distance straight line, average path velocity, curvilinear velocity, linear velocity, straightness (STR), linearity of forward progression (LIN), wobble, and average lateral head displacement (ALH). Bull groups were used to determine in vitro embryo cleavage ability after fertilization of Bali cattle. Ovaries of Bali cattle were collected by slicing, and only cytoplasmic oocytes with compact cumulus cells were used in this study. The oocytes were matured, and in vitro fertilization was performed using fertilization media with a final sperm concentration of 1.5 × 106 spermatozoa/mL. After 48 h, the embryo cleavage ability of the cultured oocytes was evaluated. Results: There were significant differences in motility values between the NFS and PFS groups; however, there were no significant differences in the volume or sperm concentration. There was a significant difference in the LIN value between the groups but no significant differences in other CASA parameters. There were no significant differences in the cleavage rate and morula between the groups, but a positive correlation was observed between the cleavage rate and the morula and between the morula and ALH. A significant negative correlation was observed between the cleavage rate and STR and between the morula and STR; no significant differences were observed for other variables. Conclusion: Despite variations in sperm characteristics, both normal and poor sperm motility demonstrated comparable in vitro embryonic development competence. These findings provide important insights into the fertility potential of Bali bulls, providing valuable information that can enhance selection strategies to improve the quality of livestock production. Keywords: Bali cattle, embryos, in vitro semen quality.

This study aimed to analyze the characteristics of the HSP70 gene and protein in spermatozoa of Bali bulls of different age groups and to examine its potential as a biomarker determining bull fertility. This study used frozen semen produced from six Bali bulls divided into two groups based on age (≤ 9 years and ≥ 12 years). Parameters of frozen semen quality analyzed included sperm motility and kinetics using computer-assisted semen analysis, sperm morphological defects using Diff-Quick staining, acrosome integrity using FITC-PNA staining, and DNA fragmentation using acridine orange staining. HSP70 gene expression characterization was analyzed using qRT-PCR, and HSP70 protein abundance was analyzed using enzyme immunoassays. Fertility field data were obtained by analyzing the percentage conception rate for each bull based on the artificial insemination service data contained in the Indonesian-integrated system of the National Animal Health Information System (iSIKHNAS). The results showed significant differences (P0.05) in different age groups. HSP70 gene expression correlated with fertility rate (P<0.05). Age affected several semen quality parameters but did not affect HSP70 gene expression and protein abundance. The HSP70 gene molecule could be a biomarker that determines the fertility of Bali bulls.

Background and Aim: Bali-polled bulls, known for their favorable traits, require advanced reproductive performance analysis to optimize breeding programs. This study aimed to evaluate sperm kinematics and seminal plasma proteomic profiles as biomarkers for sperm motility and fertility in Bali-polled bulls. Materials and Methods: Semen from five Bali-polled bulls was collected biweekly over five batches using artificial vaginas. Sperm kinematics were assessed using computer-assisted semen analysis (CASA). Fertility was evaluated through service per conception (S/C) in artificial insemination trials. Seminal plasma proteins were analyzed through liquid chromatography–tandem mass spectrometry (LC-MS/MS) and annotated using the UniProt database, PANTHER for gene ontology, and STRING database for protein interactions. Results: Post-thaw sperm kinematics showed satisfactory results, with a mean S/C of 1.52. Proteomic analysis identified 138 proteins, including six (TEX101, BSP1, PRSS55, BSP3, SPADH2, and TPPP2) linked to sperm motility. These proteins were involved in key biological processes such as sperm capacitation, motility regulation, and sperm-oocyte interaction. Conclusion: Sperm kinematics and seminal plasma proteomics provide insights into Bali-polled bull fertility. Identified proteins can serve as fertility biomarkers, aiding in superior local breed development and reproductive efficiency improvement. Keywords: artificial insemination, Bali-polled bulls, fertility biomarkers, seminal plasma proteomics, sperm kinematics.