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: 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.

Objective: To determine the correlation between the molecular weight (MW) of proteins in seminal plasma and spermatozoa and the quality of fresh and frozen semen production in Pasundan bulls. Materials and methods: Nine selected Pasundan bulls, aged 5–10 years, from the Regional Artificial Insemination Center at Ciamis, West Java, Indonesia, were used in the study, with fresh semen sperm motility ≥70% and <70%. We analyzed the motility, viability, integrity of the intact plasma membrane (IPM), and the morphological characteristics of spermatozoa. 1D-SDS-PAGE analysis was performed to determine the protein profile by assessing MW, depicted as bands on the gel. Results: The motility, viability, and IPM of spermatozoa had lower values (p < 0.05) in Pasundan bulls named Bagaskara and Kertarajasa compared to the other bulls. Proteins with MW 35–50 kDa were not detected in the seminal plasma of Pasundan bulls, exhibiting low quality in fresh semen. The correlation analysis showed that the non-detected proteins with MW 35–50 kDa in seminal plasma correlated with spermatozoa motility (r = 0.421), viability (r = 0.424), and IPM (r = 0.428) so that fresh semen quality was low in both Pasundan bulls. Analysis of semen volume, spermatozoa concentration, and spermatozoa motility showed that the average frozen semen production of Pasundan bulls per ejaculate was 128.73 ± 15.35 straws. Conclusion: Protein analysis based on MW is a predictive indicator for the quality of fresh semen and the production of frozen semen in Pasundan bulls. Evaluation parameters of fresh semen quality by MW analysis can be used to select Pasundan bulls in Indonesia.

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.

Objective: The study aimed to assess sperm motility characteristics, kinematic parameters, and sperm protein molecular weight (MW) in Indonesian buffalo to predict fertility. Materials and Methods: Frozen semen from Silangit (4 bulls), Murrah (4 bulls), and Toraya (2 bulls)—aged 7–10 years, was analyzed. Sperm motility was assessed using Computer Assisted Semen Analysis, viability and abnormality were evaluated using eosin-nigrosin staining, plasma membrane integrity was evaluated using the hypoosmotic swelling test, acrosomal status was evaluated using lectin peanut agglutinin, protamine deficiency was evaluated using chromomycin A3, and deoxyribonucleic acid (DNA) integrity was evaluated using Halomax. Protein concentra¬tion was determined using the bicinchoninic acid method and characterized with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results: The study revealed breed-specific variations in semen quality. Silangit buffaloes exhibited lower DNA integrity, while Murrah buffaloes showed elevated motility and membrane integrity. Toraya buffaloes displayed higher normal morphology and protamine status, though they had lower viability. Notable differences in protein expression included the presence of SPAG9 and the absence of IZUMO1 in Toraya buffaloes. Protein MW analysis further showed correlations with sperm characteristics. In Murrah buffaloes, proteins within the 130–125 kilodalton (kDa) range were negatively correlated with acrosome integrity, whereas in Toraya buffaloes, proteins within the 55–50 kDa range were negatively correlated with sperm abnormalities. Silangit buffaloes showed a positive correlation between proteins at 32 kDa and sperm abnormalities. Conclusion: Analyzing protein MW through SDS-PAGE provides a promising approach for assess¬ing semen quality in indigenous Indonesian buffalo bulls. Although the semen quality of the buf¬faloes in this study was variable, all bulls met the established Indonesian standards for semen quality and exhibited adequate fertilization potential. These results provide valuable insights into the reproductive biology of Indonesian buffalo bulls and form the basis for predicting fertility capacity through a comprehensive analysis of sperm characteristics and molecular profiles of sperm proteins.

Indonesia has two National Artificial Insemination Centers (AIC) and more than 15 Regional Artificial Insemination Centers (RAIC) spread across several provinces. Bulls in the AIC must have a high libido and produce good quality semen. This study examines the correlation between libido with semen quality and testosterone concentration to determine potential frozen semen production from Bali bulls in South Sulawesi RAIC. Ten Bali bull were used in this study. Semen collection was carried out twice a week with semen evaluation following the RAIC protocol. At the same time, blood samples and libido measurements were carried out from each bull. The frozen semen production potential was calculated by multiplying the semen volume, motility, and sperm concentration. The results showed that the quality of fresh semen and testosterone concentrations did not differ between high and low libido of Bali bulls. Libido has a positive correlation with semen volume (r= 0.52) and sperm motility (r= 0.62), while testosterone concentration has a negative correlation with semen volume (r= -0.65), sperm motility (r= -0.60), and libido (r= -0.48). Bulls with high and low libido have good frozen semen production potential, ranging from 19,755 – 21,640 straws per year. Bali bulls in RAIC have fresh semen quality and testosterone concentrations under normal conditions, with high potential for frozen semen production, although only 60% of Bali cattle have high libido and 40% have low libido.