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 evaluate the effect of glutathione (GSH) supplementation in maturation and adaptation media on oocyte development, embryo quality, and oocyte viability after vitrification. Methods: The GSH concentrations were classified into four groups (0, 0.5, 1.0, and 1.5 mM) which were added to the maturation medium. The maturation process was conducted in vitro for 24 h. Following maturation, oocytes were fertilized with Bali bull semen for 5-6 h and then cultured for 48 h. The morphological quality of ocytes matured with GSH addition and the vitrification method used was evaluated. Parameters assessed included maturation rate, fertilization rate, embryo development, post-vitrification oocyte morphology, and quality of post-vitrification oocytes with GSH added to the adaptation medium. Results: The addition of GSH to the maturation medium significantly improved oocyte quality and embryo development (P<0.05). Specifically, adding 1.5 mM GSH increased the percentage of oocytes reaching metaphase II from 57.6% without GSH oocytes to 79.0% with 1.5 mM GSH, two-pronuclei fertilization from 47.0% to 72.7%, embryo development from 37.1% to 57.2%, morula formation from 14.6% to 33.7%, and blastocyst formation from 8.1% to 23.8%. Additionally, the survival rate of oocytes post-vitrification increased to 75% with GSH supplementation. Conclusions: The addition of 0.5-1.5 mM of GSH to the maturation and adaptation media significantly enhanced the metaphase II stage, fertilization rates, cleavage rates, and the survival of oocytes after vitrification. Among the concentrations of 1.5 mM was the most effective in increasing oocyte development and maintaining oocytes viability post-vitrification.

The facts in the field show that seedstock of Bali cattle in South Sulawesi have decreased because the high rate of female productive Bali cattle were slaughtering and good bull maintained for fattening and trading and the price of cow Bali were more cheaper than bull. Therefore, it needs a new breakthrough to solve this problem by applying the embryo production technology in vitro. This process consists of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). These three processes have been studied using Bali cattle oocytes, the maturity level of Bali cattle oocyte reaches 91.53%, while the fertilization and division rate was 68%-70,03% and embryo in vitro culture of Bali cattle reached 32 cell blastocyst stages. The results of the study indicate that the potential of invitro embryo production can be used to maintain and improve the genetic quality of Bali cattle.

Background and Aim: Seminal plasma proteins (SPPs) significantly influence sperm quality, playing a critical role in fertility. This study aims to investigate the molecular weight (MW) profiles of SPPs in horned and polled Bali bulls and their correlation with sperm quality parameters. Materials and Methods: Semen samples were collected from six Bali bulls (3 horned, 3 polled). Sperm quality was evaluated based on motility, viability, abnormalities, intact membranes, and acrosomes. SPPs were extracted and analyzed using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine protein MWs. Pearson’s correlation was used to analyze relationships between MW profiles and sperm quality metrics. Results: SPPs were identified across a MW range of 15–165 kDa, with specific proteins showing strong correlations with sperm quality. Proteins at 50 and 46 kDa positively correlated with motility (r = –0.96), viability (r = –0.99), and intact membranes (r = –0.86). Conversely, proteins at 40 kDa negatively correlated with these parameters. A 25 kDa protein displayed a positive correlation with intact acrosomes (r = –0.93) and a negative correlation with abnormalities (r = –0.99). Differences in sperm quality metrics between horned and polled bulls were observed, with polled bulls exhibiting fewer abnormalities. Conclusion: This study highlights the potential of SPP MW profiles as biomarkers of sperm quality in Bali bulls. Proteins at 50, 46, and 25 kDa are promising markers for sperm motility, viability, and intact acrosomes, respectively. These findings could inform bull selection and reproductive management strategies. Further research is recommended to validate these biomarkers using advanced proteomic approaches. Keywords: Bali bulls, molecular weight, seminal plasma proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sperm fertility.