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.

Background: Reproductive efficiency is a critical factor in the productivity and profitability of cattle farming, especially within smallholder systems prevalent in regions like South Sulawesi, Indonesia. Bali cattle (Bos javanicus domesticus) are a local beef cattle breed originating from Indonesia, specifically from the island of Bali Indonesia. These cattle are known for their high adaptability to tropical environmental conditions and minimal feed requirements, making them ideal for small-scale farming systems. In terms of usage, Bali cattle are primarily utilized to produce high-quality beef. Reproductive disorders can lead to substantial economic losses due to reduced fertility, increased calving intervals, and higher veterinary costs. Identifying the prevalence and causes of these disorders is crucial for developing effective management strategies. Aim: The study aims to provide a comprehensive overview of the incidence of reproductive disorders in Bali cows kept under smallholder farms in South Sulawesi. Methods: The study was conducted on Bali cows aged over 2 years that raised traditionally on smallholder farms in South Sulawesi, Indonesia. A total of 283 Bali cows was selected as sampling randomly. The location selection was carried out purposively with the consideration that there were Bali cows at that location. Results: The results of this study showed that 70.3% of Bali cows had normal reproductive status and the remaining 29.7% of cows suffered from reproduction disorders. A trend is observed in the proportion of Bali cows experiencing reproductive disorders compared to normal Bali cows in relation to age, parity, and BCS. For age and parity, there is a tendency for similar proportions between reproductive disorders and normal Bali cows. Thus, there is no significant relationship between these two factors and the occurrence of reproductive disorders. On the other hand, for BCS, a different trend is observed between Bali cows experiencing reproductive disorders and normal Bali cows. Therefore, BCS influences the occurrence of reproductive disorders in these Bali cows. Conclusion: This study provides valuable insights into the prevalence of reproductive disorders within a sample population. The results underscore the importance of continued study in this area to enhance our understanding of reproductive health issues.

The fertility rate of bulls in a breeding program is not only described by the quantity and quality of semen. Factors, such as the interstice factor of the sperm and the plasma component of semen, affect the fertility rate of bulls. The fertility rate can also be determined by identifying the protein content of semen plasma. Therefore, the current study aimed to identify the relationship between seminal plasma protein molecular weight and semen quality of Bali-polled bulls. The study was conducted at the Laboratory of Semen Processing, Faculty of Animal Science, Hasanuddin University, Makassar, Indonesia, the Research Center for Applied Zoology, National Research and Innovation Agency, Cibinong, Indonesia and the Laboratory of Animal Biotechnology Center, IPB University, Bogor, Indonesia from November 2021 to January 2022. The samples came from 5 Bali-polled and 5 Bali-horned bulls. Semen collection was conducted twice a week using an artificial vagina. The concentration of seminal plasma protein was determined by the Bradford method of 1D-SDS-PAGE. The study results showed that fresh semen of Bali-polled and Bali-horned bulls was considered a normal category. Seminal plasma proteins of Bali-polled and Bali-horned bulls were classified using 8 bands to categorize molecular weight; 150 kD (IGF-1), 110 kD (A-kinase anchoring protein 3), 93 kD (A-kinase anchoring protein 4), 54-87 kD (Arylsulfatase-a), 44-62 kD (N-Acetyl-ß-Guicosaminidase), 44kD (Phosphoglycerate kinase), 15-30 kD (BSP A1/A2, BSP-A3 and BSP-30 [BSP1, BSP3, and BSP5]) and 12-14 kD (Acidic seminal fluid proteins). The findings indicated that both Bali-polled and Bali-horned bulls could have a high reproductive rate. In conclusion, protein analysis based on molecular weight using 1D-SDS-PAGE can be used as a biomarker for semen quality in Bali-polled bulls. Therefore, evaluating the semen quality with a molecular basis as an additional indicator of superior bull in the selection process is an alternative method.

The objective of this study was to determine the effect of nitrogen fertilizer level on growth, dry matter yield and nodulation of Indigofera zollingeriana plant at during the nursery. The experiments were arranged in a complete randomized design consisting of four treatments and four replications. The utilized four levels of the nitrogen fertilizer were 0 (P0), 100 (P1), 150 (P2) and 200 kg N/ha (P3) equivalent to 0, 0.15, 0.23 and 0.30 g N/polybag respectively. The variables in this study included the plant height, stem diameter, number of petioles per plant, length of petiole, number of leaves per plant, root length for each plant and dry matter yield. The results showed that the fertilizer doses increased the plant height, stem diameter, number of leaves per plant, and dry matter yield of leaf, leaf petiole, stem, root and nodule. It could be concluded that nitrogen fertilization was effective in stimulating growth, production and nodulation at early nursery stage, yet higher nitrogen doses (200 Kg N/ha) are ineffective and have inhibitory effects of growth and production. Therefore, high-dose nitrogen fertilizers in legume plants should be avoided because Indigofera zollingeriana is able to fix nitrogen from the atmosphere as in other legumes.

The Gaga chicken is an indigenous Indonesian breed that is important to preserve using semen cryopreservation technology. The study was conducted to determine the effect of adding sorbitol and butylated hydroxytoluene (BHT) in the diluent on the structural and surface ultrastructure of cryopreserved Gaga chicken sperm during cryopreservation /frozen storage. The study aimed to assess how adding sorbitol and butylated hydroxytoluene (BHT) to the diluent affects the structure and surface ultrastructure of cryopreserved Gaga chicken sperm. A completely randomized design was employed with four treatments and 10 replications including egg yolk-lactate ringer diluent (EYLR) as the control group, EYLR diluent with 3 mM BHT, EYLR diluent with 2% sorbitol, and EYLR diluent with both 3 mM BHT and 2% sorbitol. Semen was collected using a massage technique from 4 male chickens aged approximately 10 months, pooled semen was diluted, packaged in 0.25 mL straws, equilibrated for 2 hours at 5 °C, pre-freeze for 10 minutes, frozen for 24 hours, and thawed for 30 seconds at 37 °C. The parameters evaluated were sperm plasma membrane integrity, acrosome integrity, DNA damage, mitochondrial functionality, and surface ultrastructure. The results showed that the treatment had a significant effect on plasma membrane integrity and post-thawing mitochondrial functionality compared to the control, but no effect was observed on acrosome integrity or DNA damage. The results showed that the combination treatment of BHT with sorbitol had a significant effect on plasma membrane integrity and post-thawing mitochondrial function, but did not affect acrosome integrity or DNA damage when compared to the control group. Ultrastructural observations indicated that cryopreservation caused damage to the head, middle, and tail of the sperm in the control groups. However, these changes were prevented by the diluent containing a combination of BHT and sorbitol. The addition of both components (BHT 3 mM + sorbitol 2%) effectively maintained plasma membrane integrity, mitochondrial functionality, and surface ultrastructure of Gaga chicken sperm during cryopreservation. Keywords: Butylated hydroxytoluene, Chicken sperm, Cryopreservation, Sorbito, Structure, Sperm ultrastructure

| The purpose of this study was to determine the characteristics and kinematics of Bali-polled bull sperm to identify reproductive potential. Semen collection of Bali-polled bull was conducted once a week using an artificial vagina. The semen was proceeded until the frozen semen. Parameters were motility, motility progressive, kinematics sperms, viability, abnormality membrane integrity, and acrosome integrity. The Bali-polled bull semen was analyzed by using CASA with the Sperms Vision Version 3.7.5 program. The results in this study showed that sperms motility in Bali-horned bull was significantly lower (P<0.01) than Bali-polled bull, Brahman, Limousine, and Simmental. The sperms viability in Bali-horned and polled bull was significantly lower (P<0.01) compared to other bull breeds. The membrane integrity and acrosome integrity of Bali-polled bull sperms was significantly higher (P<0.01) than in Bali-horned bull. DCL, DAP, and DSL of Bali-polled bull were significantly higher (P<0.01) than Bali-horned bull. VCL, VAP, and VSL of Bali-horned bull were significantly lower (P<0.01) than Bali-polled, Simmental, Limousine, and Brahman bull. Based on the results of this study, it can be concluded that Bali-polled bull had good sperm characteristics and kinematics compared with various breeds so that the Bali-polled bull can have the potential to become superior bulls to contribute to the strategies for developing Bali bull as a national beef bull contributor.