Sperm HSP70: may not be an age-dependent gene but is associated with field fertility in Bali bulls (Bos sondaicus).

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 (P<0.05) in total and progressive motility, morphological defects of the neck and midpiece, and tail of sperm, and acrosome integrity between the age groups of Bali bulls. HSP70 gene expression and protein abundance showed no significant differences (P>0.05) 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.

Comparative developmental competence of in vitro embryos recovered from Bali cattle with normal and poor sperm motility.

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.

A proteomic approach to identifying spermatozoa proteins in Indonesian native Madura bulls.

Proteins assist sperm mature, transit the female reproductive tract, and recognise sperm oocytes. Indigenous Indonesian bulls, Madura bulls, have not been studied for reproductive proteomics. As local Indonesian beef livestock, Madura cattle assist in achieving food security; hence, their number must be improved. Thus, the identification of molecular proteomics-based bull fertility biomarkers is needed. This study aimed to characterise the sperm fertility function of the superior Madura bull (Bos indicus × Bos Javanicus) spermatozoa proteome. Frozen semen from eight Madura superior bulls (Bos indicus × Bos javanicus) aged 4-8 years was obtained from the artificial insemination centre (AIC) in Singosari and Lembang. Madura superior bulls are those that have passed the bull breeding soundness evaluation. Frozen sperm were thawed and centrifuged at 3000 × g for 30 min. Proteins in sperm were characterised through proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting gene symbols for each protein were then subjected to bioinformatics tools, including UniProt, DAVID, and STRING databases. Regarding sperm fertility, the analysis revealed that 15 proteins were identified in the sperm of Madura bulls. Amongst the identified proteins, the superior Madura bull sperm contained several motilities, energy-related proteins, and chaperone proteins. A substantial portion of characterised proteins are linked to metabolic pathways and the tricarboxylic acid (TCA) cycle, contributing to sperm energy production. In conclusion, the first in-depth proteome identification of sperm related to sperm quality and bull fertility of a unique indigenous Madura breed of Indonesia was performed using the LC-MS/MS proteomic method. These findings may serve as a reference point for further studies related to the functions of bovine sperm and biomarkers of fertility and sperm quality.

Correlation between Post-Thaw Spermatozoa Quality of the Endangered Javan Banteng with OPN Gene Expression.

The role of ex situ conservation facilities or captivity through captive breeding programs is essential in the conservation of the endangered Javan banteng. The development of semen cryopreservation may assist on one side of the conservation plan. However, the male Javan banteng reproductive capability must be considered as it influences the targeted outputs. Studying the potential biomarker for fertility such as osteopontin gene expression is also expected to help predict male fertility. Therefore, this study aimed to analyze the quality of spermatozoa after thawing to help predict the male reproductive capability of Javan banteng. Furthermore, this study investigated the potential role of osteopontin gene expression in male Javan banteng fertility. A positive reinforcement approach was used to accustom the male and female animals as we focused on establishing a collection procedure using neither sedation nor anaesthesia. Semen samples were collected at Taman Safari Indonesia, Bogor, in accordance with the female banteng receptivity. Semen samples were then evaluated and then cryopreserved under field conditions. Our study showed the different predicted reproductive capability of the Javan banteng based on the post-thaw spermatozoa quality, which showed significant differences. The OPN gene showed positive correlations with the progressive motility (r = 0.711, p = 0.048), viability (r = 0.822, p = 0.012), and acrosomal integrity (r = 0.665, p = 0.072) of Javan banteng spermatozoa after thawing. Our study demonstrated the predicted Javan banteng reproductive capability based on various post-thaw spermatozoa variables. This finding is also the first report on the OPN gene potential to be developed as the assessment tool of post-thaw spermatozoa quality of the male Javan banteng. The findings in our study may help give recommendations for future breeding programs, especially in the ex situ conservation sites.

Protein Identification of Seminal Plasma in Bali Bull (Bos javanicus).

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).

Semen quality and frozen semen production in Pasundan bulls: A molecular weight perspective on seminal plasma and spermatozoa protein.

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.

The potential of sperm bovine protamine as a protein marker of semen production and quality at the National Artificial Insemination Center of Indonesia.

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.

Comparative analysis of various step-dilution techniques on the quality of frozen Limousin bull semen.

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 <20% sperm abnormality was divided into three tubes and diluted with skim milk-egg yolk (SMEY) using three different dilution techniques: One-step dilution (100% SMEY with 8% glycerol) at room temperature ([RT] 20°C until 25°C) two-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol after 1 h stored at 5°C); and three-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol added twice at 1 h and 1.5 h after being stored at 5°C). The diluted semen was loaded into 0.25 mL mini straws, equilibrated, and frozen using a freezing machine. Sperm motility, viability, membranes, DNA integrity, and concentrations of malondialdehyde (MDA) and aspartate aminotransferase (AST) enzymes were evaluated after thawing. RESULTS: The results showed that there were no significant differences in sperm motility and DNA integrity between dilutions (p>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.