Identification of heat shock protein70-2 and protamine-1 mRNA, proteins, and analyses of their association with fertility using frozen-thawed sperm in Madura bulls

Abstract

Objective: This study aims to identify heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) mRNA and protein in Madura bull sperm and demonstrate their relation as bull fertility biomarkers.Methods: The Madura bull fertility rates were grouped based on the percentage of first service conception rate (%FSCR) as high fertility (HF) (79.04%; n = 4), and low fertility (LF) (65.84%; n = 4). mRNA of HSP70-2 and PRM1 with peptidylprolyl isomerase A (PPIA) as a housekeeping gene were determined by quantitative real-time polymerase chain reaction, while enzyme-linked immunoassay was used to measure protein abundance. In the post-thawed semen samples, sperm motility, viability, acrosome integrity, and sperm DNA fragmentation index were analyzed. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1, among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. The Pearson correlation was used to analyze the relationship between semen quality, mRNA, proteins, and fertility rate.Results: Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were detected and were found to be highly expressed in bulls with HF (p<0.05) and were associated with several parameters of semen quality.Conclusion: HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.

Sperm Head Morphology Alterations Associated with Chromatin Instability and Lack of Protamine Abundance in Frozen-Thawed Sperm of Indonesian Local Bulls

“…As reported by Nasr-Esfahani et al [33], CMA 3 staining as an indicator of protamine content, along with semen analysis, could be a useful full test for evaluating fertility status in sub-fertile cases. Although recently fertility assessments have been developed based on sperm genes and protein [34,35], considering that the CMA 3 test has good sensitivity and specificity values and is much easier and cheaper, there is nothing wrong with this test being the right choice for testing bull fertility.…”

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

“…In this study, HSPA9 and HSPA2 were found to be ubiquitously expressed. HSPA9, found in sperm mitochondria, significantly reduces the effects of heat stress in tropical cattle ( 33 , 34 ). It is also known to regulate sperm motility through metabolic processes, ATP binding, and folding of mitochondrial proteins ( 35 ).…”

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

“…The primer sequences used for HSP70 gene expression analysis (NM_174344.1) were forward 5'-TTGGGGACAAGTCAGAGAATG-3' and reverse 5'-ATCGTGGGGTTCCTTTTGATG-3'. The HSP70 primer was obtained from literature studies where this primer has been applied and used on local bull breeds, such as Qinchuan ( Bos taurus ) ( Zhang et al, 2015a ), Madura ( Bos javanicus x Bos indicus ) ( Rosyada et al, 2023b ), and Bali ( Bos sondaicus ) ( Pardede et al, 2023 ) bulls. PPIA (NM_178320.2) was used as an endogenous control, with the primary sequence being forward 5'-ATGCTGGCCCCAACACAA-3' and reverse 5'-CCCTCTCACCTTGCCAAA-3'.…”