Bovine sperm HSP-70 molecules: a potential cryo-tolerance marker associated with semen quality and fertility rate

Abstract

IntroductionFreezability is the ability of sperm to maintain its vitality and quality from various stress during the cryopreservation process, which is very important for the success of fertilization in AI programs. Heat shock proteins (HSPs) are unique proteins induced in response to various stress, including excess reactive oxygen species (ROS) and oxidative damage to intracellular enzymes that can harm cells. This study aimed to analyze the potential of HSP-70 molecules in bovine sperm as a marker of freezability or cryo-tolerance, as well as its association with semen quality and fertility rate.MethodsThe classification of bulls is based on freezability (good freezability/GF and poor freezability/PF), which is obtained from the value of post-thaw viability using the SYBR-14/PI-flow cytometry. Semen quality assessed included sperm motility and kinetics (computer-assisted sperm analyses), plasma membrane integrity (HOS test), acrosome integrity (FITC-PNA), mitochondrial membrane (JC-1), and DNA damage (Halomax kit). The bull fertility rate assessment was analyzed based on the first service conception rate of each bull derived from data on the success of artificial insemination contained in the Indonesian-integrated National Animal Health Information System (iSIKHNAS). Gene expression levels of HSP-70 bovine sperm were performed using the RT-qPCR method. The protein abundance of HSP-70 bovine sperm was determined using the enzyme immunoassay (EIA) method.ResultsBovine sperm HSP-70 molecules, at the gene and protein level, showed a higher abundance in GF (p < 0.05) than in PF bulls. The percentage of each parameter of frozen–thawed sperm quality was significantly higher in GF (p < 0.05) than in PF bulls. The HSP-70 molecules at the gene and protein levels were significantly positively correlated (p < 0.01) with the fertility rate. Furthermore, HSP-70 molecules were negatively associated (p < 0.01) with low mitochondrial membrane potential and sperm DNA damage and positively correlated (p < 0.01) with other frozen–thawed sperm quality parameters. The overall quality of frozen–thawed sperm was closely related (p < 0.01) to the fertility rate.ConclusionWe may conclude that HSP-70 molecules in bovine sperm at the gene and protein level have the potential to be developed as a marker for cryo-tolerance or freezability, which may be utilized as a predictor of fertility and frozen–thawed sperm quality in bulls.

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

“…Correlation test results between the HSP70 gene or protein and semen quality parameters did not show a strong relationship. It is different from previous studies, which found that there is a close relationship between HSP70 and motility ( Pardede et al, 2023 ; Zhang et al, 2015a ; Hung et al, 1998 ), sperm morphological defects ( Huang et al, 2000 ), acrosome integrity ( Kamaruddin et al, 2004 ), and DNA fragmentation ( Rajoriya et al, 2014 ). Varghese et al (2016) also reported that there was a significant (P<0.05) decrease in HSP70 levels before freezing and after thawing.…”

“…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'.…”