The widespread use of artificial insemination (AI) in bovine breeding is indicative of the need to select bull based on the fertility level and sperm quality [1]. Fertile bulls are expected to have sperm capable of fertilizing an oocyte and supporting embryo development, as shown by the success of AI in the form of a conception rate reaching 70% [2]. However, the selection of bulls with good fertility and sperm quality remains a major challenge for AI centers and the livestock industry [3]. The standard quality parameters such as post-thawing sperm motility, which are routinely applied in evaluation tests at several centers, are not entirely reliable in determining fertility [4]. In several countries, the standard for determining sperm quality and fertility based only on post-thawing sperm motility of at least 40% is inadequate [3]. A previous study showed that 50% of the tested bulls only had medium to low fertility rates [4], and some bulls were found to produce only 20-30% fertility rates [5], despite meeting the minimum requirements for sperm quality for the AI program. These results significantly impact the livestock industry, causing economic problems in various countries. Recent studies have reported the influence of intrinsic factors on sperm, which is genetic, incorporating various sperm-specific RNA or protein molecules. These molecules play a crucial molecular role during sperm formation and maturation in spermatogenesis, which is essential in determining fertilization success [1,2,6,7]. During spermatogenesis, including the process of condensation of chromatin, sperm generate a wide variety of RNA and protein molecules that contribute to their normal function at the beginning of the journey to the egg [3,8].

Chromatin condensation during the spermiogenesis phase is critical in protecting the integrity of genetic material. This process is facilitated by the product of spermiogenesis-specific RNA molecules or sperm nucleus proteins, including nucleoprotein transitions (TNPs) [9]. During the spermiogenesis phase, the transformation process of spermatid into sperm occurs, including the most significant and complex changes in the structure and function of chromatin [3]. These changes include elongated nuclei, the cessation of the transcription process, and the near-complete removal of histones, as well as chromatin, which transforms from delicate fiber to a highly dense form during the final process of the spermiogenesis phase [9]. Moreover, histone proteins that previously dominated the chromatin are replaced by protamine [3]. In mammals, small essential nuclear proteins play a role in the transition process after the histone proteins are removed, signifying the initiation of the chromatin condensation process, namely TNP1 and TNP2 [10]. TNP2 works first by turning off the histone transcription process, followed by TNP1 stimulating DNA repair that was broken during histone removal [11]. TNP1 is responsible for replacing histones with protamine in mammalian elongated spermatid [12]. Subsequently, TNPs are replaced by phosphorylated protamine, leading to the formation of a toroidal structure in the chromatin [11]. This process results in a six-fold increase in the levels of DNA packaging and chromatin condensation compared to somatic cells [12].

TNP1 is essential for producing healthy sperm and its absence in 60% of males has been linked to infertility due to a significant decrease in motility [13]. Meanwhile, male infertility stemming from the lack of TNP2 protein results in sperm head (head defects) anomalies characterized by acrosome deficiencies, slowed movement, and an inability to penetrate the zona pellucida [13,14]. Other studies stated that sperm with TNP1 and TNP2 null genotypes showed decreased normal morphology, motility, and chromatin condensation [15,16]. The improper regulation of sperm nucleoprotein replacement, aberrant chromatin condensation patterns, and reduction have also been linked to TNP1 deficiency in mice [13]. Although these discoveries show the importance of TNPs in male fertility, investigation of TNPs in bulls used for AI is still minimal. Hirenallur Maheshwarappa et al. [17] reported that TNP1 and TNP2 genes showed no difference in expression in sperm of Vrindavani bulls with good and poor motility. TNP1 was also reported not to affect individual motility and mass activity in male Murrah bulls [18]. These reports primarily focused on sperm motility and did not explore other quality characteristics, as well as fertility. Therefore, this study aimed to analyze mRNA transcription and protein concentrations of TNP1 and TNP2 in bulls sperm used for the AI program. The potential of TNP1 and TNP2 as candidate genes or proteins as sperm motility biomarkers and bull fertility determinants were analyzed, including their correlation with other quality traits. The results are expected to provide new insight regarding how accurate and effective the currently used quality standards are based solely on post-thawing motility. Therefore, it is assumed that a combination of conventional and molecular examinations can be incorporated to predict bulls’ fertility accurately.