PRM1 Gene Expression and Its Protein Abundance in Frozen-Thawed Spermatozoa as Potential Fertility Markers in Breeding Bulls

Abstract

Functional genes and proteins in sperm play an essential role in bulls’ reproductive processes. They are more accurate in determining bull fertility than conventional semen quality tests. Protamine-1 (PRM1) is a gene or protein crucial for packaging and protecting sperm DNA until fertilization affects normal sperm function. This study analyzes the genes and proteins potential from PRM1 as fertility markers for different breeds of bulls utilized in the artificial insemination programs, expected to be an accurate tool in interpreting bull fertility in Indonesia. This study used Limousin, Holstein, and Ongole Grade bulls divided into two groups based on fertility, high-fertility (HF) and low fertility (LF). The semen quality assessment included progressive motility (computer-assisted semen analysis), viability (eosin-nigrosine), and plasma membrane integrity (HOS test). Sperm DNA fragmentation (SDF) was assessed using the acridine orange staining and the Halomax test. Sperm PRM deficiency was evaluated with the chromomycin A3 method. Moreover, PRM1 gene expression was measured using qRT-PCR, and the PRM1 protein abundance was measured with the enzyme immunoassay method. Semen quality values, relative expression of PRM1 gene, and quantity of PRM1 protein were significantly higher (p < 0.05) in HF bulls than in LF bulls. The SDF and PRM deficiency values in LF bulls were significantly higher (p < 0.05) than HF bulls. Additionally, PRM1 at the gene and protein levels correlated significantly (p < 0.01) with fertility. Therefore, PRM1 is a potential candidate for fertility markers in bulls in Indonesia.

Keywords: breeding bulls; fertility marker; frozen-thawed spermatozoa; gene expression; protamine-1; protein abundance.

Figure 1

The photomicrograph of sperm in AO staining of HF and LF bulls (A,B); Sperm with normal DNA integrity will be stained with green fluorescence; SDF will be stained with yellow-orange fluorescence (arrow). The photomicrograph of Halomax test in HF and LF bulls (C,D); Sperm with normal DNA integrity showed a slight halo, and SDF showed a large halo (arrow). The photomicrograph of PRM deficiency by the Chromomycin A3 (CMA₃) assay (E,F); bright green fluorescence-stained sperm (CMA3+) indicated PRM deficiency (arrow), whereas dull green stained sperm (CMA3−) indicated normal PRM-stained sperm.

Figure 2

The value of SDF using AO staining (A) and Halomax test (B) on Limousin, Holstein, and Ongole Grade bulls with different fertility (HF vs. LF). * Significant difference when compared to LF (p < 0.05).

Figure 3

The PRM deficiency (CMA3) percentage (A), relative expression of PRM1 gene (B), and the abundance of PRM1 protein (C) in Limousin, Holstein, and Ongole Grade bulls with different fertility (HF vs. LF). * Significant difference when compared to LF (p < 0.05).

Figure 4

Scatter plot matrix graph of the linearity relationship between %CR and relative abundance of gene and protein of PRM1 in bulls regardless of bull grouping based on breed and fertility.