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

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.


Introduction
The artificial insemination (AI) program is a proven and successful reproductive technology in increasing population and livestock production [1]. The AI program for cattle uses frozen semen from different breeds of bulls produced at the AI center with specific standards [2]. Generally, sperm motility above 40% with spermatozoa concentration not less than 25 million per insemination dose is used for quality control of frozen semen in AI programs [3]. This quality standard should predict bull fertility and optimize AI success via a high percentage of pregnancy and calf births. Unfortunately, facts reveal that the success of AI is still comparatively low. Rosyada et al. [4] reported that the conception rate of several Holstein bulls was still classified as low fertility (LF), namely, 33.78% from >1000 AI data and 21.15% from <1000 AI data, even though the quality of frozen semen met the standards, including spermatozoa motility above 40%. Pardede et al. [5] also reported that the %CR of bulls in controlled breeding program centers was sub-optimal, at 63% from

Experimental Animals
The Indonesian National AI center produced the frozen semen used in this study. The frozen semen sample used in this study is a commercial product from the AI center, where every procedure, from the process of collecting fresh semen to producing it into frozen semen, including the management of bulls' maintenance, follows the operational standards of the AI center, which is supervised by a veterinarian and has complied with every principle of animal welfare. The semen was obtained from bulls at their sexual maturity and productive phase, at 4-5 years of age. This study used 18 bulls, including six Limousin, Holstein, and Ongole Grade bulls. Furthermore, each breed was grouped into two based on the fertility score, HF (n = 3) and LF (n = 3). The fertility score was based on each bull's first service %CR; %CR > 70% are categorized as HF bulls, and %CR < 70% Vet. Sci. 2022, 9,111 3 of 15 is classified as LF bulls [6]. The %CR data was obtained from AI data and pregnancy tests on cows for the last two years at the National Animal Health Information System (iSIKHNAS), Indonesia.
The iSIKHNAS is an animal health information system in all regions in Indonesia, including data on AI programs accompanied by pregnancy data. The results of the analysis carried out on iSIKHNAS data, the number of cows/heifers that were inseminated in each breed of bulls in the study were 143,010 (Limousin), 81,741 (Holstein), and 98,571 (Ongole Grade) cows/heifers. Meanwhile, the number of AI services for each breed of the bull in the study were 197,588 (Limousin), 106,034 (Holstein), and 136,686 (Ongole Grade) services. The percentage of pregnant cows/heifers from the first insemination service of the total AI services (%CR) was analyzed according to Pardede et al. [5].

Frozen Semen Quality
A total of 90 frozen semen straws (five straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used in this stage. First, the frozen semen was thawed in a water bath at 37 • C for 30 s, then put into a microtube for further observation. Next, the sperm's progressive motility (PM) parameters were analyzed using computer-assisted semen analysis. After that, 10 µL of thawed sperm were dropped onto a slide and covered with a coverslip. Finally, four fields were evaluated with a range of 50-250 sperm cells automatically counted using Sperm Vision (Minitube, Tiefenbach, Germany) [20].
Additionally, sperm viability was evaluated using eosin-nigrosine (0.825 g eosin Y, 5 g nigrosine, 0.375 g Na Citrate, and 100 mL aquadest) staining. First, the sperm viability was evaluated by making a smear on slides from a mixture of 10 µL semen and 10 µL eosin-nigrosine (1:1). Then, the slides were dried using a heating table, observed, and counted under a light microscope with 40× objective magnification. Two hundred sperm cells per slide were evaluated, with live sperm unstained and dead sperm with damaged membranes stained (reddish) [31]. Next, the integrity of the plasma membrane of sperm was evaluated using the HOS (hypoosmotic swelling) test method. Furthermore, 20 µL of semen was mixed into 300 µL of HOS solution (0.735 g Na citrate, 1351 g, and 100 mL aquadest) and incubated in a water bath at 37 • C for 30 min. Then, 10 µL of the mixture was dropped on a slide, covered with a coverslip, and observed under a light microscope with 40× objective magnification. Finally, 200 sperm cells per slide were counted, with a coiled tail indicating sperm of intact plasma membrane integrity [5,32].

Sperm DNA Fragmentation
Sperm DNA fragmentation (SDF) was assessed using acridine orange (AO) staining and the Halomax test. A total of 90 frozen semen straws (five straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used for AO staining. First, a smear was made 5-10 µL of semen on a slide, then fixed in Carnoy's solution (methanol: glacial acetic acid (3:1)) for 2 h. Next, the slides were stained in a new AO solution (10 mL of 1% acridine orange, 40 mL of 0.1 M citric acid, 2.5 mL Na 2 HPO 4 7H 2 O) for 5 min in a dark room. Next, a coverslip was placed on the slide after washing with distilled water. Finally, 500 sperm cells per slide were observed and counted under a fluorescent microscope using a 490 nm excitation filter and a 530 nm emission filter. The green fluorescence denoted the sperm with mature chromatin and intact DNA integrity, whereas sperm with fragmented DNA integrity was characterized by a reddish-orange fluorescence [33].
Moreover, according to the manufacturer's protocols, SDF was assessed using the Halomax test (Bos taurus, Halotech DNA SL, Madrid, Spain). A total of 90 frozen semen straws (five straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used for this test. First, 25 µL (15-20 × 10 6 sperm cells/mL) was mixed with 50 µL of liquid agarose in a microtube at 37 • C. A total of 1.5-2 µL of cell suspension was dropped into marked wells on a pre-cooled slide (4 • C), and a coverslip was placed. The slide was incubated in the refrigerator at 4 • C for 5 min. The coverslip was carefully removed from the slide at room temperature (22 • C), and a lysing solution was dripped on the slide horizontally for 5 min and later dried. Next, the slides were washed with distilled water for 5 min and re-dried. Finally, the slides were dehydrated with 70% and 90% ethanol solutions, respectively, for 2 min and dried. The staining process was done by dripping 5 µL of 0.01 mm propidium iodide dye for 5 min in a dark room. Five hundred sperm cells per slide were observed under a fluorescent microscope using a 510-560 nm excitation filter and a 590 nm emission filter. Sperm cells with the fragmented DNA were then denoted by large and dispersed spotty halos, whereas tiny, dense halos represented sperm as the unfragmented DNA [34].

Sperm PRM Deficiency Assay
Sperm PRM deficiency was evaluated using the Chromomycin A 3 (CMA 3 ) staining method. A total of 90 frozen semen straws (five straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used in this stage. Sperm PRM deficiency was assessed by smear preparations of 5-10 µL of semen on a slide, then fixed in Carnoy's solution (methanol: glacial acetic acid (3:1)) for five min at 4 • C. Then, the slides were stained in CMA 3 dye solution (0.25 mg/mL CMA 3 in McIlvane's buffer (pH 7.0) supplemented with 10 mM MgCl 2 ) for 20 min. Next, the slide was rinsed in McIlvane's buffer (pH 7.0) and air-dried. Finally, 500 sperm cells per slide were observed under a fluorescent microscope using a 470-490 nm excitation filter and a 510 nm emission filter. Sperm with deficient PRM (CMA3+ positive) was indicated by bright green fluorescence on the head, whereas dull green indicated sperm with intact PRM (CMA3−negative) [35].

PRM1 Gene Expression Analysis
A total of 144 frozen semen straws (eight straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used in this stage. Next, each frozen semen was thawed in a water bath for 30 s at 37 • C, washed three times with phosphatebuffered saline (PBS), and pelleted (about 25 × 10 6 sperm cells/mL) by centrifugation at 16,000× g for 15 min. After that, the total RNA was extracted using the TRI reagent, as recommended by the manufacturer (Zymo Research, Irvine, CA, USA). The NanoDropTM One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific, Marsiling Industrial Estate Rd 3, Singapore) determined the total RNA amount and purity (Thermo Scientific, Marsiling Industrial Estate Rd 3, Singapore). According to the manufacturer's protocols, the SensiFASTTM cDNA Synthesis Kit (Bioline Ltd., Memphis, TN, USA) was also used for cDNA synthesis. A total of 20 µL of cDNA obtained from this reaction was ready for RT-PCR. Next, the NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific, Marsiling Industrial Estate Rd 3, Singapore) was used to assess the total cDNA amount and purity. The quantitative real-time PCR (qPCR) was also used to determine the transcripts' quantity, and the reactions were performed using the SsoFastTM EvaGreen Supermix (Bio-Rad Lab, Hercules, CA, USA). The genes identified in this study were PRM1 (BC108207; forward: 5 -AGATACCGATGCTCCTCACC-3 and reverse: 5 -GCAGCACACTCTCCTCCTG-3 ) [22] with PPIA (XM_001252921.1; forward: 5 -ATGCTGGCCCCAACACAA-3 and reverse: 5 -CCCTCTTTCACCTTGCCAAA-3 ) as housekeeping gene [27]. The 2 −∆∆CT [36] was used to assess the relative expression levels of the PRM1 gene, which were normalized to the housekeeping gene PPIA expression.

PRM1 Protein Assay
A total of 108 frozen semen straws (six straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used. Each frozen sperm was thawed in a 37 • C water bath for 30 s, washed twice with PBS, and pelleted (about 25 × 10 6 sperm cells/mL) by centrifugation at 700× g for 15 min. The amount of PRM1 protein was then measured in sperm pellets using the enzyme immunoassay (EIA) technique, according to Vet. Sci. 2022, 9, 111 5 of 15 the manufacturer's protocols (Bovine PRM1, MyBioSource, Inc., San Diego, CA, USA) [20]. The prepared sperm pellets were briefly added to the appropriate wells.
Furthermore, the reaction wells were closed and hatched in an incubator (37 • C) for 90 min. After that, the EIA plate was washed twice, then 100 µL of antibody solution was added and covered with adhesive tape on each well. Next, the EIA plate was re-hatched in the incubator (37 • C) for 60 min and then washed thrice. A total enzyme solution, 100 µL, was poured into each well, closed, and re-hatched in the incubator (37 • C) for 30 min. Washing was repeated five times on the EIA plate; then, color reagent solution was added and hatched again in the incubator (37 • C) in the darkroom for 30 min. Finally, the color C reagent mixture mixed thoroughly in each well. The analysis was conducted using an EIA reader with a wavelength of 450 nm in 10 min.

Statistical Analysis
The fertility score (%CR) data for each bull breed were analyzed using Microsoft Excel (Microsoft Office Pro Plus 2019 Microsoft). The differences in the mean number of breeding, fertility score (%CR), semen quality parameters, SDF, PRM deficiency, the abundance of PRM1 protein, and relative expression of PRM1 mRNA in the FH and LF bulls were analyzed using the t-test method. The Pearson correlation analysis examined the overall association between the parameters evaluated and the bulls' percentage CR. A scatter plot matrix graph presented the relationship between %CR, PRM1 protein, and mRNA. The t-test method, Pearson correlation analysis, and scatter plot matrix were obtained using the Statistical Package for the Social Sciences (v.25.0, IBM, Armonk, NY, USA). The data were presented in the form of mean ± standard error.

Discussion
Fertile bulls are determined by the ability of a bull to produce sperm capable of fertilizing the oocytes [37] and have a new calf [38]. The fertility decline in bulls impacts cows' low conception level, resulting in low productivity and economic problems related to the livestock industry [38,39]. Chenoweth [40] and Parkinson [41] showed that fertility data and extensive progeny records in cattle prove the inability of superior bulls to produce a maximum %CR, even when the applicable standards are met by sperm motility and morphology, similar to our study's results. The semen used is produced from superior bulls that underwent the previous selection process with fertility scores that have not reached a good %CR category ( Table 1). The quality of post-thawing semen significantly resulted in PM showing a percentage exceeding 40%, quality control of frozen semen used for the AI program in each breed of bulls in the study (Table 1). This value meets the minimum PM requirements of frozen semen used for the AI program, according to the Indonesian National Standard [3] and Zewdie et al. [42]. However, the value exceeded the standard; it did not produce a maximum fertility score. Diskin [6] and Butler [43] reported that a good fertility score reached at least 70% of 100% of the first inseminated cows.
Similar results were previously reported [4], showing that the fertility score of several Holstein bulls in Indonesia was approximately 21-35%, including in the LF bulls category. This result contradicted Rosyada et al. [4], who reported no correlation between PM and %CR (fertility); however, they showed a close correlation between the two parameters (p < 0.01) ( Table 2). Moreover, it is undeniable that only the motile and progressive sperm can pass through the cervix and penetrate the cumulus and the zona pellucida for fertilization to occur [44].
In addition to PM, semen quality parameters such as viability and plasma membrane integrity showed a significant difference (p < 0.05) between HF and LF bulls (Table 1) associated with %CR (Table 2) in each breed. The outcomes of this study revealed a higher percentage value of viability and integrity of the plasma membrane than the previous research's average [4,5]; however, it has not yet reached the maximum fertility level. This result is comparable to a previously published report by Pardede et al. [5], who discovered that even though the percentage CR was less than 70%, semen quality remained a critical aspect of bull fertility. Again, however, the results of this study aligned with the previous findings that showed the inability of conventional semen quality assessment to facilitate the fertility of a bull.
Disturbances exist during the spermatogenesis process in bulls, impacting the molecular defects that affect the quality and production of sperm, consequently decreasing fertility and even causing infertility [9]. The molecular defects in uncompensated sperm and sex-steroid enzyme alteration influence sperm's capacity to fertilize the egg, contributing to the embryo's normal early development [45][46][47]. Additionally, the non-compensable factors, such as molecular defects in sperm, cannot be addressed by raising the number of sperm per insemination instead of compensable characteristics, which may be solved by increasing the number of sperm per insemination [48][49][50][51]. PRM, the primary protein packaging for sperm DNA, is a component in sperm that plays a crucial role in determining these uncompensated traits and is also associated with molecular defects in sperm [8,10,12,13,25,28]. Additionally, hypercondensation of sperm chromatin occurs, during the elongated spermatid phase of spermiogenesis, involving the replacement of histones by transient transition proteins [52], then replaced by at least 85% by PRM until maternal histones return them after fertilization [53].
Proper DNA protamination of sperm influences sperm chromatin dynamics in mammals [23]. Furthermore, disturbances in this process can affect the sperm maturation process in the epididymis, causing an impact on sperm dysfunction and male subfertility, including infertility [54]. Immature sperm contains more histone-packed chromatin regions more likely to display SDF than well-PRM sperm [55]. Our study evaluated SDF using AO staining and the Halomax test (Figure 1). Evenson [56] reported that sperm chromatin structure assay (SCSA) is the most stable SDF assessment method, whereas AO staining is the least sensitive method compared to other methods. The Halomax test is the simplest and easiest method of evaluating SDF, which is included in a kit combined with various cell dyes primarily used with light and fluorescence microscopy [56]. The instability of sperm chromatin packaging also causes SDF and dispersion, forming a large halo [57]. Garcia-Macias et al. [34] reported that evaluating SDF using the Halomax test is one among four fertility predictors. This study did not use SCSA because of its expensiveness. It is practically not affordable for application in the field and requires flow cytometry compared to the Halomax test method and AO staining.
The SDF assessment with AO staining showed a significant increase (p < 0.05) in LF bulls than in HF bulls (Figure 2A), with the threshold value reaching 3.27% in LF bulls. In a study finding, the Halomax test was used to evaluate SDF on LF bulls reporting threshold value substantially higher (p < 0.05) than on HF bulls (Figure 2A), with the value reaching 3.49% on LF bulls. Dogan et al. [13] reported that the Halomax test method showed a negative correlation with fertility in bulls, in low SDF bulls with LF. Our study also found that SDF percentage using both techniques was adversely linked (p < 0.001; p < 0.000) with %CR for each breed of bull (Table 2). However, previous studies reported the various effects of severe SDF, such as reduced fertility rates, poor embryo quality, and decreased pregnancy rates [58]. Cho et al. [59] and Boe-Hansen et al. [60] also reported early embryonic death and smaller fetal size due to severe SDF.
Several factors cause spermatozoa DNA fragmentation, such as hormones, age, infection, increased reactive oxygen species, chemical/toxic exposure, smoking, drugs, testicular hyperthermia, apoptosis, and protamine deficiency during spermatogenesis [51]. As previously discussed, the high SDF levels in sperm are related to the level of maturity of the sperm due to impaired protamination of the chromatin structure of sperm. However, Simon et al. [58] report showed a correlation of abnormal expression or low PRM content in sperm with SDF. Therefore, this study also evaluated the level of PRM deficiency indirectly using the CMA 3 method (Figure 1). The guanine-cytosine-specific fluorochromes from the CMA 3 assay competed with PRM binding sites on DNA, showing that chromatin packaging under PRM deficiency conditions will cause binding of chromomycin to DNA (CMA3+) [61]. In sperm with DNA chromatin packaging compacted and stabilized by PRM, arginine, which is rich in guanine-cytosine (CMA3−), abundant in PRM, would inhibit the dye CMA 3 [62,63]. However, using CMA 3 as an indirect assay of PRM content in sperm associated with SDF and fertility is widely used [64][65][66][67][68].
Our study's findings agreed with earlier research, where LF bulls in each bull breed exhibited a higher percentage of CMA3+ or PRM deficiency (p < 0.01) than HF bulls ( Figure 3A). A negative correlation (p < 0.000) shown between CMA3+ and the %CR in all breeds of bulls was used in this study (Table 2). In this study, the results of the indirect PRM assessment were also confirmed by identifying PRM1, both at the mRNA ( Figure 3B) and protein levels ( Figure 3C). PRM1 mRNA was highly expressed (p < 0.05) in HF bulls than in LF bulls in all bull breeds during this study ( Figure 3B). At the protein level, PRM1 protein was also seen to be significantly more abundant (p < 0.05) in HF bulls compared to LF bulls in all breeds of bulls ( Figure 3C). A positive correlation (p < 0.000; p < 0.009) ( Table 2) and linearity in the linear regression curve graph ( Figure 4) were also shown between mRNA expression and PRM1 protein abundance with %CR. Our results are similar to those reported in a previous study in Holstein bulls, showing transcription of PRM1 mRNA with low concentrations in LF bulls [12]. Additionally, Dogan et al. [13] reported that PRM1 was detected in low concentrations in bulls with LF rates with an increased SDF at the protein level.
The condition of PRM deficiency or aberrant protamination of sperm resulting in LF conditions or even infertility cases is associated with several plausible causes, including disruption of transcription, translation, and synthesis processes [69][70][71][72]. Recently, research has linked defective protein synthesis to abnormal mRNA retention, implying that abnormalities in PRM translation control may lead to PRM inadequacy in infertile males [73]. Additionally, it is reported that abnormal histone ratios resulting from the failure or complete failure of the protein remodeling process in sperm chromatin, in which PRM dominates 85% of the sperm nucleus protein, is a cause of PRM aberration that causes male infertility [74]. In other species, PRM haploinsufficiency in transgenic mice reportedly impacts changes or disruption of spermatogenesis and result in infertility [75]. However, unlike in mice and humans, where another variant of PRM, PRM2, must have a similar ratio as PRM1, and a ratio that deviates at the protein and mRNA levels, will impact infertility [76][77][78][79]. PRM1 in bulls is the only PRM variant that plays a direct role in the normal function of fertile sperm [12,13]. Ganguly et al. [27] and Pardede et al. [20] also proved that PRM1, both at the mRNA and protein levels, was the most critical PRM variant in normal sperm function in bulls.
Nevertheless, specifically, the regulation and mechanism of PRM1 in sperm remains not fully understood [29] necessary to be studied further. Steger et al. [80] stated that abnormal expression or decreased expression and concentration of PRM1 resulting from the abnormal function of transcriptional regulators, translation, or post-translational modifications affect PRM and various genes during spermatogenesis. Therefore, it is not surprising that PRM can be a "checkpoint" during spermiogenesis, and abnormal PRM expression will impact the rate of apoptosis [80]. Increased apoptosis affects poor semen quality associated with fertilization and pregnancy after IVF [81]. However, this study supports the hypotheses that have been reported so far regarding the importance of PRM1 in normal spermatozoa function and its relationship to fertility in bulls, especially bulls used for AI programs in Indonesia. As found in this study, PRM1 proved to be closely related to fertility, with bulls still classified as subfertile or LF, even though the semen quality was conventionally categorized as suitable for the AI program. Moreover, the combination of assessment with a gene or protein analysis approach with semen quality assessment, including SDF assessment, can be the best choice in determining the fertility of bulls.

Conclusions
In conclusion, inadequate or low sperm chromatin protamination at both mRNA and protein levels was associated with defects in sperm chromatin condensation, found with increased SDF, and coinciding with a decrease in the %CR (fertility score) in bulls. Therefore, considering that this research aimed to find candidate fertility markers that accurately determine bulls' fertility and can be used in the bull selection process, especially in Indonesia, PRM1 shows to be a promising candidate in terms of fertility markers. Furthermore, to the best of our knowledge, this is the first work in Indonesia to demonstrate correlations in the mRNA expression and protein content of sperm PRM1 from HF compared to LF bulls. Moreover, it is not only in one breed of cattle but also represents commodity groups of cattle breeds in Indonesia, such as exotic beef cattle (Limousin), dairy cattle (Holstein), and local cattle breeds (Ongole Grade).