Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis

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Weintraub, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4185383/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 May, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions when compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mostly overrepresented in the “metabolism” and the “metabolism of lipids” pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays were performed and revealed sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of proteins of differential abundance, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction. Biological sciences/Biotechnology/Proteomics Biological sciences/Zoology/Animal physiology Health sciences/Medical research/Translational research Stallion sperm Thoroughbred impaired acrosomal exocytosis proteomics acrosome enzymes arylsulfatase F Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The acrosomal exocytosis (AE) process involves a series of biochemical changes in the sperm, mediated mostly by an increase in intracellular pH and calcium levels, and plasma membrane destabilization due to cholesterol depletion 1 – 3 . Together, all these cellular changes will result in the fusion of the outer acrosomal membrane with the sperm plasma membrane, leading to the release of multiple enzymes at the vicinity of the cumulus-oocyte complex (COC) and facilitating the binding of sperm to the zona pellucida 1– 3 . Stallion subfertility due to impaired acrosomal exocytosis (IAE) is a condition identified thus far only in stallions of the Thoroughbred (TB) registry. Sperm from these stallions are characterized as having a lower acrosomal response after in vitro exposure to either non-physiologic (i.e., calcium ionophore A23187), or physiologic (i.e., lactate-induced) conditions known to result in acrosomal exocytosis (AE) 4 – 6 . These stallions have low fertility (< 30% per-cycle pregnancy rates), even though typical features of sperm quality and breeding management are acceptable. An association between the IAE phenotype and the presence of a double homozygous A/A-A/A genotype for SNPs chr13:11,353,372G > A (rs397316122) and chr13:11,353,436A > C (rs69101140) in FKBP6 exon 5 (EquCab3) has been identified in two separate studies 7 , 8 . The frequency of subfertile TB stallions that carry the FKBP6 A/A-A/A genotype approaches 1–3% of the TB breeding stallion population (four out of 150 stallions evaluated in Central Kentucky, USA, for the presence of the susceptibility genotype 8 ; and seven out of 1,128 stallions of various breeds evaluated during 17 years at a reference laboratory 5 ). In mice, the FKBP6 protein has been associated with the normal formation of the synaptonemal complex during spermatogenesis; thus, double knock-out male mice are sterile due to azoospermia 9 . Similar findings have also been identified in men with idiopathic azoospermia 10 , 11 , indicating that FKBP6 is related to the normal progression of meiosis in spermatocytes. These findings contrast the clinical characteristics of TB stallions with the A/A-A/A combined genotype in FKBP6 exon5 (i.e., normal sperm quality and testicular size), making a potential link between the susceptibility genotype for IAE in these stallions and the function of FKBP6 difficult to demonstrate. Proteomic technologies have been used to identify proteins of importance for sperm physiologic processes 12 – 16 and for the identification of candidate biological markers that could either be used to select males with higher fertility potential 17 – 19 or identify potential proteins that explain causes of reduced fertility 20 – 22 . Most of these studies included proteomic analysis using mass spectrometry-based technologies, mainly involving the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this method, the mass-to-charge ratio ( m/z ) of ionized molecules (in this case peptides produced by proteolytic digestion during sample preparation) are detected and the ions then fragmented, generating precursor (MS1) and product-ion (MS2) mass spectra 23 , 24 . These MS2 mass spectra are then queried against published protein sequence databases for the identification (and relative quantification) of the proteins in the sample 23 , 24 . The mass spectra produced in this way are obtained using an approach known as data-dependent acquisition (DDA), in which peptides that are detected in a precursor scan are sequentially selected based on relative abundance and fragmented to generate sequence-informative tandem mass spectra. When using DDA-MS for the analysis of complex samples, there is inevitably under-sampling (i.e., lack of detection and fragmentation of many lower-abundance peptides) even with state-of-the-art instruments that have extremely fast scan rates. This can hamper the detection of low-abundance proteins which may have biological relevance. Recently, an approach known as data-independent acquisitionmass spectrometry (DIA-MS) has gained popularity. In DIA-MS, ionized peptides in small "windows" of m/z ranges are sequentially fragmented rather than individually selected precursors, thereby permitting much more comprehensive detection of the complement of peptides in a digest. In this way, DIA-MS provides identification and relative quantification of a much larger number of proteins with greater accuracy and precision than DDA-MS. Various studies have analyzed the proteome of sperm following incubation under capacitating conditions or after attempted stimulation of AE 13 , 25 , 26 . To the best of our knowledge, such studies have not yet been conducted using stallion sperm. We recently reported a method to consistently induce AE in sperm from stallions with different in vivo fertility levels, including TB stallions that were confirmed to have IAE both by genotyping and acrosomal function testing 6 . Given the potential advantages of DIA-MS to identify candidate proteins that otherwise would not be discovered by other methods, we utilized this approach in combination with our method to induce AE in stallion sperm to identify potential candidate proteins that could explain the etiology of IAE in TB stallions. Results Sperm quality parameters in fresh and frozen/thawed semen from fertile and subfertile TB stallions (A/A-A/A) The initial sperm quality parameters in fresh semen, fertility parameters (mare book, PC-PR, and SPR), and the FKBP6 genotype results from both stallion groups are shown in Supplementary Table 1. There were no differences observed in sperm quality between stallion groups (P > 0.05). Frozen/thawed semen from both fertile and subfertile TB stallions was exposed to the Lac-MW model to determine their acrosomal response. At T0h and T2h, mean AE/Viable was similar for fertile and subfertile stallions (Fig. 1 ; P > 0.05), while at T4h and T6h, mean AE/Viable was higher for fertile than for subfertile stallions (P < 0.05). Representative scattergrams of the viability/acrosomal exocytosis assay conducted in frozen semen from a fertile stallion and a subfertile A/A-A/A stallion are also presented in Fig. 1 . The proteome of frozen/thawed sperm from fertile and subfertile TB stallions There were 2202 proteins (FDR 1.0%) identified from 18723 peptides by DIA-MS in sperm from both fertile and subfertile TB stallions. By using the stallion phenotype (fertile vs. subfertile) as the main effect to test by two-way ANOVA, a total of 298 proteins reached significance after FWER correction (FDR q-value 0.05; Fig. 2 ). After applying stringent criteria (i.e., log2fold ≤ − 0.585 or ≥ 0.585), a total of 140 proteins were identified, which identified 61% of the variance in protein abundance between stallion groups, resulting in two distinctive data point clusters (Fig. 2 ). Of these, 83 were found to be of lower relative abundance in sperm from the subfertile TB stallions [log 2 (fold change) 0.585] in sperm from these stallions when compared to sperm from fertile TB stallions. The 140 differentially abundant proteins were used for subsequent analysis. Gene ontology analysis of differential abundance sperm proteins in subfertile TB stallions. An initial analysis using g:Profiler and Equus caballus orthologs was conducted in each of the two lists of differentially abundant proteins (lower vs. higher abundance). Manhattan plots corresponding to the GO CC, BP, MF, and KEGG analyses are presented in Fig. 3 , while GO terms for each of these categories in proteins of lower and higher abundance in sperm from subfertile stallions are presented in Table 1 . A subsequent GO analysis was conducted in g:Profiler by using Homo sapiens rather than Equus caballus orthologs, to increase the coverage of GO terms. Manhattan plots corresponding to this analysis are presented in Supplementary Fig. 1, while GO terms for each of these additional protein categories are presented in Supplementary Table 2. Table 1 List of gene ontology (GO) terms related to proteins with lower and higher relative abundance in sperm from subfertile TB stallions when compared to sperm from fertile TB stallions. The proteins were queried using Equus caballus orthologs. The ID numbers correspond to the IDs represented in the Manhattan plots in Fig. 3 . GO: Gene ontology; CC: Cellular component; BP: Biological process; MF: Molecular function; KEGG: Kyoto Encyclopedia of Genes and Genomes. Proteins of lower relative abundance in sperm from subfertile TB stallions ID Source (GO term) Term ID Term Name p -value (adjusted) 1 CC GO: 0005783 Endoplasmic reticulum 2.639 x 10 − 7 2 CC GO: 0012505 Endomembrane system 1.085 x 10 − 5 3 CC GO: 0005789 Endoplasmic reticulum membrane 3.044 x 10 − 5 4 CC GO: 0031090 Organelle membrane 7.072 x 10 − 5 5 CC GO: 0005737 Cytoplasm 3.819 x 10 − 4 6 CC GO: 0031984 Organelle subcompartment 1.424 x 10 − 3 7 CC GO: 0031301 Integral component of organelle membrane 3.086 x 10 − 3 11 BP GO: 0044281 Small molecule metabolic process 9.674 x 10 − 6 12 BP GO: 0044283 Small molecule biosynthetic process 2.660 x 10 − 4 13 BP GO: 0006629 Lipid metabolic process 1.214 x 10 − 3 14 BP GO: 0016126 Sterol biosynthetic process 4.675 x 10 − 3 15 BP GO: 0044255 Cellular lipid metabolic process 3.869 x 10 − 2 16 BP GO: 0006694 Steroid biosynthetic process 4.374 x 10 − 2 17 KEGG KEGG: 00071 Fatty acid degradation 5.743 x 10 − 3 18 KEGG KEGG: 01100 Metabolic pathways 1.016 x 10 − 7 19 KEGG KEGG: 00100 Steroid biosynthesis 1.162 x 10 − 2 20 KEGG KEGG: 01212 Fatty acid metabolism 1.741 x 10 − 2 22 MF GO: 0016491 Oxidoreductase activity 4.214 x 10 − 4 23 MF GO: 0003824 Catalytic activity 4.581 x 10 − 6 24 MF GO: 0016787 Hydrolase activity 3.561 x 10 − 2 Proteins of higher relative abundance in sperm from subfertile TB stallions 8 CC GO: 0005940 Septin ring 1.486 x 10 − 2 9 CC GO: 0035686 Sperm fibrous sheath 1.486 x 10 − 2 10 CC GO: 0031105 Septin complex 3.551 x 10 − 2 21 CC GO: 0036126 Sperm flagellum 1.739 x 10 − 2 Functional network analysis of differentially abundant proteins in sperm from subfertile TB stallions. The ClueGo analysis tool was used in conjunction with Homo sapiens orthologs for GO and KEGG terms to classify the differentially abundant proteins in sperm from subfertile TB stallions in a functional network. A diagram of the functional network for the proteins of lower abundance in sperm from subfertile stallions is presented in Fig. 4 . The results indicated that these proteins were mostly involved in fatty acid metabolism, fatty acid derivative metabolism, sterol biosynthetic process, cellular aldehyde metabolic process, and endoplasmic reticulum protein-containing complex. Conversely, the network for the proteins of higher abundance in sperm from subfertile stallions was mostly associated with vesicle docking and sperm flagellum (Supplementary Fig. 2). A group of proteins associated with lipid metabolism, including two enzymes of acrosomal origin, display differential abundance between stallion groups and within time periods. Bioinformatic analyses suggested that most of the proteins of differential abundance, and particularly those of lower abundance, in sperm from the subfertile TB stallions were associated with metabolism and metabolism of lipids. Examination of the data in Scaffold DIA was utilized to identify time-point-related changes in the relative abundance of proteins belonging to these categories. These changes were also analyzed at the time periods in which AE was observed in sperm from fertile but not from subfertile TB stallions (i.e., 4 and 6 hours of incubation in Lac-MW medium). Proteins that fulfilled these criteria included: arachidonate lipoxygenase 3 (ALOX3), arachidonate 12-lipoxygenase (ALOX12), arylsulfatase F (ARSF), extracellular matrix protein 1 (ECM1), ergosterol biosynthesis 28 homolog (ERG28), Na + -dependent phosphate cotransporter 2B (SLC34A2), and zona pellucida-binding protein 2 (ZPBP2). The median relative abundance levels of these proteins between stallion groups, and within time periods are presented in Fig. 5 . The expression of the acrosome protein arylsulfatase F (ARSF) in sperm from fertile and subfertile TB stallions One of the proteins of lower abundance in sperm from subfertile TB stallions is an enzyme of acrosome origin and might have relevance during the sperm-oocyte interaction process. As such, we sought to determine if this protein was present in stallion sperm, and whether its expression and/or location were different between sperm of fertile and subfertile TB stallions. By indirect immunofluorescence, we observed that the protein arylsulfatase F (ARSF) was expressed at a higher proportion at the acrosome, mid-, and principal piece in sperm (Pattern I) from fertile TB stallions than in sperm from subfertile TB stallions (mean ± SD: 57 ± 8% vs. 17 ± 5%, respectively; P < 0.05), while at a lower proportion at the midpiece and principal piece (Pattern II) in sperm from fertile TB stallions than in sperm from subfertile TB stallions (mean ± SD: 16 ± 7% vs. 49 ± 3%, respectively; P < 0.05). Representative immunofluorescence images of the location of ARSF in stallion sperm are presented in Fig. 6 . Frozen/thawed sperm from subfertile TB stallions display a lower ability to bind to the zona pellucida. A total of 120 in vitro -matured porcine oocytes (20 oocytes per stallion; 60 for fertile TB and 60 for subfertile TB stallions, respectively) were used in this experiment. The mean number of sperm bound to the ZP was higher in fertile TB than in subfertile TB stallions (55 ± 8 sperm/ZP vs. 15 ± 4 sperm/ZP; P < 0.05; Fig. 7 ). Discussion In the current study, we identified candidate proteins to further investigate the causes of IAE in TB stallions. For this purpose, we combined the use of a method to induce AE in viable sperm (i.e., incubation in a lactate-only containing medium [Lac-MW]), and DIA-MS to identify and quantify the relative levels of proteins in the sperm of both fertile and subfertile TB stallions. Previous studies have used mass spectrometry-based approaches to investigate the stallion sperm proteome to identify potential proteins associated with higher sperm quality or fertility potential 15 , 22 , 27 , 28 , or to determine the effects of storage methods (i.e., cooled storage or freezing/thawing) on the sperm proteome 16 , 29 – 32 . To the best of our knowledge, the current study is the first to use this technology to investigate the potential cause(s) of a clinical condition in stallions that is unique due to the phenotype that these individuals present: reduced in vivo fertility associated with IAE despite having normal-to-excellent conventional sperm quality parameters. Our results indicate that most of the proteins of differential abundance in sperm from the subfertile TB stallions correspond to processes associated with either cell metabolism or metabolism of lipids, as observed in the gene ontology analyses using both Equus caballus or Homo sapiens orthologs. These results are interesting from two perspectives: 1) both sperm capacitation and AE require extensive remodeling of the sperm plasma membrane to prepare the fusion between both plasma and outer acrosomal membranes, facilitating the release of acrosomal contents 33 – 37 . This remodeling process, in turn, requires cholesterol depletion from the plasma membrane, allowing the entry of calcium at the intracellular level and resulting in the activation of second messenger pathways 38 – 41 . Some of the biochemical changes related to sperm capacitation and AE are also related to the metabolism of lipids at the sperm membrane, particularly by the activation of phospholipase A2 (PLA2), which will result in the production of arachidonic acid metabolites and lysophospholipids that enhance the initiation of AE 42 – 46 . Furthermore, both sperm capacitation and AE require considerable quantities of energy in the form of ATP and are also governed by a delicate balance between the consumption of energy and the production of reactive oxygen species (ROS) 47 – 49 ; thus, it would be expected that proteins associated with metabolic processes would be overrepresented in proteomic analysis of sperm. We did not observe an overall interaction between the stallion condition (fertile vs. subfertile) and the sperm AE during incubation periods using the Lac-MW medium. It is possible that there are differences in protein post-translational modifications (PTMs) across the various time points. Since biological PTMs were not evaluated in our DIA-MS analyses, no conclusions can be reached in this regard for this study. Nonetheless, we were still able to identify changes over time in seven proteins (ALOX3, ALOX12, ARSF, ECM1, ERG28, SLC34A2, ZPBP2) that were differentially abundant when sperm underwent AE (i.e., T4h and T6h), which also were of lesser abundance in sperm from the subfertile stallions (Fig. 5 ). These seven proteins were subjected to GO term analysis using the g:Profiler server and were found to correspond to the terms metabolism and metabolism of lipids . These findings are consistent with an earlier study from our group indicating that subfertile TB stallions that carry the susceptibility genotype for IAE have a higher cholesterol-to-phospholipid ratio in their sperm membranes and seminal plasma 50 . As such, it is possible that the acrosomal dysfunction observed in these individuals could be related to excessive levels of cholesterol in the sperm membrane that influence the ability of sperm to undergo either capacitation or AE 50 . The changes in the relative abundance of these proteins also coincided with the maximum levels of AE in viable sperm, as determined by flow cytometry. Two of these proteins, ALOX3 and ALOX12 correspond to enzymes associated with the metabolism of fatty acids into leukotrienes, which is related to an increase in the lipid peroxidation levels of biological membranes 51 . Contrasting results have indicated that lipoxygenases may 52 , 53 , or may not 54 be related to the occurrence of AE in hamster, bull, or human sperm incubated under capacitating conditions. A recent study showed that inhibition of ALOX15 in human sperm resulted in increases in sperm motility, calcium ionophore A23187-induced AE, and sperm-ZP binding compared to samples that were only treated to stimulate oxidative stress but in which ALOX15 was not inhibited 55 . Yet, these results should not be interpreted that arachidonate lipoxygenases induce only deleterious effects on sperm, but rather that a balance between the pro-oxidative effects of these lipoxygenases must exist to induce redox changes associated with sperm capacitation and AE, and not only associated with oxidative stress and cell death. Another protein of interest is ERG28. Studies in human sperm indicate that the addition of ergosterol to sperm incubated under capacitating conditions for 24 hours resulted in a reduced rate of spontaneous and progesterone induced AE 56 . In that study, the reduced rate of AE caused by the addition of ergosterol to sperm was related to its similar effects on the cholesterol-to-phospholipid ratio on the sperm membranes, as ergosterol is structurally similar to cholesterol and can be incorporated within the sperm plasma membrane similarly as cholesterol 56 . This is in contrast with our results showing a higher relative abundance of ERG28 in the sperm from fertile stallions that also had a higher rate of spontaneous AE in viable sperm. RT-PCR analysis indicated that ERG28 is highly expressed in human testicular tissue 57 , and the role of this protein in Saccharomyces cerevisiae indicates that this protein is necessary for the normal progression of the ergosterol biosynthetic pathway 58 . However, the actual localization of ERG28 within the testicular cell types or its potential relevance in male reproductive physiology has not yet been described. The role of a Na + -dependent phosphate cotransporter (SLC34A2) during sperm capacitation or AE could be explained by the requirements of membrane hyperpolarization that lead to the entry of calcium within the sperm and the increase of intracellular pH 59 , 60 ; nonetheless, this specific cotransporter has not been previously identified in sperm from any species. Arylsulfatase, particularly arylsulfatase A (ARSA), has been identified in sperm from rabbits, mice, and humans in the post-acrosomal region and plasma membrane 61 – 63 . This protein remains at the sperm post-acrosomal region after AE 61 , 62 and is implicated in sperm-oocyte binding due to its activity as a sulfatase that interacts with the sulfoglycoproteins present in the receptors ZP2 and ZP3 64 – 66 . In the present study, another member of the arylsulfatase gene family, arylsulfatase F (ARSF), was observed by immunofluorescence at three regions of the sperm from fertile stallions: acrosome, midpiece, and principal piece (Fig. 6 ). In contrast, in sperm from subfertile TB stallions, the ARSF signal was only observed at the sperm midpiece and principal piece (Fig. 6 ). Such marked difference in the localization of ARSF at the acrosome region might explain the reduced ability of sperm from subfertile stallions to both undergo AE and bind to the ZP. Further studies in which the immunolocalization of ARSF in stallion sperm is studied after incubation under capacitating conditions and after the occurrence of AE are warranted. Another protein of interest identified by DIA-MS in our study was zona pellucida-binding protein 2 (ZPBP2), which has been identified in proacrosomal vesicles that later integrate the inner acrosomal membrane in sperm from other species 67 . This protein not only is released from sperm during AE acting as a secondary receptor for sperm-ZP binding 68 , but also interacts with other proteins such as testisin, which recently has been identified in stallion sperm as an important serine protease required for normal sperm capacitation, AE, and ZP-binding 69 . Interestingly, in a recent case report involving a TB stallion with considerably low in vivo fertility, zona pellucida-binding protein was identified as one potential candidate biomarker for impaired acrosomal exocytosis, based on mass spectrometry-based analysis 21 . The stallion involved in that case report had a reduced rate of AE following stimulation with calcium ionophore A23187, a similar finding to what we have previously observed in TB stallions that carry the IAE susceptibility genotype 5 , 7 . Unfortunately, the case report by Swegen et al., 21 did not indicate whether the affected stallion also carried the IAE susceptibility genotype. Nonetheless, according to the clinical phenotype, it seems plausible that the TB stallion from that report carried the same IAE susceptibility haplotypes as the stallions used in the current study; as such, the zona pellucida-binding protein can be considered an important marker protein for IAE in TB stallions. While in our study we did not identified by immunofluorescence the presence of ZPBP2 in sperm from either fertile or subfertile TB stallions, a potential confirmation of reduced zona pellucida-binding protein function in sperm from subfertile TB stallions was their reduced ability to bind to porcine ZP after incubation under capacitating conditions (Fig. 7 ). In the present study, we utilized frozen/thawed sperm from both fertile and subfertile TB stallions, mainly due to the inability to access fresh semen from these individuals, as mentioned above. Freezing and thawing alters the proteome of stallion sperm, resulting in a lower abundance of several proteins involved in metabolism regulation and redox regulation 29 , 31 , 32 . Proteins related to sperm-oocyte interactions, namely IZUMO-4 and zona pellucida binding protein, had lower abundance in frozen/thawed stallion sperm when compared to freshly ejaculated sperm 29 . In the current study, we did not observe statistical differences in the relative abundance of any of the IZUMO proteins (1, 2, 3, or 4) between stallion groups or within periods (data not shown), and we only detected differences in the relative abundance of zona pellucida-binding protein, as described above. Since we did not compare the proteome of fresh versus frozen/thawed sperm from the stallions enrolled in the present study, we cannot compare our results with those presented by Martin-Cano et al 29 . Notably and despite the compelling evidence from several studies that the double homozygous A/A-A/A genotype in FKBP6 exon 5 is significantly associated with the occurrence of IAE in TB stallions 5 , 7 , 8 , we did not detect the presence of the FKBP6 protein in sperm from any of the stallion groups tested. This is consistent with the recent theory that FKBP6 is not the causative gene for IAE and the A/A-A/A genotype in exon 5 is rather tagging a haplotype unique to Thoroughbreds with IAE 8 . The current results might offer insight into the mechanisms underlying IAE given the reported high cholesterol-to-phospholipid ratio in the sperm membranes 70 , the reduced acrosomal function after non-physiological 4 , 5 , or physiological stimulation of AE 6 . Future investigations will be focused on identifying the dynamics (i.e., localization or patterns of expression) of some of these proteins during the initiation of capacitation and AE in stallion sperm. Conclusions The current study explored the sperm proteome in both fertile and subfertile TB stallions, with the latter being carriers of the susceptibility genotype for IAE. Using a DIA-MS approach in conjunction with a method that induces AE in viable sperm, we were able to identify a group of differentially abundant proteins associated with metabolism and metabolism of lipids that may explain the acrosomal dysfunction observed in subfertile stallions. One of these proteins, ARSF, was detected at the acrosome, midpiece, and principal piece in sperm from fertile TB stallions, but only at the midpiece, and principal piece in sperm from subfertile TB stallions. We also provide evidence that the ability of frozen/thawed sperm from subfertile TB stallions to bind to porcine ZP was decreased when compared to that of fertile TB stallions. Some of the proteins identified by mass spectrometry and immunofluorescence in the current study are candidates for further studies focused on determining the pathophysiological cause of IAE and understanding the biological processes involved in sperm capacitation and AE in stallions. Materials and methods Reagents and media Unless otherwise stated, reagents were purchased from Sigma Aldrich (St. Louis, MO, USA). Fixable Live/Dead Red Stain, rabbit anti-arylsulfatase F (ARSF) polyclonal antibody, goat anti-rabbit IgG Alexa-555-conjugated secondary antibody, 10% normal goat serum, SlowFade™ Diamond Antifade Mounting Solution with DAPI, and methanol-free 16% paraformaldehyde were acquired from Thermo Fisher Scientific (Waltham, MA, USA). Accumax® Cell Detachment Solution was purchased from Stemcell™ Technologies Inc., (Cambridge, MA, USA). Acridine Orange stain was obtained from Polysciences Inc., (Warrington, PA, USA). CryoMax Lactose-EDTA® semen freezing extender (20% egg-yolk + 2% glycerol and 3% methyl formamide) was obtained from Animal Reproduction Systems (Chino, CA, USA). A silane-coated silica particle solution (Redigrad®) for density gradient centrifugation was acquired from Global Life Sciences Solutions (Marlborough, MA, USA). The base medium, MW-HEPES, used for sperm washing by density gradient centrifugation was a modified Whitten’s medium 27 and consisted of 110 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl 2 , 1.9 mM CaCl 2 , 22 mM HEPES, and 50 µL/mL gentamicin sulfate. The medium used for sperm in vitro incubation under capacitating conditions (Lac-MW) was prepared as reported previously 6 , 27 and consisted of modified Whitten’s medium with 25 mM HCO 3 − instead of HEPES, 7 mg/mL bovine serum albumin – heat shock fraction (BSA), and 10 mM sodium-DL lactate (60% syrup). All media were adjusted with NaCl to an osmolality of 280–290 mOsm/kg. On the day of the experiment, the pH of each medium was adjusted to 7.25 using NaOH or HCl. The Lac-MW medium was maintained at 38.2°C in 5% CO 2 for a minimum of 2 hours before use. Stallions and semen collection Texas A&M University Institutional Animal Care and Use Committee (IACUC 2021-0007) approved all the procedures performed in this study. All stallions enrolled (n = 6) were Thoroughbred, sexually active, and 7–15 years old. Hair samples were procured to determine the presence of the susceptibility genotype for IAE, A/A-A/A in the gene FKBP6 exon 5, as previously reported 7 , 8 . The FKBP6 genotype, in vivo fertility rates, and conventional sperm quality parameters of the six stallions used (three fertile and three subfertile) are presented in Table 1 . Before semen collection, each stallion was exposed to an ovariectomized mare (when available), or a mare in standing estrus. Once erect, the penis was rinsed thoroughly with warm water and dried with paper towels. The ejaculates from two fertile TB stallions and one subfertile TB stallion were collected using a Colorado-type artificial vagina (Animal Reproduction Systems, Chino, CA, USA), while ejaculates from the other three TB stallions (one fertile and two subfertile) were collected using a Missouri-Model artificial vagina (Nasco, Ft. Atkinson, WI, USA). For both artificial vagina types, an in-line nylon micromesh filter (Animal Reproduction Systems) was placed between the artificial vagina and the semen collection receptacle to separate the gel fraction from the gel-free semen. Following semen collection, the gel-free semen was transported to an adjacent laboratory and placed in an incubator (37°C) before processing. Initial semen processing and analyses The gel-free semen volume was estimated based on sample weight, while sperm concentration and plasma membrane intactness (i.e., viability) were measured using a fluorescence-based cell counter (NucleoCounter SP-100™, Chemometec A/S, Allerød, Denmark), following a previously described methodology 28 . Sperm motion characteristics were determined using computer-assisted sperm analysis (CASA; Hamilton-Thorne IVOS II, Hamilton-Thorne Inc., Beverly, MA, USA), as reported previously 29 . The preset values for the instrument consisted of the following: frames acquired, 45/s; frame rate, 60 Hz; minimum contrast, 70; minimum cell size, four pixels; minimum static contrast, 30; straightness (STR) threshold for progressive motility, 50%; average path velocity (VAP) threshold for progressive motility, 30%; VAP threshold for static cells, 15 µm/s; cell intensity, 106 pixels; static head size, 0.60 to 2.00 µm; static head intensity, 0.20 to 2.01; static elongation, 40 to 85; illumination intensity, 2200. Sperm motility parameters included the percent of total motility (TMOT), progressive motility (PMOT), and the mean curvilinear velocity (µm/s; VCL). For sperm morphology analysis, samples of raw semen were fixed with buffered-formal saline (BFS; 4.75% formaldehyde) and analyzed using differential interference contrast (DIC) microscopy (1,563x, Olympus BX-60, Olympus Corporation, Melville, NY, USA). Sperm morphological classification was done as previously reported 30 . All morphologic abnormalities were counted for each spermatozoon to determine the incidence rate. A total of 100 sperm were counted for each ejaculate, and the percentage of morphologically normal sperm was recorded. Sperm DNA quality was determined in flash-frozen/thawed samples obtained from raw semen, using the Sperm Chromatin Structure Assay (SCSA), as previously described 31 . The percentage of Cells Outside the Main Population (COMP α−t ), was used as an endpoint to determine the extent of the susceptibility of sperm DNA to denaturation. Semen cryopreservation and stimulation of lactate-induced spontaneous AE in viable frozen/thawed sperm In a previous study, we determined that stallion sperm stored at 5°C for 24 hours, or frozen/thawed stallion sperm do undergo spontaneous AE in viable sperm at the same rate as fresh semen after being incubated in a Lac-MW medium 6 . Because all the subfertile TB stallions that have been identified by our group as carrying the IAE susceptibility genotype were either located far from our laboratory, had been already castrated, or their sperm were cryopreserved several years previously, in the current study we used frozen/thawed sperm from these stallions to perform the acrosome function and subsequent DIA-MS analyses. When these ejaculates were obtained, immediately after semen collection and initial sperm analysis, the raw semen was diluted 1:1 (v/v) with INRA-96® extender (IMV Technologies, L’Aigle, France) and subjected to cushioned centrifugation at 1000 x g for 20 min, as described previously 32 . After centrifugation, the supernatant was removed, and the sperm pellet was resuspended with the EZ-Freezin CryoMax LE® semen freezing extender (Animal Reproduction Systems, Chino, CA) at a final sperm concentration of 200 x 10 6 sperm/mL. Sperm diluted with the freezing extender was loaded into 0.5-mL plastic straws, sealed ultrasonically, and frozen in a controlled rate freezer (CBS 2100; Custom Biogenic Systems, Bruce Township, MI, USA) using the following cooling curve: − 2.0°C/min from 25 to 20°C; − 0.1°C/min from 20 to 5°C; hold for 5 min; − 60°C/min from 4°C to − 140°C 33 . The straws were plunged directly into liquid nitrogen and stored in a liquid nitrogen tank. Frozen straws from each stallion were thawed for 30 sec in a water bath set at 37°C and the thawed semen was processed through density gradient centrifugation using 40% Redigrad® (Global Life Science Solutions, Marlborough, MA 34 ) to remove seminal plasma, debris, and semen extender. After centrifugation, the sperm pellet was diluted to 30 x 10 6 sperm/mL in Lac-MW medium 6 , 27 and incubated for up to 6 hours at 38.2°C in 5% CO 2 . Sperm aliquots were analyzed after 0, 2, 4, and 6 h of incubation (T0h, T2h, T4h, and T6h, respectively) in Lac-MW medium for viability/acrosomal exocytosis (AE-Viable), as previously reported 6 . At each time point (T0h, T2h, T4h, and T6h), a 30 x 10 6 sperm aliquot was also flash-frozen in dry ice for further proteomic analysis using DIA-MS. Analysis of sperm viability/acrosomal exocytosis (Fixable Live/Dead Red stain + FITC-PSA) The intactness of both the plasma membranes (viability) and the acrosome membranes (AE) was evaluated simultaneously, as previously described 35 , with some modifications. An aliquot (50 µL) of frozen/thawed semen diluted (30 x 10 6 sperm/mL) in Lac-MW was added to 1 mL of Lac-MW medium. This dilution resulted in a final sperm concentration of approximately 1.5 x 10 6 sperm/mL. One µL of Fixable Live/Dead Red stain (Excitation: 488 nm, Emission: 617 nm; final concentration: 50 µg/mL) was added to the sperm sample and incubated at 38.2°C in an air atmosphere for 20 min. Then, 140 µL of methanol-free paraformaldehyde (paraformaldehyde final concentration: 1.88% v/v) was added, and the sperm sample was stored at 5°C in the dark for 30 min. Subsequently, the samples were centrifuged (400 x g x 5 min) using BSA in DPBS (2 mg/mL), permeabilized with Triton-X100 (1% v: v), centrifuged again with BSA in DPBS, diluted in 133 µL Accumax to avoid sperm clumping, and incubated with 10 µL Pisum sativum agglutinin (PSA)-FITC conjugate (excitation, 488 nm; emission, 517 nm; final concentration, 0.0375 mg/mL) for 20 min at room temperature in the dark. The Fixable Live/Dead Red stain binds to free amines both in the intracellular space and the surface of cells with a disrupted plasma membrane (“non-viable” cells) 36 . In the case of the Fixable Live/Dead Red stain, non-viable sperm will allow the internalization of this dye and emit red fluorescence 35 . PSA binds to the glycoconjugates of the acrosomal matrix, particularly to the α-D-glucosyl and the α-D-mannosyl residues at the inner acrosomal membrane 37 . When the acrosomal membrane is disrupted, either as a consequence of acrosome damage or during AE, FITC-PSA will bind to such residues and emit a green, fluorescent signal. In both fixed and permeabilized sperm, four subpopulations are identified using Fixable Live/Dead Red stain and FITC-PSA: 1) sperm with both intact plasma and acrosomal membranes [FITC-PSA (+)/Fixable Live/Dead Red (–)]; 2) sperm with intact plasma membrane and disrupted acrosomal membrane [FITC-PSA (–)/Fixable Live/Dead Red (–)]; 3) sperm with damaged plasma membrane and intact acrosomal membrane [FITC-PSA (+)/Fixable Live/Dead Red (+)]; 4) sperm with both damaged plasma and acrosomal membranes [FITC-PSA (–)/Fixable Live/Dead Red (+)]. Following incubation with FITC-PSA, the samples were diluted in 150 µL Accumax and processed immediately using a flow cytometer (FACScan, Beckton Dickinson, Mountain View, CA) equipped with a 488-nm argon laser at 20 mW and three fluorescent detectors (FL1, bandpass 530/30nm; FL2, bandpass 585/42nm; and FL3, long pass 670 nm). The voltage settings on the flow cytometer were: FSC-H, 553; SSC, 240; FL1, 741, and FL2, 821. The compensation was set on FL2 as 98% of FL1. The FITC-PSA signal was acquired using the FL1 filter, while the Fixable Live/Dead Red stain signal was acquired using the FL2 filter. The flow rate was 200–400 sperm/s and a minimum of 5000 sperm were analyzed per sample. To identify doublets and clumps that could affect the analysis and interpretation of sperm events, a manual gating strategy was applied whereby the FSC and SSC were plotted. This methodology has been validated in previous reports from our laboratory 6 , 27 . Flow cytometry data were analyzed by WinList™ software (Verity Software House, Topsham, ME, USA). The percentage of AE in viable sperm (AE/Viable) was considered as the experimental endpoint. Sperm preparation for protein extraction Aliquots of frozen/thawed sperm (30 x 10 6 sperm) incubated at either 0h, 2h, 4h, or 6h in Lac-MW medium were flash/frozen in dry ice until further processing, and then thawed for 1 min at 37°C in a water bath. Immediately after thawing, 10 µL of EDTA-free protease inhibitor cocktail (Pierce™ Protease Inhibitor Tablets, Thermo Fisher Scientific, Waltham, MA) was added per each sperm aliquot. The sperm were washed two times in calcium-free PBS at 400 x g for 5 min, pelleted, maintained frozen at − 80°C, and sent to the Institutional Mass Spectrometry Core Laboratory at the University of Texas Health Science Center at San Antonio (San Antonio, Texas) for further analysis. Protein identification and relative quantification by data-independent acquisition mass spectrometry (DIA-MS) Protein aliquots corresponding to 70 µg protein, as quantified using the EZQ™ Protein Quantitation Kit (Thermo Scientific, Waltman, MA), were reduced with tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl; Pierce™, Thermo Fisher Scientific), alkylated in the dark with iodoacetamide and applied to S-Traps spin columns (Protifi™, Farmingdale, NY) for tryptic digestion (sequencing grade; Promega) in 50 mM triethylammonium bicarbonate (TEAB; Thermo Fisher Scientific). Peptides were eluted from the S-Traps spin columns with 0.2% formic acid in 50% aqueous acetonitrile and quantified using the Pierce™ Quantitative Fluorometric Peptide Assay (Thermo Fisher Scientific). Data-independent acquisition mass spectrometry (DIA-MS) was conducted on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). On-line HPLC separation was accomplished with an RSLC NANO HPLC system (Thermo Fisher Scientific/Dionex) and a PicoFrit™ nanospray column (75 µm i.d.; New Objective, Littleton, MA) packed to 15 cm with C18 adsorbent column (218MS 5 µm, 300 Å; Vydac®, W.R. Grace & Co., Columbia, MA); mobile phase A, 0.5% acetic acid (Hac)/0.005% trifluoroacetic acid (TFA; Pierce™, Thermo Fisher Scientific) in water; mobile phase B, 90% acetonitrile/0.5% Hac/0.005% TFA/9.5% water; gradient 3 to 42% B in 120 min; flow rate: 0.4 µL/min. A pool was made of all samples, and 2 µg aliquots of the digests were analyzed using gas-phase fractionation and 4- m/z windows [three mass ranges (395-605mz, 595-805mz, 795-1005mz), staggered; 30k resolution for precursor and product ion scans, all in the orbitrap] to create a DIA chromatogram library 38 by searching against a Prosit-generated predicted spectral library 39 based on the UniProt Equus caballus protein sequence database (44,488 sequences, downloaded on 12-11-2020): peptide mass tolerance, 10.0 ppm; fragment mass tolerance, 10.0 ppm; fixed modification, carbamidomethylation (C); enzyme, trypsin with a maximum of one missed cleavage; peptide charge state, + 2 – +3; peptide length, 6–30; protein FDR, 1%; minimum of two identified peptides; peptide quantification, Encyclopedia (0.8.1) based on the five highest quality fragment ions. Experimental samples were blocked by replicate and randomized within each replicate for sample preparation and analysis, employing 2 µg of peptides and the 2-h HPLC gradient described above. MS data for experimental samples were acquired in the orbitrap using 8- m/z windows (390-1010mz, staggered; 30k resolution for precursor and product ion scans) and searched against the chromatogram library using the same parameters as described above for generation of the chromatogram library. Scaffold DIA (v3.2.1; Proteome Software) was used for all DIA-MS data processing. The data files were converted to mzML format using ProteoWizard (3.0.19254) 40 . Deconvolution of the staggered windows was performed. Peptides identified in each sample were filtered by Percolator (3.01.nightly-13-655e4c7-dirty) 41 to achieve a maximum false discovery rate (FDR) of 0.01. Individual search results were combined, and peptide identifications were assigned posterior error probabilities and filtered to an FDR threshold of 0.01 by Percolator. Peptide quantification was performed by Encyclopedia (version 1.12.31) 38 . For each peptide, the five highest-quality fragment ions were selected for quantification. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis were grouped to satisfy the principles of parsimony. Quartile normalization was applied on the log10 peptide intensities across replicates. Bioinformatic analyses Scaffold DIA was used to assess differences in the results for samples from fertile and subfertile stallions (A/A-A/A) across the selected time periods (T0h, T2h, T4h, or T6h). A two-way ANOVA design, available in the Scaffold DIA software, was used in which the primary category of analysis was the stallion phenotype (fertile vs. subfertile), while the second category of analysis was the time point (T0h vs. T2h vs. T4h vs. T6h). Significance was assessed using Benjamini-Hochberg multiple testing correction 42 , resulting in a cutoff of P < 0.0069. Proteins that exhibited log 2 (fold change) ≤ − 0.585 or ≥ 0.585 (i.e., fold change in protein abundance between fertile and subfertile stallions 1.5x up or down) were used to filter the results for further analysis. The proteins identified as differentially abundant between stallion groups and among time periods were queried for gene ontology (GO) terms using g:Profiler 43 , according to cellular component (CC), biological process (BP), and molecular function (MF). Overrepresentation of the proteins of differential abundance was queried using Equus caballus orthologs and a g:SCS threshold value of 0.05. To analyze the overrepresentation of biological pathways, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was also used in g:Profiler. In addition, pathway enrichment analyses were conducted utilizing both the g:Profiler and Reactome servers using Homo sapiens orthologs, given the increased depth of the human proteome in terms of annotation. To identify functionally grouped GO terms, a network analysis of the differentially abundant proteins was performed using the Cytoscape (version 3.9.1) plugin ClueGo (version 2.5.8) 44 . Detection of acrosome proteins of differential abundance between fertile and subfertile TB stallions by immunofluorescence Indirect immunofluorescence of the protein arylsulfatase F (ARSF) was performed, as previously described 6 , with some modifications. Briefly, frozen/thawed semen from both fertile and subfertile TB stallions (n = 3 stallions per group) were processed by density gradient centrifugation using 40% Redigrad and diluted to 30 x 10 6 sperm in Lac-MW. Next, the sperm were centrifuged (600 x g for 5 min) three times in DPBS, applied to poly-L-lysine coated slides for 2h at room temperature, and exposed to 4% paraformaldehyde in DPBS for 20 min at 4°C. As ARSF is a protein of acrosome origin, we did not expose the sperm to any permeabilizing agent (i.e., triton X-100) to avoid any loss of proteins located at the outer acrosomal membrane or acrosomal matrix. Following fixation, the slides were washed again three times using DPBS, blocked with 10% normal goat serum for 1h at room temperature, and washed again three times using DPBS. A rabbit anti-ARSF, diluted 1:100 in 10% normal goat serum, was added to the slides and incubated overnight at 4°C. A negative control was produced in which the slides were not exposed to the primary antibody (i.e., anti-ARSF). Following overnight incubation, the sets of slides were washed three times with DPBS and stained for 1h at room temperature with a goat anti-rabbit IgG Alexa-555-conjugated secondary antibody (excitation: 555 nm; emission: 572 nm) at a 1:100 dilution in 10% normal goat serum. A final washing step was performed using DPBS; then, 3.5 µL of the SlowFade Mountant with DAPI was added, to both prevent fluorescence bleaching and to counterstain the sperm nuclei. Then, a coverslip was carefully applied to the slide, and the slides were evaluated using an Olympus BX-60 fluorescence microscope at a 1,563x magnification. A total of 100 sperm were analyzed per ejaculate, and the localization of ARSF within each spermatozoon was recorded and compared between fertile and subfertile TB stallions. Heterologous zona pellucida-binding assay Due to the difficulties in establishing a repeatable method for conventional in vitro fertilization of equine oocytes using frozen/thawed stallion sperm, we performed a heterologous zona pellucida-binding assay to determine the ability of stallion sperm incubated in Lac-MW to undergo acrosomal exocytosis and bind to the zona pellucida (ZP) 45 , 46 . Porcine oocytes were used to determine the ability of frozen/thawed sperm from subfertile and fertile TB stallions to bind to the ZP. Ovaries were procured from a slaughterhouse 1 hour away from our laboratory, and the specimens were maintained at 37°C in an insulated container for transport. Upon arrival at our laboratory, the ovaries were washed with warm PBS and dried with cotton gauze. All visible follicles were sliced using a scalpel blade, and the follicular cavity was flushed using Vigro® Complete Flush Medium (Vetoquinol, Pullman, WA, USA) over a sterile Petri dish. Groups of 50 cumulus -oocyte complexes (COCs) were identified using a stereoscope and transferred into a Petri dish containing 150-µL droplets of maturation medium [M199 with Earle’s salts, supplemented with 5 mU/mL FSH (Sioux Chemicals, Sioux Center, IA, USA), 10 IU/mL hCG (Chorulon, Merck Animal Health, Rahway, NJ, USA), 10% FBS, and 25 µg/mL gentamicin] under light mineral oil, at 38.2°C in a humidified atmosphere of 5% CO 2 for 22h. After this period, the COCs were placed into a fresh droplet of maturation medium and incubated for an additional 22h, under the same conditions. Following the maturation period, COCs were denuded of cumulus by repeated pipetting in M199 medium with Hank’s salts containing 10% FBS and 0.5 mg/mL hyaluronidase. Denuded oocytes were considered as matured if a polar body was extruded and present at the periphery of the oolemma. To perform the heterologous zona-binding assay, frozen/thawed semen from three fertile and three subfertile TB stallions was thawed, processed by density gradient centrifugation using 40% Redigrad, and resuspended to 30 x 10 6 sperm/mL in Lac-MW. A total of 20 mature oocytes were placed in 50-µL droplets of Lac-MW, inseminated with 5 x 10 4 sperm from each stallion, and incubated for 4 h at 38.2°C in a humidified atmosphere of 6% CO 2 , 5% O 2 , and 89% N 2 . Following the co-incubation period, to remove any loosely bound sperm, the oocytes were washed by repeated pipetting in M199 with Hank’s salts, transferred to a 50-µL droplet of 2% paraformaldehyde in DPBS for 10 min at room temperature and placed into a 50-µL droplet of 2% BSA + 10 µM Hoechst 33342 in DPBS for 10 min at room temperature. The oocytes were loaded onto polylysine-coated slides, carefully covered with a coverslip, and examined at 400x using a BX-60 Olympus fluorescence microscope. For each oocyte, the total number of sperm bound to the ZP was quantified and compared between fertile and subfertile TB stallions. Statistical analysis Statistical analyses were performed using commercial software (SAS Version 9.4; SAS Institute, Inc., Corp., Cary, NC, USA). The Shapiro-Wilk test (PROC UNIVARIATE) was conducted to test data distribution. Within periods (T0h, T2h, T4h, or T6h), Student t -tests (PROC TTEST) or the Wilcoxon Ranked Sum test (PROC NPAR1WAY) were used to compare the percent of AE in viable sperm (AE-Viable), the immunolocalization of ARSF in sperm from fertile and subfertile stallions, and the number of sperm bound to the ZP between fertile and subfertile stallions. Group differences were set at P < 0.05. Declarations Acknowledgments Funding for this project was provided by the Legends Premier Stallion Season Auction, Texas A&M University. The authors would like to thank Ms. Crystal Howard (Rood & Riddle Equine Hospital, Lexington, KY) for her help while procuring samples from some fertile and subfertile TB stallions. Competing interests The authors declare no competing interests. Author contributions C. H.-A: study and experimental design, data collection and interpretation, Formal analysis, writing of the initial draft. L. R.-A.: study and experimental design, data collection and interpretation, text review and editing. S.T.W. study and experimental design, supervision of mass spectrometry analysis and data processing, data interpretation, text review, and editing. C.F.S.: data collection, text review, and editing. B.W.D: data interpretation, text review, and editing. T.R. : data interpretation, text review, and editing. D.D.V : study and experimental design, data interpretation, text review and editing, funding acquisition. 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Supplementary Files SupplFig1.tif SupplFig2.tif SupplFigLegends.docx Supplementarytables.docx Cite Share Download PDF Status: Published Journal Publication published 30 May, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 17 May, 2024 Reviews received at journal 08 May, 2024 Reviews received at journal 20 Apr, 2024 Reviewers agreed at journal 15 Apr, 2024 Reviewers agreed at journal 15 Apr, 2024 Reviewers invited by journal 15 Apr, 2024 Editor assigned by journal 15 Apr, 2024 Editor invited by journal 09 Apr, 2024 Submission checks completed at journal 08 Apr, 2024 First submitted to journal 28 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4185383","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":285210156,"identity":"6f13e13b-0d7c-4062-b78b-645cce47b994","order_by":0,"name":"Camilo Hernández-Avilés","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYDACCQjF2MDAfABJmI0oLWwJSHzitPAYEKeFf3bzsc8FNXdk+/nPfPzMm8NQxz/tjAHDh7LDuC25cyx59oxjz4xnNpzdLM27jUFC4naOAeOMc7i1GEjkGDPzsB1O3HCwdxvjTKAWBqAWZt42fFryPzPz/DucuP8wzzOwFnmQlr94teQwg8xM3MDGw8bwEajFAKSFEY8WiRtpxsy8fYeNZ5xhM5b4uE1CcuPttIKDPefScWrhn5H8mJnn22HZ/v7DDz8kbrPhl7udvPHBjzJrnFowbAWTB4hWPwpGwSgYBaMAKwAALcNTNKi26N0AAAAASUVORK5CYII=","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":true,"prefix":"","firstName":"Camilo","middleName":"","lastName":"Hernández-Avilés","suffix":""},{"id":285210159,"identity":"33b030a9-32b2-4b36-a122-40aa56249a22","order_by":1,"name":"Luisa Ramírez-Agámez","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Luisa","middleName":"","lastName":"Ramírez-Agámez","suffix":""},{"id":285210162,"identity":"9b392e57-5b61-45a1-82db-d55494952224","order_by":2,"name":"Susan T. 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Love","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Charles","middleName":"C.","lastName":"Love","suffix":""}],"badges":[],"createdAt":"2024-03-29 03:29:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4185383/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4185383/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-63410-3","type":"published","date":"2024-05-30T14:02:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53912572,"identity":"57a880e1-9020-478c-a830-ac899c6d785c","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":170235,"visible":true,"origin":"","legend":"\u003cp\u003ea. The mean percent of AE in viable sperm of frozen/thawed semen from three fertile Thoroughbred stallions (black dots), and three subfertile Thoroughbred stallions (red squares) that carry the susceptibility genotype for IAE. Frozen/thawed semen was processed using the Lac-MW model for induction of AE. Data are presented as mean (black bar) ± SEM. b. Representative scatterplots of the flow cytometric evaluation of viability/acrosomal exocytosis at 6 hours incubation in Lac-MW are presented. On the X-axis of the scattergrams, the fluorescent signal of FITC-PSA is represented, while on the Y-axis the fluorescent signal of the Fixable Live/Dead Red Stain is represented. Within each period (T0h, T2h, T4h, T6h), asterisk (*) indicates significant differences between stallion groups (fertile vs. subfertile) (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/7e935dcf0800049548c26fff.png"},{"id":53912565,"identity":"ee81a945-e715-4f9c-bad7-af3312a9fff1","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":228351,"visible":true,"origin":"","legend":"\u003cp\u003ea. Volcano plot illustrating the differences in relative protein abundance in sperm from fertile and subfertile Thoroughbred stallions. The \u003cem\u003ex\u003c/em\u003e-axis denotes the log\u003csub\u003e2\u003c/sub\u003e(fold change) between stallion groups (fertile vs. subfertile), while the \u003cem\u003ey\u003c/em\u003e-axis represents the –log\u003csub\u003e10\u003c/sub\u003e \u003cem\u003eP\u003c/em\u003e-value of the change in protein abundance between stallion groups (fertile vs. subfertile). b. A principal component analysis (PCA) of the proteins of differential abundance (140 proteins; two-way ANOVA, q-value 0.05; log\u003csub\u003e2\u003c/sub\u003efold \u0026lt; –0.586 \u0026gt; 0.586) between stallion groups (fertile vs. subfertile), and among time periods (T0h, T2h, T4h, T6h), is presented, whereby a clear clustering of the stallion groups can be appreciated.\u003c/p\u003e\n\u003cp\u003eGreen diamonds – Significant: two-way ANOVA with Benjamini-Hochberg multiple testing correction (FDR q-value 0.05).\u003c/p\u003e\n\u003cp\u003eYellow diamonds – Significant (P \u0026lt; 0.05) without multiple testing correction.\u003c/p\u003e\n\u003cp\u003eBlack diamonds – Not significant\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/85239c81d5cc613f4168ccdb.png"},{"id":53913079,"identity":"af2c97a3-f354-41a1-bc69-c610f5b854d4","added_by":"auto","created_at":"2024-04-02 07:04:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":78302,"visible":true,"origin":"","legend":"\u003cp\u003eg:Profiler (g:GOST) multi-query Manhattan plot demonstrating the enrichment analysis of differentially abundant proteins (lower [a.] and higher [b.] relative abundance) in sperm from subfertile TB stallions, when compared to sperm from fertile TB stallions. The proteins were queried using \u003cem\u003eEquus caballus\u003c/em\u003eorthologs. The adjusted \u003cem\u003ep\u003c/em\u003e-values for statistical overrepresentation are depicted on the y-axis of each plot. GO: Gene ontology; CC: Cellular component; BP: Biological process; MF: Molecular function; KEGG: Kyoto Encyclopedia of Genes and Genomes.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/159b717d819c07223e24e674.png"},{"id":53912554,"identity":"9c942b70-0771-44ef-a834-a7859d386180","added_by":"auto","created_at":"2024-04-02 06:56:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":496759,"visible":true,"origin":"","legend":"\u003cp\u003eClueGo network analysis of proteins of lower relative abundance in sperm from subfertile TB stallions. a. GO: BP (ellipse), GO: CC (hexagon), and Reactome pathways (rectangle). Functionally grouped networks with terms are indicated as nodes based on their kappa score level (\u0026gt; 0.4), where only the label of the most significant term per group is shown. b. Overview pie chart with functional groups including specific terms for overrepresented proteins. c. GO/pathway terms specific for overrepresented proteins. The bars represent the number of proteins associated with each term.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/9d463979fe33023bcca94aad.png"},{"id":53912557,"identity":"13e1dbe6-5950-4dd9-8b08-09264a21fec6","added_by":"auto","created_at":"2024-04-02 06:56:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":455840,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplots representing the median relative abundance of the proteins arachidonate-3-lipoxygenase (ALOX3), arachidonate-12-lipoxygenase (ALOX12), arylsulfatase F (ARSF),\u0026nbsp; extracellular matrix protein 1 (ECM1), ergosterol biosynthesis 28 homolog (ERG28), Na\u003csup\u003e+\u003c/sup\u003e-dependent phosphate cotransporter 2B (SLC34A2), and zona pellucida-binding protein 2 (ZPBP2) in sperm from fertile (left) and subfertile (right) TB stallions, in function of incubation time after exposure to Lac-MW medium.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/9b54b406bad74a50acf4b84c.png"},{"id":53912563,"identity":"7b782f7e-d82d-4d4c-8bf6-00bb78e63135","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":197628,"visible":true,"origin":"","legend":"\u003cp\u003ea. The mean (± SD) percentage of frozen/thawed sperm from fertile TB stallions (black dots) and subfertile TB stallions (red squares), that displayed two different immunofluorescence patterns of arylsulfatase F (ARSF) localization within the sperm. Pattern I: Immunofluorescence signal at the acrosome, midpiece, and principal piece; Pattern II: Immunofluorescence signal at the midpiece and principal piece. A rabbit anti-ARSF polyclonal antibody was conjugated with a goat anti-rabbit IgG Alexa-555-secondary antibody, while the sperm nucleus was counterstained with DAPI. b. Representative images of the immunolocalization patterns of arylsulfatase F (ARSF) in frozen/thawed stallion sperm are presented. Original magnification: 1,563X. **within immunofluorescence patterns, indicates significant differences between stallion groups (fertile vs. subfertile).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/32b41231f11086915d91968a.png"},{"id":53912576,"identity":"f79b4e01-b301-4f8c-ac15-e7e442ab8d59","added_by":"auto","created_at":"2024-04-02 06:56:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":635080,"visible":true,"origin":"","legend":"\u003cp\u003ea. The mean number of frozen/thawed sperm from fertile TB stallions (dark grey bar), or subfertile TB stallions (light grey bar) that bound to porcine zona pellucida following incubation in Lac-MW for 4 hours. b. Representative microphotographs obtained by phase-contrast and fluorescence microscopy of an oocyte inseminated with sperm from a fertile stallion (left) and a subfertile stallion (right) are presented (original magnification: 400X). * Indicates significant differences between groups (fertile vs. subfertile) in the mean number of sperm bound to the zona pellucida.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/fa23a42197455a7848530dbb.png"},{"id":57570065,"identity":"f314fb4d-59eb-469f-8270-91f6589cd0d7","added_by":"auto","created_at":"2024-06-02 14:02:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3608508,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/8a14cc26-cc04-4ff2-a28d-4b1c844e4c11.pdf"},{"id":53912570,"identity":"6e1576de-5a6c-42a2-82af-7ba522e0b018","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":163036,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/3ea999b874380bfd71392c8e.tif"},{"id":53912586,"identity":"90b93d9a-2992-4c16-9d3b-60b9ab02b287","added_by":"auto","created_at":"2024-04-02 06:56:14","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":239812,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFig2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/404cf806e7f8014540ccf26b.tif"},{"id":53912574,"identity":"362f3f4e-ad5c-43f7-b34d-5c1374bb6ce4","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":14778,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFigLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/74851dac5b127ed74d87746d.docx"},{"id":53912559,"identity":"c3800632-523e-4fcc-bddf-7e7817105c93","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":19544,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4185383/v1/04e6b05e51be36040bfc2b8f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe acrosomal exocytosis (AE) process involves a series of biochemical changes in the sperm, mediated mostly by an increase in intracellular pH and calcium levels, and plasma membrane destabilization due to cholesterol depletion\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Together, all these cellular changes will result in the fusion of the outer acrosomal membrane with the sperm plasma membrane, leading to the release of multiple enzymes at the vicinity of the cumulus-oocyte complex (COC) and facilitating the binding of sperm to the zona pellucida\u003csup\u003e1\u0026ndash; \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Stallion subfertility due to impaired acrosomal exocytosis (IAE) is a condition identified thus far only in stallions of the Thoroughbred (TB) registry. Sperm from these stallions are characterized as having a lower acrosomal response after \u003cem\u003ein vitro\u003c/em\u003e exposure to either non-physiologic (i.e., calcium ionophore A23187), or physiologic (i.e., lactate-induced) conditions known to result in acrosomal exocytosis (AE)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These stallions have low fertility (\u0026lt;\u0026thinsp;30% per-cycle pregnancy rates), even though typical features of sperm quality and breeding management are acceptable. An association between the IAE phenotype and the presence of a double homozygous A/A-A/A genotype for SNPs chr13:11,353,372G\u0026thinsp;\u0026gt;\u0026thinsp;A (rs397316122) and chr13:11,353,436A\u0026thinsp;\u0026gt;\u0026thinsp;C (rs69101140) in \u003cem\u003eFKBP6\u003c/em\u003e exon 5 (EquCab3) has been identified in two separate studies\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The frequency of subfertile TB stallions that carry the \u003cem\u003eFKBP6\u003c/em\u003e A/A-A/A genotype approaches 1\u0026ndash;3% of the TB breeding stallion population (four out of 150 stallions evaluated in Central Kentucky, USA, for the presence of the susceptibility genotype\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e; and seven out of 1,128 stallions of various breeds evaluated during 17 years at a reference laboratory\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e). In mice, the FKBP6 protein has been associated with the normal formation of the synaptonemal complex during spermatogenesis; thus, double knock-out male mice are sterile due to azoospermia\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Similar findings have also been identified in men with idiopathic azoospermia\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, indicating that FKBP6 is related to the normal progression of meiosis in spermatocytes. These findings contrast the clinical characteristics of TB stallions with the A/A-A/A combined genotype in \u003cem\u003eFKBP6\u003c/em\u003e exon5 (i.e., normal sperm quality and testicular size), making a potential link between the susceptibility genotype for IAE in these stallions and the function of FKBP6 difficult to demonstrate.\u003c/p\u003e \u003cp\u003eProteomic technologies have been used to identify proteins of importance for sperm physiologic processes\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and for the identification of candidate biological markers that could either be used to select males with higher fertility potential\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e or identify potential proteins that explain causes of reduced fertility\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Most of these studies included proteomic analysis using mass spectrometry-based technologies, mainly involving the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this method, the mass-to-charge ratio (\u003cem\u003em/z\u003c/em\u003e) of ionized molecules (in this case peptides produced by proteolytic digestion during sample preparation) are detected and the ions then fragmented, generating precursor (MS1) and product-ion (MS2) mass spectra\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These MS2 mass spectra are then queried against published protein sequence databases for the identification (and relative quantification) of the proteins in the sample\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The mass spectra produced in this way are obtained using an approach known as data-dependent acquisition (DDA), in which peptides that are detected in a precursor scan are sequentially selected based on relative abundance and fragmented to generate sequence-informative tandem mass spectra. When using DDA-MS for the analysis of complex samples, there is inevitably under-sampling (i.e., lack of detection and fragmentation of many lower-abundance peptides) even with state-of-the-art instruments that have extremely fast scan rates. This can hamper the detection of low-abundance proteins which may have biological relevance. Recently, an approach known as data-independent acquisitionmass spectrometry (DIA-MS) has gained popularity. In DIA-MS, ionized peptides in small \"windows\" of m/z ranges are sequentially fragmented rather than individually selected precursors, thereby permitting much more comprehensive detection of the complement of peptides in a digest. In this way, DIA-MS provides identification and relative quantification of a much larger number of proteins with greater accuracy and precision than DDA-MS.\u003c/p\u003e \u003cp\u003eVarious studies have analyzed the proteome of sperm following incubation under capacitating conditions or after attempted stimulation of AE\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. To the best of our knowledge, such studies have not yet been conducted using stallion sperm. We recently reported a method to consistently induce AE in sperm from stallions with different \u003cem\u003ein vivo\u003c/em\u003e fertility levels, including TB stallions that were confirmed to have IAE both by genotyping and acrosomal function testing\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Given the potential advantages of DIA-MS to identify candidate proteins that otherwise would not be discovered by other methods, we utilized this approach in combination with our method to induce AE in stallion sperm to identify potential candidate proteins that could explain the etiology of IAE in TB stallions.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eSperm quality parameters in fresh and frozen/thawed semen from fertile and subfertile TB stallions (A/A-A/A)\u003c/h2\u003e\n\u003cp\u003eThe initial sperm quality parameters in fresh semen, fertility parameters (mare book, PC-PR, and SPR), and the \u003cem\u003eFKBP6\u003c/em\u003e genotype results from both stallion groups are shown in Supplementary Table\u0026nbsp;1. There were no differences observed in sperm quality between stallion groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Frozen/thawed semen from both fertile and subfertile TB stallions was exposed to the Lac-MW model to determine their acrosomal response. At T0h and T2h, mean AE/Viable was similar for fertile and subfertile stallions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while at T4h and T6h, mean AE/Viable was higher for fertile than for subfertile stallions (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Representative scattergrams of the viability/acrosomal exocytosis assay conducted in frozen semen from a fertile stallion and a subfertile A/A-A/A stallion are also presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eThe proteome of frozen/thawed sperm from fertile and subfertile TB stallions\u003c/h2\u003e\n\u003cp\u003eThere were 2202 proteins (FDR 1.0%) identified from 18723 peptides by DIA-MS in sperm from both fertile and subfertile TB stallions. By using the stallion phenotype (fertile vs. subfertile) as the main effect to test by two-way ANOVA, a total of 298 proteins reached significance after FWER correction (FDR q-value 0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). After applying stringent criteria (i.e., log2fold \u0026le; \u0026minus;\u0026thinsp;0.585 or \u0026ge;\u0026thinsp;0.585), a total of 140 proteins were identified, which identified 61% of the variance in protein abundance between stallion groups, resulting in two distinctive data point clusters (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Of these, 83 were found to be of lower relative abundance in sperm from the subfertile TB stallions [log\u003csub\u003e2\u003c/sub\u003e(fold change) \u0026lt; \u0026minus;\u0026thinsp;0.585], while 57 proteins were found to have a higher relative abundance [log\u003csub\u003e2\u003c/sub\u003e(fold change)\u0026thinsp;\u0026gt;\u0026thinsp;0.585] in sperm from these stallions when compared to sperm from fertile TB stallions. The 140 differentially abundant proteins were used for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene ontology analysis of differential abundance sperm proteins in subfertile TB stallions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn initial analysis using g:Profiler and \u003cem\u003eEquus caballus\u003c/em\u003e orthologs was conducted in each of the two lists of differentially abundant proteins (lower vs. higher abundance). Manhattan plots corresponding to the GO CC, BP, MF, and KEGG analyses are presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, while GO terms for each of these categories in proteins of lower and higher abundance in sperm from subfertile stallions are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. A subsequent GO analysis was conducted in g:Profiler by using \u003cem\u003eHomo sapiens\u003c/em\u003e rather than \u003cem\u003eEquus caballus\u003c/em\u003e orthologs, to increase the coverage of GO terms. Manhattan plots corresponding to this analysis are presented in Supplementary Fig.\u0026nbsp;1, while GO terms for each of these additional protein categories are presented in Supplementary Table\u0026nbsp;2.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eList of gene ontology (GO) terms related to proteins with lower and higher relative abundance in sperm from subfertile TB stallions when compared to sperm from fertile TB stallions. The proteins were queried using \u003cem\u003eEquus caballus\u003c/em\u003e orthologs. The ID numbers correspond to the IDs represented in the Manhattan plots in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. GO: Gene ontology; CC: Cellular component; BP: Biological process; MF: Molecular function; KEGG: Kyoto Encyclopedia of Genes and Genomes.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eProteins of lower relative abundance in sperm from subfertile TB stallions\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eID\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSource\u003c/p\u003e\n\u003cp\u003e(GO term)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTerm ID\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"Underline\"\u003eTerm Name\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value (adjusted)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0005783\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEndoplasmic reticulum\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.639 x 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0012505\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEndomembrane system\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.085 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0005789\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEndoplasmic reticulum membrane\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.044 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0031090\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOrganelle membrane\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.072 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0005737\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCytoplasm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.819 x 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0031984\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOrganelle subcompartment\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.424 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0031301\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIntegral component of organelle membrane\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.086 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0044281\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSmall molecule metabolic process\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.674 x 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0044283\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSmall molecule biosynthetic process\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.660 x 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0006629\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLipid metabolic process\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.214 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0016126\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSterol biosynthetic process\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.675 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0044255\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCellular lipid metabolic process\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.869 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0006694\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSteroid biosynthetic process\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.374 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKEGG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKEGG: 00071\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFatty acid degradation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.743 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKEGG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKEGG: 01100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMetabolic pathways\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.016 x 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKEGG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKEGG: 00100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSteroid biosynthesis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.162 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKEGG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKEGG: 01212\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFatty acid metabolism\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.741 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0016491\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOxidoreductase activity\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.214 x 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0003824\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCatalytic activity\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.581 x 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0016787\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHydrolase activity\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.561 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eProteins of higher relative abundance in sperm from subfertile TB stallions\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0005940\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSeptin ring\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.486 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0035686\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSperm fibrous sheath\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.486 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0031105\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSeptin complex\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.551 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGO: 0036126\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSperm flagellum\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.739 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional network analysis of differentially abundant proteins in sperm from subfertile TB stallions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ClueGo analysis tool was used in conjunction with \u003cem\u003eHomo sapiens\u003c/em\u003e orthologs for GO and KEGG terms to classify the differentially abundant proteins in sperm from subfertile TB stallions in a functional network. A diagram of the functional network for the proteins of lower abundance in sperm from subfertile stallions is presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The results indicated that these proteins were mostly involved in fatty acid metabolism, fatty acid derivative metabolism, sterol biosynthetic process, cellular aldehyde metabolic process, and endoplasmic reticulum protein-containing complex. Conversely, the network for the proteins of higher abundance in sperm from subfertile stallions was mostly associated with vesicle docking and sperm flagellum (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA group of proteins associated with lipid metabolism, including two enzymes of acrosomal origin, display differential abundance between stallion groups and within time periods.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBioinformatic analyses suggested that most of the proteins of differential abundance, and particularly those of lower abundance, in sperm from the subfertile TB stallions were associated with metabolism and metabolism of lipids. Examination of the data in Scaffold DIA was utilized to identify time-point-related changes in the relative abundance of proteins belonging to these categories. These changes were also analyzed at the time periods in which AE was observed in sperm from fertile but not from subfertile TB stallions (i.e., 4 and 6 hours of incubation in Lac-MW medium). Proteins that fulfilled these criteria included: arachidonate lipoxygenase 3 (ALOX3), arachidonate 12-lipoxygenase (ALOX12), arylsulfatase F (ARSF), extracellular matrix protein 1 (ECM1), ergosterol biosynthesis 28 homolog (ERG28), Na\u003csup\u003e+\u003c/sup\u003e-dependent phosphate cotransporter 2B (SLC34A2), and zona pellucida-binding protein 2 (ZPBP2). The median relative abundance levels of these proteins between stallion groups, and within time periods are presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe expression of the acrosome protein arylsulfatase F (ARSF) in sperm from fertile and subfertile TB stallions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne of the proteins of lower abundance in sperm from subfertile TB stallions is an enzyme of acrosome origin and might have relevance during the sperm-oocyte interaction process. As such, we sought to determine if this protein was present in stallion sperm, and whether its expression and/or location were different between sperm of fertile and subfertile TB stallions. By indirect immunofluorescence, we observed that the protein arylsulfatase F (ARSF) was expressed at a higher proportion at the acrosome, mid-, and principal piece in sperm (Pattern I) from fertile TB stallions than in sperm from subfertile TB stallions (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD: 57\u0026thinsp;\u0026plusmn;\u0026thinsp;8% vs. 17\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, respectively; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while at a lower proportion at the midpiece and principal piece (Pattern II) in sperm from fertile TB stallions than in sperm from subfertile TB stallions (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD: 16\u0026thinsp;\u0026plusmn;\u0026thinsp;7% vs. 49\u0026thinsp;\u0026plusmn;\u0026thinsp;3%, respectively; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Representative immunofluorescence images of the location of ARSF in stallion sperm are presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFrozen/thawed sperm from subfertile TB stallions display a lower ability to bind to the zona pellucida.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 120 \u003cem\u003ein vitro\u003c/em\u003e-matured porcine oocytes (20 oocytes per stallion; 60 for fertile TB and 60 for subfertile TB stallions, respectively) were used in this experiment. The mean number of sperm bound to the ZP was higher in fertile TB than in subfertile TB stallions (55\u0026thinsp;\u0026plusmn;\u0026thinsp;8 sperm/ZP vs. 15\u0026thinsp;\u0026plusmn;\u0026thinsp;4 sperm/ZP; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the current study, we identified candidate proteins to further investigate the causes of IAE in TB stallions. For this purpose, we combined the use of a method to induce AE in viable sperm (i.e., incubation in a lactate-only containing medium [Lac-MW]), and DIA-MS to identify and quantify the relative levels of proteins in the sperm of both fertile and subfertile TB stallions. Previous studies have used mass spectrometry-based approaches to investigate the stallion sperm proteome to identify potential proteins associated with higher sperm quality or fertility potential\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, or to determine the effects of storage methods (i.e., cooled storage or freezing/thawing) on the sperm proteome\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To the best of our knowledge, the current study is the first to use this technology to investigate the potential cause(s) of a clinical condition in stallions that is unique due to the phenotype that these individuals present: reduced \u003cem\u003ein vivo\u003c/em\u003e fertility associated with IAE despite having normal-to-excellent conventional sperm quality parameters.\u003c/p\u003e \u003cp\u003eOur results indicate that most of the proteins of differential abundance in sperm from the subfertile TB stallions correspond to processes associated with either cell metabolism or metabolism of lipids, as observed in the gene ontology analyses using both \u003cem\u003eEquus caballus\u003c/em\u003e or \u003cem\u003eHomo sapiens\u003c/em\u003e orthologs. These results are interesting from two perspectives: 1) both sperm capacitation and AE require extensive remodeling of the sperm plasma membrane to prepare the fusion between both plasma and outer acrosomal membranes, facilitating the release of acrosomal contents \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This remodeling process, in turn, requires cholesterol depletion from the plasma membrane, allowing the entry of calcium at the intracellular level and resulting in the activation of second messenger pathways \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Some of the biochemical changes related to sperm capacitation and AE are also related to the metabolism of lipids at the sperm membrane, particularly by the activation of phospholipase A2 (PLA2), which will result in the production of arachidonic acid metabolites and lysophospholipids that enhance the initiation of AE \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Furthermore, both sperm capacitation and AE require considerable quantities of energy in the form of ATP and are also governed by a delicate balance between the consumption of energy and the production of reactive oxygen species (ROS) \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e; thus, it would be expected that proteins associated with metabolic processes would be overrepresented in proteomic analysis of sperm.\u003c/p\u003e \u003cp\u003eWe did not observe an overall interaction between the stallion condition (fertile vs. subfertile) and the sperm AE during incubation periods using the Lac-MW medium. It is possible that there are differences in protein post-translational modifications (PTMs) across the various time points. Since biological PTMs were not evaluated in our DIA-MS analyses, no conclusions can be reached in this regard for this study. Nonetheless, we were still able to identify changes over time in seven proteins (ALOX3, ALOX12, ARSF, ECM1, ERG28, SLC34A2, ZPBP2) that were differentially abundant when sperm underwent AE (i.e., T4h and T6h), which also were of lesser abundance in sperm from the subfertile stallions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These seven proteins were subjected to GO term analysis using the g:Profiler server and were found to correspond to the terms \u003cem\u003emetabolism\u003c/em\u003e and \u003cem\u003emetabolism of lipids\u003c/em\u003e. These findings are consistent with an earlier study from our group indicating that subfertile TB stallions that carry the susceptibility genotype for IAE have a higher cholesterol-to-phospholipid ratio in their sperm membranes and seminal plasma\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. As such, it is possible that the acrosomal dysfunction observed in these individuals could be related to excessive levels of cholesterol in the sperm membrane that influence the ability of sperm to undergo either capacitation or AE\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The changes in the relative abundance of these proteins also coincided with the maximum levels of AE in viable sperm, as determined by flow cytometry. Two of these proteins, ALOX3 and ALOX12 correspond to enzymes associated with the metabolism of fatty acids into leukotrienes, which is related to an increase in the lipid peroxidation levels of biological membranes\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Contrasting results have indicated that lipoxygenases may \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, or may not\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e be related to the occurrence of AE in hamster, bull, or human sperm incubated under capacitating conditions. A recent study showed that inhibition of ALOX15 in human sperm resulted in increases in sperm motility, calcium ionophore A23187-induced AE, and sperm-ZP binding compared to samples that were only treated to stimulate oxidative stress but in which ALOX15 was not inhibited\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Yet, these results should not be interpreted that arachidonate lipoxygenases induce only deleterious effects on sperm, but rather that a balance between the pro-oxidative effects of these lipoxygenases must exist to induce redox changes associated with sperm capacitation and AE, and not only associated with oxidative stress and cell death. Another protein of interest is ERG28. Studies in human sperm indicate that the addition of ergosterol to sperm incubated under capacitating conditions for 24 hours resulted in a reduced rate of spontaneous and progesterone induced AE\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. In that study, the reduced rate of AE caused by the addition of ergosterol to sperm was related to its similar effects on the cholesterol-to-phospholipid ratio on the sperm membranes, as ergosterol is structurally similar to cholesterol and can be incorporated within the sperm plasma membrane similarly as cholesterol\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. This is in contrast with our results showing a higher relative abundance of ERG28 in the sperm from fertile stallions that also had a higher rate of spontaneous AE in viable sperm. RT-PCR analysis indicated that ERG28 is highly expressed in human testicular tissue\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, and the role of this protein in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e indicates that this protein is necessary for the normal progression of the ergosterol biosynthetic pathway\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. However, the actual localization of ERG28 within the testicular cell types or its potential relevance in male reproductive physiology has not yet been described. The role of a Na\u003csup\u003e+\u003c/sup\u003e-dependent phosphate cotransporter (SLC34A2) during sperm capacitation or AE could be explained by the requirements of membrane hyperpolarization that lead to the entry of calcium within the sperm and the increase of intracellular pH\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e; nonetheless, this specific cotransporter has not been previously identified in sperm from any species.\u003c/p\u003e \u003cp\u003eArylsulfatase, particularly arylsulfatase A (ARSA), has been identified in sperm from rabbits, mice, and humans in the post-acrosomal region and plasma membrane \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. This protein remains at the sperm post-acrosomal region after AE\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e and is implicated in sperm-oocyte binding due to its activity as a sulfatase that interacts with the sulfoglycoproteins present in the receptors ZP2 and ZP3\u003csup\u003e64 \u0026ndash; 66\u003c/sup\u003e. In the present study, another member of the arylsulfatase gene family, arylsulfatase F (ARSF), was observed by immunofluorescence at three regions of the sperm from fertile stallions: acrosome, midpiece, and principal piece (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In contrast, in sperm from subfertile TB stallions, the ARSF signal was only observed at the sperm midpiece and principal piece (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Such marked difference in the localization of ARSF at the acrosome region might explain the reduced ability of sperm from subfertile stallions to both undergo AE and bind to the ZP. Further studies in which the immunolocalization of ARSF in stallion sperm is studied after incubation under capacitating conditions and after the occurrence of AE are warranted.\u003c/p\u003e \u003cp\u003eAnother protein of interest identified by DIA-MS in our study was zona pellucida-binding protein 2 (ZPBP2), which has been identified in proacrosomal vesicles that later integrate the inner acrosomal membrane in sperm from other species\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. This protein not only is released from sperm during AE acting as a secondary receptor for sperm-ZP binding\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, but also interacts with other proteins such as testisin, which recently has been identified in stallion sperm as an important serine protease required for normal sperm capacitation, AE, and ZP-binding\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Interestingly, in a recent case report involving a TB stallion with considerably low \u003cem\u003ein vivo\u003c/em\u003e fertility, zona pellucida-binding protein was identified as one potential candidate biomarker for impaired acrosomal exocytosis, based on mass spectrometry-based analysis\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The stallion involved in that case report had a reduced rate of AE following stimulation with calcium ionophore A23187, a similar finding to what we have previously observed in TB stallions that carry the IAE susceptibility genotype\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Unfortunately, the case report by Swegen et al.,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e did not indicate whether the affected stallion also carried the IAE susceptibility genotype. Nonetheless, according to the clinical phenotype, it seems plausible that the TB stallion from that report carried the same IAE susceptibility haplotypes as the stallions used in the current study; as such, the zona pellucida-binding protein can be considered an important marker protein for IAE in TB stallions. While in our study we did not identified by immunofluorescence the presence of ZPBP2 in sperm from either fertile or subfertile TB stallions, a potential confirmation of reduced zona pellucida-binding protein function in sperm from subfertile TB stallions was their reduced ability to bind to porcine ZP after incubation under capacitating conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we utilized frozen/thawed sperm from both fertile and subfertile TB stallions, mainly due to the inability to access fresh semen from these individuals, as mentioned above. Freezing and thawing alters the proteome of stallion sperm, resulting in a lower abundance of several proteins involved in metabolism regulation and redox regulation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Proteins related to sperm-oocyte interactions, namely IZUMO-4 and zona pellucida binding protein, had lower abundance in frozen/thawed stallion sperm when compared to freshly ejaculated sperm\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In the current study, we did not observe statistical differences in the relative abundance of any of the IZUMO proteins (1, 2, 3, or 4) between stallion groups or within periods (data not shown), and we only detected differences in the relative abundance of zona pellucida-binding protein, as described above. Since we did not compare the proteome of fresh versus frozen/thawed sperm from the stallions enrolled in the present study, we cannot compare our results with those presented by Martin-Cano \u003cem\u003eet al\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNotably and despite the compelling evidence from several studies that the double homozygous A/A-A/A genotype in \u003cem\u003eFKBP6\u003c/em\u003e exon 5 is significantly associated with the occurrence of IAE in TB stallions\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, we did not detect the presence of the FKBP6 protein in sperm from any of the stallion groups tested. This is consistent with the recent theory that \u003cem\u003eFKBP6\u003c/em\u003e is not the causative gene for IAE and the A/A-A/A genotype in exon 5 is rather tagging a haplotype unique to Thoroughbreds with IAE\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The current results might offer insight into the mechanisms underlying IAE given the reported high cholesterol-to-phospholipid ratio in the sperm membranes\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, the reduced acrosomal function after non-physiological\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, or physiological stimulation of AE\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Future investigations will be focused on identifying the dynamics (i.e., localization or patterns of expression) of some of these proteins during the initiation of capacitation and AE in stallion sperm.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe current study explored the sperm proteome in both fertile and subfertile TB stallions, with the latter being carriers of the susceptibility genotype for IAE. Using a DIA-MS approach in conjunction with a method that induces AE in viable sperm, we were able to identify a group of differentially abundant proteins associated with metabolism and metabolism of lipids that may explain the acrosomal dysfunction observed in subfertile stallions. One of these proteins, ARSF, was detected at the acrosome, midpiece, and principal piece in sperm from fertile TB stallions, but only at the midpiece, and principal piece in sperm from subfertile TB stallions. We also provide evidence that the ability of frozen/thawed sperm from subfertile TB stallions to bind to porcine ZP was decreased when compared to that of fertile TB stallions. Some of the proteins identified by mass spectrometry and immunofluorescence in the current study are candidates for further studies focused on determining the pathophysiological cause of IAE and understanding the biological processes involved in sperm capacitation and AE in stallions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReagents and media\u003c/h2\u003e \u003cp\u003eUnless otherwise stated, reagents were purchased from Sigma Aldrich (St. Louis, MO, USA). Fixable Live/Dead Red Stain, rabbit anti-arylsulfatase F (ARSF) polyclonal antibody, goat anti-rabbit IgG Alexa-555-conjugated secondary antibody, 10% normal goat serum, SlowFade\u0026trade; Diamond Antifade Mounting Solution with DAPI, and methanol-free 16% paraformaldehyde were acquired from Thermo Fisher Scientific (Waltham, MA, USA). Accumax\u0026reg; Cell Detachment Solution was purchased from Stemcell\u0026trade; Technologies Inc., (Cambridge, MA, USA). Acridine Orange stain was obtained from Polysciences Inc., (Warrington, PA, USA). CryoMax Lactose-EDTA\u0026reg; semen freezing extender (20% egg-yolk\u0026thinsp;+\u0026thinsp;2% glycerol and 3% methyl formamide) was obtained from Animal Reproduction Systems (Chino, CA, USA). A silane-coated silica particle solution (Redigrad\u0026reg;) for density gradient centrifugation was acquired from Global Life Sciences Solutions (Marlborough, MA, USA). The base medium, MW-HEPES, used for sperm washing by density gradient centrifugation was a modified Whitten\u0026rsquo;s medium\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and consisted of 110 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1.9 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 22 mM HEPES, and 50 \u0026micro;L/mL gentamicin sulfate. The medium used for sperm \u003cem\u003ein vitro\u003c/em\u003e incubation under capacitating conditions (Lac-MW) was prepared as reported previously\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and consisted of modified Whitten\u0026rsquo;s medium with 25 mM HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e instead of HEPES, 7 mg/mL bovine serum albumin \u0026ndash; heat shock fraction (BSA), and 10 mM sodium-DL lactate (60% syrup). All media were adjusted with NaCl to an osmolality of 280\u0026ndash;290 mOsm/kg. On the day of the experiment, the pH of each medium was adjusted to 7.25 using NaOH or HCl. The Lac-MW medium was maintained at 38.2\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for a minimum of 2 hours before use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStallions and semen collection\u003c/h2\u003e \u003cp\u003eTexas A\u0026amp;M University Institutional Animal Care and Use Committee (IACUC 2021-0007) approved all the procedures performed in this study. All stallions enrolled (n\u0026thinsp;=\u0026thinsp;6) were Thoroughbred, sexually active, and 7\u0026ndash;15 years old. Hair samples were procured to determine the presence of the susceptibility genotype for IAE, A/A-A/A in the gene \u003cem\u003eFKBP6\u003c/em\u003e exon 5, as previously reported\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eFKBP6\u003c/em\u003e genotype, \u003cem\u003ein vivo\u003c/em\u003e fertility rates, and conventional sperm quality parameters of the six stallions used (three fertile and three subfertile) are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Before semen collection, each stallion was exposed to an ovariectomized mare (when available), or a mare in standing estrus. Once erect, the penis was rinsed thoroughly with warm water and dried with paper towels. The ejaculates from two fertile TB stallions and one subfertile TB stallion were collected using a Colorado-type artificial vagina (Animal Reproduction Systems, Chino, CA, USA), while ejaculates from the other three TB stallions (one fertile and two subfertile) were collected using a Missouri-Model artificial vagina (Nasco, Ft. Atkinson, WI, USA). For both artificial vagina types, an in-line nylon micromesh filter (Animal Reproduction Systems) was placed between the artificial vagina and the semen collection receptacle to separate the gel fraction from the gel-free semen. Following semen collection, the gel-free semen was transported to an adjacent laboratory and placed in an incubator (37\u0026deg;C) before processing.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eInitial semen processing and analyses\u003c/h2\u003e \u003cp\u003eThe gel-free semen volume was estimated based on sample weight, while sperm concentration and plasma membrane intactness (i.e., viability) were measured using a fluorescence-based cell counter (NucleoCounter SP-100\u0026trade;, Chemometec A/S, Aller\u0026oslash;d, Denmark), following a previously described methodology\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Sperm motion characteristics were determined using computer-assisted sperm analysis (CASA; Hamilton-Thorne IVOS II, Hamilton-Thorne Inc., Beverly, MA, USA), as reported previously\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The preset values for the instrument consisted of the following: frames acquired, 45/s; frame rate, 60 Hz; minimum contrast, 70; minimum cell size, four pixels; minimum static contrast, 30; straightness (STR) threshold for progressive motility, 50%; average path velocity (VAP) threshold for progressive motility, 30%; VAP threshold for static cells, 15 \u0026micro;m/s; cell intensity, 106 pixels; static head size, 0.60 to 2.00 \u0026micro;m; static head intensity, 0.20 to 2.01; static elongation, 40 to 85; illumination intensity, 2200. Sperm motility parameters included the percent of total motility (TMOT), progressive motility (PMOT), and the mean curvilinear velocity (\u0026micro;m/s; VCL). For sperm morphology analysis, samples of raw semen were fixed with buffered-formal saline (BFS; 4.75% formaldehyde) and analyzed using differential interference contrast (DIC) microscopy (1,563x, Olympus BX-60, Olympus Corporation, Melville, NY, USA). Sperm morphological classification was done as previously reported\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. All morphologic abnormalities were counted for each spermatozoon to determine the incidence rate. A total of 100 sperm were counted for each ejaculate, and the percentage of morphologically normal sperm was recorded. Sperm DNA quality was determined in flash-frozen/thawed samples obtained from raw semen, using the Sperm Chromatin Structure Assay (SCSA), as previously described\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The percentage of Cells Outside the Main Population (COMP\u003csub\u003eα\u0026minus;t\u003c/sub\u003e), was used as an endpoint to determine the extent of the susceptibility of sperm DNA to denaturation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSemen cryopreservation and stimulation of lactate-induced spontaneous AE in viable frozen/thawed sperm\u003c/h2\u003e \u003cp\u003eIn a previous study, we determined that stallion sperm stored at 5\u0026deg;C for 24 hours, or frozen/thawed stallion sperm do undergo spontaneous AE in viable sperm at the same rate as fresh semen after being incubated in a Lac-MW medium\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Because all the subfertile TB stallions that have been identified by our group as carrying the IAE susceptibility genotype were either located far from our laboratory, had been already castrated, or their sperm were cryopreserved several years previously, in the current study we used frozen/thawed sperm from these stallions to perform the acrosome function and subsequent DIA-MS analyses. When these ejaculates were obtained, immediately after semen collection and initial sperm analysis, the raw semen was diluted 1:1 (v/v) with INRA-96\u0026reg; extender (IMV Technologies, L\u0026rsquo;Aigle, France) and subjected to cushioned centrifugation at 1000 x \u003cem\u003eg\u003c/em\u003e for 20 min, as described previously\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. After centrifugation, the supernatant was removed, and the sperm pellet was resuspended with the EZ-Freezin CryoMax LE\u0026reg; semen freezing extender (Animal Reproduction Systems, Chino, CA) at a final sperm concentration of 200 x 10\u003csup\u003e6\u003c/sup\u003e sperm/mL. Sperm diluted with the freezing extender was loaded into 0.5-mL plastic straws, sealed ultrasonically, and frozen in a controlled rate freezer (CBS 2100; Custom Biogenic Systems, Bruce Township, MI, USA) using the following cooling curve: \u0026minus;\u0026thinsp;2.0\u0026deg;C/min from 25 to 20\u0026deg;C; \u0026minus;\u0026thinsp;0.1\u0026deg;C/min from 20 to 5\u0026deg;C; hold for 5 min; \u0026minus;\u0026thinsp;60\u0026deg;C/min from 4\u0026deg;C to \u0026minus;\u0026thinsp;140\u0026deg;C\u003csup\u003e33\u003c/sup\u003e. The straws were plunged directly into liquid nitrogen and stored in a liquid nitrogen tank. Frozen straws from each stallion were thawed for 30 sec in a water bath set at 37\u0026deg;C and the thawed semen was processed through density gradient centrifugation using 40% Redigrad\u0026reg; (Global Life Science Solutions, Marlborough, MA\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e) to remove seminal plasma, debris, and semen extender. After centrifugation, the sperm pellet was diluted to 30 x 10\u003csup\u003e6\u003c/sup\u003e sperm/mL in Lac-MW medium\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and incubated for up to 6 hours at 38.2\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Sperm aliquots were analyzed after 0, 2, 4, and 6 h of incubation (T0h, T2h, T4h, and T6h, respectively) in Lac-MW medium for viability/acrosomal exocytosis (AE-Viable), as previously reported\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. At each time point (T0h, T2h, T4h, and T6h), a 30 x 10\u003csup\u003e6\u003c/sup\u003e sperm aliquot was also flash-frozen in dry ice for further proteomic analysis using DIA-MS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of sperm viability/acrosomal exocytosis (Fixable Live/Dead Red stain\u0026thinsp;+\u0026thinsp;FITC-PSA)\u003c/h2\u003e \u003cp\u003eThe intactness of both the plasma membranes (viability) and the acrosome membranes (AE) was evaluated simultaneously, as previously described\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, with some modifications. An aliquot (50 \u0026micro;L) of frozen/thawed semen diluted (30 x 10\u003csup\u003e6\u003c/sup\u003e sperm/mL) in Lac-MW was added to 1 mL of Lac-MW medium. This dilution resulted in a final sperm concentration of approximately 1.5 x 10\u003csup\u003e6\u003c/sup\u003e sperm/mL. One \u0026micro;L of Fixable Live/Dead Red stain (Excitation: 488 nm, Emission: 617 nm; final concentration: 50 \u0026micro;g/mL) was added to the sperm sample and incubated at 38.2\u0026deg;C in an air atmosphere for 20 min. Then, 140 \u0026micro;L of methanol-free paraformaldehyde (paraformaldehyde final concentration: 1.88% v/v) was added, and the sperm sample was stored at 5\u0026deg;C in the dark for 30 min. Subsequently, the samples were centrifuged (400 x \u003cem\u003eg\u003c/em\u003e x 5 min) using BSA in DPBS (2 mg/mL), permeabilized with Triton-X100 (1% v: v), centrifuged again with BSA in DPBS, diluted in 133 \u0026micro;L Accumax to avoid sperm clumping, and incubated with 10 \u0026micro;L \u003cem\u003ePisum sativum\u003c/em\u003e agglutinin (PSA)-FITC conjugate (excitation, 488 nm; emission, 517 nm; final concentration, 0.0375 mg/mL) for 20 min at room temperature in the dark. The Fixable Live/Dead Red stain binds to free amines both in the intracellular space and the surface of cells with a disrupted plasma membrane (\u0026ldquo;non-viable\u0026rdquo; cells)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In the case of the Fixable Live/Dead Red stain, non-viable sperm will allow the internalization of this dye and emit red fluorescence\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. PSA binds to the glycoconjugates of the acrosomal matrix, particularly to the α-D-glucosyl and the α-D-mannosyl residues at the inner acrosomal membrane\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. When the acrosomal membrane is disrupted, either as a consequence of acrosome damage or during AE, FITC-PSA will bind to such residues and emit a green, fluorescent signal. In both fixed and permeabilized sperm, four subpopulations are identified using Fixable Live/Dead Red stain and FITC-PSA: 1) sperm with both intact plasma and acrosomal membranes [FITC-PSA (+)/Fixable Live/Dead Red (\u0026ndash;)]; 2) sperm with intact plasma membrane and disrupted acrosomal membrane [FITC-PSA (\u0026ndash;)/Fixable Live/Dead Red (\u0026ndash;)]; 3) sperm with damaged plasma membrane and intact acrosomal membrane [FITC-PSA (+)/Fixable Live/Dead Red (+)]; 4) sperm with both damaged plasma and acrosomal membranes [FITC-PSA (\u0026ndash;)/Fixable Live/Dead Red (+)]. Following incubation with FITC-PSA, the samples were diluted in 150 \u0026micro;L Accumax and processed immediately using a flow cytometer (FACScan, Beckton Dickinson, Mountain View, CA) equipped with a 488-nm argon laser at 20 mW and three fluorescent detectors (FL1, bandpass 530/30nm; FL2, bandpass 585/42nm; and FL3, long pass 670 nm). The voltage settings on the flow cytometer were: FSC-H, 553; SSC, 240; FL1, 741, and FL2, 821. The compensation was set on FL2 as 98% of FL1. The FITC-PSA signal was acquired using the FL1 filter, while the Fixable Live/Dead Red stain signal was acquired using the FL2 filter. The flow rate was 200\u0026ndash;400 sperm/s and a minimum of 5000 sperm were analyzed per sample. To identify doublets and clumps that could affect the analysis and interpretation of sperm events, a manual gating strategy was applied whereby the FSC and SSC were plotted. This methodology has been validated in previous reports from our laboratory\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Flow cytometry data were analyzed by WinList\u0026trade; software (Verity Software House, Topsham, ME, USA). The percentage of AE in viable sperm (AE/Viable) was considered as the experimental endpoint.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSperm preparation for protein extraction\u003c/h2\u003e \u003cp\u003eAliquots of frozen/thawed sperm (30 x 10\u003csup\u003e6\u003c/sup\u003e sperm) incubated at either 0h, 2h, 4h, or 6h in Lac-MW medium were flash/frozen in dry ice until further processing, and then thawed for 1 min at 37\u0026deg;C in a water bath. Immediately after thawing, 10 \u0026micro;L of EDTA-free protease inhibitor cocktail (Pierce\u0026trade; Protease Inhibitor Tablets, Thermo Fisher Scientific, Waltham, MA) was added per each sperm aliquot. The sperm were washed two times in calcium-free PBS at 400 x \u003cem\u003eg\u003c/em\u003e for 5 min, pelleted, maintained frozen at \u0026minus;\u0026thinsp;80\u0026deg;C, and sent to the Institutional Mass Spectrometry Core Laboratory at the University of Texas Health Science Center at San Antonio (San Antonio, Texas) for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProtein identification and relative quantification by data-independent acquisition mass spectrometry (DIA-MS)\u003c/h2\u003e \u003cp\u003eProtein aliquots corresponding to 70 \u0026micro;g protein, as quantified using the EZQ\u0026trade; Protein Quantitation Kit (Thermo Scientific, Waltman, MA), were reduced with tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl; Pierce\u0026trade;, Thermo Fisher Scientific), alkylated in the dark with iodoacetamide and applied to S-Traps spin columns (Protifi\u0026trade;, Farmingdale, NY) for tryptic digestion (sequencing grade; Promega) in 50 mM triethylammonium bicarbonate (TEAB; Thermo Fisher Scientific). Peptides were eluted from the S-Traps spin columns with 0.2% formic acid in 50% aqueous acetonitrile and quantified using the Pierce\u0026trade; Quantitative Fluorometric Peptide Assay (Thermo Fisher Scientific). Data-independent acquisition mass spectrometry (DIA-MS) was conducted on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). On-line HPLC separation was accomplished with an RSLC NANO HPLC system (Thermo Fisher Scientific/Dionex) and a PicoFrit\u0026trade; nanospray column (75 \u0026micro;m i.d.; New Objective, Littleton, MA) packed to 15 cm with C18 adsorbent column (218MS 5 \u0026micro;m, 300 \u0026Aring;; Vydac\u0026reg;, W.R. Grace \u0026amp; Co., Columbia, MA); mobile phase A, 0.5% acetic acid (Hac)/0.005% trifluoroacetic acid (TFA; Pierce\u0026trade;, Thermo Fisher Scientific) in water; mobile phase B, 90% acetonitrile/0.5% Hac/0.005% TFA/9.5% water; gradient 3 to 42% B in 120 min; flow rate: 0.4 \u0026micro;L/min. A pool was made of all samples, and 2 \u0026micro;g aliquots of the digests were analyzed using gas-phase fractionation and 4-\u003cem\u003em/z\u003c/em\u003e windows [three mass ranges (395-605mz, 595-805mz, 795-1005mz), staggered; 30k resolution for precursor and product ion scans, all in the orbitrap] to create a DIA chromatogram library\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e by searching against a Prosit-generated predicted spectral library\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e based on the UniProt \u003cem\u003eEquus caballus\u003c/em\u003e protein sequence database (44,488 sequences, downloaded on 12-11-2020): peptide mass tolerance, 10.0 ppm; fragment mass tolerance, 10.0 ppm; fixed modification, carbamidomethylation (C); enzyme, trypsin with a maximum of one missed cleavage; peptide charge state, +\u0026thinsp;2 \u0026ndash; +3; peptide length, 6\u0026ndash;30; protein FDR, 1%; minimum of two identified peptides; peptide quantification, Encyclopedia (0.8.1) based on the five highest quality fragment ions. Experimental samples were blocked by replicate and randomized within each replicate for sample preparation and analysis, employing 2 \u0026micro;g of peptides and the 2-h HPLC gradient described above. MS data for experimental samples were acquired in the orbitrap using 8-\u003cem\u003em/z\u003c/em\u003e windows (390-1010mz, staggered; 30k resolution for precursor and product ion scans) and searched against the chromatogram library using the same parameters as described above for generation of the chromatogram library. Scaffold DIA (v3.2.1; Proteome Software) was used for all DIA-MS data processing. The data files were converted to mzML format using ProteoWizard (3.0.19254)\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Deconvolution of the staggered windows was performed. Peptides identified in each sample were filtered by Percolator (3.01.nightly-13-655e4c7-dirty)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e to achieve a maximum false discovery rate (FDR) of 0.01. Individual search results were combined, and peptide identifications were assigned posterior error probabilities and filtered to an FDR threshold of 0.01 by Percolator. Peptide quantification was performed by Encyclopedia (version 1.12.31)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. For each peptide, the five highest-quality fragment ions were selected for quantification. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis were grouped to satisfy the principles of parsimony. Quartile normalization was applied on the log10 peptide intensities across replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic analyses\u003c/h2\u003e \u003cp\u003eScaffold DIA was used to assess differences in the results for samples from fertile and subfertile stallions (A/A-A/A) across the selected time periods (T0h, T2h, T4h, or T6h). A two-way ANOVA design, available in the Scaffold DIA software, was used in which the primary category of analysis was the stallion phenotype (fertile vs. subfertile), while the second category of analysis was the time point (T0h vs. T2h vs. T4h vs. T6h). Significance was assessed using Benjamini-Hochberg multiple testing correction\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, resulting in a cutoff of P\u0026thinsp;\u0026lt;\u0026thinsp;0.0069. Proteins that exhibited log\u003csub\u003e2\u003c/sub\u003e(fold change) \u0026le; \u0026minus;\u0026thinsp;0.585 or \u0026ge;\u0026thinsp;0.585 (i.e., fold change in protein abundance between fertile and subfertile stallions 1.5x up or down) were used to filter the results for further analysis.\u003c/p\u003e \u003cp\u003eThe proteins identified as differentially abundant between stallion groups and among time periods were queried for gene ontology (GO) terms using g:Profiler\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, according to cellular component (CC), biological process (BP), and molecular function (MF). Overrepresentation of the proteins of differential abundance was queried using \u003cem\u003eEquus caballus\u003c/em\u003e orthologs and a g:SCS threshold value of 0.05. To analyze the overrepresentation of biological pathways, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was also used in g:Profiler. In addition, pathway enrichment analyses were conducted utilizing both the g:Profiler and Reactome servers using \u003cem\u003eHomo sapiens\u003c/em\u003e orthologs, given the increased depth of the human proteome in terms of annotation. To identify functionally grouped GO terms, a network analysis of the differentially abundant proteins was performed using the Cytoscape (version 3.9.1) plugin ClueGo (version 2.5.8)\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDetection of acrosome proteins of differential abundance between fertile and subfertile TB stallions by immunofluorescence\u003c/h2\u003e \u003cp\u003eIndirect immunofluorescence of the protein arylsulfatase F (ARSF) was performed, as previously described\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, with some modifications. Briefly, frozen/thawed semen from both fertile and subfertile TB stallions (n\u0026thinsp;=\u0026thinsp;3 stallions per group) were processed by density gradient centrifugation using 40% Redigrad and diluted to 30 x 10\u003csup\u003e6\u003c/sup\u003e sperm in Lac-MW. Next, the sperm were centrifuged (600 x \u003cem\u003eg\u003c/em\u003e for 5 min) three times in DPBS, applied to poly-L-lysine coated slides for 2h at room temperature, and exposed to 4% paraformaldehyde in DPBS for 20 min at 4\u0026deg;C. As ARSF is a protein of acrosome origin, we did not expose the sperm to any permeabilizing agent (i.e., triton X-100) to avoid any loss of proteins located at the outer acrosomal membrane or acrosomal matrix. Following fixation, the slides were washed again three times using DPBS, blocked with 10% normal goat serum for 1h at room temperature, and washed again three times using DPBS. A rabbit anti-ARSF, diluted 1:100 in 10% normal goat serum, was added to the slides and incubated overnight at 4\u0026deg;C. A negative control was produced in which the slides were not exposed to the primary antibody (i.e., anti-ARSF). Following overnight incubation, the sets of slides were washed three times with DPBS and stained for 1h at room temperature with a goat anti-rabbit IgG Alexa-555-conjugated secondary antibody (excitation: 555 nm; emission: 572 nm) at a 1:100 dilution in 10% normal goat serum. A final washing step was performed using DPBS; then, 3.5 \u0026micro;L of the SlowFade Mountant with DAPI was added, to both prevent fluorescence bleaching and to counterstain the sperm nuclei. Then, a coverslip was carefully applied to the slide, and the slides were evaluated using an Olympus BX-60 fluorescence microscope at a 1,563x magnification. A total of 100 sperm were analyzed per ejaculate, and the localization of ARSF within each spermatozoon was recorded and compared between fertile and subfertile TB stallions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHeterologous zona pellucida-binding assay\u003c/h2\u003e \u003cp\u003eDue to the difficulties in establishing a repeatable method for conventional \u003cem\u003ein vitro\u003c/em\u003e fertilization of equine oocytes using frozen/thawed stallion sperm, we performed a heterologous zona pellucida-binding assay to determine the ability of stallion sperm incubated in Lac-MW to undergo acrosomal exocytosis and bind to the zona pellucida (ZP)\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Porcine oocytes were used to determine the ability of frozen/thawed sperm from subfertile and fertile TB stallions to bind to the ZP. Ovaries were procured from a slaughterhouse 1 hour away from our laboratory, and the specimens were maintained at 37\u0026deg;C in an insulated container for transport. Upon arrival at our laboratory, the ovaries were washed with warm PBS and dried with cotton gauze. All visible follicles were sliced using a scalpel blade, and the follicular cavity was flushed using Vigro\u0026reg; Complete Flush Medium (Vetoquinol, Pullman, WA, USA) over a sterile Petri dish. Groups of 50 \u003cem\u003ecumulus\u003c/em\u003e-oocyte complexes (COCs) were identified using a stereoscope and transferred into a Petri dish containing 150-\u0026micro;L droplets of maturation medium [M199 with Earle\u0026rsquo;s salts, supplemented with 5 mU/mL FSH (Sioux Chemicals, Sioux Center, IA, USA), 10 IU/mL hCG (Chorulon, Merck Animal Health, Rahway, NJ, USA), 10% FBS, and 25 \u0026micro;g/mL gentamicin] under light mineral oil, at 38.2\u0026deg;C in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e for 22h. After this period, the COCs were placed into a fresh droplet of maturation medium and incubated for an additional 22h, under the same conditions. Following the maturation period, COCs were denuded of cumulus by repeated pipetting in M199 medium with Hank\u0026rsquo;s salts containing 10% FBS and 0.5 mg/mL hyaluronidase. Denuded oocytes were considered as matured if a polar body was extruded and present at the periphery of the oolemma. To perform the heterologous zona-binding assay, frozen/thawed semen from three fertile and three subfertile TB stallions was thawed, processed by density gradient centrifugation using 40% Redigrad, and resuspended to 30 x 10\u003csup\u003e6\u003c/sup\u003e sperm/mL in Lac-MW. A total of 20 mature oocytes were placed in 50-\u0026micro;L droplets of Lac-MW, inseminated with 5 x 10\u003csup\u003e4\u003c/sup\u003e sperm from each stallion, and incubated for 4 h at 38.2\u0026deg;C in a humidified atmosphere of 6% CO\u003csub\u003e2\u003c/sub\u003e, 5% O\u003csub\u003e2\u003c/sub\u003e, and 89% N\u003csub\u003e2\u003c/sub\u003e. Following the co-incubation period, to remove any loosely bound sperm, the oocytes were washed by repeated pipetting in M199 with Hank\u0026rsquo;s salts, transferred to a 50-\u0026micro;L droplet of 2% paraformaldehyde in DPBS for 10 min at room temperature and placed into a 50-\u0026micro;L droplet of 2% BSA\u0026thinsp;+\u0026thinsp;10 \u0026micro;M Hoechst 33342 in DPBS for 10 min at room temperature. The oocytes were loaded onto polylysine-coated slides, carefully covered with a coverslip, and examined at 400x using a BX-60 Olympus fluorescence microscope. For each oocyte, the total number of sperm bound to the ZP was quantified and compared between fertile and subfertile TB stallions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using commercial software (SAS Version 9.4; SAS Institute, Inc., Corp., Cary, NC, USA). The Shapiro-Wilk test (PROC UNIVARIATE) was conducted to test data distribution. Within periods (T0h, T2h, T4h, or T6h), Student \u003cem\u003et\u003c/em\u003e-tests (PROC TTEST) or the Wilcoxon Ranked Sum test (PROC NPAR1WAY) were used to compare the percent of AE in viable sperm (AE-Viable), the immunolocalization of ARSF in sperm from fertile and subfertile stallions, and the number of sperm bound to the ZP between fertile and subfertile stallions. Group differences were set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for this project was provided by the Legends Premier Stallion Season Auction, Texas A\u0026amp;M University. The authors would like to thank Ms. Crystal Howard (Rood \u0026amp; Riddle Equine Hospital, Lexington, KY) for her help while procuring samples from some fertile and subfertile TB stallions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. H.-A:\u0026nbsp;\u003c/strong\u003estudy and experimental design, data collection and interpretation, Formal analysis, writing of the initial draft. \u003cstrong\u003eL. R.-A.:\u0026nbsp;\u003c/strong\u003estudy and experimental design, data collection and interpretation, text review and editing. \u003cstrong\u003eS.T.W.\u0026nbsp;\u003c/strong\u003estudy and experimental design, supervision of mass spectrometry analysis and data processing, data interpretation, text review, and editing. \u003cstrong\u003eC.F.S.:\u0026nbsp;\u003c/strong\u003e data collection, text review, and editing. \u003cstrong\u003eB.W.D:\u0026nbsp;\u003c/strong\u003edata interpretation, text review, and editing. \u003cstrong\u003eT.R.\u003c/strong\u003e: data interpretation, text review, and editing. \u003cstrong\u003eD.D.V\u003c/strong\u003e: study and experimental design, data interpretation, text review and editing, funding acquisition. \u003cstrong\u003eC.C.L.:\u0026nbsp;\u003c/strong\u003estudy and experimental design, data collection and interpretation, text review and editing, funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DIA-MS data from this study is accessible at the Mass Spectrometry Interactive Virtual Environment (MASSIVE) repository, with accession number: MSV000093730.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWassarman, P.M. 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In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions when compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mostly overrepresented in the \u0026ldquo;metabolism\u0026rdquo; and the \u0026ldquo;metabolism of lipids\u0026rdquo; pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays were performed and revealed sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of proteins of differential abundance, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction.\u003c/p\u003e","manuscriptTitle":"Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-02 06:55:53","doi":"10.21203/rs.3.rs-4185383/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-17T13:17:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-08T12:44:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-20T18:13:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83d43507-ff54-4564-a57d-426314db1d94","date":"2024-04-15T16:35:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58180f29-f1a2-4bc0-bad9-95cccd2dd76d","date":"2024-04-15T12:44:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-15T12:21:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-15T12:12:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-09T12:18:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-08T12:06:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-03-29T03:15:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8d4edf27-fa16-4629-a330-9f6b68058959","owner":[],"postedDate":"April 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30026763,"name":"Biological sciences/Biotechnology/Proteomics"},{"id":30026764,"name":"Biological sciences/Zoology/Animal physiology"},{"id":30026765,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2024-06-02T14:02:02+00:00","versionOfRecord":{"articleIdentity":"rs-4185383","link":"https://doi.org/10.1038/s41598-024-63410-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-05-30 14:02:02","publishedOnDateReadable":"May 30th, 2024"},"versionCreatedAt":"2024-04-02 06:55:53","video":"","vorDoi":"10.1038/s41598-024-63410-3","vorDoiUrl":"https://doi.org/10.1038/s41598-024-63410-3","workflowStages":[]},"version":"v1","identity":"rs-4185383","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4185383","identity":"rs-4185383","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}