ProAKAP4 Concentration Is Related to Sperm Motility and Motile Sperm Subpopulations in Frozen–Thawed Horse Semen

Simple Summary ProAKAP4 is the precursor of AKAP4 protein, whose principal function is to anchor protein complexes to regulate the long-term motion and flagellum structure of mammal spermatozoa. The objective of the present study was to evaluate how proAKAP4 sperm concentrations correlate with the motion parameters of equine spermatozoa. We showed that proAKAP4 concentrations are associated with motility and velocity parameters. Low proAKAP4 concentrations reflect a loss of spermatozoa functionality. In conclusion, proAKAP4 concentrations are associated with better motion parameters in stallions. Abstract ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyzed. Spermatic motility descriptors were obtained by the CASA system, and four motile subpopulations (SP) with specific motility patterns were statistically identified. ProAKAP4 concentrations were evaluated by ELISA. The relationship between motility descriptors of sperm subpopulations and proAKAP4 concentrations was evaluated. Following a hierarchical cluster statistical analysis, ejaculates were divided into two groups according to their proAKAP4 concentrations, either having low proAKAP4 concentrations (5.06–35.61 ng/10M spz; n = 23) or high (39.92–82.23 ng/10M spz; n = 25) proAKAP4 concentrations (p < 0.001). ProAKAP4 concentrations were positively correlated (p < 0.05) with total and progressive motility, as well as with parameters of velocity. ProAKAP4 amount also showed a negative correlation (p < 0.05) with sperm motile subpopulation number 3, which was the subpopulation with the lowest velocity parameters. In conclusion, proAKAP4 concentration in stallion semen positively reflects sperm progressive motility with the functional velocity kinematic descriptors. Concentrations of proAKAP4 higher than 37.77 ng/10M spz were correlated with a very good quality frozen/thawed stallion semen.


Introduction
Spermatozoa are basic motile cells to provide fertilization capacity. Formerly, sperm analytics was subjective, but since the late 1980s, when the first CASA (computer-assisted sperm analysis) was commercialized [1], motion kinematic descriptors and morphometric characteristics have been analyzed objectively [2]. The use of the CASA system evinced

Semen Collection and Cryopreservation
The collection of the samples was performed at the Equine Reproduction Service of the Universitat Autonoma de Barcelona (Bellaterra, Cerdanyola del Vallès, Spain), which is a European centre approved to produce semen with authorization code ES09RS01E. Stallions used came at the service for commercial semen freezing. Thirteen stallions from 4 to 18 years old of several breeds (Arabian, Andalusian, and Warmblood) were used, with a total of 48 ejaculates kept counting between 1 to 5 ejaculates per stallion.
Semen collection was conducted using a Hannover artificial vagina (Minitüb GmbH, Tiefen-bach, Germany). The ejaculate without gel was diluted 1:5 in Kenney extender in 50 mL corning tubes previously warmed at 37 • C. All ejaculates were evaluated before freezing: total volume was recorded, sperm concentration was evaluated by a haemocytometer (Neubauer chamber, Paul Marienfeld GmbH and Co., KG; Lauda-Königshofen, Germany), motility by a CASA system (Section 2.2), and sperm morphology and viability using eosin-nigrosin staining [30].

Sperm Cryopreservation
Prior to cryopreservation, each extended semen sample was centrifuged at 660× g and 20 • C for 15 min (Medifriger BL-S, JP Selecta S.A., Barcelona, Spain) to remove seminal plasma. Supernatants were discarded, and pellets were resuspended in a commercial freezing medium (Botucrio ® , Botupharma Animal Biotechnology; Botucatu, Brazil). Afterwards, sperm concentration and viability were re-evaluated, and freezing medium (Botucrio ® ) was added to obtain a final concentration of 200 × 106 per mL of viable sperm. Samples were packaged into 0.5 mL straws and cryopreserved using a controlled-rate freezer (Ice-Cube 14S; Minitüb). Briefly, the cryopreservation procedure consists of three successive freezing steps: first: cooling from 20 • C to 5 • C at rate of −0.25 • C/min for 60 min; second: freezing from 5 • C to −90 • C at a rate of −4.75 • C/min for 20 min; and third: freezing step from −90 • C to −120 • C at a rate −11.11 • C/min for 2.7 min. Straws were stored into liquid nitrogen in appropriate tanks.
The samples were then thawed in a circulating water bath at 37 • C for 30 s, and the content of each straw was poured into a 10 mL conical tube.

proAKAP4 ELISA Assays
ProAKAP4 analysis was performed in the ONIRIS Laboratory in Nantes (France) using a commercialized sandwich-ELISA-based assay (Horse 4MID ® Kit, 4BioDx, Lille, France). The first step was thawing one straw of each ejaculate (See Section 2.2 for more information). Then, 100 µL of post-thawed semen was diluted in the lysis solution, mixed, then loaded onto the 96-well microplate according to the manufacturer's instructions. A secondary Animals 2022, 12, 3417 4 of 11 detection antibody was then added to the wells. After washing, the colourimetric substrate was added to each well. This blocked the enzymatic conversion of the colourimetric substrate. The color intensity was measured by a spectrophotometer plate reader, and given optical densities are proportional to proAKAP4 concentrations, based on an internal calibration curve of predetermined proAKAP4 concentration calibrators.

Statistical Analysis
Data were analyzed with the statistical package R (V 4.0.3, R Core Team; Vienna, Austria), and graphs were made with GraphPad Prism software (V 8.4.0, GraphPad Software LLC; San Diego, CA, USA). The first step was to check the normality of the data using the Shapiro-Wilk test, as well as the homoscedasticity of variances using the Levene test. Only when necessary was the function arcsin √ x applied to transform the data and, thus, obtain a normal distribution. Then, a hierarchical cluster analysis was performed to classify frozen-thawed horse ejaculates into two groups according to proAKAP4 concentration (low or high). Differences in TM, PM, and motile sperm subpopulations between the two proAKAP4 groups were analyzed using a t-test for independent samples. Consequently, A Pearson correlation was applied to obtain the correlation coefficients between proAKAP4 concentration (both groups) with sperm motility parameters (TM, PM, VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF) and with motile sperm subpopulations of frozen-thawed horse ejaculates. Finally, to analyze proAKAP4 between two quality groups and differences between breeds, a two-way ANOVA was performed.
In all cases, the minimum level of statistical significance was set at p ≤ 0.05. Results in the text were expressed as means ± error standard of the mean (SEM).

Motile Sperm Subpopulations
The procedure for calculating the motile sperm subpopulations in frozen-thawed horse ejaculates was the one proposed by Martí et al. [31]. In the first step, a principal component analysis (PCA) was performed with the values of the kinematic parameters (VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF) of each individual trajectory after thawing. The matrix obtained was rotated using the Varimax method with Kaiser normalization, where the regression scores assigned to each sperm were used to perform a non-hierarchical multivariate cluster analysis using the k-means model based on Euclidean distances calculated from the kinematic parameters. Ultimately, for each frozen-thawed horse ejaculate, the proportion of spermatozoa present in each subpopulation was calculated.

Relationship between proAKAP4 Groups and Its Distribution in Different Breeds
Taking account different breeds in our study, there are not significant different between breeds (p > 0.05). The Arabian pure breed (n = 8 ejaculates) showed a median of 53.31 ± 4.00 ng/10M spz in the high quality group (n = 6) and a median of 19.75 ± 0.96 ng/10M spz in the low quality group (n = 2). The pure Spanish breed (n = 33 ejaculates) showed a median of 57.64 ± 3.28 ng/10M spz in the high quality group (n = 15) and, in the low quality group (n = 18), a median of 19.75 ± 19.51 ng/10M spz. The Warmblood breed (n = 7 ejaculates) showed a median of 46.31 ± 3.41 ng/10M spz in the high quality group (n = 4) and, in the low quality group (n = 3), a median of 32.28 ± 2.29 ng/10M spz ( Figure 2).

Relationship between proAKAP4 Groups and Its Distribution in Different Breeds
Taking account different breeds in our study, there are not significant different between breeds (p > 0.05). The Arabian pure breed (n = 8 ejaculates) showed a median of 53.31 ± 4.00 ng/10M spz in the high quality group (n = 6) and a median of 19.75 ± 0.96 ng/10M spz in the low quality group (n = 2). The pure Spanish breed (n = 33 ejaculates) showed a median of 57.64 ± 3.28 ng/10M spz in the high quality group (n = 15) and, in the low quality group (n = 18), a median of 19.75 ± 19.51 ng/10M spz. The Warmblood breed (n = 7 ejaculates) showed a median of 46.31 ± 3.41 ng/10M spz in the high quality group (n = 4) and, in the low quality group (n = 3), a median of 32.28 ± 2.29 ng/10M spz ( Figure 2).

Figure 2.
Box-whisker plot showing the differences in proAKAP4 concentration (ng/10M spz) of frozen-thawed horse ejaculates from different breeds (Pure Arabian breed = red; Warmblood breed= blue; pure Spanish breed = green) as having low or high proAKAP4 concentration (ng/10M spz). The line indicates the median, the cross indicates the mean, the boxes enclose the 25th and 75th percentiles, and the whiskers extend to the 5th and 95th percentiles. (a,b) Different letters indicate significant difference (p ≤ 0.05) between the proAKAP4 groups within each breed. The same number indicates no significant difference (p > 0.05) between breeds within each proAKAP4 group.  Box-whisker plot showing the proAKAP4 concentration (ng/10M spz) of frozen-thawed horse ejaculates as having low (n = 23; red) or high (n = 25; green) proAKAP4 concentrations. The line indicates the median, the boxes enclose the 25th and 75th percentiles, and the whiskers extend to the 5% and 95% percentiles (*** p ≤ 0.001).

Relationship between proAKAP4 Groups and Its Distribution in Different Breeds
Taking account different breeds in our study, there are not significant different between breeds (p > 0.05). The Arabian pure breed (n = 8 ejaculates) showed a median of 53.31 ± 4.00 ng/10M spz in the high quality group (n = 6) and a median of 19.75 ± 0.96 ng/10M spz in the low quality group (n = 2). The pure Spanish breed (n = 33 ejaculates) showed a median of 57.64 ± 3.28 ng/10M spz in the high quality group (n = 15) and, in the low quality group (n = 18), a median of 19.75 ± 19.51 ng/10M spz. The Warmblood breed (n = 7 ejaculates) showed a median of 46.31 ± 3.41 ng/10M spz in the high quality group (n = 4) and, in the low quality group (n = 3), a median of 32.28 ± 2.29 ng/10M spz ( Figure 2).

Figure 2.
Box-whisker plot showing the differences in proAKAP4 concentration (ng/10M spz) of frozen-thawed horse ejaculates from different breeds (Pure Arabian breed = red; Warmblood breed= blue; pure Spanish breed = green) as having low or high proAKAP4 concentration (ng/10M spz). The line indicates the median, the cross indicates the mean, the boxes enclose the 25th and 75th percentiles, and the whiskers extend to the 5th and 95th percentiles. (a,b) Different letters indicate significant difference (p ≤ 0.05) between the proAKAP4 groups within each breed. The same number indicates no significant difference (p > 0.05) between breeds within each proAKAP4 group.

Relationship between proAKAP4 Concentration and Sperm Motility and Motile Sperm Subpopulations in Frozen-Thawed Horse Ejaculates
The percentage of total motile sperm was significantly lower (p ≤ 0.05) in ejaculates having low (62.91 ± 4.60%) proAKAP4 concentration compared to those having high (73.06 ± 2.65%) concentration (Figure 3a). On the contrary, the percentage of progressive motile sperm did not differ between frozen-thawed ejaculates with low or high proAKAP4 concentration (Figure 3b).

Relationship between proAKAP4 Concentration and Sperm Motility and Motile Sperm Subpopulations in Frozen-Thawed Horse Ejaculates
The percentage of total motile sperm was significantly lower (p ≤ 0.05) in ejaculates having low (62.91 ± 4.60%) proAKAP4 concentration compared to those having high (73.06 ± 2.65%) concentration (Figure 3a). On the contrary, the percentage of progressive motile sperm did not differ between frozen-thawed ejaculates with low or high proAKAP4 concentration (Figure 3b).     The proportion of motile sperm of SP3 identified in frozen-thawed horse ejaculates (Figure 4) was significantly higher (p < 0.05) in ejaculates having low (47.40 ± 4.46%) proAKAP4 concentration compared to those having high (37.16 ± 2.83%) concentration. On the contrary, SP1, SP2, and SP4 did not show differences between ejaculates as having low or high proAKAP4 concentrations. The proportion of motile sperm of SP3 identified in frozen-thawed horse ejaculates (Figure 4) was significantly higher (p < 0.05) in ejaculates having low (47.40 ± 4.46%) proAKAP4 concentration compared to those having high (37.16 ± 2.83%) concentration. On the contrary, SP1, SP2, and SP4 did not show differences between ejaculates as having low or high proAKAP4 concentrations.

Discussion
Classification of frozen/thawed stallion ejaculates depending on proAKAP4 concentration has been established. Stallion ejaculates with high proAKAP4 concentrations are inside the rank of 39.92-82.23 ng/10M spz. Those having low proAKAP4 concentrations are between 5.06-35.61 ng/10M spz. This can establish a mean of 37.77 ng/10M spz. In the same way, when we compare the three breeds of this study (Arabian, Spanish, and Warmblood), quality groups are consistent. Our results agree with 4BioDx commercial information [32], considering the rank 0-15 ng/10M spz bad semen, 15-40 ng/10M spz correct semen, and more than 40 ng/10M spz very good semen. The mean concentration obtained in the present study is then in agreement with the established thresholds of proAKAP4 described in other mammal species. Herein, sperm having more than 37.77 ng/10M spz could be considered as very good semen, whereas those having lower concentration, or good, or with concentration below 15 ng/10M spz, are of low quality.
As a precursor of the AKAP4 protein, proAKAP4 is positively correlated with total and progressive motility; the same results were found [24,28] in stallions, but also in bulls [33,34], men [35], mice [26], dromedaries [19], and rams [16]. A deeper analysis of the motility descriptors, proAKAP4 shows a positive correlation with velocity parameters (VCL, VSL, and VAP), but not with pathway descriptors (LIN, STR, WOB, ALH, and BCF). By the analysis of these results, we can conclude that proAKAP4 describes the capacity of spermatozoa to move forward through the velocity descriptors, but it does not describe how this movement occurs. Further studies are needed to identify how proAKAP4 affects the trajectory of the spermatozoa.
While considering the relationship between subpopulations and proAKAP4 concentration, subpopulation 3 (SP3) is higher in ejaculates of the lowest proAKAP4 concentration group. This result is supported by a negative correlation between proAKAP4 and SP3 found when taking all the ejaculates together. This means that, the slower the spermatozoa are, the lower the proAKAP4 concentration is. In consequence, one ejaculate with a high percentage of slow spermatozoa will show reduced proAKAP4 levels. Other subpopulations are positively correlated with proAKAP4 concentration, even if it is not statistically significant. This confirms that proAKAP4 is related to the motion of the spermatozoa [9,10,24]. The first time when spermatic subpopulations in stallions were described [3], four subpopulations were determined, as in our study. A low proAKAP4 concentration could be an indicator of early semen deterioration because of a high percentage of SP3. In an ejaculate, this means a descent of quality parameters such as viability or total motility [3]. This result is complementary and agrees with Griffin et al. [28], who found a positive correlation between proAKAP4 and the percentage of rapid spermatozoa in the ejaculate.
Boersma et al. [36] demonstrated that proAKAP4 is not affected by cryopreservation in epidydimary semen in mice, which is relvevant because proAKAP4 in frozen/thawed semen can be a good predictor of its quality before ejaculation. Nevertheless, it is important to keep in mind that epidydimary sperm has not been under the influence of the seminal plasma. Further studies are needed to elucidate how cryopreservation affects proAKAP4 concentration in stallion-ejaculated sperm.
The importance of spermatic motility on fertility rates has been described over the years [37][38][39][40][41][42][43], but some stallions are not fertile even if this is not reflected in motility [37,44]. The consequence of motility on fertility is not clear [45,46]. ProAKAP4 describes the motion of the spermatozoa [9,10,24], but not how they move. This may evoke a bias relating the proAKAP4 and fertility. AKAP4 has been elucidated as having a role in the phosphorylation before capacitation [28] due to protein translocation at the plasma membrane reorganization during the capacitation process, including hypermotility [47]. Ramal-Sanchez et al. [48] have shown that spermatozoa from bovine oviductal epithelial cells after progesterone influence showed increased levels of AKAP-4, and, at this moment, spermatozoa show a hypermotility pattern [48,49]. AKAP4 can be considered as a possible biomarker of fertility in bulls [48]. Further studies need to be performed with proAKAP4 to relate its action mechanism with motility patterns and the outcome of fertility.

Conclusions
The concentration of proAKAP4 after freezing/thawing in stallion sperm correlates positively with total and progressive motility, as well as with velocity kinematic parameters. In addition to this, a negative relationship was observed between ejaculates with proAKAP4 levels and SP3, which is the slowest motile sperm subpopulation. Concentrations of proAKAP4 higher than 37.77 ng/10M spz are correlated with qualified frozen/thawed semen.