ProAKAP4 as Indicator of Post-thaw Motility in Stallion

1. Introduction

The cryopreservation of sperm over extended periods is pivotal for the survival, maintenance, and enhancement of genetic diversity and improvement within species and breeds worldwide [1]. In the equine field, semen cryopreservation has been important for different reasons like spreading good genetics worldwide, minimizing disease transmission, getting offspring after the death of the stallion [2] or keeping rare genetic lines from endangered breeds [1].

The most important point about frozen-thawed semen is to maintain the capacity of fertilization after thawing. One of the major impediments to the development of the horse frozen semen industry is the lower fertility with frozen semen versus to cooled semen [3]. However, Lomis (2001) [3] concluded that acceptable fertility with frozen semen is possible with good quality semen. A good quality semen once thawed will reach the fertilization site to penetrate the oocyte [1]. Thawed semen is considered good when its progressive motility is over 35%, [4] but this is not a warranty that the semen will be fertile [4,5]. The definition of semen fertility is not easy and precise [5], it is usually related to the seminal quality parameters [4,6]. The estimate of semen fertility has been always a challenge, frozen semen appears to have shorter longevity once thawed [2] and lower conception rates [7,8]. Insemination of the mare with frozen semen is a challenge due to the short lifetime of the spermatozoa once thawed. It is also difficult to predict how long the semen will be alive and motile after thawing to adapt to the ovarian scanning routine of the mare [8] for artificial insemination with frozen-thawed semen. The most commonly employed protocol to inseminate a mare with frozen semen to obtain a high pregnancy rate, the mare has to be inseminated within a 6-hour window around ovulation [2,9]. To reach this objective the mare has to be scanned regularly to catch the ovulation time.

The traditional semen analysis (sperm concentration, subjective motility and sperm morphoabnormalities) has a reduced predictive value in the evaluation of stallion fertility. An important number of sperm tests (e.g., sperm head morphometry, the hypoosmotic swelling test, acrosome integrity, membrane integrity, progesterone receptor exposure, DNA integrity or fragmentation, intracellular Calcium, intracellular ROS, mitochondrial activity…) have been developed in order to increase the sperm fertility prediction. However, most examine only a single or a narrow range of the attributes that a sperm must possess. Flow cytometry offers the possibility of quickly and simultaneously combining many of these analyzes [6]. On the other hand, the CASA-Mot (Computer Assisted Semen Analysis for Motility) can be used to rapidly assess total and progressive motility, as well as determine values for other kinematic variables [10]. Nowadays, the prediction of seminal quality has been usually related to motility parameters analyzed by CASA systems, flow cytometry analysis and formulas combining both [1]. However, semen quality is not always correlated with good fertility rates [11]. In a practical point of view, the search for new fertility predictors is an important field of research.

ProAKAP4 is a marker of sperm quality and male fertility in different species [12], also in stallions [13,14,15,16,17]. ProAKAP4 is the precursor of an A-kinase anchor protein 4 which is the main protein of the fibrous sheath of the principal piece of the flagellum [12,18]. As an important part of flagellum [19,20], proAKAP4 is a good marker of post-meiotic stage of spermatogenesis [12,21,22] as well as, a good marker of sperm progressive and total motility in stallion [13,14,15,16] and in other mammals like bulls [23], jackass [24], dogs [25], rabbits [26], rams [27] and dromedary [28].

This study aims to correlate motility descriptors and mitochondrial membrane potential with proAKAP4 concentration in stallion frozen-thawed semen and explore its evolution over three hours to assess the potential of proAKAP4 as a long-lasting motility indicator. Another aim of this study is to test how cryoprotectant affects proAKAP4 evolution.

2. Materials and Methods

2.1. Semen Samples

Sixteen ejaculates obtained from four French warmblood stallions, housed at Haras de Hus in Petit Mars, France, were utilized for this study, intended for both reproductive and sport (showjumping and dressage) purposes. The stallions ranged in age from 3 to 8 years at the time of semen collection. These animals were selected based on their proven fertility, excellent physical condition, and negative testing for Equine Arteritis, Infectious Anemia, and Contagious Metritis, conforming to CEE health requirements prior to semen production. Each stallion provided four ejaculates, from which three straws were extracted for further analysis.

Cryopreservation of all ejaculates was conducted between 2012 and 2016 at Haras de Hus, a collection center approved by European authorities. Consistent cryopreservation protocols were followed, employing INRAFreeze® as an extender and MiniDigitcool® (IMV Technologies; France) as the automatic freezer.

2.2. Experimental Groups

Three straws for each ejaculate were thawed at 37°C in a water bath for 50 seconds. Semen samples were deposited in an Eppendorf Tube containing 500µL of INRA96® previously warmed at 37°C. The resulting mix was gently shaken split into two aliquots. One aliquot was centrifuged at 600xg for 10 minutes to remove the cryoprotectant, the other one was not centrifuged. After centrifugation, the pellet was resuspended with 500µL of INRA96® pre-warmed at 37°C. Then both aliquots were kept inside an incubator at 37°C. The analyses were done at 0 hours, 1 hour and 3 hours after thawing.

Semen analysis encompassed the evaluation of kinematic parameters utilizing Computer-Assisted Semen Analysis (CASA) (section 2.3), determination of proAKAP4 concentrations (section 2.4), and assessment of mitochondrial membrane potential using JC-1 stain (section 2.5). The semen concentration of each sample was calculated employing CASA methodology.

2.3. Motility Analysis

Sperm motility assessment was conducted utilizing a Computer-Assisted Sperm Analysis (CASA) system (IVOS II ®; Hamilton Thorne; IMV Technologies; France). Post-thawing, all samples were maintained at 37 °C in a water bath for the initial minute, followed by transfer to a 37°C incubator for the subsequent 3 hours to sustain optimal temperature conditions for analysis. For examination, 3 μL of sperm samples were dispensed onto a prewarmed Leja® slide at 37 °C. Three distinct fields were examined to assess various motility descriptors including total motility (TM, %), progressive motility (PM, %), curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity (LIN, %), straightness (STR, %), amplitude of lateral head displacement (ALH, μm), and frequency of head displacement (BCF, Hz).

The CASA parameters were set as follows: magnification of x200 with 10 randomly selected fields; acquisition of 60 images per second; capture of 30 sequences in each analysis; particle area set to 3 pixels; connectivity threshold set at 6; minimum number of images required to calculate ALH set at 10. The cut-off value for motile spermatozoa was established as VAP ≥20 µm/s, while progressive motility was defined as STR ≥80% and VAP ≥30 µm/s.

2.4. ProAKAP4 ELISA

ProAKAP4 quantification was performed using a commercially available quantitative enzyme-linked immunosorbent assay (ELISA). The analysis was conducted at the ONIRIS Laboratory in Nantes, France, utilizing the Horse 4MID® kit (SPQI, Lille, France). This kit employs a quantitative ELISA sandwich assay designed to specifically detect and quantify proAKAP4 in equine semen samples. A reference curve was generated using a provided seven-point standard solution to determine the concentration of proAKAP4 present in each stallion's thawed semen sample.

Sample preparation followed the manufacturer's instructions for cryopreserved stallion semen. Forty microliters of diluted semen from each experimental group described in section 2.2 were mixed with 160 µl of specific lysis buffer included in the kit. After vigorous vortex mixing for one minute, 200 µL of dilution buffer was added and rapidly mixed before loading into the 96-well coated plate. The plate was then incubated for 2 hours under gentle agitation with an ELISA-plate horizontal shaker.

Following three successive washing steps, horseradish-conjugated detection antibody was added to each well and further incubated for 1 hour. Subsequently, TMB substrate was added, initiating a color reaction after 10 minutes of incubation. The intensity of the color reaction, measured spectrophotometrically with a 450 nm filter, is quantitatively proportional to the amount of proAKAP4 present in each sample. The color reaction was halted by adding a stop solution to each well, and color intensities were measured using a spectrophotometer.

Optical densities were then used to determine proAKAP4 concentration with the calculation sheet provided by the manufacturer (SPQI, Lille, France). Specifically, proAKAP4 concentration was expressed in ng per 10 million spermatozoa. This concentration was calculated using the formula: proAKAP4 ng/10 million spermatozoa = (proAKAP4 in ng/mL / spermatozoa M/mL) x 10 x 16 x (2/3). The 10x factor accounts for the final concentration in 10 million spermatozoa, while the 10x dilution factor corresponds to the post-thawed sample dilution during sample preparation. The 2/3 factor corresponds to the dilution volume loaded in each well of the 96-well plate in the Horse 4MID® assay.

2.5. Mitochondrial Membrane Potential

Mitochondrial membrane potential measurement was conducted utilizing the Guava® Easycyte II™ flow cytometer (Guava Technologies Inc., Hayward, CA, USA, distributed by IMV Technologies, France) and EasyKit™2 kit 37 of ready-to-use reagents (IMV Technologies, France). The EasyKit™2 comprises a 96-well plate containing the JC-1 fluorochrome, which is specific for mitochondrial membrane potential.

At each time point (0, 1, and 3 hours), 190 μL of EasyBuffer® (IMV Technologies, France), 10 μL of DMSO, and 1.75 μL of diluted semen were deposited in each well. Subsequently, the plate was incubated in darkness for 30 minutes at 37°C. Analysis of the wells was performed using the Guava® Easycyte II™ flow cytometer, calibrated beforehand with the Easy Check® kit (Guava Technologies Inc., MA, USA), and the Express Pro® software (IMV, France).

The EasyKit™2 fluorochrome emits maximum fluorescence at 525 nm (green) and 596 nm (red). For each well, 5,000 spermatozoa were analyzed. The JC-1 fluorescent stain serves as a marker of the intermediate piece of spermatozoa with high membrane potential and low membrane potential mitochondria. Polarized, active mitochondria are colored orange, while depolarized, inactive mitochondria are colored green, as per manufacturer-defined parameters.

Results are expressed as a percentage of "polarized" mitochondria (strongly or partially) and as a percentage of "depolarized" mitochondria. In our investigation, mitochondrial membrane potential is quantified by the percentage of "polarized" mitochondria, referred to as "active" mitochondria.

2.6. Statistical Analysis

The statistical analysis commenced with the assessment of data normality and homoscedasticity using the Shapiro-Wilk and Levene tests, respectively. In instances where data deviated from normal distribution, the arcsin √x transformation was employed to fulfill parametric assumptions. Subsequently, a mixed generalized linear model of repeated measures was applied to compare proAKAP4 concentration, total and progressive motility, and mitochondrial membrane potential of equine sperm across different incubation times (0 h, 1 h, and 3 h). Treatment (with cryoprotectant and without cryoprotectant) was considered as the fixed-effects factor, while individual horses were treated as random-effects factors. Furthermore, Pearson's correlation analysis was conducted to assess the correlation coefficients (r) between ProAKAP4 concentration and sperm motility parameters, as well as mitochondrial membrane potential, within each treatment and at each time point.

Statistical analyses were conducted using the R statistical package (V 4.0.3, R Core Team; Vienna, Austria), while graphical representations were generated using GraphPad Prism software (V 8.4.0, GraphPad Software LLC; San Diego, CA, USA). The significance level was set at p < 0.05 for all analyses, and results are presented as mean ± standard error of the mean (SEM). Analyzes of the experimental data were performed with the statistical package R (V 4.0.3, R Core Team; Vienna, Austria), while the graphs were elaborated with the GraphPad Prism software (V 8.4.0, GraphPad Software LLC; San Diego, CA, USA). The minimum level of statistical significance was set at p < 0.05 for all analyses. Results are expressed as mean ± standard error of the mean (SEM).

3. Results

3.1. Correlation between proAKAP4, Motility Descriptors and Mitochondrial Membrane Potential

Total and progressive motility showed a positive correlation with proAKAP4 concentration at 0, 1 and 3 hours. However, considering the different descriptors of sperm motility, only those that refer to speed (VCL, VSL, VAP) and the sperm Lateral Head Displacement (ALH) evidenced a positive correlation with proAKAP4 levels at 0 hours (Table 1).

3.2. Evolution of proAKAP4, Motility Descriptors and Mitochondrial Membrane Potential over 3 Hours

No significant differences on proAKAP4 concentration were observed between the batch of samples with and without cryoprotectant at 0 and 1hours of incubation. However, regardless of cryoprotectant removal a significant decrease (P<0.05) of proAKAP4 levels was observed at 3 hours after thawing. The removal of the cryoprotectant had no effect on the concentration of proAKAP4 at 0 and 1h, but showed a significant decrease (P<0.05) at 3h when the cryoprotectant was eliminated (Figure 1).

The evolution of total motility (MT) and progressive motility (MP) in percentages is shown in Figure 2A,B. In terms of total motility (MT), there was no difference between samples with and without cryoprotectant and between incubation time. Regarding progressive motility (MP), no significant differences were observed between incubation time for booth type of samples, with or without cryoprotectant. Nevertheless, samples with cryoprotectant showed significantly lower progressive motility at all incubation times analyzed.

Analyzing the mitochondrial membrane potential, no significant difference was observed between samples (with or without cryoprotectant) at different incubation times. However, in both cases, removing or not the cryoprotectant, a significant (P<0.05) decrease between 0 and 1 hour of incubation was observed but not between 1 and 3 hours (Figure 3).

4. Discussion

The results of this experiment confirm a positive correlation between proAKAP4 concentration and total and progressive motility in a stallion frozen/thawed semen at any time of our study. This positive correlation has been found previously in fresh semen in stallions [13,14,16] and in other mammals like bulls [23,29,30], jackass [24], dromedaries [28] rams [27], bucks [31], pigs [32], dogs [25], mice [33,34], and even in humans [35,36]. In the present study, this correlation between proAKAP4 and motility parameters is sustained during the 3 hours post thaw in contrast with the same experiment in donkeys [24] where this correlation between motility descriptors and proAKAP4 concentration was only described at time 0 and 1 hour after thawing but not at 3 hours. Horse and donkey, although phylogenetically close, are different species with their own reproductive strategies. All kinematic parameters for donkeys are significantly higher than for horse. Donkey sperm is more rapid and linear than horse one and, possibly, the effect of proAKAP4 on the motility patterns is also different between booth species [37]. Nevertheless, obtained results confirm proAKAP4 as a good marker of sperm quality and longevity; kinematic parameters and longevity are both important for the fertilizing ability of frozen thawed spermatozoa [39,40].

However, analyzing the descriptors of the sperm trajectory only the velocity parameters (VCL, VCL and VAP) and the lateral head displacement (ALH), and only at 1 h of post thaw incubation but not later, correlate positively with proAKAP4 concentration. This correlation has been previously found in stallions [13] and in bulls [20,26,27,34]. In other mammals, like bulls, progressive motility [37] and velocity descriptors [38,39] have been described as sperm quality and even fertility markers. It seems evident that proAKAP4 has a relationship with the sperm motile ability but not an effect on the kind of movement, or trajectory description, in stallion. Further studies are needed in order to evaluate the effect on frozen/thawed semen fertility

ProAKAP4 as a reservoir of motility and energy is described in several articles and drawn as a "backpack" with the energy of spermatozoa [12,18,32]. ProAKAP4 does not significantly decrease in the first hour but diminishes between one and three hours; in this case, there is no significant difference between the groups containing or not cryoprotectants at 0 and 1h. However, at 3h the samples without cryoprotectant showed a significant decrease in the proAKAP4 concentration. On the other hand, removing the cryoprotectant higher levels of Progressive Motility were observed, indicating higher consumption of this energy “backpack”.

Total motility showed no significant difference between the centrifuged group (removing the cryoprotectant) and the non-centrifuged group (maintaining the cryoprotectant) and thus there is no non-significant difference between the 3 times studied. Centrifugation at 600G during 10 minutes has not affect significantly total motility and viability parameters [44]. However, removing the cryoprotectant the Progressive Motility evidenced higher values at all analyzed times. This may indicate a deleterious effect of the cryoprotectant on sperm motility and, as explained before, higher proAKAP4 consumption when the Progressive Motility is higher.

Comparing proAKAP4 with total and progressive motility, proAKAP4 holds well the first hour because it maintains motility and then suddenly decreases, but the motility has decreased slowly throughout the period. Comparing with data in donkeys [21], where our team carried out the same experiment, the results are very similar and comparable. It can be concluded that proAKAP4 is a structural protein that functions as the potential motility reserve of the equines spermatozoon [9] so by measuring the concentration it is probably possible to predict whether post-thaw motility will be constant over some time.

Longevity of post-thawed spermatozoa can be affected depending on the extender in different species. In deer, sperm longevity at four hours post-thawed is different depending on the cryoprotectant used [45]. In carp, supplement the extender with seminal plasma transferrin improves cryopreservation survival and longevity of the frozen-thawed semen [46]. In the present study, the used extender is an egg yolk-based extender (INRA Freeze ®). Egg yolk-based extenders are beneficial for semen preservation and post-thaw survival in tom cats [47] and significantly improve sperm thaw quality and longevity in African wild dog [48]. In stallion, cryoprotectants agents like phosphodiesterase inhibitors, caffeine, or taurine increase motility and longevity in post-thaw conditions [8].

No significant differences in pro AKAP4 concentration was observed in stallions between batches with or without cryoprotectant; however, in donkey [24], a significant difference in proAKAP4 concentration has been found between the samples with or without cryoprotectant. This difference can be explained because a different cryoprotectant was used in both experiments. Blommaert et al., [15] showed that proAKAP4 concentration differs significantly depending on the extender used in stallion thawed semen. Kowalczyk et al., [38] showed how the addition of Holothuroidea extract in the semen extender modulates positively the proAKAP4 concentration in the frozen/thawed bovine semen. In the present study, INRAFreeze ® was used to freeze all the ejaculates and in the case of donkey [24] BotuCrio ® was the election. Observed differences between donkey and horse can be explained because a different cryoprotectant has been used in both experiments; furthermore, other components could have an impact and there could be species-specific sensitivities, as explained before. Blommaert et al., [15] have shown that proAKAP4 concentration differs significantly depending on the extender used in stallion thawed semen. Further analysis comparing these 2 extenders (BotuCrio ® and INRAFreeze ®) is needed to elucidate if they exert the same effect on proAKAP4 or not. There are two hypotheses about these differences between extenders one is that a toxic effect might degrade proAKAP4 directly, and second, one extender might converse proAKAP4 more rapidly to AKAP4 than the other, dependent on the extender composition.

For mitochondrial membrane potential, using the JC1 stain, there is no significant difference between groups containing cryoprotectants or not, but the change over time is contrary to proAKAP4, decreasing rapidly between 0 and 1 hour only. The absence of a relationship between the mitochondrial membrane potential and proAKAP4 levels suggests independence between spermatozoa energy production and the transmission of motion. This can explain why in previous reports no direct relationship was observed between mitochondrial activity stimulation and spermatozoa motility increase [49,50].

5. Conclusions

In conclusion, proAKAP4 is a good marker of sperm quality correlating positively with total and progressive motility at any time in our study. It also correlates with velocity kinematic parameters at the moment of thawing. ProAKAP4 can be considered a long-lasting motility marker because it reduces at the time when motility parameters have decreased. Removing the cryoprotectant from frozen/thawed horse semen shows better maintenance of progressive sperm motility at 3h and lower proAKAP4 concentration. Greater progressive motility means more proAKAP4 consumption.