Introduction
There are currently 40 species of wild cats described and 18 of them are listed as vulnerable or threatened with extinction (IUCN, online). Therefore, a great effort is required for the conservation of these animals and assisted reproductive technologies are strong allies for this purpose. However, the establishment of protocols in wild cats is very difficult, as their biological material is scarce. Therefore, due to genetic similarities and availability, the domestic cat is often used as a research model (Klaus et al., Reference Klaus, Eder, Franz and Müller2016).
Cryopreservation of semen is an essential tool for long-term storage, allowing genetic preservation, transporting, and application in artificial insemination and in vitro fertilization, thereby contributing to the conservation of threatened species. However, cat semen cryopreservation protocols are not well established, resulting in below-expected post-thaw outcomes regarding sperm quality (Buranaamnuay, Reference Buranaamnuay2017). This process potentially induces spermatozoa damage (Luvoni, Reference Luvoni2006), resulting in a reduction in viability, motility, and structural integrity (Watson, Reference Watson2000; Isachenko et al., Reference Isachenko, Isachenko, Katkov, Dessole and Nawroth2003). So, it is important to test different substances that may have a role as a cryoprotectant to increase the overall cryopreservation efficiency.
Antifreeze proteins (AFPs) are a class of polypeptides first described in Arctic fish as an adaptive mechanism at sub-zero temperatures and then in arthropods, plants, algae, bacteria, and fungi (De Vries and Wohlschlag, Reference DeVries and Wohlschlag1969; Kim et al., Reference Kim, Lee, Hur, Lee, Park and Koo2017). Four fish-derived types of AFP (I, II, III, and IV) and AFGP (glycoprotein) have been described, these differ according to their composition and tertiary structure (Cheung et al., Reference Cheung, Ng and Wong2017). These proteins play a role in inhibiting the normal growth and morphology of ice crystals; lowering the freezing point in a non-colligative manner, at a process called thermal hysteresis; inhibiting the recrystallization; and protecting the plasma membrane (Kim et al., Reference Kim, Lee, Hur, Lee, Park and Koo2017). Therefore, the addition of AFP I to the cryopreservation medium could benefit post-thaw semen quality as it presents positive results for sperm cryoresistance, resulting in higher sperm kinetics, higher normal sperm rates and better plasma integrity, in species such as cattle (Prathalingam et al., Reference Prathalingam, Holt, Revell, Mirczuk, Fleck and Watson2006) and ram (Payne et al., Reference Payne, Oliver and Upreti1994; Upreti et al., Reference Upreti, Payne, Duganzich, Oliver and Smith1996; Correia et al., Reference Correia, Espírito-Santo, Braga, Carvalho-de-Paula, da Silva, Brandão, Freitas, Ungerfeld and Souza-Fabjan2021). To the authors’ best knowledge, there have been no data published about the use of any AFP for cat semen cryopreservation. Therefore, this study aimed to assess the use of different concentrations of AFP I added into the extender for sperm cryopreservation in domestic cats.
Materials and methods
Animals
This study did not require the Ethics Committee of Animal Use approval of the University, as the domestic male cat (n = 86) testes were collected at local veterinary clinics, as byproducts from owner-requested routine orchiectomies.
Experimental design
Cat spermatozoa were collected from the cauda epididymis; sperm were incubated in Tris buffer, and pooled. The pooled fresh sperm were evaluated and allocated into three experimental groups according to the final concentration of AFP I after its addition in both Extenders I and II: control (0 µg/ml), 0.1 µg/ml (10 µg), and 0.5 µg/ml (50 µg). The AFP I concentrations used were based on the study of Correia et al. (Reference Correia, Espírito-Santo, Braga, Carvalho-de-Paula, da Silva, Brandão, Freitas, Ungerfeld and Souza-Fabjan2021). Subsequently, the pooled sperm samples were frozen at a final concentration of 25 × 106 spermatozoa/ml (Buranaamnuay, Reference Buranaamnuay2015). Pre-freezing evaluation consisted of motility, functional membrane integrity, mitochondrial activity, vitality, chromatin condensation, and viability, and acrosome status. For thawing, the straws were placed at 37°C for 30 s and promptly evaluated for the analyses cited above, plus sperm kinetics, morphology, and binding to egg perivitelline membrane assessment.
Sperm harvesting
The removed testicles from each cat were sent to the laboratory in sterile 0.9% (w/v) NaCl solution at 4°C within 2 h after surgery. Epididymis and proximal vasa deferentia were isolated and dissected free from visible blood vessels and connective tissues. Afterward, the epididymis was transversely cut and placed for 10 min in 400 µl of Tris buffer at 37°C. After that, the tissue fragments were removed, and the fresh samples were pooled and evaluated for sperm motility, vigour, and concentration. All sperm samples used in this study exhibited values of ≥50% subjective motility and 3 for vigour. Nine replicates were conducted, ranging between seven and 13 animals each.
Preparation of medium
Tris buffer [3.025 g Tris, 1.4% citric acid, 0.8% glucose, 0.1% antibiotic and antimycotic solution (streptomycin, penicillin, and amphotericin B), in MilliQ water], Extender I (the same composition as Tris buffer, with added 3% glycerol, 20% egg yolk, and AFP I according to the experimental group) and II [same as Extender I, but containing 7% glycerol and 0.6% sodium dodecyl sulfate (SDS)] were prepared according to Chatdarong et al. (Reference Chatdarong, Thuwanut, Manee-in, Lohachit and Axnér2010) and Mizutani et al. (Reference Mizutani, Sumigama, Nagakubo, Shimizu, Oba, Hori and Tsutsui2010) (Table 1).
Table 1. Composition of sperm buffer and extenders prepared and used in this study
Abbreviations: SDS: sodium dodecyl sulfate; Tris: tris(hydroxymethyl)aminomethane.
Cryopreservation process
Sperm samples were pooled, allocated into one of the three groups according to AFP I concentrations (Figure 1), centrifuged at 700 g for 6 min (Thammapradit and Ponglowhapan, Reference Thammapradit and Ponglowhapan2018), and their supernatant was removed. The pellets were diluted in Extender I and kept at 4°C for 1 h. Thereafter, the respective Extender II was added and an aliquot from each experimental group was removed for pre-freezing analyses. The 0.25 ml straws were filled, and kept at 4°C for 1 h to equilibrate. Then, the straws were suspended horizontally 7 cm above the liquid nitrogen level for 10 min and immediately immersed (Toyonaga et al., Reference Toyonaga, Sato, Morita, Watanabe, Oba, Mizutani, Hori and Tsutsui2010).
Figure 1. Illustrative diagram of the semen cryopreservation process: 1. Incubation in Tris buffer. 2. Formation of pooled semen. 3. Centrifugation at 700 g for 6 min. 4. Supernatant removal. 5. Pellets dilution in Extender I. 6. Addition of Extender II. 7. Loading of 0.25 ml. 8. Equilibration at 4°C for 1 h. 9. Freezing in Styrofoam box for 10 min. 10. Plunge into LN2.
Sperm assessments
Sperm concentration, subjective motility and vigour
Sperm concentration was determined by Neubauer chamber. Subjective (percentage of motile spermatozoa) and vigour (scale from 0 to 5, with 0 being immotile sperm and 5 being fast, steady forward progression) were evaluated using a phase-contrast microscope at ×400 magnification. These parameters were only assessed in fresh samples.
Sperm kinetics
Sperm motility patterns were only assessed in frozen–thawed samples using computer-assisted sperm analysis (CASA) and the SCA® system (Sperm Class Analyzer Microptic, Nikon Eclipse Ci – Tokyo, Japan) connected to a phase-contrast microscope. Software settings were adjusted for cat spermatozoa. Standard configuration of parameters was the spermatic head dimension detectable by the system between 5 and 60 µm2. In total, 25 images per second were captured; measurements were performed in a 24 × 24 mm cover slide with a 10-µl volume, at ×100 magnification. The following parameters were analyzed: Total Motility (%), Progressive Motility (%), Non-progressive Motility (%), Fast Sperm (%), Medium Sperm (%), Slow Sperm (%), curvilinear velocity (VCL, µm/s), straight-line velocity (VSL, µm/s), average path velocity (VAP, µm/s), linearity (LIN, %), straightness (STR, %), WOB (mean value of the ratio between VAP and VCL, %), amplitude of lateral head displacement (ALH, µm), beat/cross frequency (BCF, Hz). Spermatozoa were classified according to their curvilinear velocity (VCL). It was considered fast when it was above 75 μm/s; medium when it was between 45 and 75 μm/s; slow when it was between 10 and 45 µm/s; and immobile when the value was less than 10 μm/s. Sperm cells showing straightness (STR) above 80% were characterized as progressive motile sperm.
Functional membrane integrity
For functional integrity, the hypo-osmotic swelling test was performed (Ramu and Jeyendran, Reference Ramu and Jeyendran2013). Briefly, sperm was mixed with a hypo-osmotic solution of 100 mOsm/kg of MilliQ water (1:9) and incubated at 37ºC for 20 min. At least 200 sperm cells were counted, through wet preparation in phase-contrast microscopy (×1000 magnification). The results were based on counting spermatozoa that folded the tail, meaning that their membrane was still intact when exposed to the hypo-osmotic environment.
Sperm viability and acrosome status
Trypan blue/Giemsa staining was applied as reported by Kovács and Foote (Reference Kovács and Foote1992), with minor modifications. Briefly, the sperm were incubated for 15 min with trypan blue 0.2% at 37ºC (1:1). Then, a smear slide was prepared, air dried and Giemsa stain (10%) was applied for at least 1 h. Ultimately, the slides were rinsed with running water, air dried, and evaluated under an optical microscope at ×1000 magnification. At least 200 sperm cells were evaluated in each smear. Sperm were classified as live sperm with intact acrosome (LSIA): purple; dead sperm with intact acrosome (DSIA): violet; live sperm with acrosome reacted (LSAR): light pink; and dead sperm with acrosome lost (DSAL): post acrosome region violet and light pink acrosome.
Chromatin condensation
The acidic aniline blue staining was applied to assess the chromatin condensation according to Dadoune et al. (Reference Dadoune, Mayaux and Guihard-Moscato1988), with modifications. The sperm smear was fixed in 4% formalin solution for 5 min. Then, it was washed and stained in 5% aniline blue solution mixed with 4% acetic acid (pH 3.5) for 5 min and once more washed. Finally, the slide was stained in a 0.5% eosin solution for 30 s, washed in water, and dried in air. In total, 200 sperm cells were evaluated under an optical microscope at ×1000 magnification and immersion. Sperm were differentiated into either intense stained (immature chromatin) or slightly stained nuclei (mature chromatin).
Sperm vitality and morphology
Eosin–nigrosin dye (3% and 8%, respectively) was mixed with the sperm sample (1:1), smeared on a slide, and immediately evaluated for sperm vitality (Cedenho and Miyamoto, Reference Cedenho and Miyamoto1990). Furthermore, the fixed sperm smears allowed the assessment of morphological abnormalities of the sperm tails, cytoplasmic droplets, detached heads, and abnormal mid-pieces (Cocchia et al., Reference Cocchia, Ciani, El-Rass, Russo, Borzacchiello, Esposito, Montagnaro, Avallone, Tortora and Lorizio2010). In the absence of any abnormality, sperm were classified as presenting normal morphology. For both analyses, 200 sperm cells were counted and evaluated under an optical microscope at ×1000 magnification.
Mitochondrial activity
Mitochondrial activity was assessed according to Rui et al. (Reference Rui, Angrimani, Losano, Bicudo, Nichi and Pereira2017). Briefly, the sperm was diluted (1:1) in a 1 mg/ml solution of 3,3′-diaminobenzidine (DAB) and incubated at 37°C for 1 h in the dark. After this period, smears were fixed in 10% formaldehyde for 10 min. At least 200 sperm cells were counted under a phase-contrast optical microscope (×1000 magnification). The sperm were categorized as: all mitochondria active (100% of the midpiece stained: DAB I); most mitochondria active (more than 50% of the midpiece stained: DAB II); most mitochondria inactive (less than 50% of the midpiece stained: DAB III); and all mitochondria inactive (absence of staining in the midpiece: DAB IV).
Sperm binding to egg perivitelline membrane test
Fresh and non-fertile chicken egg yolks were separated from the albumen for perivitelline membrane acquisition. Then, they were washed with phosphate-buffered saline (PBS) and the membranes were cut into squares (0.5 cm2). Finally, the membranes were incubated for 1 h in 1 ml of FERT-TALP and 20 μl of sperm sample at 38.5ºC and 5% of CO2. For evaluation, the membrane was washed three times in PBS, placed on a slide, and 5 μl of Hoechst 33342 stain (1 mg/ml) were added. Five fields per sample were visualized and counted under an epifluorescence microscope (Nikon Eclipse Ci, Nikon Corporation, Japan), at ×600 magnification, and the results were expressed as spermatozoa binding per mm2 of the membrane (Barbato et al., Reference Barbato, Cramer and Hammerstedt1998).
Statistical analysis
All variables were subjected to normality (Shapiro–Wilk and Kolmogorov–Smirnov tests) and homoscedasticity tests. Parametric data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s test, whereas non-parametric data were evaluated using Kruskal–Wallis test, followed by Dunn’s test. Data are presented as mean ± standard error of the mean (SEM). P-values < 0.05 were considered significant and those at 0.05 ≤ P ≤ 0.1 as a tendency. All analyses were performed using IBM SPSS 25 software version (IBM Corporation, Armonk, NY, USA).
Results
Fresh pooled spermatozoa data regarding subjective motility, concentration and total spermatozoa were: 69.4 ± 2.3%, 49.3 ± 5.7 × 106 spermatozoa/ml, and 124.7 ± 11.5 × 106 spermatozoa, respectively.
Data regarding pre-freezing evaluation are presented in Table 2. The subjective motility, vitality, functional membrane integrity, mature chromatin, and acrosome status showed no difference (P > 0.05) among groups. Furthermore, the mitochondrial activity assay indicated a tendency (P = 0.069) for the DAB I category, with the control group presenting a greater (P < 0.05) value compared with both AFP I-supplemented groups. DAB II, III, and IV categories did not exhibit any differences (P > 0.05).
Table 2. Pre-freezing and post-thawed sperm parameters related to the supplementation of 0, 0.1 or 0.5 µg/ml of antifreeze protein (AFP) I in cryopreservation medium of domestic cats. Values are expressed as mean ± standard error of the mean (SEM)
a,b Within a row, values differ significantly at the same moment (either before freezing or 0 h) (P < 0.05).
Abbreviations: DAB: 3′-diaminobenzidine (mitochondrial activity); DAB I: all mitochondria active; DAB II: majority of mitochondria active; DAB III: majority of mitochondria inactive; DAB IV: all mitochondria inactive; DSAL: dead sperm with acrosome lost; DSIA: dead sperm with intact acrosome; LSAR: live sperm with acrosome reacted; LSIA: live sperm with intact acrosome; sperm binding: sperm binding to egg perivitelline membrane.
* Subjective and objective assessments were performed to estimate pre-freezing and post-thawed total motility, respectively.
** Results based on the counting of spermatozoa that folded the tail (intact membrane).
In post-thawing assessments (Table 2), differences were detected in the acrosome status and mitochondrial activity. The LSIA percentages in the control and 0.1 µg/ml groups were greater (P < 0.05) when compared with 0.5 µg/ml. For mitochondrial activity, the DAB II category presented a tendency (P = 0.053) favouring the 0.5 µg/ml group, when compared with the control, whereas 0.1 µg/ml supplementation was similar to both treated groups. Concerning DAB III, the 0.5 µg/ml AFP I addition resulted in a lower percentage compared with the control group. Supplementation of 0.1 µg/ml showed no difference (P > 0.05) compared with the other groups. All other parameters were similar (P > 0.05) among the groups. Similarly, the presence of AFP did not affect (P > 0.05) any sperm kinetic parameter assessed by CASA and the results are shown in Table 3.
Table 3. Post-thawed sperm kinetics parameters performed by computer-assisted sperm analysis regarding the supplementation of 0, 0.1 or 0.5 µg/ml of antifreeze protein (AFP) I in cryopreservation medium of domestic cats. Values are expressed as mean ± standard error of the mean (SEM)
Abbreviations: ALH: amplitude of lateral head displacement; BCF: beat/cross frequency; LIN: linearity; STR: straightness; VAP: average path velocity; VCL: curvilinear velocity; VSL: straight-line velocity; WOB: mean value of the ratio between VAP and VCL.
Discussion
The results presented in this study were not as expected in our initial hypothesis that the supplementation of AFP I in cryopreservation extender would have a beneficial effect on the quality of frozen–thawed spermatozoa. In fact, the addition of AFP I did not result in any major effect; although it negatively affected the acrosome status (LSIA) and positively affected the mitochondrial activity in the 0.5 µg/ml group.
It is important to note that most studies using AFP I in semen cryopreservation extenders have resulted in a beneficial effect. In ram, an improvement in sperm kinetics, motility, membrane integrity, and normal morphology was reported using 0.1–10 µg/ml (Payne et al., Reference Payne, Oliver and Upreti1994; Upreti et al., Reference Upreti, Payne, Duganzich, Oliver and Smith1996; Correia et al., Reference Correia, Espírito-Santo, Braga, Carvalho-de-Paula, da Silva, Brandão, Freitas, Ungerfeld and Souza-Fabjan2021), whereas in cattle, the membrane integrity parameter was superior following supplementation with 0.1–10 µg/ml of AFP I (Prathalingam et al., Reference Prathalingam, Holt, Revell, Mirczuk, Fleck and Watson2006). However, this was not true for all species studied to date. In mice, supplementation of 0.01–100 µg/ml did not improve sperm quality, in contrast, the results showed that AFP I decreased motility (Koshimoto and Mazur, Reference Koshimoto and Mazur2002). We can hypothesize that similar effects regarding physical stress during cryopreservation are expected to occur in species presenting similar sizes of sperm heads. Similarly, this parameter might also affect binding between AFP I and the cell membrane and, consequently, the protective effects of the molecule during cryopreservation. For example, sperm from mice (6.30 and 3.29 μm) and cats (5.56 and 3.10 μm) have greater proximity in size (length and width, respectively) when compared with cattle (7.87 and 4.05 μm), and ram (7.13 and 3.79 μm). It has been proposed that the area and shape of the sperm head can cause variations in water movement, exchanges of heat, ions, and cryoprotectants (Curry, Reference Curry2000; Esteso et al., Reference Esteso, Soler, Fernández-Santos, Quintero-Moreno and Garde2013). Therefore, these differences in physical properties dependent on size and shape could be one of the factors responsible for resistance to freezability.
Another possibility is related to the composition of Extender II. It is important to note that all media used for this article were based on previous studies on domestic cats. Indeed, SDS is generally added to the cryopreservation medium (Buranaamnuay, Reference Buranaamnuay2017) as its outcomes have shown improvement in the quality and cryotolerance of the spermatozoa (Axnér et al., Reference Axnér, Hermansson and Linde-Forsberg2004; Mizutani et al., Reference Mizutani, Sumigama, Nagakubo, Shimizu, Oba, Hori and Tsutsui2010; Zambelli et al., Reference Zambelli, Iacono, Raccagni and Merlo2010). For these reasons, SDS was supplemented to our cryopreservation extender to a final concentration of 3 mg/ml, as suggested by Mizutani et al. (Reference Mizutani, Sumigama, Nagakubo, Shimizu, Oba, Hori and Tsutsui2010). However, SDS could have a detrimental effect on the structure of AFP I, by causing disturbances in the protein’s secondary structure (Kar et al., Reference Kar, Mroue, Kumar, Tejo and Bhunia2016), which could possibly explain the absence of consistent positive effects of the AFP I molecule in this study.
It can be speculated that the concentration range tested in the current study was not adequate to reach the beneficial effects of AFP. Another important factor is the origin of the sperm. Epididymal sperm seem to exhibit lower cryotolerance compared with that of ejaculated sperm (Luvoni and Morselli, Reference Luvoni and Morselli2017), which could be attributed, for example, to the absence of contact with seminal plasma (SP) proteins. As these proteins have important effects on sperm cryopreservation due to their role in membrane stabilization (Bernardini et al., Reference Bernardini, Hozbor, Sanchez, Fornés, Alberio and Cesari2011), their absence could explain why, even when adding some theoretically useful substances such as AFP, the results are still contrasting. For instance, in ram (Colás et al., Reference Colás, Junquera, Pérez-Pé, Cebrián-Pérez and Muiño-Blanco2009), cattle (Patel et al., Reference Patel, Gandotra, Cheema, Bansal and Kumar2016), and Iberian red der (Martínez-Pastor et al., Reference Martínez-Pastor, Anel, Guerra, Alvarez, Soler, Garde, Chamorro and de Paz2006), the supplementation of semen with SP was beneficial for cryopreservation. Therefore, further studies are needed that aim to detect the most appropriate concentration of AFP I and/or to assess different parameters, such as the role of SP when using epidydimal sperm.
In conclusion, the supplementation of 0.1 and 0.5 µg/ml of AFP I did not exhibit beneficial effects on the overall cryopreservation quality of epididymis spermatozoa from domestic cats. However, it remains unclear whether an adjustment to the cryopreservation protocol, such as a change in AFP I concentration, could lead to a more promising outcome.
Data availability
The data that support the study findings are available from the corresponding author upon reasonable request.
Acknowledgements
The authors thank the following organizations for financial support: Fluminense Federal University (FOPESQ), the Brazilian National Council for Scientific and Technological Development (CNPQ; project #434302/2018–0), and the Carlos Chagas Filho Foundation for Research Support in the State of Rio de Janeiro (FAPERJ). The authors also thank the Instituto Municipal de Medicina Veterinária Jorge Vaitsman for the surgical procedures and donation of traits for this study. The authors are grateful to Rachel Ferreira Braga and Viviane Lopes Brair for their technical assistance. LPA, PVSP, TAO, and LFLC are CAPES fellows (Finance code 001) and EMV was supported by CNPq. FZB and JMGS-F are both FAPERJ and CNPq fellows.
Author contributions
LPA, LFLC, and JMGS-F designed the study. LPA, PVSP, and TAO conducted the experiments. LPA, LFLC, EMV, and JMGS-F analyzed the data. LPA wrote the first manuscript, and PVSP, TAO, LFLC, EMV, FZB, and JMGS-F critically revised and approved the final version of the manuscript.
Conflicts of interest
The authors declare no conflict of interest.
Ethical standards
The authors declare that all procedures were performed according to national and institutional guides on the care and use of animals.
