Cooling of porcine semen in an extender supplemented with isoespintanolABSTRACT:
Spermatozoa experience oxidative, osmotic, chemical, and thermal stresses when cooled, which degrade the quality and fertilizing capacity of the cells. Adding antioxidants to the sperm extender mitigates these alterations. This study evaluated the effect of isoespintanol (ISO) on boar semen subjected to cooling. Fifteen ejaculates from five boars (Susscrofadomestica) were extended in Beltsville thawing solution (BTS) supplemented with 0 µM (control), 5 µM (ISO5), 10 µM (ISO10), 15 µM (ISO15), 20 µM (ISO20), 25 µM (ISO25), and 30 µM (ISO30) of ISO, which were then cooled for five days at 16 °C. Sperm kinetics, total motility (TM), and progressive motility (PM) were evaluated every 24 h using an IVOS computer-assisted sperm analysis (CASA) system. On day 1 and day 5 of cooling, a hypoosmotic test, spectrofluorometry, and flow cytometry were performed to evaluate the following: membrane functionality, measured as a function of hypoosmotic swelling (HOS); total antioxidant capacity (TAC); reactive oxygen species (ROS); and mitochondrial membrane potential (Δ¥M). Regression analysis and comparison of means using the Duncan test were performed. The ISO added had a slight impact on sperm motility, as evidenced by a reduction in TM at 24 h of cooling (but not prior) with the addition of 20 µM of ISO. Similarly, no effect of the ISO on the kinetics and functional integrity of the sperm membrane was observed at 96 h of cooling; however, the regression coefficients indicated that the ISO lowered the rate of decrease in sperm motility and the proportion of rapid spermatozoa relative to the concentration of ISO used. The ISO did not affect the TAC of the cooled semen; however, different concentrations of ISO lowered ROS production in the semen after 96 h of cooling. ISO also impacted the Δ¥M of the spermatozoa at 0 h of cooling, increasing the proportion of low Δ¥M cells and decreasing the proportion of high Δ¥M cells. In conclusion, ISO can reduce the loss of quality and oxidative stress occurring in boar semen during cooling and can modulate the mitochondrial activity of sperm.
Key words:
antioxidant; boar; preservation; semen quality; spermatozoa
RESUMO:
Durante a refrigeração, os espermatozoides sofrem estresse oxidativo, osmótico, químico e térmico, que diminuem sua qualidade e afetam sua capacidade de fertilização. A adição de antioxidantes ao diluente espermático é uma alternativa para mitigar essas alterações. O objetivo desta pesquisa foi avaliar o efeito do isospintanol (ISO) na refrigeração do sêmen suíno. Quinze ejaculados de cinco varrascos (Sus scrofa domestica) foram diluídos em BTS suplementado com ISO a 0 (controle), 5 (ISO5), 10 (ISO10), 15 (ISO15), 20 (ISO20), 25 (ISO25) e 30 (ISO30) µM e foram refrigerados por cinco dias a 16 °C. A motilidade total (MT), motilidade progressiva (MP) e cinética dos espermatozóides foram avaliadas a cada 24 h com um sistema CASA IVOS. Nos dias um e cinco de refrigeração, foram avaliadas a funcionalidade da membrana, a capacidade antioxidante total (CAT), as espécies reativas de oxigênio (ROS) e o potencial de membrana mitocondrial (Δ¥M), através do teste hiposmótico (HOS), espectrofluorimetría e citometria de fluxo. Foram realizadas análises de regressão e comparação de médias, pelo teste de Duncan. A adição de ISO teve pouca influência na motilidade espermática, apresentando apenas redução na MT em 24 h de refrigeração, devido à adição de 20 µM. Da mesma forma, não foi observada influência de ISO na cinética e integridade funcional da membrana em 96 horas de refrigeração; porém, os coeficientes de regressão mostraram que ISO produziu menor taxa de diminuição da motilidade e proporção de espermatozoides rápidos dependendo da concentração utilizada. ISO não influenciou significativamente na CAT do sêmen refrigerado; entretanto, diferentes concentrações de ISO reduziram a produção de EROs a partir do sêmen após de 96 h de refrigeração. ISO também influenciou o Δ¥M dos espermatozóides em 0 h de refrigeração, com aumento das células de baixo Δ¥M e diminuição das células de alto Δ¥M. Em conclusão, o isospintanol pode reduzir a perda da qualidade e o estresse oxidativo do sêmen suíno durante a refrigeração e pode modular a atividade mitocondrial do esperma.
Palavras-chave:
Antioxidante; suíno; preservação; qualidade do sêmen; espermatozoides
INTRODUCTION:
The cryopreservation of boar semen is a critical biotechnology in the conservation and dissemination of genetic resources (YESTE et al., 2017). However, in swine reproduction, 99% of artificial inseminations are performed using refrigerated semen (PEZO et al., 2019), with no cryopreservation. This is because cryopreservation significantly reduces the fertilizing capacity of porcine spermatozoa (KNOX, 2015), inducing the death of 30% to 50% of sperm cells via cryogenic damage (WABERSKI et al., 2019).
However, when refrigerated at 5 ºCor 15 - 17ºC, boar spermatozoa undergoes morphological and functional changes (HIDALGO, 2013) due to heat shock and oxidative stress, which affect sperm quality (TIAN et al., 2019). This oxidative stress induces cell damage and lowers sperm motility and the integrity of the spermatozoa plasma membrane, significantly shortening the sperm life span (PEZO et al., 2020).
Hence, there is a need to find a way to counteract the stress produced during cold storage, such as using antioxidant molecules via supplementation in the extender to improve the fertilizing characteristics of semen at low temperatures and to enhance the preservation of desirable sperm characteristics (CÓRDOVA et al., 2009).
Isoespintanol (ISO) is a biosynthetic analog of thymol extracted from the leaves of Oxandra cf. xylopioides (Annonaceae), a naturally occurring substance with sufficient antioxidant capacity to neutralize free radicals and decrease the degree of membrane lipid peroxidation in sperm cells (ROJANO et al., 2008). Previous studies have demonstrated the ability of ISO to reduce alterations in equine and canine spermatozoa during cryopreservation (USUGA et al., 2021; RESTREPO et al., 2022). However, no studies have been conducted on the effects of this molecule (i.e., ISO) on refrigerated semen. This study evaluated the capacity of ISO as an alternative natural antioxidant for the conservation of boar semen (Susscrofadomestica) subjected to cooling.
MATERIALS AND METHODS:
Collection, initial evaluation, and processing of the semen
A total of 15 porcine ejaculates were collected from five boars using the gloved hand method equipped with polyvinyl gloves and a solid dummy. After ejaculation, the first part of the ejaculate or gel fraction was filtered, and the seminal material was immediately incubated at 37 Cº in a water bath.
For each ejaculate, volume, concentration, and sperm motility were evaluated using a graduated cylinder, an SDM1 photometer (Minitube GmbH, Tiefenbach, Germany), and the HTM-IVOS computer-assisted semen analysis (CASA) system, version 12.3 (Hamilton Thorne, Beverly, MA, USA). Then, seven 5 mL aliquots were prepared, diluting the semen in an MR-A extender (Kubus S.A, Madrid, Spain), until a concentration of 60 × 106 spermatozoa/mL was reached. These aliquots, with a final volume of 5 mL, were supplemented with ISO (5, 10, 15, 20, 25, and 30 µM), along with a control without an antioxidant, and then stored at 16 ℃ for 96 h (5 days). Ejaculates with a minimum total motility (TM) of 80% and progressive motility (PM) of 60% were processed.
Assessment of semen quality parameters
During the five days of storage, the motility (TM and PM) and kinetics of the spermatozoa were evaluated using the HTM-IVOS CASA system. The functional integrity of the sperm plasma membrane was assessed at 0 h and 96 h of cooling using a modified hypoosmotic swelling (HOS) test methodology (NEILD et al., 2001), employing a 1.52% fructose-based solution. The resulting mixture of semen and hypoosmotic solution was then incubated at 37 ℃. A drop of this mixture was spread on a warm slide with a coverslip, and 200 spermatozoa were evaluated at 1000 magnification on an optical microscope (BUCKETT, 1997; FANG, 2017).
At the two cooling duration measurement junctures (0 h and 96 h), several complementary evaluations described subsequently were also performed via spectrofluorometry.
Total antioxidant capacity (TAC) was evaluated with the ABTS●+ test (ARTS et al., 2004), performed using 10 μl of semen and 990 μl of ABTS●+ radical solution. After 30 minutes of reaction at ambient temperature in the dark, TAC was measured in a Jenway 6405 UV/Vis spectrophotometer (Jenway Ltd., Essex, UK) as a function of the change in absorbance measured against a reference solution. The radical solution was generated via the oxidation of 3.5 mM ABTS with 1.25 mM potassium persulfate. After 24 h of reaction, the absorbance was adjusted with phosphate buffered saline (PBS) at pH 7.4 to 0.70 units at λ = 732 nm; then compared against a Troloxstandard curve.
Reactive oxygen species (ROS) detection was performed using fluorescein diacetate (FDA) (GUTHRIE & WELCH, 2006). Every sample was prepared with 30 μl of 40 mM FDA (Thermo Fisher, Inc., Waltham, USA), 240 μl of PBS (pH 7.4), and 30 μl of semen. Controlled conditions were used, including keeping temperature at 37 ℃ and pH at 7.4, with Trolox antioxidant (Merck KGaA, Darmstadt, Germany) as a reference. Readings were performed using an LS 55 spectrofluorometer (Perkin Elmer, Waltham, USA) at an excitation λ of 490 nm, with an excitation slit of 10, an emission λ of 530 nm, and an emission slit of 15. The results were expressed as relative fluorescence units (RFU).
Δ¥M was evaluated using flow cytometry. For this analysis, only the strongest ISO concentrations (ISO5 and ISO30) were used. Δ¥M was assessed using a specific JC-1 probe. In this evaluation, mitochondria that exhibit high Δ¥M are expressed as dimers with emission at 590 nm when excited at 549 nm, while in mitochondria with low Δ¥M, JC-1 are presented as monomers emitting at 525 nm when excited at 488 nm (GUO et al., 2017). The protocol described by GUO et al. (2017) was followed for this evaluation. A semen sample was diluted in HANKS balanced salt solution at a concentration of 1 x 106/mL, and JC-1 (MitoProbe, Thermo Fisher, Inc., Waltham, USA) was then added to achieve a final concentration of 0.4 μM. The Δ¥M was measured using an LSRFortessaTM flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). Specimen samples were excited using a 488 nm solid-phase laser, and JC-1 fluorescence was detected at 530/30 nm. The results were analyzed with FlowJo software, version 7.6.2 (FlowJo LLC, Ashland, OR, USA).
Statistical analysis
To evaluate the effect of the studied antioxidants on the quality of boar semen stored for 96 h, generalized linear models were fitted to determine the effect of the treatments on the dependent variables (TM, PM, TAC, ROS, Δ¥M, sperm kinetics, and the functional integrity of the plasma membrane). Comparison of means between treatments was performed using Duncan’s multiple range test. Regression analyses were conducted for each semen quality parameter. The significance level considered for all evaluations was P < 0.05. All analyses were performed with SAS 9.2 software (SAS Institute Inc., Cary, NC, USA).
RESULTS AND DISCUSSION:
Globally, a significant percentage of swine farms practice artificial insemination (AI) with cooled semen, using extenders that provide the necessary conditions to preserve the fertilizing capacity of the spermatozoa (PEZO et al., 2019). However, during preservation, spermatozoa are subjected to low temperatures, decreasing motility and other characteristics, which reduces sperm functionality (FANG et al., 2017).
In this study, sperm quality was evaluated during cooling. Results showed that TM and PM values decreased with increasing cooling time. This result pattern is consistent with the effect produced in two previous studies in which rosmarinic acid (FENG et al., 2020) and salvianic acid A (TIAN et al., 2019), both natural antioxidants, were added to cooled boar semen. In this study, however, there was no significant difference between the control and the ISO supplementation specimens (P > 0.05), except for a reduction in TM at 24 h of cooling due to supplementation with 20 µM of ISO (Figures 1 and 2). There were also no significant differences in sperm motility, sperm kinetics (Table 1), or sperm membrane functional integrity (Table 2) at 96 hours of cooling.
Total motility of cooled semen. Total motility values are expressed as percentage ± standard error of the mean. a-b: Different superscripts indicate differences between treatments within the same evaluation time (P < 0.05). 0h, 24h, 48h, 72h and 96h refer to hours of cooling at 16 °C.
Progressive motility of cooled semen. Progressive motility values are expressed as percentage ± standard error of the mean. a-b: Different superscripts indicate differences between treatments within the same evaluation time (P < 0.05). 0h, 24h, 48h, 72h and 96h refer to hours of cooling at 16 °C.
Regression coefficients were obtained from the regression analysis performed, which express the degree of change in sperm motility, sperm kinetics, and sperm membrane functionality as a function of the cooling time (Table 3). These regression coefficients indicate that ISO reduced the rate of decrease in various motility parameters and the proportion of rapid spermatozoa, depending on the concentration of ISO used. ISO25 was the treatment that yielded the strongest protective capacity during cooling, generating lower losses per day of storage in TM, PM, VAP, VSL, VCL, ALH, RAP, and HOS.
Several studies have shown that oxidative stress is one of the primary factors impacting the quality and fertilizing capacity of spermatozoa during preservation (FANG et al., 2017). This condition is induced by an excessive accumulation of ROS, which damages sperm cell membranes (PEREIRA et al., 2019), produces peroxidized lipids (BOLLWEIN & BITTNER, 2018), alters cell structure and function, damages DNA (SCHULTE et al., 2010), and reduces intracellular ATP levels (AITKEN et al., 2012), all of which explain the decrease in sperm motility that was observed in this study. Storing semen with the addition of antioxidants in the preservation extender is an alternative approach that reduces the adverse effects of oxidative stress and preserves sperm functionality for a longer period (TIAN et al., 2019).
Therefore, TAC (Figure 3), ROS (Figure 4), and Δ¥M (Figure 5) were monitored at 0 h and 96 h of cooling. Figure 3 shows that the TAC evaluated using ABTS did not decrease during cooling, except for ISO25, contrary to that reported by TIAN et al. (2019) with salvianic acid A, in which TAC decreased with storage time. In addition, that the TAC decreased with storage time shows that at 0 h of cooling, TAC evaluated using an ABTS test evidences significant differences (P < 0.05) between the control and ISO20, while at 96 h, control did not differ for either treatment. In this study, the antioxidant contribution of ISO in a porcine sperm extender was not clearly evidenced, which can be attributed to the interaction of this molecule with antioxidant enzymes (such as superoxide dismutase and glutathione peroxidase) responsible for counteracting the damage produced by ROS. In this sense, the molecules of ISO can reduce the activity of these enzymes, possibly generating alterations in TAC results. This situation has been previously reported in equine cryopreserved semen with ISO (RESTREPO & ROJANO, 2017).
Total antioxidant capacity (TAC) of cooled semen. Total antioxidant capacity (TAC) values, evaluated by the ABTS assay are expressed in μmol Trolox / L of sample ± standard error of the mean. a-c: Different superscripts indicate differences between treatments within the same evaluation time (P < 0.05). 0h and 96h refers to hours of cooling at 16 °C.
Reactive oxygen species (ROS) production of cooled semen. The production of reactive oxygen species is expressed in relative fluorescence units (RFU) ± standard error of the mean. a-d: Different superscripts indicate differences between treatments within the same evaluation time (P < 0.05). 0h and 96h refers to hours of cooling at 16 °C.
Mitochondrial membrane potential of cooled semen. Monomers: cells with low mitochondrial membrane potential (low - Δ¥M); Aggregates: cells with high mitochondrial membrane potential (high-Δ¥M). The number of cells in each population is expressed as a percentage (%) ± standard error of the mean. a-b: Different superscripts indicate differences between treatments within the same population at a given cooling time (P < 0.05).
In addition, for ROS production, ISO30 (Figure 4) showed significant differences (P < 0.05) vs. control at both cooling times (0 and 96 h), while this same concentration produced less ROS at 96 h. This decrease coincides with the results obtained in cryopreserved canine semen when ISO was added to the sperm extender, which is a fundamental aspect when trying to reduce oxidative damage (USUGA et al., 2021).
Finally, for Δ¥M evaluation, ISO5 showed similar behavior to the control during 0 and 96 h of cooling, while ISO30 decreased the proportion of dimers and increased the population of monomers (Figure 5). This demonstrated that ISO30 was able to modify the mitochondrial membrane potential in cooled porcine semen, in contrast to cryopreserved canine semen, where no effect on mitochondrial activity was evident (USUGA et al., 2021). The capacity to modify the mitochondrial membrane potential and reduce ROS production may be explained by the fact that ISO can regulate Ca2+ capture and ROS production in the mitochondria, which in turn could modify ATP synthesis and sperm motility (RESTREPO et al., 2022). Currently, there is little information about the evaluation of ISO as an antioxidant molecule. It has been studied in cryopreserved equine (RESTREPO & ROJANO, 2017; RESTREPO et al., 2022) and canine semen (USUGA et al., 2021), but no reports have been found on the addition of ISO to cooled semen and or in boar spermatozoa.
It has been reported that supplementation of ISO in doses higher than 80 µM generates cytotoxicity (MÁRQUEZ et al., 2018), while doses of 40 µM of this molecule in equine semen reduce TM (RESTREPO et al., 2022). In this study, ISO did not produce severe alterations in seminal quality; hence, it could be thought that it does not exert cytotoxic effects on boar sperm at the concentrations used in this study. Therefore, new studies are required to evaluate the cytotoxicity of ISO in sperm cells.
CONCLUSION:
Isoespintanol can reduce the loss of sperm quality and damage induced by oxidative stress in boar semen during cooling. In addition, this antioxidant can modulate the mitochondrial activity of sperm.
ACKNOWLEDGMENTS
The authors would like to thank to San Pablo Agricultural Station of Universidad Nacional de Colombia (UNAL).
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