Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

  • Print ISSN 0367-6722

  • Online ISSN 0976-0555

  • NAAS Rating 6.50

  • SJR 0.263

  • Impact Factor 0.4 (2024)

Frequency :
Monthly (January, February, March, April, May, June, July, August, September, October, November and December)
Indexing Services :
Science Citation Index Expanded, BIOSIS Preview, ISI Citation Index, Biological Abstracts, Scopus, AGRICOLA, Google Scholar, CrossRef, CAB Abstracting Journals, Chemical Abstracts, Indian Science Abstracts, EBSCO Indexing Services, Index Copernicus
Indian Journal of Animal Research, volume 55 issue 2 (february 2021) : 150-154

Pores Formation in Porcine Sperm Incubated in Streptolysin O and Its Post Thawing Viability after Trehalose Treatment

E. Jácome-Sosa1, M. Barrientos-Morales1, M.L. Juárez-Mosqueda2, B. Domínguez-Mancera1,*
1School of Veterinary Medicine and Animal Science, University of Veracruz, Veracruz, México.
2Biology of Tissular Reproduction Lab., School of Veterinary Medicine Veterinary and Animal Science, UNAM, México.
Cite article:- Jácome-Sosa E., Barrientos-Morales M., Juárez-Mosqueda M.L., Domínguez-Mancera B. (2021). Pores Formation in Porcine Sperm Incubated in Streptolysin O and Its Post Thawing Viability after Trehalose Treatment . Indian Journal of Animal Research. 55(2): 150-154. doi: 10.18805/IJAR.B-1302.
Background: Streptolysin O (SLO), a pore-forming protein in plasma membrane (PM), has been used to internalize a variety of molecules (DNA and RNA) in cells. In sperm, however, SLO has only been used to release acrosomal contents. Its possible use as biotechnology in the cryopreservation of pig semen. However, porcine sperm are very sensitive to the freeze-thaw process. The study aimed to evaluate the pore formation in the PM, the addition of trehalose and the post cryopreservation viability of porcine spermatozoa using SLO.

Methods: Research period was spring 2017- summer 2018. Thirty ejaculates from five mature boars were used. Semen was incubated in SLO 0.6 IU/ml and trehalose (added at 100, 200 and 400 ìM). Semen diluted in commercial diluent as control group. Presence of pores was checked by scanning electronic microscopy. To evaluate sperm membrane integrity and functional status the Coomassie stain with HOST test and the Chlortetracycline test were used. 

Result: It was found that SLO could form pores in the sperm cell membrane The addiction of 200 ìM trehalose to the freezing medium have different effects on the quality of boar sperm, showing highest motility and viability during the cooling process.
Conservation of sperm for a long period is one of the reproductive tools with greater potential in the area of medicine and animal production. Semen cryopreservation is a very useful technology for storing gametes and achieving massive distribution of genes allowing the amplification of desirable reproductive traits (Toker et al., 2016). Different freezing methods have been studied for conserving mammalian spermatozoa. However, according to protocols, results are still deficient due to damage by cold shock, oxidative stress and cryocapacitation, which increases cell death (Chutia et al., 2014). Freezing of porcine semen is an active area of research where many attempts have been made to understand and avoid harmful conditions during cryopreservation. As a result, the formation of ice (Córdova et al., 2001), oxidative stress (Agarwal et al., 2012) and cryocapacitation had been studied (Atroshchenko and Bragina, 2011). Many protocols (with cryoprotectants that do not penetrate the sperm membrane) to avoid or decrease these damages have been developed and tested. Even though such improvements are not negligible, more additional studies are needed (Athurupana and Funahashi, 2016).
Trehalose is a non-permeating cryoprotectant with dehydration-protection property, which minimizes the probability of formation of the critical gel phase, ice crystals and loss of cell integrity after rehydration during frozen-thawed procedure. Because this attributes, trehalose it has been used like cryoprotectant in diverse preservation and storage of biological samples protocols (Malo et al., 2010; Men et al., 2013; El-Sheshtawy et al., 2015). On the other hand, membranes permeabilization has been widely used in different mammalian cells to introduce extrinsic molecules into the intracellular compartment (Brito et al., 2008; Pocognoni et al., 2013; Sim et al., 2013). For this purpose, some cholesterol-dependent cytolysins (CDC) have been used. Some CDCs have been tested on sperm, such as Streptolysin O in mice and humans (Johnson et al., 1999; Yunes et al., 2000) and Perfringolysine O (PFO) in humans (Pocognoni et al., 2013). Results of these studies show that SLO is a molecule that shows potential for biomedical studies of the sperm cell and possess a high potential for the genetic and/or structural modification of this gamete. In this sense, trehalose has properties that make interesting its use as an internal cryoprotectant of the sperm cell. Therefore, the present study was undertaken to assess the viability of the use of Streptolysin O and trehalose into sperm cells and determine their effect on sperm cell functionality.
Semen collection and semen processing
Research period was spring 2017- summer 2018. The experiment was conducted in the Laboratory of Biology of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Veracruz, Veracruz city, Mexico. Thirty ejaculates from five mature boars were used. Semen sperm-rich fraction (80-120 ml) was collected by the gloved hand technique (Bottini-Luzardo et al., 2012). After collection, the quality of the fresh semen samples was evaluated. Sperm concentration analysis was performed by counting cells in a Neubauer chamber as described by Gutiérrez-Pérez et al., (2009). After counting, semen was diluted (1:1) using a commercial long-term boar semen diluent (Vitacem LD 40g/1L H2O format, from Megapor®, Spain) and finally storage for 24 h at 16°C until use.
Analysis of sperm motility and viability
Mass motility was evaluated by visual estimation of the percentage of spermatozoa showing progressive motility between 0 and 100% (Maxwel and Evans, 1990). Samples exhibiting values ≥70% progressive motility were selected for the study (Dziekońska and Strzezek, 2011). Viability and morphology were assessed utilizing Eosin-Nigrosin staining (Sigma-Aldrich, St. Louis MO, USA) (Björndahl et al., 2003).
After 24 h in storage (16°C), each sample (in fractions of 2 ml [1 x 10-8/ml]) received the following treatments.
T1: Incubation in a SLO (Sigma-Aldrich, St. Louis MO, USA) solution (0.6 UI/ml) and trehalose (Sigma-Aldrich, St. Louis MO, USA) at 100 µM/ml for 5 minutes at 37°C.
T2: Incubation in SLO (0.6 UI/ml) and trehalose at 200 µM/ml for 5 minutes.
T3: Incubation in SLO (0.6 UI/ml) and trehalose at 400 µM/ml for 5 minutes.
T4: Control group (no treatment just diluted semen). 
The incubation in SLO was intended to permeabilize the sperm membrane (Valdés et al., 2015).
Pores and post-thawing viability evaluation 
The observation of pores was done by scanning electronic microscopy (JEOL-JSM-5410-LV, USA) (Metkar et al., 2015). At the same time, trehalose was added during the incubation to evaluate post-thawing viability. Cryopreservation was made after pore sealing in accordance to Fawcett et al., (1998) using the two-step freezing protocol proposed by Westerndorf (Gutiérrez-Pérez et al., 2009). For the control group, the diluent contained 20% egg yolk and 230.8 mM glucose (as a replacement for trehalose). After 15 days of cryopreservation, straws were thawed for 30s in a water bath at 37°C. Then straws were held over liquid nitrogen vapors (4 cm) for 20 min before plunging them into liquid nitrogen. This procedure allowed to reach the optimal rate reported for boar sperm freezing of -30°C/min. Straw contents were placed in tepid assay tubes, previously supplemented with extender in a 1:6 v/v thawed semen/extender proportion. Samples were maintained 10 min at 37°C before evaluating the effect of temperature on motility.
Membrane integrity evaluation
Hypo-osmotic swelling test (HOST) in combination with Coomassie Bright Blue (CBB) (Sigma-Aldrich, St. Louis MO, USA) were used to evaluate the integrity of the membrane (Oliveira et al., 2013). Both tests were carried out in at least three replicates by the same researcher on a 400X magnification with a minimum of 200 cell counts per reading.
Sperm functional status 
The functional status of spermatozoa was assessed with Chlortetracycline (CTC) (Sigma-Aldrich, St. Louis MO, USA), a fluorescent antibiotic (Álvarez-Guerrero et al., 2016). This assay allows observing changes in the sperm plasma membrane-associated with the capacitation status of the sperm. The sperms were stained during 30s and then fixed with 22 μl of 0.2% glutaraldehyde. Finally, 10 μl of the fixed sperm solution was placed on a glass slide with an equal amount of DABCO® (Sigma-Aldrich, St. Louis MO, USA) and then covered with a coverslip. Samples were evaluated using an epi-fluorescence microscope (Leica DM 020-518500/LS) with filter blue at 405-455 nm, 400X magnification.
Statistical analysis 
Data in each treatment were compared using the non-parametric module of STATISTICA V10.0. Kruskal-Wallis H test was performed to determine effects among treatments and to test if a group of data came from the same population.
The analysis of scanning electron microscopy of a sperm sample incubated with SLO allows the observation of structures on the spermatic surface (Fig 1). Vesicular and circular forms are seen in the head and tail. Fig 2 shows the effect of trehalose on sperm cell membrane integrity after post-thawing, in which shows a significant difference (P<0.05) between cells with intact and damaged membrane. Fig 5 indicates the effect of the trehalose concentration on the acrosomal reaction state; there is a significant difference (P<0.05) in treatments between the percentage of no capacitated spermatozoa with intact acrosome and the percentage of cells capacitated with acrosomal reaction. During capacitation, sperm undergo a change in the motility pattern called hyperactivation and become capable to go through a physiological exocytotic process known as acrosome reaction (Puga et al., 2018), both required (and important) for successful fertilization (Darszon et al., 2011). They are indicators of the viability of frozen-thawed sperm. Numerous research studies have been carried out using the CDC as a tool of reversible permeabilization in different cell types (Fawcett et al., 1998; Brito et al., 2008), including the sperm cell (Pocognoni et al., 2013; Sim et al., 2013); however, there is little information available on SLO permeabilization in porcine spermatozoa (Sim et al., 2013). In the present study, 0.6 IU of SLO was used to permeabilize the plasma membrane of the spermatozoa, in the presence of 200 μM of trehalose. Works in mouse and porcine spermatozoa, concluded that the optimum and safe (without loss cell viability) permeabilization, was achieved at 0.6 IU/ml of SLO (Johnson et al., 1999; Valdéset_al2015).

Fig 1: Photograph of a porcine sperm cell, obtained by scanning electron microscopy 10000x.


Fig 2: Effect of Trehalose concentration on the functional membrane state.


Fig 5: Effect of Trehalose concentration on sperm capacitation status and acrosomal reaction.

The evaluation of the plasma membrane/functional integrity of cells with the HOST test indicates that the 200 μM treatment obtained the highest percentage of sperm quality (Fig 2). It has been shown that 100 μM treahalose used externally improves the viability and parameters of in vitro fertilization (Malo et al., 2010). It has also been observed in bovine semen that the addition of 50-100 μM of trehalose in the freezing medium improves its viability (El-Sheshtawy et al., 2015). The addition of trehalose in the freezing medium possess an antioxidant activity, enhancing the viability and fertility of semen (Perumal et al., 2015; Iqbal et al., 2016).
For the specific case of the acrosome status (damaged), there was a significant difference in each treatment (Fig 3). The treatment using 200 μM presented the lower percentage of damage with 35% (Fig 3). Our results are in contrast with those by Gutiérrez-Pérez et al., (2009), where they reported 10.9% acrosomal integrity in pigs and bulls respectively. Nonetheless, these studies used higher concentrations of trehalose and only freezing diluent was used without a previous cell permeabilization. This disaccharide can provide a greater stability and produce less damage when the cell is defrosted, which in turn influences the percentage of cells with integral acrosome and good motility (Fig 4). Disaccharides such as trehalose, sucrose and maltose have been reported to reduce the number of dead spermatozoa and/or damaged acrosome rates (Yildiz et al., 2000; Hu et al., 2010; Ahmad et al., 2014).

Fig 3: Effect of Trehalose on the acrosome status.


Fig 4: Effect of Trehalose concentration on sperm motility.

The capacitation status and acrosomal reaction of the spermatozoa had similar results to those of sperm with an intact acrosome. The treatment at 200 μM showed the highest percentage of spermatozoa without capacitation with 16.8% and 11.1% of sperms capacited but without acrosomal reaction (Fig 5). Silva et al., (2015) reported a 37.2% of cells with intact acrosome. They noted the relevance of using a defreezing curve in other to allow trehalose to get better results. This is a variation of the feeezing process, similar to the method utilized in the present study. The use of different extenders, the combined use of lactose, trehalose and glycerol can provide the best quality of sperm and suggests that there may be important differences between animal species (Karunakaran et al., 2018).
The use of cryoprotectants is an important parameter influencing sperm cell viability during frozen-thawed process. The addition of trehalose to the freezing medium have different effects on the quality of boar sperm. A suitable dose decreases the effects of low temperatures on sperm quality during freezing preservation compared with a control group. The T2 group has almost the same sperm motility than the control group and longer survival time than the other groups. Moreover, this research shows that SLO was able to form pores in plasmatic membrane of the porcine sperm cell.
This research was supported by CONACYT (CB 169861) from the National Council for Sciences and Technology (CONACYT), México, to Barrientos-Morales, M. (Principal Investigator). Author Jácome-Sosa, E. was supported by CONACYT whit a doctoral fellowship; this manuscript is part of her doctoral work.

  1. Agarwal, A., Aponte-Mellado, A., Premkumar, B.J., Shaman, A., Gupta, S. (2012). The effects of oxidative stress on female reproduction: a review. Reproductive Biology and Endocrinology. 10: 49.

  2. Ahmad, E., Naseer, Z., Aksoy, M., küçük, N., Uçan, U., Serin, I., Ceylan, A. (2014). Trehalose enhances osmotic tolerance and suppresses lysophosphatidylcholine-induced acrosome reaction in ram spermatozoon. Andrologia. 47(7): 1-7.

  3. Álvarez-Guerrero, A., González-Díaz, F., Medrano, A., Moreno-Mendoza, N. (2016). In vitro capacitation and acrosome reaction in sperm of the phyllostomid bat Artibeus jamaicensis. In vitro Cellular and Developmental Biology-Animal. 52: 454-465.

  4. Athurupana, R. and Funahashi, H. (2016). Milk supplements in a glycerol free trehalose freezing extender-enhanced cryosurvival of boar spermatozoa. Asian Pacific Journal of Reproduction. 5: 58-62. 

  5. Atroshchenko, M.M. and Bragina, E.E. (2011). Change in the ultrastructure of stallion spermatozoa under the effect of cryopreservation. Russian Agricultural Sciences. 37: 175-178.

  6. Björndahl, L., Söderlund, I., Kvist, U. (2003). Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment. Human Reproduction. 18: 813-816.

  7. Bottini-Luzardo, M., Centurión-Castro, F., Alfaro-Gamboa, M. (2012). Effect of addition of coconut water (Cocos nucifera) to the freezing media on post-thaw viability of boar sperm. Tropical Animal Health and Production. 45: 101-106.

  8. Brito, J.L., Davies, F.E., Gonzalez, D., Morgan, G.J. (2008). Streptolysin-O reversible permeabilisation is an effective method to transfect siRNAs into myeloma cells. Journal of Immunological Methods. 333: 147-155. 

  9. Chutia, T., Biswas, R.K., Tamuli, M.K., Sinha, S., Goswami, J., Deka, B.C., Banik, S., Kayastha, R.B. (2014). Efficacy of different extenders in preservation of liquid hampshire boar semen at 15°C. Indian Journal of Animal Research. 48(5): 496-500.

  10. Córdova, A., Pérez, J.F., Lleó, B. (2001). In vitro fertilizing capacity of deep frozen boar semen packaged in 0.5 and 5 ml straws. Reproduction in Domestic Animals. 36: 199-202.

  11. Darszon, A., Nishigaki, T., Beltran, C., Treviño, C.L. (2011). Calcium channels in the development, maturation and function of spermatozoa. Physiology Reviews. 91(4): 1305-1355. 

  12. Dziekoñska, A. and Strzezek, J. (2011). Boar variability affects sperm metabolism activity in liquid stored semen at 5°C. Polish Journal of Veterinary Science. 14: 21-27. 

  13. El-Sheshtawy, R.I, Sisy, G.A., El-Nattat, W.S. (2015). Effects of different concentrations of sucrose or trehalose on the post-thawing quality of cattle bull semen. Asian Pacific Journal of Reproduction. 4(1): 26-31.

  14. Fawcett, J.M., Harrison, S.M., Orchard, C.H. (1998). A method for reversible permeabilization of isolated rat ventricular myocytes. Experimental Physiology. 83: 293-303.

  15. Gutiérrez-Pérez, O., Juárez-Mosqueda, M.L., Carvajal, S.U., Ortega, E.M.T. (2009). Boar spermatozoa cryopreservation in low glycerol/trehalose enriched freezing media improves cellular integrity. Cryobiology. 58: 287-292. 

  16. Hu, J.H., Zan, L.S., Zhao, X.L. (2010). Effects of trehalose supplementation on semen quality and oxidative stress variables in frozen-thawed bovine semen. Journal of Animal Science. 88: 1657-1662.

  17. Iqbal, S. andrabi, S.M.H., Riaz, A. (2016). Trehalose improves semen antioxidant enzymes activity, post-thaw quality and fertility in Nili Ravi buffaloes (Bubalus bubalis). Theriogenology. 85: 954-959. 

  18. Johnson, L.R., Moss, S.B., Gerton, G.L. (1999). Maintenance of motility in mouse sperm permeabilized with Streptolysin O. Biology of Reproduction. 60: 683-690.

  19. Karunakaran, M., Konyak, P., Mandal, A., Mondal, M., Bhakat, C., Bhakat, S., Rai, C., Bhakat, M., Rai, S., Ghosh, M., Behera, R. (2018). Effect of trehalose- an impermeant cryoprotectant on cryopreservation of Black Bengal buck semen. Indian Journal of Animal Research. 53(1): 37-40.

  20. Malo, C., Gil, L., Gonzalez, N. (2010). Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender. Cryobiology. 61: 17-21.

  21. Men, N.T., Kikuchi, K., Nakai, M. (2013). Effect of trehalose on DNA integrity of freeze-dried boar sperm, fertilization and embryo development after Intracytoplasmic Sperm Injection. Theriogenology. 80: 1033-1044.

  22. Metkar, S.S., Marchioretto, M., Antonini, V., Lunelli, L., Wang, B., Gilbert, R.J. anderluh, G., Roth, R., Pooga, M., Pardo, J., Heuser, J.E., Serra, M.D., Froelich, C.J. (2015). Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes. Cell Death and Differentiation. 22(1): 74-85.

  23. Oliveira, L.Z., de Arruda, R.P., de Andrade, A.F.C. (2013). Assessment of in vitro sperm characteristics and their importance in the prediction of conception rate in a bovine timed-AI program. Animal Reproduction Science. 137: 145-155. 

  24. Perumal, P., Vupru, K., Rajkhowa, C. (2015). Effect of addition of trehalose on the liquid storage (5°C) of mithun (Bos frontalis) semen. Indian Journal of Animal Research. 49: 837-846.

  25. Pocognoni, C.A., De Blas, G.A., Heuck, A.P. (2013). Perfringolysin O as a useful tool to study human sperm physiology. Fertility and Sterility. 99: 99-106.

  26. Puga, M.L.C., Luque, G.M., Balestrini, P.A., Marín-Briggiler, C.I., Romarowski, A., Buffone, M.G. (2018). Molecular Basis of Human Sperm Capacitation. Frontiers in Cell and Developmental Biology. 6: 72.

  27. Silva, C.G., Cunha, E.R., Blume, G.R. (2015). Cryopreservation of boar sperm comparing different cryoprotectants associated in media based on powdered coconut water, lactose and trehalose. Cryobiology. 70: 90-94.

  28. Sim, B.W., Cha, J.J., Song, B.S. (2013). Efficient production of transgenic mice by Intracytoplasmic Injection of Streptolysin- O-treated spermatozoa. Molecular Reproduction and Development. 80: 233-241.

  29. Toker, M.B., Alcay, S., Gokce, E., Ustuner, B. (2016). Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience. Cryobiology. 72: 205-209. 

  30. Valdés, C.M., Barrientos-Morales, M., Domínguez, M.B. (2015). Cell viability, acrosomal status and DNA integrity in porcine sperm permeabilized with Streptolysin O. Annual Research and Review in Biology. 7: 399-407. 

  31. Yildiz, C., Kaya, A., Aksoy, M., Tekeli, T. (2000). Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing. Theriogenology. 54: 579-585.

  32. Yunes, R., Michaut, M., Tomes, C., Mayorga, L.S. (2000). Rab3A triggers the acrosome reaction in permeabilized human spermatozoa. Biology of Reproduction. 62: 1084-1089.

Editorial Board

View all (0)