As an important technique for production of domestic animal, artificial insemination (AI) and in vitro fertilization (IVF) are used for improvement of animal breed, production of transgenic animal and it require the liquid- or cryo-preserved sperm (Roca et al., 2011). Generally, liquid preservation of sperm is used in AI for piglet production. However, these preservation method is difficult to long-term preservation because spermatozoa was exposed to various stress by metabolism (Silva and Gadella, 2006). These damage in spermatozoa during liquid preservation cause to reduce viability and fertility. On the contrary, metabolism of sperm was reduced or stopped during cryopreservation and it enable to long-term preservation (Yoshida, 2000). However, ice crystal formation during freezing and thawing process damaged to spermatozoa by redistribution of phospholipid and protein in membrane, change of plasma membrane structure and function such as permeability (Lessard et al., 2000). These cryo-stress induce the generation of reactive oxygen species (ROS) in sperm and functions of frozen-thawed sperm including motility, viability and fertility were reduced (Bailey et al., 2003; Grobfeld et al., 2008). For resolution of these problem of sperm cryopreservation, a variety of supplement such as antioxidants (Kaeoket et al., 2010; Giaretta et al., 2015) and cholesterol (Mocé et al., 2010) were used during cryopreservation process.
Polyunsaturated fatty acids (PUFAs), which are one of components of phospholipid in plasma membrane of sperm, play an important role in sperm motility, viability and membrane function for oocyte-sperm membrane fusion and regulation of membrane fluidity (Aksoy et al., 2006). In human and cow, sperm motility was increased by dietary docosahexanoic acid and intake of fish oil that contain amount of omega-3 (n-3) fatty acid enhanced the motility and viability of pigs (Rooke et al., 2001), cows (Khoshvaght et al., 2016) and sheeps (Samadian et al., 2010). As a one of n-3 PUFAs, α-linolenic acid (ALA) in plasma membrane regulate the membrane protein and provide energy to plasma membrane (Shevchenoko and Simons, 2010). These function of ALA maintained the function of plasma membrane and sperm viability. We expect that decreasing of sperm function by cryopreservation process could improve by ALA supplement.
Bovine serum albumin (BSA), which is most abundant in the plasma, has play roles in preventing of damage to sperm. It stabilize a membrane and protect the plasma membrane of sperm by regulating of membrane fluidity (Cheng et al., 2015). Besides, this protein is known to eliminate the free radical through its free radical-trapping property (Roche et al., 2008) and increase the antioxidant activity during freeze-thawing process of bull semen (Schafer and Holzmann, 2000). These function of BSA improve the post-thawed motility and viability of bull (Ashrafi et al., 2013) and ram (Uysal and Bucak, 2007) sperm against heat shock during freeze-thawing process.
Mammalian sperm contain high concentration of PUFAs in plasma membrane and is sensitive to attack by ROS (Alvarez and Storey, 1995). Generation of ROS in spermatozoa induce lipid peroxidation (LPO) and produced lipid peroxides effuse PUFAs in plasma membrane via activation of phospholipase A2 (Wathes et al., 2007). We expect that supplement of ALA and BSA to freezing extender could prevent cryo-damage such as ROS generation and effusion of PUFAs in plasma membrane of sperm. Therefore, this study was conducted to evaluate effect of ALA and BSA on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm.
MATERIALS AND METHODS
All procedures that involved the use of animals were approved by the Kangwon National University Institutional Animal Care and Use Committee (KIACUC- 09-0139). The ejaculate was collected by gloved-hand methods and diluted with modified Modena B (30.0 g/ L glucose, 2.25 g/L EDTA, 2.50 g/L sodium citrate, 1.00 g/L sodium bicarbonate, 5.00g/L tris, 2.50 g/L citric acid, 0.05 g/L cysteine, and 0.30 g/L gentamicin sulfate).
Samples were transported to laboratory within 1 h and stored at 18°C for 24 h. Ejaculated semen in this study was used with more than 80% viability.
Semen Freezing and Thawing Process
1st freezing extender, which mixed 11% α-lactose solution (Sigma, St. Louis, MO, USA) with 20% eggyolk, was centrifuged for 30 min (3000 rpm, 4°C) and collected supernatant. 3 ng/mL ALA and/or 20 μg/mL BSA were added to 1st freezing extender (control: no addition; ALA: only ALA addition; BSA: only BSA addition; ALA+BSA: both of ALA and BSA addition) and boar sperm (1.5×109 spermatozoa/mL) were diluted with freezing extender. Subsequently, it were cooled down to 4°C for 2 h and 2nd freezing extender that was 1st freezing extender containing 9% Glycerol (Sigma) and 1.5% (OEP; Nova Chem, USA) mixed with diluted sperm to 1×109 spermatozoas/mL concentration. Prepared sperm samples were loaded into 0.5 mL straw and pre-freezing to —120°C for 10 min. finally, sperm samples were preserved in liquid nitrogen and frozen sperms were thawed in 37.5°C water bath for 45 sec to analyze.
Analysis of Viability, Acrosome Reaction, and Mitochondrial Intact
Analysis of sperm characteristics were carried out using methods previously described (Lee et al., 2016). Briefly, SYBR-14 (40 nM; Live/Dead sperm viability kit, Molecular probes, USA), Lectin from Arachis hypogagea (2 μM; FITC-PNA; Sigma), and Rhodamine123 (2 μM; Sigma) were added to frozen-thawed boar sperm (1×106 spermatozoas/mL) for viability, acrosome reaction, and mitochondrial intact, respectively. The sperm samples were incubated for 5 min at 38°C in dark room and propidium iodide (2 μM; PI; Sigma) was stained for 5min in same condition. After stain, total 10,000 stained sperm were measured using flow cytometry (FACSCaliber, BD, USA) and measured data from flow cytometry were analyzed by CELLQuest version 6.0(Becton Dickinson).
Date were analyzed using Statistical Analysis System software (SAS®, version 9.3). All obtained data were analyzed using general linear model (GLM) and treatment groups were compared for differences though use of Duncan’s modified multiple range test (p<0.05).
The viability of frozen-thawed boar sperm was presented in Fig. 1. Sperm viability in ALA+BSA treatment group was significantly increased than control group (p<0.05). Both of ALA group and BSA group increased viability than control (57.83% and 57.6% vs 54.57%, respectively; p>0.05). Acrosome reacted boar sperm in both of all and live sperm population were significantly reduced by treatment of ALA and/or BSA than non-treatment group (Fig. 2; p<0.05). Mitochondrial intact level in all sperm population was no difference among the all group, however, ALA and ALA+BSA treatment in live sperm population enhanced mitochondrial intact compare with control group (Fig. 3; p<0.05).
This study was conducted to confirm effects of ALA and BSA in freezing extender on characteristics of frozen- thawed boar sperm. During freeze-thawing process, viability, motility and lipid composition of sperm were negatively influenced by physical and chemical damage such as ice crystal formation, change of temperature and osmotic pressure (Watson, 2000; Martinez-Soto et al., 2013). Because mammalian sperms contain amount of PUFAs in plasma membrane, it were sensitive to LPO by ROS and PUFAs in plasma membrane were effused by LPO (Alvarez and Storey, 1995; Wathes et al., 2007). These fatty acids were reduced and generation of ROS was increased during freeze-thawing process (Parks and Lynch, 1992; Kim et al., 2011). Kaka et al. (2015b) reported that concentration of ALA in bull spermatozoa was increased by supplement of ALA in freezing extender. Thus, these absorption of ALA and antioxidant property of BSA may enhance quality of frozen-thawed sperm via reducing effusion of PUFAs and ROS generation.
The function of plasma membrane, which play important role in viability, motility and cell death in sperm, was inhibited by various stress during cryopreservation (Watson, 2000). For reducing of membrane damage, many researchers added a sugar (Bucak et al., 2007), antioxidants (Memon et al., 2011) and fatty acids (Kaka et al., 2015a) during cryopreservation process of sperm. Our previous study (2016) shown that supplement of 3 ng/mL ALA in freezing extender prevented the plasma membrane damage by ethanol in frozenthawed boar sperm. Also, this present result show that both of ALA and BSA treatment in freezing extender enhance the sperm viability compared with non-treatment group (Fig. 1; p<0.05), however, treatment of ALA or BSA were not effect. ALA supplement during cryopreservation process of sperm induced LPO that caused membrane damage (Kaka et al., 2015a) and BSA was known to effuse cholesterol of the plasma membrane in sperm, which induce the generation of ROS (Radomil et al., 2011). Despite positive effect of ALA and BSA, treatment of only ALA or BSA may not enhanced to viability of frozen-thawed sperm by LPO and effusion of cholesterol. During liquid storage of boar sperm, supplement of 3~5 g/L BSA decreased LPO level at day 3 and day 7 (Zhang et al., 2015). Thus, BSA treatment with ALA may increase sperm viability through preventing of ALA-induced LPO.
Acrosome of spermatozoa contain an enzyme including acrosin and hyaluronidase and acrosome reaction is essential phenomenon for fertilization. During cryopreservation process, acrosome reaction was induced by cryo-damage (Watson, 2000; O’Flaherty et al., 2005) and acrosome reacted sperms have low fertility. Treatment of H2O2 and sodium nitroprusside increase acrosome reaction in boar spermatozoa (Kim et al., 2009). In this study, acrosome reacted sperm in both of live and all sperm population were significantly decreased in ALA and/or BSA supplement group than control group (Fig. 2; p<0.05) and this result is similar to sperm viability. Kaka et al (2015b) reported that acrosome integrity in frozen-thawed bull sperm was enhanced by 5 ng/mL ALA supplement. Therefore, ALA could protect the acrosome membrane via preventing damage of plasma membrane and BSA decrease acrosome membrane damage by antioxidant ability.
Mitochondrial metabolism plays an important role for production of energy (Ramio-Lluch et al., 2011) and mitochondrial function in spermatozoa is important for motility, viability and fertilization of sperm. In present study, mitochondrial intact was higher in ALA and ALA+BSA treatment groups than control (Fig. 3; p< 0.05). During energy production, ROS such as superoxide (O2 · ), hydrogen peroxide (H2O2), or hydroxyl radical (OH · ) and it damaged to mitochondrial DNA (Kumar et al., 2009). ALA and BSA treatment may reduce the mitochondrial damages by ROS through reduction of damage in plasma membrane. In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.