In vitro Supplementation of Linoleic Acid Improves Quality of Cryopreserved Buffalo Semen

Cryopreservation alters plasma membrane structure and reduces sperm viability due to osmotic and thermal shocks during freezing and thawing (Holt, 2000) ultimately reducing fertility of sperm cells (Watson, 2000). The main cause of impairment of membrane function is the transformation of lipids into rigid structure during cryopreservation (Watson, 2000). The characteristics of membranes that affect their sensitivity include cholesterol/phospholipid ratio, content of non-bilayer-preferring lipids, degree of hydrocarbon chain saturation and protein/phospholipid ratio (Medeiros et al., 2002). Buffalo sperm are more susceptible to cold shock and osmotic stress during cryopreservation compared to cattle sperm (Andrabi, 2009) probably due to difference in lipid ratio of buffalo and cattle sperm.

Lipids are a basic component of semen, contributing to the membrane structure of spermatozoa, the metabolism of the sperm cells and their ability to capacitate and fertilize the female gamete (Mann and Lutwak-Mann, 1981). (Lin et al., 1993) reported high concentrations of polyunsaturated phospholipids in sperm membranes that are a major determinant of cold sensitivity and overall viability (Hammerstedt, 1993). During cooling, membrane bound phospholipids reorient themselves into a different configuration (Amann and Graham, 1993) that adversely affects sperm plasma membrane and acrosome integrity (Holt et al., 1992). Polyunsaturated fatty acids, predominant in sperm plasmamembrane, are susceptible to lipid peroxidation (Aitken et al., 1993) and cause membrane damage, intracellular enzyme leakage and inhibition of respiration. It is reported that lipids and phospholipids are lost when bull spermatozoa are subjected to cold shock and freezing (Pickett and Komarek, 1967) as lipids are being utilized in the production of energy using lipid metabolic pathways (Lenzi et al., 1996). In buffalo (Bubalus bubalis) spermatozoa, cold shock and freezing results in a significant loss of 15.8% and 34.55% of total lipids and 6.49% and 19.1% of phospholipids respectively (Sarmah et al., 1984). It is pertinent to mention that motility is negatively correlated with lipid peroxidation of bovine semen (Kasimanickam et al., 2007). Higher lipid peroxidation levels beyond the physiological level cause higher fluidity of the plasma membrane that reduces sperm plasma membrane integrity (Storey, 1997).

Dietary supplementation of polyunsaturated fatty acids has been developed as a potential strategy for enhancing the production of fertile spermatozoa in boars (Audet et al., 2004), sheep (Sarmadian et al., 2010) and bull (Gholami et al., 2010). Supplementation of sunflower oil-enriched diets improved motility and plasma membrane integrity of cryopreserved buffalo semen (Adeel et al., 2009). Present study was designed to evaluate the effect of supplementing linoleic acid (LA) at different levels in extender on post-thaw quality of cryopreserved buffalo semen.

Tris-citric acid (pH 7.0; osmotic pressure 320 mOsmol Kg-1) was used as buffer, consisted of 1.56% citric acid (Fisher Scientific, Loughborough, Leicestershire, UK), 3.0% tris (hydroxymethyl) aminomethane (Research Organics, Cleveland, OH, USA), 0.2% w/v fructose (Scharlau, Barcelona, Spain), 7.0% glycerol (Merck, Darmstadt, Germany), egg yolk 20% v/v, antibiotics; benzyl penicillin (1000 IU mL-1) and streptomycin sulphate (1000 μg mL-1) in 74 mL distilled water. Four experimental extenders were prepared having 0.0 ng mL-1 (extender I), 5.0 ng mL-1 (extender II) 10.0 ng mL-1 (extender III) and 20.0 ng mL-1 (extender IV) of LA (Sigma Chemical Co., St. Louis, MO, USA). Because of the insolubility of fatty acid in extender, ethanol 0.05% was used (Ejaz et al., 2017).
 
Experimental animals
 
Three adult Nili-Ravi buffalo bulls (Bubalus bubalis) of similar age (7-8 years) and known fertility, maintained at Semen Production Unit Qadirabad, Sahiwal, Pakistan were selected for this study.
 
Semen collection and initial evaluation
 
Two consecutive ejaculates were collected from each bull in a graduated tube with the aid of artificial vagina (42°C) at weekly intervals for a period of five weeks (replicates). After collection, semen was immediately transferred to laboratory for initial evaluation. The qualified semen ejaculates (>0.5 mL volume, >60% motility, >0.5 × 109sperm D mL concentration) were split into four aliquots and held for 15 min at 37°C in the water bath before dilution in four different experimental extenders.
 
Semen processing
 
Each semen aliquot was extended in Tris citric acid extender (37°C; 50 × 106 motile spermatozoa per mL) containing 5, 10 and 20 ng mL-1of LA (Sigma Chemical Co., St. Louis, MO, USA) while extender without LA was kept as control. Diluted semen was cooled to 4°C in 2 hours and equilibrated for 4h at 4°C. Semen was then filled in 0.5 mL French straws (IMV, France) by suction pump at 4°C in cold cabinet unit and kept on liquid nitrogen vapours (5 cm above the level of liquid nitrogen) for 10 minutes. Straws were then plunged and stored into liquid nitrogen (-196°C) container (Jindal, 1994). After 24h, semen straws were thawed in a water bath at 37°C for 30 seconds. For each treatment, semen from three straws (from same replicate) was pooled and incubated at 37°C for assessment of post-thaw semen quality after thawing.
 
Evaluation of Post-Thaw Sperm Functional Assays
 
Sperm progressive motility: A drop of thawed semen sample was placed on a pre-warmed glass slide and covered with cover slip, progressive motility was assessed under phase contrast microscope (400X; 37°C) with closed circuit television. 
 
Sperm plasma membrane integrity: Sperm plasma membrane integrity was assessed using supravital hypo-osmotic swelling test (HOST) as described by Ejaz et al., (2016). Solution for HOS assay was consisted of 0.73 g sodium citrate (Merck) and 1.35 g fructose (Scharlau, Barcelona, Spain) in 100 mL distilled water (osmotic pressure 190 mOsmolD kg). For the assessment of sperm plasma membrane integrity 50μL of the semen sample was mixed with 500μL of the pre-warmed HOS solution and incubated at 37°C for 30-40 min. After incubation a drop of mixture was placed on a slide, covered with cover slip and visualized microscopically (400X magnification), one hundred spermatozoa per experimental extender were evaluated in five different fields. Swollen tails of sperm were indicated as intact, biochemically active sperm membranes, while unswollen tails were indicated as disrupted, inactive, non functional sperm membranes.
 
Acrosome intact live sperm: Acrosome intact live sperm was assessed using trypan blue Giemsa stain as described by Akhter et al., (2008). Briefly, equal drops of trypan blue and semen were placed on a slide, mixed quickly and air-dried. The samples were fixed with formaldehyde-neutral red for 5 min, rinsed with running distilled water and followed by the application of 7.5% Giemsa stain for 4 h. Samples were air dried and mounted with Canada Balsam and under phase contrast microscope at 1000X. Trypan blue penetrates nonviable spermatozoa with disrupted membrane, which stain in blue, while live sperms with intact acrosome appeared unstained. Giemsa accumulates in spermatozoa with an intact acrosome, staining the acrosome region in purple.
 
Sperm chromatin integrity: Sperm chromatin integrity was assessed as practiced by Ejaz et al., (2014). Air dried smears of semen samples were fixed in 96% ethanol-acetone (1:1) at 4°C for 30min, hydrolyzed with 4N HCl at 25°C for 10-30 min. Smears were suspended in distilled water, three times for two minutes each. The slides were stained with toluidine blue in Mcllvaine buffer (sodium citrate-phosphate) for 10min. Samples were airdried and mounted with Canada Balsam and observed under light microscope at 1000X. Toluidine blue stain penetrates in sperms having damaged chromatin staining from dark blue to purple while spermatozoa having intact chromatin were stained light blue.
 
Sperm morphological abnormalities: To assess sperm morphological abnormalities, 100 mL of semen sample was fixed in 500 mL of 1% formal citrate (2.9 g Tris-sodiumcitrate dihydrate, 1 mL of 37% solution of formaldehyde, dissolved in 100 mL of distilled water). Samples were observed by phase contrast microscope (X1000; Olympus BX20, Tokyo, Japan) under oil immersion. Sperm abnormalities were recorded viz: head abnormalities like micro and macro heads, detached heads, double heads, pyriform heads; mid piece abnormalities including proximal droplet, distal droplet and abaxial attachment; tail abnormalities like tail coiled below the head, tail bent at mid piece, tail without head and double tail (Zafar et al., 1988).
 
Statistical analysis: Data on sperm post thaw quality parameters were analyzed using analysis of variance (ANOVA) in randomized complete block design using fixed effect model and were presented as mean (±SE). When F-ratio was found significant (P < 0.05), LSD test was used to compare treatment means.

Sperm progressive motility and plasma membrane integrity were significantly improved (P<0.05) in extender containing 10.0 ng/mL of LA compared to control (Table 1). The increased sperm progressive motility and plasma membrane integrity by supplementation of LA in extender indicates the cryoprotective effect of LA on sperm plasma membrane. Our studies are in line with previous work on bull semen (Nasiri et al., 2011) suggesting addition of n-3 PUFA at 10.0 ng/mL accompanying α-tocopherol as an antioxidant improved sperm motility. Addition of LA in extender improved motility of boar sperm (Hossain et al., 2007). It has been observed that adding linoleic-oleic acid in the presence of semen plasma proteins improves the preservation of ram sperm viability (Perez-Pe et al., 2001). The spermatozoa utilize lipids as energy resources (Lenzi et al., 1996), therefore the proportion of fatty acids decreases significantly after freezing and thawing (Cerolini et al., 2006). The reason for increased sperm motility and viability by LA supplementation to semen extender in present study may be attributed to increased fatty acid proportion in the sperm head and tail membrane during cryopreservation that would have improved the fluidity, necessary for sperm motility and overall viability (Nasiri et al., 2011). Oleic and LA supplementation of extender significantly improved the motility and viability of boar spermatozoa (Hossain et al., 2007). The mechanism of how fatty acids enhance sperm motility and plasma membrane integrity is not clear yet. The probable reason for increased sperm motility and plasma membrane integrity is that LA might have served as energy substrates and its addition might stabilize the energy metabolism of spermatozoa.



LA supplementation in extender didn’t affect chromatin integrity; number of morphologically normal sperm (Fig 1) and acrosome intact live sperm (P > 0.05). This indicates that LA might not incorporate in the acrosome region of sperm head. Moreover, sperm chromatin is tightly coiled due to presence of highly condensed nuclear proteins especially protamine-1 in sperm nucleus (Martins et al., 2007) and it prevents the sperm chromatin damage during cryopreservation. Hossain et al., (2007) reported that supplementation of oleic and LA to extender significantly improved viability of boar spermatozoa. The difference in the result might be due to differences in lipid composition of the sperm plasma membrane in both species that is a key factor for freezability of the sperm (Parks and Lynch, 1992).

LA supplementation at 10.0 ng/mL in extender improved motility and plasma membrane integrity of cryopreserved Nili-Ravi buffalo sperm. It suggests that LA might have effectively incorporated in sperm membrane and unsaturated fatty acids of the cell membrane increased at low temperature. As a result the phase transition temperature decreased which suggests that cell membrane fluidity is secured.