Effect of in-vitro supplementation of polyunsaturated fatty acids on frozen- thawed bull sperm characteristics using Bioxcell extender

Cryopreservation is considered that it decreases sperm quality about 50 percent therefore research is being conducted to increase the frozen-thawed quality of bull sperm. The object of study was to examine the effect of polyunsaturated fatty acids on the frozen-thawed bull semen sperm characteristics. Semen was collected from three bulls with an average of 8 collection from each bull at interval of 3 days. Semen ejaculates with motility and normal morphology 70% and 80% respectively were processed in extender (BioXcell) having combined supplementation 0 to 15ng/ml of alpha linolenic acid (ALA) and docosohexaenoic acid (DHA). Extended sample was cooled at 5C for 2 hours, packaged and frozen into straws and in liquid nitrogen for 24 hours. Sequentially, straws were examined for sperm cytological characteristics, lipid peroxidation, fatty acid concentration and super oxide dismutase (SOD) after thawing. Sperm cytological characteristics decreased in the supplemented groups in comparison to control group. Docosahexaenoic acid improved in all supplemented groups, whereas ALA improved at 5ng/ml of ALA. Superoxide dismutase and lipid peroxidation were improved linearly by increasing fatty acids. Conclusion, combination of ALA and DHA has detrimental effect on frozen-thawed bull sperm parameters.


Introduction
Artificial insemination (AI) mainly depends on cryopreserved semen which is achieved with semen extender. Main role of extender is to provide energy, prevent contamination from bacterial and provide nourishment to the spermatozoa during freezing. Artificial insemination (AI) is based upon the good quality frozen semen. It is reported that liveability of frozen sperm is lesser than fresh semen in bulls. Freezing decreases fifty percent survival ability and fertility is decreased by seven fold of sperm [1,2]. There are series of processes such as freezing and thawing during cryopreservation which may affect sperm physically and chemically [3]. It is unclear that how sperm damage occurs, however, it is confirmed that decrease in liveability and fertility may occur due to the scratch in spermatozoa membrane [4]. Sperm membrane is main structure which regulates cellular processes, which are important for fertilization process. Lipids are main constituent of sperm membrane, mainly polyunsaturated fatty acids (PUFAs) [5]. Formation of ice crystals reorganize lipid and modify bonds of membrane and alters membrane fluidity. Lipids are important for lipid bilayer to control ionic exchanges, help for the sperm during development and acquire viability and regulate membrane physiology. Therefore, changes in sperm membrane decrease sperm fertilizing ability [6]. In addition, reactive oxygen species (ROS) are main production during freezing, which burns PUFAs by increasing the process of oxidation [4]. Role of PUFAs during cooling and storage is still being debated. The increase in concentration of PUFAs is believed to increase membrane fluidity, decreases resistant to cold shock due to the fatty acids presence and make sperm susceptible to the oxidation of lipids [7,8]. However, higher amount of PUFAs increases the frozenthawed quality of sperm [9]. Dietary DHA improved motility of the fresh semen collected from bulls [10]. In vitro addition of DHA did not enhance the viability of frozen boars sperm [11]. Similarly, dietary addition of PUFAs produced negative effect on motility and morphology of crude and stored equine semen [12]. Likely to dietary, in vitro addition of PUFAs in semen extender decreased frozen thawed quality of bull sperm in citrate extender [4,6], however, fish oil supplemented in egg yolk-lactose extender has enhanced frozen-thawed quality of boar sperm [13]. However, less data available on in-vitro supplementation of PUFAs effects on qualityof frozen-thawed bull semen. Therefore, this study was planned, to examine the effect of combination ALA and DHA on frozen thawed sperm parameters, fatty acid concentration, Melondialdehyde (MDA) and SOD on semen of bull extended into BioXcell ® Materials and methods Collection of Semen and experimental design An electro-ejaculator was used to collect semen ejaculates from three Brangus-Simmental cross-bred bulls (Electro Jac 5, Ideal Instruments Neogen Corporation, Lansing, Michigan, USA). Before collection each bull was prepared washing and cleaning of perpetual area and rectal massage for about 15minuts before collection. Eight collection from each bull total twenty-four ejaculates were collected at interval of 3 days. After collection semen was transported immediately at 37 o C to the laboratory for evaluation and further processing. Samples having motility of ≥70% and normal morphological of ≥80 % and were used in this study.
Each ejaculate was added into BioXcell ® consist of equal concentrations (control), 3, 5, 10 and 15ng/ml levels of DHA and ALA were added (Sigma Chemical Co., St. Louis, MO, USA). Ethanol (0.05%) was used to dissolve the fatty acids in water [14]. The samples extended were kept warm at 37 o C for 15 minutes to assimilation of fatty acids into membrane of sperm [15,16]. Then, diluted samples were cooled at 5 o C (2 hours) [17], and then packed in 0.25ml straws with average of 20 x 106 and successively, frozen into liquid nitrogen for 24 hours. The frozen straws were thawed at 37oC for 30 seconds in water bath [18,15,16], and then investigated for sperm characteristics, fatty acid concentration, SOD and MDA production.

Evaluation of semen
Motility was determined by automatic semen analyzer, (Computer assisted semen analyser, CASA, IVOS Hamilton Thorne bioscience, Beverly, Massachusetts USA). Twenty µl of diluted semen was placed on glass slide CASA 2X-cell (20 µm) cover slip was placed to cover (20mm x 20mm) after Loaded on the CASA for motility calculation. An average Superoxide dismutase test was calculated by (Cayman chemicals company USA) assay kit. Two hundred fifty µl of semen were placed in Eppendorf tube and centrifuged for 10 minutes at 1000 x g. The upper (seminal plasma) layer was discarded while lower (sperm rich ) layer was vortex-for 10 seconds for 3 times with 0.5 ml of 0.1 % Triton X-100 (Fisher, Montre´al, PQ, Canada) and centrifuged again for 3 minutes at 1000 x g [19]. Other process was followed as the company recommended. Peroxidation of lipid was calculated by melondialdehyde (MDA) production using thiobarbituric acid reactive substances (TBARS) according to the method explained by [15,20].

Statistical analysis
Data of sperm cytological characteristics, Superoxide Dismutase, fatty acid concentration and melondialdehyde were analysed using the general linear model (GLM) with SAS 9.2 version. Duncan multiple range test was used to differentiate among the means. P.values were considered to be statistically significant at (P<0.05). Table 1 presents percentages of cytological characteristics of frozen-thawed spermatozoa extended in Bioxcell® consist of fatty acids.

Results
The results of this study shows that all sperm cytological characteristics decreased (P≥0.05) in supplemented groups than control. Though, SOD increased significantly in supplemented groups than control. Fatty acid concentration of frozen samples is shown in Table 2. Results of DHA concentration was higher (P<0.05) at 10 and 15ng/ml.
A non-significant difference (P>0.05) other supplemented and control groups. Alpha linolenic acid was higher at 5ng/ml. There was no significant difference among n-3 PUFAs (P>0.05). Figure 1 presents results of the MDA production. According to the graph MDA production was higher (P<0.05) in supplemented groups than control.

Discussion
Present study, addition of fatty acids reduced characteristics frozen thawed sperm such as motility, membrane integrity, morphology, acrosome integrity and viability. Earlier studies are in line with this study, which reported that PUFAs reduced quality of frozen thawed bull semen in citrate extender in [6]. Similarly, other experiment also reported that PUFAs supplementation lowered sperm viability; motility and morphology [4]. Moreover, the PUFAs with an antioxidant in ovine semen improved the frozen-thawed quality parameters [5]. It is proven before that that cryopreservation reduces the sperm quality [11]. In the current study, PUFAs reduced the quality of frozenthawed bull semen. Polyunsaturated fatty acids affects positively and negatively on fertility usually depend on only the type of fatty acids [21]. Polyunsaturated fatty acids are found in sperm membrane man, ram and bull. Fatty acids improve the membrane fluidity, and provide resistance to the sperm against cold shock [22]. However, PUFAs such as DHA are important to maintenance of the membrane fluidity and its fertilizing ability.
[23]. Nevertheless, omega-3 fatty acids also make sperm susceptible by ROS production and lipid peroxidation which compromise the functional integrity of the sperm. The present study with addition of ALA and DHA improved fatty acids the in frozen sperm of bull. Alpha linolenic acid, EPA and DHA was significant difference among treatment and control. However, total n-3 fatty acids were non-significant. This may be due to the ALA is considered precursor of DHA and other n-3 fatty acids. These results are in range of results obtained after addition of n-3 fatty acids, improved DHA concentration and total PUFAs in ram semen [5], in bulls [5,14], in goats [24]. However, about boars' results reported after of in -vivo and vitro addition of PUFAs enhanced n-3 PUFAs of frozen semen [25]. Melondialdehyde is the main concern after addition of ALA and DHA for decreases the quality of bull semen, in the present experiment the MDA increased after addition of ALA and DHA which resulted in decreased frozen thawed quality of bull semen. The supplementation of PUFAs decreased resistance against lipid peroxidation [6]. Moreover, it is reported that supplementation of PUFAs decreases resistance against cold shock and increases lipid peroxidation in the semen [4]. SOD level was improved that were similar to results reported in chicken [26], and similarly PUFAS from fish oil increased SOD [27]. In current study enzymatic (SOD) activity was enhanced but it was not enough to prevent the frozen thawed sperm damage. Being the most important defense enzyme in the sperm but due to less concentration in the cytoplasm it is unable neutralize the peroxidation in sperm during freezing [28]. Conclusion Conclusively, current study revealed that combined supplementation of DHA and ALA into BioXcel ® reduced quality of frozen thawed bull sperm. All sperm parameters were decreased in relation to control.