doi:10.5455/vetworld.2013.579-585 Sperm selection techniques and antioxidant fortification in low grade semen of bulls: Review 1

The low grade ejaculates are very common in bulls. Low grade ejaculates might be due to age or non specific factors like thermal stress, transport and vaccination stress during the dynamic life of bulls. Lipid peroxidation of membrane induced sperm damage further aggravates the situation. Researches reveal that selection of sperm and antioxidant fortification play crucial role in improving the quality of semen. Different methods used for semen up-gradation like washing, sedimentation, swim up procedure and filtration like percoll gradient, glass wool, sephadex and sephadex ion exchanger with significant improvement in motility, Hypo osmotic swelling test reactivity, viability and acrosomal integrity. Antioxidants are added directly to extender at standard dose rate with positive result. Among different filtration column used sephadex ion exchanger (FS+IE) was superior over other in improving the semen quality especially when fortified with Vitamin E. Moreover a complete protocol is required, which contain both antioxidant fortification and sperm selection simultaneously to handle the 
low grade semen from sub-fertile bulls.


Introduction
Previously different methods were tried for improving the low grade semen by reducing the number of dead or The improvement in quality of semen is an abnormal cells by trapping it in filtration column.important aspect for maximum utilization of Fertile and quality spermatozoa can be selected using genetically superior sub-fertile sire, for the reason of different processes, such as centrifugation, washing, age and or non specific factors like thermal stress, pooling, chromatography, sedimentation (bovine transport and vaccination stress during their dynamic serum albumin gradient), percoll gradient, and swim life show a low grade semen quality than previously.It up procedure, followed by glass wool, glass bead, is usual practice to feed poor quality ration, especially sephadex gel and sephadex with ion-exchanger column silage, in scarcity period to the bulls which shown an filtration [4].Sephadex with ion exchanger signifiincrease in abnormal and dead spermatozoa count in cantly improve the quality of fresh semen [5], semen due to some unknown factors [1].The metabolic especially when fortified with anti-oxidant agent like byproducts released by infertile spermatozoa are vitamin E [6].The sephadex and ion exchanger filters believed to adversely affect the fertilization potential are superior over other as it filters the extended semen of fertile male gamete, ultimately influences the efficiently.These techniques not only allow for fertility of productive bull.Peroxides, free radicals, selection of best sperm which enhances motility but various proteolytic enzymes released from may also be used to remove the extender and dead cells disintegrating spermatozoa damage the plasma present in frozen thawed sperm sample.membrane of fertile spermatozoa [2].More than 75 The success of semen preservation depends largely percent of total ejaculates are discarded due to poor on resistance of spermatozoa to ultra low temperature.initial motility even in high quality elite cross bred However, conventional technique i.e. freezing protocol bulls [3].The problems of low grade semen ejaculates without antioxidant fortification, are able to recover are common predominantly in crossbred bulls.
only small portion of the frozen germplasm on Ejaculates of semen from bulls with compen-sable thawing.Rest being destroyed or become less viable sperm defects seem to be significantly improved by moreover more viability is desired to achieve target filtration of neat or extended semen.Filtration conception and fertility.The reduction or loss in significantly improves fertility of sub-fertile bulls after viability and fertility can be attributed to many changes removal of dead and abnormal spermatozoa from takes place in the sperm cells during cryopreservation.extended ejaculates using different filtration media.
Changes in sperm cell structure and function related to cold shock have often been reported, especially in plasma membrane, acrosome and mitochondria.Plasma membrane and acrosome are important sites of the doi:10.5455/vetworld.2013.579-585changes caused by cryopreservation process.Cryo-profiles were studied before and after processing preservation affects the lipid architecture of plasma (different process including filtration and antioxidant membrane [7] and metabolism due to high amount of fortification) to access the effect of the same.polyunsaturated fatty acid with significantly less Different methods of semen upgradation cytoplasmic component containing antioxidant [8].It 1. Washing Procedure: The semen sample was is susceptible to peroxide damage with subsequent loss o centrifuged (800 x g for 10 minutes at 16 C) in a test of membrane integrity, impaired cell function and tube followed by removal of seminal plasma using a decreased motility of spermatozoa.Cold shock Pasteur pipette.Tris-citric acid was then added to enhances susceptibility of spermatozoa to lipid perosemen up to previous volume, and then pellets were xidation and structure alteration [9].Lipid perodispersed in tris buffer.The second centrifugation was xidation of membrane is considered to be the key of done in analogous condition and supernatant was reactive oxygen species (ROS) induced sperm damage removed and reconstituted to the original volume by [10], leads to infertility.Moreover, buffalo bull buffer [12].Washing of semen is almost similar to sperma-tozoa are more vulnerable to oxidative damage sephadex filtration with respect to post-thaw recovery as compared to cattle bull spermatozoa [7,11].Addition rate. of antioxidant might be helpful in preventing the damage under such condition especially ascorbic acid 2. Sperm sedimentation: Semen sample was taken in and á-tocopherol [10].It is therefore, worthwhile to special glass tube with inner cone that allow only those fortify the low grade semen especially after filtration spermatozoa capable of swimming out from the for improving the keeping quality of semen.
liquidized semen to the sediment in the inner cone.

Purpose
Here ROS production minimized because centrifugation not required here.Recovery rate is very less in Maximizing the production of high grade semen this method.i.e., without discarding too many poor qualities semen ejaculates.wool, micro fiber is gently placed at a depth of 2 cm.

Evaluation of semen
The column was vertically suspended and rinsed Semen is evaluated in terms of spermatozoa repeatedly with different media including 5 mM concentration, individual motility, membrane integrity caffeine sodium benzoate and 10 ìg/mL heparin to using Hypo osmotic swelling test (HOST), live-dead remove any loose wool fiber prior to filtration.The  be used to improve substandard semen ejaculates [18].allowed to swell overnight at 4 C.The filtration column Glass wool filtered semen was better as compared to was prepared in a 10 ml disposable plastic syringe.A sephadex and percoll gradient especially for embryo hole (1.6 mm) was drilled at 8 ml level of the syringe production [16].However the effect was not significant barrel to allow removal of air trapped in the barrel.A in buffalo semen [19].
small amount of glass wool was compressed with the After filtration, the glass wool should be gently plunger to bottom of the barrel to prevent the loss of removed from the Pasteur pipette and rinsed with trissephadex.A plastic tube was attached to the tip of the buffer in watch glass.Smear prepared from this syringe and clamped.The free end of the tubing was solution may used for calculating the percentage of inserted in the filtrate collection tube.In FS (sephadex dead and abnormal spermatozoa.filtration) column, 3 ml slurry (0.25 g) of sephadex was Glass wool filtration of semen has beneficial gently layered over the glass wool and allowed to settle effect as it showed improved motility of spermatozoa for 3-4 minutes (Fig. 4, 5 and 6).and lower incidence of abnormal spermatozoa.
Mechanism of trapping by sephadex: A barrier is Spermatozoal concentration declined because of provided by sephadex particles or allows immotile or effective retention of dead and abnormal sperm cells, dead spermatozoa to agglomerate.The protein may be especially the spermatozoa with mid-piece and tail present on capacitated spermatozoa which bind with abnormality.Capacity to withstand cold shock and sephadex particles easily.freezability reported to improved after the filtration.All beneficial effects were more pronounced in poor cellulose prepared fresh before preparation of column g Hepes in 50 ml distilled water].The 90% percoll [6].solution was obtained by diluting a stock of percoll solution with Bracket and Oliphant (BO) media.For Ion exchanger: An ion exchange resin consists of an preparing 45% percoll solution, 90 percent percoll insoluble matrix with attached covalently charge solution was mixed at a 1:1 ratio with medium.In a 15 group.Both positive and negative charged ion exchangers ml conical tube, 1.5 ml of the 90 % Percoll solution was are available.Negatively charged exchangers binds added and 1.5 ml of 45% Percoll was deposited.The with positively charged ions and vice versa.Anion average percent motility in percoll was higher as binds with one type of cation however when presented compared to sephadex and glass wool.More over with a second type of cation, this may displace or greater development rate to blastocyst stage than exchange with first one.Hence these resins are called cleaved embryo produced by percoll filtration as cation-exchange resins.compared to glass wool [16].
Mechanism of trapping in Ion exchanger: Surface 6. Preparation of sephadex (FS) columns: Tris-citric charge of sperm changes after its death, therefore on the acid buffer [24 mg/ml Tris hydroxymethyl aminobasis of difference in charge between live and dead spermatozoa can be separated by using respective scopy is also used for selection of sperm for the purpose opposite charges.
of in-vitro fertilization.

Filtration of semen:
Membrane integrity of sperm: Lipid being the main membrane constituents of cells play most important role in providing barrier and also involved in cell response to stimuli like hormone, growth factors and neurotransmitters.Peroxidation of lipids by ROS damages the important parts of spermatozoa and thus causes either cell death or altered characteristics of the sperm cells.The reactive oxygen metabolite (ROM) is oxygen centered free radical and their metabolites.Polyunsaturated fatty acid (PUFA) is present in high proportion in lipid fraction of spermatozoa, which reflects the need for maintaining high membrane fluidity and fusion with the oocytes.The main source of ROS is mitochondrial respiration in fresh semen, however in case of processed semen ROS originate Effect of filtration on semen profiles: There was from contaminating lecocytes as well as from significant improvement in motility, live spermatozoa spermatozoa with residual cytoplasm.Spermatozoa and intact acrosome after filtration of semen through also produces ROS by their flagellar motion sephadex G-15 [20], G-100, G-200.The semen volume contributes the lipid peroxidation [28].ROS at low and sperm concentration decreased after filtration concentration shows positive biological effects and significantly (P<0.01).Considerable decrease in also regulate physiological sperm function such as sperm abnormalities was also reported.The G-100 and capacitation and sperm egg fusion although it may G-200 column was proved more efficient as compared cause pathological effects with oxidative stress when to G-25 to G-75 [21].Sephadex filtration was superior the oxide exceeds the scavenging activity of over simple washing by centrifugation with respect to antioxidants [29].Production of ROS can not be different sperm profiles.FS + IE column method was prevented completely; however measures may be superior over FS and other methods [22,23].Post-thaw taken to minimize them to improve semen profile by recovery rate of motile spermatozoa was higher in incorporating various agents like antioxidants, filtered semen as compared to non-filtered (68 vs 39%) membrane stabilizer and heavy metal scavengers.semen.Semen quality was best preserved by filtration of semen in sephadex plus ion exchanger combination Oxidative stress: The oxidative stress is a medical term method fortified with vitamin E [6].Tail and total for damage to animal cells caused by ROS which abnormality of spermatozoa was also reduced include superoxide, singlet oxygen, peroxi-nitrite or significantly.The effect of traditional glass wool hydrogen peroxide free radical.It is defined as an filtration was improved by using annexin V binding imbalance between pro-oxidant and antioxidant, with which isolates apoptotic marker spermatozoa [24] and more pro-oxidant.In other words, oxidative stress is thus enhances the results of IVF.The column filtration result of imbalance between ROM productions and is effective methods to enhance sperm quality by selecting neutralizing capacity of antioxidant system.Oxidation viable and morphologically normal spermatozoa without is essential to nearly all cells in the body to provide altering DNA, plasma membrane, mitochondrial energy for vital function.Approximately 95-98 percent sheath integrity or inducing premature acrosome of oxygen reduced to water during aerobic metabolism reaction [25,26].However, in vitro fertilizing capacity and remaining fraction converted to oxidative by not differs significantly for buffalo semen [19] after product, ROS that may bring degenerative changes [9].

filtration. Selection of sperm based on sperm
Antioxidant fortification: The antioxidants are membrane maturity includes hyaluronic acid (HA) substance, able to neutralize other oxidizable substrate sperm binding, as HA binding site on sperm regarded and thus significantly delay or inhibit the oxidation of as sign of sperm maturity [27].Selection of best sperm the substrate at relatively low concentration.based on live sperm organelle morphology exami- The maintenance of sperm membrane phosphonation in inverted light microscope in magnification of lipids and its susceptibility to per-oxidation (oxidation over 6000 came into existence.Now-a-days spectro- The semen was filtered at room temperature in prepared column.The syringe was secured vertically in filtration frame.Before filtration of semen the buffer part of slurries was removed by releasing the tubing clamp, covering the hole on the syringe and forcing the plunger manually.The plunger was not allowed to pass through the hole in order to prevent the lifting of the column while removing the plunger.The extended semen sample was gently layered on to the column and filtered at rate of 1.5 ml per minutes.Fortification of antioxidant with semen before filtration improved the recovery rate of spermatozoa through the same sephadex and ion exchange column more than 90 % [6].by peroxide) depends on adequate antioxidant status, lation severely damage the cell and tissue.It is a very which are responsible for reduction in the risk efficient scavenger of free radical.Vitamin E localized associated with sperm damage during storage.If there in cell membrane, therefore it can not protect the is deficiency of these fractions spermatozoa may get cytosol from free radicals, its counterpart "selenium" damaged and motility and fertilizing ability diminishes.
present in cytosol is responsible for protection in Naturally antioxidants are present in seminal cytosol.Vitamin E increases intracellular ATP and plasma, protecting the spermatozoa against lipid decreases cell permeability and enzyme inactivation peroxidation.Antioxidants are classified as exogenous [32,33].Vitamin E was superior over vitamin C and (natural or synthetic) or endogenous compound, both vitamin E+C blend [23].Vitamin E (4.5 mM) and responsible for removal of free radical, scavenging Vitamin C (9 mM) was superior over glutathione (7 ROS, inhibiting ROS synthesis and binding metal ions mM) with respect to post-thaw motility [34].However required for catalysis of ROS generation [30].Natural in buffalo á-tocopherol addition in skim milk based antioxidant system divided into two groups enzymatic extender did not improve sperm profile significantly and non enzymatic.Enzymatic antioxidants made up of [35].limited number of proteins like catalase, glutathione Mechanism of action of Vitamin E: Vitamin E prevents peroxidase SOD (superoxide dismutase).Non cell damage by binding to the free radical and enzymatic antioxidant classified into direct acting neutralizing its unpaired electron mediated by an (lipoic acid, poly phenol and carotenoid) and indirect 'tocopheryl-quinone' formation.acting antioxidant, which binds to redox metal to prevent free radical generation (Chelating agent).An Vitamin C as antioxidants: Vitamin C required for integrated antioxidant system is provided to the bovine growth and repair of all body tissues and formation of seminal plasma by association of natural antioxidants collagen.It protects sperm cell from free radical (vitamin E, Ascorbic acid and glutathione) and antidamage.Antioxidant efficacy of vitamin C was oxidant enzymes (superoxide dismutase, glutathione superior over caffeine and chlorquine in diluter.peroxidase).This system gives the semen protection Fortification of ascorbic acid (0.02 %) or cholorquine from free radicals as well as from toxic products of diphosphate (10 M) to extender improves the semen their metabolism.The delicate balance between free quality and fertility of buffalo bull semen and vitamin radical production and antioxidant defense is an C was slightly superior over Chlorquine diphosphate important determinant of grade of semen with respect [36].to fertilizing ability.During period of semen Glutathione: Freezing of semen followed by thawing processing like filtration and storage the level of stress causes a significant reduction in glutathione content in and ROS production increases.This will cause bull sperm.Exogenous glutathione in extender at the enhanced oxidative damage by overwhelming the dose rate of 2 mM improves post-thaw quality of normal scavenging mechanism of oxygen species by buffalo bull spermatozoa [37].antioxidants in the seminal plasma.Therefore additional antioxidant competence is desired by the Cysteine fortification: Semen extender containing Lsperms.During the process of cryopreservation, cysteine (1.0 Mm) improves the catalase activity [38, irreparable damage can be prevented as well as keeping 39] plasma membrane integrity [40] and freezability of quality of the cells can be enhanced by fortification of spermatozoa [41,42].extender with antioxidants.
improvement in pentoxifylline fortified fraction [43].cation commercially so that it is constantly used for low Glass wool filtration of bull cryopreserved semen: a rapid and effective method to obtain a high percentage of grade ejaculates of elite bulls, immediately at large functional sperm.Theriogenology., 78 (1): 201-9. scale.

Figure- 4 .Figure- 5 .Figure- 6 .
Figure-4.Filtration of semen using Control vs FS, FS+IE, FS+IE+ Vit E Figure-5.Microscopic view of trapped spermatozoa in Sephadex filter Figure-6.Microscopic view of blank sephadex filter both pre-freez and post-thaw stage of semen 13.Mustafa, G., Anzar, M. and Arslan, M. (1998) Separation of preservation.Although it requires interventions and motile spermatozoa from frozen-thawed buffalo semen: swim-up vs filtration procedure.Theriogenology., 50: 205-211.additional sophisticated preparatory stages in standard 14.Chandrahasan, C., Pattabiraman, S. R. and Venkataswami, V. protocol of cryopreservation of semen, it might be (1986) Studies on the effect of Glass wool column filtration recommended for routine AI work to improve sperm on the quality of semen of cross bred bulls.Indian Vet.J., 63: viability after thawing.The washing, sedimentation 913-918.15.Vyas, S., Dhami, A. J., Mohan, G. and Sahni, K. L. (1991) and filtration can be used for sperm preparation in IVF Effect of sephadex and glass-wool column filtration on the laboratories by using zona free oocyte preparation test o quality and storage (at 5 C) of crossbred bull semen.Indian or swim up test for motile sperm separation.Filtration Journal of Animal Science., 61 (7): 702-704.technique is proved efficient and antioxidant fortifica-16.Lee, H., Kim, S., Ji, D. and Kim, Y. (2009) A comparative tion is also equally important as far as improving study of sephadex, glass wool and percoll separation techniques on sperm quality and IVF results for preservation quality of semen is concern, moreover cryopreserved bovine semen.J. Vet.Sci., 10 (3): 249-255.need of hour is, to use it as ready to use package i.e., 17.Arzondo, M. M., Caballero, J. N., Marin-Briggiler, C. L., sperm selection procedure plus antioxidant fortifi-Davit, G., Cetica, P. D. and Vezquez-Levin, M. H. (2012)

7. Preparation of Sephadex (FS) plus Ion exchanger quality
/low grade semen, as compared to high grade (IE) filtration column: In same line as sephadex semen.column, the FS +IE column was prepared.For this 1.25 ml (0.1 g) di-ethyl amino-ethane-52 (DEAE positive

5. Percoll separation procedure: Percoll solution
It was superior over used for compounds sharing the tocopherol and vitamin E and vitamin C as it has both antioxidant and tocotrienol structures, which are lipid soluble in nature.antimicrobialactivity simultaneously.Vitamin E appears to be the first line of defense against peroxidation and is important for maintaining low Vitamin E as antioxidants:Term vitamin E is generic n-Propyl gallate as antioxidants: Coenzyme Q10 and a-lipoic acid: The Coenzyme Q10 tissue concentration of peroxide, which on accumuand á-lipoic acid are antioxidants that play a role in