View of Evaluation of the antioxidant effect of vitamins C, E and Alpha lipoic acid (ALA) in improvement of Bull semen quality

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http://annalsofrscb.ro 17510

Evaluation of the antioxidant effect of vitamins C, E and Alpha lipoic acid (ALA) in improvement of

Bull semen quality

Sumaya Loay Mohamed Shams Al-Dean , Wafir Mahdi Saleh Department of Surgery & Obstetric, Collage of Vet. Medicine, University of Baghdad

Abstract

This study is designed to evaluate the effect of three chosen antioxidants (Vit.C, E and ALA) on improvement the poor semen sample collected from Holstein bull that their semen was introduced to the AI center (Cryopreservation).

Semen was collected twice daily as per AI center rule, divided to three samples according to the antioxidants number, each sample is subjected to one antioxidant that adding to the semen extender by three concentrations (treatments). The three treatments are arranged as control, cooling and freezing aliquots, semen parameters as; individual motility, dead and abnormalities % are recorded.

Upon cooling of seminal sample the motility of the control group showed 30%

motility index before treatment, to be 55% after Vit. C treatment, 50% after vit E treatment and 55% after ALA treatment. While seminal samples motility index upon freezing revealed as 20- 25% for the control group (before antioxidant treatment), Vit. C treated group gave 45% motility index, 40% upon Vit.E, to be 45% after ALA treated group.

Semen parameters after Vit. C treatment concerning dead sperm % and sperm abnormalities % through diluted, cooled and freezed sperms samples showed; dead % were 12.6 %, 11.0% and 11.6% respectively according to abnormalities % 11.6%, 10.4%

and 12.2% respectively. While the same parameters concerning Vit. E treatment revealed that; 13.3% dead sperms for diluted sample, 13.0% for cooled and 16.1% for freezing.

While that of sperms abnormalities % showed 11.6%, 12.3% and 13.2% for diluted, cooled and freezed semen samples respectively. Effect of ALA treatment concerned the seminal parameters showed 13.5% dead sperms upon diluted, 13.7% upon cooling and 12.5% upon freezing. While the percentage of same sperms parameters after ALA treatment upon sperms abnormalities % revealed 13.7% for diluted, 10.3% for cooled and 11.0% for freezing.

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The mean values of Vit. C, Vit.E and ALA treatments on individual motility in different dilutions after cooling revealed that; T1 40.7± 4.13, T2 41.4± 5.14 and for T3 47.8± 5.18 (M±SE), while those with Vit.E T1 36.2± 3.25, T2 42.5 ± 4.73 and T3 46.2 ± 5.43 (M± SE), and for ALA were T1 38± 3.61, T2 40± 3.82 and for T3 45± 4.82 (M±SE).

The mean values of Vit. C, Vit.E and ALA treatments on individual motility in different dilutions after freezing revealed that; T1 35± 2.56, T2 35.7± 3.12 and for T3 41.4± 3.56 (M±SE), while those with Vit.E T1 26.2± 2.11, T2 31.2 ± 2.85 and T3 35 ± 2.23 (M± SE), and for ALA were T1 33± 3.06, T2 34± 3.12 and for T3 39± 3.17 (M±SE).

Key words: Bull, sperm, cryopreservation, motility, antioxidant, freezing , cooling.

Introduction

Artificial insemination (AI) is one of the important assisted reproductive technologies, which causes widespread propagation of semen, limiting the spread of venereal transmitted diseases and mainly facilitating improvement on genetic makeup of animal. It suggested that spermatozoa of the mammalian species are highly sensitive to oxidative damage due to presence of high lipid bi-layer of plasma membrane (Sansone et al., 2000; Chaudhari et al., 2015).

Naturally bovine semen have defense system against oxidative damage, it is not tolerate under refrigeration and cryopreservation temperature. The extender/diluents used in preservation of semen is considered as important factor, which should has ambient pH and buffering capacity followed by appropriate osmolality to protect spermatozoa from the cryogenic injury. Oxidative damage of bull spermatozoa during preservation could be hindered by addition of suitable additives to the semen diluents/extenders (Ansari et al., 2011)

Semen evaluation is extremely important for successful artificial insemination (AI) in which a large number of straws is produced from an ejaculate (Barros et al., 2010). AI becomes a tool of disseminating superior genes for economic traits like milk production by propagating the animals with high genetic potential (Baruselli and Carvalho, 2005).

(Kjaestad et al., 1992)

Alpha lipoic acid was first isolated in 1951 , it was found to be a co- factor for many mitochondrial enzyme complex that are involved in energy production (Reed, 2001). The chemical structure of ALA is a medium chain fatty acid with two sulfur atoms that is synthesized within mitochondria by lipoic acid synthase. Lipoic acid is a disulfide compound that is found naturally in mitochondria as coenzyme for pyruvate dehydrogenase and a-ketoglutarate dehydrogenase (Shay et al., 2009).

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Antioxidant effect of ALA it was shown that lipoic acid have powerful antioxidant abilities, ALA has the unique ability to neutralize free radicals within aqueous and lipid regions of the cells as well as intracellular and extracellular environments, this ability allows lipoic acid to be easily transported across cellular membranes to neutralize the free radicles (Gurer et al., 1999). All ALA supplied by the diet is transported in the blood stream to tissue and incorporated in to cells, and then it is transferred in to the mitochondria. Studies in mammals have shown the alpha lipoic acid supplied by the diet dose not supply enough for purposes such as incorporation in to enzyme complexes (Shamsi et al., 2001).

Vitamin E appears to be the first line of defense against peroxidation and is important for maintaining low tissue concentration of peroxide, which on accumulation severely damage the cell and tissue. It is a very efficient scavenger of free radical.

Vitamin E localized in cell membrane, therefor it cannot protect the cytosol from free radicals, its counterpart “selenium” present in cytosol is responsible for protection in cytosol. Vitamin E increases intracellular ATP and decreases cell permeability and enzyme inactivation (Breininger et al., 2005). However in bull a-tocopherol addition in skim milk based extender did not improve sperm profile significantly (Akhter et al., 2011 ). Vitamin C (ascorbic acid or ascorbate) are non-enzymatic antioxidants. Vitamin C was discovered in 1912, isolated in 1928, and in 1933 was the first vitamin to be chemically produced. (Hughes et al., 1998). The addition of anti-oxidants is well known method to improve viability and motility of liquid storage or cryopreserved bull sperm cells. The most important antioxidants in seminal fluid seem to be vitamins C .The concentration of vitamin C in seminal plasma is 10 times greater than in blood plasma (Al-Guborya, et al., 2010).

Materials and Methods Experimental animals:

As this study was conducted on the AI center bulls that their semen were introduced to the dilution and cryopreservation, according to this reason we choose four bulls on each collection trails randomly to overcome the ejaculate volume. By his method and according to the AI center policy the duration of collection times are two times weekly, as well as we can inspect the semen of more than 10 bulls to get more accurate results.

Bulls are Australian Holstein breed, aged 3-4 year according to the AI center records, and are good trained for collection activities. Collection period is predominate early

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morning by artificial vagina method and from the rejected semen which is not fit for cryopreservation issue we take 5 ml for treatment.

Semen Collection

The semen was collected twice weekly from each bull by artificial vagina, the semen samples were immediately transferred to laboratory for processing and initial evaluation. Ejaculates with less than 40% of initial individual motility according to the AI center technique were considered as poor sample.

Study is started from December 2019 to May 2020, 8 semen samples weekly. As a routine work in the AI center, sample was immediately taken to the laboratory after semen collection, placed in water bath at (37-38 Cº) for macroscopic evaluation; volume (ml) and color. Microscopic evaluation included mass activity%, individual motility%, sperm concentration (x10/ml), dead%, abnormality%, acrosome integrity%, according to the following procedures.

Semen Dilution

Tris diluent was used with different concentration according to semen consistency, Tris content is shown in table (1).

Table (1) Tris content:

Substance Amount

Tris 12.12g

Citric acid 6.68g

Fructose 5g

Distal water 368 ml

Glycerol 32 ml

Egg yolk 96 ml

Gentamycin 3.1 ml

D.W up to 500 ml

(Bearden et al., 2004) Semen evaluation: Physical Evaluation

A. Macroscopic examination 1. Volume

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The volume of ejaculated semen was measured directly with graduated collection tube according to (Mortimer 2000), all results were recorded.

2. Color

Semen color has been determined grossly as watery/ opalescent, milky white, yellowish and creamy according to (Salisbury et al., 1978), all results were recorded.

3. pH

pH of semen was determined by paper strips by dipping the indicator pH paper in to the tube which contain the samples and observation changing in the color of paper and then compared with a table that has a pH for each color and according to (Salisbury et al., 1978).

4. Mass motility

The percentage of mass motility was directly recorded after semen collection a drop of raw semen on a warm slide under light microscope at 10x magnification with attached stage warmer (temperature was set at 37 Cº), The score was computed from 0 to 100 grades, as shown in table (2)

Table (2) Evaluation of mass motility.

% Wave motion

0 No wave, total immobility 10 No wave, individual movement 20-40 No wave, very slow movement

45-65 Wavy appearance, slow amplitude of wave 70-85 Wavy appearance, rapid wave motion, no eddies 90-100 Wavy appearance, rapid wave motion with eddies (Evans and Maxwell, 1990).

2. Individual motility

The Individual motility determined by mixing one drop of raw semen with two drops of 2.9% sodium citrate solution on warm slide at 37 Cº, and examined under light microscope at 40Xmagnification. Motility was scored based on percentage of sperm with normal progressive forward motion, whereas those exhibiting circling or oscillating motions at one location were regarded as immotile. The score was calculated as in table (3).

Table (3)- Estimation of individual motility score (Chemineau et al., 1991).

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% Individual motility

5 No motile sperm

10 1/5 of sperms motile

20-40 2/5 of sperms motile

3. Sperm concentration

The fresh semen evaluation by (semen analyzer) spectrophotometric analysis (spectrophotometer – IMV Photometer). Sperm cell concentration was determined automatically at 535 nm wave length by using pre-warmed and calibrated photometer was set to zero by using normal saline, then 20 ml from each semen sample was diluted in 2ml of normal saline (Atiq et al., 2011).

4. Dead sperm Percentage

Sperm viability was assessed using eosin and nigrosin stains (E + N). The preparation of staining solution: Dissolve 1.67g eosin and 0.9g of sodium chloride in 100 ml of distilled water in a glass beaker placed on a stirring hot plate heat gently and add 10.0 g nigrosin and dissolve it before bringing the stain to a boil. Remove the beaker from the hot plate and allow it to cool at room temperature. The stain must be filtered by filter paper and store at 4Cº in a dark glass bottle (Douglas and Kenneth, 2013).

A drop of semen sample and a stain were mixed on glass slide, air dried slides were examined under phase contrast microscope X100; Oil immersion. non-viable spermatozoa stained blue, while unstained spermatozoa is considered as viable. A total of one hundred spermatozoa were counted for each sample as reported by Mortimer (1994).

5. Sperm Abnormalities %:

Abnormalities detected by eosin-nigrosin staining method as discussed by (Salisbury et al., 1078). The slide which is used to calculate sperm abnormalities percent under light microscope (oil emersion x100), types of abnormalities in head, tail and any defect in normal shape of sperm which can be seen under light microscope are included (Evans and Maxwell, 1987).

Statistical analysis

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Results are indicated as Mean ± SE. Statistical analysis was conducting using General Linear Model procedures (GLM) of SPSS version 16.0 (SPSS lnc. Chicago, IL, USA).

Comparisons between values were analyzed by Duncan`s multiple range test following F- test in ANOVA (Duncan. 1955). Significant was set at (p<0.05).

Results and Discussions

This study is well designed to improve the poor or not good quality semen in regarding to the AI center rules that all the poor or not qualified semen is rejected or keep away from the manufacture or cryopreservation activities. This is introduced by studying the one important factor that affects the semen quality after cryopreservation which is the antioxidants.

The three important antioxidant agents are Vit.C, Vit. E and the lipoic acid.

1- The effect of Vit C treatment on semen individual motility after cooling of the sample showed in tab. (1), in which; as the dilution rate of the antioxidant increased the sperms individual motility will become more active and modulate, this effect is approved by(Ahmad et al., 2014), in which the increase level of Vit. C as antioxidant factor on the individual semen motility will be more effective

Table (4) effect of vitamin C on individual motility of bull after cooling No.

of bull

Dilution rate

Individual motility %

Motility after cooling control

T1 T2 T3

1 1:20 35 b A 45 a A 50 a A 55 a A 45 a

2 1:15 40 a A 45 a A 40 a A 45 a A 40 a

3 1:20 40 a A 40 a A 45 a A 50 a A 30 b

4 1:10 40 a A 35 a A 40 a A 45 a A 35 a

5 1:8 40 a A 40 a A 40 a A 45 a A 40 a

6 1:7 40 a A 40 a A 45 a A 50 a A 45 a

7 1:6 35 a A 40 a A 30 a B 45 a A 35 a

*Different small letter mean significant difference p<0.05 within group.

*Different small capital mean significant difference p<0.05 between group.

Maia et al., (2010) confirms these findings that the additional of Vit. C improved the individual motility after cooling; the more increase vitamin dilution is the more

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individual motility rate and suitability. The addition of vitamin C not only improved viability but it also protected acrosome and membrane integrity, Vitamin C acts as water- soluble antioxidant in blood plasma and seminal plasma and this positive impact of adding Vitamin C to diluted semen appears to identify with a decrease in DNA damage of spermatozoa as mentioned byMaia et al., (2010).

Papahn, et. al, (2011) agreed that the individual motility affect the semen quality and fertilization rate in which the beneficial effect of Vitamin C in improving fertilization rate was potentially because of a reduction in lipid peroxidation of bull sperm. However, addition of vitamin C to a semen extender can reduce the oxidative stress during cooling of semen thus protecting the bull semen during the final step of cryopreservation as well as improving the longevity and quality of sperms(Barati and Papahn, et al., 2011).

2- Effect of Vit. C treatment on individual motility after freezing

Results of the three treatments upon individual motility after freezing showed that; when the freezing process proceed spermatozoa movement will be arrested or stopped by the action of cryopreservation, this arrested movement then will be

accomplished by the action of the thawing process (Beconi, et al., 1993) by that time spermatozoa motility will be activated, this activity is the source of waste accumulation, one of the important factor is the ROS with its lethal effect on sperm as revealed by Wang, et al., (2002) for this reason the additional of antioxidant factor if too important specially for sperm cryopreservation.

Table (5) explains the effect of Vit. C additional to the poor semen sample as an antioxidant will improve the individual motility after cryopreservation and thawing, and as the dilution rate be restricted (minimized), the individual motility will be

modulated. Azawi and Hussein (2013) agreed with this improvement that additional of Vit.C to semen extender will overcome the harm effect of the oxidant factors,that could be due to inhibition or stopped lipid peroxidation damage by the effect of this

antioxidant.

Table (5) effect of vitamin C treatment on individual motility of bull after freezing

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http://annalsofrscb.ro 17518 No.

of bull

Motility after Freezing control

T1 T2 T3

1 1:20 35 b A 40 a A 40 a A 45 a A 35 b A 2 1:15 40 a A 40 a A 35 a A 40 a A 35 a A 3 1:20 40 a A 35 a A 40 a A 45 a A 25 b A 4 1:10 40 b A 30 b A 35 a A 40 b A 30 b A 5 1:8 40 a A 35 a A 40 a A 40 a A 35 a A 6 1:7 40 a A 30 a A 35 a A 40 a A 30 a A 7 1:6 35 a A 35 a A 25 b A 40 a A 30 a A

Raina et al., (2002) agreed with this result that supplementation of vitamin C increased progressive sperm motility in bull semen; the greater number of motile spermatozoa present in the frozen samples with natural antioxidants would increase the fertilizing potential of post thaw spermatozoa. This result approved by Breininger et al., (2005) finding which concerned the effect of Vit.C as an antioxidant, however; the differences in pre-freezing and post-freezing progressive sperm motility can be attributed to many factors like initial quality of semen, type of extender used, concentration and nature of cryoprotective agents and freezing rates of semen.

On the basis of the present results, Aitken, et al., (2001) conclude that;

cryopreservation reduces the functional integrity of bull spermatozoa, however, addition of vitamin C to a semen extender can reduce the oxidative stress during freezing and thawing of semen thus protecting the semen during cryopreservation as well as improving the longevity and quality of sperms.

3- The effect of Vit. E additional to extender of poor semen sample with restricted dilution rate showed in table (6) that the limited (minimized) dilution rate with high concentration level of the antioxidant (Vit. E) improves the individual motility of

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spermatozoa; this improvement will positively affect the fertilization index especially for this poor quality semen, this finding agreed with Breininger, et al., (2005), that the additional of α-Tocopherol which is the source of Vit.E will improve the biochemical and dynamic parameters of semen and act an antioxidant agent.

Table (6) effect of vitamin E treatment on individual motility of bull after cooling No.

of bull

Individual motility

%

T1% T2% T3%

1 ^1:20 45 a A 35 b A 45 a A 50 a A 40 a A

2 ^1:15 35 b A 40 b A 45 a A 50 a A 35 b A

3 ^1:20 40 a A 40 a A 45 a A 45 a A 35 b A

4 ^1:10 40 a A 40 a A 45 a A 50 a A 40 a A

5 ^1:8 35 a A 35 a A 40 a A 45 a A 35 a A 6 ^1:7 35 a A 35 a A 40 a A 45 a A 30 a A 7 ^1:6 35 a A 30 a B 40 a A 40 a B 35 a A

8 ^1:6 40 a A 35 a A 40 a A 45 a A 35 a A

The result agreed with Beheshti et al., (2011) findings that explain the addition of antioxidants such as vitamin E to the semen diluent, may prevents or diminishes cryo- damage process to spermatozoa metabolism and antioxidant capacities.

The three concentration treatments of Vit. E with restricted dilution rate showed that; as the concentration level increased the more motility improvement found if we compared with the T1, T2 and T3 level in which the last level gives more accurate result, these findings agreed with Anghel et al, (2009) and Andrabi et al, (2008).

Beconi et al, (2000) demonstrated that; Vit. E prevents lipid peroxidation of bovine semen, this prevention improved sperm quality and fertility in bull sperm. Dal, et al, (1998) and Upreti et al, (2001) have been documented low levels of vitamin E would not allowed the production of physiological level of ROS which affect the capacitation,

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acrosome reaction and in vitro fertilization, and have beneficial effect on sperm motility in liquid bull semen.

4- The effect of additional of Vit. E on individual motility of poor bull semen after freezing demonstrated in table (7)

Table (7) effect of vitamin E treatment on individual motility of bull after freezing No.

of bull

Motility after freezing control

T1% T2% T3%

1 1:20 45 a A 25 b A 30 b A 35 a A 20 b A

2 1:15 35 a A 30 a A 30 a A 35 a A 20 b A

3 1:20 40 a A 30 a A 35 a A 40 a A 25 b A

4 1:10 40 a A 20 b A 30 a A 35 a A 20 b A

5 1:8 35 a A 25 a A 30 a A 35 a A 25 a A

6 1:7 35 a A 25 a A 35 a A 35 a A 20 b A

7 1:6 35 a A 25 a A 30 a A 30 a A 25 a A

8 1:6 40 a A 30 a A 30 a A 35 a A 25 b A

The limited (minimized) dilution rate with high concentration level of the antioxidant (Vit. E) improves the individual motility of spermatozoa after freezing; this improvement will positively affect the fertilization index especially for this poor quality semen. The result revealed some variations on individual motility upon freezing the semen sample and thawing, these variations are related to the level of antioxidant treatment, that the high concentrations level the more individual motility index. Bansal, (2001) found the same result that the high doses of vitamin E improve the bull sperm quality parameters such as motility and viability of sperms after freezing.

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These findings were similar to the results that obtained by Ijaz et al (2009), Kheradmand et al (2006), Bansal and Bilaspuri (2009) in which; addition of Vit.E may be useful in preventing the rapid loss of motility which occurred during semen freezing.

5- Effect of lipoic acid treatment on individual motility of bull after cooling

The effect lipoic acid treatment or additional to the poor semen extender is demonstrated in tab. (8), that the more dilution rate index is the more significantly active even via cooling then thawing, for that the third treatment shows the more active individual motility if compared with the other less two dilutions rate.

1. Table (8) effect of lipoic acid treatment on individual motility of bull after cooling No.

of bull

Individual motility

%

T1% T2% T3%

1 1:20 45 a A 45 a A 50 a A 55 a A 40 a A

2 1:15 45 a A 40 a A 45 a A 45 a A 40 a A

3 1:20 45 a A 35 a A 35 a B 40 a A 30 b A

4 1:10 40 a A 35 a A 35 a B 45 a A 40 a A

5 1:8 25 b B 35 a A 35 a B 40 a A 35 a A

6 1:7 40 a A 35a A 35 a A 40 a A 40 aA

7 1:6 45 a A 40 a A 45 a A 45 a A 40 a A

8 1:6 40 aA 40 aA 40 aA 45 aA 40 aA

Semen cooling and thawing will change the sperms activity from semi-stable at cooling temperature to more active as the thawing temperature organized, this is approved by Gurer et al., (2003) that the additional of ALA to the extender media of semen allows the antioxidant (ALA) to protect sperm cell components from hard effects by creating a shield surrounding the mid-piece (aqueous layer) and within the structure itself (lipid layer).

The ability of ALA to create a robust (strong) shield on the cell membrane, along with the liquid that surrounds the sperm indirectly; enhance the ability of the sperm to

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tolerate higher volumes of free radical attack or the harm effects of the ROS, this ability will, in turn, indirectly reduce formation of deep pores and cracks on the sperm surface, thus ensuring structural integrity, the rate of sperm movement is largely dependent on the availability of its energy supply, due to this, normal active sperm usually have very active functioning mitochondrion, which in turn generates high quantities of free radicals as a by-product, this is approved by Szelag, et al., (2012) that the quantum chemical activities of the structural properties of the ALA supply it with the ability to protect the sperms wall from the harmful effects of the free radicals.

Shay et al., (2009), found that; to ensure constant generation of ATP, external and internal structural integrity of the organelle must be maintained. Since the sperm membrane and the various compartments of the organelle are highly lipid content, addition of ALA would protect these structures from the ever-increasing free radical species, which are a by-product of the Krebs cycle.

Said, et al., (2005) reported that; semen has been contributed significantly to keeping up sperm DNA help, for example that gonads are believed to be one of the main sources of sperms, protective antioxidant, but for some reason (s) this protective generative ability which possessed by the gonads to the produced sperms is interrupted by those harm effects of free radicles, ALA due to its chemical activity can protect sperm cells from that harm effects.

The lower values of the progressive motile sperms in control group may be because of excessive generation of ROS by accumulation of dead, immature and abnormal spermatozoa during sperm processing (e.g., extending, cooling, freezing and thawing process), accompanied by low scavenging and antioxidant concentrations in seminal plasma and semen extender inducing oxidative stress (Tuncer et al 2010), for that; it is important to support this scavenging and antioxidant self-action produced by the gonads by exogenous antioxidant additional, and to obtained higher values which may be attributed to the beneficial effects of different antioxidants through its protective action on the sperm cell membrane against ROS and lipid peroxidation during cryopreservation of semen (Naijian et al 2013).

6- Effect of lipoic acid treatment on individual motility of bull after Freezing

The effect of a lipoic acid additional to poor semen extender on individual motility after freezing is shown in table (9)

Table (9) effect of lipoic acid treatment on individual motility of bull after Freezing No. Dilution Individual Motility after Freezing control

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http://annalsofrscb.ro 17523 of

bull

rate motility % T1% T2% T3%

1 1:20 45 a A 40 a A 45 a A 45 a A 30 b A

2 1:15 45 a A 35 a A 35 a B 40 a A 30 b A

3 ^1:20 45 a A 30 b B 30 b B 35 a B 25 b A

4 1:10 40 a A 30 b B 30 b B 40 a A 35 a A

5 ^1:8 25 a B 30 a B 30 a B 35 a B 30 a A

6 1:7 40 a A 30 b B 35b B 40 a A 35 a A

7 ^1:6 25 a B 30 a B 30 a B 40a B 35a A

8 1:6 25 aB 30 aB 30aB 40aB 35aA

Szelag et al., (2012) agreed with the result of the effect of ALA to improve poor semen quality, in which; addition of ALA is thought to have assessment in the metabolism of oxidative decarboxylation by its action as a co-enzyme. The increase in oxidative decarboxylation would increase cytochrome C concentration and thus directly increase the mitochondria’s membrane potential, improving regulation of mitochondria function and its biogenesis.

Ibrahim et al., (2008) agreed with the phenomenon that; ALA has a modulation effect on sperms motility after freezing by its action on mitochondria, in which ALA is reported to assist the mitochondria’s citric cycle, this in turn will increase the level of reduced glutathione, ATP, TCA cycle enzyme and electron transport chain complex activities which are responsible for this modulations.

ALA regulation of metabolism is approved by Vernet et al., (2004), by which; the increase viability of mitochondrial co-enzymes will improvement the protection of free radicals and eventually lead to a reduced incidence of mitochondria dysfunction, thus ensuring sufficient ATP for sperm movement.

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Bustamante, et al., (2001) reported about the effect of ALA on the cell metabolism, sperm viability is closely associated with regulated homeostasis and sperm membrane integrity, even though that ALA is known to possess potent anti-oxidative properties.

Buyukleblebici, et al., (2014) due to his finding not agreed with this result in which;

a high concentrations of ALA may be the cause of a dramatic reduction in potentially viable sperm, but this must be followed with more investigations. ALA is recognized as a universal antioxidant, with abilities to scavenge free radicals in aqueous and non-aqueous phases as demonstrated by Kagan, et al., (2000). The main efficacy of ALA is derived from its ability to minimize peroxynitrite-induced damage efficiently; hence, a significant reduction of this component would in turn enhance sperm viability as mentioned byRoss et al., (2010).

Devasagayam, (2007) concluded that an optimal concentration of ALA was able to improve sperm motility and viability and minimize DNA damage.

7- The effect of Vit C treatment upon diluted, cooled and freezed poor semen in relation to dead and secondary abnormalities

The effect of Vit. C treatment or additional to the poor semen extender upon dead % and secondary abnormalities%, showed no significant variations except that parameters obtained after semen cooling in which cooling process gives limited dead% sperms with moderated secondary abnormalities, shown as distal droplets, coiled tail, bent head and detached head. The reasons for different values could be due to formation of intracellular ice crystals and ROS production, or some depressing in osmatic pressure which altered sperm function (Alvarez. 2012).

Table (10) Effect of Vit. C treatment on diluted, cooled and freezed semen in relation with dead, alive and semen abnormalities.

Semen parameters Dilution Cooling Freezing Dead % 12.6 a 11.0 a 11.6 a Abnormalities % 11.6 a 10.4 a 12.2 a

This result is in agreement with that mentioned by Wittayarat et al., (2012) that the additional of vit. C to the semen extender yielded highest percentage of sperm motility and viability, this high percentage comes mainly from the high and vital sperm motility.

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Vitamin C are the major antioxidants naturally present in mammalian semen that regulates ROS, protect the sperm from lipid peroxidation and provides higher integrity to plasma membrane and mitochondria as better kinematics for sperm post-cryopreservation (Akhter et al., 2011; Silva et al., 2013).

Increase ROS production and decrease antioxidant level are known to occur during sperm cryopreservation and thawing which is mainly due to increase sperm viability, so;

diluted semen need to be supplemented with natural antioxidants in semen extender for improve post thaw semen quality (Maia et al., 2010).

Sikka (2004) explained that; the lower values of the progressive motile sperm may due to excessive generation of ROS by dead, immature and abnormal spermatozoa during sperm processing (e.g., extending, freezing, thawing process), accompanied by low scavenging and antioxidant concentration in seminal plasma and semen extender inducing oxidative stress, additional of Vit. C to semen extender will overcome this process by maintain sperm motility and oxidative stress.

While the higher values may be attributed to the beneficial effects of Vit. C through its protective action on sperm cell membrane against ROS and lipid peroxidation during cryopreservation of semen (Tuncer et al., 2010; Naijian et al., 2013).

The Vitamin C concentrations may have sub- obtimum ability to preserve bull sperm viability, with changeable effect due to concentration level (Medeiros et al., 2002).

During cryopreservation, an increased level of abnormal sperms was observed with in groups at different steps during cryopreservation, the reason for this differences may be due to weaker plasma membranes that made it more susceptible to lipid peroxidation, which increased with decreases temperature (Correa and Zavos 1994), and reduce ability of mammals sperm to encountering with peroxidation during the freezing and thawing processes making sperms more vulnerable for lipid peroxidation and increase sperm abnormalities, this findings are approved with Alvarez and Storey (2005) dead and sperms abnormalities elevated after freezing and thawing .

8- Effect of Vit.E treatment on diluted, cooled and freezed semen on the sperm dead and sperm secondary abnormalities

Effect of Vit. E treatment on diluted, cooled and freezed semen in relation with dead and semen secondary abnormalities shown in table (4-8) in which the dead percentage is moderated at cooling temperature and elevated at freezing temperature in which result after freezing gives slightly elevated dead and abnormalities percentage after treated with vitamin E , this in agreement with Bansal and Bilaspuri, (2010), in which this is mainly due to that; vitamin E was reported to play a role in association with

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antioxidant enzymes, for preserving the functional competence of spermatozoa subjected to an oxidative attack.

Table (11): Effect of Vitamin E treatment on diluted, cooled and freezed semen in relation with dead and semen secondary abnormalities.

Semen parameters

Dilution Cooling Freezing

Dead % 13.3 a 13.0 a 16.1 a

Abnormalities

%

11.6 a 12.3 a 13.2 a

The results of this study concerning Vit.E treatment is approved with Therond, et al., (2000) which declared that; Vit. E has also been observed to increase sperm viability and reduced lipid peroxidation when subjected to oxidative stress inducer.

Reactive oxygen are responsible to sperm dysfunction due the lipid peroxidation of membranes (Arabi et al. 2001). Agrawal et al (2005) demonstrated that antioxidants are the major defensive mechanism against oxidative stress. It has been now documented that vitamin E is the major antioxidant agent of sperm cells which is a potent scavenger of free radicals and is able to protect plasma membrane from damages mediated by ROS and LPO (Yousef et al. 2003; Gurel et al. 2005; Sinclair, 2000).

It has been established that presence of vitamin E is necessary for normal function of male reproductive system and traditionally, vitamin E is called as anti-sterility (Momeni et al. 2009).

Bansal et al (2010) and Ball et al (2001) reported that E is a main chain-breaking antioxidant in membranes because it may directly quench the free radicals such as superoxide anion (•O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH•). The present study aimed at finding out the efficacy of E, a biological antioxidant, in reversing the free radical-mediated oxidative damage on sperm motility, acrosome integrity and viability.

Antioxidant capability in sperm cells is limited because of deficiency cytoplasmic components having antioxidant effects to expunction of reactive oxygen’s. Thus, mammal's sperm haven't enough ability to encountering with peroxidation during the

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freezing and thawing processes (Alvarez et al, 2005; Bilodeau et al, 2000 and Lapointe et al, 2003).

Reactive oxygen is known to play a major role in sperm membrane damage and directly damage sperm DNA that cause reduces of the sperm’s motility, acrosomal membrane integrity and sperm metabolic alterations. In recent years, adding antioxidants to semen extenders for improvement of sperm quality were studied. Addition of antioxidants such as vitamin E and vitamin C to the semen freezing diluent, may prevents or diminishes cryo-damage to spermatozoa metabolism and antioxidant capacities (Anghel et al, 2009; Andrabi et al, 2008; Beheshti et al, 2011).

Beconi et al (1991) demonstrated that vitamin E prevents lipid peroxidation of frozen bovine semen. It has been demonstrated that this improved sperm quality and fertility in human sperm. In some studies have been documented low levels of vitamin E would allow for production of physiological level of ROS that are necessary for capacitation, acrosome reaction and in vitro fertilization (Dal et al, 1998). Addition of vitamin E had a beneficial effect on sperm motility in liquid ram semen (Upreti et al, 2001), fresh human semen (Donnelly et al, 1999) and to little effect in equine chilled semen (Ball et al, 2001). In the present study, the inclusion of different levels of vitamin E to extender before freezing causes significant improvement in sperm quality parameters such as motility and viability of sperms.

Table (12) Effect of Lipoic acid treatment on diluted, cooled and freezed semen in relation with dead, alive and semen abnormalities.

Semen parameters Dilution Cooling Freezing

Dead % 13.5 a 13.7 a 12.5 a

Abnormalities % 13.7 a 10.3 a 11.0 a

The results in table (4-9) recorded no significant difference (p<0.05) in dilution, cooling and freezing on semen parameter (dead and abnormalities) which treated with lipoic acid ,this results is agreement with (Ross et al., 2010). Results obtained from this study show that an increase in sperm motility was caused by ALA capabilities in energy production but that decreases could be caused by the acidity of the environment that produces immobilization. This was further supported by previous findings, which

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suggested that sperm motility is severely compromised when acidity is induced by HCl.

(Devasagayam 2007).

Antioxidant effect agent impact of ALA it was demonstrated that lipoic acid have powerful antioxidant capacities, ALA has the unique capacity to neutralize free radicals inside watery and lipid regions of the cells just as intracellular and extracellular environments, this capacity permits lipoic corrosive to be handily moved across cell layers to neutralize free radicals. (Gurer et al., 2003). All ALA provided by the diet is moved in the blood stream to tissue and consolidated in to cells, at that point it is moved in to the mitochondria. Studies in warm blooded animals (mammals) have demonstrated the ALA provided by the diet dose not gracefully enough for purposes, for example, consolidation in to enzyme complexes (Shamsi et al., 2011).

Szelag et al., (2012) reported that the mechanism of action of ALA is a kind of multi- purpose non enzymatic antioxidant as a prevention agent that protects mitochondria and surrounding cell components from oxidation by the free radicals delivered by mitochondria during oxidative metabolism, It is well known that the protect integrity and accuracy of DNA in the nucleus of spermatozoa is imperative to move genetic material totally starting with one generation then onto the next, in light of the fact that genetic material issue makes imperfect transmission of genetic information to embryo.

There is general agreement about the antioxidant prevention agent properties of alpha lipoic acid, its searches hydroxyl radicals and singlet oxygen, other than ALA cross biological membranes effectively and suppresses free radicals, in light of its little size and high lipophilcity ,exogenous supplementation with its substance has been accounted for to increment unbound lipoic acid levels, which can go about as a powerful cell antioxidant and lessen oxidative stress both in vitro and in vivo (Ibrahim et al., 2008) Because of this cell antioxidant characteristics, various test and clinical investigations have been conveyed. Scarcely any investigations were made on the adequacy of ALA supplementation during freeze process of semen, it is hypothesized that secured sperm against cryopreservation-incited oxidative pressure, an effective cancer prevention agent treatment ought to be executed, blend of extender with ALA might be proper way to deal with reduce the reactions of oxidative stress (Shay et al., 2009).

They reported that semen has been contribute significantly to keeping up sperm DNA health, for example, that gonads are believed to be one of the main sources of sperm- protective antioxidants (Said et al.,2005). Adding of ALA in to sperm microenvironment causes limit oxidative assault and improving sperm quality that can be valuable and encouraging of barren couples to accomplish successful conception.

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(Ibrahim et al., 2008). there for, contingent upon the above attributes scientists called alpha lipoic acid the (Universal Antioxidant) since it can neutralize a wide range of assortment of free radicals.

Cryopreservation has a harmfully effect on sperm motility (Watson 2000 and Üstüner et al.,2014). The procedures of cooling, freezing and thawing cause osmotic and chemical stresses on the sperm membrane that diminishes sperm viability and fertilizing ability, this cold shock and freezing harms lead to increase reactive oxygen species (ROS) and oxidative stress (Wang et al., 2002). ROS stimulates lost sperm work and with peroxidative harm are both dangerous to mitochondria and plasma membrane, when produced in excessive amounts. Spermatozoa are more susceptible to peroxidative damage due to high convergence of polyunsaturated unsaturated fats and low antioxidant capacity (Vernet et al., 2004). Consumption of antioxidant prevention agent resistances or rise in free radical production can influence antioxidant prevention agent equalization and cause oxidative stress prompting cell death (Aitken et al.,2001,Drevet 2005).

Enzymatic and non-enzymatic antioxidants prevention agents assume a significant job in scavenging free radicals (Ross et al., 2010).

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