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Germ of cereal grains and seeds in association with semen parameters.


Cereal grains or young, green, and leafy plants in particular, unlike animal products, are the richest source of vitamin E activity [1]. Human sub-fertility or infertility is related to reactive oxygen species, affected acrosome reaction [2], decreased capacity for sperm-oocyste fusion [3,4] or dissection of spermatozoa [5]. Diets supplemented by selenium and vitamin E, improves sperm quality or increases concentration of spermatozoa in semen; an effect possibly linked to antioxidant properties of this vitamin [6,7]. In chicken, vitamin E tented to improve semen quality traits by increasing concentration of spermatozoa and cell viability [8]. Vitamin E is lipid-soluble mainly within cell membranes [9]. Its [alpha]-tocopherol maintains structural and functional characteristics of membranes via antioxidant capacity (inactivating reactive oxygen species and quenching free radicals) through direct interaction with membrane components [10-12]. The total amount of alpha-tocopherol in seminal plasma, or the percentage of motile spermatozoa is significantly related to sperm alpha-tocopherol content [13].

Vitamin E therapy has successfully been recommended for male sub fertility or infertility according to density of the ejaculate, motility or morphology of the spermatozoa or in-vitro function (zone binding assay),[12]. However, the prooxidative effect of vitamin E in terms of low-density lipoproteins (LDL) should be considered. In different in-vitro studies, vitamin E reacted pro-oxidative after co- antioxidants consumption in LDL such as ubiquinol -10 [14,15]. Moreover, [alpha]-tocopherol stimulated the rate of lipid per oxidation initiated by ([Cu.sup.2+]) [16]. It has been demonstrated that human peripheral blood mononuclear cells and monocyte-derived macrophages, which are proposed to be one source of reactive oxygen species [17] increased LDL oxidation despite the presence of [alpha]-tocopherol [16].

The present study as an animal model aimed to investigate the association of oral administration of cereal grain and seeds and reproductive performance of local (Kurdish) cocks with high reproduction rates and low maintenance cost.

Material And Methods

Animals And Dietary Supplements:

In this experiment cereal grains including wheat, barley, oat, red bean, white bean, alfalfa, clover, corn, pea, lentil, chick ling, and vetch were laid on cotton cloth and moisturized regularly till the germ grows up to 1-1.5 cm (the first joint stage) before leaf raising. Germs were cuts by fine seasore and dried up at the room temperature and then grinded till become powder and then were added into the diets of local (Kurdish) 3days old chicks cocks.

Chicks were selected from local poultry farms belong to the School of Veterinary Science of Ilam University. For this purpose 100 local chicks' cocks were equally divided into the five groups. Except group 1 (control), the diets of other groups were enriched by 100, 200, 300, 400 grams of germs of cereal grain respectively. Cocks were fed accordingly up to5 month's age. All birds originated from the same genetic stock of local (Kurdish) cocks. After a 5 months period, the association of cereal grain germ and biochemical parameters or pool semen samples was investigated. Weight of testes was also measured.

Semen Collection And Examination Of Ejaculates:

Sample was taken after adoptive period of growing cocks within few weeks. Semen samples from each group were collected by an abdominal massage method twice a week throughout the experimental period. For biochemical examinations, the semen samples were diluted with an equal volume of Ringer solution and frozen at -79[degrees]C.

Anthological examinations of the pooled semen sample were carried out within the first quarter of the trial and afterwards once a week. After collection, the following semen parameters were measured for each dietary group: ejaculate volume by using a graduated tube; colour, consistency and contaminations (blood or excrement) by visual examination; pH value by pH paper. Sperm motility was determined on a warmed sample (37[degrees]C) at a magnification of 400 x with an optical microscope. Total motility was determined as the percentage of spermatozoa that had any sign of motility [18] in two areas with a count of 100 cells per area. On the same day density was registered by means of a counter ('Thoma, chamber, Thoma-Zahlkammer, Glaswarenfabrik Karl Hecht AG, Sondheim, Germany). Percentage of morphologically deformed spermatozoa was determined by evaluating 400 cells of the semen samples after mixing an aliquot of 5[micro]l of the ejaculate with 300ul of formolcitrate. Total amount of spermatozoa was registered mathematically as a product of volume and density of ejaculate.

Biochemical examinations were carried out once a month. For determination of [alpha]-tocopherol in the whole semen, the ejaculate was saponified and the solvent extracted by a modified method [11]. Vitamin measurements were columns (C18) and fluorescent detection. Vitamin E was measured using a U.V. spectrophotometer (Gecil model, Germany) as previously described [1] with a brief modifications. The limit of sensitivity for measuring [alpha]-tocopherol was 0.1 [micro]g/ml. Thiobarbituric acid-reactive substances (TBARS) were measured according to previous reports [19]. Malodialdehyde, a second product of lipid per oxidation reacts with thiobarbituric acid forming a complex, whose concentration could be detected fluorophotometrically. Phospholipids were extracted from the ejaculate after homogenization with chloroform: methanol: distilled water (1:2: 0.8, v/v/v) [11]. A two-phase system (chloroform: aqueous phase, 1: 1.9, v/v) results from adding a mixture of chloroform: water (1: 1, v/v).

The phospholipids were extracted from the chloroform phase by solid phase extraction [20]. After esterification [21], the resultant fatty acid esters were measured by gas chromatography. At the end of the trial, six cocks were selected according to the mean body weight of the group, were killed and both testes were weighted.

Statistical Analysis:

Temporal relations were excluded by regression analysis as far as the test data of the pooled semen samples of each group were concerned. The Statistical Analyses System (SAS, Cory, NC, USA) was used for all analyses. All data were rested for Gaussian distribution and submitted to one-way ANOVA.

The Local Ethical Committee in Veterinary Researches as well as the Research Department in Ilam University approved the proposal of the present study.

Results and Discussions

Association With Semen Parameters Of Pooled Semen Samples:

The pooled semen samples in all dietary groups had the colour and consistency of cream. Contamination by excrement, varied from negative to slightly positive. Differences between dietary groups with reference to volume, pH or motility were not significantly associated with the germ of cereal grain and seeds. Volume, pH and motility of each sample were at physiological levels. The lowest mean volume was found in the control groups. Mean pH values for all groups ranged from 7.0 to 7.4. The highest percentage of forward motion of spermatozoa was observed in groups 2 (70.2 %) and differed significantly with levels in the control group (68.1 %) and group 5 (69.2 %), (p<0.05). Percentages of forward motion spermatozoa of the dietary groups were within these values. Density decreased from 3.12 million spermatozoa/[micro]l in the control group to 2.0 million spermatozoa/[micro]l of group 5. Densities of groups 1 and 2 differed significantly from the density of all other dietary groups (p<0.01). Groups 2 had the lowest value of morphologically deformed spermatozoa (deformed head, broken or bent neck or tail or rolled up sperm). Percentages of deformed spermatozoa of groups 3,4 and 5 were higher than of the control group and differed significantly from groups 1 and 2 (Table 2).

Testes And Body Weight Of Cocks:

The weights of the testes of group 5 were significantly lower than other groups. There were no significant differences among weights of the other treatment groups. The weights of testes in groups 2, 3 and 4 were 20.1g, 19.2g, and 20.6g respectively (Table 3).

Biochemical Parameters:

The concentration of a-tocopherol in whole semen was responsive to the germ of cereal of grains and seed as dietary vitamin E. Clover and wheat grains were the richest source of vitamin E accordingly (370 and 329 mg/1000g) at the 7th day of measurements with increasing trends from the 3rd to the 7th days of measurements (p=0.001 for both trends), (Table 4). The content of [alpha]-tocopherol in the semen samples in groups 1, 2 and 3 differed significantly from that of groups 4 and 5. Enhancing the dietary supply by germ cereal grains (100-200g of feeds) increased the concentration of [alpha]-tocopherol in whole semen the same reaction was observed by increasing dietary supply from 200-300g germ cereal grains in diets. In addition, increasing the dietary supply of 300-400g germ cereal grains and seed was indicated by increased vitamin E concentration in the semen samples (Table 5).

Dietary manipulation did not reduce TBARS production in whole semen to the same extent as the increase of a-tocopherol concentration in the semen samples. Although the TBARS concentration of the semen sample of group 3 was reduced compared to control group, increasing dietary supplementation with germ cereal grains from 200-300g did not reduced TBARS production. Only the concentration of TBARS in the semen samples of the highest supplemented group showed a reduction. The TBARS production of the moderately supplement groups differed from that of the high supplement dietary groups (p<0.05).

The phospholipids content of whole semen decreased with increasing amounts of germ of cereal grains and seeds supplement. Phospholipids concentration in the semen samples of group 5 was significantly lower compared to all other dietary groups except group 3. Increasing dietary supplementation by germ cereal grains and seed from 0-400 resulted in a significant decrease of phospholipids content in the semen samples.


Economically, the fertility of the cocks in a breeder flock is of greater importance in the male than that of the female due to responsibility for fertilizing the eggs from eight to ten hens. The sample recruited in the present study is representative as almost all cocks in the west and north of Iran are from Kurdish ethnicity and the primary calculated sample size increased by 10 % to cover the design effect based on population attributable risk (PAR), 22. The negative association of very high vitamin E dosage with the total amount of spermatozoa is more important than density, because density varies with the changing volume of ejaculate. The decreased total amount of spermatozoa might be related to the decreased weight of the testes [12]. The level of spermatozoa is dependent upon testes weight [23].

Thus, endocrine disorder was excluded as a cause of disturbed spermatogenesis. However, one study has reported no influence of supplemented vitamin E on testes of rats feeding comparatively lower vitamin E doses [24]. In chickens a vitamin D3 deficiency caused by interaction of vitamin E and D3 [25], could lead to disturbed spermatogenesis by decreasing of glutamyl transpeptidase activity of the testes and the reduction in the leydig cell count with degenerative changes in the germinal epithelium [26].

A pro-oxidative effect should also be considered, as [alpha]- tocopherol concentration in pooled semen samples does increase with graded vitamin E supplementation [11]. A pro-oxidative effect would probably be followed by an increment of radicals causing cell destruction. Therefore, alterations in the germinal epithelium are expected that might address the reason for the decreased testes weight. The concentration of TBARS in pooled semen samples through the present study decreased with increasing amounts of supplemented vitamin E, confirming a previous report [11].

Increased morphologically abnormal spermatozoa in high supplemented dietary groups in this study demonstrated a dietary influence. Primary morphological alterations of spermatozoa indicated a disorder in spermatogenesis, secondary occurs during the passage of epididymis, and tertiary alterations occur during or after ejaculation (e.g. incorrect treatment of the semen samples). Acquisition and treatment of ejaculate were standardized and the same for all dietary groups. Consequently, it could be eliminated as tertiary cause. Apart from vitamin E, an increased pH can cause morphological alterations or death of spermatozoa [27]. As significant difference of pH did not occur, pH value could be excluded as a tertiary cause.

As chicken spermatozoa are characterized by a very high concentration of long chain polyunsaturated fatty acids [28], they are highly susceptible to lipid per oxidation. Membrane stability of spermatozoa has reported to be increased by feeding comparatively low doses of vitamin E from natural source of germ of cereal grains with cocks [29]. The decreasing of TBARS values in the present study indicates membrane stability through deference from oxidative damage. The decreasing phospholipids content of the semen samples indicates displacement from of the membranes by vitamin E, which is contradictory to membrane stability. Decreasing phospholipids content either is the main cause for disturbed spermatogenesis. Therefore, decreased total amount of spermatozoa and increased morphologically spermatozoa is expected, because phospholipids are mainly located in spermatozoa instead of seminal plasma [30,31].

In conclusion, highly supplementation of vitamin E, increased [alpha]- tocopherol concentration of ejaculate. Reproductive performance of cocks was negatively associated with high level of vitamin E dosage, indicating a rise in oxidative defense of spermatozoa. Excess of vitamin E in diet from natural source of germ of cereal grains and seeds (wheat, barley, oat, red bean, white bean, alfalfa, clover, corn, pea, lentil, chickling, vetch) of cocks led to an increased level of a-tocopherol concentration in the semen samples, which in turn, exerted a negative association with semen parameters such as density, total spermatozoa and morphology. No positive association on semen volume or phorsperm motility was observed following a dietary vitamin E administration in the cock. The reproductive performance of the treated cocks was negatively influenced. These contradictory results showed that neither the exact mechanism nor site of action and the indication for a successful anticipative treatment of patient are clear.


The present study was financially supported by a research grant from the Ilam University awarded to Dr Ali M. Bahrami. The cooperation of laboratory and library personnel in that university is greatly appreciated.


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(1) Ali M. Bahrami, (2) Alizaman Doosti, (3) Salman Ahmady-Asbchin

(1)--School of Veterinary Medicine, Ilam University, Ilam, Iran.

(2)--Dpartment of Biology, Payam-e Noor University of Ilam, Iran.

(3)--Department of basic science, Ilam University, Ilam, Iran.

Corresponding Author

Ali M. Bahrami, School of Veterinary Medicine, Ilam University, Ilam, Iran.

Table 1. Composition of experimental diets

Ingredients (%)                Groups

                               Control   2       3       4       5

Corn (8.5% protein)            61/5      61/5    61/5    61/5    61/5
Soya bean meal (48% protein)   30/40     30/40   30/40   30/40   30/40
Calcium carbonate              1.35      1.35    1.35    1.35    1.35
  (38% calcium)
De-calcium phosphate           1.5       1.5     1.5     1.5     1.5
  (20% phosphate)
Animal fat                     3.5       3.5     3.5     3.5     3.5
Salt                           0.4       0.4     0.4     0.4     0.4
Premix                         1.0       1.0     1.0     1.0     1.0
De-methionine                  0.1       0.1     0.1     0.1     0.1
Lysine                         0.25      0.25    0.25    0.25    0.25
Germ of cereal grain (g)       0         100     200     300     400

Each kg premix contains: Vitamin A 4000000 IU, Vitamin D3
100000IU, Vitamin E 2000 mg, Vitamin K3 750 mg, Vitamin B1 600
mg, Vitamin B2 2000 mg, Vitamin B6 600 mg, Vitamin B 12 10000
mcg, Vitamin C 2000 mg, L--Lysine 10000 mg, DL--Methionine 25000
mg, Copper Carbonate 2500 mg, Cobalt Carbonate 550 mg, Ferrous
Carbonate 4000 mg, Manhanese Carbonate 50000 mg, Zinc Carbonate
5000 mg, potassium Iodide 150 mg, Choline Chloridi 110000mg,
Nicotinic Acid 9000 mg, Folic Acid 225 mg, Calcium pantothenate
3500 mg, B.H.T 125mg .
Symans Pharmaceuticals (Pvt.) Ltd.

Table 2 Semen parameters of pooled semen samples (mean [+ or -] SD)

Treatments    Volume (ml)                (million/[micro]l)

0 (control)   4.1 (b) [+ or -] 0.8 (b)   3.1 (a) [+ or -] 0.9
100           5.2 (a) [+ or -] 0.9 (a)   3.0 [+ or -] 0.8
200           4.3 (b) [+ or -] 0.8 (b)   2.9 [+ or -] 0.4
300           5.2 [+ or -] 0.9           2.2 [+ or -] 0.5
400           4.3 (b) [+ or -] 0.6 (b)   2.0 [+ or -] 0.0

Treatments    TS (billion/ml)         pH

0 (control)   13.3 [+ or -] 3.5 (b)   7.4 [+ or -] 0.4
100           16.7 [+ or -] 3.6 (a)   7.1 [+ or -] 0.1
200           11.8 [+ or -] 2.9 (c)   7.0 [+ or -] 0.2
300           14.3 [+ or -] 4.0 (b)   7.0 [+ or -] 0.3
400           10.9 [+ or -] 2.5 (c)   7.3 [+ or -] 0.2

Treatments    FM (%)              MDS (%)

0 (control)   68.1 [+ or -] 6.2   9.0 [+ or -] 2.1
100           70.2 [+ or -] 2.2   7.0 [+ or -] 1.4
200           62.4 [+ or -] 2.1   12.4 [+ or -] 3.8
300           61.1 [+ or -] 1.3   11.0 [+ or -] 1.3
400           69.2 [+ or -] 3.3   10.5 [+ or -] 3.2

(a-c) Values with no common superscript
within columns differ significantly (p<0.05)
TS: Total Spermatoza
FM: Forward Motion
MDS: Morphologically Deformed Spermatozoa
SD: Standard deviation

Table 3: Body weight (mean [+ or -] SD) and weight of testes (n=6)

Germ of                                                  Weight
cereal                                                   ratio of
grains/g    Body weight (g)     Weight of testes (g)     testes (g)

0 control   2520 [+ or -] 120   24.02 [+ or -] 7.2 (a)   0.9063
100         2412 [+ or -] 180   20.1 [+ or -] 4.2 (a)    0.8333
200         2121 [+ or -] 141   19.2 [+ or -] 1.42 (a)   0.9052
300         2118 [+ or -] 182   20.6 [+ or -] 4.5 (a)    0.9726
400         2324 [+ or -] 241   17.20 [+ or -] 2.8 (b)   0.7401

(a-cb) Values with no common superscript within
columns differ significantly (p<0.05)
SD: Standard deviation

Table 4. Vitamin E present in germ of cereal grains and seeds (mg/1000g)

Cereals                       Days of measurements            p value *

                      3rd     4th     5th     6th     7th

Lentil       Seeds    53.2    54.8    55.6    57.2    56.0    0.04
             Grains   74.4    102.0   116.4   87.6    86.8    0.51
Red Bean     Seeds    56.0    58.0    65.6    63.6    65.2    0.04
             Grains   90.0    91.15   90.14   98.0    101.2
White Bean   Seeds    53.2    54.8    57.2    54.0    64.4    0.05
             Grains   82.3    85.9    86.8    85.9    82.3    0.73
Barley       Seeds    44.8    44.9    78.8    113.0   117.4   0.01
             Grains   112.6   125.7   122.8   142.6   156.6   0.001
Alfalfa      Seeds    92.0    113.0   125.0   134.0   140.0   0.001
             Grains   123.0   146.0   165.0   173.0   197.0   0.001
Pea          Seeds    44.8    52.4    53.2    64.0    72.0    0.03
             Grains   74.0    92.8    94.8    124.4   131.2   0.01
Wheat        Seeds    72.0    82.5    104.0   128.0   143.0   0.04
             Grains   207.0   243.0   268.0   297.0   329.0   0.001
Clover       Seeds    113.0   134.0   147.0   170.0   182.0   0.01
             Grains   257.6   283.0   324.0   340.0   370.0   0.001

* Trends, linearity (chi-squared)

Table 5. Concentrations of [alpha]- tocopherol,
TBARS and phospholipids in semen samples

Germ of cereal        Vitamin E              TBARS
grains/gms          ([micro]l/ml)          (nmol/ml)

0 (control)      0.41 [+ or -] 0.30    3.11 [+ or -] 1.01
100              0.91 [+ or -] 0.21    1.14 [+ or -] 0.98
200              2.45 [+ or -] 1.98    1.93 [+ or -] 0.36
300              8.91 [+ or -] 4.66    1.98 [+ or -] 0.32
400              15.12 [+ or -] 9.12   1.62 [+ or -] 0.31

Germ of cereal       Phospholipids
grains/gms              (ng/ml)

0 (control)      2941.1 [+ or -] 461.0
100              2421.3 [+ or -] 421.1
200              2313.6 [+ or -] 321.0
300              2402.0 [+ or -] 411.8
400              1921.3 [+ or -] 210.1

Values with no common superscript within columns differ
significantly (p<0.05) SD: Standard deviation Mean [+ or -] SD
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Title Annotation:Original Article
Author:Bahrami, Ali M.; Doosti, Alizaman; Ahmady-Asbchin, Salman
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:Aug 1, 2011
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