The Potential Effect of Grape Seeds Extract against Lead toxicity That Induces Infertility to Male Rats

T he present study was designed to find the role, of grape seed extract against lead toxicity. Study was used 25 male rats that divided randomly to five groups (5 rats in each group) and the treatment continue four week in each group, control group administrated with normal saline, rats group treatment with (orally) lead (Pb), third group received lead (Pb) 100mg/kg and 50mg/kg seed extract, fourth group received lead (Pb) and 100mg/kg seed extract, fifth group received lead (Pb) and 200mg/kg seed extract. Rats group received lead show significant (P<0.05) decrease in counts, motility of sperm and increase the deformity of sperms. Other wise, rats group received lead show significant (P<0.05) decrease in glutathione (GSH), catalase (CAT) and increase levels of malonedialdehyied (MDA) compared with control group. The treated group with seed extract at different doses, sperm parameters and oxidative stress show gradual recovery. In 200mg/kg dose of seed extract, all parameters of study back to the normal ranges. It was concluded from this study that grape seed extract has important role against toxicity of lead in male rats.


Introduction
Grape vine (Vitis vinifera), is cultivated today in all regions with high temperature of the world [1].Seeds of grape were, containing many active components, including poly phenols, flavonoids, proanthicyanidins and procyanidines.Grape seeds extract (GSE) contain 70%-95% proanthocyanidins [2][3].Proanthocyanidins are widely located in vegetables, flowers, fruits, seeds, and grape seed.The medicinal properties of proanthocyanidins have been widely reviewed [3][4].In addition to antioxidant activity, proanthocyanidins possess anticarcinogenic, vasodilatory, immunestimulating, cardioprotective, antiinflammatory, antiviral, and estrogenic activities, as well as being inhibitors of enzymes, such as, cyclooxygenase, phospholipase, A2, and lipoxygenase [5][6].Lead is a ubiquitous environmental and industrial pollutant, that may have its toxic effects on the male [7].Lead has known to induce over production of reactive oxygen species(ROS) and enhance lipid peroxidation.Lead may inhibit spermatogenesis and reduce, a mature spermatids, spermatocytes, and mature spermatids [8][9].So, the aim of this study is to know the role, of grape seed extract against lead toxicity that induced infertility of male rats.

Materials & methods
Animal model 25 adult male rats, (wt: 225-250mg with age: 4-6 Mon) obtained from general company for manufacture pharmaceuticals and medical requirements, Samara/Iraq, and kept on standard pellet diet and water for two weeks before experiment.

Preparation of the extract
Grape seed extract was purchased by isolated the seed from grape and homogenized by machine grinder.A total of 250 g of dried grape seed extract were extracted from 5 kg of fresh grape fruit.The extract was evaporated to dryness in oven at 60 Ċ for 40 minute.The extract was suspended in distilled water and used in this study in concentrations as in recommendation [10].Experimental design 25 adult male rats were used and distributed in five groups (five rats in each group) as following and administrated treatments orally: A. Control group received normal saline and normal diet for seven days.B. Second group administrated lead (orally, 100mg/kg) for four weeks, and then killed.C. Third group administrated lead (orally, 100mg/kg) and 50mg/kg seed extract for four weeks, and then killed.D. Fourth group administrated lead (orally, 100mg/kg) and 100mg/kg seed extract for four weeks, and then killed.E. Fifth group administrated lead (orally, 100mg/kg) and 200mg/kg seed extract for four weeks, and then killed.Prepare of blood solution 2 ml of blood collected by cardiac puncture under anesthesia and put in test tubs.Then using centerfigation 5000 cycle/min for 15 min.Sera were taken and stored by deep freezing to estimate the biochemical measurement.

Semen Collection
Under anesthesia, testes and epididymides were removed.epididymides of each rat was divided in two parts.The caudal part was separated from the testes.Collected semen diluted with normal saline and put it in the tubes.Semen plasma was obtained by using centrifuge to study some parameters [11].

Sperm analysis
The content of the caudal part of epididymis was put in tube contain sodium citrate (1.9%) at (37° C). then, one drop put on slide and mixed with one drop of eosin -nigrosin stain to determining the percentage of live/dead and to abnormal/normal [12].Also, sperm forms, sperm count, motility of sperm was done.

Statistical analysis
Data of study were analyzed by using a statistical program known as Minitab.data Means were compared using Duncan's Multiple Range test.Probability levels of more than 0.05 were regarded as statistically non-significant, whereas values less than 0.05 were considered as significant [13].

Sperm counts and normality
Lead treated group showed significant (P<0.05)decrease in number of sperm counts (3.4±0.3)compared with control (5.6 ± 0.2).The sperm counts still show significant decreased in group treated with lead and seed extract (50mg/kg) and lead and seed extract (100mg/kg) (3.9±0.5and4.7±0.3respectively) compared with control group, but better than the lead group.The sperm counts in group treated with lead and seed extract (200mg/kg) group showed nonsignificant changes in the counts of sperm.The percent of normal and deformity of sperm (60 ±7 and 40 ± 5 respectively) in lead group showed significant decrease compared with control (93±5 and 7±2 respectively).The percent of normal and deformity of sperm till show significant decrease in lead and seed extract (50mg/kg) group and lead and seed extract (100mg/kg) (69±2 and 4.7±0.3respectively) compared with control group, but better than the lead group.The percent of live and deformity of sperm in 200mg extract group (88 ± 3 and 10 ± 3 respectively) showed non-significant changes as show in table (1).

Sperm deformity
The microscope examination show normal form of sperm in control group (fig: 1).Lead treated group showed a deformity in the tail of sperm, that appear as ring in some and it's disappear in other (fig: 2-3).Lead and seed extract (50mg/kg) group and lead and seed extract (100mg/kg) groups showed a deformity in the tail of sperm that also appear as ring in some and it's disappear in other(fig: 4-5).Lead and seed extract (200mg/kg) group show decreased in deformity of sperm tail and appear near to the normal state (fig: 6).

Discussion
The results of previous studies reported that the exposure of human to lead (Pb) can reduce human semen quality.Where, they suggest lead induce decrease in sperm counts and motility and alter morphology [14][15].Also, studies reported that exposure to lead (Pb) has side effects on sperm parameter (decreased counts, motile and increase sperm abnormality) [16][17], that is in agreement with results of present study.Also, in study carried by Ramah et al. (2015) referred that the lead acetate caused oxidative stress.They found that the lead acetate cause decrease in level of GSH and GST and significant increase in level of MDA in rats group administrated with lead acetate [9].Other wise, Salawu et al. (2009) referred that the lead (Pb) has effect on antioxidant factors in mice.They found that the lead caused significant (P <0.05) decrease in both plasma and testicular catalase of animals treated with Pb only compare with control group [18], that is in agreement with results of present study.
The results of current study showed a protective effect of grape seeds extract against the toxicity of lead (Pb).Where, In study of Khattab et al. (2010) referred that the grape seeds extract has a potential effects on sperm activity.Where in their study rats administrated with aluminum chloride (AlCl 3 ), they found AlCl 3 induced highly significant decrease in sperm count, motility (%) and viability (%), with increase in dead and abnormal sperm count as compared to both control groups but treatment with grape seed extract showed potential role of seed extract against the toxicity of AlCl 3 .Also, they found that AlCl 3 decrease the total antioxidant capacity in testicular tissue and serum of rats, but after treatment with grape seed extract, levels of total antioxidant capacity back to normal ranges [19].Also, the results of present study agreed with Aysun et al. (2008) who reported that oral intake of grape seed extract reduced the oxidative stress [20].Other wise, grape seed extract treatment increased the formation of antioxidant products which may be regarded to the phenolic constituents of GSE and its antioxidant activity [21].Hafsa et al. (2016) referred to the protective effect of grape seed extract against the toxicity lindane in male rabbits.They found that lindane decrease the sperm counts, sperm concentration, total sperm output, sperm motility and increase in percentage of abnormal sperm, dead sperm.After treatment with grape seed extract, all sperm parameters back to normal ranges, suggest the positive effects of dietary grape seed extract on both sperm count and sperm motility and the reduced percentage of dead sperm could be linked to the antioxidative properties of grape seed extract [22].