The cryopreservation is a complex technique including multi steps. It is mostly accepted that a lot of sperms are defecting before and post cryopreservation and lead to increases of dead sperms percentage, abnormality, lowering of motility and sperm fertilization ability after use the artificial insemination [1,2]. Generally, sperm damage may be occurring by shock of cold, ice crystal formation or toxicity and oxidative stress. The spermatic response to all type of ROS (Reactive Oxygen Species) includes both of irreversible non enzymatic reactions and reversible redox signaling which depend on concentration and nature of the ROS involved [3]. R.O.S. are secondary aerobic cells   product of metabolic. They are stay at a little level by system of antioxidant. Antioxidant sources that include some of enzymes [4] like catalase (CAT), thioredoxins (TRXs), superoxide dismutase (SOD), glutathione peroxidases (GPXs), and other molecules with scavenging properties like some vitamins (C and E). However, when the antioxidant system is disorganized, and ROS production is Aggravating, then these molecules become more harmful by-products of sperm ‘s cells metabolism [5], Because of this, some plant extracts or plants that have antioxidant properties are usually added when the scavenging enzyme concentrations are lowering in the seminal plasma [6,7]. Grape  is a fruit, botanically a berry, G.S.E. contains many compounds working as  antioxidants that has been helped to protect body against oxidative stress [8,9,10,11]. The study objectives to investigate the effect of G.S.E. powder in different levels to Tris semen extender on some rams semen trails after cooling and freezing storage.

Animals and Semen Collection

Three Iraqi Awassi rams (ages 2.5-3 years) have been selected from six rams after trained to collect semen by artificial vagina. Rams were housed and fed as a semi extensive system at the Ruminants Researches Station located in Agargof, (the station which belong to the State Board of Agricultural Researches/Ministry of Agriculture, Iraq). All rams were in a good health. The study was carried out from the 17 of January to 23 of April, 2021.Two ejaculates/ram/week were collected by artificial vagina (41-42°C) during 4 weeks. Samples were transported to the laboratory at 37°C immediately in a thermos flask. A good quality Ejaculates were used, pooled to eliminate individual differences, they kept at 37°C in water bath. Then, evaluated performed on fresh pooled semen and equally divided into five groups within the experimental treatments.

Preparation of G.S.E. Powder

Grape Vitis vinifera L. (Al-halawani cultivar) was bought from a local's market from Baghdad city. Two kilograms were cleaned, dried kneaded by hand, the seeds were separated from other pomace components (stems and skin), dried at room temperature, and pulverized to very fine powder to a particles size (mesh#250µ) .50 grams of dried grape seeds powder were extracted in 500 mL of boiled distilled water for 24 hours in shacked water bath, then filtered, dried. The extract was collect and pulverized to very fine powder to a particles size (mesh#75µ), storage at cooling, and used as grape watery extract powder in experiment [12].

Fresh Semen Samples and Diluted Semen Analysis

Sperms were counted by Chamber of a Neubauer Haemocytometer [13]. The individual motility percentage and mass activity were estimated by procedure was created by Chemineau et al. [14]. Abnormal and dead sperm was performed by Eosin – Nigrosin staining method described by AL-Sarray [15] and at less more than 200 sperm from different microscopic field were examined under a microscope (400x). The percentage of acrosomal damage was estimated by Gemsa stain by AL-Sarray [15] method. 

Semen Dilution and Experimental Processing

Semen samples were diluted with Tris basted extender (hydroxyl methyl amino methane (3.63g), glucose (0.50 g), egg yolk (14%), citric acid (1.99) glycerol (6%), penicillin (100000 IU) and streptomycin (100 mg)) dissolved in 100 mL of double distilled water (Evans and Maxwell, 1987). Semen was split into five parts and diluted with tris extender contain different concentration of G.S.E. powder as follows G0 (as a control group), 0.05, 0.1, 0.15 and 0.2 % G.S.E powder for G1, G2, G3, G4 respectively. Samples were investigated after dilution (first period) and dividing into two groups. First group was preserved at cooling (5Cº) for 0,24,48 and 72 hours, anther group was packaged into straws (IMV 0.25 ml, France), then straws of semen were cooled to +5ºC within 4-4.5 hours prior exposing to exposed to the liquid nitrogen vapor. The semen straws were placed at 5-7 centimeter above surface level of liquid nitrogen for 8-10 min, then immerged in liquid nitrogen tank and preserved for one week. Frozen straws were thawed in 37Cº water bath for three min. All procedures were used to presses of semen were described by Evans and Maxwell [16].

Data Statistical Analysis

The experimental treatments were designed as a completely randomized design (C.R.D) to study the effect of treatment (concentration of grape seeds extract powder) at time of storage. Data were analyzed by S.A.S.  computer program and, Duncan Multiple Ranges test was used to compare the significant differences between experimental groups means.

Results were indicated that there were no differences between all groups and control in the individual sperm ‘s motility percentage after dilution immediately, while a significant difference (P ≤ 0.01) were appeared between G4 group and G2, G3 on the other side no significant differences between G1, G2 compared with control treatment at 0 cooling time (Tabel 1). Treatment G3 was recorded the highest individual motility during the 24-hour period at 5Cº, followed by G1 and G2 (69.12±1.07,70.00±0.51 % respectively).  Also, results were refer to that no significant variation between G4 compared with control treatment at the same period (Table 1). On the other hand, G3 group was recorded a high percentage of motility during 48, 72 hours at cooling storage (66.87±0.89 and 57.87±1.07 % for two cooling period respectively), where G4 was recorded a lowest percentage of individual sperms motility at 48 and 72 hours at cooling storage periods. G1 has been recorded that no significant differences compared with control treatment at 72 hours' period. Treatment G3 (29.12±0.95%) was significantly superior (P≤0.01) on the percentage of cell individual motility compared with the all experimental and control groups post cryopreservation for one week at liquid nitrogen (-196 Cº) (Table 1). The results from table (2) was referring that no significant differences appeared at post dilution and 0 hour intervals at 5Cº between all experimental and control groups in percentage of dead sperm percentage. However, control group (G0) and G4 were recorded a high percentage (12.20± 0.32, 12.11±0.27 % for tow experimental treatments respectively). But, G1, G2 and G3 were recorded a lower percentage (11.76± 0.08 ,11.95±0.07 and 11.99±0.30 % for three treatments respectively). Treatment G3 were recorded a significant difference (P ≤ 0.01) compared with the other treatments and control groups at 24,48,72 hours at cooling, also, the same treatment was recorded significantly decrease (P≤0.01) on dead sperm cell percentage after cryopreservation for one week (42.60 ± 0.53%), followed by G1 and G2 (48.17 ± 0.11 and 48.37±0.07 % respectively), while G4 and G0 groups were recorded a significant increase in dead sperm percentage after storage at liquid nitrogen for one week.

Table (3) indicated that no significant differences between the control group and other experimental groups within the first period (post dilution) in the percentage of sperm abnormality. However, there were a significant difference (P≤0.01) between G1, G2, G3 compared with control and G4 groups at 0 cooling period. On the other side, G3 has been recorded a significant decrease (p≤0.01) on abnormality percentage on 24, 48 and 72 hours at cooling storage compared with control and other experimental groups where recorded 13.31±0.26, 15.32±0.17 and 18.57±0.26 % for three periods respectively, on the other side G4 group was recorded a significant increase (p≤0.01) on sperm abnormality on 24,48, and 72 hours at cooling preservation compared with control and other experimental groups (18.30±0.20, 23.36±0.40 and 29.31±0.31 % respectively). G3 treatment was recorded a significant decrease (33.40±0.39%) compared with the other treatment groups and control group at storage in liquid nitrogen for one week, followed by the G1and G2 (36.07±0.12 and 35.43±0.20 % respectively). While the G4 was recorded a significant increase of sperm abnormality (40.29±0.31%) at the same interval.

Table 1: Effect of Grape seeds extract  powder on percentage of individual sperm‘s cells motility post storage at cooling and freezing storage (mean±SEM)

(Means in each column with different letter are significantly different(p≤0.01).

Table 2: Effect of Grape seeds extract  powder on percentage of dead sperm‘s cells post storage at cooling and freezing storage (mean±SEM)

(Means in each column with different letter are significantly different(p≤0.01).

Table 3: Effect of Grape seeds extract on percentage of abnormal sperm‘s cells post storage at cooling and freezing storage (mean±SEM)

(Means in each column with different letter are significantly different(p≤0.01).

Table 4:  Effect of Grape seeds extract powder on percentage of acrosomal sperm‘s cells defect post storage at cooling and freezing storage (mean±SEM)

(Means in each column with different letter are significantly different(p≤0.01).

Results in table (4) were detecting that there was a significant difference (p≤0.01) between the experimental and control groups in sperm acrosome defect percentage at the first duration (post dilution), while the high differences (p≤0.01) were showed between control and other experimental groups at 0,24,48 and 72 hours of 5Cº storage periods. However, there were no significant differences between G1(35.85±0.07%) and G4(35.62±0.24%) after one week in liquid nitrogen. In addition, the results were showed a significant decrease (P ≤ 0.01) which is recorded by G2 (33.72±0.05%) compared with the other groups and control post freezing storage for one week, followed by the G3 (34.27 ± 0.24 %).

In the past decades, there was progress in semen preservation, however, semen quality has been decreased during this process. The quality of storage semen decreased with an increase of the storage period [17], and the damage caused by oxidative stress is considered an important factor affecting semen quality specially sperm cell membrane have a high level of polyunsaturated fatty acids (PUFA) which are attacked by ROS [18,19].  ROS accumulation during storage periods leads to sperm cells damage [20,21]. Many studies explain that antioxidants have a significant effect on sperm quality by direct effects on the ROS formation such as Opuntia Ficus Indica leaves extract [22], lycopene [6], and selenium [23]. Table No. 1 was showed a high significant (P≤0.01) effect on the individual sperm motility by adding 0.1 and 0.15 % of G.S.E. powder to the tris diluted ram semen at a different time of cooling and post cryopreservation as a compared with control group.

Our results showed a highly significant effect for decreasing of the sperm dead, abnormalities and acrosome defects percentage by using different concentrations of grape seed extract powder (0.05, 1, and 0.15 %) at cooling storage and post cryopreservation compared with the control group. These positive results may be caused by grape seed extract which added to Tris extender. Sochorova et al. [24] found that the grape seeds contain a high concentration of reactive compounds such as polyphenols, this compound has pharmacological and biochemical effects against the free radicals. Polyphenols are considered essentially all flavonoids in grape seeds, ferulic acid, quercetin, and gallic acid are a flavonoid prominent in grape seeds that have a protective role against lipid peroxidation [25,26,27]. Guo et al. [28] has been also noticed that the grape seed extract has significant effect on the DNA integrity of mice brain cells. Al-Daraji  was suggested that the grape seeds extract components especially flavonoids gave a good suppression against the detriments of lipid peroxidation during roosters' semen storage up to 14 days.

Grape seed extract has significant effects on rat sperm motility and improve most of sperm quality [29,30]. Saleh [31] was concluded that the grape seed extract has important in fertility of male rats. Wen et al. [32] refer that the acrosome integrity, motility, plasma membrane integrity, total antioxidative capacity, superoxide dismutase (SOD) and catalase (CAT) activity in the grape seed extract groups were significantly higher than the control group, however, the malondialdehyde (MDA) decreased at 4 ºC after 120 hours of cooling storage. Grape extract at lambs reproductive development stage upregulated the expression of antioxidative, steroidogenesis, and polyunsaturated fatty acid metabolism-related genes, changed the fatty acid profiles, increased the antioxidant capacity in lamb's testis, and contributed to testis development and spermatogenesis [33]. Many studies were explained that the antioxidant activity of grape seed phenolics compound could be connected by different ways, like the properties to chelate metals [34,35], the redox activity as antioxidant , and the free radical-scavenging activity. According to the grape seed extracts may be contain flavonoids constituted by three shapes of rings of aromatic compounds, whose OH side group can regulate the confers them an electron dislocation responsible for the scavenging activity of free radical [36,37]. In conclusion, using 0.1% or 0.15 % concentration of grape seeds extract powder to the tris extender of ram semen have been played a great role to improve individual motility of sperm and reduce sperm dead, abnormality, and acrosomal defect under cooling and frozen storage.

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