Effect of Addition of Zn, Cysteine, PGF2α and their Combination on Holstein Bulls Cooled Semen in vitro

نياتشلوهلا ناريثل ًايربتخم دربملا يونملا لئاسلا ىلإ اهطيلخو نيدنلاكتسوربلاو ،نيتسسلا ،كنزلا ةفاضإ ريثأت ةصلاخلا يونملا لئاسلا ىلإ امهطيلخو ةدح ىلع لٌك نيدنلاكتسوربلاو نيتسسلاو ،كنزلا نم لك ةفاضإ ريثأت ىدم ةفرعمل ةساردلا هذه تيرجأ ففخم( ففخملا Tris .ديربتلا نم ةفلتخم ددم دعب نياتشلوهلا ناريثل يونملا لئاسلا تافص يف ) ،يعانطصلاا حيقلتلا مسق يف ةساردلا تذفن ةورثلا ةرئاد يناثلا نوناك نم ةرتفلل دادغب برغ بيرغ وبأ ةقطنم يف ةعارزلا ةرازو /ةيناويحلا 2019 بآ رهش ىتحو 2019 . مدختسا نم اهرامعأ تحوارت نياتشلوه ناريث ةعبس ةساردلا هذه يف 2.5 3 ةفذق عقاوبو يعانطصلاا لبهملا ةطساوب يونملا لئاسلا عمج .تاونس لاث ةدمل ايعوبسأ روثلل ةدحاو ًاعيمج ناريثلل ةفذقلا جمد مت ،ديج ريغلا عونلا نمو جزاطلا يونملا لئاسلا مييقت تاصوحف تيرجأ .رهشأ ةث ( Pooled semen ( ىلولأا ةعومجملا ىلإ فيضأ ،ةفلتخم عيماجم ةسمخ ىلع هميسقتو ) Control ةعومجم ةباثمب دع دقف سرت ففخم ) ةفاضإ مت يذلا تقولا يف ،ةرطيس (0.576 mmol/ ml) ،ةبرجتلا نم ةيناثلا ةعومجملا تلكش يذلاو سرتلا ففخم ىلإ تيفلس كنزلا نم / نم نوكتت ةثلاثلا ةعومجملا تناك امنيب (5 mmol/ ml) سرتلا ففخم ىلإ فاضملا نيتسسلا نم 20 فيضأ دقف ةعبارلا ةعومجملا امأ ،لم 37.5 pg/ m سرتلا ففخم ىلإ نيدنلاكتسوربلا نومره نم 20 فيضأ ،لم نم ةفيلوت سرتلا ففخم ىلإ (0.576 mmol/ ml) نم و تيفلس كنزلا (5 mmol/ ml) و نيتسسلا نم 37.5 pg/ m .)طيلخلا ةعومجم( ةسماخلا ةعومجملا تربتعاو نيدنلاكتسوربلا نم مت ةرارح ةجرد دنع ديربتلا ددم للاخ يونملا لئاسلا تافص ىلع تافاضلإا ريثأت ةسارد 5 ( مْ 24 ، 48 و 72 .ةعاس ) ةفاضإ نإ جئاتنلا ترهظأ ( ةعبرلأا ديربتلا ددم نيب ةيونعم قورف ترهظأ كنزلا 5 ،مْ 24 ، 48 ، 72 ( ةعاس ) P<0.05 .فطنلل ةيدرفلا ةكرحلل ةيوئملا ةبسنلل ) تققح ( ًايونعم ًاقوفت نيتسسلاو كنزلا ةفاضإ P<0.05 يونعم ضافخنا طيلخلا ةفاضإ تققح امك .ةيحلا فطنلل ةيوئملا ةبسنلا يف ) ( P<0.05 يف ) ديربتلا ةرتف دنع ةهوشملا فطنلا نم ةبسن لقا ىلع تلصحو )ةهوشملا( ةعيبطلا ريغ فطنلا ةبسن 5 نم لك ةفاضإ تققحو .مْ ( ًايونعم ًاقوفت كنزلاو نيدنلاكتسوربلا P<0.05 ( يمزلابلا ءاشغلاا ةملاسل ةيوئملا ةبسنلا يف ) HOST ةرتف دنع فطنلا يف ) 48 نم ةعاس .ديربتلا ( ً ايونعم ً اقوفت كنزلا ةفاضإ تققح امك P<0.05 دنع ) 5 ( يف ديربتلا ددم يقاب ىلع مْ HOST ًايونعم كنزلا ةلماعم تقوفت .) ( P<0.05 دنع ) 5 ( فطنلا موسوركا ةملاسل ةيوئملا ةبسنلا يف تلاماعملا يقاب ىلع مْ AI ةدسكلأا تاداضم ةفاضإ نأ ةساردلا نم جتنتسن .) إو ،نيتسسلاو ،كنزلاك نيدنلاكتسوربلا نومره ةفاض PGF2α .ناريثلا يف يونملا لئاسلا ةيعون نم نسح دق


Introduction
Artificial insemination plays an important role in genetic improvement in dairy bulls via which a single ejaculate from bull could inseminate many cows. Cooling of semen storage facilitates semen transport for a distances and enables extension of superior genetic merit. Cooling have an exert effect on physiological as well as certain chemical stress on sperm cell (Chatterje et al., 2001). These stress may be induced by oxidative stress by free radical (Salvader et al., 2006). Sperm cells have a high content of unsaturated fatty acid but not an antioxidant in it's cell membrane, So the cell membranes is highly sensitive to the lipid peroxidation via free radical and H2O2 (Sinha et al., 1996). Cellular damage resulted from oxidative stress due to reactive oxygen species (ROS) produced from cell components of semen during cooling, It may leads to decrease in motility and fertility during storage, while it may cause low temperature on structure of membrane of sperm destabilization (Mustafa and Necmettin, 2007). The present study aimed to study the effect of addition of antioxidant such as zinc sulphate, cysteine and PGF2α on bull diluted and cooled semen.
Materials and Methods The current study was carried out to investigate the effect of addition of zinc sulphate, cysteine, Prostaglandin F2α and their combination to diluted semen on semen characteristics of Holstein bulls after different periods of cooling. The study was conducted on seven Holstein bulls, Aged between 2.5-3 years, presented at Artificial insemination centers/ Directorate of Animal Resources/ Ministry of Agriculture at Abu-Graib at the west of Baghdad during the period from December 2019 to August 2019. Semen was collected by Artificial Vagina one ejaculate per a week for three months. Fresh semen was evaluated which were of bad grade. Pooled semen were divided into five parts. The 1 st part (T1) only diluted semen (Tris-based extender) serve as a control. The 2 nd part (T2) added 0.576 mmol/ ml of zinc sulphate to diluted semen. The 3 rd part (T3) added 5 mmol/ ml of cysteine to diluted semen. The 4 th part (T4) added 37.5 pg/ ml of PGF2α to diluted semen. The 5 th part (T5) added a combination of previous components to the diluted semen. Then the semen was evaluated on 5C° and after 24, 48, and 72 hrs. of cooling. The following parameters of semen were evaluated: Mass and Individual motility according to (5). Sperm abnormalities and a live spermatozoa according to (6). Measurements of sperm concentration with spectrophotometer according to the method of (7). Spermatozoal plasma membrane integrity percent were (HOST) calculated according to the method of (8). Acrosomal integrity percent using Gemsi stain according to the method of (9). Statistical analysis were used according to SAS (10) and Duncan multiple range test (11). Table-1 showed semen parameters of fresh semen of Holstein bulls which is of bad quality. These parameters includes: ejaculate volume, mass motility, individual motility, liveability of spermatozoa, sperm concentration, sperm abnormalities, plasma membrane integrity and acrosomal integrity (AI). Table-2 showed the effect of different treatment on different periods on individual motility of Holstein bulls semen. It has been shown that there was no significant difference between the five treatment during the periods of cooling at 5C°, 24, 48 and 72 hrs. on the percent of individual motility of spermatozoa, although there was mathematical difference between treatments. While there was a significant superiority during periods for the some treatment (P<0.01) at 5C° as compared with the periods of control, PGF2α, zinc sulphat and their combination. Cysteine treatment showed no differences during different periods of cooling at the same treatment. this superiority might be due to that the best temperature of preservation in cooling at 4-5 C° for several days (12,13,14). These results a greed with (15) who claimed that the quality of semen decrease with the time of preservation till 48 hrs. according to the liveability of spermatozoa and the effect of preservation temperature. with control one. While there was no differences with PGF2α and combination treatments at 5C° of cooling. These differences has been explained by (16) who added 1 mg/ ml of zinc to the semen and observed no changes in the motility and the percent of forward movement as compared with other treatments. The results agreed with (17) who explained that zinc have the ability to protect sulfhydryl group (Sh) from oxidation enhance the synthesis of molecules rich with (Sh) which reduce glutathione and melatonin which acts as antioxidant. (18) reported that addition of zinc in a concentration of 0.8 mg/ ml to the bulls semen improve the concentration of spermatozoa and the acrosomal integrity of the sperms. The results of our study disagreed with (19) who observed that addition of high levels of zinc (50, 100 and 150) mmol/ ml to the diluted semen of Holstein bulls have no effect on morphology and motility of spermatozoa as compared with control one. It has been reported that high concentration of zinc might decrease oxygen consumption in sperm cell respiration that affect motility. These variation might be due to differences in the concentration added by the workers (20). Also the results agreed with (21) who explained that cysteine act on glutathione peroxidase that improve the content of spermatozoa from glutathione. It is also act on reduction of reactive oxygen species (ROS) and decrease lipid and nucleic acid oxidation that percent damage occurred to the head and protect the plasma membranes and mitochondria of sperm cells.

Results and Discussion
The results of cysteine disagreed with the (22) who reported the negative effect of addition of cysteine in a high concentration (12 and 15) mmol/ ml to the semen of Jersy bulls. Table-4 showed the effect of different treatments on the percent of sperm cells abnormalities of Holstein bulls semen after different periods of cooling. It has been observed that there was a significant decrease (P<0.05) in the percent of sperm abnormalities at the combination treatment and PGF2α as compared with other treatments and different periods at 5C° and 24 hrs. This superiority of combination might be due to the synergistic effect between the components. The zinc showed a great effect on growth of the testes, spermatogenesis and the activity of spermatozoa, in addition to its action as antioxidant (17,23). Cysteine acts as a reducing agent to ROS free radicals and decrease the lipid and nucleic acid oxidation with protection of head, plasma membrane and mitochondria of spermatozoa (24). (25) showed that addition of PGF2α in a concentration of 37.5 pg/ ml to the diluted and cooled semen improved semen characteristic of Holstein-Fresian bulls. Other workers showed a decrease in sperm abnormalities after addition of PGF2α to the semen of Frie-Raraan cross-Breed (26).
There was no information available on addition of this combination to the semen of bulls. It has been observed that the best temperature of preservation for cooled semen at 4-5 C° (13,14). The quality of semen reduced with the time of preservation till 48 hrs. according on the nature of diluent, temperature and the method of preparation of the diluent (15). Table-5 showed the effect of different periods of cooling on hypo-osmotic swelling test (HOST) plasma membrane sperm cell integrity. The results showed that the zinc sulphate treatment differ significantly (P<0.05) from that of the combination while showed no differences with the control, PGF2α and cysteine treatment for the same periods of preservation at 5C°. This might be due to that the zinc protect sulfhydryl group protein from oxidation and acts to synthesized molecules rich in (Sh) that decrease the glutathione and melatonin which plays a role as antioxidant (17).
Our results a greed with the (27) who explained that addition of zinc leads to increase the integrity of spermatozoal cell membrane and reduce the damage of DNA of the cells. Similar results have been reported by (19) who observed that addition of zinc in concentration of (50, 100 and 150) mmol/ ml semen diluent of Holstein bulls increase the cell membrane integrity of the spermatozoa. Also (28) observed that addition of yeast enhanced with zinc and selenium to the ram ration leads to improvement of reproductive performance of the ram's (includes: ejaculate volume, individual motility, mass motility, liveability and concentration of spermatozoa). The results also showed there was a significant difference (P<0.05) in the characteristic of HOST between zinc and cysteine as compared with the PGF2α and the combination treatments during the period of preservation at 24 hrs. The superiority of cysteine treatments might be due to its action to protect the plasma membrane of bulls spermatozoa (29). This is also in accordance with (30) who explained that addition of cysteine to Tris dilute of Holstein bulls semen leads to improvement of semen parameters.
The results also agreed with (25) on the effect of PGF2α and zinc significantly (P<0.05) during cooled preservation at 48 her. Table-6 showed the effect of different treatment after different periods of cooling on acrosomal integrity (AI) of Holstein bulls semen. The results showed that semen examined at 5C° the zinc treatments showed significant difference (P<0.05) in acrosomal integrity (AI) as compared with control, PGF2α and combination treatment.
This might be due to the effect of zinc on lipid fluidity that affect stability of biological membrane and it is also participate and play a role in sperm capacitation and acrosomal reaction (31). These results agreed with (32) who reported that zinc additives protect sperm cells from bacterial infection and prevent chromosomal damage. Similar observations have been made by (18). Conclsion It was concluded from this study that addition, of zinc sulphate, cysteine, PGF2α to the Holstein bulls semen improve the characteristics of semen parameters.