اثر موننسين و عصاره گياهي بر متابوليت‌هاي خون و شكمبه و بيان ژن ناقل اوره در اپيتليوم شكمبه بره‌هاي پرواري

Effect of Monensin and plant extract on rumen and blood metabolites and gene expression of the urea transporter gene in the rumen epithelium of fattening lambs

زمينه مطالعاتي: موننسين و عصاره گياهي مي ­تواند با تغيير در متابوليت­ هاي شكمبه و خون بر بيان ژن ناقل اوره در اپيتليوم شكمبه اثر گذارد. هدف: اين آزمايش به‌منظور تعيين روابط بين بيان ژن ناقل اوره با متغيرهاي شكمبه­ اي و خوني در بره­ هاي تغذيه‌شده با جيره­ هاي حاوي موننسين و عصاره گياهي انجام گرفت. روش كار: در اين طرح از 16 رأس بره­ نر نژاد افشاري (ميانگين وزني 5/6±41 كيلوگرم و سن 6 ماه) استفاده شد. بره­ ها به­ طور تصادفي به چهار جيره­ آزمايشي اختصاص داده شدند كه شامل: جيره پايه، جيره پايه به‌اضافه 30 ميلي­گرم موننسين در روز، جيره پايه به‌اضافه 30 ميلي ­گرم موننسين به‌صورت 2 هفته در ميان و جيره پايه به‌اضافه 2 گرم عصاره گياهي در روز براي هر رأس بود. نمونه­ هاي خون و نمونه ­هاي مايع شكمبه پيش از كشتار و نمونه ­هاي بافت شكمبه از قسمت شكمي شكمبه گرفته شدند. اسيدهاي چرب فرار، نيتروژن اوره­اي پلاسما و نيتروژن آمونياكي شكمبه اندازه­گيري شدند. ميزان بيان نسبي ژن انتقال‌دهنده نوع B اوره­ (UT-B) با استفاده از تكنيك Real-time PCR بررسي شد. نتايج: نتايج حاصل از اين پژوهش نشان داد كه ميزان بيان نسبي ژن ناقل اوره در بره هاي تغذيه‌شده با موننسين به‌صورت دوره­ اي كاهش يافت (0/05>P) و غلظت نيتروژن آمونياكي، نسبت مولي اسيد بوتيريك و اسيد والريك نيز در بين تيمارها تفاوت معني­داري داشت (0/05>P). نتيجه‌گيري كلي: درمجموع بين متغيرهاي مهم، همبستگي عكس و متوسطي بين ناقل اوره با نيتروژن آمونياكي، اسيد بوتيريك و اسيد والريك و همبستگي مثبت با اسيدهاي چرب فرار مشاهده شد

Introduction : Urea as the final product of nitrogen metabolism plays a pivotal role in ruminants’ nitrogen economic efficiency ( Marini and Van Amburgh , 2003). Ruminants are capable to recycle and return a tremendous amount of urea to rumen rather than excretion to urine; the recycled urea is hydrolyzed to CO2 and ammonia and thereafter is used for the synthesis of microbial protein ( Harmeyer and Martens, 1980). By changing ruminal and blood metabolites, M onensin and herbal extracts can change and modify the expression of urea transporter gene in rumen epithelium. Monensin is an ionophore antibiotic which changes the ratio of VFAs in the rumen in favor of propionic acid and decreases the production of methane ( Duffield and Ba gg , 2000) . The aim of present study was to investigate the effects of M onensin and herbal extracts as feed additives on type B urea transporter gene in rumen epithelium and also on ruminal ammonia, VFAs, pH and blood ammonia in fattening lambs. Material an d methods : In this experiment 16 Afshari ram lambs with initial BW of 41±5.6 kg and 6 month s of age were used. The lams were randomly assigned to four experimental dietary treatments in a completely randomized design; 1) no additive (control), 2) 30 mg mon ensin.d - 1 per lamb, 3) periodical inclusion of 30 mg monensin.d - 1 per lamb, and 4) 2 g of a commercial blend of plant extract.d - 1 per lamb. During the experiment , lambs were kept in individual boxes and the diets and fresh drinking water were offered ad li bitum . 3 weeks were considered as an adaptation to diets and 8 weeks were considered as an experimental period in which treatments and samplings were conducted. The forage: concentrate ratio was set to 20:80 and the rations were prepared daily, mixed by hand and were offered two times at 09:00 and 16:00. Two hours after morning feeding rumen samples were collected every other 2 weeks by using a special tube and electrical vacuum pump. Also, immediately after slaughtering and before skinning 2 rumen samples were collected, filtered and the pH was determined. 8 ml of the first sample was mixed with 2 ml of % 25 sulfu ric acid for measurement of ammonia and 8 ml of the second sample was mixed with %25 meta - phosphoric acid for measurement of VFAs. The mixed samples were kept in - 20ºC until assays. After thawing, rumen samples were centrifuged for 10 to 15 min and then th e supernatant w as separated and stored for FAs and ammonia measurements. Blood and rumen samples were collected before slaughtering and rumen tissue samples were taken from ventral part of the rumen . VFA in rumen samples was measured by using Gas chromatog raph (Mirzaei - Alamouti et al. 2016). The ammonia nitrogen was measured by using a spectrophotometer . Blood samples were taken at the beginning of the experiment and then every 2 weeks after the morning feeding into heparinized tubes. Blood samples were cen trifuged at 3000 rpm for 15 min and plasma samples were stored at - 20º C for plasma urea nitrogen. Blood urea nitrogen was measured with commercial kits using a spectrophotometer . The lambs were slaughtered 56 days after feeding with experimental diets and the tissue samples were taken from ventral part of the rumen . Relative gene expression of UT - B was determined by Real - time PCR technique (Mirzaei_Alamouti et al, 2016) . Experimental data were subjected to analysis of variance by the mixed procedure of SAS (9.1). Results and discussion : The relative expression of urea transporter gene in lambs fed with periodical M onensin was 0.167 in compar ison to control group. In Pfaffl’s (2001) method, the gene expression in control group is considered 1; thus, the expr ession of urea transporter gene in periodical M onensin group has been reduced (P< 0.05). In addition to this, expression of urea transporter gene in M onensin group tended to increase and in herbal extract group tended to decrease. The ammonia nitrogen leve ls in periodical M onensin group increased in compar ison to control group (P<0.05). In herbal extract group, the relative expression of urea transporter gene was not affected in compar ison to control group (P<0.05). The levels of ammonia nitrogen in herbal extract group were increased in compar ison to control group. Rumen pH was not affected by diets containing M onensin, herbal extract or control group (P<0.05). Rumen pH was higher in periodical M onensin group in compare to other groups (P<0.05). The conce ntration of total free fatty acids which stimulate the absorption of urea from rumen epithelium did not show any significant difference (P<0.05). Conclusion: all in all, the results of this study showed an inverse or average relationship between urea tran sporter gene expression with ammonia nitrogen, butric acid and valeric acid and a positive relationship with VFAs. The periodical inclusion of M onensin to the diets may change the rumen fermentation and reduce the expression of urea type B transporter. In compar ison to this, the continu ed inclusion of M onensin to the diets would improve rumen fermentation and increase the expression of the urea - type B transporter gene . The herbal extract used in this study may change the rumen fermentation but is not possib ly effective on epithelial cells which are involved in urea transportation. In this study, the rumen variables were more effective in urea transportation in compare to blood variables.