| Previous study has revealed that butyrate caused epithelial growth links to the upregulation of cyclin Dl,but the precise molecular mechanism behind this effect have not been elucidated.The aim of present study was to determine the effect of butyrate on rumen epithelial growth and this effect is hypothetically associated with the simultaneous expression of both proliferative and apoptotic genes.However,due to the lack of molecular study on these two vital cellular process regulated by butyrate in ruminants,we explored first time the effect of butyrate on regulatory genes associated with cell cycle and apoptosis in rumen epithelium of goat.Twenty four healthy goats,fixed with ruminal fistula(4-month of age;19.05±0.43kg of body weight)were randomly allocated to two groups:group B(n=12,50 ml of 0.1M potassium phosphate buffer with butyrate 0.3 g/kg BW/day infused into rumen)and group C(n=12,infused same amount of buffer,but without butyrate).The infusion was performed one hour before morning feeding for 28 days.Four goats from each group were slaughtered at time points of 5,7 and 9h after last infusion.Morphomatric study of rumen papillae was determined by sliding caliper.SCFA concentration in rumen fluid was measured by gas chromatography and the relative gene expression in rumen epithelium was quantified by real time PCR.Our results showed that in comparison to group C the ruminal butyrate concentration in group B increased by 99.77%at 1h post-infusion and remained significantly elevated by 53.40%up to 2.5h thereafter returned to the pre-infusion value.Morphometric analysis of rumen papillae shows that group B goats exhibited increased rumen papillae size in 3 tested regions of rumen i.e atrium ruminis,ventral sac and ventral blind sac from 3 time point samples.In group B,papillae length increased by 21.77%(P<0.05),19.06%(P<0.05)and 28.14%(P<0.05);at 5h,26.76%(P<0.05),14.32%(P<0.05)and 24.80%(P<0.05)at 7h and 18.85%(P<0.05),22.70%(P<0.05)and 24.36%(P<0.05)at 9h in atrium ruminis,ventral sac and ventral blind sac respectively as compared to group C.Width increased by 35.46%(P<0.05),29.28%(P<0.05),and 34.61%(P<0.05)at 5h,35.66%(P<0.05),13.12%(P<0.05)and 14.86%(P<0.05)at 7h and 36.41%(P<0.05),25.32%(P=0.05)and 33.84%(P=0.05)at 9h in atrium ruminis,ventral sac and ventral blind sac respectively in group B compared to group C samples.The density of papillae increased by 25.26%(P<0.05)18.47%(P<0.05)and 15.43%(P<0.05)at 5h,16.27%(P<0.05),23.98%(P<0.05)and 21.40(P<0.05)at 7h and 30.99%(P<0.05),22.94%,(P<0.05)and 19.64%(P=0.05)at 9h in atrium ruminis,ventral sac and ventral blind sac,respectively in group B compared to group C.Overall papillae dimension and densities led to increase in the surface area by 108.47%(P<0.05),80.45%(P<0.05)and 97.37(P<0.05)at 5h,100.66%(P<0.05),64.68%(P<0.05)and 72.52%(P<0.05)at 7h and 113.76%(P<0.05),91.67%(P<0.05)and 99.44%.(P<0.05)at 9h in atrium ruminis,ventral sac and ventral blind sac respectively in group B as compared to group C.The number of cells in stratum germinativum,SGv9148.18 ±291at 5h,SGv9096.93±264 at 7h and SGv8047.62±258 at 9h in B group were significantly higher(P<0.05)than in C group 7936.03±186 at 5h,7797.93±125 at 7h and 6883.95±197 at 9h respectively.Likewise the cell number in stratum basale,SB 292.04 ±13.2 at 5h,293.87 ± 15.0 at 7h and 283.75±12.4 at 9h in B group were significantly higher(P<0.05)than in group C,250.83±4.9 at 5h,246.58±4.8 at 7h and 242.52±4.1 at 9h respectively.Improved papillae size in B group was coupled with the upregulation of mRNA expression of G0/G1 phase regulators namely cyclinDl,(P<0.05),CDK2(P<0.01),CDK4(P<0.01),CDK6(P<0.05)and p21(P<0.05),at 5h compared with the C group.However,G1/S phase regulators namely cyclin E1,(P<0.05)at 7h and S and G2/M phase regulators namelycyclin A,(P<0.05)and CDK1(P<0.05)at 9h post infusion were significantly increased in the B group compared with the C group.Furthermore,the mRNA abundances of apoptosis i.e caspase 3(P<0.05),caspase 9(P<0.05)and Bax(P<0.05)at 5h post-infusion were upregulated in group B compared with animals of group C.The mRNA abundances of Bcl-2 and the ratio of Bcl-2 to Bax(Bcl-2/bax)were non significantly different(P>0.05)between the groups.The correlation analysis shows that the mRNA abundances of genes involved in cell proliferation,cell cycle inhibition and apoptosis was positively correlated with ruminal butyrate concentration.Eight adult healthy goats were used for invitro study.After slaughter,the rumen epithelium of goats was collected and processed enzymatically to obtain a primary culture of rumen epithelial cells(REC)after 24h cell adhesion.Thereafter the cells were treated in the following manner:at pH 7.4+Butyrate 5mM(B,n=8)and pH 7.4(C,n=8)for 24 h,later cells were collected for gene expression analysis.Our results showed that in cultured cells treated with butyrate 5mM(pH7.4)had significant effects on the expression of most of the genes tested in the present study.Butyrate increased(P<0.05)the mRNA expression of cell cycle related genes include cyclin D1,cyclin E1,cyclin B1,cyclin A,CDK2,CDK4 and CDK6 compared with C group.While the mRNA expression of CDK1 and p21 were not significantly different between groups.Furthermore,cells treated with butyrate exhibit increased(P<0.05)mRNA expression of apoptosis-related genes include caspase 3,caspase 9 and Bax compared to C group and the ratio of Bcl-2 to Bax(Bcl-2/bax)was reduced(P<0.05)in B group compared to C group.It is concluded that intraruminal butyrate infusion stimulates epithelial and papillae growth,showing time dependent expression of cell cycle and apoptotic genes is associated with ruminal butyrate concentration.The expression pattern of cell cycle regulating genes showed successfully promoting cell progression from G0/G1 to S phase or to G2/M phase which ultimately complete the cell division.Furthermore,the concurrent and time-dependent expressions of genes related to cell cycle and cell apoptosis can contributes to maintain tissue homeostasis.Invitro study showed that butyrate upregulates cell cycle and apoptotic gene expression in rumen epithelial cells.Rumen of goat is considered as vat for fermentation of food and production of volatile fatty acid.Healthy rumen is considered to be vital property ensuring better growth and production of goat.Papillae morphology is one of the markers for evaluation of healthy rumen.Since it has been proved by previous studies that papillae growth improves the overall health of goat.In Pakistan no work is done on papillae morphology of different goat.In addition,there is closed cooperation between China and Pakistan in agriculture and livestock.To be an exchange student sent by government of Pakistan,I would like to introduce 3 Pakistani goat breeds to China and to compare the breed variation in rumen papillae morphometric parameters between Pakistani goat breeds:viz Kamori,Pateri,Tapri and Yangzi river goat.The aim was to promote the cooperation between China and Pakistan in ruminant research.Twelve goats(3 in each breed)were included in this study.The stomach of total of nine goats(n=3 each Pakistani breed)were purchased from local slaughter house while the stomach of 3 Yangzi river goats(C group goats of part Ⅰ study)was used.After weighing of stomach and rumen(full and empty),the rumen epithelial sampleswere collected from atrium ruminis,ventral sac and ventral blind sac,washed with PBS and preserved in 10%neutral buffered formalin for morphometric analysis.We detected that full and empty stomach and full rumen weight among all breed were not significantly different.Whereas the empty rumen weight of kamori goat was significantly increased compared to Pateri,Tapri and Yangzi river.Morphometry revealed that the papillae size(length and width),density and surface area of kamori goat was significantly(P<0.05)increased only in atrium ruminis compared to Pateri,Tapri and Yangzi river.Papillae length increased by 39.60%,40.29%and 26.45%,width by 51.47%.56.09%and 77.77%,density by 26.32%,28.39%and 32.69%,surface area by 164.98%,166.21%and 211.22%compared to Pateri,Tapri and yangzi river goat respectively.In ventral and ventral blind sac the morphometric parameters of rumen papillae were not significant differentbetween all breeds.It is concluded that the higher empty rumen weight and papillae morphometric parameters in atrium ruminis of Kamori goat could be breed characteristic.The results of invivo study will be helpful to develop future study in continuation to first preliminary base line study on local goat breeds of Pakistan. |
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