Inhibition Of Rumen Protozoa By Select Plants Leaves To Improve Nitrogen Utilization Efficiency Potentially In Ruminants

The role of rumen ciliate protozoa is controversial and elusive because some genera especially the most predominant Entodinium has been ascribed to contribute to lower down the nitrogen utilization efficiency through ammoniagenesis and methanogenesis,while other genera such as Dasytricha,Isotricha,Ophryoscolex and Epidinium contribute to the feed digestion together with their associated prokaryotic symbionts.Several previous studies including total defaunation attempted to eliminate the whole rumen protozoa to ensure a sustainable ruminant production using chemicals but unfortunately,this is not the best approach because in all the cases,the ecological niche of the rumen protozoa should not completely be eradicated.Meanwhile,reducing a sub-group or detrimental protozoa genus while maintaining the other genera is currently technologically challenging.This research aimed to enhance nitrogen utilization efficiency through inhibiting the Entodinium rumen protozoa using phytochemical compounds.More specifically,this research developed anaerobic cultivation of the rumen protozoa as a preliminary to screen a set of six different forest plant leaves for their phytochemical analysis and their potential to manipulate the rumen protozoa without creating microbial unbalance.The most promising plant has therefore been further evaluated for its effect on the prokaryotic population,the fermentation characteristics including ammonia nitrogen concentration,methane production,volatile fatty acids and alpha-glucosidase enzyme activity.The data were statistically analyzed in a complete randomized design using thePROC GLM procedure of SAS 9.4(SAS Institute,Cary,NC,USA)to compare the meansof protozoal cell counts,the fermentation characteristics,the alpha-amylase inhibition activity as well as the phytochemical compounds among the different plants.Significance was declared at P<0.05.Spearman rank's correlation was calculated to identify the correlation between the protozoal cell counts and the fermentation characteristics using the corrplot package in R(R 3.5.3).The GC/MS Translator B.07.17 was used to convert the Chemstat HPLC data files into MassHunter files for qualitative and quantitative analysis of the compounds in the plant extracts.Results showed a maximum growth of 74.02%of the total ciliate protozoa when O2concentration is completely trapped in combination with fermentation gas release(P<0.05)and the lowest growth of 13.29%in the presence of O2 with fermentation pressure release(P<0.05).The pressure release increased the total protozoa growth to a maximum level when there is no O2(P<0.05).The most predominant genus Entodinium more than doubled for No O2 combined topressure release(P<0.05).Dasytrichaandlsotricha increased more with aerobic media(P>0.05),Epidinium increased with "No O2 no pressure release" and with "O2 pressure release" but decreased with "O2 no pressure release" and with "aerobic media pressure release"(P<0.05).Eudiplodinium,MetadiniumandOphryoscolexwere maintained(P>0.05).Total VFAs,individual VFAs,ammonia nitrogen and methane production increased compared to the initial time T0(P<0.05)but remained constant throughout the other treatments.Overall,the new culture technique could contribute to filling the existing gaps related to the rumen ciliate protozoa culture and thereby contribute to the understanding of the rumen protozoa and studies that involves in vitro cultivation of this group of unique rumen microbes.The nutritional and functional properties of another six species of forest plants(Flemingia macrophylla,Kalimeris indica,Brassica rapasubsp.chinensis,Portulaca oleracea,Calotropis gigantea,and Adansonia digitata)were further investigated fortheir nutritional and phytochemical potential to inhibit rumen protozoa,ammoniagenesis,and ?-glucosidase activity.Quercetin,anthraquinone,3-hydroxybenzoic acid,astragaloside,and myricetin were the five bioactive components found at different levels in the different plant leaves.All the tested plants reduced total rumen protozoa counts,but C gigantea and B.rapasubsp.chinensis had the most inhibitory effect(P ? 0.05),with a reduction of 50%and 70%respectively,of the initial rumen protozoa counts at the dose of 1.1 mg/mL.The cell surface of Entodinium wilted similarly across all the tested plants.Ammonia nitrogen(NH3-N)production did not change with the plant supplementation(P?0.05)except for C gigantea which decreased NH3-N concentration by 32.48%while F.macrophylla increased ammoniagenesis by 15.95%at the dose of 1.1 mg/mL(P ? 0.05).In addition,the extracts from the leaves of five of the six plants exhibited inhibition against ?-glucosidase activity in a dose-dependent manner.The tested plants showed promising results as potential functional feed additives to manipulate the rumen protozoa,but the full phytochemical screening remains to be evaluated including their effect on the other microbes of the rumen microbiome.Furthermore,the most promising plant,giant milkweed(Calotropis gigantea),was further evaluated for the potential to improve nitrogen utilization efficiency(NUE)through analyzing its effect on rumen protozoa in vitro,at the genus level.Ground leaves of giant milkweed at 1.6 and 3.2 mg/mL decreased the counts of Entodiniumcells by 41.30%and 58.89%,respectively,and damaged their cell surface structure.Dasytricha,Isotricha,Epidinium,Ophryoscolex,and Diplodiniumwere not affected,while total bacterial and archaeal populations did not decrease.Ammonia nitrogen(NH3-N)concentration decreased by 50.64%and 33.33%at 1.6 g/mL and 3.2 mg/mL,respectively.Methane production also decreased.Total Volatile fatty acids(VFAs)production remained unaffected,but except for acetate,the rest of individual VFAs increased.The giant milkweed leaves contained(per gram of dry matter)3,636?g of phenolics including 205.9 ?g of 3-hydroxybenzoic acid,2,079 ?gof flavonoids including 1,197.5 ?g of quercetin and 91.4 ?g of myricetin,and 490?gof alkaloids including 219.8 ?g of anthraquinone glycosides.The effective inhibition of Entodinium was accompanied by a decrease in NH3-N concentration and methane production did not increase except for the dose of 1.6 mg/mL.Overall,giant milkweed may be used as a new feed additive or an alternative to chemicals or antibiotics for sustainable animal husbandry enhancing NUE in ruminants without impairing the fermentation characteristics.