The impact of synbiotic administration through in ovo technology on the microstructure of a broiler chicken small intestine tissue on the 1stand 42ndday of rearing

Background

Synbiotics consist of a combination of synergistically interacting probiotics and prebiotics which can be aimed at improving the resistance and stability of health-promoting organisms in the gut of birds by providing a substrate for fermentation[1,2].Thus,synbiotics are factors that modulate the immune system of birds by acting on the bacterial flora of their gastrointestinal tract[3].To ensure the greatest protection of the immune system for newly hatched chickens,external supplementation with bioactive substances,such as synbiotics,should occur as early as possible.Application of an innovative method,such as in ovo technology,provides a means of modulating the immune system at early embryonic stages.This technology involves the introduction-on the appropriate day of embryonic development of birds-of the particular substance in solution form to the air chamber of eggs or directly into a developing embryo[4].Of course,the effectiveness of the injection and the level of use of the injected bioactive substance by the avian embryo depend on various factors[5].These can be the chemical and physical features of the injected substances,its dose and the egg surface where the injection was performed(i.e.the embryo,the amnion,the allantois,the air chamber egg or the yolk sac).A thorough understanding of the various stages of embryonic development in birds allows the optimal time of injection to be defined[6,7].According to Villaluenga et al.[8],the optimal time of prebiotic injection is on the 12thday of embryonic development.In comparison with injections on d 1,8 and 17,a significantly higher number of Bifidobacteria was observed in the colon of two-day-old chickens.A similar result was obtained by Pilarski et al.[9],who studied the effect of alphagalactosides(RFOs)administered on the 12thday of egg incubation on selected traits of chickens.

This is mainly due to the fact that on this day of bird embryonic development,allantochorion is already fully developed and vascularised.The embryo is surrounded by the amniotic fluid that remains in contact with the embryonic gastrointestinal tract,which permits the transport of substances from the air chamber into the intestine[10].Thus,a highly vascularised allantochorion enables efficient transport of bioactive substances given by injection in ovo on the 12thday of egg incubation between the air chamber of the eggs and the digestive tract of chickens.Upon hatching,the in ovo modulated profile of the gut microflora has an influence on the good condition of health of a chick,eliminating the need for antibiotics.We presume that this beneficial condition of the GI(gastrointestinal)tract would be reflected in the morphology of the intestines and might be maintained throughout the life of the chicken.

The prebiotics used in this study as components of the synbiotics are:commercially developed non-digestive transgalacto-oligosaccharides(Bi2tos,Clasado Ltd.)and raffinose family oligosaccharides(RFOs),which was obtained from lupin seeds as a white powder.

In previous projects carried out by our team,we evaluated the impact of bioactive substances in the form of pre-,pro-and synbiotics injected on the 12thday of incubation into the air chamber of the egg on production traits and the macro-and microstructure of the small intestine.The best composition of these substances and their optimal dose was selected.On the basis of these studies,we found the most beneficial effect of synbiotics on the above-mentioned parameters[11,12].

The aim of study was to determine the influence of the in ovo stimulation with synbiotics on the microstructure of the duodenum,jejunum and ileum on the 1stand 42ndday of rearing.

颈源性头痛可根据神经根的不同受累部分,分为神经源性疼痛和肌源性疼痛。神经根的感觉根纤维受到刺激引起神经源性疼痛,而其腹侧运动神经根受刺激时则以肌源性疼痛。

Methods

The experiment was conducted on hatching eggs of the Cobb 500 FF line incubated in commercial hatchery conditions(Drobex-Agro Ltd.,Solec Kujawski,Poland)in Petersime incubators and on 1-and 42-day-old broilers.On d 12 of incubation,the eggs were candled,and the infertile ones or those containing dead embryos were discarded.Eggs containing living embryos were randomly divided into 3 groups.Bioactive substances were administered in an amount of 0.2 mL into the air chamber of the egg on the 12thday of embryonic development by the in ovo technique.The hole in the shell of the egg was sealed with the use of a special automatic system[13].5000 eggs were injected.The SYN1 group received synbiotic L.salivarius IBB3154+Bi2tos,Clasado Ltd.(2 mg of Bi2tos prebiotic+105bacteria/egg),and the SYN2 group received symbiotic L.plantarum IBB3036+lupin RFOs(2 mg of RFO prebiotic+105bacteria/egg).The Control group was injected with physiological saline 0.9%NaCl.

Animals

The rearing experiment was conducted on the experimental farm of the PIAST company in Olszowa according to the technological recommendations and lasted for 42 d.All groups were fed and watered ad libitum.Commercial diets were used according to the age of the chickens:1-10 d-starter,10-20 d-grower,20-41 dfinisher(Table 1).

Abbreviations

The material for the morphological and histological assays(approx.2 cm)of the small intestine(duodenum,jejunum,ileum)were collected from 1-and 42-day-old chickens.Before slaughter,680 chickens from each group were weighed,and their mean body weight was calculated.Subsequently,15 birds per group,with a body weight similar to the mean for the group,were selected.Directly after slaughter,the small intestine was extracted,and all sections of the small intestine was excised,measured and weighed.Samples were taken from the midpoint of the duodenum,from the midpoint of the jejunum between the point of entry of the bile duct and Meckel’s diverticulum and the midpoint of the ileum between Meckel’s diverticulum and the ileocecal junction.A total of 270 samples were collected(3 groups×3 sections×15 birds×2 repetitions).The individual sections of the intestine were flushed with 0.9%saline and then fixed in 4%CaCO3buffered formalin.The fixed samples were dehydrated,cleared and permeated with paraffin in a tissue processor(Thermo Shandon,Chadwick Road,Astmoor,Runcorn,Cheshire,United Kingdom),and subsequently embedded in paraffin blocks using an embedding system(Medite,Burgdorf,Germany).Thus,the formed blocks were cut into 10 μm-thick sections using a rotary microtome(Thermo Shandon,Chadwick Road,Astmoor,Runcorn,Cheshire,United Kingdom),which in turn were placed on microscope slides coated with egg protein with the addition of glycerine.

实验在F环岛先均匀布置10个检测器节点标记为A组,每个节点的通信模块中拨码选择为00,设置为固定周期传输。A组每个检测器节点旁边同时再布置一个检测节点并标记为B组,B组10个节点通信模块中拨码选择为01,设置为变周期数据传输,变周期数据采样温度阈值设置为εc=0.5 ℃,CO2浓度阈值设置为εt=15×10-6。变周期数据传输模式只有当前后两次采集到的数据差大于相应阈值范围时,才进行当前采集数据的传输。A、B二组检测器节点硬件相同,但是在数据传输方式上不同。每个检测器的基本采样周期设置为2 min,每10个检测节点为一组搭配一个网关进行组网,运行监测软件进行实验。

Table 1　Composition of premix for starter,grower and finisher diets for chickens

Inclusion rate kg/t feed Starter diet　Grower diet　Finisher diet Vitamin/Element Declared name Additive Code UnitsQuantity per tonne of finished feed Quantity of compound per tonne of finished feed Quantity of compound per tonne of finished feed Vitamin A　Retinyl acetate Quantity per tonne of finished feed Quantity of compound per tonne of finished feed Quantity per tonne of finished feed E672　MIU　13.00　-　10.00　-　10.00　-Vitamin D3　Cholecalciferol E671　MIU　5.00　-　5.00　-　5.00　-Vitamin E　alpha Tocopherol 3a700　g　80.00　-　50.00　-　50.00　-Vitamin K　Vitamin K　-　g　3.00　-　3.00　-　3.00　-Vitamin B1　Vitamin B1　-　g　3.00　-　2.00　-　2.00　-Vitamin B2　Vitamin B2　-　g　9.00　-　8.00　-　6.00　-Vitamin B6　Vitamin B6　3a831　g　4.00　-　3.00　-　3.00　-Vitamin B12　Vitamin B12　-　mg　20.00　-　15.00　-　15.00　-Biotin　Biotin　-　g　0.15　-　0.12　-　0.12　-Cal-D-Pan　Calcium Pantothenate-　g　15.00　-　12.00　-　10.00　-Nicotinic acid Nicotinic acid　-　g　60.00　-　50.00　-　50.00　-Folic　Folic acid　-　g　2.00　-　2.00　-　1.50　-Choline　Choline chloride-　g　0.50　-　0.40　-　0.35　-Methio-nine　Methionine　3.1.1　g　3405.00　-　3018.00　-　2514.00　-Threonine　Threonine　3.3.1　g　745.00　-　726.00　-　361.00　-Lysine　Lysine　3.2.3　g　2812.00　-　2831.00　-　1779.00　-Iodine　Calcium iodate E2　g　1.00　1.59　1.00　1.59　1.00　1.59 Selenium　Sodium selenite E8　g　0.35　7.78　0.35　7.78　0.35　7.78 Iron　Ferrous sulphate E1　g　40.00　133.33　40.00　133.33　40.00　133.33 Molybdenum Sodium molybdate E7　g　0.50　1.27　0.50　1.27　0.50　1.27 Manganese　Manganous oxide E5　g　100.00　161.29　100.00　161.29　100.00　161.29 Copper　Cupric sulphate E4　g　15.00　60.00　15.00　60.00　15.00　60.00 Zinc　Zinc oxide　E6　g　100.00　138.89　100.00　138.89　100.00　138.89

Before staining,the preparations were deparaffinised and hydrated.They were subsequently subjected to PAS(Periodic acid-Schiff)staining with Schiff’s reagent to conduct morphometric analyses of the small intestine and to stain and count neutral goblet cells.Measurements were made using a DELTA EVOLUTION 300 microscope equipped with a digital camera by ToupCam™.Measurements included:height and width of intestinal villi and intestinal crypt depth,and the number of neutral goblet cells in the intestine were calculated using the MultiScan-Base v.18.03(Computer Scanning Systems II,Warsaw,Poland).In order to measure the height of the villi,ten villi per bird were randomly selected from a cross section.The length was measured from the tip of the villus to its base at the crypt-villus outlet.The perimeter length of the villus was measured to calculate the surface area using the formula cited by Sakamoto et al.[14].The depth of intestinal crypts was defined as the invagination depth between neighbouring intestinal villi and was measured between 10 villi[15].The number of PAS-positive cells was calculated per 1 mm2of the intestinal villi surface area.

The data were analysed by means of a one-way analysis of variance using STATISTICA 10.0 PL.The arithmetic mean()and the standard error of the mean(SEM)were calculated.Significant differences between the groups were tested using Duncan’s Multiple Range Test.

Results

Synbiotic L.plantarum IBB3036+lupin RFO(SYN2 group)significantly increased the body weight of chickens on the 1stday of life(P≤0.05)(Table 2),while significantly higher feed intake was found in both injected groups compared to the Control group(P≤0.05)(Table 3).FCE(Feed Conversion Ratio)did not significantly increase after synbiotics throughout the entire rearing period(Table 3).In all study groups,we also observed a very low mortality rate,which was below 2%[16].

While macroscopically evaluating the small intestines of the chickens,we found that the use of synbiotic 2 reduced the length(P≤0.05 and P≤0.01)and weight(P≤0.01)of the duodenum on the 1stday of life of the chickens in relation to the other groups(Table 4).However,it did not affect the microstructure of the analysed section at thisage,because in the duodenum,in both experimental groups(SYN1 and SYN2),a significantly higher and wider intestinal villi as well as a significantly larger absorbent surface of these villi were found in comparison with the Control group(P≤0.01)(Table 5).Simultaneously,in theSYN1 group,a significant decrease in the depth of crypts relative to the SYN2 group was reported.In the same group,a reduction in the number of goblet cells on the surface of 1 mm2of the villi was observed,while the villi were significantly larger compared to the Control group(P≤0.01)(Table 5).In turn,on the 42ndday of rearing after treatment of synbiotic 1(SYN 1 group),a greater weight of the duodenum(P≤0.01)than in the other groups and an increased length of this section(P≤0.05)compare to the Control group were observed(Table 4).This beneficial effect was reflected by the numerically higher villi(no statistical differences)with a larger surface(P≤0.01)in the duodenum in the SYN1 group compare to the Control group(Table 5).

随着国内赛事经济的红火，体育赛事转播权这一“新兴事物”引起了业界内外人士的高度关注。近有咪咕视频和优酷网从央视手中高价购得2018年世界杯的新媒体转播权；远有体奥动力体育传播有限公司斥资80亿购买5年的“中超版权”。然而本文细究后发现，无论是对体育赛事转播权的界定，还是它的保护方式都众说纷云。换言之，体奥动力等公司以80亿元天价所购得的究竟是什么东西，是否属于法律意义上的“权利”，在目前的法律语境中尚未有定论。故本文希望厘清：体育赛事转播权并非局限于知识产权领域的权利；它的法律保护路径需要在更为宏观的视野下才能绘制成型。

Table 2　Effect of synbiotics treatment injected in ovo on d 12 of incubation on the body weight of broiler chickens

a-bDifference(P≤0.05)between treatments(vertical),Means±SEM representing 680 birds per group.SYN 1-L.salivarius IBB3154+Bi2tos,Clasado Ltd.,SYN 2-L.plantarum IBB3036+lupin RFOs

Item　Day 1 42 Body weight,g Control　40.7b　3127 SYN1　40.3b　3146 SYN2　41.6a　3111 SEM　0.1803　9.7890

Table 3　Effect of synbiotics treatment injected in ovo on d 12 of incubation on the feed intake and on the FCE(the efficiency of feed conversion)of broiler chickens

a-bDifference(P≤0.05)between treatments(vertical),Means±SEM representing 680 birds per group.SYN 1-L.salivarius IBB3154+Bi2tos,Clasado Ltd.,SYN 2-L.plantarum IBB3036+lupin RFOs

Item　Day 1-10　1-41 Feed intake,g Control　247b　4930 SYN1　254a　4940 SYN2　258a　4898 SEM　1.3291　24.4097 FCE,g/g Control　1.21　1.60 SYN1　1.24　1.59 SYN2　1.29　1.60 SEM　0.0146　0.0067

Table 4　Effect of synbiotics treatment injected in ovo on d 12 of incubation on the length and on the weight of intestine of broiler chickens

a-bDifference(P≤0.05),A-Bdifference(P≤0.01)between treatments(vertical),Means±SEM representing 15 birds.SYN 1-L.salivarius IBB3154+Bi2tos,Clasado Ltd.,SYN 2-L.plantarum IBB3036+lupin RFOs

Item　Day 1 42 Length of intestine,cm Duodenum　Control　9.60±0.24Aa　28.67±0.73b SYN 1　9.03±0.24a　31.37±0.59a SYN 2　8.28±0.19Bb　30.10±0.96ab Jejunum　Control　17.20±0.65　64.00±1.93b SYN 1　16.60±0.65　70.20±2.34a SYN 2　17.27±0.49　68.50±1.86ab Ileum　Control　15.60±0.41　60.80±1.73b SYN 1　14.77±0.55　68.30±2.03a SYN 2　14.54±0.47　66.47±2.15a Weight of intestine,g Duodenum　Control　0.44±0.01a　17.44±0.36B SYN 1　0.41±0.01ab　19.38±0.39A SYN 2　0.38±0.02b　17.22±0.45B Jejunum　Control　0.47±0.03　45.31±1.39AB SYN 1　0.43±0.02　48.12±2.19A SYN 2　0.44±0.01　40.61±1.72B Ileum　Control　0.36±0.02　39.25±1.37 SYN 1　0.32±0.02　41.45±2.65 SYN 2　0.32±0.01　35.98±1.86

Table 5　Effect of synbiotics injected in ovo on the duodenum morphology of chickens at 1stand 42ndday of age

A-CDifference(P≤0.01)between treatments(vertical),Means±SEM representing 15 birds.SYN 1-L.salivarius IBB3154+Bi2tos,Clasado Ltd.,SYN 2-L.plantarum IBB3036+lupin RFOs

Item　Day 1 42 Villus height,μm Control　529.96±19.96B　1889.79±39.79 SYN1　603.47±12.39A　1940.95±36.10 SYN2　594.37±17.07A　1880.52±30.88 Villus width,μm Control　62.88±1.62B　188.59±5.13B SYN1　75.72±1.23A　216.69±8.05A SYN2　73.96±1.71A　202.13±5.45AB Villus surface area,μm2 Control　106,267±6158.5B　1123,441±39,725.1B SYN1　144,041±4238.0A　1323,359±58,298.1A SYN2　139,322±5970.8A　1187,036±32,976.4AB Crypt depth,μm Control　49.95±2.13AB　136.82±3.54B SYN1　45.81±1.89B　189.31±4.26A SYN2　53.97±1.27A　138.47±2.30B No.of neutral goblet cells/1 mm2 Control　808±49.08A　134±8.78 SYN1　526±21.30B　116±4.85 SYN2　456±14.28C　133±3.63

The above mentioned research and the results of our study present the different effects of synbiotics,depending on their composition and depending on the anatomical structure of GI tract.These two synbiotics:synbiotic 1(L.salivarius IBB3154+Bi2tos,Clasado　Ltd.)　and　synbiotic　2　(L.　plantarum IBB3036+lupin RFOs)designed based on our in vitro results[16]presented the two types of synergism,i.e.,between　the　prebiotic　and　probiotic components(synbiotic 1)and synergism between the two independent bioactive compounds and the host(synbiotic 2).In this last case RFO is less efficiently used by the probiotic bacteria,and it remains available to other indigenous stains of intestinal microbiota.In this situation,the prebiotic has a positive influence on the host organism through improvement of microbial balance in the intestines.

Table 6　Effect of synbiotics injected in ovo on the jejunum morphology of chickens at 1stand 42ndd of age

a-bDifference(P≤0.05),A-Bdifference(P≤0.01)between treatments(vertical),Means±SEM representing 15 birds.SYN 1-L.salivarius IBB3154+Bi2tos,Clasado Ltd.,SYN 2-L.plantarum IBB3036+lupin RFOs

Item　Day 1 42 Villus height,μm Control　337.88±11.01B　1334.68±46.19AB SYN1　383.86±10.01A　1190.20±50.03B SYN2　352.47±10.80AB　1517.95±54.52A Villus width,μm Control　51.64±1.06b　170.44±7.44 SYN1　56.42±1.30a　178.89±8.10 SYN2　52.55±1.46b　172.66±5.66 Villus surface area,μm2 Control　55,022.6±2410.6B　724,927.0±50,976.2 SYN1　67,746.0±2309.5A　688,644.6±51,191.4 SYN2　58,459.9±2489.5B　814,432.8±30,911.1 Crypt depth,μm Control　46.92±0.85A　150.87±7.65A SYN1　37.59±0.93B　125.41±2.87B SYN2　48.00±0.97A　112.88±3.95B No.of neutral goblet cells/1mm2 Control　743±42.21　153±20.30b SYN1　778±32.57　232±26.24a SYN2　681±22.49　185±6.49ab

The ileum is characterised by much shorter villi and less absorbent surface of these structures compared to the duodenum and jejunum,which may explain the reduction of the absorption of nutrients at this stage.Evidence of this was the fact that most of the digested food substances had already been absorbed in the upper parts of the small intestine,i.e.the duodenum and jejunum[24].Injection of the applied synbiotics slightly affected the microstructure of ileum differently compared to the duodenum and jejunum.In 1-day-old chickens,synbiotic L.plantarum IBB3036+lupin RFOs(SYN2 group)significantly increased villi width and crypt depth relative to the other groups.In the studies of Bogucka et al.[11],a synbiotic containing prebiotic Bi2tos significantly contributed to the shortening of the villi in 1-day-old chicks,whereas on the 42ndday of rearing,the width of the villi was already significantly lower in both experimental groups than in the Control group.In this section,at the end of rearing,a reduction in the absorbent surface of the villi was found in the SYN1 group in comparison to the Control group while prolonging the crypt depth in both experimental groups.A reduction of the absorbent surface area along with a deepening of the crypts was observed in the group of 35-day-old chickens treated in ovo with a synbiotic containing prebiotic Bi2tos and probiotic bacteria Lactococcus lactis spp.Cremoris in the study of Bogucka et al.[12].Different results were obtained by Awad et al.[19,20],which showed a significant extension of the villi and decreased crypt depth in the ileum after application of the synbiotic,although the substance was administered as a feed additive.

Discussion

Studying the impact of injected synbiotics on the body weight of chickens throughout their rearing,we found a positive effect of synbiotic 2 on this parameter just in 1-day-old chickens.Opposite,in the research conducted by Bogucka et al.[11],no significant effect of any bioactivesubstance injected in ovo on the 12thday of incubation on the body weight of 1-day-old chickens was shown.

Table 7　Effect of synbiotics injected in ovo on the ileum morphology of chickens at 1stand 42ndd of age

a-cDifference(P≤0.05),A-Cdifference(P≤0.01)between treatments(vertical),Means±SEM representing 15 birds.SYN 1-L.salivarius IBB3154+Bi2tos,Clasado Ltd.,SYN 2-L.plantarum IBB3036+lupin RFOs

Item　Day 1 42 Villus height,μm Control　295.04±9.16　933.31±43.10 SYN1　298.67±10.86　847.78±50.01 SYN2　289.81±9.09　949.05±38.70 Villus width,μm Control　46.42±1.29b　162.69±5.58 SYN1　46.62±1.34b　166.18±4.45 SYN2　50.85±1.66a　161.32±5.30 Villus surface area,μm2 Control　43,547.4±2124.7　471,197.5±20,933.6 SYN1　44,138.3±2318.3　445,293.0±27,184.3 SYN2　45,834.9±1735.9　484,522.9±26,619.7 Crypt depth,μm Control　46.14±0.77B　91.28±2.59B SYN1　38.55±0.85C　116.81±3.18A SYN2　50.14±0.98A　117.39±5.53A No.of neutral goblet cells/1mm2 Control　974±50.59b　263±26.05 SYN1　1129±61.42a　358±40.66 SYN2　678±19.60c　285±37.21

Our study showed a differential effect of the applied synbiotics(synbiotic 1-L.salivarius IBB3154+galactooligosaccharides　and　synbiotic　2　-　L.plantarum IBB3036+Raffinose Family Oligosaccharides)on the microstructure of individual sections of a broiler chicken small intestine.We found a positive effect for both synbiotics given in ovo on the height,width and the absorbent surface of duodenum villi in comparison to the Control group on the 1stday of life of the chickens.In turn,on the 42ndday of the life of the chickens,only synbiotic 1 demonstrated a positive impact in comparison to the Control group on the villi width and villi surface area.A similar effect of synbiotics on the width of the villi was observed by Bogucka et al.[12],however,it did not reflect on the absorbent surface of the intestine.Additionally,in birds from the same group at the end of rearing,the deepest crypts were found.The positive effect of in ovo injection of the synbiotic composed of Bi2tos and Lactococcus lactis subsp.cremoris IBB SC1 on the height of duodenum villi on the 1stday of life of chickens was also demonstrated in our previous studies[11].According to Pluske et al.[17],longer villi and their greater absorbent surface area translate into better utilisation of feed,and thereby improve the health of the birds.Deeper crypts,in turn,indicate rapid tissue regeneration processes to permit the renewal of villi to normal sloughing or inflammation due to the presence of pathogens or their toxins[18].Awad et al.[19],studying the effect of synbiotic supplementation,which is a combination of Enterococcus faecium probiotic and a prebiotic derived from chicory rich in inulin and immunomodulatory substances derived from sea algae,did not demonstrate significantly higher villi and significantly deeper crypts in the duodenum of 35-day-old broiler chickens.Similar results were obtained by Awad et al.in their further study in 2009[20].

In the jejunum of 1-day-old chickens,a beneficial impact of synbiotic 1 on the microstructure was demonstrated,but this wasn’t maintained for 42 d.Similar results in relation to the heights of villi after in ovo administration of bioactive substances(prebiotic:inulin,synbiotics:inulin　+　Lactococcuslactis　spp.lactis,Bi2tos+Lactococcus lactis spp.cremoris)in 1-day-old chickens were obtained by Bogucka et al.[11].At the end of rearing in the group,in which was applied synbiotic 2,significantly higher intestinal villi were stated in comparing to the SYN1 group and the biggest surface area of these villi(in this case no significant differences)which may have an impact on better absorption of nutrients.In our previous studies[12],there were no significant effects of both prebiotics and synbiotics on the height of intestinal villi in 35-day-old broiler chickens.

FCR:Feed conversion ratio;PAS:Periodic acid-Schiff;RFO:Raffinose family of oligosaccharides;SEM:The standard error of the mean

Analysing the crypts depth of the jejunum in examined groups of birds their significant shortening was stated in both experimental groups(SYN1 and SYN2).The crypt depth is one of the indicators of the health and functional status of the intestine in chickens,and their size can be a measure of the intensity of intestinal epithelial cell renewal processes[21,22].Xu et al.[23]indicate that“The crypt can be regarded as the villus factory,and a large crypt indicates fast tissue turnover and a high demand for new tissue”so in our study the decreased crypt depth may indicates an efficient tissue turnover and good condition of the gut.Different results were obtained after in ovo injection of a synbiotic containing the prebiotics inulin and Bi2tos.Bioactives had a significant impact on the deepening intestinal crypts of 35-day-old broiler chickens[12].Rehman et al.[18]examining the effect of the bioactive substance-inulin-on the jejunum histomorphology on the 35thday of the rearing of chickens and found significantly longer villi(P≤0.05)and significantly deeper crypts(P≤0.01)as a result of this prebiotic supplementation compared to the Control group.

In the ileum of 1-day-old chickens,the widest villi(P≤0.05)and the deepest crypts(P≤0.01)were found in the SYN2 group.In the same group,there was also the least amount of neutral goblet cells in comparison to the other groups(P≤0.05)(Table 7).Despite the lack of a significant effect of the applied synbiotics on the length and weight of the analysed sections of the small intestine on the 1stday of life,on the 42ndday of rearing,the length of the ileum(P≤0.05)was significantly greater in both treatment groups compared to the Control group(Table 4).In both experimental groups,a deepening crypt depth was observed(P≤0.01)(Table 7).Other parameters,such as villus height,villus width and villus surface area,were similar in all the groups.

The mucus layer in the small intestine is secreted by goblet cells,which permits the excretion of gastric contents and form a protective barrier against mechanical and chemical injuries(ingested food,microorganisms,pathogens)[25,26].The percentage of goblet cells in the duodenum is the lowest and increases towards the large intestine throughout the rearing period.This is because a greater number of microbial organisms is present in the proximal colon[27].This was also confirmed by our findings.On the 1stday after hatching-in groups injected with synbioticsa significantly lower number of neutral goblet cells on the surface of 1 mm2of duodenum villi was found.At the end of rearing,in the SYN1 group,there were significantly more neutral goblet cells in the jejunum compared to the other groups.In our previous studies[12],the significant effect of the symbiotic(galactooligosaccharides+L.lactis subsp.cremoris 477)on an increase in the number of goblet cells in the same segment of the intestine was also shown.However,only a several-fold increase of goblet cells may indicate an infection of the intestinal pathogens[28].According to Langhout et al.[29]and Sharma et al.[30],the effect for increasing the number of neutral goblet cells both in the jejunum and in the ileum may be due to delayed feed intake by the animals.This results in a reduction in the surface of intestinal absorption and an increase in the number of these cells,which was also confirmed in our results.

In the jejunum on the 1stday of life,similar to the duodenum,in the SYN1 group,significantly higher villi than in the Control group,with a simultaneous decrease in the depth of crypts(P≤0.01),and also the largest width of villi and their absorbent area(P≤0.01)in comparison to the other groups were found(Table 6).On the 42ndday of life,in the jejunum,an increase of the villi height whilst reducing the crypt depth in the SYN2 group was found(P≤0.01).In turn,in the SYN1 group,there weresignificantly more neutral goblet cells observed compared with the control group(P≤0.05),(Table 6).

饮食的基本功能是提供营养和健康，其次才是滋味。倘若兼而有之，那是再理想不过的。现在经济条件好了，物流很发达，想吃点东西并不难，但注意养成科学合理的饮食观和饮食习惯很重要。一味地贪图美味、口福和快感，会使“啤酒肚”、痛风、肥胖症和“三高”人群有增无减。此外因不断追求美食，上天入地求之遍，同保护生态环境也存在着矛盾，有一句话说的好，没有买卖，就没有杀戮。

However,the positive effect of injected synbiotics was mainly indicated in the duodenum and jejunum.

Conclusions/implications

Summarising our research focused on the evaluation of the impact of synbiotics given in ovo on the production　traits　and　histomorphology　ofa　broiler chicken small intestine,we observed that synbiotic L.salivarius IBB3154+Bi2tos,Clasado Ltd.and synbiotic L.plantarum IBB3036+lupin RFOs beneficially affected the examined characteristics on the 1stand 42ndday of life.The obtained results allow us to conclude that the use of synbiotics significantly affect gut structure which should contribute to improvement in nutrient absorption by the gut.

Histomorphological examination

高效准确的识别叶芽和花芽，才能够准确的运用各种修剪技术，提高冬剪的效率和质量，使树体合理负载，节约养分，有效克服树体大小年现象。

医景网总部设在美国纽约,是世界最大的医学门户网WebMD.com的组成部分(美国有4成医生都是该网站会员，2016年月度独立访客访问达到7千万人，2017年7月其母公司被美国KKR基金以28亿美元收购)。它免费为临床医生和普通大众提供医疗信息，同时它的任务也包括对医生和健康专业人士的继续教育。其主要内容来源包括医学杂志的专业文章，医学继续教育信息，国家图书馆的医学数据库，医学新闻和药物信息。

Funding

This project was funded from the European Union’s Seventh Framework Programme for research,technological development and demonstration under grant agreement n°311,794.It was co-financed from funds for science of the Polish Ministry of Science and Education allocated to an international project ECO FCE in the years 2013-2017.

Availability of data and materials

Authors do not wish to share the data due to the propriety nature of the data.

Authors’contributions

AS,JB,AD data and results interpretation,performed the statistical analysis,histological analysis,drafted the manuscript.JB histological study coordination,aided in data interpretation.GEW data and results interpretation.KS participated in study design and coordination.MB designed research.All authors read and approved the final manuscript.

Ethics approval

The use of animals reviewed and approved by the local ethics committee for the animal experiments in Bydgoszcz.

肝硬化是肝脏发生的不可逆的严重病理改变，可出现多种并发症，如上消化道出血、肝性脑病、腹水、肝肾综合征等[1] ，后果不良，生活质量也受到严重的影响。针对肝硬化患者，应给予积极的综合治疗，以控制患者的病情、改善患者的预后，在治疗时配合护理措施干预，可有效提高患者的生活质量。现将笔者针对肝硬化患者实施的优质护理体会报告如下。

[例8]In face her attitude towards this work was the same as someone with a passion for sweet-eating,indulged,indulged in solitude,or some other private pastime,like acting out scenes with an invisible alter ego,or carrying on conversation with one’s image in thelooking-glass.（1972：167）

Consent for publication

Not applicable.

细节分析有很多教学技巧。比如提炼具体信息，从上下文来推断句子的含义，识别论述的模式。对于一个特定的课文，我们应强调句子关系的学习，段落结构的技巧，而不是集中在语义的学习上，千万不可一句一句地释义。只有通过学习某一文章的梗概，才能帮助学生形成逻辑思维，这是理解课文必不可少的。

Competing interests

The authors declare that they have no competing interests.

Author details

证明 对于G=PQ,考虑Q在P上的作用,则有P=CP(Q)×[P,Q](见文献[10]中定理8.2.7).若CP(Q)＞1,则[P,Q]＜P,从而G交换,矛盾.故CP(Q)=1,P是初等交换p群,取P中的p阶子群的生成元代表A={x1,x2,···,xt},其中t=又循环群Q非正规,由Sylow第三定理可知其Sylow q-子群的个数至少∪为q+1,取出这q+1个循环群的生成元代表B={y1,y2,···,ys},其中s=q+1,显然AB即构成了势为q+1的点独立集,不等式显然成立.下面考虑等号成立条件.

1Department of Animal Biochemistry and Biotechnology,University of Science and Technology in Bydgoszcz,Mazowiecka 28,85-084 Bydgoszcz,Poland.2Department of Normal Anatomy,Ludwik Rydygier Collegium Medicum in Bydgoszcz,Nicolaus Copernicus University in Toruń,Karłowicza 24,85-092 Bydgoszcz,Poland.

华南农大教授张承林教授认为，氯化钾价格更低、养分浓度更高、溶解速度更快，在灌溉水硬度大的地区不易沉淀、堵塞喷头，价格又比水溶性好的硝酸钾及磷酸二氢钾低，要大力发展水溶肥料“氯化钾”才是首选原料。水溶性结晶硫酸钾由于其中既有硫又有钾，被称之为“黄金搭档”。随着水溶肥发展，不管资源型还是加工型硫酸钾企业都在跃跃欲试。而碳酸钾也是水溶肥的优质原料，不仅可以提供植物必须的钾元素养分，还可以通过反应释放二氧化碳，增加土壤或大棚里的二氧化碳释放量。此外，硝酸钾在南方经济作物上的表现获得认同，弥补了价格“短板”。磷酸二氢钾0.1%以下的水溶性，无论经济作物还是大田作物，均有增产增收的明显效果。

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