The differences of intestinal development and microorganism between male and female ducks

Background There are great differences in physiological and biological functions between animals of different genders. However, whether there would be a consensus in duck intestinal development and microorganisms is still unknown. Methods Male and female ducks were weighed at 2, 5 and 10 weeks, respectively, then the duodenum, jejunum, ileum and cecum were sampled and measured, and the content were analyzed by 16S RNA. Results The results showed that male duck hold shorter intestinal length with higher density and jejunum VH/CD, and the similarity, evenness and diversity of the total intestinal microorganism of male duck were higher, with 99.66% OTUs shared by both gender, while Rikenellae , Prevotella and Nocardia were only in male duck. Proeobacteria hold higher abundance than Bacteroidetes in female intestine, while the value of Firmicutes/Bacteroides was lower than male duck. Whats more, the different functional pathway of microorganisms was only showed in jejunum, among which the metabolic and disease pathways of male ducks were higher, while gene pathways was lower. Conclusions The intestines of male ducks have higher development, nutrient absorption, fat deposition and metabolic capacity while their disease resistance was relatively weak, providing a basic reference for intestinal development and microorganism symbiosis of duck in different genders. LEfSe analysis was carried out on the samples at different microbial classication levels with LDA score>4 to determine which microorganisms caused above differences in the intestinal microora. The results showed that there were different microorganisms in the other three intestine segments except ileum between male and female ducks. The differences of Firmicutes at phylum level, Clostridia at Class level, Clostridiales at order level and Lachnospiraceae and Ruminocaceae at family level exited in duodenum, and all the abundance of the them was higher in the samples of male ducks. Only the abundance of Rothis at genus level in the female ducks jejunum was higher than that of male ducks, while 22 other microorganisms were lower at different classication levels, such as Pseudomonas, Sphingomonas and Desulfovibrio at genus level. The male ducks had higher Proteobacteria abundance at phylum level than the female in the cecum (gure 5). Further analysis showed that the differential pathways were only distributed the


Introduction
Intestinal tract is an important part of the body defense system and the main place for animal to digest and absorb the nutrients of feed [1,2]. The functions of poultry intestines are mainly carried out in the small intestine, which is also the longest part of the digestive tract [3]. The nutrients in small intesnal content can be easily absorbed due to the tight blood capillaries with thin vascular wall in villi [4], and lymphatic capillaries, unstriated bers and neural net also play a certain role through accelerating blood ow by villi ex and swing. The cells at the top of the villi are differentiating from the crypt basal epithelial, and the premature mice with poor weight hold lower villus height by comparing with the premature mice with normal weight, [5]. The height of intestinal villi and the depth of crypt can re ect the body's ability to absorb nutrients and the maturation rate of crypt cells, respectively, and the value of villi height/ crypt depth (VH/CD) comprehensively re ect the nutrient absorption capacity of small intestine.
Intestinal microorganism is mainly distributed in the chyme owing in the intestinal cavity and adheres to the intestinal and related mucosa. There are lots of species and microbial communities in the intestinal tract, which are rich in genes involved in carbohydrate, microbial, amino acid and short chain fatty acids (SCFAs) metabolism. The dynamic balance of interdependence and mutual restriction between host and microorganism exit in a healthy intestinal state, and most of the microorganisms exchange energy, transmit information and symbiosis with each other, while diseases may occur when the population convert to maladjustment [6]. About 35% of the enzymes of microorganisms can be used by the host, which plays an important role in nutrient metabolism regulation, intestinal mucosal immune activation, host behavior regulation, intestinal epithelial cell repair and resistance to pathogenic microorganisms [7,8]. More than 1 × 10 6 microbial genes have been found planted in poultry gastrointestinal tract, which is 40 ~ 50 times of the whole chicken genome, while 90 ~ 95% of cecal microorganisms can not be cultured in the laboratory environment, and these microorganisms play an essential role in the absorption of diet nutrients and the immune metabolism [9]. Poultry intestinal microorganism is related to the structure of diet, age, gender and individual speci c conditions, and the composition of microbial ora in different intestinal segments also varies from others [10,11].
There are great differences in hormone secretion, energy metabolism and immune response among animals of different genders [12], including intestinal health [13,14]. It has been found that the diversity of intestinal microorganisms in female Octopus was signi cantly lower than that in males [15], and the relative abundance of anaerobes decreased with the facultative anaerobes increasing in female LTsclKO mice during the development of HCC, while Paraprevotella, Parapervotella and Prevotella increased in males [16]. Besides, 30 species of intestinal microorganisms affected by host gender signi cantly has been found in high body weight line families of chickens (P < 0.05), such as Lactobacillius, Lactococcus and Actinomyces, and 17 found in low body weight line families (P < 0.05), such as Lactobacillius, Acinetobacter and Brachycharacter [17]. Although many studies have addressed the effects of endogenous and exogenous factors on the intestinal development and microbiota compositio, only limited data are available on the intestinal development and microbiota in animal models of different genders. The study on the development and microorganisms of intestine of male and female ducks is helpful to provide basic reference for the research on the intestinal growth and microorganism symbiosis and co metabolism rules of duck in different genders, and has certain theoretical and practical signi cance for the in-depth study on the characteristics of ducks.

Laboratory Animals
The Nonghua sheldrake used in this experiment was provided by the poultry raising experimental farm of Sichuan Agricultural University. 30 healthy male ducklings and 30 healthy female ducklings were managed in a uni ed way to ensure each duck intake and drink similarly at liberty and under uni ed management during the experiment. 10 ducks of each gender were randomly selected at 2, 5 and 10 weeks, killed by cervical dislocation after fasting for 12h, then sample the intestine and its content, and subsequently treated as non-hazardous waste. All animals handling procedures were approved by Sichuan Agricultural University Animal Welfare Committee.

Measurement of Intestinal Growth
Take out the duodenum, jejunum, ileum and cecum of each duck successively , x one end of them on the glass plate moistened with distilled water after removing the pancreas, fat and other attachments attached to the intestinal tract, then straighten it slightly and measure the length with a ruler when the intestinal tract recovers to no longer retract. The lter paper was used to dry the intestinal segements after the intestinal contents were squeezed out, and the intestines were weighed by electronic balance.
The middle parts of duodenum, jejunum and ileum were cut 1 cm respectively, washed with normal saline, and xed in 4% polyformaldehyde solution, then embedded in para n, sectioned and stained with HE. The microscopic images were taken at 50x and 100x. Three replicates were collected for each sample, and three different visual elds with complete and straight direction villus were collected for each replica. Villus height (VH), crypt depth (CD) and intestinal wall thickness (IWT) of duodenum, jejunum and ileum were measured and recorded by Image-Pro Plus 6.0 software, and the ratio of VH to CD was calculated. The vertical distance from the top of villus to the opening of the crypt is VH(mm), from the bottom of the crypt to the transition area between the crypt and villus is CD(mm), and the lineal length of the longest and widest intestinal wall is IWT(mm).
Place the obtained contents of intestines in the marked EP tubes and put them into liquid nitrogen immediately. The 16S rRNA gene library was constructed by extracting total microbial DNA from intestinal contents and amplifyed 16S rRNA sequences of all microorganisms. PCR products were puri ed using Agencourt Ampure XP beads(Beckman, USA) accounting to the instructions of manufacturer nd quanti ed using the PicoGreen dsDNA Assay kit (Invitogen, USA), equally combined and followed by gel puri cation using a QIAquick Gel Extraction Kit (Qiagen, USA), and then requanti ed by PicoGreen. The prepared DNA library was then sequenced using the MiSeq platform (Illumina, USA). An OTU table was generated using the Uparse clustering method (97 % cutoff), and all samples were rare ed to the same sequencing depth by resampling OTUs prior to downstream analysis.

Data Statistic Analysis
Mothur software and QIIME software were used to evaluate the Alpha diversity and Beta diversity of the samples, and the gures was drawn by R language tool. SPSS 21.0 software (IBM, USA)was used to analyze the data and T-test was used to analyze the signi cance of the sample data. The data were expressed in the form of Mean ± S.D. . Statistically, P <0.05 represents signi cant difference and P <0.01 is extremely signi cant difference.

Results
The effect of gender on intestinal development There was no signi cant difference in RL, RW and ID between the male and female ducks at 2 weeks (P>0.05) by comparing the development of intestine between the two genders. The RL of jejunum and ileum in female ducks was signi cantly higher than that in male ducks at 5 weeks (P<0.05); nevertheless, the ID of duodenum and jejunum in female ducks were great signi cantly (P<0.01) and signi cantly lower (P<0.05) than that in male ducks, respectively. Both duodenal RW and jejunal ID of the male ducks hold signi cantly higher value than that of the female duck at 10 weeks (P<0.05) ( Figure 1).
There was no signi cant difference in the intestinal morphological structure of duodenum, jejunum and ileum between male and female ducks in appearance during the whole experiment ( Figure 2). Then the result was further studied by relevant data and it showed that the VH, VH/CD of the jejunum and the IWT of the ileum in females were signi cantly higher (P<0.01) at 2 weeks. Both VH and CD of both duodenum and jejunum of female ducks were signi cantly higher than that in males, while the IWT of the jejunum was signi cantly lower (P<0.05) at 5 weeks. The VH of duodenum and jejunum in male ducks was great signi cantly higher than that in female ducks (P<0.01) at 10 weeks, while the CD of ileum was signi cantly lower (P<0.05), and only the VH/CD of jejunum was signi cantly higher than females (P<0.01) (Figure 3).

The effect of gender on intestinal microorganisms
A total of 6 096 426 pairs of reads were obtained by sequencing the contents of the intestine from 60 ducks, and 5 101 854 clean tags were generated after splicing and ltering whith at least 22 390 clean tags produced by each sample and 4873 clean tags on average.
The number of intestinal OTUs of male and female ducks obtained by clustering was used as the vertical coordinate to draw the multy samples rarefaction curves, and each curve trend tended to be at with the increase of the number of randomly selected sequencing pieces, suggesting the sequencing quantity was enough to re ect the species diversity. The highest species richness of intestinal micro ora in both sexes was appeared in 10 weeks with the lowest in 2 weeks, and the intestinal micro ora richness of male ducks was higher than that of females except 2 weeks. In addition, the abscissas of the end point of the multy samples rarefaction curves of the female ducks in the three time periods were all larger than males ( Figure 4A). However, only the microbial abundance in the duodenum of male ducks was signi cantly higher than females at 10 weeks by comparing the Chao1 index ( Table 1). The distribution of female duck was obviously separated both at the rst and second principal component according to the PAC analysis, while the distribution of male duck is more closely clustered at the second principal component ( Figure 4B). The ** above the two bars represents the great significant difference between the two samples (P<0.01), * represents the significant difference between the two samples (P<0.05). The picture below is the same.
889 OTUs were identi ed from intestinal contents of two genders of ducks, of which 99.66% were both in male and female, while the Rikenellaceae Prevotella and Nocardia were unique to male ducks ( Figure  4C).The ominant intestinal micro ora of both genders were Firmicutes, Proeobacteria, Bacteroidetes and Actinobacteria based on the analysis at phylum level. However, the abundance of Bacteroidetes (17.2%) male duck was higher than that of Proeobacteria (14.4%), while that Proeobacteria (25.3%) of female duck was higher than that of Bacteroidetes (15.4%) ( Figure 4D). The ratio of Firmicutes to Bacteroidetes was calculated and it turned to be out that the value is signi cantly higher in the male than that in the female at 10 weeks as well as the weight of the male was signi cantly higher at the same time (P<0.05) ( Table 2). LEfSe analysis was carried out on the samples at different microbial classi cation levels with LDA score>4 to determine which microorganisms caused above differences in the intestinal micro ora. The results showed that there were different microorganisms in the other three intestine segments except ileum between male and female ducks. The differences of Firmicutes at phylum level, Clostridia at Class level, Clostridiales at order level and Lachnospiraceae and Ruminocaceae at family level exited in duodenum, and all the abundance of the them was higher in the samples of male ducks. Only the abundance of Rothis at genus level in the female ducks jejunum was higher than that of male ducks, while 22 other microorganisms were lower at different classi cation levels, such as Pseudomonas, Sphingomonas and Desulfovibrio at genus level. The male ducks had higher Proteobacteria abundance at phylum level than the female in the cecum ( gure 5). Further analysis showed that the differential pathways were only distributed in the jejunum. The viral infectious diseases, lipid metabolism, metabolism of terpenoids and polyketides, parasitic infectious diseases, xenobiotics biodegegradation and metabolism, cardiovascular disease and metabolism of other amino acids of the male duck was higher than that of female duck, while the abundance of gene folding, sorting and degradation pathway and nucleotide metabolism of female was higher than that of male ( gure 5).

Discussion
Intestinal development directly affects the digestion, absorption and metabolism of nutrients, which is very important for health and growth potential. The increase peak of small intestine length of broilers is earlier than their daily intake and daily gain, and the relative weight can reach the peak only after the relative growth speed of digestive organs reaches the peak, suggesting that the quality and index of intestine can re ect the biological and ecological conditions and functions of animals in the growth and development stage [18]. Intestinal transit tends to be slower in female for the steroidal hormones would promote intestinal hypomotility and inhibit gastric emptying by acting as a smooth muscle relaxant [19], and progesterone also could decrease gastrointestinal motility through an inhibitory effect on motilin [20]. However, a completely different angle is provided in this experiment rather than hormone. Previous studies have shown that postprandial changes in intestinal volumes are higher in males than females by using a single photon emission computed tomography [21], and the volume of post-mortem uid in the intestine were higher in men than in women after standardization by body weight [22], indicating that different gender had certain in uence on the capacity value of intestine. According to the results of this experiment, greater RL of the female duck lead the longer intestinal emptying time, while the greater ID of the male duck contribute the more effective peristalsis, suggesting that the digestion cycle of male duck is shorter than females.
The digestion and absorption ability of the intestine to nutrition depends on the joint action of pancreas, enzyme activity, surface area and nutrient transport carrier [23,24], and the surface area of intestinal villi is the key factor limiting the growth of poultry [25,26]. Another structure of the mucosal layer, crypt, transports nutrient molecules from the digestive tract to the blood for use, and the value of CD can re ect the renewal speed of the small intestine epithelium [27]. Therefore, the higher CD value of the female ducks meaned faster update of epithelial cells in intestine. VH is adjusted correspondingly with the change of animal function and intestinal nutrient demand under normal circumstances, which is realized by the increment rate and intestinal cell turnover rate of intestinal crypt cell [28]. The intestinal epithelium is a complex multicellular system, closely related to the growth potential. Sexbased differences of infection and in ammation were found in the intestine of 2-week-old C57BL/6 mice [14]. In this study, the digestive capacity of the male duck's jejunum was lower than females in the early stage due to VH/CD, and then higher than females in the later stage with the development of VH, which is similar with intestinal microbial diversity, suggesting there is consistency between intestinal development and intestinal microorganisms in ducks.
The balance and stability of intestinal microorganisms play an important role in maintaining the health of the body. Gender-related differences in intestinal microbial communities have been observed in many animals, such as mice, macaques and humans [29][30][31]. Samples from same gender wer found clustered together by Principal Component Analysis of T-RFLP and DGGE pro les from either human ora associated rats or speci c pathogen free rats [32]. Androgens could impact intesyinal microbial composition in lupus-susceptible mice and protect males against the development of lupus [33]. Besides, males have higher serum SCFAs level come from the microbial fermentation by comparing with females, such as butyrate, propionate and acetate,which have a myriad of anti-in ammatory effects [34,35]. Likely causes not only includ differing sex hormone levels in males and females but also in uenced by the intestinal microbiota.
The evenness and diversity of the intestinal micro ora of the female duck were higher by comparing with the male duck, and the distribution of the intestinal micro ora were signi cantly separated. Proteobacteria is the largest family of bacteria, including many pathogens and free-living species, such as E.coli, Salmonella and many bacteria that can carry out nitrogen xation, covered with lipopolysaccharide which can cause strong pro-in ammatory effect and secrete pro-in ammatory factors including interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) [34], and γProteobacteria increase signi cantly in intestinal in NAFLD and NASH patients [36]. Bacteroides was initially known for its pathogenicity, and then found it was a large number of normal bacteria in the body, which played an important role in the host's nutrition absorption, fat accumulation, gastrointestinal ora balance and body immunity. The difference of the abundance of Proeobacteria and Bacteroides between the male and female ducks suggested stronger metabolism ability weaker anti-in ammatory ability in male ducks. It has been found that the abundance ratio of Firmicutes to Bacteroides in intestinal microorganisms can re ect the degree of obesity to a certain extent [37,38], and the relative proportion of Bacteroides in obese and thin people is low [39], and the decrease of Bacteroides was accompanied by the increase of Firmicutes [40]. In this experiment, the difference of the intestinal Firmicutes/Bacteroides and body wight between the male and the female ducks appeared at 10 weeks, suggesting that the fat deposition potential of male ducks was stronger females.
Microorganisms in the intestine affect the health and growth performance of the host through participating in different functional pathways [41]. These was no signi cant difference in functional pathways although there were signi cant differences between male and female ducks at 2, 5 and 10 weeks. Only jejunum showed signi cant differences of function pathways between the two sexes, which was consistent with the value of VH/CD. Lipid metabolism is an important and complex biochemical reaction [42,43], processed into substances needed by the body through digestion, absorption, synthesis and decomposition with the help of various enzymes and bile salts to ensure the normal operation of physiological functions [44,45]. It has been found that the upper part of small intestine was the mainly place where fat digested and hydrolyzed to glycerol and fatty acids [46]. The higher abundance of lipid metabolism pathway in male duck's jejunum in this experiment may be related to the higher nutritional absorption capacity of male ducks. Neverthless, The disease pathway of the male duck is signi cantly higher by comparing with the female duck, which noteworthy in today's intensive aquaculture and it is necessary to strengthen the vaccine requirements and prevention and control measures for the male duck. Different from the male duck, the females have higher abundance of gene aspect. Nucleotide is the precursor of synthetic biological macromolecular RNA and DNA, which plays a dominant role in the growth, development, reproduction and heredity of organisms [47]. Congenital whey aciduria would caused by the damage of pyrimidine nucleotide metabolism, and ventilation, Lesch Nyhan syndrome and immune de ciency by the damage of purinine nucleotide metabolism [48,49]. It has been reported that the male to female ratio of gout patients is 20:1 and the incidence of obesity is higher in male, which is consistent with the high abundance of nucleotide metabolic pathway and the weak ability of fat deposition in this experiment.

Conclusion
The differences in duck intestinal development and microorganisms between different genders were found in this study. Compared with female ducks, males had shorter digestion cycle because of shorter intestines and stronger peristalsis,which allowed them to take in more energy in the same time. The intestinal digestive capacity and microbial diversity of female ducks were higher in the early stage, while lower than that of male ducks in the later stage. The difference of intestinal morphology structure mainly occured in jejunum, which was also the only intestinal segment with different microbial function pathways. The abundance of intestinal metabolism and disease pathways in male ducks is higher, while the abundance of gene pathway is lower than females. This study showed that the male ducks hold stronger metabolism ability weaker disease resistant under the common growing environment, indicating it is necessary to strengthen the fattening of female ducks and the immunity of male ducks in the actual rearing process, providing a basic reference for intestinal development and microorganism symbiosis of duck in different genders.

Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
Not applicable.  Figure 1 the development of the intestine in the male and female duck. RL, RW and ID in the gure represent the relative length, relative weight and intestinal density of intestine, respectively. 2W, 5W and 10W represent three time periods. The ** above the two bars represents the great signi cant difference between the two samples (P<0.01), * represents the signi cant difference between the two samples (P<0.05). The picture below is the same Figure 2 Structure and morphology of small intestine after HE staining. D, J and I in the rst vertical row on the left represent duodenum, jejunum and ileum, respectively. The second vertical M and F represent male duck and female duck, respectively.

Figure 3
Morphological structure of small intestine in male and female ducks. VH, CD and IWT in the gure respectively represent the villi height, crypt depth and the intestinal wall thickness.