ACCEPTED AUTHOR VERSION OF THE MANUSCRIPT: The effect of different medium chain fatty acids, calcium butyrate, and salinomycin on performance, nutrient utilization and gastrointestinal tract of chicken of Polish Green Legged Partridge hen

The present study aimed to determine the effect of diets supplemented by feed additives, on the growth performance and digestive tract functioning in chicken of green-legged partridge hen (GLPH). Two types of diets were used. The both “low-digestible” (LD) diet characterized by high level of crude fibre and lard, and “high-digestible” (HD) diet characterized by low content of crude fibre and soybean oil were supplemented by different additives such as 0,34% DM (dry matter) salinomycin, 0,30 % DM medium-chain triglycerides (MCT), 1,00 % DM calcium butyrate (CB), or 0,85 % DM medium-chain fatty acids (MCFA). The experiment was conducted on 960 one-day-old male chickens of GLPH, randomly divided into 10 treatment groups. One group consisted of 12 replications (each with 8 birds). Chickens fed HD diet presented significantly higher body weight gain (BWG) in 15-56 days and also 0-56 days of experiment and lower feed conversion ratio (FCR) during whole time (0-56 days) of experiment ( P < 0.05). Addition of CB and MCFA did not affect the differences in BWG (15-56; 0-56 days) and FCR (15-56 days) in comparison with the control diet. Significant interaction between the type of diet and additives ( P < 0.05) was observed for feed intake (FI) throughout the experiment. The highest FI was found on LD diet with CB. On LD diet, all additives resulted in significantly worse nitrogen retention, however, on HD diet, it was observed only for salinomycin. The additives had no impact on fat digestibility when chickens were fed HD diet. There was significant ( P < 0.05) lower-fat digestibility when LD diet with MCT, CB, and MCFA was used. The greatest value of nitrogen-corrected apparent metabolizable energy (AME N ) was found on HD diet ( P < 0.05). The addition of MCT, CB, and MCFA increased the percentage share of ileum, and MCT also significantly increased the percentage share of the liver. The lactic acid bacteria and C. perfringens counts were lower on HD diet (P<0,05). The addition of MCT and CB increased the count of lactic acid bacteria in ceaca (P<0,05). Many interactions between experimental factors in this experiment showed that the efficacy of using different feed additives depends on the type of diet. The results suggest that MCFA may be the most beneficial feed additive for GLPH chicken as an alternative for antibiotic growth promoters (AGP).

years there has been a decrease in the number of sick people, the situation still has to be controlled (Koutsoumanis et al. 2019). Many countries have their own monitoring systems for protecting poultry production against different serotypes of Salmonella (Kuczkowski et al. 2015). Until 2006, antibiotic growth promoters were popularly used for protection against many pathogens in poultry nutrition. However, the use of AGP is currently forbidden in Europe. The growth parameters when AGP were used, were improved by limiting the development of pathogenic microbiota in the gastrointestinal tract of birds and reduced the use of nutrients by pathogens. Using AGP limited the development of pathogenic microflora which led to improvement effectiveness of gastrointestinal tract processes, which used the positive bacterias (Brennan et al. 2003;Knarreborg et al. 2004).
Salinomycin is an ionospheric antibiotic, which exhibits anticoccidial properties. After its addition to the feed, salinomycin enters the protozoa cells, leading to high ionophore concentration inside the cells and death of the cells (Danforth et al. 1977;Bolder et al. 1999).
In general, coccidiostats are used popularly in poultry nutrition for protecting birds against Eimeria species, which cause gastrointestinal diseases. Through the control of coccidia with salinomycin and others, the poultry production is made safer, which may prevent the risk related to Eimeria and improve the economical aspect (Engberg et al. 2000;Johansen et al. 2007).
Either way, the use of coccidiostats creates some controversy among consumers, and their residues in products can affect animal or human health. However, the using of salinomycin is allow in chicken nutrition (decision of EK 2017(decision of EK /1914. Butyric acid and medium-chain fatty acids (MCFA) may have an effect on growth performance, body weight, and feed intake (FI) and can restrict the development of deleterious bacteria in the gastrointestinal tract by reducing the pH value (Evans et al. 2000;Namkung et al. 2011;Lipiński et al. 2016; Van der Aar et al. 2016). Alternative additives generally exhibit a similar mechanism of action like that of AGP with effects that could improve the growth performance and promote proper functioning of the gastrointestinal tract (Huyghebaert et al. 2011). In addition, inclusion of MCFA into the drinking water or feed yields positive results in limiting the development of Campylobacter jejuni (Solis de Los Santos et al. 2008;Hermans et al. 2010). On the other hand, other data suggest that both MCFA and butyric acid did not significantly improve chicken production (Hejdysz et al. 2012;Ocejo et al. 2017). The use of feed additives such as AGP, coccidiostats, or others creates much controversy among consumers. Most often, many consumers believe that the products from organic farms are of much higher quality. Eco-trend has gained significant attention in the past years (Grashorn and Serini. 2006). Our previous experiments were focused on fast-growing chickens that were maintained and fed conventionally (Hejdysz et al. 2018). However, the Polish breed greenlegged partridge hen (GLPH) can be successfully used in organic farms and be a source of meat and eggs free from many food additives, which have a negative effect from the consumer point of view (Grashorn and Serini. 2006). Organic acids are not forbidden in organic poultry production and could make it much safer from a microbiological aspect with regard to human health. Previously presented papers did not show sufficient clear results as to whether MCFA or butyrate may improve the growth parameters and can be treated as good alternatives for AGP or coccidiostats (Hejdysz et al. 2018). Therefore, as a continuation, the present study aimed to determine the effect of diets with different crude fiber contents and fat sources (low digestible (LD) and high digestible (HD)), which were supplemented by salinomycin, MCFA, mediumchain triglycerides (MCT), or calcium butyrate (CB), on the growth performance and digestive tract functioning in chicken of GLPH.

Material and methods
All the research methods and procedures were conducted according to the relevant European Union (EU) directives, and the study protocol was approved by the Local Ethics and Animal Experimentation Committee at Poznan University of Life Science. All the requirements concerning animal welfare were followed.

Animals and feeding program
The experiment was conducted on 960 one-day-old male chickens of GLPH, randomly divided into 10 treatments (average weight 412 g). Each group consisted of 12 replications (each with 8 birds). The experiment was performed in totally controlled environmental conditions: the birds were reared on wood shavings litter in pens of dimensions 1.2 × 0.8 m; the room temperature was gradually reduced from 30°C to 21-22°C; the lighting program comprised 24 h of light until 10 days, followed by 19 h of light and 5 h of darkness. Both LD and HD diets were supplemented with salinomycin-0,34%; triglyceride form of capric and caprylic acid (MCT; 1.38:1)-0.3%; CB-1.0%; and mixture of caproic, caprylic, and capric acids (MCFA; 1:1:1)-0.85%. Salinomycin was purchased from Huvepharma NV, while the other additives from Brenntag Polska Sp. z o.o. The chickens were fed isoprotein and isoenergetic experimental diets: in the period of 1-14 days with starter; in days 15-56-with grower mixtures based on wheat, rye, barley, rapeseed meal, and fish meal-LD (Table 1) or diet based on corn, wheat, and soybean meal-HD (Table 2). Each kind of diet was composed of five treatments. The linear optimization based on own previous dates was used to calculate the composition of feed mixtures. Chickens were fed mixtures in mashed form, ad libitum throughout the experiment, and coccidiostats were not added because all the additives used exhibit some anticoccidial properties. Nutrients met or exceeded the breeder recommendations for slow-growing chickens (Smulikowska and Rutkowski. 2018). Nonabsorbable marker such as titanium dioxide was added to all mixtures (3 g/kg) and used to calculate nitrogen retention (NR), crude fat digestibility coefficient, and nitrogen-corrected apparent metabolizable energy (AMEN) value in the excreta.

Data collection
Excreta was collected in four days at the end of the experiment, twice a day in each cage on floor covered with plastic foil ). Subsequently, excreta (pooled per cage and n = 10 for each group) were homogenized and frozen to be prepared for chemical analysis. In total, 36 birds were selected randomly (3 birds per replication) at the end of the experiment (56 days) and slaughtered by cervical dislocation. Then, the organs and digestive tract were removed at once. A part of fresh contents of the crop, ileum, and ceaca from 21 chickens per treatment was used for microbiological assays.

Analytical methods
Before chemical analysis, excreta samples were subjected to lyophilization (Christ 1825 Medizinische Apparatebau 326 Osterode/Harz, Germany) and grinding. In the starter and grower diets as well in the excreta samples, the parameters were determined in accordance with the AOAC standards (2007) Czech Republic) was used, and ninhydrin was used for 10 postcolumn derivatizations. All the samples were hydrolyzed with 6 N HCl for 24 h at 110C in advance (procedure 994.12;AOAC 2005). Methionine was determined as methionine sulfone, and cystine was determined as cysteic acid with procedure 994.12, alternative 3; 15 (AOAC 2005) after cold performic acid oxidation before hydrolysis. Calcium was analyzed according to the procedure of the AOAC (2007), and phytate was analyzed in the mixtures according to the method of Haug and Lantzsch (1983). Nonphytate-P was calculated as total P minus phytate. The level of crude fiber in the mixtures was analyzed using PN-EN ISO 6865. In both the mixtures and the excreta, the level of titanium dioxide was determined using the method of Short et al. (1996), and the samples were prepared according to the method presented by Myers et al. (2004). A calorimeter bomb standardized with benzoic acid (KL 12Mn, Precyzja-Bit PPHU, Poland) was used to evaluate the gross energy in the mixtures and samples.

Microbial status of gastrointestinal tract
For bacterial enumeration, subsamples obtained from the ileum (lactic acid bacteria and C. perfringens) and ceaca (lactic acid bacteria) of 2.5 g were diluted in 30 mL of sterile prereduced salt medium, and the suspensions were homogenized for 2 min in CO2-flushed plastic bags using a stomacher homogenizer (Interscience, France). Subsequently, the samples were serially diluted in 10-fold steps using prereduced salt medium (Miller et al. (1974)). Lactic

Calculation and statistical analysis
Total tract digestibility of crude fat (CF), nitrogen retention (NR) and AME N values were determined using the following formula: where TTD = total tract digestibility, EX = excreta, and CF = crude fat.
where GE = gross energy, N = nitrogen, and 34.4 = the energy equivalent of uric acid nitrogen (Hill and Anderson 1958).
The results were statistically analyzed using the general linear model of Statsoft (2011), according to the following general model:

Yij = μ + αi + βj + (αβ)ij + δij
where Yij was the observed variable; μ was the overall mean; αi was the effect of diet; βj was the effect of supplement; (αβ)ij was the interaction between diet and supplements; and δij was the random error.
In cases where the overall effect was significant (P≤0.05, P≤0.01), means were compared pairwise (pdiff). Results are expressed as the least squares means with pooled standard error of the mean (SEM). Analysis of variance (ANOVA) two-way and Duncan's test were used to identify significant statistical differences and compare means.

Growth performance
The mortality was low (<1%) throughout the experiment (group 1, 2, 5, 8, 9 -1,04% in each, group 6 -2,08%). Table 3 shows the effects of using different diets and different additives on performance results in chicken of GLPH. The interaction between diet and additives was observed for body weight gain (BWG) in the first period of the experiment (0-14 days). In the first period, additives in LD diets had no significant impact on BWG, but CB added to HD diet significantly reduced the BWG in compared with control diet and other additives. MCT also significantly reduced BWG when compared to salinomycin in HD diets. However, no interaction for BWG was observed in the next period of the experiment (15-56 days) and in general (0-56 days). The kind of diet (HD or LD) used had a significant effect on BWG (P < 0.05); HD diet led to greater BWG in 15-56 days and 0-56 days of the experiment. The interaction was observed for FI for each part of the experiment (0-14 days; 15-56 days; 0-56 days). FI in the first period (0-14 days) of the experiment was higher only on LD diet with the addition of CB (P < 0.05). In the second part of the experiment (15-56 days), the impact of using additives was not observed on LD diet. However, addition of CB and MCFA to HD diet significantly reduced FI compared to salinomycin included diet during 15-56 days. In this period, CB addition to HD diet resulted in lower FI when compared to control diet as well (P < 0.05). There was no interaction for FCR in the experiment. FCR was significantly higher on LD diet in every period of the experiment (P < 0.05). Addition of salinomycin led to a significantly higher (P < 0.05) FCR than the other groups (except MCT) in the second period of the experiment (15-56 days). In comparison with the control diet, the other additives had no impact on FCR in the second part (15-56 days) of the experiment. The effect of used additives on FCR was not observed in periods 14 days and 0-56 days.

NR, crude fat digestibility, and AMEN
The effect of using feed additives and different diets on NR, crude fat digestibility, and AMEN is shown in Table 4. The significant interaction (P < 0.05) between the kind of diets and additives used was observed for NR and crude fat digestibility. The additives used, except salinomycin, did not have any significant effect on NR in HD diet as they reduced nitrogen retention (%) in LD diets when compared to control group (P < 0.05). There was no significant difference (P > 0.05) in crude fat digestibility on HD diet. The additives reduced crude fat digestibility on LD diet (P < 0.05); however, salinomycin diet was not significantly different from control diet. Interaction was not observed (P = 0.084) for AMEN; however, significant difference (P < 0.05) was found only for the kind of diets. The AMEN value was significantly higher (P < 0.05) on an HD diet, and the additives used had no impact. Table 5 shows the effect of treatments on the relative weights of the liver, pancreas, The number of bacteria in the ileum (lactic acid bacteria and C. perfringens) and ceaca (only lactic acid bacteria) is shown in Table 6. No interaction between the type of diet and kind of additives was observed for bacterial counts in the digesta. However, the additives used had an effect (P<0.05) on the counts of lactic acid bacteria in the ceaca only, while MCT and CB led to an increase of the bacterial counts in comparison with the control diet. Significant differences between the kind of diets were observed for the concentration of both kinds of bacteria, and LD diet led to significantly (P<0.05) higher counts of lactic acid bacteria in both the ileum and ceaca. In addition, LD diet enhanced the counts of C. perfringens in the ileum.

Gastrointestinal tract measurements
No effects of the additives used were observed for the counts of lactic acid bacteria and C.
perfringens in the ileum.

Discussion
In presented experiment conducted on green-legged partridge hen chickens fed LD diet (barley, rye, wheat, rape seed meal) or HD diet (with corn and wheat as grain components, soybean oil), also characterized by diversified crude fiber and NSP-the fact that these contents significantly reducing the intracellular pH, and thus disrupting their functioning. In addition, it limits the development of pathogens in the intestine by lowering the pH (Khan et al. 2016). In general, young chickens are exposed to many pathogens from hatcheries that may cause deaths in the first period. No impact of the use of butyric acid on BWG was observed by Czerwiński et al. (2012). In contrast, another study  showed an increase in BWG in the first period (0-14 days) due to the use of encapsulated butyric acid, which had an effect on the more accurate use of CB. BWG was worse during the whole period (0-56 days) on the MCT diet. Lai et al. (2014) suggest that main factor affects improvement in body gain is the dose of MCT. Furuse et al. (1992) showed that reduced growth of chickens could be due to the use of energy from MCT to increase the efficiency of NR, which, however, has not been confirmed in this study. In study conducted on pigs, authors have shown that protein digestibility was higher when the dose of MCT increased, however the differences in BWG were not observed (Lai et al. (2014). Importantly, diets with MCT, CB, and MCFA were associated with significantly better FCR in contrast to diet with salinomycin. It could be partially explained by the function of organic acids as a promotor for developing of gastrointestinal tract. The surface of ileum was enhanced on MCT, CB and MCFA treatments.
The HD diet with salinomycin exhibited significantly worse NR (P < 0.05), which could lead the birds to consume more feed to obtain the necessary nutrients. In addition, the salinomycin diet exhibited a slightly lower AMEN value, which forced higher FI to meet the energy needs of the birds. The growth-depressive effect after the use of salinomycin was also shown by Harms et al. (1989). On the other hand, no effect on the production parameters of broiler chickens after the use of salinomycin was reported by Engberg et al. (2000) and Kierończyk et al. (2016). The different effects of salinomycin can partly be attributed to the conditions under which the study was conducted and whether a coccidiostats was required depending on the environmental conditions. Significantly lower FI was noted on the HD diet with MCT, CB, and MCFA when compared to salinomycin diet throughout the experiment. A possible explanation is the effect of MCFA on cholecystokinin secretion, which plays a great role in feeding behavior as shown by a study conducted on rats (Douglas et al. 1990). Contrasting result was presented by Engberg et al. (2006), who showed insignificant differences in FI. In general, an interaction was observed in every part of an experiment; however, the direct impact on FI by the use of MCFA, CB, or MTC was not observed, which was consistent with the results obtained by Khatibjoo et al. (2017). Additionally, other data present the interaction (P < 0.05) between using organic acids and crude fiber level, which had no effect on FI (Sabour et al. 2018). In the second part (15-56) of the experiment and in the entire experiment (0-56), the use of LD diet resulted in significantly worse BWG (P < 0.05). In addition, LD diet resulted in higher FCR during every part of an experiment, which was related to lower AMEN value (P < 0.05); the same result was reported by Hejdysz et al. (2018) and Hetland and Svihus. (2001). Birds first meet their energy needs and then protein, which results in increased consumption if the diet is less energetic. The LD diet was composed of ingredients that contain many fractions of NSP, which change the viscosity of the nutrient content, increase the time of passage of the content through the intestines, and as a result, worsen the digestibility, which is consistent with the reports of Shakouri et al. (2006). On the contrary, no effect on BWG was observed after using a standard poultry diet as control and three diets with a different source of crude fiber (Amerah et al. 2009).
The diet with a high level of crude fiber stimulates the development of the gastrointestinal tract.
In the present study, the percentage share of the ileum was significantly greater (P < 0.05) on LD diet, which is in accordance with other results (Hetland and Svihus. 2001;Kalmendal et. al. 2011). It suggests that the development of ileum surface could increase the time when the ileal digesta are being digested and absorbed, although the components of an LD diet can be classified as difficult to digest. Kalmendal et al. (2011)  showing a significant interaction (P < 0.05). As a result of the use of salinomycin, proliferation of bacteria in the ceaca should be limited, as reported by Knarreborg et al. (2004). Previously, it has been confirmed that butyric acid has a beneficial effect on gut development, and the working mechanism of MCFA is determined to be similar. The beneficial effects of butyrate on gut development have been reported earlier (Choct 2009;Wu et al. 2016). Enterocytes can absorb butyric acid quickly and simply, which increases the concentration of goblet cells, enhances the bowel efficiency, and changes the ratio of crypts to villi (Adil et al. 2011). Feed components and dietary constituents commonly used in feed mixtures for sensitive growing chicks on a substantial degree have an effect on the development, histological structure of ileal walls, and digestive functions of the alimentary tract (Jørgensen et al. 1966;Noy and Sklan. 1995;Uni et al. 1998;Jamroz et al. 2006). The expected reduction of the number of potential pathogens (anaerobes, C. perfringens) by the bacterial action MCFA was not observed. In many publications, the beneficial effect of these fatty acids and butyrate was confirmed (Biagi et al. 2007;Guilloteau et al. 2009Dahiya et al. 2017, especially the use of encapsulated MCFA. The accumulation of fatty acids into the bacteria cell, dissociation and change of hydrogen ions, and pH value can reduce the bacterial enzymes activity and their vitality (Guilloteau et al. 2009).
MCT and CB significantly increased the number of lactic acid bacteria in the ceaca (P < 0.05).
It is well known, that addition of butyric acid can reduce the number of pathogens and increase the number of positive baterias such as lactic acid bacterias (Abdelquader et Al-Fataftah (2016); Makled et al. (2019)). Morover, Kulkarni et al. (2007) have shown that butyric acid can limit harmful bacterias gene expression. In general, organic acids limit the development of bacteria, a phenomenon which it is difficult to explain. Despite this, lactic acid bacteria are beneficial for poultry and necessary for the proper functioning of the gastrointestinal tract; furthermore, this effect is not treated like a defect (Taheri et al. 2009). All LD diets exhibit a higher content of both beneficial lactic acid bacteria and harmful bacteria such as C. perfringens. It was suspected that the addition of organic acid would limit the development of Clostridium and enhance the development of lactic acid bacteria. Previously, similar result was observed with other additives (risk husk) on poultry gut microbiota (Abazari et al. 2016). No effect of salinomycin on the counts of lactic acid bacteria was observed in this study and by Johansen et al. (2007). Enberg et al. (2000) observed decreasing of lactic acid baterias in caeca in broiler fed diet with salinomycin. In general, salinomycin is a coccidiostat which is dedicated for protecting against Eimeria spp. and antibacterial prosperities against other pathogens is mostly not find or much poorer (Johansen et el. (2007)).
In conclusion, the alternative feed additives such as MCT, CB, and MCFA could be used as alternative for salinomycin in both "low-digestible" diet and "high-digestible" diet for light strain chickens without any negative effect on the performance. However, there was no reduction in the number of pathogens. A diet with easier-to-digest nutrients can lead to better performance. MCFA and butyrate can be good alternatives for AGP and coccidiostats in poultry nutrition, especially in an ecological system of production, when native breeds are used. The results of this study suggest that MCFA may be the most beneficial additive for GLPH nutrition.