Efficacy of lysophospholipids on growth performance, carcase, intestinal morphology, microbial population and nutrient digestibility in broiler chickens fed different dietary oil sources

Abstract An experiment was conducted to investigate the growth performance, microbial population, intestinal morphology and nutrient digestibility of broiler chickens fed diets supplemented with lysophospholipids (LPL) in combination with soybean (SO), flaxseed (FSO) or sesame seed (SSO) oil sources. A completely randomised design with a 2 × 3 factorial arrangement including two levels of LPL (0 or 0.1% Lipidol) and three different oil sources was used. A total of three hundred one-day-old broiler chicks were randomly allocated into six treatments of five pens with ten birds per pen. The results showed that body weight gain (BWG) and feed conversion ratio (FCR) significantly increased in broilers fed dietary LPL and SSO (p < .05). There was a significant interaction between the oil sources and LPL supplementation on 10 days of age (p < .05). Inclusion of SSO to the diets increased villus width and villus surface area compared with SO diet (p < .05). Broilers fed LPL supplemented diets had lower crypt depth, while villus length to crypt depth ratio was greater in broilers fed LPL supplementation (p < .05). Lactobacillus population increased in broilers fed LPL supplemented diet compared to those without dietary LPL (p < .05). Inclusion of LPL increased ileal digestibility of dry matter, crude protein and ether extract (p < .05). Broiler fed SSO diets had greater digestibility coefficient for ether extract compared with SO group (p < .05). In conclusion, dietary LPL supplementation and SSO increased growth performance, intestinal morphology, microbiota activity and nutrient digestibility in broiler chickens. HIGHLIGHTS Lysophospholipids (LPL) supplementation increased growth performance, intestinal morphology, and microbiota activity in broiler chickens. Supplemental LPL enhanced ileal nutrient digestibility coefficients in broiler chickens. Dietary sesame seed oil (SSO) increased growth performance, ether extract digestibility and intestinal morphometric variables in broiler chickens compared with soybean oil (SO).


Introduction
Lipids (oils and fats) are widely added into poultry diets to enhance the dietary energy concentration, increase the bioavailability of fat-soluble vitamins, and increase feed palatability. Lysophospholipids (LPL) are different mixtures of biosurfactants (emulsifiers) that derive from an enzymatic hydrolysis (phospholipase A1 or A2) of phospholipids and aid in the digestion and absorption of lipids (Haetinger et al. 2021). The mechanism of action of LPL has been well documented as hydrophilic compounds properties and thus a better oil-water emulsification capacity (Zampiga et al. 2016). Dietary LPL increase monoglycerides and diglycerides release by emulsion of dietary fat and promote incorporation of fatty acids into micelles and increase fat digestibility (Zhao and Kim 2017). It has been observed that LPL supplemented diets enhanced energy retention and intestinal morphometric indices including villus length or absorption area in broiler chickens (Brautigan et al. 2017). In addition, it has been documented that LPL increases gut permeability to bimolecular compounds such as proteins, enzyme activity, impact on the formation of protein channels and cause duodenal epithelial cells hypertrophy in broiler chickens (Zampiga et al. 2016). Therefore, it is assumed that enhancing lipid digestibility as a result of using LPL supplementation may increase growth performance and gut health of broiler chickens.
Various sources of lipids such as animal fats and vegetable oils are used in the broiler rations. Several factors may influence dietary lipids digestion and absorption in broiler chickens such as lipid source, fatty acid composition, the saturation degree of lipid and the chain length of fatty acids (Tancharoenrat et al. 2013). The use of vegetable oils is more important than animal fats in poultry nutrition due to their lower content of saturated fatty acids and long chain fatty acids. It is well documented that polyunsaturated fatty acids (PUFAs) are the components that have major roles in several biological activities such as metabolism and endocrine events (Zanussi et al. 2019). However, de novo synthesis of omega 3 PUFAs does not occur in birds species due to the lack of fatty acid desaturase enzymes activity in their body (Cerolini et al. 2005). Therefore, it is necessary to make the diets supplemented with omega 3 sources in the poultry species. Flaxseed oil (FO) may be a suitable source of linolenic acid (ALA) in the broiler chicken diets may lead to the incorporation of polyunsaturated fatty acids into the bird's tissues (Lopez-Ferrer et al. 2001). There are several reports on the beneficial effects of dietary flaxseed oil on the growth performance and gut health of broilers and quails (Abbasi et al. 2019;Nasir et al. 2020;Mirshekar et al. 2021). These effects may be explained by the increased digestibility of flaxseed oil that occurs with enhancing in the unsaturated grade of fatty acids. Also, a few experiments have evaluated the substitution of soybean oil by flaxseed oil on the microbiota activity and intestinal morphometric indices of broilers. Sesame (Sesamum indicum) is an important tropical or subtropical plant which is cultivated in much tropical area of the world as well as in the northeast of Iran. Sesame seed is composed of 45-50% lipids that a large portion of these seeds are used for oil production (Rezaeipour et al. 2016). Sesame oil consist of a variety of main fatty acids including oleic acid (35-50%), linoleic acid (35-50%), stearic acid (3.5-6%) and palmitic acid (7-12%) based on the species of sesame seeds (Mohammed et al. 2018). Despite the presence of about 85% unsaturated fatty acids in the sesame oil, oxidative stability of sesame oil is superior to that of other vegetable oils (Abou-Gharbia et al. 2000). A group of phytoestrogen (phenolic) compounds known as lignans in the sesame oil including sesamol and sesamolinol formed from sesamolin are the major antioxidants responsible for the reduction of sesame oil rancidity (Mohamed and Wakwak 2014). Sufficient information on the growth performance of broiler chickens with dietary sesame oil is not available. It was hypothesised that, dietary LPL in combination with different oil sources may enhance growth performance of broiler chickens through increase the gut morphology and nutrient digestibility. On the other hand, it was assumed that inclusion of LPL supplementation may have different effects for oil sources such as sesame seed oil, flaxseed oil and soybean oil in terms of nutrient digestibility coefficient, intestinal morphology and subsequently growth performance of broiler chickens.
Therefore, this experiment was aimed to evaluate the effect of LPL supplementation on growth performance, carcase traits, intestinal morphology, microbial population and nutrient digestibility in broiler chickens fed different oil sources.

Materials and methods
All experimental protocols in this study were approved and conducted under the guidelines of the Animal Care and Use Committee of Qaemshahr Branch, Islamic Azad University.

Experimental diets and birds
A total of three hundred Ross 308 broiler chickens (one d old) with an average weight of 44 ± 2 g were used in a 35-day experiment. Broilers were distributed into six experimental groups with five replicate pens of ten birds per pen with a 2 Â 3 factorial arrangement design. Factors were 2 levels of LPL (0 or 0.1% Lipidol) and 3 oil sources including soybean oil (SO), flaxseed oil (FSO) or sesame seed oil (SSO). The LPL supplementation (Lipidol, Easybio Company, Seoul, South Korea), derived from soy lecithin, was provided from a company (Gorgan, Iran). Corn-soybean meal based diets (1-10, 11-24, and 25-35 days) containing SO (Table 1) were formulated according to the nutrient recommendations for Ross 308 broiler chicken. To prepare experimental diets based on different oils, SSO and FSO were substituted for SO in the basal diets. All broilers were allowed to consume feed and water ad libitum.

Growth performance and carcase characteristics
To determine the body weight gain (BWG), all broiler chickens were weighed by pen on d 1, 10, 24 and 35. Feed intake (FI) was recorded on the same days and obtained data was then used to calculate feed conversion ratio (FCR).
At the end of the experiment (35 days of age), five broiler chickens per treatment (with a weight close to the pen average) were selected and sacrificed by cervical dislocation method. The digestive tract of each bird was gently removed and the weight of thigh, breast, liver, pancreas, heart, gizzard, and proventriculus was measured. Data were presented based on g/ 100 g body weight of bird. In addition, the length of duodenum, jejunum, ileum and caecum as different parts of intestinal tract was recorded.

Intestinal morphology and microbial population
In order to determine the intestinal morphometric indices and caecal microbiota activity, one bird per pen (five birds per treatment) was randomly chosen from each pen and euthanized by cervical dislocation on day 35. Small intestine of each bird was gently removed and a 3 cm fragment of jejunum (middle of the jejunum) was excised for morphological measurements. Jejunum was fragmented from the distal portion of duodenal loop to the Meckel's diverticulum. Taken samples were flushed with physiological saline, and then fixed in 10% buffered formalin solution (10%). tissue sample was processed was paraffin embedding technique after dehydration and then, a 5mm section was stained with haematoxylin and eosin. The jejunal morphometric variables including villus length (VL) and width (VW), crypt depth (CD), and VL/ CD ratio were recorded by a light microscope system with LED Illumination. The villus surface area (VSA) was calculated using the following formula (Sakamoto et al. 2000): The same euthanized birds were also used for microbial population study. Caecal content (3 g) from each selected bird was separately collected in a sterile tube for the enumeration of microbial population. Briefly, One gram per each sample was transferred into 9 mL sterile physiological saline solution (NaCl 85%) and was serially diluted from 10 À1 to 10 À7 . Subsequently, 0.1 ml of each dilution suspension was plated onto appropriate media. Eosin methylene blue agar (Merck, Darmstadt, Germany) was used for E. coli enumeration after incubation at 37 C for 24 h. Lactobacilli bacteria was cultured on de Man, Rogosa, Sharpe agar (Merck, Darmstadt, Germany) after incubation for 48-72 h at 37 C. Bacteria were counted using a colony counter and the results expressed as the logarithm of the number of bacteria per gram of the sample.

Nutrient digestibility
In order to determine apparent ileal digestibility (AID), chromium oxide (3 g/kg of diet) as an indigestible marker was added to each experimental treatment on 28-35 days of age. At the end of the bioassay, five broiler chickens per each experimental group were selected and euthanized by cervical dislocation. Ileum segment (Meckel's diverticulum to 1 cm proximal to the ileo-caecal junction) was chosen for each bird after the removing of the digestive tract. Samples of fresh digesta from the end half of this section were collected and dried at 55 C for 72 h, and then ground to pass through a one-mm screen. All feed and ileal digesta samples were analysed for dry matter (method 934.01), crude protein (method 988.05) and ether extract (920.39) according to AOAC (1990) procedures. Chromium oxide was determined using Fenton and Fenton (1979) procedure. AID coefficients were calculated using the following equation:

Statistical analysis
Data obtained from this study were analysed as a 2 Â 3 factorial experiment using the general linear model of SAS (2001). The means comparisons were analysed by Tukey test and differences between treatments were deemed to be significant if the probability value (p-value) was <.05.

Results
The influence of dietary LPL supplementation and oil sources on the growth performance of broiler chickens are presented in Table 2. The interactions between dietary oil sources and LPL on growth performance parameters were not significant, except for feed conversion ratio (FCR) on 10 days of age (p < .05). Inclusion of SSO to the diets increased body weight gain (BWG) during the starter, finisher, or overall phases in broiler chickens, but did not differ from FSO treatment (during finisher, or the entire trial period) that was intermediate (p < .05). BWG was significantly (p < .05) enhanced by the addition of LPL to the experimental diets. No significant differences were observed for feed intake (FI) in broiler chickens. Dietary LPL supplementation improved FCR during the entire experimental period (p < .05). In addition, FCR variable during the starter, finisher, or overall phases tended to increase as SSO added to the experimental diets (p < .05) but did not differ from FSO treatment (during starter, or the entire experimental period) that was intermediate.
No significant differences were found for carcase variables and internal organs for the broiler chickens given the different dietary oil sources or LPL supplementation (Table 3). Dietary treatments had no significant effects on the length of the different parts of small intestine of broiler chickens (Table 4).
The interactions between dietary oil sources and LPL on the intestinal morphometric variables were not significant (Table 5). Villus width (VW) and villus surface  In ileal nutrient digestibility (Table 6), the LPL supplement main effect showed that LPL supplement had beneficial impacts on the ileal digestibility coefficients of dry matter (DM), crude protein (CP) and ether extract (EE) in broilers (p < .05). Dietary SSO increased ileal digestibility of EE in broiler chickens compared SO diet (p < .05) but did not differ from FSO treatment that was intermediate.

Discussion
In the present experiment, BWG and FCR were improved by dietary LPL supplementation. There are Table 4. Effects of the dietary treatments on proventriculus and gizzard weights (g/100 g body weight of bird) and the length (cm) of different parts of intestine in broiler chickens.  several studies available on the positive influence of LPL on the growth performance of broiler chickens (Zampiga et al. 2016;An et al. 2020;Zangeneh et al. 2020). In accordance with these results, it has been reported that the synthetic LPL product improved body weight gain and FCR of broiler chickens (Haetinger et al. 2021). In contrast with the present findings, supplementation of lysolecithin as LPL additive in broiler diets had no significant influence on the growth performance during 1-21 days of age (Gheisar et al. 2015). On the other hand, several reports indicated that supplementation of the diets with LPL increased growth performance of broiler chickens mainly during the early feeding phase (Chen et al. 2019;Zhao and Kim 2017). This effect may due to the insufficient production of bile salts and lipase in young broilers, which results in a low capacity of fat digestion and absorption (Chen et al. 2019). Therefore, increase in the BWG and FCR in broiler chickens in response to LPL supplementation may be the result of enhanced fatty acid and nutrient digestibility.
In the present experiment, the LPL supplementation increased intestinal morphometric variables including crypt depth and villus length to crypt depth ratio. Increasing the villus length to crypt depth ratio is a main factor to evaluate the nutrient absorption ability in broiler chickens (Roofchaei et al. 2019). Besides, it is well documented that the status of intestinal mucosa is an important indicator for determining gastrointestinal health and function (Kolbadinejad and Rezaeipour 2020). Therefore, an increase in these morphological indices may enhance the absorption capacity and digestive enzyme activity in broilers. In consistent with our results, it was observed that addition of LPL to broilers diet not only decreased crypt depth in the jejunum but also increased jejunal villus length to crypt depth ratio (Boontiam et al. 2017).
In the present study, broiler chickens fed diets containing LPL supplementation presented a greater viable count of Lactobacillus in the caecal region. The mode of action of LPL on the proliferation of positive bacteria such as Lactobacilli has been demonstrated (Huyghebaert et al. 2011). According to these authors, the LPL additives could contribute to the decline of the growth depressing substances that secreted by positive bacteria. On the other hand, it is reported that LPL supplementation may alter the bacteria membrane permeability, subsequently to disrupt the bacteria integrity due to ionic imbalance (Polycarpo et al. 2016). A dearth of information exists in term of intestinal microbial population in response to LPL supplemented diets. Therefore, further studies are needed to better understand of the effects of LPL supplementation on the microbiota activity in broiler chickens.
The effect of the LPL supplemented diets on the ileal nutrient digestibility was significant.
In consistent with the present results, several studies demonstrated the beneficial effect of LPL feed additive on the nutrient digestibility in broiler chickens (Schwarzer and Adams 1996;Boontiam et al. 2017;Zhao and Kim 2017). The positive impacts of LPL supplemented diets on the ileal digestibility coefficients may be attributed to the emulsification property of these molecules. LPL compounds have a suitable hydrophilic-to-lipophilic balance value than lecithin, which increases water in oil emulsion (Shumilina et al. 2006). LPL substances are magnificent surfactants and can increase the mixing of intestinal digesta, decline the particle size of the emulsified droplets and subsequently increase the enzyme availability to lipids (Jansen 2015).
Inclusion of SSO into the diet increased growth performance of broiler chickens compared with dietary SO. Although several studies have been conducted on the beneficial effects of plant oils such as SO or FSO compared with animal fats in growth performance of broilers (Lai et al. 2018;Olomu and Baracos 1991;Tancharoenrat et al. 2013), limited data are available on the SO efficacy compared with other vegetable oils in broiler diets. Furthermore, Most of these researches have been on the effectiveness of sesame seeds or cakes, but not SSO (Jacob et al. 1996; Onainor et al. 2018). In contrast with our results, no significant influence of sesame seed or SSO was observed on the productive performance of laying quails (Al-Daraji et al. 2012). In parallel with the present results, it has been reported that SSO or sesame seed supplemented diets had positive effect on the body weight gain in broiler chickens (Agah et al. 2016;Onainor et al. 2018).
Dietary SSO increased morphological variables including villus width and villus surface area in the Jejunum region of broiler chickens. This result supported the study of (Agah et al. 2016) who observed that dietary SSO could increase intestinal morphometric indices such as villus absorption surface, villus length and villus width in broiler chickens. Besides, an increase in the intestinal villus height and surface area for absorption was observed in broiler chickens fed with diets supplemented by sesame seed (Adebiyi et al. 2015). The exact mechanism by which using SSO in the diet increases gut health and morphology is not well understood. However, there is evidence that SSO may increase intestinal health and function due to its antioxidant properties of lignans (Ahmad et al. 2006). In line with this mechanism, it has been documented that increased intestinal villi length and surface area in broiler chickens may be due to the antioxidant efficacy of some of dietary ingredients (Hassanpour et al. 2010).
Inclusion of SSO into the broiler diets increased lipid digestibility. In a study on the common carp (Cyprinus carpio), it has been observed that addition of SSO increased fat digestibility (Albassam and Al-Habeeb 2019). In the literature review, no information has been reported about the effect of SSO on the nutrient digestibility in poultry. Therefore, more research is needed in this field.

Conclusions
It is concluded that LPL supplementation can be used to increase growth performance, gut morphology and microbial population in broiler chickens. In view of nutrient digestibility, it is observed that the LPL additive extensively increased ileal digestibility coefficients. Furthermore, it was observed that SSO increased productive performance, intestinal morphometric variables and ether extract digestibility of broiler chickens. However, further research is needed to elucidate the mechanism of action of sesame SSO on the nutrient digestibility in broilers.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data statement availability
All data generated and analysed during this study are included in this published article.