Responses of broiler chicken to different oil levels within constant energy levels from 20 to 40 days of age under hot weather conditions

Abstract In total, 144 Arbour Acres broiler chickens were distributed among four treatment groups (six replicates per treatment; six chickens per replicate) during days 20–40 of age. The chickens were offered iso-caloric and iso-nitrogenous diets containing four dietary oil levels (DOL): 0 (oil non-supplemented diet, control), 2, 4, and 6% in a relatively low-energy diet (12.4 MJ ME/kg diet). During the experimental period, the chickens were reared under natural hot weather conditions (32.5 ± 4 °C, 54 ± 7% relative humidity). Growth, feed conversion ratio (FCR), protein (PCR), metabolisable energy (MECR) ratio, and European production efficiency index (EPEI) were similar among groups fed up to 4% DOL but raising DOL to 6% impaired these traits. Besides, DOL at 6% decreased digestibility of dry matter, crude protein, and ash. Furthermore, a 6% DOL showed the lowest digestibility of dry matter, crude protein, and ash. Dressing percentage was the highest in 2%, and abdominal fat percentage showed the same trend in 6% DOL, but the gizzard percentage was the lowest in 6% DOL. The liver percentage increased significantly with fat/oil inclusion compared to the control. Meat dry matter and either extract increased considerably due to offering different DOLs, with maximum values at 6%. The inclusion of dietary oils in diets significantly increased serum malondialdehyde (MDA) but decreased serum total antioxidant capacity (TAC)/MDA ratio compared to the 0% DOL. In conclusion, under natural summer conditions, from 20 to 40 days of age, broilers' best productive characteristics were achieved using 0-2% DOL, and the best immune response was obtained for 4–6% DOL. Highlights Hot weather negatively influences the productivity of broilers. Fats/oils are essential for animal and human nutrition for several reasons; however, they are expensive compared to other energy sources. Improving the production index is essential to keep broilers farming profits under hot weather.


Introduction
The closure of dry borders and ports and traffic restrictions in many countries to reduce the spread of COVID-19 have led to a reduction in feedstuffs for the poultry industry in some countries, with adverse effects on agricultural production, feed/food supply, and related economic activities (Hashem et al. 2020;Hussain et al. 2020;Poudel et al. 2020). One lesson learned from the COVID-19 outbreak is to depend on locally available human and animal nutrition resources to minimise imports Hafez and Attia 2020;Siche 2020). After the outbreak of COVID-19 in early 2020, the cereal and oilseed markets collapsed (FAO http://www.fao.org/2019-ncov/q-and-a/impact-on-foodand-agriculture/en/) because of a drastic drop in feed sales during the lockdown (Hafez and Attia 2020).
In some areas of the world, temperatures can reach more than 40 C, causing heat stress (HS) and substantial economic loss (Daghir 2008). Heat stress can be naturally induced due to natural heat waves and/or artificially generated by exposing animals to high temperatures (Attia and Hassan 2017). The severity of heat stress mainly depends on the temperature and/or duration of exposure to heat, resulting in different animal responses (Beckford et al. 2020). In previous studies, HS increased energy expenditure and negatively impacted energy balance, causing thermal imbalance (Daghir 2008) and, consequently, homoeostasis disturbance as well as physiological, performance, antioxidant imbalance, altered immune responses, and lower economic outputs (Al-Harthi et al. 2002;Al-Sultan et al. 2019). Besides, HS adversely affects nutrient digestibility, inducing metabolic disturbance reflected in blood and transport mechanisms and, thus, altered deposition in the tissue (Sahin et al. 2009;. Besides, HS increases the production of free radicals, hence inducing oxidative damages (Attia et al. 2009;Yang et al. 2010), adversely impacting immunological mechanisms (Mehaisen et al. 2019) and decreasing bursa, spleen, and thymus yield (Fouad et al. 2016).
In many developing countries, fats and oils are imported for human and animal food. Also, fats/oils are expensive energy sources that are mainly imported from developed countries. In some countries, some feedstuffs' trading and pricing were adversely influenced by changes in the feed market during the first wave of COVID-19 (Poudel et al. 2020). Thus, using imported feed supplies, and costly ones, requires reconsideration to minimise their inclusion in poultry diets via reformulation to decrease fat/oil levels and/or energy levels (Hafez and Attia 2020).
An effective way for feeding broilers during HS is modifying the dietary nutrient profiles (Syafwan et al. 2011;Suganya et al. 2015) to enhance feed intake (Veldkamp et al. 2000). Feed, nutrient, and metabolisable energy (ME) intake decrease during high temperature (NRC, 1994;Daghir 2008;Attia et al. 2011). There is emerging evidence that including dietary fats/ oils in broiler diets may encourage feed intake and reduce heat load while enhancing animals' productivity (Aggoor et al. 2000;Ghazalah et al. 2008). The use of fats/oils has attracted great interest in broiler nutrition and the production of fast-growing animals under normal and HS conditions due to several beneficial effects (Attia et al. 2006(Attia et al. , 2018Pesti et al. 2015). The availability of ME during HS is vital to maintain production, as energy is critical to maintaining an energy balance essential for dispersing metabolic heat (Lin et al. 2005;;Attia et al. 2006;Attia and Hassan 2017;Attia et al. 2018). In the literature, various results about dietary ME impacts on chickens subjected to HS can be found (Daghir 2008). For example, fat/oil supplementation in iso-caloric diets improved productive and economical traits of broilers raised under either normal or HS conditions (Veldkamp et al. 2000;Attia et al. 2018Attia et al. , 2020. On the contrary, fat/oil addition to broilers' diets exposed to HS did not influence performance and economic traits (Sinurat and Balnave 1985). These contradictions lead to uncertainties about the recommended levels of fats/oils in broiler nutrition due to variations in fat sources, fatty acid profiles, dietary composition, ME, crude protein, environmental conditions, and age and size of birds during heat stress (Raju et al. 2004;Pesti et al. 2015;Alagawany et al. 2019). Besides, studies about the use of relatively low ME levels supplemented with elevated concentrations of fats/oils under a constant energy level, and the impacts on broilers' productivity reared under hot conditions are scarce. Thus, this study's objective was to test broiler chicken's productive and physiological responses to different oil levels (0, 2, 4, and 6) within constant energy levels from 20 to 40 days of age under hot weather conditions.

Chickens, experimental design, and diets
This research was approved under protocol no 108-155-1441 by DSR, King Abdulaziz University, Saudi Arabia. In total, 144 Arbour Acres male broiler chickens with an initial body weight of 43.6 ± 2.7 g were randomly distributed in a straight run, completely randomised experimental design, with treatment groups during 20-40 days of age. The males were used herein to improve sample homogeneity and avoid gender differences within replicates and treatments.
The chickens were fed iso-caloric and iso-nitrogenous diets containing four levels of vegetable oils, namely 0% (basal control diet), 2, 4, and 6% (Table 1). The change in diet profiles was done by including oil instead of corn and soybean meal with a gradual increase in the sand to compensate for the differences in energy between oil and corn and soybean meal. The differences in protein levels between the two ingredients were compensated by increasing soybean meal. This was done to keep iso-caloric and nitrogenous diets among different treatments. Under commercial broilers' nutrition, broilers' diets may contain up to 6% oil depending on the diet's energy value, which usually is about 13 M.J./kg diet.
Each treatment consisted of six replicates and six male chickens per replicate. Each replicate was kept in battery brooders (35 Â 25 Â 30 cm length-Â width Â height). From 20-40 days of age, chickens were exposed to natural hot weather conditions with an outdoor temperature of 34 ± 5 C and 52 ± 8% relative humidity. The indoor temperature during the stress period, which is usually observed, started at 10 am and ended by 5 pm; the duration is about 7 hours daily. The average temperature during this period was 31.5 ± 4 C, with 54 ± 7% relative humidity (RH). During this period, broilers showed signs of heat stress, such as increasing painting and lying down. From days 1-19 of age, the broilers were reared using standard husbandry practices, according to the broiler management guide. They fed a commercial mash diet containing 220 g/kg diet crude protein (CP), 12.8 MJ/kg diet, 10 g/kg Ca, and 5 g/kg available phosphorus (Table 1).

Broiler rearing and management
During the preliminary experimental period (1-19 days of age), the broilers were reared under similar hygienic and management conditions. Mash from the corn-soybean meal based-diet was offered from 1 to 19 days of age (Table 1), following the Arbour Acres broiler breeder guidelines. Water and diets were provided ad libitum. Health care included vaccination with Hitchiner þ IB at 8 days of age, Newcastle disease virus (NDV) via Lasota at 14, 20, and 30 days of age, Gumboro at 14 and 24 days of age, and avian influenza (AI) (H5N2) at 9 days of age. The light scheme was 23:1 hour light/dark cycle. The electric light was turned off one hour every 24 hours at the end of the day, around 8 p.m.

Response measurements
As heat stress affected broilers after 3 weeks of age, causing substantial changes in metabolic profiles and loss in productive performance, we recorded data during 20 and 40 days of age, where the feed consumption was measured, and chickens were weighed (g) on a replicate basis as the experimental unit. Data for feed consumption and chemical analysis of feeds were then used for the calculation of feed (g), protein (g), and ME (kcal) intake as relative to body weight gain. Furthermore, the feed conversion ratio (FCR), protein (PCR), and energy (MECR) were estimated by dividing the consumption of feed, protein, and ME by the body weight gain. The European production efficiency index (EPEI) was reported as cited by Attia and Hassan (2017).
A digestibility trial was carried out during 40-47 days of age, using six individually caged broilers per treatment. After an adaptation period of 5 days, feed intake and excreta were collected daily at the same time for three successive days. The gut tract collection method (Total tract nutrient retention) was used for excreta collection, according to Moharrery (2011). Excreta were spiked with 1% boric acid solution to control nitrogen evaporation, dried in a forced ventilated oven at 60 C until constant weight, ground in a Wiley mill, mixed well-kept in screwed glass jars until analyses. The urine nitrogen was separated from faecal nitrogen, according to Davidson and Thomas (1969). Digestibility was calculated based on the amount of the intake and excreted of each nutrient (retained) divided by the amount consumed multiplied by 100.
According to the Islamic method, six broilers from each treatment were randomly selected to represent all treatment replicates and slaughtered at 40 days of age. Dressing percentage was calculated as the weight after slaughter (hot carcase), which is equal to empty carcase without blood, feather, intestinal, head, and legs, plus liver, empty gizzard, and heart divided by live body weight before slaughter and multiplying by 100. The abdominal fat, proventriculus, gizzard, intestines, pancreas, liver, heart, and lymphoid organs (spleen, bursa of Fabricius, and thymus) were separated, weighed, and expressed as relative to the weight of chickens before slaughtering.
Equal amounts of breast and thigh deboned meat were used for chemical analyses of meat samples (n ¼ 6 replicates per treatment). Meat samples (150 g) were collected from each slaughtered animal on day 40 of age, kept in plastic bags, and frozen at À18 C until used for analyses. The dry matter, crude protein, ether extract, and ash content were determined in meat feeds, and faecal as well as crude fibre in feed and faecal according to AOAC (2004) using the methods 925.04, 990.3, 2003.06 and 942.05, 978.10, respectively. The meat's physical characteristics were estimated using fresh meat cuts (n ¼ 6 replicates per treatment). The method of Volvoinskaia and Kelman (1962) was used to determine the water-holding capacity (WHC) and tenderness of the meat, and the colorimetric method was applied for the determination of the intensity of meat colour as the optical densities of the meat and drip (Husani et al. 1950). Meat acidity, pH values of the meat, and drip were determined according to Aitken et al. (1962).
Blood samples (n ¼ 6 per treatment) were gathered at 40 days of age in heparinised and un-heparinised tubes. The samples were centrifuged at 500 g for 15 min, and plasma and serum were collected and stored at À18 C for determination of blood biochemical and haematological contents. The haematological traits consisted of counts of red blood cells (RBCs), as indicated by Hepler (1966). We further determined blood haemoglobin (Hgb) and packed cell volume (PCV) using the method of Eilers (1969). Mean cell haemoglobin concentration (MCHC), mean cell haemoglobin (MCH), and mean cell volume (MCV) were estimated as reported by (Eilers 1967). The counts of white blood cells (WBCs) and WBC portions were determined as noticed by Lucas and Jamroz (1961), and the activity of phagocytes (PA) and the index of phagocytes (PI) were obtained according to Leijh et al. (1986). The levels of plasma glucose (Trinder 1969, total serum protein (Weichselbaum 1946), serum albumin (Doumas 1971), serum globulin (Coles 1974), and the albumin-to-globulin ratio were estimated according to (Attia and Hassan 2017).
The TAC and MDA levels were estimated according to Koracevic et al. (2001) and Richard et al. (1992), respectively. The serum antibody body titres for avian influenza (AI) and Newcastle disease virus (NDV) were measured as suggested by Kai et al. (1988) and Takatsy (1956), respectively, and infectious bursal disease (IBD) was estimated according to the method of Cosgrove (1962).

Statistical evaluation
The number of replicates was estimated using the power analyses, and 0.07 difference in FCR of chickens at market age difference in FCR (1.93 vs. 2.0 kg feed/ kg gain) was considered to be 0.07 based on published results by (30; 62; 63). The standard deviation of 0.04 kg feed/kg gain, the two-sided test, the P-value of 0.05, and the desired power of 80 was employed. The estimated number of replicates was 6 replicates, according to [https://www.stat.ubc.ca/$rollin/stats/ ssize/n2.html]. The normality of error distribution and data was tested using the Shapiro-Wilk test of normality (SAS Institute 2009). Also, the homogeneity of the variance (homoscedasticity) has been evaluated using the Levene's test (SAS, 2009 V R ). The assumptions of ANOVA were tested according to a random selection of the samples. One-way ANOVA, using SAS V R (2009), was run using the following statistical model: where Y is the dependent variable, l is the overall mean, Fi is the effect of dietary oil levels, and eij is the random error. The experimental unit was the replicate. Prior to analysis of variance, data presented in percentages were transformed to log10 to normalise data distribution. Differences among means at P .05 were examined using the Tukey's procedure. The chisquare test was used for testing the survival rate.

Results
Broiler growth Table 2 shows the effect of dietary oil level (DOL) on broiler performance during 20-40 days of age. The consumption of feed, protein, and energy did not differ among the treatments. Growth of broilers on DOL up to 4% was similar, although raising DOL to 6% significantly lowered final body weight and body weight gain (BWG) compared to the other DOL levels. The conversion of feed, protein, and ME to gain was similar among groups fed a diet containing up to 4% DOL, whereas DOL inclusion up to 6% significantly impaired these traits and EPEI. Table 3 represents the effect of DOL on nutrient digestibility. Feeding a diet with 6% DOL induced the lowest digestibility of dry matter and total tract retention of nitrogen (crude protein), and ash. Besides, other 2 and 4% DOLs significantly reduced the digestibility of ether extract and ash compared to the nonsupplemented diet. Table 4 indicates the effect of DOL on carcase traits and inner organs at 40 days of age. The dressing percentage was the highest for the 2% oil-supplemented diet. The diet supplemented with 6% oil resulted in the highest abdominal fat values; the gizzard percentage was lowest for 6% DOL. Liver percentage significantly increased similarly with increasing DOLs. There were no significant differences in the proventriculus, intestines, pancreas, and heart due to different DOLs. Table 5 shows the effect of DOL on the chemical and physical characteristics of meat of 40-day-old broilers. Meat dry matter and either extract increased Table 2. Growth, feed intake, feed, protein, and energy conversion ratio, survival rate and European production efficiency index of broilers exposed to natural high temperature and fed graded levels of oils within a constituent calorific level during 20-40 days of age.  significantly with increasing inclusion levels, with a maximum at 6% DOL. Meat crude protein and ash and physical characteristics of meat, such as meat colour measured as optical density, WHC, tenderness, and pH values, were similar among DOL groups.

Chemical and physical meat characteristics
Biochemical constituents and indices of the liver and renal function Table 6 shows the biochemical constituents and the indices of the liver and renal function of 40-day-old chickens. Serum albumin and albumin/globulin ratio levels were significantly lower in the group fed 2% DOL than in the other groups. There were no Table 4. Carcase traits and inner body organs of broilers exposed to natural high temperature and fed containing graded levels of oils within a constituent calorific level during 20-40 days of age.   Table 6. Plasma biochemical and indices of liver and renal function of broilers exposed to natural high temperature and fed containing graded levels of oils within a constituent calorific level during 20-40 days of age. significant differences in serum total protein and globulin among the different DOL groups.
Serum triglyceride and vLDL were significantly higher in groups fed 4 and 6% DOL than in the other groups, but the HDL to LDL ratio of 6% DOL was lower than that of the control. Besides, HDL was lower for 2 and 6% DOLs than in the control group. Serum total cholesterol, as well as LDL, were not significantly affected by DOL.
Plasma glucose levels decreased with increasing DOL at 6% DOL compared to the other DOLs groups. Most liver leakage enzymes and all renal function indices were not significantly affected by DOL, except for AST, which was considerably lower at 2%. We found no significant DOL effect on the renal function indices urea, creatinine, and urea-to-creatinine ratio. Table 7 displays the impacts of DOL on RBCs parameters and different leukocytes proportional. There were no significant effects of DOL on RBC characteristics and most of the WBC parameters, except for a substantial decrease in WBCs of groups fed 2% DOL compared to the higher DOLs groups.

Haematological parameters
Antioxidant status, lymphoid organs, phagocytosis, and antibody titre Table 8 represents the effect of DOL on antioxidants status, lymphoid organs, and phagocytosis, and antibody titre at 40 days of age. The inclusion of dietary oils in broiler diets significantly increased serum MDA compared to the control, whereas the TAC/MDA ratio was declined. There was no significant effect of DOL on TAC.
Spleen percentage was significantly lower in groups on 2% DOL than in the other groups. Besides, thymus percentage was markedly lower in groups fed 2% DOL than in those with 0 and 4% DOL. The phagocyte index was significantly higher at 4% DOL compared to the lower DOL groups.
Antibody titre against AI was increased in the 4 and 6% DOL groups compared to the other groups. Also, antibody titre against IBD tended to be numerically higher (P .07) for 6% DOL compared to the other groups. We found no difference due to DOL in phagocyte index, antibody titre for NC, and heterophile-tolymphocyte ratio.

Discussion
The changes in feed supply and chain during COVID-19 illustrate the needs for a new concept to reformulate broiler diets considering low-energy and low-cost diets to reduce feeding costs and minimise the demands for cereals, oilseeds, and fats/oils (Hafez and Attia 2020;Hashem et al. 2020;Hussain et al. 2020;Poudel et al. 2020;Siche 2020). A good example is the use of low-energy diets in broiler nutrition compared to diets supplemented with high DOL, especially under high ambient temperatures.
An indoor temperature of 31.5 ± 4 C during the experimental period induces heat stress in broilers, as evidenced by behavioural changes such as increased panting, lying down, and high body temperature (Daghir 2008;Attia et al. 2006). The effect of oil supplementation on broilers' growth performance is Table 7. Red and white blood cells parameters of broilers exposed to natural high temperature and fed graded levels of oils within a constituent calorific level during 20-40 days of age. complex. An isocaloric diet containing 12.4 MJ/kg (ME), supplemented up to 4% DOL, resulted in similar growth performance and survival rate, and this related to the results digestibility. However, ether extract and ash digestibility of groups receiving 2 and 4% DOL were reduced. Besides, ash digestibility was further reduced by 6% DOL. Also, increasing DOL to 6% at unchanged ME levels reduced the performance index and dressing percentage of broilers compared to 2% DOL. The reduction in growth performance due to offering 6% DOL coincided with reducing digestibility DM, CP, either extract diet or ash. The 6% DOL group's lower performance may be related to increased fat deposition in the abdominal cavity compared to 2% DOL, and the liver and gizzard decreased. These changes indicate that the extra caloric effect provided by increasing DOL to 6% was directed to fat deposition in the abdominal cavity, liver, and meat, as meat lipid percentage also increased. These changes indicate that nutrient repatriating for fat deposition rather than muscle growth when DOL increased above energy requirements for muscle growth (Attia et al. 2018). Chickens offered diets containing different oils showed alterations in the carcase's quality and composition (Baiao and Lara 2005;Attia et al. 2018). The group's decreased performance fed 6% DOL may also be due to the low digestibility of nutrients due to reduced gizzard percentage. The gizzard is essential for the mechanical digestion of coarse parts of the diet (Daghir 2008;Pesti et al. 2015).
Our results suggest that broiler diets containing 12.4 MJ ME without or with supplementation of 2% DOL are adequate for broilers' performance during hot weather conditions. The use of 2% oil could be recommended to meet broilers' essential fatty acid needs under hot weather conditions . The recommended ME for broilers of 1-6 weeks is 13.4 MJ/kg diet ; the level used herein was 7.5% lower (12.4 MJ/kg diet), saving about 2.8% of supplemented fat/oils in chicken diets. There is evidence that the DOL impacts broiler performance during hot weather conditions; a diet supplemented with fat/oil may enhance productive traits (NRC 1994;Raju et al. 2004;Baiao and Lara 2005;Attia et al. 2020). The primary energy sources for poultry nutrition are fats/oils and carbohydrates, although oils and/ fats are preferred. And, this may be due to the positive impact of fats/oils on digestibility, low heat increment, contents of fat-soluble vitamins, extra-calorific effect, and high-energy value; however, they are generally expensive (Pesti et al. 2015Abd El-Hack et al. 2019).
In the literature, supplementation of oil/fat under high ambient temperatures is complex, and there was a lack of responses in some experiments (Sinurat and Balnave 1985;Attia et al. 2003). However, in others, the influence of oils/fats was dependent on the dose (Attia et al. 2018), the fat source , and the fatty acid profiles . Besides, reducing the ME level during high ambient temperatures promotes feed intake (Baghel and Pradhan 1990;Hoffmann et al. 1991), and some authors suggest that energy requirements are affected by weather temperature (Hurwitz et al. 1980;NRC 1994;Al-Harthi et al. 2002). The lower growth performance (growth rate, FCR, and survival) of broilers observed herein can be elucidated by the effect of heat stress on growth performance, such as feed Table 8. Antioxidants status, lymphoid organs, phagocytosis, and antibody titre of broilers exposed to natural high temperature and fed containing graded levels of oils within a constituent calorific level during 20-40 days of age. intake, growth, and FCR. Performance of broilers can be affected by geographical area, region, season, type of diet, housing condition, type of house, slaughter age, nutritional level, hygiene and welfare, and epidemiology of diseases. The results recorded herein are in line with broilers' performance and survival in commercial enterprises in Saudi Arabia (Attia et al. 2018. Also, hot weather negatively affects animal performance and liveability. And, this coincided with lower feed intake, lower digestibility, impaired metabiotic profiles, and acid-base balance (Daghir 2008;Al-Sultan et al. 2019;Beckford et al. 2020).
In our study, the group's dressing percentage fed 2% DOL was improved, which reflected increased growth performance. The present results show that up to 6% DOL's inclusion levels had no adverse effects on meat physical traits. High-quality meat has an increased customer acceptance, and meat with a greater juiciness/WHC is generally preferred (Attia et al. 2014(Attia et al. , 2018.
An increase in DOL of up to 6% was associated with impaired lipid metabolism, as shown by the increased triglycerides of broilers on 4 and 6% DOL and the reduced HDL of the HDL to LDL ratio of 4 and 6% DOL. The present study revealed that lipid metabolites are the primary index of lipid metabolism; however, DOL is a valuable source of PUFA since it is mainly composed of sunflower and soybean oils. The increase in dietary fat intake can lead to cardiovascular issues, high blood pressure, and lipoedema. However, humans' dietary fat recommendations are sometimes contradictory and affected by dietary fat source, level, and fatty acid profile .
Interestingly, plasma glucose was gradually reduced with increasing DOLs, with a minimum level at 6%, indicating that oil supplementation can be used to control hyperglycaemia. In a previous study, fish oil and a PUFA-rich omega-3 source increased people's insulin sensitivity with metabolic disorders . A positive effect was noticed in AST, which was potentially decreased when 2% DOL was fed, providing further evidence for recommending 2% DOL to improve productive performance and maintain hepatic functions (Attia et al. 2018). However, low-fat diets are recommended to control Conroy heart disease and highlight the substitution of fat by carbohydrates .
We observed a substantial decrease in TAC/MDA due to the inclusion of DOL at 2% or higher. This decrease in the antioxidant balance reflected an increase in MDA, a product of lipid peroxidation, particularly PUFA ). The oils used in this study were a 1:1 mixture of soybean oil and sunflower oil, rich PUFA sources (Al-Khalaifah 2020). The impaired antioxidant status may provide further evidence for the limitation of increasing DOL in chicken diets above 4% and the need for the addition of antioxidants, preferably from natural sources, to improve the antioxidant balance (Abaza 2002;Hu et al. 2019).
The lesson learned from the COVID-19 crisis is that both humans' and animals' immunity is the front line of defense for protecting emerging and remerging diseases (Hafez and Attia 2020). The effect of oils/fats on animal immunity and health has been reported ) is affected by specific fatty acids (Gao et al. 2017; Al-Khalaifah 2020) as well as dietary levels and sources (Attia et al. 2018. In the present trial, the antibody titre for Avian influenza was markedly elevated due to feeding 4 and 6% DOL compared to the other DOLs. The antibody titre against IBD tended to be numerically higher (P .07) for 6% DOL chickens than for the other groups. The changes in antibody titres were supported by the changes in secondary lymphatic organs, such as the spleen, with a decrease in the 2% group, combined with a reduction in WBCs (cell-mediated immunity).
Furthermore, 4% DOL resulted in increased thymus (T cells) levels, which agrees with a previous study . A similar trend was observed for the phagocyte index, which is an indicator of humoral immunity. These results indicate that higher levels of oils/fats (4-6%) positively influence chickens' humoral immune response. However, the productive trait and DM and CP's digestibility were markedly reduced in broilers fed 6% DOL. This decrease is independent of the inclusion of dietary sand levels in the diet. Sand level in broilers and laying hens diets up to 10% did not adversely affect performance (Sahraei and Shariatmad 2007;van der Meulen et al. 2008;Atapattu and Silva 2016). Sand can improve the digestibility of nutrients due to improving grinding and mechanical digestion of coarse components in the gizzard and increasing the surface exposed to enzymes in the small intestinal (Sahraei and Shariatmad 2007;van der Meulen et al. 2008;Atapattu and Silva 2016). The boosting effect of fats/oils at high inclusion levels on immune function found herein under high ambient temperatures could be attributed to several nutritional benefits (Mateos et al. 1982;Pesti et al. 2015). In the literature, HS's impact on the immune system depends on the strain, the severity and the duration of HS, and animal age and size most significantly affect cell-mediated immunity (Abaza 2002). The present results show that feeding broilers' dietary oil levels under hot weather conditions depend on the production goal, as 2% can be suggested for growth performance and 4-6% for immune response. Thus, further research supports the recommended levels of dietary oils on broiler performance is essential. This should consider DOL, fatty acid profile, and ambient temperature. Due to the literature's contradictory results (Sinurat and Balnave 1985;Lou et al. 2003).
However, our study is limited, mainly because of the lack of a control group reared under normal climatic conditions (Attia and Hassan 2017;Attia et al. 2018). Also, to the well-known demonstrated literature, group reared under normal environmental temperature had no additive value to the current experiment set-up due to primary goals in which the hot weather was investigated rather than heat stress per see. However, similar studies using carbohydrate-basedenergy diets without oil supplementation compared to diets with 8-10% DOL, under heat stress, are lacking. The use of DOL may be suggested as a valuable nutritional technique to negate the harmful effects of hot weather; however, the most suitable level of oil/fat remains uncertain and depends on the production propose (Daghir 2008;Fouad et al. 2016).

Conclusion
Under natural high-temperature conditions from 20 to 40 days of age, broilers' best production performance index was achieved at 0-2% DOL. However, feeding 6% DOL to broilers markedly decreased growth performance and impaired lipid metabolism, albeit with an improved immune status. This showed that broiler feeding under natural high temperatures is a factor of the achieved goal, such as improved growth performance or enhanced immune status. The results indicate that the effect of dietary oils on broilers depends on goals of production as growth performance needs less oils (À02%) than immune response, which showed greater responses to high oil supplemented diet (4-6%) %) and/or essential fatty acids requirements.

Ethical approval
The experimental work and ethical treatment of animals in this study were approved by DSR, King Abdulaziz University, under protocol no G-108-155-1441. The procedures suggested minimal stress, ensure rights and welfare by eliminating harm or suffering to animals.