INTERNAL FATS OF ROSS 308 BROILER CHICKENS AFTER APPLICATION OF GRAPE, FLAX AND PUMPKIN POMACE INTO THEIR DIET

Poultry fat is interesting in terms of fatty acid composition, especially due to the higher content of unsaturated fatty acids compared to mammals. However, internal fats are considered as one of the wastes from poultry processing. In previous experiments, we observed the effect of grape, flax and pumpkin pomace on the meat performance and chemical composition of broiler chicken muscle. Therefore, the aim of this work was to examine the effect of these pomaces on the formation of internal fats in Ross 308 broiler chickens. The control group (C) was fed with a feed mixture (FM) without antibiotics and growth stimulators. In the experimental diets, wheat in FM was replaced by 1, 2 and 3% red grape pomace, variety Alibernet (groups E1, E2 and E3), 2 and 4% flax pomace (groups E4 and E5) and 2 and 4% pumpkin pomace (groups E6 and E7). The chickens were fed and watered ad libitum for 42 days. Amounts of internal fats are presented as % from carcass weight (CW). Of the internal fats in females, only heart fat was significantly affected (P≤0.05) (least in E5 and E7 – 0.20%). In males, on the other hand, we found significant differences (P≤0.001) in other fats: abdominal fat was highest in E2 and E3 (0.94 and 0.91%, respectively), gizzard fat was highest in E2 (0.59%) and internal fats together in E2 and E3 (1.67 and 1.48%, respectively). The results show that negatively higher internal fat content of the chicken carcass was caused mainly by the addition of 2 and 3% RGP in the diet of Ross 308 chickens.

with unsaturated fatty acids (Gioielli, Ming and Sotero Solis, 2002). However, small chicken meat producers generally dispose of these internal fats together with intestines, feathers and blood, which creates a problem for the environment (Peña-Saldarriaga, Fernández-López and Pérez-Alvarez, 2020). However, chicken fats content of polyunsaturated fatty acids is associated with poor oxidative stability (Moser, 2010). The predominant fatty acids in chicken fats are described as oleic, palmitic, and linoleic, compared to saturated fatty acids of fats (for example pig fat) that are generally used in meat products. Valorization of chicken by-products using internal fats as fat source in soft chicken meat products could be an alternative contributing to the poultry sector sustainability (Peña-Saldarriaga, Fernández-López and Pérez-Alvarez, 2020). Feed antibiotics (also known as antibiotics grow promoters -AGP) have been used as a major barrier against the spread of bacterial infections in broiler chickens. Their effect consisted in direct protection with antibacterial agents, respectively by adjusting the intestinal microflora, reducing the incidence of intestinal infections in livestock and thus their overall better physical health and increased production Ritter et al., 2019). However, the widespread use of feed antibiotics around the world has led to the development of resistant strains of bacteria against antibiotics for the treatment of human diseases. Therefore, in 2006, their use in the EU was banned by a decision of the European Commission (Haščík et al., 2018). In 2011, research found that approximately 48% of producers in the world keep broiler chickens without the use of antibiotics, using them only when necessary (MacDonald, 2014). More recent findings estimate that more than 50% of industrially bred broiler chickens are reared without the use of feed antibiotics (National Chicken Council, 2019). This type of breeding is called NAE (No Antibiotics Ever). Broiler chickens kept with this system are prohibited from administering any antibacterial agents in feed mixture, water or injections throughout whole life (Newman, 2018). The mortality of broiler chickens in NAE production has increased by 25-50% compared to conventional production (Salois, 2017) and is on average around 4.2%, while in conventional production using antibiotics approximately 2.9% (Ritter et. al, 2019). Feed costs are main part of the overall animal production cost and according to Donohue and Cunningham (2009) it could be up to 80% of production costs. Overall cost of feed can be affected by demand from other markets than animal production, such as biofuel industry and its demands for corn or wheat, which are main component in feed mixtures (Donohue and Cunningham, 2009; Masey O'Neill et al., 2012). Therefore it is necessary to find new products such as agricultural by-products with relatively high nutritional value.
Poultry fat is interesting in terms of fatty acid composition, especially due to the higher content of unsaturated fatty acids compared to mammals. However, internal fats are considered as one of the wastes from poultry processing. In previous experiments, we observed the effect of grape, flax and pumpkin pomace on the meat performance and chemical composition of broiler chicken muscle. Therefore, the aim of this work was to examine the effect of these pomaces on the formation of internal fats in Ross 308 broiler chickens. The control group (C) was fed with a feed mixture (FM) without antibiotics and growth stimulators. In the experimental diets, wheat in FM was replaced by 1, 2 and 3% red grape pomace, variety Alibernet (groups E1, E2 and E3), 2 and 4% flax pomace (groups E4 and E5) and 2 and 4% pumpkin pomace (groups E6 and E7). The chickens were fed and watered ad libitum for 42 days. Amounts of internal fats are presented as % from carcass weight (CW). Of the internal fats in females, only heart fat was significantly affected (P≤0.05) (least in E5 and E7 -0.20%). In males, on the other hand, we found significant differences (P≤0.001) in other fats: abdominal fat was highest in E2 and E3 (0.94 and 0.91%, respectively), gizzard fat was highest in E2 (0.59%) and internal fats together in E2 and E3 (1.67 and 1.48%, respectively). The results show that negatively higher internal fat content of the chicken carcass was caused mainly by the addition of 2 and 3% RGP in the diet of Ross 308 chickens.
Wine production makes a significant contribution of the agricultural and beverage industry. According to International Organization of Vine and Wine (2021), more than 270 million hectoliters of wine were produced worldwide in 2016. The wine industry generates a huge amount of waste, which consists of stems, GP, sewage waters and yeast sludge. The treatment or disposal of these can have a toxic impact on the environment (Ilyas et al., 2021). According to Mendes et al. (2014) and Garcia-Lomillo and Gonzalez (2017) for every 6 liters of wine, about 1 kg of GP is produced. Traditionally, GP is incorporated into the soil or as feed for livestock. GP can also be used to produce value-added products thanks to various organic acids (tartaric, malic and citric acid), alcohol, fiber and grape seed oil with a positive fatty acid composition (Maier et al., 2007). In addition, GP is a source of various polyphenols, including flavonoids, anthocyanins, proanthocyanidins, and phenolic acids ( The protein content of GP is about 14%, the lipid content 4 -11% thanks to the presence of oilrich seeds. They generally contain a lot of fiber, ranging from 26 to 70%, exceptionally with a lignin level of about 18 -55%. The sugar content can vary from 4 -9% in RGP up to 28 -31% in pomace from special types of white wine (Heuze and Tran, 2020). The nutrient and bioactive compounds present in the GP make them a suitable candidate for use in animal nutrition (Aditya et al., 2018). The fatty acid composition of monogastric animals is directly affected by the fatty acid composition of the feed. Flaxseeds are characterized by high concentrations of α-linolenic acid (ALA) among oilseeds. They contain 35 -45% fat, of which ALA represents 45 -52%. Therefore, the inclusion of flax seeds as a rich source of omega-3 fatty acids in the nutrition of monogastric animals makes it possible to adjust the fatty acid profile in poultry meat, in particular the omega-6:3 ratio (Newkirk, 2015). On the other side, due to their fatty acids' composition, such diets are exposed to a higher lipid oxidation and may require the use antioxidants reported that pumpkin seeds contain 30.6% crude protein, 4.5% ash, 34.5% fat, and 16.24% crude fiber; protein content in the cake after pressing the pumpkin seeds can even be up to around 54%. Production of wine, flaxseed and pumpkin oil is associated with production of voluminous by-products also known as oilcakes or pomace. These, as mentioned above, still have an interesting nutritional composition and therefore also in view of our previous researches (Haščík et al., 2020; Čech et al., 2021; Jurčaga et al., 2021) the aim of this study is to explore potential negative effect of red grape, flax and pumpkin pomace on the amount of internal fats in broiler chickens Ross 308.

Animals and experimental design
The experiment was realized in the Slovak University of Agriculture (SUA) in Nitra (Test poultry station Kolíňany) according to methodology of Haščík et al. (2020). Diets were prepared following the recommended reference levels (Bulletin of MARD SR, 2005) to meet nutritional needs of broiler chickens. Broilers were subjected to a two-phase feeding program, starter HYD-01 (1 -21 d) and grower HYD-02 (22 -42 d) diets. Their composition are presented in Table 1. The starter and grower feed mixtures were produced without any antibiotics and coccidiostatics and were prepared by Biofeed, Inc. (Kolárovo, Slovak Republic). The experimental groups were set up as follows: the control group (C) fed with complete FM; the experimental group E1 fed with basal diet plus RGP 1 kg.100 kg -1 of FM; experimental group E2 fed with a complete FM plus RGP 2 kg.100 kg -1 of FM, experimental group E3 fed with a complete FM plus RGP 3 kg.100 kg -1 of FM, experimental group E4 fed with a complete FM plus flax pomace 2 kg.100 kg -1 of FM, experimental group E5 fed with a complete FM plus flax pomace 4 kg.100 kg -1 of FM, experimental group E6 fed with a complete FM plus pumpkin pomace 2 kg.100 kg -1 of FM and experimental group E7 fed with a complete FM plus pumpkin pomace 4 kg.100 kg -1 of FM. 11.92 11.92 *active substances per kilogram of premix: vitamin A 2,500,000 IU; vitamin E 50,000 mg; vitamin D3 800,000 IU; niacin 12,000 mg; D-pantothenic acid 3,000 mg; riboflavin 1,800 mg; pyridoxine 1,200 mg; thiamine 600 mg; methadione 800 mg; ascorbic acid 50,000 mg; folic acid 400 mg; biotin 40 mg; cobalamin 10.0 mg; choline 100,000 mg; betaine 50,000 mg; Mn 20,000 mg; Zn 16,000 mg; Fe 14,000 mg; Cu 2,400 mg; Co 80 mg; I 200 mg; Se 50 mg.

Slaughter and measurements
After fattening period (42 days), 10 males and 10 males of hybrid combination Ross 308 were selected from each experimental group based on average weight. Broiler chickens were weighed for determining the live weight and then killed by conventional neck cut in the experimental slaughterhouse of the Institute of Food Science (SUA, Nitra). After bleeding out, the feathers were removed from the broiler chickens and they were dissected. The carcass weight was determined and thereafter the abdominal fat, gizzard fat, heart fat and these fats together were weighted after the dissection. By calculating from the carcass weight, we found out their percentage.

Statistical analysis
The obtained results were subjected to statistical analysis by XLSTAT program (Addinsoft, Paris, France, 2017), using analysis of variance (ANOVA) to obtain baseline values, which are presented as mean ± standard deviation. To determine the statistical evidence between the individual experimental groups, we used the Duncan test and the differences were considered significant at P≤0.05.

RESULTS AND DISCUSSION
The results of an experiment aimed at determining the percentage of internal fat from carcass weight after the addition of Alibernet variety RGP, flax and pumpkin pomace are given in Tables 2 and 3, respectively: carcass weight, % of abdominal fat, % of gizzard fat, % of heart fat and % of listed fats together. Legend: C -Control group; E1-E7 -Experimental groups 1-7 CWcarcass weight; % AFpercentual amount of abdominal fat; % GFpercentual amount of gizzard fat; % HFpercentual amount of heart fat; % FTpercentual amount of internal fats in total; a,b,cdifferent superscripts mean significant differences between experimental groups at significance level P≤0.05 or P≤0.001 In females we found the highest carcass weight after application of 3% RGP (group E3) and 4% linseed pomace to the FM (group E5), namely 1478.20 g and 1511.80 g, respectively. These differences were significant (P≤0.001) compared to the control and all the other experimental groups. On the contrary, the significantly lowest (P≤0.001) carcass weight was observed after application of 2% pumpkin pomace (group E6), namely 1277.60 g. In males, the highest carcass weight (P≤0.001) was also observed after the application of RGP in an amount of 3% (group E3) in comparison with experimental groups E5 and E6. The significantly lowest carcass weight (P≤0.001) in males was in the experimental group E5 after the application of 4% of flax pomace, in comparison with all experimental groups except E6. In females, we observed significant differences only in % of heart fat, which namely after the addition of 4% flax pomace (E5) as well as after 4% pumpkin pomace (E7), both 0.20%. These differences were significant (P≤0.05) compared to experimental groups E1, E2, E3 and E4. We did not observe any significant differences in other internal fats and their amounts ranged from 0.50% (E6) to 0.73 (E3) in abdominal fat, from 0.32% (E7) to 0.53% (E5) in gastric fat and for fats together from 1.03% (E7) to 1.37% (E5).
In males, on the other hand, we did not observe significant differences only in the % amount of the heart fat, where it was in similar values in all experimental groups, on average 0.13%. On the other hand, we observed significant differences in all other parameters. Abdominal fat was the highest (P≤0.001) in males after application of RGP in the amount of 2 and 3% (group E2 -0.94% and E3 -0.91%) compared to all other experimental groups and the least in group E6 after application 2% pumpkin pomace -0.40%. Similarly, the significantly lowest (P≤0.001) proportion of gizzard fat was also in this experimental group, namely 0.22% and the highest in experimental group E2 -0.59%. The above results were also reflected in the % of total internal fats from the carcass in males and thus the significantly highest (P≤0.001) amounts were observed after the application of RGP in the amount of 2 and 3% (group E2 -1.67% and E3 -1, 48%) compared to the other groups and the significantly lowest amount of internal fats after application of 2 and 4% pumpkin pomace (group E6 -0.90 and E7 -0.94%). In our previous researches, we found out, that addition of RGP into Ross 308 broiler chicken diet significantly increased live body weight (Haščík et al., 2020), also increased amount of desirable intramuscular fat as taste promoter ( showed that weight of total internal fat of chickens fed with the additional linoleic acid was higher (males -2.51% and females -2.75%) compared to control group without addition (males -2.38 and females -2.61%). Similar results as ours were found by Haščík et al.
(2019), when total internal fats were in the control group 1.94%, in the experimental group supplemented with bee pollen extract + probiotics 2.01% and in the experimental group supplemented with propolis extract + probiotics 1.94%.

CONCLUSION
The aim of this work was to investigate the effect of grape, flax and pumpkin pomace as voluminous agricultural by-products on the percentage of internal fats from the carcass weight of Ross 30 broiler chickens. As the solved internal fats are considered as waste in poultry processing, the application of RGP, which generally increased their amount in both females and males in experimental groups E2 and E3, seems inappropriate. On the other hand, the addition of flax (except of E5 group in females) and pumpkin pomace either did not increase or even decrease the percentage of internal fats, even significantly in comparison with either the control group or the mentioned experimental groups E2 and E3. The most significant reduction in the total fat content was observed especially in males after the application of pumpkin pomace (P≤0.001) in comparison almost with all other groups. Overall, however, it should be noted that there are too few of the same studies that could confirm or refute our findings. It would also be useful to look for ways to reintroduce chicken fat into the food chain, given its favorable fatty acid composition. On the other hand, the results also show that the experimental groups fed with RGP also had a higher carcass weight as important economical parameter, especially in males compared to the groups fed with pumpkin and flax pomace.