Influence of feed substitution with acorns on fattening performance, carcass characteristics, and meat composition of Prestice Black-Pied pigs finished in a conventional indoor system

Abstract With their considerable nutritive value, acorns have long been a natural feed source for pigs and, in recent years, fattening pigs with acorns has become very popular even in countries where it has not traditionally been customary. The meat of pigs finished with acorns has a unique flavour and fat profile as reflected by fatty acids content. We decided to determine the nutritional value of the most common species of acorns in Central Europe (Quercus robur) and examine the effects of this feed upon fattening performance, carcass value, and the composition of meat and fat produced by indoor finishing of pigs, as forest grazing is prohibited here. We conclude that the Prestice Black-Pied breed can be fattened utilizing a mixed feed containing acorns at the level of 10% under intensive conditions without significantly changing the performance parameters (average daily gain was 750 g versus 761 g in the control group). We found improvements in fatty acid profiles of intramuscular fat away from saturated toward monounsaturated. The improvements in the fatty acid profiles, similar to those seen in pigs raised in sylvan settings, create pork that may benefit human health. Acorns can constitute an alternative feedstuff even for pigs finished indoors and bring new benefits to agriculture in modern times.


Introduction
The wild boar (Sus scrofa) prefers to live in mature broadleaf or mixed forests with energy-rich plant foods, such as acorns, chestnuts, beechnuts, or others depending upon its range (Schley & Roper, 2003).Therefore, acorns have long been a natural feed source for pigs.Acorns have high nutritive value.They were also a part of human diets in the past and today they are regaining popularity among recommendations as to their health benefits.Biologically active substances enable acorns to be utilized in the preparation of functional foods (Rakić et al., 2006).Acorns obtained from certain species, such as the Quercus brantii tree, have been used in folk medicine (in Turkey), and some studies have reported their antioxidant, antimicrobial, anti-inflammatory, gastroprotective, and antitumoral properties (Dogan et al., 2015).Acorns are receiving increasing attention today for their potential as sources of essential fatty acids and of tocopherol (Akcan et al., 2017).
Fattening pigs with acorns has recently become very popular, even in countries where forest grazing is not maintained.This is due not only to efforts to raise animals in natural conditions and respect their welfare but also in connection with the increasing demand for meat produced naturally or of organic quality.The meat of pigs finished with acorns has a unique flavour and fat profile as reflected by its fatty acids content, in particular by increased oleic acid (Pugliese et al., 2008).This alters the nutritional properties of the pork products and changes the proportions of fatty acids, such as being more advantageous for human nutrition from a cardiovascular perspective (Fallola et al., 1998).Although the resulting pork has higher fat content than does that of commonly reared pigs, it is healthier for consumers due to its high content of beneficial omega-3 fatty acids and oleic acid, which, among other effects, favourably influence cholesterol levels (Petron et al., 2004).
The fact that cereals are utilized as pig feed when they could be consumed directly as human food is often criticized.Livestock consume one-third of global cereal production and use about 40% of global arable land (Mottet et al., 2017).Acorns represent an alternative for sustainable production of pig feed, particularly in the context of a possible agricultural transformation from cereal crops toward nut trees, as has been mentioned by British permaculture expert Patrick Whitefield in his book The Earth Care Manual (Whitefield, 2011).Using an agroforestry system in temperate regions can help to mitigate many of the negative impacts of agriculture by regulating soil, water, and air quality; reducing inputs; and modifying climates (Smith et al., 2013).
In the Czech Republic, the tradition of grazing has not been preserved as in the states of southern Europe, but about 60 years ago, grazing was still a common phenomenon here.This is reflected in nutrient value tables for feedstuffs from 1965 (Čvančara, Rafii et al., 1991).In the following decades, agriculture in the Czech Republic was intensified and so feedstuffs gradually changed.We have decided to update the information and determine the nutritional value of acorns commonly occurring in the landscape, as well as to evaluate the effects this feedstuff has on the growth of pigs and the quality of meat produced when feeding acorns.The indigenous Prestice Black-Pied (PBP) breed can be used for these purposes.It is very adaptable, undemanding, and capable of utilizing pasturing.Compared to improved breeds, the PBP pig is characterized by a lower growth performance and higher carcass fatness.On the other hand, characteristics such as hardiness and adaptability have been preserved in this breed.Similar to other indigenous European breeds, the pigs are suited to extensive rearing conditions.Despite these facts, most of the PBP pig population is kept within a conventional indoor system (Dostálová et al., 2020).
With the presently increasing numbers of alternative and organic farms in the Czech Republic, the use of grazing and outdoor methods of pig farming has been partially renewed.However, these methods are not suitable for the dominant modern, highly demanding genotypes and are applicable only for small concentrations of animals in the conditions of our country.Besides, forest grazing is a legally prohibited activity in most Central and Eastern European countries (Varga et al., 2020).Due to the risk of African swine fever being introduced and spread in relation to outdoor pig farms, the European Union member states and Commission currently have a general recommendation that outdoor keeping of pigs be prohibited (EFSA Panel on Animal Health and Welfare, 2021).On the other hand, there is increasing demand for meat with high nutritional value, of organic quality, and produced in a sustainable manner.From this point of view, feeding acorns to pigs is a very appealing alternative for using local raw materials produced in a natural way.Thus, the positive effect of these fruits on animal nutrition and human nutrition could be taken advantage of.The inclusion of acorns is also an option for reducing inputs to feed production.
The aims of this study were to determine the nutritional value of acorns of species Quercus robur, to evaluate the use of acorns in feed rations suitable for indoor fattening of PBP pigs, and to verify this alternative feedstuff's effect on fattening performance, carcass characteristics, and the composition of meat and fat so produced.

Material and methods
The work utilized fallen acorns from English oaks (Quercus robur), which constitute the most common oak species in Central Europe.Acorns were collected in October, at five locations within one region of the Czech Republic.A sample was taken from each site for analysis, then all the acorns were mixed and used for the experiment.Dried, unpeeled acorns were analysed according to standard methodologies used for other feedstuffs.Dry matter was determined gravimetrically after drying for 4 h at 105°C.Nitrogen was determined after converting nitrogen compounds to ammonium sulphate through mineralization with sulfuric acid and distilling ammonia through alkalimetric titration.Nitrogen substances were then determined by multiplying the amount of nitrogen by 6.25.Fat was determined using ether extraction with the extracted fat determined gravimetrically.Ash was determined gravimetrically as the residue after the complete burning of organic matter at 550°C.Fibre was determined as the solid residue after acid and alkaline hydrolysis using sulfuric acid and potassium hydroxide after deducting ash gravimetrically.Starch was determined polarimetrically after hydrolysis using a 1.125% solution of hydrochloric acid.Saccharose content was determined by extracting water and then clarifying the water extract with Carrez reagents.Carbohydrate content before and after inversion with 0.1 M HCl was determined in an aliquot of the solution using the Luff-Schoorl method.Fatty acid composition was determined after chloroform-methanol extraction of total lipids and alkaline trans-methylation of fatty acids.Gas chromatography of methyl esters was carried out using a Hewlett Packard 5890 Series II chromatograph and evaluated in accordance with the Supelco 37 Component FAME Mix standard.
The proposed mixed feed containing acorns was balanced using the Optimization of Feed Mixtures for Pigs software program (Agrokonzulta Žamberk, Žamberk, the Czech Republic) to the same levels of nutrients as contained in a control mixed feed.Metabolizable energy of the diet was calculated according to accepted standards (Hoffmann & Schiemann, 1980).
The designed acorn diet was verified in practice through a feeding experiment with 20 pigs of the PBP breed.The experimental conditions corresponded to the principles laid down in EC Directive 2010/63/EU regarding the protection of animals used for experimental and other scientific purposes.
A total of 20 pigs were divided into two groups with a balanced sex ratio (five males and five females per group).Males had been castrated at 5 days of age.The experimental group (A) was fed the acorn mixture containing 10% dried and shredded acorns.The control group (C) was fed the basic feed mix.The composition of the diets is shown in Table 1.The animals were fed ad libitum and housed indoors in group pens with full concrete flooring and bedding.The experiment was started at a mean pig weight of 74 kg.Animals were weighed individually at weekly intervals, and feed consumption was recorded at the same time.The average daily gain was calculated from the initial and final weights of individual animals.Feed intake and conversion were determined from group consumption of the mixed feed and recalculated to a per-pig value.
At the end of the trial, faecal samples were collected daily from each group for 3 days and stored at −20°C.Faecal samples were thawed, mixed, and subsamples taken for the determination of moisture, crude protein, ether extract, crude fibre, and acid-insoluble ash.The level of acidinsoluble ash in diets and faeces was determined using the method of Vogtmann et al. (1975).The apparent total tract digestibility (ATTD) of nutrients in the experimental diets was determined while using acid-insoluble ash as an indicator (McCarthy et al., 1974).The ATTD of dry matter (DM) in the experimental diets was calculated using the following equation (Biagi et al., 2016): 100 -[(100 × % acid-insoluble ash in the diet)/% acid-insoluble ash in faeces] The apparent digestibility of crude protein, ether extract, and crude fibre was calculated using the following equation: 100 -(% nutrient in faeces × (100 -DM digestibility)/% nutrient in the diet).
The pigs were slaughtered at a mean weight of 117 kg.The carcasses were classified 45 min after slaughter.Lean meat content was determined by the ZP-Method (two-point method) of the SEUROP system (European Commission, 2005).The following equation was used for the calculation: Lean meat (%) = 59.08991 − 0.433868 × S + 0.09792 × M where S is the backfat thickness (mm) with skin measured at the point of the lowest layer above the middle of the musculus gluteus medius, and M is the muscle thickness between the cranial top of the musculus gluteus medius and the dorsal edge of the spinal canal (mm).

Samples of muscle (musculus longissimus lumborum et thoracis [MLLT]
) and backfat (BF) from loin were collected between the second and third to last rib 24 h after slaughter.Drip loss value was calculated as weight loss of the fresh meat sample (150 g) after storage for 24 h at 4°C.The drip loss value was expressed as a percentage of the initial weight according to Honikel (1998).Samples of the MLLT and BF were frozen for later chemical analysis.
The data are presented in the tables as arithmetic means and standard deviation (SD).Student's t-test was used for analysis of the data, and the differences among means in the ttest were considered significant only if the F-test result was significant.Differences between the assessed groups were considered significant at P < 0.05.The experimental data were statistically processed by QC expert (TriloByte Statistical Software Ltd.).Feed intake, feed conversion, and digestibility of nutrients were measured per group (i.e., they provide descriptive information without statistical evaluation).

Results of feed analyses
The mean nutrient composition of acorns as determined by analysis is shown in Table 1 Based on the analyses, a mixed feed containing 10% dried shredded acorns was designed and balanced to the same nutrient level as was the control mixture.The proposed mixtures are composed of common components used in commercial feed mixes for fattening pigs.Ingredients and nutrient composition of the diets are given in Table 2.
The ATTD of crude protein, ether extract, and crude fibre was determined using an indicator method, where the indicator was acid insoluble ash, which occurs naturally in feed and does not affect health or natural processes in the animal.The calculated ATTD values (%) are shown in Table 2.In experimental Group A, a decrease was recorded for all monitored nutrients compared to Group C. In the case of ether extract, the mean difference between diets and pig groups was rather small, but the ATTD values for crude protein and especially for crude fibre were more substantial.

Fattening and slaughtering traits
Table 3 presents the performance parameters attained during the experiment.Pigs fed the mixture with added acorns achieved an average daily gain of 750 g over the whole period of the experiment.The control group gained 761 g.This difference was not statistically significant.Feed consumption was 3.17 kg/day for Group A and 3.08 kg for Group C, corresponding to an average feed conversion of 4.19 and 4.05, respectively.Significance levels: ns = not significant.
Carcass trait and meat quality parameters are presented in Table 4. Group A on the acorn diet had slightly lower lean meat content and higher measured BF thickness.Neither of these differences was statistically significant.The drip loss parameter for both groups was at a level common for meat of the breed (Dostálová et al., 2020;Matoušek et al., 2016).Group A had slightly higher drip loss than did the control group (2.9% vs. 2.6%), but again without being statistically significant.
Meat from Group A pigs had lower protein content and higher intramuscular fat content, but these differences were not statistically significant.The only statistically significant difference (P < 0.05) was the lower dry matter content of Group A compared to the control group (25.94% vs. 27.43%).

Fatty acid composition of feed rations, muscle, and backfat
Acorns were analysed for fatty acids composition.Thirty fatty acids were determined, including some of the most important saturated fatty acids (SFAs) and the most important representatives of the unsaturated fatty acids, as well as the total value of SFAs, monounsaturated (MUFAs), and polyunsaturated fatty acids (PUFAs).These values are given in Table 5.Among the fatty acids, oleic acid (C18:1n9) was the most abundant (35.82%), linoleic acids (C18:2n6) were found in large amounts (29.27%), and palmitic acid (C16:0) was third-most abundant (17.12%).Stearic (C18:0) and α-linolenic acids (C18:3n3) were found in smaller amounts (2-5%).Other fatty acids were found in proportions <1% or in trace amounts.The proportions of these fatty acids are partially similar to those of acorns of Quercus rotundifolia and Quercus ilex found in Spain, Portugal, or Italy (Akcan et al., 2017;Rafii et al., 1991).Table 5 also shows the results of diet analysis for fatty acids and the ratio of omega 6 and omega 3 fatty acids.This ratio was changed in a favourable direction (from 5.93 to 4.98) by adding acorns to the mixture.
Tables 6 and 7 report the fatty acids composition of MLLT and BF, respectively.Shown are the contents of five SFAs, four MUFAs, and six PUFA detected in the samples.Oleic acid (C18:1n9), palmitic acid (C16:0), stearic acid (C18:0), and linoleic acid (C18:2n6) were most abundant in the muscle and fat samples of both groups.Analysis of fatty acid composition showed some significant differences between pigs fed with the control versus acorn diet.The proportions of C18:1n9 and C20:3n6 in muscle were greater by statistically significant amounts at the P < 0.001 level when feeding the acorn diet.On the contrary, saturated acid C:20 was lower as determined at the same level of significance (P < 0.001).This indicates an overall trend, as documented by the sums of SFAs, MUFAs, and PUFAs, wherein acorn diets produced lower SFAs content in muscle (P < 0.001)  and the MUFAs content increased (P < 0.01).The difference in PUFAs content was not statistically significant.
The results observed in BF were slightly different, as the most significant difference (higher in Group A) occurred in C16:1n7 (P < 0.001) and MUFAs (P < 0.05).Again, SFA values were lower for Group A, but not significantly so (P > 0.05).The sums of PUFAs n-3 and n-6 in both groups (both diets) did not differ with statistically significance in either muscle or BF.

Discussion
Because pig grazing and the use of acorns for feed purposes were common in the Czech Republic in the middle of the twentieth century, the nutrient composition of acorns was compared with that from data in the literature at that time.Considering their high starch content (40.85%), acorns can be classified as a high-carbohydrate feed, as previously.There are slight differences in fat and fibre contents, with our analysis recording higher values in comparison to the earlier nutritional table values (Čvančara, 1965).The composition and nutritional values of acorns had also been provided in Nehring's Textbook of Animal Nutrition and Feed (1952).For dried acorns, it states the following values: dry matter 81.5%, protein 7.5%, fat 4%, fibre 9%, nitrogen-free extract 59%, and ash 2%.This gives an especially different value for protein content that is substantially higher than the value determined in the present study as well as the value stated in the historical nutritional value tables (Čvančara, 1965).Differences could be due to harvest location, year, or sampling, similarly as has been observed in other acorn species (Akcan et al., 2017;Tejerina et al., 2011).
The apparent total tract digestibility (ATTD) of nutrients in experimental diets was determined.A decrease of ATTD (%) in experimental Group A was recorded for all monitored nutrients compared to Group C. In the case of ether extract, the difference was not very substantial.The more markedly lower ATTD values for crude protein and crude fibre may have been affected by higher crude fibre levels, similarly as seen in other alternative feed components (Dong et al., 2019).The decrease was probably also due to the presence of anti-nutritional factors in the acorns, specifically tannins, which are present in the plant and fruits of oak (Nehring, 1952).The presence of tannins at high levels can cause significant reductions (by as much as 23%) in protein and amino acid digestibility in rats, poultry, and pigs (Sarwar Gilani et al., 2012).There was a reduction in the digestibility of crude protein by 5.72% compared to Group C. The reduced digestibility of nutrients was partially offset by 2.9% greater feed intake in Group A (3.17 kg/day vs 3.08 kg/day in the control group).Similarly, Prevolnik et al. (2012) had found 3% higher consumption and conversion ratio in pigs supplemented with tannins extract.Although tannins are regarded as antinutritive substances, they could have positive influence on growth at low concentration due to their effect on gut health (Goel et al., 2005).There was somewhat less weight gain in Group A (average daily gain 750 g vs. 761 g of the control group) throughout the experiment period as a whole, but the difference was not statistically significant (P > 0.05).We therefore can conclude that the feeding mixture with acorns did not affect the performance parameters in the experiment.
The finishing system with acorns did not affect carcass traits in a statistically significantly (P > 0.05) manner.The group fed an acorn diet had slightly lower lean meat content and higher measured BF thickness, but these differences, too, were not statistically significant.On the contrary, Morales et al. (2003) had found BF thickness of pigs given a diet with acorns tending to be lower than that of pigs fed a diet with maize, while there were no differences in the intramuscular fat content.
In the present experiment, the impact of diet on the chemical composition of meat was not large.This was reflected solely in lower DM content of the meat in Group A (P < 0.05).Group A also produced greater intramuscular fat, but this difference was not statistically significant.This is consistent with the finding of Almeida et al. (2018).In their study, the finishing systems (finished intensively or on pasture and acorns) did not affect the chemical composition of meat.Also, Millet et al. (2005) found in their study that organic nutrition from weaning until slaughter compared with a conventional diet did not affect growth performance or carcass quality in pigs.
In assessing overall effect of the experimental feed mix's on performance parameters, one should consider that this native breed does not perform at the same level as do modern pig genotypes but, on the other hand, is able to adapt to alternative, less highly concentrated feeds.Comparison with other native breeds is also difficult due to the different characteristics of the breeds.Especially Iberian pigs, which are traditionally fed acorns, constitute a very specific breed different from the partially improved PBP pigs.In addition, fattening by acorns usually takes place in outdoor farming under extensive conditions, where pigs have free access to acorns, often in combination with grazing.There are a number of publications on the use of acorns in fattening pigs, mostly from Spain and Italy, but these always concern free-range conditions (Almeida et al., 2018;Pugliese et al., 2009;Ventanas et al., 2007).The present experiment took place under intensive, indoor feeding conditions and showed that it is possible to use acorns even with this method of fattening.It can be used to produce meat for local markets, as an alternative in case of a feed shortage, or to reduce the price of feed in a conventional indoor system.It is the fatty acids profile that makes acorns potentially important for animal and human nutrition.This is also one of the main factors affecting the quality of meat and products from the Iberian pig that is fed extensively on this natural feedstuff (Ventanas et al., 2005(Ventanas et al., , 2007)).It is broadly agreed that acorns constitute a substantial source of oleic acid, which is predominantly present in acorns and often exceeds 60% of the total fatty acid content (Cantos et al., 2003;Ferreira Dias et al., 2007;Tejerina et al., 2011).Akcan et al. (2017) present acorns as a novel source of oleic acid and tocopherols for livestock and humans.The concentration of oleic acid in this fruit is significantly greater than in other fruits.This is important in relation to the positive effect that acorns consumption can have.The proportion of individual fatty acids in acorns varies among species of oaks and also between harvest years (Tejerina et al., 2011).In samples of Quercus robur, the oleic acid content was lower compared to that in the species growing on the Iberian Peninsula, but even here this fatty acid was the most abundant of all (35.8%).The fatty acid profile and fat content in our Q.robur samples are closest to those of Q. pyrenaica acorns.Ferreira Dias et al. ( 2007) state that oil from Q. pyrenaica acorns is rather unsaturated and different from olive oil.The highest levels of linoleic (25.1-34.3%)and linolenic acids (1.4-2.3%) and lowest oleic acid content (43.4-52.2%)were observed in the oil from this fruit compared to other types of acorns.
The meat of monogastric animals tends to reflect their food intake, and especially during the final stage of fattening, as in the case of the Iberian pig (Tejerina et al., 2011).The high oleic acid content in acorns greatly affects muscle lipid composition in animals fed these fruits, as has been confirmed by a number of studies (Petron et al., 2004;Pugliese et al., 2009;Tejeda et al., 2002).In addition to the increase in oleic acid content, these authors also noted a decrease in SFAs, especially stearic (C:16) and palmitic (C:18).Similarly, in our experiment, the changes in the fatty acid profile of the feed affected by the admixture of acorns were reflected in the presence of fatty acids in the muscle.We recorded a statistically significant increase in oleic acid content (P < 0.001) and a decrease in palmitic acid (P < 0.01).A decrease was also measured in stearic acid but without statistical confirmation (P > 0.05).Lower SFAs content in muscle was observed in the experimental group along with higher MUFAs content compared to the control group.This effect was less pronounced in BF of the experimental animals.Because the cause is not clear, it would be appropriate to obtain additional data for clarification.The lower SFAs level was not statistically significant, but there was also greater oleic acid content (P < 0.01) and the associated higher MUFAs level (P < 0.05) as already observed in muscle.The high degree of MUFAs in pig fat affects in a positive way the organoleptic sensations while also providing a healthy nutritional quality from the cardiovascular point of view (Fallola et al., 1998).
Regarding SFAs, palmitic (C16: 0) and stearic (C18: 0) acids predominate within the SFAs group in animal fat (Kasprzyk et al., 2015).This is consistent with the experimental data presented here.Due to the acorns diet, the SFAs content in intramuscular fat was significantly lower (P < 0.001), and particularly so for C16:0, C17:0, and C 20:0.Ulbricht and Southgate (1991) stated that, among the SFAs, lauric (C12:0), myristic (C14:0), and palmitic (C16:0) acids increased plasma cholesterol concentration.Excessive SFAs intake has been regarded as one out of many other factors associated with cancer and coronary heart disease (Webb & O'Neill, 2008).Significantly lower content of this group of fatty acids in meat can thus clearly be regarded as beneficial for human health.
It is further recommended that the PUFA:SFA ratio be above 0.4 (Kasprzyk et al., 2015).This ratio was higher in the pigs fed the acorn mixture compared to the control group, and it slightly exceeded the stated limit of 0.4 in the BF (Group A: 0.41 vs. Group C: 0.37).The n-6:n-3 fatty acid ratio is also important due to its influence on human health.By adding acorns to the mixed feed, this ratio in the mixture was desirably reduced.The ratio was lower in the intramuscular fat of Group A compared to Group C, while in BF it was higher.
For a better comparison of the fatty acids profile, the atherogenic index and thrombogenicity index were calculated according to Ulbricht and Southgate (1991).These results are shown in Tables 6 and 7. We found values that are consistent with the findings of Kasprzyk et al. (2015) and other authors.Indices were lower in the acorn-supplemented group, both in muscle and BF, similar to what was found by Dostálová et al. (2020) in PBP pigs in an outdoor housing system.The results are again positive in terms of human health.

Conclusions
We conclude that the PBP breed can be fattened on a mixed feed ration containing acorns at the level of 10% under intensive conditions without significantly changing the performance parameters.Acorns constitute a high-quality and natural feed with positive effect on the quality of the produced meat.Improvements in the fatty acid profiles away from SFAs towards MUFAs, similar to as seen in pigs raised in a sylvan setting, create pork that may reduce cardiovascular risk factors and prediabetes in humans (Talbott et al., 2006).Another benefit of acorns in a diet for pigs is that it can be a sustainable food source.It would also be appropriate to verify the possibility of using acorns as a feed component in larger quantities, given that Iberian pigs fed acorns consume as much as 9 kg of the feedstuff per day.Oaks could then be integrated into modern agricultural systems and contribute to a transformation towards agroforestry, which offers the benefits of reduced inputs and a better nutrient cycle, soil health, and pest control (Brodt et al., 2020).That acorns are a quality feed for pigs certainly has been proven for thousands of years, and they can produce new benefits in modern times.
The research meets the principles laid down in EC Directive 2010/63/EU regarding the protection of animals used for experimental and other scientific purposes.

Table 2 . Ingredients and nutrient composition of mixed feeds Control mixture Acorn mixture
Notes: ME-metabolizable energy, ATTD-apparent total tract digestibility.