Interannual variability and evolution during the montanera period of Holm oak ( Quercus rotundifolia Lam . ) acorns

Eighty-eight samples of Quercus rotundifolia acorns were analyzed during three consecutive years (2005/06, 2006/07 and 2007/08) to determine variations in their morphology, sanitary condition, and nutritional composition throughout the same period of montanera (free-range swine-raising system of SW Iberia) and between different years of study. Results showed that morphological characteristics remained constant throughout montanera season. Fungal damage increased, and Curculio sp. and Cydia sp. pests decreased over the course of the montanera season. Dry matter content decreased in both kernel and pericarp (P < 0.001), while the crude fiber and protein contents were not modified. The ash, nitrogen-free extract, and fat contents, however, were also affected by sampling date within a montanera season. With respect to the interannual variability, variations (P < 0.05) were observed in all the variables studied. It is therefore confirmed that there is variability in the sanitary condition and nutritional composition of acorns within the same montanera season and between different years of study. These could explain the differences in the chemical characteristics of Iberian pig tissues from animals raised in different montanera seasons, and even during the same montanera season. Additional key words: acorn sanitary condition, dehesa, feeding, Iberian pigs, proximate composition.


Introduction
Species of the genus Quercus represent the fundamental arboreal element in the «dehesa» ecosystem typical of Southwestern Europe.It is a Mediterranean forest type in which various species of Quercus coexist, and the primitive understorey has been eliminated by man to favor the growth of annual grasses and legumi-Interannual variability and evolution during the montanera period of Holm oak (Quercus rotundifolia Lam.) acorns nous herbs (Olea et al., 1989;Devesa, 1991).In this ecosystem, pigs of a local breed (Iberian) are bred in a free-range system, with a diet based on natural resources (acorns and grass), from October-November until January-February.This period is known as montanera.
The «dehesa» and the production of acorns for the feeding of the Iberian pig are intimately linked.This is reflected in the Spanish legislation by the Royal Decree 1469/2007 of 2 November (BOE, 2007) in which the Ministry of Agriculture and Fisheries directly associates the Iberian pig with the «dehesa» ecosystem.
The great social acceptance of the products derived from this system is mainly due to the high quality of the end products that result from this linkage.This quality is chiefly the result of the monounsaturated fatty acids (MUFA) that the acorn contributes to Iberian pig meat (Muriel et al., 2002).
The analysis of fatty acid composition has been the evaluation method used in Spain since the beginning of the 1980s for the quality discrimination of these products.Today, this method is widely questioned in the Iberian pig sector, and has fallen into disuse.Indeed, the lipid profile of the Holm oak (Quercus rotundifolia) acorns in Spain varies enormously (Vázquez and Doncel, 2002), and there are marked interannual variations within a single livestock rearing zone (Vázquez et al., 2001).
Preliminary studies have considered the nutritional composition and digestibility of acorns (Vázquez, 1998a;López-Carrasco et al., 2005;Rey et al., 2006) of both Q. rotundifolia and Q. suber (cork oak).Up to now, however, there has been relatively little reference to how these live food resources change over time.The research that has been previously carried out includes studies on the variations observed during the montanera season in the acorns' nutritional composition (Paredes, 1998), phytosanitary quality (Almeida et al., 1992), and their total availability, which determines the duration of the montanera (Benito et al., 1997), as well as the livestock density that the system can support (Aparicio-Macarro, 1987).
The objective of the present work was to study the temporal evolution of the morphology, sanitary condition, and proximate composition of acorns of Q. rotundifolia in a range-farming estate located in Southwestern Europe during the montanera season (November-January), and their variability throughout three consecutive years.
Valdesequera's trees are Holm oak (100%), with a 0.3-0.5% cover fraction, i.e., the fraction of ground area shaded by the vertical projection of the trees' outermost perimeters (measured as per one, units).The soil is a vertisol with a sandy texture and a pH of 5.6-6.7.
Samples were collected fortnightly during each of the montanera seasons (Table 1).For each sample, 2-3 kg of acorns were collected directly from the ground from at least 10 different randomly selected trees per plot (1 kg = 60-110 acorns).
The sampling areas were also randomly selected, and were isolated from the domestic animals bred in the farm.Wild birds could not be fully controlled, but farmers avoided their presence during the day.Acorns were collected directly from the soil and they were not cleared between each sample collection in order to reproduce as closely as possible the reality of the Iberian pig's montanera season.This was important to determine the real variation in the nutritive composition and sanitary condition of acorns, since variation could be due to the ripening stage during the three months of the montanera season.Furthermore, it could be the reason for the great variability in the quality of meat products between different years or within the same year.
The heterogeneity in the number of samples for each year was the unavoidable result of the availability of acorns and the duration of the montanera, both of which depend on such uncontrollable factors as weather conditions (Table 2) or the presence of wildlife feeding on the acorns.The total production of acorns was estimated following the method proposed by Vázquez (1998b).

Acorn analyses
After collection, samples were taken to the laboratory of the «La Orden-Valdesequera» research center of the regional government of Extremadura, for their analysis.The morphological, sanitary, and moisture content characterization was performed on the fresh material, which was then stored frozen at -20°C for the subsequent determination of nutritional parameters.
From each sample batch, 25 acorns were selected at random.After removing the acorn from its cupule of hardened bracts, the length (mm) and width (mm) were measured using an Electronic Digital Calliper (Mod.5900603, COMECTA ® ).The length was taken from the point of the base to the apex, excluding the final appendix, and the width was taken at the widest part of the acorn.
For each sampling point, 50 acorns were chosen at random.The kernel of each acorn was opened, and the percentage of acorns attacked by phytophagous insects (Curculio sp., Cydia sp.) or fungus (Penicillium sp., Aspergillus sp.) was determined visually, and guided by photographic standards to make the evaluation and classification of each type of damage easier (see Soria et al., 1999).The acorn was considered as damaged if there was any indication of eggs, larvae, the presence of the insect, putrefaction, etc.The results were expressed as percentage of unhealthy acorns relative to the total number of acorns.
For each sampling point, 5 subsamples of 10 acorns each were taken, and the pericarp was removed.The dry matter (DM) content was assayed following the official AOAC method (AOAC, 2003) were fresh acorns are oven-dried at 105°C.The results are expressed as g kg -1 fresh-acorn.
Proteins, ash, and crude fiber (CF) were also assayed following the official AOAC methods (AOAC, 2003) applied to the dry-milled sample.The results are expressed as g kg -1 DM.The fat content was assayed following the method described by Folch et al. (1957), again applied to the dry-milled sample.The results are expressed as g kg -1 DM.
The total NFE (nitrogen-free extract) content was calculated following the Weende method (Henneberg and Stohmann, 1867)

Statistical analysis
The statistical analysis of the data consisted of a multiple analysis of variance (two-way ANOVA) using the software package SPSS.PC+ ( 2005), taking into account the sampling date (evolution) within a given period, the year effect, and the interaction of the two (sampling date × year effect).Differences were compared using the means.Tukey's HSD test was applied to compare the mean values of each group.Statistical significance was set at P < 0.05.

Morphology (length and width) of the acorns
The morphological characteristics, length and width (Table 3 The average length of the acorns was also influenced by the interaction between sampling date and study year (P < 0.001).

Sanitary condition of the acorns
The percentage of unhealthy acorns due to the presence of Curculio sp. and Cydia sp.varied throughout montanera season (P < 0.001) (Table 3), being greater at the beginning and lower at the end of the season.In contrast, the percentage of acorns attacked by fungi was greater in the samples at the end than at the beginning of the montanera season (P < 0.01).
There were interannual variations in the proportion of unhealthy acorns.This was highly significant (P < 0.001) with regard to the presence of both Curculio sp. and fungi, and very significant (P < 0.01) in the case of Cydia sp.(Table 3).The year with the greatest incidence of these parasites was 2007/08, with 2005/06 and 2006/07 enjoying a greater proportion of healthy acorns.
The interaction between the sampling date and the effect of the sampling year significantly influenced the presence of Curculio sp. and Cydia sp.This has an overall impact on the final quality of the acorns in each montanera.

Proximate composition of the acorns
The proximate composition of the acorns is shown in Table 3. Dry matter content of both the kernel and the pericarp varied signif icantly (P < 0.001), both throughout the different years and during the montanera season, the highest contents being observed at the last stage.Similarly, fat and ash contents also changed during the montanera season (P < 0.01), and the highest content were also observed in the f inal stages.In contrast, the NFE declined throughout the montanera season (P < 0.01).The protein and f iber contents, however, were unaffected by the sampling date during a montanera season (P > 0.05).
There was a noticeable effect of the sampling year on the concentrations of these parameters, with significant variations observed in all of them (P < 0.001).These variations mean that the f inal quality of the acorns differed between the three years of the study.
The interaction of the year effect and the sampling date in a given year directly affected the NFE, ash, and mean dry matter content of the pericarp (P < 0.001), and to a lesser extent, the dry matter content of the kernel (P < 0.05).

Morphology (length and width) of the acorns
There were no differences in the length and width of the acorns within a montanera season.This was expected since, during the fruiting period, the trees produce acorns of certain dimensions which may vary from tree to tree (Afzal-Rafii et al., 1992).But since acorns then fall and mature on the ground, they cease to receive nutrients from the tree and these dimensions do not change (Vázquez, 2000).Both the data collected for length and width in the present study are coherent with the literature values reported by Vázquez et al. (1992) in a research regarding the characterization of acorns from Q. rotundifolia Lam. in Extremadura (Spain).
There were, however, interannual differences in these parameters.This may have been due mainly to each year's meteorological conditions (see Table 2).The variations in rainfall and temperature would have conditioned the soil properties, affecting the availability of nutrients for the trees, and therefore their fruiting capacity.A year with unfavorable weather conditions would lead to the trees being stressed, thus affecting the amount and size of the fruit they produce.As a matter of fact, this is a clear indication of the interannual variability of acorns in a given geographical zone (Almeida et al., 1992;Vázquez et al., 2001).In the year 2005/06, the total acorn production was lower and the acorns were smaller, in both length and width, than in the other two years of study.And indeed, the meteorological parameters for that year (Table 2) were the most unfavorable, with the lowest temperatures and the least rainfall.These conditions probably put the trees under greater stress, affecting the total production and the size of the fruit, since the trees would have had insufficient nutrients to produce larger fruit.

Sanitary condition of the acorns
During a montanera season, the percentage of acorns attacked by Curculio sp. and Cydia sp. was lowest during the final stages, whereas the presence of fungi was highest during this same period.This inverse relationship between the two types of attack is consistent with the explanation given by Vázquez (2000) that the greater presence of phytophagous insects in the early montanera stages facilitates subsequent fungal attack within the seeds (Vázquez, 1998a).Moreover, phytophage attacks occur when the acorns are still on the tree, and the larvae continue their life cycle while the seed matures on the ground.In contrast, fungi penetrate into the interior when the acorns are already on the ground, and therefore the presence of moisture favors their proliferation (Vázquez, 2000).
The differences observed in the three years of study may have also been due, besides the meteorological conditions, to a greater or lesser presence of plagues of phytophagous insects in the zone, since these have been observed to negatively influence the total acorn production and the phytosanitary quality of acorns (Vázquez, 1998a;Siscart et al., 1999;López-Carrasco et al., 2004).
A greater incidence of phytophages and fungi was found in the 2007/08 montanera, the year in which the percentage of unhealthy acorns was the highest.This may have been because the average temperature was higher during that year, thus favoring an increased presence of phytophagous insects.Furthermore, the average rainfall was also higher, favoring a greater proliferation of fungi.
All this confirms the variability in the phytosanitary quality of acorns during the montanera, as has been reported in other works (Almeida et al., 1992;Paredes, 1998;Vázquez, 1998a).A study of the quality of acorns and its impact on the quality of Iberian pig meat found that the latter indeed depends on the phytosanitary status of the acorns (Daza et al., 2007).

Proximate composition of the acorns
The acorn is a dynamic food source whose proximate composition evolves as the seed matures and in response to external factors such as climate and the presence of parasites (Almeida et al., 1992;Vázquez, 2000).During a montanera season, variability in its nutritive composition and sanitary condition could be due to two mechanisms: new acorns that fall into the ground and pre-existing acorns that are kept for long periods, with different levels of ripening.
Thus, a real tendency could be observed in the results presented in Table 3.As the acorns ripen on the ground during the montanera season, changes take place in their proximate composition, mainly in the dry matter content of both the kernel and the pericarp, and in the ash, fat, and NFE contents.This is because once the acorns have fallen to the ground they are subject -more or less simultaneously-to a series of, at times contrasting, processes of ripening, germination, desiccation, dampening, rotting, and attack by pests.Since all these processes can lead to changes in the acorns' physical (weight) and organoleptic (external aspect, smell, flavor) characteristics and in their proximate composition, there would thus be a continual addition of new elements to the variability experienced by the acorns that are affected by these processes, especially at the end of the montanera season (Almeida and Baptista, 1992;Fernández et al., 2004;López-Carrasco et al., 2004).These biochemical processes (especially germination) consume carbohydrates as fuel, which is why a decline in the NFE content was observed as the ripening and germination processes took place during the montanera season.
Furthermore, the ripening process on the ground itself causes a progressive loss of moisture, which would be reflected in the increasing DM content of both the kernel and the pericarp, and an even greater concentration of minerals.Nevertheless, desiccation is not a clear and constant process, but depends on the environmental humidity and the soil moisture during the montanera months, and on other factors such as adjacent vegetation, which may protect the acorns from inclement weather (Vázquez, 2000).
Variability in the fat content may be due to the presence or absence of phytophagous pests, as López-Carrasco et al. (2004) found that the crude fat of the kernel is reduced in acorns attacked by Curculio sp.Thus, at the beginning of the montanera season, when there was a greater incidence of this pest in the kernels, the fat content was lower.In agreement with the observations of Almeida and Baptista (1992), Fernández et al. (2004), López-Carrasco et al. (2004) and Olea et al. (2004) we did not observe changes in the protein and fiber contents.
The amount of feed available decreased over the course of the montanera as a consequence of the attack by phytophages and adverse weather conditions.However, acorns are a highly energetic resource and have large amounts of protein.This situation allows for two different uses of the montanera: i) the first half of the montanera season, with plenty of protein-rich food from acorns, which is useful for the growth of the Iberian pigs; and ii) the second half of montanera season, with a lower food availability, originating in acorns from the top of Quercus trees and with a higher energy content, intended for fattening of the Iberian pigs.
The interannual differences in the parameters studied (see Table 3) could be due to the fact that acorns, like any other fruit, receive their nourishment from the tree, and their chemical characteristics will therefore be influenced by factors such as the greater or lesser incidence of parasites, or by the meteorological conditions (Almeida, 1992;Vázquez, 2000;Vázquez et al., 2001).The latter will affect the soil's characteristics and the availability of nutrients that can be taken up by the tree, thus affecting the tree's fruiting capacity and the total acorn production.
Overall, chemical composition (dry matter, crude protein, fiber and fat contents) agreed with the range of values found in the literature (Cava et al., 1997;Daza et al., 2005;Rey et al., 2006) with slight variations depending upon the season studied.Results evidenced the marked differences in the proximate composition of acorns in different years of study in the same geographical area, as well as in studies in other distinct geographical areas, as has been described in the work by Almeida et al. (1992), Vázquez (2000) and Rodríguez-Estévez et al. (2008).
Variations between montanera seasons and over the course of a montanera with regard to the morphological characteristics, health state, and proximate composition of acorns are relevant factors that affect their consumption by Iberian pigs, thus affecting the final characteristics of their tissues.Recently, Rodríguez-Estévez et al. (2009) described a selective consumption by Iberian pigs for acorns with larger kernels, and a variation in acorn preferences throughout the montanera period.Additionally, the acorns' state of conservation (pest attacks or rotting) has been described to be Variation of acorn composition during montanera a determinant factor on the preference of consumption that further affects their chemical composition.(Steele et al., 1996;Rodríguez-Estévez et al., 2009).In this sense, López Carrasco et al. (2005) and Daza et al. (2007) reported that Curculio sp.infestation affects the acorns' dry matter, crude protein, fat, and crude fiber contents, as well as their fatty acids profiles.These alterations consequently modified the fat content and the fatty acids profiles of Iberian pigs.These authors reported that the consumption of healthy acorns by pigs produced a higher intramuscular fat percentage in Longissimus dorsi than in pigs fed with acorns infested with Curculio sp.Additionally, the subcutaneous backfat, and the intramuscular and liver fat from pigs fed healthy acorns had higher C18:1n-9 and MUFA, and lower C18:0 and SFA proportions than those of pigs fed acorns infested with Curculio sp.These facts confirm that the quality of acorns (size, health state and composition) is not homogeneous and varies over the years and over the course of montanera seasons.These variations could be responsible for differences in the fat content and the fatty acid prof iles of the tissues of pigs reared in different montanera seasons and even from the same montanera season.
In conclusion, the present results have shown that the acorn, as a live material, undergoes a series of changes in its sanitary condition and nutritional composition throughout its ripening stage, which will influence its final quality.
The changes observed during the montanera period and the differences between the three years of study demonstrate that the acorn is a dynamic and variable element of the «dehesa» ecosystem.These dynamic changes could be the explanation for the variability in the tissue composition of Iberian pigs and therefore on the quality of meat products from Iberian pigs raised under this unique free-range system.

Table 1 .
Experimental design: sampling distribution throughout the three years of the study

Table 2 .
) did not show signif icant variations with Meteorological data (temperature and rainfall) for the period of study

Table 3 .
Effect of date and year of sampling on morphology, sanitary condition and proximate composition of acorn samples