The Effect of Dietary Inclusion of Microalgae Schizochytrium spp. on Ewes' Milk Quality and Oxidative Status.

An unprecedented challenge for nutritionists arises during the 21st century in order to produce highly nutritious and functional food which promotes human health. Polyunsaturated fatty acids (PUFA) that are highly contained in microalgae have broadly been confirmed for preventing cardiovascular diseases and regulating immune-oxidative status. However, their optimum dietary inclusion level needs to be defined since PUFA are prone to oxidation. For this purpose, 24 cross-bred dairy ewes, were separated into four groups (n = 6) and were fed with different levels of microalgae Schizochytrium spp. [0 (CON, no microalgae), 20 (SC20), 30 (SC30) and 40 (SC40) g/ewe/day] for 60 days. The results showed that although the production parameters were not impaired, milk fat content was decreased in medium and high-level supplemented groups while protein content was suppressed only for the medium one. Concerning the fatty acids (FA) profile, the proportions of C14:0, trans C18:1, trans-11 C18:1, cis-9, trans-11 C18:2, trans-10, cis-12 C18:2, C20:5 (EPA), C22:5n-6 (DPA), C22:6n-3 (DHA), the total ω3 FA and PUFA were significantly increased, while those of C18:0, cis-9 C18:1 and C18:2n-6c were decreased in the milk of treated ewes. Additionally, in the S40 group an oxidative response was induced, observed by the increased malondialdehyde (MDA) levels in milk and blood plasma. In conclusion, the dietary inclusion of 20 g Schizochytrium spp./ewe/day, improves milks' fatty acid profile and seems to be a promising way for producing ω3 fatty acid-enriched dairy products.

Effect of a Carotenoid Extract from Citrus reticulata By-Products on the Immune-Oxidative Status of Broilers.

Although carotenoids generally possess antimicrobial and antioxidant properties, the in vivo synergistic action of carotenoid blends derived from plant-based by-products has not been thoroughly studied. Therefore, the carotenoid characterization and antimicrobial potential of Citrus reticulata extract as well as the impact of this carotenoid-rich extract (CCE) dietary supplementation on the performance, meat quality, and immune-oxidative status of broiler chickens were determined. One hundred and twenty one-day-old hatched chicks (Ross 308) were allocated to two dietary groups, with four replicate pens of 15 birds each. Birds were fed either a basal diet (CON) or the basal diet supplemented with 0.1% CCE (25 mg carotenoid extract included in 1 g of soluble starch) for 42 d. ß-Cryptoxanthin, ß-Carotene, Zeaxanthin, and Lutein were the prevailing carotenoid compounds in the Citrus reticulata extract. The CCE feed additive exerted inhibitory properties against both Gram-positive (Staphylococcus aureus) and negative (Klebsiella oxytoca, Escherichia coli, and Salmonella typhimurium) bacteria. Both the broiler performance and meat quality did not substantially differ, while the breast muscle malondialdehyde (MDA) concentration tended to decrease (p = 0.070) in the CCE-fed broilers. The inclusion of CCE decreased the alanine aminotransferase and MDA concentration, and the activity of glutathione peroxidase, while the activity of superoxide dismutase was increased in the blood. Catalase and NADPH oxidase 2 relative transcript levels were significantly downregulated in the livers of the CCE-fed broilers. Additionally, Interleukin 1ß and tumor necrosis factor (TNF) relative transcript levels were downregulated in the livers of the CCE- fed broilers, while TNF and interferon γ (IFNG) tended to decrease in the spleens and bursa of Fabricius, respectively. The present study provided new insights regarding the beneficial properties of carotenoids contained in Citrus reticulata in broilers' immune-oxidative status. These promising outcomes could be the basis for further research under field conditions.

Immune-Related Gene Expression Profiling of Broiler Chickens Fed Diets Supplemented with Vinification Byproducts: A Valorization Approach II.

The valorization of vinification byproducts portrays a promising bioprocess for the enrichment of animals' diet with bioactive compounds, such as polyphenols, which could regulate the immune response. Therefore, the impact of dietary grounded grape pomace (GGP), wine lees extract (WYC), and grape stem extract (PE) on the relative transcript level of immune related genes of broiler chickens were examined. Two hundred forty, one-day-old as hatched (male/female) chicks (Ross 308) were allocated to four dietary groups, with four replicate pens each with 15 birds. Birds were fed either a basal diet (CON) or the basal diet supplemented with 2.5% GGP, or 0.2% WYC, or 0.1% PE for 42 d. The relative expression of immune-related genes was investigated using a real-time PCR platform. The mRNA levels of Toll-like Receptor 4 (TLR4) were downregulated (p = 0.039) in the liver of broilers fed the GGP-containing diet compared to the CON, while in the spleen of PE-fed broilers, TLR4 was significantly upregulated (p = 0.043). The mRNA levels of interleukin 8 (IL8) tended to upregulate (p = 0.099) in the bursa of Fabricius and were significantly increased (p = 0.036) in the spleen of broilers fed the PE diet. Vinification byproducts depict a promising sustainable source of polyphenols for the poultry feed industry, but more research is needed under field conditions.

Effect of Farming System (Organic vs. Conventional) and Season on Composition and Fatty Acid Profile of Bovine, Caprine and Ovine Milk and Retail Halloumi Cheese Produced in Cyprus.

The present work aimed to evaluate the effect of farming practices and season on the fat and protein content and fatty acid (FA) profile of milk and Halloumi cheese produced in Cyprus. Over a year, raw bulk-tank milk samples from cow, goat, and sheep farms were collected seasonally from all organic (11) and representative conventional (44) dairy farms, whereas Fresh Halloumi cheese samples were collected monthly from retail outlets (48 organic and 48 conventional samples in total). The different farming practices did not affect the milk fat content of ruminants, while protein levels were decreased in organic bovine and caprine milk. Under organic farming practices, milk and cheese contained increased values of total mono-unsaturated FA (MUFA) and poly-unsaturated FA (PUFA), and specific FA, such as oleic, conjugated linoleic, linoleic, and α-linolenic acids. Total saturated FA (SFA) levels were particularly decreased in organic samples and, consequently, the atherogenic indices of milk and cheese were decreased. Season influenced milk and Halloumi cheese FA profile; spring samples had lower SFA and higher PUFA and MUFA concentrations. Overall, the organic farm practices improved the lipid profile of milk and Halloumi cheese, which is more likely attributed to the different feeding strategies applied in organic dairy farms.

Hesperidin and Naringin Improve Broiler Meat Fatty Acid Profile and Modulate the Expression of Genes Involved in Fatty Acid ß-oxidation and Antioxidant Defense in a Dose Dependent Manner.

The beneficial properties of the flavanones hesperidin and naringin as feed additives in poultry have lately been under investigation. In broilers, both flavanones have been shown to exhibit antioxidant properties while their individual effects on fatty acid (FA) composition and the underlying molecular mechanisms of their activity have not been explored. Here, we studied their effects on broiler meats' FA profiles and on the expression of genes related to lipid metabolism, antioxidant defense and anti-inflammatory function. The experimental design comprised six treatment groups of broilers, each supplemented from day 11 until slaughter at 42 days with hesperidin, naringin or vitamin E, as follows: the E1 group received 0.75 g of hesperidin per kg of feed, E2 received 1.5 g hesperidin/kg feed, N1 received 0.75 g naringin/kg feed, N2 received 1.5 g naringin/kg feed, vitamin E (VE) received 0.2 g a-tocopheryl acetate/kg feed, and the control group was not provided with a supplemented feed. The VE treatment group served as a positive control for antioxidant activity. An analysis of the FA profiles of the abdominal adipose tissue (fat pad), major pectoralis (breast) and biceps femoris (thigh) muscles showed that both hesperidin and naringin had significant effects on saturated FA (SFA), polyunsaturated FA (PUFA) and omega n-6 content. Both compounds reduced SFA and increased PUFA and n-6 content, as well as reducing the atherogenicity and thrombogenicity indices in the breast muscle and fat pad. The effects on the thigh muscle were limited. An analysis of gene expression in the liver revealed that naringin significantly increased peroxisome proliferator-activated receptor alpha (PPARα), Acyl-CoA oxidase 1 (ACOX1) and glutathione disulfide reductase (GSR) expression. In the breast muscle, both hesperidin and naringin increased fatty acid synthase (FASN) expression and hesperidin increased the expression of adiponectin. In brief, both hesperidin and naringin supplementation beneficially affected FA profiles in the breast meat and fat pad of broiler chicken. These effects could be attributed to an increase in FA ß-oxidation since the increased expression of related genes (PPARα and ACOX1) was observed in the liver. Furthermore, the antioxidant activity of hesperidin and naringin previously observed in the meat of broilers could be attributed, at least partly, to the regulation of antioxidant defense genes, as evidenced by the increased GSR expression in response to naringin supplementation.

Application of Olive By-Products in Livestock with Emphasis on Small Ruminants: Implications on Rumen Function, Growth Performance, Milk and Meat Quality.

The olive oil industry has a leading position in the Mediterranean countries, resulting in the production of considerable quantities of the respective by-products (OB) that constitute an important environmental issue. OB contain valuable nutrients and bioactive components that can be re-used under the bioeconomy strategy, and several chemical, physical, and biological processes have been evaluated with the intention to improve their nutritional value. One feasible application of OB is their incorporation in the diets of livestock and especially ruminants due to their high fiber content. As indicated by numerous studies, OB dietary supplementation increases the levels of monounsaturated fatty acids (MUFAs) and decreases that of saturated fatty acids (SFAs) in the milk and meat of ruminants with beneficial effects for consumers' health. At the same time, environmental impact and feeding costs are reduced without detrimental effects on ruminal fermentation, nutrients utilization, growth performance, carcass traits, milk yield and composition.

Impact of Mycotoxins on Animals' Oxidative Status.

Mycotoxins appear to be the "Achilles' heel" of the agriculture sector inducing enormous economic losses and representing a severe risk to the health of humans and animals. Although novel determination protocols have been developed and legislation has been implemented within Europe, the side effects of mycotoxins on the homeostatic mechanisms of the animals have not been extensively considered. Feed mycotoxin contamination and the effects on the antioxidant status of livestock (poultry, swine, and ruminants) are presented. The findings support the idea that the antioxidant systems in both monogastrics and ruminants are challenged under the detrimental effect of mycotoxins by increasing the toxic lipid peroxidation by-product malondialdehyde (MDA) and inhibiting the activity of antioxidant defense mechanisms. The degree of oxidative stress is related to the duration of contamination, co-contamination, the synergetic effects, toxin levels, animal age, species, and productive stage. Since the damaging effects of MDA and other by-products derived by lipid peroxidation as well as reactive oxygen species have been extensively studied on human health, a more integrated monitoring mechanism (which will take into account the oxidative stability) is urgently required to be implemented in animal products.

Quercetin and Egg Metallome.

The objective of the present study was to investigate the effect of the natural flavonoid quercetin dietary supplementation on the alteration of egg metallome by applying the basic principles of elemental metabolomics. One hundred and ninety-two laying hens were allocated into 4 treatment groups: the control (C) group that was fed with a commercial basal diet and the other experimental groups that were offered the same diet further supplemented with quercetin at 200, 400 and 800 mg per kg of feed (Q2, Q4 and Q8 group, respectively) for 28 days. The diets contained the same vitamin and mineral premix, thus all birds received the same amount of elements since no differences on feed intake existed. The egg elemental profile consisted of As, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, Pb, Sb, Se, Sr, V, Zn and was determined using inductively coupled plasma mass spectrometry (ICP-MS). Quercetin supplementation altered the elemental profile. Most notably, quercetin altered the element concentrations predominantly in egg shell and albumen. It increased the concentration of Sb while reduced that of Cr and Se in both egg shell and albumen. Moreover, it increased As, Cd in albumen and V in yolk, while compared to the control, reduced As, Cd, Cr, Cu and V and also raised Ca, Fe, Mg and Ni in egg shell. The presence of quercetin led to differentiation of the deposition of certain trace minerals in egg compartments compared to that of hens fed a basal diet, possibly indicating that tailor made eggs for specific nutritional and health requirements could be created in the future.

Elemental Metabolomics: Modulation of Egg Metallome with Flavonoids, an Exploratory Study.

The basic principles of elemental metabolomics were applied to investigate whether alteration of egg metallome could be achieved after two flavonoids addition, namely hesperidin and naringin in diets of laying hens. A total of 72 hens were divided into six groups: Control (C) (basal diet), E1 (750 mg hesperidin/kg diet), E2 (1500 mg hesperidin/kg diet), N1 (750 mg naringin/kg diet), N2 (1500 mg naringin/kg diet), and VE (200 mg vitamin E/kg diet). The same diet was provided to birds of all treatments, with the exception of added supplements. The diets had the same vitamin and mineral premix; thus, all birds received the same number of elements because no differences on feed intake existed. The egg elemental profile consisted of As, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, Pb, Sb, Se, Sr, V, Zn, and was determined using ICP-MS. Flavonoid supplementation altered the elemental profile. Most notably, in both albumen and yolk, hesperidin increased Ni, Pb, and Sr concentration while it decreased that of Co and Sb. Naringin increased Cd, Cr, Cu, Ni, and V and lowered the concentration of Co and Sb in both yolk and albumen. Vitamin E supplementation, in comparison to the control, decreased Co in both albumen and yolk and also raised Sb in albumen. Flavonoid presence led to the differences in deposition of certain trace minerals in egg compared to that of hens fed a basal diet or a diet with vitamin E supplementation.

Antioxidant status, meat oxidative stability and quality characteristics of lambs fed with hesperidin, naringin or α-tocopheryl acetate supplemented diets.

BACKGROUND: Dietary supplementation of sheep with bioactive compounds such as citrus flavonoids appears as a great alternative for improving meat antioxidant capacity and could also have beneficial effects on growth performance, carcass traits and meat quality characteristics. An experiment was therefore conducted to examine the effects of hesperidin or naringin dietary supplementation on lambs' growth performance, antioxidant status and meat quality characteristics. Forty-four male 3-month-old Chios lambs were randomly assigned to four groups: C, fed with a basal diet, or H or N or VE offered the same diet further supplemented with hesperidin at 2500 mg or naringin at 2500 mg or α-tocopheryl acetate (vitamin E) at 200 mg kg-1 feed respectively. At the end of the experiment (day 35), samples of longissimus thoracis muscle were collected for meat quality evaluation. RESULTS: Both flavonoids and vitamin E dietary supplementation reduced plasma malondialdehyde (MDA) levels on day 35 (P < 0.05). No significant differences were observed in performance traits and meat quality characteristics among the experimental groups (P > 0.05). Hesperidin and naringin also reduced MDA levels in meat stored at 4 °C for up to 8 days (P < 0.05), although to a lesser extent than α-tocopheryl acetate. CONCLUSION: Incorporation of flavonoids in sheep diets resulted in an improvement of plasma and meat antioxidant capacity. © 2018 Society of Chemical Industry.

Enrichment of Animal Diets with Essential Oils-A Great Perspective on Improving Animal Performance and Quality Characteristics of the Derived Products.

Food industry operates in a competitive market and is continually facing challenges to retain or even increase its market share. Consistent high-quality animal products are required to maintain consumer confidence and consumption. Enrichment of foods with bioactive compounds such as the essential oils appears to improve quality characteristics of the derived products and protects consumers against oxidation and bacterial spoilage effects. Synthetic additives are nowadays questioned due to their suspected carcinogenic potential, and therefore extensive research has been undertaken to identify safe and efficient alternatives. Aromatic plants and their respective essential oils belong to natural products and are generally used in pig, poultry, rabbit and ruminant nutrition. The inclusion of essential oils in livestock diets is nowadays becoming a common practice, since dietary supplementation has been proven a simple and convenient strategy to effectively inhibit the oxidative reactions or microbial spoilage at their localized sites. A wide range of essential oils contain bioactive compounds that have the potential to act as multifunctional feed supplements for animals including effects on growth performance, digestive system, pathogenic bacterial growth and lipid oxidation. However, further studies are needed to clarify their exact action and establish their regular use in animal production.

NMR Metabolomics Investigates the Influence of Flavonoid-Enriched Rations on Chicken Plasma.

The use of flavonoids as dietary supplements is well established, mainly due to their intense antioxidant and anti-inflammatory properties. In the present study, hesperidin, naringin, and vitamin E were used as additives at different concentrations in poultry rations in order to achieve meat of improved quality. NMR metabolomics was applied to chicken blood serum samples to discern whether and how the enriched rations affected the animals' metabolic profile. Variations in the metabolic patterns according to sustenance consumption were traced by orthogonal projections to latent structures discriminant analysis (OPLS-DA) models and were attributed to specific metabolites by using S-line plots. In particular, serum samples from chickens fed with vitamin E displayed higher concentrations of glycine and succinic acid compared to control samples, which were mainly characterized by betaine, formic acid, and lipoproteins. Samples from chickens fed with hesperidin were characterized by increased levels of lactic acid, citric acid, creatine, carnosine, creatinine, phosphocreatine, anserine, glucose, and alanine compared to control samples. Lastly, naringin samples exhibited increased levels of citric and acetic acids. Results verify the scalability of NMR metabolomics to highlight metabolite variations among chicken serum samples in relation to food rations.

The Influence of Naringin or Hesperidin Dietary Supplementation on Broiler Meat Quality and Oxidative Stability.

An experiment was conducted to examine the effects of supplementing broiler feed with hesperidin or naringin, on growth performance, carcass characteristics, breast meat quality and the oxidative stability of breast and thigh meat. Two hundred and forty 1-day-old Ross 308 broiler chickens were randomly assigned to 6 groups. One of the groups served as a control (C) and was given commercial basal diets, whereas the other five groups were given the same diets further supplemented with naringin at 0.75 g/kg (N1), naringin at 1.5 g/kg (N2), hesperidin at 0.75 g/kg (E1), hesperidin at 1.5 g/kg (E2) and a-tocopheryl acetate at 0.2 g/kg (E). At 42 days of age, 10 chickens per treatment group were slaughtered for meat quality and oxidative stability assessment. No significant differences were observed among groups in final body weight, carcass weight and internal organs weights (P>0.05) apart from liver that decreased linearly with increased levels of naringin (P-linear<0.05). Regarding the breast meat quality parameters, only redness (a*) value was higher in E1 and N1 group compared to VE group (P<0.05), while all the others i.e. shear values (N/mm2), pH24, cooking loss (%) and L* and b* color parameters were not significantly different among groups (P>0.05). Measurement of lipid oxidation values showed that after hesperidin and naringin dietary supplementation, malondialdehyde values decreased in tissue samples in a dose depended manner (P-linear<0.05). In conclusion, hesperidin and naringin, positively influence meat antioxidative properties without negative implications on growth performance and meat quality characteristics in poultry, thus appearing as important additives for both the consumer and the industry.

Severe maternal undernutrition and post-weaning behavior of rabbits.

The objective of the present experiment was to investigate the implications of severe maternal undernutrition on the post-weaning behavior of rabbits. Thirty two does were randomly assigned to four groups: the control group (C) that was fed 100% of the recommended energy maintenance requirements throughout pregnancy and lactation and the U1, U2 and U3 groups that were fed 50% of the recommended energy maintenance requirements between the 6th and the 19th day of pregnancy, between the 20th and the 27th day of pregnancy and between the 3rd and the 10th day of lactation, respectively. At the age of 50 and 65 days, behavior of rabbits in cages was recorded and rabbits were further subjected to an open-field test; a paradigm used as an indicator of fear and emotional distress. Significant differences were found between the U2 and the other experimental groups for the duration of eating and drinking, and duration of locomotory and investigatory behaviors (P<0.05). At the age of 65 days, duration of comfort behaviors was also lower in the U2 compared to the other groups (P<0.05). On the other hand, duration of resting appeared to be the highest in the group of rabbits born from undernourished does between the 20th and the 27th day of pregnancy (P<0.05). Duration of resting was significantly increased, although frequency and duration of eating and drinking were significantly decreased at the age of 65 days (P<0.05). Male rabbits had greater values for locomotory and investigatory behaviors, and duration of eating and drinking compared to the females (P<0.01). A decreased activity of rabbits during the light period was observed (as expected), whereas an increase in duration of locomotory and investigatory behaviors was observed during the first four and the last 4h of the light and dark periods, respectively (P<0.05). Rabbits born from the U1 and U2 group of does displayed increased rates of latency to leave the start position compared to the other groups during the implementation of the open-field test (P<0.05). No other significant differences in the exhibition of the examined behaviors were found between the treatment groups, with the exception of 'moving forelegs' activity that had the lowest value in the U3 group of rabbits. It is concluded that severe maternal undernutrition (50% of the recommended maintenance requirements) especially between the 20th and the 27th day of pregnancy has significant effects on the exhibition of rabbits' post-weaning behavior, since the offspring from the U2 undernourished does were significantly less active compared to the other experimental groups.

Effects of dietary hesperidin supplementation on lamb performance and meat characteristics.

An experiment was conducted to examine the effects of supplementing feed with hesperidin or α-tocopheryl acetate on lambs' growth performance, carcass and meat characteristics. Sixteen male lambs were randomly assigned to four groups. One of the groups served as control and was given a basal diet, whereas the other three groups were given the same diet further supplemented with hesperidin at 1500 mg/kg, or hesperidin at 3000 mg/kg, or α-tocopheryl acetate at 200 mg/kg. At the end of the experiment, lambs were fasted, weighed and slaughtered. After overnight chilling, samples of Longissimus thoracis muscle were taken and were used for meat quality evaluation. No significant differences were observed in final body weight, body weight gain and organ weights among the four groups. pH, color, water-holding capacity, shear force values and intramuscular fat concentration of Longissimus thoracis muscle were also not significantly influenced by the dietary treatments. Measurement of lipid oxidation values showed that hesperidin supplementation positively influenced meat antioxidant properties during refrigerated storage.

Feeding preferences in lambs influenced by prenatal flavour exposure.

The aim of the present study is to highlight and evaluate the role of flavour prenatal exposure in postnatal feeding preferences in sheep after weaning and until the early stages of puberty. 16 lambs were selected from two groups of ewes; the first group was fed with a control diet, consisted of concentrated feed and alfalfa hay and the second group with the same diet, with the only difference that the concentrate was supplemented with oregano essential oil (1 ml/kg), during the period of pregnancy (50th-130th day). Lambs were later individually subjected to feeding preference tests, at the age of 3, 4.5, 6, 7.5 months old. Each test lasted 25 min and it was a free choice situation between 3 different test feeds supplemented with eucalyptus or orange or oregano essential oil (1 ml/kg). Lambs born to oregano-treated ewes ate higher amounts of the oregano supplemented test feed during all feeding preference tests compared to lambs from the control group (P<0.01). Rates for occurrences and duration of eating were also greater in lambs born to oregano-treated ewes than the offspring of the control ewe group (P<0.01). On the other hand, animals without a flavour exposure precedent did not exhibit an evident strong preference for a specific test feed, although orange supplemented feed tended to be more preferable compared to the other offered feeds. Average total intake of lambs were not different between groups during all feeding preference tests at 3, 41/2, 6 and 71/2 months old. As it is concluded, prenatal exposure to oregano essential oil via maternal ingestion drastically influences lamb feeding preferences till adulthood.

Effect of breed and age on sexual behaviour of rams.

The objective of this study was to highlight the problems that arise during the reproduction between thin-tailed rams and fat-tailed ewes. At the same time, particular emphasis laid on the influence of sheep breed, sheep age, time after ram introduction and day of the ewe estrus cycle on ram and ewe sexual behaviour. Rams were subjected to sexual performance tests by being individually exposed to 12 ewes for 3 h daily, 19 consecutive days. The 16 rams of the experiment were separated according to their age (9 and 21 months old) and breed (Chios and Karagouniki), and the 96 ewes of Chios fat-tailed breed, were divided by age (9 and 21 months old). The main characteristics of courtship behaviour, like sniffing, nudging, flehmen response and following were recorded and studied in detail. Mature Chios rams, which were the only one with previous experience of Chios ewes, exhibited higher rates of sexual interest per ewe than the other rams (P < 0.05). On the other hand, rams sniffed and nudged more young than mature ewes (P < 0.05), probably due to the fact that young ewes did not express intense symptoms of estrus. Young rams exhibited substandard sexual interest towards mature ewes, when they first came in contact with them (P < 0.05). In general, Karagouniki thin-tailed rams exhibited reduced rates of mating behaviour when they courted with Chios fat-tailed ewes in comparison with Chios rams (P < 0.05). Moreover, as the time after ram introduction passed, the frequency and duration of sexual behaviour components decreased (P < 0.001). Finally, the effect of the day of the experiment was only significant in the case of sniffing, which increased during the first 2 days and then declined and stabilized (P < 0.01). As it was demonstrated, ram age and ram breed played a fundamental role in the exhibition of sexual interest elements.