Effect of Hempseed (Cannabis sativa sp.) Inclusion to the Diet on Performance, Carcass and Antioxidative Activity in Japanese Quail (Coturnix coturnix japonica)

Introduction

The cannabis sativa is an annual plant and well known as an important source of food, dietary oil and medicine for thousands of years in many western and eastern countries (Callaway, 2004; de Padua et al., 1999). Hempseed both crushed and whole, remains an important grain in traditional Asiatic foods and medicines (Callaway, 2004). Industrial hemp with low levels (<2 g/kg) of delta-9-tetrahydrocannabinoid (THC) has been permitted for cultivation in European Union countries with EU Council regulation 1782/2003 (2008). Also, EU commission has been working on regulations of hemps’ THC levels and usage in different areas (Callaway, 2008). Hempseed contains about 30% oil and 25% protein, 34% carbohydrates, dietary fibre, vitamins and minerals (Callaway, 2004; Kriese et al., 2004). Hempseed oil is high in polyunsaturated fatty acids (PUFAs, about 80%), and rich in two essential fatty acids such as linoleic acid (18:2 ω-6, 55.48%) and α-linolenic acid (18:3 ω-3 21.51%) (Bağcı et al., 2003; Uluata and Ozdemir, 2012) which are important for optimal human health (Schwab et al., 2006). Also, linoleic and linolenic fatty acids in quail (Yalcin et al., 2012) and layer hens’ (Gakhar et al., 2012; Goldberg et al., 2012; Silversides and Le Francois, 2005) diets may transfer to meat or egg via hempseed and hemp oil usage. In addition hempseed is rich in many essential amino acids, especially high levels of arginine and methi- onine and special peptides (Callaway, 2004; Tang et al., 2006). It is well known that antioxidant activities of peptides are closely related to amino acid composition and sequences of these amino acids and hempseeds’ special protein types show potential of natural antioxidant agents (Gebrekidan, 2012). The consumption of foodstuffs rich in antioxidants provides protection against cancer and cardiovascular diseases (Parker et al., 2003). Uluata and Ozdemir (2012) and Chen et al. (2012) reported that hempseed can be utilized for the production of formulations containing phytochemicals with significant antioxidant properties and health benefits and hempseed addition could increase the shelf life of the products as well. Additionally, it is reported that hempseed’s cannabidiols have antimicrobial, immunomodulatory, antioxidative, antihypertensive and mineral binding properties (Korhonen and Pihlanto, 2003). The development of bioactive protein peptides of hempseed may contribute to new economic opportunities for increased value-added utilization of hemp (Lu et al., 2010).

Cells generate small amounts of free radicals or reactive oxygen species (ROS) while performing their normal metabolic functions. Although low levels of ROS are essential in many biochemical processes, high levels of ROS may damage cells and tissue macromolecules, that is, lipids, proteins, carbohydrates and deoxyribonucleic acid (DNA) (Mates et al., 1999; Tang et al., 2006). The tocopherols, phenolic compounds and carotenoids present in vegetable oils are used against the oxidation reaction (Chen et al., 2012; Ramadan et al., 2003; Uluata and Ozdemir, 2012). Due to all these properties, in the past, hemp oil and seed had been used as a medical plant to treat some diseases (de Padua et al., 1999).

It is reported that hempseed is an excellent source of nutrition. Not only traditional Chinese medicine but also recent modern human clinical trials agree that hempseed has health promoting properties which is supported by results from nutritional analyses of the seed, oil and seed meal (Callaway, 2004). Recently, to be protected from some illnesses, natural antioxidants have attracted great attention from consumers due to their reliable properties compared to synthetic ones. As a result of healthy food production strategy, use of natural antioxidants in diets is alleviating. The aim of the present study was to determine the effects of hempseed on performance, carcass and oxidative activity parameters in the blood of Japanese quail.

Material and Methods

Animal and diets

A total of (192 unsexed seven-day-old Japanese quail chicks (Coturnix coturnix japonica) were individually weighed, wing banded and distributed into 4 treatment groups with 4 replicates and 12 chicks per cage. Each cage was furnished with a heater, two wateriers and feeders. The rearing cage dimensions were 50×90×20 cm (wide, length, height). Replicates were designated as the experimental units, and randomized with respect to the dietary treatments. The diet treatments were as follows; 1) Basal diet, hempseed not included in diet (control=C); 2) 5% hempseed inclusion (H5); 3) 10% hempseed inclusion (H10); 4) 20% hempseed inclusion (H20). The experimental diets were offered to quail for 5 wk. Maizesoybean based diets were utilized and all formulated on similar levels of nutrient composition. The hempseed was bought from a local seed shop. All diets’ compositions were prepared according to NRC (1994) recommendations. The compositions of hempseed and experimental diets used in this study were given in Table 1 and Table 2, respectively.

Analytical Methods

Results

The hempseed nutrient and fatty acids composition are presented in Table 1. The hempseed’s dry matter, crude protein, fat and ash were 95.11, 21.05, 31.45 and 8.8% respectively. The fatty acids of hempseed oil were found as linoleic acid 52.51%, linolenic acid 23.24%, ω-6/ω-3 ratio 2.26 and PUFA 87.9%. The effects of hempseed on body weight (BW), feed intake (FI) and feed conversion ratio (FCR) values were shown in Table 3. There were no differences statistically among the treatment groups in terms of FI, FCR and BW on d 7 and d 21 during the experimental period (p>0.05). However, on d 42, inclusion of 20% hempseed decreased the BW of quail (p< 0.05).

Effect of hempseed inclusion on carcass and intestinal organ traits and their relative incidence were given in Table 4. The carcass and heart weights of the H10 group were higher than those of the other treatment groups (p< 0.05). The liver and intestine weight in the C group was higher than those of the others (p<0.01) but carcass yield was lower than all hempseed supplemented groups (p< 0.05). The gizzard weight and the gizzard and heart relative incidence were not influenced by the treatments. The intestine weight and relative incidence of H20 groups was lower than those of the other groups (p<0.01). The heart weight and relative incidence were not influenced by sex. The carcass, liver, gizzard, intestine weights and liver, gizzard and intestine relative incidence were lower in male quail (p<0.05 and p<0.01). Hempseed level and sex interactions were not influenced by the treatments. In general 20% hempseed inclusion decreased the carcass and internal organ weight and relative incidence.

The effects of hempseed included diets on serum malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and nitric oxide (NO) activity showed in Table 5 and Figs. 1, 2, 3, 4 and 5. The serum protein levels were not affected by the hempseed addition into quail diets (p>0.05). The serum MDA and NO levels were decreased by hempseed addition into quail diets (p>0.05). In this study, all hempseed supplemented groups serum SOD, CAT and GSH-Px levels were increased compared to control group (p<0.001). The serum MDA, SOD, CAT and GSH-Px values in male quail were higher than female, however in female serum NO level was higher than males (p<0.01). Interactions (hempseed and sex) affected serum NO (p<0.01), SOD and CAT values (p<0.01).

Discussion

The compositional analysis showed that hempseed contained 21.05% protein and 31.45% oil. These results were similar to the results obtained by Callaway (2004) and House et al. (2010). Also the fatty acids of hempseed oil were found C18:2, 52.51%; C18:3 23.24% and ω-6/ω-3 ratio 2.26. Similar fatty acid content and ω-6/ω-3 ratio were reported by Callaway (2004) and Uluata and Ozdemir (2012). The inclusion of hempseed to layer (Gakhar et al., 2012), quail (Yalcin et al., 2012) and feedlot (Gibb et al., 2005) diets showed an increase in high ratio ω-6 and ω-3 fatty acids.

Hempseed supplementation to quail diet did not affect BW on d 7 and d 21 but inclusion of 20% hempseed decreased the BW of quail on d 42 (p<0.05). The FI and FCR values were not affected by the treatments. Silversides and Lefrancois (2005) and Gakhar et al. (2012) showed that including up to 20% hempseed meal in diets did not significantly influence the BW of laying hens. Also, similar results were obtained by Gibb et al. (2005) who reported that 0, 9 and 14% hempseed inclusion to feedlot cattle did not change BW, BWG and FI of steers. However, in contrast to our results, Khan et al. (2010) found that 20% hempseed inclusion to diets increased BW of broilers. However, as a processed product hempseed cake inclusion to broiler diets did not affect performance traits (Eriksson and Wall, 2012). Analysis results showed that hempseed contained high percentages of crude ash (8.80%, Table 1) and cellulose (House et al., 2010). This ratio is considerably high when compared to some traditional feeds such as corn, wheat and soybean meal. High crude ash and cellulose levels in diets may cause lower BW due to decreasing of energy and digestibility of feeds. On the other hand, it is reported that hempseed has high quality protein content and amino acid composition (Callaway, 2004). However, due to addition of hempseed some amino acid content may be excessively increased in diet, and imbalance of amino acid ratios in diet may have an antagonistic effect and lower amino acid bioavailability. Another reason of lower BW in H20 group may also be high related to THC content increase in the diet (Callaway, 2004). Unfortunately, there are only a few hempseed inclusion experiments in the literature to discuss and compare with our results.

Hempseed and its by-products have been used from ancient times until today as a medicinal plant (de Padua et al., 1999). Today, there are still many unknowns about hempseed and its oil’s nutritional value and effects when used in animal and human diets. The development and characterization of the bioactive contents in novel foods are required to provide scientific evidence to improve their quality and nutritional value (Yu et al., 2005). The present study was designed to evaluate the inclusion of hempseed on performance and serum lipid antioxidant properties of Japanese quail. Hempseed contains 30% oil which is high in linoleic (LA) and alpha-linolenic acids (ALA) (Callaway, 2004; Uluata and Ozdemir, 2012). The LA and ALA are necessary in body as essential fatty acids and protect cell uniformity in the diet for healthy individuals (Goldberg et al., 2012).

In the H10 group the carcass and heart weights were higher than those of other groups. However, in the C group the liver weight was higher than 5 and 20% H groups and intestinal weight were higher than those of the other groups. When considered the relative incidence of carcass in the C group’s carcass ratio was lower and liver and intestinal incidence was higher than those of the hempseed supplemented groups. The gizzard weights and incidences were not influenced by the treatments. Male quail carcass and internal organ traits without carcass incidence were lower than those of females. Khan et al. (2010) reported that 20% hempseed in broiler rations did not affect positively dressing percentage at 42 d. Callaway (2004) reported that hempseed has some beneficial effect on health in human and animals therefore it is hypothesized its inclusion may increase quail performance traits. However, in this study superior results were not observed in performance traits but increased carcass percentage.

The serum protein levels were not affected by the hempseed addition into quail diets. In this study, protein levels of all diets were adjusted at similar levels. On the other hand, hempseed protein level (21.2%, Table 1) was similar with the experimental quail diets (24%), therefore serum protein levels were not significantly affected by the hempseed inclusion. There are no enough studies results for comparison of our results with others. However, Karimi and Hayatghaibi (2006) reported that hempseed in rat diet after 20 d caused an increase in blood protein levels compared to blood protein level at first day of the experiment. Although, their experiment lack of control diet, there is a comparison with first and last day of the experiment to evaluate rat blood component.

The MDA, a carbonile group produced during lipid peroxidation, is used widely in determining oxidative stress and formed as an end product of lipid peroxidation (Sumida et al. (1989). Lipid peroxidation is a chain reaction providing a continuous supply of free radicals as it involves the oxidation of polyunsaturated fatty acids contributes to the loss of cellular functions through the inactivation of membrane enzymes and even of cytoplasmic proteins and consequences cell damage. On molecular level of lipid peroxidation, the importance of oxidative membrane damage is twofold (Stark, 2005). In the present study, the serum MDA levels were decreased by hempseed addition into quail diets. This may show that hempseed inclusion to quail diets has a positive effect against to lipid peroxidation in the serum of Japanese quail. On the other hand hempseed contains some special protein types and amino acids (Lu et al., 2010) and high ratio of ω-3 and ω-6 fatty acids which may protect the integrity of the cell membrane (Uluata and Ozdemir, 2012). Hu et al., (2008) reported that serum MDA levels were decreased in test rats those fed by diets with hemp seed oil at the end of 90-day trial period compared with control groups of contained soybean oil and the normal diet. Also, it is reported that extract of cinnamon (Faix et al., 2009; Ciftci et al., 2010), rosemary (Yesilbag et al., 2011), oregano (Botsoglou et al., 2002) decreased MDA levels in broilers and quails (Bulbul et al., 2012).

NO is produced at the cellular level from L-arginine catalyzed by nitric oxide synthase, a very important signaling molecule (Nathan, 1992). NO is a diffusible free radical that plays many roles as an effector molecule in diverse biological systems including vasodilatation, neuronal messenger, immunological response, antimicrobial and antitumor activities (Beckman and Koppenol, 1996; Balakrishnan et al., 2009). However, NO participates in several cell processes as a highly reactive free radical (Moncada et al., 1991). NO in the body is oxidized within seconds; in the presence of oxygen and the oxidation endproducts of NO can oxidize cell lipids (Hassanpour et al., 2009) consequently, cell damage occurs. In the current study, hempseed inclusion to diets caused a linear decrease in the serum NO levels. The control group serum NO level was higher than all other groups (p<0.001). These results revealed that the inclusion of hempseed to quail diets exhibited well in NO scavenging activity. The present study shows that hempseed inclusion to diet may potent nitric oxide scavenging activity. Similar results with green tea were obtained by Hassanpour et al. (2010) which showed green tea supplementation to broiler diets reduced NO metabolites as free radical group.

The SOD, GSH-Px, and CAT are antioxidative enzymes act as the first step of antioxidant defense and protection cells against damage of free radical groups (Tang et al., 2003). In the body, increase of these antioxidant enzymes enhances in scavenging activity for oxygen free radicals. In this study, all hempseed supplemented groups’ the serum SOD, CAT and GSH-Px levels were increased compared to control group. Furthermore, when hempseed inclusion rate were increased, in a linear increase in the level of serum SOD, CAT and GSH-Px was observed (p<0.001). These findings showed that hempseed inclusion increased total blood antioxidant enzyme capacity. Hu et al. (2008) found that serum SOD and GSH-Px levels increased significantly at the end of 90-day feeding with hempseed oil. Hempseed and its oil contain some phenolic compounds, n3 and n6 fatty acids (Callaway, 2004), special peptides and protein types (Lu et al., 2010) and they have protection ability against harmful effects of ROS in the body (Gebrekidan, 2012; Uluata and Ozdemir, 2012). In different experiment results showed that vegetable oils such as rosemary and garlic (Ancsin et al., 2009) and cinnamon oil (Ciftci et al., 2010) in broilers and essential oil’s mixture of different plants (Bozkurt et al., 2012) increased GSH-Px levels in layers.

Taken together, these results showed that the hempseed inclusion at 5, 10 and 15% levels to Japanese quail diet improved antioxidant status of animals. Because, hempseed has special fatty acids composition (Uluata and Ozdemir, 2012), phenolic compounds (Chen et al., 2012; Uluata and Ozdemir, 2012), special proteins and amino acids (Gebrekidan, 2012), antimicrobial, immunomodulatory, antioxidative, antihypertensive and mineral binding properties (Korhonen and Pihlanto, 2003). In addition to Yu et al. (2005) reported that hempseed oil has better oxygen radical absorbing capacity than many plant oils. Therefore it has unique material to protect against ROS and protect cell membrane integrity. The researchers claimed that findings are significant for the discovery and development of natural antioxidants from hemp by-products (Callaway, 2004; Chen et al., 2012; Korhonen and Pihlanto, 2003; Uluata and Ozdemir, 2012; Yu et al., 2005). Results of the our study are confirming the findings of Callaway (2004) who reported that hempseed oil is a functional food for animal and humans and for animal feeding long-standing utility as an important food resource.

Conclusion

In conclusion, according to our results, up to 10% hempseed may use in quail diet without negative effect on performance and carcass traits and hempseed inclusion may increase carcass yield. In this experiment hempseed inclusion did not cause any significant change in serum protein levels, but caused decrease in serum MDA and NO levels and increase in serum SOD, CAT and GSH-Px as antioxidant parameters. These parameters revealed that hempseed have good potential as a natural antioxidant material for health improvement and disease prevention. In addition, hempseed ingredients as natural antioxidant source may contribute to longer duration of shelf life of animal products. There is no enough experiment on effect of hempseed inclusion in animal diets and its nutritive and antioxidant efficacy in animals (Botsoglou et al., 2002). Further experiments are needed to determine the effects of hempseed in poultry feeding.