Conjugated linoleic acid as functional food in poultry products: A review

ABSTRACT

The conjugated linoleic acid is one of the many lipid molecules that had proven to be beneficial to the human health. Conjugated linoleic acid is reported to have anticancer and anti-diabetic properties, as well as exhibiting marked effects on the body composition. Conjugated linoleic acid is a term used to describe positional and geometric isomers of linoleic acid (C18:2n-6; LA). The two main isomers are cis-9, trans-11 and trans-10, cis-12. The mechanism by which dietary conjugated linoleic acids impart their reported effects involved the regulation of lipid mediator synthesis, and/or transcriptional regulation of gene expression through peroxisome proliferator activated receptors. Chicken has been considered as an appropriate model for nutritional research since they are highly sensitive to dietary manipulation. Therefore, the dietary inclusion of conjugated linoleic acids in poultry diets would be of great advantage to consumers since chicken meat is low in fat and cholesterol. In addition, through the consumption of conjugated linoleic acid-enriched meat and egg products, the human population would have gained conjugated linoleic acids that are crucial for health and well-being. This review attempts to provide an overview of the available data on conjugated linoleic acids in poultry meat, products originating from different poultry species, as well as the observable inclusion effects of conjugated linoleic acids in poultry.

KEYWORDS:

• Conjugated linoleic acid
• Functional food
• Poultry meat
• Poultry products
• Human

Introduction

Conjugated linoleic acid (CLA) is a collective term because all of their known isomers have double bonds with a single carbon bond in between (also known as conjugated double bonds), instead of the usual methylene-separation. The CLA can either be trans (t) or cis (c) configured, and include a wide spectrum of isomers with variations in position (7,9; 8,10; 9,11; 10,12; or 11,13), and geometry (c/c; c/t; t/t; or t/c).^[1] CLA originated naturally from bacterial isomerization or/and biohydrogenation of polyunsaturated fatty acids (PUFAs) in the rumen. CLA can also occur through the desaturation of trans-fatty acids in the adipose tissue and mammary gland.^[2] Figure 1 shows the structure of linoleic acid (LA) and its major CLA derivatives.

Figure 1. Structure of linoleic acid and CLA isomers. (1) Linoleic acid (typical n-6 PUFA); (2) cis-9, trans-11 CLA; (3) trans-10, cis-12 CLA. Adapted from Benjamin and Spener.^[13]

The isomerization/biohydrogenation of PUFA occurs when rumen microbes metabolize lipids, resulting in the formation of CLA, or other important intermediate precursors of CLA. In fact, CLA is an important intermediate when highly unsaturated C18 fatty acids are metabolized to produce the end product, stearic acid.^[4] Food sources originating from ruminants are known to have markedly higher CLA concentration than those from monogastric animals. Fish and some vegetable products also contain low CLA concentrations.^[4] In 1979, Pariza and Hargraves, from the University of Wisconsin, demonstrated that a lipid fraction has anti-mutagenic properties in a pan-fried hamburger. This lipid fraction inhibits the initiation of mouse epidermal tumors.^[5] The lipid fraction, which is responsible for this effect, was recognized as CLA.^[6] Over the last 10 years, beneficial effects of dietary CLA has been focused on body composition, lipoprotein metabolism, carcinogenesis, cardiovascular disease, immune system, and diabetes.^[7–12] The cis-9,trans-11 CLA isomer is the principal dietary form of CLA, it comprises approximately 90%, found primarily in beef and dairy products, while the trans-10,cis-12 isomer comprises the remaining 10%.^[13] In synthetic CLA preparations, the cis-9,trans-11 and trans-10,cis-12 -18:2 isomers are predominant and often present in a 1:1 ratio.^[14]

Butyrivibrio fibrisolvens, a butyrate-producing ruminal bacterium, was the first bacteria reported to produce CLA.^[16] Lacobacillus (L) acidophilus, L. bulgaricus, L. casei, L. plantarum, B. Bifidobacterium. (B) breve, B. infantis, B. longum, and Streptococcus thermophiles were also demonstrated as CLA-producing bacteria.^[17] Many study reported that the CLA-producing potentials of these bacteria has been established by adding LA to washed cells of lactic acid bacteria (LAB) or their enzyme extracts.^[18] The mechanism of CLA production from LA using Lactobacillus acidophilus AKU 1137 as a representative strain, via hydration of LA to 10-hydroxy-18:1 and the dehydrating isomerization of the hydroxy fatty acid to CLA. In this strain, the CLA isomers produced were identified as cis-9, trans-11- or trans-9, cis-11-octadecadienoic acid and trans-9, trans-11-octadecadienoic acid^[19] as shown in Fig. 2. The aim of the present review is to summarize the published data relating to CLA in poultry products as functional food. Furthermore, a short overview will be given on CLA biosynthesis, its reported beneficial properties and concentration in poultry products, as well as the factors influencing the concentration of CLA in animal tissues.

Figure 2. CLA production by Lactobacillus acidophilus. Adapted from Ogawa et al.^[19]

Metabolism of CLA

Non-ruminant meat, such as chicken and pork, contain 0.9 and 0.6 mg/g CLA, respectively.^[20] This is because monogastric animals do not have any significant amount of bacteria in their digestive system to produce CLA, hence the endogenous synthesis could be the only source of CLA.^[21] It is well-established that CLA isomers deposited in animal tissue was obtained via dietary sources. Synthetic and commercially available CLA usually has equal amount of the cis-9, trans-11-CLA, and trans-10, cis-12-CLA and significant levels of other CLA isomers.^[11] The natural sources of CLA contain mostly the cis-9, trans-11-CLA isomer.^[22] There are two biosynthetic processes responsible for the formation of CLA, which is found primarily in ruminants. The first mechanism is through the incomplete biohydrogenation of linoleic and linolenic acids that happened in the rumen.^[23] In the second mechanism, CLA can also be endogenously synthesized via Δ9 desaturation of trans-vaccenic acid in ruminant, mice, and humans,^[24–26] as shown in Fig. 3.

Figure 3. CLA biosynthesis in the ruminant’s body. Adapted from Bessa et al.^[27]

The impacts of CLA on human health

Since Ha et al.^[6] first reported the effect of CLA on epidermal neoplasia of mouse, there have been extensive amounts of research to investigate the health effects of CLA in human populations. There are many notable studies that demonstrated the beneficial effects of CLA in humans.^[28–30] A study by Whigham et al.^[31] stated that, at a dose of 3.2 g/d, CLA resulted in a significant reduction in fat mass in CLA supplemented group compared to the control. However, it should be noted that the normal concentration of CLA is typically in the range 0.1% of total fatty acid composition in the human body. This is still far from the intended effective dosage and thus warrants the supplementation of CLA in human diets. Therefore, the enrichment of CLA into poultry meat and products will prove to be a viable supplementary source of CLA for human consumption.

Overview of poultry production globally

The global poultry industry is benefiting from lower feed costs and high prices of other meats, according to the latest report from Rabobank.^[32] The continuous demand for chicken meat is increasing, and about 40% of chicken consumed in the Asia region alone. Poultry meat is high in demand, mainly due to its universal religious acceptance among the meat consumers, where pork is forbidden to Muslims, while beef is prohibited to Hindus. According to a report from the Food and Agriculture Organization (FAO),^[33] the demand for poultry meat has increased by about 30 million tonnes from 66.4 million tonnes back in 2000 to 2009. In 2013, the poultry meat demand stands at around 94 million tonnes, representing more than 90% of the global meat demand of 106 million tonnes. In view of the healthy demand for poultry meat across these regions, enriching the CLA content or modifying the fatty acid profiles of the poultry meat is a feasible way to position poultry meat as a functional food for humans.^[34] Tables 1 and 2 showed the CLA content in various poultry products and across various poultry species, respectively.

Table 1. Conjugated linoleic acid of various foods.

Food	Total CLA (mg/g) fat
Fish	0.3
Salmon	0.3
Pork	0.6
Shrimp	0.6
Chicken	0.9
Fresh ground turkey	2.5
Ice cream	3.6
Medium cheddar	4.1
Fresh ground beef	4.3
Butter	4.7
Plain yogurt	4.8
Cultured buttermilk	5.4
Homogenized milk	5.5
Lamb	5.6
Butter fat	6.1
Condensed milk	7.0

Modified and adapted from Muller and Delahoy.^[15]

Table 2. Mean CLA content in various poultry products.

Meat products	CLA content	References
Ground turkey	0.25%	Chin et al.^[Citation35]
Turkey frank	1.6 mg/fat	Chin et al.^[Citation20]
Smoked turkey	2.4 mg/fat	Chin et al.^[Citation20]
Canned food chicken breast	0.4 mg/fat	Chin et al.^[Citation20]
Chicken stick	0.6 mg/fat	Chin et al.^[Citation20]
Turkey stick	1.8 mg/fat	Chin et al.^[Citation20]

Fat deposition in poultry

The information on the mechanism that is responsible for the regulation of adipose tissue deposition, and metabolism in the chicken is relatively limited. Fat deposition in body tissues is the net result of absorption, de novo synthesis (fatty acid synthesis) and ß-oxidation (lipolysis). Lipids are absorbed by enterocytes and transported in packaged with cholesterol, lipoproteins, and other lipids into chylomicron (the largest lipoprotein) into blood circulation. The chylomicrons are rapidly dispersed and their constituent lipids utilized and stored in fat cells.^[38] Dietary lipids are synthesized by the liver and packed into very low-density lipoprotein (VLDL) and released into circulation. Free fatty acids (FFAs) are the targets of lipoprotein lipase (LPL) which incorporate into triglycerides (TAG)-rich VLDL, taken up by adipocell for storage. Only 15% of de novo lipogenesis occur in adipose tissue.^[39] De novo lipogenesis is a complex and highly regulated metabolic pathway that converts acetyl-CoA to fatty acids. There are two main enzymes involve in fatty acid synthesis in chickens namely acetyl-coenzyme (acetyl-CoA) carboxylase and fatty acid synthase (FAS).^[40] De novo lipogenesis begins with conversion of acetyl-CoA with malony-CoA and the final product with be palmitic acid (C16:0).^[41] Elongation beyond C16:0 to stearic acid (C18:0) and desaturated to oleic acid (C18:1Δ9) which is further desaturated and elongated to produce a variety of other PUFAs including arachidonic acid (C20:4n-6) and eicosapentaenoic acid (C20:5n-3).^[42] β-oxidation occurs when the supplied energy from carbohydrates is not sufficient. Mobilization of fatty acids from adipose during tissue TAG (lipolysis) occurs during times of negative energy balance or in response to stress, fasting or starvation. The initial step in lipolysis is catalyzed by hormone-sensitive TAG lipase.^[43] Once the fatty acids enter the muscle cell, they still must traverse the mitochondrial membrane where the fatty acid undergone degradation.^[44] Carnitine palmitoyl transferase is an enzyme that activates fatty acids so that they can be transported into mitochondria for oxidation.^[44] Excessive FFA supply and low oxidation resulted in more FFA to be directed toward non-adipose tissues such as the liver, skeletal muscle and the heart.^[45]

Fat a threat in the poultry industry

Current broiler chicken strains typically have about 13–14.5% of fat as part of their body weight.^[46] Faster growth rate in broiler chickens is typically accompanied by increased body fat deposition. This situation most commonly occurs among broiler chickens given feed ad libitum. There are several factors affecting fat deposition in broiler chickens including genetic, nutrition, sex, and age of the broiler chicken. Fat deposits grow by both hyperplasia and hypertrophy.^[47] During fat deposition in chicken, adipose tissue developed in three phases: the first phase is dominated by hyperplasia until 4 or 5 weeks of age, this is then followed by a second phase that featured both hyperplasia and hypertrophy until 6 or 7 weeks of age, and finally a predominant hypertrophy phase for chickens beyond 7 weeks of age.^[46] Modern emphasis on weight gain and efficiencies in poultry farming has resulted in rapid and excessive accumulation of poultry fats. This development is contrary to the increasing awareness of the importance of diet, and the detrimental effects of bad fats in the onset of human disease. Thus, it is not surprising that most health guidelines advocated reduced consumption of animal fats as a major preventive measure in preventing diseases of the cardiovascular system. In this sense, excessive fats in an animal carcass is considered a threat to the poultry industry, unless and until the health effects of the various types of fats are known. Therefore, CLA, known for its beneficial health effects, may hold a lot of promise to negate the adverse effects of fats on human health, if it is incorporated in a sufficient and efficient manner in poultry meats.

Approach to provide a nutritionally enhanced poultry meat source to consumers

In view of the immense beneficial effects of CLA, it is no surprise that there are feeding strategy in poultry production that is geared to increase CLA contents in meat. This could be achieved by means of by adding CLA in diets. There are profound interest in these efforts to enrich poultry meat with CLA, as CLA has been shown to protect against chemically induced cancer,^[6] atherosclerosis,^[48] diabetes,^[49] and obesity^[7] in rodent models. However, despite the potential for enhanced functional and nutritional properties that can be achieved by the inclusion of CLA in poultry meats, incorporation of CLA in chicken meats has yet to be commercially pursued in a large scale. Geese raised in a closed-house system and fed a standard feed that is low in LA and PUFA, while rich in saturated fatty acid (SFA), demonstrated lesser amounts of the beneficial n-3 fatty acids in their meat.^[50] The SFA and n-6 PUFA will increased the LDL cholesterol in the blood. These in turn increases the risk of atherosclerosis and cardiovascular diseases in man.^[51] Therefore, supplementation of CLA in poultry feed could produce a functional meat that provides good essential fatty acids for human consumption. Royan et al.,^[52] stated that high dosage of CLA (7%) can reduce broiler chickens performance, but their combination with soybean oil can moderate these adverse effects. In another study done by Shin et al.,^[53] it was stated that the combination of CLA and menhaden fish oil was recommended to reduce the concentrations of linoleic and arachidonic acids in broiler chicken breast and thigh muscles. This was done as it has the potential to provide consumers with a functional broiler chicken meat. It would be beneficial to the poultry industry if CLA, but less n-6 fatty acids, could be deposited in the poultry meat. Meats with high CLA but lower n-6 fatty acids may add value by creating new markets for poultry products aimed at health conscious consumers.

CLA reduces adipose tissue in poultry

During early life development, the chicken adipose tissue is characterized by hyperplasia and hypertrophy.^[54] Hyperplasia depends on the proliferation of pre-adipocytes, as mature adipocyte do not proliferate.^[55] There are many reports about the effects of CLA adipocyte proliferation and differentiation. For example, cell proliferation and differentiation were inhibited by CLA in 3T3-L1,^[56] rodents,^[57] human,^[58] and pig^[59] adipocytes. To our knowledge, the only report on the effects of CLA on adipocyte cellularity in chickens is by Ramiah et al.^[60] This study reported that 5% CLA dietary inclusion resulted in a decrease among the adipose cell numbers. According to Jiang et al.,^[61] CLA are known to regulate gene and proteins related to adipocyte proliferation and differentiation. Zhang et al.^[62] suggested that CLA reduces total body fat deposition in broilers by suppressing the activity of LPL in the plasma. LPL is an enzyme for fat absorption, which hydrolyzes fatty acids from circulating triacylglycerol, thus decrease fat uptake.^[63] CLA isomers are known to decrease the activity of stearoyl-CoA desaturase (SCD) or Δ9-desaturase.^[64] Several studies have shown that dietary CLA inhibits the activity of SCD in layers^[65] and broilers.^[66] The changes in SCD activity are associated with reductions in body fatness.^[67]

Alteration CLA on fatty acid profiles in poultry products

The purpose of modifying animal fats is to produce high quality products, which meet the dietary recommendations for a reduced intake of fat and cholesterol in the human diet, as well as to achieve an optimal ratio between SFA, monounsaturated fatty acid (MUFA) and PUFA, to minimize the risk for obesity, cancer, and cardiovascular diseases.^[68] In poultry, it is common that dietary fat has a great influence on fatty acid profiles of poultry meat.^[69] Two-thirds of poultry fat is composed of unsaturated fatty acids, and they belonged to the n-3 and n-6 fatty acids.^[70] Therefore, employing nutritional strategies including changing dietary fatty acid profile have a great potential in converting poultry meat, from a simple animal protein source to a valuable functional product. In general, meat from ruminants contains considerably more CLA than meat from non-ruminants, with the veal having the lowest, and lamb the highest (2.7 versus 5.6 mg CLA/g fat). Foods derived from non-ruminant animals were far lower in CLA content except for turkey. Turkey meat has the highest CLA content of 2.5 mg/g fat among non-ruminant species.^[20]

Effects of CLA on lipid metabolism in poultry species

The effect of CLA supplementation on the fatty acid composition in meats has been extensively studied in broilers,^[71–74] pigs,^[75,76] rabbits,^[77] and egg yolk.^[78] Kawahara et al.^[79] reported that the content of CLA in chicken breast meat significantly increased with feeding levels of dietary CLA. Similar results were reported by Aletor et al.^[80] and Bolukbasi.^[73] Du and Ahn^[72] reported that supplementing chickens with 0, 2.0, or 3.0% CLA over a 5-week period resulted in 0, 105.1, and 177.5 mg CLA/g lipids in breast muscle, respectively. Similarly, the concentration of CLA isomers (cis-9,trans-11 and trans-10,cis-12) increased linearly (p < 0.01) in leg and breast tissue samples.^[73] Apart from increased CLA concentrations in the adipose and muscle tissue, the supplementation of CLA influences tissue fatty acid composition in poultry. Several reports indicated that CLA supplementation increased the amount of SFAs (C14:0, C16:0, and C18:0), and decreases the MUFA fraction (mainly C18:1n-9) in poultry tissues by down-regulating the Δ9-desaturase activity.^[81,82] Choa et al.^[83] suggested that CLA could be included as an additive to produce meat containing rich n-3 PUFA, with a lower n-6: n-3 ratio. Similar trends were observed in layers,^[84] and it seems that CLA supplement via diets reduced the messenger RNA (mRNA) transcription of the Δ9-desaturase in a dose-dependent manner. Sisk et al.^[85] reported that SFA in liver lipids increased, whereas those of MUFA decreased with CLA supplementation and total concentration of PUFA did not change with dietary treatment. Szymczyk, et al.^[66] speculated that CLA reduced the concentration of oleic acid by inhibiting liver Δ9-desaturase activity. It was also found that dietary CLA decreased the concentrations of palmitoleic and oleic in the broiler chicken. Meanwhile, Ostrowska et al.^[86] reported an increased in C16:0 and C16:1 for intramuscular and subcutaneous fat deposits following CLA treatments. CLA production by bacteria such as Lactobacillus, Butyrivibrio and Propionibacterium strains have received special interest in the research field.^[87–89] A recent study done by Herzallah^[90] stated that supplementation of pure Lactobacillus (Lactobacillus reuteri) strains from camel, cattle, and goat increased CLA concentrations of 0.2–1.2 mg/g of fat in eggs and 0.3–1.88 mg/g of fat in broiler chicken.

CLA in laying hens

The enrichment of eggs with CLA can be considered a good supplementation strategy to increase human intake of CLA because eggs contain 30–35% fat. Study by Jones et al.^[91] showed that CLA in medium and high-fed groups (0.5 and 1.0 g CLA/kg), resulted in an increase in egg yolk CLA within 1 week of feeding. Effects of dietary CLA on egg production rate varied depending on experimental condition. Study by Kim et al.^[92] indicated that egg production rate decreased approximately at 10% when the laying hens were fed with 2% CLA. Similarly Shang et al.^[84] found that laying hen fed with 6% CLA had a noticeable reduction in egg production. Raes et al.^[78] showed that 1% CLA did not affect the rate of egg production and egg weight. Shang et al.^[84] and Watkins et al.^[93] demonstrated that dietary CLA affect the texture of egg yolk. It is reported that as dietary CLA concentration and refrigeration time increased, yolk firmness increased.^[94] The firmness of egg yolks from CLA-fed hen may be due to changes in pH, water content, and increased the content of SFAs.^[84] Supplementation of CLA to the hens’ diets had clear effects on the fatty acid composition of egg yolk, and resulted in a significant increase of SFA at the expense of MUFA.^[95] The content of SFA in yolk lipid is higher because of the increased of SFA components especially C14 and C16.^[78] Researchers found that dietary CLA was shown to induce significantly higher embryonic mortality in fertile eggs by altering the fatty acid composition in egg yolk.^[96] Oleic acid is the major fatty acids in egg yolk, and accounts for about 40% of total fatty acids in the eggs. This alteration may probably cause higher embryonic mortality in fertile eggs as oleic acid play an essential role in the survival of avian embryos.^[97] Li and Watkins^[98] reported that the decreased concentrations of linoleic and linolenic acids in yolk lipids of hens fed CLA, probably because of the competition of metabolites of dietary CLA isomers with metabolites of LA, particularly in reaction with Δ6-, Δ5-desaturases, and elongase. Table 3 summarized a number of studies on the effects of CLA on egg production in laying hens.

Table 3. Mean CLA content in various poultry species.

Reference	Broiler	Egg	Turkey
Chin et al.^[Citation20]	0.9 mg/fat	0.6 mg/fat	2.5 mg/fat
Schmid et al.^[Citation4]	1.5 mg/g FAME		0.2 mg/g FAME
Rule et al.^[Citation36]	0.7 mg/g FAME
Fritsche and Steinhardt^[Citation37]	1.5 mg/g FAME

Table 4. Effects of CLA on egg production of laying hens.

Experimental evidence	Author
Increase CLA in egg yolk after 1 week feeding	Jones et al.^[Citation91]
Decreased in egg production by 10% after fed with 2% CLA	Kim et al.^[Citation92]
Decreased in egg production by 6%	Shang et al.^[Citation84]
1% CLA did not affect the rate of egg production and egg weight	Raes et al.^[Citation78]
Increased egg yolk firmness	Choi^[Citation94]
Significant increase in SFA	Raes et al.^[Citation78]
High embryonic mortality	Aydin et al.^[Citation97], Aydin and Cook^[Citation96]

CLA in ducks

Halle et al.^[100] found that feeding 1 or 2 g CLA/kg diet increased the proportion of SFA and PUFA, whereas the MUFA was decreased in the breast muscle of ducks. The fatty acid compositions were not altered by CLA on breasts muscle of broiler chickens. Aydin and Cook^[96] measured significantly higher feed intake in broiler for the first 3 weeks of age after feeding 2 g CLA/kg, whereas CLA feeding did not depress feed intake of Peking ducks. However, no comparable results exist for body weight gain and final body weight between the duck and broiler chickens. Fesler and Peterson^[101] exhibited a 24 and 42% decrease in dissectible adipose tissue in CLA-fed Moulard duck after 3 and 6 weeks, respectively. The 20% increase in liver mass was noticed among growing ducks after 6 weeks; however, no effects on the adipose tissue was observed. Fesler and Peterson^[101] concluded that CLA elicited measurable effects on lipid metabolism in ducks, but these effects depended on physiological age.

CLA in geese

Zhang et al.^[102] reported that a significant increase of the biologically active cis-9, trans-11, and trans-10, and cis-12 CLA isomers in both liver and muscle of geese fed CLA (p < 0.01). In the same study, dietary CLA led to an increase (p < 0.01) in SFA and a reduction in MUFA (p < 0.05) concentrations in both the liver and muscle.^[102]

CLA in quails

In a study conducted in Japanese quails, feeding 0.25% CLA resulted in increased egg size. However, when the dose was increased to 2 and 3%, the egg size was reduced. In addition, liver size (%) increased in all groups except for quails fed with 0.25% CLA.^[96]

CLA as antioxidants

Whether CLA has antioxidant property is still debatable. CLA have been recognized as having antioxidative properties in several animal studies.^[103,104] A recent study by Jiang et al.^[74] reported that inclusion of 1% CLA into the diet increased the total superoxide dismutase activity, and decreased the malondialdehyde content in broiler chickens. Further to support the hypotheses, inclusion of dietary CLA at higher level (>3%) decreased lipid oxidation in tissue from rabbits and pigs.^[104,106] Bolukbasi^[73] explained that the decrease of lipid oxidation in meat lipid may be due to decreases in the overall degree of unsaturation in meat from chickens fed with CLA. In another study by Smith et al.,^[75] mixed isomers of CLA-fed pigs caused a reduction of stearoyl-coenzyme A desaturase (Δ9 desaturase) catalytic activity, and gene expression in adipose tissue which resulted in an increase of stearic acid and consequent migration of stearic acid to the sn-1/3 positions of triacylglycerols. This eventually increased the melting point and inhibits the susceptibility of lipid oxidation in pork. Yan et al.^[105] postulated that the conjugated form of CLA makes it less susceptible to free radical attack in studies of cooked chicken meat, ready-to-eat turkey breast rolls and raw turkey breasts. If it is proven that CLA have significant antioxidant properties, enrichment of meat with dietary CLA would be a useful method to control lipid oxidation.

Ip et al.^[104] found that CLA might inhibit the formation of thiobarbituric acid reactive substances (TBARS) in the mammary gland. However, TBARS is known to measure only the secondary oxidation products and may not reflect the degree of lipid oxidation. Despite repeated suggestions supporting the presence of antioxidant activities of CLA, there are few reports on CLA acting as pro-oxidant. van den Berg et al.^[106] tested whether CLA could protect membranes composed of 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) from oxidative modification under conditions of metal ion-dependent or ion-independent oxidative stress. Their results did not support CLA as an antioxidant. Yang et al.^[107] reported that CLA was very susceptible to auto-oxidation when exposed to air. This may take into account that the influence of CLA on growth performances as CLA might have lost its oxidation properties prior to feeding. Ramiah et al.^[108] reported that the propensity for lipid peroxidation was significantly higher after 6 days of meat storage indicating that CLA has no antioxidant properties. Another report by Leung and Lin^[109] stated that CLA might not possess antioxidant properties. Cis-9, trans-11 CLA possess strong pro-oxidant properties while pro-oxidant activity was not observed in trans-10,cis-12 CLA.^[111] The results of CLA as antioxidant are, therefore, inconsistent. According to Kang and Choi,^[110] the metabolic rate of CLA is slower in liver than in plasma. This indicated that CLA affected the fatty acid composition in a tissue-specific manner.

CLA as an anticancer agent

The mechanisms of apoptosis are highly complex and sophisticated. Apoptosis involved an energy-dependent cascade of molecular events.^[111] Crow et al.^[112] reported that the main apoptotic pathways comprised of extrinsic and intrinsic pathways. In the intrinsic pathway, stimulation via drugs, radiation and other stimulus leads to DNA injuries that cause changes to the mitochondrial permeability. This will activate apoptotic molecules such as cytochrome C, which then will combine with procaspase-8 to form a death-inducing signaling complex (DISC).^[113] Inhibition of mammary tumor metastasis in the murine model supplemented with as little as 0.1% CLA compared to diets with no CLA was observed by Hubbard et al.^[114] These studies indicated that even a concentration of CLA at 0.1% has evident anticarcinogenic properties in rodent models.

Tsuboyama-Kasaoka et al.^[115] demonstrated that CLA are able to reduce fat mass among female C57BL/6J mice which were fed with 1% (w/w) CLA. Results showed that the tumor necrosis factor (TNF)-α, and uncoupling protein (UCP)-2 mRNA levels increased 12- and 6-fold, respectively, in isolated adipocytes from CLA-fed mice compared with control mice. The upregulation of two genes by CLA is an important pre-requisite in apoptosis induction. Another study conducted by Palombo et al.^[116] reported that CLA might inhibit tumor growth by increasing tumor cell apoptosis on human colorectal and prostatic cancer cells. It is widely postulated that CLA may inhibit tumor growth through apoptosis.^[117,118] CLA was shown to be able to inhibit proliferation of tumor cells in human colon cancer cell line, mammary carcinogenesis in rats.^[119–121] Studies have suggested that CLA could be incorporated into tumor cell membrane lipid, and thus modulate arachidonic acid metabolism. In this sense, it was speculated that the CLA was desaturated and then elongated to a 20-carbon conjugated fatty acid. This 20-carbon fatty acid would interfere with the cellular arachidonic metabolism, and inhibit further tumor expansion.^[122] The ability of CLA to alter arachidonate levels may depend on the form of CLA (FFA versus esterified), as well as tissue- and species-specific effects.^[122] Study done by Qi et al.^[113] demonstrated increased expression of TNF-α, TNFR, and caspase-8. These suggested that both intrinsic and extrinsic apoptotic pathways were activated in the fat tissue of CLA-fed pigs (Fig. 4).

Figure 4. Regulating CLA-induced adipocyte apoptosis via intrinsic and extrinsic pathway. Adapted from Qi et al.^[115]

Transcriptional regulation of genes by CLAs

In recent years, insight into the mechanisms underlying the biological effects of fatty acids has enabled us to understand the roles of fatty acids in disease and growth. This is not surprising given the facts that fatty acids are recognized as major regulators of biological activity. Fatty acids are involved in the pathways of blood coagulation, and in blood vessels. Fatty acids are also linked to vascular resistance, sterol metabolism, signal transduction, enzyme activities, cell proliferation and differentiation, and receptor expression.^[123] Different levels of dietary fatty acids lead to changes in cell membrane function and structure. Fatty acids regulate de novo lipogenesis through their effects on gene expression.^[124] The peroxisome proliferator activated receptors (PPARs) are perhaps the best recognized “sensor” system for fatty acids. PPARs are member of the nuclear receptor superfamily and there are three subtypes of PPAR have been identified: PPAR α (alpha), PPAR β (beta), and PPAR γ (gamma).^[125] PPAR heterodimerize with retinoid X receptor and control the target gene expression at the promotor region. This stimulates gene transcription, and thus with CLA serving as ligand activator for PPAR isoforms, CLA is able to regulate cellular metabolism, differentiation, and part of carbohydrate homeostasis.^[126] In several landmark articles from the 2000s, it was demonstrated that PPARs are able to bind fatty acids with a general preference for CLA.^[127,128] Report by Konig et al.^[129] demonstrated that laying hens fed with 3% CLA failed to alter PPAR-α mRNA concentrations, but increased triacylglycerol and cholesterol concentrations in the liver. Dietary CLA was also reported to have resulted in the downregulation of PPAR-γ in abdominal adipose tissue of broiler chicken.^[108,130] In broiler chickens, dietary CLA has been shown to enhance mRNA expression of PPAR -γ in the spleen.^[131] In another report, Brown Dwarf laying hens fed with 5% CLA led to reduced SCD-1 activity and mRNA abundance (p < 0.05).^[132] Royan et al.^[133] reported that birds fed with dietary CLA showed the highest liver fat content and PPAR-α expression was low. Collectively, these evidences pointed to the anti-lipogenic effect of CLA. Therefore, it is clear that CLA may have imparted its action on the body composition of chickens through a series of mechanisms linked PPAR and their related genes.

Conclusions and future directions

The CLA content in the poultry species is low compared to the ruminants. This is unsurprising as the CLA is predominantly associated with products from ruminants. Lack of CLA content in poultry species offered a unique opportunity for producers to enrich the CLA content in meat through feeding and other biotechnological strategies. Even at such small volume (as little as 0.5%), CLA are able to alter the expression of gene and other associated mechanisms. This allowed CLA to exhibit beneficial antiobesity, and anticarcinogenic effects seen in in vivo and in vitro studies. It is, therefore, concluded that while CLA holds much promise to be incorporated in poultry meats as a functional food, a lot more has still to be done to make this technology practical during poultry production. Future direction on CLA research in poultry could thus focus on developing practical CLA incorporation method at the farm, as well as in depth study into the molecular mechanism and complex signaling pathways responsible for the beneficial effects of CLA for both poultry and in the human consumers.

Acknowledgments

The authors are very grateful to the Faculty of Veterinary Medicine, Universiti Putra Malaysia, and Institute of Tropical Agriculture. The authors declare that they have no competing interest. Suriya Kumari drafted the manuscript. Mehdi Ebrahimii revised the manuscript. Goh Yong Meng supervised the whole study and was the main reviser. All authors read and approved the final manuscripts.

Funding

This research was supported by the Malaysian Government Fundamental Research Grant Scheme (FRGS) under grant number 5450632.

Additional information

Funding

This research was supported by the Malaysian Government Fundamental Research Grant Scheme (FRGS) under grant number 5450632.