Studies in the late 1950s and 1970s showed that chicks fed a vitamin (V)-E deficient diet containing LA developed encephalomalacia,21,22,23,24,25 a neurodegenerative condition characterized by necrosis, edema, and microvasculature abnormalities that can lead to ataxia and death.26 Table 1 provides a detailed summary of all these studies, which are discussed herein.

Table 1 Effect of LA or OXLAMs on neurological outcomes in chickens. Full size table

Dam et al. reported that 2-day-old chicks fed a V-E deficient diet containing 1.5% ethyl-LA (weight percent) from the 2nd day of life onwards, died from encephalomalacia within 28 days (11 out of 12 chicks), compared to chicks fed a V-E deficient diet containing ethyl-ALA (0 out of 12) or a V-E deficient basal diet lacking both LA and ALA (0 out of 12).21 In a follow-up study, the authors found that a V-E deficient diet containing 1.5% ethyl-AA induced encephalomalacia faster than a V-E deficient diet containing 1.5% ethyl-LA or lard (mixed source of LA and AA).24 These studies demonstrated that when V-E is absent from the diet, AA and to a lesser extent LA, damage the brain of young chicks. ALA caused no damage.

Follow-up studies in chickens demonstrated that oxidized LA metabolites (i.e., OXLAMs) were the likely cause of encephalomalacia, and that this effect was linked by maternal dietary LA content. Bartov and Bornstein reported that maternal intake of a 4% soybean oil (relatively high in LA) or 4% tallow (low in LA) diet containing 5 or 25 mg/kg α-tocopherol acetate each, from 6 to 11 months of age, reduced egg-yolk LA concentration in the tallow group (independent of V-E dietary content), and decreased V-E concentration in yolk of both groups fed the tallow and soybean diets containing low α-tocopherol acetate (5 mg/kg).23 Chicks from each maternal group were then fed a V-E free diet containing 4% or 10% oxidized safflower oil high in LA (~70%) for 31 days to induce encephalomalacia. Oxidation was achieved by heating the oil at 145 °C for 24 h.

The authors reported that regardless of safflower oil content of the chick diet (4 or 10% level), chicks born to hens that were fed the 4% soybean oil diet containing low (5 mg/kg) α-tocopherol acetate levels experienced a significantly higher frequency of ataxia and mortality compared to chicks born hens fed 4% soybean oil containing 25 mg/kg α-tocopherol, or beef tallow containing either 5 or 25 mg/kg α-tocopherol.23 Hence, a high LA maternal diet low in V-E accelerated the development of ataxia and encephalomalacia-related mortality in chick offspring maintained on a V-E free diet containing oxidized safflower oil. Lowering maternal dietary LA or supplementing it with V-E was protective.

Budowski et al. performed a series of experiments to understand the cause of ataxia and mortaility linked to encephalomalacia.25 They fed a V-E free diet containing oxidized safflower oil or non-heated safflower oil methyl esters at 4% or 10% (w/w) to chicks for 2 to 3 weeks. At 4% oil content, the incidence of ataxia and mortality were higher in the group fed the non-heated safflower oil compared to the group fed oxidized safflower oil. At 10%, ataxia and mortality were higher in the group fed oxidized safflower compared to the group fed non-heated safflower oil. The findings suggest that in the absence of V-E, oxidized safflower oil at 10% (w/w) induced more neurological deficits than at 4%. Also, the fact that ataxia and death were observed in the group fed 4% non-heated safflower oil, suggests the absence of dietary V-E might induce oil auto-oxidation of LA (the main PUFA in safflower oil). It is not known whether the OXLAM species generated by auto-oxidation due to the absence of V-E in the diet, differ from those generated by heating. Notably, this study did not include a control group provided with V-E in the diet.

To test whether V-E was protective, the authors provided 1-day-old male chicks with non-heated or heated 10% (w/w) safflower oil methyl esters lacking or containing 1 µg/kg V-E for 19 days.25 Ataxia and mortality were relatively high in the groups fed non-heated oils lacking or containing V-E (65–70% ataxia; 20–30% mortality). In the groups provided with oxidized oil with or without V-E, the incidence of ataxia and mortality were much increased (90–95% ataxia, 70% mortality), irrespective of V-E content. It is possible that higher doses of V-E might have been more effective in reducing mortality and the incidence of ataxia by limiting auto-oxidation, particularly in the non-heated safflower oil group.

The authors also introduced the polar fraction (at 0.3% w/w) to the V-E free diet containing 4% safflower oil, and found that it induced ataxia and increased mortality compared to the same background diet lacking the polar extracts.25 Because oleic acid and LA are the main unsaturated fatty acids in safflower oil, the authors introduced oleic acid and LA derived ketone esters (i.e., oxidized fatty acids) at 0.2% and 0.12%, respectively, to the same V-E free diets containing 4% safflower oil. They found that the incidence of ataxia was 50% and 80% with the oleic acid and LA keto-esters, respectively, and thus higher than the diets lacking these oxidized compounds. A separate study by Kokatnur et al. showed that the addition of 0.25% (w/w) 12-oxo-cos-9-octadecenoic acid, 12-oxo-octadecanoic acid, 12-oxo-trans-10-octadecenoic acid, or 12-oxo-cos-9-octadecenoic acid methyl ester to a V-E deficient basal diet containing 10% corn oil, induced encephalomalacia in 42–100% of chickens compared to 17% in those fed the basal diet.27 The authors also determined that as little as 0.05% of ketone fatty acids induced encephalomalacia and that this effect was exacerbated by adding more corn oil to the diet (from 2.5 to 10% w/w).27 These studies provide direct evidence that OXLAMs, and to a lesser extent oxidized oleic acid metabolites, induce ataxia and mortality due to encephalomalacia in chickens.

In the Budowski et al. study, the administration of the anti-coagulant dicumarol to a V-E free diet containing 4% safflower oil reduced the incidence of ataxia and mortality,25 suggesting that the neurotoxic effects of OXLAMs may be caused by blood coagulation and disturbances in the brain’s microvasculature. This is in agreement with a study by Fischer et al. who showed morphologic changes in cerebral vasculature, characteristic of pre-ceroid formation in adult chickens fed an 8% LA diet lacking V-E, compared to chickens on a similar diet containing V-E.22 Fully developed ceroids have been linked to neurodegeneration.28