Effects of Krill Oil and Coconut Oil on Behavioral Changes and Inflammatory Markers in Rats with Chronic Unpredictable Mild Stress Induced Depression Model

Abstract

This study was conducted to determine the effects of two different types of fat (krill oil [KO] and coconut oil [CO]) on obesity, behavioral tests, and some inflammatory markers when consumed with a high-fat or control diet in rats with depression. The study was conducted mainly in two phases: the induction of depression (37 days) and the dietary intervention (60 days). After the induction of depression by chronic unpredictable mild stress, dietary intervention started. Sixty male Sprague–Dawley rats were divided into 6 groups with 10 rats in each group: (1) standard diet (SD), (2) SD + 5% KO, (3) SD + 5% medium-chain triglyceride (MCT)* (*CO to contain 5% MCT), (4) high-fat diet (HFD), (5) HFD + 5% KO, and (6) HFD + 5% MCT*. The open field test (OFT), forced swimming test (FST), and sucrose preference test were performed at baseline, end of the depression induction, and dietary intervention to observe behavioral changes in rats. After the final behavioral test, animals were sacrificed, and blood samples were collected for biochemical analyses C-reactive protein (milligram per liter), cortisol (microgram per deciliter), and insulin (micro-international units per milliliter) to assess inflammatory changes in the blood. All data were analyzed under two headings: baseline, end of depression induction, end of dietary intervention, and dietary intervention groups. Body weight gain was highest in the SD+KO and lowest in the SD+MCT group (P < .05). When behavioral tests were evaluated according to dietary intervention, it was found that the SD+MCT group spent the most time in the center, the least time in the periphery, and the lowest immobilization time (P < .05). In FST, the SD+KO with the highest weight gain was the most immobile group (P < .05). The study indicates that the weight-reducing effects of MCTs resulted in positive behavioral responses, particularly in OFT and FST. Through these properties, MCTs can be used medicinally in the prevention and treatment of behavioral changes due to depression.

INTRODUCTION

Depression, characterized by symptoms such as fatigue, guilt, lack of self-confidence, decreased appetite, and sleep disturbances, is a chronic disease that affects ∼1 in 5 of the world’s population, often with recurrent and life-threatening consequences.¹ The increasing effects of depression and related diseases on the world population have led to an increase in studies on the etiology of the disease and the establishment of new hypotheses in the etiology of the disease.¹ As a result of this, in addition to the genetic and environmental factors of depression that have been known for years, the effect of stress, inflammation, and immunological processes on depression has begun to be understood more clearly.¹

Although depression is a disease that triggers the inflammatory response, many diseases accompanied by inflammation also cause depression.¹ From another point of view, it is thought that the presence of chronic diseases accompanied by ongoing low-grade inflammation, such as obesity, may also affect the increase in depressive symptoms in the population. In summary, there is a mutual interaction between stress, inflammation, and chronic diseases.¹ In studies to determine the etiology of depression and inflammation, impaired cellular immunity and increased inflammatory biomarkers such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and C-reactive protein (CRP) have been found to play a role.¹ Some disorders in cellular immunity and the increase in inflammatory marker levels can be prevented by taking adequate amounts of antioxidants with food.²

Krill oil (KO) is an oil obtained from the ocean-dwelling Euphausia superba, rich in omega-3, eicosapentaenoic acid, and docosahexaenoic acid content.³ KO consists of omega-3 fatty acids, vitamins A and E, astaxanthin, a fat-soluble antioxidant carotenoid, phospholipids, and various fatty acids.³ The fact that the omega-3 fatty acids in KO are linked to phospholipids makes KO different from other marine sources of omega-3 fatty acids. In addition, astaxanthin, which is naturally present in its structure, is a powerful antioxidant with strong stability.³ Thanks to these properties, KO positively affects diseases characterized by inflammation, cognitive functions, and mental health, such as depression.

Coconut oil (CO) is an edible oil extracted from the mature coconut kernel harvested from the coconut palm.⁴ Although CO is often classified as a source of saturated fats because about 90% of its composition is saturated fatty acids, it is the medium-chain triglyceride (MCT) fatty acids that make up 64% of its content that distinguishes CO from other oils and make it unique in its health effects.⁴ Besides MCTs, it also contains phospholipids, tocopherols, and other bioactive components.⁴ In the literature, the health benefits of CO are associated with its MCT content.^4,5 MCTs, containing 6–12 carbons, are distinguished from other triglycerides due to their short carbon length.⁶ Unlike long-chain fatty acids, which are transported to the lymph via chylomicrons, MCTs are transported directly to the liver via the portal blood and don’t require the carnitine shuttle when transported to the mitochondrial membrane.⁶ In summary, MCT fatty acids’ transport pathways and oxidation rates differ from those of long-chain fatty acids.⁶ MCTs reduce body fat stores; they are known to accelerate thermogenesis, increase satiety, and reduce fat accumulation in adipocytes.⁶ Depression, chronic stress, obesity, and increased blood fat are processes that are related to each other.¹ Thanks to these properties, MCTs can help break the vicious circle between depression, chronic stress, and obesity.⁶

Adequate and balanced nutrition means the regular distribution of energy from macronutrients in the diet according to the body’s needs.⁷ The unbalanced distribution of energy percentages from macronutrients called carbohydrates, proteins, and fats in the diet is one of the most important factors causing obesity.⁷ In this direction, the energy coming from fats in the diet should not be ignored.⁷ Because a high-fat diet (HFD) is one of the most important triggers of obesity.⁷ Considering that obesity is a disease characterized by low-grade chronic inflammation, depression triggered by the inflammatory process and related findings are expected to be seen together with obesity.¹

KO has a protective effect on inflammation and obesity associated with inflammation, as it is rich in strong antioxidants such as vitamins A and E, astaxanthin, and omega-3 fatty acids, essential anti-inflammatory fatty acids.⁸ Based on the relationship between inflammation, obesity, and depression, it is predicted that KO will also have positive effects on depression. MCTs reduce fat accumulation in adipocytes by increasing the thermic effect and feeling of satiety. In other words, MCTs have a weight-reducing effect.⁸ These properties make MCTs unique in treating and preventing obesity.⁶ Similar to KO, MCTs are predicted to have positive effects on depression based on the relationship between inflammation, obesity, and depression. This study was conducted to determine the effects of two different oil types (KO and CO) on obesity, behavioral tests, and some inflammatory markers in the blood when consumed with a high-fat or a control diet in rats with depression.

MATERIALS AND METHODS

Animals

All animal experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals and performed under the Erciyes University Animal Experiments Local Ethics Committee, Kayseri, Türkiye (Date: 11.05.2022, Decision no: 22/088). With the one-way analysis of variance method, it was determined that a total of 48 rats initially, 8 in each group, should be studied with 80% power and 5% margin of error in line with the hypothesis that there would be an effect size of F = 0.56 between the groups. However, as a result of the literature review, studies on animals dying due to depression during the experiment were found, and this rate was found to be ∼20%^9
–11; it was finally decided that 60 rats should be studied, taking into account that there would be losses in the study. A total of 60 male Sprague–Dawley rats, aged 5 weeks and weighing 125–150 g, were obtained from the Erciyes University Experimental Researches and Application Center. The oestrus cycle in female rats affects depression-related processes and antioxidant parameters, so it was decided to study male rats in this study.¹²

Before the experiment’s start, the rats were acclimatized for 1 week in specially prepared and automatically acclimatized rooms with a constant temperature of 20–24°C, 45–65% relative humidity, and 12-h light/dark periods. All experiments were conducted at the Erciyes University Experimental Researches and Application Center.

Depression model

The chronic unpredictable mild stress (CUMS) model was used to establish the depression model in animals. CUMS is the most widely used, effective, and reliable rodent depression model today.¹³ In this model, researchers randomly apply various stressors. Application of all stressors should take at least two weeks, and the cycle should be repeated.¹³ The recommended period for the clear observation of depression symptoms is 30 days.¹³ The main theme of the model is to determine the stress state due to tension in animals based on the development of “anhedonia.”¹³

The CUMS defined by López-López et al. was used as a depression model in the study.¹⁴ The stressors applied in the study were, respectively, cage tilting (for 5 h at a 45° angle), sawdust wetting (for 12 h), swimming rats in 4°C cold water (15 min), circadian cycle change (for 24 h), water restriction (for 12 h), food restriction (for 12 h), crowded cages (for 24 h), putting a foreign object in the cage (for 5 h), isolation (for 24 h), and changing of animals between cages (for 12 h). All of the mentioned stress exposures were applied at different times of the day for 37 days, and care was taken not to apply the same stressor for two consecutive days. Rats consumed standard pellet feed during this period.

Behavioral tests were performed to determine whether the animals had developed depression. These tests were conducted at baseline, at the end of the depression model, and at the end of the dietary intervention.

Behavioral tests

The open-field test

It is a physiological evaluation method frequently used to evaluate behavioral parameters in experimental animals. It is made in a square arrangement made of plexiglass in special sizes (cca 2 × 2 m), the base of which is divided into equal squares by a line. The number of lines (locomotor activity) that the experimental animal left in a corner crossed with its four extremities, the behavior of sniffing and exploring the surroundings by rising on its hind extremities (rampancy), the duration, and the number of cleaning behaviors (grooming) were recorded and evaluated by video. As an indicator of autonomic functions, the number of defecations in 5 min was evaluated.¹⁵ The test was applied for 5 min. After each animal, the inside of the open field test (OFT) was cleaned with 20% alcohol.

The sucrose preference test

It is one of the tests frequently used to evaluate anhedonia in experimental animals. In the sucrose preference test (SPT), animals were exposed to both the test solution (20% sucrose) and drinking water for a period of 1 h following 23 h of food and water deprivation. Rats were placed in test cages, and fluid intake and sucrose consumption (20% sucrose) were recorded for 15 min. The test measured sucrose intake by reweighing a preweighed bottle. Anhedonia, a reduced sucrose consumption, is a widely accepted indicator of a depressive-like state in animals. Preference (%) for sucrose over water was calculated as: (Sucrose intake [g]/total fluid intake [g] × 100).¹⁵

The forced-swim test

It is one of the tests frequently used in creating and evaluating depression models in experimental animals. Rats were floated in a cylindrical container made of plexiglass with a height of 45 cm and a diameter of 30 cm. Rats were not allowed to jump out of the water and get support by touching their tails to the bottom of the water. The water was changed after each animal’s swim, and the temperature was kept between 23°C and 26°C. A 6-min video was recorded. The test results were evaluated based on the total time the animal was immobile.¹⁶

All behavioral tests were video recorded with a professional camera. The researchers analyzed them by watching them in slow motion.

Dietary intervention

After the depression model was formed, the animals’ body weight was re-measured, rats with similar weights were grouped into 10 rats (5 rats per cage) in each group, and dietary intervention started.

The contents of the diet were determined by the researchers by taking the contents of the feeds in a recent, similar study by Son et al.¹⁷ as an example and evaluating the contents of the ready-made feeds in the commercially available market, considering that it would affect the ease of preparation. The feeds were specially prepared.

In the literature review, it was found that the ratio of krill, coconut, or fish oil added to pellet feeds ranged between 2.5% (w/w) and 5% (w/w).^14
–16 Since this study is the most similar in terms of both timeliness and study design, it was based on the study conducted by Son et al.,¹⁷ and it was decided that the ratio of fatty acids in the final concentration should be 5% (w/w).

CO was used as an MCT source in the study. CO was added so that the final concentration in the feeds was 5% MCT. The contents of the diets were used in the experiment, and the number of rats in groups is given in Table 1.

Table 1.

Dietary Intervention and Number of Rats in Groups

n	10 (Group 1)		10 (Group 2)		10 (Group 3)		10 (Group 4)		10 (Group 5)		10 (Group 6)
Dietary intervention	Standart diet (SD)(Control Diet)		SD + 5% KO		SD + 5% MCT^b		High-fat diet (HFD)^a		HFD + 5% KO		HFD + 5% MCT^b
Ingredients		g/kg		g/kg		g/kg		g/kg		g/kg		g/kg
	Casein	200	Casein	200	Casein	200	Casein	200	Casein	200	Casein	200
	l-cystine	3	l-cystine	3	l-cystine	3	l-cystine	3	l-cystine	3	l-cystine	3
	Corn starch	150	Corn starch	150	Corn starch	150	Corn starch	150	Corn starch	150	Corn starch	150
	Sucrose	500	Sucrose	500	Sucrose	500	Sucrose	500	Sucrose	500	Sucrose	500
	Cellulose	50	Cellulose	50	Cellulose	50	Cellulose	50	Cellulose	50	Cellulose	50
	Corn oil	50	Corn oil	50	Corn oil	50	Corn oil	270	Corn oil	270	Corn oil	270
	Vitamin mix	10	Vitamin mix	10	Vitamin mix	10	Vitamin mix	10	Vitamin mix	10	Vitamin mix	10
	Mineral mix	35	Mineral mix	35	Mineral mix	35	Mineral mix	35	Mineral mix	35	Mineral mix	35
	Choline bitartrate	2	Choline bitartrate	2	Choline bitartrate	2	Choline bitartrate	2	Choline bitartrate	2	Choline bitartrate	2
			+Krill oil	50 g/kg	+Coconut oil	50 g/kg			+Krill oil	50 g/kg	+Coconut oil	50 g/kg

About 60% of the energy comes from fat.

Coconut oil to contain 5% MCTs.

KO, krill oil; MCT, medium-chain triglyceride.

Study groups are Group 1: Standard diet (SD), Group 2: SD + %5 KO, Group 3: SD + 5% MCT, Group 4: HFD, Group 5: HFD + 5% KO, Group 6: HFD + 5% MCT.

Arden Research and Experiment in Türkiye provided diets. During the dietary intervention process, the feeds were stored at −20°C. Every three days, at the same time of day (10:00–11:00 a.m.), the experimental animals’ feed consumption and body weights were recorded, and their feed was renewed.

The literature review determined that the duration of dietary intervention with fatty acid modification in experimental animals was generally between 2 and 2.5 months.^17
–19 In line with the literature, the dietary intervention was continued for 2 months in this study. The animals were sacrificed at the end of the 14-week experimental period, and blood samples were collected for biochemical analyses.

The methodology can be summarized as given in Figure 1.

FIG. 1.

Study methodology.

Statistical analysis

The SPSS 22.0 for Windows” statistical package program was used for statistical analysis. The data group of this study is 10. It is not appropriate to use parametric tests for a study with a data group of 10. Even if normal distribution tests show this, the first condition for the parametric assumption is the number of data. For this reason, all of the data were evaluated with nonparametric tests. As a descriptive statistical analysis, median (x ̃), and minimum–maximum values are used. “Kruskal–Wallis” test for nonparametric data was performed. In cases where the difference between the groups was significant (P < .05), the appropriate post hoc method, the “Dunn–Bonferroni” test, was used for Kruskal–Wallis. In repeated measurements, “Friedman” analysis was performed. In cases where the difference between the groups was significant (P < .05), the appropriate post hoc method, the “Wilcoxon Signed Ranks” test, was used for Friedman. The data were evaluated at a significance level 0.05 with a 95% confidence interval.²⁰

RESULTS

The experiment had no adverse events, and the study was completed with 60 rats. All data were analyzed under two headings: baseline, end of depression induction, end of dietary intervention, and dietary intervention groups. Table 2 shows the body weight and feed intake of rats during the dietary intervention. There was a significant difference between SD, SD+KO, HFD+KO, HFD+MCT; SD+MCT, HFD+KO, HFD+MCT, and HFD, HFD+KO, for beginning body weight (P < .05). Weight gain rate in order from high to low: SD+KO, HFD+KO, HFD+MCT, HFD, SD, SD+MCT, respectively (P < .001). The difference between groups was statistically significant.

Table 2.

Body Weight and Feed Intake of Rats During the Dietary Intervention

	SD(n = 10)	SD + KO(n = 10)	SD + MCT(n = 10)	HFD(n = 10)	HFD + KO(n = 10)	HFD + MCT(n = 10)
Parameters	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–-Max)	P
Body weight at the beginning of the dietary intervention (g)^a	191.33^a (95.03–283.00)	167.50^a (67.33–169.66)	180.00^b (72.13–180.66)	175.83^c (71.50–181.66)	153.83^a ^,b ^,c (62.63–159.33)	156.83^a ^,b (63.63–161.33)	.001
Body weight at the end of the dietary intervention (g)^a	338.30(328.20–348.40)	443.70(329.60–557.80)	367.70(353.60–381.80)	370.40(348.20–392.60)	375.30(362.20–388.40)	358.70(357.40–360.00)	.617
Weight gain (g)^a	113.20(107.60–118.80)	251.40(128.60–354.20)	151.30(136.80–165.80)	155.90(130.20–181.60)	187.40(177.60–197.20)	167.80(166.40–169.20)	.282
Weight gain rate (%)^b	69.90(10.17–71.17)	118.71(25.22–125.28)	50.22(12.15–52.59)	72.42(15.87–85.47)	100.00(21.20–110.10)	87.89(18.47–93.98)	<.001
Food intake (g animal⁻¹ day⁻¹)^a	443.45(441.47–445.47)	462.80(403.80–521.80)	411.06(405.07–417.07)	446.63(445.33–447.93)	465.36(452.33–478.40)	399.67(392.00–406.33)	.427

P was calculated by the Kruskal–Wallis test, and the Dun–Bonferonni test was performed for post hoc analysis. A significant difference exists between groups with the same letters a, b, and c in the same line.

P was calculated by the Kruskal–Wallis test, and the Dun–Bonferonni test was performed for post hoc analysis. Statistically significant differences were found in all groups.

Data shown in bold are significant at P < 0.05 level.

Table 3 summarizes the results of behavioral tests. There were statistical differences in time spent at the center and periphery in the OFT (P < .05). The time spent at the center decreased, and the time spent at the periphery increased with depression induction (P < .05). As a result of the dietary intervention, it was determined that the time spent in the periphery, which is an indicator of depressive symptoms, decreased significantly while the time spent in the center increased significantly (P < .05). The longest immobilization time in the forced swimming test (FST) was determined at the end of the depression induction, as expected. It was found that this time decreased significantly with dietary intervention (P < .05). The consumption rate of sucrose water at the end of the depression induction was 56.60 ± 0.47, the beginning was 41.55 ± 1.22, and the end of the dietary intervention was 39.41 ± 0.72. The highest consumption rate of sucrose water was determined at the end of the depression induction. There were statistical differences between the beginning and end of the depression induction; the end of the depression induction, and the end of the dietary intervention (P < .05).

Table 3.

Results of Behavioral Tests

Duration
Behavior tests	Baseline	End of depression induction	End of dietary intervention
Behavior tests	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–Max)	P
The open field test
Time spent at the center (sec/5 min)^a	1.50 (0.00–5.00)^a	0.00 (0.00–16.00)^b	7.00 (0.00–31.00)^a ^,b	.004
Time spent in the periphery (sec/5 min)^a	298.50 (295.00–300.00)^a	300.00 (284.00–300.00)^b	294.50 (269.00–396.00)^a ^,b	.004
Crossed with four extremities^a	36.00 (0.00–66.00)	25.00 (2.00–67.00)	40.00 (0.00–96.00)	.303
Rampancy behavior (number/5 min)^a	14.00 (4.00–18.00)	10.00 (2.00–20.00)	13.00 (3.00–27.00)	.197
Grooming behavior (number/5 min)^a	2.50 (1.00–7.00)	2.00 (0.00–6.00)	2.00 (0.00–6.00)	.965
Defecation (number/5 min)^a	4.00 (0.00–6.00)	3.00 (1.00–8.00)	4.00 (0.00–7.00)	.438
The Forced Swim Test
Immobilization time (sec/5 min)^a	22.00 (14.00–48.00)	35.00^a (9.00–60.00)	22.00^a (6.00–59.00)	.044
The Sucrose Preference Test
The consumption rate of sucrose intake/total fluid intake (%)^a	38.2 (15.8–54.4)^a	52.6 (41.8–71.8)^a ^, ^b	34.5 (13.2–90.4)^b	.011

P was calculated by the Friedman test and the Wilcoxon signed-rank test was performed for post hoc analysis. A significant difference exists between groups with the same letters a and b in the same column.

Data shown in bold are significant at P < 0.05 level.

The behavioral test results of rats according to dietary intervention groups are displayed in Table 4. Significant differences were found in only two of the five parameters examined in the OFT, according to the diet groups. There was time spent at the center and on the periphery (P < .05). The group with the highest time spent at the center was the SD+MCT. In line with this situation, the group with the least time spent in the periphery is SD+MCT. There were statistical differences between SD, SD+KO; SD+KO, SD+MCT; SD, HFD; SD+MCT, HFD and SD, HFD+KO in terms of time spent at the center (P < .05). There were statistical differences between SD, HFD+KO; SD+MCT, HFD+KO; SD, HFD+MCT; SD, SD+KO, and SD, HFD in terms of time spent in the periphery (P < .001). There were statistical differences between the SD, SD+KO; SD+KO, and SD+MCT groups in the forced swim test (P < .05). The longest immobilization time was found in the SD+KO group.

Table 4.

Behavioral Tests According to Dietary Intervention Groups

	SD (n = 10)	SD + KO (n = 10)	SD + MCT (n = 10)	HFD (n = 10)	HFD + KO (n = 10)	HFD + MCT (n = 10)
Behavior tests	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–Max)	$\tilde{x}$ (Min–Max)	P
The open field test
Time spent at the center (sec/5 min)^f	10.00 (3.00–28.00)^a,c,e	2.00 (0.00–20.00)^a,b	15.00 (5.00–25.00)^b,d,e	2.00 (0.00–14.00)^c,d	4.00 (0.00–11.00)^e	4.00 (0.00–31.00)	.004
Time spent in the periphery (sec/5 min)^f	288.00^a,c,d,e (272.00–297.00)	298.00^d (280.00–300.00)	285.00^b (275.00–295.00)	298.00^e (286.00–300.00)	297.00^a,b (289.00–390.00)	297.50^c (269.00–396.00)	<.001
Crossed with four extremities^f	47.00 (13.00–74.00)	20.00 (2.00–94.00)	45.00 (25.00–96.00)	35.00 (5.00–66.00)	31.50 (0.00–90.00)	29.50 (11.00–58.00)	.231
Rampancy behavior (number/5 min)^f	13.00 (5.00–21.00)	14.00 (6.00–27.00)	12.50 (5.00–22.00)	11.50 (3.00–23.00)	12.00 (5.00–27.00)	15.50 (7.00–27.00)	.546
Grooming behavior (number/5 min)^f	1.50 (0.00–5.00)	2.00 (0.00–4.00)	3.00 (0.00–6.00)	3.00 (1.00–4.00)	2.50 (0.00–5.00)	2.50 (0.00–4.00)	.659
Defecation (number/5 min)^f	5.00 (1.00–7.00)	3.00 (0.00–6.00)	4.00 (0.00–5.00)	4.00 (2.00–6.00)	3.00 (0.00–5.00)	4.00 (2.00–5.00)	.383
The Forced Swim Test
Immobilization time (sec/5 min)^f	22.50 (11.00–41.00)^a	45.50 (27.00–58.00)^a,b	21.50 (9.00–41.00)^b	41.50 (16.00–51.00)	35.50 (15.00–60.00)	40.50 (27.00–49.00)	.002
The Sucrose Preference Test
The consumption rate of sucrose intake / total fluid intake (%)^f	60.2 (49.2–74.6)	31.00 (13.2–52.2)	50.7 (23.2–68.2)	48.00 (20.4–71.8)	39.30 (15.80–90.4)	34.90 (30.4–59.8)	.115

A significant difference exists between groups with the same letters a, b, c, d, and e in the same line.

P was calculated by the Kruskal–Wallis test and the Dun–Bonferonni test was performed for post hoc analysis.

Data shown in bold are significant at P < 0.05 level.

Figure 2 shows some biochemical results of rats according to dietary intervention groups. The highest and lowest CRP (milligram per liter) values were found in the HFD and SD+KO groups, respectively. The highest and lowest cortisol (microgram per deciliter) levels were found in SD and SD+KO groups, respectively. However, there was not a significant difference between the groups in terms of CRP and cortisol (P = .330 and P = .180, respectively). In the study, the insulin (micro-international units per milliliter) values of the rats were also examined, but since the insulin value was found to be too low (<0.400) to be measured in all of the rats, it could not be evaluated.

DISCUSSION

Depression is thought to be a strong comorbidity in both humans and animals.¹ Studies on animals are of great importance in terms of elucidating the relevant mechanisms in humans. This study evaluated the effects of KO and CO on depression, obesity, behavioral tests, and the inflammation cycle. It was hypothesized that KO, with its anti-inflammatory properties, would break this cycle (depression–inflammation–behavioral tests), and CO, with its antiobesogenic effect (thanks to MCTs), would break this cycle (depression–obesity–behavioral tests). The study results showed that MCT fatty acids were more effective in this process and that the body weight-reducing effect of MCT fatty acids produced positive effects in behavioral tests, especially in the open field and FSTs. Thanks to these properties, MCT fatty acids can be used medicinally in the prevention and treatment of behavioral changes due to depression. The use of such nutritional treatment options in depression and depression-related diseases such as obesity and inflammation will contribute to the reduction of drug-dependent costs and contribute to the understanding of the medical power of nutrients, especially fatty acids.

Another aim of the study was to answer the question, “Can krill oil or coconut oil perform these positive properties even on a high-fat diet?” For this reason, a HFD group was added to each SD group in the study. However, the expected results were not achieved in both behavioral tests and biochemical parameters. It can be said that differences in the stress response made it difficult to access and interpret the predicted data at many points.

To our knowledge, this is the first report to comprehensively evaluate the relationship between depression, obesity, different medicinal foods (especially various oils and fatty acids), some inflammatory markers and behavioral parameters.

It was determined that the weight gain rate in the group fed with CO was significantly lower compared to the group fed with KO (P < .001, Table 2). In a study comparing the change in body weight of rats consuming feed containing long-chain fatty acids, or MCTs, visceral fat mass was significantly lower in the MCT group.²¹ In another recent study investigating the effects of MCTs in obese rats, it was found that MCTs had a weight-reducing effect and lowered plasma and liver lipid levels.²² MCTs reduce body fat stores by accelerating thermogenesis, increasing satiety, and reducing adipocyte fat accumulation.⁴ It is thought that the mentioned properties of MCTs play a role in this result.

The study evaluated the time spent in the center and the periphery within the OFT, the number of lines crossed with the four extremities, rampancy, grooming behavior, and the number of defecations. It is known that animals with low stress tend to spend more time in the central, open area of the box; animals with high-stress levels spend more time in corners.¹⁵ In parallel with this information, it was determined in the study that the time spent in the center increased and the time spent in the periphery decreased with the effect of dietary intervention (P < .05, Table 3).

In a recent study by Ueno et al.,²³ the researcher petted some rats daily, and some were assigned as the control group. In the OFT performed at the end of the 3-week experiment period, two parameters were evaluated and there was not a significant difference in terms of distance the animals traveled and time spent in the center. Among the parameters evaluated in our study, a significant difference was found in terms of time spent in the center and the periphery (P < .05, Table 3).

Another parameter investigated in the OFT is the exploratory behavior of rats to explore the environment and obtain information. In cases of stress, the frequency of this behavior decreases.²⁴ A study observed that the group subjected to prenatal stress did less lifting movement in the hind extremity than the control group.²⁵ In this study, it was determined that these movements decreased with depression and increased again as a result of diet intervention, but this difference was not statistically significant. The number of defecations, which is an indicator of autonomic functions, increases in animals with anxiety behavior.²⁶ In the study, the highest number of defecations was calculated during the depression period. However, this difference is not statistically significant (P > .05, Table 3).

All of the factors, such as odor, sound, and laboratory conditions in the place where the OFT is carried out, can affect the stress level of the animals and, thus, the test results. In addition, the transition of animals from the cage to the open space is a stress factor associated with a “new environment” for them. These situations can influence factors related to stress and anxiety independent of the experimental process.¹⁵ These factors may explain the statistically significant difference between results and parameters.

In this study, as expected, it was determined that as a result of the depression induction, immobilization time in the forced swim test significantly increased (P < .05, Table 3). In a recent study investigating the effects of stress on animals, there was not a significant difference between the animals in the stressed group and the control group in terms of immobilization time in the forced swim test.²³ In a study conducted in 2017, which aimed to evaluate the effects of antidepressants on mice with the FST, there was not a significant difference between the control and experimental groups in terms of immobilization time.²⁷ In another study evaluating the effect of antidepressants, it was determined that immobilization time in the experimental group decreased significantly.²⁸ The sensitivity of rats to the FST can be affected by many environmental factors, such as pretest preconditioning, treatment protocol, experimental design, and laboratory environment.¹⁵ These factors can explain the differences between the results.

The SPT is a reliable behavioral test frequently used to evaluate anhedonia, one of the main symptoms of depression.¹⁵ Rodents have an innate affinity for sweet food or solutions.¹⁵ In the case of depression, the consumption of sweet flavors is expected to decrease.¹⁵ In a study evaluating the effects of depression-related symptoms on age and gender, it was found that the sucrose preference index significantly reduced along with anhedonia.²⁹ In another study, which was carried out similarly to the design of our study, it was found that the water consumption rate with sucrose in depressed rats was significantly higher than in the control group.³⁰ In this study, the highest sucrose consumption rate was found at the end of the depression induction in parallel with the findings of the last-mentioned study (P < .05, Table 3). Here, the effect of stress on appetite should not be overlooked. Because it has been reported that mice may have increased appetite due to chronic stress.³¹ This may affect the increase in the sucrose consumption rate of rats during the depression process and the decrease as a result of dietary intervention.

When the behavioral tests were evaluated according to the dietary intervention groups, it can be said that the SD+MCT and SD groups were the two prominent groups in achieving the desired outcome in the dietary intervention (In the OFT, the highest time spent in the center and lowest time spent in the periphery; In the forced swim test, the lowest immobilization time) (P < .05, Table 4). Behavioral tests according to dietary intervention groups and weight gain data are evaluated together. It is expected that the SD+KO group with the highest weight gain was the most immobile group, and the SD+MCT group, with the lowest body weight gain, was the most mobile group.

The lowest CRP and cortisol levels were found in the SD+KO group (P > .05, Fig. 1). As mentioned, KO is an oil with antioxidant properties. However, no significant difference was found between the groups in terms of CRP and cortisol. This may be attributed to the response of animals to stress exposure. Stress causes differences in biochemical response and behavioral parameters in rodents, just like in humans.³² In summary, it can be said that the difference in the stress response prevented the expected results in biochemical parameters.

As mentioned in the study’s most important findings in the introduction to the discussion, another aim of the study was to evaluate the effect of KO and CO on the inflammatory process and weight gain, respectively, induced by an HFD. In other words, to answer the question, “Can krill oil or coconut oil perform these positive properties even on a high-fat diet?” For this reason, an HFD group was added to each SD group in the study. However, the expected results were not achieved in both behavioral tests and biochemical parameters (e.g., the HFD+KO group did not have anti-inflammatory biochemical values or the HFD+MCT group did not lose weight.) This may be explained by the individual variability of depression and stress response in rodents, just like in humans.³²

The most important limitation of the study is that the difference in response to depression and stress makes it difficult to interpret the study findings. MCT oil could also have been used as a source of MCT in the study. However, difficult and costly access to MCT oil in the region where the study was conducted led the researchers to search for an alternative oil source. This situation can be considered among the limitations of the study.

The study mostly interpreted the processes associated with depression in rats through behavioral tests. Future studies based on more objective data (more blood markers, stem cell modeling, etc.) measuring the stress level of rats will help to elucidate the processes of depression, obesity, and inflammation.

The strengths of the study can be expressed as follows: KO is labeled as a “new food” by the European Union, and its popularity is increasing daily. Available information in the literature shows that KO positively affects mental health, glucose tolerance, cardiovascular health with antiatherosclerotic activity, anti-inflammatory effect, and menstrual health. However, despite all the possible positive effects reported, expert organizations in the field (Food and Drug Administration —European Food Safety Authority) continue to be concerned about the use of KO in the treatment of diseases in humans due to a lack of studies. To determine the treatment protocols to be applied to humans, it is mandatory to conduct studies on experimental animals.³³ The planned study contributed to the literature by questioning the preventive and therapeutic use of KO in neurological diseases such as depression and using KO in these processes. Although statistically significant results could not be obtained, low CRP and cortisol levels obtained in the group receiving KO can be considered a positive indicator of inflammation.

MCT fatty acids are generally known for their effects on increasing thermogenesis and reducing weight. In addition to these properties, positive effects on behavior on the depression–obesity axis were observed concretely in this study. The prevalence of neurodegenerative diseases such as depression is increasing daily; in this direction, medical foods for the treatment of related diseases should be brought to the forefront. With the results of this study, MCTs can be considered in terms of their effects on neurodegenerative diseases, behavior, and obesity.

As aforementioned, this is, to the best of our knowledge, the first report to comprehensively evaluate the relationship between depression, obesity, different medicinal foods (especially various oils and fatty acids), some inflammatory markers, and behavioral parameters. These can be considered among the study’s strengths.

CONCLUSION

The difference in the response to depression and stress made the interpretation of the study findings very difficult. In future studies, to interpret the response to stress and inflammation more objectively, more blood findings related to inflammation markers (such as TNF-α, IL-6) can be added to the study, studies at the cell level related to the stress line, including endoplasmic reticulum stress, or immunohistochemical studies can be added to strengthen future studies.

MCT fatty acid can be used directly as a source of MCT in areas where it is available. In this study, MCT was calculated from the content and concentration of CO. The use of direct MCT oil may provide more effective results.

Another comment: factors such as the fatty acid ratio used in the dietary intervention (5%) and the diet duration (60 days) may explain why KO was ineffective. In future studies, different intervention durations and concentrations of dietary interventions can be tested.

Further studies are needed to understand better the roles of fats in depression, inflammation, and obesity and to incorporate fats into medical processes.

Footnotes

AUTHORS’ CONTRIBUTIONS

Study design: H.K.B., N.Ö., A.L.; Data collection: H.K.B.; Data analysis: H.K.B., N.Ö., A.L.; Draft preparation: H.K.B.; Critical review for content: H.K.B., N.Ö., A.L.; Final approval of the version to be published: H.K.B., N.Ö., A.L.

DATA AVAILABILITY STATEMENT

The authors declare that all data associated with the current study are available from the corresponding author upon reasonable request.

AUTHOR DISCLOSURE STATEMENT

There is no conflict of interest between the authors.

FUNDING INFORMATION

This work was supported by the Erciyes University Scientific Research Projects Coordination Unit (Project ID:12064).

References

Milaneschi

, Simmons

, van Rossum

EFC

, et al. Depression and obesity: Evidence of shared biological mechanisms. Mol Psychiatry, 2019; 24(1):18–33; doi: 10.1038/s41380-018-0017-5

Morén

, deSouza

, Giraldo

, et al. Antioxidant therapeutic strategies in neurodegenerative diseases. Int J Mol Sci, 2022; 23(16):9328; doi: 10.3390/ijms23169328

Yang

, Lee

, et al. Krill oil supplementation improves dyslipidemia and lowers body weight in mice fed a high-fat diet through activation of amp-activated protein kinase. J Med Food, 2016; 19(12):1120–1129; doi: 10.1089/jmf.2016.3720

Deen

, Visvanathan

, Wickramarachchi

, et al. Chemical composition and health benefits of coconut oil: An overview. J Sci Food Agric, 2021; 101(6):2182–2193; doi: 10.1002/jsfa.10870

Wang

, Wang

, Li

, et al. Effects of dietary coconut oil as a medium-chain fatty acid source on performance, carcass composition and serum lipids in male broilers. Asian-Australas J Anim Sci, 2015; 28(2):223–230; doi: 10.5713/ajas.14.0328

Sung

, Liao

, Chien

. Medium-chain triglycerides lower blood lipids and body weight in streptozotocin-induced type 2 diabetes rats. Nutrients, 2018; 10(8):963; doi: 10.3390/nu10080963

Kim

, Ahn

. Eating out and consumers’ health: Evidence on obesity and balanced nutrition intakes. Int J Environ Res Public Health, 2020; 17(2):586; doi: 10.3390/ijerph17020586

Ouakinin

SRS

, Barreira

, Gois

. Depression and obesity: Integrating the role of stress, neuroendocrine dysfunction and inflammatory pathways. Front Endocrinol (Lausanne), 2018; 9(9):431; doi: 10.3389/fendo.2018.00431

Strekalova

, Spanagel

, Bartsch

, et al. Stress-induced anhedonia in mice is associated with deficits in forced swimming and exploration. Neuropsychopharmacol, 2004; 29(11):2007–2017; doi: 10.1038/sj.npp.1300532

10.

Carnevali

, Mastorci

, Audero

, et al. Stress-induced susceptibility to sudden cardiac death in mice with altered serotonin homeostasis. PLoS One, 2012; 7(7):e41184; doi: 10.1371/journal.pone.0041184

11.

Sudakov Konstantin

. Sudden death: Mechanisms of resistance under acute emotional stress in rats and rabbits. Pathophysiol, 1999; 6(2):111–119; doi: 10.1016/S0928-4680(99)00007-3

12.

Rebolledo-Solleiro

, Fernández-Guasti

. Influence of sex and estrous cycle on blood glucose levels, body weight gain, and depressive-like behavior in streptozotocin-induced diabetic rats. Physiol Behav, 2018; 194:560–567; doi: 10.1016/j.physbeh.2018.06.033

13.

Antoniuk

, Bijata

, Ponimaskin

, et al. Chronic unpredictable mild stress for modeling depression in rodents: Meta-analysis of model reliability. Neurosci Biobehav Rev, 2019; 99:101–116; doi: 10.1016/j.neubiorev.2018.12.002

14.

López-López

, Jaime

, Escobar Villanueva

MDC

, et al. Chronic unpredictable mild stress generates oxidative stress and systemic inflammation in rats. Physiol Behav, 2016; 161:15–23; doi: 10.1016/j.physbeh.2016.03.017

15.

Sarkisova

, Kuznetsova

, Kulikov

, et al. Spike-wave discharges are necessary for the expression of behavioural depression-like symptoms. Epilepsia, 2010; 51(1):146–160; doi: 10.1111/j.1528-1167.2009.02260.x

16.

Yankelevitch-Yahav

, Franko

, Huly

, et al. The forced swim test as a model of depressive-like behavior. J Vis Exp, 2015(97):52587; doi: 10.3791/52587

17.

Son

, Kim

, Lee

, et al. Partial replacement of dietary fat with krill oil or coconut oil alleviates dyslipidemia by partly modulating lipid metabolism in lipopolysaccharide-injected rats on a high-fat diet. Int J Environ Res Public Health, 2022; 19(2):843; doi: 10.3390/ijerph19020843

18.

Aydin Cil

, Ghosi Ghareaghaji

, Bayir

, et al. Efficacy of krill oil versus fish oil on obesity-related parameters and lipid gene expression in rats: Randomized controlled study. PeerJ, 2021; 9:e12009; doi: 10.7717/peerj.12009

19.

Ferramosca

, Conte

, Burri

, et al. A krill oil supplemented diet suppresses hepatic steatosis in high-fat fed rats. PLoS One, 2012; 7(6):e38797; doi: 10.1371/journal.pone.0038797

20.

Panos

, Boeckler

. Statistical analysis in clinical and experimental medical research: Simplified guidance for authors and reviewers. Drug Des Devel Ther, 2023; 17:1959–1961; doi: 10.2147/DDDT.S427470

21.

Suzuki

, Oshima

, Sonotsuka

, et al. Effects of medium chain triglycerides intake on lipid metabolism and intestinal disaccharidase activities in rats. Yakugaku Zasshi, 2020; 140(8):1051–1061; doi: 10.1248/yakushi.20-00024

22.

Xia

, Yu

, Zeng

, et al. Effects of medium chain triglycerides on lipid metabolism in high-fat diet induced obese rats. Food Funct, 2022; 13(17):8998–9009; doi: 10.1039/d2fo01711c

23.

Ueno

, Takahashi

, Suemitsu

, et al. Effects of repetitive gentle handling of male C57BL/6NCrl mice on comparative behavioural test results. Sci Rep, 2020; 10(1):3509; doi: 10.1038/s41598-020-60530-4

24.

Prut

, Belzung

. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: A review. Eur J Pharmacol, 2003; 463(1–3):3–33; doi: 10.1016/s0014-2999(03)01272-x

25.

Nater

, Abbruzzese

, Krebs

, et al. Sex differences in emotional and psychophysiological responses to musical stimuli. Int J Psychophysiol, 2006; 62(2):300–308; doi: 10.1016/j.ijpsycho.2006.05.011

26.

Campos

, Fogaça

, Aguiar

, et al. Animal models of anxiety disorders and stress. Braz J Psychiatry, 2013; 35 Suppl 2:S101–S11; doi: 10.1590/1516-4446-2013-1139

27.

Suman

, Zerbinatti

, Theindl

, et al. Failure to detect the action of antidepressants in the forced swim test in Swiss mice. Acta Neuropsychiatr, 2018; 30(3):158–167; doi: 10.1017/neu.2017.33

28.

Tanyeri

, Büyükokuroğlu

, Tanyeri

, et al. Evaluation of the effects of desipramine, venlafaxine and bupropion on depression and anxiety in mice by forced swimming test and elevated plus maze test. Sakarya Med J, 2018; 8(4):820–829; doi: 10.3183/2/smj.483533

29.

Eltokhi

, Kurpiers

, Pitzer

. Baseline depression-like behaviors in wild-type adolescent mice are strain and age but not sex dependent. Front Behav Neurosci, 2021; 15:759574; doi: 10.3389/fnbeh.2021.759574

30.

Chen

, Wang

, Xu

. Chronic unpredictable mild stress induced depression-like behaviours and glutamate-glutamine cycling dysfunctions in both blood and brain of mice. Pharm Biol, 2019; 57(1):280–286; doi: 10.1080/13880209.2019.1598445

31.

Patterson

, Abizaid

. Stress induced obesity: Lessons from rodent models of stress. Front Neurosci, 2013; 7:130; doi: 10.3389/fnins.2013.00130

32.

Joëls

, Karst

, Sarabdjitsingh

. The stressed brain of humans and rodents. Acta Physiol (Oxf), 2018; 223(2):e13066; doi: 10.1111/apha.13066

33.

Adıgüzel Tel

, Işgın

, Pekcan

. Krill oil supplementation and new scientific evidences. J Nutr Dietetics, 2015; 43(3):258–263. Available from: https://beslenmevediyetdergisi.org/index.php/bdd/article/view/154