This review was not supported by grants. We thank Sanypet Forza 10 USA Corp. Orlando, FL, USA for kindly providing the ND used in this study.

The protocol was examined and approved by the Veterinary Ethical Review Committee prior to the beginning of the study. The recommendations of the ARRIVE guidelines in animal research were also consulted and considered21-25.
1. Dog Selection and Food Supplementation
<ol>
	<li>Select 24 dogs of different breeds (mean age and weight &#177; SEM: 2.9 &#177; 0.3 years and 32.01 &#177; 1.17 kg; 14 males, 10 females) with evident clinical symptoms of behavioral disturbances, such as anxiety, diffidence, irregular biorhythm, reactivity, activation, irritability, alertness, and constant environmental exploration.</li>
	<li>Randomly divide the animals into two groups and place each into a 215,278 sq ft single box. Following the manufacturer&#39;s instructions, give the appropriate dose of standard diet (SD, n = 12) or nutraceutical diet (ND, n = 12) for 10 days, according to the weights of the animals (Table 1).</li>
	<li>Complete two veterinary inspections of the dogs before (T0) and 10 days after (T1) the treatment.</li>
</ol>
2. Behavior Symptoms Acquiring and Scoring
<ol>
	<li>Have a certified veterinary behaviorist score the behavioral (marking, anxiety, diffidence, irregular biorhythm, reactivity, activation, irritability, alertness, environmental exploration and attention requirement) and clinical (dandruff, itchiness, flush, seborrhea, fur opacity, vomiting, diarrhea, flatulence, lachrymation and anal sac repletion) conditions of each dog.
	<ol>
		<li>For each dog, gather a score before and after the 10-days evaluation as follows: 1 = absence of symptom; 2 = moderate presence of symptoms; 3 = marked presence of symptoms.</li>
		<li>At the end of the evaluation summarize, for each symptom, the scores of the dogs of each group before and after 10 days. Plot the data on a statistical software.</li>
	</ol>
	</li>
</ol>
3. The Sensor
Note: The sensor has a 3-axis accelerometer, allowing it to gather movement in each direction (x, y, z). It weighs 8 g and is extremely small (41 x 28 x 11 mm). Moreover, it is compatible with every mobile device that features a global wireless communication. It has a rechargeable battery that can last for 14 days after charging. The data output is calculated in points, which are the units of time spent active and at rest by each dog during the day.
<ol>
	<li>Ensure that the collar is not wider than 30 mm for the optimal fit of the sensor.</li>
	<li>Using the procedure outlined in sections 3 and 4, evaluate the behavioral changes related to the time spent active and at rest before and after treatment with the specific diet.</li>
</ol>
4. Sensor Setting
<ol>
	<li>Open the micro USB cap on the bottom of the unit and use the cable provided to connect the sensor to the computer&#39;s USB 1x/2.0 port or to a Class 2/Limited power supply with a USB output. When an LED starts blinking, charge the sensor for at least 90 min.</li>
	<li>Download and install the dedicated free mobile app from the web store.</li>
</ol>
5. Activity Monitoring and Analysis
<ol>
	<li>Insert the plugs of a wireless router and the dedicated Wi-Fi base station into two different sockets. Wait until the router is ready and the Wi-Fi base station starts flashing.</li>
	<li>Enable Bluetooth on the mobile device and make sure that it is connected to the Internet.</li>
	<li>Open the app and sign up. Then, tap on &#34;Add new dog&#34; and follow the steps below.
	<ol>
		<li>Take a picture of the dog and provide its name.</li>
		<li>Appropriately set the gender, age, weight, neutering status, and location of the dog.</li>
		<li>Set the primary and the secondary breed of the dog.</li>
		<li>Select, if applicable, the presence of allergies (skin, ear, etc.), arthritis, brain aging, cancer, diabetes, heart disease, or excessive weight.</li>
		<li>Select one of the three available lifestyles, with their respective points, according to personal requirements (1. Average, 2. Active, or 3. Olympian). 
		NOTE: Depending on the age of the dog, the points of each lifestyle will change. Given that the aim of this evaluation was to decrease the hyperactivity and the stress of the dogs, the first lifestyle, &#34;Average,&#34; was set as the final goal to reach.</li>
		<li>Tap on &#34;Wi-Fi base station&#34; and then on &#34;Pair a base station&#34; to connect the sensor to the Wi-Fi base station. Wait until the word &#34;FitBark&#34; appears below &#34;Pair a base station.&#34;</li>
	</ol>
	</li>
	<li>Clip a charged sensor to the collar of the dog.</li>
	<li>Repeat steps 4.3 to 4.4 for each dog.</li>
</ol>
6. Bark Recording
<ol>
	<li>Fix a digital voice recorder to the wall of each box at the beginning of the study.</li>
	<li>Start recording the bark activity.
	<ol>
		<li>Every day, before acquiring new data, connect the digital voice recorder to a computer by means of a retractable USB connector provided along with the recorder.</li>
		<li>Drag the folder with voice data from the device to the computer and rename it with the current date.</li>
	</ol>
	</li>
	<li>Repeat steps 6.2.1 - 6.2.2 every day for 10 days.</li>
	<li>At the end of the evaluation transform (in s) the bark time recorded. Summarize the bark time for each group before and after 10 days. Plot the data on a statistical software.</li>
</ol>

The authors have nothing to disclose.

Both SD and ND were two commercially available diets that completely fulfill the recommendations for protein, carbohydrate, and fat content according to the nutritional guidelines for complete and complementary pet food. However, in the ND, nutraceutical substances, such as Punica granatum (0.0457%), Valeriana officinalis (0.026%), Rosmarinus officinalis (0.000044%), Tilia spp (0.0635%), tea extract (0.031%), and L-tryptophan (0.0329%), were added. It is noteworthy that this clinical evaluation was inspired by a previous trivial evaluation where 2 dogs with evident behavioral symptoms mainly ascribed to generalized anxiety showed significant improvements after 3 days of ND supplementation. Here, we successfully used the same ND with 24 dogs presenting behavioral symptoms mainly ascribed to generalized anxiety.
The only critical step that occurred in the protocol was related to the Wi-Fi connection. In some boxes, the sensor signal did not reach the Wi-Fi station and therefore did not provide any data on the activity of the dogs. Thus, a Wi-Fi range extender was used to completely cover the distance between those boxes and the Wi-Fi station. Many studies have been performed to validate the usefulness of small, lightweight, motion-sensing accelerometers for pets44 and humans46-54. The sensor used in this clinical evaluation presents some limitations with respect to the gold-standard method of using a video camera55,56, such as a lack of specificity in distinguishing rest activity from sleep and general motion from an anxiety-related one. On the other hand, the sensor allows for the easy and fast detection of movement, as well as the ability to monitor daily improvements by means of a mobile phone app. Moreover, with respect to the other commercially available devices, this new sensor has a lower weight and price, can be worn by a dog of any weight, and has a long-lasting (~ 14 days) battery life. Moreover, because of the Wi-Fi station, it does not require the owner to be close to the dog while it is registering the improvements57-59. In fact, after registration with the website of the product company, the station can gather and store information that can be seen either on a computer or on a mobile device, even at a long distance (i.e., beyond the Bluetooth and Wi-Fi range). Possible further applications of this sensor will be the monitoring of excessive movement in dogs affected by separation anxiety47-54, abnormal repetitive behaviors60, or narcolepsy61once left alone at home.
Our results pave the way for a different short-term approach to manage dogs with behavioral symptoms mainly ascribed to generalized anxiety disorders, allowing the owner to re-establish a mutual attachment relationship with the dog. In conclusion, a better understanding of dog behavior, both by pet owners and by behavioral experts able to recognize behavioral and clinical symptoms related to generalized anxiety, might be coupled with a specific diet in order to ensure a better quality of life for the animals.

Dogs are generally recognized as the most devoted domesticated animals that live with humans. They are often considered to be family members whose behavioral changes are regarded as serious concerns, especially when these changes threaten their physical integrity and general wellbeing1. Therefore, the development of adjuvant approaches to common therapies for relieving canine behavioral disturbances would help families to improve the quality of life of their dogs, avoiding unwanted phenomena such as dog abandonment and euthanasia2. Most of these behavioral disturbances are related to anxiety caused by stress, and the anxiety may become pathological without proper intervention1,3.
It has been hypothesized that anxiety disorders in dogs are caused not only by significant life changes, but also by chronic or post-traumatic stresses that can alter their homeostasis and can consequently lead to adaptation disorders1,3. This study is based on a clinical evaluation of behavioral disturbances mainly ascribed to generalized anxiety. The typical clinical symptoms of this disorder include constant or increasing reactivity, body and environmental exploration, activation, alertness, and excessive barking; it also often affects the social interactions between dog and owner1,4,5. The predisposing factors could be intrinsic, such as genetics, or extrinsic, such as environmental stimuli1,6. In fact, the aforementioned clinical symptoms can become frequent even without a triggering environmental stimulus. In this sense, the study of the origin of these factors becomes essential for proper diagnosis and subsequent therapy.
The most common therapies for generalized anxiety rely on counterconditioning and desensitization techniques, where the dog learns how to behave once faced with a stimulus that causes anxiety, or on a pharmacological approach based on anxiolytic drug administration7. Based on these considerations, 24 dogs affected by behavioral disturbances mainly ascribed to generalized anxiety received a counterconditioning and desensitization behavioral therapy combined with a nutraceutical diet for 10 days. The diet consisted of a mixed formula of fish proteins, rice carbohydrates, Punica granatum, Valeriana officinalis, Rosmarinus officinalis, Tilia spp, tea extract, and L-tryptophan, with an omega-3:-6 ratio of 1:0.8. Literature reports clearly evidenced that P. granatum is used to treat chronic anxiety and insomnia1,8, while Valeriana officinalis is used for mild sleep disorders and nervous tension9,10. Furthermore, anti-anxiety and anti-depressant effects have been observed after Rosmarinus officinalis11-14 and Tilia spp15,16 consumption. L-theanine, one of the tea constituents, has been shown to play a role in reducing stress and decreasing heart rate in chronic anxiety17,18. Conversely, many studies reported the onset of anxiety, mood, and depressive symptoms after L-tryptophan depletion and/or an omega-3 deficiency19,20.
Generalized anxiety behavior and clinical symptoms, including marking, anxiety, diffidence, irregular biorhythm, reactivity, activation, irritability, alertness, environmental exploration, body exploration, attention requirement, dandruff, itchiness, flush, seborrhea, fur opacity, vomiting, diarrhea, flatulence, lachrymation, and anal sac repletion, were also evaluated. Most of these symptoms were accompanied by signs that induced dogs to spend more time awake and active, rather than at rest or asleep. Therefore, activity and rest times spent by each dog before and after evaluation were assessed. To continuously monitor daily improvements of activity and rest time, a commercially available sensor, which was fixed to the collars of the dogs and connected to a mobile phone or to a Wi-Fi station, was used.

<table><tbody><tr><td>FitBark Activity monitor</td><td>FitBark Inc.</td><td></td><td>Sensor</td></tr><tr><td>&amp;nbsp;FitBark Wi-Fi Base Station</td><td>FitBark Inc.</td><td>7002</td><td>Wi-Fi Base Station</td></tr><tr><td>FORZA10 Behavioral Diet 6lbs</td><td>Forza10 USA Corp</td><td>E0030922007</td><td>Nutraceutical diet</td></tr><tr><td>M5, 3G Mobile Wi-Fi&amp;nbsp;</td><td>TP-LINK</td><td>M5250</td><td>Router</td></tr><tr><td>SmartBox</td><td>Laika</td><td></td><td>215,278 sq ft Dog box</td></tr><tr><td>Recorder</td><td>Olympus</td><td>WS-831</td><td>Voice recorder</td></tr></tbody></table>

behavioral disturbances: an innovative approach to monitor the modulatory effects of a nutraceutical diet

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, the traditional ways to monitor behavioral changes in dogs are complicated and not efficient. In the current clinical evaluation, a new, simple monitoring system was used to assess the effectiveness of a specific diet in positively modulating the intense and restless activity of 24 dogs of different ages and breeds. This protocol describes how to easily and rapidly evaluate improvement in a set of symptoms related to generalized anxiety by using a specific sensor, a mobile phone app, a wireless router, and a computer. The results showed that dogs treated with specific diets showed significant improvement in the times spent active and at rest after 10 days (p &lt; 0.01 and p &lt; 0.05, respectively). These dogs also showed an overall significant improvement in clinical and behavioral symptoms. A specific sensor, along with its related hardware, was demonstrated to successfully monitor behavioral changes relating to movement in dogs.

Table 1 shows the daily amount of nutraceutical diet suggested by the manufacturer. In Figure 1, the daily mean activity and rest times spent by dogs belonging to the SD and ND groups during the evaluation period are shown. For instance, a significant decrease from a T0 value of 7,343 &#177; 611.7 to a T1 value of 5,093 &#177; 526.5 was observed in the ND group after 10 days, while no significant difference was observed in the SD group (Figure 1A, *p &#60; 0.05). Conversely, the daily mean rest time significantly increased from a T0 value of 7.6 &#177; 0.3 h to a T1 value of 9.5 &#177; 0.3 h in the ND group after 10 days (**p &#60; 0.01), while no significant difference was observed in the SD group (Figure 1B).
Regarding behavioral symptoms, a reduction of the mean intensity of marking, from 2.50 to 2.41, was observed in dogs belonging to ND group, while no difference was observed in those belonging to the SD group (Figure 2A). On the contrary, anxiety, diffidence, and irregular biorhythm showed a significant reduction in dogs belonging to the ND group, from a T0 value of 2.50 &#177; 0.19 to a T1 value of 1.16 &#177; 0.11 (***p &#60; 0.001, Figure 2B), from a T0 value of 2.08 &#177; 0.28 to a T1 value of 1.17 &#177; 0.12 (Figure 2C, *p &#60; 0.05), and from a T0 value of 2.08 &#177; 0.28 to a T1 value of 1.08 &#177; 0.08 (Figure 2D, **p &#60; 0.01), respectively. No significant difference was observed in the respective SD groups. Also, reactivity, activation, irritability, alertness, environmental exploration, and attention requirement mean intensities showed significant reductions after ND supplementation. In particular, reactivity decreased from a T0 value of 2.16 &#177; 0.27 to a T1 value of 1.25 &#177; 0.13 (Figure 2E, **p &#60; 0.01), activation decreased from a T0 value of 2.25 &#177; 0.25 to a T1 value of 1.33 &#177; 0.14 (Figure 2F, *p &#60; 0.05), irritability decreased from a T0 value of 2.66 &#177; 0.18 to a T1 value of 1.66 &#177; 0.22 (Figure 2G, **p &#60; 0.01), alertness decreased from a T0 value of 2.50 &#177; 0.19 to a T1 value of 1.66 &#177; 0.22 (Figure 2H, *p &#60; 0.05), environmental exploration decreased from a T0 value of 2.33 &#177; 0.18 to a T1 value of 1.66 &#177; 0.22 (Figure 2I, **p &#60; 0.01), and attention requirement decreased from a T0 value of 2.24 &#177; 0.15 to a T1 value of 1.55 &#177; 0.21 (Figure 2J, *p &#60; 0.05). No significant difference was observed in the respective SD group.
In Figure 3, the mean intensities of clinical symptoms in dogs belonging to the SD and ND groups, before (T0) and after the 10-day evaluation period (T1), are shown. Dandruff significantly decreased from a T0 value of 2.33 &#177; 0.14 to a T1 value of 1.08 &#177; 0.08 (Figure 3A, ***p &#60; 0.001). Also, itchiness, flush, seborrhea, and fur opacity significantly decreased from a T0 value of 2.08 &#177; 0.26 to a T1 value of 1.04 &#177; 0.05, from a T0 value of 2.11 &#177; 0.24 to a T1 value of 1.16 &#177; 0.11, from a T0 value of 2.35 &#177; 0.25 to a T1 value of 1.33 &#177; 0.14, and from a T0 value of 2.22 &#177; 0.13 to a T1 value of 1.41 &#177; 0.15, respectively (Figure 3B-E, *p &#60; 0.05). A similar trend was observed for vomiting, diarrhea, flatulence, lachrymation, and anal sac repletion scores, which significantly decreased from a T0 value of 2.75 &#177; 0.10 to a T1 value of 1.58 &#177; 0.17 (Figure 3F, *p &#60; 0.001), from a T0 value of 2.69 &#177; 0.12 to a T1 value of 2.06 &#177; 0.19 (Figure 3G, *p &#60; 0.01), from a T0 value of 1.75 &#177; 0.13 to a T1 value of 1.25 &#177; 0.13 (Figure 3H, *p &#60; 0.05), from a T0 value of 2.16 &#177; 0.11 to a T1 value of 1.32 &#177; 0.03 (Figure 3I, *p &#60; 0.05), and from a T0 value of 2.28 &#177; 0.12 to a T1 value of 1.30 &#177; 0.14 (Figure 3J, *p &#60; 0.01), respectively. No significant difference was observed in dogs belonging to the SD group.
Figure 4 shows the mean time spent in barking by dogs belonging to the SD and ND groups before (T0) and after the evaluation period (T1). A significant decrease from a T0 value of 180.21 &#177; 15.35 to a T1 value of 76.02 &#177; 7.22 was observed in the ND group after 10 days. No significant difference was observed in the SD group.
The data were analyzed using graphing and statistical software. All data are presented as &#177; the standard error of the mean and were first checked for normality using the D&#39;Agostino-Pearson normality test. Differences in activity, rest and barking time as well as symptoms during the evaluation period were analyzed using a two-way ANOVA test followed by Tukey&#39;s multiple comparisons test. p &#60; 0.05 was considered significant.
It is worth noting that, at the beginning of the study, each dog was automatically assigned by the mobile app software to achieve a desired daily activity according to weight, age, and breed. After treatment, all dogs belonging to the ND group showed a significant decrease in the mean daily activity (p &#60; 0.05) with respect to SD group, which was even lower than expected, and a consequent significant increase in daily mean rest time (p &#60; 0.01). These results were also well correlated with consequent clinical symptoms and signs (Figures 2 - 3), demonstrating significant improvement as well as a significant reduction in barking time (Figure 4, p &#60; 0.001). Taken together, all these considerations strengthened the efficacy of the ND in improving the results of common behavioral therapies for generalized anxiety. These results are also in agreement with our recent paper, which described the efficacy of a similar diet in relieving some of the clinical symptoms, such as dandruff, itchiness, flush, diarrhea, and flatulence, that have all been taken into account in this study26.
It is also worth noting that clinical symptoms might also be the manifestation of an overall inflammation status, with a consequent oxidative stress imbalance. Inflammation is also known to contribute to the etiology of anxiety disorders, depression27, and neurotransmitter activity28. In this sense, we recently identified a specific compound, oxytetracycline, as a possible agent able to trigger an inflammatory condition both in vitro29,30and in vivo31,32. Oxytetracycline belongs to the class of tetracyclines, which are the most widely and legally used antibiotics in intensive farming (e.g., poultry30, livestock33, and aquaculture34) due to their low cost and high efficacy35. Unfortunately, oxytetracycline also has a high affinity for calcium-rich tissues, such as bone and teeth36, and can remain fixed in treated animals for extended periods, even respecting withdrawal time30. Moreover, pet food production relies on meat (mainly poultry) by-products, which are mechanically separated37. This kind of separation generates a bone-based meal bearing oxytetracycline residues that are present in commercially available diets (canned, semi-moist, and dry) at 20 - 30% and can accumulate within a pet&#39;s body.
Concerning the ND group, it is reasonable to hypothesize that the mean score intensity reduction of all clinical symptoms was thus a consequence of the anti-inflammatory and antioxidant effect of the nutraceutical substances Punica granatum38, Valeriana officinalis39, Rosmarinus officinalis40, Tilia spp41, tea extract42, and polyunsaturated fatty acids (PUFA) present within the diet. For instance, PUFA have been show to modulate behavioral symptoms in attention-deficit hyperactivity disorder (ADHD) patients and in aggressive dogs43. These dogs had lower docosahexaenoic acid (DHA) levels than normal, as well as a higher omega-6:omega-3 ratio44,45.
<img alt="Figure 1" src="/files/ftp_upload/54878/54878fig1.jpg" /> 
Figure 1. ND Reduces Activity Time and Increases Rest Time in Dogs. Schematic representation of the daily mean activity (A) and rest time (B) of dogs before (T0) and 10 days after (T1) SD and ND supplementation (**p &#60; 0.01). The error bars are &#177; the standard error of mean. <a href="http://cloudflare.jove.com/files/ftp_upload/54878/54878fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54878/54878fig2.jpg" /> 
Figure 2. ND Improves the Behavioral Disturbances in Affected Dogs. Schematic representations of the mean score intensities of behavioral symptoms in dogs before (T0) and 10 days after (T1) SD and ND supplementation. (A) Marking. (B) Anxiety (***p &#60; 0.001). (C) Diffidence (*p &#60; 0.05). (D) Irregular biorhythm (**p &#60; 0.01). (E) Reactivity (**p &#60; 0.01). (F) Activation (*p &#60; 0.05). (G) Irritability (**p &#60; 0.01). (H) Alertness (*p &#60; 0.05). (I) Environmental exploration (**p &#60; 0.01). (J) Attention requirement (**p &#60; 0.01). The error bars are &#177; the standard error of mean. <a href="http://cloudflare.jove.com/files/ftp_upload/54878/54878fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54878/54878fig3.jpg" /> 
Figure 3. ND Improves the Clinical Signs in Affected Dogs. Schematic representation of the mean score intensities of clinical symptoms in dogs before (T0) and 10 days after (T1) SD and ND supplementation (A) Dandruff (***p &#60; 0.001). (B) Itch (*p &#60; 0.05). (C) Flush (*p &#60; 0.05). (D) Seborrhea (*p &#60; 0.05). (E) Fur opacity (*p &#60; 0.05). (F) Vomiting (***p &#60; 0.001). (G) Diarrhea (**p &#60; 0.01). (H) Flatulence (*p &#60; 0.05). (I) Lachrymation (*p &#60; 0.05). (J) Anal sac repletion (**p &#60; 0.01). The error bars are &#177; the standard error of mean. <a href="http://cloudflare.jove.com/files/ftp_upload/54878/54878fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54878/54878fig4.jpg" /> 
Figure 4. ND Reduces the Bark Time in Supplemented Dogs. Schematic representation of the mean time spent barking in dogs before (T0) and 10 days after (T1) SD and ND supplementation (***p &#60; 0.001). The error bars are &#177; the standard error of mean. <a href="http://cloudflare.jove.com/files/ftp_upload/54878/54878fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Daily Ratio</td>
		</tr>
		<tr>
			<td>Weight (kg)</td>
			<td>Amount (g)</td>
		</tr>
		<tr>
			<td>1 - 10</td>
			<td>30 - 180</td>
		</tr>
		<tr>
			<td>11 -20</td>
			<td>190 - 300</td>
		</tr>
		<tr>
			<td>21 - 35</td>
			<td>310 - 455</td>
		</tr>
		<tr>
			<td>36 - 50</td>
			<td>465 - 595</td>
		</tr>
	</tbody>
</table>
Table 1. Daily Amount of Food Provided to the Dogs.

Watch this Scientific Journal Video about Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet at JoVE.com

Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

We report a simple approach to evaluate the effectiveness of a specific diet in positively modulating the daily activity and clinical and behavioral symptoms of dogs with evident behavioral disturbances.

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, ...

behavioral-disturbances-an-innovative-approach-to-monitor-modulatory

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

8.8K Views.  “G. d’Annunzio” University. We report a simple approach to evaluate the effectiveness of a specific diet in positively modulating the daily activity and clinical and behavioral symptoms of dogs with evident behavioral disturbances.

Video: Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

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This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and ...

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Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A challenging illness to treat, due to the lack of understanding of the underlying neurobiology, AN has the highest mortality rate among psychiatric illnesses. To address this need, neuroscientists are using an animal model to study how neural circuits may contribute toward vulnerability to AN and may be affected by AN. Activity-based anorexia (ABA) is a bio-behavioral phenomenon described in rodents that models the key symptoms of anorexia nervosa. When rodents with free access to voluntary exercise on a running wheel experience food restriction, they become hyperactive &#8211; running more than animals with free access to food. Here, we describe the procedures by which ABA is induced in adolescent female C57BL/6 mice. On postnatal day 36 (P36), the animal is housed with access to voluntary exercise on a running wheel. After 4 days of acclimation to the running wheel, on P40, all food is removed from the cage. For the next 3 days, food is returned to the cage (allowing animals free food access) for 2 hr daily. After the fourth day of food restriction, free access to food is returned and the running wheel is removed from the cage to allow the animals to recover. Continuous multi-day analysis of running wheel activity shows that mice become hyperactive within 24 hr following the onset of food restriction. The mice run even during the limited time during which they have access to food. Additionally, the circadian pattern of wheel running becomes disrupted by the experience of food restriction. We have been able to correlate neurobiological changes with various aspects of the animals&#8217; wheel running behavior to implicate particular brain regions and neurochemical changes with resilience and vulnerability to food-restriction induced hyperactivity.

Here we present a protocol to induce activity-based anorexia (ABA) in female adolescent mice. ABA is a condition of hyperactivity evoked by imposing food restriction on rodents with access to a running wheel. This phenomenon is being used as a model to study the underlying neurobiology of anorexia nervosa.

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A ...

An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression have been reported, the unpredictable chronic mild stress (UCMS) protocol is an established translationally-relevant model for inducing behavioral symptoms commonly associated with clinical depression, such as anhedonia, altered grooming behavior, and learned helplessness in rodents. The UCMS protocol also induces physiological (e.g., hypercortisolemia, hypertension) and neurological (e.g., anhedonia, learned helplessness) changes that are clinically associated with depression. Importantly, UCMS-induced depressive symptoms can be ameliorated through chronic, but not acute, treatment with common SSRIs. As such, the UCMS protocol offers many advantages over acute stress protocols or protocols that utilize more extreme stressors. Our protocol involves randomized, daily exposures to 7 distinct stressors: damp bedding, removal of bedding, cage tilt, alteration of light/dark cycles, social stresses, shallow water bath, and predator sounds/smells. By subjecting rodents 3-4 hr daily to these mild stressors for 8 weeks, we demonstrate both significant behavioral changes and poor health outcomes to the cardiovascular system. This approach allows for in-depth interrogation of the neurological, behavioral, and physiological alterations associated with chronic stress-induced depression, as well as for testing of new potential therapeutic agents or intervention strategies.

The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender.

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression ...

Assessing Activity-based Anorexia in Mice

Rodents develop activity-based anorexia (ABA) when exposed to a restricted feeding schedule and allowed free access to a running wheel. These conditions lead to a life-threatening reduction in body weight. However, rodents exposed to only one of these conditions ultimately adapt to re-establish normal body weight. Although increased running coupled with reduction in voluntary food intake appear paradoxical under ABA conditions, ABA behavior is observed across numerous mammalian species. 
The ABA paradigm provides an animal model for anorexia nervosa (AN), an eating disorder with severe dysregulation of appetite-behavior. Subjects are singly housed with free access to a running wheel. Each day, the subject is offered food for a limited amount of time. During the course of the experiment, a subject's body weight decreases from high activity and low caloric intake. The duration of the study varies based on how long food is offered daily, the type of food offered, the strain of mouse, if drugs are being tested, and environmental factors.
A lack of effective pharmacological treatments for AN patients, their low quality of life, high cost of treatment, and their high mortality rate indicate the urgency to further research AN. We provide a basic outline for performing ABA experiments with mice, offering a method to investigate AN-like behavior in order to develop novel therapies. This protocol is optimized for use in Balb/cJ mice, but can easily be manipulated for other strains, providing great flexibility in working with different questions, especially related to genetic factors of ABA.

Mice individually housed with a running wheel while given limited access to food develop reductions in food consumption and increase activity on the running wheel. This experimental phenomenon is called activity-based anorexia. This paradigm provides an experimental tool for studying the neurobiology and behaviors underlying aspects of anorexia nervosa.

Rodents develop activity-based anorexia (ABA) when exposed to a restricted feeding schedule and allowed free access to a running wheel. These conditions ...

Control of Eating Behavior Using a Novel Feedback System

Subjects eat food from a plate that sits on a scale connected to a computer that records the weight loss of the plate during the meal and makes up a curve of food intake, meal duration and rate of eating modeled by a quadratic equation. The purpose of the method is to change eating behavior by providing visual feedback on the computer screen that the subject can adapt to because her/his own rate of eating appears on the screen during the meal. The data generated by the method is automatically analyzed and fitted to the quadratic equation using a custom made algorithm. The method has the advantage of recording eating behavior objectively and offers the possibility of changing eating behavior both in experiments and in clinical practice. A limitation may be that experimental subjects are affected by the method. The same limitation may be an advantage in clinical practice, as eating behavior is more easily stabilized by the method. A treatment that uses this method has normalized body weight and restored the health of several hundred patients with anorexia nervosa and other eating disorders and has reduced the weight and improved the health of severely overweight patients.

Subjects eat food from a plate placed on a scale connected to a computer that records the weight loss of the plate during the meal. Feedback on the computer screen allows the subject to adapt her/his eating behavior to reference curves thus normalizing body weight.

Subjects eat food from a plate that sits on a scale connected to a computer that records the weight loss of the plate during the meal and makes up a curve ...

Elevated Plus Maze Test Combined with Video Tracking Software to Investigate the Anxiolytic Effect of Exogenous Ketogenic Supplements

The overall goal of this study is to describe the methodology of the elevated plus maze (EPM) test in combination with a video tracking software. The purpose of the method is to document the effect of various potential anxiolytic treatments on laboratory rodent models. The EPM test is based on the rodents&#39; proclivity toward protected, enclosed dark spaces and unconditioned fear of open spaces and heights, and their innate intense motivation to explore novel environments. The EPM test is a widely used behavioral test for investigating the anxiolytic or anxiogenic responses of rodents given drugs that are known to affect behavior. Observation demonstrating a decreased proportion of time spent on closed arms, an increased proportion of time spent on open arms, a reduced number of entries to closed arms, and an elevated number of entries to open arms measured by the EPM test may reflect reduced anxiety levels. Using this method, the effect of exogenous ketone supplements on anxiety-related behavior is tested in Sprague Dawley (SPD) rats. Exogenous ketone supplements are chronically fed to the rats for 83 days or subchronically and acutely orally gavaged, daily for 7 days, before conducting the EPM test. Behavioral data collection is performed using the SMART video tracking system by a blinded observer at the end of the treatments. The main findings indicate that the EPM test is an effective method to detect the ketone supplement-induced anxiolytic effect and can be considered a sensitive measure to assess changes in anxiety behavior associated with drug- or metabolic-based therapies.

Here, we present a protocol to investigate changes in the anxiety level of rodent animal models. The elevated plus maze (EPM) test, used together with a video tracking software, provides a reliable method to document the effect of various potential anxiolytic treatments in preclinical laboratory scenarios.

The overall goal of this study is to describe the methodology of the elevated plus maze (EPM) test in combination with a video tracking software. The ...

Investigating Binge Eating Disorder with a Palatable Food Access Mouse Model

Intermittent Binge-Intake Model in Mice

Obesity affects one in eight individuals globally. Overeating, especially high-calorie foods, plays a significant role in obesity. Binge eating disorder (BED) is a complex condition caused by a combination of biological, psychological, and social factors. It involves excessive consumption of high-calorie foods in a short period and is often linked to anxiety and cravings.
Here, we present a protocol employing continuous and intermittent access to a new highly palatable food (HPF) -- M&amp;M&#39;s -- to develop a mouse model for investigating binge eating disorder (BED). The HPF, chosen for its high fat and sugar content, represents an optimal food source to study due to its strong palatability, which makes it particularly suited for examining compulsive eating behaviors. Using C57BL/6 mice, we provided continuous or intermittent access to M&amp;M&#39;s while allowing unrestricted access to standard chow and water. By the 8th day, the mice in the intermittent access group exhibited pronounced binge-eating behaviors, which persisted through the 26th day. These mice also consumed significantly more calories -- predominantly from the confectionary -- than those in the continuous access and control groups. The contrast between continuous and intermittent access underscores the critical role of feeding schedules in promoting the overconsumption of palatable foods.
Furthermore, behavioral assessments revealed intermittent access to the HPF-induced anxiety-like behaviors, highlighting the psychological impact of access patterns on both eating behavior and emotional states. By incorporating these two distinct feeding paradigms, this study offers valuable insights into how the availability of highly palatable foods can exacerbate binge-eating tendencies. This model provides a more realistic approach to studying binge-eating behaviors and their metabolic consequences. It highlights the importance of standardized models in biomedical research. Our findings offer insights into the physiological and neural mechanisms underlying BED, which could pave the way for developing effective therapeutic interventions for BED and obesity.

Author Spotlight: Accessible M&amp;M-Based Mouse Model for Investigating Binge Eating Disorder - Insights into Eating Behaviors, Anxiety, and Neural Mechanisms

A binge-eating model was established in mice using intermittent access to M&amp;M's (a highly palatable food made of fat and sugar). Mice with intermittent access to this high-fat, high-sugar food showed stress-like behaviors, increased caloric intake, and a preference for that food over standard chow compared to mice with continuous or no access to the food.

Obesity affects one in eight individuals globally. Overeating, especially high-calorie foods, plays a significant role in obesity. Binge eating disorder ...

Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral studies in animal models. In this protocol, to study depression-like and anhedonic behavior in rats, the established sucrose preference and novelty-induced hypophagia tests are combined with an automated food and liquid intake monitoring system. Prior to testing, in the sucrose preference paradigm, male rats are trained for at least 2 days to consume a sucrose solution in addition to tap water. During the test, rats are again exposed to water and sucrose solution. Consumption is registered every second by the automated system. The ratio of sucrose to total water intake (sucrose preference ratio) is a surrogate parameter for anhedonia. In the novelty-induced hypophagia test, male rats undergo a training period in which they are exposed to a palatable snack. During training, rodents show a stable baseline snack intake. On test day, the animals are transferred from home cages into a fresh, empty cage representing a novel unknown environment with access to the known palatable snack. The automated system records the total intake and its underlying microstructure (e.g., latency to approaching the snack), providing insight into anhedonic and anxious behaviors. The combination of these paradigms with an automated measuring system provides more detailed information, along with higher accuracy by reducing measuring errors. However, the tests use surrogate parameters and only depict depression and anhedonia in an indirect manner.

Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters.

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral ...

Medicine

Behavioral and Network Pharmacology-Based Analyses for the Traditional Mongolian Medicine Zadi-5 in a Rat Model of Depression

Zadi-5 is a traditional Mongolian medicine that is widely used for the treatment of depression and symptoms of irritation. Although the therapeutic effects of Zadi-5 against depression have been indicated in previously reported clinical studies, the identity and impact of the active pharmaceutical compounds present in the drug have not been fully elucidated.&#160;This study used network pharmacology to predict the drug composition and identify the therapeutically active compounds in Zadi-5 pills. Here, we established a rat model of chronic unpredicted mild stress (CUMS) and conducted an open field test (OFT), Morris water maze (MWM) analysis, and sucrose consumption test (SCT) to investigate the potential therapeutic efficacy of Zadi-5 in depression. This study aimed to demonstrate Zadi-5&#39;s therapeutic effects for depression and predict the critical pathway of the action of Zadi-5 against the disorder. The vertical and horizontal scores (OFT), SCT, and zone crossing numbers of the fluoxetine (positive control) and Zadi-5 groups were significantly higher (P &#60; 0.05) than those of the CUMS group rats without treatment. According to the results of network pharmacology analysis, the PI3K-AKT pathway was found to be essential for the antidepressant effect of Zadi-5.

The present protocol describes a method for the behavioral test validation and bioinformatical prediction of the therapeutic efficacy of Zadi-5, a traditional Mongolian medicine, in depression.

Zadi-5 is a traditional Mongolian medicine that is widely used for the treatment of depression and symptoms of irritation. Although the therapeutic ...