We thank Lahav CRO for conducting the research in their farm and to the farm&#39;s team for providing valuable technical help throughout the study. The study was funded by the Israel Chief Scientist, Ministry of Agriculture and Rural Development.

The study protocol was ethically approved by the Hebrew University&#39;s Institutional Animal Care and Use Committee (MD-16-14754-2). The study was conducted during 2017, at Lahav Animal Research Institute and the Hebrew University of Jerusalem, Israel.
1. Environmental enrichment protocol during farrowing and lactation period
NOTE: In this study, sows were housed in farrowing/lactation pens, under restraint for a limited time, from 3-5 days prior to the expected farrowing date and up to 10 days post-farrowing; thereafter, confinement bars were removed, to allow free movement and interaction between the sow and its piglets (illustrated in Figure 1A). However, the following protocol is also suitable for conventional farrowing crates, in which the sow is restrained throughout the entire lactation period.
<ol>
	<li>Hang the enrichment materials (jute and cotton ropes) in the farrowing pen before the sow is moved into it, in order to allow the sows&#39; nesting-like behavior prior to farrowing.</li>
	<li>For each farrowing pen/crate, prepare two pieces of jute (20 cm wide, one-meter-long), and two pieces of 100% natural, uncolored cotton ropes (2-3 cm in diameter), approximately one-meter-long each.</li>
	<li>Tie the jute and cotton ropes (double overhand knot) to the pen/crates&#39; bars, as illustrated in Figure 1, from its middle, allowing the two free ends of each piece to be hung towards the floor. The height of the jute and cotton ropes knots should be just lower than the sows&#39; shoulder, while its free ends are just above the floor (Figure 1B-C).</li>
	<li>Sows are typically interested in the jute and cotton ropes; they often pull and chew it. Therefore, replace or refill materials when dirty or finished. 
	NOTE: In this study, materials were inspected every 4-5 days, and replaced or refilled when needed.</li>
	<li>After piglets are born, hang the jute and cotton ropes so that they are reachable for both the sow and its piglets, in a safe place. Typically, when the materials are reachable for both, piglets imitate their dams from a very early age. 
	NOTE: As an addition, shredded paper can also be provided as environmental enrichment for the farrowing sows and piglets, on slatted floor. However, the compatibility of the drainage and sewage systems, should be critically examined on the specific farm.</li>
</ol>
2. Environmental enrichment protocol for weaners (when straw cannot be provided)
NOTE: In this study, after weaning, each two litters were grouped into one pen of weaners. Optimally, groups should remain static until slaughter, without mixing or introducing new pen mates into the pen, to avoid hierarchy-related struggles and injuries. Space allowance in the pen was according to European Council Directive 2008/120/EC and the Israeli legislation, with approximately 20 pigs per group, and 0.3 m2 of unobstructed floor area available per pig. For technical reasons, straw could not be provided for weaners, not even in racks. When straw can be provided, use the finishers&#39; protocol from weaning to slaughter, as detailed in protocol section 3.
<ol>
	<li>For each pen, prepare one-meter-long pieces of 100% natural, uncolored cotton ropes, 2-3 cm in diameter; at least one piece for every 10 pigs. Tie the cotton ropes to a chain hung from the ceiling or to a hanging pole (to distance it from the wall), as demonstrated in Figure 2B.
	<ol>
		<li>Position the ropes preferably at the center of the &#34;active&#34; area of the pen, close to the feeder and drinkers, and far from the pens&#39; wall (as marked in Figure 2A) to allow 360&#730; access, which is expected to prevent aggressive behavior due to competition.</li>
	</ol>
	</li>
	<li>Hang the ropes at a height which allows the pigs to reach it with their mouths easily. Typically, the knot should be lower than the pigs&#39; shoulder, while the free ends of the ropes should be just above the floor, without touching it (Figure 2).</li>
	<li>Replace or refill ropes when dirty or finished. 
	NOTE: In this study materials were inspected every 4-5 days, and replaced or refilled when needed.</li>
	<li>In addition to the cotton ropes, provide chewable silicone sticks or similar chewable devices (Figure 2). The device used (see the Table of Materials) contains four chewable sticks which allows at least four pigs to interact with the device simultaneously. Provide one device for every 10 to 15 pigs.</li>
	<li>Hang the chewable device with a chain from the ceiling (included in the kit) to allow the device swinging movement; this increases the activity and challenge to the pigs, and also allows more pigs to participate at once.
	<ol>
		<li>Position the device at the center of the active area of the pen, far from the pen&#39;s walls to allow 360&#730; access, which is expected to prevent aggressive behavior due to competition. The end of the silicone sticks should be at a height of 10 to 20 cm above the floor.</li>
	</ol>
	</li>
	<li>Change the silicone sticks when finished or if dirty. 
	NOTE: In this study silicone sticks required to be changed only when moved from the weaners&#39; pens to the finishers&#39; pens.</li>
</ol>
3. Environmental enrichment protocol for finishers
NOTE: When straw can be provided during the whole period, start using this protocol from weaning until slaughter, instead of in the finishers&#39; pens only.
<ol>
	<li>Provide each pen with straw placed in a rack, in a hanged net, or in a tower, as illustrated in Figure 2C. When provided in such devices, only small amounts of the straw are available for the pigs at a time, and most of it is being chewed. Therefore, this enrichment can be used on slatted floors, when straw cannot be provided as bedding. 
	NOTE: In this study, straw was provided in commercial racks (Figure 2C).</li>
	<li>Hang the straw racks at a height which is reachable for all pigs, usually just below the shoulder level. Attach them to the wall with the easiest access for as many pigs as possible, preferably within the &#34;active area&#34;. Make sure the racks are properly and safely secured to the wall to avoid device breakage and injuries.</li>
	<li>Make sure racks are completely filled with straw at the beginning; an addition small amount of fresh straw can be added every 1-3 days. Alternatively refill when the rack is empty, typically every 7-10 days. 
	NOTE: Long straw is better than chopped straw to reduce aggressiveness in the group and to meet pigs behavioral needs for rooting and exploring20.</li>
	<li>When applicable, consider placing a collecting tray or a mat underneath the rack, when the floor is slatted, to avoid straw from being scattered and blocking the drainage and sewage systems (Figure 2C). Since straw is provided for pigs mainly to fulfill their rooting and exploratory behavior, a mat can allow the presence of some free straw to be explored at the ground level.</li>
	<li>In addition to the straw, provide chewable devices, as detailed in section 2.</li>
	<li>When cotton ropes are used for saliva collection, as detailed in the next section, leave the ropes in the pens after saliva collection, as an additional enrichment. Other cotton ropes can also be provided, as detailed in section 2.</li>
</ol>
4. Saliva samples collection for cortisol concentrations analysis
NOTE: This section provides information how to non-invasively collect saliva for cortisol concentration analysis, as a biomarker for acute stress; however, saliva samples can also be used for analysis of other biomarkers and even for screening of potential pathogens. In addition, hair cortisol should be measured when applicable.
<ol>
	<li>Plan saliva sampling for the same time of the day, to avoid bias, as cortisol secretion changes along the day due to physiologic circadian rhythm, or due to feeding21. Sampling is typically easier before feeding, preferably before the second feeding of the day.</li>
	<li>Use uncolored, 100% cotton rope (2-3 cm in diameter). One rope is typically needed per 12-15 pigs. 
	NOTE: In this study, commercial, ready to use kits were used for collection of saliva samples.</li>
	<li>Tie the rope close to the center of the pen, far from the pens&#39; wall. Make sure that the cord which is used for hanging (between the cotton rope and the bar) is not reachable for chewing by the pigs, and that the free ends of the cotton ropes do not touch the floor, for hygiene reasons (Figure 3A).</li>
	<li>Unravel the free end of the rope, to increase the available chewable surface (Figure 3B).</li>
	<li>Leave the rope hanging for 15-30 min. Typically, pigs are interested in newly provided ropes and would chew it during this time. Typically, the vast majority of the pigs will approach the rope.</li>
	<li>To extract the saliva from the rope, put the free part of the rope into a sterile plastic bag (as illustrated in Figure 3D) and squeeze the rope by applying pressure from the outside of the bag, from top to bottom, until enough liquid (suggested at least 2 mL) accumulates at the bottom of the bag (Figure 3E), and then remove the rope from the bag. 
	NOTE: In this study a 30 cm x 40 cm bag was included in the kit. If possible, this step should be performed in the pen without cutting the hanging cord. In this way, the saliva extraction is usually easier, and the ropes can be left in the pen as an enrichment. If there is not enough saliva, a wringer can be used (was not required in this study).</li>
	<li>To transfer the saliva from the bag into a sterile tube, pull the rope out and carefully cut the bottom corner of the bag above a 50 mL sterile plastic tube (although only 400 &#181;L is usually required for each analysis in duplicates, at least 2 ml are recommended for collection; Figure 3F). Seal the tube, and mark it with a permanent marker or a pre-prepared sticker (animals/pen, date, time, farm, etc.).</li>
	<li>Store saliva samples in an insulated container with ice packs until delivered to the lab.</li>
	<li>In the lab, centrifuge saliva samples until dirt can be separated (for 8 min at 3000 x g). The clean saliva should be transferred with a clean disposable Pasteur pipette into pre-marked sterile tubes of smaller volume (e.g., 2 mL screw cap freezer tubes) for convenience. Samples can be analyzed immediately, or can be kept frozen at a temperature of -20 &#176;C or below until analysis.</li>
	<li>Prior to cortisol concentration analysis, the relevant samples identity should be confirmed, and samples should be thawed at room temperature if frozen. Gently mix each sample, and then centrifuge it again before the analysis to remove possible leftover dirt that may interfere with the assay (8 min at 3000 x g).</li>
	<li>Perform cortisol concentrations analysis with a validated assay, according to the specific kit protocol. 
	NOTE: In this study, saliva cortisol samples were measured in duplicates by enzyme-linked immunosorbent assay according to the manufacturer guidelines. For pigs that were previously provided with ropes as enrichments, collecting saliva would be very easy as pigs show high interest in the ropes. However, for pigs that are unfamiliar with the ropes, it might take time for training; thus, collecting adequate saliva may not be possible from the first time. If needed, ropes can be soaked in apple juice for training (obviously, in such case, saliva cannot be used for analysis). If saliva samples are needed from individually housed pigs, the collection may be more challenging than from a group, and training sessions with apple juice-soaked ropes are typically required. Chewing of collection ropes may also be encouraged by sampling individuals one next to each other due to mimicking.</li>
</ol>

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The authors have nothing to disclose.

Herein, we describe protocols detailing how to provide simple and practical environmental enrichment for pigs which are housed on slatted floor&#160;during different stages of their life, and how to non-invasively collect saliva samples for the measurement of cortisol concentrations, as a biomarker for acute stress. Straw as a bedding is considered as one of the most suitable environmental enrichment for pigs, but may be impractical on slatted floors. However, relatively inexpensive alternatives such as jute, cotton ropes, straw in racks, and chewable devices can practically and safely be applied.
The range of saliva cortisol concentrations in this study are comparable to previous publications17,22,23. The significant differences in saliva cortisol concentrations between enriched- and non-enriched animals, is in accordance with the hypothesis that environmental enrichment may reduce acute stress in pigs at a point sample, as has been shown in previous studies, at least up to a certain age, as long as the circadian rhythm is not damaged by the prolonged chronic stress24,25. As noted, cortisol can be measured in blood, urine, and saliva as markers for acute stress, and in hair as a marker for chronic stress. A protocol for measuring hair cortisol was previously described by Meyers et al. and may be relevant when assessing animal welfare19. However, when looking for markers for acute stress, one should consider that any sampling, by itself, may increase cortisol release due to animal handling or pain caused during the procedure16,26. Hence, non-invasive saliva sampling, as suggested in the current study, is a relevant method, which can be used for cortisol analysis, as well as for the analysis of other biomarkers and even for screening of potential pathogens27.
The main challenges in collecting saliva according to this protocol may be when collecting it from pigs that are unfamiliar with the ropes, as they may avoid chewing the ropes, and therefore, training may be needed. Training can be performed by using ropes which are soaked in apple juice (in such cases, those saliva samples cannot be used for analyses). If saliva samples are needed from individually housed pigs, the collection may be even more challenging than from a group, and training sessions with apple juice-soaked ropes are typically required. Chewing the ropes may also be encouraged by sampling individuals next to each other due to mimicking. For the training of young piglets, ropes should be hung to be reachable for their mother to encourage learning and early chewing. Another possible obstacle when using cotton ropes for enrichment or for saliva collection is that it can be caught in ear tags of certain brands. If it occurs, either the ear tags or the enrichment method has to be replaced.
Using cotton ropes allows measuring saliva cortisol concentration at the group level, which provides applicable screening for this hormone. A possible disadvantage of this collection method is that it may not be a representative measurement of all individuals in the group, as it is affected by the amount of saliva absorbed in the rope from each individual pig. In order to minimize this disadvantage, it is advised to provide adequate number of ropes, to allow sufficient collection time, as well as to perform several repeated collections. As compared to the existing method of individually sampling, the collection itself is not stressful for the pigs, since it is given as an environmental enrichment, without handling of the pigs.
In summary, pigs can be environmentally enriched at all life stages, even on slatted floor, using jute, cotton ropes, straw in racks and chewable devices. The type of enrichment, the hanging method, as well as the height and location within the pen, are important for their effectiveness. Regarding saliva collection by cotton topes, the timing and the hanging recommendations are crucial for successful samplings. These protocols are relevant for professionals aiming to improve and monitor animal welfare, in both research and industrial swine farming.

Pork is the most consumed meat worldwide, with over 1.3 billion pigs being raised and slaughtered annually1,2. In recent years, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes substantial pressure, coming from customers, legislators and other stakeholders, to make management changes to improve the well-being of these animals. Several studies demonstrated that providing environmental enrichment to pigs is associated with reduced stress and improved production and welfare, as it allows the pigs to express their natural behavior, such as rooting and exploring, as well as nesting prior to farrowing3,4,5,6,7.
Pigs are considered to be intelligent animals, and have a highly inquisitive nature; thus, if a suitable environment is not provided, pigs will likely demonstrate stereotypic behavior and direct manipulative social behavior towards pen mates, which may lead to tail biting, as well as other injuries and stress8,9. Therefore, providing a meaningful environmental enrichment is advised by professionals, and in some countries even imposed by regulations and legislations, such as the European Union Council Directive 2008/120/EC5.
Providing meaningful environmental enrichment can be challenging; it should fulfill the natural behavioral needs of the pigs in each stage of their lives, as well as take into consideration practical and technical limitations. Prior to farrowing, providing nesting material is associated with shorter farrowing duration as well as higher survival rate of the newborn piglets during farrowing and throughout the lactation period. Moreover, in free pens with environmental enrichment, maternal behavior is improved, as well as the cognitive performance and weight gain of the piglets3,10,11,12,13. After weaning, grouping pigs (weaners, growing or finishers) from different litters or pens can be stressful and cause aggressiveness towards other pen mates, which may lead to injuries14,15. Therefore, when mixing unfamiliar pigs, providing meaningful environmental enrichment can potentially reduce the occurrence of undesired behavior resulting from frustration and aggressiveness or redirection of rooting behavior.
According to the European Union Council Directive 2008/120/EC (established in 2001/93/EC), it is required that pigs have permanent access to a sufficient quantity of material, to enable proper investigation and manipulation activities, such as straw, hay, wood, sawdust, mushroom compost, peat or a mixture of these5,6. However, the use of these materials may be unmanageable on many farms, as it may cause blockage of the drainage and sewage systems, particularly on farms with slatted flooring. Still, according to the EU Council Directive, farmers can provide alternative enrichment materials, as long as it allows the pigs to perform proper investigation and manipulation activities.
Lack of environmental enrichment may potentially lead to frustration and stress3, which may activate the hypothalamic pituitary adrenal (HPA) axis. In pigs, as well as in humans and other animals, stress typically leads to the secretion of adrenocorticotropic hormone (ACTH) from the pituitary gland. ACTH binds to its receptors on the adrenal cortex and stimulates adrenal release of the glucocorticoid cortisol, which is considered as a major biomarker of stress and can be measured in blood, urine, saliva and hair. Saliva cortisol is a marker for acute stress, since it reflects its biologically active concentration in the blood4. It has the advantage of a non-invasive sample collection, without handling of the animals, by means of cotton ropes provided as environmental enrichment16,17. Hair cortisol is being used as a marker for chronic stress, since cortisol accumulates in the hair over time and can be extracted and measured18,19; however, it requires animal handling, the number of repeated sampling is limited by the hair growth rate, and the analysis is more cumbersome as it require long extraction process. Still, both saliva and hair cortisol may provide complementary information to asses animal welfare.
The objectives of this paper are (1) to give methodologic information on how to provide relatively simple and practical meaningful environmental enrichment for pigs which are housed on slatted floor&#160;during different stages of their lives (farrowing and lactation, weaners to finishers), and (2) to demonstrate how to non-invasively collect saliva samples for the measurement of cortisol concentrations, as a biomarker for acute stress.

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providing meaningful environmental enrichment and measuring saliva cortisol in pigs housed on slatted flooring

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a substantial pressure coming from customers, legislators and other stakeholders, to make management changes to improve the well-being of these animals. Several studies have demonstrated that providing environmental enrichment to pigs allows them to express their natural behavior, such as rooting and exploring, as well as nesting prior to farrowing, and is associated with reduced stress and improved production and welfare. However, many considerations should be taken into account when providing environmental enrichment, such as the type of floor, drainage and sewage systems, the pigs&#39; stage in life, the material, as well as its hanging method, height and location within the pen. The objectives of this paper are (1) to give methodologic information on how to provide a relatively simple and practical meaningful environmental enrichment for pigs which are housed on slatted floors during the different stages of their life, and (2) to demonstrate how to collect saliva samples for the measurement of cortisol concentrations, as a biomarker for acute stress. Protocols include information regarding the use of jute, cotton ropes, straw in racks, as well as chewable silicone sticks devices as environmental enrichment in pens of farrowing and lactation, weaners and finishers. In addition, the use of cotton rope for a non-invasive saliva samples collection for cortisol concentrations analysis is detailed. The protocols provided are relevant for professionals aiming to improve and monitor animal welfare, in both research and industrial swine farming.

In the current study, 16 litters (170 piglets) were allocated randomly into two treatment groups; in one group, environmental enrichment was provided to eight litters (&#34;Enriched Group&#34;, as described in protocol sections 1-3), while it was not provided to the other eight litters (&#34;Non-enriched Group&#34;). After weaning, each two litters were grouped into one group of about 20 pigs. Saliva samples were collected and analyzed for cortisol concentrations every two weeks in each pen, as described in protocol section 4.
In this study, when provided properly, sows, piglets and pigs showed interest in all enrichment devices. Sows used the jute and cotton ropes prior to farrowing, when provided. During the lactation period, when jute and cotton ropes were reachable for both the dam and her piglets, piglets imitated their dams from the age of 2-3 days old; however, when materials were not reachable for the sow, piglets started using the material later, typically after more than one week of age. In lactation and weaners&#39; pens, the jute and cotton ropes were usually replaced or refilled every 4-5 days, while the chewable silicone sticks were replaced only once, when pigs were moved from weaners&#39; pen to finishers&#39; pen, at the age of 70 days. In the finishers&#39; pens, no drainage and sewage blockages were recorded.
The odds to die during the whole period until slaughter was significantly higher among piglets/pigs in the non-enriched group as compared to the enriched group (OR = 3.38, 95% CI = 1.05-10.83; P = 0.004). Saliva cortisol samples were successfully collected during the study every two weeks, just before the second feeding of the day. The pigs in the enriched group showed high interest in the saliva collecting cotton ropes from the beginning; however, in the non-enriched group saliva volumes in the initial collection were typically lower (2-10 mL), but from the second or third collection sessions it was similar between the groups. Typically, a volume larger than 30-40 mL was easily extracted from each rope. Samples were stored frozen (-80 &#176;C) until analysis. Cortisol concentrations, as measured by ELISA, were overall significantly lower in the enriched pigs, as compared to the non-enriched pigs (P = 0.0044; Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60070/60070fig1.jpg" /> 
Figure 1: Environmental enrichment during farrowing and lactation period. (A) Designed farrowing pen illustration which demonstrates two stages of the confinement bars, either when the sow is restrained (stage R) or after confinement bars are removed (stage F) to allow free movement and interaction between the sow and her piglets. Suggested locations for the jute and cotton ropes before and after farrowing are marked in green and blue, respectively. (B) Images of a sow and her piglets interact with cotton ropes (CR) and jute (J) provided as environmental enrichment in a farrowing/lactation pen. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60070/60070fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60070/60070fig2.jpg" /> 
Figure 2: Environmental enrichment for weaners and finishers. (A) A schematic illustration of a pen demonstrating the suggested locations for hanging the environmental materials in the &#34;active area&#34; (blue X). (B-C) Images of pigs enriched as suggested. (B) In the weaners&#39; pen, cotton ropes (CR) provided as environmental enrichment or for saliva samples collection hung from the ceiling or from a pole (P) to distance it from the pen&#39;s wall, chewable silicone sticks device (BR) hung&#160;by a chain from the ceiling (HC). (C) In the finishers&#39; pen, straw is provided in a rack (SR), a straw tower (ST) or a straw basket (SB); a mat (M) can cover the slatted floor underneath each of these straw devices. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60070/60070fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60070/60070fig3.jpg" /> 
Figure 3: Saliva sampling process using cotton ropes. (A-F) The six main stages for the saliva sampling are illustrated: hanging the rope, unraveling the free end of the rope, allowing the pigs to chew it, saliva extraction from the rope into a plastic bag and then transferring it to a plastic tube. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60070/60070fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60070/60070fig4.jpg" /> 
Figure 4: Saliva cortisol concentrations in enriched and non-enriched pigs. Saliva samples were collected every two weeks using cotton ropes, at the pens&#39; level, from weaning to slaughter, from enriched and non-enriched pigs; cortisol concentrations were measured by ELISA. For this analysis, all samples were clustered together per group, without taking the sampling&#160;date into consideration. Cortisol concentrations were significantly lower in the Enriched Group, as compared to the Non-enriched Group (Mann Whitney test; P = 0.0044). Results are presented as mean &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60070/60070fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring at JoVE.com

Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring

This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress.

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a ...

providing-meaningful-environmental-enrichment-measuring-saliva

Nanyang Technological University

Research

JoVE Journal

Behavior

7.1K Views.  The Hebrew University of Jerusalem. This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress.

Video: Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

A Protocol for Housing Mice in an Enriched Environment

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent studies demonstrate that environmental enrichment decreases adiposity, increases energy expenditure, resists diet induced obesity, and causes cancer remission and inhibition in mice. EE typically consists of larger living space, a variety of &#8216;toys&#8217; to interact with, running wheels, and can include a number of other novel environmental changes. All of this fosters a more complex social engagement, cognitive and physical stimulations. Importantly, the toy location and type of toy is changed regularly, which encourages the mice to adapt to a frequently changing and novel environment. Many variables can be manipulated in EE to promote health effects in mice. Thus these approaches are difficult to control and must be properly managed to successfully replicate the associated phenotypes. Therefore, the goal of this video is to demonstrate how EE is properly set up and maintained to assure a complex, challenging, and controlled environment so that other researchers can easily reproduce the protective effects of EE against obesity and cancer.

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent ...

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 ...

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

Biochemistry

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 &#177; 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80&#160;rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 &#176;C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 &#177; 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 &#176;C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

In this study, we present a protocol for cortisol extraction from the fin and jawbone of sturgeon species. Fin and jawbone cortisol levels were further examined by comparing two washing solvents followed by ELISA assays. This study piloted the feasibility of jawbone cortisol as a novel stress indicator.

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

Environment

Noninvasive, In-pen Approach Test for Laboratory-housed Pigs

Traumatic brain injury (TBI) incidences have increased in both civilian and military populations, and many researchers are adopting a porcine model for TBI. Unlike rodent models for TBI, there are few behavioral tests that have been standardized. A larger animal requires more invasive handling in test areas than rodents, which potentially adds stress and variation to the animals&#39; responses. Here, the human approach test (HAT) is described, which was developed to be performed in front of laboratory pigs&#39; home pen. It is noninvasive, but flexible enough that it allows for differences in housing set-ups.
During the HAT, three behavioral ethograms were developed and then a formula was applied to create an approach index (AI). Results indicate that the HAT and its index, AI, are sensitive enough to detect mild and temporary alterations in pigs&#39; behavior after a mild TBI (mTBI). In addition, although specific behavior outcomes are housing-dependent, the use of an AI reduces variation and allows for consistent measurements across laboratories. This test is reliable and valid; HAT can be used across many laboratories and for various types of porcine models of injury, sickness, and distress. This test was developed for an optimized manual timestamping method such that the observer consistently spends no more than 9 min on each sample.

This protocol describes a new behavioral test&#8212;the human approach test in the pigs&#39; home pen&#8212;to detect functional deficits in laboratory pigs after subconcussive traumatic brain injury.

Traumatic brain injury (TBI) incidences have increased in both civilian and military populations, and many researchers are adopting a porcine model for ...

Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report formats, a growing number of studies now incorporate biological stress-related hormones and catalysts, such as cortisol and &#945;-amylase, to measure levels of stress. Presented here is a protocol to examine the effects on levels of biophysical and psychological stress following visitation to three different locations with differing levels of nature. Biophysical and self-reported psychological stress levels are measured immediately upon entering the selected locations and just prior to the visitors leaving the site. Using a &#34;drool&#34;&#160;method, the biophysical measure consists of 1-2 mL samples of saliva provided by study subjects upon entry to one of three study locations. As prescribed by extant literature, the saliva is collected within a 45 minute time frame following the end of the visitor&#8217;s engagement at the location. Following saliva collection, the samples are labeled and transported to a biological lab. Cortisol is the biophysical variable of interest in this study and measured using an ELISA process with a TECAN plate reader. To measure self-reported stress, the Perceived Stress Questionnaire (PSQ), which reports levels of worry, tension, joy, and perceived demands. Data are collected at all three sites in the late afternoon through early evening. When compared across all three settings, stress levels, as measured by both the biological markers and self-reports, are significantly lower after visitation to the most natural setting.

The purpose of this paper is to identify changes in stress levels after visitation to three different settings and to describe the methods used in identifying stress levels based on measures of salivary cortisol, &#945;-amylase, and a psychological self-report instrument.&#160;

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Medicine

Transition of Farm Pigs to Research Pigs using a Designated Checklist followed by Initiation of Clicker Training - a Refinement Initiative

Implementation of 3R initiatives is important. To refine the life of laboratory pigs, we focused on the time period prior to inclusion of animals in experiments. We improved the checklist during the development. By using the checklist each individual pig&#39;s progress was documented. This aids the caretakers so they remain focused on the actions necessary to socialize the animals and limit the animal&#39;s stress and anxiety level. During this phase, the pigs become calm and ready to commence additional clicker training to further limit stress during the experimental period. The pigs will readily allow basic outcome measures to be collected without sedation or fixation.
The protocol is divided in two. First the transition phase that begins as the pigs arrive at the research facility. Introducing treats and gradually closer contact to the caretakers are the first steps. Then, the caretaker introduces actions to be able to touch the head and hind, to mimic e.g. manipulating the ear to take a blood sample or the hind to obtain a rectal temperature. A strong foundation is established in the animal-caretaker relationship and this swiftly minimizes the stress the pigs express after arrival. Secondly, the positive reinforcement training commences to teach the animal how to carry out more complex tasks without becoming stressed. A clicker is introduced as a secondary reinforcer.
Time, structure, education and thorough communication seem to be the most important criteria to succeed in the application of this protocol. In addition to having calm and compliant pigs, the caretakers report that they enjoy the focus they are allowed to give each individual pig, adding to their overall job satisfaction. Having caretakers with a strong commitment, and a focus on the transition progress and welfare of each pig, additionally strengthens the department&#39;s Culture of Care.

Refinement of porcine studies is achieved by introducing a standardized checklist and positive reinforcement training using a clicker. This work supports the collection of samples and the conduct of daily chores related to the animals.

Implementation of 3R initiatives is important. To refine the life of laboratory pigs, we focused on the time period prior to inclusion of animals in ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...