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>

<ol>
	<li>McGlone, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Future+of+Pork+Production+in+the+World:+Towards+Sustainable,+Welfare-Positive+Systems.">The Future of Pork Production in the World: Towards Sustainable, Welfare-Positive Systems.</a> Animals (Basel. 3, 401-415 (2013).</li><li>Kuberka, L., Cozzens, T., Mezoughem, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Livestock+and+Poultry:+World+Markets+and+Trade.+Available+at:+https://apps.fas.usda.gov/psdonline/circulars/livestock_poultry.pdf+[accessed).">Livestock and Poultry: World Markets and Trade. Available at: https://apps.fas.usda.gov/psdonline/circulars/livestock_poultry.pdf [accessed).</a> USDA-FAS, Office of Global Analysis, Global Commodity Analysis Division. , (2018).</li><li>Oostindjer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+enrichment+and+loose+housing+of+lactating+sows+on+piglet+performance+before+and+after+weaning.">Effects of environmental enrichment and loose housing of lactating sows on piglet performance before and after weaning.</a> Journal of Animal Science. 88, 3554-3562 (2010).</li><li>Munsterhjelm, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+in+early+life+affects+cortisol+patterns+in+growing+pigs.">Environmental enrichment in early life affects cortisol patterns in growing pigs.</a> Animal. 4, 242-249 (2010).</li><li>Bracke, M. B. M., Koene, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expert+opinion+on+metal+chains+and+other+indestructible+objects+as+proper+enrichment+for+intensively-farmed+pigs.">Expert opinion on metal chains and other indestructible objects as proper enrichment for intensively-farmed pigs.</a> PLoS One. 14, (2019).</li><li>Marcet Rius, ., M, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Providing+straw+to+allow+exploratory+behaviour+in+a+pig+experimental+system+does+not+modify+putative+indicators+of+positive+welfare:+peripheral+oxytocin+and+serotonin.">Providing straw to allow exploratory behaviour in a pig experimental system does not modify putative indicators of positive welfare: peripheral oxytocin and serotonin.</a> Animal. 12, 2138-2146 (2018).</li><li>Giuliotti, L., Benvenuti, M. N., Giannarelli, A., Mariti, C., Gazzano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Different+Environment+Enrichments+on+Behaviour+and+Social+Interactions+in+Growing+Pigs.">Effect of Different Environment Enrichments on Behaviour and Social Interactions in Growing Pigs.</a> Animals. 9, (2019).</li><li>Stafford, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tail+biting:+an+important+and+undesirable+behaviour+of+growing+pigs.">Tail biting: an important and undesirable behaviour of growing pigs.</a> Veterinary journal. 186, 131-132 (2010).</li><li>Sutherland, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Welfare+implications+of+invasive+piglet+husbandry+procedures,+methods+of+alleviation+and+alternatives:+a+review.">Welfare implications of invasive piglet husbandry procedures, methods of alleviation and alternatives: a review.</a> New Zealand Veterinary Journal. 63, 52-57 (2015).</li><li>Herskin, M. S., Jensen, K. H., Thodberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+environmental+stimuli+on+nursing+and+suckling+behaviour+in+domestic+sows+and+piglets.">Influence of environmental stimuli on nursing and suckling behaviour in domestic sows and piglets.</a> Animal Science. 68, 27-34 (1999).</li><li>Lawrence, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Environment+on+Behavior,+Plasma-Cortisol+and+Prolactin+in+Parturient+Sows.">The Effect of Environment on Behavior, Plasma-Cortisol and Prolactin in Parturient Sows.</a> Applied Animal Behaviour Science. 39 (94), 313-330 (1994).</li><li>Baxter, E. M., Lawrence, A. B., Edwards, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+farrowing+accommodation:+welfare+and+economic+aspects+of+existing+farrowing+and+lactation+systems+for+pigs.">Alternative farrowing accommodation: welfare and economic aspects of existing farrowing and lactation systems for pigs.</a> Animal. 6, 96-117 (2012).</li><li>Oliviero, C., Heinonen, M., Valros, A., Halli, O., Peltoniemi, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+environment+on+the+physiology+of+the+sow+during+late+pregnancy,+farrowing+and+early+lactation.">Effect of the environment on the physiology of the sow during late pregnancy, farrowing and early lactation.</a> Animal Reproduction Science. , 365-377 (2008).</li><li>Thomsen, R., Edwards, S. A., Rousing, T., Labouriau, R., Sorensen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+social+mixing+and+group+size+on+skin+lesions+and+mounting+in+organic+entire+male+pigs.">Influence of social mixing and group size on skin lesions and mounting in organic entire male pigs.</a> Animal. 10, 1225-1233 (2016).</li><li>Rydhmer, L., Hansson, M., Lundstrom, K., Brunius, C., Andersson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Welfare+of+entire+male+pigs+is+improved+by+socialising+piglets+and+keeping+intact+groups+until+slaughter.">Welfare of entire male pigs is improved by socialising piglets and keeping intact groups until slaughter.</a> Animal. 7, 1532-1541 (2013).</li><li>Heimburge, S., Kanitz, E., Otten, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+hair+cortisol+for+the+assessment+of+stress+in+animals.">The use of hair cortisol for the assessment of stress in animals.</a> General and Comparative Endocrinology. , 10-17 (2019).</li><li>Morgan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+group+housing+on+reproductive+performance,+lameness,+injuries+and+saliva+cortisol+in+gestating+sows.">Effects of group housing on reproductive performance, lameness, injuries and saliva cortisol in gestating sows.</a> Preventive Veterinary Medicine. , 10-17 (2018).</li><li>Meyer, J. S., Novak, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+Hair+cortisol:+a+novel+biomarker+of+hypothalamic-pituitary-adrenocortical+activity.">Minireview: Hair cortisol: a novel biomarker of hypothalamic-pituitary-adrenocortical activity.</a> Endocrinology. 153, 4120-4127 (2012).</li><li>Meyer, J., Novak, M., Hamel, A., Rosenberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+analysis+of+cortisol+from+human+and+monkey+hair.">Extraction and analysis of cortisol from human and monkey hair.</a> Journal of Visualized Experiments : JoVE. , (2014).</li><li>Lahrmann, H. P., Oxholm, L. C., Steinmetz, H., Nielsen, M. B., D'Eath, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+long+or+chopped+straw+on+pig+behaviour.">The effect of long or chopped straw on pig behaviour.</a> Animal. 9, 862-870 (2015).</li><li>Boumans, I., de Boer, I. J. M., Hofstede, G. J., Fleur, l. a., E, S., Bokkers, E. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+hormonal+circadian+rhythms+in+daily+feeding+patterns:+An+illustration+with+simulated+pigs.">The importance of hormonal circadian rhythms in daily feeding patterns: An illustration with simulated pigs.</a> Hormones and Behavior. 93, 82-93 (2017).</li><li>Schonreiter, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+cortisol+as+a+stress+parameter+in+piglets.">Salivary cortisol as a stress parameter in piglets.</a> Tierarztliche Praxis Ausgabe G: Grosstiere - Nutztiere. 27, 175-179 (1999).</li><li>Cook, N. J., Hayne, S. M., Rioja-Lang, F. C., Schaefer, A. L., Gonyou, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+collection+of+multiple+saliva+samples+from+pigs+and+the+effect+on+adrenocortical+activity.">The collection of multiple saliva samples from pigs and the effect on adrenocortical activity.</a> Canadian Journal of Animal Science. 93, 329-333 (2013).</li><li>Reimert, I., Rodenburg, T. B., Ursinus, W. W., Kemp, B., Bolhuis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+to+novel+situations+of+female+and+castrated+male+pigs+with+divergent+social+breeding+values+and+different+backtest+classifications+in+barren+and+straw-enriched+housing.">Responses to novel situations of female and castrated male pigs with divergent social breeding values and different backtest classifications in barren and straw-enriched housing.</a> Applied Animal Behaviour Science. , 24-35 (2014).</li><li>Avan de Weerd, H., Day, J. E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+environmental+enrichment+for+pigs+housed+in+intensive+housing+systems.">A review of environmental enrichment for pigs housed in intensive housing systems.</a> Applied Animal Behaviour Science. 116, 1-20 (2009).</li><li>Wright, K. D., Hickman, R., Laudenslager, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+Cortisol+Analysis:+A+Promising+Biomarker+of+HPA+Activation+in+Older+Adults.">Hair Cortisol Analysis: A Promising Biomarker of HPA Activation in Older Adults.</a> Gerontologist. 55 Suppl 1, S140-S145 (2015).</li><li>Decorte, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+total+and+PRRSV-specific+antibodies+in+oral+fluids+collected+with+different+rope+types+from+PRRSV-vaccinated+and+experimentally+infected+pigs.">Detection of total and PRRSV-specific antibodies in oral fluids collected with different rope types from PRRSV-vaccinated and experimentally infected pigs.</a> BMC Veterinary Research. 10, 134 (2014).</li></ol>

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 rope...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bite-Rite</td>
			<td>Ikadan System USA Inc.</td>
			<td></td>
			<td>Consider ordering additional replaceable silicone sticks</td>
		</tr>
		<tr>
			<td>ELISA; Saliva Cortisol Kit</td>
			<td>DRG International Inc. NJ, USA</td>
			<td>Slv2930</td>
			<td></td>
		</tr>
		<tr>
			<td>HALM 60/80 CM</td>
			<td>W. Domino A/S, DK</td>
			<td>49084/ 85</td>
			<td></td>
		</tr>
		<tr>
			<td>TEGO Swine Oral Fluids Kit</td>
			<td>ITL BioMedical, USA</td>
			<td>A100930</td>
			<td>Including everything needed for the saliva sampling protcol</td>
		</tr>
	</tbody>
</table>

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 p...

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

Research

JoVE Journal

Behavior

7.0K 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

Eye Tracking, Cortisol, and a Sleep vs. Wake Consolidation Delay: Combining Methods to Uncover an Interactive Effect of Sleep and Cortisol on Memory

Although rises in cortisol can benefit memory consolidation, as can sleep soon after encoding, there is currently a paucity of literature as to how these two factors may interact to influence consolidation. Here we present a protocol to examine the interactive influence of cortisol and sleep on memory consolidation, by combining three methods: eye tracking, salivary cortisol analysis, and behavioral memory testing across sleep and wake delays. To assess resting cortisol levels, participants gave a saliva sample before viewing negative and neutral objects within scenes. To measure overt attention, participants&#8217; eye gaze was tracked during encoding. To manipulate whether sleep occurred during the consolidation window, participants either encoded scenes in the evening, slept overnight, and took a recognition test the next morning, or encoded scenes in the morning and remained awake during a comparably long retention interval. Additional control groups were tested after a 20&#160;min&#160;delay in the morning or evening, to control for time-of-day effects. Together, results showed that there is a direct relation between resting cortisol at encoding and subsequent memory, only following a period of sleep. Through eye tracking, it was further determined that for negative stimuli, this beneficial effect of cortisol on subsequent memory may be due to cortisol strengthening the relation between where participants look during encoding and what they are later able to remember. Overall, results obtained by a combination of these methods uncovered an interactive effect of sleep and cortisol on memory consolidation.

We present a protocol used to discover an interactive effect between sleep and cortisol on memory consolidation, particularly for negative arousing images. Specifically, the experimental design utilizes eye tracking, salivary cortisol analysis, and behavioral memory testing &#8211; methods that can be used with both healthy and clinical participants.

Although rises in cortisol can benefit memory consolidation, as can sleep soon after encoding, there is currently a paucity of literature as to how these ...

The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment

Environmental enrichment can dramatically influence the development and function of neural circuits. Further, enrichment has been shown to successfully delay the onset of symptoms in models of Huntington&#8217;s disease 1-4, suggesting environmental factors can evoke a neuroprotective effect against the progressive, cellular level damage observed in neurodegenerative disorders. The ways in which an animal can be environmentally enriched, however, can vary considerably. Further, there is no straightforward manner in which the effects of environmental enrichment can be assessed: most methods require either fairly complicated behavioral paradigms and/or postmortem anatomical/physiological analyses. This protocol describes the use of a simple and inexpensive behavioral assay, the Puzzle Box 5-7 as a robust means of determining the efficacy of increased social, sensory and motor stimulation on mice compared to cohorts raised in standard laboratory conditions. This simple problem solving task takes advantage of a rodent&#8217;s innate desire to avoid open enclosures by seeking shelter. Cognitive ability is assessed by adding increasingly complex impediments to the shelter&#8217;s entrance. The time a given subject takes to successfully remove the obstructions and enter the shelter serves as the primary metric for task performance. This method could provide a reliable means of rapidly assessing the efficacy of different enrichment protocols on cognitive function, thus paving the way for systematically determining the role specific environmental factors play in delaying the onset of neurodevelopmental and neurodegenerative disease.

Environmental enrichment provides a potential protective effect against neurodegenerative disorders. Currently, however, there is no easy way of determining the efficacy of enrichment procedures. This protocol describes a simple &#8220;Puzzle Box&#8221; method for assessing an animal&#8217;s cognitive function, in order to reveal the effectiveness of environmental enrichment.

Environmental enrichment can dramatically influence the development and function of neural circuits.  Further, enrichment has been shown to successfully ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Feeding Experimentation Device (FED): Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in mice: the Feeding Experimentation Device (FED). FED is an open-source system that was designed to facilitate flexibility in food intake studies. Due to its compact and battery powered design, FED can be placed within standard home cages or other experimental equipment. Food intake measurements can also be synchronized with other equipment in real-time via FED's transistor-transistor logic (TTL) digital output, or in post-acquisition processing as FED timestamps every event with a real-time clock. When in use, a food pellet sits within FED's food well where it is monitored via an infrared beam. When the pellet is removed by the mouse, FED logs the timestamp onto its internal secure digital (SD) card and dispenses another pellet. FED can run for up to 5 days before it is necessary to charge the battery and refill the pellet hopper, minimizing human interference in data collection. Assembly of FED requires minimal engineering background, and off-the-shelf materials and electronics were prioritized in its construction. We also provide scripts for analysis of food intake and meal patterns. Finally, FED is open-source and all design and construction files are online, to facilitate modifications and improvements by other researchers.

Feeding Experimentation Device FED: Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Feeding Experimentation Device (FED) is an open-source device for measuring food intake in mice. FED can also synchronize food intake measurements with other techniques via a real-time digital output. Here, we provide a step-by-step tutorial for the construction, validation, and usage of FED.

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral science, conflicting outcomes are often provided from different laboratories. To identify reasons for the different outcomes, we previously focused on arm features, which differ between laboratories, most notably the presence/absence of ledges at the sides of open arms and the transparency/opaqueness of closed arm walls. In a previous report, we used a custom designed container to compare rat behavior on different combinations of open and closed arm designs under otherwise identical experimental conditions, and showed that differences in arm features interfere with experimental outcomes. In brief, open arm ledges significantly increased anxiety-like behavior in rats, while transparent arms may also have decreased this behavior. Furthermore, we verified a higher detection sensitivity of the effect of an anxiolytic drug in a combination of no-ledged open arm + opaque walled closed arm compared with a combination of ledged open arm + transparent walled closed arm. In this report, we introduce our protocol for the elevated plus maze test, together with discussion of the key results from the previous report and our experimental experience. We believe this report will provide useful information for researchers who have employed or who plan to use the elevated plus maze in their studies.

Rats' anxiety-like behavior tested by the elevated plus maze varies highly depending on the testing environment. Here we show how apparatus settings, especially arm structure, affect behavior on the maze, and we explain a reliable protocol to detect effects of anxiolytic drugs. This report will contribute to anxiety research.

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral ...

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit numerous benefits to being housed accordingly. Although these rabbits are innately gregarious, certain behaviors can still arise when kept in captivity, which if left unchecked, can confound research results or lead to wounding, which in extreme cases can be severe. To prevent these issues, there must be a well-structured plan for the monitoring and maintenance of paired laboratory rabbits. The purpose of this protocol is to present effective procedures for establishing newly paired NZW rabbits as well as methods for successful maintenance. Multiple methods have been tested for the creation of newly paired female rabbits from the vendor, but the most efficacious technique emphasizes capitalizing on the stress bonding from transport, urine marking, pairing in a neutral cage with no forced sharing of resources and a system of monitoring and intervention. To determine the best method of housing paired rabbits in a standard caging environment, data were collected to generate a behavioral ethogram. Behaviors were then quantified as positive, neutral or negative and were tracked across the lifespan of the pair to determine which behaviors indicated pair success or failure. With the newfound knowledge of socially housed laboratory NZW rabbit behavior, enrichment intervention was applied to alleviate aggression and prevent wounding, thus resulting in a higher percentage of successful pairs. Through several years of trialing different pairing methods, the development of the ethogram and the resulting enrichment interventions, understanding of the highly complex social constructs that dominate pair housed rabbit behavior has dramatically increased and allowed for the provision of more species-specific care and increased standards of welfare.

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits Oryctolagus Cuniculus Via Behavioral Ethogram, Monitoring, and Interventions

Though European rabbits are a social species, socially housing them can be challenging. Therefore, there must be a thorough understanding of behaviors and social structures of pair-housed laboratory rabbits. Here we present a protocol to identify pairing methods, species-typical hierarchy establishment behaviors and behaviors that warrant appropriate intervention.

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit ...

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as a useful biological indicator of water quality. Even though crayfish cannot directly specify the substances that cause water quality deterioration, they can immediately (within a few seconds) warn humans of water quality deterioration via acute changes in their cardiac and behavioral activities.
In this study, we present a noninvasive method that is simple enough to be implemented under various conditions due to a combination of simplicity and reliability in one model.
This approach, in which the biological organisms are implemented into environmental evaluation processes, provides a reliable and timely alarm for warning of and preventing acute water deterioration in an ambient environment. Therefore, this noninvasive system based on crayfish physiological and ethological parameter recordings was investigated for the detection of changes in an aquatic environment. This system is now applied at a local brewery for controlling&#160;quality of the water used for beverage production, but it can be used at any water treatment and supply facility for continuous, real-time water quality evaluation and for regular laboratory investigations of crayfish cardiac physiology and behavior.

This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as ...

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 ...