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

Summary

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.

Abstract

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' 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.

Introduction

Pork is the most consumed meat worldwide, with over 1.3 billion pigs being raised and slaughtered annually^1,2. 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 farrowing^3,4,5,6,7.

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 stress^8,9. 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 piglets^{3,10,11,12,13}. 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 injuries^14,15. 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 these^5,6. 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 enrichment^16,17. Hair cortisol is being used as a marker for chronic stress, since cortisol accumulates in the hair over time and can be extracted and measured^18,19; 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 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.

Protocol

The study protocol was ethically approved by the Hebrew University'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.

1. 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' nesting-like behavior prior to farrowing.
2. 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.
3. Tie the jute and cotton ropes (double overhand knot) to the pen/crates' 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' shoulder, while its free ends are just above the floor (Figure 1B-C).
4. 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.
5. 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.

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' protocol from weaning to slaughter, as detailed in protocol section 3.

1. 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.
   1. Position the ropes preferably at the center of the "active" area of the pen, close to the feeder and drinkers, and far from the pens' wall (as marked in Figure 2A) to allow 360˚ access, which is expected to prevent aggressive behavior due to competition.
2. 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' shoulder, while the free ends of the ropes should be just above the floor, without touching it (Figure 2).
3. 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.
4. 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.
5. 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.
   1. Position the device at the center of the active area of the pen, far from the pen's walls to allow 360˚ 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.
6. 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' pens to the finishers' pens.

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' pens only.

1. 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).
2. 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 "active area". Make sure the racks are properly and safely secured to the wall to avoid device breakage and injuries.
3. 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.
4. 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.
5. In addition to the straw, provide chewable devices, as detailed in section 2.
6. 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.

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.

1. 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.
2. 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.
3. Tie the rope close to the center of the pen, far from the pens' 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).
4. Unravel the free end of the rope, to increase the available chewable surface (Figure 3B).
5. 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.
6. 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).
7. 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 µL 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.).
8. Store saliva samples in an insulated container with ice packs until delivered to the lab.
9. 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 °C or below until analysis.
10. 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).
11. 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.

Results

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 ("Enriched Group", as described in protocol sections 1-3), while it was not provided to the other eight litters ("Non-enriched Group"). 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...

Discussion

Herein, we describe protocols detailing how to provide simple and practical environmental enrichment for pigs which are housed on slatted floor 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...

Materials

Name	Company	Catalog Number	Comments
Bite-Rite	Ikadan System USA Inc.		Consider ordering additional replaceable silicone sticks
ELISA; Saliva Cortisol Kit	DRG International Inc. NJ, USA	Slv2930
HALM 60/80 CM	W. Domino A/S, DK	49084/ 85
TEGO Swine Oral Fluids Kit	ITL BioMedical, USA	A100930	Including everything needed for the saliva sampling protcol