Cortisol as an Indicator of Stress in Animals Under Different Grazing Systems

Final report for GS19-202

Project Type: Graduate Student
Funds awarded in 2019: $13,500.00
Projected End Date: 08/31/2021
Grant Recipient: Virginia Tech
Region: Southern
State: Virginia
Graduate Student:
Major Professor:
John Fike
school of Plant and Environmental Sciences, Va Tech
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Project Information


Heat stress can compromise animal welfare and productivity, causing significant economic consequences. Many producers in the southeastern United States purchase expensive shade systems or allow livestock access to shaded streams to reduce heat load. Managing livestock in silvopasture systems can help reduce heat stress and improve animal behavior and productivity. Although gain typically is the default measure of animal well-being, it may not fully assess the effects of acute and chronic exposure to high temperature environments. Prolonged stress elevates cortisol in animals, and cortisol may be a good indicator of animal stress in different grazing systems. Blood is a common matrix for accessing cortisol levels in animals, but the sampling procedure requires capturing and restraining animals that itself increase the cortisol level, potentially confounding the reliability of the assessment. Hair cortisol is a relatively non-invasive and reliable measure of chronic stress, but it has received limited use especially in pasture systems. Hair cortisol reflects long-term chronic stress levels in animals ranging from several weeks to months depending on the length of hair and growth rate. It is a more reliable method of assessing stress as it is not influenced by the handling and restraining of animals during sampling procedures. In addition to being a relatively less invasive procedure, samples can be store at room temperature for a long time. In this study, we compared behavioral and physiological (temperature, hair, and blood cortisol) responses of ewes that grazed open pasture or black walnut (Juglans nigra) or honeylocust (Gleditsia triacanthos) silvopastures.  Thirty-six (36) Katahdin ewes were stratified by age, weight, and coat color, assigned to one of the treatments, and were rotationally stocked for a 6-week summer grazing trial. Among systems there was no difference in body weight gains for these mature animals. However, ewes grazing OP showed signs of heat stress as they spent more time loafing and less time lying down compared to ewes on silvopasture treatments. This was measurable, too, as ewes on open pasture had 0.5-1.0°C (0.9-1.8°F) hotter intravaginal temperatures between 1200h-1700h than ewes on silvopasture treatments. Measures of plasma cortisol were not different by treatment. However, measures of hair cortisol worked well for determining differences in this stress hormone. Ewes in the open pastures had greater hair cortisol levels than ewes in silvopasture treatments. Trees within the silvopastures moderated ambient conditions, thus reducing stress and improving the behavioral and physiological responses. Hair cortisol can be a reliable and relatively non-invasive method of assessing long-term chronic stress.

Project Objectives:

To compare the behavioral and physiological responses of ewes in black walnut- (Juglans nigra) and honeylocust- (Gleditsia triacanthos) based silvopastures with that of ewes in open pasture using minimally-invasive measures – specifically, hair cortisol and intravaginal temperature loggers.


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Materials and methods:

This 6-week summer (July-September) grazing trial was carried out at the Whitethorne Agroforestry Demonstration Center at Virginia Tech's Kentland Farm in Blacksburg, Virginia (37°12'00.6"N 80°34'34.8"W). The study site consists of three experimental systems- open pastures (OP), and black walnut (BW; Juglans nigra), and honeylocust (HL; Gleditsia triacanthos) silvopastures replicated three times across the site in a randomized complete block design (nine experimental units; EU). Thirty-six (36) Katahdin ewes were stratified based on their body weight (BW), age, and body color and were randomly assigned to one of the nine experimental units with 4 ewes in each EU. Ewes were rotationally stocked by dividing each EU into 4 sub paddocks for the 6-week grazing period.

Pre- and post-grazing forage biomass within each pasture type were estimated with a double-sample technique using a rising plate meter and quadrats. Animal body weights (BW) were taken at the beginning and end of the study period and used to calculate the average daily gain (ADG) of the animals. Hair samples were collected from an approximate 15 cm x 15 cm site in the rump region at the beginning (D0), mid (D21), and end (D42) of the study period and were used to analyze hair cortisol concentration (HCC). Along with the hair samples, blood samples were also collected by jugular venipuncture into tubes through standard operating procedures without sedation. Plasma was extracted from blood samples by centrifuging for 15 min at 3,550×g (22°C) and then stored at -70°C until analysis for cortisol. Both HCC and plasma cortisol levels were quantified with a commercial cortisol ELISA (Enzyme-Linked Immunosorbent Assay) kit (Item No. 500360, Caymans Chemical, Ann Arbor, MI), according to the manufacturer’s instructions.

Trail cameras (Moultrie D-500, EBSCO Industries, Inc., Birmingham, AL) were installed in each EU to capture time-lapse imagery for two consecutive days at three-week intervals (i.e., twice). The time-lapse imagery captured was later processed sequentially by manually recording the animal’s behavior at one-minute intervals. Behaviors were categorized as grazing, lying, loafing (standing but not grazing), drinking water, and eating minerals. The total time in each behavior was calculated as the sum of the total minutes engaged in each behavior. Intra-vaginal temperatures of ewes were collected with a Star Oddi Data Storage Tag micro-T temperature logger (Star Oddi, Iceland) secured inside a blank controlled internal drug release (CIDR) device. These temperature loggers were inserted into the vagina of lambs for two days within every 3 weeks interval period and set to collect temperature data every 10 minutes.

Research results and discussion:

The ADG of ewes was 12.6, 30.0, 19.8 g (0.03, 0.07, and 0.04 lb) day-1 in BW, HL, and OP systems, respectively with no difference in ADG among treatments. Total gain over the six-week study was 2.1, 5.0, and 3.3 kg (4.6, 11, and 7.3 lb) for ewes in BW, HL, and OP respectively, with no differences among treatments. Lack of gain differences are not surprising, as this was a short-duration study, and the ewes used for this research were 4 to 6 years old. Mature females at maintenance have less potential for gain or loss than young, growing animals. Pre- and post-grazing forage biomass were least in BW (3048 and 2138 kg ha-1; 2715 and 1904 lb ac-1), intermediate in HL (3339 and 2430 kg ha-1; 2974 and 2164 lb ac-1), and greatest in OP (3984 and 3170 kg ha-1; 3549 and 2823 lb ac-1)  treatments. However, dry matter intake by ewes on BW, HL, and OP was 910, 909, and 815 kg ha-1 respectively (811, 810, and 725 lb ac-1) and not different among treatments. Lower biomass in silvopasture treatments might have been the result of lower light penetration to the understory forages. Greater competition for resources in silvopasture compared to open pasture is also possible; however, the BW trees are generally larger and have a thicker canopy cover which casts more shade compared to HL trees. Other studies from this site have found similar forage production results. These data suggest greater stocking rate may be possible in open systems, in part because of greater forage production and perhaps due to lower forage intake. This can be affected by tree stand density and canopy management. In particular, forage production in BW-based systems may benefit with lower tree density or perhaps greater pruning in the lower layer of the tree canopy.  

Sheep physiological response to silvopasture

Results from intravaginal temperature loggers showed that ewes on OP had greater intravaginal temperatures compared to ewes on silvopasture treatments. Ewes on open pasture were 0.5-1.0°C (0.9-1.8°F) hotter between 1200h-1700h compared to ewes on silvopasture treatments. Silvopasture with tree shade moderated ambient conditions, thus reducing the core body temperature of ewes on silvopasture treatments compared to ewes on open pasture, and these temperature differences were most pronounced during the hottest portion of the day. Other studies have also reported reduced body temperature of livestock with shade in silvopastures.

Plasma cortisol levels did not differ by treatment on D-21 (BW: 19.8 ng ml-1; HL: 19.2 ng ml-1; OP: 18.2 ng ml-1) or D-42 (BW: 20.7 ng ml-1; HL: 19.3 ng ml-1; OP: 24.1 ng ml-1). However,  plasma cortisol level is easily influenced by handling and restraining of animals during the sampling procedure. This results in highly variable data which confounds the reliability of the assessment. Hair cortisol levels were higher in ewes on OP compared to silvopasture treatments on D-21 (BW: 22.1 pg mg-1, HL: 22.8 pg mg-1, OP: 42.5 pg mg-1) and D-42 (BW: 20.8 pg mg-1, HL: 22.8 pg mg-1, OP- 35.3 pg mg-1). Hair cortisol reflects long-term chronic stress levels in animals as it is not influenced by the handling and restraining of animals during the sampling procedure. Ewes on open pastures had limited shade and were exposed to higher ambient conditions and greater radiant heat load. This likely increased their stress level compared to ewes on silvopasture treatments, which had opportunity to follow shade during the day.

Ewes on OP spent 5X more diurnal time standing than ewes on silvopasture treatments. More time spent loafing/standing has been suggested as a means of lowering heat stress, as standing increases the effectiveness of convective heat loss through improved airflow. Standing/loafing is also considered as a general response of livestock to heat stress. Ewes on OP also spent 45% of their diurnal time lying down which was less than the percent time spent by ewes on BW (54%) and HL (62%) silvopastures. Time spent lying down is a traditional metric of animal comfort and may be a more effective means of conductive heat loss in silvopastures as soil surface temperatures are generally lower than in open pastures.

Trees modulated the pasture environment with benefits for animal behavioral and physiological responses. It is interesting to note that the study animals were hair sheep, which shed their wool during summer as a means of mitigating heat stress. Had these animals been traditional wool breeds, it is possible that the benefits of silvopasture may have been even more pronounced.

Participation Summary

Educational & Outreach Activities

4 Webinars / talks / presentations

Participation Summary:

Education/outreach description:

Sheep temperatures and cortisol in open pasture and silvopasture

Findings from the study have been already shared among producers, research, and agriculture professionals at national and regional conferences- 2021 ASAS-CSAS Annual Meeting and Trade Show, July 14th-July 18th, 2021 (Poster Presentation); 2021 North American Agroforestry Conference (NAAC), June 28th-July 2nd, 2021 (Oral Presentation); Virtual Spring Conference of the Virginia Academy of Sciences, May 17th-May 21st, 2021 (Oral Presentation); Savannah Institute's Perennial Farm Gathering, December 2020 (Oral Presentation). We further plan to share the findings in other upcoming regional and national conferences, meetings, and extension events. A manuscript based on the findings will also be prepared for submission to a journal for publication.

Project Outcomes

Project outcomes:

This work has established that silvopasture systems (which integrate hardwood tree species - in this case honeylocust and black walnut trees) reduce stress levels in animals, improving their behavioral and physiological responses. Animals in open pastures spent more time engaged in "loafing" behaviors - i.e., neither grazing nor resting (lying down). They also had hotter intravaginal temperatures than animals in silvopastures through much of the afternoon. This work also demonstrated that cortisol can be used as a biomarker of animal stress in extensively managed grazing systems. Hair cortisol seems to be a more reliable, less variable, and relatively less invasive method of assessing long-term chronic stress in grazing animals compared to plasma cortisol. Short-term acute stress associated with handling and restraining of animals during the sampling procedure can easily and rapidly elevate plasma cortisol, which may confound results. Utilizing the less invasive hair sampling method to assess this stress response proved essential, as we did not measure any differences in plasma cortisol among treatments.  This research study was conducted for two consecutive summers and the samples from the second year are being processed and remain to be analyzed. These data will be included in the graduate student’s dissertation and findings from the study will be published in a scientific journal and shared in extension publications and field days.

Knowledge Gained:

Findings from this project gave us useful insights on how a well-managed silvopasture system can help improve animals’ behavioral and physiological responses and overall welfare. Sampling and analyzing hair for cortisol proved to be an easy, relatively less invasive, and more reliable method of assessing cortisol (and long-term chronic stress levels) in grazing animals than plasma cortisol.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.