HHS Public Access Author manuscript Author Manuscript
Toxicol Pathol. Author manuscript; available in PMC 2017 February 12. Published in final edited form as: Toxicol Pathol. 2016 February ; 44(2): 233–245. doi:10.1177/0192623315625330.
Impact of Environmental Enrichment Devices on NTP In Vivo Studies Sheba R. Churchill1, Daniel L. Morgan1, Grace E. Kissling1, Gregory S. Travlos1, and Angela P. King-Herbert1 1National
Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
Author Manuscript
Abstract
Author Manuscript
The goal of this study was to determine whether the use of nesting material or polycarbonate shelters, as enrichment devices would have an impact on endpoints commonly measured during the conduct of the National Toxicology Program (NTP) 13-week studies. The study design was consistent with the NTP 13-week toxicity studies. Harlan Sprague Dawley (HSD) rats and their offspring, and B6C3F1/N mice were assigned to control (unenriched) and enriched experimental groups. Body weight, food and water consumption, behavioral observations, fecal content, clinical pathology, gross pathology, organ weights, and histopathology were evaluated. Enriched male mice and male and female rats exhibited decreased feed intake without a subsequent decrease in body weight; this may have been the result of the nesting material reducing the effect of cold stress thereby allowing for more efficient use of feed. There were statistical differences in some hematological parameters, however these were not considered physiologically relevant since all values were within the normal range. Gross pathology and histopathological findings were background changes and were not considered enrichment-related. Nesting material and shelters were used frequently and consistently and allowed animals to display species typical behavior. There was no significant impact on commonly measured endpoints in HSD rats and B6C3F1/N mice given enrichment devices.
Keywords environmental enrichment; animal welfare; laboratory animals; rats; mice
Author Manuscript
INTRODUCTION Animal welfare is a prevailing and important issue in biomedical research. Much advancement has been made with regard to providing adequate care, environments, and husbandry in which laboratory animals can thrive. The research community endeavors to fulfill and improve the three Rs: replacement, refinement, and reduction (Russell and Burch, 1959). One aspect currently in the forefront of animal welfare is a method of refinement referred to as environmental enrichment.
Sheba Churchill, NIEHS, P.O. Box 12233, MD B3-06, Research Triangle Park, NC 27709, USA, [email protected], 919-541-2919.
Churchill et al.
Page 2
Author Manuscript
The most recent edition of the Guide for the Care and Use of Laboratory Animals (the Guide, NRC, 2011) encourages biomedical research programs to facilitate the development and use of enrichment programs. Environmental enrichment is defined as “a measure and/or resources designed to promote expression of natural, species typical behaviors”. The goals of environmental enrichment include decreasing abnormal behaviors and providing a certain degree of control over its environment to the animal, hence improving animal welfare through the reduction of stress (Brinkman, 1996). Multiple studies have shown that environmental enrichment can lead to improvements in animal welfare such as decreased maladaptive behaviors and decreased aggression (Van der Meer et al. 2004). According to The Guide, investigators, animal care staff, and IACUC members are encouraged to design enrichment programs after consulting current and applicable literature. Environmental enrichment is a variable that must be evaluated on a case-by-case basis with each experiment and constructed on the specific needs of the program (NRC, 2011).
Author Manuscript
The Guide recommends the use of appropriate enrichment methods for vertebrates used in research. Users of research animals have a responsibility to address enrichment from an animal welfare perspective. There are challenges associated with adding environmental enrichment to an animal study, including, 1) enrichment devices may introduce a potential variable, 2) the composition of the enrichment device may impact the study, 3) the physiological effects on the study animals, and 4) the practicality of implementing devices in a research program. Ultimately, it is the responsibility of the researchers, veterinary team, and IACUCs to test and support environmental enrichment that is deemed appropriate for the facility and study. There is ample literature on the use and comparison of various enrichment devices in non-regulatory rodent studies, thus increasing the likelihood of finding enrichment suitable for the needs of a specific regulatory study (Dean, 1999).
Author Manuscript Author Manuscript
The goals of this study were to evaluate whether or not the animals used the enrichment devices provided, and to determine whether studies conducted by the National Toxicology Program (NTP) were positively or negatively impacted by the use of enrichment devices. We hypothesized that the use of enrichment in NTP studies will have no detrimental impact. Currently in NTP studies, animals are provided social enrichment through group housing of male rats, female rats, and female mice in feed, water and gavage studies. Male mice and pregnant rats are housed individually and have visual and olfactory contact with conspecifics. Animals used for inhalation and dermal studies are housed individually as well. Our study evaluated the impact of the use of nesting material (Crink-l'nest™, The Andersons, Inc., Maumee, OH) in mouse studies, and nesting material (Crink-l'nest™) or rectangular polycarbonate shelters (Rat Retreats™, Bio-Serv, Frenchtown, NJ) for pregnant dams and their weanlings in rat studies. The endpoints evaluated included body weight, food and water consumption, behavioral observations, fecal content, clinical pathology, gross pathology, organ weights, and histopathology.
MATERIALS AND METHODS Animals Eleven to fourteen-week old time-mated female Harlan Sprague-Dawley rats, Hsd: Sprague Dawley® SD® were obtained from Harlan, Inc. (Indianapolis, IN) on gestation day 2 (GD2). Toxicol Pathol. Author manuscript; available in PMC 2017 February 12.
Churchill et al.
Page 3
Author Manuscript
The dams were provided NIH-07 irradiated pelleted feed, (Zeigler Brothers, Inc., Gardners, PA) and tap water ad libitum during the gestation and lactation period. Post weaning, NTP-2000 irradiated pelleted feed (Zeigler Brothers, Inc., Gardners, PA) and tap water were provided ad libitum to the rat weanlings. The pregnant dams were housed individually during the study and with their litters after gestation. On postnatal day 28, the pups were weaned and randomized into group housing. Male rat weanlings were housed in pairs and female rat weanlings were housed in groups of three. Sentinel rats assigned to the study (6/ sex) were evaluated and were negative for lymphocytic choriomeningitis virus, Mycoplasma pulmonis bacteria, rat parvovirus 1, rat minute virus, Kilham's rat virus, Toolan's H-1 virus, pneumonia virus of mice, rat coronavirus, sialodacryoadenitis virus, reovirus type 3, rat theilovirus, and sendai virus.
Author Manuscript
Three to four week-old B6C3F1/N mice were obtained from the NTP colony maintained at Taconic Biosciences Inc. (Germantown, NY). The mice were provided irradiated NTP-2000 diet (Zeigler Bros. Inc, Gardners, PA) and tap water, ad libitum. Male mice were single housed and female mice group housed, 5 per cage. Vendor surveillance records indicated that the mice were free of common murine pathogens. Sentinel mice (5/sex) assigned to the study were evaluated and were negative for the bacterial agent Mycoplasma pulmonis and the following viruses: ectromelia virus, murine rotavirus, mouse hepatitis virus, minute virus of mice, mouse norovirus, mouse parvovirus, pneumonia virus of mice, reovirus 3, Sendai virus, Theiler's mouse encephalomyelitis virus, and lymphocytic choriomeningitis. Housing and Environmental Conditions
Author Manuscript
Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited facility and in accordance with the Guide for the Care and Use of Laboratory Animals (NRC, 2011). Animals were quarantined and acclimated for 10 days upon arrival. The housing rooms were maintained on an alternating 12-hour light/dark cycle with a temperature range of 68-73 °F and 35 -65% humidity. Solid bottom polycarbonate cages (Lab Products, Inc., Seaford, DE) with certified, irradiated hardwood bedding (Beta-Chips, Northeastern Products Corp., Montville, NJ) were used to house the animals. Procedures performed during the study were reviewed and approved by the facility's Institutional Animal Care and Use Committee. Study Design
Author Manuscript
The study design was consistent with the NTP 13-week (subchronic) toxicology studies (including perinatal exposure in rats), which are designed to characterize the subchronic toxicity of chemical, physical or biological agents. The endpoints evaluated included body weight, food and water consumption, behavioral observations, fecal content, clinical pathology, gross pathology, organ weights, and histopathology. The study was staggered by one week to accommodate handling of a large number rat of pups. In rats, each pregnant dam was randomly assigned to a treatment (enriched, n=32) or control (unenriched, n=18) group by body weight after identification via tail tattoos (Table 1). Pregnant dams were weighed on GD5 and every three days thereafter until parturition; GD5 was the first day nesting material was added to the cage and behavioral observations
Toxicol Pathol. Author manuscript; available in PMC 2017 February 12.
Churchill et al.
Page 4
Author Manuscript
were started. Pups were weighed every three days and rat weanlings weighed weekly. Food and water consumption were measured weekly, at postnatal day 28 (PND28), the litters were weaned and randomly assigned to three groups (n=30/sex) which included unenriched, enriched with nesting material, or enriched with shelters (Table 2). Weaned rats were held for up to 13-weeks. In the 13-week mouse study, each animal was randomly assigned to the treatment (enriched) or control (unenriched) groups (n=40/sex) following subcutaneous implantation of transponders for identification (Table 3). Mice were weighed and behavioral observations were started on Day 1 (D1) of the study; D1 was the first day nesting material was added to the cage. Mice were weighed weekly. Additionally, food and water consumption were measured weekly.
Author Manuscript
Enrichment Natural, non-bleached nesting material (approximately 8 g/cage; Crink-l'Nest™, The Andersons, Inc., Maumee, OH) was added to the cages of the pregnant rat dams in the enriched group through out pregnancy and lactation. When the rat pups from the enriched group were weaned, the weanling rats were divided to receive either the nesting material or shelters until the end of the study. The pregnant dams in the unenriched group were kept in the same housing with no enrichment; rat weanlings from these dams continued to be housed without enrichment. For mice, approximately 6 g of nesting material was added to the cages at each cage change until the end of the study. The unenriched mice were kept in identical housing with no enrichment. Behavioral Observations
Author Manuscript Author Manuscript
Behavioral observations were performed prior to and 24 h after the addition of fresh enrichment (clean nesting material or clean shelters) to the cages. Observations were performed to determine the frequency of use and activity involving the enrichment devices. Cage changes were performed twice weekly for the rats and female mice with fresh nesting material (rats and mice) or clean shelters (rats only) added at each change. Cage changes were performed once a week for male mice with fresh nesting material added at each change. Observations occurred on the day of cage change before the enrichment was changed and 24 h later to view the interactions with the enrichment. Observations for all study animals were performed between 06:00 and 10:00 h and took approximately one hour (12 hr light cycle began at 06:00 h). The observer recorded the activity of the animals and the animal's interaction with the enrichment device (nest building or use of the shelters) on an ethogram driven tally sheet specifically adapted for this study (Ethogram adapted from Winnicker et al., 2012). The ethogram was adapted in the following way: 1)
Rodent activity in each cage was divided into 3 main categories a.
Active - activities that included 1) rearing, 2) ambulating, 3) climbing
b. Maintenance - activities that included 1) grooming, 2) feeding/drinking, 3) nest building
Toxicol Pathol. Author manuscript; available in PMC 2017 February 12.
Churchill et al.
Page 5
c.
Author Manuscript
2)
Inactive - activities that included 1) sleeping, 2) resting while awake and motionless
Use of enrichment device a.
Rodent did not touch enrichment material
b. Rodent only scattered enrichment material c.
Rodent made nest with enrichment material or used shelters
Examination of Fecal Content
Author Manuscript
Fecal samples of 5-6 pellets per cage were collected weekly to examine for remnants of ingested nesting material (mice and rats) and plastic from the shelters (rats). The fecal samples were placed in a petri dish with water, crushed, placed on white filter absorbent paper (Nalgene® VERSI-DRY®, Waltham, MA) and viewed under a dissecting microscope to aid in identifying enrichment material. Clinical Pathology
Author Manuscript
For mice and rats, K2EDTA-anticoagulated whole blood samples were collected at necropsy under isoflurane anesthesia from the retro-orbital venous sinus (mice) and retro-orbital plexus (rats) for a complete blood count (CBC) evaluation. The CBC included: a red blood cell (erythrocyte) count (RBCs count), hemoglobin (HB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total white blood cell (leukocyte) count (WBCs count), leukocyte differential (both % and absolute): (neutrophils [NEU], lymphocytes [LYM], monocytes [MON], eosinophils [EOS], basophils [BAS] and large unstained cells [LUC]), reticulocytes (RET% and RET). The CBCs were analyzed for mice using the ProCyte Dx hematology analyzer (IDEXX Laboratories, Westbrook, Maine) and for rats using the ADVIA 2120 hematology system (Siemens Medical Solutions, Malvern, PA). Blood smears were prepared for each specimen and stained with Romanowsky-stained and examined to assess RBC, WBC and platelet morphology. Plasma and feces were collected at necropsy and corticosterone concentrations were analyzed using an Apex Automatic Gamma Counter (ICN/Micromedic Systems, Huntsville, AL) using a double antibody, 125I radioimmunoassay method obtained from MP Biomedicals, LLC. (Orangeburg, NY). The plasma samples were analyzed following the procedures provided by the reagent manufacturer. The fecal samples were analyzed following methods described previously by Touma et al. (2004).
Author Manuscript
Anatomical Pathology Following euthanasia with 100% carbon dioxide and death confirmed by opening the chest, a complete necropsy was performed. All gross lesions collected and were documented. Liver, spleen, thymus, adrenal glands, lung, kidneys, and gastrointestinal tract were collected and weighed. The gastrointestinal tract was opened and examined. The collected tissues were fixed by immersion in 10% neutral-buffered formalin for 24 h and transferred to the histopathology laboratory for processing.
Toxicol Pathol. Author manuscript; available in PMC 2017 February 12.
Churchill et al.
Page 6
Statistical Methods
Author Manuscript Author Manuscript
For animals that were multiple-housed, feed and water consumption was estimated as amounts per animal per day, based on consumption totals for each cage. For pregnant HSD dams, feed consumption, water consumption, and body and organ weights were compared between enriched and unenriched groups using two-sample t-tests (significance = p < 0.05). Because not all dams produced litters, a one-sided Fisher's exact test was used to compare littering rates between the enriched and unenriched groups. For rat weanlings, feed and water consumption and body and organ weights were compared among the three groups (unenriched, nesting material, and shelters) using analysis of variance followed by Tukey's test to adjust for conducting multiple tests. Corticosterone values were not normally distributed (skewed to the right), so analyses were conducted by two-sample t-tests on logtransformed values. Prior to analysis of the hematology data, extreme values were identified by the outlier test of Dixon and Massey (1951) and implausible values, i.e., extreme values from causes other than treatment and values that the laboratory indicated as being inadequate due to measurement problems, were eliminated from the analysis. There were eight such values excluded from the statistical analyses. Hematology data, which typically has a skewed distribution, were analyzed using the two-sided nonparametric multiple comparison method (Dunn, 1964) in which the unenriched group was considered the comparator group. Litter effects were not considered in the analysis of the rat weanling data.
Author Manuscript
For the mouse data, two approaches were employed to assess the significance of pairwise comparisons between enriched and unenriched groups in the analysis of continuous variables. Parametric multiple comparison methods (Williams, 1971, 1972; Dunnett, 1955) were used to analyze organ and body weight data that have approximately normal distribution. Nonparametric multiple comparison methods (Shirley, 1977; Dunn, 1964) were used to analyze hematology and hormone data which typically have skewed distributions. Jonckheere's test (Jonckheere, 1954) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams’ or Shirley's test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic doseresponse (Dunnett's or Dunn's test). Trend-sensitive tests were used when Jonckheere's test was significant at p
Toxicol Pathol. Author manuscript; available in PMC 2017 February 12. Published in final edited form as: Toxicol Pathol. 2016 February ; 44(2): 233–245. doi:10.1177/0192623315625330.
Impact of Environmental Enrichment Devices on NTP In Vivo Studies Sheba R. Churchill1, Daniel L. Morgan1, Grace E. Kissling1, Gregory S. Travlos1, and Angela P. King-Herbert1 1National
Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
Author Manuscript
Abstract
Author Manuscript
The goal of this study was to determine whether the use of nesting material or polycarbonate shelters, as enrichment devices would have an impact on endpoints commonly measured during the conduct of the National Toxicology Program (NTP) 13-week studies. The study design was consistent with the NTP 13-week toxicity studies. Harlan Sprague Dawley (HSD) rats and their offspring, and B6C3F1/N mice were assigned to control (unenriched) and enriched experimental groups. Body weight, food and water consumption, behavioral observations, fecal content, clinical pathology, gross pathology, organ weights, and histopathology were evaluated. Enriched male mice and male and female rats exhibited decreased feed intake without a subsequent decrease in body weight; this may have been the result of the nesting material reducing the effect of cold stress thereby allowing for more efficient use of feed. There were statistical differences in some hematological parameters, however these were not considered physiologically relevant since all values were within the normal range. Gross pathology and histopathological findings were background changes and were not considered enrichment-related. Nesting material and shelters were used frequently and consistently and allowed animals to display species typical behavior. There was no significant impact on commonly measured endpoints in HSD rats and B6C3F1/N mice given enrichment devices.
Keywords environmental enrichment; animal welfare; laboratory animals; rats; mice
Author Manuscript
INTRODUCTION Animal welfare is a prevailing and important issue in biomedical research. Much advancement has been made with regard to providing adequate care, environments, and husbandry in which laboratory animals can thrive. The research community endeavors to fulfill and improve the three Rs: replacement, refinement, and reduction (Russell and Burch, 1959). One aspect currently in the forefront of animal welfare is a method of refinement referred to as environmental enrichment.
Sheba Churchill, NIEHS, P.O. Box 12233, MD B3-06, Research Triangle Park, NC 27709, USA, [email protected], 919-541-2919.
Churchill et al.
Page 2
Author Manuscript
The most recent edition of the Guide for the Care and Use of Laboratory Animals (the Guide, NRC, 2011) encourages biomedical research programs to facilitate the development and use of enrichment programs. Environmental enrichment is defined as “a measure and/or resources designed to promote expression of natural, species typical behaviors”. The goals of environmental enrichment include decreasing abnormal behaviors and providing a certain degree of control over its environment to the animal, hence improving animal welfare through the reduction of stress (Brinkman, 1996). Multiple studies have shown that environmental enrichment can lead to improvements in animal welfare such as decreased maladaptive behaviors and decreased aggression (Van der Meer et al. 2004). According to The Guide, investigators, animal care staff, and IACUC members are encouraged to design enrichment programs after consulting current and applicable literature. Environmental enrichment is a variable that must be evaluated on a case-by-case basis with each experiment and constructed on the specific needs of the program (NRC, 2011).
Author Manuscript
The Guide recommends the use of appropriate enrichment methods for vertebrates used in research. Users of research animals have a responsibility to address enrichment from an animal welfare perspective. There are challenges associated with adding environmental enrichment to an animal study, including, 1) enrichment devices may introduce a potential variable, 2) the composition of the enrichment device may impact the study, 3) the physiological effects on the study animals, and 4) the practicality of implementing devices in a research program. Ultimately, it is the responsibility of the researchers, veterinary team, and IACUCs to test and support environmental enrichment that is deemed appropriate for the facility and study. There is ample literature on the use and comparison of various enrichment devices in non-regulatory rodent studies, thus increasing the likelihood of finding enrichment suitable for the needs of a specific regulatory study (Dean, 1999).
Author Manuscript Author Manuscript
The goals of this study were to evaluate whether or not the animals used the enrichment devices provided, and to determine whether studies conducted by the National Toxicology Program (NTP) were positively or negatively impacted by the use of enrichment devices. We hypothesized that the use of enrichment in NTP studies will have no detrimental impact. Currently in NTP studies, animals are provided social enrichment through group housing of male rats, female rats, and female mice in feed, water and gavage studies. Male mice and pregnant rats are housed individually and have visual and olfactory contact with conspecifics. Animals used for inhalation and dermal studies are housed individually as well. Our study evaluated the impact of the use of nesting material (Crink-l'nest™, The Andersons, Inc., Maumee, OH) in mouse studies, and nesting material (Crink-l'nest™) or rectangular polycarbonate shelters (Rat Retreats™, Bio-Serv, Frenchtown, NJ) for pregnant dams and their weanlings in rat studies. The endpoints evaluated included body weight, food and water consumption, behavioral observations, fecal content, clinical pathology, gross pathology, organ weights, and histopathology.
MATERIALS AND METHODS Animals Eleven to fourteen-week old time-mated female Harlan Sprague-Dawley rats, Hsd: Sprague Dawley® SD® were obtained from Harlan, Inc. (Indianapolis, IN) on gestation day 2 (GD2). Toxicol Pathol. Author manuscript; available in PMC 2017 February 12.
Churchill et al.
Page 3
Author Manuscript
The dams were provided NIH-07 irradiated pelleted feed, (Zeigler Brothers, Inc., Gardners, PA) and tap water ad libitum during the gestation and lactation period. Post weaning, NTP-2000 irradiated pelleted feed (Zeigler Brothers, Inc., Gardners, PA) and tap water were provided ad libitum to the rat weanlings. The pregnant dams were housed individually during the study and with their litters after gestation. On postnatal day 28, the pups were weaned and randomized into group housing. Male rat weanlings were housed in pairs and female rat weanlings were housed in groups of three. Sentinel rats assigned to the study (6/ sex) were evaluated and were negative for lymphocytic choriomeningitis virus, Mycoplasma pulmonis bacteria, rat parvovirus 1, rat minute virus, Kilham's rat virus, Toolan's H-1 virus, pneumonia virus of mice, rat coronavirus, sialodacryoadenitis virus, reovirus type 3, rat theilovirus, and sendai virus.
Author Manuscript
Three to four week-old B6C3F1/N mice were obtained from the NTP colony maintained at Taconic Biosciences Inc. (Germantown, NY). The mice were provided irradiated NTP-2000 diet (Zeigler Bros. Inc, Gardners, PA) and tap water, ad libitum. Male mice were single housed and female mice group housed, 5 per cage. Vendor surveillance records indicated that the mice were free of common murine pathogens. Sentinel mice (5/sex) assigned to the study were evaluated and were negative for the bacterial agent Mycoplasma pulmonis and the following viruses: ectromelia virus, murine rotavirus, mouse hepatitis virus, minute virus of mice, mouse norovirus, mouse parvovirus, pneumonia virus of mice, reovirus 3, Sendai virus, Theiler's mouse encephalomyelitis virus, and lymphocytic choriomeningitis. Housing and Environmental Conditions
Author Manuscript
Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited facility and in accordance with the Guide for the Care and Use of Laboratory Animals (NRC, 2011). Animals were quarantined and acclimated for 10 days upon arrival. The housing rooms were maintained on an alternating 12-hour light/dark cycle with a temperature range of 68-73 °F and 35 -65% humidity. Solid bottom polycarbonate cages (Lab Products, Inc., Seaford, DE) with certified, irradiated hardwood bedding (Beta-Chips, Northeastern Products Corp., Montville, NJ) were used to house the animals. Procedures performed during the study were reviewed and approved by the facility's Institutional Animal Care and Use Committee. Study Design
Author Manuscript
The study design was consistent with the NTP 13-week (subchronic) toxicology studies (including perinatal exposure in rats), which are designed to characterize the subchronic toxicity of chemical, physical or biological agents. The endpoints evaluated included body weight, food and water consumption, behavioral observations, fecal content, clinical pathology, gross pathology, organ weights, and histopathology. The study was staggered by one week to accommodate handling of a large number rat of pups. In rats, each pregnant dam was randomly assigned to a treatment (enriched, n=32) or control (unenriched, n=18) group by body weight after identification via tail tattoos (Table 1). Pregnant dams were weighed on GD5 and every three days thereafter until parturition; GD5 was the first day nesting material was added to the cage and behavioral observations
Toxicol Pathol. Author manuscript; available in PMC 2017 February 12.
Churchill et al.
Page 4
Author Manuscript
were started. Pups were weighed every three days and rat weanlings weighed weekly. Food and water consumption were measured weekly, at postnatal day 28 (PND28), the litters were weaned and randomly assigned to three groups (n=30/sex) which included unenriched, enriched with nesting material, or enriched with shelters (Table 2). Weaned rats were held for up to 13-weeks. In the 13-week mouse study, each animal was randomly assigned to the treatment (enriched) or control (unenriched) groups (n=40/sex) following subcutaneous implantation of transponders for identification (Table 3). Mice were weighed and behavioral observations were started on Day 1 (D1) of the study; D1 was the first day nesting material was added to the cage. Mice were weighed weekly. Additionally, food and water consumption were measured weekly.
Author Manuscript
Enrichment Natural, non-bleached nesting material (approximately 8 g/cage; Crink-l'Nest™, The Andersons, Inc., Maumee, OH) was added to the cages of the pregnant rat dams in the enriched group through out pregnancy and lactation. When the rat pups from the enriched group were weaned, the weanling rats were divided to receive either the nesting material or shelters until the end of the study. The pregnant dams in the unenriched group were kept in the same housing with no enrichment; rat weanlings from these dams continued to be housed without enrichment. For mice, approximately 6 g of nesting material was added to the cages at each cage change until the end of the study. The unenriched mice were kept in identical housing with no enrichment. Behavioral Observations
Author Manuscript Author Manuscript
Behavioral observations were performed prior to and 24 h after the addition of fresh enrichment (clean nesting material or clean shelters) to the cages. Observations were performed to determine the frequency of use and activity involving the enrichment devices. Cage changes were performed twice weekly for the rats and female mice with fresh nesting material (rats and mice) or clean shelters (rats only) added at each change. Cage changes were performed once a week for male mice with fresh nesting material added at each change. Observations occurred on the day of cage change before the enrichment was changed and 24 h later to view the interactions with the enrichment. Observations for all study animals were performed between 06:00 and 10:00 h and took approximately one hour (12 hr light cycle began at 06:00 h). The observer recorded the activity of the animals and the animal's interaction with the enrichment device (nest building or use of the shelters) on an ethogram driven tally sheet specifically adapted for this study (Ethogram adapted from Winnicker et al., 2012). The ethogram was adapted in the following way: 1)
Rodent activity in each cage was divided into 3 main categories a.
Active - activities that included 1) rearing, 2) ambulating, 3) climbing
b. Maintenance - activities that included 1) grooming, 2) feeding/drinking, 3) nest building
Toxicol Pathol. Author manuscript; available in PMC 2017 February 12.
Churchill et al.
Page 5
c.
Author Manuscript
2)
Inactive - activities that included 1) sleeping, 2) resting while awake and motionless
Use of enrichment device a.
Rodent did not touch enrichment material
b. Rodent only scattered enrichment material c.
Rodent made nest with enrichment material or used shelters
Examination of Fecal Content
Author Manuscript
Fecal samples of 5-6 pellets per cage were collected weekly to examine for remnants of ingested nesting material (mice and rats) and plastic from the shelters (rats). The fecal samples were placed in a petri dish with water, crushed, placed on white filter absorbent paper (Nalgene® VERSI-DRY®, Waltham, MA) and viewed under a dissecting microscope to aid in identifying enrichment material. Clinical Pathology
Author Manuscript
For mice and rats, K2EDTA-anticoagulated whole blood samples were collected at necropsy under isoflurane anesthesia from the retro-orbital venous sinus (mice) and retro-orbital plexus (rats) for a complete blood count (CBC) evaluation. The CBC included: a red blood cell (erythrocyte) count (RBCs count), hemoglobin (HB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total white blood cell (leukocyte) count (WBCs count), leukocyte differential (both % and absolute): (neutrophils [NEU], lymphocytes [LYM], monocytes [MON], eosinophils [EOS], basophils [BAS] and large unstained cells [LUC]), reticulocytes (RET% and RET). The CBCs were analyzed for mice using the ProCyte Dx hematology analyzer (IDEXX Laboratories, Westbrook, Maine) and for rats using the ADVIA 2120 hematology system (Siemens Medical Solutions, Malvern, PA). Blood smears were prepared for each specimen and stained with Romanowsky-stained and examined to assess RBC, WBC and platelet morphology. Plasma and feces were collected at necropsy and corticosterone concentrations were analyzed using an Apex Automatic Gamma Counter (ICN/Micromedic Systems, Huntsville, AL) using a double antibody, 125I radioimmunoassay method obtained from MP Biomedicals, LLC. (Orangeburg, NY). The plasma samples were analyzed following the procedures provided by the reagent manufacturer. The fecal samples were analyzed following methods described previously by Touma et al. (2004).
Author Manuscript
Anatomical Pathology Following euthanasia with 100% carbon dioxide and death confirmed by opening the chest, a complete necropsy was performed. All gross lesions collected and were documented. Liver, spleen, thymus, adrenal glands, lung, kidneys, and gastrointestinal tract were collected and weighed. The gastrointestinal tract was opened and examined. The collected tissues were fixed by immersion in 10% neutral-buffered formalin for 24 h and transferred to the histopathology laboratory for processing.
Toxicol Pathol. Author manuscript; available in PMC 2017 February 12.
Churchill et al.
Page 6
Statistical Methods
Author Manuscript Author Manuscript
For animals that were multiple-housed, feed and water consumption was estimated as amounts per animal per day, based on consumption totals for each cage. For pregnant HSD dams, feed consumption, water consumption, and body and organ weights were compared between enriched and unenriched groups using two-sample t-tests (significance = p < 0.05). Because not all dams produced litters, a one-sided Fisher's exact test was used to compare littering rates between the enriched and unenriched groups. For rat weanlings, feed and water consumption and body and organ weights were compared among the three groups (unenriched, nesting material, and shelters) using analysis of variance followed by Tukey's test to adjust for conducting multiple tests. Corticosterone values were not normally distributed (skewed to the right), so analyses were conducted by two-sample t-tests on logtransformed values. Prior to analysis of the hematology data, extreme values were identified by the outlier test of Dixon and Massey (1951) and implausible values, i.e., extreme values from causes other than treatment and values that the laboratory indicated as being inadequate due to measurement problems, were eliminated from the analysis. There were eight such values excluded from the statistical analyses. Hematology data, which typically has a skewed distribution, were analyzed using the two-sided nonparametric multiple comparison method (Dunn, 1964) in which the unenriched group was considered the comparator group. Litter effects were not considered in the analysis of the rat weanling data.
Author Manuscript
For the mouse data, two approaches were employed to assess the significance of pairwise comparisons between enriched and unenriched groups in the analysis of continuous variables. Parametric multiple comparison methods (Williams, 1971, 1972; Dunnett, 1955) were used to analyze organ and body weight data that have approximately normal distribution. Nonparametric multiple comparison methods (Shirley, 1977; Dunn, 1964) were used to analyze hematology and hormone data which typically have skewed distributions. Jonckheere's test (Jonckheere, 1954) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams’ or Shirley's test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic doseresponse (Dunnett's or Dunn's test). Trend-sensitive tests were used when Jonckheere's test was significant at p
