Research Note
Enrichment materials do not negatively affect reproductive success and offspring survival and weight in mice
© 2012 Nature America, Inc. All rights reserved.
Harry N. Shair, PhD1,2, Yasmin Nunez3 & Mohamed M. Osman, DVM, PhD 2,4
Environmental enrichment is designed to improve the overall welfare of laboratory animals, including mice. Few studies have directly assessed the effects of different types of enrichment on mouse offspring survival and growth. The authors examined how survival and growth of C57BL/6 mouse pups are affected by three kinds of cage enrichment materials: compressed cotton squares, two-ply tissues and plastic igloos. During the last week of gestation and the first two weeks postpartum, the authors observed cages with litters and noted use of the enrichment materials, quality of nest construction, number of pups per litter and weight of pups. Both the first and second litters were evaluated for each dam. Dams and pups had continuous contact with the enrichment materials, especially cotton squares and tissues. Neither the presence nor the type of enrichment material influenced the survival and weight of offspring, suggesting that the use of such materials does not negatively impact reproductive success or offspring survival. Environmental enrichment, as a part of normal husbandry procedures, is intended to improve the welfare of laboratory animals by allowing them to engage in natural behaviors. For mice, one of the most common forms of enrichment is the provision of a quantity of nest material that can be manipulated. This material, often made of paper, wood or cotton, provides an opportunity for laboratory mice to engage in behaviors similar to those observed in wild mice, such as gathering, chewing, nest building and burrowing1,2. Mice show a preference for nest materials and for cages that contain them3,4. Many studies have shown that use of enrichment materials can have beneficial effects, including improved cognition5,6, reduced alopecia7, decreased aggression and stress levels8 and halted progression of neurodegeneration in animal models of disease9. Mice not only prefer more enriched environments, but also will work to obtain access to enriched conditions10. While enrichment materials may be beneficial to adult mice, they may present a danger to newborn pups. Newborn mice are not able to ambulate well or
disentangle themselves from constricting conditions. Few studies have directly assessed the effects of different enrichment materials on offspring survival and growth. In one study, two nest materials, aspen wood-wool and paper towel, did not significantly differ in their effects on the number of litters produced; litter size; neonatal mortality; or the age, sex and weight of weanlings11. In another study, adding four types of enrichment material to a cage (compressed cotton squares, plastic tunnels, nylon rings or running wheels) significantly improved reproductive performance as measured by increased pup survival to weaning age and greater pup body weight12. The effects of other kinds of cage enrichment materials on parturition and on early pup development have not yet been investigated. The goal of the present study was to determine whether enrichment materials are safe for use in mouse cages during the perinatal period. We bred and reared C57BL/6J mice under different enrichment conditions to examine the effects of three enrichment materials on perinatal success in mice. Two of these materials,
1Division
of Developmental Neuroscience, New York State Psychiatric Institute, New York, NY. 2Department of Psychiatry, Columbia College of Physicians and Surgeons, New York, NY. 3School of Health and Natural Sciences, Mercy College, Dobbs Ferry, NY. 4Division of Comparative Medicine, New York State Psychiatric Institute, New York, NY. Correspondence should be addressed to H.N.S. (
[email protected]).
14 Volume 41, No. 1 | JANUARY 2012
www.labanimal.com
Research Note
compressed cotton squares (Nestlets) and two-ply tissues (Kleenex), could be torn and shredded, whereas the third, a plastic igloo, could not. All three materials could potentially be used in nest building. Because maternal nest building begins in the period before birth13, we observed each cage from the last week of breeding until 2 weeks after parturition. Before the birth of a litter, we examined the interaction of the dams with the enrichment material and the quality of the nests built. Litter size, pup weight and pup survival rate were evaluated after the birth of a litter. Additionally, we evaluated whether enrichment affected nest quality, average pup weight and pup survival rate differently for dams’ first and second litters. © 2012 Nature America, Inc. All rights reserved.
METHODS Animals We obtained 10-week-old C57BL/6J mice (48 female and 24 male; Jackson Laboratory, Bar Harbor, ME) and housed them in same-sex groups for 4 weeks to allow them to acclimate to our husbandry conditions. The females were housed in pairs, and the males were housed in groups of four. Six pairs of females were exposed to each enrichment condition. All experimental procedures were carried out in the animal care facility at New York State Psychiatric Institute (accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International) and were consistent with both federal and state regulations. The experimental protocol was reviewed and approved by the New York State Psychiatric Institute IACUC before any procedures were carried out. Mice were housed in polycarbonate cages (28 cm × 17 cm × 14 cm; Thoren Caging Systems, Inc., Hazelton, PA) on non-ventilated racks. To facilitate observations, cages were placed in a group and oriented with one long side facing forward. We placed 4 oz (19.03 in3 in volume) of corn cob bedding material (eighth inch Bed-o’Cobs; The Andersons, Inc., Maumee, OH) into each cage, resulting in a layer approximately 0.25 in deep. Food (Purina 5015; LabDiet, St. Louis, MO) and autoclaved tap water were available ad libitum. The colony room was maintained on a 12-h:12-h light:dark cycle with lights on at 7:00 a.m. Cages were changed once a week, but not within 2 d before the expected birth or 2 d after the birth of a litter. Enrichment materials were introduced into the cages on the day that breeding began and were changed at the same time as the cages. Each male was placed in a cage with two females for breeding. After 14 d, the male was removed and the two females remained in their home cage. Each maternity cage thus contained two adult females and two litters, if both dams gave birth. Before the start of the study, none of the females had had any pups. LAB ANIMAL
Enrichment materials We used four different enrichment conditions: the nonenriched control group received only the standard bedding material, and each cage in the three experimental groups received either a compressed cotton square (Nestlets; Ancare, Bellmore, NY), two two-ply tissues (Kleenex; Kimberly-Clark Corporation, Dallas, TX) or a plastic igloo (Mouse Igloo, Bio-Serv, Frenchtown, NJ) along with the standard bedding. The amounts of the first two enrichment materials were roughly matched by weight: one Nestlet, a 2-in × 2-in square, weighed 2.1 g and two Kleenex tissues weighed 2.4 g. Plastic igloos (with diameter of 4.25 in and height of 2.25 in) were translucent and were either red or yellow. Igloos were changed throughout the experiment and the colors were distributed randomly. We did not keep track of which color igloo was provided to which cage during the experiment. We are unaware of any reports that igloo color affects enrichment, and we observed no differences in the animals’ behavior that made us consider that the color might be important. We feel that the lack of consistency in igloo color more closely mimics the enrichment practices of most facilities. Observations of dams, cages, nests and litters We examined the influence of enrichment materials on reproductive success during the gestational and postnatal periods, by observing the mice and their cages every day from the last week of breeding until 2 weeks after parturition of both the first and second litters of each dam. Informal observations included whether dams appeared pregnant, whether a litter had been born and whether there were any dead pups in the cage. Formal observations were made on gestational day (GD) 16, GD 21, postnatal day (PND) 1, PND 7 and PND 14 (± 1 day for all observations) for cages with surviving litters. The number of dams with surviving litters on PND 1, PND 7 and PND 14 were recorded. Formal observations were made using a ‘snapshot’ approach. Between 3:00 p.m. and 5:00 p.m. on each day of observation, the experimenter looked into each cage for approximately 2 min and made a sketch showing the locations of the dam, litter, nest and enrichment materials. For mice in the three enrichment groups, it was noted whether or not the mice were in direct contact with the enrichment material during each observation period. After the births of litters, direct contact of the pups with the enrichment material, defined as at least half of the litter being in contact with the material at the same time, was also noted. We also recorded whether or not the enrichment materials appeared to have been manipulated by the dams since the last observation. Evidence of manipulation included shredding of the cotton or tissues, movement of the corn cob bedding into or out of the igloo and movement of any material from the place where it had last Volume 41, No. 1 | JANUARY 2012 1 5
Research Note
Rating
Nest structure
1
No depression in bedding with no walls
2
Slight depression in bedding with no walls
3
Flat with low walls
4
Crater with medium-height walls
5
Deep crater with high walls
© 2012 Nature America, Inc. All rights reserved.
Use of the igloo as a nest was given a rating of 3, 4 or 5 depending on the depth of the crater, if there was one.
been seen to a new quadrant of the cage. Our observations of interaction with the enrichment materials are quantified as the percentage of the total number of observations made in which the behavior of interest was observed. The quality of each nest (as estimated by crater formation and wall height) was rated on a scale of 1 to 5 (Table 1). On PND 1, PND 7 and PND 14, litter size and average pup weight were measured for each litter. Dams were removed after the observations of the home cage were completed, and pups were counted and litters weighed. The average individual pup weight for each litter was calculated by dividing the litter weight by the number of pups. Mice that were never visibly pregnant and litters that were completely lost by PND 1 were excluded from all analyses. Litters that were alive on PND 1 but did not survive to PND 14 were excluded from calculations of average litter size and all other analyses. After all observations were made on each dam’s first litter, the first litters were euthanized after PND 15. Dams were maintained in their cages for approximately 2 to 4 weeks afterwards and were then bred a second time. Males were introduced into the cage between 2 and 4 weeks after the first litters were weaned and remained in the cages as described for the first litter. Dams received the same enrichment materials that they had received during their first pregnancies. The same experimenter repeated all observations and ratings during the gestational and postnatal periods of the second litters. Statistical analyses To compare litter survival rate and average pup weight between the first and second litters, we used threeway analysis of variance (ANOVA) tests (litter by enrichment condition by day, with repeated measures on day) using SYSTAT 11 software (Systat Software, Inc., Chicago, IL). Survival rates for first and second litters were calculated as the percentage of pups counted on PND 1 that survived to PND 14. We carried out ANOVA tests to compare observations of animal contact with the enrichment materials and dams’ mani pulation of enrichment materials between the three 16 Volume 41, No. 1 | JANUARY 2012
enrichment groups and across observational days. To analyze the effects of enrichment condition on nest quality, surviving litter size and average pup weight for all groups, we combined data from the two sets of litters and used two-way ANOVA tests (enrichment condition by day, with repeated measures on days). Enrichment condition was a between-subjects factor and day was a within-subject factor. In all cases in which parametric ANOVA results are reported, we tested the data for normality and homogeneity of variance using the Kolmogorov–Smirnov test and Bartlett’s test, respectively. Homogeneity of variance was verified for all results. In the few cases in which the assumption of normality was violated, we used non-parametric Kruskal–Wallis tests to verify the results of the ANOVA tests. In all cases, the results of the parametric and non-parametric tests were in agreement. We present the results of the parametric ANOVA tests here because of the added power given by the repeated measures design. For observations of manipulation of enrichment material, there was no variability in the observations of the compressed cotton and tissue groups, which prevented the use of parametric ANOVA. The results of the Kruskal–Wallis tests are reported for these observations. RESULTS Comparison of first and second litters We compared pup survival from PND 1 to PND 14 and average pup weight for dams’ first and second litters. Dams’ previous pregnancy had a significant positive effect on pup survival rate (f(1,32) = 5.271, P
