Abstract
Environmental enrichment is the provision of different substrates to mimic an animal’s natural environment and encourage natural, species-specific behavior. However, the use of enrichment to improve breeding efficiency in mouse models for neurologic conditions is not well described. There are reports that diminished environmental stimuli and chronic isolation can result in the early expression of the Parkinson phenotype in mice with a genetic predisposition to the disease. In this study, we compared the provision of crinkle paper, DietGel, and their combination on reproductive parameters in B6.Cg-Tg(THY1-SNCA*A53T)M53Sud mice. We found that enhanced enrichment combined with enhanced nutrition increased dam weight and decreased the interlitter intervals. In addition, enhanced enrichment increased the production index, number of pups born, pups weaned, and the percent survival of pups. This study underscores the importance of incorporating enrichment to enhance the reproductive parameters in mice that are models of Parkinson disease.
Introduction
A53T mice are a transgenic mouse model that expresses a mutated form of the human α-synuclein protein associated with and used in studying human neuronal α-synucleinopathies, such as familial Parkinson disease.3,13,14,19 These mice are characterized as developing age-related disease progression in their locomotor activity, olfaction, as well as fine motor deficits.3,13,14 Initial signs of disease include poor nest-building scores, incoordination, and hyperactivity, followed by decreased locomotor activity and inability to right themselves.13,14
Although A53T mice are reported to be fertile, these mice are difficult to reproduce in the laboratory setting for several reasons. A53T mice have been reported to have a low breeding efficiency demonstrated by a difficulty to obtain sufficient numbers of offspring from breeding pairs.14 This can be due to various factors such as poor colony management, consequences of their genotype, or environmental factors. For example, the Hualpha-Syn(A53T) strain of A53T mice is reported to be infertile when homozygous for the A53T transgene.14 One report found A53T mice developed poor nesting behavior after one month of age and chronic mild stress resulted in profound motor deficits.18,19
Researchers have identified that there are negative consequences from chronic stress. Stressors such as isolation or reduced environmental stimuli resulted in the progression of Parkinson disease and impaired gastrointestinal motor activity compared with group-housed A53T mice.4,18 Overall, these factors contribute to the difficulty in reproducing A53T mice in the laboratory setting, which can limit their usefulness as a model for Parkinson disease research.
The Guide for the Care and Use of Laboratory Animals emphasizes the importance of providing micro- and macroenvironments that encourage normal behaviors in animals.6 Environmental enrichment aims to enhance the well-being of the animals.2,10 Examples of such opportunities would be increasing the complexity of the mouse’s living environment by adding various types of environmental structures, such as tunnels, platforms, ramps, or hiding places. These structures provide opportunities for exploration, physical exercise, spatial complexity, and decreased stress-induced anxiety-like behavior.1,2,11,12,15 There has been some evidence that the provision of extra enrichment results in positive reproductive performance and decreased pup mortality.8–10,16,17 Diverse types of nesting materials such as shredded paper or compressed cotton nesting squares enable mice to engage in natural nesting behaviors and create comfortable nests, which include a rigid component for structural integrity and a soft component that lines the inside of the nest to keep the pups warm and cushioned.2,17 The incorporation of enhanced nutrition, such as scattered food or providing additional nutritional sources, engages mice in natural foraging behaviors, provides mental stimulation, and may also serve to reduce cannibalization of pups by the dam.8 It has been noted that providing an enriched environment can prevent disturbances to the transcription of neurogenesis via α-synuclein overexpression in transgenic mice expressing human SNCA.15
The purpose of this study is to investigate whether environmental enrichment and enhanced nutrition attenuate reproductive malperformance in a mouse model of Parkinson disease. We hypothesize that the addition of extra enrichment and a supplemental source of nutrition will improve reproductive parameters (number of pups born, number of pups weaned, percent survival, pup weights, dam weights, production index, interlitter intervals, and nest-building scores) in A53T mice.
Materials and Methods
Humane care and use of animals.
This study was performed in an AAALAC-accredited facility and approved by the Yale University IACUC.
Animal housing.
Mice were housed in individually ventilated cages; GM500 mouse caging (77.66 in.2) on DGM80 racks (Tecniplast, West Chester, PA) containing 1/8-in. corncob bedding (Teklad 7092; Inotiv, Madison, WI), pelleted rodent diet (Teklad Global Rodent Diet 2018S; Inotiv), and compressed cotton nesting material (Nestlet; Ancare, Bellmore, NY). Cages and their components (wire tops, feed, bedding, and filter tops) were preassembled and autoclaved before use. Mice were provided ad libitum access to hyperchlorinated (4 to 6 ppm) water delivered via an automated watering system (Avidity, Waterford, WI). To mitigate the effect of light influencing the breeding parameters, all mice were housed on the same rack in the middle row. Room temperature and relative humidity were maintained at 72 ± 2 °F and 50% ± 10%, respectively, with 10 to 15 air changes hourly and a 12:12-h light:dark cycle. Mice were checked daily via visual inspection by the researchers for health status and the presence of pups. DietGel (ClearH2O, Westbrook, ME) was placed in the assigned cages and replenished every 2 d. Cage changes were performed by the researcher every 2 wk, and a veterinarian provided nest-building scores to control for researcher influences on our outcome variables. At each cage change, a portion of the nesting material from the soiled cage was transferred into the new cage, as is standard practice at the authors’ institution to minimize the stress associated with the clean cage.
Mice.
Two-month-old female heterozygous B6.Cg-Tg(THY1-SNCA*A53T)M53Sud mice (Mus musculus) were acquired from a laboratory within the institution, and 8-wk-old male C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Heterozygote A53T mice were paired with wildtype C57BL/6J after consultation with both The Jackson Laboratory and the laboratory that previously owned the mice. The lab advised against breeding homozygotes and heterozygotes together due to the production of nonviable offspring. Based on vendor health reports and in-house sentinel testing, the mice were free of the following agents: Ectromelia virus, K virus, Lactate dehydrogenase elevating virus (LDEV), lymphocytic choriomeningitis virus (LCMV), mouse adenovirus (MAV), murine chapparvovirus (MuCPV), mouse hepatitis virus (MHV), mouse minute virus (MMV), mouse norovirus (MNV), mouse parvovirus (MPV), mouse thymic virus (MTV), polyoma virus, pneumonia virus of mice (PVM), Sendai virus, Reovirus 3, Bordetella, Citrobacter rodentium, Clostridium piliforme, CAR bacillus, Mycoplasma spp., Salmonella spp, Helicobacter spp., Streptobacillus moniliformis, pinworms, and fur mites. During the study, one of the dams in group C (enhanced nutrition group) was euthanized due to nonspecific signs of illness and replaced with a new female. At the time, the dam was caring for 9 pups from her third litter that were ultimately excluded from the analysis to prevent its influence on the percent survival and number weaned results. Postmortem examination of the gravid uterus with histology revealed malformed early-stage pups with inflammation of the endometrium.
Experimental design.
A total of 8 cages with each containing a single breeding pair (heterozygous A53T female and C57BL/6J male) were randomly paired and assigned to 4 experimental groups, with a final count of 2 breeding pairs per experimental group: group A: standard cage setup (standard chow and cotton square); group B: enhanced enrichment (standard chow, cotton square, and crinkle paper); group C: enhanced nutrition (standard chow, DietGel, and cotton square); and group D: both enhanced enrichment and nutrition (standard chow, DietGel, cotton square, and crinkle paper; Figure 1). Male and female mice were continuously paired for breeding and the pups were weaned at 21 d of age. The targeted number of litters produced per cage was 3 in total; however, 2 cages produced 5 litters and were maintained within the analysis apart from calculating the average interlitter interval by group assignment. Interlitter interval is calculated as the days between one litter to the next. When a new litter was identified, pups were counted by visual inspection so as to not disturb the nest and stress the dam. Before the study, the body weights of females ranged from 26.4 to 27 g. The males were not weighed and were not analyzed in this study. At the time of weaning, pups, and dams were weighed to the nearest centigram using a Precision-Balance digital analytic scale (U.S. Solid, Cleveland, OH). In the interest of the 3Rs (Replacement, Reduction, and Refinement), all pups weaned were offered to other researchers within the institution to be used experimentally.
Figure 1.
Diagram depicting the experimental design. WT: wild-type C57BL/6J male mice; HET: heterozygous A53T female mice.
Genotyping.
Mice were genotyped at time of weaning to not disturb the cage more than necessary and impact our results. At 21 d of age, ear punch samples were collected from each pup and sent to Transnetyx (Cordova, TN) for genotyping.
Nest-building scores.
Nest-building scores were calculated at each cage change by the same clinical veterinarian. The scoring system used has been described in previous literature, graded on a 0 to 5 scale5: 0 = nest material is untouched; 1 = nest material spread throughout the cage; 2 = nest is present but flat with no walls; 3 = nest wall height is less than half the height required to cover the mouse; 4 = nest wall is half the height required to cover a mouse; and 5 = nest wall height may enclose the nest.
Statistical methods.
A priori, omnibus, one-way sample size estimates using G*Power software and internal colony data from the donating lab using percent survival as the predictor revealed a total of 120 pups were necessary to reach an achieved power of 80% with an effect size (f) of 0.25. Interlitter interval was calculated as the number of days between the birth of one litter to the next in days. The production index is calculated as the number of pups weaned per dam per week. Data were analyzed using commercially available SPSS software (SPSS 28; IBM, Armonk, NY). Normality tests were conducted using Shapiro-Wilk tests, and homogeneity was examined with a Levene test. One-way ANOVA with a post hoc Tukey honestly significant difference (HSD) test was implemented to determine if there was a statistically significant difference between dam weights, percent survival of pups, and interlitter interval. Analysis of covariance (ANCOVA) was used to assess a potential interaction between the group assignment, number of pups within the litter, and pup weights. Adjusted means, test values (F), effect sizes (η2), and the significance level (P) are reported. The experimental groups were compared against each other using Bonferroni post hoc tests. When the data were determined to follow a nonparametric distribution, Kruskal-Wallis tests with post hoc pairwise comparisons were employed when analyzing the number of pups born, and number of pups weaned. Nonparametric Mann-Whitney U tests were used to identify the difference in weight by sex. When examining the difference in pup weight by genotype, independent sample t tests were employed. A Welch one-way ANOVA was used to compare the production index across the groups. A P value <0.05 at 95% CI is considered statistically significant unless otherwise stated.
Results
Both enhanced enrichment and nutrition increased dam body weight.
The weight of the dams was measured in grams during each weaning session. We analyzed the total weights based on group assignment to minimize the influence of pregnancy status on weight. This is because not all dams were pregnant when weaning their current litter. Dam weight in grams across each group was normally distributed, as examined by the Shapiro-Wilk test of normality (P > 0.05). Mean and SDs are reported. Weights increased from the standard cage setup (group A; 29.53 ± 4.68), the enhanced nutrition group (group C; 31.39 ± 2.07), the enhanced enrichment group, (group B; 33.42 ± 4.47), and the enhanced enrichment and nutrition group (group D; 38.55 ± 5.46), which was statistically significant (F[3,319.482] = 5.158, P = 0.007). Post hoc Tukey HSD test for multiple comparisons with the adjusted significance at P < 0.05 revealed a statistically significant difference between the enhanced enrichment and nutrition group (group D) as compared with the standard cage setup (group A) (P = 0.006) but no other combination (Figure 2).
Figure 2.
The average weight of the dams by group presented in ascending mean values. (A) Standard cage setup (standard chow and cotton square). (B) Enhanced enrichment (standard chow, cotton square, and crinkle paper). (C) Enhanced nutrition (standard chow, diet gel, and cotton square). (D) Both enhanced enrichment and nutrition (standard chow, diet gel, cotton square, and crinkle paper). Error bars are ±2 SD. *, Significance.
Dam weight was not different between successive litters.
Dam weight across all litters was normally distributed, as examined by the Shapiro-Wilk test of normality (P > 0.05) with no violation of homogeneity of variance using a Levene test (P > 0.05). Mean and SDs are presented. The overall mean weight of the dams increased from litter 5 (29.2 ± 0.0041), litter 4 (31.66 ± 5.32), litter 1 (33.68 ± 6.45), litter 2 (34.43 ± 5.33), and litter 3 (35.57 ± 5.60); however, the increases are not statistically significantly different (F[4,82.5] = 0.648 P = 0.634; Table 1).
Table 1.
Average dam weight expressed in grams by litter number
| Litter number | Mean | SD |
|---|---|---|
| 1 | 33.68 | 6.45 |
| 2 | 34.43 | 5.33 |
| 3 | 35.57 | 5.60 |
| 4 | 31.66 | 5.32 |
| 5 | 29.20 | 0.0041 |
| Total | 33.69 | 5.49 |
Dam weight was not statistically significantly different between successive litters (F[4,82.5] = 0.648, P = 0.634).
Enhanced nutrition increased pup body weight after adjusting for number of pups in the litter.
ANCOVA was used to assess a potential interaction between the group assignment, litter number, and the number of pups within the litter. Test values (F), effect sizes (η2), and the significance level (P) are reported. The effect of the number of pups born within the litter on pup weights is not statistically significantly different (F[131,1] = 0.474, P = 0.493). The main effects for group assignment and litter number are statistically significant (P = 0.007 and P < 0.001, respectively). The means presented are adjusted for the covariate litter number unless otherwise stated. There was a statistically significant difference in pup weights between the groups (F[3,141] = 5.121, P = 0.002, partial η2 = 0.10). Post hoc analysis was performed with a Bonferroni adjustment. Pup weights were statistically significantly greater in group D compared with group B (Mdiff = 0.865 95% CI [0.026 to 1.704], P = 0.039) and to group C and B (Mdiff = 1.554, 95% CI [0.302 to 2.806], P = 0.007; Table 2).
Table 2.
Adjusted mean pup weights expressed in grams at weaning by group assignment
| Pup weight (g) | ||||
|---|---|---|---|---|
| Group | Mean | SE | 95% CI | |
| Lower bound | Upper bound | |||
| A | 9.77a,b | 0.325 | 9.13 | 10.41 |
| B | 8.91b | 0.199 | 8.52 | 9.31 |
| C | 10.47a | 0.415 | 9.65 | 11.29 |
| D | 9.78a | 0.246 | 9.29 | 10.27 |
Covariates appearing in the model are evaluated at the following values: litter number = 2.18. Group A: Standard cage setup (n = 24). Group B: Enhanced enrichment (n = 65). Group C: Enhanced nutrition (n = 17). Group D: Both enhanced enrichment and nutrition (n = 39). Means with different superscripts indicate statistical significance (a,b, P < 0.05).
Genotype and sex did not influence body weight.
The weight at the time of weaning of the pups by genotype is expressed in grams. Means with standard deviations are reported. The weight across genotypes was normally distributed, as examined by the Shapiro-Wilk test of normality (P > 0.05). Homogeneity of variance was not violated as examined by the Levene test (P = 0.433). The mean weight of the wild-type pups was 9.25 ± 1.532 g (n = 91) and for heterozygous A53T pups was 9.15 ± 1.472 g (n = 55); however, this difference was not statistically significant (95% CI [−0.53 to 0.72], t[93] = 0.282, P = 0.779; Figure 3).
Figure 3.
Average pup weight expressed in grams by genotype. Wild type: n = 91; A53T: n = 55. Error bars are ± 2 SD. This difference in weight was not statistically significant (95% CI [−0.53 to 0.72], t[93] = 0.282, P = 0.779).
A Mann-Whitney U test was run to determine if there were differences in body weight of the pups between males and females at the time of weaning. Distributions of the weight for males and females were similar. Median weight was higher for males (9.80 g; n = 60) as compared with females (9.29 g; n = 86), but the result was not statistically significantly different (U = 2112, z = −1.861, P = 0.063; Figure 4).
Figure 4.
Average pup weight expressed in grams by sex. Male: n = 60; female: n = 86. Error bars are ±2 SD. Weight by sex was not statistically significantly different (U = 2112, z = −1.861, P = 0.063).
Enhanced enrichment alone or in combination with enhanced nutrition decreased the interlitter interval.
Interlitter interval is expressed in days between one litter to the next. The interlitter interval followed a normal distribution with homogeneity of variance not violated according to the Levene test (P > 0.05). Intervals 4 and 5 were excluded from the analysis since only groups B and D produced beyond litter interval 3. Means and SD are reported. The mean interlitter interval increased from the D group (24.5 ± 4.2) to the B group (24.5 ± 2.38) to the A group (30.7 ± 6.02) and C group (36.0 ± 6.93). One-way ANOVA revealed a statistically significant difference (F[3,128.193] = 5.160, P = 0.014]. Post hoc Tukey HSD test with a corrected significance of P < 0.05 revealed a difference between groups B and C (P = 0.021) and D and C (P = 0.028) but no other combination (Table 3).
Table 3.
Interlitter interval expressed in days from one litter to the next by group assignment
| Group | Mean | SD |
|---|---|---|
| A | 30.70a,b | 6.02 |
| B | 24.50b | 2.38 |
| C | 36.00a | 6.93 |
| D | 24.50b | 4.20 |
| Total | 28.71 | 6.65 |
Group A: Standard cage setup. Group B: Enhanced enrichment. Group C: Enhanced nutrition. Group D: Both enhanced enrichment and nutrition. Means with different superscripts indicate statistical significance (a,b, P < 0.05).
Extra enrichment increased the number of pups born.
A total of 158 pups were included in the analysis. Group A produced a minimum of 1 and a maximum of 9 pups per litter (n = 27). Group B produced a minimum of 3 and a maximum of 9 pups in a litter (n = 66). Group C produced a minimum of 2 and a maximum of 9 pups (n = 22). Group D produced a minimum of 2 and a maximum of 10 pups in a litter (n = 43). Median number of pups born were statistically significantly different between the different groups (χ2[3] = 14.078, P = 0.003). A Bonferroni correction for multiple comparisons was conducted with adjusted P values presented. This post hoc analysis revealed statistically significant differences in the median number of pups born between group B median = 9.00 and group D median = 6.00 but not between any other group combination (Table 4).
Table 4.
Median and total number of pups born and weaned by group assignment
| Group | Median | 95% CI for the median | Total litters | Minimum n | Maximum n | Total n | |
|---|---|---|---|---|---|---|---|
| Lower bound | Upper bound | ||||||
| Number born | |||||||
| A | 7.00a,b | 5.00 | 9.00 | 4 | 1 | 9 | 27 |
| B | 9.00a | 8.00 | 9.00 | 8 | 3 | 9 | 66 |
| C | 6.00a,b | 5.00 | 7.00 | 4 | 2 | 9 | 22 |
| D | 6.00b | 6.00 | 9.00 | 7 | 2 | 10 | 43 |
| Total | 7.00 | 7.00 | 8.00 | 23 | 1 | 10 | 158 |
| Number weaned | |||||||
| A | 7.00b | 5.00 | 9.00 | 4 | 1 | 9 | 24 |
| B | 9.00a | 8.00 | 9.00 | 8 | 3 | 9 | 65 |
| C | 6.00b | 6.00 | 7.00 | 3 | 0 | 7 | 17 |
| D | 6.00b | 6.00 | 7.00 | 6 | 3 | 9 | 39 |
| Total | 7.00 | 7.00 | 8.00 | 21 | 0 | 9 | 146 |
Group A: Standard cage setup. Group B: Enhanced enrichment. Group C: Enhanced nutrition. Group D: Both enhanced enrichment and nutrition. Means with different superscripts indicate statistical significance (a,b, P < 0.05).
Enhanced enrichment resulted in a greater number of pups weaned.
Group B weaned the greatest number of pups (n = 65) followed by group D (n = 39), group A (n = 24), and group C (n = 17). The overall median litter size was 7.0 pups weaned per litter across all 4 groups. A Kruskal-Wallis test with post hoc Bonferroni correction was conducted to determine if there were differences in number of pups born between groups. The median number of pups weaned per litter decreased from group B (9.00) to group A (7.00) with groups D and C having the same median (6.00), which were statistically significantly different (χ2[3] = 33.112, P = 0.001). Post hoc Bonferroni correction revealed a statistically significant difference in number of pups weaned per litter by group between B and C (P < 0.001), B and A (P = 0.024), and B and D (P < 0.001) but not any other combination (Table 4).
Enhanced enrichment increased the percent survival of pups.
Means with SD are reported. Percent survival increased from group C (mean = 79.09 ± 31.64), group D (mean = 90.98 ± 22.6), group A (mean = 96.30 ± 19.25), and group B (mean = 97.48 ± 0.00). One-way ANOVA revealed a statistically significant difference (F[3,1,682] = 4.368, P = 0.006). Post hoc Tukey HSD test with a corrected significance of P < 0.05 revealed a difference between groups B and C (P = 0.004) and A and C (P = 0.027) but no other combination. (Table 5).
Table 5.
Mean and median percent survival by group assignment
| Group | Median | Mean | SD |
|---|---|---|---|
| A | 100.0 | 96.30a | 19.25 |
| B | 100.0 | 97.98a | 0.00 |
| C | 77.78 | 79.09b | 31.64 |
| D | 100.0 | 90.98a,b | 22.60 |
| Total | 100.0 | 93.79 | 19.62 |
Group A: Standard cage setup. Group B: Enhanced enrichment. Group C: Enhanced nutrition. Group D: Both enhanced enrichment and nutrition. Means with different superscripts indicate statistical significance (a,b, P < 0.05).
Enhanced enrichment improved the production index but is not statistically different.
A one-way Welch ANOVA was conducted to determine if the production index was different for the 4 groups. Data are presented as means ± SD. Mean production index increased from group A (1.29 ± 0.65) to group C (1.31 ± 0.26), group D (1.73 ± 0.22), and group B (2.8 ± 0.63), in that order, but the differences between the groups were not statistically significant (Welch F[3,3.115] = 4.420, P = 0.093).
Enhanced enrichment improved nest-building scores.
A total of 64 nest-building scores were collected for analysis. A Kruskal-Wallis test was conducted to determine if there were differences in overall nest-building scores between groups. Median nest-building scores increased from group C (2.00) to group A (3.00) with groups D and B having the same median (4.00) which were statistically significantly different (χ2[3] = 36.156, P < 0.001). Post hoc Bonferroni correction for multiple tests revealed a statistically significant difference in nest-building scores between groups B and C (P < 0.001), B and A (P < 0.001), D and A (P < 0.001), and C and D (P < 0.0005) but not any other combination.
Discussion
To our knowledge, this report is the first to compare nutritional and enrichment interventions on reproductive parameters in female heterozygous A53T mice. We found that dams receiving crinkle paper nesting material in addition to compressed cotton nesting material (Nestlets) and DietGel were 30.55% heavier compared with mice in the standard cage setup (standard diet and Nestlets only). As expected, we found no significant difference in dam weight over time. Interestingly, both enhanced enrichment and nutrition resulted in the shortest interlitter intervals and heaviest dams. However, of note, DietGel alone resulted in an increased interlitter interval, which could suggest that the synergistic effects of crinkle paper and increased nutritional supplementation promote reproductive success. We also hypothesize that placing DietGel in the cage may act as a source of novel object anxiety. A downfall of the DietGel is that it requires frequent disruption of the cage to replace the cups. To this end, we also suspect that the reason for the shorter interlitter intervals in our groups receiving crinkle paper is that it allows the mice to build a more complete nest, providing a place to hide away from the view of the handler and thus reduce stress. It is also possible that the crinkle paper may improve the environment to meet natural behaviors, reduce stress, and improve breeding. These findings are consistent with those of other authors using different mouse strains.7,8,9
In contrast to previous reports regarding the effects of extra enrichment,7,10 we found no difference in pup weight by genotype or sex but did measure an effect of DietGel, in which the pups born to dams receiving enhanced nutrition were approximately 10% to 11% heavier than those in the standard cage even when corrected for the litter number. Environmental enrichment resulted in increased litter size, but this effect is lost with DietGel alone. This result is surprising given that we hypothesized that enhanced nutrition would increase litter size. As previously stated, we hypothesize that the disruption caused to the cage when changing out the DietGel contributed to this outcome. This effect might have been mitigated in the cages with both diet gel and crinkle paper since these mice were able to build more complete nests which provided better shelter. Most notably, the DietGel-only group had by far the lowest pup survival, and the group receiving both enhanced enrichment and nutrition had the second lowest survival (same for litter sizes), suggesting that in both cases the addition of DietGel decreased litter size and pup survival. It appears that the standard setup was significantly better than either of the groups receiving enhanced nutrition, but the addition of crinkle paper only marginally improved this. We suggest that a better way to improve perinatal nutrition would be to provide a breeder diet chow, which does not require opening the cages every 2 d. The rationale behind using DietGel in this study is that it is widely used within our institution for support and that A53T mice develop age-dependent motor deficits that have been reported within our institution to impair their ability to reach both the water and food hopper, contributing to dehydration and poor body condition scores. The authors report that cages belonging to the standard cage setup group only yielded 2 litters, after which no litters were produced. This coincides with the anecdotal evidence research staff have reported to our veterinary staff. One theory is that these mice were not provided sufficient nesting substrate compared with the groups that were provided crinkle paper, resulting in prolonged interlitter intervals and fewer litters achieved by the time they reached reproductive senescence. We hypothesize that because the standard cage group was less frequently disturbed than the mice that only received DietGel, this resulted in less stress and contributed to their higher percent survival compared with the mice that only received DietGel.
As expected, the groups without the crinkle paper scored lower in their nest-building scores than the groups with the addition of crinkle paper, presumably due to the inherent structural deficiency of the compressed cotton squares. As such, comparing nest-building scores between groups provided different nesting materials makes it difficult to differentiate whether the differences in scores are secondary to impaired fine motor or cognitive abilities as this was not directly examined in this study. One limitation of this assessment is that it was impossible to blind the veterinarian performing the score, since it is evident which groups had the interventions. The group with DietGel alone continuously scored low in their nest-building scores over time. One recommendation moving forward would be to have 2 individuals scoring the nests and taking the average of those scores.
The increasing trend for use of transgenic mouse models in research has resulted in behavioral differences and their responses to their immediate environment. As outlined previously, the A53T mouse model is sensitive to their environment, and unenriched environments can result in the early expression of the phenotype.4,18 In our study, crinkle paper alone slightly increased survival (97% compared with 96%), whereas the addition of DietGel with or without crinkle paper greatly decreased survival (91% and 79%, respectively). However, the interlitter intervals were the shortest for those groups receiving crinkle paper, with our standard cage groups having the longest interlitter intervals. Our results contradict the anecdotal evidence of benefits from enhanced feeding but demonstrate some benefits from an enhanced environment. Our results also suggest that while pups and dams were slightly larger in the DietGel groups, pup survival was significantly lower. Directions of research should investigate enrichment on the longitudinal outcome and expression of the Parkinson phenotype. Moving forward, we recommend that, at a minimum, transgenic mouse models for Parkinson disease should receive both structural material (for example, crinkle paper) and soft lining (for example, cotton nesting material) to allow for the construction of a complete nest and improve their breeding efficiency and that the implementation of the enrichment should take into consideration the long-term study goals. Future work should assess the effects of alternative enrichments and nutritional supplementation that does not require frequent disturbance of the cage on the development and progression of neurologic signs, as well as the development of histologic lesions in the brains of AT53 mice.
Acknowledgment
We thank Jennifer Terrebonne of ClearH2O for providing the DietGel used in this study.
Conflict of Interest
The authors have no conflicts of interest to declare.
Funding
This work was internally funded.
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