Retrospective Analysis of Reproductive Performance of Pair-bred Compared with Trio-bred Mice

Abstract

Federal guidelines provide recommendations regarding the minimum of floor space that should be allotted for breeding laboratory rodents. Mouse mating systems used by a variety of institutions frequently deviate from these recommendations. Regulatory agencies suggest that deviations from established guidelines should be assessed on an institutional basis and recommend periodic review by the local IACUC. Mouse breeding data, collected in a laboratory animal management software program at a single institution, were retrospectively analyzed to determine the effects of 2 breeding schemes on reproductive performance. Data were analyzed over a 20-mo period from all inbred strains of mice breeding in the vivarium. Variables included total number of pups per litter, pups per female, and litters per female. Data indicated that the numbers of pups and litters per dam do not differ between trio-bred (one male, 2 female) and pair-bred (one male, one female) mice. However, more pups were weaned per litter in trio-bred (mean, 5.8 pups) when-compared with pair-bred (mean, 4.7 pups) mice. These results suggest that allocating less than the recommended amount of floor space is not detrimental to the overall breeding success of the strains of mice examined.

The Guide for the Care and Use of Laboratory Animals is the primary reference used to assist institutions in the care and use of research animals in ways judged to be “scientifically, technically and humanely appropriate.”¹³ As a guide, this publication provides recommendations for the development of a comprehensive animal care program at the local institutional level. This 8th revision attempts to balance the prescriptive engineering tendencies of the past with performance standards that include “professional input, sound judgement, and a team approach” to meet the requirements of this guide. In addition to listing the recommended floor space for group-housed rodents, this latest revision includes a new metric for the minimum floor space that should be allotted for a female mouse or rat plus her litter.

At the University of California Merced, the IACUC approves mouse protocols with trio breeding schemes based largely on the veterinarian's professional judgement and experience. In this vivarium it is not uncommon to see multiple litters—and multiple litters of different ages—in these continuously breeding cages. Despite this situation, trio-bred mice appear to produce as many healthy pups and as many litters as do pair-bred mice. This retrospective analysis of mouse breeding data from the vivarium was performed in an attempt to corroborate professional judgement and observations with performance-based data.

Materials and Methods

Animal work in this study was done in accordance with the Guide and was IACUC-approved. The University of California Merced holds an assurance from the Office of Laboratory Animal Welfare and is AAALAC-accredited.

Mice in this study were obtained from a variety of sources or were bred inhouse. Mice were housed in 1 of 4 animal rooms within the vivarium. Room temperatures ranged from 20 to 26 °C, humidity was between 30% to 70%, and the light cycle was a 12:12-h light:dark photoperiod. Two sentinels were exposed to dirty bedding from a maximum of 70 cages for 3 mo prior to submission. On a quarterly basis, 2 sentinels from each room were submitted to an academic diagnostic laboratory (Comparative Pathology Laboratory—UC Davis; http://cpl.ucdavis.edu). Rodent health surveillance consistently identified Helicobacter hepaticus and murine norovirus in 1 of the 4 mouse-breeding rooms.

Mice were housed in IVC (Lab Products Super Mouse 750TM ventilated caging systems; floor space, 75 in² [483 cm²]). Cage bedding consisted of irradiated 1/8-in. corncob (Bed-o'Cobs, Andersons Lab Bedding) and nesting material (Cotton squares, Ancare, Bellmore, NY; and Enviro-dri, Shepherd Specialty Papers, Watertown, TN). Reverse-osmosis–purified water and rodent chow (diet 2919, Teklad Global 19% Protein Extruded Irradiated Rodent Diet, Envigo, Derby, United Kingdom) were provided without restriction. Breeding cages were changed every 2 wk or more frequently based on daily welfare checks performed by vivarium staff. Technicians and laboratory staff were given leeway to use clinical judgement when weaning; if pups were deemed too small to wean at 21 d, they were allowed to stay with parents for additional days as needed.³² When new litters were recorded, cage changing and other disturbances to the cage were postponed for 3 to 5 d. Genotyping samples were collected from pups (if needed) between 7 and 10 d of age.

Breeding data were retrieved from a commercial laboratory animal management software program (Mosaic Vivarium, Virtual Chemistry). Data were analyzed using SAS software (SAS Institute). Performance metrics were characterized by the number of pups weaned per litter and the number of litters per dam. Frequency distribution and variance were determined by kurtosis and skewness of the data, and square-root transformation was performed to normalize the data. The Satterthwaite approximation t test was used to determine the central tendency of 2 populations based on samples of independent data with unequal variances.²⁷ The α value was set at 0.05; the null hypothesis was rejected when the P value was less than 0.05 and retained when the P value was at least 0.05.

Results

Data were collected from December 2013 and through August 2015. Breeding data were available from 53 genetically unique lines of inbred mice on 3 different background strains and consisting of 275 breeding units during this period (37 strains on C57BL/6; 14 on BALB/c; and 2 on C3H/HeOu inbred strains). Briefly, data were aggregated at the cage level and categorized into 2 ‘treatment’ groups (trio breeding compared with pair breeding). Breeding units with 0 pups (111 units; data not shown) were excluded from the data because either the units were determined to be nonproductive 45 d after set-up and therefore were replaced with a new set of breeders (n = 82) or because units had been established for less than 21 d when data were collected and therefore litters had not yet been reported (n = 29).

Mice were cohoused for breeding between 6 and 8 wk of age and remained together for an average of 144 d (minimum, 23 d; maximum, 386 d; data not shown). Breeding units were left undisturbed (no adult mice removed or added) throughout the lifespan of the breeding unit. Mice were mated in breeding pairs or trios depending on 1) the investigator's experimental needs regarding a specific number, sex, or genotype of offspring; 2) historical reproductive characteristics of mouse line; 3) availability of replacement male or female mice for a specific line; and 4) whether a specific mouse line was needed for production or maintenance. In many cases, both types of mating units and multiple breeding units were established for each mouse line.

Descriptive statistics of reproductive variables are shown in Table 1. Analysis of stem-and-leaf as well as typical quartile–quartile plots of the variables compared with transformed variables indicated that squared-root transformation of each variable normalized the data appropriately for statistical analysis. Transformed data are shown in Table 2. The Satterthwaite approximation t test for independent samples with unequal variance was performed (Table 3). The data suggested that neither the number of pups weaned per dam nor the number litters per dam differed between pair and trio breeding configurations. However, the number of pups weaned per litter was approximately one pup greater for trio-bred litters compared with pair-bred litters when means were examined (Table 1).

Table 1.

Descriptive statistics of nontransformed data (P≤ 0.05)

	n	Mean	1 SD	SE Mean
No. of pups weaned per litter
Pair	75	4. 7	2.3	0.27
Trio	200	5. 8	2.6	0.18
No of pups weaned per dam
Pair	75	10.9	7.8	0.90
Trio	200	12.4	8.5	0.60
No. of litters per dam
Pair	75	2.3	1.3	0.15
Trio	200	2.2	1.4	0.10

Table 2.

Descriptive statistics of square root-transformed data (P≤ 0.05)

	n	Mean	1 SD	SE mean
No. of pups weaned per litter
Pair	75	2.1	0.6	0.07
Trio	200	2.4	0.5	0.04
No. of pups weaned per dam
Pair	75	3.1	1.3	0.15
Trio	200	3.3	1.2	0.08
No. of litters per dam
Pair	75	1.5	0.4	0.09
Trio	200	1.4	0.5	0.03

Table 3.

Satterthwaite approximation ttest of transformed data; 2-tailed p (P≤ 0.10)

	DF	T	Pr t	Significant?
No. of pups weaned per litter	119.25	−3.63	0.00	yes
No. of pups weaned per dam	126.94	−1.51	0.13	no
No. of litters per dam	146.35	0.58	0.56	no

Discussion

Mice have evolved several survival strategies that contribute to their value as a model research organism. Feral and commensal mice live in small, quiet, undisturbed locations within range of food and water.² They live in demes composed of a dominant male, a hierarchy of 2 to 5 breeding females, and an assortment of subordinate males and females.²⁶ Reproduction is remarkably fast and efficient in mice: females have short gestation periods of approximately 21 d; pups are capable of independent survival from the dam within 21 to 28 d after birth and are sexually mature as early as 4 wk of age.²⁴ In addition, females undergo a postpartum estrus within 12 h of parturition, thus facilitating continuous generation of offspring.²⁴ Mice practice communal nesting, by which all adults (including the male and nonpregnant females) tend to the young in a single nest. Communal nesting allows for thermoregulation of altricial pups, enhances food supply, and most likely provides innumerable neurobiological and developmental benefits due to tactile, auditory, and olfactory interactions between adults and young.^9,12,15,30 All of these factors occur without the usual genetic bottle neck seen in isolated populations. This situation has resulted in an animal model whose high reproductive capacity, minimal husbandry needs, genetic pliability, and natural tendency to inbreed have been used to science's advantage. Common sense indicates that outbred mice (for example, Swiss Webster and related substrains), which normally nurture and wean large litters, do not need to be set up in trio breeding units to maximize productivity.⁵ However inbred, genetically modified mice may benefit from a more biologically relevant breeding deme structure to maximize reproductive output. In either case, total floor space does not appear to be a limiting factor.

The brand of IVC used in the study vivarium provides approximately 75 in.² (483 cm²) of floor space. With the exception of a single breeding pair with one litter (one adult, one female with litter = 15 + 51 = 66 in.² [427 cm²]), none of the remainder of our breeding schemes fell within the Guide’s recommendations (for example, 2 adults, one female with litter = 15 + 15 + 51= 90 in.² [524 cm²]; one adult, 2 females with litters = 15 + 51 +51 = 117 in.² [757 cm²]). Data analysis revealed no statistically significant differences in the number of pups weaned per female nor in the number of litters a female produces during her breeding lifespan, regardless of whether that female was in a breeding pair or trio. A closer look at the descriptive statistics (Table 1) suggests that one additional pup per litter is produced in trio-bred as compared with pair-bred mice (5.8 compared with 4.7, respectively). The biologic significance of this difference is unknown. Most of the genetically unique lines in this data set were on a C57BL/6 background. Commercial vendor statistics indicate that this inbred strain has good reproductive performance, averaging 6.2 pups per litter.⁸ This report is consistent with our overall findings regarding the number of pups per litter. Taken together, our data suggest that the amount of available floor space is not a critical factor for optimal reproduction over the lifespan of the breeding unit and that the reproductive performance of trio-bred mice is similar to—or no worse than—pair-bred mice. These results are consistent with those of other studies examining the interactions of cage size and configuration,^5,11,36,37 caging type or rack systems,^6,33,34 breeding format,¹⁴ cage changing or disturbance frequency,^23,25,28 culling or early weaning of litters,^5,20,22 and the use of communal nesting^{1,7,9,10,12,29-31,35} on mouse reproductive performance. Others have shown that stable, nonreproducing mouse demes—no mature mice added, with mice removed only through attrition—can cohabitate without any apparent negative behavioral or physiologic effects, even at cage densities 2 to 3 times those recommended in the Guide.^18,19,21 This information suggests that given sufficient food, water, and nesting material, compatible cohorts, and the ability of offspring to disperse or be weaned in a timely fashion and if left relatively undisturbed, mice will reproduce to the extent of their genetic potential, regardless of the constraints of captivity, with no or few apparent ill effects on their overall wellbeing.^3,4,17,26

Several limitations to this study must be acknowledged. This study was a retrospective study of data collected on an on-going basis, so prospective randomization of the groups was not performed. No attempt was made to analyze data by individual genetic mutation(s) or to control for parity or litter size of each female. Weaning—as compared with preweaning—data were used, because in our experience, preweaning data are less reliable and more difficult to collect due to reluctance to disturb new litters. Although the mice in this study were housed in a single style of IVC within the same vivarium, no control for variation of cage location in animal room, room location within the vivarium, or husbandry personnel differences was performed. In addition, performance data—for example, maternal or paternal behavioral observations, nest building activity, stereotypy, weaning and adult body weights, behavioral testing of adult offspring, physiologic parameters, organ weights—were not collected. Review of the literature suggests that continuous mating schemes,¹⁶ available breeding cage space,¹¹ and housing density of same-sex, inbred, or F1 hybrid mice¹⁸ have little effect on normal mouse biology, ecology, and behavior. However, this study did not require the acquisition of additional experimental animals and therefore did not “unnecessarily duplicate previous experiments”¹³. Mouse breeding was covered under each principal investigator's IACUC-approved protocol. Colony data were collected on a daily basis as part of standard vivarium management. These data were analyzed to determine whether breeding production variables were statistically significant between trio-bred and pair-bred mice housed in one particular cage model. Results suggest that they were not.