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Social, Maternal and Aggressive Behaviors in Rodents

Many standard behavioral tests exist for the study of interactive behavior in mice and rats. In order to choose the most appropriate test for a research study it is important to understand something about the range of rodent social behaviors and what, specifically, behavioral tests are attempting to measure.

Rats and mice used in research are considered social species, meaning, in general, they prefer some form of group living. Species that live together must interact and so have evolved various behaviors that allow and facilitate group living. Environmental conditions and individual characteristics (e.g., sex, age, reproductive status, genetic background, etc…) are important in determining the form and amount of social interaction that occur within a group. In addition, sensory and motor abilities and health status can influence the expression of social behavior in individual animals. For example, an animal may be less willing to interact with others if it is ill or in pain. In another example, the sense of smell (olfaction) is extremely important in mouse communication and mice with olfactory deficiencies may behave quite differently than normal mice.

Before performing behavioral tests on rodents, especially when using unfamiliar strains or mutants, investigators must evaluate overall health and specific sensory and motor capabilities of the animals to avoid biased and inaccurate interpretations of the role of genetics in behavior.

General Categories of Rodent Social Behavior


Aggression and Social Dominance Behaviors

Specific Tests:

Aggressive behaviors are usually related to either territorial or maternal defensive actions or the establishment and maintenance of social status within a group. Males tend to show more territorial and social dominance behaviors than females but there are exceptions. Predatory behaviors (behavior oriented toward catching and killing of prey) are not included in this category. Rodents who bite humans are also not displaying true aggressive behavior but rather, fear induced defensive behavior.

Rats and mice differ in their social organization and use of aggressive behaviors. Male mice are territorial and do not tolerate unfamiliar males within their home range (or cage). Females may establish territories but tend not to defend them with aggressive behavior. Male (and female) mice mark territorial boundaries with urine; this is an important method of avoiding unnecessary aggression and its consequences in this species. In contrast, rats have evolved to live in multi-male/multi-female groups and tend to coexist peacefully if group composition is stable.

Although both mice and rats establish social dominance hierarchies within groups, they differ in important characteristics. Male social hierarchies in stable rat groups tend to stay the same despite changes in weight and/or size of individuals. In these types of groups, age may be the best predictor of social status. Male mice also establish social dominance hierarchies in a group but they will continuously compete for dominance. This often results in fighting and subsequent injuries. Changes in group composition, the presence of female mice in the room (olfactory stimulation) or manipulation of the mice (e.g., cage changing, temporary removal for experimental procedures) may increase fighting. If multiple mice are in the cage, removal of the dominant mouse will not necessarily stop the injuries, as the remaining mice will fight to reestablish a social order.

Female mice and rats also establish social dominance hierarchies but tend not to fight. This makes it easier to group house them but harder to study social organization.

Behavioral Tests:

Standard Opponent Test

This test evaluates male aggression and social dominance in a test animal placed with an unfamiliar conspecific in a neutral area. The test subject is confined with a ‘standard opponent’ partner for a specific time in an unfamiliar cage or other defined space.

Important considerations for this test:

  1. The standard opponent(s) males are selected for highly replicable behavior as either submissive or dominant males in repeated tests with other males. Standard opponent partners are usually chosen from mouse strains known for either high or low levels of aggression. The selected mice are then used as standard opponent test partners in pairings with experimental mice.
  2. Differences in weight, age and size between the test mouse and the standard opponent may also influence test results.
  3. Keep in mind that it will be necessary to keep track of which mouse is the test subject and which is the ‘standard opponent’ during the test session. Mice with different coat colors will make this easier to do.
  4. Test sessions are often 5 minutes in length but are terminated early if attacks and biting are severe.
  5. The presence of humans can influence animal behavior during the test. The observer should be screened from the animals and/or the mice may be videotaped for scoring later.
  6. The frequencies of specific (predetermined) behaviors are scored. Examples of behaviors include:
      • Body sniffing
      • Anogenital sniffing
      • Following
      • Chasing
      • Tail rattling
      • Number and location of bites

More information on standard opponent testing may be found in this and other references:

Crawley, JN. What’s Wrong With My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice, 2nd ed. Wiley-Interscience, 2007.

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Isolation-Induced Fighting

This is a modification of the standard opponent test in which male mice are singly housed for a specific time period (e.g., four weeks) prior to placement with an unfamiliar male mouse into a test arena or cage.  Isolation of male mice tends to increase the frequency of fighting and attack behaviors.

 

Resident-Intruder Test

Another modification of the standard opponent test, the resident-intruder test is conducted in the home cage of the test mouse. The unfamiliar male mouse is the ‘intruder’. The test mouse (‘resident’) will attempt to defend its home cage from the intruder. Isolation is not needed prior to this test. Aggression in the resident mouse will be higher if he is living with a female and her litter (sired by him). However, the female and her pups must be separated from the fighting area, as the female will also display aggression toward the intruder (maternal defense).

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Tube-Test for Social Dominance

This test measures dominant/submissive behavior in mice without allowing them to fight and injure each other. Both male and female mice may be tested with the Tube-Test. In the test, two mice of the same gender are placed at opposite ends of a clear, cylindrical tube and allowed to explore toward the tube center. At the point where the mice meet, the submissive mouse will tend to back up as the dominant mouse continues moving forward. The mouse that leaves the tube first (‘pushed out’) is the loser and the other mouse (dominant) is the winner. Automated equipment for this test exists that can measure additional parameters such as duration of match, latency to enter the tube, etc… This test can be used for determining social dominance relationships within a group of mice.

Additional information on an automated version of the tube-test may be found here: http://www.noldus.com/animal-behavior-research/products/automated-tube-test.

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Rat and Mouse Parental and Maternal Behavior

Parental behaviors can be classified as direct (having an immediate physical impact on offspring and their survival) or indirect ((behaviors that do not involve physical contact but still affect offspring survival). Examples of direct behaviors include nursing, grooming or licking, retrieving and huddling. Some direct behaviors may be performed by males (i.e., the sire). Examples of indirect behaviors include nest building, defense against conspecifics or predators, acquiring and defending critical resources and care for pregnant or lactating females. Indirect behaviors may be performed by either parent and by other (non-parent) adults, which is referred to as alloparental care.


Although some laboratory studies indicate that adult male mice and rats are capable of parental behaviors, these occur at a low level and care of the young is primarily left to the female. Studies of wild mice and rats have shown that males are not involved in care of the young and will kill young that are not their own. Males will also kill unrelated or unfamiliar young under laboratory conditions. While the presence of the male sire in the breeding cage is generally not harmful to pups, there is no evidence that the male benefits pup growth and development. Adult males other than the sire, however, should not have access to young other than their own. See references 1 and 2 below for more information.


Maternal behavior typically refers to all aspects of behavior of the dam between parturition and weaning of the offspring and includes both direct and indirect behaviors. Some aspects of maternal behavior (e.g., nestbuilding) may begin prior to birth of the young. Laboratory studies with rodents have shown that hormonal changes (e.g., oxytocin) are important triggers for onset of maternal behavior. As hormonal influence decreases after parturition, infant stimulation increases in importance in this regard. Stimuli from pups, including ultrasonic vocalizations (USV), are needed to maintain maternal care after about 5 days postpartum (2, 3, 4). Infant rats and mice emit a variety of sonic and ultrasonic vocalizations that attract the dam’s attention. In mice, inbred strain differences in hearing ability and the number of USV emitted by pups have been found. USV have been extensively studied in rodents and various protocols for are available for experimental research (2,4).


In rats and mice, a postpartum estrus occurs within 24 hours after parturition. Laboratory studies have suggested that postpartum mating activities are shorter in duration than during normal estrus periods and do not significantly reduce maternal time spent with the litter (2). After the postpartum estrus period the female will not come into estrus again until after the pups are weaned. If she mated and conceived during the postpartum estrus, the second gestation may be prolonged by a week or more.


Young rats and mice are altricial, which means they are born in a relatively undeveloped state and cannot move, maintain body heat, see or hear on their own. Extensive maternal care is required for the young to survive. Rats and mice have evolved specific behaviors that contribute to the survival of altricial offspring. Both rats and mice will actively build nests in which to rear their young. These nests are built by the female and may be complex, multi-entrance enclosures if the dams are provided with appropriate building material. Significant strain differences in nest building skills have been shown in mice.


Both rats and mice will nest communally (multiple females rear their young in the same nest) and nurse offspring that are not their own. Two DBA mouse dams with their litters in a communal nestLaboratory studies have indicated that pup survival to weaning is higher for rats who rear their litters alone rather than in a communal nest. The opposite may be true in mice. Multiple studies have shown that mouse pups reared in communal nests had higher growth rates and better survival than pups reared alone with their dam (4). However, communal nesting/nursing may not be successful if the age difference between litters is greater than 5-7 days. In this situation, dams may be aggressive toward pups that are not their own.


Lactating females will display aggressive behavior to defend their offspring from others of their own species. The presence of pups appears to be the primary trigger for female postpartum aggression. The presence of unfamiliar male or female conspecifics will provoke maternal aggression although the likelihood and expression of maternal aggression varies with strain, individual and location (e.g., home cage versus test arena) (2,4).


There are a number of events and experiences that will influence the behavior of both the mother and the pups. These include the effects of handling of the dam and/or pups and disturbance of the cage environment by the researcher. Depending on the experimental objective these could be confounding factors and must be considered. Maternal behavior during lactation will also be affected by changes in the pups as they grow and mature and by the evolving physiological state of the dam.


Laboratory studies have shown that the main components of rodent maternal behavior (nursing, licking and grooming, pup retrieval and nest building) are present at high levels in almost all rats and mice after giving birth (2,4). Time spent in these behaviors typically declines gradually during the first two weeks of lactation and then decreases further or disappears during the third or fourth week after parturition. Consumption of food and water by the dam increases dramatically over the first two weeks of lactation and may influence the amount of time spent on maternal behaviors. Although commonly used as experimental measures of maternal behavior, nest building and pup retrieval do not normally occur at high frequencies in undisturbed conditions. Mice and rats build nests if material is available but once made, the nest is not rebuilt from scratch unless disturbed. Pup retrieval is also infrequently necessary under normal conditions.


Rat and mouse pups start eating solid food around 15-17 days of age and nursing by the dam ends by four weeks after gestation. Weaning of a litter is normally a gradual process that can stretch well beyond the third week. The typical abrupt weaning that takes place in the laboratory when the pups are 3-4 weeks of age provides another example of experimental manipulation influencing normal behavior.

References:

  1. Brown, R. E. (1986). Paternal behavior in the male long-evans rat (rattus norvegicus). Journal of Comparative Psychology, 100(2), 162-172. doi:http://dx.doi.org/10.1037/0735-7036.100.2.162
  2. Elwood, R.W. (Ed.). (1983). Parental Behaviour of Rodents. Chichester: Wiley and Sons.
  3. Kazutaka, M., Nagasawa, M., Kikusui, T. (2011). Developmental consequences and biological significance of mother-infant bonding. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 35, 1232-1241.
  4. Weber, E.M. & Olsson, A.S. (2008). Maternal Behaviour in Mus musculus sp.: An ethological review. Applied Animal Behaviour Science, 114, 1-22.

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