Breeding Programme Results

In the first breeding round 21 of 22 females produced litters, 13 females assigned to the half- sibling group and all eight females assigned to the full sibling group (Table 3.1). Two pups died or were killed as a result of infanticide in the half-sibling group between birth and weaning. Mean time between dam and sire pairing and the birth of litters was 22.0 days in both the full sibling and half-sibling groups, with a range between 19 and 25 days.

In the second breeding round, 22 out of 22 dams produced litters, 14 in the half-sibling group and eight in the full sibling group (Table 3.1). Six pups died between birth and weaning in the half-sibling group. The dams that lost pups from their first litters kept all pups in the second litters, suggesting that pup death was not due to infanticidal dams. Mean time between dam and sire (re)pairing and the birth of second litters was 22.7 days in the half-sibling group and 20.6 days in the full sibling group, with a range between 19 and 32 days.

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Figure 3.1: Breeding programme for half and full siblings.

Males and females paired for the first litters. When females appeared pregnant males were removed from breeding cages. After the first litters were weaned, paired of males and females were either re-established to produce second litters that were full siblings of first litters (grey symbols), or new pairs were established to produce second litters that were maternal and paternal half-siblings of first litters (open and black symbols).

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Table 3.1: Breeding success of first and second breeding sessions for full and half-siblings.

First Litter Second Litter

Total number of females

Half-Siblings 37 54

Full Siblings 24 34

Total number of males

Half-Siblings 38 50

Full Siblings 25 30

Number of females per litter (mean ± standard error)

Half-Siblings 2.6 ± 0.5 3.9 ± 0.3

Full Siblings 3.0 ± 0.4 4.3 ± 0.6

Number of males per litter (mean ± standard error)

Half-Siblings 2.7 ±0.4 3.6 ± 0.3

Full Siblings 3.1 ± 0.5 3.8 ± 0.7

Litter size at birth (mean ± standard error)

Half-Siblings 5.5 ± 0.5 7.8 ± 0.5

Full Siblings 6.1 ± 0.4 8.0 ± 0.4

Litter size at weaning (mean ± standard error)

Half-Siblings 5.4 ± 0.5 7.4 ± 0.5

Full Siblings 6.1 ± 0.4 8.0 ± 0.4

Mass (g) of pup on day of birth (mean ± standard error)

Half-Siblings 1.5 ± 0.04 1.5 ± 0.07

Full Siblings 1.3 ± 0.08 1.4 ± 0.04

Mass (g) of pup at weaning (mean ± standard error)

Half-Siblings 12.2 ± 0.4 11.8 ± 0.3

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3.3.3 Behavioural Assay Schedule

Subject females were given three behavioural assays and the same individual triads of females (subject and two stimulus females) were used in each test. First, subject females were given a short scent discrimination and attraction assay designed to test female response to urine from related (either maternal or paternal half-sisters) or unrelated stimulus females. Subject females were then given a nest partner choice assay to establish whether subjects prefer to nest with related or unrelated stimulus females. Finally, subject females were given two consecutive female-female interaction assays, one with a related stimulus female and one with an unrelated stimulus female. This tested whether females behave differently with related or unrelated stimulus females when allowed to interact in a neutral arena over a short time period.

The different behavioural assays were arranged to follow after one another in order of increasing female interaction: females were presented with urine, then given a nest partner choice with limited interaction occurring through a mesh barrier, and finally pairs of females were given a free behaviour interaction trial in a neutral arena. Scent discrimination and attraction assays were run in the morning of the first test day and females were placed in the nest partner choice cages at approximately 4 – 5 pm of that same day. On the morning of the third test day females were removed from the nest partner choice cages and returned to their home cages. Four days later (on the morning of the seventh test day) subject females were given the first interaction trial with either a related or unrelated stimulus female. The following day (test day eight) subject females were given a second interaction trial with the remaining stimulus female.

On completion of all the previous behavioural assays an odour-genes covariance assay tested for whether neutral female house mice can detect a similarity between the urine of half-sisters (maternal or paternal) compared to the urine of unrelated females. Subject females for the odour-genes covariance assay were unrelated and unfamiliar with any of the stimulus females. Urine samples were provided by a triad of stimulus females: an habituation female, a related discrimination female and an unrelated discrimination female. These triads were the same as those tested previously in the scent discrimination and attraction, nest partner preference and female-female interaction assays – previous subject females provided the habituation urine and the previous corresponding related and unrelated stimulus females provided the related and unrelated discrimination urine respectively.

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3.3.4 Subject and Stimulus Females

Subject females were six months old when behavioural assays began, and stimulus females were eight months old. Stimulus females were weight-matched to within 2 g (1.08 ± 0.09, mean ± standard error). Subject females were weight-matched to within 3 g of both stimulus females (1.72 ± 0.17, mean ± standard error).

The scent discrimination and attraction assay requires that subject females directly sniff both stimulus urine marks. A number of females often do not sniff both scents and therefore have to be excluded from the analysis. For the nest cage partner choice and female-female interaction assays 44 subject females were tested (maternal n = 22, paternal n = 22). However, to increase the scent discrimination and attraction assay sample size an additional eight triads of females were tested in each lineage group (maternal = 30, paternal = 30). Triads of these additional subject and stimulus females were established according to the same conditions as before. The subject females were only tested in the scent assay. Of the 16 additional subject females in the scent discrimination and attraction assay, seven had previously been used in the scent assay before. Scent assays for the subject females were performed at least four weeks following the completion of the second female-female interaction assay, providing a large amount of time between repeated scent assays for those seven females that had been used previously. Repeated females were not used as subject females in the same lineage group.

All subject females were fur marked at least two days prior to the start of behavioural assays. This allowed for identification of subject females from stimulus females during the female- female interactions. Fur marking was completed using Jerome Russell b Blonde hair dye (Jerome Russell, CO, USA). Powder bleach and peroxide solution was mixed according to the manufacturer’s instructions and lightly applied onto the centre of the lower back while the subject was held in a handling tube. Females were then transferred to an empty M3 cage for ten minutes, after which the dye solution was carefully washed off using warm, damp cotton pads. A dry cotton pad was then gently applied to soak up any excess water from the fur. Females were checked to ensure all dye had been washed off before being returned to their home cage. By using a handling tube this process could be completed without having to physically restrain the females and therefore was considered less stressful.

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