The occurrence of ultrasonic vocalisations during sexual interactions is closely related to male sexual arousal in mice (Nyby, 1983) and females typically prefer to associate with males that vocalise at a high rate over individuals that fail to emit any ultrasounds (Pomerantz et al., 1983). To determine whether the motivation of individuals to interact was linked with male signalling investment, the behaviour of males and females when separated by a barrier was recorded.
75 Figure 2.7 Urinary protein and creatinine concentrations and scent mark investment during ‘barrier’ trials.
Male scent marking was (a) negatively correlated with urinary protein concentration (rs
= -0.48, p = 0.046 and (b) negatively correlated with creatinine concentration (rs = -0.53,
p = 0.03). Males that deposited a relatively high number of scent marks had very dilute urine containing a low concentration of protein.
76 During these trials the amount of time females and males spent sniffing, chewing and pulling at the mesh section of the barrier was recorded as an indicator of interest and attraction; this was positively correlated with male investment in ultrasonic calls (female: Spearman’s rho; rs = 0.48, p = 0.04; male:Spearman’s rho; rs = 0.49, p =
0.04; Figure 2.8).
In addition, male and female behaviours were recorded during interactions; primary patterns of behaviour were obtained via principal components analysis. The three components from the PCA on male behaviour with an Eigenvalue > 1 are shown in Table 2.2. The first component accounted for 45% of variance in the data and contrasted affiliative behaviours and mount latency with the number of mounting attempts. This component was positively correlated with male calling investment (Spearman’s rho; rs = 0.62, p = 0.008; Figure 2.9a), but negatively correlated with scent
marking (Spearman’s rho; rs = -0.50, p = 0.04; Figure 2.9b). Males that displayed a high
level of affiliative behaviour called at a high rate, deposited relatively few scent marks and made few mounting attempts. The second component accounted for 19% of variance in the data and contrasted following the female with grooming the female; the third component accounted for 18% of variance of the data and contrasted sniffing female genitals and mounting attempts with mount latency. Neither of these components correlated with male signalling investment.
The principal components analysis for female behaviour revealed two components with an Eigenvalue > 1; these are shown in Table 2.3. The first component accounted for 45% of variance in the data. This component contrasted sniffing and grooming the male with rejection of mounting attempts and was negatively correlated with the number of scent marks deposited by males (Spearman’s rho: rs = - 0.66, p = 0.004; Figure 2.10). This component was also correlated with the first component obtained from the PCA on male behaviour (Spearman’s rho: rs = 0.49, p =
0.048; Figure 2.11). Females therefore sniffed and groomed males more when relatively few scent marks were deposited and males displayed a high level of affiliative behaviour. The second PCA component accounted for 39% of variance, contrasting following the male and sniffing male genitals with rejection of mounting attempts; this component was not correlated with male signalling investment. Despite comprising behaviours similar to those in PCA component 3 for male behaviour, the two were not correlated (Spearman’s rho: rs = 0.02, p = 0.94).
77 Figure 2.8Male calling and behaviour at the mesh partition of barrier.
Male (black squares) and female (open circles) behaviour was positively correlated with male calling investment when individuals were separated by a barrier (Male: rs = 0.49, p
= 0.04; female: rs = 0.48, p = 0.04). Behaviours included in this analysis were sniffing, chewing and pulling on the mesh area of the divider.
78
PC1
PC2
PC3
Sniff female genitals 0.55 0.01 0.76
Sniff female face 0.69 0.43 -0.18
Groom female 0.71 -0.48 0.33
Follow female 0.39 0.83 0.12
Mount latency 0.89 -0.21 -0.21
Mounting attempts -0.71 0.15 0.55
Variance 45% 19% 18%
Table 2.2 Principal component weights describing male behaviour exhibited during interactions.
Component 1 contrasted affiliative behaviours and mount latency with the number of mounting attempts, component 2 contrasted sniffing and following with grooming, and component 3 contrasted sniffing genitals and mounting attempts with mount latency.
79 Figure 2.9Male signalling and behaviour during interactions.
The main patterns of male behaviour were obtained from a principal components analysis as shown in Table 2.1; individual scores for principal component 1 contrasting affiliative behaviour and mount latency with mounting attempts were positively correlated with (a) male calling and (b) scent marking (calling: rs = 0.62, p = 0.008; scent marking: rs = -0.50, p = 0.04). Males that invested heavily in calling typically displayed a high level of affiliative behaviour towards females but also tended to make fewer mounting attempts. Males that invested heavily in scent marking typically spent less time grooming and sniffing the female but made a high number of mounting attempts.
80
PC1
PC2
Sniff male genitals -0.24 0.89
Sniff male face 0.96 0.01
Groom male 0.96 0.05
Follow male -0.22 0.89
Flee mounting -0.55 -0.63
Variance 45% 39%
Table 2.3 Principal component weights describing female sexual behaviour during interactions.
Component 1 contrasted sniffing and grooming with fleeing from mounting attempts and component 2 contrasted sniffing and following with fleeing from mounting attempts.
81 Figure 2.10Male scent mark investment and female behaviour.
The main patterns of female behaviour were obtained from a principal components analysis as shown in Table 2.3. Principal component 1, contrasting sniffing and grooming the male with rejection of mounting attempts, was negatively correlated with male scent marking (rs = -0.66, p = 0.004). Females exhibited a high level of affiliative
82 Figure 2.11Male and female behaviour during interactions.
The main patterns of behaviour were derived from separate principal components analyses on female and male behaviour during interactions; the components obtained are shown in Tables 2.2 and 2.3. Individual scores for principal component 1 describing male behaviour were correlated with individual scores for principal component 1 describing female behaviour (rs = 0.48, p = 0.048). Males displaying a high
level of affiliative behaviour interacted with females that spent the greatest amount of time sniffing and grooming the male.
83
EXPERIMENT 2
The second experiment was conducted to extend the findings of interaction trials in the first experiment and to investigate other factors that may be linked to male signalling investment. As a result of single sexual interactions in experiment one, only correlations between male signalling investment and the behaviour of males and females could be identified. In this second experiment, subjects participated in multiple sexual interactions to establish causality and assess whether male investment in signalling is a stable trait or influenced by the behaviour of different individual females. As in experiment one, in addition to measuring the number of scent marks and call rate, during interactions the total duration of expression of affiliative behaviours (e.g. sniffing and following the other animal) and male mounting activity was recorded. Additionally, the links between male signalling investment, corticosterone and male reproductive physiology were assessed.
2.4 METHODS
2.4.1 Subject and stimulus animals
Subject males (n = 18) were CD-1 laboratory mice bred in house (parents obtained from Harlan UK) and aged 12 months at the start of the experiment. 18 adult female C57BL/6JOlaHsd laboratory mice bred in house (parents obtained from Harlan UK) and aged 12 months acted as stimulus animals. Males were housed singly in 48cm x 11.5cm x 12cm cages (M3, North Kent Plastics Ltd., UK) and females were housed in same-sex pairs in 48cm x 11.5cm x 12cm cages (M3, North Kent Plastics Ltd., UK); pairs were maintained throughout the study. Mice in this experiment were different to individuals used in the first experiment and had no prior sexual experience but had previously been exposed to conspecific odours. Soiled male nesting material was added to the home cages of females three days prior to each interaction to induce oestrus (Cheetham et al., 2007). Throughout the experiment all animals were maintained on a reversed 12:12 light cycle (lights off at 08:00h) and on Corn Cob Absorb 10/14 substrate with paper wool bedding material and ad libitum access to water and food (Lab Diet 5002 Certified Rodent Diet, Purina Mills, MO, USA). Acrylic tunnels (5cm x
84 15cm) and red plastic houses (Techniplast, NJ, USA) were provided for home cage enrichment.
2.4.2 Experimental procedure
Male subjects and female stimulus animals participated in three interactions, each with a different individual of the opposite sex. All trials were separated by between 5 and 7 days. All interactions took place during the dark phase of the light cycle, under red lighting at a consistent level of illumination and location, and in clean test arenas measuring 45cm x 28cm x 13cm (MB1, North Kent Plastics Ltd., UK). Animals were all handled and transferred between test arenas and home cages using clear acyclic tunnels to minimise stress and anxiety during testing (Hurst and West, 2010). Interactions lasted 30 minutes and took place in a room separate from the rest of the colony. Benchkote paper (Fisher Scientific, Nottingham, UK) was placed on the base of each test arena to assist in the recording of scent marks.