CAPÍTULO IV: CONCLUSIONES Y RECOMENDACIONES
6.1 Organización interna
Subordinate Female Naked Mdle-Rats
3.2.1 Aim and In troduction Aim
In v estig a te th e role of odour cues from th e p a re n t colony in conjunction with behavioural cues from colony members in th e su p p re ssio n of rep ro d u ctio n in female naked m ole-rats
In tro d u ctio n
Within th e naked m ole-rat colony th e re is only one breeding female (Faulkes e t al., 1990a). The rem aining n o n -b re e d e rs a re rep ro d u ctiv ely q u iescen t w hilst in th e colony environm ent b u t a re all capable of rep ro d u cin g as shown by th e rap id o n set of te stic u la r and ovarian a ctiv ity in n o n -b reed in g males and females when removed from th e ir colony and housed singly, or with a member of th e opposite sex (Faulkes and Abbott, 1993). This implied th a t a mechanism involving co n tact with th e colony was responsible for th e rep ro d u c tiv e su p p ressio n . Social fa c to rs have been shown to s u p p re ss rep ro d u ctio n of conspecifics in mammalian species via two mechanisms (i) Behavioural dominance and (ii) chemical cues (’prim er pherom ones’). This p re s e n t section in v estig ated th e role of th e se two fac to rs in th e social su p p ressio n of rep ro d u ctio n in su b ô rd in a te female naked m ole-rats.
In o rd e r to in v estig ate w hether chemical signals contained in soiled litte r from th e toilet cham ber (i.e. u rine, faeces, body chemicals) were resp o n sib le fo r th e su p p ressio n of rep ro d u ctio n in male and female naked m ole-rats, Faulkes and Abbott, (1993) tra n s fe rre d , on a daily basis, soiled litte r and bedding m aterial from th e p a re n t colony to sin g ly -h o u sed n o n -b reed in g male and female naked m ole-rats. T ran sfer of chemical signals from th e p a re n t colony n e ith er delayed nor
p rev e n ted th e o n set of rep ro d u c tiv e activation in male and female naked m ole-rats compared to a control group. The re s u lts su g g ested th a t chemical signals alone were not a major cause of rep ro d u c tiv e su p p re ssio n in th e naked m ole-rat colony (see also Marsden and Bronson, 1964; Bruce, 1969; Drickamer, 1974; Lawton and Whitset, 1979 fo r examples of ’odour tr a n s f e r ’ experim ents in ro d en ts assessin g th e effect of odour cues alone from one individual o r a group of individuals on th e rep ro d u c tiv e physiology and behaviour of conspecifics).
Previous stu d ies have shown th a t although chemical cues may reg u la te rep ro d u ctio n in ro d en ts, th e reg u latin g effect is often more effective if th e chemical cues were p resen te d in conjunction with additional se n so ry cues such as tactile or visual cues. In ro d en ts, chemical signals from an unfam iliar male have been shown to cause im plantation failu re in newly-m ated female field voles and meadow voles, b u t th e implantation failu re was g re a te r if physical contact with th e male was allowed in addition to th e chemical cues (Milligan, 1976; Storey, 1986, 1990). Similarly, in ju v en ile female mice th e combination of tactile cues with chemical cues from male mice, was shown to be more effective in promoting th e o n set of p u b e rty th an chemical cues alone (Bronson and Maruniak, 1975). The p re se n t stu d y in v estig ated th e role of odour cues in conjunction with behavioural cues from colony members in th e su p p ressio n of female rep roduction. The p re s e n t experim ent was divided into two phases. Phase I was a control phase d u rin g which th e behaviour and ovarian activ ity of all females in th e home colony was monitored. The second phase was an experim ental phase d u rin g which e ig h t n o n -rep ro d u ctiv e females were removed from th e ir p a re n t colony, housed in th e ir own mini-colony and maintained in olfactory contact with th e p a re n t colony whilst a t th e same time being in social contact with all members of th e p a re n t colony except th e b reeding pair. In th is way th e e ffe ct of b eh av io u ral/co n tact cues from th e breeding p air on su p p ressio n of rep ro d u ctio n in th e sep arated females was a d d re ssed as d istin c t from th e effect of behavioural cues from th e n o n -b ree d ers. Behavioural and ovarian a ctiv ity of th e perm anently sep arated females was monitored d u rin g th e experim ental phase in o rd e r to in v estig ate th e effect of only sep aratin g females from d irec t physical and behavioural contact with th e breeding pair w hilst maintaining chemical contact with
th e p a re n t colony and physical and behavioural contact with all th e n o n -b re e d e rs.
3 .2 .2 Methods 3.2.2.1 Animals
A to tal of 72 animals were used from two naked m ole-rat colonies 2200, (n=36) and 1800 (n=36).
3.2.2.2 Determination of ovarian s ta tu s in females
Ovarian a ctiv ity and p reg n an cy was assessed in all females th ro u g h o u t th e experim ent by determ ining concentrations of u rin a ry p ro g estero n e (ng/m g Cr; see C hapter 2, sections 2.3.1 and 2.3.2). U rine samples were collected a t least twice weekly as d escribed in C hapter 2, section
2.2.1.
3.2.2.3 Experimental protocol
The experim ent was divided into two phases:
Phase I (Control p hase - 80 days) The animals were u n d istu rb e d in th e home colony th ro u g h o u t th e control phase I except fo r m easuring body w eights. The body weight of all males and females was reco rd ed e v e ry two weeks. Behavioural ob serv atio n s were made on all animals u sing th e same observation protocol and time schedule as in phase II. The re s u lts were used to c o n stru c t a dominance h iera rch y fo r animals in th e colony (see section 3.2.2.3) On th e basis of th e dominance h ierarch y , th e animals were divided into th re e equal groups: Group (I) contained th e top th ird most dominant animals, group (II) th e next th ird most dominant animals, and group (III) th e least dominant animals. N on-breeding females for separation were chosen from g ro u p s (II) and (III).
Determination of th e b reeding p air
B reeding Female : T here was only breeding female in both colonies stu d ied . This animal was th e only female to have a p e rfo ra te vagina, to exhibit ovarian activ ity as reflected in u rin a ry p ro g estero n e levels (mean concentration + /- s.e.m. of u rin a ry p ro g estero n e fo r p hases I and Ilfo r th e two queens was 31.42 + /- 4.8 ng/m g Cr) and to give b irth d u rin g th e experim ent (see F ig u res 3.2.1a and 3.2.1b). Each queen was also th e most dominant animal in th e colony as determ ined by behavioural o b serv atio n s of over / u n d e r passage sco res (Faulkes, unpublished re su lts). As described previously by Reeve and Sherman, (1991), th e queen also exhibited th e h ig h est freq u e n cy of shoving behaviour (1.8 p er hour v e rs u s 0.1 p er hour fo r th e n o n -b ree d ers).
Male : The breeding male was identified by th re e methods: (i) th e male which showed th e h ig h est ra te s of ano-genital nuzzling with th e breed in g female d u rin g phase one of th e experim ent (rate s of ano genital nuzzling were 1.2 per h our for th e breeding male v e rs u s for 0 n o n -b reed in g males; Ja rv is 1991; Lacey and Sherman, 1991), (ii) from previous o b serv atio n s of mating with th e breeding female and (iii) u rin a ry testo ste ro n e determ inations made previously. In both colonies, th e breeding male was th e second most dominant animal in th e colony a fte r th e queen, as computed from th e dominance m atrices.
All o th e r animals were defined as n o n -b ree d ers. R eproductive quiescence was confirmed in females by levels of u rin a ry p ro g estero n e < 2ng/mg Cr Phase II (Experimental phase - 80 days) (see F igure 3.2.2) Eight non breeding females were perm anently sep arated from th e ir p a re n t colonies on day 0. Of th ese su b je c t females, four were from group (II; num bers 25, 30, 40, 53) and fo u r from group (III; num bers 38, 41, 45, 46) in th e ir p a re n t colony as determ ined from th e dominance h ierarch y . At any one time, two se p ara ted females from one p a re n t colony were involved in experim ental phase II. Each perm anently sep arated female was housed in an individual mini-colony (experim ental colony) and k ep t in chemical and social contact with th e ir p a re n t colony, excluding d irec t contact with th e b reeding pair. The mini-colony comprised 2m of perspex tu b in g linking th re e perspex cham bers (15 x 15 x 15cm).
u CD
i
c F ig . 3 .2 .1 .a Q u e e n 1 8 0 0 I II 100 n Birth Birth 8 0- 6 0- 40 - 2 0- 20 40 60 80 -80 -60 -40 -20 0Days from Start o f experimental phase II
ea o a. >» F ig . 3 .2 .1 .b Q u e e n 2 2 0 0 100-, 8 0- Birth 6 0- 40 - 2 0- 20 40 60 80 -80 -60 -40 -20 0
Days from start of experimental phase II
F ig u re 3.2.1 U rin ary p ro g estero n e co n cen tratio n s (ng/m g Cr) fo r th e Queen in colony (a) 1800 and (b) 2200 d u rin g control p hase I and experim ental p h ase II
Chemical contact was maintained by th e daily ro tatio n of approxim ately 500ml soiled bedding and litte r material between th e nest, food and toilet cham bers from th e p a re n t colony, and th e experim ental colony, between 8-9am. All naked m ole-rats typically u rin a te and defaecate in th e designated toilet chamber. By tra n s fe rrin g soiled m aterial from th is cham ber of th e p a re n t colony in p articu lar, it was th o u g h t th a t any possible u rin a ry or faecal ’prim er pherom one’ would be tra n s fe rre d to th e perm anently sep arated females.
Social contact was maintained by th e rotation of pre-d eterm in ed and fixed g ro u p s of 4 n o n -breeding animals (balanced for g en d er and social sta tu s ), e v e ry a lte rn a te day, between th e p a re n t and experim ental colony. The non -b reed in g animals in th e rotation gro u p s were re fe rre d to as n o n -b re e d e rs ’on ro tatio n ’. Excluding th e breeding pair, th e re were 32 animals, ie. e ig h t groups of fo u r animals available fo r rotation from each of th e two p a re n t colonies. As a group rotation o ccu rred e v e ry a lte rn a te day, it allowed each ro tated group to be in th e experim ental colony with th e perm anently sep arated female fo r 48 hours. Over a period of 16 days, th e perm anently sep arated female th e re fo re en countered e v e ry member of h er colony except th e breeding pair. Each such 16-day period was defined as one complete animal rotation. Since th e re were two su b je c t females perm anently sep arated from one p a re n t colony a t any one time, each n o n -b reed in g female ’on ro tatio n ’ would spend 48 hour in th e mini-colony of a se p arated female, and th en 6 days later, 48hr in th e mini-colony of th e o th er sep arated female, per 16-d ay period.
Phase II continued for five animal ro tatio n s (80 days), or until fighting or death of th e perm anently sep arated females forced th e experim ent to term inate.
Figure 3.2.2 Protocol of experim ental phase II, where day 0 is th e f ir s t day of phase II and AR = Animal rotation, O = Behavioural observations,
ZÆCZ2Ü3 = soiled litter and ^ ^ ^ = g roups of naked m ole-rats on rotation*.
DAY PROCEDURE PARENT COLONY COLONY OF
SEPARATED E c a E 8
o
AR FEMALE - — — 0 -> AR 0 ARThe body weight, behaviour and ovarian a ctiv ity of all eig h t perm anently sep arated females were monitored th ro u g h o u t phase II. The body w eight of all n o n -b reed in g females ’on ro tatio n ’ was reco rd ed e v e ry two weeks and th e ir ovarian activ ity monitored as outlined in section 3.2.2.2. Behavioural o b serv atio n s were made on ro tated females as d escrib ed in section 3.2.2.4.
3.2.2.4 Behavioural analysis
3.2.2.4(a) D istribution of behaviour o bservations
Behavioural ob serv atio n s employed a focal animal sampling tech n iq u e of 10 m inute observation periods between 8 - 10am, and 3.30 - 5.30pm. Each focal animal was ob serv ed fo r 10 m inutes am and 10 minutes pm and th e scores combined to give a 20 minute ’daily sco re’ balanced for time of day. The freq u en cy and duration of th e behaviours listed in section 3.2.2.4 (b) as perform ed by th e focal animal were reco rd ed on a check sh eet to g e th e r with th e id e n tity of an y in te rac tin g animal.
PHASE I : th e d istrib u tio n of behavioural ob serv atio n s fo r each female in control phase I, were equivalent to th o se o ccu rrin g in th e experim ental phase II. By quantifying reco rd ed over / u n d e r passage sco res a dominance matrix was c o n stru cted fo r all animals in each p a re n t colony (C.G. Faulkes, unpublished re s u lts ). An animal was given a sco re of 1 each time it passed over th e top of a n o th er animal, initiated a shove, initiated a jaw lock or d rag g ed an o th er animal by th e tail (see section 3.2.2.3(b). The position of th e animals in th e dominance matrix was re -a rra n g e d so th a t th e animals were o rd ered in such a way so th a t th e sco res above th e diagonal (top left to bottom rig h t), were maximised, whilst placing each animal as high as possible. Using th is method, for colony 2200, 81 % of th e scores fell above th e diagonal and fo r colony 1800, 83 % of th e scores. From such dominance m atrices th e position of each animal in a colony’s social h iera rch y was determ ined.
PHASE II : Focal animal o b serv atio n s were c arried o u t in th e morning and afternoon on th e a lte rn a te days when an animal rotation
was not o ccu rrin g (see F igure 3.2.2). Focal animal o bservations were c arried out on all females which were in th e experim ental colony of th e perm anently sep arated female on th e p a rticu la r observation day. O bservations were no longer c arried o u t on females when th ey were in th e p a re n t colony. The perm anently sep ara ted female was th e re fo re