Protocols for field and laboratory rodent studies - HAL

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Protocols for field and laboratory rodent studies3.1.2. Sex determinationMale and female can be differentiated byobserving their external reproductiveorgans: presence of scrotum coveringtesticles in adult males and of the vaginain females (Fig. 37). Nevertheless, juvenilemales have their testes inside the bodyand can be confused with juvenilefemales. Differentiation can be done byobserving the distance between the anusand the genitals, which is greater in malesthan in females. If the identification cannotbe done using external morphologicalfeatures, the internal sexual organs shouldbe observed at the end of the dissection.Figure 37: External comparison of male and female reproductive organs(Photo: Herbreteau V.)● Females:- Vagina: the vaginal opening is covered by a translucent skin in juvenile females. Record if the vagina is“not perforated” (Code = 1), or “perforated” (Code = 2).- Teats: record if teats are “barelyvisible” (hardly distinguished andenumerated, Code = 1) or“prominent” (Code = 2).- Mammary formula (Fig. 38): itcorresponds to three parts of thebody, from the top to the bottom:pectoral + post-axillary + inguinalThis formula can help to distinguishdifferent species (Aplin et al., 2003;Marshall, 1988).- If the female is pregnant, mentionthe number of embryos on eachside. Figure 38: Position of teats, example of Bandicota indica (1+2+3)(Photo: Herbreteau V.)● Males:- Indicate if the testicles are “inside” (i.e.abdominal = juvenile, Code = 1), or outside(i.e. descended into the scrotal sac = adult,Code = 2).- Measure the testicle length after thedissection (Fig. 39). If the testicles are in thescrotum, pull one out of it before measuring.The size of testicles differs between speciesand regarding the sexual activity.- Indicate if the seminal vesicles aredeveloped (visible) or undeveloped (Fig.40). This informs on the sexual activity ofmales.Figure 39: Measurement of testiclelength (Photo: Herbreteau P.)Figure 40: Developedseminal vesicles in an adultmale (Photo: Herbreteau P.)223.1.3. Maturity determination and development stageThe determination of sexual maturity (immature / mature) and that of the development stage (juvenile / sub-adult /adult) are both necessary to understand the ecological traits of an animal as well as to interpret epidemiologicalresults. Also it can help to correctly identify an animal to the species level. In some case, juveniles could bemisidentified with adults from another species. Figure 41 shows such difficulty with an adult Rattus exulans and ajuvenile Rattus tanezumi, which look like similar in size and shape. However external observation can help todistinguish juveniles from adult individuals. Juveniles usually have a soft fur and their skull, ears and feet arelarger (relative to their body size) than those of adults.The observation of the sexual organs is highly informative to assess their sexual maturity:● Juveniles:- Juvenile females: the vagina is not perforated and teats are barely visible.- Juvenile males: testes are abdominal (inside the body cavity), the scrotal sac is not apparent.
3- Identifying rodents● Sexually mature individuals:- Females: the vagina is perforated and teats areeasily visible. Verify the presence of embryos orscars in the uterus.- Males: testes are descended into the scrotalsac; seminal vesicles are well developed whenthe animal is sexually active. In rare cases,testicles can be inside the body even if thescrotum and the seminal vesicles aredeveloped: note these observations in the datasheet.Finally, the close inspection of the dentition is alsoindicative of the development stage of a rodent.However it can only be performed on a skull perfectlycleaned up. In particular the growth of the thirdmandibular molar (M3) is a useful criteria. The M3growth can be divided in at least three stages (Fig. 42).This information is interesting because in some casesrodents start to reproduce before the end of theirmorphological development. The stages subadult/adultare thus not fully congruent with sexual maturity (Aplinet al., 2003). The observation of the M3 growth gives agood idea of the age structure of a population and thenof its demography. Another popular method to reachthis goal is the use of the weight of the crystalline lens(Rowe et al., 1985). We then recommend estimatingseparately the sexual maturity and the developmentstage of the rodents (Fig. 43).Figure 41: Comparison between an adult female Rattusexulans (C0207, above) and a juvenile female Rattustanezumi (C0206, below), from Veal Renh in SouthCambodia (Photo: Herbreteau V.)Figure 42: Different stages of the thirdmolar (M3) growth (Photo: Chaval Y.)Figure 43: Decision tree to determine the maturity of a rodent, by consideringthe third molar growth and sexual organs externally3.1.4. Data recording and species identificationExternal description of each animal should be recorded during the dissection work on a paper sheet. Then recordthe data in the computer as soon as possible. This will allow a safe storing of the data, on two different supports.Notice that recording the data directly on the computer may be risky as a power outage or a computer failure isnot unlikely. In such case, one can get back the paper support and data are not lost.We provide in appendices two models of data sheet that can be used during measurement and dissection ofrodents. Appendix 2 aims at a detailed description of each animal, while Appendix 3 allows description of 10individuals with less information. First, identification numbers should be attributed to the animal and the trappingplace. The name of species should be indicated with a question mark if the identification is dubious. Differentdates should be given to indicate the days of capture, of death and of dissection. A short description of thetrapping place should be given, in addition to its detailed description in another data sheet (Appendix 1).Measurements and every sample collected (organs, helminths and ectoparasites) should also be indicated.23
Page 3 and 4: Protocols for field and laboratory
Page 7: Background: CERoPath projectCERoPat
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Page 21 and 22: 2- Dissecting rodentsTris-EDTA buff
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Page 27 and 28: 2- Dissecting rodentsCryostabilates
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Protocols for field and laboratory rodent studies - HAL

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