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Mastozoología Neotropical ISSN: 0327-9383 [email protected] Sociedad Argentina para el Estudio de los Mamíferos Argentina

Villamizar-Ramírez, Ángela M.; Serrano-Cardozo, Víctor H.; Ramírez-Pinilla, Martha P. REPRODUCTIVE ACTIVITY OF A POPULATION OF meridensis (RODENTIA: ) IN COLOMBIA Mastozoología Neotropical, vol. 24, núm. 1, julio, 2017, pp. 177-189 Sociedad Argentina para el Estudio de los Mamíferos Tucumán, Argentina

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Artículo

REPRODUCTIVE ACTIVITY OF A POPULATION OF Nephelomys meridensis (RODENTIA: CRICETIDAE) IN COLOMBIA

Ángela M. Villamizar-Ramírez1, Víctor H. Serrano-Cardozo1, 3, and Martha P. Ramírez-Pinilla2, 3

1 Laboratorio de Ecología, Escuela de Biología, Universidad Industrial de Santander, Bucaramanga, Santander, Colombia. [Correspondence: Víctor H. Serrano-Cardozo ] 2 Laboratorio de Biología Reproductiva de Vertebrados, Escuela de Biología, Universidad Industrial de Santander, Bucaramanga, Santander, Colombia. 3 Grupo de Estudios en Biodiversidad, Escuela de Biología, Facultad de Ciencias, Universidad Industrial de Santander, Bucaramanga, Santander, Colombia.

ABSTRACT. We studied the annual reproductive activity of a population of Nephelomys meridensis in an Andean oak forest in the Cordillera Oriental of Colombia. Monthly during a year, Sherman live traps were established in 5 fixed stations (20 traps per station) during 4 nights per month, along an altitudinal rangeof 2530-2657 m. Reproductive condition was established in adult by external morphology including nipple development, distension, and features of the vaginal opening. Adult females were categorized as pregnant or in . Additionally, reproductive condition was complemented with the analysis of vaginal smears of each female with open and with a general description of the for some females in captivity. Females of this population of N. meridensis reproduce continuously throughout the year, and females were pregnant, in lactation or in estrous several months. The presence of postpartum estrus both in captivity and in the field was observed. Under captivity conditions, the estrous cycle can last more than5 days because some phases were extended more than 2 days. No relationship was found between the frequency of reproductive females in each reproductive state and the monthly average of rainfall or with the variation in the environmental availability of food ( abundance and oak fructification). Therefore, in this popula- tion of N. meridensis females have an aseasonal polyestric pattern suggesting a constant offer of environmental resources for reproduction.

RESUMEN. Actividad reproductiva de una población de Nephelomys meridensis (Rodentia: Cricetidae) en Colombia. Estudiamos la actividad reproductiva anual de una población de Nephelomys meridensis en un bosque andino de roble en la Cordillera Oriental de Colombia y otros aspectos reproductivos de esta población. Mensualmente y por un año se establecieron 5 estaciones fijas en un intervalo altitudinal de 2530 a 2657 m; en cada una se ubicaron de manera aleatoria 20 trampas Sherman. La condición reproductiva se estableció en hembras adultas por morfología externa del desarrollo de las mamas, la distensión abdominal y la de la abertura vaginal; las mismas se categorizaron como preñadas o en lactancia. Adicionalmente se complementó el análisis de la condición reproductiva con el análisis de frotis vaginales realizados a cada hembra con vagina abierta y con la descripción a manera general del ciclo estral de hembras en cautiverio. Las hembras de esta población de N. meridensis se reproducen continuamente a lo largo del año. Se encontraron hembras preñadas y en lactancia casi todos los meses y hembras en estro en varios meses. Se determinó la presencia de estro post-parto tanto en cautiverio como en campo. Bajo las condiciones de cautiverio, el ciclo estral puede durar más de 5 días

Recibido 13 julio 2016. Aceptado 20 marzo 2017. Editor asociado: M Busch 178 Mastozoología Neotropical, 24(1):177-189, Mendoza, 2017 AM Villamizar et al. http://www.sarem.org.ar - http://www.sbmz.com.br debido a que algunas fases se extienden por más de 2 días. No se encontró una relación entre la frecuencia de hembras reproductivas en cada estado y la variación en el promedio mensual de lluvias, ni tampoco con la variación en la oferta ambiental de alimento (abundancia de artrópodos y fructificación del roble). Por tanto, en esta población de N. meridensis las hembras tienen un patrón de poliestría no estacional lo que sugiere una oferta ambiental constante de recursos para la reproducción.

Key words: Andean forest. Environmental factors. Nephelomys meridensis. Reproduction.

Palabras clave: Bosque andino. Factores ambientales. Nephelomys meridensis. Reproducción.

INTRODUCTION al., 2005; Yener et al., 2007; Caligioni, 2009; Byers et al., 2012; McLean et al., 2012), but is Reproductive activity in small has poorly known in wild . However, it has been documented mainly for species living been studied under laboratory conditions in in temperate latitudes, where factors such as Mastomys natalensis (Johnston and Oliff, 1954), photoperiod, resource availability, environ- Baiomys taylori (Hudson, 1974), Peromyscus mental temperature and precipitation influence californicus (Gubernick, 1988), and Proechimys reproductive patterns (Bronson, 1985). chrysaeolus (Sabogal-Guáqueta et al., 2013), There are few studies in the tropics where among others. The estrous cycle consists of most of the mammals live; therefore, a big four phases called proestrus, estrus, metestrus gap in the understanding of the regulation of and diestrus; each distinguished by the type reproduction by environmental factors exists for and amount of vaginal epithelial cells, and the most of the tropical mammals (Bronson, 2009). presence or absence of leukocytes in the vagina Several studies suggest that reproduction in (Yener et al., 2007; Byers et al., 2012.). tropical rodents is continuous (Lacher, 1992). Nephelomys meridensis is a of the This is the case in capito (Fleming, Cricetidae family, formerly in the “albigularis” 1971), O. nigripes, O. trinitatis (Fonseca and group of Oryzomys (Weksler et al., 2006), Kierulff, 1989), Proechimys semispinosus, Hoplo- N. meridensis is distributed from the Northeast mys gymnurus (Alberico and González-M., of the Cordillera Oriental of Colombia in the 1993) and Nyctomys sumichrasti (Romero and Department of Boyacá to the North of the Timm, 2013), among others. However, there is Sierra de Mérida in , at elevations of evidence of reproductive peaks during either 1100-4000 m (Percequillo, 2015). Individuals the highest or the lowest precipitation seasons of the Nephelomys are terrestrial, so- (Lacher, 1992; Bronson, 2009); for example, cial and nocturnal; they take refuge inside Oryzomys intermedius, Trinomys iheringi, fallen trees and rock formations covered with squamipes have their reproductive moss, and food includes fruits, peaks during the peak of the rainy season and seeds (Rivas, 1997; Gibson, 2014). Díaz (Bergallo and Magnusson, 1999); in Akodon de Pascual (1981) mentioned that the diet of cursor the reproductive peak starts at the end O. albigularis includes 50% endocarp of guama of the rainy season and goes until the end of fruit (Inga sp.), 20% larvae, 20% palm the dry season (Fonseca and Kierulff, 1989). seeds, 2% fungi, and 2% flowers of Heliconia sp. Then, seasonal reproductive activity in tropi- No additional detailed information is available cal rodents would be related to the intensity on the natural history of this species. and duration of the rainy and dry seasons and We studied the annual reproductive activity its relation to the availability of resources for of females and other aspects of the reproduc- reproduction. tion of a population of Nephelomys meridensis The estrous cycle has been widely studied in living in an Andean forest with a bimodal laboratory mice and rats (e. g. in Hubscher et regimen of rains. The study contributes to the Nephelomys meridensis REPRODUCTIVE BIOLOGY 179 knowledge of reproduction and its relationship mula C.S. = (I.C / C.E) x 100, where: S. C = capture with environmental factors in small mammals success, I.C. = Number of individuals captured and that inhabit upland forests in the Neotropics. C.E = capture effort (Steinmann et al., 2003; Gallina et al., 2008). MATERIALS AND METHODS Reproductive activity This work was done in an Andean oak forest in the Cordillera Oriental of Colombia at an altitude of From morphological measurements, weight and 2600 m, in Santa Barbara municipality, Santander, head-body length (HBL), and hair dorsal color of the Colombia (7º 01’ 9.9’’ N, 72º 53’ 33.6’’ W). The individuals captured in this study, we defined three forest is characterized by the dominance of trees categories of age: adults, sub-adults, and juveniles of Quercus humboldtii, followed by Clusiaceae and (Table 1); the latter two were taken as a category Rubiaceae and the presence of trees with an average of immature individuals. Reproductive condition height of 5 m and a diameter of 7.3 cm. The area has was established only in adult females by observ- a bimodal rainfall regime, with two periods of high ing external morphological characteristics such as rainfall from April to June and from September to nipples development and the state of the vagina November, and two periods of low rains, from July (closed, open or vaginal plug), and the analysis of to August and from December to March; the mean samples of vaginal swabs for determining the phases annual rainfall is 1214 mm (Worldclim - Global of the estrous cycle. Climate Data). Adult females were categorized as pregnant (dis- A monthly sampling was conducted from October tended , partially open vagina or vaginal 2014 to September 2015 (except February). For each plug with developed nipples) and lactating (open sampling, 5 fixed stations spaced approximately every vagina with developed nipples and milk production). 100 m were established along one transect in an al- The vaginal plug is a mixture of , secretions titudinal range from 2530-2657 m, covering an area from the accessory sex glands in males and vaginal of 1.84 hectares. In each station 20 Sherman traps secretions in the vagina that coagulate forming a (23 x 9 x 7.7 cm) were randomly placed above the “plug” of dry texture and relatively hard; this cap ground, for a total of 100 traps, which were active seems to prevent the female to be copulated by for 4 consecutive nights each month. Every day, traps another male (Benavides and Guénet, 2003). The were baited with a mixture of ground peanuts, oat presence of vaginal plug in females was seen as an flakes, ground peel corn, vegetable fat and essence indication of recent intercourse. of vanilla, shortbread or banana, and revised in the Vaginal smears were made on each female with morning and evening. open vagina by vaginal washing with 4.5 μl of saline For each individual captured we recorded sex, solution (0.9%) introduced into the vaginal canal weight (body mass) with a Pesola (100 g spring with a micropipette Accumax Pro (0.1-10μl). The scale ± 0.5 g), and standard external measurements solution was collected and placed on a glass slide, with a dial caliper calibrated to the nearest 0.1 mm was dried at ambient temperature and fixed by (Total length [TL], head-body length [HBL], tail adding a few drops of ethanol (70%). The samples length [TL], ear length [EL] and hind foot length were stained with eosin and methylene blue (10%), [HFL]). Mice were labeled by injection in the back observed under a microscope (Nikon Eclipse 55i®) of an intradermal microchip 5 mm (Biomark®) and photographed with a Canon EOS Rebel XS® disinfected with iodine solution. The were camera. A duplicate of each wash was performed released in the same place of capture; however, some to ensure best results. Those females that showed individuals were collected to corroborate taxonomic no morphological features of or lactation determination with the proposed key of Percequillo states were characterized by the results of vaginal (2015) for the genus Nephelomys. These individuals epithelium analyses. They were found in proestrus, were deposited in the collection of mammals of estrus or metestrus and were considered as repro- the Museo de Historia Natural of the Universidad ductive females since there is hormonal stimulation Industrial de Santander. This research followed ASM for each phase and a clear indication that they are guidelines (Sikes et al., 2011). sexually receptive. The capture effort was calculated by multiplying To determine the estrous cycle, during each the number of traps installed in the study area sampling month we took 1 or 2 females with open by the number of nights per sampling month. In and one adult male. They were kept in addition, capture success was calculated by the for- captivity for a period of 4-6 days. Each individual 180 Mastozoología Neotropical, 24(1):177-189, Mendoza, 2017 AM Villamizar et al. http://www.sarem.org.ar - http://www.sbmz.com.br

Table 1 Categories of age in males and females of Nephelomys meridensis based on sex, Head Body Length (HBL), body weight and fur description of the individuals captured in this study.

Age category Sex HBL (mm) Body Weight (g) Dorsal fur Males 148.6±10.4 74.1±9.8 (N=40) Abundant and brown Adults Females with ocher tips 143.5±6.5 65.7±9.3 (N=28)

Males 126.3±3.2 52.6±10.1 (N=3) Less abundant and brown Sub-adults Females with some ocher tips 124.6±4.5 45±6.2 (N=3)

Males 109±8 35.6±5.5 (N=3) Sparse and dark gray Juveniles Females without ocher tips 106.5±9 34.3±12.4 (N=10) remained separately in transparent plastic contain- by Montenegro-Díaz et al. (1991), among others. ers (34.5; 24; 24.5 cm), which were adapted with a However, males were removed from the analysis layer of dry leaves on the bottom, and 80-90 holes because the position of scrotal testes changed rapidly opened in the lid to allow air flow. Individuals were to abdominal during manipulation; therefore, fed with few grains of almonds, unsalted peanuts, it was determined that the position of the testes is and small oak acorns and provided with water ad an unreliable indicator of male reproductive activity. libitum. Every other day each female was placed next to the male (with no chance of intercourse). Environmental food resources During the captivity, every day a vaginal smear was The collection of food resources (arthropods) was obtained from each female; the samples were treated done monthly; for this, pitfall traps were installed in the same manner as described above. After the at each station, made up of 10 plastic cups of 16 oz days of captivity, individuals were released at the with a mixture of soap and water during 96 hours site of capture. each month. The individuals collected were stored The phases of the estrous cycle differed quali- in ethanol (70%); they were subsequently identified tatively considering the type of vaginal epithelial to the taxonomic level of order and weighed for wet cells present in each sample (Hubscher et al., 2005; biomass and then dried for obtaining dry weight Yener et al., 2007; Caligioni, 2009, Byers et al., (Pesola® M500, 500g d = 0.1g). Evaluation of food 2012; McLean et al., 2012). Having identified the resources also included the vegetable supply; we only estrous phase in each female, their characteristics included the fruits of the most common tree, the and duration were determined. In addition to the oak Quercus humboldtii. We made visual tracking of recording and analysis of vaginal smears, we took their fructification time during the sampling visits; photographs (Nikon Coolpix l330® Camera) to the we consider the peak of fructification when most of vaginal opening of every female to observe changes the trees in the study area have fruits. The hypothesis related to the estrous cycle (Byers et al., 2012). we have was that the peak of reproductive events For adult males we tried to determine the re- concurs or is close in time with the peak of food productive activity by the position of the offer (arthropod abundance and oak fructification). as has been considered in other studies (reproduc- tive males with scrotal testes and non-reproductive Data analysis males with abdominal testes), like in Hoplomys gymnurus and Proechimys semispinosus by Alberico A binomial test was used to determine significant and González-M (1993) and Rhipidomys latimanus differences from a 1:1 sex ratio (software R v3.3.1, Nephelomys meridensis REPRODUCTIVE BIOLOGY 181

2016); to establish whether there was significant 2015). Because the relationship between environmen- difference between the abundance of males and tal and resource factors may influence reproduction females over time, we used a G-test. To evaluate later on, we used Spearman correlations (software R the possible existence of significant differences v3.3.1, 2016; Hmisc package, Harrell and Charles, between sexes in body size in adult individuals, 6 2015) with the corresponding month and the pre- variables were compared: body weight and 5 standard vious month (Antunes et al., 2009). Statistical tests morphological measurements, these were tested by (Mann-Whitney U, t-student and Kruskal-Wallis) t-student test or nonparametric Mann-Whitney U were performed in STATISTICA 7.0 (StatSoft, 2004) test. A index was calculated for software with a significance level of 0.05. G-tests were variables that showed significant differences between implemented using the software R v3.3.1 (2016) with adult males and females (Schulte-Hostedde, 2007). RVAideMemoire package (Hervé, 2017). In neither In addition, we investigated whether there was sig- of these tests June was included because only one nificant variation in body weight over time using a individual was captured. Kruskal-Wallis test. To determine whether there were significant differ- RESULTS ences between the number of adults with respect to immatures obtained for the entire sampling period A total of 87 individuals were captured in 130 we used a Mann-Whitney U test, and to determine capture events. Seventeen individuals were whether there were significant differences in their recaptured, including 13 adults (8 males and distribution between months a G-test was employed. 5 females), 1 sub-adult female and 2 juvenile The monthly reproductive activity was taken as the females. The number of times in which an number of pregnant, lactating, and reproductive individual was recaptured ranged from 1 to 7. females (according to the characterization of the The total sampling effort was 4400 trap nights ). Significant differences in the and the success capture was 1.97%. Table 2 abundance of each reproductive state over time shows the reproductive stage of captured and was tested by G-test (June and August were not recaptured females; based on these data the included because only one individual was captured period can be estimated between 28 each month). To determine whether there was an association be- and 32 days, and sexual maturity in females tween the monthly average of rains and the monthly can be achieved at approximately 100 days. average of arthropods biomass with reproductive Adults were captured in larger numbers than activity, we used a Spearman correlation (software juveniles throughout the sampling period (68 R v3.3.1, 2016; Hmisc package, Harrell and Charles, adults, 40 males and 28 females; 19 immatures,

Table 2 Reproductive status found in recaptured females of Nephelomys meridensis during sampling. (**, female with post-partum estrus).

Reproductive state Reproductive state Age category # Female Days passed in the capture in the recapture 1 Early pregnant 27 Late pregnant 2 Early pregnant 27 Late pregnant Adults 3 Early pregnant 32 Lactating 4 Estrus 26 Estrus 5** Late pregnant 27 Late pregnant

Sub-adults 6 ----- 30 Adult, estrus

7 ----- 47 Early pregnant Juveniles 8 ----- 65 Lactating 182 Mastozoología Neotropical, 24(1):177-189, Mendoza, 2017 AM Villamizar et al. http://www.sarem.org.ar - http://www.sbmz.com.br

6 males and 13 females); this difference was the period in which late pregnant females significant (U = 11; P = 0.001). Significant dif- were found, while their lower weight was in ferences were also found over time, as adults September, October and November (the second were s more frequently captured between rainfall peak of the year, Fig. 1). October and March (G = 31.42; df = 9; n = 108; Sexual dimorphism showed significant dif- P < 0.0003) and immatures were found mostly ferences, with higher values in males than between April and September (G = 23.13; df = 9; in females in total length and body weight n = 21; P < 0.006, Fig. 1). (Table 3). The index of sexual dimorphism The sex ratio (46 males: 41 females) for the (F:M) was 0.97 for total length and 0.88 for entire sample was not significantly different body mass. from 1:1 (P = 0.66, binomial test). The abun- Reproductive activity dance of females remained constant during sampling (G = 7.98; df = 9; n = 62; P < 0.54) All adult females were found to be reproduc- with a slight decrease in January, March, and tively active throughout the year, either in September; the number of males varied sig- pregnancy (51%), lactating (20%), or reproduc- nificantly over time (G = 21.64; df = 9; n = 67; tive (29%, in the estrus or proestrus phases of P < 0.01), increasing between September and the reproductive cycle). Reproductive females March (Fig. 1). The mean body weight did not (as observed by vaginal smears) were found change over time in adult males (H = 14.25; between September and January, and its oc- df = 9; n = 61; P = 0.11), but varied in adult currence was significantly different between females (H = 26.76; df = 9; n = 47; P = 0.0015). months (G = 18.63; df = 8; n = 14; P < 0.02) Females tended to be heavier in March, April (Fig. 2). Seven females were in estrus in Sep- and May (the first rainfall peak of the year), tember, November, December, and January, and

Fig. 1. Body weight of adult females, males and immature individuals (juveniles and sub-adults) of Nephelomys meridensis. The shaded background area shows average monthly precipitation in the study area. Nephelomys meridensis REPRODUCTIVE BIOLOGY 183

Table 3 Sexual dimorphism. Comparison between sexes in body sizes (mm) and weight (g) of adults of all the sample of Nephelomys meridensis. Mean ± standard deviation, higher and lower intervals in parentheses, and probability of significance (P) of the t test or nonparametric homologous U Mann-Whitney. (* Significant probability).

Variable Male adults Female adults P Test

318.8±12.7 309±14.5 Total Length (287-345) (288-337) 0.01* T-student (n=38) (n=25)

148.6±10.4 143.5±6.5 Head Body Length (134-168) (132-155) 0.06 U Mann-Whitney (n=40) (n=28)

170.9±6.9 166.5±11.5 Tail Length (153-183) (147-186) 0.07 U Mann-Whitney (n=38) (n=25)

19.3±1.6 19.4±1.7 Ear Length (15,4-24,9) (16-22.8) 0.9 U Mann-Whitney (n=40) (n=28)

33.3±1.6 32.8±1.6 Hind Foot Length (30-37) (29-36) 0.17 T-student (n=40) (n=28)

74.1±9.8 65.7±9.3 Body weight (58-88) (50-86) 0.0004* T-student (n=40) (n=28)

Fig. 2. Proportion of pregnant, lactating, and reproductive females of Nephelomys meridensis and average monthly rain- fall throughout the sampling period. Numbers above bars denote the number of females in that month (captures and recaptures). Reproductive females captured in estrus (stars) and proestrus (circle). June and August were not included because only one individual was captured. 184 Mastozoología Neotropical, 24(1):177-189, Mendoza, 2017 AM Villamizar et al. http://www.sarem.org.ar - http://www.sbmz.com.br one female in proestrus in October (Fig. 2). predominate. Pregnant females were in the Pregnant females were in the metestrus phase. metestrus phase. In the vaginal smears no sperm was observed. A complete estrous cycle was observed in Pregnant females were found in almost every only 2 females, in which the three basic stages month except in June, August and September, of the cycle (proestrus, estrus, and metestrus) with no significant differences in their oc- were observed (Fig. 3). currence over time (G = 8.18; df = 8; n = 25; In estrus (Fig. 5 A and B) the vaginal open- P < 0.42). A higher proportion of late pregnant ing is wide and bordered by an inflamed tis- females was observed in April and May, when sue that has striations at the edges, while for their body weight ranged between 81 and 86 g; females in metestrus-diestrus (Fig. 5C and the remaining mid-pregnant females weighted D) the opening is small, smooth and without between 60 and 75 g. Lactating females were tissue inflammation. also captured during most sampling of the Reproductive activity and its relationship year without significant differences over time with rainfall and food resources (G = 7.43; df = 8; n = 8; P < 0.5). We found no correlations of reproductive Estrous cycle condition (number of females in different Nine females were maintained in captivity, of reproductive phases) and precipitation in the which 4 were pregnant and 5 reproductive fe- corresponding or the previous month (results males. In the latter, it was possible to observe not shown); among these, only the number of the different phases of the estrous cycle (Figs. 3 lactating females was marginally and negatively and 4). Each phase is clearly distinguishable correlated with precipitation (r = -0.61, P = 0.06). by the type and amount of cells of the vaginal Similarly, no significant associations were found epithelium, also by the presence or absence of between the dry weight of arthropods and the leukocytes (Fig. 4). numbers of pregnant, lactating and reproduc- Clusters of nucleated epithelial cells were tive females. found in proestrus (Fig. 4A); they can be para- Fructification of the oakQuercus humboldtii basal, intermediate and superficial. Cornified, in the study area showed two peaks, one from enucleated cells were also found but in smaller April to June and the other from August to proportion. In a female, in December, clusters December, which correspond to the two peaks of parabasal nucleated cells and some leuko- of rainfall. Few fruits were observed in the other cytes were observed, which was considered an months, suggesting a marked seasonal regime early proestrus. of fructification for this tree in the study area. Cornified cells predominated in the estrous The greatest proportion of late pregnant females phase although few superficial nucleated cells was found during the first peak of fructifica- were found. One female of November was con- tion of the year (April and May), but no other sidered as in early estrus since some cornified relationships of reproductive events and oak cells and nucleated cells were in similar pro- fructification peaks are apparent. portions. In captivity, the estrous phase lasted DISCUSSION up to five days, followed by metestrus of up to 2 days. In March, a female in captivity gave This paper describes a pattern of continuous to three pups and the vaginal smear taken polyestrous reproduction for a population of in the following day showed estrous features, N. meridensis in an Andean oak forest in the thus representing a postpartum estrus (Fig. 3). Cordillera Oriental of Colombia, this being the Metestrus (Fig. 4C) is distinguished by first report to the species. The studied popula- the presence of three cell types: nucleated tion of N. meridensis has continuous reproduc- cells, mostly cornified cells, and leukocytes. tion throughout the year. Females in different In contrast, during diestrus (Fig. 4D) the reproductive stages (receptive, pregnant, and same cell types are found but leukocytes in lactation) were found throughout the year. Nephelomys meridensis REPRODUCTIVE BIOLOGY 185

Fig. 3. Phases of the estrous cycle found in each female of Nephelomys meridensis kept in captivity. Female 1 to 5 with no evidence of pregnancy or lactation, female 6 pregnant. Recaptured female (*); pregnant female who had postpartum estrus (**). A complete estrous cycle for females 4 and 1* (proestrus, estrus, and metestrus or diestrus) was observed.

Fig. 4. Phases of the estrous cycle from vaginal smears in females of Nephelomys meridensis. (A) proestrus, (B) estrus, (C) metestrus, (D) diestrus. Nucleated cells (black arrow), cornified cells (black triangle) and leukocytes (white arrow). Scale bar 50 μm. 186 Mastozoología Neotropical, 24(1):177-189, Mendoza, 2017 AM Villamizar et al. http://www.sarem.org.ar - http://www.sbmz.com.br

Fig. 5. Morphology of the vaginal opening in the phases of the estrous cycle in Nephelomys meridensis. (A and B) estrus, (C and D) mestaestrus-diestrus.

Tracking successive recaptures of individu- The higher capture of mature individuals als in the field revealed a time to reach sexual than juveniles and their unequal distribution maturity in females of about 100 days and throughout the year observed in this population a gestation period between 28 and 32 days. of N. meridendis has also been seen in other These observations compare well with other Neotropical rodents (Rhipidomys latimanus, studies of tropical rodents, such as for example, Montenegro-Díaz et al., 1991; Peromyscus Rhipidomys latimanus (maturity 95 days, ges- mexicanus, Rojas and Rodríguez, 2007; tation 30 days; Montenegro-Díaz et al., 1991) , Oryzomys intermedius, and Thomasomys laniger (maturity 3.5 months, Trinomys iheringi and Akodon cursor, Bergallo gestation 24 days, López-Arévalo et al., 1993). and Magnusson, 1999; Nephelomys caracolus, However, Gentile et al. (2000) reported the age García et al., 2013). This pattern might re- of sexual maturity in Nectomys squamipes to be flect the more limited motility of juveniles, between 31 and 51 days, and 38.8 to 51.2 days as pointed out by Alberico and González-M in Akodon cursor. (1993), as well as short times from birth to These times are also variable when observed sexual maturity. in studies of wild rodents in captivity. For ex- The sex ratio observed here forN. meridensis is ample in Peromyscus californicus (maturity 44 nearly 1:1, as in some other Neotropical rodents days, gestation 31-33 days, Gubernick, 1988) (Rhipidomys latimanus, Montenegro-Díaz et al., and in Proechimys chrysaeolus (gestation 50-60 1991; Peromyscus mexicanus, Rojas and Rodríguez, days, Sabogal-Guáqueta et al., 2013). Within the 2007). There are several reports of deviations from genus Nephelomys, N. caracolus reaches matu- a 1:1 sex ratio in temperate rodents, such as the rity between 26 and 93 days, and gestation time Argentine rodent Salinomys delicatus (Rodríguez is 26.65 days (Moscarella and Aguilera, 1999), et al., 2012) and the North American Peromyscus values that are similar to those reported here. maniculatus borealis (Havelka and Millar, 1997). Nephelomys meridensis REPRODUCTIVE BIOLOGY 187

Generally, uneven sex ratios are associated receptivity (estrus) in those females that could with size differences. Often, male biased sex be considered as inactive when only external ratios are linked to larger males, in turn suggest- morphology is used to establish the female ing male-male competition and polygyny (e.g. reproductive state. Cornely and Baker, 1986; Schulte-Hostedde, Some studies suggest that many rodents in 2007). There are also reports of larger body the tropics have continuous reproduction due to sizes in females (Schulte-Hostedde, 2007), as favorable habitat conditions and opportunistic in the case of Salinomys delicatus (Rodríguez et life strategies, characteristic of rodents that have al., 2012) and Peromyscus maniculatus (Schulte- a short life expectancy, rapid sexual maturity, Hostedde et al., 2001). In N. meridensis males short gestation period and the presence of are larger than females in total body length and postpartum estrus (Bronson, 1985; Bronson weight, raising the possibility of polygyny and and Perrigo, 1987; Lacher, 1992; Gentile et male competition for the opportunity to mate. al., 2000). Some of these characteristics are Phases of the estrous cycle of rodents often determined from this study for the population change daily in captivity; however, colony com- of N. meridensis; the gestation period could position and density may change this pattern be between 28 and 32 days, sexual maturity (e.g. McLean et al., 2012; Sabogal-Guáqueta et in females could be reached at approximately al., 2013). Females of N. meridensis maintained 100 days of age, and females are continuous in captivity showed prolonged phases of estrus polyestrous and may have postpartum estrus. and metestrus. These phases were extended This is a common pattern in Neotropical in females even in presence of a male, but rodents. Oryzomys nigripes and O. trinitatisse was not allowed. Postpartum estrus breed throughout the year in the Atlantic forest was observed in a female in captivity about 12 of Brazil (Fonseca and Kierulff, 1989). In Pan- hours after giving birth, consistent with those ama, in a less seasonal tropical rainforest than reported in other rodents (Dewsbury et al., the Atlantic forest of Brazil, O. capito reproduc- 1977; Gubernick, 1988). es throughout the year (Fleming, 1971). In the Caligioni (2009) and McLean et al. (2012) plains of Venezuela, which are seasonally flood- noted that vaginal smears through 2-3 cycles ed savannas with a dry season from December were needed to determine the duration of the to April, O’Connell (1982) and Vivas (1986) estrous cycle. Our observations are indicative mentioned that Oryzomys bicolor ( of estrous cycles longer than 5 days, but these bicolor), Rhipidomys mastacalis (Rhipidomys results need to be corroborated with additional nitela) and brevicauda reproduce sampling efforts. Many studies of rodents in all year. Finally, Romero and Timm (2013) captivity show cycles to vary between 4 and found that in humid tropical lowland forests 20 days (e.g., Dewsbury et al., 1977; Guber- of Costa Rica, Nyctomys sumichrasti reproduces nick, 1988). throughout the year. In Colombia, other rodents In this study, the vaginal opening varied also reproduce continuously throughout the between estrus and metestrus-diestrus phases year as Rhipidomys latimanus and Thomasomys in a way similar to that found in laboratory laniger in high Andean forests (Montenegro- mice (Byers et al., 2012). Therefore, both tools Díaz et al., 1991; López-Arévalo et al., 1993) (vaginal smears and morphology of vaginal and Proechimys semispinosus and Hoplomys opening) are useful in distinguishing the dif- gymnurus in a lowland rainforest (Alberico ferent phases of the estrous cycle and to easily and González-M., 1993). determine the state of receptivity of the female. However, for some tropical rodents, increased This study is the first to correlate the results reproductive activity is reported in seasons with of both analyses and to implement the vaginal higher rainfall, and others in dry seasons in epithelium study as a tool to assess the repro- habitats where seasonality of precipitation is ductive status of females in wild rodents. This very strong and influences the availability of re- analysis, along with the parallel observation of sources (Lacher, 1992; Gentile et al., 2000; Bron- the vaginal opening, allowed detecting sexual son, 2009). 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