ARTÍCULO

Reproductive activity of a population of Nephelomys meridensis (Rodentia: Cricetidae) in Colombia

 

Ángela M. Villamizar-Ramírez¹, Víctor H. Serrano-Cardozo^{1, 3}, and Martha P. Ramírez-Pinilla^{2, 3}

¹ Laboratorio de Ecología, Escuela de Biología, Universidad Industrial de Santander, Bucaramanga, Santander, Colombia. [Correspondence: Víctor H. Serrano-Cardozo <vserrano@uis.edu.co>]
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.

Recibido 13 julio 2016.
Aceptado 20 marzo 2017.
Editor asociado: M Busch

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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 range of 2530-2657 m. Reproductive condition was established in adult females by external morphology including nipple development, abdomen distension, and features of the vaginal opening. Adult females were categorized as pregnant or in lactation. Additionally, female reproductive condition was complemented with the analysis of vaginal smears of each female with open vagina and with a general description of the estrous cycle 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 than 5 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 (arthropod abundance and oak fructification). Therefore, in this population 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 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.

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INTRODUCTION

Reproductive activity in small mammals has been documented mainly for species living in temperate latitudes, where factors such as photoperiod, resource availability, environmental temperature and precipitation influence reproductive patterns (Bronson, 1985).

There are few studies in the tropics where most of the mammals live; therefore, a big gap in the understanding of the regulation of reproduction by environmental factors exists for most of the tropical mammals (Bronson, 2009).

Several studies suggest that reproduction in tropical rodents is continuous (Lacher, 1992). This is the case in Oryzomys capito (Fleming, 1971), O. nigripes, O. trinitatis (Fonseca and Kierulff, 1989), Proechimys semispinosus, Hoplomys gymnurus (Alberico and González-M., 1993) and Nyctomys sumichrasti (Romero and Timm, 2013), among others. However, there is evidence of reproductive peaks during either the highest or the lowest precipitation seasons (Lacher, 1992; Bronson, 2009); for example, Oryzomys intermedius, Trinomys iheringi, Nectomys squamipes have their reproductive peaks during the peak of the rainy season (Bergallo and Magnusson, 1999); in Akodon cursor the reproductive peak starts at the end of the rainy season and goes until the end of the dry season (Fonseca and Kierulff, 1989). Then, seasonal reproductive activity in tropical rodents would be related to the intensity and duration of the rainy and dry seasons and its relation to the availability of resources for reproduction.

The estrous cycle has been widely studied in laboratory mice and rats (e. g. in Hubscher et al., 2005; Yener et al., 2007; Caligioni, 2009; Byers et al., 2012; McLean et al., 2012), but is poorly known in wild rodents. However, it has been studied under laboratory conditions in Mastomys natalensis (Johnston and Oliff, 1954), Baiomys taylori (Hudson, 1974), Peromyscus californicus (Gubernick, 1988), and Proechimys chrysaeolus (Sabogal-Guáqueta et al., 2013), among others. The estrous cycle consists of four phases called proestrus, estrus, metestrus and diestrus; each distinguished by the type and amount of vaginal epithelial cells, and the presence or absence of leukocytes in the vagina (Yener et al., 2007; Byers et al., 2012.).

Nephelomys meridensis is a rodent of the Cricetidae family, formerly in the “albigularis” group of Oryzomys (Weksler et al., 2006), N. meridensis is distributed from the Northeast of the Cordillera Oriental of Colombia in the Department of Boyacá to the North of the Sierra de Mérida in Venezuela, at elevations of 1100-4000 m (Percequillo, 2015). Individuals of the genus Nephelomys are terrestrial, social and nocturnal; they take refuge inside fallen trees and rock formations covered with moss, and food includes fruits, arthropods and seeds (Rivas, 1997; Gibson, 2014). Díaz de Pascual (1981) mentioned that the diet of O. albigularis includes 50% endocarp of guama fruit (Inga sp.), 20% insect larvae, 20% palm seeds, 2% fungi, and 2% flowers of Heliconia sp. No additional detailed information is available on the natural history of this species.

We studied the annual reproductive activity of females and other aspects of the reproduction of a population of Nephelomys meridensis living in an Andean forest with a bimodal regimen of rains. The study contributes to the knowledge of reproduction and its relationship with environmental factors in small mammals that inhabit upland forests in the Neotropics.

MATERIALS AND METHODS

This work was done in an Andean oak forest in the Cordillera Oriental of Colombia at an altitude of 2600 m, in Santa Barbara municipality, Santander, Colombia (7º 01’ 9.9’’ N, 72º 53’ 33.6’’ W). The forest is characterized by the dominance of trees of Quercus humboldtii, followed by Clusiaceae and Rubiaceae and the presence of trees with an average height of 5 m and a diameter of 7.3 cm. The area has a bimodal rainfall regime, with two periods of high rainfall from April to June and from September to November, and two periods of low rains, from July to August and from December to March; the mean annual rainfall is 1214 mm (Worldclim - Global Climate Data).

A monthly sampling was conducted from October 2014 to September 2015 (except February). For each sampling, 5 fixed stations spaced approximately every 100 m were established along one transect in an altitudinal range from 2530-2657 m, covering an area of 1.84 hectares. In each station 20 Sherman traps (23 x 9 x 7.7 cm) were randomly placed above the ground, for a total of 100 traps, which were active for 4 consecutive nights each month. Every day, traps were baited with a mixture of ground peanuts, oat flakes, ground peel corn, vegetable fat and essence of vanilla, shortbread or banana, and revised in the morning and evening.

For each individual captured we recorded sex, weight (body mass) with a Pesola (100 g spring scale ± 0.5 g), and standard external measurements with a dial caliper calibrated to the nearest 0.1 mm (Total length [TL], head-body length [HBL], tail length [TL], ear length [EL] and hind foot length [HFL]). Mice were labeled by injection in the back of an intradermal microchip 5 mm (Biomark^®) disinfected with iodine solution. The animals were released in the same place of capture; however, some individuals were collected to corroborate taxonomic determination with the proposed key of Percequillo (2015) for the genus Nephelomys. These individuals were deposited in the collection of mammals of the Museo de Historia Natural of the Universidad Industrial de Santander. This research followed ASM guidelines (Sikes et al., 2011).

The capture effort was calculated by multiplying the number of traps installed in the study area by the number of nights per sampling month. In addition, capture success was calculated by the formula C.S. = (I.C / C.E) x 100, where: S. C = capture success, I.C. = Number of individuals captured and C.E = capture effort (Steinmann et al., 2003; Gallina et al., 2008).

Reproductive activity

From morphological measurements, weight and head-body length (HBL), and hair dorsal color of the individuals captured in this study, we defined three categories of age: adults, sub-adults, and juveniles (Table 1); the latter two were taken as a category of immature individuals. Reproductive condition was established only in adult females by observing external morphological characteristics such as nipples development and the state of the vagina (closed, open or vaginal plug), and the analysis of samples of vaginal swabs for determining the phases of the estrous cycle.

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.

Adult females were categorized as pregnant (distended abdomens, partially open vagina or vaginal plug with developed nipples) and lactating (open vagina with developed nipples and milk production). The vaginal plug is a mixture of sperm, secretions from the accessory sex glands in males and vaginal secretions in the vagina that coagulate forming a “plug” of dry texture and relatively hard; this cap seems to prevent the female to be copulated by another male (Benavides and Guénet, 2003). The presence of vaginal plug in females was seen as an indication of recent intercourse.

Vaginal smears were made on each female with open vagina by vaginal washing with 4.5 μl of saline solution (0.9%) introduced into the vaginal canal with a micropipette Accumax Pro (0.1-10μl). The solution was collected and placed on a glass slide, was dried at ambient temperature and fixed by adding a few drops of ethanol (70%). The samples were stained with eosin and methylene blue (10%), observed under a microscope (Nikon Eclipse 55i^®) and photographed with a Canon EOS Rebel XS^® camera. A duplicate of each wash was performed to ensure best results. Those females that showed no morphological features of pregnancy or lactation states were characterized by the results of vaginal epithelium analyses. They were found in proestrus, estrus or metestrus and were considered as reproductive females since there is hormonal stimulation for each phase and a clear indication that they are sexually receptive.

To determine the estrous cycle, during each sampling month we took 1 or 2 females with open vaginas and one adult male. They were kept in captivity for a period of 4-6 days. Each individual remained separately in transparent plastic containers (34.5; 24; 24.5 cm), which were adapted with a layer of dry leaves on the bottom, and 80-90 holes opened in the lid to allow air flow. Individuals were fed with few grains of almonds, unsalted peanuts, and small oak acorns and provided with water ad libitum. Every other day each female was placed next to the male (with no chance of intercourse). During the captivity, every day a vaginal smear was obtained from each female; the samples were treated in the same manner as described above. After the days of captivity, individuals were released at the site of capture.

The phases of the estrous cycle differed qualitatively considering the type of vaginal epithelial cells present in each sample (Hubscher et al., 2005; Yener et al., 2007; Caligioni, 2009, Byers et al., 2012; McLean et al., 2012). Having identified the estrous phase in each female, their characteristics and duration were determined. In addition to the recording and analysis of vaginal smears, we took photographs (Nikon Coolpix l330^® Camera) to the vaginal opening of every female to observe changes related to the estrous cycle (Byers et al., 2012).

For adult males we tried to determine the reproductive activity by the position of the testicles as has been considered in other studies (reproductive males with scrotal testes and non-reproductive males with abdominal testes), like in Hoplomys gymnurus and Proechimys semispinosus by Alberico and González-M (1993) and Rhipidomys latimanus by Montenegro-Díaz et al. (1991), among others. However, males were removed from the analysis because the position of scrotal testes changed rapidly to abdominal during animal manipulation; therefore, it was determined that the position of the testes is an unreliable indicator of male reproductive activity.

Environmental food resources

The collection of food resources (arthropods) was done monthly; for this, pitfall traps were installed at each station, made up of 10 plastic cups of 16 oz with a mixture of soap and water during 96 hours each month. The individuals collected were stored in ethanol (70%); they were subsequently identified to the taxonomic level of order and weighed for wet biomass and then dried for obtaining dry weight (Pesola^® M500, 500g d = 0.1g). Evaluation of food resources also included the vegetable supply; we only included the fruits of the most common tree, the oak Quercus humboldtii. We made visual tracking of their fructification time during the sampling visits; we consider the peak of fructification when most of the trees in the study area have fruits. The hypothesis we have was that the peak of reproductive events concurs or is close in time with the peak of food offer (arthropod abundance and oak fructification).

Data analysis

A binomial test was used to determine significant differences from a 1:1 sex ratio (software R v3.3.1, 2016); to establish whether there was significant difference between the abundance of males and females over time, we used a G-test. To evaluate the possible existence of significant differences between sexes in body size in adult individuals, 6 variables were compared: body weight and 5 standard morphological measurements, these were tested by t-student test or nonparametric Mann-Whitney U test. A sexual dimorphism index was calculated for variables that showed significant differences between adult males and females (Schulte-Hostedde, 2007). In addition, we investigated whether there was significant variation in body weight over time using a Kruskal-Wallis test.

To determine whether there were significant differences between the number of adults with respect to immatures obtained for the entire sampling period we used a Mann-Whitney U test, and to determine whether there were significant differences in their distribution between months a G-test was employed. The monthly reproductive activity was taken as the number of pregnant, lactating, and reproductive females (according to the characterization of the vaginal epithelium). Significant differences in the abundance of each reproductive state over time was tested by G-test (June and August were not included because only one individual was captured each month).

To determine whether there was an association between the monthly average of rains and the monthly average of arthropods biomass with reproductive activity, we used a Spearman correlation (software R v3.3.1, 2016; Hmisc package, Harrell and Charles, 2015). Because the relationship between environmental and resource factors may influence reproduction later on, we used Spearman correlations (software R v3.3.1, 2016; Hmisc package, Harrell and Charles, 2015) with the corresponding month and the previous month (Antunes et al., 2009). Statistical tests (Mann-Whitney U, t-student and Kruskal-Wallis) were performed in STATISTICA 7.0 (StatSoft, 2004) software with a significance level of 0.05. G-tests were implemented using the software R v3.3.1 (2016) with RVAideMemoire package (Hervé, 2017). In neither of these tests June was included because only one individual was captured.

RESULTS

A total of 87 individuals were captured in 130 capture events. Seventeen individuals were recaptured, including 13 adults (8 males and 5 females), 1 sub-adult female and 2 juvenile females. The number of times in which an individual was recaptured ranged from 1 to 7. The total sampling effort was 4400 trap nights and the success capture was 1.97%. Table 2 shows the reproductive stage of captured and recaptured females; based on these data the gestation period can be estimated between 28 and 32 days, and sexual maturity in females can be achieved at approximately 100 days.

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

Adults were captured in larger numbers than juveniles throughout the sampling period (68 adults, 40 males and 28 females; 19 immatures, 6 males and 13 females); this difference was significant (U = 11; P = 0.001). Significant differences were also found over time, as adults were s more frequently captured between October and March (G = 31.42; df = 9; n = 108; P < 0.0003) and immatures were found mostly between April and September (G = 23.13; df = 9; n = 21; P < 0.006, Fig. 1).

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.

The sex ratio (46 males: 41 females) for the entire sample was not significantly different from 1:1 (P = 0.66, binomial test). The abundance of females remained constant during sampling (G = 7.98; df = 9; n = 62; P < 0.54) with a slight decrease in January, March, and September; the number of males varied significantly over time (G = 21.64; df = 9; n = 67; P < 0.01), increasing between September and March (Fig. 1). The mean body weight did not change over time in adult males (H = 14.25; df = 9; n = 61; P = 0.11), but varied in adult females (H = 26.76; df = 9; n = 47; P = 0.0015). Females tended to be heavier in March, April and May (the first rainfall peak of the year), the period in which late pregnant females were found, while their lower weight was in September, October and November (the second rainfall peak of the year, Fig. 1).

Sexual dimorphism showed significant differences, with higher values in males than in females in total length and body weight (Table 3). The index of sexual dimorphism (F:M) was 0.97 for total length and 0.88 for body mass.

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).

Reproductive activity

All adult females were found to be reproductively active throughout the year, either in pregnancy (51%), lactating (20%), or reproductive (29%, in the estrus or proestrus phases of the reproductive cycle). Reproductive females (as observed by vaginal smears) were found between September and January, and its occurrence was significantly different between months (G = 18.63; df = 8; n = 14; P < 0.02) (Fig. 2). Seven females were in estrus in September, November, December, and January, and one female in proestrus in October (Fig. 2). Pregnant females were in the metestrus phase. In the vaginal smears no sperm was observed.

Fig. 2. Proportion of pregnant, lactating, and reproductive females of Nephelomys meridensis and average monthly rainfall 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.

Pregnant females were found in almost every month except in June, August and September, with no significant differences in their occurrence over time (G = 8.18; df = 8; n = 25; P < 0.42). A higher proportion of late pregnant females was observed in April and May, when their body weight ranged between 81 and 86 g; the remaining mid-pregnant females weighted between 60 and 75 g. Lactating females were also captured during most sampling of the year without significant differences over time (G = 7.43; df = 8; n = 8; P < 0.5).

Estrous cycle

Nine females were maintained in captivity, of which 4 were pregnant and 5 reproductive females. In the latter, it was possible to observe the different phases of the estrous cycle (Figs. 3 and 4). Each phase is clearly distinguishable by the type and amount of cells of the vaginal epithelium, also by the presence or absence of leukocytes (Fig. 4).

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.

Clusters of nucleated epithelial cells were found in proestrus (Fig. 4A); they can be parabasal, intermediate and superficial. Cornified, enucleated cells were also found but in smaller proportion. In a female, in December, clusters of parabasal nucleated cells and some leukocytes were observed, which was considered an early proestrus.

Cornified cells predominated in the estrous phase although few superficial nucleated cells were found. One female of November was considered as in early estrus since some cornified cells and nucleated cells were in similar proportions. In captivity, the estrous phase lasted up to five days, followed by metestrus of up to 2 days. In March, a female in captivity gave birth to three pups and the vaginal smear taken in the following day showed estrous features, thus representing a postpartum estrus (Fig. 3).

Metestrus (Fig. 4C) is distinguished by the presence of three cell types: nucleated cells, mostly cornified cells, and leukocytes. In contrast, during diestrus (Fig. 4D) the same cell types are found but leukocytes predominate. Pregnant females were in the metestrus phase.

A complete estrous cycle was observed in only 2 females, in which the three basic stages of the cycle (proestrus, estrus, and metestrus) were observed (Fig. 3).

In estrus (Fig. 5 A and B) the vaginal opening is wide and bordered by an inflamed tissue that has striations at the edges, while for females in metestrus-diestrus (Fig. 5C and D) the opening is small, smooth and without tissue inflammation.

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.

Reproductive activity and its relationship with rainfall and food resources

We found no correlations of reproductive condition (number of females in different reproductive phases) and precipitation in the corresponding or the previous month (results not shown); among these, only the number of lactating females was marginally and negatively correlated with precipitation (r = -0.61, P = 0.06). Similarly, no significant associations were found between the dry weight of arthropods and the numbers of pregnant, lactating and reproductive females.

Fructification of the oak Quercus humboldtii in the study area showed two peaks, one from April to June and the other from August to December, which correspond to the two peaks of rainfall. Few fruits were observed in the other months, suggesting a marked seasonal regime of fructification for this tree in the study area. The greatest proportion of late pregnant females was found during the first peak of fructification of the year (April and May), but no other relationships of reproductive events and oak fructification peaks are apparent.

DISCUSSION

This paper describes a pattern of continuous polyestrous reproduction for a population of N. meridensis in an Andean oak forest in the Cordillera Oriental of Colombia, this being the first report to the species. The studied population of N. meridensis has continuous reproduction throughout the year. Females in different reproductive stages (receptive, pregnant, and in lactation) were found throughout the year.

Tracking successive recaptures of individuals in the field revealed a time to reach sexual maturity in females of about 100 days and a gestation period between 28 and 32 days. These observations compare well with other studies of tropical rodents, such as for example, Rhipidomys latimanus (maturity 95 days, gestation 30 days; Montenegro-Díaz et al., 1991) and Thomasomys laniger (maturity 3.5 months, gestation 24 days, López-Arévalo et al., 1993). However, Gentile et al. (2000) reported the age of sexual maturity in Nectomys squamipes to be between 31 and 51 days, and 38.8 to 51.2 days in Akodon cursor.

These times are also variable when observed in studies of wild rodents in captivity. For example in Peromyscus californicus (maturity 44 days, gestation 31-33 days, Gubernick, 1988) and in Proechimys chrysaeolus (gestation 50-60 days, Sabogal-Guáqueta et al., 2013). Within the genus Nephelomys, N. caracolus reaches maturity between 26 and 93 days, and gestation time is 26.65 days (Moscarella and Aguilera, 1999), values that are similar to those reported here.

The higher capture of mature individuals than juveniles and their unequal distribution throughout the year observed in this population of N. meridendis has also been seen in other Neotropical rodents (Rhipidomys latimanus, Montenegro-Díaz et al., 1991; Peromyscus mexicanus, Rojas and Rodríguez, 2007; Nectomys squamipes, Oryzomys intermedius, Trinomys iheringi and Akodon cursor, Bergallo and Magnusson, 1999; Nephelomys caracolus, García et al., 2013). This pattern might reflect the more limited motility of juveniles, as pointed out by Alberico and González-M (1993), as well as short times from birth to sexual maturity.

The sex ratio observed here for N. meridensis is nearly 1:1, as in some other Neotropical rodents (Rhipidomys latimanus, Montenegro-Díaz et al., 1991; Peromyscus mexicanus, Rojas and Rodríguez, 2007). There are several reports of deviations from a 1:1 sex ratio in temperate rodents, such as the Argentine rodent Salinomys delicatus (Rodríguez et al., 2012) and the North American Peromyscus maniculatus borealis (Havelka and Millar, 1997).

Generally, uneven sex ratios are associated with size differences. Often, male biased sex ratios are linked to larger males, in turn suggesting male-male competition and polygyny (e.g. Cornely and Baker, 1986; Schulte-Hostedde, 2007). There are also reports of larger body sizes in females (Schulte-Hostedde, 2007), as in the case of Salinomys delicatus (Rodríguez et al., 2012) and Peromyscus maniculatus (Schulte- Hostedde et al., 2001). In N. meridensis males are larger than females in total body length and weight, raising the possibility of polygyny and male competition for the opportunity to mate.

Phases of the estrous cycle of rodents often change daily in captivity; however, colony composition and density may change this pattern (e.g. McLean et al., 2012; Sabogal-Guáqueta et al., 2013). Females of N. meridensis maintained in captivity showed prolonged phases of estrus and metestrus. These phases were extended in females even in presence of a male, but copulation was not allowed. Postpartum estrus was observed in a female in captivity about 12 hours after giving birth, consistent with those reported in other rodents (Dewsbury et al., 1977; Gubernick, 1988).

Caligioni (2009) and McLean et al. (2012) noted that vaginal smears through 2-3 cycles were needed to determine the duration of the estrous cycle. Our observations are indicative of estrous cycles longer than 5 days, but these results need to be corroborated with additional sampling efforts. Many studies of rodents in captivity show cycles to vary between 4 and 20 days (e.g., Dewsbury et al., 1977; Gubernick, 1988).

In this study, the vaginal opening varied between estrus and metestrus-diestrus phases in a way similar to that found in laboratory mice (Byers et al., 2012). Therefore, both tools (vaginal smears and morphology of vaginal opening) are useful in distinguishing the different phases of the estrous cycle and to easily determine the state of receptivity of the female. This study is the first to correlate the results of both analyses and to implement the vaginal epithelium study as a tool to assess the reproductive status of females in wild rodents. This analysis, along with the parallel observation of the vaginal opening, allowed detecting sexual receptivity (estrus) in those females that could be considered as inactive when only external morphology is used to establish the female reproductive state.

Some studies suggest that many rodents in the tropics have continuous reproduction due to favorable habitat conditions and opportunistic life strategies, characteristic of rodents that have a short life expectancy, rapid sexual maturity, short gestation period and the presence of postpartum estrus (Bronson, 1985; Bronson and Perrigo, 1987; Lacher, 1992; Gentile et al., 2000). Some of these characteristics are determined from this study for the population of N. meridensis; the gestation period could be between 28 and 32 days, sexual maturity in females could be reached at approximately 100 days of age, and females are continuous polyestrous and may have postpartum estrus.

This is a common pattern in Neotropical rodents. Oryzomys nigripes and O. trinitatisse breed throughout the year in the Atlantic forest of Brazil (Fonseca and Kierulff, 1989). In Panama, in a less seasonal tropical rainforest than the Atlantic forest of Brazil, O. capito reproduces throughout the year (Fleming, 1971). In the plains of Venezuela, which are seasonally flooded savannas with a dry season from December to April, O’Connell (1982) and Vivas (1986) mentioned that Oryzomys bicolor (Oecomys bicolor), Rhipidomys mastacalis (Rhipidomys nitela) and Zygodontomys brevicauda reproduce all year. Finally, Romero and Timm (2013) found that in humid tropical lowland forests of Costa Rica, Nyctomys sumichrasti reproduces throughout the year. In Colombia, other rodents also reproduce continuously throughout the year as Rhipidomys latimanus and Thomasomys laniger in high Andean forests (Montenegro- Díaz et al., 1991; López-Arévalo et al., 1993) and Proechimys semispinosus and Hoplomys gymnurus in a lowland rainforest (Alberico and González-M., 1993).

However, for some tropical rodents, increased reproductive activity is reported in seasons with higher rainfall, and others in dry seasons in habitats where seasonality of precipitation is very strong and influences the availability of resources (Lacher, 1992; Gentile et al., 2000; Bronson, 2009). Nitikman and Mares (1987), Lacher et al. (1989) and Mares et al. (1989) found that in the Brazilian Cerrado (seasonal savannas) many species of Sigmodontine (Akodon cursor, A. reinhardti, A. cerradensis, A. lindbergi, Bolomys lasiurus, Calomys callosas, C. tener, Holochilus brasiliensis, Oryzomys bicolor (=Oecomys bicolor), O. capito, O. chacoensis, O. concolor, O. fomesi, O. nigripes, O. subfiavus, and Oxymycterus roberti) reproduce seasonally, starting in the dry season and ending in the middle of the rainy season. In the Atlantic Forest of Brazil, Akodon cursor has a reproductive peak that starts at the end of the rainy season until the end of the dry season (Fonseca and Kierulff, 1989). In IIha do Cardoso (Atlantic forest, Brazil), Oryzomys intermedius and Trinomys iheringi have a reproductive peak in times of increased rainfall, while Nectomys squamipes reproduces seasonally (Bergallo and Magnusson, 1999).

Despite the existence of a bimodal rainfall regime in the study area of the population of N. meridensis, and variation in arthropod and oak fruit abundance, there is limited variation in female reproductive activity throughout the year. Some associations can be made between occurrences of juveniles vs. adults and of late pregnant females with some months of the year, but these do not determine reproductive peaks or seasons.

ACKNOWLEDGMENTS

Laura C. Vargas, Christian F. Cacua, Andrés Quiñones, Omar Reyes, Julián Villamizar and Sebastian Solis provided support in the field phase. Javier Colmenares and Julián E. Lozano-Flórez helped with their knowledge of taxonomy and capture methods of small mammals. Grupo de Estudios en Biodiversidad (UIS) provided access to facilities and provided financial support.

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