Embryonic and fetal development of the red brocket deer (Mazama americana)

Theriogenology 134 (2019) 53e64

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Embryonic and fetal development of the red brocket deer (Mazama americana) Pedro Mayor a, b, c, d, e, *, Thyago Habner de Souza Pereira e, Rafael dos Santos de Andrade e,
lez-Benavent a, Frederico Ozanan Barros Monteiro e, Richard Bodmer b, c, d, Elena Gonza ~o Valsecchi c, d, f, Hani Rocha El Bizri c, d, f, g Joa ria, Barcelona, Spain noma de Barcelona (UAB), Departament de Sanitat i d’Anatomia Animals, Facultat de Veterina Universitat Auto
nicas, Iquitos, Loreto, Peru Museo de Culturas Indígenas Amazo ComFauna, Comunidad de Manejo de Fauna Silvestre en la Amazonía y en Latinoam
erica, 332 Malecon Tarapaca, Iquitos, Peru d ~o e Uso da Fauna na Amazo ^nia (REDEFAUNA), Manaus, Amazonas, Brazil Rede de Pesquisa em Diversidade, Conservaça e
, Brazil Federal Rural University of the Amazon (UFRA), Postgraduate Program in Animal Health and Production in Amazonia (PPGSPAA), Bel
em, Para f
Sustainable Development Institute (IDSM), Tef
Mamiraua e, Amazonas, Brazil g School of Science and the Environment, Manchester Metropolitan University, Oxford Road, M15 6BH, Manchester, United Kingdom a

b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 February 2019 Received in revised form 22 April 2019 Accepted 16 May 2019 Available online 21 May 2019

The red brocket deer (Mazama americana), a medium-sized Neotropical ungulate, is one of the most hunted mammals in the Amazon. This study analyzes the intrauterine development in the red brocket deer through the description of the external and internal morphology in one embryo and 38 fetuses collected from animals hunted for subsistence in the Amazon. The chronological order of occurrence of external characteristics in relation to the total dorsal length (TDL) was: differentiated genitalia, limbs and eyelid buds (TDL ¼ 3.9 cm), fusioned eyelids, outer ear and hooves (TDL  9.5 cm), skin (TDL  20.4 cm), tactile pelage and nasal pigmentation (TDL  21.5 cm), covering pelage and skin spots (TDL  31.3 cm), and teeth eruption and opened eyelids (TDL  34.2 cm). The formula of fetal age was ∛W ¼ 0.072 (t e 42), with a high linear relationship between TDL and gestational age. Multiple linear and non-linear regressions showed strong positive associations between biometric measures and absolute visceral weights with TDL. The relative weight of the tubular gastrointestinal organs, spleen and thymus increased during the fetal development; in contrast, the liver and kidneys’ relative weight diminished during the fetal development. Advanced fetuses (44.0 cm TDL) had lower proportion of liver and larger tubular gastrointestinal organs within the visceral weight than adults. The chronology of appearance of the main events of the fetal development suggests that the red brocket deer adopt some precocial features, such as the early development of the sensorial function, including the early development of eyelids, outer ear and tactile pelage, the early development of the covering pelage which acts in thermoregulation and the early teeth eruption which allows the early foraging. Nevertheless, the precocial level of the red brocket deer is apparently lower than other species more frequently predated by large felids, such as peccaries and the paca. © 2019 Elsevier Inc. All rights reserved.

Keywords: Cervidae Reproduction Fetal features Gestational development Amazon

1. Introduction The red brocket deer (Mazama americana) is a medium-sized artiodactyl, weighting from 20 to 30 kg [1], and one of the most

noma de Barcelona (UAB), Departament * Corresponding author. Universitat Auto ria, Barcelona, Spain. de Sanitat i d’Anatomia Animals, Facultat de Veterina E-mail addresses: [email protected], [email protected] (P. Mayor). https://doi.org/10.1016/j.theriogenology.2019.05.015 0093-691X/© 2019 Elsevier Inc. All rights reserved.

frequently hunted species in the Amazon region [2]. This species has a wide distribution ranging from southern Mexico to Amazonia and the Chaco, and through eastern Paraguay to northern Argentina [3]. The red brocket deer is currently considered as “Data Deficient” by the International Union for Conservation of Nature (IUCN) and main threats are predatory hunting and habitat loss by agricultural expansion [4]. According to Duarte and Vogliotti [4], further studies on the life history of the species are needed, to develop in situ and

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ex-situ conservation actions for the species. The definition of the conservation status of a game species and the implementation of conservation programs are often guided by assessments of the vulnerability to extinction or sustainability of hunting of the target species in a given area [5,6]. Although there is a considerable variation on reproductive patterns in mammalian vertebrates, reproduction is the most important strategy in the life history to beget offspring and thereby perpetuate the species survival through time [7]. The reproductive strategy is an evolutionary response to disturbances, influencing the dynamics of wildlife populations and indicating how the species will withstand different predation and harvest rates [8]. Nevertheless, the reproductive biology of most wild species, including the red brocket deer is still largely unknown [9]. In the Amazon region, the female red brocket deer is a nonseasonal polyestrous species, apparently breeding year-round [9,10], but some studies reported peaks of conceptions and births [11,12]. This species has a mean gestation length of 210 days [13] and each reproductive cycle has a mean ovulation rate of 1.14 ovulations per female [9] and a litter size of one young per parturition, but twins may occasionally occur [10]. Estimated yearly reproductive production in the wild is 0.74 young per adult female [14]. Gestation length in mammals is correlated with the degree of development of the young at birth, and parturition must occur at a time when conditions are most likely to be optimal for survival of newborns [7]. The embryonic and fetal development comprises the process of individual's intrauterine maturation from fecundation to parturition [15]. Therefore, the study of the intrauterine development allows understanding and comparing the strategies adopted by different mammal species to maximize their maternal and neonatal survival [16,17]. Ultrasonography is a minimally invasive and low-cost technique, which has already been proved useful for the pregnancy monitoring and to evaluate the stages of embryonic and fetal developments in wild species [18,19]. Nevertheless, those studies are usually difficult to be conducted in wild species because researchers are often unable to access a large number of individuals these species (i.e. through live captures), which limits the sample size. This constraint may be solved through the collection of samples originated from hunting activities by local people [14]. This study aims to describe the embryonic and fetal development of the red brocket deer using embryos and fetuses collected through the collaboration of Amazon dwellers in rural communities whose protein income depends on the subsistence hunting. Participatory collection of females’ reproductive tracts with the aid of local people has been proved efficient to provide sufficient samples and accurate reproductive assessments, allowing the standardization of parameters for application of ultrasonography and other imaging techniques [14,20e22]. 2. Material and methods 2.1. Study sites This study was conducted in two areas in the Amazon rainforest. The first area, the Yavarí-Mirín River (YMR, S 0419.53; W 7157.33), is in Northeastern Peruvian Amazon and is a continuous area of 107,000 ha of predominantly upland forests. A single indigenous community of 307 inhabitants is found in the region. The second ~ Sustainable Development Reserve (ASDR, S site, the Amana 0154.00; W 64 22.00), is a protected area of 2,313,000 ha in the
rivers, Central Brazilian Amazon, between the Negro and Japura and consists primarily of upland forests. The ASDR has a population of approximately 4,000 riverine people, found in 23 communities

and some isolated settlements. In both areas, local communities rely mainly on agriculture for income and on hunting and fishing for subsistence. The climate in both study areas is typically equatorial with annual temperatures ranging from 22  C to 36  C, a relative humidity of 80%, and annual rainfall between 1500 and 3000 mm, comprising dry and wet/flooded seasons. 2.2. Biological sample collection and processing From 2002 to 2015, local hunters collected and voluntarily donated reproductive tracts from 39 pregnant red brocket deer females, 32 (82.0%) in the YMR and 7 (18.0%) in the ASDR. All pregnant females had yielded a total sample of one embryo and 38 fetuses; no twin gestations were recorded. Hunters were trained to remove all abdominal and pelvic organs complete with the perineal region and to store these in buffered 4% formaldehyde solution (v/ v). Since hunters do not consume these materials, any invasive procedure or additional mortality for the purpose of the study was avoided [14]. The research protocol was approved by the Research Ethics Committee for Experimentation in Wildlife at the Direccion General de Flora y Fauna Silvestre from Peru (License 0350-2012DGFFS-DGEFFS), by the Instituto Chico Mendes for Biodiversity Conservation from Brazil (License SISBIO No 29092) and by the Committee on Ethics in Research with Animals of the Federal Rural University of the Amazon (CEUA/UFRA protocol 008/2016). Samples
m, Par
were sent to UFRA, Bele a, Brazil, under the export license CITES/IBAMA (No 14BR015991/DF). We externally examined the only embryo and the 38 fetus to describe the presence of the following morphological features: 1) differentiated genitalia, 2) differentiated limbs, 3) eyelids, 4) skin, 5) skin spots, 6) covering and tactile pelage, 7) erupted teeth, 8) hooves, 9) nasal pigmentation and 10) outer ear. The embryo/fetal stage was determined according to the International Committee on Veterinary Embryological Nomenclature [23], and the embryo characterized by the presence of buds of eyelids, genitalia and limbs. We performed biometry measurements in the embryo/fetuses to describe the external biometry. These measurements included body mass, total dorsal length (TDL), crown-rump length (CRL), biparietal diameter (BPD), occipital-frontal diameter (OFD), cranial circumference (CC), femur and humerus length (FL and HL), length of thoracic and pelvic limbs (TL and PL), thoracic diameter and circumference (TD and TC), as well as abdominal diameter and circumference (AD and AC). Thoracic and abdominal measurements were obtained from the last rib and the insertion of the umbilical cord, respectively. The biometry conducted in the embryo included the same measurements at analogous anatomical regions, but we did not measured FL and HL. We eviscerated and weighed the thoracic and abdominal organs (heart, lungs, thymus, liver, spleen, kidneys, and tubular gastrointestinal organs). These measurements were done in 31 fetuses, after excluding the embryo and fetuses that were crushed or in poor conservation conditions of internal organs. The summative weight of all organs was considered as the total visceral weight. The difference between the total fetal weight and the total visceral weight was considered as a proxy measure of the musculoskeletal system. The relative weight of each fetal organ was calculated as a percentage of the total visceral weight. In parallel, the weight of the same organs was also measured in 2 adult (one male and one female) red brocket deers hunted in the YMR to compare the relative weight of fetal organs in advanced pregnancy stages with that in adults. Adult organ samples were collected and stored through the same procedures as those described for the reproductive organs (buffered 4% formaldehyde solution). We also measured the longitudinal diameter of the cotyledonary placenta and counted and measured the longitudinal and width

P. Mayor et al. / Theriogenology 134 (2019) 53e64

diameter of all dome-shaped caruncles per pregnant female. The mass of the body and organs was measured in grams using a digital weighting scale (0.1 g accuracy), while a tape measure (0.1 mm accuracy) and a metal caliper (full measurement capability 300 mm) were employed for body measurements. 2.3. Statistical analysis The gestational age was estimated using the formula proposed by Huggett and Widdas [24], ∛W ¼ a (t-t0), where W is the fetal weight, a is the specific fetal growth velocity, t is the fetal age in days, and t0 is the calculated interception on the age axis. According to those authors, t0 is equal to 20% of gestation length in species that present between 100 and 400 days of pregnancy. We considered a mean gestation length of 210 days [13]. Since we did not find available information in literature on the newborn weight, we considered a mean weight of 1,800 g at birth, considering the weight stabilization in fetuses in more advanced stages of gestation (44.0 cm TDL, n ¼ 3). For the estimation of fetal age t (in days), we used the weight of each fetus and the value of a and t0 calculated previously. Logistic regressions were applied to estimate the probability of occurrence of each external morphological characteristic in relation to TDL using the software Statistica 8.0 (StatSoft Inc., Tulsa, USA). Multiple regression modeling relationships between TDL and biometric measures of the fetus, the placenta and absolute and relative organ weights were conducted using the software CurveExpert 2.4 (© Copyright 2017, Daniel G. Hyams), which defined those functions that best fitted to the plots. Regressions were also used to assess allometric relationships between BPD and OFD, TC and AC, HL and FL, TL and PL, and to assess the trends in the relative weight of each organ according to the total visceral weight, considering both fetuses and adults. For absolute measurements, we forced linear regressions to origin and only considered those functions with a starting point on or near zero, since we expected both internal and external measurements to be zero on day 0 of fetal development. We compared the relative weight of visceral organs of larger fetuses (44.0 cm TDL) with those of adults by means of T-student tests. Differences with a probability value of 0.05 or less were considered significant. All values are expressed as the mean ± standard deviation (S.D.). 3. Results In the studied embryo/fetuses, the average TDL was 28.6 ± 10.9 SD cm (range 3.9e50.8 cm). The mean body mass was 548.1 ± 502.1

55

SD g (range 9.5e2.100 g). The growth formula used to determine fetal age was ∛W ¼ 0.072 (t e 42). Both associations between gestational age and TDL (r2 ¼ 0.96, P < 0.001) and CRL (r2 ¼ 0.96, P < 0.001) presented high positive linear relationships (Fig. 1). Fig. 2 shows the probability curves for the occurrence of external morphological features according to TDL, and Table 1 shows the regression models. The earliest fetus (TDL ¼ 3.9 cm and CRL ¼ 3.1 cm) presented differentiated genitalia and limbs, and eyelid buds at initial stage of development. Presence of fusioned eyelids, outer ear and hooves were observed in fetuses with TDL 9.5 cm (CRL  6.6 cm). First signs of skin were observed in fetuses 20.4 cm TDL (13.5 cm CRL), tactile pelage and nasal pigmentation from 21.5 cm TDL onwards (12.5 cm CRL) and covering pelage and skin spots from 31.3 cm TDL onwards (24.3 cm CRL). Tooth eruption and opened eyelids were the last characteristics observed in advanced fetuses, from 34.2 cm TDL (28.1 cm CRL) (Fig. 3). All associations between TDL and external biometric measures had high and significant coefficients of determination (r2 > 0.86, P < 0.0001; Fig. 4). The CC and BPD (r2 ¼ 0.96, P < 0.0001) and OFD, TC and TL (r2 ¼ 0.95, P < 0,0001) presented the best relationships with TDL. The allometric relationships showed strong interactions among all analyzed parameters (r2  0.93, P < 0.0001), but while the relationship between HL and FL showed a 1:1 proportion of growth (r2 ¼ 0.96, P < 0.01), the PL was longer than TL during the whole fetal development (r2 ¼ 0.97, P < 0.01, Fig. 5). All associations between TDL and the absolute weight of internal organs showed high and significant coefficients of determination (r2  0.55, P < 0.01; Fig. 6). The best associations were found for the total visceral weight, lungs and the tubular gastrointestinal organs (r2 ¼ 0.87, P < 0.0001), heart (r2 ¼ 0.86, P < 0.0001), and spleen (r2 ¼ 0.85, P < 0.0001). During the fetal development, associations between the relative weight of the spleen (r2 ¼ 0.46, r ¼ 0.68, P < 0.0001), the liver (r2 ¼ 0.46, r ¼ 0.83, P ¼ 0.001), the thymus (r2 ¼ 0.46, r ¼ 0.68, P ¼ 0.003), the kidneys (r2 ¼ 0.26, r ¼ 0.51, P ¼ 0.011) and the tubular gastrointestinal organs (r2 ¼ 0.71, r ¼ 0.84, P ¼ 0.039) showed high and significant coefficients of determination. The relative weight of the tubular gastrointestinal organs and spleen increased during both fetal and post-natal development; in contrast, the relative weight of the liver and kidneys diminished during the fetal and post-natal development (Fig. 7). The relative weight of thymus increased during the fetal development and diminished during the post-natal development. The relative weight of the heart (7.7 ± 1.3 SD %; P ¼ 0.139), lungs (21.4 ± 3.3 SD %; P ¼ 0.495) and musculoskeletal system (88.6 ± 4.2 SD %; P ¼ 0.498) maintained constant along the whole development. Comparisons

Fig. 1. Relationship between gestational age and total dorsal length (TDL) and crown-rump length (CRL) in 39 red brocket deer (Mazama americana) embryo/fetuses. The red line represents an expected linear trend for CRL (y ¼ 0.149x) and TDL (y ¼ 0.205x).

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Fig. 2. Probability curves for external morphological features along the increase in total dorsal length (TDL) in 39 embryo/fetuses of red brocket deer (Mazama americana).

Table 1 Logistic equations for the external morphological features parameters in 39 embryo/fetuses of red brocket deers (Mazama americana). Morphological features

Equation

Fusioned eyes, hooves and outer ear Skin Tactile pelage Covering pelage Nasal pigmentation Skin spots Opened eyelids Teeth eruption

y y y y y y y y

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

exp(-6.6757þ(0.858337)*x)/(1 þ exp(-6.6757þ(0.858337)*x)) exp(-14.298þ(0.64016)*x)/(1 þ exp(-14.298þ(0.64016)*x)) exp(-19.828þ(0.89125)*x)/(1 þ exp(-19.828þ(0.89125)*x)) exp(-35.854þ(1.14449)*x)/(1 þ exp(-35.854þ(1.14449)*x)) exp(-27.534þ(1.20364)*x)/(1 þ exp(-27.534þ(1.20364)*x)) exp(-24.916þ(0.763286)*x)/(1 þ exp(-24.916þ(0.763286)*x)) exp(-14.886þ(0.389759)*x)/(1 þ exp(-14.886þ(0.389759)*x)) exp(-10.779þ(0.270678)*x)/(1 þ exp(-10.779þ(0.270678)*x))

of the relative weight of organs between advanced fetuses (44.0 cm TDL, n ¼ 3) and adults (n ¼ 2) showed minor proportionof liver and higher proportion of tubular gastrointestinal organs in adults (Table 2). Thymus was not observed in both studied adult red brocket deers. The longitudinal diameter of the cotyledonary placenta showed a positive relationship with TDL (r2 > 0.68, P < 0.01; Figs. 8 and 9). The uterus of all pregnant females had 6 dome-shaped caruncles, which average diameter showed a progressive increase (r2 > 0.80, P < 0.0001) along pregnancy. 4. Discussion This study describes important events of the fetal development in the red brocket deer, which may be useful to develop appropriate reproductive in situ and ex situ management practices and conservation for this species. Understanding the chronological appearance of fetal developmental landmarks is also useful to clarify the evolutionary strategies of mammalian intrauterine development, classifying species into precocial or altricial [25]. As most ungulates, red brocket deer neonates present precocial characteristics, with developed structures for the autonomous post-natal survival and the low dependence on parental care. The methodology used herein has been also used in similar studies conducted in the paca (Cuniculus paca) [20], the woolly monkey (Lagothrix poeppigii) [21] and the white-lipped peccary

Chi-square (Df)

P value

8.859 (1) 37.769 (1) 40.759 (1) 45.653 (1) 44.975 (1) 41.165 (1) 26.281 (1) 19.154 (1)

0.003 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

(Tayassu pecari) [22]. In all these studies, results were congruent with research conducted using B-mode ultrasound and with measures conducted in neonates born in captivity, suggesting that the measures obtained here can be used as accurate standard parameters for the application of imaging techniques in the red brocket deer. The initial development of eyelids buds was observed in embryos at 55 days (26.0% gestation length), which fused in fetuses at 75 days (35.8% gestation length); the pre-birth opening of eyelids occurred on around pregnancy day 166 (79.0% gestation length). The opening of eyelids in the pre-natal phase is also observed in other Neotropical precocious species such as the white-lipped peccary and the lowland paca, which show opened eyelids at the final phase of gestation (78.2% and 95% of gestational length, respectively) [20,22]. In contrast, in altricial species such as primates, open eyelids occur only during the post-natal period [21,26]. In the red brocket deer, the early sensory development is also perceived by the early formation of the outer ear on pregnancy day 62 (39.0% gestation length), and the tactile pelage on pregnancy day 119 (56.8% gestation length). The early development of the sensory system in wild species has direct implications for the independence of the newborn, determining its ability to receive stimuli from the environment to locate imminent dangers, food sources and socialize with individuals from the same group [27,28]. In mammals, the skin and the covering pelage act in thermoregulation and as external protection [29]. In the red brocket deer,

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Fig. 3. An embryo and fetuses of red brocket deer (Mazama americana) at different stages of development according to total dorsal length (TDL): (a) Embryo of 3.5 cm TDL and 9.5 g, presenting differentiated limb buds and initial growth of eyelid buds (bar: 1 cm); (b) Fetus of 9.5 cm TDL and 15.0 g, presenting fusioned eyelids, differentiated limbs and the initial development of hooves and the outer ear (bar: 3 cm); (c) Fetus with 21.5 cm TDL and 175 g, presenting nasal pigmentation and the initial development of skin and tactile pelage (bar: 3.5 cm); (d) Fetus with 24.5 cm TDL and 240 g, presenting developing skin, nasal pigmentation and differentiated tactile pelage (bar: 4 cm); (e) Fetus with 31.3 cm TDL and 615 g, presenting the initial development of coverage pelage and skin spots (bar: 6 cm); (f) Fetus of 50.3 cm TDL and 2,100 g, showing all fetal external characteristics, including opened eyelids (bar: 10 cm). Fetus (b) includes the performed biometry measurements of TDL (curve white dashed line) and CRL (straight white line).

the initial growth of the skin and covering pelage was observed around the day 115 (54.9% gestation length) and 155 of pregnancy (74.0% gestation length), respectively; both structures are fully formed at the end of gestation. In peccaries, first signs of covering pelage were observed on the 100-120th day of gestation (72.5e75.5% gestation length, respectively), and are also fully formed at the end of gestation [20,22,29]. In contrast, in the woolly monkey, an altricial species, the covering pelage was not fully developed during the gestation [22], which completes its formation around the day 112 of the post-natal development, and during the first 4 months of age thermoregulation depends on the maternal contact [30]. In terms of teeth development, incisives were the first teeth to erupt on around pregnancy day 166 (79.0% gestation length). Similarly, the northern white-tailed deer (Odocoileus virginatus

borealis) and the grey brocket deer (Mazama gouazoubira) had their first teeth eruption on pregnancy day 181 and 190 of pregnancy (90% and 95% gestation length), respectively [31,32]. In peccaries, canines are the first teeth to erupt on pregnancy day 116e120 (72.9e86.9% gestation length, respectively) [22,33]. Dentition in newborns allows the early foraging, and young ruminants begin to consume solid foods in three post-natal weeks and regularly graze with adults at 4e6 months of age [34]. In contrast, dental eruption was not observed during the intrauterine development of altricial species, such as woolly monkey [22]. The reproductive pattern of any species aims to maximize neonatal survival and depends on the selective pressures of predation on the mother and the newborn [35]. Species less frequently predated are usually altricial, while those highly predated are usually precocial [35]. Precocial species usually have longer

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Fig. 4. Relationship between the total dorsal length (TDL) and (a) the body mass, (b) biparieral diameter, (c) occipital-frontal diameter, (d) cranial circumference, (e) humerus length, (f) femur length, (g) length of thoracic limbs, (h) length of pelvic limbs, (i) thorax diameter, (j) thorax circumference, (k) abdominal diameter and (l) abdominal circumference in 39 red brocket deer (Mazama americana) embryo/fetuses. The green line represents the model best fitted to the plots, while the red line represents an expected linear trend with no intercept.

gestations with smaller litter sizes, allocating most of the reproductive energy in the fetal development. On the other hand, altricial species have shorter gestations, deliver a larger number of offspring and allocate most energy in the post-natal care of poorly developed newborns [36]. The early independence of precocial newborns avoids prolonged energetic costs for the mother and results in shorter intervals between births compared to altricial species [19,21]. The red brocket deer has been reported as a common prey in the diet of large felines (Puma concolor and Panthera onca) [37], which preferentially kill youngs and individuals with diminished locomotor capabilities [38,39], avoiding the predation of adult individuals [40]. Since newborns are vulnerable prey for natural predators, the red brocket deer produces precocial newborns with an early autonomous functionality to allow the early afterbirth control of thermoregulation, nutrition, and locomotion and sensorial processes to better detect and respond against predation. Ultrasound examination has generated important reproductive knowledge for the detection of pathological processes related to gestation and for the reproductive management of wild species [19,41,42]. However, this reproductive information is still scarce in most wild species. The present study provides fetal measures in the red brocket deer that can be used as standard estimates for the

ultrasound assessment of fetal development. The present study also describes the fetal development of main visceral organs. In the red brocket deer, the relative weight of the liver decreased from 50.0% to 27.2% between pregnancy days 87 and 210 (41.7% and 100.0% gestation length, respectively), and kept decreasing during the post-natal development, reaching a 17.0% relative weight in adults. The development of the liver was very similar to that observed in both altricial [21] and precocial species [20,22,43]. The liver is the first organ responsible for the erythropoietic function during the intrauterine development [44], which explains the presence of a large liver in fetuses of early age. The deceleration in growth of the relative weight of the liver along gestation and post-natal development is associated with the substitution of the hepatic pre-natal erythropoiesis by the bone marrow erythropoiesis in the post-natal phase [45]. In contrast, the spleen had a constant relative growth during the fetal phase, from 0.6% at 87 day of gestation to 3.7% at the end of gestation and maintained a similar relative weight (4.8%) in adulthood. The increase of the fetal spleen is related to the erythropoietic function, shared with the liver in this period, while in the post-natal phase the spleen function is related to the control of erythrocyte cell activities and the induction of immune reactions against systemic [46]. The deceleration in the spleen relative

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Fig. 5. Allometric relationship of (a) the total dorsal length versus crown-rump length, (b) biparietal diameter versus the occipital-frontal diameter, (c) humerus length versus femur length, (d) length of thoracic limbs versus length of pelvic limbs, (e) thorax diameter versus abdominal diameter and (f) thorax circumference versus abdominal circumference in 39 red brocket deer (Mazama americana) embryo/fetuses. The green line represents the model best fitted to the plots, while the red line represents an expected linear trend with no intercept.

growth in the post-natal phase could also be associated to the starting of the bone marrow erythropoiesis [45]. The gastrointestinal tract in the red brocket deer presented a relative increase from 17.6% to 35.9%, reaching a 50.9% relative weight in adulthood. In the domestic pig, a similar growth was observed during the last third of gestation [45]. In early stages of gestation, the growth of the digestive tract is favored by the ingestion of amniotic fluid [47]. The development of the digestive tract in final stages of gestation is explained by the neonate's need to perform the digestion of breast milk and solid food after weaning [48]. The rumen of newborns is physically underdeveloped and physiologically nonfunctional. During the post-natal development, inoculation and establishment of the anaerobic microbial ecosystem, initial solid food intake, and the accompanying

fermentation processes are all needed to trigger the development of the multicavity stomach [34,47,48]. Similar to other ungulates [22,43], the relative weight of the respiratory tract in the red brocket reed remained constant, ranging from 19.5% to 22.0% from day 70e159 of gestation, respectively; and diminishing slightly during the post-natal development. During the late fetal development, the pulmonary epithelium modifies and produces surfactant [49]. The post-natal development of the lungs is related to the onset of respiratory function (absent in the fetal period), the maturation of the alveolar system and the epithelium decrease to improve the gas exchange in adulthood [49,50]. The relative weight of kidneys decreased during fetal phase from 7.9% at pregnancy day 87 (41.7% gestation length) to 2.2% at the end of gestation; the adult kidneys represented 1.9% of the total

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Fig. 6. Relationship between the absolute weight of the (a) heart, (b) lungs, (c) liver, (d) tubular gastrointestinal organs, (e) kidneys, (f) spleen, (g) thymus, (h) total visceral tissues and (i) proxy weight of the musculoskeletal system, with the total dorsal length (TDL) in 31 red brocket deer (Mazama americana) fetuses. The green line represents the model best fitted to the plots, while the red line represents an expected linear trend with no intercept.

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Fig. 7. Relationship between the relative weight of the (a) heart, (b) lungs, (c) liver, (d) tubular gastrointestinal organs, (e) kidneys, (f) spleen, (g) thymus, with the log of the total visceral weight, and relationship between the relative weight of the (h) total visceral tissues and (i) proxy weight of the musculoskeletal system with the total dorsal length (TDL) in 31 red brocket deer (Mazama americana) fetuses.

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Table 2 Absolute and relative volume of the visceral organs of the red brocket deer (Mazama americana) in advanced pregnancy stage (TDL > 44.0 cm; n ¼ 03) and adulthood (n ¼ 02). Organ

Heart Thymus Lungs Liver Tubular gastrointestinal organs Kidneys Spleen Total visceral volume

Absolute volume ± SD (ml)

Relative volume ± SD (%)

Fetus

Adult

Fetus

Adult

13.1 ± 3.0 3.2 ± 1.0 34.6 ± 7.38 43.2 ± 3.3 59.2 ± 22.2 3.3 ± 0.1 5.4 ± 0.2 161.7 ± 30.2

187.3 ± 67.9 0.0 385.1 ± 106.6 383.0 ± 123.0 1142.5 ± 335.9 40.5 ± 0.7 11.0 ± 60.8 2250.0 ± 694.4

8.2 ± 1.5 2.11 ± 0.2 21.4 ± 2.4 27.2 ± 3.9 35.9 ± 7.7 2.2 ± 0.3 3.7 ± 0.9 -

8.2 ± 0.2 0 17.2 ± 0.6 17.0 ± 0.5 50.9 ± 0.8 1.9 ± 0.6 4.8 ± 1.2 -

T value

F value

df

P value

0.078 12.818 2.320 3.494 2.621 0.72 1.03 -

2.35 2.91 2.35 2.35 2.35 2.91 2.91

3 2 3 3 3 2 2 -

0.471 0.003 0.051 0.019 0.039 0.272 0.205 -

Fig. 8. Relationship between the total dorsal length (TDL) and the longitudinal diameter of the cotyledonary placenta (a), and diameter of dome-shaped caruncles (b) in 39 red brocket deer (Mazama americana) embryo/fetuses.

visceral weight. During the fetal phase, the kidneys perform the excretion of hypotonic urine inside the aminiotic cavity [51]. In the domestic pig, although the kidneys undergo the continuous formation of nephrons along the pre-natal development, their relative weight remains constant [43]. From the 21th day post-natal onwards the differentiation process of the existing nephrons initiates [52]. The thymus is involved in the maturation of the immune system. In the red brocket deer, the thymus showed a constant growth during the fetal period and started its regression in the early postnatal development when the bone marrow becomes the main responsible organ for the animals’ immunity [45]. The thymus disappeared in the adulthood [53]. The musculoskeletal system maintained a high and constant relative weight along the pre-natal development. Nevertheless, the greatest growth occurs in late stages of gestation with the ossification and the muscular development [54]. The reproductive performance of any species is related to its particular level of harvest [8,35]. Although large felids have little coincidence of activity times with the red brocket deer [55], this species is a common prey in their diets [37]. Large felids inhabiting tropical moist forests have often been classified as opportunistic predators [56], predating a large variety of prey, but mainly medium-to large-sized mammalian prey [1,57]. Preys could be classified into two groups: group-living species with a high cost of predation due to potential injury, such as the capybara and the white-lipped peccary [58,59], and solitary species associated with lower injury risk to jaguars, but that can reach large body sizes and are difficult to capture, such as deers [57]. Jaguars prefer collared peccary (Pecari tajacu), nine-banded armadillo (Dasypus novemcinctus) and paca as prey, while exhibit moderate avoidance of white-lipped peccary and red brocket deer, and complete

avoidance of tapir and small-sized species [60]. Although body size has an overwhelming importance in prey selection [61], large sizes help to reduce predation risk [60]. As response to such level of natural harvest, the red brocket deer adopted a precocial strategy to deliver an early autonomous and functional newborn in terms of thermoregulation, nutrition, locomotion and sensorial process, and consequently, with a good ability to detect and escape from predators, reducing the predation rate of young animals. Similar to other large mammals, the red brocket deer produces well-developed neonates that are relatively heavy, as a consequence of a longer gestation period [25]. In concordance, all external morphological characteristics are already present in fetuses at the end of gestation in the red brocket deer. Nevertheless, the chronology of appearance of these external characteristics suggests that the precociality level of the red brocket deer is apparently lower than other species more frequently predated by large felids, such as peccaries and the paca. For instance, the specific fetal growth velocity in the red brocket deer (a ¼ 0.072), is slightly lower than that in the paca (a ¼ 0.077) [20] and the white-lipped peccary (a ¼ 0.084) [22], but considerably higher than that found in altricial primates such as the woolly monkey (a ¼ 0.042) [21]. Classification of the mammal development into precocial and altricial species is a difficult task. Derrickson [25], Case [35] and Eisenberg [62] tended to focus on differences among neonates to determine the degrees of altriciality. However, the study on the relative timing of appearance of the main events of the fetal development may clarify the comparative development of species and the consequences on life-history traits [25]. This study presents useful information to develop more appropriate reproductive management practices and provides standard measures for the application of imaging techniques, which may improve the success

P. Mayor et al. / Theriogenology 134 (2019) 53e64

63

Fig. 9. View of (a) the cotyledonary placenta in the red brocket deer, showing the cotyledons (black asterisk) (bar: 1.5 cm); and (b) the uterine horns of a pregnant female with dome-shaped caruncules (white asterisk) (bar: 4 cm).

of captive breeding of the species, as well as the understanding of the different mammalian reproductive strategies that guarantee the newborn survival. Acknowledgments We sincerely thank all the people from the community of Nueva Esperanza in the Yavarí-Mirín River, and from the communities of
m, Boa Esperança, Bom Jesus do Bare
, Sa ~o Jose
do Urini Nova Jerusale and Belo Monte in the Aman~ a Sustainable Development Reserve, who actively participated in data collection, showing that

communal participation is an important step in the development of wildlife management. The authors have no conflict of interest to declare. We are especially thankful for the institutional support
pico y de Altura, provided by the Instituto de Investigaciones de Tro the Museo de la Universidad Nacional de la Amazonía Peruana and
n General de Flora y Fauna Silvestre from Peru. This the Direccio work was supported by the National Council of Technological and Scientific Development (CNPq, protocol 452908/2016-7, 441435/ 2007-3, 201475/2017-0 and Edital nº 016/2014 PPP-CNPq),  Pesquisa do Estado do Amazonas (FAPEAM, Fundaç~ ao de Amparo a Edital nº 016/2014 PPP-CNPq) and the Earthwatch Institute.

64

P. Mayor et al. / Theriogenology 134 (2019) 53e64

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