Functional Morphology of the Female Genital Organs in the Peruvian ...

American Journal of Primatology 75:545–554 (2013)

RESEARCH ARTICLE Functional Morphology of the Female Genital Organs in the Peruvian Red Uakari Monkey (Cacajao Calvus Ucayalii)* PEDRO MAYOR1,2*, MARK BOWLER2,3,4, AND CARLOS LÓPEZ‐PLANA1 1 Department of Animal Health and Anatomy, Faculty of Veterinary, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain 2 YAVACUS, Yavarí Conservación y Uso Sostenible, Iquitos, Peru 3 San Diego Zoo Global Institute for Conservation Research, Behavioural Biology Unit, Escondido, California 4 Centre for Social Learning and Cognitive Evolution, School of Psychology, University of St. Andrews, Scotland, United Kingdom

Functional morphology may provide important information that could improve methodologies for the diagnosis of the reproductive phase of females, and develop assisted breeding techniques for wildlife. This study examined features of genital organs in 19 Peruvian red uakari monkey (Cacajao calvus ucayalii) females in different reproductive stages, collected from wild animals hunted for food by rural communities in the North‐eastern Peruvian Amazon, in order to provide knowledge on the reproductive physiology of this species. The observed mean ovulation rate was 1.4 follicles, and the observed maximal follicle diameter was 0.8 cm. After ovulation, the matured follicle luteinizes resulting in functional CL. In case of oocyte fertilization, the pregnancy CL grows to a maximum of 1.2 cm in diameter, and luteal volume per female decreases related to the advance of pregnancy. Pregnant females also present follicular activity until late pregnancy, but non‐ovulated follicles do not undergo atretic processes and apparently transform to accessory CL, resulting in a contribution of 30% of the total luteal volume. All pregnant females delivered a single fetus at term, resulting in a rate of reproductive wastage of 20% of oocytes or embryos. The endometrium and the endometrial glands in non‐pregnant females in the follicular phase show a significant increase related to the follicular growth, reaching a high proliferation in non‐pregnant females in the luteal phase. The red uakari monkey showed different vaginal epithelium features in accordance with the reproductive state of the female, suggesting that vaginal cytology could be a successful methodology with which to characterize the estrous cycle of this species. The present reproductive evaluation of the Peruvian red uakari monkey provides important information that could improve the development of assisted reproductive techniques in non‐human primates. Am. J. Primatol. 75:545–554, 2013. © 2013 Wiley Periodicals, Inc. Key words:

red uakari; reproduction; Cacajao calvus; ovary; tubular genital organs

INTRODUCTION The Peruvian red uakari monkey (Cacajao calvus ucayalii) is a medium‐bodied platyrrhine considered Vulnerable by the IUCN [Veiga et al., 2008]. Uakari monkeys (genus Cacajao) generally occur at low densities and have patchy distributions [Bowler et al., 2009; Heyman & Aquino, 2010], and their populations may be apparently declining due to hunting and habitat loss [Puertas & Bodmer, 1993]. Functional morphology of the reproductive organs is a key component for the better understanding of reproductive patterns useful in maximizing reproductive efficiency in captivity and to develop assisted breeding techniques. However, assisted breeding techniques that are routine in domesticated species are not easily adapted to wildlife [Andrabi & Maxwell, 2007]. One of the major problems with the implementation of in situ and ex situ conservation

© 2013 Wiley Periodicals, Inc.

programs is the lack of availability of the biological material which is required for a better understanding of reproductive patterns [Pukazhenthi & Wildt, 2004]. Since the red uakari is difficult to breed in captivity [Fontaine, 1981] and because there are few data available on reproductive rates in wild

Contract grant sponsor: Universitat Autònoma de Barcelona; Contract grant sponsor: The Dirección General de Fauna y Flora Silvestre (DGFFS) of Perú. The authors have no conflict of interest to declare. * Correspondence to: Pedro Mayor, Department of Animal Health and Anatomy, Universitat Autònoma de Barcelona, E‐ 08193, Bellaterra, Spain. E‐mail: [email protected] Received 21 November 2012; revised 23 December 2012; revision accepted 7 January 2013 DOI 10.1002/ajp.22132 Published online 25 February 2013 in Wiley Online Library (wileyonlinelibrary.com).

546 / Mayor et al.

populations [Bowler & Bodmer, 2009; 2011; Bowler et al., 2013], the reproductive biology of the red uakari is practically unknown. This study improves the reproductive knowledge of the Peruvian red uakari monkey, characterizing the functionality of the female genital organs. METHODS Study Area This study was conducted on the Yavari‐Mirín River, in the Northeastern Peruvian Amazon. The study area spans 107,000 ha of continuous forest, predominantly non‐flooding terra firme forest. The climate in the region is typically equatorial with an annual temperature from 22 to 36°C, a relative humidity from 80% to 100%, and an annual rainfall from 1,500 to 3,000 mm. Seasons are defined as dry (January–February and July–September) and wet (March–June and October–December). Animal Collection and Sample Analysis Between 2005 and 2012, hunters living in the area where the study was conducted collected genital organs from 19 adult Peruvian red uakari monkey females, as part of an ongoing participatory conservation program that involves local hunters in implementing community‐based wildlife management. The research protocol was approved by the Research Ethics Committee for Experimentation in Wildlife at the Dirección General de Flora y Fauna Silvestre from Peru (0229‐2011‐DGFFS‐DGEFFS). No animals were killed specifically for the research and hunters were not paid for the sample collection. The hunters were trained to remove all the abdominal and pelvic organs complete with the perineal region to avoid damage to the material. Researchers were responsible for the dissection of the reproductive organs. The genital organs of adult females were examined for evidence of embryos or fetuses. Females with one embryo or fetus were considered pregnant, and the pregnancy stage was defined as embryonic or fetal [International Committee on Veterinary Embryological Nomenclature, 1994]. Non‐pregnant adult animals with ovaries containing active cyclic corpora lutea (CL) were described as being in the luteal phase of the estrous cycle, while females with ovaries bearing large antral follicles and lacking cyclic CL were considered to be in the follicular phase of the estrous cycle. In the absence of either large antral follicles or CL, the ovaries were considered inactive. The macroscopic features of the fixed genital organs were analyzed in all the experimental animals. Ovarian sizes (r1, r2, and r3) were calculated and the ovarian volume estimated by means of the formula for the volume of ovoid bodies: V ¼ (4/3)p (r1r2r3). Measurements of tubular organs included mean outer diameter and length of the uterine tubes,

Am. J. Primatol.

uterine body, cervix, vagina, and size of the external urogenital opening. All measurements were determined in three fields per tubular organ and female. Samples of the reproductive organs were dehydrated and embedded in paraffin wax. Ovaries were serially sectioned at 3 mm sections along the longitudinal axis. Every sixth section was mounted on a glass microscope slide. Three sections from the tubular organs of each female were mounted onto a glass microscope slide. Periodic acid‐Schiff (PAS) stain was used to stain connective tissues, mucus and basal laminae, and hematoxylin or Masson’s trichrome stains were used to distinguish collagen, keratine, and muscle fibers. Sections were examined under a light microscope. Ovarian CL were considered active after a pregnancy was established or as indicated by luteal cell morphology. Depending on the diameter, viability of luteal cells, the presence or absence of an oocyte, and the reproductive state of females, CL were classified as cyclic CL, pregnancy CL, accessory CL, and atretic CL [Mayor et al., 2013]. Based on the number of pregnancy CL, we determined the ovulation rate, expressed as the number of CL per female with ovulations. We calculated the rate of ovum or embryo mortality as the difference between the number of CL and the observed embryos or fetuses. Measurements of antral follicles and CL were taken using a micrometric ocular. Diameters (D) were measured as the mean length of the two maximal perpendicular axes. Luteal volumes were calculated using the formula: V ¼ (4/3)p(D/2)3. Recorded ovarian variables were number and diameter of antral follicles, and number, diameter, volume and type of CL. Luteal volume per female was calculated as the sum of volumes from active luteal structures. Recorded tubular organ variables were microscopic features and measurements of the epithelium, the tunica mucosa and the tunica muscularis of the uterine tubes and uterus, and epithelium thickness and number of cell layers of the vaginal epithelium. The density of endometrial glands was measured and ranked from 1 to 5, according the average number of glands countered in random 1‐mm2 fields at 100. Significance scoring of density of endometrial glands was 1 (0–5 glands/mm2), 2 (5–10 glands/mm2), 3 (10– 15 glands/mm2), 4 (15–20 glands/mm2), and 5 (20– 25 glands/mm2). Cornification of the vaginal epithelium was calculated as the thickness of the cornified layers respect to the total epithelial thickness in each female. These features were correlated to the reproductive state of the females. All variables were determined in five randomly selected fields. Diameters (D) of the materno‐fetal attachment were measured as the mean length of the two maximal perpendicular axes, and the area of the materno‐fetal attachment was calculated using the formula: V ¼ (4/3)p(D/2)3.

Female Genital Organs of the Red Uakari Monkey / 547

TABLE I. Average Volume of Ovaries in the Red Uakari (N ¼ 19) n Inactive females Non‐pregnant females in the follicular phase Non‐pregnant females in the luteal phase Pregnant females

Left ovary (cm3)

4 4 5 6 F3,15

Right ovary (cm3)

0.50 0.71 3.13 1.93 4.48 2.76 3.59 4.78 ¼ 1.207; P ¼ 0.341

F3,15

0.67 0.94 3.52 1.87 3.56 1.41 3.54 4.33 ¼ 1.134; P ¼ 0.367

Differences in ovarian volume among females in different reproductive states were tested using 1‐way ANOVA and Tukey–Kramer multiple comparisons test.

Statistical Analysis Assessments of the relationships between macroscopic and microscopic features of the female genital organs and reproductive state were tested by ANOVA. Differences between means were tested using the Tukey–Kramer multiple comparisons test. We used linear regression and Pearson’s correlation in order to determine the relationships between the largest follicle and number of antral follicles, between the largest follicle and density of glands of endometrium, between fetal length and total luteal volume, and between fetal length and area of the materno‐ fetal attachment. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA: www.graphpad.com). Differences with a probability value of 0.05 or less were considered significant. All values are expressed as the mean standard deviation (SD). All research conducted was in compliance with American Society of Primatologists’ guidelines for the ethical treatment of non‐human primates and adhered to the legal requirements of Perú. RESULTS Of the 19 adult Peruvian red uakari monkey females included in this study, 9 were active non‐ pregnant females (4 in the follicular and 5 in the luteal phase), and 6 were pregnant females at

different stages of pregnancy (2 at the embryonic and 4 at the fetal stage of pregnancy). Four inactive females lacked large antral follicles and CL. Ovaries The creamy white ovaries of adult red uakari monkey females were ovoid bodies with a smooth surface. The ovarian size did not show significant differences related to the reproductive state and the side of the body (Table I). Only large antral follicles were visible as transparent bodies but did not protrude above the surface. Ovarian follicles were observed in both pregnant and non‐pregnant females. Non‐pregnant females in the follicular phase showed a higher number of antral follicles >2 mm (F3,211 ¼ 21.612; P < 0.0001) and a larger antral‐follicle diameter (F3,211 ¼ 30.926; P < 0.0001) than other females (Table II). The largest antral follicle observed had a diameter of 8.06 mm. Non‐pregnant females in the follicular phase showed a non‐statistical decrease in the number of antral follicles related to the largest antral follicle diameter (r2 ¼ 0.446; P ¼ 0.332). Females in the luteal phase had fewer antral follicles than other females (F3,211 ¼ 28.892; P < 0.0001). Atretic follicles and thecal‐type interstitial tissue were widely present in the ovary. The CL did not protrude above the surface, presented a very lobed appearance and was

TABLE II. Follicular Characterization According to Reproductive Status of the Female Red Uakari (N ¼ 19)

Reproductive status

n

Number of antral follicles per female

Number of large antral follicles >2 mm

Average largest antral‐follicle diameter (mm)

Largest antral follicle diameter (mm)

Inactive females Non‐pregnant females in the follicular phase Non‐pregnant females in the luteal phase Pregnant females

85 49

21.2 9.4a 12.2 9.5a

0.00 0.00a 0.75 0.96b

1.19 0.05a 3.70 3.10b

1.25 8.06

27

5.2 5.7b

0.20 0.45a

1.64 0.68a

2.51

54

9.0 11.4a,b F3,211 ¼ 28.892; P < 0.0001

0.17 0.41a F3,211 ¼ 21.612; P < 0.0001

1.31 0.76a F3,211 ¼ 30.926; P < 0.0001

2.11

Values appearing in columns with different superscripts are significantly different (P < 0.05). Differences in number and diameter of antral follicles were tested using 1‐way ANOVA and Tukey–Kramer multiple comparisons test.

a,b

Am. J. Primatol.

548 / Mayor et al.

Figure 1. Total luteal volume (cm3) in the pregnant red uakari (N ¼ 6) according to the fetal length (cm). Relationship between the luteal volume and the fetal length was tested using linear regression and Pearson’s correlation test.

externally bounded by a zone of condensed stromal tissue representing the theca externa of the ovulatory follicle. Largest CL occupied almost the whole ovarian stroma. All (100%, 5/5) females in the luteal phase and most (83.3%; 5/6) pregnant females had at least one cyclic or pregnant CL, respectively, and the total luteal volume of pregnant females diminished according to the fetal length (r2 ¼ 0.7711; P ¼ 0.021; Fig. 1). The pregnant female in the most advanced pregnancy stage, with a fetal length of 23 cm, lacked pregnancy CL. There was no difference in CL characteristics between pregnant and non‐pregnant females in the luteal phase (Table III). Accessory CL were observed in the 20.0% (1/5) and the 50.0% (3/6) of females in the luteal phase and pregnant females, respectively. The accessory CL was characterized by the presence of remnants of zona pelucida, the lack of stromal tissue surrounding the CL and the smaller‐size compared to cyclic and

pregnancy CL. The average diameter of the accessory CL was 1.94 0.79 mm. Based on the number of CL in pregnant females with at least one CL, the mean ovulation rate was 1.44 0.55 CL/female. Since all pregnant females had one embryo or fetus (1.00 0.00 embryo or fetus/ female), mean oocyte mortality was 0.44 0.55 (20.0 27.4%) oocytes or embryos per pregnancy. Uterine Tubes The uterine tube was very simple and mesosalpinx formed no bursa ovarica. Length of the uterine tubes was 3.46 0.75 cm and did not differ with the reproductive state of the female. The infundibulum had a thinner tunica muscularis (F2,54 ¼ 63.811, P < 0.0001) and a larger lumen (F2,54 ¼ 67.892; P < 0.0001) compared to the ampulla and the isthmus (Table IV). No significant difference was observed according to the reproductive state of the

TABLE III. Number, Mean Diameter, and Volume of Accessory, Cyclic and Pregnancy Corpus Lutea, and Mean Luteal Volume Observed in the Ovaries of the Red Uakari (N ¼ 11)

Accesory CL Number Diameter (mm) Largest diameter (mm) Volume per accessory CL (mm3) Total accessory CL volume (mm3) per female Cyclic or pregnancy CL Number Diameter (mm) Largest diameter (mm) Volume per CL (mm3) Total true CL volume (mm3) per female Luteal volume per female (mm3) % True CL respect luteal volume per female % Acc CL respect luteal volume per female

Luteal phase

Pregnant

1.80 4.02 1.7 1.3 4.5 8.3 16.8 14.9 33.4

6.80 11.97 2.7 0.9 4.3 8.2 11.5 34.9 65.6

1.00 0.00 8.3 1.4 10.5 317.4 177.1 317.4 177.1 332.4 177.1 95.3 10.6 4.7 10.6

1.17 0.75 8.7 2.1 12.0 390.9 289.6 496.1 569.9 524.9 565.0 70.6 43.9 29.4 43.9

CL: corpus lutea. Differences in number and diameter of antral follicles were tested using two‐tailed ANOVA and t‐test.

Am. J. Primatol.

t9 ¼ 0.886; P ¼ 0.398 t9 ¼ 1.507; P ¼ 0.166 t9 ¼ 0.012; P ¼ 0.991 t9 ¼ 0.615; P ¼ 0.553 t9 ¼ 0.362; P ¼ 0.725 t9 t9 t9 t9 t9

¼ ¼ ¼ ¼ ¼

0.493; 0.669; 0.727; 1.219; 1.219;

P P P P P

¼ ¼ ¼ ¼ ¼

0.634 0.520 0.486 0.254 0.254


TABLE IV. Microscopic Measurements of the Uterine Tubes in the Female Red Uakari (N ¼ 19) Uterine tubes

Epithelium thickness (mm)

Infundibulum Ampulla Isthmus F2,54

14.7 14.2 15.7 ¼ 2.093;

Muscular thickness (mm)

Lumen thickness (mm)

53.1 18.2 86.1 34.6b 191.3 55.9c ¼ 63.811; P < 0.0001 a

2.4 2.3 2.2 P ¼ 0.133

F2,54

F2,54

1.45 0.57a 0.54 0.11b 0.22 0.07c ¼ 67.892; P < 0.0001

Values appearing in columns with different superscripts are significantly different (P < 0.05). Differences in microscopic measurements of the uterine tube were tested using 1‐way ANOVA and Tukey–Kramer multiple comparisons test.

a,b,c

located inside the pelvic cavity, but in pregnant females the uterine body extended to abdominal cavity. Table V shows macroscopic measurements of the uterine body and cervix. Figure 3 shows the changes in the endometrial glands related to the reproductive phase of the female. In non‐pregnant females in the follicular phase, the endometrial glands showed a significant increase related to the follicular growth (r2 ¼ 0.771; P ¼ 0.021). Non‐pregnant females in the luteal phase showed well‐developed endometrial glands and endometrium. Non‐pregnant females in the follicular

female (Table V). Whereas the mucosal lining of the fimbriated infundibulum presented a labyrinth of branching, longitudinal folds with primary, secondary, and tertiary folds, only primary folds were observed in the isthmus. Uterus The simplex uterus was an elongated organ characterized by a globular fundic portion and a long cervix that protruded into the vaginal canal (Fig. 2). The uterus of non‐pregnant females was completely

TABLE V. Macroscopic and Microscopic Measurements of the Uterine Tubes, Uterus, Vagina, and External Urogenital Opening in the Female Uakari (N ¼ 19)

n Uterine tubes Infundibulum diameter (mm) Ampulla diameter (mm) Isthmus diameter (mm) Total length (cm) Uterine body Length (cm) Diameter (cm) Total thickness (mm) Endometrium (mm) Density of endometrial glands Myometrium (mm) Uterine cervix Length (cm) Diameter (cm) Density of cervical glands Myometrium (mm) Vagina Length (cm) Diameter (cm) Thickness epithelium (mm) Number of epithelial cell layers Cornification of the epithelium External urogenital opening. Dorso‐ventral diameter (cm)

Inactive

Non‐pregnant in the follicular phase

Non‐pregnant in the luteal phase

Pregnant

4

4

5

6

2.43 1.86 1.93 3.60

0.87 0.55 0.23 1.39

2.53 1.98 1.78 3.03

0.67 0.54 0.61 0.84

2.68 2.37 2.15 3.60

0.65 0.92 0.81 0.42

2.23 2.05 1.85 3.12

0.73 0.76 0.75 0.56

F3,15 ¼ 0.365; P ¼ 0.779 F3,15 ¼ 0.409; P ¼ 0.749 F3,15 ¼ 0.276; P ¼ 0.842 F3,15 ¼ 0.629; P ¼ 0.608

1.72 1.48 1.23 0.42 0.7 0.80

0.86 0.97a 0.18a,b 0.25a,b 0.3a 0.07

1.47 1.36 2.12 1.35 4.0 0.76

0.53 0.36a 1.23a,b 0.96a,b 1.4b,c 0.27

1.86 1.37 2.58 1.78 4.5 0.55

0.85 0.48a 1.45a 1.22a 0.6c 0.14

6.83 4.22 0.83 0.28 1.6 0.78

5.65 2.29b 0.62b 0.54b 2.3a,b 0.26

F3,15 F3,15 F3,15 F3,15 F3,15 F3,15

¼ ¼ ¼ ¼ ¼ ¼

2.906; 5.381; 3.323; 3.808; 6.788; 1.524;

P P P P P P

¼ ¼ ¼ ¼ ¼ ¼

0.069 0.010 0.048 0.033 0.0041 0.241

0.93 1.22 1.33 0.68

0.38 0.49 0.58a 0.03

0.98 1.38 3.00 0.69

0.19 0.64 0.87a,b 0.04

1.09 1.37 3.25 0.69

0.21 0.48 1.50a,b 0.15

1.15 1.36 3.60 0.71

0.42 0.29 0.89b 0.30

F3,15 F3,15 F3,15 F3,15

¼ ¼ ¼ ¼

0.453; 0.107; 4.141; 0.022;

P P P P

¼ ¼ ¼ ¼

0.719 0.954 0.025 0.996

3.18 1.41 55.0 6.7 0.0 2.44

0.86 0.88 16.7a 0.8 0.0a 0.76

3.31 1.05 132.0 14.5 3.3 2.13

0.30 0.31 54.6b 10.8 1.1b 0.64

3.32 1.25 120.7 11.0 0.9 2.51

0.38 0.24 21.7b 2.7 0.7a 0.42

3.43 1.15 67.8 5.5 0.5 2.45

0.81 0.49 51.8a 2.4 0.7a 0.38

F3,15 F3,15 F3,15 F3,15 F3,15 F3,15

¼ ¼ ¼ ¼ ¼ ¼

0.124; P ¼ 0.947 0.361; P ¼ 0.782 3.888; P ¼ 0.031 2.87; P ¼ 0.071 16.51; P < 0.0001 0.425; P ¼ 0.738

Values appearing in columns with different superscripts are significantly different (P < 0.05). Differences in macroscopic and microscopic measurements of tubular organs were tested using 1‐way ANOVA and Tukey–Kramer multiple comparisons test.

a,b,c

Am. J. Primatol.

550 / Mayor et al.

difference related to the reproductive state of active females. Pregnant red uakari monkey females had a discoid‐in‐shape, hemochorial and deciduate placenta, with two discs as a region of maternal–fetal attachment (Fig. 6), showing a progressive surface increase related to the fetal length (r2 ¼ 0.9944; P ¼ 0.0028; Fig. 7). Vagina The vaginal process of the cervix formed a 0.38 0.28 cm enfolding protruding into the vagina. Macroscopic features did not show significant differences according to the reproductive state of the female (Table V). The vaginal mucosa was characterized by a stratified squamous epithelium with numerous lamina propria papilla. Non‐pregnant females in the follicular phase and in the luteal phase had a thicker epithelium than pregnant and inactive females (F3,15 ¼ 3.888; P ¼ 0.031). Non‐ pregnant females in the follicular phase had a greater epithelial cornification compared to other females (F3,15 ¼ 16.51; P < 0.0001; Fig. 8). Figure 2. Dorsal view of the genital organs of a non‐pregnant female red uakari. Ovaries (O), Oviduct (Ov), uterine body (UB), cervix (CX), vagina (VG), perineal region (Pr), and urinary bladder (Ub) (bar: 2 cm).

and the luteal phase had a greater proliferation of endometrial glands (F3,15 ¼ 6.788; P ¼ 0.0041) and a thicker endometrium (F3,15 ¼ 3.808; P ¼ 0.033), compared to pregnant females (Fig. 4). In pregnant females at the embryonic stage, the embryo implantation occupied the secretive endometriun (Fig. 5) and pregnant females at the fetal stage presented an almost non‐existent endometrium. Females in the follicular phase did not present amounts of collagen beneath the endometrial epithelium. The mucosa of the endocervix had many primary folds, and the epithelium was monostratified with large columnar PAS‐stained mucous‐secreting cells. Although glandular development in pregnant females was greater than that in inactive females (F3,15 ¼ 4.141; P ¼ 0.025), there was no significant

DISCUSSION One of the major problems with the implementation of in situ and ex situ conservation programs is the poor knowledge of reproductive performance in wild species [Andrabi & Maxwell, 2007]. Studying reproduction in Vulnerable and endangered species is difficult due to the access to a large enough sample size. In this study, genital organs were recovered from 19 female uakari monkeys to provide new information on the reproductive physiology of the Peruvian red uakari monkey. Non‐pregnant uakari females in the follicular phase showed an apparent decrease in the number of antral follicles related to the largest antral follicle diameter. The determination of follicular waves requires frequent evaluations of follicular growth within individuals. Although these evaluations were not conducted in the present study, the results of this study suggest a synchronous growth of a cohort of

Figure 3. Changes in the endometrial glands related to the reproductive phase of the red uakari female (N ¼ 15). Relationship between the largest follicle and the density of endometrial glands was tested using linear regression and Pearson’s correlation test.

Am. J. Primatol.


Figure 4. Sections of the uterine body of (A) a non‐pregnant female in the follicular phase in the early follicular growth, (B) a non‐pregnant female in the follicular phase in the late follicular growth, and (C) a non‐pregnant female in the luteal phase of the estrous cycle. H&E (bar: 500 mm). In females in the luteal stage, secretion of endometrial glands was observed with respect to females in the follicular phase.

follicles. In the Poeppig' woolly monkey, Mayor et al. [2013] suggested the synchronous growth of a cohort of follicles through the different stages of development, and Baerwald et al. [2012] suggested that multiple waves of antral follicles develop during the human menstrual cycle, developing ovarian follicular waves comparable with those documented in several animal species. Due to a process of follicular selection, in the red uakari monkey only one or two follicles survive and continue development reaching maturity with a

diameter of 1 cm. As suggested by Armstrong & Webb [1997] in domestic species, at the time of selection, the largest follicle is probably secreting a compound with a paracrine inhibitory effect on the growth of the other follicles of the cohort. Although not confirmed, this inhibitory effect on growth of the non‐selected follicles could also occur in the red uakari monkey, resulting in the high correspondence between the number of selected follicles for

Figure 5. Section of the uterine body of a pregnant red uakari female in implantational stage. The embryonary implantation (*) occupies the secretive endometriun. H&E (bar: 1 mm).

Figure 6. View of the discoid‐in‐shape, hemochorial and deciduate placenta of a pregnant red uakari with two areas of materno‐ fetal attachment (bar: 3 cm).

Am. J. Primatol.

552 / Mayor et al.

Figure 7. Total area (cm2) of the materno‐fetal attachment according to the fetal length (cm) in the pregnant red uakari (N ¼ 4). Relationship between the fetal length and the area of the materno‐fetal attachment was tested using linear regression and Pearson’s correlation test.

further development and the ovulation rate [Webb et al., 2003]. Follicular growth also occurs in pregnant females and females in the luteal phase. In common with observations in the Poeppig' woolly monkey [Mayor et al., 2013], in the red uakari monkey the largest antral follicles observed in these females had diameters between 2 and 3 mm, a diameter significantly smaller than that of the pre‐ovulatory follicle (8 mm). As in the Poeppig's woolly monkey [Mayor et al., 2013], all non‐pregnant red uakari females in the luteal phase and most pregnant females present at least one CL, and the luteal volume decreased as pregnancies progressed. Progesterone, the sexual hormone responsible for the adaptation of the endometrium to receive the implantation of the embryo [Spenser & Bazer, 2004], is primarily

synthesized in the functional CL until a functional placenta is formed. In some primates, including humans, the CL does not outlast gestation [Funk & de Mayo, 1999; Stouffer, 1999] and the placenta assumes the primary role of steroid production during the mid‐ and late stages of pregnancy as the CL gradually regresses [Funk & de Mayo, 1999; Stouffer, 1999]. Nonetheless, the need for progesterone is probably filled by the unusual occurrence of follicular development and the further formation of accessory CL [Weir & Rowlands, 1973]. In our red uakari monkey samples, the only female lacking pregnancy CL was the female in the most advanced stage of pregnancy, with a fetal length of 23 cm, suggesting the probable CL regression in advanced pregnancy. The maximal antral follicular diameter (0.8 cm) and the maximal diameter of the pregnancy CL

Figure 8. Sections of the vagina of two non‐pregnant red uakari females: (A) in the follicular phase, showing a pattern of developed cornification (bar: 50 mm); and (B) in the luteal phase, showing non‐developed cornification of the vaginal epithelium. H&E (bar: 30 mm).

Am. J. Primatol.


(1.2 cm) resulted in an approximately 1.5‐fold increase between both ovarian structures. Zuckerman & Weir [1977] concluded that the final size of the CL is related to the diameter of the mature follicle from which it arose, but there is variation from near equality to a twofold increase over that of the follicle. Formation of accessory CL occurred both in pregnant and non‐pregnant females in the luteal phase. Some follicles are apparently so sensitive to the luteinizing factors that they respond before ovulation takes place, developing the luteinization of non‐ovulated follicles [Stansfield & Allen, 2012]. In the pregnant red uakari monkey, the presence of accessory CL resulted in a contribution up to 30% of the total luteal volume but was not related to the progress of pregnancy, suggesting a probable relation with the particular activity of the functional CL in each pregnant female [Rowlands et al., 1970]. The observed mean ovulation rate was 1.44 oocytes and the litter size was 1.00 fetus per pregnant female, resulting in a rate of reproductive wastage averaging 20.0% oocytes or embryos per pregnancy, similar to the 33.3% observed in the Poeppig’s woolly monkey [Mayor et al., 2013]. This apparently high percentage of reproductive wastage means the loss of a maximal one oocyte or embryo. The reduced ovulation rate seems to be compatible with the large uterine spatial requirements of the placenta and the embryo, resulting in a lower rate of oocyte mortality [Mayor et al., 2004]. As in other New World primates [Mayor et al., 2012; Veras et al., 2009], the uterus of the red uakari monkey is composed of a globular fundic portion and a long cervix that protrudes into the vaginal canal. During the estrous cycle, changes in the endometrium proceed through two distinct phases: the proliferative or follicular phase, and the secretory or luteal phase [Burton, 1980; Dempsey, 1939; Kraemer et al., 1977; Mayor et al., 2012; Veras et al., 2009; Zuckerman & Parkes, 1932]. In the proliferative or follicular phase, the endometrium prepares the epithelium and stroma for implantation, showing a progressive development of endometrial glands related to the follicular growth. In the luteal phase, the endometrium transforms from a proliferative to a secretory type, showing a thicker endometrium with proliferation and active secretion of endometrial uterine glands. Under the influence of progesterone, the non‐pregnant uterus transforms into an enriched environment suited for the developing embryo and for maintenance of the pregnant state [Lyngset, 1968a]. In the absence of fertilization, the endometrium cannot be maintained, collapses and probably most of it is shed during a period of bleeding, resulting in the onset of menstruation. New World primates apparently show important differences related to the physiology of uterine changes. Whereas Veras et al. [2009] suggested

that the howler monkey does not exhibit visible menstrual bleeding, Mayor et al., [2012] reported in the Poeppig’s woolly monkey a dramatic loss of the functional zone of the endometrium and a menstrual bleeding that could stand as an external sign for the diagnosis of the reproductive phase. In the red uakari, the small sample size does not permit us to draw conclusive results, but the lack of large amounts of collagen, as endometrial remodeling [Tanaka et al., 2009], suggests a non‐visible menstrual bleeding. In the case of fertilization, the discoid‐in‐shape, hemochorial and deciduate placenta progressively collapses the endometrial glands and the endometrium disappears. As in most New World platyrrhine monkeys, and contrary to observations in the howler monkey [Benirschke & Miller, 1982; Miller & Benirschke, 1985] and the woolly monkey [Mayor et al., 2012], the red uakari monkey presents a bidiscoid placenta. The region of maternal–fetal attachment shows a progressive increase in surface area as pregnancy progresses. As in the brown howler monkey [Veras et al., 2009] and the woolly monkey [Mayor et al., 2012], the pregnant red uakari female presents an evident mucous secretion from the endocervical glands, closing the endocervical canal so that foreign material cannot enter the uterus during gestation [Senger, 1997]. Females in the follicular phase also presented a high density and secretion of endocervical glands. We could not quantify the endocervical secretion, but rising levels of estrogens during the follicular phase probably promote a fluid secretion so that spermatozoa may enter into the uterus around the time of ovulation [Lyngset, 1968b]. In the red uakari, the non‐glandular, stratified and squamous vaginal epithelium shows changing features in accordance with the reproductive state of the female. Whereas females in the follicular phase show a thick vaginal epithelium and a clear pattern of cornification, females in the luteal phase maintain the epithelial thickness but lose the epithelial cornification. Finally, pregnant and inactive females have a thin and non‐cornified epithelium. As observed in other New World monkeys [Dempsey, 1939; Gluckman et al., 2004; Goodman & Wislocki, 1935; Mayor et al., 2012] but not in the howler monkey [Veras et al., 2009], the vaginal features observed suggest that vaginal cytology could be a successful methodology in order to characterize the estrous cycle of the red uakari monkey. In practice, current reproductive biotechnologies are inefficient for many endangered animals due to insufficient knowledge of basic reproductive factors [Andrabi & Maxwell, 2007]. The present reproductive evaluation of the Peruvian red uakari monkey provides important morphological and physiological information that could be directed towards the development of management strategies for the species. Furthermore, this study may improve the

Am. J. Primatol.

554 / Mayor et al.

development of imaging diagnoses for monitoring females. ACKNOWLEDGMENTS We sincerely thank all the people from the community of Nueva Esperanza in the Yavarí‐Mirín River, who actively participated in data collection, which shows that communal participation is an important step in the development of wildlife management. We also extend our thanks to Dr. Richard Bodmer and the Wildlife Conservation Society for their kind assistance during the fieldwork. REFERENCES Andrabi SMH, Maxwell WMC. 2007. A review on reproductive biotechnologies for conservation of endangered mammalian species. Anim Reprod Sci 99:223–243. Armstrong DG, Webb R. 1997. Ovarian follicular dominance: the role of intraovarian growth factors and novel proteins. Rev Reprod 2:139–146. Baerwald R, Adams GP, Pierson RA. 2012. Ovarian antral folliculogenesis during the human menstrual cycle: a review. Hum Reprod Update 18:73–91. Benirschke K, Miller CJ. 1982. Anatomical and functional differences in the placenta of primates. Biol Reprod 26: 29–53. Bowler M, Barton C, McCann‐Wood S, Puertas P, Bodmer R. 2013. Annual variation in breeding success and changes in population density of Cacajao calvus ucayalii in the Lago Preto conservation concession, Peru. In: Veiga LM, Barnett AA, Ferrari SF, Norconk MA, editors. Evolutionary biology and conservation of Titis, Sakis and Uacaris. Cambridge, UK: Cambridge University Press. p 173–178. Bowler M, Bodmer R. 2009. Social behavior in fission–fusion groups of red uakari monkeys (Cacajao calvus ucayalii). Am J Primatol 71:976–987. Bowler M, Bodmer RE. 2011. Diet and food choice in Peruvian Red Uakaris (Cacajao calvus ucayalii): selective or opportunistic seed predation? Int J Primatol 32:1109–1112. Bowler M, Noriega J, Recharte M, Puertas PE, Bodmer RE. 2009. Peruvian red uakari monkeys (Cacajao calvus ucayalii) in the Pacaya‐Samiria National Reserve—a range extension across a major river barrier. Neotrop Primates 16:34–37. Burton GJ. 1980. Light microscopy of the endometrium of the dusky leaf‐monkey (Presbytis obscura). J Reprod Fert 59:107–111. Dempsey EW. 1939. The reproductive cycle of the new world monkeys. Am J Anat 64:381–405. Fontaine R. 1981. The Uakaris, genus Cacajao. In: Coimbra‐ Filho AF, Mittermeier RA, editors. Ecology and behavior of neotropical primates. Rio de Janeiro: Academia Brasileira de Ciencias. p 443–493. Funk CR, de Mayo FJ. 1999. Progesterone actions on reproductive tract. In: Knobil E, Neill JD, editors. Encyclopedia of reproduction. San Diego, CA: Academic Press. p 6–16. Gluckman TL, Walz SE, Schultz‐Darken N, Bolton ID. 2004. Cytologic assessment of the vaginal epithelium in the common marmoset (Callithrix jacchus): a preliminary new approach to reproductive screening. Contemp Top Lab Anim Sci 43:28–31. Goodman L, Wislocki GB. 1935. Cyclical uterine bleeding in a New world monkey (Ateles geoffroyi). Anat Rec 61:379–387.

Am. J. Primatol.

Heymann EW, Aquino R. 2010. Peruvian red uakaris (Cacajao calvus ucayalii) are not flooded‐forest specialists. Int J Primatol 31:751–758. International Committee on Veterinary Embryological Nomenclature. 1994. Nomina embryologica veterinaria. New York: World Association of Veterinary Anatomists. Kraemer DC, Maqueo M, Hendrickx AG, Vera‐Cruz NC. 1977. Histology of the baboon endometrium during the menstrual cycle and pregnancy. Fert Steril 28:482–487. Lyngset O. 1968a. Studies on reproduction in the goat. I. The normal genital organs of the pregnant goat. Acta Vet Scand 9:242–256. Lyngset O. 1968b. Studies on reproduction in the goat. I. The normal genital organs of the non‐pregnant goat. Acta Vet Scand 9:208–222. Mayor P, Bowler M, López‐Plana C. 2013. Ovarian functionality in Poeppig’s woolly monkey (Lagothrix poeppigii). Anim Reprod Sci 136:310–316. Mayor P, Bowler M, López‐Plana C. 2012. Anatomicohistological characteristics of the tubular genital organs of the female woolly monkey (Lagothrix poeppigii). Am J Primatol 74:1006–1016. Mayor P, Jori F, López‐Béjar M. 2004. Anatomicohistological characteristics of the tubular genital organs of the female collared peccary (Tayassu tajacu) from north‐eastern Amazon. Anat Histol Embryol 33:65–74. Miller PW, Benirschke K. 1985. A large allantoic sac in the placenta of the spider monkey Ateles geoffroyi. Placenta 6:423–426. Puertas P, Bodmer RE. 1993. Conservation of a high diversity primate assemblage. Biodivers Conserv 2:586–593. Pukazhenthi BS, Wildt DE. 2004. Which reproductive technologies are most relevant to studying, managing and conserving wildlife? Reprod Fertil Dev 16:33–46. Rowlands IW, Tam WH, Kleiman DG. 1970. Histological and biochemical studies on the ovary and of progesterone levels in the systemic blood of the green acouchi (Myoprocta pratti). J Fertil 22:533–545. Senger PL. 1997. Pathways to pregnancy and parturition. Pullman; WA: Current Conceptions Inc. Spenser TE, Bazer FW. 2004. Conceptus signals for the establishment and maintenance of pregnancy. Reprod Biol Endocrinol 2:49. Stansfield FJ, Allen WR. 2012. Luteal maintenance of pregnancy in the African elephant (Loxodonta africana). Reproduction 143:845–854. Stouffer RL. 1999. Corpus luteum of pregnancy. In: Knobil E, Neill JD, editors. Encyclopedia of reproduction. San Diego, CA: Academic Press. p 709–717. Tanaka T, Wang C, Umesaki N. 2009. Remodelling of the human endometrial is regulated by laminin and type IV collagen. Int J Mol Med 23:173–180. Veiga LM, Bowler M, Silva JS, et al. 2008. Cacajao calvus. In: IUCN 2011. IUCN Red list of threatened species. Version 2011.2. http://www.iucnredlist.org Accessed May 30, 2012. Veras MM, Marqués KV, Miglino MA, García E. 2009. Observations on the female internal reproductive organs of the brown howler monkey (Alouatta guariba clamitans). Am J Primatol 71:145–152. Webb R, Nicholas B, Gong JG, et al. 2003. Mechanisms regulating follicular development and selection of the dominant follicle. Reprod Suppl 61:71–90. Weir BJ, Rowlands IW. 1973. Reproductive strategies of mammals. Annu Rev Ecol Syst 4:139–163. Zuckerman L, Weir JJ. 1997. The ovary. New York: Academic Press. Zuckerman S, Parkes AS. 1932. The menstrual cycle of the primates: the cycle of the baboon. Proc Zool Soc London 102:139–192.