Functional anatomy of the female genital organs of the wild black agouti (Dasyprocta fuliginosa) female in the Peruvian Amazon

Animal Reproduction Science 123 (2011) 249–257

Contents lists available at ScienceDirect

Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Functional anatomy of the female genital organs of the wild black agouti (Dasyprocta fuliginosa) female in the Peruvian Amazon P. Mayor a,∗ , R.E. Bodmer b , M. Lopez-Bejar a a b

Department of Animal Health and Anatomy, Faculty of Veterinary, Universitat Autònoma de Barcelona, Bellaterra, E-08193 Barcelona, Spain Durrell Institute of Conservation and Ecology, University of Kent, Canterbury, Kent CT2 7NS, UK

a r t i c l e

i n f o

Article history: Received 28 June 2010 Received in revised form 22 November 2010 Accepted 10 December 2010 Available online 17 December 2010 Keywords: Reproduction Anatomy Histology Female genital tract Black agouti Dasyprocta

a b s t r a c t This study examined anatomical and histological characteristics of genital organs of 38 black agouti females in the wild in different reproductive stages, collected by rural hunters in the North-eastern Peruvian Amazon. Females in the follicular phase of the estrous cycle had greater antral follicle sizes than other females, the largest antral follicle measuring 2.34 mm. Antral follicles in pregnant females and females in luteal phase of the estrous cycle had an average maximum diameter smaller than 1 mm. In black agouti females in follicular phase, some antral follicles are selected to continue to growth, reaching a pre-ovulatory diameter of 2 mm. Mean ovulation rate was 2.5 follicles and litter size was 2.1 embryos or fetuses per pregnant female, resulting in a rate of ovum mortality of 20.8%. Many follicles from which ovulation did not occur of 1-mm maximum diameter luteinize forming accessory CL. The constituent active luteal tissues of the ovary are functional and accessory CL. Although all females had accessory CL, transformation of follicles into accessory CL occurred especially in pregnant females, resulting in a contribution from 9% to 23% of the total luteal volume as pregnancy advances. The persistence of functional CL throughout pregnancy might reflect the importance for the maintenance of gestation and may be essential for the continuous hormonal production. The duplex uterus of the agouti female is composed by two completely independent uterine horns with correspondent separate cervices opening into the vagina. In pregnant females, most remarkable observed uterine adaptations were induced by the progressive enlargement caused by the normal pregnancy evolution. The wild black agouti showed different vaginal epithelium features in accordance with the reproductive state of the female. © 2011 Elsevier B.V. All rights reserved.

1. Introduction In the Amazon region, wildlife subsistence hunting has been a traditional source of food, animal skins, and other essential items for local people (Fitz Gibbon, 1998). Hunting, however, has led to a situation in which mammalian species in forested habitats are becoming locally or even widely extinct (Redford, 1993). The agouti (Dasyprocta spp.) is one of the most frequently hunted species in the

∗ Corresponding author. Tel.: +34 3 581 2482; fax: +34 3 581 2006. E-mail address: [email protected] (P. Mayor). 0378-4320/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2010.12.006

Amazon region, representing an important source of meat for local human populations (Redford, 1993; Puertas and Bodmer, 2004). The black agouti (Dasyprocta fuliginosa) is a mediumsized hystricognath rodent, weighing from 3.5 to 7 kg. This important seed spreader has a wide distribution range from southern Mexico to Northern Argentina (Emmons, 1997). The agouti female is considered to be aseasonally polyestrus in the Amazon region, apparently breeding year-round (Ojasti, 1996). Length of the estrous cycle is 30–34 days (Weir, 1971a; Guimaraes, 2000) and estrus lasts 24 h (Guimaraes, 2000). Contrary to other rodents, the agouti has spontaneous ovulations (Weir, 1974). This

250

P. Mayor et al. / Animal Reproduction Science 123 (2011) 249–257

species has a mean gestation period length of 104 days and a litter size of 2 fetuses or newborns (Brown, 1936; Weir, 1967; Nieuwendijk, 1980; Guimaraes, 2000). Reproductive information is critical for the development of management strategies and to address conservation concerns regarding the sustainable use of any species. However, reproductive variables for the agouti in the wild are still largely unknown, and most of the data comes from animals maintained in captivity (Weir, 1967, 1971a; Guimaraes, 2000). The maintenance of the animal in captivity could produce reproductive variations due to different factors, such as the stress of captivity and diet (Quesnel et al., 1998). Therefore, it is necessary to further investigate the current reproductive performance of the wild agouti to create management tools and model population dynamics. The present report characterizes the reproductive physiology of the black agouti through the description of macroscopic and microscopic features of the female genital organs in the North-eastern Peruvian Amazon. 2. Materials and methods 2.1. Study area This study was conducted in the Tamshiyacu–Tahuayo Communal Reserve, in the Northeastern Peruvian Amazon. The reserve spans 322,500 ha of continuous forest, predominantly non-flooded terra firme forest, with a harvest per km2 and year between 1.1 and 2.7 mammals. The climate in that region is typically equatorial with an annual temperature of 22–36◦ C, a relative humidity of 80–100%, and an annual rainfall of 1500–3000 mm. Seasons are defined as dry (January–February and July–September) and wet (March–June and October–December). 2.2. Animal collection and sample analysis From 2003 to 2006, hunters living in the area where the study was conducted within the reserve collected 38 genital organs from adult female black agouties, as part of an ongoing participatory conservation program that involves local hunters in implementing community-based wildlife management. This metholodgy assured the absence of any unnecessariy cruelty in the experimental animals. Whole body and genital organs weights were recorded, and tissues were maintained in buffered 4% formaldehyde solution (v/v), and were stored at the Museo de Zoologia de la Universidad Nacional de la Amazonía Peruana (UNAP) until analysis. The genital organs of adult females were examined for evidence of embryos or fetuses. Females with at least one embryo or fetus were considered to be pregnant, and the pregnancy stage was defined as embryonic or fetal (Nomina Embryologica Veterinaria, 1994). Non-pregnant adult animals with ovaries containing active true corpora lutea (CL) were described to be in the luteal phase of the estrous cycle, while females with ovaries bearing large antral follicles and lacking true 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. Monthly

pregnancy rate was determined according the date when the agouti females were collected. Ovarian sizes (r1 , r2 and r3 ) were calculated and volume estimated by means of the formula for volume of ovoid bodies: V = 4/3(r1 r2 r3 ). Measurements of tubular organs included mean diameter and length of the uterine tubes, uterine horns, cervix and vagina, and size of the external vaginal opening. Samples of the ovaries, uterine tubes, uterus and vagina were dehydrated, embedded in paraffin wax, cut into 3␮m sections, stained with haematoxylin and eosin, PAShaematoxylin or Masson’s trichrome stains and examined under a light microscope. Ovarian follicles were classified using the optical plane through which the oocyte nucleolus was visible, according to a modified classification of Jori et al. (2002). Morphometric features of primordial, primary, small pre-antral, large pre-antral, small antral and large antral follicles were recorded. The classification of luteal tissue for hystricognath rodents was used to classify corpora lutea (Weir, 1974; Jori et al., 2002). Ovarian corpora lutea were considered active after a pregnancy was established or as indicated by luteal cell morphology (Jori et al., 2002). Depending on the diameter, viability of luteal cells and presence or absence of an oocyte, corpora lutea were classified as: true corpora lutea, differentiated as cyclic or pregnancy CL; accessory CL, luteinized non-ovulated follicles; and atretic CL. Measurements of ovarian structures were taken using a micrometric ocular. Diameters were measured as the mean length of the 2 maximum perpendicular axes. Luteal volumes were calculated using the formula: V = 4/3(D/2)3 , according to Mayor et al. (2006). Luteal volume per female was calculated as the sum of volumes from active luteal structures. Recorded ovarian variables were number and type of antral follicles, number and type of CL, luteal volume, and size of luteal cells. Recorded tubular organ variables were microscopic features and measurements of the tunica mucosa and tunica muscularis of the uterine tubes and uterus, and quantification of cornified layers and epithelium thickness of the vaginal epithelium. Density of endometrial glands was measured ranking from 1 (low density) to 5 (high density). These features were correlated to the reproductive state of the females. All variables were determined in 5 randomly selected fields at 1000×. Based on the number of true CL, ovulation rate was determined, which is expressed as the number of CL per female with ovulations. The rate of ovum mortality was calculated as the difference between the number of CL and the observed embryos or fetuses. Average litter size was determined as the total number of embryos or fetuses per total number of pregnant females. 2.3. Statistical analysis The relationships among the reproductive state and the carcass mean weight, and absolute and relative weights of the genital organs were estimated by analysis of variance. Assessments of the relationships among macroscopic

P. Mayor et al. / Animal Reproduction Science 123 (2011) 249–257

251

Fig. 1. Average distribution of antral follicles according to the follicle diameter and the reproductive state of the agouti female (n = 38).

and microscopic features of the genital organs and the reproductive state of the female were performed by analysis of variance. Means were tested for differences using the Tukey–Kramer Multiple Comparisons Test. To test the hypothesis of seasonal reproduction, monthly pregnancy rate by season was used and the chi-square test was applied. Statistical analyses were performed using GraphPad Instat (version 3.01 for Windows 95, GraphPad Software Inc., San Diego, CA, USA, 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). 3. Results Among the 38 adult black agouti females included in this study, 16 were non-pregnant females (5 in follicular and 11 in luteal phase) and 22 were pregnant females at different stages of pregnancy (Table 1). Four pregnant females were considered to be at the embryonic stage of pregnancy, with an embryo from 0.5 to 1 cm in size and limb buds present. Eighteen pregnant females were considered to be at the fetal stage of pregnancy. In the fetal stage, the fetuses showed developed eyelids, fingers and external genitalia, and all the vital organs in place. In pregnant females in the late pregnancy stage, absolute and relative weight of genital organs were larger as compared with other females (P < 0.05). Pregnancies were homogenously distributed in every month of the year, and pregnancy rate were not significantly different during the dry and wet season (55.6% and 60.0%, respectively).

3.1. Ovarian features Although the mesosalpinx partially surrounded the ovary, there is not an evident bursa ovarica. Mean ovarian volume was not significantly different in morphometric measures with varying reproductive stages of females (non-pregnant females in follicular phase, 0.69 ± 0.62 cm3 ; non-pregnant females in luteal phase, 0.47 ± 0.27 cm3 ; pregnant females, 0.74 ± 0.43 cm3 ). There were not differences in ovarian morphometric measures according the side of the ovary. In the ovary, growing follicles or CL were not externally discernible. Ovarian follicles were observed in both pregnant and non-pregnant agouti females. Fig. 1 shows the follicular distribution of antral follicles per female according the reproductive state of the female. Females in the follicular phase had a greater average number and diameter of antral follicles >1 mm (P < 0.05) than the other females (Table 2). The smallest and largest antral follicles were of a diameter of 0.25 mm and 2.34 mm, respectively. Only one pregnant female and another female in luteal phase had a single follicle with a diameter larger than 1 mm. All pregnant females and females in luteal phase had at least one functional CL. According to the number of CL of pregnancy, the mean ovulation rate was 2.50 ± 1.40 corpora lutea/female (n = 22). The number of functional CL found in pregnant females and females in luteal phase was not different. There was no difference in number of functional CL between the left and right ovary (55.7% compared to 44.3%). Pregnant agouti females had an average litter size of 2.10 ± 0.32 embryo or fetuses (n = 22) and a mean ovum

Table 1 Reproductive state, carcass and genital mean weight, and relative genital weight of the ovaries of the female agouti (n = 38). Reproductive state Non-pregnant in folicular phase Non-pregnant in luteal phase Pregnant (0–1 cm embryo) Pregnant (1–10 cm embryo) Pregnant (10–20 cm embryo)

n 5 11 4 5 13

Carcass mean weight (kg) 4.83 4.50 4.90 4.75 5.31

± ± ± ± ±

1.04 0.50 0.89 0.96 1.53

Values appearing in rows with different superscripts (a and b) are different (P < 0.05).

Genital organs mean weight (g) 54.50 40.40 51.50 50.33 117.50

± ± ± ± ±

a

2.12 15.68a 24.35a 20.11a 51.91b

Relative genital weight (%) 1.06 0.90 1.13 1.15 2.49

± ± ± ± ±

0.26a 0.32a 0.71a 0.39a 0.88b

252

P. Mayor et al. / Animal Reproduction Science 123 (2011) 249–257

Table 2 Ovarian follicular characterization according to the reproductive state of the agouti female (n = 38). Reproductive status

Number of antral follicles per female

Number of large antral follicles >1.0 mm

Antral-follicle diameter (mm)

Average largest antral follicle diameter (mm)

Largest diameter of large antral follicle (mm)

Non-pregnant females in follicular phase Non-pregnant females in luteal phase Pregnant females

10.5 ± 0.5 10.6 ± 7.2 6.4 ± 5.2

6.5 ± 3.5a 0.8 ± 1.6b 0.1 ± 0.3b

1.03 ± 0.55a (n = 52) 0.65 ± 0.23b (n = 58) 0.47 ± 0.15c (n = 84)

1.99 ± 0.27a 0.80 ± 0.30b 0.65 ± 0.23b

2.34 1.25 1.11

Values appearing in rows with different superscripts (a–c) are different (P < 0.05).

as pregnancy advanced, total luteal volume did not differ (Table 4). Luteal cells in true and accessory CL had a similar diameter (20.5 ± 6.23 compared to 22.5 ± 4.5 ␮m, respectively). Atretic CL were present in all females and presented degenerating signs in luteal cells. No corpora albicans were observed. Interstitial tissue was widely present in the agouti ovary. 3.2. Uterine tubes

Fig. 2. Detailed image of several accessory corpora lutea with remnants of zona pelucida of non-ovulated oocytes (arrowheads) (bar: 40 mm).

mortality of 0.40 ± 0.64 (20.8 ± 32.0%) oocytes or embryo per pregnancy. Table 3 shows the number, diameter and volume of CL, and luteal volume in the agouti females. There was no difference in functional CL or luteal volume per female between pregnant and non-pregnant females in luteal phase. The largest functional CL was 6.30 mm in diameter and the smallest was 2.92 mm. All females had accessory CL (Fig. 2), characterized by the presence of an inner oocyte or remnants of zona pelucida and its smaller-size (P < 0.001) compared to functional CL, and were situated peripherally displacing the CL of pregnancy towards the center. None of the accessory CL was greater than 2.5 mm in diameter, and many nonovulated folicles were observed in luteinizing processes. Although the volume of accessory CL per female increased

Length of the uterine tubes was 5.04 ± 1.23 mm and did not differ with reproductive state of the female. Table 5 shows macroscopic and microscopic measurements of the anatomical portions (infundibulum, ampulla and isthmus) of the uterine tubes. Oviduct diameter was greater in the infundibulum, relative to the ampulla and the isthmus (P < 0.0001). The epithelial mucosa had an intermittently ciliated columnar epithelium and was highly folded, with primary, secondary and tertiary folds. The tunica muscularis of the uterine tubes consisted of an inner circular layer and outer layer with longitudinal smooth muscle cells. Tunica mucosa and muscularis were thinner in the infundibulum (P < 0.0001). There were no significant differences with reproductive state of females. 3.3. Uterus A duplex uterus characterized the female agouti. The uterine horns were completely independent from each other and had separate cervices opening into the vagina (Figs. 3 and 4). Tables 6 and 7 show macro and micrometrical measurements of the uterine horns and cervix of

Table 3 Number and mean diameter (mm) and volume (mm3 ) of accessory, estrous cyclic (functional) and pregnancy CL, and mean luteal volume (mm3 ) observed in the ovaries of agouti females (n = 38). Follicular phase Accesory CL

Functional CL

Number Diameter (mm) Largest diameter (mm) Volume per accessory CL (mm3 ) Total accessory CL volume (mm3 ) per female Number Diameter (mm) Largest diameter (mm) Volume per CL (mm3 ) Total true CL volume (mm3 ) per female

Luteal volume per female %Acc CL respect luteal volume per female Values appearing in rows with different superscripts (a and b) are different (P < 0.05).

7.05 ± 1.48 ± 2.40 2.32 ± 8.32 ±

4.64 0.48

2.40 11.76 – – – – – 8.32 ± 11.76a 100.0 ± 0.0a

Luteal phase

Pregnant

8.79 ± 1.23 ± 1.80 1.52 ± 10.88 ± 2.25 ± 4.34 ± 6.30 9.20 ± 126.32 ± 136.32 ± 6.3 ±

12.90 ± 1.26 ± 2.18 1.36 ± 21.36 ± 2.50 ± 5.06 ± 6.10 8.08 ± 108.64 ± 132.64 ± 15.14 ±

11.33 0.36 2.08 16.56 0.43 1.50 2.16 120.40 138.64b 6.4b

10.79 0.40 0.88 19.44 1.40 2.12 2.08 104.96 67.84b 8.05b

P. Mayor et al. / Animal Reproduction Science 123 (2011) 249–257

253

Table 4 Comparisons of total luteal volume (mm3 ) and average volumes (in mm3 and % respect total luteal volume of the female) of accessory and pregnancy CL observed in the ovaries of pregnant agouties related to the pregnancy stage. Pregnant females were classified in early, mid and last pregnancy stage in accordance with the fetal crown-rump-length (0–1 cm, 1–10 cm and 10–20 cm, respectively). Pregnancy stage

Early pregnancy stage

Mid pregnancy stage

Last pregnancy stage

n Total luteal volume per female Volume of accessory CL per female (%) Volume pregnancy CL per female (%) Luteal volume per pregnancy CL

4 101.68 1.12 11.59 39.76

5 131.04 2.15 14.23 40.72

13 165.20 4.74 15.91 51.92

± ± ± ±

27.20 0.49a (9.11%) 3.38 (90.89%) 6.96

± ± ± ±

66.96 2.09a,b (13.69%) 8.05 (86.31%) 17.52

± ± ± ±

100.72 2.95b (22.64%) 9.65 (77.36%) 20.56

Values appearing in rows with different superscripts (a–c) are different (P < 0.05).

Table 5 Macroscopic and microscopic measurements of the uterine tube (classified in infundibulum, ampulla and isthmus portions) in the female agouti (n = 38).

Infundibulum Ampulla Isthmus

Diameter(mm)

Epithelium(␮m)

Muscular(␮m)

Lumen(mm)

0.19 ± 0.06a 0.12 ± 0.03b 0.08 ± 0.02c

18.5 ± 2.8 17.5 ± 5.9 17.9 ± 6.0

32.9 ± 21.2a 117.9 ± 50.3b 205.7 ± 52.6c

1.10 ± 0.36a 0.33 ± 0.18b 0.25 ± 0.19b

Values appearing in rows with different superscripts (a–c) are different (P < 0.05).

Table 6 Macroscopic measurements (in cm) of the uterine horns and cervix in the female agouti (n = 38). n

Non-pregnant in follicular phase Non-pregnant in luteal phase Pregnant

5 11 22

Uterine horns

Uterine cervix

Length

Diameter

Length

Diameter

12.87 ± 3.49a 10.26 ± 2.72a 16.15 ± 6.67b

0.69 ± 0.27a 0.75 ± 0.17a 0.67 ± 0.32A ,a 3.60 ± 2.51B ,b

2.02 ± 0.62 2.03 ± 0.77 1.98 ± 1.02

1.73 ± 0.43 1.85 ± 0.62 1.68 ± 0.43

Values appearing in rows with different superscripts (a and b) are different (P < 0.05). A Non-pregnant. B Pregnant uterine horns.

Table 7 Thickness (in mm) of the endometrium and myometrium of the uterine horns and cervix in the female agouti (n = 38). n

Non-pregnant in follicular phase Non-pregnant in luteal phase Pregnant

5 11 22

Uterine horns

Uterine cervix

Total thickness

Endometrium

Endometrial glands

Myometrium

Endometrium

Myometrium

1.45 ± 0.85a 1.35 ± 0.40a 0.95 ± 0.62b

1.02 ± 0.80a 0.92 ± 0.35a 0.56 ± 0.46b

2.2 ± 2.5 2.8 ± 1.3 2.3 ± 1.4

0.43 ± 0.11 0.43 ± 0.27 0.40 ± 0.28

0.08 ± 0.03 0.15 ± 0.16 0.23 ± 0.21

1.23 ± 0.23 0.70 ± 0.63 0.89 ± 0.39

Values appearing in rows with different superscripts (a and b) are different (P < 0.05).

Fig. 3. Dorsal view of the genital organs of a non-pregnant agouti female. Ovaries (Ov), uterine tube (UT), uterine horn (UH), cervix (UC), vagina (VG), perineal region (Pr), and urinary bladder (B) (bar: 4 cm).

the female agouti. In the uterine horns the endometrial epithelium was monostratified and columnar ciliated. The endometrium of the agouti female was shallow and the simple tubular glands were sparse. The myometrium consisted of two muscular layers composed of smooth muscle cells: a circular inner and a longitudinal outer layer. A vascular layer was located between both muscle layers. All pregnancies involved both uterine horns. Right and left uterine horns contained a similar number of fetuses (54.3% and 45.7%, respectively). Pregnant agouti females had a discoidal haemochorial placenta with a characteristic subplacenta (Fig. 5). Placentae were present in only one specific region of the uterus and the rest of the uterus did not show signs of pregnancy. The pregnant uterine horn had a larger length and diameter, and thinner endometrium and myometrium in the region of the placenta (P < 0.01). The uterine cervix presented a muscular, thick and firm wall. The mucosa of the endocervix had many high, thin longitudinal folds, and the epithelium was monostratified

254

P. Mayor et al. / Animal Reproduction Science 123 (2011) 249–257

Fig. 6. Detailed image of the mucosa of the endocervix composed of large columnar mucous-secreting cells with PAS-positive contents (arrowheads). PAS (bar: 200 ␮m).

Fig. 4. View of the open uterine cervix. Uterine horns and cervix are completely independent from each other through an inner gross septum. H & E (bar: 1 cm).

3.4. Vagina Table 8 shows measurements of the vagina in the female agouti. The vagina was a thin-wall tubular organ with long longitudinal folds. On the cranial side, the vaginal process of the cervix there was a protrusion into the vagina in the form of a 0.50 ± 0.03 cm enfolding. The epithelium of the vagina was covered by a stratified squamous epithelium and had significant variations depending on the reproductive state of the female (Fig. 7; P < 0.001). Vaginal epithelium in females in follicular phase was thicker (88.3 ± 40.6 ␮m), and included more layers (8.0 ± 3.9 cell layers) than females in the luteal phase (19.8 ± 1.8 ␮m and 1.8 ± 0.5 cell layers) and pregnant females (22.8 ± 1.9 ␮m and 1.9 ± 0.5 cell layers). A clear increase in cornification of the vaginal epithelium was observed in females in the follicular phase. 4. Discussion

Fig. 5. View of the discoidal placenta with a characteristic subplacenta (SP) of a pregnant agouti female. H & E (bar: 3 cm).

and columnar with large columnar mucous-secreting cells. A larger proportion of secretory cells with PAS-positive contents was observed in pregnant females and females in the luteal phase of the estrous cycle than in females in the follicular phase (Fig. 6). In pregnant females, abundant mucous secretion occupied the lumen of the endocervical canal. The myometrium was very well-developed, having an outer longitudinal layer and an inner circular layer of smooth muscle.

The aim of the present study was to provide knowledge on the reproductive physiology of the black agouti inhabiting the Amazon region, based on the anatomical and histological description of female genital organs in different reproductive states. Despite the diversity of reproductive patterns observed on the sudorder Hystricomorpha (Weir and Rowlands, 1974), the general morphology of the reproductive organs of the agouti is similar to the one observed in other South American hystricognath rodents (Erethizon sp., Mossman and Judas, 1949; Lagidium sp., Pearson, 1949; Chinchilla laniger, Weir, 1966, 1970; Myoprocta pratti, Rowlands et al., 1970; Lagos-

Table 8 Macroscopic measurements (in cm) of the vagina and external vaginal opening in the female agouti (n = 38). n

Non-pregnant in follicular phase Non-pregnant in luteal phase Pregnant

5 11 22

Vagina

Vaginal opening

Length

Diameter

Perimeter

Dorso-ventral diameter

7.77 ± 2.70 7.20 ± 0.71 8.14 ± 1.68

1.63 ± 1.00 1.45 ± 1.01 1.69 ± 0.50

4.97 ± 2.12 4.44 ± 1.61 4.96 ± 1.13

0.41 ± 0.11 0.43 ± 0.08 0.40 ± 0.18

P. Mayor et al. / Animal Reproduction Science 123 (2011) 249–257

255

Fig. 7. Section of the vagina of two non-pregnant agouti females: (A) in follicular phase, showing a pattern of developed stratification and cornification; and (B) in luteal phase, showing non-developed stratification of the vaginal epithelium. White and black arrowheads present the basal zone of the tunica mucosa. H & E (bar: 50 ␮m).

tomus maximus, Weir, 1971b; and Myocastor coypus, Felipe et al., 1998). In the present study, pregnant females were homogenously distributed year-around. In agreement with the findings of Ojasti (1996), the present study suggests that the agouti apparently breeds year round in the Northeastern Peruvian Amazon, probably due to the lack of huge seasonal differences in resources in the tropical environment that result in a sufficient food supply to sustain a pregnancy regardless of the month of the year (Gottdenker and Bodmer, 1998). Females in follicular phase of the estrous cycle had more folicles >1 mm and a greater follicle size than other females with the largest antral folicle size of 2.34 mm. Antral follicles in pregnant females and females in luteal phase had an average maximum diameter considerably smaller than 1 mm, and had many follicles from which ovulation did not occur that were in luteinizing processes. The mean ovulation rate was 2.5 follicles and litter size was 2.1 embryo or fetuses per pregnant female, resulting in a small rate of ovum mortality, averaging 20.8% oocytes or embryos per pregnancy. In this rodent, the reduction in litter size, compatible with the spatial requirement of the placenta and embryo in the uterus, is paralleled by a reduction in ovulation rate, resulting in a lesser rate of ovum mortality (Weir and Rowlands, 1974). Pregnant females and females in luteal phase had the same number of functional CL and total luteal volume. The presence of CL in pregnant females in advanced stages of pregnancy suggests the persistence of CL throughout gestation. All females had a number of previously formed accessory CL. Formation of accessory CL occurred especially during pregnancy and tended to increase as pregnancy advanced. As reported by Weir (1971a), accessory CL were distinguished from functional CL by the smaller size and occasional presence of a degenerating oocyte or remnants of the zona pellucida. The largest accessory CL was 2.4 mm

in diameter and this was smaller than any of the functional CL. Luteal cells in functional and accessory CL were uniform in appearance and indistinguishable. The accessory CL were situated peripherally and tended to displace the CL of pregnancy towards the center. Accesory CL, developed by luteinization of follicles from which ovulation did not occur, have been described in most hystricognath rodents, mares, elephants, cetaceans and some primates (Weir and Rowlands, 1974; Harrison and Weir, 1977; Almeida et al., 2003). In other hystricognaths, Rowlands et al. (1970) and Tam (1972, 1974) reported the progesterone content and probable progesterone secretory activity of the accessory CL, the sexual hormone responsible for the adaptation of the endometrium to implantation of the embryo. In the agouti, the probably progesterone requirements for pregnancy development seem to be met through the development of accessory CL. The basic pattern of ovarian events in the agouti seems to be as follows. In agouti females in follicular phase of the estrous cycle, some antral follicles are selected to complete its growth, reaching a pre-ovulatory diameter of 2 mm. After ovulation, these follicles luteinize resulting in functional CL. In case of oocyte fertilization, the functional CL (now pregnancy CL) grows to a maximum of 6.30 mm in diameter and persists throughout pregnancy. Probably, as observed in the South American plains viscacha (Jensen et al., 2006), due to the suppression of follicular apoptosis, many follicles from which ovulation did not occur of 1mm maximum diameter do not undergo atretic processes and transform to accessory CL by luteinization of the membrane granulosa. Although accessory CL are observed in all reproductive states, luteinization occurs especially in pregnant females, resulting in a contribution from 9% to 23% of the total luteal volume as pregnancy advances. In view of the existence of accessory CL, the total amount of lutein tissue in the ovary should be taken into account when considering the progesterone requirements of pregnancy. In the green acouchi (Rowlands et al., 1970) the contribu-

256

P. Mayor et al. / Animal Reproduction Science 123 (2011) 249–257

tion of the accessory CL to the concentration of ovarian progesterone is considerable at all stages of gestation and increases to more than 50% at mid-pregnancy. The persistence of functional CL throughout pregnancy and the presence of accessory CL might reflect the importance for the maintenance of gestation and may be essential for the continuous hormonal production. As in other hystricognaths (Jori et al., 2002), the presence of atretic CL in all females suggests the slow regression of functional CL. Similar to other hystricognath rodents (Weir, 1970, 1971a,b, 1974; Rowlands et al., 1970; Kleiman et al., 1979; Felipe et al., 1998; Jori et al., 2002), the duplex uterus is composed by two completely independent uterine horns with separate cervices opening into the vagina. Both uteri were completely independent from each other through an inner gross septum. In pregnant females, fetal implantation involved a specific area of the uterine horn. This area had a thinner endometrium and myometrium probably due to the progressive enlargement caused by the pregnancy. On the contrary, in pregnant females, the non-pregnant uterine horn was histologically similar with respect to the uterine horns of non-pregnant females. The endometrium had a lesser density of endometrial uterine glands independent of the reproductive state of the female. In pregnant females and females in luteal phase of the estrous cycle, abundant mucous secretion occupies the lumen of the endocervical canal. These females had a larger proportion of secretory cells with PAS-positive contents with respect to females in follicular phase of the estrous cycle. Under the influence of progesterone, the cervical mucus becomes quite viscous (Mullins and Saacke, 1989) and closes the canal so that foreign material cannot enter the uterus during gestation (Senger, 1997). Pregnant agouti females had a discoidal chorioallantoic and haemochorial placenta with a characteristic subplacenta. The agouti placenta can, therefore, be classified as deciduate. The sub-placenta is a unique structure that distinguishes hystricognath rodents from all other mammals (Luckett and Mossman, 1981; Mess, 2003). Although the subplacenta is a very obvious structure, its function is poorly understood. The function that has been ascribed to the subplacenta includes hormone production (Rodrigues et al., 2006). Other studies (Mess, 2003) suggest that the function of the subplacenta is to secrete growth factors, hormones or cytokines into fetal circulation. Consequently, the subplacenta could act as an important control-system of the reproduction of the agouti female. As reported by Weir (1971a), the vaginal cycle of the agouti follows the usual rodent pattern of mucification and cornification, and shows different vaginal epithelium features in accordance with the reproductive state of the female. Females in the follicular phase of the estrous cycle had a thicker and more cornified epithelium than other females. This feature suggests that vaginal cytological patterns may be useful indicators of the estrous phase and period of mating in the agouti. The ability of any species to support intense hunting pressures depends on its reproductive efficiency. Reproductive knowledge of species is fundamental to understand the dynamics of wild mammal populations

and to determine the sustainability of hunting using production models. Efficient reproducing mammals are more suitable as bush meat species, because they can withstand greater hunting intensity than slower reproducing species. Therefore, based on the reproductive information, species availability can be studied to withstand moderate harvesting intensity, and predictions of how particular preying species respond to different harvesting intensities (Puertas and Bodmer, 2004). Furthermore, knowledge of the reproductive biology of this species is of paramount importance to optimize the zootechnical performances of the captive agouti, and to develop appropriate management practices for this species. 5. Conclusions In conclusion, in black agouti females in follicular phase of the estrous cycle antral follicles are selected to continue to grow, reaching a pre-ovulatory diameter of 2 mm. Mean ovulation rate was 2.5 follicles and litter size was 2.1 embryos or fetuses per pregnant female, resulting in a rate of reproductive wastage of 20.8%. In pregnant females, transformation of follicles from which ovulation did not occur into accessory CL resulted in a contribution from 9% to 23% of the total luteal volume as pregnancy advances. Finally, the wild black agouti showed different vaginal epithelium features relative to the reproductive state of the female. Acknowledgements We sincerely thank all the people from the TamshiyacuTahuayo Communal Reserve (El Chino, San Pedro, 7 de Julio, Diamante and Nueva Esperanza) 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 P. Puertas, E. Puertas, M. Antúnez and P. Pérez for their kind assistance during the fieldwork. We are specially thankful for the institutional support provided by the Universidad Nacional de la Amazonía Peruana and the Dirección General de Flora y Fauna (DGFF) of Perú. References Almeida, M.M., Carvalho, M.A.M., Cavalcante-Filho, M.F., Miglino, M.A., Menezes, D.J.A., 2003. Morfological and morfometric study of the ovary in agoutis (Dasyprocta aguti, Linnaeus, 1766). Braz. J. Vet. Res. Anim. Sci. 40, 55–62. Brown, C., 1936. Rearing wild animals in captivity, and gestation periods. J. Mammal. 17, 10–13. Emmons, L.H., 1997. Neotropical Rainforest Mammals: A field Guide, third ed. University of Chicago Press, Chicago. Felipe, A., Calleja, J., Cabodevila, J., 1998. Anatomicohistological characteristics of female genital tubular organs of the South American Nutria (Myocastor coypus). Anat. Histol. Embryol. 27, 245–250. Fitz Gibbon, C., 1998. The management of subsistence harvesting: behavioural ecology of hunter and their mammalian prey. In: Caro, T. (Ed.), Behavioural Ecology and Conservation Biology. Oxford University Press, Oxford, pp. 449–474. Gottdenker, N., Bodmer, R., 1998. Reproduction and productivity of whitelipped and collared peccaries in the Peruvian Amazon. J. Zool. 245, 423–430. Guimaraes, D.A., 2000. Aspectos reproductivos e endocrinos da pubertade, ciclo estral, gestac¸ao e cio pós-parto de cutias (Rodentia: Dasyproctidae), criadas en cativerio. PhD Thesis. Belém, Brasil.

P. Mayor et al. / Animal Reproduction Science 123 (2011) 249–257 Harrison, R.J., Weir, B.J., 1977. Structure of the mammalian ovary. In: Zuckerman, P.L., Weir, B.J. (Eds.), The Ovary. Academic Press, London, pp. 112–217. Jensen, F., Willis, M.A., Abramonte, M.S., Espinosa, M.B., Vitullo, A.D., 2006. Naturally suppressed apoptosis prevents follicular atresia and oocyte reserve decline in the adult ovary of Lagostomus maximus (Rodentia, Caviomorpha). Reproduction 132, 301–308. Jori, F., López-Béjar, M., Mayor, P., López, C., 2002. Functional anatomy of the ovaries of the wild brush-tailed porcupine (Atherurus africanus, Gray 1842) from Gabon. J. Zool. 256, 35–43. Kleiman, D.G., Eisenberg, J.F., Maliniak, E., 1979. Reproductive parameters and productivity of Caviomorph rodents. In: Eisenberg, J. (Ed.), Vertebrate Ecology in the Northern Neotropics. Smithsonian Institution Press, Washington, pp. 73–183. Luckett, W.P., Mossman, H.W., 1981. Development and phylogenetic significance of the fetal membranes and placenta of the African hystricognathous rodents Bathyergus and Hystrix. Am. J. Anat. 162, 265–285. Mayor, P., Fenech, M., Lopez-Bejar, M., 2006. Ovarian features of the wild Collared Peccary (Tayassu tajacu) from Peruvian Northeastern Amazon. Gen. Comp. Endocrinol. 147, 268–275. Mess, A., 2003. Evolutionary transformations of chorioallantoic placental characters in rodentia with special reference to hystricognath species. J. Exp. Zool. A: Comp. Exp. Biol. 299, 78–98. Mossman, H.W., Judas, I., 1949. Accessory corpora lutea, lutein cell origin and the ovarian cycle in the Canadian porcupine. Am. J. Anat. 85, 1–39. Mullins, K.J., Saacke, R.G., 1989. Study of the functional anatomy of bovine cervical mucosa with special reference to mucus secretion and sperm transport. Anat. Rec. 225, 106–117. Nieuwendijk, J.G., 1980. Agoeti’s. Artis, Amst. 25, 147–151. Nomina Embryologica Veterinaria, 1994. International Committee on Veterinary Embryological Nomenclature. Zurich and Ithaca, New York. Ojasti, J., 1996. Wildlife Utilization in Latin America: Current Situation and Prospects for Sustainable Management. Food and Agriculture Organization of the United Nations, Rome. Pearson, O.P., 1949. Reproduction of a South American rodent, the mountain viscacha. Am. J. Anat. 84, 143–174. Puertas, P., Bodmer, R.E., 2004. Hunting effort as a tool for communitybased wildlife management in Amazonia. In: Silvius, K., Bodmer, R.E.,

257

Fragoso, J.M.V. (Eds.), People in Nature. Columbia University Press, New York, pp. 123–137. Quesnel, H., Pasquier, A., Mounier, A.M., Prunier, A., 1998. Influence of feed restriction during lactation on gonadotropic hormones and ovarian development in primiparous sows. J. Anim. Sci. 76, 856–863. Redford, K.H., 1993. Hunting in neotropical forests: a subsidy from nature. In: Hladik, C.M., Hladik, A., Linares, O.F., Pagezy, H., Semple, A., Hadley, M. (Eds.), Tropical Forests, People and Food: Biocultural Interactions and Applications to Development. Partenon Pub. Group, Paris, pp. 227–246. Rodrigues, R.F., Carter, A.M., Ambrosio, C.E., dos Santos, T.C., Miglino, M.A., 2006. The subplacenta of the red-rumped agouti (Dasyprocta leporina L.). Reprod. Biol. Endocrinol. 4, 31. Rowlands, I.W., Tam, W.H., Kleiman, D.G., 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, P.L., 1997. Pathways to Pregnancy and Parturition. Current Conceptions Inc., Pullman. Tam, W.H., 1972. Steroid metabolic pathways in the ovary of the chinchilla (Chinchilla laniger). J. Endocrinol. 52, 37–50. Tam, W.H., 1974. The synthesis of progesterone in some hystricognaths rodents. Symp. Zool. Soc. Lond. 34, 363–384. Weir, B.J., 1966. Aspects of reproduction in chinchilla. J. Reprod. Fertil. 12, 410–411. Weir, B.J., 1967. The care and management of laboratory hystricomorph rodents. Lab. Anim. 1, 95–104. Weir, B.J., 1970. The management and breeding of some more hystricomorph rodents. Lab. Anim. 4, 83–97. Weir, B.J., 1971a. Some observations on reproduction in the female agouti (Dasyprocta aguti). J. Reprod. Fertil. 24, 203–211. Weir, B.J., 1971b. The reproductive organs of the female plains viscacha, Lagostomus maximus. J. Reprod. Fertil. 25, 365–373. Weir, B.J., 1974. Reproductive characteristics of hystricomorph rodents. Symp. Zool. Soc. Lond. 34, 265–301. Weir, B.J., Rowlands, I.W., 1974. Functional anatomy of hystricognath rodents. In: Rowlands, I.W., Weir, B.J. (Eds.), The Biology of Hystricomorph Rodents. Zool. Soc. Lond., Academic Press, London, pp. 264–299.