Histomorphometric evaluation of the neotropical brown brocket deer Mazama gouazoubira testis, with an emphasis on cell population indexes of spermatogenic yield

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Animal Reproduction Science 127 (2011) 202– 212

Contents lists available at ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

istomorphometric evaluation of the neotropical brown brocket deerazama gouazoubira testis, with an emphasis on cell population

ndexes of spermatogenic yield

yvia Lugate Cardoso Costaa, Sérgio Luis Pinto da Mattaa,∗, Marcos de Lucca Moreiraomesb, Tarcízio Antônio Rêgo de Paulac, Karine Moura de Freitasb, Fabíola de Araújoesende Carvalhoa, Juliana de Assis Silveiraa, Heidi Dolderb, S.M.L. Chamindraniendis-Handagamad

General Biology Department, Federal University of Vic osa, Minas Gerais 36570-000, BrazilDABCF, Biology Institute, Unicamp, São Paulo 13083-863, CP 6109, BrazilVeterinary Department, Federal University of Vic osa, Minas Gerais 36570-000, BrazilDepartment of Comparative Medicine, University of Tennessee, TN 37996, USA

r t i c l e i n f o

rticle history:eceived 25 March 2010eceived in revised form 11 July 2011ccepted 22 July 2011vailable online 16 August 2011

eywords:permatogenesisestiseminiferous epitheliumertoli cellervids

a b s t r a c t

Information on the reproductive biology of neotropical cervids is scarce. Therefore, the aimof this study was to perform biometric, histologic and stereologic analyses of the brownbrocket deer Mazama gouazoubira testis, with an emphasis on the intrinsic yield and theSertoli cell index. Seven adult males kept in captivity were used. The animals were immo-bilized; anesthetized and testicle fragments were obtained by biopsy incision. The materialwas fixed, processed and examined by routine histological methods for light microscopy.The average body weight was 17.2 kg, from which 0.40% were allocated in gonads and 0.33%in seminiferous tubules, which represented 85.9% of the testis parenchyma. The meanalbuginea width and volume were 345.7 �m and 3.5 mL (5.3% of the testicular weight),respectively. The mean mediastinum volume of both testicles was 1.0 mL (1.5% of thetesticular weight) and the testicular parenchyma volume corresponded to 93.1% of totaltesticular weight (64.9 g). The seminiferous tubules diameter was 224.4 �m, while theepithelium height was 69.6 �m. On average, an adult brown brocket deer showed a totalof 1418 m of seminiferous tubules in both testicles (21.5 m per gram of testis). Each stageI seminiferous tubular cross section contained 1.10 type A spermatogonia, 13.4 primaryspermatocytes in pre-leptotene/leptotene, 13.7 spermatocytes in pachytene, 48.8 roundspermatids and 3.7 Sertoli cells. The general yield of spermatogenesis was 44.7 cells and

the Sertoli cell index was 13.2. The qualitative and quantitative description of testicular his-tology of brown brocket deer help to understand its spermatogenic process and to establishparameters for the reproductive biology of this wild species. Furthermore, the data from thepresent research will help further studies using other species of Brazilian cervids, especiallyendangered ones, making an additional effort to the species preservation.

∗ Corresponding author at: Federal University of Vic osa, General Biol-gy Department (CCBS), CEP 36570-000, Vic osa, Minas Gerais, Brazil. Tel.:55 31 3899 3361; fax: +55 31 3899 2549.

E-mail address: [email protected] (S.L.P. da Matta).

378-4320/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.anireprosci.2011.07.016

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

The species of deer in Brazil have been showing sharppopulation decline, mainly due to the habitat loss, diseaseand indiscriminate hunting (Duarte, 1997). Although less

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numerous than the domesticated ruminants, wild rumi-nants are also important for the maintenance of differentecological systems (Verme and Ullrey, 1984).

There is a growing interest in the study of cervids, eitherfor maintaining and preserving natural areas for speciesconservation or for the possibility of captive breeding(Berndt, 2005). The brown brocket deer (Mazama goua-zoubira) is a species of small deer, weighing about 18 kg andaveraging the height of 50 cm at the withers. This species iswidely distributed, covering areas from southern Uruguayto the north of Mato Grosso State (Brazil), and from theAndes mountains to the Atlantic coast (Duarte, 2006). Infor-mation on the reproductive cycle of this species is limited.Data from behavioral studies indicate that hinds are polye-strous (Pereira et al., 2006) and have a post-partum estrus(Sadlier, 1987). Based on annual analysis of testosteronelevels, testicular diameter and characteristics of the ejacu-late, the brown brocket deer is considered a non-seasonalbreeder, giving birth at all times of the year (Stallings, 1986;Eisenberg, 1989; Duarte and Garcia, 1997).

An important contribution towards conservation ofany species is the knowledge of its reproductive biology,especially in its basic aspects (Guião-Leite et al., 2006).Histological quantification of the testicular parenchyma isa key requirement for studies involving male reproduc-tive parameters (Paula et al., 2002). Its composition alongwith the relative size of the testicles is able to providevaluable information on the reproductive physiology andeven on the mating system of a given species (Kenagyand Trombulak, 1986; Paula et al., 2002). Thus, qualita-tive and quantitative study of the spermatogenic processis essential to understand the physiological patterns withwhich parameters for reproductive biology can be estab-lished (Morrow and Monfort, 1998; Azevedo et al., 2010).Therefore, the present study was designed to perform bio-metric, histologic and stereologic analyses of the brownbrocket deer M. gouazoubira testis, with an emphasis onthe intrinsic yield and the Sertoli cell index.

2. Materials and methods

2.1. Animals and tissue preparation

Seven adult males of brown brocket deer (M. goua-zoubira) were used, three from a private animal and poultryconservation station belonging to the Engenho D’águaFarm, in Ouro Preto (MG) (20◦23′28′′S, 43◦30′20′′W), threefrom the Center of Research and Conservation of Cervids(NUPECCE), in Jaboticabal (SP) (21◦15′22′′S, 48◦18′58′′W)and one maintained by the Zoobotanic Foundation, in BeloHorizonte (MG) (19◦55′15′′S, 43◦56′16′′W). All institutionsare located in Southeast Brazil.

Testis samples were collected in October. The testicularfragments were collected by incisional testicular biopsy. Todo so, the animals were restrained and kept under disso-ciative anesthesia (ketamine and xylazine; 10:1 mg/kg). Allbiopsies were previously authorized by the Brazilian Insti-

tute for the Environment and Natural Renewable Resources(IBAMA, protocol number 1946480).

The animals were weighed and both testes were mea-sured in width, thickness and length for the calculation

n Science 127 (2011) 202– 212 203

of volumes using the formula: 4/3 (�) (ABC) (A = half thewidth, B = half the thickness and C = half the length) (Guião-Leite and Paula, 2003; Mascarenhas et al., 2006).

As the density of testes in mammals is close to 1 g/mL(1.046), the weight in grams was considered equivalentto its volume in millilitre (Johnson et al., 1981; Mendis-Handagama and Ewing, 1990; Paula et al., 2002; Tae et al.,2005; Leal and Franc a, 2006). The thickness of the scrotumskin was also measured and subtracted from testicular size.After local cleansing, the scrotum skin and the fibrous tunic(spermatic tunic) were cut to expose the tunica albuginea.

Each fragment measured 4 mm in diameter and wasremoved from the middle region of the left testicle using acircular scalpel and immediately immersed in Karnovsky’sfixative (Karnovsky, 1965). The testicular albuginea, thefibrous tunica and skin were sutured separately with intra-dermal continuous irreversible end stitches.

The thickness of the tunica albuginea was mea-sured using a digital caliper (King Caliper Tools; accu-racy = 10 �m). To calculate the volume of the testicularparenchyma, the formula for the volume of an ellipsoid [4/3(�) (ABC)] was used, considering the testicular dimensions(length, width and thickness) as well as the thickness ofthe tunica albuginea (Bittencourt et al., 2004; Mascarenhaset al., 2006). The volume of testicular albuginea wasobtained by the difference between the total testicularvolume and the testicular volume without the albuginea.The mediastinum testis is a network of fibrous connec-tive tissue that extends from the upper to near the lowerextremity of the testis, and is wider above than below. Itsupports the rete testis (irregular anastomosing channels)and blood vessels of the testis in their passage to and fromthe substance of the gland (Wrobel and Bergmann, 2006).Subsequently, the volumes of the mediastinum and albug-inea were subtracted from the volume of the testis to obtainthe volume of testicular parenchyma.

Due to the natural death of two animals in a conser-vation farm in Minas Gerais State, the testes of both deerwere only used to obtain data from the mediastinum, tocomplement this study. The mediastinum was removedand weighed, and the mean value was discounted from thetesticular volume in all animals used in the present study.

The fixed material was processed routinely for infil-tration in glycolmethacrylate resin (Historesin® – Leica).Semi-serial sections (3 �m) were made using a microtomeand stained with toluidine blue/sodium borate 1%.

Gonadosomatic index (GSI), which is defined by theratio body weight and testis weight, was calculated usingthe formula TW/BW × 100 (TW = testis weight; BW = bodyweight) (Amann, 1970).

2.2. Testis stereology

Ten digital images of the testicular parenchyma weretaken randomly to calculate the volumetric proportionoccupied by the seminiferous tubules and the inter-stitium (400× magnifications). A 475 points grid was

placed on each image, in a total of 4750 points peranimal. Then, the percentage of points on the seminif-erous tubules and the interstitial tissue was calculated.The volume (mL) of each compartment of the testicular

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Table 1Biometric data of adult male brown brocket deer (Mazama gouazoubira)kept in captivity (mean ± standard error; CV = coefficient of variation).

Variables Mean ± SEM CV (%)

Body weight (kg) 17.2 ± 0.38 5.88Length of right testis (cm) 5.9 ± 0.30 13.32Width of the right testis (cm) 3.4 ± 0.11 8.99Height of the right testis (cm) 3.2 ± 0.10 8.25Length of left testis (cm) 6.1 ± 0.20 8.81Width of the left testis (cm) 3.3 ± 0.16 12.82Height of the left testis (cm) 3.3 ± 0.11 8.42Weight of right testis (g) 33.8 ± 3.40 26.53Weight of left testis (g) 35.6 ± 3.66 27.12Total weight of testes (g) 69.4 ± 6.80 25.88Gonadosomatic index (%) 0.40 ± 0.03 22.40Tunica albuginea width (�m) 345.7 ± 40.67 31.06Tunica albuginea volume (mL) 3.5 ± 0.37 27.96Mediastinum volume (mL) 1.00 ± 0.01 3.24

04 K.L.C. Costa et al. / Animal Repr

arenchyma was estimated using the formula: (% ofubule or interstitium/100) × total volume of the testiculararenchyma. The tubulesomatic index (TSI) was also cal-ulated: STV/BW × 100 (STV = seminiferous tubule volume;W = body weight) (Bittencourt et al., 2004; Sarti, 2006;arreta Junior, 2008).

The average diameter of seminiferous tubules wasbtained from 30 circular cross-sections in each animal.he seminiferous epithelium height was measured in theame sections in which the tubule diameter was obtained.o do so, the distance from the basement membrane tohe luminal edge was considered. Two diametrically oppo-ite readings were taken using a digital ruler on each crossection, using their mean value. All the morphometricalnalyses listed above were performed using digital images200× magnification) with the software Image Pro Plus,.6.0.

The total length (m) of the seminiferous tubules wasstimated from the volume occupied by seminiferousubules within the testes and the tubular diameter averageor each animal, using the formula: STV/�R2 (STV = totaleminiferous tubule volume; �R2 = area of the seminifer-us tubules cross section; R = tubular diameter/2) (Attal andourot, 1963; Dorst and Sajonski, 1974). The length of theeminiferous tubules per gram of testis was calculated byividing the total length by the testes weight.

.3. Stages of the seminiferous epithelium cycle

Stages of the seminiferous epithelium cycle were char-cterized based on the shape and location of spermatiduclei, presence of meiotic divisions, and overall semi-iferous epithelium composition (Amann and Almquist,962; Courot et al., 1970; Leal and Franc a, 2006). Thisethod, known as tubular morphology method, provides

ight stages of the seminiferous epithelium (Berndtson,977; Paula et al., 1999).

The relative stage frequencies were determined fromhe characterization and counting of 250 seminiferousubule cross-sections for each animal at 400× magnifica-ion, randomly chosen.

.4. Cell counts and cell numbers

The germ cell and Sertoli cell populations were esti-ated following the tubular morphology method, by

ounting 10 cross sections of seminiferous tubules in Stage, from 5 animals, because the biopsies made in two ofhem did not contain enough tubules in Stage 1. The type Apermatogonia (SPTGA), the primary spermatocytes in pre-eptotene (PL) and pachytene (PQ), the round spermatidsRS) and the Sertoli cells (S), with well defined nucleoli,ere counted.

The mean nuclei and nucleoli diameters, the latteror Sertoli cells, were obtained by measuring 30 nucleif each germ cell cited above. The measurements wereerformed on digital images using the software Image

ro Plus (v. 6.0). Due to variations in the size, countingf the different cell types was corrected by the formularoposed by Abercrombie (1946), further modified bymann and Almquist (1962). Based on the corrected val-

Testicular parenchyma volume (mL) 64.9 ± 6.78 27.61Volumetric proportion of T. albuginea (%) 5.3 ± 0.69 34.08Volumetric proportion of mediastinum (%) 1.5 ± 0.15 26.24

ues, it was possible to quantify the following coefficients:spermatogonial mitosis (PL/SPTGA), meiotic yield (RS/PQ),spermatogenic yield (RS/SPTGA) and Sertoli cell index((SPTGA + PL + PQ + RS)/S). The total number of Sertoli cellswas determined from the corrected counts of Sertoli cellnucleoli per seminiferous tubule cross section and the totallength of seminiferous tubules (Hochereau-de Reviers andLincoln, 1978).

Student’s t-test was employed to evaluate possiblechanges between left and right testicles weight. The soft-ware BIOESTAT 5.0 (Ayres et al., 2007) was used forstatistical analysis. Significance was set at an alpha levelequal to 0.05. All data are reported as mean ± standarderror of the mean (SEM).

3. Results

3.1. Biometric data and testis volume density

All biometric data can be found in Table 1. The bodyweight varied from 16.4 to 17.9 kg, with an average weightof 17.2 kg. The weights of right and left testis were, respec-tively, 33.8 g and 35.6 g (Table 1). These values did not varysignificantly (t = −0.37; n = 7; P = 0.72). The testis weightwas 69.4 g, leading to a 0.40% GSI (Fig. 1a and Table 1).The albuginea width (Fig. 1b) and volume were 345.7 �mand 3.5 mL (5.3% of the testicular weight), respectively.The mediastinum volume was 1.0 g (1.5% of the testic-ular weight). The value for testicular parenchyma wasobtained by subtracting albuginea and mediastinum vol-umes from total testicular volume, therefore resulting in64.9 mL, which corresponded to 93.1% of total testicularweight (Table 1).

3.2. Testis stereology

The tubular compartment of M. gouazoubira occupied

85.9% (56.03 mL) of the testicular parenchyma, whereasthe intertubular compartment performed 14.1% (8.85 mL)of the parenchyma (Table 2). The mean diameter of theseminiferous tubules was 224.4 �m, while the epithelium

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Fig. 1. Brown brocket deer (Mazama gouazoubira) testicle – (a) macroscopic view (arrow heads: mediastinum; stars: epididymis; C and Cd: capitata andcaudata regions. Histology – (b) testicular edge showing the seminiferous tubules (ST) within the parenchyma, the tunica albuginea (star) and the tunicavaginalis (multi point star); (c) a transverse section of a seminiferous tubule and a model showing how the diameter (bigger arrows) and the epithelium

elium,

s, star: p

height (smaller arrows) were taken; (d) Stage 1 of the seminiferous epithtubules, Lu: lumen, In: intertubular compartment), RS: round spermatidspermatocytes); bars = A: 1.5 cm; B–D: 50 �m and detail: 25 �m.

height was 69.6 �m. An adult animal showed a total of1418 m of seminiferous tubules. On average, there was21.5 m of seminiferous tubules per gram of testis (Fig. 1cand Table 2). The tubulesomatic index (TSI) was 0.33%(Table 2).

3.3. Stages of the seminiferous epithelium cycle and

relative stage frequencies

In M. gouazoubira, the nuclei of the Sertoli cells exhib-ited a nucleolus with approximately 2.2 �m diameter and

Table 2Volumetric proportion, seminiferous tubule morphometry and tubulesomatic

(mean ± standard error; CV = coefficient of variation %).

Variables

Seminiferous tubule volumetric proportion (%)

Interstitial tissue volumetric proportion (%)Seminiferous tubule volume (mL)

Interstitial tissue volume (mL)

Tubular diameter (�m)

Epithelium height (�m)

Total length of the seminiferous tubules in testes (m)Length of seminiferous tubules per gram of testis (m/g)

Tubulesomatic index (%)

showing the cell population within the tubule (detail) (ST: seminiferousre-leptotene/leptotene, arrow: Sertoli cell nucleus, arrow heads: type I

a loose chromatin. Spermatogonia A-type was observedfrom Stage 1 through Stage 8, near the basal lamina(Figs. 2 and 3). In Stages 5 and 6 some intermediatespermatogonia were observed. In Stage 6 the B-type sper-matogonia were observed, presenting a round or ovoidnucleus and greater content of heterochromatin (Fig. 3).Stage 1 was characterized by the presence of round

spermatids distributed into three or four cells layers.The primary spermatocytes pre-leptotene were observedclose to the basal membrane. The spermatocytes inpachytene were located between the round spermatids and

index of brown brocket deer (Mazama gouazoubira) kept in captivity

Mean ± SEM CV (%)

85.9 ± 2.15 6.6014.1 ± 2.15 40.1056.0 ± 6.64 31.27

8.9 ± 1.24 36.90224.4 ± 14.85 17.47

69.6 ± 3.66 13.901,418 ± 98.12 18.26

21.5 ± 2.30 28.230.33 ± 0.03 28.03

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Fig. 2. Histological cross-section of seminiferous tubules showing Stages 1–4 of the seminiferous epithelium cycle in M. gouazoubira (tubular morphologym type A s( lotene

s

ssctnts

ethod) – (a) Stage 1; (b) Stage 2; (c) Stage 3; (d) Stage 4. Sertoli cell (S);

Z); primary spermatocyte in pachytene (P); primary spermatocyte in dippermatids (El). toluidine blue. Bar: 30 �m.

permatocytes in pre-leptotene (Fig. 2a). Stage 2 containedpermatids in an initial phase of nuclear elongation andhromatin condensation. Primary spermatocytes under

ransition from pre-leptotene to leptotene were locatedear the basal lamina, as well as some primary sperma-ocytes in pachytene (Fig. 2b). In Stage 3, the elongatedpermatids were grouped into bunches with their heads

permatogonia (A); pre-leptotene (Pl); primary spermatocyte in zygotene(D); metaphase figure (M); round spermatids (Rs); elongating/elongated

oriented towards the basal compartment. Two gener-ations of primary spermatocytes were present at thisstage: spermatocytes in zygotene and diplotene with their

characteristically large nuclei (Fig. 2c). In Stage 4, the occur-rence of two meiotic divisions was the most characteristicaspect, regarding to observations made on metaphaseplates. The spermatocytes in diplotene stage generate the

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Fig. 3. Histological cross-section of seminiferous tubules showing Stages 5–8 of the seminiferous epithelium cycle in M. gouazoubira (tubular morphologype A spe

sperm

method) – (a) Stage 5; (b) Stage 6; (c) Stage 7; (d) Stage 8. Sertoli cell (S); ty(B); pre-leptotene (Pl); primary spermatocyte in zygotene (Z); primaryspermatids (El) and residual bodies (Rb); toluidine blue; bar: 30 �m.

secondary spermatocytes, which divide to produce theround spermatids. Some bunches of elongated spermatidsand the primary spermatocytes undergoing transition from

zygotene to pachytene were also observed (Fig. 2d). Twodistinct spermatid generations were observed in Stage 5:the recently formed round and the elongated spermatids.Some bunches of elongated spermatids were located in

rmatogonia (A); intermediate spermatogonia (In); type B spermatogoniaatocyte in pachytene (P); round spermatids (Rs); elongating/elongated

crypts of the Sertoli cells. Primary spermatocytes undergo-ing transition from zygotene to pachytene were observedbetween the round spermatids and the basal compart-

ment. Nuclei of the Sertoli cells with prominent nucleolusgenerally exhibited their longitudinal axis perpendicularlypositioned at the basal lamina (Fig. 3a). In Stage 6, all cel-lular types observed at the previous stage were present,

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0

5

10

15

20

25

30

35

40

1 2 3 4

Freq

uenc

ies

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Sta

Pre M M

F lium cyct c phase

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ig. 4. Mean percentage (±SEM) of each stage of the seminiferous epithehe cycle. PreM: pre-meiotic phase; M: meiotic phase; PosM: post-meioti

xcept for the spermatocytes in zygotene. In general, thepermatid bunches were closer to the tubular lumen, whichs a characteristic aspect for this stage. Primary spermato-ytes in pachytene were lying in the middle region of theeminiferous epithelium (Fig. 3b). In Stage 7, the bunchedroups of the elongated spermatids were dissociated fromach other and located close to the tubular lumen. Someuclei of the primary spermatocytes in pachytene werebserved in the mid-region of the seminiferous epitheliumFig. 3c). In Stage 8, the location of the elongated sper-

atids was present as these cells are at a developmentaltage ready to be delivered from the seminiferous epithe-ium. Residual bodies were lying in the luminal border ofhe seminiferous epithelium. Spermatocytes in pachytenend round spermatids were also observed. Some spermato-ytes in pre-leptotene were observed near the basal laminaFig. 3d).

The mean relative frequencies of the eight stages in. gouazoubira are shown in Fig. 4. Stages with greatest

nd least frequencies were Stages 3 (29.9%) and 7 (5.5%),espectively. The pre-meiotic phase (Stages 1–3) repre-ented 60.1%, the meiotic phase (Stage 4) represented 7.5%nd the post-meiotic phase (Stages 5–8) 35.5% of the cycleFig. 4).

.4. Cell counts and cell numbers

On average, each Stage 1 of the seminiferous tubuleross section (Fig. 1d) contained 1.10 type A spermatogonia,3.4 primary spermatocytes in pre-leptotene, 13.7 sperma-ocytes in pachytene, 48.8 round spermatids and 3.7 Sertoliells (Table 3). On average, 12.0 primary spermatocytesn pre leptotene were produced by type A spermatogonia

Table 3).

Theoretically, four spermatids should be formed fromach primary spermatocyte in a 100% efficient testis. In theresent investigation, a 9.5% loss was detected because

n Science 127 (2011) 202– 212

5 6 7 8

ges

PosM

le characterized according to tubular morphology system, and phases of.

each primary spermatocyte formed 3.62 spermatids. Thegeneral yield of spermatogenesis in M. gouazoubira semi-niferous epithelium was 44.7 cells, which means that 44.7round spermatids were produced from each spermatogo-nia (Table 3). The Sertoli cell index, which indicates theSertoli cell capacity to support germ cells in the seminif-erous epithelium, was 13.2 germ line cells (Table 3). Thenumber of Sertoli cells per gram of testis was 25.4 × 106,and this number per testis was 1.7 × 109 (Table 3).

4. Discussion

The present study is the first on brown brocket deer,regarding the description of the spermatogenic processand its features. The description of the spermatogenic pro-cess could be valuable in further studies and/or in newreproductive strategies in order to protect this endangeredspecies. The process of testicular biopsy has been used as avaluable tool in several studies on the reproductive phys-iology of the testis of wild animals, avoiding the need forcastration (Bittencourt et al., 2004; Caldeira, 2005; Azevedoet al., 2010; Guião-Leite et al., 2006; Sarti, 2006; Barroset al., 2007). It is considered a conservative method forevaluating reproductive ability because it does not causedeleterious effects to the testes, except for the removal of afragment (Galina, 1971; Lopate et al., 1989; Freneau, 1996;Feldman and Nelson, 1996; Munson et al., 1996; Faber andRoser, 2001; Mascarenhas et al., 2006).

The organization pattern of the tubular compartmentbrown brocket deer testis was similar to that describedfor most mammalian species studied (Russell et al., 1990;Franc a and Russell, 1998; Hess and Franc a, 2008). Also, themean body weight agreed with the standards described by

Duarte (2006), who reported the average weight of 18.0 kgfor animals of the same species.

The weight of the left and right testis did not show sig-nificant variation. Thus, the use of unilateral biopsy would

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Table 3Corrected cell population, by tubular cross section, at Stage I of the seminiferous epithelium and intrinsic spermatogenesis yield and Sertoli cell index ofbrown brocket deer (Mazama gouazoubira) (mean ± standard error).

Variables Animals Mean ± SEM

1 2 3 4 5

SPTG A 0.79 1.06 1.57 1.07 0.97 1.1 ± 0.13SPTC PL 8.03 9.17 19.64 19.11 10.68 13.4 ± 2.51SPTC PC 8.84 9.91 19.29 19.25 11.28 13.7 ± 2.31SPTD RS 33.15 36.09 67.00 62.93 44.86 48.8 ± 6.92SC 3.95 3.65 3.32 4.17 3.57 3.7 ± 0.15CESM 10.14 8.67 12.39 17.81 11.05 12.0 ± 1.58MY 3.75 3.64 3.47 3.27 3.98 3.6 ± 0.12GSY 41.83 34.12 42.25 58.64 46.44 44.7 ± 4.03SCI 8.39 9.90 20.18 15.09 12.58 13.2 ± 2.09SC/g T (×106) 24.22 35.61 14.66 23.06 29.20 25.4 ± 7.75SC/T (×109) 1.29 1.65 1.33 2.14 2.02 1.7 ± 0.38

ptotene): meiot

SPTG A: type A spermatogonia; SPTC PL: primary spermatocytes (pre-leSertoli cells; CESM: coefficient of efficiency of spermatogonia mitosis; MYSertoli cell per gram of testis; SC/T: Sertoli cell number per testis.

be adequate to show the dynamic state of both organs whenthe testicles of an individual have similar sizes (Lopateet al., 1989; Hunt and Foote, 1997). Therefore, the choiceof testicular biopsy method proved to be relevant in thisstudy.

The gonadosomatic index of M. gouazoubira was 0.40%,being greater than those obtained for some members of theorder Artiodactyla, such as bull (0.1%) and buffalo (0.04%)(Franc a and Russell, 1998), white-lipped peccaries (0.19%,Costa et al., 2007a), collared peccary (0.21%) (Costa et al.,2009) and wild boars (0.31%, Almeida et al., 2006). Thisvalue was less, however, than that found for sheep (0.70%,Franc a and Russell, 1998) and other domesticated breedsof sheep such as “Australian Merinos” (0.84%), “British Clunforest” (0.60%) and “Ile de France” (0.66%) (Setchell andBreed, 2006). However, the values found for the brownbrocket deer were similar to those found for domestic pigs(0.40%; Godinho and Cardoso, 1979; Okwun et al., 1996;Franc a et al., 2005) and goats (0.35%; Leal et al., 2004).

The brown brocket deer is considered a small deer withpolygynous mating system (Black-Décima, 2000; Duarte,2006). Small species have a greater allocation and energyexpenditure in testicular mass, which could explain thelarge investment of this species in testicular mass, directlyreflected in its reproductive behavior. In this case, the mat-ing system is multi-type males, which also determines thelarge testicular size. This fact is also evidenced in otherspecies of deer, being different when there is only onebreeding male whose testicles might be smaller (Kenagyand Trombulak, 1986).

The tunica albuginea and the mediastinum are partof the testicular morphology, although not participatingdirectly either in spermatogenesis or androgen secretion.In several physiological studies these structures are dis-counted from the testicular weight, to obtain the testicularparenchyma weight (Johnson et al., 1981). The brownbrocket deer had a lesser percentage of albuginea andmediastinum than most domestic species, in which the

volume ratio is around 10% (Franc a and Russell, 1998).

The testicular parenchyma is the functional part of thetestes and contains two compartments: the tubular andinterstitial (Fawcett et al., 1973; Russell et al., 1990). The

; SPTC PC: spermatocytes (pachytene); SPTD RS: round spermatids; SC:ic yield; GSY: general spermatogenic yield; SCI: Sertoli cell index; SC/g T:

ratio between these compartments varies considerablybetween species, being responsible for the difference inefficiency of sperm production (Hess and Franc a, 2008).The tubular compartment constitutes most of the testis,occupying 61–86% of mammalian testicular parenchyma(Franc a and Russell, 1998). The volumetric proportion ofseminiferous tubules in M. gouazoubira was 85.9%, simi-lar to the values found for buffalo and goats (80%, Franc aand Russell, 1998), boar (82.1%, Costa and Silva, 2006), pig(82.9%, Franc a and Cardoso, 1998) and collared peccary(83.6%, Costa et al., 2007b).

The tubulesomatic index (TSI), which is a parameter tomeasure the investment in tubules in relation to the weightof the animal, was also investigated. This index is of greatimportance in evaluating the influence of reproductivebehavior on the tubular morphology (Paula et al., 2002).The brown brocket deer allocated 0.33% of body weightin seminiferous tubules, one of the highest ever reportedfor wild animals: African lion (0.009%; Barros et al., 2007),puma (0.02%; Guião-Leite et al., 2006), jaguar (0.022%;Azevedo et al., 2010), maned wolf (0.03%; Bittencourt etal., 2004), crab-eating fox (0.042%; Caldeira, 2005), ocelot(0.074%; Sarti, 2006) and paca (0.24%; Costa et al., 2010).The largest investment in sperm production reinforcesthe trend described by Kenagy and Trombulak (1986)for smaller animals that showed polygynous behavior ormulti-male mating system.

The tubular measurement is one of the approachesused as spermatogenic activity indicators in experimentsrelated to testicular function (Godinho and Cardoso, 1979;Russell et al., 1994; Franc a and Cardoso, 1998). Althoughthe average diameter can reach 550 �m in some speciesof marsupials (Woolley, 1975), the observed value formost amniotes ranges from 180 to 300 �m (Roosen-Runge, 1977). The value found for the brown brocketdeer (224.4 �m) is within the range observed for othermammalian species, being closer to those found in pigs(224.0 �m; Franc a and Russell, 1998), white-lipped pecca-

ries (225.6 �m; Costa et al., 2007a) and goats (237.0 �m;Leal et al., 2004), while slightly less than those reportedfor boars (249.20 �m; Costa and Silva, 2006) and collaredpeccary (255.00 �m; Costa et al., 2009).

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The height of the seminiferous epithelium is essen-ial for the evaluation of sperm production (Wing andhristensen, 1982). In M. gouazoubira, the average thick-ess of the seminiferous epithelium (69.6 �m) is within theange observed for domestic animals (60–100 �m) (Hessnd Franc a, 2008). Similar values were observed in buf-aloes (60–65 �m; Hess and Franc a, 2008), collared peccary66.3 �m; Costa et al., 2004) and boar (67.5 �m; Costa andilva, 2006).

Species that have large testes and high volumetric pro-ortion of seminiferous tubules have an advantage overhose with lesser testicular weight. Thus, comparisonsetween species with different testicular weights are irrel-vant. However, when the total length of the seminiferousubules is converted to total length per gram of testis, suchomparisons become possible. The brown brocket deerhowed 21.5 m of seminiferous tubules per gram of testis.uch value was situated above the range of 10–15 m/gescribed for most livestock animals (Franc a and Russell,998), although under the values observed for Felis catus23 m/g; Godinho, 1999), Cuniculus paca (35 m/g; Costat al., 2010) and Molossus molossus (48 m/g; Morais, 2008).imilar values were reported for Capra hircus (20 m/g; Lealt al., 2004) and Sus scrofa scrofa (19.3 m/g, Costa and Silva,006 and 18.3 m/g, Almeida et al., 2006). In contrast, lessereans were observed for Tayassu pecari (15.8 m/g; Costa

t al., 2007a), Syncerus caffer (16.3 m/g; Franc a and Russell,998), Sus domesticus (11–12 m/g; Franc a and Russell,998), Bos taurus (10–18 m/g; Franc a and Russell, 1998) andayassu tajacu (10.3 m/g; Costa et al., 2004).

The eight stages of the seminiferous epithelium cyclef the brow brocket deer were similar to those found forild boar (Almeida et al., 2006), landrace boars (Garcia-Gil

t al., 2002), white-lipped peccaries (Costa et al., 2007a),ollared peccary (Costa et al., 2010) and goats (Franc at al., 1999). Usually, only one stage of the seminiferouspithelium cycle was observed in each cross section of theeminiferous tubule in brown brocket deer. These resultsre similar to those reported in capybaras (Paula et al.,999) and domestic animals (Franc a and Cardoso, 1998;ranc a and Russell, 1998; Franc a et al., 1999). In primates,permatogenesis is distributed asymmetrically resulting inhe presence of two or more stages per tubular cross sectionSharpe, 1994; Franc a and Cardoso, 1998).

Different stages may be grouped into three phasessing meiosis as a reference point, as follows: the pre-eiotic phase (after spermiation and prior to metaphase

f meiosis); the meiotic phase (two meiotic divisionsccur and the secondary spermatocytes are present); andhe post-meiotic phase (after the completion of meiosisntil spermiation) (Franc a and Russell, 1998; Hess andranc a, 2008). Relative frequencies of the eight stages ofhe seminiferous epithelium cycle in brown brocket deerere characterized and grouped into premeiotic, mei-

tic and posmeiotic phases, all of them similar to buffaloMcCool et al., 1989), ram (Cardoso and Queiroz, 1988),oat (Franc a et al., 1999), collared peccary (Costa et al.,

010), white-lipped peccaries (Costa et al., 2007a) andild boars (Almeida et al., 2006). In general, ruminantsave greater pre-meiotic stage frequencies than the com-only observed for post-meiotic stages, while meiotic

n Science 127 (2011) 202– 212

stage frequencies account for approximately 10% of thecycle (Franc a and Russell, 1998; Franc a et al., 1999). Similarvalues were also found in the present study.

The relative frequency of stages of the seminiferousepithelium cycle is essential for estimating the durationof each stage. A less frequent stage probably has a shorterduration while the stages found more frequently have agreater relative duration. Stage 3 was the most frequent(29.9%), while Stage 7 was least frequent (5.5%), very similarto those reported for goats (Franc a et al., 1999).

Efficiency of the spermatogenic process is determinedby the analysis of numeric ratios between type A sper-matogonia and the other cell types within the seminiferousepithelium (Costa et al., 2004; Franc a et al., 2005). Besidesallowing the comparison of different species, these ratiosprovide quantification of losses occurring during cell divi-sion and differentiation (Costa et al., 2007a).

The coefficient of efficiency of spermatogonial mito-sis represents the number of primary spermatocytesin pre-leptotene/leptotene derived from each type Aspermatogonia (Castro et al., 1997). The number of sper-matogonial generations in M. gouazoubira is not known,but considering that most mammalian species have sixgenerations and a 100% yield in mitotic divisions, eachtype A spermatogonia produces 64 primary spermatocytes(Franc a and Russell, 1998; Costa et al., 2007a). In that case,12.01 cells would represent a 18.8% efficiently relative tothe maximal possible number, suggesting a loss of 81.3%during this phase. In most domestic animals, the cell lossesoccur during spermatogonial mitosis, ranging from 60% to90% (Franc a and Russell, 1998).

During meiotic division, the theoretically expectednumber of round spermatids produced by each primaryspermatocyte is four cells (1:4). In the testis of the brocketbrown deer an average percentage of 90.5% of round sper-matids was observed (1:3.62). During meiotic division indomestic mammalian spermatogenesis, losses generallyrange from 10% to 30% (Franc a and Russell, 1998), but insome wild species such as capybara this number can bemuch greater (Paula et al., 2002).

The general spermatogenic yield (GSY) measures theefficiency in the spermatogenesis process as a whole.Its reliability as an evaluation index of the spermatozoaproductions is based on the fact that losses during thespermiogenic process are considered small and insignifi-cant (Amann, 1970; Russell and Peterson, 1984). The GSYof brocket brown deer was greater than that observed inwhite-lipped peccaries (25.9; Costa et al., 2007a), with thegreatest efficiency value among the Artiodactyla domesticspecies.

The relative mass of tubular tissue determines howmuch space is devoted to sperm production. In general,species with testes that have a greater proportion of theseminiferous tubule compartment produce more spermper mass unit, mainly when this aspect is associated with agreater number of Sertoli cells (SC) per testis and a greaternumber of germ cells per Sertoli cell (Sertoli cell efficiency)

(Franc a and Russell, 1998; Hess and Franc a, 2008). EachSertoli cell supports a limited number of germ cells in aspecies-specific manner (Russell and Peterson, 1984). Thenumber of Sertoli cells established before puberty deter-

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mines the rate of sperm production in sexually matureanimals (Orth et al., 1988; Franc a and Russell, 1998). TheSertoli cell efficiency of the brown brocket deer was slightlygreater than the rate observed in domestic boar (12.4)but more than two times greater than in wild boars (6.6)(Franc a and Russell, 1998; Franc a et al., 2005). The num-ber of Sertoli cells found per testis and per gram of testiswas 25.35 × 106 and 1.68 × 109, respectively. The value ofSertoli cell per gram of testis was similar to those foundfor boars (20 × 106), stallions (28 × 106), bulls (29 × 106),goats (21 × 106) and collared peccary (28 × 106) (Hess andFranc a, 2008; Leal et al., 2004; Costa et al., 2009).

The description of testicular histology of brown brocketdeer helps to understand its spermatogenic process, andto establish parameters of the reproductive biology ofthis wild species. Furthermore, the data from the presentstudy could help improving the knowledge of reproduc-tive parameters of this species, and could have implicationson subsequent research using other species of Braziliancervids, especially the endangered ones and contributingto their preservation.

Acknowledgements

The authors wish to thank the Engenho D’água Farm,in Ouro Preto (MG), the Center of Research and Conser-vation of Cervids (NUPECCE), in Jaboticabal (SP) and theZoobotanic Foundation, in Belo Horizonte (MG). Also, tothe State Forest Institute (IEF-MG), for the Master’s schol-arship, as well as the ONG/OSCIP Ambiente Brasil Centro deEstudos, for the logistics.

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