Testis morphometry and kinetics of spermatogenesis in the feral pig (Sus scrofa)

Accepted Manuscript

Title: Testis morphometry and kinetics of spermatogenesis inthe feral pig (Sus scrofa)

Author: Deiler S. Costa Fabio J.C. Faria Carlos A.C.Fernandes Juliana C.B. Silva Sarah A. Auharek

PII: S0378-4320(13)00271-6DOI: http://dx.doi.org/doi:10.1016/j.anireprosci.2013.09.007Reference: ANIREP 4836

To appear in: Animal Reproduction Science

Received date: 19-7-2013Revised date: 11-9-2013Accepted date: 13-9-2013

Please cite this article as: Costa, D.S., Faria, F.J.C., Fernandes, C.A.C., Silva,J.C.B., Auharek, S.A., Testis morphometry and kinetics of spermatogenesisin the feral pig (Sus scrofa), Animal Reproduction Science (2013),http://dx.doi.org/10.1016/j.anireprosci.2013.09.007

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http://dx.doi.org/doi:10.1016/j.anireprosci.2013.09.007

http://dx.doi.org/10.1016/j.anireprosci.2013.09.007

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Testis morphometry and kinetics of spermatogenesis in the feral pig 1

(Sus scrofa) 2

Deiler S. Costaa*, Fábio J.C. Fariaa, Carlos A.C. Fernandesb, Juliana C.B. Silvac, Sarah 3

A. Auharekd 4

5

aFaculty of Veterinary Medicine and Animal Science, Federal University of Mato 6

Grosso do Sul, Av. Filinto Muller, 2443, Vila Ipiranga, Campo Grande, MS, 79070-7

900, Brazil. bBiotran, Alfenas, MG, Brazil. cESALQ, Piracicaba, SP, Brazil. dCentre of 8

Biological Science and Health UFMS 9

10

*Corresponding author. Tel.: +55 067 3345 3632; fax: +55 067 3345 3600; 11

e-mail address: [email protected] 12

13

ABSTRACT 14

The feral pig (Sus scrofa sp) also known as Monteiro pig, originated from a domestic 15

pig breed that was introduced into Pantanal region in Brazil in the eighteenth century. 16

Although the feral pig has commercial potential, there are few reports in the literature 17

concerning the reproductive biology of this species. Therefore, the aim of this study was 18

to further describe the feral pig testis parenchyma as well as characterize the stages of 19

the seminiferous epithelium cycle by tubular morphology method, and to evaluate the 20

number of differentiated spermatogonia generations in this species. Eight sexually 21

mature feral pigs were analyzed. Fragments of testes were embedded in plastic resin and 22

used to prepare slides for morphometrical studies. It was concluded that the feral pig has 23

six generations of differentiated spermatogonials (A1, A2, A3, A4, In, B) and that the 24

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cellular composition in the eight stages of the seminiferous epithelium cycle of these 25

animals were very similar to those reported in species of suidae and tayssuidae already 26

studied. 27

28

Keywords: Testis, Stages, Seminiferous epithelium cycle, Spermatogonia 29

30

1. Introduction 31

The feral pig (Sus scrofa sp), also known as Monteiro pig, originated from a 32

domestic pig breed that was introduced into Pantanal region in Brazil in the second half 33

of the eighteenth century (Cavalcanti, 1985) coexisting with native peccaries 34

(Tayassuidae) and this co-existence could be the cause of the decrease of the peccaries 35

population (Alho & Lacher, 1991). Feral pigs are major contributors to biomass of 36

mammals in the Pantanal, reflecting the ecological importance of this species to the 37

region (Lacher et al., 1986). Previous studies showed that feral pig meat has less fat and 38

cholesterol compared with the meat of most domestic animals and it is 39

socioeconomically important to the local population as subsistence hunting (Sollero, 40

2006). Although the feral pig has commercial potential, there are few reports in the 41

literature concerning the reproductive biology of this species (Costa et al., 2011; 42

Macedo et al., 2011). 43

Spermatogenesis is a cyclical and highly organized process that occurs in the 44

seminiferous tubules, where a diploid cell differentiates into a haploid cell, the 45

spermatozoid. This process is made up of different cell associations called stages, which 46

are established before puberty and classified based on changes in the shape of spermatid 47

nucleus - occurrence of meiotic divisions and the arrangement of spermatids within the 48

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germinal epithelium. Spermatogenesis last from 30 to 75 days in mammals and this 49

period is under the control of the germ cell genotype (Russell et al., 1990; Johnson, 50

1991; França et al., 2008; França and Russell, 1998; França et al, 2005). 51

The spermatogenesis involves three classes of germ cells: spermatogonia, 52

spermatocytes and spermatids. This process can be divided into three functional and 53

morphologically distinct phases named spermatogonial (proliferative or mitotic), 54

spermatocytic (meiotic) and spermiogenic (differentiation) phases, each one 55

characterized by morphological and biochemical changes in the components of the 56

cytoplasm and cellular nucleus of the germ cells (Courot et al., 1970; Russell et al., 57

1990; Sharpe, 1994). 58

The spermatogenic cells (germ cells) are well arranged in the seminiferous 59

tubules, consisting in cellular associations that characterize stages of the seminiferous 60

epithelium cycle, which are segmental (only one stage per tubular cross-section) in most 61

domestic mammals already investigated and helicoidal in some primates, including 62

humans (Leblond & Clermont, 1952, Russel et al., 1990; França et al., 2005). Then, 63

germ cells within each layer of the seminiferous epithelium change in synchrony with 64

the other layers over time. The cells do not migrate laterally along the length of the 65

seminiferous tubule. A coordinate order of the stages is observed, whereby sequential 66

stages occur with repetition along the length of the tubules, in a wave of the 67

seminiferous epithelium (Castro et al., 1997; Hess & França, 2008). In addition, the 68

identification of the different stages of the seminiferous epithelium is essential to 69

perform quantitative studies of the spermatogenesis, which is important to understand 70

the normal spermatogenesis, as well as to determine the specific stages of the process 71

that could be affected by treatment or drug administration (Berndtson, 1977). 72

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Therefore, the aim of this study was to further characterize the sexually mature 73

feral pig testis parenchyma as well as characterize the stages of the seminiferous 74

epithelium cycle by tubular morphology method, and to evaluate the number of 75

differentiated spermatogonia generations in free-ranging feral pigs (Sus scrofa sp). 76

77

2. Material and methods 78

2.1. Animals 79

Eight fully sexually mature male free-ranging feral pigs were used in the present 80

study. The animals were captured in Pantanal do Rio Negro, Mato Grosso do Sul, Brazil 81

(IBAMA license for collection #1916054). After capture, the animals were sedated with 82

intramuscular azaperone (Stresnil® Janssen Animal Health) 1.0 mL/20kg associated 83

with 10 mg of Diazepan® and submitted to bilateral orchiectomy. Then, the animals 84

were monitored until complete recovery and returned to their natural environment. All 85

surgical procedures were performed by a veterinarian and followed approved guidelines 86

for ethical treatment of animals. 87

88

2.2. Tissue preparation 89

The testes were fixed by gravity-fed perfusion through the testicular artery with 90

0.9% saline containing 5000 IU of Liquemine® for 15 minutes at room temperature and, 91

subsequently, with 4% buffered glutaraldehyde for 20 minutes (Costa et al. 2007). After 92

fixation, testes were trimmed from the epididymis, weighed, and cut longitudinally with 93

a razor blade. Tissue samples with dimensions of approximately 3.0 mm in diameter, 94

5.0 in width and 8.0 mm in length were obtained and the fragments were immediately 95

re-fixated by immersion, in a new glutaraldehyde solution at 4% in phosphate buffer 0.1 96

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M (pH 7.4), for at least 2 hours. Testis fragments were routinely processed and 97

embedded in glycol methacrylate (Leica Historesin Embedding Kit®). Subsequently, 4-98

µm-thick sections were obtained and stained with toluidine blue – 1% sodium borate 99

solution. 100

101

2.3. Testis Morphometry 102

To perform light microscopic investigations, images were obtained using a 103

digital camera (Leica DFC400) attached to a light microscope (Leica DM 2500) at 400 104

and 1000 x magnification, and these images were analyzed with the aid of morphometry 105

software ImageJ 1.34 (Rasband, 2005). To estimate the tubular diameter of the 106

seminiferous tubules, at least 20 tubular profiles that were round or nearly round were 107

chosen randomly and measured for each animal. The volume densities of the testis 108

tissue components were determine using a 560-intersection grid in each image. A total 109

of 6720 points were scored for each animal. 110

Points were classified as one of the following: seminiferous tubule (comprising tunica 111

propria, epithelium and lumen), Leydig cell, connective tissue, blood and lymphatic 112

vessels. The volume of each testis component was determined as the product of its 113

volume density and testis volume. Artifacts were rarely seen and were not included in 114

the data. 115

116

2.4. Cell Counts 117

The seminiferous epithelium cycle was staged according to the tubular 118

morphology method (Courot et al., 1970, Ortavant et al., 1977, Swierstra, 1968). The 119

number of germ and Sertoli cells, for each animal, was estimated by the analysis of cell 120

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populations in 20 cross-sections of seminiferous tubules of circular profile that included 121

different stages of the cycle. The following cellular types were counted: type A 122

spermatogonia, in all eight stages; intermediate- type spermatogonia, in stage 6; type B 123

spermatogonia, in stage 7; pre-leptotene/leptotene primary spermatocytes, in stages 8, 1 124

and 2; zygotene primary spermatocytes, in stages 3, 4 and 5; pachytene primary 125

spermatocytes, in stage 3; round spermatids, in stages 5, 6, 7, 8 and 1; and Sertoli cells, 126

in all eight stages. 127

The count obtained for each cellular type was corrected for the mean nuclear 128

diameter and thickness of the section, using the Abercrombie (1946) formula modified 129

by Amann (1962). Because Sertoli cells have irregular nuclei, the correction was made 130

from the mean nucleolar diameter. Then, only nuclei with evident nucleolus were 131

counted. 132

The mean nuclear diameter (MND) was obtained by the means value from the 133

analysis of 10 nuclei in each cell type, per stage of the seminiferous epithelium cycle, in 134

each animal. For type A spermatogonia, which presents ovoid or slightly elongated 135

nuclei, the mean values were obtained from the largest and smallest nuclear diameter. 136

Because the Leydig cell nucleus is round or nearly round, its volume was 137

determined from its mean nuclear diameter. For this purpose, 30 nuclei where a 138

nucleolus was observed were measured for each animal. Leydig cell nuclear volume 139

was expressed in µm3 and obtained by the formula 4 ⁄ 3πR3, where R is nuclear diameter 140

⁄ 2. To calculate the proportion between nucleus and cytoplasm, a 560-point square 141

lattice was placed over the captured image at 1000x magnification. One thousand points 142

over Leydig cell per testis were counted for each animal. The number of Leydig cell per 143

testis was estimated from Leydig cell the individual volume (nuclear volume plus 144

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cytoplasmatic volume) and the volume density of Leydig cell in the testis parenchyma. 145

146

2.5. Statistical analysis 147

All data were expressed as mean ± standard deviation, and the means, when 148

necessary, were evaluated by analysis of variance and compared by the Tukey test at a 149

5% significance level. 150

151

3. Results 152

3.1. Biometric, testis volume density and Leydig cell number 153

The biometric and morphometric data in sexually mature Sus scrofa are 154

presented in Table 1. The mean testis weight in feral pig was approximately 125 g. 155

Volume density of the seminiferous tubules and Leydig cells was 79.8 ± 1.7% and 12.9 156

± 1.5%, respectively. Therefore, Leydig cells occupied nearly 64% of the intertubular 157

compartment. The mean tubular diameter was 243 ± 8 µm. Based on the volume of the 158

testis parenchyma (testis weight minus tunica albuginea weight) and the volume 159

occupied by seminiferous tubules in the testis and tubule diameter, there were 17.8 and 160

1953 m of seminiferous tubules per testis gram and per testis, respectively (Table 1). 161

Data for Leydig cell morphometry in sexually mature free-ranging feral pigs are 162

presented in Table 2. The estimated Leydig cell nuclear volume was 116 µm3 and the 163

cell size was 841 µm3.The mean number of Leydig cell per testis and per gram of testis 164

was, respectively, 18.8 billion and 170 million. 165

166

3.2. Cell population of the seminiferous tubules 167

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Cell populations of the eight stages of the seminiferous epithelium cycle in 168

sexually mature free-ranging feral pigs are presented in Table 3. The number registered 169

for each cell type was corrected according Abercrombie (1946) modified by Amann 170

(1962). Furthermore, when numbers of cells by transversal section of seminiferous 171

tubule are considered in absolute terms, there is a great variability among the species, 172

even among the different populations within the same species (Costa et al., 2011). 173

174

3.3. Stages of the seminiferous epithelium cycle 175

Based on the tubular morphology system, eight stages of the cycle were 176

characterized, as follows (Figure 1 and 2): 177

Stage 1 (Fig. 1A): was characterized by the presence of a generation of spermatids with 178

dark and round nuclei, which formed four to six layers in the upper part of the 179

seminiferous epithelium. The nuclei of the Sertoli cells were well-developed nucleolus 180

and loose chromatin. Type A spermatogonia and primary spermatocytes, in the 181

transition from pre-leptotene to leptotene were detected close to the basal membrane. 182

Pachytene spermatocytes were located between the pre-leptotene/leptotene 183

spermatocytes and the round spermatids. 184

Stage 2 (Fig. 1B): spermatid nuclei began elongation in the direction of the Sertoli cell 185

nuclei located at the base of the tubule. Primary spermatocytes in pre-186

leptotene/leptotene were observed near the basal lamina and pachytene primary 187

spermatocytes were detected in transition to diplotene spermatocytes. Moreover, type A 188

spermatogonia nuclei and the nucleolus of Sertoli cell were of similar morphology to 189

those of the previous stage. 190

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Stage 3 (Fig. 1C): elongated spermatids formed bundles composed of few nuclei. Two 191

generations of primary spermatocytes were present in this stage, spermatocytes in 192

zygotene and diplotene stages with large nuclei. The nucleolus of ertoli cell and type A 193

spermatogonia were observed close to the basal lamina. 194

Stage 4 (Fig. 1D): the main feature of this stage was the presence of meiotic cells. 195

Diplotene spermatocytes formed secondary spermatocytes, which divided and produced 196

round spermatids. Batches of elongated spermatids and zygotene spermatocytes were 197

also observed. The type A spermatogonia population was increased in comparison to the 198

previous stage. The nucleolus of Sertoli cell was similar to those already described for 199

the other stages. 200

Stage 5 (Fig. 2A): two generations of spermatids were present in this stage- round 201

spermatids recently formed and elongated spermatids. Although these cells had a 202

smaller diameter, the round spermatid nucleus morphology was similar to that observed 203

for secondary spermatocytes. Batches of elongated spermatids were located in Sertoli 204

cell crypts with common locations of some nuclei being present deeply within the 205

seminiferous epithelium. Zygotene spermatocytes in the transition to pachytene were 206

detected between the round spermatids and the basal compartment. Type A 207

spermatogonia were present in the base of the tubule. The nucleus of Sertoli cell was 208

located on the longitudinal axis, generally, perpendicular to the basal lamina. 209

Stage 6 (Fig. 2B): except to the zygotene spermatocyte, all cell types observed in stage 5 210

were present in this stage. The spermatid batches were, generally, closer to the tubular 211

lumen. Intermediate spermatogonia were also observed and had a smaller and darker 212

nucleus in comparison to the type A spermatogonia. Pachytenes spermatocytes were 213

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detected in the medium region of the seminiferous epithelium. The nucleus of Sertoli 214

cells and type A spermatogonia were present near the basal lamina. 215

Stage 7 (Fig. 2C): elongated spermatid bundles had dissociated and spermatids nuclei 216

were located close to the tubular lumen. Pachytene spermatocyte nuclei were larger than 217

those in the previous stages. Type B spermatogonia had round or ovoid nuclei with 218

abundant heterochromatin. The other cell types present in this stage were the type A 219

spermatogonia, round spermatids and Sertoli cells. 220

Stage 8 (Fig. 2D): the most characteristic aspect of this stage was the location of 221

elongated spermatids ready to be released from the seminiferous epithelium. 222

Cytoplasmic lobes of the elongated spermatids and residual bodies were fewer in 223

number, and were situated in the luminal edge of the epithelium. Type A 224

spermatogonia, Pachytene spermatocytes, round spermatids, and Sertoli cells were also 225

present. Pre-leptotene spermatocytes were seen close to the basal lamina. 226

227

4. Discussion 228

Recent studies showed the intrinsic rate of spermatogenesis in free ranging feral 229

pigs. Those data indicated that the supporting capacity of Sertoli cells in free-ranging 230

feral pigs is among the greatest values reported for most domestic animals, and the 231

overall yield of spermatogenesis is comparable to that reported in wild boars (Costa et 232

al., 2011). Based on these findings, the aim of the present study was to further 233

characterize the feral pig testis parenchyma as well as to evaluate the number of 234

differentiated spermatogonia generations in free-ranging feral pigs (Sus scrofa sp). 235

The relative mass of seminiferous tissue determines the amount of space in the 236

testis for sperm production (Costa et al., 2008) because the main causes of different 237

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spermatogenic efficiencies observed among mammalian species seems to be the 238

variation in the proportion of tubular and intertubular compartments (França et al., 239

2005). The mean percentage of the intertubular compartment in feral pigs was 240

approximately 35% greater than the previously reported value for wild boars (Almeida 241

et al., 2006). This difference may be related to the greater percentage of Leydig cells in 242

the feral pig testis (~13%) compared with that in wild boar (6%) (Almeida et al., 2006) 243

which is among the greatest values reported for most mammals (França et al., 2005; 244

Hess & França, 2008). Although the number of Leydig cell per testis was greater in 245

feral pigs (~50%) in comparison to wild boars, the number of Leydig cell per testis 246

gram was similar between these species (Almeida et al., 2006). 247

According to the tubular morphology method, which characterize the stages of 248

the seminiferous epithelium, based on the observation of modifications in the form and 249

position of the nuclei in the spermatids, and the occurrence of meiotic division figures 250

(Courot et al., 1970, Ortavant et al., 1977, Berndtson, 1977), it was possible to identify 251

typical cellular associations in the eight stages of the seminiferous epithelium in feral 252

pigs. Moreover, there were similar patterns observed in most mammals, the transversal 253

sections of the seminiferous tubules in feral pigs was a single stage in the cycle, in 254

contrast to what was observed in primates, where a single transversal section is 255

occupied by several stages (Clermont, 1963, Guerra, 1981). 256

At the light microscope level, the Sertoli cells in feral pig testes showed classical 257

morphology with elusive boundaries, an indented nucleus with characteristic tripartite 258

nucleolus with a mean nuclear diameter very similar to that found in suidae and 259

tayssuidae (Russell et al., 1990; Costa et al., 2004, França et al., 2005, Costa and Silva, 260

2006, Costa et al., 2011). The Sertoli cells were detected in all of the transversal 261

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sections of seminiferous tubules of feral pigs in the eight stages of the seminiferous 262

epithelium cycle. 263

The spermatogenic cells in feral pigs were characterized based on the nuclear 264

morphology and topographic position in relation to the other cells and the basal lamina. 265

Therefore, three types of spermatogonia were identified: type A spermatogonia, 266

intermediate (In) spermatogonia and type B spermatogonia. The morphology of these 267

cells, as well as the primary spermatocytes in meiotic prophase, the secondary 268

spermatocytes and the spermatids in feral pigs, did not differ substantially from that 269

described for mammals in general (Courot et al., 1970, Clermont, 1972, Ortavant et al., 270

1977) and the mean nuclear diameter was very similar to that described for cathetus 271

(Tayassu tajacu), wild boars (Tayassu pecari), boars (Sus scrofa scrofa) and Piau suines 272

(França et al., 2005, Costa et al., 2004, Costa and Silva, 2006, Costa et al., 2011). 273

The population of differentiated spermatogonia in the seminiferous tubules 274

varied considerably between stages 1 and 5, however, it was relatively constant in stages 275

6, 7 and 8. In stage 2 the population of type A spermatogonia was 58% greater than in 276

stage 1 (P<0.05), suggesting a first peak of mitotic division for this cell type. The 277

population of spermatogonias A in stage 3 was 34% greater P<0.05) than those in stage 278

2, suggesting that the second peak of mitosis occurs in stage 3. In relation to the 279

population of spermatogonia A in stage 3, a numerical reduction of 3.2% was verified in 280

comparison to stage 4, although it was not significant (P>0.05). The population of type 281

A spermatogonia increased 17.4% (P<0.05) from stage 4 to stadium 5, suggesting that 282

the third peak of mitotic divisions occurs in stage 5. 283

There was no difference (P>0.05) among the number of spermatognia A found 284

in stages 6, 7 and 8, although a reduction in this cell type was observed in these stages, 285

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in comparison to the previous stage. The spermatogonia could not be observed by light 286

microscopy with the tissue preparation used in the present study (Chiarini-Garcia and 287

Russell, 2001), [DJK1]these cells probably comprise the different types of undifferentiated 288

spermatogonia, as described in rats and domestic pigs (Huckins, 1971; Frankenhuis et 289

al., 1982). Cells are spermatogonia As (single), Apr (paired) and Aal (aligned). The As 290

spermatogonia are the stem cells of spermatogenesis. By mitotic division, normally half 291

these cells originate the Apr spermatogonia and the other half will constitute the renewal 292

population of As spermatogonia (Oakberg, 1971). The Apr spermatogonia divide to form 293

four, eight or 16 Aal spermatogonia. These differ in spermatogonia A1, which is the first 294

generation of differentiated spermatogonia (De Rooij e Grootegoed, 1998). 295

In stage 6 the intermediate spermatogonia was identified for the first time, 296

formed from the mitotic division of the last generation of type A spermatogonia. This 297

cell type had a smaller and darker nucleus when compared to those from type A 298

spermatogonia. The total population of intermediate spermatogonia was 35% greater 299

than those for type A spermatogonia, in stage 5. In stage 7, besides the type A 300

spermatogonia, a new cellular type identified as type B spermatogonia was observed. 301

The population of B spermatogonia was 31.2% greater than the population of In 302

spermatogonia in previous stages. Generally, spermatogonia may be differentiated by 303

the amount of chromatin lying along the inner aspect of the nuclear envelope; type A 304

spermatogonia have very little, In spermatogonia have moderate amounts, and type B 305

spermatogonia possess a large amount (Russell et al., 1990). 306

Taking this into account, it was possible to infer that there are at least four 307

generations of type A differentiated spermatogonia in feral pigs, that is, type A1 (stage 308

1), type A2 (stage 2), type A3 (stages 3 and 4) and type A4 (stages 5). This is different 309

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from what was reported in rabbits, for example, where there are two generations of 310

intermediate spermatogonia (In1 and In2), and in ruminants, where there are two types of 311

B spermatogonia (B1 and B2) (Guraya, 1987, França and Russell, 1998), no new 312

generations were detected in In and B spermatogonia in feral pigs. Furthermore, the 313

process of spermatogonial divisions is still one of the most complex and controversial 314

aspects in kinetic studies of spermatogenesis in mammals, because in most of the 315

species the standard of multiplication and renewal of spermatogonia is still not entirely 316

elucidated (Castro et al., 1997). The estimated number of spermatogonial generations 317

vary from four to six in most species, with those having six generations being the 318

cathetus, wild boar, boar, bull, lamb and the dog, and five generations the rabbit and 319

horse (Frankenhuis et al., 1982; França e Russell, 1998; Costa e Paula, 2006). While the 320

spermatogonial kinetics is still not well defined for most mammals, the meiotic phase 321

(spermatocytes) and the differentiation (spermiogenic) phase is well established in 322

mammals regarding the number of cellular generations. In all the species there is only 323

one generation of primary spermatocyte, secondary spermatocytes and spermatids 324

(Clermont, 1972; Ortavant et al., 1977). 325

The proliferative or spermatogonial phase is defined by a greater mitotic rate 326

and, consequently, is more susceptible to agents that affect spermatogenesis. In rats, the 327

spermatogonia divide approximately nine times a week (Russell et al., 1990). 328

Considering a species that has only six generations of spermatogonia, the yield of the 329

spermatogenesis would be 100% if there were not cellular losses during the entire 330

spermatogenic process. These divisions would result from the eight divisions of type A1 331

spermatogonia (A1→A2 →A3 →A4 →In →B →primary spermatocyte → secondary 332

spermatocyte → spermatid) that would occur until the formation of spermatozoids. 333

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Taking this into account, one A1 spermatogonia should generate two A2, four A3, eight 334

A4, 16 intermediate spermatogonias, 32 B spermatogonias, 64 primary spermatocytes, 335

128 secondary spermatocytes and 256 spermatids, which would differentiate into an 336

equal number of spermatozoids (Russell et al., 1990). 337

However, in feral pigs, that have six generations of differentiated 338

spermatogonias, one A1 spermatogonia generated 1.6 A2, 2.2 A3, 2.4 A4, 3.2 In 339

spermatogonia, 4.3 B spermatogonia, 8.0 spermatocytes and 21.5 round spermatids. 340

This result indicates that feral pigs have a 91.6% total cellular loss during the 341

spermatogenic process. From these losses 80.7% occurred during the mitotic divisions 342

and only 10.9% was from meiotic division. Moreover, cellular loss during 343

spermatogenesis in healthy animals is considered normal, and it has been reported in all 344

of the species (Amann, 1962, Berndtson and Desjardins, 1974, França and Russell, 345

1998, Costa and Paula, 2003). These losses generally vary from 60% to 90% in most 346

animals (França and Russell, 1998; Roosen-Runge, 1973). 347

Additionally cellular losses during the spermatogenic process have a 348

chromosomal nature and have an important role in eliminating cells with drastic 349

chromosomal aberrations and genetic alterations that may occur during cell growth and 350

multiplication. Noteworthy, this degenerating process of cellular lysis is rapid and 351

difficult to detect (Oakberg, 1956). Besides, the density-dependent theory suggests that 352

germ cell loss, in the spermatogenic process, could be a homeostatic mechanism to limit 353

the germ cells to a number that can be supported by Sertoli cells (Huckins, 1978; 354

Sharpe, 1994; De Rooij, 1998). According to Costa and Paula (2003), this mechanism is 355

probably a competition by cells for growth factors and other important factors. 356

However, it is important to note that, for each species, the Sertoli cells support a limited 357

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number of germ cells, therefore, inter-specie comparisons must be made carefully 358

(Costa and Paula 2003). 359

360

5. Conclusions 361

In conclusion, the present study shows that there are six differentiated 362

spermatogonial generations in feral pigs, and that the cellular composition of the eight 363

stages of the seminiferous epithelium cycle was very similar to that described for others 364

species of suidae and taysuidae. 365

366

References[DJK2][DJK3] 367

Abercrombie, M., 1946. Estimation of nuclear populations from microtome sections. 368

Anat. Rec. 94, 238-248. 369

Alho, C.J.R., Lacher T.E.J. 1991. Mammalian conservation in the Pantanal of Brazil. In: 370

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Fig.1. Stages of the seminiferous epithelium cycle in feral pigs – (A) stage 1; (B) stage 475

2; (C) stage 3 and (D) stage 4. A: spermatogonial A, Pl/L: primary spermatocyte in 476

preleptotene/leptotene, P: primary spermatocyte in pachytene, Z: primary spermatocyte 477

in zygotene, D: primary spermatocyte in diplotene, M: figures of meiosis, II: secondary 478

spermatocyte, Rs: round spermatid, Es; elongated spermatid. Toluidine blue + sodium 479

borate (1%). 400X magnification 480

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Fig.2. Stages of the seminiferous epithelium cycle in feral pigs – (A) stage 5; (B) stage 481

6; (C) stage 7 and (D) stage 8. A: A spermatogonia; B: B spermatogonia; In: 482

intermediary spermatogonia; P: primary spermatocyte in pachytene; Pl: spermatocytes 483

in recently formed preleptotene; Z: primary spermatocyte in zygotene; Rs: round 484

spermatid; Es: elongated spermatid; S: Sertoli cell; Rb: residual bodies. Toluidine blue 485

and sodium borate (1%). 400X magnification 486

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Table 1 487

Biometric and morphometric data in sexually mature free-ranging feral pigs (mean ± 488

sem) 489

Parameter (n = 8)

Body weight (g) 60 ± 2.8

Testis weight (g)* 124.6 ± 15

Testis parenchyma volume density (%)

Seminiferous tubules 79.8 ± 1.7

Tunica propria 1.8 ± 0.09

Seminiferous epithelium 67.6 ± 1.6

Lume 10.4 ± 1.2

Intertubular compartment 20.2 ± 1.7

Leydig cell 12.9 ± 1.5

Blood vessel 4.2 ± 0.5

Lymphatic space 2.3 ± 0.6

Connective tissue 0.8 ± 0.2

Tubular diameter (μm) 243 ± 8

*Right testis plus left testis divided by two

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Table 2 491

Leydig cell morphometry in sexually mature free‐ranging feral pigs (mean ± sem) 492

Parameter (n = 8)

Leydig cell nuclear diameter (µm) 6.0 ± 0.1

Leydig cell volume (µm3) 841.0 ± 108.0

Nuclear volume (µm3) 116.0, ± 8.0

Cytoplasm volume (µm3) 725.0 ± 102.0

Leydig cell no. per gram of testis (106) 170.0 ± 23.0

Leydig cell no. per testis (109) 18.8 ± 2.4

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Table 3 494

Cell populations of the eight stages of the seminiferous epithelium cycle in sexually 495

mature free-ranging feral pigs 496

Cellular types Stages Number of cells* (mean±sem)

Sertoli Cells 1 5.00 ± 1.09a








Type A spermatogonia 1 4.63 ± 0.92A

Type A spermatogonia 2 7.32 ± 0.97B

Type A spermatogonia 3 9.82 ± 0.58C

Type A spermatogonia 4 9.50 ± 1.12C

Type A spermatogonia 5 11.15 ± 0.95D




Type In spermatogonia 6 15.03 ± 1.41

Type B spermatogonia 7 19.71 ± 2.13

PL/L spermatocyte I 1 35.12 ± 7.80a



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Z spermatocyte I 3 37.51 ± 5.55A

Z spermatocyte I 4 36.18 ± 4.65A

P spermatocyte I 1 36.59 ± 9.21a






D spermatocyte I 3 37.15 ± 5.80

Rs spermatid 1 111.02 ± 23.07A





* Numbers corrected according to Amann (1962). Means followed by different letters for the same 497

cellular type were different - Tukey test (P < 0.05) 498

** In, intermediate; PL/L pre-leptotene/leptotene; Z, zygotene; P, pachytene; D, diplotene; Rs, 499

round 500

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Testis morphometry and kinetics of spermatogenesis in the feral pig (Sus scrofa)

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