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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
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
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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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475
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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
481
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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
487
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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
490
491
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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
493
494
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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
Sertoli Cells 2 4.87 ± 0.78a
Sertoli Cells 3 4.73 ± 1.13a
Sertoli Cells 4 4.51 ± 0.82a
Sertoli Cells 5 4.62 ± 1.00a
Sertoli Cells 6 5.19 ± 1.13a
Sertoli Cells 7 5.21 ± 1.47a
Sertoli Cells 8 4.62 ± 1.31a
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 A spermatogonia 6 4.73 ± 0.74A
Type A spermatogonia 7 4.57 ± 0.86A
Type A spermatogonia 8 4.88 ± 0.86A
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
PL/L spermatocyte I 2 37.12 ± 6.24a
PL/L spermatocyte I 8 35.33 ± 4.72a
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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
P spermatocyte I 2 37.55 ± 9.28a
P spermatocyte I 5 38.77 ± 7.94a
P spermatocyte I 6 37.94 ± 6.71a
P spermatocyte I 7 36.47 ± 8.75a
P spermatocyte I 8 37.73 ± 8.74a
D spermatocyte I 3 37.15 ± 5.80
Rs spermatid 1 111.02 ± 23.07A
Rs spermatid 5 99.69 ± 29.99A
Rs spermatid 6 106.15 ± 19.51A
Rs spermatid 7 119.7 ± 23.24A
Rs spermatid 8 93.76 ± 21.55A
* 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
501
502
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Fig 2