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Journal of Equine Veterinary Science 50 (2017) 20–26

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Journal of Equine Veterinary Science

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Original Research

Morphology and Morphometry of Seminiferous Tubules inCrioulo Horses

Giuliano M. Figueiró a, Mara I.B. Rubin b, Fábio J.C. Faria c, Marcelo D. Santos d,Carlos A.C. Fernandes e, Fernando H.G. Furtado c, Deiler S. Costa c,*

aDepartment of Veterinary Medicine, Federal University of Santa Catarina, Curitibanos, SC, BrazilbDepartment of Veterinary Medicine, Laboratory of Animal Embryology, Federal University of Santa Maria, Santa Maria, RS, BrazilcDepartment of Veterinary Medicine, Laboratory of Assisted Reproduction, Federal University of Mato Grosso do Sul, Campo Grande, MS, BrazildDepartment of Veterinary Medicine, University of Cuiabá, Cuiabá, MT, BrazileDepartment of Veterinary Medicine, University of Alfenas, Alfenas, MG, Brazil

a r t i c l e i n f o

Article history:Received 14 June 2016Received in revised form 3 November 2016Accepted 9 November 2016Available online 21 November 2016

Keywords:EquineTestisSpermatogenesis

* Corresponding author at: Deiler S. Costa, FacVeterinária e Zootecnia, Universidade Federal de MFilinto Muller, 2443, 79074-460, Campo Grande, MS

E-mail address: deiler.costa@outlook.com (D.S. C

0737-0806/$ – see front matter � 2016 Elsevier Inchttp://dx.doi.org/10.1016/j.jevs.2016.11.003

a b s t r a c t

This study aimed to assess the biometrics of the testes and the morphology of the semi-niferous tubules of Crioulo horses. We studied 10 sexually mature stallions (3–6 years ofage). After orchiectomy, testes were perfused with Karnovsky’s solution and thenembedded in glycol methacrylate. Testis sections (4 mm) were cut and stained with tolu-idine blue and a solution of 1% sodium borate. The histological images were digitized, andthe morphometric analysis was performed using ImageJ software. The average weight ofthe stallions was 377.5 kg, and the average weight of both testicles was 162.9 g. Thepercentage of testicular parenchyma occupied by the seminiferous tubules and theintertubular tissue was 77.97% � 6.34% and 22.03% � 6.34%, respectively. The averagetubular diameter was 205.00 � 36.91 mm, whereas the average height of the seminiferousepithelium was 70.56 � 2.82 mm. Average tubular length per testicle and average tubularlength per gram of testicle were 4,085.10 � 1,170.68 m and 26.09 � 10.63 m/g, respec-tively. The characteristics of the eight stages of the seminiferous epithelium cycle weresimilar to those reported in other horse breeds. We conclude that the morphometry of theseminiferous tubules of Crioulo horse resembles what has been reported in other horsebreeds. The volumetric proportion of the seminiferous tubules and the Leydig cells of theCrioulo horse is one of the highest ever reported for stallions.

� 2016 Elsevier Inc. All rights reserved.

1. Introduction

The Crioulo horse breed is found in several countries inSouth America, such as Brazil, Argentina, Uruguay Chile,and Paraguay. This breed underwent a process of naturalselection over the course of four centuries, which resultedin a strong adaptation to the environmental conditions ofSouth America, generating animals with great rusticity.

uldade de Medicinaato Grosso do Sul, Av, Brazil.osta).

. All rights reserved.

This rusticity was accompanied by high fertility andlongevity. Crioulo horses stand out for their ability towithstand adverse climate conditions and to survive inbarren native lands because of their low nutritional de-mands [1]. However, despite the economic and culturalimportance of Crioulo horses, there are only few studiesthat characterize some basic principles of their reproduc-tive physiology, such as testes functional morphology.

Spermatogonia are found inwell-organized and definedpatterns in the seminiferous tubules, constituting cellularassociations that characterize the stages of the seminifer-ous epithelium cycle (SEC). In the most mammals, thestages of the SEC present a segmented pattern, and

G.M. Figueiró et al. / Journal of Equine Veterinary Science 50 (2017) 20–26 21

normally there is only one stage per transversal section oftubule [2,3].

A stage in the SEC is considered as a set of definedgenerations of germ cells found, at a particular moment, ina transversely sectioned seminiferous tubule [4]. Becausethe spermatogenic cells develop at the same time and inclose relationship with each other, over the course of timeparticular transversal section of seminiferous tubule beginto have a constant and progressive stage development,constituting the SEC. Identifying the various stages of theSEC is necessary for conducting quantitative studies onspermatogenesis; this is an important factor for under-standing normal spermatogenesis, as well as for deter-mining specific phases that might be affected by certaintreatments or drugs [5].

The main methodology used to study the stages of theSEC in mammals is called the tubular morphology method,which is based on the variations in the shape of the nucleusof spermatogenic cells, the detection of meiotic divisions,and in the disposition of spermatids in the seminiferousepithelium. This method has enabled the description ofeight stages in all species studied [5,6].

The testicular parenchyma of mammals is morpho-functionally constituted by two basic compartments: (1)the interstitial or intertubular compartment, also known asthe intertubular space, which is responsible for producingsteroids, and is constituted by blood and lymph vessels,nerves, connective tissue fibers, macrophages, mastocytes,and Leydig cells; and (2) the tubular compartment, whichcontains the seminiferous tubules that produce spermato-zoids. These tubules are constituted by the tunica propria,the seminiferous epithelium, and the lumen. Myoid orperitubular cells, the basal membrane, and the collagenousfibers are found in the tunic. Sertoli cells and the malegerm-line cells are in the seminiferous epithelium. Thefluid secreted by the Sertoli cells and the spermatozoids arefound in the tubular lumen [2,3].

The aim of this study was to evaluate the biometrics ofthe testes and the morphology of the seminiferous tubulesin Crioulo horses, focusing on the volumetric proportion oftesticular parenchyma, the characterization of the stages inthe SEC, the diameter of the seminiferous tubules, thetubular length, and the height of the seminiferousepithelium.

2. Materials and Methods

2.1. Collection of Testes and Histological Processing

We collected the testes of 10 Crioulo horses between 4and 6 years of age. Sampled animals were from thewesternborder of the central region of Rio Grande do Sul State andthe northern area of Espírito Santo State, during the winterseason. All the animals used have been maintained grazingin native pastures of the corresponding region, and sup-plemented with minerals.

The animals were castrated under general anesthesiaaccording to protocol no. 23081.013329/2009-20, approvedby the Ethics and Animal Experimentation Committee ofthe Federal University of Santa Maria. Testicles wereremoved from the epididymis and weighed on a digital

scale. The left testicular arterywas cannulated for perfusionwith 0.9% saline solution containing 5,000 IU/L of heparinfor at least 15 minutes at room temperature. Immediatelyafter this procedure, testes were perfused with Karnovsky’sfixative solution (4% paraformaldehyde, 5% glutaraldehydein a 0.1 M and pH 7.4 phosphate buffer) for 20minutes, alsoat room temperature. The vials containing the perfusionsolution were kept at 120 cm above the testis to ensure apressure of approximately 80 mmHg [7].

Fragments of testicular parenchyma (approximately 8.0� 5.0 � 3.0 mm) were cut from the middle third of theorgan, and were then refixed in a new Karnovsky’s solutionfor 24 hours. Following this, they were stored in a refrig-erated phosphate buffer for 5 days until they were pro-cessed. The right testis of each animal was frozen, and usedfor further analysis of the volumetric percentage occupiedby the albugineous tunic and mediastinum.

Fragments were dehydrated with increasing concen-trations of ethanol, and embedded in a glycol methacrylatesolution, following the methods of Costa et al [7]. Finally,using a glass blade microtome, 4 mm sections were cut andstained with toluidine blue and a 1% sodium borate solu-tion, sections were thenmounted on Entellan (Merck) glassslides, according to the manufacturer’s instructions.

2.2. Morphometric Analyses of the Testes

The volumetric proportion occupied by different com-ponents of the testicular parenchyma was determined bymeans of the ImageJ 1.34s software [8], considering agraticule with 494 intersections as a point. The coincidentpoints were computed with the seminiferous tubules(tunic, seminiferous epithelium, tubular lumen) and theintertubular tissue (Leydig cells, connective tissue, bloodand lymph vessels) in 30 fields per animal. The fields werechosen randomly by means of a horizontal scan of thehistological sections. The volumetric proportions wereexpressed as percentages, and estimated based on 14,820points per testis. Analysis was performed at 400� magni-fication, under a light microscope.

The volume of the testicular parenchymawas calculatedby subtracting the weight of the albuginea and the medi-astinum, which were determined through the desiccationof the stored testes, from the weight of the correspondingtestis. Testis density was considered to be very close to one[9]; therefore, the weight was considered to be equal to thevolume.

The diameter of the seminiferous tubules was calculatedby measuring 20 cross-sections of tubules, as circular-shaped as possible, per animal. For this, we consideredthe average between the greatest and the smallest diam-eter of each section. The average height of the seminiferousepithelium was measured from the basal membrane to theadluminal edge. Based on the same image used for calcu-lating the tubular diameter and the height of the seminif-erous epithelium, we determined the cross-sectional areaof the seminiferous tubule. These three assessments wereperformed using ImageJ 1.34s software [8].

The total length of the seminiferous tubules was calcu-lated according to the method described by Attal et al [10];we divided the corrected value of the total volume of the

G.M. Figueiró et al. / Journal of Equine Veterinary Science 50 (2017) 20–2622

seminiferous tubules by the cross-sectional area. Thevalues of the cross-sectional areas and the total correctedvolume of the seminiferous tubules were estimated byconsidering a linear shrinkage factor of 5% [11]. The totalcorrected volume was obtained by multiplying the pro-portion of the seminiferous tubule by the weight of thetesticular parenchyma.

The total length of the tubules was expressed in meters(m), and the calculation of the length per gram (m/g) wascarried out by dividing this length value by the weight ofthe testis.

We characterized the stages of the SEC by means ofusing the tubular morphology method, which assesses thevariations in the shape of the nucleus of the spermatogeniccells, the occurrence of meiotic divisions, and thearrangement of the spermatids in the seminiferousepithelium [5,6].

2.3. Statistical Analysis

Statistical analysis of the results was mainly descriptive.The averages and standard deviations of the data obtainedin this experiment were calculated using BioEstat 3.0software [12].

3. Results and Discussion

The animals included in this experiment are a sampleobtained from a homogeneous and representative popu-lation of young and sexually mature stallions (4–6 years ofage, 377.5 kg). All the animals included in this study werebred and raised in the field with mineral supplements. Inour results, we found that the average body weight(Table 1) was very similar to a previous report by Mendes[13], who studied Crioulo horses of the same age-group(>3 years of age, 389 kg), and our results were consistentwith the weight range reported for the majority of adultstallions of different breeds (between 150 and 400 g) [14].

Testis weight of the animals in the present study(Table 1) was also consistent with the values reported byMendes [13], which was 190 g for the left testis and 150 gfor the right testis. In stallions, the left testis is usually 5%

Table 1Total body and testicular measurements of 10 sexually mature Crioulo horses.

Horse Body Weight, kg Testis Weight, g TePa

g

1 440.0 140.0 112 440.0 190.0 153 325.0 200.0 174 390.0 199.7 175 370.0 242.2 206 435.0 197.8 157 330.0 138.8 118 350.0 101.3 89 325.0 160.0 1310 370.0 121.5 10Average 377.5 169.13 14Standard deviation 45.8 43.72 3

heavier than the right testis because of the early develop-ment of left testis [15].

The percentage represented by the albuginea and themediastinum (Table 1) in relation to the testicular weightwas similar to that described by Swierstra et al [16] andNeves et al [17], who studied undefined breeds. This pro-portion tends to be conservative within species in animalsof similar ages, maintaining a value close to 10% [18].

The tubular compartment is the main component of thetestis in almost all mammals studied. Differences observedbetween breeds and individuals are directly reflected intheir sperm production [18]. In Crioulo horses, the semi-niferous tubules occupied 78% of the testicular parenchyma(Table 2). This proportion falls within the range reported formost species, which is between 60% and 90% [19]. However,compared with other horse breeds, this is one of thehighest proportions ever reported. For example, the pro-portion of tubular tissue in the Pantaneiro (Table 2) is 72.5%[20], and in crossbred horses, it has been reported to rangefrom 62% to 72% [9,16,21]. On the other hand, the pro-portions occupied by the seminiferous epithelium, tunic,and lumen in the seminiferous tubules do not vary greatlybetween the above-mentioned breeds (crossbred andPantaneiro), displaying highly similar values.

The intertubular tissue occupied 22% of the testicularparenchyma in Crioulo horses, a proportion that fallswithin the 10%–30% average range reported for mostbreeds [18]. In this intertubular compartment, Leydig cellscomprise the greatest proportion, at 9.0%–16.2% in mostmammals [22]. In the animals in the present study, theLeydig cells comprised 7.96% of the testicular parenchyma.This value is greatly higher compared with that reportedfor the Brazilian Sport Horse breed (1.5%; Jones [15]), andwith the 3.1% reported for donkeys [23]. However, thisdifference does not necessarily imply that Crioulo horseshave a greater production or need for testosterone to pro-duce sperm and to maintain peripheral levels of androgens.Several factors may influence the number of Leydig cells ineach animal, such as the quantity of available luteinizinghormone (LH), the number of LH receptors per cell, thequantity of testosterone that the Leydig cell is capable tosecrete at a given time, the speed at which the testosteroneleaves the testis via lymph vessels, blood vessels, and

sticularrenchyma

Albuginea Mediastinum

% g % g %

4.2 81.57 19.6 14.00 6.2 4.439.6 84.00 23.6 12.42 6.8 3.582.8 86.39 20.46 10.23 6.8 3.394.7 87.48 19.8 9.91 5.2 2.607.3 85.59 26.8 11.07 8.1 3.348.1 79.93 28.9 14.61 10.8 5.462.8 81.27 18.7 13.47 7.3 5.262.0 80.95 14.5 14.31 4.8 4.746.4 85.25 17.6 11.00 6.0 3.751.2 83.29 14.6 12.02 5.7 4.691.91 83.57 20.46 12.30 6.8 4.129.1 2.58 4.76 1.73 1.72 0.93

Table 2Volumetric proportion occupied by different components of the testicularparenchyma (%) and morphometry of the seminiferous tubules of 10sexually mature Crioulo horses.

Parameter Average � StandardDeviation

Seminiferous tubule, % 77.97 � 6.34Tunic, % 7.54 � 2.54Seminiferous tubule epithelium, % 63.74 � 6.21Seminiferous tubule lumen, % 6.69 � 3.54

Intertubular tissue, % 22.03 � 6.34Leydig cells, % 7.96 � 4.12Connective tissue, % 11.97 � 3.75Lymph and blood vessels, % 2.10 � 0.57

Tubular diametera, mm 205.00 � 36.91Height of the seminiferous epitheliuma, mm 70.56 � 2.82Length of the seminiferous tubulea, m/g 26.09 � 10.63Total length of the seminiferous tubulea, m 4,085.10 � 1,170.68

a Corrected according to the linear shrinkage factor of 5% [11].

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seminal fluid, the animal’s blood volume, and the meta-bolism rate of the testosterone [24,25].

The average diameter of the seminiferous tubules ofCrioulo horses was 205 mm (Table 2), which falls within thenormal range reported for most amniotes (180–300 mm)[26]. In sexually mature animals, with no reproductiveseasonality, and under normal conditions, the averagetubular diameter does not undergo any significant

Fig. 1. Cross-sections of seminiferous tubules characterizing stages 1 (A), 2 (B), 3 (Cduring nonreproductive season. Figures show the following cellular groups: A: typePQ: primary spermatocytes in pachytene, Ar: round spermatids, L: primary spermazygotene, D: primary spermatocytes in diplotene, M: figures in meiosis, II: secondaand Al are forming bundles. Toluidine blue, 400� magnification.

variations [18]. However, in species with seasonal repro-duction, major differencesmay be observed in the diameterof the seminiferous tubules between different breeds andlineages, even during the nonreproductive season [27].

Besides differences due to genetic selection, which favoranimals with greater sperm production per gram of testis,other factors can also contribute to the large rangeof tubulardiameters described in mammals. For example, methodol-ogies in some studies do not consider the tissue shrinkageduring histological processing, particularly when applyingparaffin-embedded methods. According to Amann [11],tissue shrinkage can reach up to 15%. Other explanations forthe large range of tubular diameters are the inclusion ofsexually immature animals in the studies, variations in thenumber of myoid cell layers that constitute the tunic, dif-ferences in the Sertoli and spermatogenic cell populations,and different fluid secretion patterns of the Sertoli cells,which determines the size of the tubular lumen [27].

Regarding the height of the seminiferous epithelium(Table 2), we reported values (70.6 mm) that fall within therange reported for domesticated animals (60–100 mm) [18].The values found in Crioulo horses were very similar tothose reported for crossbred horses ranging from 3 to10 years of age (76 � 2 mm; [28]), and in donkeys (78 mm;[23]). On the other hand, Varoni [20] reported that inPantaneiro horses (3–6 years of age), the seminiferousepithelium height was 59.6� 6.2 mm. This difference seems

), and 4 (D) of the seminiferous epithelium of orchiectomied Crioulo horsesA spermatogonia, S: Sertoli cells, PL: primary spermatocytes in preleptotene,tocytes in leptotene, Al: elongating spermatids, Z: primary spermatocytes inry spermatocytes. Al forming bundles are observed in part C. In part D, II, Ar,

Fig. 2. Cross-sections of seminiferous tubules characterizing stages 5 (A), 6 (B), 7 (C), and 8 (D) of the seminiferous epithelium of orchiectomied Crioulo horsesduring nonreproductive season. Figures show the following cellular groups: A: type A spermatogonia, S: Sertoli cells, PQ: primary spermatocytes in pachytene, Ar:round spermatids, AI: elongated spermatids. B: type B spermatogonia, CR: residual bodies, PL: spermatocytes in pre-leptotene. In part A, AI are forming compactbundles. In part B, PQ, Ar, and Al bundles are migrating toward the tubular lumen. In part C, El are no longer forming bundles and are close to the lumen. All thestages present type A spermatogonia and Sertoli cells. In part D, Al have a palisade-shape on the luminal edge. Toluidine blue, 400� magnification.

G.M. Figueiró et al. / Journal of Equine Veterinary Science 50 (2017) 20–2624

to be more related to the inclusion of a greater number ofyoung immature animals, and would not reflect a low ef-ficiency of spermatogenesis in Pantaneiro horses comparedwith other breeds.

Despite the aforementioned variations found betweendifferent species and horse breeds, these differences werenot observed between different stages of the SEC, regard-less of the different cellular associations and the differentSertoli cell volumes at each stage [29,30].

In the present study, the total tubular length in Crioulohorses was 4,085.10 � 1,170.68 m (Table 2), which is verysimilar to a previous study by Varoni [20] for Pantaneirohorses (4,849.67 m); however, our value is higher than the3,800 m reported by Neves [23] for donkeys, 2,419 m re-ported by Swierstra et al [16] for crossbred horses, and2,808 m reported by Jones [15] for 30-month-old BrazilianSport Horse. However, the determination of this parameterhas some limitations, considering that it depends on thetesticular volume of each animal. Thus, an animal with agreater testicular volume could have a greater tubularlength than another animal with lesser volume; however,this difference does not necessarily reflect different levelsof sperm production. Therefore, these comparisons aremore reliable when comparing tubular length per gram oftestis. In this case, the calculation is independent of thedifferences in testicular volume among animals.

The length of seminiferous tubules per gram of testis inCrioulo horses (26.1 �10.6 m) was slightly higher than the10–20 m/g reported for most mammals [18]. However, ourstudy showed lower values than those reported for Pan-taneira (37.7 m/g), suggesting a greater potential of spermproduction in this latter species compared with the Crioulohorse. However, the pattern observed in the present studydiffers from that in other studies; the length of seminifer-ous tubules per gram obtained in the present study issimilar to the values reported for crossbred horses at17.0 m/g [16] and 16.6 m/g [9], and 22m/g for donkeys [23].

The characteristic cellular associations of the eightstages of the SEC in Crioulo horses are described as follows:

Stage 1 (Fig. 1A): At this stage, the presence of type A (A)spermatogonia and primary spermatocytes in pre-leptotene/leptotene (PL/L) were observed in the basalcompartment of the seminiferous epithelium. Primarypachytene (PC) spermatocytes and round spermatids (Rd)forming several layers were observed in the adluminalcompartment.Stage 2 (Fig 1B): This stage was characterized by the pres-ence of type A spermatogonia and primary leptotene (L)spermatocytes close to the basal membrane. The rest of theseminiferous epithelium was constituted by PC spermato-cytes and spermatids in the elongation phase (EL).

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Stage 3 (Fig 1C): Type A primary spermatogonia in zygotene(Z) and in diplotene (D) were found in the basal compart-ment. In the rest of the compartments, we found elongatedspermatids forming clearly defined bundles in theepithelium.Stage 4 (Fig 1D): In this stage, the most characteristicfeature was the presence of meiotic elements. Type Aspermatogonia were found in the basal compartment, andprimary spermatocytes were found in transition fromzygotene to pachytene. We also observed spermatocytes indiplotene and secondary spermatocytes (II). In addition, weobserved recently formed round spermatids, and theelongated spermatids were forming compact bundles.Stage 5 (Fig 2A): The type A spermatogonia were found inthe basal compartment, agreeing with previous studies;however, the mitotic division for forming type B1 sper-matogonia was also visible. Primary spermatocytes inpachytene, and the recently formed round spermatids werevisible in the adluminal compartment. This compartmentalso contained elongated spermatids, which were formingdeeper in the epithelium, presumably inside the crypts ofthe Sertoli cells.Stage6 (Fig2B): TypeAspermatogoniawerevisibleagain,butB1 spermatogoniawereprominent in thebasal compartment.Primary spermatocytes in pachytene, round spermatids, andelongated spermatids forming bundles heading toward theluminal edge were observed in the adluminal compartment.B1 spermatogonia were occasionally seen in division tofurther develop into B2 spermatogonia.Stage 7 (Fig 2C): Several B2 spermatogonia and few type Aspermatogonia were seen in the basal compartment at thisstage. In the adluminal compartment, we observed primaryspermatocytes in pachytene, round spermatids, and elon-gated spermatids. The elongated spermatids, whencompared with the previous stage, were forming bundleslocated even closer to the luminal edge. Small residualbodies, resulting from the elongated spermatids’ cytoplasmcould also be observed.Stage 8 (Fig 2D): Themost characteristic aspect of this stagewas the presence of elongated spermatids on the luminaledge forming a palisade-shape. Some of the elongatedspermatids were in the process of release toward thetubular lumen. The residual bodies were larger and darkerthan those observed in the previous stage. Types A and B2

spermatogonia could be seen intermingled with primaryspermatocytes in PL. Finally, spermatocytes in pachyteneand round spermatids could be seen in the adluminalcompartment.

Sertoli cells could be seen in all the eight stages, andwerecharacterized by their undefined shape, irregular nucleus,and clearlyapparent nucleolus. TypeA spermatogonia couldalso be observed in all the stages; however, because of themethodology used, their morphological differences couldnot be classified as differentiated or undifferentiated.

4. Conclusions

We conclude that the morphometry of seminiferoustubules in Crioulo horses are similar to that of previous

studies on other horse breeds. To our knowledge, thevolumetric proportions of the seminiferous tubules andLeydig cells in Crioulo horses in the present study are one ofthe highest reported in stallions.

Acknowledgments

The authors would like to thank the following famrs forproviding the animals included in this study: CabanhaNossaSenhora da Penha and Fazenda Pantanal in the State ofEspírito Santo, and Cabanha Trevo Branco, Cabanha Infinito,and Estância Artigas in the State of Rio Grande do Sul.

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