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Table of Contents
ORIGINAL ARTICLE
Year : 2017  |  Volume : 6  |  Issue : 1  |  Page : 23-28

Ultrasound guided testicular fine needle aspiration in buck (Capra hircus)-An animal model


1 Department of Surgery and Radiology, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh
2 Department of Surgery and Obstetrics, Faculty of Veterinary Science, Agricultural University, Mymensingh, Bangladesh
3 Department of Anatomy and Histology, Faculty of Veterinary Science, Agricultural University, Mymensingh, Bangladesh
4 Department of Pathology, Faculty of Veterinary Science, Agricultural University, Mymensingh, Bangladesh

Date of Web Publication25-Sep-2017

Correspondence Address:
Sattwikesh Paul
Assistant Professor, Department of Surgery and Radiology, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh MujiburRahman Agricultural University, Gazipur-1706
Bangladesh
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Source of Support: None, Conflict of Interest: None


DOI: 10.12980/apjr.6.20170105

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  Abstract 


Objective: To establish ultrasound guided testicular fine needle aspiration (TFNA) as well as to assess the effectiveness of uni-directional (UD) and multi-directional (MD) TFNA in buck according to testicular cells, echotexture and gross changes of testicle, age of buck was considered. Methods: A total of 120 samples were collected with both directions (UD, n=60) and (MD, n=60) suction from testis of 10 apparently healthy bucks. All slides were stained with May-Grünwald-Giemsa and examined under light microscope with 1 000 × magnifications to count spermatogenic cells, spermatozoa and sertoli cells. The percentage of spermatozoa and sertoli cells were expressed as spermatic index and sertoli cell index. Results: Results revealed no difference in the presence of various spermatogenic and sertoli cells in cell cluster of slides made either unidirectional TFNA or multidirectional TFNA. Early spermatids were the most numerous, followed by late spermatids, primary spermatocytes, spermatogonia. Sertoli cell index was higher in TFNA smears of young bucks prepared 7-13 mo of age and spermatic index was higher in adult bucks 14-24 mo of age. No echogenic change was observed in the echotexture of testisafter TFNA. Conclusions: It seems that TFNA has no serious ill effect on the buck testis when uni-direction aspiration is performed. Moreover, the possibility to standardize this method might provide a greater impulse to the clinical diagnostics of male animal infertility.

Keywords: Testicular fine needle aspiration, Spermatic index, Sertoli cell index


How to cite this article:
Paul S, Juyena NS, Das SK, Ferdousy RN, Sarker RD, Mou SJ. Ultrasound guided testicular fine needle aspiration in buck (Capra hircus)-An animal model. Asian Pac J Reprod 2017;6:23-8

How to cite this URL:
Paul S, Juyena NS, Das SK, Ferdousy RN, Sarker RD, Mou SJ. Ultrasound guided testicular fine needle aspiration in buck (Capra hircus)-An animal model. Asian Pac J Reprod [serial online] 2017 [cited 2020 Oct 24];6:23-8. Available from: http://www.apjr.net/text.asp?2017/6/1/23/215597




  1. Introduction Top


Goats among the first domesticated species for the production of meat, milk, skin, and fiber,are an integral part of rural's symbiotic system of crop and livestock production in Bangladesh. Bangladesh possesses 34.5 million goats at present, which constitute about 4.49% of the total population in the world[1]. In Bangladesh the Black Bengal goat having variation in color, size and weight comprised more than 90% of goat population[2]. Fertility assessment is one of the most talks of the topics in livestock sector now a day. Sub-fertile sires can easily be identified and eliminated by evaluating reproductive soundness. Identification of fertility problems in bucks requires accurate diagnostic techniques. Moreover, various chemical castration procedures are followed to control breeding male and fattening purposes, for which study of spermatogenesis and semen production are necessary to evaluate the efficacy of castration method. Testicular Fine Needle Aspiration (TFNA) cytology and trans-scrotal ultrasonography can be employed in assessing fertility in bucks, as well as the diagnosis of testicular lesions. Recent technical advances of ultrasonogram applications and post processing developments have enabled new aspects in the structural and functional analysis of testicular tissue and therefore male fertility[3]. Accurate measurement of scrotal circumference can be done bytrans-scrotal scanning through visualization of tissue interfaces within the scrotum[4]. Moreover, palpable and nonpalpable testicular lesions can easily be assessed by this method[5].

TFNA is a painless, minimally invasive and cost-effective procedure. It is a less time consuming procedure which have the ability to present all spermatogenic cells with their defined characteristics. TFNA has established itself as the useful diagnostic tool for male infertility[6],[7],[8]. It is advantageous to use TFNA as this technique avoids the problems of post-operative hemorrhage, fibrosis, adhesions, and risk of development of anti-sperm antibodies. However, all of the conditions may appear when open biopsy of testes is performed[9]. In addition, TFNA has recognized widely in Intra Cytoplasmic Sperm Injection (ICSI) in assisted reproduction technique (ART)[10]. But, there are few reports of TFNA in domestic animals, such as, in dog[11] and[12], bull[13], stallion[14] and alpacas[15] rather than in man. Studies on TFNA in buck are very scanty. Therefore, this study was designed, to establish ultrasonoguided testicular fine needle aspiration in bucks, to assess the effectiveness of uni-directional and multidirectional TFNA in buck according to qualitative and quantitative identification of testicular cells; echotexture of testicle and gross changes in testicular size (wide and length) and architecture after TFNA.


  2. Materials and methods Top


2.1. Animals

Ten bucks were divided into two groups according to age and each group contained 5 bucks. Samples (n=60) were collected through unidirectional TFNA from both right and left testes. After one month multidirectional TFNA was performed to collect samples (n=60) from all bucks. All TFNA samples were monitored with trans-scrotal ultrasonography.

2.2. Ultrasonography of testis

B-mode digital Ultrasound system (Vet Eickemeyer Magic 5 000) with transrectal probe 5 MHz, probe type: C20615S) was used for ultrasonographic scanning. Buck was restrained in lateral recumbency by an assistant. The buck was cleaned over the inguinal area. The testis and epididymis were grasped at the scrotal neck and the transducer was progressed cranially along the neck of the testis to the cauda epididymis. Local infiltration of 2% Lidocaine Hydrochloride (Jasocaine®, Jayson Pharmaceuticals Ltd., Bangladesh) was performed in spermatic cord. Desensitization was confirmed by needle pricking. The testis and scrotum were grasped with one hand so that the scrotal skin tensed over the site to be aspirated. The ultrasonography machine was activated and the probe was placed over the site of fine needle insertion to the testis. A 21 gauge 2.3´´ butterfly needle attached to a 50 mL syringe was advanced at right angles to the skin and inserted smoothly into the testes using a method similar to that described by Larkin[16]. Inserted needle was observed ultrasonographycally for proper placement and direction of needle in the testis. The ultrasound monitor and operator's eye were at comparable level for accurate reading of ultrasound images. The technique used for scanning was similar to that described by Ahmad et al.[17] in small ruminants. During TFNA probe was set on the testes to monitor sample aspiration.

2.3. Aspiration of testicular tissue

Suction pressure was applied to aspirate testicular tissue. The aspirate was then expelled onto a clean glass slide and spread using the edge of a second glass slide with a 45 degree angle. For multi directional technique fine needle was inserted into the testis in 3-4 different directions. All slides (n=120) were air-dried, stained with modified May-Grünwald-Giemsa (MGG)[18]. Briefly, smears were immersed into May-Grünwald stain for 3 min, rinsed with water, then immersed into Giemsa (diluted 1:2 in distilled water) for 10 min. Then the extra stain was rinsed and samples were again air-dried. The cytological smears were examined under light microscope with 1 000 magnification with immersion oil. An adequate and informative TFNA specimen was defined by observing several clusters or cords of cells that contained 10 or more cells, and for interpretation at least 200 well-dispersed spermatogenic cells and sertoli cells were counted from different clusters on each slide. Various cell types were identified by their distinctive morphology and criteria described by Schenck et al.[19]. Spermatogonia (Sg), primary spermatocytes (Ps), secondary spermatocytes (Ss), early spermatids (Es), late spermatids (Ls), spermatozoa and sertoli cells were identified and counted. Wide and length of the testis were measured before and after needle insertion with the help of slide calipers. Before taking the measurement gentle pressure was given at the base of the scrotum for apposition of the testis. Post operated data were collected for at least five consecutive days. All data were recorded.

2.4. Castration of buck

Open method castration was done in four bucks (2 for each direction) following aseptical method after 6 d of TFNA. After castration each testis was sectioned to observe any tract or mark of TFNA with two directions.

2.5. Statistical analysis

The results were given as mean±SEM. The spermatozoa were expressed as the spermatic index (SI=the number of spermatozoa/ the number of total spermatogenic cells), and the Sertoli cells were expressed as Sertoli cell index (SEI= the number of Sertoli cell/ the number of total spermatogenetic cells). Statistical analysis was performed using SPSS version 17.0. Paired t-test was performed to analyze significant variations among cellular types in two methods of TFNA and two age groups. ANOVA was carried out to analyze significant variation among testicular size (wide and length) recorded before and after TFNA. P<0.05 value was considered as significant.


  3. Results Top


3.1. Qualitative and quantitative identification of testicular cells

The study was conducted to establish the ultrasonoguided fine needle aspiration technique in buck. It is assumable that multi-direction suction of tissue gives diversified cells than uni-direction suction during TFNA. The cytological appearance of spermatogenic cells in an air-dried MGG stained smear showed various transitional forms, from spermatogonia to spermatozoa, characterized by diminution of nuclear size and condensation of chromatin.

There was no difference in the presence of various spermatogenic cells and sertoli cells in cell cluster of TFNA slides prepared after two directions suction [Figure 1].
Figure 1: Presence of different spermatogenic cells and sertoli cells in cell cluster considered for cell counting.

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In the cytological smear, sertoli cells were identified as having a nude and large round or little oval shape, vesicular nucleus which shows finely granular chromatin and usually contains a large nucleolus. Nucleolus always took different color from nucleus (e.g. Red colored nucleus, blue colored nucleolus) Cytoplasm was not always seen [Figure 2]a. The spermatogonia were usually uninucleate but occasionally are binucleate or multinucleate. The nuclei were round or oval, slightly eccentric and dark or pale, depending on their chromatin density. The chromatin frequently had a denser area peripherally, with a fine homogenous chromatin pattern. Pale colored spermatogonia were found in this study [Figure 2]b. Primary spermatocytes had a large nucleus with thread-like or coarse chromatin. As chromosomal structures became very prominent, the nuclear outline may become irregular. The cytoplasm, if present, was basophilic; it was more deeply stained in the periphery of the cell [Figure 2]c. Secondary spermatocytes were rarely identified because of their short lifespan and immediate transformation into spermatids. Nucleus was smaller than primary spermatocytes and granular and eccentric. Nearly red or violet colored granules were seen in red colored nucleus. Nucleolus was not normally seen [Figure 2]d. Spermatids were of two types-Es and Ls. Early spermatids had a much smaller, round or irregularly triangular nucleus. Chromatin was finely, homogenously granular and doubly (some) colored. Granular nucleus was always seen in the central of cytoplasm. Cytoplasm was basophilic, granular and vacular [Figure 2]e. The nucleus of Ls was small, homogenous and eccentric in basophilic granular cytoplasm [Figure 2]f. Spermatozoa had oval nuclei with very dense chromatin with or without tail [Figure 2]g. Leydig cells were not detected or, possibly, could not recognize.
Figure 2: Photographic demonstration of different types of cells in TFNA samples; a) Sertoli cells b) spermatogonia c) primary spermatocyte d) Secondary spermatocyte e) early spermatid f) late spermatid g) spermatozoa

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Quantitative analysis of testicular cytology was performed. The number (mean±SEM) of various spermatogenic cells were shown in [Table 1]. In this study, both age and TFNA direction were considered to describe numerosity of cells. Early spermatids were the most numerous, followed by Ls, Ps, Sg in bucks of two age groups although significant variation was existed in numbers of cells [Table 1]. There was no significant difference in Sg and Ss between age groups as well as between TFNA directions. But Sg in right testis varied significantly (P<0.05) between age groups. Similarly, significant (P<0.01) variation was existed in number of Ps, Es and Ls. Regarding the needle direction, we found significant (P<0.01) variations in number of spermatids Es and Ls.
Table 1: Numerosity of spermatogenic cells (mean±SEM) in smear of TFNA regarding age and needle direction in bucks.

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SI and SEI are considered for demonstration of the efficacy of spermatogenesis and are presented in [Table 2]. SEI was higher in TFNA smears of bucks of 7-13 mo of age. Whereas, SI was higher in TFNA smears of bucks in 14-24 mo age group. Insignificant variation was found in SEI and SI when needle direction was considered.
Table 2: Spermatic index (SI) and sertoli cell index (SEI) of TFNA smears in bucks.

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3.2. Observation

3.2.1. Trans-scrotal ultrasonography

After needle insertion into the testis a hyper-echoic area was seen [Figure 3]a. A wavy hyper-echoic area was also identified in ultrasonography picture during aspiration [Figure 3]b. There was no echogenic change in the echotexture of testis during monitoring after both uni-direction and multi-direction suction [Figure 3]c. However one buck was identified having several hyper-echoic areas within the testis from the beginning [Figure 4] which might be microlith.
Figure 3: Presence of needle head within testis (a); a wavy hyper-echoic area (yellow) circle during aspiration (b); normal echotexture of testis after TFNA (c).

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Figure 4: Presence of several hyperechoic areas in testis (Arrow head).

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3.2.2. Testicular width and length

Testicular size was changed due to scrotal skin swelling, although there was no echotextural change observed in ultrasonophy monitoring. Swelling was not marked in testes after uni-direction suction, for this data were excluded. But, more swelling with pain was present after multi-direction suction, which disappeared within 2-4 d. [Table 3] represents the changes in testicular size (length and width) which indicated the swelling after TFNA. Variations among the observations in testicular width and length were insignificant.
Table 3: Changes in testicular size after TFNA.

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3.2.3 Cross sectional observation of testes after TFNA

After castration testes were examined carefully. No change of the testicular tissue was observed in testis of bucks after uni-direction suction of tissue. In contrast, marked bloody needle line was present in cross section of testis having multi-direction suction for TFNA .


  4. Discussion Top


Reproductive soundness evaluation facilitates the elimination of sub-fertile sires from breeding programs. Proper clinical examination and reproductive analysis of male animals are necessary for evaluation of breeding soundness and infertility. Several techniques, e.g. testicular palpation, ultrasonography of reproductive organs and testicular biopsy can be performed during sire selection[20]. Generally, testicular biopsy has been used to evaluate and classify males with varying degrees of testicular failure[21] and it provides a definitive assessment of seminiferous tubule and interstitial cellular architecture[22]. Among different biopsy techniques, TFNA has gained increasing popularity as a painless, minimally invasive and cost-effective procedure, which can provide a more representative sample of the testis in much shorter time as compared to open biopsy[15]. There is limited study on the use TFNA in animals. The study was concentrated to evaluate ultrasound guided TFNA technique with uni and multi directions of tissue collection through qualitative and quantitative study of testicular tissues, echotexture observation of testes in buck.

In this study, spermatogenic cells were well characterized and were identified by their distinctive morphology described by Schenck and Schill[19]. Percentages from spermatogonia to late spermatids were increased showing a normal evolution of the spermatogenic process in smears. Considering the numerosity of cells, early spermatids were the most numerous in buck TFNA. We are unknown of data regarding testicular cytology in buck. However, Stelletta et al.[15] have reported higher number of early spernatocytes in Alpaca. In a study, Foresta and Varotto[23] reported greater numbers of late spermatids in cytological smears of men with normal fertility. The testis is an ideal organ for evaluation by FNA because of its uniform cellularity and easy accessibility[24]. The identified patterns represent histological diagnoses but are based on relative numbers of three easily identified germ cell types on cytologic assessment: primary spermatocytes, spermatids, and spermatozoa. The number of secondary spermatocytes was very small. This finding is in agreement with Stelletta et al.[15]. After production secondary spermatocytes rapidly divide to produce haploid spermatids (Meiosis II). The brevity of this stage means that secondary spermatocytes are rarely seen in histological studies and the small number might be due to their short life span[21]. Leydig cell was not observed in our study. Aspiration of interstitial tissue is difficult and hence Leydig cells are usually not visualized in the cytological smears[11],[15].

Regarding the needle direction, authors found significant (P<0.01) variations in number of early and late spermatids. It might be a result of technical process of preparing aspiration smears, which could be attributable to the usual counting technique. These findings may be a normal cytological finding for buck's testes, which needs a further confirmation. Spermatic Index demonstrates the efficacy of the spermiogenesis[23]. The relationship between spermatogenic and sertoli cell numbers or SEI has been studied as an indicator of tubular germ cell potential or the spermatogenesis efficiency[25],[26]. In both testes, there were significant differences (P>0.05) between SEI obtained from bucks of two age groups. In both humans and horses, the number of sertoli cells is related to the level of spermatogenesis as measured as daily sperm production per testis This relationship is higher for horses (r2 =0.68) than for humans (r2 =0.39). The spermatogenic cells are in close contact with sertoli cells which are thought to provide structural and metabolic support to the developing sperm cells. Proliferation of sertoli cells is greatly reduced in the adult compared to the early proliferation rates prior to puberty[27].

In trans-scrotal ultrasonography, the testis of bucks appeared as a homogenously hypoechogenic structure. The mediastinum testis was represented as a hyperechoic line in longitudinal images. These observations are similar to those described for boar[28], dog[29], bull[30], rams and male goats[17] and camels[31]. Ahmad et al.[32] recorded an increase in the echogenicity of testicular parenchyma with advancement in age of the bull; the mean number of pixels of testicular ultrasonograms increased significantly up to 24 mo of age and then leveled off as the age increased. One buck was identified having several hyperechoic areas within the testis from the beginning [Figure 4]. These hyperechoic areas were assumed to represent mineralization in the testes. Ahmad et al.[33] and Ahmad and Noakes[5] observed hyperechoic areas in the testicular parenchyma of infertile bucks. According to Lenz and Giwercman[34], such bright echogenic spots were representing microlithiasis or microcalcifications.

Ultrasonography demonstrated hypoechoic areas inside the testicular parenchyma just afteraspiration of the buck testis. Carpi et al.[35] also revealed similar hypoechoic areas in human testes during ultrasonography at the time of TFNA and denoted these areas as intra-testicular bleeding. This intra-testicular hemorrhage was evident in testicular cross section after the multi-directional suction has taken place. In this study, there was no echogenic change in the echotexture of testis during monitoring after both uni-direction and multi-direction suction [Figure 3]c.

Testicular ultrasonography can be used as a diagnostic tool for acute scrotal pain to more chronic and nonspecific symptoms[36],[37]. In animal studies trans-scrotal ultrasonography has been shown to demonstrate the ultrasonic morphology of testes of bulls[38], rams and boars[28]; to assess testicular trauma, evaluation of testicular swelling or masses[39], infertility[40], male hypogonadism[41], testicular interventions etc. It is also helpful not only for diagnosis of testicular torsion, cryptorchidism, scrotal hernia, varicocoele but also follow-up of testicular microlithiasis[42].

Sperm production ability and fertility is largely depends on testicular size[43],[45]. Length and width of testis or the total scrotal diameter is used to determine the size of the testis. Testicular size (wide and lengthy) was measured up to 5th day of post-aspiration. Swelling was not observed in testes after uni-direction suction, for this data were excluded. But, more swelling with pain was present after multi-direction TFNA suction, which disappeared within 2-4 d. Following FNA in 7 dogs, there was no pain or local trauma[12]. The extent of testicular damage by multiple punctures and negative pressure applied during aspiration is still unknown[45],[46]. The number of punctures and of excursions at each puncture site issignificant technical aspects of FNA[46]. Gouletsou et al.[46] observed skin hemorrhage and mild swelling of the scrotum immediately after multi-direction FNA at insertion point which lasted for 2-5 d post-aspirationdays in cat. When castration was performed at 6th day post-aspiration, the entrance point of the needle could be detected on the surface of the testis. In multi-direction TFNA after testicular cross section, hemorrhagic areas were evident indicating the path of the needle inside the testicular parenchyma. Similar finding was reported by Gouletsou et al.[46].

This study confirms that TFNA can evaluate all classically defined histological types of spermatogenic cells. Though, swelling with pain and marked haemorrhagic areas was present in cross section of testis having multi direction suction for TFNA, there is no echogenic change in the echotexture of testis during monitoring after both uni-direction and multi direction suction. During this study, semen could not be collected and evaluated to correlate TFNA cytology with sperm production. However, further study should be performed to standardize the numerosity of testicular cells from TFNA smears in both fertile and infertile bucks considering age, breeding activities, seasonality and pathological influences. This study could help us for the assessment of function of testicular cells in determining breeding soundness of not only buck but also other male animals more effectively and efficiently.

The cytological appearance of testicular cells after TFNA can be well defined and early spermatids are the most numerous, followed by late spermatids, primary spermatocytes, spermatogonia in bucks. In case of indices Sertoli cell index is higher in TFNA smears of bucks of 7-13 mo of age and Spermatic index is higher in TFNA smears of bucks in 14-24 mo age. Insignificant variation is found in testicular cells when needle direction was considered. Testicular fine needle aspiration (TFNA) has no serious ill effect on the buck testis and uni-direction aspiration is advisable to study testicular cytology.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgements

The author is thankful to University Grant Commission (UGC) for National Science and Technology (NST) Fellowship and financial assistance. The author is also thankful to Dr. Md. Musharraf Uddin Bhuiyan, Professor, Department of Surgery and Obstetrics, Bangladesh Agricultural University, Mymensingh and honorable Incharge, Community-based Dairy Veterinary Foundation (CDVF), Bangladesh Agricultural University, Mymensingh for his affectionate encouragement and for the opportunity of examining prepared slide samples used in the research work. The author is also thankful to the Department of Surgery and Obstetrics to give ethical approval of the research work.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

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Abstract
1. Introduction
2. Materials and...
3. Results
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