Testicular cytology of alpaca: Comparison between impressed and smeared slides

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Abstract

Testicular fine needle aspiration (TFNA) has proven to be a simple and minimally invasive procedure, which allows assessments of cytological parameters of seminiferous epithelium/tubules more accurately in a short time. Though this technique does not cause negative effects on sperm quality or any damage to testicular tissue, its use is very limited in male animal infertility diagnostics. Report on the use of this technique in South American Camelids (SAC) is very limited. Therefore, the aim of this study was to evaluate the efficacy of TFNA for identification of different testicular cells and cell indices, and their correlation with that of impression cytology. A total of 98 slides were prepared from testes of six adult alpaca males, collected immediately after slaughter. Aspiration samples were performed by inserting a fine butterfly needle (21 G) connected to a 50 ml syringe into a testicle and multiple plane aspirations were carried out to obtain the materials destined to the smear. Three different imprints on slides were taken from each testicle. All slides were air-dried, stained with modified May–Grünwald–Giemsa (MGG) stain and then examined under light microscope with 1000× magnifications. Spermatogenic cells such as, spermatogonia (Sg), primary spermatocytes, secondary spermatocytes, early spermatids (ab), late spermatids (cd) and spermatozoa, and Sertoli cells were counted. The spermatozoa percentage was expressed as spermatic index (SI) and the number of Sertoli cells, counted apart, was expressed as sertoli cell index (SEI). There was not any significant difference between the spermatogenic cell parameters obtained from the two types of slides, but SEI were significantly different in two types of smears. The results of the study provide support for the use of TFNA as a useful minimally invasive modality to identify different spermatogenetic cell classes in alpaca. Moreover, the possibility to standardize this method might provide a greater impulse to the clinical diagnostics of SAC male infertility.

Introduction

The reproductive system of the male camelid presents several anatomical and physiological peculiarities (Tibary and Vaughan, 2006) and they are necessarily bred at older ages than most domestic species. Testicular growth in these species is slow and maximum size is not reached until 3 years of age (Bravo and Johnson, 1994). Though sperm production starts as early as 10–12 months of age in a few male alpacas (Smith et al., 1994), male alpacas do not reach full maturity until 5 years of age. Therefore, proper clinical examination and reproductive analysis of male alpacas are necessary for evaluation of breeding soundness and infertility, and several techniques such as testicular palpation, ultrasonography of reproductive organs and testicular biopsy have been performed during sire selection (Tibary and Vaughan, 2006). Generally, testicular biopsy has been used to evaluate and classify males with varying degrees of testicular failure (Pàpic et al., 1988) and it provides a definitive assessment of seminiferous tubule and interstitial cellular architecture (Gottschalk-Sabag et al., 1993, Turek et al., 1997). 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. TFNA has proved to be useful diagnostic tool for male infertility (Mahajan et al., 1999), testicular tumors as well as in non neoplastic and inflammatory conditions of testes (Al-Jitawi et al., 1997). In addition to its usefulness and accuracy in diagnosis of the state of spermatogenesis in the infertile male, TFNA offers many advantages over open testicular biopsy as this technique avoids the problems of post-operative haemorrhage, fibrosis, adhesions, and risk of development of anti-sperm antibodies resulting from the breach of blood–testis barrier during testicular biopsy (Agarwal et al., 2004). Moreover, in assisted reproduction technique (ART), TFNA has gained wide acceptance in Intra Cytoplasmic Sperm Injection (ICSI) for the management of azoospermic patient (Arïdoğan et al., 2003). Though this effective technique is widely used in man, there are few reports on TFNA in domestic animals, such as, in dog (Dahlbom et al., 1997, Romagnoli1 et al., 2009), bull (Chapwanya et al., 2008) and stallion (Leme and Papa, 2000). Moreover, report on the use of this technique in male alpacas is very limited and standardization of this technique may have great impact on assessment of male alpacas for breeding suitability. Therefore, the purpose of this study was to compare percentage variability of different testicular cells obtained from two slide types (TFNA and impression/IMPRINT smears) to assess the effectiveness of TFNA in male alpacas for qualitative and quantitative identification of testicular cells.

Section snippets

Materials and methods

Testes from six alpaca males were collected immediately after slaughter. A fine butterfly (21 G) needle connected to a 50 ml syringe was inserted into a testicle and multiple plane aspirations were carried out to obtain materials destined to the smear from different areas to obviate sampling errors. Five slides were prepared from aspiration of each testicle. After making aspirated smears, all testes were incised to make impression slides. The impression slides were taken into consideration as a

Results

In this study, it was possible to obtain enough material from all testes to identify and quantify the spermatogenic cells and Sertoli cells with fine needle aspiration. In TFNA smears, different types of spermatogenic cells were scattered over the slide and normal cell-to-cell contacts were less in most cases in contrast to impression smears (Fig. 1a and b). The cytological appearance of spermatogenic cells in an air-dried MGG stained smear showed various transitional forms, from spermatogonia

Discussion

The aim of this experiment was concentrated on the quantitative study of spermatogenic cells and Sertoli cells in TFNA and impression smears, and to define SI and SEI, which quantify testis activity of alpacas. Interest in TFNA has picked up in recent years following characterization of different cell types in cytological smears and demonstration of good correlation of cytological diagnosis with histological categories by the pioneering works of some researchers (Obrant and Persson, 1965, Pàpic

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