Introduction

Male contribution to feline reproductive efficiency is of great importance, given that, besides the genetic donation, it is possible to apply a greater selective pressure on males than on females. Therefore, males should be adequately examined and evaluated to allow for better selection. The study of the semen obtained from tomcats is fundamental to safeguard their genetic potential and aid in the biotechniques for animal reproduction. Domestic cats have been used as experimental models for the study of reproduction in wild felids in risk of extinction, as well as primates and humans. Knowledge obtained from research in this area can be used as support for advances in innovative or even revolutionary therapeutic methodologies with application on reproductive disorders, as well as for the introduction, development and improvement of advanced biotechniques (França & Godinho, 2003, Pukazhernthi et al. 2006, Saake, 2008). Electroejaculation is the ideal method for obtaining feline semen (Johnston et al.2001, Axner et al.2007, Vieira et al.2007). Semen evaluation can be used in cases of suspected infertility, for the confirmation of normal spermatogenesis in young males before they initiate their reproductive life, or as a part of artificial insemination and freezing program; it can also be used to evaluate semen production following certain systemic diseases or drug therapies. In semen evaluation, a sample should be analyzed immediately after collection in order to determine its macroscopic characteristics such as volume, color, odor and pH, and its microscopic features such as motility, vigor, and sperm morphology (Pineda et al.1984, Feldman & Nelson, 1996, Johnston et al.2001). Any alteration in the endocrine or exocrine function of the testis will reflect in some way on modifications of the spermiogram, including morphological as well as functional aspects. Due to these alterations, semen analysis is of great importance in evaluating feline reproductive capacity. The objectives of this work were to evaluate the technique for semen collection in domestic cats using the electroejaculation method, as well as the characteristics of the ejaculate obtained from domestic cats with no history of reproductive disorders.

Materials and Methods

Fifteen domestic cats (Felis catus), of mixed breeds, with no history of reproductive disorders, between one and three years of age, coming from the Castelo Branco Veterinary University Clinic in Rio de Janeiro, were evaluated. The animals were sedated and anesthetized for better clinical evaluation and semen collection with the purpose of decreasing stress and guaranteeing the animals' well-being. All cats were submitted to a complete andrological evaluation including: clinical examination, testicular biometry as well as macroscopic and microscopic semen analysis. The animals were kept under general anesthesia during the clinical examination of the male reproductive system with the use of ketamine (5 mg/kg), xylazine (0.5 mg/kg) and atropine (0.04 mg/kg) as well as epidural anesthesia with 2% lidocaine without vasoconstrictor (6 mg/kg). The animals were monitored throughout the whole procedure, with careful observation of their body temperature, breathing movements and cardiac rhythm. Data obtained from anamnesis in addition to the animal's reproductive history was recorded in the animal's individual chart. Clinical examination included evaluation of the scrotum, as well as size, shape and presence or absence of one or both testicles inside the scrotum, evaluation of size and conformation of the epididymis and spermatic cord, and examination of the penis and prepuce. Both testicles (right and left) were palpated to determine their consistency and mobility. Testicular biometry was performed through the evaluation of scrotal circumference as well as width and length of the testicles. In order to measure scrotal circumference, the testicles were pulled towards the lower end of the scrotum and a reading was obtained (in centimeters) using a measuring tape placed around the widest portion of the scrotum. A caliper was used to measure the length and width of each testicle. Semen collection was obtained using an electroejaculator (Eletrovet), a cylindrical transrectal electrode (probe) measuring 12.0 cm x 1.0 cm in diameter, containing three longitudinal strips of stainless steel each measuring 5.0 cm in length (Figure 1). The penis was extruded for evaluation of the mucous membrane, which should be light pink in color, and a collecting tube (Eppendorf tube) was applied over the penis. The series of electrical shocks followed a 60 stimuli protocol divided into two series. Following semen collection, the ejaculate was immediately evaluated regarding its macroscopic characteristics (volume, color, odor and pH) and microscopic characteristics, which corresponds to motility and vigor analysis, to be classified as 0-100% and 0-5, respectively. The analysis of sperm cell morphology is aimed at identifying any structural abnormalities of the spermatozoa and was performed using Giemsa-stained sperm samples on microscope slides, where 200 spermatozoa were evaluated under a light microscope at 400x magnification. The structural abnormalities observed were divided into two categories: primary defects (major) and secondary defects (minor). Two hundred spermatozoa were evaluated, and semen containing more than 60% morphologically normal spermatozoa was considered ideal, given that it contained no more than 10% of total primary defects (individuals) and a maximum of 20% secondary defects. Data obtained for each cat was recorded in their individual charts.

Figure 1. Semen collection using an electroejaculator.

Results

All of the cats included in this study presented no abnormalities in their clinical examination. No alterations were observed in the animals' ejaculate following the previously mentioned anesthetic protocol. A small volume of semen was obtained from each of the 15 cats used in this study through the electroejaculation method. Regarding testicular biometry, scrotal perimeter varied between 6.0 and 9.0 cm and the width of the right testicles ranged from 0.8 cm to 1.2 cm, whereas the length ranged from 0.8 to 1.5 cm. The width of the left testicles varied between 0.8 cm and 1.2 cm whereas its length ranged from 0.8 to 1.5 cm. The macroscopic aspects of all the ejaculates were milky white in color, with no alterations regarding their odor, and a seminal pH that ranged from 6.0 to 7.0. Sperm motility varied between 0 and 90% in all evaluated animals, while sperm vigor ranged from 0 to 4. Five of the studied animals were found to have no morphologically normal spermatozoa, with total defects that ranged from 56 to 76% (Table 1). The morphological defects found in these animals were: tightly coiled flagellum, bent flagellum, abnormal mid-piece formation, detached heads, piriform head, abaxial, narrow, underdeveloped, distal retained cytoplasmic droplet and irregular contour. (Table 1)

Table 1. Semen evaluation in 15 domestic cats, obtained by the electroejaculation method.

Discussion and Conclusions

Regarding the animals' clinical examination, no clinical alteration was found in any of the studied animals, probably because they had no history of reproductive disorders and were all young animals, with a maximum of three years of age. The anesthetic protocol used in this study allowed for the use of the electroejaculation method without causing any discomfort to the animals and without altering the animal's ejaculate, according to Pires et al. 2007. The electroejaculation method, which is commonly used for feline semen collection, was proved to be efficient for obtaining and evaluating this species' semen (Dooley & Pineda, 1986, Axner, 2006, Vieira et al. 2007). Axner & Forsberg, 2007, also found small volumes of ejaculate obtained from cats following the electroejaculation method. Color and odor of the ejaculate obtained from all the animals were similar to those found by Vannuchi & Santos, 1997 and Vieira et al. 2007. Cats presenting spermatozoa with high motility (70 to 90%) and vigor (scale of 3 to 4) were also reported by Root & Johnston, 1994, Feldman & Nelson, 1996, Johnston et al. 2001, Zambelli & Cunto, 2006. Cats with sperm motility below 20 and vigor below 2 were considered to have low fertility, as proposed by Johnston et al. 2001, Zambelli & Cunto, 2006, Pukazhenthi et al. 2001, Pukazhenthi et al. 2006. Ten cats presented morphologically normal spermatozoa that ranged from 69 to 80%, characterizing good quality semen (Figure 2). Results obtained regarding sperm cell morphology are similar to those reported by Pineda et al.1984, Johnston et al. 2001, Zambelli & Cunto, 2006, Vieira et al. 2007. The remaining animals presented over 60% of abnormal spermatozoa, and the defects observed, such as tightly coiled flagellum or bent flagellum, mid-piece abnormalities, abaxial, underdeveloped, distal retained cytoplasmic droplet and irregular contour, are in agreement with those reported by other authors (Axner & Forsberg 1997, Zambelli & Cunto 2006, Axner & Forsberg 2007) (Figure 3). Penfold, et al. 2003, Pukazhenthi et al. 2006 have demonstrated in their study that although domestic cats are usually normospermic, some males can produce high concentrations of morphologically abnormal spermatozoa, in other words teratospermic, and the data agrees with that obtained in this study. The present work concludes that semen analysis is essential for the evaluation of fertility in the domestic cat; that the anesthetic protocol used was adequate for the proposed procedures; that the electroejaculation technique allows for collection of good quality ejaculates for macroscopic and microscopic analysis of the semen; and that 70% of the evaluated animals were suitable for reproduction.

Figure 2. Normal sperm.

Figure 3. Bent or coiled flagellum (small arrow), swollen mid-piece (medium arrow) and tightly coiled flagellum (large arrow).

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