Ultrasonography is an imaging technique that allows the diagnosis of changes in several organs. The obstacle to its use is  the  disagreement  in  the  interpretation  of  the findings under subjective and individual analysis (Nyland and Matton, 2004). To avoid such divergences, methods have been developed to quantify echogenicity and echotexture, such as the gray-scale histogram (Feeney et al., 2008). This allows quantification with the measurement of gray levels, with the determination of echogenicity by quantifying the echoes that return from the transducer and ecotexture by the luminous points that appear most frequently (Santos et al., 2009). This is efficient in the evaluation of testicular echogenicity during physiological changes in the reproductive phases (Silva et al., 2015).

Ultrasound evaluation can provide data that can aid in the diagnosis of disorders in the male reproductive tract, which can course in testicular dysfunction and subfetility (Ortiz-Rodriguez et al., 2017; Segabinazzi et al., 2018). 

In animal andrological evaluation, testicular ultrasound is the technique of choice to evaluate the testicular parenchyma and mediastinum (Clark et al., 2003) at different stages of maturation (Ahmad and Noakes, 1995). In this context, testicular biometrics is one of the components of andrological evaluation, constituting an important parameter in the choice of a breeder (Pimentel and Silva, 2010). In Brazil, the main interest in donkeys is summarized in the production of mules and the use as work animals. Although some studies have demonstrated reproductive evaluation techniques for donkeys (Costa et al., 1991), they do not yet have well-established andrological standards and do not present a standardized quantitative analysis of echotexture and testicular biometrics (Canisso, 2008).

In recent years, there have been many studies evaluating the reproductive tract of cattle through ultrasound, however, studies related to donkeys are scarce (Gacem et al., 2020). The difficulty in performing studies on donkeys is also due to the limited number of individuals that meet the selection criteria and the reduced breeding and reproduction of these individuals (Navas et al., 2017). This study aims to quantitatively determine the testicular echogenicity of asinines without a defined racial pattern, as well as to evaluate testicular biometrics and ultrasound changes observed in the testicles of these animals.  

There was no difference between the biometric variables and echogenicity of the testicular parenchyma and mediastinum of the right and left testicles (P > 0.05), with a testicular index (TI) of 2.98. The testicular volume results did not have a statistically significant difference between the antimers, maintaining bilateral symmetry (Table 1).

When analyzing testicular biometric variables according to age groups, a statistically significant difference was found for length (P < 0.05), width (P < 0.05) and testicular height (P < 0.05) between AG1 and AG2 for both the right testicle as for the left, as observed in Table 2. The testicular index and volume also showed statistically significant differences (P < 0.05) between the first and the second group of animals.

However, the echogenicity of the testicular parenchyma measured by the gray-scale histogram ranged from 70.25 to 75.08 pixels with homogeneous echotexture between animals, there was no correlation between echogenicity and  age  group.  Regarding  the  testicular  mediastinum, identified in all animals, an event similar to the testicular parenchyma was noted, with no statistical difference between the age groups in the left testicle. However, there was a statistical difference between the age groups when comparing the echogenicity of the right testicular mediastinum with increased echogenicity in the right testicular mediastinum correlated with age. After ultrasound scans of the testicles, the testicular parenchyma of homogeneous echotexture was evident, varying from low to moderate intensity, the hyperechogenic testicular mediastinum in relation to the testicular parenchyma and the hyperechogenic tunica surrounding the testicular parenchyma.

In the evaluation of the spermatic cord, the hypoechoic pampiniform  plexus was observed in relation to the testicular parenchyma and the presence of anechoic circular structures with anechoic content, visualizing the lumen of the pampiniform plexus veins. Ultrasonographic changes were observed in four animals in AG1 and in five in AG2, with heterogeneous testicular parenchyma, with the presence of microlithiasis (hyperechoic foci not formers of acoustic shadow), suggesting points of fibrosis (Figure 1).

Symmetry between the gonads was also observed in studies carried out with Nordestina asinines (Gastal, 1991). In  Campolina stallions, it was observed that the biometric values ??of the testicles showed no difference between the variables (Borges et al., 2010). These similar results demonstrate that even with different management and without an adequate nutritional condition, testicular symmetry was maintained, having no relationship with the development of testicular antimers.

However, this symmetry was not observed when studying the testicular biometry of asinines, observing in these animals’ different biometric values ??for right and left testicles, with significant difference in testicular length, as well as the height and width of the left testicle, smaller than that of the right one (El Wishy, 1974; Morais, 1990). In horses, this difference can be attributed to the early growth of the right testicle, in relation to the left one, during the initial reproductive development phase of the asinine (Gastal, 1991).

The biometric values ??of the animals evaluated in the present study were lower than the descriptions made for

asinines by El Wishy (1974) (LEN: 8.39 cm; HEI: 5.8 cm; WID: 4.6 cm), Kreuchauf (1984) (LEN: 8.58 cm; WID: 5.78 cm; HEI: 6.30 cm), Morais (1990) (LEN: 10.2 cm; WID: 7.0 cm; HEI: 7.4 cm), Gebbers (1995) (LEN: 8.7 cm, HEI: 7.4 cm; WID: 6.7 cm), Gastal (1991) (LEN: 7.6 cm; HEI: 5.5 cm; WID: 5.1 cm) Canisso et al. (2009) (LEN: 8.8 cm; HEI: 6.2 cm; WID: 68.35 cm). This difference in values ??is due to the difference in body weight between the animals evaluated since body weight directly influences testicular volume and size. In this study the average weight was 100 kg, while in the other studies the average weight varied between 150 to 275 kg.

However, in a study conducted by Aissanou and Ayad (2020), with testicular biometry of donkeys from Algeria, with an average weight of 193 kg and 10 years of age, they observed that the length (7.1 cm), width (4.87 cm), height (3.65 cm) and volume (73.6 cm³). This result is close to the values of the present study, even with the variation in body weight among the animals studied, this value in testicular biometry may be influenced by the arid location, with food scarcity and seasonal changes that these animals are subjected to.

The testicular index (TI) in the present study was lower than that recommended for stallions (Morais, 1990) and Pêga donkeys (Morais, 1990; Costa, 1991; Gebbers, 1995; Canisso et al., 2009). This fact can be explained by the body difference between the animals evaluated. In addition, management conditions can influence testicular and TI biometric characteristics. According to several studies, animals with TI less than or greater than eight proved to be reproductively efficient (Canisso et al., 2009; Costa, 1991; Gebbers, 1995). Asinines, even with an TI below that recommended for horses, have among domestic animals the highest spermatogenic efficiency per gram of testicular parenchyma (Neves, 2001).

The present testicular volume results are lower than those found by El Wishy in 1974. This author studied the testicles of nine donkeys (averages: age 5.1 years and weight 257 kg) and developed a formula for evaluating testicular volume and he obtained the volumes of 103.44 and 103.89 ml for the right and left testicles, respectively. The difference in testicular volume can be attributed by age and management conditions (Gacem et al., 2020).

The relationship between biometric values ??and age observed in the present study is similar to that obtained for donkeys of the Pêga breed (Canisso et al., 2009), sheep of the Santa Inês breed (Jucá et al., 2009), bubalus (Ayala, 2011), and Creole stallions (Mendes, 2012). In these studies, the authors observed that the biometric values ??in young animals are lower, with a proportional increase in the biometric characteristics with age. A progressive increase in testicular width was observed after 10 months in donkeys, more pronounced after 16 months of age, even in patients before puberty (Rota et al., 2018). This relationship is influenced by testicular and body development in young animals, since in adult animals’ testicular conformation, as well as sperm and hormonal production are already established (Canisso et al., 2009).

The measurement of testicular volume in different age groups can be a useful tool in andrological evaluation and in the prediction quality of sperm production, since it provides global dimensions of the organ and would facilitate the comparison between animals under different conditions of creation (Canisso et al., 2009; Gacem et al., 2020). When comparing the results of the testicular biometric characteristics of Pêga donkeys with the other breeds, it was observed that these, because they are medium-large animals within the species, present testicular biometric characteristics superior to those reported for Nordestina donkeys (Gastal, 1991) and for crossbred animals of African origin (Kreuchauf ,1984).

The echogenicity of the testicular parenchyma with homogeneous echotexture without alteration of echogenicity among the age groups, for this study, differed from the results found for bubalus (Ayala, 2011), in which a progressive growth of testicular echogenicity was observed between the age groups. It is justified by the fact that the testicular parenchyma of young animals is homogeneous and with low echogenicity, increasing in direct proportion to the age of the animals (Cardilli et al., 2009a, 2009b, 2010; Pastore, 2008).

Although it has been described that the male's reproductive organ is more resistant to nutritional changes in immature animals (Leathem, 1975), this fact was not evidenced in the present study, since the evaluated asinines did not have any nutritional control and the echogenicity remained without statistical difference, even among animals in the pubertal phase.

The increase in echogenicity may be associated with the increase in daily sperm production (Brito et al., 2002).

The similar echogenicity between the age groups in the present study can be influenced by the full function of the gonads in both groups, because in the pubertal phase the animals already produce sperm and hormones.

It was believed that the changes in the echogenicity of the testicular parenchyma of animals in sexual development would be due to the differentiation of the Sertoli cells (CS) and the formation of the hematotesticular barrier (Brito et al., 2012). However, in a study carried out with pantaneiro horses (Varoni, 2014), no correlation of pixel intensity with CS was observed, suggesting that the change in the echogenicity of the testicular parenchyma of animals in sexual development would be caused by the formation of the tubular lumen.

The echogenicity of the testicular parenchyma was not influenced by the production of fluids inside the seminiferous tubules during maturation. According to Evans et al. (1996), the beginning of fluid production promotes a decrease in the pixel average, which was also observed in bunalos in the age group from 36 to 60 months of age (Ayala, 2011). Young asinines in the present study had testicular echogenicity similar to mature animals, differing from previous studies (Aravindakshan et al., 2000; Brito et al., 2004; Chandolia et al., 1997), which described that the initial reduction in numerical pixel values ??in prepubertal animals probably reflects the production of liquid in testicles, commonly seen before the onset of spermatogenesis or explained by the significant increase in tubule volume.

The evaluation of the spermatic cord was similar to that observed in sheep, which visualized the vas deferens with hyperechoic walls and hypoechoic lumen. As the age group increases, the testicles become more pendulous, providing better ultrasound access to the pampiniform plexus and the vas deferens, allowing for better assessments (Andrade et al., 2012). As in the present study, in cattle and sheep, a higher frequency of fibrosis lesions was observed in the testicular parenchyma (Pinho et al., 2013a, b, Silva et al., 2015). In cattle, this fact was attributed to the intense proliferation of fibroblasts in the testicular parenchyma (Varoni, 2014).

Through testicular ultrasonography, it was possible to quantitatively determine the testicular echogenicity of asinines without a defined racial pattern, used in animal-drawn vehicles in the city of São Luís, Maranhão state, as well as the evaluation of testicular biometry and observed ultrasound changes. The gray scale histogram technique proved to be efficient in determining testicular echogenicity for the studied groups, allowing the observation of testicular parenchyma and mediastinal echogenicity among the studied age groups.

The study was approved by the Animal Ethics and Experimentation Committee of the Veterinary Medicine Course, State University of Maranhão (SUMA), with protocol number 13/2015, approved on 11/24/2015.

The authors have not declared any conflict of interests.

The author express gratitude to FAPEMA, the institution that finances the research; the State University of Maranhão, for the availability of the necessary equipment and infrastructure, and to the asinine breeders, for the availability in giving the animals for the experiment.