Mohamed Gamal Hedia, Mohamed Shehata El-Belely, Sayed Taha Ismail, Amal Mahmoud Abo El-Maaty

1Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt

2Department of Animal Reproduction and AI, Veterinary Division, National Research Center, Dokki, Giza, Egypt

ABSTRACT Objective: To establish reference values for pixel intensity of testicular ultrasonogram of rams in breeding and non-breeding seasons, and to investigate if the seasonal changes in testicular echogenicity and heterogeneity are associated with semen characteristics.Methods: Five Awassi rams aged 3-5 years old and weighing 45-60 kg were subjected to ultrasonographic scanning of the testes twice monthly for one year (from January 2018 to December 2018), together with semen collection and evaluation of ejaculate volume, sperm motility, sperm concentration, sperm morphology and viability. The ejaculate volume was determined in a graduated collection tube (scale of 0.1 mL). Mass and individual sperm motility was expressed in percentage of motile spermatozoa under optical microscope equipped with a warm stage. Sperm concentration was determined by using a Neubauer chamber.Semen smears stained with eosin-nigrosin were used to determine the percentage of live spermatozoa and sperm cell morphology by using a light microscope.Results: The mean testicular pixel intensity was the lowest in winter and increased gradually from breeding season to nonbreeding season, reaching its maximum value in summer (P＜0.05).Pixel intensity was found to have a significant negative correlation with progressive motility (r=-0.605, P＜0.05), and sperm concentration (r=-0.619, P＜0.05). It is also positively correlated with the percentage of sperm morphological abnormalities(r=0.666, P＜0.05).Conclusions: Pixel intensity values of testicular ultrasonogram in rams undergo marked seasonal changes that are associated with fluctuations in photoperiod and ambient temperature. The resulting values of testicular echogenicity (pixel intensity) throughout the year provide useful reference values for predicting the testicular function in Awassi rams.

KEYWORDS: Testes; Echotexture; Pixel intensity; Semen;Season; Rams

1. Introduction

Reproductive performance in rams is widely dependent on the seasonal changes in average ambient temperature and photoperiod length in tropical and subtropical localities[1-4], where rams show a superior reproductive capacity during the breeding seasons(autumn and winter).

For maximal animal production, producers tend to rear a limited number of rams with high merit of reproductive efficiency, which permit them to impregnate a large number of ewes in the same flock[5]. Breeding soundness examination is used for ram selection based on a series of sequential steps, which are conducted to indirectly ensure the reproductive performance of rams through evaluation of certain physical, reproductive and ultrasonographical parameters in accordance with semen quality and plasma hormonal profile[3,6].

Ultrasound imaging is a valuable, non invasive technique, which is used to safely evaluate the external and internal reproductive organs in males[7]. Recently, the use of grey-scale ultrasound has widely been conducted in different species to assess the testicular functionality[8-10]. Interestingly, there are various methodologies available based on ultrasonographic evaluation of the testicular parenchyma and epididymis in rams[11-13]. In the first reports,ultrasound has been exclusively used for scanning of testicular parenchyma and differentiating normal testes from those with pathologies[14,15], which is followed by the application of pulsedwave Doppler analysis of testicular artery[6]. Currently, the testicular ultrasound imaging is performed by using computerassisted software in rams, which provides visual image analysis system of previously saved B-mode ultrasound images of testicular parenchyma, and this is known as testicular ultrasonogram[11,13,16].

Ultrasonogram is composed of an array of pixels, where the variation in pixel intensities displayed in a range of shades of gray is used to determine tissue density[13,17]. Several authors have demonstrated that the changes in pixel intensity are correlated with changes that occur during the development of the reproductive organs in rams[18], bucks[10] and bulls[16]. In addition to this clear association, the changes in pixel intensity values appear to be also correlated with semen quality parameters[12,19]. Estimation of the numerical “pixel intensity” values of testicular echogenicity using computer-assisted image analysis, is expressed on a scale of 0 to 255; where 0 represents absolute black and 255 represents absolute white. Testicular heterogeneity is estimated by calculating the pixel standard deviation[10,16,17,19,20].

To the best of our knowledge, the available data to date concerning the seasonal changes in testicular echogenicity and heterogeneity are not widely investigated in rams. The rationale that seasonal variations in testicular ultrasonogram values may be associated with other seasonal changes in semen quality was checked for further functional validation. Thus, the current study aimed to determine seasonal reference values for testicular echogenicity and heterogeneity during the breeding and nonbreeding seasons in fat-tailed Awassi rams, and to investigate the possible correlation between seasonal changes of these values and semen characteristics.

2. Materials and methods

2.1. Animals and management

Five adult fat-tailed Awassi rams (a native breed), weighing 45-60 kg and aged 3-5 years, were kept under natural environmental conditions. Animals received a maintenance ration and had a free access to fresh drinking water. They were previously vaccinated and received treatments for both external and internal parasites.Animals were selected after thorough clinical and andrological examination and proved to be free from any reproductive problems. Means of seasonal temperature, temperature-humidity index and sunny hours for the study period (from January 2018 to December 2018) were obtained from the Egyptian Meteorological Auyhority, Kobry El-Kobba, Cairo, Egypt. Seasonal average temperature, temperature-humidity index and sunny hours,respectively, were (20.5 ℃, 20, 11 h) during winter, (31.5 ℃,29, 14 h) during spring, (33.0 ℃, 31, 13 h) during summer and (25.0 ℃, 23, 11 h) during autumn. The studied seasons were defined as the following: winter: January, February, and March; spring: April, May, and June; summer: July, August, and September; and autumn: October, November, and December. The breeding season was the period from early autumn to late winter.

2.2. Study design

All rams were subjected to semen collection followed by ultrasonographical examination of the testes twice monthly (every two weeks) for one year (January to December 2018) (i.e. 24 measures for each parameter/animal). Examinations were always performed between 8 and 9 am.

2.3. Semen collection and evaluation

Semen was collected by using an artificial vagina of ram(40 ℃-42 ℃). Fresh semen samples were directly transferred to the Seminalysis Laboratory, Faculty of Veterinary Medicine,Cairo University for immediate evaluation. Semen was evaluated according to Evans and Maxwell[21] for the following characteristics: ejaculate volume, sperm motility (mass and individual sperm motility), sperm concentration, sperm viability and sperm morphology. The ejaculate volume was determined in a graduated collection tube (scale of 0.1 mL). Mass and individual sperm motility was expressed in percentage of motile spermatozoa under optical microscope (Olympus BH-2, Olympos Optical Co. Ltd., Japan) equipped with a warm stage (100× and 400×,respectively). Sperm concentration was determined by using a Neubauer chamber. Semen smears stained with eosin-nigrosin were used to determine the percentage of live spermatozoa (400×)and sperm cell morphology by using a light microscope (under oil immersion lens, 1 000×).

2.4. Testicular ultrasonogram

All examinations were carried out by using ultrasound scanner(EXAGO, Echo Control Medical, France) equipped with a linear array transducer (7.5 MHz). The ultrasound settings (focus, gain,and brightness) were standardized and kept constant for the whole study period. All ultrasonographic examinations were performed by the same operator. The rams were restrained without sedation.To eliminate the presence of air spaces, the fine wool on both sides of the scrotum were shaved, and the transducer was covered with a copious amount of gel as a coupling material to facilitate ultrasonographic imaging. With a minimum pressure, each testis was imaged by placing the transducer vertically on the caudal aspect of the scrotum and parallel to the long axis of the testis.

Figure 1. Ultrasonogram of the testis of Awassi rams. The figure shows placement of rectangle 0.5 to 1.0 cm deep into the parenchyma.

Table 1. Testicular ultrasonogram (pixel intensity and pixel standard deviation) as well as semen characteristics in breeding and non-breeding seasons of Awassi rams.

The image was aligned until the mediastinum of the testis was clearly visible. All ultrasonograms were captured and downloaded to a computer for further analysis. Computer analysis of each image was performed by using image analysis software(ImageJ, U. S. National Institutes of Health, Maryland, USA)[22]. Testicular echogenicity (pixel intensity) and heterogeneity(pixels standard deviation) were computed on one selected area of 1 cm2per testis by drawing a rectangle 0.5 to 1.0 cm deep into the parenchyma (Figure 1) where it appeared homogenous[16]. Testicular pixel intensity represented the average value attributed to each pixel within the selected area according to the shade of gray[13].

2.5. Statistical analysis

Seasonal results of fresh semen characteristics were presented as mean±standard deviation (mean±SD). Data were analyzed by analysis of variance using general linear model procedures for repeated measurements (SPSS, version 16.0)[23]. Dependent variables in the mathematical model were pixel intensity, pixel standard deviation and semen traits. Pearson's correlation coefficients among ultrasonogram parameters and semen parameters were calculated (SPSS, version 16.0)[23]. Differences were considered significant atα= 0.05.

2.6. Ethical approval

The present experiment was carried out at the Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University,Egypt. All the experimental protocols were approved by the Ethics Committee of Cairo University (IACUC, Institutional Animal Care and Use Committee, CUⅡS 5 18).

3. Results

3.1. Impact of testicular laterality on echotexture assessment

There were no significant differences in testicular echogenicity and heterogeneity measurements between the right and left testis of the same ram at each time point. Thus, the means values of the right and left testis were used for further analysis.

3.2. Semen characteristics and testicular ultrasonogram

As depicted in Table 1, semen quality parameters showed a higher values during the breeding season as compared to the non-breeding season (P＜0.05). The overall averages of the ejaculate volume,sperm progressive motility and sperm cell concentration were significantly (P＜0.05) higher in the breeding season. Testicular ultrasonogram showed marked changes in the measures of pixel intensity in breeding season compared with those recorded in the non-breeding season (P＜0.05), whereas the values of pixel standard deviation (testicular heterogeneity) were not different between breeding season and non-breeding season (P＞0.05).

3.3. Correlation coefficients

As shown in Table 2, testicular pixel intensity exhibited a moderate significant negative correlation with the percentage of progressively motile sperm (r=-0.605, P＜0.05) as well as sperm cell concentration (r=-0.619, P＜0.05), while it showed a positive association with the percentage of total sperm abnormalities(r=0.666, P＜0.05) (Figure 2, 3).

Table 2. Correlation coefficients between testicular echhotextural characteristics and parameters analyzed in fat-tailed rams.

Figure 2. Correlation between testicular pixel intensity and sperm parameters.

Figure 3. Correlation between testicular pixel standard deviation and sperm parameters.

4. Discussion

In the current study, significant seasonal changes in semen characteristics were noticed in Awassi fat-tailed rams. These changes were previously described in several reports concerning the Egyptian native breeds of fat-tailed rams[24,25]. These studies showed a better semen quality in autumn and winter (breeding season) compared with the spring and summer (non-breeding season).

Our results showed that the overall semen volume was similar to the value that was previously reported for the same native Egyptian breeds of fat-tailed ram[26,27], Pampinta rams[1] and Algerian rams[28]. Considering that autumn and winter are seasons with decreasing daylight, it is obvious that the present fat-tailed rams are sensitive to photoperiodism.

In the current study, semen mass activity scores (mass motility) and individual motility percentages were higher in autumn and summer compared to the other seasons. This finding was in agreement with previous several studies conducted on fat-tailed rams in different localities[25,29,30]. However, our results contradict the observation of Salem et al[26] and Belkhiri et al[28] who found that sperm motility parameters were higher in breeding season (autumn and winter) than in the non-breeding season (spring and summer).

In our study, the percentage of live sperm cells was higher in nonbreeding season compared with the breeding season. These results are in line with the findings of Aller et al[1], Kafi et al[29] and Pourseif et al[31]. However, other reports demonstrated a higher value of live sperm cells in breeding season as compared to the non-breeding season[30,32]. These different trends may be, at least in part, due to breed differences and geographical location.

In the present study, sperm concentration exhibited higher values during the breeding season as compared with the non-breeding season, which is in agreement with several previous reports[25,31].However, these results are different from those obtained by Aller et al[1] and Sàrlos et al[2], who recorded a higher range than those found in the present study. This may be due to the frequency of semen collection and the local average seasonal temperature.

As far as we know, there is very limited number of reports that describe testicular ultrasonogram and its value as a predictor of reproductive capacity in rams. One study has suggested a possible correlation between semen characteristics as well as scrotal circumference on one hand, and testicular echotexture on the other hand[11] with emphasis on season effects in mature rams.

In the present study, testicular pixel intensity showed marked higher values during the non-breeding season compared with values recorded in the breeding season. Our results were in agreement with values obtained by Ahmadi et al[11] in mature, healthy rams. Interestingly, several studies showed that the testicular echotexture measures had a considerable association with testicular histopathological changes in adult rams, where Ahmadi et al[11] and Giffin et al[33] recorded a positive correlation between testicular pixel intensity with seminiferous tubules and epithelium area. In addition,testicular pixel intensity measurement revealed the mean value depicted to each pixel in the selected area of assessment within the testicular parenchyma, and exhibited as shade of gray on a range of 1 to 255, where 1 recorded as black and 255 recorded as white[16]. In a previous study, Al-Ghetaa[34] demonstrated that the seminiferous tubules showed degenerative changes during the non-breeding season owing to the higher air temperature and photoperiod,which ultrasonographically[35] exhibited as white spots within the homogenous testicular parenchyma of rams.

In the present study, testicular pixel intensity showed a negative correlation with sperm cells concentration and sperm progressive motility, and a positive association with the percentage of abnormal sperm cells.

However, testicular heterogeneity (pixel standard deviation) showed no differences between the breeding and non-breeding seasons. In their report, Ahmadi et al[11] recorded higher significant measures of testicular heterogeneity in the non-breeding season (August), but these measures did not change in the breeding season (December and February). This discrepancy might be, at least in part, due to the native ambient temperature.

Several results of other studies, such as Ntemka et al[12], reported that the percentages of sperm cells viability were positively associated with testicular echotexture in rams (r=0.6). In addition,Ahmadi et al[11] investigated a linear relationship between testicular heterogeneity and future semen quality. In contrast, Tomlinson et al[36] recorded no significant association between the testicular echotexture and the sperm quality parameters.

In conclusion, seasonal assessment of testicular echogenicity and heterogeneity measures combined with semen characteristics in the present study show evidence that testicular echogenicity and heterogeneity is a potential indicator of reproductive performance in rams. Therefore, image analysis of testicular parenchyma can be used as prognostic tool to determine ram fertility as a step during breeding soundness examination practices in rams.

Conflict of interest statement

The authors declare that they don’t have any conflict of interest.

Authors’ contributions

Mohamed Hedia was the main participant during conducting the experimental protocol, Mohamed El-Belely and Sayed Ismail are the main contributors in designing the protocol and monitoring,while Amal Abo-El-Maaty was the main person during ultrasound examinations and data analysis.