Hormonal and Histopathological Fertility Assessments of Male Dogs in Kosovo

Animals
 
In September 2017, we conducted a scientific study within a castration involving histopathological and biochemical investigations in Kosovo (supported by the Ministry of Education, Science and Technology of the Republic of Kosovo under grant number 2-2615). One hundred male dogs aged 3 months to 11 years were obtained from shelters, streets or owners. All client-owned dogs were enrolled with informed consent from their owners. All dogs were physically examined and lacked a recent history of illness or steroid administration. The approval granted for this study allowed the collection of 7 different breeds of dogs (Hunting, Labrador, mixed, Belgian Malinois, Illyrian Sheepdog, Pitbull and Terrier) from 8 different regions in Kosovo (Malisheva, Deçan, Fush Kosova, Kamenica, Mitrovica, Prishtina, Rahovec and Vushtrria). The study was performed according to the welfare principles in Kosovo (Animal Welfare Law no. 02/L10) and ethical and professional principles (FVA-0360019).
 
Stray dogs from shelters and streets were randomly selected for this study. Dogs with owners were randomly selected for this study based on breed, age and region and the owners provided written informed consent for participation in this project. The dogs were brought to a clinic the day before castration and identified individually by ear tag. Before the castration procedure, a veterinarian confirmed that the animal was clinically healthy and took blood for biochemical and hormone analyses. After castration, the dogs recovered at the clinic in 1.2 x 1.8 m cages with bedding under standard conditions, including adequate ventilation, lighting and temperature. The air temperature on the premises where the animals were kept was not below +5°C during summer and was compatible with the outside temperature.
 
The animals were fed according to standard procedures in Kosovo (Administrative Instruction (MAFRD) No 04/2017 on technical conditions to be met by temporary care centres for stray animals). The animals were returned to the shelters, streets and owners. Blood samples were collected from the right cephalic vein.
 
Hormone analyses of testosterone, LH and FSH levels
 
Blood samples (10 ml) were centrifuged at 3000 rpm for 15 minutes and the serum was stored at -20°C for biochemical hormone analyses. Testosterone, LH and FSH were measured by enzyme-linked immunosorbent assay (ELISA) using kits from Innova Biotech Company (Keyuan Road, Industry Development Zone, Daxing District, Beijing, China): Canine Testosterone ELISA Kit (In-Cn0019), Canine Luteinizing Hormone (LH) ELISA Kit (In-Cn0024) and Canine Follicle-Stimulating Hormone (FSH) ELISA Kit (In-Cn0015) (Mialot et al., 1988). The analysis of samples was done at Institute of Biochemistry, Faculty of Medicine, University of Prishtina “Hasan Prishtina”.
 
Castration
 
Surgical castration was performed at Private Veterinary Clinics in Kamenica, Prishtina, Mitrovica, Fushë Kosova and Rahovec using Ket-A-Xyl anaesthesia (Agro Vet market S. A, Lima, Peru, company name of the chemicals; 0.5 - 1 ml/10 kg bw; i.m.). After the operation, all dogs were monitored closely for 72 hours and then returned to shelters, streets or owners.
 
Histopathological studies
 
Tissue samples were obtained from both testes immediately after organ removal, fixed for 48 hours in 10% neutral buffered formalin, dehydrated in ascending concentrations of ethyl alcohol, cleared in xylene and embedded in paraffin. Sections (5-6 µm thick) were prepared and stained with haematoxylin and eosin (H&E) (Bancroft et al., 2012) for histopathological examination with a light microscope (Olympus BX50, Japan).
 
Tissue Samples were done at the Institutes of Pathology, Faculty of Medicine, University of Prishtina “Hasan Prishtina” in Kosovo and Department of Pathology, Faculty of Veterinary Medicine, Cairo University in Egypt.
 
Statistical analysis
 
The data are presented as the mean ± SEM. Comparisons were conducted by one-way analysis of variance (ANOVA) followed by Tukey’s comparisons test. The level of significance was set at P<0.05 and data were analysed using GraphPad software (GraphPad, CA, USA).

Testosterone, LH and FSH levels in the studied animals of all breeds are shown in Table 2. Overall, 10 dogs (10% of the study population) showed alterations in hormone levels. Seven of these 10 dogs had hormone levels above the normal range, whereas 3 had values below the normal range. The hormone assessments of the affected cases from different regions in Kosovo revealed that dogs from Deçan and Malisheva recorded high levels of testosterone, FSH and LH; dogs from Prishtina recorded low levels of both testosterone and FSH and dogs from Mitrovica showed low levels of only LH (Table 1).
 

 

 
For each tested hormone (testosterone, LH and FSH), the number of dogs with levels above or below the normal range was determined. Our study identified more dogs with higher hormone levels but only one dog with lower levels of each hormone (Table 2).
 
Hormone assessments of the tested dogs of different breeds demonstrated changes in only mixed-breed dogs (Table 3).
 

 
As shown in Table 4, the mean testosterone value was 557.73 pg/ml in dogs with normal testosterone levels and 4014.8 pg/ml in dogs with levels outside the normal range; the difference in mean testosterone level between these two groups was significant (p<0.0001).
 

 
As shown in Table 5, the mean LH level was 1.401 ng/ml in the dogs with normal LH values and 9.17 ng/ml in those with LH levels outside the normal range; the difference between groups was significant (p<0.0001).
 

 
As shown in Table 6, the mean FSH level was 6.598 ng/ml in dogs with values within the normal range and 60.64 ng/ml in dogs with values outside the normal range; the difference between groups was significant (p<0.0001).
 

 
Microscopic examination of testicular tissues from dogs in various regions of Kosovo and of various breeds revealed histological changes that ranged from normal tissue structure to mild, moderate and severe structural alterations.
 
Testicular tissues of mixed-breed dogs from the Malisheva, Fushë Kosova, Kamenica and Vushtrria regions showed no histological changes, with apparently normal seminiferous tubules, spermatogonia and spermatozoa (Fig 1a and b).
 

 
Mild histological alterations were detected in testes of mixed-breed dogs from the Mitrovica and Rahovec regions; such alterations included oedema between seminiferous tubules and congestion in the interstitial blood vessels (Fig 1c and d).
 
Moderate changes were reported in testes of mixed-breed dogs from the Prishtina region; these changes were represented by the initiation of fibrous connective tissue proliferation, oedema between seminiferous tubules and congested blood vessels (Fig 1e and f).
 
Severe testicular histological changes were noted in mixed-breed dogs from the Deçan and Malisheva regions, including severe fibrosis with atrophied seminiferous tubules (Fig 2a); hyalinized (Fig 2b), degenerated and necrosed seminiferous tubules (Fig 2c) and congested blood vessels with a thickened muscular wall (Fig 2d).
 

 
One hundred male dogs of various breeds from different regions of Kosovo were assessed for hormone levels and histopathological indicators of fertility (De Gier et al., 2012). After analysing the levels of hormones such as testosterone, LH and FSH, we found that only 10% of the dogs had hormone levels outside the normal range (Frank et al., 2003). Of the 10 breeds that were evaluated in this study, only mixed-breed dogs showed hormonal changes. Dogs from Deçan and Malisheva had high levels of testosterone, FSH and LH and these changes were confirmed histopathologically by the presence of severe testicular pathology, including severe fibrosis with atrophied seminiferous tubules; hyalinized, degenerated and necrosed seminiferous tubules and congested blood vessels with a thickened muscular wall (Fukuda et al., 2000). Dogs from Prishtina had low levels of both testosterone and FSH, which was reflected histopathologically as slight fibrous connective tissue proliferation, oedema between seminiferous tubules and congested blood vessels (Mendis-Handagama et al., 2005). Testicular degeneration and leydig cell atrophy have been observed (Shahar et al., 2017). Moreover, dogs from Mitrovica had low levels of only LH, which appeared histologically as oedema between seminiferous tubules and congestion in the interstitial blood vessels (Kellom and O’Conner, 1991). The mean testosterone level in dogs with values within the normal range was 557.73, while that for dogs with values outside the normal range was 4014.8. Moreover, the mean LH level was 1.401 in dogs with values within the normal range and 9.17 in those with values outside the normal range (Nett et al., 2002). Lastly, the mean FSH level was 6.598 in dogs with values within the normal range and 60.64 in those with values outside the normal range. In comparisons of the mean value of each of the three hormones in the dogs within and outside normal, the two groups were significantly different in all three comparisons (p<0.0001) (Ortega-Pacheco et al., 2006).
 
Our study concluded that hormonal analysis and histopathological examination are good parameters for assessing fertility and the obtained results provide valuable guidance for fertility determinations in male dogs in Kosovo.

In our study, we found high levels of testosterone, FSH and LH and these changes were confirmed histopathologically by the presence of severe testicular pathology, including severe fibrosis with atrophied seminiferous tubules; hyalinized, degenerated and necrosed seminiferous tubules. This is a pioneer work which could be explored for evaluating biochemical and histophalogical alterations in male dogs in Kosovo.

This study was supported by the Ministry of Education, Science and Technology of the Republic of Kosovo with grant number 2-2615.