4.1 Ethical approval
Three independent Brazilian committees approved this study: the National Management System for Genetic Heritage and Associated Traditional Knowledge (#A1B0E09), Chico Mendes Institute for Biodiversity Conservation (#65487) and the ethics committee on the use of animals in research (CEUA) at Sao Paulo State University (#0002/2018). All postmortem sampling collection was done at the Department of Veterinary Pathology Service from Sao Paulo State University (UNESP).
4.2 Samples and morphological analysis
4.2.1 Samples collection
Thirteen free-living giant anteaters were autopsied within six hours interval after death due to blunt trauma force. Fresh prostate glands initially were macroscopically evaluated based on anatomical measurements and organ weight. For microscopic examination, the prostatic tissue samples and testis were fixed in neutral-buffered 10% formalin for 24 hours, embedded in paraffin, and routinely processed for histologic examination (FFPE). Additionally, samples were fixed in pentane – 1,5-dial glutaraldehyde for 24 hours at 4°C for scanning electron microscopy. The individual subjects’ information, including body weight and prostate measurement, are shown in Table 1.
Table 1. Giant anteaters autopsied information.
|
|
|
|
|
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Identification
|
Age
|
Weight
(Kg)
|
Central Zone Size*
|
Peripheral zone
|
Left Zone Size (cm)
|
Right Zine Size (cm)
|
Subject 1
|
Young
|
19
|
3.1x3.5x1.9
|
3.5x4.1.3.1*
|
NA
|
Subject 2
|
Young
|
22
|
3.9x3.2x2.5
|
3.9x4.0x2.9*
|
NA
|
Subject 3
|
Adult
|
38
|
5.6x6.1x4.3
|
7.1x3.7x5.6
|
7.2x3.5x5.4
|
Subject 4
|
Adult
|
40
|
6.1x5.4x4.9
|
6.9x4.1x5.4
|
6.7x4.4x5.6
|
Subject 5
|
Adult
|
37
|
5.6x5.5x4x2
|
7.3x4.7x5.1
|
7.1x4.9x5.1
|
Subject 6
|
Adult
|
35
|
5.4x4.9x4.1
|
5.9x2.7x4.9
|
6.2x3.1x4.8
|
Subject 7
|
Adult
|
34
|
5.8x5.1x3.9
|
6.1x4.3x4.8
|
6.3x4.2x4.7
|
Subject 8
|
Adult
|
37
|
5.6x4.9x4.1
|
6.5x4.1x5.0
|
6.3x4.1x5.1
|
Subject 9
|
Adult
|
38
|
5.9x3.9x3.8
|
6.8x4.0x4.9
|
6.9x4.2x4.7
|
Subject 10
|
Adult
|
39
|
6.1x5.7x4.5
|
7.3x4.2x5.3
|
7.1x4.4x5.2
|
Subject 11
|
Adult
|
33
|
5.3x5.1x5.2
|
6.5x2.9x5.0
|
6.6x2.9x4.8
|
Subject 12
|
Adult
|
38
|
5.4x4.9x4.7
|
6.9x4.7x4.2
|
7.0x4.7x4.5
|
Subject 13
|
Adult
|
39
|
5.7x5.1x4.9
|
7.1x3.3x5.0
|
7.1x3.3x5.0
|
The sexual maturity estimation of studied free-living giant anteaters was based on the criteria reported by Smith (2007), Baugher (2004), and Jerex and Haloy (20003) resulting into two studied groups: adult vs young. In brief, the young category includes animals with at least 10-month-old, when the female halts are still carrying it in her back up to two-year-old, before the sexual maturity be reached. The adult category includes males with ≥ 30 kg of body weight associated with sexual maturity. The testicular maturity was determined by the presence of spermatogenesis in FFPE testis tissue samples.
4.2.2 Histochemistry
All the histological sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome stain, and Periodic acid-Schiff stain (PAS). Morphological analysis of the giant anteater’s prostatic gland followed the standards established by Rossi et al. (2013)7.
4.2.3 Scanning electron microscopy
The prostate sample selected for scanning electron microscopy analysis (MEV-EDX, FEI Company, Quanta 200 e EDX da Oxford, 51-XMX1119) was previously fixed in pentane – 1,5-dial glutaraldehyde for 24 hours at 4ºC.
4.2.4 Immunohistochemistry
The tissue sections obtained from the FFPE blocks were placed on charged slides (Starfrost, Knitell, Bielefeld, Germany). For the antigen retrieval, the slides were incubated in a citrate solution (pH 6,0) in a pressure cooker (Pascal, Dako, Carpinteria, CA, EUA) for 45 minutes. Endogenous peroxidase was blocked by hydrogen peroxide 8% diluted in methyl alcohol for 15 minutes and treated with skim milk 8%, at room temperature. The sections were incubated overnight at 4ºC with primary antibodies anti-AR (1:50; clone ab77557; Abcam), anti-PSA (1:1500; polyclonal; Bioss), anti-P63 (1:100; clone 4A4; Dako, Carpinteria, USA) and anti-uroplakin III (UPIII; 1: 50; clone AU1, Researches technologies). A polymer detection system (Envision, Dako, Carpinteria- USA) was applied as a secondary antibody, and immunoreactive cells were visualized by colorimetric detection (3,3ʹ- diaminobenzidine). The sections were counterstained with Harris hematoxylin (Dinamica-Diadema, Brazil). A canine prostate was used as a positive control for all antibodies. Negative controls were applied to one section of canine prostate
The immunostaining evaluation for all antibodies was classified as negative or positive expression in the membrane, cytoplasm, or nuclear immunolabelling.