**2.2 Sperm motility**

*Animal Models in Medicine and Biology*

cardiovascular diseases [5, 6].

vitality of ZDF rat reproductive cells.

**2. Material and methods**

**2.1 Biological material**

and Ethics Committee.

may cause higher levels of adipose tissue in the fetus and an increased child birth weight. Also a few studies reported that DM3 is associated with higher levels of abdominal fat and an increased risk for visceral adiposity which could be linked to other consequences following birth such as future development of DM2 and

Another factor that negatively affects male fertility is obesity. Obesity has been shown to negatively affect the male reproductive potential not only by decreasing sperm quality but mainly by altering the germ cell molecular structure in the testes [7]. Animal models play a pivotal role in monitoring and understanding DM pathogenesis due to a combination of their genetic and functional characterization [8]. Suitable models to demonstrate the effects of DM2 on the organism are Zucker diabetic fatty (ZDF) rats. This type of rats had been discovered in 1961 following cross-breeding of Merck (M-strain) and Sherman rats. ZDF rats have the unique ability to simulate symptoms and contraindications of DM2. These rats have insulin resistance caused by the presence of homozygous mutation of the leptin hormone receptor (*fa gene*) which causes obesity and an increased insulin secretion [9, 10]. The aim of this chapter was to evaluate the effect of diabetes mellitus type 2 on the

For the experiment three separate groups (**Figure 1**) of adult ZDF and Wistar rats at the age of 120 days were used. The animals were obtained from the Institute of Experimental Pharmacology (Slovak Academy of Sciences, Slovakia) and kept in plastic cages at 24 ± 1°C and 12 h light/12 h dark photoperiod. The animals were provided with water ad libitum*.* Institutional and national guidelines for the care and use of laboratory animals were followed, and all procedures were approved by the State Veterinary and Food Institute of the Slovak Republic (no. 493/18-221/3)

The first group consisting of 15 Wistar rats (15) was the healthy control group. The second group consisted of 15 ZDF rats on a normal diet (ZDN), while the third group comprised 16 ZDF rats on a special high-caloric diet (Purina 5000; ZDF).

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**Figure 1.**

*Distribution of the tested animals (source: Author).*

Sperm motility is one of the most important parameters examining the fertilizing ability of male gametes. This parameter has an important role in the fertilization process. We examined the motility manually. Rats are known for a high level of motile spermatozoa, generally within a range of 85–96% [11]. After sample collection we applied 10 μl of the epididymal sperm suspension into Makler's counting chamber and counted motile spermatozoa using a light microscope (Olympus, Tokyo, Japan) and a magnification of 40×.

#### **2.3 Membrane integrity**

Membrane integrity is a parameter which defines the quality and condition of the reproductive cells. For the determination of membrane integrity, we used a combination of eosin and nigrosin dye. This protocol follows a differential staining method for the analysis of vital and damaged cytoplasmatic membrane in sperm cells of mammals and birds. Eosin is one of the most common dyes to stain the cytoplasm and cytoplasmic proteins of cells. After application of eosin, damaged cytoplasmatic membrane of sperm cells absorbed the dye and changes color into light-red or light-pink. The vital spermatozoa stayed without any change of color. Nigrosin (Sigma-Aldrich, St. Louis, USA) provides background for the smear as a contrast dye for a better differentiation [12–14]. The slides for the analyses were prepared as follows: we applied a drop of semen on the slide and dyed it with 4 μl of eosin solution (Sigma-Aldrich, St. Louis, USA). After application of eosin, we did the same with the nigrosin solution (Sigma-Aldrich, St. Louis, USA), and using another slide glass, we did a smear on the slide. The samples were air-dried at room temperature and observed using a light microscope (Olympus, Tokyo, Japan) and a magnification of 40×. At least 100 cells were evaluated in each slide.

#### **2.4 Acrosome integrity**

For the analysis of the acrosome integrity, we used a double fast green–rose bengal stain. After application of 10 μl of semen in a glass slide, we added the same volume of fast green–rose bengal mixture (Sigma-Aldrich, St. Louis, USA), incubated for 60 seconds, and used another slide glass to prepare a smear which was subsequently air-dried at room temperature. With the help of a light microscope (Olympus, Tokyo, Japan), we observed the integrity and compactness of the sperm acrosome. Damage to the acrosome was observed as a disruption of the membrane and cluster of stain localized in the sperm head. We evaluated at least 100 cells in each slide and calculated the percentage of cells with a normal or a damaged acrosome.
