A78075gc

Fresh sperm smears were prepared for morphometric analysis by placing 5 μl of the fresh semen on the clear end of a frosted slide by dragging the drop across the slide. The smears were air-dried before staining. Three semen smears were prepared and stained with Spermblue® (Microptic Automatic Diagnostic System, Barcelona, Spain) according to Van der Horst and Maree (2009) [52 (link)]. Stained slides were used to perform morphology evaluation using the morphometry module of the Sperm Class Analyzer version 5.4.0.1 software (Microptic SL, Barcelona, Spain). The machine was equipped with a Nikon Eclipse model 50i (Nikon Corporation, Tokyo, Japan) microscope with a 60x bright-field objective and a video camera (Basler, A78075gc, Germany). A total of 200 sperms/animal were analyzed. The morphometric parameters of head and tail were determined, and abnormal sperms were detected based on previous criteria [46 (link)–48 (link), 53 (link)–57 (link)]. Sperms with banana-shaped head, amorphous head, bent neck, or two-headed and headless sperms were classified as sperms with head abnormalities, whereas sperms with a bent or broken tail were classified as sperms with tail abnormalities (Figure 1).

Five microliters of the sperm suspension was placed on a slide and smeared with another slide. The sperm smears (three samples/animal) were totally dried at room temperature and stained with Spermblue^® (Microptic S. L., Barcelona, Spain) according to the method described by [20 (link)]. For morphology analysis, 200 spermatozoa were analyzed with the morphometry module of Sperm-Class Analyzer^® version 5.4.0.1 software (Microptic S. L., Barcelona, Spain). The machine was equipped with a Nikon Eclipse model 50i (Nikon Corporation, Tokyo, Japan) microscope with a 60× bright-field objective and a video camera (Basler, A78075gc, Germany). The abnormalities of the spermatozoa were evaluated according to the criteria from previous studies [21 –25 (link)].

Five microliters of concentrated spermatozoa cloud was collected and placed on a Leja slide (Leja Products BV, Nieuw Vennep, Netherlands). The Leja slide was placed onto a temperature-controlled stage of the Nikon E200 microscope (37°C). A 4x negative phase-contrast objective in conjunction with a phase-contrast condenser was used to determine sperm motility and concentration via the motility/concentration module of the Sperm Class Analyzer® version 5.4.0.1 software (Microptic SL, Barcelona, Spain) at 50 frames/s. Data were collected by capturing images with a digital camera (Basler, A78075gc, Germany). For motility analysis, eight fields were captured with the SCA system until 200 motile spermatozoa were analyzed, as recommended by WHO (1999) [46 (link)–48 (link), 51 (link)].