Easykit 5

The acrosomal membrane integrity was evaluated with EasyKit 5 (ref. 025293; IMV Technologies) according to the manufacturer’s instructions with minor modifications, as previously described [33 (link)]. The kit is based on fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) that binds to the inner surface of the outer acrosomal membrane, which is accessible after an acrosome reaction, i.e., after the membrane has been ruptured [35 (link)]. According to the FITC-PNA labeling, a damaged acrosomal membrane expresses a positive green fluorescence signal, whereas an intact acrosomal membrane does not (Fig 2D). A volume of 2 ΔL of homogeneous spermatozoa at 57 x 10⁶/mL was added to each well of a 96-well plate containing 199 ΔL EasyBuffer B (ref. 023826; IMV Technologies) (38.5°C). The contents of each well were homogenized by pipetting, and the plate was covered and placed in an oven at 38.5°C, and protected from light for 45 min. Then, the plate was loaded into the flow cytometer for signal reading. A signal from 5000 spermatozoa was counted, and the results were expressed as the percentage spermatozoa with a damaged acrosomal membrane.

The acrosome membrane integrity was evaluated with EasyKit 5 (ref. 025293; IMV Technologies) according to the manufacturer’s instructions with minor modifications, as previously described [95 ,96 (link)]. The kit enables one to distinguish between spermatozoa with intact (intense green fluorescence), reacted (a low fluorescence signal), or damaged acrosome (no fluorescence). A volume of 2 µL of homogeneous spermatozoa at 57 × 106/mL was added to each well of a 96-well plate containing 199 µL EasyBuffer B (ref. 023826; IMV Technologies). The plate was covered to protect the samples from light and placed in an oven at 38.5 °C for 45 min. Then, the plate was loaded into the flow cytometer for signal reading. A signal from 5000 spermatozoa was counted, and the results were expressed as the spermatozoa percentage with an intact or damaged acrosomal membrane.

Acrosomal membrane integrity was assessed using Easykit 5 (ref. 025293; IMV Technologies). This kit is used to measure the level of disrupted acrosome within viable or dead sperm populations. The ready-to-use 96-well plate was filled with 200 mL of embryonic holding solution (ref. 019449; IMV Technologies), and 40,000 sperm were added during 45 min at 37°C protected from light in basal condition. The signal was read with the Guava^® EasyCyte^™ 5HT microcapillary flow cytometer, and a total of 5,000 events were acquired. Percentages of sperm with either intact or damaged plasma membrane as well as intact or damaged acrosome were computed. Spermatozoa with disrupted acrosomes were labeled with a green probe. Dead spermatozoa with damaged plasma membrane were labeled with a red fluorochrome.