Easybuffer b

Plasma membrane integrity was evaluated using the EasyKit 1 Viability and Concentration (ref. 024708; IMV Technologies) according to the manufacturer’s instructions and as previously reported [33 (link)]. The kit is based on two fluorescent probes: SYBR and propidium iodide (PI). SYBR stains nucleic acid and labels all spermatozoon heads green, whereas PI penetrates only membrane-damaged spermatozoa and labels spermatozoon heads red (Fig 2A). From each sample, 2 ΔL of homogeneous spermatozoa at 57 x 10⁶/mL was added into the well of a 96-well plate containing 199 ΔL EasyBuffer B (ref. 023826; IMV Technologies). 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 10 min. A total of 5000 spermatozoa were counted in the flow cytometry reading. The results are expressed as the percentage of viable spermatozoa.

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.

Frozen semen straws were thawed in a water bath set at 35 °C for 30 seconds. Motility and membrane viability staining were performed using computer assisted semen analysis (CASA). Briefly, for motility staining 20 μL of semen was gently mixed with 30 μL Easybuffer B (IMV Technologies, Maple Grove, MN) and 50 μL of 1 mg/ml Hoechst 33342. Samples were incubated at 35 °C for 20 minutes, loaded onto pre-warmed four chamber slides (Leja, Nieuw-Vennep, The Netherlands) and imaged using the Animal Motility package on an IVOS II (Hamilton Thorne, Beverly, MA) system using version 1.5 of the CASA II sperm analysis software and the manufacturer’s recommended settings. For membrane viability staining, semen was thawed as previously described. 20 μL of semen was gently mixed with 30 μL Easybuffer B and 50 μL of 1 mg/ml Hoechst 33258. Samples were then incubated at 35 °C for 2 minutes, loaded into pre-warmed four chamber slides and imaged using the Animal Motility Viadent package on the same IVOS II system and software.

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.

For in vitro experiments, bovine semen was purchased from Accelerated Genetics (Baraboo, WI) and All West Select Sires, Inc. (Burlington, WA). The purchased bovine semen used in all in vitro experiments were single ejaculates from three Holstein Bulls, diluted in egg yolk-citrate extender and packaged in 0.5-mL French straws. Easy Buffer B and Leja 4-chamber slides were purchased from IMV Technologies (Maple Grove, MN). LCO₂ was purchased from Airgas (Radnor, PA) and stored in 180-L microbulk tanks from Chart Industries, (Ball Ground, GA). Tanks storage pressure was set to 2,400 kPA. Unless otherwise indicated, all other reagents used in this study were purchased from Sigma (St. Louis, MO).

Plasma membrane integrity was evaluated using the EasyKit 1 Viability and Concentration (ref. 024708; IMV Technologies) according to the manufacturer’s instructions and as previously reported [95 ,96 (link)]. The kit contains two fluorochromes and allows one to distinguish between live and dead spermatozoa. From each sample, 2 µL of homogeneous spermatozoa at 57 × 106/mL was added to the well of a 96-well plate containing 199 µL EasyBuffer B (ref. 023826; IMV Technologies). 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 10 min. A total of 5000 spermatozoa were counted in the flow cytometry reading. The results are expressed as the percentage of viable spermatozoa.