1198 Elena Ruggeri et al.
division. In our study, we used confocal microscopy to examine equine zygote development at timed intervals after ICSI of IVO or IVM.
MATERIALS ANDMETHODS
Oocyte Collections IVO were collected between April and August in Fort Collins, CO, USA (40° latitude) from light-horse mares between 4 and 16 years (mean±SEM of 10.8±0.7 year). Reproductive tracts were imaged using ultrasonography to evaluate follicular growth. Oocytes were collected from the dominant follicle(s) during the follicular phase and between 18 and 25 h (21±0.3 h) after administration of human chorionic gonadotropin (1,500 IU, iv; Intervet Inc.,Millsboro, DE, USA) and deslorelin acetate (SucroMate™, 0.75mg, im; Bioniche Life Sciences Inc., Belleville, Ontario, Canada). Oocytes were collected by ultrasound-guided, transvaginal follicle aspirations as previously described (Carnevale et al., 2000), but using a commercial embryo flush solution (ViGRO™ Complete Flush, Bioniche Animal Health USA, Inc., Pullman, WA, USA) to lavage the follicle. Upon collec- tion, the oocytes were cultured for 19.5–27.0h (22.0±0.3h) in Tissue Culture Medium 199 with Earle’s salts (Gibco, Life Technologies, Grand Island, NY, USA) with additions of 10% fetal calf serum (FCS, Cell Generation LLC, Fort Collins, CO, USA), 0.2mM sodium pyruvate, and 25μg/mL gentamicin sulfate (Sigma Aldrich, St. Louis,MO,USA) at 38 or 38.5°C in ahumidified atmosphere of 6% CO2 and air. IVM were collected from excised ovaries in Cremona,
Italy (45° latitude) during the natural breeding season. Ovaries were obtained from mares of diverse breeds and unknown ages from a local abattoir and transported at 24°C for <4 h before collection of oocytes at the laboratory. Retrieved oocytes were placed in culture medium [Dulbecco’s modified Eagle’s medium (DMEM)/F12 (D8900; Sigma Aldrich, Milan, Italy) with 10% serum replacement (Life Technologies, Monza, Italy) and 0.1 IU/ mL of human menopausal gonadotropin (Menopur 75, Ferring, Milan, Italy)] for 28 h at 38.5°C in humidified atmosphere of 5% CO2 and air.
ICSI and Zygote Culture
Prior to ICSI of IVO or IVM, cumulus cells were removed and extrusion of the first polar body was confirmed. For both labs, ICSI was performed using a piezo drill. Frozen-thawed semen from one stallion in each laboratory was used for all sperm injections, and a motile sperm with normal morphology was selected for each injection. After ICSI, potential zygotes were cultured under standard conditions for each laboratory. For IVO, potential zygotes were cultured individually in 30-μL drops of medium[DMEM/F12 (Sigma Aldrich, St. Louis) with 10% FCS] under mineral oil at 38.5°C and in an atmosphere of 5%CO2,5%O2, and 90%N2. Potential zygotes from IVM were placed as a group into 300 μL of a modified synthetic oviductal fluid medium with
bovine serum albumin (BSA; Sigma Aldrich, Milan) and modified Eagle Medium amino acids (Sigma Aldrich, Milan) in four-well dishes at 38.5°C and in 5% CO2,5%O2, and 90% N2 (Lazzari et al., 2002; Colleoni et al., 2011).
Samples Fixation and Immunostaining
Presumptive zygotes were fixed at room temperature in a microtubule stabilizing buffer (MTSB-XF, 0.1M Pipes, 5mM MgCl2.6H2O, 2.5mM EGTA at pH 6.9) containing 2% formaldehyde, 1 µM taxol, 10 units/mL aprotinin, 50% deuterium oxide and modified with 0.1% Triton X-100, and 1mM 1,4-Dithiothreitol (Messinger & Albertini, 1991). Zygotes were fixed at 4 (n=5), 6 (n=6), 8 (n=5), 12 (n=5), and 16 h (n=7) (ICSI=0 h) for IVO and at the same time points 4 (n=8), 6 (n=8), 8 (n=8), 12 (n=10) and 16 h (n=10) for IVM. After fixation, oocytes were rinsed in wash solution [(phosphate buffered saline containing 0.2% powdered milk, 2% normal goat serum, 0.1M glycine, and modified to contain 1% BSA and 0.1% Triton X-100 (Messinger & Albertini, 1991)] for IVO or a modified wash solution (phosphate buffered saline containing 1% BSA and 0.1% Triton X-100) for IVM, and stored at 4°C until immunostaining. The same procedure for immunostaining was used for IVO and IVM. Oocytes were incubated with diluted primary antibodies
in wash solution and positioned in four-well plates on rotating platformshaker for 4h at 37°Cat the following concentrations: α/β tubulin cocktail (1:100, mouse; Sigma Aldrich, St. Louis) and human-anti centromere antibody-CREST/ACA (1:100; Life Technologies, Grand Island). After primary incubation, oocytes were rinsed in wash solution for aminimumof 12h at 4°C, then incubated with secondary antibodies conjugated to either Alexa 488 or Alexa 647 (1:100; Jackson ImmunoR- esearch Laboratories, West Grove, PA, USA) diluted in wash solution for 4 h.When secondary incubationwas complete, the oocytes were held in wash solution for 5h and then incubated with phalloidin (Alexa 561; Life Technologies) and Hoechst 33258 (1μg/mL; Life Technologies, Grand Island, NY, USA) for another 5h at 37°C. For confocal imaging, samples were mounted onto coverslips in 50% glycerol in phosphate buf- fered saline with 25mg/mL sodium azide and 1μg/mL of Hoechst 33258 (Barrett & Albertini, 2007). Confocal images were acquired on an Olympus IX81
microscope (Waltham, MA, USA) fitted with a Yokogawa spinning disk (CSU22 head) using either a 60X/1.42 NA, DIC Planapochromatic or a 40X/1.35NA planapochromatic oil lens. Entire oocytes were imaged in 1-µmintervals at 40× and 0.2-µm intervals at 60× . Images were captured with a Photometrics Cascade II EM CCD camera (Tucson, AZ, USA) and analyzed using SlideBook software (Intelligent Imaging Innovations, Denver, CO, USA).
Determination of Zygote Development Stages and Abnormalities After ICSI Five sequential developmental events were categorized for presumptive equine zygotes: (1) condensed spermchromatin
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