The inability of stallion sperm to capacitate in an in vitro setting has limited many equine assisted reproductive technologies such as gamete intrafallopian transfer (GIFT) and IVF. These experiments were conducted to develop methods for artificially capacitating frozen/thawed stallion sperm, using dilauroylphosphatidylcholine (PC-12), calcium ionophore A23187 (IONO), or methyl-β-cyclodextrins (MBC) so that sperm could be used in equine assisted reproductive technologies. Assisted reproductive technologies need to be established to maximize the number of offspring produced with cryopreserved spermfrom stallions with low cryosurvival rates or that have a very limited number of frozen sperm. Low concentrations of lysophosphatidylcholine (LPC) induce the acrosome reaction in capacitated sperm and, therefore, were used to detect fully capacitated sperm treated with PC-12, IONO, or MBC. Ejaculates from 8 stallions were diluted in a modified Tyrode's medium and centrifuged (1000g for 11 min). The sperm pellets were suspended to 400 million sperm mL–1 in EZ-Freezin LE, packaged into 0.5-mL straws, and frozen in liquid nitrogen vapor. Straws were thawed in a 37�C water bath for 30 s and washed through 35% Percoll (400g for 4.5 min) to remove egg yolk particles and seminal plasma. The sperm pellets were suspended in Medium 199 (0.6% BSA and 5 mm calcium chloride) to 50 million sperm mL–1, and stained with propidium iodide and FITC-peanut agglutinin (PNA) (1 mg mL–1 each; viability and acrosome reaction detection, respectively). Sperm were treated with PC-12 (13, 17, 20, 30, 40 µm), IONO (0.5, 1, 1.5, 2 µm), or MBC (0.4, 0.6, 0.8, 1 µm) and incubated in a 37�C water bath (20, 15, 20 min) to artificially capacitate sperm. Sperm were then challenged with LPC (2 or 5 µg), for 10 additional min, and analyzed by flow cytometry to determine the percentages of acrosome-reacted sperm. Data were analyzed by ANOVA and treatments were separated by Student-Newman-Keul's test. The PC-12 (20, 30, 40 µm), IONO (1.5, 2 µm), and MBC (0.4, 0.6, 0.8, 1 µm) artificially capacitated sperm equally well (59 to 70%, 52 to 59%, and 48 to 55%, respectively) and were higher than control sperm (13%, SEM � 9; P < 0.05). Sperm viability was similar at all IONO or MBC concentrations; however, PC-12-treated sperm (17 to 40 µm) had significantly lower sperm viability when compared with control (24 to 28% and 35%, respectively, SEM � 3; P < 0.05). In conclusion, frozen/thawed stallion sperm can be artificially capacitated by treating with PC-12, IONO, or MBC, and this may lead to practical applications for in vitro equine assisted reproductive technologies.
In vitro fertilization in the horse does not work reliably. Several methods of capacitating sperm in other species fail in the horse. The goal of this experiment was to develop a method to capacitate equine spermatozoa using calcium ionophore A23187 or phosphatidylcholine 12 (PC12). We also studied effects of maturing bovine oocytes for 24 or 28 h on fertilizability by capacitated equine sperm, hypothesizing that longer maturation would yield oocytes more easily fertilized by equine spermatozoa. Two sets of bovine oocytes were aspirated from 3 to 8 mm follicles of abattoir ovaries 4 h apart, but fertilized at the same time. On the day of fertilization, semen from 1 of 3 stallions was collected, evaluated, and centrifuged through 33% Percoll to remove seminal plasma. The resultant pellet was extended to 5 × 107 cells mL–1 in M199 containing 0.6% BSA, 2 mm caffeine, and 5 mm CaCl2. Sperm were treated with A23187 (1 or 3 μm) or PC12 (40 or 70 μm) or both A23187 and PC12 (1 μm/40 μm) in 500- μL aliquots. Sperm were incubated at 39°C for 10 min (for A23187 and combination treatments) or 15 min (for PC12 treatments), and then diluted 1:20 for fertilization. Oocytes from each maturation time were fertilized using the same semen preparation for each treatment. Oocytes and sperm were incubated together for 18 h in FCDM in 5% CO2 at 39°C (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596). Presumptive zygotes were cultured for 30 h in CDM-1, vortexed to remove cumulus cells, and evaluated for cleavage. Oocytes were also co-incubated with killed sperm to determine the level of parthenogenesis. Cleaved embryos were stained with orcein to ensure that each cell had a nucleus. Number of cell divisions were recorded as 0 for a 1-cell, 1 for a 2-cell, 1.5 for a 3-cell, etc. More oocytes cleaved after 28 h (18%) than 24 h (14%) maturation (P < 0.01). Sperm of Stallion 1 resulted in higher overall cleavage (24%) than Stallions 2 or 3 (11 and 12%; P < 0.01). Highest cleavage was seen with 28 h maturation and 70 μm PC12 and 3 μm A23187 (27 and 24%, respectively). The most cell divisions were seen with 28 h maturation and 70 μm PC12 (0.48); 28 of the 49 cleaved in this treatment reached ≥4-cell stage. In conclusion, both A23187 and PC12 were able to capacitate equine sperm in a dose-dependent manner as determined from cleavage of bovine oocytes matured for 28 h; maturation for the conventional 24 h was an inferior model for this purpose. Table 1. Mean responses of bovine oocytes fertilized by equine sperm
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