216 in Vitro Capacitation of Frozen/Thawed Stallion Sperm

Abstract

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.

Pre-selection by double layer density gradient centrifugation improves the fertilising capacity of frozen–thawed, capacitated stallion sperm

“…Likewise, Landim-Alvarenga et al (2001) obtained similar penetration rates (13.3%) when treating stallion sperm samples with dilauroylphosphatidylcholine (PC-12) or the calcium ionophore A23187. Actually, other authors have tried to capacitate stallion spermatozoa using other different compounds and protocols (Campos-Chillòn et al, 2007;Choi et al, 2002;Klewitz et al, 2010;McPartlin et al, 2006McPartlin et al, , 2009Spizziri et al, 2010;Wilhelm et al, 1996) however the results are poor and not repeatable (reviewed by Hinrichs, 2013). Higher than our results and of great interest were the results obtained by Taberner et al (2010) using the 0.1 M ionomycin treatment to capacitate frozen-thawed donkey spermatozoa (85.4%).…”

“…Many authors have tried to capacitate stallion sperm (Campos-Chillòn et al, 2007;Choi et al, 2003;Klewitz et al, 2010;McPartlin et al, 2006McPartlin et al, , 2009Spizziri et al, 2010;Wilhelm et al, 1996) but the results have always been poor and unrepeatable (reviewed by Hinrichs, 2013). However, Taberner et al (2010) achieved oocyte penetration rates of 85.4% after capacitating frozen-thawed donkey spermatozoa with 0.1 M ionomycin.…”

In vitro capacitation of stallion spermatozoa

“…Addition of cholesterol, using CLCs, to sperm plasma membranes increases membrane stability, and this increases the number of sperm that survive cryopreservation . However, fertility of CLC-treated stallion spermatozoa was not better than untreated sperm, even though more viable sperm were inseminated into each mare (Spizziri et al, 2008). A potential that may have affected these CLC-treated spermatozoa experienced a delay in the timing of capacitation since these sperm have 3 times more cholesterol than untreated sperm, and therefore by the time they were effectively capacitated, the oocytes were becoming too mature.…”

“…Sperm capacitation and the subsequent acrosome reaction can be induced in vitro in many species (McPartlin et al, 2008), and penetration by "capacitated" equine sperm into zona free and zona intact oocytes has been achieved (Spizziri et al, 2008;Landim-Alvarenga et al, 2001). However, equine sperm capacitation does not routinely lead to offspring produced via IVF.…”

“…The complexity of capacitation makes a single test for assessing capacitation difficult. Several compounds induce sufficient capacitation for stallion sperm to penetrate oocytes in vitro, but we are still unable to produce IVF offspring (McPartlin et al, 2008, Spizziri et al, 2008.…”