Controlled cooling during semen cryopreservation does not induce capacitation of spermatozoa from two portions of the boar ejaculate

Cryopreservation imposes dramatic changes in boar sperm survivability but it is as yet unclear which part of the process affects the spermatozoa the most. The present study monitored, along the entire process of cryopreservation, the stability (PMS) of the architecture of the lipid plasma membrane and its integrity (PMI), as well as the kinetics of the processed spermatozoa using two portions from the boar ejaculate (P1 = the first 10 mL of the sperm-rich fraction, SRF; P2 = the rest of the ejaculate), frozen in a recently developed package, the MiniFlatPack (MFPs, 0.5 x 10(9) sperm/dose). Evaluation was made at four specific stages, viz. S1 = after collection (suspended in Beltsville thawing solution, BTS); S2 = at 15 degrees C (suspended in lactose-egg yolk, LEY); S3 = at 5 degrees C (suspended in LEY plus glycerol); and S4 = post-thaw. Both sperm kinetics (using computer-assisted sperm analysis, CASA) and PMS [i.e. the degree of lipid disorder and of the exteriorization of phosphatidylserine (PS) in the plasma membrane, measured by flow cytometry using Merocyanine-540 (M-540), and Annexin-V (AV) respectively], as well as plasma membrane integrity [PMI, i.e. the degree of membrane damage, measured using Yo-Pro-1 or propidium iodide (PI)] were assessed after incubation in BTS at 38 degrees C. Moreover, spermatozoa were challenged by incubation in modified Brackett-Oliphant medium (mBO+) with 37 mm of bicarbonate at 38 degrees C for 30 min, and their PMS and PMI further explored. Total sperm motility was significantly higher in P1 than in P2 along the entire process (S1-S4; p < 0.01), decreasing significantly at S4 for both fractions (p < 0.0001). The proportion of spermatozoa showing linear motility (LinM) was similar between ejaculate portions (P1 and P2), with a significant increase post-thaw (S4; p < 0.0001). During cooling (S1-S3) but not post-thaw (S4), lateral head displacement (LHD) differed between portions and changed along the stages (p < 0.01). Sperm velocity differed between portions in S1 (p < 0.01), but remained similar, independently of the portion, thereafter (S2-S4). Both PMS and the total number of live spermatozoa remained similar between S1 and S3 while incubated in BTS for both ejaculate portions. Sperm mortality increased post-thaw (S4) in both portions but the degree of lipid disorder remained low in the live cells (1.28% for P1; 1.55% for P2). Exposure to mBO+, on the other hand, significantly increased membrane lipid disorder along cooling (S1-S3; p < 0.0001), increasing the percentages of dead spermatozoa, especially post-thaw (around 70%, both portions). PS-exteriorization (AV) was not evident along the cryopreservation process in control (BTS) samples and exposure to mBO+ only induced minor variations. The data showed that kinetics, PMS and PMI of boar spermatozoa suspended in BTS (S1), LEY (S2) or LEY plus glycerol (S3) were maintained during controlled cooling but were altered by thawing, showing more characteristics of cell injury than of sperm capacitation. The spermatozoa were able to capacitate but the bicarbonate challenge destabilized the plasma membrane during initial cooling and accelerated membrane changes post-thaw. We conclude that capacitation of boar spermatozoa does not occur during controlled cooling.