Premature Capacitation of Bovine Spermatozoa Is Initiated by Cryopreservation

Abstract

Poor motility and abnormal acrosomal morphology only partially explain the reduced fertility of cryopreserved bovine spermatozoa. To test the hypothesis that cryopreservation procedures (dilution, cooling, freeze‐thaw) induce capacitation in bovine spermatozoa, two experiments were conducted using semen diluted in egg yolk—Tris—glycerol extender (EYTG) (Tris, tris(hydroxymethyl)aminomethane). Capacitation was determined prior to and following incubation with various concentrations of heparin using the chlortetracycline (CTC) fluorescence assay or after preexposure to EYTG using in vitro fertilization (IVF) of bovine cumulus—oocyte complexes (COC) in the absence of heparin. Fresh ejaculates were divided into four treatments and the first was diluted with noncapacitating medium, NCM (+ 0.3% polyvinyl alcohol (PVA); control), then maintained at 23°C for 4 hours. The remaining semen was diluted with EYTG; the second treatment was held at 4°C (EYTG‐4), and the third treatment was held at 23°C (EYTG‐23) for 4 hours. The fourth treatment was cooled to 4°C over 4 hours, as per the normal industry protocol, cryopreserved, and thawed (frozen‐thawed). After the 4‐hour maintenance periods or thawing, all treatments were resuspended either in capacitating medium (CM; + 0.6% BSA) for the CTC experiment (n = 3) or in NCM for the IVF experiment (n = 9–11). Prior to incubation in conditions that support capacitation, the percentage of cells exhibiting pattern B (capacitated according to the CTC assay) was similar for all treatments with fresh‐extended spermatozoa. Immediately following the addition of heparin (0, 2, or 10 μg/ml), three times more frozen‐thawed than fresh‐extended spermatozoa exhibited pattern B (P < 0.05). After 3 or 6 hours of incubation, however, the percentages of cells displaying pattern B did not differ among treatments. In the absence of heparin, spermatozoa preexposed to EYTG‐4 fertilized 2.6x more COC than did control cells (P < 0.001) and 9.2 x more than spermatozoa preexposed to EYTG but held at 23°C (EYTG‐23; P < 0.0001). No differences were observed among fertilization rates for fresh‐extended (EYTG‐4) and frozen‐thawed spermatozoa. This study provides evidence that premature capacitation occurs in partially (extended and cooled) and fully cryopreserved bovine spermatozoa.

Prognostic value of spermatological parameters as predictors of in vitro fertility of frozen-thawed bull semen

“…Zhang et al [3] demonstrated significant correlations among in vitro semen tests (total sperm concentration and motile sperm concentration), zona pellucidabinding assay, and in vitro blastocyst production. The integrity of the plasma membrane reflects sperm viability and cryopreservation damages this membrane [4][5][6][7][8]. The damage includes swelling and disruption of plasma and acrosome membranes, changes in fluidity [9][10][11], altered influx of calcium [12,13], and changes in enzyme activity [8,11,[14][15][16].…”

“…Furthermore, capacitation, acrosome reaction, and binding of spermatozoa to the oocyte surface require a biochemically active membrane [14,17,18]. Destabilization of sperm membranes following cooling resembles that of the physiological sperm capacitation [4,12,19]. Considering that capacitated and/or acrosome-reacted spermatozoa have a limited life span [20], this would result in impaired fertility.…”

Motility Subpopulations with Distinct Motility Characteristics Using Swim-Up-Selected Sperm Cells from Norwegian Red Bulls: Effects of Freezing–Thawing and Between-Bull Variation

“…However, the pattern of FT semen was more alike the pattern of fresh sperm cells incubated over time at a physiological temperature. This could be caused by changes in motility complying to capacitation, as it is well known that cryopreservation leads to changes in sperm cells that resembles capacitation [ 25 , 26 ]. Moreover, one could speculate whether fresh sperm cells might have the same changes due to stress induced during incubation at body temperature.…”