Frozen storage of ram semen I. Processing, freezing, thawing and fertility after cervical insemination

Frozen storage of ram semen II. Causes of low fertility after cervical insemination and methods of improvement

Ejaculated ram spermatozoa, freed from seminal plasma by a dextran/swim-up procedure and exposed to cold shock, were incubated with ram seminal plasma proteins and analyzed by fluorescence markers and scanning electron microscopy. Seminal plasma proteins bound to the sperm plasma membrane modified the functional characteristics of damaged spermatozoa, reproducing those of live cells. Scanning electron microscopy showed that the dramatic structural damage induced by cooling reverted after incubation with seminal plasma proteins. Assessment of membrane integrity by fluorescence markers also indicated a restoration of intact-membrane cells. This protein adsorption is a concentration-dependent process that induces cell surface restoration in relation to the amount of protein in the incubation medium. Fractionation of ram seminal plasma proteins by exclusion chromatography provided three fractions able to reverse the cold shock effect. Scanning electron microscopy also confirmed the high activity of one fraction, because approximately 50% of cold-shocked sperm plasma membrane surface was restored to its original appearance after incubation. Differences in composition between the three separated fractions mainly resulted from one major band of approximately 20 kDa, which must be responsible for recovering the sperm membrane permeability characteristic of a live cell.

https://bioone.org/journals/biology-of-reproduction/volume-63/issue-5/biolreprod63.5.1531/Seminal-Plasma-Proteins-Revert-the-Cold-Shock-Damage-on-Ram/10.1095/biolreprod63.5.1531.pdf

Seminal plasma proteins revert the cold-shock damage on ram sperm membrane.

Semen plasma proteins prevent cold-shock membrane damage to ram spermatozoa

The role of seminal plasma (SP) in mammalian sperm function remains largely a matter of speculation as both inhibitory and stimulating effects have been found. Specific components of SP, particularly proteins, are adsorbed onto the surface of ejaculated sperm as they pass through the male and female reproductive tracts. These sperm coating components seem to have the important function of maintaining the stability of the membrane up to the process of capacitation (decapacitation factors). Therefore, they must be removed, modified or masked before the spermatozoa undergo the acrosome reaction, an essential process for successful fertilization. It is well known that low temperatures alter the function of spermatozoa. Cold shock results in the destabilization of sperm membranes and impairment of sperm function, and it is also well known that ram spermatozoa are more sensitive to cold-shock stress than those of other species. The addition of SP proteins to spermatozoa before and/or after cooling is able to minimize cryoinjury effects. The major proteins in ram SP which are able to protect and repair the cold-shock damage to sperm contain fibronectin-II domains. The significance of this domain and the role of these proteins in sperm capacitation and gamete interaction are discussed.

S.P. Anderson

Papers

The effect of dialysis of extended ram semen prior to freezing on post-thaw survival and fertility.