Effects of glycerol and sugar mixing temperature on the morphologic and functional integrity of cryopreserved ram sperm.

Sperm deep freezing procedures for ram semen have considerable variations regarding the steps being employed for cooling, freezing, and addition of cryoprotectants. In this work, we evaluated the effects of the addition of glycerol and/or the disaccharides sucrose and trehalose to hypertonic diluents either before or after cooling from 30 °C to 5 °C in Merino Australian ram semen cryopreservation. Using optical and transmission electron microscopy techniques, we assessed that glycerol was beneficial to the cooling process independently of its addition at 30 °C or 5 °C in terms of sperm membrane integrity in different regions of the plasma membrane (acrosomal region, 14.5% higher integrity; postacrosomal region, 8.0% higher integrity [P < 0.01]; hypoosmotic swelling test [HOST], 10.8% higher integrity [P < 0.001]). Disaccharides were necessary for a better cryopreservation in liquid nitrogen, and the best procedure was their addition after cooling at 5 °C (12% higher sperm motility [P < 0.001]; 8% higher acrosome integrity, [P < 0.05]; 9.5% higher plasma membrane integrity assessed by HOST [P < 0.001]). Trehalose showed a greater preservation cryoprotectant capacity than sucrose, as indicated by sperm motility after thawing (8.1% greater [P < 0.01]) and by the integrity of the intermediate piece (20% greater [P < 0.05]). From these results, we conclude that the best procedure for ram semen cryopreservation in hypertonic disaccharide-containing diluents is the addition of glycerol and trehalose after the cooling process, at 5 °C.

A quantitative ultramorphological approach for systematic assessment of sperm head regions: an example in rams.

Examination of the type and frequency of damage to the head of spermatozoa using electron microscopy can be used to evaluate the quality of differently treated sperm. This report describes a systematic approach based on 29 morphological categories of sperm heads assessed from discrete regions in raw, chilled and frozen-thawed spermatozoa. Injury occurred principally at the plasma membrane and could be present or absent in all regions. In the anterior segment, when the plasma membrane is present, it can be intact, dilated, very dilated, disrupted, or contain vesicles characteristic of acrosomal reaction-like capacitation changes. When the plasma membrane is absent, the acrosome may be intact, exhibit a complete loss of contents, or retain some contents of the apical ridge and present a very dilated outer acrosomal membrane. The plasma membrane in the equatorial segment and the boundary between regions can be intact, dilated, very dilated or disrupted. The post-acrosomal plasma membrane is classified as intact, dilated or very dilated, whereas the dense lamina is intact, dilated or fragmented. The morphology of the heads most frequently observed in chilled spermatozoa consists of anterior and equatorial segments with a dilated, or dilated and disrupted plasma membrane; a boundary between regions with an intact and dilated plasma membrane; and a post-acrosomal region with an intact plasma membrane and dense lamina, both dilated. In frozen-thawed spermatozoa, the morphology of the heads is more frequently characterised by no plasma membrane and an acrosome showing complete or some loss of contents in the apical ridge and very dilated outer acrosomal membrane, presenting mostly dilated and fragmented dense lamina in the post-acrosomal region. These findings are consistent with the conclusion that the freezing process produces an increase in the degree of damage to the cells when they are subjected to increasing degrees of cold shock. There are still difficulties in developing a good diluent and process for preserving the plasma membrane in ram spermatozoa. This systematisation, using different categories, allows characterisation of multiple transmission electron microscopy images. Thus, the different changes observed due to cryopreservation may be correlated.