Effects of Trehalose Supplementation on Lipid Composition of Rooster Spermatozoa Membranes in a Freeze/Thaw Protocol.

The plasma membrane of spermatozoa plays an important role in the formation and maintenance of many functions of spermatozoa, including during cryopreservation. As a result of chromatographic analysis, the content of lipids and fatty acids in the membranes of spermatozoa of roosters of two breeds was determined under the influence of cryoprotective media containing trehalose LCM-control (0 mM), Treh20 (9.5 mM), and Treh30 (13.4 mM). The use of the cryoprotective diluent Treh20 made it possible to maintain a dynamic balance between the synthesis and degradation of phospholipids and sterols in the plasma membranes of frozen/thawed spermatozoa, close to that of native spermatozoa. This contributed to an increase in the preservation of frozen/thawed spermatozoa membranes from 48.3% to 52.2% in the egg breed and from 30.0% to 35.1% in the meat- and-egg breed. It was also noted that their kinetic apparatus (mobility indicators) remained at the level of 45.6% (egg breed) and 52.4% (meat-and-egg breed). An increase in the concentration of trehalose to 13.4 mM in a cryoprotective diluent for rooster sperm resulted in a decrease in the morphofunctional parameters of frozen/thawed spermatozoa.

Role of Mono- and Disaccharide Combination in Cryoprotective Medium for Rooster Semen to Ensure Cryoresistance of Spermatozoa.

The combination of saccharides in the composition of a cryopreservation medium may represent a promising method for the preservation of the reproductive cells of male birds. In the current study, cryoprotective media with a combined composition of mono- and di-saccharides were developed. The degree of penetration of reducing saccharide molecules (maltose-Mal20 medium) and non-reducing disaccharide molecules (trehalose-Treh20 medium) from the cryoprotective medium into the cytosol of rooster spermatozoa was studied. LCM control media without disaccharides were used as the control. The number of maltose molecules penetrating from the outside into the cytosol of the spermatozoon was 1.06 × 104, and the number of trehalose molecules was 3.98 × 104. Using a combination of maltose and fructose, the progressive motility of frozen/thawed semen and the fertility rates of eggs were significantly higher ((p < 0.05) 40.2% and 68.5%, respectively) than when using a combination of trehalose and fructose in a cryoprotective diluent (33.4% and 62.4%, respectively). A higher rate of chromatin integrity at the level of 92.4% was obtained when using Treh20 versus 74.5% Mal20 (p < 0.05). Maltose positively affected the preservation of frozen/thawed sperm in the genital tract of hens. On the seventh day from the last insemination when using Mal20, the fertilization of eggs was 42.6% and only 27.3% when using Treh20. Despite the same molecular weight, maltose and trehalose have different physicochemical and biological properties that determine their function and effectiveness as components of cryoprotective media.

Effects of Saccharides Supplementation in the Extender of Cryopreserved Rooster (Gallus domesticus) Semen on the Fertility of Frozen/Thawed Spermatozoa.

The aim of this study was to create balanced media for the cryopreservation of rooster semen in pellets to maintain the functional state of the sperm after thawing. Fructose was replaced by trehalose in experimental media in proportions of 10% (LCM-T10) and 20% (LCM-T20), while LCM was used as a control. After artificial insemination of the hens, the eggs were incubated (n = 400). To determine the functional safety of spermatozoa in the genital tract of hens after 5, 10, and 15 days from the last insemination, we used a method for assessing the interaction of sperm with the perivitelline membrane. Significantly higher rates of egg fertilization (82-86%) were obtained when using LCM-T10 and LCM-T20 compared to control (79%, p < 0.05). Egg fertility on the 5th day from the last insemination with the LCM-T20 diluent reached 100% versus 86% in the control; on the 10th day, the fertility rates were 55% versus 20%, respectively. The best results for fertility duration were obtained by freezing spermatozoa with LCM-T20 medium. The numbers of interaction points of spermatozoa with the perivitelline membrane were as follows: on the 5th day from the last insemination with LCM-T20-461.5 ± 11.5 holes/cm2 (LCM-control-13.7 ± 2.7 holes/cm2), p < 0.01; on the 10th day with LCM-T20-319.3 ± 12.9 holes/cm2 (LCM-control-14.9 ± 3.5 holes/cm2); and on the 15th day with LCM-T20-345.2 ± 11.1 holes/cm2 (LCM-control-0 holes/cm2). In conclusion, the use of trehalose in LCM diluent medium can increase the fertility of frozen/thawed sperm and the duration of their fertility in the genital tract of hens.