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

SIMPLE SUMMARY: This study was devoted to the task of developing new balanced media for cryopreservation of rooster semen, which is of key importance for maintaining the functionality of spermatozoa after thawing. Trehalose in the medium increases the stability of various macromolecules under various physical influences and is capable of forming a stable vitreous matrix in cells during low-temperature stress, which maximally neutralizes the damage and metabolic activity in a dehydrated state. Also, the role of trehalose is to stabilize phospholipids during cooling and prevent disruption of the bilayer structure of cell membranes. The developed LCM-T10 and LCM-T20 media provided significantly higher rates of egg fertilization (82–86%) compared to the LCM (control) medium (79%, p < 0.05). The fertility of eggs on the 5th day from the last insemination in the LCM-T20 group had the best indicators of 100% versus 86% in the control, with 55% versus 20% in the control on the 10th day. When using test media, high results of egg fertilization were achieved and the functionality of frozen/thawed spermatozoa in the genital tract of the chicken was prolonged up to the 15th day. The results are intended to be used to preserve rare and endangered chicken breeds in vitro. ABSTRACT: 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/cm(2) (LCM-control—13.7 ± 2.7 holes/cm(2)), p < 0.01; on the 10th day with LCM-T20—319.3 ± 12.9 holes/cm(2) (LCM-control—14.9 ± 3.5 holes/cm(2)); and on the 15th day with LCM-T20—345.2 ± 11.1 holes/cm(2) (LCM-control—0 holes/cm(2)). 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.