Effects of different cryopreservation methods on canine isolated preantral follicles
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Evaluation of the effectiveness of gamete preservation in roe deer
Background: European roe deer (Capreolus capreolus) is a widely distributed deer species, therefore we used it as a model to develop artificial reproductive technology (ART), which can be used for endangered deer species. Semen cryopreservation and oocyte vitrification in roe deer have not been established yet, and good quality gametes are necessary for further steps of ART, such as insemination and in vitro fertilization (IVF). The aim of this study was to evaluate the effectiveness of the methods of semen cryopreservation and oocyte vitrification in roe deer. The testes and ovaries were collected post mortem from free-ranging adult males and females immediately after they were hunted (N=24; age: 3–5 years; males—from 15 July to 20 August 2022; females—from 5 November 2022 to 5 January 2023) in Poland. Sperm samples were collected directly from the cauda epididymis and pulled from the two testes of each individual (N=8). The fresh sperm was diluted to a concentration of 1×109 spermatozoa/mL. The motility parameters (CASA: total motility, progressive motility) and morphology in the fresh semen (FS) and in the semen after cryopreservation (AC) were compared. Additionally, hyaluronic binding assays (HBAs) were carried out for the FS, and the mitochondrial membrane potential of the sperm in the frozen–thawed semen suspension (flow cytometry) was determined for the AC. Half of the oocytes were fertilized (N=8), and the other half underwent viability measurement (MTT) and vitrification (N=8). After ten days, the oocytes were thawed and assessed for their viability. The fresh oocytes were fertilized with thawed semen, and the embryos were cultured until reaching the blastocyst stage. The numbers of isolated oocytes, cumulus–oocyte complexes (COCs), cleaved embryos, expanded blastocysts, and embryos collected from day 6 to 9 of the culture were evaluated. Results: For the FS, the HBA showed a viability rate of 61.9%. Higher percentages of the morphology parameters were observed in the FS compared to the AC, whereas the motility and progressive movement were greater in the AC semen (P ≤ 0.001). The viability of the AC semen was 50.5%, and the mitochondrial membrane potential of the thawed semen was 40.6%. In total, 311 oocytes from 8 does were collected, with an average of 38.9 oocytes per individual. From 150 COCs, 125 blastocysts developed. The viability rate of the fresh oocytes was 98%, whereas after vitrification, it was 81% (P ≤ 0.001). Conclusions: The methods developed for oocyte vitrification and cryopreservation of roe deer semen are effective and can be implemented into ART for other deer species. The comparison of the morphology, motility, progressive movement, and viability of the FS and AC semen indicates that this process did not disturb the quality of the semen. The viability of the oocytes was high before vitrification as well as after this process, which means that an effective freezing methodology was established. Moreover, the semen and oocytes were effectively used for IVF.
Evaluation of the effectiveness of gamete preservation in roe deer
Background: European roe deer (Capreolus capreolus) is a widely distributed deer species, therefore we used it as a model to develop artificial reproductive technology (ART), which can be used for endangered deer species. Semen cryopreservation and oocyte vitrification in roe deer have not been established yet, and good quality gametes are necessary for further steps of ART, such as insemination and in vitro fertilization (IVF). The aim of this study was to evaluate the effectiveness of the methods of semen cryopreservation and oocyte vitrification in roe deer. The testes and ovaries were collected post mortem from free-ranging adult males and females immediately after they were hunted (N=24; age: 3–5 years; males—from 15 July to 20 August 2022; females—from 5 November 2022 to 5 January 2023) in Poland. Sperm samples were collected directly from the cauda epididymis and pulled from the two testes of each individual (N=8). The fresh sperm was diluted to a concentration of 1×109 spermatozoa/mL. The motility parameters (CASA: total motility, progressive motility) and morphology in the fresh semen (FS) and in the semen after cryopreservation (AC) were compared. Additionally, hyaluronic binding assays (HBAs) were carried out for the FS, and the mitochondrial membrane potential of the sperm in the frozen–thawed semen suspension (flow cytometry) was determined for the AC. Half of the oocytes were fertilized (N=8), and the other half underwent viability measurement (MTT) and vitrification (N=8). After ten days, the oocytes were thawed and assessed for their viability. The fresh oocytes were fertilized with thawed semen, and the embryos were cultured until reaching the blastocyst stage. The numbers of isolated oocytes, cumulus–oocyte complexes (COCs), cleaved embryos, expanded blastocysts, and embryos collected from day 6 to 9 of the culture were evaluated. Results: For the FS, the HBA showed a viability rate of 61.9%. Higher percentages of the morphology parameters were observed in the FS compared to the AC, whereas the motility and progressive movement were greater in the AC semen (P ≤ 0.001). The viability of the AC semen was 50.5%, and the mitochondrial membrane potential of the thawed semen was 40.6%. In total, 311 oocytes from 8 does were collected, with an average of 38.9 oocytes per individual. From 150 COCs, 125 blastocysts developed. The viability rate of the fresh oocytes was 98%, whereas after vitrification, it was 81% (P ≤ 0.001). Conclusions: The methods developed for oocyte vitrification and cryopreservation of roe deer semen are effective and can be implemented into ART for other deer species. The comparison of the morphology, motility, progressive movement, and viability of the FS and AC semen indicates that this process did not disturb the quality of the semen. The viability of the oocytes was high before vitrification as well as after this process, which means that an effective freezing methodology was established. Moreover, the semen and oocytes were effectively used for IVF.
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