As an alternative method for preserving female genetic material, the cryopreservation and orthotopic transplantation of day-old gonadal tissue have become well-developed techniques in a few poultry species. The aim of the present study was to apply these methods for the first time to domestic geese. Cryopreservation was accomplished using the previously developed vitrification technique. Native and frozen/thawed White Hungarian geese ovarian tissue was transplanted onto Grey Landes geese recipients. Both donors and recipients were less than 24 h old. The surgical protocol previously used in chickens needed to be modified regarding anaesthesia; ketamine, xylazine, and midazolam were administered partly intramuscularly and partly intravenously. After sexual maturation, the Grey Landes recipients were paired with White Hungarian ganders, and eggs were collected and hatched individually. The origin of the offspring was determined by phenotype- and microsatellite markers. Donor-derived offspring were obtained from native and frozen/thawed ovarian tissue transplantation, which had not been published earlier. The ratio of donor-derived progeny producing layers was 40% and 58% in native and frozen/thawed gonadal tissue transplantation, respectively. The highest rate of donor-originated progeny was 78.9%. Consequently, gonadal tissue transplantation appears to be a suitable method for preserving female genetic material in domestic geese; the technique is already implemented in Hungarian gene bank practice.

It is well established that semen freezability varies considerably among rabbit breeds and lines, therefore, breed-specific cryopreservation protocols are required. In the study, three experiments were conducted. Semen quality was assessed based on sperm motility, the proportion of live/dead cells and morphological abnormalities. In Experiment 1, modified Lake's, BotuCrio and INRAFreeze extenders were compared using a simple static cryopreservation protocol. Post-thaw sperm motility was significantly higher with modified Lake's and BotuCrio than with INRAFreeze. However, no significant differences were found in the proportion of live, intact cells, therefore, modified Lake's extender was selected for further studies. In Experiment 2, the timing of cryoprotectant addition was evaluated. In half of the samples, 8% dimethyl sulfoxide (DMSO) was added at the beginning of the 45-minute equilibration period, while in the other half it was added only during the final 5 min. Both the percentage of live, intact cells and total motility were significantly higher when sperm were exposed to the cryoprotectant for only 5 min. In Experiment 3, modified Lake's extender supplemented with 1% glycerol and 4% dimethylformamide (DMF) was compared with BotuCrio using a rapid freezing protocol. Sperm motility and the proportion of live, intact cells were significantly higher with the modified Lake's containing glycerol and DMF than with the BotuCrio. In conclusion, the most effective method identified in this study for rabbit semen cryopreservation was the use of modified Lake's extender supplemented with 1% glycerol and 4% DMF.

Cryopreservation of rabbit semen represents an important tool for the conservation of genetic resources; however, reduced post-thaw sperm motility may limit its practical application in artificial insemination (AI) programmes. The present stud...

This study evaluated the effects of antifreeze proteins AFPI, AFPIII, and AFGP during fresh processing, and freezing-thawing of chicken semen from a commercial broiler breed (BB) and the local breed Yellow Hungarian (YH), using 0.6 mol/L DMA as cryoprotectant. AFPI had small but significant (p<0.05) positive effects on post-thaw sperm viability and motility in BB. For YH semen, three freezing protocols with different dilution rates (4×, 2.3×, 2.1×) were compared. Protocol 1 (dilution rate 4×) gave the best post-thaw viability (P<0.001) and was used further. Fertility rates (FR) of pre-freezing steps were tested by inseminating WL hens (4 AI/hen in 2 weeks, 100 × 10L sp/dose). FRs for A) raw semen, B) chilled semen, C) B + DMA, D) B + AFPI, and E) B + DMA + AFPI were 48.2, 3.7, 31.4, 21.4, and 16.1%, respectively. B was lower than A (p<0.001), while C–E were higher than B (p<0.005). AFPI during freezing gave no advantage compared with DMA, except for improved post-thaw DNA integrity. Inseminations with frozen-thawed semen (8 AI/hen in 3 weeks, 100 × 10L sp/dose) gave low FR with YH semen, regardless of hen type. FR with YH semen was 0% without AFPI and 1.5% with AFPI. The oviduct embryo mortality (18.0%±1.4) observed across all the different hen groups suggested insufficient number of spermatozoa inseminated. In conclusion, AFPI improved some post-thaw traits, but fertility outcomes remain inconclusive. Preventing pre-freezing fertility loss and increasing sperm dose concentration are required.

Sperm cryopreservation is the most effective method for the long-term preservation of genetic resources. However, rabbit sperm cryopreservation remains challenging. One of the key factors influencing freezing success is the choice of semen diluent; however, no specific diluent has been established for rabbit semen. In this study, we compared the effectiveness of modified Lake’s diluent and evaluated the cryosurvival of semen from the native Hungarian breed and a native Slovak rabbit breed. Both breeds were subjected to the same static freezing procedure. The following parameters were evaluated: motility, viability, plasma membrane integrity, mitochondrial activity, acrosomal status, and intracellular reactive oxygen species. In fresh samples, viability (93.5% vs. 77.4%), total motility (84.6% vs. 67.0%), and progressive motility (76.1% vs. 47.0%) were significantly higher for the Hungarian Giant than in the Chrabrany rabbit samples. Following cryopreservation, viability (56.4% vs. 43.4%), total (39.2% vs. 20.5%) and progressive (20.6% vs. 8.5%) motility, acrosomal status (72.2% vs. 44.9%) and ROS activity (33.8% vs. 24.6%) were higher in samples from the Chrabrany breed. The results suggest that the modified Lake’s diluent may also be suitable for other rabbit breeds and highlight the importance of developing breed-specific cryopreservation protocols.

Orthotopic transplantation of ovarian tissue at one day of age is a promising solution for preserving female genetic material in avian species; using sterile recipients can ensure that all offspring are donor-derived. This study focuses on the suitability of the Mulard duck as a sterile recipient for Pekin duck donors and provides an investigation of the hormonal background. Firstly, native Pekin ovarian tissue was grafted into Mulard duck recipients, resulting in a 40% adhesion rate and follicular development in 50% of the adhered grafts. Secondly, the transplantation of cryopreserved ovarian tissue resulted in a 66% adhesion rate, with 33% of the adhered grafts showing follicular development. Ovulation occurred in 16% of the recipients with adhered grafts, but the eggs did not move into the oviduct. Estrogen levels were elevated in the recipients with adherence but were lower than in the control Pekin group, while progesterone levels remained unchanged. Consequently, recipients received buserelin acetate, a GnRH analogue, to stimulate follicular and oviductal activity. In this group, graft adhesion occurred in 31% of animals, and primordial follicle development in 25%. The hormonal levels of the recipients with adhered ovaries were elevated, but the GnRH analogue treatment did not affect the ovulation process. We conclude that while the Mulard duck shows potential as a sterile recipient in ovarian transplantation, several questions remain unanswered regarding the adequacy of follicular maturation and ovulation.