Krisztina Liptói

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.

The fertilization capacity of cryopreserved chicken sperm varies widely, and the effects of warming within the female reproductive tract remain poorly understood. This study investigated the effects of warming chilled and frozen-thawed sperm to temperatures approaching those encountered within the hen reproductive tract on acrosomal integrity (AcrI) and fertility. In Experiment 1, broiler-breeder semen extended with ASG poultry extender, with or without 0.6 M DMA, was stored at 5 °C and evaluated before and after warming to 37 °C. In Experiment 2, cryopreserved semen (0.6 M DMA) was thawed at 5 °C and evaluated at 5, 20, or 37 °C. AcrI was assessed by methyl blue staining. Experiment 3 extended a previous study (Bernal et al., 2026) evaluating the effect of chilling on fertility in Yellow Hungarian (YH) sperm. In the present study, White Leghorn (WL) hens receiving YH chilled semen received three additional inseminations with room-temperature extended semen (RTES). In Experiment 4, YH hens were inseminated with neat semen (NS-AI), chilled semen (CES), or CES warmed to 20 °C (CES+W). Warming did not affect AcrI of chilled semen, regardless of DMA. In contrast, frozen-thawed sperm showed a marked reduction in Acr% after warming. Fertility increased from 3.7% with CES to 54.0% with RTES, whereas NS-AI reached 71.4%. In YH hens, fertility decreased from 45.2% with NS-AI to 19.5% with CES despite no differences in motility, viability, morphology, or DNA integrity. However, warming significantly reduced AcrI. These findings highlight acrosomal integrity as a relevant indicator of sperm fertility.

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.

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.

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.

The Slovak National Agricultural and Food Centre (NPPC) in cooperation with the National Centre for Biodiversity and Gene Conservation (NBGK) has been awarded a grant under the Interreg VI-A Slovakia-Hungary Cooperation Program for the implementation of its project "Support for cross-border biodiversity conservation in ex situ conditions" (HUSK/2302/1.2/018, #Biodiverzity). Its total budget is 998 683,56 EUR, of which 798946,84 EUR is Community funding for the period from 1 May 2024 to 31 October 2026. In the Danube region, due to similar environmental factors, the established indigenous species share common characteristics. Both countries are facing the loss of native varieties and a significant decline in their populations as intensive farming systems become more widespread. Without conscious genetic conservation programs, these varieties could disappear permanently. Therefore, it is a priority for both nations to preserve and integrate the genetic material of these varieties into production. This call for proposals aims to establish the foundations for long-term cooperation between the two countries. By pooling expertise from different fields, sharing knowledge, best practices and innovative solutions, we can jointly achieve our objectives. The project will be implemented collaboratively, led by the Slovak partner. Both countries will contribute resources - financial, infrastructural, and human - to ensure the successful implementation of the tasks.

Although numerous studies reported the effects of heat stress in chickens, it was not investigated in the Transylvanian Naked Neck breed. In our research, Transylvanian Naked Neck chickens, 24 h after hatching, were heat-treated at 38.5 °C for 12 h. We compared the control and heat-treated adult chickens’ productivity parameters following 12 weeks of heat-stress at 30 °C. We found that the heat-treated layers had significantly higher egg production in heat stress, but in cockerels, the sperm quality did not differ significantly between the two groups. To detect the effect of heat-treatment on a molecular level, the expression of two heat-shock proteins and four heat-shock factors were analysed in the gonads of control and heat-treated chickens. We found that the expression level of HSP90 and HSF4 increased significantly in heat-treated female chicken gonads. Still, in adult females, the expression of HSF2 and HSF3 were substantially lower compared to the control. In adult heat-treated males, the HSP70, HSF1 and HSF3 expression levels showed a significant increase in both gonads compared to the control. We think that the presented significant differences in egg production might be related to the increased expression level of HSP90 and HSF4 in heat-treated female gonads.

In the last decade, avian gene preservation research has focused on the use of the early precursors of the reproductive cells, the primordial germ cells (PGCs). This is because avian PGCs have a unique migration route through the vascular system which offers easy accessibility. Furthermore, culturing of the cells in vitro, freezing/thawing, reintegration into a recipient embryo and the development of the germ cells can be carried out in well-defined laboratory circumstances. The efficient recovery of the donor genotype and the frequency of germline transmission from the surrogate host animals are still areas which need further development. Thus, the aim of the present study was to investigate an infertile interspecific hybrid (recipient) as an appropriate host for primordial germ cells from native poultry breeds. Guinea fowl × chicken hybrids were produced, the crossing was repeated inversely. The phenotype, the hatching time, the hatching rate, the sex ratio, the presence of own germ cells, the fertility and the phenotype of viable hybrids and the incidence of chromosomal abnormalities of dead hybrid embryos were described. 6.65% viable offspring was obtained with crossing of Guinea fowl females with domestic fowl males. Crossing of domestic fowl hens with Guinea fowl male resulted in lower fertility, 0.14% viable offspring. Based on the investigations, the observed offspring from the successful crossing were sterile male hybrids, thus an extreme form of Haldane’s rule was manifested. The sterile hybrid male embryos were tested by injecting fluorescently labeled chicken PGCs. The integration rate of labeled PGCs was measured in 7.5-day, 14.5-day and 18.5-day old embryonic gonads. 50%, 5.3% and 2.4% of the injected hybrid embryos survived and 40%, 5.3% and 2.4% of the examined gonads contained fluorescent labeled donor PGCs. Therefore, these sterile hybrid males may be suitable recipients for male PGCs and possibly for female PGCs although with lower efficiency. This research work shows that the sterility of hybrids can be used in gene conservation to be a universal host for PGCs of different avian species.