Elaboration of massage technique for semen collection and examination of semen characteristics in chinchilla (Chinchilla lanigera).

The practice of artificial insemination for the long-tailed chinchilla has not been fully elaborated to date, and existing data available regarding their reproduction properties is contradictory. Until now, the collection of semen for chinchillas has been most-commonly obtained using electro-ejaculation methods exclusively. The primary objective of this study was the development of a manual technique for semen collection which meets all animal welfare requirements. An additional aim was to determine the basic spermatological parameters, such as motility, concentration, type and ratio of morphological abnormalities and live/dead cell ratio, under typical northern-hemisphere conditions, in Hungary. Over a 3 month period, a special massage technique was developed for the study, and using this method, the sperm parameters of 46 males were subsequently analyzed weekly for a period of one year. Approximately 66% of chinchillas responded positively to this technique, with the success rate of semen-collection attempts showing no variation between seasons. Average sperm concentration for the whole year was 935.17 million/ml using this method. Total cell motility was the highest in winter (90.3%), and the lowest in spring (84.3%). The proportion of live, intact cells were above 80% on average for the year, while the ratios of live, morphologically abnormal and dead cells were 6% and 14%, respectively. We found that midpiece abnormalities occurred in the highest proportion (0.95%-3.38%), while the head abnormalities showed the lowest ratio (0.01%-0.15%). Standard deviation among the parameters was relatively high, with the spring season proving to be the weakest in terms of sperm quality. This study has demonstrated that, semen can be successfully collected without the use of electro-ejaculation or anesthesia. Furthermore, although spermatological parameters do exhibit some fluctuation for the different times of the year, semen collected is nonetheless suitable for the purpose of artificial insemination of chinchillas at any time.

Mycoplasma species in the male reproductive organs and the fresh and frozen semen of the Hungarian native goose.

The objective of this study was to clarify whether the most common species of Mycoplasma can be detected in the reproductive organs and the cloaca, as well as in the semen of asymptomatic native Hungarian male geese. As it is necessary for the semen of that breed to be preserved pathogen-free in an in vitro gene-conservation programme, the presence of and sources of infection, as well as prevention of the survival of pathogens following semen cryopreservation, are key issues. Ten asymptomatic, 2-year-old ganders were tested. For the detection of mycoplasmas, samples were taken from both fresh and frozen/thawed semen, cloaca, phallus lymph, testes and vas deferens; that is five samples from each of the 10 ganders. The semen was statically frozen using dimethyl-formamide as a cryoprotectant and stored in liquid nitrogen at -196°C. Species-specific PCR systems targeting M. anserisalpingitidis, M. anseris and M. cloacale were used for screening and identification. Results of this study have shown, for the first time, that (1) among the three Mycoplasma species examined, all were detectable in the indigenous Hungarian ganders, with no clinical signs; (2) the pathogens could be detected in the cloaca, in both fresh and cryopreserved semen samples, but remained undetected within the inner reproductive organs; and (3) as pathogens were able to survive the freezing/storing/thawing procedures, the possibility of vertical transmission of the pathogens during artificial inseminations does exist, which causes problems in the in vitro gene-conservation programmes for this breed.

Successful cryopreservation and regeneration of a partridge colored Hungarian native chicken breed using primordial germ cells.

Primordial germ cells (PGCs) are the precursors of germline cells that generate sperm and ova in adults. Thus, they are promising tools for gene editing and genetic preservation, especially in avian species. In this study, we established stable male and female PGC lines from 6Hungarian indigenous chicken breeds with derivation rates ranging from 37.5 to 50 percent. We characterized the PGCs for expression of the germ cell-specific markers during prolonged culture in vitro. An in vivo colonization test was performed on PGCs from four Hungarian chicken breeds and the colonization rates were between 76 and 100%. Cryopreserved PGCs of the donor breed (Partridge color Hungarian) were injected into Black Transylvanian Naked Neck host embryos to form chimeric progeny that, after backcrossing, would permit reconstitution of the donor breed. For 24 presumptive chimeras 13 were male and 11 were female. In the course of backcrossing, 340 chicks were hatched and 17 of them (5%) were pure Partridge colored. Based on the backcrossing 1 hen and 3 roosters of the 24 presumptive chimeras (16.6%) have proven to be germline chimeras. Therefore, it was proven that the original breed can be recovered from primordial germ cells which are stored in the gene bank. To our knowledge, our study is a first that applied feeder free culturing conditions for both male and female cell lines successfully and used multiple indigenous chicken breeds to create a gene bank representing a region (Carpathian Basin).

Improvement of the application of gonadal tissue allotransplantation in the in vitro conservation of chicken genetic lines.

In avian species, the surgical technique for ovarian allotransplantation has been developed for domestic chickens; however, not all genotypes can be effectively used as recipients. The aims of the present study were to ascertain donor/recipient combinations for production of offspring from frozen/thawed ovarian tissues. The development of the technique is important because domestic chicken offspring have only been produced from fresh (never frozen) ovarian and from frozen-thawed testicular tissues. Information obtained from evaluating genetic differences of intensively selected lines in which there was successful pairing was compared in the indigenous breeds. Results indicate donor/recipient combinations were created which could be effectively used for gonadal tissue allotransplantations. Gonadal tissues of Yellow, Speckled and Partridge-color Hungarian, Black and Speckled Transylvanian Naked Neck chicken breeds were allotransplanted into White Leghorn or Novogen White breeds for offspring production. The gonadal tissues of these indigenous breeds were cryopreserved using vitrification procedures. There was successful allografting of frozen/thawed gonadal tissues at a rate between 20 % and 100 % depending on the genotype and sex, and histological examination and microsatellite marker analysis provided evidence that the donor ovarian and testicular tissues had the capacity for producing gametes. The hens of Speckled Transylvanian Naked Neck/White Leghorn combination using frozen/thawed ovarian tissues were produced for progeny tests. Of these, 58 % produced eggs and 9.1 % produced donor-derived offspring, based on data for both feather color markers and genetic analysis.

Investigation of the Guinea fowl and domestic fowl hybrids as potential surrogate hosts for avian cryopreservation programmes.

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.

Cryopreservation of gander semen in cryovials - Comparative study.

The aim of the study was to find a practical and inexpensive method for freezing goose semen for use in routine inseminations under farm conditions. Two basic freezing protocols [(1) dynamic, programmable freezing and (2) static, nitrogen vapour method] were evaluated with varying concentrations of dimethylformamide (DMF) plus additional osmoprotectants such as betaine, trehalose, and sucrose, using cryovials as containers. Altogether eight different treatments were compared. sperm viability before freezing and after thawing was examined by in vitro tests and, in the case of the simplest effective method, also by in vivo fertility test. There were no significant differences in sperm survival either in the dynamic (48-50%) or in the static protocol (43-46%), except for the treatment where the lowest DMF concentration was used without any osmoprotectant in the dynamic protocol (42.6%). The addition of osmoprotectants did not improve thawed sperm viability in any case. Fertility with frozen/thawed sperm using the simplest method was 58.5%, while that obtained with fresh, diluted semen was 66.9%. The study proved that the simple freezing of gander semen in nitrogen vapour with 9% DMF in cryovials could produce acceptable fertility. The newly elaborated method can be successfully used for routine inseminations by small- and large-scale goose breeders.