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).

Effect of Post-Hatch Heat-Treatment in Heat-Stressed Transylvanian Naked Neck Chicken.

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.

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.

Successful chimera production in the Hungarian goose (Anser anser domestica) by intracardiac injection of blastodermal cells in 3-day-old embryos.

The conservation of genetic resources of avian species has become increasingly important over the past decade. The aim of the present study was to develop a genome preservation technique for the Hungarian goose Anser anser domestica. To this end, we developed a novel approach combining the simplicity of isolating a blastodermal cell suspension, which includes forming primordial germ cells (PGCs), with the efficiency of targeting future gonads by injecting these cells into the cardiac vein of the developing host embryo. First, we determined that the migratory period of PGCs in goose embryos was between 69 and 84h of development. Then, we injected the blastodermal cell suspension into the bloodstream of recipient embryos at this stage of development and monitored donor cell transmission into the genital tract. In all, 249 embryos were injected; three were found to be chimeras in gonadal tissues, whereas only one was a chimera in other tissues. Based on these results, it is concluded that this method is suitable for producing chimeras in the domestic goose. The optimal time of cell injection was found to be between 74 and 76h. The present study is the first report of the generation of chimeras in the domestic goose using intracardiac transplantation of embryonic cells.

Effect of sex ratios, spiking and extra artificial insemination on the breeding efficiency of broiler breeders.

Since early fertility decline is a permanent problem of broiler breeders, the aim of this study was to test the effects of various sex ratios, spiking strategies and additional artificial inseminations (AI) on their breeding efficiency. Six breeder flocks were analysed during the whole reproduction cycle. In Flock A the sex ratio was maintained at 10% during the whole cycle (control), while in Flock B the number of males was increased to a final ratio of 16%. In Flocks C (technological control), D, E and F the ratio of males was gradually decreased from 10% to 6.5% until the end of the cycle. Moreover, at the age of 44 weeks in Flocks D and E 50 and 100% of cockerels were replaced by young ones, respectively, while in Flock F additional artificial inseminations were applied in the second half of the reproduction cycle. The increase of sperm transport was successful only in Groups B (increase in male numbers) and D (50% replacement of old cockerels with young ones); however, it was not sufficient for increasing the fertility rates in either group. Nor did additional artificial inseminations (Flock F) have an effect on fertility. As a conclusion, it can be established that increasing the sperm count in the hens' oviducts in any way could not improve fertility in the last third of the production cycle. The results also suggest that the expensive and labour-intensive spiking technique used in broiler breeder management is useless. The prime factor responsible for the shortened persistence of fertility may be the reduced ability of the female oviduct to accept and store sperm.

Methods for cryopreservation of guinea fowl sperm.

Conservation of indigenous poultry species is an important part of the new Hungarian agricultural strategy. Semen cryopreservation is the most practical method for the long term storage of poultry genetic material. The objective was to compare four protocols for cryopreservation of guinea fowl sperm (slow and fast programmable, freezing in nitrogen vapor, and pellet) and three cryoprotectants (10% ethylene glycol, 6% dimethyl-formamide and 6% dimethyl-acetamide). The efficiency of the methods was examined by in vitro tests (subjective motility scoring, sperm concentration, morphological and live/dead sperm analysis with eosin-aniline staining). Thereafter, the two most promising methods were tested by artificial insemination of frozen-thawed semen (3 times a week for 3 weeks using 300 million spermatozoa/hen), followed by candling of incubated eggs, assessment of fertilization, embryonic death, and hatching rate. The survival rate of live, intact spermatozoa was greatest (p≤0.05) in pellet method and the slow programmable protocol (with 10% ethylene glycol) (28.6 and 23.5%). The two best protocols (based on in vitro assessment of post-thaw semen quality) were subsequently tested in vivo with artificial insemination. The pellet method yielded a 64% fertility rate compared to slow protocol with only 30% fertility. Regardless, both freezing protocols significantly increased embryonic deaths compared to the control group (16,7; 9,1 and 8,3%, respectively). During the 3-week in vivo trial, fertility increased and early embryonic death decreased over time. According to the results the guinea fowl sperm could tolerate the fast freezing in pellet better than the slower freezing rates and resulted acceptable fertility rate.

Seasonal changes in plasma dehydro-epiandrosterone (DHEA) levels of domestic geese.

Changes in plasma DHEA, testosterone (T) and 17-B-oestradiol (E2) levels were examined in domestic geese of both sexes in the fall and winter. The levels of steroid hormones were determined in blood plasma by means of radio-immunoassay (RIA). A so-called second (autumn) cycle was induced in geese via a dark-room preparation and natural keeping conditions. The plasma levels of DHEA showed a minor peak at onset of the autumn breeding and a major one prior to the more intense spring reproduction in both sexes. The seasonal curves of plasma DHEA appeared fairly similar in ganders and layers and without considerable differences between the absolute values. In ganders, plasma DHEA peaks preceded the elevations in T levels in the fall and spring alike. With layers, in turn, the autumn and spring peaks of plasma DHEA appeared after the peaks in E2 levels. With ganders, the concentration of plasma T seemed to predominate between the two androgens throughout the experimental period. With layers, in turn, the concentration of DHEA surpassed the level of plasma E2 at the time of the peak periods and other times during the study, as well. In domestic geese, DHEA is probably involved in the autumn physiological processes and the induction of reproduction during fall and early spring periods, alike.