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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.

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.

Background One of the most critical global problem nowadays is the increased environmental temperature. Agriculture is very susceptible to this adverse effect because the productivity of animals and poultry decreased. Although several studies reported the effects of heat-stress in chicken, the expression profile of heat-shock proteins and heat shock factors was not investigated in the gonads and germ cells of Transylvanian Naked Neck chickens. Methods In the first experiment, 24 hours after hatching 80 chicks were heat treated on 38.5oC ambient temperature with 60% humidity for 12 hours. After maturation, their primary productivity parameters, such as egg production, abnormalities in embryo development, sperm quantity, concentration, and motility were studied following two weeks of heat-stress on 30 °C room temperature. In the second experiment, the thermal manipulation of 60 chicks was the same but 15 treated and 15 control chicks were sacrificed immediately after the treatment. The other 15–15 chickens were raised to maturity. Expression levels for two heat-shock proteins and four heat shock factors were determined by real-time PCR in the gonads of heat-treated and heat-stressed chickens. Results We found that the heat-treated layers had significantly higher egg production than the control group in heat-stressed conditions. In cockerels, the sperm quality did not differ significantly between the heat-treated and heat-stressed group and the heat-stressed but not heat-treated group. We examined the expression pattern of HSPs and HSFs in the gonads. We found that the expression of HSP90 and HSF4 increased significantly (p HSF2 and HSF3 were significantly lower compared to the control. In case of adult heat-treated males, the HSP70, HSF1 and HSF3 expression levels showed a significant increase in both gonads, compared to the control expression levels (P HSP90 and HSF4 in heat-treated chickens.

Assisted reproductive techniques such as artificial insemination, cryopreservation of avian spermatozoa, in vitro fertilization, intracytoplasmic sperm injection, early gonad manipulations, and embryonic cell manipulation have all been attempted in various avian species. Most of the related studies, though, are available on the domesticated poultry species such as domestic fowl, turkey, guinea fowl, quail, duck, and geese. Some of these reproductive technologies are already used in breeding practice, whereas others are still under development. In this chapter, an overview of the reproductive technologies implemented in avian species will be presented, while a continuous improvement is still in progress.

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.

Semen cryopreservation is a practical method for banking germplasm from valuable indigenous species which have an increasing risk of extinction. Many scientific publications have described different protocols of avian sperm cryopreservation for both domesticated and non-domesticated avian species, involving cryoprotectant type and packaging method, as well as freezing and thawing rates. In this research, a comparative approach was used to evaluate two freezing protocols for guinea fowls: the modified slow programmable method using 10% ethylene glycol (EG), and the newly applied fast freezing method (pellet formation) with 6% dimethylacetamide (DMA). The efficiency of two protocols is measured by both in vitro sperm evaluation assay (determination of sperm concentration and motility, morphological and live/dead sperm analysis) and in vivo sperm evaluation assay (artificial insemination, determination of fertility rate and embryonic death). For fertility determination, candling of incubated eggs was used, extended by checking of the ratio of early embryonic mortality. In vitro qualification showed that the survival rate of live and intact spermatozoa was significantly higher after pelletation than after slow protocol (28.59% vs 23.53%, p=0.022, Mann-Whitney U test). In vivo qualification showed that artificial insemination of frozenthawed semen yielded 50.2% fertility rate with the pellet, while only 25.5% with the slow programmable method (while it was 84.8% in control, p=0.027, Kruskal-Wallis ANOVA test). The simple in vitro

The proportion of spermatozoa with elongated nuclei in ejaculates from a strain of guinea fowl was estimated, subjectively, to range from approximately 1 to 6%. It was confirmed by image analysis that in an ejaculate from one male, the distribution of nuclear lengths was bimodal, with a distinct population comprising 10% of spermatozoa having a mean nuclear length that was 52% greater than that of the remaining 90%. Furthermore, the mean DNA content of the 'large-nuclei' population was 1.85 times (not significantly different from twice) that of the main sperm population. The proportion of large-nuclei spermatozoa that was motile was less than that of normal sperm (31% versus 59%) and the velocity of motile spermatozoa was also less (24 microm/s versus 72 microm/s). The poor motility of the large-nuclei spermatozoa in vitro was reflected in their limited performance in vivo, since only 1.1% were found associated with the egg outer perivitelline layer. This is the first report to quantify the occurrence of, presumed, polyploid spermatozoa in a domestic bird. The incidence of such spermatozoa in commercial guinea fowl and other domestic poultry and the genesis and effects on fertility of such spermatozoa may be significant.

Although cryopreservation of avian semen is only applicable for singlegene traits, cryopreservation of avian blastodermal cells could facilitate preservation of the entire genome of endangered or rare-breed poultry. Slow freezing methods result in acceptable survival rates; however, there are apparently no reports regarding the use of vitrification. The aim of the study was to establish methods for chicken embryonic cell vitrification, including development of a container which supported cryopreservation of large numbers of cells (to increase the probability of chimera production). Based on a preliminary study, vitrification seemed to be practical for avian blastodermal cell preservation. Pieces of mosquito net as carrier increased live cell rates compared to pellet form in media containing two macromolecules. Furthermore, we concluded that fetal calf serum in the vitrification medium could be replaced by polyvinylpyrrolidone, a chemically defined substance free of unwanted growth factors and potential pathogens.

The aim of the study was to devise a technique to allow transfer of ovarian and testicular tissues obtained from one day old chicks to recipient animals. The following combinations of chicken breeds were used: Tetra SL/Tetra SL, Tetra SL/Harco, Tetra SL/Black Transylvanian Naked Neck, Godollo New Hampshire/Speckled Transylvanian Naked Neck (recipient/donor), respectively. Only animals less than 24 h old from hatching were used as either recipient or donor. Eggs yielding recipients were treated with busulfan to hinder the development of the recipient's own gonads. Gonads were transferred surgically to the new host which was then immunosuppressed for two months. The animals were checked at 8 or 16 weeks for the existence of implanted gonads, and if found, the gonads were removed for histology examination. Tetra SL/Tetra SL pairing resulted in successfully adhered and functioning gonads in most cases. In other combinations, no gonads originating from the donors were found in the recipients. We conclude that not all breeds seem to be suitable recipients; some breeds or individuals may show incompatibility to each other and further examination is needed to find the cause of incompatibility and to establish a suitable breed combination, which can be used for gonad transfer.

Embryonic mortality is a significant problem plaguing the hatching success. Its early forms are especially hardly distinguishable from true infertility. Propidium iodide (PI) staining of the germinal disc combined with outer perivitelline layer (OPVL) sperm counting was used for the determination of 'true' fertility of duck eggs in two different experiments: fertility investigation on fresh, unincubated eggs of Hungarian ducks and on incubated eggs of a crossbred, selected as 'infertile' at the 7th day of incubation. Examination of the relationship between OPVL sperm count and fertility seems to be an adequate tool for checking the effectiveness of insemination programmes and the fertilising capacity of poultry spermatozoa. The proportion of fertile eggs was around 50% when the number of OPVL sperm was between 0.1 and 0.2 spermatozoa/mm2. Ninety-nine percent of the eggs containing ? 0.3 OPVL sperm/mm2 were fertile and all of the eggs containing < 0.05 sperm/mm2 were infertile. To assure the accuracy of fertility prediction by OPVL sperm counting, PI staining of the germinal disc was used to determine fertility in uncertain cases. Identification of very early embryonic mortality, i.e. that occurring before oviposition, is very difficult. The use of a dissecting microscope for the assessment of real fertility is suitable in most of the cases, while PI staining of the germinal discs proved to be more reliable for detecting very early embryonic death. The combination of the two methods proved to be a useful tool for detecting the 'true' fertility of duck eggs of different breeds.

In most bird species, pairs copulate many times before egg laying. The exact function of repeated inseminations (i.e. successful copulations) is unknown, but several suggestions have been made. We tested the hypothesis that repeated inseminations are required to ensure fertilization of eggs, by using an experimental method where free-ranging male collared flycatchers (Ficedula albicollis) were prevented from inseminating their mates. We show that egg fertility was lower when females had not copulated during the studied part of their fertile period. By counting sperm on the inner perivitelline layer of eggs, we estimated that a minimum of 86 sperm must reach the site of fertilization to ensure average fertility. Using the timing of inseminations and the numbers of sperm on successive eggs, we show that repeated copulations are necessary to achieve an average rate of fertilization of a single clutch. Our results thus provide evidence that repeated inseminations function to ensure fertilization success. We discuss possible constraints on sperm production and utilization that may have contributed to this pattern.

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.