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.

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.

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.

Parallel palaeogenomic transects reveal complex genetic history of early European farmers.

Ancient DNA studies have established that Neolithic European populations were descended from Anatolian migrants who received a limited amount of admixture from resident hunter-gatherers. Many open questions remain, however, about the spatial and temporal dynamics of population interactions and admixture during the Neolithic period. Here we investigate the population dynamics of Neolithization across Europe using a high-resolution genome-wide ancient DNA dataset with a total of 180 samples, of which 130 are newly reported here, from the Neolithic and Chalcolithic periods of Hungary (6000-2900 bc, n = 100), Germany (5500-3000 bc, n = 42) and Spain (5500-2200 bc, n = 38). We find that genetic diversity was shaped predominantly by local processes, with varied sources and proportions of hunter-gatherer ancestry among the three regions and through time. Admixture between groups with different ancestry profiles was pervasive and resulted in observable population transformation across almost all cultural transitions. Our results shed new light on the ways in which gene flow reshaped European populations throughout the Neolithic period and demonstrate the potential of time-series-based sampling and modelling approaches to elucidate multiple dimensions of historical population interactions.

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.

Preliminary results of the application of gonadal tissue transfer in various chicken breeds in the poultry gene conservation.

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 24h 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.

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.

Vitrification of early avian blastodermal cells with a new type of cryocontainer.

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.

Determination of the rate of true fertility in duck breeds by the combination of two in vitro methods.

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.

Repeated inseminations required for natural fertility in a wild bird population.

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.

Excess nuclear DNA in spermatozoa of guinea fowl.

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.