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The paper described a novel technique for semen collection in large psittacines (patent pending), a procedure which was not routinely possible before. For the first time, a large set of semen samples is now available for analysis as well as for artificial insemination. Semen samples of more than 100 psittacine taxa were collected and analysed; data...
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... the first egg was laid. Oviposition was expected due to observed breeding behaviour or typical abdominal extension. If possible, females were inseminated with semen from males other than their partners. The females were turned on their back and the oviduct opening was everted within the cloaca, followed by semen deposition into the oviduct (see Fig. 2). For this, the semen was pushed directly from the capillary into the oviduct by a plunger as previously described 11 . After 8-10 days of incubation in eggs which were laid after insemination, fertilisation was assessed by candling. Eggs with visual embryonic development were classified as ...
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... Therefore, the cloacal massage method has been widely used in various species, including passerines and non-passerines (Owen, 1941;Wolfson, 1952;Briskie and Montgomerie, 1992;Hemberger et al., 2001), although its effectiveness can vary among species with success rates ranging from 5 to 90% depending on the species (Sontakke et al., 2004;Della et al., 2011;Humann-Guilleminot et al., 2018). Alternatively, other attempts have been made to collect sperm, such as feces collection, wherein sperm may be obtained from feces during the breeding season (Immler and Birkhead, 2005;Calhim et al., 2007), and electro-stimulation, which induces ejaculation using weak electrical stimulation (Watanabe, 1957;Setioko and Hetzel, 1984;Lierz et al., 2013;Frediani et al., 2019). ...
Sperm competition has been studied in numerous species as a representative example of postcopulatory sexual selection, where sampling sperm from male is the most basic and important step. Sperm collection can be tricky in birds, however, because unlike mammals, the genitals of birds are generally latent in the cloacal region and their characteristics vary among species. Various methods to collect sperm from different birds have been tested, such as cloacal massage, feces collection, and electro-stimulation, but their applicability varies depending on species. In this study, we introduced the urodeum stimulation method (UroS method) to collect sperm from Cuculus cuckoos, such as the Common Cuckoo (C. canorus). These species are expected to have interesting patterns of pair bonding and sperm competition because of their unique breeding strategy called brood parasitism; however, it remains unexplored. In this study, we described the application of our new method to expel semen from male common cuckoos, measured the volume of semen collected, checked the presence of sperm in the semen sample, and finally estimated its success rate among 82 males. Samples were successfully collected from 76 cuckoos (approximately 93%) and the colors and volumes of the samples were very diverse. Sperm was present in 43 of these samples (57%), showing a sperm observation rate approximately twice as high as that of the conventional cloacal massage method. We believe that this novel method will contribute to a better understanding of postcopulatory sexual selection in avian brood parasites and facilitate the process of sperm collection and artificial insemination in other medium-sized birds.
... However, this noninvasive method requires frequent handling of birds and a training period to get them used to stimulation and milking. Electrostimulation was suggested as novel method to collect semen in large parrots providing 67% of successful attempts [12]. However, when it was applied to Spix's Macaws (Cyanopsitta spixii, very rare and endangered species probably surviving only in captivity), the success rate was decreased to 44% [13]. ...
In order to preserve endangered psittacine species, more basic and applied research in reproductive biology is required. Assisted reproductive technologies such as artificial insemination play an important role in parrots species conservation programs to overcome the problem of infertile eggs and male infertility. The aim of this study was to define an effective in vitro protocol in order to standardize the sperm quality evaluation in psittacines, studying Melopsittacus undulatus as model species. Semen was collected from twenty adult males by massage technique from May to June. Sperm concentration was measured by the spectrophotometric method. Sperm quality (sperm membrane integrity (SMI), motility, and kinetic parameters) was assessed on fresh semen. Three different experimental protocols were performed to compare the effects of various processing conditions on SMI, motility, and kinetic parameters. In protocol 1, test was performed by Lake extender with three different pH, 7.4 versus 8.2 versus 8.4, and two different equilibration temperatures after dilution of fresh semen (4°C versus 25°C). In protocol 2, two dilution rates of semen after collection were valuated, 1 : 3 versus 1 : 4, as well as three different semen storage temperatures (4°C versus 25°C versus 38°C) before sperm motility analysis with the computer-assisted sperm analysis (CASA). In protocol 3, two different Makler chamber temperatures (38 versus 41°C) during motility analysis were tested. A significant progressive improvement in spermatozoa motility and kinetic parameters was registered with pH 8.4. Progressive motility and all kinetic parameters were higher at 4°C equilibration temperature. Straightness (STR) kinetic parameter was better with 1 : 4 dilution rate. Total motile sperm was higher in 41°C Makler chamber. In this study, for the first time, the effects of different processing protocols on psittacines seminal quality analysis were investigated. Significant differences conditioning the effectiveness of analysis protocols have been described.
... If semen could not be collected by massage, electro-stimulation was used as described in large parrots [39,40,46]. For harpy eagles, two specifically designed probes were used (length: 5 cm (L) or 8 cm (XL); diameter: 5 mm). ...
... Legend: Sperm density was classified as follows: Category 1 (low, single sperm inside the glass capillary), Category 2 (few, single (1e3) sperm regularly at least in every second field of view), Category 3 (moderate, some (4e10) sperm in each field of view), Category 4 (more, multiple (~10e50) sperm in each field of view), and Category 5 (high, many (>50) sperm in each field of view). similar to previous findings for many large parrot species in which massage regularly fails and for which the application of electrostimulation is necessary to collect semen [40]. The electric current needed for successful semen collection had a maximum of 2.88 V and was comparable to the settings used in sharp-tailed grouse [49]. ...
... However, the maximum values in harpy eagles (22,500 sperm/ml) fell within the range of some of the above-mentioned raptors. It remains unclear whether this is just an example of variation in raptors, as demonstrated within the order psittaciformes [40,50], or if there are different underlying reasons. Besides spermatozoa, few pleomorphic round bodies were present in semen samples of harpy eagles, a finding that has previously been described in several other raptor species [54e57]. ...
Currently, 52% of all raptor species demonstrate a decreasing population tendency, and the American harpy eagle (Harpia harpyja) has been categorized as “near threatened” by the IUCN. Habitat loss, persecution, and subsequent reduction of genetic diversity are regarded as major threats to the world's strongest eagle. Captive breeding and reintroduction into protected habitats are approaches of species conservation projects, but captive propagation is difficult due to low ex-situ numbers and scarce successful breeding pairs.
The aim of the present study was to collect, analyze, and store semen from harpy eagles and to use aliquots for artificial insemination to increase the number of offspring and to include more individuals into the ex-situ gene pool. First, semen collection and semen availability were assessed in four males during the course of 1 year in European zoos. Second, these experiences were transferred to ex-situ breeding programs in Brazilian zoos to attempt semen collection in 13 male eagles.
Semen collection was successful in 51.7% of the attempts and in 8/13 males (individual success rates 20–100%) using electro-stimulation. Most commonly, whey-like to milky, whitish semen samples were collected, regularly containing urate impurities (67.7%). The median semen volume was 106 μl and the median sperm concentration 5000 sperm/μl (750–22,500 sperm/μl). Mean values for pH were 6.7, for sperm motility 27.7 ± 22.6%, for progressive motility 2.9 ± 5.6%, and for sperm viability 46.6 ± 16.3%. Using semen extenders, a sperm motility of 8% was maintained for 27 h in the refrigerator. Artificial insemination was performed in one female, but the success of fertilization could not be assessed due to egg destruction.
In this study, methods for assisted reproduction were refined for use in harpy eagles, and the first semen samples were evaluated as a start to establish species-specific orientation values.
... Fertility rates after AI were elevated up to 80% in quail, being equivalent to natural mating results [117]. AI in large parrots proved a milestone in a species conservation program [118]. AI in poultry species results in efficient use of best sires at pedigree farms, i.e., one male and ten to twelve females compared to the conventional mating ratio of one male and three to four females [38]. ...
The goose is a popular poultry species, and in the past two decades the goose industry has become highly profitable across the globe. Ganders low reproductive performance remains a barrier to achieving high fertility and hatchability in subsequent flocks. To address the global demand for cheaper animal protein, various methodologies for improving avian (re)production should be explored. A large amount of literature is available on reproduction traits and techniques for commercial chicken breeder flocks, while research on improved reproduction in ganders has been carried out to a lesser extent. The present review aims to provide a comprehensive literature overview focusing on recent advancements/techniques used in improving gander reproductive efficacy in the context of ensuring a globally sustainable goose industry.
... In the end, artificial insemination is possibly an excellent tool for specific situations, such as recovery programs of endangered avian species, when an increased number of offspring is desired and also mating specific birds is mandatory. 20 Egg necropsy This topic can be a question of mortality or of breeding failure. In any case, it is of paramount importance to necropsy all the eggs that do not hatch. ...
The management of a breeding facility of exotic species is challenging and managing these on a large scale can be a daunting task for general practitioners or novice exotic animal veterinarians. They are usually not trained to work with flocks or herds, and even most exotic animal veterinarians are used to working with single patients. This article gives some suggestions and tips on how to proceed when facing an exotic animal collection and especially when breeding is not as successful as expected.
... In some vertebrates, including humans, semen can be collected by massaging, applying pressure, either with or without anaesthesia, or using an artificial vagina or a dummy female (Birkhead et al. 2009;Lifjeld et al. 2010;Wasden et al. 2017;Humann-Guilleminot et al. 2018). In other vertebrate species, electroejaculation can be feasible (Lierz et al. 2013), though it can affect semen characteristics (Birkhead et al. 2009). Nonetheless, surgical or destructive semen collection is the only option in many taxa, rendering them unsuitable for longitudinal studies. ...
Physiological and biochemical traits hold great promise for demographic research as potential proxies (biomarkers) of various biotic and environmental variables that determine individual fitness and ultimately demographic rates. Integrating such biomarkers into demographic models can thus provide insights into drivers of population dynamics or increase predictive power of the models by refining estimation of vital rates. Biomarkers also represent promising means to characterise population structure and dynamics on much shorter time-scales compared to classical demographic approaches. Functional traits further emerge as direct targets of conservation efforts directed towards conserving functional diversity. Yet, biomarkers and functional traits remain underutilised in demography and population ecology, indicating that their benefits still await wider recognition. This chapter briefly reviews the most prominent physiological and biochemical traits (e.g. metabolic rates, hormones, oxidative stress markers, telomeres) that may be of interest in animal and plant demographic research, including the methods for collection, storage, and analysis, and the criteria to be met before the trait is validated as a biomarker. Hopefully, this effort will stimulate further integration of physiological and biochemical data into demographic framework.
... The vent and cloaca were cleaned with a Kimwipe (Kimberly-Clark Professional, Corinth, MO, USA) to remove debris. While anesthetized, each animal was electrostimulated using a 360 • circumferential metallic probe (20 mm length, 3 mm diameter) connected to a variable amperage power source [13,41,43]. An intromission was defined as the process of fully inserting the metallic portion of the probe into the vent and directing it cranially. ...
Reptiles are highly susceptible to anthropogenic activities as a result of their narrow geographical ranges and habitat specialization, making them a conservation concern. Geckos represent one of the mega-diverse reptile lineages under pressure; however, limited assisted reproductive technologies currently exist for these animals. Exogenous pregnant mare serum gonadotropin (PMSG) has been found to exhibit follicle stimulating hormone-like action and has been routinely used to alter reproductive hormones of vertebrates in assisted reproductive protocols. The purpose of this study was to determine the effects of serial injections of 20 IU and 50 IU PMSG on circulating testosterone concentrations, testicular dynamics, and semen production in a model species of gecko. Twenty-four captive-bred, adult, male leopard geckos (Eublepharis macularius) were divided into three treatment groups and administered a once-weekly injection of either PMSG or saline for a total of nine weeks. Ultrasonographic testicular measurements, electrostimulation for semen collection, and venipuncture were performed on days 0, 21, 42, and 63. Right unilateral orchidectomies and epididymectomies were performed in all animals on day 63; tissues were submitted for histopathology. PMSG treated geckos had significantly higher testicular volumes and weights, spermatozoa motility, and spermatozoa concentrations compared with controls. However, there were no significant differences in testosterone concentrations by treatment or time. Under the conditions outlined, PMSG is effective at stimulating spermatogenesis and increasing testicular size, but not effective at increasing testosterone concentrations in the leopard gecko between October–December in the Northern hemisphere.
... However, various stains and numerous staining protocols have been used for semen analysis in birds. Conventional live/dead stains, such as eosin blue [EB] (Behncke, 2002, Fischer et al., 2014b, Stelzer et al., 2005, Stelzer et al., 2009, Lierz et al., 2013, Schneider et al., 2017, Schneider et al., 2018, eosin-nigrosin [EBN, EYN] (Bailey, 2002, Madeddu et al., 2009, Blanco et al., 2008, Chalah and Brillard, 1998, Hartley et al., 1999, Saint Jalme et al., 2003, eosin-blue aniline [EBA] (Bailey, 2002, Varga et al., 2003 and bromphenol blue-nigrosin [BBN] (Kamar, 1959, Wilson et al., 1969 have been used beside several fluorescent probes, such as SYBR® 14/Green-propidium iodide [SYBR-PI] and SYBR® 14/Green-Ethidium Homodimer 1 [SYBR-EthD-1] (Garner and Johnson, 1995, Garner et al., 1986, Garner et al., 1994, Donoghue et al., 1995, Klimowicz-Bodys et al., 2012. The latter, dual colorimetric fluorescent methods allow the coloration of intracellular structures of cells with damaged outer cellular membranes in one color and of the genome of vital spermatozoa in a different color (Chalah and Brillard, 1998). ...
... To this end, all semen samples were diluted to equal parts (1:2) with 6-Hour SemAid (PHL Associates Inc., Davis, CA, USA) prior to motility and to viability analysis. EB was used as 2 % standard mixture according to studies in psittacines (Neumann et al., 2013, Stelzer et al., 2005. Additionally, 3 g tri-sodium citrate hydrate (Merck KgaA) ...
... Using eosin blue, the differentiation of dead from alive sperm was effectively possible, as reported from viability analysis in other avian species (Behncke, 2002, Fischer et al., 2014a, Lierz et al., 2013, Neumann et al., 2013, Schneider et al., 2018, Schneider et al., 2019, Stelzer et al., 2005, Stelzer et al., 2009 (Schneider et al., 2017, Schneider et al., 2018. The addition of sodium citrate (EB2), as suggested in some studies (Behncke, 2002) did not improve the staining properties for viability analysis. ...
Viability assessment is an important part of semen analysis and various live/dead staining protocols have been used in semen of avian species. Results of live/dead count differed between dyes, staining protocols and bird species, impeding comparability between studies and requiring species specific comparisons of viability stains. In raptor semen similar comparisons are absent. Thus, the aim of the present study was to compare eight conventional viability stains. Eosin blue 2 % [EB], eosin blue 2 % with the addition of 3 % sodium citrate [EB2], eosin blue nigrosin 5 % [EBN5], eosin yellow nigrosin 5 % [EYN5], eosin yellow nigrosin 10 % [EYN10], eosin blue aniline blue [EBA], eosin yellow aniline blue [EYA], and bromphenol blue nigrosin [BBN] were evaluated in comparison to the fluorescence stain SYBR® Green ‐ propidium iodide [SYBR‐PI] in spermatozoa of falcons. The comparison was performed using conventional light microscopy which is applicable in breeding centers, veterinary practices and field studies. Additionally, live/dead stains were correlated to motility values of the same samples to validate sperm viability.
Light microscopy using EB and using SYBR‐PI enabled an effective and clear differentiation between alive and dead spermatozoa of falcons. Motility values correlated significantly and strong with EB only (r = 0.629; p < 0.001), but not with any other stain used in the study. Therefore, our results suggest EB as the most suitable stain for viability assessment in the semen of large falcons.
... Assisted reproduction has been practiced in the domestic fowl since the 1930s [20] and was extended to non-domesticated birds as well [21e23]. In psittacines, semen collection and analysis have been performed successfully [24e28] and especially artificial insemination has been described as a useful tool in captive breeding of parrots [26,28,29]. However, in amazon parrots studies revealed that the success of conventional massage method of semen collection had been inconsistent and low. ...
... Therefore, assisted reproduction seemed less promising [25,30]. In 2011, the application of a novel method of electric stimulated semen collection increased the success rates of semen collection in psittacines [26]. Semen collection was successful in 68.7% (68 out of 97 attempts) in 20/26 amazon species, including 3/3 successful attempts in St. Vincent amazons. ...
... As detailed semen analysis has been performed in 11 amazons of different species and successful artificial insemination has been reported in 3 amazon species (A. pretrei, A. xantholora, A. finschii) the technique seemed suitable for the St. Vincent amazon [26]. ...
... In avian reproductive medicine, the functional evaluation of spermatozoa is less common, compared to mammalian species. Improvement in assisted-reproductive technologies is important in terms of species conservation [2,3], to reveal causes of infertile clutches and to enable the sorting of infertile males from breeding programs. An interspecific assay would be very useful, especially to test semen of endangered species where eggs of the same species are rarely available. ...
The aim of this study was to adapt an inner perivitelline membrane (IPVM) test as an interspecies penetration assay for avian spermatozoa. The IPVM of different bird species was evaluated to test the penetrating ability of avian spermatozoa in an intra- and interspecies design. Isolation of the IPVM via acid hydrolysis was tested in pre-incubated chicken eggs and in six other avian species. The separation protocol was modified (time, acid concentration) to facilitate practicability. Separated membranes were evaluated with dark field microscopy for the presence of holes produced by penetrating spermatozoa. In chicken eggs, the influence of different membrane storage conditions was tested. In the penetration assay, the IPVM of chicken eggs was used as a model for fresh and frozen–thawed rooster sperm and for fresh spermatozoa of cockatiels and falcons. Results demonstrated that the time of egg-incubation had a significantly negative influence on the isolation ability of the IPVM (p < 0.0001). IPVM-separation was successful for a maximum of two days after preincubation. In the experiments with eggs from other avian species, results were heterogenous: there was no isolation in geese and cockatiels, 20% in the European kestrel, and 40% in pheasant, quail, and duck. In the penetration assay, holes were found in 100% of the IPVM of chicken eggs after incubation with native and frozen–thawed rooster semen and in 10% with fresh cockatiel semen. Falcon spermatozoa failed to produce visible holes. In conclusion, the IPVM of chicken eggs seems to be unsuitable to establish a functional sperm assay in other species tested but is suitable for quality evaluation of cryopreserved rooster sperm.
