Lab partners: oocytes, embryos and company. A personal view on aspects of oocyte maturation and the development of monozygotic twins.

The present review addresses the oocyte and the preimplantation embryo, and is intended to highlight the underlying principle of the "nature versus/and nurture" question. Given the diversity in mammalian oocyte maturation, this review will not be comprehensive but instead will focus on the porcine oocyte. Historically, oogenesis was seen as the development of a passive cell nursed and determined by its somatic compartment. Currently, the advanced analysis of the cross-talk between the maternal environment and the oocyte shows a more balanced relationship: Granulosa cells nurse the oocyte, whereas the latter secretes diffusible factors that regulate proliferation and differentiation of the granulosa cells. Signal molecules of the granulosa cells either prevent the precocious initiation of meiotic maturation or enable oocyte maturation following hormonal stimulation. A similar question emerges in research on monozygotic twins or multiples: In Greek and medieval times, twins were not seen as the result of the common course of nature but were classified as faults. This seems still valid today for the rare and until now mainly unknown genesis of facultative monozygotic twins in mammals. Monozygotic twins are unique subjects for studies of the conceptus-maternal dialogue, the intra-pair similarity and dissimilarity, and the elucidation of the interplay between nature and nurture. In the course of in vivo collections of preimplantation sheep embryos and experiments on embryo splitting and other microsurgical interventions we recorded observations on double blastocysts within a single zona pellucida, double inner cell masses in zona-enclosed blastocysts and double germinal discs in elongating embryos. On the basis of these observations we add some pieces to the puzzle of the post-zygotic genesis of monozygotic twins and on maternal influences on the developing conceptus.

Computer-assisted semen analysis in large falcons (Falco spp.) and comparison to results of conventional semen analysis

OBJECTIVE: Numerous raptor species including some falcon species are facing continuous population decline in the wild and some are threatened by extinction. To support these species, captive breeding and reintroduction programs are attempted. Besides conservation, some large falcon species are commonly used in falconry and therefore bred commercially. Assisted reproduction is established in falcon breeding since the 1970s and semen analysis is an integral part to enable assessment of breeding males, inclusion or exclusion of semen donors and quality control of semen prior to artificial insemination. Conventional methods for semen analysis are widely used, but are time consuming and depend on the investigator's experience and ability. Computer-assisted semen analysis (CASA) would offer an objective, fast and reproducible alternative, but as they have not been established in large falcon species, this was the aim of this study. MATERIAL AND METHODS: To this end, we examined in 3 breeding seasons 109 semen samples of gyr-saker hybrid falcons (n=2) and peregrine falcons (n=4) in 940 fields of view using the Minitube CASA SpermVision and compared these results to results of conventional methods of semen analysis. We used a preprogrammed setting and adapted two settings of CASA according to specific semen characteristics of falcons. RESULTS: Sperm velocity, motility and viability parameters were recorded successfully using CASA. Correlation of conventional and computer-assisted motility analysis improved during the process of adaptation of CASA settings, but both methods differed significantly due to misinterpretation of round bodies and semen impurities by CASA. Viability values of conventional and computer assisted viability analysis using SYBR-PI correlated significantly while sperm concentration did not at all. CONCLUSION: CASA failed to replace conventional semen analysis regarding sperm motility and sperm concentration using 3 different settings, as a reliable differentiation between spermatozoa, spermatids and round bodies was not achieved. CLINICAL RELEVANCE: Using CASA, sperm velocity parameters were measured in spermatozoa of captive-bred large falcons for the first time and may be used as orientation values.

Viability assessment of spermatozoa in large falcons (Falco spp.) using various staining protocols.

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 bromophenol blue-nigrosin [BBN] were evaluated in comparison with 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 centres, 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 strongly with EB only (r = .629; p < .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.

Cycle-specific female preferences for visual and non-visual cues in the horse (Equus caballus).

Although female preferences are well studied in many mammals, the possible effects of the oestrous cycle are not yet sufficiently understood. Here we investigate female preferences for visual and non-visual male traits relative to the periodically cycling of sexual proceptivity (oestrus) and inactivity (dioestrus), respectively, in the polygynous horse (Equus caballus). We individually exposed mares to stallions in four experimental situations: (i) mares in oestrus and visual contact to stallions allowed, (ii) mares in oestrus, with blinds (wooden partitions preventing visual contact but allowing for acoustic and olfactory communication), (iii) mares in dioestrus, no blinds, and (iv) mares in dioestrus, with blinds. Contact times of the mares with each stallion, defined as the cumulative amount of time a mare was in the vicinity of an individual stallion and actively searching contact, were used to rank stallions according to each mare's preferences. We found that preferences based on visual traits differed significantly from preferences based on non-visual traits in dioestrous mares. The mares then showed a preference for older and larger males, but only if visual cues were available. In contrast, oestrous mares showed consistent preferences with or without blinds, i.e. their preferences were mainly based on non-visual traits and could not be predicted by male age or size. Stallions who were generally preferred displayed a high libido that may have positively influenced female interest or may have been a consequence of it. We conclude that the oestrous cycle has a significant influence on female preferences for visual and non-visual male traits in the horse.

Simultaneous umbilical blood flow during normal pregnancy in sheep and goat foetuses using non-invasive colour Doppler ultrasound.

The characteristics of umbilical blood flow (UM) was investigated using 18 (25 foetuses) pregnant ewes and 20 (41 foetus) pregnant goats transrectal non-invasive color Doppler ultrasonographic examinations were done frequently between 2 and 8 week after breeding and then transabdominally until parturition. Colour Doppler velocimetery includes blood flow volume (BFV), time averaged maximum velocity (TAMV), resistance index (RI), pulsatility index (PI), time average of mean (TAMEAN) and impedance of blood flow (PS/ED or AB ratio). Also a qualitative evaluation of UM blood flow indicating increases (P < 0.001) in BFV, TAMV and TAMEAN were observed until 19 week of pregnancy in foetuses of sheep and goats and then those values decreased (P < 0.001) from 19 week until parturition. Conversely, UM-PI, RI and PS/ED decreased (P < 0.002-0.01) until 19 week and then increased (P < 0.01-0.0001). The umbilical artery BFV increased (P < 0.0001) during pregnancy from 7.27 ± 0.82 ml/min in sheep vs. 4.96 ± 0.54 ml/min in goats at 6 week of gestation to 700.51 ± 31.05 ml/min (~100 fold) in sheep vs. 665.56 ± 48.22 ml/min (~133 fold) in goats at 19 week and then decreased (P < 0.0001) to 350.561 ± 72.15 ml/min in sheep vs. 215.17 ± 35.06 ml/min in goats at 20 week. The absence of end diastolic velocity (EDV) of umbilical artery blood flow was detected in both species between 4 and 12 week of pregnancy. Results of this study clearly show that the non-invasive colour Doppler sonography can be used successfully to assess umbilical blood flow in foetuses of pregnant sheep and goats. These may provide guidelines for assessing the state of intrauterine fetal growth retardation in pregnancies of sheep and goats.

Exfoliative Endometrial Cytology in Embryo Donor Cows-Comparison of Sampling Localizations for the Diagnosis of Subclinical Endometritis.

Subclinical endometritis has a major effect on the reproductive performance of dairy cows and also on the success of embryo collection. Thus it is important to minimize the number of false-negative diagnoses. In order to evaluate the question of whether or not a single cytobrush sample is representative of the whole endometrium, 53 German Holstein embryo donor cows in the northwest of Germany were examined via the cytobrush method at three different localizations of the uterus: the uterine body about 0.5 cm cranial of the cervical canal and both uterine horns about 1.5 cm cranial of the bifurcation. Although the mean percentage of polymorphonuclear neutrophils at the three locations is not significantly different (p = 0.64), the individual variations lead to the conclusion that more than one sample of the endometrium should be taken into account when diagnosing subclinical endometritis in embryo donor cows.

The use of semen evaluation and assisted reproduction in Spix's macaws in terms of species conservation.

The Spix's macaw (Cyanopsitta spixii) is the rarest parrot on earth. The remaining captive population consists of 79 individuals. Captive propagation is ongoing to increase the number of individuals for future reintroduction back into the wild. Unfortunately, from 2004 to 2012, only 33 chicks hatched from 331 eggs. Semen evaluation and assisted reproduction might help to overcome this problem. Therefore, a recently developed electro-stimulated semen collection technique was used in Spix's macaws. Semen collection was successful in 39 of 78 attempts in 10 out of 17 males. Examination of the semen included evaluation of volume, color, consistency, contaminations and pH, as well as determination of motility, viability, morphology, concentration, and total count of spermatozoa. The median volume of semen samples was 5.6 µl. On average, 34.7 ± 21.9% (median 30%) of the sperm were motile and 23.1 ± 22.1% (median 16.5%) were progressively motile. In addition to spermatozoa, round cells were detected in the samples. Median sperm concentration was 15,500/µl (range 500-97,500/µl) and median viability was 50% (range 5-87%). Morphological examination revealed in 26.5% normal spermatozoa, high numbers of malformations of the head (50.2%) and tail region (20.5%), with 29% of all sperm showing multiple abnormalities. Artificial insemination was performed in three females; two eggs laid after artificial insemination had spermatozoa present on the perivitelline layer, suggesting the possible success of the insemination technique. Although no fertilization could be demonstrated, these preliminary results are promising, as they indicate that assisted reproduction might be a tool for species conservation in the Spix's macaw.

Preparation and ultrastructure of spermatozoa from green poison frogs, Dendrobates auratus, following hormonal induced spermiation (Amphibia, Anura, Dendrobatidae).

Few ultrastructural studies have been performed on members of the Dendrobatidae, although such investigations can be useful for the understanding of reproductive patterns, as a diagnostic method for males in breeding programs for endangered amphibians and for phylogenetic analysis. The sperm ultrastructure of the Green Poison Frog, Dendrobates auratus, from Panama is described following induced spermiation in living animals. To date only testicular spermatozoa in other dendrobatid frogs have been analysed. Moreover, an electron microscopic preparation method (transmission and scanning electron microscopy) for dendrobatid sperm cells in low concentration is presented. Sperm cells from stimulated frogs (100 IU human chorionic gonadotropin, hCG, twice at an interval of 1h) were recovered via cloaca lavage using 600 microl isotonic phosphate-free amphibian saline (IPS). Centrifuged flushings (5 min, 173 x g) were deposited on microscopic slides. Adherent spermatozoa were treated with Karnovsky fixative (overnight, 4 degrees C). After postfixation (2h, 1% osmium tetroxide), samples were dehydrated in series of ascending acetones (30-100%). For transmission electron microscopy sperm cells were encapsulated using Epon and 1.5% 2,4,6-tris(dimethylaminomethyl)phenol (DMP 30). Ultrathin sections (70 nm) were cut and stained with uranyl acetate (30 min) and lead citrate (5 min). Sperm cells are filiform with a 21.1+/-2.7 microm long and arcuated head and a single tail (35.0+/-4.2 microm length). Their acrosomal complex is located at the anterior portion of the head and consists of the acrosomal vesicle which has low electron density, and the subjacent electron-dense subacrosomal cone. In transverse section, the nucleus is circular (1.9+/-0.2 microm diameter) and conical in longitudinal section. It is surrounded by several groups of mitochondria. The chromatin is highly condensed and electron-dense but shows numerous electron-lucent inclusions. A short midpiece has a mitochondrial collar with a proximal and a distal centriole. The latter gives rise to the axoneme which alone forms the flagellum. The sperm ultrastructure of D. auratus differs from that of other Dendrobatidae because of the absence of a nuclear space and the absence of the undulating membrane associated with an axial fibre. This tail conformation is found in the Ranoidea but not in the Bufonoidea. These results show that the spermatozoa of D. auratus are the first within the Dendrobatidae without accessory tail structures. Methods of using sperm samples from hormonal treated frogs for ultrastructural studies is not only reasonable to examine e.g. amphibian phylogeny without killing frogs threatened with extinction but allows investigations in the field of assisted reproduction and male fertility for example in conservation programs for endangered amphibians.