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Understanding the reproductive biology of giant pandas is crucial for their breeding success and conservation. Pregnancy monitoring, however, is challenging due to delayed implantation and obligatory pseudopregnancy, which limits the effectiveness of traditional immunoassays (IA). To remedy this, we combined polar metabolomics and steroidomics to enable a comprehensive view of the urinary molecular composition across six different reproductive phases spanning six pregnant and seven pseudopregnant cycles. Statistical comparisons revealed 696 discriminative features, including 174 features in the early luteal stages, well before the current pregnancy diagnostic window. Pregnant and pseudopregnant cycles showed differences in amino acid, energy, and steroid metabolism before and after CL reactivation, with androgen levels being significantly elevated in pregnant females specifically, suggesting a role in embryo implantation. Interestingly, we detected only one existing IA target metabolite, but identified other discriminative metabolites that may underlie IA signal detection. Finally, we demonstrated that classification models comprising biomarker panels may improve (early) pregnancy diagnosis with accuracies ranging from 0.763 to 1.000 across reproductive phases. These findings offer possibilities for assigning new biomarkers and optimizing IA target selection, thereby enhancing pregnancy monitoring sensitivity and reliability while improving our understanding of giant panda reproductive biology to support conservation efforts.

Background There is a dearth of literature on pseudopregnancy in the bitch, with only a few treatment-based studies published since the 1990s. Pseudopregnancy may be under-recognised in bitches and may account for a proportion of behavioural cases seen in veterinary practices including aggression. Little is known about commonly used treatments for overtly pseudopregnant bitches and it is possible that current regimes may not be prescribed for a sufficient duration to control any clinical signs including, physical and behavioural changes. To investigate current trends in diagnosis and treatment of canine pseudopregnancy, a postal survey was sent to 2000 randomly selected veterinary surgeons in UK veterinary practices. The questionnaire queried how often vets recognise cases of pseudopregnancy in spayed and entire bitches, which physical or behavioural signs are commonly recognised for diagnosis, and which management or treatment protocols are used. Results The response rate was 19.8% (397/2000). Ninety-six percent of veterinary surgeons reported seeing pseudopregnant bitches showing behavioural changes without any physical changes within the last 12 months. Of those behavioural changes, collecting and mothering objects was the most frequently reported behavioural sign (96%). Ninety-seven percent of vets had seen aggression in pseudopregnant bitches. Nevertheless, only 52% of vets routinely asked owners about behavioural changes during consultations. Forty-nine percent of respondents reported seeing pseudopregnancy in spayed bitches. The most commonly reported physical sign was enlarged mammary glands and/or milk production (89%). Treatment options varied (surgical, medical or none) and depended on duration and severity of physical and behavioural signs, owners’ preference, cost, concurrent disease, drug availability and previous history. Conclusions This is the largest epidemiological study of canine pseudopregnancy in the UK. The prevalence and severity of clinical signs in dogs with pseudopregnancy are variable and possibly under-estimated. Dogs with overt pseudopregnancy experience diverse physical and behavioural changes and information on standard treatment protocols are lacking. Although, progress on our understanding of diagnosis and treatment of pseudopregnancy in spayed and entire bitches has been made, further studies are warranted.

Kisspeptin (KiSS) and its related receptors (KiSS1R) have a critical role in the reproduction of mammals. The KiSS/KiSS1R system is expressed in numerous reproductive organs including the ovary. Here, we studied the expression of the KiSS/KiSS1R system and its functional role in rabbit corpora lutea (CL) at days 4 (early-), 9 (mid-), and 13 (late-stage) of pseudopregnancy. In vitro progesterone, prostaglandin (PG) F2α (PGF2α) and E2 (PGE2) productions and prostaglandin-endoperoxide synthase 1 (PTGS1) and 2 (PTGS2) activities were evaluated. Immune reactivity (IR) for KiSS and KiSS1R were detected in luteal cells at nuclear and cytoplasmic level at all luteal stage for KiSS and only at early- and mid-stage for KiSS1R; IR decreased from early- to later stages of pseudopregnancy. The KiSS-10 augmented progesterone and PGE2 and diminished PGF2α secretions by early- and mid-CL; KiSS-10 reduced PTGS2 activity at early- and mid-stages, but did not affect PTGS1 at any luteal stages. The antagonist KiSS-234 counteracted all KiSS-10 effects. This study shows that the KiSS/KiSS1R system is expressed in CL of pseudopregnant rabbits and exerts a luteotropic action by down-regulating PTGS2, which decreases PGF2α and increases PGE2 and progesterone.

For the preparation of embryo transfer recipients, surgically vasectomized mice are commonly used, generated by procedures associated with pain and discomfort. Sterile transgenic strains provide a nonsurgical replacement, but their maintenance requires breeding and genotyping procedures. We have previously reported the use of naturally sterile STUSB6F1 hybrids for the production of embryo transfer recipients and found the behavior of these recipients to be indistinguishable from those generated by vasectomized males. The method provides two substantial 3R impacts: refinement (when compared with surgical vasectomy) and reduction in breeding procedures (compared with sterile transgenic lines). Despite initial promise, the 3Rs impact of this innovation was limited by difficulties in breeding the parental STUS/Fore strain, which precluded the wider distribution of the sterile hybrid. The value of a 3R initiative is only as good as the uptake in the community. Here we, thus, select a different naturally sterile hybrid, generated from strains that are widely available: the B6SPRTF1 hybrid between C57BL/6J and Mus spretus . We first confirmed its sterility by sperm counting and testes weight and then trialed the recovery of cryopreserved embryos and germplasm within three UK facilities. Distribution of sperm for the generation of these hybrids by in vitro fertilization was found to be the most robust distribution method and avoided the need to maintain a live M. spretus colony. We then tested the suitability of B6SPRTF1 sterile hybrids for the generation of embryo transfer recipients at these same three UK facilities and found the hybrids to be suitable when compared with surgical vasectomized mice and a sterile transgenic strain. In conclusion, the potential 3Rs impact of this method was confirmed by the ease of distribution and the utility of sterile B6SPRTF1 hybrids at independent production facilities. Preece et al. show that B6SPRTF1 hybrid males between C57BL/6J and Mus spretus are suitable for the generation of pseudopregnant female mice for embryo transfer. By providing an alternative to vasectomized males, the method shows clear 3R benefits.

The efficiency of producing timed pregnant or pseudopregnant mice can be increased by identifying those in proestrus or estrus. Visual observation of the vagina is the quickest method, requires no special equipment, and is best used when only proestrus or estrus stages need to be identified. Strain to strain differences, especially in coat color can make it difficult to determine the stage of the estrous cycle accurately by visual observation. Presented here are a series of images of the vaginal opening at each stage of the estrous cycle for 3 mouse strains of different coat colors: black (C57BL/6J), agouti (CByB6F1/J) and albino (BALB/cByJ). When all 4 stages (proestrus, estrus, metestrus, and diestrus) need to be identified, vaginal cytology is regarded as the most accurate method. An identification tool is presented to aid the user in determining the stage of estrous when using vaginal cytology. These images and descriptions are an excellent resource for learning how to determine the stage of the estrous cycle by visual observation or vaginal cytology.

Vasectomized mice play a key role in the production of transgenic mice. However, vasectomy can cause great physical and psychological suffering to mice. Therefore, there is an urgent need to find a suitable replacement for vasectomized mice in the production of transgenic mice. In this study, we generated C57BL/6J mice ( Piwil1 D633A-INS99, Piwil1 mt/mt ) with a 99-base insertion in the Miwi ( Piwil1 ) gene using CRISPR/Cas9 technology and showed that Piwil1 mt/+ heterozygous mice were normally fertile and that homozygous Piwil1 mt/mt males were sterile and females were fertile. Transplantation of normal fertilized eggs into wild pseudopregnant females following mating with Piwil1 mt/mt males produced no Piwil1 mt/mt genotype offspring, and the number of offspring did not differ significantly from that of pseudopregnant mice following mating and breeding with ligated males. The CRISPR‒Cas9 system is available for generating Miwi -modified mice, and provides a powerful resource to replace ligated males in assisted reproduction research.

Spatially ordered embryo-like structures self-assembled from blastocyst-derived stem cells can be generated to mimic embryogenesis in vitro. However, the assembly system and developmental potential of such structures needs to be further studied. Here, we devise a nonadherent-suspension-shaking system to generate self-assembled embryo-like structures (ETX-embryoids) using mouse embryonic, trophoblast and extra-embryonic endoderm stem cells. When cultured together, the three cell types aggregate and sort into lineage-specific compartments. Signaling among these compartments results in molecular and morphogenic events that closely mimic those observed in wild-type embryos. These ETX-embryoids exhibit lumenogenesis, asymmetric patterns of gene expression for markers of mesoderm and primordial germ cell precursors, and formation of anterior visceral endoderm-like tissues. After transplantation into the pseudopregnant mouse uterus, ETX-embryoids efficiently initiate implantation and trigger the formation of decidual tissues. The ability of the three cell types to self-assemble into an embryo-like structure in vitro provides a powerful model system for studying embryogenesis.

IntroductionDelusion of pregnancy has been described as a false and fixed belief of being pregnant despite factual evidence to the contrary. Pseudocyesis is a condition in which the patient has all signs and symptoms of pregnancy except for the confirmation of the presence of a fetus. There has been described symptoms as abdominal distention, cessation of menses, morning sickness, etc.ObjectivesThe aim of this work was to distinguish between pseudocyesis and pseudopregnancy. This case presents a single 49-year-old woman, who developed the delusion of being pregnant after months of lonely confinement during Covid-19 quarantine.MethodsShe had missed her period for 10 months. She was convinced that she could feel the fetal movements. Her thought content revealed delusion of persecution, reference, and delusion of being pregnant. She did not reveal any hallucinations. Blood tests and brain imaging revealed no abnormalities. □The treatment was started with Paliperidone 100mg/month, and clonazepam 2mg/d.ResultsThe patient showed a substantial improvement within 10 weeks of treatment. Pseudocyesis and delusional pregnancy have been rarely described on scientific bibliography. A distinction has been demonstrated based on the consideration of the associated psychotic features that might be present in delusions of pregnancy, which were described in the current case. On the other hand, pseudocyesis clinical presentation is centered on the false signs and symptoms of pregnancy.ConclusionsAntipsychotics played a key role in the delusion of pregnancy. Psychodynamic and supportive psychotherapy could play a pivotal role in the management of pseudocyesis.DisclosureNo significant relationships.