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• First-ever mitogenome assembly of golden jackal samples from Turkey and Thailand. • Phylogenetic analysis revealed distinct subspecific status of Indochinese jackal. • Larger cytochrome b dataset further confirms distinctness of Indochinese jackal. • Findings can inform golden jackal evolutionary history and conservation status. The golden jackal (Canis aureus) is a canid species found across southern Eurasia. Several subspecies of this animal have been genetically studied in regions such as Europe, the Middle East, and India. However, one subspecies that lacks current research is the Indochinese jackal (Canis aureus cruesemanni), which is primarily found in Southeast Asia. Using a genome skimming approach, we assembled the first complete mitochondrial genome for an Indochinese jackal from Thailand. To expand the number of available Canis aureus mitogenomes, we also assembled and sequenced the first complete mitochondrial genome of a golden jackal from Turkey, representing the C. a. moreotica subspecies. The mitogenomes contained 37 annotated genes and are 16,729 bps (C. a. cruesemanni) and 16,669 bps (C. a. moreotica) in length. Phylogenetic analysis with 26 additional canid mitogenomes and analyses of a cytochrome b gene-only data set together support the Indochinese jackal as a distinct and early-branching lineage among golden jackals, thereby supporting its recognition as a possible subspecies. These analyses also demonstrate that the golden jackal from Turkey is likely not a distinct lineage due to close genetic relationships with golden jackals from India and Israel. [ABSTRACT FROM AUTHOR]

David E. Wildt - An Inspiring Leader in the Conservation of Wild Species. [Extracted from the article]

Objective: To determine the effects of chilling on the organization and distribution of tubulin and chromosomes in rhesus monkey oocytes.Design: Comparative laboratory study.Setting: Academic research laboratory.Animal(s): Eight adult female rhesus monkeys (Macaca mulatta) aged 6-16 years.Intervention(s): A total of 171 oocytes retrieved from eight rhesus monkeys were separated into nine groups. One group of control oocytes was held at 37 degrees C during the experiment. Four groups of oocytes were rapidly cooled to 0 degrees C and held for 1, 5, 10, or 30 minutes and then fixed and stained. Four other groups of oocytes were cooled to 0 degrees C, held for 1, 5, 10, or 30 minutes, warmed and incubated at 37 degrees C for 60 minutes, and then fixed and stained.Main Outcome Measure(s): Organization of cytoskeleton and chromosomes.Result(s): Exposure of rhesus oocytes to 0 degrees C for only 1 minute resulted in complete depolymerization of tubulin. Incubation of chilled oocytes at 37 degrees C for 60 minutes caused partial restoration of tubulin, although most oocytes exhibited abnormal alignment of chromosomes and disorganized meiotic spindles.Conclusion(s): We conclude that rhesus monkey oocytes are extremely sensitive to chilling injury. Their successful cryopreservation may require rapid cooling to outpace this injury. [ABSTRACT FROM AUTHOR]

The Special Issue features one review and three research articles focusing on mechanisms regulating dog oocyte maturation and development. A review paper presented in this Special Issue summarizes the latest findings on germ cell development, the methods available for obtaining germ cells in vitro, and their potential applications in canid species. Germ cell development: Primordial germ cells are specialized cells that give rise to gametes (sperm and eggs). [Extracted from the article]

Simple Summary: The family Canidae, composed of dog-like species such as wolves, foxes, and jackals, demonstrates a significant variety in reproductive biology. In general, female canids experience very long periods of ovarian inactivity during the year; however, there are diverse patterns with regard to seasonality between species, as well as within an individual species depending on geographic region or housing status. Understanding of these differences is critical to the development of assisted reproductive technologies for canid conservation efforts. This review summarizes the current knowledge of canid reproduction, including reproductive cyclicity, seasonal breeding, male sperm traits, and recent developments in assisted reproductive technologies for canids. The reproductive physiology of canids is unique compared to other mammalian species. Specifically, the reproductive cycle of female canids is characterized by extended periods of proestrus and estrus followed by obligatory diestrus and protracted ovarian inactivity (anestrus). Although canid reproduction follows this general pattern, studies have shown variations in reproductive biology among species and geographic regions. Understanding of these differences is critical to the development of assisted reproductive technologies including estrus induction, gamete rescue, and embryo production techniques for canid conservation efforts. This review summarizes current knowledge of canid reproduction, including estrus cyclicity, seasonality, and seminal traits, with the emphasis on species diversity. The application of reproductive technologies in wild canid conservation will also be discussed. [ABSTRACT FROM AUTHOR]

The importance of the ovarian extracellular environment and tissue rigidity on follicle survival and development has gained attention in recent years. Our laboratory has anecdotally observed differences in the rigidity of domestic cat and dog ovarian cortical tissues, which have been postulated to underlie the differences in in vitro culture responses between the species, wherein cat ovarian tissues display higher survival in extended incubation. Here, the tensile strengths of cat and dog ovarian cortical tissues were compared via micropipette aspiration. The underlying collagen patterns, including fiber length, thickness, alignment, curvature, branch points and end points, and overall tissue lacunary and high-density matrix (HDM) were quantified via picrosirius red staining and TWOMBLI analysis. Finally, we explored the potential of MMP (−1 and −9) and TIMP1 supplementation in modulating tissue rigidity, collagen structure, and follicle activation in vitro. No differences in stiffness were observed between cat or dog cortical tissues, or pre- versus post-pubertal status. Cat ovarian collagen was characterized by an increased number of branch points, thinner fibers, and lower HDM compared with dog ovarian collagen, and cat tissues exposed to MMP9 in vitro displayed a reduced Young's modulus. Yet, MMP exposure had a minor impact on follicle development in vitro in either species. This study contributes to our growing understanding of the interactions among the physical properties of the ovarian microenvironment, collagen patterns, and follicle development in vitro. [ABSTRACT FROM AUTHOR]

The remarkable radiation of South American (SA) canids produced 10 extant species distributed across diverse habitats, including disparate forms such as the short-legged, hypercarnivorous bush dog and the long-legged, largely frugivorous maned wolf. Despite considerable research spanning nearly two centuries, many aspects of their evolutionary history remain unknown. Here, we analyzed 31 whole genomes encompassing all extant SA canid species to assess phylogenetic relationships, interspecific hybridization, historical demography, current genetic diversity, and the molecular bases of adaptations in the bush dog and maned wolf. We found that SA canids originated from a single ancestor that colonized South America 3.9 to 3.5 Mya, followed by diversification east of the Andes and then a single colonization event and radiation of Lycalopex species west of the Andes. We detected extensive historical gene flow between recently diverged lineages and observed distinct patterns of genomic diversity and demographic history in SA canids, likely induced by past climatic cycles compounded by human-induced population declines. Genome-wide scans of selection showed that disparate limb proportions in the bush dog and maned wolf may derive from mutations in genes regulating chondrocyte proliferation and enlargement. Further, frugivory in the maned wolf may have been enabled by variants in genes associated with energy intake from short-chain fatty acids. In contrast, unique genetic variants detected in the bush dog may underlie interdigital webbing and dental adaptations for hypercarnivory. Our analyses shed light on the evolution of a unique carnivoran radiation and how it was shaped by South American topography and climate change. [ABSTRACT FROM AUTHOR]

The maned wolf (Chrysocyon brachyurus) is an induced ovulator. Though the mechanism of ovulation induction remains unknown, it is suspected to be urinary chemical signals excreted by males. This study assessed volatile organic compounds (VOCs) in weekly urine samples across 5 months from 13 maned wolves (6 intact males, 1 neutered male, 6 females) with the goal of identifying VOCs that are differentially expressed across sex, reproductive status, and pairing status. Solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) were used to extract and separate VOCs that were identified via spectral matching with authentic standards, with spectral libraries, or with new software that further matches molecular fragment structures with mass spectral peaks. Two VOCs were present across all 317 urine samples: 2,5-dimethyl pyrazine and 2-methyl-6-(1-propenyl)-pyrazine. Fifteen VOCs differed significantly (Adj. P < 0.001 and |log2 fold change| >2.0) between intact males and females. Using partial least squares-discriminant analysis, the compounds with the highest importance to the sex classification were delta-decalactone, delta-dodecalactone, and bis(prenyl) sulfide. Sixty-two VOCs differed between intact males and the neutered male. Important classifier compounds were 3-ethyl 2,5-dimethyl pyrazine, 2-methyl-6-(1-propenyl)-pyrazine, and tetrahydro-2-isopentyl-5-propyl furan. Several VOCs established as important here have been implicated in reproductive communication in other mammals. This study is the most robust examination of differential expression in the maned wolf thus far and provides the most comprehensive analysis of maned wolf urinary VOCs to date, increasing the sample size substantially over previous chemical communication studies in this species. New data analysis software allowed for the identification of compounds in the hormone-producing mevalonate pathway which were previously unreported in maned wolf urine. Several putative semiochemicals were identified as good candidates for behavioral bioassays to determine their role in maned wolf reproduction, and specifically in ovulation induction. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) contain multiple factors that regulate cell and tissue function. However, understanding of their influence on gametes, including communication with the oocyte, remains limited. In the present study, we characterized the proteome of domestic cat (Felis catus) follicular fluid EVs (ffEV). To determine the influence of follicular fluid EVs on gamete cryosurvival and the ability to undergo in vitro maturation, cat oocytes were vitrified using the Cryotop method in the presence or absence of ffEV. Vitrified oocytes were thawed with or without ffEVs, assessed for survival, in vitro cultured for 26 hours and then evaluated for viability and meiotic status. Cat ffEVs had an average size of 129.3 ± 61.7 nm (mean ± SD) and characteristic doughnut shaped circular vesicles in transmission electron microscopy. Proteomic analyses of the ffEVs identified a total of 674 protein groups out of 1,974 proteins, which were classified as being involved in regulation of oxidative phosphorylation, extracellular matrix formation, oocyte meiosis, cholesterol metabolism, glycolysis/gluconeogenesis, and MAPK, PI3K-AKT, HIPPO and calcium signaling pathways. Furthermore, several chaperone proteins associated with the responses to osmotic and thermal stresses were also identified. There were no differences in the oocyte survival among fresh and vitrified oocyte; however, the addition of ffEVs to vitrification and/or thawing media enhanced the ability of frozen-thawed oocytes to resume meiosis. In summary, this study is the first to characterize protein content of cat ffEVs and their potential roles in sustaining meiotic competence of cryopreserved oocytes. [ABSTRACT FROM AUTHOR]

Ovarian cancer is the fifth cause of cancer-related mortality in women, with an expected 5-year survival rate of only 47%. High-grade serous carcinoma (HGSC), an epithelial cancer phenotype, is the most common malignant ovarian cancer. It is known that the precursors of HGSC originate from secretory epithelial cells within the Fallopian tube, which first develops as serous tubal intraepithelial carcinoma (STIC). Here, we used gene editing by CRISPR-Cas9 to knock out the oncogene p53 in dog oviductal epithelia cultured in a dynamic microfluidic chip to create an in vitro model that recapitulated human STIC. Similar to human STIC, the gene-edited oviduct-on-a-chip, exhibited loss of cell polarization and had reduced ciliation, increased cell atypia and proliferation, with multilayered epithelium, increased Ki67, PAX8 and Myc and decreased PTEN and RB1 mRNA expression. This study provides a biomimetic in vitro model to study STIC progression and to identify potential biomarkers for early detection of HGSC. [ABSTRACT FROM AUTHOR]

Ovarian cancer is the fifth cause of cancer-related mortality in women, with an expected 5-year survival rate of only 47%. High-grade serous carcinoma (HGSC), an epithelial cancer phenotype, is the most common malignant ovarian cancer. It is known that the precursors of HGSC originate from secretory epithelial cells within the Fallopian tube, which first develops as serous tubal intraepithelial carcinoma (STIC). Here, we used gene editing by CRISPR-Cas9 to knock out the oncogene p53 in dog oviductal epithelia cultured in a dynamic microfluidic chip to create an in vitro model that recapitulated human STIC. Similar to human STIC, the gene-edited oviduct-on-a-chip, exhibited loss of cell polarization and had reduced ciliation, increased cell atypia and proliferation, with multilayered epithelium, increased Ki67, PAX8 and Myc and decreased PTEN and RB1 mRNA expression. This study provides a biomimetic in vitro model to study STIC progression and to identify potential biomarkers for early detection of HGSC. [ABSTRACT FROM AUTHOR]

The dhole (Cuon alpinus) is one of the least frequent studied endangered canid species and many aspects of ecological knowledge about this species are lacking. The objectives of this study were to investigate the spatial movement of dholes, prey abundance, prey selection, and prey overlaps with other large carnivorous species in the Huai Kha Khaeng Wildlife Sanctuary, Thailand, during November, 2017 and October, 2018. Two adult female dholes were captured and fitted with GPS collars. Twenty camera trap sets were systematically used to survey the area. Scat collection was conducted along forest roads and trails. The home range sizes and activity radii of the two dholes were 3,151.63 ha. and 1,442.84 m, and 33.39 ha and 331.56 m, respectively. The sambar deer (Rusa unicolor) was the most abundant prey species (30.93%). However, dhole fecal analysis showed that the monitored dholes preferred red muntjac (Muntiacus muntjak) (57.1%). There was a high degree of prey overlap between dholes and leopards (98%), indicating very high prey competition. The dholes in this study represent movement patterns in richly abundant prey habitats, but with the presence of other predators that can affect prey selection and movement patterns of the dhole in the area. [ABSTRACT FROM AUTHOR]

Purpose: Increasing intracellular energy storage by chemically activating adenosine monophosphate–activated protein kinase (AMPKα) prior to sperm cryopreservation may improve post-thawed sperm function. Using the domestic cat as a biomedical model, the objectives were to (1) confirm the expression of AMPKα and its regulatory kinases in epididymal spermatozoa and (2) assess the influence of AMPK activator, 5′-aminoimidasole-4-carboxamide-1-β-d-ribofuranoside (AICAR) on epididymal sperm function before and after cryopreservation. Methods: In study I, sperm samples of different qualities were obtained from cauda epididymides of domestic cats and evaluated for AMPKα expression. In study II, epididymal spermatozoa were equilibrated for either 30 or 60 min in the presence of 0 (control), 0.5, 2.0, and 5.0 mM AICAR and sperm functions were assessed before and after cryopreservation. In study III, epididymal spermatozoa were treated as in study II and evaluated for AMPKα signaling protein expressions (phospho-AMPKα Thr172 and GLUT1) as well as ATP levels. Results: AMPKα protein expression was higher in high-motility vs poor-motility samples. Thirty-minute equilibration with 0.5 mM AICAR improved motion characteristics and fertilizing ability of cryopreserved sperm to the control. Increased expressions of phospho-AMPKα Thr172 and GLUT1 as well as intracellular ATP level were confirmed in sperm samples equilibrated with 0.5 or 2.0 mM AICAR for 30 min. Conclusions: Presence and role of AMPKα protein in cat regulating sperm function were demonstrated before and after cryopreservation. Findings could be used to potentially enhance cryopreserved sperm function in sub-fertile men. [ABSTRACT FROM AUTHOR]

Retinoic acid (RA) facilitates tissue morphogenesis by regulating matrix matalloproteinase (MMPs) expression. Our objective was to examine the influence of RA on in vitro development of follicles enclosed within domestic cat ovarian tissues. Ovarian cortices from 9 prepubertal and 13 adult cats were incubated for 7 d in medium containing 0 (control), 1 or 5 μM RA and then analyzed for viability. Cortices from additional three animals of each age group were cultured in the same condition and follicle morphology, stage and size were histologically evaluated. In a separate study, cortices from 14 donors (7 prepubertal; 7 adult cats) were incubated in 0 or 5 μM RA for 7 d and assessed for (1) MMP1, 2, 3, 7, 9 and TIMP1 expression by qPCR and (2) protein expression of MMP9 by immunohistochemistry. Donor age did not influence follicle response to RA. Collective data from both age groups revealed that percentages of primordial follicles in 5 μM RA treatment were lower (P < 0.05; 40.5 ± 4.5%) than in fresh cortices (66.7 ± 5.3%) or controls (60.1 ± 4.0%) with 1 μM-RA treatment producing intermediate (56.3 ± 4.0%) results. Proportion of primary follicles in 5 μM RA (21.7 ± 3.3%) was higher than in fresh cortices (4.9 ± 2.9%) and controls (9.0 ± 2.8%) with 1 μM-RA treatment producing an intermediate value (13.8 ± 2.0%). Furthermore, proportion of secondary follicles increased after 7 d in the presence of 5 μM RA (9.5 ± 2.7%) compared to other groups (fresh, 1.9 ± 0.8%; control, 2.6 ± 1.1%; 1 μM RA, 2.5 ± 0.2%). MMP9 transcript and protein were upregulated, whereas MMP7 mRNA was suppressed by 5 μM-RA treatment compared to fresh counterparts. RA did not impact MMP1, 2, 3, 13 or TIMP1 expression. In summary, RA activated cat primordial follicle growth likely via a mechanism related to upregulation of MMP9 and down-regulation of MMP7 transcripts. [ABSTRACT FROM AUTHOR]

Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies—an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4–5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species. [ABSTRACT FROM AUTHOR]

The behaviour of wild cats is poorly understood. Using camera-trapping, we quantifi ed temporal overlap among seven species of Asian wild cats, including tiger Panthera tigris and leopard Panthera pardus. Based on time stamp data from 780 camera-traps and 24 study sites from 14 protected areas across Thailand, we assessed terrestrial activity patterns and temporal overlap in habitat use. For quantifying overlap, we used a coeffi cient estimator that allows for calculation of confi dence intervals. Our study provided insight into temporal interactions among species of wild cats, particularly between small cats and their larger cat relatives. We found temporal habitat segregation in several small cats with some species being strongly nocturnal (=85% records between 1800 and 0600 hours - leopard cat Prionailurus bengalensis), mostly (>50%) nocturnal (clouded leopard Neofelis nebulosa), mostly diurnal (>50% records between 0600 and 1800 hours - Asiatic golden cat Catopuma temminckii), or strongly (=85%) diurnal (marbled cat Pardofelis marmorata). We found high temporal overlap (Δ1 = 0.80) between leopard cat and clouded leopard (95% CI = 0.77-0.91), Asiatic golden cat and leopard (95% CI = 0.69-0.87), Asiatic golden cat and tiger (95% CI = 0.72-0.90), and clouded leopard and tiger (95% CI = 0.69-0.85). Our research demonstrates that temporal habitat or niche segregation may be an important process in maintaining the functioning of diverse predator guilds in tropical forests. We developed several avoidance or overlap hypotheses that can explain the patterns observed in our study and that should be further tested. [ABSTRACT FROM AUTHOR]

Background: One of the major obstacles in using artificial insemination to manage genetics of elephant population in captivity is the large variations in semen quality among ejaculates within the same and among individuals. The objectives of this study were to determine the influences of (1) age (2) seasonality (3) and circulating testosterone (SrTest), triiodothyronine (SrT3) and tetraiodothyronine (SrT4), as well as seminal (4) testosterone (SpTest), zinc (SpZn) and protein (SpTP) on semen quality in the Asian elephant Methods: Analyses, including motility, viability and morphology were performed in semen samples collected twice monthly from 13 elephant bulls (age range, 10-to 72-years) by manual stimulation between July 2004 and June 2005. Serum samples obtained monthly were assessed for SrTest, SrT3, SrT4, and seminal plasma samples were evaluated for, SpTest, SpZn and SpTP. Results: The highest semen quality was observed at age 23 to 43 years. Percentages of progressive motility and viable sperm were lowest at age 51 to 70 years (P < 0.05); on the other hand, sperm concentration was lowest at age 10 to 19 years (P < 0.05). Percentage of sperm with normal morphology was highest at age 23 to 43 years. The levels of SrT3, SrTest, SpTest and SpZn were lowest at age 51 to 70 years, whereas SrT4 was lowest at age 23 to 43 years. Seasonality significantly affected semen characteristics in which percentage of viable sperm and cell concentration were highest during rainy season and lowest during summer months (P < 0.05). However, percentage of sperm with normal morphology was highest in summer and lowest in rainy season (P < 0.05). Seasonality significantly influenced SrTest with elevated concentrations observed in rainy season and winter (P < 0.05). Conclusion: This study indicates that age and seasonality had influence on semen characteristics in the Asian elephant. The knowledge obtained in this study will improve our understanding of the reproductive biology of this species. [ABSTRACT FROM AUTHOR]

Simple Summary: Nowadays, the scientific literature lists several parameters and formulas for the prediction of parturition date in dogs, and their accuracy is affected by various factors: pregnancy phase, litter and also maternal size. The selection of the most appropriate parameter set for ultrasound assessment of gestational age and fetal organ development is essential to predict parturition date with high accuracy, but the high number of factors influencing these parameters challenges their clinical interpretation. In this review, the variables of interest are grouped as (i) maternal parameters, (ii) fetal parameters, (iii) ultrasonographic assessment of maternal and fetal heart rate and blood flow, and (iv) markers of fetal maturity. The aim of this review is to analyze the parameters that predict parturition date in the canine species, especially their accuracy, and finally propose to consider the evaluation of fetal maturity as mark of the readiness for parturition combined with the other most studied parameters. An accurate parturition timing is of key importance for breeders and veterinarians in order to give professional assistance to parturition in dogs. However, pregnancy length calculated from the breeding date has a wide variability. Different parameters and formulas have been described and calculated, as well as their accuracy which is affected by various factors: stage of pregnancy, litter and maternal size. Therefore, the selection of the most appropriate parameter panel poses the challenge of weighing their influences and impact on the overall accuracy. The aim of this review is to analyze the parameters useful for parturition timing, especially their accuracy, and to propose the addition of fetal maturity and criteria for its evaluation to detect readiness for parturition. Parameters, as described in literature, are classified as: (i) maternal parameters, (ii) fetal parameters, (iii) ultrasonographic assessment of maternal and fetal heart rate and blood flow, (iv) parameters indicating fetal maturity. A focus on recently described parameters—such as fetal gastrointestinal motility and fetal lung development detected by quantitative ultrasound—is reported. Currently, the most accurate way to predict parturition day is represented by a prepartum progesterone drop, but the identification of a panel of ultrasonographic parameters combining their significance and their accuracy throughout pregnancy is still needed. [ABSTRACT FROM AUTHOR]

Simple Summary: The article presents the results of the clinical evaluation and umbilical cord blood analysis obtained from newborn pups delivered by the elective Caesarean section. In human medicine both the umbilical cord blood and Apgar score were proved to provide valuable information on neonatal status. In veterinary medicine very few reports concerning the relation between the clinical neonatal assessment (Apgar score) and umbilical cord blood parameters exist. All puppies show mild respiratory acidemia regardless the Apgar scores result. The lactates were not elevated whereas all the puppies with low Apgar sores had higher glucose and mortality rates. Only pups with low initial Apgar scores were at risk of death within first 24 h. Adaptation to the extra-uterine life is crucial and any practical improvement in neonatal diagnostics and care would be beneficial for newborn puppy survival. The article presents the results of the clinical evaluation (Apgar scores, AS) and umbilical cord blood gas analysis (UCBGA) obtained from clamped umbilical cords of newborn pups delivered by the elective Caesarean section. The study was planned as a controlled clinical study, the newborns were allocated into one of the groups, I—critical neonates (severe distress, AS ≤ 3), II—weak neonates (moderate distress, AS 4–6) and III—healthy neonates (no distress, AS ≥ 7). The following parameters were evaluated: pH (pH units), carbon dioxide partial pressure (pCO2; mmHg), oxygen partial pressure (pO2; mmHg), actual bicarbonate (cHCO3−; mmol/L), total carbon dioxide (cTCO2; mmol/L), base excess of extracellular fluid (BE(ecf); mmol/L), base excess of blood (BE(b); mmol/L), oxygen saturation (csO2; %), lactate (Lac; mg/dL), hematocrit (Hct; %PCV), hemoglobin (cHgb; g/dL), glucose (Glu; mg/dL), ions (Na, K, Ca, Cl). The majority of puppies had low AS at birth (AS 4–6 in 38.1% and AS ≤ 3 in 57.1% of the neonates), but most of them (85.7%) improved by the 20th min. reaching AS of 7 and more. Moreover, puppies with lower AS (≤ 3) were at higher risk of death within the first 24h (20.8% did not survive). The positive correlation was found between Apgar score measured at 0 min and pH (r = 0.46, p = 0.01), and between Apgar score (at 0 min) and base excess in whole blood measured [BE(b)] r = 0.36, p = 0.03). Whereas, a negative correlation was detected between Apgar score at 0 and 5th min and glycemia (r = −0.42, p = 0.05, r = −0.34, p = 0.02 respectively. Overall, the puppies with higher glucose levels had lower Apgar scores and were at higher risk of death. Furthermore, in our study, the newborn puppies had mild acidemia with elevated pCO2 levels and the HCO3 at the lower range of normal limits, suggesting the mixed component in the acidemic state. Adaptation to extra-uterine life is crucial and any practical improvement in neonatal diagnostics and care would be beneficial for newborn puppy survival. [ABSTRACT FROM AUTHOR]

Simple Summary: The progression of germ cells is a remarkable event that allows biological discovery in the differ-entiation process during in vivo and in vitro development. This is crucial for understanding one toward making oogenesis and spermatogenesis. Companion animals, such as canine, could offer new animal models for experimental and clinical testing for translation to human models. In this review, we describe the latest and more relevant findings on germ cell development. In addition, we showed the methods available for obtaining germ cells in vitro and the characterization of pri-mordial germ cells and spermatogonial stem cells. However, it is necessary to further conduct basic research in canine to clarify the beginning of germ cell development. Primordial germ cells (PGCs) have been described as precursors of gametes and provide a connection within generations, passing on the genome to the next generation. Failures in the formation of gametes/germ cells can compromise the maintenance and conservation of species. Most of the studies with PGCs have been carried out in mice, but this species is not always the best study model when transposing this knowledge to humans. Domestic animals, such as canines (canine), have become a valuable translational research model for stem cells and therapy. Furthermore, the study of canine germ cells opens new avenues for veterinary reproduction. In this review, the objective is to provide a comprehensive overview of the current knowledge on canine germ cells. The aspects of canine development and germ cells have been discussed since the origin, specifications, and development of spermatogonial canine were first discussed. Additionally, we discussed and explored some in vitro aspects of canine reproduction with germ cells, such as embryonic germ cells and spermatogonial stem cells. [ABSTRACT FROM AUTHOR]

Simple Summary: As the dog shows unique and peculiar reproductive characteristics, assisted reproductive techniques such as in vitro maturation and in vitro fertilization have not been well-established compared with those of other mammals. Our recent work demonstrated the interplay between in vitro oviductal cell-derived extracellular vesicles (OC-EVs) and cumulus-oocyte complexes in dogs. Here, we provided for the first time a comprehensive proteomic analysis of OC-EVs. A total of 398 proteins were identified in all OC-EVs samples. A functional enrichment analysis indicated that these core proteins were involved in the key cellular metabolic process related to oocyte maturation and embryonic development. The current comprehensive description of the canine OC-EVs proteome would provide a fundamental resource for further understanding canine reproductive physiology, the interaction of sperms with female counterparts during fertilization, early pregnancy, and establishing an efficient system of in vitro embryo production. Dogs (Canis lupus familiaris) have unique and peculiar reproductive characteristics. While the interplay between in vitro oviductal cell-derived extracellular vesicles (OC-EVs) and cumulus-oocyte complexes in dogs has begun to be elucidated, no study has yet provided extensive information on the biological content and physiological function of OC-EVs and their role in canine oocyte development. Here, we aimed to provide the first comprehensive proteomic analysis of OC-EVs. We identified 398 proteins as present in all OC-EVs samples. The functional enrichment analysis using Gene Ontology terms and an Ingenuity Pathway Analysis revealed that the identified proteins were involved in several cellular metabolic processes, including translation, synthesis, expression, and protein metabolism. Notably, the proteins were also involved in critical canonical pathways with essential functions in oocyte and embryo development, such as ERK/MAPK, EIF2, PI3K/AKT, and mTOR signaling. These data would be an important resource for studying canine reproductive physiology and establishing a successful in vitro embryo production system in dogs. [ABSTRACT FROM AUTHOR]

Simple Summary: Shear wave elastography (SWE) is a new diagnostic method allowing for a non-invasive differential diagnosis between benign and malignant mammary tumors. Mammary tumors in dogs are often hormone-dependent, and a surgical approach is currently the most recommended method for the treatment of mammary neoplasia. However, preoperative, pharmacological therapy eliminating hormonal stimulation can sometimes be indicated to reduce the size of the tumor and facilitate subsequent surgery. The aim of this study was to evaluate the influence of the reduction in the benign tumor size observed after treatment with the use of aglepristone on the shear wave elastography results, obtained before and after therapy. We noticed that a fast and significant reduction in tumor size, as a consequence of preoperative pharmacological treatment, did not influence the diagnosis performed using the SWE technique. These results seem to indicate the accuracy and usefulness of this new diagnostic method, and confirm the hypothesis that tumor tissue density is a valuable parameter when determining the benignancy of mammary tumors in dogs. Shear wave elastography (SWE) can be useful to discriminate between malignant and benign mammary tumors. In dogs with elevated progesterone levels compared to the baseline and fast-growing tumors, treatment with the use of aglepristone allows for tumor size reduction, which facilitates surgery. This study aimed to evaluate the influence of the preoperative treatment of benign mammary tumors (BMTs), performed with the use of aglepristone, on the density of the tumor tissue measured by SWE. Twelve female dogs with diagnosed BMTs and increased levels of progesterone were treated with aglepristone (Alizine, Virbac, France) at 10 mg/kg s.c. (Subcutaneous injection). twice, with a 24 h interval. The density of the tumor was evaluated by SWE before and after the treatment. The type of tumor was evaluated by fine needle aspiration cytology before treatment, and a histopathological examination was made after surgical removal, performed after the aglepristone treatment. In all the cases, a significant reduction in the mammary tumor's size was observed following treatment, with no influence on the density of the tumor's tissue measured by SWE. Similar studies on malignant mammary tumors are warranted to verify if in these cases, density will also be a constant parameter that is not dependent on the tumor size. [ABSTRACT FROM AUTHOR]

Artificial insemination (AI) is a valuable tool for ex situ wildlife conservation, allowing the re-infusion and dissemination of genetic material, even after death of the donor. However, the application of AI to species conservation is still limited, due mainly to the poor survival of cryopreserved sperm. Recent work demonstrated that oviductal extracellular vesicles (oEVs) improved cat sperm motility and reduced premature acrosomal exocytosis. Here, we build on these findings by describing the protein content of dog and cat oEVs and investigating whether the incubation of cryopreserved red wolf and cheetah sperm with oEVs during thawing improves sperm function. Both red wolf and cheetah sperm thawed with dog and cat oEVs, respectively, had more intact acrosomes than the non-EV controls. Moreover, red wolf sperm thawed in the presence of dog oEVs better maintained sperm motility over time (>15%) though such an improvement was not observed in cheetah sperm. Our work demonstrates that dog and cat oEVs carry proteins important for sperm function and improve post-thaw motility and/or acrosome integrity of red wolf and cheetah sperm in vitro. The findings show how oEVs can be a valuable tool for improving the success of AI with cryopreserved sperm in threatened species. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]