Evaluation of short‐term safety of ultrasound‐guided foetal fluid sampling in the dog (Canis lupus familiaris)

Background: In humans, analysis of amniotic fluid is widely used for diagnostic and prognostic purposes. Amniocentesis has scarcely been used in veterinary medicine to date, despite a tremendous potential for clinical and research applications in dogs. Our study aimed to establish a safe method for foetal fluid sampling in female dogs. Methods: Two transabdominal ultrasound-guided methods were assessed: the "free hand" and the needle-guided bracket sampling. In addition, through a subsequent routinely scheduled ovariohysterectomy, fluid was directly collected. Samples from 98 conceptuses were collected at day 46.7 +/- 7.5 of pregnancy. Results: The amount of fluid retrieved varied between 0.5 and 5.0 ml per collection. Macroscopic examination of the uterus and conceptuses identified 53% of the puncture sites. Neither fluid leakage nor foetal injury was detected, and six hematomas (5.8%) were visible. Ultrasound-guided foetal fluid collection was found to be potentially safe, and it can be performed by using either transabdominal method. Conclusion: Foetal fluid collection is possible with relative ease and low short-term risk, and may open paths for diagnostic, therapeutic and research purposes in dogs. The procedure can provide new insights into prenatal clinical medicine, including diagnostics of foetal deaths, early identification of heritable diseases and so on

Faecal steroid analysis in two captive female cheetahs

Introduction and aim. Cheetahs (Acinonyx jubatus), as most wild felid species, is an induced ovulator and reproduces poorly in captivity, a problem attributed to inappropriate husbandry (1). Monitoring endocrine activity is essential for assessing the reproductive potential, but hormonal determination trough blood serum sampling is an unpractical procedure in these species, as it requires anaesthesia and restraint. It has been demonstrated in the domestic cat that estrogens and progesterone are secreted into faeces (1,2) and some Authors already applied this method to other wild felids (1,3,4), as well as to cheetahs (5,6). However, cheetah management, i.e. social groups and caging situations, was not standardized throughout the period of the study or a low number of samples were collected during short time periods. The aim of the present work was to evaluate the dynamic of faecal estrogens and progesterone throughout an entire year in two cheetahs constantly housed with conspecific males in the Northern of Italy. Materials and methods. Two female cheetahs (Geijsha and Duchessa, 2 years of age) maintained at Le Cornelle Animal Park (Valbrembo, BG, Italy) and housed with 3 conspecific males were included in this study. Faecal samples were collected daily for one year (from August 2007 to July 2008) and immediately frozen and stored at -20\ub0C. Faecal oestradiol (E2) and progesterone (P4) metabolites were extracted from fresh samples as previously described in dry samples (1). The metabolites were quantified using radioimmunoassay. Basal E2 and P4 values and the duration of the estrous cycle on the whole sampling period were calculated according to the method proposed by Brown et al., 1996 (6). Data are resumed as mean \ub1SD. Results. Faecal E2 basal values were 21.6+5.8 ng/g and 19.3+5.1 ng/g with peak concentrations ranging from 30.6 to 73.8 ng/g and from 29.6 to 164.8 in Geijsha and Duchessa, respectively. Estrous cycle duration was 10.4\ub13 (n=13, range, 6-15 days) and 11.3 \ub1 6.9 days (n=10, range, 5-23 days) in the two females. The males did not show any interest in the females which did not display evident behavioural signs of estrus throughout the year. Faecal P4 basal values of 112.6+41.5 ng/g and 100.6+23.4 ng/g remained at baseline values all year. In addition, none of the female were continuously cyclic: follicular activity was interrupted by anestrous periods of 1 to 4 months in duration and not associated with season. Conclusions. These data suggest that captive cheethas show a discontinuos ovarian ciclicity which duration and anestrus periods are similar to those described in the literature (6) in which estrous cycle duration from 10.4\ub11 to 19\ub12.2 days and anestrous periods of 2 to 5 months are reported. In the present study extraction of steroid metabolites was performed from fresh faecal sample, thus a comparison with hormonal concentrations in dry samples reported by other authors is not possible. It is generally accepted that behavioural signs of estrous are difficult to interpret in cheetahs, however lack of mating and of subsequent luteal phase were likely due to the continous presence of males which sexual interest was inhibited. Thus, adequacy of husbandry conditions is necessary and enforced social living should be avoided in order to increase reproductive activity of captive cheethas. Aknowledgements. We thank E. Benedetti, N. Benedetti and Dr. R. Schneider of Le Cornelle Animal Park for logistical support and sample collection. This study was supported by PUR 2008. References. 1) Brown et al., Biol Reprod 1994;51:776-86; 2) Shille et al., Zoo Biol 1984;3:201-09; 3) Brown et al., J Reprod Fertil 2001;Suppl 57:71-82; 4) Graham et al., Zoo Biol 1995;14:233-37; 5) Czekala et al., Zoo Biol 1994;13: 119-28; 6) Brown et al., J Reprod Fertil 1996;106:337-46

Bioenergetic/oxidative balance in feline morulae and blastocysts produced in vitro after temporary oocyte holding at room temperature versus cold ovary storage.

Maternal aging is associated with an increase in embryonic aneuploidy and early miscarriage, as a result of errors in chromosome segregation [1]. Failure to achieve correct alignment of the chromosomes on the spindle is an important factor contributing to mis-segregation errors in oocytes [2,3]. Since little data is available in horses, the aim of this study was to evaluate the effect of maternal aging on spindle morphology and the incidence of chromosomes misalignment. Cumulus oocyte complexes (COCs) were recovered from slaughtered mares, divided into groups depending on mare age (young, < 15 years; old, ≥15 years) and matured in vitro for 26h. After maturation and denudation, only the oocytes that had reached MII were used and these were further subdivided into Control and Nocodazole groups (total = 4 groups; n=20 per group). Oocytes in the Nocodazole groups were incubated for 10 min in medium containing 20 μM Nocodazole, to induce tubulin depolymerization, washed and then incubated for 2 hrs in maturation medium. In order to destabilize any tubulin fibers not properly attached to the kinetochores samples were subjected to cold shock, fixed and stored at 4 ̊C prior to immunofluorescent staining for DNA and alphatubulin. Spindle morphology and the incidence of chromosome misalignment were evaluated by confocal microscopy and 3D image analysis (Imaris 8.3). Spindle morphology was scored as normal (fusiform, bipolar) or abnormal (tri- or tetra-polar, severely misshapen), chromosome misalignment was scored as absent, mild (1-5 misaligned chromosomes) or severe (>5 misaligned chromosomes). Oocytes from aged mares showed higher rates of mild and severe chromosome misalignments when compared to those from young mares, both in normal condition (mild 37 vs 5%; severe 11 vs 0%) and after Nocodazole treatment (mild 42 vs 15%; severe 21 vs 0%). Moreover, oocytes from old mares were more likely to show abnormal spindle morphology both under control conditions (5 vs 0%) and after Nocodazole treatment (10 vs 0%). Although nocodazole treatment did not result in a significant increase in chromosome misalignment and spindle abnormalities, the incidence of chromosomal misalignments increased numerically in both aged and young groups (total % of misalignment without treatment 47.4 and 4.5% vs 63.2 and 15% after nocodazole treatment). We suggest that the compromised ability to form a normal meiotic spindle and correctly align the chromosomes observed in MII oocytes from aged mares might reflect an impaired function of the spindle assembly check point components, and explain the age-related reduction in oocyte developmental competence

Matrix‐assisted laser desorption/ionization imaging mass spectrometry for the spatial location of feline oviductal proteins

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Contents With the purpose of identifying factors involved in early stages of embryo development in the domestic cat, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was used for the first time to describe the spatial localization of proteins in the oviducts of queens. Oviducts were obtained from two 2 and 4years old cross-bred queens, divided into three segments, snap-frozen in liquid nitrogen and then stored at -80 degrees C until use. Next, they were sectioned in a cryostat, fixed on ITO (indium tin oxide) conductive glass slides for MALDI-IMS and serial sections were collected on microscope slides for histology. As confirmed by histology, MALDI-IMS was able to show contrasting protein distributions in the oviductal infundibulum, ampulla and isthmus. Mass spectra were characterized by abundant ions of m/z 1,259, 4,939, 4,960 and 10,626, which have been tentatively attributed to keratin, thymosin 10, thymosin 4 and S100, respectively. Keratin and thymosins are involved in the biological response to tissue damage. S100 proteins are calcium-modulated proteins implicated in a variety of cellular activities, including cell differentiation and regulation of cell motility. These results suggest that protein composition differs between segments of the cat oviduct, which corresponds to morphological changes within these sections. Further functional studies could elucidate the effects of these proteins on feline reproductive physiology.With the purpose of identifying factors involved in early stages of embryo development in the domestic cat, matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI‐IMS) was used for the first time to describe the spatial localization52S28892CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)501059/2013‐0sem informação8th International Symposium on Canine and Feline ReproductionJUN 22-25, 2016Paris, FRANC