NMR characterization of animals’ follicular fluids

Follicular Fluid (FF) provides a special environment to the oocyte during its maturation in vivo. The FF is derived from the sanguineous plasma and secretions, synthesised in the follicle wall that contain a large variety of metabolites (1). These metabolites are probably involved in the physiology of the oocytes (1). The chemical composition of follicular fluids is important because it is an indicator of the secretory activities and metabolism of follicular cells and thus could be related to the follicular quality. It could also provide a useful indication of the oocyte growth and maturation (2). High Resolution Nuclear Magnetic Resonance (NMR) spectroscopy provides a unique tool for studying metabolites. Initially, NMR spectroscopy was used mainly in biomedicine but it is found now in many physiological applications (3). As the NMR spectroscopy provides opportunities for obtaining qualitative and quantitative data from body fluids, it was hypothesized that this technique could provide information on mammals’ follicular fluid and on intrafollicular changes that occur during follicular growth and ovulation. As some of these changes are probably of crucial importance for oocyte developmental competence, a better knowledge of the mammals’ follicular fluid composition by 1H NMR analysis should help to resolve some of the problems encountered during in vitro procedures in the mammals. The characterization of the chemical composition of mammals follicular fluids, namely sheep, cattle, mare and pork, and the study of the changes observed during follicular growth and maturation using NMR spectroscopy will be presented. FF samples were collected from antral follicle of different dimensions. One-dimensional (1D) 1H experiments (CPMG, DOSY) were obtained for all the FF samples. In addition, several two dimensional (2D) (homo and heteronuclear) experiments (DQF-COSY, TOCSY, JRES, 1H-13C HSQC) were performed to aid in the assignment of the signals and in the identification of the metabolites in FF. A direct evaluation of the lipids, carbohydrates and metabolites were obtained from the combination of the 1D and 2D NMR experiments

Hypoluteoidism in a dog associated with recurrent mammary fibroadenoma stimulated by progestin therapy

Abstract Background Hypoluteoidism in the bitch is characterized by insufficient production and secretion of progesterone by the corpora lutea. It is a rare pathologic condition and during pregnancy, it leads to embryonic resorption or fetal abortion. Supplementary therapy with progestins is indicated during pregnancy to obtain delivery of vital puppies but unwarranted side effects of such treatment are poorly documented. Case presentation A 4-year-old, nulliparous, female Istrian Shorthaired Hound dog had been mated repeatedly in six heats with different dogs of proven fertility but signs of pregnancy did not develop. Estrous cycles, mating and pregnancies were monitored as hypoluteoidism or genital disease was suspected. During the first monitored estrus, the bitch was mated and on day 18 [day 0, day of estimated peak of luteinizing hormone (LH)], ultrasound examination showed three amniotic vesicles that were however found to be resorbed between day 20 and 23. Progesterone concentrations, measured by ELISA, were >8 ng/mL until day 12 and 1–2.5 ng/mL on days 20, 23 and 26. Primary hypoluteoidism was therefore suspected. In the second monitored estrus, the bitch was mated and during pregnancy, progesterone concentrations were >8 ng/mL until day 17 and 1–2.5 ng/mL on day 19. On days 20 and 22, two out of three embryonic vesicles had been resorbed. The bitch was treated with progesterone in oil from day 19 to day 58. Increase in the size of 2nd left thoracic mammary gland (T2-L) was observed and on day 46, ultrasound evaluation and biopsy were performed revealing a low-cellularity fibroadenoma. Parturition started spontaneously at day 65 but due to dystocia caused by fetal macrosomia, a Caesarean section was performed. During the next (third) monitored estrus, the bitch was bred again and during pregnancy, early decrease in progesterone concentration confirmed the diagnosis of primary hypoluteoidism. The bitch was treated with synthetic progestin (altrenogest) from day 8 to day 57. Five amniotic vesicles were detected by ultrasonography. Recurrence of swelling of T2-L was observed. On day 60, the bitch whelped five pups, two males and three females. As reported later by the owner, the latter did not show any sign of heat over the past 3 years. In one of them, clitoral hypertrophy and a blind ending vagina were diagnosed. Conclusions This is the first description of early hypoluteoidism in a pregnant bitch developing a mammary fibroadenoma under progestin treatment

Reproductive Performance Following Transcervical Insemination with Frozen Thawed Semen in Ewes Submitted to Surgical Incision of Cervical Folds (SICF): Comparison with Laparoscopic Artificial Insemination

Transcervical artificial insemination (AI) after the surgical incision of cervical folds (SICF) could represent a valid alternative to laparoscopic AI when frozen thawed semen is used. The aim of this experiment was to compare pregnancy (PR) and lambing rates (LR) of ewes submitted either to transcervical AI after SICF or to laparoscopic AI using frozen thawed semen. Pregnant at term ewes (n = 80) were allocated in two experimental groups. After lambing, one group (n = 39) was submitted to SICF. The remaining ewes that were regularly lambed were allocated to the group of laparoscopic AI (n = 40). Six months later, oestrous cycle of both experimental groups was synchronised and all ewes were artificially inseminated with frozen thawed semen. Ewes submitted to SICF underwent transcervical insemination and intrauterine deposition of semen was recorded. The remaining animals were submitted to laparoscopic AI. Pregnancy and LR were recorded. Intrauterine deposition of semen was possible in 89.7% pf ewes submitted to SICF. This group showed similar PR and LR compared to the laparoscopic group (respectively: PR, 71.8% vs. 70% and LR, 64.1% vs. 65%; p > 0.05). Transcervical AI after SICF may represent a valid alternative to laparoscopy in AI protocols requiring the use of frozen thawed semen

The COVID-19 Outbreak May Be Associated to a Reduced Level of Care for Breast Cancer. A Comparative Study with the Pre-COVID Era in an Italian Breast Unit

The recent COVID-19 pandemic has caused profound changes to healthcare systems as well as had deleterious repercussions on the care of cancer patients. In this comparative study, we sought to evaluate the effects of the COVID-19 pandemic on the surgical management of breast cancer in a breast unit in an Italian region with a low incidence of COVID-19 infection. Eighty-three patients were included, of whom 41 received surgery during the height of the pandemic (Group A, operated on between March and April 2020), and 42 during the same period of the year in 2019 (Group B). Clinicopathological characteristics and surgical outcomes were compared between the two groups. There were no significant differences in the baseline characteristics of the two groups with regard to age (p = 0.62), tumour size (p = 0.25), grade (p = 0.27), histology (p = 0.43), positive lymph node status (p = 0.35), and ER positive status (0.35). Waiting time for surgery was slightly longer in Group A (49.11 vs. 46.39 days, p = 0.38). Patients receiving immediate breast reconstruction were significantly less in Group A (p < 0.001). The use of sentinel node biopsy was similar in the two groups (p = 0.84). Hospital stay was longer in patients of Group B (p = 0.008). The use of regional nerve blocks was lower in Group A (p < 0.001). Patients operated on during the height of the pandemic were less likely to receive immediate reconstruction and regional nerve blocks during surgery. These features configure a situation of reduced level of care for patients with breast cancer. Efforts should be taken by the healthcare systems to maintain standard of care, even in case of a new peak in the coronavirus outbreak

Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 °C in the domestic cat model

Abstract Background Storage conditions during transportation of explanted ovaries are a critical step in setting up fertility preservation protocols in both animal and human fields. Here, we evaluated the effects of ovary storage at 4 °C on the preservation of preantral follicles and oocytes retrieved from antral follicles using the domestic cat as model. Methods Ovaries were harvested from fifty-five healthy domestic queens during ovariectomy and stored at 4 °C for 0 (control), 24, 48, 72 and 96 h. In Experiment 1, the effects of the storage period at 4 °C on the morphology, cytoskeleton (α/β tubulin) and DNA integrity (phosphorylation of histone H2AX) of preantral follicles were investigated. In Experiment 2, oocytes recovered from antral follicles were matured and fertilized in vitro to evaluate their meiotic and developmental competence. Reactive oxygen species (ROS), glutathione (GSH) and lipid peroxidation were measured in matured oocytes. Results The results showed that: a) storage up to 24 h did not affect the morphology and the DNA integrity of preantral follicles; b) extended storage times caused progressive morphological abnormalities, disassembling of microtubules and DNA damage; c) storage up to 48 h did not influence in vitro meiotic maturation of oocytes nor cleavage after in vitro fertilization. However, only oocytes stored within the ovary for 24 h produced blastocysts in a percentage similar to control oocytes; d) GSH levels of in vitro matured oocytes did not change at any time during ovary storage; a progressive increase in ROS levels was detected from 48 h associated with elevated lipid peroxidation at 72 and 96 h of storage. Conclusions Storage of cat ovaries for up to 24 h caused minimal alteration of preantral follicles and oocytes. The extension of the storage period beyond 24 h progressively impaired the structure of follicles, and modified the oxidative status of in vitro matured oocytes and their developmental competence after in vitro fertilization. This information may help when setting up programs for fertility conservation, especially for wild feline species which die in geographic areas located far away from ARTs centers